Handbook of Oxidative Stress in Cancer: Mechanistic Aspects 9811594104, 9789811594106

Table of contents :
Preface
Contents
About the Editors
About the Section Editors
Contributors
1 Reactive Species and ER-Mitochondrial Performance for Glioblastoma Multiforme Treatment Strategy
Introduction
Current Status of Glioblastoma Multiforme (GBM)
Reactive Species and [Ca2+]i on ER Stress and Mitochondrial Performance
ER Stress, Mitochondria, and GBM
Genetic Alteration by Reactive Species in GBM
Angiogenesis, NADPH Oxidase, and GBM
Implications in the Treatment of Gliomas
Maintenance of Reactive Species Steady State
Targeting ER Components and Autophagy
Other Molecular Targets
Conclusion
References
2 Oxidative Stress and Thyroid Disorders
Introduction
Hyperthyroidism and OS
Papillary Thyroid Carcinoma and OS
Medullary Thyroid Carcinoma and OS
Anaplastic Thyroid Carcinoma and OS
Conclusion
References
3 Skin Cancer Induced by Pollution-Mediated ROS
Introduction
Solar Ultraviolet (UV) Radiation
Synergy: UVA Photo-Chemo Pollution
Ozone
Particulate Matter (PM)
Tobacco Smoke
The Aryl Hydrocarbon Receptor (AHR)
The Aryl Hydrocarbon Receptor (AHR) and Skin Cancer
Squamous Cell Carcinoma (SCC)
Basal Cell Carcinoma (BCC)
Malignant Melanoma
Topical Antioxidants to Protect from Environmental Pollution
Conclusion
Cross-References
References
4 Roles of β-Glucans in Oxidative Stress and Cancer
Introduction
Cancer
Carcinogenesis and Cachexia-Anorexia Syndrome
Risk Factors
Cancer Therapy
Oxidative Stress
Sources and Effects of Reactive Oxygen Species
ROS and Antioxidant Mechanisms
Cancer and Oxidative Stress
β-Glucans
Structural Characterization of β-D-Glucans
Anticancer Effects of β-Glucans
β-Glucans, Oxidative Stress, and Cancer
Conclusions
References
5 Oral Cancer and Oxidative Stress
Introduction
Oral Carcinogenesis
Oxidative Stress
Oxidative Stress and Oral Carcinogenesis
Conclusion
Cross-References
References
6 Oxidative Stress in Genitourinary Cancer
Introduction
Kidney Cancer
Urothelial Cancer
Prostate Cancer
Anticancer Therapy for Genitourinary Cancer and Oxidative Stress
Conclusions
References
7 Oxidative Stress, Microenvironment, and Oral Cancer
Introduction
Inflammation, Reactive Oxygen Species, and Cancer
Reactive Oxygen Species and Inflammation
Inflammation, Reactive Oxygen Species, and Cancer
Tumor Cell Survival
Tumor Cell Proliferation
Tumor Cell Invasion
Angiogenesis
Oral Squamous Cell Carcinoma and Reactive Oxygen Species
Tumor Microenvironment and Reactive Oxygen Species
Reactive Oxygen Species in Oral Cancer
Oncoviruses, Oral Squamous Cell Carcinoma, and Reactive Oxygen Species
Reactive Oxygen Species as an Appealing Target for Intervention (Table 8)
Reactive Oxygen Species as Alluring Targets for Therapeutic Intervention
Reactive Oxygen Species and Oral Potentially Malignant Disorders
References
8 Oxidative Stress and Glyoxalase Pathway in Cancer
Introduction
Glyoxalase System
Glyoxalase I (GLOI)
Glyoxalase II (GLOII)
Glyoxalase III (GLOIII)
Methyglyoxal and Cancer
Methylglyoxal-Induced Reactive Oxygen Species (ROS)
Glyoxalase Enzymes and Cancer
Tumor Hypoxia, Cancer Cell Stemness, and Glyoxalase Expression
Dual Role of Glyoxalase I in Cancer
Modulation of Cellular Pathways by GLOI Overexpression
Effect of Ionizing Radiation on Glyoxalase Expression
Therapeutic Potential of Glyoxalase Pathway in Cancer
Exogenous Application of Methylgloxal for Cancer Therapy
Inhibition of Glyoxalase Enzymes as Treatment Modality in Cancer
Phytochemicals as Modulators of Glyoxalase Pathway
Naringin
Galangin
Curcumin
Oleuropein (OP)
Conclusions and Future Directions
References
9 The Implication of ROS Homeostasis in the Modulation of EMT Signaling and Its Role in Manipulating Tumor Microenvironment
Introduction
The Implication of Reactive Oxygen Species in Favoring Epithelial-to-Mesenchymal Transition (EMT)
ROS as a Signaling Molecule Controlling Pro-metastatic Responses
Regulation of Cytoskeletal Proteins by Reactive Oxygen Species: Consequences on Cellular Motility
Manipulations of Tumor Microenvironment by Reactive Oxygen Species to Expedite Metastasis
The Integrative Role of ROS During Hypoxia in Facilitating Tumor Metastasis and Angiogenesis
Conclusion
References
10 Functional Regulation Between Matrix Metalloproteases and Cell Junction Proteins in Gastric Cancer
Introduction
Matrix Metalloproteinases (MMPs) and Gastric Cancer
Cell Junction Proteins in Gastric Cancer
Tight Junctions
Gap Junction
Adherens Junction
Crosstalk Between of MMPs and Cell Junction Proteins
MMP and Adherence Junction Proteins
MMPs and Tight Junction Proteins
MMPs and Gap Junction Proteins
Conclusion and Future Directions
References
11 Association of Oxidative Stress and Mitochondrial Dysfunction to Gynecological Malignancies
Introduction
Oxidative Stress, ROS, and Antioxidants
ROS Paradox: Dual Effect on Cancer Cells
Mitochondria as a Hub of Intrinsic ROS Generation
mtROS: Prime Oncogenic Factor
ROS: A Key Driver of Gynecological Cancer Progression
ROS-Driven Metabolic Reprogramming: A Prime Hallmark of Cancer
Implications of ROS-Induction on Signaling Aberrations in Cancer
ROS Synchronizes Tumor Microenvironment Components in Gynecological Cancers
ROS-Based Therapeutic Implications in Cancer
Conclusion
References
12 Impact of Caenorhabditis elegans in Cancer Drug Resistance Development
Introduction
Hormesis
Cancer and Chemoresistance
Hormetic Stressors
Conclusion
References
13 Scaffold-Based Selective ROS Generation as Viable Therapeutic Strategies Against Cancer
Introduction
Background
Molecular Basis of ROS Production
ROS Paradox in Cancer
Modulation of ROS as a Therapeutic Target
Reduction of ROS Levels: Antioxidants and Nutraceuticals
Induction of ROS Levels: Scaffold-Based Chemotherapeutics
Quinone
Nitrogen Mustard
Nitrosoureas
Organic Endoperoxides
Organic Di- and Polysulfides: Diallyl Trisulfide and Varacin
Organosulfur Isothiocyanate: Sulforaphane and β-Phenylethylisothiocyanate
Peptide and Nucleoside
Taxane, Alkaloid, and Steroid
Non-metal and Metal
Miscellaneous Scaffolds (Procarbazine, Elesclomol, Erastin, Celecoxib)
Prodrug (Masking-Demasking)
Conclusion and Future Direction
Cross-References
References
14 Targeting Oxidative Stress in Cancer
Introduction
Mechanism of Oxidative Stress Toward Carcinogenesis
Amelioration of Oxidative Stress Toward Prevention of Cancer
Natural Compounds
Some Natural Compounds Act as Antioxidants (Table 1)
Role of Synthetic Compounds
Biomimic or Nanoflowers
Advanced Therapeutics
Targeted Therapy
Conclusion
References
15 Targeting Mitochondria as a Novel Disease-Modifying Therapeutic Strategy in Cancer
Introduction
Etiology of Cancer
Tobacco
Environmental Carcinogens
Diet, Obesity, and Physical Activity
Mitochondrion-Structural Components and Functions
Structure of Mitochondria
Mitochondrial DNA
Functions of Mitochondria
Electron Transport Chain in Mitochondria
Reactive Oxygen Species in Mitochondria
Mitochondrial Dysfunction
Therapeutic Targets in Cancer Pathology
Mitochondrial Medicine in Cancer
Bioenergetic Therapy in Cancer
Conclusion and Future Perspective
References
16 Cutaneous Unfolded Protein Response (UPR) and Endoplasmic Reticulum (ER) Stress
Introduction
Sensor-Transducers of the Cutaneous UPR: A Mechanistic Overview
The Cutaneous UPR and Environmental Stressors: An Overview
The UPR and Skin Photodamage
The Cutaneous UPR and Ionizing Radiations
The Cutaneous UPR and Toxic Metals
The Cutaneous UPR and Paraquat
The Cutaneous UPR and Cigarette Smoking
The UPR and Particulate Matter
The UPR and Chlorine Exposure
The UPR and Phenolics
The Cutaneous UPR and Cosmetic Products
The UPR and the Lewisite
The Cutaneous UPR and Sulfur Mustard
The Cutaneous UPR and Formaldehyde Exposure
Viruses and Cutaneous UPR
Bacteria and Cutaneous UPR
The UPR Link with Cutaneous Pathologies
Role of UPR in Skin Keratinization and Darier´s Disease
Role of UPR in Keratosis Linearis with Ichthyosis Cogenita and Keratoderma (KLICK) Syndrome
Role of UPR in Erythrokeratoderma Variabilis (EKV)
Role of UPR in Ichthyosis Follicularis with Atrichia and Photophobia (IFAP) Syndrome
Role of UPR in Keratosis Follicularisspinulosa Decalvans (KFSD)
Role of UPR in Rosacea
Role of UPR in Epidermolysis Bullosa Simplex
Targeting ER Stress for Therapy
Therapeutic Targeting of Abnormal UPR in Hereditary Keratosis
Enhancing Protein Folding Capacity
Targeting Individual UPR Pathways
IRE1α Inhibitors
PERK Inhibitors
ERAD Modulators
Modulators of ER Chaperones of ER Stress Signaling
Concluding Remarks and Future Directions
References
17 Iron Sulfur Clusters and ROS in Cancer
Introduction
Iron Sulfur Clusters Proteins: Types of Fe-S
Biochemistry of Fe-S Proteins
Major Proteins Containing Iron Sulfur Clusters in Biological Systems
Fe-S Cluster Biosynthetic Machinery
Mitochondrial Iron-Sulfur Cluster (ISC) Assembly Machinery: Proteins and Steps
Oxidative Stress and Fe-S Cluster Damage
How Iron-Sulfur Clusters Generate Reactive Oxygen Species
Iron Sulfur Cluster Proteins Linked with Cancer
MitoNEET- the Redox Sensor
Iron-Sulfur Cluster Assembly Enzyme
Aconitase -the Fe-S Regulator
Frataxin
Nutrient-Deprivation Autophagy Factor-1 (NAF-1)
Ferredoxin Reductase
Therapeutic Drugs for Cancer That Use or Target Fe-S Clusters
Drugs That Generate ROS Via Fe-S Clusters
Conclusion
References
18 Free Radicals, Reactive Oxygen Species, and Their Biomarkers
Introduction
Free Radicals and Reactive Oxygen Species
Radicals and Free Radicals
Reactive Oxygen Species and Oxygen Free Radicals
Metal Free Radicals
Iron
Copper
Manganese
Zinc
Oxidative Stress Biomarkers in Cancer
Single Oxidation or Antioxidant
Total Oxidant Status, Total Antioxidant Status, and Oxidant Stress Index
Measurement of Total Antioxidant Status
Measurement of Total Oxidant Status
Calculation of Oxidant Stress Index
End Products of Lipid Hydroperoxide
Conclusions
References
19 Zymographic Techniques
Introduction
Matrix Metalloproteinases in Cancer
Types of Zymographic Techniques
Basic Protocol of Zymography to Map MMP Activities
Materials for Zymography Experiment
Sample Preparation
Preparation of Gel
Sample Loading and Gel Electrophoresis
Analysis
Antioxidative Enzymes as Cancer Target
Zymographic Techniques for Antioxidative Enzymes
Zymography Protocol for Catalase Enzyme
Zymography Method for SOD Enzyme
Conclusion
References
20 Biomarkers of Oxidative Stress and Its Dynamics in Cancer
Introduction
Mitochondrial Biomarkers of Oxidative Stress
Regulation of Mitochondrial Dynamics and Its Biogenesis
Oxidative Stress in Mitochondrial Dynamics and Its Pathophysiology
Pharmacological Strategies to Target Oxidative Stress Biomarkers
Impact of Oxidative Stress in Mitochondrial Dynamics
Conclusions
References
21 Glutathione as Oxidative Stress Marker in Cancer
Introduction
Reduced and Oxidized Glutathione
Prodrugs and Co-drugs of GSH
Cancer and Oxidative Stress Mechanism
Phase 1 and Phase 2 Detoxification Pathways
Phase 1 Pathway
Phase 2 Pathway
Metal Homeostasis
Chromium
Copper
Iron
Glutathione, Metallothionein, and Cancer
Glutathione, Free Radicals, and Antioxidants
Free Radicals and Reactive Oxygen
Antioxidants
ROS and Antioxidants Balance
Cancer and Glutathione
Glutathione, Cancer, and Oxidative Stress
Type of Mechanism on Glutathione as a Marker for Cancer
Other Oxidative Stress Markers Resembling Glutathione (Benedette 2018; Payal et al. 2016) (Table 3)
The Role of Selenoproteins and Glutathione in Cancer
Antioxidants May Make Cancer Worse
Conclusion
Cross-References
References
22 Salivary Oxidative Stress Biomarkers in Oral Potentially Malignant Disorders and Squamous Cell Carcinoma
Introduction
Salivary Diagnostics
Tumor Biomarkers and Its Significance
Oral Squamous Cell Carcinoma and Oral Potentially Malignant Disorders
Oxidative Stress Biomarkers
Salivary Oxidative Stress Biomarkers in Oral Cancer
Conclusion
Cross-References
References
23 Recent Development of Monoclonal Antibodies Targeting Tyrosine Kinase in ROS-Mediated Cancer
Introduction
Reactive Oxygen Species (ROS) and Its Importance in Cancer and Its Treatment
MAbs-Based Therapy for ROS-Mediated Cancer
Recent Development of mAbs Targeting Tyrosine Kinase of ROS-Mediated Cancer
Anti-EGFR Monoclonal Antibodies
Anti-HER2 Monoclonal Antibodies
Future Scope and Conclusion
References
24 Fluoride as a Carcinogen: A Myth or Fact?
Introduction
Fluoride and Cancer
Fluoride and Apoptosis
Conclusions
References
25 The Role of ROS in Chemical Carcinogenesis Induced by Lead, Nickel, and Chromium
Introduction
The Role of ROS in Chemical Carcinogenesis Induced by Lead
The Role of ROS in Chemical Carcinogenesis Induced by Nickel
The Role of ROS in Chemical Carcinogenesis Induced by Chromium
Concluding Remarks
References
26 Environmental Contaminants, Oxidative Stress, and Reproductive Cancer
Introduction
Epidemiological Aspect of Reproductive Cancer
Environmental Causes of Female Reproductive Cancer
Environmental Causes of Male Reproductive Cancer
Endocrine Disruptors and Reproductive Cancer
Pesticides and Reproductive Cancer
Obesogens and Reproductive Cancer
Oxidative Stress and Reproductive Cancer
Antioxidants and Reproductive Cancer
Conclusions
References
27 Environmental Toxicants and Carcinogenicity: Role of Oxidative Stress
Introduction
Heavy Metals, Oxidative Stress, and Cancer
Pesticides, Oxidative Stress, and Cancer
Conclusion
References
28 Environmental and Occupational Exposure to Pesticides and Cancer Development
Introduction
Environmental and Occupational Exposure to Pesticides
Herbicides
Organochlorine Pesticides
Organophosphate Pesticides
Conclusion
References
29 Benzo(a)Pyrene-Induced ROS-Mediated Lung Cancer
Introduction
Benzo(a)Pyrene and Lung Cancer
Source and Route of B(a)P
B(a)P Metabolism
B(a)P and Oxidative Stress
Radical Cations and B(a)P
Redox-Active Quinone Radical
DNA Adducts
Oxygen Free Radicals and DNA Adducts
Role of Free Radicals in Cigarette Smoking
Role of Oxygen Radicals in Initiating Tumors
Oxygen Radicals as Tumor Promoters
Conclusion
References
30 Essential Role of Occupational Hazards in Cancer Among Women
Introduction
Breast Cancer
Colon Cancer
Gastrointestinal Cancers
Ovarian and Uterine Cancers
Lung Cancer
Skin Cancer
Conclusion
References
31 Arsenic: An Environmental Toxicant-Induced Oxidative Stress and Carcinogenesis
Introduction
Skin Cancer
Lung Cancer
Bladder Cancer
Liver Cancer
Kidney Cancer
Cellular Metabolism of Arsenic
Role of ROS in Arsenic Carcinogenicity
Conclusion
Cross-References
References
32 ROS in Apoptosis of Cancer Cells
Introduction
Definitions for Cancer Cells, ROS, and Apoptosis
Relationship Between Apoptosis and ROS
ROS in Regulation of the Mitochondrial or Intrinsic Apoptotic Pathway
ROS in Regulation of the Death Receptor or Extrinsic Apoptotic Pathway
Apoptosis of Cancer Cells Triggered by ROS
Conclusion
References
33 Role of ROS in Triggering Death Receptor-Mediated Apoptosis
Introduction to Apoptosis
Apoptosis Signaling Pathways
General Features of Apoptosis
Caspase
Main Mechanisms of Apoptosis Regulation and Execution
The Mitochondrial or Intrinsic Pathway
The Death Receptor or Extrinsic Pathway
Endoplasmic Reticulum Pathway
ROS and Apoptosis
The Intrinsic Pathway and ROS
ROS and the Extrinsic Pathway
The ER Pathway and ROS
ROS in Apoptosis and Cell Survival in Cancer Cells
ROS Mediator and Executor of Apoptosis
Conclusions
References
34 Advanced Glycation End Products-Mediated Oxidative Stress and Regulated Cell Death Signaling in Cancer
Introduction
Receptor for Advanced Glycation End Products (RAGE)
AGES and RAGE in Cancer
Oxidative Stress and AGE/RAGE Signaling in Cancer
AGE/RAGE and Regulated Cell Death Signaling in Cancer
Conclusion
References
35 Helping Leukemia Cells to Die with Natural or Chemical Compounds Through H2O2 Signaling
Introduction
An Enemy Within Myself
Making Fun of Death
Oxidative Distress: The Dose Makes Life or Death Signals
H2O2: ``A match that starts bush fires´´
Quo Vadis?
Conclusion
References
36 Microtubule-Targeting Agents Induce ROS-Mediated Apoptosis in Cancer
Introduction
Importance of Microtubules in Anticancer Therapy
ROS: A Friend or Foe
ROS in Cancer Development and Metastasis
Cytotoxic Role of ROS in Cancer Cells
Mitochondria: The Missing Link Between MTAs and ROS Generation in Cancer Cells
Modulation of ROS-Mediated Apoptotic Signaling by MTAs
Microtubule Targeting Agent Kills Cancer Cells by Modulating ROS Induced Autophagy
Conclusion
References
37 ROS Induced by Chemo- and Targeted Therapy Promote Apoptosis in Cancer Cells
Introduction
ROS: A Double-Edged Sword
ROS as an Oncogene
ROS as a Tumor Suppressor
ROS-Mediated Apoptosis in Malignant Cells by Targeted Therapy
Tyrosine Kinase Inhibitors
PIM Kinase Inhibitors
ROS-Mediated Apoptosis in Malignant Cells by Chemotherapy
Strategies and Drugs Targeted to Increases ROS-Mediated Apoptosis in Malignant Cells
Conclusion
Cross-References
References
38 ROS-Mediated Apoptosis in Cancer
Introduction
ROS in the Mitochondria-Mediated Intrinsic Apoptotic Pathway
ROS in the Death Receptor-Mediated Apoptotic Pathway
ROS in p53-Mediated Apoptosis
ROS in ER Stress-Induced Apoptosis
ROS in Calcium-Mediated Apoptosis
Prooxidant-Based Cancer Therapy
Conclusion
Cross-References
References
39 Genomic Instability in Carcinogenesis
Introduction
Genomic Instability - An Overview
Genomic Instability and Cancer
The Role of Oxidative Stress in Genomic Instability in Cancer
Oxidative Stress Induced Genomic Instability in Carcinogenesis: Mechanism and Evidences
The Role of Oxidative Stress-Induced Lipid Peroxidation in Genomic Instability
Cellular Defense Mechanism against Oxidative Stress-Induced Genomic Instability
Conclusion and Future Perspectives
References
40 Impact of Environmental and Occupational Exposures in Reactive Oxygen Species-Induced Pancreatic Cancer
Introduction
Epidemiology of Pancreatic Cancer with Incidence and Prevalence
Prevalence/Geographic Distribution of Pancreatic Cancer
Environmental, Occupational, Lifestyle, and Genetic Risk Factors for Pancreatic Cancer
Age, Gender, and Race
Smoking and Tobacco
Alcohol
Dietary Factors
Vitamin D and UVB Irradiation
Obesity
Occupational Exposures of Heavy Metals
Chronic Pancreatitis
Infections
Genetic Risk Factors
Current Treatment of Pancreatic Cancer
Reactive Oxygen Species (ROS)/Oxidative Stress Promotes Cancer Development
Reactive Oxygen Species (ROS) in Relation to Pancreatic Adenocarcinoma
Environmental Risk Factor of Pancreatic Cancer: Tobacco Smoking in Relation to Reactive Oxygen Species
Occupational Risk Factor of Pancreatic Cancer: Cadmium Heavy Metal in Pesticide in Relation to Reactive Oxygen Species (ROS)
Cadmium
Conclusions
References
41 Reactive Oxygen Species: Central Regulators of the Tumor Microenvironment
Introduction
The Origin of ROS: Mitochondrial and Nonmitochondrial Sources
Major Producers of ROS in the TME
Molecular Events Triggered by ROS: Impact on Tumorigenesis
Modulation of Immune Cells by Tumor Microenvironment-Associated Oxidative Stress
Natural Killer Cells
Dendritic Cells
T-Cells
Treg Cells
ROS Involvement on Immunomodulation by Antitumor Therapeutics Immunotherapy
Conclusion
References
42 Biomarkers of Oxidative Stress-Induced Cancer
Introduction
Oxidative Stress and Cancer
Biomarkers of Oxidative Stress and Role in Cancer Diagnosis and Treatment
Oxidative Stress Biomarker Research in Breast Cancer
Oxidative Stress Biomarker Research in Gastro-Intestinal and Colorectal Cancer
Oxidative Stress Biomarker Research in Prostate Cancer
Biomarkers of Oxidative Stress Research in Lung Cancer
Biomarkers of Oxidative Stress Research in Ovarian Cancer
Biomarker of Oxidative Stress in Cervical Cancer
Conclusion
References
43 Reactive Oxygen Species in Male Reproductive Cancers
Introduction
ROS in Prostate Cancer Development
Imbalance in ROS Generation and Antioxidant Protection in Prostate Leads to mtDNA Mutations
Mutations in Mitochondrial Genome and Correlation with Prostate Cancer
NADPH Oxidase Is a Source of ROS for Prostatic Cancer Cells
Androgens in Regulation of ROS Generation in Prostate Cancer
Ageing and ROS in Incidence of Prostate Cancer
Cancer Prevention by Dietary Antioxidants?
Testicular Cancer
Regulation of ROS and Aerobic Glycolysis in Testicular Cancer
Heat Stress and Testicular Cancers
Penile Cancer
SOD2 as a Marker for Penile Cancer Metastasis
Targeting ROS for Cancer Therapy
Conclusions and Future Directions
Cross-References
References
44 Biomarkers of Oxidative Stress in Cancer and Their Clinical Implications
Introduction
Reactive Oxygen Species and Oxidative Stress
Cellular Sources of ROS
Cellular Signaling Mediated by ROS
The Antioxidant System
ROS Influences Signaling Pathways in Cancer
Inflammation and Oxidative Stress in Cancer
Biomarkers of Oxidative Stress
End Products of Lipid Oxidation
By-products of DNA Oxidation
By-products of Protein Oxidation
Other Biomarkers of Oxidative Stress in Cancer
ROS Markers: Methods of Measurement
Direct Measurement of ROS
Superoxide
Hydrogen Peroxide
Nitric Oxide
Peroxy-Nitrite
Assessing the Damage to Macromolecules Such as DNA, Proteins, and Lipids
Protein Damage
Lipid Damage
DNA Damage
Measurement of Antioxidant Levels
Enzymatic Antioxidants
Superoxide Dismutase
Catalase
Glutathione Peroxidase
Nonenzymatic Antioxidants
Glutathione
Vitamin-C
Vitamin-E
Clinical Implications of Oxidative Stress Markers in Cancer
ROS-Induced Apoptosis
ROS-Induced Autophagy and Necrosis
ROS-Induced Necrosis
ROS-Mediated Resistance to Cancer Therapy
ROS as a Marker of Risk Prediction and Prognosis in Cancer
Conclusion
References
45 The Role of Reactive Oxygen Species on Cellular Fate and Function of Tumor-Infiltrating Lymphocytes
Introduction
Role of ROS as Signaling Molecules in Cancer
ROS and the Immune System
ROS and Innate Immunity
ROS in Adaptive Immunity
ROS Production in the Tumor Microenvironment
MDSCs
Tumor-Associated Macrophages
Tumor Cells
ROS and Tumor-Infiltrating Lymphocytes
Mechanisms of Action of ROS on Tumor-Infiltrating T Lymphocytes
T Cell Hyporesponsiveness
Altered T Cell Activation
T Cell Death
Therapeutic Strategies that Affect ROS and Influence Anti-tumor Immunity
Conclusions
References
46 ROS-Mediated Inflammatory Response in Cancer
Introduction
Production of ROS
ROS from Mitochondria
ROS from Oxidase Activity
ROS from Peroxisomes
Chronic Inflammation and Cancer
Oxidative-Stress-Induced Inflammation
Molecular Mechanisms of ROS-Induced Carcinogenesis
DNA Damage
Role of ROS in DNA Damage Induced by Replication Stress/Other Factors
Cell Signaling Cascades in ROS-Mediated Inflammation and Cancer
Transcription Factors - NF-κB, STAT, AP-1, HIF-1
Apoptosis and Survival
Inflammatory Markers and ROS
Tumor Microenvironment
Components and Characteristics of TME
Hypoxia, Angiogenesis, and Metastasis
ROS in Cancer Metastasis
Angiogenesis and ROS
Regulation of ROS
Conclusion
References
47 Food Colors and Associated Oxidative Stress in Chemical Carcinogenesis
Introduction
Use of Synthetic Dyes
International Legislations
Regulation in the European Union
Regulation in the USA, Japan, and China
Regulatory Measures in India
Carcinogenicity of Food Dyes
Oxidative Stress
Genotoxicity
Neurotoxicity
Biotransformation
Oxidative Effects and Associated Health Risks of Synthetic Dyes
Health Concerns of Approved Dyes
Health Concerns of Illegal Dyes
Conclusions
Cross-References
References
48 Benzo(a)Pyrene-Induced Oxidative Stress During Lung Cancer and Treatment with Baicalein
Introduction
Lung Cancer
Epidemiology and Incidence Statistics
Known Risk Factors
Polycyclic Aromatic Hydrocarbons (PAHs)
Benzo(a)pyrene [B(a)P]
B(a)P: A Potent Inducer of Oxidative Stress
B(a)P-Induced Inflammatory Responses
Mechanisms of B(a)P-Induced Lung Cancer
Lung Cancer Progression Stages
Traditional Treatment for Oxidative Damage and Lung Cancer
Free Radical Scavenging and Antioxidant Activity of Baicalein
Anti-Inflammatory Activity of Baicalein
Conclusion
References
49 Assessing the Contributions of Lipid Profile and Oxidative Lipid Damage to Carcinogenesis
Introduction
Lipids: Key Macromolecules with Structural and Functional Diversities
Body
Carcinogenesis Through Inflammation
Redox Homeostasis: Oxidative Stress and Antioxidant Defense
ROS and Lipid Peroxidation in Physiological and Pathophysiological Conditions
Dietary, Blood, and Biomembrane Lipid Composition and LPO Products
Mechanism of Carcinogenesis Through LPO and Inflammation
Organelle Dysfunction Caused by LPO Products
Lipid Peroxidation and Its Products: Structure, Signalling, and Cellular Biochemical Effects
Conclusion
References
50 Oxidative Stress in Hepatocarcinogenesis and Role of Antioxidant Therapy
Introduction
Liver Cells and Inflammatory Cytokines
Mitochondrial Roles in ROS-Induced Hepatocarcinogenesis
Viral Hepatitis Increases Free Radicals: Toward HCC Induction
Alcohol and Oxidants Cause HCC
Warburg Effect Increases Oxidative Stress in HCC Cells
Current Preventive and Treatment Modalities of HCC and Related Limitations
Antioxidant Therapy for HCC
Natural Honey
Nigella sativa
Ajwa Date Fruit
Costus
Fennel (Foeniculum vulgare Mill)
Conclusion
References
51 Oxidative Stress in Orchestrating Genomic Instability-Associated Cancer Progression
Introduction
Oxidative Stress: Its Generation and Implication in Cancer
Oxidative Stress-Mediated DNA Damage Leads to Genomic Instability
Chromosomal Instability
Telomere Shortening
Centrosome Amplification, Multipolarity, and Centrosome Clustering
Microsatellite Instability
Epigenetic Modifications
Oncogenic Replication Stress
Oxidative Stress Manipulates DNA Damage Response to Facilitate Genomic Instability
Therapeutic Strategies to Mitigate Genomic Instability in Cancer Progression
Amelioration of Oxidative Stress to Prohibit Establishment of Genomic Instability
Increasing Oxidative Stress to Kill Cancer Cells Via Comprehensive Genomic Degradation
Conclusion
References
52 Hypoxic Stress Perturb DNA Repair Mechanisms Leading to Genetic Instability
Introduction: The Hypoxic Tumor Microenvironment
Hypoxia and Reoxygenation Induces Oxidative Stress Through Reactive Oxygen Species (ROS)
Hypoxia and the Activation of DNA Damage Response
Downregulation of DNA Repair Pathways Under Hypoxia
Base Excision Repair (BER)
Nucleotide Excision Repair (NER)
Mismatch Repair (MMR) Pathway
Homology-Directed Repair (HDR)
Non-Homologous End Joining (NHEJ)
Fanconi Anemia (FA) Pathway
Hypoxia Induced Replication Stress
Hypoxia Induced Genetic Instability
Conclusion
References
53 DNA Lesions Induced by Lipid Peroxidation Products in Cancer Progression
Introduction
DNA Lesions Induced by Reactive Lipid Peroxidation Products
Malondialdehyde
α,β-Unsaturated Aldehydes
Propano Adducts
Etheno Adducts
Ketoaldehydes
DNA Lesions from Lipid Peroxidation in Cancer Development
Conclusion
Cross-References
References
54 Understanding ROS-Induced DNA Damage for Therapeutics
Introduction
Relevance of ROS-Induced Oxidation in Pathogenesis of Cancer
ROS Interaction with Lipids
Generation Cytosolic ROS from Proteins
Ras Signaling
PI3-K/Akt Pathway
IKK/NF-κB Pathway Regulation by ROS
ROS and Nuclear Signaling
Oxidative Stress Promotes Cancer
ROS Serves Dual Purpose in Cancer
Cancer Progression and Metastasis Are Promoted by CAFs
ROS and Cellular Death Pathways
Apoptosis
Caspases
Autophagy and ROS (Programmed Cell Death Type II)
Necrosis and ROS
Necroptosis (Programmed Cell Death: Type III)
Ferroptosis and ROS
Therapeutics and ROS
ROS and Multidrug Resistance
Nuclear ROS Induces DNA Damage
ROS Targeted Nanotherapeutic Drugs
Conclusion
References
55 Mitochondrial Metabolism, Oxidative Stress, and the Microenvironment in Breast Cancer Development and Progression
Introduction
The Role of Mitochondrial Metabolism in Breast Cancer Development and Progression with Focus on Mitochondria and ROS Production
The Importance of Mitochondria for Cancer Cell Metabolism
Mitochondrial Functions in Non-cancer Cells and Changes in Cancer Cells
The Breast Tumor Microenvironment, Metabolic Interactions and Crosstalk Between Stroma and Tumor Cells
Oxidative Stress as an Essential Factor in the Pathogenesis and Progression of Breast Cancer
ROS and Oxidative Stress
Triggers for ROS Production and Oxidative Stress
Oxidative Stress Affecting Sites Distal from Tumor Tissue
Conclusions and Perspectives
References
56 Role of Oxidative Stress and DNA Damage/Repair in Lung Cancer
Introduction
Inducers of Lung Cancer
Genetic Factors
Epigenetics and Lung Cancer
Environmental Factors
Link Between Inflammation, Oxidative Stress, and DNA Damage
Vicious Cycle Between Inflammation and DNA Damage in Development of Lung Cancer (Fig. 1)
Relation Between Various Chronic Inflammatory Lung Diseases and Lung Cancer
Sources of Reactive Oxidative Species (ROS) in Lung Cancer
DNA Repair Mechanisms in Lung Cancer
Oxidative Stress/DNA Damage Markers in Lung Cancer
Therapeutic Targets in Lung Cancer in Oxidative Stress, DNA Damage, and DNA Repair
Controversial Role of Oxidative Stress in Lung Cancer
Important Clinical Trials Targeting Redox Candidates in Lung Cancer
Conclusions and Future Direction
References
57 Oxidative Stress and Cancer: Role of the Nrf2-Antioxidant Response Element Signaling Pathway
Introduction
Oxidative Stress and Cancer
Oxidative Stress
Oxidative Stress and Carcinogenesis
The Nrf2-Antioxidant Response Element (Nrf2-ARE) Signaling Pathway
The Double Role of Nrf2 in Cancer
Pro-Oncogenic Effects of Nrf2: Nrf2 as a Proto-Oncogene
Nrf2 as Tumor Suppressor
Mechanisms of Nrf2 Activation in Cancer Cells
Somatic Mutations of Keap1, Nrf2, and Cul3
Epigenetic Modifications of Keap1
Disruption of Nrf2/Keap1 Interactions by Other Signaling Pathways
Role of Nrf2 in Chemoresistance
Conclusions
Cross-References
References
58 Role of Macrophages in Oxidative Stress-Induced Inflammatory Tumor Microenvironment
Introduction
Oxidative Stress and Chronic Inflammation: Important Regulators of Tumor Progression
Source of Oxidative Stress Within the Tumor Microenvironment
Cancer Cells
Microenvironmental Factors
Stromal Cells
Relationship Between Oxidative Stress, Tumor, and Chronic Inflammation
Cancer Cells and the Inflammatory Response
Maintenance of Chronic Smoldering Inflammation in the Tumor Microenvironment
Oxidative Stress in Tumors Prevents Suppression of Inflammation, Leading to Chronic Inflammatory Conditions
Recruitment and Oxidative Stress Mediated Differentiation of Leucocytes into Pro-tumorigenic Type
Recruitment of Monocytes to the Tumor Microenvironment
TME Mediated Activation of Recruited Monocytes
Both Tumor and TAM Derived ROS Influence Macrophages Function
Macrophage Differentiation and Oxidative Stress
Conclusion
References
59 The Triad, Hypoxia-ROS-Inflammation
Introduction
Oxidative Stress, Inflammation, and Hypoxia: Vicious Cycle
Effects of Oxidative Stress, Hypoxia, and Inflammation on Host Immune System
Tumor-Oxidative Stress, Hypoxia, and Inflammatory Mediators Directly Affect T Cell Survival and Function
Tumor Microenvironment and Oxidative Stress Cause Phenotypic Alteration of Macrophages and Immature Myeloid Lineage Cells
Regulatory T Cells and Tumor Microenvironment Factors
Targeted Immunotherapy of Cancer and Oxidative-Inflammatory Barrier
Conclusion
Cross-References
References
60 Interplay Between Reactive Oxygen Species and Key Players in the DNA Damage Response Signaling Network
Introduction
Components of the DDR Signaling System
The MRN Complex
ATM/ATR
H2AX
53BP1
DDR and Redox-Sensitive Transcription Factors
p53
NF-κB
NFE2L2
ROS at the Crossroads of Cell Proliferation and Apoptosis
Therapeutic Implications of DDR-ROS Interplay
Conclusions
References
61 ROS at the Intersection of Inflammation and Immunity in Cancer
Introduction
Oxidative Stress: Generation and Impact
An Essential Pillar of Innate Immunity: Nod-Like Receptors
ROS, Inflammation, and Immunity
The Interplay Between Oxidative Stress, Inflammation, and Immunity in Cancer
Therapeutic Implications
Conclusions
References
62 Sestrin-2 Connects Autophagy: Gatekeepers Against Tumor Progression
Introduction
Sestrin Family of Genes
Oxidative Stress and Sestrins
Cellular Effects of Sestrins
Sestrins: Gatekeepers Against Cancer
p53-Sestrin-AMPK Connection
Sestrin Regulation of Nrf2/Keap1 and ER Stress Pathways
Mechanism of Autophagy
Sestrin Connects Autophagy in Cancer
Sestrin 2 and Mitophagy
Conclusion
References
63 Insights into the Role of NRf2 Pathway in Cadmium-Induced Carcinogenesis
Introduction
Mechanism of Cadmium-Mediated Carcinogenesis
Role of ROS in Cadmium-Induced Cancer
Modulation of Nrf2-Mediated Pathway in Cadmium-Induced Cancer
Conclusion and Future Prospects
References
64 Interplay Between Redox Homeostasis and Oxidative Stress in the Perspective of Ovarian and Cervical Cancer Immunopathogenes...
Introduction
Nexus Between ROS, Cancer, and Immune Cells
Chronic Inflammation-Related Oxidative Stress in the Pathogenesis of Ovarian and Cervical Cancers
Oxidative Stress in Ovarian and Cervical Cancer Initiation
Oxidative Stress in Ovarian and Cervical Cancer Promotion
Oxidative Stress in Cell Proliferation and Survival
Oxidative Stress in Angiogenesis
Oxidative Stress in Immune Suppression
Oxidative Stress in Ovarian and Cervical Cancer Progression
Modulation of ROS as Cancer Therapy
Chemotherapy
Doxorubicin
Platinum-Based Drugs (Cisplatin, Carboplatin)
Paclitaxel
Poly (ADP-Ribose) Polymerase (PARP) Inhibitors
Immunotherapy
Conclusion
Cross-References
References
65 Oxidative Dyshomeostasis in the Mitochondria
Introduction
Oxidative Stress and Cancer
Mitochondrial Alterations in Cancer
Origins of Oxidative Stress
What Happens to Mitochondrial Antioxidants in Cancer Cells?
Metabolic Modulations in the Tumor Microenvironment
mtDNA Mutations in Cancer
Cancer Cells Manipulate Mitochondrial Functions to Resist Apoptosis and Favor Autophagy
Mitochondrial Oxidative Stress in Tumor Growth and Metastasis
Applications of Mitochondrial Variability in Diagnosis and Staging of Cancer
mtDNA Markers of Tumors
Mitochondrial Metabolic Markers for Tumors
Mitochondrial Oxidative Stress-Associated Cancer Therapy Approaches
Conclusion
References
66 The Double-Edged Sword Role of ROS in Cancer
Introduction
Role of ROS in Tumor Promotion
Tumorigenesis Via Cell Signaling
Tumorigenesis in Response to ROS-Mediated Activation of Factors and Other Genetic Changes
Angiogenesis
Metastasis
Role of ROS in Tumor Suppression
Apoptosis
Autophagy
Necrosis
Ferroptosis
Chemosensitization
ROS in Cancer Stem Cells
Role of ROS in Cancer Therapies
Roles of ROS in Molecular Targeted Therapies
Roles of ROS in Targeted Tyrosine Kinase Therapies
Roles of ROS in Cancer Chemotherapy
Roles of ROS in Radiotherapy
Conclusion
References
67 ROS Modulation on Apical Junctional Complex
Introduction
Metastatic Signaling and ROS Modulation
Tight Junctions and Oxidative Stress
Tight Junctions Proteins
Oxidative Stress, Tight Junctions, and Metastasis
Adherent Junctions and Oxidative Stress
Conclusions
References
68 The Interdependence of Inflammation and ROS in Cancer
Introduction
Overview the Role of Main Cell-Components in TME
The Roles of CAFs and TAMs in TME
Immunosuppressive Cell Types in TME
NK and B Cells in the TME
TME Network
Interdependence of ROS and Inflammation in Cancer
Inflammation-Induced ROS in Cancer
Mechanisms of ROS-Induced Inflammation in Cancer
ROS and Inflammation Promote EMT in Cancer
ROS Mediated Interactions in TME
ROS and CAFs in TME
ROS and TAMs in TME
ROS and MDSCs in TME
ROS and Tregs in TME
Therapeutic Approaches to Remodel the TME
Approaches Targeting ROS in TME
Approaches Targeting the Inflammatory Signals in TME
Approaches Targeting the Immunosuppressive Signals in TME
Perspectives and Conclusion
References
69 Interplay Between Oxidative Stress and Endoplasmic Reticulum Stress in the Metastasis of Colon Cancer
Introduction
Incidence and Risk Factors of Colon Cancer
Stages and Pathological Features of Colon Cancer
Impact of Oxidative Stress on Colorectal Cancer
Endoplasmic Reticulum Stress and Colon Cancer
ER Stress and Colon Cancer
Role of ER Stress in Mediating Epithelial-Mesenchymal Transition
Role of ER Stress in EMT
ER Stress in Cell Fate Decision
Conclusion
References
70 Mitochondria-Targeted Antioxidants and Cancer
Introduction
Antioxidants and Cancer Therapy
Mitochondria and Cancer
Mitochondrial Antioxidant Defense System
Dismutation of Superoxide Radicals
Removal of H2O2
Targeting Mitochondrial Oxidative Stress
Antitumor Effects of Mitochondria-Targeted Antioxidants
MitoQ
SkQs
Mito-Vitamin E
Mitochondrial-Targeted Nitroxides
Mito-Carboxy PROXYL (Mito-CP)
TPP+-Conjugated TEMPO Derivatives
SS Tetrapeptide
Conclusion
Cross-References
References
71 ROS-Mediated Genome Alterations at Cellular Microenvironment During Cancer Metastasis: A Curtain Raiser
Introduction
Aneuploidy: The Key Factor in Tumerogenesis
Effects of Aneuploidy on Cellular Metabolism (Cancer Microenvironment)
Oxidative Stress and Molecular Damage
Nucleic Acids Damage (DNA and RNA)
Role of ROS in DNA Damage by Oncogenic Replication Stress
Protein Damage
Lipid Damage
Conclusion
References
72 Oxidative Stress in Cancer and Its Influence on Amoeboidal Migration
Introduction
Cancer and Cellular Migration
Epithelial Migration
Multicellular Streaming
Tumor Budding
Single Cell Migration
Mesenchymal Migration
Amoeboid Migration
Components of Amoeboidal Migration
Relationship Between Amoeboidal Migration and Oxidative Stress
Oxidative Stress and Migration Plasticity
Mesenchymal to Amoeboid Transition (MAT)
Amoeboid to Mesenchymal Transition (AMT)
Collective to Amoeboid Transition (CAT)
Conclusion
Reference
73 The Intricacy of ROS in Cancer Therapy Resistance
Introduction
Introduction to Reactive Oxygen Species (ROS)
Sources and Activation of ROS
Regulation of ROS Production
ROS Regulating Cancer and Therapy Resistance: Highlighting the Dichotomous Role of ROS
Cancer-Promoting Roles of ROS
Increase Genomic Instability by ROS
Alteration of Metabolic Pathways by ROS
Regulation of Metastasis by ROS
Triggering of Angiogenesis by ROS
Epigenetic Regulation by ROS
Cancer-Killing Roles of ROS
Induction of Apoptosis by ROS
Effect on Cancer Stem Cells by ROS
ROS-Induced Senescence
ROS-Induced Necrosis
ROS-Induced Cell Cycle Arrest
Molecular Pathways Regulated by ROS in the Cancer Therapy Resistance
The Impact of ROS and Antioxidant Defense System to Promote Cancer Therapy Resistance
P-glycoprotein Regulation by ROS to Promote Cancer Therapy Resistance
The Interplay Between Autophagy and ROS in the Development of Cancer Therapy Resistance
Conclusions and Future Perspectives
References
74 Oxidative Stress
Introduction
Free Radicals: Precursors of Oxidative Stress
Oxidative Stress in Breast Cancer
Oxidative Stress in Breast Tumor Growth
Role of Oxidative Stress in Modulation of Breast Tumor Microenvironment
Role of Oxidative Stress in Breast Tumor Metastasis
Role of Oxidative Stress in Breast Tumor Angiogenesis
Role of Oxidative Stress in Metabolic Reprogramming in Breast Cancer
Drug Resistance Due to Oxidative Stress in Breast Cancer
Oxidative Stress and Breast Cancer Therapy
Conclusion
References
75 Relationship Between ROS, Autophagy, and Cancer
Introduction
Reactive Oxygen Species
Signaling by ROS
Autophagy
Initiation
Nucleation
Phagophore Expansion and Maturation
Lysosomal Fusion
Degradation
Signals that Trigger Autophagy
Activation of Autophagy by ROS
Protective Effect of Autophagy Against Ischemia
The Conjugation Complex ATG3, ATG4, ATG7, and ATG8 (LC3)
ROS Induce Transcription Factors of Autophagy Genes
The Transcription Factor EB (TFEB)
Transcription Factors FoxO1 and FoxO3
Activation of the Nuclear Factor Erythroid 2-Related Factor 2 (NRF2)
c-Jun/c-Fos-Beclin 1
Negative Feedback Against ROS
Hypoxia Inducible Factor HIF1-BNIP3
Autophagy Activating Complexes ATF4/LC3 and CHOP/ATG5
Conclusions
References
76 TRP Channels, Oxidative Stress, and Cancer
Introduction
Present Knowledge of TRP Channels, Cancer, and Oxidative Stress
Altered Expression of TRP Channels and Cancer Pathology
TRP Channels: Tumor Enhancer or Suppressor
TRP Channels, Oxidative Stress, and Angiogenesis
Functional Role of TRP Channels in Cancer Under Oxidative Stress
TRPA1
TRPM2
Advanced Understanding of Redox-Sensitive TRP Channels in Relation to Specific Cancer Pathology
Reactive Electrophiles, TRP Channels, and Lung Cancer
Acute Myeloid Lymphoma (AML) and TRPM2 Channels
Altered Mitochondrial Bioenergetics, TRPM2 Channels and Gastric Cancer
Glioblastoma and TRP Channels
Oxidative Stress, Other TRP Channels, and Their Role in Breast and Prostate Cancer
Involvement of TRP Channels in Other Types of Cancer
Conclusions and Future Directions
References
77 Wnt Signaling in Cancer
Introduction
Wnt Signaling Pathways and Machinery
Wnt Ligands and Receptors
Wnt Canonical Pathway
Noncanonical Wnt Pathways
Aberration in Wnt Signaling in Cancer
Colorectal Cancer (CRC)
Leukemia
Liver Cancer
Lung Cancer
Multiple Myeloma
Ovarian Cancer
Breast Cancer
Other Cancers
Targeting of Wnt Signaling in Cancer Therapeutics
Conclusion
References
78 Two-Faced Role of ROS in the Regulation of Cancer Cell Signaling
Introduction
Significance of ROS in Cancer
Cellular ROS
Oxidant Sources
Oxidant Types
Commanding Role of ROS in Cancer
ROS Regulation
ROS Regulation by the Antioxidant Defense System
Regulation of ROS by Tumor Suppressor Genes
ROS as a Signaling Molecule
Impact of Two-Faced ROS on Cancer Signaling
One Face: ROS Enhances Protumorigenic Signaling in Cancer
Second Face: ROS Regulates Anti-tumorigenic Signaling in Cancer
Conclusion
Cross-References
References
79 Oxidative Stress and Notch Signaling
Introduction
Notch Signaling in Cancer
Oxidative Stress in Cancer
Cross Talk Between Oxidative Stress and Notch Signaling in Cancer
Oxidative Stress Regulated Notch Signaling in Cancer
Notch Signaling Regulates Oxidative Stress in Cancer
Implications of the Cross Talk Between Notch Signaling and Oxidative Stress in Cancer
Proliferation and Survival
Apoptosis
Epithelial to Mesenchymal Transition (EMT), Migration, and Invasion
Cancer Stem Cells (CSCs)
Therapeutic Targeting of Oxidative Stress/Notch Signaling in Cancer
Conclusion
References
80 ROS-Induced Regulatory Crosstalk with Autophagy and AKT/mTOR Signaling in Cancer Cells
Introduction
Understanding ROS-Induced Autophagy Response During Cancer
Relation Between ROS and Autophagy During Malignancy
Specific Signaling Pathways Linking ROS and Autophagy
Impact of ROS-Responsive Autophagy in Chemotherapeutic Applications
Role of ROS-Responsive PI3K/AKT/mTOR Signaling During Cancer
Conclusion
References
81 Redox Regulation of Estrogen Signaling in Human Breast Cancer
Introduction
Estrogen and Breast Cancer
Estrogen Actions by Oxidative Stress-Mediated Signaling
Estrogen Synthesis and Its Metabolizing Pathways
Molecular Mechanism of Estrogen Biosynthesis
Estrogen-Metabolizing Enzymes and Its Receptor
Estrogen Sulfotransferase
Steroid Sulfatase
Receptors
Involvement of SULT1E1 in Breast Carcinogenesis
Involvement of NFκB in Breast Carcinogenesis
Involvement of Nrf-2 in Breast Carcinogenesis
Discussion
Conclusion
References
82 ROS Impacts on Cell Cycle Checkpoint Signaling in Carcinogenesis
Introduction
ROS Impacts on G1 Phase Regulators and Carcinogenesis Risk
G1 Phase Events
G1 Tumor Suppressor Genes and ROS
p14
p53
p21
p27 and p57
RB
TGF-β
G1 Oncogenes and ROS
Mdm-2
CDK4/Cyclin D
CDK2/Cyclin E-A
ROS Impacts on S Phase Signaling Proteins and Carcinogenesis Risk
S Phase Events
S Tumor Suppressor Genes and ROS
p21
ATM and ATR
S Oncogenes and ROS
CDC25A
CDK2/Cyclin A
ROS Impacts on G2 Phase Regulators and Carcinogenesis Risk
G2 Phase Events
G2 Tumor Suppressor Gene and ROS
p21
G2 Oncogenes and ROS
CDC25C
CDK1/Cyclin B
ROS Impacts on M Phase Regulators and Carcinogenesis Risk
M Phase Events
M Tumor Suppressor Genes and ROS
p53
APC/C
M Oncogene and ROS
CDC20
Conclusion and Future Direction
References
83 Modulation of JAK/STAT Pathways in Cancer by Phytochemicals
Introduction
Pathophysiology of Cancer
Phytochemicals and Cancer
JAK/STAT Signaling Pathway in Oncogenesis
Regulation of JAK/STAT Signaling In Vitro by Phytochemicals
Inhibition of JAK/STAT Signaling in Animal Models by Phytochemicals
The Interaction of Phytochemicals Regulating JAK/STAT with Chemotherapeutic Drugs
Conclusion
References
84 Role of Carotenoids on Oxidative Stress-Mediated Signaling in Cancer Cells
Introduction
Oxidative Stress and Cancer
Carotenoids and Cancer
Effect of Carotenoids on Cell Cycle Regulation in Cancer Cells
Role of Carotenoids on Apoptosis Induction in Cancer Cells
Role of Carotenoids on Redox-Sensitive Protein-Mediated Cell Death Progression in Cancer Cells
Effect of Carotenoids on Growth Factors and Regulation of Cell signaling in Cancer Cells
Role of Carotenoids on the Regulation of Cell Signaling
Redox-Related Modulation of Transcription Factors
Conclusion
References
85 Cross Talk Between Oxidative Stress and p53 Family Members in Regulating Cancer
Introduction
Role of p53 Family Members in Genomic Integrity Maintenance Under Oxidative Stress
P53 Family and ROS Cross Talk in Cellular Metabolism
ROS and p53 Family in Cell Death
Conclusion
References
86 Mutant K-Ras-Mediated Oxidative Stress in Pancreatic Cancer
Introduction
K-ras Mutation: The Driving Force of PDAC Tumorigenesis
Oxidative Stress in Pancreatic Cancer
K-rasG12D and Redox Balance in PDAC Cells
Conclusion
References
87 mRNA Stabilizing Factor HuR: A Crucial Player in ROS-Mediated Cancer Progression
Introduction: mRNA Half-life, RNA-Binding Proteins, and Oncology
HuR and Its Nuts and Bolts
HuR Linked in Tumorigenicity and Therapeutics
HuR, the Effective Modulator of ROS Generation in Cancerous Traits
Discussion and Future Directions
References
88 Redox State and Gene Regulation in Breast Cancer
Introduction
Origin of ROS in Cells
ROS Mediated Signaling Pathways
ROS and Breast Cancer
Additional Mechanisms of ROS Generation in Cancer Cells
Modulation of Cancer Pathways by ROS
DNA Damage Response
Epigenetics and Gene Regulation
Alterations in Signaling Pathways
Cellular Proliferation
Cell Survival
Autophagy
Angiogenesis
Motility and Metastasis
Conclusion
References
89 Glutathione Peroxidase and Lung Cancer: An Unravel Story
Introduction
GPX and Its Characterisation
GPX and Its Involving Pathways
GPX and Its Co-expressed and Interacting Genes
GPX and Lung Cancer
Conclusions and Future Direction
References
90 Importance of Silencing RNAs in Cancer Research
Introduction
Silencing RNA (siRNA) in RNA Interference
Concise Introduction About siRNA
Applications of siRNA
SiRNA Mediated Drug Delivery Methods
Narrowing Down to the Intervening Drug Delivery Methods of siRNA Specific to Cancer Treatment
SiRNAs Delivery System for Neurogenerative Disease Conditions
Limitations of siRNA Delivery to the Targeted Site for Actions
Versatility of siRNA Employed for Therapeutic Application of Cancer
Chemical Alterations of siRNA
Lipid Mediated siRNA Delivery System
Polymer-Enhanced siRNA Delivery System
Different Modes of Administration by the Delivery of siRNA
Emphasizing the Systemic Delivery Approaches of siRNA
Conclusion
Future Perspective
Reference
91 Cellular Redox Status and Modifiable Behaviors
Introduction
Excess Body Weight
Poor Nutrition
Cigarette Smoking
Conclusion
Cross-References
References
92 Yin-Yang of Oxidative Stress in Pancreatic Cancers
Introduction
ROS-Induced Epigenetic Regulation in Cancer
DNA Methylation
Histone Modification
Transcriptional Control
Posttranscriptional Control
Translational and Posttranslational Control
MAPK/ERK1/2 Pathway
AKT Pathway
JAK-STAT Pathway
NRF2-KEAP Signaling Pathway
Conclusion
Summary
Cross-References
References
93 Epigenetic Therapy as a Potential Approach for Targeting Oxidative Stress-Induced Non-small-Cell Lung Cancer
Introduction
Etiology of Lung Cancer
Smoking
Genetic Factors
Gender
Age
COPD and Other Pulmonary Conditions
Diet and Obesity
Environmental Air Pollution
Role of Reactive Oxygen Species (ROS) in Lung Cancer
Oxidative Stress-Mediated Non-Small-Cell Lung Cancer (NSCLC)
Role of Cigarette Smoking in Oxidative Stress-Induced LC (NSCLC)
Epigenetic Changes Due to OS in NSCLC
DNA Modification/Mutation and Lung Cancer
HDACi as Potential Epigenetic Therapy in NSCLC
Conclusion
Reference
94 Free Radicals-Mediated Epigenetic Changes and Breast Cancer Progression
Introduction
Epigenetics and Control of Gene Expression
DNA Methylation
Histone Modifications in Epigenetic Regulation
Noncoding RNAs (Long Noncoding RNA and MicroRNAs) and ATP-Dependent Chromatin Remodeling Complexes in Epigenetic Regulation
Biology of Breast Cancer
Oxidative Stress, Reactive Oxygen Species (ROS) Generation, and Epigenetic Mechanisms in Breast Cancer
Free Radicals in Health and Cancer
Free Radicals Induced Epigenetic Alterations and Affected Pathways
Antioxidant Enzymes and Redox Homeostasis
ROS-MicroRNAs Interactions and Epigenetic Alterations in Breast Cancer
Oxidative Stress in Breast Cancer Epigenetics, and Scope of Treatment
Dietary Agents with Antioxidant Properties as Epigenetic Modulators in Breast Cancer
Curcumin
Resveratrol
Quercetin
The Interplay Between Oxidative Stress and Epigenetic Drugs in Breast Cancer
Conclusive Remarks
Cross-References
References
95 Phytochemicals in ROS-Mediated Epigenetic Modulation of Cancer
Introduction
Epigenetic Control Mechanisms in Cells
Histone Modification
DNA Methylation
Epigenetic Control by microRNA
Oxidative Stress and Inflammation
Keap1-Nrf2 Pathway
Epigenetic Control by Phytochemicals
Roles of Phytochemicals in Epigenetic Regulation
Conclusion
References
96 Noncoding RNAs Regulation of Redox Balance in Cancer
Introduction
MiRNAs Regulation of Redox Homeostasis in Cancer
Breast Cancer
Classical Hodgkin Lymphomas
Colon Cancer
Colorectal Cancer
Gastric Cancer
Glioma
Hepatocellular Carcinoma
Lung Cancer
Medulloblastoma
Neuroblastoma
Oral Carcinoma
Ovarian Cancer
p53 Wild-Type Cancers
Pancreatic Cancer
lncRNAs Regulation of Redox Homeostasis in Cancer
snoRNA Regulation of Redox Homeostasis in Cancer
circRNA Regulation of Redox Homeostasis in Cancer
Conclusion
References
97 Oxidized DNA Base Damage Repair and Transcription
Introduction
Oxidative DNA Base Damage Shaping the Mutation Burden in Cancer
Oxidative DNA Base Damage Repair
The Interplay Between Posttranslational Modification of BER Proteins and Transcriptional Regulation
8-OxoG and OGG1 as a Novel Epigenetic Regulator for Controlling Gene Expression
BER in Hypoxia-Induced VEGF mRNA Expression
Role of 8-OxoG and OGG1 in the Regulation of Inflammatory Response Genes
Histone Demethylation, Oxidation of DNA, BER, and Gene Expression
Regulatory Roles of G Oxidation and BER Proteins in G4 Structure Formation in the Genome to Regulate Gene Expression
Conclusion
References
98 Epigenetic Instability Caused by Oxidative Stress Triggers Tumorigenesis
Introduction
Oxidative Stress-Induced Epigenetic Changes
Role of Oxidative Stress in Regional Hypermethylation in the Promoter Region of TSGs
Oxidative Stress-Induced Global Hypomethylation
Therapeutic Strategies to Regulate Epigenetic Alterations
Polyphenols
HDAC or DNMT Inhibitors
Histone Deacetylase Inhibitors (HDi)
Nuclear Factor Erythroid 2-Related Factor 2 Inhibitors
Conclusion and Future Directions
Cross-References
References
99 Implications of ROS in Cancer Stem Cells Mechanism of Action
Introduction
CSCs Inside Stem Cells Spectrum
CSCs Heterogeneity and Metabolic Landscape
Plasticity, Quiescence, and Embryonic Signature of CSCs
Tumor Microenvironment: CSC Relationship
The Signaling Pathways Involved in CSCs Maintenance
ROS, Carcinogenesis Theories, and CSCs
Oxidative Stress in Cancer Initiation and Development
Direct Damage of Structural Components and Metabolism with Induction of Gene Mutations
Modulation of Signal Transduction Pathways or Gene Transcription Factors with Alterations of Oncogene Expression and Tumor Pro...
The Association Between Oxidative Stress and CSCs
Single Nucleotide Polymorphisms Results in Cancer Risk Increase
Pathologic Conditions Related to Oxidative Stress and Carcinogenesis Promoters
ROS and Tumor Status
CSCs and Metastasis
Conclusions
References
100 Metabolism-Redox Interplay in Tumor Stem Cell Signaling
Introduction
Cancer Stem Cell Signaling
Cancer Stem Cell Metabolism
Redox Regulation in Cancer Stem Cells
Intracellular Signaling - Redox State Crosstalk: Metabolism Interplay
Conclusions
Cross-References
References
101 Association of ROS with Epithelial-Mesenchymal Transition and Acquisition of Stemness During Carcinogenesis
Introduction
Epithelial-Mesenchymal Transition
What Is EMT?
Types of EMT
Type-1 EMT
Type-2 EMT
Type-3 EMT
EMT and Cytoskeleton
Transcriptional Regulation of EMT
EMT and Carcinogenesis
Invasion-Metastasis Cascade
Step-1: Invasion
Step-2: Intravasation
Step-3: Extravasation
Step-4: Colonization
EMT and Cancer Stem Cells
Oxidative Stress: Regulator of EMT and CSC
What Is Oxidative Stress?
ROS and EMT
Redox Regulation over EMC Remodeling
ROS and Cytoskeletal Rearrangement
Oxidative Stress and Cell-Cell Junction Regulation
Redox Regulation over Cell Mobility
Association of ROS with EMT and Stemness
Differential Level of ROS in CSCs and Non-CSCs
Conclusion
References
102 Therapeutic Effect of Natural Compounds in Targeting ROS-Induced Cancer
Introduction
Natural Compounds in Regulation of ROS Generation
Polyphenols
Flavonoids
Proanthocyanidins
Anthocyanidin or Anthocyanin
Flavones
Isoflavones
Flavonol
Flavan-3-ol
Flavanone
Nonflavonoids
Stilbenes or Stillbenoid
Phenolic Acid
Hydroxycinnamic Acids and Hydroxybenzoic Acids
Flavonolignans
Coumarins
Lignans
Other Polyphenols
Diarylheptanoid
Curcumin
Vitamins
Carotenoid (Vitamin A or Retinol, Fucoxanthin)
Vitamin C
Dehydroascorbic Acid (DHA)
Vitamin E
Vitamin K3
Alkaloids
Sampangine
Boldine
Quinoline
Pyridocarbazole
Isoquinoline
Terpenoids
Triterpenoid
Lactone Sesquiterpenoid
Saponins
Carotenoid
Diterpenoids
Quinones
Cribrostatin 6
Naphthoquinone
Thymoquinone
Toluquinones
Miscellaneous Natural Compounds and Products
Essential Oils (EOs)
Boswellia Carteri (Frankincense Oil) EO
Artemisia Lavandulaefolia EO
Salvia Libanotica EO
Boswellia Sacra EO
Aniba Rosaeodora (Rosewood) EOs
Zanthoxylum Schinifolium EO
Minerals
Zinc
Selenium
Isothiocyanates
BITC
PEITC
Sulforaphane
Conclusion
References
103 Therapeutics of Oxidative Stress and Stemness in Breast Cancer
Introduction
Oxidative Stress, Tumor Heterogeneity, and Cancer Progression
Role of NO in Stem Cells and DNA Damage Response
Role of ROS in Cancer Cells
Role of Oxidative Stress in Evolving Cancer Cells
Role of Oxidative Stress in Microenvironment
Therapeutic Targeting of Oxidative Stress
Future Directions
Conclusion
Cross-References
References
104 Reactive Oxygen Species in Stem Cell Proliferation and Cancer
Introduction
Stem Cells and Their Classification
Types of Stem Cells
Reactive Oxygen Species
Role of Reactive Oxygen Species (ROS)
Chemistry of Reactive Oxygen Species
Cellular Generation of ROS
Role of ROS in Cell Proliferation
Signaling Through Cell Cycle
Role of Hypoxia in Induction of Proliferation
Hypoxia/ROS in Stem Cell Differentiation
Cancer Stem Cells (CSCs)
Development of Cancer from CSCs
ROS-Dependent Signaling Pathways in CSCs
Antioxidant Mechanism of ROS Scavenge
ROS-Associated Diseases Through Lifestyle
Conclusion
References
105 Targeting Redox Signaling and ROS Metabolism in Cancer Treatment
Introduction
Generation, Pathophysiological, and Regulatory Functions of Reactive Oxygen Species
Sources of ROS
ROS in the Regulation of Physiological and Pathophysiological Signal Transduction Pathways
Excess ROS Perturbs the Cellular Homeostasis
Reactive Oxygen Species and Cancer
ROS in Cancer Progression
ROS in Cancer Inhibition
Application of ROS Induction for Anticancer Strategies
Direct ROS Generation
Antioxidant Process Inhibition
Combination Therapy
Immunotherapies
Conclusions and Future Direction
References
106 Modulatory Role of Adipocytes and Their Stem Nature in the ROS Signaling Within a Tumor Micro-environment
Introduction
Lipid and ROS-Signaling in the Adipocyte-Mediated Tissue Dynamics?
Conjugated Linoleic Acids as Switching Molecules
Mechanism of CLA-Induced Delipidation/Dedifferentiation in the Adipocyte
Role of ROS in the CLAs Switching Mechanism
The Role in Cancer
Conclusions
References
107 Hypoxia-Induced Stress Responses in Cancer and Cancer Stem Cells
Introduction
HIF-1α a Key Player in Tumor Hypoxia
Metabolic Adaptation to Hypoxia
Antioxidative Responses
ER-Stress Response to Hypoxia
Autophagy
Hypoxia and Cancer Stem Cells (CSCs)
Hypoxia and Cancer Therapy
Conclusion
References
108 Oxidative Stress-Targeted Therapies for the Treatment of Acute Myeloid Leukemia
Introduction
Oxidative Stress Targeted Therapies
Prooxidant Therapy
Antioxidant Therapy
ROS-Mediated Differentiation Therapy
Conclusion
Cross-References
References
109 Role of ROS in Cancer Stem Cells
Introduction
Stem Cells and Their Role in Cancer
Cancer Stem Cells
Biomarkers of Ovarian CSCs
Surface Markers
Functional Markers
Ovarian CSC Microenvironment
Growth Factor-Mediated Proliferative Signaling
Cytokines and Inflammatory Network
miRNA Mediated Stemness Maintenance
Effect of ROS on Cancer Stem Cells: Avenues for Therapy
Stem Cells and ROS
Reactive Oxygen Species in Cancer and Cancer Stem Cells
Regulation of ROS in Malignancy and CSCs
ROS-Dependent Fundamental Transcription Factors in CSCs
HIF-1α
NF-kB
p53
NRF2
ROS-Dependent Fundamental Signal Transduction Pathways
ATM Pathway
PI3K/AKT Pathway
PTEN
FOXOs
Notch Pathway
Wnt Pathway
Clinical Relevance of CSCs
Recent Developments (Drug Candidates)
Conclusion
References
110 Reactive Oxygen Species-Dependent Signaling Pathways in Cancer Stem Cells
Introduction
Cancer Stem Cells
ROS-Mediated Signaling in CSCs
Ataxia Telangiectasia Mutation (ATM) Pathway in CSCs
PI3K/AKT Pathway in CSCs
Notch Pathway in CSCs
Wnt Pathways in CSCs
STAT Pathway in CSCs
ROS-Dependent Transcription Factors in CSCs
Hypoxia Inducing Factor (HIF) in CSCs
NF-κB in CSCs
Tumor Suppressor p53 in CSCs
The Nuclear Factor Erythroid 2-Related Factor (Nrf2) in CSCs
Role of ROS in Epithelial-Mesenchymal Transition in CSCs
CSCs and Drug Resistance
Conclusion
References
111 System Biology and Network Analysis Approaches on Oxidative Stress in Cancer
Introduction
Relation Between Hypoxia and ROS in Development and Progression of Cancer
Computational Systems Biology in Revealing Complex Molecular Interconnectivity Network in Cancer
In Silico Approaches in Oxidative Stress-Induced Cancer
Construction of Disease-Specific Interaction Network
Identification of IIP and Bottlenecks
Construction of Signaling to Metabolic Crosstalk Pathway and Connectivity Analysis
Discussion
Conclusion
Cross-References
References
112 Reactive Oxygen Species–Mediated Cancer Progression and Metastasis
Introduction
Involvement of ROS in Progression and Metastasis of Cancer
Sources of ROS in Cancer Cells
Mechanisms Involved in ROS-Mediated Cancer Progression and Metastasis
Botanicals Inhibit Cancer Progression and Metastasis
Conclusion
References
113 Anthocyanins and Flavonols: Therapeutic Implications of Natural Compounds on Cancer
Introduction
Anthocyanins and Anthocyanidins: General Overview
Cyanidin and Cyanidin-3-O-Glicoside
Delphinidin and Delphinidin-3-O-Glucoside
Flavonols: General Overview
Kaempferol
Quercetin
Conclusion
References
114 Implication of Nanomedicine in Therapy of Oxidative Stress-Induced Cancer
Introduction
ROS and Cancer
ROS and Its Biological Functions
Role and Mechanism of ROS in Cancer Development
ROS Induced Transcription Factor and Regulation
ROS Altered Biomolecules Functions
Oxidative Stress Adaptation in Cancer
Therapeutics Targeting Oxidative Stress Alteration in Cancer
Nanoparticulate Carriers
Nanovesicular Carriers
Liposomes
Micelles
Nanoemulsions
Conclusion
References
115 Metabolic Oxidative Stress in Initiation, Progression, and Therapy of Cancer
Introduction
Metabolic Reprogramming and Cancer
Metabolic Reprogramming and Enhanced Glycolysis
Metabolic Reprogramming and Altered Cell Signaling
Subcellular Organelles Adaptation to Metabolic Reprogramming
Metabolic Oxidative Stress in Cancer Cells
Molecular Drivers of Metabolic Oxidative Stress
Reactive Oxygen Species in Cancer
Glucose Metabolism and Oxidative Stress
Antioxidant Defense against Metabolic Oxidative Stress
Metabolic Oxidative Stress, Tumor Growth and Metastasis
Metabolic Oxidative Stress and Drug Resistance
Modifiers of Metabolic Oxidative Stress
Modifiers of Tri Carboxylic Acid (TCA) Cycle
Modifiers of Glutamine Metabolism
Modifiers of Pentose Phosphate Pathway (PPP)
Modifiers of Serine-Glycine One-Carbon (SGOC) Metabolism
Modifiers of Glycolytic Pathway
Inhibitors of Redox Regulating Proteins
Inhibitors of Glycolysis (2-Deoxy-D-Glucose) and Oxidative Stress
Clinical Applications/Implications
Pro-oxidants and Combinational Therapies
Antioxidants and Combinational Therapies
Limitations of Metabolic Oxidative Stress Targeting Agents or Drugs/Therapeutics and Strategies for Improving Efficacy
Conclusion
References
116 Mitochondrial ROS: A Reactive Species Targeted in Cancer Therapy
Introduction
ROS
Mitochondria Produce ROS
Tight Modulation of Mitochondrial ROS
Physiological Targets of ROS
Mitochondrial ROS Act as Signaling Molecules
Physiological mROS in Cellular Regulation
Cell Proliferation During Hypoxia
mROS During Angiotensin II Signaling
mROS in Cancer Cells Proliferation
Mitochondrial Antioxidant Systems
SOD2/MnSOD2
GPx-1 and GPx-4
Prx3/TrxR2/Trx2
Targeting mROS in Cancer Therapy
Conclusion and Future Perspective
References
117 Oxidative Stress and Hypoxia in Cancer: Implications for Radiation Therapy
Introduction
Reactive Oxygen Species (ROS) and Oxidative Stress
Cellular Origin of ROS
The ROS Detoxifying Systems
Effect of ROS
ROS and Cell Proliferation
ROS and Cell Death
ROS and Metabolic Remodeling of Cancer Cell
ROS and Metastasis
Hypoxia
Effects of Hypoxia on Tumor Progression
The Effect of Metabolic Remodeling by Hypoxia
Maintaining the Anabolic Flux Through High Glycolysis
Hypoxia and HIF
HIF-1 and Tumor Progression
HIF-1 and Metabolic Remodeling
HIF-1 and Regulation of ROS Production by Cancer Cells
Oxidative Stress, Hypoxia, and RT
Manipulation of ROS Homeostasis, Hypoxia-Affecting Factors, and Tumor Hypoxia for Improving RT Response
Manipulation of ROS Homeostasis
Manipulation of Factors Affecting the Tumor Hypoxia
Manipulation of Tumor Hypoxia
Conclusions
Cross-References
References
118 Oxidative Stress in Cancer
Introduction
Inorganic Selenium and Its Implecation in Cancer Prevention and Therapy
Anticancer Potential of Organoselenium Compounds
Heterocyclic Se-Compounds
Organoselenocyanate
Isoselenocyanate
Methylseleninic Acid (MSC)
Nanoformulted Se-NPs Based Cancer-Therapy
Se-NPs as Carrier for the Anti-Cancer Drug Delivery
Conclusion and Expert Opinion
References
119 Phytocompounds-Based Approaches to Combat Oxidative Stress in Cancer
Introduction
Oxidative Stress in Cancer and Its Modulation by Phytocompounds
Phytocompounds-Based Therapeutic/Preventive Approach for the Oxidative Stress-Induced Cancer
Flavonoids
Kaempferol
Biochanin-A
Luteolin
Triterpenoids
Lupeol
Betulinic Acid
Oleanolic Acid
Ursolic Acid
Cucurbitane
Phytoestrogens (Daidzein and Genistein)
Conclusion
References
120 Phytoestrogens Modulate Oxidative Stress
Introduction
Phytoestrogen Structure and Classification
Shared Mechanisms of Estrogens and Phytoestrogens
Phytoestrogens as Cancer Treatment
Concluding Remarks
Cross-References
References
121 Prospective Application of Natural and Synthetic Redox Modulators in Oxidative Stress-Targeted Cancer Therapy
Introduction
ROS-Manipulation Strategies in Cancer Treatment
ROS-Depleting Strategy in Cancer Treatment
ROS-Elevating Strategy in Cancer Treatment
Combination Therapy
Conclusion
References
122 ROS Modulation by Iron Chelators and Lipids: A Developing Anticancer Strategy
Introduction
Cancer Metabolism and Its Link with ROS Production
Cancer Is Caused by Genomic Instability but Also by Biophysical and Chemical Changes
Cancer Metabolism
The Unique Bioenergetics of Cancer Cells
Cancer Metabolism and Bioenergetics and Their Relation to ROS Production
ROS in Cancer
Iron, ROS Production, and Iron Chelators
Iron Chelators as Anticancer Agents
Fe Chelators: ROS Scavengers or Proliferation Inhibitors?
Iron Chelators and ROS Production
Cancer Cell Iron Addiction and Iron Chelation
ROS Modulation by Lipids as an Anticancer Strategy
Membranes of Cancer Cells
Membranes (General)
Phosphatidylcholine (PC)
Phosphatidic Acid (PA)
Phosphatidylethanolamine (PE)
Lipid Droplets (LD)
Biophysics of Membranes
Importance of Membrane Functions
Membranes in Cancer
ROS Production and Lipids
Lipids as Antioxidants: Vitamin E
Lipids as Antioxidants: 2-Hydroxyoleic Acid (2OHOA)
Conclusion
Cross-References
References
123 ROS, Cancer, Stem Cells
Introduction
Functional Significance of ROS in Cancer Cells
Does Antioxidants Ameliorate Cancer Progression?
Does Prooxidants Promote Killing of Cancer Cells?
Factors Affecting ROS-Induced Cancer Progression
ROS-Induced by Non-targeted Cancer Therapies
ROS-Induced by Targeted Cancer Therapy
Dynamic Role of ROS in Cancer Stem Cells
The beneficial and harmful ROS in Cancer
Conclusions and Viewpoints
Cross-References
References
124 Modulators of ROS/NF-κB Signaling in Cancer Therapy
Introduction
NF-κB Signaling Pathway
ROS/NF-κB Signaling
Modulators f ROS/NF-κB Signaling
Phenols
Alkaloids
Terpenoids
Steroids and Saponins
Polyketides
Clinical Trials of NF-κB/ROS Signaling Modulators
Conclusions
Cross-References
References
125 Hyperglycolysis-Inflammation Connect as a Mechanistic Hot Spot in Oxidatively Compromised Cancer
Introduction
Searching Hot Spots from Tumor Cell Metabolism
Tumor Cell ROS Metabolism as a Poor Mechanistic Target
The Hyperglycolysis and the Tumor Growth
The Key Glycolytic Players
Hexokinases
Phosphofructokinases: PFK1 and PFK2
Pyruvate Kinase M2
Lactate Dehydrogenase (LDH)
M4-LDH: Cancer-Related Inflammation and Tumor Microenvironment
Epithelial-Mesenchymal Transition (EMT) and the Process of Metastasis and Angiogenesis
Tumor-Associated Macrophages (TAMs): Negotiator Between the Cancer Cell and Tumor Matrix
TME and TAM Suppress Tc Cell Activation Against the Tumor Cells
TAM, TME, and Angiogenesis
Major Hot Spots in the Hyperglycolysis-Inflammation Connect
Conclusion
References
126 Constraint-Based Modeling to Understand ROS-Mediated Effects in Cancer
Introduction
Constraint-Based Modeling (CBM) Principle and Methodology
Genome-Scale Metabolic Models (GSMMs)
Mathematical Representation of GSMM
COBRA Toolbox
Flux Balance Analysis (FBA)
Flux Variability Analysis (FVA)
Context-Specific Metabolic Models Reconstruction and Analysis
Context-Specific Metabolic Model Generation
Cancer-Specific Metabolic Reconstructions to Model Cancer Metabolism
PAN Cancer Analysis of Human Metabolic Reconstruction
Retinoblastoma GEM
Prostate Adenocarcinoma-Specific Metabolic Model
Hepatocellular Carcinoma GEMs
Colon Cancer-Specific Metabolic Models
Scope of CBM in Studying the Role of ROS in Cancer Treatment and Prevention
Conclusion
References
127 Dynamical Methods to Study Interaction in Proteins Facilitating Molecular Understanding of Cancer
Introduction
Importance of Protein-Protein Interaction (PPI) for the Molecular Understanding of Function
Dynamics Are Key in Determining PPIs
Methods to Recover Protein Dynamics
Dynamics Analysis of Proteins by Molecular Dynamics Simulations
Normal Mode Analysis: An Approach to Recover Native Dynamics of Proteins
Application of Advanced Dynamical Techniques in Cancer-Related Proteins: A Case Study of CDK2
Protein-Protein Binding Inhibition Using Advanced MD Simulations
Normal Mode Analysis for Protein-Protein Complex Elucidating Conformational Change
Conclusion
References
128 Molecular Insights into the Roles of E3-Ligases in ROS-Mediated Cancer from a Bioinformatics Perspective
Introduction
E3-Ligases
RING E3-Ligases
HECT E3-Ligase
RBR E3-Ligase
Reactive Oxygen Species
Ubiquitin Proteasomal System (UPS) and ROS
Role of ROS and E3 Ubiquitin Ligase in Cancer
System Biology and Computational Aspects
Conclusion
Reference
129 Insulin Resistance and Cancer
Introduction
Insulin
Insulin Signaling
Insulin Resistance
Insulin Resistance and Oxidative Stress
Insulin Resistance and Hyperinsulinemia
Cancer
Clinical Findings
Obesity, Prediabetes, and Type 2 Diabetes
Cancer and Diabetes Therapies
Conclusion
Cross-References
References
130 Meprins: Ancient Enzymes Newly Discovered in Cancer Progression
Introduction
Historical Background and General Enzyme Characteristics
Structure of Meprins
Primary Structure
3D Structure of Meprins
Domains and Oligomeric Structure
Meprins and Disease
Catalytic Properties
Meprins and Drug Design
Future Perspectives
References
131 Molecular Analysis of the Involvements of lncRNA in Cancer Development
Introduction
Bladder Cancer
Brain Cancer
Breast Cancer
Colorectal Cancer
Gastric Cancer
Lung Cancer
Osteosarcoma
Ovary Cancer
Prostate Cancer
Analyses of Cancer Associated lncRNAs from a Bioinformatics Point of View
Conclusion
References
132 Proteomics and Metabolomics in Cancer Diagnosis and Therapy
Introduction
Cancer Proteomics
Proteomic Workflow
Proteomics Platforms
MS Based Proteomic Approaches in Cancer
Electrospray Ionization (ESI) and Matrix-Assisted Laser Desorption/Ionization (MALDI)
SELDI-TOF
QUEST-MS
SPR-MS
Chromatography Coupled with MS
Non-MS Based Proteomic Approaches in Cancer Biology
Antibody Based Platform
Immuno-sensors Based on Lateral flow Assay (LFA)
Tissue Micro Array (TMA)
Enzyme-Linked Immunosorbent Assay (ELISA)
Antibody Array
Reverse Phase Protein Array (RPPA)
Single Cell Proteomics in Cancer
Cancer Metabolomics
Metabolomics Workflow
Metabolomics Analytical Platforms
NMR
MS
GC-MS
LC-MS
Supercritical Fluid Chromatography
Capillary Electrophoresis
Ion Mobility Spectrometry
DI-MS
MALDI-MS
Direct Analysis in Real Time (DART)-MS
Mass Spectrometry Imaging
Challenges and Opportunities
Conclusions
References
133 Systems Biology and Bioinformatics Insights into the Role of Free Radical-Mediated Oxidative Damage in the Pathophysiology...
Introduction
Computational Approaches in Systems Biology
Artificial Intelligence (AI) Predicting Cancer Susceptibility Based on Redox Data
Artificial Intelligence (AI) Predicting Cancer Prognosis Based on Redox Data
Artificial Intelligence (AI) Predicting Therapeutic Outcome Based on Redox Data
Bayesian Networks
Boolean Networks
Multi-omics Analysis
Network Analyses Showing Association of Redox Proteome with Cancer Susceptibility
Network Analyses Showing Association of Redox Proteome with Cancer Prognosis
Network Analyses Showing Association of Redox Proteome with Cancer Therapy
Conclusions
References
134 Systems Biology Resources and Their Applications to Understand the Cancer
Introduction
Systems Biology Databases Used in Cancer Research
Expression and Variation Databases
Immune System and Personalized Medicine Databases Used in Cancer Drug Designing
Databases Used for Interaction and Pathway Analyses
Tumor Databases Used for Drug Designing in Experimental and Clinical Studies
Systems Biology Approaches and Tools to Cancer
Expression and Variation-Based Systems Biology Tools and Approaches for Cancer Prediction
Common Immunoinformatic and Bioinformatics Tools to Cancer Drug Discovery
Biomolecular Networks Tools in Cancer
Text Mining Tools Used in Cancer Research
Mathematical Modeling and Simulation Tools to Model Cancer Pathways and Networks
Clinical Applications of Systems Biology Tools and Approaches
Conclusion
References
135 Targeting Reactive Oxygen Species Homeostasis and Metabolism in Cancer Stem Cells
Introduction
ROS and Cancer Stem Cells
Cross Talk Between Signaling Pathways and ROS in CSCs
PI3K/AKT/mTOR Pathway
Notch Pathway
Wnt Pathway
JAK-STAT Pathway
Metabolic Control of ROS in CSCs
Glucose Metabolism
Lipid Metabolism
Amino Acid Metabolism
Therapeutically Targeting ROS Dynamics in CSCs
Conclusion and Perspective
References
136 Thioredoxin and Glutathione Systems
Introduction
ROS and Redox Control Systems
Glutathione and Thioredoxin Systems in Cancers
Targeting Antioxidant Systems for Cancer Therapies
Conclusions
Cross-References
References
137 Vitamin C in Cancer Management: Clinical Evidence and Involvement of Redox Role
Introduction
Physiological Redox Functions
Role in Cancer Prevention
Role in Cancer Treatment: Clinical Evidence
Mechanisms: Redox Role
Conclusion
References
138 Oxidative Stress, Inflammasome, and Cancer
Introduction
Free Radicals in Oxidative Stress
Classes of ROS
Superoxides (O2-)
Hydrogen Peroxide (H2O2)
Hydroxyl Radical (OH)
Reactive Nitrogen Species (RNS)
Oxidative Stress and Cell Signaling
Oxidative Stress and Inflammasome
Inflammasomes and Cancer
Proliferation and Survival
Immunosuppression
Angiogenesis
Metastasis
Polyphenols
Conclusion
References
139 Preventive Role of Carotenoids in Oxidative Stress-Induced Cancer
Introduction
Oxidative Stress
Antioxidants
Carotenoids
Sources of Carotenoids
Carotenoid Biosynthesis
Structural Features of Carotenoids
General Functions of Carotenoids
β-Carotene
Lycopene
Lutein
Zeaxanthin
β-Cryptoxanthin
Astaxanthin
Canthaxanthin
Fucoxanthin
Conclusion
References
140 Personalized Vaccine as a Therapeutic Approach Toward Cancer
Introduction
Genome as a Target for Personalized Cancer Vaccine
Personalized Cancer Immunotherapy
microRNAs (miRNAs) in Personalized Radiotherapy
Conclusion
References
141 Enolase
Introduction
Structure of Enolase
Enolase Isoforms
Enolases and ROS
Enolases and Stem Cells
Enolases and Cancer
Conclusions
Cross-References
References
142 Analytical and Omics Approaches in the Identification of Oxidative Stress-Induced Cancer Biomarkers
Introduction
Cancer Relation with Oxidative Stress
Types of Biomarkers
Diagnostic Biomarkers
Predictive Biomarkers
Prognostic Biomarkers
Therapeutic Biomarkers
Omics Approaches in the Identification of Biomarkers
Genomics
Epigenomics
Transcriptomics
Proteomics
Metabolomics
Analytical Platforms for Biomarker Identification
NMR-Based Approach for Biomarker Identification
MS-Based Hyphenated Techniques for Biomarker Identification
LC-MS Based Approach for Biomarker Identification
GC-MS Approach for Biomarker Identification
CE-MS Approach for Biomarker Identification
Clinically Significant Biomarkers in Oxidative Stress-Induced Cancer
Malondialdehyde (MDA)
8-Hydroxy-2-Deoxyguanosine (8-OHdG)
Isoprostanes
Enzymatic Antioxidants
Chromogranin A
Lysozyme
MicroRNAs (miRNAs)
Challenges Associated with the Cancer Biomarkers Identification
Conclusion
References
143 Reactive Oxygen Species in Cancer Stem Cell Tumorigenesis, Metastasis, and Treatment Resistance
Introduction
ROS Signaling Pathways
PI3K/Akt/mTOR Signaling
Wnt Signaling
MAPK Signaling
STAT3 Signaling
TGF-β Signaling
ROS Transcription Factors
Hypoxia Inducible Factors
Nuclear Factor Erythroid 2-Related Factor 2
Nuclear Factor Kappa B
Nanog
ROS and Oncogene Activation in CSCs
RAS Oncogene
MYC Oncogene
ROS and Anticancer Treatment Resistance
Radioresistance
Chemoresistance
ROS-Dependent Antitumor Strategies
Conclusion
References
144 Dynamics of Cobalt Oxide Nanoparticles in the Activation of Reactive Oxygen Species-Induced Inflammation and Immunomodulat...
Introduction
Nanoparticles and Nanotechnology
Application of Engineering in Medication
Nanoparticle Induced ROS Mediated Inhibition of Cancer
Cobalt Oxide Nanoparticles for Cancer Therapy
Surface Modification Reduces the Toxicity of Cobalt Oxide Nanoparticles
Mechanism of Surface-Modified Cobalt Oxide Nanoparticles on Cells
Surface Modified Cobalt Oxide Nanoparticles Are Potent Immune Stimulator
Cobalt Oxide Nanoparticles Are Potent Antigen Delivery Vehicle
Adjuvanticity of Cobalt Oxide Nanoparticles
Cobalt Oxide Induced ROS Augment Immunostimulation
Conclusions
References
145 Mechanism of Gallic Acid Anticancer Activity Through Copper-Mediated Cell Death
Introduction
DNA Damage Induced by Gallic Acid in Intact and Permeabilized Cells
Determination of DNA Damage by Gallic Acid Along with Metal Chelators in Intact and Permeabilized Cells
H2O2 Generation by Gallic Acid Inside Incubation Medium
Determination of Cell Growth by Gallic Acid in Human Breast Cancer Cells
Gallic Acid and Cancer Chemoprevention
A Copper-Dependent Anticancer Mechanism
Conclusion
Future Direction
References
146 Nitrogen- and Sulfur-Containing Heterocycles as Dual Anti-oxidant and Anti-cancer Agents
Introduction
Thiazoles and Isothiazoles
Benzothiazoles and Other Hybrids
Thiazolidine and Their Derivatives
1,3,4-Thiadiazoles and 1,2,3-Dithiazoles
Thiazines and Benzothiazines
Saccharin-and Acesulfame-Based Anti-Cancer Agents
Phenothiazines
S-Methyl-Isothiosemicarbazones and Their Ruthenium Complexes as Molecular Simplification Products
Thiazepines
Natural Compounds
Conclusion
References
147 Retinoids and Reactive Oxygen Species in Cancer Cell Death and Therapeutics
Introduction
Natural Retinoids Impact on ROS
Synthetic Retinoids Impact on ROS
Conclusion
References
148 Impact of ROS on Cancer and Stem Cell Growth and Therapeutics
Introduction
Reactive Oxygen Species in Cancer Cells
Stem Cells
Origin and Hierarchy of Stem Cells
Embryonic Stem Cells (ESCs)
Adult Stem Cells
Induced Pluripotent Stem Cells (iPSCs)
Cancer Stem Cells (CSCs)
Origin of Cancer Stem Cells
Interplay Between Signaling Pathways and ROS in Cancer and Stem Cells
Oxidative Stress in Somatic Reprogramming
Therapeutics of ROS Induced Cancer and Cancer Stem Cells
Stem Cells, Tissue Engineering and Oxidative Stress
Conclusion
References
Index

Citation preview

Handbook of Oxidative Stress in Cancer: Mechanistic Aspects

Sajal Chakraborti • Bimal K. Ray • Susanta Roychoudhury Editors

Handbook of Oxidative Stress in Cancer: Mechanistic Aspects With 388 Figures and 101 Tables

Editors Sajal Chakraborti Department of Biochemistry and Biophysics University of Kalyani Kalyani, West Bengal, India

Bimal K. Ray Department of Veterinary Pathobiology University of Missouri Columbia, MO, USA

Susanta Roychoudhury CSIR-Indian Institute of Chemical Biology Kolkata, India

ISBN 978-981-15-9410-6 ISBN 978-981-15-9411-3 (eBook) ISBN 978-981-15-9412-0 (print and electronic bundle) https://doi.org/10.1007/978-981-15-9411-3 © Springer Nature Singapore Pte Ltd. 2022 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Springer imprint is published by the registered company Springer Nature Singapore Pte Ltd. The registered company address is: 152 Beach Road, #21-01/04 Gateway East, Singapore 189721, Singapore

Mr. Ratan Naval Tata, born on December 28, 1937, is an Indian industrialist, philanthropist, and head of the charitable trusts founded by the Tata Group. He is recipient of two of the highest civilian awards of India, the Padma Bhushan (2000) and Padma Vibhushan (2008). Mr. Ratan Tata graduated from Riverdale Country School in New York City in 1955. He received a degree in architecture from Cornell University in 1959. In 1975, he participated in the 7-week Advanced Management Program of Harvard Business School, an institution which he has since endowed. Tata Education and Development Trust, a philanthropic affiliate of the Tata Group under the leadership of Mr. Ratan Tata, endowed a $28 million Tata Scholarship Fund, allowing Cornell University to provide financial aid to undergraduate students from India. In 2010, the Tata Group and Tata Trusts donated $50 million for the construction of an executive

center at Harvard Business School. In 2014, the Tata Group donated 950 million rupees to IIT Bombay for the formation of the Tata Centre for Technology and Design to develop design and engineering principles suited to the needs of people and communities with limited resources. The group formed the MIT Tata Centre of Technology and Design at the Massachusetts Institute of Technology (MIT) with a mission to address the challenges of resource-constrained communities, with an initial focus on India. In 2020, Mr. Ratan Tata donated `1,500 crore to fight the Covid-19 pandemic. Over 65% of his shares in Tata Group are invested in charitable trusts. The Tata Trusts’ entry into cancer care dates back to 1941 when the Tata Memorial Hospital opened in Mumbai. The management of the hospital was handed over to the Ministry of Health, Govt. of India, in 1962. In 2012, Tata Trusts launched the Tata Medical Centre in Kolkata to address the high prevalence of cancer and the lack of suitable facilities in the eastern and north-eastern regions of India. Tata Trusts have developed cancer research and treatment centers in Varanasi, Tirupati, Bhubaneshwar, Ranchi, Allahabad, and Mangalore. They are also partnering state governments in building state-wide cancer facility networks in Assam, Odisha, Jharkhand, Telangana, and Nagaland. Mr. Ratan Tata is undoubtedly a living legendary figure in promoting higher education and research notably in cancer research and treatment facilities in India. He has the ability to inspire and motivate young scholars and entrepreneurs. We feel honored to dedicate this book to Mr. Ratan Naval Tata and wish him good health and success in all his future endeavors.

Preface

After I finished the Atlantic swim I said never again, but it didn’t take long for me to change my mind. I like to push my limits. I want to raise money for cancer research and to inspire others to follow their dreams. Benoit Lecomte

Cancer seems to be a uniquely unsolved problem in biology, and there are many fundamental aspects of multicellular dynamics in cancer which are currently unknown. In the recent past, different key topics have emerged in cancer research such as modulation of molecular targets by gene expression, epigenetic processes, and stem cell functions. There is little doubt that part of the increase in incidence of cancer is also due to environmental pollution. The carcinogenic effect of certain chemicals is well understood at a biological level. Positive correlation has been established between environmental quality and health for large populations. For instance, the 5-year study of water pollution and cancer in the Huai river valley of China has demonstrated a clear relationship between water pollution and high rates of digestive tract cancer at the county level (Atlas of the Huai River Basin Water Environment: Digestive Cancer Mortality. Eds: Gonghuan Yang, Dafang Zhuang; Springer, New York, 2014). It has also long been recognized that abnormal regulation of protein phosphorylation plays an important role in the initiation and progression of cancer. This book provides reviews devoted to the emerging roles of signal transduction events associated with cell transformation and tumor progression. The authors suggest how the activities of certain key signal transduction proteins might be manipulated in cancer therapy. Non-coding RNAs (ncRNAs) are capable of reprogramming multiple oncogenic cascades and, therefore, can be used as target agents. Recently, profiling and sequencing of ncRNAs showed discernible genomic deregulation, which leads to human cancers mostly due to aberrant mechanisms of ncRNAs biogenesis, such as amplification, deletion, and abnormal epigenetic or transcriptional regulation. Given the emerging importance of ncRNAs in malignancy, some chapters of this book have been devoted towards understanding the role of non-coding transcription in cancer progression and metastasis with an eye on rising clinical relevance of ncRNAs.

vii

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Preface

Nanotechnology as applied to cancer research is by nature multidisciplinary and relies on research mostly by chemists, cell biologists, and pharmacologists. Nanotechnology provides novel tools to manipulate cellular machineries and possesses the ability to bring a paradigm shift in how cancer is currently imaged or treated. The chapters in this book range from enumerating basic research to clinical applications of nanoparticles in the field of nanotechnology. Additionally, the development of bioinformatic tools and studies on the correlation between computational analyses and experimental observations shed light on new mechanisms on ROS-induced cancer initiation and progression and also therapeutics of ROS-induced cancer. The motivation for organizing this book stems primarily from an urge to present readers with the latest research on the mechanisms associated with ROS-induced cancer. This book does not claim to cover the entire field of ROS-induced cancer. However, it provides a plethora of reviews that will enable readers to have a glimpse of the basic mechanisms associated with ROS-induced initiation and progression of different types of cancer with global perspectives. Thanks are due to all contributors for the considerable energy, time, and effort that they spent in making this book an advancement of knowledge for understanding the mechanisms associated with ROS-induced cancer. Thanks are also due to Dr. (Ms.) Mokshika Gaur, Dr. (Ms.) Madhurima Kahali, Ms. Divya Nithyanandam, and Ms. Ilakkia Sathiyaseelan of Springer Nature for their understanding, cooperation, and support during the preparation of the book. Sajal Chakraborti Corresponding Editor

Contents

Volume 1 Part I 1

Oxidative Stress in Carcinogenesis . . . . . . . . . . . . . . . . . . . . .

1

Reactive Species and ER-Mitochondrial Performance for Glioblastoma Multiforme Treatment Strategy . . . . . . . . . . . . . . . . . Tina Nasrin, Sajal Chakraborti, and Soni Shaikh

3

2

Oxidative Stress and Thyroid Disorders . . . . . . . . . . . . . . . . . . . . . . Loganayaki Periyasamy, Kokelavani Nampalli Babu, Sneha Krishnamoorthy, Jonathan Behlen, Sridhar Muthusami, and Jone A. Stanley

23

3

Skin Cancer Induced by Pollution-Mediated ROS . . . . . . . . . . . . . . Karen E. Burke

35

4

Roles of β-Glucans in Oxidative Stress and Cancer . . . . . . . . . . . . . Eveline A. I. F. Queiroz, Pâmela Alegranci, Aneli M. Barbosa-Dekker, and Robert F. H. Dekker

57

5

Oral Cancer and Oxidative Stress Gokul Sridharan

..........................

77

6

Oxidative Stress in Genitourinary Cancer . . . . . . . . . . . . . . . . . . . . Masaki Shiota

87

7

Oxidative Stress, Microenvironment, and Oral Cancer . . . . . . . . . . Gargi Sarode, Nikunj Maniyar, Sachin Sarode, and Mamatha G. S.

99

8

Oxidative Stress and Glyoxalase Pathway in Cancer . . . . . . . . . . . . Nupurand A. B. Tiku

119

9

The Implication of ROS Homeostasis in the Modulation of EMT Signaling and Its Role in Manipulating Tumor Microenvironment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Souneek Chakraborty and Anindya Goswami

137 ix

x

10

11

12

13

Contents

Functional Regulation Between Matrix Metalloproteases and Cell Junction Proteins in Gastric Cancer . . . . . . . . . . . . . . . . . . . . . . . . Tapasi Roy, Vineet Kumar Mishra, Sudipta Mallick, and Snehasikta Swarnakar Association of Oxidative Stress and Mitochondrial Dysfunction to Gynecological Malignancies . . . . . . . . . . . . . . . . . . . . . . . . . . . . Deepshikha Ghosh, Priti Chatterjee, Tulika Mitra, and Sib Sankar Roy

165

Impact of Caenorhabditis elegans in Cancer Drug Resistance Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G. Shanmugam, S. Kannan, and K. Senthilkumar

185

Scaffold-Based Selective ROS Generation as Viable Therapeutic Strategies Against Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Md Yousuf, Mohammed Tanveer Ahmed, and Rajkumar Banerjee

197

14

Targeting Oxidative Stress in Cancer . . . . . . . . . . . . . . . . . . . . . . . Paramita Mandal, Anindita Goswami, Sarmistha Adhikari, and Subham Sarkar

15

Targeting Mitochondria as a Novel Disease-Modifying Therapeutic Strategy in Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Gurjit Kaur Bhatti, Paras Pahwa, Anshika Gupta, Uma Shanker Navik, P. Hemachandra Reddy, and Jasvinder Singh Bhatti

16

153

Cutaneous Unfolded Protein Response (UPR) and Endoplasmic Reticulum (ER) Stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Rather A. Rafiq, Ram A. Vishwakarma, and Sheikh A. Tasduq

217

241

263

17

Iron Sulfur Clusters and ROS in Cancer . . . . . . . . . . . . . . . . . . . . Joel James, Daniel Andrew M. Gideon, Debasish Roy, and Amritlal Mandal

291

18

Free Radicals, Reactive Oxygen Species, and Their Biomarkers . . . Jiafu Feng

307

19

Zymographic Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Vibha Rani, Kanishka Aggarwal, Sristis Varshney, and Neha Atale

327

20

Biomarkers of Oxidative Stress and Its Dynamics in Cancer A. K. Chaudhary and P. K. Gupta

....

341

21

Glutathione as Oxidative Stress Marker in Cancer . . . . . . . . . . . . N. Thirumoorthy, R. Senthilkumaran, L. Panayappan, Babu Thandapani, and K. Ranganathan

353

22

Salivary Oxidative Stress Biomarkers in Oral Potentially Malignant Disorders and Squamous Cell Carcinoma . . . . . . . . . . Gokul Sridharan

373

Contents

23

xi

Recent Development of Monoclonal Antibodies Targeting Tyrosine Kinase in ROS-Mediated Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . Yashodhara Dalal and Maushmi S. Kumar

24

Fluoride as a Carcinogen: A Myth or Fact? . . . . . . . . . . . . . . . . . . Arnadi Ramachandrayya Shivashankara and Manjeshwar Shrinath Baliga

25

The Role of ROS in Chemical Carcinogenesis Induced by Lead, Nickel, and Chromium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Aleksandra Buha Djordjevic, Milena Andjelkovic, Dragana Javorac, Luka Manic, Zorica Bulat, Yasmeen Talab, Emiliano Panieri, Luciano Saso, and David Wallace

26

27

28

Environmental Contaminants, Oxidative Stress, and Reproductive Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Latchoumycandane, Meenu Maniradhan, and P. P. Mathur Environmental Toxicants and Carcinogenicity: Role of Oxidative Stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sanjay Saini, Jagdish Gopal Paithankar, Anurag Sharma, and Debapratim Kar Chowdhuri Environmental and Occupational Exposure to Pesticides and Cancer Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Tamara Lazarević-Pašti

29

Benzo(a)Pyrene-Induced ROS-Mediated Lung Cancer . . . . . . . . . Rebai Ben Ammar, Fatma J. Al Saeedi, Emad A. Ahmed, and Peramaiyan Rajendran

30

Essential Role of Occupational Hazards in Cancer Among Women . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Saritha, K. Grace Theodora, and K. Vijaya Rachel

31

Arsenic: An Environmental Toxicant-Induced Oxidative Stress and Carcinogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Abhilash M, Prathapan Ayyappan, Harikumaran Nair R, and Mathews Valuparampil Varghese

383 399

405

423

437

451 463

477

491

32

ROS in Apoptosis of Cancer Cells Ayse Günes-Bayir

.........................

503

33

Role of ROS in Triggering Death Receptor-Mediated Apoptosis . . . Samaneh H. Shabani and Azam Bolhassani

517

34

Advanced Glycation End Products-Mediated Oxidative Stress and Regulated Cell Death Signaling in Cancer . . . . . . . . . . . . . . . . . . . Chandramani Pathak, Foram U. Vaidya, Bhargav N. Waghela, Abu Sufiyan Chhipa, Budhi Sagar Tiwari, and Kishu Ranjan

535

xii

35

36

37

Contents

Helping Leukemia Cells to Die with Natural or Chemical Compounds Through H2O2 Signaling . . . . . . . . . . . . . . . . . . . . . . Carlos Velez-Pardo and Marlene Jimenez-Del-Rio Microtubule-Targeting Agents Induce ROS-Mediated Apoptosis in Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Amlan Das, Santanu Paul, Subhendu Chakrabarty, Moumita Dasgupta, and Gopal Chakrabarti ROS Induced by Chemo- and Targeted Therapy Promote Apoptosis in Cancer Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sathish Kumar Reddy Padi, Shailender S. Chauhan, and Neha Singh

551

565

583

38

ROS-Mediated Apoptosis in Cancer . . . . . . . . . . . . . . . . . . . . . . . . Saranya NavaneethaKrishnan, Jesusa L. Rosales, and Ki-Young Lee

599

39

Genomic Instability in Carcinogenesis . . . . . . . . . . . . . . . . . . . . . . Somsubhra Nath and Stuti Roy

619

40

Impact of Environmental and Occupational Exposures in Reactive Oxygen Species-Induced Pancreatic Cancer . . . . . . . . . . . . . . . . . . Nilabja Sikdar, Subhankar Dey, and Sudeep Banerjee

41

Reactive Oxygen Species: Central Regulators of the Tumor Microenvironment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . María Julia Lamberti, Renzo Emanuel Vera, Martín Ernesto Fernández-Zapico, and Natalia Belén Rumie Vittar

637

663

42

Biomarkers of Oxidative Stress-Induced Cancer . . . . . . . . . . . . . . Pankaj Dixit and Dinesh Kumar Mishra

681

43

Reactive Oxygen Species in Male Reproductive Cancers . . . . . . . . Nomesh Yadu and Pradeep G. Kumar

695

44

Biomarkers of Oxidative Stress in Cancer and Their Clinical Implications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Palanivel Gajalakshmi and Thanemozhi G. Natarajan

45

The Role of Reactive Oxygen Species on Cellular Fate and Function of Tumor-Infiltrating Lymphocytes . . . . . . . . . . . . . . . . . Lakshmi R. Perumalsamy, Sanjana Rajgopal, Tapasya K., Sherine Joanna Fredrick, and Arun Dharmarajan

46

ROS-Mediated Inflammatory Response in Cancer . . . . . . . . . . . . Shibi Muralidar, Gayathri Gopal, and Senthil Visaga Ambi

47

Food Colors and Associated Oxidative Stress in Chemical Carcinogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Debadutta Mishra

711

731

751

773

Contents

48

49

50

xiii

Benzo(a)Pyrene-Induced Oxidative Stress During Lung Cancer and Treatment with Baicalein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Naveenkumar Chandrashekar

787

Assessing the Contributions of Lipid Profile and Oxidative Lipid Damage to Carcinogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Daniel Andrew M. Gideon and Joel James

805

Oxidative Stress in Hepatocarcinogenesis and Role of Antioxidant Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Salah Mohamed El Sayed

821

Volume 2 Part II 51

52

53

ROS- Induced Cancer Progression and Metastasis . . . . . . .

Oxidative Stress in Orchestrating Genomic Instability-Associated Cancer Progression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Dipita Bhakta-Guha and Gunjan Guha

841

Hypoxic Stress Perturb DNA Repair Mechanisms Leading to Genetic Instability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Goutham Hassan Venkatesh

859

DNA Lesions Induced by Lipid Peroxidation Products in Cancer Progression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ana Paula de Melo Loureiro

875

...

897

54

Understanding ROS-Induced DNA Damage for Therapeutics Imran Moin, Disha Mittal, and Anita K. Verma

55

Mitochondrial Metabolism, Oxidative Stress, and the Microenvironment in Breast Cancer Development and Progression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Heide Schatten

56

57

58

839

Role of Oxidative Stress and DNA Damage/Repair in Lung Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Joytri Dutta, Sabita Singh, Ashish Jaiswal, Archita Ray, Pamelika Das, and Ulaganathan Mabalirajan

919

937

Oxidative Stress and Cancer: Role of the Nrf2-Antioxidant Response Element Signaling Pathway . . . . . . . . . . . . . . . . . . . . . . Munindra Ruwali and Rahul Shukla

957

Role of Macrophages in Oxidative Stress-Induced Inflammatory Tumor Microenvironment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Abhishek Teli, Disha Kshirsagar, Saurav Doshi, and Tuli Dey

975

xiv

Contents

59

The Triad, Hypoxia–ROS–Inflammation . . . . . . . . . . . . . . . . . . . . Sankar Bhattacharyya

991

60

Interplay Between Reactive Oxygen Species and Key Players in the DNA Damage Response Signaling Network . . . . . . . . . . . . . . . 1005 Siddavaram Nagini, Paranthaman Thiyagarajan, and Kunchala Sridhar Rao

61

ROS at the Intersection of Inflammation and Immunity in Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1023 Shivanjali Saxena and Sushmita Jha

62

Sestrin-2 Connects Autophagy: Gatekeepers Against Tumor Progression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1041 Ganapasam Sudhandiran

63

Insights into the Role of NRf2 Pathway in Cadmium-Induced Carcinogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1055 K. B. Arun, Mathews Valuparampil Varghese, and Prathapan Ayyappan

64

Interplay Between Redox Homeostasis and Oxidative Stress in the Perspective of Ovarian and Cervical Cancer Immunopathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1065 Saurav Kumar, Vaishali Mulchandani, Anurag Banerjee, and Jayasri Das Sarma

65

Oxidative Dyshomeostasis in the Mitochondria . . . . . . . . . . . . . . . 1083 Gunjan Guha and Dipita Bhakta-Guha

66

The Double-Edged Sword Role of ROS in Cancer . . . . . . . . . . . . . 1103 Rishabh Kumar, Himanshu K. Prasad, and Munish Kumar

67

ROS Modulation on Apical Junctional Complex . . . . . . . . . . . . . . 1121 Bruno S. Gonçalves, Duane G. Pereira, Israel J. P. Garcia, Jessica M. M. Valadares, Lilian N. D. Silva, Rubén G. Contreras, and Leandro A. Barbosa

68

The Interdependence of Inflammation and ROS in Cancer . . . . . . 1135 Haijie Wu, Mingyue Zhong, and Yuzhen Wang

69

Interplay Between Oxidative Stress and Endoplasmic Reticulum Stress in the Metastasis of Colon Cancer . . . . . . . . . . . . . . . . . . . . 1153 Ganapasam Sudhandiran, Vadivel Dinesh Babu, Alagesan Seetha, and Balaraman Santhosh

70

Mitochondria-Targeted Antioxidants and Cancer . . . . . . . . . . . . . 1167 Sanjay Bharati and Sachin Shetty

Contents

xv

71

ROS-Mediated Genome Alterations at Cellular Microenvironment During Cancer Metastasis: A Curtain Raiser . . . . . . . . . . . . . . . . . 1189 Tuhin Ghosh and Dipan Adhikari

72

Oxidative Stress in Cancer and Its Influence on Amoeboidal Migration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1203 Sukanya Gayan, Pooja Sanjay Ghuge, Malhar Sojwal Chitnis, and Tuli Dey

73

The Intricacy of ROS in Cancer Therapy Resistance . . . . . . . . . . 1217 Chandan Kanta Das, Ranabir Majumder, Pritam Roy, and Mahitosh Mandal

74

Oxidative Stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1239 N. N. V. Radharani, Ipsita G. Kundu, Amit S. Yadav, and Gopal C. Kundu

75

Relationship Between ROS, Autophagy, and Cancer . . . . . . . . . . . 1253 Jessica Campos-Blázquez, Catalina Flores-Maldonado, Alan A. Pedraza-Ramírez, Octavio López-Méndez, Juan M. Gallardo, Leandro A. Barbosa, and Rubén G. Contreras

Part III ROS- Induced Modulation of Signal Transduction Mechanisms and Gene Expression in Cancer . . . . . . . . . . . . . . . . . .

1269

76

TRP Channels, Oxidative Stress, and Cancer . . . . . . . . . . . . . . . . 1271 Amritlal Mandal, Mathews Valuparampil Varghese, Joel James, and Sajal Chakraborti

77

Wnt Signaling in Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1289 Minakshi Prasad, Mayukh Ghosh, Rajesh Kumar, Lukumoni Buragohain, Ankur Kumari, and Gaya Prasad

78

Two-Faced Role of ROS in the Regulation of Cancer Cell Signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1311 Banudevi Sivanantham

79

Oxidative Stress and Notch Signaling . . . . . . . . . . . . . . . . . . . . . . . 1327 Vivek Kumar, Mohit Vashishta, and Bilikere S. Dwarakanath

80

ROS-Induced Regulatory Crosstalk with Autophagy and AKT/mTOR Signaling in Cancer Cells . . . . . . . . . . . . . . . . . . . . . 1345 Piyanki Das, Koustav Chatterjee, and Tathagata Choudhuri

81

Redox Regulation of Estrogen Signaling in Human Breast Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1359 Aarifa Nazmeen and Smarajit Maiti

xvi

Contents

82

ROS Impacts on Cell Cycle Checkpoint Signaling in Carcinogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1375 Seyed Isaac Hashemy and Seyed Mohammad Reza Seyedi

83

Modulation of JAK/STAT Pathways in Cancer by Phytochemicals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1395 Olorunfemi R. Molehin, Olusola O. Elekofehinti, Ajibade O. Oyeyemi, Oluwatosin B. Olusakin, Aderonke E. Fakayode, Ezekiel T. Ige, and Oluwatumininu O. Adesua

84

Role of Carotenoids on Oxidative Stress–Mediated Signaling in Cancer Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1407 Poorigali Raghavendra-Rao Sowmya, Rudrappa Ambedkar, and Rangaswamy Lakshminarayana

85

Cross Talk Between Oxidative Stress and p53 Family Members in Regulating Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1427 Sumiran Kumar Gurung, Lokesh Nigam, Kunwar Somesh Vikramdeo, and Neelima Mondal

86

Mutant K-Ras-Mediated Oxidative Stress in Pancreatic Cancer . . . 1443 Divya Thomas, Satish Sagar, Tristan Caffrey, and Prakash Radhakrishnan

87

mRNA Stabilizing Factor HuR: A Crucial Player in ROS-Mediated Cancer Progression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1455 Soumasree De and Kuntal Dey

88

Redox State and Gene Regulation in Breast Cancer Aritra Gupta, Shayantani Chakraborty, Partha Das, Animesh Chowdhury, and Kartiki V. Desai

89

Glutathione Peroxidase and Lung Cancer: An Unravel Story . . . . 1481 Animesh Chowdhury

90

Importance of Silencing RNAs in Cancer Research . . . . . . . . . . . . 1493 Antara Banerjee, Janani Gopi, Francesco Marotta, Secunda Rupert, Rosy Vennila, and Surajit Pathak

91

Cellular Redox Status and Modifiable Behaviors . . . . . . . . . . . . . . 1507 Mary Figueroa and Joya Chandra

92

Yin-Yang of Oxidative Stress in Pancreatic Cancers . . . . . . . . . . . 1521 Sonali Choudhury, Afreen Asif Ali Sayed, Prasad Dandawate, and Shrikant Anant

. . . . . . . . . . . 1461

Contents

xvii

93

Epigenetic Therapy as a Potential Approach for Targeting Oxidative Stress–Induced Non-small-Cell Lung Cancer . . . . . . . . 1545 Ridhima Wadhwa, Keshav Raj Paudel, Shakti Shukla, Madhur Shastri, Gaurav Gupta, Hari Prasad Devkota, Mary Bebawy, Dinesh Kumar Chellappan, Philip Michael Hansbro, and Kamal Dua

94

Free Radicals–Mediated Epigenetic Changes and Breast Cancer Progression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1561 Padmanaban S. Suresh, Nivedita Nanda, and Sanu Thankachan

95

Phytochemicals in ROS-Mediated Epigenetic Modulation of Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1583 Madhumita Roy and Amitava Datta

96

Noncoding RNAs Regulation of Redox Balance in Cancer . . . . . . 1601 Azhwar Raghunath, Raju Nagarajan, Kiruthika Sundarraj, and Lakshmikanthan Panneerselvam

97

Oxidized DNA Base Damage Repair and Transcription . . . . . . . . 1621 Suravi Pramanik, Shrabasti Roychoudhury, and Kishor K. Bhakat

98

Epigenetic Instability Caused by Oxidative Stress Triggers Tumorigenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1639 Raman Preet Kaur, Prabhsimran Kaur, and Anjana Munshi

Volume 3 Part IV

Prevention and Therapeutics of ROS Induced Cancer . . . .

1657

99

Implications of ROS in Cancer Stem Cells Mechanism of Action . . . 1659 Cornelia Amalinei, Raluca Anca Balan, Adriana Grigoras, Ludmila Lozneanu, Elena Roxana Avadanei, Simona Eliza Giusca, and Irina Draga Caruntu

100

Metabolism-Redox Interplay in Tumor Stem Cell Signaling . . . . 1681 Vanesa Martin, Maria Turos-Cabal, Ana Maria Sanchez-Sanchez, and Carmen Rodríguez

101

Association of ROS with Epithelial-Mesenchymal Transition and Acquisition of Stemness During Carcinogenesis . . . . . . . . . . . . . . 1703 Sujata Law and Ritam Chatterjee

102

Therapeutic Effect of Natural Compounds in Targeting ROS-Induced Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1717 Pawan Kumar Raghav, Zoya Mann, Vishnu Krishnakumar, and Sujata Mohanty

xviii

Contents

103

Therapeutics of Oxidative Stress and Stemness in Breast Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1765 Balraj Singh, Kalpana Mujoo, and Anthony Lucci

104

Reactive Oxygen Species in Stem Cell Proliferation and Cancer . . . 1777 Yogesh Kumar Verma, Subodh Kumar, Nishant Tyagi, and Gurudutta Gangenahalli

105

Targeting Redox Signaling and ROS Metabolism in Cancer Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1791 Eunus S. Ali, David Barua, Subbroto Kumar Saha, Maizbha Uddin Ahmed, Siddhartha Kumar Mishra, and Mohammad S. Mubarak

106

Modulatory Role of Adipocytes and Their Stem Nature in the ROS Signaling Within a Tumor Micro-environment . . . . . . . . . . 1819 Salvatore Chirumbolo, Geir Bjørklund, and Antonio Vella

107

Hypoxia-Induced Stress Responses in Cancer and Cancer Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1829 Sandhya Chipurupalli, Snehlata Kumari, Vincenzo Desiderio, and Nirmal Robinson

108

Oxidative Stress-Targeted Therapies for the Treatment of Acute Myeloid Leukemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1845 Ajit Kumar Rai and Neeraj Kumar Satija

109

Role of ROS in Cancer Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . 1855 Sharmistha Chatterjee, Abhishek Kumar Das, and Parames C. Sil

110

Reactive Oxygen Species-Dependent Signaling Pathways in Cancer Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1885 Vijay Kumar Kutala and Shaik Mohammad Naushad

111

System Biology and Network Analysis Approaches on Oxidative Stress in Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1901 Sarpita Bose, Krishna Kumar, and Saikat Chakrabarti

112

Reactive Oxygen Species–Mediated Cancer Progression and Metastasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1919 N. A. Chugh and A. Koul

113

Anthocyanins and Flavonols: Therapeutic Implications of Natural Compounds on Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . 1933 Charles Elias Assmann, Grazielle Castagna Cezimbra Weis, Jéssica Righi da Rosa, Beatriz da Silva Rosa Bonadiman, Audrei de Oliveira Alves, Felipe Tecchio Borsoi, and Margarete Dulce Bagatini

Contents

xix

114

Implication of Nanomedicine in Therapy of Oxidative Stress-Induced Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1947 Tanweer Haider, Vikas Pandey, Kamalpreet Kaur Sandha, Prem N. Gupta, and Vandana Soni

115

Metabolic Oxidative Stress in Initiation, Progression, and Therapy of Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1969 Soumen Bera, Amit Verma, Anant N. Bhatt, and Bilikere S. Dwarakanath

116

Mitochondrial ROS: A Reactive Species Targeted in Cancer Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2005 Bee Ling Tan and Mohd Esa Norhaizan

117

Oxidative Stress and Hypoxia in Cancer: Implications for Radiation Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2023 Amrita Roy, Slavisa Tubin, Bilikere S. Dwarakanath, and Seema Gupta

118

Oxidative Stress in Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2049 Ugir Hossain Sk and Sudin Bhattacharya

119

Phytocompounds-Based Approaches to Combat Oxidative Stress in Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2073 Thakur Uttam Singh, Madhu Cholenahalli Lingaraju, Govind Garg, Meemansha Sharma, Subhashree Parida, and Dinesh Kumar

120

Phytoestrogens Modulate Oxidative Stress . . . . . . . . . . . . . . . . . . 2089 Margalida Torrens-Mas and Pilar Roca

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Prospective Application of Natural and Synthetic Redox Modulators in Oxidative Stress-Targeted Cancer Therapy . . . . . 2101 Sandra Petrovic and Andreja Leskovac

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ROS Modulation by Iron Chelators and Lipids: A Developing Anticancer Strategy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2123 Or Kakhlon

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ROS, Cancer, Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2147 Sajan George and Heidi Abrahamse

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Modulators of ROS/NF-κB Signaling in Cancer Therapy . . . . . . 2165 Maria Voura, Eleni Sflakidou, and Vasiliki Sarli

125

Hyperglycolysis-Inflammation Connect as a Mechanistic Hot Spot in Oxidatively Compromised Cancer . . . . . . . . . . . . . . . . . . . . . . 2185 Brajesh Kumar Maurya, Akanksha Pandey, and Surendra Kumar Trigun

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Contents

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Constraint-Based Modeling to Understand ROS-Mediated Effects in Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2209 Prerna Bhalla, Swagatika Sahoo, Raghunathan Rengaswamy, Devarajan Karunagaran, and G. K. Suraishkumar

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Dynamical Methods to Study Interaction in Proteins Facilitating Molecular Understanding of Cancer . . . . . . . . . . . . . . . . . . . . . . . 2231 Bhaskar Dasgupta, Gert-Jan Bekker, and Narutoshi Kamiya

128

Molecular Insights into the Roles of E3-Ligases in ROS-Mediated Cancer from a Bioinformatics Perspective . . . . . . . . . . . . . . . . . . 2249 Sima Biswas and Angshuman Bagchi

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Insulin Resistance and Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . 2265 Adriana Monroy, Laura Gómez-Laguna, Carlos E. Aranda-Flores, and Silvestre Alavez

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Meprins: Ancient Enzymes Newly Discovered in Cancer Progression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2283 Sibani Sen Chakraborty, Ankur Chaudhuri, Yuthika Dholey, and Asim K. Bera

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Molecular Analysis of the Involvements of lncRNA in Cancer Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2295 Angshuman Bagchi

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Proteomics and Metabolomics in Cancer Diagnosis and Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2309 Minakshi Prasad, Somesh Banerjee, Suman, Rajesh Kumar, Lukumoni Buragohain, and Mayukh Ghosh

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Systems Biology and Bioinformatics Insights into the Role of Free Radical-Mediated Oxidative Damage in the Pathophysiology of Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2339 Shaik Mohammad Naushad and Vijay Kumar Kutala

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Systems Biology Resources and Their Applications to Understand the Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2349 Pawan Kumar Raghav, Zoya Mann, Pranav K. Pandey, and Sujata Mohanty

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Targeting Reactive Oxygen Species Homeostasis and Metabolism in Cancer Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2385 Stacy Grieve and Dipsikha Biswas

136

Thioredoxin and Glutathione Systems . . . . . . . . . . . . . . . . . . . . . 2407 Yezhou Yu, Giovanna Di Trapani, and Kathryn F. Tonissen

Contents

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137

Vitamin C in Cancer Management: Clinical Evidence and Involvement of Redox Role . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2421 Sanjib Bhattacharya

138

Oxidative Stress, Inflammasome, and Cancer . . . . . . . . . . . . . . . 2435 Biswatrish Sarkar, Prasanta Kumar Deb, and Sugato Banerjee

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Preventive Role of Carotenoids in Oxidative Stress-Induced Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2449 Edakkadath Raghavan Sindhu, Antholi Keloth Kavya, and Ponnamparambil Purushothaman Binitha

140

Personalized Vaccine as a Therapeutic Approach Toward Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2463 Shikha Mohan

141

Enolase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2473 Wasia Rizwani

142

Analytical and Omics Approaches in the Identification of Oxidative Stress-Induced Cancer Biomarkers . . . . . . . . . . . . . . . 2493 Siva Nageswara Rao Gajula, Devi Naga Jyothi Bale, and Satheesh Kumar Nanjappan

143

Reactive Oxygen Species in Cancer Stem Cell Tumorigenesis, Metastasis, and Treatment Resistance . . . . . . . . . . . . . . . . . . . . . 2517 Naomi Brook and Arun Dharmarajan

144

Dynamics of Cobalt Oxide Nanoparticles in the Activation of Reactive Oxygen Species-Induced Inflammation and Immunomodulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2541 Sourav Chattopadhyay and Somenath Roy

145

Mechanism of Gallic Acid Anticancer Activity Through Copper-Mediated Cell Death . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2559 Mohd Farhan, Mohammad Aatif, Sheikh Mumtaz Hadi, and Aamir Ahmad

146

Nitrogen- and Sulfur-Containing Heterocycles as Dual Anti-oxidant and Anti-cancer Agents . . . . . . . . . . . . . . . . . . . . . . 2571 Simone Carradori, Paolo Guglielmi, Grazia Luisi, and Daniela Secci

147

Retinoids and Reactive Oxygen Species in Cancer Cell Death and Therapeutics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2589 Chirine El-Baba, Ali H. Eid, Abdallah Shaito, Firas Kobeissy, and Nadine Darwiche

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148

Contents

Impact of ROS on Cancer and Stem Cell Growth and Therapeutics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2611 Aadil Qadir Bhat, Mir Owais Ayaz, Md Mehedi Hossain, Aalim Maqsood, and Mohd Jamal Dar

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2629

About the Editors

Dr. Sajal Chakraborti is Professor of Biochemistry at the University of Kalyani, West Bengal, India. His research covers the role of oxidant-mediated signaling in the pathogenesis of a variety of diseases. Prof. Chakraborti did is PhD from Calcutta University (1982) and DSc from Kalyani University (2004). He did postdoctoral research at the Johns Hopkins University, Baltimore; University of Utah Health Sciences Center, Salt Lake City; and New York Medical College, New York as a Fulbright Fellow. He received DBT (Govt. of India) senior overseas research award for his research at the University of Florida, Gainesville (1998– 1999). He has been engaged in teaching and research in biochemistry for the past 40 years. He has published over 120 original research papers, 22 book chapters, and 15 review articles. He also edited 12 books (Springer). Dr. Bimal K. Ray is Professor of Pathology at the University of Missouri, Columbia, Missouri, USA. He has more than 40 years of teaching and research experience in different universities in the USA. One of his research interests is the understanding the basic mechanisms associated with inflammation, the consequence of which is linked to abnormal gene expression that may lead to cancer. Recent findings of Dr. Ray’s laboratory suggest that serum amyloid A-activating factor-1 (SAF-1) activates superoxide dismutase and few other reactive species sensitive antioxidant enzymes in breast cancer and thereby allowing cancer cell proliferation. Dr. Ray has published over 100 original research papers and review articles in different international journals of repute. He has also published several chapters in books published by reputed publishers.

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About the Editors

Dr. Susanta Roychoudhury obtained his PhD degree from Calcutta University in 1985. He did postdoctoral research at the University of Pennsylvania, USA (1985– 1990). Then, he joined the CSIR-Indian Institute of Chemical Biology, Kolkata, India, as a scientist (1991– 2015). Subsequently, he was associated with Saroj Gupta Cancer Centre & Research Institute, Kolkata, India, as Chief of basic research (2015–2021). Currently, he is the ICMR Emeritus Scientist at CSIR-Indian Institute of Chemical Biology, Kolkata, India. As a cancer geneticist, Dr. Roychoudhury made significant contributions on the molecular understanding of the genomic instabilities and functional consequences of gain-of-function mutations of TP53 gene in human cancer. He has contributed in the area of cancer stem cells using oral cancer model and developed a microarray-based gene expression signature that distinguishes the aggressive subset of early stage oral cancer from non-aggressive one. He has published over 150 original research articles in several international journals of repute and several review articles and book chapters. Dr. Roychoudhury is Fellow of all the three science academies of India. He has been elected President of the Indian Association for Cancer Research in 2016 and is a recipient of Sir J C Bose National Fellowship by Department of Science and Technology, Govt. of India, in 2017.

About the Section Editors

Section: Oxidative Stress in Carcinogenesis Tanya Das Bose Institute, Bidhannagar, Kolkata, India Amit Pal National Institute of Cholera and Enteric Diseases, Kolkata, India Amritlal Mandal Department of Physiology, University of Arizona, Tucson, USA Section: ROS-Induced Cancer Progression and Metastasis Gaurisankar Sa Bose Institute, Kolkata, India Suvro Chatterjee Department of Biotechnology, Anna University, Chennai, India Section: ROS-Induced Modulation of Signal Transduction Mechanisms and Gene Expression in Cancer Snehasikta Swarnakar CSIR-Indian Institute of Chemical Biology, Kolkata, India Urmi Chatterji Department of Zoology, University of Calcutta, Kolkata, India Prosenjit Sen Indian Association for the Cultivation of Science, Kolkata, India Animesh Chowdhury Animesh Chowdhury National Institute of Biomedical Genomics, Kalyani, West Bengal, India

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About the Section Editors

Section: Prevention and Therapeutics of ROS Induced Cancer Sujata Mohanty Stem Cell Facility, DBT-Centre of Excellence for Stem Cell Research, All India Institute of Medical Sciences, New Delhi, India Parames C. Sil Division of Molecular Medicine, Bose Institute, Kolkata, India Angshuman Bagchi Department of Biochemistry and Biophysics, University of Kalyani, West Bengal, India

Contributors

Mohammad Aatif Department of Public Health, College of Applied Medical Sciences, King Faisal University, Al Ahsa, Kingdom of Saudi Arabia Heidi Abrahamse Laser Research Center, University of Johannesburg, Johannesburg, South Africa Oluwatumininu O. Adesua Department of Biochemistry, Ekiti State University, Ado-Ekiti, Nigeria Dipan Adhikari Department of Botany (UG & PG), Hooghly Mohsin College, Chinsurah, West Bengal, India Sarmistha Adhikari Biomedical Genetics Laboratory, Department of Zoology, The University of Burdwan, Bardhaman, West Bengal, India Kanishka Aggarwal Department of Biotechnology, Jaypee Institute of Information Technology, Noida, Uttar Pradesh, India Aamir Ahmad Department of Medicine, University of Alabama, Birmingham, AL, USA Emad A. Ahmed Department of Biological Sciences, College of Science, King Faisal University, Al Ahsa, Saudi Arabia Maizbha Uddin Ahmed Department of Industrial and Physical Pharmacy (IPPH), Purdue University, West Lafayette, IN, USA Mohammed Tanveer Ahmed Applied Biology Division, CSIR-Indian Institute of Chemical Technology, Hyderabad, India Academy of Scientific and Innovation Research (AcSIR), Ghaziabad, India NanoBiotechnology Research Laboratory (NBRL), School of Science, RMIT University, Melbourne, VIC, Australia Silvestre Alavez Metropolitan Autonomous University, Lerma, Health Sciences Department, Mexico City, Mexico Pâmela Alegranci Núcleo de Pesquisa e Apoio Didático em Saúde (NUPADS), Instituto de Ciências da Saúde, Câmpus Universitário de Sinop, Universidade Federal de Mato Grosso, Sinop-MT, Brazil xxvii

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Contributors

Eunus S. Ali College of Medicine and Public Health, Flinders University, Bedford Park, Australia Simpson Querrey Institute for Epigenetics, Northwestern University Feinberg School of Medicine, Chicago, IL, USA Department of Biochemistry and Molecular Genetics, Northwestern University Feinberg School of Medicine, Chicago, IL, USA Audrei de Oliveira Alves Federal University of Santa Maria, Santa Maria, RS, Brazil Cornelia Amalinei Department of Morphofunctional Sciences I, “Grigore T. Popa” University of Medicine and Pharmacy, Iasi, Romania Rudrappa Ambedkar Department of Microbiology and Biotechnology, Jnana Bharathi Campus, Bangalore University, Bengaluru, India Senthil Visaga Ambi Biopharmaceutical Research Lab, Anusandhan Kendra-1, SASTRA Deemed-to-be-University, Thanjavur, Tamil Nadu, India Department of Bioengineering, School of Chemical and Biotechnology, SASTRA Deemed-to-be-University, Thanjavur, Tamil Nadu, India Rebai Ben Ammar Department of Biological Sciences, College of Science, King Faisal University, Al Ahsa, Saudi Arabia Laboratory of Aromatic and Medicinal Plants, Center of Biotechnology, Technopole of Borj-Cedria, Hammam-Lif, Tunisia Shrikant Anant Department of Cancer Biology, University of Kansas Cancer Center, Kansas City, KS, USA Milena Andjelkovic Department of Toxicology “Akademik Danilo Soldatović”, University of Belgrade-Faculty of Pharmacy, Belgrade, Serbia Carlos E. Aranda-Flores Hospital General de México “Dr. Eduardo Liceaga”, Oncology Service, Ciudad de México, México K. B. Arun Rajiv Gandhi Center for Biotechnology, Trivandrum, Kerala, India Charles Elias Assmann Federal University of Santa Maria, Santa Maria, RS, Brazil Neha Atale Department of Biotechnology, Jaypee Institute of Information Technology, Noida, Uttar Pradesh, India Elena Roxana Avadanei Department of Morphofunctional Sciences I, “Grigore T. Popa” University of Medicine and Pharmacy, Iasi, Romania Mir Owais Ayaz Academy of Scientific & Innovative Research, Ghaziabad Uttar Pradesh, India Cancer Pharmacology Division, CSIR-Indian Institute of Integrative Medicine, Jammu, India

Contributors

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Prathapan Ayyappan Department of Surgery-Transplant, University of Nebraska Medical Center, Omaha, NE, USA Kokelavani Nampalli Babu Department of Biochemistry, Karpagam Academy of Higher Education, Coimbatore, Tamil Nadu, India Vadivel Dinesh Babu Department of Biochemistry, University of Madras, Chennai, India Margarete Dulce Bagatini Federal University of the South Border, Chapecó, SC, Brazil Angshuman Bagchi Department of Biochemistry and Biophysics, University of Kalyani, Kalyani, West Bengal, India Raluca Anca Balan Department of Morphofunctional Sciences I, “Grigore T. Popa” University of Medicine and Pharmacy, Iasi, Romania Devi Naga Jyothi Bale Department of Pharmaceutical Analysis, National Institute of Pharmaceutical Education and Research (NIPER), Hyderabad, Balanagar, Telangana, India Manjeshwar Shrinath Baliga Senior Radiobiologist and Incharge of Research, Mangalore Institute of Oncology, Mangalore, Karnataka, India Antara Banerjee Faculty of Allied Health Sciences, Chettinad Academy of Research, and Education, Chettinad Hospital and Research Institute (CHRI), Chennai, India Anurag Banerjee Department of Biological Sciences, Indian Institute of Science Education and Research Kolkata, Kolkata, India Rajkumar Banerjee Applied Biology Division, CSIR-Indian Institute of Chemical Technology, Hyderabad, India Academy of Scientific and Innovation Research (AcSIR), Ghaziabad, India Somesh Banerjee Department of Veterinary Microbiology, Immunology Section, Lala Lajpat Rai University of Veterinary and Animal Sciences, Hisar, India Sudeep Banerjee Department of Gastrointestinal Surgery, Tata Medical Center, Rajarhat, Kolkata, WB, India Sugato Banerjee National Institute of Pharmaceutical Education and Research, Division of Pharmacology and Toxicology, Kolkata, India Leandro A. Barbosa Laboratório de Bioquímica Celular, Universidades Federal de São João del Rei, Divinópolis, Brazil Laboratório de Membranas e ATPases, Universidades Federal de São João del Rei, Divinópolis, Brazil

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Contributors

Aneli M. Barbosa-Dekker Beta-Glucan Produtos Farmoquímicos EIRELI, Universidade Tecnológica Federal do Paraná, Câmpus Londrina, Londrina-PR, Brazil David Barua Department of Pathology, National University of Ireland Galway, Galway, Ireland Mary Bebawy Discipline of Pharmacy, Graduate School of Health, University of Technology Sydney, Ultimo, NSW, Australia Jonathan Behlen College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Stattion, TX, USA Gert-Jan Bekker Institute for Protein Research, Osaka University, Suita, Japan Asim K. Bera Institute for Protein Design, University of Washington, Seattle, WA, USA Soumen Bera B. S. Abdur Rahman Crescent Institute of Science and Technology, Chennai, India Kishor K. Bhakat Department of Genetics, Cell Biology and Anatomy, University of Nebraska Medical Center, Omaha, NE, USA Dipita Bhakta-Guha Cellular Dyshomeostasis Laboratory (CDHL), Department of Biotechnology, School of Chemical and Bio Technology, SASTRA University, Thanjavur, Tamil Nadu, India Prerna Bhalla Department of Biotechnology, Bhupat and Jyoti Mehta School of Biosciences building, Indian Institute of Technology Madras, Chennai, India Sanjay Bharati Department of Nuclear Medicine, Manipal College of Health Professions (MCHP), Manipal Academy of Higher Education (MAHE), Manipal, Karnataka, India Aadil Qadir Bhat Academy of Scientific & Innovative Research, Ghaziabad Uttar Pradesh, India Cancer Pharmacology Division, CSIR-Indian Institute of Integrative Medicine, Jammu, India Anant N. Bhatt Institute of Nuclear Medicine and Allied Sciences, Delhi, India Sanjib Bhattacharya West Bengal Medical Services Corporation Ltd., Kolkata, West Bengal, India Sudin Bhattacharya Kolkata, West Bengal, India Sankar Bhattacharyya Department of Zoology, Sidho Kanho Birsha University, Purulia, India Gurjit Kaur Bhatti Department of Medical Lab Technology, University Institute of Applied Health Sciences, Chandigarh University, Mohali, Punjab, India

Contributors

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Jasvinder Singh Bhatti Department of Biotechnology, Sri Guru Gobind Singh College, Chandigarh, India Department of Human Genetics and Molecular Medicine, School of Health Sciences, Central University of Punjab, Bathinda, India Ponnamparambil Purushothaman Binitha Royal Dental College, Challissery, Kerala, India Dipsikha Biswas Department of Biochemistry and Molecular Biology, Dalhousie University, Dalhousie Medicine New Brunswick, Saint John, NB, Canada Novo Nordisk Foundation Center for Basic Metabolic Research, University of Copenhagen, Copenhagen, Denmark Sima Biswas Department of Biochemistry and Biophysics, University of Kalyani, Kalyani, West Bengal, India Geir Bjørklund Council for Nutritional and Environmental Medicine (CONEM), Mo i Rana, Norway Azam Bolhassani Department of Hepatitis and AIDS, Pasteur Institute of Iran, Tehran, Iran Beatriz da Silva Rosa Bonadiman Federal University of Santa Catarina, Florianópolis, SC, Brazil Felipe Tecchio Borsoi University of the State of Santa Catarina, Pinhalzinho, SC, Brazil Sarpita Bose Structural Biology and Bioinformatics Division, Indian Institute of Chemical Biology, Kolkata, India Naomi Brook School of Pharmacy and Biomedical Science, Curtin University, Bentley, Australia Curtin Health Innovation Research Institute, Bentley, Australia Aleksandra Buha Djordjevic Department of Toxicology “Akademik Danilo Soldatović”, University of Belgrade, Belgrade, Serbia Zorica Bulat Department of Toxicology “Akademik Danilo Soldatović”, University of Belgrade, Belgrade, Serbia Lukumoni Buragohain Department of Animal Biotechnology, College of Veterinary Science, Assam Agricultural University, Guwahati, Assam, India Karen E. Burke Department of Dermatology, The Mount Sinai Icahn School of Medicine, New York, NY, USA Tristan Caffrey Iowa State University, Iowa, USA

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Contributors

Jessica Campos-Blázquez Department of Physiology, Biophysics and Neurosciences, Center for Research and Advanced Studies of the IPN (Cinvestav-IPN), México City, Mexico Simone Carradori Department of Pharmacy, “G. d’Annunzio” University of Chieti-Pescara, Chieti, Italy Irina Draga Caruntu Department of Morphofunctional Sciences I, “Grigore T. Popa” University of Medicine and Pharmacy, Iasi, Romania Gopal Chakrabarti Department of Biotechnology and Dr. B.C. Guha Centre for Genetic Engineering and Biotechnology, University of Calcutta, Kolkata, WB, India Saikat Chakrabarti Structural Biology and Bioinformatics Division, Indian Institute of Chemical Biology, Kolkata, India Subhendu Chakrabarty Department of Biotechnology and Dr. B.C. Guha Centre for Genetic Engineering and Biotechnology, University of Calcutta, Kolkata, WB, India Department of Microbiology, M.U.C Women’s College, Burdwan, India Sajal Chakraborti Department of Biochemistry and Biophysics, University of Kalyani, Kalyani, West Bengal, India Shayantani Chakraborty National Institute of Biomedical Genomics, Kalyani, West Bengal, India Sibani Sen Chakraborty Department of Microbiology, West Bengal State University, Barasat, West Bengal, India Souneek Chakraborty Cancer Pharmacology Division, CSIR-Indian Institute of Integrative Medicine, Jammu, India Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, India Joya Chandra Department of Pediatrics-Research, UT MD Anderson Cancer Center, Houston, TX, USA Department of Epigenetics and Molecular Carcinogenesis, UT MD Anderson Cancer Center, Houston, TX, USA University of Texas MD Anderson, Cancer Center UTHealth Graduate School of Biomedical Sciences, Houston, TX, USA Cancer Center for Energy Balance in Cancer Prevention and Survivorship, University of Texas MD Anderson, Houston, TX, USA Naveenkumar Chandrashekar Department of Biochemistry, Indian Academy Degree College – Autonomous, Bengaluru, Karnataka, India Koustav Chatterjee Department of Biotechnology, Siksha Bhavana, Visva Bharati, Santinikatan, Bolpur, West Bengal, India

Contributors

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Priti Chatterjee Cell Biology and Physiology Division, CSIR-Indian Institute of Chemical Biology, Kolkata, India Ritam Chatterjee School of Medical Science and Technology, Indian Institute of Technology Kharagpur, Kharagpur, West Bengal, India Sharmistha Chatterjee Division of Molecular Medicine, Bose Institute, Kolkata, India Sourav Chattopadhyay Center for Bio-medical Engineering (CBME), Indian Institute of Technology Ropar, Rupnagar, Punjab, India A. K. Chaudhary Tata Memorial Hospital Staff Qtrs, Mumbai, India Ankur Chaudhuri Department of Microbiology, West Bengal State University, Barasat, West Bengal, India Shailender S. Chauhan Department of Cellular and Molecular Medicine, The University of Arizona, Tucson, AZ, USA Dinesh Kumar Chellappan Department of Life Sciences, School of Pharmacy, International Medical University, Kuala Lumpur, Malaysia Abu Sufiyan Chhipa School of Biotechnology, Indian Institute of Advanced Research, Gandhinagar, Gujarat, India Sandhya Chipurupalli Department of Pharmacology, JSS College of Pharmacy, JSS Academy of Higher Education & Research, Ooty, India Salvatore Chirumbolo Department of Neurosciences, Biomedicine and Movement Sciences-University of Verona, Verona, Italy Malhar Sojwal Chitnis Insititute of Bioinformatics and Biotechnology, Savitribai Phule Pune University, Pune, India Tathagata Choudhuri Department of Biotechnology, Siksha Bhavana, Visva Bharati, Santinikatan, Bolpur, West Bengal, India Sonali Choudhury Department of Cancer Biology, University of Kansas Cancer Center, Kansas City, KS, USA Animesh Chowdhury National Institute of Biomedical Genomics, Kalyani, WB, India N. A. Chugh Department of Biophysics, Panjab University, Chandigarh, India Rubén G. Contreras Department of Physiology, Biophysics and Neurosciences, Center for Research and Advanced Studies of the IPN (Cinvestav-IPN), México City, Mexico Jéssica Righi da Rosa Federal University of Santa Maria, Santa Maria, RS, Brazil Yashodhara Dalal SVKM’s NMIMS, Shobhaben Pratapbhai Patel School of Pharmacy and Technology Management, Mumbai, Maharashtra, India

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Contributors

Prasad Dandawate Department of Cancer Biology, University of Kansas Cancer Center, Kansas City, KS, USA Mohd Jamal Dar Academy of Scientific & Innovative Research, Ghaziabad Uttar Pradesh, India Cancer Pharmacology Division, CSIR-Indian Institute of Integrative Medicine, Jammu, India Nadine Darwiche Department of Biochemistry and Molecular Genetics, American University of Lebanon, Beirut, Lebanon Abhishek Kumar Das Division of Molecular Medicine, Bose Institute, Kolkata, India Amlan Das Department of Biotechnology and Dr. B.C. Guha Centre for Genetic Engineering and Biotechnology, University of Calcutta, Kolkata, WB, India National Institute of Biomedical Genomics, Kalyani, Nadia, WB, India Chandan Kanta Das School of Medical Science and Technology, Indian Institute of Technology Kharagpur, Kharagpur, West Bengal, India Pamelika Das Molecular Pathobiology of Respiratory Diseases, Cell Biology and Physiology Division, CSIR-Indian Institute of Chemical Biology, Kolkata, India Partha Das National Institute of Biomedical Genomics, Kalyani, West Bengal, India Piyanki Das Department of Biotechnology, Siksha Bhavana, Visva Bharati, Santinikatan, Bolpur, West Bengal, India Jayasri Das Sarma Department of Biological Sciences, Indian Institute of Science Education and Research Kolkata, Kolkata, India Bhaskar Dasgupta RIKEN Center for Computational Sciences, RIKEN, Kobe, Japan Moumita Dasgupta Department of Biotechnology and Dr. B.C. Guha Centre for Genetic Engineering and Biotechnology, University of Calcutta, Kolkata, WB, India Amitava Datta Department of Computer Science & Software Engineering, The University of Western Australia, Perth, WA, Australia Soumasree De Department of Chemistry & Biochemistry, University of Bern, Bern, Switzerland Ana Paula de Melo Loureiro Departamento de Análises Clínicas e Toxicológicas, Faculdade de Ciências Farmacêuticas, Universidade de São Paulo, São Paulo, Brazil Prasanta Kumar Deb Department of Pharmaceutical Sciences and Technology, Birla Institute of Technology, Mesra, Ranchi, India Robert F. H. Dekker Universidade Tecnológica Federal do Paraná, Programa de Pós-Graduação em Engenharia Ambiental, Câmpus Londrina, Londrina-PR, Brazil

Contributors

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Kartiki V. Desai National Institute of Biomedical Genomics, Kalyani, West Bengal, India Vincenzo Desiderio Department of Experimental Medicine, University of Campania “L. Vanvitelli”, Naples, Italy Hari Prasad Devkota Graduate School of Pharmaceutical Sciences, Kumamoto University, Kumamoto, Japan Tuli Dey Insititute of Bioinformatics and Biotechnology, Savitribai Phule Pune University, Pune, India Kuntal Dey Institute of Veterinary Physiology, University of Zurich, Zurich, Switzerland Subhankar Dey Department of Zoology, New Alipore College, University of Calcutta, Kolkata, WB, India Arun Dharmarajan Department of Biomedical Sciences, Sri Ramachandra Institute of Higher Education and Research, Chennai, India School of Pharmacy and Biomedical Science, Curtin University, Bentley, WA, Australia Curtin Health Innovation Research Institute, Bentley, WA, Australia Yuthika Dholey Department of Microbiology, West Bengal State University, Barasat, West Bengal, India Giovanna Di Trapani School of Environment and Science, Griffith University, Nathan, Australia Pankaj Dixit Indore Institute of Pharmacy, Indore, Madhya Pradesh, India Saurav Doshi Institute of Bioinformatics and Biotechnology, Savitribai Phule Pune University, Pune, India Kamal Dua Centre for Inflammation, Centenary Institute, Sydney, NSW, Australia Discipline of Pharmacy, Graduate School of Health, University of Technology Sydney, Ultimo, NSW, Australia Priority Research Centre for Healthy Lungs, Hunter Medical Research Institute (HMRI) & School of Biomedical Sciences and Pharmacy, University of Newcastle, Callaghan, NSW, Australia Joytri Dutta Molecular Pathobiology of Respiratory Diseases, Cell Biology and Physiology Division, CSIR-Indian Institute of Chemical Biology, Kolkata, India Bilikere S. Dwarakanath Department of Research & Development, Shanghai Proton and Heavy Ion Center (SPHIC), Shanghai, China Shanghai Key Laboratory of Radiation Oncology, Shanghai, China Shanghai Engineering Research Center of Proton and Heavy Ion Radiation Therapy, Shanghai, China

xxxvi

Contributors

Present address: Central Research Facility, Sri Ramachandra Institute of Higher Education and Research, Chennai, India Ali H. Eid Department of Basic Medical Sciences and Biomedical and Pharmaceutical Research Unit, College of Medicine, QU Health, Qatar University, Doha, Qatar Department of Pharmacology and Toxicology, American University of Lebanon, Beirut, Lebanon Chirine El-Baba Department of Biochemistry and Molecular Genetics, American University of Lebanon, Beirut, Lebanon Olusola O. Elekofehinti Bioinformatics and Molecular Biology Unit, Department of Biochemistry, Federal University of Technology Akure, Akure, Nigeria Aderonke E. Fakayode Department of Biochemistry and Molecular Biology, Faculty of Science, Obafemi Awolowo University, Ile-Ife, Nigeria Mohd Farhan Department of Basic Sciences, King Faisal University, Al Ahsa, Kingdom of Saudi Arabia Jiafu Feng Mianyang Central Hospital, School of Medicine, University of Electronic Science and Technology of China, Mianyang, China Martín Ernesto Fernández-Zapico Division of Oncology Research, Schulze Center for Novel Therapeutics, Rochester, MN, USA Mary Figueroa Department of Pediatrics-Research, UT MD Anderson Cancer Center, Houston, TX, USA University of Texas MD Anderson, Cancer Center UTHealth Graduate School of Biomedical Sciences, Houston, TX, USA Catalina Flores-Maldonado Department of Physiology, Biophysics and Neurosciences, Center for Research and Advanced Studies of the IPN (Cinvestav-IPN), México City, Mexico Sherine Joanna Fredrick Department of Biomedical Sciences, Sri Ramachandra Institute of Higher Education and Research, Chennai, India Mamatha G. S. Department of Oral Pathology and Microbiology, Dr. D. Y. Patil Dental College and Hospital, Dr. D. Y. Patil Vidyapeeth, Pune, Maharashtra, India Palanivel Gajalakshmi Department of Life Sciences, AU-KBC Research Centre, MIT Campus of Anna University, Chennai, India Siva Nageswara Rao Gajula Department of Pharmaceutical Analysis, National Institute of Pharmaceutical Education and Research (NIPER), Hyderabad, Balanagar, Telangana, India Juan M. Gallardo Unidad de Investigación Médica en Enfermedades Nefrológicas, Hospital de Especialidades, Centro Médico Nacional “Siglo XXI” Instituto Mexicano del Seguro Social, México City, Mexico

Contributors

xxxvii

Gurudutta Gangenahalli Division of Stem Cells & Gene Therapy Research, Institute of Nuclear Medicine & Allied Sciences (INMAS), Defence Research and Development Organisation (DRDO), New Delhi, India Israel J. P. Garcia Laboratório de Bioquímica Celular, Universidades Federal de São João del Rei, Divinópolis, Brazil Laboratório de Membranas e ATPases, Universidades Federal de São João del Rei, Divinópolis, Brazil Govind Garg Division of Pharmacology and Toxicology, ICAR-Indian Veterinary Research Institute, Bareilly, Uttar Pradesh, India Sukanya Gayan Insititute of Bioinformatics and Biotechnology, Savitribai Phule Pune University, Pune, India Sajan George Laser Research Center, University of Johannesburg, Johannesburg, South Africa Deepshikha Ghosh Cell Biology and Physiology Division, CSIR-Indian Institute of Chemical Biology, Kolkata, India Mayukh Ghosh Department of Veterinary Physiology and Biochemistry, RGSC, Banaras Hindu University, Mirzapur, India Tuhin Ghosh Department of Chemistry (UG & PG), Durgapur Government College, Durgapur, West Bengal, India Pooja Sanjay Ghuge Insititute of Bioinformatics and Biotechnology, Savitribai Phule Pune University, Pune, India Daniel Andrew M. Gideon Department of Biotechnology and Bioinformatics, Bishop Heber College (Autonomous), Tiruchirappalli, Tamil Nadu, India Simona Eliza Giusca Department of Morphofunctional Sciences I, “Grigore T. Popa” University of Medicine and Pharmacy, Iasi, Romania Laura Gómez-Laguna Hospital General de México “Dr. Eduardo Liceaga”, Oncology Service, Ciudad de México, México Bruno S. Gonçalves Laboratório de Bioquímica Celular, Universidades Federal de São João del Rei, Divinópolis, Brazil Laboratório de Membranas e ATPases, Universidades Federal de São João del Rei, Divinópolis, Brazil Gayathri Gopal Biopharmaceutical Research Lab, Anusandhan Kendra-1, SASTRA Deemed-to-be-University, Thanjavur, Tamil Nadu, India School of Chemical and Biotechnology, SASTRA Deemed-to-be-University, Thanjavur, Tamil Nadu, India Department of Bioengineering, School of Chemical and Biotechnology, SASTRA Deemed-to-be-University, Thanjavur, Tamil Nadu, India

xxxviii

Contributors

Janani Gopi Faculty of Allied Health Sciences, Chettinad Academy of Research, and Education, Chettinad Hospital and Research Institute (CHRI), Chennai, India Anindya Goswami Cancer Pharmacology Division, CSIR-Indian Institute of Integrative Medicine, Jammu, India Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, India Anindita Goswami Biomedical Genetics Laboratory, Department of Zoology, The University of Burdwan, Bardhaman, West Bengal, India K. Grace Theodora Department of Political Science, Delhi University, New Delhi, India Stacy Grieve Department of Biology, University of New Brunswick, Saint John, NB, Canada Adriana Grigoras Department of Morphofunctional Sciences I, “Grigore T. Popa” University of Medicine and Pharmacy, Iasi, Romania Paolo Guglielmi Department of Drug Chemistry and Technologies, Sapienza University of Rome, Rome, Italy Gunjan Guha Cellular Dyshomeostasis Laboratory (CDHL), Department of Biotechnology, School of Chemical and Bio Technology, SASTRA University, Thanjavur, Tamil Nadu, India Ayse Günes-Bayir Department of Nutrition and Dietetics, Bezmialem Vakif University, Istanbul, Turkey Anshika Gupta Department of Biotechnology, Sri Guru Gobind Singh College, Chandigarh, India Aritra Gupta National Institute of Biomedical Genomics, Kalyani, West Bengal, India Gaurav Gupta School of Pharmacy, Suresh Gyan Vihar University, Jaipur, India P. K. Gupta Tuberculosis Immunology and Immunoassay Development Section, Radiation Medicine Centre, Bhabha Atomic Research Centre, Parel, Mumbai, India Prem N. Gupta Formulation & Drug Delivery Division, CSIR-Indian Institute of Integrative Medicine, Jammu, India Seema Gupta The Loop Immuno-Oncology Laboratory, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC, USA Sumiran Kumar Gurung School of Life Sciences, Jawaharlal Nehru University, New Delhi, India Sheikh Mumtaz Hadi Department of Biochemistry, Aligarh Muslim University, Aligarh, India Tanweer Haider Department of Pharmaceutical Sciences, Dr. Harisingh Gour University, Sagar, Madhya Pradesh, India

Contributors

xxxix

Philip Michael Hansbro Centre for Inflammation, Centenary Institute, Sydney, NSW, Australia Faculty of Science, University of Technology Sydney, Ultimo, NSW, Australia Priority Research Centre for Healthy Lungs, Hunter Medical Research Institute (HMRI) & School of Biomedical Sciences and Pharmacy, University of Newcastle, Callaghan, NSW, Australia Seyed Isaac Hashemy Department of Clinical Biochemistry, Mashhad University of Medical Sciences, Mashhad, Iran Surgical Oncology Research Center, Mashhad University of Medical Sciences, Mashhad, Iran Md Mehedi Hossain Academy of Scientific & Innovative Research, Ghaziabad Uttar Pradesh, India Cancer Pharmacology Division, CSIR-Indian Institute of Integrative Medicine, Jammu, India Ezekiel T. Ige Department of Pharmacology and Therapeutics, College of Medicine, Ekiti State University, Ado-Ekiti, Nigeria Ashish Jaiswal Molecular Pathobiology of Respiratory Diseases, Cell Biology and Physiology Division, CSIR-Indian Institute of Chemical Biology, Kolkata, India Joel James Department of Medicine, Division of Endocrinology, University of Arizona College of Medicine, Tucson, AZ, USA Dragana Javorac Department of Toxicology “Akademik Danilo Soldatović”, University of Belgrade, Belgrade, Serbia Sushmita Jha Department of Bioscience and Bioengineering, Indian Institute of Technology Jodhpur, Jodhpur, Rajasthan, India Marlene Jimenez-Del-Rio Neuroscience Research Group, Medical Research Institute, Faculty of Medicine, University of Antioquia (UdeA), Medellin, Colombia Tapasya K. Department of Biomedical Sciences, Sri Ramachandra Institute of Higher Education and Research, Chennai, India Or Kakhlon Department of Neurology, Hadassah-Hebrew University Medical Center, Jerusalem, Israel Narutoshi Kamiya Graduate School of Simulation Studies, University of Hyogo, Kobe, Japan S. Kannan Nanomedicine Division, Department of Zoology, Periyar University, Salem, TN, India Debapratim Kar Chowdhuri Embryotoxicology Laboratory, Environmental Toxicology Group, CSIR-Indian Institute of Toxicology Research (CSIR-IITR), Lucknow, India

xl

Contributors

Devarajan Karunagaran Department of Biotechnology, Bhupat and Jyoti Mehta School of Biosciences building, Indian Institute of Technology Madras, Chennai, India Prabhsimran Kaur Department of Human Genetics and Molecular Medicine, School of Health Sciences, Central University of Punjab, Bathinda, India Raman Preet Kaur Department of Otolaryngology, School of Medicine, Johns Hopkins University, Baltimore, MD, USA Antholi Keloth Kavya Department of Biochemistry, Co-operative Institute of Health Sciences, Kannur, Kerala, India Firas Kobeissy Department of Biochemistry and Molecular Genetics, American University of Lebanon, Beirut, Lebanon A. Koul Department of Biophysics, Panjab University, Chandigarh, India Vishnu Krishnakumar Stem Cell Facility, DBT-Centre of Excellence for Stem Cell Research, All India Institute of Medical Sciences, New Delhi, India Sneha Krishnamoorthy Department of Biochemistry, Karpagam Academy of Higher Education, Coimbatore, Tamil Nadu, India Disha Kshirsagar Institute of Bioinformatics and Biotechnology, Savitribai Phule Pune University, Pune, India Dinesh Kumar Division of Pharmacology and Toxicology, ICAR-Indian Veterinary Research Institute, Bareilly, Uttar Pradesh, India Krishna Kumar Structural Biology and Bioinformatics Division, Indian Institute of Chemical Biology, Kolkata, India Maushmi S. Kumar SVKM’s NMIMS, Shobhaben Pratapbhai Patel School of Pharmacy and Technology Management, Mumbai, Maharashtra, India Munish Kumar Department of Biochemistry, University of Allahabad, Allahabad, India Pradeep G. Kumar Division of Molecular Reproduction, Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram, Kerala, India Rajesh Kumar Department of Veterinary Physiology and Biochemistry, College of Veterinary Science, Lala Lajpat Rai University of Veterinary and Animal Sciences, Hisar, India Rishabh Kumar Department of Biochemistry, University of Allahabad, Allahabad, India Saurav Kumar Department of Biological Sciences, Indian Institute of Science Education and Research Kolkata, Kolkata, India

Contributors

xli

Subodh Kumar Division of Stem Cells & Gene Therapy Research, Institute of Nuclear Medicine & Allied Sciences (INMAS), Defence Research and Development Organisation (DRDO), New Delhi, India Vivek Kumar R&D Department, Shanghai Proton and Heavy Ion Center (SPHIC), Shanghai, China Shanghai Key Laboratory of Radiation Oncology, Shanghai, China Shanghai Engineering Research Center of Proton and Heavy Ion Radiation Therapy, Shanghai, China Ankur Kumari Department of Zoology CBLU, Bhiwani, Haryana, India Snehlata Kumari Diamantina Woolloongabba, Australia

Institute,

The

University

of

Queensland,

Gopal C. Kundu School of Biotechnology, KIIT Deemed to be University, Institute of Eminence, Bhubaneswar, India Ipsita G. Kundu Department of Pharmacy, Birla Institute of Science and Technology, Pilani, Hyderabad Campus, Institute of Eminence, Hyderabad, India Vijay Kumar Kutala Department of Clinical Pharmacology and Therapeutics, Nizam’s Institute of Medical Sciences, Hyderabad, India Rangaswamy Lakshminarayana Department of Microbiology and Biotechnology, Jnana Bharathi Campus, Bangalore University, Bengaluru, India María Julia Lamberti Division of Oncology Research, Schulze Center for Novel Therapeutics, Rochester, MN, USA Molecular Biology Department, National University of Río Cuarto, Río Cuarto, Córdoba, Argentina INBIAS, CONICET-UNRC, Río Cuarto, Córdoba, Argentina C. Latchoumycandane Molecular Pharmacology and Toxicology Laboratory, Department of Life Sciences, School of Life Sciences, Central University of Tamil Nadu, Thiruvarur, India Sujata Law Department of Biochemistry and Medical Biotechnology, Calcutta School of Tropical Medicine, Kolkata, West Bengal, India Tamara Lazarević-Pašti “VINČA” Institute of Nuclear Sciences – National Institute of thе Republic of Serbia, University of Belgrade, Belgrade, Serbia Ki-Young Lee Department of Cell Biology and Anatomy, Arnie Charbonneau Cancer Institute and Alberta Children’s Hospital Research Institute, Cumming School of Medicine, University of Calgary, Calgary, AB, Canada Andreja Leskovac Department of Physical Chemistry, Vinca Institute of Nuclear Sciences – National Institute of the Republic of Serbia, University of Belgrade, Belgrade, Serbia

xlii

Contributors

Madhu Cholenahalli Lingaraju Division of Pharmacology and Toxicology, ICAR-Indian Veterinary Research Institute, Bareilly, Uttar Pradesh, India Octavio López-Méndez Department of Physiology, Biophysics and Neurosciences, Center for Research and Advanced Studies of the IPN (Cinvestav-IPN), México City, Mexico Ludmila Lozneanu Department of Morphofunctional Sciences I, “Grigore T. Popa” University of Medicine and Pharmacy, Iasi, Romania Anthony Lucci Department of Breast Surgical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Morgan Welch Inflammatory Breast Cancer Research Program and Clinic, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Grazia Luisi Department of Pharmacy, “G. d’Annunzio” University of ChietiPescara, Chieti, Italy Abhilash M. Government Arts and Science College, Thiruvananthapuram, Kerala, India Ulaganathan Mabalirajan Molecular Pathobiology of Respiratory Diseases, Cell Biology and Physiology Division, CSIR-Indian Institute of Chemical Biology, Kolkata, India Smarajit Maiti Department of Biochemistry and Biotechnology, Cell & Molecular Therapeutics Lab, Oriental Institute of Science and Technology, Midnapore, West Bengal, India Ranabir Majumder School of Medical Science and Technology, Indian Institute of Technology Kharagpur, Kharagpur, West Bengal, India Sudipta Mallick CSIR-Indian Institute of Chemical Biology, Kolkata, India Amritlal Mandal Department of Physiology, University of Arizona, Tucson, AZ, USA Mahitosh Mandal School of Medical Science and Technology, Indian Institute of Technology Kharagpur, Kharagpur, West Bengal, India Paramita Mandal Biomedical Genetics Laboratory, Department of Zoology, The University of Burdwan, Bardhaman, West Bengal, India Luka Manic Department of Toxicology “Akademik Danilo Soldatović”, University of Belgrade-Faculty of Pharmacy, Belgrade, Serbia Meenu Maniradhan Molecular Pharmacology and Toxicology Laboratory, Department of Life Sciences, School of Life Sciences, Central University of Tamil Nadu, Thiruvarur, India Nikunj Maniyar Department of Orthodontics and Dentofacial Orthopaedics, Bapuji Dental College and Hospital, Davangere, Karnataka, India

Contributors

xliii

Zoya Mann Stem Cell Facility, DBT-Centre of Excellence for Stem Cell Research, All India Institute of Medical Sciences, New Delhi, India Aalim Maqsood Academy of Scientific & Innovative Research, Ghaziabad Uttar Pradesh, India Cancer Pharmacology Division, CSIR-Indian Institute of Integrative Medicine, Jammu, India Francesco Marotta ReGenera R&D International for Aging Intervention and San Babila Clinic, Vitality Therapeutics, Milan, Italy Vanesa Martin Departamento de Morfología y Biología Celular, Facultad de Medicina, Universidad de Oviedo, Oviedo, Spain Instituto Universitario de Oncología del Principado de Asturias, Oviedo, Spain Instituto de Investigación Sanitaria del Principado de Asturias, Oviedo, Spain P. P. Mathur Department of Biochemistry & Molecular Biology, School of Life Sciences, Pondicherry University, Pondicherry, India Birla Global University, Odisha, India Brajesh Kumar Maurya Department of Zoology, Govt PG College, Magaraha, Mirzapur, India Debadutta Mishra Department of Microbiology, National Food Laboratory, Kolkata, India Dinesh Kumar Mishra Indore Institute of Pharmacy, Indore, Madhya Pradesh, India Vineet Kumar Mishra CSIR-Indian Institute of Chemical Biology, Kolkata, India Siddhartha Kumar Mishra Department of Life Sciences, Chhatrapati Shahu Ji Maharaj University, Kanpur, India Tulika Mitra Cell Biology and Physiology Division, CSIR-Indian Institute of Chemical Biology, Kolkata, India Department of Biochemistry, School of Basic and Applied Sciences, Dayananda Sagar University, Bangalore, India Disha Mittal Nano-Biotech Lab, Department of Zoology, Kirori Mal College, University of Delhi, Delhi, India Shikha Mohan Life Sciences/Radiation Bioscience, New Delhi, India Sujata Mohanty Stem Cell Facility, DBT-Centre of Excellence for Stem Cell Research, All India Institute of Medical Sciences, New Delhi, India Imran Moin Nano-Biotech Lab, Department of Zoology, Kirori Mal College, University of Delhi, Delhi, India

xliv

Contributors

Olorunfemi R. Molehin Department of Biochemistry, Ekiti State University, Ado-Ekiti, Nigeria Neelima Mondal School of Life Sciences, Jawaharlal Nehru University, New Delhi, India Adriana Monroy Hospital General de México “Dr. Eduardo Liceaga”, Oncology Service, Ciudad de México, México Mohammad S. Mubarak Department of Chemistry, The University of Jordan, Amman, Jordan Kalpana Mujoo Institute of Molecular Medicine, UT Health at Houston, Houston, TX, USA Vaishali Mulchandani Department of Biological Sciences, Indian Institute of Science Education and Research Kolkata, Kolkata, India Anjana Munshi Department of Human Genetics and Molecular Medicine, School of Health Sciences, Central University of Punjab, Bathinda, India Shibi Muralidar Biopharmaceutical Research Lab, Anusandhan Kendra-1, SASTRA Deemed-to-be-University, Thanjavur, Tamil Nadu, India School of Chemical and Biotechnology, SASTRA Deemed-to-be-University, Thanjavur, Tamil Nadu, India Sridhar Muthusami Department of Biochemistry, Karpagam Academy of Higher Education, Coimbatore, Tamil Nadu, India Karpagam Cancer Research Centre, Karpagam Academy of Higher Education, Coimbatore, Tamilnadu, India Raju Nagarajan Department of Biotechnology, Indian Institute of Technology Madras, Chennai, Tamilnadu, India Siddavaram Nagini Department of Biochemistry and Biotechnology, Annamalai University, Annamalainagar, India Nivedita Nanda Department of Biochemistry, Jawaharlal Institute of Post-graduate Medical Education & Research (JIPMER), Puducherry, India Satheesh Kumar Nanjappan Department of Natural Products, National Institute of Pharmaceutical Education and Research (NIPER Kolkata), Chunilal Bhawan, Manicktala, Kolkata, West Bengal, India Tina Nasrin Department of Biochemistry and Biophysics, University of Kalyani, Kalyani, West Bengal, India Thanemozhi G. Natarajan Georgetown University Medical Center, Washington, DC, USA Queromatics.org, Phoenix, AZ, USA

Contributors

xlv

Somsubhra Nath Department of Basic and Translational Research, Saroj Gupta Cancer Centre and Research Institute, Kolkata, India Shaik Mohammad Naushad Department of Biochemical Genetics and Pharmacogenomics, Sandor Speciality Diagnostics Pvt Ltd, Hyderabad, India Saranya NavaneethaKrishnan Department of Cell Biology and Anatomy, Arnie Charbonneau Cancer Institute and Alberta Children’s Hospital Research Institute, Cumming School of Medicine, University of Calgary, Calgary, AB, Canada Uma Shanker Navik Department of Pharmacology, Central University of Punjab, Bathinda, India Aarifa Nazmeen Department of Biochemistry and Biotechnology, Cell & Molecular Therapeutics Lab, Oriental Institute of Science and Technology, Midnapore, West Bengal, India Lokesh Nigam School of Computational and Integrative Sciences, Jawaharlal Nehru University, New Delhi, India Mohd Esa Norhaizan Department of Nutrition, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, Serdang, Selangor, Malaysia Natural Medicines and Products Research Laboratory (NaturMeds), Universiti Putra Malaysia, Serdang, Selangor, Malaysia Oluwatosin B. Olusakin Na Moda Naturals, Federal Housing Estate, Ibadan, Nigeria Ajibade O. Oyeyemi Department of Biochemistry, Faculty of Science, Ekiti State University, Ado-Ekiti, Nigeria Sathish Kumar Reddy Padi Department of Molecular Biology and Biophysics, UConn Health Center, Farmington, CT, USA University of Arizona Cancer Center, The University of Arizona, Tucson, AZ, USA Paras Pahwa Department of Biotechnology, Sri Guru Gobind Singh College, Chandigarh, India Jagdish Gopal Paithankar Division of Environmental Health and Toxicology, Nitte University Centre for Science Education and Research (NUCSER), Deralakatte, India L. Panayappan St.James College of Pharmacy, Chalakudi, Kerala, India Akanksha Pandey Biochemistry Section, Department of Zoology, Institute of Science, Banaras Hindu University, Varanasi, India Vikas Pandey Department of Pharmaceutical Sciences, Dr. Harisingh Gour University, Sagar, Madhya Pradesh, India

xlvi

Contributors

Pranav K. Pandey Department of Ophthalmic Sciences, Dr. Rajendra Prasad Centre, All India Institute of Medical Sciences, New Delhi, India Emiliano Panieri Department of Physiology and Pharmacology “Vittorio Erspamer”, Sapienza University, Rome, Italy Lakshmikanthan Panneerselvam Department of Biotechnology, Bharathiar University, Coimbatore, Tamilnadu, India Subhashree Parida Division of Pharmacology and Toxicology, ICAR-Indian Veterinary Research Institute, Bareilly, Uttar Pradesh, India Chandramani Pathak Amity Insitute of Biotechnology, Amity University Haryana, Gurgaon, India School of Biotechnology, Indian Institute of Advanced Research, Gandhinagar, Gujarat, India Surajit Pathak Faculty of Allied Health Sciences, Chettinad Academy of Research, and Education, Chettinad Hospital and Research Institute (CHRI), Chennai, India Keshav Raj Paudel Centre for Inflammation, Centenary Institute, Sydney, NSW, Australia Faculty of Science, University of Technology Sydney, Ultimo, NSW, Australia Santanu Paul Department of Biotechnology and Dr. B.C. Guha Centre for Genetic Engineering and Biotechnology, University of Calcutta, Kolkata, WB, India Alan A. Pedraza-Ramírez Department of Physiology, Biophysics and Neurosciences, Center for Research and Advanced Studies of the IPN (Cinvestav-IPN), México City, Mexico Duane G. Pereira Laboratório de Bioquímica Celular, Universidades Federal de São João del Rei, Divinópolis, Brazil Laboratório de Membranas e ATPases, Universidades Federal de São João del Rei, Divinópolis, Brazil Loganayaki Periyasamy Department of Biochemistry, Karpagam Academy of Higher Education, Coimbatore, Tamil Nadu, India Lakshmi R. Perumalsamy Department of Biomedical Sciences, Sri Ramachandra Institute of Higher Education and Research, Chennai, India Sandra Petrovic Department of Physical Chemistry, Vinca Institute of Nuclear Sciences – National Institute of the Republic of Serbia, University of Belgrade, Belgrade, Serbia

Contributors

xlvii

Suravi Pramanik Department of Genetics, Cell Biology and Anatomy, University of Nebraska Medical Center, Omaha, NE, USA Gaya Prasad International Institute of Veterinary Education and Research, Rohtak, India Himanshu K. Prasad Department of Life Science and Bioinformatics, Assam University, Silchar, India Minakshi Prasad Department of Animal Biotechnology, Lala Lajpat Rai University of Veterinary and Animal Sciences, Hisar, India Eveline A. I. F. Queiroz Núcleo de Pesquisa e Apoio Didático em Saúde (NUPADS), Instituto de Ciências da Saúde, Câmpus Universitário de Sinop, Universidade Federal de Mato Grosso, Sinop-MT, Brazil Harikumaran Nair R. School of Biosciences, Mahatma Gandhi University, Kottayam, Kerala, India Prakash Radhakrishnan Eppley Institute for Research in Cancer and Allied Diseases, Fred & Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha, NE, USA Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE, USA Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, NE, USA Department of Genetics, Cell Biology and Anatomy, University of Nebraska Medical Center, Omaha, NE, USA N. N. V. Radharani School of Biotechnology, KIIT Deemed to be University, Institute of Eminence, Bhubaneswar, India Rather A. Rafiq PK-PD and Toxicology Division, CSIR-Indian Institute of Integrative Medicine, Jammu and Kashmir, India Pawan Kumar Raghav Stem Cell Facility, DBT-Centre of Excellence for Stem Cell Research, All India Institute of Medical Sciences, New Delhi, India Azhwar Raghunath Department of Biotechnology, Bharathiar University, Coimbatore, Tamilnadu, India Department of Pediatrics, University of Michigan, Ann Arbor, MI, USA Ajit Kumar Rai Systems Toxicology and Health Risk Assessment Group, CSIRIndian Institute of Toxicology Research (CSIR-IITR), Lucknow, India Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, India

xlviii

Contributors

Peramaiyan Rajendran Department of Biological Sciences, College of Science, King Faisal University, Al Ahsa, Saudi Arabia Sanjana Rajgopal Department of Biomedical Sciences, Sri Ramachandra Institute of Higher Education and Research, Chennai, India K. Ranganathan Sree Abirami College of Pharmacy, Coimbatore, Tamilnadu, India Vibha Rani Department of Biotechnology, Jaypee Institute of Information Technology, Noida, Uttar Pradesh, India Kishu Ranjan Department of Internal Medicine, Section of Digestive Diseases, Yale University, New Haven, CT, USA Kunchala Sridhar Rao Oncosimis Biotech Private Limited, Hyderabad, India Archita Ray Molecular Pathobiology of Respiratory Diseases, Cell Biology and Physiology Division, CSIR-Indian Institute of Chemical Biology, Kolkata, India P. Hemachandra Reddy Department of Internal Medicine, Texas Tech University Health Sciences Center, Lubbock, TX, USA Raghunathan Rengaswamy Department of Chemical Engineering, Indian Institute of Technology Madras, Chennai, India Initiative for Biological Systems Engineering, Indian Institute of Technology Madras, Chennai, India Wasia Rizwani Genzir Technologies Pvt. Ltd., Hyderabad, Telangana, India Nirmal Robinson Cellular-Stress and Immune Response Laboratory, Center for Cancer Biology, University of South Australia and SA Pathology, Adelaide, Australia Pilar Roca Grupo Multidisciplinar de Oncología Traslacional, Institut Universitari d’Investigació en Ciències de la Salut (IUNICS), Universitat de les Illes Balears, Palma de Mallorca, Spain Instituto de Investigación Sanitaria de las Islas Baleares (IdISBa), Palma de Mallorca, Spain Ciber Fisiopatología Obesidad y Nutrición (CB06/03), Instituto Salud Carlos III, Madrid, Spain Carmen Rodríguez Departamento de Morfología y Biología Celular, Universidad de Oviedo, Oviedo, Spain Instituto Universitario de Oncología del Principado de Asturias, Oviedo, Spain Instituto de Investigación Sanitaria del Principado de Asturias, Oviedo, Spain Jesusa L. Rosales Department of Cell Biology and Anatomy, Arnie Charbonneau Cancer Institute and Alberta Children’s Hospital Research Institute, Cumming School of Medicine, University of Calgary, Calgary, AB, Canada

Contributors

xlix

Amrita Roy Department of Biotechnology, Indian Academy Degree College (Autonomous), Bengaluru, Karnataka, India Debasish Roy Department of Neuroscience, Wexner Medical Center, The Ohio State University, Columbus, OH, USA Madhumita Roy Department of Environmental Carcinogenesis & Toxicology, Chittaranjan National Cancer Institute, Kolkata, India Pritam Roy School of Medical Science and Technology, Indian Institute of Technology Kharagpur, Kharagpur, West Bengal, India Sib Sankar Roy Cell Biology and Physiology Division, CSIR-Indian Institute of Chemical Biology, Kolkata, India Academy of Scientific and Innovative Research, CSIR- Indian Institute of Chemical Biology Campus, Kolkata, India Somenath Roy Department of Human Physiology with Community Health, Vidyasagar University, Midnapore, West Bengal, India Stuti Roy Department of Basic and Translational Research, Saroj Gupta Cancer Centre and Research Institute, Kolkata, India Tapasi Roy CSIR-Indian Institute of Chemical Biology, Kolkata, India Shrabasti Roychoudhury Department of Genetics, Cell Biology and Anatomy, University of Nebraska Medical Center, Omaha, NE, USA Natalia Belén Rumie Vittar Molecular Biology Department, National University of Río Cuarto, Río Cuarto, Córdoba, Argentina INBIAS, CONICET-UNRC, Río Cuarto, Córdoba, Argentina Secunda Rupert Stanley Medical College and Hospital, Chennai, India Munindra Ruwali Amity Institute of Biotechnology, Amity University Haryana, Gurgaon, India Fatma J. Al Saeedi Department of Nuclear Medicine, Kuwait University, Kuwait City, Kuwait Satish Sagar Eppley Institute for Research in Cancer and Allied Diseases, Fred & Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha, NE, USA Subbroto Kumar Saha Department of Biochemistry and Molecular Medicine, University of California, Davis, Sacramento, CA, USA Swagatika Sahoo Department of Chemical Engineering, Indian Institute of Technology Madras, Chennai, India Initiative for Biological Systems Engineering, Indian Institute of Technology Madras, Chennai, India

l

Contributors

Sanjay Saini Embryotoxicology Laboratory, Environmental Toxicology Group, CSIR-Indian Institute of Toxicology Research (CSIR-IITR), Lucknow, India Ana Maria Sanchez-Sanchez Departamento de Morfología y Biología Celular, Facultad de Medicina, Universidad de Oviedo, Oviedo, Spain Instituto Universitario de Oncología del Principado de Asturias, Oviedo, Spain Instituto de Investigación Sanitaria del Principado de Asturias, Oviedo, Spain Kamalpreet Kaur Sandha Formulation & Drug Delivery Division, CSIR-Indian Institute of Integrative Medicine, Jammu, India Balaraman Santhosh Department of Biochemistry, University of Madras, Guindy Campus, Chennai, India V. Saritha Department of Environmental Science, GITAM Deemed to be University, Visakhapatnam, AP, India Biswatrish Sarkar Department of Pharmaceutical Sciences and Technology, Birla Institute of Technology, Mesra, Ranchi, India Subham Sarkar Biomedical Genetics Laboratory, Department of Zoology, The University of Burdwan, Bardhaman, West Bengal, India Vasiliki Sarli Department of Chemistry, Aristotle University of Thessaloniki, Thessaloniki, Greece Gargi Sarode Department of Oral Pathology and Microbiology, Dr. D. Y. Patil Dental College and Hospital, Dr. D. Y. Patil Vidyapeeth, Pune, Maharashtra, India Sachin Sarode Department of Oral Pathology and Microbiology, Dr. D. Y. Patil Dental College and Hospital, Dr. D. Y. Patil Vidyapeeth, Pune, Maharashtra, India Luciano Saso Department of Physiology and Pharmacology “Vittorio Erspamer”, Sapienza University, Rome, Italy Neeraj Kumar Satija Systems Toxicology and Health Risk Assessment Group, CSIR-Indian Institute of Toxicology Research (CSIR-IITR), Lucknow, India Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, India Shivanjali Saxena Department of Bioscience and Bioengineering, Indian Institute of Technology Jodhpur, Jodhpur, Rajasthan, India Afreen Asif Ali Sayed Department of Cancer Biology, University of Kansas Cancer Center, Kansas City, KS, USA Salah Mohamed El Sayed Department of Clinical Biochemistry and Molecular Medicine, Taibah Faculty of Medicine, Taibah University, Al-Madinah AlMunawwarah, Saudi Arabia Department of Medical Biochemistry, Sohag Faculty of Medicine, Sohag University, Sohag, Egypt

Contributors

li

Heide Schatten Department of Veterinary Pathobiology, University of MissouriColumbia, Columbia, MO, USA Daniela Secci Department of Drug Chemistry and Technologies, Sapienza University of Rome, Rome, Italy Alagesan Seetha Department of Biochemistry, University of Madras, Guindy Campus, Chennai, India K. Senthilkumar Nanomedicine Division, Department of Zoology, Periyar University, Salem, TN, India National Institute for Research in Tuberculosis, Indian Council of Medical Research, Madurai, TN, India R. Senthilkumaran Medex Healthcare Group, Dubai, UAE Seyed Mohammad Reza Seyedi Department of Biology, Faculty of Sciences, Ferdowsi University of Mashhad, Mashhad, Iran Department of Chemistry, Ferdowsi University of Mashhad, Mashhad, Iran Eleni Sflakidou Department of Chemistry, Aristotle University of Thessaloniki, Thessaloniki, Greece Samaneh H. Shabani Department of Hepatitis and AIDS, Pasteur Institute of Iran, Tehran, Iran Soni Shaikh Laboratory of Histopathology, Tata Medical Center, MAR (E-W), New Town, Kolkata, India Abdallah Shaito Department of Biological and Chemical Sciences, Lebanese International University, Beirut, Lebanon G. Shanmugam Nanomedicine Division, Department of Zoology, Periyar University, Salem, TN, India Anurag Sharma Division of Environmental Health and Toxicology, Nitte University Centre for Science Education and Research (NUCSER), Deralakatte, India Meemansha Sharma Division of Pharmacology and Toxicology, ICAR-Indian Veterinary Research Institute, Bareilly, Uttar Pradesh, India Madhur Shastri School of Health Sciences, College of Health and Medicine, University of Tasmania, Launceston, Australia Sachin Shetty Department of Nuclear Medicine, Manipal College of Health Professions (MCHP), Manipal Academy of Higher Education (MAHE), Manipal, Karnataka, India Masaki Shiota Department of Urology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan

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Contributors

Arnadi Ramachandrayya Shivashankara Department of Biochemistry, Father Muller Medical College, Mangalore, Karnataka, India Rahul Shukla Department of Pharmaceutics, National Institute of Pharmaceutical Education and Research (NIPER-Raebareli), Lucknow, India Shakti Shukla Priority Research Centre for Healthy Lungs, Hunter Medical Research Institute (HMRI) & School of Biomedical Sciences and Pharmacy, University of Newcastle, Callaghan, NSW, Australia Nilabja Sikdar Human Genetics Unit, Indian Statistical Institute, Kolkata, WB, India Parames C. Sil Division of Molecular Medicine, Bose Institute, Kolkata, India Lilian N. D. Silva Laboratório de Bioquímica Celular, Universidades Federal de São João del Rei, Divinópolis, Brazil Laboratório de Membranas e ATPases, Universidades Federal de São João del Rei, Divinópolis, Brazil Edakkadath Raghavan Sindhu Division of Biochemistry, Malabar Cancer Centre, Kannur, Kerala, India Balraj Singh Department of Breast Surgical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Morgan Welch Inflammatory Breast Cancer Research Program and Clinic, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Neha Singh University of Arizona Cancer Center, The University of Arizona, Tucson, AZ, USA Sabita Singh Molecular Pathobiology of Respiratory Diseases, Cell Biology and Physiology Division, CSIR-Indian Institute of Chemical Biology, Kolkata, India Thakur Uttam Singh Division of Pharmacology and Toxicology, ICAR-Indian Veterinary Research Institute, Bareilly, Uttar Pradesh, India Banudevi Sivanantham Department of Bioengineering, School of Chemical & Biotechnology, SASTRA Deemed-to-be University, Thanjavur, Tamilnadu, India Ugir Hossain Sk Department of Clinical and Translational Research, Chittaranjan National Cancer Institute, Kolkata, West Bengal, India Vandana Soni Department of Pharmaceutical Sciences, Dr. Harisingh Gour University, Sagar, Madhya Pradesh, India Poorigali Raghavendra-Rao Sowmya Department of Microbiology and Biotechnology, Jnana Bharathi Campus, Bangalore University, Bengaluru, India Gokul Sridharan Department of Oral Pathology and Microbiology, Dr. G. D. Pol Foundation YMT Dental College and Hospital, Navi Mumbai, India

Contributors

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Jone A. Stanley College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Stattion, TX, USA Ganapasam Sudhandiran Cell Biology Laboratory, Department of Biochemistry, University of Madras, Guindy Campus, Chennai, India Suman APR Division, ICAR – Central Institute for Research on Buffaloes, Hisar, India Kiruthika Sundarraj Department of Biotechnology, Bharathiar University, Coimbatore, Tamilnadu, India G. K. Suraishkumar Department of Biotechnology, Bhupat and Jyoti Mehta School of Biosciences building, Indian Institute of Technology Madras, Chennai, India Padmanaban S. Suresh School of Biotechnology, National Institute of Technology, Calicut, Kerala, India Snehasikta Swarnakar CSIR-Indian Institute of Chemical Biology, Kolkata, India Yasmeen Talab Forensic Medicine and Clinical Toxicology Department, Faculty of Medicine, Mansoura University, Mansoura, Egypt Institute of Forensic and Traffic Medicine, University of Heidelberg, Heidelberg, Germany Bee Ling Tan Department of Nutrition, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, Serdang, Selangor, Malaysia Sheikh A. Tasduq PK-PD and Toxicology Division, CSIR-Indian Institute of Integrative Medicine, Jammu and Kashmir, India Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, India Abhishek Teli Institute of Bioinformatics and Biotechnology, Savitribai Phule Pune University, Pune, India Babu Thandapani Ultra College of Pharmacy, Madurai, Tamilnadu, India Sanu Thankachan School of Biotechnology, National Institute of Technology, Calicut, Kerala, India N. Thirumoorthy Caritas College of Pharmacy, Caritas Educity, Kottayam, Kerala, India. Paranthaman Thiyagarajan Department of Biochemistry and Biotechnology, Annamalai University, Annamalainagar, India Divya Thomas Eppley Institute for Research in Cancer and Allied Diseases, Fred & Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha, NE, USA Nupurand A. B. Tiku Radiation and Cancer Therapeutics Lab, School of Life Sciences, Jawaharlal Nehru University, New Delhi, India

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Contributors

Budhi Sagar Tiwari School of Biotechnology, Indian Institute of Advanced Research, Gandhinagar, Gujarat, India Kathryn F. Tonissen School of Environment and Science, Griffith University, Nathan, Australia Griffith Institute for Drug Discovery, Griffith University, Nathan, Australia Margalida Torrens-Mas Grupo Multidisciplinar de Oncología Traslacional, Institut Universitari d’Investigació en Ciències de la Salut (IUNICS), Universitat de les Illes Balears, Palma de Mallorca, Spain Instituto de Investigación Sanitaria de las Islas Baleares (IdISBa), Palma de Mallorca, Spain Surendra Kumar Trigun Biochemistry Section, Department of Zoology, Institute of Science, Banaras Hindu University, Varanasi, India Slavisa Tubin MedAustron Center for Ion Therapy and Research, Wiener Neustadt, Austria Maria Turos-Cabal Departamento de Morfología y Biología Celular, Facultad de Medicina, Universidad de Oviedo, Oviedo, Spain Instituto Universitario de Oncología del Principado de Asturias, Oviedo, Spain Instituto de Investigación Sanitaria del Principado de Asturias, Oviedo, Spain Nishant Tyagi Division of Stem Cells & Gene Therapy Research, Institute of Nuclear Medicine & Allied Sciences (INMAS), Defence Research and Development Organisation (DRDO), New Delhi, India Foram U. Vaidya School of Biotechnology, Indian Institute of Advanced Research, Gandhinagar, Gujarat, India Jessica M. M. Valadares Laboratório de Bioquímica Celular, Universidades Federal de São João del Rei, Divinópolis, Brazil Laboratório de Membranas e ATPases, Universidades Federal de São João del Rei, Divinópolis, Brazil Mathews Valuparampil Varghese Department of Medicine, Division of Endocrinology, The University of Arizona College of Medicine, Tucson, AZ, USA Sristis Varshney Department of Biotechnology, Jaypee Institute of Information Technology, Noida, Uttar Pradesh, India Mohit Vashishta R&D Department, Shanghai Proton and Heavy Ion Center (SPHIC), Shanghai, China Shanghai Key Laboratory of Radiation Oncology, Shanghai, China Shanghai Engineering Research Center of Proton and Heavy Ion Radiation Therapy, Shanghai, China

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Carlos Velez-Pardo Neuroscience Research Group, Medical Research Institute, Faculty of Medicine, University of Antioquia (UdeA), Medellin, Colombia Antonio Vella Unit of Immunology-Azienda Ospedaliera Universitaria Integrata, Verona, Italy Goutham Hassan Venkatesh Thumbay Research Institute for Precision Medicine, Gulf Medical University, Ajman, United Arab Emirates Rosy Vennila Government Medical College and Hospital, Karur, Tamil Nadu, India Renzo Emanuel Vera Division of Oncology Research, Schulze Center for Novel Therapeutics, Rochester, MN, USA Amit Verma PACT & Health LLC, Branford, CT, USA Anita K. Verma Nano-Biotech Lab, Department of Zoology, Kirori Mal College, University of Delhi, Delhi, India Yogesh Kumar Verma Division of Stem Cells & Gene Therapy Research, Institute of Nuclear Medicine & Allied Sciences (INMAS), Defence Research and Development Organisation (DRDO), New Delhi, India K. Vijaya Rachel Department of Biochemistry and Bioinformatics, GITAM Deemed to be University, Visakhapatnam, AP, India Kunwar Somesh Vikramdeo School of Life Sciences, Jawaharlal Nehru University, New Delhi, India Ram A. Vishwakarma PK-PD and Toxicology Division, CSIR-Indian Institute of Integrative Medicine, Jammu and Kashmir, India Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, India Maria Voura Department of Chemistry, Aristotle University of Thessaloniki, Thessaloniki, Greece Ridhima Wadhwa Centre for Inflammation, Centenary Institute, Sydney, NSW, Australia Discipline of Pharmacy, Graduate School of Health, University of Technology Sydney, Ultimo, NSW, Australia Bhargav N. Waghela School of Biotechnology, Indian Institute of Advanced Research, Gandhinagar, Gujarat, India David Wallace Department of Pharmacology & Toxicology, Oklahoma State University Center for Health Sciences, Tulsa, OK, USA Yuzhen Wang Department of Pharmaceutical Engineering, College of Life Science, Inner Mongolia Agricultural University, Hohhot, China

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Contributors

Grazielle Castagna Cezimbra Weis Federal University of Santa Maria, Santa Maria, RS, Brazil Haijie Wu Department of Pharmaceutical Engineering, College of Life Science, Inner Mongolia Agricultural University, Hohhot, China Amit S. Yadav School of Biotechnology, KIIT Deemed to be University, Institute of Eminence, Bhubaneswar, India Nomesh Yadu Division of Molecular Reproduction, Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram, Kerala, India Md Yousuf Applied Biology Division, CSIR-Indian Institute of Chemical Technology, Hyderabad, India Department of Chemistry, Ramnagar College, Purba Medinipur, West Bengal, India Yezhou Yu School of Environment and Science, Griffith University, Nathan, Australia Griffith Institute for Drug Discovery, Griffith University, Nathan, Australia Mingyue Zhong Department of Pharmaceutical Engineering, College of Life Science, Inner Mongolia Agricultural University, Hohhot, China

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Reactive Species and ER-Mitochondrial Performance for Glioblastoma Multiforme Treatment Strategy Tina Nasrin, Sajal Chakraborti, and Soni Shaikh

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Current Status of Glioblastoma Multiforme (GBM) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Reactive Species and [Ca2+]i on ER Stress and Mitochondrial Performance . . . . . . . . . . . . . . . . . . . ER Stress, Mitochondria, and GBM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genetic Alteration by Reactive Species in GBM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Angiogenesis, NADPH Oxidase, and GBM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Implications in the Treatment of Gliomas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Maintenance of Reactive Species Steady State . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Targeting ER Components and Autophagy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Other Molecular Targets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract

Glioblastoma multiforme (GBM: also called glioblastoma) often occurs in the frontal and temporal lobes of the brain and develops from the glial cells. It is the most devastating and biologically aggressive tumor with challenging treatment strategies, in fact, with limited treatment protocols usually surgery, radiation, and chemotherapy with temozolomide (TMZ). The complex pathology appears to occur due to cellular stress produced by intra-/extracellular stimuli. Furthermore, it has been observed that the stress-induced reactive oxygen/nitrogen species (ROS/RNS) generation increases during GBM progression. Actually, such reactive species distort the activity of the antioxidant system as well as intracellular [Ca2+]i signaling of the glioma cells. In addition, it can induce the posttranslational modifications of cellular proteins and thereby result in the initiation and progression of GBM. Thus, the T. Nasrin · S. Chakraborti Department of Biochemistry and Biophysics, University of Kalyani, Kalyani, West Bengal, India S. Shaikh (*) Laboratory of Histopathology, Tata Medical Center, MAR (E-W), New Town, Kolkata, India © Springer Nature Singapore Pte Ltd. 2022 S. Chakraborti et al. (eds.), Handbook of Oxidative Stress in Cancer: Mechanistic Aspects, https://doi.org/10.1007/978-981-15-9411-3_20

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therapeutic values of the intra-/extracellular agents to modulate the redox status of GBM cells are under investigation. Herein, we summarized the salient features of the GBM and the role of ROS/RNS system in its progression along with the currently known strategies for its treatment. Keywords

Glioblastoma multiforme · Cancer · RNS · ROS · Calcium · ER stress · Mitochondria

Introduction There are two major types of reactive species that are known to dysregulate metabolisms in the eukaryotic cells: one is reactive oxygen species (ROS), and the other is reactive nitrogen species (RNS). Both of them possess uncoupled electrons in their outermost electron shell, which makes them unstable, and therefore, a free radical entity is created (Table 1). Along with such free radicals, other reactive molecules derived from ROS and RNS have also been shown to have their impact on the biological system. Therefore, the term oxidative stress can be broadened to nitro-oxidative stress (Cipak Gasparovic et al. 2017). Many extracellular factors are known for inducing the ROS/RNS production in the living cells, such as air/water pollution, ionizing radiation, heavy metal exposure, alcohol, pesticides, cigarette smoking, and so on. The mitochondrial electron transport chain has been considered as the main site for such species production. Other sites are: endoplasmic reticulum, microsomal mixed-function oxidase, cell membrane-associated lipoxygenases, prostaglandin endoperoxidases, NADPH oxidase, xanthine oxidase, nitric oxide synthase, lysosomal myeloperoxidase, etc. (Cipak Gasparovic et al. 2017; Hameister et al. 2020). Cellular oxidative stress is widely reported for its involvement in the cell signaling cascades such as apoptosis, autophagy, enhanced metabolism, Table 1 Some biologically important ROS/RNS and their characteristics. R Organic alkyl groups, s seconds Name Super oxide anion Hydroxyl radical Alkoxyl radical Peroxyl radical Nitric oxide Nitrogen dioxide Hydrogen peroxide Organic peroxides Peroxynitrite Nitrous acid Peroxy nitrous acid

Symbol O2˙– OH• RO. ROO. NO. NOO. H2O2 R2O2 ONOO
HNO2 ONNOH

Types Radical Radical Radical Radical Radical Radical Nonradical Nonradical Nonradical Nonradical Nonradical

Stability (t1/2) in S 10
6 10
10 10
6 17 Environment dependent s Stable Stable 10
3 s Fairly stable

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mitochondrial defaults, and oncogene expression (Valko et al. 2006; Kaminskyy and Zhivotovsky 2014; Kruk and Aboul-Enein 2017). In fact, in cancer models, such pathological conditions dysregulate the functional activities of macromolecules such as DNA, protein, and lipid (Fig. 1). Some of the free radicals are reported for causing the damage to cells and tissues, thereby facilitating the tumor growth and invasion. Recent studies also support this concept for fostering of proliferation, survival and migration of the cancer cell types including GBM. The GBMs are made by such stressed conditions, resulting in unregulated cell growth with stepped up intracellular biological reactions (Fig. 1) (Valko et al. 2006; Trachootham et al. 2009; Cholia et al. 2018).

Current Status of Glioblastoma Multiforme (GBM) Gliomas are the primary brain tumor occurring mostly in the cerebral hemispheres and observed in the frontal and temporal lobes of the brain. These are subgrouped on the basis of surgical specimen and morphological features such as atypia, necrosis,

Fig. 1 Mitochondrial reactive species production mechanism and their mode of participation in the glioblastoma multiforme (GBM). Production of ROS/RNS through mitochondrial cycles leads GBM progression through the cell macromolecules (DNA, Proteins, and Lipids) distortion. GBM Glioblastoma multiforme, GSH Glutathione, NADPH Nicotinamide adenine dinucleotide phosphate hydrogen, NOS Nitric oxide synthase, NOX NADPH oxidase, RNS Reactive oxygen species, ROS Reactive oxygen species, SOD Super oxide dismutase, UPR Unfolded protein response

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and endothelial proliferation. The subgroups are accordingly diffuse astrocytoma (grade II), anaplastic astrocytoma (grade III), and glioblastoma multiforme (GBM; grade IV) (World Health Organization, WHO). Among them, the grade IV glioma (GBM) is the most common and aggressive brain cancer occurring from the heterogenous astrocytes and oligodendrocytes. Cancer in such glial cells (supports the other brain cells) rapidly grows and spreads into the proximate brain tissues (Nàger et al. 2018). According to the American Association of Neurological Surgeons (AANS), it accounts for 17% of all brain tumors. According to Mayo Clinic (USA), age seems to be one of the vital parameters for the disease occurrence, and mostly adults between 45 and 65 years are possible victims. GBM is so devastating that it causes death with the median survival period of 15 months after the diagnosis, although 10% of cases show a survival probability up to 5 years depending on the treatment strategies. Like other cancers, GBM also develops rapidly under the immune-suppression (Cholia et al. 2018; Nàger et al. 2018). The disease symptoms are unspecific, which often depends on the location of the tumor within the brain. Some of the observable symptoms may be the persistence of headaches, double or blurred vision, loss of appetite, vomiting, seizures, and changes in the personality. Unfortunately, there is no complete treatment protocol for GBM. The common treatment strategy is surgery, but in major instances, it cannot prevent the recurrence of GBM. Although some reports confirm the modest benefits with surgery, currently surgery followed by the chemotherapy and radiation therapy is the most accountable protocol. However, a combined treatment strategy is more acceptable for a good outcome nowadays (Batash et al. 2017). The main problem for such disease treatment is that despite different treatment strategies such as surgery, radiotherapy, and chemotherapy with temozolomide (TMZ)-like drugs, recurrence of GBM is very common. Therefore, a second line of management for GBM is very important. Herein, we tried to describe the involvement of cell signaling pathways, which can prevent the recurrence of the GBM formation. Researchers are trying to find out the mechanism(s) of intraorganelles like ER, mitochondria, and membrane communications to differentiate the wild type and drug, especially TMZ-resistant cells in the context of GBM (Nàger et al. 2018; Visa et al. 2019; Wang et al. 2017).

Reactive Species and [Ca2+]i on ER Stress and Mitochondrial Performance The role of ER stress has been discussed in several pieces of literatures in the context of the solid tumor microenvironment. Basically, ER, known as the intracellular [Ca2+]i storage hub, plays an important role in several signal transduction pathways (He et al. 2019). Although the ER stress is associated with different types of cancer, the underlying molecular mechanisms are quite complex. The pathophysiological conditions such as hypoxia, calcium depletion, and oxidative stress can directly regulate the cellular reactive species such as ROS/RNS, which could result in alteration of the protein-folding capacities of the ER. In cancer-like high-energy demanding cells, ROS such as hydroxyl (OH•) and superoxide (O2.) radicals are

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endogenously formed in the mitochondria with higher concentrations. The superoxide (O2.) radical in turn forms peroxynitrite (ONOO
), an overreactive molecule that can cause oxidation or nitration of proteins and DNAs (Lin et al. 2019; Kabiraj et al. 2015). ER, the main site for protein synthesis, is prone to be affected under such conditions (Fig. 1). It is, therefore, apparent that the [Ca2+]i depletion and oxidative stress are the main causes of the disruption of normal ER activities. This causes an alteration of protein-folding capacity leading to the formation of misfolded proteins and thereby dysregulation of normal cell functions (Lin et al. 2019; Kabiraj et al. 2015; Peñaranda Fajardo et al. 2016). Importantly, an individual and/or cooperative function of mitochondria and ER is essential to maintain the cell physiology. Disturbance of their mechanistic steady-state often leads to protumorigenic conditions. ER stress initiates an integral signal transduction pathway called unfolded protein response (UPR). The UPR is mainly controlled by: (a) protein kinase R (PKR)-like ER kinase (PERK); (b) inositol-requiring protein 1α (IRE1α); (c) activating transcription factor (ATF); and (d) binding immunoglobulin protein (BiP/GRP78). In fact, these signaling molecules are mainly activated to neutralize ER stress (Fig. 2a). All of the said proteins are, however, connected to the mitochondrial signaling cascade and observed to be crucial for changing the microenvironment of the GBM cells (Peñaranda Fajardo et al. 2016; Papaioannou and Chevet 2018). ER also consecutively supplies [Ca2+]i to the mitochondria for ATP production. High energy demands of the cancer cells accordingly recruit calcium for ATP production in the mitochondria. In the cancer cells, overproduction of the reactive species in the mitochondria is also a common feature, which causes ER-mitochondrial miscommunication and subsequently disrupts the basal autophagy mechanism, leading to apoptosis and necrosis or both (Senft and Ronai 2015; Pinton et al. 2008). The importance of the [Ca2+] i like second messengers has been illustrated in a variety of cellular functions including metabolism, gene expression, cell survival, and cell death that are directly or indirectly linked to the glioblastoma like cancer formation (Peñaranda Fajardo et al. 2016). Additionally, [Ca2+]i regulatory components such as sarco-/endoplasmic reticulum pump (SERCA), inositol 1,4,5-triphosphate receptors (IP3R), ryanodine receptors (RyRs), mitochondria-associated ER membrane (MAM), and sodium-calcium exchanger (NCX) are localized in the ER, and that in turn is involved in the mitochondrial [Ca2+]i regulation (Fig. 2a). Mitochondrial electrogenic (ΔΨm) [Ca2+]i entry is known to play an important role in cell physiology and pathology. As stated earlier, mitochondrial [Ca2+]i contributes to the energy production (ATP) through the electron transport chain, and like other products, a marked increase in ROS/RNS is also generated as by-products during enhanced activities of the mitochondrial respiratory chain. Additionally, through the other extramitochondrial enzymes, like NADPH oxidase, xanthine oxidase, nitric oxide synthase, and cytochrome P450, the cells can also modulate [Ca2+]i for their regulatory activities (Görlach et al. 2015). In their mechanistic way, the reactive species, irrespective of their production resources, have a direct effect on the posttranslational modifications and the activity of ER [Ca2+]i regulators. For example, RyRs can increase the [Ca2+]i leak from the ER store and, thereby, modulates the NADPH oxidase activities. ROS

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Fig. 2 (a) ER-mitochondrial communication in the reactive species scenario, where autophagy and calcium signaling regulators were found to play the key roles. ATF, Activating transcription factor; Bip/GRP78, Binding immunoglobin proteins; HK2, Hexokinase; IP3R, Inositol 1,4,5-triphosphate (IP3) receptor; MAM, Mitochondria associated ER membrane; MCU, Mitochondrial calcium

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derived from the xanthine oxidase has been observed to impair the thiol residues of the IP3R. Furthermore, correlations of NOX with IP3R and [Ca2+]i are also available in the literature (Görlach et al. 2015). Therefore, a fine-tuning of redox state is important for the intracellular [Ca2+]i regulation, which could imply the involvement of the SERCA enzymatic functions. The ROS actually retard the binding of ATP to the SERCA. All the above [Ca2+]i regulatory activities are somehow connected to the mitochondrial [Ca2+]i homeostasis. For example, mitochondrial ROS has been observed for its RyR2-mediated [Ca2+]i sparking. In the cancer cells, mitochondrial ROS is produced in excess amount, and it exerts a bidirectional-mode of [Ca2+]i sparking, supporting mitochondrial control on [Ca2+]i release from the ER (Görlach et al. 2015; More et al. 2018; Xu and Krukoff 2005). Therefore, in an oxidative environment, intracellular [Ca2+]i regulation is made up of both mitochondria and the ER in a bidirectional mode. Alongside, in the tumor cells, the mitochondrial permeability transition pore (mPTP) is desensitized to [Ca2+]i and reactive species, resulting in their death resistance activity. On the other hand, loss of cyclindependent kinases (Cdk5) in cancer cells increases ROS production. Dinaciclib, an inhibitor of Cdk, inhibits the proliferation of human glioma cells. The Cdk molecule has been observed to be colocalized with MAM of the brain cells, which can cause the ER-mitochondrial tethering for [Ca2+]i transfer from ER. Cdk5 is also associated with the ATP-driven [Ca2+]i uptake from ER (Seidlmayer et al. 2015; NavaneethaKrishnan et al. 2020; Lubanska and Porter 2017). Since the intracellular reactive species and [Ca2+]i are the key regulators of normal ER activities, especially the UPR and mitochondrial activities in the cancer cells, a fine state of balance is very important for it. Thus, the molecular mechanism for such interactive pathways appears to be important for better understanding of the GBM disease progression.

ER Stress, Mitochondria, and GBM About 20% of the mitochondria have direct contact with ER along with the close proximity of mitochondria-associated ER membrane (MAM) (Fig. 2a). Such association of ER and mitochondria is important for GBM as dysfunctional mitochondria and stress-induced ER signaling pathways are involved in the tumorigenesis, migration, invasion, and survival of GBM, which eventually correlates with subcellular ä Fig. 2 (continued) uniporter; mPTP, Mitochondrial permeability transition pore; NCX, Sodium calcium exchanger; PERK, Protein kinase R-like ER kinase; RyR, Ryanodine receptor; SERCA, Sarcoendoplasmic reticulum (SR) calcium transport ATPase; UPR, Unfolded protein response; VDAC, Voltage-dependent anion channel. (b) Major targets for the GBM therapeutic strategies, where the breakdown of threshold point of ROS/RNS and oxidant-antioxidant balance in the cell are the key features for the treatment strategies. The mitochondrial activity plays key role by its increased activity. (c) Gene targets for GBM-therapeutic strategies, where dependency of reactive spices on EGRR expression could be a hint. (d) miRNAs-based treatment also would be a possibility through the increased miRNA expression or producing the analog of miRNAs

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[Ca2+]i concentration (Peñaranda Fajardo et al. 2016; Navaneetha Krishnan et al. 2020; Liu et al. 2013). A similar report stating that the reactive species-mediated induction of apoptosis in the glioblastoma cells is calcium-dependent is also available. Although it does not describe the direct role of the mitochondria or ER-mitochondrial contact, ROS/RNS has been suggested to promote caspasedependent apoptosis, where mitochondria and ER meet in a common point of the signaling pathway to promote the apoptosis (Das et al. 2007). In addition, cell migration and invasion in the GBM are critically dependent on the increase in [Ca2+]i that results from the ER [Ca2+]i release. In the GBM cells, a variety of molecules participate in the tumor microenvironment to activate the PLC/IP3 pathway and decrease the mitochondrial membrane potential (ΔΨm); therefore, ROS induces the ER stress and the intrinsic (mitochondrial) pathways in the GBM cells (Catacuzzeno and Franciolini 2018; Chou et al. 2015). On the other hand, it is well documented that in the cancer cells, ER stress made by ROS/RNS expresses the unfolded protein response (UPR) system to cope with the GBM progression (Peñaranda Fajardo et al. 2016; Papaioannou and Chevet 2018). GBM cell lines revealed an increase in the level of ER chaperones, activate transcription factors (e.g., ATF4), and binding with Bip/GRP78 such as UPR components, whose expression is positively correlated with the cell proliferation rate. In other instances, a UPR component, protein kinase RNA-like endoplasmic reticulum kinase (PERK), showed activation in grade III and grade IV GBM. Interrelation of ATF, Bip/GRP78, and PERK like autophagic molecules is known for the progression of GBM progression (Peñaranda Fajardo et al. 2016; Papaioannou and Chevet 2018). In starved condition, PERK has been observed to be important for cell survival. In such condition, PERK phosphorylates serine/threonine kinase (Akt), a key player of the PI3K-signaling pathway, which promotes the translocation of hexokinase 2 (HK2) toward the mitochondria. In addition, epidermal growth factor receptor (EGFR) can also cause the activation of the Akt and HK2 for the cell survival in autophagic conditions. This is relevant to the UPR system of the autophagy mechanism (Peñaranda Fajardo et al. 2016; Papaioannou and Chevet 2018). In addition to a special mitochondrial engulfment called mitophagy, mitochondria are also directly participated in the classical autophagy mechanisms. Focusing on the mitochondriaassociated ER membrane (MAM) has been evident for the association with the functional and metabolic state of the human glioma cells that actually controls the mitochondrial integrity as well as autophagy. Notably, mTORC2 (mammalian target of rapamycin complex) together with serine/threonine kinase (Akt) is able to control the mitochondrial activity and thereby the cell survival via MAM-associated proteins such as IP3R and HK2 that are already described for their contribution in PERKmediated glioblastoma progression during autophagy. mTORC2 markedly increases the glycolysis in glioblastoma through MAM-localized HK2 indicating MAM-UPR correlation and thereby suggesting an important role in this perspective (Fig. 2a). Of note, MAM proteins regulated by reactive species are involved in the intracellular [Ca2+]i homeostasis and superoxide production (Papaioannou and Chevet 2018; Arismendi-Morillo et al. 2017). It has been suggested that alteration of the activity of ER membrane sodium/calcium exchanger (NCX) under stress condition inhibits

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glioblastoma cell growth; however, no direct evidence has yet been made for NCX expression in the human glioblastoma cells (Song et al. 2014). Therefore, ER/mitochondrial NCXs would be an important research interest for better understanding the mechanism of GBM occurrence.

Genetic Alteration by Reactive Species in GBM It has already become evident that metabolic oxidative stress persists in the cancer microenvironment due to the high metabolic rate of mitochondria, ER, and cell membranes. In such conditions, the production of RNS and ROS are very known as the usual effectors (Lin et al. 2019; Kabiraj et al. 2015). The production of a high level of reactive species in the cancer cells leads to DNA mutation or inhibits the DNA repair system, a crucial event for the cancer initiation and progression (Fig. 1) due to the activation of oncogenes, gene amplification, DNA strand breaks, miscoding, rearrangement, and modification of the DNA sequences. This does not imply only genomic DNA alteration, but also the mitochondrial DNA alteration due to its close proximity to the electron transport chain (Kumari et al. 2018). Like other cancer models, GBM is also characterized by oxidative stress-induced DNA damages including N-hydroxylation and nitrosation, activate aryl amines to generate ROS and induce 7,8-dihydro-8-oxo-20 -deoxyguanosine (8-oxodG) formation (a guanine modification). ROS-mediated DNA oxidation results in 8-oxo-dG at the gene promoters (Jha et al. 2014), and the demethylation of DNA occurs frequently in the GBM upon ROS induction. In addition, the ROS-related genes NCF1 (neutrophil cytosolic factor 1) and NOX4 (NADPH oxidase subunit 4) regulate the cell proliferation and also upregulate GBM formation (Jha et al. 2014; Barciszewska et al. 2019). Functional NCF1 encodes the regulatory p47phox subunit that requires the activation of NOX complex. Complex formation of ROS-NCF is somehow related to the mTOR (mammalian target of rapamycin) and thereby autophagy-mediated cell death. Therefore, activation of NOX complex is an important event in GBM progression. The biological function of NOX4 in the context of cancer is well documented, which in turn modulates vascular endothelial growth factor (VEGF), mitogen-activated protein kinase (MAPK), phosphoinositide 3-kinase/AKT(PI3K/ AKT), and epidermal growth factor receptor (EGFR) such as classical signaling pathways. Knockdown of NOX4 by shRNA has been shown to reduce significantly the ROS production in the GBM cells, which eventually suppress the glioblastoma cell proliferation, invasion, and tumor-induced angiogenesis, but increases its radiosensitivity (Li et al. 2014). On the other hand, superoxide dismutase (SOD2) plays a vital role in controlling ROS production and mostly plays a defensive mechanism in the TMZ-resistant GBM cells. It has been observed that the downregulation of SOD2 by RNA interference (RNAi) allows the TMZ to kill the genetically modified cells again (Chien et al. 2019). Peroxynitrite (ONOO
) produced by the action of mitochondrial superoxide dismutase (SOD2) can inhibit the specific DNA-binding ability of transcription factor p53 function in malignant glioma cells. Actually, the

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concentration of ONOO
is associated with the tumor inflammatory environment caused by dysregulation of wild-type p53 transcription and downstream p21 expression (Cobbs et al. 2003). It has been observed that the mutant p53-associated autophagy mechanism sensitized the cancer cells, which inhibits mTOR activity. An increase in mTORC1 expression may lead to the initiation of a cross-talk between nuclear factor erythroid 2-related factor 2 (Nrf2) and p62 resulting in ROS-mediated cytotoxicity. Nrf2 is known to control the cellular redox homeostasis. The role of Nrf2 in cancer cell proliferation in multiple glioma cell lines has also been described in the literature (Cordani et al. 2016; Gilardini Montani et al. 2019; Massi et al. 2004). The noncoding part of the genome produces miRNA like small molecules, which are known to be actively involved in cancer development. ROS-mediated signaling axis appears to be the key regulatory mechanism for such cancer progression. Limited research demonstrated the possibility of the involvement of nitrogen species in the regulation of miRNAs in different cancer models (Fig. 1) (D’Souza et al. 2020; Yakovlev 2015). Like other cancers, a number of miRNAs have been identified in glioblastoma as the biomarkers. A list of predicted miRNAs involved in the GBM progression including redox mechanism are retrieved from the HMDD v3.0: a database for human microRNA–disease associations (Huang et al. 2019) (Table 2A). Knockdown of miR-155, a microRNA that is encoded by the MIR155 host gene or MIR155HG in the human, has been observed to enhance the anticancer effect of TMZ on glioma cells, which in turn lower the accumulation of reactive oxygen species (ROS) and slowed down the progression of cancer (Liu et al. 2015; Shea et al. 2016). The prediction tool (HMDD v3.0) revealed that miR-155 can downregulate the EGFR, CDK, PIK3R1, PIK3CA, and HK2 like intracellular reactive species regulatory genes and some autophagy regulatory genes in the glioma cells. Another miRNA, miR-302b could also be targeted to the EGFR and CDK like genes in GBM (Huang et al. 2019) (Table 2B). On the basis of the literature survey, miR-302b has been found to act on the gene named E2F3 that produces the transcription factor E2F3. Such a transcription factor is well known for its importance in the cell cycle regulation and action on the tumor suppressor molecules. miR-302b-mediated direct targeting of E2F3 gene has been described for its functional dependency on mitochondrial dysfunction and ER stress in glioma cells. Therefore, it seems that both the expressions of miR-155 and miR-302 increase with GBM progression that affects the cell proliferation, apoptosis, invasion, and chemoresistance like activities that are associated with the autophagic genes (Shea et al. 2016; Chen et al. 2014).

Angiogenesis, NADPH Oxidase, and GBM Angiogenesis and necrosis are the phenotypes of glioblastoma (Beyer et al. 2017; Fukai and Ushio-Fukai 2020). Thus, enhancing or inhibiting angiogenesis has great cancer therapeutic consequences. The common ROS sources such as NADPH oxidases (NOX), xanthine oxidase (XO), endothelial nitric oxide synthase (eNOS),

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Table 2 Prediction of microRNAs (miRNAs) from HMDDv3.0 database for glioblastoma (A) Genes related to reactive species-mediated cell activities and their mode of regulation by miRNAs in the glioma cells Gene miRNA Regulation CDK6 hsa-mir-1, hsa-mir-320a, hsa-mir-340, hsa-mir-302b, hsa-mirDownregulation 124, hsa-mir-15a, hsa-mir-193b, hsa-mir-155, hsa-mir-15b, hsa-mir-491, hsa-mir-195, hsa-mir-342, hsa-mir-222, hsa-let-7b, hsa-mir-885, hsa-mir-218, hsa-mir-145, hsa-mir-183, hsa-mir29a, hsa-mir-205, hsa-mir-22, hsa-mir-504, hsa-mir-191, hsa-mir-452, hsa-mir-34c, hsa-mir-506, hsa-let-7a, hsa-mir-21, hsa-mir-603, hsa-mir-449a, hsa-mir-30a, hsa-mir-34a, hsa-mir302c, hsa-mir-20b, hsa-mir-221 PIK3CA hsa-mir-124, hsa-mir-155, hsa-mir-17, hsa-mir-375, hsa-mir-139, Downregulation hsa-mir-19a VEGFA hsa-mir-17, hsa-mir-423, hsa-mir-503, hsa-mir-320a, hsa-mirDownregulation 598, hsa-mir-195, hsa-mir-299, hsa-mir-429, hsa-mir-20a, hsa-mir-200b, hsa-mir-145, hsa-mir-205, hsa-mir-106a, hsa-mir125a, hsa-mir-200c, hsa-mir-302d, hsa-mir-504, hsa-mir-3163, hsa-mir-296, hsa-mir-297, hsa-mir-93, hsa-mir-27a, hsa-mir-34a, hsa-mir-330, hsa-mir-20b, hsa-mir-126 EGFR hsa-mir-302b, hsa-mir-146b, hsa-mir-155, hsa-mir-491, hsa-mir- Downregulation 7, hsa-mir-146a, hsa-mir-125a, hsa-mir-30a EGFR hsa-mir-21 Upregulation (B) Mostly reported miRNAs mediated the regulation of autophagic, intracellular calcium regulatory and stress-induced genes in the glioma cells miRNA Genes Regulation hsa-mirPIK3R1, EGFR, CDK6, FOXO3, HK2, PIK3CA, SMAD4 and Downregulation 155 3, CDKN2A, RAC1, FADD, NFKB1, KCNN3, SIRT1 hsa-mirCDK6, EGFR, CLCN3, ESR2, BMI1, EIF2S1 Downregulation 302b

and electron transport chain (ETC) have been observed to promote the angiogenesis mechanism. However, the most prominent among them is the increase in NOXs expressions (Fukai and Ushio-Fukai 2020). Available literature suggests that endothelial cells are sensitized by a signal from resting tumor cells to a rapid growth state (Beyer et al. 2017; Folkman 1971). The poor structural and functional development of the tumor blood vessels is often hypoxic in nature, and it is supposed that the high ROS production is the cause of such pathological conditions like glioblastoma as well. NOXs, specially NOX4, participated in the tumor angiogenesis and carcinogenesis (Vimalraj et al. 2018; Weyemi et al. 2013). The NOX4 overexpression regulates the Akt-mediated survival mechanism of glioblastoma cells. NOX4 inhibition is important because it has a direct role in the glycolytic phenotype of glioblastoma cells. For example, NOX4 inhibitor 2-Deoxy-D-Glucose (2DG) increases the glycolytic phenotype and thereby migration, invasion, and angiogenesis. Combined treatment of 2DG plus shikonin, a naturally occurring naphthoquinone compound, has been shown to increase the sensitivity to 2DG and decreases in glycolytic phenotype in terms of hexokinase, ATP, O2 consumption,

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and glucose uptake (Gupta et al. 2015). Due to the complex nature of the angiogenic system, it may target multiple signaling pathways, and possibly resist the activity of selective therapeutic agents. For example, bevacizumab-like agent targets VEGF (Beyer et al. 2017; Hayes et al. 2016). As stated earlier, like other cancers, an increase in ROS production is characteristic of GBM progression. In that context, cell hypoxia produces ROS in GBM due to a marked increase in the expression of NOX4, which has been shown to play an important role in the cycling of hypoxiamediated hypoxia-inducible factor-1 (HIF-1) activation and thereby promotes tumor progression in glioblastoma, indicating that NOX4 is a critical mediator of radioresistance in GBM since the hypoxia resists the tumor cells from the effects of radiation. Knockdown of NOX4 results in reduced ROS production and suppresses the glioblastoma cell proliferation, invasion, and tumor-induced angiogenesis along with increased radiosensitivity. Therefore, such consequences of inhibition of NOX4 by shRNA or other means would be strategically significant to overcome the radioresistance as well as therapeutic efficacy for glioblastoma (Li et al. 2014). In addition to NOX, mitochondria-derived ROS regulates cellular redox status that is important for angiogenesis. In hypoxic condition, H2O2 produced from mitochondria triggers HIF-1 stabilization, which increases the transcription of VEGF or upstream of angiogenic genes, for example, STAT3. Thus, mitochondria regulate the angiogenic responses by tuning the ROS-associated cell metabolism. The hypoxic ROS-mediated STAT3 activation through induction of NOX4 expression in human glioblastoma leads to angiogenesis by VEGF, HIF-1, MMPs, and fibroblast growth factors. But among them, the VEGF is considered the most significant mediator for angiogenesis progression. Along with VEGF, fibroblast growth factor-2 (FGF-2), interleukin-8 (IL-8), transforming growth factor-β (TGF-β), and platelet-derived growth factor (PDGF) can also act as positive regulators (Yu et al. 2015; Wang et al. 2015). Some miRNAs also take part in regulating the expression of angiogenic genes. Among them, miR-296 and miR-7 are the most studied miRNAs to promote and inhibit angiogenesis. It has been observed that VEGF significantly induces miR-296 expression in glioma cells, while miR-7 targets the EGFR- and PI3K-signaling pathways (Beyer et al. 2017).

Implications in the Treatment of Gliomas Maintenance of Reactive Species Steady State In order to develop the new drugs, one should keep in mind the following: (i) autophagy mechanism; (ii) ER-mitochondrial activities; (iii) oxidant-antioxidant balance; and (iv) threshold level of reactive species production (Fig. 2b). Due to the high metabolic rate of the glioma cells in comparison to the normal cells, redox status and its corresponding elements are more susceptible as the therapeutic target. Overproduction of chemically active species causes cell damage, thereby providing the self-killing opportunity of the tumor cells (Görlach et al. 2015; More et al. 2018; Xu and Krukoff 2005). Accordingly, a moderate increase of reactive species in the

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cells leads to proliferation and differentiation; whereas, a certain threshold level of increase in ROS triggers cell death (Fig. 2b). As stated previously, mitochondria are the primary hub for the reactive species production and known as the greatest oxygen consumer in the glioma cells. Therefore, the direct involvement of the mitochondrial pathways in cell death mechanisms has been examined (Das et al. 2007; Catacuzzeno and Franciolini 2018; Chou et al. 2015). So, a drug that can increase the mitochondrial activities by impairing ER-mitochondrial communicative molecules and thereby generating an increase in the level of reactive species leading to cell death would be a treatment strategy. There are two possible ways for the high reactive species’ steady state in the GBM cell types: (i) either through an increase in reactive species production (ii) or through inhibition of the function of cellular antioxidants. Such an anticancer strategy is known as oxidative therapy and provides the body with excess oxygen burden (Fig. 2b). The possibility of implementation of such therapy can be implemented when the mitochondrial ROS production is induced by TMZ, which also activates the chaperone-mediated autophagy process (Lo Dico et al. 2019). It is known that some GBM cell types are resistant to the TMZ-like drugs. In some tumor cells, it has been demonstrated that such drug-induced oxidative stress can compensate with the activation of antioxidant enzymes such as superoxide dismutase (SOD), catalase, glutathione peroxidase, and peroxiredoxin (Fig. 2b). The drug resistance may occur in various ways that include elevated drug efflux, mutation of the targets, and by altering the drug metabolism. Furthermore, the reactive speciesmediated inactivation of death signaling also showed drug resistance activity (Cipak Gasparovic et al. 2017; Hameister et al. 2020; Cui et al. 2018).

Targeting ER Components and Autophagy Inhibition of ER component IP3R also leads to the suppression of the migration and invasion of the glioblastoma cells that are somehow linked to the oxidant-antioxidant balance mechanism. It has been observed that the IP3R variant IP3R3 level is significantly higher in glioblastoma cells than in normal cells. The connection of IP3R and mitochondrial-reactive species production mechanism is described in the previous subheading of this chapter, mostly in the background of intracellular calcium regulation (Catacuzzeno and Franciolini 2018; Chou et al. 2015). In the cancer cells, PI3K/Akt signaling contributes to ROS level through the modulation of mitochondrial activity and NADPH oxidase (NOX) directly, whereas ROS is also produced as the metabolic by-product (Koundouros and Poulogiannis 2018). It has been also observed that the inhibition of PI3K/Akt pathway restored ER calcium release by the disruption of the stomal-interacting molecule (STIM1)-IP3R complex (Santoso et al. 2011). A number of miRNAs (hsa-mir-124, hsa-mir-155, hsa-mir-17, hsa-mir-375, hsa-mir-139, and hsa-mir-19a) have been found to be the possible regulator of PI3K-catalytic subunit at the genetic level of the glioma cells (Huang et al. 2019) (Table 2A). Implying that, IP3R or its upstream intracellular calcium regulators would be a promising therapeutic target for the glioblastoma cells. As ER is a vital organelle in the eukaryotic cells in terms of communication to mitochondria

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for energy production as well as the regulation of basal autophagy of the cells (He et al. 2019; Senft and Ronai 2015; Pinton et al. 2008), the contribution of ER molecules such as IP3R in autophagy progression is important in this aspect. Alongside, the functional aspect of the UPR system is also well described for the regulation of autophagy mechanism and ER-mitochondrial connections (Peñaranda Fajardo et al. 2016; Papaioannou and Chevet 2018). Some studies found that the UPR components are actively involved in the PERK pathway and contribute to ER stress-induced cytotoxicity in GBM. This indicates the possible role of a noncanonical PERK-dependent mechanism in the regulation of stem cells renewal and its differentiation in the posttranscriptional regulation of SOX2 expression. Therefore, the development of an ER stress inducer as well as PERK inhibitors could be a promise for therapeutic strategies of GBM, and understanding of the glioblastoma UPR response also has a great chance to be a therapeutic target for glioblastoma (Peñaranda Fajardo et al. 2016, 2019; Papaioannou and Chevet 2018). In genetic consequences, both ROS and RNS may interfere with the DNA mutation, DNA damage, genomic instability, and cell proliferation like the cellular events. However, different reactive species exert different functions. Some studies demonstrated that knockdown of Nrf2 prevented glioma cell proliferation by ATP depletion, a mitochondrial yield, and consequently inhibited the mammalian target of rapamycin (mTOR) pathway. mTOR pathway has been suggested to play an important role in the autophagy progression, and initiation and termination of nuclear factor erythroid 2-related factor 2 (Nrf2) functions, and also activity at the stem cells, which eventually inhibits the differentiation of glioma at stem cells (Massi et al. 2004; Akhavan et al. 2010). Thus, Nfr2pathway could provide important information for the survival of tumor cells under oxidative stress (Akhavan et al. 2010). Overall, in chemoresistance glioblastoma cells, Nrf2 inhibitory approach could be a promising therapeutic approach. As stated before, mTOR and Nfr2 together play a crucial role in glioblastoma cells; however, the therapeutic strategies to target Nrf2/mTOR are in their infancy.

Other Molecular Targets Although EGFR-mediated regulation is known for cell growth and differentiation such as cellular events, limited research demonstrated EGFR-mediated therapies. Interestingly, the expression of the oncogenic variant of EGFR also causes an increase in ROS production that leads to alterations of the GBM genome (Pudełek et al. 2020; Nitta et al. 2011). Additional information suggests a crucial cross-talk between oxidative stress and poly(ADP-ribose)polymerase-1 (PARP-1) in GBM. PARP-1 a.k.a. NAD+ ADP-ribosyltransferase-1 is produced by the PARP-1 gene and acts mainly as a damaged DNA sensor. Concerning its function, the event of ROS accumulation and EGFR overactivation depends on PARP-1-mediated base excision repair genes (Nitta et al. 2011). This could be one of the possibilities for the development of an effective anticancer drug for GBM (Fig. 2c). Moreover, miRNAs retrieved from HMDD v3.0 database show an interesting output that hsa-mir-

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21could possibly upregulate the EGFR expression during the GBM, whereas numerous miRNAs such as hsa-mir-155, hsa-mir-15b, hsa-mir-491, hsa-mir-195, and hsa-mir-342 can downregulate the EGFR expression (Huang et al. 2019) (Table 2A). Strategically, this functional contradiction of miRNAs would be helpful for controlling the GBM progression. On the other hand, it has been suggested that prolonged hypoxia promotes the expression and activation of VEGF in glioma cells. Therefore, suppression of VEGF signaling at the different molecular steps perhaps represents a strategy to control the GBM progression (Rinaldi et al. 2016). As NOX4 regulates the intracellular ROS level as well as the VEGF signaling in cancer models such as GBM, NOX4 inhibition could be a promising treatment strategy to overcome the radioresistance of the glioblastoma. Although NOX4 has been well established as the causative agent for angiogenesis and glioblastoma multiforme progression, highly isoformspecific NOX inhibitors are still unavailable. Therefore, chemical and genetic inhibitor(s) screening would be a future direction for finding isoform-specific NOX inhibitors. Some NOX inhibitors such as VAS2870 and GKT136901 have been reported for their concentration-dependent target selectivity, but somehow they are not target specific. A GTK136901 derivative, GTK137831, is also under the clinical trials in other models of the disease, where NOX4 is the most abundant isoform (Li et al. 2014; Weyemi et al. 2013; Xu et al. 2013). It has also been suggested that collective numbers of miRNA are expressed in the glioma cells for the downregulation of the VEGF gene (Table 2A). The inhibition of brain tissue-specific Cdk5 has been observed to retard the proliferation of human glioma cells via increasing the reactive species production (Lubanska and Porter 2017). Thus, in addition to the potential pharmaceutical agents, various miRNAs can also downregulate the Cdk activities (Table 2A). The choice of Cdk molecule appears to be prominent as it is associated with MAM region of ER-mitochondrial junction. Therefore, drug targeting the NOX4, VEGF, and Cdk is taken into consideration. In addition to the classical drug-targeting methods, current research focuses on the miRNA-based target, since some of such miRNA molecules are associated with oxidative stress-mediated cancer regulation and have the ability to target several genes through the same or different pathways. In the context of glioma cells, there are several reactive species, which are regulated by miRNAs (Luo et al. 2015). Therefore, specific miRNA expression could be a biomarker of gliomas for the treatment strategy, although these observations are just in the prediction stage. So, further research on miRNAs seems important for a better understanding of the therapeutics of glioma. MiRNAs can act as an oncogene or tumor suppressors and targeting approaches that can proceed through two ways: first, by increasing the expression of miRNAs; and second, by producing the analog of miRNAs to restore the target of miRNAs activity (Shea et al. 2016) (Fig. 2d). Reported suppression of hsa-mir-155 and hsa-mir-302b on PIK3R1, EGFR, CDK6, FOXO3, HK2, PIK3CA, SMAD4 and 3, CDKN2A, RAC1, FADD, NFKB1, KCNN3, SIRT1, ESR2, BMI1, and EIF2S1 genes toward evaluating GBM therapy is underway (Huang et al. 2019) (Table 2).

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Conclusion Although considerable improvements in the conventional treatment protocol and surgical approaches can lower the high rate of morbidity and mortality of the malignant gliomas, abnormal vascular structure formation and intensive cell proliferation seem to be the key burden for the standard treatment strategy. Therefore, an alternative therapeutic strategy for GBM treatment is always in the hunt. Since metabolic oxidative stress persists in the GBM microenvironment, redox balance in the GBM cells has gained interest in the protective measure as the marker that could lead to a better understanding of gliomagenesis and drug designing (Fig. 2b). ROS/RNS can react with the cellular macromolecules such as carbohydrates, proteins, and lipids, thereby disrupting the redox homeostasis that subsequently alters the DNA repair mechanism. Many initial or intermediate signaling molecules, especially ER-mitochondrial communicative molecules, are associated with the ROS/RNS production mechanism in the cancer cells. Controlling their cytotoxicity, treatment strategy for the selective modulation of inactive glioma cells may be useful. In addition, targeted therapies by conventional pharmaceuticals or miRNAs in the context of reactive species-mediated cell networks could prove effective for drug development.

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Oxidative Stress and Thyroid Disorders Loganayaki Periyasamy, Kokelavani Nampalli Babu, Sneha Krishnamoorthy, Jonathan Behlen, Sridhar Muthusami, and Jone A. Stanley

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Hyperthyroidism and OS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Papillary Thyroid Carcinoma and OS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Medullary Thyroid Carcinoma and OS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Anaplastic Thyroid Carcinoma and OS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract

Physiological role of reactive oxygen species (ROS) in the biosynthesis of thyroid hormone (TH) is well known; however, ROS deregulation is noted in hyper- and hypothyroidism which could be targeted pharmaceutically using different drugs. Further, excessive generation of ROS due to reduced elimination and/or enhanced production could significantly result in the carcinogenesis of thyroid cancer through the activation of multiple signaling mechanisms. The role of ROS in the development and progression of papillary thyroid carcinoma, medullary L. Periyasamy · K. N. Babu · S. Krishnamoorthy Department of Biochemistry, Karpagam Academy of Higher Education, Coimbatore, Tamil Nadu, India J. Behlen · J. A. Stanley (*) College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Stattion, TX, USA e-mail: [email protected] S. Muthusami (*) Department of Biochemistry, Karpagam Academy of Higher Education, Coimbatore, Tamil Nadu, India Karpagam Cancer Research Centre, Karpagam Academy of Higher Education, Coimbatore, Tamilnadu, India e-mail: [email protected] © Springer Nature Singapore Pte Ltd. 2022 S. Chakraborti et al. (eds.), Handbook of Oxidative Stress in Cancer: Mechanistic Aspects, https://doi.org/10.1007/978-981-15-9411-3_1

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thyroid carcinoma, and anaplastic carcinoma was discussed in detail. The molecular mechanisms associated with ROS-mediated thyroid disorders are consolidated. Several risk factors such as radiation exposure in the development of thyroid cancer are associated with ROS generation. The role of enzymatic antioxidants such as superoxide dismutase (SOD), glutathione peroxidase (GPx), and non-enzymatic antioxidants such as vitamin C, reduced glutathione deregulation is also discussed elaborately. This chapter is focused on consolidating the role of ROS in different thyroid disorders and to identify prospective therapeutic targets that can be used for the treatment. Keywords

ROS · Oxidative stress · Hyperthyroidism · Thyroid cancer

Introduction The discrepancy between the production of free radicals and antioxidant defense results in the oxidative stress (OS) (Betteridge 2000). The free radicals like Reactive Oxygen Species (ROS) are the molecules with minimum of one oxygen and one or more unpaired electrons in their outermost valence shell, with strong oxidizing potency (Jakubczyk et al. 2020). Mitochondria play a vital role in the production, metabolism, and signaling of ROS. It has been theoretically stated that the ROS emission can be changed via regulating the generation of ROS or by altering the ROS scavenging mechanism in the ROS production sites within mitochondria (Starkov 2008). Superoxide radicals, hydrogen peroxide (H2O2), singlet oxygen, and hydroxyl radicals are some of the examples of ROS molecules that are capable of attacking the polyunsaturated fatty acid (PUFA) molecules (Young and Woodside 2001; Lobo et al. 2010). This results in the degradation of lipids and affects the membrane fluidity, precisely known as lipid peroxidation. The lipid peroxidation ends up in morphological changes of the membrane, membranedependent signaling disruptions, nucleic acid damage, autophagy, ferroptosis, and mutagenesis. When the lipid peroxidation is highly accelerated, the rate of cell death increases or membrane flipping takes place (Park and Chung 2019). These free radicals like ROS are produced by the human system during normal aerobic conditions. In order to neutralize the effect of ROS, our human system is gifted with defensive free radical scavengers such as SOD, catalases (CAT), and GPx (Pham-Huy et al. 2008). SOD catalyzes the conversion of a superoxide into H2O2 and molecular oxygen with the help of copper, zinc, and manganese. The metals located at the active site of SOD determine its classification. CuSOD and ZnSOD are widely produced in eukaryotes and few prokaryotes. NiSOD and FeSOD are widely present in prokaryotes and MnSOD is located in mitochondria of eukaryotes (Perry et al. 2010; Azadmanesh and Borgstahl 2018). The H2O2 generated can be detoxified by CAT and GPx. They involve in the conversion of the H2O2 molecule into a water molecule and oxygen (Day 2009). If the cells are rich in

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reduced glutathione (GSH), the reaction can be taken over by GPx. The GSH is an intracellular antioxidant tripeptide containing γ-glutamic acid, cysteine, and glycine. In cells with the deficient GSH, CAT will execute the function of detoxification. Exogenous antioxidants also play a crucial role in scavenging free radicals. This may include vitamin C (Ascorbic acid) and vitamin E (Tocopherol) which are responsible in scavenging hydroxyl, superoxide radicals and degradation of lipids, respectively. There are other antioxidants such as phenolic acids, resveratrol, flavonoids, oil lecithins, and other minerals and trace elements (Pisoschi and Pop 2015). Overproduction of free radicals may end up in recruiting oxidative stress resulting in acute to chronic diseases or disorders. An increased OS results in underactive thyroid disorder (Chakrabarti et al. 2016). A study shows that inflammation due to OS will additionally reduce the deiodinase enzyme expression bringing about low triiodothyronine hormone (T3), a condition called hypothyroidism (Segni et al. 2016). Some enzyme activity is dependable on the TH stimulation such as enzyme (SOD) which upturns the TH activation besides ROS production whereas CAT and GPx enzymes inhibit the ROS production and devitalize the antioxidant activity (Villanueva et al. 2013). The role of ROS such as H2O2 is very crucial for the biosynthesis of TH. H2O2 production is a prerequisite for the production of TH. H2O2 is produced by the enzymatic action of dual oxidases 1 and 2 (DOX 1 and 2) which belong to Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase family. DOX 1 and 2 are abundantly present on the surface of thyrocytes (Karbownik-Lewińska and Kokoszko-Bilska 2012). During TH biosynthesis, thyroxine iodination is catalyzed by thyroperoxidase enzyme which requires H2O2 as an electron acceptor in endoplasmic reticulum (ER). However, excessive production of H2O2 could be deleterious to the thyroid gland and impair functioning. For instance, excessive production of free radicals is associated with the initiation and establishment of thyroid cancer (Karbownik and Lewinski 2003). The initiation of cancer is determined by several causative factors that induce ROS generation and OS. Documented pieces of evidence report that the ability of ROS to activate mitogenic signaling such as MAP kinase and PI3K leads to enhanced production of cyclins and cyclin-dependent kinases (CDK) resulting in carcinogenesis (Xing 2012). Further, ROS can potentiate the action of tumor growth factor-beta 1(TGF-β1) and matrix metalloproteases (MMP). For example, a BRAF mutant thyroid cancer microenvironment possesses increased inflammatory cytokines and MMPs. In rat hepatocytes, TGF-β1 has been shown to attenuate the suppression of SOD such as Mn-SOD, Cu-SOD, Zn-SOD, and CAT (Nucera et al. 2011). Oxidative stress is a condition caused by the imbalance of ROS production and neutralization duo to the internal and external pathological factors. The imbalance may occur in the excess production of free radicals or depleted action of enzymatic/nonenzymatic antioxidants. So, it is crucial to balance the redox homeostasis to maintain the normal physiological process. Superoxide ion (O2˙
) is commonly produced in mitochondria due to the addition of one electron to molecular oxygen (Valko et al. 2007). Complex I and complex III of electron transport chain (ETC) leak 1–3% of all electrons to generate O2˙
(Muller et al. 2004). Oxidative damage induces damage in the nucleotides of DNA which may initiates mutations

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and carcinogenesis. Activation of epidermal growth factor (EGF) receptor, vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), and cytokine receptors results in the production of ROS in non-phagocytic cells (Sundaresan et al. 1996; Chapple 1997). Moreover, ROS such as H2O2 and O2˙
potentially activates protein tyrosine kinase (PKC) by phosphorylation of tyrosine residue in immune cells such as B and T lymphocytes, macrophages, and fibroblasts (Abe and Berk 1999). In normal physiological conditions the minimum to maximum range of H2O2 is 0.001–0.7 μm. In mammalian cells, H2O2 treatment at low doses breaks the single standard DNA and produces DNA adducts whereas in high doses of H2O2 breaks the double stranded DNA (Cantoni et al. 1989). TH is essentially involved in the regulation of energy metabolism, development and differentiation, and reproduction by binding with its specific ligand on the surface of target cells. TH increases the heat production and oxygen consumption thereby; basal metabolism increases during hyperthyroidism whereas decreases during hypothyroidism. The protein synthesis and function via transcription and translation require energy which is mediated by binding of TH on the mitochondrial membrane. The plasma membrane and ER could also be considered as a binding site for TH (Wilson and Foster 1985).

Hyperthyroidism and OS Increased metabolic rate in hyperthyroidism potentially stimulates the release of ROS from mitochondria which is the primary target of free radicals (Napolitano et al. 2019). These are evidenced by several experimental studies. In vivo administration of tetraiodothyronine (T4) (Swaroop and Ramasarma 1985) and T3 (Fernandez and Videla 1993) increased the H2O2 and O2•– in liver mitochondria of experimental rats, respectively. Another in vivo study demonstrated that the administration of 10 μg of T3 per 100 g body weight for 10 days in hypothyroid rats stimulated the excess release of liver, heart, and muscle tissues (Napolitano et al. 2019). T3 administration in rats induced the free radical release from the Kupffer cells, thereby activating hepatic nuclear factor kappa B (NF-κb) for transcriptional activity of cytokines. In hypothyroid rats, liver tissues increase the mRNA expression of tumor necrosis factor alpha (TNF-α) which is known to enhance ROS generation (Tapia et al. 2003). In bal b/c female mice, treatment of T4 (12 mg/liter in drinking water) significantly increases the mRNA expression of CAT, GPx, and ROS production. Likewise, treatment of 250 μm H2O2 increases the CAT and GPx in lymphoid cells isolated from euthyroid mice. Transcription of antioxidant enzymesgenes were mediated by increased phosphorylation of nuclear factor erythroid 2–related factor 2 (Nrf2) and increased kinase activity of protein kinase C (PKC) in lymphoid cells of hyperthyroid mice (Costilla et al. 2019). T3 treatment upregulates the lectin-like oxidized low-density lipoprotein (LOX-1) which is shown to be involved in atherosclerosis mediated by increased ROS production (Balzan et al. 2017). OS-induced by T3 treatment stimulates unfold protein response (UPR) involved in ER stress (Videla et al. 2015). Kong et al. (2015) documented the influence of TH on the oxidative state in uterus. A significant increase in serum T3, T4, and estradiol was observed in hyperthyroid rats than hypothyroid rats. Similarly, nitric oxide (NO), nitric oxide

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synthase (NOS), CAT, and GPx were increased in hyperthyroid rats. Further, immunohistochemical analysis of uterine tissue revealed the expression of TH nuclear receptor α/β (TR α/β) which indicates the role of TH in the oxidative stress in the uterus (Kong et al. 2015).

Papillary Thyroid Carcinoma and OS Thyroid cancer is the most common endocrine cancer that occurs worldwide. Differentiated thyroid cancer (DTC) is a more common thyroid cancer subtype that accounts for 95% and arises from follicular epithelial cells. DTC includes papillary thyroid cancer (PTC) and anaplastic thyroid cancer (ATC). Among these types, PTC is the most common thyroid cancer and tends to metastasize into cervical lymph nodes and lungs (Cabanillas et al. 2016). Thyroid cancer initiation and development comprises several mechanisms like harboring a mutation in signaling pathways, epigenetic alterations, and environmental factors like the exposure of thyroid gland to radiation. In TC, the mitogen-activated protein kinase (MAPK) pathway is commonly mutated which regulates cell proliferation. For the initiation of thyroid cancer, the MAPK pathway downstream molecule such as RAS, RAF, MEK, and ERK plays a crucial role whereas in the cancer progression PI3K-mTOR pathway is involved. During the advanced stages of TC, the tumor suppressor P53 gene is also reported to be mutated which is failed to regulate cell cycle, DNA damage and apoptotic machinery (Cabanillas et al. 2016). The thyroid gland is more vulnerable to radiation because of its iodine absorbing nature and location of gland. Exposure of radiation at a younger age increases the risk of getting TC especially PTC (Liu et al. 2017). In PTC, the common mutation is B-type Raf kinase (BRAF) and accounts for more than 90% in cancer individuals. In BRAF mutations, the transverse mutation (thymidine to adenine) in exon 15 at 1799 nucleotide leads to the replacement of valine to glutamic acid at 600th position (BRAFV600E) (Tang and Lee 2010). In thyroid rat cells, the overexpression of BRAFV600E mutation increases the migration and invasion was evidenced using matrigel assay (Mitsutake et al. 2006). BRAFV600E mutation is associated with the increased expression of matrix metalloproteinases (MMPs) such as MMP3, MMP9, and MMP13 (Tang and Lee 2010). Telomerase reverse transcriptase (TERT) maintain the end of the telomerase in chromosomes and has been reported to be highly expressed in cancer patients which involved in proliferation. The localization of TERT in mitochondria was higher in tumor tissues. The intracellular H2O2 and GPx levels in PTC tissues were significantly increased than normal tissues (Muzza et al. 2016; Stanley et al. 2016). Preoperative PTC patients reported to have higher 8-hydroxy-20 -deoxyguanosine (8-OHdG) in serum which is an indicator of OS induced DNA damage than postoperative and healthy individuals (Tabur et al. 2015). A comparative study of oxidative status between Graves’ disease (GD), Hashimoto thyroiditis (HT), and PTC showed the increased activity of SOD and malondialdehyde levels (MDA) in plasma samples. Also, the increased activity of CAT and the decreased activity of GPx were observed in the plasma of PTC patients (Lassoued et al. 2010). Gerić et al. (2016) also reported the altered oxidative profile in thyroid disease individuals. Increased elevation of

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MDA ((40.66  16.79 μmol/L) and protein carbonyl (11.27  3.53 μmol/L), however, decreased the level of GSH (76.04  33.53 μmol/L), and decreased activity of CAT (28.69  18.49 U/mL) was detected in thyroid disease individuals compared to healthy controls (Gerić et al. 2016). The translocator protein (TSPO) involved in the regulation of mitochondrial membrane potential, which supports OS induced apoptosis, also have been reported to be expressed in various types of carcinomas. OS induced by H2O2 in TSPO silenced thyroid cells loses its mitochondrial membrane potential, whereas in TPSO treated cells potentially attenuated the OS induced depolarization. Therefore, TSPO expression could prevent the oxidative stress induced apoptosis in PTC cells (Klubo-Gwiezdzinska et al. 2012). Nrf2 is a transcription factor mediated by OS and has been shown to be elevated in PTC tissues than benign and normal tissues. Also, the abundant expression of oxidative stress 4-hydroxynonenal (4-HNE) was observed in PTC tissues representing the oxidative stress (Ziros et al. 2013). TGF-β aberrant expression has been reported in various cancers including PTC and could repress the expression of thyroglobulin, thyroperoxidase, and sodium/iodide symporter (Nicolussi et al. 2003). BRAFV600E expression stimulates the bioactive TGF-β production and leads to the epithelial mesenchymal transition (EMT), migration, and invasion in thyroid cells and mice model (Riesco-Eizaguirre et al. 2009; Knauf et al. 2011). BRAFV600E regulated the expression of NADPH Oxidase 4 (NOX4) encoded for NADPH oxidase enzyme via TGFβ-Smad3-dependent pathway and leads to the generation of ROS in thyroid cells. These free radicals downregulate the expression of sodium/iodide symporter in thyrocytes. Therefore, this experiment clearly shows that the BRAFV600E expression induces the ROS generation through NADPH oxidases (Azouzi et al. 2017). Under hypoxic conditions, transcriptional and translational increased expression of NOX4 has been reported and the interaction between Hypoxia inducible factor -1α(HIF-1α) and NOX4 was evidenced by chromatin immunoprecipitation assay. These results suggest the role of hypoxia in NOX4-mediated ROS generation (Diebold et al. 2010). Silencing of NOX4 in TPC-1 cells reduced the cell proliferation, mitochondrial ROS generation, and HIF-1α destabilization and leads to the diminishment of glycolysis, thereby reducing the cancer cell growth (Tang et al. 2018). In the tumor microenvironment, NOX4 gene recruiting tumor-associated M2-macrophages by ROS/PI3K signaling produces cytokines such as Interleukin-8 (IL-8), Colony stimulating factor (CSF-1), Chemokine (C-C motif) ligand 7 (CCL7), and Vascular endothelial growth factor C (VEGF-C) which supports the tumor growth (Szanto et al. 2019). In PTC individuals, estrogen receptor α (ER α) expression was highly increased compared to adjacent thyroid tissue. Besides, ER α potentially activates ERK1/2 signaling and enhances the ROS production which plays a crucial role in autophagy of PTC cells (Fan et al. 2015).

Medullary Thyroid Carcinoma and OS Medullary thyroid carcinoma is usually a rare form of cancer and it is an infiltrative tumor containing solid cell nests of non-cohesive cells along with amyloid accumulations (Thomas et al. 2019). This type of cancer has upregulated levels of calcitonin,

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produced from C cells or parafollicular cells, which have the activity of reducing calcium levels. Calcitonin levels have a functional role in the assessment of tumor proportions and development (Toledo et al. 2009). Sporadic carcinoma is delineated to be 48–86% and hereditary carcinoma types to be about 14–52% in patients with medullary thyroid carcinoma (Stamatakos et al. 2011). RET (Rearrangement during transfection) gene is a protein-coding gene which plays a consequent role in the human neuroendocrine tumors likely in medullary thyroid carcinoma and mutations in the amino acid cysteine and leads to an unpaired amino acid form along with a change in a position resulting to the activation of oncogene RET (Taccaliti et al. 2011). A biomarker called malondialdehyde (MDA) is used in analyzing OS and it is the end product of lipid peroxidation where its increase may cause high levels of free radicals in the thyroid carcinoma (Hosseini-Zijoud et al. 2016; Stanley et al. 2016). The RET tyrosine kinase following mutational activation has been resulted in triggering the initial stage of tumor progression mainly in medullary thyroid carcinoma (Ha et al. 2000).

Anaplastic Thyroid Carcinoma and OS Anaplastic thyroid carcinoma (ATC) is a primitive type of thyroid cancer with an aggressive clinical course resulting in early malignancy. These undifferentiated follicular cells signify 2–5% of all the thyroid tumors and they have a poor survival rate of approximately 3 months (O’Neill and Shaha 2013; Seshadri 2019). About 14–39% of mortality rate have been determined to occur due to this rare anaplastic cancer. The origination of ATC occurs in follicular cells of thyroid gland due to de novo or preexisting well-differentiated thyroid cancer (WDTCs) (Rashid et al. 2019). This conversion of well-differentiated cells into undifferentiated cells involves a wide spectrum of genetic alterations and varied cell signaling resulting in cytological and histological changes. The tissue dissection of ATC was found to be highly necrotic with a huge mass of hemorrhage. These cells infiltrate the adjacent tissues, most often thyroid gland parenchyma resulting in the structural deformity of the neck. Histologic configurations of ATC are composed of the giant spindle, squamoid, and polymorphic multinucleated cells with bizarre hyperchromatic nuclei. Clinical researchers have shown that ATC cells resemble the cells of undifferentiated pleomorphic sarcoma, giant bone tumor, and fibrosarcoma, and a trace of papillary or follicular thyroid carcinoma was also found in few ATC patients (Deeken-Draisey et al. 2018). ATC patients can be diagnosed via fine-needle aspiration biopsy (FNAB), ultrasonography, intraoperative frozen section, tomography, magnetic resonance imaging (MRI), and F-fluorodeoxyglucose positron emission tomography (FDG-PET). Each diagnosis method comes up with unique way of determining the ATC presence and progression by defining tumor morphology, cytological findings, etc. (Takashima et al. 1990; Suh et al. 2013; Loh and Zulkiflee 2018). Various genetic variations are responsible for ATC formation and progression. More than 90% of thyroid cancer associates with the MAPK pathway mutation along with the genetic aberrations in the Ras, BRAF, PIK3CA, TERT, CTNNB1, p53, PTEN, EGFR, PDGFR, VEGFR, KIT, and MET genes (Ragazzi et al. 2014). In

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most of cancer cells, an increased amount of ROS production results in OS leading to apoptotic cell signaling (Yokoyama et al. 2017). Zheng et al. performed an experiment using THJ-16 and THJ-11 T cells which are derived from ATC malignant patients. When those cells were treated with resveratrol and after incubating for 48 h, it showed subsequent elevation in the ROS level in THJ-16 cells along with the mitochondrial swelling and declining of cristae and the ROS production was nullified in the THJ-11 T cells. The apoptotic key players, caspase 3 and caspase 9, were found to be increased in THJ-16 cells. This research speculates that resveratrol has the potency to influence OS in the ATC cell lines and can cause ATC cell retardation and apoptosis. The resveratrol had been targeting and inhibiting the production of SOD and CAT antioxidant enzyme (Zheng et al. 2018). The fact why THJ-11 T is resistant to resveratrol remains unknown. Human thyroid cancer cell line ARO was used in another study and it was carried on with the juglone drug treatment in combination with ascorbate. The drug combination increased the OS by overproduction of free radicals in the ATC cells, and it was shown to directly inhibit the SOD and CAT enzymes and the higher amount of H2O2 leads to the increased activation of c-jun N-Terminal kinase (JNK), p53, DNA damage. The increased free radicals can also aid in the membrane attack of mitochondria leading to apoptosis and necrosis via caspase 3 and caspase 9 activation (Gaikwad et al. 2018).

Conclusion Considering thyroid cancer, the known established cancer risk factors are few like radiation exposure and family history of thyroid cancer. Even though the involvement of OS is a known risk factor associated with various thyroid diseases, the source of oxidative stress/conditions in thyroid cancer remains to be investigated.

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Skin Cancer Induced by Pollution-Mediated ROS Karen E. Burke

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Solar Ultraviolet (UV) Radiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Synergy: UVA Photo-Chemo Pollution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ozone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Particulate Matter (PM) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Tobacco Smoke . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Aryl Hydrocarbon Receptor (AHR) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Aryl Hydrocarbon Receptor (AHR) and Skin Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Squamous Cell Carcinoma (SCC) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Basal Cell Carcinoma (BCC) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Malignant Melanoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Topical Antioxidants to Protect from Environmental Pollution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cross-References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract

The incidence of skin cancer is indeed alarming. More people are diagnosed with skin cancer each year than all other cancers combined. In the USA, one in five will develop skin cancer by the age of 70; each year, more than 24,000 Americans die of skin cancers – the most lethal of which are increased by environmental pollution. Physicians have realized for decades that UVB initiates skin cancer, but only recently have medical researchers learned that UVA interacts with airborne pollutants synergistically to initiate and promote skin cancer. Also, ubiquitous environmental pollutants – including ozone, polycyclic aromatic hydrocarbons (PAHs), nitrogen oxides, volatile organic compounds (VOCs) – generate reactive oxygen species (ROS) which oxidize epidermal lipids, inducing a cascade of cellular stress reactions that can initiate skin cancer. Furthermore, environmental K. E. Burke (*) Department of Dermatology, The Mount Sinai Icahn School of Medicine, New York, NY, USA © Springer Nature Singapore Pte Ltd. 2022 S. Chakraborti et al. (eds.), Handbook of Oxidative Stress in Cancer: Mechanistic Aspects, https://doi.org/10.1007/978-981-15-9411-3_2

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toxins are carried on the surface and within the core of particulate matter (PM). This PM can be absorbed cutaneously to deep epidermal layers and can enter the dermis through hair follicles as well as via blood flow after respiratory pulmonary absorption. The xenobiotic pollutants act by binding to the aryl hydrocarbon receptor (AHR), a ligand-activated transcription factor found in all types of skin cells – keratinocytes, melanocytes, fibroblasts, and dermal dendritic cells. For full protection from the environmental damage (extrinsic aging and skin cancer), certainly sunscreens that filter UVA as well as UVB are necessary, but sunscreens are not enough! To combat the dangerous oxidative damage induced by airborne pollutants, correctly formulated topical antioxidants can effectively protect. Keywords

Skin cancer · Air pollution · UV radiation · Polycyclic aromatic hydrocarbons (PAH) · Ozone · Particulate matter · Aryl hydrocarbon receptor (AHR) · Reactive oxygen species (ROS) Abbreviations

4-ABP ARNT AHR AHRR BaP BCC bFGF BPDE CAP CO COX-1,
2 CPD cSRC CYP EGFR ERK-1,-2 FICZ GPx HNE H2O2 HSP-27,-32,-70,-90 ICAM IcdP I‘-1α,-1b,-8 LDH MAPK

4-aminobiphenyl AHR nuclear translocator aryl hydrocarbon receptor AHR repressor benzo[a]pyrene basal cell carcinoma basic fibroblast growth factor BaP diol epoxide concentrated ambient particles carbon monoxide cyclooxygenase-1,
2 cyclobutane pyrimidine dimer “cellular sarcoma,” photo oncogene tyrosine-protein kinase cytochrome P450 epidermal growth factor receptor extracellular signal-regulated kinase-1,-2 6-formylindolo[3,2b]carbazole glutathione peroxidase 4-hydroxy-2-nonenal (protein adduct) hydrogen peroxide heat shock proteins-27,-32,-70,-90 intercellular adhesion molecule indole [1,2,3-cd]pyrene interleukin-1α,-1b,-8 lactate dehydrogenase mitogen-activated protein kinases

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MCP-1 MED MMP NF-κB NNN NOx NO2 NRF2 8-OHdG O3 PAH PM PM10, PM2.5 ppm PR PRE PRP PUVA RHE ROS SCC SOD-1 TCDD TGF-β TIMP TNF-α,-β Trp UFP UV VCAM VOC WHO

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monocyte chemoattractant protein-1 minimal erythema dose matrix metalloproteinases nuclear factor of kappa-light-chain polypeptide gene enhancer in B-cells N-nitrosonornicotine nitrous oxide nitrogen dioxide nuclear transcription factor (erythroid-derived 2)-like2 8-hydroxy deoxyguanosine ozone polycyclic aromatic hydrocarbons particulate matter PM with diameter 10 μm, 2.5 μm parts per million progesterone receptor progesterone response element progesterone receptor + progesterone complex psoralen plus UVA reconstructed human epidermis reactive oxygen species squamous cell carcinoma superoxide dismutase-1 tetrachlorodibenzo-p-dioxin transforming growth factor β tissue inhibitors of metalloproteinases tumor necrosis factor-α,-β tryptophan ultrafine particles (1.0 cm diameter) BCCs and “giant” BCCs was noted in smokers (Smith and Randle 2001). Smoking may increase aggressive growth, particularly of the highly invasive morpheaform type of BCCs (Erbagci and Erkilic 2002). Overall, the incidence of melanoma in smokers is about double that of nonsmokers, though the data is conflicting. Some research proposes that smoking has no influence on the risk of melanoma (De Hertog et al. 2001), while another study proposes that smoking is protective, especially in lowering the occurrence of acral melanomas (Green et al. 1999). This possible protection may be due to the nicotine which can act as a precursor to melanin synthesis and can irreversibly bind to melanin (Claffey et al. 2001). Nicotine accumulates in tissues containing melanin, possibly blocking the binding of strong carcinogens in tobacco such as N-nitrosonornicotine (NNN) and 4-(methyl nitrosamino)-I-(3-pyridyl)-I-butanone (NNK) which also have a strong affinity for melanin. On the other hand, nicotine is known to increase tumor vascularity (which enhances growth) (Heeschen et al. 2001) and through fibroblasts, alters the stroma in ways that enhance tumor growth and invasion (Coppe et al. 2008). Retrospective analysis of data from patients with metastatic melanoma demonstrated that on initial diagnosis (1) smokers presented with more metastases, (2) smokers showed more visceral metastases, and (3) smokers developed more metastases within the first 2 years of diagnosis (Smith and Fenske 1996). Significantly, more male nonsmokers were free of melanoma 5 years after diagnosis than were nonsmokers (Smith and Fenske 1996).

The Aryl Hydrocarbon Receptor (AHR) The aryl hydrocarbon receptor (AHR) is a ligand-activated cytoplasmic transcription factor which functions in the detoxification of pollutants. The AHR recognizes and binds to the pollutant, then signals gene transcription to synthesize enzymes that metabolize the xenobiotic substances (Larigot et al. 2018). The AHR is highly expressed on all types of skin cells – keratinocytes, fibroblasts, and melanocytes – and on regulatory T cells and dendritic cells. Very many kinds of both exogenous and endogenous ligands can bind to the AHR, each turning on different specific genes that determine the precise cellular metabolic response. The AHR can stimulate tumor repressor genes that activate antiproliferative mechanisms or, alternatively, can stimulate cancer promoter genes to enhance cell proliferation. The AHR can regulate cell adhesion and extracellular matrix interactions. It is involved in maintaining the

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epidermal barrier function, in altering pigmentation by effecting melanocyte proliferation and melanin synthesis in response to exogenous xenobiotics, and in regulating skin immunity and immune suppression. Independent of xenobiotic metabolism, the AHR plays a key role in cellular physiology, especially in embryo and organ development. The AHR resides in the cellular cytoplasm as a multiprotein complex composed of heat shock proteins (such as HSP-90), the AHR interacting protein, the co-chaperone p23 protein, and soluble tyrosine kinase (cSrc) (Vogeley et al. 2019). Ligand binding leads to conformational change with dissociation of the complex and translocation to the nucleus where AHR forms a dimer with AHR nuclear translocator (ARNT), as shown in Fig. 3. This dimer then binds to genes that induce transcription of RNA polymerases. The prototype gene target codes for the monooxygenase cytochrome P450 family (CYP1A1, 1A2, 1B1) – drug-metabolizing enzymes (which act primarily to detoxify hydrocarbons) used as biomarkers for AHR activity. The AHR can selectively induce transcription of other genes including NRF2 (which activates antioxidant responses), NF-κB (leading to inflammatory cytokines), immune modulators, proliferation inducers, cell cycle modulators, protein kinases, and estrogen receptor signaling. Another AHR key target gene is the AHR repressor (AHRR) which cannot bind ligands, but rather transcribes co-repressors to modulate AHR, thereby discontinuing the subsequent transcription of other AHR-dependent genes (Larigot et al. 2018). The other inhibitory regulation of AHR results from ligand-AHR being expelled from the nucleus and degraded in the cytoplasm by the proteasome. Both of these mechanisms prevent overstimulation by the pollutant ligand-AHR complex. As shown in Fig. 3, dissociation of the initial cytoplasmic AHR complex after ligand binding has further consequences. The co-chaperone p23 becomes a chaperone to the cytoplasmic protein the progesterone receptor (PR): This receptorprogesterone (PRP) complex moves into the nucleus to bind to the progesterone response element (PRE), leading to transcription of progesterone-induced genes. The AHR-complex co-chaperone p23 also becomes a chaperone to heat shock proteins. The family of heat shock proteins are expressed after cellular stress – including not only after burns (hence their name) but also after exposure to cold or UV and during wound healing or tissue remodeling. These heat shock proteins are chaperoned by p23. The chaperone cSRC is released with subsequent binding to the epidermal growth factor receptor (EGFR) which activates the oncogene cSRC protein kinase to induce cellular proliferation and carcinogenesis. cSRC also activates both focal adhesion kinase that acts to disrupt cellular focal adhesion points and mitogen-activated protein kinases (MAPKs) that regulate inflammation (Larigot et al. 2018). Among the most potent ligands are the PAHs of urban air pollution and tobacco smoke as well as tetrachlorodibenzo-p-dioxin (TCDD), a component of pesticide (the main contaminant of Agent Orange) which can give exposure through diet as well as through cutaneous contact. Also skin-resident indole metabolites produced by certain cruciferous vegetables (broccoli and Brussels sprouts) as well as by bacteria (e.g., nonpathogenic Staphylococci) and yeasts (such as Malassezia furfur)

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Fig. 3 The aryl hydrocarbon receptor (AHR). In the cytoplasm, the AHR resides within a multiprotein complex with chaperone molecules, including tyrosine kinase cSRC, heat shock protein-90, and the p23 protein. Binding of a xenobiotic ligand (or the UVA-induced ligand metabolite (Lm)) to the AHR results in conformational changes which dissemble the multimolecular complex so that the ligand-AHR entity translocates into the nucleus where it dimerizes with ARNT. The ligand-AHR-ARNT complex induces transcription of target genes. The particular genes transcribed are determined by each specific xenobiotic ligand. Overstimulation by the ligandAHR complex is prevented (1) by RNA transcription of the AHR repressor (AHRR) gene which leads to synthesis of co-repressors that bind to inactivate further AHR-ARNT transcription, and (2) by expulsion from the nucleus followed by degradation in the cytoplasm. The dissociation of the original cytoplasmic AHR complex after ligand binding leads to further elegant biologic cascades involving heat shock proteins co-chaperoned by p23, activation of the progesterone receptor (PR) by p23 binding, and binding of cSRC to the epidermal growth factor receptor (EGFR) to activate the cSRC protein kinase which induces carcinogenesis. (Modified from Vogeley et al. (2019) with consent of authors)

bind to the AHR (Sowada et al. 2014). In the epidermis, UVB is absorbed by the aromatic amino acid tryptophan to form photo-products, particularly 6-formylindolo (3,2-b)carbazole (FICZ) and indoles, which bind to AHR with high affinity to induce signaling as described above.

The Aryl Hydrocarbon Receptor (AHR) and Skin Cancer The regulation of cellular physiology by the AHR is highly complex, especially with respect to influencing the initiation and propagation of skin cancer, as excellently reviewed by Vogeley et al. (2019). Functioning to protect against xenobiotic

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pollutants, the AHR influences cellular proliferation, adhesion, and migration as well as extracellular matrix integrity.

Squamous Cell Carcinoma (SCC) Both the initiation and the promotion of SCC by environmental pollutants are highly regulated by AHR signaling. To invoke the AHR, UVB radiation energy is absorbed by tryptophan to generate FICZ and other photoproducts. These metabolites serve as chemical ligands that bind to the AHR to activate gene transcription of proteins which direct downstream signaling. The AHR-FICZ complex upregulates genes coding for CYP1. CYP1A1 in turn initiates rapid metabolism of FICZ to decrease the AHR-signaling as soon as UVB radiation is terminated, thus limiting exposure that would increase carcinogenesis. On the other hand, intense UVB radiation further triggers CYP1 to generate ROS with subsequent oxidative damage and enhancement of SCC growth. The AHR-FICZ also leads to gene transcription of COX-2 that generates inflammation as well as anti-apoptotic metabolites which promote SCC tumor growth. MMPs are activated with breakdown of extracellular collagen and elastic tissue, effects known to enhance tumor cell migration and invasion. Studies from the Krutmann and Haarmann-Stemmann laboratory (Vogeley et al. 2019) showed that after UVB-exposure of epidermal keratinocytes, AHR inhibited nucleotide excision repair of DNA CPDs and apoptosis of mutated cells. AHR activation and signaling resulted in proteolysis of tumor suppressor protein p27KIPI and cyclindependent kinase inhibitor – both normal cellular defense mechanisms that are thus suppressed by AHR. These researchers demonstrated that inhibition of AHR in UVB-exposed Skh-1 hairless mice increased nucleotide excision repair of CPDs, stabilized cutaneous levels of suppressor protein p27KIPI, and increased apoptosis of injured keratinocytes, thereby decreasing SCC incidence by 50%. This certainly demonstrates that the AHR is an important determinant of cutaneous carcinogenesis. Also since immunosuppression is triggered by CPDs, DNA mutation repair in AHR-null mice would increase immune response, further lowering SCC incidence. These scientists further demonstrated that AHR activation by both UVB and chemical xenobiotic ligands changed antigen-presenting dendritic cells from stimulating to regulating, so that regulatory T cells were induced without signaling by DNA damage, thereby enhancing the immunosuppression induced by the AHR. SCCs are initiated not only by UVB but also by exposure to concentrations of PAHs higher than those measured in urban environments, concentrations only encountered with industrial exposure or by smoking tobacco with exposure to nitrosamines and aromatic amines as well as PAHs – all of which activate AHR signaling. AHR activates the cutaneous monooxygenases CYP1A1, CYP1A2, and CYP1B1. CYP1A1 leads to metabolism of BaP to highly carcinogenic epoxides, so AHR-null mice have no BaP cutaneous carcinogenicity. Similarly gene-targeting inactivation of epidermal ARNT prevents BaP-induced skin cancer (Shimizu et al. 2000). AHR-PAH stimulation of CYP1B1 metabolizes PAH to genotoxins, a

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reaction that is not altered in AHR-null mice. Interestingly, keratinocytes express CYP1A1 more than CYP1B1, whereas CYP1B1 is more predominantly expressed by Langerhans cells (Modi et al. 2012). CYP1B1 is controlled not only by AHR but also by other transcription factors such as the estrogen receptor-α. As described above, the cellular and therefore clinical responses to exposure to environmental pollutant ligands which influence cutaneous SCC development by activation of the AHR are indeed complex, depending upon the precise ligand and its concentration, the cell type most exposed, the possible synergistic effect of UVA and/or UVB with the specific pollutants, the timing of that UV exposure with respect to the timing of xenobiotic contact, and contributions by other transcription modulators. The net result is indeed a balancing act!

Basal Cell Carcinoma (BCC) Interestingly, although cutaneous BCCs are definitely caused by UV exposure, there is no reported association with AHR in the occurrence of BCC initiation or promotion. Thus BCC are initiated directly by UVB-induced genetic mutations in keratinocyte basal cells. UVA contributes to BCC proliferation by generating ROS which oxidize DNA to form mutagenic 8-OHdG, by directly inducing mutations in the tumor suppressor p53 gene in the stem cell-rich basal layer of the epithelium, by immunosuppression, and by activating MMP destruction of extracellular matrix. The AHR has no known interaction with the sonic hedgehog signaling and other BCC signaling pathways. As of now, no environmental pollutants are implicated in influencing BCC initiation, though possibly cigarette smoking increases the incidence of large and “giant” BCCs (Smith and Randle 2001) and renders BCC growth more aggressive (Erbagci and Erkilic 2002).

Malignant Melanoma Contradicting effects have been reported in studying the effects of AHR activation and malignant melanoma. Some in vitro melanoma cell lines are shown by gene expression to have highly expressed AHR (O’Donnell et al. 2012). TCDD exposure was shown to increase incidence of melanoma in Vietnam War veterans (Akhtar et al. 2004), probably as a tumor promoter, increasing proliferation of pre-initiated malignant melanoma cells. In vitro, exposure of human A2058 melanoma cells to TCDD increased invasive growth by activating MMPs (MMP-1, MMP-2, and MMP-9) (Villano et al. 2006). Tumor transplantation studies in mice with a B16 melanoma cell line confirmed that the AHR influenced microenvironmental stroma by signaling activation of mediators that induce angiogenesis and cancer cell motility (Contador-Troca et al. 2013). However, the AHR had anti-tumorigenic activities in the B16 tumor cells themselves and in other in vitro experiments with highly migratory and invasive C8161 malignant melanoma cells (Contador-Troca et al. 2013). Currently research using targeting therapies to modulate AHR

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expression are being investigated to inhibit malignant melanoma and curtail metastasis (Vogeley et al. 2019).

Topical Antioxidants to Protect from Environmental Pollution Although sunscreen that filters not only UVB but also UVA does protect against solar-induced skin cancer as well as against the recently recognized, synergistic damage induced by simultaneous exposure to UVA and environmental pollutants, sunscreen alone cannot prevent the damage induced by the airborne toxins carried on PM, as well as by the VOCs, PAHs, ozone, and many other chemicals liberated from cigarette smoke and fossil fuels. This damage is largely oxidative, so clearly antioxidants provide some protection. All layers of the skin’s surface have endogenous antioxidants, but these are depleted by exposure to UV and urban pollutants. The epidermal concentration of vitamin C (L-ascorbic acid) is depleted by 34% and vitamin E (d-α-tocopherol) by 25% after exposure to only 1.6 minimal erythema dose (MED) of UVB (Shindo et al. 1994) with surface stratum corneum levels of vitamin E decreasing by 49% and 77% after exposure to only 0.3 and 0.5 MED, respectively (Valacchi et al. 2000). Exposure to high levels of O3 (10 parts per million (ppm)) decreases epidermal vitamin C by 55% and vitamin E by 25% (Thiele et al. 1997), and Valacchi et al. (2000) showed that O3 levels of 0.5 or 1.0 ppm, comparable to levels in polluted cities, diminished stratum corneum vitamin E by 23% and 54%, respectively, with simultaneous exposure to both UV (MED ¼ 0.3) and O3 (0.5 ppm), thus synergistically lowering stratum corneum vitamin E levels even more. Probably these epidermal antioxidants are depleted as they act to protect the skin with the surface stratum corneum as the most exposed, suffering the maximal decrease. The optimal way to deliver high concentrations of antioxidants to the skin is by topical application. Though oral intake does raise levels in the skin, topical application of correct formulations can increase the concentration of vitamin C by a factor of 27–40 more than attained by high oral intake (Pinnell et al. 2001), vitamin E, by 12-fold (Burke et al. 2000), and selenium, by eight-fold (Burke 2017). Since antioxidants are very labile molecules, there is difficulty in creating a formulation that (1) maintains stability for the shelf-life required after manufacturing, packaging, and storage before sale and application; (2) is absorbed transepidermally; and (3) is active as an antioxidant in epidermal and dermal layers of the skin. Vitamin C is often provided for topical application as ascorbyl-6-palmitate or magnesium ascorbyl palmitate and vitamin E, as vitamin E acetate-esters. None of these esters are absorbed or metabolized by the skin to act as antioxidants. Furthermore, topical vitamin E acetate has been reported to cause urticaria and one case of erythema multiforme. Also only d-α-tocopherol – one of the 32 isomers of vitamin E (eight “dl” isomer configurations and four α, β, γ, δ forms) – is effective when applied topically (Burke et al. 2000). High concentrations are required, optimally 15–20% L-ascorbic acid and 2–5% d-α-tocopherol. Of the multiple valence forms of selenium in nature, the optimal form of the antioxidant selenium that is absorbed and

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active when delivered topically is the natural biologic L-selenomethionine at concentrations of 0.02–0.05%. (“Selenium-yeast” does contain selenomethionine, but the exact concentration is not precise.) There is extensive literature demonstrating that formulations of L-ascorbic acid, d-α-tocopherol, and L-selenomethionine do protect from the UV-induced skin damage of erythema, tanning, premature aging (hyperpigmentation and rhytides) and skin cancer, as reviewed by Burke (2017). New formulations combining antioxidants (such as vitamin C (15%) + α-tocopherol (1%)) and/or adding other antioxidants (such as the powerful plant antioxidant ferulic acid (0.5%)) increase the stability and shelf-life of the formulation, reinforcing longer-term activity of the topical product. These combinations also synergistically enhance efficacy in protecting against UV skin damage – by four-fold with L-ascorbic acid combined with α-tocopherol and by eight-fold when ferulic acid is added to the vitamin C + E serum (Murray et al. 2008). These combinations as well as another new formulation containing L-ascorbic acid (10%) and ferulic acid (0.5%) with another plant antioxidant phloretin (2%) have been shown by Valacchi et al. (2015) to prevent O3-induced damage to in vitro human keratinocytes. Pretreatment with each of these antioxidant combinations for 24 h prior to O3 exposure decreases the cytotoxicity, lowers cell proliferation, decreases formation of carbonyl protein and HNE adducts, and lowers ROS production induced by O3. There is also inhibition of translocation of NF-κB to the nucleus, thereby decreasing the release of inflammatory cytokines. Increased NRF2 activation protected against O3-induced oxidative stress. Protection by pretreatment with topical antioxidant formulations was similarly demonstrated in reconstructed human epidermis (RHE) (Valacchi et al. 2017). In both experiments, the formulation containing the α-tocopherol had greater efficacy than that with phloretin. Other new combination formulations using liposomes and microemulsions are currently under investigation (Burke, unpublished data 2020).

Conclusion Sixty-eight percent of the world’s population (and 85% of Americans) live in evermore crowded urban environments. We must protect especially our skin against the ubiquitous airborne pollutants (including O3, PAHs, VOCs, and PM) that cause direct damage by generating ROS which stimulate a cascade of inflammatory reactions that can lead to skin cancer. Also, the synergistic interaction by simultaneous exposure to solar UV and pollutants directly incites cellular genetic mutations and/or oxidation causing skin cancer. The stepwise development of environmentally induced skin precancers and cancers (from initiation to promotion to recognition and suppression by the body’s immune response) is, to a great extent, regulated by AHR-dependent reactions. By understanding the cellular and molecular mechanisms involved in processing xenobiotic insults (both from UV radiation and from airborne chemicals) by the AHR, we are learning how to inhibit, to treat, and even to prevent most skin cancers, the majority of which are indeed environmentally induced.

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Promising new research on topical antioxidants shows protection not only against UV-induced skin cancer but also against other urban environmental insults and against the dangerous synergy of simultaneous exposure to UV and pollutants. Other new topical antioxidant formulations with different antioxidants such as lycopene (Zhou et al. 2019), genistein (Wei et al. 2003), plant-derived phenolic compounds (such as cocoa, green tea, grape, and pomegranate) (Boo 2019) and some marine algae are currently being studied to inhibit ROS production and possibly modulate the AHR processing and downstream cascades.

Cross-References ▶ Benzo(a)Pyrene-Induced Oxidative Stress During Lung Cancer and Treatment with Baicalein ▶ Impact of Environmental and Occupational Exposures in Reactive Oxygen Species-Induced Pancreatic Cancer ▶ Oxidative Stress in Cancer ▶ Oxidative Stress, Inflammasome, and Cancer ▶ Phytocompounds-Based Approaches to Combat Oxidative Stress in Cancer ▶ Phytoestrogens Modulate Oxidative Stress ▶ Preventive Role of Carotenoids in Oxidative Stress-Induced Cancer ▶ Vitamin C in Cancer Management: Clinical Evidence and Involvement of Redox Role Acknowledgments The author thanks Xueyan Zhou, MD, MS, for research assistance and Heather Nolan, MA, for excellent artistic rendition of the figures, research assistance, editing, and typing. Conflicts of Interest The author declares no conflict of interest with any aspect of the publication.

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Roles of β-Glucans in Oxidative Stress and Cancer

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Eveline A. I. F. Queiroz, Paˆmela Alegranci, Aneli M. Barbosa-Dekker, and Robert F. H. Dekker

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Carcinogenesis and Cachexia-Anorexia Syndrome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Risk Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cancer Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Oxidative Stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sources and Effects of Reactive Oxygen Species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ROS and Antioxidant Mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cancer and Oxidative Stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . β-Glucans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Structural Characterization of β-D-Glucans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Anticancer Effects of β-Glucans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . β-Glucans, Oxidative Stress, and Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract

Cancer is a multifactorial disease characterized by an uncontrolled growth of cells that can invade other tissues causing metastasis. Its incidence is increasing at an alarming rate worldwide, and is the second leading cause of death in the world,

E. A. I. F. Queiroz (*) · P. Alegranci Núcleo de Pesquisa e Apoio Didático em Saúde (NUPADS), Instituto de Ciências da Saúde, Câmpus Universitário de Sinop, Universidade Federal de Mato Grosso, Sinop-MT, Brazil A. M. Barbosa-Dekker Beta-Glucan Produtos Farmoquímicos EIRELI, Universidade Tecnológica Federal do Paraná, Câmpus Londrina, Londrina-PR, Brazil R. F. H. Dekker Universidade Tecnológica Federal do Paraná, Programa de Pós-Graduação em Engenharia Ambiental, Câmpus Londrina, Londrina-PR, Brazil © Springer Nature Singapore Pte Ltd. 2022 S. Chakraborti et al. (eds.), Handbook of Oxidative Stress in Cancer: Mechanistic Aspects, https://doi.org/10.1007/978-981-15-9411-3_3

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being considered a public health problem. Several factors like genetics, hormonal, radiation, viral, chemicals, obesity, and oxidative stress can all contribute to the development of cancer. Furthermore, studies have demonstrated that oxidative stress is an important factor for cancer progression and therapy. Oxidative stress is a state wherein high levels of reactive oxygen species (ROS) and free radicals are produced, and is characterized by an imbalance between antioxidant defense and the levels of prooxidant agents produced. ROS can contribute to carcinogenesis since they can stimulate cancer initiation promoting DNA mutations, damage to the cells, and activate pro-oncogenic signaling pathways. On the other hand, high levels of ROS can contribute to cancer cell death, promoting apoptosis and necrosis. β-Glucans, such as the (1 ! 3)-β-D-glucans, have been described as effective in treating dyslipidemia, diabetes, cardiovascular-related diseases, and cancer. Studies have demonstrated that β-glucans present direct and indirect antitumoral effects that can be mediated by the action of an antioxidant or prooxidant. In this chapter, we provide an overview of the relationship between cancer, oxidative stress, and β-glucans, and discuss the effects of β-glucans on conditions of cancer and oxidative stress. Keywords

Cancer · Oxidative stress · ROS · Antioxidants · β-Glucans · (1 ! 3)(1 ! 6)-βD-Glucans · Botryosphaeran

Introduction Cancers are multifactorial diseases as several factors, such as environmental exposure (chemicals, radiation), obesity, diet, and oxidative stress, are known to contribute significantly to tumor development through acting in specific phases of carcinogenesis (WHO 2020; Oliveira et al. 2007). Oxidative stress is characterized by an imbalance between the generation of reactive oxygen and nitrogen species (ROS; RNS), and the antioxidant defenses when the levels of ROS/RNS are higher relevant to the action of antioxidants. These reactive species can act in promoting chromosomal abnormalities, genetic instability, and damage to nucleic acids, proteins, and lipids, which consequently can activate different signaling pathways that contribute to cell proliferation and decrease apoptosis, leading to the development of several diseases, including cancer (Moloney and Cotter 2018; Oliveira et al. 2007). ROS/RNS can also contribute to cell mutation, cell proliferation, angiogenesis, invasion, and metastasis of tumor cells, as well as leading to resistance of apoptosis and drug therapy. Toxic levels of ROS, however, can contribute to cell death producing an anti-tumorigenic effect that is associated with the mechanism of action of several antineoplastic drugs, which can be natural or synthetic (Moloney and Cotter 2018; Saha et al. 2017). β-D-Glucans are carbohydrate biopolymers composed of D-glucose residues that are present in cereals (oats and barley), fungi (fruiting bodies such as mushrooms

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and brackets), and are found as components of the cell wall matrix of fungi including yeasts. They can also be secreted exocellularly by several microorganisms during submerged fermentation processes, in which case they are known as exopolysaccharides (EPS). β-D-Glucans have been described as effective in treating several conditions including dyslipidemia, diabetes, and cancer (Chen and Seviour 2007; Geraldelli et al. 2020a, b; Joyce et al. 2019; Queiroz et al. 2015). In addition, studies have demonstrated that β-glucans present direct and indirect antitumoral effects, and that the antitumor effect can be mediated by an antioxidant or prooxidant effect (Choromanska et al. 2018; Kaya et al. 2016; Queiroz et al. 2015). In this chapter, we provide an overview of the relationships between cancer, oxidative stress, and the (1 ! 3)-β-D-glucans of different sources, and evaluate the effects of β-glucans on the conditions of cancer and oxidative stress.

Cancer Cancer is characterized by uncontrolled growth of mutated cells that can invade other tissues via the blood or lymphoid vessels causing metastasis (WHO 2020). Studies have demonstrated that cancer is increasing at an alarming rate worldwide, and has been considered the second cause of deaths globally, accounting for an estimated 9.6 million deaths (WHO 2020). In Brazil, the estimate for the triennium, 2020–2022, is that approximately six hundred and twenty five thousand new cases of cancer will occur each year (INCA 2020). A cancer cell condition is derived from a normal cell that suffers some form of mutations in its DNA that is promoted by carcinogenic agents (carcinogens), and leads to the formation of malignant tumors (WHO 2020). The cancer-initiated cell multiplies clonally, accumulates genetic and/or epigenetic alterations, and escapes to apoptosis. These events, in turn, convert the cell into a neoplastic cell that leads to the development of a tumor classified as benign or malignant in accordance with cellular characteristics of the tumor (Oliveira et al. 2007).

Carcinogenesis and Cachexia-Anorexia Syndrome The process of carcinogenesis frequently develops slowly, and is subdivided into three stages: initiation, promotion, and progression (Fig. 1) (Oliveira et al. 2007; Belitskiy et al. 2020). Initiation is the first stage of this process and occurs when normal cells undergo genetic changes due to the action of the carcinogens (Fig. 1). Thus, progressive accumulation of mutations occur transforming the cells that potentially gives rise to a cancer (Oliveira et al. 2007). This cancerogenic stage, however, is not able to develop into a tumor, but is responsible in promoting permanent damage of the DNA, which is essential for the development of the neoplasia, and has a “memory” characteristic (Oliveira et al. 2007).

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Fig. 1 Stages of carcinogenesis and substances that influence each stage of tumor development. ROS reactive oxygen species, IGF-1 insulin like growth factor 1

Promotion is the second stage of carcinogenesis, and is characterized by the action of promoter agents directed at the initiated cells (mutated cells). The promoter agents stimulate cell proliferation contributing to the growth of the tumor, and amplification of the mutated cells, establishing mutations, without interacting directly with the DNA. The promoters can, nevertheless, still damage the DNA via an indirect mechanism promoting oxidation (Fig. 1; Oliveira et al. 2007). This stage progresses slowly and gradually, and the formation of malignant cells requires a long and continuous contact with the promoter agent. Furthermore, the promoter effect is time and concentration-dependent in the specific tissue where the cancer is developed (Oliveira et al. 2007). Certain growth factors, such as insulin, estrogen, insulin-like growth factor-1 (IGF-1), and tumor necrosis factor-alpha (TNF-α), are important examples of promoter agents that contribute to the growth of tumors (Fig. 1; Avgerinos et al. 2019; Gallagher and LeRoith 2015). The final stage of carcinogenesis named progression occurs when there is continuous contact with the promoter agents. Progression is characterized by the disordered and irreversible multiplication of cells that have been genetically altered, and this phenomenon leads to genetic instability, rapid cell growth, invasion of tissues that leads to metastasis (Fig. 1; Belitskiy et al. 2020; Oliveira et al. 2007). Tumor progression is related to successive modification of phenotypes, that is, changes in gene expression of neoplastic cells, making them more aggressive and invasive, as well as altering biochemical, metabolic, endocrine, and/or morphological characteristics of the transformed cells (Oliveira et al. 2007). In addition, in the progression phase, a cancer phenotype is acquired through genetic and epigenetic mechanisms (Belitskiy et al. 2020; Oliveira et al. 2007). The last stage of carcinogenesis is represented by the manifestation of tumor signals, where symptoms may take years to emerge, and depending upon the

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location of the tumor and the affected organs, these symptoms may vary, such as anemia, weight loss, pain, among others. In addition, tumor growth can also contribute to the development of the cachexia-anorexia syndrome, which can lead to death of the patient (Avgerinos et al. 2019 ;Argilés et al. 2018). The development of cachexia-anorexia syndrome is associated with the advanced stages of cancer, and is characterized by weight loss, depletion of adipose and muscle tissue mass, immunosuppression, anemia, asthenia, and accompanied by several modifications in the metabolism of lipids, proteins, and carbohydrates, among others (Argilés et al. 2018).

Risk Factors Since cancer is a multifactorial disease, many factors can contribute to the risk of developing tumors. The risk factors can be divided into (i) internal/endogenous factors (usually predetermined genetically), and (ii) external/exogenous factors (related to the environment and to the lifestyle habits of the individual), both of which are interrelated (Avgerinos et al. 2019; Belitskiy et al. 2020; Oliveira et al. 2007). Furthermore, studies have demonstrated that 80–90% of cancers are associated with environmental factors, with rare cases of cancers that are due exclusively to hereditary factors (INCA 2020). Recently, several studies in the literature have demonstrated that obesity is considered a major risk factor for several types of cancers, for example, breast, colorectal, kidney, liver, stomach, esophagus, ovarian, among others (Avgerinos et al. 2019; Gallagher and LeRoith 2015). A recent epidemiological survey in Brazil in collaboration with the United States (Harvard University) demonstrated that at least 15,000 cancer cases per year could be avoided by controlling the overweight condition (obesity), and is estimated that this number could increase to 29,000 new cases by 2025 (Rezende et al. 2018). A study by the International Agency of Research on Cancer (IARC) from the World Health Organization in 2002 reported that normal body weight (BMI 18.5– 24.9 Kg/m2) decreases the risk of the development of five types of cancer (colon and rectum, esophagus (adenocarcinoma), kidney (renal-cell), breast (post-menopausal), and corpus uteri) (Lauby-Secretan et al. 2016). In 2016, a new study by IARC conducted an evaluation on the preventive effects of body weight control (reduction) on the risks of cancer development, and analyzed data from a systematic review of the literature (>1000 studies) on the mechanisms associated with the accumulation of adipose tissue (obesity) and cancer. The observations reported the same association for an extra eight types of tumors (gastric cardia, liver, gallbladder, pancreas, ovary, meningioma, thyroid, and multiple myeloma), concluding that the absence of the accumulation of adipose tissue mass decreased the risk for a majority of cancers, as esophagus (adenocarcinoma), gastric cardia, colon and rectum, liver, gallbladder, pancreas, breast (post-menopausal), corpus uteri, ovary, kidney (renal-cell), meningioma, thyroid, and multiple myeloma (LaubySecretan et al. 2016).

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Insulin resistance, hyperinsulinemia, low-grade inflammation, and oxidative stress, frequently present in the obesity condition, have been postulated as the mechanisms by which obesity can promote tumorigenesis (Avgerinos et al. 2019; Gallagher and LeRoith 2015; Lauby-Secretan et al. 2016; Rezende et al. 2018). Moreover, the strong association between excessive body weight and cancer-risk confirmed the importance of a better quality of life, with a healthy food intake and nutrition to prevent, and or control, the development of these diseases (obesity and cancer). It is well known that cellular oxidative stress, especially generated by ROS and RNS, contributes positively to the initiation of tumor development leading to cell mutation (Moloney and Cotter 2018). These reactive species can damage DNA, RNA, lipid, and proteins by different chemical reactions such as oxidation, nitration/ nitrosation, and peroxidation, which contribute to cell mutation and physiological alterations (Oliveira et al. 2007). In addition, oxidative stress can contribute to the promotion and progression stages of carcinogenesis by leading the cells to proliferation and stimulating angiogenesis through a mechanism fostered by genotoxicity (Moloney and Cotter 2018; Oliveira et al. 2007). Moreover, oxidative stress can alter cell signaling pathways, contributing to tumor development in breast, lung, liver, colon, prostate, ovary, and brain (Saha et al. 2017).

Cancer Therapy There are different types of therapies for treating cancers that can include surgery, various therapies (radiation, chemical, immunological, hormonal), and bone marrow transplantation. Depending upon the type and severity of the cancer, a combination of these techniques can be used (Belitskiy et al. 2020). Chemotherapy uses chemical agents, called antineoplastic drugs, to destroy tumor cells by preserving healthy cells (Belitskiy et al. 2020). Moreover, many of antineoplastic compounds can be activated by oxidative stress in cancer cells, which leads to cell death through peroxidation of lipids and the thiol groups of proteins. Presently, there are several chemotherapeutic agents for the treatment of cancer; however, most antineoplastic agents act in a nonspecific manner, causing damage to both types of cells (normal and cancer), which lead to adverse side effects, including nausea, vomiting, anemia, immunosuppression, alopecia, and secondary neoplasia, which increases a major obstacle in the cures for cancer (Belitskiy et al. 2020). Thus, the search for new antineoplastic drugs with low side effects is of fundamental importance. Natural products (microbial, plant, animal) have demonstrated important antineoplastic, and/or adjuvant, therapies in the treatment of several types of cancers (Pan et al. 2019). In addition, studies have shown that many natural compounds, such as plant-derived carotenoids, curcumin, resveratrol, and epigallocatechin-3-gallate, as well as various β-glucans (microbial, plant), present a chemo-protective effect alone, or in association with standard cancer therapies, thereby improving and modulating

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the immune system of humans and model lab animals with tumors (Chen and Seviour 2007; Comiran et al. 2020; Pan et al. 2019).

Oxidative Stress Sources and Effects of Reactive Oxygen Species ROS are produced by the metabolic activity of normal cells and participate in signaling pathways. They can be produced by several sources such as organelles, for example, mitochondria, peroxisomes, and the endoplasmic reticulum, as well as through the catabolism of polyamines and thymidines. Furthermore, enzymatic systems, such as the NADPH oxidases (NOXs), cyclooxygenases, lipoxygenases, xanthine oxidases, nitric oxide synthases (NOS), and cytochrome P450 enzymes, contribute significantly to ROS production (Moloney and Cotter 2018; Snezhkina et al. 2019). Another source of ROS are exogenous factors, such as radiation, drugs, chemicals, heavy metals, and pollutants (Galadari et al. 2017). The main source of ROS are the mitochondria through the actions of the membranebound NOXs. During the metabolism of oxygen, molecular oxygen (O2) receives electrons from NADPH generating the superoxide anion radical (O2-●). This radical species can in turn react with nitric oxide (NO) to generate peroxynitrite (ONOO) (another potential reactive specie). Through the action of the enzyme, superoxide dismutase (SOD), O2-● is converted into hydrogen peroxide (H2O2) in a dismutationcatalyzed reaction (Fig. 2; Snezhkina et al. 2019; Holley et al. 2011). H2O2 in turn is metabolized through the action of catalase to water and oxygen, but the Fenton reaction

Fig. 2 Reactive oxygen species (ROS) and reactive nitrogen species (RNS) production and regulation by the antioxidant system. O2 molecular oxygen, NOX NADPH oxidase, NADPH nicotinamide adenine dinucleotide phosphate, O2˙ superoxide anion radical, SOD superoxide dismutase, H2O2 hydrogen peroxide, H2O2 water, NOS nitric oxide synthase, NO nitric oxide, ONOO peroxynitrite, NO3 nitrate, OH• hydroxyl radical, Fe+2 (ferrous), and Fe+3 (ferric) states

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can also participate to produce hydroxyl radicals (OH•). The damage induced by the latter radical leads to further increased levels of H2O2, and this can form a vicious cycle. A point of note, H2O2, HOCl (hypochlorous acid), and ONOO are not free radicals, but they also cause cellular damage (Fig. 2; Moloney and Cotter 2018). The NOX family of enzymes consists of transmembrane proteins that transport electrons across cell membranes to reduce oxygen to superoxide. The NOX family consists of seven isoforms, NOX 1–5 and DUOX 1–2, that present a NADPH binding site, a flavin adenine dinucleotide (FAD)-binding site, six transmembrane domains and four heme-binding histidines, being expressed in different tissues, for example, NOX4 is highly expressed in the kidneys and pancreas (Moloney and Cotter 2018). Among these, the NOX2 complex, fully assembled and activated, leads to the generation of O2-● through the transfer of an electron from NADPH to oxygen in the extracellular space. In humans, NOX2 is widely distributed, being found in phagocytes and other cells such as hepatocytes and vascular endothelium (Bedard and Krause 2007). ROS can also be produced in the endoplasmic reticulum, through the action of enzymes, such as protein disulfide isomerase (PDI). PDI is oxidized by endoplasmic reticulum oxireduction-1 (ERO1), and thus transfers electrons to molecular oxygen leading to the formation of H2O2. In addition, the microsomal monooxidase system (MMO), which catalyzes the oxygenation of exogenous hydrophobic compounds and some endogenous substrates, results in the production of superoxide and H2O2 (Snezhkina et al. 2019). Metals, such as iron and copper, are catalysts and contribute to the production of ROS, as observed by the decomposition of H2O2 via the Fenton and/or Haber-Weiss reactions that lead to the formation of ●OH and OH (Xu et al. 2017). As already mentioned, there are several sites that produce ROS in the body, and, these products participate in normal cell activities and in signaling pathways of the cells. Trouble can occur when an excess of ROS is not controlled through antioxidant mechanisms, then these species become harmful to the body generating the socalled oxidative stress condition.

ROS and Antioxidant Mechanisms Enzymatic and nonenzymatic systems constitute the antioxidant mechanisms. The enzymatic system includes several enzymes, such as catalase (CAT), SOD, glutathione peroxidase (GPx), epoxide hydrolase 2 (EPHX2), and others (Fig. 2). The nonenzymatic system includes glutathione, various plant products (flavonoids, polyphenols, tocopherols, carotenoids, curcumin), vitamins A, C and E, and the βglucans (Figs. 2 and 3). SOD is dependent upon co-factors such as copper (Cu), zinc (Zn), and manganese (Mn), with Cu and Zn acting in the cytoplasm of the cell, while Mn in the mitochondria. These enzymes are classified as SOD1 (Cu/ZnSOD), SOD2 (MnSOD) and SOD3 (Cu/ZnSOD; extracellular) (Moloney and Cotter 2018). MnSOD is the main guardian enzyme present in the mitochondria, and changes in

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Fig. 3 Schematic diagram of oxidative stress, cancer, and β-glucans. Sources of ROS and antioxidants that regulate the ROS/RNS levels influencing tumor development or promoting antitumor effects. ROS reactive oxygen species, RNS reactive nitrogen species, DNA deoxyribonucleic acid, RNA ribonucleic acid

its function can lead to the development of disease (Holley et al. 2011). Once H2O2 is produced, it also undergoes enzymatic catalysis by CAT into water and oxygen (Fig. 2) (Moloney and Cotter 2018). Accumulation of H2O2 leads to the production of ●OH, and the glutathione enzyme system (that includes glutathione (GSH), glutathione reductase, GPx and glutathione-s-transferase, GST) converts ●OH into H2O (Fig. 2; Arteel and Sies 2001; Moloney and Cotter 2018). Peroxiredoxin (Prx) and thioredoxin (Trx) are other enzymes that can act as antioxidants eliminating ROS by decomposing peroxides (Moloney and Cotter 2018). Vitamins (A, C, and E) and the carotenoids contribute to antioxidant effects. Vitamin E occurs in eight natural forms as tocopherols (alpha, beta, gamma, and delta) and tocotrienols (alpha, beta, gamma, and delta), all of which possess potent antioxidant properties. The reaction of α-tocopherol with peroxyl radicals prevents the oxidation of unsaturated fatty acids (Carini et al. 2017). Another example is vitamin C, which acts by decreasing the formation of superoxide and peroxynitrite, and directly scavenges superoxide (Carini et al. 2017). Thus, it is possible to observe that the antioxidants present in the body, in foods and in dietary supplements, present important and significant beneficial effects on health, protecting the body from damage produced by oxidative stress, decreasing the genotoxic effects of ROS, and slowing the cancer progression (Carini et al. 2017; Saha et al. 2017). The success of antioxidant effects depends upon the levels of ROS present in the cell. In normal cells oxidative homeostasis is maintained, and ROS contribute to

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metabolic and signaling pathways, but when ROS levels rise, the mechanisms of cell-death are activated.

Cancer and Oxidative Stress The effects of ROS in cancer are controversial. Due to the higher metabolic activity of cancer cells, it is possible to observe a higher production rate of ROS by these cells, which can lead to a positive or negative effect on the development of tumors. It is still unclear whether ROS contributes to the tumor development, or if it is contributing to cancer cell apoptosis and an antitumoral effect (Galadari et al. 2017). Moloney and Cotter (2018) described in their review the important influence of reactive oxygen species in the biology of cancer. Several cancers present high levels of ROS, and these reactive species have been shown to demonstrate several roles in tumor tissues, as they can activate pro-tumorigenic signaling pathways, such as the MAPK/ERK1/2 and PI3K/Akt pathways that increase cell survival and enhance cell proliferation, as well as leading to DNA damage and genetic instability. Moreover, tumor cells can also modulate the expression of antioxidant enzymes, for example, by increasing the expression of SOD2 (MnSOD) and inactivating the enzymes CAT and PRx, establishing a redox balance. They, nevertheless, still maintain higher levels of H2O2, and pro-tumorigenic signaling and resistance to apoptosis (Fig. 3). Oxidative stress can possibly significantly contribute to all stages of carcinogenesis, inducing cell mutation, cell proliferation, and angiogenesis, and contribute to tumor growth, invasion and metastasis (Fig. 3; Moloney and Cotter 2018; Oliveira et al. 2007; Saha et al. 2017). Toxic levels of ROS, however, can also promote an anti-tumorigenic effect, promoting tumor cell-death mediated by oxidative stress, as the higher levels of ROS decrease the GSH levels, thereby loosing redox homeostasis (Moloney and Cotter 2018). Saha et al. (2017) in their review reported that several nutrients, such as carbohydrates and lipids, can contribute to increase the ROS levels following a meal. For example, the high levels of glucose following a meal intake leads to ROS generation, as well as contributing to insulin secretion by the β cells of the pancreas. Insulin, consequently, can raise the levels of H2O2 in cells and increase cell proliferation, including the proliferation of tumor cells. It is well known that cancer cells consume a higher amount of glucose as a main source of energy to produce ATP (Warburg effect) (Argilés et al. 2018; Avgerinos et al. 2019; Moloney and Cotter 2018). Thus, both glucose concentration and ATP generation in the tumor cells can contribute to ROS production creating a vicious cycle associated with ROS and metabolism, and the proliferation of cancer cells that lead to increase tumor growth. Furthermore, it is known that antineoplastic drugs, such as anthracyclines (daunorubicin, doxorubicin), cisplatin, bleomycin, and arsenic trioxide, promote the death of cancer cells by increasing ROS production. However, ROS can also contribute to cell mutation leading to several side effects, and even development of other types of cancers following chemotherapeutic treatment (Belitskiy et al. 2020; Moloney and Cotter 2018; Saha et al. 2017).

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Therefore the development of new therapies to eliminate ROS through the actions of antioxidants, or by acting to significantly elevate the ROS levels, may be potentially effective as antitumor therapies, as the antioxidants may contribute to decrease cell mutation, initiation and progression of cancer, and toxic levels of ROS can contribute to cancer cell death.

β-Glucans Structural Characterization of β-D-Glucans Among the various β-D-glucans that exist is the group of (1 ! 3)-β-D-glucans; polysaccharides composed of D-glucose units linked through β-(1 ! 3)-D-glucosidic bonds in a backbone chain that carry D-glucose-bearing appendages at the C-6 positions through β-(1 ! 6)-linkages. They constitute the most common form of the β-D-glucan group (Bacic et al. 2009). An example of the chemical structure of a fungal branched chain (1 ! 3)-β-D-glucan is botryosphaeran, a (1 ! 3)(1 ! 6)-βD-glucan (Fig. 4). There are exceptions in which the branched β-glucans can possess different linkages in both their backbone chains and in their substituents (Synytsya and Novák 2013). The β-glucans are present in cereals (oats and barley), fungi (fruiting bodies such as mushrooms and brackets), and as components of the cell wall matrix of fungi that includes bakers/brewers yeasts. They can also be secreted exocellularly by bacteria, fungi, and microalgae during submerged fermentation processes, whence they are known as exopolysaccharides (EPS). The fungal β-D-glucans can differ in fine structure, molecular weight, extent of branching, and conformation (Wang et al. 2017), features that confer a crucial role on their biological response activities (Bohn and BeMiller 1995). Several types of (1 ! 3)-β-D-glucans are produced by different fungi, for example, lentinan from Lentinula edodes, schizophyllan from Schizophyllum

Fig. 4 Chemical structure of a fungal (1 ! 3)(1 ! 6)-β-D-glucan. Botryosphaeran, produced by the fungus Botryosphaeria rhodina MAMB-05

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commune, and scleroglucan from Sclerotium rolfsii are from basidiomyceteous fungi; while botryosphaeran from Botryosphaeria rhodina (Dekker et al. 2019) and lasiodiplodan from Lasiodiplodia theobromae (Cunha et al. 2019) are examples from ascomycetes. The β-glucans present in cereals have a different chemical structure, they contain (1 ! 3)(1 ! 4)-β-D-glucosidic linkages, and comprise a linear structure of Dglucose residues interconnected by (1 ! 4)- and (1 ! 3)-linkages (Clarke and Stone 1966). They do, however, exhibit similar biological functions to the fungal (1 ! 3)β-glucans.

Anticancer Effects of β-Glucans The fungal (1 ! 3)-β-D-glucans are well-recognized in possessing medicinal properties effective in treating human disease conditions including inflammation, diabetes, cardiovascular diseases, dyslipidemia, and cancers (Batbayar et al. 2012; Chen and Seviour 2007; Joyce et al. 2019; Theuwissen and Mensink 2008). Studies have also demonstrated that β-glucans can present a direct cytotoxic effect (Queiroz et al. 2015), and indirect antitumor effect mediated by immunomodulatory and metabolic effects (Chen and Seviour 2007; Comiran et al. 2020; Geraldelli et al. 2020). The indirect effects can be mediated by activation of the immune system against the tumor, and by the improvement of metabolic functions, which contribute to decrease the stimulus for cell proliferation, such as improving the insulin sensitivity and correcting hyperglycemia (Chen and Seviour 2007; Comiran et al. 2020). Epidemiological studies have shown that foods, particularly cereals (oats and barley) and mushrooms, which are a rich source of β-glucans, may prevent or decrease tumor development, reducing, for example, the development of gastric, colorectal and breast cancers (Pan et al. 2019). β-Glucans can also improve the quality of life, and the survival of patients with cancer (Aleem 2013). The (1 ! 3)(1 ! 6)-β-D-glucans, as well as the (1 ! 3)(1 ! 4)-β-D-glucans, are known as biological response modifiers (Bohn and BeMiller 1995), demonstrating immunomodulation effects against infectious diseases and cancer, by stimulating immune cells (neutrophils, eosinophils, monocytes, macrophages, and NK cells) (Ahn et al. 2004; Batbayar et al. 2012; Chen and Seviour 2007; Kano et al. 1996). According to Daou and Zhang (2012), β-glucans have been used since 1980 as an immunoadjuvant for cancer therapy. In addition, the antitumor efficacy of β-glucans from oat (the (1 ! 3)(1 ! 4)-β-D-glucans) appears to be associated with the tumor type, tumor burden, dose, method of administration, and the moment of administration, being capable of stimulating the immune system by modulating granulocytes and other components of the innate immune system (Daou and Zhang 2012). Several studies, in vitro and in vivo, have been developed to demonstrate the beneficial effects of β-glucans on tumor development, and many studies were realized in humans and animals (Comiran et al. 2020; Geraldelli et al. 2020a, b; Pan et al. 2019; Queiroz et al. 2015).

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Pan et al. (2019) reported in their review article that many β-glucans, such as lentinan (shiitake mushroom, Lentinus edodes), schizophyllan (Schizophyllum commune), Krestin (Trametes (formerly named Coriolus or Polyporus) versicolor), maitake (Grifola frondosa), and the dietary Agaricales mushrooms, Agaricus blazei and A. sylvaticus, have been trialed in human clinical studies, and demonstrated beneficial effects on different types of cancers. Another study demonstrated that an extract from Agaricus blazei Murill Kyowa increased the number and/or activity of natural killer cells in patients with cancers of the cervix, ovary, and endometrium (Ahn et al. 2004). In addition Kano et al. (1996) demonstrated that schizophyllan could increase the number of CD4+ T-helper cells and the production of interleukin 2 (IL-2) in patients with head and neck cancer. Previous studies from our research group have demonstrated the potential antitumoral effects of botryosphaeran, a (1 ! 3)(1 ! 6)-β-D-glucan, in rats bearing Walker-256 tumors (Comiran et al. 2020; Geraldelli et al. 2020), in human tumorigenic (Jurkat) T-lymphocytes (Malini et al. 2015), and in breast carcinoma cell line (MCF-7 cells) (Queiroz et al. 2015). Queiroz et al. (2015) demonstrated an important antiproliferative effect of botryosphaeran against MCF-7 cells in vitro, which was associated with apoptosis, necrosis, and oxidative stress, and was mediated by AMPactivated protein kinase (AMPK) and the Forkhead transcription factor 3a (FOXO3a), thereby increasing the expression of tumor suppressor proteins (p53 and p27), and the pro-apoptotic proteins, bax, and caspase-3. Malini et al. (2015) demonstrated that botryosphaeran in vitro could modulate gene expression and regulate cell cycle progression through repressing genes related to the G1 phase of the cell cycle; effects related to the inhibition of the CCR-5 receptor, expressed in human Jurkat T lymphocytes; a receptor that is associated with malignancy and tumor growth. Recently, Geraldelli et al. (2020a) demonstrated that botryosphaeran treatment (30 mg/kg b.w./day for 15-days) of Walker-256 tumor-bearing Wistar nonobese male rats presented a significant antitumor effect reducing significantly tumor development and the cancer cachexia syndrome. The mechanism associated with this effect is thought to be, in part, due to metabolic and hematological events, that is, through maintaining glycemia within the normal limits, correcting hypertriglyceridemia and macrocytic anemia, as well as by increasing the apoptosis of the tumor cells. Furthermore, in Walker-256 tumor-bearing obese rats, botryosphaeran (30 mg/kg b.w./day for 15-days) also significantly decreased tumor development and the cancer cachexia syndrome. The mechanism is thought to be associated, at least in part, with the reduction of visceral adipose tissue (mesenteric adipose tissue), improvement of insulin sensitivity, and the correction of macrocytic anemia, as well as, by increasing considerably the activity of FOXO3a (Geraldelli et al. 2020b). Other work from our lab demonstrated that the treatment of Walker-256 tumorbearing obese and nonobese rats with botryosphaeran at a dose of 12 mg/Kg b.w. for 15 days attenuated tumor development and the cancer cachexia syndrome in both groups. The mechanism is associated with the metabolic and immunologic effects of botryosphaeran, correcting insulin resistance and hyperglycemia, modulating the cholesterol levels, and increasing the leukocyte and lymphocytes in obese rats

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(Comiran et al. 2020). Thus, as demonstrated by Comiran et al. (2020) and Geraldelli et al. (2020a, b), it is possible to observe that the antitumor effect of botryosphaeran occurs in a concentration-dependent manner, as a dose of 12 mg botryosphaeran/kg b.w. attenuated tumor development, while at a higher dose of 30 mg/kg b.w., botryosphaeran significantly decreased tumor growth and the cachexia syndrome in both obese and nonobese Wistar rats by mechanisms associated with the direct and indirect antitumoral effect of botryosphaeran.

β-Glucans, Oxidative Stress, and Cancer β-Glucans are known to possess strong antioxidant activity through scavenging of free radicals thereby avoiding damage by reactive oxygen species (ROS) (Wang et al. 2013), and thus protecting the cells through the protective effects of their antioxidant status, and inhibiting the peroxidation of membrane lipids (Ciecierska et al. 2019; Kofuji et al. 2012; Slameňová et al. 2003). The scavenging activity of βglucans was detected against different free radicals, such as H2O2, OH●., O2-●, DPPH radicals, and nitric oxide, thus protecting against ROS damage and diseases like diabetes and cardiovascular diseases (Erkol et al. 2011; Slameňová et al. 2003; Ciecierska et al. 2019; Saha et al. 2017). Botryosphaeran likewise exhibited in vitro free-radical scavenging properties and antioxidant activities (Giese et al. 2015). Giese et al. (2015) demonstrated that botryosphaeran presented a total antioxidant activity of 80%, as well as the capacity to scavenge hydroxyl radicals at the level of 90.6%, and nitric oxide of 90%, in the in vitro model. Studies have demonstrated that the chemical structure of different β-glucans can have an influence on the biological effects of these β-glucans, for example, increasing or not its antioxidant activity (Ciecierska et al. 2019; Kofuji et al. 2012). Kofuji et al. (2012) demonstrated that barley β-glucan has a higher antioxidant activity than that of oat and yeast β-glucans. As mentioned by Ciecierska et al. (2019) in their review, the antioxidant effects of β-glucans may be mediated by the presence of anomeric hydrogen atoms in their molecules, and may be mediated by the polymeric structure of these polysaccharides, which contribute to remove free radicals. Several studies have demonstrated that oral consumption of β-glucans in humans and animals can be effective in preventing oxidative stress and damage to several types of cells including erythrocytes, platelets, and lung cells (Choromanska et al. 2018; Slameňová et al. 2003). Slameňová et al. (2003) demonstrated that yeastderived β-glucans presented a significant antigenotoxic and antioxidant effects in Chinese hamster lung fibroblast cells V79 (in vitro), and this effect was associated with the scavenging of singlet oxygen and OH● radicals. Erkol et al. (2011) also demonstrated that yeast-derived β-glucans presented a protective effect against oxidative damage in the liver of Wistar albino rats. They observed that treatment with yeast β-glucan reduced the levels of lipid peroxide and malondialdehyde in the liver, and significantly increased glutathione and superoxide dismutase levels.

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In addition, Pillai and Uma Devi (2013) demonstrated that in Swiss albino mice, the β-glucans from Ganoderma lucidum exerted an antioxidant and radioprotective effect on the animals. A β-glucan from Antrodia camphorata demonstrated a significant antioxidant effect in human hepatocytes, decreasing ROS production induced by bacterial lipopolysaccharide (LPS) (Yücel et al. 2017). This particular β-glucan also decreased the expression of some proteins of the NADPH oxidase homologues NOX1, NOX2, and NOX4 that are associated with the enzyme-catalyzed production of superoxide anion radical. Furthermore, it is known that treatment with β-glucan can inhibit several signaling pathways: for example, ERK (extracellular signalregulated kinase), p38 (mitogen-activated protein kinase), and Akt (serine/threonine-specific protein kinase) pathways (Galadari et al. 2017), which can contribute to decrease the ROS levels and consequently, cell damage and reduce tumor growth. Kaya et al. (2016) demonstrated that treatment of Sprague Dawley rats with baker’s yeast β-glucan (50 mg/kg b.w./day) over 14 days together with cisplatin (a therapeutic anticancer drug) significantly decreased elevated levels of TBARS (thiobarbituric acid reactive substance) and increased the levels of several antioxidant agents (SOD, CAT, GPx, and GSH) in brain tissue when compared with the group of rats treated with cisplatin alone. Moreover, treatment with β-glucan alone did not damage brain tissues, as evidenced for histopathological analysis of the neurons of the cerebral cortex, as well as other parts of nervous system, showed normal appearance in both the control and β-glucan-treated animal groups. β-Glucan also significantly reduced the histopathological alterations promoted by cisplatin. Jumes et al. (2010) demonstrated that powdered basidiocarp and extracts rich in β-glucans derived from Agaricus brasiliensis, presented significant antitumor effects against Walker-256 tumor-bearing Wistar rats, and the antitumor effect was primarily associated with the β-glucan’s antioxidant activity. The treatment of Walker-256 tumor-bearing Wistar rats with the preparations from A. brasiliensis, furthermore, reduced tumor growth and the cancer cachexia syndrome, as well as increasing hepatic catalase and SOD activities. Similarly, recent data from our laboratory demonstrated that botryosphaeran, a (1 ! 3)(1 ! 6)-β-D-glucan from Botryosphaeria rhodina MAMB 05, presented a significant antioxidant effect on Walker-256 tumor-bearing obese and nonobese rats (Pereira da Silva, unpublished data). Botryosphaeran decreased the levels of TBARS in the liver and adipose tissue of obese rats, and increased hepatic GST and GSH activities, and the levels of hepatic vitamin C. Furthermore, this fungal (1 ! 3) (1 ! 6)-β-D-glucan increased SOD activity in the tumor tissue of nonobese rats and increased the vitamin C levels in the tumor tissue of obese rats (Pereira da Silva, unpublished data). Although several studies have demonstrated the antioxidant effect of β-glucans, some studies have also demonstrated that antitumor and other biological effects of βglucans can be mediated by an increase in oxidative stress (Choromanska et al. 2018; Queiroz et al. 2015). Choromanska et al. (2018) demonstrated that both high- and low-molecular weight (1 ! 3)(1 ! 4)-β-D-glucan fractions derived from oats presented a

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significant cytotoxic effect against lung cancer A549 and H69AR cell-lines, decreasing cell viability, increasing apoptosis, and inducing oxidative stress in the cancer cells compared to normal cells (HaCaT, normal human keratinocytes), as well as increasing the expression of MnSOD that protected human erythrocytes against hemolysis induced by a hypotonic medium. As aforementioned, Queiroz et al. (2015) demonstrated a significant antiproliferative effect of botryosphaeran against breast cancer MCF-7 cells (in vitro); an effect that was associated with oxidative stress. The treatment of MCF-7 cells with botryosphaeran decreased the viability of the MCF-7 cells and increased the generation of ROS in a time-dependent manner. Yang et al. (2006) demonstrated that the exopolysaccharides (heteropolysaccharides rich in galactose) from Antrodia camphorata promoted apoptosis of MCF-7 breast cancer cells in a dose- and timedependent manner, and this effect was associated with increased ROS production. Studies have suggested that the mechanism associated with the increased production of ROS by β-glucans may be attributable to the activation of cell surface receptors (dectin-1 and Toll-like Receptor) upon the binding with β-glucan, as the activation of these receptors leads to the activation of spleen tyrosine kinase and NOXs proteins, consequently increasing the production of ROS (Batbayar et al. 2012).

Conclusions In summary, oxidative stress can contribute to tumor development promoting cell mutation, cell proliferation, angiogenesis, invasion, and metastasis of tumor cells, as well as contributing to the resistance of apoptosis and drug therapy. Depending upon the concentration of the levels of ROS, apoptosis can be activated that leads to cancer cell-death, and this may be the mechanism of action of some compounds, such as antineoplastic drugs and other natural and synthetic agents. Antioxidants, such as the β-glucans, have demonstrated direct and indirect antitumoral effects, protecting the normal cells from damage by oxidative stress. Thus, β-glucans have shown a promising effect as an adjunct for applications in treating cancers.

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Oral Cancer and Oxidative Stress Gokul Sridharan

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Oral Carcinogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Oxidative Stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Oxidative Stress and Oral Carcinogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cross-References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract

Oral squamous cell carcinoma (OSCC) is one of the common forms of oral cancer associated with significant morbidity and mortality. Over several years, attempts have been made to understand the mechanism of oral carcinogenesis with an intention for early diagnosis and implementation of appropriate therapeutic strategies. OSCC is generally characterized by a complex interplay of environment and genetic factors resulting in its initiation, promotion, and subsequent progression. Etiological factors such as tobacco, smoking, and viruses along with genetic and epigenetic factors are the major contributors of oral carcinomas. Oxidative stress is a general term used to indicate the imbalance associated with either an increased reactive oxygen species or deficient antioxidant defense mechanisms or both. While literature evidence suggests a causal role of oxidative stress in oral carcinogenesis, the effect of oxidative stress and the antioxidants on oral carcinogenesis is still unclear. The purpose of this chapter is to discuss the various effects of oxidative stress on OSCC and enable the understanding of its potential role in oral carcinogenesis.

G. Sridharan (*) Department of Oral Pathology and Microbiology, Dr. G. D. Pol Foundation YMT Dental College and Hospital, Navi Mumbai, India © Springer Nature Singapore Pte Ltd. 2022 S. Chakraborti et al. (eds.), Handbook of Oxidative Stress in Cancer: Mechanistic Aspects, https://doi.org/10.1007/978-981-15-9411-3_7

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Keywords

Oral cancer · Oral potentially malignant disorders · Carcinogenesis · Oxidative stress · Reactive oxygen species · Antioxidants · Cancer diagnostics

Introduction Cancer is the second leading cause of death in the world after cardiovascular diseases (Murthy and Mathew 2004). Urbanization, industrialization, changes in lifestyle, population growth, and aging have all contributed to the epidemiological transition of cancer worldwide. Cancers of lung, esophagus, stomach, oral cavity, and pharynx are highly prevalent in males while carcinoma of cervix and breast is common in women (Murthy and Mathew 2004). It is estimated that 80–90% of human cancer may be attributed to environmental and lifestyle habits such as tobacco, alcohol, and dietary factors (Sudhakar 2009). Over the years, several factors were identified as potential carcinogens, and as of 2014 World Health Organization’s International agency for research on cancer (IARC) has identified more than 100 chemical, physical, and biological carcinogens. The discovery of exact chemical structure of DNA further helped in understanding the complex problems of chemistry and biology of human cancer. The identification of oncogenes and tumor suppressor genes played a pivotal role in decrypting the mutational changes of DNA that could occur in a cancerous cell. Despite the tremendous advancements in the field of diagnostics and therapeutics, the burden of cancer is still increasing worldwide. One of the important forms of cancer is those affecting the oral and pharyngeal sites, which has a high prevalence in developing countries of the world. Carcinomas account for 96% of all oral cancers, and 90% of these are squamous cell carcinoma (Feller and Lemmer 2012). Oral squamous cell carcinoma is a neoplasm of epithelial origin associated with poor survival rate. OSCC is a multifactorial disorder characterized by a complex interplay of environmental and genetic factors. The causative agents of OSCC known as carcinogens may be chemical, physical, and biological which influence the process of carcinogenesis. The role of tobacco as a primary risk factor is well known with other contributing factors being alcohol, nutrition, oxidative stress, and microorganisms. These factors either independently or in coordination result in genetic mutations in the form of inhibition of tumor suppressor genes and promotion of proto-oncogenes thereby leading to unrestrained tumor growth (Gleich and Salamone 2002). An important factor associated with oral carcinogenesis is the induction of oxidative stress that results in cellular and molecular damage affecting DNA, proteins, and lipids. Oxidative stress is characterized by an imbalance in the equilibrium between the reactive oxygen species and the antioxidant defense mechanism (Bhattacharya et al. 2007). This imbalance may result due to overproduction of free radicals and/or an inefficient antioxidant defense mechanism leading to uncontrolled damage to DNA, proteins, and lipids that are potential hallmarks of carcinogenesis. While the role of oxidative stress in oral carcinogenesis has been

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scientifically documented, arguments do exist on their exact role and utility as a diagnostic, therapeutic, and prognostic marker. The purpose of this chapter is to provide an overview of oral squamous cell carcinoma, the role of oxidative stress in oral carcinogenesis, and their potential implications in diagnosis, treatment, and prognosis of OSCC.

Oral Carcinogenesis The process of oral carcinogenesis is characterized by a sequence of genetic defects brought about by various environmental factors which results in mutated cell followed by clonal expansion. The genetic alterations that may occur during carcinogenesis include point mutations, amplifications, rearrangements, and deletions. The most common and earliest genetic change is the mutation in p53 and k-ras gene. These genetic alterations in OSCC are mainly due to oncogene activation and inactivation of tumor suppressor genes leading to deregulation of cell proliferation and death (Mehrotra and Yadav 2006). There are eight defined hallmarks of carcinogenesis: inhibition of tumor suppressor genes; stimulation of proto-oncogenes; evasion of apoptosis; failure of DNA repair mechanisms; uncontrolled proliferation, sustained angiogenesis, invasion, and metastasis; evasion of host immune system (Fouad and Aanei 2017). The changes occurring at the molecular level are multitude in nature and usually occur because of the other known risk factors. However, these changes play an important role in the promotion and progression of oral cancer. OSCC initiates in a multistep process in which normal cells are transformed into a preneoplastic cell followed by its malignant transformation, or they may arise de novo. These lesions that have the propensity to develop into malignancy are collectively referred to as oral potentially malignant disorders (Villa and Gohel 2014). Oral cancer is an important malignancy caused by tobacco chewing and betel quid mainly in India and other Asian countries (Mehrotra and Yadav 2006). In India, tobacco use in various forms and alcoholism are some of the common social habits which may contribute to increased prevalence of oral cancer (Sridharan 2014). Chronic use of smokeless tobacco can cause nicotine addiction, oral potentially malignant disorders, and OSCC, among other diseases (Stepanov et al. 2008). There are two main types of smokeless tobacco in use, namely, chewing tobacco and snuff. Chewing tobacco is in the form of loose leaf, cut or shredded tobacco, while snuff is either in dry or moist form. An important factor that acts as a risk factor for oral squamous cell carcinoma is the excessive generation of reactive oxygen species (ROS) especially in tobacco and betel quid chewers. The overproduction of ROS associated with tobacco consumption results in DNA damage which gets accumulated over a period ultimately leading to derangement of cell cycle (Kolanjiappan et al. 2003). DNA damage is a prime factor in initiating the genetic damage that is evident in oral carcinogenesis. Other factors that contribute to oral carcinogenesis include viruses, chemicals, irradiation, and the genetic make-up of an individual (Beevi et al. 2004). It is increasingly evident that potentially cytotoxic agents important in the etiology of cancer are

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generated by the interaction of nitric oxide and reactive oxygen species (Rasheed et al. 2007).

Oxidative Stress Cell cycle is a dynamic process characterized by a continuous release of free electrons and production of free radicals. Experimental data emphasizes that continuous and excessive production of free radicals, particularly ROS, may attack biomolecules, producing alterations in DNA, proteins, and lipids and is thus implicated in the pathogenesis of age-related disease and cancer (Van Wijk et al. 2008). Oxidative stress is defined as excess formation and/or insufficient removal of highly reactive molecules such as ROS & RNS (Johansen et al. 2005). A free radical is an atom, group of atoms, or molecules containing one unpaired electron within an outer orbit (Van Wijk et al. 2008). The products of partial reduction of oxygen through oxidation reactions are highly reactive, and these are termed as reactive oxygen species which includes hydroxyl (HO•), nitric oxide (NO), superoxide (O2•-), hydrogen peroxide (H2O2), and peroxynitrite (ONOO ) (Miricescu et al. 2011). Aerobic organisms possess antioxidant defense system that counteracts the effect of ROS and free radicals produced through aerobic respiration and substrate oxidation (Mates et al. 1999). The antioxidant system includes several enzymes and lowmolecular weight compounds which function by either inhibiting the production of free radicals or inactivation of the formed free radicals. The major intracellular enzymes involved are glutathione, glutathione peroxidase, glutathione reductase, superoxide dismutase (SOD), and catalase (Gutteridge 1995). In addition, external agents such as vitamin A, vitamin E, and minerals such as selenium function as effective antioxidant agents (Yang et al. 2002). The antioxidant system is responsible to protect cells against ROS by preventing excessive accumulation of ROS. Failure to maintain an appropriate balance between the ROS and antioxidant system results in cellular damage and ultimately leads to various human diseases including malignancies. While under physiological conditions, ROS are involved in various actions such as signaling molecules and defense mechanisms; excessive generation in oxidative stress has pathological consequences that may lead to proteins, lipids, and DNA damage (Johansen et al. 2005). ROS are postulated to be involved in various stages of carcinogenesis particularly in the stages of initiation and promotion. The primary targets of lipid peroxidation by ROS are the polyunsaturated fatty acids in the membrane lipids (Beevi et al. 2004). The structure and function of cell surface are altered by changes in membrane lipids which are important aspects for development of malignancies (Kolanjiappan et al. 2003). Lipid peroxidation results in the release of various end products such as lipid hydroperoxides (LHP) and Malondialdehyde (MDA) which may serve as markers of cellular damage (Beevi et al. 2004). Highly reactive oxygen species and free-radical-induced damage are implicated in carcinogenic processes. The damage caused by free radicals is known to cause the malignant transformation of normal cells (Seven et al. 1999). However, the ability of

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the free-radical-induced damage causing malignant transformation depends on the innate ability of the body’s defense mechanisms by various cellular antioxidants (Van Wijk et al. 2008). Disruption of this delicate oxidant-antioxidant balance in the body seems to play a causative role in carcinogenesis (Beevi et al. 2004).

Oxidative Stress and Oral Carcinogenesis Cancer cells show a variety of alterations that include the cell surface, the cytoskeletal components, and cell cycle dysregulation when compared with their normal counterparts. It has been observed that the cellular responses occurring in response to the external stimuli are mediated by membrane lipids which have been further implicated in tumor development (Kolanjiappan et al. 2003). Active oxygen species and other reactive free radicals found in the human diet can also mediate other phenotypic and genotypic alterations that lead to neoplasia. In order to counteract the effect of free radicals, various antioxidants such as vitamins A, E, C, and β-carotene derived from foods have been used as chemopreventive agents against oral cancers (Garewal 1995). The mechanism of action of these antioxidants includes neutralizing the reactive oxygen species, interfering with the activation of procarcinogens, preventing binding of carcinogens to DNA, etc. Some of the chemopreventive agents with antioxidant capacities have been demonstrated to reverse the cellular and morphological changes characteristic of various oral precancerous lesions (Enwonwu and Meeks 1995). Tumor cells during their process of transformation create an environment conducive for excessive production of ROS for the cancer cell survival further leading to enhanced lipid peroxidation in HNSCC patients. Another mechanism by which lipid peroxidation takes place is through prostaglandin synthesis, activation of signal transduction pathways, decomposition of polyunsaturated fatty acids present in membranes, and poor antioxidant system (Gupta et al. 2004; Metkari et al. 2007). Overall, it can be hypothesized that elevated levels of lipid peroxidation could be a possible link between initiation and promotion in carcinogenesis (Rasheed et al. 2007; Beevi et al. 2004). Several literature evidences indicate the significant contribution of increased levels of free radicals with or without an associated depletion of antioxidant system in oral carcinogenesis (Manoharan et al. 2005; Nisha and Lal 2008; Khanna et al. 2005). It was also established that the lipid peroxidation increases with severity of the disease reflecting the extent of tissue injury. The general understanding is that oxidative stress that occurs as a result of an excessive generation of free radicals and insufficient antioxidant levels is one of the contributory factors of oral carcinogenesis. While literature data indicates to these changes consistently in circulating fluids such as blood, the same may not always hold true with respect to the tumor tissue. There are indications that the products of lipid peroxidation are less susceptible on the malignant cells which could be due to the low availability of polyunsaturated fatty acid substrate. Several studies have found that the lipid peroxidation was significantly decreased in the tumor tissue. These findings suggested a decreased susceptibility of oral tumor tissue to lipid

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peroxidation. Lipid peroxides have been suggested to be involved in the control of cell division. Tumor progression is associated with low levels of MDA. An inverse relationship has been observed between levels of lipid peroxidation and the rate of cell proliferation. The low levels were thought to be related to cell proliferation occurring in oral carcinogenesis (Nagini et al. 1998). The reduction in MDA levels was thought to be related to cell proliferation. In contrast to decreased lipid peroxidation in tumor tissues, blood samples showed enhanced lipid peroxidation in OSCC patients in comparison to healthy samples. The increase in lipid peroxides may be related to deficiency of superoxide dismutase in tumor tissue. This can result in accumulation of superoxide anion causing deleterious effects away from the tumor sites (Subapriya et al. 2002). Significantly decreased levels of lipid peroxidation products were demonstrated in tumor tissue of squamous cell carcinoma patients in comparison to tissue from healthy controls. The reduction in the lipid peroxidation of the tumor tissue may be related to the increase in cholesterol, decrease in phospholipids, and increase in C/P ratio which further may lead to membrane rigidity and decreased oxygen supply (Kolanjiappan et al. 2003). Cells can decrease or limit the oxidative destruction of free oxygen radicals. It is possible that antioxidants destroy the formed reactive oxygen species or prevent their production or in some cases repair the targeted molecules after the hazard has taken place (Topdag et al. 2005). Various antioxidants act by different mechanisms and affect the different stages of carcinogenesis (Beevi et al. 2004). Antioxidants such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) are the first line of cellular defense against oxidative injury, leading to decomposition of superoxide radicals and H2O2. Among the antioxidants, glutathione S- transferase (GST) play an important protective role by conjugating electrophilic agent to glutathione (GSH) and the reduction of lipid hydroxides (Burlakova et al. 2010). A decrease in the activities of SOD and catalase in the cancer patients as compared to control subjects was observed in several studies (Subapriya et al. 2002). These low activities of antioxidants may be due to the depletion of the antioxidant defense system or because of overwhelming free radicals. A weak antioxidant defense system with resultant failure to counteract the effects of free radicals alters the intracellular redox balance that favors a DNA damage and disease progression (Beevi et al. 2004). The constant endeavor to comprehend the exact role of reactive oxygen species and antioxidants in carcinogenesis has resulted in the current understanding of the dual facet of the ROS-induced oxidative stress. ROS-induced oxidative stress by causing damage to DNA, proteins, and lipids is associated with its oncogenic potential, promoting genomic instability and tumorigenesis. On the other hand, there is evidence that the ROS may act as signaling molecule to promote cancer cell proliferation, survival, angiogenesis, and metastasis (Sabharwal and Schumacker 2014). Excessive generation of ROS can lead to increase in oxidative stress that may further lead to oxidative stress-induced cancer cell death (Nogueira et al. 2008). The effects of increased ROS on cell cycle arrest, senescence, and

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cancer cell death could probably occur through the activation of the ASK1/JNK- and ASK1/p38-signaling pathways (Ichijo et al. 1997; Moon et al. 2010). Additionally, the cancer cell also increases their antioxidant capacity in order to maintain a redox balance so as to prevent ROS-induced cell death (Reczek and Chandel 2017). Thus, it is imperative that oxidative-stress-directed therapy could be of help in management of oral cancer. Cancer therapy aimed at the oxidative stress parameters generally works on the principal that ROS are protumorigenic, and antioxidants by negating the effect of ROS could serve as a potential suppressor. Based on this observation, multiple clinical trials have evaluated the clinical efficacy of the dietary antioxidants in cancer therapeutics but have met with conflicting results. One of the reasons for the inefficient defense mechanisms of dietary antioxidants on ROS is that they are unable to efficiently scavenge the protumorigenic ROS. On the other hand, these antioxidants may probably contribute to promote tumor progression by decreasing the toxicity of ROS at the tumor site (Chandel and Tuveson 2014). One possibility to overcome this is by attempting to use a targeted therapy that can access and detoxify the locally generated ROS involved in protumorigenic signaling. Based on the fact that ROS possess an antitumorigenic effect on the cancer cell, it is possible to consider different treatment strategies that aim to enhance ROS generation in cancer cell that will further lead to oxidative stress-induced death of the cancer cells. Novel treatment strategies that attempt to elevate ROS levels either by increasing ROS production or decreasing ROS scavenging that may affect cancer cells while sparing the normal cells have been researched (Postovit et al. 2018). Various compounds such as high dose of vitamin C (Yun et al. 2015), lanperisone (Shaw et al. 2011), and buthiomine sulfoximine that depletes glutathione production (Andringa et al. 2006) have been tried with reasonable success in anticancer treatment. The mechanism of action of cancer chemotherapy and its effect on oxidative stress should be understood for better implementation of oxidative stress-related therapy. If the mechanism of action of the cancer chemotherapeutic agent is by generation of free radicals, then antioxidants may probably impede the antineoplastic activity of the chemotherapeutic activity. However, if the reactive species are responsible only for the drug’s adverse effects, antioxidants may reduce the severity of such effects without interfering with the drug’s antineoplastic activity. Thus, it is important to determine the exact nature of the chemotherapy before initiating the antioxidant treatment (Conklin 2004).

Conclusion Oxidative stress characterized by an imbalance between reactive oxygen species and antioxidants is an important factor in the process of oral carcinogenesis (Waris and Ahsan 2006). The debate on whether they play a causal role in oral squamous cell carcinoma is still unsettled. However, much information has been unraveled regarding the molecular pathogenesis and, vis-à-vis, has helped us to understand the potential impact of oxidative stress mediators in various stages of oral

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carcinogenesis. It is now proven that the reactive oxygen species possess both protumorigenic and antitumorigenic activity (Yang et al. 2016). Hence, the implementation of antioxidant therapy may not always be beneficial. The current treatment modality thus is aimed at the use of targeted antioxidant therapy as well as the use of compounds that results in oxidative stress-induced cancer cell death. Further research and clinical trials are necessary to elucidate the complete therapeutic benefit of this method which will further improve prognosis and reduce mortality.

Cross-References ▶ Oral Cancer and Oxidative Stress ▶ Salivary Oxidative Stress Biomarkers in Oral Potentially Malignant Disorders and Squamous Cell Carcinoma

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Oxidative Stress in Genitourinary Cancer Masaki Shiota

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Kidney Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Urothelial Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Prostate Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Anticancer Therapy for Genitourinary Cancer and Oxidative Stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract

Oxidative stress is caused by reactive oxygen species (ROS) and reactive nitrogen species (RNS), and affects various cellular functions in both cancer and non-cancer cells. While ROS/RNS are required for physiological processes such as signal transduction, excessive ROS/RNS causes oxidative stress, resulting in gene mutation and epigenetic changes due to damage or modification of nucleic acids, lipids, and proteins, leading to the dysregulation of various cellular signaling pathways. Findings from various experimental models as well as human studies support the key roles of oxidative stress in carcinogenesis and cancer progression in various cancers, including genitourinary cancer. Oxidative stress is also closely involved in effects of anticancer therapeutics in genitourinary cancer. Although efforts are being made to prevent the development and progression of genitourinary cancers by suppressing oxidative stress as well as improve therapeutic efficacy and adverse effects by modulating oxidative stress, no clinical efficacy of these strategies has yet been established. Here we summarize the relationship between oxidative stress and the development and progression of genitourinary cancer, with a focus on kidney cancer, urothelial cancer, and prostate cancer. M. Shiota (*) Department of Urology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan e-mail: [email protected] © Springer Nature Singapore Pte Ltd. 2022 S. Chakraborti et al. (eds.), Handbook of Oxidative Stress in Cancer: Mechanistic Aspects, https://doi.org/10.1007/978-981-15-9411-3_9

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Keywords

Antioxidant · Bladder cancer · Oxidative stress · Prostate cancer · Kidney cancer · Reactive oxygen species · Urothelial cancer

Introduction Impairments of prooxidant and antioxidant systems resulting in increased oxidative stress and oxidation damage have been identified in various cancers. Oxidative stress has thus been proposed to contribute to the pathogenesis of cancer. Oxidative stress is caused by an imbalance between the production of reactive oxygen species (ROS)/ reactive nitrogen species (RNS) and the extinction of ROS/RNS caused by decreased antioxidant capacity (Fig. 1). Various factors induce oxidative stress, such as irritation, ultraviolet irradiation, chemical substances, inflammation, excessive calorie intake, and senescence (Fig. 1). Cause of oxidative stress • Senescence • Renal failure • Infection • Inflammation • Smoking • Chemical substance • Irradiation • Ultraviolet-ray • Metabolic syndrome • Excessive calorie intake ROS/RNS generation • Mitochondria • NADPH oxidase • Xanthine oxidase • Cyclooxygenase • Hypoxia • Nitric oxide synthase Anti-oxidative ability • Anti-oxidant enzyme • Anti-oxidant substance

Oxidative stress Fig. 1 Causes of oxidative stress. Various factors induce oxidative stress through increases of pro-oxidative properties and decreases of anti-oxidative properties

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Oxidative stress

High

Low

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Cell death

Damage and modification to

Genotoxicity

•

Nucleic acids

•

•

Lipids

Non-genotoxicity

•

Proteins

•

Altered epigenetics

•

Dysregulated signal transduction

Cell survival

Genomic alterations

Carcinogenesis Cancer progression Treatment resistance

Fig. 2 Molecular mechanism of carcinogenesis and cancer progression by oxidative stress. Oxidative stress induces damage to and modification of nucleic acids, lipids, and proteins, resulting in genotoxicity such as genomic alterations and non-genotoxicity such as altered epigenetics and dysregulated signal transduction

Oxidative stress is caused by ROS, such as hydroxyl radicals, superoxides, and hydrogen peroxide, as well as RNS, such as nitrogen dioxide, dinitrogen trioxide, and nitrogen peroxide, and affects various cellular functions in both cancer and non-cancer cells. ROS are induced mainly by endogenous factors such as ATP production in mitochondria, NADPH oxidase, xanthine oxidase, cyclooxygenase, and hypoxia, as well as by various exogenous factors. RNS is generated from the reaction of nitric oxide (NO), which is produced by nitric oxide synthase (NOS), with ROS and metals. While ROS/RNS are required for physiological processes such as signal transduction, excessive ROS/RNS causes oxidative stress, resulting in gene mutation and epigenetic changes due to damage or modification of nucleic acids, lipids, and proteins, leading to the dysregulation of various cellular signaling pathways (Fig. 2). Oxidative stress plays key roles in the development and progression of various cancers including genitourinary cancer through promoting cancer initiation to cancer progression (Shiota et al. 2014). Since the urinary tract is exposed to urine that is concentrated and excreted from the kidney, where condensed ROS/RNS exist, the urinary tract is supposed to be exposed to higher level of oxidative stress than other organs. Accordingly, genitourinary cancer is suggested to be closely affected by oxidative stress. Several efforts have been made to prevent the onset and progression of cancer by suppressing oxidative stress using compounds with antioxidant properties. Many studies have been conducted on the prevention of carcinogenesis in genitourinary cancer using antioxidants, and promising results have been reported, at least in preclinical studies. In addition, various anticancer treatments such as chemotherapy agents and radiation treatments exert antitumor effects by inducing oxidative stress (Shiota et al. 2012a). Oxidative stress is therefore not only a cause of cancer but can also be applied for the treatment of cancer, and thus oxidative stress is considered a double-edged sword.

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In this chapter, we summarize the relationship between oxidative stress and the development and progression of genitourinary cancer, with a focus on kidney cancer, urothelial cancer, and prostate cancer.

Kidney Cancer The prevalence of kidney cancer has been rising in recent years in part due to advances in diagnostic imaging. Hereditary diseases such as von Hippel-Lindau (VHL) disease accompanied by hemangioblastoma and pheochromocytoma of the brain, spinal cord, and retina, pancreatic endocrine tumor, and renal failure, as well as lifestyle factors such as smoking, obesity, and hypertension, have also been linked to the development of kidney cancer (Capitanio et al. 2019; Al-Bayati et al. 2019). Renal failure and smoking are associated with increased oxidative stress, and obesity and hypertension are also pathological metabolic conditions that also show a close relationship with oxidative stress. Thus, the increased oxidative stress due to these factors may contribute to the development of kidney cancer (Table 1). Genomic alterations such as deletions and mutations in the VHL gene have been reported in many kidney cancers (Shen and Kaelin Jr. 2013). Recent studies using next-generation sequencing revealed numerous novel gene mutations in various pathological subtypes of kidney cancer (Sato et al. 2013; Davis et al. 2014; Scelo et al. 2014; Wang et al. 2017). Thus, oxidative stress may be involved in the Table 1 The relationship between risk factors in genitourinary cancer with oxidative stress Cancer type Kidney cancer

Urothelial cancer

Prostate cancer

Risk factors Hereditary diseases Renal failure Smoking Obesity Hypertension Aging Smoking Occupation Medication Infection/inflammation Irradiation Gender Aging Race Family history Diet Lifestyle Sex hormone Inflammation

Relationship with oxidative stress Unknown Presence Presence Presence Presence Presence Presence Presence Unknown Presence Presence Unknown Presence Unknown Unknown Presence Unknown Unknown Presence

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development of kidney cancer through increasing genomic alterations such as gene mutations (such as G:C to T:A transverse mutations) due to oxidized bases such as 8-oxo-guanine. In addition, oxidative stress leads to the activation of a key molecule for kidney cancer HIF-1, which may promote the development and progression of kidney cancer from changes in signal transduction pathways (Sudarshan et al. 2009). In a study of animal experiments using rats, iron nitrilotriacetic acid caused gene mutations due to oxidative stress, and the treated rats developed kidney cancer (Toyokuni 2009). In addition, loss of VHL has been reported to result in increased iron uptake via HIF-1, where VHL mutation is suggested to be involved in carcinogenesis in kidney through oxidative stress by iron metabolism (Alberghini et al. 2005). In dialysis-associated kidney cancer, increased oxidative stress is observed in cancer tissues (Hori et al. 2007). Previous studies in non-dialysis-associated kidney cancer reported the associations among oxidative stress, changes in expression of antioxidant enzymes and the development and progression of cancer, suggesting the involvement of oxidative stress in renal carcinogenesis (Miyake et al. 2004).

Urothelial Cancer Urothelial cancers include upper urinary tract cancers, which develop in the upper urinary tract such as the renal pelvis and ureter, and bladder cancer. Risk factors for the development of bladder cancer include aging, smoking, exposure to drugs such as cyclophosphamide or phenacetin, occupational exposure to chemical dyes, and secondary carcinogenesis after radiation therapy. In addition, a high incidence rate of squamous cell carcinoma is observed in patients with urinary calculi, chronic urinary tract infections, and Schistosoma haematobium, suggesting an association between inflammation and bladder cancer (Cumberbatch et al. 2018). Furthermore, while the morbidity rate of bladder cancers in males is high, the prognosis for invasive bladder cancer in females is poor, suggesting that sex hormones may also affect the development and progression of bladder cancer (Inoue et al. 2018). Oxidative stress due to aging, inflammation, exposure to chemical substances and irradiation is thought to be involved in the development of bladder cancer (Table 1). Mutations in oncogenes such as Ras and FGFR and tumor suppressor genes such as p53 and RB1 are frequently detected in bladder cancer (Audenet et al. 2018). In addition, various mutations in genes involved in chromosomal stability and DNA repair have recently been reported using next-generation sequencing (Guo et al. 2013; Cancer Genome Atlas Research Network 2014). Oxidative stress is suggested to be involved in the development and progression of bladder cancer through these genomic alterations, which is induced by oxidative stress, at least in part. A previous study reported that hydrogen peroxide in addition to carcinogens such as N-methylN-nitrosourea induced bladder carcinogenesis in an animal model (Crallan et al. 2006). Single nucleotide polymorphisms (SNPs) in antioxidant enzyme genes GPX1, NQO1, myeloperoxidase, and MnSOD and deletion of GSTM1 have been correlated with an increased incidence of bladder cancer (Cao et al. 2014; Gong et al. 2013;

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Simic et al. 2009). Thus, it is possible that the difference of antioxidant capacity in an individual may affect the development of bladder cancer (Golka et al. 2011). However, in patients with bladder cancer, a decrease in antioxidant capacity and an increase in oxidative stress have been shown (Badjatia et al. 2010). In addition, a SNP in NOS correlates with the recurrence and progression of non-muscle-invasive bladder cancer (Amasyali et al. 2012), and oxidative stress is increased in bladder cancer patients with poor prognosis (Soini et al. 2011). Thus, oxidative stress is thought to be involved in both development and progression of bladder cancer.

Prostate Cancer Prostate cancer is the second most prevalent cancer in men in Europe and the United States and affects 1 in 6 men (Bray et al. 2018). Aging and hereditary predisposition such as race and family history are established risk factors for prostate cancer, while diet, lifestyle, sex hormones, and inflammation have been proposed as possible risk factors (Table 1) (Shiota et al. 2014). Aging, diet, and inflammation are closely related to oxidative stress, suggesting the development of prostate cancer may be due, in part, to oxidative stress. TMPRESS/ERG fusion genes, which was shown to promote prostate carcinogenesis through androgen-regulated ERG expression, are found in approximately one-fourth to half of prostate cancers (Zhou et al. 2017). In addition, mutations in tumor suppressor genes such as PTEN, p53 and RB1 have been identified in castration-sensitive prostate cancer, and mutations and amplification in androgen receptor (AR), which plays an important role in the survival and growth of prostate cancer, are frequently observed in castration-resistant prostate cancer (Beltran et al. 2013). Various mutations in genes involved in AR-related signals as well as oncogenic and DNA repair processes such as SPOP, FOXA1, PIK3CA, CTNNB1, and BRCA2 have also been identified in prostate cancer using next-generation sequencing (Grasso et al. 2012; Baca et al. 2013; Lindquist et al. 2016; Ren et al. 2018; Armenia et al. 2018). Oxidative stress may thus be involved in the development and progression of prostate cancer through these genomic alterations, which is induced by oxidative stress, at least in part. Oxidative stress is also thought to be involved in the development and progression of prostate cancer by activating AR signals through changes in epigenetic mechanisms and signal transduction systems (Shiota et al. 2011a). In animal experiments using rats, 2-amino-1-methyl-6-phenylimidazo [4,5-b] pyridine, which is one of the most abundant mutagenic heterocyclic amines in N-methyl-N-nitrosourea and heated fish meat products, was shown to cause prostate carcinogenesis (Nakai and Nonomura 2013), suggesting that oxidative stress caused by these chemicals may promote carcinogenesis. SNPs in genes such as MnSOD, catalase, and GPX4 have also been reported to be correlated with morbidity and progression of prostate cancer (Wang et al. 2009; Geybels et al. 2015; Van Blarigan et al. 2014) and diminished expression of GSTP1 by promoter methylation is observed in many prostate cancers, suggesting that a

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decrease in antioxidant capacity results in oxidative stress (Perry et al. 2006). Actually, a decrease in antioxidant capacity and an increase in oxidative stress have been reported in prostate cancer patients (Battisti et al. 2011). Various antioxidants have been shown to suppress the development and progression of prostate cancer in preclinical studies (Lin et al. 2019; Shiota et al. 2012b). The SELECT test was conducted to examine the prevention of prostate cancer development in human patients using selenium and vitamin E, but no significant results were observed (Klein et al. 2011; Thompson et al. 2014a, b).

Anticancer Therapy for Genitourinary Cancer and Oxidative Stress Various anticancer therapies have been used for genitourinary cancers. Radiotherapy and chemotherapy in addition to surgical treatment have been the main treatments for several decades (Table 2). Both radiotherapy and cytotoxic chemotherapy are well known to exert oxidative stress (Glebova et al. 2015), which is thought to play an important role in the therapeutic effect and toxicity in both cancer and non-cancer cells. Oxidative stress induced by cytotoxic modalities, in turn, also induces a variety

Table 2 Summary of therapeutic options for genitourinary cancer Therapeutic modalities Surgery

Kidney cancer Radical nephrectomy Partial nephrectomy

Radiotherapy

Chemotherapy

Urothelial cancer Transurethral resection Radical cystectomy

Prostate cancer Radical prostatectomy

External beam therapy

External beam therapy

Platinum-based therapy

Hormone therapy

Molecular-targeted therapy Immunotherapy

Brachytherapy Radium-223 Taxanes Androgen deprivation therapy Antiandrogen CYP17 inhibitor

Tyrosine kinase inhibitor mTOR inhibitor PD-1 inhibitor CTLA-4 inhibitor

Intravesical BCG therapy PD-1/PD-L1 inhibitor

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of anti-oxidative and anti-apoptotic signal transduction pathways, resulting in cellular resistance to cytotoxic therapies (Shiota et al. 2012a). Androgen deprivation therapy is the backbone therapy for recurrent and advanced prostate cancer. However, most prostate cancers recur as castrationresistant prostate cancer, which is attributable mostly to aberrant AR activation (Shiota et al. 2011b). Previous studies reported that androgen deprivation therapy causes oxidative stress, which in turn activates AR signaling through various mechanisms, where oxidative stress mediates castration resistance (Shiota et al. 2011a). Molecular-targeted agents such as tyrosine kinase inhibitors and mammalian target of rapamycin (mTOR) inhibitors have been used for advanced kidney cancers. Although the association between mTOR inhibitors and oxidative stress has not been fully explored, several studies on tyrosine kinase inhibitors and oxidative stress have been reported, although the results have not been conclusive. However, one study showed that the tyrosine kinase inhibitor sorafenib may exert anticancer effects through oxidative stress (Teppo et al. 2017). Intravesical therapy using Bacillus Calmette-Guérin (BCG) has been used for non-muscle-invasive bladder cancer. Intravesical BCG therapy, which exerts anticancer effects through immune-related functions, has been reported to induce oxidative stress (Shah et al. 2014a, b). Actually, it is reported that a SNP in NOS correlates with the recurrence and progression of non-muscle-invasive bladder cancer after intravesical BCG therapy, supporting the involvement of oxidative stress in its therapeutic effect (Wei et al. 2012). Although immune-checkpoint inhibitors targeting PD-1/PD-L1 and CTLA-4 have been used for treatment of advanced kidney cancer and urothelial cancer, the role of oxidative stress in the treatment effects of these agents has not been explored and should be investigated in future studies. Together these studies indicate that oxidative stress induced by anticancer therapies for genitourinary cancer is a two-edged sword, in which oxidative stress exerts anti-survival and pro-apoptotic effects, but augments anti-apoptotic pathways and aggressive phenotypes in cancer cells, indicating the importance of regulating oxidative stress during anticancer therapies.

Conclusions Oxidative stress caused by various factors induces cell damage, resulting in carcinogenesis, cancer development, and treatment resistance in genitourinary cancer. Efforts are currently being made to prevent the development and progression of genitourinary cancer by suppressing oxidative stress, as well as improve the efficacy and adverse effects of therapeutics by modulating oxidative stress. However, no clinical efficacy of these strategies has yet been established. Therefore, elucidating the relationship between oxidative stress and the development and progression of genitourinary cancer and the development of effective antioxidant compounds should be examined in future studies.

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Oxidative Stress, Microenvironment, and Oral Cancer Gargi Sarode, Nikunj Maniyar, Sachin Sarode, and Mamatha G. S.

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Inflammation, Reactive Oxygen Species, and Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Tumor Cell Survival . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Tumor Cell Proliferation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Tumor Cell Invasion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Angiogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Oral Squamous Cell Carcinoma and Reactive Oxygen Species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Tumor Microenvironment and Reactive Oxygen Species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Reactive Oxygen Species in Oral Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Oncoviruses, Oral Squamous Cell Carcinoma, and Reactive Oxygen Species . . . . . . . . . . . . . . . . Reactive Oxygen Species as an Appealing Target for Intervention (Table 8) . . . . . . . . . . . . . . . . . Reactive Oxygen Species as Alluring Targets for Therapeutic Intervention . . . . . . . . . . . . . . . . . . . Reactive Oxygen Species and Oral Potentially Malignant Disorders . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

100 102 104 104 105 106 107 107 110 110 112 113 114 115

Abstract

Oral cancer or oral squamous cell carcinoma (OSCC) is one of the most common malignancies worldwide especially in the Asian countries. It is considered as a unique type of malignancy because of various reasons like diverse tissue composition, microbiome, exposure to outer environment, etc. Thus, it separates oral cancer from other malignancies in terms of the microenvironment and tumoral heterogeneity. Chronic inflammation plays a major role in altering of OSCC microenvironment and its etiopathogenesis. This chapter deals with the role of

G. Sarode (*) · S. Sarode · M. G. S. Department of Oral Pathology and Microbiology, Dr. D. Y. Patil Dental College and Hospital, Dr. D. Y. Patil Vidyapeeth, Pune, Maharashtra, India N. Maniyar Department of Orthodontics and Dentofacial Orthopaedics, Bapuji Dental College and Hospital, Davangere, Karnataka, India © Springer Nature Singapore Pte Ltd. 2022 S. Chakraborti et al. (eds.), Handbook of Oxidative Stress in Cancer: Mechanistic Aspects, https://doi.org/10.1007/978-981-15-9411-3_10

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oxidative stress in the etiopathogenesis and progression of OSCC in context with the modification of its microenvironment in detail. It also emphasizes on the role of antioxidants in OSCC and oral potentially malignant disorders (OPMDs) as therapeutic option and as an adjuvant in OSCC treatment. Keywords

Microenvironment · Oral cancer · Oxidative stress · Oral squamous cell carcinoma · Oral potentially malignant disorders · OSCC · OPMDs · Reactive oxygen species

Introduction Head and neck cancer (HNC) is found to be the sixth most common malignancy in the world. It is predominantly seen in South-central Asian region and stands at third position in the list of most common types (Warnakulasuriya 2009; Petersen 2003). In all the cases of HNCs, oral squamous cell carcinoma (OSCC) is the most pervasive cancer type and is a main reason for death worldwide. It is known as “oral cancer” and is significantly common in developing countries (Krishna Rao et al. 2013). An increase in the OSCC cases in developed countries is noted nowadays because of various reasons. OSCC is the most common type of cancer in South Asian countries (Sarode et al. 2020). Around 400,000 new cases of OSCC annually have been detected worldwide (Khan et al. 2010). OSCC is a multistep process with a multifactorial etiology mainly involving chewable tobacco. Advances in understanding of mechanism of oral carcinogenesis are necessary to improve survival of the OSCC patients. The oral cavity has remained among the worst of all the cancer sites since a few decades (Khan et al. 2010). OSCC is also considered as a unique type of malignancy. There is a conceivable rationale behind the uniqueness of OSCC because of certain reasons (Patel et al. 2016). Sarode et al. (2017) have discussed OSCC as different from other malignancies based on the following factors: • • • • • • •

Oral cavity’s distinctive microenvironment ascribed to diverse tissue composition Microbial flora Varied carcinogenic exposures Oral potentially malignant disorders (OPMDs) Epithelial turnover rate Saliva Exposure to outer environment Thus the oral microenvironment is likely to temper the etiopathogenesis of OSCC. The above-mentioned factors are exclusive for OSCC. The tumoral heterogeneity is evident in OSCC and differentiates it from other malignancies.

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Owing to the above unique environment of the oral cavity, it should be considered and treated differently from other malignancies. All the factors mentioned above point toward the inflammatory-mediated carcinogenesis involvement in the etiopathogenesis of OSCC. Thus, inflammation is considered as the major hallmark in oral carcinogenesis. Since nineteenth century it has been believed that cancer is associated with inflammation. For a significant period, this belief was being neglected (Colotta et al. 2009). Epidemiological studies have shown that the chronic inflammation theaters an obvious role in the OSCC etiopathogenesis. Various reports have shown the function of different inflammatory mediators like nuclear factor kappa B, vascular endothelial growth factor, inflammatory cytokines, prostaglandins, p53, nitric oxide, reactive oxygen species (ROS) and nitrogen species (RNS), and microRNAs (miRNAs) are major key players in the pathogenesis of OSCC (Patel et al. 2016). Chronic inflammation can be regarded to effectuate persistent tissue damage and alterations in the inflammatory cells and cytokines within the tissue microenvironment. Several epidemiological and molecular studies have concluded that inflammation can greatly increase the risk of cancer development. Sufficient evidence can be found in the literature that proposes that chronic inflammation by obstinate bacteria, viruses, or chemicals imposes as risk factors for malignant transformation. Certain specific transcription factors serve as important link between cancer and inflammation, which once stimulated holds the potential to boost the genes expression responsible for regulating and producing the inflammatory mediators and also regulation of the survival and proliferation of cancer cells. Chronic inflammationmediated activation of cellular pathways forms the risk factors for initial stages like cell transformation and cancer progression. In later stages of cancer, the cells provoke amplified inflammatory state in the stroma leading to proliferation, invasion and metastasis (Grivennikov and Karin 2010). Microenvironment of tissues affected by inflammation witnesses an increased accumulation of prostaglandin, chemokines, cytokines, and free radicals of ROS and RNS (Feller et al. 2013). Oxidative stress generated as an outcome of discrepancy between free radical production and the capability of antioxidant system to counteract it, has been related to many human disorders (Coussens and Werb 2002). Comprehensive research during the past decade has unveiled several pathways through which oxidative stress can result in chronic inflammation leading to several chronic diseases like cardiovascular diseases, neurodegenerative disorders, cancer, etc. (Reuter et al. 2010). The defilement caused by ROS to the cell is not only dependent on their concentration but also on the equilibrium between the ROS and the antioxidants. Loss of this balance between prooxidants and antioxidants leads to generation of oxidative stress that alters and damages several intracellular molecules, including DNA, RNA, proteins, and lipids. Alteration of the molecules can upsurge the possibility of mutagenic alterations. Persistent environmental strain causes continuous and prolonged generation of free radicals that significantly damage

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cellular structure and function. It may stimulate somatic mutations and malignant change. Indeed, cancer initiation and progression can be associated with oxidative stress increasing DNA mutations, genetic instability, and cellular growth (Reuter et al. 2010).

Inflammation, Reactive Oxygen Species, and Cancer Reactive Oxygen Species and Inflammation Inflammation can be accounted as the primary immune system response that eliminates the stimuli in an attempt to reestablish the normal cells or substitute the damaged cells. This communication of the immune system with antigens results in ROS generation resulting in activation of signaling pathways triggering the formation of pro-inflammatory cytokines and chemokines. Through this process of antigen removal, macrophages generate ROS including hydrogen peroxide, superoxide, nitric oxide, hydroxyl radical, hydrochlorous acid, peroxynitrite etc. (Khansari et al. 2009). A sustained active inflammatory reaction leads to cell damage following ROS overproduction via cells of innate immune system. Furthermore, inflammatory cells release metabolites of arachidonic acid, cytokines, chemokines, etc. recruiting inflammatory cells producing added free radicals. These crucial mediators trigger signal transduction cascades inducing alterations in transcription factors, which intercede instant cellular stress reactions (Reuter et al. 2010; Hussain and Harris 2007). Activation of protein kinase signaling cascade by pro-inflammatory cytokines in phagocytic and non-phagocytic cells produces ROS and RNS. Involvement of cyclooxygenase-2 (COX-2), inducible nitric oxide synthase, aberrant expression of inflammatory cytokines along with changes in few microRNAs has a major role to play in oxidative stress-induced inflammation (Hussain and Harris 2007). Tumor necrosis factor alpha (TNF-α) increases the production of ROS while interleukin 1 beta (IL1-β), TNF-α, and interferon gamma (INF-γ) uphold the nitric oxide synthase expression in inflammatory as well as epithelial cells (Federico et al. 2007). Hinson et al. (1996) demonstrated in one of their experimental animal studies that pristineinduced plasma cell tumors need inflammatory mediator IL-6 for their development which is produced by increased levels of prostagandin E2 (PGE2) in inflammatory macrophages. Prostaglandin induces the inflammatory cytokine production, leading to enhanced production of ROS and RNS. COX-2 is regarded as the crucial enzyme that retaliates to inflammation by synthesizing prostaglandins in monocytes and macrophages. Reports suggest that COX-2 is upregulated in several cancer types (carcinoma of breast, lung, colon, pancreas, esophagus, and head & neck) (Lin et al. 2002). Remarkably, prostaglandin synthesis can also be triggered by peroxynitrite (toxic radical derived from nitric oxide). This continual oxidative environment leads to damage of healthy neighboring cells and its chronic presence can trigger carcinogenesis (Federico et al. 2007).

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Table 1 How oxidative stress corresponds with the stages of neoplastic transformation Stages of neoplastic transformation Initiation

Promotion

Progression

Corresponding functions of oxidative stress DNA strand breakage Point mutations Abnormal DNA cross-linking Mutations in proto-oncogenes and tumor suppressor genes Unusual gene expression Impediment of cell-to-cell communication Alteration of secondary messenger system Upregulated transcriptional factors Upturned cellular proliferation and/or a decrease in the apoptosis Further addition of DNA modifications

Inflammation, Reactive Oxygen Species, and Cancer Several chronic inflammatory conditions dispose normal but potential cells to malignant transformation. Chronic inflammation is associated with various carcinogenesis steps (Coussens and Werb 2002). Pathogenesis of cancer development can be described under three headings: initiation, promotion, and progression. Oxidative stress corresponds with all the three stages of neoplastic transformation. In the first stage of cancer initiation, ROS may cause damage to DNA, thus fostering neoplastic development (Meira et al. 2008) (Table 1). Following chronic inflammation this initiation of carcinogenesis, propagated by ROS and RNS, is direct or indirect. During promotion, ROS can be attributed to various abnormal activities (Table 1). Transcriptional factors involved in carcinogenesis like c-fos and c-jun oncogenes are upregulated by ROS (Khansari et al. 2009). This facilitates in an upturned cellular proliferation and/or reduced apoptosis of the initiated cell population. Lastly, by further adding DNA modifications in the initiated cells, cancer progression is promoted by oxidative stress (Reuter et al. 2010). A noteworthy feature of cancer promoters is their capability to employee inflammatory cells so that they can generate ROS and facilitate cancer growth. Further damage to DNA is increased by inflammatory cells through activation of procarcinogens to DNA-damaging species. Cytokines like TNF-α along with ROS and RNS activates nuclear factor kappa light chain enhancer of activated B cells (NF-κB) through phosphorylation and consequent proteasomal degradation. The activated NF-κB relocates to the nucleus and further activates specific gene transcription. NF-κB thus incites regulation of genes associated with cellular proliferation and apoptosis and also enhances production of pro-inflammatory cytokines that will further increase the inflammatory responses (Khansari et al. 2009). Neutrophils may trigger aromatic amines, alfa-toxins, estrogens, phenols, and polycyclic aromatic hydrocarbons through pathways involving ROS. In addition, large amount of superoxide, hydrogen peroxide, and hydroxyl radical is released by neutrophils and macrophages following activation of their redox metabolism. Results of some earlier

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experiments had revealed that oxidative stress upsurge the rate of mutation and acts as a DNA-damaging factor leading to malignant transformation. Studies have disclosed that ROS can lead to tumor development, growth, angiogenesis, and metastasis by activating explicit signaling pathways (Reuter et al. 2010). Nitrosative stress triggers activator protein-1 (AP-1) leading to inflammation-related oncogenesis thus upholding transformation and proliferation (Cerutti and Trump 1991).

Tumor Cell Survival A key characteristic of tumor cells that out marks those from normal cells are their ability to survive within host microenvironment. Animal and human models have shown role of ROS in facilitating cancer survival by facilitating cellular signal transduction pathways. The pathways are associated with the channeling of intercellular or intracellular information and support tumor cell survival and cell providence. NADPH oxidase (NOX) family is a likely source of ROS and their reduced level is reported to uphold cancer cell survival and growth (Reuter et al. 2010). The serine-threonine kinase Akt is delineated in downregulation of antioxidant defenses and encouragement of tumor cell survival. By restraining phosphatase and tensin homolog which is deleted from chromosome 10 (PTEN), ROS can activate Akt, which may advance carcinogenesis by several ways, including stabilization of c-Myc and cyclin D1 or by provoking destruction of the cyclindependent kinase (CDK) inhibitor, p27 kinase inhibitor protein (p27kip1) (Reuter et al. 2010; Manning and Cantley 2007). By inactivating pro-apoptotic molecules caspase-9 and the Bcl-2 homology 3 (BH3) and prompting the action of the transcription factor NFκB, Akt inhibits apoptosis of cancer cells and thus facilitates their survival. Also, Akt assists mouse double minute 2 homolog (MDM2), which counters apoptosis mediated by p53. Averting apoptosis and enhancing oxidative metabolism, Akt stays at the core of the intricate signaling systems that amalgamate several potentially carcinogenic signals (Reuter et al. 2010; Manning and Cantley 2007).

Tumor Cell Proliferation Unrestrained propagation of cancer cells necessitates the upregulation of multiple intracellular signaling pathways. Orientation of redox-sensitive pathways becomes necessary for tumor cell multiplication as mitosis requires great amount of energy. The metabolites produced from such energy-generating reactions need to be safeguarded in order to avoid oxidative harm and thus cell death. The considerable effect of oxidants on signaling pathways is perceived in the mitogen-activated protein (MAP kinase)/activator protein 1 (AP-1) and NF-κB pathways. MAP kinase family controls expression of genes by phosphorylation of a variety of transcription

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factors (Müller et al. 1997). Of this, the extracellular signal-regulated kinase (ERK) pathway is frequently related with the modulation of cellular multiplication. Any fluctuations in the redox equilibrium of the cell can trigger stimulation of ERK, c-Jun N-terminal kinase (JNK), and p38 subfamilies, thus promoting proliferation of malignant cells (Xia et al. 1995). JNK and p38 MAP kinase pathways can mediate initiation of AP-1 through hydrogen peroxide, cytokines, and many other stressors. Once triggered, JNK proteins develop transcriptional activities through the translocation to the nucleus and phosphorylation of c-Jun and activation of transcription factor-2 (ATF-2) (Reuter et al. 2010). NF-κB modulates numerous genes concerning with cellular alteration, propagation, and angiogenesis. It has been found that expression of NF-κB can potentiate the proliferation of cells, while its inactivation blocks it. Redox status influences regulation of NF-κB. Many cancers including squamous cell carcinomas are believed to constitutively prompt NF-κB inactivated form (Reuter et al. 2010). Gloire et al. (2006) concluded that even a slight oxidative stress can meek NF-κB activation whereas large oxidative stress impedes the same. ROS are the second messengers that intricate in NF-κB activation via TNF and IL-1. Certainly, downregulation of TNF and IL-1 were shown to suppress active NF-κB and constrain lymphoma proliferation and myelogenous leukemia cells (Giri and Aggarwal 1998).

Tumor Cell Invasion Several phases in the metastatic cascade are monitored by redox signaling. By raising the rate of cancer cell migration, free radicals may advance invasion and metastasis of the cancer. When any malignancies of epithelial origin transforms into invasive carcinoma, the cancerous epithelial cells undergoes intense changes in terms of morphology and adhesiveness that results in a loss of normal epithelial differentiation and polarity, with a swap to a more motile and invasive phenotype. Oxidative stress monitors the intercellular adhesion protein-1 (ICAM-1) expression by NF-B activation. ICAM-1 along with IL-8 controls the transendothelial relocation of neutrophils and plays a major role in tumor metastasis (Roebuck 1999). Role of matrix metalloproteins (MMPs) in invasion and metastasis of cancer cells of diverse histologic origin is another area of interest in carcinogenesis. Mori et al. (2004) found marked upregulation of MMP-13, MMP-3, and MMP-10 by the oxidants which can be inculpated in the invasion potential in NMuMG cells. MMPs like gelatinases (MMP-2 and -9) play a vital role in tumor invasion and metastasis, and are triggered by prolonged oxidative treatment. ROS play a role in MMP gene expression (Reuter et al. 2010). Moreover, many studies have stated the contribution of chemokines and chemokines receptors in the cancer metastasis by inducing the expression of different MMPs that aids cancer cell invasion. Inhibition of cellular proliferation, adhesion, chemotaxis, and invasion was observed by Wen

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et al. in mucoepidermoid carcinoma when CXCR4 gene was sliced endogenously. Recent data suggests the role of the small guanosine triphosphatase Rac1 (GTPase Rac1) along with ROS in cancer cell motility and invasion by varying cell-cell and cell-matrix adhesion (van Wetering et al. 2002).

Angiogenesis Several solid tumors are known to stimulate angiogenic reaction by the host blood supply for neoangiogenesis to derive oxygen and other nutrients. This neoangiogenesis is somewhat responsible for cancer growth and metastasis. Hypoxia, nutrient deprivation and ROS are some of the key stimulants for angiogenic signaling. Overexpression of Ras has been related to tumor angiogenesis (Reuter et al. 2010). Distinctly, transformation by Ras alleviates hypoxia-inducible factor-1 (HIF-1) and upregulates the transcription of vascular endothelial growth factor A (VEGF-A). In addition to HIF, several other mechanisms involving a number of other molecules like NF-κB and p53 are reported. VEGF provokes a vital role in stimulating angiogenesis, which leads to activation of signaling enzymes and helps in angiogenic-related responses in endothelial cells (Ushio-Fukai and Alexander 2004) (Fig. 1). Apart from this, several oncogenes and tumor suppressor genes that are usually linked with cell transformation such as Ras, cellular myelocytomatosis (c-Myc), murine sarcoma 3611 oncogene (RAF), human epidermal growth factor receptor-2 VEGF Receptor tyrosine kinase VEGF receptor-2, fetal liver kinase 1/ kinase insert domain receptor (Flk1/KDR)

Endothelial cell proliferation and migration

Tyrosine phosphorylation of KDR

Activated downstream signaling enzymes

Angiogenic-related responses in endothelial cells

Angiogenesis Fig. 1 VEGF and Angiogenesis Stimulation

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(HER-2/neu), c-Jun, and steroid receptor co-activator (SRC) may control tumor vascularization by either expression of VEGF or downregulation of angiogenesis suppressor thrombospondin-1 (Mazure et al. 1996). As a response to ROS, cancer cells and/or inflammatory cells releases angiogenic factors like VEGF, fibroblast growth actors (FGF), and platelet-derived growth factor (PDGF) within the tumor microenvironment. New blood vessels are formed by endothelial cells activate by these factors. Monte et al. (1997) have confirmed that lymphocyte-induced angiogenesis is activated by oxidative stress within the tumor.

Oral Squamous Cell Carcinoma and Reactive Oxygen Species Several studies have been carried out on serum levels of antioxidants of OSCC patients. Studies have suggested that serum antioxidant capacity is a disposing feature in OSCC pathogenesis. It is not a pathological feature and stays unchanged even if the cancer is cured. Srivastava et al. (2012) testified that ROS and their toxic outcomes are related to the pathogenesis of OSCC. High lipid peroxidation levels with a low antioxidant level in the serum of OSCC patients. They also found an increased tissue levels of free radicals and decreased levels of superoxide dismutase (SOD), glutathione (GSH), glutathione peroxidase (GPx), and catalase in various stages of OSCC (II, III, and IV). Metgud and Bajaj (2014) stated the saliva as a diagnostic tool in assessing malondialdehyde (MDA) and GSH levels in OSCC patients with a habit of smoking (Fig. 2). Factors related to regulation of ROS and antioxidant defense system (oxidant sources):

Tumor Microenvironment and Reactive Oxygen Species Genetically transformed cancer cells in presence of stromal cells from the microenvironment are well established. Mainly, two mechanisms play a vital role in deciding the biological behavior of malignancy and metastasis (Fig. 3 and Table 2).

Fig. 2 MDA and GSH levels in OSCC (Metgud and Bajaj 2014)

Tobacco (exogenous source of ROS) ------Æoxidative stress

Healthy OPMDs OSCC

-MDA -GSH

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Mechanisms

Structure and function basedCell-based Matrix configuration, hypoxia, acidity

CAFs, macrophages, CAMs, endothelial precursors

Stromal fibroblasts

TGFβ1 Increased intracellular ROS level

Changed gene expression

HGF,Ilk-6,VEGF

signals for tumour cell migration stromal oxidative stress Senescence senescent fibroblasts senescence-activated secretory pathways, SASPs proinflammatory cytokines and proteases inflammatory environment promote tumor progression Fig. 3 Mechanisms involving ROS in biological behavior of malignancy (Kalluri and Zeisberg 2006; Davalos et al. 2010)

Cancer associated macrophages (CAMs) synchronize with cancer associated fibroblasts (CAFs) promoting a prooxidant environment by different mechanisms (Coussens and Werb 2002; Grivennikov et al. 2010) (Table 3). Cells as a response to hypoxia activate stress signal reaction, triggering hypoxiainducible factor (HIF-) 1 and -2 transcription. Hypoxic cells result in prevention of

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Table 2 Components of senescence-activated secretory pathways (Davalsos et al. 2010)

1 2 3 4 5 6 7

109 Soluble signaling factors Chemokines IGF-1 Proteases Plasminogen activators: tissue-type uPA receptor Inhibitors of plasminogen activator

Table 3 Various mechanisms involving CAMs and CAFs Mechanism Stimulation of macrophage NOX-2 and inducible NOS CAMs produce pro-inflammatory cytokines Hypoxia

Action Constant ROS production Manage inflammation in stromal and cancer cells Increased mitochondrial ROS

Result CAFs recruitment or MMPs activation Directly promote invasion and metastasis Cancer cells dissemination (Coussens and Werb 2002; Grivennikov et al. 2010) Uphold tumor progression (UshioFukai and Urao 2009)

Table 4 Effects on cancer cells due to intra-tumoral hypoxia (Harris 2002; Pani et al. 2010; Storz 2005) 1 2 3 4 5 6 7 8 9

Metabolic reprogramming concerning a glycolytic phenotype Over production of ABC transporters Assortment of mutated cells with deficient apoptosis Defense from apoptotic inducers Cancer cells show invasiveness Resistant to apoptosis Resistant to chemotherapy and radiation therapy Adaptive tactics to survive Enhance cells’ antioxidant capacity

HIF resulting in protein equilibrium and triggers off its transcriptional activity (Ushio-Fukai and Urao 2009). Ubiquinone cycle of complex III is the basis of generation of ROS in hypoxic environment (Table 4). Gao et al. (2007) have described the effects of hypoxia in the following manner (Fig. 4 and Table 5): Epithelial mesenchymal transition (EMT) plays a major role in cancer development and progression. There are certain factors, which have been described associated with EMT and stemness, that make the tumor environment redox sensitive and thus responsible for progression of tumor and developing resistance to chemotherapies in several cancer models (Thiery 2002; Giannoni et al. 2011) (Fig. 5 and Table 6).

110 Fig. 4 Hypoxia effects

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Transformed ROS + continual oxidative stress

Activated HIF-1/NF-B

Antitumorigenic effect of antioxidants

Table 5 Factors of tumor microenvironment contributing in cancer development Sr. No. 1 2 3 4 5 6

Factors of tumor microenvironment playing role in cancer progression CAFs Hypoxia Acidity Heightened motility Endurance to stressful environment Realignment of metabolism

Stromal elements are also known to strengthen hypoxic stimuli. 1. CAFs imitate the hypoxic stimuli 2. HIF-1 formation because of oxidative stress but without real oxygen deficit

Reactive Oxygen Species in Oral Cancer ROS are significant to both the etiopathogenesis and management of OSCC via ROS-based strategies. Risk factors, which include deteriorating habits, increase oxidative DNA damage after ROS production (Table 7).

Oncoviruses, Oral Squamous Cell Carcinoma, and Reactive Oxygen Species Oncoviruses like Epstein-Barr virus (EBV) and human papilloma virus (HPV) have been associated with OSCC in various studies and have been implied to act via ROSbased mechanisms (Cao et al. 2012) (Fig. 6).

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EMT by intratumoral hypoxia: a biphasic manner Early mitochondrial ROS delivery

Cell polarization and orientation migration

II delayed phase

ROS acts on HIF-1α stabilization and VEGF

Sustained active motility Stromal elements are also known to strengthen hypoxic stimuli. 1. CAFs imitate the hypoxic stimuli 2. HIF-1 formation because of oxidative stress but without real oxygen deficit Fig. 5 EMT and ROS (Novo et al. 2012)

Table 6 EMT factors affecting tumor microenvironment (Thiery 2002; Giannoni et al. 2011) Sr. No. 1 2 3 4 5 6 7 8 9

Factors correlated with EMT Epigenetic transcriptional program Lose epithelial features Mesenchymal-like motility Achievement of stem-cell like cues Increase in the ratio of expression of CD44 and CD24 Increase in CD133 expression Enhancement of anchorage-independent growth Spheroid formation Selection of tumor initiating cells able to disseminate metastases

EMT and stemness are redox-sensitive and play a role in: Exploiting prooxidant environment Metastatic distribution Resistance to chemotherapies in several cancer models

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Table 7 ROS in OSCC Authors Liu et al. (2014) Lu et al. (2010)

Lee et al. (2013)

ROS in OSCC Increased SOD2 rs4880 in smokers with OSCC Areca nut extract treated OSCC cells leads to: Upregulated MKP-1 Increased autophagy Inhibition of apoptosis Increased levels of ROS ROS-induced Snail family of transcription factors elevated in OSCC patients having areca quid chewing habit and lymph node metastasis

High-risk oncoviruses HPV, HPV 16 & 18 move between nucleus and cytoplasm

Attachment of E2 to mitochondrial membranes

Short isoform of protein E6 Increased ROS in HPV-positive and negative cells

Increased mitochondrial ROS production

Decreased SOD2 and GPX

Fig. 6 Oncoviruses and ROS

Reactive Oxygen Species as an Appealing Target for Intervention (Table 8)

Table 8 Various molecules associated with ROS have been targeted to modify growth and/or development of HNCs or OSCC Author Shrotriya et al. (2012)

Compound/Molecule Extract of grape seed

Hua et al. (2013)

Polyenolpyrrole auxarconjugatin B

Action Constrained growth Arrest of cell cycle Apoptosis DNA damage Effect on DNA repair resulting in irreversible injury and apoptosis ROS generation via cytochrome P450 1A1 DNA damage Apoptosis Inhibition of OSCC xenograft growth, by increased caspase-3 activity (continued)

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Table 8 (continued) Author Chang et al. (2013)

Compound/Molecule Curcumin nanoparticles

Park et al. (2015) Kim et al. (2013)

Endocannabinoid anandamide

Kang et al. (2014)

Non-thermal plasma (a mixture by ionization of neutral gas)

Xanthorrhizol

Action Increased ROS caspase-3 Increased apoptotic proteins caspase 9, cytochrome c, apoptotic protease activating factor 1, apoptosis inducing factor, Bcl2associated X protein Cell proliferation inhibition by increased ROS via a receptor-independent mechanism ROS-mediated p38 MAP kinase and JNK activation Decreased cell viability Apoptosis MAP kinase-mediated increase in mitochondrial ROS Induced apoptosis

Reactive Oxygen Species as Alluring Targets for Therapeutic Intervention There are numerous side effects of radiation therapy on OSCC which can also be changed by interference in the pathways involving redox reactions. It has been shown that concurrent glycolysis inhibition and hanging up of pentose phosphate pathway results in metabolic oxidative stress. It leads to radio-sensitization resulting in apoptosis via JNK/p38 MAP kinase signaling (Sharma et al. 2012). Analogous mechanisms have been seen in various following studies involving alteration of cellular redox environment in reducing side effects of radiation therapy (Table 9): Table 9 Altered ROS as a therapeutic adjunct Author Boivin et al. (2011) Sasse et al. (2006)

Mechanism Depletion of glutathione before radiotherapy Detoxification of reactive metabolites Scavenging of free radicals

Cotrim et al. (2012)

Stable nitroxide, Tempol prior to radiation  cisplatin

Bennett et al. (2012) Moen and Stuhr 2012)

Hyperbaric oxygen treatment for osteoradionecrosis Activation of the MAP kinase pathway Apoptosis subsequent to hyperbaric oxygen

Result Radiation-induced JNK activation Apoptosis Alleviated oral side effects (xerostomia, mucositis, etc.) E.g., Amifostine decreases oral side effects associated with radiation therapy Protection of normal cells from radiation damage No oral ulceration

Decreased cell proliferation

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Reactive Oxygen Species and Oral Potentially Malignant Disorders Recently, the role of oxidants and antioxidants has been established in oral potentially malignant disorders (OPMDs) in explaining their pathogenesis and therapeutic options. These disorders include oral leukoplakia, erythroplakia, and oral submucous fibrosis (OSMF). There are comparative studies available where oxidants and antioxidant levels were compared between OPMDs and OSCC. It has also been reported that in OPMDs not only serum but also saliva has been used to assess the ROS status, an oxidative stress marker. Vlkovà et al. (2012) have found increased levels of salivary lipo-peroxidation, carbonyl stress markers, thiobarbituric acid reacting substances, and advanced glycation end products in patients with oral leukoplakia, and on the other hand, salivary SOD and total antioxidant capacity were found to be low. The disease process from normal mucosa to leukoplakia and then to oral cancer involves a major role of ROS. Srivastava et al. (2016) found a notably reduction in GSH, GPx, catalase, and SOD levels in patients

Table 10 Levels of oxidants and antioxidants in oral potentially malignant and oral cancer patients Authors Vlková et al. (2012)

Conditions Oral Leukoplakia

Srivastava and Shrivastava (2016)

Oral Leukoplakia

Increased levels Lipo-peroxidation Carbonyl stress markers thiobarbituric acid-reacting substances (TBARS) Advanced glycation end-products TBARS and nitrates

Avinash Tejasvi et al. (2014) ChethanAradhya et al. (2018) Sadaksharam (2018) Misra et al. (2016)

OSMF

MDA

GSH GPx Catalase SOD All antioxidant enzymes SOD

OSMF

Serum MDA

Serum catalase

OSMF and OSCC

Serum nitric oxide

Serum SOD

OSMF, Oral Leukoplakia, and OSCC Oral Leukoplakia, OSMF, and OSCC

Serum MDA

Serum vitamin A, vitamin E, and SOD Serum beta carotene, vitamin E, and SOD E-SOD and GPx levels

Rai et al. (2015)

Gurudath et al. (n.d.)

OSCC BID ¼ tBID –a BH3-only activator> BAX/BAK > CASP9) activates CASP3 (Galluzzi et al. 2018). Therefore, CASP3 represents an executer protein which converges the intrinsic and extrinsic apoptosis. Leukemia cell can avoid apoptosis by several mechanisms including overexpression of antiapoptotic protein BCL2 (McBride et al. 2019), dysfunctional expression of the cell cycle gatekeeper protein p53 (Prokocimer et al. 2017), expression of mutated CASPASE 8, and overexpression of X-linked inhibitor of

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apoptosis protein (XIAP, Sung et al. 2009). Although the molecularly targeted drugs and chemotherapy aiming at restoring apoptosis in leukemic cells have shown encouraging activity, some leukemic patients are still refractory to treatment (e.g., venetoclax Birkinshaw et al. 2019). Therefore, further research efforts are necessary to develop new and more efficient treatments against leukemia wherein activation of apoptosis pathways prevails.

Oxidative Distress: The Dose Makes Life or Death Signals Metabolic reprogramming is another prominent strategy used by leukemia cells to evade apoptosis. Consequently, leukemia cells generate higher concentrations of reactive oxygen species (ROS, e.g., hydrogen peroxide, H2O2) than nonleukemic cells (Samimi et al. 2018). Specifically, abnormal mitochondrial metabolism has been identified as the key source for abundant ROS yield. Therefore, depending on the concentration, H2O2 has a dual role in leukemia cells (Reczek and Chandel 2017). At low concentrations, H2O2 can promote protumorigenic signaling (cell proliferation, survival, and adaptation to hypoxia), whereas at high concentrations, it can promote antitumorigenic signaling by triggering apoptosis via oxidative stress (OS). This term is understood as an imbalance between oxidants and antioxidants in favor of the oxidants, leading to a disruption of redox signaling and control/or molecular damage (Sies 2015). Furthermore, OS can be classified as oxidative eustress (OeS), wherein low-level (physiological) OS is used in redox signaling and redox regulation, and as oxidative distress (OdS), wherein high-level (supraphysiological) OS disturbs redox signaling ensuing oxidative damage to biological molecules (Sies 2018). Therefore, the idea of treating leukemia cells with drugs that either directly or indirectly generates H2O2 as an effective therapeutic strategy for selectively pushing them over ROS threshold, i.e., oxidative therapy, and induce apoptosis has ultimately gained scientific momentum (e.g., Harris et al. 2015; Galadari et al. 2017; Chio and Tuveson 2017; Zou et al. 2017; Zhang et al. 2018). Indeed, some antileukemic drugs currently used in clinic (e.g., arsenic trioxide, As2O3) effectively kill leukemic cells by inducing ROS generation. However, the exact mechanism by which some therapeutic molecules generate H2O2 is not yet fully established. Accordingly, our research group has focused on unrevealing the molecular mechanism of OdS-induced apoptosis in leukemia cells by some selected natural or chemical molecules (Fig. 1) including vitamins (e.g., vitamin C (VC) and K3 (VK3) Bonilla-Porras et al. 2011; vitamin E D-α-tocopheryl polyethylene glycol succinate (TPGS) Ruiz-Moreno et al. 2016), snake venoms (e.g., metalloproteinase nasulysin-1 Bonilla-Porras et al. 2016; l-amino oxidase MipLAAO Bedoya-Medina et al. 2019), avocado extracts (Bonilla-Porras et al. 2014), mitochondrial complex I inhibitor rotenone (ROT, Mendivil-Perez et al. 2014), metal chelator TPEN (Mendivil-Perez et al. 2012; Rojas-Valencia et al. 2017); antibiotic doxorubicin (DXR, Mendivil-Perez et al. 2015), and minocycline (MC, Ruiz-Moreno et al. 2018). In addition, we have also investigated the effect of starvation, i.e., glucose starvation (Mendivil-Perez et al. 2013), and fructose as sole energetic source (Diaz-

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Fig. 1 Schematic representation of natural and chemical molecules as source of prooxidant agent H2O2

Aguirre et al. 2016) associated with OdS and apoptosis. We have used the cell line Jurkat, as model of ALL, and K562, as model of CML.

H2O2: “A match that starts bush fires” Because of its oxidant chemical nature, H2O2 has emerged as a central redox signaling molecule (Di Marzo et al. 2018). Specifically, H2O2 has been shown to directly or indirectly activating kinases, redox sensor proteins, and/ or transcription factors through mainly oxidation of cysteine amino acid residues (Marinho et al. 2014; Poole 2015). Interestingly, metabolic alterations (e.g., glucose starvation, fructose as sole energetic source), natural or chemical molecules, generate H2O2 (Fig. 1) through different mechanisms, involving cellular cytoplasmic redox reactions (e.g., VC/VK3, avocado extracts; DOX; MC; TPEN), inhibition of mitochondria complex I and/or II (e.g., ROT; TPGS; MC), metabolic shift from aerobic glycolysis to oxidative phosphorylation (e.g., by fructose), and enzymatic reactions (e.g., MipLAAO). All those reactions result in the reduction of molecular dioxygen (O2) into O2.and the subsequent (enzymatic or spontaneous) dismutation of O2.- into H2O2 (Fig. 2, step1). One of such sensor molecules that reacts specifically with H2O2 is

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Fig. 2 Schematic representation of H2O2-induced molecular signaling of cell death in leukemia cells

protein DJ-1 (step 2), a multifunctional OS-sensor protein with antioxidant activity (Oh and Maral Mouradian 2018). As shown, the Cys106 residue of DJ-1 is the residue most sensitive to H2O2-mediated oxidation (Kinumi et al. 2004). In fact, DJ-1

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(i.e., DJ-1 Cys106 thiol residue) is sequentially oxidized by H2O2 to Cys106-sulfenate (SO), Cys106-sulfinate (SO2), and then Cys106-sulfonate (SO3). Currently, DJ-1 constitutes an important intracellular antioxidant and/or sensor protein accepted as a valid marker of H2O2 redox signaling (e.g., Fig. 3a). Remarkably, H2O2 is also capable of triggering the activation of two independent but complementary intrinsic death subroutines. The first one suggests that once H2O2 has been produce by prooxidant agents (e.g., Fig. 2), it might indirectly activate the “master regulator” of OS transcription factor NF-κB (Fig. 3b) via phosphorylation of IκBα either by (i) the Syk kinase (Fig. 2, step 3) at the tyrosine42 residue, (ii) the SHIP-1 phosphatase (step 4)/IKK complex pathway at the serine32 and serine36 residues, or (iii) MEKK1 (step 5) via IKK. Once NF-κB is free from its inhibitor, IκBα, the NF-κB dimer (step 6) translocates into the nucleus and transcribes several antiapoptotic and proapoptotic genes, including p53 (step 7 and

Fig. 3 (a) Minocycline induces reactive oxygen species and DJ-1 oxidation in Jurkat cells. Cells (1  106 cells per well/mL) were left untreated or treated with Mino (100, 200 μM) or H2O2 (1 mM) for 24 h at 37 °C. Proteins in the nuclear extracts were blotted with anti-DJ-1 Cys106 -SO3 (sulfonic) (upper panel, oxidized DJ-1), anti-DJ-1 (middle panel, total DJ-1) and anti-ACTIN (lower panel) antibodies. Numbers on the top of representative blots represent the fold increase (fi) in DJ-1 oxidation with respect to DJ-1 oxidation in the control. (b–d) Minocycline induces activation of apoptotic-related transcription factors NF-κB, P53, and c-JUN; (b) induces overexpression of the apoptotic-related mitochondrial maintenance PINK1/PARKIN system and BAX/PUMA proteins; (c) and induces activation of apoptotic-related nuclei-dismantling CASPASE-3 and AIF proteins in Jurkat cells (d). Cells (1  106 cells per well/mL) were left untreated or treated with Mino (50, 100, 200 μM) for 24 h at 37 °C. Protein extracts were blotted with anti-NF-κB, anti-P53, anti-c-JUN, anti-PINK1, anti-PARKIN, anti-BAX, anti-PUMA, anti-CASPASE-3, anti-AIF, and anti-ACTIN antibodies. (Figures a-d are reproduced from Ruiz-Moreno et al. 2018 under a Creative Commons attribution-type BY-NC)

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Fig. 3). In turn, transcription factor p53 transcribes proapoptotic genes such as PUMA (step 8 and Fig. 3c) and BAX (step 9 and Fig. 3c). Both BAX and PUMA contribute to MOMP (step 10), and loss of ΔΨm, thereby opening of the mitochondrial transition pore (by a mechanism not yet fully understood). Consequently, release of apoptogenic proteins such as AIF (step 11 and Fig. 3d) and cytochrome C, which together with Apaf-1, dATP, and procaspase-9 (i.e., the apoptosome) elicit CASPASE-3 protease activation (step 12 and Fig. 3d), is responsible for causing the stage I nuclear morphology (step 13 and Fig. 4a,b) and stage II nuclear morphology (step 14 and Fig. 4a versus 4c), respectively, typical of apoptosis (Fig. 4d versus 4e-k). The second death subroutine initiates with the dissociation of thioredoxin (Trx, a small redox protein) from the ASK1/Trx complex through H2O2 oxidation of the redox-active Cys32 and Cys35 thiol residues of Trx. Once ASK1 is free from its inhibitor, Trx, ASK1 autophosphorylated (i.e., p-ASK1 Thr 845, Fig. 2, step 15) and signals downstream JNK kinase activation (step 16) through MKK4, which in turn activates the transcription factor c-JUN (step 17 and Fig. 3b), and then c-JUN upregulates PUMA (Bonilla-Porras et al. 2018) (step 8). Noticeably, PUMA represents a convergent factor of both the ASK1/JNK and NF-κB/p53 pathways. Since MEKK1 kinase phosphorylates both the IKK/NF-κB and MKK4/JNK/c-Jun pathways, it also represents a crosstalk between the JNK and NF-κB pathways. During the last years, it has been shown that PINK1 (step 18 and Fig. 3c)/ PARKIN (step 19 and Fig. 3c) pathway play an important role in maintaining mitochondrial function and in preventing OS. How these two proteins are related with OS is a matter of research. However, it has been shown that p53 upregulates PARKIN expression through transcriptional regulation. Then, PARKIN can either directly interact with H2O2 (Kitada et al. 2016), probably serving as antioxidant molecules, or translocate from the cytosol to damaged mitochondria. Because dissipation of ΔΨm activates PINK1, this kinase activates the E3 ligase function of PARKIN, which in turn activates NF-κB signaling to the IκB kinase/NF-κB pathway. The PINK1/PARKIN interaction might amplify the NF-κB/p53 death signal axis (blue lines). Clearly, H2O2 induces a vicious cycle wherein it indirectly triggers PINK1, which in turn activates (>) PARKIN > NF-κB > p53 > PARKIN. Our findings suggest that PINK1/PARKIN might be important target proteins to increase leukemia cell death upon exposure to OS stimuli (Mendivil-Perez et al. 2014; Ruiz-Moreno et al. 2018).

Quo Vadis? The idea of using mega doses of VC (ascorbic acid ¼ ascorbate) to fight cancer was first postulated by Linus Pauling (Nobel Prize of Chemistry, 1954 and Peace, 1962) and Ewan Cameron (M.D.) in 1974. After providing theoretical as well as experimental support for their claim, they ignited an almost endless controversial issue: Whether or not high doses of VC can be used to treat cancer, and specifically, for the treatment of leukemia. Despite historical controversy, it has recently been

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Fig. 4 Representative fluorescence microphotography of the chromatin condensation and nuclear fragmentation in Jurkat cells untreated (a, d) or treated (b, c, e–k) with natural and/or chemical agents depicted in Fig. 1. (Figures a, b, and c are reproduced from Bonilla-Porras et al. 2014 with permission from Informa Health UK Ltd., and Fig. j is reproduced from Ruiz-Moreno et al. 2016 under a Creative Commons attribution-type BY-NC)

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demonstrated that IV VC administered twice weekly at a dose of 70 g/infusion initiated in 2014 to a 52-year-old female with relapsed AML remain in complete remission until recently (Foster et al. 2018). Although this is a single encouraging case report, it is to be hoped that further clinical trials will yield more information about the high-dose IV VC treatment in leukemia patients. Furthermore, in agreement with our previous observations (Bonilla-Porras et al. 2011), it has been demonstrated that VC and VK3 are redox system highly toxic for leukemia lymphocytes, and sensitizer when used in conjunction with other anticancer drugs. Taken together, these data strengthen the view that pharmacological doses of VC alone or in combinations with clinically used drugs (Blaszczak et al. 2019) might exert beneficial effects in the treatment of leukemia. The antibiotic doxorubicin (DOX) is a drug approved by FDA for the treatment of ALL, AML (https://www.cancer.gov/about-cancer/treatment/drugs/leukemia), and other types of cancer (http://chemocare.com/chemotherapy/drug-info/doxorubicin. aspx). However, DOX has been proved for cardiac toxicity, e.g., in children. Therefore, the search of breakthrough analogs is urgently required. Recently, we have demonstrated that the antibiotic minocycline (MC) specifically induces apoptosis in ALL Jurkat cells (Ruiz-Moreno et al. 2018). Because MC has wellestablished clinical pharmacokinetic and pharmacodynamics properties, this tetracycline is a promising antileukemic drug deserving further clinical trial investigation.

Conclusion On the condition that new natural and/or chemical agents demonstrated cell-type selectivity, we anticipate that such agent(s), as described in the present review, e.g., MipLAAO (Bedoya-Medina et al. 2019), TPEN (Mendivil-Perez et al. 2012; RojasValencia et al. 2017), TPGS (Ruiz-Moreno et al. 2016), and MC (Ruiz-Moreno et al. 2018), endowed with prooxidant activity might have an important therapeutic impact on leukemia cells. However, further preclinical (i.e., ex vivo and in vivo) and clinical research is required to establish their original therapeutic benefit in leukemic patients. Acknowledgements This work was supported by the University of Antioquia, UdeA, and “Fundación Alfonso Moreno Jaramillo” grants #2017-16748 and #2018-20454 to CV-P and MJ-Del-R. The funders had no role, data collection and analysis, decision to publish, or preparation of the manuscript.

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Amlan Das, Santanu Paul, Subhendu Chakrabarty, Moumita Dasgupta, and Gopal Chakrabarti

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Importance of Microtubules in Anticancer Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ROS: A Friend or Foe . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ROS in Cancer Development and Metastasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cytotoxic Role of ROS in Cancer Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mitochondria: The Missing Link Between MTAs and ROS Generation in Cancer Cells . . . . . Modulation of ROS-Mediated Apoptotic Signaling by MTAs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Microtubule Targeting Agent Kills Cancer Cells by Modulating ROS Induced Autophagy . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract

Cancer is a devastating disease worldwide and is characterized by elevated reactive oxygen species (ROS). ROS overproduction has been identified in Amlan Das, Santanu Paul and Subhendu Chakrabarty contributed equally with all other contributors. A. Das Department of Biotechnology and Dr. B.C. Guha Centre for Genetic Engineering and Biotechnology, University of Calcutta, Kolkata, WB, India National Institute of Biomedical Genomics, Kalyani, Nadia, WB, India S. Paul · M. Dasgupta · G. Chakrabarti (*) Department of Biotechnology and Dr. B.C. Guha Centre for Genetic Engineering and Biotechnology, University of Calcutta, Kolkata, WB, India e-mail: [email protected] S. Chakrabarty Department of Biotechnology and Dr. B.C. Guha Centre for Genetic Engineering and Biotechnology, University of Calcutta, Kolkata, WB, India Department of Microbiology, M.U.C Women’s College, Burdwan, India © Springer Nature Singapore Pte Ltd. 2022 S. Chakraborti et al. (eds.), Handbook of Oxidative Stress in Cancer: Mechanistic Aspects, https://doi.org/10.1007/978-981-15-9411-3_46

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various cancers which serves numerous roles in cancer progression and tumorigenesis. Microtubule targeting agents can be natural or synthetic small molecules which can target cellular microtubule dynamics. Microtubule targeting agents are widely studied and are used in clinical interventions as well for different cancer therapeutics. Microtubule dynamics and ROS both are interrelated in caner formation and progression ROS can activate pro-tumorigenic signaling to enhance genetic instability, DNA damage, cell proliferation, metastasis, angiogenesis, and tumor development. However, counter intuitively ROS can also promote antitumorigenic property, which can initiate oxidative stress-mediated cell death. Many cellular microtubule targeting chemotherapeutic drugs can produce ROS to promote cancer cell death by primarily through apoptosis and autophagy. This book chapter discusses about how microtubule targeting agents showing anticancer properties with the generation of ROS-mediated apoptosis by activating different cellular signaling pathways. Keywords

Reactive oxygen species · Microtubule targeting agents · Cancer · Mitochondria · Apoptosis · Autophagy · Therapeutics

Introduction Reactive species of oxygen are known to be essential for living organisms since they participate in various physiological processes such as regulation of cell signaling, cell growth and differentiation, apoptosis, modulation of enzyme activities, stimulation of cytokine production, and also the elimination of foreign pathogens (Liou and Storz 2010; Redza-Dutordoir and Averill-Bates 2016). Reactive oxygen species (ROS) are short-lived, highly reactive molecules, which are formed by one or more unpaired electrons on oxygen, resulting in its incomplete reduction. These highly unstable and partially reduced reactive oxygen species may be broadly classified into two types: free radicals and nonradical species. The reactive oxygen free radicals include a wide spectrum of highly reactive radicals including superoxide anions (O2•ˉ), hydroxyl radical (OH•), peroxyl radicals (ROO•), nitric oxide (NO•), alkoxyl radicals (RO•), thiyl radicals (RS•), thiylperoxyl radicals (RSOO•), and sulfonyl radicals (ROS•) to name a few. The nonradical entities include hydrogen peroxide (H2O2), singlet oxygen (1O2), ozone (O3), organic hydroperoxides (ROOH), and hypochlorous acid (HOCl) (Ray et al. 2012). Similar to ROS, there is another family of highly reactive molecules, which are formed from the chemical reactions involving Nitric Oxide (NO). These reactive species are known as Reactive Nitrogen Species or RNS. These RNS include a variety of nitrogen-containing reactive species such as peroxynitrite (ONOOˉ), nitrosoniumcation (NO+), nitroxyl (NOˉ), higher oxides of nitrogen, S-nitrosothiols (RSNOs), and also dinitrosyl iron complexes (Martinez and Andriantsitohaina 2009). Both these ROS and RNS can act as strong oxidants, reacting with the biological macromolecules and causing extensive cellular damage (Ping et al. 2020).

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Microtubule targeting agents (MTAs) comprise of a variety of natural and synthetic small molecules which target the cellular microtubule dynamics and architecture to induce apoptosis in the cancer cells (Cermak et al. 2020; Parker et al. 2014). Microtubules are dynamic cytoskeletal proteins which participate in a variety of physiological processes of which cell division and intracellular transport deserve special mention (Parker et al. 2014; Steinmetz and Prota 2018). These functions are possible only due to the dynamic behavior of cellular microtubules. The beauty of MTAs is that they can inhibit the growth of cancer cells in a doseresponsive fashion. At lower doses, MTAs can perturb microtubule dynamics without damaging the microtubule architecture and by this property they can induce cell cycle arrest at different phases of the cell cycle (Das Mukherjee et al. 2016). Although there is no direct mechanism by which the MTAs can produce ROS, there are substantial experimental shreds of evidence that disruption of cellular microtubules can induce significant ROS generation in cancer cells which in turn activates the apoptotic signaling (Rovini et al. 2011).

Importance of Microtubules in Anticancer Therapy Microtubules are cytoskeletal polymers composed of tubulin heterodimers (α and β), which are present in all eukaryotic cells and facilitate numerous physiological processes such as cellular structure, function, motility, transport, segregation of chromosome during cell division, etc. (Cermak et al. 2020; Parker et al. 2014). Microtubules exhibit two interesting dynamic properties known as dynamic instability and treadmilling, which play crucial roles in modulating microtubule functions inside the cell. Dynamic instability is the intrinsic property of the microtubule, which refers to the ability of the individual microtubules to oscillate between the states of growth and shortening (Cermak et al. 2020). At high concentrations of GTP-tubulin dimmers, the rate of addition of tubulin is faster than the rate of dissociation of GTP-tubulin from microtubule ends. Thus, the microtubule grows, leading to the formation of a stable GTP cap at the (+) end of the microtubules, while at low concentrations of GTP-tubulin, the rate of addition of tubulin to (+) end is diminished, and thus, the rate of GTP hydrolysis surpasses the rate of addition of tubulin subunits. Consequently, an unstable GDP cap is formed, which leads to the disintegration of microtubules in tubulin subunits (Parker et al. 2014). In addition to dynamic instability, microtubules possess another dynamic characteristic known as the “treadmilling,” which is defined as the ability of the microtubules to grow at their “+” ends and shorten at the “
” ends. Treadmilling involves the flow or “flux” of tubulin subunits from the “+” end of the microtubules to the “
” end. Treadmilling occurs due to the difference in the critical protein concentrations at the two ends (Steinmetz and Prota 2018). The microtubule dynamics maintain a chemical equilibrium between the intracellular pool of tubulin heterodimers and microtubule polymers that regulate the cell cycle and cytoskeleton architecture. In dividing cells undergoing mitosis, the microtubule assembles to form a mitotic spindle at the metaphase. During anaphase, the

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shortening of the spindle microtubules takes place, which results in the segregation of the sister chromatids. The rate of microtubule elongation and shortening increases rapidly during mitosis, which facilitates the whole process of chromosomal division. This dynamic property of microtubules makes them potential targets for chemotherapy and anticancer drugs. The majority of microtubule-binding agents (MTAs) bind to the α/β interface of the β-tubulin subunit and suppress microtubule dynamics, resulting in the delay or blockade at the metaphase-anaphase transition during mitosis. Due to the inhibition of the cell cycle progression, apoptotic signaling gets activated, resulting in cell death. Various kinds of agents mainly isolated from the plant products or chemically synthesized are reported to bind tubulin-microtubule and modulate microtubule dynamics either by promoting the assembly or disassembly of the microtubule network (Verma et al. 2017). On the basis of their functions, microtubule targeting drugs may be classified into two categories: microtubule-destabilizing and microtubule-stabilizing drugs (Fig. 1). Microtubule-destabilizing agents bind primarily to α/β-tubulin heterodimers and thus inhibit polymerization of microtubule, whereas microtubule-stabilizing drugs have the capacity to bind microtubule polymer which acts mainly by stabilizing microtubules. The first class of compounds includes the colchicine and vinca alkaloid types of drugs such as vimblastin and vincristine, and several other drugs like podophyllotoxin and the second class of compounds are the microtubule-stabilizing drugs exemplified by the group of drugs, including paclitaxel, epothilones, eleutherobin, discodermolide, parthenolide (Zhao et al. 2016). However, based on the binding site on tubulin heterodimer, the microtubule targeting agents (MTA) may be classified into three types (Fig. 1):

Fig. 1 Disruption of Microtubule dynamics by the MT targeting agents. Three different binding site in MT structure, namely, Taxane, Colchicine, and Vinca alkaloids binding dough

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(i) Agents binding at the colchicine-binding site on tubulin: Colchicine, a tricyclic alkaloid, extracted from the plant Colchicum autumnale, binds to a specific domain located at the interface of α and β-subunits. The colchicine binding site or CBS on tubulin heterodimer is composed of helix 7 (H7) containing Cys-241, loop 7 (T7) and helix 8 (H8), in which the trimethoxyphenyl group of colchicine forms a hydrogen bond with Cys-241on the β-subunit, and the residues Thr-179 and Val-181 on α-subunit form hydrogen bonds with the tropolone ring of colchicine. A wide variety of compounds are known to interfere with the CBS on tubulin and induce microtubule depolymerization. They include Combretastatin A-4 and its analogs such as chalcone derivatives, phenstatin derivatives, dihydronapthalene derivatives, quinazolinone derivatives, pyrazole analogues, and 1,2,4-Triazole-derivatives to mention a few; Podophyllotoxin and its analogues; curacin A; Benzimidazoles like Nocodazole; 2-methoxyestradiol; Avanbulin; Nitrobenzoate IMB5046; Imidazole BZML; Crolibulin; Tivantinib; Plinabulin; Lisavanbulin; Crolibulin, etc. (Cermak et al. 2020; McLoughlin and O’Boyle 2020). All these compounds bind tubulin dimer at or near CBS and inhibit tubulin polymerization and promote microtubule disassembly. A few of them had also entered clinical trials against several carcinomas. Our groups had also reported a wide variety of natural and synthetic compounds that can induce cell cycle arrest and apoptosis in cancer cells by binding at or near CBS of the tubulin dimer. They include natural compounds such as Genistein, Vitamin K3, Thymoquinone, EGCG, Apocynin and also chemically synthesized novel anticancer compounds such as bis(indolyl)-hydrazide-hydrazone derivatives-NMK-BH3 and NMK-BH2, and 5-(3-indolyl)-2-substituted-1,3,4-thiadiazole derivatives or NMK-TD100). (ii) Compounds binding at the vinblastin-binding site on tubulin: Vinblastine or vincaleukoblastine, isolated from the alkaloids of Madagascar periwinkle (Catharanthus roseus) plant, is a potent anticancer drug that binds to a specific binding site on tubulin, known as the vinca domain. The vinca domain is located at the α/β interface site between two tubulin heterodimers. The binding of vinblastin to tubulin is quite fast, and there are one or two high-affinity sites and several low affinity-binding sites available on the tubulin dimer interface. It has been observed that vinblastine and colchicine did not affect each other’s binding to tubulin rather; vinblastine protects the colchicine binding site against decay (Verma et al. 2017). The vinblastine binding site was located near the taxol-binding site on tubulin (β217–231), and hence, taxol binding was found to be inhibited by vinblastine. Apart from the vinca alkaloids such as vinblastine, vincristine, vinorelbine, many other drugs are known to bind tubulin at or near the vinca domain. These include eribulin, rhizoxin, maytansine, the marine sponge-derived Hemiasterlin, Spongistatin, Halichondrin-B, Dolastatin 10, etc., the cyanobacterium-derived Cryptophycin (Nostoc sp.), and the black tea component theaflavin to name a few (Cermak et al. 2020). (iii) Compounds binding at the taxol-binding site on tubulin: The third well-studied binding pocket is the taxane site, found on β-tubulin in the lumen of

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microtubules (Alushin et al. 2014). Paclitaxel (Ptx), also known as taxol (isolated from the bark of the Pacific Yew tree, Taxus brevifolia), is a very commonly used drug in cancer chemotherapy. Taxol promotes microtubule assembly, and hyper stabilizes the structure of microtubules and thus inhibits microtubule dynamics. Notable mentions of this group include paclitaxel, epothilones, eleutherobin, discodermolide, etc., which stabilize the microtubule structure by perturbing its dynamic properties (Fig. 1).

ROS: A Friend or Foe ROS plays a crucial role in the regulation of various cellular processes, including cellular proliferation, differentiation, and death. The fate of the cell is dependent on the critical balance of ROS levels maintained intracellularly or influenced by external factors. Cells generally tend to overcome the oxidative burden by maintaining a delicate balance to confine the ROS levels within a threshold limit, by a process known as redox homeostasis. Redox homeostasis in cancer cells is mainly achieved by the intracellular antioxidant proteins, which generally remain elevated compared to noncancerous cells (He et al. 2017). Within the threshold limit of redox homeostasis, cancer cells may utilize a highly controlled ROS for aiding their pathophysiological activities such as proliferation, differentiation, and even migration. But any disturbance of redox homeostasis leads to the elevation of ROS levels in the cells, which can cause irreversible oxidative damage of cellular macromolecules, thus leading to cell death (Acharya et al. 2010) (Fig. 2).

Fig. 2 Balance of Reactive oxygen species (ROS) in normal and cancer cell. ROS homeostatic in normal cell, elevated ROS in cancer cell, and chemotherapeutic targeting drugs develops more elevated ROS inside the cancer cells for inducing its death

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ROS in Cancer Development and Metastasis ROS plays a vital role in tumor initiation, promotion, and progression (Waris and Ahsan 2006). At levels below the threshold, ROS can activate several oncogenes such as Ras, c-Myc, TP53 in cancer cells. Activation of these oncogenes results in an increased mutation load leading to a series of events such as deregulated signal transduction, inhibition of apoptosis, and enhanced generation of intracellular ROS. Enhanced ROS generation further activates the inflammatory cytokines and chemokines such tumor necrosis factor (TNF-α), interleukin 6 (IL-6), transforming growth factor β (TGF-β), and interleukin 6 (IL-6) to name a few (Reuter 2010). These inflammatory cytokines and chemokines, in turn, can modulate certain signaling proteins which can significantly contribute in the development of tumor microenvironment (Aggarwal et al. 2019). ROS has been known to activate the phosphoinositide-3-kinase (PI3K)/protein kinase B (AKT) pathway resulting in the transcriptional inhibition of the apoptosis-inducing genes such as glycogen synthase kinase 3 (GSK3), BCL-2-associated death promoter (BAD) and forkhead box O (FOXO) along with the simultaneous activation of the oncogenic modulator mammalian target of rapamycin (mTOR1) (Kim et al. 2018; Byun et al. 2012). Thus, it can be inferred that intracellular ROS in cancer cells modulate particular signaling cascades, which play pivotal roles in the maintenance of tumor microenvironment, the resistance of available anticancer regimes, and enhanced proliferation by inhibiting the apoptotic cascade. Migration or invasive potential of cancer cells is facilitated by a series of coordinated events known as epithelial to mesenchymal transition (EMT). EMT is a collection of physiological process by which an epithelial cell loses its polarity and the ability of cell-cell adhesion, which is known to be the hallmark of cancer metastasis (Heerboth 2015, Fig. 2).

Cytotoxic Role of ROS in Cancer Cells When the oxidative burden inside the cell exceeds the threshold limit, then the pro-tumorigenic role of ROS may be reverted into a cytotoxic mode. Most chemotherapeutic drugs kill the cancer cells by exploiting the ROS homeostasis via an increased generation of ROS. This chemotherapeutic amplification of intracellular ROS levels significantly increases the oxidative burden far beyond the threshold limit, eventually leading to cell death. A wide array anticancer regimes including platinum-based drugs such as cisplatin, oxaliplatin, and carboplatin; alkylating agents; topoisomerase inhibitors including irinotecan, topotecan, camptothecin, etc., for topoisomerase-I and etoposide; doxorubicin, epirubicin, epipodophyllotoxin, etc., for topoisomerase-II; anthracyclines (cancer chemotherapy drugs isolated from Streptomyces sp.), such as doxorubicin, daunorubicin and epirubicin; microtubule targeting drugs such as taxanes, vinca alkaloids, colchicine, etc.; antimetabolites such as azathioprine, mercaptopurine, and thioguanine (purine antagonist) and fluorouracil and floxuridine (pyrimidine antagonists) are

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responsible for the generation of high levels of ROS in cancer cells (Fig. 2, Ivanova et al. 2016).

Mitochondria: The Missing Link Between MTAs and ROS Generation in Cancer Cells In the seminal work, Bhalla K et al. first demonstrated the apoptotic potential of microtubule targeting drug paclitaxel in myeloid leukemia cells via mitochondriadependent apoptosis. As discussed earlier, ROS plays a pivotal role in cellular apoptosis by triggering various signaling cascades. Among the two well-studied apoptotic pathways, the death receptor-mediated pathway or the extrinsic pathway was generally excluded from MTA-induced apoptosis, rather the role of the mitochondria-mediated intrinsic pathway in MTA-induced cytotoxicity is well documented (Carre et al. 2002). This unique mechanism can be explained with the fact that tubulin was reported to be an integral component of the mitochondrial membrane and regulates the voltage-dependent anion channel (Carre et al. 2002). Interestingly it was also observed that the βIII-tubulin, which is a key regulator of tumor aggressiveness, chemoresistance, and metastasis, is the major tubulin isotype that remained associated with the mitochondrial membrane (Cicchillitti et al. 2008). Since most of the MTAs preferably bind to the β-tubulin, mitochondrial membrane thus became a potential target for these drugs. When targeted by MTAs in cancer cells, disruption of mitochondrial membrane takes place that further resulted in the depletion of MMP and further activation of caspase-3 mediated intrinsic apoptosis pathway via the cytosolic release of cytochrome C (Evtodienko et al. 1996). Microtubules are also known to regulate intracellular trafficking, including the movement mitochondria in the cells. It has been shown that the microtubule-stabilizing drug paclitaxel increased the speed of mitochondrial movement, whereas microtubuledepolymerizing agent colchicine and nocodazole retarded the mitochondrial transport (Carre et al. 2002; Evtodienko et al. 1996). Thus drugs interfering with microtubule dynamics also alter mitochondrial transport and equilibrium in cancer cells. As discussed earlier, mitochondrial dysfunction is the primary source of intracellular ROS, which is employed by a variety of anticancer drugs to inhibit cancer cell growth (Zhang et al. 2016). Distortion of mitochondrial ETC leads to the ROS-generation coupled with the leakage of cytochrome C into the cytosol. Also, it had been reported that MTA could suppress the closure of mitochondrial permeable pores in tumor cells (Evtodienko et al. 1996), thus enhancing the caspase-3 mediated apoptosis. In a series of reports published by our group, we have demonstrated the role of mitochondria-mediated apoptosis induced by several MTAs against various cancer cells (Ganguli et al. 2014). Extensive studies have been performed previously to establish the connection of ROS with MT disruption. In one of the seminal works, Lin HL et al. showed that the MT targeting drugs 2-methoxyestradiol, docetaxel, and paclitaxel induced significant ROS generation in hepatoma cells which is further attenuated by the antioxidant treatment (Lin et al. 2000). Similar observations were noticed in Ptx-treated

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MDA-MB-435 breast cancer cells, where significant ROS-generation and apoptosis were observed upon paclitaxel treatment. Application of GSH significantly attenuated paclitaxel-induced apoptosis, thus confirming the role of ROS in MTA-induced cytotoxicity. Alexandre J et al. further showed that in A549 human lung adenocarcinoma cells treated with Ptx, accumulation of H2O2 takes place as an early response event and contribute to the apoptotic potential of taxanes (Alexandre et al. 2006). Recently it was observed that ROS generation could take place in a biphasic fashion when the cancer cells are exposed to Ptx. The biphasic response of ROS was reflected in its consistent increase with the increased cell death due to Ptx-treatment and later normalization with the development of Ptx-resistance (Lin et al. 2000). Thus, MTA-induced ROS can also contribute to the development of chemoresistance in cancer cells. The other two types of microtubule-targeting drugs colchicine and vinblastine were also reported to generate ROS and apoptosis in different cancer cells and solid tumors (Lin et al. 2000). The naturally occurring dietary isothiocyanates (ITCs) group of compounds consisting of an isothiocyanate (-N¼C¼S) moiety have been reported to induce microtubule depolymerization in cancer cells by covalently binding to tubulin subunits (Pocasap et al. 2018). However, further studies had revealed that the isothiocyanates occurring in cruciferous vegetables such as erucin, erysolin, sulforaphene, sulforaphane, alyssin, iberin, and phenethyl isothiocyanate induced apoptosis in hepatocellular carcinoma cells via microtubule disruption and subsequent ROS generation (Pocasap et al. 2018). Thus mitochondrial ROS plays an important role in MTA-induced apoptosis in cancer cells.

Modulation of ROS-Mediated Apoptotic Signaling by MTAs ROS-balance or redox homeostasis plays a crucial role in determining the fate of the cancer cells destined for survival or death. As mentioned earlier, mitochondrial ROS produced as a consequence of microtubule disruption by MTAs resulted in the activation of cytochrome C mediated or intrinsic pathway for apoptosis. ROS-mediated apoptosis is initiated by the disruption of mitochondrial integrity, thereby activating intrinsic apoptotic signaling via death receptors. Increased ROS-production results in depolarization of the mitochondrial membrane, which in turn reduces the mitochondrial membrane potential (MMP), produced by the change in proton gradient across mitochondrial inner membrane. Reduction of MMP leads to the release of cytochrome C to the inter-membrane space, allowing the entry of cytochrome C into the mitochondrial cytosol. Cytosolic cytochrome C then activates caspase-9 by triggering apoptotic protein-activating factor-1 (APAF-1), which then leads to the production of caspase-3, the central executioner of apoptosis. Several antiapoptotic proteins such as BCL-2 and BCL-XL and pro-apoptotic proteins like BAD, BAK, BAX, BID, and BIM are also known to contribute to mitochondrial membrane depolarization (Fig. 3). Many chemotherapeutic strategies were designed to specifically enhance the cellular ROS levels to trigger apoptosis in cancer cells, either by chemotherapy or radiation therapy (Pocasap et al. 2018). For example, different

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Fig. 3 Mechanism of cell death of a cancer cell by MT targeting agents. Disruption of MT by compounds induces disruption of mitochondrial membrane potential (MMP) and subsequently activates mitochondrial mediated apoptotic process with activation of different signaling pathways

small molecules such as gemcitabine, trichostatin A, capsaicin, epigallocatechin-3gallate, isothiocyanates either alone or in combination are known to induce ROS-mediated apoptosis in aggressive cancers like pancreatic cancer and other carcinomas (Verma et al. 2017). A wide range of ROS-causing MTAs has been already discussed in the previous section. All of these compounds share the same mechanism of targeting the cellular microtubules as ROS-causing event. The major ROS-activated signaling cascade resulting in the cellular apoptosis may be classified as follows (Fig. 3): I. p53 signaling pathway is well-known tumor suppressor gene which can transcriptionally activate a wide array of apoptotic genes and simultaneously downregulate the pro-apoptotic genes in cancer cells. The activation of p53 leads to growth arrest, senescence, and apoptosis in cancer cells (Haupt et al. 2003). Under normal physiological conditions, p53 is a short-lived protein, tightly regulated by its inhibitor Mdm2, which represses p53-mediated activation of apoptosis in cancer cells. Mdm2 inhibits the transcriptional activity of p53 and perturbed p53 stability by promoting its proteasomal degradation. However, when ROS is generated in cancer cells due to chemotherapy or radiotherapy, the status of p53 is significantly altered. ROS-generation in cancer cells leads to p53 activation via its stabilization and enhancement of the DNA binding and transcriptional activity. Interestingly, Ginnakakou P et al. demonstrated that p53 is associated with cellular microtubules and is translocated to the nucleus

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by a microtubule (
) end oriented motor protein called dynein. Moreover, the same group further demonstrated that suppression of microtubule dynamics by MTAs resulted in the enhanced nuclear translocation of p53 (Giannakakou et al. 2000). Thus, a wide variety of MTAs which can suppress microtubule dynamics at lower doses without disruption of cellular microtubule organization can also efficiently induce the nuclear translocation of P53. In the nucleus, P53 can act as a transcriptional activator or inhibitor of a wide array of genes which participate in cell cycle and apoptosis. The apoptotic genes which get transactivated by P53 include Bax, Bad, Bak, Puma, Noxa, Bid, p53AIP1, Apaf1 while it can also result in the transrepression of antiapoptotic genes like as Bcl-2 and Survivin (Haupt et al. 2003). Interestingly, it was also reported that P53 can induce mitochondria-dependent apoptosis in cancer cells independent of its nuclear translocation and transcription regulation property (Zhao et al. 2005). ROS, generated by different chemotherapeutic drugs or external stress, resulted in the translocation of P53 as a preceding event than its nuclear translocation (Zhao et al. 2005). Upon translocation to mitochondria, P53 interacts with the Bcl-2 protein family members causing their activation or inhibition. This resulted the permealization of mitochondrial outer membrane (MOM) which further triggers release of cytochrome C into the cytoplasm enabling activation of the apoptotic complex. Furthermore, upon activation, Bax and Bak which reside in the MOM are activated and polymerized to form dynamic lipid pores that mediate the cytochrome C release as well. Another member of Bcl-2 family comprises of the BH3-only class having a single BH3 domain. These include tBid and Bim which facilitate the oligomerization of Bax and Bak oligomerization, and the repressors like Puma, Noxa, and Bad which inhibit the antiapoptotic Bcl-2, Bcl-xL, and Mcl-1 proteins. Thus, P53 can orchestrate the mitochondrial-dependent apoptosis independent of its transcription regulatory activity. II. Mitogen-activated protein kinases (MAPKs) signaling pathway are ROS-responsive signaling pathways consisting of the c-Jun N-terminal kinases (JNK), p38 kinase, extracellular signal-related kinases (ERK1/2), and the MAP kinase 1 (BMK1/ERK5) pathways. These are major intracellular signaling cascades that play a key role in various cellular metabolism including cell survival, cell cycle, differentiation, and apoptosis (Zhang et al. 2016). These signaling pathways (JNK, ERK, p38, and BMK1) belong to the family of serine/threonine kinases, which are activated by intracellular or extracellular ROS. Initially, a MAP kinase kinase (MAPKK) which in turn phosphorylates and activates a MAPKK. The activated MAPKK then phosphorylates and activates MAPK which further phosphorylates various substrate proteins required for various physiological functions. III. JNK pathway is activated by oxidative stress and cytokines such as tumor necrosis factor (TNF) and FAS. The pathway is triggered by the activated MAPKK inducing JNK phosphorylation on threonine and tyrosine residues. Activated JNK translocate from cytoplasm to the nucleus where it facilitates activation and phosphorylation of the transcription factor c-Jun (Dhanasekaran

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and Reddy 2008). Phosphorylation of c-Jun leads to its binding with the protein called c-Fos, and both of them dimerize to form the Activator Protein (AP-1). AP-1 is involved in the transcription of various proteins, including the pro-apoptotic proteins like TNF-α, Fas-L, and Bak. Also, the mitochondrial Bim, an activator of the pro-apoptotic Bax, was also found to be phosphorylated by JNK. In addition to the canonical nuclear signaling, JNKs also plays an essential role in regulating the mitochondrial pro- and antiapoptotic proteins upon its translocation to the mitochondria as a response to the apoptotic stimuli. Several MTAs were known to activate this pathway for the induction of apoptosis in cancer cells. IV. ERK pathway is triggered by the various growth factors such as epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and cytokines like TNF-α and IL-1β, with the involvement of tyrosine kinase receptors (RTKs). Interaction of these receptors with specific ligands leads to the activation of Ras, an intracellular GTPase that acts downstream from most RTKs (Zhang et al. 2016). The activated Ras further recruits cytoplasmic kinase Raf (acting as MAPKKK) to the cell membrane, which in turn phosphorylates MEK1/2 (acting as MAPKK). Activated MEK1/2 finally phosphorylates ERK1/2 (MAPK), which gets activated and translocated in the nucleus. Activated ERK1/2 modulates the transcription of various gene products in the nucleus. This pathway remains highly activated in tumors and it has been observed that MTA s, such as paclitaxel alone or in combination, can induce apoptosis in cancer cells by targeting this pathway. Similar effects were observed with the MTAs vinblastine, vincristine, and colchicine in cervical cancer cells (Stone and Chambers 2000). On the contrary, the pro-apoptotic function of Ras/Raf/ ERK–the pathway is well documented. This pro-apoptotic switch is triggered in the response of ROS-induced DNA damage by a variety of MTAs such as etoposide, doxorubicin, resveratrol, phenethyl isothiocyanate, quercetin, apigenin, miltefosine, and paclitaxel to name a few (Cagnol and Chambard 2010). V. The p38 pathway is activated by extracellular stresses or ROS, growth factor, and cytokines like IL-1β and TNF-α, with the involvement of the sequential activation of the downstream effector Ras and Rac1. MTAs were known to have a differential effect on p38 signaling, depending on the cancer types. The MTAs like vincristine, vinblastine, and colchicine reduced phosphor p38 levels in cervical carcinoma cells but had no impact in leukemia and lymphoma cells. Hence, the role of this pathway in MTA-induced apoptosis is not very conclusive. VI. Phosphatidylinositol-3-kinases (PI3Ks)/Akt signaling pathway have been known to be involved in many crucial cellular functions, such as proliferation, invasion, cell cycle, autophagy, apoptosis, and cancer-chemoresistance (Zhang et al. 2016). PI3Ks can be classified into three subtypes based on their structures and substrate specificities, namely, class I, class II and class III, among which the class I PI3Ks are activated by cell surface receptors. The class I PI3Ks are heterodimeric proteins consist of the regulatory subunit p85 (p85α,

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p85β, and p85γ) and the catalytic subunit p110 (p110α, p110β, p110δ, and p110γ), respectively. Upon activation, the p85 regulatory subunit binds directly to receptor tyrosine kinases on the cell membrane, which then facilitates the activation of the catalytic subunit p110. The activated p110 subunit catalyzes the reaction converting phosphatidylinositol 4,5-bisphosphate (PIP2) to phosphatidylinositol 3,4,5-triphosphate (PIP3), which acts as a second messenger to induce many downstream signaling. The membrane-bound PIP3 further triggers proteins containing the pleckstrin homology (PH) domain including phosphoinositide-dependent protein kinase (PDK) and protein kinase B (Akt) serine/threonine kinases. The subsequent activation of PDK and Akt leads to the activation and transcription of various target genes which are essential for cell growth and apoptosis, such as FOXO, GSK3β, BAD, mTOR1, PTEN, and p53 (Xu et al. 2020). This pathway remains hyperactivated in various aggressive tumors and carcinomas and hence acts as one of the most important therapeutic targets for anticancer drugs. Due to its pivotal role in chemoresistance and tumor malignancies, PI3K/Akt/mTOR axis serves as a potential target for many anticancer drugs, including the MTAs. Long back in early 2000, a few reports first demonstrated that the anticancer potential of paclitaxel and vinca alkaloids was enhanced in combination with PI3K inhibitors like LY294002 in different cancer models (Shingu et al. 2003). Later numerous reports had revealed the effectiveness of this signaling as an important drug target. Quite a few reports from our group demonstrating the affinity of different MTAs to target this pathway in cancer cells deserved special mention in this field of research (Chakrabarty 2019; Acharya et al. 2011). VII. The transcription factor nuclear factor-κ light chain enhancer of activated B cells (NFκB) controls a various cellular processes including proliferation, differentiation, inflammation, autophagy, and apoptosis. Just like the decisive role of ROS in cancer, NFκB signaling can act as both pro-tumorigenic and apoptosis activator in cancer cells, depending on the type of the stimuli (Zhang et al. 2016). NFκB encompasses a family of transcription factors divided into two classes: Class I, including NFκB1 (p50/p105) and NFκB2 (p52/p100), and Class II, including RelA/p65, RelB, and c-Rel. In the absence of any stimulus, NF-κB is sequestered in the cytoplasm by its inhibitor IκBs (Inhibitor of κB). The IκBs consists of multiple copies of a specific sequence called ankyrin repeats, which mask the nuclear localization signal (NLS) of NF-κB and keep them sequestered in the cytosol in an inactivated state. Degradation of IκB required the activation of a kinase called IκB kinase (IKK), which is composed of IKKα and IKKβ subunits. Upon phosphorylation of NFκB1 by stress-responsive kinases, the IkBα subunit undergoes ubiquitination followed by the proteasomal degradation. This results in the dimerization of p50 and p65/RelA and subsequent translocation of this heterodimer to the nucleus. In the nucleus, it regulates the transcription of various genes involved in inflammation, cell proliferation, chemoresistance, and apoptosis (Ghoneum et al. 2020). Previous reports had

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revealed that suppression of microtubule dynamics resulted in the activation of NFκB signaling leading to apoptosis in cancer cells (Rai et al. 2015).

Microtubule Targeting Agent Kills Cancer Cells by Modulating ROS Induced Autophagy Autophagy is an essential and highly regulated self-destructive or pro-survival process that plays a housekeeping role in various physiological processes such as removal of aggregated, mis-folded proteins, damaged organelles, growth regulation, and cell death to name a few (Ravanan et al. 2017). The process involves the engulfment of cytoplasmic proteins or organelles into a specialized pseudo-organelle called the autophagosome, which is the fusion of lysosome and late endosome, where complex cellular materials are degraded by acidic hydrolases (Ravanan et al. 2017). The degradation products, including amino acids, fatty acids nucleotides, or carbohydrates, can then be transported back and recycled into general cell metabolism. Intracellular ROS is known to one of the key regulators of autophagy, which may be protective or detrimental to the cancer cells (Li et al. 2015; Galadari 2017). The internal regulatory mechanisms of autophagy by ROS include various pathways like ROS–FOXO3–LC3/BNIP3–autophagy, ROS– HIF1–BNIP3/NIX–autophagy, ROS–NRF2–P62–autophagy, and ROS–TIGAR–autophagy, respectively (Li et al. 2015). Generation of reactive oxygen species (ROS) is one of the most common mechanisms by which the anticancer drugs induce cytotoxicity in cancer cells by targeting the mitochondrial integrity. But in some instances, the mitochondrial ROS formed due to drug-treatment had resulted in the protective autophagic responses in cancer cells (Mackeh et al. 2013). Recent studies had also revealed that microtubuletargeting agents could induce autophagy as a ROS-mediated phenomenon (Mackeh et al. 2013; Karna et al. 2010; Datta 2019).Thus, the link between MTAs, mitochondrial ROS generation, and autophagy is complex. As we have discussed previously, due to the presence of the β-tubulin, mitochondria becomes a target for the MTAs and this mitochondrial damage occurs in a gradient wise fashion. Perhaps the initial mitochondrial damage by the MTAs leads to ROS generation, which in turn provoked an autophagy response. This autophagy further mediated the clearance of the damaged mitochondria and opposed further ROS generation and promote cell survival. However, with aggravation of stress, more and more mitochondria get damaged, which finally shifted the cellular fate to apoptosis by overcoming the protective autophagy (Paul 2020; Acharya et al. 2011).

Conclusion ROS is an indispensable event in the induction of apoptosis by chemotherapy agents in cancer cells. As discussed earlier, there are various sources of intracellular ROS, but the mitochondrial ROS is the major contributor in this process, formed due to the targeting of mitochondria by chemotherapeutic agents. Interestingly more or less most of the microtubule targeting agents had been reported to generate ROS or

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modulate ROS-induced signaling cascades. Still, only a few reports had directly correlated these parallel events. In this chapter, we have correlated those events and revealed how microtubule disruption or suppression of microtubule dynamics could orchestrate ROS generation and subsequent activation of apoptotic signaling in cancer cells. Mitochondria were found to be the missing link in those events. Microtubule was reported to be the integral component of the mitochondrial membrane which also controlled the opening of the voltage-dependent mitochondrial pores. Interestingly most of the mitochondria-associated tubulin was found to be the β-isotypes or predominantly the β-III isotype that is overexpressed in most of the aggressive cancers. The β-subunit in the tubulin dimer also formed the cavity for binding the MTAs. Hence, any MTA having an affinity for tubulin might bind to the β-subunit both in the interphase microtubule at the cytosol and also to the mitochondrial tubulin. Perturbation of the microtubule dynamics will inhibit different cellular processes and also will distort the mitochondrial integrity, resulting in a decrease of mitochondrial membrane potential. This will eventually result in the perturbation of mitochondrial ETC resulting in ROS generation and also release of cytochrome C in the cytosol. All the above-mentioned signaling pathways will be activated by ROS, which will further aggravate the caspase-dependent apoptosis in the cancer cells. Thus, the microtubule targeting agents can modulate mitochondrial ROS to induce apoptosis in cancer cells.

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Sathish Kumar Reddy Padi, Shailender S. Chauhan, and Neha Singh

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ROS: A Double-Edged Sword . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ROS as an Oncogene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ROS as a Tumor Suppressor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cross-References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract

Reactive oxygen species (ROS) are highly reactive molecules that play an important role in cellular homeostasis. Their levels are maintained in equilibrium by redox enzymes and reduced factors. At low to moderate levels, ROS is crucial for the regulation of various biological functions such as cell cycle progression, proliferation, and differentiation involved during the development of mammalian cells. However, excessive ROS levels can damage intracellular biomacromolecules leading to the activation of cell death machinery involving apoptosis. Relative to a normal cell, cancer cells have high ROS content due to abnormal metabolic activity and decreased antioxidant capacity. It is believed that the higher endogenous ROS levels exhibited by cancer cells are vital for S. K. R. Padi (*) Department of Molecular Biology and Biophysics, UConn Health Center, Farmington, CT, USA University of Arizona Cancer Center, The University of Arizona, Tucson, AZ, USA e-mail: [email protected] S. S. Chauhan Department of Cellular and Molecular Medicine, The University of Arizona, Tucson, AZ, USA e-mail: [email protected] N. Singh University of Arizona Cancer Center, The University of Arizona, Tucson, AZ, USA e-mail: [email protected] © Springer Nature Singapore Pte Ltd. 2022 S. Chakraborti et al. (eds.), Handbook of Oxidative Stress in Cancer: Mechanistic Aspects, https://doi.org/10.1007/978-981-15-9411-3_47

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their functions. Since cancer cells are dependent on high endogenous ROS, they are more susceptible to ROS-inducing anticancer agents. Indeed, highly effective targeted drugs or chemotherapeutic agents currently in the clinic kill cancer cells by exceeding the ROS threshold. Regulation of oxidative stress by targeted cancer therapy is still emerging as compared to traditional chemotherapy, where ROS has been directly linked to the cytotoxic effects. Thus, combining ROS-inducing agents with either targeted therapy or traditional chemotherapy seems to be one of the promising strategies in cancer treatment. In this book chapter, we focus on the current evidence of ROS-mediated apoptosis in cancer cells by targeted and chemotherapy. Keywords

Reactive oxygen species · Targeted therapy · Chemotherapy · Apoptosis · Oxidative stress

Introduction Normal metabolic processes often generate reactive molecules called reactive oxygen species (ROS) which are broadly categorized as free radical (hydroxyl radicals, (.OH); superoxide anions, (O2
); nitric oxide, (NO.); peroxyl radicals, (ROO.); and alkoxyl radicals, (RO.)) and non-radical (peroxide, (H2O2); delta state singlet oxygen, (1O2); and ozone, (O3)) oxygen species. ROS acts as a second messenger in cell signaling and plays a crucial role in both, physiological functions as well as cancer development (Chio and Tuveson 2017). ROS production is elevated in cancer cells as compared to normal cells, due to increased metabolic rate, activation of oncogenes, activation of cell signaling, loss of functional p53, hypoxia, nutrient deprivation, and increased activities of cyclooxygenases and oxidases. The implication of ROS tumor initiation, progression, and metastasis highlights its oncogenic potential displayed by malignant cells (Takeuchi et al. 1996). In contrast, normal cells exhibit fine equilibrium between generation and removal to maintain low basal ROS levels. Irrespective of the cell type, antioxidant enzymes are responsible for maintaining intracellular ROS balance favoring vital cell functions. Indeed, many studies have suggested that cancer cells may upregulate the expression of antioxidants to counteract increased ROS induction (Castaldo et al. 2016; Karihtala and Soini 2007). Antioxidants such as superoxide dismutase (SOD), glutathione (GSH), and thioredoxin-1 (Trx-1) are major players in quenching ROS species and keep the system in check (Fig. 1) (He et al. 2017). The expression of antioxidant enzymes is largely controlled by a transcription factor, nuclear factor erythroid 2-related factor 2 (NFE2L2 or NRF2), which is also described as a major antioxidant response regulator (Castaldo et al. 2016). A growing number of studies now suggest deregulation of the Kelch-like ECH-associated protein 1 (KEAP1)-NRF2 pathway in various cancer cells, due to mutations, loss of heterozygosity, and epigenetic changes in the KEAP1 promoter region (Tong et al. 2015).

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ROS Induced by Chemo- and Targeted Therapy Promote Apoptosis in Cancer. . .

Fig. 1 ROS levels in Cancer cells. (Cancer cells accumulate excess ROS due to enhanced metabolic activity, nutrient deprivation, and hypoxia. To balance out this oxidative stress, cancer cells activate the levels of Nrf2 (nuclear factor erythroid 2–related factor 2) to promote the expression of various antioxidants, like glutathione peroxidases (GPXs), superoxide dismutases (Sods). This critical balance of ROS levels promotes tumorigenesis)

ROS Scavengers

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Substantial cancer-related investigations have demonstrated that ROS participate in numerous cell signaling processes, such as immune signaling, hypoxic signaling, apoptosis, metabolism, and aging (Zhang et al. 2016). ROS function via oxidating sulfhydryl (SH) groups of cysteine residues in different protein kinases such as receptor tyrosine kinase (RTK), protein kinase A (PKA), and protein kinase C (PKC) which then phosphorylate their targets to regulate different cellular signaling mechanisms. RTKs such as epidermal growth factor receptor (EGFR), platelet-derived growth factor receptor (PDGFR), and insulin receptor have all been reported to undergo direct oxidation on their cysteine residues (Truong and Carroll 2013). Although malignant cells express higher levels of ROS than normal cells, their level is still below a toxic threshold and thus compatible with cellular homeostasis. However, when the ROS levels exceed the optimal concentration, due to the imbalance between intracellular ROS and antioxidant enzymes, this can evoke irreversible oxidative damage and induce cell death via various mechanisms including apoptosis, autophagy, ferroptosis, and necrosis. Several studies showed that increased ROS levels by redox modulation can selectively kill malignant cells without causing significant toxicity to normal cells (Nogueira et al. 2008; Trachootham et al. 2006; Zou et al. 2017). Indeed, numerous anticancer approaches currently applicable to patients involve the use of targeted therapeutic drugs and chemotherapeutic agents which can effectively suppress tumor growth by inducing ROS production beyond a toxic threshold (Fig. 2). The effectiveness of chemotherapy is largely based on the generation of ROS and an increase in oxidative stress, leading to cancer cell death (Castaldo et al. 2016). While the regulation of oxidative

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Cell Death

Relative ROS levels

Fig. 2 ROS levels in Normal versus Tumor cells. (Increased ROS levels lead to deregulated cell signaling and induce Cancer. Chemotherapy or Targeted therapy induces uncontrolled ROS levels, results in oxidative stress and cell death. Hence, an optimal ROS level is required for maintaining cellular homeostasis)

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Survival Advantage

Normal Homeostasis Normal Cell

Cancer Cell Cancer + Chemotherapy (or) Cell Targeted therapy

stress by targeted therapy is still emerging, there is some evidence of ROS-mediated cell death caused by kinase inhibitors and monoclonal antibodies (Bellosillo et al. 2001; Okon et al. 2015). In this book chapter, we will review the current evidence of ROS-mediated apoptosis induced by chemotherapy and targeted therapy in solid tumors and hematopoietic malignancies.

ROS: A Double-Edged Sword ROS as an Oncogene As mentioned in the above section, NRF2 is an important transcription factor that is responsible for maintaining oxidative homeostasis in cancer cells. Under normal conditions, NRF2 is bound to KEAP1 and undergoes proteasomal degradation (Bryan et al. 2013). Whereas, in response to oxidative stress, NRF2 releases from KEAP1, escapes proteasomal degradation, and translocate into the nucleus. Nuclear NRF2 transactivates the expression of genes related to cell survival and homeostasis, by binding to the antioxidant response element (Pham et al.) in their promoter region (Nguyen et al. 2009). For example, it binds to the promoter region of Bcl-2, an antiapoptotic protein in cancer cells, and upregulates its expression. This leads to a decrease in pro-apoptotic protein Bax, cytochrome c release from mitochondria, activation of caspases, and increased cell survival (Niture and Jaiswal 2012). NF-kB is another essential transcription factor that promotes cell survival under conditions of oxidative stress by suppressing ROS accumulation. Though modulation of NF-kB pathway by oxidative stress is cell- and context-specific, it has been shown to moderate ROS levels by regulating many anti-oxidant targets, such as manganese superoxide dismutase (MnSOD), ferritin heavy chain, heme oxygenase 1, and glutathione peroxidase-1 (Pham et al. 2004). Recent studies have reported a critical

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role for NF-kB in the development of drug resistance in tumor cells during therapy (Fukuoka et al. 2018; Lim et al. 2020). Also, the role of ROS can be regarded as oncogenic as most molecular mechanisms studied so far depend on ROS to promote cancer initiation, progression, invasion, and metastasis (Boonstra and Post 2004; Zhang et al. 2016). There is induction in ROS production following growth factor (PDGF, EGF, FGF, etc.) binding to receptor tyrosine kinases (Truong and Carroll 2013). For instance, H2O2 inhibits protein tyrosine phosphatase 1B which leads to increased phosphorylation of growth factors at tyrosine residues ultimately resulting in the activation of downstream pathways including MAPK/Erk1/2, STAT, and PI3K/AKT/mTOR. Multiple studies have shown that the activation of Erk1/2 through ROS increases cell survival and motility (McCubrey et al. 2007; Zhou et al. 2008). Silva et al., demonstrated the crucial role of ROS in T-cell acute lymphoblastic leukemia (T-ALL) cell viability mediated through IL-7 involving PI3K/Akt/mTOR crosstalk (Silva et al. 2011). Recently, it has been shown that ROS contributes to ICAM-1 and PD-L1 induction in salivary gland epithelial cells through activation of the JAK/STAT pathway PMID: 31867003.

ROS as a Tumor Suppressor As mentioned earlier, malignant cells exhibit higher ROS levels than normal cells. However, this increased ROS production in malignant cells is counteracted by enhanced activity of antioxidant enzymes. An imbalance between intracellular ROS and antioxidant enzymes, push malignant cells beyond the breaking point, leading to irreversible oxidative damage and induces cell death by processes such as apoptosis, autophagy, ferroptosis, and necrosis (Nogueira et al. 2008; Trachootham et al. 2006; Zou et al. 2017). Interestingly, numerous anticancer agents effectively eliminate malignant cells and sensitize them to chemotherapeutic agents or targeted therapeutic drugs via modulating ROS levels beyond a toxic threshold to induce apoptosis (Bellosillo et al. 2001; Castaldo et al. 2016; Okon et al. 2015). Apoptosis also referred as programmed cell death, is the most common form of cell death that is a tightly regulated and highly conserved process. Two major well-studied apoptotic pathways are death receptor-dependent (extrinsic) and mitochondria-dependent (intrinsic). Death receptor-mediated apoptosis is initiated by binding of the death-inducing ligands such as FasL, TNF-α, and TRAIL to their specific receptors FasR, TNFR, and DR4/DR5, respectively. The ligandreceptor interaction is a key step for the recruitment of the adapter protein (FADD) and pro-caspase. This is followed by the death-inducing signaling complex (DISC) formation, caspase activation, and apoptosis. The intrinsic apoptotic pathway involves increased mitochondrial permeability leading to cytochrome-c release and apoptosis subsequently. These factors form an apoptosome complex with pro-caspase 9, leading to the caspase activation and induces apoptosis (Hengartner 2000).

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ROS-Mediated Apoptosis in Malignant Cells by Targeted Therapy Targeted therapy in cancers mostly comprises of monoclonal antibodies, kinase inhibitors, and immunotherapies. In this book chapter, we will review the ROS-mediated effects of kinase inhibitors used for the treatment of solid and hematopoietic malignancies. About 30% of the novel drugs approved for cancer treatment by the Food and Drug Administration (FDA) from 2015–2019 were kinase inhibitors. These inhibitors are currently preferred over chemotherapy by the scientific community because they can more precisely target cancer cells. The main disadvantage of traditional chemotherapy as of now is the damage to normal cells. Whereas, kinase inhibitors inhibit specific signaling pathways activated in various cancer cells. Due to this specificity, targeted therapies result in mild side effects and a shorter time of hospitalization for the patients. In most cases, tumor biopsies from cancer patients could be screened for any mutation that can be targeted by specific drugs. To name a few, kinase inhibitors are being used for targeting CDK4/6, MEK, BRAF, FLT3, JAK, ALK, and EGFR.

Tyrosine Kinase Inhibitors Tyrosine kinase inhibitors (TKIs) inhibit or block one or more enzymes from a family of tyrosine protein kinases. Deregulated RTKs result in uncontrolled growth and cell division which ultimately could lead to cancer. Mutations in RTKs are often oncogenic, giving rise to genes that transform a normal healthy cell into cancerous. TKIs are believed to treat cancer by rectifying this deregulation. More than 30 TKIs have been approved by the FDA in the United States for cancer treatment. Drugs that are targeting Tyrosine kinases for cancer treatment include monoclonal antibodies (Trastuzumab, Pertuzumab, Cetuximab, and Bevacizumab) and small molecule inhibitors (Sunitinib, Gefitinib, Pazopanib, Sorafenib, Erlotinib, and Axitinib). Trastuzumab (Herceptin) is a humanized monoclonal antibody directed against the extracellular domain IV of HER2. It is the first anti-HER2 antibody approved by the FDA for HER2-amplified metastatic breast cancer patients. Yang et al. showed that Trastuzumab can induce ROS production in breast cancer and promote cell death (Yang et al. 2017). Bevacizumab is also a humanized monoclonal antibody directed against vascular endothelial growth factor (VEGF). It was first approved in the United States in February 2004 and later approved by the European Medicines Agency (Amjad et al.) in January 2005 for colorectal cancer. Bevacizumab has been reported to increase oxidative stress by reducing l-cysteine and GSH levels in cancer cells (Fack et al. 2015). Erlotinib and Gefitinib are epidermal growth factor receptor (EGFR) inhibitors, approved for the treatment of non-small cell lung cancer (NSCLC). Leone et al., demonstrated that Vorinostat, an HDAC inhibitor, in combination with either Erlotinib or Gefitinib induced a synergistic pro-apoptotic effect by targeting c-MYC-NRF2-KEAP1 signaling pathway. As mentioned earlier, NRF2 and KEAP1 are crucial for redox stress response, and these genes are often deregulated and associated with EGFR-TKI resistance in NSCLC (Leone et al. 2015). The mechanism of action of a pan-TKI, Pazopanib involves effective blocking of

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VEGF receptors (VEGFRs), PDGFRs, fibroblast growth factor receptors (FGFRs), and c-Kit. A recent study reported that Pazopanib induces eryptosis (suicidal erythrocyte death), an effect in part dependent on oxidative stress (Signoretto et al. 2016). Sorafenib is also a multi-kinase inhibitor, which blocks VEGFRs, PDGFRs, and RAF kinases. Hepatocellular carcinoma (HCC) cells treated with Sorafenib showed a significant increase in mitochondrial ROS levels (H2O2, O2.ˉ, and NO.) followed by decreased mitochondrial, cytoplasmic, and nuclear GSH levels (Chiou et al. 2009; Coriat et al. 2012). PIM Kinase Inhibitors A kinase that takes the limelight in recent years is a serine/threonine PIM kinase, implicated in driving cancer cell growth, proliferation, and survival. It is known to be overexpressed in prostate cancer, breast cancer, and hematological malignancies. PIM kinase upregulation is usually associated with resistance to chemotherapy, radiotherapy, PI3K inhibitors, and other therapeutic approaches (Chauhan et al. 2020; Le et al. 2016; Song et al. 2018). Various clinical trials are being conducted on co-targeting PIM kinase with other therapeutic approaches currently. Adenosine triphosphate (ATP) competitive PIM inhibitors such as LGH447 and INCB053914 have been in a clinical trial for the treatment of patients with myelofibrosis (NCT02370706), relapsed and/or refractory multiple myeloma (NCT02144038, NCT04355039), relapsed/refractory acute myeloid leukemia (AML) or high-risk myelodysplastic syndrome (NCT02078609, NCT02587598), and replaced/refractory diffuse large B-cell lymphoma (NCT03688152). Mouse embryonic fibroblasts (MEFs) lacking all three PIM isoforms (TKO) are vulnerable to activated K-Ras (K-Ras12V)-mediated ROS and apoptosis because they exhibit a significant decrease in the expression of antioxidant enzymes. PIM kinase inhibition can alter the cellular redox state and lead to abnormal mitochondrial oxidative phosphorylation and decreased level of metabolic intermediates in the glycolytic and pentose phosphate pathways. Interestingly, these metabolic perturbations and cytotoxic effects caused due to PIM loss or pharmacological inhibition were reversed by the addition of a ROS scavenger, N-acetyl cysteine (NAC) which indicates these effects are dependent on free-radical production (Fig. 3) (Song et al. 2015). Malignant cells exhibiting upregulated PIM kinase expression are protected from oxidative damage in hypoxia due to increased nuclear factor-erythroid 2 p45-related factor 2 (NRF2) transcriptional activity. Precisely, PIM kinase regulates nuclear localization of NRF2 and transcription of target genes associated with cytoprotection. PIM kinase inhibition blocks NRF2 transcriptional activity and sensitizes malignant cells to ROS/oxidative damage in hypoxia. NAC and Sod successfully blocked the toxic effect of Pan-PIM inhibitors, suggesting the potential use of PIM kinase inhibitors to oppose hypoxia-mediated therapeutic resistance and induce apoptosis in the hypoxic tumor microenvironment (Warfel et al. 2016). PIM kinase modulates elF4B and mTORC1 signaling to induces tumor cell resistance to PI3K/AKT inhibitors by augmenting NRF2 transcriptional activity. Thus, a combination approach targeting PIM and PI3K together was evident to

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A Stress Oncogenic, Kras, G12V

NRF2

PI3K Inhibitor

Fusion

PIM

ROS

Cytoplasm Survival Therapeutic resistance

Nucleus NRF2

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ARE

B Stress Oncogenic, Kras, G12V PIM

NRF2

Fission

PIM Inhibitor

PI3K Inhibitor ROS

ROS

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Cytoplasm Apoptotic Cell Death

Nucleus Cytoprotective genes ARE

Fig. 3 PIM kinases regulate ROS levels. ((a) The upregulation of PIM kinases promotes survival and therapeutic resistance. This is achieved in part due to the activation of cytoprotective genes following nuclear translocation of NRF2 and induction in mitochondrial fusion, both resulting in decreased cellular ROS accumulation. Under these conditions, stress signals (oncogenic, Kras, G12V, etc.) and/or PI3K inhibitor produce low levels of ROS favoring survival/therapeutic resistance. (b) PIM inhibition promotes apoptotic cell death and sensitizes cancer cells to chemotherapy. This is achieved by in part due to the inactivation/absence of cytoprotective genes as NRF2 is restricted to cytoplasm and induction in mitochondrial fragmentation, both resulting in increased cellular ROS accumulation. Under these conditions, stress signals (oncogenic, Kras, G12V, etc.) and/or PI3K inhibitor produce ROS levels above a toxic threshold favoring apoptotic cell death or inhibition of therapeutic resistance)

retard cell proliferation more effectively. The observed inhibition in proliferation was attributed to ROS accumulation (Fig. 3). Similar effects of combination treatment were noticed in vivo (Song et al. 2018).

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Dual inhibition of PI3K and PIM kinases achieved by IBL-202 is one of the effective strategies reported for targeting chronic lymphocytic leukemia (CLL) cells rAnti-caecently. IBL-202 blocked the activity of the PI3K/AKT pathway and induced mitochondrial ROS production in conditions that mimic the hypoxic tumor microenvironment. Both cytotoxic and cytostatic effects caused by dual inhibition were significant which lead to the downregulation of crucial proteins involved in the proliferation and migration of CLL cells to the tumor microenvironment (Crassini et al. 2018). PIM Kinase alleviates oxidative stress by regulating mitochondrial dynamics, limiting acute mitochondrial superoxide production and cellular ROS accumulation. At the same time, increased PIM expression stabilizes NRF2 levels to augment the antioxidant capacity of the cell. In contrast, PIM kinase inhibition leads to increased DRP1 expression, recruitment to mitochondria, and induction in mitochondrial fragmentation with a concomitant surge in superoxide production and cellular ROS accumulation (Fig. 3). In summary, the compromised NRF2-related cytoprotection and elevated ROS production upon PIM1 inhibition leads to persistent oxidative damage in malignant cells and sensitize them to anti-cancer therapy (Chauhan et al. 2020). These preclinical studies suggest that PIM kinase inhibitors have the potential to induce ROS mediated apoptosis in various cancer cell types.

ROS-Mediated Apoptosis in Malignant Cells by Chemotherapy It is well established that most chemotherapeutic agents cause cell death by inducing ROS production over a toxic threshold and this happens to be one of the general mechanisms by which different chemotherapies are believed to suppress tumor progression. The increased ROS accumulation during chemotherapy could be attributed to enhanced generation and compromised antioxidant defense system. Indeed, several anti-cancer drugs marketed currently are characterized by the induction of oxidative stress (ROS surge) causing cellular and DNA damage. The highest levels of cellular ROS are achieved by anthracyclines, such as Doxorubicin, Daunorubicin, and Epirubicin. Similarly, cancer cells exhibit higher ROS levels following treatment with platinum complexes, such as Cisplatin and Carboplatin. Whereas anti-cancer drugs including Vincristine, Paclitaxel, and Docetaxel interfere with the electron transport chain, augment superoxide radical production, and promote cytochrome c release from the mitochondria to cause cell death, see Table 1 (Conklin 2004). Procarbazine was the first ROS inducing alkylating agent used in anti-cancer therapy. Azo-derivatives produced from Procarbazines increase ROS levels to induce oxidative DNA damage and apoptosis in Hodgkin’s lymphoma and brain tumors. A natural metabolite of estradiol, 2-methoxyestradiol, was shown to promote apoptosis in human neuroblastoma cells in vitro through ROS induction and loss of mitochondrial membrane potential (Poprac et al. 2017). Vinca alkaloids have been reported to cause aberrant ROS-mediated c-Jun N-terminal kinase (JNK) activation, decreased Mcl-1 levels, and DNA damage followed by mitochondrial dysfunction related apoptosis in lung cancer cells (Chiu et al. 2012). Arsenic trioxide (As2O3) induces apoptotic cell death through elevated ROS production and damage to the mitochondrial membrane. As2O3 related apoptosis induction has been reported

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Table 1 ROS induction by Chemotherapeutic agents – mechanisms of action I. Alter Mitochondrial Function/ROS generation Topoisomerase inhibitors: Topotecan and Irinotecan Platinum compounds: Cisplatin, carboplatin, and Oxaliplatin Anthracyclines: Doxorubicin and Daunorubicin Alkylating agents: Dacrabazine, Procarbazine, and Temozolomide Vinca alkaloids: Vinorelbine and vincristine DNA methyltransferase inhibitor: Decitabine and 5-Azacytidine II. Regulate Antioxidants Curcuminoids: Curcumin, demethoxycurcumin, and bisdemethoxycurcumin Arsenic agents: AS2O3 Taxanes: Paclitaxel, and Docetaxel

in myeloma, leukemia, lung cancer, and breast cancer. Doxorubicin facilitates its cytotoxic effects through induced production of hydroxyl radicals and the treatment has shown significant success in various malignancies. Capsaicin is the principal ingredient of chili peppers in the plant genus Capsicum. Recent studies have shown that Capsaicin exhibit profound antineoplastic activity by inducing ROS-mediated apoptosis in prostate, liver, and breast cancer. However, clinical applications of Capsaicin are limited due to its unfavorable adverse effects. This has led to the development of second-generation Capsaicin analogs, such as Capsiates, Resiniferatoxin, and Dihydrocapsaicin. These analogs share a similar mechanism of action with Capsaicin, dependent on elevated ROS levels and loss of mitochondrial membrane potential in cancer cells (Friedman et al. 2018). Curcumin is a phytopolyphenol that is mainly found in turmeric (Curcuma longa) and has been reported to exhibit anti-tumorigenic activity. Larasati et al., showed that in chronic myeloid leukemia (CML) cells, curcumin binds to several enzymes involved in ROS metabolism including, glyoxylase 1, NAD(P)H dehydrogenase [quinone]1 (NQO1), and aldoketoreductase family 1 member 1 (AKR1C1) to suppress tumor growth by enhancing ROS levels over the threshold (Larasati et al. 2018). Sulforaphane (SFN) is a biologically active phytochemical found in high concentrations in cruciferous vegetables like broccoli. It has been studied broadly for its anti-neoplastic efficacy and the basic mechanisms using human cancer cell lines and preclinical mouse models. SFN is a cancer preventive agent that has shown activity in diverse cancer models, including colon cancer, breast cancer, prostate cancer, and leukemia. Following SFN treatment, cancer cells exhibit apoptotic cell death initiated by ROS generation, disruption of mitochondrial membrane potential, and release of cytochrome c (Amjad et al. 2015). Topotecan is a semi-synthetic derivative of Camptothecin, and an FDA-approved chemotherapeutic agent for the treatment of cervical, ovarian, and SCLC patients. It inhibits Topoisomerase-1 involved in the correction of topological DNA errors and the resulting Topotecan-DNA complex leads to double-strand cuts and apoptosis in the cancer cells. Topotecan also induces cell death by ROS induction and causing irreversible DNA damage (Hormann et al. 2012).

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Strategies and Drugs Targeted to Increases ROS-Mediated Apoptosis in Malignant Cells Despite tremendous progress in the treatment strategies for different malignancies, intrinsic/acquired resistance results in treatment failure at the metastatic stage, and deters long-term use of the drug. Likewise, the major reason why chemotherapy stops working at one point is the development of multidrug resistance in cancer cells. To improve the clinical outcome in patients, several drug combination strategies are rationally designed to target the resistant cancer cell population resulting from the activation of a compensatory signaling pathway. Doshi et al., demonstrated that inhibition of PIM kinase sensitizes internal tandem duplication of fms-like tyrosine kinase-3 (FLT3-ITD) positive acute myeloid leukemia (AML) cell lines to apoptosis activated by various chemotherapeutic drugs such as Daunorubicin, Etoposide, and Mitoxantrone. The trigger for apoptosis in this study following combination treatment was associated with enhanced DNA doublestrand breaks and increased ROS levels. Most importantly, NAC, a ROS scavenger rescued FLT-ITD positive AML cells from apoptosis induced by PIM kinase inhibitor and Daunorubicin combination treatment (Doshi et al. 2016). In NSCLC, inhibition of PIM kinases leads to increased mitochondrial fragmentation accompanied by a surge in cellular ROS accumulation which then sensitizes cancer cells to chemotherapy (Chauhan et al. 2020). A recent study showed that melanoma cells developing resistance to MAPK inhibitors through various mechanisms (PDGFRB overexpression, BRAF amplification or splice site mutations, mutations in KRAS, and NRAS) reactivating MAPK pathway become sensitive to HDAC inhibitor. Increased ROS levels in BRAF resistant melanoma due to hyperactive MAPK signaling makes them more vulnerable to oxidative stress. Further stimulation of ROS levels by HDAC inhibitor (Vorinostat) through suppression of SLC7A11 (gene encoding glutathione precursor) leads to significant DNA damage and apoptotic cell death in hyperactive MAPK-resistant cells (Wang et al. 2018). Yang et al., demonstrated that a combination of HER2 (Trastuzumab) and EGFR (Nimotuzumab) inhibitors exhibit a synergistic anti-tumor activity in HER2 overexpressing breast cancer cells. More precisely, increased ROS production and compromised NRF2 signaling was considered responsible for the inhibition of breast cancer growth (Yang et al. 2017). Triptolide, a diterpenoid triepoxide, exhibits preclinical anti-cancer activity in a broad range of cancers including AML, prostate cancer, breast cancer, lung cancer, and neuroblastoma (Noel et al. 2019). However, the clinical use of Triptolide is limited because it offers a narrow therapeutic window and has severe toxicity on the digestive, urogenital, and blood circulatory systems. Yet, numerous investigations have revealed that a relatively low dose of Triptolide could sensitize cancer cells to chemotherapy. Most importantly, liver cancer cells treated with Triptolide in combination with 5-FU or Cisplatin exhibit a marked induction of ROS levels and increased caspase-3 activity. Combination treatment also induced Bax expression and inhibited Bcl-2 levels, leading to apoptosis in liver cancer cells (Li and Hu 2014).

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ABT-737, a small molecule inhibitor from Abbott laboratories target anti-apoptotic Bcl-2 family proteins (Bcl-2, Bcl-Xl, and Bcl-w) and activate Bak/Bax-mediated apoptosis in cancer cells. Many studies have shown promising preclinical activity of ABT-737 as a single agent or in combination with various chemotherapeutic agents against AML, lymphoma, CLL, NSCLC, and acute lymphoblastic leukemia. However, ABT-737 has a very weak affinity for Mcl-1, a Bcl-2 family member, whose upregulation is considered responsible for causing ABT-737 resistance in cancer cells. Interestingly, a synthetic cytotoxic retinoid N-(4-hydroxyphenyl) retinamide (4HPR) is known to generate ROS, and increased ROS have been shown to activate c-Jun kinase (JNK), which in turn inhibits Mcl-1 expression through phosphorylation. The combination of ABT-737 and 4HPR was shown to enhance mitochondrial apoptotic cascade in acute lymphoblastic leukemia cells. Similarly, combining ABT-737 with arsenic trioxide, or cyclin-dependent kinase inhibitor have achieved synergy via Mcl-1 inhibition (Trachootham et al. 2009). Thymoquinone, the active ingredient of Nigella sativa black seed, exhibits promising therapeutic potential against various cancers. Thymoquinone in combination with Doxorubicin induces apoptosis by increasing ROS and triggering disruption of mitochondrial membrane potential. Importantly, pretreatment with NAC or pan-caspase inhibitor significantly repressed the apoptosis suggesting that the cell death is ROS- and caspase-dependent. The combined treatment of Thymoquinone and Doxorubicin also suppressed tumor volume in mice more significantly than single drug treatments through enhanced apoptosis (Fatfat et al. 2019).

Conclusion Over the years, studies have established a fundamental role of ROS in maintaining cellular homeostasis and activating cell signaling events. Also, it is believed that decreasing cellular ROS levels below a particular threshold could negatively affect vital processes including proliferation and differentiation. Malignant cells have adapted to the increased rate of ROS production and exhibit abnormal mechanisms to regulate their exceptional redox status. The likely reasons contributing to increased ROS generation in malignant cells are oncogenic signaling, increased energy metabolism to support uncontrolled cell proliferation, and compromised mitochondrial respiration (Castaldo et al. 2016). The composite interconnection between ROS levels and malignancy is fundamentally based on the precise finetuning between production and elimination. Upon ROS induction, malignant cells undergo molecular changes crucial for tumorigenesis and chemoresistance. However, an increase in ROS levels to a toxic threshold using agents that either directly surge ROS production or inhibit antioxidant defenses, or their combinations can selectively kill malignant cells without causing significant toxicity to normal cells. Several studies have shown that chemotherapeutic agents and targeted drugs increase cellular ROS levels to induce irreparable damages, which results in tumor cell apoptosis (Castaldo et al. 2016).

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Long-term use of anti-cancer drugs acting through ROS induction can lead to chemoresistance in cancer cells by augmenting antioxidant defense mechanisms, such as activation and stabilization of NRF2. For example, Oxaliplatin-resistant colon cancer cells show altered expression of antioxidant proteins, such as Sod1 and Sod2. Also, this resistant phenotype is associated with increased NRF2 expression (Kelley et al. 2014). Various investigations in the past have reported ROS-mediated cell death in different cancers exhibiting compromised antioxidant defense system. Targeted therapeutic agents such as TKIs and PIM kinase inhibitors increase the oxidative stress burden to toxic levels that surpass the reduction capacity of tumor cells. Suggesting these targeted agents possess anti-cancer efficacy in addition to their targeted effect (Song et al. 2018; Yang et al. 2017). Although targeted therapy is advancing by leaps and bounds in the treatment of solid tumors and lymphomas, there is a subset of patients who respond initially but then develop resistance. The genetic and phenotypic variability of cancer patients poses a hurdle to effective therapy. Heterogeneity within-tumors and between-tumors among cancer patients makes it difficult to predict the effectiveness of drug therapy. To overcome this variability, several therapeutic combination approaches are rationally designed to target the resistant cancer cell population resulting from feedback activation of the compensatory survival signaling pathways. Initial efforts to target PIM kinase alone have been hampered by the activation of compensatory signaling pathways (Lim et al. 2020). Later multiple studies have suggested that co-targeting PIM kinases with other treatment approaches such as chemotherapy or targeted therapy could potentially be used as a novel strategy, to limit the acquired drug resistance (Crassini et al. 2018; Lim et al. 2020; Song et al. 2018). For example, a recent study demonstrated that inhibition of PIM kinases caused excessive mitochondrial fragmentation along with a significant increase in intracellular ROS accumulation to combat chemoresistance in lung cancer (Chauhan et al. 2020). Considering the important role of ROS in tumor progression and drug resistance, designing therapeutic strategies to manipulate ROS levels may improve clinical outcomes in cancer patients.

Cross-References ▶ Microtubule-Targeting Agents Induce ROS-Mediated Apoptosis in Cancer ▶ Role of ROS in Triggering Death Receptor-Mediated Apoptosis ▶ ROS in Apoptosis of Cancer Cells ▶ ROS-Mediated Apoptosis in Cancer

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ROS-Mediated Apoptosis in Cancer

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Saranya NavaneethaKrishnan, Jesusa L. Rosales, and Ki-Young Lee

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ROS in the Mitochondria-Mediated Intrinsic Apoptotic Pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ROS in the Death Receptor-Mediated Apoptotic Pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ROS in p53-Mediated Apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ROS in ER Stress-Induced Apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ROS in Calcium-Mediated Apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Prooxidant-Based Cancer Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cross-References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract

Redox homeostasis is defined as a balance between reactive oxygen species (ROS) production and ROS elimination. Intracellular ROS are produced during endoplasmic reticulum (ER) stress, oxidative phosphorylation of mitochondria and by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) family members. ROS are eliminated by antioxidant enzymes such as superoxide dismutase, catalase, and peroxiredoxins as well as by nonenzymatic antioxidants. Moderate levels of ROS activate signaling pathways for cancer cell proliferation and survival, whereas high levels of ROS trigger apoptotic signals. Redox imbalance in cancer cells toward accelerated ROS production makes them more vulnerable to oxidative stress-mediated apoptosis. Therefore, pro-oxidative molecules are exploited for the development of anticancer drugs. In this chapter, we discuss ROS-induced (i) mitochondria-, (ii) death receptor-, (iii) p53-, (iv) ER S. NavaneethaKrishnan · J. L. Rosales · K.-Y. Lee (*) Department of Cell Biology and Anatomy, Arnie Charbonneau Cancer Institute and Alberta Children’s Hospital Research Institute, Cumming School of Medicine, University of Calgary, Calgary, AB, Canada e-mail: [email protected]; [email protected]; [email protected] © Springer Nature Singapore Pte Ltd. 2022 S. Chakraborti et al. (eds.), Handbook of Oxidative Stress in Cancer: Mechanistic Aspects, https://doi.org/10.1007/978-981-15-9411-3_48

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stress-, and (v) calcium-mediated apoptotic pathways in cancer. We also discuss (vi) prooxidant-based cancer therapy. Keywords

ROS · Apoptosis · Cancer · Mitochondria · ER-stress · Calcium

Introduction Reactive oxygen species (ROS) act as secondary messengers and play a major role in cancer development and progression (Liou and Storz 2010). At low levels, ROS stimulate cancer cell survival and proliferation, while at high levels apoptosis is induced in a variety of cancer types (Vilema-Enriquez et al. 2016). For example, low concentration of H2O2 increases the proliferation of hepatoma (at 1–10 μM) (Liu et al. 2002) and colon cancer (at 10 μM) cells (Park et al. 2006), but high concentration induces apoptosis in colon cancer (at 1000 μM) (Park et al. 2006) and breast cancer (at 100–200 μM) (Chua et al. 2009) cells and cell cycle arrest in lung cancer cells (at 50 μM) (Upadhyay et al. 2007). Cancer cells are known to exhibit increased ROS levels compared to normal cells. Thus, further increases in ROS levels generated by prooxidant chemotherapeutic drugs render increased susceptibility of cancer cells to oxidative stress-induced apoptosis (Trachootham et al. 2009). Several studies have shown that ROS oxidize cellular glutathione (GSH) and initiate apoptosis through post-translational S-glutathiolation of redox-sensitive cysteines. For example, S-glutathiolation of Fas at Cys294 enhances its binding to FasL, promoting an apoptosis signaling cascade (Anathy et al. 2009). ROS have also been shown to induce apoptotic signaling cascade by activating extracellular regulated kinase 1/2 (ERK1/2), p38 kinase, and c-Jun N-terminal kinase (JNK) (Zhang et al. 2016; NavaneethaKrishnan et al. 2019) pathways. For instance, exogenous H2O2 activates ERK1/2-mediated cell death in glioma cells. However, catalase and U0126, an inhibitor of the ERK upstream kinase, MEK1/2, completely inhibit H2O2-induced ERK activation and cell death in these cells (Lee et al. 2005). In addition, ROS oxidation of thioredoxin, a redox-regulated protein that binds and inhibits apoptosis signal-regulating kinase-1 (ASK-1) (Katagiri et al. 2010; Saitoh et al. 1998), causes the release and activation of ASK-1, which in turn results in the activation of JNK and p38 (Liou and Storz 2010; Saitoh et al. 1998; Watanabe et al. 2015). ROS-dependent ERK, JNK, and p38 activation upregulates pro-apoptotic proteins while phosphorylating and downregulating antiapoptotic proteins (Lu and Xu 2006; Dhanasekaran and Reddy 2008). In hepatoma cells, ROS-dependent oxidation of glutathione S-transferase pi (GST-pi) disrupts the GST-pi-JNK complex, resulting in sustained activation of JNK and its downstream gene targets (Lu et al. 2007a). It is also known that ROS induce apoptosis mediated by the forkhead transcription factors, p66Shc and p53 (Liou and Storz 2010; Redza-Dutordoir and Averill-Bates 1863). Our focus in this chapter is to discuss ROS as they relate to various cancer cell apoptotic pathways (Figs. 1 and 2) and prooxidant-based cancer therapy.

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Fig. 1 ROS-induced intrinsic and extrinsic apoptotic pathways. mPTP: mitochondrial permeability transition pore; ΔΨm: mitochondrial membrane potential; CL: cardiolipin; JNK: c-Jun N-terminal kinase; TRAIL: TNF-related apoptosis-inducing ligand; FADD: Fas-associated death domain; TNF: tumor necrosis factor; TRADD: TNFR-associated death domain protein; TRAF2: TNFassociated factor-2; MOMP: mitochondrial outer membrane permeabilization; Bcl-2: B-cell lymphoma 2; Bad: Bcl-2-associated agonist of cell death; Bax: Bcl-2-associated X protein; APAF-1: apoptotic protein-activating factor 1; IAP: inhibitor of apoptosis; AIF: apoptosis inducing factor; ASK-1: apoptosis signal-regulating kinase-1; Cyt C: cytochrome C; FLIP: FLICE inhibitory protein; DISC is a complex of FAS/FASL/FADD/Pro-caspase-8

ROS in the Mitochondria-Mediated Intrinsic Apoptotic Pathway Intrinsic apoptotic stimuli such as overwhelming levels of ROS, dissipates mitochondrial transmembrane potential (ΔΨm), inducing permeabilization of the mitochondrial outer and inner membranes. Disruption of mitochondrial structural integrity from mitochondrial outer membrane permeabilization (MOMP) permits the release of the two main groups of mitochondrial pro-apoptotic proteins into the cytosol (Uren et al. 2005; Newmeyer and Ferguson-Miller 2003) (Fig. 1). The first group includes cytochrome c (cyt c), Smac/DIABLO and the serine protease, HtrA2/Omi. Upon release from the mitochondrial intermembrane space, cyt c binds to the apoptotic protease activating factor-1 (APAF-1) and forms apoptosome that dimerizes with and activates caspase-9, which in turn activates caspase 3 (Zou et al. 1999; Jiang and Wang 2000). On the other hand, Smac/DIABLO and HtrA2/Omi

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Fig. 2 ROS-mediated ER stress-activated apoptotic pathway. IRE1: inositol-requiring enzyme 1; eIF2α: eukaryotic translation initiation factor 2α; PERK: protein kinase RNA-like endoplasmic reticulum kinase; ATF4: activating transcription factor 4; DR: death receptor; ERO1α: endoplasmic reticulum oxidoreductin-1α; IP3R: Inositol trisphosphate receptor; CaMKII: calcium-dependent protein kinase; CHOP: C/EBP homologous protein transcription factor; NOX2: NADPH oxidase 2; ASK-1: apoptosis signal-regulating kinase-1; JNK: c-Jun N-terminal kinase; Cyt C: cytochrome C

promote apoptosis by inhibiting the activity of the inhibitors of apoptosis proteins (IAP) (Verhagen et al. 2000; Du et al. 2000; Suzuki et al. 2001). The second group consists of AIF and endonuclease G, which appears to function independent of caspases. They translocate into the nucleus and cause nuclear condensation and DNA fragmentation (Susin et al. 1999; Li et al. 2001). Intracellular oxidative stress also leads to mitochondrial DNA (mtDNA) damage and induces mitochondria-mediated apoptosis by causing the release of cyt c, AIF, or Smac/Diablo from the mitochondria, which then triggers caspase-dependent or independent apoptosis (Van Houten et al. 2016; Toyokuni et al. 1995; Sullivan and Chandel 2014) (Fig. 1). mtDNA damage impairs the respiratory chain function,

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resulting in a further increase in ROS production, which induces mitochondrial depolarization and subsequently, apoptosis. Consistent with the concept described above, ROS-mediated apoptotic signaling in human promyelocytic leukemia is associated with decreased GSH levels, loss of mitochondrial membrane potential, cyt c release, and caspase-3 activation (Hileman et al. 2004; Fan et al. 2016). Similarly, in Jurkat cells, H2O2 (50μM) induces dissipation of mitochondrial membrane potential, cyt c release, and caspase-9 activation (Stridh et al. 1998; Hampton and Orrenius 1997). In HeLa cells, treatment with H2O2 (25–100μM) causes significant dose-dependent cyt c release and caspase-9 and -3 activation (Pallepati and Averill-Bates 2010). H2O2 at 50μM was also found to increase p53 phosphorylation at Ser46 and Ser15 and upregulate p53 upregulated modulator of apoptosis (PUMA) and B-cell lymphoma 2 (Bcl-2)-associated X protein (Bax) (Pallepati and AverillBates 2010), which both promote apoptosis. Katoh et al. (Katoh et al. 2004) also showed that H2O2 (0.2 mM) causes disulfide bridge-mediated caspase-9 activation in human leukemic U937 cells. Furthermore, it was shown that treatment of HeLa cells with H2O2 (125μM) triggers apoptosis via the mitochondrial pathway by upregulating p73 and Bax. This H2O2 effect is accompanied by the upregulation of ERK, JNK, c-Myc, and Hsp-70 and downregulation of the anti-apoptotic B-cell lymphoma-extra large (Bcl-XL), resulting in cyt c release and caspases-9 and -3 activation (Singh et al. 2007). In bladder cancer, treatment with exogenous H2O2 combined with the histone deacetylase inhibitor, FK228, induces activation of MEK1/2 and ERK1/2, increasing NADPH oxidase 1 (NOX-1) level, intracellular ROS production, caspase activation, and cell death (Choudhary et al. 2011). Cyt c is bound to the inner mitochondrial membrane (IMM) by the mitochondriaspecific phospholipid, cardiolipin (CL). Mitochondrial ROS (mtROS) oxidize CL and facilitate cyt c detachment and release through the outer mitochondrial membrane (OMM), resulting in the initiation of apoptosis (Kagan et al. 2005; Kagan et al. 2009; Kagan et al. 2014; Zhong et al. 2017). Jiang et al. (Jiang et al. 2008) showed that Bax translocation to mitochondria is upstream of ROS production and CL peroxidation, which induces apoptosis in human acute lymphoblastoid leukemia (Asumendi et al. 2002) and pancreatic cancers (Wan et al. 2018). Lipid peroxidationderived products such as 4-hydroxy-2-nonenals (4-HNE) are highly mutagenic and involved in the initiation and progression of cancers, including liver, pancreatic, kidney, gastric, and colon cancers (Shoeb et al. 2014; Ma et al. 2013). However, high concentrations of 4-HNE induce cancer cell apoptosis. Thus, manipulation of mitochondrial ROS generation, lipid peroxidation, and levels of lipid peroxidationderived products such as 4-HNE could be viewed as a therapeutic strategy to prevent or treat cancer (Zhong and Yin 2015). Mitochondrial permeability transition pore (mPTP) is another key participant in oxidative stress-induced mitochondrial apoptosis. The mPTP is a cyclosporine A (CsA)-sensitive high-conductance channel, triggered by mitochondrial calcium overload, leading to mitochondrial depolarization and swelling followed by the release of apoptotic mediators and cell death (Lemasters et al. 1787; Batandier et al. 2004). ROS-triggered mPTP opening stimulates further ROS formation or ROS-induced ROS release (RIRR) (Zorov et al. 2014). In breast cancer cells, loss of

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cdk5 (cyclin dependent kinase 5) induces ROS elevation, mitochondrial depolarization, fragmentation, and mPTP opening that is associated with increased Ca2+ level (NavaneethaKrishnan et al. 2018). Conversely, erastin (in colorectal cancer cells (Huo et al. 2016), Oroxylin A (in human hepatoma cells (Xin-Eng Huang et al. 2016)) and Hirsutine (in human lung cancer (Zhang et al. 2018)) were shown to induce ROS production and mPTP opening.

ROS in the Death Receptor-Mediated Apoptotic Pathway Activation of death receptors (DRS) by tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) triggers receptor oligomerization and recruits Fasassociated death domain (FADD) the adaptor molecule that prompts the formation of the death-inducing signaling complex (DISC), which in turn activates caspase8 and its downstream effector, caspase-3 (Fig. 1). Caspase-8 can also activate the intrinsic mitochondrial apoptotic pathway by cleaving the BH3-only protein, Bid, to produce truncated Bid (t-Bid) (Kantari and Walczak 1813). t-Bid then translocates into the OMM and induces Bax translocation to the OMM as well as Bax/Bak oligomerization and MOMP (Yi et al. 2003). TRAIL-mediated apoptosis is very similar to apoptosis mediated by Fas/FasL, except Fas serves as the death ligand in the latter. FLICE inhibitory protein (FLIP) is a key regulatory protein in the Fas death pathway. FLIP prevents binding of procaspase-8 to FADD and interferes with DISC formation and activation of caspase-8, inhibiting apoptosis (Krueger et al. 2001). In contrast, TNF receptor (TNFα/TNF-R1)-induced signaling results in the recruitment of TNFR-associated death domain (TRADD) protein and TNF-associated factor-2 (TRAF2), which in turn activates ASK1- and JNK-mediated apoptotic signaling (Xiong et al. 2011). Intracellular ROS mediate apoptosis induced by TRAIL. Several studies have shown crosstalk between mROS and mitochondrial membrane depolarization in TRAIL-induced apoptosis and mutual regulation occurs through death receptor 5 (DR5) expression (Suzuki-Karasaki et al. 2014a; Suzuki-Karasaki et al. 2014b). In Jurkat cells, TRAIL induces mitochondrial O2•- generation, CL oxidation, and ER stress responses such as caspase-12 activation. Treatment of these cells with FCCP (uncoupler of oxidative phosphorylation), rotenone (complex I inhibitor), or antimycin A (complex III inhibitor) promotes TRAIL-induced activation of caspase-3/7 and X-box-binding protein-1 (XBP-1), and pro-apoptotic events are inhibited by the superoxide scavenger, MnTBaP [Mn(III) tetrakis (4-benzoic acid) porphyrin chloride] (Inoue and Suzuki-Karasaki 2013). It is notable that mitochondrial membrane depolarization potentiates the TRAIL-induced activation of XBP-1, which in turn regulates DR5 expression (Liu et al. 2009). Similarly, human colon carcinoma cell lines are sensitized to TRAIL-induced apoptosis by carbonyl cyanide m-chlorophenylhydrazone (CCCP) through promotion of ROS production, resulting in caspase8 and Bax activation, Smac/DIABLO release into the cytosol and X-linked inhibitor of apoptosis (XIAP) degradation (Izeradjene et al. 2005). In TRAIL-resistant human melanoma cells, H2O2 (30–100μM) induces cell death via superoxide generation

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followed by increased mitochondrial membrane depolarization, caspase-3/7 activation and subsequent activation of ER stress responses, including caspase-12 and XBP-1 (Tochigi et al. 2013). ROS also activate Fas-mediated apoptosis in various cell types. In HeLa cells, H2O2 (5–50μM) triggers upregulation of FasL and activation of caspase-8 and caspase-2, resulting in cleavage of Bid, t-Bid translocation to mitochondria and caspase-9 activation (Pallepati and Averill-Bates 2011a). In HL-60 cells, singlet oxygen induces caspase-8-mediated caspase-3 activation (Zhuang et al. 1999) through phosphorylation of p38 (Zhuang et al. 2000), whereas H2O2 only activates the caspase8-mediated apoptotic pathway (Zhuang et al. 2000). In human gastric carcinoma cells, H2O2 (0.4 mM) causes Bax upregulation and Bcl-2 downregulation, which are accompanied by p53-dependent Fas and FasL upregulation (Mao et al. 2006). Activation of Fas receptor has been linked to ROS production (Vercammen et al. 1998; Gulbins et al. 1996), and H2O2 was found to downregulate FLIP through the ubiquitin-mediated proteasome pathway in Fas-induced apoptosis (Wang et al. 2008). Interestingly, Fas stimulation in Jurkat cells causes early onset of ROS production by NADPH oxidase and/or NO synthase (Beltran et al. 2002) and late onset of ROS production by mPTP, inducing apoptosome formation and caspasemediated apoptosis (Sato et al. 2004). Knocking down of either CD95 or CD95L (Fas or FasL) gene causes sustained death induced by CD95R/L elimination (DICE) by triggering ROS production, DNA damage, and caspase activation, which are independent of caspase-8, receptor interacting protein kinases 1/mixed lineage kinase domain-like (RIPK1/MLKL), and p53 (Hadji et al. 2014).

ROS in p53-Mediated Apoptosis Oxidative stress has also been associated with p53-dependent cell cycle arrest, DNA repair, and apoptosis (Vousden and Lane 2007; He and Simon 2013). p53, a transcription factor, acts as both a downstream target of ROS regulators and an upstream regulator of ROS production. Oxidative modification of the cysteine residues in p53 causes conformational changes that result in loss of function of the protein (Maillet and Pervaiz 2012). p53 is known to upregulate several antioxidant genes, including manganese superoxide dismutase (MnSOD), GSH peroxidase 1 (Gpx1), and p53-induced genes (PIGs), and repress the expression of cyclooxygenase 2 (COX2) and inducible nitric oxide synthase 2 (NOS2) (Maillet and Pervaiz 2012). However, p53 has also been reported to have a repressive effect on MnSOD at the promoter level during induction of p53mediated apoptosis (Drane et al. 2001). In fact, p53 plays a dual role in activating prooxidant or antioxidant genes, depending on ROS intensity (Sablina et al. 2005; Humpton and Vousden 2016). With increased ROS accumulation, p53 suppresses the nuclear factor erythroid 2-related factor 2 (Nrf2)-dependent transcription of ARE-containing promoters of the subunit of the cystine/glutamate transporter (x-CT), NAD(P)H quinone oxidoreductase (NQO1) and glutathione S-transferase1 (GST-α1) genes (Faraonio et al. 2006). In cancer cells, elevated ROS levels

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achieved by inhibition of thioredoxin reductase and pharmacological activation of p53 promotes JNK-mediated p53 activation and downstream upregulation of the prooxidant genes, PUMA, and PIGs, leading to synthetic lethality (Shi et al. 2014). We note that p53 also targets p66Shc (Trinei et al. 2002), a redox enzyme that produces H2O2 to induce mPTP opening and apoptosis (Giorgio et al. 2005).

ROS in ER Stress-Induced Apoptosis The electron coupling systems, including protein disulfide isomerase (PDI) and endoplasmic reticulum oxidoreductin-1 (ERO1), intra ER-glutathione disulfide (GSSG)/GSH, and the microsomal monooxygenase (MMO) system, are the major sources of ROS on the ER membrane (Zeeshan et al. 2016). During disulfide bond formation, molecular oxygen oxidizes ERO1, which then oxidizes PDI, which in turn, forms disulfides in ER proteins. In the presence of FAD, the reactivation of ERO1 by molecular oxygen causes H2O2 generation and production of oxidized GSH (Tu and Weissman 2004). About 25% of ROS are produced by disulfide bonds in the ER during oxidative protein folding (Tu and Weissman 2004). The MMO system consists of cytochrome P450 (P450) and NADPH-P450 reductase (NPR), and produces superoxide anion radicals and H2O2 as by products (Davydov 2001). Thus, increased protein folding and disulfide bond formation in the ER trigger ROS accumulation. The unbalanced redox state and impaired antioxidant mechanisms could cause ER stress-mediated cell death. When ER stress exceeds the threshold levels, the unfolded protein response (UPR)-induced signals, resulting from the accumulation of unfolded or misfolded proteins, activate both the extrinsic and intrinsic apoptotic pathways by modulating (i) the protein kinase RNA-like endoplasmic reticulum kinase (PERK)/eukaryotic translation initiation factor 2α (eIF2α)mediated activation of the transcription factor, C/EBP homologous protein transcription factor (CHOP), which upregulates DR5, Bcl-2-interacting mediator of cell death (Bim) and PUMA (Sano and Reed 1833; Hu et al. 2018), and (ii) the inositolrequiring enzyme 1 (IRE-1)-dependent TRAF2 activation and stimulation of the ASK-1/JNK signaling pathway (Sano and Reed 1833) (Fig. 2). In HeLa cells, short exposure (15 min) to H2O2 (15-50μM) activates the UPR by increasing the expression of p-PERK, p-eIF2α, and p-IRE1α, whereas long exposure (1-3 h) increases CHOP expression, calpain activity, and caspase-7,
4,
12 and
9 levels, and induces ER-mediated apoptosis. The calcium chelator, BAPTA-AM, and inhibitors of calpain and caspase-7 decrease the activation of caspase-4 and -12, confirming the roles of calcium, calpain, and caspase-7 in H2O2-induced ER stress-mediated apoptosis in HeLa cells (Pallepati and Averill-Bates 2011b). Similarly, H2O2 (0.3 mM) in oral cancer cells triggers apoptosis via the mitochondrial and ER stress pathways by upregulating CHOP and the 78-kDa glucose-regulated protein (GRP78), and activating caspase-3 and -9 (Min et al. 2008). In MDA-MB468 and T47D breast cancer cells, depletion of PERK reduces Nrf2 activity that results in ROS accumulation and oxidative DNA damage (Bobrovnikova-Marjon et al. 2010). In A94 fibroblast cells, overexpression of CHOP downregulates Bcl-2 expression and depletes GSH, causing an increase in

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ROS levels (McCullough et al. 2001). However, CHOP overexpression alone in these cells is not sufficient to trigger cell death, although it increases cell sensitivity to ER stress inducing agents. CHOP also promotes the expression of the growth arrest and DNA-damage-inducible 45 protein (GADD45), triggering apoptosis by inhibiting protein synthesis (Lei et al. 2017). In leukemia and lymphoma cells, the chemotherapeutic drugs, bortezomib and dipyridamole enhance ER stress by GSH depletion and elevation of ROS generation, subsequently downregulating myeloid cell leukemia 1 (Mcl-1), Bcl-XL, Bcl-2, and XIAP and causing cell death via the mitochondrial apoptotic pathway (Goda et al. 2015). Bortezomib also triggers ER stress and ROS-induced apoptosis in head and neck squamous cell carcinoma (Fribley et al. 2004). In gastric cancer, bortezomib induces apoptosis by inhibiting nuclear factor κB (NF-κB) activation and subsequent ROS production and JNK activation (Nakata et al. 2011).

ROS in Calcium-Mediated Apoptosis Oxidative stress induces rapid calcium release from the ER and triggers the Ca2+mediated mitochondrial cell death pathway (Gorlach et al. 2015). The ER is closely tethered to mitochondria and they interact through mitochondria-associated membranes (MAMs) (Vance 2014). MAMs control the transport of lipids, calcium, and other small molecules between the ER and mitochondria (Hayashi et al. 2009; Wiedemann et al. 2009). Due to their close juxtaposition (Liu and Zhu 2017), mitochondria incorporates some of the Ca2+ released from the ER (Rizzuto et al. 1993; Rizzuto et al. 1998). Increased levels of mitochondrial calcium [Ca2+]mt accelerate the activity of the electron transport chain complex I and III, inducing mitochondrial ROS production (Adam-Vizi and Starkov 2010). Accumulation of [Ca2+]mt also results in mPTP opening, which further increases ROS production due to transient mitochondrial depolarization (Adam-Vizi and Starkov 2010). Mutual interplay between Ca2+ and ROS occurs during the onset of apoptosis (Hempel and Trebak 2017). Under oxidizing and normoxic conditions in the ER, it was found in HeLa cells that ERO1α localizes in MAMs (Gilady et al. 2010; Anelli et al. 2012) and regulates calcium release from the ER by inositol 1,4,5- trisphosphate receptor 1 (IP3R1) during apoptosis initiation (Gilady et al. 2010). In ER-stressed cells, CHOP activates ERO1α-IP3R1-mediated calcium release (Li et al. 2009), subsequently activating calcium/calmodulin-dependent protein kinase II (CaMKII), which in turn induces NOX2-mediated ROS production (Rozpedek et al. 2016). ROS generated through NOX2 further enhance the activation of CHOP and promote apoptosis (Vandewynckel et al. 2013). PERK has also been shown to localize in MAMs and maintain ER-mitochondrial tethering and calcium transfer to induce ROS-mediated mitochondrial apoptosis (Verfaillie et al. 2012). Apoptosis induced by altered calcium signaling around MAMs has been reported in different cancer types (Morciano et al. 2018) but this has not been related to oxidative stress-mediated Ca2+ regulation. We note that ROS also modulate plasma membrane calcium channels, including voltage-dependent Ca2+ channels (VDCC), store-operated Ca2+ entry (SOCE), and

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transient receptor potential (TRP) channels by oxidizing their cysteine residues (Gorlach et al. 2015).

Prooxidant-Based Cancer Therapy Most of the current cancer chemotherapeutic drugs elevate ROS levels in cancer cells either by increasing ROS production or inhibiting antioxidant defense systems. The commonly used doxorubicin, daunorubicin, and epirubicin as well as camptothecins, arsenic agents, and topoisomerase inhibitors generate the highest levels of ROS. For example, arsenic trioxide (Trisenox™), an FDA-approved drug for the treatment of relapsed or refractory acute promyelocytic leukemia (APL) (Antman 2001), induces cancer cell death by ROS production (Chen et al. 1998; Jing et al. 1999), mitochondrial membrane depolarization (Woo et al. 2002), mitochondrial aggregation (Shen et al. 2000), and mPTP opening (Kroemer and de The 1999), which lead to further ROS generation. Arsenic trioxide also irreversibly inhibits thioredoxin reductase in breast cancer cells (Lu et al. 2007b) and diminishes intracellular GSH levels in neuroblastoma cells (Akao et al. 2000). NADPH oxidase was further shown as the main target of arsenic-induced ROS production in APL (Chou et al. 2004). In addition, arsenic trioxide triggers mitochondrial calcium overload in esophageal carcinoma cells (Shen et al. 2002) and ER stress in hepatocellular carcinoma cells (Zhang et al. 2015). Newer chemotherapy drugs/adjuvants that are being developed and work by altering ROS levels in cancer cells include motexafin gadolinium (MGd, Xcytrin), imexon, elesclomol (STA-4783), mangafodipir, and ATN-224. Motexafin gadolinium is a tumor-selective anticancer drug that generates superoxide and other ROS through redox cycling (Evens 2004). It oxidizes thiol groups in intracellular metabolites, such as GSH, ascorbate, and nicotinamide adenine dinucleotide phosphate to produce hydrogen peroxide (Magda et al. 2001). MGd serves as an inhibitor of thioredoxin reductase and ribonucleotide reductase. Studies have shown that MGd induces ROS-mediated apoptosis in multiple myeloma, non-Hodgkin lymphoma, and chronic lymphocytic leukemia (Lin et al. 2009; Naumovski et al. 2004). MGd combined with radiation and/or other chemotherapeutic drug has been shown to have increased effectiveness against solid tumors (Evens 2004). In human uterine sarcoma cells, MGd enhances the effectiveness of doxorubicin and bleomycin in vitro and in vivo (Miller et al. 2001). Similarly, MGd increases the antitumor activity of carboplatin, paclitaxel, cisplatin, and docetaxel in lung cancer cell lines (William Jr. et al. 2007; Magda and Miller 2006). Imexon is a prooxidant molecule that increases ROS levels by reducing cellular GSH level (Dvorakova et al. 2000). It causes mitochondrial swelling, mitochondrial depolarization, cyt c release, and activation of caspase-3,
9, and
8 in multiple myeloma tumor cells (Dvorakova et al. 2001; Evens et al. 2004; Samulitis et al. 2006). In pancreatic cancer cells, imexon induces G2 arrest, ROS accumulation, and apoptosis. At a dose of 100 mg/kg/day, imexon inhibits tumor growth by 27% in 9 days, and delays tumor growth by 21 days in SCID mice (Dorr et al. 2005). Imexon has also been shown to have synergistic effect with docetaxel in breast, prostate, and

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non-small-cell lung cancer (Moulder et al. 2010). Phase I dose escalation study revealed that imexon is well-tolerated at a dose of 875 mg/m2/day for 5 days every other week for 2 months in patients with refractory lymphomas and advanced cancer (Dragovich et al. 2007). Elesclomol (STA-4783) is a chemotherapy drug/adjuvant that promotes apoptosis in cancer cells by inducing oxidative stress (Modica-Napolitano et al. 2019). In melanoma and leukemic cells, elesclomol causes rapid accumulation of ROS and induces a transcriptional gene profile characteristic of an oxidative stress response in vitro, but pretreatment with N-acetylcysteine blocks ROS production and apoptosis in these cells. Elesclomol was also shown to selectively kill cisplatin-resistant lung cancer cell lines by inducing ROS production (Wangpaichitr et al. 2009). Elesclomol combined with paclitaxel enhances antitumor activity in tumor xenografts of breast cancer, lung cancer, and lymphoma cell lines (Berkenblit et al. 2007). In phase II clinical trials, elesclomol with paclitaxel significantly increases the progression-free survival (PFS) of patients with metastatic melanoma (Tuma 2008). A randomized, double-blind, controlled phase III SYMMETRY study in advanced melanoma showed that elesclomol and paclitaxel combination improves PFS in patients with normal baseline lactate dehydrogenase (O’Day et al. 2013). Mangafodipir is a superoxide dismutase (SOD) mimic. In colon cancer cells, mangafodipir potentiates the cytotoxic activity of paclitaxel, oxaliplatin, and 5fluorouracil in vitro and in vivo by increasing H2O2 levels while having no effect in normal cells (Alexandre et al. 2006). ATN-224 is an orally bioavailable copper chelating inhibitor of SOD1 and has antitumor effects in vitro and in vivo. In nonsmall-cell lung cancer, ATN-224 increases ROS levels, induces H2O2-dependent activation of P38 MAPK, and decreases the expression of the anti-apoptotic factor, Mcl-1 (Glasauer et al. 2014). In phase I clinical trials, oral ATN-224 is well-tolerated at a dose of 300 mg/day in patients with advanced solid tumors (Lowndes et al. 2008). In a phase II clinical trial, low-dose ATN-224 (30 mg daily) exhibits bioactivity in patients with biochemically recurrent prostate cancer (Lin et al. 2013). ATN224 combined with bortezomib resensitizes bortezomib-resistant myeloma cells (Richard et al. 2006). ATN-224 combined with temozolomide exhibits additive cytotoxic effects in melanoma cells (Trapp et al. 2009). Several phytochemicals, such as quercetin, curcumin, capsaicin, and phenethyl isothiocyanate, have also been shown to induce ROS-mediated apoptosis in various cancers, and there have been successes in human clinical trials for cancer therapy (NavaneethaKrishnan et al. 2019). Nonetheless, more studies are needed to assess their effectiveness in various cancer situations.

Conclusion ROS implications in cancer cell death include modulation of various intracellular cancer apoptotic signaling pathways such as ERK, JNK, ASK-1, and p53. Oxidative stress in cancer cells potentiates the cancer killing activity of redox modulating drugs, contributing to the therapeutic activity of these drugs.

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However, ROS have also been shown to promote cancer cell progression and metastasis in cancer cell models, and cancer cells develop redox-mediated chemotherapy resistance via regulation of ER stress-mediated autophagy, cell proliferation, and promotion of epithelial-mesenchymal transition (Kim et al. 2019). Furthermore, increased ROS may inhibit apoptosis in cancer cells (Clement and Stamenkovic 1996). Thus, it is critical to understand the dual role of ROS in malignancies in order to develop novel and effective anticancer therapy.

Cross-References ▶ Cutaneous Unfolded Protein Response (UPR) and Endoplasmic Reticulum (ER) Stress ▶ Role of ROS in Triggering Death Receptor-Mediated Apoptosis ▶ Targeting Mitochondria as a Novel Disease-Modifying Therapeutic Strategy in Cancer Acknowledgments This work was supported in part by grants from the CIHR (MOP-123400) and NSERC (RGPIN/06270-2019) to KYL and an Alberta Cancer Foundation graduate studentship to SN. Conflicts of Interest The authors declare no conflict of interest. Authors’ Contributions SN wrote the draft. JR and KYL revised the manuscript.

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Genomic Instability in Carcinogenesis The Role of Oxidative Stress

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Somsubhra Nath and Stuti Roy

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genomic Instability – An Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genomic Instability and Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Role of Oxidative Stress in Genomic Instability in Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Oxidative Stress Induced Genomic Instability in Carcinogenesis: Mechanism and Evidences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Role of Oxidative Stress-Induced Lipid Peroxidation in Genomic Instability . . . . . . . . . . . . Cellular Defense Mechanism against Oxidative Stress-Induced Genomic Instability . . . . . . . . . Conclusion and Future Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract

Genomic instability is one of the hallmarks of cancer. Several theories have been put forth to ascertain this departure from the normal state as being the driver of tumorigenesis or as its final manifestation. Two such theories, the mutator phenotype hypothesis and the oncogene induced replication stress model, have attempted to resolve this dichotomy. However, the source of the mutations implicit in these theories has remained unaddressed in the context of genomic instability. One likely source is the oxidative stress caused by an uncounterable surge of reactive chemical species like reactive oxygen species (ROS) and reactive nitrogen species (RNS). Such species undergo various chemical reactions with different biomolecules present in the cell, most importantly with the DNA and the proteins involved in the maintenance of the genome, leading to the loss of cellular homeostasis and genomic instability. This chapter aims to explicate how oxidative stress drives genetic instability and thus cancer development through interactions with DNA, proteins, and lipids. It also touches upon the S. Nath (*) · S. Roy Department of Basic and Translational Research, Saroj Gupta Cancer Centre and Research Institute, Kolkata, India © Springer Nature Singapore Pte Ltd. 2022 S. Chakraborti et al. (eds.), Handbook of Oxidative Stress in Cancer: Mechanistic Aspects, https://doi.org/10.1007/978-981-15-9411-3_155

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mechanisms the stressed cells employ to reset the homeostasis and how the failure of such means ultimately leads to the loss of genetic integrity and the onset of carcinogenesis. Keywords

Genomic instability · Carcinogenesis · Oxidative stress · Oxidative DNA damage · Mutation · Aneuploidy · DNA damage repair · ROS · Lipid peroxidation · Autophagy

Introduction Genomic instability is a hallmark of cancer (Hanahan and Weinberg 2011). Established theories are there to validate the direct association of genomic instability to carcinogenesis. These instabilities vary in their nature and therefore, categorized into several types. Individually or in combinations, these anomalies initiate as well as augment the mutagenic load in a cellular genome, pushing the cell towards malignancy. In search of pinpointing the source(s) (endogenous factors and exogenous agents) contributing to genomic instability, the role of oxidative stress came forward. An intracellular inequity between the level of pro-oxidant(s) and antioxidant(s) often ends up in oxidative stress, a cellular catastrophe aided by accumulation of reactive chemical species (reactive oxygen species/ROS and reactive nitrogen species/RNS). This chapter will elaborate the role of this oxidative stress in genomic instability and carcinogenesis; the sections will sequentially highlight on the nature of genomic instability, its role in carcinogenesis, the contribution of oxidative stress in genomic instability and carcinogenesis, and the cellular defense mechanism involved there.

Genomic Instability – An Overview Genomic instability is the structural alteration(s) of the genetic constituent (known as “genome”) of a cell. Decoding this, “the genetic constituent” or “genome” of a cell is consisting of DNA (deoxyribonucleic acid), neatly organized in copies of chromosomes in a cellular milieu. Structurally, the DNA range over various lengths, where four bases, namely, adenine/A, guanine/G, cytosine/C, and thymine/T, act as building blocks. The genome of each cell of a particular eukaryotic organism is defined by two identical copies or sets of a fixed number of chromosomes (2 N, where N can be any fixed number for a particular organism). Each set of chromosomes is termed as “ploidy.” Barring a few exceptions, the cellular genome of the majority of eukaryotes is comprised of two sets of chromosomes and hence, diploid. This diploid state of cellular chromosomes is termed as “euploidy.” Moreover, the DNA of each pair of chromosomes has a defined structural organization (the combination of A, T, G, and C) over a particular length and this organization gives each pair of chromosomes of eukaryotic cells a unique structural identity, termed as the karyotype. Maintenance of this karyotype of euploid genome, over the rounds of cell divisions, ensures the

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genomic identity of an organism, where any deviation from it brings upon genomic instability. As discussed above, the genomic instability is a structural change in the chromosomal DNA. As identified, these structural changes are of different natures and are broadly classified into three types, namely, (a) Chromosomal instability (CIN) (b) Microsatellite instability (MSI/MIN) (c) Nucleotide instability (NIN) The chromosomal instability (CIN) represents major group of genomic instability. In CIN, a whole chromosome or a part of it gets duplicated or deleted. The genome is duplicated during the replication or S phase of the cell cycle. During cell division, the spindle assembly checkpoint (SAC) monitors an even and bipolar segregation of duplicated chromosomes and thus, maintains euploidy in two progeny cells. However, defects in SAC give rise to progeny cells that differ from the mother cell in chromosomal constitution. Upon the loss or gain of a whole chromosome, a cell deviates from euploidy, thereby resulting in the onset of aneuploidy; from a diploid mother cell, two aneuploid progeny cells are generated with a change in karyotype. Although the loss or gain of a part of a chromosome does not fit in the classical definition of aneuploidy, it also contributes to the onset of CIN as well as change in karyotype. The microsatellite instability (MSI or MIN) is confined to the microsatellite, a region of genomic DNA characterized by a continuous repeat sequence of 1–6 nucleotides. There are many such regions (50,000 to 1,00,000 microsatellites) spanning the human genome. All the cells in a human body carry the identical pattern of such sequences. To note, during replication, efficiency errors of DNA polymerase often allow mismatches to incorporate in the stretch of a microsatellite region and the cellular mismatch repair (MMR) mechanism removes these mismatches by an enzymatic process. However, this MMR system is found inactive in several diseased conditions, like cancer. This, in turn, results in the persistece of the incorporated mismatches in the microsatellite region, establishing MSI in the cellular genome. The nucleotide instability (NIN), often known as single nucleotide instability, is considered as any deletion, substitution, or insertion of one or a few nucleotides in the cellular genome. This is the least occurring type of genomic instability. The base excision repair (BER) and the nucleotide excision repair (NER) systems are responsible for recognizing such nucleotide incorporation errors during replication. Indeed, impairment of these systems allows NIN to accumulate in the cellular genome.

Genomic Instability and Cancer Genomic instability is a hallmark of cancer; more than 90% of solid tumors and 75% of hematopoietic malignancies show aneuploidy and other forms of instability in the genetic backbone. The advent of advanced cutting-edge technologies has revealed

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numerous mutations in still numerous genes, chromosomal rearrangements, as well as chromosomal deletions and duplications. But most of these cannot be certainly held accountable as the cause of a given event of carcinogenesis. Hence, a validation is required to differentiate between the “drivers” and the “passengers.” The tumor cell population is genetically unstable than normal cells, owing to the presence of different mutations, changes in chromosome number, and microsatellite instability. Such an unstable genome confers these cells greater growth potential and, faster replication, as well as allows evasion of cell cycle and physiological checkpoints, thus giving these cells an advantage in natural selection in the cellular microenvironment or in their niche. Tumor initiation occurs when originally healthy cells undergo genetic changes; those changes when provide a selective advantage drive tumor progression (Yao and Dai 2014). However, it still remains inconclusive if genomic instability is an early step or a manifestation of cancer progression. Indeed, the mechanisms underlying genomic instability remain unclear but several hypotheses have been proposed to explain what drives tumor initiation and progression from the perspective of genomic instability. The two noteworthy ones are (1) the mutator phenotype hypothesis and (2) oncogene induced DNA replication stress model. There is a wide difference in the number of mutations found in normal cells and the ones associated with various malignant diseases. Whereas the occurrence of mutations is rare in normal cells, malignant cells present a whole range of mutations. This disparity is accounted by the mutator phenotype hypothesis. Mutations in the genes that maintain the genetic integrity confer transformed cells a mutator phenotype (Fig. 1). The manifestation of such a phenotype is the increase in genetic instability – higher frequency of mutations which confers a selective advantage, allowing evolution of such cells driving tumor progression (Loeb 2001). This hypothesis suggests that genomic instability is already present in precancerous lesions, which acts as a driving force of tumorigenesis by increasing the rate of spontaneous mutations. That genomic instability is an early driver of tumorigenesis is supported by findings from various elegant studies. One such study found significant chromosomal derangements in nondysplastic and dysplastic colonic epithelia, in addition to chromosomal aberrations found in cancerous cells in hereditary colorectal cancers (Willenbucher et al. 1999). Findings like this suggest chromosomal changes and their instability precedes cancer development. Such chromosomal arrangements are not cancer specific but, are a result of the underlying genetic instability in such cells (Loeb 2001). The genes, mutations in which have been implicated in genomic instability, are also called genome caretaker genes. These are involved in DNA damage repair pathway, DNA damage checkpoint pathway, mitotic checkpoint, and a few more processes required to maintain the integrity of the genome (Loeb 2001; Yao and Dai 2014). Examples of these genes include: (a) DNA damage repair pathway genes – all the genes that participate in nucleotide excision repair (NER), base excision repair (BER), mismatch repair (MMR), and DNA double strand break repair (DSBR).

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Fig. 1 Mutations in multiple pathways in tumor cells result in a mutator phenotype. Reprinted with permission from Keith R. Loeb, Genetic instability and the mutator phenotype, American Journal of Pathology, Vol.154, No.6, June 1999. (Loeb and Loeb 1999)

(b) DNA damage checkpoint genes – genes like ATM, ATR, CHEK2, CDKN2A, etc. (c) Mitotic checkpoint genes – BUB1, BUB3, MAD1, MAD2, MAD3, SGO1, AURORA B, PLK1, PPP2CA, etc. (d) DNA polymerases genes – DNA POL α/β/δ. (e) DNA helicase genes – mutations in DNA helicase genes have been implicated in the development of Bloom’s syndrome and Werner’s syndrome. Thus, inherited mutations in DNA helicases are linked with diseases that are associated with high susceptibility to cancer. (f) In addition to these genes, other genes whose products participate in the ancillary processes of the aforementioned pathways can also be classified as caretaker genes. Mutation in these genes will, as well, contribute to genomic instability as the main pathways would then become nonfunctional. Genomic instability in different inherited or familial cancers can be explained on the basis of the mutator phenotype hypothesis by attributing to the germline mutations in the caretaker genes. A mere second hit to the remaining wild type allele of such genes in the lifetime of an individual is thus capable of driving genomic instability and tumorigenesis. However, in sporadic cancers, mutations in such genes are infrequently encountered. According to various studies, most of the cancers do not have mutations in caretaker genes. Mutation in the mitotic checkpoint gene BUB1 leads to chromosomal instability in experimental models but such mutations are rare in human cancers (Moon et al. 2019). Thus, it is apparent that genomic instability in sporadic cancers is not due to inactivation of caretaker genes. Such a conclusion is, however, not infallible. Since many genes

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are repressed by epigenetic mechanisms, there remains a chance of underestimation of frequency of gene inactivation by mutation frequency analyses. Nevertheless, most of the caretaker genes are recessive in nature; whose loss of function warrants mutations in both the alleles. However, two such events in nonhereditary format are very rare to occur. This argument thus works against the mutator phenotype hypothesis in sporadic cancers but at the same time explains why mutations in caretaker genes lead to genomic instability in hereditary cancers. Since mutations in caretaker genes are not implicated in causing genomic instability in sporadic cancers, mutations in other genes might be held responsible. Thus, it can be said that the most frequently mutated genes are the ones responsible for genomic instability. High throughput studies have indicated high mutation frequencies in TP53 tumor suppressor and in the genes regulating cellular growth (oncogenes). Between these two, mutations in TP53 gene in experimental models do not lead to aneuploidy, and also, in human precancerous lesions, genomic instability is detectable before the occurrence of TP53 mutations. But on the other hand, activation of oncogenes, which stimulate the growth signaling pathways, has been found to induce genomic instability in experimental systems. Taking a cue from this fact, the oncogene induced DNA replication stress model was proposed. This model explains the occurrence of genomic instability from the perspective of DNA replication stress caused by activation of oncogenes in various cancers. This hypothesis also explains the high frequency of TP53 mutations in sporadic cancers despite their apparent inability to induce genomic instability in experimental systems. Oncogene-induced DNA replication stress and thus damage elicits the p53 dependent DNA damage response which limits the growth of the lesion. But when the function of p53 is lost, the cells can escape the restrictions imposed by it. This loss of function is exactly what gets selected in the precancerous/cancer microenvironment (Moon et al. 2019). Replication stress is defined as the slowing and stalling of the replication fork progression and/or DNA synthesis. It, thus, ultimately leads to the replication fork collapse and DNA damage. It usually results in physical structures such as stretches of single stranded DNA (ssDNA). This ssDNA forms when the helicase continues to unwind the parental DNA even after DNA polymerase has stalled (Zeman and Cimprich 2014). Replication stress consequently results in DNA base pair mutations, changes in chromosome structures and chromosome number, ultimately leading to the loss of genomic integrity. Replication stress can be marked or identified by certain biomarkers like the activation of the ATR-CHK1 kinase pathway components, phosphorylated H2AX at serine 139 (γH2AX), POLD3, phosphorylation of replication protein A (RPA) at serine 133, elevated cyclin E levels, etc. (Ren et al. 2017). There are a number of ways in which the replication stress can be induced. Notable and the one relevant here is the oncogene-induced replication stress. As oncogene activation is one of the hallmarks of cancer, constitutive activation of such genes is capable of inducing replication stress. Some of the ways in which oncogenes induce replication stress are listed as follows: (Kerem 2017; Primo and Teixeira 2019).

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(a) Reduction in the number of functional origins – uncontrolled DNA replication due to oncogene activation results in improper assembly of prereplication (preRC) complex proteins, resulting in decrease in the number of functional origins of replication (ORIs). Oncoproteins are capable of directly inhibiting the loading of minichromosome maintenance protein complex (MCM) proteins onto the chromatin. Excess of Cyclin E has been reported to impair the loading of MCM2–7 to chromatin, resulting in a reduced number of activated origins and thus unsuccessful replication. (b) Increased firing of replication origins – Activation of multiple ORIs in a specific region of DNA might lead to DNA re-replication. Oncogene activation may result in the increased expression of pre-RC proteins resulting in increased origin firing. (c) Perturbation of replication fork – Oncogene activation also leads to fork deceleration which causes fork stalling and/or collapse. Arrested replication forks are deleterious to genome integrity. (d) Collisions between replication and transcription machineries – uncontrolled replication may result in the collision of the replication and transcription machineries leading to various DNA topological constraints and the collapse of replication forks. (e) Nucleotide depletion – uncontrolled S phase entry due to oncogene activation leads to exhaustion of dNTP levels resulting in fork stalling and collapse.

The Role of Oxidative Stress in Genomic Instability in Cancer As discussed so far, these two hypotheses, the mutator phenotype and the oncogeneinduced replication stress model do not address the source of mutations that initiate carcinogenesis and thus genomic instability. Whatever be the source of mutation, it starts its manifestation by altering the chemical nature of the DNA. The deoxyribonucleic acid is not an inert chemical entity. It is capable of undergoing various chemical reactions with both exogenous and endogenous chemical assailants. Such assaults on DNA lead to DNA damage and if the damages are left unrepaired, they give rise to various mutations. These very mutations thus ultimately lead to loss of cellular homeostasis and various malignancies. The DNA damaging agents are broadly classified into exogenous and endogenous agents, as mentioned below: 1. Exogenous agents – These agents are sourced from the external environment. The examples include acrolein, formaldehyde, acrylonitrile, 1, 3-butadiene, acetaldehyde, ethylene oxide, isoprene, pyrolysis compounds generated in food, Nnitroso compounds, bile acids (deoxycholic acid and lithocholic acid), saturated fat, ionising radiation, ultra-violet radiation, aflatoxin B and various plant and viral toxins. 2. Endogenous agents – Such agents are formed as by-products of various metabolic reactions inside the body. These include reactive oxygen species (ROS), reactive

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nitrogen species (RNS), various estrogen metabolites (e.g., catechol estrogens), endogenous alkylating agents like S-adenosylmethionine (SAM) and various processes and agents causing hydrolysis of the DNA. Theses endogenous damaging agents inflict several types of damage to the DNA such as: (a) Oxidation of bases (b) Alkylation of bases (c) Hydrolysis of bases, such as deamination, depurination, depyrimidination (d) Bulky adduct formation (e) Single stranded breaks (f) Double stranded breaks Of these, the most frequent type of DNA damage is the oxidative DNA damage, a by-product of cellular oxidative stress, caused by the reactive chemical species [reactive oxygen species (ROS) and reactive nitrogen species (RNS)]. ROS are produced as an unavoidable consequence of the aerobic respiration and by some environmental DNA damaging agents as well. ROS include free radicals like .OH (hydroxyl radical) and non-radical molecules like H2O2, singlet oxygen, etc. ROS attack both base and the sugar moieties of the DNA. Oxidative attack on the DNA bases involves .OH addition to the double bonds. Guanine is the most frequently oxidized base as it has the lowest reduction potential amongst the four bases. Here, 8-oxo-20 -deoxyguanosine (8-oxo-dG) is the most commonly occurring oxidative lesion. The principal source of such oxidants in a cell is the mitochondrial respiratory chain which transports electrons from reduced equivalents to molecular oxygen forming H2O, coupled with ATP synthesis. In this process, the mitochondrial complexes (mainly I and II) can leak electrons leading to partial reduction of molecular oxygen to O2.- (superoxide radical anion) which spontaneously or by the action of superoxide dismutase disproportionates into H2O2. It is estimated that 1–2% of the total O2 consumed by mitochondria is converted to reactive oxygen species. Mitochondria consume 2.2  1010 molecules of O2 every day, which takes the amount of ROS produced intracellularly to the scale of about 1 billion. Multiplying this with the number of cells in our body, which is in the order of trillions, we get an estimate of the ROS produced in our body (Filomeni et al. 2015). The amount of ROS varies with the metabolic demand, age, mitochondrial health, and various other factors. O2. and H2O2, thus produced, react further with other intracellular molecules giving rise to still more reactive species. These reactive species ultimately target the various biomolecules in the cell, primarily the DNA. ROS are neutralized by certain scavengers in the cell known as antioxidants. The eukaryotic cells likewise have thus evolved to possess various antioxidant enzymes and mechanisms. This allows for the maintenance of a balance between the prooxidants and the anti-oxidants. But whenever this balance shifts towards the prooxidants, the cells experience what is known as the oxidative stress. The oxidative

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stress increases the frequency of oxidative damage to DNA, proteins, and lipids. The cells employ the various DNA damage repair pathways which get underway to repair the damaged DNA. But in the circumstance of oxidative stress, the scale of the oxidative damage overwhelms the cellular DNA repair machinery. Add to that a defect (mutation) in any of the repair pathways or oxidation of any protein (impairing its ability to interconvert between active and inactive forms) which regulates the integrity of the genome, this oxidative stress then finally manifests as genomic instability.

Oxidative Stress Induced Genomic Instability in Carcinogenesis: Mechanism and Evidences ROS generates DNA damage through oxidation of nucleotide bases, generating a variety of alterations in the original sequence. As estimated, ROS can cause a total of approximately 100 types of oxidative base lesions and 2-deoxyribose modifications (Chatterjee and Walker 2017). These, in turn, result in base modifications, single strand breaks, and double strand breaks. Here comes the most pertinent question as to how oxidative stress-induced DNA damage perpetuates into genomic instability (GI). Mechanistically, ROS can induce GI by (a) directly damaging the DNA or (b) compromising the cellular surveillance system. As an instance of damage-dependent effect, ROS-mediated attacks often target guanine (G) and converts it into 8-oxo-7,8-dihydroguanine (8-oxoG). Indeed, during replication, 8-oxoG is paired with adenine (A), instead of cytosine (C), and thus, propagates the nucleotide instability (NIN). To repair 8-oxoG or any such base modifications, cells employ base excision repair (BER) system. Similarly, single and double strand breaks are repaired by single strand break (SSB) repair and double strand break (DSB) repair systems, respectively. However, insufficient functioning of these repair pathways, as seen frequently in dividing cells, leads to accumulation of damages and, in turn, contributes to the perpetuation of genomic instability. Evidences, especially in leukemic malignancies, support that the defect in a main DSB repair system, namely, nonhomologous end joining (NHEJ), brings upon alternative inefficient repair mechanism, which culminates into chromosomal aberrations and GI. Towards that, several elegant research findings could be cited to make this notion easier (Sallmyr et al. 2008). Acute myeloid leukemia (AML) is characterized by the presence of the internal tandem duplication (ITD) of the juxtamembrane domain of FLT3 (known as FLT3-ITD). The resultant onco-protein is involved in a signaling cascade which culminates into elevated levels of H2O2 under the enzymatic activity of NADPH oxidase 4 (or NOX4). This elevation in ROS results in the significant increase of a base modification, in the form of 8-hydroxy, 20 -deoxyguanosine (8-OHdG), as well as DSB. Indeed, aided by the increased activation of the unfaithful alternative end-joining pathway and decreased activity of the efficient NHEJ pathway, these damages contribute to genomic instability in the cells of AML-affected individuals. Similarly, in chronic myeloid leukemia (CML), the signature onco-protein, BCR-ABL, is

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involved in perpetuation of GI by inducing ROS production, which causes DNA damage, including DSB. Apart from this ROS-mediated direct DNA damage, the other compromised key cellular mechanisms have also been cited in ROSgenerated genomic instability. One such study pointed towards the involvement of a compromised spindle assembly checkpoint (SAC) activity in ROS-driven GI. SAC is a surveillance mechanism which ensures equal partitioning of the mitotic chromosomes in progeny cells during the course of cell division, thereby maintaining cellular ploidy. At the molecular level, SAC blocks ubiquitination-mediated degradation of anaphase inhibitors, Securin and Cyclin B1, until the bipolar attachment of chromosomes is achieved at the metaphase plane. On the other hand, defects in SAC functioning lead to untimely and faulty chromosome segregation, generating chromosomal instability (CIN). Research findings have showed that H2O2 treatment could override SAC by facilitating premature degradation of anaphase inhibitors, suggesting a link between oxidative stress and chromosomal instability (D’Angiolella et al. 2007). Another study identified Cyclin D1 as a target molecule of ROS towards generation of genomic instability. Continuous release of ROS from mitochondria oxidizes nuclear PP2A phosphatase resulting in the loss of its enzymatic activity. This, in turn, culminates in the loss of the negative feedback control on the AKT pathway. The resultant constitutively active AKT signaling promotes abnormal nuclear retention of Cyclin D1 by blocking its nuclear export signal. This abnormal accumulation in S-phase blocks the DNA re-replication and replication fork synthesis, leading towards double strand breaks in DNA and thus, aiding in GI (Shimura et al. 2016). The idea that there is a relationship between oxidative stress and genomic instability comes from the earliest work in this regard where researchers subjected mammalian cells to exogenous oxidative stresses and subsequently assayed for genomic instability. When the human-hamster hybrid line GM10115 was subjected to oxidative stress by treatment with H2O2 or glucose oxidase (GO), clastogenic effects were analyzed by tracking the single copy of the human chromosome 4, which serves as a marker to monitor chromosomal instability. Exposure to both H2O2 and GO led to a significant increase in the percentage of metaphases where the chromosome 4 was either rearranged or absent (Limoli and Giedzinski 2003). The human-hamster hybrid line could exhibit such an aberrant phenotype only when it was exposed to an oxidative insult for a prolonged period of time (1 h daily for 30 days). Acute oxidative stress (once for 30 min) failed to elicit any such instability (Limoli et al. 1997). Another study showed that when the Chinese hamster fibroblast cell line HA1 was subjected to chronic exposure to H2O2, a 20 to 30 fold increase in the catalase gene activity was observed (catalase decomposes H2O2 to water and oxygen). However, correspondingly only a four to six fold increase in catalase gene copy number was observed. It was then hypothesized that this 20 to 30 fold increase in the catalase gene activity is due to the chromosomal translocations, caused by the activation of the fragile sites by oxidative stress. This altered the gene expression by bringing new regulatory elements such as enhancers in the vicinity of the catalase gene. Development of such a phenotype could also be attributed to ROS induced

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mutations in respective transcription factors or breaks and translocations of negative regulatory elements away from the concerned gene (Hunt et al. 1998). Since the mitochondrial respiratory chain is inextricably linked to ROS generation, mutations in the genes encoding the components of the electron transport complexes can result in the generation of oxidative stress and thereby genomic instability. The B9 Chinese hamster fibroblast cells, which harbor a mutation in the succinate dehydrogenase subunit C (SDHC), were shown to have an increase in the steady state level of O2. and thus metabolic oxidative stress. These cells were found to be highly aneuploid as compared to the parental B1 cells with wild type SDHC (Slane et al. 2006). Such results also advocate the mutator phenotype hypothesis wherein such genes can be enlisted as caretaker genes. By uncoupling the mitochondrial electron transport and disrupting the mitochondrial function in mouse zygotes and early embryos, the protonophore, carbonyl cynide p-trifluoromethoxyphenylhydrazone (FCCP), treatment caused telomere loss, chromosome fusion and breakage. Further treatment of the embryos with the antioxidant Nacetylcysteine (NAC), in addition to FCCP, reverted the state of the chromosomes to the control levels (Liu et al. 2002). Eukaryotic cells have evolved a repertoire of antioxidant defense mechanisms. One of these is the enzyme manganese superoxide dismutase (SOD2). It is the principle defense against superoxide in mitochondria. One such study showed that SOD2 null mouse embryonic fibroblasts (MEFs) had an expected increase in superoxide production. These cells showed a significant increase in the number of double stranded breaks (DSBs), fragments of chromosomes, end to end fusions, and a nine-fold increase in translocations of chromosomal materials per cell as compared to the wild type controls (Samper et al. 2003). Since genomic instability is one of the hallmarks of cancer, many proteins implicated in carcinogenesis have been investigated for their role in eliciting genomic instability through the generation of ROS. Matrix metalloproteases (MMP) are important components of the tumor microenvironment. These proteins remodel the extracellular matrix (ECM) of the tumor niche by their proteolytic activity, allowing tumor invasion and metastasis. An elegant study reported that MMP-3 induced the expression of an alternatively spliced variant of RacI, namely, RacIb, in mouse mammary epithelial cells. RacIb was found to induce the production and the release of the mitochondrial superoxide into cytoplasm. This increase in ROS consequently led to the increase in genomic rearrangements as assayed by the increased resistance of these cells to N-(phosphonacetyl)-L-aspartate (PALA) and in additional genomic amplifications and deletions in MMP-3 treated cells (Radisky et al. 2005). Studying the pathophysiology of the inflammatory diseases, which eventually lead to cancer, further corroborates the link between oxidative stress and genomic instability. It is known that damage associated with inflammatory diseases is significantly contributed by H2O2 induced oxidative stress. One such study reported significant increase in chromosomal abnormalities in normal looking pancreatic epithelial cells derived from patients with chronic pancreatitis and the cells derived from the neoplastic epithelia of patients with pancreatic adenocarcinoma as compared to normal pancreatic epithelia. However, no significant difference in chromosomal abnormalities was found between cells derived from the chronic pancreatitis

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and pancreatic cancer patients, respectively (Moskovitz et al. 2003). This also strongly suggests that genomic instability initiates tumorigenesis. Development of colorectal cancer in patients with ulcerative colitis further supports this observation. Significant alterations in chromosome number were found in the nondysplastic, dysplastic, and cancer specimens from the ulcerative colitis-related epithelium when the respective biopsies were analyzed by fluorescence in situ hybridization and comparative genomic hybridization (Willenbucher et al. 1999). Analysis of the esophageal epithelia from the patients with esophageal squamous cell carcinoma (ESCC) further substantiates this relationship, where development of ESCC is attributed to chronic inflammation of the esophageal epithelia. Presence of double strand breaks in the DNA is marked by phosphorylation of serine 139 in the Cterminal tail of γH2AX. Accumulation of the same indicates the onset and/or prevalence of genomic instability. Dysplastic epithelial cells show significantly higher staining for this marker as compared to the histologically normal tissues (Lin et al. 2016). In vitro culture of the hematopoietic stem cells (HSCs) exemplifies the causal relationship between oxidative stress and genomic instability. Mouse HSCs when cultured under normoxic conditions (20% O2) showed a significant increase in the percentage of aneuploid cells as compared to the same cells when cultured under hypoxic conditions (3% O2). The increase in the number of aneuploid cells as a result of oxidative stress could be reverted to the level of aneuploidy observed under hypoxic conditions by treatment with N-acetyl cysteine, thus, confirming the link between oxidative stress and genomic instability (Liu et al. 2012). Recent studies in polyploid fungal cells have further shed light on the oxidative stress and genomic integrity link. The fungal pathogen, Candida albicans, displays dynamism at the ploidy level. The tetraploid form of the fungus (heterozygous for GAL1 gene on chromosome 1), when grown in a glucose rich medium, shows signs of chromosomal instability as assayed by their viability on 2deoxygalactose (2-DOG) media. The 2-DOG media selects for those cells which have lost the two GAL alleles and undergone a reduction in ploidy. Glucose rich media stimulates oxidative stress. In addition to the reduction in ploidy, there was a significant increase in the DSB, expression of DNA damage repair genes, and oxidative stress induced genes like DDR48 and SOD3 (Thomson et al. 2019).

The Role of Oxidative Stress-Induced Lipid Peroxidation in Genomic Instability In addition to the direct DNA damage, ROS and RNS are also capable of inducing peroxidation of cellular lipids giving rise to the lipid peroxidation (LPO) products. Most common lipid peroxidation products include malondialdehyde (MDA), 4-hydroxy, 2-nonenal (HNE), and acrolein among others. Between these, MDA and HNE are the most studied LPO products. HNE primarily cross links various proteins leading to their modification and thus resulting in changes in gene expression, cell signaling cascades, and signal transduction (Liu et al. 1999; Grune and Davies 2003; West and Marnett 2005).

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MDA interacts with nucleic acids and thus induces various mutations. It reacts with guanine and adenine nucleosides to form various LPO adducts (Marnett 1999b). The major LPO adduct of DNA is a pyrimidopurinone called pyrimido (1,2-α) purin-10 (3H)-one (M1dG) (Marnett 1999a). M1dG may induce base pair mutations like transversions to A or frameshift mutations (Cellai et al. 2017). HNE can react with all the four DNA bases with the highest reactivity with G and C. It can also induce base substitutions like C:T transitions and G:C and A:C transversions (Kowalczyk et al. 2004). HNE also reacts with the amino groups of the proteins and with the amine groups of the DNA bases forming Schiff’s bases. Such reactions lead to cross linking of proteins, protein-DNA, DNA-DNA (intra- and interstrand) (Schaur 2003; Huang et al. 2008). The ethenoadducts like 1, N(6)-ethanoadenine and 1, N(6)-ethenoadenine) are capable of blocking DNA polymerases due to their bulky molecular structure (Wolfle et al. 2006). The DNA polymerases which do make it past these adducts mis-incorporate the bases (incorporating C opposite an A adduct) (Hang et al. 2003). Ethenocytosine induces C to T transitions and C to A transversions in addition to base pair deletions (Basu et al. 1993). Propano adducts of guanosine induce transversions to T and transitions to A (Moriya et al. 1994). Such DNA adducts have been found to be positively correlated with the occurrence of chromosomal aberrations (Talaska et al. 1987). Additionally, ethenoadducts have been found to increase the DNA scission activity of topoisomerase II, leading to DNA strand breaks (Sabourin and Osheroff 2000). Eukaryotic cells are equipped with defense mechanisms to repair the DNA-LPO adducts. However, under the conditions of oxidative stress, this cellular defense is overwhelmed and becomes insufficient to maintain the homeostasis. Additionally, DNA repair proteins are also targeted by LPO products, thus inhibiting the repair of bulky adducts formed leading to the induction of genomic instability. This phenomenon further sensitizes such cells to other carcinogenic exposures.

Cellular Defense Mechanism against Oxidative Stress-Induced Genomic Instability When the elevated levels of ROS and RNS overwhelm the cellular antioxidant systems, damage to DNA and other biomolecules gets induced as has been discussed previously. Concomitantly, it activates a lysosome dependent degradation process called autophagy. Oxidative stress is an important mediator of autophagy. It gets rid of the oxidized biomolecules, hence can be said to be a part of the antioxidant and DNA repair systems (Filomeni et al. 2015). Treatment of cells with antioxidants reverts the process. Autophagy plays an important role in the maintenance of chromosomal integrity and in such an oxidized environment mediates DNA damage response (DDR), both directly and indirectly (Fig. 2). The autophagy reporter protein p62, involved in the ubiquitin proteasomal pathway, physically interacts with a protein called Keap1. Keap1 is the adaptor subunit of the Cul3-ubiquitin E3 ligase complex, which is responsible for the

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Fig. 2 Induction of autophagy by oxidative stress – Increase in cellular ROS leads to activation of autophagy which induces downstream pathways to re-establish cellular homeostasis

degradation of a transcription factor called Nrf2 (nuclear factor erythroid derived 2-like 2) protein. The physical interaction between p62 and Keap1 interferes with the Keap1-Nrf2 binding, thus, resulting in the release of Nrf2 in the cytosol (Komatsu et al. 2010; Ichimura et al. 2013). This uninhibited and thus accumulated Nrf2 then translocate to the nucleus where it regulates the expression of genes involved in oxidant defense like glutathione peroxidase, peroxiredoxin, etc. (Ma 2013). Nrf2 also induces the transcription of the DNA damage repair gene, 8-oxoguanine DNA glycosylase, which removes 8-OHdG from DNA (Singh et al. 2013). Additionally, uninhibited Nrf2 leads to the transcriptional activation of ATM and ATR kinases, the central components of the DNA damage response pathway (Khalil and Deeni 2015). Thus, in this manner, autophagy directly induces DDR. Autophagy can also indirectly induce DNA damage response. It induces the production of ATP (ATP surge) required to meet the energy demands of DNA repair (Katayama et al. 2007). It also controls the dNTP levels in the cell needed for DNA damage repair (Dyavaiah et al. 2011). Once DDR is initiated (directly by DNA damage or by autophagy), it again positively induces autophagy, mainly via p53 which transcriptionally activates various autophagy modulators like DRAM (damage-regulated autophagy modulator) (Crighton et al. 2006) among various others (Scherz-Shouval et al. 2010), thus creating a positive feedback loop ensuring chromosomal integrity. However, deficiency in any of the pathways which participates in mitigating the effects of oxidative stress on the cellular genome leads to genomic instability.

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Conclusion and Future Perspectives The role of oxidative stress in genomic instability as well as its role in carcinogenesis is well documented. Indeed, the direct involvement of ROS in causing DNA damage is validated and supported by experimental evidences. Interestingly, whether these events crosslink each other towards the promotion of malignancy remains a matter of discussion. This chapter analyzed whether the ROS-induced DNA damage perpetuates into genomic instability and cancer promotion (Fig. 3). In this endeavor, initially we summarized evidences for ROS in bringing upon DNA damages in the form of base modifications, single strand as well as double strand DNA breaks. As per the normal functioning of a cell, these damages must be recognized and repaired by DNA damage repair machineries, to avoid instability to pass onto the next cellular cycle. However, as we have discussed in the “genomic instability and cancer” section, the “mutator phenotype model” points towards prior mutations in the DNA damage repair pathway genes and the resulting anomalies in DNA damage repair systems in precancerous lesions. This, in turn, leads to accumulating genomic instability and the carcinogenic transformation of a cell. Interestingly, ROS-induced DNA damages fall prey to this nonfunctional damage repair and majorly contribute

Fig. 3 A schematic diagram on the effect of oxidative stress induced DNA damage on cellular fate. The left-sided arrow displays the onset of genomic instability aided by inefficient or faulty DNA damage repair and its downstream effect on malignant transformation of healthy euploid cells. The right-sided arrow displays the maintenance of genomic stability aided by cellular DNA damage repair system and its effect on propagation of healthy euploid cells and maintenance of cellular homeostasis

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towards genomic instability and carcinogenesis. Moreover, we hypothesize that oxidative stress mediated damage can directly affect the genomic structure of cellular caretaker genes, which might initiate as well as fuel the mutator phenotype model. Together, these explain the involvement of ROS in carcinogenesis through DNA damage mediated genomic instability. Similarly, our hypothesis also extends towards the “oncogene-induced replication stress” model. ROS-induced damage might bring mutations in proto-oncogene(s) and the resultant onco-protein(s) might lead towards replication stress and contribute towards carcinogenesis. Additionally, evidences have also been presented where oxidative stress mediated alteration of protein structure/function bringing upon genomic instability. Oxidative stress has long been associated with different events of cancer. In this chapter, we tried to document and explain the role of oxidative stress in promoting genomic instability and carcinogenesis. Genomic instability is a hallmark of cancer and the molecular association of oxidative stress in this event highlights one of the crucial leads in the field of oxidative stress and cancer. The ongoing as well as future researches will definitely shed light on re-purposing the cellular events like antioxidation and others, in combating the deadly disease, named cancer. Acknowledgment SN is supported by Early Career Award, Science & Engineering Research Board (SERB)-Dept. of Science and Technology (DST), Govt Of India (File No. ECR/2015/ 000206) and Grant-in-Aid, Department of Science & Technology and Biotechnology (DSTBT), Govt. of West Bengal (FST/P/S&T/9G-21/2016) and, Extra mural research grant, Science & Engineering Research Board (SERB)-Dept. of Science and Technology (DST), Govt Of India (File No.EMR/2015/001835). SR is supported by University Grants Commission- Junior Research Fellowship (UGC Ref. No.:771/CSIR-UGC NET-2017).

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Impact of Environmental and Occupational Exposures in Reactive Oxygen Species-Induced Pancreatic Cancer

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Nilabja Sikdar, Subhankar Dey, and Sudeep Banerjee

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Epidemiology of Pancreatic Cancer with Incidence and Prevalence . . . . . . . . . . . . . . . . . . . . . . . Prevalence/Geographic Distribution of Pancreatic Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Environmental, Occupational, Lifestyle, and Genetic Risk Factors for Pancreatic Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Current Treatment of Pancreatic Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Reactive Oxygen Species (ROS)/Oxidative Stress Promotes Cancer Development . . . . . . . . . . Reactive Oxygen Species (ROS) in Relation to Pancreatic Adenocarcinoma . . . . . . . . . . . . . . . . . Environmental Risk Factor of Pancreatic Cancer: Tobacco Smoking in Relation to Reactive Oxygen Species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Occupational Risk Factor of Pancreatic Cancer: Cadmium Heavy Metal in Pesticide in Relation to Reactive Oxygen Species (ROS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cadmium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract

One of the deadliest types of human cancer is Pancreatic Cancer (PanCa), owing to its late stage at presentation and pervasive therapeutic resistance. In the USA, by 2020, PanCa is expected to be the second deadliest carcinogenesis. However, this cancer is usually asymptomatic in nature. Chronic pancreatitis, diabetes, and some infectious diseases are important risk factors. Besides these, acquired risk habits, such as smoking and high alcohol intake increase the chance of N. Sikdar (*) Human Genetics Unit, Indian Statistical Institute, Kolkata, WB, India e-mail: [email protected] S. Dey Department of Zoology, New Alipore College, University of Calcutta, Kolkata, WB, India S. Banerjee Department of Gastrointestinal Surgery, Tata Medical Center, Rajarhat, Kolkata, WB, India © Springer Nature Singapore Pte Ltd. 2022 S. Chakraborti et al. (eds.), Handbook of Oxidative Stress in Cancer: Mechanistic Aspects, https://doi.org/10.1007/978-981-15-9411-3_157

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developing PanCa. Current meta-analysis suggests that the risk of PanCa may be increased by occupational exposures to chlorinated hydrocarbon solvents, organochlorine pesticides, polycyclic aromatic hydrocarbons (PAHs), nickel (Ni), cadmium (Cd), and chromium (Cr). In the digestive system, oxidative stress is known to initiate cancer response. Mitochondrial respiration is one of the key generation machinery, and other environmental and occupational exposures/ chemical reagents that induce damage to the DNA, proteins, lipids, and produce many toxic and high mutagenic metabolites that could result in tumor behavior, changing it into a malignant phenotype, and finally in cancer cell transformation. Numerous toxic, carcinogenic, and mutagenic chemical substances along with stable and unstable free radicals and reactive oxygen species (ROS), in the particulate and gas phase of tobacco smoke have the potential of biological oxidation. Several research documentations suggest that carcinogenic compounds in cigarette smoke stimulate cellular proliferation and progression of PanCa by inducing inflammation and fibrosis which results in genetic factors leading to the inhibition of cell death. It has been hypothesized that exposure to one of the occupational risk factors, Cd, a ubiquitous metal, is one of the potential cause of PanCa due to its toxic and carcinogenic properties. Evidence of involvement of Cd in PanCa development has been recently mentioned in our observational studies, meta-analyses, and experimental animal and in vitro studies. Gathering all these previous data, it is clear that oxidative stress-related responses are affected during Cd stress, but the apparent discrepancies observed in between the different research findings point towards the necessity to increase our knowledge on the spatial and temporal ROS signature under Cd stress which is present in different kinds of occupational exposures. Our present effort is to summarize findings in a systematic approach, as to how environmental and occupational risk factors induce ROS generation in PanCa, in addition to its mechanism of action, signalling cascade in the prognosis of the disease. Keywords

Pancreatic cancer · Environmental risk factors · Occupational exposures · Oxidative toxic stress · Heavy metal ions · Oxidative damage Abbreviations

1-(N-methyl-N-nitrosamino)-1-(3-pyridinyl)-4-butanal): NNAA,4-(methylnitrosamine)-1-(3-pyridyl)-1-butanone: NNK,4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol: NNAL,4-hydroxynonenal Alkoxyl radicals Aluminum Aqueous Cigarette Tar Arsenic Base Excision Repair Benzyl IsoThioCyanate Body Mass Index

4-HNE

RO• Al ACT As BER BITC BMI

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Cadmium Carbon-centered radicals Catalase Chromium Hepato Cellular Cancer Chronic Hepatitis c-Jun N-terminal Kinase Coenzyme Q10 Copper Cyclic AMP Cytochrome P450 Dinitrogen dioxide Disulfides Electron Paramagnetic Resonance Endoplasmic Reticulum Epigallocate-3-gallate Epithelial-to-Mesenchymal Transition European Prospective Investigation into Cancer and Nutrition Extra Cellular Matrix Extracellular-Regulated kinase ½ Familial Pancreatic Cancer Glutathione Peroxidase Glutathione-S transferase Hydrogen Peroxide Hydroquinone Hydroxyl radical Hypochloride Hypoxia Inducible Factor-1 Inflammatory Bowel Disease Insulin-like growth factor I Interleukin International Agency for Research in Cancer International Pancreatic Cancer Cohort Consortium Iron Kelch-like protein 19 Lactate dehydrogenase Lead Lipid Peroxidation Malonaldehyde Mammalian Target of Rapamycin Mercury Metal regulatory transcription factor 1 Metallothioneins Mitochondrial matrix Mitogen-activated kinase Mitogen-Activated Protein Kinase

Cd -C• CAT Cr HCC CH JNK CoQ Cu cAMP CYPs 450 N2O2 RSSR EPR ER EGCG EMT EPICN ECM ERK1/2 FPC GPXs GST H2O2 QH2 •OH HOCl HIF-1 IBD IGF1 IL IARC IPCCC Fe KEAP1 LDH Pb LPO MDA mTOR Hg Mtf1 MT MnSOD MEK MAPK

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N0 - nitrosonornicotine NADPH oxidase National Toxicology Program NFE2 related factor Nicotinamide Adenine Dinucleotide Phosphate Reduced Nicotine Acetylcholine Receptors Nitric Oxide radical Nitrocarbonate anion Nitronium Nitrosoperoxycarbonate anion Nuclear Excision Repair Nuclear factor erythroid 2-related factor 2 Organic Hydroperoxides Organic radicals Oxidative Toxic Stress Ozone/trioxygen Pancreatic Cancer Pancreatic Ductal AdenoCarcinoma Pancreatic Stellate Cells Peroxiredoxins Peroxyl radicals Peroxynitrite Peroxynitrite Phosphatidylinositol 3-kinase Poly Aromatic Hydrocarbons Protein Kinase C Quinone Reactive Chloride Species Reactive Nitrogen Species Reactive Oxygen Species Reactive Sulfur Species Receptor Tyrosine Kinase Semiquinone Singlet oxygen Sulfonyl radicals Superoxide dismutase Superoxide Thioredoxins Third National Health and Nutritional Examination Survey Thiyl radicals Thiol peroxyl radicals Upstream stimulator factor Vascular Endothelial Growth Factor Zinc Γ-glutamylcysteine synthetase

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NNN NOX NTP Nrf2 NADPH nAChR NO• O2NOCO2 NO2+ O¼NOOCO2 NER NRF2 ROOH R• OTS O3 PanCa PDAC PSC PRXs ROO• ONOO Ο¼ΝΝOΟ PI3K/Akt PAHs PKC Q RCS RNS ROS RSS RTK QH• 1O2 ROS• SOD O2• TRXs NHANES III RS• RSOO• USF VEGF Zn γ GCS

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Introduction Epidemiology of Pancreatic Cancer with Incidence and Prevalence The pancreas is an endoderm-derived glandular organ and popularly known for its mixed secretory nature. It secretes several metabolic hormones and digestive enzymes for carbohydrate and protein homeostasis. The gland largely (about 80%) comprises of acinar cells which are exocrine in nature and secrete zymogens. Secretion from the exocrine tissue carries outside the gland through branched ductal system and ultimately leads into the duodenum by ampulla of Vater. Pancreatic ductal adenocarcinoma (PDAC) is the most common type of pancreatic neoplasm and accounts for >85% of all PanCa. PDAC may develop from any part of the pancreas and also from periampullary region (Maitra and Hruban 2008). Although the exact reasons of development of sporadic PDAC are still unknown, there are, however, several factors that have been duly identified in association with the occurrence of PanCa. These factors include the following: long-term exposure to heavy metals; occupational hazards; lifestyle factors such as smoking, drinking, and diet; metabolic conditions such as diabetes mellitus; and inflammatory conditions such as pancreatitis. DNA damage due to generation of reactive oxygen species (ROS) is a well-established cause of different types of cancer, including PanCa, and all of these above mentioned factors show the ability of generating oxidative stress and DNA damage (Djordjevic et al. 2019).

Prevalence/Geographic Distribution of Pancreatic Cancer Globally, PanCa could be considered as a rare form of cancer with around 2.5% of all types of cancer. However, if we look at the last 10 years data of PanCa incidence, it clearly indicates the drastic rising pattern of PanCa. In 2008 and 2018 world wide 2,77,000 and 4,58,918 new PanCa cases were recorded respectively, for both sexes. According to many workers, this extreme rise in PanCa cases in recent days is due to precision in PanCa diagnosis. In spite of low frequency of occurrence, due to poor prognosis, vague symptomatic features, rapid metastasizing nature, low survival rate (median 5 year survival rates of 6%), and profound resistance to chemo- and radiotherapy, PanCa ranks the seventh most serious cause of cancer death globally and fourth in the developing countries (Fig. 1) (Raimondi et al. 2009). In comparison to the less-developed regions such as Africa and South Asia, more PanCa incidence has been recorded from the developed regions such as Northern America, Europe, Australia, and New Zealand; however, moderate cases have been recorded from South and Central America and East and West Asia (Lewis et al. 2009). In the northern countries, the incidence rate is three to four fold higher for both sexes than the countries that lie near the equator (Fig. 2). This increase in the incidence of PanCa in higher latitudes may be correlated with decrease in exposure

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Fig. 1 Seventh position of PanCa among all other types of cancer based on age-standardized rate (World standard population, per 100,000) of death for both sexes. (GLOBOCAN 2018 estimates)

to sunlight and synthesis of vitamin D. This could be a possible reason of variation in the rates of PanCa in the countries spanning a wide range of latitude such as Japan (Fig. 3) (Lewis et al. 2009). Since 1990, irrespective of the rapid increase of PanCa cases, the mortality rate for both sexes (7–9 per 100,000/year in men and 4–6 per 100,000/year in women) is almost stable in developing countries. The reason could be the increase in the accuracy of PanCa diagnosis and better life support (Rawla et al. 2019).

Environmental, Occupational, Lifestyle, and Genetic Risk Factors for Pancreatic Cancer Age, Gender, and Race The occurrence of PanCa is strongly age dependent like other adult tumors. In the USA, PanCa incidence has been recorded as one of the highest in the world, and it mostly occurs after 70 years. However, many PanCa cases have been recorded at an early age or before the age of 50 years old which is only 5–10% of all PanCa

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Fig. 2 Estimated age-standardized pancreatic cancer incidence rate per 100,000 for 2018 (both sexes, all ages). (GLOBOCAN 2018)

incidences (Maitra and Hruban 2008; Orth et al. 2019). Pancreatic cancer in the early age group may include those having underlying predisposing genetic alterations or who had previous exposure to radiation therapy for cancer treatment. Commonly, the incidence of PanCa in females is less than in males (Fig. 4). This is probably due to the influence of male-specific hormonal activity, predominant smoking habit or higher rate of alcohol consumption in males. The lifetime cumulative probability of developing PanCa is about 1% for males, whereas in females it is comparatively less (Capasso et al. 2018). A 5-year-long (2001–2005) study by Arnold et al. 2009, demonstrated a higher rate (>48%) of PanCa incidence in the Black people than the Whites (Arnold et al. 2009). The mortality rate is also much higher (>35%) in the Black people as compared to the Whites, and this disparity persists across genders. The mortality rate of PanCa is approximately 27% greater in Black men and 38% greater in Black women in comparison with the White men and women. This striking difference in the frequency of PanCa in different human races is not clear; however, it is believed to have some non-genetic risk factors, such as diabetes, body mass index, smoking, alcohol consumption, and vitamin D deficiency, or some genetic risk factors, such as race-specific genetic disability to detoxify tobacco products, production of vitamin D, etc. (Hassan et al. 2007).

Smoking and Tobacco Almost all the research studies have positively correlated smoking habit with PanCa. Smoking causes a great damage to the pancreas immediately after the lungs and raises the chance of PanCa by 70–100%. Active smoking may double the risk of

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Fig. 3 Pancreatic cancer mortality time trends in males and females respectively for selected countries upto 2005 estimates in relation to demographic factors. [upper panel: age-standardized rate (world) for males of all ages] [lower panel: age-standardized rate (world) for females of all ages] [International Agency for Research on Cancer (IARC) 26.5.2010]

PanCa in comparison to the nonsmokers, and the total elimination of smoking habit may decrease the chance of PanCa to approximately 25%. Passive smoking also has a great impact especially on children. Many pooled data analyses from the International Pancreatic Cancer Cohort Consortium (IPCCC) and the European Prospective Investigation into Cancer and Nutrition (EPICN) have clearly indicated the increased chance of PanCa in smokers to 77%. Besides smoking, the smokeless tobacco

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Fig. 4 Continent-wise age-standardized rate (World standard population, per 100,000) of incidence and mortality in men and women due to pancreatic cancer for all the ages. (GLOBOCAN 2018 estimates)

products also have detrimental impact on the pancreas and increase the risk of PanCa, although few recent meta-analyses have provided ambiguous results (Chowdhury and Rayford 2000; Edderkaoui and Thrower 2013).

Alcohol Alcohol is found to be one of the known risk factors in several organs, especially in the esophagus and liver, although earlier studies have revealed either little or no effect of alcohol on the pancreas. The pooled data studies of 14 cohorts with highest alcohol consuming (30 g alcohol/day) women category have exposed a weak relationship between alcohol consumption and PanCa; however, meta-analyses have indicated a dose-dependent relationship. Consumption of repeated (3 or more drinks per day) or high amount (30–40 g alcohol/day) of alcohol may increase the chance of PanCa to 22.0% (Go et al. 2005). Dietary Factors No direct relationship has been observed between diet and PanCa; however, some studies have indicated an increased risk of PanCa in the persons having a diet rich in meat (especially red meat), cholesterol, and nitrosamines. Consumption of grilled or

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fried foods may equally cause high risk of PanCa, although many studies have suggested a preventive role of a healthy diet (rich with vegetables and citrus fruits) or so-called Mediterranean diet against many cancers including PanCa (Silverman et al. 1994).

Vitamin D and UVB Irradiation Vitamin D is considered as one of the potential preventive factors of PanCa. Since vitamin D comes from daily diet as well as the body can synthesize it under the influence of sunlight, the direct assessment of the preventive nature of vitamin D-rich diet against PanCa is difficult. In spite of that, some studies have suggested a protective effect of both dietary and body-stored vitamin D on PanCa (Hassan et al. 2007). Obesity In many previous studies, no such relation has been observed between obesity and PanCa; but almost all recent studies have indicated the deleterious impact of high caloric intake or obesity or both on the pancreas. Although the mechanism of PanCa development due to obesity is not clearly understood, there may be a direct link in tumor formation or indirect link via inflammatory responses. Recent meta-analyses have revealed that the increase in body mass index (BMI) is closely related to the increase in the risk of PanCa (Hassan et al. 2007). Occupational Exposures of Heavy Metals The relationship between the risks of PanCa and other occupational exposures remains contentious. However, people engaged in some professional sectors may come in contact with potential carcinogens or teratogens on a regular basis; many professional fields, such as laundry, dry cleaning, painting, printing, and paper manufacturing; chemical, petroleum, rubber, and leather industries; transformer manufacturing plants; the production of pesticides; and mechanic and metallurgical occupations, as well as miners, shot-firers, stonecutters, and carvers, machinery mechanics and filters, building trade workers and motor vehicle drivers in men, office clerks in women, and waiters in both sexes have the highest adverse effect on human health that includes high risk of PanCa (Alguacil et al. 2003; Ji et al. 2001). Numerous studies have marked the occupational exposure to organochlorine, chlorinated hydrocarbons, formaldehyde, PAHs, styrene, pesticides, and heavy metals, such as lead (Pb), nickel (Ni), chromium (Cr), and Cd, as a high chance of causing PanCa (Fritschi et al. 2015). PanCa incidence is also linked to Cd, arsenic (As), and Pb exposures in observational studies. Highest incidence of PanCa with the highest levels of As (more than 10 ug/L, values recommended by the World Health Organization) has been observed in Baltic countries and Central and Eastern European countries, such as Austria, Czech Republic, Slovakia, and Hungary (Martinez et al. 2017). Some of these agents are believed to be directly genotoxic, while others might enhance the mutagenicity and carcinogenicity of directly acting genotoxic agents (Andreotti and Silverman 2012; Ojajarvi et al. 2000). Increased risk of PanCa was associated with some exposures, but most of the research found weak or moderate

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results, often associated with the very less number of participants. Studies that have established the association by type of occupation did not usually determined the specific agents to which the worker is exposed, and the ultimate effect is to establish specific associations. Gene-environment interactions are important to progress the study related to the association with K-ras gene in search of more end results (Lo et al. 2007; Santibanez et al. 2010). Well-established carcinogens PAHs are generally produced by the combustion of organic matters found in soot, carbon black, coal tar and pitch, bitumen, asphalt, and mineral oils. Many epidemiological studies conducted in Norway, Italy, and Canada have pointed out the risk of PanCa incidence as well as high mortality rate in the professional groups who are exposed to PAHs in aluminum (Al) production and asphalt industries. People who are routinely in contact with diesel exhaust (e.g., motor vehicle drivers and railroad workers) may also have high risk of PanCa. Higher PanCa incidences in embalmer and pathologist group also indicate the noxious effect of formaldehyde on pancreas (Li et al. 2006).

Chronic Pancreatitis Although chronic pancreatitis is a rare incidence ( regulatory T cells > naive T cells > memory T cells. CD8+ effector memory T cells are the most sensitive to ROS among T cell subsets (Mougiakakos et al. 2009). ROS also manipulate factors that participate in T cell proliferation and survival. For instance, the nuclear factor of activated T cell 5 (NFAT5), which plays a role in T cell proliferation and survival, is inhibited in its binding to IL-6 promoter by ROS (Trama et al. 2000).

Therapeutic Strategies that Affect ROS and Influence Anti-tumor Immunity ROS signaling is critical for cancer initiation, progression, and metastasis. Some of the drugs that regulate ROS levels in the TME exhibit antitumor immune modulation (Table 1). Bortezomib, an inhibitor of the ubiquitin-proteasome proteolytic pathway, enhanced cytotoxic T lymphocyte responses against immune-resistant melanoma (Shanker et al. 2015). Doxorubicin induces ROS generation and shows selective MDSC cytotoxicity (Alizadeh et al. 2014). Treatment with this drug results in increased effector T cells and NK cells. 5-flurouracil and cisplatin have been individually shown in murine models of breast and colorectal cancers to induce ROS production and subsequent MDSC apoptosis. This treatment also generates enhanced CD8+ T cell and NKT cell responses. Photodynamic therapy (PDT) or photochemotherapy results in death of tumor cells by generating excess ROS. Through a localized oxidative stress–induced strong inflammatory reaction, PDT has been shown to modulate the host immune response to the tumor (Castano et al. 2006). In contrast, drugs that cause a reduction of ROS have resulted in increased antitumor immunity. Celecoxib, a COX-2 inhibitor used for the treatment of

Drug Metformin

Doxorubicin

5fluorouracil (5FU)

Cisplatin

Bortezomib

Celecoxib

SI. No 1.

2.

4.

5.

6.

7.

MDSC

CD4+

Tregs and MDSC

MDSC

Tumor-induced MDSC

Immune cell type Tumor-infiltrating CD4+ CD25+ regulatory T cells (Ti-Treg)

BALB/c mice

Peripheral blood samples from 53 patients with multiple myeloma treated with bortezomib

Female C57BL/6 mice (5– 8 week old)

Six- to eight-week-old Balb/c and C57BL/6 mice. Six- to eight-week-old gp91phox / (C57Bl6-Cybbtm1Din) Nude mice and TLR4 / C57BL/6 mice

Model systems BALB/c and C57BL/6 (B6) mice

Table 1 Immune cell responses to ROS modulators in cancer treatment

Selectively induces MDSC apoptotic cell death and enhances antitumor CD8+ T cells, NK cells, and B lymphocyte cell functions Cisplatin and CRT/E7 DNA vaccine generated E7-specific CD8+ T cell immune responses, which reduced MDSC and Tregs The selective and reversible proteasome inhibitor targets the catalytic 20S core of the proteasome and induces apoptosis in myeloma and lymphoma cells Impaired function of all MDSC subtypes due to the reduction in ROS and NO levels

Phenotype Differentiation of naïve CD4+ T cells into inducible Tregs (iTregs) is inhibited by reducing forkhead box P3 (Foxp3) protein expression Selective cytotoxic effects on MDSC and increase in effector lymphocytes and NK cells

Prostate cancer

Head and neck cancers and multiple myelomas

Breast, ovarian, lung, and gastric cancers

Hematological malignancies, soft tissue sarcomas, and breast cancer Colorectal and breast cancers

Cancer treated Pancreatic and gastric cancers

Veltman et al. (2010)

Shanker et al. (2015)

Reviewed in De Biasi et al. (2014)

Vincent et al. (2010)

Alizadeh et al. (2014)

References Kunisada et al. (2017)

744 L. R. Perumalsamy et al.

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colorectal cancer, resulted in impaired MDSC function with reduced ROS levels (Veltman et al. 2010). Similarly, metformin inhibits ROS production and prevents formation of inducible Tregs (Kunisada et al. 2017). The addition of ROS scavengers resulted in enhanced activation of CD8+ TILs in kidney tumors by activation of SOD2 (Siska et al. 2017). Collectively, these studies suggest that modulating the redox homeostasis in TME can induce antitumor immunity. Tumor immunotherapy including T cell adoptive immunotherapy (ACT) and checkpoint inhibitors functions by augmenting or reinforcing the host’s immune system. Recent studies have demonstrated that combining immunotherapy with ROS modulation increases antitumor activity. Combining the use of chemo-photodynamic therapy with ROS-sensitive nanoparticle augmented the antitumor activity of the anti-PD-L1 (checkpoint) antibody (Hu et al. 2019). Inhibition of NOX4 resulted in an increased recruitment of CD8þ cells and susceptibility to different immune-based therapies in mouse models (Ford et al. 2020). Treatment of tumor-specific CD4-positive T cells with Cytoxan (ROS inducer) results production of inflammatory cytokines (such as TNF-α, interferon γ). Adoptive transfer of these cells promoted the decay of vessel-intensive tumor. In addition, the Cytoxantreated CD4+ T cells altered the tumor metabolism resulting in deficiency of antioxidant GSH and enhanced antitumor activity. Cytotoxic T lymphocytes engineered with T cell receptors that coexpressed catalase showed protection from oxidative stress. Subsequently, they also exhibited increased antitumor activity (Ligtenberg et al. 2016). A summary of the effect of modulating ROS in TME on immune cell function is summarized in Fig. 2.

Conclusions Redox homeostasis plays an essential role in maintaining diverse cellular processes. The immune response to the cancer is tightly regulated by the redox state in the TME. Increased ROS levels are both NOX-2 dependent and mtROS, and result in an immunosuppressive environment in the TME. In addition to the tumor cells, several immune cells including MDSCs, Tregs, and TAMs contribute to this immunosuppression. Elevated ROS in the TME helps in recruiting, activating, and promoting the function of these suppressive cells. This results in attenuation of the cellmediated immune response to the tumor. In particular, the activation and function of effector T cells are compromised. Given its dichotomous nature, the maintenance of ROS levels is essential for potent cell–mediated immunity. Within the TME, the ROS levels are maintained to promote immunosuppression. Several lines of evidence have shown that disruption of this steady-state levels within the TME result in mounting successful antitumor host immune response. Emerging research suggests that combining ROS modulation of TIL during adoptive immunotherapy would augment their antitumor activity. Thus, further studies should focus on mapping the relationship of ROS to their

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Fig. 2 Immune response to ROS modulating drugs

immunosuppressive effects in T cells across cancer types. This approach would combine the targeting of ROS with immune-based treatment of cancer patients to provide a more promising clinical outcome.

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Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Production of ROS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ROS from Mitochondria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ROS from Oxidase Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ROS from Peroxisomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chronic Inflammation and Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Oxidative-Stress-Induced Inflammation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Molecular Mechanisms of ROS-Induced Carcinogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cell Signaling Cascades in ROS-Mediated Inflammation and Cancer . . . . . . . . . . . . . . . . . . . . .

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Shibi Muralidar and Gayathri Gopal contributed equally with all other contributors. S. Muralidar Biopharmaceutical Research Lab, Anusandhan Kendra-1, SASTRA Deemed-to-be-University, Thanjavur, Tamil Nadu, India School of Chemical and Biotechnology, SASTRA Deemed-to-be-University, Thanjavur, Tamil Nadu, India G. Gopal Biopharmaceutical Research Lab, Anusandhan Kendra-1, SASTRA Deemed-to-be-University, Thanjavur, Tamil Nadu, India School of Chemical and Biotechnology, SASTRA Deemed-to-be-University, Thanjavur, Tamil Nadu, India Department of Bioengineering, School of Chemical and Biotechnology, SASTRA Deemed-to-beUniversity, Thanjavur, Tamil Nadu, India S. V. Ambi (*) Biopharmaceutical Research Lab, Anusandhan Kendra-1, SASTRA Deemed-to-be-University, Thanjavur, Tamil Nadu, India Department of Bioengineering, School of Chemical and Biotechnology, SASTRA Deemed-to-beUniversity, Thanjavur, Tamil Nadu, India e-mail: [email protected] © Springer Nature Singapore Pte Ltd. 2022 S. Chakraborti et al. (eds.), Handbook of Oxidative Stress in Cancer: Mechanistic Aspects, https://doi.org/10.1007/978-981-15-9411-3_181

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Apoptosis and Survival . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Inflammatory Markers and ROS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Tumor Microenvironment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Components and Characteristics of TME . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Hypoxia, Angiogenesis, and Metastasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ROS in Cancer Metastasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Angiogenesis and ROS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Regulation of ROS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract

Cancer still exists as one of the most alarming disease throughout the world. Predictions put forward a count of 13 million fatalities due to cancer by the year 2030 which shows the indispensable need for scientific interventions against cancer. In recent years, dysregulation of the redox balance has been demonstrated as an important cause for cancer development, progression, and subsequent metastasis in the human cells. This disruption in redox homeostasis is mediated through the upregulation of free radicals which are predominantly found to be reactive oxygen species (ROS). ROS plays a crucial role in tissue homeostasis, regulation of cellular signaling, differentiation, and survival. In addition, ROS regulates cellular homeostasis and also acts as a chief modulator in the process of cellular dysfunction resulting in disease pathophysiology. Elevated levels of the dysregulated ROS subsidize the detrimental processes like tumorigenesis, cancer progression, and spreading. On the other hand, excessive levels of ROS will subsequently result in an anti-tumorigenic effect facilitated by the promotion of cell-death, induction of cell-cycle arrest, and senescence. Thus, ROS acts as a double-edged sword with both pro and anti-tumorigenic effects leading to a duo of detrimental and beneficial role in cancer biology. Chronic inflammation is often associated with elevated levels of ROS and RNS that give rise to several epigenetic changes, DNA mutations, and genomic instability which in turn promotes tumor initiation, development, progression, metastatic dissemination, and treatment resistance. This chapter will summarize in detail the production of ROS, the link between oxidative-stressinduced inflammation in cancer, chronic inflammation and cancer, the role of ROS in the tumor microenvironment, and regulation of ROS. Keywords

Chronic inflammation · Oxidative-stress · RNS · Antioxidants · Transcription factors · Tumor microenvironment

Introduction Regardless of continuous efforts in advancements and development of novel treatment strategies, cancer still exists as one of the most alarming disease throughout the world. Being a dreadful disease, cancer remains an unconquerable elusive target for scientists in both developing and developed countries. Predictions put forward a count of

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13 million fatalities due to cancer by the year 2030 which shows the indispensable need for scientific interventions against cancer. Also, the multifaceted presentation of cancer further worsens the complexity of the disease. In recent years, dysregulation of the redox balance has been demonstrated as an important cause for cancer development, progression, and subsequent metastasis in the human cells. This disruption in redox homeostasis is mediated through the upregulation of free radicals which are predominantly found to be reactive oxygen species (ROS) (The Lancet 2018; Aggarwal et al. 2019). ROS is a group of highly reactive molecules that is produced as a result of aerobic metabolism. ROS plays a crucial role in tissue homeostasis, regulation of cellular signaling, differentiation, and survival. In addition, ROS regulates cellular homeostasis and also acts as a chief modulator in the process of cellular dysfunction resulting in disease pathophysiology (Forrester et al. 2018; Harris and Denicola 2020; Jia et al. 2020; Perillo et al. 2020). Disparities in the basal levels of ROS lead to detrimental effects in cells, provoking several disease conditions. Substantial research during the past two decades has unraveled the essentiality of ROS for the process of initiation, progression, angiogenesis, and metastasis of cancer. Elevated levels of the dysregulated ROS subsidize the detrimental processes like tumorigenesis, cancer progression, and spreading. On the other hand, excessive levels of ROS will subsequently result in an anti-tumorigenic effect facilitated by the promotion of cell death, induction of cell-cycle arrest, and senescence. Thus, ROS acts as a double-edged sword with both pro- and anti-tumorigenic effects leading to a duo of detrimental and beneficial role in cancer biology (Liao et al. 2019; Harris and Denicola 2020; Kirtonia et al. 2020). This chapter will summarize in detail about the production of ROS, the link between oxidative-stress-induced inflammation in cancer, chronic inflammation and cancer, the role of ROS in the tumor microenvironment, and regulation of ROS.

Production of ROS ROS can potentially mediate both pathophysiological and physiological signal transduction. Production of ROS is dependent upon several enzymes and subcellular compartments that are linked to numerous metabolic regulations. Further, any changes in the redox balance can directly influence the diseases that are associated with metabolic dysfunction, proving the importance of controlled levels of ROS. ROS are series of molecules that include superoxide (O2 ) – which is moderately reactive and short-lived, hydrogen peroxide (H2O2) – moderately reactive and longlived, hydroxyl radical (•OH) – extremely short-lived, and the most potent oxidizing species, and peroxyl radical (ROO•) – the most reactive ROS (Mittal et al. 2014; Forrester et al. 2018; Harris and Denicola 2020).

ROS from Mitochondria Mitochondria are the central source of endogenous ROS. A subtle balance between pro- and antioxidants is a necessitating factor for the proper functioning of the

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respiratory chain. Mitochondrial respiration, which is relied on a proton gradient and electron transfer, plays a pivotal role in oxidative ATP production. During this process, water is acquired as a resultant product by the reduction of molecular oxygen (O2) in the electron transport chain (ETC). Improper mitochondrial ROS (mitoROS) productions are associated with several detrimental metabolic diseases and inflammatory reactions. MitoROS production generates intracellular ROS like O2˙ and H2O2 at various sites of mitochondria (Forrester et al. 2018; Snezhkina et al. 2019). For example, O2˙ are produced at complex I, complex III, pyruvate dehydrogenase, glycerol 3-phosphate dehydrogenase, 2-oxoglutarate dehydrogenase, and Q oxidoreductase (Brand 2010). Complex I act as an access point for electrons from NADH to get into the mitochondrial respiratory chain. Interaction of O2 with flavin mononucleotide (FMN) in the presence of a high ratio of matrix NADH/NAD+ results in the production and subsequent release of O2˙ into the mitochondrial matrix (MM). Further, mitoROS are also produced through a two-step process of reverse electron transfer (RET) in complex I. Complex III provides another crucial platform for mitoROS production. Even though complex III produces a low amount of O2˙ , the Qi site of mitochondrial complex III gets inhibited in the presence of antimycin A, which in turn upregulates the production of O2˙ from Qo site. This upregulation results from the interaction of O2 with ubisemiquinone bound to the Qo site. Apart from complex I and III, complex II flavin site also assists in mitoROS production. ROS generated from complex III and glycerol 3-phosphate dehydrogenase is mainly released into intermembrane mitochondrial space (IMS), but further dismutation of O2˙ into H2O2 by Manganese superoxide dismutase (Mn-SOD) leads to the diffusion of H2O2 into MM. Further, mitochondrial aconitase converts H2O2 into •OH through a Fenton reaction in MM (Forrester et al. 2018; Snezhkina et al. 2019). Thus, the indecorous functioning of the powerhouse of the cell powers-up the production of detrimental ROS.

ROS from Oxidase Activity Apart from mitochondria, numerous oxidases acts as pivotal producers of ROS, among which NADPH oxidases (NOX) play the chief role. One of the most common sources of cytoplasmic ROS (cytoROS) is the NOX family, which includes seven members, namely, NOX1, NOX2, NOX3, NOX4, NOX5, dual oxidase 1 (DUOX1), and DUOX2 (Landry and Cotter 2014). As integral membrane proteins, NOX proteins generate O2 via NADPH electron exchange (transference of electron from NADPH to FAD). Dysfunctional NOX activity can potentially uplift ROS production which in turn enhances cell transformation, tumorigenesis, angiogenesis, tumor growth, and metastasis. Together with NOX activity, enzymes like xanthine oxidase (XOD), nitric oxide synthase (NOS), cyclooxygenase (COX), lipoxygenase (LOX), monoamine oxidases A and B (MAOA, MOAB), diamine oxidase (DAO), acetylpolyamine oxidase (APOA), spermine oxidase (SMO), cytochrome P450 (CYP) oxidase, and lysyl oxidase also produce ROS in their own respective

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mechanism (Forrester et al. 2018; Liao et al. 2019; Jia et al. 2020; Snezhkina et al. 2019). The aforementioned enzymatic production of ROS plays an indispensable disadvantageous role that leads to several diseases and inflammatory responses.

ROS from Peroxisomes Peroxisomes play wide-ranging important roles in the living cells which include fatty acid β-oxidation, α-oxidation, amino acid catabolism, ketogenesis, pentose phosphate pathway (PPP), polyamine oxidation, glyoxylate metabolism, and cholesterol and isoprenoid metabolism (Wanders and Waterham 2006). Peroxisomes play an indispensable role in the maintenance of cellular oxidative balance; any disruption or dysfunctionality in its role will facilitate carcinogenesis. Peroxisomes generate a broad range of ROS: O2˙ , H2O2, •OH, and reactive nitrogen species (RNS): nitric oxide (NO•), peroxynitrite (ONOO ). Peroxisomes are one of the chief producers of H2O2 despite the presence of catalase (CAT, which detoxifies H2O2). Disparate to mitochondria, electron transfer in peroxisome will not result in ATP generation, instead, H2O2 is produced from the transfer of the free electrons to H2O. Further, the catalytic activity of various peroxisomal enzymes and spontaneous dismutation of the O2˙ leads to the generation of H2O2 (Forrester et al. 2018; Snezhkina et al. 2019). Peroxisome generates O2˙ in both membrane and matrix. Xanthine oxidoreductase (XOR) and urate oxidase (UO) are the two enzymes that are responsible for the generation of O2˙ in the matrix and ETC in the peroxisomal membrane acts as an alternative source for O2˙ . XOR catalyzes the reduction of nitrites and nitrates to NO• which reacts with O2˙ to produce the highly reactive ONOO . Thus, dysregulation of the peroxisome activity and the reduced CAT activity can potentially promote the generation of ROS and oxidative-stress leading to genome instability and DNA damage which in turn facilitates cancer development (Forrester et al. 2018; Snezhkina et al. 2019).

Chronic Inflammation and Cancer Chronic inflammation is often associated with elevated levels of ROS and RNS that give rise to several epigenetic changes, DNA mutations, and genomic instability which in turn promotes tumor initiation, development, progression, metastatic dissemination, and treatment resistance. Inflammation has various tumor-promoting effects in the tumor microenvironment and is also considered as a recognized hallmark of cancer. This crucial correlation of inflammation and cancer was first documented by Rudolf Virchow which got numerous mechanistic and epidemiological supports in the past decades (Salman and Ashraf 2013; Shalapour et al. 2015). Inflammation is caused by wide-spread sources including viral and microbial infections, exposure to allergens, toxic chemicals, radiation, consumption of tobacco, and alcohol. In general, inflammation is a sort of protective mechanism,

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but prolonged persistent inflammation can potentially cause harmful effects and damages to the body cells and tissues. The two phases of inflammation are acute and chronic inflammation. Acute inflammation is often a short-time beneficial inflammatory response mediated by the innate immune system. Whereas, in the second phase, prolonged inflammation referred to chronic inflammation can potentially persuade chronic illness like cancer. In the process of inflammation, recruitment of mast cells and leukocytes to the damage-site leads to a condition of “respiratory burst” which is a resultant event of elevated uptake of oxygen that further upshots an upregulated release and buildup of ROS at the damage site (Reuter et al. 2010; Salman and Ashraf 2013). ROS is involved in all three stages of cancer – cancer initiation, promotion, and progression. In the initial stage of cancer (initiation), ROS causes gene mutation, structural alterations, and damages to DNA. During the promotion stage, ROS leads to upregulated cell proliferation or a reduction in apoptosis due to its detrimental contribution to the blockade of cell-to-cell communication, abnormal gene expression, alteration of second-messenger systems. Toward the final stage, ROS worsens the initiated cell population by adding DNA alternations. Along with this, ROS potentially triggers some of the signaling pathways including numerous transcription factors like nuclear factor κB (NF-κB), activator protein-1 (AP-1), specificity protein (Sp-1), p53, and mitogen-activated protein kinase phosphatases (MKP). Activation of these transcriptional factors leads to angiogenesis, cell proliferation, and metastasis. Similarly, inflammatory cells also release a diverse set of soluble mediators and metabolites like arachidonic acid, chemokines, and cytokines which further recruit more inflammatory cells to the damage-site and ultimately produce an elevated level of reactive species. These inflammatory mediators trigger changes in transcriptional factors and activate several deleterious signal transduction cascades. Numerous transcriptional factors are involved in this process which includes NF-κB, hypoxia-inducible factor-1α (HIF1-α), signal transducer and activator of transcription 3 (STAT3), nuclear factor of activated T cells (NFAT), AP-1, and NF-E2-related factor-2 (Nrf2), which are responsible for facilitating instantaneous cellular responses. Additionally, abnormal expressions of inflammatory chemokines (CXC chemokine receptor-4 (CXCR4), Interleukin-8 (IL-8)), and cytokines (IL-1, IL-6, tumor necrosis factor (TNF)) have also been reported for its critical role in this process of oxidative-stress induced inflammation. Thus, the scenario of sustained oxidative-inflammatory environment leads to a pernicious cycle of events which not only damages the same cell but also damages the healthy neighboring cells and thereby leads to carcinogenesis (Reuter et al. 2010; Salman and Ashraf 2015).

Oxidative-Stress-Induced Inflammation Inflammation is a normal host response as a result of infections or other stimuli. It is a primary reaction of a tissue to eliminate pathogen or infected tissue components to restore the normal physiological functions (Wu et al. 2014). Immune cells such as phagocytic macrophages, Polymorph nuclear neutrophils (PMNs), eosinophil are the

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integral part of innate immune response and play a vital role in fighting against the infection through the generation of various reactive species such as Hydrogen Peroxide (H2O2), Superoxide, Nitrous oxide, Hydroxyl radical, and peroxynitrite (Azad et al. 2008). The species help in invading and eliminating the pathogen. Often, inflammation subsides after the removal of infectious agents or on the completion of tissue repair mechanism. However, continuous tissue injuries and regeneration will increase the production of ROS from the inflammatory cells causing damage to the healthy cells (Walser et al. 2008). ROS interacts with epithelial DNA resulting in mutation and chromosomal alterations. As a response to DNA damage, the cells activate p53 genes associated with cell cycle and DNA repair process. But when the rate of ROS is increased, it leads to chronic inflammation. The chronic inflammation provides a platform for recurring DNA damage, rich inflammatory cells, increased ROS, proliferating growth factors, and other growth-inducing factors that ultimately increase the frequency of mutation. All put together facilitates the progression of transforming the cells into their malignant state therby increasing the risk of tumorigenesis (Li et al. 2013).

Molecular Mechanisms of ROS-Induced Carcinogenesis ROS was found to be the key component in inducing inflammation. Over the past few years, it was reported that the expression and synthesis of nitric oxide were upregulated by various cytotoxins during cell division and cell repair, further complicating the process of inflammation (Azad et al. 2008). With all the comprehensive understanding and available studies, it can be stated that the effects of ROS over inflammation may be beneficial or harmful depending on the cell type and physiological conditions involved (Walser et al. 2008). The molecular mechanisms involved in inflammation-induced carcinogenesis are analogous to the ROS generating potential of the cells. The elevated risk of developing cancer is due to the behavior of damaged cells with an imbalance in cell division and cell repair mechanisms (Salman and Ashraf 2013). Generally, all cancer cells develop permanent functional changes to the DNA leading to oxidative damage in the bases, also attacking the proteins and lipids of the cells. Oxidative modification of DNA polymerase or inhibition of DNA repair enzymes indirectly promotes mutagenesis. DNA adduct formation mediated by ROS elevates the risk of carcinogenesis, further stimulating the oncogenes such as jun and fos (Azad et al. 2008). The inflammatory cells produce certain growth factors and transcription factors including NF-κB, STAT3, AP-1, hypoxia-inducible factor-1 (HIF-1), as well as altered expression of specific microRNAs in cancer cells that promote the expression of genes related to cell growth, apoptosis, and invasion (Li et al. 2013).

DNA Damage Extensive studies have been conducted to show that DNA damage including alterations in the native structure such as base pair insertion/deletion, base modifications,

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chromosomal changes, and microsatellite instability and translocation of segments was due to the direct interaction of ROS to the DNA. Other than ROS-induced damage, DNA is subjected to more than 100 different oxidative modifications (Walser et al. 2008). Most importantly, the hydroxyl radical (OH•) is the major source of DNA damage that influences the phosphates, bases, and deoxyribose ultimately resulting in strand damage. Interaction of hydroxyl radicals with the deoxyribose of the DNA can give rise to single and double-stranded breaks. The most frequent base modifications observed in DNA are 8-oxo-7,8-dihydroguanine (8-oxoG) 2,6-diamino-4-hydroxy-5-formamidopyrimidine resulting from the addition of Hydroxyl radical to the eighth position of the guanine ring producing oxidized end products (Srinivas et al. 2018). The single-strand breaks and the oxidized bases of DNA give rise to chromosomal instability and can act as major contributors in tumorigenesis. Further, alterations in the DNA methylation patterns of genes suggest that ROS is also responsible for the epigenetic changes and tumorigenic effects due to the oxidative stress undergone by various sites in the genome (Reuter et al. 2010).

Role of ROS in DNA Damage Induced by Replication Stress/Other Factors Among the various sources of endogenous oxidative DNA damage, replication stress induced by oncogenes plays a key role. Any unusual replication fork during DNA replication results in replication stress to which the oncogenes are often associated (Salman and Ashraf 2013). Frequent alterations in the pro-oncogenes cause replication stress in the DNA inducing genomic instability that helps in tumor development (Sesti et al. 2012). As the oncogene is activated and modified, the risk of ROS is hiked to influence the DNA replication. ROS rich environment does not allow the cells to recruit antioxidant proteins for the recovery of cellular stress (Srinivas et al. 2018). Recent studies suggest that the source of most ROS accompanying acute or chronic inflammation is the NADPH Oxidase (NOX) factor. In a study performed with human tumor cell lines, it was found that the expression of NOX protein to generate ROS increases the risk of inflammation by recruitment of several inflammatory mediators (Wu et al. 2014). ROS production by NOX has a critical role in modifying the inflammatory responses and DNA damage with respect to cancer progression. It was also implicated that a multifold increase in p53 mutation causing loss of DNA repair mechanism is due to the overexpression of NOX1 complex (Yang et al. 2018).

Cell Signaling Cascades in ROS-Mediated Inflammation and Cancer ROS functions as an important modulator of signaling mechanisms in cancer initiation and progression. A series of pathological events including cell proliferation, apoptosis, infiltration of inflammatory mediators and growth factors, and transcription of genes that are encoded for inflammatory proteins are activated as a result of oxidative stress mediated by ROS and RNS (Reuter et al. 2010). The signaling cascade mediated by ROS induces the activation and phosphorylation

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of mitogen-activated protein kinase (MAPK), which results in the activation of the NF-κB, AP-1, and STAT. These factors ultimately make the genes such as jun and fos (immediate early genes) to get activated, which involves inflammatory influx, cell proliferation, transformation, and differentiation. When the mechanisms associated with the oxidative upregulation of these factors are persistent, the molecular changes resulting from these pathways elicit the progression of cells eventually leading to cancer (Gào and Schöttker 2017). Endogenous free radical arises as secondary messengers form the inflammatory neutrophils or macrophage activation during various cell signaling pathways or as by-products of P50 metabolism, NADPH oxidase activity, and peroxisome activities. Besides the aforementioned transcription factors, ROS also finds a role in regulating the growth factors and certain Kinases or phosphates. The effect of ROS is partly mediated by a subfamily of MAKPs – Extracellular signal-regulated Kinases (ERKs) that mediate the cell proliferation, stress responses, and apoptosis throughout the signaling cascade (Sesti et al. 2012).

Transcription Factors – NF-kB, STAT, AP-1, HIF-1 AP-1 and NF-κB are the most important factors which are sensitive to ROS. Oxidants such as H2O2 and inflammatory cytokines such as TNF-α activate AP-1 and NF- κB and were reported to modulate the expression of pro-inflammatory genes leading to initiation of neoplastic transformation and recruitment of several genes involved in cell proliferation, differentiation, inflammation, and tumor development (Azad et al. 2008). Activation of NF-κB by phosphorylation-dependent proteasome degradation of IκBα facilitates the accumulation of the NF-κB in the nucleus. Upon accumulation, NF-κB binds to the κB elements in the promoter regions of genes encoding pro-inflammatory cytokines, INOX, and COX-2, which are involved in the inflammation-associated carcinogenesis. NF-κB regulated genes were found to play a vital role in the modulation of intracellular levels. Conversely, ROS was shown to activate NF-κB by direct oxidation of the transcription factor (Zhang et al. 2016). STAT is a redox-sensitive factor that mainly acts as a switch between the inflammation and cancer. Once activated, STAT-3 dimers get translocated to the nucleus and bind to the promoter regions of genes encoding inflammatory and cell cycle regulatory proteins. Oxidation of STAT-3 by H2O2 enhances the proliferation of tumor-initiating cells; protects the normal and premalignant cells from apoptosis thereby promoting tumor progression (Wu et al. 2014). Hypoxia Inducing Factor-1 (HIF-1) mediates the responses to chronic hypoxia characterized by reduced oxygen availability to the cells. NOX-derived ROS have been shown to influence the hypoxic conditions by increasing the HIF-1α synthesis and involving in the regulation o inflammatory cytokines (VEGF and IL-6), ultimately resulting in cell aggregation and tumor development (Aggarwal et al. 2019) (Fig. 1).

Apoptosis and Survival Apoptosis is a normal physiological process by which unwanted or damaged cells are eliminated from the body during normal biological processes. It is characterized

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Pathogenic aack

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Fig. 1 Schematic illustration of key molecular events and cell signaling cascades induced by ROS, leading chronic inflammation and cancer

by cell shrinkage, membrane blebbing, and breakdown of DNA (Sesti et al. 2012). Imbalance in the rate of cell division and cell death in normal tissues favors the neoplastic development and cancer cells use increased survival and decreased death strategies to sustain proliferation and evade apoptosis (Azad et al. 2008). ROS such as H2O2 has been associated with the cell survival responses at lower doses whereas higher doses of ROS activate apoptosis. High ROS doses activate tumor suppressor gene p53 which plays a key role in oxidative-induced stress or other cellular stress responses. This induces cell cycle arrest to promote DNA repair and cell death by apoptosis (Ivanova et al. 2016). Apoptosis induced by H2O2 is linked with the increased protein expression of p53, PUMA, NOXA, and Bax and phosphorylation of p53 in several cancer types. The mitochondrial pathway of apoptosis is induced as a response to cellular stresses including DNA damage, growth factor deprivation, hypoxia, and oxidative-stress. ROS have been strictly associated with the mitochondrial apoptosis. Mitochondria are the habitat of intracellular ROS and are produced by leakage from the respiratory

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ETC (Kuwabara et al. 2008). Mitochondrial ROS targets the mtDNA and causes impaired transcription of proteins in the ETC which further increases ROS generation leading to loss of membrane potential and loss of ATP synthesis (RedzaDutordoir and Averill-Bates 2016). These series of events finally result in apoptosis. Superoxide and H2O2 can cause cytochrome release from the mitochondria and initiates the mitochondrial apoptosis. In a study with HeLa cells, oxidative-stress induced apoptosis was mediated by the upstream regulation of p53 through caspase-dependent and caspase-independent mechanisms involving loss of membrane permeability and release of apoptosis-inducing factor from the mitochondria, respectively (Redza-Dutordoir and Averill-Bates 2016). ROS function both as supporting and opposing factor in tumor development. Activation of pro-apoptotic signaling molecules such as apoptosis regulating kinase (ASK1), c-jun N-terminal Kinase (JNK), and p38, mediated by ROS can initiate apoptosis. As a denial, NF-κB induced by ROS comes into play to inhibit the process of apoptosis leading to neoplastic development in various cell types (Kuwabara et al. 2008). Different ROS with varying doses involves antiapoptotic roles by inactivating caspases, induction of p53 gene expression, and upregulation of proteins such as Flip, Bcl-2, and Bcl-X. Hence, ROS exhibits pro and antiapoptotic roles depending on the cellular redox state and the types of cells involved. ERK, a member of MAPK, and Protein kinase B (PKB) are the leading factors in cell survival induced by ROS (Redza-Dutordoir and Averill-Bates 2016). It has been reported that when cells are exposed to H2O2, phosphoinositide 3-kinase (PI3K) is upregulated, and PKB is activated bringing about the cell survival pathways. Several studies have shown that the oxidative stress by different sources of ROS stimulates two opposite pathways leading to cell death and cell survival and can simultaneously be suppressed by antioxidizing agents (Kuwabara et al. 2008) (Fig. 2).

Inflammatory Markers and ROS Some of the notable inflammatory proteins associated with the NOX activation and ROS overproduction are matrix metalloproteinase-9 (MMP-9), intracellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule (VAM), COX2, cytosolic phospholipase 2 (cPLA2). It has been known that cells release inflammatory mediators, cytokines, and chemokines such as IL-1β, IL-6, IL-8, and TNF-α, as a response to oxidative stress of various sources. The inflammatory mediators have a crucial role to play in the process of chronic inflammation and are capable of directing the nature of the inflammatory responses by selective recruitment and activation of inflammatory cells. Direct or indirect sources of ROS activate the epithelial cells to induce pro-inflammatory cells. The genes of the inflammatory mediators are regulated by the redox-sensitive transcription factors including AP-1 and NF-κB (Lee and Yang 2012). The relationship between the role of ROS in cancer and prolonged inflammation has been experimentally validated by several studies. The experimental data showed

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ROS

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Fig. 2 Oxidative-stress induced by ROS leading to apoptosis and cell survival by activation of the MAPK pathway

that the inflammatory responses regulate cancer based on the release of cytokines, chemokines in the tumor microenvironment either by providing an antitumor response or by inducing cell transformation and malignancy (Forrester et al. 2018). ROS can influence the inflammatory mediators creating crosstalk between the chronic inflammation and cancer progression through their endogenous accumulation. The inflammatory cells will eventually lead to a massive generation of ROS by upregulating oxidant-associated enzymes (Policastro and Notcovich 2013). In general, ROS activates NF-κB in response to inflammatory signals through amino acid phosphorylation mechanisms. Specific makers of inflammation use ROS as a part of the signaling pathways. IL-1β involves NOX2-derived ROS that induces the recruitment of endosomal recruitment and TNF-α induced NF-κB activation increases antioxidant expression, thereby lowering the apoptotic signaling through the JNK pathway (Lee and Yang 2012).

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Many evidences prove the role of mitochondrial ROS in mediating the inflammatory signaling on pathogen exposure and also during the malignant state of tumors. Mitochondrial H2O2 production contributes to the activation of NF-κB and inhibits Hypoxia-induced NF-κB activation and IL-6 secretion leading to tumor growth. Mitochondrial ROS signaling is considered primary in regulating the inflammasome activation involving IL-1β and IL-18 transcription and apoptosis regulating Caspase1 (Lee and Yang 2012). Fully activated inflammasome with the lysosomal destabilization governs the IL-1β maturation, which in turn reflects in the apoptosis of damaged cells. Lysosomal fusion requires translocation of the microtubule-associated protein to the phagosome, induced by mitochondrial ROS. The TLR signaling induces the recruitment of mitochondrial proteins to the phagosome where ROS kills phagocytosed pathogens and increases the NOX-dependent ROS production. ROS production also plays a role in the activation of NF-κB in T-cells and regulates their metabolic programming. Overall, ROS acts as primary regulators of inflammatory response with respect to NF-κB activation and inflammasome signaling (Forrester et al. 2018).

Tumor Microenvironment The tumor microenvironment is a complex mixture of multiple cell types, tumor supporting matrix, and several additional factors that can aid and assist in tumor growth. Recruitment of fibroblasts, immune cells, and vasculature associated cells by the malignant cells initiate tumors and drive tumor progression (Gu et al. 2018). As the tumor progresses, the extracellular matrix (ECM) being a part of the tumor mass, provides structural support for tumor development and modulates the tumor microenvironment as well. The components of tumor microenvironment include malignant cells and non-cancer stromal cells, ECM components, tumor lymphatic and vessels, and inflammatory cells as well. The characteristics of the tumor microenvironment may vary according to the redox state of the cells including Hypoxia, angiogenesis, tumor metabolism, and cell signaling (Weinberg et al. 2019). It is widely recognized that tumor microenvironments are thoroughly influenced by ten important characteristics of cancer. The characteristics include the following: (i) (ii) (iii) (iv) (v) (vi) (vii) (viii) (ix) (x)

Unlimited multiplication Escaping from growth suppressors Maintaining proliferative signaling Resisting apoptosis Genome instability and mutation Promoting invasion and metastasis Stimulating angiogenesis Eliminating cell energy limitations Tumor-enhanced inflammation Evading immune destruction of foreign bodies

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Components and Characteristics of TME ECM and stroma form a basement membrane that can serve as a storage reservoir of growth factors and chemokines to stimulate tumorigenesis. Normal fibroblasts are responsible for tissue homeostasis and are primarily in the process of wound healing. In contrast, cancer-associated fibroblasts (CAFs) are infiltrating into tumor cells establishing crucial roles in cancer initiation, progression, and metastasis. Alpha smooth muscle actin myofibroblasts are found to be the major subtype of CAFs present in tumors. CAFs are more proliferative than the normal fibroblasts and can activate specific signaling pathways significant for the promotion and progression of cancer (Whiteside 2008; Wang et al. 2017; Weinberg et al. 2019). Desmoplasia is an important marker of tumor growth and can generate mechanical forces that will limit the blood supply to the tumor by compressing the vessels thereby creating a hypoxic environment. Excess production of CAFs identified in the aggressive tumors express smooth muscle actin (α-SMA) and is termed as myofibroblasts whose main function is wound healing and tissue repair. ROS is an important factor in the differentiation of fibroblasts to myofibroblasts. TGF-β1 also plays a major role in the transition of fibroblast to myofibroblasts (Wang et al. 2017). It is known that the mitochondrial ROS activates TGF-β1 in the signaling of inflammation-induced cancer. When fibroblasts become devoid of mitochondrial ROS, the expression of TGF-β1 is reduced, wherein cancer progression is eventually subsided. Several studies have suggested that ROS shows an impact on the subtypes of fibroblasts. One such subtype affected by ROS is the platelet-derived growth factor β (PDGF- β) and ROS could be an integral part of the fibroblast proliferation and migration. ROS produced by the tumor cell can facilitate the reprogramming of CAFs. ROS finds a role in disturbing the routine of tumor-infiltrating T cells depending on the levels of ROS (Policastro and Notcovich 2013). The main function of immune cells is to maintain the tissue homeostasis, to protect against the invading pathogens and to eliminate damaged cells. However, the immune-inflammatory cells persist in the sites of chronic inflammation leading to diverse tissue pathologies and neoplasia (Whiteside 2008). Few studies on immunesystem research have suggested that the infiltration of immune-inflammatory cells may be the early initiation of cancer. The involvement of immune cells in cancer development can be divided into 3 stages: elimination, equilibrium, and escape. In the elimination phase, the immune system defeats nascent tumors, achieved with the help of several signaling molecules and inflammatory factors. Once the cancer cells are eliminated, the active immune cells have an additional role called “immunoediting” wherein the equilibrium stage keeps the tumor growth under control. However, the tumor cells are completely eliminated, and in order to escape the immune surveillance, cancer cells tend to adapt certain phenotypic changes including EMT (Endothelial mesenchyme transition). With the advantage of surviving, the cancer cells would develop into solid tumors. The immune system helps the cancer cells to accompany the dominant cells so that they grow at the fastest rate in

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the limited environment. In the escape phase, immune-inflammatory cells can help the cancer cells in altering the tumor immune escape mechanisms and can reduce the antitumor protein or cytokines to promote tumor development (Wang et al. 2017; Gu et al. 2018).

Hypoxia, Angiogenesis, and Metastasis ROS is viewed as an important signaling molecule rather than merely being a byproduct of cellular metabolism (Wu et al. 2014). At low levels, ROS participate in the process of hypoxia adaptation by regulating the stability of HIF-1α. Moderate levels of ROS are involved in the production of inflammatory cytokines and their regulation by directly inactivating MAKP and higher levels of ROS are capable of inducing apoptosis and autophagy (Waris and Ahsan 2006). Oxygen radicals and insufficiency (hypoxia) cooperatively promote tumor angiogenesis (Xia et al. 2007). Tumor cells generally outgrow their blood supply leading to oxygen deprivation causing hypoxia, which leads to degradation of DNA to its constituent bases. The release of thymidine is catabolized by thymidine phosphorylase which is an important overexpressing enzyme in tumor cells and can cause oxygen radical production (Navaneetha Krishnan et al. 2019). The aforementioned fact was substantiated in Breast Cancer wherein reoxygenation of the tumor after hypoxia will drive additional oxygen radical formation and they are stressed by metabolic alteration and macrophage infiltrations. The accumulation of HIF1 in hypoxia promotes the transcription of Vascular endothelial growth factor (VEGF) resulting in angiogenesis. Oxygen radicals increase the production of VEGF and HIF-1 (Kumari et al. 2018) (Fig. 3).

ROS in Cancer Metastasis Metastasis is the state where primary tumor cells spread to distant organs and are considered to be the main cause of cancer morbidity and mortality (Aggarwal et al. 2019). Studies on tumor microenvironment have revealed that the tumor metastasis is not an autonomous process but a complex event, occurring due to the bidirectional interaction between the malignant and nonmalignant cells and the mutational burden of cancer cells. The upregulation of NF-κB, metalloproteases, and transforming growth factor beta (TGF-β) causes tumor metastasis (Brown and Bicknell 2001). Epithelial to mesenchymal transition (EMT) is the key component in causing metastasis wherein the epithelial cells tend to lose their cell ashesion, polarity and gain entry into the circulation thereby reaching different tissues at distant site. It has been proved that ROS is majorly involved in causing EMT. TGFβ facilitates cell migration and invasion through ROS-dependent mechanisms. Another study has revealed that ROS can increase tumor migration by inducing hypoxia-mediated protein expression leading to the rapid mobility of the malignant cells (Liao et al. 2019).

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NF-κB

VEGF ANGIOGENESIS Acvaon of VEGF growth factor

MMP2

Acvaon of NF-κB TLR acvaon

MMP9

ROS aacking lipids of cell membrane

Acvaon of matrix metalloproteases HIF-1α

Increased stabilizaon of HIF-1α

Hypoxia independent pathway

Hypoxia dependent pathway

EMT

ROS

Acvaon of matrix metalloproteases MMP2 MMP9 Inducon of metastac state METASTASIS

Fig. 3 High levels of ROS leading to cancer metastasis and induction of angiogenesis by ROS via hypoxia-dependent and -independent pathways

Angiogenesis and ROS In the initial stages of tumorigenesis, new blood vessels are formed from the preexisting vasculatures, popularly called as angiogenesis (Xia et al. 2007). ROSmediated angiogenesis is initiated by cancer proliferation thereby increasing the metabolic rate leading to increased ROS levels. Elevation of ROS levels in the tumor microenvironment causes oxidative-stress, initiating secretion of angiogenic modulators (Aggarwal et al. 2019). Endogenous and exogenous ROS stimulates growth factors such as VEGF- and HIF-1α-promoting tumor migration and ROS-dependent cellular signaling. ROS mediates the VEGF secretion and activates the PI3K pathway and is additionally modulating the cancer progression (Xia et al. 2007). It has been reported that the epidermal growth factor (EGF) leads to increased production of H2O2 activating the PI3K pathway resulting in overexpression of VEGF. Further NADPH oxidase 2 (NOX-2)-derived ROS was reported to induce cancer progression and migration that are regulated by the proto-oncogene tyrosine protein kinase (tpk) pathway, inducing angiogenesis (Ushio-Fukai and Nakamura 2008). In human endothelial cells, it was reported that Angiotensin 1 induced the release of ROS by

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activating the endothelial-specific tyrosine kinase receptor leading to vascular remodeling. The hypoxia-dependent pathway mediated by the phosphoinositide-3kinase regulatory subunit or serine-threonine kinase mechanisms increase the expression of VEGF via the activation of MAPK involving HIF-1α and causes upregulation of matrix metalloproteinase (MMP2 & MMP9) ultimately leading to angiogenesis. Hypoxia independent pathway results in angiogenesis through the activation of NF-κB via Toll-like receptors (TLRs) where the membrane lipid ligands are oxidized exogenous ROS (Aggarwal et al. 2019).

Regulation of ROS ROS homeostasis is essential for proper cell signaling and cell survival, where the optimal levels of ROS trigger numerous signaling pathways that are responsible for the regulation of several crucial functions like differentiation, cellular proliferation, and metabolic adaptations in an organized manner. The increased production of spatially localized ROS in the cancerous cells hyperactivates various cell signaling pathways, which are required for detrimental cell transformation and tumorigenesis. In the process of ROS production, levels of ROS are delimited by several factors in the tumor microenvironment. Accumulation of ROS can either be the result of increased ROS production or decreased ROS elimination (Trachootham et al. 2009; Sena and Chandel 2012; Sabharwal and Schumacker 2014; Chen et al. 2016). ROS production is restricted by several checkpoints of NOXs, following the activation of receptors through ligands like insulin, transforming growth factor, platelet-derived growth factor, fibroblast growth factor, nerve growth factor, epidermal growth factor, and tumor necrosis factor-α (TNF-α). A tumor tissue becomes hypoxic when the tumor diameter reaches 200 μm, wherein the state of hypoxia regulates the transcription of Nrf2 and thereby reduces the ROS accumulation. Matrix metalloproteinases (MMPs) are found to be a crucial regulator of the mitochondrial respiratory chain and intracellular ROS production. Furthermore, cellular metabolism, glucose metabolism, and mitochondrial respiratory chain are also associated with regulation and generation of ROS (Chen et al. 2016). To maintain the level of ROS and to protect the cells from ROS-induced damages, cells use their extensive antioxidant defense system, either enzymatic (peroxidases, dismutases, and catalases) or nonenzymatic (glutathione (GSH), vitamin A, C, and E). Superoxide dismutases (SODs) are placed in several cellular compartments and play a crucial role in the rapid conversion of O2 to H2O2. In order to avoid cellular toxicity, levels of H2O2 are maintained in an optimal range for proper cell signaling. Numerous antioxidants like peroxiredoxins (PRXs), catalases (CAT), and glutathione peroxidases (GPXs) are responsible for this task, wherein these antioxidants convert the intracellular H2O2 into water (H2O). Thus, for the purpose of the regulation of intracellular ROS levels, cells have a robust antioxidant system where SODs are responsible for the dismutation of O2 into H2O2 which in turn removed/converted by CAT, PRXs, and GPXs to generate H2O (Chen et al. 2016; Reczek and Chandel 2017).

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Both, generation and detoxification of ROS can be promoted by NADPH, where NADPH can be generated through multiple generative mechanisms in both cytosol and mitochondria. In that line, one-carbon metabolism, a pathway centered around folate, where carbon units from serine and glycine feed to the folate cycle generates NADPH in both cytosol and mitochondria. Unsurprisingly, cancer cell proliferation is enhanced by the upregulated one-carbon metabolism, where the serine catabolism mediated through mitochondrial one-carbon metabolism sustains the redox balance and thereby allows cancer cell proliferation in tumor hypoxia. Hypoxia-inducible factor (HIF) collaborates with Myc in the Myc-transformed cells during the process of hypoxia to persuade the expression of serine hydroxymethyltransferase 2 (SHMT2, mitochondrial one-carbon metabolism enzyme) which in turn elevates the production of NADPH. Furthermore, this increase of NADPH generation leads to the counterbalance of hypoxia-induced escalation in mitoROS by maintaining the antioxidant capacity (Reczek and Chandel 2017).

Conclusion ROS can influence the inflammatory mediators creating crosstalk between the chronic inflammation and cancer progression through their endogenous accumulation. The inflammatory cells will eventually lead to a massive generation of ROS by upregulating oxidant-associated enzymes. The relationship between the role of ROS in cancer and prolonged inflammation has been experimentally validated by several studies. The inflammatory mediators selectively recruit inflammatory cells and serves as the key players in mediating the inflammatory responses. During chronic inflammation, the immune-inflammatory cells persist in the sites of inflammation causing intense tissue pathologies and neoplasia. Several studies have been conducted to understand the infiltration of inflammatory cells, when ROS causes obstruction to the normal cellular signaling, which would eventually result in the early initiation of cancer. Low levels of ROS induce the activation of HIF-1α and regulates it stability to participate in the adaptation of hypoxia. On the other hand, moderate levels of ROS are involved in transcription of inflammatory genes resulting in cell survival and cancer development and high levels of ROS are responsible for inducing apoptosis and autophagy. Oxygen radicals and insufficiency (hypoxia) cooperatively promote tumor angiogenesis. Overall, the extensive research during the past two decades have substantially revealed the role of oxidative stress, and its mediators can lead to chronic inflammation, which in turn can activate multiple inflammation pathways suggesting a close link between oxidative stress, chronic inflammation, and cancer. Acknowledgment The authors express their gratitude to SASTRA-Deemed-to-be-University, Tamil Nadu, India, for infrastructure and financial support.

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Food Colors and Associated Oxidative Stress in Chemical Carcinogenesis

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Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Use of Synthetic Dyes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . International Legislations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Regulation in the European Union . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Regulation in the USA, Japan, and China . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Regulatory Measures in India . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Carcinogenicity of Food Dyes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Oxidative Stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genotoxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Neurotoxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Biotransformation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Oxidative Effects and Associated Health Risks of Synthetic Dyes . . . . . . . . . . . . . . . . . . . . . . . . . . . . Health Concerns of Approved Dyes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Health Concerns of Illegal Dyes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cross-References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract

“Whatever may be the father of a disease, all ill-diet is the mother” as was rightly pronounced by Dr. George Herbert. Unhealthy processed foods have become a part of our daily diet. Such foods are often added with illegal food dyes specifically synthetic ones as coloring agents having genotoxic or carcinogenic properties and thus is a matter of public health concern. Consumption of artificial food

D. Mishra (*) Department of Microbiology, National Food Laboratory, Kolkata, India © Springer Nature Singapore Pte Ltd. 2022 S. Chakraborti et al. (eds.), Handbook of Oxidative Stress in Cancer: Mechanistic Aspects, https://doi.org/10.1007/978-981-15-9411-3_182

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dye has increased many folds in the last 50 years, and children are the biggest consumers. Some dyes may contain cancer-causing contaminants. Food dyes such as Erythrosine, Carmoisine, and Tartrazine are the leading causes of liver carcinogenesis. Azo dyes and triphenylmethanes are the most common classes of illegally added food dyes. The toxic activity of azo dyes, which are widely used in industry, is a result of their metabolism. The enzyme-mediated azo reduction leads to formation of active aromatic amines attacking DNA. Intake of these agents in amounts higher than the advised daily intake (ADI) has been shown to have adverse effects in mammalian models. An understanding of the causal mechanisms that link food color and cancer is still evolving. In this chapter, adverse health effects of food colors are presented. Keywords

Food dyes · Synthetic colors · Carcinogens · Oxidative stress · Carcinogenesis · Cancer · Safety concerns

Introduction Toxic chemicals have become a part of our daily diet. These chemical compounds on one hand benefit society in a number of ways, pesticides, for instance, enhance food productivity. On the other hand, they have several disadvantages, notably their toxic side effects, especially for the young (Gadah et al. 2020). Risk of cancer and chronic diseases from consumption of unsafe food is a global problem. Followed by cardiovascular disease, cancer is a leading cause of mortality worldwide. The genetic mutations acquired by body’s cells either accidentally during replication or elicited by carcinogen often results in their uncontrolled growth affecting normal cells (Pitot and Loeb 2002). A plethora of carcinogens occur in food, exposure to which leads either to immediate death or to the process of chemical carcinogenesis. The carcinogenic chemical compounds damages the genome and can induce cancer; some of them directly attack the DNA, while others affect DNA indirectly by affecting cellular constituents other than DNA. This induces mutations in some genes critical to biological processes (Soliman 2018). A categorization of carcinogens based on their origin has been introduced. Carcinogens can have a synthetic chemical origin (e.g., synthetic food colors) including synthetic azo dyes or can occur from natural substances (e.g., hydrazine present in mushroom) including naturally occurring pigments (Leo et al. 2018). Carcinogenesis can develop from external (exogenous) sources or from internal (endogenous) sources. Exogenous substances are any of the physical, chemical, or biological agents having mutagenic effects with their introduction into the cell (Badger 1956). Endogenous substances are the reactive oxygen species (ROS) and reactive nitrogen species (RNS) produced due to metabolism. Finally, based on the involvement of carcinogens in a particular stage of carcinogenesis, they either initiate the cancer process or play role in cancer progression (Baer-Dubowska et al. 2005).

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Use of Synthetic Dyes Initially, the food dyes were derived from coal tar but were subsequently produced from petroleum. These synthetic chemicals have always remained a matter of concern due to the safety issues associated with them either due to adverse effects on laboratory animals or due to unavailability of adequate laboratory studies (Kobylewski and Jacobson 2012). The synthetic dyes are commonly used by food industries because they are often cheaper, more stable, and brighter than most natural colorings. Foods with vibrant color attract consumer attention; adding to their sensory and aesthetic quality attributes. Additionally, synthetic colors mask unattractive look of the basic food ingredients, their un-uniformity and presence of food additives (Mpountoukas et al. 2010). Current production of synthetic food color has been estimated to be eight million tons per year (Revankar and Lele 2007). Recent data show a dramatic increase in consumption of dyes in the past few years. Although many are identified as potential carcinogens, they are still in common use due to the need to extended food usability goal vis-à-vis keeping balance with increased risk of cancer (Ames et al. 1987; Ferguson 1999). There is no nutritional value associated with synthetic food colors. Effects of color additives on human physiology have emerged many times in past few years. Several color additives have been removed from use in the past several years due to safety concerns or industry disinterest (Food and Drug Administration 2012). With the increased health concerns, the food industries are now voluntarily replacing synthetic colors with natural products.

International Legislations The legislation concerning food color varies in different countries (Table 1). The criticalities in the field of food colors worldwide lies with unavailability of uniform regulations concerning legal food colors specifically for food imports and exports. Some food colors are legally allowed in one country but declared illegal in another (Oplatowska-Stachowiak and Elliott 2017). For example, Red 40 (Allura Red) allowed in the EU, the USA, Japan, and China is banned in India, whereas Red 105 (Rose Bengal B) permitted to be used in Japan is banned in the EU, the USA, China, and India. Similarly, use of Red 2 (Amaranth) is banned in the USA and India but permitted in the EU, Japan, and China.

Regulation in the European Union Introduced in 2008, the EU, Regulation (EC) No 1333/2008 has harmonized the use of all food additives. Currently there are 25 colors of natural (or nature identical) origin and 15 synthetic on the list, each associated with a corresponding E number. The regulation specifies the approved color additives, foods in which food color

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Table 1 Comparison of the synthetic dyes authorized for use in food in different global regions Japan Food yellow No. 4

India Tartrazine

Food yellow No. 5

Sunset yellow FCF Carmoisine

Food red No. 2 Food red No. 102 Food red No. 3

Ponceau 4R Erythrosine

Food red No. 40

Food blue No. 2 Food blue No. 1

Food green No. 3

Indigo carmine Brilliant blue FCF

China Tartrazine Quinoline yellow Sunset yellow Carmoisine Amaranth Ponceau 4R Erythrosine

EU E102 Tartrazine E104 quinoline yellow E110 sunset yellow FCF E122 azorubine E123 amaranth E124 ponceau 4R E127 erythrosine

Allura red

E129 allura red AC E131 patent blue V E132 indigotine

Indigotine Brilliant blue

Fast green FCF

E133 brilliant blue FCF E142 green S E151 brilliant black BN E155 brown HT E180 litholrubine BK

USA FD&C yellow No. 5 FD&C yellow No. 6

FD&C red No. 3 FD&C red No. 40

FD&C blue No. 2 FD&C blue No. 1

FD&C green No. 3 orange B citrus red No. 2

Food red No. 104 (phloxine B) Food red No. 105 (rose bengal) Food red No. 106 (acid red 52) New red

presence is not permitted, and the conditions of use of each additive in different foods. In the ingredients list of the product, the color number or name must be declared. When used according to regulations of European Food Safety Authority (EFSA), these synthetic food color compounds did not raise substantial safety issues (EFSA 2009a, b, c, d, e, f). However for six dyes, viz., tatrazine, quinoline yellow, sunset yellow, ponceau 4R, allura red, and carmoisine, there is a legal requirement that the food be labeled with a warning that they might have an adverse effect on the attention in children, that they have been linked to hyperactivity (EC No 1333 2008).

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Regulation in the USA, Japan, and China In the USA, regulations concerning food additives are contained in the title 21 of the Code of Federal Regulations (e-CFR 2009). Food dyes are not permitted to be used unless the US Food & Drug Administration (FDA) has tested and certified that each batch meets the legal specifications. The Federal Food, Drug, and Cosmetic Act (FD&C) made the certification of some food color additives mandatory. Nine synthetic food colors are allowed subject to certification (F&DA 2012, 2013). In Japan, 12 synthetic colors in food are allowed under the legislation (JETO 2006) whereas Chinese legislation permits 11 synthetic colors in food (Ministry of Health, People’s Republic of China 2011).

Regulatory Measures in India The specifications for “Colouring Matter” have been laid down in the Food Safety and Standards (Food Products Standards and Food Additives) Regulations 2011. The label declaration about the food colors are in accordance with the FSS (Packaging & Labeling) Regulations 2011. The use of natural colors, viz., Carotene and Carotenoids, Canthaxanthin; Chlorophyll; Caramel; Riboflavin; Annatto (permitted in edible oil); Saffron; Curcumin or turmeric; and artificial colors, viz., Red from: Ponceau 4R (E124 red No. 102), Carmoisine (E122 azorubine), and Erythrosine (E127 red No. 3); Yellow from: Tartrazine (E102 yellow No. 4, yellow No. 5) and Sunset Yellow FCF (E110 yellow No. 5, yellow No. 6); Blue from: Indigo Carmine (E132 indigotine blue No. 2) and Brilliant Blue FCF (E133 blue No. 1); Green from: Fast Green FCF (green No. 3) are permitted by the Food Safety and Standards Authority of India (FSSAI)

Carcinogenicity of Food Dyes Among others, use of harmful illegal dyes, hyperactive behavior due to new as well as known artificial colors, and use of natural colors as replacement to synthetic ones are emerging as major food safety challenges. Recent long-term animal feeding studies have confirmed that in comparison to other food additives, exposure to food dyes pose more health risks including cancer or other effects (Kobylewski and Jacobson 2012). In such studies, a dye is considered as safe if it does not produce more than one cancer in one million people. The problem with most of the carcinogenicity studies is that it targets only the “free” forms and does not consider (except possibly aniline) contaminants that are bound to other molecules (Kobylewski and Jacobson 2012). Carcinogenesis of food colors usually follow initiation, promotion, or progression stages, though these stages are difficult to define in experimental studies, specifically those involving human studies and studies involving mixtures of colors. However, in general, initiation often means genotoxicity and promotion means nongenotoxic events.

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Oxidative Stress In response to environmental stress, Reactive oxygen or nitrogen species (ROS/ RNS) are generated endogenously and cause tissue injury. Generation of these reactive molecule leads to oxidative stress that ultimately controls the process of nongenotoxic chemical carcinogenesis (Miyata et al. 2017). Oxidative stress results from an imbalance between the production of oxidative radicals and the antioxidant defense due to either excess oxidative load or inadequate cell antioxidant defense and/or the inadequate supply of antioxidants supplements or via nutrients, which can lead to cell and tissue damage. The resultant oxidative stress radicals target proteins, DNA, lipids, and vitamins, resulting in various diseases, tissue injury, and cell death. Increased oxidative stress causes harmful effects by alteration of many signaling and metabolic pathways and the fate of the cell would be dictated by whether the cell is able to accommodate such an insult or be overwhelmed leading to the development of tissue injury and cell death.

Genotoxicity Genotoxic chemicals either form DNA adducts directly or interact with DNA after metabolic activation, mostly leading to mutations (Williams and Weisburger 1986; Tennant et al. 1987; Reitz et al. 1988; Harris 1990; Rosenkrantz and Klopman 1990). For example, Aflatoxin B2, a potent hepato-carcinogen, attacks guanine sites in DNA and produces mutations in specific genes (Harris 1993). Formation of specific DNA adducts are often used as tools for studying genotoxic effect of chemicals (Choy 1993; Weinstein et al. 1995). Chromosomal aberrations assay, Ames test and micronucleus are the common tests to study genotoxicity of food additives. Nearly all azo chemical colors and their oxidative end products have carcinogenic or mutagenic potential and can cause modification of DNA (Chequer et al. 2011). The food azo dye sunset yellow was evaluated for genotoxicity to Brassica campestris root meristematic cells; highly significant reduction in mitotic index and increase in chromosomal aberrations was observed (Dwivedi and Kumar 2015). Metanil Yellow when orally administered in rats at dose concentration of 430 mg/kg body weight, activates detoxification enzymes and cytochrome P-450 (Das et al. 1997). Carmoisine produces carcinogenicity and biochemical toxicity in mice by increasing the concentration of some serum marker enzymes and downregulating the expression of defensive genes (Al Reza et al. 2019). Nongenotoxic processes on the other hand affect cell replication through oxidative damage affecting DNA indirectly either through specific receptors or through nonreceptor-mediated events.

Neurotoxicity Exposure to excessive colorants resulting in oxidative stress poses serious neurobehavioral problems in young children (Gadah et al. 2020). The advised

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daily intake (ADI) of artificial color is 100 mg/kg body weight. Majority of food manufacturers use color that exceeds this value, which is an alarming issue particularly in most of the developing countries. There are ever-growing evidences linking their use to hyperactive behavior (Bateman et al. 2004; McCann et al. 2007). Intake of these agents in amounts higher than the ADI has been shown to have adverse effects in mammalian models often resulting in many ailments particularly in children as they have low body weights (Dixit et al. 2011). The food color intake of youngsters most often exceeds ADI values prescribed (Husain et al. 2006; Rao et al. 2004). Although intake of synthetic food color exceeds the prescribed ADI limits in almost all age groups, children, mostly in developing countries, are the major sufferers as they consume mostly colored food and are thus the major sufferers from use of legally permitted synthetic colors. Carmoisine is the major color that often exceeds average intake of color in food products. In a study conducted in India, it was found that the use of a mixture of sunset yellow FCF and tartrazine exceeded the prescribed limit by many-folds in food products (Dixit et al. 2010, 2011). A randomized trial study has shown hyperactivity due to consumption of foods containing artificial colors in children aged between 3 and 9 years (McCann et al. 2007). Uptake of synthetic food color has shown neurobehavioral changes during pregnancy and teratogenicity in the newborns (Weiss 2012). Perinatal exposure to azo dyes even within the ADI range has recently been shown to have neurobehavioral effect in mice offspring due to redox imbalance (Gadah et al. 2020). Such redox imbalances leads to brain damage due to oxidative damage in its polyunsaturated fatty acids, reduced GSH content, and SOD activity in cerebral, cerebellar, and medullary regions. Exposure to azo dye elicited lipid peroxidation in different brain regions of the newborns although the exact mechanisms have not been fully elucidated (Gadah et al. 2020).

Biotransformation The azo reduction of dyes in intestinal microbiota leads to formation of aromatic amines (Moutinho et al. 2007), which leads to carcinogenic effects (Chung 2000) involving phase I (oxidation, reduction, and hydrolysis) or phase II (conjugative) reactions. The metabolic pathways of biotransformation are similar for both synthetic and natural dyes producing metabolites that alkylate DNA causing initiation of carcinogenic process. Other common reactions include deamination and dehydroxylation. Dehydroxylation is carried out by gut bacteria. Many other reactions of lesser importance can occur.

Oxidative Effects and Associated Health Risks of Synthetic Dyes Several toxicity studies have established the carcinogenic and mutagenic effects of food colors. Based on these research data, they have been removed from the regulated list of “acceptable food colors” (Demirkol et al. 2012). In spite of

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stringent regulatory provisions, surveillance studies have shown that common industrial dyes used in textile and industrial dyes that are meant for coloring fabrics and plastics are widely used illegally as food color. Metanil yellow, Lead chromate, Rhodamine, Sudan red (III and IV), Orange II, and Malachite green are some of the common non-permitted synthetic colors posing serious health hazards (Bhat and Mathur 1998). The scientific data’s on health risks of food colors are collected and evaluated by international organizations such as IARC (International Agency for Research on Cancer) (IARC 1975, 1978), NTP (National Toxicology Program), and JECFA (Joint Food and Agriculture Organization of the United Nation/World Health Organization Expert Committee on Food Additives 2009).

Health Concerns of Approved Dyes The three most widely used dyes – Red 40 (Allura Red), Yellow 5 (Tartrazine), and Yellow 6 (Sunset Yellow) – are contaminated with known carcinogens. The granddaddy of them all, Red 3 (Fast Green), is recognized by the Food and Drug Administration as a carcinogen. 1. Hyperactivity and other behavioral effects in children are mostly linked to exposure to Tartrazine (Yellow 5) and Red 40. The reduction of Yellow 5 to Sulfanilic acid metabolite occurs via the GI flora. Tartrazine causes chromosome aberrations in cultured cells in vivo. Yellow 5 may be contaminated with several carcinogens, including benzidine and 4-aminobiphenyl. The one generally accepted concern about Yellow 5 is its mild to severe hypersensitivity reactions, mostly in children. Due to serious adverse effect, posing some risks, while serving no nutritional or safety purpose, Yellow 5 should not be allowed in foods. 2. The water-soluble sulfonated azo dye Sunset yellow FCF (Yellow 6) is commonly used in bakery industries. Yellow 6 reductions happen primarily in the gut by intestinal microflora producing reactive metabolites. Yellow 6 causes mild to severe hypersensitivity reactions and may cause hyperactivity in some children. It is often contaminated with several cancer-causing chemicals that occasionally cause severe hypersensitivity reactions. Yellow 6 adds an unnecessary risk to the food supply as some children may be consuming amounts about five times as much dye as an average above the ADI over their lifetimes. It caused adrenal tumors in animals, though it is disputed by industry. 3. Amaranth (Red 2) used for coloring the skins of oranges not used for processing, is toxic to rodents at modest levels and caused tumors of the urinary bladder and possibly other organs. This water insoluble dye is broken down in the GI tract by intestinal bacteria. The dye poses minimal human risk because it is only used at minuscule levels and only on orange peels, but it still has no place in the food supply. 4. Erythrosine B (Red 3) is a water-soluble dye with about 58% iodine content. About 200,000 pounds of the dye being used annually is an animal

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carcinogen when administered in the diet. Several studies demonstrated the genotoxic potential of the dye using mammalian cells or an in vivo method (comet assay). It acts as a secondary carcinogen exerting its carcinogenicity via an indirect pathway. Allura Red (Red 40) is one of the most commonly used dye that often causes hypersensitivity and hyperactivity behavior through azo reduction by gut microflora. Toxicity studies on both male and female mice have shown DNA damaging effects of this dye. This dye is often contaminated with other cancer-causing chemicals adding to its health risks. Serious safety concerns have been raised due to positive results in comet assays, hypersensitivity reactions, and hyperactivity in children. Health effects of Fast green FCF (Green 3) is linked to tumor in bladder and testes in male rats. Further testings will confirm its other health effects. Brilliant blue FCF (Blue 1) has been shown to induce kidney tumors and affect nerve cells specifically in fetuses and babies under the age of 6 months, though confirmatory studies on this is lacking. The dye also plays role in hypersensitivity reactions. Further research needs to be conducted before this dye can be considered safe. It is poorly absorbed by the GI tract and is not susceptible to breakdown by intestinal microbiota. Indigo Carmine (Blue 2) is not readily absorbed by rats. 5-sulfoanthranilic acid, its final breakdown product, is absorbed more readily by the GI tract than is the intact dye. Blue 2 cannot be considered safe given that toxicity/carcinogenicity study shows statistically significant incidence of tumors, particularly brain gliomas and malignant mammary gland tumors, in male rats. It cannot be considered safe for human consumption and should not be used in foods.

Health Concerns of Illegal Dyes Many dyes have been declared as banned by regulatory bodies because of their adverse effects on laboratory animals. These include: Green 1; Orange 1, 2 and B; Red 1, 2, 4, Red 32 (Citrus Red 2) and Sudan 1; Violet 1; Yellow 1, 2, 3, and 4. 1. The azo dyes Auramine, Metanil yellow, Lead chromate, Sudan dyes, rhodamine B, methyl yellow, and malachite green have been receiving significant consideration since these synthetic agents can pose a health risk and exert negative effects on the liver, kidney, and nervous system (Alsalman et al. 2019; El-Desoky et al. 2017; Meyer et al. 2017; Amin et al. 2010). The unifying mechanism for their toxicity is based on oxidative stress. Azo dyes elicit electron transfer, reactive oxygen species, and oxidative stress (El-Desoky et al. 2017; Meyer et al. 2017) and pro-inflammatory effects (Leo et al. 2018). 2. Auramine: Yellow or orange in color, it is also commonly used to stain laboratory slides. Owing to its bright yellow color, it is used to color beverages. Auramine is not a permitted food color and is also potentially carcinogenic. It is known to retard growth and damage kidneys and liver.

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3. Metanil yellow: In India, it is commonly used to color sweets like jalebis and ladoos. Its common use is a very serious issue as it causes degenerative changes in the lining of the stomach, kidneys, and liver. It also adversely affects the ovaries and testes, proving to be dangerous to reproductive organs. It is commonly used to add color to ladoos, and biryanis and consumption of this color has reported symptoms of giddiness, weakness, and food poisoning. 4. Rhodamine: Green in powder form turning a vivid fluorescent pink in water, it is commonly used to stain slides in laboratories and in the sewage industry to test for leaks in drains. A major component of sweets and bright red-colored drinks, it is known to break down red blood cells and adversely affect the immune system. It is not a permitted food color and is considered to be potentially carcinogenic. It also causes growth retardation and damages the liver and kidneys. 5. Lead chromate: Bright yellow in color, it is also known as chrome yellow. It is commonly added to turmeric powder, for imparting a bright color. Lead chromate is highly dangerous as it causes anemia, abdominal pain, neurological problem, hypertension, fetal distress all leading to lead poisoning. 6. Sudan dyes: An industrial azo dye, Sudan is commonly used to color plastics and other materials, including leather, fabrics, fats, oils, waxes, polystyrene, cellulose, and synthetic lacquers and polishes. Sudan dyes are also commonly used as the bright red color is highly attractive. Sudan dyes are used illicitly to enhance and maintain the color of food, especially chili and chili-derived products. In 2003, the dyes were found in chili and chili products imported from India (Tripathi et al. 2007). Since then, Sudan dyes have been detected in foods, including chili and curry powders and the processed foods that contain them, sumac, curcuma, and palm oil. It is not only highly toxic to the liver but also known to cause kidney lesions and is a probable carcinogen. Sudan dyes are classified by the IARC as Group 3 carcinogens and are banned as food additives worldwide (EFSA 2005). In 2005, the European Food Safety Authority (EFSA) initiated a review of the toxicology of a number of dyes found illegally in food in the EU. The EFSA came to the conclusion that, especially for Sudan I, there is strong evidence for both genotoxicity and carcinogenicity. Because of structural similarities between Sudan I and the other Sudan dyes, the larger group is presumed to have the same deleterious effects (EFSA 2005a; IARC 2008). Their presence in food entering the EU, for example, has been reported over a number of years on the Rapid Alert System for Food and Feed (RASFF). The EU issued Decision 2003/460/EC requiring that all hot chili and hot chili products imported to Europe be tested for Sudan I. The Decision was amended in January of 2004 (2004/92/EC) to include Sudan II, III, and IV. This requirement remains in effect. Although regulations do not restrict analysts to a particular analytical method, permissible levels of these dyes have generally followed the limit of quantification (LOQ) which is currently 0.5–1.0 mg/kg using HPLC-UV.

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Conclusions Food dyes cannot be considered safe because of their toxic effects, including carcinogenicity, hypersensitivity reactions, and behavioral effects. The banned synthetic dyes available for industrial applications should be regularly monitored in market places to restrict their entry into food systems. With stricter legislation, more comprehensive monitoring programs, and by employing rapid, cost-effective testing methods capable of detecting multiple hazards, such unacceptable practices can be reduced. Meanwhile, companies voluntarily should replace dyes with other safer natural alternatives.

Cross-References ▶ Environmental Toxicants and Carcinogenicity: Role of Oxidative Stress ▶ Reactive Oxygen Species–Mediated Cancer Progression and Metastasis

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Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Lung Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Epidemiology and Incidence Statistics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Known Risk Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Polycyclic Aromatic Hydrocarbons (PAHs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Benzo(a)pyrene [B(a)P] . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B(a)P: A Potent Inducer of Oxidative Stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B(a)P-Induced Inflammatory Responses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mechanisms of B(a)P-Induced Lung Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Lung Cancer Progression Stages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Traditional Treatment for Oxidative Damage and Lung Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Free Radical Scavenging and Antioxidant Activity of Baicalein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Anti-Inflammatory Activity of Baicalein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract

Toxicity of benzo(a)pyrene [B(a)P] is frequently intervened by oxidative metabolism to reactive intermediates that interrelate with macromolecules prompting changes in the structure and function of target cells. Hence, the human body is continually under oxidative stress merging from exogenous sources mainly through tobacco smoke and cellular endogenous roots which involve mitochondria. Where such oxidative stress surpasses the limit of the body’s oxidationreduction system, gene mutations may result, or intracellular signal transduction and transcription factors might be influenced legitimately or through antioxidants, leading to carcinogenesis. Increasing evidence indicates that oncogenic incitement has expanded metabolic action, and malfunction of mitochondria is the N. Chandrashekar (*) Department of Biochemistry, Indian Academy Degree College – Autonomous, Bengaluru, Karnataka, India © Springer Nature Singapore Pte Ltd. 2022 S. Chakraborti et al. (eds.), Handbook of Oxidative Stress in Cancer: Mechanistic Aspects, https://doi.org/10.1007/978-981-15-9411-3_183

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main reason for increased intrinsic reactive oxygen species (ROS) stress in cancer cells. In cancer cells, the enormous production of endogenous ROS is the major source of DNA-damaging agents, which leads to genetic instability, and as a consequence, the cancer cells become drug resistant. Mitochondrial respiratory chain (electron transport complexes) is the major source of ROS generation in the cell which causes damages in the mitochondrial DNA. ROS-interceded damage appears to be a mechanism for augmenting ROS stress in cancer cells. This present chapter discusses the role of oxidative stress derived from B(a)P metabolism relating to lung cancer. Keywords

Benzo(a)pyrene · Oxidative stress · Reactive oxygen species · Pulmonary dysfunction · Mitochondrial dysfunction · Lung cancer · Baicalein

Introduction Lung Cancer Many diseases, including cancer, have involved oxidative stress in the pathophysiology. Cancer is a group of illnesses characterized by cell growth that is out of control; when this uncontrolled cell growth starts in one or both lungs, it develops into lung cancer (Hecht 1999). Instead of developing into healthy, normal lung tissue, these anomalous cells continue to divide and form lumps or tissue masses called tumors (Hecht 1999). Tumors interfere with the main function of the lung, which is to provide oxygen to the whole body via the bloodstream. If a tumor remains at one point and shows limited growth, it is generally regarded as benign (Hart and Saini 1992). If a tumor cell effectively spreads and expands to other areas of the body, it damages and kills other healthy tissues. When tumor cells spread to other areas of the body via the lymphatic system or through blood circulation and become malignant tumors, it is said to have metastasized. This process itself is called metastasis, resulting in a more serious condition which is very hard to treat (Hart and Saini 1992).

Epidemiology and Incidence Statistics In the world today, the most widely recognized types of cancer are lung cancer, with an estimation of 80–90% emerging from tobacco smoke (Haugen 2002). A lifetime smoker has a 20 to 30 times higher chance of developing lung cancer compared to a nonsmoker (Dela Cruz et al. 2011). Lung cancer has a high prevalence in both developed countries and rapidly developing areas such as China and India (Dela Cruz et al. 2011). Lung cancers have a high incidence of regional differences that could be explained by genetic differences among populations, lifestyle variations, environmental exposures, and clinical practices such as screening and are likewise

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liable to be significant determinants of malignant growth chance (Rastogi et al. 2004). After a lag period of ~7 years, smoking cessation results in the reduced risk of lung cancer and the utmost prevention of all cancers (Peto et al. 1992). In 2011, it was estimated that there would be 1,350,000 new cases of lung cancer and 1,180,000 deaths worldwide, or 17.6% total lung cancer deaths and 12.4% total lung cancer diagnosis combined in men and women (Dela Cruz et al. 2011). Lung cancer now has the highest mortality rate of all cancers in most nations, with the highest levels in developed regions such as Central Asia, Europe, and North America (Barta et al. 2019). There are many variations in the incidence of lung cancer by geographic area such as in the UK and Poland with the highest occurrence (> 100 cases per 100,000 population per year) (Jemal et al. 2011) and Senegal and Nigeria with the lowest occurrence (< 1 case per 100,000 populations per year) (Jemal et al. 2011). According to the World Health Organization (WHO), increased smoking in developing countries in China and India is expected to increase the incidence in the coming years (Perez-Warnisher et al. 2018).

Known Risk Factors A variety of occupational and environmental exposures were concerned as potential risk factors for lung cancer development (Shankar et al. 2019). An increased risk of lung cancer is directly associated with smoking and also with passive smoking. Approximately, 80% of lung cancers are associated with smoking (Bialous and Sarna 2017). In smokers it is 20–30 times more evident than in nonsmokers (Peto et al. 1992). To understand how doctors assess this risk it is important to discuss pack-years or the number of cigarettes (or other smoked substances such as cigars, pipes, marijuana, or herbal cigarettes) every day for 1 year. Many smokers report a variable number of cigarettes consumed throughout their lives (Shiffman 2009). Cigarette smoke compounds are metabolized and produce various types of active quinines, which further generate free radicals and cause oxidative DNA damage and DNA adduct formation. The generation of reactive oxygen species (ROS) induced by cigarette smoke causes oxidative damage to proteins and lipids and can act as an initiator and promoter of chemical carcinogenesis (Witschi et al. 2000). The risk can be modified because research has shown that if a person stops smoking, his or her risk of developing cancer is significantly lower than those who continue smoking (Witschi et al. 2000). Although former smokers still have a higher risk than nonsmokers, the increased number of years spent on nonsmoking gives the body time to repair lung damage and regenerate healthy tissue, thus reducing the overall risk of developing lung cancer (Shiffman 2009).

Polycyclic Aromatic Hydrocarbons (PAHs) A polycyclic aromatic hydrocarbon (PAH) comprises a huge class of chemicals with widespread presence of environmental concoctions in which humans are exposed

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and are believed to contribute significantly to human cancers. PAHs are produced during the incomplete combustion phase of organic materials and are commonly present in the atmosphere, e.g., in diesel exhaust, cigarette smoke, etc., (Phillips 1999). Like several other carcinogens, PAHs are metabolized, breaking down enzymatically to different metabolites, some of which are reactive. The most studied member of the PAH class of compounds is benzo(a)pyrene [B(a)P] because of its mutagenic properties and is one of the common environmental PAHs that have been tested for their toxic effects in laboratory animals (Travis et al. 1995). The oxidation of unsubstituted PAHs rings is the metabolic activation of B(a)P in which an epoxide intermediate is formed that leads to essential chemical reactivity to bind covalently with DNA and to serve as the ultimate carcinogenic form (Cavalieri and Rogan 1995). The 44% of 6-OH B(a)P oxidation constitutes B(a)P-3,6-quinone (Lesko et al. 1975) and the binding position of 1, 3, and 6- of B(a)P-3,6-quinone to DNA is associated with a free radical toxic action mechanism (Rogan et al. 1978).

Benzo(a)pyrene [B(a)P] B(a)P has a five-ring polycyclic aromatic hydrocarbon (Fig. 1), also known as 1,4benzo(a)pyrene, with a chemical formula of C20H12 and a molecular weight of 252.31 g/mol. It exists as crystalline yellow solid plaques or needles that are mutagenic and highly carcinogenic in tobacco smoke, coal tar, diesel exhaust, wood smoke, etc., (Wynder and Hoffmann 1994). B(a)P is readily absorbed by exposure routes in the nasal, inhalation, oral, and dermal regions. B(a)P are considered as procarcinogens that require activation to exert genotoxic effects in electrophilic forms (King et al. 1979). The highly reactive quinone form of B(a)P reaction leads to radical cation mainly with deoxy-guanine (dG) to yield DNA adducts. The intercalation in DNA is the preliminary step to induce cancer (Ewa and Danuta 2017).

Fig. 1 The chemical structure of B(a)P (https:// chem.nlm.nih.gov/ chemidplus/rn/50-32-8)

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12 11

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B(a)P: A Potent Inducer of Oxidative Stress Useful indicators of tumor progression during carcinogenic processes are overexpression of glycoprotein content and these changes alter the rigidity of the cell membrane (Selvam and Nagini 1995). In one of our study, B(a)P-induced lung cancer in animals were found to be significantly elevated in the levels of hexose, hexosamine, and sialic acid, i.e., components of glycoproteins (Naveenkumar et al. 2012b). This inference could be because the changes in surface carbohydrates observed in B(a)P-induced lung cancer–bearing mice during cell differentiation and increased cell proliferation may result in the promotion of reactive oxygeninitiated cells, thereby increasing the possibility of neoplastic changes (Selvendiran et al. 2006). Accumulation of free radicals oxidizes the protein and inactivates the enzymes which lead to altering cellular functions and death (Muralidhara Kumar 2007). Exposure to B(a)P in mice induces a marked rise in pulmonary oxidative stress which leads to a marked increase in the levels of protein carbonyls as a reflection of an oxidative protein disruption (Naveenkumar et al. 2012b). Hence, protein carbonyls are also used as a valuable marker of oxidative stress (Dalle-Donne et al. 2003). The content of carbonyl is measured as a protein oxidation index where the amino acids are converted to carbonyl derivatives (Levine et al. 1990). Additionally, mice exposure to B(a)P induces an obvious upsurge in the components of the extracellular matrix and proteases during lung carcinogenesis (Anandakumar et al. 2015). The bioeffects of some plant phenolic compounds are reported to regulate iron homeostasis in the body by strong iron-binding capacity and neutralizing it (Guo et al. 2007). Here, we find that exposure in mice to B(a)P induces a substantial increase in the lung iron levels (Naveenkumar et al. 2012b). In the body, an abnormal accumulation of iron and other metals has contributed to oxidative stress by redox reactions associated with a large number of diseases, such as cancer (Wei and Guo 2007). Oxidative damage induction by ROS causes a decrease in the efficiency of the antioxidant defense mechanism (Navarro et al. 1999). Approximately 1–3% of the total mitochondrial oxygen expended is incompletely decreased in contributing to ROS production. Therefore, mitochondria might also be the main targets vulnerable to ROS attacks (Desagher and Martinou 2000). ROS can provoke extensive oxidative DNA damage, DNA strand breaks, and chromosomal aberrations (Loft and Poulsen 1996). Substantial DNA damage resulting from endogenous free radical attacks has already been suggested to contribute to cancer pathology (Panandiker et al. 1994). Studies on ROS generation is a valuable method in assessing oxidative stress caused by carcinogenesis. In our study, B(a)P-administered mice shows an upsurge in the endogenous production of ROS levels which lead to cellular DNA unwinding and mitochondrial (mt) DNA unwinding in the mice lung (Naveenkumar et al. 2012b; Naveenkumar et al. 2013). Particularly, hydroxyl radicals (OH•) can cause substantial chemical changes in DNA leading to unwinding and can lead to carcinogenic progression. The B(a)P is an effective chemical with the ability to

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induce huge quantities of free radicals, which react with lipids that cause lipid peroxidation (LPO) (Asokkumar et al. 2012). LPO is initiated by free radicals through peroxidative degradation of polyunsaturated fatty acid (PUFA) richly present in membrane lipids and ultimately ends in the formation of stable products such as malondialdehyde (MDA) and hydroperoxides (OH) (Nayak and Pinto 2007). Hence, analysis of the lipid peroxidation marker, i.e., MDA status in terms of TBARS and OH was suggested as a useful indicator of the oxidative damage response. There was an increase in the lung TBARS and OH in both cytosolic and mitochondrial B(a)P administered for animals, which may be due to the excessive free radicals created by the administration of B(a)P (Naveenkumar et al. 2012b, 2013). ROS and oxygen-derived radicals will attack cell membranes together with vital cell components, resulting in LPOs spreading (Padmavathi et al. 2006). Previous studies reported that a high number of white blood cells (WBC) and their subtype neutrophils were also increased. Concomitantly, other subtypes such as monocytes and lymphocytes show a low number. Besides, other components such as red blood count (RBC) and hemoglobin percentage was decreased supportively. Hence, these components are used as an indicator of anemic condition. Anemia complications resulting from hypoxia of virtually all organs due to oxidative stress are often seen as a potential therapeutic problem (Anandakumar et al. 2012). In cells, antioxidant enzymes exist to protect against the toxic effects of oxygenderived species released during normal cell metabolism or oxidative stress (Padmavathi et al. 2006). Moreover, oxidative damage induced by ROS leads to a decrease in antioxidant defense mechanism efficiency (Subashini et al. 2006). Cellular and subcellular antioxidants such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione-S-transferase (GST), and glutathione reductase (GR) are critical in combating the ROS–induced cell death and tissue injury. The balance between these enzymes is an essential process for successful oxygen stress removal in intracellular organelles (Subashini et al. 2006). A cellular and mitochondrial enzymic antioxidant such as SOD, CAT, GPx, GST, and GR plays an essential role in reducing ROS and in defending against oxidative stress. According to the current results, the large decrease in SOD and CAT activity was attributed to enzyme fatigue due to oxidative stress and increased LPO caused by B (a)P. Also, the decreased GPx, GST, and GR activity may be due to the decreased availability of substrate, reduced glutathione (GSH), and increased peroxide rates in mice administered with B(a)P (Naveenkumar et al. 2012b, 2013). To protect the cell from oxidative damage, cellular and mitochondrial nonenzymic antioxidants such as GSH, vitamin E, vitamin C, and vitamin A are closely interlinked which play an excellent function in tumor cells activity levels which have decreased significantly due to the accumulation of H2O2 and O2 (Naveenkumar et al. 2012b, 2013). In another study, B(a)P supplementation increased the production of phase I microsomal enzymes in the lung and liver from their baseline levels, revealing the metabolic activation of xenobiotics, resulting in their cytotoxic, mutagenic, and carcinogenic activities (Anandakumar et al. 2009). Mitochondria have the inimitable ability to allow various components to selectively move through their membrane permeability transition pore (MPTP) opening

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(Rodriguez-Enriquez et al. 2004). During illness, increasing the generation of ROS and dysregulation of antioxidant mechanisms lead to mitochondrial stress. As a consequence, mitochondrial MPTP dysfunction, respiratory chain impairment, energy failure, and apoptosis/necrosis take place (Rodriguez-Enriquez et al. 2004). In our study, B(a)P-induced mice showed an increase in MPTP activities and a decrease in the mRNA expression of adenine nucleotide translocase (ANT) and voltage-dependent anion channel (VDAC) (Naveenkumar et al. 2013). This indicates that the MPTP structural protein is disorganized with massive mitochondrial swelling. The permeabilization of the outer mitochondrial membrane is regulated by Bcl-2 family proteins and in conjunction with other variables, such as Ca2+ overload, inorganic phosphate, low pH, and relative mitochondrial ATP insufficiency, which lead to the formation of abnormal MPTP which is caused by oxidative stress (Gateau-Roesch et al. 2006). ROS-induced oxidative damage in mitochondria contributes to decreased mitochondrial enzyme function efficiency (Wang et al. 2004). Previous studies reported a decrease in activities of the tricarboxylic acid (TCA) cycle enzymes and electron transport chain enzymes. This confirms respiratory chain impairment and energy failure in lung cancer–bearing mice induced by B(a)P (Naveenkumar et al. 2013). This impaired enzyme activity can be characterized by the accumulation of oxidized lipids, proteins, and DNA due to mitochondrial dysfunction resulting in disorganization of the mitochondrial structure and systolic failure that can lead to cancer pathogenesis (Naveenkumar et al. 2013). Excessive production of MDA leads to membrane lipid peroxidation that causes loss of activity of the membrane integrity, and in turn membrane-bound enzymes which lead to cellular homeostasis disruption (Hussain et al. 2003). Enhanced oxidative stress during lung cancer conditions causes membrane damage or loss of membrane integrity which leads to repress in the activity of membrane-bound ATPases such as Na+/K+-ATPase, Mg2+-ATPase, and Ca2+-ATPase (Bean 1992). Previous studies reported reduced levels of membrane-bound ATPases activity such as Ca2+-ATPase, Mg2+-ATPase, and Na+/K+-ATPase in B(a)P-induced lung cancer–bearing animals (Naveenkumar et al. 2012b). This is because it has been shown that the activity of ATPases is strongly inhibited by the concentration of high Ca2+ overload. Inhibition of Ca2+-ATPases activity can also be reduced due to GSH levels and also on exposure to activated oxygen and thiol depletion status (Rauchova et al. 1995).

B(a)P-Induced Inflammatory Responses The inflammatory neoplastic tissue microenvironment is characterized by host leukocyte involvement in supporting stroma as well as among tumor cells, with differential distribution of dendritic cells, mast cells (MCs), macrophages, and T cells. (Coussens and Werb 2002). In most types of tumors, MCs are found and also overlooked in many tumor inflammations studies. Mast cells have been shown to have essential proangiogenic effects and to cause tumor growth in some cancers (Tomita et al. 2000). In our study, we found that the increased frequency of immature MC precursors (toluidine blue staining), and mature MC (alcianblue+safranin+

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staining) was substantially greater in the lung tissue of animals induced by B(a)P than in control animals (Naveenkumar et al. 2012c). It suggested that there was ongoing penetration of MCs during lung cancer. A tumor microenvironment that represents a persistent inflammatory state and thus fosters tumor progression is produced by increasing the amount of inflammatory MCs that infiltrates. This causes an “angiogenic turn” in which normal stroma-epithelium contact is disrupted by matrix degradation at the tumor invasion sites and angiogenesis facilitation (Coussens et al. 1999). Inflammation plays a significant part in the growth of tumors (Coussens and Werb 2002). One of our findings indicates that inflammatory cell counts showed a substantial increase in granulocyte (neutrophils and eosinophils) percentage and a substantial decrease in macrophage and lymphocyte percentage in bronchoalveolar lavage fluid (BALF) samples. This supports B(a)P-induced inflammation of the airway and was attributed to inflammatory cell infiltration with a rise in the total number of BALF cells in the lung (Naveenkumar et al. 2012c). Malignancy and metastasis have been promoted by acute inflammation under certain conditions, which is caused by exogenous administration of interleukin-1 (IL-1), tumor necrosis factor (TNF), and inducible nitric oxide synthase (i-NOS) (Balkwill and Mantovani 2001). IL-1 is a proinflammatory cytokine that initiates and propagates inflammation, induced primarily by myeloid cells. The effects of IL-1α and IL-1β on tumor microenvironment revealed that both molecules are necessary for angiogenesis, leading to metastasis and invasiveness of the tumor (Voronov et al. 2003). TNF is a major proinflammatory cytokine that plays a pivotal role in the inflammatory responses and serves to bridge inflammation and carcinogenesis as an endogenous tumor promoter (Balkwill 2002). Previous studies reported in animals with B(a)Pinduced lung cancer showed that the IL-1 concentration, TNF concentration, and iNOS concentration of lung tissue were significantly increased, which was a wellcorrelated predictor of inflammatory responses during lung cancer progression (Naveenkumar et al. 2012c).

Mechanisms of B(a)P-Induced Lung Cancer B(a)P is an environmental pollutant found in grilled meat and cigarette smoke, detoxified by activation of aryl hydrocarbon receptors (AHR) and their enzyme induction abilities (Miller and Ramos 2001). Abnormal activation of AHR and sequential metabolic activation of B(a)P principally by phase-I enzyme CYP1A1 generates benzo(a)pyrene-diol-epoxide molecule, i.e., (+) benzopyrene-7,8dihydrodiol-9,10 epoxide (BPDE) (Shou et al. 1996). This BPDE is believed to be the ultimate carcinogenic metabolite of B(a)P that leads to intercalate with DNA, covalently bonding to the nucleophilic guanine nucleobases at the N2 position and formation of DNA adducts (Szeliga and Dipple 1998). This disrupts the normal DNA copying cycle, induces mutations, and induces toxicity leading to malignancy and inflammatory diseases (Uno and Makishima 2009). The p53 protein is a transcription factor in which the transactivation of the p53 gene is therefore known

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to play a significant role in its tumor suppressor function. B(a)P might provoke p53 gene mutation and the defensive gene p53 is directly targeting the benzo(a)pyrenediol-epoxide (Pfeifer et al. 2002). The predominance of G (guanine) to T (thymidine) transversions in tumors is consistent with B(a)P-adduct formation at CpG sites (Pfeifer et al. 2002). Figure 2 shows that B(a)P quinones, such as BPQ, generate free radical reactions to ROS in cells (Shimada 2006; Miller and Ramos 2001). Autoxidation of 6-OH B (a)P leads to the formation of quinone by subsequent production of radical growth, including superoxide and hydroxyl radicals, and by redox cycling. These oxygen radicals can react to produce H2O2 (Miller and Ramos 2001). B(a)P quinones take part in one-electron redox cycles between their corresponding hydroquinone’s [B(a) P diols] and semiquinone radicals. The combination of these NADPH-cytochrome P450 reductases catalyzed cycles with molecular oxygen that creates ROS in the form of superoxide radicals (O2˙ ) and H2O2 (Lorentzen and Ts’o 1977). These redox cycles occur under physiological conditions, and cellular respiratory enzymes may assist. Ultimately, excessive oxidant generation can disturb the antioxidant/ oxidant balance in target cells to alter redox status and induce cell injury (Raghunandhakumar et al. 2013). Enormous amounts of ROS released by B(a)P induction plays a role as a second messenger in the activation of nuclear factor-κB (NF-κB) and priming of inflammatory genes such as interferon-γ (IFN-γ), IL-1β, and TNF-α (Naveenkumar et al. 2012c; Anandakumar et al. 2012).

Fig. 2 Metabolism of B(a)P by xenobiotic-metabolizing enzymes (Shimada 2006) (P450 cytochrome P450, EH epoxide hydrolase, GST glutathione S-transferase, UGT UDP-glucuronosyltransferase, SULT sulfotransferase, NQO1 NADP(H)-quinone oxidoreductase-1, AKR aldo-keto reductase)

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Fig. 3 Histopathological characterization of pulmonary neoplastic changes during B(a)P-induced progression of lung cancer (H & E staining-10X & 40X) (Naveenkumar et al. 2012a). (a). Control mouse lung section showing normal appearance of alveolus with normal intact architecture; bronchiole lined by single-layered uniform epithelial cells with basement membrane indicated by black arrows (40X). (b). B(a)P-administered mouse lung section after 6th week showing the appearance of anaplastic changes in the bronchiolar region indicated by black arrow (10X). (c). Mouse lung section after 10th week of B(a)P exposure showing an appearance of severe hyperplasia, in a bronchiolar and alveolar region indicated by black arrows (10X). (d). Mouse lung section after 12th week of B(a)P exposure showing the progression of changes towards dysplasia, which is characterized by a tangled growth of bronchiolar epithelial cells with large hyperchromatic nuclei indicated by black arrows (10X). (e). Mouse lung section after 14th week of B(a)P exposure

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Lung Cancer Progression Stages Generally, a series of progressive molecular alterations followed by morphological changes in the cells that histologically instigate the normal epithelium is the cause of lung cancer (Maggiore et al. 2004). It is therefore conceivable that mutations lead to more proliferation if DNA-damaged cells no longer respond to apoptosis, which can result in the accumulation of malignant cells and eventually in the genesis of neoplasia. Such cells are premalignant (pre-cancerous) and no longer look or function like normal cells (Banerjee et al. 2006a). Late-stage aggregation of irregular cells forms a mass of anomalous tissue known as a tumor. B(a)P-induced lung cancer group analysis showed the stepwise occurrence of neoplastic lesions up to adenocarcinoma (AC) in Fig. 3 (Naveenkumar et al. 2012c). Lung cancer typically begins in the lining of one of the bronchial tubes, or the alveoli (air sacs). Such tissues are exposed directly to air and/or carcinogens which render them especially vulnerable (Banerjee et al. 2006b). This AC ascends from the progenitor cells of the bronchioles (Clara cells) and alveoli (Type II pneumocytes) or the mucin-producing cells (Minna et al. 2002).

Traditional Treatment for Oxidative Damage and Lung Cancer Generally, traditional Chinese medicines (TCM) have been familiar as a potential source of anticancer drugs and as potential adjuvant chemotherapy to increase its efficacy and decrease the side effects, but their healing mechanisms remain largely unknown. The effective components of the dry root of Scutellaria are important for traditional Chinese medicine prescriptions due to its extensive biological and pharmacological activities (Tang and Eisenbrand 1992). One of the main flavonoids in the root of the Scutellaria baicalensis Georgi plant is baicalein (BE) (Fig. 4), a flavone that is readily absorbed into the bloodstream, where a wide spectrum of important biological activities has been demonstrated (Nishioka et al. 1992). One of the major subclasses of dietary polyphenols is flavonoids, and dietary flavonoid consumption is associated with a decreased risk of various forms of cancer since it has strong antioxidant properties (Middleton Jr et al. 2000). The literature demonstrates that flavonoids have an unsaturated-2,3 bond (shaded yellow) in combination with the C-ring 4-oxo group (shaded red) and the Aring 5-hydroxy group (Morel et al. 1994) (Fig. 4) as important structural features ä Fig. 3 (continued) showing severe dysplasia, indicating a complete loss of bronchiolar and alveolar region with an accumulation of tumor cells represented by black arrows (10X). (f). Appearance of mouse lung section after 16th week of B(a)P exposure, showing the characteristics of AC. Arrow headed showing the complete loss of bronchiole and alveoli structure with nuclear overcrowding and presence of lung tumor nodule (AC) (10X)

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Fig. 4 The chemical structure of BE (Li-Weber 2009)

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in the classical antioxidant potency concept of flavonoids and their ability to chelate redox-active metal ions such as copper and iron (shaded green) (Li-Weber 2009; Rice-Evans et al. 1996). The antitumor functions of BE flavone may be due to its ability to enhance antioxidant mechanisms by scavenging ROS and preventing viral infections. Furthermore, BE suppresses the cyclo-oxygenase-2 (COX-2) gene and controls the cell cycle by attenuating the function of NF-κB (Li-Weber 2009).

Free Radical Scavenging and Antioxidant Activity of Baicalein ROS is continuously produced by the cells as natural by-products during aerobic metabolism. Under physiological conditions, ROS is a minor product of the mitochondrial oxidative respiratory chain, in which molecular oxygen is reduced to water during ATP production (Curtin et al. 2002). During endogenous ROS production, cytosolic superoxide dismutase (SOD) catalyzes the dismutation reaction in which metabolic conversion of O2˙ into nonradical H2O2 takes place (Binuclara et al. 2013). ROS also serves to regulate biological and physiological processes as signaling molecules with inherent chemical properties that confer reactivity to different biological targets, inducing oxidative stress that leads to cancer (Storz 2005). BE supplementation prevents protein damage by decreasing carbonyl tissue levels and functioned as an anti-lipid-propagating oxidative damage by decreasing the levels of thiobarbituric acid reactive substances (TBARS) and hydroperoxides (OOH ) by stopping peroxyl radical-mediated reaction (Naveenkumar et al. 2012b). Under physiological conditions, BE may be a strong iron chelator that can inhibit radical damage induced by Fenton reaction and can thus modulate iron homeostasis in the body (Perez et al. 2009). This suggests that BE can fight the oxidative damage caused by ROS. Free radicals can cause substantial chemical changes in DNA, leading to unwinding. BE supplementation showed a marked reduction in nuclear DNA damages and mt-DNA damage (unwinding). Therefore, it is suggested that the BE treatment might have protected the lung from oxidative stress caused by B(a)P damage to DNA. The defensive antioxidant system plays an important role in eliminating ROS and protects against oxidative stress (Naveenkumar et al. 2012b).

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Furthermore, BE dynamically decreases the microsomal xenobiotic-metabolizing enzyme activities such as phase I enzymes NADPH-cytochrome P450 reductase, cytochrome b5, cytochrome P4501A1 (CYP1A1), and cytochrome P4501B1 (CYP1B1) in the cancer-bearing animals with concomitant increases in the activities of all these three important phase-II enzymes such as UDP-glucuronyl transferase (UDP-GT), GST, and DT-diaphorase (DTD). This may be due to its hydroxyl group in flavonoid structure, which reduces the activity of cytochrome-P450 either by increasing Vmax or decreasing Km for microsomal monooxygenase (Naveenkumar et al. 2014). Through the oxygen reduction cycle intermediates are scavenged by antioxidant enzymes and this will provide the primary protection against cytotoxic oxygen radicals in the tissue (Madankumar et al. 2011). BE treatment may have protected the normal cell/tissue from the cytotoxic effects of B(a)P-induced oxidative stress by bolstering the antioxidant protection mechanism as demonstrated by the increased cytosolic and mitochondrial levels of both enzymatic and nonenzymatic antioxidants (Naveenkumar et al. 2012b). Also, B(a)P-induced oxidative stress inhibits enzyme activities of membrane-bound ATPases (Na+/K+ ATPase, Mg2+ ATPase, and Ca2+ ATPase) which stabilize and restore to normalcy in BE-treated mice (Naveenkumar et al. 2012b). BE treatment dramatically brought glycoprotein levels back to normalcy. This lessening in glycoprotein component levels indicates that BE can resolve malignancy by decreasing the degree of lung cancer by modulating cell proliferation and differentiation (Naveenkumar et al. 2012b). An increase in VDAC and ANT proteins during BE care have a direct effect on MPTP function and BE supplementation has also shown substantially increased levels of major TCA cycle enzymes (ICDH, α-KDH, SDH, and MDH) and respiratory marker enzyme (Cyt-C oxidase and NADH dehydrogenase) activities in the lungs, which indicate BE’s capacity for restoration and membrane-stabilizing ability in mitochondria (Naveenkumar et al. 2013).

Anti-Inflammatory Activity of Baicalein Inflammation plays a major role in tumor growth (Coussens and Werb 2002). It has been estimated that about 20% of all human cancers result from chronic inflammation in adults that may lead to the development of cancer. In various animal models, Scutellaria flavone baicalein has been shown to protect tissues from in vitro and in vivo inflammation. BE’s anti-inflammatory activity is at least in part due to its inhibition of producing nitric oxide (NO) through the downregulation of several inflammation-associated genes such as iNOS, COXs, and LOXs. NO is an extremely responsive free radical that is produced in an enzymatic reaction by NO synthase (NOS) utilizing L-arginine, NADPH, and oxygen. In malignant tumors, NO and NOS are persistent and have both pro- and antitumor effects (Fukumura et al. 2006). A key pathway in inflammatory-mediated carcinogenesis is the association between NO and p53 axis. Baicalein can inhibit the expression of iNOS genes and

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downregulate the production of NO by various in vitro and in vivo inflammatory stimuli in animal models. In our results, inflammatory cell counts in BALF samples showed that BE treatment dramatically decreases in the lung neutrophils and eosinophils and a substantial decrease in the lung macrophages and lymphocytes. The supposition was that BE would have worked by preventing the buildup of lung inflammatory cells (Naveenkumar et al. 2012c). However, BE treatment substantially decreases mast cell density (MCD), and this may be due to its anti-inflammatory properties by reducing vascular permeability by restricting the induction of downregulation of matrix metalloproteinases (MMPs) and COX-2 (Naveenkumar et al. 2012c).

Conclusion PAHs are all-encompassing environmental pollutants that come from various sources, predominantly from vehicle exhaust pollution, cigarette smoke, and food cooking. B(a)P is a typical member of the chemical class PAH, widely considered for its toxic properties in laboratory animals and humans. ROS is constantly produced and eliminated in the biological system and plays an important role in several biochemical functions and abnormal pathological processes. B(a)P is oxidized to reactive intermediates and radical species which compromise cell functions through phase I and II metabolism. Among these, the most reactive are the hydroxy metabolites, epoxides, and quinones, and that undergo further metabolism for ROS development. This chapter addresses oxidative stress in cancer cells, its underlying mechanisms, and relationship with mitochondrial dysfunction and proposes new therapeutic approaches that leverage increased ROS in cancer cells to improve therapeutic activity and selectivity.

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Assessing the Contributions of Lipid Profile and Oxidative Lipid Damage to Carcinogenesis

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Daniel Andrew M. Gideon and Joel James

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Lipids: Key Macromolecules with Structural and Functional Diversities . . . . . . . . . . . . . . . . . . Body . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Carcinogenesis Through Inflammation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Redox Homeostasis: Oxidative Stress and Antioxidant Defense . . . . . . . . . . . . . . . . . . . . . . . . . . . ROS and Lipid Peroxidation in Physiological and Pathophysiological Conditions . . . . . . . Dietary, Blood, and Biomembrane Lipid Composition and LPO Products . . . . . . . . . . . . . . . . Mechanism of Carcinogenesis Through LPO and Inflammation . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract

Lipids are integral components in the body, performing various structural and functional roles – as energy reservoirs, insulators, cell membrane components, hormone precursors, and as key signalling agents which affect cellular metabolism and gene expression. Dysregulation of redox proteins/enzymes involved in the process of redox homeostasis and generation of nonphysiological levels of free radicals causes oxidative stress, an important nexus in the pathogenesis and pathophysiology of several diseases, including cancer. Oxidative damage to lipids causes lipid peroxidation, a process which leads to the formation of toxic aldehydes and other secondary products, which sometimes have beneficial roles at physiological levels. However, oxidative stress-mediated lipid peroxidation D. A. M. Gideon (*) Department of Biotechnology and Bioinformatics, Bishop Heber College (Autonomous), Tiruchirappalli, Tamil Nadu, India J. James Department of Medicine, Division of Endocrinology, University of Arizona College of Medicine, Tucson, AZ, USA e-mail: [email protected] © Springer Nature Singapore Pte Ltd. 2022 S. Chakraborti et al. (eds.), Handbook of Oxidative Stress in Cancer: Mechanistic Aspects, https://doi.org/10.1007/978-981-15-9411-3_185

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products initiate carcinogenesis through macromolecular damage (DNA/RNA/ protein adduction), inhibition of DNA repair, and alteration of the cellular transcriptome. The problem of lipid damage poses a conundrum, where lipid damage/oxidation products can be both beneficial as well as harmful in a dosedependent manner. This chapter is aimed at deciphering the mechanisms of lipid oxidation and the cellular signalling events mediated by lipid damage products which determine important cellular outcomes. The toxic products cause a plethora of effects such as impairment of mitochondrial energy metabolism, membrane protein dysfunction, alteration of the cellular transcriptome, upregulation of antioxidant gene transcription, depletion of the cellular thiol pool, genomic instability, ferroptosis, etc., and thereby, pathogenesis of several cancers. The roles of dietary lipids, blood lipid profile, cellular distribution of diverse endogenous and/or dietary lipids, and finally, the currently known mechanisms of oxidative lipid damage in carcinogenesis/tumorigenesis are reviewed. Keywords

Lipid peroxidation · Inflammation · Carcinogenesis · Oxidative stress · Lipid profile · LPO products · Cell signalling · Base adducts Abbreviations

4HNE/HNE AA ALA ALOX ARE CAT CLs COQ10/UQ COX-2 CROALD CYP450/CYPs DHA DODE DOOE EBV ECM EDE EDGF EGFR EHN/EH EPA ERK ESRE ETC

4-hydroxynonenal Arachidonic acid Alpha-linolenic acid Lipoxygenase Antioxidant response element Catalase Cardiolipins CoenzymeQ10/ubiquinone Cyclooxygenase Crotonaldehyde Cytochrome P450 Docosahexaenoic acid 9,12-dioxo-(10E)-dodecenoic acid 5,8-dioxo10(E)-octenoic acid Epstein–Barr virus Extracellular matrix 4,5-epoxy-(2E)-decenal Epidermal growth factor Epidermal growth factor receptor 2,3-epoxy-4-hydroxynonanal Eicosapentaenoic acid Extracellular signal-regulated kinase Ethanol and stress response element Electron transfer chain

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Assessing the Contributions of Lipid Profile and Oxidative Lipid Damage to. . .

FA/FAs FRs GA GCL GLA GPx GSK3β GST HBV HCV HDHA HEPE HETE HHE HHV HO HODE HPHE HPNE HSE HTLV HUE HX iNOS Keap1 LA LOOHs LOX LPO LT MAPK MaR MDA MPO MUFA NA NFκB NOS NOX Nrf-2 NSRE OA ODC ONE OXPHOS

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Fatty acids Free radicals Gondoic acid γ-glutamylcysteine ligase/glutamate-cysteine ligase Gamma-linolenic acid Glutathione peroxidase Glycogen synthase kinase-3 beta Glutathione S-transferases Hepatitis B virus Hepatitis C virus Hydroxy-docosahexaenoic acid Hydroxyeicosapentaenoic acid Hydroxyeicosatetraenoic acid 4-hydroxy-2-hexenal Human herpes virus Heme oxygenase Hydroxyoctadecadienoic acid 4-hydroperoxy-2-heptenal 4-hydroperoxy-2-nonenal Heat-stress-responsive element Human T-lymphotropic virus 4-hydroxyundecenal Hepoxilins Inducible nitric oxide synthase Kelch-like ECH-associated protein 1 Linoleic acid Lipid hydroperoxides Lipoxygenase Lipid peroxidation Leukotrienes Mitogen-activated protein kinase Maresins Malondialdehyde Myeloperoxidase Monounsaturated fatty acids Nervonic acid Nuclear factor kappa-light-chain-enhancer of activated B cells Nitric oxide synthases NADPH oxidase Nuclear factor erythroid 2–related factor 2 Nutrient-sensing response element Oleic acid Ornithine decarboxylase 4-oxo-(2E)-nonenal Oxidative phosphorylation

808

PA PAHs PD PGH2 PI3K PLs PMRS PPAR PUFA RNS ROI ROS RSS Rv SDHA SLs SOD SSBs/DSBs TBARS TGs TNFα TX UCP VA XO

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Palmitic acid Polyaromatic hydrocarbons Protectin Prostaglandin Phosphatidylinositol-3-kinase Phospholipids Plasma membrane redox system Peroxisome proliferator-activated receptor Polyunsaturated fatty acids Reactive nitrogen species Reactive oxygen intermediates Reactive oxygen species Reactive sulfur species Resolvins Succinate dehydrogenase subunit-A Sphingolipids Superoxide dismutase Single/double-strand breaks Thiobarbituric acid reactive substances Triglycerides Tumor necrosis factor Thromboxane Uncoupler protein Vaccenic acid Xanthine oxidase

Introduction Lipids: Key Macromolecules with Structural and Functional Diversities Cells contain hundreds of lipid types with phenomenal structural variations to facilitate the diverse functions of lipids in cellular biochemistry and physiology (Muro et al. 2014; Sämfors and Fletcher 2020). It is believed that mammals have even evolved a special lipid taste. Since lipids are known to be as structurally diverse as proteins, cells express several thousands of proteins which can regulate fatty acid transport and metabolism, hence expending significant energy to produce, alter, distribute, and modify lipids (Muro et al. 2014). Mass spectrometry-based shotgun lipidomics is useful in comprehensive and simultaneous quantification of several classes of lipids and their constituent FAs in a single experiment (Klose et al. 2012). About ten different lipid constituents are found in limiting structures/membranes such as the plasma membrane and the endomembranes. The ratios of diverse structural/membrane lipid components such as glycerophospholipids, sphingolipids, cholesteryl esters, cholesterol, and glycolipids often differ, enabling various lipid membranes to have phenomenal structural

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and functional diversities (Harayama and Riezman 2018). Since lipids are amphipathic, they can interact with their immediate environment to form – (a) the more aqueous/ protic media which surround the membrane (e.g., cytosol/extracellular fluid), (b) the two dimensional and nonpolar, aprotic media which form the lipid phase of membranes (Domene et al. 2003), and (c) an aqueous-lipid interface, which provides ambience for interfacial biochemical reactions in biomembranes (Sugihara et al. 2008). In a way, plasma membranes can be considered to be “the primary and critical regulators of stress and disease adaptation” (Murphy 2009). Triglycerides serve as storage fuel in lipid depots of adipocytes; they are mobilized via blood and utilized for cellular energy needs in other tissues. Dietary lipids are absorbed in the small intestine and are transported throughout the body in the form of lipoproteins. Both saturated and unsaturated fatty acids of varying carbon lengths, extent of unsaturation (i.e., Δ1–5 or more), and type of unsaturation – ω-3/ω-6 PUFAs (or ω-7/ω-9 FAs/MUFAs) are present in TGs, PLs, and SLs. PUFAs are more prone to attack by ROS generated during routine cellular electron transfer reactions. Lipid membranes are akin to enclosures/boundaries – wherein, cellular/organelle-specific redox reactions are compartmentalized and redox homeostasis is maintained (Jones and Go 2010). The lipidome of a cell can determine the extent of LPO and the resulting concentrations of toxic primary LOOHs/secondary aldehyde products during oxidative stress (and their downstream effects, such as carcinogenesis). Hence, FA composition of dietary lipids and circulating blood plasma lipoproteins can determine the difference between health and disease. Also, LPO is a key contributor to an array of cancer-related processes such as carcinogenesis, tumor growth, angiogenesis, ECM remodelling, and metastasis.

Body Carcinogenesis Through Inflammation Inflammation-related carcinogenesis triggered by (a) infectious diseases caused by viruses/bacteria/parasites (HBV, EBV, HCV, HHV-8, HTLV-1, H. pylori, Schistosoma sp., etc.) and (b) physical and chemical factors (e.g., xenobiotics, tobacco, asbestos, UV, ionizing radiation, etc.) can be influenced by membrane and dietary lipid composition. All these contributing factors have a common precedent: inflammation via oxidative stress. A range of factors such as – circulating hormone levels in blood, immune system hyperactivation and production of pro-inflammatory mediators (e.g., NFκB, iNOS, COX-2, TNFα, IL-6, etc.), dynamic changes in membrane lipid composition, altered mitochondrial metabolism, cellular responses to key stimuli (cell signalling cascades), and transcriptional/epigenetic changes during oxidative stress-sponsored LPO can determine the degree of inflammation. Indeed, factors like chronic inflammation, oncogene-induced inflammation, predisposing risk factors like obesity and diabetes, and bad dietary choices can influence carcinogenesis (Gonzalez 1992). Chronic inflammation is often compared to the “fire burning within,” because it drives immune dysfunction.

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Redox Homeostasis: Oxidative Stress and Antioxidant Defense Cells contain thousands of proteins, small molecules/metabolites, gaseous messengers, as well as ions. “The order of life processes is maintained by several stochastic routes which enable the spatiotemporal generation of desired products” and hence, cells regulate the outcomes through transcription, translation and allosteric modification/regulation of key proteins in metabolic pathways (Parashar et al. 2018). Redox biology is indeed the fundamental theme of aerobic life (Halliwell 2006); this is because redox reactions are involved in orchestrating elemental cellular processes such as mitochondrial OXPHOS, ATP synthesis, signal transduction, cellular senescence, and gene expression. Formation of ROS is a natural consequence of aerobic respiration (Murphy 2009).

ROS and Lipid Peroxidation in Physiological and Pathophysiological Conditions The “redox code” encompasses a set of principles wherein, key redox molecules such as reduced/oxidized nicotinamide coenzymes (NAD, NADP), reduced/oxidized flavins (FMN/FAD), total thiol/disulfide, and other redox coenzymes are distributed within biological systems in both space and time (Jones and Sies 2015). An excess of transition metal ions such as Fe(II), Cr(II), Pb(II), Cd(II) has been shown to cause LPO. Initiation of LPO is caused by O2•–, •OH, and •OOH (hydroperoxyl radical, HO2•). Radical products of various small molecules (endobiotics as well as xenobiotics) are also known to initiate LPO (Gutteridge and Halliwell 2000). Since most of the antioxidant defense system (SOD, CAT, GPx, thiol redox systems) comprises of cytosolic/soluble factors, LPO (which occurs in the lipid phase) can go unabated through chain propagation, until lipophilic antioxidants such as tocopherol can halt the chain propagation reactions. Lipids act as free radical sinks and modulators of cellular ROS production (Schönfeld and Wojtczak 2008; Ray et al. 2016). Alterations in redox homeostasis, viz., oxidative as well as reductive stress, can lead to both pathogenesis and progression of disease (Franco and Vargas 2018). Higher concentrations of free iron and high cellular glucose levels tend to increase ROS generation and cause ferroptosis. Inflammation and oxidative stress are two key processes which are responsible for many diseases, including cancer. During oxidative stress, ROS production is escalated in the mitochondrial membrane and sequentially, e pass from triplet/ ground state oxygen (3O2) to form superoxide (O2•–) and later on – H2O2, •OH and finally, H2O. Singlet oxygen (1O2) is produced through photooxidation/photosensitization reactions; while 1O2 is not involved in initiation of LPO, it reacts with unsaturated lipids to form lipid hydroperoxides (LOOH). Most of the cellular ROS/FRs are produced in the mitochondria and have limited half-life at physiological pH. In mitochondria and other cellular locations, some ROS traverse very finite (nm to C > A >T. The physiological cellular concentrations of LPO products are in nanomolar ranges (up to 0.3 mM); in human blood and serum, the range of 4-HNE is 0.05–0.15 μM (Dalleau et al. 2013). Different concentrations of HNE (1 μM vs. 10 μM and higher), a key carcinogenic metabolite of LPO (Dianzani 1998), were found to exert dosedependent effects (Ayala et al. 2014). At low/physiological concentrations, activation of the antioxidant response element occurs via Nrf2 and thereby, ARE-dependent antioxidants are transcriptionally activated. However, slightly higher (or medium) HNE levels reportedly cause cells to persist through autophagy, senescence, and cell cycle arrest. Very high HNE concentrations triggered HSE, NSRE, and also mildly increased ESRE (Dianzani 1998), paving the way for formation of DNA/RNA/protein adducts

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of HNE and subsequently, pathogenesis of diseases. Also, LPO products form adducts with redox buffers such as glutathione; moreover, GS-HNE and other such glutathione adducts activate redox sensitive mediators – NFκB and AP-1, through which cell proliferation, differentiation, and cell survival is enhanced. The various reaction types of HNE – Michael additions, reduction, epoxidation, acetal/thioacetal formation, Schiff’s base formation, and other oxidation/reduction reactions have been reviewed in detail elsewhere (Schaur et al. 2015). At very low and very high concentrations, ROS can react with themselves to form autocollapse products. In lieu of these factors (and other potential contributing factors), we can surmise that LPO products are responsible for driving the carcinogenesis of different cancers, in conjunction with the simultaneous actions of FRs/ROS. The physiological roles of ROS, RNS, RSS, and RHS have come to light in more recent decades of redox biology research. During oxidative stress, NO and RNS (as well as other reactive species such as RSS and RHS) are also involved in manifesting pro-carcinogenic effects, and thereby, elevating DNA adduction and inhibiting DNA repair pathways (Feng et al. 2004). When the FRs levels overwhelm the cellular antioxidant defense system, the effects of LPO products are amplified. During oxidative stress, antioxidants themselves, at instances, can have prooxidant capabilities (Halliwell 2008). Furthermore, both endobiotics and xenobiotics are metabolized by CYP450s, which convert procarcinogens into carcinogens. CYP450s rely on DROS for substrate oxidations; hence, nonphysiological ROS concentrations (in oxidative stress) affect the in milieu reaction networks and ET reactions occurring in cellular milieu, often causing alternate product formation and depletion of precious redox equivalents (Manoj et al. 2016). Dysregulation of heme-containing enzymes and protein/lipid nitration by ROS as well as miscellaneous RNS – (a) radical RNS, NO•, and NO•2 as well as (b) non-radical RNS (which can give rise to radical RNS) – OONO, ONOOH, NO+, N2O3, N2O4, HNO2, and NO2Cl – drives inflammation (Dedon and Tannenbaum 2004). NO• irreversibly binds to heme iron and regulates the activity of several cellular enzymes (including NOX, NOS, and CYP450). Besides, RNS react with FeS proteins to form dinitrosyl iron complexes (DNICs) and cause protein tyrosine nitration (Toledo Jr and Augusto 2012). This reveals the connection between LPO and lipid nitration by nitroso compounds/PAHs found in red meat/charred foods and the pathogenesis of stomach, small intestinal, and colon cancer (Steinberg 2019).

Organelle Dysfunction Caused by LPO Products Apart from reacting with nucleic acid bases, the products of LPO cause organelle dysfunction. In mitochondria, LOOHs are the primary LPO products. The LOOHs are largely short-lived and can be degraded to form stable molecules, which later on, diffuse to different sites and thereby, act at sites quite distinct from the site of generation. When the levels of FRs are excessively high, the radicals form nonspecific autocollapse products, and in this regard, lipid radical/LOO• dimers (L-L, LO-OL), lipid radical-ROS/lipid radical-RNS reaction products, as well as carboxylated peroxynitrite (OONOCO2 ) have been identified (Toledo Jr and Augusto 2012). Upon decomposition, LOOHs give rise to reactive electrophiles which react

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with nucleophilic compounds and form adducts with macromolecules. While HNE is mostly derived from plasma membrane and endomembrane PLs, it is also produced from mitochondria-specific CLs (Zhong and Yin 2015). Moderate mitochondrial impairment is another important driving force in LPO-mediated carcinogenesis. CLs are known to be associated with key mitochondrial electron transfer machinery complexes such as complex III, IV, and V (ATP synthase). Hence, depletion of PUFAs from CL and formation of HNE has been shown to cause structural changes in the membrane, leading to membrane depolarization and altered mitochondrial transmembrane potential, ΔΨm. 4-HNE was found to form adducts with key mitochondrial proteins such as ATP5B, SDHA, NADH dehydrogenase, NDUFS2, and HADHA. 4-HNE, at constitutively expressed levels, is opined to have protective roles; however, at higher concentrations (during oxidative stress), it inhibits OXPHOS and TCA cycle, whereby it acts as a feedback system to mitigate high ROS levels. In the process, 4-HNE impairs mitochondrial function and leads to upregulation and activation of UCP-1, UCP-2, UCP-3, and ANT; thus, HNE is involved in the physiological control of mitochondrial uncoupling (Echtay et al. 2003). ALDH2, which metabolizes endogenous aldehydic products (for example, conversion of HNE to its aldehyde), is susceptible to inactivation by LPO products. In alcohol-induced carcinogenesis of several cancer types, this enzyme is inactivated by toxic LPO aldehydes and this increases the risk for upper aerodigestive tract, liver, colorectal, and breast cancers (Zhong and Yin 2015). Extremely high HNE concentrations cause ferroptosis, apoptosis, and necrotic cell death. Since many fundamental cellular processes – e.g., electron transfer reactions tied to ATP synthesis, ion transport, protein trafficking, and metabolite transport are dependent on membrane-embedded proteins, damage to lipid as well as membrane proteins (and the ensuing alteration of membrane structure) significantly affects these processes. ER stress, dysfunction of peroxisomes, Golgi bodies, and lysosomes occurs when key biochemical reactions in these organelles are inhibited, leading to carcinogenesis (Britton et al. 1987). Decreased activity of key metabolic enzymes such as G6PDH, CYP450s, galactosyltransferase, ODC, and lysosomal enzymes through adduction by LPO products can stifle routine cellular metabolism. Sustained LPO propagation causes loss of membrane integrity.

Lipid Peroxidation and Its Products: Structure, Signalling, and Cellular Biochemical Effects Different LPO products have been implicated in the pathogenesis of diverse cancer types. The most well-known LPO products like acrolein, CROALD, DODE, DOOE, EDE, EH, HNE, HHE, HPHE, HPNE, MDA, NDE, OHE, and ONE are responsible for driving carcinogenesis in different tissues (Dianzani 1998; Gentile et al. 2017); see Fig. 3. These cell/tissue-specific effects are also contingent upon diversities in membrane lipid composition of cells in different tissues. The diversities in cancers are perhaps due to – differential expression of LPO product-specific receptor expression, tissue-specific differences in metabolism/gene expression, and change in levels of key oxidoreductases in a given LPO reaction milieu (plasma membrane, mitochondrial membrane, or any other endomembrane). For example, MDA is

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considered to be the most important LPO product in laryngeal and breast cancer (Wang et al. 1996), while colon, bladder, and lung cancer is attributed mainly to acrolein (Zarkovic et al. 2006). ONE is a major player in gastric carcinoma. These LPO products are involved in pathogenesis and pathophysiology (progression, angiogenesis, and metastasis) of cancers. RHS such as HOCl (generated through MPO action) (Dryden et al. 2005) and RSS like disulfide radical anion (formed during oxidative stress) were found to cause LPO by increasing LOOH formation and production of TBARS. Antioxidant proteins which minimize oxidative stress and inhibit these potentially carcinogenic events are shown in Fig. 3. Advancements in biophysics and molecular biology techniques such as GC-MS, LC-MS, NMR, and EPR have led to identification various LPO products and their respective lipid precursors (and FA type undergoing LPO). In toto, these effects together orchestrate genomic instability, trigger apoptosis/ferroptosis, or alternatively, induce carcinogenesis. As shown in Figs. 2 and 3, a range of cellular functions are impacted by the gene expression changes resulting from HNE signalling, as documented in vitro (West and Marnett 2005).

ROS in LPO

Physiological ROS levels

ROS

Superoxide

P

X

O2•-

Plasma membrane lipid components

Blockage of apoptosis in HL-60 cells lower c-myc and c-myb gene expression Reduced cyclin D1, D2 and A expression

Hydrogen peroxide

H2 O 2 Adenylate Cyclase activation (hepatocytes) Protein Kinase-C activation Stimulated heat shock protein response Chemokines release increased in neutrophils Higher aldose reductase activity Increased TGFß expression in macrophages Stimulated chemotaxis in polymorphonuclear leukocytes

Singlet oxygen

1

O2

Hydroxyl radical

HO• Hydroperoxyl radical

HO2•

ROS X

high antioxidant defense

Endomembrane lipid components accumulated macromolecular damage

ROS

ROS

lipid droplets

low antioxidant defense

ti da xi ) o r O pe P d (L pi i L ROS

Non-physiological ROS levels (OXIDATIVE STRESS)

ROS

Decreased glucose 6-phosphate dehydrogenase activity Low galactosyltransferase activity in golgi apparatus Decreased lipoprotein/protein secretion Decreased tubulin polymerization Low ornithine decarboxylase activity Decreased cell viability Low plasma membrane 5'-nucleotidase and adenylate cyclase activity Low phagocytosis Low lysosomal enzyme activity Decreased P450 activity in microsomes Higher flux of extracellular Ca2+ in hepatocytes Increased ER stress Dose-dependent activation of ARE, HSE, NSRE Moderate induction of ESRE

on

MDA EH

DODE 2-butenal/CROALD

DDE

DOOE

ONE

HNE

H2N N N N N H P

Salient LPO products

HPNE

Acrolein

Protein/enzyme adduction and inactivation (enzymatic lipid peroxidation)

base adduction

decreased X

[lipid peroxidized products], eg., 10µM of 4-HNE

Mutations and high cancer risk

membrane fluidity

low macromolecular damage

Natural process of lipid degradation Reduce cell growth and promote cell differentiation Inhibit platelet aggregation Induction of autophagy

X

Lipid damage Chain reaction

Organelle dysfunction

EDE

NDE

CONSEQUENCES Genomic instability Carcinogenesis Apoptosis/ferroptosis Necrosis Pathogenesis of other diseases

Fig. 2 LPO products from biomembranes – their structure and physiological roles in physiological/ nonphysiological conditions (oxidative stress). Unsaturated FAs in both plasma membrane as well as endomembranes are attacked by ROS such as •OH and xenobiotic radicals (R•) to initiate LPO. Products of LPO at (a) physiological levels and (b) pathophysiological conditions have different signalling and biochemical routes. Cellular processes affected by LPO products (in both normal and pathophysiological scenarios) are summarized within boxes. Structures of a few salient LPO products are shown

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Fig. 3 LPO products, DNA and RNA adductions and specific gene expression changes orchestrated by LPO products with HNE as an example – Various LPO products bind to diverse membrane-embedded and soluble cytosolic (nuclear) receptors. Direct adduction of the LPO products with DNA/RNA, depurination and release of oxidative damage products are simultaneously known to occur through attack of ROS as well as LPO products. DNA damage products of LPO (base adducts) are shown in the box to the right. HNE is the most important LPO product and hence, the gene expression changes reported (upregulated and downregulated genes) in HNE-treated cells are summarized in the boxes at the bottom

Conclusion Due to greater ROS solubility in biomembranes, lipid peroxidation is a very probable event at both physiological and nonphysiological (high) concentrations of FRs. ROS and lipids are not villains but are crucial protagonists which orchestrate life processes (Jacob and Manoj 2019). The species of ROS produced and the relative concentrations of the various ROS, the cellular concentrations of participating oxidoreductases, LPO products, the position of the proteins, etc. determine the cellular outcomes such as cellular proliferation/longevity, apoptosis, and ferroptosis (Auten and Davis 2009). Through LPO, lipids are involved in ROS-mediated protective feedback mechanisms which would protect cells from carcinogenesis. Alternatively, excess ROS/FRs can thwart these protective functions and lead to undesirable biochemical events. Hence, we need to direct more research efforts to (a) find as yet unknown LPO products, their receptors and dose-response relationships, (b) identify the protective roles of specific antioxidants in averting

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LPO-induced carcinogenesis, (c) discover specific pharmaceutical targets, and (d) determine the optimal dietary regimes or blood lipid profiles which can minimize the risk of LPO-mediated carcinogenesis.

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Oxidative Stress in Hepatocarcinogenesis and Role of Antioxidant Therapy

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Salah Mohamed El Sayed

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Liver Cells and Inflammatory Cytokines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mitochondrial Roles in ROS-Induced Hepatocarcinogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Viral Hepatitis Increases Free Radicals: Toward HCC Induction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Alcohol and Oxidants Cause HCC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Warburg Effect Increases Oxidative Stress in HCC Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Current Preventive and Treatment Modalities of HCC and Related Limitations . . . . . . . . . . . . . . Antioxidant Therapy for HCC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Natural Honey . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Nigella sativa . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ajwa Date Fruit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Costus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Fennel (Foeniculum vulgare Mill) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract

Oxidative stress plays important roles in hepatocarcinogenesis and subsequent progression of hepatocytes to hepatocellular carcinoma (HCC) phenotype (Warburg effect) where glucose is converted into lactate. In previous publications, we declared that pyruvate is a strong antioxidant formed from cytoplasmic glucose oxidation (glycolysis) in normal cellular metabolism that is distinguished from lactate (not antioxidant) formed during cancer metabolism. Lactate increases endogenous oxidative stress and favors generation of reactive oxygen species (ROS). Subsequent ROS-induced genotoxic effects increase mutagenesis and S. M. El Sayed (*) Department of Clinical Biochemistry and Molecular Medicine, Taibah Faculty of Medicine, Taibah University, Al-Madinah Al-Munawwarah, Saudi Arabia Department of Medical Biochemistry, Sohag Faculty of Medicine, Sohag University, Sohag, Egypt e-mail: [email protected]; [email protected] © Springer Nature Singapore Pte Ltd. 2022 S. Chakraborti et al. (eds.), Handbook of Oxidative Stress in Cancer: Mechanistic Aspects, https://doi.org/10.1007/978-981-15-9411-3_187

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tumorigenesis. Hepatitis viruses, chronic alcohol consumption, and hepatotoxins are causes of hepatitis increasing ROS production and favoring hepatocarcinogenesis, invasion, and metastasis. Inflammatory mediators and cytokines are common in hepatocarcinogenesis, fibrogenesis, and insulin resistance. Remedies of prophetic medicine are natural products and medicinal plants that are rich in many active ingredients all of which are antioxidants, e.g., thymoquinone (of Nigella sativa), anethol (of fennel), dehydrocostus lactone and costunolide (of costus, Saussurea lappa), flavonoids (of natural honey and ajwa date fruit), and others. In this chapter, Ajwa date fruit extract caused exciting reversion of malignant cells into normal behavior that was reported in study done in Saudi Arabia. β-D-glucan (in Ajwa dates) causes a dose- and time-dependent inhibition of HCC cells viability and growth. Natural honey suppresses TNF-α that mediates tumor initiation, promotion, and progression. Natural antioxidant ingredients in Nigella sativa protect liver cells against malignant transformation via different mechanisms, e.g., suppressing oxidative stress-induced tissue damage. Nigella sativa includes thymoquinone, thymol, and α-hederin exert potent antioxidant effects. Thymoquinone, thymol, and alpha-hederin protect liver cells from a wide range of injurious agents. Costus enhanced the protein levels of many proapoptotic factors and suppressed the protein levels of antiapoptotic factors. Fennel exerts antimutagenic effects. Essential oil of fennel exerts potent protective effects against chemotherapy-induced genotoxicity in experimental animals. Keywords

HCC · Hepatocarcinogenesis · Mitochondria · Antioxidants · Oxidative stress · Ajwa dates · Nigella sativa · Natural honey · Costus · Fennel

Introduction Hepatocellular carcinoma (HCC) is the most common liver malignancy predisposed to and highly associated with hepatic cirrhosis. HCC is the second major factor in cancer-induced mortality globally. HCC is usually discovered late and kills patients several months after diagnosis. Unfortunately, HCC exhibits a high annual recurrence rate (about 20%). That is quite high and may hinder treatment efforts and lessen disease-free survival. HCC incidence is still high in the USA and Europe possibly due to alcohol intake, HCV, obesity, diabetes mellitus, alcoholic liver disease, and nonalcoholic liver diseases. With the discovery of efficient treatments of HCV and better HBV immunization, the outcomes improve. However, HCC prognosis is still dismal with a very low survival rate that is less than 9%. Unfortunately, HCC is resistant to chemotherapy and has dismal outcomes after radiotherapy and after surgical interventions particularly in nonresectable lesions. Oxidative stress is a state of increased oxidants and related tissue-damaging effects versus decreased antioxidants and related tissue-protective effects. Oxidative stress results from harmful endogenous or exogenous factors, e.g., oxidants as reactive oxygen species

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(ROS) and reactive nitrogen species (RNS). Oxidative stress ultimately causes oxidative damage to genes and DNA and disturbed protein expression, causing the tissues in vulnerable conditions. This augments the pathogenesis of so many disease conditions, e.g., diabetes mellitus, cancer, and cardiovascular and nervous system. Oxidative stress is implicated in the pathogenesis (both initiating malignancy and enhancing progression) due to different causative pathological substances. Mitochondria are the principal intracellular source of ROS produced by cellular respiration (Jaeschke and Mitchell 1989).

Liver Cells and Inflammatory Cytokines Different immune mediators, cytokines, and chemokines can be produced by hepatic stellate cells, liver sinusoidal endothelial cells, Kupffer cells, and dendritic cells. For example, interleukin- (IL-) 6 is a proinflammatory cytokine that can be produced in this context (Yu et al. 2009). Tumor necrosis factor alpha (TNF-α) is an inflammatory mediator that enhances cellular damage, supports inflammatory cells, and enhances fibrogenesis. TGF-β increase reflects severity of tissue injury and liver fibrosis. Such cytokines affect liver cells inflammation, apoptosis, and fibrosis and enhance alcoholic steatohepatitis versus nonalcoholic steatohepatitis in addition to governing related metabolic derangements, e.g., insulin resistance (Severi et al. 2010).

Mitochondrial Roles in ROS-Induced Hepatocarcinogenesis Oxidative stress causes increased mitochondrial transcription and replication. ROS include superoxide (O2˙ ), hydrogen peroxide (H2O2), and hydroxyl radical (OH•), and RNS include peroxynitrite (ONOO ), nitric oxide (NO•), and others. All are oxidative stress agents which cause structural and functional damage to cellular proteins, lipids, and DNA. Liver cells tend to enhance defense systems against oxidative stress-induced damage via utilizing antioxidant agents such as superoxide dismutase and catalase, peroxidase in addition to antioxidant molecules as tocopherol, ascorbic acid, and polyphenols. In addition, oxidative stress-induced mitochondrial damage afflicts the electron transport chain resulting in ROS accumulation with subsequent increase in mitochondrial damage, and a vicious circle arises. TNF-α (from Kupffer cells) directly damages the mitochondrial respiratory chain and mitochondrial complexes and components, e.g., cytochrome oxidase. Furthermore, damage of any component of the respiratory chain drives ROS increase and increased oxidative lipid deposition with more subsequent lipid peroxidation. This ultimately inhibits the respiratory electron transport chain and so on (Wang et al. 2016). Unfortunately, reserves of antioxidants progressively decrease in a vicious cycle also. Fatty degeneration results in lipid peroxidation where ROS deplete the cellular antioxidant systems: antioxidant enzymes, reduced glutathione, and vitamin E. Unfortunately, this adds more ROS-induced deleterious effects on the

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mitochondria (Wang et al. 2016), e.g., increased permeability of the mitochondrial membrane, increased release of apoptotic factors, and increased damage of mitochondrial DNA (Malik and Czajka 2013). Cytochrome P4502E1 (CYP2E1) is a microsomal enzyme that scavenges prooxidants. CYP2E1 damage causes oxidative stress if not antagonized by antioxidants. In a study in animals exhibiting nonalcoholic steatohepatitis, CYP2E1 expression correlated with increased serious damage in liver cells, i.e., oxidative stress in microsomes is strongly related to cell injury (Zeng et al. 2008). Telomeres are DNA repeats that are vital in the occurrence of carcinogenesis. Telomere shortening may facilitate aging and apoptosis. Oxidative stress can fasten the process of telomere shortening resulting in enhancement of oxidative damage. In recent reports, a chronic oxidative stress resulted in migration of reverse transcriptase subunits of telomerase in the cytoplasm and decreased its activity. Decreasing apoptosis signals in cells or tissues enhances carcinogenesis (Calado et al. 2011). Pathogenesis of HCC involves chronic inflammation, oxidative/nitrosative stress (causing lipid peroxidation, mutagenesis, genomic instability, decreased DNA repair and genetic damage, and promutagenic DNA adducts). HCC causes 5.5% of all cancer cases internationally (Rahib et al. 2014). HCC mostly occurs on top of liver cirrhosis where hepatatocarcinogenesis utilizes chronic inflammation and severe oxidative stress. In the early beginning, ROS may afflict DNA directly resulting in damaging specific genes that control cell growth and differentiation and enhance carcinogenicity-induced xenobiotics via enhancing their reactivity. Hepatitis B virus (HBV), hepatitis C virus (HCV), alcohol ingestion, obesity, type 2 diabetes mellitus, and aflatoxins are inborn errors of metabolism. Then, carcinogenesis progresses where ROS stimulate the growth of cancer cells. Hydroxyl radicals are the most reactive, and the most damaging radicals – in addition to other ROS intermediates – share in inducing the inflammatory process. Hydroxyl radicals result in forming thymine base mutations, thymidine glycol, 8-hydroxydeoxyguanosine, and 5-hydroxylmethyluracil. Major risk factors for HCC include chronic hepatitis B and C viruses (accounting for 80% of HCC cases), in addition, aflatoxin B1 consumption, cigarette smoking, and heavy drinking. Ethanol ingestion increases both ROS/RNS and peroxidation of lipids, DNA, and proteins resulting in activation of hepatic stellate cells involved in increased production of extracellular matrix and accelerated cellular proliferation, i.e., ROS accelerate fibrogenesis and cirrhotic changes. Such pathogenesis favors hepatatocarcinogenesis (Table 1). Postoperative HCC recurrence may occur de novo or be metastatic (occurs in the first postoperative year reflecting intrahepatic metastasis) (Schwartz et al. 2002).

Viral Hepatitis Increases Free Radicals: Toward HCC Induction HCV infection is closely associated with chronic inflammation, free radical generation, chronic liver damage, and carcinogenesis. Lipid peroxides and markers of oxidation of nucleic acids causing DNA mutations are increased in both serum and hepatic tissues of patients infected with HCV (Sumida et al. 2000). Mitochondrial

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Table 1 ROS causes and effects in HCC * Factors implicated in increased oxidative stress in hepatic tissue Cigarette smoking Aflatoxins Alcohol ingestion (heavy wine drinking) Nonalcoholic fatty liver disease Ischemia–reperfusion injury Drugs, e.g., paracetamol depletes glutathione Toxins and hepatocarcinogens Iron overload * Deleterious ROS effects on liver cells: toward carcinogenesis Promote pathologic polyploidization Augment inflammatory processes (chronic hepatitis) Enhance insulin resistance Enhance migration, invasion, and metastasis of HCC Change gene expression, cell cycle, cell metabolism, cell adhesion, and cell death Induce oxidative DNA damage, i.e., ROS are mutagens Increase chromosomal aberrations Activate cellular signaling pathways, e.g., PI3K, p53, MAPK, NF-kB, β-catenin/Wnt, and angiogenesis pathways

damage, endoplasmic reticulum damage, and immune cell-mediated oxidative stress effects participate in HCV-associated oxidative stress. HCV transgenic mice undergo hepatocarcinogenesis that follows hepatic steatosis (Moriya et al. 2001). Hepatic steatosis results in chronic hepatic inflammation, ROS, and DNA damage in experimental animals. Oxidative stress was reported to occur with no inflammation in animal models of hepatitis C virus-associated hepatocarcinogenesis (Moriya et al. 2001). Mitochondrial damage, oxidative effects on genes, and changes in antioxidant gene functions were all affected by hepatitis C virus core proteins. Compared to liver tissue from HCC patients (with or without cirrhosis), both HBV or HCV can cause genetic damage impeding cell cycle control, DNA repair, pathways of signal transduction, and apoptosis (Ozen et al. 2013). Viral hepatitis causes liver cirrhosis that is exacerbated by chemicals exposure. Hepatotoxins are ubiquitous in nature and can be genotoxic and nongenotoxic modes of action as aflatoxin, ethanol. Their toxicity is enhanced in those having diminished antioxidants reserves, e.g., selenium and Vitamin E. The same picture is encountered with excess oxidants, e.g., iron excess. Integration of HBV DNA into chromosomal DNA occurs near the loci of the genes encoding retinoic acid receptor and cyclin A. Genetics and epigenetics of liver cancer genetics and epigenetics of liver cancer (Dejean 1990) enhance cells replication and division, i.e., HBV was reported to be an insertional mutagen in a some HCC patients on top of HBV infection (Dejean 1990). Compared to tumors from HBV-infected patients, HCC cells based on HCV exhibited enhanced expression of specific enzymes (e.g., cytochrome p450IIE) that converts procarcinogens into more active products. Likewise, HBV tumor cells had diminished levels of antioxidant enzymes, e.g., glutathione peroxidase that scavenges the oxidant effects of chemical carcinogens. Oxidative stress predisposes to HCC development in transgenic mice

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that increases the production of intracellular HBsAg. Accumulation of mutagenic reactive oxygen intermediates occurs on top of inflammatory chronic HCV or HBV infections, prolonged aflatoxin exposure, and chronic alcoholism (Guyton and Kensler 1993). Reactive oxygen intermediates also result in upregulation of signal transduction pathways and related genes, e.g., c-jun, c-fos, and c-myc causing abnormal host gene expression and subsequently affect the regulation of cell growth (Guyton and Kensler 1993). HCV proteins-induced cytokines storm and free radical production usually result in oxidative genomic injury that eventually causes HCC. HCV infection enhances the levels of ROS/RNS while decreasing antioxidant levels in hepatitis patients (Jain et al. 2002). Oxidative stress induced by ROS/RNS is responsible for HCV-induced hepatocarcinogenesis. HCV core proteins activate inducible nitric oxide synthase that forms RNS causing DNA mutations, immunoglobulin mutations, and tumor suppressor genes mutations (Machida et al. 2004). HCV core proteins are highly significant in hepatocarcinogenesis induced by HCV. HCV core proteins cause increased ROS generation and lipid peroxidation. HCV core gene expression results in synthesis of core proteins that decrease intracellular/mitochondrial reduced glutathione levels and mitochondrial NADPH levels. This results in increased calcium uptake and ROS generation at mitochondrial complex I. HCV Core proteins also induce endoplasmic reticulum stress and enhance the production of cytokines augmenting ROS generation. Inducible nitric oxide synthase and cyclooxygenase-2 are increased in this respect. HCV infection enhances mitochondrial permeability transition resulting in the generation of massive ROS amount. That subsequently enhances DNA mutations and double-stranded DNA breaks. Transgenic mice strains expressing HCV core protein resulted in steatosis (after few months) and HCC in one-third of transgenic animals (after 18 months). In hepatocytes, inflammatory cytokines, e.g., IL-1β, TNF-α, and interferon-gamma, cause generation of ROS. Transgenic mice expressing HCV polyprotein exhibited hepatocarcinogenesis more than animals that expressed structural proteins only (Lerat et al. 2002) (Table 2). Table 2 Metabolic and oxidative sequelae of HCV infection Increased inflammatory cytokines Increased chronic inflammation, e.g., increased phagocytic NAD(P)H oxidase activation) Increased ROS/RNS levels Increased lipid peroxidation products in serum, liver tissues, and peripheral blood mononuclear cells Increased oxidative stress markers (as 4-hydroxynonenal and 8-hydroxydeoxyguanosine) Increase sensitivity to toxins such as ethanol and CCl4 Decreased antioxidants levels (e.g., reduced glutathione decreases while oxidized glutathione increases) Decreased intracellular GSH levels and the mitochondrial NADPH content Activation of Kupffer cells (produces proinflammatory cytokines, e.g., TNF-α and IL-1 and profibrotic cytokines, e.g., TGF-β) Altered proto-oncogenes and tumor suppressor genes Antiapoptotic effects

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Alcohol and Oxidants Cause HCC WHO had set alcohol-induced morbidities as the third cause of morbidity and mortality in developed countries and non-Muslim developing countries. Alcohol induces peroxidation of lipids, DNA, and proteins (Cederbaum 1991). Alcoholism results in direct DNA damage due to acetaldehyde that binds to DNA molecules, inhibits DNA repair mechanisms, and causes the formation of carcinogenic DNA molecules, e.g., adducts. Chronic alcohol ingestion prevents methyl group transfer and may change gene expression. High levels of oxidative stress among obese and diabetic are implicated in hepatatocarcinogenesis (Matsuzawa-Nagata et al. 2008). Nutritional antioxidants, e.g., high fruit and vegetable consumption result in low cancer incidence with improved DNA repair mechanisms (Block et al. 1992). Knocking out antioxidant enzyme systems in experimental animals caused increased hepatatocarcinogenesis, e.g., knocking out the antioxidant copper-/zinc-dependent superoxide dismutase resulted in augmented oxidative stress and diminished antioxidant scavenging of ROS that correlated with increased hepatatocarcinogenesis (Halliwell 2007). Oxodihydro-deoxyguanosine (8-oxo-dG) is a promutagenic DNA adduct that occurs during the process of hepatocarcinognesis confirming the occurrence of DNA lesions during HCC transformation (Jüngst et al. 2004). 8-Hydroxydeoxyguanosine is a guanine base change resulting in a point mutation of new daughter strand of DNA and acts as an indicator of DNA damage (Kuchino et al. 1987). HCV infection causes inflammatory lesions with subsequent oxidative DNA damage (8-oxo-dG). Moreover, propano DNA adducts (large DNA adducts), e.g., formation of γ-hydroxy-propanodeoxyguanosine (γ-OHPdG, caused by peroxidation of lipids) are causative of hepatatocarcinogenesis. Furthermore, lipid peroxidation gives rise to acrolein that consequently enhances the formation of γ-OHPdG that causes DNA base transition mutations, i.e., cause mutations of G to T and mutations of G to A. This affects vital genes, e.g., p53. GC > TA mutations were ubiquitous (90% of reported mutations). GC > TA mutations may indicate that γ-OHPdG plays an important role in hepatatocarcinogenesis (VanderVeen et al. 2001). Diagnostic evaluation of ROS in hepatocarcinogenesis aims at estimating ROS derivatives and serum hydrogen peroxide (for prognosis of HCC recurrence after surgery or radiofrequency).

Warburg Effect Increases Oxidative Stress in HCC Cells Unlike normally dividing mature cells that depend on oxidative phosphorylation to get energetics, actively proliferating malignant cells possess a characteristic metabolic behavior that drives them to preferentially consume glucose through aerobic glycolysis converting it into lactate (fermentation) even in the presence of oxygen. This is termed the “Warburg effect.” Cancer cells dependence on cytoplasmic glucose consumption differentiates malignant cells from their normal nonmalignant counterparts. HCC cells exhibit a reprogramming of glucose metabolism that correlates with the progression of liver disease to HCC. The major isozyme hexokinase II

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is exclusively upregulated in many types of cancer cells but not normal adult tissues, e.g., hepatocytes. Hexokinase II helps aerobic glycolysis via accelerating glucose flux. MicroRNAs were confirmed to negatively regulate the Warburg effect in HCC cells via silencing hexokinase II enzyme (Xu et al. 2019). In a previous report, I explained how Warburg effect enhances ROS quantities in cancer cells via suppressing antioxidant capabilities (El Sayed et al. 2013). In malignant cells exhibiting the Warburg effect, glucose is converted into lactic acid that is effluxed outside cancer cells. In normal cells, glucose is converted into pyruvic acid that is not effluxed extracellularly but is further catabolized into acetyl CoA to initiate Krebs cycle. Pyruvic acid is a potent antioxidant, while lactic acid is not an antioxidant. Pyruvic acid is converted to acetyl CoA in normal cells that begin Krebs cycle producing antioxidant biomolecules, e.g., reducing equivalents (NADH.H+), oxaloacetate, citrate, and malate. NADH.H+ can regenerate NADPH.H+ (via activity of the enzyme mitochondrial transhydrogenase) that subsequently activates glutathione reductase to restore the active form of glutathione (reduced glutathione, potent antioxidant). This adds further antioxidant benefits to normal cells while malignant cells are deprived of these antioxidant benefits. That is because Warburg effect consumes pyruvate in favor of lactate production (not antioxidant). This subsequently minimizes antioxidant effects and enhances endogenous oxidative stress in cancer cells. Glycolytic behavior in malignant cells causes a relatively increased noncytotoxic oxidative stress that may enhance the cancerous behavior. Glycolysis enhances the energetic arm, the mitotic arm, and the metastatic arms of carcinogenesis (El Sayed et al. 2013).

Current Preventive and Treatment Modalities of HCC and Related Limitations There is no currently approved drug by FDA against HCC recurrence. However, three main preventive pathways may work well: 1. HBV and HBV eradication using interferon therapy. However, HCV-induced damage in hepatocytes is irreversible. 2. Anticancer chemotherapy, i.e., the STROM trial (sorafenib and Tegafur-uracil). 3. Inducing differentiation of liver cancer cells, e.g., using pertinoin causing apoptosis and differentiation of cancer cells.

Antioxidant Therapy for HCC Antioxidants administration (as reduced glutathione) or antioxidant strategies (as iron chelators) minimize the toxic effects of ethanol. Intake of antioxidants, e.g., substrates of reduced glutathione, vitamin E, superoxide dismutase, and ebselen,blocked alcohol-induced hepatic tissue damage in experimental animals.

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Antioxidants usually work through hydrogen donation, free radical scavenging, chelators of metallic ions, and precursors of flavonoids for hydroxyl and superoxide radical actions. Antioxidants were reported to suppress γ-OHPdG, i.e., may prevent its roles in hepatocarcinogenesis. Ethanol metabolism using the enzyme alcohol dehydrogenase caused enhanced ROS production, hepatocyte damage, and apoptosis. All that was collectively inhibited by antioxidants. Unfortunately, many drugs and chemicals induce oxidative damage via generating ROS. Hence, it is quite vital to use antioxidants to combat ROS-induced deleterious effects.

Natural Honey Natural honey exerts potent antimutagenic activity, i.e., inhibits genetic mutations that are interlinked with carcinogenicity. Different types of honey cause inhibition of mutagens as Trp-p-1. Phenolics and flavonoids exhibit potent antioxidant activities that exert potent tissue-protective and anticancer effects. Honey intake at 1.2 g/kg/ day increased serum levels of antioxidants, e.g., vitamin C, beta-carotene, and glutathione reductase (Ahmed and Othman 2013). The nuclear protein Ki-67 is a marker of cellular proliferation. Honey affects cell cycle arrest and markedly decreases expression of Ki67-LI in tumor cells (Scholzen and Gerdes 2000), i.e., honey therapy decreases tumor cell proliferation by arresting cell cycle, e.g., honey blocks the cell cycle of colon, glioma, and melanoma cancer cells in G0/G1 phase. Honey downregulated many signaling pathways as ornithine decarboxylase, tyrosine cyclooxygenase, and different kinases. Moreover, honey modulates p53 regulation. Honey suppresses TNF-α that mediates tumor initiation, promotion, and progression. TNF-α exerts proinflammatory effects via activating NF-kB causing activation of inflammatory genes, e.g., cyclooxygenase-2, lipoxygenase-2, chemokines, cell-adhesion molecules, iNOS, and other inflammatory cytokines. TNF-α release regulates important cellular processes such as apoptosis, cell proliferation, and inflammation (Ahmed and Othman 2013).

Nigella sativa Nigella sativa is recommended among other herbs as a spice for prevention and treatment of cancers. Nigella sativa is a natural pharmacy rich in so many active pharmacological agents potentiating each other in exerting antioxidant and anticancer effects. Nigella sativa contains thymoquinone, α-pinene, β-pinene, α-phellandrene, α-longipinene, gamma-terpinene, trans-anethole, longifolene, alpha-terpinene, alphaterpineol, citral, p-cymene, cis-carveol, carvacrol, thymol, terpinen-4-ol, and 1,8-cineole. Thymoquinone is the most abundant constituent in Nigella sativa and is famous for its tissue-protective and antiviral effects. Thymoquinone powerfully suppressed Epstein–Barr virus-infected B cells (Zihlif et al. 2013). Nigella sativa oil intake caused tumor-suppressed metastasis of the liver that also resulted in increasing the mouse’s survival (Ait Mbarek et al. 2007).

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Natural ingredients in Nigella sativa protect liver cells against malignant transformation via different mechanisms, e.g., suppressing oxidative stressinduced tissue damage. Thymoquinone, thymol, and α-hederin exert potent antioxidant effects. Thymoquinone, thymol, and alpha-hederin protect liver cells from a wide range of injurious agents. Nigella sativa ingredients inhibit iron-induced cellular lipid peroxides formation causing increased total thiols content and reduced glutathione level. Nigella sativa ingredients cause free radical scavenging. Nigella sativa contents enhance the antioxidant enzyme systems, e.g., superoxide dismutase, quinone reductase, glutathione peroxidase, glutathione transferase, and catalase. Nigella sativa ingredients exert significant antiinflammatory benefits that suppress common inflammatory and carcinogenic pathways, e.g., NF-κB pathway (Tabassum et al. 2018). Thymoquinone (most ubiquitous ingredient in Nigella sativa) suppresses hepatic fibrogenesis via suppressing hepatic stellate cells (responsible for secreting collagen fibers). In liver stellate cells, thymoquinone suppresses the expression of CD14 and Toll-like receptor-4. Moreover, thymoquinone stops signaling pathways for hepatic carcinogenesis as phosphatidylinositol 3-kinase phosphorylation and serine/threonine kinase-protein kinase B. Expressions of collagen-1 and α-SMA got a significant suppression by thymoquinone. Thymoquinone minimized the expression of many antiapoptotic proteins (cellular FLIP and XIAP) that may be associated with malignant cells immortalization (Bai et al. 2013). Nigella sativa exerted potent antiproliferative effects against malignant hepatoma HepG2 cells. About 88% of suppressive effect occurred 24 hours after incubation with different concentrations (0–50 mg/ml) of the Nigella sativa extract. Oral thymoquinone was effective in enhancing the activities of antioxidant enzymes, e.g., glutathione transferase, glutathione peroxidase, and quinone reductase. This makes thymoquinone a promising antihepatoma ingredient to guard against chemical carcinogenesis and mutagenesis in hepatic cancer (Khan et al. 2011).

Ajwa Date Fruit Ajwa dates (Phoenix dactylifera L.) are native to Al-Madinah, Saudi Arabia. In prophetic medicine (medical knowledge gained from sayings and teachings of Prophet Muhammad peace be upon him), Ajwa date fruit is described as a preventive and therapeutic nutrient to guard against toxins. Interestingly, carcinogenesis and malignancy are largely initiated by toxins particularly carcinogenic toxins. Ajwa dates are a rich in carbohydrates (fructose and glucose), vitamins, high dietary fibers, sugars, proteins, minerals, and fats. Ajwa date fruit has a lot of useful plant foodstuffs as sterols, polyphenols, flavonoids, and glycosides. Diverse tissueprotective effects are attributed to Ajwa date fruits, e.g., hepatoprotective, nephroprotective, cardioprotective, hypolipidemic, antioxidant, and antiinflammatory effects (Yasin et al. 2015). Anticancer effects of Ajwa date fruit are evident in many studies. Ajwa date fruit contains concentrated polyphenols that suppressed both growth and division of

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colon cancer cells (Eid et al. 2014). Ajwa polysaccharide, β-D-glucan, significantly inhibited sarcoma-180 cells in vivo in experimental animals. Moreover, the methanol extract of Ajwa date fruits suppressed human breast adenocarcinoma cells through causing cell cycle inhibition and apoptosis to cancer cells. Effects of ethanolic extract of Ajwa date pulp were studied where Ajwa date pulp extract caused morphological apoptotic changes on human HCC cell line HepG2 cells that were evident under phase contrast microscopy. Moreover, β-D-glucan (in Ajwa dates) caused a dose- and time-dependent inhibition of HCC cells viability and growth. Ajwa date pulp extract induced elevation of S and G2/M phases of cell cycle. Moreover, Ajwa date pulp extract induced apoptotic cell death to HCC cells unrelated to tumor suppressor genes. Ajwa date pulp did not exhibit any toxic effects on normal cell lines (Khan et al. 2017). Based on that, Ajwa date pulp extract can be looked at as a safe and natural potential drug candidate against HCC. In an exciting study, Khan et al. reported that the antioxidant effects of Ajwa date extract outweighed the prooxidant and hepatocarcinogenic effects of diethylnitrosamine (DENA) and reverted the malignant behavior of DENA-induced hepatocarcinogenesis into a benign outcome. Moreover, liver function tests (serum ALT and AST) and HCC tumor marker (alpha fetoprotein) were markedly elevated by DENA (in line with hepatocarcinogenesis) and were then restored to near normal levels upon treatment with Ajwa date fruit extract. Antioxidant enzyme systems were also disturbed by DENA (in line with malignant transformation) and were then restored to near normal levels upon treatment with Ajwa date fruit extract. Inflammatory cytokines levels were also disturbed by DENA (in line with hepatocarcinogenesis) and were restored to near normal levels upon treatment with Ajwa date fruit extract (Khan et al. 2017). Exposure to hepatocarcinogens, e.g., DENA may occur accidentally resulting in genotoxicity, nucleic acid damage, and disturbed DNA repair owing to generation of massive ROS and related oxidative damage that entail cell membranes and genetic make-up. DENA-induced oxidative stress causes cell membrane damage, inflammatory cells infiltration, loss of cells differentiation (atypia), and genotoxicity ending in hepatocarcinogenesis (Loeppky 1994). Ajwa date extract helped in the reversal of DENA-induced carcinogenesis toward a normal behavior. Regaining the carcinogenesis-induced lost functions of liver enzymes activities, antioxidant enzyme activities, near-normal cytokines equilibrium, and gene expression to normal levels following Ajwa date extract treatment frankly proved that Ajwa date extract reverses the prooxidant effects and their sequelae that take place during carcinogenesis. Based on that, Ajwa date extract is a strongly recommended nutritional supplement that may potentiate conventional current therapeutics for HCC. Ajwa date fruit can be looked at as a large pool of powerful antioxidants that may counteract oxidative stress-induced initiation and propagation of carcinogenesis. Luteolin is an efficient flavonoid available in fruits and vegetables. Luteolin is also available in Ajwa date fruit (106). Luteolin was evident in suppressing oxidative stress and regaining near normal histological pictures following DENA-induced toxicity (Loeppky 1994) (Figs. 1 and 2).

Fig. 1 Khan et al. report (Khan et al. 2017). Improvement of serum antioxidant enzymes to near normal levels after treatment with Ajwa date fruit. Antioxidant enzyme activities assayed were (a) superoxide dismutase (SOD); (b) glutathione reductase (GR); (c) glutathione peroxidase (GPx); (d) catalase (CAT); and (e) lipid peroxidase levels were analyzed. DENA-induced changes in antioxidant enzymes were in line with DENA-induced hepatocarcinogenesis. Reversal of serum antioxidant enzymes to near normal values was in line with Ajwa date fruit-induced restoration of normal histology and biochemistry

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Fig. 2 Khan et al. report (Khan et al. 2017). Hematoxylin and eosin-stained images of the liver sections of different experimental conditions of rats treated with Ajwa date extract following administration of the hepatocarcinogen diethylnitrosamine and induction of HCC (a) Negative control: exhibits healthy hepatocytes having intact cell membrane and central vein; (b) Rats that administered Diethylnitrosamine only. Liver shows altered hepatocyte morphology, lost cellular membranes, increased nucleocytoplasmic ratio, and connective tissue infiltration; (c) Rats received a small dose of Ajwa date extract (0.5 g/kg body weight) showing a moderately normal hepatocytes architecture with normally oriented hepatocytes similar to that of the control; (d) Rats received a large dose of Ajwa date extract (1 g/kg body weight) showing an almost normal hepatocytes architecture with normally oriented hepatocytes similar to that of the control. Magnification (40X). Inset (e) exhibits the spot magnification of region (b) (DENA-treated rats), with evident cellular morphological changes, nuclear changes, and connective tissue infiltration. Inset (f) exhibits the spot magnification of region (d) in rats receiving both DENA and Ajwa (1 g/kg body weight). High-dose Ajwa administration resulted in normal microscopic structure of the liver. Thin black arrows show normal hepatocyte shape. Thick black arrows show infiltration of the connective tissue. Black arrow heads indicate the nuclei (exhibiting abnormal morphology)

Khan et al. (Khan et al. 2017) also reported improvement of serum cytokines to near normal levels after treatment with Ajwa date fruit. The cytokines, IL-12, IL-10, IL-4, GM-CSF, IL-2, IL-1α, and IL-1β, were elevated after DENA administration indicating oxidative tissue-damaging effects. DENA-induced changes in inflammatory cytokines were in line with DENA-induced hepatocarcinogenesis. Reversal of serum cytokines to near normal values was in line with Ajwa date fruit-induced restoration of normal histology and biochemistry. Interestingly, there was improvement of alpha-fetoprotein (hepatoma marker) and ilterleukin-6 to near normal levels after treatment with Ajwa date fruit. Gene expression analysis of hepatic tissue of rats treated with Ajwa date extract after induction of HCC using DENA proved anticancer effects of Ajwa date fruit through restoration of serum levels.

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Costus Costus (Saussurea lappa) and related herbs in the same species are promising anticancer medicinal plants. Volatile oil from Saussurea lappa root suppressed HCC cell proliferation via stopping cell cycle progression at S and G2/M phases, and causing HCC cell apoptosis by activating Caspase3 pathway. Volatile oil from Saussurea lappa root also decreased the ability of HCC cells to migrate and invade via suppressing matrix metalloproteinase-9 (Lin et al. 2016). Costus enhanced the protein levels of many proapoptotic factors and suppressed the protein levels of antiapoptotic factors (Shati et al. 2020). Saussurea involucrata is another medicinal plant of the same genus Costus. Saussurea involucrata exerts potent anticancer activities, e.g., apoptosis and cell cycle arrest. Saussurea involucrata-induced suppression of metastatic cells growth was dose-dependent and time-dependent. Saussurea involucrata significantly suppressed malignant HCC cells migration, invasion, and adhesion to gelatin-coated substrates (Byambaragchaa et al. 2013). Moreover, methanolic extract of Costus pictus significantly induced histone deacetylase inhibition-mediated cytotoxicity of HCC cells (Neethu et al. 2017). Methanol leaf extract of Costus speciosus exerted confirmed apoptotic and inhibitory effects on proliferation of experimental HCC (HepG2 cells) (Nair et al. 2014).

Fennel (Foeniculum vulgare Mill) Fennel exerts potent antioxidant activities being an enormous and wonderful source of many naturally occurring antioxidants that may satisfy to the daily antioxidant dietary requirements. Fennel exhibits a free radical scavenging activity with a high content of phenolics and flavonoids as confirmed by the high DPPH scavenging activity. Fennel is a medicinal plant rich in phenolics (potent antioxidants), e.g., hesperidin, cinnamic acid, ferulic acid, hydroxyl cinnamic acid derivatives, rosmarinic acid, quercetin, apigenin, chlorogenic acid, caffeic acid, p-coumaric acid, gallic acid, chlorogenic acid, flavonoid glycosides, and flavonoid aglycones, neochlorogenic, ferulic acid-7-o-glucoside, and quercetin-7-o-glucoside. Fennel is also rich in flavonoids (potent antioxidants), e.g., quercetin, rutin, and isoquercitrin that exert immunomodulatory effects (Badgujar et al. 2014). Essential oil of fennel seeds had proved strong hepatoprotective effect against carbon tetrachloride-induced acute hepatotoxicity acute liver toxicity in experimental animals. Oral intake of fennel oil proved effective in decreasing the levels of serum liver enzymes, e.g., alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, and bilirubin compared to the control group. Fennel active ingredients as D-limonene and β-myrcene in fennel essential oil may be quite responsible for fennel-induced protection of liver from CCl4 toxicity. Fennel exerts antimutagenic effects. Essential oil of fennel exerts potent protective effects against chemotherapy-induced genotoxicity in experimental animals, e.g., cyclophosphamide-induced genotoxicity. Cyclophosphamide causes cellular genotoxicity with subsequent cytotoxicity that was confirmed using sperm

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abnormality assays, bone marrow chromosomal aberration, and micronuclei. Essential oil of fennel significantly inhibited cyclophosphamide-induced genotoxic effects. Fennel oil suppressed cyclophosphamide-induced chromosomal aberrations, micronuclei formation, aberrant metaphases, and related cytotoxicity in mouse bone marrow cells. Moreover, fennel essential oil caused a significant decrease in abnormal sperm counts. Interestingly, fennel essential oil raised levels of the antioxidant enzymes, e.g., glutathione peroxidase, glutathione reductase, superoxide dismutase, and catalase in addition to enhancing antioxidant biomolecules as glutathione. Likewise, fennel exerted potent tissue-protective effects via decreasing malondialdehyde levels (marker of oxidative stress) in the liver, i.e., fennelsuppressed cyclophosphamide-induced oxidative stress and tissue damage. Fennel exerts potent cellular protection and anticancer effects. Anethole is the main active ingredient in fennel seeds that has excellent anticancer effects. Anethole exerted potent anticancer effects against Ehrlich ascites carcinoma induced in experimental animals. Anethole use was associated with decreased tumor weight, prolonged survival time, and decreased volume and body weight of the Ehrlich ascites tumor-bearing. Anethole exerted significant cytotoxic effects in mice bearing Ehrlich ascites tumor cells. That was associated with increasing the antioxidant/oxidant effects via increasing reduced glutathione concentrations and decreasing the levels of nucleic acids and malondialdehyde (Badgujar et al. 2014).

Conclusion Oxidative stress is the hallmark of HCC cells and hepatocarcinogenesis. Oxidative stress is both mutagenic and genotoxic to hepatocytes favoring their transformation and carcinogenesis. Natural antioxidants are enormously available and recommended in all types of natural medicine particularly prophetic medicine. Natural products and medicinal plants are quite safe even at high doses. Nigella sativa was safe at 20 g/kg in broiler chicken (Kumar et al. 2017). Costus is tissueprotective with no mutagenic, toxic, or teratogenic effects and is quite safe at a dose of 1.5 g/kg/day in animal studies (Subasinghe et al. 2014). Fennel extract was safe at 3 g/kg orally with no toxic or mutagenic effects (Badgujar et al. 2014). I strongly recommend HCC patients to add to their regular diet an antioxidant nutritional supplement (twice daily if possible) including grinded Nigella sativa (3 grams), grinded costus (1 gram), grinded fennel (1 gram), Ajwa date fruit (7 dates), and one large spoonful of natural honey (15 ml) to be mixed and added to yoghurt or any other food to make use of their medicinal and antioxidant benefits.

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Oxidative Stress in Orchestrating Genomic Instability-Associated Cancer Progression

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Dipita Bhakta-Guha and Gunjan Guha

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Oxidative Stress: Its Generation and Implication in Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Oxidative Stress-Mediated DNA Damage Leads to Genomic Instability . . . . . . . . . . . . . . . . . . . . . . Chromosomal Instability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Microsatellite Instability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Epigenetic Modifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Oncogenic Replication Stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Oxidative Stress Manipulates DNA Damage Response to Facilitate Genomic Instability . . . Therapeutic Strategies to Mitigate Genomic Instability in Cancer Progression . . . . . . . . . . . . . . . Amelioration of Oxidative Stress to Prohibit Establishment of Genomic Instability . . . . . . Increasing Oxidative Stress to Kill Cancer Cells Via Comprehensive Genomic Degradation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract

Cancer is a grievous challenge to human health owing to a colossal heterogeneity in its etiology. Initiation of tumorigenesis is facilitated by genomic instability that, in turn, is promoted by accumulation of damage in the DNA. Oxidative stress, a chief contributor of such damage, therefore, plays a crucial role in the onset of genomic instability. By altering structural and functional integrity of DNA, oxidative stress paves a path for onset of comprehensive genomic dyshomeostasis, which snowballs into neoplastic transformation. These alterations, exhibited as the chromosomal instability, microsatellite instability, oncogenic replication stress, and epigenetic modifications, are exploited by cancer cells to effectuate

D. Bhakta-Guha (*) · G. Guha Cellular Dyshomeostasis Laboratory (CDHL), Department of Biotechnology, School of Chemical and Bio Technology, SASTRA University, Thanjavur, Tamil Nadu, India e-mail: [email protected]; [email protected] © Springer Nature Singapore Pte Ltd. 2022 S. Chakraborti et al. (eds.), Handbook of Oxidative Stress in Cancer: Mechanistic Aspects, https://doi.org/10.1007/978-981-15-9411-3_50

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unencumbered proliferation. Furthermore, oxidative stress tends to damage critical components of the endogenous cellular repair machineries that otherwise could have inhibited such anomalous progressions. In the ambit of the critical role that oxidative stress plays in governing genomic instability in different types of cancers, this chapter provides an insight into the variegated molecular mechanisms that are involved in the process. Also, the therapeutic strategies aimed at ameliorating oxidative stress to annihilate cancer cells have been discussed here. Overall, the chapter aims at highlighting the mechanistic significance of oxidative stress in orchestrating genomic instability-associated cancer progression. Keywords

Cancer · Oxidative stress · Genomic instability · Aneuploidy · DNA damage response · Therapeutic strategies

Introduction The global burden of cancer has amassed an alarming expanse over the last few decades. Characterized by an unhindered proliferation of abnormal cells, cancer continues to plague mankind, owing to its heterogeneity, variegated etiology, and convoluted cross-talks between multiple signaling cascades. The complex network of these pathways not only differs across the types of cancer, or the stages of aggressiveness, but also varies among affected individuals. It is well-established that specialized capabilities are acquired by cancer cells, as a result of which they are able to escape any form of molecular/cellular surveillance, and flourish unencumbered. These distinctive capabilities are designated as the hallmarks of cancer (Hanahan and Weinberg 2000), understanding which presents a logical framework for deciphering tumorigenesis and neoplastic progression. Genomic instability, replicative immortality, evasion of anti-growth signaling, modulation of metabolism, immune evasion, angiogenesis, resistance to apoptosis, sustained proliferation, tumor-promoting inflammation, tissue invasion and metastasis are the chief hallmarks of cancer (Block et al. 2015). Among all the hallmarks, genomic instability is the primary critical phenomenon that ensures cancer establishment. Often referred to as the “fundamental characteristic” that promotes onset of other cancer hallmarks, genomic instability is characterized by alterations in the genetic makeup of a cell (Ferguson et al. 2015). These alterations include specific mutations, deletions, additions, rearrangements of chromosomal segments, as well as complete loss or gain of chromosomes (Shen 2011). Over the years, several studies have revealed varied factors that might contribute to genomic instability. A major contributor is oxidative stress, that is generated by excess amounts of reactive oxygen species (ROS), which cannot be neutralized by a cell’s endogenous antioxidant machinery. ROS can react aggressively with major macromolecules (lipids, carbohydrates, proteins, and nucleic acids) of the cell and consequently disrupt their structural and functional integrity. If oxidative stress increases massively, such damage gets

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Fig. 1 Fate of a cell is defined by a critical intracellular antioxidant/ROS balance. Equilibrium between ROS and antioxidants sustains cellular homeostasis. Increased oxidative stress promotes accumulation of DNA damage that leads to tumorigenesis via genomic instability (GI). Excessive degrees of ROS, however, result in severe DNA damage and GI, thereby propelling a cell toward its death

enhanced severely, and consequently pushes cells toward death. However, it is to be noted that ROS are indispensable for a cell, since they serve as signaling molecules for various obligatory physiological events, such as cell differentiation, transcription, and immune response (Liguori et al. 2018). This makes ROS a tricky component owing to its “necessary evil” status. Interestingly, it is the moderately high degree of ROS that shifts the balance toward dyshomeostasis and tumorigenesis. As seen in Fig. 1, the swing in the balance between ROS and antioxidant typically defines the state and fate of a cell. Oxidative stress not only alters the chemistry of a cell but also triggers the onset of a cascade of events that are involved in the etiology of various pathologies, such as cancer. It has been shown that oxidative stress in precancerous cells can cause severe damage to nucleic acids, which can further snowball into genomic instability, eventually causing cancer. It is, therefore, pertinent to elucidate the impact of ROS-induced damage to cellular DNA, that can illustrate the tripartite nexus between oxidative stress, genomic instability and cancer.

Oxidative Stress: Its Generation and Implication in Cancer Cells carrying out metabolism produce a plethora of free radicals called reactive oxygen species (ROS) (Li et al. 2016). To state a few examples, superoxide ions (O2•-), singlet oxygen (1O2), hydroxyl radicals (•OH), hydrogen peroxide (H2O2),

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nitric oxide radicals (NO•), radicals of nitrogen dioxide (NO2•) and peroxynitrite (ONOO
) are the most common forms of ROS (Phaniendra et al. 2015; Pizzino et al. 2017). Primarily, cellular ROS are generated endogenously in mitochondria as by-products of cellular metabolism through oxidative phosphorylation. Electrons generated in the process are transferred across a range of electron donors and acceptors, which constitute the electron transport chain (ETC) located in the inner mitochondrial membrane (Reczek and Chandel 2017). In the course of this transfer, electrons tend to leak out and react with O2, thereby producing superoxide radicals (a form of ROS). These are then either released into the intermembrane space or escape into the cytosol. Commonly, the generated superoxides get converted into H2O2 by superoxide dismutase (SOD; MnSOD or SOD2 in the mitochondrial matrix and Cu/Zn-SOD or SOD1 in the cytosol) (Kumari and Badana 2018). In addition to mitochondria, peroxisomes and smooth endoplasmic reticulum also promote ROS generation via myriad enzymatic and nonenzymatic redox reactions. Another source of ROS is the family of NADPH oxidases or NOX (transmembrane enzymes found in different tissue types). The well-understood isoforms of the NOX family include NOX1, NOX2/gp91phox, NOX3, NOX4, NOX5, dual oxidase 1 (DUOX1), and DUOX2 (Bedard and Krause 2007). Hence, it is evident that multiple sources within the cell contribute to an expanding gamut of ROS as a by-product of metabolic activities. Endogenous antioxidants, such as catalase, SOD1/2, reduced glutathione (GSH) and glutathione peroxidase, scavenge these reactive species, and thus prohibit them from disrupting important cellular features. In case the adequacy of antioxidants falls short vis-à-vis augmented levels of prooxidants (such as ROS), oxidative stress might prevail and escalate. It is the fine balance between the levels of antioxidants and the prooxidants that defines cellular homeostasis. The shift in this balance toward ROS causes oxidative stress, thereby facilitating DNA damage, and even tumorigenesis (Fig. 1).

Oxidative Stress-Mediated DNA Damage Leads to Genomic Instability The impact of oxidative stress on the etiology of cancer is multipronged, as is evidenced by its involvement in the initiation and progression of cancer. For example, oxidative stress can inflict injuries to the DNA, which can hinder replication and jeopardize genomic integrity (Fig. 2). Consequently, the overall central dogma of cells can get comprehensively altered, thereby leading to holistic cellular dyshomeostasis. While in normal cellular physiology, endogenous DNA damage repair systems are designed to rectify such damage/errors, these repair machineries falter in case of a malignant transformation. It still remains to be better understood whether malignant transformation makes the reparative potential defective or the faulty repair system induces malignancy. Nevertheless, accumulation of oxidative stress can impact both nuclear and mitochondrial DNA (mtDNA), with the latter being more vulnerable (mitochondria being the primary seat of oxidative metabolism). In the

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Fig. 2 The origins and oxidative impact of ROS. Oxidative stress can build up within the cell due to accumulation of ROS that are generated by endogenous sources, such as the mitochondria, through the electron transport chain (ETC). Peroxisome, smooth endoplasmic reticulum, and NOX family of enzymes also endogenously produce ROS. A cell’s microenvironment might also cause oxidative insult (exogenous ROS) to its machinery. ROS, thus produced, damage DNA through varied mechanisms, thereby contributing to genomic instability

course of this chapter, we will focus on nuclear DNA damage in order to correlate with genomic instability-driven neoplastic transformations. There are two primary mechanisms that confer DNA-damaging potential to oxidative stress. These are: (a) the Fenton reaction and (b) increased concentration of intracellular Ca+2. Fenton reaction is characterized by the generation of •OH from H2O2. On the other hand, increased ROS leads to an increase in intracellular Ca+2, that in turn, has been reported to activate nucleases that degrade DNA (Davalli et al. 2018). Consequent to these mechanisms, a plethora of products are known to be generated due to oxidative stress-induced damage. For example, the most common product, 8-oxo-7,8-dihydrodeoxyguanosine (8-oxodG) (a common biomarker of oxidative stress) is generated due to the reaction of ROS with nitrogen of the DNA bases (preferentially guanine). These modified bases can pair with adenine or cytosine. In case of an unrepaired mismatch, G!T transversion ensues. This feature is widespread across varied types of cancers (Klaunig et al. 2010). Similar transversion is seen when accumulation of ONOO
generates 8-nitroguanine that produces apurinic sites (due to spontaneous deamination) in the DNA. Presence of such apurinic sites appoints DNA polymerase ζ (facilitates error-prone translesion DNA synthesis) that allows replication in spite of the errors still incorporated. Progression

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in replication, despite errors, abundantly manifests into genomic instability. In addition to these, interaction of DNA with electrophiles (that have been generated through lipid peroxidation) produces yet another DNA damage product, the etheno adducts. Furthermore, hydroxylated thymine (5-hydroxymethyl uracil) and M1dG (formed by the reaction of malondialdehyde and propenals derived from oxidative damage of DNA bases) are some of the other DNA damage products (Chan and Dedon 2010). Grossly, these reactions/products evoke base modifications, crosslinks, stalled replication fork, single-stranded breaks (SSBs) or double-stranded breaks (DSBs). These injuries further give rise to translocation, deletion, insertion, and holistic alteration in the chromosomal number and/or structure, that in turn, pave the path for genomic instability (Helm and Rudel 2020). Common contributors to this instability are centrosome amplification leading to supernumerary centrosomes (SNCs), telomeric damage, microsatellite instability, epigenetic modifications, and oncogenic replication stress, among other factors. In the following sections, we will be looking into different cellular facets that contribute to genomic instability. It would be interesting to understand the role played by oxidative stress in regulating each of these facets.

Chromosomal Instability Chromosomal instability (CIN) is the most recurrent form of genomic instability that is primarily characterized by a structural or numerical aberration in chromosomes. Numerical aberration is defined by addition or deletion of whole chromosome (s) (aneuploidy), whereas structural instability refers to partial loss or gain of segments of chromosome(s). These erroneous chromosomal modifications lead to genome chaos in cancer cells and have often been reported to contribute to intratumoral heterogeneity. More often than not, aneuploidy (a very common feature of almost all cancers) is a consequence of inappropriate segregation (missegregation) of chromosomes. This is commonly caused due to improper microtubulekinetochore attachments. These erroneous attachments are further classified as monotely, syntely and merotely. Briefly, monotely is a condition where only one of the two kinetochores per chromosome is attached to the spindle fiber. Syntely is characterized by the attachment of fibers emerging from the same pole to both the kinetochores. Faulty microtubule-kinetochore attachment is also seen in case of merotely where the same kinetochore attaches to different spindle fibers emerging from opposite poles. In fact, merotely has been reported in around 85% of all carcinomas (Vargas-Rondón et al. 2017). Several factors (as mentioned below) are known to contribute to CIN.

Telomere Shortening Telomeres are small (5–10 kilo base pairs), repetitive stretches of noncoding sequences [(TTAGGG)n] at the ends of each chromosome. These protective sequences tend to lose out around 100 base pairs per cycle of division, leading to

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shortening of the length of chromosome after each replication cycle. In normal cells, moderate decline in the chromosomal length triggers replicative senescence (permanent cell cycle arrest) or cell death (mostly through apoptosis). This ensures prohibition of continuous divisions, thus inhibiting transformation of normal cells. However, in case that the surveillance machinery that is ought to check the length reduction becomes dysfunctional, the prevention of telomere shortening beyond moderate degrees is compromised. Eventually, the critically reduced length of telomere leads to CIN that paves the path to neoplastic transformations. Thus, the crucial telomere shortening-associated CIN might be one of the prerequisites for cancer inception (Ferguson et al. 2015). Oxidative stress has been reported to enhance the rate of telomere shortening. This is primarily due to the augmented formation of DNA strand breaks that are easily formed in the telomeric region, which is rich in guanine bases (thus forming colossal amounts of 8-oxodG). Oxidative DNA lesions can block telomeric repeat binding factors 1 and 2 (TRF1 and TRF2), which sustain the homeostasis of the telomere. These factors inhibit phosphorylation of ataxia telangiectasia mutated (ATM) kinase, which is required for the repair of DNA breaks (Opresko et al. 2005). Telomeric oxidative stress also leads to dysfunctional G-quadruplex folds (G-rich repeats of telomeres that stack together to generate stable DNA folds), which are supposed to protect the telomeres in normal cells (Jansson et al. 2019). In addition to this, accumulation of such elevated ROS-associated SSBs depletes the reparative potential of the telomeric region(s) as well as the functions of telomerase. In fact, increased levels of telomeric 8-oxodG and malondialdehyde-DNA adducts have been widely reported in breast cancer (Shen et al. 2009), thus suggesting a causal role of telomeric oxidative injury in tumor initiation/progression.

Centrosome Amplification, Multipolarity, and Centrosome Clustering Centrosomes are the microtubule organizing centers (MTOCs) from where microtubules arise (nucleate) in animal cells. Comprising of two orthogonally placed centrioles embedded in the pericentriolar material, a centrosome is responsible for orchestrating the spindle formation. In each normal cell, the number of centrosomes is tightly regulated via the centrosome duplication cycle. Various licensing factors that constitute the centrosome duplication machinery ensure that each centrosome divides only once per cell cycle. This asserts the presence of a single centrosome in a non-dividing cell and two in case of a cell that is undergoing division. Guided by proteins like polo-like kinases (Plks), NIMA-related kinases (Neks), Aurora kinase A, various CDK/cyclin complexes, Cep 152/192 and others, centrosome duplication, maturation and separation eventuate through the various stages of the cell cycle. The centrosomes then move in opposite directions, consequently forming the two poles of the future bipolar spindle. In most cancers, however, several of these regulatory proteins are erroneously expressed, thereby leading to centrosome amplification (CA) that results in supernumerary (multiple) centrosomes (SNCs) (Wang et al. 2014). The occurrence of SNCs, in principle, promotes formation of multipolar spindle, since each centrosome forms a pole of the spindle apparatus. Microtubules emerging

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out of such a multipolar spindle can erroneously attach to kinetochores and pull chromatids with unequal forces. Consequently, these unequal forces alert the spindle assembly checkpoint (SAC) that is associated with checking correct microtubulekinetochore attachment. Activation of the SAC either delays the onset of anaphase while waiting for the attachment to be rectified, or, in an irreparable juncture, triggers cell death. Interestingly, despite CA, cancer cells have an escape strategy that does not activate the SAC. This is achieved by gathering the SNCs into opposite poles and feigning bipolarity (pseudo-bipolar spindle formation) through the evasive strategy of centrosome clustering (CC). Several types of cancer (such as breast adenocarcinoma, head and neck squamous cell carcinoma, pancreatic adenocarcinoma, etc.) abundantly exhibit this phenomenon. CA-led CC mimics a bipolar spindle apparatus and escapes cell death, while inadvertently causing aneuploidy (CIN) in the daughter cells (Bhakta-Guha et al. 2015). Studies have revealed that oxidative stress might have a role in the centrosomal dyshomeostasis mentioned above. Long-term exposure to elevated oxidative stress was observed to reduce the duration of S phase and hasten G2/M phase of the cell cycle. This reportedly leads to a sped-up centrosome duplication cycle to the extent that SNCs are generated in addition to multinucleated cells (Xie et al. 2008). While oxidative stress promotes CA, the latter is known to augment ROS levels as well. This is caused due to activation of NOX that, in turn, increases interleukin-8 (IL-8) expression, which is necessary for paracrine invasion of breast adenocarcinoma cells (Arnandis et al. 2018). Oxidative stress generated by 2-chloroethyl ethylsulfide (analog of mustard gas) was seen to induce monoadduct formation in the DNA, thus leading to CA and aneuploidy in osteosarcoma cells (Bennett et al. 2014). In prostate cancer (PC-3 cells), ROS-inflicted DNA damage leads to increased expressions of Plk4 and Cdk2 which are known to promote CA and multipolar spindle formation (Pannu et al. 2012). Additionally, aneuploidy often leads to altered expressions of various proteins that cause proteotoxic stress response. Proteotoxic stress is defined as an excess accumulation of proteins due to abnormally high translation and low degradation mechanisms, which further jeopardizes the redox balance. Under such a circumstance, more ROS get generated and the vicious cycle of ROS-CIN-ROS continues (Simonetti et al. 2019). Thus, ROS behave both as the source and product of CIN through centrosome dyshomeostasis.

Microsatellite Instability Microsatellite instability (MSI) is caused due to mutations in the microsatellites (short tandem repeats spread across the DNA). Such alterations in the DNA sequence are prominently observed in different forms of cancer. This type of instability is generally attributed to mismatches during replication. In fact, MSI promotes the mutator phenotype that allows cancer cells to possess significantly higher number of mutations than a normal cell. Depending on the frequency, MSI can be further categorized as low-MSI and high-MSI. In a study carried out with colorectal cancer cells, it was reported that generation of oxidative stress by heavy

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metals like arsenic and cadmium reduced the expression of mismatch repair (MMR) proteins. MMR proteins are responsible for repairing incorrect base pairs, and therefore, reduction in their expressions subsequently increased MSI (Wu et al. 2017). In fact, 15–17% of all cancers have been reported to harbor MMR deficiency (Ferguson et al. 2015). In two discrete studies employing colon (HCT116) and lung cancer (HCC-15 and NCI-H2009) cells, increase in frameshift mutations were observed on treatment with moderate ROS inducers (Zienolddiny et al. 2000; Gasche et al. 2001). Elevated frequency of mutations has been reported in several hereditary cancers with rampant loss of DNA repair genes (Yao and Dai 2014).

Epigenetic Modifications Epigenetic modifications constitute the heritable changes that do not alter the primary DNA sequence, but modify the pattern of gene expression and functionality of the DNA damage repair machinery. These modulations are achieved through diverse mechanisms, such as methylation, histone modification, chromatin remodeling, etc. Varied levels of sequence-specific methylation colossally contribute to genomic instability. For example, hypomethylation of oncogene promoters augments the expression of gene products. Hypermethylation silences several cell cycle regulators as well as repair proteins. Methylation of the CpG islands in the promoter of the MMR gene induces the mutator phenotype. In addition, modifications such as acetylation, deacetylation, sumoylation, phosphorylation and ubiquitination of histones can cast a diversified influence on transcription. Interestingly, oxidative stress plays a paramount role in controlling several of these epigenetic regulators, subsequently promoting carcinogenesis via genomic instability. In hepatocellular carcinoma, presence of 8-oxodG reportedly inhibits methylation of adjacent cytosine residues and reduces the binding efficacy of methyl CpG binding protein (MBP) to the DNA. It is to be noted that MBP is responsible for recruitment of DNA methyltransferase (DNMT) and histone deacetylase (HDAC). Such reduced binding efficacy promotes global hypomethylation, leading to silencing of the tumor suppressor genes. Similarly, in colorectal cancer, DNA damage incurred due to oxidative stress can promote formation of the DNMT1/DNMT3B/polycomb repressive complex (PRC4) assembly at the CpG islands that results in DNA methylation and subsequent silencing of the gene, thereby orchestrating tumorigenesis (O’Hagan et al. 2011). In prostate cancer, elevated ROS level is associated with hypermethylation of GSTP1 gene, thus causing loss of its expression. This is critical, since GSTP1 encodes for the antioxidant enzyme glutathione-s-transferase P1 and has been observed to be associated with high-grade intraepithelial neoplasia (Donkena et al. 2010). Similarly, oxidative stress-mediated histone modifications, including increased expression of HDAC1 and decreased acetylation of H3 at lysine residues (9, 14, 18, and 27) were found to be prominent indicators of progression of renal cell carcinoma (Mahalingaiah et al. 2017).

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Oncogenic Replication Stress In most types of cancer, cell cycle witnesses defective progression of the replication fork characterized by slowing or stalling, which is known as replication stress. This is usually caused due to either a physical hindrance to the fork or decoupling of DNA polymerase and helicase, or both. Irrespective of the cause, the anomalous progression of the fork impacts DNA synthesis. Replication stress inflicts genomic instability that further catapults into accumulation of additional carcinogenic changes. Oxidative stress has been observed to play an important role in generation of replication stress through diverse mechanisms. Elevated ROS levels can slow down the velocity of the replication fork by oxidizing the deoxyribonucleotide triphosphates (dNTPs), or, by dissociating peroxiredoxin2 (PRDX2) from the replication fork accelerator protein, TIMELESS. In noncancerous conditions, PRDX2 is supposed to form a replisome that binds to TIMELESS. Thus, the dissociation of PRDX2 and TIMELESS in cancer cells slows down the fork, thereby causing replication stress (Srinivas et al. 2019). Furthermore, oxidation of bases leads to various conformational changes, which in turn, can cause obstructions for the moving replication fork. Such physical impediments often lead to the breakdown of the replication fork thereby generating DSBs. In the absence or evasion of repair systems, presence of DSBs ensues accumulation of defective DNA, consequently giving rise to genomic instability.

Oxidative Stress Manipulates DNA Damage Response to Facilitate Genomic Instability It is to be noted that ROS are a necessary component of normal cellular functioning. This means that a normal cell possesses the mechanisms to consistently counter the negative impact of ROS on cellular homeostasis. It is only the increased ROS levels that pose a threat to the structural and functional integrity of a cell. In normal cells, integrity of the genome is tightly regulated at all times owing to stringent surveillance by DNA damage checkpoints as well as mitotic checkpoints. Any anomaly in DNA gets rectified using the cell’s endogenous repair machineries, such as the base excision repair (BER), mismatch repair (MMR), nucleotide excision repair (NER), translesion synthesis (TLS), nonhomologous end joining (NHEJ), and homologous recombination (HR). BER is responsible for rectifying modified bases, while mismatched base pairs are repaired by the MMR system. The NER gets involved in correcting crosslinks, paused replication forks and SSBs. DSBs usually get rectified by HR or NHEJ (when homologous chromosomes are not available). In principle, during an ongoing DNA repair, a complex network of signaling cascades, called DNA damage response (DDR), impedes cell division, cell cycle and DNA duplication, by inhibiting transcription. This prohibits the transmission of defective genetic content to the daughter cells, thereby asserting genomic stability. Cancer cells, however, continue to proliferate even with genetic anomalies due to either complete absence of the genes associated with these repair systems, or by staying elusive from the surveillance of the repair machineries. This is attained by harboring mutant

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proteins that render the DDR machinery highly ineffective. As a result, the cancer cells keep proliferating without a check by means of accumulated mutations, and consequently produce lineages of abnormal cells. At this juncture, it becomes crucial to understand that there are several ROS-sensitive proteins that are associated with DDR. These proteins get damaged and amass mutations in the presence of oxidative stress (as in cancer cells), thereby rendering the DDR inefficient, and consequently inducing genomic instability. For example, cysteine-rich proteins, such as checkpoint kinase1 (Chk1), checkpoint kinase2 (Chk2), Wee1, polo-like kinase 1 (Plk1) and caspases, get oxidized by ROS at their cysteine residues. This leads to altered structures of these proteins that hinder their respective functions. In their unaltered forms, these proteins are associated with different cellular homeostatic processes, such as cell cycle progression, apoptosis, and activation of p53 and p21 pathways. In principle, ATM, ATR (ataxia telangiectasia and Rad3-related), and DNA-dependent protein kinase catalytic subunits (DNA-PKcs) are the primary kinases enabled with sensing ROS-mediated DNA damage and activation of DDR. Oxidative stress-mediated ATM’s activation in cancer cells stimulates the formation of disulfide homodimer that is associated with transcription of pro-survival and anti-apoptotic factors. Inactivation of Cdc25 by oxidative stress allows the neoplastic cells to escape DNA damage-associated G2 arrest, thereby persuading these cells to divide. Similarly, oxidative damage inflicted on PTEN (phosphatase and tensin homolog) leads to its inactivation, that in turn, activates Akt through PI3K (phosphatidylinositol 3 kinase) in cancer cells. This enhances production of endogenous antioxidants to scavenge excess ROS in order to sustain pro-survival signals (Davalli et al. 2018). Another critical cellular player that diminishes oxidative genotoxicity is p53, often termed the “guardian of the genome.” It activates the transcription of several genes that are involved in the regulation of oxidative stress, such as sestrin, glutathione peroxidase (GPX), aldehyde dehydrogenase (ALDH), tumor protein 53-induced nuclear protein 1 (TP53INP1), tumor protein 53-induced glycolysis, and apoptosis regulator (TIGAR) and SOD2. However, since p53 itself is present in mutant state in diverse tumors, cancer cells easily circumvent this scrutiny (Liu and Xu 2011). It has also been observed that oxidative stress depletes the expression of ARID1A (AT-rich interaction domain 1A) in ovarian cancer by methylation of its promoter. It is to be noted that ARID1A, which is a subunit of chromatin remodeling complex BAF (BRG1/BRM-associated factor), is necessary for activation of ATR-led DDR. Again, phosphorylated ARID1A is ubiquitinated by β-TrCP (an E3 ubiquitin ligase) on being triggered by oxidative stress-inactivated ATM in gastric cancer cells (Jiang et al. 2019).

Therapeutic Strategies to Mitigate Genomic Instability in Cancer Progression From the above sections, it is evident that genomic instability is the fundamental basis of cancer (Fig. 3). Therefore, targeting it might be a lucrative intervention strategy. One of the possible strategies to avoid the causation of genomic instability

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Fig. 3 Role of oxidative stress in orchestrating genomic instability-associated cancer progression. Oxidative stress can induce DNA damage by forming a range of products obtained through Fenton reaction or intracellular Ca+2. These products can inflict structural/functional alterations in DNA via chromosomal instability, microsatellite instability, epigenetic modifications, and/or oncogenic replication stress. These modifications can lead to genomic instability, subsequently aiding in cancer initiation and progression. These alterations might also be facilitated by ROS-induced inactivation of ROS-sensitive DDR factors, thereby inhibiting the functions of the DNA damage response machinery. Incompetent DDR eventually leads to genomic instability

is to prevent or ameliorate oxidative stress. This emphasizes on the use of therapeutics that can mitigate oxidative stress, thereby ensuring that no damage to DNA is incurred. The other strategy, conversely, is to increase oxidative stress to such a massive scale that the genomic content of the cancer cells is comprehensively degraded, leading to cell death.

Amelioration of Oxidative Stress to Prohibit Establishment of Genomic Instability Several cell-, animal-, and human trial-based studies have probed into the role of potential therapeutic agents that can decrease oxidative stress, thereby prohibiting DNA damage, which otherwise could have manifested into genomic instability. For instance, β-cryptoxanthin can induce BER of 8-oxodG in HeLa and Caco2

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cells. Selenium can protect the genome against chromosomal breaks and telomere shortening. Dietary antioxidants have also shown promising results in safeguarding genomic stability by scavenging ROS. For example, vitamin A can quench 1O2, while vitamin C can react with •OH and convert it into water. Vitamin E, on the other hand, inhibits lipid peroxidation. Various NOX inhibitors, such as diphenyleneiodonium chloride (DPI), fulvene-5, celastrol, imipramine blue, GKT136901, and GKT137831 have also been reported to prevent excessive ROS formation. Nanoparticles, such as cerium oxide, fullerene, platinum, mesoporous silica, either independently or in combination with chemotherapeutic drugs, can scavenge free radicals and subsequently assuage tumor growth and proliferation (Morry et al. 2017). Again, N-acetyl cysteine, curcumin and eugenol can curb MSI in cancer cells (Wu et al. 2017). Administration of 5-fluorouracil to colon cancer (lacking p53) reportedly promotes centrosome amplification. Such cells could be chemosensitized to apoptosis by treatment with the antioxidant resveratrol. In colorectal cancer cells, administration of aspirin can quench •OH and reduce the cancer progression.

Increasing Oxidative Stress to Kill Cancer Cells Via Comprehensive Genomic Degradation While alterations in the genome brought about by oxidative damage lay the foundation for cancer cells to flourish, there exists a threshold till which the damages are beneficial and sustainable for the cancer cells. Excessive oxidative stress can inflict tremendously amplified assaults to the DNA – to such an extent that the degree of genomic instability surpasses the limits of genomic integrity. This causes comprehensive genomic degradation, which results in the cancer cells to succumb (Andor et al. 2017). Thus, it is judicious to exploit this “savior-turned-slayer” status of oxidative stress in cancer cells. This paves a path for a potential therapeutic strategy involving the use of agents that can increase ROS levels beyond the “tolerance limit” in cancer cells. Over the years, a colossal number of studies have employed different natural/synthetic/semi-synthetic derivatives that can successfully obliterate cancer cells by augmenting the degree of oxidative stress. It is practically impossible to enlist all the agents that have been reported till date in a single book chapter. Table 1 enlists some of the common agents that have shown promising anticancer effects via increased oxidative stress. In addition to the ones listed in Table 1, several conventional chemotherapeutics also fit the stated strategy. For example, “oxidation therapy” employs the use of therapeutics, such as cisplatin, mitomycin C, camptothecin, and doxorubicin, to induce ROS-dependent apoptosis in a wide range of cancers. Vinca alkaloids promote ROS formation and downregulate Mcl-1 in a JNK-dependent fashion, subsequently leading to mitochondrial dysfunction-associated apoptosis. Oxidative stress generated due to cisplatin administration increases lipid peroxidation, as well as oxidation of proteins. Such modified molecules contribute to more ROS that

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Table 1 List of agents that increase ROS-mediated DNA damage by critically increasing genomic instability, and thus causing cancer cell death Therapeutic ATN-224

Cancer type Non-small cell lung cancer (NSCLC)

Study model A549 cells

Butein

Mouse neuroblastoma

Neuro-2A

Cribrostatin 6

Breast, cervical, melanoma, lymphoma, leukemia Leukemia

MCF-7, HeLa, SK-MEL-5, U-937, HL-60 cells HL-60

D,Lsulforaphane

Leukemia

HL-60, K562 cells

Gallic acid

Prostrate

LNCaP cells

Parthenolide analogs

Breast, leukemia

Phx-3

Mouse melanoma

Cyanidin

Lung

B16 cells, C56BL/6 mice A549 cells

Romidepsin

Leukemia, colon

HL-60, Caco-2 cells BJAB cells HL-60 cells

Rotenone

B-cell lymphoma Promyelocytic leukemia

Silibinin

Breast

MCF-7 cells

Tanshinone

Cervical, colon, liver

Hela, Colo-205, Hep-2 cells

Mode of action Increases H2O2 by inhibiting SOD1 leading to activation of p38 MAPK, that in turn reduces antiapoptotic Mcl-1 pushing the cells to apoptosis Increases ROS, decreases Bcl-2/Bax leading to cleavage of poly (ADP-ribose) polymerase (PARP) followed by apoptosis Reduces HO1 (antioxidant) expression, increases ROS subsequently promoting apoptosis Induces ROS-mediated p38 MAPK and JNK activation that activates Bim-mediated apoptosis Increases 4-hydroxynonenal (lipid peroxidation product) leading to G2/M arrest and apoptosis Gets auto-oxidized leading to increased ROS, loss of mitochondrial membrane potential, cytochrome c release followed by apoptosis Induces oxidative stress, activates p53 and inhibits NFκB/STAT3, reduces GSH leading to G2/M arrest followed by apoptosis Upregulates expression of Fas and induces apoptosis Reduces intracellular pH promoting mitochondrial dysfunction-led increased ROS production, nuclear translocation of NFκB to trigger apoptosis Promotes H2O2 generation leading to apoptosis Binds and inhibits complex I of ETC generating massive amount of ROS, leading to DNA fragmentation and apoptosis induction Induces apoptosis through increased NO• Interacts with DNA and produces ROS in close proximity to the bases, thereby inflicting DNA damage

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ultimately promote apoptosis. Tetrathiomolybdate was seen to inhibit Cu/Zn-SOD, thereby driving the cancer cells to excess ROS-associated apoptosis. Doxorubicin was reported to inflict direct DNA damage by activating PARP and NAD(P)H oxidase to produce H2O2 (Barrera 2012). Use of methotrexate can increase oxidized nucleotides in MutS homolog 2 (MSH2, an MMR gene)-deficient metastatic colon cancer cells. This consequently drives them to apoptosis due to accumulation of excessive number of damaged bases. Platinum-based chemotherapy has also shown positive results especially in cancers harboring loss of one or more DDR genes. For example, in non-small cell lung cancer (NSCLS), cisplatin treatment leads to the formation of cisplatin-DNA adducts that can eventually cause apoptosis (Martin et al. 2010). Several combinatorial therapies aimed at more effectively killing cancer cells have emerged over the years. Combinations of DDR inhibitors along with conventional chemotherapeutics/radiation therapy are used to attain enhanced efficacy. For example, in irradiated NSCLC cells, combination of olaparib (inhibitor of PARP) and cisplatin can activate NOX and lead to massive amounts of ROS. Similarly, carboplatin along with AZD1775 (Wee kinase inhibitor) amplify oxidative stress driving cancer cells to death. In pancreatic cancer patients, UCN-01 and 5-fluorouracil can sensitize gemcitabine-refractory cases by generating ROS-associated DNA injury. Again, combination of ABT-888 (inhibitor of PARP) and auranofin (inhibits thioredoxin) in mantle cell lymphoma can massively increase oxidative stress leading to downregulation of phosphorylated Chk1, that in turn, can induce apoptosis (Davalli et al. 2018). While the combinations change, the objective continues to remain to be able to sensitize cancer cells to a therapy that can effectively cause genomic degradation-induced death of cancer cells by massively escalating oxidative stress.

Conclusion Cancer has intrigued researchers for more than two centuries owing to the vast gamut of molecules/signaling cascades and their interplays that orchestrate its etiology. Genomic instability finds a superlative position due to its role in aiding tumorigenesis. Oxidative stress is the chief contributor to this hallmark. It thus becomes imperative to understand the myriad strategies through which oxidative stress inflicts DNA damage. Apropos to cancer inception, the accrual of oxidative injuries triggers the onset of DNA damage-associated instabilities, compounded with faulty cellular repair/surveillance systems, thus favoring initiation and progression of cancer cells. Therefore, understanding the precise mechanisms via which oxidative stress orchestrates genomic instability as a modality of cancer progression is of prominent significance. This will not only endow us with holistic understanding of cancerspecific molecular targets, but will also provide a road map for developing efficacious cancer intervention strategies.

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Hypoxic Stress Perturb DNA Repair Mechanisms Leading to Genetic Instability

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Goutham Hassan Venkatesh

Contents Introduction: The Hypoxic Tumor Microenvironment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Hypoxia and Reoxygenation Induces Oxidative Stress Through Reactive Oxygen Species (ROS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Hypoxia and the Activation of DNA Damage Response . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Downregulation of DNA Repair Pathways Under Hypoxia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Base Excision Repair (BER) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Nucleotide Excision Repair (NER) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mismatch Repair (MMR) Pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Homology-Directed Repair (HDR) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Non-Homologous End Joining (NHEJ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Fanconi Anemia (FA) Pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Hypoxia Induced Replication Stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Hypoxia Induced Genetic Instability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract

Hypoxia contributes to the growth and development of the tumors by facilitating the formation of stem cells, epithelial to mesenchymal transition, heterogeneity, and immune escape. Hypoxia inducible factors (HIF-1α and HIF-1β) regulate several mechanisms like cell survival, proliferation, metabolism, pH regulation, and angiogenesis in the tumor microenvironment. Currently, we understand that genomic instability leads to accumulation of mutations and contributes to heterogeneity and evolution, metastases, and resistance to treatment. Hypoxia contributes to genomic instability in several ways. Tumor hypoxia downregulate the DNA damage and repair pathways at transcriptional and translational level. Hypoxia induced reduction G. H. Venkatesh (*) Thumbay Research Institute for Precision Medicine, Gulf Medical University, Ajman, United Arab Emirates e-mail: [email protected] © Springer Nature Singapore Pte Ltd. 2022 S. Chakraborti et al. (eds.), Handbook of Oxidative Stress in Cancer: Mechanistic Aspects, https://doi.org/10.1007/978-981-15-9411-3_51

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in nucleotide availability can lead to replication stress. In addition, hypoxia exerts selectional pressure on genetically transformed cells and is one of the causative factor for genetic heterogeneity. Hence understanding the functionality of DNA damage response and repair mechanisms play an important role in deciphering the key properties of tumor development under hypoxic conditions. In this chapter, we discuss about the influence of hypoxia on the DNA damage response and the key DNA repair pathways, oxidative stress, replication control, and plethora of genetic changes that arise in the cellular model systems. Keywords

Hypoxia · DNA damage response · Oxidative stress · Replication stress

Introduction: The Hypoxic Tumor Microenvironment The tumor microenvironment is broadly composed of two major components, viz., cellular factors and chemical factors. The cellular tumor microenvironment is a hodgepodge of cancer cells, fibroblasts, immune cells, blood, and lymphatic cells. The chemical factors like metabolites (glucose, lactate, etc.), oxygen levels, pH, and other small molecules (e.g., NO, chemokines, etc.) play an important role in the tumor development (Chouaib et al. 2017). The spatiotemporal interaction of these cells determines the tumor development and treatment outcome. As a consequence of accelerated tumor growth, there is reduced supply of oxygen in the regions that are distant from blood vessels, thus forming a hypoxic region. Further, three types of hypoxia exist in the tumor microenvironment, namely, acute or shorter durations of hypoxia, chronic or permanent hypoxia, characterized by longer duration of hypoxic conditions, and intermittent hypoxia characterized by transient and fluctuating hypoxic conditions and complete anoxia with no oxygen supply in the regions which are up to 150 μM away from the nearest blood vessel (Scanlon and Glazer 2015). Estimates have shown that approximately 50% of solid tumors experience hypoxia and/or anoxia. Through genetic and adaptive changes, hypoxic cancer cells acquire the ability to survive under insufficient oxygen. Hypoxia is responsible for inducing intratumoral heterogeneity and can promote stemness, altered metabolism, genetic instability, angiogenesis, and metastases. Data from clinical studies suggest that tumor hypoxia is associated with tumor resistance to radiation therapy as well as chemotherapy and shows poor prognosis in several cancer types (Dewhirst et al. 2008). Hypoxia inducible factors (HIFs) are the key transcription factors responsible for signaling in anoxic conditions and regulate several genes related to glucose metabolism, angiogenesis, cell cycle, and apoptosis. HIF is a heterodimer composed of one hypoxia induced alpha sub-unit (HIF-1α, HIF-2α, HIF-3α) and a constitutively expressed beta sub-unit (HIF-β) (Petrova et al. 2018). Under normoxic conditions, the alpha subunit undergoes proteosomal degradation by the tumor suppressor von-Hippel-Lindau (VHL) in an oxygen-dependent manner. Enzymes with prolyl4-hydroxylase domain (PHD) utilize oxygen and mediate the VHL protein binding and degradation. Of all the 3 alpha subunit, HIF-1α is one of the highly studied

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protein with respect to its functional significance in tumorigenesis. Under hypoxic conditions, the alpha (HIF-1, -2, -3) subunit translocates to the nucleus and dimerize with the beta subunit. The stable heterodimer then binds to regulatory region (also called Hypoxia Responsive Elements) of HIF target genes and induces gene expression upon binding to its cofactors – p300 and cAMP response element-binding protein (CBP). HIF-1 expression has been associated with poor response to treatment in breast, colorectal, cervical, glioblastoma, head and neck, nasopharyngeal, osteosarcoma, and pancreatic carcinomas. Similarly, HIF-2 expression correlates with poor response in melanoma, colorectal, ovarian, and lung carcinomas (Murphy 2012; Semenza 2012; Petrova et al. 2018).

Hypoxia and Reoxygenation Induces Oxidative Stress Through Reactive Oxygen Species (ROS) The generation of ROS in the cells happens through NADPH oxidases in the plasma membrane, formation of di-sulfide bonds in the endoplasmic reticulum, and oxidation process in mitochondria. The role of reactive oxygen species (ROS) in controlling the HIF-α has been a subject of controversy. Several reports demonstrate the increase in intracellular ROS levels under hypoxia (Kondoh et al. 2013; Azimi et al. 2017). However, contradictory reports also exist about the downregulation of ROS under hypoxic conditions (Sgarbi et al. 2018). It is hypothesized that the mitochondrial electron transport system is the source of ROS under hypoxic conditions. However, much importance has been given to the production of ROS during reoxygenation with the involvement of all three cellular mechanisms (Koritzinsky and Wouters 2013). Reoxygenation after hypoxic phase results in sudden burst of free radicals in the tumor microenvironment. The increase in ROS and its regulation of HIF-α was established under oxygenated conditions. ROS plays an important role in regulating the HIF-α levels and mimics hypoxic regulation of HIF-α. Similar to hypoxia, ROS mediates HIF-α stability by affecting the hydroxylation and pVHL binding. The availability of Fe(II) is very important for the hydroxylation of HIF-α under oxygenated conditions and increased ROS results in oxidation of Fe(II) to Fe(III). Further, intracellular ROS can also increase the transcription of HIF-1α through the involvement of phosphatidylinositol 3-kinase (PI3K)-Akt, protein kinase C (PKC), and histone deacetylase (HDAC) (Koshikawa et al. 2009). Extreme hypoxia followed by reoxygenation can cause the activation of DNA damage response mechanisms and also induce cell cycle arrest (Kim et al. 2007). Reoxygenation during cycling hypoxia may induce varied cellular responses in comparison to chronic hypoxic conditions and can also induce different transcriptomic programs (Michiels et al. 2016).

Hypoxia and the Activation of DNA Damage Response DNA damage response (DDR) is a downstream signaling cascade responsible for sensing the DNA damage and activation of appropriate repair pathway. Essentially, they play a major role in maintaining the genome integrity (Jackson and Bartek

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2009). One of the earliest studies representing p53 stabilization and activation in response to hypoxia provided the evidence for the involvement of DNA damage response (Graeber et al. 1996). Under severe hypoxia (0.1% O2), there is accumulation of single stranded DNA due to nucleotide deprivation and leads to replication fork stalling. These single stranded fragments are coated with Replication protein A (RPA) and acts as a signal for DDR. Both, Ataxia Telangiectasia Mutated (ATM), and ATM and Rad3-related (ATR) signaling mechanisms are activated due to replication stress under hypoxic conditions. Severe and longterm hypoxia-induced p53 initiates apoptosis as against cell cycle arrest. Another interesting thing to note is that hypoxia as such, does not induce any DNA damages that can activate DDR. Evidence for this came from elegant showing the absence of DNA damages under hypoxia using detection methods like alkaline comet assay, pulsed field gel electrophoresis and γH2AX/53BP1 foci analysis (Bencokova et al. 2009; Olcina et al. 2013). However, there was pan-nuclear distribution of γH2AX in response to hypoxia (Bencokova et al. 2009). ATM has been shown to be phosphorylated in response to extreme hypoxia (Pires et al. 2010a). Further, reoxygenation after hypoxia leads to accumulation of Reactive Oxygen Species (ROS), in turn resulting in detectable genomic damage. This leads to activation of ATM-dependent DDR. Also, reoxygenation after hypoxia is known to induce the activation of DNA damage response and cell cycle arrest through phosphorylation in p53 (Ser15), Checkpoint Kinase 1 (Chk 1 – Ser345), and Chk2 (Thr68) (Kim et al. 2007). Importantly, direct link for the interaction of HIF-1 and the DDR has been shown in several studies (Cam et al. 2010; Pires et al. 2012; Fallone et al. 2013). Even under mild hypoxic conditions (0.2–1% O2), ATM can phosphorylate HIF-1α and came stimulate REDD1, eventually down-regulating the mTORC signaling pathway (Cam et al. 2010). Also, it was shown that ATR inhibitors can delay the stabilization of HIF-1α under hypoxic conditions (Pires et al. 2012). It was shown that HIF-1 translation is dependent on ATR kinase activity (PMID) (Fallone et al. 2013). Altogether, these findings demonstrate the activation of sensors (RPA), transducers (ATM and ATR), and effector (γH2AX/53BP1 and p53) molecules under hypoxic conditions.

Downregulation of DNA Repair Pathways Under Hypoxia Hypoxia has been shown to downregulate DNA repair pathways and is a principal cause for genetic instability in tumors (Fig. 1). A few DNA repair genes also carry the hypoxia responsive element making them the direct target of HIF-1α (Filippi et al. 2008; Scanlon and Glazer 2015). Six different DNA repair pathways like base excision repair, nucleotide excision repair, mismatch repair, Fanconi anemia pathway, homologous recombination, and non-homologous end joining exist in mammalians. Although, the presence of DNA damage under hypoxic conditions has not been evidenced by the common DNA repair assays (Filippi et al. 2008), reoxygenation after hypoxia leads to accumulation of DNA damage and activates DNA repair pathways (Kim et al. 2007; Pires et al. 2010b).

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Fig. 1 Hypoxia leads to downregulation of DNA repair pathways at epigenetic, transcription, and translational levels. Through selectional pressure, hypoxia increases the susceptibility for acquiring genetic changes and induces genetic heterogeneity in tumors

Base Excision Repair (BER) BER plays an important role in repairing endogenous oxidative, alkylated and deaminated base damages (Wallace 2014). Many BER factors like 8-Oxoguanine glycosylase (OGG1), MUTYH glycosylase (MUTYH), DNA polymerase beta (POLB), Apurinic/apyrimidinic (AP) endonuclease 1 (APE1), Replication Protein A (RPA), and Proliferating cell nuclear antigen (PCNA) has been downregulated after prolonged hypoxic exposure (Chan et al. 2014). Hypoxia also induces a functional decrease in BER activity (Chan et al. 2014). A recent retrospective study demonstrated that high hypoxia score (measured from 52 hypoxia responding genes) displayed increased expression in the BER gene Nei Endonuclease VIII-Like 3 (NEIL3) and was associated with poor survival rates (Chang and Lai 2019). However, further studies confirming the effect of hypoxia on BER pathway would be necessary to confirm the findings.

Nucleotide Excision Repair (NER) NER is mainly responsible for the repair of bulky adducts, DNA crosslinks and ultraviolet (UV) radiation induced damages. In mammals, NER acts by two different mechanisms, viz., transcription coupled repair and global genome repair. The global genome repair mechanism functions through any region of the genome and requires XPC-RAD23B for the initiation. As the name suggests, transcription coupled repair functions only at the transcription site and is initiated by RNA polymerase along with Cockayne Syndrome A (CSA), CSB, and XPA Binding Protein-2 (Schärer 2013). The effect of hypoxia on NER repair and the expression of NER proteins has been explained in several studies. Earlier studies demonstrated that the reactivation of plasmids after UV exposure was inhibited by hypoxia and there was elevated

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levels of UV mutagenesis (Yuan et al. 2000). Similar findings demonstrated that hypoxia had a delayed effect on the repair of UV damaged DNA in fibroblast cells as measured by host cell reactivation assay (Dregoesc and Rainbow 2009). However, contradictory reports exist about the increased activity of NER under hypoxia (Madan et al. 2012). Seminal studies demonstrated that HIF-1α regulated the expression of NER proteins – Xeroderma Pigmentosum B (XPB), Xeroderma Pigmentosum G (XPG), Excision Repair Cross-Complementing 8 (ERCC8), and CSB (Filippi et al. 2008; Rezvani et al. 2010). However, hypoxia did not induce any change in the expression of NER proteins (Yuan et al. 2000; Bindra et al. 2004). The current data regarding the role of hypoxia on the NER pathway remains inconclusive and warrants further studies.

Mismatch Repair (MMR) Pathway Mismatch repair has an important role in correcting the errors formed during DNA replication and also to avoid recombination events between two distinct sequences (Harfe and Jinks-Robertson 2000; Kunkel and Erie 2015). Hypoxia affects MMR pathway through transcriptional and epigenetic downregulation of MMR genes. Initial studies by Mihaylova and coworkers demonstrated that MutL Homolog 1 (MLH1) and Postmeiotic Segregation Increased 2 (PMS2) were significantly downregulated under hypoxic conditions leading to the hypermutability of reporter genes (Mihaylova et al. 2003). Another study reported the downregulation of MutS Homolog 2 (MSH2) and MutS Homolog 6 (MSH6) under hypoxic conditions through HIF-1α-MYC pathway (Koshiji et al. 2005). The MYC-dependent regulation of MSH2 and MSH6 under normoxia was replaced by HIF-1α-dependent regulation under hypoxic conditions (Nakamura et al. 2008; Yoo et al. 2011). Also, hypoxia leads to downregulation of MLH1, through transcriptional factors – DEC1 and DEC2 (Nakamura et al. 2008). Further, the transient repression of MSH1 and MLH2 by change in promoter binding of the complex c-Myc/Max to Mnt/Max and Mad1/Max, respectively (Bindra and Glazer 2007a). Further, studying the protein expression in mouse models also demonstrated the downregulation of MSH2 and MLH1 under hypoxic conditions (Shahrzad et al. 2005; Edwards et al. 2009; Lu et al. 2014). Also, miRNA-mediated downregulation of MMR genes and epigenetic repression of MMR genes under hypoxic conditions has been characterized (Rodríguez-Jiménez et al. 2008; Valeri et al. 2010).

Homology-Directed Repair (HDR) The repair of double strand breaks formed during S and G2 phases of the cell cycle are repaired through homologous sequence directed repair process and thus represents an error-free mechanism of repair (Aparicio et al. 2014). The downregulation of HDR process by hypoxia happens at transcriptional, translational, and epigenetic mechanisms. Studies using quantitative PCR has shown a decrease in RNA expression of several HDR factors (Meng et al. 2005; Fanale et al. 2013). However,

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microarray analysis has specifically shown the downregulation of RAD51 (RAD51 Recombinase) and Breast cancer type 1 susceptibility protein (BRCA1) (Bindra et al. 2004). Under hypoxia, HIF-1α as a transcription factor competes with MYC and repress the expression of BRCA1 and NBS (Koshiji et al. 2005; To et al. 2006). The transcriptional repression of BRCA1 and RAD51 is mediated through E2F4/p300 complex binding at the promoter site of these genes (Bindra et al. 2005; Bindra and Glazer 2007b). The expression of several key proteins of HDR like BRCA1, BRCA2, Fanconi Anemia Complementation D2 (FANCD2), RAD51 (all sub types), RAD54, and XRCC3 goes-down under hypoxic conditions (Chan et al. 2008). MicroRNAs (miR-210 and miR-373) expressed during hypoxia can suppress the expression of HR proteins (RAD52 and RAD23) (Crosby et al. 2009). Shuttle vector-based DSB analysis has also shown that the HDR activity is functionally compromised under hypoxic conditions (Bindra et al. 2004). All these studies clearly point out the impact of hypoxia on downregulation of some HDR genes with functional decrease in the repair process.

Non-Homologous End Joining (NHEJ) Mammalian cells possess an alternate DSB repair process which is active during the G0/G1 phase of the cell cycle. This repair mechanism is called NHEJ and does not depend on a homologous sequence, and hence, the error rate can be higher (Chang et al. 2017). The effect of hypoxia on the regulation of NHEJ has remained inconclusive. It was shown that hypoxia induced a decrease in RNA expression levels for XRCC6 (Ku70), DNA Protein Kinase (DNA-PK), DNA Ligase IV, XRCC4 in prostate cancer cells (Meng et al. 2005; Fanale et al. 2013). Although, studies have shown the downregulation of NHEJ components, there is no reduction in expression at protein levels. For instance, there is no change in the expression of Ku70 even in hypoxic conditions (Meng et al. 2005). On the contrary, a study also demonstrated that DNA-PK gets auto-phosphorylated at Ser2056 and is activated in mild hypoxia (Bouquet et al. 2011). Also, HIF-1 mediated upregulation of NHEJ factors PRKDC and XRCC6 has been recorded (Um et al. 2004). In contrast, an analysis from a small cohort of cervical carcinomas displayed reduced expression of Ku70/Ku80 in hypoxic regions (Lara et al. 2008). Contradictory reports also exist about the NHEJ activity under hypoxic conditions. One study has demonstrated that hypoxic cells display increased tendency to acquire chromosomal instability due to decreased NHEJ activity (Kumareswaran et al. 2012). Conversely, an enhanced NHEJ activity under the influence of hypoxia has also been reported (Bindra et al. 2005; Madan et al. 2012). Further studies would be required to conclusively determine the effect of hypoxia on NHEJ pathway.

Fanconi Anemia (FA) Pathway FA pathway plays critical role in the removal of inter-strand crosslinks that hinders replication and transcription process (Ceccaldi et al. 2016). FANC genes have

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important roles in other DNA repair pathways like HR and NER (Kelsall et al. 2012; Michl et al. 2016). Mutation in any of the 21 FANC genes can lead to genomic instability and cancer predisposition (Sumpter and Levine 2017). The knowledge about the effect of hypoxia on the FA proteins and functional activity is very little. Earlier studies demonstrated that fibroblasts deficient of FANCD2 showed extreme radiosensitivity under hypoxic conditions in comparison to normoxia (Kuhnert et al. 2009). Further, Ramaekers and colleagues showed that hypoxia downregulated FA pathway and renders the cells sensitive to inter-strand crosslink inducing agent (Mitomycin) (Ramaekers et al. 2011). Prolonged exposure to hypoxia can downregulate the FANCD2 protein expression and can contribute to genomic instability (Scanlon and Glazer 2014).

Hypoxia Induced Replication Stress DNA replication ensures the correct duplication of genome and plays a major role in maintaining the stability of the genome. Any delay in the timing of initiation of replication fork and as well as progression of replication fork can have severe consequences on the genome stability (Donley 2013; Zeman and Cimprich 2014). Replication stress is the slowing or halting of replication fork with stretches of long single stranded DNA (ssDNA). These long stretches of ssDNA are stabilized by the binding of Replication Protein A (RPA) and activation of ATR-dependent replication stress response. ATR eventually ensures the restart of replication forks by accumulating histones, deoxyribonucleotides (dNTPs), and polymerases and also activates cell-cycle checkpoints to prevent the cell cycle progression. Replication fork progression can be delayed or completely stalled due to DNA damages, repetitive DNA sequences, reduced nucleotide availability, fragile sites, ribonucleotide incorporation, and transcription process (Zeman and Cimprich 2014). If the stalled forks fail to restart, then double strand breaks can be formed due to fork breakage or through the breakage of ssDNA by nucleases like Mus81. The compromise in replication under hypoxic conditions was evidenced by decrease in 5-bromo-20 -deoxyuridine incorporation assay (Hammond et al. 2002). Hypoxia treatment also resulted in phosphorylation of RPA (serine 33), γ-H2AX and CHK-1 (serine 345), and replication fork structures evidenced through DNA fiber analysis (Pires et al. 2010a; Forskolin et al. 2016). Hypoxia led to an increase in number of stalled forks, slower replication rate at the active fork sites, and also decrease in new origin firing (Bencokova et al. 2009; Pires et al. 2010a). Even in the absence of DNA damage, ATM activation was noticed in hypoxic cells indicating the activation of DNA damage response (Foskolou et al. 2016). Shorter durations of severe hypoxia of 0.1% and eventual reoxygenation led to the restart of replication fork. However no replication restart process was noticed in cells treated with hypoxia for longer durations and the collapse in replication forks were noted (Pires et al. 2010a; Pires et al. 2010b). The functional activity of the enzyme – Ribonucleotide Reductase (responsible for the biosynthesis of dNTPs) is reduced under hypoxia (Foskolou et al. 2017). Hence under hypoxic conditions, the

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availability of dNTPs for the DNA synthesis is greatly reduced (Pires et al. 2010b). Altogether, hypoxia contributes to genomic instability through increased replication stress.

Hypoxia Induced Genetic Instability Genetic instability is the tendency of the cells to acquire mutations with each cell division. Cells maintain their genome integrity by activating several surveillance mechanisms like DNA damage response, DNA repair, and mitotic checkpoints. Genetic instability contributes to genetic heterogeneity and clonal segregation of tumors (Negrini et al. 2010). Duplication of the entire genome due to cell fusion defects or due to metaphase or anaphase failure can cause euploidy conditions (Burrell et al. 2013). Major reasons for the loss/gain in a segment of the genome or even whole chromosomes can be due to defects in DNA replication, mitotic defects, telomere dysfunction, and replication stress. Microsatellites are short tandem repeats in DNA of 1–6 nucleotides and represent 3% of our genome and are highly susceptible to mutation (Gadgil et al. 2017). Microsatellites are the hotspots for replication fork stalling and eventual double strand break formation. All these changes can arise under hypoxia due to loss of DNA repair activity as well as increased accumulation of mutations due to DNA repair defects. Hypoxia-induced replication stress can also cause genomic instability through replication fork stalling at microsatellite regions. Several studies report the hypoxia-induced increase in mutation rate through reporter gene assays, locus-specific assays, and through chromosomal analysis (Table 1). An important feature of hypoxic stress is to induce selection pressure on genetically transformed cells, thereby causing the proliferation of mutant clones within the tumor. Graeber and colleagues demonstrated the hypoxia led to the expansion of p53 mutant clones through reduced apoptotic activity (Graeber et al. 1996). Further, mathematical models were derived to calculate the selection pressure on p53 mutant cells for their growth and survival in comparison to p53 wild-type cells (Thompson and Royds 1999; Gammack et al. 2001). It was also hypothesized that increased genomic instability can also arise due to contextual loss of heterozygosity, wherein one allele carries the mutation in the DNA repair gene and the other allele expression can be downregulated by hypoxia (Chan and Bristow 2010).

Conclusion In summary, the evidence from current literature survey indicates that hypoxia greatly contribute to genomic instability through several mechanisms. One of the important mechanisms is the downregulation of DNA repair mechanisms through epigenetic, transcriptional, and translational process. Another important mechanism of hypoxia induced genomic instability is through induction of replication stress. A plethora of genomic changes like euploidy, structural changes like translocation,

Copy number changes (gene amplifications, deletions of large fragments)

Ring chromosomes, translocations, telomere fusion and anaphase bridges

Fragile sites and double minutes and sister chromatid exchanges

Oxidative base damages and base changes

3

4

5

Type of genetic instability Euploidy

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Sl No 1

Rat pulmonary artery endothelial cells

Fragile site induction and FISH Sister chromatid exchange assay Chromatin immunoprecipitation and PCR

Multicolor FISH

Human fibroblast cell line

GMA32 cell line (Chinese hamster cells) Human lymphocytes

Fluorescent in-situ hybridization (FISH)

Flow cytometry based mutation assay

Flow cytometry for cell cycle analysis and copy number analysis by slot-blot

Assay Chromosome analysis

Human glioblastoma cell line – TX3868

Chinese hamster ovary cells

Cell type BEX-c (human melanoma cell line) SAX-c Chinese hamster ovary cells Increased over-replication and increased frequency of dihydrofolate reductase gene amplifications Frequency of large deletion was higher in hypoxic cells in comparison to normoxia Severe hypoxia induces fragile sites, double minutes and anaphase-bridge-like structures Formation of anaphase bridges, increased incidence of chromatid breaks, ring chromosomes, fragmented DNA and telomeric fusions in hypoxic cells following ionizing radiation exposure Induction of fragile sites and double minutes Gradual increases in sister chromatid exchange Formation of 8-oxoguanine in the HRE regions was higher in hypoxia treated cells

Observed changes in genetic instability 10% of the cells displayed tetraploidy

Coquelle et al. (1998) Lee et al. (2010) Pastukh et al. (2015)

Kumareswaran et al. (2012)

Fischer et al. (2008)

Keysar et al. (2010)

Rice et al. (1986)

Reference Rofstad et al. (1996)

Table 1 The above table illustrates the type of genetic changes, the model system, and the assays used to observe genetic instability under hypoxic conditions

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Microsatellite instability

Point mutations

6

7

Increase in microsatellite instability Upto four-fold increase in point mutational frequency Upto two-fold increase in mutational frequency Hypoxia induces an increase in supFG1 and lacZ mutations Insertions or deletions in poly (CA) repeats in β-gal reporter.

Host cell microsatellite instability assay PCR and DNA sequence analysis cII mutagenicity analysis and DNA sequencing β-Galactosidase and supFG1 mutation assays Host cell replication error assay

RodríguezJiménez et al. (2008)

RodríguezJiménez et al. (2008) Kondo et al. (2001) Reynolds et al. (1996) Papp-Szabó et al. (2005) Mihaylova et al. (2003)

Increase in microsatellite instability

Human colon carcinoma – HCT116 Tumorigenic mouse cell line LN2 Epithelial and fibroblast cells Cervical carcinoma –HeLa, breast carcinoma – EMT6 and fibroblasts Mouse neural and human mesenchymal stem cell

Koshiji et al. (2005)

Hypoxia increases mutational frequency in microsatellite regions

β-Galactosidase mutation assay and microsatellite analysis Sequencing of microsatellite markers

Human Colon carcinoma – HCT116 and endometrial carcinoma – HEC59 Mouse neural and human mesenchymal stem cell

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deletion, duplications, microsatellite instability, fragile site induction, oxidative base damages, and point mutations has been recognized in cells exposed to hypoxia. Further, hypoxia can also be detrimental by inducing selection pressure on transformed cells for their survival, thereby leading to intratumoral heterogeneity. Targeting the DNA damage pathway alone in hypoxic cells might not prove to be effective. Therapeutic strategies that combines hypoxic targeting drugs with irradiation or anti-angiogenic factors can be effective in developing improved chemotherapy regimens.

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DNA Lesions Induced by Lipid Peroxidation Products in Cancer Progression

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Ana Paula de Melo Loureiro

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . DNA Lesions Induced by Reactive Lipid Peroxidation Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Malondialdehyde . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . α,β-Unsaturated Aldehydes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ketoaldehydes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . DNA Lesions from Lipid Peroxidation in Cancer Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cross-References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract

Lipid peroxidation, initiated by reactive oxygen species or enzymes, originates a variety of electrophiles able to modify biomolecules, including DNA. The best studied lipid peroxidation electrophiles are malondialdehyde, 2-alkenals, 4hydroxy-2-alkenals, and 4-oxo-2-alkenals. We provide an overview of the DNA adducts formed by these aldehydes, their mutagenic properties, the mechanisms involved in their removal from DNA, their occurrence in vivo, and the associations between their in vivo formation and the carcinogenesis process. The DNA adducts most extensively quantified in carcinogenesis conditions in vivo are the malondialdehyde adducts and the etheno adducts, primarily the unsubstituted etheno adducts, but there is an upward trend in the exploitation of substituted etheno adducts formed by 4-oxo-alkenals in cancer-prone inflammatory conditions. The development of mass spectrometry–based analytical methods has taken advantage of the high sensitivity and high resolution of modern instruments to provide promising advances in a DNA adductomics approach. This approach will A. P. de Melo Loureiro (*) Departamento de Análises Clínicas e Toxicológicas, Faculdade de Ciências Farmacêuticas, Universidade de São Paulo, São Paulo, Brazil e-mail: [email protected] © Springer Nature Singapore Pte Ltd. 2022 S. Chakraborti et al. (eds.), Handbook of Oxidative Stress in Cancer: Mechanistic Aspects, https://doi.org/10.1007/978-981-15-9411-3_52

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expand our view of the endogenous DNA lesions that may be associated with different phases of carcinogenesis and provide useful biomarkers for assessment of disease risk, preventive and treatment interventions, and for diagnosis and prognosis purposes. There is also a need to investigate the relationships between the occurrence of the lipid peroxidation–derived DNA adducts, mutations in critical genes, altered gene expression, and changes in cell signaling, metabolism, and epigenetics. Examination of the global picture of changes and their interrelationships will improve our understanding of the role of lipid peroxidation– derived DNA adducts in carcinogenesis. Keywords

Lipid peroxidation · α β-unsaturated aldehydes · Bifunctional electrophiles · DNA adducts · Etheno adducts · Propano adducts · Malondialdehyde adducts · 4-oxo-2-alkenals adducts

Introduction Lipid peroxidation is an important chain reaction that is primarily initiated by the oxidation of polyunsaturated fatty acids (PUFAs). Reactive oxygen and nitrogen species (ROS and RNS, respectively), such as the highly reactive hydroxyl (•OH) and nitrogen dioxide (NO2•) free radicals and the nonradical singlet oxygen (1O2) react in different ways with PUFAs to yield a variety of oxidized products (Augusto et al. 2002; Ronsein et al. 2006; Medeiros 2019). Among these products are lipid radicals, lipid peroxyl and alcoxyl radicals, lipid hydroperoxides, and cyclic peroxides that may undergo rearrangement, fragmentation, or oxidation to alkanes, alkenes, and a series of electrophilic carbonyls, such as malondialdehyde (MDA), glyoxal, methylglyoxal, acrolein, crotonaldehyde, trans-2-hexenal, 4-hydroxy-trans-2-hexenal (HHE), 5,8dioxo-(6E)-octenoic acid (DOOE), 4-hydroxy-trans-2-nonenal (HNE), 4hydroperoxy-(2E)-nonenal (HPNE), 4-oxo-(2E)-nonenal (ONE), 4-oxo-(2E)-hexenal (OHE), 2,4-decadienal (DDE), 4,5-epoxy-(2E)-decenal (EDE), 9,12-dioxo-(10E)dodecenoic acid (DODE), isolevuglandins (formerly called isoketals), and A2- and J2-isoprostanes, as illustrated in Fig. 1 (Higdon et al. 2012; Vistoli et al. 2013; Yu et al. 2016; Kawai and Nuka 2018; Medeiros 2019; Davies et al. 2020). PUFAs, such as arachidonic acid, may also undergo enzymatic oxidation by cyclooxygenases (COX-1, COX-2) and lipoxygenases (5-, 12- and 15-LOX) to form prostaglandins, thromboxane A2, leukotrienes, and lipoxins, which interact reversibly with cellular receptors and regulate various physiological processes, including inflammation (Higdon et al. 2012). Among the products of the enzymatic oxidation of arachidonic acid are the electrophilic leukotriene A4, cyclopentenones, 15-deoxyprostaglandin J2, prostaglandin A2, and prostaglandin J2 (Higdon et al. 2012). The enzymatic oxidation of ω-3 fatty acids provides oxidized products with anti-inflammatory properties, including electrophilic oxo-derivatives (Higdon et al. 2012).

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The electrophilic products formed from lipid peroxidation may react with nucleophilic sites in macromolecules and modulate cell function, differentiation, survival, proliferation, or death as part of the development of pathophysiological conditions (Higdon et al. 2012). We focused our attention on the formation of DNA lesions caused by reactive lipid peroxidation products and the role of these lesions in cancer development.

DNA Lesions Induced by Reactive Lipid Peroxidation Products Among the reactive lipid peroxidation products, short-chain reactive carbonyls are the most studied in the induction of DNA lesions. This class of reactive species is comprised of aldehydes that are classified as α,β-unsaturated aldehydes (e.g., acrolein, crotonaldehyde, hexenal, HHE, HNE, HPNE, DDE, and EDE), dialdehydes (e.g., MDA, glyoxal), and ketoaldehydes (e.g., methylglyoxal, OHE, ONE, DODE, DOOE, and isolevuglandins) (Vistoli et al. 2013) (see Fig. 1 for structures). These compounds exhibit different degrees of reactivity towards cellular nucleophiles, including thiols (e.g., cysteine), imidazoles (e.g., histidine) and amino groups in peptides, proteins, nucleic acid bases, or lipids (Medeiros 2019; Davies et al. 2020), which partially modulate the rates at which they induce DNA damage. Reactions with DNA typically involve the exocyclic and/or the ring amino groups of the bases guanine, cytosine, adenine, and, less often, thymine, which does not have an exocyclic amino group, to yield cyclic or acyclic adducts (Medeiros 2019). Brief discussions of the main DNA lesions induced by the most studied aldehydes are provided below.

Malondialdehyde MDA is a major aldehyde that results from the peroxidation of PUFAs with at least three double bonds (ω-3 and ω-6 PUFAs). MDA is also formed during prostaglandin biosynthesis. Its enol tautomer (β-hydroxyacrolein) reacts with deoxyguanosine (dG), deoxyadenosine (dA), and deoxycytidine (dC) to yield the adducts shown in Fig. 2 (Marnett 1994; Loureiro et al. 2002). The reaction occurs between the C-3 of the aldehyde and the exocyclic amino group of the bases, followed by H2O elimination. In the case of guanine, cyclization to the N1 position occurs and forms the pyrimidopurinone 3-(2-deoxy-β-D-erythro-pentafuranosyl)pyrimido-[1,2-α]purine10(3H)-one (M1dG) adduct after the elimination of another H2O molecule (Marnett 1994; Medeiros 2019). The main adduct formed in the reactions of MDA with DNA is M1dG, followed by N6-(3-oxo-1-propenyl)-20 -deoxyadenosine (M1dA) and N4-(3oxo-1-propenyl)-20 -deoxycytidine (M1dC) at much lower levels (Marnett 1999). Ring-opening of M1dG to N2-oxopropenyl-dG occurs when it is positioned opposite to cytosine in double-stranded DNA. This opening provides reactive functional groups within the DNA molecule that may lead to inter- and intra-strand DNADNA cross-links and DNA-protein cross-links (Mao et al. 1999).

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Fig. 1 Some electrophilic carbonyls generated from lipid peroxidation. MDA, malondialdehyde; HHE, 4-hydroxy-trans-2-hexenal; DOOE, 5,8-dioxo-(6E)-octenoic acid; HNE, 4-hydroxy-trans-2nonenal; HPNE, 4-hydroperoxy-(2E)-nonenal; ONE, 4-oxo-(2E)-nonenal; OHE, 4-oxo-(2E)hexenal; DDE, 2,4-decadienal; EDE, 4,5-epoxy-(2E)-decenal; and DODE, 9,12-dioxo-(10E)dodecenoic acid

M1dG is detected in DNA extracted from tissues of human subjects and experimental animals submitted or not to conditions that increase oxidative stress (Wang and Liehr 1995; Nair et al. 2007). M1dG was a mutagenic lesion in double-stranded vectors replicated in E. coli and primarily induced G!A and G!T mutations at an

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Fig. 2 DNA adducts formed by MDA. dR ¼ deoxyribose

approximate total mutation frequency of 18%. M1dG was also a strong block to replication (Fink et al. 1997). Oligodeoxynucleotides containing a single M1dG, either incorporated in reiterated CG-rich sequences (models for microsatellites) or in a nonreiterated sequence, were inserted in a single-stranded vector that was

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replicated in COS-7 simian kidney cells. Mutations opposite the lesion and at nearby sites were observed for both sequence contexts, and frameshift mutations were found in reiterated sequences (VanderVeen et al. 2003). The nucleotide excision repair complex may remove M1dG from DNA (Fink et al. 1997). M1dG was also a substrate for oxidation and repair by the E. coli direct reversal DNA repair enzyme AlkB, which is an α-ketoglutarate/Fe(II)-dependent dioxygenase (Singh et al. 2014). Whether the AlkB mammalian homologues are involved in the repair of this and related DNA lesions in mammalian cells is not known (Singh et al. 2014). Notably, MDA is not the exclusive source of M1dG, or likely M1dA and M1dC, in DNA. The abstraction of the 40 -hydrogen atom of 20 -deoxyribose by oxidants leads to the formation of base propenals (Fig. 3), which are oxopropenyl derivatives of DNA bases and analogues to β-substituted acrolein (Dedon et al. 1998). Base propenals are more reactive with DNA bases than β-hydroxyacrolein (Zhou et al. 2005).

α,β-Unsaturated Aldehydes Many α,β-unsaturated aldehydes are more electrophilic than MDA at physiological pH and may contribute significantly to the induction of DNA damage. These α,βunsaturated aldehydes include 4-hydroxy-trans-2-alkenals and trans-2-alkenals (Burcham 1999). The 4-hydroxy-trans-2-alkenals that induce DNA lesions are primarily represented by HNE and HHE, which are products of ω-6 (linoleic acid, arachidonic acid, γ-linolenic acid) and ω-3 (α-linolenic acid, eicosapentaenoic acid,

Fig. 3 Formation of M1dG by a base propenal. dR ¼ deoxyribose

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docosahexaenoic acid) PUFAs peroxidation, respectively (Kawai and Nuka 2018). HNE is one of the major aldehydes that results from lipid peroxidation (Benedetti et al. 1980). 4-Hydroxy-trans-2-alkenals are highly reactive towards nucleophilic sites (preferentially thiolate residues) due to the presence of three functional groups: the aldehyde at C-1, the double bond between C-2 and C-3, and the hydroxyl group at C-4 (Esterbauer et al. 1991). They induce two main groups of lesions in reactions with DNA, the propano and etheno adducts (Figs. 4 and 5). Several trans-2-alkenals also induce the formation of propano or etheno adducts. For example, acrolein, crotonaldehyde, and hexenal yield propano or etheno adducts in reactions with DNA bases (Chung et al. 1996), and HPNE, DDE, and EDE form etheno adducts (Loureiro et al. 2000; Lee et al. 2005a; Blair 2008).

Propano Adducts Propano adducts are formed by the Michael addition of the exocyclic amino group of dG, dA, or dC to C-3 of the aldehyde, followed by ring closure via nucleophilic attack of the heterocyclic nitrogen of the base at the aldehyde C-1 (Fig. 4). Mixtures of diastereomers are formed (Minko et al. 2009). The Michael addition and ring

Fig. 4 Propano adducts formed by reactions of α,β-unsaturated aldehydes with dG. dR deoxyribose

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Fig. 5 Etheno adducts formed by reactions of oxidized α,β-unsaturated aldehydes with dA, dG, and dC. dR ¼ deoxyribose

closure may also occur in the opposite direction, as observed for the reactions of acrolein with dG (Minko et al. 2009). In this reaction, a new saturated ring containing three carbon atoms of the aldehyde molecule is added to a DNA base, except thymine due to the absence of the exocyclic amino group. Major propano

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adducts are formed with dG and are primarily 6-substituted, 8-hydroxy-1-N2-propano-dG adducts. The side chain at C-6 varies according to the aldehyde that generates the adduct (Burcham 1999). Ring-opening to the N2-dG aldehydes was detected when the propano adducts were placed opposite cytosine in double-stranded DNA (Huang et al. 2010). The N2dG aldehydes form inter-strand cross-links at rates that depend on the source aldehyde (acrolein > crotonaldehyde > HNE) and the stereochemistry of the propano adduct (Huang et al. 2010). They may also form intra-strand DNA and DNA-protein cross-links (Minko et al. 2009). Regarding the four diastereomeric propano-dG adducts formed by HNE, the ring-opened N2-dG aldehydes exist in equilibrium with cyclic hemiacetals, which predominate and mask the aldehydes, slowing the rate of cross-linking compared to the propano-dG adducts of acrolein and crotonaldehyde (Huang et al. 2010). Acrolein-, crotonaldehyde-, and HNE-derived propano-dG adducts were detected in DNA extracted from tissues of human subjects and experimental animals submitted or not to conditions that increase oxidative stress (reviewed by Nair et al. 2007; Minko et al. 2009). Vector DNAs containing site specifically inserted acrolein-, crotonaldehyde-, and HNE-derived propano-dG adducts were replicated in E. coli and mammalian cells to assess their mutagenic properties (Minko et al. 2009). The site-specific mutations of the propano-dG adducts were primarily G!T tranversions, and the total mutation frequencies varied from less than 1% to 12% depending on the source aldehyde, the adduct stereochemistry, the biological host (mammalian cell line, bacteria), the sequence context, and the vector (single-stranded or double-stranded DNA). Overall, these data indicate that the propano-dG adducts are not strongly miscoding (Minko et al. 2009). The eukaryotic DNA polymerases pol η, pol ι, pol κ, and Rev1/pol ζ catalyze error-free DNA synthesis past the ring-opened lesions. The error-free DNA synthesis past the ring-closed adducts is performed by pol ι and Rev1. Mutagenic bypass of the adducts may also occur via reactions catalyzed by pol η, pol ι, and pol δ (Minko et al. 2009). The propano-dG adducts are substrates for repair by the nucleotide excision repair complex, but the rate of repair is influenced by the adduct stereochemistry (Minko et al. 2009). The two regioisomers of the propano-dG adducts formed by acrolein [6(or α)-hydroxy-1,N2-propano-dG and 8(or γ)-hydroxy-1,N2-propano-dG] are substrates for oxidation and repair by the E. coli direct reversal DNA repair enzyme AlkB (Singh et al. 2014). The processing of the inter-strand cross-links formed by the ring-opened N2-dG aldehydes was also studied in site-specific modified oligodeoxynucleotides. The data showed that the cross-links were processed in mammalian cells with rare generation of mutations. DNA-protein or DNA-peptide cross-links may be more mutagenic than the monoadduct (Minko et al. 2009).

Etheno Adducts The α,β-unsaturated aldehydes may be oxidized to epoxyaldehydes in the presence of hydrogen peroxide, fatty acid hydroperoxides, and other organic peroxides, as

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demonstrated in vitro (Chen and Chung 1996). The autoxidation of α,β-unsaturated aldehydes may also occur, in addition to the suggested epoxidation by cytochrome P450s and prostaglandin synthase enzymes (Chung et al. 1996). The oxidized aldehydes are very reactive towards DNA bases and yield etheno adducts (Chen and Chung 1996; Ham et al. 2000; Loureiro et al. 2000). The exocyclic amino groups of dG, dA, or dC typically attack the epoxyaldehyde C-1, which is followed by ring closure via the nucleophilic attack of the heterocyclic nitrogen of the base at the C-2 (epoxy group) of the epoxyaldehyde. The elimination of H2O yields mixtures of diastereomers of the alkyl-substituted etheno adducts with side chains that vary according to the source aldehydes. Further elimination of the side chain gives the unsubstituted etheno adducts 1,N2-etheno-20 -deoxyguanosine (1,N2-εdG), N2,3-etheno-20 -deoxyguanosine (N2,3-εdG), 1,N6-etheno-20 -deoxyadenosine (1,N6εdA), and 3,N4-etheno-20 -deoxycytidine (3,N4-εdC) (Fig. 5) (Esterbauer et al. 1991; Chung et al. 1996; Ham et al. 2000; Loureiro et al. 2000; Lee et al. 2005a). The etheno adducts are characterized by the addition of a new five-membered unsaturated ring containing two carbon atoms of the aldehyde molecule to a DNA base, except thymine due to the absence of the exocyclic amino group. It has been shown that the unsubstituted etheno adducts present some chemical lability (Martinez et al. 2018 and reviewed studies). Unsubstituted etheno adducts were detected in DNA extracted from tissues of human subjects and experimental animals submitted or not to conditions that increase oxidative stress (Loureiro et al. 2002; Nair et al. 2007; Oliveira et al. 2018; Medeiros 2019). Of the substituted etheno adducts, only the 7-(10 ,20 dihydroxyheptyl)-1,N6-etheno-20 -deoxyadenosine (DHHεdA), which is formed by the reaction of the HNE epoxide (2,3-epoxy-4-hydroxynonanal) with dA, was detected as a background DNA lesion in rodent and human tissues. Its levels were higher than the propano-dG adduct formed by HNE (Fu et al. 2014). The mutagenic properties of the unsubstituted etheno adducts were investigated in site-specific modified oligonucleotides inserted in vector DNAs replicated in E. coli and mammalian cells (reviewed by Loureiro et al. 2002; Yu et al. 2016). 1,N6-εdA induced a very high mutation frequency (70%) in single-stranded vectors replicated in COS7 simian kidney cells, which was comprised of A!G transitions (63%) and A!T (6%) and A!C (1%) transversions, but a marginal mutation frequency was observed in E. coli (0.53% in SOS-induced and less than 0.18% in noninduced cells) (Pandya and Moriya 1996). In another mammalian cell line, HeLa human cervical cancer cells, the targeted mutation frequency of 1,N6-εdA was 14% (7% A!T, 5% A!C, 2% A!G) when present in the leading strand of a double-stranded vector, 10% (5% A!G, 4% A!T, 1% A!C) when in the lagging strand, and 11% (8% A!T, 2% A!G, 1% A!C) when in a single-stranded vector (Levine et al. 2000). The sequence context, location of 1,N6-εdA relative to the replication origin, and the different host cells can account for the observed differences between studies (Levine et al. 2000). The mutation frequency induced by 3,N4-εdC was also very high (81%) in a single-stranded vector replicated in COS7 simian kidney cells, but lower mutation frequencies were observed when the replication occurred in E. coli (32% in SOS-

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induced and 2% in noninduced cells). The mutations observed in COS7 cells were C!A (49.5%), C!T (29%) and C!G (2.5%) (Moriya et al. 1994). N2,3-εdG primarily induced G!A transitions during DNA replication in E. coli, with a total mutation frequency of 13% (Cheng et al. 1991). Mutations induced by 1,N2-εdG were investigated in E. coli (Langouët et al. 1998) and mammalian cells (Akasaka and Guengerich 1999) using double-strand vectors. Mutations in E. coli were observed in approximately 3% of the viral progeny and primarily consisted of G!A transitions, followed by G!T and G!C transversions (Langouët et al. 1998). An intra-chromosomal site-specific mutagenesis system was used to assess the mutation frequency in mammalian cells, which was 4.6%. Sequence analyses of 21 clones derived from the mutant fraction revealed 5 that corresponded to mutations directly at the 1,N2-εdG incorporation site (primarily G!A transitions). The other mutants included deletions, rearrangements, double mutants, and base pair substitutions at sites close to the adduct site (Akasaka and Guengerich 1999). The etheno adducts are excised from DNA by different DNA glycosylases of the base excision repair (BER) pathway (reviewed by Gros et al. 2003). Transcriptioncoupled nucleotide excision repair also contributed to the removal of 3,N4-εdC from DNA (Chaim et al. 2017). 1,N6-εdA, 3,N4-εdC, and 1,N2-εdG are also substrates for AlkB dioxygenases, including the human homologs AlkBH2 and AlkBH3 (reviewed by Fedeles et al. 2015). Besides the aldehydes that result from lipid peroxidation, the carcinogens vinyl chloride and ethyl carbamate react with DNA bases after biotransformation to their respective oxiranes and also yield unsubstituted etheno adducts. Increased levels of the etheno adducts 1,N6-εdA, 3,N4-εdC, and N2,3-εdG were found in DNA extracted from the liver, lung, and kidney of rats exposed to vinyl chloride (reviewed by Yu et al. 2016). Therefore, the unsubstituted etheno adducts cannot be used as specific biomarkers of lipid peroxidation–induced DNA damage.

Ketoaldehydes This group is comprised of bifunctional electrophiles from lipid peroxidation whose importance in the induction of DNA damage was revealed more recently. We focused on the α,β-unsaturated aldehydes that contain an oxo group at the C-4 position (e.g., the 4-oxo-2-alkenals OHE, ONE, DODE, and DOOE). 4-Oxo-2-alkenals (e.g., ONE) originate from the corresponding 4-hydroperoxy2-alkenals (e.g., HPNE), which result from the decomposition of PUFA hydroperoxides during lipid peroxidation. They are also formed from metal-catalyzed autoxidation of the corresponding 2-alkenals, with the formation of 4-hydroperoxy-2alkenals as intermediates (Kawai and Nuka 2018). Interest in the formation of DNA adducts by 4-oxo-2-alkenals has grown since 1999, with the discovery of ONE as the principal breakdown product of the linoleic acid hydroperoxide 13(S)-hydroperoxy9,10-(Z,E)-octadecadienoic acid (13-HPODE) (Rindgen et al. 1999). Vitamin C– mediated decomposition of the arachidonic acid hydroperoxide 15-hydroperoxy-

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5,8,11,13-(Z,Z,Z,E)-eicosatetraenoic acid (15-HPETE) also yields ONE, but in reduced amounts compared to 13-HPODE. Other bifunctional electrophiles produced from the decomposition of 13-HPODE and 15-HPETE are HPNE, trans4,5-epoxy-2(E)-decenal (t-EDE), cis-4,5-epoxy-2(E)-decenal (c-EDE), and HNE (Williams et al. 2005). HPNE is the direct precursor of HNE and ONE (Williams et al. 2006). HPNE and EDE react with DNA bases to yield the unsubstituted etheno adducts discussed above (Lee et al. 2005a). The decomposition of 13-HPODE produces, additionally, DODE (Williams et al. 2005). Decomposition of the arachidonic acid hydroperoxide 5(S)-hydroperoxy-6,8,11,14-(E,Z,Z,Z)-eicosatetraenoic acid (5-HPETE) yields DOOE, ONE, and HPNE (Jian et al. 2005). 13HPODE and 15-HPETE are products of COX-1-, COX-2-, and 15-LOX-mediated oxidation of linoleic acid and arachidonic acid, respectively (Williams et al. 2005). 5-HPETE is the precursor of leukotriene A4, and it is formed by 5-LOX-mediated oxidation of arachidonic acid (Jian et al. 2009) (Fig. 6). This line of studies revealed pathways by which the enzymatic oxidation of PUFAs contributes to the generation of electrophiles able to induce DNA damage. The reaction of ONE directly with dG (Rindgen et al. 1999), dA (Lee et al. 2000), and dC (Pollack et al. 2006) leads to the formation of substituted etheno adducts containing a carbonyl group in the side chain, i.e., the 2-oxo-heptyl-substituted etheno adducts HεdG, HεdA, and HεdC. The reaction of DODE with dG and dC yields the 9-carboxy-2-oxo-nonyl-substituted etheno adducts CNεdG and CNεdC (Lee et al. 2005b), and DOOE reacts with dG to form the 5-carboxy-2-oxo-pentylsubstituted etheno adduct CPεdG (Jian et al. 2005) (Fig. 6). An analogous reaction between 4-oxo-2-pentenal (product of toluene degradation, solvolysis of α-acetoxyN-nitrosopiperidine, and metabolite of 2-methylfuran) and dG was previously described (Hecht et al. 1992). 4-Oxo-2(E)-hexenal (OHE) results from the peroxidation of ω-3 PUFAs and reacts with dC, 5-methyl-dC, dG, and dA to yield the 2-oxo-butyl-substituted etheno adducts BεdC, BεmedC, BεdG, and BεdA, respectively (Kawai et al. 2010). In general, the reaction begins with the nucleophilic addition of the exocyclic amino group of dG, dA, or dC to C-1 of the 4-oxo-2alkenal, which is followed by ring closure via reaction of the heterocyclic nitrogen of the base to C-2 of the 4-oxo-2-alkenal. The elimination of H2O produces mixtures of diastereomers of the oxo-alkyl-substituted etheno adducts (Kawai and Nuka 2018). The substituted etheno adducts formed by ONE and OHE were detected in DNA extracted from tissues of human subjects and experimental animals submitted or not to conditions that increase oxidative stress (Williams et al. 2006; Chou et al. 2010; Kawai et al. 2010). The mutagenic properties of HεdC were investigated in site-specific modified oligonucleotides inserted in vector DNAs replicated in E. coli and human cells (Pollack et al. 2006). HεdC was a strong block to DNA synthesis, which can lead to DNA double-strand breaks and chromosomal aberrations. It was also a highly mutagenic lesion. The mutation frequency observed in E. coli was 40–50% (primarily C!G transversions), and it was more than 90% in human cell lines (primarily C!A transversions and C!T transitions) (Pollack et al. 2006). These types of lesions may be removed from DNA via the base excision repair pathway (Blair 2008).

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Fig. 6 Substituted etheno adducts formed by 4-oxo-2-alkenals. The structures of DOOE, DODE, HPNE, HNE, ONE, and EDE are shown in Fig. 1. The structures of 1,N6-εdA, 3,N4-εdC, and 1,N2εdG are shown in Fig. 5. dR ¼ deoxyribose

DNA Lesions from Lipid Peroxidation in Cancer Development Cancer development occurs via a complex acquisition of alterations that lead to uncontrolled cellular proliferation, tissue invasion, and metastasis (Hanahan and Weinberg 2011). Three distinct phases were proposed in the course of cell malignant transformation: initiation, meaning the occurrence of mutations in normal cells that may sensitize the cells to proliferative signals; promotion, when there is a selective proliferation and clonal expansion of the initiated cells; and progression, when genetic instability is evident, the tumor cells are genetically and phenotypically

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heterogeneous and create their own ways to survive, proliferate, and migrate (Sun 1990). Based on the role of gene mutations in the development of cancer, it is reasonable to consider that mutagenic DNA lesions induced by electrophilic products of lipid peroxidation contribute to the initiation and progression of carcinogenesis. To obtain evidence of this association, we focused on some conditions that predispose individuals to redox stress and the development of cancer, in which DNA lesions induced by lipid peroxidation were investigated. Patients with Wilson’s disease (WD) and primary hemochromatosis present with hepatic copper and iron overload, respectively. These genetic metal storage disorders increase the risk of development of liver cancer (Nair et al. 1998a). The excess hepatic load of transition metal ions accelerates lipid peroxidation. Therefore, etheno adducts were quantified in liver DNA of these patients (Nair et al. 1998a). Increased levels of 1,N6-εdA and 3,N4-εdC were found in the livers of WD and primary hemochromatosis patients compared to liver of healthy subjects (Nair et al. 1998a). Moreover, increased levels of 1,N6-εdA were observed in liver samples of WD and primary hemochromatosis patients and liver biopsies of patients diagnosed with the cancer-prone diseases fatty liver, cirrhosis, and fibrosis, predominantly due to alcohol abuse, which induces hepatic inflammation, redox stress, and increases the risk of liver cancer (Frank et al. 2004). Tumor and non-tumor liver tissues of patients with hepatocellular carcinoma (HCC) were compared to each other and control liver samples of non-HCC patients for the levels of 1,N6-εdA, mutated tumor suppressor protein p53, proliferating cell nuclear antigen (PCNA), total antioxidant capacity (T-AOC), and total superoxide dismutase (SOD) activity (Zhou et al. 2013). 1,N6-εdA levels were higher in HCC compared to non-tumor and control liver tissues, and correlated well with mutated p53 expression. HCC tissues exhibited lower T-AOC and SOD activity than the other tissues, which suggests the occurrence of redox stress. A high cellular proliferation in HCC tissues was demonstrated in the positive rate of PCNA. The amount of 1,N6-εdA was associated with liver inflammation and fibrosis (Zhou et al. 2013). Further evidence relating redox stress with mutations in p53 comes from the investigation of the mutation frequency of the tumor suppressor gene TP53 at codons 249 and 250 in non-tumor liver tissues from WD and hemochromatosis patients compared to normal controls (Hussain et al. 2000a). WD patients exhibited higher frequencies of G:C to T:A transversions at codon 249 and C:G to A:T transversions and C:G to T:A transitions at codon 250, and primary hemochromatosis patients exhibited a higher frequency of G:C to T:A transversions at codon 249 (Hussain et al. 2000a). Inducible nitric oxide synthase (iNOS) was found overexpressed in the livers of 60% of the WD and 28% of hemochromatosis cases, which indicates the occurrence of inflammation and redox stress (Hussain et al. 2000a). Furthermore, the TK-6 lymphoblastoid cell line exposed to HNE presented increased frequencies of TP53 G:C to T:A transversions at the third base (AGG to AGT) of codon 249 (Hussain et al. 2000a). The propano-dG adducts of HNE were preferentially formed at the third base of codon 249 of the TP53 gene, which is a mutational hotspot in human cancers, particularly HCC (Chung et al. 2003). As described in the sections above, C!A and C!T mutations may be induced by 3,N4-εdC (Moriya et al. 1994)

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and HεdC (Pollack et al. 2006), G!A transitions may be induced by N2,3-εdG (Cheng et al. 1991), 1,N2-εdG (Akasaka and Guengerich 1999), and M1dG (Fink et al. 1997), and G!T transversions may be induced by M1dG (Fink et al. 1997) and propano-dG adducts (Minko et al. 2009). However, other DNA lesions induced under oxidative stress conditions may also account for the observed mutations. Chronic inflammation is a risk factor for cancer development. Among the several pathways involved, excessive ROS and RNS generation increases the rate of damage to biomolecules (Bartsch and Nair 2006). iNOS is upregulated or overexpressed in inflamed tissues, which increases the rate of generation of nitric oxide (•NO) (Bartsch and Nair 2006). Several ROS and RNS are generated from reactions of • NO with superoxide radical (O2•-), with the intermediacy of peroxynitrite (Augusto et al. 2002). Among the generated species are highly reactive free radicals that may initiate lipid peroxidation. In a mouse model of •NO overproduction, the levels of 1, N6-εdA and 3,N4-εdC increased approximately sixfold in spleen DNA compared to controls. The etheno adduct levels in spleen DNA of the animals that received the iNOS inhibitor NG-methyl-L-arginine were close to control levels (Nair et al. 1998b), which supports the role of excess •NO in the generation of the etheno adducts. 1,N6-εdA and/or 3,N4-εdC were also increased severalfold in cancerprone organs and urine of humans with chronic inflammatory disorders, such as chronic pancreatitis, Crohn’s disease, ulcerative colitis, H. pylori infection/high salt intake, chronic viral hepatitis, alcohol-related hepatitis, and cirrhosis (reviewed by Bartsch and Nair 2006). Similarly to the cancer-prone liver tissue of WD and primary hemochromatosis patients, ulcerative colitis patients exhibited a high frequency of TP53 mutated alleles (G:C to A:T transitions at the CpG site of codon 248 and C:G to T:A transitions at codon 247) in inflamed lesional regions of noncancerous colon tissue, which may predispose these regions to tumor development (Hussain et al. 2000b). As a mouse model for colorectal cancer, C57DL/6JAPCmin (multiple intestinal neoplasia) mice exhibit an upregulation of COX-2 in the small intestine and increased susceptibility to the development of polyps due to a mutated adenomatous polyposis coli (APC) gene. DNA extracted from the small intestine of these mice showed the formation of HεdG and HεdC. The levels of HεdG and HεdC were 3.1-fold and 10.7fold higher in C57BL/6JAPCmin mice compared to control mice (C57BL/6J), which do not exhibit upregulated COX-2 (Williams et al. 2006). As explained in Section “Ketoaldehydes,” COX-2 oxidizes arachidonic acid to 15-HPETE in the first step of prostaglandin biosynthesis. Under redox stress, 15-HPETE survives long enough to decompose to ONE, which is the bifunctional electrophile that generates HεdG and HεdC (Williams et al. 2005). Colon adenoma tissue of C57BL/6JAPCmin mice also showed increased levels of the malondialdehyde adduct M1dG compared to normal mucosa. Mice treatment with an inhibitor of adenoma growth, curcumin, reduced COX-2 expression and M1dG levels (Tunstall et al. 2006). COX-2 is inducible by numerous factors, such as pro-inflammatory cytokines and growth factors, and it is upregulated in tumor tissues (Williams et al. 2006; Bartsch and Nair 2006). Individuals with inherited familial adenomatous polyposis (FAP) develop multiple polyps with upregulated COX-2 in the intestine and are at high risk of

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development of colon adenocarcinoma (Bartsch and Nair 2006). Treatment of these individuals with nonsteroidal anti-inflammatory drugs (NSAIDs), which are COX inhibitors, decreases polyp number and size (Williams et al. 2006). The etheno adducts 1,N6-εdA and 3,N4-εdC were increased approximately two-fold in DNA from colonic polyps of FAP patients compared to the epithelium of normal colon (Schmid et al. 2000). A correlation was observed between the levels of arachidonic acid metabolites, e.g., the leukotriene precursors 8-hydroxyeicosatetraenoic acid (8-HETE) and 12-hydroxyieicosatetraenoic acid (12-HETE), and the formation of 1,N6-εdA and 3,N4-εdC in a mouse skin carcinogenesis model initiated with 7,12dimethylbenzanthracene (DMBA) and promoted by 12-O-tetradecanoylforbol-13acetate (TPA) (Nair et al. 2000). In the papillomas that formed 20 weeks after exposure to TPA, the levels of 8-HETE and 12-HETE increased 15- and 68-fold, respectively, which showed amplified LOX-catalyzed lipid peroxidation. Twelveand ninefold increases in 1,N6-εdA and 3,N4-εdC levels were also observed (Nair et al. 2000). Another chemical carcinogenesis model demonstrated a two- to threefold increase in M1dG levels in kidneys of Syrian hamsters exposed to kidney cancer– inducing doses of diethylstilbestrol or estradiol (Wang and Liehr 1995). Some additional data on the levels of M1dG and other malondialdehyde-derived DNA adducts in human cancer tissues compared to normal tissues reinforce the increased formation of these types of DNA lesions in carcinogenesis (Munnia et al. 2004, 2006; Peluso et al. 2011). Larynx cancer tissues exhibited increased levels of M1dG compared to controls. Cigarette smoking and alcohol intake were associated with increased levels of the MDA-DNA adducts (Munnia et al. 2004). Another study that investigated the relationship between tobacco smoking, bronchial MDA-DNA adducts, and lung cancer found that the levels of the MDA-DNA adducts were higher in current smokers than never smokers and higher in cancer cases compared to controls among smokers. Worse survival was observed for cancer cases with the highest levels of the DNA adducts. Polymorphisms of the cyclin D1 gene affected the MDA-DNA adduct levels (Munnia et al. 2006). Significantly higher levels of M1dG were also found in breast fine needle aspirates from breast cancer patients compared to healthy controls. A trend of increasing M1dG levels was observed for increased tumor grade and pathological diameter (Peluso et al. 2011).

Conclusion A variety of electrophilic products are formed during the process of lipid peroxidation. Some electrophiles were identified, for which adducts with different DNA bases were structurally characterized and their miscoding properties elucidated. Among the different types of DNA adducts originating from lipid peroxidation, the most extensively quantified in carcinogenesis conditions in vivo are the malondialdehyde adducts and the etheno adducts, primarily the unsubstituted etheno adducts, but there is an upward trend in the exploitation of substituted etheno adducts formed by 4-oxo-alkenals in cancer-prone inflammatory conditions. The

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quantification of DNA adducts in vivo is hindered by the need for ultrasensitive and selective methods that can detect levels as low as 1 lesion per 109 normal parent deoxynucleosides in a few micrograms of DNA without interference of similar molecules. There is also the need to examine several known and unknown DNA adducts simultaneously because individual factors may affect the levels of particular lesions and prevent conclusions when only a few lesions are investigated. The DNA adductomics approach using mass spectrometry–based techniques is being developed for this purpose (Villalta and Balbo 2017). The possibility to obtain a global picture of DNA adducts and associate groups of lesions to specific conditions, like cancer-prone redox stress conditions, will open a field of research to reveal useful biomarkers for assessments of cancer risk, preventive and treatment interventions, and diagnosis and prognosis purposes. Besides identifying and quantifying the DNA adducts in the carcinogenesis process, there is a need for experimental models and strategies to elucidate relationships between the occurrence of the DNA adducts, mutations in critical genes (e.g., tumor suppressor genes and proto-oncogenes), altered gene expression, and changes of cell signaling, metabolism, and epigenetics. Observation of the global picture of changes and their interrelationships will improve our understanding of the role of lipid peroxidation–derived DNA adducts in carcinogenesis.

Cross-References ▶ Analytical and Omics Approaches in the Identification of Oxidative StressInduced Cancer Biomarkers ▶ Biomarkers of Oxidative Stress and Its Dynamics in Cancer ▶ Biomarkers of Oxidative Stress in Cancer and Their Clinical Implications ▶ Free Radicals–Mediated Epigenetic Changes and Breast Cancer Progression ▶ Genomic Instability in Carcinogenesis ▶ Oxidative Stress in Orchestrating Genomic Instability-Associated Cancer Progression ▶ The Interdependence of Inflammation and ROS in Cancer

References Akasaka S, Guengerich FP (1999) Mutagenicity of site-specifically located 1,N2-ethenoguanine in Chinese hamster ovary cell chromosomal DNA. Chem Res Toxicol 12:501–507. https://doi.org/ 10.1021/tx980259j Augusto O, Bonini MG, Amanso AM, Linares E, Santos CCX, De Menezes SL (2002) Nitrogen dioxide and carbonate radical anion: two emerging radicals in biology. Free Radic Biol Med 32:841–859. https://doi.org/10.1016/s0891-5849(02)00786-4 Bartsch H, Nair J (2006) Chronic inflammation and oxidative stress in the genesis and perpetuation of cancer: role of lipid peroxidation, DNA damage, and repair. Langenbeck’s Arch Surg 391:499–510. https://doi.org/10.1007/s00423-006-0073-1

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Imran Moin, Disha Mittal, and Anita K. Verma

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Relevance of ROS-Induced Oxidation in Pathogenesis of Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ROS Interaction with Lipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Generation Cytosolic ROS from Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ROS and Nuclear Signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Oxidative Stress Promotes Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ROS Serves Dual Purpose in Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cancer Progression and Metastasis Are Promoted by CAFs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ROS and Cellular Death Pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Caspases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Autophagy and ROS (Programmed Cell Death Type II) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Necrosis and ROS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Necroptosis (Programmed Cell Death: Type III) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ferroptosis and ROS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Therapeutics and ROS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ROS and Multidrug Resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Nuclear ROS Induces DNA Damage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ROS Targeted Nanotherapeutic Drugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

900 901 902 902 904 904 905 905 907 907 908 908 910 910 910 911 913 914 914 915 916

Abstract

A cluster of extremely reactive moieties that have advanced as key regulators of significant signaling pathways, termed as reactive oxygen species (ROS) are quintessential. Cellular functions including gene expression are maintained by modest levels of ROS, the levels are constitutively higher in cancer cells owing to enhanced metabolic rate causing hypoxia as well as gene mutation. Elevated ROS I. Moin · D. Mittal · A. K. Verma (*) Nano-Biotech Lab, Department of Zoology, Kirori Mal College, University of Delhi, Delhi, India e-mail: [email protected] © Springer Nature Singapore Pte Ltd. 2022 S. Chakraborti et al. (eds.), Handbook of Oxidative Stress in Cancer: Mechanistic Aspects, https://doi.org/10.1007/978-981-15-9411-3_53

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are satiated by enhanced antioxidant enzymatic and nonenzymatic pathways occurring in the individual cells. Modest increase of ROS has been associated with many pathologies including advancement of and progression of tumor. Since, ROS perturbations can cause mutations in DNA and alter various signaling pathways. Also, ROS are responsible for activating programmed cell death (PCD). The chapter summarizes the mechanistic analysis of molecular pathways essential for understanding and evolving a holistic approach to develop therapeutic strategies to treat cancer by modulating the ROS levels. Especially, we highlight the “double-edged sword effect,” where we evaluate the varied metabolic pathways generating ROS, and how these molecular mechanisms can be exploited to act as therapeutic moieties, to eradicate ROS-induced initiation and progression of cancer. Keywords

ROS · DNA damage · Molecular pathways · PCD · Therapeutic strategies Abbreviations

ABC Akt/PKB AML AMPK ANT APAF APE1 ARE ASK1 ATG4 ATP BAX Bcl-2 Bcl-xL Bid BRCA CAFs CAT c-FLIP DISC DNA DNAMT EGFR EMT

Adenosine triphosphate (ATP)-Binding Cassette Protein Kinase-B Acute Myeloid Leukemia AMP-activated Protein Kinases Adenine Nucleotide Translocator Apoptotic Protease Activating Factor Apurinic/apyrimidinic Endonuclease 1 Anti-oxidant Response Elements Apoptosis Signal-regulating Kinase 1 Autophagy-related Gene-4 Adenosine triphosphate BCL2 Associated X Apoptosis Regulator B-cell lymphoma 2 B-cell lymphoma-extra large BH3 interacting-domain death BReast CAncer gene Cancer-Associated Fibroblasts Catalase Cellular FLICE (FADD-like IL-1β-converting enzyme)inhibitory protein Death-inducing Signaling Complex Deoxyribonucleic acid DNA methyltransferase Epidermal Growth Factor Receptor Epithelial-Mesenchymal Transition ERK Extracellular Signal-Regulated Kinase

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ETC FADH FOXO GCL GPX GR GSH GSSG HDAC HIF HMOX1 HSP IAP IκB IKK JNK Keap1 LSD1 MAPK MEK MMP MKK4/7 mTOR NAC NADPH NEMO

NIK NO NOXA/PMAIP1 NOXs Nrf2 NSAID OXPHOS PARP PCD PDGFR PERK PI3Ks PKC

899

Electron Transport Chain Flavin Adenine Dinucleotide Forkhead Box-O Glutamate-cysteine Ligase Glutathione Peroxidase Glutathione Reductase Glutathione Glutathione disulfide Histone deacetylase Hypoxia-Inducible Factor Heme Oxygenase Heat Shock Proteins Inhibitors of Apoptosis Proteins Inhibitory kappa B IκB Kinase c-Jun N-terminal kinase Kelch-like ECH-associated protein-1 Lysine-Specific histone Demethylase 1A Mitogen-Activated Protein Kinase MAPK/ERK Kinase Mitochondrial Membrane Potential Mitogen-activated protein Kinase (MAPK) Kinase (MKK)7 and MKK4 mammalian Target Of Rapamycin N-acetylcysteine Nicotinamide Adenine Dinucleotide Phosphate Hydrogen NF-kappa B Essential Modulator NF-κB Nuclear Factor Kappa-light-chain-enhancer of activated B cells NF-κB-inducing kinase Nitric Oxide Phorbol-12-myristate-13-acetate-induced protein 1 Nicotinamide Adenine Dinucleotide Phosphate Hydrogen Oxidases Nuclear Factor Erythroid-related Factor 2 Non-Steroidal Anti-Inflammatory Drug Oxidative Phosphorylation Poly (ADP-ribose) polymerase Program Cell Death Platelet-derived Growth Factor Receptors Protein kinase RNA-like Endoplasmic Reticulum Kinase Phosphoinositide 3-kinases Protein Kinase-C

900

PRX PTEN PTP PUMA PUFA RAS RCS RNS ROS RSS O2•
SOD TME TRADD Trx VDAC RIPK1/RIPK3 Z-VAD-FMK

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Peroxiredoxin Phosphatase and TENsin Homolog Protein tyrosine phosphatases p53 upregulated modulator of apoptosis polyunsaturated fatty acids RAt Sarcoma Reactive Chloride Species Reactive Nitrogen Species Reactive Oxygen Species Reactive Sulfur Species Superoxide Superoxide Dismutase Tumor Microenvironment TNFRSF1A Associated via Death Domain Thioredoxin Voltage-dependent Anion Channels Receptor-interacting serine/threonine-protein kinase 1/3 selective Channel carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]fluoromethyl ketone

Introduction In eukaryotic cells, the most neglected by-products of anaerobic cellular metabolism have advanced as key regulators of diverse signaling pathways. Reactive Oxygen Species (ROS) are oxygen-containing molecules with high reactivity. Depending on the key molecules involved, the reactive species may be divided into ROS, reactive sulfur species (RSS), reactive chloride species (RCS), and reactive nitrogen species (RNS). Ions/molecules with a single unpaired electron in their outermost shells make them extremely reactive and hence so termed. ROS is the most abundantly formed of all the compounds derived from oxidative metabolism, usually small, short-lived, their half-lives depending on the molecule’s stability vary from a few nanoseconds to hours, and comprise of free radicals derived from oxygen. The most investigated ROS molecules for cancer are H2O2, O2•
, and OH• (Liou GY Storz 2010). Although, H2O2 is nonreactive as compared to other ROS species, it can penetrate any cellular compartment before getting transformed by glutathione peroxidases and peroxiredoxins into oxygen and water. Actually, H2O2 acts as a secondary messenger in various signal transduction pathways, or as extracellular indicators, thereby controlling gene expression and contributing to redox signaling (Forman et al. 2014). During mitochondrial respiration via the electron transport chain (ETC), ~1–5% of the oxygen undergoes single electron transfer, creating O2•
anion, that exhibits partial reactivity, evident by the enzymatic conversion of O2•
to H2O2 by

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superoxide dismutase (SOD), β-oxidation of fatty acids in peroxisomes and protein oxidation in endoplasmic reticulum (ER). In mitochondria, oxidative phosphorylation encompasses four major electron transport multiplexes, constituting ATP synthase that translocates the proton, directly derived primarily from the oxidation of FADH2 and NADPH, including the chemiosmotic cascade that culminates in pumping of protons outside the mitochondria. Enzymatic reactions directly accountable for ROS generation involve cyclooxygenases, lipoxygenases, xanthine oxidases, and NADPH oxidases (NOXs) by Fenton reaction, catalyzed by iron; NOXs are conservative molecules that have initially evolved to produce ROS (Bedard and Krause 2007). Exposure to exogenous factors like ultraviolet rays and heat generates ROS, including chemotherapeutic and radiotherapeutic interventions in cancer. Cellular metabolic activity meticulously regulates the ROS levels, as base levels of ROS are critical for proliferation and differentiation in cells. In addition to being a vascular-relaxant and neurotransmitter, nitric oxide (NO) is also a physiologically free radical released by phagocytes. While NO and O2•
are not highly reactive, peroxy-nitrite, the result of their mutual reaction, can create hydroxyl radicals, too. When ROS generation is in excess amount and the antioxidant system’s scavenging ability is compromised and unable to compete with the physiological levels of ROS, it leads to tilting of balance in favor of ROS, referred to as “oxidative stress.” Oxidative stress induces cellular injury or adaptation through the upregulation of antioxidant defense systems, which restores the antioxidant/prooxidant balance. Inability to scavenge the excess ROS in tissues causes cell injury, as it targets the nucleophiles locally including proteins, lipids, and DNA. Inside the cell, ROS oxidizes amino acids and DNA bases, forms DNA adducts, causes lipid peroxidation that is characterized by loss of integrity, cellular dysfunction, and survivability of the cells, based on the location and generation of oxidative stress. Therefore, eukaryotic cells take advantage from the scavengers comprising of the extracellular matrix and mitochondria; the cytosolic SODs, glutathione reductase (GR), glutathione peroxidase (GPX), thioredoxin, peroxiredoxin, and catalases that convert O2•
to H2O and antioxidants that are recycled in their reduced state. The degree to which oxidative DNA damage contributes to the process of carcinogenesis is not yet clear, but DNA damage is quintessential for initiation of damage. DNA mutation is the perilous stage in carcinogenesis and preeminent oxidative stress levels induced DNA lesions have been reported in several cancers, inevitably implicating the damage to the etiology of cancer. The chapter summarizes the relevance of ROS generation in pathogenesis of cancer, cellular death pathways, and therapeutic approaches targeting intracellular ROS-induced DNA damage.

Relevance of ROS-Induced Oxidation in Pathogenesis of Cancer There are a distinct number of oxidative hits that a cell is exposed to per day. Any imbalance in ROS generation vs scavenged ROS pushes the cells toward oxidative stress. Oxidative stress levels largely regulate the pathogenesis of cancer as

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progression depends on the excess production of ROS during aerobic glycolysis, with subsequent oxidation of pyruvate within the mitochondria, also termed as “Warburg effect,” enhanced receptor activity, oncogene expression, and triggering of oxidizing enzymes that induce genetic instability and various growth factordependent pathways (Finkel 2011). Furthermore, the disproportionate intracellular ROS levels damage the cellular lipids, cytosolic proteins, and nuclear DNA. This very ability to modulate ROS has been the therapeutic strategy exploited for making anticancer drugs.

ROS Interaction with Lipids Oxidative stress induced by ROS can interplay with lipids via a feedback ring originated by fatty acid peroxidation that effectively modifies the lipid bilayer of cell membranes to generate free radicals. Generation of free radicals causing peroxidation of lipid have short half-lives, yet is potentially hazardous to cells as loss of integrity of cell membrane gets affected by mitochondrial phospholipid peroxidation, that forms transition pores and disrupts the respiratory chain, targeting complexes I and III causing leakage of electrons within intramembranous mitochondrial space (Forman et al. 2014).

Generation Cytosolic ROS from Proteins The primary signaling pathway entails the oxidation of protein moieties, that is, redox-sensitive tyrosine and/or cysteine residues present adjacent to or within the active site, which makes inter-protein and intra-protein connections that alter functions of protein (Roy et al. 2012). Since, these alterations are revocable, they modify many cellular responses.

Ras Signaling Oxidative stress in cancer relies on Ras pathway, wherein Ras-gene family comprises of G-proteins, namely N-, Ki-, and Ha-Ras, which contribute in cell signaling. Mutant Ras often enhances ROS levels, causing cell transformation by induced DNA damage (Rai et al. 2011). Roughly 30% of solid tumors in humans have mutations in the Ras oncogene family. Overall, ROS may negatively regulate the phosphatases, but kinases can either be stimulated or inhibited, especially the nonreceptor protein kinases akin to the family of Src, which belongs to a small G protein family, for example. Ras is activated by ROS and so are the growth factor tyrosine kinase receptors, along with the parts of p38 kinase (p38MAPK) pathways and c-Jun N-terminal kinases (JNK) that trigger apoptosis (Corcoran and Cotter 2013). The upstream activator for Erk1/2 is Ras, which may be activated precisely by the oxidative modifications on its cysteine residue 118, resulting in GDP/GTP interchange inhibition. Erk1/2 stimulation by ROS improves cell survival, motility, and anchorage-independent growth of cells (Fig. 1).

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Fig. 1 ROS activates RAS, PI3/AKT, and IKK/NF-κB protein signaling pathways. ((Black arrows signify forward pathways, and red lines signify inhibition. Binding of growth factors to EGFR generates ROS via mitochondria and NOXs. Ros activates RAS/MAPK and PI3/AKT/mTOR pathways by direct oxidation of the kinases or via PTEN inactivation or PTP inhibition at the cysteine residues. ROS-induced oxidative stress can impede mTOR activity, although it can trigger the AMP-activated protein kinase (AMPK). Initiation of NF-kB is an alternative signaling pathway. Antioxidants reduce cancer initiation but enhance progression during tumorigenesis)

PI3-K/Akt Pathway Negligible elevation of ROS preferentially activates the PI3-K/Akt pathway, and sustained increase would predictably stimulate the p38MAPK-dependent PCD/apoptosis. Akt/PKB (protein kinase-B) facilitates survival of cell via substrate level phosphorylation causing inhibition of FOXO transcription factors along with the pro-apoptotic proteins Bax, Bad, and Bim. Akt activity is closely regulated via a cascade of signals that includes the PDK-1 (30 -phospho-inositide dependent kinase1), PI3K mTOR, along with PTEN (phosphatases with the TENsin homolog). mTOR and PDK-1 mediate the activation of phosphorylate in S473 and T308 while PI3 K produces phosphatidylinositol-3,4,5-triphosphate (PIP3), which aids as an anchor of the cell membrane. PIP3 levels are negatively regulated by PTEN thereby decreasing the activity of Akt. Formation of the di-sulfide bonds between H2O2, the catalytic cysteines together inactivate PTEN and releases the phosphoinositide-3-kinase (PI3-K)-dependent enrolment of its downstream messengers, by oxidation of redox protein-thioredoxin, thereby suppressing the inhibitory effect on p38MAPK signaling cascade (Latimer and Veal 2016). PTEN is reversibly inactivated by H2O2. The calcium channel activity is further influenced by ROS levels as they trigger intracellular calcium release that subsequently stimulates the kinases, for example, protein kinase-C(PKC), which significantly regulates cellular proliferation in cancer (Weinberg et al. 2010).

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IKK/NF-kB Pathway Regulation by ROS NF-κB is considered to be a redox-sensor that requires H2O2 for activation but in extremely low doses. Transcription of NF-kB has a vital role in cell cycle control, cell survival, proliferation, and cell adhesion. When NF-κB is inactive, it is firmly linked to its IκB inhibitor which sequesters the cytosol transcription factor. The IκB kinases (IKK) and NF-κB-inducing kinase (NIK), comprising IKK-α, IKK-β, and NEMO, mediate canonical activation of the NFκB. Active IKK gets degraded by phosphorylation of IμB. tIκB depletion causes NF-κB to be translocated into the nucleus, as a transcription factor that may stimulate the anti-inflammatory gene expression along with the expression of the anti-apoptotic genes (Perillo et al. 2014).

ROS and Nuclear Signaling Whether endogenous/exogenous source of ROS, once in cytoplasm the ROS pathways transmit the signals to nucleus, thereby influencing the function of transcription factors that regulate gene expression. To combat the unwarranted rise in intracellular ROS, the cancer cells stimulate the synthesis of antioxidant enzymes, reiterating the significance of a comprehensive understanding of the metabolic pathways to augment strategies that modulate ROS levels. Nuclear factor erythroid-2-related factor-2(Nrf2) is the rudimentary redox-sensitive transcriptional factor, which is known to control the antioxidant response in tumor cells. Typically, Nrf2 is metabolized by the Kelch-like ECH-associated protein-1(Keap1), but gene overexpression is induced by oxidation of Keap1 and transfer of Nrf2 to the nucleus during oxidative stress. Further, within the cells, Nrf2 regulates the generation of glutathione (GSH), the foremost antioxidant molecule, by the enzymatic reaction of glutamate-cysteine ligase (GCL), that catalyzes GSH synthesis that is a rate-limiting reaction, enables utilization of GSH and recycling. Similarly, it regulates the homeostasis of free Fe(II) by upregulating heme-oxygenase HO-1, that in-turn is responsible for generating free Fe(II) resulting in collapse of the heme molecules. Fenton reaction is catalyzed by Fe(II) to generate OH• free radical from H2O2, its enhancement represents a paradox: Gene expression encoding numerous parts of ferritin multicomplex that are responsible for detoxifying Fe(II) and storing after altering it to Fe(III). Unusually, enhanced sera levels of ferritin have also been indicated in many tumors with poor prognosis. Another family of transcription factors, the forkhead box-O (FOXO), is further stimulated by JNK as a result of ROS elevation, inducing the expression of catalase and SODs (Valko et al. 2016).

Oxidative Stress Promotes Cancer Cancer is categorized by specific hallmarks (Hanahan and Weinberg 2011), namely transformation of cell, genome instability, enhanced proliferation, angiogenesis, immortalization, metastasis, and epithelial-mesenchymal transition (EMT), which are all predisposed by intracellular ROS in many ways.

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ROS Serves Dual Purpose in Cancer Noncancerous cells like cancer-associated fibroblasts (CAFs) are generally associated with tumors, especially those present in tumor microenvironment (TME), dynamically regulate the homeostasis in tumor, enhancing tumor progression as well as incursion of cancer cells. ROS and CAFs participate paradoxically: either ROS targets the fibroblasts, principally H2O2, which transforms the fibroblasts to active CAFs via the upregulation of HIF1α; or CAFs are pivotal for elevating levels of ROS in tumors (Chan et al. 2018). Figure 3 indicates that enhanced levels of ROS caused by high cellular metabolic activity or chronic inflammation lead to pro-oncogenic mutations promoting tumor progression, neo-vascularization, and even metastasis. Further, excessive ROS pushes the cell to senescence and apoptosis, leading to tumor regression.

Cancer Progression and Metastasis Are Promoted by CAFs ROS and CAFS are interlinked by increasing ROS-produced CAFs, to which majority of cancers are sensitive to and respond by enhancing the antioxidant gene expression. It is rather strange as to why certain tumor cells favor this comparatively unproductive pathway for production of energy. Tumor cells prefer enhanced glucose metabolic activity that leads to lactate generation and release into the surrounding tumor-stroma cross-border, creating an acidic tumor microenvironment. Tumor metabolic activity requires a complex interplay, as solid tumors consist of heterogeneous cells, both cancerous and stromal, with some cells having a glycolytic phenotype, whereas others primarily exploit the oxidative phosphorylation (OXPHOS). Metabolites produced by CAFs further complicate the microenvironment that are utilized by cancer cells to quench the metabolic stresses, preserve production of ATP, and offer an alternative source of carbon as fuel for cancer metabolism (Wu et al. 2017). The “reverse Warburg effect” elucidates a cancer metabolism model, which is based on two-cell model, wherein CAFs and tumor cells are coupled metabolically (Fig. 2). Recycling of tumor-derived lactate has been detected in breast cancer stromal CAFs that may be for self-benefit for energetic requirements or changed to pyruvate, that was released enabling formation of metabolites via glycolysis in tumor cells (Patel et al. 2017). Cancer metabolism can be interpreted as a dynamic equilibrium. The major complexities in cancer cells necessitate flexibility of metabolic activities that favor on-going metabolic requirements in cells in tandem with the metabolites secreted by tumor microenvironment. Figure 3 summarizes that Nrf2 gene targets like HO-1 promote development of cancer, as they offset the oxidative stress effects in transformed cells (Zimta et al. 2019). Furthermore, conventional oncogenes such as c-myc and K-Ras that also stimulate cytosolic ROS are known to stabilize Nrf2. Hence, NRF2 mutations along with its controller KEAP1 are pronounced in tumors, which verify the fundamental role of antioxidant genes in cancer progression (Lignitto et al. 2019). Actually, breast cancer susceptibility-1 (BRCA-1) gene triggers the expression of Nrf2 promoting cell survival. Hormonal stimulus with

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Fig. 2 The dual-purpose role of ROS in tumors

Fig. 3 Illustration of ROS activated Nrf2-ARE signaling cascade. (In cytosol, Nrf2 is bound to Keap1 protein under normal conditions. Keap1 hinders Nrf2 stimulation by triggering ubiquitination of Nrf2 and ensuing destruction via proteasome. Moderate oxidative stress and activators of Nrf2 disassociate the complex-Nrf2-keap, phosphorylate Nrf2 causing its translocation to nucleus, where Nrf2 triggers antioxidant transcriptional factors like catalase, haemeoxygenase-1 [HO-1],SOD,NAD(P)H: quinone oxidoreductase-1[NQO1], and other detoxifying enzymes that bind to the promoter regions of target genes, i.e., the Anti-oxidant Response Elements (ARE). HO-1 plays a double-edged role in ferroptosis. HO-1 also catalyzes heme degradation to generate Fe2+, which is highly reactive as prooxidant, thereby generating ROS. Excess ROS damages protein, DNA as well as lipid peroxidation and causes cell death via Ferroptosis. Nrf2 stimulates ferritin expression to chelate Fe2+, circumventing the ROS surplus)

estrogen in breast cancer cells promotes cell survival, although they lack expression of BRCA-1, they exhibit elevated intracellular ROS levels, which effectively salvage NRF2 transcription. Moreover, FOXO transcription factors are associated with

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Fig. 4 Oxidative stress and Redox Signaling Cascade in CAFs and Tumor cells. (The key signaling pathways initiated by growth factor-triggered ROS are emphasized on CAFs. Equivalent pathways influencing cell cycle, affecting the interplay of cellular responses to hypoxic tumor microenvironment (TME) by modulating the activity of transcription genes and factors. ROS further stimulates lipid peroxidation with proportionate loss of MMP causing electron leakage followed by Ca2+ release from intracellular stores. In cancer cells, the key oxidative stress consequences are indicated on the right panel. Modestly enhanced ROS levels trigger oncogenes and hinder tumor suppressor genes, thereby elevating ROS levels. Release of Ca2+ induces PKC, whereas expression of angiogenic genes and a robust antioxidant system is maintained. ROS further activates chromatin-HDACs and has a twin-effect on the pro-oncogenic and tumor-suppressor gene expression via the DNMTs. Oxidation of bases activates mutagenesis and recruits DNA repair mechanisms)

carcinogenesis: FOXO genes exhibit enhanced mutations in rhabdomyosarcomas that make them unresponsive to blocking by AKT signaling (Gorrini et al. 2013). While increased intracellular GSH levels are necessary for both initiation leading to progression, GR inhibitors perform as targets for anticancer therapy in various tumors (Harris et al. 2015). Elevated NADPH levels promote the metastatic activity in melanoma cells (Fig. 4).

ROS and Cellular Death Pathways Apoptosis Apoptosis is an essential, tightly regulated mechanism that eliminates redundant or potentially DNA damaged cells, thereby preventing cellular growth vital for maintenance of cellular homeostasis. Apoptosis exhibits distinguished morphological characteristics like cell shrinkage, blebbing of membrane, condensation of chromatin, nuclear fragmentation, formation, and release of apoptotic bodies, which are phagocytosed by neighboring macrophages, thereby reducing inflammatory response. Both extrinsic and intrinsic factors trigger apoptosis, which include ROS, DNA-damaging agents, RNS, heat shock, hypoxia, etc. (Pallepati and Averill-Bates 2012). ROS like H2O2 are associated with activation of survival responses by cell at lower doses, while higher doses trigger apoptosis (Kaminskyy

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and Zhivotovsky 2014). Death inducing ligands like Fas ligand and TNFα facilitate the extrinsic pathway by binding to cognate receptors that sequentially recruit procaspases along with receptor proteins making an assorted assemblage of the deathinducing signaling complex (DISC), followed by triggering of effector caspases. This interface can be effectively blocked with cellular-FLICE-inhibitory protein (cFLIP). Downregulation of c-FLIP induced by ROS by degrading its ubiquitinproteasome, thereby further enhancing the extrinsic pathway (Wilkie-Grantham et al. 2013). Plethora of evidence proposes that apoptosis relies on triggering of intrinsic pathway, involving mitochondria, and this approach helps in developing most ROS-related anticancer therapeutics. Since, permeability of mitochondrial PTPs increases when pro-apoptotic factors like cytochrome-c are released in cytosol to make a complex with pro-caspase-9, and apoptotic protease activating factor-1 (Apaf-1) to make an apoptosome, which sequentially induces other effector caspases (Fig. 5a). Intriguingly, ROS induces three key mechanisms pivotal for PTPs opening, adenine nucleotide translocase (ANT), the voltage-dependent anion selective channel (VDAC) along with cyclophilin D, through cysteine oxidation of their exact active sites. ROS may stimulate apoptosis by enhancing the ubiquitination or inactivating of Bcl-2, a critical anti-apoptotic protein and subsequently reducing cytosolic Bad and Bax levels (Luanpitpong et al. 2013). Elevated ROS levels trigger apoptosis as the crucial mechanistic pathway responsible for the therapeutic efficacy of tyrosine kinase inhibitors and monoclonal antibodies and represent the very basis for targeted cancer treatment (Teppo et al. 2017). Erlotinib – an EGFR inhibitor – and imatinib – a PDGFR inhibitor – are tyrosine kinase inhibitors, which are responsible for inducing ROS-induced apoptosis in non-small-cell lung carcinoma and melanoma, via disturbance of the mitochondrial membrane potential (MMP), post-activation by phosphorylation of p38 and JNK. Contrarily, vemurafenib – a BRAF inhibitor, enhances the generation of O2•
ions with proportionate loss of MMP in melanoma cells (Bauer et al. 2017).

Caspases Post-translational modifications on the cysteine residues of their catalytic site regulate redox activity of the caspases by oxidation, whereas S-glutathiolation is susceptible to procaspase-9, procaspase-3, and caspase-3. Oxidants like H2O2 irreversibly trigger caspases and inactivates caspases-3, -8, and -9 (Circu and Aw 2010). Intracellular GSH levels monitor the S-glutathiolation-mediated caspase3 activity, which decreases proteolytic cleaving accessibility and contributes to apoptosis resistance.

Autophagy and ROS (Programmed Cell Death Type II) Another significant therapeutic strategy to destroy cancer cells is ROS-induced autophagy. Autophagy is an environmental stress defense adaptation pathway, which acts as a survival mechanism under starvation. It involves the formation of

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Fig 5 Oxidative stress guides the mechanisms of cell death. (Loss in Mitochondrial potential (MMP) is driven by low stress in endoplasmic reticulum (ER) that controls the cell death cascade: (i) reduced stress in ER leads to (ii) momentary perturbation causing inadequate release of Ca2+ (iii) as a result of fractional loss of MMP occurs. Release of pro-apoptotic molecules are thus stimulated, (iv) causing leakage of cyt C, (v) that trigger effector caspases that may induce (a) APOPTOSIS. (vi) Continued reduced stress and minute amounts of ATP triggers mitochondrial membrane permeabilization, causing necrosis. ROS accumulation stimulates caspase-independent apoptosis too. Alternatively, (a) enhanced stress of ER and (b) continued trepidation results in surplus release of Ca2+, causing complete loss of MMP (c) which in-turn generates (d) more ROS, leading to (e) depletion of ATP, pushing the cell toward (b) NECROSIS. Dysfunction of mitochondrial causes release of (1) O2
°, which contributes to (2) accumulation of ROS and (3) membrane degradation of lysosome inducing additional vacuolation in cytosol pushing the cell to undertake cell death by programmed necrosis termed as (c) NECROPTOSIS. Sustained metabolic insults coupled with (f) hypoxia enhances mitochondrial dysfunction causing excess (g) “ATP depletion.” Exogenous ROS triggers (d) ROS-induced autophagy that involves destruction of [A]cytosolic proteins and organelles, [B]AMPK, and [C]PI-3 K, formation of phagosomes and autolysosomes)

a double membraned vacuole – autophagosome (Fig. 5) The autophagosome’s outer membrane fuses with the lysosome membrane forming an autolysosome, encapsulating lysosomal enzymes like cathepsins B, D, and L that degrade cytoplasmic contents. Degradation of the cytoplasmic content during autophagy under conditions of nutrient deficiency produces amino acids and energy for the survival of cell (Redza-Dutordoir et al. 2016). Inhibition of autophagy-related gene-4(ATG4) that is H2O2-dependent enhances formation of autophagosome. ROS levels regulate the induction of autophagy in cells via AMP-activated-protein-kinases (AMPK). Deprivation of oxygen along with nutrients causes the accumulation of AMP/ATP which then activates AMPK, initiating inhibition of mTORC1, a crucial negative regulator. Undeniably, autophagy, termed as type-II programmed cell death, is a cell survival as well as a tumor suppressor pathway that triggers elimination of cancer cells. Incidentally, glioma cells have shown H2O2-induced autophagic death, posttreatment with polycyclic ammonium ion-sanguinarine that enhances mitochondrial leakage of electrons, thereby stimulating NOXs (Li et al. 2012). Transcription factors

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like NF-κB further regulate gene expression of autophagy-linked genes like: BECLIN1/ATG6 or SQSTM1/p62 in cancer cells. Various chemotherapeutic drugs trigger ROS-dependent autophagy, which may cause drug resistance, apoptosis, or both. Mitochondrial dysfunction and elevated ROS levels can be correlated to cell death by autophagy (Poillet-Perez et al. 2015).

Necrosis and ROS Necrosis is sensitive to severe stress and involves acute cellular dysfunction and characteristic ATP depletion. Excess H2O2 causes necrosis, and can possibly shift process of cell death from apoptosis to necrosis. Without adequate ATP, cells infused with toxic levels of therapeutic agents will die as a result of necrosis rather than apoptosis. Hence, ATP is another critical parameter that regulates necrosis. Mitochondrial ROS is the key contributor of ROS for mediation of necrosis, whereby NOX1, a large subunit of NADPH oxidase, is essential for the production of TNFαstimulated superoxide in mouse fibroblasts, and is triggered by RIP1-induced signaling complex comprising NOX1, NOXO1, TRADD along with GTPase-Rac1, targeted by TNFα therapy (Wang and Yi 2008).

Necroptosis (Programmed Cell Death: Type III) Conventionally, necrosis was described as unregulated cell death, but ROS-induced necrosis can be documented as necroptosis-type-III programmed cell death. Necroptosis is characterized by cell rounding-off, granulation of cytoplasm, swelling, and cell-membrane rupture, and the contents of the cell are released in the surrounding tissue generating an inflammatory response that maybe triggered by TNF-α and a pan-caspase inhibitor (z-VAD-FMK). Toll-receptors, Death-receptors, viruses along with DNA damage lead to necroptosis activation. Elevation in metabolism of energy caused by ceramide induced ROS generation can be stimulated by many receptor-interacting protein kinases (RIPs), in NOXs and ETC that enhance necroptosis (Dixon and Stockwell 2014). RIP1 and RIP3 are the main signaling molecules involved in necroptosis and RIP3 acts as a switch between apoptosis and necroptosis (Buchheit et al. 2012). Nrf2, a transcription factor, activates antioxidant responses and detoxifies intracellular ROS. Nrf2 proves to be a molecular link between necroptosis and ROS. ROS actively induces and executes cell death by necroptosis (Vanden Berghe et al. 2015).

Ferroptosis and ROS Ferroptosis has distinctive morphological and biochemical characteristics and can easily be differentiated from other programmed cell death like apoptosis, necroptosis, necrosis, and autophagy (Jiang et al. 2015). Ferroptosis depends on

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lipid ROS and iron metabolism. Ferroptosis may be initiated by cysteine depletion, loss of lipid repair enzyme: glutathione peroxidase-4 (GPX4). Peroxide accumulation in lipid membranes is associated with loss of GPX4, which causes accumulation of lipid-induced ROS that triggers peroxidation of poly-unsaturated fatty acids (PUFAs) via long-chain fatty acid-CoA ligase-4 (ACSL4) along with their collection in the membrane lyso-phospholipids. Reports suggest that PUFA peroxidation catalyzes lipoxygenases (LOXs) (Hayano et al. 2016) (Fig. 2). Suppression of system Xc
Na+-dependent cysteine-glutamate membrane exchange transporter prevents influx of extracellular cysteine and reduces intracellular levels of GSH. System Xc
represents the antiporter system-glutamate/cystine, that is further correlated with influx of extracellular cystine to exchange intracellular glutamate (Hayano et al. 2016). Cysteine – the reduced form of cystine, behaves as a predecessor in glutathione (GSH) synthesis. GPX4 catalyzes lipid peroxide inhibition having GSH as a cofactor. The inhibition of system Xc
by ultra-fine molecules exhibits an accumulation of ROS and fatal lipid peroxides initiating ferroptosis. Membrane-porin proteins are transmembrane voltage-dependent anion channels (VDACs) for exchange of ions and metabolites generally dispersed on the outer mitochondrial membrane. Therefore, any mitochondrial dysfunction or release of oxidants will inevitably cause an oxidation-dependent non-apoptotic cell death. Tumor-suppression by p53 impedes uptake of cystine characteristically arbitrated by the suppression of a crucial factor of the cystine-glutamate antiporter (Jiang et al. 2015). A probable paradox is that cell survival is encouraged by p53 via averting unwarranted increased ROS levels during modest oxidative stress. But, if ROS levels cross the threshold, it transforms as ROS-inducer, stimulating cell death. p53 elevates intracellular ROS levels by stimulating ROS-induced stress response, eventually increasing susceptibility of cancer cells to ferroptosis. Since, ferroptosis is apparently an oxidation-induced death pathway, numerous drugs have undergone trials to elicit the ferroptotic pathway (Louandre et al. 2013).

Therapeutics and ROS Chemotherapeutic enhancement of ROS production rates is expected to drive the already increased levels of ROS in cancer cells above the threshold causing apoptosis through intrinsic or extrinsic pathways (Brenneisen and Reichert 2018). Targeting ROS is one of the most hypothesized therapeutic pathways for tumor regression. Two key factors are responsible for enhanced cellular ROS generation induced by chemotherapy, either mitochondrial ROS or inhibition of the antioxidant system (Fig. 6). Highest levels of cellular ROS are generated by anthracyclines such as alkylating agents, Doxorubicin, Daunorubicin, Epirubicin, platinum coordination complexes, and camptothecins. A majority of chemotherapeutic agents like cisplatin, doxorubicin, and arsenic trigger cell death via ROS-dependent or ROS-independent pathways. For example, the cytotoxicity of cisplatin is mediated by nuclear DNA adducts, which hamper DNA replication and are unable to get repaired causing DNA damage,

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Fig. 6 Overview of chemotherapeutic drugs and nanoparticles as Pro- and antioxidants. (Drugs as antioxidants block the detoxification enzymes by cells. Modulation of ROS generation in cancer and elimination by Chemotherapeutic intervention decides the ultimate ROS levels and their final therapeutic efficacy. Effect of surface functionalization can alter behavior and characteristics of nanoparticles. A variety of ligands used as functionalizing agents to increase the solubility and specificity (pro-oxidant) toward target organs. The ligand functionalization may confer numerous interesting physicochemical properties essential for biomedical applications)

generating more intracellular ROS (Yang et al. 2018). Drugs are designed to cause oxidative stress to achieve successful anticancer therapy. OH• are generated by triggering the Fenton reaction in Doxorubicin-dependent cytotoxicity in breast cancer, Kaposi’s sarcoma, bladder cancer, and acute lymphocytic leukemia. Conventional therapeutic strategy targets the tumor suppressor genes and oncogenes, the success of which is a strategy that appears challenging owing to the intricate involvement of genes modulating the compensatory pathways (Wiel et al. 2019). Rituximab, a monoclonal antibody that specifically binds to calcium-channel protein-CD20, present on B-cell surface and on mature plasma cells elevates ROS levels, triggering apoptosis by inhibiting Bcl-2 and p38MAPK signaling cascade, which may be exploited for B-cell lymphoma therapy. Arabino-cytosine therapy curbs replication of DNA, followed by anthracyclines to elevate ROS levels, indicating PCD, with advantageous responses for acute myeloid leukemia (AML) patients (Prieto-Bermejo et al. 2018). Arsenic trioxide may stimulate leakage of electrons in the ETC by inducing apoptosis in lung cancer, myeloma, and leukemia. p53 plays an important role in regulating stress responses by induction of cell cycle arrest that facilitates DNA repair and survival, or cell death by apoptosis. During stress or DNA disruption, p53 cleaves from Mdm2 to be stabilized by post-translation modifications, thereby preventing the degradation of proteasomes. During low stress, p53 induces arrest of

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the cell cycle, reparation of DNA and senescence, high stress enables p53 to regulate apoptosis by lowering the expression of antiapoptotic proteins, like Bcl-2, IAPs, BclXL. Further, increased expression of proapoptotic members like Bax, Puma, Bid, Noxa, and Apaf regulates mitochondrial-signaling pathways of apoptosis. H2O2induced apoptosis has been linked to upregulated p53, Noxa, Puma, Bax, and Ser15 and Ser46 p53 phosphorylation in several cell types like rat neural AF5, glioma, colon cancer, and HeLa cells (Pallepati and Averill-Bates 2010). However, 5-fluorouracil, the analogue of pyrimidine, generates ROS via p53-dependent pathways that trigger apoptosis in rectal and colon cancer cells. Also, ROS levels are elevated by platinum-based drugs that promote PCD; administration protocols for these compounds are often combined with poly (ADP-ribose) polymerase (PARP) inhibitors that maintain and protect DNA integrity have indicated growth arrest in breast cancer. PARP can block the DNA-damage repair mechanism that facilitates cancer cells to be more responsive to oxidative stress induced by platinum-based drugs. Elevated levels of ROS mediate programmed cell death (PCD) by acting on sphingomyelinase to produce ceramide from sphingomyelin, which enables them to adhere to the death-receptors expressed on surface of membranes of cancer cells. UVirradiation of lymphoma cells activates this pathway. Drugs targeting mitochondria triggers generation of ROS that appears as a plausible approach to reach PCD in cancer by inducing oxidative stress (Caino and Altieri 2016). Excess ER stress triggers apoptosis in cells, which is supposedly induced by dysfunctional protein folding in the ER. Currently, new drugs are being designed to heighten ER stress in cancer cells by inducting oxidative stress. Inhibitor of proteasome – bortezomib – stimulates ROS production coupled with ER stress in squamous-cell carcinoma. A nonsteroidal anti-inflammatory drug (NSAID) – celecoxib, intensifies the ER stress and triggers PCD by modulating the pro- and anti-apoptotic markers, Bax/Bcl-2 ratio leading to elevated ROS levels in prostate cancers. Therapies based on GSH depletion (aziridine derivatives and isothiocyanates bind to GSH), or by inhibiting the binding of a precursor of its synthesis that is rate limiting (XCT-inhibitors of glutamate/cysteine antiporter), significantly influence survival of cancer cell (Perillo et al. 2020a, b). Sulfasalazine, a specific XCT inhibitor, is apparently very useful in small-cell lung and pancreatic cancer cell therapy. Thioredoxin plays a pivotal role in these redox systems, where it is reduced by NADPH to translocate the electrons enabling signal transduction, synthesis of DNA, and redox homeostasis. Another thioredoxin inhibitor – Auranofin, has also been effectively used in therapy of head and neck carcinoma cell lines; inhibition by N-acetylcysteine (NAC), a ROS scavenger, reiterates the important part played by ROS in cancers.

ROS and Multidrug Resistance Multidrug resistance in cancer cells can be impaired by enhanced ROS levels can further promote tumor development and even metastasis, during or after chemotherapy. Membrane efflux pumps in tumor cells are vital for maintaining extracellular efflux of antitumor drugs. Pgp pumps are part of ATP-binding cassette (ABC)

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transporter super-family and are reliant on the intracellular ATP supplies. The accumulation of ATP is due to synthase, promoted by a proton-gradient produced via NADH-dependent ETC in mitochondria; ATP synthesis can possibly be blocked by converting NADH to NAD via novel that target the mitochondria to generate ROS with instantaneous quenching of NADH (Steeg 2016). Enhanced ROS-triggered apoptosis has been suggested in tumor cells subsequent to reduction of ATP molecule recruited from glycolytic enzymes, radiotherapy, or chemotherapy; these findings reiterate the prominent function of modulating ROS and evolving an alternative anticancer combinatorial therapy (Sies et al. 2017).

Nuclear ROS Induces DNA Damage Nuclear function of ROS-dependent transcriptional expression has been emphasized. Cells undergo a stringent programmed mechanism for differentiation explicitly based on the arranged order of gene expression. Owing to the spatial restraints, the genes must involve an intricate unfolding process to recognize the transcriptional mechanisms that are triggered via the post-translational alterations at the N-terminal ends of core histones. Collectively, modifications of the core histones that are triggered by synchronized directing of the transcription factors are denoted as epigenetic markers, which confine to a meticulous code with precise time constraints to regulate the entire gene expression. Estrogen-induced transcription can be triggered by catalytic cleavage of LSD1causing lysine-9 de-methylation that is present in the histone H3 at H3K9 position. The reaction can be initiated by binding of the ligand to the estrogen receptor, leading to ROS generation through the FADH2 oxidation, initiated by demethylase, and subsequent oxidation of the neighboring guanines – 8-oxo-Gs, the DNA-repair pathway enrolment enzymes like APE1, which causes the single-strand cleavage in DNA and allows formation of loops among the enhancer/promoter, with the poly-adenylation target sites of genes having prolific transcription. Naturally, ROS generation in this progression must be appropriately and spatially regulated to restrict excess DNA damage (Tsang et al. 2014). H3-histone (H3S10) phosphorylation at the serine-10 being hormone-dependent may inhibit the instant re-methylation of earlier lysine moiety, which serves as an intermediary giving DNA damage repair mechanism adequate period to remove the oxidized nucleotides in the vicinity by DNA (Perillo et al. 2014). Inhibiting the phosphorylation of serine-10 in the signaling cascade simultaneously tested with estradiol in breast cancer cells exhibits ROS over-production, followed by enhanced DNA oxidation, which overpowers the DNA repair machinery in a variety of cells by inducing PCD.

ROS Targeted Nanotherapeutic Drugs Nanotechnological innovations are exploring several nanomaterials having ROSregulating properties to chaperon the dynamic behavior of ROS in biological systems. This forms the basis of a neo-nanomaterial based therapeutic strategy for

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ROS modulation. This interdependence on biology, chemistry, and nano-therapy forms the very basis of evolution of “ROS Science” that studies the biochemical reactions occurring in the biological milieu by the chemically synthesized nanoparticles that are ultrafine in size, possessing large surface area to volume ratio. These unique physicochemical characteristics result in radically altered chemicobiological properties when compared to the bulk material. Nanotherapeutics basically focuses on ROS-related nano-chem interfaces which include ROS production, transition, and scavenging for increased efficacy and also ROS-related nano-bio interactions that include biocompatibility, biodegradability, and bioavailability for good therapeutic outcomes. Interaction between nanoparticle-biomacromolecules influence nanoparticle-cell interplay, like nanoparticles-mediated cellular sub-structural alterations and biochemical perturbations. ROS generating nanoplatforms include TiO2 loaded nanoplatforms that generate ROS in response to exogenous generation of light. Also, FeS and CuS nanoparticles can convert biological H2O2 to highly toxic OH-free radical for antibacterial treatment. ROS scavenging platforms include CeO2 nanoparticles that can scavenge O2•
and H2O2 showing CAT and SOD like activities, hence proves to play an essential role in antioxidative therapeutics (Yang et al. 2019).

Conclusion ROS generation due to aerobic life is unavoidable. ROS and RNS are persistent source of endogenous or exogenous metabolic insults on genetic material that may be effectively modulated by hormonal, environmental, or nutritional influences, leading to superfluous ROS/RNS generation triggering oxidative or nitrosative stress to living organisms. The multifaceted interplay between cancer and ROS levels primarily depends on precise refinement amid ROS generation and scavenging. Cancer initiation, proliferation, and advancement influence moderate elevation in levels of ROS. Hence, cancer cells flourish when ROS levels are discreetly higher than the normal cells, because normal cells have functional antioxidant systems to counterbalance the prooxidants. This characteristic aspect of cancer cells renders them more sensitive to exogenous trigger that further increases ROS production. Cellular responses that increase ROS need to be understood and visualized as an interplay of several stages, wherein their relative concentration, nature, and their specific location has a significant role. Mitochondrial ROS promotes cell death by redox signaling that relies on movement of electrons, because of which this pathway has diffused signaling. In nature, ROS behavior has been adjudged as a two-edged sword, although literature documents the advantages of antioxidant agents as drugs for treating cancer. A plethora of literature suggests that blocking the antioxidant enzymes ensues killing of cancer cells, specifically when combinatorial therapy elevating ROS levels is used. NP-induced biotoxicity generating ROS are not yet clear, hence there is need to explore the mechanisms that can provide verifiable evidence to alter the chemico-physico topographies of NPs to modulate ROS levels. Essentially, the shared mechanisms prompted by oncogenes to endorse the adaptation to stress conditions are regarding the redox balance. In conclusion, the explosion

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of information on ROS targeting strengthens our belief that ROS targeting will epitomize the base for imminent strategies for molecular anticancer therapy.

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Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Role of Mitochondrial Metabolism in Breast Cancer Development and Progression with Focus on Mitochondria and ROS Production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Importance of Mitochondria for Cancer Cell Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mitochondrial Functions in Non-cancer Cells and Changes in Cancer Cells . . . . . . . . . . . . . . The Breast Tumor Microenvironment, Metabolic Interactions and Crosstalk Between Stroma and Tumor Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Oxidative Stress as an Essential Factor in the Pathogenesis and Progression of Breast Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ROS and Oxidative Stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Triggers for ROS Production and Oxidative Stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Oxidative Stress Affecting Sites Distal from Tumor Tissue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions and Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Oxidative stress has been implicated in a variety of diseases including initiation and progression of cancer. This review will focus on the role of oxidative stress in breast cancer with specific focus on mitochondrial metabolism and mitochondriaderived oxidative stress that affects the breast cancer microenvironment. The review will detail the significant effects of oxidative stress on components of the stroma that in turn plays a critical role in communications with cancer cells, thereby promoting breast cancer cell alterations and progression to solid tumors. Three specific sections will cover (1) The role of mitochondrial metabolism in breast cancer development and progression with focus on mitochondria and ROS production; (2) The breast tumor microenvironment, metabolic interactions and H. Schatten (*) Department of Veterinary Pathobiology, University of Missouri-Columbia, Columbia, MO, USA e-mail: [email protected] © Springer Nature Singapore Pte Ltd. 2022 S. Chakraborti et al. (eds.), Handbook of Oxidative Stress in Cancer: Mechanistic Aspects, https://doi.org/10.1007/978-981-15-9411-3_56

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crosstalk between stroma and tumor cells; (3) Oxidative stress as an essential factor in the pathogenesis and progression of breast cancer. Keywords

Oxidative stress · Breast cancer · Mitochondria · Metabolism · Microenvironment · ROS production · Stroma · Cancer cells

Introduction Breast cancer is still among the most frequently diagnosed cancers worldwide and accounts for an average of 30% newly diagnosed cases per year of all new cancers among women in the United States. Statistically, one in 8 women in the USA will develop invasive breast cancer. For 2020, according to current estimation, about 276,480 new cases of invasive breast cancer will be diagnosed along with 48,530 new cases of non-invasive (in situ) breast cancers in women; 42,170 women are expected to die from the disease in 2020. In addition, in men, about 2620 new cases of invasive breast cancer are expected to be diagnosed in 2020 which accounts for 1 in 883 males (American Cancer Society 2019, 2020a, b; National Cancer Institute 2018). Of these cases, less than 15% of breast cancer cases in women have a family history and 5–10% of breast cancers in women relate to known genetic mutations inherited from either father or mother. While we know that aging is significantly related to breast cancer development and certain other risk factors such as alcohol consumption, smoking, and environmental pollutants have been identified we do not yet fully understand the molecular mechanisms underlying initiation of breast cancer development and progression in an individual. However, research in recent years has established that breast cancer is not only the result of mutated somatic cells but that the tumor microenvironment plays a contributing role in breast cancer manifestation. The breast cancer stromal microenvironment contains cells such as fibroblasts and adipocytes as well as immune and endothelial cells; cancer cells and stroma interact closely which contributes to regulation of breast cancer cell pathways. Oxidative stress has been implicated in the initiation steps. Oxidative stress is a major contributor to the cellular changes that take place in cells comprising the tumor environment as well as in the developing tumor itself. Oxidative stress promotes activation of fibroblasts to become myofibroblasts. In breast cancer, approximately 80% of stromal cells become activated resulting in secretion of elevated levels of growth factors, cytokines, and metalloproteinases, and production of hydrogen peroxide, a major contributor of oxidative stress. Oxidative stress in the tumor stroma facilitates tumor cell proliferation and tumor growth. This chapter will have 3 sections to cover (1) The role of mitochondrial metabolism in breast cancer development and progression with focus on mitochondria and

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ROS production; (2) The breast tumor microenvironment, metabolic interactions and crosstalk between stroma and tumor cells; and (3) Oxidative stress as an essential factor in the pathogenesis and progression of breast cancer.

The Role of Mitochondrial Metabolism in Breast Cancer Development and Progression with Focus on Mitochondria and ROS Production The Importance of Mitochondria for Cancer Cell Metabolism An important aspect of understanding altered metabolism in cancer cells came from early experiments by Otto Warburg (Warburg et al. 1927), opening up numerous subsequent studies to explore the “Warburg Effect” in more detail. The Warburg Effect was based on the major finding that mitochondria in cancer cells switch from oxidative phosphorylation (OXPHOS) to aerobic glycolysis using glucose as major energy source with an increase in glucose uptake and fermentation of glucose to lactate. However, while excellent new data have been reported on the Warburg Effect in cancer cells, we still do not yet completely understand the functional implications of the Warburg Effect on cancer initiation, progression, and cell proliferation (reviewed in Liberti and Locasale 2016; Potter et al. 2016; Bubici and Papa 2019). In more recent years, more detailed studies led to important new insights showing that oxidative stress and the cancer environment both contribute to the changes in metabolism turning non-cancer cells into cancer cells with subsequent tumor progression (Balliet et al. 2011a, b). Cancer cells utilize higher levels of glucose in the presence of oxygen with an associated increase in lactate production. The phenomenon of aerobic glycolysis, as described by the Warburg Effect has been elaborated for several cancers including breast cancer (Grover-McKay et al. 1998). However, it has later been shown that a dynamic interplay is likely to exist between oxidative metabolism and glycolysis (reviewed in Potter et al. 2016) which is an important aspect for the design of potential therapies considering that subpopulations within a tumor exist that may have different metabolic activities and different bioenergetics demands. Although the bulk of tumor cells depend on aerobic glycolysis tumors are metabolically less homogeneous than previously conceived containing subpopulations with dependency on mitochondrial respiration rather than aerobic glycolysis. Based on studies in recent years, we now know that cancer cells display a remarkable ability to either use glycolysis, oxidative phosphorylation (OXPHOS), and fatty acid oxidation as the source of energy, depending on different microenvironmental conditions (addressed in section “The Breast Tumor Microenvironment, Metabolic Interactions and Crosstalk Between Stroma and Tumor Cells”) including local acidosis, which inhibits glycolysis (Corbet et al. 2016; reviewed in Porporato et al. 2018). In this regard, while positron emission tomography (PET) is still the most commonly used imaging tool to diagnose cancer its detection is limited to the

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majority of cancer cell populations that display elevated uptake of glucose but it may miss subpopulations that utilize different metabolic activities based on metabolic plasticity. Because of the heterogeneity of a solid tumor PET imaging may not be optimal and new imaging modalities are being explored (Wang 2017), some of which are based on biomarkers.

Mitochondrial Functions in Non-cancer Cells and Changes in Cancer Cells Mitochondria play an essential role in various critical cellular functions including the main functions of ATP generation for cellular energy supply, calcium storage, transport and signaling, cellular quality control to maintain control of the cell cycle and cell growth, cellular differentiation, programmed cell death, and various others. Mitochondria are also regarded as important biosynthetic and signaling organelles (Chandel 2014). Mitochondria synthesize 95% of cellular metabolic energy and 1200 nuclear genes are involved in mitochondrial function. The mitochondrial genome contains 37 genes of which 24 genes are involved in processing 13 genes within the mitochondrial genome, producing the subunits essential for electron transport. These 13 key genes communicate closely with 93 nuclear proteins. Mutations in mitochondrial DNA are major drivers of increasing pools of reactive oxygen species (ROS) which contributes to proliferative advantages and tumor growth. Mitochondria are generally known as a cell’s most powerful organelles for energy production, which in normal cells oxidize glucose to produce energy. In normal cells, glucose is a major macronutrient needed for the production of cellular energy in form of ATP production through oxidation of its carbon bonds. Mitochondrial metabolism is essential for all mammalian life with end products being either lactate or CO2, through full oxidation of glucose via mitochondrial respiration. In contrast to mitochondrial respiration in non-cancer cells, in tumors as well as in other fast proliferating or developing cells, the rate of glucose uptake increases, resulting in the production of lactate, even when oxygen is present. In the absence of oxygen, glucose is converted into lactate, a highly efficient fuel that is also an important signaling molecule (Goodwin et al. 2015). Lactate can stabilize the hypoxia-inducing factor and increase vascular endothelial growth factor expression (Goodwin et al. 2015). The mechanisms driving mitochondrial respiration include the tricarboxylic acid (TCA) cycle and fatty acid β-oxidation enzymes in the mitochondrial matrix, thereby generating electron donors to support respiration, which involves electron transport chain (ETC) complexes and ATP synthase in the inner mitochondrial membrane (IMM) that play a role in oxidative phosphorylation. The mitochondrial respiratory chain (MRC), localized to the inner mitochondrial membrane (IM), contains a series of enzyme complexes (reviewed in detail in Bhattacharyya et al. 2014), and includes complexes I-IV (NADH-ubiquinone oxidoreductase, succinate dehydrogenase, ubiquinol-cytochrome c oxidoreductase, and cytochrome c oxidase), coenzyme Q (CoQ), and cytochrome c. Radicals are

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generated in part by electron leakage from MRC complexes I and III, which results in reduction of molecular oxygen. The amount of cellular ATP is regulated by mitochondrial and glycolytic ATP synthesis, which involves an electron flow through the respiratory chain, converting the energy released into an H+ gradient through the inner mitochondrial membrane. This gradient is responsible for the ATP synthesis through the adenosine triphosphate (ATP) synthase complex (complex V). Mitochondria are important building blocks for tumor anabolism; their functions are complex and include control of redox and calcium homeostasis, a role in transcriptional regulation, and in cell death. Mitochondria are significantly involved in all steps of oncogenesis through cancer cell-intrinsic and cell-extrinsic mechanisms (reviewed in Porporato et al. 2018). Cancer cell mitochondria are distinguished from non-cancer cell mitochondria by displaying several cancer-specific characteristics. Among the significant differences is the resistance to apoptosis. In non-cancer cells, the mitochondrial outer membrane permeabilization (MOMP) plays a critical role in the execution of regulated cell death (RCD) but cancer cells display resistance to RCD (Hanahan and Weinberg 2000) which initially presented difficulties in targeting mitochondria to eliminate cancer cells by inducing cancer cell death. It is now well recognized that some mitochondrial metabolites are sufficient to drive oncogenesis, and that some mitochondrial modifications can take place to adapt to bioenergetic or anabolic requirements of cancer cells, thereby enabling malignant cells to have metabolic plasticity. Because of the differences in mitochondrial metabolism in cancer and non-cancer cells, several therapeutic approaches have been proposed to target cancer cell mitochondria in order to eliminate specific cancer cell populations (reviewed in Porporato et al. 2018). As research on mitochondria has accelerated in recent years it is now also clear that tumor cell mitochondria have a broad spectrum of bioenergetic phenotypes and both aerobic glycolysis and mitochondrial metabolism take place in cancer cells which indicates that complex therapies are needed if tumor cell metabolism is to be targeted. Based on a large body of new research mitochondrial metabolism is now being explored as a promising target for the development of novel antineoplastic agents (Martinez-Outschoorn et al. 2017; Galluzzi et al. 2013). By now, cancer cell metabolism has become an increasingly promising target for new cancer therapies, as metabolic reprogramming/deregulated cellular energetics has become one of the hallmarks of cancer along with other hallmarks such as uncontrolled proliferative signaling, resistance to apoptosis, neo-angiogenesis, replicative immortality, invasion and metastasis, and evading growth suppressors (Hanahan and Weinberg 2000, 2011). While the exact reasons underlying the metabolic switch are not yet known, mitochondria are central in gaining a better understanding considering that mitochondria play an essential role in cell functions for cells to live or undergo apoptosis. Several drugs are available to affect mitochondrial metabolism, which includes the drug metformin. Metformin is an inhibitor of OXPHOS metabolism and of oxidative stress, and it can directly impair complex I of the respiratory chain (Owen et al. 2000). It induces tumor cell apoptosis and rescues stromal Cav-1

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(Cav-1 is addressed in section “The Breast Tumor Microenvironment, Metabolic Interactions and Crosstalk Between Stroma and Tumor Cells”). Modifications in mitochondrial metabolism plays a critical role in “malignant transformation,” a term used to refer to the conversion of a normal cell into a neoplastic precursor that is further modified to acquire uncontrolled proliferation potential, eventually resulting in dispersion and tumor progression with formation of distant macro-metastases (Weinberg et al. 2010). Mitochondria-derived malignant transformation involves several contributing factors (reviewed in Porporato et al. 2018) including mitochondrial reactive oxygen species (ROS). ROS can drive accumulation of potentially oncogenic DNA defects and it can activate potentially oncogenic signaling pathways. Abnormal accumulation of specific mitochondrial metabolites include fumarate, succinate, and 2-hydroxyglutarate (2-HG) (Gaude and Frezza 2014). Functional defects in MOMP or mitochondrial permeability transition (MPT) favor survival rather than RCD. To overcome and neutralize mitochondrial damage by ROS mitochondria have multiple antioxidant pathways including superoxide dismutase (SOD2), glutathione, thioredoxin and peroxiredoxins. Specific pathways of ROS generation as contributors to oncogenesis are reviewed in more detail by Vyas et al. (2016). We do not yet fully understand why cancer cells undergo altered metabolic changes although we are beginning to uncover the mechanisms leading to some of the changes. Aerobic glycolysis is generally an inefficient way to generate ATP compared to the per unit amount of glucose through mitochondrial respiration (Locasale and Cantley 2011) but the rate of glucose metabolism through aerobic glycolysis is higher; the production of lactate from glucose is about 10–100 times faster than the complete oxidation of glucose. These data show that the speed of producing ATP may present an advantage for cancer cells but we still do not yet have sufficient insights into the varied metabolisms employed by cancer cell mitochondria. At this time we only know that the increased glucose consumption is needed as a carbon source for anabolic processes that favor rapid cell proliferation (Boroughs and DeBerardinis 2015) which includes de novo generation of nucleotides, lipids, proteins, a variety of cellular building blocks and a diversity of pathways needed for cell cycle progression. There are other considerations in regard to why cancer cells might undergo aerobic glycolysis. Lactate production creates acidification of the microenvironment (discussed in section “The Breast Tumor Microenvironment, Metabolic Interactions and Crosstalk Between Stroma and Tumor Cells”) and allows different metabolic cross-talks. It may also allow communication with cells in the immune system to support pro-tumor immunity. Because signaling functions are important for generation and modulation of reactive oxygen species (ROS) (Liberti and Locasale 2016), it is important to maintain the appropriate balance of ROS (Sena and Chandel 2012). While aerobic glycolysis results in alterations in mitochondrial redox potential and changes in ROS generation (Locasale and Cantley 2011), excessive ROS damages cellular components including cell membranes and nucleic acids while insufficient ROS affects

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signaling processes that are needed for cell proliferation including inactivation of phosphatase and tensin homolog (PTEN) and tyrosine phosphatases. Mitochondrial DNA (mtDNA) is extremely sensitive to oxidative damage, as mitochondria lack protective histones and do not have efficient repair mechanisms, resulting in mitochondrial ROS-promoting malignant cell transformation. One of the hallmarks of tumor progression is the ability of cancer cells to metastasize into different organs which is known as metastatic dissemination. The metastatic cascade starts when cancer cells acquire the ability to form macroscopic lesions at distant sites (Lopez-Soto et al. 2017) which is a multi-step process most notably characterized by the epithelial-to-mesenchymal transition (EMT). Mitochondrial metabolism plays a role in this transition in that optimal mitochondrial biogenesis and OXPHOS are required for metastatic dissemination, as shown in models of breast cancer (LeBleu et al. 2014) and ovarian cancer. Mitophagic defects promote metastatic potential which is associated with mild ROS overproduction (Ishikawa et al. 2008; Porporato et al. 2014) that activates several signal transduction cascades including SRC and protein tyrosine kinase 2 beta (PTK2B) signaling (Porporato et al. 2014; Park et al. 2016). The cytoskeleton becomes significantly modified during EMT which facilitates cellular motility and migration of tumor cells to distant sites. Oxidative mitochondrial metabolism at the cellular leading edge is involved in this process (Caino et al. 2016).

The Breast Tumor Microenvironment, Metabolic Interactions and Crosstalk Between Stroma and Tumor Cells Because metabolic flexibility of cancer cells can be linked to environmental factors and because different environmental conditions affect different types of cancerassociated mutations that impact metabolism, it is important to gain a detailed understanding of the cancer cell microenvironment, the effects of mutations on cancer cell metabolism and the associated signaling pathways. Numerous studies on breast cancer have been performed on cell cultures but most of these studies represent a homotypic cell population and do not reflect the complexities of heterogeneous cell types within solid tumors that are affected by the tumor cell microenvironment. Such heterogeneous cell populations can have different mitochondrial metabolisms and it is now clear that different metabolic compartments exist within solid tumors which may require different treatment strategies. In breast cancer, tumor growth is promoted by glycolytic stromal cells in which the hypoxia-inducible factor (HIF)-1α drives glycolysis as a key transcription factor. We now also know that cancer cell metabolism is linked to oxidative stress and autophagy and that there is crosstalk between autophagy, ROS, and inflammation. It is further known that increased autophagy in the stroma promotes breast cancer progression and drives breast cancer aggressiveness. The tumor microenvironment, composed of non-cancer cells and their stroma, has increasingly become recognized as a major factor to influence and support tumor growth in complex interactions and metastatic synergy (reviewed in Li et al. 2007).

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Cells within the stroma include fibroblasts, immune cells, vascular cells, extracellular molecules of the extracellular matrix (ECM), and others. Fibroblasts are of particular interest, as they change from their normal state in normal tissue to an irreversible activated state in the tumor-surrounding stroma which is different from temporarily activated fibroblasts that are involved in wound healing. In normal tissue, a major function of fibroblasts is to produce the extracellular matrix (ECM) with type I, type III, and type V collagen and fibronectin, which allows the formation of the basement membrane through secretion of type IV collagen and laminin. In the tumor microenvironment, cancer-associated fibroblasts (CAFs) are the major cell types in the stroma and display an activated phenotype that has lost the ability to convert to a normal phenotype and to undergo apoptosis. The mechanisms for these changes are not yet fully understood but it is clear that they play a major role in supporting the tumorigenic environment and exert several functions that include promoting tumor growth. CAFs can generate glutamine that is induced by cancer cells. It shows how cancer cells can reprogram fibroblasts to play a role in the altered complex cancer cell mitochondrial metabolism. While reactive oxygen species are produced in all cell types (Tobar et al. 2010) and play a role in cellular signaling, an abnormally high amount of ROS will lead to cellular senescence, resistance to apoptosis, transformation into tumor cells, and increased aggressiveness of cells to promote breast cancer (Graham et al. 2010). As mentioned above, ROS can drive differentiation of normal fibroblasts into myofibroblasts, which will further increase oxidative stress in the microenvironment (Comito et al. 2012; Toullec et al. 2010; Waghray et al. 2005). In breast cancer, almost 80% of fibroblasts undergo activation (Kalluri and Zeisberg 2006) resulting in modified functions including secretion of type I collagen and metalloproteinases (MMPs) such as MMP-2, MMP-3, and MMP-9 which results in extracellular matrix turnover (Koch et al. 2009; Radisky et al. 2005). The activation of fibroblasts in the stroma results in significant amounts of ROS generation and subsequent tumor growth. Other cells in the tumor-surrounding stroma include immune cells such as monocytes/macrophages, neutrophils, and lymphocytes that are recruited to the tumor stroma. Monocytes differentiate into tumor-associated macrophages (TAMs) where their phenotype becomes altered and where they upregulate hypoxia-induced transcription factors. For neovascularization, progenitor cells are recruited into the stroma. In addition, cytokines and growth factors are secreted by cells of the stroma, further supporting tumor cell proliferation and tumor growth and creating a chronic inflammation environment and immune tolerance that allows tumor cells to grow while avoiding immune-mediated elimination. As such, the stroma is essential for maintaining a favorable environment for tumor growth. As stromal cells can influence tumor cells, tumor cells can reprogram stromal cells to generate and release inflammatory cytokines and increase oxidative stress in the microenvironment. Taken together, there is significant synergy and crosstalk between the tumor and its stromal microenvironment. Tumor progression requires a high degree of cellular plasticity to interact with components of the non-tumor microenvironment (Hensley et al. 2016; Tabassum

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and Polyak 2015; McGranahan and Swanton 2017). Aside from oncogenic drivers, the tumor microenvironment particularly influences metabolic activities in tumor cells which includes a role for cancer stem cells (CSCs) (Mayers et al. 2016; Davidson et al. 2016). The cancer stem cell population predominantly utilize a glycolytic metabolism (Palorini et al. 2014; Ciavardelli et al. 2014) which plays a role in influencing the various tumor cell populations. However, as addressed above, solid tumors contain highly diverse and heterogeneous cell populations and heterogeneity may also be present in the CSC populations to influence metabolic plasticity (Nakajima and Van Houten 2013; MartinezOutschoorn et al. 2011). Several cell signaling cascades are stimulated by oxidative stress in cancer stem cells. Elevated ROS have been considered as mutagens in cancer stem cells which influences their proper self-renewal ability. The complex signaling interactions between tumor cells and the microenvironment are still being explored and the multiple pathways need to be considered when designing drug regiments, especially since the tumor microenvironment regulates chemotherapy resistance that involves soluble factors, cell-cell interactions and induction of certain stem cell phenotypes. The question on how drug treatment is associated with oxidative stress and may affect the tumor microenvironment is also worth consideration in light of the fact that drugs frequently used in breast cancer patients including doxorubicin and paclitaxel caused mitochondrial loss and increased ROS production, as studied in murine myotube cell cultures (Guigni et al. 2018). These studies included findings that chemotherapy-derived oxidative stress also affects mitochondria in muscle leading to the observed skeletal muscle atrophy following chemotherapy administered during breast cancer treatment. As mentioned above and addressed in more detail in section “Oxidative Stress as an Essential Factor in the Pathogenesis and Progression of Breast Cancer,” oxidative stress and the cancer environment contribute to cancer progression (Balliet et al. 2011a, b). Oxidative stress results in a number of cellular metabolic changes, which in turn leads to further molecular changes that favor tumorigenesis. Such changes include inducing loss of stromal caveolin-1 (Cav-1). Loss of fibroblast Cav-1 has several consequences, as it promotes tumor growth and autophagy; it further results in secretion of inflammatory cytokines. Stromal loss of Cav-1 drives glycolysis. Based on these findings, the level of Cav-1 in the microenvironment can serve as marker for oxidative stress, hypoxia, and autophagy (Martinez-Outschoorn et al. 2010a, b, 2014). Cav-1 is a caveolin protein that is localized in caveolae in the lipid rafts of the plasma membrane; it plays an important role in cell signaling regulation, autophagy, and oxidative stress. In normal cells, Cav-1 is predominantly expressed in differentiated stromal cells such as normal fibroblasts. Research has shown that loss of Cav-1 in stromal cells results in tumor formation and progression while restoring Cav-1 can result in tumor regression. Loss of Cav-1 induces high levels of ROS, autophagy and glycolysis. Loss of stromal Cav-1 and upregulation of monocarboxylate transporter 4 (MCT4) are markers of progression from ductal carcinoma in situ to invasive ductal carcinoma (Martins et al. 2013).

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Oxidative stress has a fundamental impact on the tumor microenvironment as discussed above and includes breast cancer initiation and progression. As mentioned above, the tumor stroma contributes to tumor initiation, progression, and metastasis by inducing genetic instability through signaling pathways that can result in increased DNA damage results in increased mutagenesis and impaired DNA damage pathways that further affect tumor formation and progression (reviewed by Bindra and Glazer 2005). For example, BRCA1 mutations in breast cancer cells are associated with oxidative stress in the breast cancer stroma. They drive downregulation of Cav-1 and upregulation of MCT4 in stromal breast cancer cells (reviewed in Martinez-Outschoorn et al. 2014).

Oxidative Stress as an Essential Factor in the Pathogenesis and Progression of Breast Cancer ROS and Oxidative Stress Oxidative stress refers to a disruption of the redox balance toward an increase in prooxidants resulting in an imbalance that favors unstable reactive oxygen species (ROS). ROS are highly reactive, unstable, and short-lived molecules that are important players in both health and disease. They are derived from molecular oxygen lacking one or more unpaired electrons resulting from a diversity of endogenous and exogenous influences such as ultraviolet light exposure, ionizing radiation, carcinogen exposure, and various other factors that will be addressed below. While small concentrations of ROS are needed for essential physiological mechanisms including cellular signaling (Droge 2002), proliferation, and differentiation, abnormal increases of ROS concentrations will result in free radical-mediated chain reactions, and negatively affect proteins (Stadtman and Levine 2000), lipids, polysaccharides and DNA. ROS are established genotoxins that cause mutagenesis, and ROS can also trigger oncogenic signal transduction cascades such as signaling through the mitogen-activated protein kinase (MAPK), the epidermal growth factor receptor (EGFR) and other signaling cascades that are altered as a result of excessive ROS. As addressed in section “The Role of Mitochondrial Metabolism in Breast Cancer Development and Progression with Focus on Mitochondria and ROS Production,” mitochondria are the main site of oxygen metabolism, accounting for about 85–90% of the cellular oxygen consumption. ROS are continuously produced in all aerobic organisms as by-products of oxygen metabolism associated with mitochondrial respiration. While oxidative stress is the result of an imbalance and excessive ROS production, cellular antioxidant defense systems are available to counteract excessive ROS production. ROS and oxidative stress is more pronounced in the stroma of tumors, as tumor cells display higher antioxidant responses and increased antioxidant expression which can counteract the excessive ROS production to some extent. Oxidative stress in stroma can result in mitochondrial impairment that induces breast cancer growth (Wang et al. 2013).

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Diseases linked to oxidative stress related to mitochondrial dysfunctions aside from cancer are numerous and diverse and include Alzheimer’s disease, Parkinson’s disease, dementia, schizophrenia, bipolar disorder, epilepsy, stroke cardiovascular disease, chronic fatigue syndrome, retinitis pigmentosa, diabetes mellitus, autoimmune diseases, as well as fertility disorders and infertility among others. Several factors can play a role in increasing oxidative stress including oncogenes and external factors such as ionizing radiation, heat stress (reviewed in Slimen et al. 2014), imbalanced dietary consumption, hydrogen peroxide (H2O2; product of superoxide detoxification by MnSOD), and several others. Hydrogen peroxide (H2O2) is produced by a normal epithelium during normal wound healing and to convert normal fibroblasts to activated myofibroblasts that produce H2O2 to enhance the inflammatory signal. Heat stress increases H2O2 and hydroxyl radical formation. Production of H2O2 is particularly damaging, as it can diffuse freely across cell membranes, potentially mediating toxic effects at sites other than at the site of ROS production. ROS are also generated by NADPH oxidase while converting NADPH to NADP+ (Segal and Abo 1993). Oxidative stress is one of the major conditions resulting in enhanced ROS production and modifications of lipids, proteins and nucleic acids. This causes non-specific bioenergetic dysfunction. Mitochondrial membranes are particularly vulnerable to oxidative damage by ROS, as they are composed of phospholipid components and unsaturated fatty acids that can undergo peroxidation through a chain of oxidative reactions. Decreased mitochondrial functions and/or dysfunctions are the result of mitochondrial damage through oxidative stress caused by ROS (Agarwal and Prabhakaran 2005). Mitochondrial protein denaturation triggered by ROS include damage of subunits of the pyruvate decarboxylase complex, subunits of the ATP synthase, and enzymes of the tricarboxylic acid (TCA) cycle (Rhoads et al. 2006). As detailed in section “The Role of Mitochondrial Metabolism in Breast Cancer Development and Progression with Focus on Mitochondria and ROS Production,” in most mammalian cells, the mitochondrial electron transport chain is the major site of ROS production (Poyton et al. 2009). Enzymes that are involved in catalyzing the ROS-generating chemical reactions include peroxidases, NADPH oxidase, NADPH oxidase isoforms (NOX), xanthine oxidase (XO), lipoxygenases (LOXs), glucose oxidase, myeloperoxidase (MPO), nitric oxide synthase, and cyclooxygenases (COXs). As mentioned above, ROS are generated as by-products of normal cellular metabolic activities, primarily being specific byproducts of the mitochondrial electron transport chain or activation of the NADPH oxidases (NOX). In healthy conditions, the enzymes superoxide dismutase, glutathione peroxidase, and catalase protect cells from the damaging effects of ROS. In excess amounts, the oxygencentered small molecules such as superoxide, hydrogen peroxide, hypochlorous acid, chloramines and ozone can interact with lipids, carbohydrates, and nucleic acids with irreversible effects and inactivation of the target molecules. If the balance of ROS production and a cell’s capacity to rapidly detoxify reactive intermediates is disturbed, oxidative stress is initiated and progression to disease states follows.

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Oxidative stress on mtDNA has severe consequences, as several proteins of the ETC are encoded by mtDNA, and as ETC is the main source of ROS. Damage of mtDNA is more extensive than damage on nuclear DNA (nDNA) (Melegh et al. 1996; Yakes and Van Houten 1997), leading to ETC complex dysfunction, further increased production of ROS, thereby resulting in further oxidative damage, with cycles of no return to normal conditions. Based on the significant number of studies on oxidative stress and ROS-related cancers (well over 7500 original articles) numerous studies have been performed to investigate whether antioxidants can be applied as cancer therapies. However, this area of research is complex and highly controversial and the use of antioxidants as cancer therapy to counteract oxidative stress has been debated extensively with varying results (discussed in Sullivan and Chandel 2014; Slimen et al. 2014). The results are still not clear at this time and may vary for different stages of tumor progression and different types of cancer, and in some cases, it may even increase incidences of deaths (discussed in Sarmiento-Salinas et al. 2019).

Triggers for ROS Production and Oxidative Stress While a large body of research has shown that oxidative stress is implicated in the pathogenesis of cancer we do not yet specifically understand how oxidative stress causes transformation of normal cells to tumor cells. We do know that ROS can be produced in response to ultraviolet radiation, excessive alcohol consumption, cigarette smoking, nonsteroidal anti-inflammatory drugs, ischemia-reperfusion injury, chronic infections, and inflammatory disorders as well as inadequate or excessive nutrient consumption. It is estimated that inadequate or excessive nutrient consumption is associated with 30–35% of cancer cases and that more than 65% of all cancers occurs as a result of exposure to harmful substances that do not exist naturally in the environment and significantly involves the pathogenesis of breast cancer. Inadequate nutrition triggers oxidative stress at the cellular level and causes dysfunctions in the biological signaling cascades with increases in oxidative stress during tissue metabolism. Excessive alcohol consumption is prominently linked to increased breast cancer risk. The underlying mechanisms include disruption of intra- and extra-cellular network functions which are associated with chromosomal abnormalities, DNA methylation modification, DNA damage, signaling pathway alteration, tumor necrosis factor α (TNF-α) release, and retinoid metabolism impairment. Excessive consumption of low-quality carbohydrates plays a role in developing breast cancer by influencing plasma levels of glucose and insulin. Increased dietary carbohydrate and glycemic burden have been shown to be associated with an increase in ER /PR and ER breast cancer and consumption of food with high sugar or excessive carbohydrate content may increase the risk of breast cancer (reviewed in Saha et al. 2017). Insulin-related oxidative stress is associated with

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raises in H2O2 which augments tumor cell proliferation (reviewed in Saha et al. 2017). It is known that disruption of normal cellular homeostasis by redox signaling can affect all organs and may result in cardiovascular, neurodegenerative diseases and various cancers among others. Dissecting the signaling cascades at the cellular level initiated by oxidative free radicals as well as the physiological responses to stress is important to better understand the early changes for potential prevention and cures at the early stages of cancer.

Oxidative Stress Affecting Sites Distal from Tumor Tissue As much as early stage cancers, late stage cancers are equally affected by oxidative stress. Inflammation, cell signaling pathways and ROS molecular crosstalk communication all affect cells close to the tumor as well as cells in distal areas. Cancer cachexia is one of several little understood phenomena that typically presents severe complications associated with late stages of cancer including breast cancer. Although not as frequent in breast cancer as in other cancers it can occur and is characterized by serious weight loss. Cancer cachexia is the result of systemic effects of cancer cells mediated by oxidative stress, inflammation and autophagy causing metabolic reprogramming of fat, muscle, and liver resulting in loss of muscle mass. Up to 80% of patients with advanced cancer can develop some degree of cachexia. The mechanisms underlying the development of cancer cachexia includes the release of cytokines by tumor that promotes the formation of ROS, which then can influence catabolic pathways leading to severe muscle atrophy.

Conclusions and Perspectives Nearly 100 years have passed since Otto Warburg reported his pioneering discovery that cancer cell mitochondria primarily employ aerobic glycolysis using glucose as major energy source with an increase in glucose uptake and fermentation of glucose to lactate which is different from non-cancer cells that employ oxidative phosphorylation (OXPHOS). Since then a wealth of new data have been generated on cell, molecular and tissue levels showing that solid tumors contain heterogeneous cell populations being metabolically less homogeneous than previously conceived, containing various subpopulations with dependency on mitochondrial respiration rather than glycolysis. A dynamic interplay is likely to exist between oxidative metabolism and aerobic glycolysis which needs to be taken into consideration when designing therapies to control tumors. Although the bulk of tumor cells depend on aerobic glycolysis the different metabolic activities in subpopulations are thought to be in part the result of different interactions with the tumor microenvironment. We now know that cancer cells display a remarkable ability to either use glycolysis, oxidative phosphorylation (OXPHOS) and fatty acid oxidation as the source of energy in response to

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fluctuating micro-environmental conditions. We further know that cancer is not only the result of mutated somatic cells but that the tumor microenvironment plays a significant contributing role in tumor manifestation. The tumor microenvironment, composed of non-cancer cells and their stroma, has increasingly become recognized as a major factor to influence and support tumor growth in complex interactions and metastatic synergy. Reactive oxygen species (ROS) are major contributors that cause mutations in mitochondrial DNA (mtDNA) which in turn contributes to increases in the pool of ROS, thereby feeding a cycle of no return to normal conditions, thus increasing proliferative advantages and tumor growth. While small concentrations of ROS are needed for essential physiological mechanisms including cellular signaling, proliferation and differentiation, abnormal increases of ROS concentrations result in free radical-mediated chain reactions, thereby adversely targeting proteins, lipids, polysaccharides, and DNA. ROS are established genotoxins and aside from favoring mutagenesis, ROS can trigger potentially oncogenic signal transduction cascades. Excessive ROS damages cellular components including cell membranes and nucleic acids, while insufficient ROS affects signaling processes that are needed for cell proliferation. Mitochondrial DNA (mtDNA) is extremely sensitive to oxidative damage, as mitochondria lack protective histones and do not have efficient repair mechanisms, resulting in mitochondrial ROS promoting malignant cell transformation. Mitochondrial membranes are particularly vulnerable to oxidative damage by ROS, as they are composed of phospholipid components and unsaturated fatty acids whose double bonds can undergo peroxidation through a chain of oxidative reactions resulting in decreased mitochondrial functions and/or dysfunctions. Initiation, propagation and termination are the steps involved in damaging mitochondria through oxidative stress caused by ROS. Oxidative stress has a fundamental impact on the tumor microenvironment. Oxidative stress in the tumor stroma contributes to tumor initiation, progression, and metastasis by inducing genetic instability through signaling pathways that can result in increased DNA damage with subsequent enhanced mutagenesis and impaired DNA damage pathways. Oxidative stress in the stroma promotes activation of fibroblasts to become myofibroblasts. In breast cancer, approximately 80% of stromal cells become activated resulting in secretion of elevated levels of growth factors, cytokines, and metalloproteinases, and production of hydrogen peroxide. While we do not yet specifically understand how oxidative stress causes transformation of normal cells to tumor cells we know that ROS are produced in response to various endogenous as well as exogenous factors including ultraviolet radiation, cigarette smoking, excessive alcohol consumption, nonsteroidal anti-inflammatory drugs, ischemia-reperfusion injury, chronic infections, inflammatory disorders, and inadequate or excessive nutrient consumption. Excellent new studies uncovered the complex interactions and cross-signaling between the tumor cells and the microenvironment and showed that it is influenced by ROS and by changes in mitochondrial metabolism with significant effects on molecular cascades leading to cycles of no return to normal functions as known for non-cancer cells. New therapies take into consideration targeting the tumor cell microenvironment as well as the various tumor cell populations that require

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individualized and personalized treatment regimens. With these new insights new approaches are now possible to target the various and multiple abnormalities associated with breast cancer.

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Joytri Dutta, Sabita Singh, Ashish Jaiswal, Archita Ray, Pamelika Das, and Ulaganathan Mabalirajan

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Inducers of Lung Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genetic Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Epigenetics and Lung Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Environmental Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Link Between Inflammation, Oxidative Stress, and DNA Damage . . . . . . . . . . . . . . . . . . . . . . . . . . . . Vicious Cycle Between Inflammation and DNA Damage in Development of Lung Cancer (Fig. 1) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Relation Between Various Chronic Inflammatory Lung Diseases and Lung Cancer . . . . . . Sources of Reactive Oxidative Species (ROS) in Lung Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . DNA Repair Mechanisms in Lung Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Oxidative Stress/DNA Damage Markers in Lung Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Therapeutic Targets in Lung Cancer in Oxidative Stress, DNA Damage, and DNA Repair . . . Controversial Role of Oxidative Stress in Lung Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Important Clinical Trials Targeting Redox Candidates in Lung Cancer . . . . . . . . . . . . . . . . . . . . . . . Conclusions and Future Direction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract

The incidence, mortality, and morbidity rates of lung cancer vary from country to country due to variations in the quality of air, cigarette smoke habits, exposure to biomass fuels, and occupational exposure to environmental toxicants. The 5-year survival rate is just 3–6% for advanced stages of lung cancer. This is the warning sign that forces us to revisit the pathophysiology of lung cancer. Almost every etiological agent for the development of lung cancer has oxidative potential to cause oxidative stress by impairing the endogenous antioxidant defense mechanisms. Further, the resultant oxidative stress damages DNA and accumulated J. Dutta · S. Singh · A. Jaiswal · A. Ray · P. Das · U. Mabalirajan (*) Molecular Pathobiology of Respiratory Diseases, Cell Biology and Physiology Division, CSIR-Indian Institute of Chemical Biology, Kolkata, India © Springer Nature Singapore Pte Ltd. 2022 S. Chakraborti et al. (eds.), Handbook of Oxidative Stress in Cancer: Mechanistic Aspects, https://doi.org/10.1007/978-981-15-9411-3_57

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mutations lead to the development of lung cancer. Based on this view, finding potent antioxidants seems to be an attractive therapeutic target in lung cancer. However, recent evidence had demonstrated a controversial role of oxidative free radicals in lung cancer metastasis. As antioxidants have been shown to aggravate the lung cancer, drugs that target redox balance and prooxidants are being tried. Keywords

Oxidative stress · DNA damage · DNA repair · Lung cancer

Introduction The respiratory system is having numerous defense mechanisms against environmental pollutants like anatomical barriers (hair, cilia, and mucus), physiological barriers (sneezing, coughing, and even airway narrowing that might restrict the maximal entry of allergens/toxicants), immunogenic responses, antioxidant system, DNA repair mechanisms, etc. These defense mechanisms are continuously operational as there is an entry of air every second to our lungs. This could partially explain why different proteins involved in defense mechanisms are expressed constitutively in lung structural cells. Indeed, the constitutive expressions of various DNA repair proteins such as Ku70, Ku86, DNA-PKcs, etc. in normal human lungs indicate that these constitutive expressions may be required to correct the baseline DNA damage that might occur when the lungs are exposed to a variety of environmental pollutants (Caramori et al. 2011). Similarly, our lab had demonstrated that knocking down of various key proteins is sufficient to cause spontaneous asthma and lung injury features. All these indicate that structural cells of the lung are continuously fighting against environmental pollutants to maintain lung homeostasis. Thus, the airway does not just act as conducting air pipe but also as an effective protective force to protect the alveoli, which are the vital units of the respiratory system. However, if these protective mechanisms fail or exhaust, the structural cells become helpless victims of exogenous/endogenous pollutants/toxicants and thus leads to a variety of lung diseases including lung cancer. After prostate and breast cancers in both men and women, respectively, lung cancer is the next common cancer (de Groot et al. 2018). Lung cancer is further subclassified into various types based on cellular origin and morphology like adenocarcinoma. Among the two major types of lung cancer, NSCLC is very common (Adcock et al. 2011). Both genetic and environmental factors play crucial roles that cause an imbalance between oncogenes and tumor suppressor genes to initiate the development of lung cancer. As a result, there would be an overexpression of oncogenes and downregulation of tumor suppressor genes. In addition to this imbalance, various other imbalances like oxidant/antioxidant, anti-inflammatory/pro-inflammatory, and DNA damage/DNA repair have been linked to the lung cancer development. However, all these imbalances are tightly interconnected (Fig. 1). The pulmonary inflammation

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Fig. 1 The interconnection between inflammation, oxidative stress, DNA damage, and lung cancer. The exposure of lungs to cigarette smoke and environmental and indoor air pollutants lead to inflammation. This triggers the generation of reactive oxygen and nitrogen species in the inflammatory milieu. If this oxidative stress is not controlled by antioxidant enzymes, it hurls damage to the DNA. This leads to mutation and genomic instability resulting in lung cancer. Lung inflammation can also promote cell survival and proliferation that leads to lung cancer. The DNA damage response generated due to DNA damage can also promote lung inflammation. Oxidative stress and subsequent lipid peroxidation can activate pro-survival and detoxification pathways resulting in chemoresistance

caused by endogenous/exogenous insults leads to the release of a variety of oxidative free radicals that generate oxidative stress and could further could activate various signaling pathways and induce the expression of oncogenes which initiate lung cancer. The imbalance between the generation of oxidative free radicals and endogenous antioxidants leads to oxidative stress that affects various biomolecules. Reactive oxygen species (ROS) produced during endogenous metabolic reactions are mostly hydroxyl radical (OH•), superoxide anion, hydrogen peroxide (H2O2), and organic peroxides. Oxidative free radicals are involved in every stage of cancer. For example, ROS introduce mutations at initiation stage that cause an imbalance between cell proliferation and death at the stage of promotion, and further add DNA alterations at the stage of progression. Thus, ROS has a vital role in every stage in cancer

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development and progression. In this chapter, we will be traveling through the axis of pulmonary oxidative stress like how it is associated with pulmonary inflammation and how it leads to DNA damage towards the development of lung cancer. In addition, we will be reviewing about the oxidative stress/DNA damage markers in lung cancer along with an exploration of possible therapeutic targets that have a link with oxidative stress. Further, we have emphasized the controversial role of oxidative stress in lung cancer and this property could prohibit us to use antioxidants for lung cancer therapy.

Inducers of Lung Cancer Genetic Factors Since only 15% of all-time smokers develop lung cancer and 10% of the never smokers also develop lung cancer indicates the involvement of susceptible or resistant genetic factors in addition to the environmental factors (Adcock et al. 2011). Lung cancer in nonsmokers (LCINS) is a great concern and is the seventh leading cause of cancer deaths. While there are many factors like exposure to secondhand smoke, use of biomass fuels in the developing countries, occupational toxicant exposure, etc. to cause LCINS, one cannot rule out the involvement of genetic factors (de Groot et al. 2018). Three genetic loci, 15q25-26, 5p15, and 6q21, were found to be linked with heightened incidences of lung cancer through genome-wide scan studies. These loci encode telomerase reverse transcriptase and G-protein signaling. In addition to these genome-wide association study (GWAS) associated genes, most of the lung cancer– related mutations are found in genes that are crucial in cell signaling and tumor suppressors like K-ras, ErbB family genes, etc. Genomic studies in SCLC have revealed that TP53 (tumor protein 53) and RB1 (retinoblastoma) protein are inactivated and abnormal activation of PI3K/mTOR pathway causes aberrant activation of oncogene MYC (Fang et al. 2018). The most prominent mutations at individual gene level in SCLC are found in tumor suppressor genes TP53 and RB1 with nearly uniform loss of function. The drastic reduction in the expression of TP53 is very common phenomena in SCLC (Semenova et al. 2015). Exogenous or endogenous oxidative stress cause cellular DNA damage or hypoxia which activate TP53 to maintain genomic integrity through cell cycle arrest or apoptosis. Recent studies have demonstrated the occurrence of mutant TP53 in healthy bronchial epithelium accompanying SCLC. These indicate that mutations in TP53 could be an initiating event in developing SCLC. RB1 (retinoblastoma susceptibility gene), a tumor suppressor gene, is also found to be inactivated in majority of the SCLC forms. The central role of RB1 in cell cycle regulation is to mediate the G1 to S phase transition of cells in the cell cycle and regulate cellular differentiation. The mutant form of RB1 is unable to inhibit cell cycle progression while it can still promote cellular differentiation (Semenova et al. 2015).

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The metabolites of harmful chemicals (by xenobiotic metabolizing enzymes) present in the cigarette smoke can be toxic in some instances, indicating the role of xenobiotic metabolism in developing lung cancer. Human lung cells express major xenobiotic metabolism enzymes, both phase I [cytochromes P450 (CYPs), myeloperoxidase (MPO), flavin monooxygenases, and microsomal epoxide hydrolases (EPHX)] and phase II enzymes [such as glutathione S-transferases (GSTs)]. Genetic polymorphism in these enzymes including EPHX, CYPs, and GST that activate or detoxify tobacco smoke carcinogen might serve as a risk factor for developing squamous cell carcinoma (Adcock et al. 2011).

Epigenetics and Lung Cancer In addition to the genetic factors, epigenetic also play critical role in the regulation of the genes that are linked to oxidative stress like superoxide dismutase (SOD) and their downstream signaling pathways. Modulation of these genes manifests the disease and thus epigenetics can serve as potential therapeutic target in the treatment strategy. Histone deacetylases (HDACs) are known to form a protein complex by deacetylating the ε-N acetyl lysine amino acid on histone, thereby inhibiting transcription. An increased expression HDAC 1 was found in NSCLC stage III or IV, whereas mSin3A, a corepressor of HDAC multiprotein complex, was downregulated in NSCLC. Therefore, HDAC inhibitors can be considered as a new therapeutic strategy, which blocks the suppression of antioxidant genes and tumor suppressor genes. Oxidative stress induces increased acetylation of histone by histone acetyltransferase (HAT), which in turn stimulates NF-κB binding to DNA. This binding triggers a hike in the levels of pro-inflammatory IL-8, IL-6 to cause lung inflammation which may ultimately lead to lung cancer. Thus, epigenetic therapeutic strategies targeting DNA methylation by DNA methyltransferase inhibitors and histone acetylation by HDAC inhibitors for the treatment of NSCLC seem to be beneficial in lung cancer (Reuter et al. 2010; Shtivelman et al. 2014; Lawless et al. 2009).

Environmental Factors Involvement of genetic factors alone cannot explain the increased incidence of lung cancer worldwide. On the other hand, environmental factors like increase in traffic emission, pollution-causing factories, industrialization, and release of both natural and human-made toxicants also are leading to increase in the lung cancer incidence. Everyday our lungs are exposed to indoor and outdoor pollutants/oxidants. Various studies have shown that exposure to metal powder, tobacco smoke, mineral fibers, airborne particulate matter (PM10 and PM2.5), and pollution from diesel and petrol engines are involved in producing oxidative stress and can be risk factors for developing lung cancer (Valavanidis et al. 2013). The environmental factors have more or less same mechanism to induce lung cancer, as these may be involved in

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generation of free radicals that can further oxidize DNA, lipid, and proteins in cell and lead to lung injury and inflammation (Valavanidis et al. 2013). Cigarette smoke is the major driving cause for the development of lung cancer. Tobacco smoke contains hundreds of carcinogens/oxidants that can either be directly involved in generating oxidative stress or leads to mutation in oncogenes and tumor suppressor genes and ultimately result in lung cancer (Paz-Elizur et al. 2003). The oxidative stress generated by cigarette smoke increases the expression of cytoplasmic p21WAP/CIP1 to promote cell cycle from G1 to G2/M phase; this may in turn disturb the balance between apoptosis/proliferation towards hyper-proliferation in lung epithelial cells, resulting in transition of normal epithelia to carcinomatous status in smokers (Adcock et al. 2011). While air pollution leads to highest mortality worldwide, the participation of traffic emission–mediated air pollution is reducible. The magnitude of problem was very much felt when the lockdown was implemented in most of the countries during the coronavirus pandemic in 2019–2020. The major traffic emission pollutants are carbon monoxide, carbon dioxide, and nitrogen dioxide and one report had demonstrated that more than 50% cumulative CO, CO2, and NO2 is due to traffic emission (Salvi and Salim 2019). These three gaseous pollutants are having capability to induce oxidative stress along with an elevation of various pro-inflammatory cytokines. Though developed countries and most of the cities in developing countries use huge electricity, vehicles, and industries for fulfilling the modern needs, the people in villages of developing countries also have numerous environmental induced respiratory diseases like COPD and lung cancer. This indicates that the use of biomass fuels in economically deprived population in developing countries is responsible for the same. In addition to the developing countries, people from developed countries also use wood fires for room heating. The smoke from biomass contains more than 200 chemicals like polycyclic aromatic hydrocarbons (PAH). In addition to numerous toxic compounds, biomass smoke also contains potent oxidants. In addition to biomass smoke–derived oxidants, host immune cells also produce numerous oxidants in response to biomass smoke exposure (Capistrano et al. 2017). These cumulative oxidants could be responsible for the development of COPD and lung cancer.

Link Between Inflammation, Oxidative Stress, and DNA Damage Vicious Cycle Between Inflammation and DNA Damage in Development of Lung Cancer (Fig. 1) A cause and effect relationship between inflammation and cancer was initially put forward by Galen. After that, Virchow observed that leucocytes infiltrate malignant tissues and proposed that cancers develop at areas of chronic inflammation. With the persistence of inflammation, the risk of associated carcinogenesis increases (Mouronte-Roibas et al. 2018; Azad et al. 2008; Kawanishi et al. 2017). Moreover, recent evidence indicates that infections in the regions of chronic inflammation are responsible for one-third of all the known cancers (Azad et al. 2008).

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Certain chemical, physical, and immunological factors can induce chronic inflammation. Inhalation of particulate matters (such as quartz particles, diesel engine exhaust, and mineral asbestos) and cigarette smoke can cause chronic inflammation in respiratory system and may lead to lung cancer. During inflammation, inflammatory cells (including macrophages and neutrophils) and epithelial cells produce reactive oxygen species. During the normal cellular metabolism, moderate concentration of oxygen-free radicals or more precisely ROS and reactive nitrogen species (RNS) are generated but higher concentrations of ROS and RNS are detrimental for the living organisms as these hurl oxidative damage to biomolecules like DNA, proteins, lipids, etc. (Valavanidis et al. 2013). Excessive ROS if not controlled by enzymatic or nonenzymatic antioxidant system can lead to chronic inflammation (Valavanidis et al. 2013). Under inflammatory conditions, inflammatory cells and epithelial cells also produce NO by inducible nitric oxide synthase (iNOS). NO can diffuse to neighboring cells via plasma membrane and enter into the nucleus. NO can react with superoxide to form peroxynitrite (ONOO
) which leads to guanine nitration to form 8-nitroguanine. Thus, both ROS and RNS can damage DNA and can form 8-oxo-7, 8-dihydro-2-o-guanosine, and 8-nitroguanine, which further form apurinic site in DNA and thus 8-nitroguanine leads to single-base substitution (Kawanishi et al. 2017). Inflammation not only induces DNA mutation but also impairs DNA repair machinery by inhibiting a number of DNA repair enzymes including O-6methylguanine-DNA methyltransferase (MGMT) (Kawanishi et al. 2017). Moreover, DNA damage response can also induce inflammatory microenvironment which is mediated by hypoxia (Kawanishi et al. 2017). Thus, both DNA damage response and inflammation form a vicious cycle (Fig. 1). DNA damage response can promote inflammation by accelerating senescence and necrosis. Further, some DNA repair proteins are involved in NF-kB-mediated transcription of some inflammatory genes. There are many DNA repair proteins involved in inflammation including PARP1, OGG1, and ATM/ATR. PARP1 (poly ADP-ribose polymerase 1) detects and binds to the single-strand DNA breaks. In many studies, PARP1 has been shown to act as a pro-inflammatory protein. Similar to PARP1, OGG1 (BER glycosylase) can amplify inflammation via NF-kB. The double-strand break sensors ATM and ATR both can promote NF-kB signaling independent of downstream DNA damage response (Kay et al. 2019). DNA damage can also promote inflammation indirectly via Fas ligand– mediated apoptosis (Kay et al. 2019). All these evidences suggest that the inflammatory microenvironment could induce DNA damage and mutation and thus can lead to carcinogenesis by genomic instability.

Relation Between Various Chronic Inflammatory Lung Diseases and Lung Cancer In chronic obstructive pulmonary disease (COPD), chronic inflammation and oxidative stress are well-known drivers of carcinogenesis (Kwong et al. 2017). Various immune cells like neutrophils and macrophages lead to increased oxidative stress

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and also increased release of protease and thus lead to DNA damage and potentiate tumor migration, respectively. Moreover, pulmonary macrophages release supportive factors in the tumor niche. Also there is an exhaustion of the T cell population. Consequently, the clearance of cancer cells by cytotoxic T cells is suppressed that eventually leads to tumor evasion. IL-17A, a cytokine secreted by nonconventional T cells in COPD, is increasingly being recognized as a mediator of tumor development (Bozinovski et al. 2016). If the resultant DNA damage in COPD conditions is not properly repaired, it leads to accumulation of mutations. RNOS activity inhibits mechanisms that are able to prevent mutations like DNA repair and apoptosis (Durham and Adcock 2015). Failure in DNA repair, mutations that cause increased expression and function of oncogenes, and mutations that cause decreased expression and function of tumor suppressor genes ultimately leads to cancer (Azad et al. 2008). RNOS can also inhibit protective proteins like anti-proteases (Durham and Adcock 2015). Damage to the alveolar cells is repaired by proliferation of basal alveolar stem cells (BASC). However, recurrent induction of BASC proliferation makes the cells susceptible to malignancy. Asthma is another chronic inflammatory disease of the lungs (Houghton et al. 2008). Few studies have found that there is a positive association between asthma and the risk of lung cancer (Qu et al. 2017), but the mechanisms for such association were not clearly investigated. In silicosis, physiological host response to chronic inflammation minimizes the immune stimulation in the lungs. This leads to cause the suppression of immune response and inhibit the activity of T cells leading to malignant transformation of cells (Sato et al. 2018). In asbestosis, a chronic inflammatory milieu is created in the lungs where the cells receive prosurvival signals like TNF-α. Moreover, both direct and ROS-mediated indirect DNA damage occur. Collectively, these factors lead to the development of cancer (Napolitano et al. 2014).

Sources of Reactive Oxidative Species (ROS) in Lung Cancer As lungs are exposed to high oxygen levels continuously, the oxidative stress in the lungs is an inevitable phenomenon (Di Rosanna and Salvatore 2012). Oxidative stress is a condition of imbalance between ROS production and the efficacy of various endogenous antioxidants to eliminate them. ROS, chemically reactive molecules, damages almost every major biomolecule like nucleic acids, lipids, and proteins and altering their functions. Potential endogenous sources for the formation of ROS are cell organelles such as mitochondria, peroxisomes, and endoplasmic reticulum. In addition to these organelles, enzymatic systems like cytochrome P450 metabolism and nonenzymatic systems can also induce the formation of oxidative free radicals (Jelic et al. 2021; Villegas et al. 2014). In the mitochondria, during the electron transport chain reaction, a large percentage of oxygen is converted to water, whereas about 5% anion is converted to superoxide (Jelic et al. 2021). There are several nonmitochondrial sources of ROS within the cell (Di Rosanna and Salvatore

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2012). Peroxisomes generate huge amounts of hydrogen peroxide and superoxide anions (Jelic et al. 2021). In the endoplasmic reticulum, the resident cytochrome P-450 oxidizes pollutants, drugs, toxins, and unsaturated fatty acids to produce •O2 - and H2O2. When the ROS production surpasses the endogenous antioxidant response, major biomolecules like lipids, proteins, and nucleic acids are oxidized. This may cause genetic and/or epigenetic changes that lead to dysregulation of oncogenes and tumor suppressor genes that eventually lead to cancer (Di Rosanna and Salvatore 2012). Enzymatic systems like NADPH oxidase (NOX), cytochrome P450, cyclooxygenase, aldehyde oxidase, dihydroorotate dehydrogenase, tryptophan dioxygenase, xanthine oxidase, and nitric oxide synthase are major sources of intracellular ROS (Filaire et al. 2013). The NOX generates superoxide by reducing oxygen (Prasad et al. 2017; Ilonen et al. 2009). Various NOX isoforms are expressed in lung tissues like NOX2, dual oxidase, and NOX4 (Ilonen et al. 2009). In addition to enzymemediated generation of reactive oxygen and nitrogen species, Fenton reaction generate oxidative free radicals without enzyme (Villegas et al. 2014). Exogenous sources of ROS inducers are various pollutants and also radiation as ionizing radiation generates ROS by interacting with water (Prasad et al. 2017). Particulate matter from the environment also can induce oxidative DNA damage in lung epithelial cells (Jelic et al. 2021). Cigarette smoke (CS), the main causal agent of NSCLC, contains numerous oxidants and oxidant-producing compounds. In addition, CS leads to oxidative stress via various immune cells (Ilonen et al. 2009).

DNA Repair Mechanisms in Lung Cancer DNA is the integral framework of human body. Damage to the DNA plays a driving role in lung cancer and DNA damage has been demonstrated in most of the lung cancer patients who had smoking habit, though all smokers do not suffer from lung cancer. Thus, cigarette smoking plays a causative role in lung cancer (Mattson et al. 1987). Various studies performed worldwide have shown that tobacco smoke contains some harmful chemicals and carcinogens that cause mutations or significant damage in the DNA strands. The different types of DNA damage include single or double strand break and addition of unwanted DNA adducts. DNA damage response (DDR) is a cellular homeostatic mechanism to detect DNA repair and initiate signaling to recruit DNA repair proteins to repair damaged DNA. The activation of DDR results in the repair of the broken DNA strand or cell cycle arrest and eventually cell death. The unwanted DNA adducts which goes unnoticed results in mutations in the oncogenes and tumor suppressor genes leading to lung cancer. By removing and preventing unwanted DNA mutations, the various DNA repair pathways thus play a regulatory role in preventing lung cancer (Cooke et al. 2003). Studies show that in contrast to healthy individuals, the score of DNA repairing is low in patient suffering from lung cancer. In other words, impaired DNA repair mechanism could increase cigarette smokers’ susceptibility towards lung cancer. Apart from this, inflammation indirectly also leads to lung cancer. Upon exposure to

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tobacco smoke, ROS and RNS are released from the inflammatory cells which cause oxidative stress by disturbing the redox homeostasis and increasing DNA damage (Reuter et al. 2010). The major DNA repair pathways include mismatch repair for single-base mismatch errors, nucleotide excision repair for pyrimidine dimers, base excision repair for small DNA lesions, homologous recombination repair, and nonhomologous end joining for double-strand break repair. The oxidative damage lesions are repaired by base excision repair mechanism present in nucleus and also in mitochondria. Excision repair cross-complementation group 1(ERCC1), a major protein involved in NER pathway, is found to be deficient in NSCLC patients (Simon et al. 2007). Deficiency in DNA repair genes increases mutation rate to convert normal cell to cancer cell. The curative treatment for NSCLC is radiotherapy and chemotherapy. However, sometimes these cancer treatments become resistant and can pose some DNA damaging effect (Chang 2011). With advancement in medicine, many studies came up which target the DDR pathway for cancer therapy. In the aggressive malignant SCLC, there is a huge reduction in expressions of Tp53, RB1 proteins resulting in uncontrolled cell proliferation and genomic instability. DNA repair mediators like PARP, ATM, ATR, and CHEK1 are found to be higher in SCLC patients compared to normal individuals. To minimize these harmful effects, some epigenetic modulators like poly ADP ribose polymerase-1 (PARP-1) inhibitor and CHK1 inhibitor are given alongside to prevent the unchecked growth of tumors (Sen et al. 2018). However, PARP-1 inhibition stops the repair of single DNA strand and lead to accumulation of double-strand breaks. It will be more damaging if the homologous recombination (HR) repair mechanism for correcting the double strand break is deficient in cells. Thus, to prevent this, epigenetic regulators targeting homologous recombination pathway in HR-deficient NSCLC cells is another approach (Ji et al. 2020).

Oxidative Stress/DNA Damage Markers in Lung Cancer ROS and other free radicals accumulate during the state of oxidative stress. This can cause modifications in the DNA bases. The most occurring ubiquitous oxidized DNA damage by-product is 8-oxo-2’deoxyguanine (8-oxo-dG) or its tautomer 8hydroxy-2-deoxyguanine (8-OHdG). It occurs when hydroxyl radical acts on the C8 carbon atom of the guanosine nucleoside residue, causing G to T base transversion and mutations, and can alter gene methylation pattern (Valavanidis et al. 2009). Another ROS-induced DNA modification is the formation of O6-methylguanine which is mispaired with thymine base and causes mutation (Valavanidis et al. 2009). 8-oxo-dG or its tautomeric form 8-OHdG serves as a prognostic biomarker in lung cancer. Unlike nonsmokers, the level of 8-OHdG level is found to be higher among smokers. In various diseases, the positive correlation was found between 8-OHdG and severity of the disease (Yano et al. 2009). 8-oxoguanine DNA N-glycosylase (OGG) is a base excision repair (BER) enzyme which

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removes the N-glycosyl bond to repair the DNA adduct 8-oxo-dG. Compared to healthy individuals, the OGG activity in blood PBMC is found to be reduced in patients suffering from NSCLC. Thus, a correlation could be found between reduced OGG and cancer risk factor, making it a marker for lung cancer (PazElizur et al. 2003). The DNA repair enzyme O6-methylguanine methyl transferase (MGMT) eliminates the DNA alkylation adducts caused by carcinogens. The amount of MGMT enzyme can be correlated with the amount of DNA damage caused by alkylating agents. It is seen that in NSCLC patients, the MGMT promoter is methylated. Divergent methylation in the promoter region can thus be a potential biomarker in detecting lung cancer in the clinical samples of BAL fluid and serum (Nakagawachi et al. 2003). Apart from this, enzyme MPG (methylpurine DNA glycosylase) that acts on hypoxanthine and APE1 (apurinic/apyrimidinic endonuclease 1) for furanyl abasic sites are possible biomarkers in detecting lung cancer. Thus, in lung cancer, along with low DNA repair score, a lot of DNA repair genes are upregulated. The DNA repair score correlates with the enzyme activity assay values (Paz-Elizur et al. 2019). However, as the levels of 8-OHdG, OGG, and MGMT can also be modulated in various chronic inflammatory lung diseases with increased oxidative stress, these cannot be used as pure biomarkers of lung cancer. In any event, these can be useful to assess the prognosis of lung cancer. Enhanced lipid peroxidation is seen in lung cancer patients along with alterations in the serum parameters. Studies show that there is a significant enhancement of 4 hydroxynonenal (4-HNE), derived from peroxidation of omega-6 polyunsaturated fatty acid, in lung cancer. Codon polymorphism in the GPX-1 gene that encodes for the antioxidant enzyme glutathione peroxidase is also linked with an increased risk of lung cancer. The GPx is known for its property of converting detrimental lipid peroxides to less toxic products like hydroxyl fatty acids. 8-isoprostane is another marker that indicates lipid peroxidation. Studies have shown that the level of 8isoprostane is higher in NSCLC patient and in response to chemotherapy treatment, the level decreases. Thus, 8-isoprostane serves as a good biomarker as it can also be used to assess patient’s response to the treatment (Pelclova et al. 2008).

Therapeutic Targets in Lung Cancer in Oxidative Stress, DNA Damage, and DNA Repair A) Therapeutic Targets for Tumor Cell Survival 1. NADPH Oxidase Enzymes The NADPH oxidase or NOX are a class of enzymatic family that significantly contributes in promoting the survivability of tumor cells in lung cancer, especially the expression of NOX-5 has been elucidated to stimulate cellular proliferation. NOX enzymes are known to increase tumor cell survival via their capabilities to generate more ROS-free radicals (Chakraborti et al. 2020; Reuter et al. 2010; Lawless et al. 2009)

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2. Serine Threonine Kinase Akt It downregulates antioxidant defenses and enhances the survival of tumor cells. The expression of the oncogene c-Myc (cellular myelocytomatosis) and cyclin D1 are stabilized by the Akt protein, thus promoting tumorigenesis. Akt also counteracts apoptosis by inactivating the proapoptotic proteins like caspase-9, BH3 (Bcl-2 homology-3), and BAD protein (Bcl-xl/Bcl-2associated death promoter), and enhancing the activity of NF-κB. The translocation of MDM2 into the nucleus, an ubiquitin ligase which antagonizes the action of p53 in inducing apoptosis, is assisted by the Akt. Thus, by enhancing the oxidative metabolism and opposing apoptosis, Akt has a prominent role in integrating a myriad of decisive oncogenic signals (Chakraborti et al. 2020). B) Therapeutic Targets for Tumor Cell Proliferation 1. Mitogen-Activated Protein Kinase (MAPK) Uncontrolled cellular proliferation in lung cancer is promoted by upregulation of various proteins involved in cellular survival, proliferation, and progression. Oxidants have shown a significant impact on the MAPK/ AP-1 pathways. The family of MAP kinases phosphorylates a variety of TFs (transcription factors), thus altering the expression of genes. Out of all these, the ERK pathway has the most common linkage with cellular proliferation. The activated ERK, JNK (c-Jun N-terminal kinase), and subfamilies of p38 MAPK by the cellular redox balance induces the activity of stressors like AP-1, cytokines, etc. Activated JNK in turn translocates to the nucleus to increase the transcription of key proteins such as ATF-2 (activating transcription factor-2), c-Jun, etc. towards tumor cell survival and proliferation (Chakraborti et al. 2020; Reuter et al. 2010). 2. NF-κB Various tumor-promoting carcinogens like UV rays, asbestos, phorbol, and chemicals present in cigarette smoke are found to activate NF-κB which in turn regulates angiogenesis, cell transformation, and proliferation. Mild oxidative stress leads to moderate activation of NF-κB, whereas extensive oxidative stress can inhibit the same. Hence, a complex relation exists between ROS and NF-κB and the mechanism underlying is not clear. Also, cellular protection is mediated by NF-κB against oxidative stress by inducing ferritin heavy chain and SOD-2 genes. NF-κB is activated via TNF and IL-1 by the participation of ROS as a second messenger. Thus, it can be concluded that inhibition of the ROS leads to the suppression of NF-κB and thus inhibiting tumor cell proliferation. The involvement of ROS in activating NF-κB has been implicated by the fact that majority of the stimulus are capable of activating NF-κB which are in turn counteracted by antioxidants like thiols, green tea polyphenols, etc. This might not be much convincing as antioxidants have multiple targets (Reuter et al. 2010; Shtivelman et al. 2014).

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C) Therapeutic Targets for Tumor Cell Invasion 1. Intracellular Adhesion Molecule-1 (ICAM-1) Oxygen-free radicals may enhance metastasis and tumor invasion by regulating the rates of cell migration. This could possibly be because of enhanced expression of cell motility regulating proteins and abated adhesion of tumorous cells to the basement membrane. ROS regulates the expression of ICAM-1, a cell surface glycoprotein, mostly by triggering NF-κB. ICAM1 conjointly with IL-8 has a prospective role in regulating tumor metastasis (Reuter et al. 2010). 2. Matrix Metalloproteinases MMP is accredited to drive tumor cell invasion and their increased expressions regulate the cell invasion and spread of the malignant tumors. MMP-3, MMP-13, and MMP-10 are found to be regulated by oxidants directly and are often implicated in the invasive potential of malignant tumors. Gelatinases (MMP-2 and 9) play prominent role in tumor cell metastasis by degrading type-IV collagen. These are activated post-transcriptionally by persistent oxidative stress. Most of the components of basement membrane and extracellular matrix are cleaved by MMPs. ROS is one of the key regulators of MMP activation as oxidative free radicals react with thiol group of protease catalytic domain and activate MMPs like MMP-2. Hydrogen peroxide, nitric acid donors, and iNOS induce the expression of various MMPs (Reuter et al. 2010). D) Therapeutic Targets for Tumor Cell Angiogenesis 1. Oncogenic RAS (Rat Sarcoma Virus) An angiogenic response by host is induced by solid tumors for meeting the cellular nutrients and oxygen requirements. This sort of response transforms the poorly vascularized, less invasive tumors into angiogenic and extremely invasive tumors. HIF-1α (hypoxia inducible factor-1α) is stabilized by the overexpressed RAS which in turn enhances the VEGF-A (vascular endothelial growth factor-A) to increase vascularity of the cancer tissue. A number of chemical antioxidants have been found to block the mitogenic activity of RAS, implicating the direct involvement of ROS in cancerous transformation (Reuter et al. 2010). 2. Hypoxia Inducible Factor-1α Cellular stress factors like hypoxia, nutrient deficiency, and ROS induce angiogenic response. HIF-1α, VEGF, and oxidation of DNA are found to be upregulated due to the overproduction of ROS which is induced by the oxidative stress in hypoxic conditions (Reuter et al. 2010; Lawless et al. 2009). 3. Heme Oxygenases-1 A cellular stress response gene, heme oxygenase 1(HO-1), protects the lungs from oxidative stress–based insults like cigarette smoke by breaking down heme to biliverdin, iron, and carbon monoxide. Both biliverdin and its

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derivative bilirubin have antioxidant effects. Carbon monoxide itself in low concentration can confer similar tissue-protective and cytoprotective effects like anti-inflammatory, antiproliferative, and antiapoptotic effects (Reuter et al. 2010; Lawless et al. 2009). Though iron has a prooxidant effect, ferritin that gets induced by increased iron possesses antioxidant effects. Thus, overall HO-1 and its downstream molecules have a protective effect on lung. 4. Epidermal Growth Factor Receptor (EGFR) Various growth factors bind to EGFR, a receptor tyrosine kinase, and activated EGFR which in turn activates downstream signaling pathways like MAP kinase and PI3K to cause cellular proliferation. Oxidative stress activates EFGR and oxidative free radicals also regulate the oxidation status of EGFR and its subsequent function. In any event, oxidative stress has dose-dependent controversial role in EGFR activation. Low concentration of oxidative free radicals promotes EGFR activation, whereas higher concentration of radicals indeed inhibits EGFR activation. This could maintain homeostasis and acts as a negative feedback. Mutations in EGFR has been the suspected cause in many types of cancer especially adenocarcinoma. Frequency of mutation of EGFR is high in nonmucinous tumors, nonsmokers, and women. Amplification, mutation, or overexpression either of these had been the causes of deregulation of EGFR. In 60% of the cases of NSCLC, EGFR overexpression has occurred, probably at a higher rate in adenocarcinoma than SQCC. Cigarette smoke contains benzo[a]pyrene which induces the activation of EGFR signaling pathway via ROS. It increases the proliferative potential by upregulation of phosphorylated EGFR as also it induces expression of EGFR ligands like amphiregulin, epiregulin, etc. Hence, EGFR has a major contribution towards uncontrolled cell growth and proliferation (Chakraborti et al. 2020). Studies have shown that patients with mutated EGFR when treated with EGFR inhibitors in contrast to the traditional chemotherapeutic regimen had boosted PFS (progression free survival) and OS (overall survival). Adenocarcinoma patients and “large cell histology” NSCLC patients if diagnosed with EGFR mutations are benefited with EGFR inhibitors (Shtivelman et al. 2014) 5. Natural Products While natural products and nutritional supplements are rich sources of antioxidants, these have not been used as therapeutics for lung cancer actively. However, various studies have found a correlation between the regular consumption of these nutritional supplements and risk of cancer. Most of these products are having potent antioxidant capacity and this might be attributable to their protection against lung cancer. The resveratrol, curcumin, lycopene, diallyl disulfide (DADS), epigallocatechin-3 gallate, genistein, quercetin, and sulforaphane are few natural products that have been demonstrated to neutralize oxidative stress to cause protection against cancer development.

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Controversial Role of Oxidative Stress in Lung Cancer Antioxidants are chemical compounds that scavenges the free, unstable molecules ROS (reactive oxygen species). These ROS targets the DNA leading to damages that can culminate into mutations, which can further lead to cancer. Our body generates endogenous antioxidants in order to quench these free radicals. However, our body system preferably counts upon the external sources of antioxidants, referred to as the “exogenous antioxidants.” These exogenous sources include dietary antioxidants like Vitamin A, C, and E, beta-carotene, etc. (Machlin and Bendich 1987). These antioxidants are believed to bestow a panacea of benefits. Lung cancer commencing in the lungs starts metastasizing, advancing from the lymph nodes to the brain and other body parts, making it the deadliest of all cancers. The metastatic cascade involves a multitude of steps which include epithelial– mesenchymal transition (EMT), intravasation, evading the immune system, survival in circulation, extravasation, and distant organ colonization. Cancerous cells generate enormous proportions of ROS by altering the various pathways involved in signaling and metabolism. Over a period of time, the notion that ROS is responsible for initiating and progress of tumor has inclined the mindset of the people and patients suffering from cancer to consume antioxidants as a supplement. In recent decades, scientists have been shoving up antioxidants in cell cultures, humans, mice, rats, etc. to determine the beneficial role of antioxidants. A series of studies have cranked up with the controversial roles of antioxidants. Antioxidants once endorsed as a cancer preventive are now anticipated in spurring up the progression of the disease which was evident from some randomized clinical trials. A cancer prevention study carried out in 1994, entitled as “The Alpha-Tocopherol (vitamin E)/ Beta-Carotene Cancer Prevention Study” exemplified that daily intake of beta-carotene-enhanced lung cancer rates significantly in Finnish male smokers; however, intake of vitamin E showed no affect (Alpha-Tocopherol Beta Carotene Cancer Prevention Study Group, 1994). The blanket knowledge regarding antioxidants that they can help fight cancer has been gainsaid when researchers recently discovered that antioxidants hasten the progression of cancerous cells. In 2014, two independent research teams, one led by Dr. Martin Bergo at the Karolinska Institute and Gothenburg University in Sweden and the other at New York University, USA, showed that vitamin E used as an antioxidant supplement fueled up the tumor growth. Since then, follow-up researches providing compelling evidence that antioxidants have pro-tumorigenic activity have been conducted. In 2019, the same group conducted a study to further explain how antioxidants boost cell growth and promote lung cancer metastasis. The study involves the role of heme or “iron protoporphyrin IX”. Various forms of oxidative stress kindle the heme-containing proteins to liberate heme that aggravates the cellular oxidative stress because free heme is responsible for generating more number of ROS molecules. The intracellular levels of free heme are strongly regulated by HO-1. Dr. Bergo’s team unveiled a new idea that supplementation of antioxidants stabilizes BACH1 (BTB and CNC homology 1), a transcription factor (Wiel et al. 2019). BACH1, belonging to the cap’n’collar (CNC) b-Zip family of proteins is a crucial

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element responsible for sensing cellular oxidative stress. BACH1 straightaway coheres to the antioxidant response elements (AREs) present in the promoter region of antioxidant genes and ascertains the heme levels present in the cell (Ogawa et al. 2001). Physiologically, KEAP1 (Kelch-like ECH-associated protein 1) causes degradation of Nrf2 (Menegon et al. 2016). However, deactivated Keap1 causes accumulation of Nrf2 under the situation of oxidative stress. The enhanced amount of free heme leads to the stimulation of HO-1 expression which binds to the AREs on the promoter region of HO-1 (Chiang et al. 2019). Eventually the proteosomalmediated degradation of BACH-1 occurs which is promoted by the elevated levels of free heme. However, excess supplementation of antioxidants reduces the oxidative stress which causes low levels of heme and thus stabilization of BACH1. BACH1 is considered to displace NRF2 from ARE and causes the transcriptional repression of HO-1. Stabilized BACH1 and its accumulation contribute to metastasis by activating the transcription of Hexokinase 2 and Gapdh which results in enhanced glycolysis rate, glucose uptake, and lactate secretion (Wiel et al. 2019; Lignitto et al. 2019). It has been demonstrated that BACH1 which is stabilized by the antioxidants elevates the proficiency of the metastasis. In order to demolish the progression of the cancerous cell, two novel targets for therapeutic intervention have been identified which includes either destabilization of BACH1 or by inhibiting HO-1, which is associated to BACH1, which will equally restraint the process of metastasis.

Important Clinical Trials Targeting Redox Candidates in Lung Cancer Above-mentioned controversial aspect of oxidative stress in the lung cancer did not stop the researchers to find novel strategy in lung cancer in this direction. The aggravation of lung cancer progression with increased metastasis with antioxidants forced us to redefine the existing role of ROS in cancer pathophysiology. Though ROS is needed for cellular proliferation, higher ROS also could kill cancer cells and so cancer cells should have antioxidant mechanisms to survive. This could attribute to aggravation of cancer with antioxidants treatment. While this could be surprising, one of the mechanims for radiotherapy in cancer is generating oxidants. Indeed, various reports have demonstrated the beneficial effects of prooxidants in lung cancer therapeutics. Now, researchers are coming up with cutting-edge technologies to harvest the prominence of the redox state in cancer, aiming at the vulnerable nodes to suppress the development and progression of the malignant cells (Chaiswing et al. 2018). Modulation of the redox state may selectively target the cancer cells without inducing toxicity to the normal cells as altered redox homeostasis is often observed in patients suffering from lung cancer. There are numerous ongoing clinical trials which focus on studying the therapeutic efficacy of redox drugs in patients suffering from lung cancer (Marengo et al. 2016; Chaiswing et al. 2018). The redox system comprises main participants like reactive oxygen and nitrogen species, antioxidants, and redox thiol couples and secondary participants that regulate the redox system like various transcription factors, DNA repair proteins, heat shock

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proteins, HIF-1 alpha, and other proteins that involve in maintaining the redox system (Chaiswing et al. 2018). Among predominant redox couples (cysteine/cystine and GSH/glutathione disulfide (GSSG), peroxiredoxin/sulfiredoxin, thioredoxin/ thioredoxin disulfide, NADH/NAD, and NADPH/NADP), GSH/GSSG is dominant. Indeed, GSH/GSSG is being used as a scale to assess the redox status of the cells (Chaiswing et al. 2018). Combinatorial therapy has emerged to suppress the cancerous cells that have become resistant to chemotherapeutic agents. A multicenter phase II clinical trial performed for determining the efficacy of triapine that causes chelation of iron, and gemcitabine has been completed in patients with advanced-stage NSCLC (Ma et al. 2008). Another combinatorial phase I trial of GSH-depleting agent Imexon (pro-oxidant small compound) with docetaxel in lung cancer had promoted apoptosis in NSCLC (Montero and Jassem 2011). Menadione, a vitamin K analogue that was used for the treatment of lung cancer, induces dose-dependent oxidative stress and cell death through depleting GSH. As a phase II trial, in combination with mitomycin C, menadione was used for chemo modulation (Marengo et al. 2016). Another potent target in the line of thwarting the progression of lung cancer includes the thioredoxin (Trx) system involved in maintaining the cellular redox system. Dimesna (BNP7787), another untested chemo-protective disulfide compound, targets the Trx and Grx system (Montero and Jassem 2011). When used in combination with chemotherapeutic drugs like docetaxel and cisplatin, BNP7787 proves to be favorable in diminishing the harmful effects of chemotherapy in patients who are in stage IIIB or IV of NSCLC (Montero and Jassem 2011). BNP7787 in combination with taxane and platinum chemotherapy was clinically evaluated in patients suffering from advanced stage of NSCLC (Montero and Jassem 2011). Motexafin Gadolinium (MGd), a Trx inhibitor, falls under the status of completed randomized clinical trial phase 2. This is a second-line approach for the treatment of NSCLC. MGd acts specifically on the tumor cell by inducing mitochondrial pathway and promotes apoptosis. This is used in combination with various other drugs like pemetrexed in medicating lung cancer patients (Edelman et al. 2011). Vitamin C, an antioxidant, and its various redox forms like ascorbate, targets the redox disparity found in the cancer cells who suffer from more oxidative stress. Mutations in the L-gulonolactone oxidase (GULO) gene impair the endogenous synthesizing proficiency of Vitamin C in the human body. Vitamin C is always considered to have antioxidant. Recent evidences indicate that micromolar concentrations of vitamin C could reduce the effects of ROS, whereas it could also function as a prooxidant when it is given intravenously and when it reaches millimolor concentrations in plasma (Ngo et al. 2019). As it is less costly and fluently available, various studies all across the globe focused on the anticancer therapy obtained from Vitamin C. Beside the dose level, mode of administration also plays the role of deciding factor in the efficacy of treatment. Though pharmacokinetic study of ascorbate showed that it has a capacity to reduce tumor growth alone, mouse cancer models relied on the combinational therapy for the tumor microenvironment. High dose of ascorbate given intravenously with other combining chemotherapeutic drugs like paclitaxel and carboplatin was used as anticancer therapy for NSCLC, under phase II clinical trial.

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Conclusions and Future Direction For a long time, researches have substantiated a strong relationship between oxidative stress and genesis of lung cancer. However, the failure of potent antioxidants in clinical trials not only in lung cancer but also in various other diseases indicated the existence of some missing links. One such theoretical missing link is the promotion of cell growth and lung cancer metastasis by antioxidants. This dark side of antioxidants in lung cancer needs more detailed studies so that we will be able to understand the exact part played by the oxidative stress in driving the development of lung cancer. The controversial role of oxidative stress in the lung cancer have captivated the attention of researchers and opened up a new direction for thinking about the underlying pathophysiology of lung cancer. Now that the researchers are familiar with the pleiotropic effects of redox drugs, they have shifted their focus on exploiting the redox balance to potentiate the armamentarium of chemotherapeutic agents. The impressive number of clinical trials has validated the effectiveness of various molecular cancer targets amenable to redox intervention.

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Oxidative Stress and Cancer: Role of the Nrf2-Antioxidant Response Element Signaling Pathway

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Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Oxidative Stress and Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Oxidative Stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Oxidative Stress and Carcinogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Nrf2-Antioxidant Response Element (Nrf2-ARE) Signaling Pathway . . . . . . . . . . . . . . . . . . . . The Double Role of Nrf2 in Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pro-Oncogenic Effects of Nrf2: Nrf2 as a Proto-Oncogene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Nrf2 as Tumor Suppressor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mechanisms of Nrf2 Activation in Cancer Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Somatic Mutations of Keap1, Nrf2, and Cul3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Epigenetic Modifications of Keap1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Disruption of Nrf2/Keap1 Interactions by Other Signaling Pathways . . . . . . . . . . . . . . . . . . . . . Role of Nrf2 in Chemoresistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cross-References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract

Oxidative stress is the outcome of the imbalances between the generation and disposal of reactive oxygen species (ROS) and is a major causative factor for several chronic diseases such as cancer. Nuclear factor erythroid 2-related factor 2 (Nrf2), a transcription factor, mediates the activation of gene transcription through the ARE and plays an important role in antioxidant response of the M. Ruwali (*) Amity Institute of Biotechnology, Amity University Haryana, Gurgaon, India e-mail: [email protected] R. Shukla Department of Pharmaceutics, National Institute of Pharmaceutical Education and Research (NIPER-Raebareli), Lucknow, India e-mail: [email protected] © Springer Nature Singapore Pte Ltd. 2022 S. Chakraborti et al. (eds.), Handbook of Oxidative Stress in Cancer: Mechanistic Aspects, https://doi.org/10.1007/978-981-15-9411-3_60

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cells. In case of normal cellular functioning, Nrf2 is degraded by proteasomes via poly-ubiquitination by Kelch-like ECH-associated protein 1 (Keap1). When cells are exposed to oxidative stress, there is an activation of Nrf2 which is accompanied by its detachment from Keap1 and movement to the nucleus followed by upregulation of the cytoprotective genes. Interestingly, accumulating evidence from several recent studies suggests that Nrf2 performs opposing roles in cancer development. Under normal cellular conditions, Nrf2 works as an anti-tumor molecule due to its induction of cytoprotective genes which protect cells from electrophilic and oxidative damage. However, several mechanisms have been identified which lead to the constitutive activation of Nrf2 causing overexpression of protective Nrf2 target genes. This provides advantage to the cancer cells as they can maintain a moderate oxidative intracellular niche which promotes survival and rapid proliferation. Besides having a role in cancer development and progression, Nrf2 pathway is also a major contributor in acquired chemoresistance. Several studies have implicated Nrf2 in chemoresistance in response to some of the most commonly used chemotherapeutic agents such as cisplatin, doxorubicin and 5-fluorouracil. Thus, targeting the Nrf2 pathway can be employed as a promising method for increasing the effectiveness of drugs used in chemotherapy. Keywords

Oxidative stress · Cancer · Nrf2 · Keap1 · Antioxidant response element

Introduction Antioxidant response element (ARE) is a regulatory element that controls the expression of phase II genes at transcriptional level. Several transcription factors interact with AREs to regulate the expression of genes involved in protecting the cells from several toxic and damaging exposures (Raghunath et al. 2018). Oxidative stress has now been widely recognized as an important causative factor for development of several diseases such as atherosclerosis, rheumatoid arthritis, neurological deficiencies, cancer, etc. Oxidative stress is the outcome of the imbalances between the generation and disposal of reactive oxygen species (ROS) which are produced after exposure to environmental pollutants, tobacco exposure, and xenobiotics. There are several sources for the production of ROS within cells like cytosolic NAPDH oxidases (NOXs), oxidative phosphorylation in mitochondria, xanthine oxidases as well as oxidation processes in peroxisomes. ROS is not always detrimental to the cells, as low levels of ROS is involved in physiological functions like signal transduction and in providing defense against microorganisms. However, higher levels can be deleterious for the cells as it causes oxidative damage of some of the very important cellular components (Pizzino et al. 2017). Activation of gene transcription of phase II genes takes place by binding of nuclear factor erythroid 2 (NFE2)-related factor 2 (Nrf2) to the ARE. Nrf2 is a

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transcription factor, first isolated through cloning experiments (Moi et al. 1994), and later recognized to be binding to the ARE of phase II gene NQO1 and a number of other genes. The findings were also supported by studies in which Nrf2 deficiency led to the downregulation of target genes in mouse models and in vitro studies (Nguyen et al. 2005). Kelch-like ECH-associated protein 1 (KEAP1) was discovered as a novel binding partner of Nrf2 by yeast 2-hybrid assay. Further studies involving Nrf2 discovered that the Keap1/Nrf2/ARE pathway is a required molecular mechanism in the metabolism of oxidizing agents such as electrophiles and oxidants.

Oxidative Stress and Cancer Oxidative Stress Oxidative stress can be defined as an imbalance that results when reactive oxygen species (ROS) are not eliminated at the same rate by the antioxidants as their production. The resulting excess of oxidants leads to the cellular damage which can have deleterious effects on the whole organism (Duracková 2010). The respiratory process in the mitochondria of eukaryotic cells not only generates energy needed for the functioning of the cell but also generates the oxidative compounds. Though a majority of these compounds are useful for the cell, a small percentage ( ˙OH + OH ). However, Complexes I and III majorly contribute to generating mitochondrial ROS (Quinlan et al. 2013). It releases 80% of superoxide-free radicals of mitochondria into the intermembrane space and the remaining 20% into the mitochondrial matrix. The superoxide radicals are transported into the cytoplasm through mitochondrial permeability transition pore located at the mitochondrion outer membrane. In the cytoplasm, superoxide (O2˙─) is dismutated in the presence of superoxide dismutase (Cu/ZnSOD) to hydrogen peroxide (H2O2), a highly diffusible secondary messenger (Crompton 1999). Moreover, hydrogen peroxide is released from cell membranes by the aquaporin 8 channel. The other major source of ROS generation is called the peroxisome (Schrader and Fahimi 2006). Xanthine oxidase located in the peroxisomal membrane and matrix produces superoxide (O2˙─) and hydrogen peroxide (H2O2). Other miscellaneous sources of ROS are exogenous elements such as flavorings, drugs, coloring agents, etc., and endogenous substances such as prostaglandins, fatty acids, etc. Smooth endoplasmic reticulum refines these elements and converts them into free radicals, majorly in ˙OH. Besides that, leucocytes and macrophages can also generate free radicals during the process of the immune response (Schrader and Fahimi 2006).

Regulation of ROS Production ROS production is highly regulated by various protective mechanisms or antioxidants that scavenge ROS. Misbalance in ROS production and elimination can lead to a damaging effect on cellular components including proteins, lipids, and DNA. These types of impairment can increase the risk of mutagenesis (Durackova 2010). Most of the superoxide (O2˙─) radicals are transported to the mitochondrial matrix via electron transport chain, and here superoxide (O2˙─) radicals are dismutated to H2O2 via the superoxide dismutase (MnSOD or SOD2) (Kumari et al. 2018). Few superoxide radicals are allowed to transfer into the cytoplasm through the mitochondrial permeability transition pore in the mitochondrial outer membrane (Kumari et al. 2018). After passing into the cytoplasm, these superoxide radicals are dismutated into an extremely diffusive secondary messenger, the hydrogen peroxide (H2O2), by cytosolic SOD (Nogueira and Hay 2013). The other antioxidant systems are catalase, glutathione systems such as glutathione transferase, glutathione peroxidase (GPX), glutathione reductase (GR), reduced glutathione (GSH), etc., and thioredoxin systems such as peroxiredoxins (PRX), thioredoxin peroxidase, thioredoxins (Trx), etc., and vitamin E and C. In the antioxidant defense system, GSH, a major nonenzymatic constituent contributes a key role in the maintenance of ROS balancing (NADP/ NADPH and GSSG/GSH ratios) with the highly controlled intracellular level of NADPH. The intracellular NADPH is mainly generated by the mitochondrial

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metabolism and the pentose phosphate pathway (PPP), and it is majorly utilized for the detoxification of H2O2 by GPX, PRX, and fatty acid synthesis (Schafer and Buettner 2001). Thus, the generation and utilization of NADPH maintain the homeostasis of cellular NADPH. This kind of high regulation maintains a moderate level of intracellular ROS which helps to form the solid tumor and accelerate metastasis during the energetic stress of cancer cells.

ROS Regulating Cancer and Therapy Resistance: Highlighting the Dichotomous Role of ROS ROS is snatching the attention of the scientific community not only due to its potential role as a signal transducer in various pathways that regulate its effect in cell death but also its role in the regulation of tumor initiation, proliferation, promotion, and therapy resistance. Regulation of ROS in cancer cells is very crucial because it can determine the fate of the cancer cells. For instance, cancer cells have an increased level of ROS in comparison to the noncancerous cells, but after a certain limit, ROS promotes cell death in the cancerous cells. Most of the anticancer agents elevate the ROS level or suppress the antioxidant defense system of cancer cells to treat cancer. ROS has been generated due to increased metabolic activity, mitochondrial deregulation, and increased enzymatic activity such as oxygenase, lipoxygenase, and different kinases, and increased cell signaling which can activate many oncogenic pathways in favor of tumor promotion and therapy resistance. While doing these, cancer cells maintain a strong antioxidant defense mechanism including enzymes such as superoxide dismutase (SOD), catalase, and glutathione peroxidase (GSH) which gives strong evidence that too much ROS can negatively affect the physiology of cancer. In the following part, we are describing the dual nature of the ROS in cancer initiation and progression (Fig. 2).

Cancer-Promoting Roles of ROS An elevated level of ROS production along with defective antioxidant mechanisms increases the oxidative stress level assisting in various pathophysiological conditions including cancer. In the following sections, we have discussed how ROS helps in the progression of cancer by modulating various mechanisms accountable for the progression of cancer and therapy resistance.

Increase Genomic Instability by ROS Genomic instability is a major driving force of developing cancer from healthy cells. The introduction of various new mutations and the failure of the defense mechanism of the cell cause harmful changes in the physiology of the cell and give detrimental effects. ROS is a major factor that can modify DNA base pairs by chemical changes and subsequently may change the gene sequence to promote mutation or DNA damage. Oxidative damages contain a bunch of DNA lesions including DNA strand

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Fig. 2 Dichotomous role of ROS in cancer. ROS helps both in the progression and the inhibition of cancer

breaks, base oxidation, and modification, gene copy number amplification, and DNA miscoding lesions. ROS is generally produced from the ETC of intracellular mitochondria due to its improper function. Oxidation of the guanine bases is the most common modification of the DNA done by various ROS. OH˙ radical has the most detrimental effect on cellular DNA as it involves oxidation, addition, and hydrogen elimination (Cadet et al. 1999). OH˙ attacks the C4, C5, and C8 position of the guanine although its most favorable position is C8 and makes 8-oxoguanine (8-OG) which is a tautomer of 8-hydroxyguanine (8OHG). It is a two-step process wherein the first step OH attaches to the C8 and makes a transition state of CH8OH˙. In the next step, this transition molecule reacts with another OH˙ to subtract the H from the position of C8 and produce C8-OH (enol) or 8OG (keto); other C4 and C5 adducts are not so stable because they break the five-member imidazole ring of guanine (Shukla et al. 2004). These modifications of guanine lead to G:C-T: A transversion due to its affinity to adenine as well as cytosine which mutate the gene and can promote cancer. Cells are well equipped with the various repair mechanisms to fight against this damage such as OGG1 (8-oxoguanine DNA glycosylase 1) which eliminates the 8-OG from the DNA. OGG1 polymorphisms are associated with several cancers such as the lung, esophagus, and stomach cancer (Mahjabeen et al. 2012). ROS accumulation can adversely affect the mitochondrial genome which successively increases the ROS in the cells

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(Shokolenko et al. 2009). Telomeres comprise tandem repeats of (TTAGGG)n, and the transversion mutation in that portion diminishes the binding of telomeric protein and eventually causes telomere shortening and chromosomal instability (Shokolenko et al. 2009). Double-strand break (DSB) is another important mechanism to promote genomic instability and causes faulty replication which gives rise to oncogenic replication stress and heads the normal cells to cancer (MayaMendoza et al. 2015). ROS slows down the replication fork progression by oxidizing the dNTPs and induces DSB at the fragile sites resulting in over- or underreplication of the genes. ROS dissociates peroxiredoxin 2 oligomer (PDRX) which halts the fork elongation. DNA damage response (DDR) is activated by the H2O2 which helps to activate ATR response. Defects in the repair system such as mismatch repair system gene (MSH1), and homologous recombination gene (BRCA1) lead to the development of many cancers such as glioblastoma, breast cancer, and myeloid malignancy (Sallmyr et al. 2008).

Alteration of Metabolic Pathways by ROS Oxidative stress is a key facilitator of metabolic reprogramming in the cancer cell which successively helps in the generation of a significant amount of ROS and leads to tumor promotion (Kim et al. 2016). Accumulation of ROS in the tumor is adjusted by the increase of antioxidant mechanism, directly linked to the cellular metabolism. There is prominent crosstalk between redox balance and metabolic pathways. Despite the presence of abundant oxygen, tumor cells rely on the glycolytic pathway to produce various intermediary products, and NADPH as a byproduct is known as the Warburg effect (Liberti and Locasale 2016). It is noticed that the major ATP production is done by the increased glycolytic shift. In highly aggressive cancer, activation of the oncogene and inactivation of the tumor suppressor gene lead to increased glycolytic efficacy. Redox homeostasis in the cancer cell is greatly regulated by PPP-derived NADPH and glutaminolysis-derived GSH. So, suppression of these two pathways shows an excellent antiproliferative effect on the breast and pancreatic cancer cells due to oxidative stress (Li et al. 2015). It has been studied that in a nutrient-deprived condition, an increase in the metabolism of the glucose restricts the H2O2induced cell death. Lactate dehydrogenase A (LDHA) acts as a tumor promoter by reducing the pyruvate into lactate (Das et al. 2019); suppression of this enzyme by small-molecule Fx11 enhances oxidative stress and limits the development of human lymphoma and pancreatic cancer xenografts (Le et al. 2010). Fatty acid oxidation (FAO) in the mammalian mitochondria is another promising method to maintain cellular ROS. Long-chain fatty acids break into short chains and generate acetyl-co-A, FADH2, and NADH to facilitate the biosynthetic pathway and ATP generation. In the cancer cells, these acetyl-co-As increase cellular NADPH through isocitrate dehydrogenase (IDH1) and the malic enzyme (ME) (Carracedo et al. 2013). FAO prevents cell death upon matrix detachment and metabolic stress through the LKB1/AMPK axis (Jeon et al. 2012). In glioblastoma cells, pharmacological inhibition of FAO by Etomoxir leads to proapoptotic induction due to NADPH impairment and ATP depletion (Pike et al.

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2011). The pentose phosphate pathway (PPP) is a biosynthetic pathway of ribose sugar which is the backbone of nucleic acid. Besides the production of ribose-5phosphate, it is a major source of NADPH and GSH production which regulates the redox balance in the cells. G6PD is the rate-limiting enzyme of the PPP which is shown to be highly expressed in osteosarcoma and leads to PPP-derived NADPH. Mucin1 promotes TIGAR-dependent glucose entry into PPP and gives resistance to Bortezomib due to ROS depletion via GSH in multiple myeloma cells (Yin et al. 2014).

Regulation of Metastasis by ROS Cell migration and invasion are the crucial hallmarks of cancer progression. Various cancers such as breast cancer cell line MCF7 show an elevated level of ROS from their normal counterpart which further augments CXCL14, a novel chemokine that leads to invasion of various parts of the body such as the liver, spleen, and lungs (Pelicano et al. 2009). Cell motility is dependent on the integrinbinding with the extracellular matrix, and ROS can activate various cellular integrin signaling which leads to invasion. Besides that, intracellular ROS produced from the cytoplasm acts with the actin fibers and remodels the cytoskeleton for further movement (Chiarugi and Fiaschi 2007). Normal cells cannot survive without the attachment to the extracellular matrix, and upon detachment, they die. This phenomenon is known as anoikis. Integrins give survival signals to the attached cells not to die, and this mechanism is mimicked by the increased ROS such as in pancreatic cancer cells; ROS synergistically activates Src and EGF signaling to avoid anoikis (Chiarugi and Fiaschi 2007). ROS activates various phosphatases such as LMW-PTP, Src, and signaling such as FAK which forms focal adhesion, promote cell motility, and prevent anoikis (Gilmore 2005). Before the invasion, cells change their fate from epithelial to mesenchymal state (EMT) to move freely, and for this, various transcription factors such as the snail, slug, and some metalloproteinases (MMP) are upregulated. Change of E-cadherin to N-cadherin is a prominent indication of EMT; H2O2 lessens the expression of E-cadherin by methylating the promoter with histone deacetylase by upregulating snail expression which is a crucial marker of EMT in hepatocellular carcinoma (Lim et al. 2008). Src modulates NADPH oxidase1 (NOX1) which is essential to keep the transformed state and the migratory property of cells (Gianni et al. 2008). It is seen that MMP3-mediated activation of ROS induces EMT in the mammary tumor by the Rac1 (Radisky et al. 2005). Rac1 can upregulate MMP1 production and NF-kB activation which lead to migration of the MCF7 and T47D breast cancer cell line (Tobar et al. 2008). Cancer cells invade other organs through the blood vessel by a mechanism known as transendothelial migration. Various adhesion molecules such as I-CAM, CD54, and several cytokine-like IL-8 expressed by ROS induce NF-kB which helps in the invasion and migration process (Roebuck 1999). Besides, ROS-induced signaling molecules such as p38, MAPK/ERK, and heat shock proteins can change the actin dynamics and facilitate the invasion (Huot et al. 1997).

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Triggering of Angiogenesis by ROS Nutrient availability and oxygen enrichment become less with the increasing mass of the tumor due to excessive cell proliferation. To overcome these adverse conditions, tumor cells start to elicit the formation of new blood vessels through sprouting or branching from the existing one. Proliferation, migration, and tube formation are the major mechanisms to produce new blood vessels or angiogenesis. ROS modulates VEGF signaling by various pathways followed by HIF1 activation. Growth factors such as VEGF promote HIF1α signaling by the downstream molecule such as MAPK and PI3K/Akt. So, inhibition of ROS via mitochondrial inhibitors or glutathione peroxidase, HIF1α as well as VEGF signaling, are downregulated (Liu et al. 2006). ROS-induced MMP1, MMP3, and MMP9 can also upregulate the capillarylike structure formation, EC migration (Ushio-Fukai and Nakamura 2008). Vasodilation in the tumor territory is triggered by the ROS-induced heme oxygenase-1 enzyme (Milligan et al. 1996) that meets up the nutrient deprivation and favors the proliferation. Epigenetic Regulation by ROS ROS can regulate the expression of various tumor suppressor genes by subjecting them to epigenetic modification and promoter methylation. The production of ROS can influence the activity of the DNA methyltransferase 1 (DNMT1) (Wu and Ni 2015). RUNX3, a tumor suppressor protein, has been suppressed by the promoter methylation by H2O2 in the SNU-407 human colorectal cancer cell line (Kang et al. 2012). Other tumor suppressor genes such as caudal type homeobox-1 (CDX1), breast cancer 1 (BRCA1), Von Hippel-Lindau (VHL), retinoblastoma (Rb), and cyclin-dependent kinase inhibitor 2A (CDKN2A) have also been suppressed by the ROS (Toyokuni 2008; Zhang et al. 2013). MicroRNAs (miRNAs) are one of the important candidates of epigenetic regulations. miR-135 (tumor promoter) accumulates in the glutamine (endogenous antioxidant)-deprived cancer cells. It requires ROS-regulated activation of the mutant p53 which binds to the miR-135 promoter region and upregulates miR-135 expression leading to cancer progression (Yang et al. 2019).

Cancer-Killing Roles of ROS ROS performs an important role in cancer proliferation, invasion, and survival; however, beyond a certain limit, an uncontrollable increase in ROS provides a remarkable increase in cancer cell death. ROS is highly induced by most of the anticancer agents to eliminate the cancer cells. In the following section, we have discussed the anticancer effects of ROS.

Induction of Apoptosis by ROS Apoptosis is known as programmed cell death type I, and it is believed to be one of the major hurdles in the path of cancer progression, proliferation, and execution of

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the devastating effects on human beings. This process is regulated by different ligands such as FAS ligand, TNFα, and TNF-related apoptosis-inducing ligand (TRAIL), and death receptors such as DR1 and DR2. FAS receptor (FASR) and FAS-related death domain (FADD) which produce the FLICE complex ultimately activate the caspase cascade (Galadari et al. 2015). ROS has been shown to suppress the FLICE-inhibitory protein (c-FLIP) by preventing proteasomal degradation which increases FLICE and leads to apoptosis (Safa 2012). Mitochondria membrane permeabilization by O2 triggers the release of the Cyt-c that leads to cell death in a VDAC-dependent manner. Cyt-c remains binded with the cardiolipin to be activated, and this complex has to be dissociated. However, ROS oxidizes these complexes to rescue the Cyt-c and promote cell death (Iverson and Orrenius 2004). Cyt-c makes a complex with the Apaf-1 and procaspase-9 to form an apoptosome to facilitate cell death intrinsically. ROS can also diminish the activity of the antiapoptotic proteins such as Bcl-2 and decrease the degradation of proapoptotic proteins such as Bid and Bad (Luanpitpong et al. 2013). Intrinsic ROS generated from the ETC or lipid peroxidation can also promote other methods of cell death-like ferroptosis and necroptosis (Dixon and Stockwell 2014).

Effect on Cancer Stem Cells by ROS Tumor progression and recurrence are established to execute by a small group of dedifferentiated primary cancer cells known as a cancer stem cell (CSC). ROS is shown to provide a negative effect on the CSC populations as a study by Diehn et al. showed a decrease in ROS in CSC (Diehn et al. 2009). The difference in the ROS level between CSC and peripheral tissue helps to maintain stem cell function. Various ROS scavenging pathways are highly upregulated in the CSC population to provide strong cancer-promoting capabilities and recurrence ability. Leukemia stem cells (LSCs) are adversely affected by the increased ROS level in cells (Kim et al. 2013). Depletion of glutathione synthase (GSH) by L-S, R-buthionine sulphoximine (BSO) depletes the CSC in epithelial cancer. ROS is greatly reduced in the case of gastrointestinal CSC due to enhanced activity of the GSH and Xcactivation (Ishimoto et al. 2011). FOXO1 regulates various scavenging enzymes such as SOD and catalase in CSC to promote CSC proliferation and prevent the effect of ROS on CSC. Moreover, ROS promotes DNA damage and instability which leads to CSC depletion upon radiation treatment, and targeting CSC via ROS promotion may provide an important therapeutic strategy to fight against cancer. ROS-Induced Senescence Senescence is a condition where stable proliferation is arrested in cells. Senescent cells have a flattened and expanded morphology with the upregulated activity of senescence-associated β-galactosidase (SA-β-gal) and hypophosphorylated- p16, p21, p53, and Rb proteins. It can be induced by several stimuli such as dysfunction of the telomere, DNA damage, activation of the oncogene, dysfunction of mitochondria, hypoxia, and disruption of chromatin. But increased oxidative stress is the

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key factor to initiate cellular senescence in cancer cells as oxidative stress is induced due to the higher production or insufficient scavenging of ROS (Tsai et al. 2016). Cell and nuclear membranes are disrupted by increased ROS which leads to an increase in membrane permeability and DNA/cellular protein damages. These initiate the activation of p16 and the acceleration of telomere shortening (Ben-Porath and Weinberg 2005). In senescence, the p53 and p16-Rb pathways play a major role. In the p53 pathway, ROS-induced DNA damage upregulates either ATM/ATR and Chk1/Chk2 or the ARF proteins expression to stimulate p53 which activates the transcriptional target p21 and leads to senescence (Ben-Porath and Weinberg 2005). The transcriptional target p21 is the main key factor in cellular senescence as it is the mediator of the pRB pathway. It is reported that telomere-induced senescence can be suppressed by inhibiting p21 protein expression (Campisi and d’Adda di Fagagna 2007). In the p16-Rb pathway, ROS-induced DNA damage activates oncogenic RAS which upregulates p16 expression and activates pRB to downregulate the E2F-governed transcription of cell growth gene targets, thus leading to senescence (Campisi and d’Adda di Fagagna 2007). As ROS-induced senescence prevents abnormal uncontrolled cancer cell growth, it can be a novel therapeutic strategy to suppress cancer progression.

ROS-Induced Necrosis Necrosis is a bioenergetic catastrophe and a chaotic cell death. It is defined by the cellular organelles swelling, loss of plasma membrane integrity, arbitrary DNA damage, and unrestrained discharge of molecules such as HMGB1 and LDH from the damaged cells into the extracellular space, which activate immune response or wound repair (Zong and Thompson 2006). Higher production of ROS in the cancer cells can damage the intracellular molecules and organelles due to the generation of oxidative stress, which further initiates necrosis. ROS-mediated DNA damage occurs due to cleaving of DNA, oxidation of purines, and disruption of DNA-protein cross-linking. ROS may also oxidize cellular lipid membrane by altering various double bonds of polyunsaturated fatty acids. Further, it directs to the disruption of cellular membranes and causes the inflow of Ca2+ or leakages of noncaspase proteases, which can initiate necrosis (Gorlach et al. 2015). ROS-Induced Cell Cycle Arrest Cell growth is mostly controlled by the cell cycle, and it consists of four consecutive phases such as G0/G1, S, G2, and M phases. A precise successive initiation of cyclins (CCNS), cyclin-dependent kinases (CDKs), and cyclin-dependent kinase inhibitors (CKIs) tightly regulates the cell cycle progression via phosphoinositide 3-kinase (PI3K)/Akt pathway. Increased ROS in the cancer cells can promote cell cycle arrest in the G1 phase by downregulating PI3K/Akt protein (Wang et al. 2018); as a result, due to the lack of direct phosphorylation of p16, p21, p27, and GSK3 β (tumor suppressor protein) via PI3K/Akt, these tumor suppressor proteins have been upregulated and initiate cell cycle arrest in the G1 phase via suppression of cyclin D1 and CDK 4.

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Molecular Pathways Regulated by ROS in the Cancer Therapy Resistance ROS can regulate various signaling molecules and pathways which are very essential for cancer development, promotion, and resistance to conventional therapy. Receptor tyrosine kinase (RTK) is one of the most important pathways which regulates the growth and development of cancer. Exogenous ROS helps in the dimerization and activation of RTKs in a ligand-independent manner. Prolonged activation of the EGF, PDGF-α, and PDGF-β receptors occurred by Exogenous H2O2 via phosphorylation (Gamou and Shimizu 1995). ROS can modulate several signaling cascades such as the NF-ĸB pathway, PI3K/Akt pathway, MAPK pathway, etc., and can be regulated by an antioxidant pathway such as the Keap1-Nrf2 pathway. Multidrug resistance (MDR) is one of the principal causes of developing therapy resistance in cancer which is greatly influenced by increased ROS concentration. In the case of vestibular schwannomas, oxidative stress is induced and persisted in respect of increased recurrence and leads to survival (Robinett et al. 2018). ROS-induced HIF-1 promotes the secretion of VEGF and MIF from the multinucleated cells (MNCs) which give chemoresistance both in vitro and in vivo conditions (Parekh et al. 2018). Increased ROS in various cancers such as ovarian, breast, and glioma induces several antioxidant mechanisms that favor the resistance. In ovarian cancer, mitochondrial metabolic alteration induces ROS followed by increased PGC1α induction which stimulates cisplatin and paclitaxel resistance leading to metastasis (Kim et al. 2017). ROS activates DDH (dihydrodiol dehydrogenase), an enzyme of aldo-ketoreductases (AKRs) family which generates a lot of semiquinone anion molecules during the conversion of PAH (polycyclic aromatic hydrocarbon) to PAH-o semiquinone and causes mutagenic changes to acquire resistance (Chen et al. 2008). In the following sections, we have elaborately explained the principal molecular pathways induced by ROS for the development of cancer therapy resistance.

The Impact of ROS and Antioxidant Defense System to Promote Cancer Therapy Resistance The antioxidant defense system is a powerful cellular defense mechanism against oxidative stress (ROS), which originated from several physiological and chemical stresses. In normal conditions, increased ROS stress in the cellular environment activates nuclear factor erythroid 2–related factor 2 (Nrf2), which promotes the expression of various antioxidant defense systems such as catalase, glutathione peroxidase (GPX), and Superoxide dismutase (SOD), and they downregulate ROS to cope up with stress and help in the progression of cancer and therapy resistance (Kumari et al. 2018). In such a way, ROS has been maintained within a narrow boundary level, and the cell is protected from DNA damages, genomic instability, cell death, carcinogenesis, and even cancer. On the other hand, chemotherapeutic agent-induced ROS plays the same role in stimulating antioxidant mechanisms.

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Fig. 3 Impact of ROS on antioxidant mechanism regarding the development of therapy resistance in the cancer cells. Chemotherapy-induced ROS activates Nfr2, which helps to generate endogenous antioxidants such as Superoxide dismutase (SOD), glutathione peroxidase (GPX), etc. These endogenous antioxidants protect the cancer cells from oxidative stress by regulating ROS negatively and lead to chemotherapeutics resistance

Nrf2, a leucine zipper transcription factor is one of the major regulators of the antioxidant defense pathway (Nguyen et al. 2003). This remains bound to the ECH-associated protein 1 (Keap1), and upon activation, it detaches from the complex and goes to the nucleus, and binds to antioxidant responsive element (ARE) of antioxidant genes (Dinkova-Kostova et al. 2005). Upon binding, it enhances the expression of antioxidant enzymes such as SOD, GPX, Glucose 6 phosphate dehydrogenase (G6PD), and Cyclo-oxygenase 2 (COX2) (Zhang et al. 2012). Such kind of activation of Nfr2 in cancer cells provides cytoprotection from higher oxidative stress. The basic mechanism of many chemotherapeutic drugs is to generate oxidative stress (e.g., ROS) to destroy cancer cells. However, the Nrf2 pathway creates an atmosphere that promotes survival and proliferation of cancer cells as well as gives resistance against oxidative stress induced by chemotherapy (Fig. 3).

P-glycoprotein Regulation by ROS to Promote Cancer Therapy Resistance ROS can control the expression of P-glycoprotein (P-gp) in a moderate concentration and increases its expression to promote resistance to chemotherapy (Fig. 4). P-gp, ABCB1, or MDR1 is the ATP-binding cassette (ABC) transporters, mediate the efflux of therapeutic agents used in chemotherapy such as doxorubicin, vincristine, taxanes, etoposide, teniposide, and actinomycin D (Deng et al. 2002). In such a way, P-gp detoxifies chemotherapeutic drugs in cancer cells. The overexpression of

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Fig. 4 Interplay between P-glycoprotein (P-gp) and ROS in the development of therapy resistance in the cancer cells. A high level of ROS in cancer cells can upregulate P-gp, and it increases the efflux of therapeutic agents used in chemotherapy. Also, P-gp can lead to lysosomal degradation of chemotherapeutic agents. These can mediate chemotherapy resistance in the cancer cells

P-gp has been observed in several types of cancer cells, such as leukemia, neuroblastomas, ovarian, and breast cancer cells which prove the contribution of P-gp to chemoresistance. ROS also stimulates NF-ĸB that further activates HIF-1α (Seebacher et al. 2015. It activates the MDR1 gene and generates P-gp. Thus, ROS-induced NF-ĸB upregulates P-gp expression in the cancer cell and increases the efflux of chemotherapeutic agents which leads to therapy resistance.

The Interplay Between Autophagy and ROS in the Development of Cancer Therapy Resistance Autophagy is a catabolic process, activated by the various types of cellular stresses such as ROS upregulation, hypoxia, nutritional deprivation, etc. The word autophagy has come from the Greek words “auto” meaning self and “phagy” meaning eating. The function of autophagy is to degrade and recycle various cytoplasmic elements such as damaged organelles, proteins, and lipids to maintain cellular homeostasis. Such kind of tight regulation facilitates cell survival under cellular stress conditions (Das et al. 2018b, 2020). In conventional chemotherapy (cisplatin, doxorubicin, and vincristine), anticancer drugs generate a high level of oxidative stress via ROS to destroy cellular components, and it leads to cell death via apoptosis, necrosis, etc. In the presence of elevated level cellular oxidative stress, autophagy is activated, and it is needed to transform the cells by the RAS oncogene for the promotion of cell stress tolerance (Lock et al. 2011; Poillet-Perez et al. 2015). It has been observed that inhibition of autophagy suppresses the

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transformation and the proliferation of mouse embryonic fibroblasts (MEF) transformed with HRAS and MDA-MB-231 breast cancer cells ectopically expressing KRAS (Lock et al. 2011). Furthermore, other studies have exhibited that in the presence of high basal level autophagy, oncogenic RAS is upregulated in pancreatic ductal adenocarcinoma (PDAC), MCF-10A, and immortalized baby mouse kidney (iBMK) cell lines, and suppression of autophagy (via deleting ATG5 or ATG7) suppresses Ras-induced cell proliferation (Guo et al. 2011). Recent studies have shown that anticancer drugs activate autophagy in response to the generated ROS to defend the cellular stresses that further initiates the development of cancer cell drug resistance. Therefore, several clinical trials have been developed to estimate the effect of autophagy inhibition in cancer treatment such as hydroxychloroquine, which has been already in use with other anticancer drugs, as an autophagy inhibitor to evaluate the therapeutic effect in cancer treatment (Carew et al. 2012). Of note, it has been found that anticancer drugs stimulate ROS–induced autophagy which initiates either cancer cell drug resistance or induce apoptosis or both. Therefore, autophagy is considered as a very complex system in cancer therapy. Cytoprotective autophagy helps in the survival of the cancer cells leading to therapy resistance as we have noticed earlier (Das et al. 2018a, 2019). In such a condition, the application of autophagy inhibitors resensitizes the cancer cells to therapy. Honokiol, a promising anticancer natural product that is currently under the process of drug development for the treatment of prostate cancer, induces ROS-mediated autophagy by upregulating LC3B-II protein level which leads to cancer cell drug resistance. In this case, the use of 3-methyladenine (3-MA) or ATG5 siRNA helps to inhibit drug-induced autophagy which restores the effect of anticancer drugs and facilitates cancer cell death through apoptosis (Hahm et al. 2014). Similarly, it has been noticed that Rocaglamide A (Roc-A), a bioactive molecule, generates ROS which further stimulates the induction of protective autophagy and mitophagy (mitochondrial autophagy) through PINK1/Parkin signaling in prostate cancer cells. Interestingly, inhibition of autophagy/ mitophagy and scavenging of ROS by NAC resensitize the prostate cancer cells to ROC-A (Zhao et al. 2019). Furthermore, the application of antioxidants (catalase, N-acetylcysteine, and superoxide dismutase) suppresses autophagy in some cases which shows the direct connection between ROS and autophagy in the induction of cancer therapy resistance (Fig. 5).

Conclusions and Future Perspectives Therapy resistance is a major hurdle in cancer treatment. There are several mechanisms involved in the development of therapy resistance. Among them, ROS has a significant role in the progression of cancer and giving rise to therapy resistance. ROS normally behaves as a signaling molecule that induces cytotoxicity by activating oxidative stress. However, studies also reveal that ROS generating oxidative stress can trigger either cell death or cell survival mechanisms, depending on the intensity and extent of exposure. ROS stimulates the cell

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Fig. 5 Role of ROS-induced autophagy in chemotherapy resistance in the cancer cells. In highly aggressive cancers, therapeutic agents generate ROS which activates cytoprotective autophagy that further negatively regulates the level of ROS in cancer cells and leads to therapy resistance

proliferation and survival of cancer cells at low concentrations, typically at submicromolar concentrations. ROS encourages cell differentiation by prompting temporary or permanent cell cycle arrest at intermediate concentrations. Moreover, at higher concentrations, ROS causes mutations by damaging various biomolecules and thus develops cancerous progression in normal cells and also develops multidrug resistance in cancer cells as an adaptive mechanism to various therapies (Aggarwal et al. 2019). Interestingly, due to intrinsic oxidative stress, most of the cancer cells remain alive and become resistant to various therapeutics by hindering apoptosis. Overall, in this chapter, we have discussed the potential role of ROS in the regulation of cancer leading to therapy resistance. In particular, we have discussed the generation, activation, and regulation of ROS, the dichotomous role of ROS in the progression and the inhibition of cancer, and the molecular pathways regulated by ROS for the development of cancer and therapy resistance. However, some key concerns need to be focused in the future for the better treatment of cancer and therapy resistance by targeting ROS. ROS has a dichotomous role in cancer, it may be prosurvival or prodeath. So, attempts must be taken either to attenuate or to augment ROS for sensitizing and killing the cancer cells. Nevertheless, the strategy of augmenting ROS levels may have some adverse effects on the normal cells, especially stem cells which are much more sensitive to ROS levels. So, it is crucial to distinguish and target the selective antioxidant pathways employed by cancer and therapy-resistant cells for redox balancing. Finally, searching for the combination of specific ROS modulators along with novel anticancer drugs may contribute better therapeutic avenues against cancer therapy resistance.

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Acknowledgment We would like to acknowledge Department of Science and Technology (DSTINSPIRE-IF130677), University Grants Commission (UGC), Council of Scientific and Industrial Research (CSIR), Indian Council of Medical Research (ICMR), Science and Engineering Research Board (SERB-J C Bose National Fellowship), Ministry of Human Resource and Development (MHRD), Government of India; Indian Institute of Technology Kharagpur, India; and German Academic Exchange Service (DAAD), Germany, for providing financial support.

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Oxidative Stress A Key Regulator of Breast Cancer Progression and Drug Resistance

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N. N. V. Radharani, Ipsita G. Kundu, Amit S. Yadav, and Gopal C. Kundu

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Free Radicals: Precursors of Oxidative Stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Oxidative Stress in Breast Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Oxidative Stress in Breast Tumor Growth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Role of Oxidative Stress in Modulation of Breast Tumor Microenvironment . . . . . . . . . . . . . . . . Role of Oxidative Stress in Breast Tumor Metastasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Role of Oxidative Stress in Breast Tumor Angiogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Role of Oxidative Stress in Metabolic Reprogramming in Breast Cancer . . . . . . . . . . . . . . . . . . . . Drug Resistance Due to Oxidative Stress in Breast Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Oxidative Stress and Breast Cancer Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract

Oxidative stress is a product of redox imbalance in which there is an enhancement of free radicals in the body which can cause tissue damage. ROS is the most important free radical species which can be generated in breast cancer by various conditions such as hypoxia, altered metabolism, and drug treatment. ROS perform a pivotal role in breast tumor initiation and progression by inducing DNA damage and upregulating various cell survival and oncogenic pathways. Higher oxidative stress N. N. V. Radharani, Ipsita G. Kundu and Amit S. Yadav contributed equally with all other contributors. N. N. V. Radharani · A. S. Yadav · G. C. Kundu (*) School of Biotechnology, KIIT Deemed to be University, Institute of Eminence, Bhubaneswar, India e-mail: [email protected] I. G. Kundu Department of Pharmacy, Birla Institute of Science and Technology, Pilani, Hyderabad Campus, Institute of Eminence, Hyderabad, India © Springer Nature Singapore Pte Ltd. 2022 S. Chakraborti et al. (eds.), Handbook of Oxidative Stress in Cancer: Mechanistic Aspects, https://doi.org/10.1007/978-981-15-9411-3_164

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can inhibit antioxidant defense against angiogenesis and metastasis by regulating the expression of pro-metastatic and pro-angiogenic factors in breast tumor microenvironment. ROS is also a product and key regulator of altered cancer metabolism. Furthermore, oxidative stress is also associated with chemotherapeutic drug resistance by inducing the expression of various drug transporters in breast cancer. These processes play critical role in the development and progression of breast cancer. In this chapter, we have discussed free radicals as precursors of oxidative stress and the role of oxidative stress in breast cancer progression, metastasis, angiogenesis, metabolic reprogramming, and drug resistance for better identification of the potential targets for redox dependent breast cancer therapy. Keywords

Breast cancer · Oxidative stress · Reactive oxygen species (ROS) · Tumor progression · Drug resistance

Introduction Cancer is one of the leading causes of death worldwide. Breast cancer is the most common cancer among women globally (Bray et al. 2018). Multiple risk factors are associated with development of breast cancer such as late marriage, late pregnancy, alcohol consumption, genetic factors, obesity, etc. Most of these risk factors are associated with oxidative stress. Oxidation and redox reactions are regulatory mechanisms that occur in our body naturally, but any imbalance between the oxidants like free radicals and antioxidants leads to the oxidative stress (Tas et al. 2005). In mammalian system, because of aerobic respiration, there is a continuous generation of free radicals. In mitochondrial electron transport system, superoxides are generated and reduced to hydrogen peroxide and hydroxyl radicals. These free radicals if imbalanced leads to the generation of oxidative stress which stimulates various disorders in the body like Parkinson’s, Alzheimer’s diseases (neurological disorders), inflammation, arthritis, and cancers including breast. Studies have shown that oxidative stress can affects the lipids, proteins, and nucleic acids regulation which further leads to the tissue damage (Sosa et al. 2013). In cancer, ROS has been shown to have ambiguous behavior depending upon stage of cancer progression. In initial stage of tumor generation, moderate levels of ROS seem to have pro-tumorigenic effect while in late stage of tumor progression toxic levels of ROS induce cell death and senescence (Assi 2017). Oxidative stress is found to be associated with all major hallmarks of cancer (Fig. 1). Hence, ROS-mediated oxidative stress functions as a double-edged sword and need to be studied extensively.

Free Radicals: Precursors of Oxidative Stress Free radicals like reactive oxygen species (ROS), reactive nitrogen species (RNS), reactive chloride species (RCS), and sulfur are produced during its utilization in various metabolic reactions, and ROS is found to be the major oxidant responsible

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Fig. 1 Association of oxidative stress with various hallmarks in breast cancer

for inducing oxidative damage to the cells (Sosa et al. 2013). ROS includes nonradical molecules like hydrogen peroxide (H2O2), free radicals like super oxide anion (O2˙ ), and hydroxyl radicals (OH•) (Sosa et al. 2013; Klaunig et al. 2010). The source for ROS includes internal factors like cellular mitochondria, peroxisomes, endoplasmic reticulum (ER), and immune cells like macrophages, neutrophils, and eosinophils, and external factors like alcohol, drugs, smoking, pollution, anti-cancer drugs, radiation, etc. The major source for ROS production is observed in mitochondria, where 2% of the oxygen consumed is used to form superoxides. The first ROS superoxide ion O2 is produced in inner mitochondrial membrane from oxidative phosphorylation and electron transport chain that in turn reacts with other molecules and produces free radicals like reactive oxygen and nitrogen. Peroxisomes and ER also produces ROS where peroxisomes produce ROS (flavin oxidase activity and oxidation of fatty acids) and can also catalyze decomposition of H2O2 thereby acting as scavengers for ROS. ROS can be generated either by enzymatic or nonenzymatic reactions. The nonenzymatic source of ROS production is mitochondrial respiration and the enzymatic source includes xanthine oxidase, NADPH

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oxidase, arachidonic acid, uncoupled endothelial nitric oxide synthase (eNOS), and metabolism specific enzymes like lipoxygenase, cyclooxygenase, and cytochrome P450 enzymes (Sosa et al. 2013; Assi 2017; Nourazarian et al. 2014). To maintain cellular homeostasis, it is necessary to maintain a constant level of intracellular ROS, as different amounts of ROS affect biological responses. Low to moderate levels of ROS helps in inducing proliferation and differentiation of cells and for activation of survival pathways, whereas higher levels of ROS can induce DNA damage. Thus, in order to maintain the redox balance, antioxidants comes into play that includes several antioxidant enzymes like super oxide dismutase (SOD), catalase glutathione peroxidase, and nonenzymatic oxidants like vitamins A, C, and E, which can be obtained by consuming antioxidant-rich food and metal ion chelators, flavonoids, glutathione, albumin, and uric acid (Nourazarian et al. 2014; Feng et al. 2012). However, when the redox system is dysregulated, ROS induces oxidative stress which further affects cell-specific mechanisms like cell proliferation, metabolism of the cell, cell signaling pathways, and apoptosis. Superoxides can further induce mutations and converts proto-oncogenes to oncogenes, also changes transcriptional and cell signaling pathways thereby ultimately initiating tumor growth and progression leading to the development of cancers (Sosa et al. 2013; Assi 2017; Klaunig et al. 2010; Nourazarian et al. 2014; Feng et al. 2012).

Oxidative Stress in Breast Cancer There are various physical and cellular factors which contribute to generation of oxidative stress in cancer. Hypoxia is one of the main physical factors responsible for oxidative stress in cancer (Brown and Bicknell 2001). When the diameter of the tumor reaches more than 200 μm diameter, the angiogenic blood supply will reduce towards the core of the tumor thus creating a low oxygen tension milieu termed hypoxia. Thus, hypoxia became a characteristic feature for solid tumors (Raja et al. 2014). Extensive studies have showed that in hypoxic environment, cancer cells exhibit Warburg effect (increased aerobic glycolysis) which is considered as one of the hallmarks of cancer. There exists a need for higher amounts of ATP, as the proliferation rate of cancer cells is high, and the downside of this increased energy production leads to enhanced enrichment of ROS. Thus, in tumor, the levels of ROS were found to be upregulated compared to normal cells. Further, hypoxia also contribute to generation of ROS and aggravation of oxidative stress in breast cancer by impairing the activity of complex III (cytochrome b oxidoreductase) which is a component of mitochondrial respiratory chain and the activity of NADPH oxidase of macrophages (Nourazarian et al. 2014; Fiaschi and Chiarugi 2012). Within the tumor microenvironment, cellular factors like tumor-associated macrophages (TAMs), cancer-associated fibroblasts (CAFs), and other immune cells contributes to the enrichment of ROS within the breast cancer (Nourazarian et al. 2014; JezierskaDrutel et al. 2013). Thymidine phosphorylase, an enzyme highly expressed in breast carcinoma, induces oxidative stress by catalyzing thymidine to thymine and deoxide ribose phosphate, the latter which glycates various proteins thereby producing

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free radicals in breast tumor (Brown and Bicknell 2001; Brown et al. 2000). In breast cancer, oxidative stress can also be caused due to the lactoperoxidase produced in mammary glands. Lactoperoxidase catalyzes oxidation of estradiol and produces panoxyl free radicals (Brown and Bicknell 2001; Sipe et al. 1994). Several markers for oxidative stress have been observed in breast tumor samples. These markers include ROS-mediated lipid peroxidation product malondialdehyde (MDA), DNA damage product 8-hydroxydeoxy guanosine (8-OHdG) and glutathione, etc. (Nourazarian et al. 2014).

Oxidative Stress in Breast Tumor Growth ROS induces breast tumor formation by mutating the DNA, by altering the transcriptional mechanisms, or by altering various tumor-promoting pathways like mitogen-activated protein kinase (MAPK), protein kinase C, phospholipase C, and phosphatidylinositol 3-kinase (PI3-K). High amount of oxygen free radical and reduced catalase activity has been found in breast cancer specimens (Tas et al. 2005; Nourazarian et al. 2014; Brown and Bicknell 2001; Wiseman and Halliwell 1996). It has been shown that overexpression of NADPH oxidase 4 (NOX4), a ROSproducing enzyme, results in resistance to apoptosis in breast cancer cells which leads to enhanced tumorigenic potential and aggressiveness (Graham et al. 2010). Hypoxia and low glucose levels are associated with oxidative stress-mediated increased breast tumor growth. In MCF-7 cells, low glucose level induces ROS and oxidative stress (Brown and Bicknell 2001). The hypoxic niche further promotes tumor progression by activating various tumor promoting oncogenes and signaling pathways through hypoxia inducible factor (HIF1), a transcription factor. HIF1 comprises of two subunits: HIF1α, an oxygen responsive subunit, and HIF1β, which is a constitutively expressed subunit. In the presence of oxygen, prolyl 4 hydroxylase domain proteins (PHDs) hydroxylates prolyl residues of HIF1α, the hydroxylated HIF1α binds to the von Hippel-Lindau proteins (pVHLs) which induces proteosomal degradation of HIF1α by recruiting E3 ubiquitin ligase. In the absence of oxygen, the unhydroxylated HIF1α translocates to the nucleus further dimerizes with HIF1β and binds to the promoter regions of hypoxia-response element thereby activating gene expression of various genes involved in tumor growth, angiogenesis, and metastasis. The role of hypoxia in inducing carcinogenesis has been studied extensively at both cellular and molecular levels. Further the hypoxic environment activates ROS production which in turn activates HIF1α by inactivating PHDs (Yang et al. 2014). Alteration in redox state can modulate various genes and transcription factors associated with cell proliferation and DNA repair. Increased ROS further activates various genes involved in malignancy. Hydrogen peroxide triggers proliferation in breast cancer cells by activating MAPK pathway. Additionally, ROS can induce mitosis via Ras-driven pathways. Stress-activated protein kinase (SAPK) which includes p38 and JNK and mitogenic stimulated kinase ERK hyper phosphorylates cJun and cFos leading to induction of AP-1 activation thereby promoting cell proliferation under oxidative stress. In MCF-7

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cells, free radical superoxide concentration affects the proliferation of cancer cells. In breast cancer cells, it was observed that arsenate can induce cell proliferation by increasing ROS levels and activating signaling molecules like NF-κB and increased c-Myc (Sosa et al. 2013; Aggarwal et al. 2019).

Role of Oxidative Stress in Modulation of Breast Tumor Microenvironment The interaction between cancer cells and stromal microenvironment regulates breast tumor progression. Stromal compartment within a tumor comprises of cancer-associated fibroblasts (CAFs), immune cells like macrophages, neutrophils, endothelial cells, pericytes, etc. ROS produced from stromal cells can promote tumor progression. It has been reported that migration of MCF-7 cells can be induced by ROS synthesized by NADPH oxidase in stromal cells (Tobar et al. 2010). Generation of ROS by breast cancer cells lead to mitochondrial injury in CAFs leading to organelle autophagy which results in release of nutrients for glycolysis and hence inducing Warburg effect (Lozy and Karantza 2012). Studies have shown the role of hydrogen peroxide in inducing senescence in fibroblasts and cancer cells. In fibroblasts, senescence induces a transition from senescence to autophagy (SAT). These fibroblasts express senescence specific markers like p21, p16, and beta-galactosidase thereby secreting various growth factors, cytokines which promotes cancer cell migration (Capparelli et al. 2012). Additionally, increase in number of senescent fibroblasts lead to senescence-activated secretory phenotype (SASP) resulting in inflammatory environment due to secretion of proinflammatory cytokines such as IL-6, IL-8, insulin-like growth factors, chemokines, proteases, etc. which in turn promotes breast tumor progression and endothelial to mesenchymal transition (EMT) (Capparelli et al. 2012; Coppe et al. 2010). Reports suggest that one of the byproducts of oxidative stress is mitochondrial dysfunction. Further, studies have shown that ROS is sufficient to induce differentiation of fibroblast to myofibroblasts which further promote tumor proliferation, metastasis, and angiogenesis by secreting high levels of growth factors, cytokines, and metalloproteinases (Fig. 2). These myofibroblasts produce ROS thereby increasing oxidative stress in tumors (Toullec et al. 2010; Giannoni et al. 2011). It has been studied that breast tumor-mediated oxidative stress induces lysosomal degradation of caveolin-1 (Cav-1) by activation of NF-κB and HIF-1α in differentiated fibroblasts (Martinez-Outschoorn et al. 2010a). Loss of stromal Cav-1 reflects lethal condition in breast cancer as it is associated with decreased overall survival, enhanced tumor recurrence, higher lymph node metastasis, and drug resistance. Loss of Cav-1 has been reported in both hormone receptor positive and negative breast cancer; however, it is more predominant in hormone receptor negative subtype (Witkiewicz et al. 2010). Immune cells like macrophages are one of the main sources for the production of ROS. In breast tumor, several studies have shown the increased infiltration of macrophages in tumor. Macrophages or monocytes that are present in the blood are attracted to the tumor site because of the factors secreted by tumor cells like MCP-1, M-CSF, GM-CSF, etc. Once recruited, these macrophages produce various growth factors and cytokines that control tumor growth and metastasis.

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Fig. 2 ROS-mediated signaling in promotion of breast tumor growth, metastasis, angiogenesis, and drug resistance

In order to perform phagocytic and bactericidal activity, macrophages and neutrophils generate ROS through plasma membrane-specific NOX enzyme that is regulated by Rac1. The Rac1 is encoded by proto-oncogene, RAS and its GTPase activity produces superoxide ions which encourages tumor growth. Coso et al. have shown that NOXderived ROS further induces endothelial cell proliferation and tube formation. Additionally, tumor-associated macrophages (TAMs) secrete tumor necrosis factor (TNF) which induces oxidative stress (Condeelis and Pollard 2006; Grivennikov et al. 2010).

Role of Oxidative Stress in Breast Tumor Metastasis Metastasis is a complex and multifaceted process where malignant cells disseminate from the primary tumor growth to the nearby tissues and to other distant organs. It is triggered by the activation of various transcriptional factors such as nuclear factor-κ B (NF-κB), ETS proto-oncogene 1 (ETS-1, transcription factor), various EMT regulators (Twist, Snail, etc.); metalloproteases such as MMP-9 and MMP-2; and a diverse variety of chemokines or cytokines including transforming growth factor beta (TGF-β) (Seyfried and Huysentruyt 2013). ROS-dependent oxidative stress is a crucial regulator of the migration and invasion of breast cancer cells (Liao et al. 2019). EMT is the main event in tumor metastasis process characterized by loss in their polarity, cell-cell adhesion, and gain mobility in epithelial cells (Yadav et al.

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2018). Various studies have identified ROS as a major inducer of EMT. ROS facilitate cell migration and invasion by inducing TGF-β1-mediated regulation of uPA (urokinase-type plasminogen activator) and MMP-9 (Liao et al. 2019). Another study demonstrated that ROS enhances breast cancer cell migration, hypoxia-dependent expression of MMPs, and cathepsin (Shin et al. 2015). Further, it has been observed that TGF β1 induces EMT in MDA-MB-231 and MCF-10A cells in response to ROS produced by NADPH oxidase 4 (NOX4) (Zhang et al. 2013). Moreover, C-X-C motif chemokine 14 (CXCL14) expression was found to be upregulated by ROS via AP-1 signaling pathway which further induced breast cancer cell migration through elevation of cytosolic Ca2+ levels (Pelicano et al. 2009). ROS-dependent activation of fibroblast induces secretion of MMPs and other cytokines that aid in EMT and eventually breast cancer metastasis (Fig. 2) (Toullec et al. 2010; Giannoni et al. 2011).

Role of Oxidative Stress in Breast Tumor Angiogenesis Generation of neo-vasculature from the preexisting vasculatures in initial phase of tumor generation is known as angiogenesis. Angiogenesis plays crucial role in tumor proliferation and survival. Tumor proliferation leads to higher metabolic rate which in turn generates high amount of ROS leading to oxidative stress which ultimately modulates tumor microenvironment in secreting pro-angiogenic molecules. VEGF is one of the key pro-angiogenic molecule that is enriched in hypoxia and oxidative stress (Aggarwal et al. 2019). Several studies have shown that within the tumors, hypoxia and oxidative stress may exist together, and the increased levels of VEGF reciprocate the synergistic effect of hypoxia and oxidative stress. Further the oxidative stress-enriched hypoxia induces VEGF expression. Oxygen radicals induce tumor cells-mediated secretion of VEGF, IL-8, and matrix metalloproteinase-1 (MMP-1) which helps in vessel growth and tube formation (Dewhirst et al. 2008). It has been shown that ROS and hypoxia collaborate in promotion of angiogenesis in various cancers including breast through HIF-1α (Liu et al. n.d.). Exogenous or endogenous ROS activates various pathways such as PI3K/Akt/mTOR, PTEN, and MAPK which through HIF-1α induce VEGF, MMPs, and various cytokine expression leading to angiogenesis (Karar and Maity 2011). It has been shown that ROS can promote expression of VEGF, HIF-1α, and G-protein coupled estrogen receptor (GPER) in breast cancer cells by activating EGFR/ERK/c-Fos signaling (Rigiracciolo et al. 2015). ROS may also induce angiogenesis by activation of NF-κB via TLRs in a hypoxia independent pathway (Fig. 2) (Aggarwal et al. 2019).

Role of Oxidative Stress in Metabolic Reprogramming in Breast Cancer Aberrant metabolism is a hallmark of cancer. Compared to normal cells, cancer cells exhibit enhanced steady state levels of ROS which in turn is compensated by enhanced glucose metabolism. Evidences have suggested that ROS can alter the

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metabolic settings in tumor microenvironment towards aerobic glycolysis. ROS can modulate the activity of metabolic enzymes by posttranslational modifications. Oxidative stress affects the functioning of main metabolic enzymes such as PDH, IDH, fumarase, GAPDH, malic enzyme, and citrate synthase and alter the levels of various metabolites generated from glycolysis and Kreb’s cycle (Wang et al. 2019). Hypoxia-dependent O-GlcNAcylation of phosphofructokinase-1 (PFK-1) has been shown to inhibit PFK1 activity. This glycosylation happens at serine529 of PFK-1 which diverts glucose metabolism toward oxidative PPP, leading to increased NADPH production for cell survival under stress (Yi et al. 2012). Further, ROSdependent Akt-mediated phosphorylation of PFK-2 leads to activation of PFK-1 by fructose 2, 6 bis-phosphate, a metabolic product of PFK-2 (Deprez et al. 1997). Moreover, ROS-mediated oxidation of cysteine 358 residue of PKM2 inhibits its activity leading to generation of lactate (Anastasiou et al. 2011). Interestingly, it has been observed that SUMOylation of core metabolic enzymes under oxidative stress may redirect the cellular metabolism towards increased flux through the glycolytic pathway (Agbor et al. 2011). Moreover, higher ROS levels can impair different mechanisms such as HIF-1α stabilization, posttranslational modifications of complex I proteins, along with inhibiting the activity of PTP1b, PTEN, and MAPK phosphatases leading to shift towards aerobic glycolysis and survival (Kim et al. 2016). ROS also make changes in stromal component of cancer metabolism to favor tumor progression (Jezierska-Drutel et al. 2013). Loss of Cav-1 by ROSdependent lysosomal degradation in CAFs seems to affect aerobic glycolysis. Loss of Cav-1 in CAFs results in lower levels of pyruvate dehydrogenase, an enzyme which converts pyruvate to acetyl-coA. It may inhibit progression of metabolic cycle from glycolysis to oxidative phosphorylation leading to shift towards aerobic glycolysis (Jezierska-Drutel et al. 2013; Martinez-Outschoorn et al. 2010b). Cav-1 degradation is also associated with the expression of two monocarboxylate transporters MCT 1 and MCT4 on breast cancer cells and CAFs, respectively, which assist in efficient import of lactate from CAFs to breast cancer cells to fulfill energy needs and material metabolism (Jezierska-Drutel et al. 2013; Whitaker-Menezes et al. 2011).

Drug Resistance Due to Oxidative Stress in Breast Cancer Cancer cells can survive and proliferate in conditions that would lead to death of normal cells. Deregulation of cellular energetics is a hallmark of cancer. When tumors are below 1% oxygen concentration, they are considered to be hypoxic which leads to alteration of cellular metabolism. A consequence of this is higher levels of reactive oxygen species (ROS) with collateral alterations in antioxidants pathway. Targeting this hypoxia-actuated tumor metabolism is a propitious strategy for anticancer treatment. However, the effect of hypoxia on cells is multifaceted. ROS in below micromolar level regulates many usual cellular processes because normoxic cells maintain a redox homeostasis between cellular oxidants and antioxidants. But exorbitant levels of ROS lead to oxidative stress and cause tumor cell death. This is used as an ingenious strategy to induce oxidative stress via anticancer

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drugs. Various chemotherapeutic drugs like taxanes, platinum compounds, and anthracycline trigger cell death by causing oxidative stress. However, cancer cells rendered a way to acclimatize to the ROS-dependent cytotoxicity through “redox resetting” in which tumors acquire a new redox homeostasis at higher accumulation of ROS and stronger antioxidants system (Liu et al. 2016). This redox resetting has been implicated in drug resistance through enhanced drug efflux, modulated drug metabolism, and altered drug targets. ROS-dependent drug resistance could be due to various factors such as genetic instability, enhanced drug efflux, mutation of drug targets, activation of cell survival pathways, inactivation of downstream signaling pathways, and thus failure to induce cell death (Liu et al. 2016). ROS regulates various proteins such as HIF1α, NRF2 (nuclear factor-erythroid-2–related factor 2), Keap1 (Kelch-like ECH-associated protein 1), ATM (ataxia telangiectasia); transport protein like ABC (ATP-binding cassette), MDR1 (multidrug resistance protein 1), BCRP (breast cancer resistance protein), P-glycoprotein; transcription factors like FOXO (Forkhead box O), ARE (antioxidant response element) which play a significant role in breast cancer drug resistance (Liu et al. 2016). In relation to multidrug resistance, MDR1 and BCRP have been studied extensively. It has been observed that ROS can regulate the necessary conformational changes required by drug transporters and enhance the drug efflux resulting in drug resistance (Liu et al. 2016). ROS can also mediate in overexpression of drug efflux pumps for the export of drugs. It has been observed that after anticancer drug treatment, resulting redox signaling can control the transporter expression at transcriptional, translational, posttranslational, and epigenetic levels. NRF2, a redox sensing transcription factor, can bind to AREs which can be found in promoter region of drug efflux transporters such as BCRP and MRPs and induce their expression. KEAP1 binds to NRF2 in cytoplasm and leads to its ubiquitination and proteasomal degradation has important target residues for oxidation. Redox changes break this complex allowing NRF2 to translocate into the nucleus facilitating expression of drug transporters (Fig. 2). Enhanced NRF2 expression has been correlated with taxol resistance in breast cancer (Hayes and McMahon 2006). Moreover, its target genes are linked to taxol resistance. FOXO proteins are family of transcription factors which are found to be deregulated in various cancer including breast. Anticancer drugs induced stress leads to FOXO-dependent overexpression of MDR1 and other relevant genes to cause drug efflux and antioxidant defense in adriamycin-resistant breast cancer cells (Fig. 2) (Han et al. 2008). Further, MDR1 acts as a substrate of various protein kinases (PK) including PKC. Evidences suggest that redox alteration can enhance the MDR1 activity by modulating PKC catalytic activity (Giorgi et al. 2010). Moreover, oxidation of TYMS (thymidylate synthase) sequels in reduction of translational repressor function that leads to 5-FU (5 fluorouracil) resistance in cancer cells (Field et al. 2015). Oxidative stress created by hypoxia inhibits apoptosis in cells treated with paclitaxel by activation of c-Jun N-terminal kinase pathway. Furthermore, hypoxia induces acidification in MCF7 cells which results in mitoxantrone resistance due to decreased cellular uptake (Greijer et al. 2005). Cancer cells attune to oxidative stress via many mechanisms and hence survive in hypoxia and become drug resistance.

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Oxidative Stress and Breast Cancer Therapy As discussed earlier that ROS in oxidative stress works as a double-edged sword in cancer. Moderate increase in ROS levels promotes tumor progression, while at very high levels, it causes cell death in cancer. Based on this, various approaches have been developed for ROS-dependent cancer therapy. One approach is to disrupt antioxidant system by inhibiting core antioxidant enzymes like SOD, glutathione (GSH), and thioredoxin 1 (Trx) to facilitate higher ROS production in malignant cells leading to cell death (Fig. 3) (Aggarwal et al. 2019). Various therapeutic candidates have been developed to block antioxidant activity in cancer cells in order to induce cell death and sensitivity to chemotherapeutic drugs. Phenethyl isothiocyanate (PICT) and buthionine sulfoximine (BSO) are two such candidates which have been shown to induce chemo-sensitivity and cell death in various cancers by inhibiting GSH activity (Trachootham et al. 2006; Gana et al. 2019). Another approach is to develop ROS producing agents to enhance ROS levels and eventually induce cellular damage. Such agents include antifolates, alkaloids, and taxanes which can induce ROS production by impairing the mitochondrial electron transport chain (ETC) (Aggarwal et al. 2019). Many chemotherapeutic drugs such as doxorubicin and cisplatin also enhance ROS production leading to cytotoxicity. The first ROS-dependent drug used for anticancer therapy was procarbazine which could generate ROS by producing azo-derivatives (Parasramka et al. 2017). In the last two decades, multiple ROS-inducing therapeutic agents have been tested extensively for their antitumor efficacy and safety. Doxorubicin and anthracyclines are the examples of ROS-inducing drugs which are commonly employed in the treatment of various cancers including breast (Fig. 3) (Pilco-Ferreto and Calaf 2016).

Conclusion The relation between oxidative stress and breast cancer progression is intricate and diverse depending upon its intensity and duration of exposure. It has been observed that higher oxidative stress mostly induces death in breast cancer cells of different subtypes. However, highly aggressive cell lines possess resistance against oxidativemediated cell death. Oxidative stress in cancer can be generated by higher production of free radical moieties such as ROS by cancer and stromal cells in response to hypoxia, altered metabolism, and drug treatment which leads to impairment of oxidant/antioxidant homeostasis. Oxidative stress can influence breast tumor initiation by causing DNA damage. It helps in breast tumor growth by upregulation of various growth factors, cytokines, chemokines, oncogenic signaling pathway, and pro-inflammatory environment. It aids in breast tumor metastasis and angiogenesis by secretion of pro-metastatic and pro-angiogenic molecules such as MMPs, VEGF, etc. in tumor microenvironment. ROS mediates in metabolic alteration of tumor microenvironment to fulfill the energy and metabolite requirement of cancer cells. It further induces drug resistance in treated tumors by regulating expression of drug

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Fig. 3 ROS-dependent therapeutic approaches in breast cancer treatment

transporters and altering drug targets. Hence, the role of oxidative stress in various process involved in breast tumor initiation, progression, and treatment resistance must be thoroughly investigated which will lead to identify more effective therapeutic approaches for management of breast cancer.

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Jessica Campos-Bla´zquez, Catalina Flores-Maldonado, Alan A. Pedraza-Ramírez, Octavio Lo´pez-Me´ndez, Juan M. Gallardo, Leandro A. Barbosa, and Rube´n G. Contreras

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Reactive Oxygen Species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Signaling by ROS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Autophagy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Signals that Trigger Autophagy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Activation of Autophagy by ROS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Protective Effect of Autophagy Against Ischemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Conjugation Complex ATG3, ATG4, ATG7, and ATG8 (LC3) . . . . . . . . . . . . . . . . . . . . . . ROS Induce Transcription Factors of Autophagy Genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Negative Feedback Against ROS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract

Reactive oxygen species (ROS) used to be perceived as harmful cellular weaponry designed to eliminate pathogens, whereas autophagy as the process for the disintegration and re-utilization of cytoplasm. Both pathways respond to cellular stress and are evolutionary conserved, ubiquitous in modern organisms, J. Campos-Blázquez · C. Flores-Maldonado · A. A. Pedraza-Ramírez · O. López-Méndez · R. G. Contreras (*) Department of Physiology, Biophysics and Neurosciences, Center for Research and Advanced Studies of the IPN (Cinvestav-IPN), México City, Mexico e-mail: [email protected]; rcontrer@fisio.cinvestav.mx J. M. Gallardo Unidad de Investigación Médica en Enfermedades Nefrológicas, Hospital de Especialidades, Centro Médico Nacional “Siglo XXI” Instituto Mexicano del Seguro Social, México City, Mexico L. A. Barbosa (*) Laboratório de Bioquímica Celular, Universidades Federal de São João del Rei, Divinópolis, Brazil Laboratório de Membranas e ATPases, Universidades Federal de São João del Rei, Divinópolis, Brazil © Springer Nature Singapore Pte Ltd. 2022 S. Chakraborti et al. (eds.), Handbook of Oxidative Stress in Cancer: Mechanistic Aspects, https://doi.org/10.1007/978-981-15-9411-3_167

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necessary for development and important in the pathogeny of chronic diseases. The activity of the ROS pathway depends on spatial confinement of its proteins, the exposition of sensor’s cysteines to the solvent of regulatory proteins, the oxidationdriven conformational and oligomeric change of these proteins, and the limitation of the diffusion of ROS. ROS determine autophagy pathway activity exerting similar changes on crucial autophagy proteins, namely, ATG4, 3, 7, and 8, as well as the regulatory proteins AMPK and mTOR. The intricated relationship of ROS and autophagy pathways is determinant for the proper functioning of both pathways. Keywords

ATG4 · Nox · Cysteine · Prx · Oxidation

Introduction Reactive Oxygen Species Life origin and evolution relied on chemical instability for the formation of primitive biomolecules, and oxygen is one of the most important molecules for most of today’s cells. In the early earth, characterized by reducing conditions, volcanic eruptions produced carbon dioxide and, around 2.5 billion years ago, cyanobacteria evolved the photosynthesis that liberated oxygen from its association with carbon and enriched progressively the atmosphere with oxygen, in a process called “great oxidation event” (Planavsky et al. 2020). Along with the built-up of an oxidizing atmosphere, cells evolved aerobic metabolism that includes the controlled production of the oxygen-derived substances, more prone to chemical reaction than the relatively stable molecular oxygen. Modern cells have a group of oxygen-derived substances known as reactive oxygen species (ROS) that include the superoxide (O2
•) hydroxyl (OH•), alkoxy (RO•), and peroxy (ROO•) free radicals, as well as hydrogen (H2O2) and organic (ROOH) peroxides (Bartosz 2009) (Table 1, Fig. 1a). In today’s earth, physical and chemical environmental factors, such as pollution, radiation, light, thermal shock, and drugs, as well as the enzymes NADPH (Nox) and dual (Duox) NADPH oxidases, expressed on the plasma membrane, produce exogenous ROS. Complexes I and III of the respiratory chain, the cytochrome P450, and the glycerol-3-phosphate dehydrogenase (GPDH) of the mitochondrial inner membrane, monoaminoxidase, and cytochrome b5R in the outer mitochondrial membrane, aconitase, pyruvate dehydrogenase (PDH), and α-ketoglutarate dehydrogenase (α-KGDH) in the mitochondrial matrix produce endogenous ROS. The cells introduce the extracellular H2O2 produced by Nox and Duox through aquaporins 11 and 8 expressed at the plasma membrane and can also transport it to neighboring cells using communication junctions (D’Autréaux and Toledano 2007; Finkel 2011). To balance the chemical reactivity of ROS, cells need antioxidant systems, which include the mitochondrial matrix enzymes thiorredoxins (Trx), superoxide dismutases (MnSOD), glutathione peroxidase (GPA), catalase (CAT), and

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Table 1 Reactive oxygen species and their regulatory enzymes

ROS name Hydroxyl OH•

Superoxide O2‾•

Hydrogen peroxide H2O2

Redox potential (V) pH7 Redox couple Half reaction 0.38 H2O2/OH• H2O2.+H++e
! H2O2+OH• 2.32 H2O+OH• H2O.+h++! H++OH• -0.16 O2/ O2‾• O2+e
! O2‾•

0.89 O2
•/ H2O2 H2O2 O
•2.+2H++e
!H2O2 0.28 O2/H2O2 O2.+2H++2e
!H2O2

Oxidant enzymes Non

Cytochrome b5 (Cb5R) Reductase CI Reductase CIII

Monoamino oxidase-A (MAO-A) MAO-B Dehydroorotate dehydrogenase (DHODH, in absence of Co-enzyme Q) Dlicerol 3-phosphate dehydrogenase (GDPH) α-ketarate dehidrogenase (α-KGDH) Complexes I and III of the respiratory chain (CI, CIII) Succinate dehydrogenases (SDH)

Antioxidant enzymes Non

Cytochrome C (Cc) MnSuperoxyde dismutase (Mn-SOD) Catalase (Cat) Pyredoxins (Prx) Glutathion peroxidase (GP)

Characteristics Impermeable to the membrane Very reactive Diffuses short distances Radical anion Impermeable to the membrane

Peroxide. Permeable to the membrane Not very reactive Diffuses long distances

After Bartosz et al. 2009 and Li et al. 2018 (Bartosz 2009; Li et al. 2019). Importance of ROS in the cellular physiology depends on the reactivity of the specific ROS, evaluated by the Redox potential for a specific reaction, and on its diffusion capacity across the membranes

peroxirredoxins (Prx), as well as a variety of cytosolic enzymes and small compounds like vitamin E, ascorbic acid, and glutathione (GSH). An unbalance between the oxidant and the antioxidant cellular systems induces “oxidative stress” (Bartosz 2009), a concept that refers to the accumulation of free radicals and peroxides synthesized by aerobic cells and under the influence of epigenetic modifications or the incapacity of the antioxidant system to reduce the free radical species (Sohal and

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Fig. 1 Reactive oxygen species (ROS) and autophagy pathways. (a) ROS synthesis and consume pathways. Superoxide free radical (O2
•) is mainly produced by the complexes I and III of the respiratory chain, a minor amount is a product of pollution, radiation, and drugs. Cells produce O2
• at the plasma membrane by the NADPH (Nox) and dual (Duox) NADPH oxidases. Extracellular H2O2 enters the cytoplasm through aquaporines 11 and 8. Mitochondrial enzymes such as superoxide dismutase (SOD), catalase (CAT) and peroxiredoxins (Prx), or the small molecule glutathione (GSH) neutralize ROS. The oxidation of GSH produces oxidized glutathione (GSSG). ROS either have harmful effects or participate in cell signaling. (b) Cysteine containing proteins are the target of ROS, which work in association with other signals like phosphorylation, glutathionylation, and acetylation (Ac). Acetylation and/or phosphorylation provokes the exposition of cryptic cysteine SH groups, hidden inside the protein, to the solvent, that react according to the degree of oxidation to produce sulfenic (RSOH), sulfinic (RSO2H), or sulfonic (RSO3H) forms. Protein can also form disulfide bridges (R-S-S-R). (c) Autophagy pathway. Representation of the main protein components of the different autophagy stages. For explanation see the text. Created with BioRender.com

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Allen 1990). The accumulation of ROS leads to a disorder of the physiological oxidation system and produces DNA hydroxylation, protein and lipid degradation, and tissue damage (Sheng and Qin 2019). Although antioxidant enzymes reside usually in the cytosol and mitochondrial matrix, it can be also unconventionally secreted outside the cells in an active form upon starvation, suggesting that cells regulate also environmental conditions, probably to favor posterior recovery from stress (Cruz-Garcia et al. 2020) (Fig. 1a). The chemical reactivity and diffusion through membranes determine the impact of ROS on cell physiology. OH• free radical has the highest reactivity, indicated by its high redox potential, but diffuses only short distances and reacts readily with any molecule in the proximity, condition that limits its effects and decreases its importance as a messenger in cells. O2
• free radical has a low reactivity but can be converted to H2O2 and exert then its effects on cells. On the other hand, H2O2 is middling reactive but diffuses longer distances and, therefore, exerts a more perdurable effect, therefore, is considered the most important intracellular messenger (Table 1). ROS oxidize proteins in two ways: they interfere with iron and sulfur ([Fe-S]) clusters and the oxidize cysteine residues exposed to the solvent. The electrical charge of [Fe-S] clusters attracts to O2
• free radicals, which provokes the liberation of the iron from the cluster and, consequently, the inactivation of the protein. H2O2 oxidizes exposed cysteine residues to a variety of sequential states, depending on the oxidation conditions of the solvent. Under mild oxidation conditions, cysteine is oxidized to the sulfenic state (RSOH), which is rapidly converted to its sulfinic (RSO2H) form. If the solvent has very oxidizing conditions, RSO2H is further oxidized to the very stable sulphonic (RSO3H) form. Given that the sulfenic is comparatively more active than the RSO3H form, it may react to form intramolecular or intermolecular disulfide bridges that change the conformation and/or dimerization of the oxidable cysteine-containing proteins (D’Autréaux and Toledano 2007; Finkel 2011) (Fig. 1b). To adapt to and survive the oxidative stress, cells have evolved compensatory and regulatory mechanisms. The most studied is GSH, an abundant tripeptidyl molecule, necessary for cell survival in mammalians cells that plays an important antioxidant role. GSH is synthesized by most eukaryotes and some bacteria. GSH is a stable molecule and ROS force it to become oxidized glutathione (GSSG), which is reduced back to GSH by the glutathione reductases (GR) dependent on nicotinamide adenine dinucleotide phosphate (NADPH). Besides an oxidation buffer, GSH helps to detoxify cells and metabolize drugs (Homma and Fujii 2015). The increment of oxidative stress is related to aging, male infertility, synaptic dysfunction, and Alzheimer’s disease (Bisht et al. 2017; Sheng and Qin 2019; Tönnies and Trushina 2017). Interestingly, the production of ROS and cellular antioxidants compensate each other in circadian rhythms. Peroxiredoxins (Prxs), which are highly conserved, specific, and efficient antioxidant proteins, are expressed since cyanobacteria up to vertebrates, in so precise oscillations that behave like a cellular timer. The acquisition of this efficient oscillatory redox system constituted an evolutive advantage for successful species that allowed the emergence of aerobic respiration (Edgar et al. 2012).

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Signaling by ROS The high reactivity of ROS, their capacity to destroy vital molecules of cells, the discovery of SOD, and the clear relation of the increment of ROS with aging, advocated for a harmful effect on cells. Recent evidence has demonstrated that ROS are not only harmful to cells but a kind of second messengers with important participation in signaling. Current models propose that the specificity of ROS signaling results from the exposition of cryptic cysteines hidden in the interior of the protein to the solvent, the spatial confinement of the participant proteins, and the interaction with other protein modifications, such as phosphorylation and acetylation (Behring et al. 2020; D’Autréaux and Toledano 2007). In this respect, Klebsiella pneumoniae binding to specific receptors in mice neutrophils triggers the Src Family Kinases (SFK) and Src Kinase Associated Phosphoprotein-2 (SKAP2) signaling cascade that results in the degranulation of neutrophils and a controlled liberation of a lethal amount of ROS that kills invading bacteria (Nguyen et al. 2020). H2O2 activates a survival pathway involving the sequential phosphorylation of Src and alb SFKs, protein kinase D (PKD), the inhibitor of nuclear factor kappa-B kinase subunit beta (IKKβ), and the transcription factor known as nuclear factor kappalight-chain-enhancer of activated B (NF-κB) in human cervical cancer cells (HeLa) (Storz 2003). H2O2 likewise activates the spleen tyrosine kinase (Syk) that in turn phosphorylates and induces the degradation of the nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor (I-κB). Active NF-κB induces survival though the increases in the expression of the antiapoptotic protein Bcl-2 and the apoptotic inhibitor IAP (Storz 2003). When the epidermal growth factor (EGF) binds its receptor (EGFR) in skin carcinoma cells, A431 induces the oxidation of 4200 cysteine residues in three different waves. The early one spans the first 15 minutes and oxidizes signal initiators including proteins that increment cell migration, adhesion, and the activation of small GTP binding proteins Rho, Rac, Cdc42. The intermediate or stimulator’s wave lasts from the 15 to the 30 following minutes after EGF treatment and oxidates proteins of the cell cycle, glycolysis, ER-Golgi, and Golgi-ER vesicular transport, among many others. The terminal effector’s wave encompasses the period between the 30th to the 60th min and shows an active ROS metabolic process, activation of Prxs, and cytosolic calcium signaling. EGF specifies which cysteine residues are to be oxidized by inducing its exposition to the solvent after phosphorylation of the corresponding proteins and incrementing the concentration of nucleotide substrates (Behring et al. 2020). Wnt ligands control several differentiation processes in normal tissues. In Wnt ligands absence, β-catenin is part of the “degradation complex” together with glycogen synthase kinase-3β (GSK3β), Axin2, and the adenomatous polyposis coli scaffold protein (APC). In this complex, GSK3β phosphorylates β-catenin, then the ligase β-TcRP ubiquitinates the phosphorylated β-catenin, which then goes into the proteasome for degradation. Wnt ligands’ receptor consists of frizzled and its coreceptor, the low-density lipoprotein receptor-related protein 5/6 (LRPS/6). Binding of Wnt to its receptor recruits and activates the intracellular protein

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Dishevelled (Dvl), which also recruits Axin2, inducing β-catenin degradation complex disassembly. In these conditions, β-catenin accumulates in the cytoplasm where it binds to the T-cell factor/ lymphoid enhancer factor (TCF/LEF) family of transcription factors, being TCF4 the best characterized. β-catenin/TCF4 complex, finally, enters inside the nucleus and promotes the transcription of genes that regulate cell polarity, proliferation, and differentiation, including c-Jun, c-Myc, and others (http://www.stanford.edu/~rnusse/pathways/targets.html). The reduced form of nucleoredoxin (Nrx), a thioredoxin-related protein, associates with Dvl, impairing its phosphorylation and posterior proteasomal degradation targeted by the Kelch-like 12 (KLHL12) ubiquitin ligase. This inactive Dvl accumulation favors robust activation of Wnt/β-catenin signaling upon Wnt stimulation (Funato et al. 2010). The ROS pathway is important for many cellular functions and it is not strange that regulates also autophagy, a crucial catabolic pathway that participates also in a great number of cellular functions.

Autophagy Autophagy is a degradative process mainly activated by cellular stress, such as nutrient deprivation, hypoxia, and oxidative stress (de Duve and Wattiaux 1966; Mizushima 2007; Tsukada and Ohsumi 1993); during these conditions, autophagy provides molecular components needed for cell survival from a nonselective cytosolic material degradation. However, autophagy is also essential to cell homeostasis by clearing selectively protein aggregates and damaged organelles. According to the cargo sequestration and its delivery to the lysosomes, autophagy is classified into (a) Microautophagy, in which cytosolic components are directly engulfed into the lysosome (Li et al. 2012); (b) Chaperone-mediated autophagy, in which specific proteins are recognized by chaperones like Hsc70 and internalized into the lysosome through a membrane pore (Massey et al. 2004), and (c) Macroautophagy, commonly referred just as autophagy, characterized by the formation of a double membrane organelle denominated phagophore (Baba et al. 1994), that engulfs cytosolic material and targets it to the lysosome. This chapter is focused on macroautophagy, hereafter termed autophagy. This process consists in a five steps pathway denominated autophagy flux, summarized below (Fig. 1c).

Initiation Autophagy initiation relays on a group of proteins known as induction complex, consisting of Ser/Thr kinase ULK1 (Unc-51-like kinase), FIP200, ATG13, and ATG101 (Jung et al. 2009; Sinha et al. 2015). ULK1 is the main regulatory protein within this complex and autophagy regulation mechanisms act through ULK1 activity. AMPK (AMP-activated protein kinase), the major autophagy promoter, activates ULK1 through phosphorylation at Ser555 and in consequence induces the assembly of the ULK complex and autophagy, whereas mTORC1 (mechanistic target of rapamycin complex 1), the major autophagy inhibitor, inactivates ULK1 through phosphorylation at Ser757 and blocks autophagy (Kim et al. 2011).

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Nucleation Once ULK1 is activated and the induction complex assembled, ULK1 phosphorylates and activates proteins within the PI3K class III complex (PI3KC3), consisting of the lipid kinase VPS34 (vacuolar protein sorting 34), Beclin 1, AMBRA (activating molecule in Beclin 1-regulated autophagy protein 1), ATG14, and p115. This complex promotes phosphatidylinositol-3-phosphate (PI3P) production at the rough endoplasmic reticulum (RER) membrane (He and Levine 2010; Nishimura et al. 2017), which is recognized by DPCP1 (zinc-finger FYVE domain-containing protein 1) and WIPIs (WD repeat domain phosphoinositide-interacting proteins); these proteins bind to and deform the membrane, forming a structure known as omegasome (Dooley et al. 2014). ATG9 also contributes to the omegasome formation by delivering vesicles from the Golgi apparatus, the mitochondria, or the endocytic route. The omegasome releasing mechanism from the RER to form the phagophore is not well understood. Phagophore Expansion and Maturation This step depends on the activity of two ubiquitin-like proteins and their activation and conjugation system. The first one is ATG5, which covalently binds to ATG12 through the activity of the E1-like enzyme ATG7 and the E2-like enzyme ATG10. Then, the ATG5-ATG12 conjugate binds to ATG16L; this complex (ATG5-ATG12ATG16L) binds to PI3P at the external phagophore membrane and directs the elongation (Kuma et al. 2002). The second ubiquitin-like protein is Atg8 (also known as LC3, from “microtubule associated protein light chain 3”), when this is newly synthesized, LC3 is recognized by the cysteine protease ATG4 to produce LC3 I, the inactive form of LC3. This is conjugated with the membrane phosphatidylethanolamine (PE) to produce LC3 II, the active form that attaches to the inner and outer phagophore membrane through the PE; this conjugation depends on the E2-like enzyme ATG3 and the E3-like enzyme ATG5-ATG12-ATG16L. LC3 attachment is essential to the phagophore expansion (Rawet Slobodkin and Elazar 2013). The maturation refers to the mechanism of phagophore closure to form the autophagosome; this mechanism is not well understood, although the activity of these ubiquitin-like proteins is also necessary for maturation. Lysosomal Fusion As typical membrane trafficking, lysosomal fusion is dependent on Rab and SNARE proteins. Rab7 is responsible for aiding the autophagosome to the lysosome; SNARE proteins sintaxin17, SNAP29 (synaptosomal associated Protein 29), and lysosomal VAMP8 (vesicle-associated membrane protein 8) are in charge of the fusion. Fusion also implicates the protein ATG14 to facilitate the interaction between sintaxin17 (STX17) and SNAP29 (Nakamura and Yoshimori 2017). Degradation Fusion occurs between the autophagosome external membrane and lysosome membrane, so the inner membrane components attached to it, and material internalized in the autophagosome is recognized and degraded by lysosomal hydrolases.

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Phagophore expansion engulfs a nonspecific portion of cytoplasm; however, autophagy also mediates selective degradation by specific material recognition; this is known as selective autophagy, in which receptors such as p62 (also known as sequestosome1/SQSTM1), OPTN (optineurin), NDP52 (nuclear domain 10 protein 52), and NBR1 (neighbor of BRCA1 gene 1) bind to K-63 polyubiquitinated substrates through their ubiquitin association domain (UBA); these receptors also contain an LC3 interacting region (LIR) motif recognized by LC3 to approach the ubiquitinated substrates to the phagophore membrane (Pankiv et al. 2007). Selective autophagy regarding mitochondria is known as mitophagy. Mitochondria accounts the main cellular ROS production, as it may take part during ATP synthesis; thus, these organelles are the most susceptible for oxidative damage. During mitophagy, the Ser/Thr kinase PINK1 (PTEN-induced putative kinase 1) accumulates at the outer membrane of damaged mitochondria, recruiting and activating the ubiquitin ligase (E3) Parkin; thus, Parkin ubiquitinates mitochondrial outer membrane proteins that can be further recognized by selective autophagy receptors mentioned before, therefore promoting specific autophagy of damaged mitochondria (Gegg et al. 2010).

Signals that Trigger Autophagy Hormones and growth factors regulate autophagy through the classical target of rapamycin (mTOR) pathway. Insulin activates class I phosphatidylinositol 3-kinases (Class I PI3K), promoting the synthesis of phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P3) which activates the pathway phosphoinositide-dependent kinase-1 (PDK1), the protein kinase B (PKB, also known as Akt), the tuberous sclerosis 1 and 2 (TSC1/2), and the homolog enriched in brain (Rheb) proteins, allowing mTOR activation which blocks autophagy. The phosphatase and tensin homolog (PTEN) dephosphorylates PtdIns(3,4,5)P3 PDK1/Akt/mTOR pathway, therefore releasing the inhibitory effect of class I PI3Ks on autophagy (Errafiy et al. 2013). The absence of nutrients (glucose, amino acids) activates a variety of pathways like the mitogen-activated protein kinases (extracellular regulated kinase 1/2, ERK1/ 2, c-Jun N-terminal kinases (JNK), and P38), and AMP-activated protein kinase (AMPK) pathways are particularly illustrative and well-studied. Under starving conditions, ERK1/2 is activated, travels into the nucleus, and activates the transcription of autophagy genes. AMPK is a sensor and regulator of cellular energy metabolism that detects AMP/ATP ratio in the cell and is constituted by one α, a β, and a γ subunits. The α-subunit has the kinase activity which is promoted by the phosphorylation of its threonine 172 by the liver kinase B1 (LKB1) and other kinases. The activation of AMPK is terminated by the dephosphorylation of α-subunit by protein phosphatases. The β-subunit has a glycogen-binding site necessary for the localization of the enzyme at glycogen rich regions, near the GSK3β. The γ-subunit has the AMP binding site. Under starving conditions, in a media pour in ATP and rich in AMP, the AMP/ATP ratio increases and AMP binds to the AMPK γ-subunit, provoking an

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allosteric activation of the enzyme, a conformational change of the protein that makes the γ-subunit a better substrate for LKB1 and insensitive to the phosphatases that inactivate AMPK. All these changes result in the promotion of autophagy (Hinchy et al. 2018; Klionsky 2007).

Activation of Autophagy by ROS There is ample evidence that ROS and autophagy pathways interact in a complex way, depending on the context, species, physiological state, age, and other factors. This interaction has important implications for many natural and pathophysiological conditions. In this respect, recent evidence shows that secreted factors from hepatoma cells activate Nox2, increment cellular H2O2, and induce autophagy in macrophages (Shiau et al. 2020) and that the microtubules depolymerizing drug CYT997 induce mitochondrial ROS accumulation and autophagy in gastric cancer (Cao et al. 2020). A sustained excess of ROS is harmful to rats brain cells after injury and autophagy functions as a protective mechanism that reduces oxidative stress (Xu et al. 2018). It is necessary to know the molecular mechanisms of the relationship between ROS and autophagy (Fig. 2).

Protective Effect of Autophagy Against Ischemia Patients that underwent heart surgery often suffer from myocardial ischemia/reperfusion. The application of the anesthetic sevoflurane, shortly before ischemia, protects patients from this malfunction. This recovery depends on increases of ROS species, the activation of AMPK, and an increase of the autophagic flux (Hong et al. 2020).

The Conjugation Complex ATG3, ATG4, ATG7, and ATG8 (LC3) After performing its function, ATG4 delipidates ATG8-PE and ATG8 and PE are recycled (Nakatogawa 2020). The cysteine 572 of ATG7, cysteine 264 of ATG3, and cysteine 81 of ATG4 are all crucial parts of the corresponding redox sensors susceptible to oxidation by ROS. During starvation, cells produce mitochondrial H2O2, a phenomenon that generates an oxidative microenvironment that oxidizes ATG7, ATG3, and ATG4 redox sensors. Mutagenesis of these cysteines, for example, the conserved cysteine 81 residue substituted by alanine or serine, renders these ATG proteins incapable to oxidize by ROS and impairs the formation of autophagosomes in cells (Scherz-Shouval et al. 2007a, b; Scherz-Shouval and Elazar 2007, 2011). Besides oxidation of cysteines, ROS induces specific acetylation of ATG7 and ATG3 and these postransductional modifications proceed in concert with oxidation to promote autophagy (Sedlackova and Korolchuk 2020).

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Fig. 2 Autophagy-ROS relationship. ROS regulates autophagy at several levels. ROS induce signaling pathways, like the lysosomal channel mucolipin I that, through the liberation of lysosomal Ca2+ and the consequent increase of cytosolic Ca2+, induces the activation of the TFEB that, in turn, autophagy genes; ROS can also activate FOXO3, NRF, and JNK transcription factors. At the cytoplasmic level, ROS oxidize ATG4, ATG3, and ATG7 that in conjunction with specific acetylations and phosphorylations activate ATG autophagy proteins. Created with BioRender.com

ROS Induce Transcription Factors of Autophagy Genes The Transcription Factor EB (TFEB) The transcription factor EB (TFEB) belongs to the microphthalmia family of bHLHLZ transcription factors (Mit/TFE), implicated in lysosomal biogenesis and autophagy. Active TFEB is a homodimer that induces the transcription of autophagy genes ATG4, LC3B, and SQSTM1, among others. The acetylation of TFEB induces its monomerization, condition that renders it incapable to bind DNA and to induce autophagy’s protein expression. Mucolipin1, a lysosomal Ca2+ channel, detects oxidative stress and liberates lysosomal Ca2+ that activates the cytosolic Ca2+ dependent phosphatase calcineurin 1. This phosphatase dephosphorylates cytosolic TFEB, thus promoting its translocation into the nucleus to induce the transcription of

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autophagy genes. ROS also oxidizes TFEB’s cysteine 212 to favor nuclear localization (Sedlackova and Korolchuk 2020).

Transcription Factors FoxO1 and FoxO3 Forkhead box class O (FOXO) transcription factors regulate protein autophagy. Upon exposition to growth factors, for example, insulin-like growth factor, mammalian cells activate their EGFR/PI3K/Akt and Ras/Raf/ERK1/2 pathways, promoting the translocation of the kinases Akt and ERK1/2 into the nucleus. These kinases phosphorylate nuclear FoxO1 thus inducing FoxO1 detachment from the DNA and transcriptional inactivity. This inhibitory effect requires a concurrent acetylation that exposes the cryptic Ser252 residue to be phosphorylated thus generating docking sites for 14–3-3 protein binding. The resulting heterodimer exits to the cytoplasm thus ending autophagy (Matsuzaki et al. 2005). Transcription factor forkhead box FO3 (FOXO3) activation can induce expression of several autophagy related proteins like, LC3, BNIP3, ULK1, ATG12, and ATG4 (Zhou et al. 2015). FOXO3 can be activated by an increase of ROS like H2O2, in addition AMPK, which is sensitive to the cellular oxidative state can also phosphorylate and activate FOXO3, finally JNK, whose oxidative stress activation is explained below, is also able to activate this transcription factor, thus inducing autophagy (Sedlackova and Korolchuk 2020). Activation of the Nuclear Factor Erythroid 2-Related Factor 2 (NRF2) NFR2 belongs to the basic leucine zipper (bZIP) family of transcription factors and is the master regulator of antioxidative response. In normal conditions, NRF2 binds to KEAP1. The heterodimer is then integrated into a degradation complex that consists of CUL3, RBX1, and E2, a ubiquitin-conjugating enzyme. NRF2 is constantly polyubiquitinated in the lysine 48 and, consequently, degraded in the proteasome. KEAP1 has a cysteine redox sensor that, once oxidized by inducers or chronic diseases like diabetes or cancer, provokes a conformational change on KEAP1 that impairs NRF2 ubiquitination. In these conditions, NRF2 enters into the nucleus and promotes the transcription of survival genes (Baird and Yamamoto 2020). There is a second path to activate NRF2 by ROS. In response to an increase in H2O2, NRF2 specifically binds the antioxidant-responsive element (ARE motif) located in p62 promoter, thus increasing p62 expression, therefore increasing autophagy (Puissant et al. 2012). p62 binds to KEAP1 inhibiting its interaction with NRF2 and promoting its accumulation, and the autophagy degradation of KEAP1. In this way, p62 contributes to its own expression through a positive feedback (Jain et al. 2010). c-Jun/c-Fos-Beclin 1 c-Jun is a transcription factor activated via phosphorylation by the kinase JNK, c-Jun active form a dimer with the transcription factor c-Fos. The dimer promotes Beclin 1 transcription, thus contributing to autophagy. JNK activation by oxidative stress depends on the inactivation of Trx-1 that binds and inhibits ASK-1, the kinase

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responsible of JNK activation, so in an oxidative environment, Trx-1 oxidized form loses its ability to bind ASK-1; this leads to the JNK activation (Han et al. 2009; Zhou et al. 2015).

Negative Feedback Against ROS Oxidative stress and autophagy are related by negative feedback; this means that an increase in ROS induces autophagy through transcriptional or posttranscriptional regulation of autophagy proteins; thus, autophagy will reduce oxidative damage and ROS levels through removal of protein aggregates and damaged organelles such as mitochondria. Here we summarize the transcriptional mechanisms by which oxidative stress induces autophagy.

Hypoxia Inducible Factor HIF1-BNIP3 Hypoxia-inducible factor (HIF) is crucial for cell survival to hypoxia. In normoxia, HIF1 is hydroxylated and susceptible to ubiquitination and proteasomal degradation. Hypoxia, though, induces the accumulation of the active nonhydroxylated form of HIF1, which in turn induces the expression of the protein BNIP3, a member of the Bcl-2 family. The growing amount of BNIP3 releases Beclin 1 bound to Bcl2 and induces the association of the free Beclin 1 to the PI3KC3, a complex that triggers mitophagy and, consequently, reduces ROS production (Semenza 2011). Autophagy Activating Complexes ATF4/LC3 and CHOP/ATG5 In cancer cells, hypoxia activates the kinase PERK that in turn, stimulates the activating transcription factor 4 (ATF4) and CCAAT-enhancer-binding-protein homologous protein (CHOP), which increases the transcription of LC3 and ATG5, respectively (Avivar-Valderas et al. 2011). In this way, hypoxia-induced autophagy contributes to resistance to cancer cells treatment.

Conclusions ROS are not just harmful substances to defend from, but second messengers necessary for cell homeostasis and development, and are implicated in the pathogeny of illnesses. The ROS and autophagy signaling intermingle in nodal points, characterized by the presence of proteins with cysteine-rich oxidation sensors that, following exposition to the solvent and chemical reaction with ROS, change its conformation and oligomerization, thus determining the extent and type of autophagy. Cysteine oxidation, protein phosphorylation, acetylation, and glutathionylation operate coordinately to trigger and regulate autophagy. ROS produced in the mitochondria and the plasma membrane, together with kinases, phosphatases, acetylases, and deacetylases of both routes, determine the activity of AMPK, mTOR, ATG4, ATG8, ATG3, and ATG7, to promote autophagy and determine its degree and expression. In the absence of stressors, localized and moderated ROS bursts are

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necessary for autophagy progression and the normal turnover of cellular structures. Under endoplasmic reticulum stress and mitochondrial malfunction, ROS increase excessively and keep high for longer times, but autophagy eliminates the excess and the damaged organelles.

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TRP Channels, Oxidative Stress, and Cancer Amritlal Mandal, Mathews Valuparampil Varghese, Joel James, and Sajal Chakraborti

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Present Knowledge of TRP Channels, Cancer, and Oxidative Stress . . . . . . . . . . . . . . . . . . . . . . . . . Altered Expression of TRP Channels and Cancer Pathology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . TRP Channels: Tumor Enhancer or Suppressor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . TRP Channels, Oxidative Stress, and Angiogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Functional Role of TRP Channels in Cancer Under Oxidative Stress . . . . . . . . . . . . . . . . . . . . TRPA1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . TRPM2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Advanced Understanding of Redox-Sensitive TRP Channels in Relation to Specific Cancer Pathology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Reactive Electrophiles, TRP Channels, and Lung Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acute Myeloid Lymphoma (AML) and TRPM2 Channels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Altered Mitochondrial Bioenergetics, TRPM2 Channels and Gastric Cancer . . . . . . . . . . . . . Glioblastoma and TRP Channels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Oxidative Stress, Other TRP Channels, and Their Role in Breast and Prostate Cancer . . . Involvement of TRP Channels in Other Types of Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions and Future Directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract

Transient receptor potential (TRP) channels have evolved as trigger for many physiological and pathophysiological conditions including cancer. These are nonA. Mandal (*) Department of Physiology, University of Arizona, Tucson, AZ, USA e-mail: [email protected] M. V. Varghese · J. James Department of Medicine, Division of Endocrinology, University of Arizona College of Medicine, Tucson, AZ, USA S. Chakraborti Department of Biochemistry and Biophysics, University of Kalyani, Kalyani, West Bengal, India © Springer Nature Singapore Pte Ltd. 2022 S. Chakraborti et al. (eds.), Handbook of Oxidative Stress in Cancer: Mechanistic Aspects, https://doi.org/10.1007/978-981-15-9411-3_80

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selective cation-specific ion channels and are activated by many endogenous and exogenous stimuli including certain chemicals, changes in osmotic condition, and temperature. Until now, studies on TRP channels regarding its role in cancer progression are only handful. Most of the studies in this specific area have been done using either in vitro or in vivo models. Recent development in bioinformatics and big data analysis provided an advanced platform to study tumor biology, target identification, genetic analysis, and anti-cancer drug development. Exploiting this combination of strategies new research is being carried out to link TRP channels, oxidative stress, and cancer pathology. Certain TRP channel family members have been identified to be up- or downregulated and play either tumor enhancer or suppressor functions. TRPM2 and TRPA1 are the two main redox-sensor ion channels that have been relatively well-investigated in relation to cancer and the present chapter highlights the major roles played by these channels in cancer pathophysiology. The chapter briefly discusses certain tumor-specific expression pattern of TRP channels, their biological role under oxidative stress, and possible use of some proof of concepts for developing therapeutic strategies for cancer management. Keywords

TRP channels · Cancer · ROS · Oxidative stress Abbreviations

AKT ATP bFGF Ca2+ [Ca2+]i CADPR CaM DAG ECM ERK FOXO3a GSH GSH-Px H2O2 HIF IC50 ICGC: InsP3 IR-injury JNK KO

Protein kinase B (PKB) also known as AKT Adenosine triphosphate Basic fibroblast growth factor Calcium Intracellular calcium Cyclic ADP ribose Calmodulin Diacylglycerol Extracellular matrix Extracellular signal-regulated kinase Fork head box transcription factor 3a Glutathione Glutathione peroxidase Hydrogen peroxide Hypoxia-inducible factor Half maximal inhibitory concentration International cancer genome consortium Inositol 1,4,5-triphosphate Ischemia-reperfusion injury c-Jun N-terminal kinase Knock out

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MCL-1 mTOR NAC NRF2 OCR PARG PARP PDGF PI3K PLC PYK2 RAS SCLC SOD TGF TRP channels VEGF VEGFR WT

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Myeloid leukemia cell-1 The mammalian target of rapamycin N-acetyl-L-cysteine Nuclear factor erythroid 2-related factor 2 Oxygen consumption rate Poly (ADP-ribose) glycohydrolase Poly (ADP-ribose) polymerase Platelet-derived growth factor Phosphoinositide 3-kinase Phospholipase C Proline-rich tyrosine kinase-2 Rat sarcoma; a family of oncogene Small cell lung carcinoma Superoxide dismutase Transforming growth factor Transient receptor potential channels Vascular endothelial growth factor Vascular endothelial growth factor receptor Wild type

Introduction Transient receptor potential (TRP) channels are a family of non-selective cationspecific channels and responsible for generating electrochemical signals by modulating the transmembrane potential or by changing the intracellular calcium [Ca2+]i concentration as a result of various endogenous and exogenous stimuli (Moran et al. 2011; Nilius and Szallasi 2014). Those channels are widely expressed across the species (mammalian, non-mammalian, and insect) and broadly consist of 28 members in human, and those channels are categorized further into six subfamilies (Nilius and Owsianik 2011): ankyrin (TRPA), canonical (TRPC), melastatin (TRPM), vanniloid (TRPV), polycystin (TRPP), and mucolipin (TRPML). In recent past, significant research interest has been drawn towards TRP channel-related pathologies and novel information are evolving on a regular basis that show the involvement and functional role of different TRP channel family members in human health and disease including cancer. Aberrant expression of different TRP channels has been identified as hallmark for different types of cancer initiation and progression. The clinical roles played by the different TRP channel family members have not been widely investigated in cancer patients. Advancement in bioinformatics and big data analysis have influenced significantly towards understanding tumor biology, target identification, genetic analysis, and anti-cancer drug development. Those combined strategies are now in a verge for providing hope for early diagnosis of cancer by developing companion diagnostics and subsequent treatment plans for the patients suffering from different types of cancer. More recently, the data-driven approaches have been found to be useful to link the functional roles of TRP channel

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proteins in cancer. In a recent study (Park et al. 2016), clinical significance of TRP channel family members has been investigated using the dataset obtained from the International Cancer Genome Consortium (ICGC). This study provided seminal implication for developing conceptual framework for extending biological information of TRP channel proteins from bench to bedside. Oxidative stress plays a pivotal role in several physiological processes and in disease conditions including neurodegenerative disorders, aging, ischemia-reperfusion (IR)-injury, autoimmune diseases, diabetes, and cancer. Reactive oxygen species (ROS) not only cause oxidative stress and cell damage; it also acts as signaling molecules. TRP channels have gained overwhelming attention in past decades due to their calcium permeability that sense and respond to environmental conditions including oxidative stress. TRPA1, TRPV1, TRPC5, TRPM2, and TRPM7 were identified as ROS sensors (Kashio and Tominaga 2017). The calcium signaling mechanisms due to the activation of specific TRP channel proteins under pathological conditions is currently considered to be an important area of research. Selective inhibition of different ROS-sensitive TRP channels are emerging as potential therapeutic targets in ROS-mediated disease progression including cancer (Yamamoto and Shimizu 2016). Figure 1 summarizes the scheme of some ROS-sensitive TRP channel activation mechanisms.

Fig. 1 ROS-induced activation mechanisms of ROS-sensitive TRP channels. The cartoon shows the differences in the activation mechanisms among TRP channels. Four TRP channel family members (TRPM2, TRPC5, TRPV1, and TRPA1) have been found to be ROS-sensitive. TRPA1 has the greatest ROS sensitivity among those channels. Trigger for the TRPM2 activation is the binding of ADP-Ribose to the C-terminal cytosolic NUDT9-homology (NUDT9H) domain. ROS such as Hydrogen peroxide (H2O2) is a known activator of TRPM2 and ROS-induced activation of TRPM2 channel is triggered by the biosynthesis of ADP-ribose in the nucleus involving PARP1. Interestingly, other ROS sensitive TRP channels (TRPC5, TRPV1 and TRPA1) become activated by ROS-induced modifications of the cysteine residues. (Taken from Yamamoto and Shimizu (2016) with permission)

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Present Knowledge of TRP Channels, Cancer, and Oxidative Stress Altered Expression of TRP Channels and Cancer Pathology The involvement of TRP channel proteins in a wide variety of tumor development has been implicated due to their role in cellular calcium influx mechanisms, which is pivotal in cell proliferation, differentiation, and apoptosis. Several chemosensory TRP channels have been found to be associated with cancer development and reviewed in detailed by Büch et al. (2018). Those TRP channels have been described to be either up- or downregulated in the cancer cells and tissues with associated changes in function(s). Importantly, the knowledge gained about TRP channels in association with cancer did not point towards any critical mutations in those proteins affecting channel activity, rather an altered expression of mRNA/protein levels has been indicated in the cancer cells as opposed to the wild type (WT) counterparts (Gkika and Prevarskaya 2009).

TRP Channels: Tumor Enhancer or Suppressor TRP channels have known effects showing either tumor enhancer or suppressor activities in different instances. TRPM channels, an oxidant sensor, have been indicated as tumor suppressor (Hantute-Ghesquier et al. 2018). Downregulation of TRPM1 gene has been reported in melanoma cells (Duncan et al. 1998). Upregulated TRPM8 transcripts have been found in prostate cancer tissues (Tsavaler et al. 2001). In line with these finding another TRP family of proteins (TRPV) has been implicated in cancer pathophysiology. Especially TRPV1 has been a very well-researched member and linked to the regulation of several apoptotic pathways induced by cannabinoids in gynecologic carcinoma (Fonseca et al. 2018; Ligresti et al. 2006). Overexpression of TRPV6 has been reported in prostate cancer (Wissenbach et al. 2001). The level of TRPV6 gene expression has been correlated with severity of tumor grading, indicated a potential role of TRPV6 in tumor growth. TRPA1 channel has been indicated for having a protective mechanism in lung cancer progression (Jaquemar et al. 1999; Schenker and Trueb 1998).

TRP Channels, Oxidative Stress, and Angiogenesis Angiogenesis is a biological process that forms new blood vessels involving endothelial cell proliferation, migration, and spatial reorganization. This process is regulated by a delicate balance of some stimulatory and inhibitory factors. Proangiogenic vascular growth factors such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), transforming growth factor (TGF), and the inhibitory factors like thrombospondin and endostatin are the key components of angiogenesis. Phosphorylation of tyrosine residues in the VEGFR activates phospholipase C (PLC), inositol

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1,4,5-triphosphate (InsP3) and DAG production and Ca2+ entry following calcium store release that modulates key signaling events resulting angiogenesis (Simons et al. 2016). Significant amount of research have provided evidence indicating that VEGF-induced cellular Ca2+ influx occurs via different TRP channels in a cell typespecific manner. Angiogenesis usually results an increase in vascular permeability with concomitant digestion of extracellular matrix (ECM), which could facilitate endothelial cell migration and proliferation. Endothelial cells are known to express multiple TRP channel proteins (TRPC, TRPV, TRPP, TRPA, and TRPM families) and their role in angiogenesis has been recently reviewed by Smani et al. (2018) and Kwan et al. (2007). Cellular Ca2+ mobilization through the TRP channels play critical roles in normal physiological and pathophysiological processes. Increased TRPC1 expression has been observed to cause vascular/endothelial barrier dysfunction (Paria et al. 2006; Paria and Vogel 2004; Tiruppathi et al. 2002), intimal wall thickening, atherosclerosis, and leads to angiogenesis (Pocock et al. 2004; Antoniotti et al. 2002; Cheng et al. 2006; Fantozzi et al. 2003). TRPC3 has been shown to be activated by oxidants, diacylglycerol (DAG), and VEGF and thereby produces oxidative stress-induced endothelial damage (Balzer et al. 1999; Poteser et al. 2006) that eventually leads to essential hypertension (Liu et al. 2006), idiopathic pulmonary arterial hypertension (Yu et al. 2004), and angiogenesis (Pocock et al. 2004; Cheng et al. 2006; Fantozzi et al. 2003). TRPC6 channel activation has also been observed to be associated with endothelial barrier dysfunction (Pocock et al. 2004) and angiogenesis (Pocock et al. 2004; Cheng et al. 2006; Fantozzi et al. 2003). Oxidant-induced TRPM7 channel activation was found to cause angiogenesis (Yao and Garland 2005) and oxidative stress-induced cell death (Aarts et al. 2003). Pathological conditions including carcinogenesis result upregulation of angiogenic factors and predominates over anti-angiogenic factors. Members of the TRP channel family like TRPC3 and TRPC6 (Hamdollah Zadeh et al. 2008; Andrikopoulos et al. 2017), ROS-sensitive TRPM2 (Mittal et al. 2015) and TRPV1 (Garreis et al. 2016) play important role in this scenario. TRPV1 has been described as pro-angiogenic. Intraperitoneal injection of evodiamine, a TRPV1 ligand, has been observed to produce angiogenesis and vascularization in wild-type (WT) mice and significant degree of reduced angiogenesis has been observed upon evodiamine induction in TRV1-KO experimental mice model (Ching et al. 2011).

Functional Role of TRP Channels in Cancer Under Oxidative Stress Oxidants, in normal cells, play significant roles which ultimately lead to ROSinduced apoptotic pathways to limit unrestricted cell proliferation. In cancer cells, the metabolic pathways have been found to be altered in such a way, that the cells can increase their antioxidant defense mechanisms to evade cellular apoptosis and the ultimate result is the uncontrolled cell proliferation. The basic metabolic shift for such a pathway mainly involves the Warburg mechanism, where instead of producing energy (ATP) from glucose by oxidative phosphorylation, the glycolytic pathway is redirected towards anaerobic glycolysis, that readily produces faster and more

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Fig. 2 ROS-induced and TRPA1 activation-dependent calcium signaling mechanisms facilitate the proliferation and survival of cancer cells. Cancer cells are found to be adapted to handle high ROS level. NRF2, a transcription factor, induces expression of TRPA1 channels in cancer cells. ROS, such as H2O2, in mitochondria oxidizes target cysteine residues on the TRPA1 to activate the channel resulting a rise of [Ca2+]i which essentially activates many Ca2+-dependent pro-survival and anti-apoptotic signaling pathways involving the MCL-1 protein. (Taken from Reczek and Chandel (2018) with permission)

ATPs by increasing glucose consumption and shifting the energy metabolism of the cells that favor growth of the tumor. Takahashi et al. (2018) has identified a noncanonical antioxidant-defense mechanism involving TRPA1 channel, which is found to be a redox-sensitive Ca2+ channel and up regulates the anti-apoptotic pathways that facilitate cancer cell survival. Figure 2 is a schematic representation of upregulation of ROS-sensitive TRPA1 channels and their anti-apoptotic activities by promoting resistance to ROS. When ROS levels are relatively very high, senescence-induced cancer cell death happens through anti-tumorigenic signaling mechanisms (Reczek and Chandel 2017). Maintenance of optimal ROS level is very critical for the cancer cells to proliferate. To scavenge excessive ROS accumulation, cancer cells are capable of increasing antioxidant capacity (Gorrini et al. 2013). Upregulation of nuclear factor erythroid 2-related factor 2 (NRF2) is one such mechanism by which cancer cells achieve ROS neutralization. Stabilization of NRF2 in the oxidative stress condition induces several antioxidants including the enzymes involved in glutathione (GSH) synthesis and utilization (Gorrini et al. 2013). Since ROS homeostasis in cancer cells are finely tuned, anti-cancer therapies that alter ROS levels appear to be particularly

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important from a clinical perspective. Calculated inhibition of antioxidant enzymes in cancer cells may have potential to stop cancer cell proliferation. Though theoretically this approach appears interesting, in practical situation therapeutic targeting of the drugs specifically to the cancer cells is virtually a big hurdle, because normal cells are also very much susceptible to high ROS levels.

TRPA1 Takahashi et al. (2018) reported a novel defense mechanism to overcome oxidative stress in the cancer cells involving TRPA1-mediated upregulation of Ca2+-dependent apoptotic resistance to evade ROS-induced cell death. TRPA1 is known as a chemosensor and widely investigated in pain and inflammation pathophysiology (Paulsen et al. 2015). TRPA1 has been reported to become activated by hydrogen peroxide (H2O2) and oxidizes hyper-reactive cysteine residue of the target protein from the cytoplasmic side (Paulsen et al. 2015). TRPA1 activation causes Ca2+ influx into the cells that serves as a second messenger and induces cell proliferation and survival (Paulsen et al. 2015). In the stromal cells of human prostate cancer, triclosan, an antibacterial agent has been reported to cause TRPA1 activationinduced Ca2+ influx causing vascular endothelial growth factor (VEGF) secretion and increased cell growth (Derouiche et al. 2017). Takahashi et al. (2018) shed light in a systematic way regarding ROS-induced TRPA1 activation in cancer cells following Ca2+ entry, affecting the apoptosisdefense mechanisms, leading to cell survival and proliferation. Their research reported for the first time an increased TRPA1 gene and protein expression in breast and lung tumors. The authors have further demonstrated that increased TRPA1 activation-induced Ca2+-influx is a required step for the cancer cells to gain survival ability upon exposure to H2O2. The authors have shown that the inner cells of cancer spheroids are responsible for high level of ROS production and that triggers the TRPA1-induced increased Ca2+-influx to the inner cells to survive. Interestingly TRPA1 inhibition did not have any effect on the inner cancer cells of the spheroids. That indicates a novel mechanism operating in the core of the tumor that involves ROS-induced activation of TRPA1 and subsequent Ca2+entry-induced signaling mechanisms that promote cell survival and proliferation. The mechanistic role of TRPA1 channel in cancer cell survival has been further investigated by the same group by examining the role of TRPA1 in anchorageindependent cellular growth, which is a hallmark of anoikis (anchorage-dependent programmed cell death) resistance. By employing an antioxidant N-acetyl-L-cysteine (NAC), the authors have shown that the extra cellular matrix (ECM) detachmentinduced ROS generation is a required step for the cancer cells to increase the intracellular Ca2+ through TRPA1 channel. The authors have also reported the available therapies (e.g., carboplatin) that produce ROS, make the cancer cells resistant involving TRPA1 activation-induced cellular Ca2+ influx. The authors have further extended their study by employing a mice model either by TRPA1

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gene knock down or by pharmacological inhibition and had shown a suppression of tumor growth in vivo. By employing reverse-phase protein array, the authors identified TRPA1 activation-induced Ca2+ entry, which leads to Ca2+-dependent prosurvival and anti-apoptotic pathways involving myeloid leukemia cell differentiation protein (MCL-1) to mediate oxidative stress tolerance in cancer cells. This specific study also provided evidence that shows TRPA1 is responsible for an increased phosphorylation of RAS-ERK, PI3K/AKT, and mTOR prosurvival signaling molecules that lead to an increased expression of anti-apoptotic protein MCL-1 in a CaM/ PYK2-dependent manner. When the cells were exposed to H2O2, translocation of NRF2 has been observed from cytoplasm to nucleus and then the expression of canonical (antioxidants) and non-canonical (TRPA1) proteins of oxidative defense mechanism were observed. TRPA1 expression in cancer cells act under direct regulation of redox-sensitive regulator protein NRF2. In the presence of high ROS level, specific cysteine residues of TRPA1 protein get phosphorylated, which does not necessarily reduce the antioxidant capacity of the cancer cells but increase the cell survival mechanisms by employing Ca2+ influx. TRPA1 channels have been found to be overexpressed in different cancer cell types compared to the normal cells (Takahashi et al. 2018). ROS-induced cancer cell survival has been discussed in detail by Reczek and Chandel (2018). The mechanisms of ROS-mediated TRPA1 activation-induced calcium signaling mechanisms promoting cancer cell survival has been summarized in Fig. 3.

TRPM2 Increased TRPM2 expression has been reported by Chen et al. (2014) in many cancer cell types. During oxidative stress, an increased ADP-ribose (ADPR) production eventually activates full-length TRPM2 (TRPM2-L) (Hara et al. 2002). Chen et al. (2014) have shown the functional expression of TRPM2-L in neuroblastoma cells and reported TRPM2 channel provides protection for the cancer cells from moderate oxidative stress through increased expression of fork head box transcription factor 3a (FOXO3a) and SOD2. This specific study has shown cells expressing dominant negative short form of TRPM2 (TRPM2-S) to be associated with reduced FOXO3a and SOD2 expression levels. Those cells have also been found having reduced Ca2+ influx in response to oxidative stress, increased ROS level and decreased cell viability. Reduced expression of hypoxiainducible factor (HIF)-1/2α has been reported in the neuroblastoma cells expressing TRPM2-S compared to the tumor cells expressing TRPM2-L. Activation of TRPM2 channels have been reported as an effect of hypoxia (Turlova et al. 2018). Functional mutation of the pore region of TRPM2 channel increased mitochondrial ROS production (Bao et al. 2016). In neuroblastoma cells, a protective effect of TRPM2 under hypoxia condition has been attributed due to an increased activity SOD2 and reduced ROS levels (Chen et al. 2014; Bao et al. 2016).

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Fig. 3 Scheme showing the overexpression of TRPA1 channel in the cancer cells under direct control of NRF2. The figure summarizes the TRPA1 activation-induced increase in [Ca2+]i and the downstream signaling mechanisms involving different kinase pathways that essentially target MCL-1 protein for developing increased tolerance against oxidative stress. (Taken from Takahashi et al. (2018) with permission)

A recent study by Chen et al. (2014) has shown treating neuroblastoma cells (SHSY5Y) having stable expression of TRPM2 channels, with clotrimazole, a TRPM2L inhibitor, or by overexpressing TRPM2-S increased the doxorubicin (a chemotherapy drug) sensitivity, resulting reduced cell survival. Doxorubicin treatment to the cancer cells has been reported to cause the gain of HIF-1/2α function and promote cell death. The data provides evidence that TRPM2 activity is important for tumor growth, cell viability, and survival followed by doxorubicin treatment by interfering with TRPM2-L function. This signaling pathway could be an important route to reduce tumor growth by modulating HIF-1/2α protein, resulting mitochondrial dysfunction and mitophagy.

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Advanced Understanding of Redox-Sensitive TRP Channels in Relation to Specific Cancer Pathology Reactive Electrophiles, TRP Channels, and Lung Cancer Sensory TRP channels (TRPA1, TRPV1 and TRPM8) are known to be expressed in the lung cancer cells. Functional expression of TRPA1 has also been identified in the small cell lung carcinoma (SCLC) cell lines and has been linked to the survival pathway (Schaefer et al. 2013). In Lewis lung carcinoma cells, both TRPA1 and TRPM8 are functionally expressed and regulate various critical cellular functions including energy metabolism, autophagy, and metastasis (Du et al. 2014). TRPV1 expression has been demonstrated in lung adenosarcoma cells (Ramer and Hinz 2008) and in lung fibroblasts including normal lung epithelium and in the sensory nerves (Sadofsky et al. 2012). TRP channels in the airway epithelium potentially act as a sensor for inhaled carcinogens, e.g., reactive electrophiles.

Acute Myeloid Lymphoma (AML) and TRPM2 Channels Elevated ROS level is associated with AML (Farquhar and Bowen 2003). Mitochondria are the main sources of ROS production and elevated ROS has been found to be responsible for cellular and tissue damage involving numerous mechanisms including oxidation, peroxidation, and mutagenesis (Miller and Zhang 2011). Malignant cells have been shown to become proliferative and metastatic upon moderate ROS exposure and excessive increase in ROS causes cell death (Hole et al. 2011). Therapy involving pro-oxidant drugs or by inhibiting the endogenous anti-oxidant mechanisms to raise the cellular ROS to a cytotoxic level has been proposed to optimize anti-cancer drug activity (Hole et al. 2011). TRPM2 channels are highly expressed in AML cells (Chen et al. 2020). Effect of TRPM2 depletion has been studied in U937 (an AML model) cells (Chen et al. 2020). In this specific study, both in vitro and xenograft models have shown an inhibition of cell proliferation upon TRPM2 depletion.

Altered Mitochondrial Bioenergetics, TRPM2 Channels and Gastric Cancer Study by Almasi et al. (2018) has demonstrated the expression of TRPM2 and its critical role in gastric cancer cells bioenergetics and survival. Further research by the same group has provided evidence for the role of TRPM2 ion channels in promoting gastric cancer migration, invasion, and tumor growth through the AKT signaling pathway (Almasi et al. 2019). This study provided evidence for TRPM2 downregulation-dependent impairment of JNK signaling mechanisms causing autophagy, mitochondrial damage, and cell death in gastric cancer cells. Lack of effective treatment regime for controlling the progression of gastric cancer and associated

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death makes TRPM2 particularly a very well sought area (Almasi et al. 2018). These studies have provided evidence showing gene knockdown of TRPM2 in AGS and MKN-45 cells (human gastric cancer cell lines) could make the cells apoptotic with decrease in cell proliferation. Impairment of mitochondrial metabolism has also been observed upon TRPM2 knockdown, as indicated by a reduction of basal mitochondrial oxygen consumption rate (OCR) and ATP production. Further research has identified TRPM2 as a modulator of autophagy that works through JNK-dependent and mTOR independent pathway. More evidence in this specific area has provided new information regarding the ability of TRPM2 downregulation to increase the efficacy of the chemotherapy involving paclitaxel and doxorubicin in gastric cancer cells.

Glioblastoma and TRP Channels A172 cells (human glioblastoma cell line) showed an increased cell death due to H2O2 exposure when TRPM2 channels have been genetically inserted (Ishii et al. 2007). Consistent with this observation, this study reported a H2O2-induced cellular Ca2+ increase in the TRPM2 expressing cells and this phenomenon was absent in the WT counterparts. Altered cell proliferation, migration, and invasion have also been noted in those cells and TRPM2 appears to be an interesting candidate for gene therapy in glioblastoma cells. TRPC6 upregulation has been reported in glioma cells as an effect of hypoxia that results proliferation and invasion (Chigurupati et al. 2010). TRPC6 channels upon activation stabilizes HIF-1/2α in hypoxic glioma cells and facilitates hypoxic glucose metabolism via GLUT1 transporter (Li et al. 2015).

Oxidative Stress, Other TRP Channels, and Their Role in Breast and Prostate Cancer TRPM8 has been indicated as a potential target in many cancer types including breast and prostate cancer cell lines (Nazıroğlu et al. 2018). TRPM8 function has been linked to the survival pathways in prostate cancer cells, whereas TRPM8 knockdown renders the cells with oxidative stress and apoptosis (Bidaux et al. 2016). High metabolic activity and energy demand of the tumor cells coupled with insufficient blood supply to the tumor tissue is often associated with reduced oxygen supply and make the stroma of the tumor hypoxic (Muz et al. 2015). In the breast cancer cells, hypoxia-induced expression of TRPC1 channel has been observed which results hypoxia-induced signaling pathways (Azimi et al. 2017). ROS production is associated with increased hypoxia and has been shown to have both proand antitumor effects (Yang et al. 2013). ROS-mediated activation of TRPC6, TRPV1, TRPM2/4/7, and TRPA1 are reported and their function has been linked to the regulatory steps associated with developing metastatic cascade (Nilius and Szallasi 2014).

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Involvement of TRP Channels in Other Types of Cancer The role of TRPM7 in cancer cell migration has recently been identified (Meng et al. 2013). Reduced expression of HIF-1α has been found upon TRPC1 knockdown in follicular thyroid cancer cell line ML-1 (Asghar et al. 2015). Activation of TRPC6 has been reported in both pancreatic- and hepatic stellate cells under hypoxia condition (Iyer et al. 2015). The role of TRP channels in the cell signaling mechanisms for metastatic events have been reviewed in detail by Fels et al. (2018).

Conclusions and Future Directions Till now though several potential TRP channel antagonists have been developed, yet they seriously lack the target-specific inhibitory activity. Several chemical compounds including herbal extracts of St John’s wort (inhibition of TRPM2/TRPV1) (Uslusoy et al. 2017), cinnamon, ginger, garlic, clove oil, pepper (activation of TRPA1/TRPM8) (Bharate and Bharate 2012) demonstrated promising results as ROS-activated TRP channel modulators. Antioxidant enzymes glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), and elemental cofactors (selenium and copper) may also play promising effects due to their ROS-scavenging activity could indirectly inhibit ROS sensitive TRP channel activation (Öz et al. 2017). TRPA1 has been observed to be responsible for developing resistance against ROS-producing chemotherapies. Accordingly, targeted inhibition of TRPA1 was able to suppress the xenograft tumor growth and increases chemotherapeutic sensitivity to the tumor cells. TRPA1 did not affect the redox status of the tumor cells, but responsible for upregulating calcium-dependent anti-apoptotic pathways. The transcription factor NRF2, which has been implicated as an important player against oxidant-induced damage may directly controls TRPA1 expression and eventually acts as ROS-neutralizing mechanism in the cancer cells. Those findings could be useful to develop novel cancer therapies by targeting TRPA1 ion channel. So far, many TRPA1 modulator drugs have been developed as novel therapeutics for pain and respiratory diseases such as asthma and chronic obstructive pulmonary disease. However, due to poor pharmacokinetics, their uses as a prospective anticancer therapy remain limited. There has been plenty of information available regarding clinical trials of TRP channel targeting drugs in treating different pathophysiological conditions associated with pain including neuropathic pain, acute migraine, and osteoarthritis. Limited information is available regarding successful clinical trials of TRP channel modulators as therapeutics of different types of cancer. TRPM8 agonist, D-3263 (Dendreon Corp. Seal Beach, CA) has entered the phase 1 clinical trial (ClinicalTrials.gov Identifier: NCT00839631) for treating cancer and solid tumors. A recent review on TRP channels (Mandal 2020) by the corresponding author has extensively listed the clinical trials that has been executed using TRP channel targeting drugs in a wide variety of disease conditions. Future research is needed in this area to improve the efficacy of TRPA1 inhibitors as possible anticancer therapy. Combinational therapy by inhibiting TRPA1 along with a reduction

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of ROS production by carboplatin showed promising results to treat the cancer cells with the drugs alone. Since TRPA1 has been shown to be activated by ROS in an NRF2-dependent manner, there is prospective scope of employing TRPA1 inhibitors in hypoxic tumor cells that possesses constitutively active pool of NRF2. The candidate tumor for such a therapeutic approach is the lung adenocarcinoma cells. TRPM2 is also emerging as a potential target for many cancer therapies and further investigation is needed on this channel.

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Wnt Signaling in Cancer

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Minakshi Prasad, Mayukh Ghosh, Rajesh Kumar, Lukumoni Buragohain, Ankur Kumari, and Gaya Prasad

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Wnt Signaling Pathways and Machinery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Wnt Ligands and Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Wnt Canonical Pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Noncanonical Wnt Pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Aberration in Wnt Signaling in Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Colorectal Cancer (CRC) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Leukemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Liver Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Lung Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Multiple Myeloma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ovarian Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Breast Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Other Cancers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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M. Prasad (*) Department of Animal Biotechnology, Lala Lajpat Rai University of Veterinary and Animal Sciences, Hisar, India M. Ghosh Department of Veterinary Physiology and Biochemistry, RGSC, Banaras Hindu University, Mirzapur, India R. Kumar Department of Veterinary Physiology and Biochemistry, College of Veterinary Science, Lala Lajpat Rai University of Veterinary and Animal Sciences, Hisar, India L. Buragohain Department of Animal Biotechnology, College of Veterinary Science, Assam Agricultural University, Guwahati, Assam, India A. Kumari Department of Zoology CBLU, Bhiwani, Haryana, India G. Prasad International Institute of Veterinary Education and Research, Rohtak, India © Springer Nature Singapore Pte Ltd. 2022 S. Chakraborti et al. (eds.), Handbook of Oxidative Stress in Cancer: Mechanistic Aspects, https://doi.org/10.1007/978-981-15-9411-3_81

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Targeting of Wnt Signaling in Cancer Therapeutics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1301 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1304 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1305

Abstract

Stringent regulation of the Wnt pathways components and modulators is indispensable for the restoration of tissue homeostasis and development. The Wnt/β-catenin-dependent canonical pathway is the most comprehensively introspected Wnt signaling pathway which relies upon Frizzled receptor activation and cytosolic β-catenin stabilization followed by their nuclear transport and subsequent transcriptional activation of multiple target genes to influence multiple cellular processes. The β-catenin-independent noncanonical pathway is the alternative Wnt signaling pathway which operates through either planar cell polarity [PCP] or calcium-signaling mechanism to modulate gene regulation. These pathways are highly branched and interconnected to modulate downstream mechanisms individually or in a concerted fashion. Aberrant Wnt signaling induced by genetic or epigenetic impetus leads to carcinogenesis and metastasis; evidently, several cancer types have depicted alterations in multiple Wnt pathway components to render them as potential targets for anticancer chemotherapy. Several small molecules and biologicals antagonizing the Wnt signaling are currently undergoing through different stages of clinical trial for customizing efficacious as well as safe anticancer therapeutics. However, the healthy cells are also susceptible to blockage of Wnt signaling pathways as potential side effects that pose inherent challenge to the strategy which needs to be dealt with utmost precaution. Cancer-specific Wnt markers and combination of multiple therapeutic strategies can overcome the limitation which lies at the focus of the ongoing oncotherapeutic introspections. Keywords

Wnt signaling · β-catenin · Canonical pathway · Noncanonical pathway · Cancer therapeutics

Introduction The Wnt signaling pathway regulates a variety of crucial cellular processes which include but not limited to cell differentiation, proliferation, cell migration, cell polarity, neural patterning, stem cell pluripotency, and cell fate (Komiya and Habas 2008; Pohl et al. 2017). The existence of Wnt signaling was first elucidated in murine mammary carcinogenesis when activation of the murine int-1 gene (Wnt1) was encountered in murine mammary tumor and subsequently its role was identified in embryogenesis. In 1982, Roel Nusse and Harold Varmus discovered a new protooncogene in mouse named as int1 (integration 1) while conducting an experiment in which they infected mice with mouse mammary tumor virus (MMTV) to detect the

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mutated genes in mammary tumors (Nusse 2005; Nusse et al. 1984). The gene was found to be orthologous to the wingless gene of Drosophila melanogaster which underlines key developmental patterning processes in the common fruit fly (Rijsewijk et al. 1987). The name Wnt was derived from the fusion of the Drosophila wingless gene and the vertebrate gene integration 1 (int-1) (Wodarz and Nusse 1998). The Wnt signaling pathway regulates numerous development processes and tissue homeostasis in vertebrates. Although it comprises of several overlapping and interconnected signal transduction pathways but broadly sub-branched as β-catenin-dependent canonical pathway and β-catenin-independent noncanonical pathway. The noncanonical pathway can be operated through either planar cell polarity [PCP] or calcium-mediated pathway. In homeostatic condition, β-cateninmediated gene regulation is stringently controlled by β-catenin destruction complex comprised of adenomatous polyposis coli (APC), glycogen synthase kinase 3α/β (GSK-3α/β), casein kinase 1 (CK1), and AXIN1. The destruction complex downregulates β-catenin by primary phosphorylation through CK1 and subsequent multiple phosphorylation by GSK3 followed by degradation of the phosphorylated β-catenin through E3 ubiquitin ligase (β-TrCP). This averts the nuclear translocation of β-catenin preventing T-cell factor (TCF)4/β-catenin-mediated activation of Wnt target genes. Genetic aberrations in β-catenin or destruction complex components may strip-off the tight regulation leading to the release of several Wnt ligands. The WNT ligands family contains 19 yet elucidated secreted glycoprotein homologs which interacts with the N-terminal extracellular cysteine-rich domain of a Frizzled (FZ) family receptor and low-density lipoprotein receptor-related proteins 5,6 (LRP5/6) co-receptors by either paracrine or autocrine mode to activate the β-catenin-dependent canonical pathway (Rao and Kühl 2010). The FZ receptors (FZDs) are distinct family of seven-pass G-protein coupled transmembrane receptors (GPCRs) (Schulte and Bryja 2007); 10 of such different FZDs have been identified in humans. The initiation of β-catenin-independent noncanonical pathway takes place through the interaction of Wnt ligands with the FZDs along with the ROR 1/2 or RYK co-receptors leading to activation of PCP or Wnt/Ca+2 pathways. Activation of the FZDs recruits phosphoprotein Dishevelled (Dsh/DVL in mammals) which is present in the cytoplasm. The Dsh proteins are present in most of the organisms and share three highly conserved domains: a DIX domain at aminoterminal, a PDZ domain at center, and a DEP domain at carboxy-terminal. The different domains of Dsh protein helps to branch off the Wnt signal into multiple pathways and each pathway activates different downstream molecules that elicit different cellular responses (Habas and Dawid 2005). The clinical importance of Wnt signaling was elucidated after identification of association between mutations in multiple key elements of this pathway with several diseases such as colorectal cancer, leukemia, glioblastoma and diverse other solid and liquid cancers, type II diabetes, and degenerative disorders (Logan and Nusse 2004; Komiya and Habas 2008). Aberrant and tissue-specific Wnt signaling has been depicted in diverse types of cancers including but not restricted to gastrointestinal cancers, melanoma, leukemia, pancreatic ductal adenocarcinoma, mammary

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carcinoma, lung cancer, prostate cancer, ovarian cancer, cervical cancer, and cancer of central nervous system (Spaan et al. 2018). Familial adenomatous polyposis (FAP), an autosomal, dominantly inherited cancer syndrome characterized by formation of polyps in the colon and rectum, is often underlined by truncated adenomatosis polyposis coli (APC) protein (Kinzler et al. 1991; Nishisho et al. 1991). As APC is the key element of β-catenin destruction complex, thus dysfunctional APC results into β-catenin stabilization and abnormal trans-activation of the Wnt pathway which in turn increases cell proliferation leading to adenomatous lesions. Mutations in β-catenin and APC were found not only to be associated with colon cancers but also etiologically relevant to several other types of cancers (Giles et al. 2003). Similarly, mutations in Axin, another key component of the β-catenin destruction complex, has also depicted to be associated with hepatocellular carcinomas (Satoh et al. 2000). Evidently, aberrations, either genetic or epigenetic, in key molecules of Wnt signaling often predispose the subject with diverse cancer types. The Wnt signaling pathway is extremely complex as compared to other pathways and extensively regulated by feedback mechanisms. Despite Wnt signals are being extremely pleiotropic and regulate numerous pathophysiological and developmental processes, still it offers several potential checkpoints for targeted cancer therapy. Several Wnt signaling inhibitors are already undergoing through different phases of clinical trial as potential therapeutics against diverse cancer types. Further exhaustive analysis of Wnt signaling pathway may solve several lasting oncology conundrums and facilitate strategic devising of efficient, targeted, and personalized cancer therapeutics.

Wnt Signaling Pathways and Machinery Wnt Ligands and Receptors The Wnt ligands are lipid-modified glycoproteins and the length of these proteins ranges from 350–400 amino acids (Cadigan and Nusse R 1997). The lipid modification that occurs in Wnts is palmitoleoylation of conserved serine residue (Hannoush 2015) which is necessary for their interaction with carrier protein Wntless (WLS) leading to transportation of the Wnts to the plasma membrane followed by their secretion (Yu et al. 2014). It also facilitates the binding of Wnts to their cognate Frizzled receptor (Janda et al. 2012; Hosseini et al. 2018). The glycosylation of Wnt ligands takes place in the endoplasmic reticulum (ER) along with acylation by the O-acetyltransferase Porcupine (PRCN) (Kurayoshi et al. 2007). Subsequently, WLS-mediated transportation of the lipid-conjugated Wnt ligands to the plasma membrane takes place en-routed through the Golgi complex facilitated by p24 proteins. Extracellular secretion of the Wnt ligands is mediated by membrane vesicles like exosome as per the glycosylation and acylation signal. In human and mouse, 19 Wnts have been identified yet, while 7 and 5 Wnts have been documented in Drosophila and C. Elegans, respectively. The Wnts are highly conserved from invertebrate to vertebrates (Nusse 2005).

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The major receptors involved in Wnt signaling are family of Frizzled (FZD) receptors that interacts with Wnt ligands and co-receptors (Martin-Orozco et al. 2019). Lipoprotein receptor-related protein (LRP)-5/6 takes part as the most common co-receptor predominantly in the β-catenin-dependent canonical Wnt pathway (He et al. 2004). However, LPR-5/6 co-receptor in association with FZDs also carries out the Wnt/STOP signaling which is a β-catenin-independent pathway for protein stabilization during mitosis (Acebron et al. 2014). The other co-receptors such as receptor tyrosine kinase (RTK) and receptor tyrosine kinase-like orphan receptors (ROR1/ ROR2) also participate but in the noncanonical Wnt signaling pathways (Komiya and Habas 2008).

Wnt Canonical Pathway The β-catenin protein is the central molecule of the WNT canonical pathway which remains attached at the cytoplasmic face of the plasma membrane through E-cadherin and along with actin through α-catenin as a crucial modulator of cytoskeletal remodeling. At the homeostatic environment, β-catenin level is strictly regulated by the β-catenin destruction complex comprised of APC and AXIN1 as scaffolding proteins, CK1 and GSK 3β as Ser/Thr kinases, Yes-associated protein (YAP) an transcriptional co-activator with a PDZ-binding domain (TAZ) as transcriptional modulators. During inactive state, β-catenin is continuously synthesized followed by sequential phosphorylation through CK1 and GSK 3β, and degradation by E3 ubiquitin ligase (β-TrCP). Thus β-catenin stabilization in the cytoplasm and their nuclear transport is deranged. As a consequence, TLE/Groucho repressors entangle with transcription factor/lymphoid enhancer-binding factor (TCF/LEF) in the nucleus and bind with HDACs (histone deacetylases HDAC1 and HDAC2) to arrest the transcriptional activation of the Wnt genes and Wnt-canonical pathway remains inactive. Any genetic or epigenetic impetus which downplays the activity of the β-catenin destruction complex and promotes cytosolic β-catenin stabilization turns on the pathway as nuclear transport of β-catenin disengage the TLE/Groucho repressors to form active complex with TCF/LEF along with several co-activators (CBP/p300, BRG1, BCL9, and PYGO) leading to transactivation of Wnt genes. Wnt stimulation or binding of Wnt ligands to the Frizzled receptors and the LRP5/6 co-receptors triggers a series of events that destabilizes the β-catenin destruction complex required for degradation of β-catenin. Once the Wnt ligands bind to its receptor complex, LRP5/6 receptors are phosphorylated by CK1α and GSK3, as a result Dishevelled (DVL/Dsh) proteins are recruited to the plasma membrane where they polymerize and get activated. The activated DVL inhibits the activity of the GSK3 enzyme and triggers a complex series of events that prevents degradation of β-catenin (Fagotto et al. 1998). Again, phosphorylated LRP5/6 interacts with Axin and this interaction destabilizes the β-catenin destruction complex. Consequently, β-catenin is stabilized and gets accumulated in cytoplasm. Then the stabilized β-catenin translocate into the nucleus and initiate the transcription of specific target genes to modulate multiple cellular responses (Fig. 1) (Komiya and Habas 2008; Pohl et al. 2017; Zhan et al. 2017; Martin-Orozco et al. 2019).

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Fig. 1 Overview of canonical WNT pathway. (a) In absence of Wnt ligands, synthesized β-catenin is phosphorylated by GSK3β of the destruction complex which is comprised with protein Axin, APC, GSK3β, and CK1α. The phosphorylated β-catenin is further ubiquitinated by β-TrCP leading to its degradation in proteasome. Due to the absence of nuclear β-catenin, TCF/LEF and TLE/Groucho complex (a repressive complex) employs HDACs which in turn arrest the transcriptional activation of the Wnt genes and thereby canonical pathway remains inactive. (b) Canonical pathway is activated when Wnt ligands bind to the receptors and co-receptors, FZD and LRP5/6, respectively. Binding of Wnt ligands to its receptor complex activates a cascade of events that destabilizes the β-catenin destruction complex. As a consequence, the β-catenin gets accumulated in the cytoplasm and finally gets translocate into the nucleus. By displacing TLE/Groucho complex, β-catenin forms complex with TCF/LEF and recruits many co-activators such as Pygo, CBP/p300, BCL9, and BRG1 leading to active transcription of Wnt pathway-related genes resulting different cellular responses

Noncanonical Wnt Pathways Noncanonical Wnt pathways are independent of β-catenin and the attachment of Wnt ligands with the FZD receptors initiate the signaling cascade. The Wnt/PCP pathway which regulates cytoskeletal organization for cellular shape, polarity,

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Fig. 2 Overview of noncanonical Wnt pathway: Noncanonical Wnt pathway is β-catenin independent and it is activated upon binding of Wnt ligands to receptor complex (ROR-FZD receptor). The activated receptor complex employs and activates DVL protein which gets associated with DAAM1 and activates RHO. Activated RHO stimulates ROCK which regulates cytoskeletal rearrangement. The DVL also stimulates RAC which in turn activates JNK leading to cytoskeletal rearrangement and transcriptional activation of genes via activating transcription factor 2 (ATF2). Again, stimulated receptor complex interacts with G-protein along with DVL. The activated G-protein stimulates PLC leading to production of IP3 and DAG. IP3 causes the release of Ca2+ from ER whereas DAG stimulates PKC. There is also activation of CaMKII and calcineurin which stimulates various transcription factors (NFAT) leading to transcription of genes and several cellular responses

migration, etc. and the Wnt/calcium pathway which regulates calcium inside the cell are the best understood noncanonical Wnt pathways (Fig. 2) (Zhan et al. 2017). The noncanonical Wnt/PCP pathway begins with the binding of Wnt ligands to the FZ receptors while co-receptors such as pseudo tyrosine kinase 7 (PTK7), receptor-like tyrosine kinase (RYK), receptor tyrosine kinase-like orphan receptor (ROR1/2), etc. facilitate downstream propagation of the activation signal through adaptor proteins like DVL. The activated DVL entangles with

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Dishevelled-associated activator of morphogenesis 1 (DAAM1) to trans-activate a small GTPase Ras homolog family member A (RhoA) via Rho guanine nucleotide exchange factor. Rho attaches with the α-helical domain of the Rhoassociated kinase (ROCK) to remove the autoinhibition leading to its activation which regulates cellular actin and cytoskeletal rearrangement. DVL also interacts with Ras-related C3 toxin substrate (Rac) and activates Rac which in turn activates c-Jun N-terminal kinase (JNK) leading to actin polymerization and transcriptional modulation (Komiya and Habas 2008; Pohl et al. 2017; Zhan et al. 2017; Gruszka et al. 2019). The Wnt/Ca2+ pathway is another major β-catenin-independent noncanonical pathway which regulates cytosolic Ca2+ concentration either by inducing release of Ca2+ from the endoplasmic reticulum (ER) or by influx from outside the cell. The Wnt/Ca2+signaling get activated upon binding of Wnt ligands to FZD receptors. The stimulated receptor interacts with DVL and G proteins leading to activation of phospholipase C-beta (PLC-beta), which in turn hydrolyzes the phosphatidylinositol 4,5-bisphosphate (PIP2), an indispensable glycerophospholipid of the plasma membrane into two cleavage components 1,2-diacylglycerol (DAG) and inositol 1,4,5-triphosphate (IP3). Subsequently, IP3 induces Ca2+ release from the endoplasmic reticulum whereas DAG and IP3 trigger downstream signaling of protein kinase C (PKC), the Ca2+/calmodulindependent protein kinase type II (CaMKII), calpain-1, and calcineurin in a concerted effort. The CaMKII activates the transcription factors, nuclear factor kappa B (NFκB), and nuclear factor of activated T-cells (NFAT) which modulates cell adhesion, migration, and tissue separation. The ligand Wnt5a stimulates activation of CaMKIIdependent Wnt/Ca2+ signaling. The TGFβ-activated kinase (TAK1) and Nemo-like kinase (NLK) activated by CamKII can antagonize β-catenin/TCF signaling and prevents the binding of β-catenin/TCF4 complex to DNA. Genes activated via the Wnt/Ca2+signaling regulates cell fate and cell migration (Komiya and Habas 2008; Pohl et al. 2017; Zhan et al. 2017; Gruszka et al. 2019).

Aberration in Wnt Signaling in Cancer Colorectal Cancer (CRC) Among all types of cancer, the etiological association of Wnt signaling in colorectal cancer is probably the most comprehensively elucidated as hyperactivation of Wnt pathway has been observed in majority of colon cancer (Polakis 2012; Zhan et al. 2017; Schatoff et al. 2017). Aberrations in several components (at gene or protein level) of Wnt signaling pathway has been identified leading to colorectal cancer. The Cancer Genome Atlas (TCGA) consortium stated that up to 92% CRCs have one or other form of aberrations in Wnt signaling factors (Schatoff et al. 2017). APC is a tumor suppressor gene and key component of β-catenin destruction complex; dysfunction of APC has been highlighted as one of the major causes of colorectal cancer. Different forms of mutations in APC leads to different types of canonical Wnt pathway activity which have been associated with tumor locations in large

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intestine (Christie et al. 2013; Buchert et al. 2010). Besides APC, alterations in AXIN, CTNNB1, and TCF7L2 genes have also been reported to be responsible for CRCs (Cuilliere-Dartigues et al. 2006; Polakis 2012; Schatoff et al. 2017). Defunct AXIN downplays the activity of β-catenin destruction complex leading to activation of the Wnt pathway causing sporadic CRCs (Novellasdemunt et al. 2015). The key modulators of Wnt signaling ZNRF3/RNF43 which serves as E3 ubiquitin ligases facilitate lysosomal degradation of the Frizzled receptors and suppress the downstream pathway activation. In contrary, R-spondin ligand family members (RSPO) bind with the LGR5 co-receptor to inhibit the ZNRF3/RNF43 activity leading to accretion of the FZ receptors and persistence of the Wnt signaling. Mutational inactivation of RNF43 leads to suppression of negative feedback regulation of the R-spondin/Lgr5/RNF43 axis underlining several CRC subtypes including endometrial and pancreatic ductal adenocarcinoma. Similarly, mutations in TCF and Wnt/STOP signaling have also been reported in CRC. Besides these, several other under-characterized factors and poorly understood mechanisms also exist which are directly or indirectly connected to Wnt pathway leading to CRC and other gastrointestinal cancers (Novellasdemunt et al. 2015; Schatoff et al. 2017).

Leukemia Wnt/β-catenin signaling regulates and maintains the population of hematopoietic stem cells (HSCs) (Luis et al. 2012), but the activity of Wnt was found to be increased in leukemia (Lento et al. 2013). The exact mechanism of Wnt/β-catenin signaling in HSCs and leukemia is still debatable as several conflicting reports are available. However, the conflicting data are suggestive of inhibition of Wnt/β-catenin axis compromises proper hematopoiesis, but mild or slight activation of the pathway leads to development of myeloid and increases clonogenicity of HSCs, intermediary high-level intensity of Wnt/β-catenin activity results in development of T-cells, whereas hyperactivation of Wnt/β-catenin cause impaired hematopoiesis and development of leukemia (Chiarini et al. 2020). Deregulation of Wnt/β-catenin pathway leads to conversion of healthy HSCs in leukemic stem cells (LSCs) (Lento et al. 2013). Aberration in Wnt/β-catenin signaling is connected to different forms of hematological malignancies which include, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphoid leukemia, and chronic myeloid leukemia (Chiarini et al. 2020). Multiple factors such as differential expression of Wnt proteins, mutations in β-catenin, APC, and Axin, imbalance of the TCF/LEF complex, and epigenetic alterations may lead to overstimulation of the Wnt/β-catenin axis in ALL (Chiarini et al. 2020). Higher level of β-catenin was encountered in about 80% of T-cell acute lymphoblastic leukemia (T-ALL) pediatric patients leading to activation of β-catenin-dependent genes such as C-MYC, TCF1, LEF, and BIRC5 (Arensman et al. 2014; Fernandes et al. 2017). In LSCs of mouse and human T-ALL, the Wnt/β-catenin pathway is activated and contributes to drug resistance (Chiarini et al. 2020). A member of the LEF/TCF complex, “LEF1” interacts with the nuclear

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β-catenin to activate c-MYC and Cyclin D1 and 25% of adult T-ALL subjects exhibit a significant increase of LEF1 (Yu et al. 2012; Guo et al. 2015). Wnt3a-mediated activation of Wnt/β-catenin pathway and accumulation of β-catenin have been depicted to be associated with the development of B-cell acute lymphoblastic leukemia (B-ALL) (Nygren et al. 2007). Further, alteration in Wnt/β-catenin pathway components through genetic alterations, convergence with other noncanonical Wnt pathways or β-catenin localization may increase the susceptibility towards development of B-ALL (Nygren et al. 2009). Occurrence of B-ALL has also been observed in mice that exhibit hyper-expression of a constitutively active LEF1 mutant (Petropoulos et al. 2008), and LEF1 overexpression has been reported in nearly 25% of adult B-ALL patients (Kühnl et al. 2011). Acute myeloid leukemia (AML) is a clonal hematopoietic disease characterized by abnormal accumulation of nonfunctional myeloblasts (or blasts) which is due to transformation of a hematopoietic stem or progenitor cell into malignant cells (Saultz and Garzon 2016). Abnormal activation of Wnt pathway and its downstream events has been exhibited in acute myeloid leukemia (Mikesch et al. 2007). Mutant Fms-like tyrosine kinase 3 (FLT3, receptor tyrosine kinases), promyelocytic leukemia-retinoic acid receptor-α, and acute myeloid leukemia1-ETO are involved in downstream events of Wnt pathway in acute myeloid leukemia (Mikesch et al. 2007). Translocation products such as AML1-ETO, MLL-AF9, and PML-RARα affect canonical Wnt signaling in AML patients and derived cell lines (Zhan et al. 2017). Clinical samples of AML patients showed several mutations associated with high levels of β-catenin leading to a poor overall survival rate of AML patients (Staal et al. 2016). Aberration in Wnt signaling due to the epigenetic inactivation of Wnt pathway inhibitors has been demonstrated in several AML leukemic patients. The activation of the Wnt pathway in AML cells has found to be associated with methylation status of Wnt antagonists, such as DKK1 and SFRP-1, 3, 4, and correlated with poor prognosis (Staal et al. 2016). Moreover, aberrant expressions of certain other Wnt ligands (i.e., WNT2B, WNT6, WNT10A, and WNT10B) have also been observed in AML (Majeti et al. 2009; Staal et al. 2016). Chronic lymphocytic leukemia (CLL) is commonly encountered in adults characterized by accumulation of dysfunctional but mature CD5+and CD19+ B cells (Lu et al. 2004; Staal et al. 2016). Canonical Wnt pathway and its genes have been found to be active in CLL cells (Lu et al. 2004). Selective and high-level expression of WNT3 was observed in in CLL cells but not in other types of B cell malignancies (Staal et al. 2016). Again, overexpression of LEF1 was encountered in CLL cells (Gandhirajan et al. 2010). Besides these, other molecules such as Wnt5b, Wnt6, Wnt10a, Wnt14, and Wnt16, and Wnt receptor Fzd3 has found to be highly expressed in CLL cells (Lu et al. 2004). Indeed, methylation status of the WNT-antagonists also contributes to enhanced WNT-activity in CLL cells (Staal et al. 2016). Chronic myeloid leukemia or chronic myelogenous leukemia (CML) is a myeloproliferative neoplastic disorder designated by the presence of BCR-ABL fusion gene and Philadelphia (Ph+) chromosome, which is due to reciprocal translocation of

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chromosomes 9 and 22 (Heidel et al. 2012; Staal et al. 2016; Grassi et al. 2019). The fusion gene, BCR-ABL (results from translocation), encodes a fusion oncogenic protein, “BCR-ABL.” This fusion protein exhibits tyrosine kinase activity which activates various transduction pathways involved in cell differentiation and growth leading to transformation of HSCs into neoplastic clones (Staal et al. 2016; Grassi et al. 2019). The expression of BCR-ABL protein was found to have correlation with high levels of active β-catenin in CML (Jamieson et al. 2004). Further, the presence of β-catenin has been depicted to be vital for LSC survival and maintenance in CML (Heidel et al. 2012). In a CML murine model, BCR-ABL was found to stimulate β-catenin via the phosphoinositide 3-kinase (PI3K/AKT) pathway leading to leukemic progression (Arrigoni et al. 2018). Although a little less explored but the connection of noncanonical Wnt signaling in the pathogenesis of CML as well as ALL has also been reported (Gregory et al. 2010).

Liver Cancer Ample evidences of hyperactivated canonical Wnt pathway have been documented in hepatocellular carcinoma (HCC), cholangiocarcinoma (CCA), or hepatoblastoma (Takigawa and Brown 2008; Perugorria et al. 2019). In hepatobiliary tumors, there are aberrations in key genes of Wnt-β-catenin signaling that promotes growth, dedifferentiation, and dissemination of tumor cells (Perugorria et al. 2019). Different types and frequencies of mutations have been reported in key genes of Wnt pathways (Wnt ligands, Wnt receptors, DVL proteins, β-catenin destruction complex, β-catenin and co-activators, and regulators of the Wnt-β-catenin pathway) in hepatobiliary tumors (Perugorria et al. 2019). Epigenetic alterations such as DNA methylation, histone modification, and long noncoding RNAs are involved in abnormal activation of Wnt-β-catenin signaling in liver carcinomas (Carotenuto et al. 2017; Perugorria et al. 2019). Aberrant Wnt-β-catenin signaling is a striking feature of hepatoblastoma (most common pediatric liver cancer) and highest rate of mutation is observed in CTNNB1 gene (Forbes et al. 2017). Frequent mutations (deletions or missense) have been encountered at the GSK3β phosphorylation motif of β-catenin (Wei et al. 2000). Besides CTNNB1, mutations (loss-of-function) in APC, AXIN1, AXIN2, and LGR6 were detected in hepatoblastoma (Forbes et al. 2017). Like hepatoblastoma, activating mutations in CTNNB1 dominated by missense substitutions has been detected in HCC (Perugorria et al. 2019). Again, mutation in AXIN1 and AXIN2 were also observed in HCC (Takigawa and Brown 2008; Polakis 2012; Perugorria et al. 2019). Overexpression of FZD7 has also been observed in HCC tumors and cell lines (Merle et al. 2004). In CCA, activation of Wnt-β-catenin is mainly regulated by epigenetic mechanism (Perugorria et al. 2019). However, mutations in the primary components of Wnt pathway have also been detected but to lesser degree than HCC. Mutations in AXIN1, APC, and CTNBB1 have been predominantly observed in CCA but with lesser frequencies in DVL2, DVL3, FZD10, WNT10B, and WNT8B (Perugorria et al. 2019).

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Lung Cancer Lung cancer (LC) is one of the common and most lethal types of cancer encountered globally. Broadly LC can be categorized into two types, small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC) (Rapp et al. 2017). Key components of Wnt pathway play a significant role in lung carcinogenesis (Rapp et al. 2017); however, genetic mutations are uncommon in Wnt signaling components in LC (Stewart 2014; Rapp et al. 2017). In LC, crucial elements of Wnt pathways such as Wnt1, Wnt2, Wnt3, Wnt5a, Wnt7b, Wnt11, FZD-8, TCF-4, and Dishevelled proteins have been reported to be overexpressed, whereas APC, AXIN, Wnt inhibitor Dickkopf-3 (DKK3) and secreted frizzle-related protein-1 (SFRP1) have been reported to be downregulated in LC (Tennis et al. 2007; Stewart 2014; Rapp et al. 2017). However, missense mutation in CTNNB1 (substitutions of Ser/Thr) which activates Wnt pathway has also been reported leading to development or progression of LC (Sunaga et al. 2001).

Multiple Myeloma Multiple myeloma (MM) is a cancer of antibody-secreting plasma cells (PCs) characterized by clonal proliferation and infiltration of PCs in the bone marrow (Raab et al. 2009; Spaan et al. 2018). The Wnt signaling is abnormally activated in a considerable fraction of MMs; however, intrinsic mutations in the vital components of the Wnt pathway is rarely encountered (Kim et al. 2011; Spaan et al. 2018). Activation of Wnt signaling results from genetic and epigenetic lesions of the Wnt modulators such as deletion of the tumor suppressor CYLD, overexpression of the co-transcriptional activator BCL9 and the R-spondin receptor LGR4, as well as promoter methylation of the Wnt antagonists DKK1, DKK3, WIF1, sFRP1, sFRP2, sFRP4 and sFRP5 (van Andel et al. 2019).

Ovarian Cancer Ovarian cancer is considered as one of the deadliest gynecologic cancer. The Wnt/β-catenin pathway promotes epithelial-to-mesenchymal transition (EMT) towards enhanced metastasis and found to be in active state during ovarian carcinogenesis (Arend et al. 2013). EMT is the process by which transition of the polarized epithelial cells take place to migratory mesenchymal cells having invasive properties. Even noncanonical Wnt signaling associated EMT has also been depicted in other cancer types like colon cancer. The most common genetic aberrations observed in the Wnt/β-catenin pathway involved in epithelial ovarian cancer (EOC) is the mutation in CTNNB1 gene (Wu et al. 2001). Mutations have also been encountered in β-catenin destruction complex which includes AXIN, GSK3β, and APC in ovarian cancer (Nguyen et al. 2019). Besides these, Wnt signaling is also abnormally activated in ovarian cancer due to differential expression of Wnt ligands and receptors, abnormal activation of intracellular mediators, aberrant promotion of the

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β-catenin/TCF transcriptional activity, and inhibition of the association of β-catenin/ E-cadherin on the cell membrane (Nguyen et al. 2019). Further, several instance of various noncoding RNAs (miRNA and long noncoding RNA) modulating the Wnt pathway to actuate the ovarian cancer also exists (Nguyen et al. 2019).

Breast Cancer More than 50% of the breast cancer patient exhibit enhanced Wnt signaling activity (Lin et al. 2000). The involvement of canonical Wnt signaling in development of triple negative breast cancer has been studied extensively (Zhan et al. 2017) but a strong link between Wnt signaling and breast cancer is yet to be established vividly (van Schie and van Amerongen 2020). However, reports are available depicting the activation of downstream components of the Wnt signaling in a significant proportion of breast tumors (Brown 2001). Although the association between Wnt signaling and breast cancer has been explored nearly 40 years back as Wnt1 was detected as a proto-oncogene capable of causing mammary tumor in mice but ample knowledge gap still exists due to lack of basic understanding regarding the role of WNT pathway in breast development under homeostatic condition, thus, the precise molecular deviations altering the WNT pathway in different subtypes of breast cancer has remained obscure (van Schie and van Amerongen 2020). Several Wnt ligands, receptors and antagonists such as Wnt7b, Wnt10b along with Wnt receptors LRP6 and FZD7 have been identified to be spiked in breast cancer (Yin et al. 2018). Although Wnt1 has been observed to be upregulated in mice model but in human breast cancer, it is rarely overexpressed (Meyers et al. 1990). In addition, DNA methylation causing downregulation of SFRP1, SFRP2, SFRP5, DKK, and WIF-1 has been observed in breast cancer (Yin et al. 2018).

Other Cancers The Wnt signaling is also hyperactivated in several other types of cancers. Mutations in key Wnt signaling components such as APC, Axin, β-catenin, Wnt ligands and receptors, critical downstream mediators have been observed in pancreatic ductal adenocarcinoma, prostate cancer, cervical cancer, and tumors of the central nervous system (Spaan et al. 2018). In addition, several epigenetic mechanisms particularly DNA methylation and histone modification of the key Wnt signaling regulatory genes also contribute significantly to the aberrant activation of Wnt pathway leading to different types of cancer such as human medulloblastoma, HCC, colon, breast, and pancreatic cancer, AML, melanoma, etc.

Targeting of Wnt Signaling in Cancer Therapeutics Although Wnt signaling molecules provide lucrative checkpoints for anticancer therapy but targeting of such signaling components to precisely destroy the malignant cells is challenging, since these key molecules of Wnt pathways are also

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expressed in healthy cells. Therefore, while drug designing against any Wnt molecules, utmost precautions should be taken to curtail the undesired side effects in normal tissues. In this regard, therapeutic Wnt inhibitors can be manipulated or combined with other therapies so that their efficiency and specificity get enhanced. Although extensive research is going on to develop potent WNT/β-catenin pathway inhibitors as antineoplastic therapeutics against diverse hematologic and solid malignancies but none have made headway in the clinical use yet (Martin-Orozco et al. 2019). However, there are certain FDA-approved generic category drugs (nonsteroidal anti-inflammatory drugs, cyclooxygenases inhibitor, vitamins, etc.) that exhibits anticancer effects by nonspecific and indirect targeting of Wnt signaling. Nonsteroidal anti-inflammatory drugs (NSAIDs) such as aspirin, sulindac, and indomethacin and specific cyclooxygenases 2 (COX2) inhibitors, like celecoxib can inhibit Wnt/β-catenin and thwart polyp formation in FAP mouse models including patients (Novellasdemunt et al. 2015; Duchartre et al. 2016). Further, several introspections have revealed that vitamins, particularly vitamin A and D, can be effective against cancer by suppressing Wnt/β-catenin signaling at various levels (Duchartre et al. 2016; Novellasdemunt et al. 2015). Several specific therapeutics as inhibitors of the Wnt pathways have been developed to treat different types of cancer; quite a few of them are in preclinical stage or entered different phase of clinical trials (Table 1). In this regard, a wide variety of therapeutic agents such as small-molecule compounds, monoclonal antibodies (mAbs), peptides, recombinant proteins, and other inhibitors that modulate the Wnt signaling have been reported. The current therapeutic strategies predominantly target the Wnt ligand-receptor complex, the β-catenin destruction complex and nuclear/transcription factor complexes (Novellasdemunt et al. 2015; Schatoff et al. 2017). For cancer treatment, the Wnt ligands or receptors can be suppressed by using agents that are Porcupine (PORCN) inhibitors, Wnt ligand antagonists, and FZD antagonists/monoclonal antibodies (Jung and Park 2020). Porcupine is a membranebound O-acyltransferase (MBOAT) which adds the palmitoyl group to Wnt proteins necessary for processing Wnt ligand secretion (Novellasdemunt et al. 2015). Two novel classes of small molecules which include inhibitors of Wnt response (IWR) and inhibitors of Wnt production (IWP) have been identified for cancer therapy (Chen et al. 2009). The IWP inhibits the activity of PORCN, while IWR abrogates Axin protein turnover destruction and thereby suppresses Wnt/β-catenin activity. Another effective porcupine inhibitor is LGK974 that exhibited tumor regression in mice without any adverse effect in healthy tissue (Liu et al. 2013). Secreted frizzledrelated proteins (SFRPs) and SFRP-derived peptides function as antagonists that capture Wnt ligands and render them unavailable for activation of Wnt signaling. Both SFRPs and SFRP peptides have depicted tumor suppressive activity in preclinical models (Jung and Park 2020). Monoclonal antibody, such as Vantictumab (OMP-18R5) and OSTA101, directly targets the FZD receptors and inhibits Wnt signaling in cancer (Jung and Park 2020). Ipafricept (OMP-54F28) is a recombinant Fc-fusion protein which can bind native FZD8 receptor’s ligands and thereby inhibit

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Table 1 Antagonists of Wnt signaling molecules as potential cancer therapeutics Name WNT974 (LGK974)

CGX1321

OMP-18R5

OMP-54F28

DKN-01

Cancer type Squamous cell cancer; CRC; pancreatic cancer; melanoma; breast cancer Gastrointestinal cancer; HCC; bile duct carcinoma; pancreatic and gastric adenocarcinoma Solid tumors; breast cancer; pancreatic cancer Liver cancer; pancreatic cancer; ovarian cancer; solid tumors Gallbladder cancer; cholangiocarcinoma; other cancers

BHQ880

Myeloma

OTSA101 OMP-131R10 SM08502

Synovial sarcoma Solid tumors Solid tumors

PRI-724

Solid tumors; pancreatic cancer AML; CML

PRI-724 alone or in combination with oxaliplatin, leucovorin calcium, or fluorouracil Foxy-5 CWP232291

LY2090314

Ad5-SGEREIC/Dkk3

CRC; breast cancer; prostate cancer Acute myeloid leukemia; chronic myelomonocytic leukemia Pancreatic cancer; leukemia; other metastatic cancer Prostate cancer

Target/function PORCN inhibitor

Trial stage Phase I

PORCN inhibitor

Phase I

Mab against FZD receptor

Phase I

FZD8 decoy receptor

Phase I

Mab against DKK1 (Dickkopf-related protein 1) Mab against DKK1 FZD10 antagonist R-spondin 3 β-catenincontrolled gene expression inhibitor CBP/β-catenin antagonist CBP/β-catenin antagonist

Phase I/II

Phase I/II Phase I Phase I Phase I

Phase I Phase II

Wnt5a mimetic

Phase I

β-catenin degradation

Phase I/II

GSK3β inhibitor

Phase I/II

LRP5, LRP6

Phase II

References Jung, and Park (2020); Gruszka et al. (2019); Perugorria et al. (2019); van Andel et al. (2019); Zhan et al. (2017)

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Wnt signaling (Duchartre et al. 2016; Jung and Park 2020). Clinical trials of OMP-54F28 conjugated with many drugs are underway to treat different types of cancer (Jung and Park 2020). Rottlerin, salinomycin, and monensin induces degradation of LRP6 by inducing phosphorylation of LRP6, resulting in suppression of Wnt signaling (Jung and Park 2020). Various agents (NSAID or non-NSAIDs) were identified that blocks FZD and DVL interaction which in turn blocks the signal transduction cascade of Wnt pathway. Because binding of DVL to the carboxyl terminal end of the FZD receptors is essential for activation of Wnt signaling (Martin-Orozco et al. 2019). Many tankyrase inhibitors (XAV939, JW-55, RK-287107, and G007-LK) have been demonstrated to produce Wnt-antagonizing effects in cancer cells by Axin-mediated downregulation of β-catenin stabilization (Jung and Park 2020). Library screening of natural compounds have yielded identification of several potent small molecule antagonists which can disrupt the β-catenin/TCF complex formation and provides opportunity for anticancer therapy (Lepourcelet et al. 2004). For instance, small molecule ICG-001 has been depicted to precisely bind with the cAMP-response element-binding protein-binding protein (CBP) which acts as a co-activator of β-catenin/TCF-mediated transcriptional activation of the target genes, thus antagonizing the β-catenin/TCF dependent Wnt pathway (Emami et al. 2004). Another small molecule methyl 3-[(4-methylphenyl) sulfonyl] aminobenzoate (MSAB) interacts with β-catenin to promote proteasomal degradation through ubiquitination, thus yielded potent antitumor effect in Wnt-dependent cancer (Hwang et al. 2016; Lyou et al. 2017).

Conclusion The Wnt signaling, β-catenin dependent or independent pathways deliver significant input either individually or in a concerted manner in diverse homeostatic and developmental processes. Deranged Wnt signaling evidently predisposes the individuals towards varied oncogenic developments. Drug resistance and immune evasion have been proficiently promoted by various genetic or epigenetic alterations of the Wnt signaling components leading to different cancer types as well as deciding the prognostic outcome. Thus, Wnt pathway molecules promisingly lure for diligent introspection as key chemotherapeutic targets, but simultaneously pose inherent challenge of potential side effects on the healthy cells. In this conundrum, cancer-specific Wnt pathway modulators can extend safer targets besides exploring the potential of a combinatorial approach of several promising strategies to deliver any safe as well as effective remedial. Oncological research has currently panned towards extensive exploration of the key Wnt signaling targets with several small molecules and biologicals are already undergoing through different preclinical and clinical phase of trial to ensure the likely elucidation of suitable Wnt-associated oncotherapeutics imminently.

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Emerging Concepts Banudevi Sivanantham

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Significance of ROS in Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cellular ROS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Oxidant Sources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Oxidant Types . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Commanding Role of ROS in Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ROS Regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ROS as a Signaling Molecule . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Impact of Two-Faced ROS on Cancer Signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . One Face: ROS Enhances Protumorigenic Signaling in Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . Second Face: ROS Regulates Anti-tumorigenic Signaling in Cancer . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cross-References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract

Cancer has become a prolonged health burden and a common group of diseases leading to cancer-related deaths in the global scenario. The major players of this dreadful disease are multifactorial such as DNA mutation, genomic instability, age, lifestyle, diet, family history, obesity, etc. These risk factors impart their effects through the superfluous production of reactive oxygen species (ROS) which ends up in oxidative cellular damage by a process called oxidative stress. The oxidants or ROS, the by-products of normal aerobic metabolism, are in close association with cancer. Oxidative stress is likely to affect most of the essential cellular activities such as cell signaling pathways, various metabolic routes, pathways responsible for gene regulation, cell proliferation, and apoptotic B. Sivanantham (*) Department of Bioengineering, School of Chemical & Biotechnology, SASTRA Deemed-to-be University, Thanjavur, Tamilnadu, India e-mail: [email protected] © Springer Nature Singapore Pte Ltd. 2022 S. Chakraborti et al. (eds.), Handbook of Oxidative Stress in Cancer: Mechanistic Aspects, https://doi.org/10.1007/978-981-15-9411-3_82

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pathways. Moreover, ROS does not play a one-sided role in cancer; rather, it behaves as a double-edged sword in the regulation of various cancer signaling pathways. This chapter explores the emerging and interesting facts of two-faced ROS in the regulation of cancer cell signaling. Keywords

Antioxidants · Cancer · DNA damage · Oxidants · Oxidative stress · ROS

Introduction Oxidative stress is caused due to the disturbance in redox signaling, i.e., disparity between reactive oxygen species (ROS) or oxidant generation and their eradication by defense mechanisms or antioxidants. Such discrepancy leads to impairment of essential cellular biomolecules including proteins, lipids, and DNA, with a higher impact on the whole entity and with an increased probability of oncogenesis (Sosa et al. 2013). ROS displays its greater impact on the modulation of signaling pathways with relevance to several amendments both intracellular and extracellular environmental insults in the mammalian system (Durackova 2010). These disturbances in redox balance contribute to cancer development (Sabharwal and Schumacker 2014).

Significance of ROS in Cancer Cellular ROS Who are the major players responsible for the regulation of cancer cell signaling? Among the vast array of molecular players, ROS grabs attention as an essential player in the regulation of cancer cell signaling. Oxidants or free radicals are capable of reacting with the other molecules and behave as oxidizing agents or electron acceptors. Important oxidants are ROS, reactive nitrogen species (RNS), highly reactive sulfur, and chloride species. Among the oxidants, ROS seem to be the major oxidants and can cause oxidative damage to biomolecules that regulate the entire cellular metabolism. Besides, ROS influences the generation of other reactive species including RNS (Gonenc et al. 2006).

Oxidant Sources Where do these ROS players get ready to stimulate or inhibit cancer cell signaling? There are two major sources of ROS: exogenous and endogenous sources. Cellular endogenous sources include mitochondria, membrane-bound organelles such as peroxisome, phagosomes, endosomes, or exosomes, extracellular fluid-plasma, and

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inflammatory cells (neutrophils, eosinophils, and macrophages); other endogenous sources are NADPH oxidase (NOX), cytochrome P450 (Cyt P450), xanthine oxidase, adrenaline and dopamine, flavin, and quinones. Exogenous ROS sources include environmental adulterants, tobacco smoke, alcohol, iron salts, radiation, and chemical factors including anticancer drugs (Gorrini et al. 2013b).

Oxidant Types ROS are generally categorized into two major groups: (i) Free radical oxidants comprise one or more unpaired electrons in their outermost orbitals such as hydroxyl radicals (OH•) and superoxide radicals (O2
). (ii) Non-radical oxidants comprise chemically reactive molecules and in turn can be transformed into a radical form of ROS due to any metabolic insults. Such oxidants do not possess unpaired electrons, for example, hydrogen peroxide (H2O2). In both cases, ROS can be generated either by enzymatic reactions like NOX, lipoxygenase, and cyclooxygenase (COX), Cyt P450 enzymes, or non-enzymatic reactions, i.e., oxidants generated during electron transport chain which confirms the heterogeneous nature of ROS. Oxidants like O2
, H2O2, and OH
are the highly reactive free radicals, and other oxidants are also generated from H2O2 and metals like iron and copper through Fenton chemical reaction (Barrera 2012).

Commanding Role of ROS in Cancer How does ROS enroll its commanding role in cancer as an essential biomarker? The doom of ROS either as a signaling molecule or a toxin is completely based on the concentration of oxidants generated, type of oxidants, and availability of antioxidants. Several studies have evidenced the existence of a very close association of ROS with different stages of cancer development, i.e., initiation, promotion, and progression, via induction of DNA damage/mutations, genetic instability, and finally cell proliferation (Reuter et al. 2010). A persistent pro-oxidative state, a vital characteristic of cancer cells over normal cells, can cause ingrained oxidative stress (Toyokuni et al. 1995). Increased oxidative stress during cancer conditions is caused by two main reasons; one is due to excessive ROS formation, and the other is decreased levels of antioxidant systems required to defend against effects caused by ROS. Cancer cell survival is highly dependent on its ability to regulate the endogenous emergence of antioxidants to withstand a steady-state level of ROS that in turn causes cancer cell death. Hence, cancer cells created a smart adaptive system that efficiently involves the antioxidant function rearrangement and upregulation of prosurvival molecules to protect them from ingrained oxidative stress (Farber and Rubin 1991; Huang et al. 2000). Also, ROS promotes cancer initiation through the

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Fig. 1 (continued)

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stimulation of protumorigenic signaling molecules. This is achieved by changing the activity of target proteins by oxidizing the cysteine residue through oxidation of the disulfide or sulfenic amide bonds residing in the target protein molecules, particularly the tyrosine phosphatase family members (Tonks 2006).

ROS Regulation ROS homeostasis is a very crucial process required for cell survival and proper signaling events during normal cellular functioning. The contribution of oxidants is protean in the initiation and further progression of cancer cells. Generally, ROS at lower concentrations acts as a mitogen promoting cell proliferation and cell survival, thereby resulting in the development of cancer (Sena and Chandel 2012). At intermediate concentration, it causes a fleeting or enduring cell cycle arrest and differentiation, whereas at higher concentration, it triggers tumor cell death via extrinsic or intrinsic apoptosis, autophagy, and necroptosis (del Rio 1992). ROS homeostasis in cancer cells is regulated mainly in two ways; one is through an antioxidant defense system, and the other is by tumor suppressor gene regulation.

ROS Regulation by the Antioxidant Defense System The antioxidant defense system encloses both enzymatic and non-enzymatic antioxidants, which are quite actively entailed in the maintenance of intracellular ROS levels in cancer cells. Mitochondrial and NOX-induced production of O2
causes cellular damage through inactivation of iron (Fe2+)-sulfur-containing proteins. So formed O2
is effectively scavenged by superoxide dismutase (SOD) that is rapidly transformed into H2O2 and then converted to water (H2O) by different antioxidants such as catalase (CAT), glutathione peroxidases (GPXs), and peroxiredoxins (PRXs). PRXs seem to be good H2O2 scavengers whose cysteine residues undergo H2O2-mediated oxidation to detoxify H2O2. The oxidized form of PRXs is subsequently reduced by thioredoxin (TRX) resulting in the formation of an oxidized form of TRX that is restored to reduce TRX by thioredoxin reductase and NADPH (Berndt et al. 2007). In addition to PRXs, GPX also imparts its effects to eliminate H2O2 by oxidizing reduced glutathione (GSH) to glutathione disulfide (GSSG) which in turn retrieves back to GSH by glutathione reductase (GR) which utilizes NADPH. CAT implements its activity to scavenge H2O2 in the absence of cofactors in peroxisomes (Fig. 1). Thus, the intracellular ROS is robustly regulated by both enzymatic and non-enzymatic antioxidant defense systems (Berndt et al. 2007).

ä Fig. 1 ROS regulation by antioxidant defense system and tumor suppressor genes. ROS homeostasis is regulated in two major mechanisms: antioxidant defense system and tumor suppressor gene. In cancer, the steady-state balance of ROS is maintained by different antioxidant enzymes like SOD, CAT, GPX, GR, TRX, PRX, and non-enzymatic GSH and tumor suppressor genes including p53, FOXO, and mSIRT3

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Regulation of ROS by Tumor Suppressor Genes Several scientific reports have emphazised the implication of tumor suppressor protein p53 in the regulation of antioxidants and metabolic genes, and its involvement in induction of cell-cycle arrest and cell death. Increased p53 expression highly targets TP53-inducible glycolysis and apoptosis regulator (TIGAR), a positive regulator of phosphofructokinase-1 to modulate antioxidant system in cancer cells by decreasing glycolytic flux and glucose shunting via pentose phosphate pathway (PPP) to generate NADPH, thereby decreasing ROS accumulation (Cheung et al. 2012). p53 also downregulates ROS accumulation by regulation of expression of antioxidant genes like SOD2, GPX1, and CAT through the induction of TIGAR (Cheung et al. 2016). Other tumor suppressor genes such as forkhead box transcription factors (FOXO) repress tumorigenesis by influencing the antioxidant expression (Dansen and Burgering 2008). ROS accumulation is decreased significantly by breast cancer gene1 (BRCA1) by enhancing the stabilization and activation of nuclear factor erythroid 2-related factors (NRF2) (Gorrini et al. 2013a), and mitochondrial sirtuin 3 (SIRT3) also regulates the ROS shunting activity by deacetylating SOD2 (Perillo et al. 2020) (Fig. 1). ROS homeostasis is further regulated by triggering off transcription factors such as NRF2. Increased ROS reversibly oxidizes the cysteine moiety of Kelch-like ECH-associated protein 1 (KEAP1) averting it from proteasomal deterioration and causes KEAP1 dissociation from NRF2; therefore, NRF2 entries the nucleus and dimerizes with musculoaponeurotic fibrosarcoma (MAF) proteins and finally binds to antioxidant response element (ARE) to transcribe antioxidants such as CAT, GPXs, and PRXs (Gorrini et al. 2013a; Reczek and Chandel 2017). Apart from elevated ROS levels, antioxidant defense by NRF2 is also contributed by increased ERK/MAPK and PI3K/Akt signaling, KEAP1 mutation, and loss of NRF2 (Reczek and Chandel 2017) (Fig. 1). Besides these two ways of ROS homeostasis, NADPH effectively imparts its role in both ROS production and detoxification in the mammalian system. There are multiple sources of NADPH generation in the cytosol and mitochondria that include glucose-6-phosphate through PPP, cytosolic isocitrate and mitochondrial malate of tricarboxylic acid (TCA) cycle intermediates, the activity of malic and isocitrate dehydrogenase enzymes (IDH1, IDH2), and serine one-carbon metabolism via the folate cycle (Ye et al. 2014).

ROS as a Signaling Molecule H2O2 seems to be a central mediator in inducing or suppressing the molecular players involved in cancer cell signaling among other ROS. ROS-induced oxidative damage causes redox signaling, i.e., altering the cysteine residues of target proteins responsible for various cellular signaling pathways. In particular, H2O2 is the one that mediates the oxidation of cysteine moiety that resides in the target protein. The cysteine residue exists in thiolate form (Cys-S
) at physiological pH and is more

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prone to oxidation than the protonated form of cysteine thiol moiety (Cys-SH). During the redox signaling, hydrogen peroxide reversibly oxidizes the cysteine thiol group into sulfenic form (Cys-SOH), which in turn forms reversible disulfide (Cys-S-S-Cys) or sulfenic amide (Cys-S-N) bond formation to change protein conformation and its functional activity, thereby safeguarding the target proteins from irreversible oxidation. If the H2O2 levels are increased further, Cys-SOH can be reduced to Cys-SH form by additional H2O2 to form sulfinic (SO2
) and sulfonic acids (SO3
) by thioredoxin (TRX), glutaredoxin (GRX), and disulfide reductase to restore the functional activity of the target proteins (Berasain 2009) (Fig. 2). In addition to its role in altering protein conformation, H2O2 also mediates prolyl hydroxylase domain protein 2 (PHD2) oxidation resulting in hypoxia-inducible factor 1 (HIF-1) stabilization during hypoxia condition in cancer cells. Stabilized HIF-1 in turn promotes tumor angiogenesis by increasing the proangiogenic gene expression like vascular endothelial growth factor (VEGF) (Lee et al. 2016). H2O2 acts as a signaling molecule in cancer cell survival pathways through two major mechanisms: the redox-relay mechanism and the floodgate model mechanism. These two mechanisms are dependent upon the capability of ROS scavenging enzymes to recognize and change over the signal to H2O2 (Winterbourn 2013). In the redox-relay mechanism, glutathione peroxidase (GPX) or peroxiredoxin (PXR) accepts the H2O2 oxidation signal and transfers those signals to the target protein. The redox-relay mechanism has been well established in the mammalian system, e.g., PRX2-signal transducer and activator of transcription protein (STAT3) redox relay (Sobotta et al. 2015), and even glyceraldehyde 3-phosphate dehydrogenase

Fig. 2 H2O2 as a signaling molecule in redox signaling. H2O2 oxidizes the cysteine moiety of target protein during oxidative stress resulting in the regulation of enzyme activity. The sulfenic form is readily reversible, and at the higher oxidation state, it results in irreversible modification

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(GAPDH) sensitivity to H2O2 has been increased by a proton-relay mechanism (D’Autreaux and Toledano 2007). In the floodgate model mechanism, hyperoxidation or phosphorylation inactivates the scavenging enzymes causing an increase in H2O2 level leading to oxidation of target protein (Woo et al. 2010). For instance, cytochrome P450-induced mitochondrial H2O2 was hyperoxidized and inactivated by PRX3 during corticosterone generation in vivo (Kil et al. 2012). Increased production of NOX4-derived H2O2 that is stimulated by a growth factor receptor activates Src kinase and suppresses the activity of phosphatases, thereby inactivating PRXl through posttranslational modification, permitting H2O2-mediated signal transduction in the cancer cell (Woo et al. 2010). In other way, H2O2 mediates the regulation of cellular processes like cell cycle regulation, transcription, and cell metabolism through this similar mechanism by activating mitochondrial Lyn and Syk kinase and Src family protein pathway (Patterson et al. 2015). Besides the H2O2 implication as a signaling molecule, ROS also induces oxidative stress through lipid peroxidation by changing the lipid bilayer, particularly polyunsaturated fatty acid. The lipid peroxidation process results in the formation of lipoperoxyl radical that reacts with lipids to generate lipid-based radical and lipid hydroperoxides that are minute and short-lived. It further forms peroxyl and alkoxyl radicals and is finally degraded to secondary products, and its local effects are very minimal. However, breakdown products of lipid peroxidation like malondialdehyde and 4-hydroxynonenal (HNE) serve as oxidative stress second messengers that possess a strong potentiality to move from the origin because of their prolonged half-life. In particular, HEN is a highly reactive electrophile that reacts with nonenzymatic antioxidant glutathione (GSH), DNA, and other proteins resulting in oxidative damage (Ye et al. 2014). These lipid peroxides may in turn influence the mitochondrial permeability transition pore (mPTP) integrity and affect electron transport chain complexes I and II and in turn increase electron leakage within the mitochondrial intermembrane region (Petrosillo et al. 2003).

Impact of Two-Faced ROS on Cancer Signaling One Face: ROS Enhances Protumorigenic Signaling in Cancer How does ROS make its impression as a protumorigenic factor in cancer progression through regulation of cancer cell signaling? The understanding of the ROS role in the development of cancer is obscure until the 1990s. A vast array of studies thereafter evolved to confirm its strong relationship between ROS and cancer initiation, progression, and metastasis. H2O2 oxidizes the cysteine thiol moiety of protein phosphatase 2A (PP2A), phosphatase and tensin homolog (PTEN), and protein tyrosine phosphatase 1B (PTP1B) which results in the disruption of their functional activities and, in turn, hyperactivates PI3K/Akt/mTOR cell survival pathway (Brewer et al. 2015) in various cancers such as glioblastoma, melanoma, prostate, breast, and endometrial cancers (Wu et al. 2003). ROS-induced cancer cell

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proliferation signaling has been activated in most of the cancers through the inactivation of mitogen-activated protein kinase phosphatases (MAPKP), thereby enhancing the growth factor receptor expression and finally promoting MAPK/ extracellular-regulated kinase (ERK) signaling (Son et al. 2011). Oncogenic activation of Akt and Ras mutation results in increased generation of NOX4-derived ROS that further promotes cancer cell survival and proliferation (Los et al. 2009). In breast cancer, ROS-mediated upregulation of Akt stimulates the neoplastic growth, and also k-Ras-derived mitochondrial ROS was found to upregulate the epidermal growth factor (EGF) proliferative signaling through the activation of nuclear factor-kappa B cell (NFκ-B) via polycystin-1 (PDK1) in lung and pancreatic cancers (Weinberg et al. 2010; Liou et al. 2016) (Fig. 3). Downregulation of the tumor suppressor gene, adenomatous polyposis coli (APC), has been evidenced to mediate abnormal wingless int-1 protein (Wnt) signaling and intestinal cell proliferation via the upregulation of Ras-related C3 botulinum toxin substrate 1 (RAC1) and TIGAR (Cheung et al. 2016). Remarkably, RAC1 instigates increased production of NOX-derived ROS, whereas TIGAR decreases ROS levels by upregulating NADPH. Thus, TIGAR-induced NADPH retrieves the antioxidant activity through the shunting of ROS, which in turn allows RAC1 to promote upregulation of ROS to perform pro-proliferative signaling function (Cheung et al. 2016). Besides the ROS-induced cancer initiation, it also promotes angiogenesis and metastasis through activation of hypoxia-inducible factor (HIF) and 50 -adenosine monophosphate-activated protein kinase (AMPK) signaling (Jeon et al. 2012). ROS enrolls its role in regulating the growth factor signaling to power cancer cell proliferation and seems to be an integral factor in response to the metabolic stress that has been experienced when highly proliferative tumors outgrow their blood supply, i.e., hypoxia (Gatenby and Gillies 2004). This hypoxic and glucose-deprived condition successively increases ROS generation in cancer cells. Increased ROS is likely to activate the proangiogenic signaling pathway through induction of HIF-1α stabilization and also promotes the genes responsible for cancer cell survival, metastasis, and glycolysis (Jeon et al. 2012). ROS reduces NADPH formed via the HMP shunt pathway because of the decreased supply of glucose (Ye et al. 2014). However, ROS in other way regulates NADPH production by increasing one-carbon metabolism enzymes and serine hydroxymethyltransferase 2 (SHMT2) and enhances serine degradation that is the HIF-dependent process and maintains the antioxidant ability to redeem the hypoxia-induced upregulation of mitochondrial ROS (mROS) (Ye et al. 2014). In cancer cells, ROS-induced activation of AMPK is likely to increase the production of NADPH and prevent synthetic metabolic processes that need NADPH to avert ROS-induced cancer cell death and to enhance antioxidant ability consumption (Jeon et al. 2012). ROS also impacts its effects on the regulation of genes involved in cancer invasion and metastasis through matrix metalloproteinase (MMP). MMPs are the required factors that boost the epithelial-mesenchymal transition (EMT), a predictive event in metastatic cancer. E-cadherin downregulation causes increased MMP-2, 3, 9, and 28 expressions in EMT resulting in enhanced motility and invasion of tumor cells and decreased epithelial marker and increases stemness property in

Fig. 3 Two-faced ROS in the regulation of cancer cell signaling. ROS acts as a protumorigenic molecule (purple side) implementing its regulatory activity on cell survival and cell proliferation signaling pathways. At a very high rate of ROS, it acts as an anti-tumorigenic molecule (black side) regulating the tumor cell death signaling. AP-1, Activator protein 1; APE-1, Apurinic or apyrimidinic endonuclease 1; SP-1, Specificity protein 1

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cancer cells (Illman et al. 2006). Studies have shown that the integrin-Racb1 pathway responsible for the cytoskeletal rearrangement process is involved in increasing the intracellular ROS that leads to cancer cell migration and invasion through stimulation of MMP-3-mediated EMT (Radisky et al. 2005). The ROS quenching effect was studied using N-acetyl cysteine, which efficiently suppressed the activity of MMP-3-activated EMT, thereby reducing tumor aggressiveness (Seema et al. 2017). ROS induces epigenetic changes by hypermethylation of the E-cadherin promoter region because of enhanced Snail protein that is likely to promote DNA methylation by increased histone deacetylase 1 (HDAC1) and DNA methyltransferase 1 (DNMT1) enzymes (Lim et al. 2008). NOX-derived ROS helps in the formation of invadopodia and protrusion of cell membranes through the regulation of HIF-1α-induced vimentin gene expression during cancer cell invasion and migration (Kidd et al. 2014). Also, ROS mediates oxidation of v-Src, and their activation evidences the enhanced invasion potentiality and anchorage independency of Src-transformed cells (Giannoni et al. 2005). Thus, ROS-mediated Src activation triggers the ligand-independent EGFR signaling, thereby conferring anoikis resistance (matrix-detachment-induced cell death) to cancer cells (Giannoni et al. 2009) (Fig. 3, purple side).

Second Face: ROS Regulates Anti-tumorigenic Signaling in Cancer In general, there will be a very high rate of ROS generation in cancer cells that is normally redeemed analogously by a high rate of antioxidant system to achieve balanced redox signaling. If the cancer cells are not able to regulate the increased ROS level, then they are likely to induce oxidative stress-mediated cell cycle arrest, cell senescence, and finally apoptosis (Gorrini et al. 2013b). Increased level of ROS was found to stimulate cancer cell death via apoptosis signal-regulating kinase 1 (ASK1) or MAP 3 K5/c-Jun N-terminal kinase (JNK) and ASK1/P38 signaling pathways. The inactive form of ASK1 usually interacts with the reduced form of TRX. In case of highly increased ROS-mediated oxidative stress, H2O2 oxidizes TRX resulting in sequestration and activation of ASK1, thereby triggering downregulation of anti-apoptotic proteins via sustained stimulation of MAPKK (MKK4)/MKK7/JNK and MKK3/MKK6/p38 signaling pathways and finally ending up in cell cycle arrest and cell death in numerous cancers (Bauer et al. 2017; Banudevi et al. 2018) (Fig. 3). ROS also induces cancer cell death either by inactivating or by upregulating the ubiquitination of B-cell lymphoma-2 (Bcl-2) and by downregulating the intracellular pro-apoptotic proteins such as Bcl-2 associated death promoter (BAD) and Bcl-2-associated X protein (BAX) (Luanpitpong et al. 2013). A very high rate of ROS in cancer cell was found to address both extrinsic and intrinsic programmed cell death through downregulation of cellular FLICE-inhibitory protein (c-FLIP) half-life by promoting ubiquitin-mediated proteasomal degradation (Wilkie-Grantham et al. 2013) and increased cytoplasmic release of pro-apoptotic proteins, cytochrome C, apoptotic protease-activating factor 1 (APAF-1), and caspase 9 (Zuo et al. 2009).

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H2O2-mediated oxidation of autophagy-related 4A cysteine peptidase (ATG4) enzymes, a prerequisite for ATG8 delipidation, in turn, inactivates ATG4, which subsequently increases microtubule-associated protein 1A/1B-light chain 3 (LC-3)mediated autophagosome formation resulting in cancer autophagy (Poillet-perez et al. 2015). H2O2 also increases ataxia-telangiectasia-mutated protein (ATM)dependent AMPK oxidation that suppresses the mammalian target of rapamycin complex 1 (mTORC1) activity resulting in enhanced cancer autophagy (Perillo et al. 2020). NFκ-B also regulates ATG6/Beclin or p62/sequestosome 1 (SQSTM1) expression that governs ROS-mediated cancer autophagy (Lin et al. 2019). Interestingly, ROS also induces necroptosis, a non-apoptotic form of cancer cell death. Decreased p53 level confers p53-mediated ferroptosis that results in cysteine– glutamate antiporter and SLC7A11 transcriptional repression, leading to reduced uptake of cysteine residues and subsequent GSH synthesis levels (Jiang et al. 2015). Increased NADPH ensures its negative correlation with susceptibility to ferroptosisinfluencing agents (Shimada et al. 2016). Thus, several human cancers, including liver and breast cancers, overexpress cysteine–glutamate antiporter to regulate their antioxidant defense ability, thereby evading ferroptosis (Jiang et al. 2015). In cancer metastasis, the tumor cells encounter metabolic changes to enhance antioxidant ability to underpin the anchorage-independent cancer cell growth and prevent cell death (Piskounova et al. 2015; Jiang et al. 2016). In particular, isocitrate dehydrogenase (IDH-1)-dependent reductive carboxylation encourages anchorage-independent cancer growth by upregulating NADPH levels in mitochondria and downregulates mROS levels (Piskounova et al. 2015). In melanoma, cancer cells need SHMT2 and NADPHproducing enzymes to promote metastasis via the folate cycle (Piskounova et al. 2015). Thus, perplexing these ROS-alleviating pathways may be a feasible therapeutic perspective to vanquish cancer cell proliferation, invasion, and metastasis.

Conclusion Emerging studies on ROS explicate the double-edged sword role of ROS in cancer cells. ROS is no longer considered as a molecule invoking oxidative stress; rather, it plays a role in the regulation of cell signaling pathways that influence the physiological and various cellular responses through redox signaling. The shreds of evidence comprehend that the redox signaling, not oxidative stress, minds the regulation of cancer cell signaling pathways and the concentration of ROS to decide the doom of cancer cells. Hence, the dual role of ROS seems to be a challenging target for cancer therapeutics, and extensive research is needed for the understanding of ROS and its redox state that helps in creating a greater avenue to lessen the cancer burden.

Cross-References ▶ ROS-Mediated Apoptosis in Cancer Acknowledgments This work was supported in part by the TRR research fund, SASTRA Deemed-to-be University, Thanjavur, Tamil Nadu, India.

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Oxidative Stress and Notch Signaling Implications in Cancer

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Vivek Kumar, Mohit Vashishta, and Bilikere S. Dwarakanath

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Notch Signaling in Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Oxidative Stress in Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cross Talk Between Oxidative Stress and Notch Signaling in Cancer . . . . . . . . . . . . . . . . . . . . . . . . Oxidative Stress Regulated Notch Signaling in Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Notch Signaling Regulates Oxidative Stress in Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Implications of the Cross Talk Between Notch Signaling and Oxidative Stress in Cancer . . . Proliferation and Survival . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Epithelial to Mesenchymal Transition (EMT), Migration, and Invasion . . . . . . . . . . . . . . . . . . Cancer Stem Cells (CSCs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Therapeutic Targeting of Oxidative Stress/Notch Signaling in Cancer . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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V. Kumar (*) · M. Vashishta R&D Department, Shanghai Proton and Heavy Ion Center (SPHIC), Shanghai, China Shanghai Key Laboratory of Radiation Oncology, Shanghai, China Shanghai Engineering Research Center of Proton and Heavy Ion Radiation Therapy, Shanghai, China e-mail: [email protected] B. S. Dwarakanath Department of Research & Development, Shanghai Proton and Heavy Ion Center (SPHIC), Shanghai, China Shanghai Key Laboratory of Radiation Oncology, Shanghai, China Shanghai Engineering Research Center of Proton and Heavy Ion Radiation Therapy, Shanghai, China Present address: Central Research Facility, Sri Ramachandra Institute of Higher Education and Research, Chennai, India © Springer Nature Singapore Pte Ltd. 2022 S. Chakraborti et al. (eds.), Handbook of Oxidative Stress in Cancer: Mechanistic Aspects, https://doi.org/10.1007/978-981-15-9411-3_83

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Abstract

Both Notch signaling and oxidative stress have pivotal roles in health and diseases. Emerging evidences indicate that they both can regulate each other to influence crucial processes necessary for the tumorigenesis. Notch signaling can modulate oxidative stress by directly regulating some of the critical components related to the generation of oxidative stress or indirectly by downregulating the antioxidant defense system. Meanwhile, oxidative stress can also regulate Notch signaling at various stages of tumor development. Therefore, understanding the complex interplay between the Notch signaling and oxidative stress in cancer may lead to the development of effective therapeutic strategies for the treatment of cancer. This chapter presents an overview of the Notch signaling and oxidative stress in cancer, how they interact and regulate each other, and what are the implications of this cross talk in cancer. Further, various anticancer therapeutics which target oxidative stress and Notch signaling in cancer are also discussed. Keywords

Notch signaling · Oxidative stress · Epithelial-mesenchymal transition · Cancer stem cells · Therapeutic targeting

Introduction Oxidative stress is the state of disturbed balance between the production of reactive oxygen species (ROS) or reactive nitrogen species (RNS) and the efficiency of antioxidants. Intracellular accumulation of ROS/RNS can contribute to the conversion of normal cells into cancer cells. In addition, oxidative stress–induced events may affect all aspects of tumor development and progression; thus, elevated oxidative stress levels are characteristics of several cancers (Hayes et al. 2020). Tumor cells often upregulate the antioxidant systems, which can counter the increased ROS/RNS, thus avoiding the damaging effects of the excess oxidative stress. There are several mechanisms by which the cells upregulate the antioxidant machinery, and one of them is by regulating the signaling pathways (Marinho et al. 2014). It is now established that ROS generated during oxidative stress often upregulate various signaling pathways (involved in the development and tumorigenesis) to regulate the redox status of the cells. One among them is the Notch signaling pathway. Notch signaling is an evolutionarily conserved signaling pathway that plays a pivotal role in embryonic development and tissue homeostasis by regulating cell fate decisions and cellular processes (Hori et al. 2013). Dysregulated Notch signaling is associated with several human diseases (Siebel and Lendahl 2017) and also leads to tumor growth by altering the cell’s developmental state and thereby maintaining the cells in an undifferentiated or proliferative fate. Notch signaling thus plays a key role in the development of tumors by causing cells to adopt a proliferative cell fate. Emerging evidences suggest that Notch signaling and oxidative stress can interact and modulate each other in several ways (Wakabayashi et al. 2015; Small et al.

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2014). Notch signaling can either directly modulate various factors involved in oxidative stress or indirectly, by altering the intermediate molecules involved in oxidative stress. Likewise, various components of oxidative stress can regulate Notch signaling. A deeper understanding of the rationale behind this cross talk, which cancer cells adapt, will lead to the development of effective therapeutic strategies for the treatment of cancer. This chapter presents an overview of the Notch signaling and oxidative stress in cancer and how they interact and regulate each other in cancer. Moreover, various anticancer therapeutics that target oxidative stress and Notch signaling in cancer are also discussed.

Notch Signaling in Cancer Notch signaling is an evolutionarily conserved signaling pathway that mediates short-range cellular communication through interaction of the Notch receptors with ligands presented on neighboring cells, thereby inducing the response to the environmental cues in a multicellular organism. Notch pathway in mammals consists of four Notch receptors (Notch 1–4) and five ligands (Jagged-1, -2, and Delta-like-1, -3, and -4), which are all type I transmembrane protein. Structurally, the Notch receptors are composed of the extracellular region consisting of epidermal growth factor (EGF) like repeats (at N-terminal region), which varies among Notch receptors. 36 EGF-like repeats are present in Notch1 and Notch2, while Notch3 has 34, and Notch4 has 29. This is followed by a negative regulatory region (NRR) consisting of 3 LNR (Lin-12 and Notch repeats) domains and a heterodimerization domain (HD). The intracellular region of the Notch receptors is composed of RAM domain, Ankyrin domain, a transcriptional activation domain (TAD), and a C-terminal PEST domain. Notch3 and Notch4 lack the TAD domain (Fig. 1) (Kovall et al. 2017; Gordon et al. 2008). Notch ligands are composed of an extracellular region consisting of module of N-terminus of Notch ligands (MNNL), followed by DSL (Delta, Serrate, and Lag-2) and EGF-like repeats, which range in number from 5 to 9 in the Delta family and 16 in Jagged family. The intracellular region has a PDZL domain, which was identified in Jagged1, Delta-like-1, and Delta-like-4 (Fig. 1) (D’Souza et al. 2008; Lobry et al. 2014). Notch receptors are synthesized as single precursor proteins that undergo cleavage by a furin-like protease in the Golgi apparatus at the S1 site, forming a noncovalently linked heterodimer consisting of a Notch extracellular domain and a transmembrane domain. The resulting mature receptor is then translocated to the membrane. Notch signaling is triggered by the interaction between neighboring cells by the ligand-receptor. This leads to the S2 cleavage by ADAMs metalloproteases, producing a short-lived transmembrane form of Notch that is rapidly cleaved (S3) within its transmembrane segment by γ-secretase, releasing the Notch intracellular domain (NICD). The free NICD translocates to the nucleus, where it binds to the CBF-1/Su(H)/LAG1 (CSL) transcription factor. This results in the displacement of repressive factors and recruitment of coactivators, thus activating the transcription of the target genes (Kopan 2012) (Fig. 2).

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Fig. 1 Notch ligands and receptors. Mammals have 4 Notch receptors; Notch1 to Notch4. The extracellular domain of the receptors have EGF-like repeats (varies from 36 to 29 in number in different receptors) and negative regulatory region (NRR), which consists of 3 LNR (Lin-12 and Notch repeats) domains. The cytoplasmic portion of the receptor contains a RBPJκ associated molecule (RAM) domain, Ankyrin (ANK) repeats, and a Proline-glutamate-serine-threonine-rich (PEST) domain. Notch1 and Notch 2 have transactivation domain (TAD) which is absent in Notch3 and Notch4. Mammals have 5 Notch ligands: Jagged1, Jagged2, Delta-like1 (Dll1), Delta-like3 (Dll3), and Delta-like4 (Dll4). All ligands have an extracellular domain consisting of module of N-terminus of Notch ligands (MNNL), followed by DSL (Delta, Serrate, and Lag-2). The EGF-like repeats varies in numbers in Delta and Jagged family ligands

Apart from having an important role in embryonic development, the Notch signaling pathway is recognized as a major player in the cancer cells. Notch signaling can act as both, oncogene or tumor suppressor in cancer (Aster et al. 2017). Its role as an oncogene has been well described in T cell acute lymphoblastic leukemia (T-ALL), where activating mutations in Notch1 receptor occurs in more than 50% of the cases. The mutation that often leads to the activation of Notch genes have also been identified in other hematological malignancies such as splenic marginal zone lymphoma, mantle cell lymphoma, and CLL (B cell chronic lymphocytic leukemia) (Lobry et al. 2011). Apart from having an oncogenic role, the Notch signaling has also been shown to function as a tumor suppressor in tissue such as pancreatic epithelium, skin, hematopoietic cells, as well as in hepatocytes (Dotto 2008).

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Fig. 2 Simplified overview of canonical Notch signaling. Notch receptor is synthesized as a single precursor protein, which is cleaved by furin-like convertase (S1 cleavage) in Golgi, and exits as noncovalently linked heterodimer which translocates to the cell surface. As Notch ligand (in signal sending cell) binds to the Notch receptor (in signal receiving cell), two consecutive proteolytic cleavages of the Notch receptor are initiated. S2 cleavage by ADAMs generates substrate for the S3 cleavage by the γ-secretase complex. The Notch intracellular domain (NICD) is then released from the membrane and translocate to the nucleus, where it forms a transcriptional activation complex with CSL and coactivators (CoA), thereby inducing the transcription of target genes

Oxidative Stress in Cancer Oxidative stress is an imbalance between the production of reactive oxygen species (ROS) and the antioxidant defense. It is an important factor that promotes cell survival, proliferation, and progression of cancer. ROS are oxygen-derived chemical species found predominantly in mitochondria, peroxisomes, and the endoplasmic reticulum (ER). These species include superoxide (O2˙ ) and hydroxyl (HO•) free radicals, as well as nonradical molecules such as hydrogen peroxide (H2O2), which are formed as a by-product of aerobic respiration (Fig. 3). Upregulation of various

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Fig. 3 (continued)

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enzymes such as NADPH oxidase, xanthine oxidase, lipoxygenase, endothelial nitric oxide synthase (eNOS), arachidonic acid, and metabolic enzymes (cytochrome P450), and cyclooxygenase also catalyzes the ROS generation (Fig. 3). The antioxidant defense has mainly two arms; enzymatic and nonenzymatic. The enzymatic arm comprises superoxide dismutases, catalase, and peroxidases, while the nonenzymatic arm consists of glutathione, thioredoxin as well intermediates of glycolysis such as pyruvate, lactate, and NADPH (Fig. 3). ROS species targets various biomacromolecules like DNA, protein, lipids, and other cellular components and plays an important role in cellular homeostasis as it stimulates and regulates various intracellular signaling pathways. It also serves as a sensor and activator for various immune and cell survival signaling pathways (Gorrini et al. 2013). The precise role of ROS in cancer is not clear. Physiological levels of ROS functions as a signaling molecule and contributes to the tumorigenesis by promoting the mutation in genomic DNA at low to moderate levels. It also activates various proteins responsible for tumor growth and survival, like phosphorylation of MAPK, ERK, cyclin D1 expression, and JUN N-1 terminal kinase (JNK) activation. In general, cancer cells possess a higher level of ROS than normal cells that leads to DNA damage and impaired signaling. In the early stages of cancer, cells have high antioxidant levels to combat the enhanced ROS levels. In the case of liver and breast cancer stem cells, ROS levels are low due to increased anti-ROS defense. The levels of ROS further increases upon radiotherapy and chemotherapy. However, owing to their ability to enhance the antioxidant defense system, the cancer cells become resistant and result in the expansion of resistant cells (Gorrini et al. 2013). High or pathological levels of ROS generated beyond the threshold of the cellular defense system results in the induction of cell death mainly through regulated and unregulated interphase death (apoptosis and necrosis). ROS serves as a modulator and sensor in the various signaling pathway. One such pathway that is important in cancer is the Keap-1-Nrf2 system. Nrf2 regulates many antioxidant genes expression that is involved in ROS detoxification (McGrathMorrow et al. 2009). ROS generation results in the upregulation of Nrf2, which leads to the activation of HMOX1 and GCLM involved in cell survival and proliferation. Further, Nrf2 stabilization is regulated by putative oncogenes like PARK7, KRAS, and MYC that promote the antioxidant response to oxidative stress. ROS generated during oxidative stress is known to regulate tumor suppressor genes. For example, the tumor suppresser gene such as phosphatase and tensin homolog (PTEN) and protein tyrosine phosphatases (PTPs) were shown to get ä Fig. 3 Generation of reactive oxygen species and antioxidant defense system in cancer cells. Mitochondria, endoplasmic reticulum, peroxisomes, and lipoxisomes are the major sources of generation of reactive oxygen species (ROS), while the antioxidant defense (in Red) is composed of enzymatic (superoxide dismutase (SODs), catalase, glutathione peroxidase lipoxigenases, and NADPH oxidase) and nonenzymatic (GSH, thioredoxin, uric acid, vitamins, and CoEnzQ) processes. Upregulation of the expression of antioxidant enzymes limits the oxidative stress caused by ROS generated from endogenous sources and exogenous stimulations

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reversibly inactivated by ROS, as their catalytic center had redox-sensitive cysteine residues. These modulations lead to the suppression of antioxidant genes (Leslie et al. 2003; Xu et al. 2002). Likewise, other tumor suppressors like p53 and FOXO are also affected by ROS, but their response to the oxidative stress is controversial, as emerging reports shows that they can be either activated or inhibited after ROS levels are increased. In turn, tumor suppressors are also shown to regulate oxidative stress in a context-dependent manner. They can either activate or suppress the expression of antioxidant genes, which affects oxidative stress. For example, the inactivation of FOXO due to the loss of PTEN leads to an overall increase in oxidative stress (Nogueira et al. 2008). Similarly, BRCA1 is implicated in the regulation of oxidative stress by controlling the expression of Nrf2-regulated antioxidant genes (Bae et al. 2004). Moreover, loss of the ATM gene that regulates ROS levels and autophagy through mTORC1 results in chronic oxidative stress in mice and cancer patients (Barzilai et al. 2002). Certain key metabolic reprogramming molecules are also influenced by oxidative stress. Pyruvate kinase M2 (PKM2) is a tumor-specific metabolic enzyme that causes a paradigm shift of glycolysis to NADPH-reducing equivalents as it is less efficient than PKM1. It plays an important role in the initiation of tumors following detachment from the matrix and enhanced oxidative stress (Schafer et al. 2009).

Cross Talk Between Oxidative Stress and Notch Signaling in Cancer Oxidative Stress Regulated Notch Signaling in Cancer Different factors that are involved in the oxidative stress can regulate Notch signaling at various levels, starting from transcription of the Notch receptor to its activation. One of these factors, which has a prominent role in regulating Notch signaling in cancer, is the nuclear factor erythroid 2-related factor 2 (Nrf2). The Nrf2 transcription factor is the master regulator of the cellular antioxidant response. Several studies have illustrated the important role played by Nrf2 in tumorigenesis (Rojo de la Vega et al. 2018). Nrf2 recognizes antioxidant response elements (AREs) in the regulatory regions of genes and activates their expression. The first report, which suggested that Notch1 signaling may be regulated by Nrf2, came from the initial gene expression analysis on immortalized mouse embryonic fibroblast (MEF) from Keap1 / , Nrf2 / , Keap1 / :: Nrf2 / , and wild type mice (Kwak et al. 2003). Further analysis of the promoter region of the mouse Notch1 gene revealed that it possessed the functional ARE in its proximal region, thus strengthening the proposition that Nrf2 can transcriptionally regulate the Notch1 gene (Fig. 4) (Kensler and Wakabayashi 2010). This was also observed in vivo, where the Notch1 transcripts were reduced in the liver of adult Nrf2-null mice compared to the wild-type counterpart (Kensler and Wakabayashi 2010). Since both Notch and Nrf2 cross talk with many other signaling pathways involved in cancer, it is not surprising that Nrf2 can also indirectly regulate Notch signaling via other signaling pathways.

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Fig. 4 Cross talk between oxidative stress and Notch signaling in cancer. Oxidative stress induces intracellular events that interact/regulate Notch signaling at various level starting from activation of transcription of Notch receptors by Nrf2. Notch signaling in turn regulates the mitochondrial integrity and function (which alters the generation of radical species) besides regulating other components of antioxidant defense, which modulates oxidative stress in cancer

In this context, it is pertinent to note that overexpression of Nrf2 in breast cancer upregulates the expression of Notch1 via G6PD/HIF-1α pathway (Zhang et al. 2019). Besides regulating Notch signaling indirectly via other signaling pathways such as Nrf2, NF-κB, p53, etc., ROS generated during oxidative stress can also regulate Notch signaling directly by modulating some of the components involved in Notch signaling pathways (Hayes et al. 2020). The synthesis of the metalloprotease ADAM17 can be induced by ROS. ADMA17 is involved in the cleavage of the ectodomain of the Notch receptor, thus activating the Notch signaling (Fig. 4) (Kavian et al. 2010). Moreover, upregulation of TACE (ADAM17) activity by LPS was mediated by ROS via activation of the p38 signaling pathway (Scott et al. 2011). Therefore, ROS-mediated upregulation of ADAM17 could be another major factor for the activation of the Notch pathway in cancer. Redox reagents such as H2O2 can also regulate ADAM17 by modifying the sulfhydryl groups of ADAM17 (Wang et al. 2009). Mechanistically it is shown that two vicinal cysteine sulfhydryl motifs (C522XXC and C600XXC) in the disintegrin/cysteine-rich region of ADAM17 contain redox-sensitive sites which upon oxidation by H2O2 causes disulfide bond formation, thereby switching the conformation of ADAM17 from less active to a fully active state (Wang et al. 2009). This modification within the extracellular portion of ADAM17 may be important for the activation of Notch signaling in cancer as it is seen in the shedding of L-selectin ectodomain after neutrophil activation (Wang et al. 2009). Another level at which the oxidative stress can modulate Notch signaling is through post-translational modification of the Notch receptor. The reactive chemical species generated by oxidative stress are known to modify protein, thereby

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impacting their functions. Mass spectrometry using nanoelectrospray ionization for proteomic analysis of protein nitration in aging skeletal identified 3-nitrotyrosinecontaining sequences in rat Notch homolog (Kanski et al. 2005), indicating that tyrosine residue of Notch receptor can be modified by nitration (Fig. 4). The functional consequence of this modification is indicted by evidence showing that nitration of the Notch intracellular domain by LPS-induced NO may repress Notch signaling, as LPS-treated cells secrete NO into the culture medium, which represses the Notch reporters, and pharmacological inhibitors of NO synthesis block the LPS-induced repression of these reporters (Kim et al. 2008). An inverse relationship between the extracellular NO concentration and the amount of Notch intracellular domain (NICD) bound to the reporter promoters has been observed with high NO concentrations preventing NICD from associating with its DNA-binding partner RBPJk (Kim et al. 2008). Furthermore, these studies have also indicated that nitration represses NICD activity by rendering it incapable of binding to its transcriptional coactivator(s) (Kim et al. 2008). In contrast to this, NO has also been found to activate Notch signaling, but this is mediated indirectly through NO/cGMP/ PKG pathway (Charles et al. 2010). These opposing roles of NO in Notch signaling need to be further investigated. In addition, the physical association of Presenilins (which is a part of gamma-secretase complex) with two antioxidant enzymes, thiolspecific antioxidant (TSA) and proliferation-associated gene (PAG), of the peroxiredoxin family has been shown to result in phenotypes typical of Notch signaling loss-of-function mutations (Fig. 4) (Wangler et al. 2011).

Notch Signaling Regulates Oxidative Stress in Cancer Notch signaling can modulate oxidative stress in cancer by directly regulating the transcription of some of the components involved in oxidative stress, and one of them is Nrf2. The proximal gene regulatory region in mammalian Nrf2 genes contains functional RBPJκ core-binding sequence. The activated Notch induced by ligands or constitutive active form can bind to this region and regulate the transcription of the Nrf2 gene (Wakabayashi et al. 2014). Mitochondria are considered as the major source of ROS in cancer (Hayes et al. 2020). Therefore, any entity that can affect the mitochondrial integrity and function can alter the ROS generation and thereby the oxidative stress. Notch signaling can alter the proteome of mitochondria, which results in an alteration of its function. Indeed emerging evidences suggest that Notch plays a key role in the metabolism and biogenesis of mitochondria (Hossain et al. 2018). The cross talk between HIF-1 and the Notch signaling pathways is involved in the regulation of cancer cell’s mitochondrial biogenesis to maintain REDOX balance (Kung-Chun Chiu et al. 2019). PINK1, which is an essential gene for mitochondrial biogenesis, was found to be a novel transcriptional target of HEY1 (one of the Notch targets) (Kung-Chun Chiu et al. 2019). An inverse correlation between HEY1 and PINK1 expressions has been observed in human HCC (Kung-Chun Chiu et al. 2019). Furthermore, HEY1 overexpression and PINK1 underexpression are often associated with poor clinical outcomes (Kung-Chun Chiu et al. 2019). Moreover, overexpression of HEY1 or

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knockdown of PINK1 consistently decreases mitochondrial cristae, mitochondrial mass, oxidative stress level, and increased HCC growth (Kung-Chun Chiu et al. 2019). Notch signaling can also regulate the oxidative stress by directly or indirectly regulating the molecules that either generate or scavenge the reactive radicals. MnSOD, a crucial ROS scavenger in mitochondria, is regulated by Notch signaling at the transcription level via Jak2-STAT3 signaling (Yu et al. 2011). Disruption of Notch signaling results in decreased Hes5-STAT3 complex, leading to increased ROS and apoptosis of hepatocyte during the ischemia-reperfusion injury (Yu et al. 2011). However, the consequence of this mechanism in carcinogenesis remains to be investigated. Nox4, which is a member of the NOX family of NADPH oxidases, is responsible for the superoxide (O2 ) production. Nox4 is generally involved in transferring electrons across the membrane from NAD(P)H to molecular oxygen. Notch signaling negatively regulates the production of ROS by suppressing Nox4 expression in endothelial cells, and inhibition of Notch signaling leads to enhanced expression of Nox4 (Cai et al. 2014). Moreover, bioinformatics analysis has revealed that there are two binding sites for RBPJκ and one binding site for Hes1 at around 700 bp upstream of the Nox4 gene transcription start site, further supporting that Notch signaling can directly regulate Nox4 expression (Cai et al. 2014). In contrast to this, Notch signaling has been shown to positively regulate Nox4 expression in high glucose-treated human retinal endothelial cells, resulting in increased ROS production and cell death (Jiao et al. 2019). Whether this mechanism also prevails in cancer cells needs to be investigated. Nitric oxide (NO) generated by the tumor, stromal, and endothelial cells play a multifaceted role in tumor biology. Notch can activate the NOS (nitric oxide synthase) in endothelial cells, which induce the production of NO in the endothelial cells of the tumor vasculature, thereby affecting the blood vessel function and consequently the tumor growth. Specific inhibition of Notch signaling in the endothelial cell decreases eNOS activity and NO production, thus affecting tumor growth (Patenaude et al. 2014).

Implications of the Cross Talk Between Notch Signaling and Oxidative Stress in Cancer Notch signaling and oxidative stress both play a crucial role in different stages of cancer development, progression, and also in the resistance to therapy. Therefore, the regulation of Notch signaling by oxidative stress and vice versa could have a major impact on the crucial processes that are required for tumorigenesis.

Proliferation and Survival The main characteristics of tumor cells are increased ability to survive and uncontrolled proliferation. ROS can activate several cellular signal transduction

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pathways that have a prominent role in the survival and proliferation of cancer cells (Hayes et al. 2020), while the Notch involvement in the survival and proliferation of cancer cells has also been well known (Aster et al. 2017). As described earlier, various components of the oxidative stress regulate Notch signaling; this, in turn, can control the survival and proliferation of cancer cells. The positive regulation of Notch signaling by Nrf2 in this context has been associated with cancer cell survival and proliferation. Activation of Nrf2 in Keap1-/- mice resulted in the activation of Notch signaling in squamous epithelial cells in the mouse tongue, which was associated with the hyperproliferation of these cells (Fan et al. 2017). In addition, it was found that overexpression of Nrf2 upregulates the expression of Notch1, which in turn influences the proliferation of breast cancer cells (Zhang et al. 2019).

Apoptosis Defects in apoptosis are seen as characteristics of most cancers (Wong 2011). Emerging data show that Notch signaling protects cancer cells from apoptosis by maintaining low ROS levels. In this regard, Notch inhibitors potentiate druginduced apoptosis by increasing the production of ROS and decreasing the levels of mTOR, NF-κB, and ERK (Takam Kamga et al. 2019). The suppression of apoptosis signal-regulating kinase (ASK)1 signaling pathway is another mechanism by which Notch signaling controls oxidative stress–induced cell death. Notch1-ICD interacted directly with ASK1 and blocked its homodimerization and activity. In addition, it promotes the translocation of the activated ASK1 to the nucleus and thus prevents the oxidative stress-induced cell death through apoptosis. In this context, the knockdown of Notch1 has been found to induce oxidative stress–linked cell death (Mo et al. 2013). In addition, Nrf2’s knockdown facilitates radiation-induced apoptosis, which is mediated by Notch1 signaling in NSCLC cells (Zhao et al. 2017).

Epithelial to Mesenchymal Transition (EMT), Migration, and Invasion EMT is a transdifferentiation program in which epithelial cancer cells lose cell-cell adhesion and acquire mesenchymal features of migration and invasion. Both oxidative stress and Notch signaling can directly regulate EMT as well as migration and invasion. At the same time, they can also influence each other to modulate the EMT and migratory behavior of cancer (Tam et al. 2020). In breast cancer, for example, Nrf2 upregulates Notch1’s expression through G6PD/HIF-1α pathway, which in turn regulates the migration of breast cancer cells by influencing the EMT pathway (Zhang et al. 2019). In another instance, Nrf2 and Notch1 downregulation has been found to synergistically inhibit radiation-induced migratory and invasive properties of NSCLC cells (Zhao et al. 2017).

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Cancer Stem Cells (CSCs) Cancer stem cells play a significant role in tumor resistance and cancer recurrence. They constitute the subpopulation of transformed cells and have the intrinsic ability to undergo self-renewal and differentiation. Moreover, it has been suggested that CSCs possess enhanced protection against ROS-induced oxidative stress when compared with non-stem-like cancer cells (Zhou et al. 2014). Indeed CSC maintains lower ROS levels. To this point, the Notch pathway is critical to regulate the ROS level in CSCs, and one potential target is the PI3K/AKT pathway. In glioma stem cells, Notch upregulates PI3K/AKT pathway, which in turn activates the ROS scavenging enzymes (Ding et al. 2015). On the other hand, ROS/RNS can also activate the Notch signaling pathway in order to sustain the CSCs. For example, the nitric oxide released by endothelial cells can activate Notch signaling and promote the stemness of the PDGF-induced glioma cells (Charles et al. 2010). Moreover, the activation of Notch signaling by nitric oxide has been shown to enhance the side population phenotype (CSCs) in cultured human glioma cells (Charles et al. 2010). In addition, the activation of Notch signaling by HO-1-derived CO stimulates the formation of mammospheres in breast cancer cells (Kim et al. 2018). Mechanistically it was shown that HO-derived CO promotes Jagged-1 expression in breast stem-like cancer cells and induces Notch-1 activation, which in turn upregulates Hes1 expression. This confers self-renewal activity and tumor sphere formation capacity in breast cancer stem-like cells (Kim et al. 2018).

Therapeutic Targeting of Oxidative Stress/Notch Signaling in Cancer In the last few decades, many drugs that are used as an effective anticancer therapeutics were shown to have a direct and indirect effect on ROS. For example, drugs such as taxanes (paclitaxel and docetaxel), vinca alkaloids (vincristine and vinblastine), and antimetabolites (used as chemotherapeutics) are known to induce oxidative stress. These drugs interfere with the electron transport chain (ETC), which results in the generation of superoxide radicals (Simunek et al. 2009). Moreover, drugs such as platinum coordination complexes (e.g., cisplatin, carboplatin, and oxaliplatin), arsenic trioxide (As2O3), and anthracyclines (e.g., doxorubicin, epirubicin, and daunorubicin) were shown to induce apoptosis in various cancer cells, including lung cancer, leukemia, and myeloma via producing extremely high levels of ROS (Hwang et al. 2007). Similarly, 5-fluorouracil (5-FU), a pyrimidine analog that is used in the treatment of colon, rectal, and head and neck cancer, generates mitochondrial ROS via a p53-dependent pathway (Hwang et al. 2007). However, tumor cells often develop resistance to most of these drugs. Interestingly, Notch signaling (known to mediate resistance to these anticancer therapies) also gets upregulated by these agents. Therefore, the inhibition of Notch signaling is shown to reverse the resistance phenotype gained by cancer cells. The preclinical studies show

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that many natural compounds that possess antioxidant properties can modulate Notch signaling in cancer. Among them, the most widely studied compound is curcumin, which is widely investigated for its anticancer effects. The downregulation of Notch signaling by curcumin is associated with inhibition of cell proliferation and the induction of apoptosis in pancreatic cancer cells and human hepatocellular carcinoma (Liu et al. 2014). Likewise, ganoderic acid, which is a lanosterol triterpene, is isolated from the fungus Ganoderma lucidum with a wide range of medicinal values. The molecular docking studies have shown that ganoderic acid A and curcumin are best docked among different compounds and downregulate Notch-1 mRNA expression in a neuroblastoma cell line (IMR-32 cells) and inhibits proliferation, viability, and ROS activity (Gill and Navgeet 2019). Asiatic acid (AA) (triterpenoid isolated from Centella Asiatica) is a novel small molecule inhibitor of the Notch signaling pathway that exerts antioxidant, antitumor, and antiinflammatory effects (Yuyun et al. 2018). Similarly, N-acetylcysteine (NAC), which is a precursor of intracellular glutathione (GSH), has been extensively explored for the prevention and therapy of several cancers. NAC promotes the degradation of Notch2 via the lysosomal pathway in an antioxidant-independent manner. This leads to a decrease in pro-tumorigenic Notch2 malignant signaling in GBM cells, thereby decreasing the malignant characteristics of glioblastoma cells (Deng et al. 2019). Drugs targeting Notch signaling (inhibitors of gamma-secretase as well as antibodies against various components of Notch signaling) have been evaluated either as a monotherapeutic agent or as a part of combinational strategies with the standard of care therapeutics like gemcitabine, cisplatin/carboplatin, folate antimetabolites, taxol, etc. Phase I and II clinical trials have shown that Notch inhibitors alone (as a monotherapeutic) or in combination with other anticancer therapeutics is well tolerated without any additional toxicities with modest clinical benefits (Du et al. 2019). However, unequivocal clinical benefits of drugs targeting Notch signaling are yet to be established in Phase III clinical trials. Moreover, the possible contribution of oxidative stress in the clinical effects of these Notch targeting drugs remains to be established. Interestingly, hyperactivation of Notch1 due to mutations does not show a good correlation with the outcome of chemotherapy (a combination of methotrexate and cyclophosphamide) in children with T-cell acute lymphoblastic leukemia (T-ALL), although a favorable early response was noted (Clappier et al. 2010).

Conclusion Notch signaling and oxidative stress play an important role in various stages of cancer. Both can have an oncogenic role or tumor suppressor role in cancer. Emerging evidence suggests that they both interact and modulate each other, which can support the cancer cell to adapt to the tumor microenvironment. Nonetheless, more details regarding the interplay between Notch signaling and oxidative stress in cancer remain to be elucidated. A deeper insight into the Notch/oxidative

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stress network in cancer will provide the rationale for suitably combining the existing therapies with Notch inhibitors and designing new ones in the future.

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ROS-Induced Regulatory Crosstalk with Autophagy and AKT/mTOR Signaling in Cancer Cells

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mTOR Signaling in Cancer Piyanki Das, Koustav Chatterjee, and Tathagata Choudhuri

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Understanding ROS-Induced Autophagy Response During Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . Relation Between ROS and Autophagy During Malignancy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Specific Signaling Pathways Linking ROS and Autophagy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Impact of ROS-Responsive Autophagy in Chemotherapeutic Applications . . . . . . . . . . . . . . Role of ROS-Responsive PI3K/AKT/mTOR Signaling During Cancer . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract

ROS is not only a metabolic by-product but also a cellular alarm whose varied level determines the oxidative stress response of the cell in response to several stress stimuli. Autophagy is the major cellular catabolic machinery which acts as the damage repairing system in response to high oxidative stress. High level of ROS is associated with different pathological conditions due to the pathogenesis as well as toxic therapeutic applications. High oxidative stress acts as the signal transducer for autophagy which is supposed to eliminate the stress by clearing the damaged particles, maintaining the energy balance, or inducing the damage repair system and sometimes induction of the cell death process. When this cellular stress balance cannot be managed and the cell utilize the stress management system and hijack the autophagy, it results in cancer. During the process of cancer, the cell is evolved in such a way where it modulates several signaling pathway which maintains an oncogenic stress response system mainly utilizing the autophagy. Here we have critically

P. Das · K. Chatterjee · T. Choudhuri (*) Department of Biotechnology, Siksha Bhavana, Visva Bharati, Santinikatan, Bolpur, West Bengal, India e-mail: [email protected] © Springer Nature Singapore Pte Ltd. 2022 S. Chakraborti et al. (eds.), Handbook of Oxidative Stress in Cancer: Mechanistic Aspects, https://doi.org/10.1007/978-981-15-9411-3_84

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analyzed how autophagy helps at different stages of malignancy by its stress balance mechanism. Along with the understanding of different ROS-responsive signaling pathway between autophagy and oxidative stress response system, we have specifically emphasized the main ROS-responsive autophagy regulatory pathway, PI3K/AKT/mTOR during cancer. Interestingly, we have also tried to reveal the impact of the complex oncogenic autophagy-mediated stress balance system during antioxidant application as treatment modality (the modulators for ROS) against cancer. Keywords

ROS · Modulated autophagy · Cancer cell stress response · Cross-linking regulatory pathways · Therapeutic interpretation · Major regulatory kinase – PI3K

Introduction In normal cell, a well-balanced oxidative response goes on, which spontaneously and enzymatically balance the level of oxygen-centric metabolic by-products or the free radicals of the cell. When the cell experiences any kind of external stress stimulus like nutrient deprivation, genotoxic stress, hypoxia, pathogenic infection, etc. or any kind of endogenous stress, mainly generated from the cell’s energy source, the mitochondria, the metabolic balance is disturbed and high level of metabolic by-products generates reactive oxygen species (ROS) inside the cell, which leads to oxidative stress response (Liou and Storz 2010). Interestingly, the cell has its own inbuilt physiological stress management system, the autophagy, which acts as the repair mechanism by eliminating these unwanted stuffs and returning back the cell to its normal functional state with a balanced metabolic status. When the damage is irreparable and it is hard to maintain the cell, autophagy can also direct the cell towards death. ROS helps the cell to sustain this important conserved “catabolic damage repair system” for maintenance of the cellular stress homeostasis (Scherz-Shouval and Elazar 2011). Parallelly, all the abovementioned stress stimulus can trigger both high level of ROS and autophagy expression indicating towards a regulatory and molecular interactive crosstalk between these two. Precisely, the high oxidative stress promotes the transcription factors for autophagic gene expression leading to autophagy induction which degrade the damaged mitochondria by lysosomal degradation, particularly known as mitophagy (Lemasters 2005). Cancer cell is associated with high ROS level which helps in the pathogenesis. In response to this ROS, the cancer cell very strategically utilizes the ROS balancing autophagy mechanism to utilize this imbalance. In the following discussion, we will try to figure out how the cancer cell utilizes the ROS responsive stress management system and its associated regulatory signaling pathways during cancer.

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Understanding ROS-Induced Autophagy Response During Cancer Relation Between ROS and Autophagy During Malignancy The relation of ROS, autophagy, and cancer is not univocal, hence a complex phenomena. When the cell cannot handle the metabolic imbalance and stress, utilizing autophagy, it shows damaging effect resulting aging and different diseases including cancer. The cancer cells are highly proliferative with unrecognized defective cellular mechanisms. To maintain this unregulated cell division and defend the host cell protective mechanisms during cancer, a high level of metabolic need is needed inside the cell. Thus, the cells are also associated with a high level of metabolic by-product, damaged particles, etc. resulting increased ROS. Cancer cell can utilize both high ROS as well as minimize the elivated ROS level according to its need. The principal technique which participates in this process is autophagy. Autophagy can meet the high metabolic requirement of the cancer cell, it can repair the damage, recycle different cellular products, and has control over several cellular mechanisms like cell cycle regulation, and cell death process. During cancer, this autophagy becomes the central strategy for the oncogenic cell for its stress management tactics and act in response to varied ROS level. If we dissect different phases of cancer, we can observe the following phenomena. During initial stage of cancer, sometimes cells are associated with high level of chronic inflammation, like during liver cancer. For the maintenance of normal liver homeostasis, a basal level of autophagy is needed. The autophagy, in response to high ROS, supposed to control chronic inflammation by leukocyte infiltration, but during cancerous condition, chronic inflammation release high oxidative stress and ROS, and the sustained impaired autophagy helps in accumulation of damaged mitochondria and metabolic by-products which creates an inflammatory environment which provoke tumor initiation with a hypoxic resistance condition (Cursio et al. 2015). miRNA suppressing autophagy leads to decreased tumor growth. On the other hand, hepatocellular carcinoma with high constant inflammatory environment can be halted by the constitutive autophagy of HCC cells with dominating role of Beclin-1 autophagic protein. Therapeutic recovery of autophagy with significant inflammation suppressive role has been observed during liver cancer initiation. (Yazdani et al. 2019). On later stage of cancer, autophagy is associated with cellular transformation. Cancer cells are associated with high level of metabolic energy demand during stress-induced cellular transformation. Autophagy helps to manage or minimize cellular stress and helps in cellular transformation by increasing the stress tolerance which results in modulation of several signaling pathways and promote unregulated cell proliferation. At this stage, temporary autophagic arrest or triggering its oncosuppressive molecular activity creates a cellular stress homeostatic barrier against cellular transformation (Galluzzi et al. 2015). In the ultimate stage, autophagy helps in pro-metastatic maintenance of the cell by supporting the cell survival in hypoxic condition. In the next step, increased autophagy is associated with epithelial-mesenchymal transition (EMT) and cellular

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mobility by balancing the stress level of the cell. Activated autophagy play crucial role in the cellular detachment from primary tumor site and inhibit the detachment induced cell death process (anoikis). Active autophagy is needed where it helps in minimizing the high ATP and ROS production in the detached motile oncogenic epithelial cells acquiring mesenchymal properties during hepatocellular and mammary cell carcinoma. Angiogenesis is an important step during metastasis. Autophagy can help in the metastatic process and also able to stop it, by inhibiting the EMT. Metastatic hypoxic cells are associated with high inflammation regulated angiogenic factors where angiogenic inhibitors as well as the stimulators both can induce autophagy and apoptosis (Poillet-Perez et al. 2015). Thus, during cancer, the autophagic mechanism, in response to ROS, that is supposed to help in managing the stress balance of the cell, helps the cancer cell with the similar mechanism to propagate. The cancer cell gets the control and modulate the autophagy and its regulatory signaling pathways in such a way where it gets the wrong signals and by its repair mechanism, it eliminate or minimize the oncogenic stress and supports the unregulated cell proliferation. Now an interesting aspect of the relationship between ROS and autophagy is observed during pathogen-associated cancers specifically the viral cancers. During virus-associated malignancies, the virus is harbored inside the target cells of the host body and it modulates or mimics several host regulatory pathways for its maintenance. These alterations in cellular oncogenic signaling pathways results in the unregulated cell proliferations. Varied level of ROS is also associated with these cancers and responsible for the oncogenic development. But it is not that simple as it stated, because viral persistence inside host body depends on both of its dual life cycle phases, the fine balance of latency and lytic cycles are associated with creating and maintenance of a malignant environment. Both virus-infected latent and lytic cells experience different level of oxidative stress, and in case of a viral cancer, it is always associated with a high level of oncogenic stress. The intelligent virusinduced oncogenic stress management system balance this and helps in the malignancy process. When the host cell is infected with oncogenic virus, high level of ROS helps in the infection process and establishment, and then the cellular stress management system is hijacked by the virus (Mao et al. 2019). Then the cancer cell oxidative stress management helps in minimizing the high ROS level and maintains the virus in latent form which allows the unregulated cell proliferation. The subverted autophagy is the main stress balancing mechanism where the latent cell constitutively maintains a basal level of autophagic expression (Leidal et al. 2012; Oh 2010). When these latent cells experience slight high ROS due to any cellular metabolism, nutrient deprivation, or therapeutic- or drug-induced stress, it carefully chooses to convert themselves towards lytic reactivation (Wen et al. 2010) This is the better way for the virus-infected oncogenic cell to avoid the complete cell destruction. The reactive virus again utilizing the high ROS inside normal cell, infect them and establish itself and later converted into latency phase by minimizing the ROS. In the final step of viral replication, the autophagic flux is again minimized which probably controls the excessive high ROS which may damage the newly produced replicative virus (Granato et al. 2014). This is how virus hijacks the

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cellular stress responsive autophagic system during different minute phases of viral oncogenetic process which involves modulation of autophagic signaling and its several associated regulatory signaling pathways helping in maintenance the malignancy during both latent and lytic stages (Vescovo et al. 2020).

Specific Signaling Pathways Linking ROS and Autophagy Now it is clear that ROS is the regulator of autophagy. Autophagy responds to this alarm in cancer cell and regulates the stress balance for maintenance of malignancy. But how ROS can regulate the autophagic response, with different ROS level in cancer cell, we need to intersect the interlinked molecular scenario between ROS and autophagy during different cancer manifestations. Mitochondria is the major source of ROS. During stress condition, mitochondria is depolarized and various ubiqutinized proteins are exposed on outer side of mitochondria. There are cargo adaptor proteins, that act as the bridge between these damage mitochondria, the autophagic substrate and autophagy. Cargo adaptor proteins have two domains, one ubiqutinized protein-binding domain and another autophagy-related protein-binding domain. Autophagy detects and selectively degrades its substrates. This degradation leads to induction of antioxidative pathways helping in elimination of the oxidative stress. There are mainly of two major pathways by which the above process is mediated. First, the cargo adaptor protein NIX/BNIP3L is induced after ROS activation and interacts with the autophagyrelated ATG-8 family proteins (LC3, GARABAP-1/GARABAP-2) and the ubiquitin proteins. This interaction helps in the degradation of the cargo molecules subjected for degradation inside the autophagosome. It has been found that, NIX-mediated mitophagy helps in cancer progression (Humpton et al. 2019). On another pathway, active ROS activates PTEN-induced putative kinase (PINK1) and translocates in outer side of the mitochondrial membrane. PINK1 induce PARKIN (Ubiquitin E3 Ligase) which ubiquitinize lots of mitochondrial outer membrane proteins after depolarization of the mitochondria. The ubiquitinized proteins are recognized by another cargo adaptor protein p62/SQSTM1. p62 also have autophagy protein LC3 binding affinity. Thus, inside activated autophagosome, p62 interacts with KEAP1 and degrades it and thus releases Nrf-2. Nrf-2 is the transcription factor of antioxidative enzymatic signaling pathways which helps in eliminating the cellular stress. Regulation of PINK1/Parkin-mediated mitophagy found protective during pancreatic cancer and several multidrug-resistant cancers. Inhibition of this mitophagy helps in restricting the cancer (Zhao et al. 2019; Yao et al. 2019). Now there are several interconnecting signaling circuits which act as regulator or modulator for ROS-induced autophagic regulation which controls the tumorigenesis. ROS-responsive autophagy and Nf-kB may cross-regulate each other during cancer. The ROS-responsive autophagy and Nf-kB signaling works in TNF-alpha-mediated mechanism. TNF-alpha-responsive Nf-kB activation leads to ROS accumulation and inhibition of autophagy via the activation of the negative regulator of autophagy, the mTOR signalling during various cancers like leukemia, breast cancer, Ewing’s

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sarcoma, etc. TNF-mediated Nf-kB active ROS accumulation provides cancer cell survival. This TNF-alpha mediated Nf-kB suppression results in hypoxia or starvation-induced cell death and autophagic promotion (Djavaheri-Mergny et al. 2006; Verzella et al. 2020). Another important phenomena is observed in involvement of DNA damage and repair systems in response to ROS-mediated autophagy during cancer manifestation. ROS affects the integrity of DNA and thus it activates the DNA damage response, and subsequently activates the repair mechanisms, cell cyclecheck points to make up the injury or prevent the cell survival in case of unrepaired cellular conditions. The repair system activates autophagy which eliminates the source of ROS and toxic accumulation as well as it provides energy for the damage-repairing process by recycling the metabolic precursors. Cytoplasm to vacuole targeting (Cvt), PolyADP-ribose polymerase 1 (PARP-1) ATM, p53 and its family member protein p63 and p73 are directly involved in the DNA damage-induced autophagic upregulation after ROS. Highly active DNA damage signaling and ROS is balanced by autophagy and parallelly ROS-induced accumulated DNA damage by defective autophagy during tumorigenesis. Highly active DNA damage response in tumor cells can be impaired by therapeutically induced autophagy and cell death is also reported which acts as tumor suppressive role (Filomeni et al. 2015). Except the mitochondria, the endoplasmic reticulum (ER) can also experience stress during pathological condition and utilize the autophagic mechanism for its proliferation during cancer. The function of ER is also regulated by membraneassociated Ca2+-dependent oxidation-reduction process. Under external stress stimulus, this balance is hampered and toxic by-products are released into the ER causing damage to the organelle. The ER then behaves abnormally by uncontrolled protein folding resulting misfolded protein response (UPR) and resulting ROS or imbalanced oxidation reduction state. The UPR is supposed to induce the apoptotic pathway to eliminate the damaged ER. During cancerous condition, UPR induces autophagy and by means of this autophagic regulation, the ER stress response is balanced and the cell gets the signal for normal cell proliferation. Thus, ROS-induced autophagy is helping the cancer cell to proliferate in this case (Cao and Kaufman 2014; Wu et al. 2018; Lin et al. 2019). There are several other cross-linking pathways which regulate ROSresponsive autophagy. Upon stress conditions, AMP (Adenosine monophosphate) accumulation occurs which leads to phosphorylation and AMPK (AMP-activated protein kinase) activation. Active AMPK activates autophagy through phosphorylation of ULK1/ATG1 or through mTOR-mediated pathway (Li et al. 2013). During collateral cancer, the activation of mitochondria-mediated apoptosis and ROS-dependent AMPK-mTOR activated autophagic cell death is observed (Sun et al. 2019). Oncogenic RAS regulates autophagy and the autophagy controls the RAS-mediated oncogenesis. Breast cancer cells, pancreatic ductal adenocarcinoma cells show RAS-induced oncogenesis which is totally dependent on autophagic induction. On the other hand, RAS-mediated tumorigenesis can be

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inhibited by autophagy-mediated cell death in bladder cancer. RAS-induced autophagy determines the pro- or anti-tumorigenic fate of the cell after stress stimulus (Schmukler et al. 2014). FAK (focal adhesion kinase) is a tyrosine kinase which is induced by several extracellular stimuli including ROS during cancer (Ben Mahdi et al. 2000). Amplified FAK production is associated with breast cancer tumorigenesis. Autophagy plays an important role in balancing the stress induced cellular transformation during breast cancer by interacting with FAK family protein. Targeting FAK familyinteracting protein and inhibition of autophagy helps in restricting this type of tumor progression (Luo and Guan 2010). Mitogen-activated protein kinase (MAPK)/p38 and JNK (an important member of this family) is an important stress-stimulated signaling pathway which is associated with the malignant transformation. Cisplatin resistance during cancer can be well explained by activated MAPK and autophagic pathway which act as cancer cell survival during osteoblastoma. In osteoblastoma, Escin-induced apoptotic cell death is observed where MAPK and autophagy are activated and act cross-regulately in the cancer prevention. Inhibitor of MAPK shows elevated apoptosis in these type of cancers. On the other hand, brucine D-induced apoptosis in lung cancer is associated with activated MAPK and autophagic pathway where inhibitor of autophagy and ROS scavengers helps in triggering the apoptosis process (Fan et al. 2020; Zhu et al. 2017; Mukherjee et al. 2017; Dhillon et al. 2007). It is clear from the above study that several ROS-induced cancer-responsive regulatory signaling pathways interacts and have regulatory role with autophagy and maintains a modulated stress balance system which determines the fate of the cell during the cancer. Now we will try to figure out how this ROS-responsive autophagy behaves during different therapeutic applications. Promising treatment strategies can be developed if we can understand the therapeutic scenario which can specifically targets this modulated autophagy-dependent ROS-responsive cellular stress management system, specifically in terms of antioxidants, which are the major player against cellular stress.

Impact of ROS-Responsive Autophagy in Chemotherapeutic Applications First of all, the ROS-autophagy interaction is well observed during “chemotherapeutic after effect” due to prolonged application of drugs against cancer. In relation to the harmful cytotoxic side effects of chemotherapy, ROS-autophagy conjoint mechanism plays major role. Various anthracycline group of anti-cancerous drugs, mainly doxorubicin (DOX), is known for showing such drug induced ROS responsive cytotoxic aftereffect. During the metabolic processing and accumulation of DOX inside mitochondria, several toxic substances is released resulting high level of ROS. High ROS leads to cytotoxicity to the normal cells and parallelly, it triggers the autophagic machinery to eliminate this toxicity. The activated lysosomal autophagy machinery eliminates important useful proteins from the cell and thus

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hampers the normal functioning of the cell leading to various deleterious effects which confers cell death. Chemotherapeutic side effects resulting myotoxicity such as cardiovascular muscle disfunction which is a very common example of such mechanism that results in sudden compelled stop of drug treatment and hindrance in cancer therapy. It has also been proved that mutation in autophagosome forming gene interaction prior DOX treatment can minimize ROS and increase proper mitochondrial metabolism. Though there has been no such promising report of drugs that can ensure DOX-induced tumor cell death and parallelly minimizing its cytotoxicity but there are research which is showing its increased effect, we just have to analyze the ROS-autophagic scenario in those conditions (Koleini and Kardami 2017; Xiao et al. 2019). On the other hand, “anti-oxidative stress” is another phenomenon that happens after application of several synthetic antioxidants in form of dietary substitutes to minimize the chemotherapeutic side effects. But antioxidants cannot differentiate between the useful and harmful free radicals of the cell and thus they function as downregulating the normal cellular antioxidative stress response utilizing the autophagy. Thus, intake of these supplementary antioxidants results in hampering the normal cellular physiological functioning which culminates into cancer. Moreover, the use of antioxidants as adjuvant therapy with the anti-cancerous drugs is well practiced to increase the therapeutic efficacy of the drugs and eliminating their toxic effect, but this might be harmful as well because the antioxidants might reduce the oxidative stress-induced cell death potential of the chemotherapeutic drugs by its anti-oxidative effect (Villanueva and Kross 2012). Chemoresistance is a phenomenon where profound role of ROS-responsive autophagy dominated mechanism has been found. For example, different autophagy-related genes like activated ATG3 has been reported to play chemoprotective role in cisplatin-resistant lung cancer. Beclin-1 mediated autophagy was found to increase in osteosarcoma chemoresistance. ATG5-induced enhanced autophagy shows doxorubicin resistance in gallbladder cancer, whereas in case of gastric cancer chemoresistance, ATG-12 mediated upregulated autophagy plays significant role. On the other hand, complete knockdown of these autophagy-related genes sometimes shows drug resistance which minimizes the cellular sensitivity of cell death process, indicating toward the intrinsic role of autophagy in against cancer protection. For example, cross-linking regulatory signaling pathway of autophagy,the p62 plays prominent role in cisplatin resistance cancer where inhibition of autophagy promotes cell death. thus it is clearly found that, both autophagic upregulation and downregulation are associated with drug resistance. On this note, it was observed that,several profound anti-cancerous drugs which are less effective or completely resistant against certain cancers when combined with autophagy modulatory ROS balancing metabolic regulators, were found more efficacious. For example, sorafenib drug resistance is minimized by combining it with chloroquine, an autophagy inhibitor. The resistance property of this drug is increased when combined with rapamycin, an autophagy upregulator (Li et al. 2019).

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Role of ROS-Responsive PI3K/AKT/mTOR Signaling During Cancer Different kinase cascades are the major regulator of the stress management system of the cell followed by ROS stimulus. Moreover, autophagy is the central determinant mechanism which balances the stress response and decides the cell’s fate. Among these kinase regulatory systems, mTOR kinase is the major and principal one. So, understanding the ROS responsive mTOR signaling in detail during cancer is important. The accumulation of ROS is comparatively higher in tumor cell than normal cell. But the stress management system cannot revive the normal cellular environment utilizing the mechanism. Moreover, cancer cell has evolved several mechanistic approaches to stabilize the ROS and sustain a redox homeostasis for its survival. Cancer cell utilize this ROS balancing mechanism for supporting the cancer process. ATK, also known as protein kinase B (PKB), is a downstream effector of the phosphoinositide 3-kinase (PI3K) signaling and interacts with different signaling molecules, thus regulate several cellular metabolic pathways, inhibit apoptosis, and activate mTOR signaling. It is evolutionarily conserved for glucose uptake and metabolism, especially through the activation of GLUT1, phosphofructokinase (PFK), and hexokinase (HK) (Fruman et al. 2017). AKT play a dual role depending on the glucose availability and the concentration of ROS for the proliferation and the survival of cancer. Under glucose deprivation, AKT induce mitochondrial oxidative phosphorylation and other metabolic pathways to maintain the growth of the cell which generate ROS as the metabolic side effect. NOX, an enzyme stimulated by AKT, which serves in the respiratory burst through the production of superoxide when electron is transferred from NADPH to oxygen and generates ROS as by-product (Chatterjee et al. 2012). PI3K/AKT also activates another rate-limiting enzyme, cyclooxygenase (COX), involved in the biosynthesis of prostaglandins (PEG2) from arachidonic acid. COX has two isoforms, COX1 and COX2 where the upregulation of COX2 is reported in several cancers. PI3K/AKT can activate COX1 and COX2 through NF-κB/IκB mediated pathway, the peroxidase activity leads to the generation of superoxide radicals (Bhattacharyya et al. 2014). Phosphatase and TENsin homolog (PTEN) is known as a potent tumor suppressor and a negative regulator of PI3K signaling pathway. Inactivation of PTEN or its mutation have been reported in many cancers. Accumulated ROS can promote the hyper activation of PI3K/AKT by facilitating posttranscriptional modification, followed by ubiquitylation and subsequent degradation of PTEN. ROS also regulates the protein, tyrosine phosphatases (PTPs), which inhibits receptor tyrosine kinases such as PDGFR and EGFR and activate PI3K/AKT signaling (Koundouros and Poulogiannis 2018). From a different aspect, PI3K/AKT signaling also repoted to prevent the adverse effect of ROS on cell viability by controlling several signaling pathways. The activation of Keap1-Nrf2 pathway and the modulation of glutathione metabolism are the two major regulatory mechanism governed by PI3K/AKT to reduce the oxidative stress. In normal condition, the transcriptional regulation of Nrf2 is

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facilitated by the Keap1. Higher concentration of ROS oxidize the cysteine residue of Keap1, allowing Nrf2 to translocate to the nucleus to induce the antioxidative gene expression, inge to detoxify ROS and improve cancer cell survival. PI3K/AKT play an essential task in the nuclear translocation of Nrf2 (Mitsuishi et al. 2012). Antioxidative pathways are relied on NADPH, which is synthesized from the enzymes involved in the pentose phosphate pathway (PPP) such as 6-phosphogluconate dehydrogenase (6PGD) and glucose-6-phosphate dehydrogenase (G6PD). Upon activation, Nrf2 induces the PPP through an AKT-dependent manner, also with the activation of malic enzyme 1 (ME1) and isocitrate dehydrogenase (IDH) for NADPH synthesis. As AKT is a potent activator of glycolysis through the GLUT1-mediated glucose uptake, PI3K/AKT activated in tumor could shunt the glycolysis product glucose-6-phosphate (G6P) to PPP induced by Nrf2, therefore sustaining a pool of NADPH, which can reduce the ROS and enhance anabolism to promote the cancer cell survival and growth. PI3K/AKT and Nrf2 with the increase of NADPH regulate glutathione synthesis, which exist between reduced (GSH) and oxidized (GSSG) form. At the higher level of ROS, GSH transferase (GSH Tr) and GSH peroxidase oxidize GSH to GSSG and reduce ROS, including H2O2 (Koundouros and Poulogiannis 2018; Lu 2009). mTOR is a downstream effector molecule of PI3K/AKT signaling pathway. It s regulates glucose and amino acid metabolism and plays important roles such as biosynthesis of lipid, protein, mitochondrial metabolism, autophagy, etc. It is a serine-threonine kinase complex, comprises two multi-protein components, mTORC1 and mTORC2. mTORC1 regulate the activation of eukaryotic initiation factors (eIFs) and elongation factors (eEFs) by phosphorylating ribosomal protein S6 kinase eukaryotic translation initiation factors 4E binding proteins (4E-BP), therefore starts protein synthesis (Zhao et al. 2017). It also inhibits autophagy by inactivating UNC51-like kinase-1 (ULK1) at ser-757. Under glucose deficiency, AKT stimulate mTORC1 either by inhibiting TSC2, allowing Rheb-GAP to phosphorylate mTORC1, or by inactivating PRAS40, which promote oxygen consumption and ROS production (Kelly et al. 2014). Study showed that activated mTORC1 can also modulate antioxidant mechanism in solid tumor to maintain redox homeostasis. mTORC1 can enhance the expression of Nrf2 in different ways, it is shown to phosphorylate serine 351 of SQSTM1/p62 (a Keap1 interacting region), thus promote degradation of Keap1 and activate Nrf2 gene expression. The transcription of Nrf2 is depended on the activity of elongation factor eIF4F. Activated mTORC1 altered the inhibitory effect of 4E-BP on eIF4E and could promote the Nrf2mediated reduction of excess ROS.

Conclusion The immune system in our body, whose activation is needed for fighting against any disease condition and disbalance of the same is also responsible for creating the disease environment. Similarly, the oxidative stress management system acts as double-edged sword which is pleotropic in nature during cancer. That means, the

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intrinsic stress balance system is needed for maintaining a proper balance of reactive oxygen species during different stress stimulus, but the same is mimicked or hijacked in a cancer cell and behaves in the same way to support the cancer by influencing several master regulatory signaling pathways. Cancer cell actually evolves an oncogenic stress management system of its own utilizing the intrinsic stress regulation. ROS is the major cellular indicator for the stress response mechanisms and autophagy is the main determinant of its balance. From the above study it is clear that, autophagy can act both as pro-survival as well as pro-death mechanism during different stress circumstances. Several interconnecting ROS alarming signaling pathways found to regulate the changing level of autophagy at different stage and

Fig. 1 ROS-responsive autophagic balance is the principal determinant of the cell’s fate

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different types of cancer. It is also found that ROS responsive PI3K is the major negative regulator of autophagy, which is targeted during cancer, to balance the ROS. Interestingly it has been found that, in the process of oncogenesis and oncogenic stress management, ROS responsive autophagy and PI3K pathways are well interactive with each other. Nrf2 regulatory signaling is the major interactive partner between PI3K/mTOR and autophagy during this oncogenic stress situation. Discussion over therapeutic applications shows that proper flexible control over both inhibition and upregulation of autophagy by targeting these regulatory standpoints in detail is needed to exert a proper cell death process depending on varied stress responses of the oncogenic cell (Fig. 1).

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Redox Regulation of Estrogen Signaling in Human Breast Cancer

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Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Estrogen and Breast Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Estrogen Actions by Oxidative Stress-Mediated Signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Estrogen Synthesis and Its Metabolizing Pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Molecular Mechanism of Estrogen Biosynthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Estrogen-Metabolizing Enzymes and Its Receptor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Estrogen Sulfotransferase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Steroid Sulfatase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Involvement of SULT1E1 in Breast Carcinogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Involvement of NFκB in Breast Carcinogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Involvement of Nrf-2 in Breast Carcinogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract

Estrogen and oxidative stress are interconnected in a very delicate fashion, leading to redox regulation of estrogen and its regulatory molecules. On the other hand, estrogen regulates the environmental oxidative stress via its intermediate metabolites. Transcription factors such as Nrf2 and nuclear factors such as NFκB intertwined with estrogen and oxidative stress. The current study shapes a possible associative pathway involving NFκB, Nrf2, SULT1E1, estrogen, and oxidative stress to hypothesize possible stimulators and inhibitors and the concerned targets, which may either prevent cancer initiation or be possible therapeutics for late-stage cancer. Cancer cells possess stem cell property, which helps in disease recurrence and tumor resistance. G6PD and HIF-1α A. Nazmeen · S. Maiti (*) Department of Biochemistry and Biotechnology, Cell & Molecular Therapeutics Lab, Oriental Institute of Science and Technology, Midnapore, West Bengal, India © Springer Nature Singapore Pte Ltd. 2022 S. Chakraborti et al. (eds.), Handbook of Oxidative Stress in Cancer: Mechanistic Aspects, https://doi.org/10.1007/978-981-15-9411-3_85

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were upregulated in MCF-7 and MDA-MB-231 cells by Nrf2 overexpression. HIF-1α coordinates the metabolic adaptation in hypoxic cancer cells. Nrf2 may upregulate GCLC and GSH, which decreases intracellular ROS, leading to a strong reducing environment, which may upregulate nuclear FoxO3a and its binding to the Bmi-1 promoter. Nuclear FoxO3a-mediated transcription is responsible for the self-renewal activity of breast cancer stemlike cells. Our earlier study conferred that estrogen partially regulates MMP and metastasis along with simultaneous regulations of NFκβ, SULT1E1, and Nrf2 via oxidative stress under the influence of estradiol. Overall, these activities of estrogen under redox regulation may contribute to the severity of human breast carcinogenesis. Keywords

Breast cancer · Estrogen · Sulfotransferase · Oxidative stress · Nrf2 · NFκβ

Introduction Stress is part of the daily life of a human. Stress are generated and exerted in one of many forms such as oxidative stress. Steroids have a great role in cellular metabolism and physiology. Several of those like estrogens participate during early embryogenesis and organogenesis gender independently and influence in female adolescence and reproductive ages. Estrogen has been involved in different physiological process in female, and it acts as an antiaging compound. It can protect from different disease-causing factors. Estrogen deficiency has been linked to osteoporosis mechanism in both women and men. Estrogen deficiency-related bone aging is linked to reactive oxygen species (ROS). This emerging evidence also provides link between age-related changes in other organs and tissues, such as ovaries and breast tissues. Estrogen is a major molecule in the making of female physiological system. If production of estrogen fails, development of the mammary glands and ductal system is hampered or remains insufficient. Estrogen deficiency is associated with primary ovarian failure or with the development of hypogonadotropism (Sun et al. 2018). Both preneoplastic and malignant growth are promoted by estrogen via interaction with its cognate receptors, i.e., estrogen nuclear receptor alpha and beta (ERα and ERβ), at certain physiological conditions. Endocrine therapy forms a central modality in the treatment of estrogen receptor-positive breast cancer.

Estrogen and Breast Cancer Estrogen receptors conduct transcription of estrogen response elements, or other elements via non-genomic pathways, such as the activation of cellular signaling pathways including MAPK, protein kinase A and C, and calcium pathways. These

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genomic and non-genomic pathways together initiate cell signaling cascades, leading to proliferation processes (Marino et al. 2006). The ER can also directly bind to AP-1, leading to its activation. An earlier study explored a mutation on the DNA-binding domain of mouse ERα, which provoked it to interact with AP-1 coactivator, Jun (Farooq 2015). Nevertheless, estrogen activates the growth regulatory gene cyclin D1 cells, which upregulate c-Jun in MCF-7 cell line. These combine and bind AP-1 sites on DNA (Sabbah et al. 1999), leading to increased AP-1 activity without the required or expected c-Fos and c-Jun synthesis. This implies that estrogen directly activates AP-1, without having any impact on the synthesis of coactivator proteins (Farooq 2015; Sabbah et al. 1999). Estrogen also possesses roles in activating transcription factors such as NFκB and CREB (Wade and Dorsa 2003). Estrogen-mediated transactivation of several signaling molecules both at cellular and nuclear levels and their synergism control over the cellular proliferative functions.

Estrogen Actions by Oxidative Stress-Mediated Signaling The redox cycling of stilbene and catechol estrogens produces free radicals (Liehr and Roy 1990). These estrogen-induced free radicals can lead to DNA strand breakage, 8-hydroxylation of purine bases of DNA, and lipid hydroperoxidemediated DNA modification (De Bont and van Larebeke 2004). Both ROS and NO levels were elevated in OVCAR-3 cells treated with E2. Increased ROS production parallel increased the cell viability, indicating that estrogen can participate in cancer progression by inducing ROS (Maleki et al. 2015). An elevated level (~threefold higher) of 8-OHdG was observed in ER+ when compared with ER-malignant tissues. 8-OHdG level in ER+ MCF-7 cell line was significantly higher (~ninefold higher) than ER
MDA-MB 231 cell line (Musarrat et al. 1996). This suggests that ER-mediated pathway, that is, the nuclear regulations, has some more implications in E2-mediated carcinogenesis. ROS-mediated DNA damages are reported. Abnormal DNA metabolite products are the good markers of oxidative stress at molecular level. Higher amount of 8-OHdG was found in the DNA of early-stage cancer tissue as compared to the late-stage cancer tissue. 8-OHdG being a product of oxidative stress, ROS thus plays a vital role in the initial phase of carcinogenesis (Yamamoto et al. 1996). Abnormal estrogen metabolism and catechol derivatives are the one of the important sources of oxidative stress. The study revealing the ER-dependent growth-inducing function of estrogen infers that ROS plays a critical role in mediating estrogen signaling. Estrogen-induced mitochondrial ROS, which act as signal-transducing messengers, are responsible for growth and proliferation. Positive energy balance is the prime requirement for cell growth and proliferation. A higher rate of energy production via ETC induction may increase ROS production. Estrogen-regulated ROS signaling is the result of higher energy balance, thus promoting cellular growth. Further explorations and analyses are required to reveal more about estrogen-induced mitochondrial ROS formation (Felty et al. 2005).

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Estrogen Synthesis and Its Metabolizing Pathways Production of estrogen is systemically and tightly regulated by the hypothalamicpituitary-ovarian hormone through a feedback regulation. Neurons in the hypothalamus, at first, sense the serum levels of estrogens and control it. Nuclear estrogen receptors (ERs) get activated, and negative feedback regulation is triggered, when the serum estrogens are detected to be above normal physiological levels. Gonadotropin-releasing hormone (GnRH) secreted by the hypothalamus induces anterior pituitary to produce gonadotropins, such as luteinizing hormone (LH) and follicle-stimulating hormone (FSH) (Fig. 1). LH stimulates ovarian theca cells and granulosa cells, while FSH stimulates granulosa cells to synthesize steroid hormones. ERα mediates the suppression of hypothalamic GnRH production, when serum estrogen level goes beyond the physiological threshold levels, thereby decreasing the production of gonadotropin by the pituitary. If this negative feedback regulation pathway is disturbed, it may result in endocrine disorders or abnormal proliferation (Kato et al. 2020). And this has been discussed elaborately in the rest of the chapter. Disruptions of the metabolic pathways, abnormal production, and uneven distribution of estrogen in different tissues result in aberrant signaling functions of this molecule.

Fig. 1 Regulation of E2 by SULTE1 and ST under oxidative stress

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Molecular Mechanism of Estrogen Biosynthesis Estrogen biosynthesis is mainly conducted by three enzymes in human, i.e., cytochrome P450 aromatase, 17b-hydroxysteroid dehydrogenase type 1, and steroid sulfatase. An effective way to control estrogen is the selective inhibition of these three enzymes in breast tumors to inhibit cell proliferation. In contrary, imbalance of the functions among these three enzymes creates estrogen to remain higher in the female body. In certain physiological conditions, especially in postmenopausal condition, this extra E2 may perform some functions other than reproductive processes. Aromatase is an endoplasmic reticulum-based membrane-bound hemeprotein. Aromatase belongs to the cytochrome P450 family and is encoded by the CYP19 gene. Aromatase converts androgens into estrogens by aromatization. For the treatment of advanced breast cancer in postmenopausal women, a variety of potent and selective nonsteroidal aromatase inhibitors are now available, such as anastrozole, which seems to be a major benefit (Buzdar 2000). Human 17beta-hydroxysteroid dehydrogenase type 1 (17β-HSD1) is capable of converting steroid from one to another and is known to play critical roles in the synthesis of estradiol. 17β-HSD1 preferentially reduces the weak estrogen, i.e., estrone, yielding a potent estrogen 17β-estradiol by utilizing NADH or NADPH as a cofactor (Aka et al. 2012). Studies also report that 17β-HSD1 has a role in the migration of breast cancer cell, though it has some positive regulatory effect on NM23, which is an antimetastatic gene. This feature of 17β-HSD1 is correlated with its growth stimulatory effect on breast cancer cell. These studies confirm the 17β-HSD1 targeting in ER-positive breast cancer. These findings suggest multitude of directions for future research on 17β-HSD1 in breast cancer progression and target-based treatment (Jerome et al. 2014). The major determinants of E2 signaling are ER, estrogen sulfotransferase (SULT1E1), sulfatase (STS), and 17β-HSD1 along with formylglycine-generating enzyme (FGE), which regulates STS activity. Our earlier report and reviews show that oxidative stress targets ER, SULT1E1, STS, and FGE and may alter its function due to various oxidizing and reducing environmental intensities (Maiti and Nazmeen 2019). These studies conclude the need for further investigation on the critical events that connect oxidative stress and regulation of estrogen-associated molecules (Figs. 1 and 2).

Estrogen-Metabolizing Enzymes and Its Receptor Estrogen Sulfotransferase Mouse estrogen sulfotransferase (mSult1e1) was the first sulfotransferase, of which a crystal structure was solved. The important structural features of this protein include a single α/β globular protein with a five-stranded parallel β-sheet at the center, α-helical regions flanking the β-sheet, and a PSB loop which interacts with the 50 -phosphate of 30 -phosphoadenosine-50 -phosphosulfate (PAPS) (Kakuta et al.

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Fig. 2 Role of E2 alone or in combination with oxidative stress in the initiation and progression of breast cancer

1997). Further studies revealed structures of various SULTs, including hSULT1E1 (Pedersen et al. 2002; Duffel 2010). SULT1E1 is one among the family of enzymes that introduce the sulfonate (SO3
) anion from an activated donor, PAPS, to the hydroxyl acceptor of a steroid, leading to the inactivation of the hormone. SULT1E1 is encoded by a gene on chromosome 4q13.1. Several tissues show the expression of SULT1E1, including the adrenal gland, kidneys, liver, fat, muscle, and uterus. An increase in the concentration of progesterone increases its activity in the endometrium, thus elevating the level of estradiol sulfate in the secretory phase and inhibiting estradiol-mediated functions. This inactivated estradiol needs to be activated when required, and the human system has evolved other enzymes, such as steroid sulfatase, which reverse the sulfonation process, resulting in activated estradiol (Mueller et al. 2015).

Steroid Sulfatase The sulfonate group added to steroids by sulfotransferase is cleaved by steroid sulfatase. This enzyme is encoded by the sulfatase gene on chromosome Xp 22.3. This enzyme plays a key role in activating steroids from their inactive sulfated forms such as estrone sulfate and DHEA sulfate. Sulfated steroids are not membrane permeable and hydrophilic, which are transported via specific transport proteins, such as sodium-dependent anion transporter (SLC10A6) (Mueller et al. 2015).

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Receptors The biological functions of estradiol are mainly carried out by nuclear receptors, i.e., ERα and ERβ. These receptors act as ligand-dependent transcriptional factors, which bind to estrogen response elements (ERE) on the DNA with the recruitment of other protein complexes required to enhance or repress transcription. ERα and ERβ are both expressed in multiple tissues and organs, including the brain. The T lymphocytes and other cells of the immune system show high expression of ERα (Paterni et al. 2014).

Involvement of SULT1E1 in Breast Carcinogenesis Strategies, such as the avoidance of estrogen binding to ER or the inhibition of aromatase-based synthesis of estrogen or sulfatase inhibition, have been used in breast cancer therapeutics. SULT induction effectively inactivates estrogen, though this method is rarely being used against breast cancer. The superfamilies of nuclear receptors (NR) are sensors for xenobiotic and endogenous molecules and can regulate the expression of the SULT. Targeting SULT induction may become a new means of treating estrogen-dependent breast cancer. There are two different SULTs which catalyze the estrogen sulfation. Sulfotransferase 1A1 (SULT1A1) detects estrogen at a higher concentration, and SULT1E1 catalyzes sulfation at a very low estrogen concentration (Ji et al. 2015a). Expression of SULT1A1 gene is tissue specific, and normal breast epithelia expresses least or no SULT1A1. Breast tumors do express SULT1A1, but their transcriptional regulation is rarely understood. Nuclear factor I (NFI) has been identified as a transcription factor family, which regulates SULT1A1 expression. Both normal breast MCF-10A cells and breast cancer ZR-75-1 cells have shown SULT1A1 expression (Yao-Borengasser et al. 2014a). Increased aromatase was commonly found in the epithelial and stromal cells of the malignant tumor. Lymph node metastases show the expression of estrogen receptor α and stromal aromatase in an associated fashion. This study hypothesizes that decreasing the expression of aromatase and estrogen receptors α and β in lymphatic metastases lowers the local estrogen’s activity and thus makes metastatic stages hormone insensitive, contributing to the poor response to endocrine therapy that is often seen in nodal-positive tumors (Yao-Borengasser et al. 2014b). SULT1A1 polymorphism at Arg213His is one of the high risk factors for breast cancer in Asian women and postmenopausal women of all races (Gschwantler-Kaulich et al. 2011). Clinical advantages have been noticed by inhibition of intra-tumoral STS in estrogen-dependent breast cancer patients. This infers that an important primary source of intra-tumoral estrogen is estrogen sulfate reactivated by STS (Jiang et al. 2010). Inhibiting the overexpression of STS in carcinomas may theoretically or possibly inhibit the development of cancer. The effects of the STS inhibition were associated with ER-α and ER-β production, progesterone receptor A (PR-A) and B (PR-B), and CDC47

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proliferation marker. An important correlation tends to occur between SULT1E1 and ER-β, or PR-β. Receptors of estrogen and progesterone are transcription activators, which seem to control the defensive enzyme such as SULT1E1 expression (Sasano et al. 2009). Studies demonstrate that both aromatase and SULT1E1 are present in malignant epithelium and adjacent stromal fibroblasts in ductal carcinoma in situ (DCIS). Preinvasive breast cancer displays lower expression of stromal aromatase, whereas SULT1E1 expression was lower in highgrade DCIS (Poisson Paré et al. 2009). Our earlier study revealed that the increase in the serum E2 level as noted with impairment in the hepatic estrogen sulfotransferase (SULT1E1) protein expression with interfered hepatic free-thiols only in ENU-treated (potent carcinogen) and xenograft-E2 group compared to a group of animals traded with arsenic (Hudelist et al. 2008). This indicates that carcinogen or carcinogenic process targets to lower the E2 modulator enzymes, such as SULT1E1, at the initiation of cancer, and the SULT1E1 expression may increase in later stages. In further studies from our lab, we noticed that SULT1E1 expression and E2 level were increased in tumor tissue compared to their corresponding surrounding tissues (Nazmeen and Maiti 2018a). The confusion is if SULT1E1 is increased, then E2 may decrease, and this was not the actual case. Further, we found that higher malondialdehyde (MDA) nonprotein-soluble thiol (NPSH) elevated superoxide dismutase, glutathione peroxidase, and catalase activity prevails in tumor as compared to the corresponding surrounding tissue, which infers that an oxidative stress environment captivates the tumor (Nazmeen et al. 2020a). These studies led us into investigating the role of oxidative stress on SULT1E1, and we found that there exists dependence between SULT1E1 and nuclear receptor factor-2 (Nrf2), which is responsible for transcription of antioxidant and antioxidative genes, such as SOD, Cat, and GPx (Nazmeen et al. 2020b). Finally, it was uncovered that oxidative stress induces SULT1E1 via Nrf2/NFκβ cooperation in breast tumor (Fig. 2). Since SULT1E1 is induced by Nrf2, which declares an increased oxidative stress, therefore, it infers that SULT1E1 remains inactive (Fig. 1) as described by one of our earlier studies, which says that Cys at the estradiol-binding site of the SULT1E1 gets blocked due to disulfide bond formation (Maiti et al. 2007) and hence incapable of estradiol sulfation (Fig. 2). The induction of SULT1E1 action of TM208 and tamoxifen showed reduction of estrogen concentration, which contributes to the anti-breast cancer effect (Ji et al. 2015b). Hence, reduction of E2 with the induction of SULT1E1 probably needs a reducing environment or the enzyme in the reduced state.

Involvement of NFkB in Breast Carcinogenesis The nuclear factor-kappa B (NFκB), which is a pro-inflammatory transcription factor, remains activated in breast cancer. NFκB increases the magnitude of the disease progression, resulting into a high-grade or late-stage tumor phenotype by promoting breast cancer hormone independence. This study states a possible

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pathway of involving NFκB, Nrf2, and SULT1E1. Where possible stimulators and inhibitors and the concerned targets may either prevent cancer or be possible therapeutics (Wang et al. 2015) (Fig. 2). NFκB plays an important role in cancer initiation and progression. Bay-11-7082 repressed NFκB and resulted in decreased expression of CD44. The cell surface glycoprotein, such as CD44, is involved in cell adhesion, migration, and proliferation. Upregulation of CD44 is considered a marker of tumor-initiating breast cancer cells and other cancer types. Repression of CD44 via inhibition of NFκB consequently reduces cell proliferation and invasiveness in breast cancer (Smith et al. 2014). These findings provide potential therapeutic targets that may help eliminate chemo- and radiation-resistant cancer cells. Stem cells in breast cancer possess self-renewing ability and are crucial players in propagating tumor and in treatment failures. The pathways that disable or trigger NFκB are strongly involved in the breast cancer development and target different progenitor cells (Shostak and Chariot 2011). Elevated oxidative stress and NFκB expression at the protein level were noticed in the breast tumor tissue as compared to the surrounding tissue (Nazmeen et al. 2020a, b). The fusion peptide, a cellpermeable antennapedia-NBD (wild-type NEMO-binding domain), exhibits an anti-inflammatory activity in a mouse model of acute inflammation. A selective inhibitor of the IKK complex, known as NBD, blocks activation of heregulinmediated NFκB and induces apoptosis in proliferating cancer cells. This explains that the classical pathway largely contributes to tumor development (Biswas et al. 2004). The classical NFκB-activating pathway is not the key benefactor of breast cancer development. NFκB protein p52 is also expressed in breast cancer samples; this demonstrates that both classical and alternative NFκB-activating pathways play a crucial role in breast cancer development (Dejardin et al. 1995). Uncontrolled activation of NFκB results in chronic nuclear localization of the proteins p50, p52, p65, cRel, and RelB. The nuclear localization of these proteins breaks the balance between cell proliferation and death by antiapoptotic protein regulation (Karin and Lin 2002). The immortalized MCF10A cell line, derived from normal mammary epithelial cells, undergoes epithelial-mesenchymal transition (EMT) when NFκB protein p65 is overexpressed. The expression of epithelial markers such as E-cadherin and desmoplakin protein is suppressed, but mesenchymal markers such as vimentin are induced. This process may occur through the NFκB-dependent expression of two transcriptional regulators ZEB-1/ZFHX1A and ZEB-2/ZFHX1B/Smad-interacting protein (SIP1), which repress E-cadherin expression and eventually promote EMT. These studies suggest stronger involvement of NFκB on breast tumor development and progression (Chua et al. 2007). NFκB is one of the critical connections between cancer and inflammatory process. It is still unknown whether its activation is regulated by long noncoding RNAs (lncRNAs). A study identified an NFκB-interacting lncRNA (NKILA), which was upregulated by NFκB itself. NKILA masks the phosphorylation motifs of IκB and NFκB, thus inhibits their activation. Therefore, lncRNA acts as a modulator and interacts with functional domains of signaling proteins, which can potentially suppress cancer metastasis (Liu et al. 2015).

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Involvement of Nrf-2 in Breast Carcinogenesis A number of studies suggest that oxidative stress can also be utilized as a therapeutic agent to treat breast cancer. A bioactive compound isolated from Rubus fairholmianus was able to increase the intrinsic apoptotic cell death via increasing the reactive oxygen species and elevating the expression of apoptotic proteins (George and Abrahamse 2019). Thus, compounds with pro-oxidant properties can treat breast cancer. In contrast oxidative stress is already a known inducer of cancer, or it can help cancer cells thrive against stress adversities via induction of Nrf2 (Antunes and Brito 2017). The transcription factor Nrf2 is one among the leucine zipper family. Kelch-like ECH-associated protein 1 (Keap1) is the repressor of the Nrf2, which sequesters Nrf2 in the cytoplasm (Itoh et al. 1999). Keap1 led Nrf2 into proteasomal degradation pathway (Kobayashi et al. 2006). Both Nrf2 and Keap1 proteins deliver cellular response to oxidative stress and to xenobiotics (electrophilic nature) (Osburn and Kensler 2008). Nrf2 controls antioxidant enzymes, electrophilic conjugation, glutathione homeostasis, induced synthesis of reducing equivalents, proteasomal activity, etc. (Hayes and McMahon 2009). Nrf2 regulates several levels of function such as transcription, translation, and posttranslational modifications, such as phosphorylation, degradation, and translocation (Nioi and Hayes 2004). Inhibition of Nrf2 may cause cell death in oxidative stress-resistant breast cancers. A study has shown the influence of micro-ribonucleic acid (miR)-101 on the proliferation of breast cancer cell via Nrf2 signaling. The expression level of Nrf2 was significantly lowered in the cell in the miR-101 mimic. Thus, suppression of Nrf2 may theoretically reverse the tumor resistance (Yi et al. 2019). Cancer cells possess stem cell property, which helps in disease recurrence and tumor resistance. A report reveals association between Nrf2 and self-renewal of cancer stem cells. Over-activation of Nrf2 may upregulate GCLC and GSH biosynthesis, which reduces intracellular ROS accumulation, provoking the reductive stress. Nuclear FoxO3a is upregulated and its binding to the Bmi-1 promoter is enhanced. These upregulated molecules are may be responsible for the self-renewal activity of breast cancer stemlike cells as evident from growth in a xenograft mouse model (Kim et al. 2020). Nrf2 silencing through miR-181c-mediated hypoxiainducing factor α (HIF-1α) dysregulation causes inhibition of hypoxia-induced metabolic changes in glycolysis and autophagy. Taken together, targeting Nrf2/ miR-181c could be an efficient approach to offset HIF-1α-coordinated metabolic adaptation in hypoxic cancer cells (Lee et al. 2019). Both glucose-6-phosphate dehydrogenase (G6PD) and hypoxia-inducing factor 1α (HIF-1α) were upregulated in MCF-7 and MDA-MB-231 cells by Nrf2 overexpression. The expression of Notch1 was also promoted via G6PD/HIF-1α pathway. Thus, Nrf2 proves itself as a potential treatment target in breast cancer (Zhang et al. 2019). Nrf2 and BRCA1 have interactive roles; BRCA1 C-terminal (BRCT) domain cross talk with Nrf2 activates the Nrf2/antioxidant response element signaling pathway. This seems to prevent neural damage from I/R injury through Nrf2-mediated antioxidant pathway (Xu et al. 2018a). The interrelations of NFκB pathways, Nrf2, SULT1E1, and E2

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signaling, do exist and together mediate proliferative activity, but the mechanism still needs further investigation (Nazmeen et al. 2020a). In human breast cancer cell, a consistent positive correlation existed between Nrf2 or HIF1α and several key glycolytic genes. Nrf2 is capable of promoting glycolysis through coactivation of HIF1α. Eventually both Nrf2 and HIF1α are potential molecular targets for breast cancer treatment (Zhang et al. 2018). Studies have shown that Nrf2 serves as a significant regulator in chemotherapeutic resistance under hypoxia via ROS-Nrf2-GCLC-GSH pathway (Syu et al. 2016). Studies show that cancer stem cells (CSCs) sustain decreased rates of ROS by elevating the production of antioxidant enzymes and ROS-scavenging receptors, which specifically benefit the CSC longevity and chemoresistance. Brusatol, an Nrf2 inhibitor, will become a novel chemotherapeutic drug to combat refractory tumor that initiates cancer stem cell proliferation (Wu et al. 2015). Possibilities of estrogen being partially in charge of matrix metalloproteinases (MMP) and metastasis have also been conferred from one of our studies, which proposes that regulations of NFκB, SULT1E1, and Nrf2 via oxidative stress under the influence of estradiol may execute MMP function, leading to the severity of human breast carcinogenesis (Nazmeen et al. 2020a, b).

Discussion The implications to regulate ROS in breast carcinogenesis are noteworthy for cancer therapy because radio- and chemotherapeutic drugs influence tumor outcome through ROS modulation. When both ER alpha-positive and alpha-negative MCF-7 cells were treated with E2, the peroxide-metabolizing property of the cells was shown to reduce. And that is paralleled with the decrease in the cellular catalase activity and total glutathione level. Peroxide-induced DNA damage was also increased in these cells. All these effects were opposed by antiestrogens. E2 is capable of inducing an increase in sensitivity to oxidative DNA damage through an ER-mediated mechanism (Mobley and Brueggemeier 2004). Thus, E2-metabolizing protein may hold significance in the therapeutic strategies. Activation of peroxisome proliferator-activated receptor gamma (PPARγ) was required for SULT1E1-mediated downregulation of C-myc, cyclin D1, MMP-2, and MMP-9 as well as for cell apoptosis, migration, and invasion (Xu et al. 2018b). This clearly suggests the role of E2 and its metabolizing enzyme SULT1E1 regulations in cell cycle controlling and extracellular matrix architecture. Overexpression of SULT1E1 and PAPSS1 also retarded MCF-7 cell growth in vivo and in vitro by the same strategy of cell cycle arrest and induction of apoptosis. Thus, targeting SULT1E1 and PAPSS expressions might be an important approach for estrogen-associated cancers (Xu et al. 2012). Breast cancer subtypes are based on the presence or absence of steroid receptors such as ER, PR, and HER2. Other causative factors are also responsible such as BRCA1/2 mutation, estradiol (E2), carcinogenic substrates, and extrinsic or intrinsic stress. E2 plays a pivotal role in breast cancer, despite being different disease types and occurred due to different factors (Lakhani et al.

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2010). Earlier report from our lab reveals that a synergistic mechanism occurs between BRCA1 mutation and impaired estrogen signaling. Modulation of oxidative stresses by estrogen or vice versa makes only the estrogen-responsive tissues (breast) more susceptible to develop cancer in the presence of BRCA1 mutation (Nazmeen and Maiti 2018b). This suggests that interactive roles among several causative factors can make the situation more complicated. So, the therapeutic approach should be formulated, taking into account the roles of integrative factors. Reactive oxygen species (ROS) signaling is the most complex network in the physiological system, where there is no quantitative demarcation for determining the toxic or threshold ROS level. The ROS has both physiological and pathological implications. Epidermal growth factor receptor (EGFR)-associated signaling pathway can be mentioned here, in which proteins such as the nuclear factor erythroid 2-related factor 2 (Nrf2) and RAF are involved. Furthermore, the mitogen-activated protein kinases (MAPKs), phosphatidylinositol 3-kinase (PI3K), phospholipase C, and protein kinase C are affected by oxidative stress. ROS also alters the expression of the p53 suppressor gene that is a key molecule in apoptosis. ROS in tumor cells causes increased expression of the hypoxia-inducing factor (HIF-1α) (Barrera 2012). Activation of NFκB pathways is extremely involved in the process of breast cancer progression. NFκB targets a few particular progenitor cells for induction of cancer (Shostak and Chariot 2011).

Conclusion Regulation of all the above factors via oxidative stress either may favor cancer development or may hinder cancer cell growth. G-protein Rac-1 regulates all these enzymes. Ras proto-oncogene encodes G-protein; Ras and Rac proteins possess GTPase activity and produce superoxide anion as a ROS. This ROS causes distortion of cells and fibroblasts (Brown and Bicknell 2001). These studies evidently support the elevated MDA level in breast tumors as compared to their surroundings. SULT1E1, Nrf2, NFκB, and MMP exhibit cross talk that strongly leads to disease enhancement.

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ROS Impacts on Cell Cycle Checkpoint Signaling in Carcinogenesis Seyed Isaac Hashemy and Seyed Mohammad Reza Seyedi

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ROS Impacts on G1 Phase Regulators and Carcinogenesis Risk . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G1 Phase Events . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G1 Oncogenes and ROS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ROS Impacts on S Phase Signaling Proteins and Carcinogenesis Risk . . . . . . . . . . . . . . . . . . . . . . . S Phase Events . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S Tumor Suppressor Genes and ROS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S Oncogenes and ROS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ROS Impacts on G2 Phase Regulators and Carcinogenesis Risk . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G2 Phase Events . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G2 Tumor Suppressor Gene and ROS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G2 Oncogenes and ROS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ROS Impacts on M Phase Regulators and Carcinogenesis Risk . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . M Phase Events . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . M Tumor Suppressor Genes and ROS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . M Oncogene and ROS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion and Future Direction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract

There are several types of human disorders that are not as intelligent as cancers. Cancerous cells gain novel abilities for being alive, grow, and proliferate out of control during the carcinogenesis events. They have different cell cycle regulation S. I. Hashemy (*) Department of Clinical Biochemistry, Mashhad University of Medical Sciences, Mashhad, Iran Surgical Oncology Research Center, Mashhad University of Medical Sciences, Mashhad, Iran e-mail: [email protected] S. M. R. Seyedi Department of Biology, Faculty of Sciences, Ferdowsi University of Mashhad, Mashhad, Iran Department of Chemistry, Ferdowsi University of Mashhad, Mashhad, Iran © Springer Nature Singapore Pte Ltd. 2022 S. Chakraborti et al. (eds.), Handbook of Oxidative Stress in Cancer: Mechanistic Aspects, https://doi.org/10.1007/978-981-15-9411-3_86

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compared with normal cells, which makes them distinguish in cellular response against extra- and intra-based oxidative stresses. In the current chapter, we have described the cell cycle events based on both key oncogenic and tumor-suppressive regulators during the oxidative storms. Keywords

Cancerous cells · Carcinogenesis · Oxidative stress · Cell cycle regulation

Introduction Carcinogenesis is mainly based on disregulations in the cell cycle procedure, which is caused by various environmental and genetic risk factors. Depending on the cellular response, the generated reactive oxygen specious (ROS) induces oxidative stress (OS) and leads to cellular death or cancer transformation by induction DNA and protein damages. There are various types of cell cycle checkpoint proteins regulating the cells’ proliferation, which guarantees the accuracy of proliferation. The loss of cell cycle regulator proteins or their disfunctionality allows the abnormal cells to complete cell cycle steps along with not-repaired genetical mistakes. Therefore, the cells get closer to cancerous types (Parascandolo and Laukkanen 2019). In the current chapter, we are going to explain the impact of ROS on the cell cycle regulators. To clarify the protein effectors which can be influenced by excessive ROS levels and then finding their relationship with carcinogenesis induction, it is crucial first to improve our knowledge about the individual protein profile and their regulators expressing in cell cycle phases.

ROS Impacts on G1 Phase Regulators and Carcinogenesis Risk G1 Phase Events The first gap (G1) is generally considered as the cell proliferation base, which is tightly regulated in normal eukaryotic cell cycle procedure. The G1 interphase could be divided into two optional and mandatory phases which refer to the dormant G0 and mitotic G1 statuses, respectively, in the cell’s life cycle (Fig. 1). The newborn cells are not committed to continuing the proliferation cycle prior to the restriction point (RP). They could exit the cell cycle to the G0 intra-phase. Depending on the type of extrinsic factors including growth factors (mitogens), cytotoxic stresses, and various differentiators they chose one of the three sub-G0 conditions: • Quiescent: The reversible resting condition with low levels of RNA contents. • Senescent: The live/death decision condition. Since the quiescent cells lose their functionality following the stressful condition, the cells cannot come back into the G1 phase.

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Fig. 1 Cell cycle regulation under normal condition. Dash lines arrows: weaken or cancelled functions; line arrows: the induction or activation; blue shape: oncogenes; green shape: tumor suppressors, influenced factors, and functions; P circle: phosphate group

• Differentiated: The maturation/commitment condition. The cells respond to the differentiators and get their individual commitments. There is a restriction point (RP) appointing whether cells continue the G0 phase or get ready for division. Since the cells are not committed to proliferation, the presence of mitogenic factors is required to promote division before the G1 interphase restriction point. Upon the cells pass the RP, cyclin-dependent kinases (CDK) and cyclin proteins regulate the other regulatory proteins by making phosphorylation-based modifications and then initiate the preparations of cell division, which ultimately commits the cell to exit dormancy and complete the division cycle. One of the most important regulatory pathways in crossing the RP point is the retinoblastoma tumor suppressor protein (RB) interactions. RB protein interacts with the E2F family transcription factors in two hypo- and hyperphosphorylated states. In this regard, phosphorylation of the hypophosphorylated RB protein by CDK4/cyclin D

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complex activates the required genes’ transcription and thus allows crossing through the RP. The division-committed cells can continue the cycle interphase without the presence of mitogens. The amounts of CDK2/cyclin E complex gets rise at the G1 end and lead to passing G1 to S phase. There are the individual phase-depended regulators playing an exclusive role in promoting cell cycle division, which can be at the mutation risks. In this regard, we study the phase-specific tumor suppressor and oncogenes. They can be influenced by several inter- and extracellular stresses and thus change into modified undesirable isoforms, which are the main reason for normal cell transformation into cancerous type.

G1 Tumor Suppressor Genes and ROS The tumor suppressor genes are called to those that could be led to induce a cancer cell if their inhibition increases or their function be lost. In this regard, there are several types of tumor suppressor genes influencing the G1 phase suppression or even progression such as p14, p53, p21, RB, p27, p57, and TGF-β. In the following, we are going to briefly know the function of each mentioned proteins in the G1 phase of the cell cycle events in cells before and after different conditions of oxidative stresses (Figs. 2 and 3). p14 The p14 has been known to inhibit p53 degradation, which is mediated by Mdm2. Therefore, it indirectly promotes the cell cycle–suppressive activity of p53 to increase the accuracy of proliferation. The p14 as a key tumor suppressor is mainly induced under oncogenic stimulation (Weber et al. 2002). The p14 expression is induced by various oncogenic genes such as E2F and Myc. p14 expression is silenced by p53 upregulation, which is required for its accurate regulation. The question is that how p14 initiates its tumor-suppressive activity. Regarding Sergio Menendez et al. findings, the functional p14 is its stabled form. They showed that the p14 stabilization can be occurred by oxidants-mediated oligomerization (Menéndez et al. 2003). In other words, oxidative stress prolongs the p14 inhibitory impact on Mdm2 and thus increases the p53 halftime to recall the DNA repair systems. It is logical to say the chronic oxidative stress due to its mutative impacts on tumor-suppressive genes and other types of post-transcriptional modifications on p53 and p14 through attenuating the DNA repair systems and bypassing apoptotic defense pathways shifts the cellsuppressive genes activity to progressive version and thus transforms normal cells into cancerous types. p53 The p53 is known as a canonical regulation point of several pathophysiological cellular responses including cell cycle, apoptosis, and growth. The tumor-suppressive function of p53 induces either cell cycle arrest or apoptosis (Vousden and Prives 2009). In cells without any tensions due to the presence of a healthy proteasomal degradation system (Mdm2), the levels of p53 protein are at the minimum status. Upon cells meet an unexpected stressful condition such as OS, the p53 levels are

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Fig. 2 Cell cycle regulation under TAOS condition; TAOS: temporary acute oxidative stress. Dash lines arrows: weaken or cancelled functions; line arrows: the induction or activation; blue shape: oncogenes; brown shape: tumor suppressors, influenced factors, and functions; P circle: phosphate group

elevated indirectly by p14 inhibitory impact on Mdm2 (Fig. 1) to decrease the cell cycle progression rate and give the cell a survival chance if the following damages were reparable, but if they were not reparable the elevated p53 levels have to finish the cell cycle by inducing apoptosis. The OS may lead to various types of transcriptional mutations on the p53 gene sequence or post-translational modifications, which can be revealed as the lack of performance or gain of new functions. The p53 due to its conserved cysteine (-SH groups) residue is considered as a cellular redox sensor. Depending on the redox regulation the presence of several cysteine sites makes p53 as the strong zinc-binding and DNA-binding protein (Hainaut and Mann 2001). Depending on the types of OS (acute or chronic), there are two expected destinies for p53 functions: cell cycle–suppressive modifications and cell cycle–progressive modifications.

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Mdm2-Nuclear Translocation Inactivated PTEN Mdm2

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Fig. 3 Cell cycle regulation under CCOS condition; CCOS: chronic-constant oxidative stress; dash lines arrows: weaken or cancelled functions; line arrows: the induction or activation; yellow arrows: the unknown mechanism; star shape: modified protein; blue shape: oncogenes; black shape: tumor suppressors, influenced factors, and functions; P circle: phosphate group

Under acute oxidative stress, p53 upregulation exhibits the cell cycle–suppressive property to activate DNA repair systems and increase the proliferation accuracy. The p53 can also act as a cell cycle–progressive gene if the oxidative stress shifts into a chronic mood. This occurs whenever the Cys 182 and 277 of p53 forms the reversible disulfide bonds with GSH, which inhibits the p53 DNA-binding activity. On the other words, the S-glutathionylation makes p53 unable in binding to the individual DNA sequences and its expression will be arrested. The modified p53 would not be able to contribute to DNA repair signaling functions, while its ability in binding to p21 remains unchanged (Buzek et al. 2002). Therefore, the cell cycle is arrested by p21 to make enough time to compensate for the tension or to be exposed more by several OS-induced mutations in a lack of efficient DNA repair systems (Chang et al. 2000). The lack of DNA repair systems causes the second destiny (cell cycle progression) and leads to some critical mutations in tumor suppressor genes to

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enforce them to act as the tumor-progressive type by modifying their protein structures and thus interaction modulations with other oncogenic partners.

p21 The p21 as the p53 key partner arrests the G1 phase by inhibiting the CDK4/cyclin D complex. The inhibited complex is not able to phosphorylate RB protein and thus the transcription of required phase-promoter genes will significantly be suppressed. This pathway is often occurred for arriving cells in senescence or differentiated G0 phase depending on the cells’ type. However, it sometimes temporarily arrests the cell cycle after a short acute oxidative stress to let them come back in a healthy mood. On the other hand, it is considered as the effective mitochondrial gene regulator (Chang et al. 2000), which is significantly associated with mitochondrial depolarization and thus apoptosis induction under temporary acute oxidative stress (Masgras et al. 2012). Under chronic oxidative stress the induction of cytoplasmic expression of p21 acts as the protective protein against the OS by suppressing ER-dependent death pathways in mitochondria (Vitiello et al. 2009). Moreover, Rawad Hodeify et al. indicated p21 phosphorylation at serine 78 reduces its CDK inhibitory activity under chronic oxidative stress (Hodeify et al. 2011). Therefore, p21 can indirectly neutralize the OS distractive impacts on DNA replication and fix the cell cycle suspension problems under chronic oxidative stress conditions. It is rational to conclude that p21 activity will be changed from cell cycle inhibitory to cell cycle acceleratory. However, further cell signaling studies are required to verify its new functions under chronic oxidative stress conditions. p27 and p57 The p27 inhibits cyclin (E, A, B)-CDK(1,2) activity as well as p57 does and thus induces cell cycle arrest in cells. It is a nuclear protein in quiescent cells, while it can be transferred to the cytoplasm in response to proliferating signals. The p57 has been found as a multifunctional protein, which not only regulates cell cycle events but also involves in differentiation, development, apoptosis, and migration. The p57 and p27 are required for G1-S and G2-M transitions and have a key role in cell differentiation and proliferation. The p27 protein levels are increased in the quiescent G0 phase and suddenly decrease after mitogen stimulation. Its constitutive expression condemns cells to be arrested in the G1 phase (Toyoshima and Hunter 1994). On the other hand, p57 has a wide range of paradoxical activities such as tumor-suppressive and tumor-progressive roles (Besson et al. 2008). Therefore, exposing these proteins with ROS in intracellular oxidative stress can effectively affect the cell cycle regulation and even cause the induction of carcinogenesis. In this regard, p27 and p57 have been shown to be affected by oxidative stress. Irene L. Ibañez et al. showed that H2O2 scavenging (oxidative stress preventing) prevents the p27 nuclear exportation, while excessive H2O2 (chronic-constant OS) induces p27 phosphorylation at serine 10 and tyrosine 198, which increases its nuclear exportation leading to lacking its cell-suppressive task. It should be mentioned that cytoplasmic P27 mediates apoptosis inhibition, whereas its nuclear exportation prevention is required for arresting cell proliferation (Ibanez et al.

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2012). On the other hand, oxidative stress initially activates p38, which has a mission to phosphorylate p57 at T143 and thus enhance its cyclin-CDK inhibitory activity (Joaquin et al. 2012). Therefore, temporary oxidative stress arrests the cell cycle by enhancing the p57 cyclin-CDK inhibitory activity. However, p57 can bind via its QT domain (aa 238–316) to apoptotic kinase JNK1/SAPK complex (activated by stress) and thus inhibits its kinase activity (Besson et al. 2008). This can exhibit the p57 anti-apoptotic activity under chronic-constant oxidative stress.

RB There are various types of intracellular and extracellular stresses leading to cell cycle checkpoints activation such as deprivation of growth factor, nucleotide depletion, and DNA damage. Retinoblastoma (RB) as a key central tumor suppressor enforces the others in response to the mentioned stresses. Its role in S-phase entrance, E2Fresponsive genes activation, and apoptosis has been well studied. RB activation (dephosphorylation) in response to DNA damage and induction of cell cycle arrest are well studied (Almasan et al. 1995). We know that the tumor-suppressive activity of RB is quenched in the majority of various types of human cancers either through gene modifications or deregulation of INK4A family (CDK inhibitors) and D-type cyclins (Weinberg 1995). Phosphorylation of Rb by CDKs leads to Rb inactivation and thus cell cycle promotion. On the other hand, under temporary acute oxidative stress, p38 kinase is activated, which leads to CDK-independent phosphorylation of Rb protein at serine 567. This causes the E2F1-Rb disruption and initiating the E2F1-mediated apoptosis (Delston et al. 2011, Chen et al. 2010). According to cells’ response procedure in temporary acute oxidative stress (TAOS) and Rb-mediated induction of the anti-apoptotic activity of BAG-1 protein, it is supposed that Rb upregulation (CCOS) can neutralize the E2F1-mediated apoptosis in chronic-constant oxidative stress. However, further experimental studies are required to clarify the CCOS-mediated anti-apoptotic role of Rb. TGF-β Transforming growth factor-beta (TGF-beta) is considered as the cell cycle progression inhibitor. TGF-β blocks the activation of G1 cyclin-dependent kinases and thus inhibits the Rb phosphorylation, which ultimately prevents G1-S transition. TGF-β normal regulation guarantees faultless cell proliferation. This has been approved by studying its lacking or deregulation impacts in several types of cancers (Hocevar and Howe 1998). Oxidative stress increases the expression of TGF-β. This is while TGF-β can regulate ROS levels by both reducing the activity of the antioxidant defense system and enhancing their generation (Krstić et al. 2015). On the other hand, TGF-β has been postulated to play a dual role depending on the tumor stage: tumor suppressor and tumor promoter in early stages and metastatic phases, respectively (Roberts and Wakefield 2003). Briefly, under temporary acute oxidative stress, TGF-β upregulation blocks the G1-S transition. It also induces the ROS generation and pro-apoptotic genes (Bim) expression to initiate a strong apoptotic response (Schuster and Krieglstein 2002). Regarding the role of chronic-constant oxidative

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stress in increasing the growth and tumorigenic potential of breast cancer cells (Mahalingaiah and Singh 2014) and mentioned anti-apoptotic activity of TGF-β in colon cancer cells, it can be assumed that such regulations can be conducted by TGFβ under CCOS conditions. However, further complementary researches on several types of cancer cells have to be done to verify the exact TGF-β anti-apoptotic mechanisms.

G1 Oncogenes and ROS The oncogenes are called to those that could be led to induce a cancer cell if to be upregulated or mutated and gaining new functions. In this regard, there are several types of oncogenes influencing the G1 phase progression, such as Mdm-2, CDK4/ cyclin D, CDK2/ cyclin E, and E2F. In the following, we are going to briefly know the function of each mentioned proteins in the G1 phase of the cell cycle events in cells before and after different conditions of ROS-induced oxidative stresses (Figs. 2 and 3).

Mdm-2 The murine double minute 2 (Mdm-2) has an axial role in regulating the cell cycle promotion by inhibiting p53 and Rb activities. Therefore, Mdm-2 activity promotes the cell cycle procedure by inhibiting p53-mediated P21 activation and inducing E2F-mediated transcription of required G1-S transition genes (Iwakuma and Lozano 2003). Under TAOS condition Mdm-2 is enforced to be inactivated by p14, which leads to cell cycle arrest and senescent. This is while the chronic-constant oxidative stress condition may promote the Mdm-2 nuclear translocation by inducing the AKT-mediated phosphorylation of Mdm-2 at serine 183 (Chibaya et al. 2018). Interestingly, under such conditions, the AKT signaling pathway is indirectly activated by OS-mediated inactivation of phosphatase and tensin homolog (PTEN) protein, which also leads to the mitochondrial-mediated ROS generation (Leslie et al. 2003, Dolado and Nebreda 2008). CDK4/Cyclin D CDK4/cycline D complex is considered as a key G1 phase promoter oncogene, whose activity and cellular levels determine the G1 phase length and cell cycle progression, respectively (Dong et al. 2018). Regarding the positive regulation of the nuclear factor erythroid 2-related factor 2 (NRf-2) signaling pathway at the early stage of oxidative stress and its blocking impact on Cycline D(1) (Márton et al. 2018), it is supposed that cell cycle arrest is the first dominant response to TAOS condition. On the one hand, the prolonged Nrf2 activation (constant oxidative stress) has been detected in the progression of several types of cancers (Jaramillo and Zhang 2013). On the other hand, oncogenic mutations leading the enhancement of proliferative signals have the potential to convert the NRF-2 cell-protective role to a cancer promoter (Kitamura and Motohashi 2018). In conclusion, the chronic-constant oxidative stress condition can convert the NRF-2 activity by inducing

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oncogenic mutations and maybe post-transcriptional modification of NRF-2. This event suppresses the G1-phase tumor suppressor proteins and supports cell cycle progression, which causes the cancerous transformation.

CDK2/Cyclin E-A Cyclin E binds to Cdk2 and conducts the G1-S transition. The CDK2/cyclin E complex tags p27 to make it ready for degradation and promote the expression of Cyclin A for S phase entrance (Sheaff et al. 1997). Also, it phosphorylates Rb to keep on G1 progression (Weinberg 1995). It is well known that overexpression of cyclin E is associated with tumorigenesis (Keck et al. 2007). However, its activity dysregulation has been attributed to cell linage–depended abnormalities such as impaired maturation, senescence, and apoptosis (Minella et al. 2008). The CDK2/ cyclin E complex is phosphorylated by p27-mediated phosphorylation at early stages of oxidative stress and leads to cell cycle arrest (Masgras et al. 2012). On the other hand, the chronic-constant endogenous ROS generation leads to inactivate the anaphase-promoting complex (APC). The APC activity is required for ubiquitinating cyclin A and thus degrading before the G1-S transition phase. The APC inactivation allows cyclin A to be early accumulated and thus accelerate the S phase transition (Havens et al. 2006), which can approve the role of CCOS condition in cancerous cell progression.

ROS Impacts on S Phase Signaling Proteins and Carcinogenesis Risk S Phase Events The S phase of the cell cycle procedure is mainly assigned to DNA replication, which is tightly regulated (David 2007). Generally, there are three checkpoints scrutinizing the genome duplication during the cellular S phase, which are programmed to arrest or delay the cell cycle progression in response to the DNA damage detection (Bartek et al. 2004). • The replication checkpoint (RCP): Responsible for detecting the stalled replication forks. It is activated by localization of RPA, RAD17, ATR, and ATRIP proteins to intracellular foci. The activated RCP temporarily suppresses the replication and improves the nucleotide biosynthesis to fix and rescue the stalled replication forks. • The intra-S phase checkpoint (ISCP): It is programmed to detect double-strand breaks (DSBs) by activating the ATR and ATM kinases. The ISCP also facilitates the DNA repair and arrests the cell cycle progression by indirectly preventing the CDK activation (CDC25A degradation). • The S-M checkpoint (SMCP): The activated ATR-mediated SMCP is supposed to prevent mitosis until the entire genome duplication. SMCP arrests the G2-M transition by inhibiting the rapid CDK1/cyclin B accumulation to ensure the

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accuracy of replication before the mitosis entrance (Eykelenboom et al. 2013) (Fig. 1).

S Tumor Suppressor Genes and ROS There are several types of tumor suppressors regulating the S phase events such as p21, ATM, and ATR genes, which have been found to regulate cell death and survival depending on the various types of cellular stresses (Figs. 2 and 3).

p21 Interacting p21 with proliferating cell nuclear antigen (PCNA) is required for regulating DNA replication (Waga et al. 1994). The p21 inhibits DNA replication by blocking the PCNA-mediated DNA synthesis (via interaction with the PIP-box region of PCNA) and induces the PCNA-mediated nucleotide excision repair (NER) (Warbrick et al. 1997). In TAOS condition the caspase-mediated p21 cleavage inhibits its nuclear localization and PCNA interaction abilities. The cleaved p21 dramatically induces the CDK2 activation and initiates the undergoing apoptosis in growth-arrested cells via phosphorylation of Bcl-xL and its conversion to a pro-apoptotic protein (Levkau et al. 1998). On the other hand, regarding p21 phosphorylation at Thr 145 by ROS-mediated AKT1 activation and thus losing its PCNA binding activity, p21 is exported to the cytoplasm. The cytoplasmic p21 accumulation promotes cell survival via the inhibition of apoptosis-related proteins existing in the cytoplasm and induces cellular proliferation (Abbas and Dutta 2009). This may be useful in interpreting the cause of cancer cell survival against CCOS conditions. ATM and ATR Ataxia-telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3-related (ATR) are involved in DNA damage response (DDR). Also, they induce the G1-S, S phase, and G2-M cell cycle checkpoints to ensure the accuracy of DNA repair and DNA replication procedures prior to M phase entrance. In response to TAOS condition DDR induces the cell cycle arrest, DNA repair, senescence, and apoptosis by activating both checkpoint kinases Chk1 and Chk2 for subsequent ATM/ ATR upregulation. The ATM-Chk2 is primarily responsible for DNA doublestrand breaks (DSBs), whereas the ATR-Chk1 is programmed to protect the chromosomes’ replication integrity (Paull 2015; Yan et al. 2014). On the one hand, it has been found Chk2 has been downregulated in various types of human tumors, indicating its fundamental rate-limiting tumor-suppressive activity in many tissues (Bartek and Lukas 2003). On the other hand, the probably oxidative stress–mediated ATR activation leads to phosphorylate the Chk1 at serines 317 and 345 and thus getting higher intrinsic protein kinase activity (Zhao and PiwnicaWorms 2001), which may protect the integrity of chromosomes’ replication (Yan 2014) under CCOS condition in cancer cells, just as the recent research approving the effective role of ATR-Chk1 inhibitors in cancer therapy (Qiu et al. 2018). In

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conclusion, the Chk2 downregulation and Chk1 hyperactivation in the governing CCOS conditions in cancer cells have the potential to promote tumor progression in spite of the existence of distractive impacts of ROS.

S Oncogenes and ROS There are several types of oncogenes influencing the S phase progression such as CDC25A and CDK2/Cyclin A. In the following, we are going to briefly know the function of each mentioned proteins in S phase of the cell cycle events in cells in TAOS and CCOS conditions (Figs. 2 and 3).

CDC25A CDC25A a phosphatase removing the inhibitory phosphate residues from CDKs and thus activating their kinase activity is considered as the key S phase oncogene and G1-S transition protein (Shen and Huang 2012). CDC25A needs to be activated by CDK2/cyclin E-mediated phosphorylation to induce the G1-S transition. It is negatively phosphorylated in a cell cycle checkpoint-dependent manner at serine 123, which induces SCF-mediated ubiquitylation of CDC25A. Also, CDC25A is become degraded in response to DNA damage and stalled replication forks (Molinari et al. 2000), which are usually occurred in acute oxidative stresses. It has been documented that Cdc25A overexpression is significantly associated with numerous human cancers and significantly associates with cancer cell genome hyperinstability (Ray and Kiyokawa 2008). Moreover, it has been shown that CDC25A can be hyperphosphorylated by Chk2 and thus more stabled in cellular DDR regulations (Busino et al. 2004). Overall, regarding the mentioned interprets of the Chk2 status under CCOS condition and its certain role in CDC25A phosphorylation, it is rational to attribute the carcinogenesis induction into the undegradable CDC25A protein. However, further studies are required to verify the CDC25A tumor-progressive role. CDK2/Cyclin A During the S phase nuclear cyclin A is involved in the replication of DNA. Just after the S phase entrance cyclin E/CDK2 is responsible in assembling pre-replication complex, the complex that facilitates the chromatin replication. The increasing nuclear cyclin A is replaced with cyclin E and forms the CDK2/cyclin A, which terminates the pre-replication complex assembling and initiates the DNA replication (Coverley et al. 2002). CDK2 is inactivated by p21 in response to irreparable DNA damage (in TAOS condition) (Bačević et al. 2017). On the other hand, CDC25A role in removing the inhibitory phosphorylation of CDK2 has been proved, which indicates the tumorigenic impact of CDC25A in various types of cancers (Shen and Huang 2012). Moreover, the CDK2 overexpression has been suggested to be associated with cancer cells’ hyperproliferation (Chohan et al. 2015). Therefore, it is rational to introduce CDC25A-mediated phosphorylation of CDK2 as the tumorprogressive protein in CCOS condition, which significantly exists in cancer cells.

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ROS Impacts on G2 Phase Regulators and Carcinogenesis Risk G2 Phase Events During the G2 phase, the cell gets ready for initiating mitosis division by continuing growth and synthesizing the required proteins. This phase is tightly regulated by a complex genetic network. A network comprising tumor suppressors and oncogenes has been briefly described in the following sections (Fig. 1).

G2 Tumor Suppressor Gene and ROS There are several types of tumor suppressors regulating the G2 phase events. p21 is one of the most important gene regulating G2 phase progression and suppression depending on the types of cellular stresses.

p21 It has been suggested that p21 can regulate G2 arrest mediated by DNA damage induction. The DNA damage–induced upregulation of p21 causes cell cycle arrest at the G2 phase (Niculescu et al. 1998). In TAOS conditions p21 blocks Rb phosphorylation and prevents mitosis by inactivating the CDK/cyclin complexes, which then leads to cyclins degradation and permanent G2 arrest (Charrier-Savournin et al. 2004). On the other hand, as mentioned in the previous section “Mdm-2,” the cytoplasmic accumulation of AKT-mediated phosphorylated p21 suppresses the MYC- and E2F-induced pro-apoptotic genes (Dotto 2000).

G2 Oncogenes and ROS There are several types of oncogenes regulating the G2 phase progression such as CDC25C and CDK1/Cyclin B. Herein, their function in G2 phase is studied regarding both the TAOS and CCOS conditions.

CDC25C Cdc25C is considered as the G2/M phase controller phosphatase. It causes entry into mitosis by CDK1/cyclin B dephosphorylation. Upon the induction of the TAOS condition, Cdc25C is transcriptionally inhibited by p53 upregulation. Therefore, the CDK1/cyclin B levels do not reach into the required G2-M transition threshold, and the G2 phase will be arrested prior to mitosis entrance. Moreover, the Cdc25C stability is decreased by Mdm2-mediated ubiquitination procedure and thus induces the G2 phase delay (Giono et al. 2017). In cancer cells the CCOS may lead to CDC25C phosphorylation at serine 216 and nuclear exportation, which induces dephosphorylation of apoptosis signal–regulating kinase 1 (ASK1) and thus its suppression. Therefore, the overexpression of CDC25C inhibits the ASK1-mediated apoptosis (Wang et al. 2010; Cho et al. 2015).

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CDK1/Cyclin B CDK1/cyclin B, a master mitotic regulator, is dephosphorylated at Tyr 15 and thus activated by CDC25C at the end of S phase. The activated complex is transferred into the nucleus to be triggered for the G2-M transition. Whenever DNA damage occurs (TAOS condition) the CDK1/cyclin B activation will be lost due to the CDC25C inhibitory phosphorylation. Therefore, the CDK1/cyclin B nuclear accumulation is suppressed and thus G2-M transition is failed (Huang et al. 2013). In cancer cells, which are utilizing various types of survival strategies against the governing cellular CCOS conditions, there are several types of oncogenic protein deregulation such as cyclin B and CDC25C. As mentioned in the previous section “CDC25A” and regarding the carcinogenic impact of the elevated cyclin B expression in various types of cancers (Ye et al. 2017), the CDC25C-mediated cyclin B dephosphorylation keeps it active. Therefore, the G2-M transition prematurely occurs in spite of several unrepaired DNA damages.

ROS Impacts on M Phase Regulators and Carcinogenesis Risk M Phase Events During M phase the nucleus is divided, which is called karyokinesis. There are quintet phases promoting the M phase events, which are sequentially known as: • Prophase: The chromatin condensation and nucleolus disappearance • Prometaphase: The nuclear membrane degradation and kinetochore-microtubules attachment and development • Metaphase: Lining up chromosome along metaphase stage • Anaphase: Chromosome breaking at centromere sites and sister chromatid migration • Telophase: Nucleus membrane formation and chromatin reforming There is a key critical checkpoint in the M phase called the spindle assembly checkpoint (SAC). Also, it is known as the metaphase–anaphase checkpoint, which determines the accuracy of all sister chromatids attachments to the spindle microtubules. In the other word, SAC prevents the onset of anaphase until the correct attachment of chromosomes to spindle (Lara-Gonzalez et al. 2012). In the following we describe the role of tumor suppressors and oncogenes in regulating the M phase progression (Fig. 1).

M Tumor Suppressor Genes and ROS There are several types of tumor suppressors regulating the M phase events. The p53 and anaphase-promoting complex (APC/C) is one of the most important genes regulating M phase suppression through the SAC checkpoint activation under different types of stressful conditions (Figs. 2 and 3).

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p53 The p53 has been known to prevent endoreduplication by cooperating with an intact SAC checkpoint upon mitotic failure. It is activated during SAC-mediated mitotic arrest, which approves the G1 checkpoint functionality. SAC checkpoint complex contains several types of proteins such as CDC20 and cyclin B, which are tightly regulated during M phase. The cells in nonefficient SAC functionality status have insufficient shortened mitotic arrest, resulting in subsequent polyploidy. Moreover, in TAOS condition the delayed or abnormal formation of the mitotic spindle is responsible for ROS-induced mitotic arrest (Vogel et al. 2004), which is necessary to induce apoptosis in order to avoid genome instability. In this regard, it can be interpreted that the oxidative stress–mediated abnormal formation of the mitotic spindle may induce the spindle checkpoint protein (Bub1) interaction with p53 to initiate p53-dependent apoptosis in metaphase arrested cells (Beeharry and Yen 2009). This is while in cancer cells (CCOS condition) the inactivating p53 mutations are common (Baker et al. 1990), which may enhance the apoptotic induction threshold during SAC-mediated mitotic arrest and promote mitosis in spite of chromosomal instability existence. APC/C APC/C, the ubiquitin-protein ligase, manages the metaphase–anaphase transition utilizing a network of regulatory partners such as cell division cycle protein (CDC20), cadherin 1 (CDH1), and MAD2L1. It is activated by CDC20/CDH1 and inhibited by MADL1. During the SAC activation MADL1 prevents the APC/C activation by forming the mitotic checkpoint complex (MCC) consisting of a ternary complex with CDC20 and BubR1. Therefore, the cell is arrested at prometaphase (Fang et al. 1999). It has been showed that under TAOS condition the OS-induced unattached kinetochore activates the MAD2L1 nuclear translocation and enrichment and thus promotes SAC activation (Waters et al. 1998), which mediates mitosis arrest. This is while under the dominant CCOS condition in various types of cancer cells, the upregulated CDC20-mediated APC/C activation overcomes the MADL1mediated APC/C inactivation and thus leads to chromosomal instability, the cancer hallmark (Kim et al. 2014; Chang et al. 2012).

M Oncogene and ROS There are several types of oncogenes regulating the M phase progression such as CDC20. Herein, its function in the M phase is studied regarding both the TAOS and CCOS conditions (Figs. 2 and 3).

CDC20 During the metaphase–anaphase transition, CDC20 is required for the intact SAC activation in preventing the reduplication failures caused at the TAOS condition (Fang et al. 1998). There are various types of cancer cells which have been detected to exhibit CDC20 upregulation (Kim et al. 2014; Chang et al. 2012), which possibly could be attributed to their dominant cellular CCOS condition.

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Conclusion and Future Direction In the current chapter we have just transiently indicated maybe the most known cell cycle regulatory factors and tried to organize them as the presented model. There are several types of phase-depended tumor suppressors and oncogenes that mediate different responses to intra and/or extracellular tensions (mainly oxidative stress) during the cell cycle progression, which are tightly being regulated. Different molecular pathway functions in response to various cellular tensions such as degrees of oxidative stresses have potential to be classified in more detail. The presented classification of the cell cycle regulators based on their response to different oxidative stress conditions can reveal the hopeful horizons in targeted cancer therapy strategies. However, further complementary and challenging data are still required to precisely organize the cell cycle regulation network in details.

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Olorunfemi R. Molehin, Olusola O. Elekofehinti, Ajibade O. Oyeyemi, Oluwatosin B. Olusakin, Aderonke E. Fakayode, Ezekiel T. Ige, and Oluwatumininu O. Adesua

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pathophysiology of Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Phytochemicals and Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . JAK/STAT Signaling Pathway in Oncogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Regulation of JAK/STAT Signaling In Vitro by Phytochemicals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Inhibition of JAK/STAT Signaling in Animal Models by Phytochemicals . . . . . . . . . . . . . . . . . . . The Interaction of Phytochemicals Regulating JAK/STAT with Chemotherapeutic Drugs . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract

Cancer remains a global major health burden. Activation of certain signaling pathways such as Janus kinase (JAK)/signal transducer and activator of transcription O. R. Molehin (*) · O. O. Adesua Department of Biochemistry, Ekiti State University, Ado-Ekiti, Nigeria e-mail: [email protected] O. O. Elekofehinti Bioinformatics and Molecular Biology Unit, Department of Biochemistry, Federal University of Technology Akure, Akure, Nigeria A. O. Oyeyemi Department of Biochemistry, Faculty of Science, Ekiti State University, Ado-Ekiti, Nigeria O. B. Olusakin Na Moda Naturals, Federal Housing Estate, Ibadan, Nigeria A. E. Fakayode Department of Biochemistry and Molecular Biology, Faculty of Science, Obafemi Awolowo University, Ile-Ife, Nigeria E. T. Ige Department of Pharmacology and Therapeutics, College of Medicine, Ekiti State University, Ado-Ekiti, Nigeria © Springer Nature Singapore Pte Ltd. 2022 S. Chakraborti et al. (eds.), Handbook of Oxidative Stress in Cancer: Mechanistic Aspects, https://doi.org/10.1007/978-981-15-9411-3_88

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(STAT) is found to be an important regulatory defect present in most cancerous cells amongst other factors. The signaling of Cytokine causes the activation of “STAT proteins” in regulating the proliferation and differentiation of cells, more so its role in the modulation of targeted genes cannot be over emphasized. Phytochemicals play significant role in the inhibition of the JAK/STAT signaling in antigen-presenting cells by targeting different pathways. In this review, Resveratrol, cucurbitacin, curcumin, Epigallocatechin-3-gallate and others are promising phytochemicals discussed. Extensive studies have now shown that phytochemicals perform crucial functions in inhibiting the multiplication and proliferation of cancerous cell thus providing novel channels for targeting cancer therapeutically. Keywords

Cancer · Chemoprevention · JAK/STAT · Phytochemicals · Epigenentics

Introduction Cancer still remains the frontline cause of death globally, with a statistics of 8.2 million deaths in only the year 2012 (Ferlay et al. 2013). Reports from GLOBOCAN 2012 reveal that “32.6 million people go about their daily life, existing with cancer in 2012,” and fresh cancer diagnosed cases of 14.1 million people were reported in the same year (Ferlay et al. 2013; Jemal et al. 2011). Lung cancer, among several cases, dominates the headlines in male patients with about 16.7% of the entire novel tumor cases and 23.6% of the entire novel tumor demises in 2012.” In contrast, in women, breast cancer accounts for about “25.2% of the new cancer cases and 14.7% of the cancer deaths in 2012” (Ferlay et al. 2013). It is projected that the global health concern of cancer is expected to be twice this current amount in 2030, reaching a new heights of “21.3 million new cancer cases and 13.1 million cancer deaths. Recent advances in sciences and present experimental studies have resulted to massive advancement in cancer therapeutics despite the significant increase in “cancer” incidence and death rates. The easily known types of cancer treatment include: immunotherapy, hormonal therapy, chemotherapy, and targeted therapy (Siegel et al. 2012; Zhang 2012). Chemotherapy comes with its attendant adverse side effects on nontargeted normal cells in the body though it has played a crucial therapeutic role in killing cancer cells (Zhang 2012; Ahles and Saykin 2007). The use of compounds from natural sources with potentials for anticancer activities in the deployment of “cancer prevention and therapy” will be a therapeutic approach producing a desired effect of decreasing the rate and number of cancer occurrences as well as “mortality in times ahead.

Pathophysiology of Cancer Cancer is characterized by irregularities in cell proliferation, invasion, as well as its potential to enlarge in auxiliary parts of the body. Research has reported diversity within each part to be responsible for the complication of “single-targeted

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therapeutics” thus resulting in cancer recurrence as it is a multi-targeted pathway. The formation of tumor is initiated as a result of chemicals that are agents that cause cancer, and these chemicals can either cause damage to the genetic information present in a cell resulting in mutation or not pose any damage to the genetic information present in a cell (Arumuggam et al. 2015; Hayashi 1992). Genotoxic carcinogens damage DNA directly by causing miss-aggregation or union of DNA, breakage of chromosome, DNA deletion, and addition of compounds to DNA (Luch 2005) resulting in mutation and possibly growth of tumors if not repaired. On the other hand, the cancer-causing chemical agents that pose no threat to the genetic information of a cell, do not interact directly with DNA, does that through processes such as suppression of the immune response, which may be complete or partial, reddening and swelling of the cell, ROS formation, or regulation of expression of genes by modifying DNA, RNA, or histone proteins (Luch 2005). “These cancer-causing chemical compounds” that can either cause mutation of genetic information or not can alter the signal transduction pathway accelerating the development process of cancerous cells (Fig. 1). Cancer is a disease condition that involves the regulation of tissue growth. The transformation of normal cell to a cancerous cell involves the alteration or mutation in the genes that are responsible for cell differentiation or growth (Pitot et al. 1981). These genes are classified into two broad categories, namely “Oncogenes” which are responsible for cell growth and reproduction and tumor suppressor genes which prevent the occurrence of cell division thus inhibiting the cell survival. Transformation of normal cells to malignant cells could be as a result of over expression of oncogenic genes or deactivation of tumor suppressor gene, however, alterations in multiple genes are responsible for the formation of cancerous cells (Vlahopoulos et al. 2015). The multistep process of carcinogenesis can be broadly classified into the initiation of the cancerous growth, its development, and its metamorphosis and advancement. The crucial step involved in the inception of a malignant cell is an unrepairable

Carcinogen exposure PROMOTION

INITIATION

Cell Proliferation

DNA repair Normal cells

Cells with adducts

Cell Proliferation Initiated cells PROGRESSION

Cell Proliferation

APOPTOSIS

CANCER

Fig. 1 Stages involve in carcinogenesis. (Oliveira et al. 2007)

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genotoxicity by cancer-causing chemical compounds that act directly on the DNA (Pitot et al. 1981). An inadequate rectification of the genotoxicity might cause alterations in either gene responsible for protecting cells on the path to being cancerous or normal gene responsible for the regulation of cell growth and differentiation, converting these genes into oncogenes. The ratio of proto-oncogenes to oncogenes is a key determinant of one of the initial phases involving a solid cell turning into a malignant cell. Proto-oncogenes are essential codes that are imperative in a typical cell development. Oncogene is formed as a result of a defective protooncogene and this oncogene promotes abnormal cell proliferation. Tumor suppressor protein that has been mutated also avoids its physiological ability, hence facilitates the growth of tumors. The second stage of the carcinogenic process involves the formation of pre-neoplastic (precancerous) lesion (Arumuggam et al. 2015; Pitot et al. 1981). This is done by “non-genotoxic” “carcinogens,” and its effects are reversible unlike genotoxic chemicals (Luch 2005). Prolonged exposure may result in permanent damage, and this occurs only after a cell has experienced an initiating mutation (Arumuggam et al. 2015; Luch 2005). The next phase involves the transformation of preneoplastic lesion into a malignant tumor; this process is termed as malignant conversion. In the fourth phase which is the progressive phase, the cancerous growth develops to an increased assaulting phase and the cancerous cells begin to go through the phase of metastasis, moving into nearby tissues, while spreading to distant secondary sites (Arumuggam et al. 2015; Pitot et al. 1981). In the series of events required for the development of a tumor, it has been reported that anomalous cells possess some general characteristics which defines the complication of a cancerous growth. These general characteristics (Arumuggam et al. 2015; Vlahopoulos et al. 2015) include: independent amplification of cells, unaffected by growth constraints, defiance to apoptosis, unlimited reproduction, the formation of new blood vessels from already existing vessels, invasion of neighbouring tissue and the spread of cancer cells into tissues and organs at a different site other than the initial location, mutations of the genetic information of a cell, swelling and redness of the cell as a result of cancerous growth, metabolic changes associated with cancerous cells, and subterfuge of the recognition and destruction of cancerous cells, are the safe guarding mechanisms escaped by anomalous cells in the formation process of cancerous growth’ and its transformation into a malignant cell.

Phytochemicals and Cancer Research has proven that the occurrence of cancer is via a cascade of reactions caused by disturbance of several signaling pathways. Lots of anticancer drugs that have emerged over the years have been known to modulate only single targets, targeting only one of these multiple pathways and thus limiting their efficiency in cancer management, making it almost impossible to achieve disease control. These anticancer drugs have generated a lot of concern because of their cytotoxicity and costly prices. The limitations of these available anticancer drugs have led to the quest

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of identification of pharmacological agents that can modulate multiple targets, innocuous yet inexpensive and handy for cancer therapy and prevention. The use of natural products from plants as a therapeutic tool has been of considerable increase with research establishing a strong connection between consumption of some fruits, vegetables, and certain spices to cancer risk reduction (Hanahan and Weinberg 2011). A vast variety of phytochemicals found in foods and medicinal plants have demonstrated preventive or protective effects against the tumor in different organs of experimental animals as well as arresting growth of neoplastic cells (Hanahan and Weinberg 2011). Some phytochemicals have been reported by research to demonstrate its ability to decline cancerous growth in animals by preventing the increased expression of the differentiation and replication of cells, anti-programmed cell death, and the formation of new blood vessels from existing ones, and this is done by interfering with the molecular signal transduction pathways of “tumor cells that answer to various information outside the cell (Arumuggam et al. 2015). These phytochemicals act as either preventing agents inhibiting the step of “tumor initiation through stopping carcinogen activation, or suppressing agents restraining tumor mobile proliferation for the duration of the “promotion” and metastasis stages of tumorigenesis either by inciting or declining specific cellular “anti-inflammatory” events and the related molecular signaling pathways (Loveth et al. 2017; Nandakumar et al. 2008). It has also been reported that phytochemicals can be used for prevention and treatment of cancers due to their antioxidant activity, anti-inflammatory activity, ability to induct Phase II enzyme, their significant role in cell cycle arrest, ability to induce apoptosis and autophagy, and their significant role shaping the composition and function of the gut microbiota (Zhao et al. 2018).

JAK/STAT Signaling Pathway in Oncogenesis The discovery of the “JAK/STAT” was brought about by the study of the linkage of interferon-responsive genes to signal transduction (Zhao et al. 2018; Pencik et al. 2016; Wilks et al. 1991). “JAK/STAT pathway is one of the crucial ‘signaling pathway’ that have been reported to be involved in ‘inflammation’ and ‘cancer’. ‘Signal Transducers and Activators of Transcription’ ‘(STAT)’ are ‘transcription factors’ that play a key role in ‘normal physiological functions’ such as ‘cell proliferation’, ‘apoptosis’ and differentiation (Bousoik and Montazeri Aliabadi 2018). The ‘STAT proteins’ are a group of dormant ‘transcription factors’ present in the ‘cytoplasm’, which becomes ‘activated’ by ‘JAKs’ through ‘phosphorylation’ of tyrosine residues” (Arumuggam et al. 2015; Darnell et al. 1994). The JAK/STAT pathway transmits messages from “chemical signals” present at the external part of a cell to a cell nucleus, leading to the “activation of genes” through a series of events known as “transcription” (Aaronson and Horvath 2002). JAK-STAT pathway is primarily associated with cytokine signaling, such as the ‘signaling’ incited by ‘erythropoietin’, ‘thrombopoietin’, ‘interferons’, ‘interleukins’, and ‘granulocyte-colony- stimulating factors’. ‘JAKs’ ‘interact’ firstly with ‘cytokine receptors’ in the latent form [23]. On ligand binding to the ‘corresponding

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cytokine receptor’, ‘JAK’ ‘transphosphorylation’ and ‘activation’ are ‘induced’. Activated JAKs incite a conformational change in the cognate cytokine receptor to create a ‘STAT docking site’ through the ‘SH2 domain’, thereby enlisting ‘STAT family members’ to the ‘JAK-cytokine receptor complex’. After enlistment, the ‘STATs’ become active and form ‘homo’- or ‘heterodimers’ [23].” Once the dimeric form is in place, STATs move to the nucleus and bind the promoter of target genes that are involved in various cell processes described above (Luo et al. 2019; O’Shea et al. 2015; Thomas et al. 2015). The JAK family is a four-membered group that includes “JAK1, JAK2, JAK3, and TYK2, each of which contains ‘seven conserved JAK homology domains’ (i.e., ‘JH1–7’), the ‘N-terminus of JAKs’ (‘JH5–7’) constitutes a 4.1 protein, ezrin, radixin, moesin” (FERM) domain which functions in the association between JAKs and cytokine receptors as well as other kinases (Pencik et al. 2016; Luo et al. 2019). The STAT family comprises of seven members: STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B, and STAT6. Some regulators of the “JAK-STAT pathway” that restrain “signaling” at “multiple levels” have been reported and they include “cytokine signaling (SOCS) suppressors” which contend with STAT binding to the cytokine receptor; tyrosine phosphatases (SHP1 and SHP2) which dephosphorylate JAKs; and the family of “protein inhibitors” of “activated STATs” (“PIAS”) which gets involved with STAT binding to DNA. Negative regulators of the JAK-STAT pathway turn off the signaling cascade, thereby hindering the JAK-STAT pathway and regulating amplitude and a brief control of pathway signaling (Pencik et al. 2016; Luo et al. 2019; Thomas et al. 2015). Their significant role in cell proliferation, apoptosis, and differentiation suggests that “JAK/STAT signaling axis” is crucial in the proliferation and survival of a range of “cancer cells,” and it may even play a role in the “resistance mechanisms” against “molecularly targeted drugs” (Bousoik and Montazeri Aliabadi 2018). This is usually as a result of aberrant regulation of the pathway. “JAKSTAT signaling pathway” plays a significant function in the “transcription” of “genes” involved in cell division, during the series of events associated with carcinogenesis, a range of pathological processes result in the established kindling of the Janus Kinase/Signal Transducers and Activators of Transcription signaling pathway (Fig. 2). Studies have earlier shown that tumors growing at the original site where it first arose and established and proliferating cells deduced from cancerous growth point out an anomalous “kindling” of distinct “STAT proteins” at increased rate of occurrence in most reported cancer cases in man (Horvath 2000). “JAK/STAT signaling” may be downregulated through aberrant expression of signal transducer and “activator of transcription 3” (“STAT3”). Increased levels of “STAT activation” have specifically been reported to be linked to some dangerous tumors such as melanoma (skin cancer) and prostate cancer (Thomas et al. 2015; Messina et al. 2008). Dysfunction in JAK-STAT signaling has been implicated in developing breast cancer, and research has proved that “JAK-STAT signalling” in glands located within

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Fig. 2 The JAK/STAT signaling pathway and its regulation (Seif et al. 2017). (a) The Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway. JAKs are activated upon cytokine stimulation and phosphorylate STATs which results in dimerization and translocation of STATs to the nucleus in order to activate or suppress the transcription of genes. (b) Regulation of the JAK–STAT pathway. A schematic of negative regulators that modulate the JAK-STAT pathway. Negative regulators consist of suppressor of cytokine signaling (SOCS) proteins, Protein Inhibitors of Activated STATs (PIAS), protein tyrosine phosphatases (PTPs), such as SRC homology 2 (SH2)domain-containing PTP1 (SHP1), SHP2, CD45, LNK, T-cell PTP (TCPTP), and protein-tyrosine phosphatase 1B (PTP1B)

the breasts can encourage cell division and decrease cell apoptosis during pregnancy and puberty, resulting in cancer formation upon excessive activation” (Groner and von Manstein 2017). “JAKs commonly interact usually with tyrosine receptors and remain in a latent state until ‘ligand binding’ happens. The abnormal ‘activation’ of ‘JAK-STAT pathway’ as a result of mutations in genetic materials leads to constant ‘activation’ of ‘JAKs’ in the absence of ‘cytokine signaling’, resulting in tumorigenesis or oncogenic activity” (Darnell et al. 1994).

Regulation of JAK/STAT Signaling In Vitro by Phytochemicals JAK/STAT pathway is a multi-targeted pathway thus might be marked by phytochemicals or other ‘drugs used for therapeutic purposes. Three possible targets by which naturally occurring chemical compounds present in plants hinder the ‘JAK/STAT pathway’ has been reported; by reducing the expression of JAK/ STAT proteins activators which also hinders JAK phosphorylation and the upstream of STAT and these activators include cytokines or growth hormones; hindering the dissociation of STAT from the receptor complex and its translocation to the nucleus; suppressing the binding of STAT to DNA and this binding

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plays a crucial role in the genes that mediate the transcription of JAK/STAT” (Arumuggam et al. 2015). JAK/STAT pathway can either be targeted by phytochemicals by inhibiting one target site or modifying it at various sites (Arumuggam et al. 2015). The following phytochemicals have been reported by different researchers to have an effect on the pathway in vivo: resveratrol, chalcone, cryptotanshinone, curcumin, EGCG, Caffeic acid, Thymoquinone, Diosgenin, Ergosterol peroxide, β-escin, Guggulsterone, Silibinin, Capsaicin, Galiellalactone, Butein, and Ursolic acid (Arumuggam et al. 2015). Different studies carried out by various researchers on the effect of these phytochemicals on the JAK/STAT pathway have shown that these phytochemicals affect the JAK/STAT pathway using one or more mechanisms (Arumuggam et al. 2015). These mechanisms can be summarized as the following: • • • • •

“Inhibiting/decreasing the phosphorylation of JAK and STAT” “Inhibiting both the constitutive and inducible STAT 3 activation” “Inhibiting Src tyrosine kinase activity” “Inducing or increasing the expression of SHP-1” “Inducing apoptosis”

Epigallocatechin-3-gallate (EGCG) is a main constituent in green tea and its protective effects are associated with various human malignancies; this has been associated with its interesting effect via inhibition of tumor sphere formation, suppression of proliferation, and induction of apoptosis (Fujiki et al. 2017). Resveratrol is a protective ingredient widely spread in the traditional Mediterranean diet which is considered to lower risk of cancer. It is characterized as polyphenolic stilbene derivative found in the skin of grapes and berries which possess antioxidant, anti-inflammatory, and anticarcinogenic properties. Ptrostilbene is ringing attention due to its chemopreventive effects in a variety of cancer types. Quercetin has been discovered to have the capability to actively inhibit the JAKSTAT signaling pathway in various inflammatory disorders, as well as inhibit interleukin-12 (IL-12)-induced phosphorylation of JAK2, tyrosine kinase-2 (TYK2), STAT3, and “STAT4” in activated T cells, which diminished degree of T cell propagation and “Th1 variation” (Seif et al. 2017). Since inflammation and apoptosis have been implicated in cancer, “these ‘anti-inflammatory’ and ‘antiapoptotic’ attributes of quercetin therefore is crucial in cancer therapy by regulating the ‘toll-like receptor-2’ (‘TLR2’) and ‘JAK2/STAT3 pathway thus responsible for the inhibition of STAT3 tyrosine phosphorylation’ within inflammatory cells (Khan et al. 2016). Likewise, it has been reported that “preceding treatment of cholangiocarcinoma cells with quercetin inhibited the cytokine-mediated’ over expression of inducible nitric oxide synthase (iNOS) and expression of intercellular adhesion molecule-1 (ICAM-1) in the JAK/STAT cascade pathway [30]. Quercetin also hindered the activation of inflammatory cytokine interleukin-6 and interferon-γ” (Khan et al. 2016; Senggunprai et al. 2014).

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Inhibition of JAK/STAT Signaling in Animal Models by Phytochemicals Studies have been carried out to observe the significant role these naturally occurring chemical compounds in plants play in the obstruction of the Janus Kinase/Signal Transducers and Activators of Transcription signaling pathway. These roles have been reported by different researchers. “The replication of diverse colon cancer cell lines was reported to be impeded by Ursolic acid by subduing the initiation of many protein expressions which includes STAT3 in mice (Arumuggam et al. 2015). Penta1,2, 3,4,6-O-galloylbeta-D-glucose (“PGG”) present in certain oriental herbs has been revealed to play a major role in inhibiting prostate cancer growth in mouse models (Arumuggam et al. 2015). This potential is connected with the capacity of PGG to obstruct the addition of phosphate group(s) to STAT3 Tyr705, causing reduced expression of Bcl-XL and Mcl-1 which are the target of STAT3 transcription (Arumuggam et al. 2015). In A/J mice with urethane-induced lung tumors, Silibinin has been reported to impede the addition of phosphate group(s) to STAT3 thus a decreased expression of its transcriptional capacity (Arumuggam et al. 2015). Caffeic acid and its derivative CADPE were found to repress STAT3 phosphorylation thus inhibiting cancerous growth in mice with human renal carcinoma (Arumuggam et al. 2015). C-28 methyl ester of 2-cyano-3, 12-dioxoolen-1, 9dien-28-oic acid was reported to have completely eradicated cancerous growth in the mammary glands of mice and to an extent lung metastasis by blocking the activation of STAT3” (Arumuggam et al. 2015). Cancerous growth in a mouse cancer xenograft exemplary of human lung carcinoma was reported to be suppressed by Cucurbitacin Q, suggesting that for inhibition of tumor growth to occur, not just JAK2 inhibition is needful but also obstruction of STAT3 activity is also expedient” (Arumuggam et al. 2015).

The Interaction of Phytochemicals Regulating JAK/STAT with Chemotherapeutic Drugs Naturally occurring chemical compounds in plants do not meddle with chemotherapy; rather these compounds strengthen the results of a range of chemical compounds used for therapeutic in a manner that is dependent on the dose [6]. When EGCG is used alongside gemcitabine a compound used in advanced and metastatic pancreatic cancer therapy, heightened the ‘inhibition of genes marked by STAT by impeding STAT3 in cancer cells’ (Arumuggam et al. 2015). The sustainability of pancreatic cancerous cells and programmed cell death of pancreatic cancerous cells by JAK3 have been reported to be impeded when the effects of EGCG is added with CP690550 (Arumuggam et al. 2015). More so, Curcumin was found to be effective in impeding the phosphorylation of STAT3 than AG490, a widely-known compound known to impede JAK2 [6]. The synergistic combination of Flavopiridol with AG490 has been discovered to have an effect on the JAK/STAT3 signaling in cancer cells of the human lungs [6]. The ability of Thymoquinone to improve the actions of

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chemotherapeutic agents used in the therapy of multiple myeloma patients such as thalidomide from 8% to 32%, and bortezomib from 20% to 75% has been reported (Arumuggam et al. 2015). Certain compounds such as Diosgenin [6], γ-tocotrienol [6], and β-escin [6] have been reported to strengthen the effects of doxorubicin and paclitaxel’, drugs used in treatment of hepatocellular cancer (Arumuggam et al. 2015). The multiple replications of diverse myeloma cells that were either affected or resistant to other typically used agents have been reported to be inhibited by Celastrol (Arumuggam et al. 2015). “Celastrol” also had synchronously enhanced the apoptotic effects of thalidomide and bortezomib (Arumuggam et al. 2015). The programmed cell death effects of Velcade and thalidomide in multiple myeloma cells have been shown to be significantly potentiated by Capsaicin (Arumuggam et al. 2015). Capecitabine is a chemotherapeutic drug used in colon cancer therapy; it was observed that its action was further improved when used in conjunction with ursolic acid (Arumuggam et al. 2015).

Conclusion JAK/STAT signaling pathway plays significant role in “modifying the survival of cell, cell multiplication, cell division, and apoptosis (Arumuggam et al. 2015). It was observed in this text that the JAK/STAT3 signaling, particularly STAT3, and IDO expression are the two major phases that regulate the conversion of pre-neoplastic lesion to cancerous cell. The IDO expression is majorly controlled by the JAK/STAT signaling pathway and thus serves as a means of preventing the occurrence of cancerous cells. Chemical compounds obtained from plants are also effective in regulating the JAK/ STAT signaling pathway. The use of plant derived compounds known as phytochemicals is implicated in little or no toxicity. Phytochemicals that present encouraging anticancer properties both in vitro and in vivo should be put in practice.

References Aaronson DS, Horvath CM (2002) A road map for those who don’t know JAK-STAT. Science 296 (5573):1653–1655 Ahles TA, Saykin AJ (2007) Candidate mechanisms for chemotherapy-induced cognitive changes. Nat Rev Cancer 7:192–201 Arumuggam N, Bhowmick Neil A, Rupasinghe H, Vasantha P (2015) Phytochemicals targeting JAK/STAT Signaling and IDO expression in Cancer. Phytother Res Rev 29(6):805–817 Bousoik E, Montazeri Aliabadi H (2018) “Do we know Jack” about JAK? A closer look at JAK/ STAT signaling pathway. Front Oncol 8:287 Darnell J, Kerr I, Stark G (1994) Jak-STAT pathways and transcriptional activation in response to IFNs and other extracellular signaling proteins. Science 264:1415–1421 erlay J, Soerjomataram I, Ervik M, Dikshit R, Eser S, Mathers C, Rebelo M, Parkin DM, Forman D, Bray F (2013) Cancer incidence and mortality worldwide. International Agency for Research on Cancer: GLOBOCAN 2012 v1.0. Available from http://globocan.iarc.fr. Accessed on 15/07/2014

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Role of Carotenoids on Oxidative Stress–Mediated Signaling in Cancer Cells Poorigali Raghavendra-Rao Sowmya, Rudrappa Ambedkar, and Rangaswamy Lakshminarayana

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Oxidative Stress and Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Carotenoids and Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Effect of Carotenoids on Cell Cycle Regulation in Cancer Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Role of Carotenoids on Apoptosis Induction in Cancer Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Role of Carotenoids on Redox-Sensitive Protein-Mediated Cell Death Progression in Cancer Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Effect of Carotenoids on Growth Factors and Regulation of Cell signaling in Cancer Cells . . . Role of Carotenoids on the Regulation of Cell Signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Redox-Related Modulation of Transcription Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract

Carotenoids are natural pigments that are naturally occurring in green vegetables, fruits, seaweeds, and microorganisms. Carotenoids belong to the category of tetraterpenoids; structurally, they are in the form of a polyene chain, which is sometimes terminated by rings. In general, carotenoids are recognized as a frontline against certain cancers, as well as cardiovascular diseases. Focus on anticancer activity reveals carotenoids are involved in regulating the cell cycle, enhancing gap junctional communications, inhibiting the malignant transformation, and induction of apoptosis and differentiation. It has suggested that apoptosis inhibited cancer cells by activation of certain natural anticancer compounds, including carotenoids. Dietary or pharmacological manipulation of apoptosis induction may underlie novel treatment strategies to protect the cancers.

P. R.-R. Sowmya · R. Ambedkar · R. Lakshminarayana (*) Department of Microbiology and Biotechnology, Jnana Bharathi Campus, Bangalore University, Bengaluru, India © Springer Nature Singapore Pte Ltd. 2022 S. Chakraborti et al. (eds.), Handbook of Oxidative Stress in Cancer: Mechanistic Aspects, https://doi.org/10.1007/978-981-15-9411-3_91

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However, it is still unresolved how carotenoids are potentially affecting the cancer proliferation in humans. Epidemiological studies have suggested that complete characterization of carotenoids role at cellular and molecular levels may help in understanding the functional properties. The antioxidant and prooxidant activities of carotenoids elucidated in healthy and cancer cells are partially understood. However, regulating the redox balance mechanism in healthy and cancer cells by carotenoids is not much discussed. In this regard, this chapter emphasizes the role of different carotenoids on oxidative stress–mediated cell signaling in cancer cells. Keywords

Carotenoids · Oxidative stress · Reactive oxygen species · Apoptosis · Cancer cells

Introduction The cells have undergone above optimal levels of oxidative stress through physiological and xenobiotics exposure (prooxidants) that damages biologically relevant molecules, like DNA, proteins, and lipids. Naturally, cells organize various endogenous defense mechanisms to manage protection from oxidative stress. However, when reaching the threshold levels, among natural compounds, carotenoids are most likely to be involved in the reduction of such oxidative insult in the cells. Further, they are efficiently neutralized by electronically excited photosensitizer molecules and inhibit the generation of free radicals and reactive oxygen species (ROS) (Truscott 1990). Also, carotenoids are known to involve physical quenching of 1O2 by transfer of energy of singlet oxygen to the carotenoid molecule and yielding ground state oxygen. However, carotenoid returns to the ground state, dissipating its heat by interaction with the surrounding solvent instead of further reaction. Since the carotenoids remain intact during physical quenching of 1O2/excited molecule, they can reutilize in such quenching cycles. Carotenes and certain xanthophylls proved to be efficient quenchers of singlet oxygen and control the oxidative reaction rates (Britton 1995; El-Agamey et al. 2004). The scavenging efficiency of singlet molecular oxygen (1O2) and peroxyl radicals (ROO•) by carotenoids is associated with the number of double bonds and type of functional groups. Major carotenoids such as α- carotene, β-carotene, β-cryptoxanthin, lutein, and zeaxanthin detected in human serum and tissues belong to highly active quenchers of ROS. Among carotenoids, acyclic carotenoid lycopene is considered to be potent in quenching 1O2 (Britton 1995; El-Agamey et al. 2004). Besides, carotenoids act effectively in the inhibition of ROO• and mediated oxidation by disrupting the chain reaction in the lipophilic cell compartments. Due to the lipophilicity of carotenoids with specific properties to scavenge ROO•, they play a crucial role in protecting membranes and lipoproteins from oxidative damage (El-Agamey et al. 2004). The

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antioxidant action of carotenoids particularly deactivation of ROO• depends on the formation of radical adducts, and a resonance-stabilized carbon-centered structural modification and oxidative cleavages. A variety of carotenoid oxidative products have been detected subsequently, including epoxides and apocarotenoids as a result of the interaction of free radicals and ROS (Lakshminarayana et al. 2013). The antioxidant activity of carotenoids depends on the oxygen tension in the cellular environment (Burton and Ingold 1984). It was known that these compounds might possess an impact on biological functions involved in signaling pathways (Vijay et al. 2018). In physiological conditions under the low oxygen pressure, carotenoids showed superior antioxidant activity (Burton and Ingold 1984). The potential involvement of each carotenoid is also dependent on their accumulation levels in particular organs or tissues. Carotenoids interact with free radicals and ROS in three main ways, namely electron transfer [1], hydrogen abstraction [2], and addition of a radical species [3]. In the human, scavenging rate of free radicals and ROS (1O2, OH•, •O
2, and H2O2) by carotenoids is strongly dependent on the nature of the ROS (Britton 1995). CAR + R•

R- CAR• [1]

CAR + R•

CAR• + RH [2]

CAR + R•

CAR•+ + R• [3]

In contrast to the antioxidant mechanism, under certain conditions like higher oxygen pressure in the cellular environment, carotenoids may act as prooxidant and increase oxidative stress (Burton and Ingold 1984). However, carotenoids activity under the above extreme condition may not necessarily be like prooxidants. Further, the antioxidant/prooxidant properties of carotenoids are complex and vary with other chemical environment of the cells, and a kind of carotenoid species interact (El-Agamey et al. 2004). Studies had shown that β-carotene acts as prooxidant and induces apoptosis by increasing intracellular ROS levels when cells are treated at higher concentrations (Palozza et al. 2001; Cui et al. 2007). Vijay et al. (2018) hypothesized that generation of ROS and its levels might profoundly alter the proapoptotic proteins. In general, increased oxidative stress plays a key role in the development of age-related complications, including cancer. In cancer cells, the metabolic rate is profoundly higher than healthy cells, and they are associated with higher oxidative stress. However, such levels of oxidative stress are less harmful to the cancer cells. The cancer cells can resist the optimal degree of oxidative stress or ROS levels by adopting a new redox balance mechanism. Though oxidative stress may promote tumor growth, it can also increase the sensitivity to treatments (Sowmya et al. 2017; Vijay et al. 2018). A high steady-state of oxidative stress in cancer cells may defeat the capability of endogenous antioxidants and become susceptible to oxidative damage–mediated death. Hence, the implications of carotenoids on ROS regulation and its situational activity are considered to be significant for cancer treatment. The antiproliferation of cancer cells is not only related to the antioxidant or prooxidant properties of carotenoids but also due to the involvement in the signaling pathway (Lakshminarayana et al. 2013; Palozza et al. 2004).

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Oxidative Stress and Cancer Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are recognized to play a dual role as both favorable and detrimental actions. ROS nature corroborated that they were acting as secondary messengers in intracellular signaling cascades, influencing the oncogenic properties of cancer cells. Nevertheless, ROS can also lead a function as antitumorigenic species involved in cellular senescence and apoptosis induction. The increased generations of ROS or RNS through endogenous or exogenous insults are considered as oxidative stress. Several types of cancers are allied with altered redox regulation and mediated cellular signaling. Oxidative stress induces the redox imbalance in cancer cells as compared with healthy cells. The redox imbalance may be related to oncogenic stimulation. Many studies examined the biochemical and molecular aspects of oxidative stress in the carcinogenesis process. Further, damages of lipids, proteins, and DNA increase oxidative stress in cancer cells. The mechanism of carcinogenesis, such as the role of signaling cascades by ROS activation of activator protein-1 and nuclear factor kappa B factors, and transcription of genes are well explained in various in vivo and in vitro models. Earlier, the influence of enzymatic and nonenzymatic antioxidants in cancer and healthy cells are well reviewed; however, the influence of carotenoids and their physiological concentration on the oxidative stress–mediated signaling in the cancer cells are not well studied.

Carotenoids and Cancer Studies have consistently evidenced that dietary intake of carotenoids is allied with a reduced risk of cancer. Therefore, carotenoids consider potential anticancer molecules among the natural compounds. In this regard, several investigations made to evaluate the role of carotenoids on progression and inhibition of different cancers (Tanaka et al. 2012). Approximately 25% of cancer is potentially preventable via modified lifestyle and consumption of nutrition-rich food, notably carotenoids and vitamins. Generally, one-third of the overall risk of cancer is due to less consumption of a healthy diet (fruits and green vegetables). Many dietary compounds have tested their anticarcinogenesis ability in different animal models. The antioxidant activity of carotenoids has been studied extensively in various cancerous and noncancerous cells (Tanaka et al. 2012). Several prospective cohort studies have shown that a high intake of lycopene reduced incidence of prostate cancer development (Tanaka et al. 2012). Apart from these, xanthophylls such as lutein inhibited mammary tumor growth by regulating angiogenesis and apoptosis in mouse (Chew et al. 2003). Yasui et al. (2011) demonstrated the effect of astaxanthin on modulation inflammatory cytokines related to cancer in mouse. Nagendraprabhu and Sudhandiran (2011) have shown the role of astaxanthin on the regulation of nuclear factor kappaB (NF-kB), cyclooxygenase2 (COX-2), matrix metalloproteinases (MMP), extracellular signalregulated kinase (ERK)-2, and protein kinase B (Akt) pathways in colon cancer cells. Wang et al. (2012) demonstrated the role of ROS-mediated Bcl-xL, Janus

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Modulation of peroxisome-proliferator activated receptors Modulation of hormone action

Modulation of apoptosis

Modulation of the cell cycle and apoptosis via redox-sensitive proteins

Modulation of cell cycle

Modulation of growth factors

Carotenoids

Redox-related modulation of transcription factors Modulation of cell differentiation

Modulation of retinoid receptors Modulation of xenobiotic and other orphan nuclear receptors Modulation of gap junction communication

Modulation of adhesion molecules and cytokines

Fig. 1 Proposed action mechanism of carotenoids on inhibition of cancer cell proliferation

kinase/signal transducer and activator of transcription (JAK-STAT) pathway, and downregulation of epidermal growth factor receptor (EGFR) in fucoxanthin-treated sarcoma xenograft–bearing mice. However, carotenoid research focused on tumor suppression in vivo is not much explored, though their precise role on the particular cell type of cancer is explored extensively in the cellular model. Sowmya et al. (2017) observed selective killing of carotenoids on breast cancer cells as compared to the normal cells; the reason may due to balanced redox status (glutathione, lipid peroxides, and ROS levels) and defense mechanism in normal cells than distorted redox mechanism in tumor cells. Generally, normal cells have shown lower oxidative stress and balanced antioxidant defense system as compared to the cancerous cells. In this regard, Vijay et al. (2018) showed that increased oxidative stress and depletion of endogenous glutathione could be toxic to the cancer cells. The primary mechanism of cancer prevention by carotenoids is the regulatory mechanisms of cell cycle progression, apoptosis-mediated signaling events (intrinsic and extrinsic), and gap junction communication (Palozza et al. 2004; Arathi et al. 2018) (Tables 1 and 2 & Fig. 1).

Effect of Carotenoids on Cell Cycle Regulation in Cancer Cells Carotenoids are believed to control the progression of cell cycle regulation by arresting at different stages through modulation of cell cycle–related proteins (Table 2). Amir et al. (1999) observed cell cycle arrest at the G0/G1 phase in HL-60 cells treated with lycopene. Further, Nahum et al. (2001) demonstrated that lycopene’s growth inhibitory effect is due to cell cycle arrest at the G0/G1 phase through decreased expression of cyclins D1 and D3 in mammary and endometrial

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Table 1 Role of carotenoids on cell cycle arrest in different cancer cell models

Carotenoids β-carotene

Lycopene

Cell lines COLO 320 HSR AtT20 DU145, LNCaP, PC-3

HL-60 Hep3B HT-29

Neoxanthin β-cryptoxanthin

Capsanthin, bixin Crocin

Crocetin

Astaxanthin

Cell cycle arrest

G2/M, S G2/M, G0/G1, and G2/M G0/G1 G0/G1 and S G1

Protein expression Downregulation of cyclin A Downregulation of Skp2 Decreased cyclins D1 and E and cdk4, and suppression of pRB phosphorylation

Reduced expression of cyclin D1, phosphorylation of pRB, and increased abundance of p27KIP Downregulation of cyclin D and altered expression of β-tubulin, CK8/1 and CK19 Downregulation of proliferating cell nuclear antigen and cyclin D1

MCF-7, MDA-MB468, SK-BR-3

G0/G1 & G1/S

KB-1

G0/G1

C3H10T1/ 2 A549

S G0/G1

Decreased cyclin D and E, and increased expression of p21

K562

G0/G1

Downregulation of cyclin D1 and upregulation of p21 and Nrf2 expression

BxPC-3, MIA PaCa-2 BxPC-3, MIA PaCa-2

G1

HCT-116

G0/G1

G2/M

References Palozza et al. (2002) Haddad et al. (2013) Hwang and Bowen (2004), Ivanov et al. (2007), Ford et al. (2011), Soares et al. (2013) Amir et al. (1999) Park et al. (2005) Tang et al. (2008)

Nahum et al. (2001), Teodoro et al. (2012), Takeshima et al. (2014), Uppala et al. (2013) Cheng et al. (2007)

Chang and Lin (1993) Lian et al. (2006),

Wu et al. (2010) Zhang et al. (2011)

Bakshi et al. (2010)

Altered expression of Cdc-2, Cdc-25C, Cyclin-B1, and EGF receptor Downregulation of cyclin D with concomitant increased expression of p53, p21, and p27

Dhar et al. (2009)

Palozza et al. (2009)

(continued)

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Table 1 (continued)

Carotenoids Fucoxanthin

Cell lines MGC-803

Cell cycle arrest G2/M

WiDr B16F-10 HepG2,

G0/G1 G0/G1 G1

DU145

G1

SK-Hep-1

G1

LNCaP, DU145

Siphonaxanthin

HL-60

–

Protein expression Downregulation of cyclin B1 and survivin –

References Yu et al. (2011)

Decreased pRb, cyclin D1, cyclin D2, and CDK4 with increased p15INK4B and p27KIP1 levels Upregulation of GADD45α (growth arrest and DNA damage gene) and GADD153, and downregulation of cyclin D1 Enhanced expression of Cx43 and Cx32 Upregulates the SAPK/JNK-mediated expression of GADD45α Upregulated expression of GADD45α

Kim et al. (2013)

Das et al. (2008), Yoshiko and Hoyoku (2007)

Liu et al. (2009) Satomi, (2012)

Sugawara et al. (2014)

cancer cells. These regulatory effects are accompanied by a reduction in the cyclindependent kinase activity (Cdk4 and Cdk2) and hypophosphorylation of regulatory protein (Rb). Further, they elucidated inhibitory activity of cdk2 kinase activity which is due to the cyclin D downregulation and retaining protein p27 in the cyclin Ecdk2 complex. Park et al. (2005) observed that lycopene treatment inhibited cell growth efficiently and induced the cell cycle arrest at G0/G1 and S phases in human hepatoma cells. Later, Ivanov et al. (2007) shown mitotic arrest in prostate cancer cells treated with lycopene-based agents. G1/S transition mediated cell cycle arrest accompanied by decreased cyclins D1 and E, kinase Cdk4, and Rb phosphorylation. Palozza et al. (2007) reported that tomato subjected to in vitro digestion procedure added to cultured colon (HT-29 and HCT-116) cancer cells showed cell cycle arrest at the G0/G1 phase through the decrease in the expression of cyclin D1. Subsequently, Tang et al. (2008) reveals the antiproliferative effect of lycopene via reduced expression of cyclin D1 and inhibition of phosphorylation of pRB protein, and increased p27KIP abundance in human colon cancer cells. Others have shown that carotenoids such as β-carotene, lycopene, and lutein inhibit proliferation of oral cancer cells by arresting various cell cycle stages through reduced expression of proliferating cell nuclear antigen and cyclin D1 in Livny et al. (2002) and Cheng et al. (2007).

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Table 2 Role of carotenoids on the induction of apoptosis in various cancer cells Carotenoids β-Carotene

Cell lines WiDr HL-60, HT-29 SKMEL-2 MCF-7 AGS

Lycopene

LNCaP, DU145

MDAMB-468

Neoxanthin

Mechanisms of apoptosis induction Enhanced ROS and blocked expression of Bcl-2 Induced DNA fragmentation NF-kB activation

References Palozza et al. (2001 2003)

Activation of PPAR-γ and production of ROS Increased p53 and decreased Bcl-2 expression Nuclear ATM levels Reduced mitochondrial membrane permeability, increased release of cytochrome c, and enhanced expression of Bax and downregulation of Bcl-2 Upregulation of Bax and increased PARP cleavage

Cui et al. (2007) Jang et al. (2009)

HT-29, T-84

DNA fragmentation and membrane blebbing

A549

Activation of caspase-3

PC-3

Activation of caspase-3 and PARP cleavage

HCT116 Lutein/Zeaxanthin

MCF-7

Loss of mitochondrial transmembrane potential and increased release of cytochrome c and apoptosis-inducing factor Activation of the intrinsic pathway

Violaxanthin

MCF-7

Induction of early apoptosis

Canthaxanthin

WiDr

Induction of DNA fragmentation

Astaxanthin

SK MEL-2 K562, TE-4

HCT116

Upregulated PPAR-γ and repressed activation of Akt and increased activation of caspase-3

The altered ratio of Bax/Bcl-2 and Bcl-xL level, and increased phosphorylation of p38, JNK, and ERK1/2

Soares et al. (2013) Takeshima et al. (2014) Teodoro et al. (2012) Wu et al. (2010) KotakeNara et al. (2005) Terasaki et al. (2007) Chew et al. (2003) Pasquet et al. (2011) Palozza et al. (2004)

Zhang et al. (2011), Lim et al. (2011) Palozza et al. (2009) (continued)

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Table 2 (continued) Carotenoids Fucoxanthin

Cell lines HL-60

Siphonaxanthin

Mechanisms of apoptosis induction ROS generation and enhanced activation of caspase-9 and 3 Cleavage of PARP and downregulation of Bcl-2/BclxL and Bax level

EJ-1

Activation of caspase-3

Halocynthiaxanthin

HL-60

DNA fragmentation; decreased expression of Bcl-2 in HL-

Fucoxanthinol

MCF-7 CaCo-2 DLD-1

60 cells

Halocynthiaxanthin Peridinin

Upregulated expression of TNF-α, DR5, cleaved PARP, and cleaved form of caspase-8, 9, 10 and
3

References Kim et al. (2010) KotakeNara et al. (2005) Sugawara et al. (2014) Zhang et al. (2008) Konishi et al. (2006)

Sugawara et al. (2007) Yoshida et al. (2007)

Studies reported that lycopene metabolite apo-120 -lycopenal suppressed prostate cancer cells proliferation through alteration in the cell cycle at different phases (Hwang and Bowen 2004; Ford et al. 2011; Arathi et al. 2018; Soares et al. 2013). In breast cancer cells, lycopene arrested cell cycle at G0/G1 and S phases accompanied by decreasing the number of cells in the G2/M phase. Cell cycle arrest and antiproliferation activity of lycopene are associated with decreases in cyclin D1/D3 and pRB protein expression, and subsequently increased p21 and altered expression of β- tubulin, CK8/18, and CK19 (Nahum et al. 2001; Teodoro et al. 2012; Uppala et al. 2013; Takeshima et al. 2014). Palozza et al. (2002) showed the β-carotene-induced cell cycle arrest at the G2/M phase in human colon adenocarcinoma cells by reducing the cyclin A expression. Tibaduiza et al. (2002) reported the role of β-carotene cleavage products on the downregulation of E2F1 and Rb and inhibition of AP-1 factors in breast cancer cells. Recently, Haddad et al. (2013) observed that β-carotene induced the cells in S and G2/M phases by the downregulation of S-phase kinase-associated protein 2 (Skp2) in pituitary adenoma cells. Likewise, in lung cancer cells, β-cryptoxanthin exhibited an antiproliferative effect through cell cycle arrest at the G0/G1 phase and associated with decreased cyclin D and E, and increased expression of p21 protein (Lian et al. 2006; Wu et al. 2010). Chang and Lin (1993) showed S phase cell cycle arrest in neoxanthin-treated C3H10T1/2 cells (mouse embryonic mesenchymal cells) by suppressing the synthesis of DNA. Zhang et al. (2011) suggested the antiproliferation effect of capsanthin and bixin

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on decreased cyclin D1 and upregulation of p21 and Nrf2 in leukemia (K562) cells. Crocetin and crocin, major carotenoid compounds found in saffron (Crocus sativus), were showed to inhibit the human pancreatic cancer cells growth through cell cycle arresting at G2/M and G1 phases, respectively (Dhar et al. 2009; Bakshi et al. 2010). They also attributed that crocetins arrested the cell cycle by altered expression of Cdc-2, Cdc-25C, Cyclin-B1, and EGFR. Palozza et al. (2009) shown that astaxanthin induces cell cycle arrest at the G0/G1 phase which is may be due to the decreased cyclin D with a concomitant increase of p21, p53, and p27 expression in colon cancer cells. Later, Yasui et al. (2011) disclose the influence of astaxanthin on reduction of colon adenocarcinoma incidence through the suppression of PCNA and survivin. Fucoxanthin, an epoxy carotenoids, was also shown to induce cell cycle arrest at the G1, G2/M, and G0 phases in several tumor cells (Das et al. 2008; Yoshiko and Hoyoku 2007; Kim et al. 2010). Das et al. (2008) demonstrated the influence of fucoxanthin on the mechanism of cell cycle arrest at the GO /G1 phase in colon cancer cells which is due to the upregulation of the p21WAF1/Cip1. Likewise, Yu et al. (2011) observed that fucoxanthin affects the cycle arrest at the G2/M phase by downregulation of cyclin B1 and survivin in gastric adenocarcinoma cells. Whereas Kim et al. (2013) observed that G0/G1 phase cell cycle arrest in melanoma cells (B16F10) was associated with decreased pRB, cyclin D1, cyclin D2, and Cdk4, and increased p15INK4B and p27KIP1 expressions. Likewise, others have shown the fucoxanthin affects the cell cycle arrest at the G1 phase by upregulation of stress-inducible genes (growth arrest and DNA damage genes; GADD45α and GADD153) and downregulation of cyclin D1 in cancer cells (Yoshiko and Hoyoku 2007; Das et al. 2008). Liu et al. (2009) shown the influence of fucoxanthin on cell cycle arrest which is associated with enhanced gap junction communication by upregulation of Cx43 and Cx32 expression and downregulation of c- Jun N-terminal kinase (JNK) and extracellular signalregulated kinase (ERK) activation in hepatocarcinoma cells. Subsequently, Satomi (2012) demonstrated the role of fucoxanthin on enhanced cell cycle arrest via SAPK/JNK-mediated upregulation of GADD45α in prostate cancer cells. Sugawara et al. (2014) explored the role of a novel carotenoid siphonaxanthin on the upregulation of GADD45α and cell cycle arrest in the human leukemia cells. The overview of carotenoid’s role in cell cycle regulation in different cancer cells is shown in Table 1.

Role of Carotenoids on Apoptosis Induction in Cancer Cells Several studies equivocally demonstrated that carotenoids could induce cell death in various cancer cell lines and partially explained possible pathways (Table 2). Generally, cancer cells may acquire resistance against cell death progression by modulation of the enhanced expression of antiapoptotic proteins or by mutation in the proapoptotic genes. In general, most of the reports have claimed that chemopreventive mechanism of carotenoids is due to the apoptosis. In vivo and in vitro research

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results show that carotenoids may alter apoptosis-related Bcl-2 and caspase family proteins. Chew et al. (2003) demonstrated that the inhibition of mouse mammary tumor growth by dietary lutein is attributed to increased expression of Bax and decreased expression of antiapoptotic Bcl-2. Liu et al. (2003) described the role of lycopene on phosphorylation of Bcl-2 associated death promoter (BAD) and activating caspase-3 in smoke-induced lung squamous carcinogenesis in ferrets. Subsequently, Hantz et al. (2005) demonstrated that physiologically relevant concentrations of lycopene considerably reduced mitochondrial transmembrane potential, induced the release of cytochrome c, and promote the apoptosis induction in human prostate cancer cells. Also, apoptosis induction by downregulation of cyclin D1 and Bcl-2, and upregulation of Bax expression in PC-3 cells is observed by Soares et al. (2013). Wu et al. (2010) observed a remarkable increase in the percentage of apoptotic cells treated with lycopene and its association with a substantial increase in caspase-3 activation in lung cancer cells. Further, Teodoro et al. (2012) observed a two- to fourfold increase in apoptosis when colon cancer cells were exposed to lycopene. Similarly, lycopene induced apoptosis in breast cancer cells by upregulation of proapoptotic protein Bax by increasing poly-ADP ribose polymerase cleavage (Takeshima et al. 2014). Palozza et al. (2001) demonstrated that β-carotene (> 50 μM) induced apoptosis through increased levels of intracellular ROS in colon adenocarcinoma cells. Consequently, Palozza et al. (2003) showed that carotenoids induces apoptosis through the activation of caspase cascade in tumor cells. Further, they demonstrated the activation of NF-κB which plays a vital role in cell death progression induced by carotenoids and NF-kB activation. Cui et al. (2007) reported the effect of β-carotene on induction of apoptosis by the activation of peroxisome proliferator–activated receptor gamma (PPAR-γ) and ROS production in breast cancer cells. Jang et al. (2009) showed that higher concentration (100 μM) of β-carotene increases the expression of p53 and decreases Bcl-2 levels and nuclear ataxia telangiectasia mutated a sensor for DNA-damaging agents in gastric cancer cells. Kotake-Nara et al. (2005) investigated the possible influence of neoxanthin and fucoxanthin on reduced cell viability by activating caspase-3 and PARP in human prostate cancer. Terasaki et al. (2007) reveal the role of polar xanthophyll neoxanthin on the loss of mitochondrial transmembrane potential and the release of cytochrome c and apoptosis-inducing factor in colon cancer cells. Pasquet et al. (2011) assessed the violaxanthin antiproliferative activity in breast cancer cells. Bi et al. (2013) revealed that zeaxanthin induced the apoptosis via activation of intrinsic signaling pathway mediated by the activation of caspase-9 and -8, and increased release of cytochrome c in human uveal melanoma cell lines. Lim et al. (2011) observed the apoptosis-inducing property of astaxanthin through downregulated phosphorylation of Akt and increased activation of caspase-3 in the esophageal cancer cell line. Palozza et al. (2009) observed that astaxanthin activity is associated with the ratio of Bax/Bcl-2 and Bcl-xL and phosphorylation of p38, JNK, and ERK1/2 in colon cancer cells. Also, β-carotene, astaxanthin, capsanthin, and bixin affected the cell proliferation which is partly due to the upregulation of PPAR-γ expression in K562 cells (Zhang et al. 2011). Likewise, the influence of marine carotenoids and its

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derivatives demonstrated the apoptosis-inducing activity in several cancer cell lines (Konishi et al. 2006; Liu et al. 2009; Zhang et al. 2008). In leukemia cells, downregulation of Bcl-2 expression and concomitant increase in caspase activation are found in siphanoxanthin- and fucoxanthin-treated cells (Sugawara et al. 2014). Besides, the ROS-mediated Bcl-xL pathway and cleavage of PARP showed the induction of apoptosis (Kim et al. 2010). Zhang et al. (2008) documented the apoptosis-inducing effect of fucoxanthin by DNA fragmentation, increased number of hypodiploid cells, and activation of caspase-3 activity. Sugawara et al. (2014) reported that siphonaxanthin activates apoptosis by downregulating Bcl-2 and increasing caspase-3 activity in human leukemia cells. Peridinin exerted a potent antiproliferative effect, through the activation of caspase-8 and -9 and mitochondrial-mediated pathways in human colon cancer cells (Sugawara et al. 2007). Correspondingly, Yoshida et al. (2007) reported that halocynthiaxanthin and peridinin enhanced TNF-related apoptosis-inducing ligand by upregulating death receptor 5 (DR5) expressions in colon cancer cell lines (Table 2).

Role of Carotenoids on Redox-Sensitive Protein-Mediated Cell Death Progression in Cancer Cells It is suggested that carotenoids may modulate the expression of redox-sensitive regulatory protein expression. Kane et al. (1993) reported the association of β-carotene on Bcl-2 expression and cell death progression by increased ROS production and lipid peroxidation. Chlichlia et al. (1998) and Zhuang et al. (1999) observed the influence of nitric oxide and singlet oxygen on the activation of caspase-8, a crucial protein-degrading enzyme involved in the apoptotic cascade. Further, β-carotene induces caspases-3 activity by interacting with a signal complex located on the cell membrane (Palozza et al. 2002, 2003). Besides, in the cytoplasm, a nonreceptor signaling pathway induces caspase-9 activation by releasing the truncated form of the protein Bid. This protein translocated to the mitochondria and acted as a potent inducer of apoptosis via releasing cytochrome c and activation of caspase-9 (Palozza et al. 2003). Palozza et al. (2005) demonstrated that carotenoids might modulate apoptosis-related proteins (p53 and p21WAF1) by a redox mechanism. It has known that mitochondrion is critical for integrating and processing of proapoptotic signals. Diverse apoptotic stimuli can cause mitochondrial dysfunction, which leads to oxidative stress and changes redox homeostasis. Previously, the role of carotenoids on the proapoptotic effects of mitochondria was systematically demonstrated (Terasaki et al. 2007). However, treatment of specific cancer cells with a combination of β-carotene and cigarette smoke condensate induced DNA damage and enhanced the 8-hydroxydeoxyguanosine (8-OH-G) levels. Also, an increased proportion of proliferating cells accompanied by the DNA damage is due to a deregulation of p53 expression (Palozza et al. 2004). Further, these complications may associate with mutagenesis and carcinogenesis (Palozza et al. 2004). Apart from these, a dose-dependent decrease in the expression

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of Cox-2 and ROS production was observed in β-carotene-treated cancer cells (Palozza et al. 2005). Since the generation of ROS by Cox-2 and peroxidase activity, the inhibition of this step by β-carotene is considered to be a potential mechanism to control cancer growth. Further, they proposed the two distinct redox-sensitive mechanisms pathways of cancer cell death: such as i) increased production of ROS at higher β-carotene concentration and ii) Cox-2 expression at low carotenoid levels. Furthermore, the type of carotenoid and its respective functional groups, concentration presence, and absence of other antioxidants (including other carotenoids) and their cleavage products and synergism are not detailed for mechanisms mentioned above.

Effect of Carotenoids on Growth Factors and Regulation of Cell signaling in Cancer Cells The dividing cells received the signal from growth factors delivered in the bloodstream which recognizes specific receptors on the cell surface. Carotenoids are known to involved in the expression of growth factors and their receptors. Liu et al. (2003) revealed that lycopene decreased the insulin-like growth factor (IGF-1) levels in the lungs of ferrets exposed to cigarette smoke. Similarly, lycopene increased the insulin-like growth factor–binding protein-3 (IGFBP-3) levels, which is reported as a potent inhibitor of Akt and mitogen-activated protein kinase (MAPK) signaling pathways Moreover, lycopene induced proapoptotic signals through a decreased phosphorylation of Bad (Muto et al. 1995). Khalil et al. (2003) revealed the role of β-carotene on the downregulation of EGFR in premalignant cervical dysplastic cells. The elevated serum concentration of IGF-1 was found to associate with increased risk for cancer, including breast, prostate, colorectal, and lung cancers (Hankinson et al. 1998). Herzog et al. (2005) observed that lycopene decreased IGF-1 expression in young rats’ prostate tissue. Uchiyama and Yamaguchi (2005) documented the role of β-cryptoxanthin and β-carotene on increased expression of IGF-1 and transforming growth factor (TGF)-β1 with respect to the inhibition of osteoporosis. In vivo studies have shown that β-carotene and canthaxanthin increase vascular growth and levels of TGFα (Schwartz and Shklar 1997). Besides, exposure of HUVEC cells to β-carotene modulates the expression of fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF), which are two vital proteins involved in endothelial cell maturation and vascular repair (Dembinska-Kiec et al. 2005). Also, carotenoids affect cellular differentiation, a critical mechanism involved in chemoprevention of chronic diseases. Lycopene, β-carotene, and lutein influence the differentiation of promyelocytic leukemia cells associated with the expression of cell surface antigen CD14, oxygen burst oxidase, and chemotactic peptide receptors (Amir et al. 1999). Although the carotenoid mechanism on cell differentiation is not clearly understood, a reasonable hypothesis is that the carotenoid activates nuclear hormone and retinoid receptors (Sharoni et al. 2002).

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Role of Carotenoids on the Regulation of Cell Signaling Several studies have shown that the biological function of retinoids involves the regulation of transcription through nuclear receptors such as retinoic acid receptors (RARs) and retinoid X receptors (RXRs). In this regard, reports have revealed the influence of β-carotene on the upregulation of retinoid receptors in mouse skin (Ponnamperuma et al. 2000). Goralczyk (2009) investigated a combination of smoke and a high dose of β-carotene influence on the reduction of retinoic acid levels and downregulation of RARβ, but not of RARα and RARγ expression in the lung of rodents. The decreased expression of RARβ is shown with enhanced cell proliferation. Likewise, lycopene metabolites on the regulation of nuclear receptor and inhibition of human mammary cancer cell growth by transactivation of the RAR reporter gene compared with lycopene and retinoic acid. Stahl et al. (2000) demonstrated that retinoic acid is much more potent than acyclo-retinoic acid in the transactivation of the retinoic acid-responsive promoters of the receptor RAR-β2.

Redox-Related Modulation of Transcription Factors The nuclear transcription factor is known to be associated with chronic inflammation for cancer proliferation; this plays a crucial role in regulating the immune response. NF-κB is responsive to oxidative stress in the cells with higher H2O2. The ROS act as essential mediators of NF-κB activation. Palozzo et al. (2003, 2004) demonstrated the increased ROS production and glutathione levels accompanied by sustained elevation of NF-κB and inhibition of cancer cell growth. Furthermore, increased expression of c-myc in carotenoids-treated cells is supported to postulate the hypothesis that redox regulation of NF-κB is involved in the growth-inhibitory and proapoptotic effects of the carotenoid in tumor cells (Palozza et al. 2003). The efficiency of carotenoids on the activation of NF-κB and the production of pro-inflammatory cytokines is observed (interleukin IL-6 and TNF-α). Huang et al. (2007) reported that lycopene suppresses invasive ability by inhibition of matrix metalloprotein-9 (MMP-9) in human hepatoma cells. He also elucidated the influence of carotenoid on the decreased expression of the IGF-1 receptor and ROS. De Stefano et al. (2007) suggested that carotenoid molecules are involved in controlling the pro-inflammatory genes through NF-κB and lowered the nitric oxide synthase and Cox-2 enzymes. Also, they demontrated the inhibition of macrophage activation by induction of gliadin and interferon IF-γ through the inhibition of NF-κB, STAT-1α (signal transducer and activator of transcription-1α), and IRF-1 (interferon regulatory factor-1). Likewise, Dorai and Aggarwal (2004) elucidated the role of redox-sensitive transcription factor, AP-1, on the regulation of cell growth. Tibaduiza et al. (2002) illustrated the influence of β-carotene and its cleavage products on inhibition of AP-1 transcription factor in mammary tumor cells. Karas et al. (2000) studied the lycopene treatment on inhibition of AP-1 and reduced induction of the IGF-1, implying an inhibitory effect of mammary cancer cell growth. Liu et al. (2000) revealed the influence of β- carotene on AP-1 proteins (c-fos and c-jun) in tobacco smoke–exposed ferrets. The activation of AP-1 at high

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doses of the carotenoid explains the increased risk of lung cancer among smokers, as per the observation conducted in clinical trials (Omenn et al. 1996). Carotenoids modulate tumor growth by acting as potent inducers of phase II enzymes in detoxifying and combating xenobiotics. Cui et al. (2007) demonstrated the apoptosisinducing effects of β-carotene by increased PPARγ mRNA and protein levels associated with increased production of ROS in MCF-7 cancer cells. DembinskaKiec et al. (2005) reported that β-carotene could modulate the expression of proteins involved in cell-cell adhesion and regulate cell-matrix interaction. Loss of gap junctional communication is a hallmark of cell malignant transformation. Hanusch et al. (1995) demonstrated that carotenoids could induce synthesis of the protein connexin 43 and increase gap junctional cell communication (GJC). Livny et al. (2002) revealed that the influence of lycopene and its oxidation products enhanced the GJC modulation by upregulation of connexin 43 expressions in cultured cells. However, molecular mechanisms are not the same for provitamin A carotenoids and non-provitamin A carotenoids. The retinoic acid receptor antagonist suppresses the expression of connexin 43 induced by retinoids compared to provitamin A carotenoid treatment. Apart from these studies, few reports, including our research, documented the prominent role carotenoids and their oxidized products play on selective oxidative stress–mediated antiproliferation of various cancer cells (Lakshminarayana et al. 2013; Arathi et al. 2016, 2018; Vijay et al. 2018).

Conclusion Three decades of carotenoid research have extensively focused on the influence of a few major carotenoids to target specific chronic health problems in various in vitro and in vivo models. The differential effect of structurally diverse carotenoids and their oxidative products on inhibition of specific health-associated problems like cancer is not understood, instead of evaluating the antiproliferation of cancer cells. Despite the structural and chemical properties of carotenoids, they involve in the antioxidant mechanism, cell signaling, and modulation of the immune system. Since carotenoids have dual characteristics under oxidative stress, exploring the pro- and antioxidant mechanisms and their selectivity on the cancer cell death mechanism is also not examined. Moreover, epidemiological studies have evidenced the biological action and its efficiency that may pronounce in combination with other phytochemicals, including carotenoids. Furthermore, the combined influence of carotenoids’ different properties on specific cancer cell proliferation and an underlying principle mechanism are yet to be detailed.

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Cross Talk Between Oxidative Stress and p53 Family Members in Regulating Cancer

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Sumiran Kumar Gurung, Lokesh Nigam, Kunwar Somesh Vikramdeo, and Neelima Mondal

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Role of p53 Family Members in Genomic Integrity Maintenance Under Oxidative Stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . P53 Family and ROS Cross Talk in Cellular Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ROS and p53 Family in Cell Death . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract

p53 family members form one of the most versatile groups of proteins with diverse functions including tumor suppression, regulation of embryonic development and cellular metabolism, and cell death. The p53 family namely p53, p63, and p73 exhibits considerable similarity and thus is able to cross-regulate their target genes. Further complications arise with expression of multiple isoforms of these p53 family members from usage of different promoters and alternative splicing. The isoforms, specially the N terminally truncated isoforms, have been reported to play very crucial role in the regulation of p53 family members itself and their target genes. Oxidative stress, a two-edged sword, plays vital role in cellular environment to regulate cell survival, death, metabolism, and ultimately diseases such as cancer. The fates of p53 family and oxidative stress are complexly intertwined with cross-regulation at many levels. The intricacies of this cross-regulation are further complicated by the presence of numerous isoforms of each p53 family member. Consequently, it is imperative to delineate the S. K. Gurung · K. S. Vikramdeo · N. Mondal (*) School of Life Sciences, Jawaharlal Nehru University, New Delhi, India e-mail: [email protected] L. Nigam School of Computational and Integrative Sciences, Jawaharlal Nehru University, New Delhi, India © Springer Nature Singapore Pte Ltd. 2022 S. Chakraborti et al. (eds.), Handbook of Oxidative Stress in Cancer: Mechanistic Aspects, https://doi.org/10.1007/978-981-15-9411-3_92

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involvement and cross-regulation of p53 family isoforms and oxidative stress in diseases such as cancer. This chapter focuses on deciphering the involvement and intricate cross-regulation of p53 family members and oxidative stress in genomic integrity, cellular metabolism, and cell death in the light of cancer. Keywords

p53 family · p63 · p73 · Oxidative stress · Cellular metabolism · Genomic integrity · Cell death

Introduction Modulation of cell cycle and induction of apoptosis are two of the most common consequences of tumor suppressor protein, p53, and activation. The transcription factor p53 is activated by various stimuli and responds accordingly in a tightly regulated manner (Brooks and Gu 2010). Often p53 is activated in response to genotoxic stresses induced by both intrinsic and extrinsic factors. It also responds to other stimuli including metabolic stress, hypoxic conditions, and activation of oncogenes. The stress response of p53 is dynamic and depends upon the extent and context of stress-induced damage. Upon encountering minor stresses, p53 alters the cellular division cycles by inducing cell cycle arrest and providing a time window for the cell to overcome the damage and ultimately reenter the division cycles (Brooks and Gu 2010). However, under conditions of extreme and irreparable damages, p53 diverts the affected cells toward cell death or senescence. p53 is one of the most studied protein owing to its tumor-suppressive functions. Several studies have reported loss of p53 tumor suppressor function through either mutations or deletion in approximately 50% of cancers. Transcriptionally active p53 induces its tumor suppressor functions through transcriptional target genes such as p21, PUMA, NOXA, etc. (Brooks and Gu 2010). It may also indirectly induce transcriptional repression of various genes involved in cell proliferation and antiapoptotic genes (Chen 2016). p53-induced cell cycle arrest is mediated through transcriptional activation of its bonafide target gene p21. The G1 phase cell cycle arrest induced by p53 is brought about by binding and inhibition of cyclin E/Cdk2 complex with p21. Moreover, p53-mediated p21 transcription induces S phase and G2/M phase arrest through cyclin A/CDK2 and cyclin B/Cdk1 inhibition, respectively. Involvement of another p53 target 14-3-3σ is also implicated in G2/M cell cycle arrest (Chen 2016). p53-DREAM pathway-mediated transcriptional repression of target genes (Engeland 2018) has also been reported to control the cell cycle regulation. Nontranscriptional function of p53 includes interaction of p53 and apoptosis-related factors such as Bcl2 proteins, and localization of p53 into mitochondria modulating mitochondria-mediated functions (Chen 2016). Along with this, p53 is also capable of regulating components involved in metabolism thus affectively inducing metabolic reprogramming of cells to overcome the stress (Chen 2016). The ability of p53 to respond to multifaceted stimuli endows it with great power and responsibility of maintaining the cellular and genomic integrity of cells and organism thereof.

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Fig. 1 Human p53 showing alternative promoter sites and various isoforms

P53 protein shares structural homology with two other proteins, namely p63 and p73, constituting the p53 family. The p53 family members, sharing structural and functional homology, can activate p53 target genes by binding to p53 response elements. Like p53, the family members consist of multiple structural domains: the DNA binding domain (DBD), oligomerization domain (OD), and transactivation domain (TAD) (Ferraiuolo et al. 2016). In addition, p63 and p73 also harbor Sterile Alpha Motif (SAM). The TAD domains of p63 and p73 bear a 22% and 30% similarity with p53; OD domain exhibits 38% similarity with p53 while their DBD domains exhibit higher 60% and 63% similarity with p53, respectively (Pflaum et al. 2014). Each of the p53 family members comprises of numerous isoforms generated through transcription from alternate promoters and alternative splicing (Figs. 1 and 2). The major isoforms of p53 family members include the full length TA isoforms and N-terminally truncated ΔN isoforms. Owing to the diverse range of isoforms, there is a tremendous diversity in the functions of p53 family members. The expression of p63 isoforms depends upon the cell and the types of tissues with prominent roles in embryonic development. Studies have linked its importance to bone morphogenesis and epithelial cells development (Costanzo et al. 2014). Specifically mice with full-length TAp63 isoform deletion (p63 / ) have been reported to exhibit developmental syndromes with absence of skin and limbs; however, its relevance in carcinogenesis is not well documented due to embryonic lethality. On the other hand, the ΔNp63 isoforms have been found to be overexpressed in cancers such as squamous cell carcinoma, breast cancer, gastric cancer, prostate cancer, and bladder cancer (Candi et al. 2007). Owing to its homology with p53 proteins, ΔNp63 isoforms have been implicated to modulate the expression of p53 as well as TAp63-responsive genes (Pflaum et al. 2014). P53 homolog, p73, has been observed to be highly expressed in tumors; however, its mutation in cancers has been rarely found (Pflaum et al. 2014). Early confusion pertaining to tumorsuppressive or -promoting properties of p73 is attributed to contrasting functionality of full-length TA isoforms and N terminally truncated ΔN isoforms. In mice-related studies, loss of full-length TAp73 increased cancer predisposition while loss of ΔNp73 isoforms reduced tumor growth. Similar to p63, the relative ratio of TAp73 and ΔNp73 dictates the outcome of p73 function in cancer cells (Costanzo et al. 2014). Paradoxically, though p73 expression drives apoptosis in cancer cells, it has been found to be upregulated in many cancers with higher malignant behavior and

Fig. 2 p63 and p73 genes along with their various promoter sites and isoforms

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poor survival (Costanzo et al. 2014). Transcriptional upregulation of TAp73 was observed upon p53 knockdown in breast cancer cells or p53 null colon cancer cells (Tophkhane et al. 2012). The upregulation of TAp73 in absence of p53 highlights the presence of operational coping mechanism of cells to p53 loss. p53 family members and oxidative stress are intricately intertwined in maintaining the cellular homeostasis. The sophisticated inter regulation of p53 family members further increases the complexity of their interaction with oxidative stress caused due to the imbalance of the reactive oxygen species (ROS) (Liu et al. 2008). ROS are a class of highly reactive molecules such as superoxide anion, H2O2, OH radical, etc., which are abundantly present in cellular environments, rather in cellular compartments. Decades of study have led to a substantial accumulation of information regarding the involvement of ROS in various biological processes including cellular immunity, death, proliferation, aging, and cancer (Reuter et al. 2010). The source of ROS can be both external and internal. External sources of ROS include environmental factors such as radiation and chemicals, while intracellularly, ROS can be generated by mitochondria, ER, peroxisomes, NADPH oxidase, etc. (Reuter et al. 2010). As the involvement of ROS in initiating or mediating various signaling cascades is crucial, its interaction with other components of the signaling cascade bears equally significant prominence.

Role of p53 Family Members in Genomic Integrity Maintenance Under Oxidative Stress DNA damage is a major event occurring under oxidative stress. ROS generated through either internal or external sources can induce single strand DNA damage as well as more deleterious DNA double strand breaks. Upon DNA damage, sensor ATM/ATR gets activated by auto phosphorylation which further activates the downstream mediators of DNA repair components including p53 (Blackford and Jackson 2017). Depending upon the stimulus, p53 undergoes various posttranslational modifications such as phosphorylation, ubiquitylylation, methylation, acetylation, and sumoylation (Meek and Anderson 2009). P53 ubiquitylation and sumoylation maintains the cellular homeostatic levels of p53 by proteasomal degradation, while p53 stabilization and activation is promoted by phosphorylation, methylation, and acetylation. Upon DNA damage, p53 is phosphorylated by ATM/ATR-activated kinases Chk2/1 leading to its activation and stabilization. In response to DNA damage, a cascade of events are initiated that determine the fate of the cell, and p53 plays a vital role in this regard. Under DNA-damaged conditions, p53-cathepsin axis has also been reported to be involved in the ROS-mediated programmed necrotic cell death (Tu et al. 2009). Signal transduction pathways leading to cell death and mode of cell killing due to DNA damage depend on the status of p53. Kutuk et al. showed the involvement of BIK (BCL2 Interacting Killer) in cisplatin and UV-induced mitochondrial apoptosis in p53 wild-type HCT116 cells. While in p53 null HCT116 cells, cisplatin and UV-induced necrotic cell death were independent of BIK proteins (Kutuk et al. 2017). Aoubala et al. showed that low-dose doxorubicin-induced

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DNA damage led to activation of p53, which in turn transactivated Δ133p53α expression. Δ133p53α inhibited p53-mediated G1 arrest and apoptosis without affecting G2/M arrest (Aoubala et al. 2011). This study highlighted the involvement of Δ133p53α in cellular response to DNA damage. P53 isoform Δ133p53 and its zebrafish orthologue Δ113p53 have been quite extensively studied in the context of DNA damage response as compared to other p53 isoforms. Gong et al. reported the involvement of Δ133p53/Δ113p53 in the DNA double strand break repair. Δ133p53 as well as Δ113p53 was significantly upregulated upon γ-irradiation but not in UV-irradiated or heat shock-treated cells. Upon DNA damage, Δ133p53 was reported to coordinate with p73 to transcribe DNA damage repair-associated genes RAD51, RAD52, and LIG4 thus promoting DNA repair and cell survival (Gong et al. 2018). There are compelling number of evidences which suggest that the activity of p63 and its isoforms increases in response to DNA damage (Bretz et al. 2016) and can induce DNA repair or apoptosis depending on the extent of DNA damage and the cell type. P63 activation can have differential outcome in the context of DNA-damagerelated genes. Ribonucleoside-diphosphate reductase subunit M2 B (RRM2B), reported to be induced by both p53 and p63 in primary neonatal human foreskin keratinocytes (HFKs), suggested that both the members of p53 family can activate DNA repair mechanism. However, there seems to be opposing roles in response to genotoxic stress where binding of p53 to RRM2B is increased while binding of p63 decreases (McDade et al. 2014) leading to its downregulation. DNA-damage-binding protein 2 (DDB2) and xeroderma pigmentosum group C (XPC) protein are key molecules involved in nucleotide exchange repair (NER). Both p53 and p63 can modulate the expression of DDB2 and XPC gene depending on cell condition, where p53 binding increases while p63 binding decreases in cells undergoing genotoxic stress. Moreover, cells with deleted p53 and/or p63, when treated with genotoxic agents, show that p53 depletion results in downregulation of these DNA repair genes while p63 depletion shows significant upregulation of these genes, suggesting that in response to genotoxic stress p53 activity is upregulated because of rescue from p63 (McDade et al. 2014). p63 plays crucial role in protection of DNA in both male and female germline cells and can thus be referred to as “guardian of the human reproduction” (Amelio et al. 2012). Phosphorylated form of p63 binds to DNA-binding sites with higher efficiency. The phosphorylation of p63 can be triggered by many kinases including c-ABL, ATM, and CDK2, which sense DNA damage (Amelio et al. 2012). Once phosphorylated, TAp63 induces expression of PUMA and NOXA which induces apoptotic death in primordial follicle oocyte (Kerr et al. 2012). The GTAp63 (Germline specific Transactivating p63), one of the isoforms of p63, is functionally significant in human testis, where, in response to genotoxic stress followed by caspase cleavage, it induces expression of proapoptotic genes PUMA, NOXA, and CD95L and thus maintains genomic integrity of spermatozoa (Beyer et al. 2011). Thus, p63 maintains human germ cell DNA integrity in response to genotoxic stress by inducing p53 response proapoptotic genes, and both p63 and p53 share a common response pathway upon genotoxic stress in different genome settings.

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Apart from germline cells, p63 plays vital role in addressing the genotoxic insults encountered by cancerous cells. Petitjean et al. expressed various isoforms of p63 in p53 null (p53 / ) hepatocellular carcinoma cell line HepG2 and challenged it with genotoxic stress. They observed that upon genotoxic stress by doxorubicin, p63 protein accumulated in the cells and underwent phosphorylation. In addition, expression of full-length TAp63 isoform led to accumulation of ΔNp73 under genotoxic stress (Petitjean et al. 2008). ΔNp63 has shown involvement in DNA repair induced by cisplatin treatment in squamous cell carcinoma (SCC) through transcriptional upregulation of FANCD2 and RAD 18. Thus, depletion of ΔNp63 or FANCD2 led to sensitization of SCC cells to cisplatin (Bretz et al. 2016). P63 constitutively induces the expression of BRCA1, BRCA2, and FANCD2 genes and, thus, promotes the repair of dsDNA breaks occurring during normal cellular processes. However, in response to genotoxic insults, p53 represses the expression of these p63 binding genes and attenuates associated DNA repair (McDade et al. 2014). P73 is phosphorylated and stabilized like p53 upon DNA damage leading to apoptosis of affected cells; however, the phosphorylation is mediated by c-Abl kinase (Costanzo et al. 2014). p73 and p53 have similar binding sites and can regulate expression of common transcriptional targets (Pflaum et al. 2014). Moreover, in cases of DNA damage, p73 has been reported to be regulated by p53 and p73 itself. DNA damage induced by bile acids induces activation of p73 but not of p53 and p63 in a c-abl kinase-dependent manner (Zaika et al. 2011). P73 induced DNA repair through transcriptional activation of SMUG1 and MUTYH. p73 deficiency was shown to reduce DNA repair efficiency under bile acid-induced DNA damage conditions (Zaika et al. 2011). Checkpoint kinases, Chk1 and Chk2, which mediate various cellular outcomes such as cell cycle arrest, DNA repair, and cell death play a crucial role in transcriptional activation of p73 through p53. Interestingly in absence of p53, it was shown that Chk1, Chk2, E2F1, and p73-mediated cytotoxic drugs induced cell death (Urist et al. 2004). Furthermore, E2F1, a target of Chk1 and Chk2, was shown to regulate expression of p73 through removal of C-EBPα repression on E2F1 (Costanzo et al. 2014). Δ Np73, the dominant negative isoform of p73, is activated in a p53-dependent manner under DNA damage conditions and regulates p53-mediated cell cycle arrest. Δ Np73 has been reported to antagonize p53 and TAp73, thus promoting cell proliferation, and inhibit apoptosis. p73 isoform ΔNp73β inhibited p53 activation through direct interaction with p53BP1. ΔNp73β being a target of both p53 and p73 has been reasoned to provide a negative feedback regulatory role through inhibition of p53BP1consequently reducing activated ATM and stabilizing MDM2 leading to p53 inhibition (Vernersson-Lindahl and Mills 2010).

P53 Family and ROS Cross Talk in Cellular Metabolism The carbohydrate metabolism is a crucial pathway involved in the energy generation and maintaining homeostasis of various cellular components. The p53 family members play a significant role in the glucose metabolism through the regulation of HK2,

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TIGAR, G6PD, Sirt1, GLS2, AMPKα2, etc. (Costanzo et al. 2014). The p53 transcriptional targets HK2 (Hexokinase 2) and TIGAR (TP53-induced glycolysis and apoptosis regulator) regulate glycolysis and pentose phosphate pathway (PPP) through glucose 6 phosphate which acts as substrate in both of the pathways (Costanzo et al. 2014). These glucose metabolism pathways generate reducing agents such as NAPDH that are involved in maintenance of redox balance in cellular environment. TIGAR induces NADPH production through PPP, which in turn produces GSH. TIGAR overexpression led to rescue of cell from p53-mediated apoptosis through ROS quenching (Bensaad et al. 2006). TIGAR also increases DNA Repair. It is evident that TIGAR regulates p53-mediated effector functions initiated due to genotoxic stress through modulation of glucose metabolism by inhibiting glycolysis and promoting PPP ultimately leading to ROS inhibition. Both p63 and p73 family members are involved in regulation of glucose metabolism and consequently in the ROS homoeostasis (Berkers et al. 2013). TAp63 also favors the shift toward the pentose phosphate pathway by reducing flux of glycolytic intermediates toward Krebs cycle resulting in their pooling up in the cytoplasm (D’Alessandro et al. 2014) leading to modulation of carbon metabolites involved in glycolysis, PPP, and nucleotide biosynthesis. Moreover, p73 can promote PPP through induction of glucose-6-phosphate dehydrogenase and support cell proliferation (Costanzo et al. 2014). Thioredoxin-interacting protein (TXNIP), a ROS sensor, can inhibit MDM2p53 interaction thus rescuing p53 from degradation leading to activation of p53-mediated antioxidant processes (Humpton and Vousden 2016). However, contrastingly under extreme oxidative stress, p53-mediated prooxidant activities are activated thus diverting cells toward oxidative stress-induced cell death. For example, p53 can inhibit PPP pathway through inhibition of glucose-6-phosphate dehydrogenase function leading to shortage of antioxidants causing oxidative stressinduced cell death (Humpton and Vousden 2016). Similarly, p73 can inhibit G6P formation through induction of IAPP leading to inhibition of PPP thus consequently depriving cells of antioxidants (Napoli and Flores 2017). Depletion of ΔN isoforms of p63 and p73 was reported to induce IAPP leading to regression of p53-deficient tumors (Venkatanarayan et al. 2015). SLC7A 11 is a p53 target protein involved in the maintenance of GSH levels by controlling cysteine supply which is a key component in the GSH synthesis, and the tumor suppressor function of p53-SLC7A11 is intact in p533KR mutant (Jiang et al. 2015). p53 regulates the expression of SESN1/2 and glutathione peroxidase under normal physiologic conditions and thus maintains ROS homeostasis (Liu et al. 2008). Under stressed conditions, p53 is capable of directly activating genes coding for redox active proteins such as quinone oxidoreductase (NQO1) and proline oxidase (POX) leading to oxidative stress and ultimately causing ROS-induced apoptosis (Liu et al. 2008). In addition, p53 can also activate other oxidative stress-inducing proteins such as PUMA and BAX which when localized to mitochondria can initiate ROS production. The mTOR proteins in association with other components consisting of mTOR, Deptor, mLST8 [GbL], PRAS40, and Raptor that form the complete mTORC1 complex are involved in cellular metabolism and cell

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proliferation (Hasty et al. 2013). Having a significant role in the maintenance of cellular energy requirements and proliferation, mTORC1 is a target of multiplesignaling pathways including p53 family (Hasty et al. 2013). Normally, p53 exerts inhibitory effect on mTORC1 leading to suppression of cell proliferation. Particularly, p53 exerts the inhibitory effect through the expression of sestrin, which activates AMPK leading to inhibition of mTORC1. p53 can also inhibit mTORC1 directly or indirectly through REDD1, TSC2, and PTEN (Hasty et al. 2013). The inhibition of mTORC1 is context dependent. Ischemic conditions induced ROS, observed frequently in solid tumors with insufficient vasculature, suppressing mTORC1. These ischemic conditions induce SOD1 (super oxide dismutase 1) activity through mTORC1 inhibition, consequently allowing the cancer cells to survive nutrient-deprived and oxidative stress-prone tumor microenvironments (Kong and Chandel 2018). TAp63 induces the expression of Sirt1, AMPK, and LKB1 in response to calorie restriction and thus decreases oxidation of fatty acids and decreases oxidative stress during calorie restriction and starvation (Su et al. 2012). In addition, LKB1 regulates the activity of mammalian target of rapamycin (mTOR) during energy restriction. mTOR is a key regulator of translation which consumes excessive energy. Thus, by inhibiting LKB1-mediated mTOR activity, p63 reduces oxidative stress by lowering down cellular activity in response to starvation/calorie restriction (Wolff et al. 2011). Sirt1 serves as the connecting link between the p63 and p53 crosstalk, where in response to TAp63 induction, Sirt1 deacetylates p53 at Lys-382, and under such conditions, p53 translocates into mitochondria where it induces transcriptionindependent apoptosis. In tumor cell lines as well as primary cells, p63 has been shown to regulate cell metabolism through mitochondrial glutaminase 2 (GLS2). TAp63-mediated expression of Sirt1 regulates expression of mitochondrial glutaminase 2 upon conditions of calorie restrictions (Su et al. 2012). It was also observed that p63 and GLS2 expression were elevated under oxidative stress conditions in cancer cells. Interestingly, downregulation of GLS2-sensitized cancer cells to oxidative stress induced damage. Besides activation by Sirt1, GLS2 is also a bona fide target of TAp63 (Costanzo et al. 2014) suggesting that the TAp63-Sirt1-GLS2 axis is a major player in regulating cellular ROS. GLUT1-mediated glucose uptake maintains its high levels which are metabolized to generate antioxidant NAPDH and GSH thus increasing cancer cells’ defense against oxidative stress. Transcription factors p63 and SOX2 (Synthesis of cytochrome c oxidase 2) were found to be involved in transactivation of intronic enhancer cluster of SLC2A1 (Hsieh et al. 2019). Further strengthening the role of P63 protein particularly ΔNp63, its involvement in glucose metabolism was highlighted through regulation of the primary enzyme Hexokinase II by ΔNp63. This provides a direct evidence of p63 involvement in maintaining cellular energy levels and protection of cells from oxidative stress. Consequently, the presence of ΔNp63-Hexokinase II axis in cancer cells indicates that p63 plays an important role in metabolic programming and oxidative regulation (Viticchiè et al. 2015). Mitochondria lies at the heart of energy production through oxidative phosphorylation (OXPHOS). p53 family members are well equipped to modulate metabolic

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activities such as glucose metabolism and fatty acid oxidations which provide the fuel for ATP, NADPH, and FADH2 production (Berkers et al. 2013). The p53 targets, synthesis of cytochrome c oxidase 2 (SCO2) and mitochondrial encoded cytochrome c oxidase 1 (MT-CO1) as well as TAp73 target cytochrome c oxidase 4 isoform 1 (COX4i1) are involved in OXPHOS maintenance (Costanzo et al. 2014). p53 proteins can also localize in the mitochondria and alter the mitochondrial genomic and membrane integrity. Beyond its transcriptional role, p73 was shown to regulate oxidative stress through control of protein synthesis. Depletion of p73 increases ROS sensitivity along with reduction in ATP levels, AMP hyperactivation, and defect in the translational procedures. P73 promotes mitochondrial activity and redox homeostasis through proper translation of mitochondrial transcripts. Marini et al. reported the role of p73 in oxidative stress response through regulation of translational activity of cells (Marini et al. 2018). Ferredoxin reductase (FR) is an important component of electron transport chain machinery and transfers electron from NADPH to cytochrome P450. All the members of p53 family can bind to the FR protein-encoding FDXR gene via p53RE. Besides, p53, isoform α of p63, and p73 can increase acetylation of histones H3 and H4 leading to accessibility to FDXR promoter in response to oxidative stress and also leading to overexpression of ferredoxin reductase which then initiates ROS-mediated cellular apoptosis (Liu and Chen 2002). Cellular metabolism, therefore, is a common target of both p53 family members and oxidative stress inducers through which they maintain the cellular homoeostsis (Fig. 3).

ROS and p53 Family in Cell Death ROS modulates various cellular responses and the outcome of these signal transductions; either cell survival or death depends on ROS levels. Cancer cell can tolerate and maintain relatively higher levels of ROS than normal cells for survival. Deviation from this ROS homeostatic level exerts tremendous pressure on redox regulatory machinery of cancer cells. Therefore, alteration in levels of ROS diverts cancer cells toward cell death. Many cancer chemotherapeutic agents exploit this property by inducing ROS generation in cancer cells leading to oxidative stress-induced cellular response such as cell cycle arrest or cell death. Oxidative stress is one of the main causes of DNA damage causing threat to genomic integrity that leads to activation of p53 family members, mainly p53. The activated p53 further dictates the consequence of the cell fate leading to either cell cycle arrest, senescence, or cell death. Multiple signaling pathways emanating from various insults converge into p53, and simultaneously an array of signaling pathways diverge from p53 depending upon the type and severity of the insult (Brooks and Gu 2010). The association of ROS generation and p53 induction was correlated to senescence or apoptosis induction in cancer as well as normal cells. In addition, further increase in ROS levels through external sources at physiological p53 levels led to conversion of senescence to apoptosis (Macip et al. 2003). Moreover, PUMA and BAX play a critical role in p53-induced ROS and

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Fig. 3 Modulation of oxidative stress by p53 family through metabolic reprogramming

apoptosis. p53 family members can also indirectly affect the ROS levels in response to genotoxic damage by promoting expression of autophagy inducing genes such as ATG-2, ULK-1, TSC2, and UVRAG (Filomeni et al. 2015). Induction of autophagy leads to increase in energy-rich nutrients and macromolecules, which may then eventually lead to decrease in ROS levels and cessation of oxidative stress. Thus, p53 provides a link between DNA damage response, ROS, and autophagy which may be beneficial in understanding the behavior of the cells in adverse conditions. Δ133p53 the N terminal-truncated isoform of p53 exerts antagonistic effects toward p53-induced apoptosis. Δ133p53 is regulated by p53 through transcriptional activation of internal TP53 P2 promoter (Aoubala et al. 2011). Nutthasirikul et al. showed that silencing of Δ133p53 restored 5FU drug sensitivity in cholangiocarcinoma cell lines leading to apoptosis characterized by increase in bax/bcl2 ratio (Nutthasirikul et al. 2015). Apart from apoptosis, autophagy, and senescence, p53 finds its role in regulation of necrotic mode of cell death. Montero et al. showed that ROS-induced stress led to PARP1-mediated necrotic cell death which was regulated by p53. They observed that human colorectal and breast cancer cell lines as well as mouse embryonic

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fibroblasts were resistant to PARP1-mediated necrosis induced by H2O2 treatment upon p53 loss (Montero et al. 2013). Tu et al. reported that necrotic cell death due to ROS-generated DNA damage can be induced by p53 through lysosomal cysteine protease cathepsin Q activation (Tu et al. 2009). Interestingly, blocking of necrosis mediated by PARP1 led to activation of p53-mediated apoptosis. p63 can influence the endogenous levels of ROS by regulating REDD1 (regulated in development and DNA damage response), a protein associated with increasing ROS in the cells in response to ionizing radiation (Ellisen et al. 2002). DNA damageinducible transcript 4 protein (DDIT4), encoded by REDD1 gene, regulates cell response to hypoxia by mTORC1 (mammalian target of rapamycin complex 1) activity (Brugarolas et al. 2004). P53 as well as p63 regulates DDIT4 expression. However, interestingly it can produce prooxidative and antioxidative response depending upon cell types. The N terminally truncated p63 isoforms, ΔNp63α, have been reported to rescue cells from oxidative stress-induced death arising as a consequence of DNA damage, ferroptosis-inducing agents, and anoikis (Wang et al. 2017). From the study of “The Cancer Genome Atlas,” ΔNp63α was shown to modulate glutathione metabolism pathway in human tumors. Wang et al. also reported that ΔNp63α cooperated with Bcl2 family proteins to promote cancer cell survival and metastasis. The epidermis lining the lungs is under constant threats of oxidative stress generated during aerobic metabolism. Recently, role of ΔNp63α has been highlighted in protecting the epidermal cells from oxidative stress through transcription of cytoglobin. The member of the globin protein family, cytoglobin, which is involved in facilitation of oxygen diffusion in tissues and scavenging of ROS has been identified as the direct transcriptional target of ΔNp63α (Latina et al. 2016). The identification of ΔNp63α-cytoglobin axis in lung and breast cancers further highlights their role in these tumors. GPX2, a member of glutathione peroxidase, is a target of p63 whose overexpression has been shown to alleviate cells of oxidative stress-mediated cell death. Conversely, the repression of GPX2 has been shown to increase the susceptibility of cells to oxidative stress-induced damage. The protective function of GPX2 is p53 dependent. Tumor cells expressing elevated levels of various isoforms of p63 specially the ΔN isoforms of which GPX2 is a target suggest that p63 isoforms act antagonistically to p53-dependent oxidative stress response (Yan and Chen 2006). p63 was identified as the transcriptional regulator of FOXM1 which is a member of Forkhead superfamily transcription factors in normal keratinocytes. Depletion of FOXM1 induces ROS production and reduction of ROS-regulating genes such as SOD2, catalase, and GPX leading to increase in sensitization of keratinocytes and squamous carcinoma cells to oxidative stress-induced apoptosis. It suggests that cancer cells may utilize p63/FOXM1 axis to avert oxidative stress-induced effects (Smirnov et al. 2016). In another study, Huang et al. showed that ΔNp63α is rapidly phosphorylated by ATM, CDK2, or p70s6K during DNA damage leading to degradation of ΔNp63α thus paving way for cells to undergo apoptosis (Huang et al. 2008). Proapoptotic role of p73 in cancer cells has been realized through overexpression studies of TAp73α, TAp73β, and TAp73γ where it was found to induce

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transcription of PUMA and Bax. Moreover, TAp73β isoform was also observed to induce p57kip2, a CDK inhibitor (Vikhreva et al. 2018). p73 transcribed PUMA induced the translocation of Bax into the mitochondrial membrane and led to cytochrome c release and consequently apoptosis (Vikhreva et al. 2018). ΔNp73 isoform has been reported to exhibit dominant negative characteristics by inhibiting TAp73 and p53-induced apoptosis. p73-transcribed proapoptotic protein, GRAmD4, promotes apoptosis by binding to bcl2 and facilitating Bax release and translocation to mitochondria (Yoon et al. 2015). Furthermore, interaction of p73 and RanBp9 has been implicated in apoptosis. P73 and RanBp9 expression in HT22 cells led to ROS generation, Bax oligomerization, and ultimately mitochondrial apoptosis (Yoon et al. 2015). P73 transactivates CD95 which bears p53-binding sequence leading to its upregulation and ultimately extrinsic apoptosis while ΔNp73 isoform inhibits CD95 transactivation thus inhibiting p53 and TAp73β-induced apoptosis (Yoon et al. 2015). Kostecka et al. reported combinatorial usage of ROS inducer, and proteasome inhibitor induced JNK-mediated p73 phosphorylation causing disruption of p73/MDM2 leading to stabilization of p73 which led to induction of apoptosis (Kostecka et al. 2014). Upon DNA damage ΔNp73, the dominant negative form of p73, which negatively regulates the function of p53 and p73, is rapidly degraded. The degradation of ΔNp73 relieves the block on p53 and p73 thus leading to activation of p53- and p73-mediated functions such as cell cycle arrest and apoptosis upon DNA damage (Maisse et al. 2004). As evident, cross regulation between p53 family members and oxidative stress affects various aspects of cellular processes thus determining the fate of the cells (Fig. 4).

Conclusion There is overwhelming evidence pertaining to involvement of p53 family members and oxidative stress in dictating cellular fate. The intricate cross talk among members of p53 family in regulating cellular metabolism, genotoxic stress, and cell fate is well documented through decades of studies. Emerging evidences point toward equally significant cross-regulation of oxidative stress and p53 family members under various spatiotemporal conditions. The tremendous variations in the isoforms of p53 family members further complicate the cross talk occurring among p53 family members and oxidative stress inducers. Particularly, in cancer which bears considerably higher levels of oxidative stress and overexpression of N terminally truncated isoforms of p53 family members, their interactions play significant role in proliferation and death evasion of cancer cells. Therefore, meaningful dissection of interaction between oxidative stress and p53 family members may extend our understanding of their role in cellular environments. As evidence suggests the role of p53 family members and oxidative stress is crucial in cancer, further studies deciphering the key points in p53 family and ROS cross regulation may be helpful in development of better therapeutic measures.

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Fig. 4 Illustration depicting modulation of various cellular processes by p53 family and oxidative stress

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Mutant K-Ras-Mediated Oxidative Stress in Pancreatic Cancer Divya Thomas, Satish Sagar, Tristan Caffrey, and Prakash Radhakrishnan

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . K-ras Mutation: The Driving Force of PDAC Tumorigenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Oxidative Stress in Pancreatic Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . K-rasG12D and Redox Balance in PDAC Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract

Pancreatic ductal adenocarcinoma (PDAC) is one of the highly lethal cancers with dismal prognosis due to lack of effective targeted therapies and resistance to chemotherapeutic drugs. Significant genetic alterations have been identified as the foremost cause for the instigation and propagation of PDAC, among which K-ras mutations are known as the driver mutation. Oncogenic expression of D. Thomas · S. Sagar Eppley Institute for Research in Cancer and Allied Diseases, Fred & Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha, NE, USA T. Caffrey Iowa State University, Iowa, USA P. Radhakrishnan (*) Eppley Institute for Research in Cancer and Allied Diseases, Fred & Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha, NE, USA Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE, USA Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, NE, USA Department of Genetics, Cell Biology and Anatomy, University of Nebraska Medical Center, Omaha, NE, USA e-mail: [email protected] © Springer Nature Singapore Pte Ltd. 2022 S. Chakraborti et al. (eds.), Handbook of Oxidative Stress in Cancer: Mechanistic Aspects, https://doi.org/10.1007/978-981-15-9411-3_94

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mutated K-ras results in metabolic changes and altered intracellular signaling pathways that can upsurge the level of cellular reactive oxygen species (ROS). Increase in ROS production further drives the progression of pre-cancerous intraepithelial lesions (PanIN) in pancreas. In order to keep ROS at a level where they are pro-tumorigenic and proliferative but not toxic, antioxidant defense systems are also upregulated in mutant K-ras driven neoplasia. This chapter brings a broad view on mutant K-ras mediated generation of ROS and the mechanisms of overcoming the deleterious effects of ROS through ROS detoxification systems during PDAC tumorigenesis. Keywords

Pancreatic cancer · Oxidative stress · K-ras mutation · NRF2 and ROS

Introduction Pancreatic ductal adenocarcinoma (PDAC) is among the highly lethal cancers with a 5-year survival rate below 10% and is predicted to become the second leading cause of cancer-associated mortality within the next decade in the United States (Rahib et al. 2014). According to GLOBOCAN database, the incidence of PDAC is 458,918 with high mortality of about 432,242 globally in 2018 (Ferlay et al. 2019). Regardless of recent advancement in the knowledge of potential risk factors associated with PDAC tumorigenicity and novel tools for the early disease diagnosis, PDAC incidence is predicted to increase with 350,000 new cases within 2030. The low survival rate is ascribed to several factors, of which later stage disease diagnosis stands as the major factor. Most of the patients with PDAC remain asymptomatic until the disease that develops to an advanced stage endures major challenges for the researchers (Kosmidis et al. 2016). The etiology of PDAC is poorly understood; however, cigarette smoking, alcohol, obesity, dietary factors, and age have been reliably identified as major risk factors for PDAC tumorigenesis (Lowenfels and Maisonneuve 2006). Virtually 70% of PDAC rise in the head of the pancreas with the rest 30% originate in the body and tail. At diagnosis stage, most of the PDAC spread beyond the pancreas and nodal metastases are quite common in PDAC patients (Luchini et al. 2016). Generally, PDAC progresses through a series of precursor lesions which are commonly known as pancreatic intraepithelial neoplasia (PanIN). Tumor progression is driven by specific genetic changes and each stage of PDAC progression is to be associated with specific mutations. The association of K-ras with PDAC was first identified three decades ago where Almoguera et al. have found a mutated oncogenic form of K-ras (KrasG12D) in most PDAC tumors (Almoguera et al. 1988). In a parallel study, Smit et al. have reported a mutation in codon 12 of the KRAS gene in 28 out of 30 patients (Smit et al. 1988). The K-ras oncogene encodes small GTPase (21KDa) which functions as binary “on-off” switches between guanosine triphosphate (GTP)-dependent active and guanosine diphosphate (GDP)-dependent

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inactive states. In normal inactive cells, K-ras is predominantly GDP-bound and inactive. Upon stimuli, K-ras transiently binds to GTP and regulates a myriad of intracellular signaling cascades (Cox and Der 2010). The binding interaction of K-ras to GTP is facilitated by guanine nucleotide exchange factors (GEFs). Cellular growth factors, cytokines, and chemokine receptors are capable to activate RAS either directly or indirectly by increasing access of GEFs. Whenever a cell has responded to an intracellular signal, GTP is hydrolyzed to GDP through interaction of K-ras with specific GTPase-activating proteins. The number of active K-ras molecules (K-ras+GTP) in a cell governs the levels of resulting signaling cascade (Spaargaren et al. 1995).

K-ras Mutation: The Driving Force of PDAC Tumorigenesis The RAS family consist of three genes in which KRAS mutations (84% of RAS mutation) predominate in pancreatic, lung, and colorectal cancer; NRAS mutations (12% of RAS mutation) predominate in acute leukemia, thyroid carcinoma, and cutaneous melanoma, and HRAS mutations (4% of RAS mutation) are found in head, neck, mammary gland, and bladder squamous cell carcinomas (Cox et al. 2014). Typically, K-ras mutation is the instigating event for PDAC. In a recent study, a cohort of more than 10,000 patients with advanced cancer have been studied and the sequence data were compiled using comprehensive MSK-IMPACT assay from tumor and matched sets normal tissues. In that, the percentage of K-ras mutation in a total of 491 Memorial Sloan Kettering Cancer Center (MSKCC) pancreatic cancer patients was found as 74.3 (Zehir et al. 2017). Among the site-specific mutations, G12D was found in more than 30% of total samples. The percentage of each site-specific K-ras mutations is outlined in Fig. 1. The transition from normal pancreatic tissue to PDAC is a stepwise genetic process that usually takes a span of 18 to 20 years (Iacobuzio-Donahue 2012). More than 95% of PanIN, the earliest pre-neoplastic stage of PDAC, is found with K-ras mutation. PanINs are graded from stage 1 to 3, and along this scale they exhibit several nuclear abnormalities and increasing cellular disorganization, where high-grade PanINs eventually transforming into PDAC. During PanIN formation, pancreatic epithelial cells acquire a cuboidal shape, increase mucin production and take on various degrees of cytological and architectural atypia. PanIN-3 lesions are characterized by papillary morphology (Distler et al. 2014). Molecular profile studies have demonstrated that K-ras mutation is followed by subsequent inactivation of the tumor-suppressor gene cyclin-dependent kinase inhibitor 2A (CDKN2A), which is further followed by inactivation of two other tumor-suppressor genes SMAD4 and tumor protein 53 (TP53) during the transition process from normal pancreas to PDAC (Fig. 2). Decades of research have laid foundation of our understanding of the vital role of K-ras cellular signaling in PDAC. KC (LSL-KrasG12D; Pdx1-Cre) and KPC (LSL-KrasG12D;LSL-Trp53R172H; Pdx1-Cre) mouse models of PDAC are widely used to recapitulate the initiation and progression of human pancreatic cancer

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Fig. 1 Most frequently mutated K-ras codons in a cohort of 491 MSKCC pancreatic cancer patients. In a cohort of 491 pancreatic cancer patients [study conducted in Memorial Sloan Kettering Cancer Center (MSKCC)], the frequency of mutated K-ras codons was found as follows: G12D (30.5%), G12V (24.8%), G12R (10.2%), Q61H (3.9%), G12C (1.8%), G12A (0.6%), G13D (0.4%), Q61L (0.4%), F28L (0.2%), S65N (0.2%)

CDKN2A

TP53 SMAD4

G12 G13 Q61

K-ras

Normal pancreatic cell

PanIN-1

PanIN-2

PanIN-3

PDAC

Fig. 2 Molecular changes occur during pancreatic cancer progression: K-ras gene mutation (expression of K-rasG12D) in normal pancreatic cells triggers low grade PanINs that progress to higher grade PanINs and PDAC. Mutational/epigenetical inactivation of tumor suppressor genes CDKN2A, TP53, and SMAD4 facilitates progression of PanIN to PDAC

(Aguirre et al. 2003; Hingorani et al. 2005). However, once induced, the expression of K-ras is irreversible in these models, which limits investigation of the activity of K-ras beyond tumorigenesis. Recently, Collins et al. have demonstrated that K-ras mutation (K-rasG12D) is required for all stages of PDAC carcinogenesis including PanIN inception, PanIN maintenance, and invasive metastasis using tissue specific,

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temporarily regulated, and reversible genetic mouse models (Collins et al. 2012). Although research attempts to find different routes either to silence or to target K-ras, these strategies are yet to transit any effective anti-Ras drug regimens into the clinic. The unsuccessful efforts in developing effective and selective direct inhibitors of K-ras leading to a common perception that K-ras is “undruggable,” and recent studies claim that this strategy may still hold promise though (Maurer et al. 2012; Sun et al. 2012; Shima et al. 2013).

Oxidative Stress in Pancreatic Cancer Cancer cells are always under insistent oxidative stress. Oncogenic transformation such as K-ras mutation and other metabolic variations leads to increased oxidative stress in PDAC cells. Cancer cells acclimatize to such persistent oxidative stress by activating several transcription factors that increase the production of endogenous antioxidants. Increased production of reactive oxygen species (ROS), a by-product of aerobic metabolism is a hallmark of pancreatic cancer. ROS is anti-apoptotic and is considered as a pro-survival mechanism in pancreatic cancer cells (Afanas’ev 2011). On the one hand, ROS-mediated DNA damage and genomic instability alters intracellular signal transduction that initiates tumorigenesis and the malignant transformation of cells (Hoeijmakers 2009). Parallelly, excessive ROS triggers apoptosis and autophagy in PDAC cells (Donadelli et al. 2011). In this way, ROS act as a doubleedged sword in PDAC. Several oncogenes participate in the enhanced production of intracellular ROS by altering signaling pathways. Weinberg et al. have reported that mutant K-ras upregulate mitochondrial ROS generation that is essential for the malignant transformation of PDAC cells through regulation of ERK-MAPK signaling pathway (Weinberg et al. 2010). Overexpression of oncogenes such as Myc, cyclin E, and Raf can silence the function of tumor suppressor genes which could further enhance the ROS production in cells (Sabharwal and Schumacker 2014). The increased intracellular ROS level could make PDAC cells more susceptible to oxidative stress induced cell death than normal cells. However, the ability of cancer cells to adapt to oxidative stress and to develop resistance against therapeutics by inducing the antioxidant defense mechanisms such as activation and stabilization of transcription factor nuclear factor erythroid-2-related factor 2 (Nrf2) would help the cells to survive. Thus, molecules that arbitrate oxidative stress adaptation could be better targets for the development of therapeutics against PDAC.

K-rasG12D and Redox Balance in PDAC Cells It has been demonstrated that oxidative stress collaborates with mutant K-ras (K-rasG12D) to initiate and promote tumorigenesis in murine pancreas model (Al Saati et al. 2013). It is essential for a cancer cell to retain ROS at a level where they are favorable for cell proliferation and survival, but below the threshold level, cells may undergo senescence or cell death (Liou and Storz 2010). Cancer cells often maintain

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ROS threshold level by an additional increase in the antioxidant defense system. A previous study has demonstrated that mutant K-ras driven carcinogenesis tightly regulates ROS levels that are accompanied by Nrf2-dependent antioxidant signature, a master switch in the antioxidant network (DeNicola et al. 2011). In order to achieve this, K-rasG12D is believed to promote beta oxidation of fatty acids that maintain intracellular NADPH/NADP+ ratio (Khasawneh et al. 2009). In an earlier study it was shown that ectopic overexpression of oncogenic Ras increased intracellular ROS production through pathways involving a flavoprotein, NADPH oxidase, and Rac1 (Irani et al. 1997). K-ras transfected PDAC cells have shown increased superoxide levels and NADPH oxidase (NOX) activity as compared to parental cells (Wang et al. 2015). Similarly, K-ras facilitated PDAC cell proliferation through the activation of Rac1-dependent NOX (Du et al. 2011); and NOX was found to be activated by K-ras through the translocation of p47(phox), the cytosolic regulatory subunit of NOX (Park et al. 2014). However, this notion has been contradicted by the previous study, which validated that ROS production was actually suppressed by endogenous expression of the K-rasG12D allele in NIH3T3 and MEFs cell lines (DeNicola et al. 2011). Consistently, human pancreatic pre-neoplastic cells and cancer cells exhibit higher Nrf2 to maintain lower levels of intracellular ROS, which significantly contribute to oncogenic K-ras mediated PDAC progression (Fig. 3). As illustrated above, K-ras driven tumors alleviate the damaging effects of ROS through the upregulation of ROS detoxifying antioxidant enzymes. This can be achieved by the activation of Nrf2, a transcription factor that regulates a number of ROS-detoxifying antioxidant genes. DeNicola et al. have provided evidences that primary PDAC cells and mice expressing KrasG12D mutations increase the transcription of Nrf2 (DeNicola et al. 2011). In another study, it was confirmed that oncogenic K-ras increases the expression of nuclear Nrf2 in acinar cells and in PanIN cells as a result of oxidative stress, as it is lost in pancreas of mutant K-ras expressing mice treated with mitochondria-targeted antioxidant (Liou et al. 2016). Nrf2 overexpression results in low, but pro-tumorigenic threshold levels of ROS in conditional KrasG12D Normal pancreatic cell

PDAC

PanIN-1

Oncogenic K-ras mutation

PanIN-2

PanIN-3

Cell death

Oxidative stress

ROS Anti-oxidants

Fig. 3 Maintenance of redox balance in normal vs. PDAC cells. Under normal conditions, cellular homeostasis is maintained by producing enough antioxidants to balance ROS generation. Genetic and metabolic alterations in cancer cells enhance ROS generation that is counteracted by abundant production of antioxidants. However, persistent oxidative stress in cancer cells disrupts the redox balance, and high ROS level can disturb normal cellular machinery, promoting cell death

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mice model (Al Saati et al. 2013). In addition to Nrf2, Pim kinases also contribute to K-RasG12V driven variation of intracellular ROS levels by regulating the expression and cellular metabolism of manganese superoxide dismutase (MnSOD), glutathione peroxidase 4 (Gpx4), and peroxiredoxin 3 (Prdx3) (Song et al. 2015). Further, Storz et al. have demonstrated that release of mitochondrial ROS activates a signal relay pathway that leads to induction of SOD2 through protein kinase D mediated activation of transcription factor NF-κB (Storz et al. 2005). In agreement with these reports, we also observed an upregulation of protein expression of Nrf2, SOD1, SOD2, and SOD3 in K-ras mutant cells. We have used parental hTERT-immortalized human pancreas nestin expressing cell line (hTERT-HPNE), HPNE cells with mutant K-rasG12D (HPNE K-ras), HPNE cells modified to express papillomavirus E6/E7 protein (HPNE 6/7), HPNE cells expressing E6/E7 in conjunction with oncogenic K-Ras (HPNE 6/7 K-ras), HPNE cells with SV40 small T-antigen (HPNE smt), and HPNE cells silenced with SV40 small T-antigen in conjunction with oncogenic K-Ras (HPNE 6/7 K-ras smt) (Campbell et al. 2007; Zhao et al. 2010) to examine whether expression of oncogenic K-ras with different transforming capacity may contribute to aberrant expression Nrf2 mediated ROS detoxifying antioxidant genes. As anticipated, expressions of Nrf2, SOD1 (Cu-Zn SOD), SOD2 (mitochondrial or manganese dependent SOD), SOD3 (extracellular SOD), and thioredoxin proteins were significantly high in HPNE cells expressing K-ras alone, HPNE 6/7 K-ras, and HPNE 6/7 K-ras smt cells as compared to parental HPNE cells (Fig. 4). Altogether, these studies have highlighted the concept that increased expression of Nrf2 can go along with increased generation of ROS by maintaining the enhanced expressions of ROS detoxifying antioxidants in mutant K-ras expressing pancreatic cancer.

Conclusion During tumorigenesis and progression of PDAC, oncogenic K-ras brings metabolic changes that lead to increased production of intracellular ROS. Oncogenic K-ras also upregulates the expression of ROS detoxifying antioxidant defense systems to balance ROS to a threshold level at which they drive other major signaling cascades that contribute to oncogenic transformation and tumor progression (Ardito et al. 2012; Navas et al. 2012). With these research findings, a key question may arise whether this can be applied for the development of novel therapeutic regimens against PDAC or not. From the available reports, it is obvious that inhibition of Nrf2 activity helps to reduce the induction of antioxidant defense system that drives oncogenic K-ras generated intracellular ROS to levels where they induce cellular senescence or cell death. This Nrf2-mediated antioxidant targeted approach may be most efficient in combination with chemotherapeutic drugs that additionally increase cellular ROS production. In spite of lacking sufficient clinical trials to date, research attempts for the better understanding of oncogenic K-ras driven tumor progression continue to move in a promising direction, which is hoped for the benefits of PDAC patients.

Fig. 4 Oncogenic K-ras induces Nrf2-mediated antioxidant proteins expression in immortalized HPNE cells. Exponentially growing cells were lysed with RIPA buffer and 30μg of cell lysates were analyzed by Western blotting with indicated antibodies. β-actin was used as a protein loading control

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HPNE HPNE K-ras HPNE6/7 HPNE6/7 Kras HPNE6/7smt HPNE6/7Kras smt

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SOD2

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SMA SOD1 Thioredoxin SOD3 β-Actin

42KDa 16kDa 12kDa

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mRNA Stabilizing Factor HuR: A Crucial Player in ROS-Mediated Cancer Progression

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Soumasree De and Kuntal Dey

Contents Introduction: mRNA Half-life, RNA-Binding Proteins, and Oncology . . . . . . . . . . . . . . . . . . . . . . . HuR and Its Nuts and Bolts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . HuR Linked in Tumorigenicity and Therapeutics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . HuR, the Effective Modulator of ROS Generation in Cancerous Traits . . . . . . . . . . . . . . . . . . . . . . Discussion and Future Directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract

Post-transcriptional regulation optimizes the balance between cellular mRNA flux and functional protein expression. Several RNABPs and noncoding RNAs participate majorly, among them HuR is one of the crucial proteins that plays significant role in mRNA stability and translation. HuR also tightens enough cellcycle and proliferation systems by maintaining the stability of associated genes. Thus HuR becomes a central molecular gear in cancer metabolism. This chapter mainly focuses on the involvement of HuR in ROS-driven carcinogenesis development and progression. Keywords

HuR · mRNA stability · Post-transcriptional regulation · ROS Abbreviations

ARE ECM

AU-rich element extracellular matrix

S. De (*) Department of Chemistry & Biochemistry, University of Bern, Bern, Switzerland K. Dey Institute of Veterinary Physiology, University of Zurich, Zurich, Switzerland © Springer Nature Singapore Pte Ltd. 2022 S. Chakraborti et al. (eds.), Handbook of Oxidative Stress in Cancer: Mechanistic Aspects, https://doi.org/10.1007/978-981-15-9411-3_97

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ELAV EMT mRNA RBP ROS RRM UTR

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embryonic lethal abnormal vision Epithelial mesenchymal transition messenger RNA RNA binding protein reactive oxygen species RNA recognition motif untranslated region

Introduction: mRNA Half-life, RNA-Binding Proteins, and Oncology Regulation of messenger RNAs (mRNAs) decay is crucial for controlling the abundance of cellular transcripts and, sequentially, in protein expressions (Shyu et al. 2008). mRNAs are directly responsible for cellular protein expression; thus, the quantity of functional and intact protein production per mRNA molecule is proportional of mRNA’s half-life and efficient translational initiation with proper elongation of coding sequence (Chen et al. 2008; Chávez et al. 2016). Moreover, any transcript can be regulated post-transcriptionally at multiple levels, including nuclear hnRNA splicing, cytosolic transport of rnRNA, stability, organelle specific localization, and translation (Orphanides and Reinberg 2002; Emerson 2002). Hence, the way from mRNA to protein is quite challenging and wellregulated. Both the mRNAs functional half-life and translational efficiency are driven by features encoded in the specific mRNA sequence, called cis-elements. The specific proteins that can bind in these cis-elements are trans-factors or RNA-binding proteins (RBPs) (Ross 1995). Turnover and translation regulatory RBPs and noncoding RNAs (particularly microRNAs) are the key trans-factors that hybridized to specific cis-elements in 30 -UTR of mRNAs and modulate their stability and translation (Valencia-Sanchez et al. 2006; Pullmann et al. 2007; Keene 2007). There are numerous of RBPs in eukaryotes, mainly with unspecified functions. Sometimes, RBPs and noncoding RNAs synergistically regulate mRNAs in a coordinated way to adjust their localization, half-life time, or protein synthesis (Keene 2007). Transacting proteins include AUF1, HuR, nucleolin, PCBP1, PCBP2, TTP, KSRP, etc. (Gerstberger et al. 2014) HuR, nucleolin, PCBP, etc. act as stabilizing factor, whereas TTP, AUF1, KSRP, etc. are destabilizing protein (García-Mauriño et al. 2017). mRNA stability and translation are energy-driven procedures, thus directly connected with cellular metabolic activity (Leibovitch and Topisirovic 2018). In a way, protein synthesis and metabolism perform critical positions in the maintenance of cell growth and proliferation homeostasis, but dysregulation leads to cancers, like, HuR regulates cell cycle and proliferation network by increasing the stability of mRNAs coding for prominent anti-apoptotic factors, thus promoting toward cancer (Burkhart et al. 2013). In this chapter, we focus on the involvement of HuR in cancer progression.

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HuR and Its Nuts and Bolts The Hu/ELAV (embryonic lethal abnormal vision) family of RNA binding protein is conserved across metazoans and has varied roles in mRNA dynamics (Hinman and Lou 2008). HuR (or HuA) is ubiquitously expressed across various tissues (Lu and Schneider 2004; Ma et al. 1996; Good 1995), initially detected in Drosophila melanogaster as crucial for neural development (Campos et al. 1985). While the other members, HuB, HuC, and HuD are exclusively expressed in the nervous system or in the germline (Ma et al. 1996; Campos et al. 1985), required for nervous system development (Kasashima et al. 1999; Akamatsu et al. 1999). HuR comprises three RNA recognition motifs (RRMs) (Wang et al. 2013a). The first two tandem RRMs directly interact with the specific AU-rich cis-elements (or AREs) in the 30 -UTR of many mRNAs. The third RRM is connected to the second RRM by a hinge region that is essential for nucleocytoplasmic shuttling of activated HuR (Brennan and Steitz 2001). Although HuR is predominantly nuclear in unstimulated cells, often it shuttles to the cytoplasm in response to various stimuli, including stress signals and mitogens. Subsequently, HuR association to the AREs leads to compartmentalization, stabilization, or translational regulation of the targeted mRNAs (Keene 1999). Presently, HuR regulates more than 90 functionally diverse ARE genes that are reported, whereas many more are expected. Given its main role in post-transcriptional gene regulation by AREs, HuR is believed as a predominant protein in the ARE pathway (Benoit et al. 2010; Lebedeva et al. 2011).

HuR Linked in Tumorigenicity and Therapeutics HuR regulates a wide subset of target mRNAs stability and translation, whose complementary proteins are involved in different pathologies, predominantly in cancer and inflammation (Srikantan and Gorospe 2012). A number of cancer-related transcripts containing AREs, including mRNAs for proto-oncogenes, cytokines, growth factors, invasion factors, etc., have been categorized as HuR targets. Thus HuR is recommended as a central regulator of tumorigenic activity in multiple cancer phenotypes (Wang et al. 2013b; Abdelmohsen 2013). Post-transcriptionally HuR regulates the expression of several tumorigenic proteins that promote cell growth and proliferation and aid the oncogenic cells to evade immune recognition for local angiogenesis and ultimately in metastasis. Henceforth, HuR is considering as one of the basal players in tumor development. Being the master regulator of mRNA processing and translation, possibly HuR is also a novel potential target for cancer therapy. For instance, HuR modulates many genes (ESR1, GATA3, CDKN1A, CXCL8, MMP9, ERBB2, and many more) and the importance of these target genes in breast cancer has already demonstrated directly in vitro and in vivo. Moreover, clinical studies revealed the association of HuR with aggressive forms of breast cancer in the prognosis of patients’ survival indicating HuR as a promising drug target for breast cancer therapy (Kotta-Loizou et al. 2016; Zhang et al. 2017).

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HuR, the Effective Modulator of ROS Generation in Cancerous Traits ROS (reactive oxygen species) promotes tumor development and progression in almost all types of cancer. Nevertheless, several therapeutic strategies generate elevated levels of antioxidant proteins to neutralize ROS by inducing apoptotic cascades. As a consequence, a quite obvious challenge is generated for novel therapeutic strategies to make a proper balance between intracellular ROS signaling and programmed cell death (Liou and Storz 2010). Emerging evidence has suggested that ROS is indispensable for metastasis by promoting epithelial mesenchymal transition (EMT) process through extracellular matrix (ECM) remodeling and cell mobility (Jiang et al. 2017). Urokinase plasminogen activator (uPA) is an extracellular serine protease that cleaves plasminogen, required for ECM degradation and MMP activation in metastasis. uPA is activated by binding to its specific receptor uPAR. High uPA and uPAR expression is linked with multiple types of cancer prognosis. It has been reported that ROS can induce the transcription of both uPA and uPAR and HuR genes. Additionally, HuR binds to the 3’-UTR of uPA and uPAR to stabilize their mRNA expression. On the contrary, HuR silencing leads to increased ROS levels in triple-negative breast cancer (TNBC), though, the actual mechanisms underlying ROS-mediated association of HuR at ARE-uPA need more investigation (Jiang et al. 2017). Triple Negative Breast Cancer (TNBC) is a discrete subset of breast cancer cell line categorized by aggressive clinical behavior with limited treatment options (Kyndi et al. 2008). HuR overexpression induces chemoresistance in TNBC. Whether Mehta M et al showed that siHuR treatment was very effective in TNBC cells to ionizing radiation at 2 Gy compared to control. The output was measured by higher levels of DNA damage and a significant reduction in cell survival. They conclude that HuR knockdown in TNBC cells elicits oxidative stress and DNA damage resulting in radio-sensitization (Mehta et al. 2016). Often, knockdown of HuR generates ROS in cancer cells, like pancreatic or colorectal cancers (Lin et al. 2017; Zarei et al. 2017). siHuR reduced transcriptional expressions of galectin-3, β-catenin, cyclin D1, Bcl-2, P-gp, MRP1, and MRP2 in epirubicin-treated colon cancer cells. Epirubicin is an anticancer drug used for chemotherapy, which generates free radicals that cause cell and DNA damage. HuR silencing increased the intracellular accumulation of epirubicin in colon cancer cells and substantially diminished the expression of Bcl-2. Moreover, increased expression of Bax, caspase-3 and -9, and their enhanced activity induce apoptosis (Lin et al. 2017). Another investigation based on RNAi or CRISPR approaches concluded that HuR promotes cell-death in drug-resistant pancreatic cell lines, MiaPaCa2, Panc-1, BxPC3, and HS-766T PDAC with or without gemcitabine treatment. The chemotherapeutic drug, gemcitabine is used in various carcinomas. RNA deep sequencing and functional analyses in HuR-deficient PDAC cell lines identified isocitrate dehydrogenase 1 (IDH1) as the sole antioxidant enzyme under HuR regulation, which highlights a new therapeutic target in pancreatic cancer (Zarei et al. 2017). All the above-mentioned studies have illuminated the

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significance of HuR in overall tumorigenesis and become an indispensable target for cancer treatment. Knock-down of HuR using siRNA revealed promising attributes to diminish oncogenic features in several studies that require more clinical investigations. However, tumor-specific siHuR delivery is another challenging issue in the lipid-based nano-formulation system.

Discussion and Future Directions Being a central molecular switch, HuR is in the focus of many researchers for the last two decades. By regulating several mRNA dynamics, HuR can effectively modify the associated cellular physiology and obviously join in many disease conditions like cancers, cardiac, neurological diseases, etc. Condition-dependent nucleus to cytosolic drift of HuR turns it more alluring to translational biologists, and people are fascinated to understand the global scenario using recently developed highthroughput technologies. Hopefully, that helps to open up several closed doors of HuR function.

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Redox State and Gene Regulation in Breast Cancer

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Aritra Gupta, Shayantani Chakraborty, Partha Das, Animesh Chowdhury, and Kartiki V. Desai

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Origin of ROS in Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ROS Mediated Signaling Pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ROS and Breast Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Additional Mechanisms of ROS Generation in Cancer Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Modulation of Cancer Pathways by ROS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . DNA Damage Response . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Epigenetics and Gene Regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Alterations in Signaling Pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cellular Proliferation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cell Survival . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Autophagy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Angiogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Motility and Metastasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract

Elevated levels of reactive oxygen species (ROS) support tumor formation and cancer progression. Since treatment efficacies in breast cancer need to be improved beyond the existing levels, targeting ROS pathways could be a novel strategy. In this chapter, we study the origin of ROS, ROS mediated gene regulation, and signaling in normal cells and review information about known ROS functions in cancer in support of its candidacy as a pathway enriched in potential novel targets. However, the titer of ROS in cancer cells is most critical, since excessive ROS leads to cancer cell apoptosis, whereas a controlled

A. Gupta · S. Chakraborty · P. Das · A. Chowdhury · K. V. Desai (*) National Institute of Biomedical Genomics, Kalyani, West Bengal, India e-mail: [email protected]; [email protected]; [email protected]; [email protected] © Springer Nature Singapore Pte Ltd. 2022 S. Chakraborti et al. (eds.), Handbook of Oxidative Stress in Cancer: Mechanistic Aspects, https://doi.org/10.1007/978-981-15-9411-3_98

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intermediate production of ROS promotes proliferation, motility, invasiveness, and angiogenesis at the site of cancer. Therapeutic strategies against ROS pathways that take into account this challenge of carefully tipping the balance towards cancer cell apoptosis would be most effective in treating certain types of breast cancer. Keywords

ROS · Hydrogen peroxide · DNA repair · Tumor progression · Cell metabolism

Introduction Breast cancer (BrCa) accounted for about 2 million cases globally and is the most commonly occurring cancer in women. In India, it accounts for 14% of all cancers found in women (https://www.who.int/cancer/prevention/diagnosis-screening/ breast-cancer/en/). The rate of onset in women in their early thirties is on the rise in India, and it peaks at ages 50–64 years (https://www.nhp.gov.in/breast-cancerawareness-month2019_pg). BrCas are highly heterogeneous in nature and contain a spectrum of diseases that can be classified on the basis of clinicopathological, histological, and molecular basis with varying prognostic and clinical outcomes. Classically, BrCas are classified into four stages (I–IV) and three grades. The breast is a female secondary sex organ required for milk production in higher mammals. It contains both hormone receptor positive and hormone receptor negative cells. Therefore, immunostaining for estrogen receptors (ER) and progesterone receptors (PR) emerged as the first molecular histopathological classifier of breast tumors and BrCas were divided into ER+ and ER
tumors. Similarly, they are also classified into PR+ and PR
tumors. The discovery of the amplification of an epidermal growth factor family member (Her2) gene residing on Chromosome 17q21 and subsequent demonstration that it is also overexpressed at the protein level led to the addition of another biomarker for BrCa (denoted as Her2+ or Her2
). The advent of genomic technologies such as microarray analysis and next generation sequencing allowed the cataloguing of the total transcriptome changes in RNA expression and genomic aberrations at the structural and single nucleotide level in over thousands of BrCa samples (https://www.cancer.gov/about-nci/ organization/ccg/research/structural-genomics/tcga). These studies recapitulated the original ER/PR/Her2 classes but also led to the discovery of additional subclasses. Gene based analysis now stratified BrCa into more than 6 subtypes (Fig. 1). About 70% of breast cancers are ER+, and these are further subdivided into Luminal A (LumA) and Luminal B (LumB) groups, with LumA having the best survival rates (Perou et al. 2000). The Her2+ tumors are mostly ER- and form another subtype. Targeted therapies against each of these receptors have improved survival and decreased mortality in women suffering from receptor positive breast cancer. However, triple negative breast cancers that are ER/PR/Her2 negative (TNBCs) are the

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Fig. 1 Molecular classification of breast cancer

most aggressive form of disease and associate with poorer prognosis. TNBCs are routinely treated with chemotherapy due to lack of available targeted therapies, exposing patients to extreme side effects and poor quality of life. Recently, at least 30–40% of TNBCs were found to express AR dividing this group in Luminal AR positive TNBCs (LARs) and AR-, that is quadruple receptor negative TNBCs (QNBCs) (Burstein et al. 2015). Despite these classifications and availability of targeted therapies, response to treatment can be improved further. About 30–40% of the women relapse following treatment and their cancer is refractory to the first line of therapy. These cancers require additional and newer drugs to achieve remission. A way to discover additional therapeutic targets is to consider targeting various hallmarks of cancer including angiogenesis and cellular metabolism (Hanahan and Weinberg 2000). Recent literature suggests that reactive oxygen species (ROS) generated due to mitochondrial dysfunction is also a key player in cancer progression. Production of ROS and the oxidative stress that follows affects many cellular signaling pathways due to nonmaintenance of cellular homeostasis. In this chapter, we summarize the ROS pathways and explore their potential as new targets in treating breast cancer. We describe the mechanisms of ROS generation and the ROS pathway in normal cells and distinguish it from that seen in cancer cells.

Origin of ROS in Cells Oxidative radicals can be divided into four groups: reactive oxygen species (ROS), reactive nitrogen species (RNS), reactive sulfur species (RSS), and reactive chloride species (RCS). Of these, ROS are the most abundant of species. Depending on their molecular stability, these compounds can have short or long half-lives. ROS includes superoxide anion (O2˙
), singlet oxygen (1O2), hydroxyl radical (OH•), hydrogen peroxide (H2O2), and ozone (O3) (Table 1). The next abundant compounds are RNS, in particular nitric oxide (NO
), and this has the ability to react with certain ROS to

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Table 1 Reactive oxygen species Type Super oxide

Chemical formula O2


Per oxide

O2


Hydrogen Per oxides Hydroxyl ion Hydroxyl radical Singlet oxygen Peroxyl radicals Alkoxyl radical Thiyl radical

H2O2 OH
OH˙ 1

O2

ROO. RO. RS.

Generation Reduction of molecular oxygen in the electron transport chain of mitochondria and other enzymatic routes: monooxygenase, NADPH oxidase, xanthine oxidase Converted from O
2 by enzyme superoxide dismutase (SOD) Converted from O
2 by enzyme superoxide dismutase (SOD) Formed by the loss of a proton from a water molecule. Produced in Haber-Weiss reaction from O-2 and H2O2 Produced in reaction of hypochlorous acid (HOCl) and H2O2 Lipid peroxidation in presence of oxygen centered molecule Lipid peroxidation in presence of oxygen centered molecule Generated by redox process of thiols and disulfide radicals

Detoxification Super oxide dismutase

Catalase Catalase Flavonoids Flavonoids Multiple pathways Multiple pathways Multiple pathways Multiple pathways

produce peroxynitrite anion (ONOO
). RNS are found to be lethal for cells and tissues. The RNS and their activities are studied elsewhere (Hanukoglu 2006; Jones 2008). ROS production in cell predominantly occurs in the mitochondria during aerobic metabolism, primarily by reduction of oxygen through electron transport chain (ETC) (Liu et al. 2002). During the course of ETC, electrons are transported in a downhill manner from one complex to the next, and leaking electrons, particularly from complex I and complex III, are the main players to reduce oxygen and produce ROS. Reduction of FMN (complex- II) by NADH/NADPH takes place and is followed by transfer of electrons to co-enzyme Q (CoQ) via iron dependent electron acceptors. A large amount of electrons are released during this process of intermembranous transfer (Starkov et al. 2004). Production of electrons was confirmed by blocking ETC using inhibitors such as Rotenone and Antimycin (Hanukoglu 2006). Apart from ETC, ROS is also generated using two oxoglutarate (2OG) as substrate. 2OG inhibits complex I resulting in the production of reasonable amount of ROS. In addition, ROS can be produced by two enzymes primarily known for their action in cholesterol pathway and steroid biosynthesis pathways: 5 alphaketo-glutarate dehydrogenase of the Kreb’s cycle and cytochrome P450 enzymes. The enzyme ATP synthase and xanthine oxidoreductase generate ROS, the latter enzyme produces these species during the course of amino acid generation (Bayir 2005).

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In smooth muscle cells of the vascular tissue, growth factors including platelet derived growth factor (PDGF), angiotensin II, vascular endothelial growth factor (VEGF), thrombin, and tumor necrosis factor alpha (TNFα) generate ROS (Brandes and Kreuzer 2005; Fujiki et al. 2005). These factors activate the membrane bound enzyme, NADPH oxidase, a member of the Nitrogen Oxide family (NOx) family that ultimately leads to production of H2O2. Similar pathways of ROS generation are observed in Neutrophils (polymorpho nulcear cells, PMNs) and they release a large amount ROS at the site of inflammation (Nguyen et al. 2017). Macrophages also produce nitric-oxide (NO)-based ROS not only in vascular tissue but also at the site of inflammation by cytokine signaling pathways (Ray et al. 2012).

ROS Mediated Signaling Pathways The phenotypic changes in the cellular behavior are exclusively dependent on the intra- and intercellular communication through signal transduction pathways. The discovery of specific genes and signaling pathways and their activation mediated by ROS led to the hypothesis that ROS could serve as the crucial subcellular messengers in gene regulatory and signal transduction pathways (Colletta et al. 1990; Roy et al. 2007). For example, hydrogen peroxide (H2O2) generated by NADPH oxidase enters the cell through aquaporin channels found on the plasma membrane (Bienert et al. 2007; Miller et al. 2010). This regulated entry of H2O2 could be designed to affect an intended target, leading to the regulatory activation of different signaling transduction pathways. Homeostasis of ROS and thus level of ROS dictates its nature of effect in a system. Usually, low-level of these reactive compounds act as essential triggers in normal cell function before their elimination. For example, in ER+ cells of the normal breast, estrogen action induces cell proliferation and a small amount of ROS production (Johar et al. 2015b). The ROS generated by the action of Estrogen can influence cell cycle, apoptosis, protein stability, and/or many other intracellular biochemical cycles. ROS directly affects MAPK pathway, induces cyclin D1, AKT, and promotes cell-cycle in breast cells. Some inhibitors like N-Acetyl L- cysteine (NAC) and Tiron inhibit estrogen induced cell proliferation by blocking phosphorylation of ERKs and JNKs pathway and some chemicals like Rotenone, Surfactin also induce ROS dependent apoptosis in breast cancer using the ERK and JNK pathways suggesting that ROS mediated signaling has profound effects on cell survival and function (Cao et al. 2010; Choi et al. 2010). The p53 tumor suppressor protein executes its function via the ROS generation, and this ROS in turn can modulate p53 by phosphorylation, ultimately leading to apoptosis and elimination of cells (Ostrakhovitch and Cherian 2005). In addition, H2O2 also acts as a second messenger for platelet-derived growth factor (PDGF), epidermal growth factor (EGF), fibroblast growth factor (bFGF), transforming growth factor-β (TGF-β1), interleukin-1, interleukin-3, and tumor necrosis factor-α (TNF-α) (Bae et al. 1997; Krieger-Brauer and Kather 1995; Lo and Cruz 1995; Sies and Jones 2020). H2O2 not only acts as a second messenger but

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also it phosphorylates many cell surface receptors such as receptor tyrosine kinases (RTKs). It also can activate mitogen-activated protein kinases (MAPKs) via the RAS pathway, protein kinase C (PKC) through activation of phospholipase C, protein kinase B (PKB) thorough the ERK cascade, and so on. IκB kinase (IKK) is activated by the nuclear factor κ-B (NF-κB) pathways (De Deken et al. 2000). H2O2 phosphorylates protein either on serine-threonine or tyrosine residues (Fetrow et al. 1999; Liu et al. 2000). Interestingly, in some cells, ROS activates JNK and p38MAPK to activate the apoptotic signal kinase, which results in the phosphorylation of MAPKK at the cysteine residue (Machado et al. 2017). This allows the propagation of the apoptotic signal, resulting in ROS induced apoptosis as opposed to ROS induced proliferation. ROS also modulates cGMP and PKA and promotes cellular proliferation since it has the potential to modulate cyclin titer in the system. In macrophages and neutrophils, Toll like receptors (TLRs) are activated by ROS. ROS activates heat shock protein like HSP70, HSP 27 via JAK, STAT pathway and takes part in protein refolding. Thus, lack of ROS production causes disease like hereditary chronic granulomatous disease (CGD) that is characterized by increased susceptibility to infections, since immune cells fail to synthesize H2O2. H2O2 is essential to fight bacterial infections and hence risk of opportunistic diseases like pneumonia increases in patients with CGD. ROS also increases the level of NF-kB, which in turn activates downstream genes such as monocyte chemotactic protein (MCP1), and IL6 which help in elucidating an inflammatory reaction as a part of host defense mechanism (Kohchi et al. 2009). As higher ROS levels have harmful effect on cells, cells have evolved protective mechanisms to neutralize ROS production. Such proteins are considered as scavengers of ROS. The transcription factor NRF2 is activated by ROS and is a master regulator of antioxidant pathway gene expression. This prevents any deleterious effects of excess ROS within the cells. Genes including phase 2 detoxifying enzymes, antioxidants, and transporters that protect cells from toxic and carcinogenic chemicals are regulated by NRF2 (Gorrini et al. 2013). With its co-factors, nuclear NRF2 binds to a cis-acting enhancer sequence called the antioxidant response element (ARE) and upregulates the transcription of cytoprotective genes. Key enzymes induced by NRF-2 are super oxide dismutase (SOD), catalase (CAT), Glutathione peroxidase (GPX), Glutathione S transferase (GST), ascorbate per oxidase (APX), Mono de-hydro ascorbate reductase (MDHAR), and dehydro ascorbate reductase (DHAR) (Gorrini et al. 2013).

ROS and Breast Cancer The initiation and progression of cancer occur through an imbalance in proteins that control cellular pathways such as cell proliferation, movement, escape from apoptosis, increased potential for neo-vascularization, and so on, collectively termed as the hallmarks of cancer (Hanahan and Weinberg 2011). Excessive ROS

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Fig. 2 Cellular response to ROS. The figure shows generation of ROS by mitochondrial electron transport chain, via action of estrogen leading to proliferation of breast cells and/or uptake of H2O2 from the extracellular milieu. ROS induces various signal transduction pathways involving oncogenic proteins such as RAS/MAPK pathway, stabilization of HIF1α and NRF2 proteins. In a cancer cell, these events lead to increased proliferation, re-organization of the actin skeleton to promote motility, induction of tolerance to oxidative stress by increase in NRF2 mediated antioxidant gene expression, and synthesis of HIF1α induced VEGF/PDGF to initiate angiogenesis. These events favor overall cancer progression and metastasis

production results in chronic oxidative stress ultimately causing changes in cellular integrity and function (summarized in Fig. 2). Basal increase in oxidative stress levels is characteristic of transformed cancer cells and affects breast cancer progression in multiple ways. In cancer, ROS are generated by increase in cancer cell metabolism, mitochondrial dysfunction, increased oncogenic/receptor activity, increased expression of cyclooxygenases, peroxisome activity, oxidases or by signals received by altered tumor microenvironment (Liou and Storz 2010). Interestingly, ROS titer determines cellular response to oxidative stress, either promoting or sabotaging tumorigenesis. For example, low ROS activates hypoxia inducible factor alpha (HIF1α), which in turn promotes growth of new blood vessels that eventually support tumor growth (Li et al. 2013a). Medium level of ROS activates MAPK causing dysregulation of normal cell cycle and results in proinflammatory cytokine production and chronic inflammation (Li et al. 2013b). Excessive ROS activates one of the key players of intrinsic apoptotic pathway Apaf1 thus resulting in cellular apoptosis. Higher ROS level damage DNA and

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cancer cells with compromised DNA repair pathway accumulate cancer promoting mutations. Excess ROS beyond tolerable range causes oxidative damage to proteins, DNA, and lipids and is reviewed elsewhere (Schieber and Chandel 2014).

Additional Mechanisms of ROS Generation in Cancer Cells In addition to ROS generation by the usual pathways described above, cancer cells are under excessive oxidative stress as ROS originates from alternate mechanisms. The expression of NADPH-oxidase is high in human cancer cell lines leading to high production of H2O2. Estrogen, the primary female steroidogenic hormone and its metabolism by lactoperoxidase to a reactive phenonyl radical is one of the most important sources of ROS generation specific to ER+ breast cancer (Brown and Bicknell 2001). This is supported by high rate of glucose metabolism and hypoxia (Johar et al. 2015a). However, TNBCs generate higher amount of ROS than ER+ tumors and are dependent on ROS for their survival (Sarmiento-Salinas et al. 2019). The enzyme thymidine phosphorylase is highly expressed in breast tumor samples. It converts Thymidine to Thymine and 2-deoxy-D-ribose 1 phosphate. The latter is a strong reducing sugar that glycates protein to generate oxygen radicals leading to increased levels of ROS in breast tumors. Further, breast cancers are often infiltrated with macrophages, known as tumor associated macrophages (TAMs) which increase ROS causing oxidative stress (Kundu et al. 1995). Usually increased ROS is detrimental to the tissue but in tumors, the process of ROS generation is delayed and made slower and the total titer remains in a sublethal concentration for a prolonged period of time (Johar et al. 2015a). To keep ROS at this concentration, several antioxidant mechanisms are altered in the breast tumors. For example, in TNBC cell lines like MDA MB 453 show at least 3 times less expression of SOD2 than MCF10A cells (breast noncancerous cells). Though SOD1 levels increase, this enzyme gets sequestered in the mitochondria instead of remaining in its cytosolic localization. This protects cancer cells from oxidative stress (Lim et al. 2011; Papa et al. 2014). Moreover, higher expression of NRF2 is characteristic of tumors and leads to an increase in the synthesis of antioxidant pathway enzymes such as GSH, HO-1, GSTA, and GSTP in breast cancer cell lines further limiting ROS production. SEPP1 or selenoprotein 1 is also a target of the NRF2 family and has antioxidant activity. SEPP1 is responsible for incorporation of selenium into cells from plasma. Oncorepressor BRCA1 modulates this NRF2 target SEPP1 transcriptionally and thus regulates NRF2 activity and reduces oxidative stress inside cell (Leone et al. 2017). BRCA1 mutation is one of the most commonly observed aberrations in hereditary breast and ovary cancers. Loss in BRCA1 helps in increasing ROS production and leads to a decrease in NRF2 production (Saha et al. 2009). ROS once generated and maintained at optimal levels promotes tumorigenesis. Its effect on hallmark pathways in cancer is summarized below.

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Modulation of Cancer Pathways by ROS DNA Damage Response As shown in Fig. 3, the most well-studied impact of oxidative stress within a cell is the DNA damage response. DNA damage occurs due to ROS induced modification of DNA bases, strand breakage, and generation of DNA-protein crosslinks. Reactive oxygen species are a constant threat to DNA and they disrupt genome function by perpetuating mutations. These mutations get fixed if not repaired, eventually building up to genome instability and cancer. Enzymatic reactions in the nucleus generate ROS. For example, lysine specific demethylase 1 (LSD1) is an enzyme of epigenetic machinery which removes the methyl groups from histones and generates ROS. This mechanism also has an effect on estrogen mediated gene regulation. DNA glycosylase OGG1 and topoisomerase IIb play important role in this mechanism by remodeling the structure at the promoter region (Poetsch 2020). In breast cancer, natural and synthetic estrogenic compounds possess pro-oxidant activity. Estrogen drives hydroxyl radical production via increasing the activity of NADPH oxidase, and this radical produces 8-Oxo-20 -deoxyguanosine (8OHDG), an oxidized derivative of deoxyguanosine. This leads to DNA damage due to increased GC to TA transversions, unless DNA repair intervenes before replication. 8OHDG is

Fig. 3 ROS and DNA damage response. In normal cells, excessive oxidative stress leads to cell death. However, chronic oxidative stress due to abolition of apoptotic mechanisms or increase antioxidant production in cancer cells can lead to DNA damage. The figure outlines ROS action and resulting pathways that are activated to control DNA damage

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highly expressed ER+ breast tissue, and it is 3.35 fold more expressed in carcinogenic breast tissue than stromal cells and 9.3 fold more in ER+ MCF7 than in ER-MDA MB 231 cells (Musarrat et al. 1996). At the same time, it is more prevalent in early stage of breast cancer; thus, it can be inferred that ROS has role in initiation of breast cancer as well. 8OHDG therefore has been exploited as a marker of oxidative DNA damage due to ROS production in several cancers. 8-oxo-dG affects the binding of transcription factor to DNA and changes secondary structure of DNA to reduce or increase access of DNA binding proteins (Poetsch 2020). The repair mechanism of choice to correct such lesions is base excision repair (BER). However, persistence of oxidative stress leads to increase in the by-products of BER reaction that may subsequently induce double strand breaks (DSBs). BER inhibits the DNA glycosylase OGG1 that removes the 8-oxo-dG from the DNA in order to decrease the possibility of DSB formation. ROS also results in oxidative modification of DNA repair proteins on cysteine residues, and this modification inhibits DNA repair, fixing ROS induced DNA lesions and this eventually permits genomic instability (Alnajjar and Sweasy 2019). Guanine lesions are usually lethal and DNA strand breaks and DNA-protein crosslinks, which are promoted by guanine modification, are mutagenic (Jena 2012). In cancer, typically a proto-oncogene is converted to an oncogene, and the uncontrolled proliferation due to oncogenic cell cycles exhibits replication stress. Several agents, notably ATR and WEE1 inhibitors, have been developed to target this increased replication stress in tumors (Srinivas et al. 2019). Oncogene activation leads to an increase in ROS, which adds to the existing replication stress. ROS oxidizes dNTPs to affect DNA polymerase activity and reduces replication fork velocity in vitro. Molecularly, the TIMELESS protein is released from the replisome complex, slowing down the fork speed and affecting the replication fork progression (Srinivas et al. 2019). These observations suggest that since cancer cells proliferate and undergo DNA replication more often than their normal counterparts, therapeutic intervention against ROS will be of value in multiple tumors. ROS can also sense DNA double strand breaks (DSBs) by sensor kinases. The sensor kinases ATM/ATR and DNA-PK along with sensor proteins detect the initial DNA damage and DSBs. The noncanonical pathway of ATM leads to an increase in the level of ROS. Accumulation of ROS inhibits another sensor kinase DNA-PKCs by altering its interaction with KU70/80 and accumulates even higher ROS and oxidative stress (Srinivas et al. 2019). Oxidative stress directly activates ATM and leads to its auto phosphorylation and subsequent downstream activation of the DNA damage response (DDR) pathway. DDR pathway is further activated by H2O2. γ-H2AX, a variant of histone 2A family, is phosphorylated by Ataxia telangiectasia mutated (ATM), Ataxia telangiectasia, and RAD3 related protein (ATR) or PI3K, and it accumulates on DSBs (Srinivas et al. 2019). Next, ROS also affects chromatin remodelers such as Brahma-related gene1 (BRG1) associated complex (BAF) and it can modulate the ATR activation in DDR pathways. The expression of the AT-rich component of this complex, AT-rich interacting domain 1A (ARID1A), is lowered

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by ROS. This loss increases reliance on OXPHOS, causing a further increase in ROS. The major result of the DDR is DNA repair which includes the activation of related pathways like the Fanconi Anaemia (FA) repair complex, Homologous Recombination (HR), Non-Homologous End Joining (NHEJ), and Micro homology Mediated End Joining (MMEJ). In dividing cell lines, transcription is ongoing and at clash points of the replication-transcription in cells, an R-loop of DNA-RNA hybrid is formed. This can aid genomic instability, unless it is resolved by Homologous Recombination (HR). ROS induced R-loops are required for enabling transcription coupled homologous recombination repair during active transcription. Other pathways involved in DNA damage repair involve genes such as BRCA1, PARP1, and BRIT1 (Davis and Lin 2011). When oxidative stress in the cell prevails, PARP1 plays an important role by either initiating DNA repair mechanism(s) or by inducing the apoptotic pathways. To date, generation of H2O2 by macrophages and by sites of inflammation was thought to induce oxidative stress driven gene expression changes in neighboring cells, more recently, uptake of exosomes or secreted exocytic vesicles (EVs) by cells was shown to propagate the oxidative stress related signals (Fig. 4). In human primary mammary epithelial cells (HMECs), exosomes derived from breast cancer cells induced the production of ROS and led to DNA damage and activation of p53 and the DDR pathway. The continuous induction of p53 led to autophagy, apoptosis, and senescence (Dutta et al. 2014). These data suggest that in breast cancer, long range interactions mediated by secreted exosomes can impact ROS mediated DNA damage response in BrCa cells/tissues.

Fig. 4 Alternate mechanism of ROS generation: Possible impact over long distances. Representative figure showing secretion of exocytic vesicles (EVs) by cancer cells that can drive induction of ROS in neighboring normal or distal cells. ROS induces specific DNA damage response in normal cells following uptake of EVs released from cancer cells

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Interestingly, anticancer drugs can increase accumulation of ROS, which further depletes H2AX protein levels and increases the sensitivity of cancer cells to anticancer drugs. However, under chronic oxidative stress, H2AX interacts with E3 ubiquitin ligase RNF168, resulting in degradation of H2AX. H2AX decrease is also associated with the impairment of NRF2 antioxidant response. In TNBC patients, the cycles of chemotherapy increase ROS and consequently decrease the H2AX protein level. This low level of H2AX protein is associated with apoptosis of tumor cells in TNBCs and can be used to monitor their response to treatment and patient survival (Gruosso et al. 2016).

Epigenetics and Gene Regulation Epigenetic alterations involve the changes at the nuclear and mitochondrial DNA (nDNA and mtDNA) by modulating their structure or conformation, without any changes in DNA sequence. Epigenetic modification commonly includes DNA methylation, histone modifications, ATP-dependent alterations to chromatin, and transcription factors mediated gene transcription. ROS are involved in epigenetic modifications both as pre- or postregulators. As preregulators, increased level of ROS can lead to changes in DNA methylation, histone modification, or transcription factor mediated gene transcription. On the other hand, all of these epigenetic modifications can lead to differential transcriptional regulation of ROS genes and here they are considered as postregulators. These phenomena are supported by many examples such as: (i) superoxide and hydrogen peroxide were found to be associated with the regulation of DNA methylation involving DNA methyl transferases (DNMTs) after radiation treatment (Shrishrimal et al. 2019); (ii) ROS-induced abnormality in DNA methylation has been observed to play a crucial role in malignant transformation leading to progression of different types of cancer, including breast cancer (Menezo et al. 2016; O’Hagan et al. 2011); (iii) ROS mediated histone acetylation induces EMT gene expression in breast cancer (Kamiya et al. 2016); (iv) ROS-induced triterpenoid inhibits rhabdomyosarcoma cell and tumor growth through targeting Sp transcription factors (Kasiappan et al. 2019); (vi) NRF2 binding with antioxidant response element (ARE) is activated by ROS leading to transcription of genes (Nguyen et al. 2009); (vii) CHOP-10/ GADD153 regulated gene expression is induced by mitochondrial ROS (Carrière et al. 2004); (viii) the basal DNA binding of the zinc finger TFs Sp1 and Sp3 is weak, but glutathione depletion-induced or hydrogen peroxideinduced oxidative stress in cells greatly induced their affinity of DNA (Ryu et al. 2003). On the other hand, ROS mediated regulation is exemplified by: (i) TF NKX6.3 reduces ROS production by regulating the expression of antioxidant genes, including Hace1 (Yoon et al. 2017); (ii) FOXO3 mediated overproduction of ROS regulates its target gene BIM expression (Hagenbuchner et al. 2012a).

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Alterations in Signaling Pathways That ROS mediates signaling in cells by acting as a second messenger is well documented in literature. The sublethal ROS levels maintained alter a portion of these signal transduction mechanisms to promote tumorigenesis. Signaling molecules such as p21ras, NF-κB, MAPK, KEAP1-NRF2-ARE, and PI3K-AKT have been shown to be direct targets of reactive oxygen and were found directly or indirectly to be involved in the progression of breast cancer (Finkel 2011). Another report revealed that elevated H2O2 levels caused nuclear accumulation of full-length c-MET and the activated cMET interacted with PARP1 and affected DNA repair in breast cancer cells (Chen et al. 2019). Additionally, mitochondrial oxidants also appear to participate in signaling events and in autophagy through the direct regulation of Atg4 activity in breast cancer (Scherz-Shouval et al. 2007). These oxidants form NLRP3 (NOD-like receptor pyrin domain-containing 3) inflammasomes which are also known to be associated with breast cancer (Zhou et al. 2011).

Cellular Proliferation Estrogen action is a major risk factor for initiation and progression of different types of cancers including breast, cervical, and endometrial carcinoma (Hagenbuchner et al. 2012b; Sundaresan et al. 1995). Interestingly, under prolonged oxidative stress, estrogen generates a higher amount of ROS through mitochondria (mROS) which helps in conversation of normal cells to cancer cells (Fu et al. 2019). Estrogen derived ROS induces Cyclin D1 and Cdc2 and increases cell proliferation, whereas inhibition of ROS production inhibits estrogen induced cell cycle genes such as cyclin B, PRC1, and PCNA expression that contain NRF-1 binding sites (Felty et al. 2005). In breast tissue, higher ROS causes hyper phosphorylation of c-JUN, a member of the activator protein complex (AP-1), that regulates several cell cycle inducing genes and causes increased cell proliferation (Xanthoudakis et al. 1992). ROS in the breast tissue can also ablate the action of the tumor suppressor PTEN and hyperactivate AKT and FOXO proteins or ERK1/2 further allowing cell survival or rapid cell proliferation (Leslie et al. 2003). Mutant Ras protein promotes activation of Ras-related C3 botulinum toxin substrate 1 (RAC1) and NADPH oxidase causing higher level of ROS generation, which in turn activate more RAS and thus cells undergo proliferation in a MAPK independent manner (Machado et al. 2017). Mutation in mitochondrial ROS dismutase produces higher ROS affecting ERK and MAPK activity, induction of cyclin gene expression and thus increased cell proliferation. Methionine sulfoxide reductase-A (MsrA), an enzyme that has a protective role against protein oxidation and is a ROS scavenger, is decreased by high ROS levels leading to loss in PTEN and increased production of vascular endothelial growth factor (VEGF), increasing cell proliferation, and angiogenesis in MDA MB 231 cells and its mouse xenograft model (De Luca et al. 2010). Overall, high ROS levels lead to increased cell proliferation in breast cancer cells using various signaling pathways.

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Cell Survival The sustained tolerable but high ROS levels eventually help in evasion of apoptosis. However, initially, ROS activates p53 and promotes apoptosis in breast tissue due to prolonged oxidative stress (Yin et al. 1999). This leads to clonal selection of breast cancer cells that have compromised p53 function. Cancer tissues often display higher expression of antioxidants like GSH, SOD1, which may allow these cells to gain an adaptive advantage over normal cells (Portakal et al. 2000). H2O2 treatment of breast cancer cells led to the oxidation of PTEN and protein tyrosine phosphatase 1B (PTP1B) resulting in the activation of the AKT pathway (Satooka and Hara-Chikuma 2016). Further, breast cancer cells gain resistance to drugs due to altered antioxidant machinery. As previously stated, NRF2 is highly activated in breast tissue. The NRF2 regulated downstream proteins Cyp3A4, HO, MRP, GSTA2, GSTP2 increase drug efflux from MCF-7 cells increasing their resistance to commonly used drugs such as Doxorubicin (Zhong et al. 2013). ROS also leads to loss in ER alpha expression resulting in tamoxifen resistance. In MCF7 cells, treatment with H2O2 for 6 months shows decrease in ER alpha due to epigenetic hyper methylation in CpG islands present in its promoter region (Mahalingaiah et al. 2015). H2O2 phosphorylates JNK and activates antiapoptotic signaling molecule Bcl2 and ROS activates the p38 MAPK pathway to modulate apoptotic signaling, increasing overall survival of breast cancer cells (Redza-Dutordoir and Averill-Bates 2016).

Autophagy Autophagy is a multistep process involving more than 40 proteins that maintains cellular homeostasis by degrading and recycling long lived organelles and molecules within the cells (Dikic and Elazar 2018). In normal cells, nutrient starvation, infection, hypoxia increase H2O2 production leading to the oxidation of the enzyme, autophagy related protein 4 (ATG4), and formation of autophagosomes. Increased ROS in cancer cells induces autophagosomes that engulf the aberrant, transformed cells, and prevent mutant cells from initiating tumor formation. This prevents genome instability. It also removes dysfunctional mitochondria to decrease ROS production. Autophagy can release NRF2 from its partner, Kelch-like ECH-associated protein 1 (KEAP1), degrading this protein by ubiquitination. NRF2 now can enter the nucleus and induce its antioxidant gene targets. This suggests that autophagy has a tumor suppressor function. However, in later stages of tumorigenesis, autophagy performs a pro-tumorigenic function since blocking this pathway prevents transformation of cells. This is observed during transformation of cells by the RAS oncogene, loss in tumorigenic potential of cells like MDA MB 231, decreased tumor formation in rodent models of breast cancer, subdued metastasis, and decreased angiogenesis in tumors (Poillet-Perez et al. 2015).

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Angiogenesis Due to high rates of cell growth and division, tumor cells expand rapidly and these continuous sheets of epithelial cells eventually are deprived of nutrients and oxygen. Often this leads to tumor collapse due to cell death; however, some tumor lesions survive since they acquire the property of co-operating with tumor stroma and endothelial cells to induce formation of new blood vessels, a process called angiogenesis. Low oxygen presence before angiogenesis, termed as hypoxia, causes a positive shift of metabolic cycles towards glycolysis and production of lactate from pyruvate. This occurs despite the presence of high glucose levels and such aerobic glycolysis is termed as the Warburg effect (Brown and Bicknell 2001; Liou and Storz 2010). Clearly, neovascularization is important to sustain the growth and progression of the tumor cells. Autophagy is necessary for stress tolerance in cancer cells and high levels of autophagy are seen in hypoxic and glucose deprived (ischemic) regions in tumors. Autophagy increases ROS generation increasing HIF1α production and the process of angiogenesis is controlled by HIF1α mediated gene regulation. High oxidative stress in breast tissue results in higher amount of HIF1α stabilization, which in turn causes an increase in VEGF and platelet derived growth factor (PDGF) production leading to effective neo-vascularization of tumors (Chandel et al. 2000). High ROS in breast malignant tissue shows higher Matrix Metalloprotease 1 (MMP1) levels, a collagenase that serves to increase blood vessel growth. In addition, H2O2 increases vasodilation via the production of nitric oxide and activation of iNOS pathway (Brown et al. 2000). Carbon monoxide (CO) also increases permeability of endothelial cells by inducing hemoxygenase 1 (HO1), which breaks CO in to biliverdin and iNOS aiding increased angiogenesis.

Motility and Metastasis Hypoxic environment of inner tumor mass results in higher ROS production which facilitates tumor invasiveness (Sosa et al. 2013). Primary tumor cells growing in situ acquire the property of cell movement, breakdown of basement membrane (BM), invasion into surrounding stroma, and eventual dissemination into blood circulation to promote tumor metastasis. In MCF7 cells, higher level of endogenous ROS increases cell motility by changing actin dynamics. An increase in Ras-GTPase activity leads to RAC1 activation downstream and increasing H2O2 synthesis (Brown and Bicknell 2001). Enzymes such as matrix metalloproteinases (MMPs) that are capable of degrading extracellular matrix proteins for BM breakdown and stromal invasion are elevated by ROS (Kessenbrock et al. 2010). Conditioned media from macrophages induce Transforming Growth Factor beta (TGFβ) inducing cell death in some MCF-7 cells but it also enhances ROS/RNS production in neighboring cells due to DNA damage. Ultimately, DNA damage induces CREB (cAMP response element binding protein) allowing cell migration and epithelial mesenchymal transition (EMT) in the stressed cells (Singh et al. 2014). In stromal cells of breast, production of NOX4 is aggravated, which in turn increases ROS production

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and promotes cell migration in ER+ cell lines (Joyce and Pollard 2009). In ER- cells such as MDA MB 231, high ROS led to more signaling molecules in stromal cells due to NOX production as compared to noninvasive MCF10A cells (Boudreau et al. 2012). ROS induced p38 MAPK and HSP27 promoted cell migration in MDAMB231 (Huot et al. 1997). Higher ROS also increased the expression of Gelatinase (MMP2) in tumor tissues (Duffy et al. 2000). In culture, mouse mammary cells show lower attachment with basement membrane components if cultured in a hypoxic environment. When tumor cells were exposed to hypoxia or H2O2 prior to injection in the mouse tail vein, they displayed higher number of metastatic lesions in the lung. This suggests that high ROS also helps in seeding of migratory cells into distal organs increasing the propensity of a tumor to be metastatic (Kundu et al. 1995).

Conclusion In cancer the titer of ROS determines if ROS is beneficial or detrimental to the process of oncogenesis. Low levels of ROS increase cancer cell proliferation by activation of various signal transduction pathways, whereas ROS accumulation can lead to cellular stress, cell cycle arrest, senescence, or apoptosis. To treat cancer cells, ROS accumulation needs to be increased either by compounds that disrupt the ETC such as taxanes or alkaloids, abrogation of antioxidant pathways, or by increasing ROS generation by immune cells. Many chemotherapeutic drugs used in cancer treatment induce ROS and cause cancer cell apoptosis. However, ROS is also known to promote chemotherapy related drug resistance in cancer cells by enhanced secretion of inflammatory cytokines. Therefore, both the amount of ROS and the cellular pathways governed by ROS need to be comprehensively studied further in cancer, to carefully design the most therapeutically beneficial ROS based treatment modality.

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Glutathione Peroxidase and Lung Cancer: An Unravel Story

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Animesh Chowdhury

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . GPX and Its Characterisation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . GPX and Its Involving Pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . GPX and Its Co-expressed and Interacting Genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . GPX and Lung Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions and Future Direction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract

Around 200 types of cancer are noted till date, among which lung cancer is the most commonly occurring cancer in men and the third most commonly occurring cancer in women. Glutathione peroxidase (GPX) acts as one of the most defensive systems to protect cells from oxidative damages. Whilst GPX has been shown to be associated with many diseases, GPX involvement in the development of lung cancer is obscure. This short writing will bring to the reader’s knowledge a brief information of all GPX isozymes and their characterisation, leading to their possible role in lung cancer, especially cigarette smoke-induced lung cancer. Keywords

Glutathione peroxidase (GPX) · Antioxidant · Lung squamous cell carcinoma · Lung adeno carcinoma · Overall survival

A. Chowdhury (*) National Institute of Biomedical Genomics, Kalyani, West Bengal, India © Springer Nature Singapore Pte Ltd. 2022 S. Chakraborti et al. (eds.), Handbook of Oxidative Stress in Cancer: Mechanistic Aspects, https://doi.org/10.1007/978-981-15-9411-3_99

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Introduction Glutathione peroxidase (GPX), a class of antioxidant enzyme, was first identified by Mills in 1957 in erythrocytes (MILLS GC 1957). In 1960 onwards, GPX activity was found to be noted in all other tissues in mammalians. Eight human and mouse GPX family members have been identified by sequence similarity and the presence of a conserved redox site till date. Four of those eight GPX contain selenocysteine at the active site in both genomes of human and mouse, and two contain cysteine in both. The other two GPX contain selenocysteine at the active site in humans but cysteine in mice. GPX has a high degree of affinity for H2O2 compared with catalase. GPX reduces and breaks down not only H2O2 but also lipid peroxide (LOOH) by catalysing a redox reaction with reduced glutathione (GSH), which serves as an electron donor: 2GSH þ H2 O2 ! GSSG þ 2H2 O 2GSH þ LOOH ! GSSG þ LOH þ H2 O Thus, GPX acts as one of the most defensive systems to protect cells from oxidative damages. Although GPX has been shown to be connected with many diseases either showing as pro or anti role like Crohn’s disease, kidney diseases, neurodegenerative diseases including cancer (Krzystek-Korpacka et al. 2010; Pinto et al. 2013; El-Far et al. 2005; Crawford et al. 2011; Vera et al. 2018; Sindhu et al. 2005; Kish et al. 1986; Aoyama and Nakaki 2013; Mason et al. 2013; Jiao et al. 2017; Chang et al. 2020; Zhang et al. 2020). This short write-up will bring a sight to the reader’s eye about GPX isozymes and their possible involvement in lung cancer.

GPX and Its Characterisation Eight GPX isozymes have been discovered till date. GPX1 gene is located on chromosome 3, and the most abundant cellular localisation of GPX1 protein is cytosol and mitochondria, whereas GPX2 gene is located on chromosome 14 and its protein found abundantly in cytosol. On the other side, GPX3 gene is located on chromosome 5 and found abundantly as an extracellular protein, whilst GPX4 gene is located on chromosome 19 and the most abundant intracellular localisation of GPX4 protein is cytosol, mitochondria and nucleus. However, it is also found as an extracellular protein. GPX5 gene is located on chromosome 6 and abundantly found in extracellular location. Further away, GPX6 gene is located on chromosome 6 and found extracellular, whereas GPX7 gene is located on chromosome 1 and found abundantly in endoplasmic reticulum and extracellular. Finally, GPX8 gene is located on chromosome 5 and found abundantly in endoplasmic reticulum. The subcellular localisation of all GPX isozymes is depicted in Fig. 1. All of the human GPX isozyme structures were evaluated except GPX6 by X-ray crystallography. The PDB structures of GPX1 (2F8A, 1.50 Å), GPX2 (2HE3, 2.10 Å), GPX3 (2R37, 1.85 Å), GPX4 (2GS3, 1.90 Å), GPX5 (2I3Y, 2.00 Å), GPX7 (2P31, 2.00 Å) and GPX8 (3CYN, 2.00 Å) are depicted in Fig. 2.

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Fig. 1 Subcellular localisation of GPX isozymes

Fig. 2 PDB structures of GPX isozymes

GPX and Its Involving Pathways GPX isozymes were found to be linked with different types of intracellular pathways in molecular level. The pathway database analysis with the Gene Analyticts tool brought the involvement of GPX isozymes with multiple pathways, among which glutathione metabolism, detoxification of reactive oxygen species, amyotrophic lateral sclerosis, linoleic acid metabolism and aldosterone synthesis and secretion

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Fig. 3 GPX-associated pathways

were found to be the top five most pathways with a high score value of 66.25, 61.14, 56.70, 55.12 and 40.87, respectively (Fig. 3). Additionally, GPX isozyme association were also observed with cellular senescence, folate metabolism, arachidonic acid metabolism, NRF2 pathway, doxorubicin pathway, ferroptosis and some others with a low score value (Fig. 3). GPX isozymes were also found to be connected with a various kind of diseases, among which anaemia, ileocolitis, glutathione peroxidase deficiency, photokeratitis and spondylometaphyseal dysplasia are the top five most diseases with a high score value of 7.30, 4.90, 4.17, 4.4 and 2.83, respectively. In addition, many other diseases such as Barett esophagus, cataract, inflammatory bowel disease 12, vulvar leiomyosarcoma, diabetes mellitus, haemoglobin D disease and ovarian clear cell carcinoma were also noticed with a low score value.

GPX and Its Co-expressed and Interacting Genes Multiple functionally different genes were found as the co-expressed and interacting partner of GPX isozymes by co-expression and protein-protein interaction analysis using two very useful Web tools Genevestigator and String, and they are depicted in Figs. 4 and 5.

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Fig. 4 GPX and their co-expressed genes

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Fig. 5 GPX and their interacting proteins

GPX and Lung Cancer Cancer is well known as one of the deadliest diseases nowadays, and it is the primary cause of morbidity and mortality worldwide. Around 200 types of cancer are noted till date, among which lung cancer is the most commonly occurring cancer in men and the third most commonly occurring cancer in women. Lung cancer is divided into two subtypes depending on histology: small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). SCLC accounts for about 15%, whereas NSCLC accounts for about 85% of all lung cancer. Different local and systemic factors such as oncogenes, tumour suppressor genes and signalling molecules were found to be associated with lung cancer by transcriptomic and proteomic analysis of the lung cancer patient’s tissue or various lung cancer cell lines; however, there is no brief report regarding the involvement of all GPX isozymes in lung cancer as per my knowledge. This prompted me to perform an analysis to give a brief report of GPX association with lung cancer. TCGA database transcriptomic analysis led an interesting outcome, and GPX2 and GPX3 were found significantly to be overexpressed and under expressed, respectively, both in the tumour tissue of lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) patients; however, other GPX isozyme mRNA expressions were found not to be changed significantly in tumour of patients in LUAD and LUSC (Figs. 6, 7, and 8) (Tang et al. 2017). Interestingly, GPX2 and GPX3 mRNA were also found to be overexpressed and under expressed, respectively, in different lung cancer cell lines compared to the normal lung epithelial cell line (Hruz et al. 2008) (Fig. 9). Later, whether GPX2 and GPX3 can truly be involved in lung cancer, we have performed overall survival analysis with GPX2 and GPX3 gene. Overall survival analysis using KM plotter found overexpression of GPX2 and lower expression of GPX3 could be associated with poor prognosis of lung cancer (Fig. 10).

Glutathione Peroxidase and Lung Cancer: An Unravel Story

Fig. 6 Heat map of the expression of GPX isozymes in LUAD and LUSC

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Fig. 7 Box plot of the expression of GPX isozymes in LUAD

Fig. 8 Box plot of the expression of GPX isozymes in LUSC

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Fig. 9 Expression of GPX isozymes in different lung cancer cell lines

Conclusions and Future Direction The dataset-based bioinformatics study signifies GPX2 and GPX3 as a plausible oncogene and tumour suppressor gene in lung cancer. However, their actual role and to get an insight how they are involved and which subsequent molecular mechanism like signalling pathways is associated or if tumour micro environment is affected by them in the development of lung cancer; a detail study dealt with GPX2 and GPX3 is

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Fig. 10 KM plot of GPX2 and GPX3 in lung cancer

required in future. Of note, GPX2 and GPX3 expressions were observed to be upregulated and downregulated, respectively, by cigarette smoking in lung epithelial cells (Singh et al. 2006; Bazzini et al. 2013). Therefore, GPX2 and GPX3 could elicit a major role in the progression of cigarette smoke-induced lung cancer.

References Aoyama K, Nakaki T (2013) Impaired glutathione synthesis in neurodegeneration. Int J Mol Sci 14: 21021–21044 Bazzini C, Rossetti V, Civello DA, Sassone F, Vezzoli V, Persani L, Tiberio L, Lanata L, Bagnasco M, Paulmichl M, Meyer G, Garavaglia ML (2013) Short- and long- term effects of cigarette smoke exposure on glutathione homeostasis in human bronchial epithelial cells. Cell Physiol Biochem 32:129 Chang C, Worley BL, Phaëton R, Hempel N (2020) Extracellular glutathione peroxidase GPx3 and its role in cancer. Cancers (Basel) 12:2197 Crawford A, Fassett RG, Coombes JS, Kunde DA, Ahuja KD, Robertson IK, Ball MJ, Geraghty DP (2011) Glutathione peroxidase, superoxide dismutase and catalase genotypes and activities and the progression of chronic kidney disease. Nephrol Dial Transplant 26:2806–2813 El-Far MA, Bakr MA, Farahat SE, Abd El-Fattah EA (2005) Glutathione peroxidase activity in patients with renal disorders. Clin Exp Nephrol 9:127–131 Hruz T, Laule O, Szabo G, Wessendorp F, Bleuler S, Oertle L, Widmayer P, Gruissem W, Zimmermann P (2008) Genevestigator v3: a reference expression database for the meta-analysis of transcriptomes. Adv Bioinforma 2008:420747 Jiao Y, Wang Y, Guo S, Wang G (2017) Glutathione peroxidases as oncotargets. Oncotarget 8: 80093–80102 Kish SJ, Morito CL, Hornykiewicz O (1986) Brain glutathione peroxidase in neurodegenerative disorders. Neurochem Pathol 4:23–28 Krzystek-Korpacka M, Neubauer K, Berdowska I, Zielinski B, Paradowski L, Gamian A (2010) Impaired erythrocyte antioxidant defense in active inflammatory bowel disease: impact of anemia and treatment. Inflamm Bowel Dis 16:1467–1475

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Mason RP, Casu M, Butler N, Breda C, Campesan S, Clapp J, Green EW, Dhulkhed D, Kyriacou CP, Giorgini F (2013) Glutathione peroxidase activity is neuroprotective in models of Huntington's disease. Nat Genet 45:1249–1254 MILLS GC (1957) Glutathione peroxidase, an erythrocyte enzyme which protects hemoglobin from oxidative breakdown. J Biol Chem 229:189–197 Pinto MA, Lopes MS, Bastos ST, Reigada CL, Dantas RF, Neto JC, Luna AS, Madi K, Nunes T, Zaltman C (2013) Does active Crohn’s disease have decreased intestinal antioxidant capacity? J Crohns Colitis 7:e358–e366 Sindhu RK, Ehdaie A, Farmand F, Dhaliwal KK, Nguyen T, Zhan C-D, Roberts CK, Vaziri ND (2005) Expression of catalase and glutathione peroxidase in renal insufficiency. Biochim Biophys Acta 1743:86–92 Singh A, Rangasamy T, Thimmulappa RK, Lee H, Osburn WO, Brigelius-Flohé R, Kensler TW, Yamamoto M, Biswal S (2006) Glutathione peroxidase 2, the major cigarette smoke-inducible isoform of GPX in lungs, is regulated by Nrf2. Am J Respir Cell Mol Biol 35:639–650 Tang Z, Li C, Kang B, Gao G, Li C, Zhang Z (2017) GEPIA: a web server for cancer and normal gene expression profiling and interactive analyses. Nucleic Acids Res 45:98–102 Vera M, Torramade-Moix S, Martin-Rodriguez S, Cases A, Cruzado JM, Rivera J, Escolar G, Palomo M, Diaz-Ricart M (2018) Antioxidant and anti-inflammatory strategies based on the potentiation of glutathione peroxidase activity prevent endothelial dysfunction in chronic kidney disease. Cell Physiol Biochem 51:1287–1300 Zhang ML, Wu HT, Chen WJ, Xu Y, Ye QQ, Shen JX, Liu J (2020) Involvement of glutathione peroxidases in the occurrence and development of breast cancers. J Transl Med 18:247

Importance of Silencing RNAs in Cancer Research

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Antara Banerjee, Janani Gopi, Francesco Marotta, Secunda Rupert, Rosy Vennila, and Surajit Pathak

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Silencing RNA (siRNA) in RNA Interference . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Concise Introduction About siRNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Applications of siRNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Limitations of siRNA Delivery to the Targeted Site for Actions . . . . . . . . . . . . . . . . . . . . . . . . . . Versatility of siRNA Employed for Therapeutic Application of Cancer . . . . . . . . . . . . . . . . . . Chemical Alterations of siRNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Different Modes of Administration by the Delivery of siRNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . Emphasizing the Systemic Delivery Approaches of siRNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Future Perspective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Reference . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Silencing RNA (siRNA) are generally about 20–22 nucleotides long that are known to significantly enhance the delivery of RNA drugs and its therapeutic approach are mostly concentrated on cancer treatment, where the siRNA is doped with anti-cancer drug for delivery to the metastatic site that are known to restrain the oncogenes such as c-Myc, K-Ras, Wnt, and also variations in the nucleotide A. Banerjee · J. Gopi · S. Pathak (*) Faculty of Allied Health Sciences, Chettinad Academy of Research, and Education, Chettinad Hospital and Research Institute (CHRI), Chennai, India e-mail: [email protected] F. Marotta ReGenera R&D International for Aging Intervention and San Babila Clinic, Vitality Therapeutics, Milan, Italy S. Rupert Stanley Medical College and Hospital, Chennai, India R. Vennila Government Medical College and Hospital, Karur, Tamil Nadu, India © Springer Nature Singapore Pte Ltd. 2022 S. Chakraborti et al. (eds.), Handbook of Oxidative Stress in Cancer: Mechanistic Aspects, https://doi.org/10.1007/978-981-15-9411-3_100

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sequence of tumor suppressor genes such as p53, APC, etc. which regulates the cellular pathways in tumor progression leading to personalized medicine. In-vivo siRNA administration is hindered by low cellular uptake, unstable under physiological conditions, immunogenicity, as well as off target site. Along with the ligand conjugated siRNA, the development of nanotechnology-based siRNA drug delivery system is the most recent approaches to treat cancer. Consequently, the forthcoming criteria of RNA-based drug will need some biochemical alteration that will increase the pharmacokinetic activities and reduce the toxicity level for clinically safe drug delivery system. Keywords

siRNA · Drug · Delivery system · Tumor · Metastasis

Introduction Silencing RNA (siRNA) in RNA Interference RNA interference (RNAi) is a cellular process which facilitates in constraining the gene expression at the phase of protein synthesis or hampers the transcription of definite gene. The major objective for RNAi exists in viruses, mobile genetic elements, and genome surveillance. Noncoding RNAs such as small interfering RNA (siRNA) and microRNA (miRNA) generated from transcribing long non-coding RNA (i.e.,) introns. They are about 20–25 nucleotides long which are corresponding to their template RNA strand. The two types of spliced RNA are miRNA and siRNA generated by incising the double-stranded RNA along with precise RNAi proteins mainly the argonaute, which attach and assist in the splicing of the intended messenger RNA, in turn, distorting the gene expression and prohibiting the protein synthesis (Fig. 1). In the past, prior to the finding of RNA interference process, consecutively they were recognized by the invariable process

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Fig. 1 Functional impact of RNA interference in the central dogma of molecular biology

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such as trans-gene silencing, post transcriptional gene suppression. Initially, there were so many erroneous assumptions on the RNA interference process (Meister and Tuschl 2004). As a consequence, the RNAi pathway is instigated by hacking the long double-stranded RNA into small portion in which the anti-sense strand is integrated into the RNA induced silencing complex that attaches to their corresponding sequences. This is well observed in the post-transcriptional gene suppression. As a result, the deprivation of messenger RNA is stimulated by the argonaute proteins, when the mRNA particularly attaches with the complementary sequences of antisense strand. Altogether, they form the RNA-inducing silencing complex (RISC) and in turn the extent of transcription is influenced by the DNA methylation, histone alteration and epigenetic modification (Mizuno et al. 1984). The subsequent procedures entailed in RNA interference process involves the double-stranded RNA is delivered into the cell by distinct techniques such as liposome-mediated dsRNA, viral transfection (Bernstein et al. 2001). An endonuclease enzyme called dicer plays a significant role in splicing the dsRNA into siRNA or miRNA which are about 20–25 nucleotides. Dicer enzyme belongs to RNAase family III approximately 200 kilo Daltons. The sense strand of siRNA or miRNA is destroyed by the cytoplasmic enzymes while antisense strand of spliced siRNA or miRNA are coalesced with RNA-induced silencing complex (RISC). Therefore, RISC complex is comprised of argonaute proteins and other proteins essential for degradation. So, the RISC complex all together with siRNA or miRNA binds with the complementary sequences in the messenger RNA and disintegrates appropriately with the help of other proteins necessary for decomposing the mRNA appropriately (Cogoni and Macino 1999). Therefore, the targeted mRNA is broken down into short segments and thus the further translation process is restrained and gene expression is impeded which causes the gene silencing appropriately (Hammond 2005).

Concise Introduction About siRNA Silencing Ribonucleic Acid (siRNA) is an RNA duplex strand which is synthesized to explicitly target the messenger RNA leading to further degradation of that gene by enduring an extensive molecular process to inhibit the production of non-functional RNAs. Therefore, the method is known as siRNA gene knockdown or siRNA gene silencing which is recognized to be employed for a wide variety of therapeutic applications. siRNA exist as the small interfering RNA or silencing Ribonucleic Acid which is a double stranded RNA molecule consisting of 20–22 nucleotides long with a 50 phosphate group and 30 hydroxyl group. As soon as, the siRNA enters the cell, it is split up by means of the restriction enzyme using RNase III enzyme called Dicer. Consequently, the siRNA is inserted into a protein complex known as the RNAi- induced silencing complex (RISC) which is eventually leading to the silencing gene expression. Several diseases can probably be treated by restraining the gene expression. Thus, the pattern of synthetic siRNA for beneficial medical uses has become a popular objective for many biopharmaceutical companies (Elbashir et al. 2001).Succeeding the contrivance of RNA interference, it was delineated that

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Fig. 2 Functional performance of RNA interference process in the biological level leads to the gene silencing by the siRNA

the post transcriptional gene silencing in plants is associated to the existence of small non-coding RNA approximately 25 nucleotides (Hamilton and Baulcombe 1999). RNA was recognized as the mainstream of the post transcriptional gene silencing process (Waterhouse et al. 1998). In 2004, Mello and Fire executed their further research in an in-vitro model of Drosophila melanogaster embryo extract (Mello and Conte Jr 2004), where the double-stranded RNA is incised into small interfering RNA that aids in the excision of messenger RNA through the attachment with the complementary sequences which ultimately disintegrates the mRNA and inhibiting their gene expression Fig. 2 (Mello and Conte Jr 2004; Parrish et al. 2000).

Applications of siRNA SiRNA Mediated Drug Delivery Methods They play a major role in restoring the normal functioning of a cell by focusing on the over expression of the gene as well as mutation noticed in case of post-

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transcriptional gene regulatory process for a wide range of pathophysiological state, namely, cancer, genetic diseases, viral infections, and other auto immune disease (Tatiparti et al. 2017), where it majorly targets the gene of interest at the cellular level via molecular tools precisely. Thus, the mode of delivery of the siRNA into the cell is by various molecular biology techniques such as in Fig. 3 (Elbashir et al. 2001). SiRNA is recognized to be an innovative class of therapeutics which is able to alter the drug targets and design appropriately for the drug delivery system facilitating the personalized medicine (Young et al. 2016). Most probably the siRNA is expected to be a promising feature in contrast to other RNA interference method as it can be directly loaded into the RISC assembly by simplifying the dose control as well as avoid the need for the introduction into the nucleus. Thus, the delivery of siRNA into the targeted tissue involves different modes of administration such as local therapies as well as systemic therapies (Dogini et al. 2014). The ultimate goal of the treatment with siRNA is to ensure safe and effective dose range of the drugs doped with the silencing RNA leading to targeted drug delivery mechanism in case of various disease conditions. The mutation or mismatch nucleotide in the genes may be due to the inherited genetic characters or else due to the epigenetic alterations that will pass on to the protein synthesized from that particular portion of the gene (Dykxhoorn et al. 2003). This paves the way for inhibiting the transcription of non-functional genes such as the non-coding RNAs (i.e. intron) which may lead to the synthesis of a new protein manufactured from the mutated segment of that gene. Consequently, the key factors contributing to the siRNA therapeutic emerging in the clinical pipeline is the development of siRNA molecular fabrication principles with enhanced delivery method helps to promote the siRNA mediated delivery systems (Davidson and McCray 2011). First and foremost part of the silencing RNA is the

Fig. 3 Various methods used for the deliverance of siRNA

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trepidation of the kinetics involved in the liberation of silencing RNA which relays on the time period of gene knockdown for a sustained delivery in the targeted site and is identified to have finite half-life that will fade out over time in the circulatory system. The siRNA fabricated with the nanotechnology related biomaterials that should be without much toxicity and immunogenicity of the delivering siRNA material that are biodegradable at the end of the process. Hence, establishing the delivery platforms for sustained release profiles for the silencing of gene will be capable of tailoring the beneficial medical applications for their outcome in tissues for remodeling (Samantha and et.al 2015). The sequential liberation profile of siRNA is used to monitor the rate at which the siRNA conjugates the drug delivery. Besides the modification of the kinetics for the diffusion associated release is also feasible. Conclusively, the naked siRNA is not stable for silencing the gene expression and thus drain into the renal filtration for a poor pharmacokinetic property whereas the chemical modifications of the siRNA conjugated with other nanocarriers for a targeted drug delivery system for a better biocompatible availability. For instance, focusing on the scaffolds and micro-particle system known to be hydrolytically degradable polyesters, namely, poly lactic-co-glycolic acid (PGLA). Scaffolds are a synthetic matrix known to be engineered to bring about cellular connections. The experimentally determined by the scaffold assisted delivery to have restrained siRNA liberation kinetics to the exploitation of the collagen extracellular matrix encumbered with siRNA devised with PAMAM dendrimer. This extracellular matrix accomplished more than 50% gene silencing at seven days experimentation with fibroblast leading to controlled suppression through scaffold related drug delivery. (Samantha and et.al 2015).

Narrowing Down to the Intervening Drug Delivery Methods of siRNA Specific to Cancer Treatment Cancer is acquainted to be a life threatening disease affecting a wide range of population which is mostly predominant to mortality and morbidity worldwide. Physiologically, tumor is caused due to abnormal proliferations of cells which have the capability to infuse into the parenchyma of normal tissue disrupting their microenvironment. Cancer is characterized into distinctive types such as sarcoma, melanoma, leukemia, etc. based on the site of residence of the tumor cells and their distribution to other body sites leading to malignancy. Thus, the advancement in the medical field has led to the development of personalized medicine by gene sequencing of tumor cells. Subsequently, it is revealed by the fabrication of artificial siRNA through increased specificity along with minimal modification followed by limited therapeutic targets are known to pave way for greater efficiency of siRNA derived drugs against cancer cells (Fig. 4). SiRNAs Delivery System for Neurogenerative Disease Conditions Alzheimer’s disease, Huntington’s disease and Parkinson’s disease are some of the commonly known neurodegenerative diseases which are delineated as a diverse group of disease conditions that are exemplified by advancing in the degeneration of the structure as well as the functionality of central or peripheral nervous system.

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Fig. 4 Unique characteristics of siRNA delivery

Currently, the systemic delivery of the synthetic siRNAs to the central nervous system is excluded due to their inability to traverse through the blood brain barrier via endonucleases as well as the filtration in the kidney which needs further modification of siRNA, so that it can intricately cross the membrane precisely. Experimental evidences have illustrated about the combination of an elevated dose of siRNA along the dopamine transporter gene through the ventricular cerebrospinal fluid that end results in the considerable knockdown of the disease causing genes in the dorsoventral and mediolateral regions. SiRNA have also been delivering to the brain through electroporation to the targeted site, for instance, hippocampus, visual cortex and also other parts of the brain resulting in gene knockdown significantly. Further, research should keenly focus on the delivery strategies of fabricated siRNA using the nanotechnology based methods to easily penetrate the blood brain barrier and overcome the obstacles during the administration of siRNA (Jagannath and Wood 2007).

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Limitations of siRNA Delivery to the Targeted Site for Actions Unbound siRNA is hydrophilic and anionic in nature with a double stranded ribonucleotide (dsRNA) and are not instantly seized by the cell and inhibit them from traversing the cell membrane. The limitations of delivering a free siRNA focus on serum solidity, material of outsized molecular weight, increase cytotoxicity, ligand receptor interface, vascular accessibility to grasp tumor tissues and exonerate from kidney are some of the shortcomings to overcome in the future of the fabrication of siRNA (Czauderna et al. 2003).Therefore, sufficient modifications are needed to trounce the inadequacy of siRNA during their movement to reach their targeted site of action or else might lead tribulations in human body precisely. For instance, employing nano-materials or nanocomposites for the fabrication of siRNA should have the stability to overcome the serum opsonins in the circulation without much demise of the siRNA coated particle during their migration to the targeted site of action (Li and Shen 2009). The substantial fidelity of siRNA is achieved by an enhanced bioavailability. Naked siRNA is known to have a half-life of not as much of ten minutes in cent percent human serum because of the nucleases disintegration. Thus, the fabrication of siRNA with nanotechnology based methodologies which are to be intended for a gene knockdown and various disease conditions by withholding their resistance towards nuclease throughout their migration to the destined region in case of systemic administration.

Versatility of siRNA Employed for Therapeutic Application of Cancer Direct conjugation of siRNA in conjunction with molecules such as polymers, peptides, lipids, antibodies and aptamers are the loading materials which are known to possess efficient pharmacokinetic properties which enable for efficient delivery system (Singh and et.al 2018).Not only had the liposomes mediated siRNA, aptamer-siRNA and carbon nanotubes with siRNA as well as various nanotechnology related fabrication techniques are employed for cancer therapeutics precisely. Advancement of tissue culture technology for the development of scaffold intervened delivery to regulate the siRNA liberation in the target site. Directly, collagen based scaffolds are encumbered with PAMAM dendrimers devised along with siRNA for their liberation in the tumor site precisely. Experimentally performed in-vitro studies demonstrate about the scaffolds with fibroblasts, which are known to attain the suppression of genes partially in seven days by limited delivery of siRNA. The therapeutic technique was keenly practiced in cancer treatment because siRNA was known to silence the oncogenes and concentrates on the mutations in the tumor suppressor genes as well as determine the minuscule signaling pathways (Tatiparti et al. 2017) in cancer Fig. 5 (Singh and et.al 2018).

Chemical Alterations of siRNA For a successful deliverance of fabricated siRNA to the malignant tissue is mediated by certain chemical alterations to reach the predestined tumor site by reducing the

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Fig. 5 Different types of siRNA for cancer therapeutics

hindrance of the serum opsonins, immunological reactions, as well as serum nucleases (Liao and Wang 2005). The presentation of siRNA is significantly improved after the chemical alteration of siRNA strands involve the incorporation of sugar, base, phosphorous acid, strand end or backbone of each sense and antisense strands. Frequently, the substitution of the phosphodiester group with phosphorothioate at 3’ends. The substitution of phosphorothioate (PS) in place of phosphodiester (PO4) at the 30 end constructs resistant siRNAwhich prevents the enzymatic deprivation by the exonucleases. The induction of an O-methyl group (2’-O-Me), a fluoro group (2’-F) or a 2-methoxyethyl group (2’-O-MOE) are the chemical derivatives which is known to extend the half-life and RNA interference activity in cultured cells and plasma significantly. The variation of siRNA with 2, 4-dinitrophenol (DNP) advances in the improved nuclease opposition alongside with an increased expansion in membrane penetrability of the synthetic siRNA. Certainly, boranophosphate siRNA demonstrated about an improved resistance to the deprivation of nuclease regardless of

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decreased RNAi activity. The dilapidation of synthetic siRNA into a non-natural molecule which end results in decreased toxicity production (Hall et al. 2004).

Lipid Mediated siRNA Delivery System A number of lipid mediated siRNA distribution have been developed to reach the tumor cell involves the following nanotechnology methods such as liposomes, stable nucleic acid particle and neutralnanoliposomes.1,2-dioleoyl-3trimethylammonium propane (DOTAP) and N-{1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl sulfate (DOTMA) are positively charged lipid module about 100 to 300 nanometer in size doped along with an anionic siRNA molecule act as a hauler which hijacks the obstacles during circulation. Mode of deliverance is by transfection that leads to an efficient drug delivery system to reach the cancer tissue without mislaying their bioavailability. The neutral nanoliposomes as well as stable nucleic acid particle follows the above explained process with slight variation in their carrier and fabrication methods using siRNA precisely (Elouahabi and Ruysshaert 2005). Polymer-Enhanced siRNA Delivery System In polymer enhanced siRNA drug deliverance, it involves diverse varieties of positively charged polymers like chitosan, polyethyleneimine, dendrimer, and cyclodextrins which are employed for the deliverance of siRNA to the target site. Dendrimers involve polycationic dendrimers like poly (amidoamine) (PAMAM) and poly(propylamine) (PPI) dendrimers which is a synthetic three dimensional nanostructure with distinct features like tunable size, accessible terminal functional groups and cargo encapsulation in a nanometer size append to their capability as a drug carrier with siRNA drug delivery system (Singh and et.al. 2017).

Different Modes of Administration by the Delivery of siRNA Naked siRNA has not much efficacy when compared to the fabricated siRNAs for the delivery systems in case of different types of disease. Free siRNA has been injected in the vicinity to the target organs such as heart, ear, lungs, eye, and brain for silencing the superfluous expression of the gene involved in the progression of the disease condition. Different formulations of siRNA involves liposome mediated siRNA, viral mediated siRNA, chemically altered siRNA are employed for targeted deliveries in case of cancerous condition by various modes of administration (Meng and et.al. 2015). The detailed description of siRNA deliverance by different modes of administration is illustrated in Fig. 6.

Emphasizing the Systemic Delivery Approaches of siRNA Systemic deliverance of silencing RNA to the destined site in the cytoplasm is known to be hindered by various barriers for exerting the gene silencing property. Therefore, the intravenous mode of systemic delivery of siRNA confronts various disputes while migrating from the region of administration to the targeted site. The

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Fig. 6 Targeted siRNA local delivery for various disease conditions

following part delineates the mechanism involved during the siRNA delivery system in the human body. Initially, the siRNA is dispersed to organs through the circulatory system of blood as well as undergoes elimination. SiRNA penetrates to the tissue by traversing the interstitial space in the targeted cell. SiRNA must hijack from the endosomes and the liberation from its carrier to the cytoplasm which is packed onto the RISC complex. The designing of carriers such as polymers, lipids, and conjugates will be critical for the systemic delivery of siRNA therapeutics. The incorporation of surface pegylation and cell-specific targeting ligands in the carriers may improve the pharmacokinetics, biodistribution, and selectivity of siRNA therapeutics. The safety, effectiveness, and ease of production and manufacturing are important considerations for selecting the appropriate siRNA carriers. Evaluation of the efficacy of siRNA therapeutics requires considerations of off-target effects and innate immune response (Wang and et al. 2010).The manifestation of the therapeutic capability of siRNAs in silencing specific disease correlated genes which is delivered systemically by rapid high pressure IV known by the technique called hydrodynamic delivery. The hydrodynamic delivery involves the transitory migration towards the right sided heart failures wherein the venous pressure one way or another facilitate the siRNA to enter the highly vascularized organs such as lungs, liver and pancreas precisely.

Conclusion Ultimately, an improved explication of the molecular mechanism of siRNA and their influence on chemical alterations that alleviate as well as decrease unspecific interactions of siRNA molecules. The main theme is entangled with their effective

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deliverance system which may head towards the initiation of siRNA as therapeutic option clinically. So, the promising factor of siRNA to co-ordinate with the cellular reaction through effectual suppression of gene by diverse biological modifications that enables to construct an efficient siRNA for medical applications. Furthermore, confound assessment of the therapeutic approaches for a well-organized delivery system using siRNA is the upcoming production of multifaceted and cost-effective siRNA formulations. The up-and-coming clinical researches have set out from the utilization of naked siRNA which is known to cause serious immunogenic alterations in the body. Thus, the fabricating siRNA molecule with other nano-carriers to enable efficient delivery system and the designed product is used in the market by patenting along with industry for commercialization which can benefit for a better therapeutic option.

Future Perspective More studies are warranted in establishing a controlled liberation of siRNA to the targeted tumor by enhancing their resistance towards nuclease and circumventing the immunological reaction. Immense potential of the siRNA molecule doped with an anti-cancer drugs followed by coating with nanomaterials which are determined for a novel therapeutic application by enabling their safety and efficacy during their drug delivery to the intended site. Thus, the future prospective of siRNA formulated drug delivery system should establish as an efficient technique in case of personalized medicine depending on their pharmacogenomics profile of the patient affected with tumor. Acknowledgment This study was supported by a grant from Department of Science and Technology (DST) – Science and Engineering Research Board (SERB) (EMR/2017/001877). Author Contribution Surajit Pathak conceived the idea of the article. Antara Banerjee and Janani Gopi, Surajit Pathak, and Francesco Marotta, Secunda Rupert and Rossy Vanilla had written the manuscript. All the authors read and approved the final version of the manuscript.

Reference Bernstein E, Caudy AA, Hammond SM, Hannon GJ (2001) Role for a bidentateribonuclease in the initiation step of RNA interference. Nature 409:363–366 Cogoni C, Macino G (1999) Gene silencing in Neurosporacrassa requires a protein homologus to RNA-depednet RNA polymerase. Nature 399:166–169 Czauderna F, Fechtner M, Dames S et al (2003) Structural variations and stabilizing modifications of synthetic siRNAs in mammalian cells. Nucleic Acids Res 31:2705–2716 Davidson BL, McCray PB (2011) Current prospects for RNA interference-based therapies. Nat Rev Genet 12:329–340 Dogini DB, Pascoal VDAB, Avansini SH, Vieira AS, Pereira TC, Lopes-Cendes I (2014) The new world of RNAs. Genet Mol Biol 37:285–293

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Dykxhoorn DM, Novina CD, Sharp PA (2003) Killing the messenger: short RNAs that silence gene expression. Nat Rev Mol Cell Biol 4:457–467 Elbashir SM et al (2001) Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411:494–498 Elouahabi A, Ruysshaert JM (2005) Formulation and intracellular trafficking of lipoplexes and polyplexes. Mol Ther 11:336–347 Hall AHS, Wan J, Shaughnessy EE et al (2004) RNA interference using boranophosphatesiRNAs: structure-activity relationships. Nucleic Acids Res 32:5991–6000 Hamilton AJ, Baulcombe DC (1999) A species of small antisense RNA in posttranscriptional gene silencing in plants. Science 286:950–952 Hammond SM (2005) Dicing and slicing. The core machinery of the RNA interference pathway. FEBS Lett 579:5822–5829 Jagannath A, Wood M (2007) RNA interference based gene therapy for neurological disease. Brief Funct Genomics 6(1):40–49 Li L, Shen Y (2009) Overcoming obstacles to develop effective and safe siRNA therapeutics. Expert Opin Biol Ther 9:609–619 Liao H, Wang JH (2005) Biomembrane-permeable and ribonucleaseresistant siRNA with enhanced activity. Oligonucleotides 15:196–205 Meister G, Tuschl T (2004) Mechanisms of gene silencing by double-stranded RNA. Nature 431: 343–349 Mello CC, Conte D Jr (2004) Revealing the world of RNA interference. Nature 431:338–342 Meng P et al (2015) New paradigms on siRNA local application. BMB Rep 48(3):147–152 Mizuno T, Chou M-Y, Inouye M (1984) A unique mechanism regulating gene expression: translational inhibition by a complementary RNA transcript (miRNA). Proc Natl Acad Sci 81: 1966–1970 Parrish S, Fleenor J, Xu S, Mello C, Fire A (2000) Functional anatomy of a dsRNA trigger: differential requirements for the two trigger strands in RNA interference. Mol Cell 6:1077–1087 Samantha MS et al (2015) Technologies for Controlled, local delivery of siRNA. J Control Release 218:94–113 Singh A et al (2017) Advances in siRNA delivery in cancer therapy. Artif Cells Nanomed Biotechnol 46(2):274–283 Singh A et al (2018) Advances in siRNA delivery in cancer therapy. Artif Cells Nanomed Biotechnol 46(2):274–283 Tatiparti K et al (2017) siRNA delivery strategies: a comprehensive review of recent development. Nano 7:77 Wang J et al (2010) Delivery of siRNA therapeutics: barriers and carriers. AAPS J 4:12 Waterhouse PM, Graham MW, Wang M-B (1998) Virusa resistance and gene silencing in plats can be induced by simultaneous expression of sense and antisense RNA. Proc Natl Acad Sci 95: 13959–13964 Young SWS, Stenzel M, Yang JL (2016) Nanoparticle-siRNA: a potential cancer therapy? Crit Rev Oncol Hematol 98:159–169

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Cellular Redox Status and Modifiable Behaviors Effects on Cancer Progression and Treatment Mary Figueroa and Joya Chandra

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Excess Body Weight . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Poor Nutrition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cigarette Smoking . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cross-References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract

Risk factors for cancer include various modifiable health behaviors such as poor diet, poor energy balance, and cigarette smoking; however, less is known regarding the influence of these behaviors on cancer progression and treatment after diagnosis, particularly from a molecular perspective. This chapter explores how obesity, poor nutrition, and cigarette smoking have been attributed to worsening M. Figueroa Department of Pediatrics-Research, UT MD Anderson Cancer Center, Houston, TX, USA University of Texas MD Anderson, Cancer Center UTHealth Graduate School of Biomedical Sciences, Houston, TX, USA e-mail: mfi[email protected] J. Chandra (*) Department of Pediatrics-Research, UT MD Anderson Cancer Center, Houston, TX, USA Department of Epigenetics and Molecular Carcinogenesis, UT MD Anderson Cancer Center, Houston, TX, USA University of Texas MD Anderson, Cancer Center UTHealth Graduate School of Biomedical Sciences, Houston, TX, USA Cancer Center for Energy Balance in Cancer Prevention and Survivorship, University of Texas MD Anderson, Houston, TX, USA e-mail: [email protected] © Springer Nature Singapore Pte Ltd. 2022 S. Chakraborti et al. (eds.), Handbook of Oxidative Stress in Cancer: Mechanistic Aspects, https://doi.org/10.1007/978-981-15-9411-3_101

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cancer progression and outcomes, and all share a common theme of perturbing cellular redox. The molecular route or source of redox imbalances stemming from each of these conditions and influencing cancer progression and refractory disease is distinct and will be discussed in detail. Obesity or increased adiposity is linked to worse outcomes for several cancer types due to adipocyte and cancer cell interactions involving redox pathways. A role for dietary influences on cancer outcome is emerging as metabolic vulnerabilities of various cancers are identified which can be targeted through redox pathways modulated by dietary components. Cigarette smoking, a leading cause of cancer, is also linked to poor treatment outcomes and increased oxidative stress in smokers has been well-documented. Enhanced understanding of the mechanism by which modifiable health behaviors can worsen patient outcomes has potential to improve or enhance treatment options for cancer patients. Keywords

Adiposity · Nutrition · Cigarette smoking · Health behaviors

Introduction Both genetic and lifestyle factors are attributed to increased risk of developing cancer, although genetic factors may be a greater contributor (Vogelstein et al. 2013). Nonetheless, many lifestyle factors are strongly associated with cancer and are modifiable behaviors. In a recent review, 1,570,975 cancer cases were analyzed for modifiable risk factors that impacted cancer development or survival across 26 different cancer types (Islami et al. 2018). Approximately 42% of cancer cases were associated with 1 of 18 modifiable risk factors. The most attributed modifiable risk factor for all cancers was cigarette smoking, second was UV radiation, third was excess body weight, and among the top 20 there were various factors related to improper nutritional intake, including low fruit and vegetable consumption, low fiber, and red meat consumption (Islami et al. 2018). Cigarette smoking, excess body weight, and poor nutrition are all leading risk factors that can be modified through behavioral interventions, and are studied extensively in the context of cancer prevention. However, there is also ample evidence that these behaviors that increase risk of cancer development are able to worsen survival in patients if these poor health behaviors are carried on after a cancer diagnosis. Although preemptive avoidance of these cancer promoting behaviors would be the best solution, many of these modifiable behaviors can be altered after a cancer diagnosis to improve patient prognoses. A common denominator between these lifestyle-related risk factors, excess body weight, poor nutrition, and cigarette smoking, is that they can contribute to redox alterations leading to oxidative stress (Fig. 1). Redox status is negotiated by the presence of numerous oxidants and antioxidants, which are carefully balanced in the body under healthy conditions. Normal metabolic processes mandate that molecular

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Fig. 1 Impact of modifiable behaviors on redox. Image depicting examples of different unhealthy behaviors and their relation to redox imbalance

oxygen is broken down into superoxide and hydrogen peroxide in the presence of key catalytic enzymes, and are the basis for formation of reactive oxygen species (ROS). Sources of ROS in cells include intracellular superoxides from the mitochondria as a byproduct of the electron transport chain. ROS are also produced through NADPH oxidase (NOX) protein complexes that when assembled, localize to the cellular membrane where NADPH is reduced to NADP+ and releases ROS extracellularly. NOX complexes are found in different cell types, but are best known for their function in neutrophils where they produce an oxidative burst as a safeguard against bacteria and viruses. Additional examples of homeostatic process that produce ROS includes the beta-oxidation of fatty acids that are used to produce acetyl-CoA for use in the citric acid cycle. Imbalance of redox status triggers the activation of proteins and signaling pathways to help restore the balance. Increased and unchecked ROS within cells can damage virtually every macromolecule. For example, oxidative DNA damage leads to the formation of the DNA lesion 8hydroxydeoxyguanosine. However, more frequently, when oxidant levels are increased through normal metabolic processes, there are antioxidant defenses that act to reduce oxidative damage to the cell. There are different types of antioxidants

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that help regulate ROS levels. The most abundant nonenzymatic antioxidant in cells is glutathione and it is produced within cells. Various enzymatic antioxidants, such as superoxide dismutase, catalase, and glutathione peroxidase, catalyze redox reactions that disable ROS into less reactive ROS or into water. NRF2, a transcription factor that drives the transcription of many enzymatic antioxidant genes, plays a critical role in defending against oxidative stress. In the context of several cancer types, increased levels of oxidative stress have been noted, and are capable of promoting cancer growth by activating proliferative signaling pathways. Also, in cancer, several antioxidant genes have been linked to drug resistance, prompting their targeting as a therapeutic strategy. Cancer progression and treatment resistance have been attributed to oxidative stress in several cancer types; however, the source of the altered redox is generally attributed to oncogenic signaling and cellular metabolic effects independent of environmental influences. Unhealthy modifiable behaviors are also able to disrupt redox balance, which can in turn promote cancer progression and hinder effective treatment response, but this is not seen universally across all cancer types. Specific cancer types, exclusively in adults, are associated with poor lifestyle related habits (Table 1). This chapter will describe molecular aspects of redox that contribute to cancer progression and treatment response for three modifiable behaviors associated with cancer risk: obesity, poor diet, and cigarette smoking.

Excess Body Weight Over 120,000 incidences of cancer were attributed to excess body weight of the patients in a single year, thus there is evidence to suggest that adiposity is promoting cancer risk (Islami et al. 2018). Excess body weight is defined as having a body mass index of at least 30 kg/m^2 and is attributed to increased adiposity; however, this narrow definition of obesity has been challenged. Here we will examine how adiposity plays a role in cancer progression. Under non-cancerous conditions, adipose tissue has been linked to systemic redox changes. Adipose tissue serves important roles within the body, including nutrient storage and adipokine signaling. At least two models indicate a role for ROS in normal adipocyte function. The release of pro-inflammatory cytokines from adipocytes can lead to inflammation and an increase in oxidative stress externally from the adipose tissue (Fig. 2). Also, differentiation of adipocytes and insulin response has been shown to require regulation of the NOX4 complex, which extracellularly produces hydrogen peroxides. Table 1 Cancer types that are linked to cancer-associated lifestyle behaviors discussed in this chapter Unhealthy behavior Excess body mass Poor nutrition Cigarette smoking

Impacted cancer type Gastric, breast, pancreatic, liver Lung, gastric, head and neck, pancreatic Lung, liver, acute myeloid leukemia, head and neck, prostate, breast

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Fig. 2 Tumor-associated neutrophils being recruited by increased IL-1beta released from hypertrophic adipocytes

Therefore, proper functioning of adipocytes contribute to ROS which are not pathogenic. However an overabundance of adipose tissue can contribute to systemic oxidative stress with potential for altering signaling in distal organs. Since excess body weight is attributed to several types of cancer, it is important to understand that adipose tissue does not function the same way in those with excess body weight compared to those with normal body weight. As mentioned above, this has been attributed to adipocyte quantity in overweight or obese individuals as well as the concept that adipocytes in individuals with obesity are functionally different from normal adipocytes. In support of the latter, adipose tissue from obese patients have been shown to possess higher levels of intracellular ROS compared to adipose in nonobese individuals and animals (Furukawa et al. 2004; Savini et al. 2013). In adipocytes from obese individuals, mitochondrial metabolism is affected and increases in ROS can increase the activity of NADPH oxidase 4 (NOX4), a protein involved in a protein complex that generates extracellular superoxides, which can lead to a positive feedback loop of oxidative stress. Additionally, obese adipocytes can inappropriately release fatty acids leading to increased fatty acid beta-oxidation in surrounding cells. These mechanisms can alter redox status in obese adipose in the absence of cancer. An important model used to study excess weight in mice is the KKaγ mouse that is heterozygous for a mutation of the yellow agouti gene, which results in yellow hair and increased fat cell growth. In overweight KKaγ mice and in mice with diet-induced obesity, adipose tissue had higher levels of lipid peroxidation and hydrogen peroxide production compared to normal weight mice. Increased triglycerides in the plasma of the obese mice, as well as decreased expression of antioxidant genes with increased expression of NOX complex proteins in the fat tissue, was also noted in these mice with potential to exert systemic effects. Similarly, the same study found that markers of lipid peroxidation in the plasma and urine of humans positively correlated with the BMI of the participants (Furukawa et al.

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2004). Inflammation in the microenvironment of adipose tissue in individuals with obesity has also been documented. Within inflamed adipose tissue, crown-like structures that are composed of macrophages surrounding dying adipocytes have been described. The number of crown-like structures have been found to be significantly increased in mice that are fed a high-fat diet for 12 weeks (Miyazawa et al. 2018). In the context of cancer, oxidative stress stemming from adipocytes has been shown to influence cancer progression and treatment response in different cancers (Moldogazieva et al. 2018). A study in breast cancer patients showed that patients whose tumors had increased expression of mRNA or protein of CPT1B, (carnitine palmitoyltransferase 1B), measured by quantitative PCR and immunofluorescence staining, led to increased fatty acid β-oxidation had poor survival and were significantly more likely to be chemotherapy resistant (Wang et al. 2018). They also showed in vitro that the release of fatty acids and leptin from human breast adipocytes could upregulate fatty acid oxidation, increase hydrogen peroxide production, and lead to chemoresistance in human breast cancer cell lines (Wang et al. 2018). In prostate cancer patients, it has been seen that periprostatic fat is inflamed in patients with high-grade prostate cancer. High-grade prostate cancer patients had higher numbers of crown-like structures and increased adipocyte diameter (Gucalp et al. 2017). Thus, adipose inflammation that is associated with excess weight and obesity was concluded to promote prostate cancer into advanced stages. Although an association was absent between obese patients and high-grade prostate cancer, the periprostatic adipose was also found to be inflamed in normal-weighted, high-grade prostate patients. An elegant study using multiple mouse models of pancreatic cancer has linked obesity to tumor growth and metastasis. Using mouse pancreatic cell lines (PAN02 and AK4.4) in high-fat diet-induced obese mice, genetically modified obese mice who lacked leptin, or genetically modified mice who developed pancreatic tumors who were on high-fat diets this study found that tumors formed earlier, grew faster, and that there was an increased number of metastatic lesions in the obese mice (Incio et al. 2016). Cancer metastasis is a frequent cause of mortality more often than the primary tumor, and has also been linked to high adiposity. A study discovered that obese mice bearing implanted, orthotopic pancreatic tumors had more tumor-associated neutrophils in their tumor microenvironment and that this was due to increased secretion of IL1β by adipocytes (Fig. 2). Using an antibody that targets IL1β, MM425B, they observed that there was a decreased number of neutrophils recruited to the tumor and that in their PAN02 tumor model, there was a significant decreased in tumor growth. The same study also found that normal weight pancreatic ductal adenocarcinoma patients had better response to adjuvant chemotherapy independent of other tumor characteristics as compared to overweight and obese patients (Incio et al. 2016). Thus, excess body weight increased cancer progression and decreased survival across numerous cancer models and patients. Additionally, the location of adipose tissue impacts different organ systems and has been shown to influence cancer growth. Visceral adipose tissue is located within the torso of the body and physically around organ systems compared to subcutaneous adipose that is located beneath the skin throughout the body. In a retrospective

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study which included over 1200 hepatocellular carcinoma patients, obesity was associated with decreased survival, and having high amounts of visceral adipose tissue was a negative prognostic factor to survival independent of obesity (Fujiwara et al. 2015). A separate study examined survival of 3460 postmenopausal breast cancer patients in relation to the amount of fat tissue in their torso, as measured by dual-energy x-ray absorptiometry (Iyengar et al. 2019). Here they found that the amount of torso fat had an inverse relationship with patient survival, even if the patient had normal BMI measurements throughout their diagnosis. From this study, it can be inferred that the amount of adipose tissue and its location is important even if the cancer patient does not overall qualify as having excess body weight. Coculturing an ovarian cancer cell line, SKOV3ip1, with human primary visceral adipocytes increased the expression of fatty acid binding proteins, increased invasiveness, and increased clonogenicity of the cancer cells (Ladanyi et al. 2018). Using an human ovarian cancer xenograft mouse and subcutaneous adipose tissue from 72 participants, there was elevated lipid peroxidation, increased mitochondrial hydrogen peroxide production, and decreased expression of antioxidant genes in subcutaneous adipose from obese individuals compared to normal weight participants (Chattopadhyay et al. 2015). Cumulatively, these data illustrate how having excess body weight can influence cancer progression, survival, and redox levels.

Poor Nutrition With the global rise in consumption of Western diets that are rich in fats and sugars, it is becoming increasingly important to consider the ties between cancer and dietary nutrition (Kanarek et al. 2020; Mittelman 2020). Poor diets can promote increased adipose tissue, but this is not always the case, and poor nutrition may exert cancerpromoting properties independent of high adiposity. People whose weight is considered normal can lack different nutrients that can inhibit or disregulate proper cellular functions, such as maintaining redox status, within the body. Food can be a source of oxidants and antioxidants that impact systemic bodily redox balance (Saha et al. 2017). A study conducted in mice compared diets containing either 5.9% or 40% fat compared the expression of genes involved in oxidative stress in tissues of mice. After 6 weeks of being on high-fat diets, the mice did not increase in weight, but microarray analysis revealed that the mice had significantly increased expression of oxidative stress genes in the liver and adipocytes of mice (Matsuzawa-Nagata et al. 2008). Notably, there was increased expression of fatty acid oxidation genes in liver tissue and increased NADPH oxidase complex genes in adipocytes, thus systemic oxidative stress from high-fat diets may arise from diverse sources in different tissues. The most important take away from this study was that high-fat diets can increase oxidative stress signaling in the body before the onset of obesity, highlighting the importance of healthy food consumption. Since diet can also produce oxidative stress, it is important to understand how this can impact cancer progression for individuals who fall within normal body weight ranges and consume nutritionally poor diets.

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There are foods whose intake is correlated with increased cancer risk, including the consumption of red meat and processed meat (Islami et al. 2018). Beyond just food intake, the preparation of the food is also an important consideration for bodily redox status. A study investigated how meat consumption and cooking method impacted levels of plasma levels of malondialdehyde (MDA), a product of lipid peroxidation that was used as an indicator of oxidative stress, and heterocyclic amines in over 600 participants (Carvalho et al. 2015). Heterocyclic amines can be produced in meat when cooked and when metabolized, produce ROS. They found that beef consumption significantly correlated with increased plasma levels of MDA. Another finding from that study was that the method of cooking and the more “done” beef was cooked both impacted levels of MDA and heterocyclic amines (Carvalho et al. 2015). Food naturally rich in antioxidants are often found in fruits, vegetables, and some spices that have been attributed to decreased cancer risk. A 13-year study that followed over 2000 middle-aged men to observe their dietary habits and any development of cancer found that there was an inverse correlation between the fruit and vegetable intake and mortality from any cancers in their participants (Michael et al. 1996). There was also a trend for low dietary fiber, more alcohol consumption, and less fruit and vegetable consumption in participants who developed cancer anywhere in their digestive tracts. This study showed this impressive correlation of fruit and vegetable consumption to decreased cancer development and decreased cancer mortality, but did not include any molecular basis for their findings. Other cancer studies have focused on using vitamins or pharmacologic derivatives of natural products that are antioxidants, which are equated by many as drugs, instead of whole food diets. There have been many confounding preclinical studies and clinical studies that have investigated how antioxidant supplement use can influence cancer progression and treatment response. A study that used added N-acetylcysteine in water or Vitamin E into diets in four different genetic mouse models for lung cancer found increased tumor burden and decreased survival compared to control mice that received neither of these antioxidants (Sayin et al. 2014). With antioxidant supplementation, the tumor cells had increased ROS levels and decreased expression of genes involved in oxidative stress defense. This group believed that decreased DNA damage with antioxidant treatment was leading to the enhanced cancer cell growth. There are many variables to consider with non-whole food-based antioxidant use, such as, if that antioxidant is able to reach the tumor and if it will be at a concentration that can impact redox status of the cancer cells. Even with confounding findings, there is still interest in using antioxidants to help cancer patients. Another study injected ascorbate twice daily into mice bearing pancreatic cancer cells, MIA PaCa-2, showed sensitivity to radiation treatment and reduced toxicity in the intestines of the mice (Alexander et al. 2018). They measured increased glutathione and decreased 4-hydroxynonenal, a byproduct of lipid peroxidation, in the small intestine compared to the pancreatic tumor, which suggested that the ascorbate acted as a protective antioxidant in normal tissue that protected against the radiotherapy. This was similarly seen in a phase I clinical trial where there was a significant increase in the overall survival of pancreatic adenocarcinoma patients treated with gemcitabine, with intravenous infusion of ascorbate before and after

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receiving radiotherapy. A separate study conducted in 540 head and neck cancer patients who received radiation therapy were given one or both antioxidant supplements, α-tocopherol or β-carotene, or placebo pills (Bairati et al. 2005). The patients were randomly assigned to the three groups and were provided with supplements starting on the first day of their radiation therapy and for the following 3 years. Those that received antioxidant supplements recorded having decreased adverse effects from the radiation therapy and there were no significant differences in the survival of patients in any of the treatment arms. While these studies were promising in these patient populations, the use of antioxidants during cancer treatment requires further investigation until there is strong evidence to support or discredit its use. Even without solid evidence for the efficacy of antioxidants, cancer patients want to increase their chances of survival as much as possible and many may take it upon themselves to take antioxidants without clear guidelines on when, how, or the quantity of antioxidants to take. A study with over 600 breast cancer patients in New York surveyed if they were self-administering antioxidant supplements (Greenlee et al. 2009). About 60% of patients reported taking any supplement, ranging from Vitamin C to multivitamins in a wide range of doses. While the vast differences in dosing and treatment in these patients made it impossible for the researchers to accurately find changes in treatment response, it did find a correlation between patients who took antioxidant supplements having healthier lifestyles before developing cancer, for example, high fruit and vegetable consumption and lower BMI. Since many of the studies cited above had conflicting results, it is important to acknowledge that their methodologies varied greatly. Antioxidant supplements may have adverse events, but clinical outcomes of many cancer patients who consumed antioxidant-rich whole food has had more promising outcomes. The long-term effects of consuming antioxidants from whole foods versus dietary supplements during cancer treatment to slow progression and to augment therapy warrants further investigation.

Cigarette Smoking Cigarette smoking and cigarette smoke exposure are associated with many different types of cancer beyond lung cancer. Cigarette smoke exposes the body to thousands of chemicals when inhaled through first- or second-hand exposure. Cigarette smoking can cause redox imbalance within the body through the introduction of chemical oxidants as well as through reactive oxygen species produced by the high heat combustion of cigarettes (Biondi-Zoccai et al. 2019). Although there is evidence of redox imbalance from smoking, there are few studies that have been conducted looking at how altered redox status from cigarette smoking may impact cancer treatment and therapy response. Some key examples of redox perturbations noted in smokers are described below. In a cohort of 88 participants, of which 54 were smokers, cigarette smoking was shown to increase the levels of oxidative DNA damage; the levels of oxidative DNA damage in the lungs, and increase the levels of reactive oxygen species in plasma of

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smokers (Cao et al. 2016). Oxidative DNA damage lesions, 8-hydroxydeoxyguanosine, can be induced in the lungs of mice through exposure to cigarette smoke. Moreover, the amount of oxidative DNA lesions were elevated in lung cancer patient bronchoalveolar lavage fluid samples compared non-cancer patients and that these lesions were found at higher levels in smoking lung cancer patients (Cao et al. 2016). Similarly, a study that used lung cancer patient serum to create an oxidative stress index, from a ratio of antioxidant and oxidants, found that former smoking patients had a higher oxidative stress index. Furthermore, this study discovered that patients with metastatic disease also displayed the highest oxidative stress indexes (An et al. 2012). Even if they had quit smoking, traces of the oxidative DNA damage were still detectable in these cancer patients, and this could negatively impact their survival. This study also observed altered glucose metabolism in the smoking patients, which could have implications for the efficacy of cancer treatments. While these studies do not directly demonstrate that oxidative stress from smoking is promoting cancer, this compelling data warrants further investigation. Beyond just damaging lungs, cigarette smoking can cause systemic changes in redox. One example is a study that found serum levels of free radicals and peroxides positively correlated with the number of daily cigarettes smoked in a cohort of 252 participants (Hayashi et al. 2007). This study also showed that all current smokers had higher levels of serum ROS than nonsmokers in their cohort. Another study found that there was increased hydrogen peroxide levels and higher levels of activated NOX2 in serum of people immediately after they smoked a cigarette (Biondi-Zoccai et al. 2019), highlighting systemic effects of smoking. While increased cancer risk from smoking is widely acknowledged, the ways that cancer progression is impacted in patients by smoking is less well known. A retrospective study that compared the survival outcomes of over 5000 patients, inclusive of 13 different cancer types, analyzed how smoking impacted patient survival. Participants in this study answered a smoking questionnaire within 1 month of their cancer diagnosis, to gauge their smoking status around the time they were diagnosed. In head and neck cancer patients, there was the expected result that current smokers had the worst survival, followed by former smokers, and then never smokers. In prostate cancer, it was found that there was only a significant increase in mortality with current smoking versus never smoking, but that former smoking did not greatly change survival compared to current smoking. Unexpectedly with male bladder, colon/rectum or melanoma patients, current smoking was not found to significantly alter their overall survival. In female patients, current smoking did negatively impact their survival if they had breast, melanoma, ovarian, or uterine cancer. Many cancer patients continue to smoke after being diagnosed with cancer, although this may negatively impact their survival prognosis. In the previous study, they found that postmenopausal breast cancer patients had improved survival if they recently quit versus if they continued smoking, but this was not found in premenopausal patients (Warren et al. 2013). This is evidence that can be used to persuade patients to quit smoking even if they already have cancer. A separate study focused on how cigarette smoking histories impacted the progression of acute myeloid leukemia (AML). They found that not only do former and current cigarette smokers have increased risk of developing AML, but when they have AML, their survival is significantly decreased compared to patients who had never

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smoked (Fircanis et al. 2014). Collectively all of these studies looked at thousands of cancer patient cases to find these correlations between cigarette smoking and its impact on cancer patients, but none of them looked for a mechanism that explains the worse survival from cigarette smoking. Challenges of further study include the fact that each cancer type has different standard treatment regimens, thus the findings of smoking impacting survival in patients with different cancer types can mean that smoking may also influence treatment response; therefore it will be important for mechanistic studies to focus on specific cancer types with well characterized and standardized therapies. However, knowledge gained from understanding how redox changes from cigarette smoking promotes treatment resistance or enhanced cancer growth can inform cancer biology and therapeutics broadly.

Conclusion This chapter has explored how redox changes attributed to three lifestyle-related topics associated with cancer risk may contribute to worse outcomes post diagnosis. Despite the fact that there are many studies that have investigated redox changes from modifiable behaviors and that modifiable health behaviors impact cancer progression, there have been few that have looked at them together. Correlations between these health behaviors and cancer survival have been observed in patients without direct connections to the molecular changes that are leading to the prognosis; thus, this is an area where there is great therapeutic potential to help cancer patients. By continuing to gain understanding of redox changes from modifiable health behaviors associated with cancer, there will be increased opportunities to improve tailored treatment for cancer patients, as well as consider implementing lifestyle changes into cancer therapy regimens.

Cross-References ▶ Oxidative Stress, Inflammasome, and Cancer ▶ Preventive Role of Carotenoids in Oxidative Stress-Induced Cancer ▶ Phytochemicals in ROS-Mediated Epigenetic Modulation of Cancer ▶ The Intricacy of ROS in Cancer Therapy Resistance ▶ Vitamin C in Cancer Management: Clinical Evidence and Involvement of Redox Role

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Sonali Choudhury, Afreen Asif Ali Sayed, Prasad Dandawate, and Shrikant Anant

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ROS-Induced Epigenetic Regulation in Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . DNA Methylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Histone Modification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Transcriptional Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Posttranscriptional Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Translational and Posttranslational Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MAPK/ERK1/2 Pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . AKT Pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . JAK-STAT Pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . NRF2-KEAP Signaling Pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cross-References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract

Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancerrelated deaths in the United States. PDAC is characterized by a poor prognosis, delayed diagnosis, early metastasis, malignant phenotype, and drug resistance, resulted in poor patient outcomes. Hence, it is essential to understand the biology of the disease to explore novel pathways involved in PDAC growth and develop novel therapeutics for the treatment of the disease. Recent studies showed that reactive oxygen species is increased in PDAC cells and plays an important role in numerous cellular activities, such as proliferation, apoptosis, invasion, and drug resistance. Depending on the concentration, ROS has a dual role in PDAC. ROS promotes carcinogenesis with low to moderate levels, while at higher S. Choudhury · A. A. A. Sayed · P. Dandawate · S. Anant (*) Department of Cancer Biology, University of Kansas Cancer Center, Kansas City, KS, USA e-mail: [email protected] © Springer Nature Singapore Pte Ltd. 2022 S. Chakraborti et al. (eds.), Handbook of Oxidative Stress in Cancer: Mechanistic Aspects, https://doi.org/10.1007/978-981-15-9411-3_104

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concentrations it induces cell death in PDAC. Cells activate their antioxidant enzyme system, containing superoxide dismutase, catalase, glutathione, as well as tumor suppressor genes (TP53, P21, P16, BRCA1, FOXO), to overcome ROS-induced oxidative stress. ROS activates the mitogen-activated phosphokinase in PDAC, and RAS gene is a key activator of this pathway. Moreover, mutations in RAS result in upregulation of ROS levels, leading to DNA damage and malignant transformation in PDAC. KRAS is a predominant mutation in most PDAC tumors; hence, ROS is a major factor affecting tumor growth. In this review, we have summarized the studies of various gene regulatory mechanisms during oxidative stress in PDAC. Keywords

Oxidative stress · Reactive oxygen species · Pancreatic ductal adenocarcinoma · Gene regulation

Introduction Pancreatic ductal adenocarcinoma (PDAC) is a major problem worldwide. The World Health Organization estimated 495,773 new cases and 466,003 deaths to occur in 2020 (GCO 2020). PDAC is the fourth leading cause of cancer-related deaths in both males and females in the United States (Siegel et al. 2021). According to the recent report of the American Cancer Society, 60,430 new cases and 42,220 deaths are estimated to occur due to PDAC in 2021 (Siegel et al. 2021). The disease is projected to be the second leading cause of cancer-related deaths by 2030 (Rahib et al. 2014). It has an abysmal 5-year survival rate of about 10% (Siegel et al. 2021). PDAC is characterized by a poor prognosis, delayed diagnosis, early metastasis, malignant phenotype, and drug resistance, resulted in poor patient outcomes. Hence, it is essential to understand the biology of the disease to explore novel pathways involved in PDAC growth and develop novel therapeutics for the treatment of the disease. Recently, several studies showed the role of reactive oxygen species (ROS) in PDAC growth. Nicotinamide adenine dinucleotide (NADPH) oxidase is reported to be a major source of intracellular ROS in PDAC cell lines (Vaquero et al. 2004). Increased ROS is also a hallmark of PDAC, and it is involved in pro-survival and antiapoptotic abilities (Afanas’ev 2011) as well as chemoresistance (Donadelli et al. 2011) in PDAC cells. Pancreatitis, a risk factor for PDAC, can lead to increased inflammation and release of reactive oxygen and nitrogen species (ROS/RNS), contributing to oxidative stress. Cytokines and chemokines produced during inflammation recruit immune cells, and these cells produce reactive species. In turn, the reactive species can activate transcription factors that bind to pro-inflammatory genes, leading to increased inflammation. This vicious cycle of inflammation and oxidative stress can damage epithelial and stromal cells, leading to tumorigenesis

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over a period of time. As such inflammatory cytokines and ROS are important in PDAC pathogenesis (Yu and Kim 2014). Oxidative stress is an imbalance of levels of ROS and antioxidant enzymes. Oxygen is a highly reactive element and a robust-oxidizing agent and has high electronegativity compared to other reactive elements. In the cellular environment, mitochondria produce ROS (Zorov et al. 2014). The reactive species differ based on the main atom involved, namely, reactive oxygen, nitrogen sulfur, and chloride species. ROS include superoxide (O2•
) and nitric oxide (NO•), along with radicals of organic (R•), hydroxyl (•OH), alkoxyl (RO•), peroxyl (ROO•), thiyl (RS•), and disulfides (RSSR) (Liou and Storz 2010). The species of ROS that are most studied in cancer are superoxide, hydrogen peroxide, and hydroxyl radicals (Liou and Storz 2010). Superoxide ion is produced in the mitochondrial inner membrane and is generated in the cytosol by the reduction of O2, catalyzed by NADPH oxidases (NOXs). It is usually short-lived and is rapidly converted to hydrogen peroxide (H2O2) by the enzymatic reaction by superoxide dismutase 1 (SOD1), both in the cytosol and in mitochondria. H2O2, in turn, can be partly reduced to hydroxyl radical (OH˙) or completely reduced to H2O by catalase (Lowenfels et al. 1993; Chari et al. 2008; Maisonneuve and Lowenfels 2015). ROS along with exogenous insults, such as chemicals and radiations to the cell, causes repairable and irreparable damages such as DNA double-stranded break (DSB) (Liou and Storz 2010). These all create an environment of oxidative stress in the cell. To overcome stress, these cells have specific repair pathways (Srinivas et al. 2019). Superoxide dismutase (SOD), catalase (CAT), glutathione peroxidases (GPXs), thioredoxins (TRXs), and peroxiredoxins (PRXs) are some of the antioxidant enzymes, which protect cells from oxidative stress. In cancer cells, ROS can promote multiple pathways that increase tumor progression by regulating proliferation, apoptosis, and invasion. However, ROS plays both proapoptotic and antiapoptotic role in PDAC (Afanas’ev 2011). On the one hand, ROS induces DNA damage, promotes tumor initiation and transformation, and facilitate cell survival and cancer progression. On the other hand, the excessive production of ROS triggers apoptotic cell death, although these effects of ROS are highly concentration dependent. In normal cells, tumor suppressor genes, such as p53, FoxO, retinoblastoma (Rb), p21, p16, and breast cancer susceptibility gene 1 and 2 (BRCA 1 and 2) (Vurusaner et al. 2012), modulate cells to adapt to oxidative stress, hence preventing lipid peroxidation and DNA damage by promoting antioxidant genes (Bishayee et al. 2015). Mutant KRAS expression also leads to increased ROS levels, resulting in DNA damage and malignant transformation (Jose et al. 2011). In addition, the expression of oncogenes, such as Raf and Myc, can also silence tumor suppressor genes that could lead to a further increase in ROS levels. Hence, ROS plays an important role in PDAC pathogenesis. In the present review, we are summarizing the role of ROS in histone and DNA modifications, transcriptional and posttranscriptional control (microRNAs, lncRNAs, RNA-binding proteins), and translational and posttranslational pathways (Fig. 1).

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Fig. 1 Reactive oxygen species (ROS)-mediated gene regulation at epigenetic, transcriptional, posttranscriptional, translational, and posttranslational regulation in pancreatic cancer cell

ROS-Induced Epigenetic Regulation in Cancer ROS can change the epigenetic landscape by affecting chromatin remodeling in part through DNA methylation and histone modifications. The two most common DNA methylation changes that occur in the development of cancer are global hypomethylation during the progress of carcinogenesis and regional hypermethylation of CpG islands, which are normally unmethylated. This results in transcriptional suppression of tumor suppressor genes and stimulation of oncogenes (Franco et al. 2008). ROS can oxidize methionine to form methionine sulfoxide, which in turn can methylate cytosine and guanine residues in the DNA. Generation of methylated nucleotides interferes with normal DNA methylation at CpG island (Kawai et al. 2010). Oxidation of guanine to 8-oxoguanine can also result in activation of base excision repair mechanisms, resulting in the apurinic site. Thousands of such sites can accumulate, affecting the sequence of DNA-binding proteins and also the structural integrity of DNA (Lewandowska and Bartoszek 2011). Epigenetics is linked to metabolism as many histone-modifying enzymes use metabolites, such as glutarate, acetyl-CoA, NAD+, and S-adenosylmethionine, which are affected by ROS production (Simpson et al. 2012). Therefore, oxidative stress can universally influence the cell on multiple stages, from DNA and histones to their modifiers, ultimately changing the epigenetic landscape of the cell.

DNA Methylation DNA methylation is the biological process of adding methyl groups to 50 -cytosinephosphate-guanine-30 (CpG) dinucleotide regions in the promoter of genes by DNA

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methyltransferases (DNMTs). There are four types of DNMTs, including DNMT3A and 3B, which are involved in de novo DNA methylation during development; DNMT1 is responsible for methylation of patterns after replication of DNA, and DNMT2 has no catalytic site but acts through de novo DNMTs to recruit complexes for chromatin remodeling (Deplus et al. 2002; Moore et al. 2013). This process results in the hypermethylation of DNA at gene promoters, the result of which is suppressing transcription of that gene. An increase in oxidative stress can enhance the levels and activity of DNMTs. A recent study involving global DNA methylation and gene expression in PDAC found more than 23-thousand CpG sites, differentially methylated with a majority of CpG sites being hypermethylated in cancer. The changes in the methylation status of genes are reported to be involved in cell proliferation, differentiation, epigenetic regulation, and development of the pancreas, all contributing to PDAC. The methylation of CpG sites of DNMT1, DNMT3A, and DNMT3B was also changed in PDAC (Mishra and Guda 2017). Several genes, such as the cyclin D2 (CCND2) gene, are known to be hypermethylated in PDAC and are currently also used as clinical markers to diagnose the disease (12684418). Furthermore, Tan et al. have also identified five imprinting genes (CPA4, MEST, MAGEL2, NDN, and SLC22A3) and four X-linked genes (BGN, GPRP, GUCY2F, and MAGEA1), which are hypomethylated in PDAC as compared to the normal tissue (Tan et al. 2009). Moreover, cyclin D1 and D3 genes are highly expressed in PDAC (Ebert et al. 2001; Gansauge et al. 1997) and associated with the loss of methylation (Tan et al. 2009). Hence, it is important to study the effect of DNA methylation on oxidative phosphorylation in PDAC. ROS can activate the transforming growth factor (TGF)-β signaling pathway, which sequentially can lead to increased levels of DNMTs, resulting in DNA methylation at specific gene promoters (Mahmoud et al. 2019). SOD2 is a tumor suppressor gene that codes for the antioxidant enzyme manganese superoxide dismutase (MnSOD), and DNA methylation at the SOD2 gene promoter modulates its expression. In PDAC cell lines, the SOD2 promoter is hypermethylated, resulting in reduced SOD2 expression (Cullen et al. 2003), causing ROS to build up in the cells. This alters the expression of several target genes (fstl5, man1a1, efhd1, prkacb, and kcnip3) involved in calcium binding, cell adhesion, and steroid metabolism in the PDAC cell line. Moreover, SOD2 expression in MiaPaCa-2 cell-induced dephosphorylation of VEGFR2 (Franco et al. 2008; D’Oto et al. 2016). These data, taken together, demonstrate the power of DNA methylation, regulated in part by ROS to play a significant role in PDAC progression.

Histone Modification Histones undergo posttranslational modifications, which are important for the chromatin structure and gene expression. The modifications comprise histone acetylation, methylation, phosphorylation, SUMOylation, and ubiquitylation. Histone lysine demethylases (KDM) are an important class of proteins that have a Jumonji domain, which is a catalytically active site and can demethylate mono-, di-, and

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trimethylated substrates. KDMs such as Ndy1/KDM2B protect cells from DNA damage response or senescence. It was previously shown that Ndy1 inhibits DNA damage and ROS-mediated signaling and protects cells from G2/M arrest and H2O2induced apoptosis (Polytarchou et al. 2008). Ndy1 protects the cells from ROS-induced oxidative stress by upregulating the expression of antioxidant enzymes, NAD(P)H quinone oxidoreductase-1 (Nqo1) and peroxiredoxin-4 (Prdx4). Ndy1 also binds to the promoter of its target genes and acts by demethylating H3K36me2 and H3K4me3 (Polytarchou et al. 2008; D’Oto et al. 2016). A recent study has shown that Ndy1 is overexpressed in PDAC and can drive tumorigenicity. It can bind to the polycomb group (PcG) protein complex at the transcriptional start sites and activate the expression of metabolic genes involved in mitochondrial function and protein synthesis. Ndy1 can also regulate EZH2 levels by epigenetic silencing of microRNAs miR-101 and let7b in PDAC cell lines (Tzatsos et al. 2013). Moreover, we recently showed that KDM3A is overexpressed in PDAC during hypoxia and contributes to the growth and stemness by interacting with doublecortin like kinase-1 (DCLK1) in orthotopic PDAC model in mice (Dandawate et al. 2019). Acetylation and deacetylation of histone residues are another important set of modifications that affect gene expression. Histone acetyltransferases (HATs) are responsible for adding acetyl residues to histone, while deacetylases (HDACs) remove the acetyl marks. In cancer, the combination of inflammation and oxidative stress enhances redox signaling by inactivating HDACs (Yang et al. 2006). During acute pancreatitis, inflammation and oxidative stress result in the transcription of the pro-inflammatory genes by regulating the expression of transcription factors by histone and chromatin modifications. Phosphatases and transcription factors such as NF-κB and HDACs are also major targets of ROS in inflammation. More research is needed to identify the mechanism of oxidative stress in histone acetylation during acute pancreatitis (Escobar et al. 2012).

Transcriptional Control Oxidative stress plays a vital role in the transcription of specific genes through the activation of redox-sensitive transcription factors. ROS affects nuclear transcription factors by modulating upstream signaling such as protein kinase and phosphatase along with calcium signaling. Transcription factors can either be tumor suppressors or promote tumor growth by changing the expression of target genes involved in the cell cycle and progression of tumors. The effect of ROS on several transcription factors has been studied in PDAC. KRAS gene is mutated in more than 90% of PDAC patients. KRAS increases ROS by multiple mechanisms. KRAS can increase ROS production by modulating mitochondrial metabolism through the hypoxia-inducible factors (HIFs) HIF-1α and HIF-2α. KRAS is involved in suppressing respiratory chain complex I and III, leading to mitochondrial dysfunction and ROS production. KRAS also alters NADPH oxidase activities to elevate the intracellular levels of ROS (Storz 2017).

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KRAS also increases the expression of antioxidant systems to balance ROS. Therefore, detoxifying ROS to maintain low levels of KRAS helps cancer cells to avoid apoptosis and increase proliferation, leading to cancer progression (Kong et al. 2013; Son et al. 2013). In PDAC cells mutant KRAS mediates the increase of mitochondrial ROS, resulting in the progression of PanIN through protein kinase D1 (PKD1) and transcription factors such as NF-κB1/B2. NF-κB1/B2 increases the expression of epidermal growth factor receptor (EGFR) and its ligands and aids in the formation of precancerous lesions in the pancreas (Liou et al. 2016). FOXM1 (forkhead box protein M1) is highly expressed in proliferating cells and is central for cell-cycle progression. FOXM1 is overexpressed in many different cancers, including prostate, pancreatic, stomach, liver, bladder, etc. It is a vital regulator of oxidative stress in a tumor. One study showed that FOXM1 induction by oncogenic Ras is dependent on ROS. This increased FOXM1 can, in turn, decrease ROS by inducing antioxidant genes such as catalase, MnSOD, and thioredoxin-dependent peroxide reductase (PRDX3). Tumor cells control oxidative stress and escape early death and apoptosis with FOXM1 expression. Studies reported that in human PDAC cells reduced VDR (vitamin D receptor) mediates expression of FOXM1 and its targets and suppresses stemness in PDAC cells (Park et al. 2009; Li et al. 2015b). The NF-κB transcription factor is a vital modulator for inflammation, inducing the expression of numerous cytokines and chemokines in macrophages and stroma, which can then increase tumor growth and angiogenesis. NF-κB also regulates angiogenesis and EMT by acting on the promoter of genes such as HIF1-α and VEGF in PDAC (Prabhu et al. 2014). NF-κB is also important for drug resistance especially to gemcitabine used for PDAC treatment (Pramanik et al. 2018). Zhang et al. showed that ROS induced by gemcitabine contributed to chemoresistance in PDAC cells. Gemcitabine promoted the phosphor STAT3 binding to the promoters of Bmi1, Nanog, and SOX2 genes to induce spheroid formation, migration, and chemoresistance. These effects were found to be NADPH oxidase (Nox) generated and ROS dependent, suggesting that gemcitabine resistance in PDAC stem cells is mediated through the Nox/ROS/NF-κB/STAT3 signaling pathway (Zhang et al. 2016b). The transcription factor p53 termed as guardian of the genome is a tumor suppressor that is regularly mutated in cancer cells. It plays a central role in DNA repair, cell proliferation, cell-cycle regulation, and stimulation of apoptosis. Under normal conditions wild-type p53 reduces ROS production. In cancer cells, mutant p53 increases the production of ROS by regulation of redox-related pathways. In turn, ROS can stabilize mutant p53, contributing to its oncogenic function (Cordani et al. 2020). Oxidative stress can induce the activity of p53 by SUMOylation. TP53INP1 (tumor protein p53-induced nuclear protein 1) is a redox sensor and suppresses tumor function. It mediates the transcription of p53 target genes and promotes its antioxidant function. TP53INP1 expression is deregulated in PDAC early in preneoplastic development. Upon DNA damage, due to oxidative stress, p53 activity is enhanced by SUMOylation of TP53INP1 (Peuget et al. 2014). In human cells, TIGAR (TP53-inducible glycolysis and apoptosis regulator) regulates glucose

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breakdown, DNA repair, and degradation of organelles (▶ Chap. 51, “Oxidative Stress in Orchestrating Genomic Instability-Associated Cancer Progression”). TIGAR promoter can be bound by p53 to activate its expression. TIGAR is activated under low levels of oxidative stress, and high levels of stress decrease its expression (Madan et al. 2012). In PDAC mouse models TIGAR levels are higher in premalignant lesions and decreased in metastatic tumors. TIGAR is upregulated in cancer cells, and at low ROS levels, it plays role in metastasis (Cheung et al. 2020). The data suggest that ROS regulation by TIGAR supports premalignant tumor initiation while restricting the metastasis in PDAC. Hypoxia-inducible factor (HIF)-1α is a part of the transcription factor HIF1 encoded by the HIF1A gene. It is a transcriptional regulator of developmental and cellular response to hypoxia. Pancreatic stellate cells (PSCs) give rise to a desmoplastic response in PDAC. Hypoxia and ROS together activate PSCs through induction of HIF1α and Gli1 expression. This promotes PSCs to secrete soluble factors, such as interleukin (IL)-6, vascular endothelial growth factor A (VEGFA), and stromal cell-derived factor 1 (SDF1), which favor PDAC invasion and promote malignant phenotype (Zhang et al. 2016a).

Posttranscriptional Control The regulation of gene expression at the RNA level after transcription but before the translation is called posttranscriptional regulation. Posttranscriptional control of mRNA transcripts is mediated by multiple factors, including RNA-binding proteins (RBPs) and miRNAs. The action of these factors can result in alternative splicing, nuclear export, stabilization, degradation, modification, etc. All these events ultimately regulate the translation of the transcript and its expression. In many types of cancer, ROS and noncoding RNAs (ncRNAs) interact to regulate the expression of genes and signaling pathways. For example, lncRNA H19 regulates many Nrf2 target genes such as GSTP1 and NQO1. NRF2 regulates the expression of antioxidant proteins, which protect the cell against inflammation and injury caused by oxidative stress or damage. H19 may have a role in antioxidant defense (Siegel et al. 2020). About 70% of the human genome gets transcribed, out of which only 2% codes for known protein-coding genes. The rest are noncoding RNAs (ncRNAs), which are small RNAs that play roles in epigenetic regulation, translation, replication of DNA, and splicing RNA. They do not code for proteins. Different ncRNAs comprise transfer RNA (tRNA), small-interfering RNA (siRNA), ribosomal RNA (rRNA), Piwi-interacting RNA (piRNA), microRNA (miRNA), and long noncoding RNA (lncRNA). Mutations in ncRNA genes are associated with several diseases including cancer. miRNAs and lncRNAs have shown successful therapeutic potential in PDAC patients through guided delivery of therapeutic nucleotides (Taucher et al. 2016). MicroRNAs (miRNAs) are small RNAs that are 18- to 25-nucleotide and noncoding and involved in regulating gene expression. They bind to a

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complementary sequence in the target mRNA and degrade the mRNA or inhibit translation of the mRNA. miRNAs are important for PDAC pathogenesis by regulating tumor growth and metastasis (Tan et al. 2018). ROS can affect the biogenesis of miRNA by reducing dicer and drosha, which are two key enzymes required for miRNA processing (Ungvari et al. 2013). Oxidative stress can also regulate miRNAs by modulating transcription factors such as Myc, p53, and NF-κB. ROS also affects DNA methylation, which in turn can affect the expression of miRNAs, which in turn also regulate the production of ROS. miRNAs can act on redox sensors and antioxidant proteins such as NRF2, AKT, Myc, and p53 to regulate redox signaling (Lin 2019). Therefore, it is important to study miRNA and ROS cross talk in tumorigenesis. miR-200a and miR-200b belong to the miR-200 family and are important in epithelial-to-mesenchymal transition (EMT). They are frequently found to have hypomethylated promoters, leading to their overexpressed in PDAC. One of their downstream targets, Smad-interacting protein 1 (SIP1) is also hypermethylated and silenced, which reduces the expression of E-cadherin, contributing to an EMT phenotype (Li et al. 2010). miRNAs can be detected in the circulation, and increased levels of miR-200c in serum can be measured as a marker for better survival of patients after surgery (Yu et al. 2010). miR-200c miRNA stimulates cell proliferation but inhibits invasion in PDAC cell lines. miR-155 is also highly upregulated by KRAS in pancreatic cell lines through the activation of the NF-κB and MAPKAP1 pathway. Overexpression of miR-155 inhibited FOXO3A expression, which decreased the level of antioxidants SOD2 and catalase. This leads to increased ROS generation and in turn cell proliferation (Wang et al. 2015). One microRNA, miR-146a, is a powerful tumor suppressor, and low expression of miR-146a is shown to have a bad prognosis in PDAC (Dando et al. 2015). The miR-210 has also been proposed to have an important part in ROS homeostasis, DNA damage repair pathway, stem cell survival, and cellular adaptation during hypoxia. miR-210 is activated by HIF-1α, HIF-2, and NF-kB in hypoxia-related ROS production. miR-210 levels are elevated in PDAC and are correlated with poor prognosis (Dando et al. 2015). miR-23b is important for regulating autophagy by acting on ATG12. In PDAC, the miR-23b expression is reduced, resulting in increased levels of ATG12. Moreover, overexpression of miR-23b blocks the induction of autophagy and sensitizes PDAC cells to radiation treatment (Wang et al. 2013). miR-135b is also a critical biomarker in PDAC and is upregulated by glutamine deficiency. Low levels of glutamine, which triggers ROS levels, are found in PDAC patient samples. Under the condition of glutamine deprivation, mutp53 induced miR-135 expression by binding directly to its promoter region. Moreover, during glutamine deprivation glycolysis was downregulated by miR-135 to promote PDAC cell survival (Yang et al. 2019). LncRNAs or long ncRNAs are more than 200 nucleotides long. The mechanisms by which the lncRNAs modulate gene expression are not fully understood, but they can regulate gene expression at several steps from transcription to translation. They can act as molecular indicators or signals of transcriptional activity. They can also act as scaffolds and provide a platform for bringing two or more proteins or RNAs to

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discreet locations. They act as decoys or molecular sponges by binding with microRNAs or other proteins and regulate gene expression. LncRNA can guide and direct the localization of the nuclear proteins and chromatin complexes to specific targets (Sun et al. 2018). Many studies have shown the role of lncRNAs in tumor progression and tumorigenesis. Aberrant lncRNA expression may follow immune escape, DNA damage, and cellular metabolic disorders in cancer cells. LncRNAs are also associated with EMT and the regulation of stemness in cancer cells. This suggests an important role of lncRNAs in carcinogenesis and disease progression (Jiang et al. 2019). LncRNAs are aberrantly expressed in various diseases and can act through the oxidationantioxidant system. They have been shown to exert their effects and act through the Nrf2/Keap1/ARE pathway (Wang et al. 2019). LncRNA Metastasis-associated lung adenocarcinoma transcript-1 (MALAT1). In response to oxidative stress, MALAT1 plays an important role. When MALAT1 was overexpressed in hydrogen peroxide (H2O2)-induced HUVECs (human umbilical vein endothelial cells), it activated and stabilized the nuclear factor erythroid 2-related factor 2 (Nrf2) protein by lowering the Keap1 level. Therefore, it can increase antioxidant capacity to oxidative stress and DNA damage. MALAT1 can also bind to Nrf2 before it binds to antioxidant response element (ARE) and acts as a regulator of Nrf2 (Wang et al. 2019). MALAT1 expression is high in PDAC cells, and it is a negative prognostic factor (Cheng et al. 2018). MALAT1 can bind to specificity protein 1 (SP1), which is a transcription factor, and modulates apoptosis and oxidative stress through the p38MAPK (p38 mitogenactivated protein kinases) pathway (Wang et al. 2019). Specificity proteins Sp1, Sp3, and Sp4 are transcription factors (TFs) (Cullen et al.). They belong to the Sp/Krüppel-like family (KLF) and are important in embryonic growth and early development, expression of SP1 decreases with age, and overexpression of SP1 or SP3 in pancreatic and other cancers is a negative prognostic factor (Hedrick et al. 2016). ROS-inducing anticancer agents that target SP 1/2/3 also target MALAT1 expression and pro-oncogenic effects of MALAT1 along with other SP-regulated genes (Cheng et al. 2018). These studies show that MALAT1 relates to antioxidant pathways through KEAP/NRF2/ARE pathway, it also modulates oxidative stress through the p38-MAPK pathway by binding through the SP protein transcription factor. Further studies are needed to investigate an oxidative stress-related role of MALAT1 in gene regulation in PDAC. LncRNA UCA1 (urothelial cancer associated 1) and tumor metabolism. Unlike normal cells, cancer cells reprogram their energy metabolism with glycolysis. Due to this cancer cells exhibit high levels of ROS, and to compensate for oxidative damage, they use the antioxidant system. Several studies have shown the possible role of ncRNA and ROS in the expression of metabolic genes (Zhang et al. 2018). Glutaminolysis is very important for cancer cells to maintain redox balance and reduce ROS levels. Glutaminase is an important enzyme in glutamine metabolism. Glutaminase is a known target of miR-16 and miR-16 regulates the levels of tumor suppressors or oncogenes after mRNA transcription. In bladder cancer cells,

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lncRNA UCA1 controls GLS2 (isoform of glutaminase) expression by acting as a sponge for miR-16, thereby increasing ROS production (Li et al. 2015a). The expression of UCA1 has been significantly upregulated in PDAC and is involved in promoting tumor growth, invasion, and migration. In PDAC cells, overexpression of UCA1 increased YAP (yes-associated protein 1) expression and promoted its stabilization and nuclear localization. YAP is an important protein Hippo signaling pathway that controls organ size through cell proliferation and apoptosis. UCA1 regulates PDAC progression through modulating the Hippo signaling pathway (Zhang et al. 2018). These studies show that UCA1 has roles in oxidative stress-related metabolic reprogramming and it is also implicated in PDAC progression. Further studies are needed to investigate the connection between UCA1-ROS and metabolic programming related to gene expression in PDAC. LncRNA H19. LncRNA H19 is 2.3 kb in size and expressed from maternal allele early during embryogenesis. Depending upon the cell types, H19 has various functions and is associated with the development and progression of PDAC. H19 can act as a sponge for miRNA and has been shown to regulate oxidative stress, fibrosis, and inflammation. In PDAC, H19 behaves as an oncogenic factor and targets miR-675 and regulates the expression of E2F-1, which is a transcription factor crucial for cell-cycle control and tumor suppressor proteins. H19 also acts as a sponger for miR-194 and regulates serine/threonine-protein kinase PFTAIRE-1 (PFTK1) expression, which is an important player in the Wnt/β catenin pathway, which regulates growth and metastasis in PDAC (Wang et al. 2020). H19 can activate the NF-κB pathway to protect cancerous cells from oxidative stress. H19 is highly expressed in HCC (hepatocellular carcinoma). Studies show that inhibition of H19 induces oxidative stress and reduces cell apoptosis and chemoresistance of CD133+ cancer stem cells. H19 can act by blocking the MAPK/ERK signaling pathway for the above function (Ding et al. 2018). Another study has shown an essential role of H19 in high-grade serous ovarian cancer. H19 contributes to cisplatin resistance in ovarian cancer by regulating glutathione metabolism. The study showed an important role of lncRNA H19 in activating antioxidant defense through the NRF2 pathway (Zheng et al. 2016). These reports suggest that H19 might be a potential candidate in the connection between oxidative stress and gene regulation in PDAC. Further studies are warranted to investigate the role of lncRNA H19 along with transcription factors, which are regulated by the redox pathway. LncRNA regulator of reprogramming (ROR). Hypoxia is a characteristic feature of solid tumors. Hypoxia produces ROS, which activates HIF1A (hypoxiainducible factor 1A). Hypoxia-driven ROS also activates NRF2, which regulates antioxidant defense genes by reducing ROS levels. These two pathways may act together or separately. ROS produced during hypoxia activates and stabilizes HIF1A, which triggers molecular mechanisms to sustain growth, survival, metastasis, and metabolic changes (Tafani et al. 2016). LncRNA ROR is upregulated in response to hypoxia and in turn increases HIF1A protein. During hypoxia, lncROR is released in HCC. LncROR is 2.6 kb long and plays oncogenic roles in cancer. It is involved in the differentiation of cells to iPSCs and the maintenance of embryonic

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stem cells. The expression of lncROR is dysregulated in several cancers and also in PDAC. One study showed lncROR as a prognostic factor in PDAC. They showed that lncROR mediates metastasis and migration partly by inhibition of p53. This leads to activation of ZEB1, which represses the members of the miR-200 family and results in increased EMT and stemness (Pan et al. 2016). So far, the relation between lncRNA and oxidative stress in PDAC is understudied. Many lncRNAs have been studied for their roles in mitochondrial metabolism. For example, TUG1 is associated with mitochondrial activity and energy metabolism of cell growth. LncRNAs, lncND5, lncND6, and lncCytB, generated from the mitochondrial genome contribute to the expression of a mitochondrial gene. Several lncRNAs, such as HOTAIR, influence mitochondrial OXPHOS activity. In addition, lncRNAs, such as MALAT1, ANRIL, H19, and HOTAIR, downregulate the apoptotic process initiated by mitochondria and positively regulate cancer cell survival (De Paepe et al. 2018). Given the important role of lncRNAs in cancer development and progression, it is crucial to understand and connect the dots between ROS, lncRNA, and aberrant gene expression in PDAC. RNA-binding proteins (RBP). RBPs play an important role in the posttranscriptional control of gene expression, thereby regulating cell proliferation, death, development, and differentiation. RBPs can either bind to single- or double-stranded RNA and are involved in pre-mRNA splicing, translation, and turnover. Dysregulation of RBPs is seen in many diseases including cancer. The RBP TTR (turnover and translation regulatory) protein can modulate the expression genes in response to oxidative stress. The human antigen R (HuR) can bind and regulate the expression of RNAs involved in cell growth and survival. HuR plays a protective part in DNA damage response and oxidative stress by regulating HIF-1α, p53, Myc, and VEGF mRNAs. HuR is upregulated in PDAC and responsible for gemcitabine sensitivity. Studies have shown that exposure to gemcitabine in cells causes HuR to translocate to the nucleus and bind to doxycycline kinase (DCK1) mRNA to increase metabolism. In glucose-deprived PDAC, HuR is involved in ROS clearance. HuR also regulates IDH1 mRNA, which codes for the antioxidant isocitrate dehydrogenase and promotes cell survival in PDAC lines (Zarei et al. 2017; Kim et al. 2017; Brody and Dixon 2018). YBX1 (Y box-binding protein 1) is known to be a transcriptional and translational regulator involved in mRNA splicing, centromere maturation, and DNA repair. Satellite RNA MajSAT binds to YBX1, under stress conditions, and this interaction inhibits nuclear translocation of YBX1 and reduces DNA damage repair function. YBX1 translocates to the nucleus under oxidative stress. In PanIN-derived cells MajSAT is aberrantly expressed and increases mutations in mitochondrial and genomic DNA and contributes to malignant transformation (Kishikawa et al. 2016).

Translational and Posttranslational Control Signaling pathways in cells heavily depend on reversible posttranscriptional modification (PTM) of proteins. PTMs are reversible and can reprogram protein functions. PTMs regulate many functions of cells including cell growth and

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differentiation to programmed cell death (Theillet et al. 2012). ROS can modify the proteins and their functions involved in cell signaling, and these functions are indicated in many diseases such as diabetes, uncontrolled growth, and inflammation. ROS along with PTMs renders a posttranscriptional control on various signaling pathways involved in many cancers such as PDAC. Many studies have shown the impact of ROS on signaling pathways and their action on the cell signaling proteins; detailed studies are needed to find out how ROS is generated, how the homeostasis is maintained, and how ROS targets and impacts the cell signaling proteins, such as NF-kB, MAPKs, Keap1-Nrf2-ARE, and PI3KAkt. Signaling pathways that are sensitive to ROS are elevated in many cancer types. These pathways can play an important role in cell proliferation, differentiation, and survival. They are also involved in inflammation, glucose metabolism, and protein synthesis (Storz 2005). Among reactive oxygen species, H2O2 plays an important role in cellular signaling; it can oxidate and regulates the activity of its target proteins such as tyrosine phosphatases, receptor tyrosine kinases, protein tyrosine kinases, and transcription factors (Liou and Storz 2010). We have summarized here some of the signaling pathways, which have shown some activation by ROS. More detailed studies are needed to understand the mechanisms.

MAPK/ERK1/2 Pathway ROS or H2O2 can act as a secondary signal to activate protein kinase cascades. H2O2 oxidizes the cysteine residues present in proteins to cysteine sulfenic acid or disulfide. The protein tyrosine phosphatases (PTPs) have cysteine residues in their active sites and can be inactivated by a low level of oxidation. In PDAC, the MAPK (mitogen-activated protein kinase)/Erk1/2 (extracellular regulated kinase 1/2) pathway is constitutively activated and mediated through KRAS and growth factors, which are important for cell proliferation. Oxidative stress can inactivate the MAPKspecific phosphatase, therefore activating MAPK/ERK1/2 pathway. Studies showed that MAPK family proteins, such as ERK1/2, p38, and JNK along with activated Ras, can regulate the expression of various matrix metalloproteinases (MMP). Oxidative stress-induced redox-sensitive NF-kB also regulates the expression of the MMP gene. Oxidative stress can regulate cell motility by activating various pathways and inactivating protease inhibitors (Liou and Storz 2010; Shi et al. 2008).

AKT Pathway AKT or protein kinase B (AKT/PKB) is involved in the cell survival pathway by inactivating proapoptotic proteins and decreasing apoptosis (Liou and Storz 2010). ROS can modulate the expression of AKT by acting on its upstream kinases, such as PI3K, PDK-1, and mTOR, or by ablating the activity of phosphatase and tensin homolog (PTEN) phosphatase (Storz and Toker 2002). AKT activity is negatively regulated by PTEN, and H2O2 reversibly inactivates PTEN. A study showed that oxidative stress can be induced in cells with exogenous H2O2, which in turn activates

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AKT and PDK-1 activity (Higaki et al. 2008; Prasad et al. 2000; Lee et al. 2002). The loss of PTEN is also accompanied by a decrease in the expression of many antioxidant enzymes, such as peroxiredoxins and superoxide dismutase, which in turn increases the basal levels of superoxide and H2O2. ROS induces AKT by inactivating PTEN and activating its upstream kinases (Liou and Storz 2010). In PDAC, it was seen that ROS increased the cell survival signals by activating AKT and inhibiting superoxide production (Mochizuki et al. 2006).

JAK-STAT Pathway During oxidative stress, the STAT family of transcription factors are activated by various growth factors and cytokines. In human PDAC cells, PANC-1 and MiaPaCa2, the enzyme NADPH oxidase 4 (NOX4) produces ROS after stimulation by growth factors. ROS then can oxidize and inactivate the protein tyrosine phosphatase (PTP), leading to enhanced and sustained phosphorylation of JAK2. This activates the JAK/STAT pathway, leading to decreased apoptotic cell death of PDAC cells (Vaquero et al. 2004). JAK2 activation can also induce ROS production in some cells, showing a feedback mechanism between JAK2 and intracellular ROS (Duhe 2013). In PDAC cell lines, the inflammatory cytokine interferon-γ can regulate the transcription of Duox2 belonging to the NADPH oxidase family and its maturation factor DuoxA2. This activation is achieved through STAT-1 and p38-MAPK signaling pathways. Duox2/DuoxA2 upregulation increases both extracellular H2O2 and intracellular ROS production, which creates a pro-angiogenic environment that favors tumor growth (Wu et al. 2011).

NRF2-KEAP Signaling Pathway The nuclear factor erythroid derived 2 (NRF2) is a transcription factor involved in the expression of antioxidant proteins to regulate redox balance in cells. It induces several detoxification enzymes such as superoxide dismutase (SOD), peroxidases, and glutathione S-transferase (GST). Nrf2 is normally bound to its regulator Kelchlike ECH-associated protein 1 (KEAP1) that can lead to its proteasomal degradation in the cytoplasm. During oxidative stress, this interaction is perturbed, and Nrf2 localizes to the nucleus and induces many genes by interacting with the ARE (antioxidant response element) sequence on their promoter region. In PDAC, the ARE-driven genes controlled by Nrf2 are upregulated, and Nrf2 upregulation in part is driven by KRAS. This contributes to chemosensitivity, proliferation, and reduced survival (Ma 2013; Lister et al. 2011; Hayes et al. 2015). In the oncogenic KRAS mouse model, inhibition of Nrf2 expression in the pancreas decreased PanIN lesions and also affected proliferate by upregulating senescence markers (DeNicola et al. 2011). The oxidative regulation of Nrf2 promotes EGFR signaling through AKT, leading to cap-dependent translation initiation. This promotes protein synthesis on a global level in PDAC. NRF2 can also directly induce translation by maintaining the reduced state of cysteine residues in proteins (Chio et al. 2016).

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Alternative splicing creates various mRNAs from a single gene and generates proteomic diversity. ESRPs (epithelial splicing regulatory protein), which plays a role in metastasis, regulates the splicing of mesenchymal-specific and epithelial proteins such as FGFR2 and CD44. Expression of CD44V, which is a splice variant of CD44, has been indicated in promoting the aggressive behavior of pancreatic along with other cancer types (Wang et al. 1999). CD44 is expressed on the surface of cells and is a known cancer stem cell (CSC) marker that is highly expressed in several cancers, including PDAC. CD44 and its isoforms aid in ROS defense through synthesis and upregulation of GSH (reduced glutathione), which is an intracellular antioxidant. CD44 splice variants can be a good target in reducing chemo- and radiotherapy resistance to the aggressiveness of the disease (MatzkeOgi et al. 2016; Nagano et al. 2013).

Conclusion Low-level ROS has been implicated in the modulation of signal transduction, which can lead to cancer development. Abnormal production of ROS during oxidative stress is a hallmark of cancer progression. In normal cells, oxidative stress is induced due to normal mitochondrial metabolism. Oxidative stress is reduced by the cell’s antioxidant program, which consists of enzymes that scavenge ROS. When these antioxidants are depleted in the cells, it undergoes apoptosis, and a redox balance is achieved. In PDAC cells, ROS works as a double-edged sword (▶ Chap. 66, “The Double-Edged Sword Role of ROS in Cancer”). Low levels of ROS are beneficial for cancer growth, but high levels of ROS can induce cell death. ROS production is increased in cancer cells, but the levels of ROS are maintained by the various antioxidant machinery to a sustained low level. This low-level ROS can then act as a secondary messenger to increase cell proliferation and survival. This modulation of ROS in PDAC is thought to be antiapoptotic and pro-survival; it also renders the cancer resistant to chemo- and radiotherapy. The cells have a unique ability to avoid cell death by maintaining ROS levels and by modulating different gene expression at various levels. Reprogramming of cellular metabolism through aberrant gene expression under oxidative stress is one of the common features of cancer cells (Fig. 2). Cancer cells exploit the cell’s redox balance to their favor by regulating genes at epigenetic, transcriptional, posttranscriptional, translational, and posttranslational levels in PDAC. In PDAC, ROS can cause epigenetic changes to DNA and histones. ROS can also affect histone-modifying enzymes by affecting the accumulation of metabolism intermediates, such as glutarate, acetyl-CoA, NAD+, and S-adenosylmethionine, that act as substrates for the histone-modifying enzymes. ROS can affect the activation of several transcription factors, which can, in turn, modulate ROS production to a low level by activating antioxidant genes. This feedback mechanism helps maintain a low level of ROS necessary for cell proliferation and survival. In PDAC the transcription factor FOXM1 suppresses stemness, P53 regulates TP53INP1, and TIGAR mediates tumorigenesis

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Fig. 2 Cancer cells hijack the antioxidant program of a normal cell to achieve balance for proliferation and survival by keeping reactive oxygen species (ROS) in balance (Galadari et al. 2017)

and metastasis. HIF1-α promotes a malignant phenotype. Mutant KRAS also has a critical role in gene regulation during oxidative stress. Many microRNAs are found to regulate the expression of genes in PDAC as a posttranscriptional control during oxidative stress. The miR-200 family, miR-155, miR-146a, miR-210, miR-23b, and miR-135b, has roles in EMT, invasion, proliferation, autophagy, and cellular adaptation during hypoxia. In PDAC RNA-binding proteins such as HuR and satellite RNAs such as MajSAT mediate cell survival and malignant transformation through the aberrant expression of many genes during oxidative stress. Many lncRNAs, such as MALAT1, H19, and HOTAIR, have also shown to influence the gene expression during cancer progression and pathogenesis under oxidative stress. ROS generation in cancer can activate various kinase cascades. ROS can regulate various signaling pathways by activating their upstream modulators or by inhibiting the phosphatase. Many phosphatases are affected by ROS, which oxidizes the cysteine residues present in their active site, constitutively activating several pathways that contribute to tumor progression. Several signaling pathways, such as MAPK/ERK-1, PI3K-AKT, NF-κB, Jak-stat, and Keap/Nrf2, are upregulated by

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ROS. Splice variants of genes such as CD44 are also indicated in gene regulation during oxidative stress and contribute to chemo- and radiotherapy resistance. These pathways play important roles in cell death, apoptosis, and chemoresistance.

Summary In summary, a moderate level of ROS favors cancer progression, and this redox balance in cancer cells is highly regulated at epigenetic, transcriptional, posttranscriptional, translational, and posttranslational levels. The oxidative stress in PDAC plays an important role in the progression and cell survival. Understanding the role and physiology of oxidative stress in PDAC can help find new therapeutic targets for the disease.

Cross-References ▶ Oxidative Stress in Orchestrating Genomic Instability-Associated Cancer Progression ▶ The Double-Edged Sword Role of ROS in Cancer

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Ridhima Wadhwa, Keshav Raj Paudel, Shakti Shukla, Madhur Shastri, Gaurav Gupta, Hari Prasad Devkota, Mary Bebawy, Dinesh Kumar Chellappan, Philip Michael Hansbro, and Kamal Dua

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Etiology of Lung Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Smoking . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genetic Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Gender . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Age . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . COPD and Other Pulmonary Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Diet and Obesity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Environmental Air Pollution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Role of Reactive Oxygen Species (ROS) in Lung Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Oxidative Stress–Mediated Non-Small-Cell Lung Cancer (NSCLC) . . . . . . . . . . . . . . . . . . . . . . . . . Role of Cigarette Smoking in Oxidative Stress–Induced LC (NSCLC) . . . . . . . . . . . . . . . . . . . . . . Epigenetic Changes Due to OS in NSCLC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . DNA Modification/Mutation and Lung Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . HDACi as Potential Epigenetic Therapy in NSCLC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Reference . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Ridhima Wadhwa and Keshav Raj Paudel contributed equally with all other contributors. R. Wadhwa Centre for Inflammation, Centenary Institute, Sydney, NSW, Australia Discipline of Pharmacy, Graduate School of Health, University of Technology Sydney, Ultimo, NSW, Australia K. R. Paudel Centre for Inflammation, Centenary Institute, Sydney, NSW, Australia Faculty of Science, University of Technology Sydney, Ultimo, NSW, Australia S. Shukla Priority Research Centre for Healthy Lungs, Hunter Medical Research Institute (HMRI) & School of Biomedical Sciences and Pharmacy, University of Newcastle, Callaghan, NSW, Australia © Crown 2022 S. Chakraborti et al. (eds.), Handbook of Oxidative Stress in Cancer: Mechanistic Aspects, https://doi.org/10.1007/978-981-15-9411-3_106

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Abstract

Lung cancer is considered as the leading cause of cancer-related deaths globally. Etiology of lung cancer include smoking, genetic factor, age, gender, ethnicity, race, diet, obesity, infections, environmental air pollution, occupational exposure, and chronic respiratory diseases. Oxidative stress mediated by cigarette smoking facilitates DNA mutation, the proliferation of lung epithelial cells, and as a result of oncogenic activation leading to the progression of lung cancer. Cigarette smoke also induces microRNA-mediated stress response, genetic expression, and apoptosis. Epigenetic regulations such as chromatin remodeling, DNA methylation, chromatin modifications, microRNA modulations, and histone modification are associated with carcinogenesis. The precise identification of genetic mutations that primarily drive tumor initiation and/or disease progression could lead to the development of targeted treatments that may improve the disease outcomes along with increasing life expectancy for patients with lung cancer. Histone deacetylase inhibitors (HDACi) are emerging as a potential epigenetic therapy for non-small-cell lung cancer (NSCLC). HDACi such as vorinostat, panobinostat, istodax, or entinostat may hold the potential to restore the downstream pathway involved in the lung cancer pathogenesis. Furthermore, HDACi M. Shastri School of Health Sciences, College of Health and Medicine, University of Tasmania, Launceston, Australia G. Gupta School of Pharmacy, Suresh Gyan Vihar University, Jaipur, India H. P. Devkota Graduate School of Pharmaceutical Sciences, Kumamoto University, Kumamoto, Japan M. Bebawy Discipline of Pharmacy, Graduate School of Health, University of Technology Sydney, Ultimo, NSW, Australia D. K. Chellappan Department of Life Sciences, School of Pharmacy, International Medical University, Kuala Lumpur, Malaysia P. M. Hansbro Centre for Inflammation, Centenary Institute, Sydney, NSW, Australia Faculty of Science, University of Technology Sydney, Ultimo, NSW, Australia Priority Research Centre for Healthy Lungs, Hunter Medical Research Institute (HMRI) & School of Biomedical Sciences and Pharmacy, University of Newcastle, Callaghan, NSW, Australia K. Dua (*) Centre for Inflammation, Centenary Institute, Sydney, NSW, Australia Discipline of Pharmacy, Graduate School of Health, University of Technology Sydney, Ultimo, NSW, Australia Priority Research Centre for Healthy Lungs, Hunter Medical Research Institute (HMRI) & School of Biomedical Sciences and Pharmacy, University of Newcastle, Callaghan, NSW, Australia e-mail: [email protected]

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has also been used in NSCLC clinical trials, either as monotherapy or as an adjuvant with radiotherapy or chemotherapy. HDACi as an adjuvant exerts potent antitumor efficacy in treating NSCLC. A combination of vorinostat, carboplatin, and paclitaxel has high efficacy in treating advanced NSCLC. In this review, we have highlighted the role of oxidative stress–mediated epigenetic changes leading to lung cancer progression and the potential of epigenetic therapy for lung cancer. Keywords

Non-small-cell lung cancer · Oxidative stress · Epigenetic therapy · DNA methylation · Histone modification

Introduction Lung cancer is among the leading causes of death worldwide, accounting for 2 million patients and 1.7 million deaths (Malyla et al. 2020). Lung cancer patients are mostly diagnosed at advanced stages, constituting more than 60% of the patients at stage III or IV, hence the 5-year survival rate of patients remains low with 16.8% and < 5% with metastatic cancer (Foegle et al. 2005). Based on histological studies, lung cancer can be categorized as small-cell lung cancer (SCLC) and non-small-cell lung cancer (NSCLC). Furthermore, NSCLC comprises of 85% of the cases and is classified into adenocarcinoma, squamous cell carcinoma, and large cell carcinoma (Malyla et al. 2020). The interaction of genes and environmental factors results in the development of lung cancer, which includes tobacco consumption mainly in the form of cigarette smoke, exposure to radiations, and environmental toxins such as asbestos, nickel, arsenic, chromium, and others. Cigarette smoke, an exogenous factor, plays a key role in the generation of reactive oxygen species (ROS)/reactive nitrogen species (RNS) resulting in chronic inflammation and injury in the lungs. Despite smoking status, chronic lung inflammation is considered as an endogenous risk factor for lung cancer. Cigarette smoke and redox imbalance has been massively studied and is known to contribute to oxidative stress in lung cancer patients (Goldkorn et al. 2014).

Etiology of Lung Cancer Smoking Cigarette smoking is a common cause for cancer growth. The direct exposure of the respiratory tract to carcinogenic substances is especially linked with smoking. Smoking can also lead to the concurrent development of several cancers at any stage after primary cancer tumor resection (Goldkorn et al. 2014). Lung cancer is associated with smoking in the vast majority of cases (85–90%), which also includes passive smoking by nonsmokers. Besides, former smokers are diagnosed with lung

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cancer in many cases, as the risk of this malignancy continues to rise many years after the cessation of smoking (Cao et al. 2019).

Genetic Factors Some studies indicate that over 80% of the incidences of lung cancer are associated with smoking habits, but less than 20% of smokers may suffer from lung cancer, which means the occurrence of lung cancer is likely to be genetically susceptible. Recent large-scale genome-wide association studies have identified many novel lung cancer genes, including those on chromosomes 5p15.33, 6p21, 15q24–25.1, 6q23–25, and 13q31.3 (Yokota et al. 2010). The disease risk can be structured as representing the relationship between the exposure to etiologic agents and their vulnerability to individual agents. In lung cancer, some gene-environment significances have been established. For example, the 15q25 region contains 3 subunit genes of nicotine acetylcholine receptor, and nicotine addiction is indirectly associated with lung cancer risk by increasing tobacco carcinogen intake.

Gender It has been shown that higher prevalence and death rates exist in men than women. Lung cancer is higher in nonsmoking women than in nonsmoking men, the epidermal growth factor (EGFR) mutations are lower in NSCLC, and lepidic adenocarcinoma in women is more severe (Rudin et al. 2009). Many genetically modified mutations are found to be more prevalent in females, including the overexpression of the CYP1A1 gene; glutathione S–transferase M1 mutation; p53 tumor suppressor gene mutations; and the overexpression of the X-linked gastrin-deleting peptide receptor, which has been shown to be more common in female smokers. Females are also at greater family risk of lung cancer. The estrogen receptor (ER)α, which is not present in healthy lung tissues, has been shown to be overexpressed in female lung adenocarcinoma, but some studies have also shown overexpression in male cancers. Generally, women have specific lung cancer risk factors compared to men, and lung tumors in women have different clinical behavior, outcomes, and prognosis compared to lung cancer in men. The incidence of lung cancer has not yet been addressed in transgender men and women. The rate of cigarette smoking among transgender people was 35.5% above the general public (Buchting et al. 2017).

Age Older age is linked to the development of cancer because of several biological factors including damage to DNA over time and the shortness of the telomere. Therefore, the median age of diagnosis for lung cancer for men and women is 70 years. Around 53% of people between 55 and 74 years of age and 37% of people

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over 75 years of age were affected. The highest incidence of lung cancer in men is 585.9 per 100,000 in 85–89 years of age, while the highest incidence in women is 365.8 per 100,000 in 75–79 years. For men over 40, and women over 59, lung cancer is, by all means, the leading cause of death (de Groot et al. 2018).

COPD and Other Pulmonary Conditions Epidemiological studies show that some 20–30% of smokers have COPD and 10–15 per 100 have lung cancer, but with a variation in prevalence between 30 and 70%, COPD is by far the common comorbidity in lung cancer patients. More studies have found that additional factors have been associated with diagnosis of lung cancer. For example, increased airway obstruction, increased age, lower body mass index, and diffuse carbon monoxide lung capability ¼ 200 nt). The ncRNAs such as miRNA, siRNA, piRNA, and lncRNA regulate gene expression by inducing histone modifications and targeting DNA methylation leading to an alteration in chromatin structure. LncRNAs regulate DNA methylation, alter acetylation, ubiquitination, methylation of histones, and change the conformation of chromatin to regulate target gene transcription. All these mechanisms explain the actions of lncRNAs at the epigenetic level (Zhang et al. 2019). MicroRNAs (miRNAs) are short noncoding RNAs containing 18–25 nucleotides that affect the protein levels by targetting mRNAs without modifying the gene sequences. They are also subjected to get regulated by epigenetic modifications providing a complex miRNA- and epigenetic feedback loop (Yao et al. 2019). Chromatin structure is modified to allow interaction of DNA with various enzymes during the process of DNA transcription, replication, and repair. ATPdependent chromatin complexes use ATP hydrolysis to alter the structure of chromatin by repositioning, assembling, restructuring, and mobilizing the nucleosomes and thereby contribute its regulatory roles in epigenetic modulation. There are four families (SWI/SNF, CHD/Mi-2, ISWI/SNF2L, and INO80) defined by the presence of conserved SNF2-like and catalytic ATPase subunit (Tyagi et al. 2016). Oxidative stress influences the activities of these ATP-dependent chromatin remodeling complexes, both in health and disease (Kietzmann et al. 2017). Many growth stimulation factors that contribute to breast cancer pathogenesis may work through ROS and in this connection, and many studies have reported the role of ROS in modulating the epigenetic changes (Fig. 2) remodeling.

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Fig. 2 Breast cancer risk factors can modulate ROS levels. ROS can modulate histone modifications (acetylation, methylation, phosphorylation, ubiquitination, etc.), DNA modifications (cytosine methylation (5mC), hydroxymethylation (5hmC) or 8-Oxo-20 deoxyguanosine (8OG), miRNAs, noncoding RNAs expression, ATP-dependent chromatin remodeling (See text)

Biology of Breast Cancer A cell acquires cancerous properties when the delicate balance between tumor suppressor genes and oncogenes is shifted in favor of the latter. Cancer features prominently among all heterogeneous diseases due to a considerable burden of morbidity and mortality associated with it. Among all types of cancer, breast cancer has remained as foremost cancer in women. Thus it is also the most frequent cause of cancer-related death in women. National Cancer Institute, USA, estimates 2,76,480 new cases of breast cancer and 42,170 deaths in the year 2020. Several risk factors have been recognized for breast cancer, such as family history, age, race, reproductive patterns, hormone use, history of alcohol use, and tobacco use. The most common mutations in BRCA1 and BRCA2 tumor suppressor genes explain the genetic basis of breast cancer. Many genes undergo epigenetic modification, which leads to alteration in their normal physiological function leading to breast cancer. Thus, several markers of breast cancer have been identified, and still, a large number of studies are ongoing in search of new markers. Based on the presence or absence of

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molecular markers, breast cancer is classified into three types: hormone receptorpositive/ERBB2 negative, ERBB2 positive, and triple-negative. Though the poor prognosis is usually typical in all types of cancer, in breast cancer, the survival rate depends on the stage and molecular subtype of breast cancer. As metastasis occurs at the later stage of cancer, more than 90% of cases in breast cancer are not metastatic at the time of diagnosis, thus making surgery (lumpectomy, mastectomy) as the primary treatment option in many cases. However, for those cases where the cancer cells have escaped the breast, and other regional lymph nodes, or metastasis has not set in, adjuvant therapy in the form of radiation or systemic therapy is administered. Systemic treatment includes a variety of targeted therapy, chemotherapy, immunotherapy, and hormone therapy. Nevertheless, elevated oxidative stress (OS), high metabolic rate, and the persistent hypoxic state are some of the consistent biochemical features in breast cancer progression. They are also associated with resistance to treatment. Many epigenetic changes alter the expression of genes involved in breast cancer growth. In this connection, many drugs that modulate epigenetic activities are undergoing clinical trials for breast cancer treatment (Shukla et al. 2019).

Oxidative Stress, Reactive Oxygen Species (ROS) Generation, and Epigenetic Mechanisms in Breast Cancer Free Radicals in Health and Cancer OS is a state of ·), hydroxyl radical (OH•), organic hydroperoxides (ROOH), hydrogen peroxide (H), and oxygen metabolites such as hypochlorous acid (HOCl), peroxynitrite (HONO), etc. There are several sources wherein endogenous ROS are produced, such as in mitochondria during electron transport chain and by enzymes xanthine oxidase, lipoxygenase, myeloperoxidase, etc. The most common source for ROS generation is mitochondria. is normally generated due to electron leakage from mitochondria during respiration, and it is quenched by the intracellular antioxidant system. If mitochondrial permeability transition pore allows leakage of superoxide to the cytoplasm, it can dismutate to generate H, which is a highly diffusible and more potent ROS (Crompton 1999). The peroxisome is another source of ROS, where both superoxide and H are generated. Apart from these two organelles, the normal metabolism of fatty acids, prostaglandins, etc. also produces ROS. Therefore, considering several sources of ROS in eukaryotes by both enzymatic and nonenzymatic machinery, it is to be noted that they also have an important physiological role in cell survival and defense. For example, ROS generated during respiratory burst is used for bactericidal action within phagocyte as part of innate immunity (Babior et al. 2002). At low concentration (physiological levels), ROS can act as intracellular signal transduction molecules involved in the regulation of responses and cell survival. Membrane-bound NADPH oxidases (NOXs) are another important ROS generating signaling pathway useful for immune response by macrophages and neutrophils and in many inflammatory reactions. The family of Nox and dual oxidase (Duox) enzymes produce ROS for a variety of physiological

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functions such as innate immunity, signal transduction (calcium signaling regulating smooth muscle contraction, regulation of protein tyrosine phosphatase activity), thyroid hormone production as well as cell differentiation, etc. (Vignais 2002). However, when their production exceeds the physiological requirement, the same ROS can initiate inflammation, cellular injury at its vicinity, specifically targeting DNA, proteins, and lipids (Plaine 1955), ultimately resulting in cell death or carcinogenesis. Therefore, ROS has emerged to be a common factor associated with several diseases such as cardiovascular disease, neurodegeneration, and cancer. Especially in cancer, these ROS play a distinct role in stimulating cell proliferation (Burdon and Rice-Evans 1989), angiogenesis, invasiveness, and metastasis, while averting apoptosis and immune surveillance (Hecht et al. 2016). Usually, cancer cells show a higher quantity of ROS compared to healthy cells. ROS generation accelerates in cancer due to several triggers such as higher metabolic rate, mitochondrial dysfunction, altered cell signaling, increased activity of enzymes such as oxidases, lipoxygenases, cyclooxygenases and oncogene activation, etc. (Rohrmann et al. 2013). Therefore, many investigations have been done to validate the role of ROS in the causation and progression of various types of cancer. Almost all types of cancer have been shown to be associated with a higher level of OS. Nevertheless, cancer cells also demonstrate an amplified antioxidant defense response, which somehow balances this out. Therefore, it is suggested that ROS levels might be either another mechanism to prevent tumorigenesis or to induce carcinogenesis. Therefore, cancer cell function requires a very subtle balance between ROS and antioxidants. Thus, modern therapeutic strategies face the challenge of modulating the tumor-promoting effect of intracellular ROS to ROS-induced apoptotic signaling in cancer patients.imbalance between ROS and antioxidant defense system in the body. The term ROS is used to denote a group of extremely reactive oxygen-related free radicals such as superoxide anion (O2˙), organic radicals (R·), alkoxyl radicals (RO·), peroxyl radicals (ROO·), thiyl radicals (RS·), etc. ROS also refers to molecules or non-radical entities such as singlet oxygen (O·22O2Superoxide anion2O22O2

Free Radicals Induced Epigenetic Alterations and Affected Pathways ROS modifies DNA bases and affects epigenetic mechanisms. It has also been suggested that superoxide can mediate cytosine methylation by direct transfer of the methyl group from SAM, which is independent of DNMT. The other base that gets modified by ROS is guanosine, and 8-oxo-20 deoxyguanosine is the oxidized product formed after the ROS attack. Carbon 8 of the imidazole ring of deoxyguanosine(dG) generates the 8-oxo-dG. The adjacent cytosines of 8-oxo-20 deoxyguanosine cannot be methylated, and this hypomethylation has been reported for transcriptional activation of many putative oncogenes such as the KRAS and VEGF. This seems to be the most common epigenetic effect mediated by 8-oxo-dG. 8-oxo-dG can also induce GC-TA transversion mutation, and if these lesions are not repaired, they become mutagenic (Lewis et al. 2005). Various studies evaluated the levels of oxidative stress-induced damage of DNA and correlated with the prognosis

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of the patients. In this connection, an earlier study by Matsui et al. (2000) reported high levels of 8-oxo-dG in the DNA of breast cancer patients and suggested that primary prevention of breast cancer requires a reduction of oxidative stress. However, Sova et al. (2010) report from the study on 173 breast cancer patients indicated an association of low serum 8-oxo-dG levels and low immunohistochemical 8-oxodG with an aggressive breast cancer phenotype (Sova et al. 2010). While both studies are contradictory, a meta-analysis study on the prognostic significance of 8-oxo-dG in solid tumors suggested high and low 8-oxo-dG is a predictor of poor prognosis in most of the solid tumors and breast cancer respectively (Qing et al. 2019). The increased antioxidant mechanisms could attribute to the low levels of 8oxo-dG. The most important affected pathways linked to ROS/RNS are the Kelchlike ECH-associated protein 1-Nuclear factor erythroid2-related factor2 (KEAP1Nrf2) pathway, inflammatory pathways, and the metal ion pathways. All these affected pathways play a role in breast cancer progression and are affected through epigenetic mechanisms. ROS can enhance inflammation through the activation of the redox-sensitive transcription factor, NF-kB, and lipid peroxidation compounds. Oxidative stress reduces HDAC2 activity and also affects the levels of HDAC3, HDAC1 and NF-kB repressors through post-translational modifications (Rajendrasozhan et al. 2008). ROS/RNS favors the conversion of 5-mC to 5-hmC. 5hmC inhibits DNA methylation by inhibiting the DNMT1 (Valinluck and Sowers 2007). It also prevents the binding of MECP2 via its methyl-binding domain and dysregulates the methylation (Valinluck et al. 2004). ROS/RNS also generates 5chlorocytosine via inflammation pathways. 5-chlorocytosine, a reactive halogen compound, affects the methylation of DNA and causes aberrant epigenetic alterations (Valinluck and Sowers 2007). In metal ion pathways, metal compounds such as copper, nickel, and iron induce oxidative stress, epigenetic changes, and DNA damage. In this regard, various investigations point out the epigenetic marks of arsenic, nickel, chromium, cadmium, and mercury (Martinez-Zamudio and Ha 2011). Induction of oxidative stress, DNA repair inhibition, and deregulation of cell proliferation are general mechanisms leading to metal ion-induced carcinogenicity in breast cancer along with epigenetic dysregulation (Mulware 2013). The other oxidative stress responses pathways that have been linked to epigenetic alterations either by direct or indirect means include Bach 1, hepcidin, SOD1, HIF-1a, PPAR-g coactivator 1a, heme oxygenase, etc. (Baird et al. 2014).

Antioxidant Enzymes and Redox Homeostasis In tissue, there exists an intricate collection of both enzymatic and nonenzymatic antioxidants. Examples of enzymatic antioxidants are superoxide dismutase (SOD), catalase, glutathione S transferase (GST), glutathione peroxidase (GPx), thioredoxin, peroxiredoxin, and nonenzymatic antioxidants are glutathione (GSH) vitamins A, C, and E, melatonin, and flavonoids. (SOD) breaks down superoxide into oxygen and H. Catalase and GPx convert H to oxygen and water. Among nonenzymatic antioxidants, the major ones are GSH, vitamin E, and vitamin C. Another Nrf2, which is a transcription factor, also plays a major role in antioxidant defense. Nrf2 is a redox-

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sensitive transcription factor that plays a crucial role in the resolution of inflammation by upregulation of transcription of phase II defense enzymes and antioxidant stress proteins, thereby boosting cellular defense mechanisms. Together both types of antioxidants are employed with the task of preventing unwanted oxidation of cellular macromolecules. However, the production of ROS and its controlled neutralization by antioxidants is effectively used by the cell to carry out its intricate functions. For example, the Nox enzyme system, which is physiologically associated with cell proliferation, migration, and survival, is also linked to the overproduction of ROS, especially in rapidly proliferating cells and cancer tissue. Interestingly recent evidence has emerged showing that certain antioxidants are very precisely controlled by the Nox-driven signaling mechanism in order to channel H for co-localization of target proteins (Toledano et al. 2010). Similarly, Nrf2, known for its role in the resolution of inflammation and uplifting antioxidant reserve, is also linked to cancer onset and its progress due to its mutation and overexpression (Wang et al. 2008). Therefore, when this delicate balance between ROS generation and its handling by antioxidants is disrupted, it can lead to abnormal cell proliferation and hence tumorigenesisSuperoxide dismutase2O22O2molecule 2O2.

ROS-MicroRNAs Interactions and Epigenetic Alterations in Breast Cancer HDACs and DNMTs are two major enzyme families involved in epigenetic mechanisms and can be regulated by ROS. By altering the levels or activities of these enzymes, ROS can influence gene expression, including the miRNAs. In this regard, several investigations studied the role of ROS in affected HDACs and DNMTs that altered miRNAs expression in cancer progression. Several transcription factors, such as NF-κB, c-Myc, and HIF-1α, that are redox-sensitive regulate the expression of miRNAs suggesting the importance of transcriptional factors-ROS-miRNAs axis in carcinogenesis. The enzymes, such as the DGCR8 and Dicer involved in miRNAs biosynthesis and maturation pathways, are also affected by the intracellular redox homeostasis suggesting the role of ROS in modulating the expression of oncogenic and or tumor-suppressive miRNAs in cancer. He et al. (2012) showed that ROS downregulates miR-199a and miR-125b that target ERBB2 and ERBB3 expression in ovarian cancer cells. The downregulation of miR-199a and miR-125b is through promoter methylation by DNMT1 (He et al. 2012). This study gave insights into the ROS-miRNAs-epigenetic regulation axis in the control of ovarian cancer progression. Nrf2 is a redox-sensitive transcription factor that induces transcription of antioxidant enzymes such as catalase and SOD. MicroRNAs that regulate Nrf2 will also have an impact on ROS levels intracellularly. A study reported the downregulation of Nrf2 by miR-28, which increases the colony formation of breast cancer cells (Yang et al. 2011). The miRNAs mediated regulation of Nrf2 is represented in Fig. 3. The nuclear factor-kB another redox master regulator is well studied for its role in regulating many oncogenic miRNAs, including miR-21in breast cancer (Niu et al. 2012). The miRNAs mediated regulation of NF- kB is represented in Fig. 4.

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Fig. 3 ROS/miRNA mediated regulation of NRF2 in breast cancer. NRF2 is a transcription factor that binds with Keap1 repressor in cytoplasm. Keap1 anchor Cullin3 (CUL3) and forms an E2 ubiquitin ligase complex. This complex directs the NRF2 to undergo proteasomal degradation. Increased ROS concentrations dissociate the NRF2 and NRF2 translocate to the nucleus, bind to antioxidant response element (ARE) sequences and increase the transcription of antioxidant genes. miR200a and miR28 have been shown to inhibit keap1 and NRF2, respectively, in breast cancer cells. The diagram is based on review by Cosentino et al. (2019). Abbreviations used Kelch-like ECH-associated protein 1 (Keap1); nuclear factor erythroid 2-related factor 2 (Nrf2)

The expression of HER2 influenced the oxidative profile in breast cancer patients, and a study by Victorino et al. (2014) demonstrated that HER2 overexpressing breast cancer cells exhibited high oxidative stress with altered antioxidants parameters (Victorino et al. 2014). Possibly, the effect of HER2 on the oxidative profile and or antioxidant settings could be explained by the HER2 potential in interacting with Nrf2 as demonstrated by Kang et al. (2014).

Oxidative Stress in Breast Cancer Epigenetics, and Scope of Treatment OS is a common mediator in various mechanisms implicated in breast cancer, such as activation of NOX and Wnt/β-catenin pathways, stabilization of HIF-1 α, activation of RAS and growth factor receptors, PI3K/Akt/mTOR, translationally

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Fig. 4 ROS and or miRNA mediated regulation of NF-κB in breast cancer. ROS have inhibitory or stimulatory roles on NF-κB signaling. Oncogenic miR-21 is transcriptionally induced by NF-κB signaling. miRNAs 520/373, 31 and 30c-2-3p inhibit NF-κB and miR-1246 induce the signaling of NF-κB in breast cancer. The diagram is based on the review by Cosentino et al. 2019

controlled tumor protein (TCTP), etc. The comprehensive schematic diagram depicting the ROS affected pathways in breast cancer is presented as Fig. 5. Lot of research is still going on targeting various checkpoints to overcome the challenges of resistance to chemotherapy and its relapse. Epigenetic changes are relatively reversible processes, thus making it a much sought after target for newer therapies. The major challenge of epigenetics targeted therapy is its effective translation from preclinical cell line study to its success in a clinical trial and then in a larger patient population. The genetic and epigenetic alterations have been directly linked to breast cancer pathogenesis via OS. One of the studies done by Sawczuk et al. (Sawczuk et al. 2019) in the saliva samples collected from breast cancer patients with the BRCA1 mutation demonstrated higher antioxidant capacity, yet a higher degree of oxidative damage to the proteins and lipid. They suggested that BRCA1 mutation can cause a predisposition to early salivary gland dysfunction (Sawczuk et al. 2019). Indeed, BRCA1 is needed for post-transcriptional repair following oxidative damage signifying the role played by oxidative stress (Ambrosone 2000). Likewise, several epigenetic changes too are implicated in breast cancer pathogenesis. For example, promoter hyper-methylation of genes such as O-6-Methylguanine-DNA Methyltransferase (MGMT) and Low-density lipoprotein

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Fig. 5 Schematic representation of ROS and its role in breast cancer pathogenesis. Abbreviations used in this chart. HIF- Hypoxia-inducible factor; VEGF- Vascular endothelial growth factor; PTEN- phosphatase and tensin homolog; PI3K- phosphoinositide 3-kinase; pAkt- phosphorylated protein kinase B; mTOR- mammalian target of rapamycin; Bcl-2- B cell lymphoma-2; BNIP3BCL2 and adenovirus E1B 19-kDa-interacting protein 3; MAPK- mitogen-activated protein kinase

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receptor adapter protein 1 (LDLRAP1 or ARH1) genes involved in DNA repair and cellular homeostasis, respectively, is implicated in breast cancer. Similarly, hypomethylation of Breast Cancer Specific Gene 1 (BCSG1) linked to cell invasion/ metastasis is observed in breast cancer. Hyper-methylation has been pointed out as a possible mechanism behind the absence of ER mRNA (ER loss) as demonstrated in ER- negative breast cancer cells (Weigel and deConinck 1993). FDA-approved tamoxifen has been used for metastatic estrogen receptor-positive breast cancer treatment since 1977. However, endocrine resistance to tamoxifen and related drugs pose a challenge due to altered expression of estrogen receptors (ER). DNMTs mediate DNA methylation by acting in association with HDACs and methyl-CpG-binding domain (MBD) family of proteins. Therefore, various DNMT inhibitors, such as 5-azacytidine (5-aza) and 5-aza-20 -deoxycytidine (decitabine), zebularine, and 5-fluoro-20 -deoxycitine, were investigated in human breast cancer cell lines to reverse methylation of ER CpG island and to cause reexpression of ER mRNA. Addition of inhibitors to HDACs was found to have a positive synergistic action (Yang et al. 2001) in cell lines. Both drugs could be beneficial especially in ER-negative breast cancers. So, HDAC inhibitors, DNMT inhibitors in combination with endocrine therapy, chemotherapy, and/or HER2 directed therapy are in clinical trials. In this context, additional nutrition in the form of dietary agents rich in antioxidants can also add to existing treatment regimen as a form of supportive therapy.

Dietary Agents with Antioxidant Properties as Epigenetic Modulators in Breast Cancer Natural phytochemicals from seeds, vegetables, spices, herb, food, etc., act as a powerful antioxidant, and many studies unraveled the anti-tumorigenic properties of these compounds in various cancers. Safety, general availability, and low toxicity are some of the key features of the phytochemicals and dietary compounds for its use in the treatment of cancer. Curcumin (turmeric), resveratrol (grapes, mulberries, peanuts, vines, and pines), quercetin (phytoalexin, the skin of red grapes and red wine), and genistein (soybeans, soy products, red clover) are few of the most important phytochemicals that are well studied for their anticancer activities, anti-inflammatory properties and also have great potential to act as an epigenetic modulator.

Curcumin Curcumin is a yellow polyphenol and is the active component of Curcuma longa that contains 80% curcuminoid complex, 17% dimethoxy curcumin, and 3% bisdemethoxycurcumin (Hassan et al. 2019). Its antioxidant activity mediates the

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essential effects of curcumin. It scavenges reactive oxygen species and suppresses lipid peroxidation and thereby alleviates disease severity due to oxidative stresses. Many studies have characterized the potent role of curcumin as an epigenetic modulator. Herein, we discuss the critical findings that explore its anti-breast cancer properties through epigenetic modulation. HDACs inhibition alters epigenetic mechanisms and cancer cell activities. So many different substances, including curcumin, are being investigated as an HDAC inhibitor to target cancer therapeutically. It has been shown that oxidative stress can stimulate NF- kB and increase the expression of pro-inflammatory mediators by the activation of HAT activity and suppression of HDAC activity. Curcumin, as an antioxidant, can regulate both HAT and HDAC through modulation of oxidative stress and by its scavenging action on ROS. Curcumin can also affect DNA methylation, and in a recent study, Al-Yousef et al. (2020) demonstrated the dual function of curcumin in re-expressing hypermethylated ). BRCA1 and suppressing the expression of hypomethylated protooncogene γ synuclein (SNCG) in the estrogen receptor-negative/progesterone receptor-negative (ER-/PR-) cell line UACC-3199, TNBC cell line HCC-38, and ER+/PR + cell line T47D. The hypomethylating effect for the BRCA1 gene and hypermethylation effect for SNCG was mediated through the upregulation of the teneleven translocation 1 (TET1) gene and DNA methyltransferase 3(DNMT3) gene, respectively. These results suggest the dual effect of curcumin on DNA methylation via specific genes (Al-Yousef et al. 2020). A study also showed that curcumin treatment decreases the expression levels of DNMT1, 3A, and 3B in MCF7 and MDA MB231 breast cancer cells (Mirza et al. 2013

Resveratrol Resveratrol is another naturally occurring polyphenol found in peanuts and the skin of berries and grapes. Resveratrol is known for its antioxidant properties, and many studies have investigated its chemopreventive ability. It also produces reactive oxygen species under specific conditions and thereby acting both as an antioxidant and prooxidant. Both properties make it an active anticancer agent. Numerous investigations focused on the role of resveratrol in cellular epigenetic modulation that affects cancer signaling pathways (Kala and Tollefsbol 2016). Medina-Aguilar et al. (2016) studied genome-wide DNA methylation-based on promoter DNA microarrays in MDA-MB-231 cells after treatment with resveratrol. The results of their study represent a catalog of cellular pathways and genes with differential DNA methylation in MDA-MB-231 cells at 24 h and 48 h after treatment with resveratrol. Based on the correlation between cancer-related mRNAs expression and DNA methylation, the investigators proposed resveratrol as a dietary epidrug in breast cancer (Medina-Aguilar et al. 2016). Epigenetic regulation of genes by resveratrol may be mediated through chromatin remodeling and/or altered epigenetic enzymes, and these can further add to the antioxidant properties of resveratrol. Moreover, decreased oxidative stress after resveratrol treatment can itself impact DNA methylation linking the antioxidant effects and epigenetic mechanisms similar to many

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dietary antioxidants (Beetch et al. 2020). Kala and Tollefsbol (2016) demonstrated a significant decrease in 5-methylcytosine levels and DNMT activity in MDA-MB157 breast cancer cells after treatment with resveratrol in combination with another dietary compound pterostilbene. This epigenetic effect restored ER-α levels and sensitized MDA-MB-157 breast cancer cells to 17β-estradiol and 4-hydroxy tamoxifen.

Quercetin Quercetin is a flavonoid found in vegetables (onions) and fruits (red wine, apples). It is a potent natural antioxidant and targets multiple intracellular proteins/pathways as an anticancer agent. Quercetin targets multiple kinases that are the positive regulator of the cell cycle and act as an antiproliferative agent. Apart from this, quercetin alters histone acetylation and influences the transcriptional regulation of COX-2 (Stepanić et al. 2014). The results from the investigations of Tao et al. (2015) reveal the miR146a upregulating effect of quercetin in human breast cancer cells. Through this upregulation, quercetin induces apoptosis by activating caspase-3 (Tao et al. 2015). The actions of quercetin as an epidrug have been demonstrated in the liver, lung, gastric, and colon cancer (Arora et al. 2019).

The Interplay Between Oxidative Stress and Epigenetic Drugs in Breast Cancer ROS producing or degrading genes can be regulated by therapeutic epigenetic enzyme inhibitors (HDACi, HMTi, DNMTi) (Hervouet et al. 2016). The by-products of epigenetic drugs, either ROS or antioxidants, alter the cytotoxic effect. Thereby combining with ROS or antioxidants along with epigenetic drugs will potentiate the therapeutic effect (Fig. 6). In breast carcinomas, HDAC expression is dysregulated, and particularly the levels of HDAC6 has been shown as a marker of endocrine responsiveness and prognosis (Zhang et al. 2004). Redox regulation by histone deacetylase inhibitor (HDACi) (vorinostat) alters its therapeutic effect on breast cancer cells, and the addition of buthionine sulfoximine that depletes the GSH potentiates the vorinostat effect (Chiaradonna et al. 2015). From the above study, it is clear that inhibiting antioxidant response and inducting ROS levels along with epigenetic drugs can potentiate the anticancer activity. Therapeutic drugs that target the redox pathway would also affect epigenetic modifications. It has been shown that cambogin drug causes mitochondria-dependent apoptosis in breast cancer cells, and mass spectrophotometry analysis revealed ROS pathways as the most affected one following cambogin treatment. The increase in ROS, activation of JNK pathway after cambogin treatment correlated with the induction of ATF-2 activity and trimethylation of H3K9 mark in the AP-1 binding region of the promoter region of anti-apoptotic Bcl-2 gene (Shen et al. 2015).

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Fig. 6 Epigenetic targeting drugs (HDAC inhibitors, DNMT inhibitors, HMT inhibitors) and their influence on ROS producing genes and ROS neutralizing antioxidant genes. The therapeutic efficiency of epigenetic-targeting drugs is determined by the ability of its regulation of ROS producing and or antioxidant genes. DNMT inhibitors can increase the transcription of ROS producing genes. HDAC and HMT inhibitors can influence the expression of antioxidant genes. A combination of ROS or antioxidants, along with epigenetic drugs depending upon the context, can increase the therapeutic efficiency. This figure is modified and redrawn from Hervouet et al. (2016) . HDAC- histone deacetylase; DNMT- DNA methyltransferase; HMT- histone methyltransferase; ROS- reactive oxygen species

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Conclusive Remarks In this chapter, we have illustrated the critical role played by oxidative stress in the pathogenesis of breast cancer. We also reviewed the general concept of epigenetics and associated changes such as histone modifications, DNA methylation, and regulation by microRNAs (Figs. 1a, b, c and 2). We also discussed the participation of miRNAs in the management of oxidative stress and thereby its epigenetic influence on essential genes affecting the pathways in breast tumors (Figs. 3 and 4). ROS-induced epigenetic changes and epigenetic control of genes that regulate the production of ROS have been described. This brings an exciting note on whether ROS production is a cause or effect of epigenetic dysregulation in breast cancer. The fundamental understanding of the interplay between epigenetic drugs and oxidative stress in breast cancer patients can help devise personalized treatment by antioxidant therapeutics (Fig. 6). We also discussed the role of dietary agents, which are antioxidants and or epigenetic regulators, providing a note on nutritional therapy for cancer. It appears that oxidative stress contributes significantly to the progression and of breast cancers through epigenetic modulation. The physicians should ensure to achieve redox homeostasis, the balance between oxidants and antioxidants to obtain maximum therapeutic benefits in breast cancer management.

Cross-References ▶ Mitochondrial Metabolism, Oxidative Stress, and the Microenvironment in Breast Cancer Development and Progression ▶ Redox State and Gene Regulation in Breast Cancer ▶ Therapeutics of Oxidative Stress and Stemness in Breast Cancer Acknowledgments PSS acknowledge Department of Biotechnology, Govt of India (BT/ PR16307/MED/30/1729/2016) and National Institute of Technology, Calicut, Kerala, India (Faculty research seed grant) for their support and encouragement. ST thanks MHRD, GOVT of India and NIT Calicut for fellowship. We apologize to many scientists whose work were not cited due to page and space limit for the manuscript. PSS thank Dr. Shama Prasada, SOLS, Manipal, India for active discussions. Conflicts of Interest Authors declare no conflict of interest.

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Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Epigenetic Control Mechanisms in Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Histone Modification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . DNA Methylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Epigenetic Control by microRNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Oxidative Stress and Inflammation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Keap1-Nrf2 Pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Epigenetic Control by Phytochemicals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Roles of Phytochemicals in Epigenetic Regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract

It is a fundamental understanding in molecular biology that changes in DNA are reflected in transcription of aberrant messenger RNA (mRNA) and translation to malfunctioning proteins. Proteins that malfunction in turn inhibit normal metabolic processes and even initiate dangerous traits like immortality in cells. However, changes in DNA are not the only factors that can affect the normal metabolic processes in cells. Most genes are organized into pathways, and the over- or underexpression of an upstream gene in a pathway often results in altered expression of downstream genes and concentrations of the corresponding proteins. There are molecular mechanisms in cells that may affect the expression M. Roy (*) Department of Environmental Carcinogenesis & Toxicology, Chittaranjan National Cancer Institute, Kolkata, India A. Datta Department of Computer Science & Software Engineering, The University of Western Australia, Perth, WA, Australia e-mail: [email protected] © Springer Nature Singapore Pte Ltd. 2022 S. Chakraborti et al. (eds.), Handbook of Oxidative Stress in Cancer: Mechanistic Aspects, https://doi.org/10.1007/978-981-15-9411-3_108

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levels of genes even though there is no change in the genetic code in the DNA. These mechanisms are collectively called epigenetic control mechanisms and our aim in this chapter is to discuss in general these mechanisms and in particular how these mechanisms are triggered by reactive oxygen species (ROS) and how phytochemicals can be effective for aberrant epigenetic modulation in cancer. Keywords

ROS · Epigenetics · Histone modification · DNA methylation · miRNA · Carcinogenesis · Phytochemicals

Introduction During carcinogenesis normal cells are transformed into cancer cells through different steps, namely, initiation, promotion, progression, and finally metastasis. It may take time to develop cancer; the latency period may be 20 years or more. During this developmental process, cells may have mutations of a number of genes (Sporn 2011). One of the major goals of cancer control is its prevention. One of the ways to achieve cancer prevention is to target and reverse epigenetic modifications, as these are reversible unlike mutations. DNA methylation, chromatin remodeling, histone post translational modifications, microRNAs, etc. are the epigenetic mechanisms that regulate gene expressions. All these epigenetic mechanisms are governed by proteins that modify some chemical groups involved in epigenetics for activation/inactivation of gene transcription. There has been a crosstalk between epigenetics and cancer metabolism. The metabolite like S-Adenosyl methionine (SAM) is a common co-substrate responsible for transfer of methyl group. Other metabolites are acetyl-coA and Adenosine monophosphate (AMP). These metabolites are essential for epigenetic mechanisms such as methylation of DNA, acetylation or phosphorylation of histone (Donohoe and Bultman 2012). These metabolic pathways and the enzymes are important for the epigenomic maintenance and adaptation. The food we eat plays an important role. Methionine deficient diet diminishes the level of SAM, culminating in reduced methylation of DNA and histone; this leaves an impact on the gene expression (Donohoe and Bultman 2012; Parasramka et al. 2012). Sometimes, deficiency of one particular nutrient can influence metabolism and functionality of the other as the metabolism are interrelated, for example, metabolism of folate, betaine, choline, methione are linked. A methyl donor deficient diet affects the global methylation pattern. DNA hypomethylation leads to elevated level of mRNA for oncogenes like c-fos, c-myc, etc., in rats given a diet devoid of methionine and choline for a short period. DNA methylation pattern gets restored by methyl donor proficient diet (Niculescu and Zeisel 2002) in the same rats. However, for a prolonged deficiency, epigenetic alteration cannot be reversed. Therefore, consumption of abundance of methyl donors may impart protection against carcinogenesis and paves a way for cancer prevention. We will discuss the epigenetic modulation mechanisms and their aberration in cancer in this chapter, with an emphasis on the aberrations due to the generation of

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reactive oxygen species (ROS) and inflammation. We will also discuss how phytochemicals can help in reversing or stopping harmful epigenetic modulations.

Epigenetic Control Mechanisms in Cells Epigenetic control works in two ways, either through transcriptional or posttranscriptional control. Transcriptional control works by preventing access to the genes by transcription factors (proteins that help in transcription from gene to mRNA). There are again two different ways this can happen: histone modification is a transcription inhibiting process whereby the histone coiling around the DNA is modified through histone acetylation or deacetylation, modifying the access of transcription factors for initiating transcription. DNA methylation on the other hand works by inhibiting access to transcription factors to the promoter regions close to the transcription start sites. One of the most important posttranscriptional control mechanisms is microRNA (miRNA) silencing. These miRNAs are nothing but small RNA molecules (about 22 nt long), which are noncoding and bind to the 3’UTR (untranslated region) of mRNAs and prevent the translation of proteins from mRNAs. These three epigenetic control mechanisms have been discussed in detail.

Histone Modification The DNA is packed tightly in eukaryote cells, and the access to specific genes by transcription factors is restricted and controlled tightly. Any modification in packaging of DNA results in either less or more access to genes by transcription factors. There are two classes of proteins responsible for packaging of DNAs: chromosomal proteins and histones. These proteins bind to the DNA forming chromosomes. The complex of histones, chromosomal proteins, and DNA is named as chromatin. Eight histone proteins constitute a nucleosome core particle, and the DNA is coiled around the nucleosome core particles. Linker DNA connects two consecutive nucleosome core particles. Chromatin is a very important structure for controlling transcription and usually the weights of histone and DNA are almost equal in eukaryote cells. The details of histone packing can be found in the book by Alberts et al. (2015). Our main aim here is to understand histone acetylation first and then histone methylation and phosphorylation (Fig. 1). There are two components of histone important for our discussion: the histone core fold and the N-terminal tail. The modifications in histone packaging occur through acetylation, phosphorylation, and methylation of histone tails. These modifications in turn change the chromatin structure and packaging, giving better access to genes by transcription factors. Histone N-terminal ends contain COOH and NH2 tails, and histone acetyltransferases (HAT) add acetyl groups to the NH2 tail. Hence, histone cannot neutralize the charges on the DNA which is wrapped around it as the lysine amino acid in the tail loses its positive charge. This loosens the DNA and gives access to transcription factors. While HATs act as transcriptional activators,

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Fig. 1 Illustration for histone acetylation and methylation that occur at the histone tails

histone deacetylases (HDAC) remove the acetyl group from the lysine residue and act as transcriptional repressors, restricting entry of transcription factors to the DNA, as the DNA packaging tightens. Histone methylation is a finer control for accessing the DNA by transcription factors. It can either enhance or decrease the transcription of genes by methylating different number of amino acids, as well as by attaching different number of methyl groups. Histone methyltransferases (HMT) do the methylation and histone demethylases perform demethylation. Histone phosphorylation is the third process for controlling access of transcription factors to the DNA. Cellular response to the damage in DNA is controlled by histone phosphatases, the proteins involved in histone phosphorylation. Histone phosphatases separate the chromatin domains around the breakage sites of DNA.

DNA Methylation Transcription factors bind to a specific DNA sequence in the promoter site of DNA close to the transcription start sites. Transcription factors have DNA binding domains for this purpose, and DNA methylation is the process that prevents the access of DNA binding domains of transcription factors to these binding sites. Transcription factors recruit RNA polymerases, which are the proteins that are instrumental for transcription. Hence, transcription cannot start unless transcription factors can bind to the DNA (Fig. 2).

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Fig. 2 Gene expression is repressed by methylation of CpG islands

The targets for DNA methylation are CpG islands, which are repetitive sequences of cytosine and guanine from the 50 to the 30 end of the DNA. Cytosines in CpG islands are methylated. It is worth noting that CpG islands may occur anywhere in the genome; however, their presence in the promoter regions of genes makes them targets for epigenetic control through DNA methylation, and over 70% of human genes contain CpG islands in the promoter regions. Most such CpG islands are generally hypomethylated, or methylated at a level lower than normal. However, the CpG islands in promoter regions of genes in tumors show different trends. Whereas the CpG islands of tumor suppressor genes are hypermethylated, the CpG islands of oncogenes are hypomethylated. Hence, the expressions of oncogenes are increased and the expressions of tumor suppressing genes are decreased, which is a trait of tumor cells. Several cancer drugs target methyltransferases so that the CpG islands of tumor suppressor genes are not hypermethylated.

Epigenetic Control by microRNA miRNAs are small RNA molecules about 18–25 nt long, which do not code for any protein (noncoding RNA). They are responsible for posttranscriptional control of mRNAs by silencing them, so that mRNAs are not translated into proteins. Many miRNAs may target the same mRNA, and a single miRNA may target multiple mRNAs. Most of the miRNA families are highly conserved across species and that indicates that epigenetic control by miRNAs is of ancient origin. Many miRNA genes are targets for histone modification and DNA methylation, and miRNAs regulate the translations of methyltransferases and HDACs. This indicates that different epigenetic control mechanisms are highly correlated. miRNAs can behave as tumor suppressor genes and also oncogenes. Metastasis is the most advanced

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Fig. 3 Inhibition of mRNA translation by miRNA

stage of most cancers, when tumor cells migrate from their original locations to distant organs. Epithelial-mesenchymal transition (EMT) is one of the main causes of metastasis, and miRNAs play major roles in EMT (Li et al. 2016) (Fig. 3).

Oxidative Stress and Inflammation Excessive concentration of reactive oxygen species (ROS) molecules can cause damage to important biological molecules like proteins, lipids, DNA, and RNA. Usually the concentrations of ROS and antioxidants (that neutralize ROS) balance each other in normal cells, as ROS is produced in normal levels due to metabolic processes. However, environmental carcinogens can elevate ROS levels and it is now firmly established that elevated ROS levels trigger carcinogenesis. Higher concentrations of ROS can cause damage to DNA at a rate much higher than the DNA repair mechanisms and the cells cannot cope with that. Inflammation is also another important cause of oxidative stress and higher concentrations of ROS in cells. ROS can damage DNA in several ways, including point mutations, chromosomal translocations, and insertions and deletions of DNA fragments. It is now known that ROS can be responsible for all stages of cancer, starting from initiation to progression and metastasis. As the theme of this chapter is epigenetic modulation mediated by ROS, we need to examine the role of ROS in different epigenetic controls mentioned above. One of the main effects of elevated ROS in cells is its intervention in epigenetic modulation

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like DNA methylation and histone acetylation. ROS has been implicated in aging and reproductive disorders. For example, Ito et al. (2017) established a connection between endometriosis, a reproductive disorder, and elevated ROS levels. The mechanism for this disorder is through aberrant histone modification and DNA methylation. Elevated ROS levels have been implicated in several cancers, including breast, human renal tubular epithelial cell, and intestinal cancers. H2O2 is one of the agents that generates ROS in cells. When estrogendependent breast cancer cells that are nonaggressive were incubated with H2O2, transformation into estrogen-independent aggressive forms was observed (Mahalingaiah et al. 2015). Human renal tubular epithelial cells were reported to have malignant transformations due to oxidative stress. Several genes associated with DNA methylation and histone modification had aberrant expressions (Mahalingaiah et al. 2017). The homeobox gene Cdx 1 transcribes a tumor suppressor protein and is expressed in the intestine. It has been reported that the expression of histone deacetylase 1 and DNA methyltransferase 1 were increased due to H2O2 intervention and these two proteins inhibited the expression of Cdx 1 in colorectal cancer cells. Inflammation may result in DNA damage and epigenetic changes that may cause tumor initiation and progression. Inflammation is self-limiting in normal circumstances, as the agents responsible for healing inflammation do not accumulate in high concentration. However, a higher concentration of healing agents may cause pathogenesis and even neoplastic progression. Though DNA mutations are corrected by DNA repair machinery in cells, these machineries do not work well under inflammatory conditions. The failure to recover from DNA mutations and the accumulation of an excessive rate of mutation may cause carcinogenesis. Cytokines are peptides that participate in extracellular immunological signaling process. Cytokines are one of the main agents that participates in the healing process during inflammation. However, it is known that cytokines are associated with cancer initiation and proliferation of cells, migration, and angiogenesis. TNF-alpha, TGFbeta1, IL-6, and interleukin-1beta are some of the important cytokines (Thejass and Kuttan 2007). Nitric oxide (NO) plays an anti-inflammatory role in normal inflammations; however, this role reverses if NO is produced at a higher rate and it becomes a proinflammatory agent. Nitric oxide synthase (NOS) is a family of enzymes that produce NO in endothelial cells. Inducible NOS or iNOS is one of the important enzymes in this family. It is now well established that the Nrf2 pathway is involved in inflammation and healing. It was reported that Nrf2-deficient mice die in hyperoxia conditions that are sublethal (Reddy et al. 2009). Another hallmark of chronic inflammation is epigenetic alterations, mainly due to histone modifications and DNA methylation. Expressions of many genes associated with inflammation are affected due to epigenetic aberrations. For example, some inflammation related diseases like cystic fibrosis and obesity have connections with DNA hypo and hypermethylation (Donohoe and Bultman 2012). Similarly, the aberrant expressions of histone acetyltransferase have been implicated in inflammation related diseases (Li et al. 2016).

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Keap1-Nrf2 Pathway The Keap1-Nrf2 pathway gets activated due to oxidative stress. The main participants in this pathway are the kelch-like ECH-associated protein (Keap1) and antioxidant response elements (AREs). AREs are transcriptional regulatory elements that are usually found in the untranslated regions of many genes. It is therefore worthwhile to understand the details of this pathway for our purpose and we review it briefly based on two papers (Raghunath et al. 2018; Nguyen et al. 2009) (Fig. 4). One of the important steps in reducing oxidative stress is detoxification of toxic chemicals and foreign chemicals (xenobiotics) accumulated in cells. There are three steps in detoxification in cells. The xenobiotics are metabolized by Cytochrome P450 superfamily of enzymes in the first step. The second step involves conjugating glutathione (GSH) with reactive electrophile species. Finally in the third step the GSH conjugates are eliminated by transporter proteins. Many of the proteins involved in these three steps have AREs as transcriptional regulatory elements. Under oxidative stress, the transcription factor Nrf2 increases the transcription of these proteins by acting on the AREs.

Fig. 4 Illustration for the Keap1-Nrf2 pathway

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The NFE2L2 gene encodes the Nrf2 protein. In normal conditions when there is no oxidative stress or when there is no excessive amount of xenobiotics in the cell, Nrf2 is produced in the cytoplasm and degraded at a steady rate. In fact the average lifetime of Nrf2 is quite short in unstressed conditions as it is degraded through ubiquitination. A group of proteins including Cullin-3 and Keap1 keep Nrf2 in the cytoplasm and participate in its ubiquitination. However, oxidative stress destabilizes the Keap1-Cullin-3 complex and the concentration of Nrf2 increases in the cytoplasm. Transporter proteins transport Nrf2 to the nucleus and it binds to the ARE in the promoter regions of many genes that participate in antioxidative functions. This initiates the increased transcription of these genes and starts the three-step process for the removal of xenobiotics mentioned above.

Epigenetic Control by Phytochemicals The plant based molecules or phytochemicals are important for chemoprevention and they do so by indirect modulation of DNA methyltransferase (DNMTs) via alteration of the SAM/SAH ratio. Phytochemicals mean plant (phyto) chemicals that aid in to the color, odor, and taste of fruits and vegetables. Phytochemicals are abundantly found in edible parts of a plant, especially the skin or peel. Scientific studies reveal that these active plant biomolecules can have an inhibitory effect on growth of tumor by altering epigenetic signaling pathways (Remely et al. 2015). Depending on the modulatory action on epigenetics, these phytochemicals can be grouped as (i) those that are direct donors of methyl group and act as co-substrates in DNA methylation process, (ii) those that modulate DNMT activity indirectly by affecting methyl pool, and (iii) those that inhibit DNMT enzyme directly (Pop et al. 2019). Therefore, it is quite clear that a healthy platter containing fruits and vegetables, along with a healthy lifestyle, can impart protection against carcinogenesis. One way of preventing cancer could be targeting and reversing the early epigenetic alterations. Epidemiological surveys reveal an inverse correlation between diets rich in bioactive compounds and the occurrence of different types of cancer. Alteration in gene expression or phenotype in cell that happens without any change in DNA sequence is epigenetics, and dietary factors have been found to influence this. Nutri-epigenetics therefore is gaining in importance day by day. A number of plant biomolecules have been found to modulate epigenetics, which is reversible as we have seen in section “Epigenetic Control Mechanisms in Cells.” Due to the reversible nature, epigenetic modifications have become targets for cancer prevention. According to the central dogma in biology, genetic information in the genetic material percolates from a DNA to a protein product within the cell (Roy and Datta 2019). This process of translation of the information from DNA to RNA and then to protein is coined as gene expression. As we have discussed in section “Epigenetic Control Mechanisms in Cells,” methylation of DNA, histone modifications, and

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miRNAs are the three important epigenetic mechanisms that regulate transcription (Thakur et al. 2014). Influence of phytochemicals in regulation of epigenetic alterations is an important area. Drugs have been developed to target histone deacetylases (HDACs) and DNA methyltransferase, which are emerging as promising anticancer therapy. Inhibition of epigenetics markers is an effective anticancer strategy. These drugs have good clinical outcomes, but there are a number of pitfalls. Remediation by natural means may be an alternative to this problem. There have been many investigations in epigenomics and it is the need of the hour to develop effective methods to improve drug delivery, minimizing side effects of the drugs and to achieve a better therapeutic index (Patnaik and Anupriya 2019). It is not an easy task to design a drug which is not toxic to normal cells. Plant derived molecules are nontoxic or have minimal toxicity. The mechanisms of action of these molecules are mostly known and they are more tolerable and acceptable to the patients. Therefore, they are better candidates for targeting epigenetic alterations (Patnaik and Anupriya 2019). These phytochemicals are secondary metabolites in the fruits and vegetables that we consume. They have a number of characteristics that make them so important. These biomolecules are capable of tackling oxidative stress, they are potent inducers of detoxification enzymes, and they prevent formation of nitrosamine, improve binding of carcinogens in the GI tract, and modulate hormone metabolism and a number of important signaling events. These phytochemicals can modulate epigenome and therefore may be considered as important candidates in cancer control (Guo et al. 2015a, b).

Roles of Phytochemicals in Epigenetic Regulation Some phytochemicals and their roles in regulation of epigenetic mechanisms have been discussed below. Polyphenols Polyphenols present abundantly in fruits, vegetables, and some beverages derived from plants have one or more polyphenolic structures. These may be categorized into phenolic acids, flavonoids, polyphenolic amides, stilbenes, and lignans. Of these, flavonoids are the most well-characterized polyphenols comprising of flavonols, flavones, flavanones, catechins, isoflavonoids, and anthocyanidins (Jayasinghe et al. 2015). Tea, the most popular beverage is known for its anticarcinogenic potential, good antioxidant, and anti-inflammatory properties. Catechin compounds in green tea and theaflavin and thearubigin in black tea impart protection against several cancers (Jayasinghe et al. 2015). The most important catechin Epigallocatechin gallate (EGCG) is an inhibitor of DNMT and facilitates reactivation of methylation silenced genes, namely, p16, Retinoic acid receptor beta (RARβ), Methylguanine Methyltransferase (MGMT), and hMLH1 in a number of cancer types, and also in cancer cell lines. Besides, reduction of CDX2 and BMP-2 genes by catechin

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compound has been reported. DNA methylation has been found to be reduced by EGCG. The transcription factor Foxp3, a master switch, controlling the regulatory T cell function gets induced by EGCG. EGCG has an impact on histone modification. EGCG also negatively regulates Enhancer of Zeste 2 Polycomb Repressive Complex 2 Subunit (EZH2) and HDAC1 levels. It has been found that EGCG increases the transcription activation of tissue inhibitor of metalloproteinases-3 (TIMP-3) gene. EGCG has been found to inhibit the activity of Histone deacetylases (HDACs) and enhances the levels of acetylated histone H3. This molecule influences H4 acetylation and increases GSTP1 promoter transcription. EGCG is therefore an important biomolecule which is both an inhibitor of DNMT and remodels chromatin (Jayasinghe et al. 2015). EGCG binds to the enzymatic substrates of HDAC1 and DNMT3b, resulting in their inhibition; as a consequence, reactivation of tumor suppressor genes results (Carlos-Reyes et al. 2019). EGCG has been found to repress acetylation of androgen receptor (AR), resulting in inhibition of cell proliferation and promotion of cell death (Carlos-Reyes et al. 2019). In skin and cervical cancer, EGCG acts as a potential epigenetic modifier of HDACs and DNMTs, restoring epigenetically silenced genes. This catechin molecule reactivates the expression of Wnt inhibitory factor-1 (WIF1) via demethylation of the promoter region. Inhibition of cell growth thus results via downregulation of the Wnt canonical pathway (Gao et al. 2009). EGCG aids in chemosensitization as well. In triple negative MDA-MB231 breast cancer cells, a combination treatment of a HDAC inhibitor trichostatin A (TSA) and EGCG, reactivation of the ERα expression has been observed, which is due to altered acetylation and methylation of histones and remodeling of the chromatin structure. In estrogen receptor negative (ER-) breast cancer cells, EGCG and SFN have been found to sensitize tamoxifen treatment, thus inhibiting cell proliferation (Li et al. 2010, 2017). EGCG in green tea can inhibit DNMT activity on one hand and on the other has the potential to reactivate the genes which are methylation-silenced. By inhibition of DNMT1 activity, this polyphenol can revert the hypermethylation of p16INK4a, RARβ, MGMT, and hMLH1 genes. Inhibition of the DNMT1 activity by EGCG may be due to its binding to the catalytic pocket of DNMT1 (Fang et al. 2003; Shankar et al. 2013). Reactivation of tumor suppressor genes may be achieved by promoter demethylation. Black tea polyphenols reduced the incidence of hepatomas, via alteration of metastatic related proteins, suppression of Hypoxia-inducible factors (HIF1α), vascular endothelial growth factor (VEGF), which is correlated with HDAC1 level (Murugan et al. 2009; Shankar et al. 2013). In colon and prostate cancer cells, EGCG, the green tea polyphenol has been found to inhibit DNMT activity and reactivate methylation silenced RAR β gene (Lee et al. 2005). In many cancers, B-lymphoma Moloney murine leukemia virus insertion region-1 (BMI-1) is overexpressed, which is downregulated by EGCG by global reduction in H3. The proteins intricately related to cell cycle like cyclin A, cyclin B1, cyclin E, cyclin dependent kinases like cdk1, cdk2, cdk4 are inhibited, concomitantly the proteins that are responsible for inhibition of cell cycle like p21, p27 get overexpressed. EGCG influences the expression of miRNAs in human hepatocellular carcinoma cells; some of the miRNAs are upregulated, some are downregulated. Some of the miRNAs like miRNA-21 and miRNA-27 which are overexpressed in

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breast cancer get downregulated by EGCG. In hormone dependent prostate cancer cells, transcriptional activation of androgen receptor (AR) gets repressed, by inhibiting the nuclear translocation of AR, which is facilitated by inhibition of androgen regulated miRNA-21. Application of EGCG, by oral as well as topical, restricts UV-induced skin carcinogenesis in murine model, through induction of cytokines (Shankar et al. 2013). EGCG from green tea plays a lead role in cancer chemoprevention through epigenetic control. Considering the poor bioavailability of EGCG, further studies are warranted to improve absorption and biotransformation of this molecule to establish its efficacy in cancer control via epigenetic regulation (Pop et al. 2019). No Indian cuisine is complete without a pinch of turmeric. The active ingredient of turmeric is curcumin. This molecule is a modulator of epigenetic mechanisms. It can demethylate DNA methylation. First 5 CpGs in the promoter site of Nrf2 is demethylated by curcumin, leading to epigenetic silencing during progression of prostate cancer in murine model. The expression of Neurog1, which is a cancer methylation marker, has been found to be altered in prostate cancer cells. Curcumin has been found to reverse CpG methylation in promoter region of Neurog1. HDACs, particularly HDAC 1, HDAC 3, and HDAC 8, have been found to be ameliorated by Curcumin, with an increase in acetylation of histone H4. HAT p300/CREB-binding protein (CBP) is also influenced by curcumin. Curcumin is an inducer of apoptosis, and it does so via FOXO1 pathway. This active biomolecule also induces methylation of partially methylated genes. Modulation of a number of miRNAs namely miR15a, miR-16, miR-20iRNA0, miR-21, miR-22, miR-26, miR-101, miR146, miR-203, and let-7 is facilitated by curcumin. Curcumin has been found to inhibit DNMT1 expression and restore the function of RASSF1A by promoter hypomethylation. Curcumin can induce histone hypoacetylation as well in brain cancer cells. Curcumin has been found to downregulate the expression of DNMT1 in the acute myeloid leukemia (AML). This active molecule can block the positive regulators of DNMT1, p65, and Sp1, thereby their activities are decreased. Thus, p65 and Sp1 can no longer bind to the promoter region of DNMT1. Curcumin in cooperation with bromo domain inhibitor JQ-1 can suppress development of prostate cancer (Zhao et al. 2018). This bioactive molecule can downregulate DNMT1, DNMT3a, DNMT3b, and HDAC4/5/6/8 proteins (Guo et al. 2015a, b). In silico studies reveal that curcumin is capable of inhibiting DNMT1 activity (Medina-Franco et al. 2011). It causes global hypomethylation in leukemia cells. This diferuloyl methane modulates histones and the HATs and HDACs enzymes activity (Shankar et al. 2013). Curcumin binds to HAT covalently. Curcumin has been shown to promote proteasomal degradation of p300 and other CBP proteins without affecting histone acetyltransferase (HATs). This activity of curcumin therefore blocks histone hyperacetylation in prostate cancer cells. An in vitro study reveals that curcumin can inhibit HAT activity of p300/CBP. Through inhibition of HDAC1, HDAC3, and p300/CBP, curcumin can repress the activity of NF-κB and Notch1 in Raji cells. This important molecule inhibits the expression of class I HDACs like HDAC1, HDAC3, and HDAC8 and can upregulate the expression of Ac-histone H4 in Raji cells. Treatment with curcumin can cause upregulation of miR-22. Induction of

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programmed cell death in a multidrug resistant lung cancer cell may be caused via downregulation of miR-186, which causes an increase in caspase 10 activity (Zhang et al. 2010). Through upregulation of miR-15a and miR-16, expression of Bcl-2 is greatly reduced (Yang et al. 2010). miR-21 is overexpressed in colon cancer cell and curcumin can diminish the level. The active ingredient in blueberries, mulberries, grapes, peanuts eta are resveratrol, which is famous due to antiaging, anti-inflammatory, anticancer, and antioxidant properties. Besides, resveratrol is a potential epigenetic modifier, particularly for class III histone deacetylases. A study reveals that epigenetic effect of resveratrol can be through estrogen receptor (ER). Recruitment of HDAC1, DNMT1, Methyl CpG binding domain protein 2MBD2, and mMH3K9 leads to the epigenetic silencing of BRCA-1 and resveratrol has been found to antagonize them. Metastasis associated proteins 1 (MTA1), which is highly expressed in prostate cancer, gets downregulated by resveratrol, which destabilizes the protein, finally allowing acetylation of p53. This aceylation of p53 causes cell cycle arrest and hence induction of apoptosis of prostate cancer cells is facilitated. This phytochemical inhibits all 4 classes of human HDACs dose dependently, as revealed from an in vitro study. Resveratrol inhibits DNMT activity and is able to revert methylation of several tumor suppressor genes. In combination with adenosine analogues, resveratrol inhibits methylation of the promoter of RARb2 gene, whereas alone it did not show much efficacy. This phytochemical target class III HDAC, SIRTs (1, 2, 3 and p300). Once sirtuins (SIRT1) gets activated, it downregulates survivin expression via deacetylase activity. Resveratrol can upregulate the expression of BRCA1 by modulating acetylation of H3. By deacetylation of the transcription factor FOXO, resveratrol regulates cell survival (Shankar et al. 2013). Resveratrol increases the expression of 22 miRNAs and diminishes the expression of 26 miRNAs, for example, miR-17, miR-21, miR-25, miR-92a2. All these miRNAs have been found to be highly expressed in colon cancer. miRNA 663 has a tumor suppressing property and resveratrol increases the level of miR 663; hence, the antitumor attribute of resveratrol may be due to miR 663. Also, resveratrol in co-operation with tea polyphenols inhibits skin carcinogenesis through activation of MAPKs and p53 pathway (George et al. 2011). A flavanol quercetin, abundantly found in citrus fruits, is a very good antioxidant, having enormous antitumor potential. Quercetin has been found to activate histone acetyl transferase (HAT) and inhibit HDACs. Quercetin-induced apoptosis is FasL related and through activation of extracellular signal-regulated kinase (ERK) and jun N-terminus kinase (JNK) pathway. IP-10, which is a TNF-induced interferon gamma inducible protein is linked with inhibition of CBP/p300 activity and phosphorylation and acetylation of histone H3. In leukemia cells, FasL mediated apoptosis is achieved by c-jun/AP-1 activation and promotion of histone H3 acetylation (Lee et al. 2011). Via inhibition of HDAC-1 and DNMT1, Quercetin can lead to cell cycle arrest and apoptosis and can block invasion and angiogenesis (Priyadarsini et al. 2011). Quercetin, along with green tea polyphenol EGCG, can prevent prostate cancer (Tang et al. 2010). Anacardic acids (AA) are phenolic lipids, found in the shell of cashew nuts. In vitro studies show that AA inhibits Tip60 histone acetyltransferase (HAT) in Ataxia

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telangiectasia mutant (ATM). DNA damage leads to the rapid acetylation of ATM, which is dependent on the Tip60 (HAT). AA has been found to inhibit acetylation of H3. Caffeic acid is mainly found in coffee. Caffeic acid inhibits methylation of the promoter region of Retinoic acid receptor β (RARβ) gene in breast cancer cells. S-Adenosyl homocysteine (SAH), an inhibitor of DNA methylation, has been found to be increased by Caffeic acid. Gallic acid, a 3,4,5-trihydroxybenzoicacid compound, is found in nuts, tea, apple peels, berries, pineapples, bananas, lemons, and in wine, both red and white. This compound inhibits p300-induced acetylation hyperacetylation of p65. Genistein, an isoflavone, first isolated from dyer’s broom, Genista tinctorial is found abundantly in soya beans. This compound hindered the growth of breast cancer cells by induction of apoptosis, which is mediated via inhibition of human telomerase reverse transcriptase (hTERT), by epigenetic mechanisms. Genistein regulates transcription of genes by modification of epigenetic events like DNA methylation, histone modification, etc. It is a potent DNMT inhibitor (Pop et al. 2019). Other flavonoids from soy products like biochanin A, diadzein may reverse DNA hypermethylation and can reactivate methylation silenced genes including RARβ, p16INK4a, and MGMT genes in esophageal cancer cells. Promoter region of a tumor suppressor gene BTG3 can be demethylated by Genistein. HAT activity can be increased by Genistein. Alteration of DNA methylation by Genistein gave opposite results in in vivo and in vitro studies; in cell culture studies, inhibition of DNA methylation has been found, whereas in in vivo it is the opposite finding. Compared to other isoflavones, Genistein possesses maximum histone modifying activity. Genistein, daidzein, and the daidzein metabolite (equol) elicit their effect by increase in histone acetylation via modulation of HAT activity (Shankar et al. 2013). Expression of p21WAF1/CIP1 and p16INK4a tumor suppressor genes is induced by Genistein through epigenetic mechanisms. Genistein causes demethylation, at the same time causes acetylation of histone H3-K9 at PTEN promoter and causes acetylation of same histone on p53 and FOXO3a promoter through decrease in SIRT1 activity. It was shown that the target of the antiestrogenic activity of this soy phytochemical is HDAC6. Genistein also modulates miRNAs. In a drug resistant pancreatic cell line, Genistein causes downregulation of miRNA-200, which leads to reversal of epithelial–mesenchymal transition (EMT) (Li et al. 2009). Isothiocyanates are compounds containing sulfur, isolated from cruciferous vegetables. Sulforaphane, belonging to this group, suppresses DNA methylation of Nrf2 promoter through downregulation of DNMTs and HDACs. Sulforaphane (SFN) modulates the miR-9-3 expression via epigenetic regulation, diminishing the DNMT1 activity and expression of DNMT3a, HDAC1, HDAC3, HDAC6, and CDH1. Also, SFN inhibited hTERT in MCF-7 and MDA-MB-231 cancer cells, decreasing DNMT1 and DNMT3a. SFN causes CpG demethylation of hTERT, and this is due to downregulation of DNMTs. A combinatorial treatment of SFN and withaferin A causes inhibition of HDAC activities, DNMT1 and DNMT3a, and leading to death of breast cancer cells (Royston et al. 2017). Also, SFN has been found to inhibit the expression of hTERT, by causing changes in histone acetylation

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and chromatin structure in prostate cancer cells. In androgen-sensitive human prostate adenocarcinoma cells, SFN has been reported to modify the methylation by negative regulation of DNMT1, DNMT3a, and DNMT3b. Demethylation of CpG islands of the promoter of Nrf2 gene is caused by SFN. SFN has been found to decrease DNMT1 and DNMT3a levels. Besides, downregulation of HDACs 1, 4, 5, and 7 is facilitated with a corresponding increase in the levels of the active H3ac chromatin marker (Zhang et al. 2013). In cervical cancer cell HeLa, SFN diminishes DNMT1 and HDAC1 activities. SNF has been shown to increase the expression of CDH1, RARβ, DAPK1, and GSTP1 via restoration of the methylation status of CpG island in cervical cancer (Khan et al. 2015; Carlos-Reyes et al. 2019). In nature more than 40,000 terpenoid compounds have been found. One among these is parthenolide which has been isolated from shoots of feverfew. This parthenolide inhibits DNMT1. Lycopene is another terpenoid found in tomatoes. It aids in demethylation of the promoter region of glutathione S-transferase pi gene (GSTP1), which is a tumor suppressor gene. Lycopene treatment may lead to demethylation of the RARβ2 and HIN-1 genes. It is quite possible that lycopene may have DNA demethylating potential. Phytochemicals that contain a nitrogen atom are known as alkaloids. Many drugs belong to this group, for example, Mahanine, Sanguinarine, Matrine. Of these, Mahanine has been shown to facilitate degradation of DNMT1 and DNMT3B through activation of protein kinase B. It also demethylates Ras Associated Domain Family 1A (RASSF1) promoter. The hypermethylation of RASSF1A gene has been considered as a marker of lung cancer (Carlos-Reyes et al. 2019) and Mahanine demethylates the RASSF1 promoter (Jayasinghe et al. 2015). Organosulfur compounds consisting of Allium vegetables like garlic had been used as a traditional medicine since ages. Organosulfur compounds (OCS) are generated upon conversion of alliin to allicin, via the action of the enzyme alliinase. The products diallyl sulfide [DAS], diallyl disulfide [DADS], and diallyl trisulfide [DATS] are highly unstable. Generous consumption of allium vegetables reduces the risk of a number of cancers. The active metabolite of DADS is S-allylmercaptocysteine [SAMC]. DADS and SAMC induce acetylation of histone and facilitate inhibition of cell growth. DADS caused increased global acetylation of histone H3 and H4. Binding of acetylated H3 to the promoter of p21 gene leads to upregulation of the latter and cell cycle arrest is caused along with HDAC inhibition. Hyperacetylation of histones has been found to be induced by DADS (Shankar et al. 2013). Indole-3-carbinol (I3C) is found in brassica vegetables as a hydrolyzed product of glucosinolate. I3C is converted in the stomach to many diindolylmethane condensation products. This molecule can induce proteasomal degradation of class I histone deacetylases, like HDAC1, HDAC2, HDAC3, and HDAC8; however, class II HDACs remain unaffected. Due to this degradation, p21 and p27 genes get affected, leading to cell cycle arrest and DNA damage in tumor cells. Invasive potential gets affected by miRNA regulated mechanism. Symptoms of cigarette smoking in rats are attenuated by I3C in rate; this is due to altered miRNA-34b, which is involved in p53 functions. Alteration in miR involved in TGF-β, for example, miR-26a, that is involved in angiogenesis, for example, miR-10a gets altered (Izzotti et al. 2010).

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Conclusion The main epigenetic control mechanisms in cells, including transcriptional mechanisms like methylation, histone acetylation, phosphorylation, DNA methylation, and posttranscriptional mechanisms like miRNA silencing of mRNA, have been discussed. As these mechanisms are reversible to some extent, they have important attributes in cancer prevention and treatment. The roles of reactive oxygen species (ROS) and inflammation in cancer initiation, progression, and metastasis have been discussed. The important roles that some of the phytochemicals play in epigenetic control mechanisms of several cancers have been covered. Phytochemicals can reverse the aberrant epigenetic control due to excessive concentration of ROS and inflammation. Hence, it is important to investigate the roles of phytochemicals further in epigenetic control of cancer.

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Lee WJ, Chen YR et al (2011) Quercetin induces FasL-related apoptosis, in part, through promotion of histone H3 acetylation in human leukemia HL-60 cells. Oncol Rep 25(2): 583–591 Li Y, VandenBoom TG et al (2009) Up-regulation of miR-200 and let-7 by natural agents leads to the reversal of epithelial-to-mesenchymal transition in gemcitabine-resistant pancreatic cancer cells. Cancer Res 69(16):6704–6712 Li Y, Yuan YY, Meeran SM, Tollefsbol TO (2010) Synergistic epigenetic reactivation of estrogen receptor-α (ERα) by combined green tea polyphenol and histone deacetylase inhibitor in ERαnegative breast cancer cells. Mol Cancer 9:274 Li W, Yue G, Zhang C, Wu R, Yang AY, Gasper J, Kong A-NT (2016) Dietary phytochemicals and cancer chemoprevention: a perspective on oxidative stress, inflammation, and epigenetics. Chem Res Toxicol 29:2071–2095 Li Y, Meeran SM, Tollefsbol TO (2017) Combinatorial bioactive botanicals re-sensitize tamoxifen treatment in ER-negative breast cancer via epigenetic reactivation of ERα expression. Sci Rep 7: 9345 Mahalingaiah PK, Ponnusamy L, Singh KP (2015) Chronic oxidative stress causes estrogenindependent aggressive phenotype, and epigenetic inactivation of estrogen receptor alpha in MCF-7 breast cancer cells. Breast Cancer Res Treat 153:41–56 Mahalingaiah PK, Ponnusamy L, Singh KP (2017) Oxidative stress-induced epigenetic changes associated with malignant transformation of human kidney epithelial cells. Oncotarget 8(7): 11127–11143 Medina-Franco JL, Lopez-Vallejo F et al (2011) Natural products as DNA methyltransferase inhibitors: a computer-aided discovery approach. Mol Divers 15(2):293–304 Murugan RS, Vinothini G et al (2009) Black tea polyphenols target matrix metalloproteinases, RECK, proangiogenic molecules and histone deacetylase in a rat hepatocarcinogenesis model. Anticancer Res 29(6):2301–2305 Nguyen T, Nioi P, Pickett CB (2009) The Nrf2-antioxidant response element signaling pathway and its activation by oxidative stress. J Biol Chem 284(20):13291–13295 Niculescu MD, Zeisel SH (2002) Diet, methyl donors and DNA methylation: interactions between dietary folate, methionine and choline. J Nutr 132(8 Suppl):2333S–2335S Parasramka MA, Ho E, Williams DE et al (2012) MicroRNAs, diet, and cancer: new mechanistic insights on the epigenetic actions of phytochemicals. Mol Carcinog 51:213–230 Patnaik S, Anupriya (2019) Drugs targeting epigenetic modifications and plausible therapeutic strategies against colorectal Cancer. Front Pharmacol 10(588):1–15 Pop S, Enciu AM, Tarcomnicu I, Gille E, Tanase C (2019) Phytochemicals in cancer prevention: modulating epigenetic alterations of DNA methylation. Phytochem Rev 18: 1005–1024 Priyadarsini RV, Vinothini G et al (2011) The flavonoid quercetin modulates the hallmark capabilities of hamster buccal pouch tumors. Nutr Cancer 63(2):218–226 Raghunath A, Sundarraj K, Nagarajan R, Arfuso F, Bian J, Kumar AP, Sethi G, Perumal E (2018) Antioxidant response elements: discovery, classes, regulation and potential applications. Redox Biol 17:297–314 Reddy NM, Kleeberger SR, Kensler TW, Yamamoto M, Hassoun PM, Reddy SP (2009) Disruption of Nrf2 impairs the resolution of hyperoxia-induced acute lung injury and inflammation in mice. J Immunol 182:7264–7271 Remely M, Lovrecic L, de la Garza AL et al (2015) Therapeutic perspectives of epigenetically active nutrients. Br J Pharmacol 172:2756–2768 Roy M, Datta A (2019) Cancer genetics and therapeutics: focus on phytochemicals. Singapore: Springer Royston KJ, Udayakumar N, Lewis K, Tollefsbol TO (2017) A novel combination of withaferin a and sulforaphane inhibits epigenetic machinery, cellular viability and induces apoptosis of breast cancer cells. Int J Mol Sci 18:1092 Shankar S, Kumar D, Srivastava RK (2013) Epigenetic modifications by dietary phytochemicals: implications for personalized nutrition. Pharmacol Ther 138(1):1–17 Sporn MB (2011) Perspective: the big C – for chemoprevention. Nature 471:S10–S11

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Tang SN, Singh C et al (2010) The dietary bioflavonoid quercetin synergizes with epigallocathechin gallate (EGCG) to inhibit prostate cancer stem cell characteristics, invasion, migration and epithelialmesenchymal transition. J Mol Signal 5:14 Thakur VS, Deb G, Babcook MA, Gupta S (2014) Plant phytochemicals as epigenetic modulators: role in cancer chemoprevention. AAPS J 16(1):151–163 Thejass P, Kuttan G (2007) Inhibition of endothelial cell differentiation and proinflammatory cytokine production during angiogenesis by allyl isothiocyanate and phenyl isothiocyanate. Integr Cancer Ther 6:389–399 Yang J, Cao Y et al (2010) Curcumin reduces the expression of Bcl-2 by upregulating miR-15a and miR-16 in MCF-7 cells. Med Oncol 27(4):1114–1118 Zhang J, Zhang T et al (2010) Curcumin promotes apoptosis in A549/DDP multidrug-resistant human lung adenocarcinoma cells through an miRNA signaling pathway. Biochem Biophys Res Commun 399(1):1–6 Zhang C, Su ZY, Khor TO, Shu L, Kong ANT (2013) Sulforaphane enhances Nrf2 expression in prostate cancer TRAMP C1 cells through epigenetic regulation. Biochem Pharmacol 85: 1398–1404 Zhao W, Zhou X, Qi G, Guo Y (2018) Curcumin suppressed the prostate cancer by inhibiting JNK pathways via epigenetic regulation. J Biochem Mol Toxicol 32:e22049

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Azhwar Raghunath, Raju Nagarajan, Kiruthika Sundarraj, and Lakshmikanthan Panneerselvam

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MiRNAs Regulation of Redox Homeostasis in Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Breast Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Classical Hodgkin Lymphomas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Colon Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Colorectal Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Gastric Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Glioma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Hepatocellular Carcinoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Lung Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Medulloblastoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Neuroblastoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Oral Carcinoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ovarian Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . p53 Wild-Type Cancers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pancreatic Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . lncRNAs Regulation of Redox Homeostasis in Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . snoRNA Regulation of Redox Homeostasis in Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . circRNA Regulation of Redox Homeostasis in Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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A. Raghunath (*) Department of Biotechnology, Bharathiar University, Coimbatore, Tamilnadu, India Department of Pediatrics, University of Michigan, Ann Arbor, MI, USA e-mail: [email protected]; [email protected] R. Nagarajan Department of Biotechnology, Indian Institute of Technology Madras, Chennai, Tamilnadu, India K. Sundarraj · L. Panneerselvam Department of Biotechnology, Bharathiar University, Coimbatore, Tamilnadu, India © Springer Nature Singapore Pte Ltd. 2022 S. Chakraborti et al. (eds.), Handbook of Oxidative Stress in Cancer: Mechanistic Aspects, https://doi.org/10.1007/978-981-15-9411-3_111

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Abstract

The noncoding RNAs (ncRNAs) role in carcinogenesis received widespread attention in the last decade due to their vast regulatory roles and functional diversity. Dysregulation in reactive oxygen species (ROS) levels and ncRNAs expression is demonstrated in cancer development, metastasis, and response to chemotherapy and radiotherapy. Research in the last decade explored the crosstalk between ROS signaling and ncRNAs in cancer. The reciprocal interaction between ROS and microRNAs (miRNA) contributed to tumorigenesis and antitumorigenesis. The levels of ROS regulate miRNAs expression, in turn, miRNAs govern the redox state in cancer. miRNAs can exhibit either tumorpromoting or tumor-suppressive effects through the regulation of redox homeostasis. Investigation on the dynamic regulation of redox balance by ncRNAs is warranted in the establishment of effective management of different types of cancers. This chapter highlights the current knowledge of the regulation of redox homeostasis by ncRNAs in cancer. The intricate network of dynamic interaction between ncRNAs and redox regulation is unraveling numerous opportunities in cancer. The versatility of ncRNAs and ROS makes them not only excellent orchestrators in signaling pathways but also core components in cancer. The knowledge of these mechanisms might be valuable for the development of targeted therapies against different cancer types. Keywords

Cancer · Circular RNA · Long noncoding RNA · MicroRNA · Noncoding RNAs · Redox homeostasis · Small nucleolar RNA

Introduction Dynamic cellular redox processes maintain redox homeostasis and thus ensure cellular proliferation and viability. Reactive oxygen species (ROS) are natural components of the cell produced as end products of aerobic metabolism (Forrester et al. 2018). ROS are reactive oxygen intermediates and comprised of hydroxyl radical (OH
), hydrogen peroxide (H2O2), peroxynitrite (ONOO
), nitric oxide (NO), nitrogen dioxide (NO2), singlet oxygen (1O2), and superoxide anion-radical (O2˙
) (Ursini et al. 2016). The levels of these ROS define the fate of the cell. Endogenous ROS production is determined by the endoplasmic reticulum, mitochondrial, and peroxisomal metabolic events, whereas exogenous factors such as drugs, radiation, and xenobiotics may also contribute to the generation of ROS (Phaniendra et al. 2015). At optimal or low levels, they act as the second messenger and orchestrate signal transduction pathways eventually regulating a myriad of processes like cell proliferation, cell differentiation, and cell survival (Schieber and Chandel 2014). In contrast, elevated ROS levels impart cytotoxicity and genotoxicity leading to cell death events such as apoptosis and necroptosis. In cancer, undue and perpetual ROS induction promotes tumorigenesis. Such

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sustainable ROS generation triggers signal transduction pathways contributing tumorigenic potential, metastasis, and even resistance to radiotherapy and chemotherapeutic agents. In cancer, ROS exhibits dual roles inducing both antiapoptotic and proapoptotic events. The ROS signaling mechanisms are different for different types of cancer. Oxidative stress not only promotes tumorigenesis but also sensitizes tumors to therapy. This dual effect of oxidative stress curtails the favorable outcome of antioxidants against cancer. The comprehensive understanding of the redox balance in cancer has not been elucidated. Redox homeostasis is regulated through processes, which are under the governance of different proteins and enzymes. Besides these players, a group of untranslated RNAs called noncoding RNAs (ncRNAs) are involved in the redox regulation in cancer (Slack and Chinnaiyan 2019). Research findings in the last two decades unraveled the role of different ncRNAs modulating the redox homeostasis in cancer. miRNAs are the prevalent subset of ncRNAs that received much focus in cancer research over the recent years. Of the ncRNAs, majority research findings unraveled the interaction between miRNAs and ROS in carcinogenesis. ROS levels regulate the miRNAs expression, and in contrast, certain miRNAs alter the ROS levels targeting redox genes (Lan et al. 2018), thereby ROS acts as transcriptional activators and downstream mediators of miRNAs. MiRNAs play a significant role in the regulation of the Nrf2 pathway and their target antioxidant enzymes thereby offer chemotherapy and radiotherapy resistance in cancer. Based on the type of interaction, they may either suppress or promote tumorigenesis. Besides miRNAs, lncRNA, snoRNA, and circRNA modulation of redox balance in cancer were explored by scientists around the globe. In this chapter, the role of different ncRNAs in the regulation of redox stability in cancer is discussed. This chapter chronicles the different mechanisms exhibited by different ncRNAs modulating the redox regulation in cancer leading to tumorigenesis, antitumorigenesis, and resistance/susceptibility of cancers against chemotherapy/radiotherapy.

MiRNAs Regulation of Redox Homeostasis in Cancer In the last two decades, the interplay of miRNAs and ROS in tumorigenesis and antitumorigenesis was discovered (He and Jiang 2016). The reciprocal relationship between miRNAs and ROS was observed in different types of cancer (Fig. 1), where ROS regulates the expression of miRNAs and vice versa miRNAs control redox homeostasis (Table 1). Cancer treatment such as chemotherapy and radiotherapy interferes with both redox homeostasis and miRNAs expression. ROS may either induce or repress miRNA expression in cancer.

Breast Cancer Radiotherapy remains one of the mainstays of therapy in solid tumors, which includes breast cancer. miR-139-5p sensitizes radiotherapy targeting genes involved in the deoxyribonucleic acid (DNA) repair and ROS defense mechanisms

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Fig. 1 Interaction network of miRNAs and mRNAs in different cancers

(Pajic et al. 2018). miR-139-5p targets key genes namely DNA polymerase theta (POLQ), RAD54L, DNA topoisomerase I (TOP1), DNA topoisomerase 2-alpha (TOP2A), and X-ray repair cross-complementing 5 (XRCC5), which are required for DNA maintenance and repair. Also, miR-139-5p targets an important ROS defense gene, methionine adenosyltransferase 2A (MAT2A). By targeting these genes, miR-139-5p interferes with ROS defense and DNA repair processes directing irradiated breast cancer cells to apoptosis. miR-139-5p along with these DNA repair and ROS defense genes serve as predictive biomarkers for breast cancer radiotherapy. A network of interaction with a different set of genes was exhibited by miR139-5p, which remain as the potent regulator of radiotherapy response in breast cancer. miR-526b and miR-655 have contributed to cancer progression, angiogenesis, metastasis, and cancer stem cell regulation in breast cancer (Hunter et al. 2019). Also, elevated levels of these two miRs were reported not only in breast cancer patients but also in advanced grades of disease (Majumder et al. 2018). The overexpression of miR-526b and miR-655 increased the ROS production in breast cancer cells (Shin et al. 2019). Thioredoxin reductase 1 (TXNRD1) is induced as a result of the increased expression of miR-526b and miR-655. The transcription factors – polybromo 1 (PBRM1) and transcription factor 21 (TCF21), which regulate TXNRD1 and are targets of miR-526b and miR-655. The suppression of PBRM1 and TCF21 by miR-526b and miR-655 upregulate TXNRD1 in breast cancer cells. Such upregulation of TXNRD1 induces ROS and eventually oxidative stress in breast cancer. The increased oxidative stress elevated the expression of miR-526b

–

Sirt1

miR-206b3p

miR-34a

miRNAs miR-143

Target mRNA –

p53 wild-type cancers

Ehrlich acid solid tumor – male BALB/c mice

Cancer type (cell line/in vivo model) Colon cancer – HCT116

Inhibits tumorigenesis

Induces tumorigenesis

Increases oxidative stress

Increases oxidative stress

Oxidative Tumorigenesis stress status Inhibits Increases tumorigenesis oxidative stress

Table 1 Summary of miRNAs that modulate redox homeostasis in cancer

TQ downregulated miR-206b-3p, decreased oxidative stress, prevented necroptosis, and thus the regeneration of liver tissue Metformin upregulation of miR-34a sensitized cancer cells to TRAILinduced apoptosis

Response to therapy miR-143 overexpression induced oxidative stress and influenced the sensitivity of tumor cells to chemotherapy

Inhibited Sirt1/Pgc1α/Nrf2 axis and triggered TRAILinduced apoptosis

Necroptotic signaling pathway

Mechanisms/ pathway Apoptotic signaling pathway

Upregulation of miR-34a downregulated sirt1 and enhanced the sensitivity of cancer cells to apoptosis

Key findings miR-143 expression sensitizes colon cancer cells and ROS generation resulted in oxidative stressmediated apoptosis miR-206b-3p expression increased oxidative stress and necrosis in liver tissue

Noncoding RNAs Regulation of Redox Balance in Cancer (continued)

Do et al. (2014)

Meral et al. (2018)

References Gomes et al. (2018)

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Oral carcinomaOSCC cells

Pancreatic cancer – Capan-2 and Aspc-1

Sirt3

–

miRNAs miR-128a

miR-31

miR-155

Cancer type (cell line/in vivo model) Medulloblastoma – Daoy cells

Target mRNA Bmi-1

Table 1 (continued)

Induces tumorigenesis

Progresses tumorigenesis

Induces ROS production

Increases ROS

Oxidative Tumorigenesis stress status Inhibits Increases tumorigenesis intracellular ROS

K-Ras oncogenic signaling pathway

miR-31SIRT3 cascade

–

–

Mechanisms/ pathway Increased senescencesignaling pathway

Response to therapy Upregulation of miR-128a specifically inhibits the Bmi1 oncogene Key findings Upregulation of miR-128a downregulate the expression of Bmi-1 oncogene, promoted senescence signaling pathway, and inhibited cell growth Upregulation of miR-31 disrupted the mitochondrial function, altered energy metabolism, and promote growth miR-155 expression was induced by KRas oncogenic signal and increased ROS in prostate cancer cell proliferation

Wang et al. (2015)

Kao et al. (2019)

References Venkataraman et al. (2010)

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Induces tumorigenesis

Promotes Induces tumorigenesis ROS and epithelialmesenchymal transition

Neuroblastoma – SH-SY5Y and SK-N-BE(2C)

Colon cancer – DLD1

–

PKM2 and H6PD

miR-661

Inhibits oxidative stress

Induces ROS stress

miR-494

Inhibits tumorigenesis

Colorectal cancer – HCT116, SW480, and SW707

–

Induces ROS stress

miR-210

Inhibits tumorigenesis

Glioma – U87 and SHG44

Nrf2

miR-153

Upregulation of miR-661 increased ROS and mitochondrial superoxide anions

–

–

Upregulation of miR-153 increases the ROS production and radiosensitization

Pentose phosphate pathway

HO-1 pathway

Apoptotic signaling pathway

Nrf-2/Gpx1 and apoptotic pathway Increased expression of miR-153 leads to upregulation of Nrf2 and GPx1 pathway, decreased neurosphere formation and stem cell marker expression Increased expression of miR-210 induced ROS production and inhibited the cancer cell proliferation miR-494 induced HO-1 and promoted cell survival miR-661 decreased aerobic glycolysis, pentose phosphate metabolism, and interrupted redox homeostasis

Noncoding RNAs Regulation of Redox Balance in Cancer (continued)

Gomez de Cedron et al. (2017)

Piras et al. (2018)

Tagscherer et al. (2016)

Yang et al. (2015)

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Prxl2A

miRNAs miR-9-3p

miR-125b

miR-139-5p MAT2A, POLQ, RAD541, TOP1, TOP2A, and XRCC5 miR-200a P38α MAP

Target mRNA Herpud1

Table 1 (continued)

Increased oxidative stress

Induces oxidative stress response

Inhibits tumorigenesis

Increases tumor growth

Breast cancer – MCF7 cells, human breast tumor specimens, and 8–12 weeks old female Balb/c nude mice Ovarian cancer – mouse cell lines – CT26, NMUMG, human cell lines (MDA-MB-435S, 293 T, MDA-MB-

Induces oxidative stress

Suppresses tumorigenesis

Oxidative Tumorigenesis stress status Inhibits Increases tumorigenesis oxidative stress

Oral carcinoma – oral squamous cell carcinoma (OSCC) cell lines

Cancer type (cell line/in vivo model) Glioma – Glioma stem cells

Enhanced expression of miR-200a increases oxidative stress response and

Expression of miR-125b in OSCC cells increased oxidative stress and sensitizes cisplatin against OSCC Expression of miR-139-5p increased oxidative stress and sensitized radiotherapy

Response to therapy Upregulation of miR-9-3p induces apoptosis through ROS

miR-139-5p is a potential regulator of radiotherapy response

Overexpression of miR-200a target p38α and modulate oxidative stress response and

–

Key findings Upregulation of miR-9-3p induces apoptosis through downregulation of Herpud1 Downregulation of miR-125b suppressor induces upregulation of PRXL2A in OSCC

Apoptotic pathway

–

Mechanisms/ pathway Apoptotic pathway

Mateescu et al. (2011)

Pajic et al. (2018)

Chen et al. (2019a)

References Yang et al. (2017)

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TXNRD1 and NFE2L2

TCF21 and PBRM1

TCF21 and PBRM1

miR-500a5p

miR-526b

miR-655

Breast cancer cell line- MCF

Induces tumorigenesis

Induces tumorigenesis

Breast cancer – MCF

Enhances ROS and SO production

Enhances ROS and SO production

Survival of ER Inhibits + breast oxidative cancer stress

436, and BT-549), ovarian adenocarcinoma cell line (SKOV3) and xenograft nude mice ER+ breast cancer cell lines – MCF7 and T47D

Upregulation of miR-655 induces the oxidative stress response

Upregulation of miR-526b induces the oxidative stress response

Overexpression of miR-500a-5p inhibits oxidative stress response genes

sensitizes paclitaxel against ovarian cancer

–

–

– Induction of oxidative stress by H2O2 increases the expression of miR-500a-5p, which inhibits the expression of oxidative stress response genes miR-526b regulates ROS generation and the expression of miR-526b increases during oxidative stress miR-655 regulates ROS generation and the expression of miR-526b increases during oxidative stress

increases tumor growth

Shin et al. (2019)

Shin et al. (2019)

Degli Esposti et al. (2017)

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and miR-655 creating a positive feedback loop in breast cancer. miR-500a-5p are known to target more than 300 genes directly or indirectly in estrogen-receptorpositive (ER+) breast cancer cell lines (Degli Esposti et al. 2017). These putative targets are oxidative stress response genes. The expression of miR-500a-5p is itself modified by oxidative stress. In breast cancer, miR-500a-5p regulates the expression of manifold oxidative stress genes, and in turn, the expression of miR-500a-5p is also triggered by oxidative stress. In breast cancer cells, miR-28 promotes cell growth through the suppression of Nrf2 (Yang et al. 2011). miR-28 binds the 30 -UTR regions of Nrf2 mRNA and causes their degradation and inhibits Nrf2 protein synthesis. This regulation of the Nrf2 pathway by miR-28 is independent of Keap1, an inhibitor of Nrf2. The inhibition of Nrf2 by miR-28 enhanced colony formation of breast cancer cells through anchorage-independent growth. miR-28 regulates Nrf2 expression in both breast epithelial and cancer cells.

Classical Hodgkin Lymphomas In the advanced Hodgkin lymphomas, multiple redoxmiRs and redox mRNA levels are associated with disease outcome and can be of prognostic value (Karihtala et al. 2017).

Colon Cancer The stable transduction of miR-143 caused over 100 proteins to differentially express in colon cancer cells (Gomes et al. 2018). These 100 proteins regulate protein folding, oxidative stress, and cell death processes. miR-143 targets superoxide dismutase 1 (SOD1) and thereby aggravates oxidative stress in cancer leading to apoptosis. The increased expression of miR-143 sensitizes oxaliplatin against colon cancer cells through the generation of ROS and induction of apoptosis. miR-661 promoted epithelial-mesenchymal transition (EMT) via increasing the expression of N-cadherin, slug, and vimentin in non-metastatic colon cancer cells (Gómez de Cedrón et al. 2017). miR-661 overexpression diverted aerobic glycolysis, inhibited pentose phosphate metabolism, and disrupted redox homeostasis. Pyruvate kinase M2 (PKM2) and hexose-6-phosphate dehydrogenase (H6PD) are the targets of miR661 in colon cancer cells. PKM2 inhibits ATP production and reroutes glycolytic intermediates into anabolic pathways (Anastasiou et al. 2011). H6PD regulates NADPH dehydrogenase and redox homeostasis. miR-661 increases superoxide anions and mitochondrial membrane potential in CC cells.

Colorectal Cancer In colorectal cancer (CRC), the overexpression of miR-210 inhibited the clonogenicity with a decreased number of cells in the G1 phase and with an increased number of cells in the G2/M phases of the cell cycle (Tagscherer et al.

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2016). In addition, miR-210 induced apoptosis with increased levels of Bcl2 interacting mediator of cell death (Bim) (proapoptotic protein) and decreased levels of myeloid cell lymphoma 1 (Mcl-1) (antiapoptotic protein). The overexpression of miR-210 led to the increased generation of ROS activating caspase 2 independent of caspase 3. N-acetylcysteine (NAC) – the ROS scavenger confirmed the role of ROS in the miR-210 mediated cell death in CRC cells. ROS production is the key event in the miR-210 mediated apoptosis in CRC cells.

Gastric Cancer In gastric cancer MKN45 cells, miR-210 regulates the H2O2-induced apoptosis (Xu et al. 2017). The H2O2 treatment reduced cell viability in MKN45 cells with the upregulation of miR-210. miR-210 inhibited the expression of Bcl-2 (antiapoptotic protein) and enhanced the caspase 3 cleavage thus triggered apoptosis. miR-210 plays an important role in the oxidative damage-induced apoptosis in gastric cancer cells.

Glioma Glioma stem cells (GSCs) exhibit reduced expression of miR-153, a similar condition was not observed in non-GSCs (Yang et al. 2015). miR-153 targets Nrf2 and with reduced expression of miR-153 in GSCs, the elevated Nrf2 levels resulted in the increased transcription of glutathione peroxidase 1 (Gpx1). This eventually led to the decreased ROS levels bringing out radioresistance in GSCs through the Nrf-2/GPx1/ ROS pathway. In contrast, overexpression of miR-153 resulted in increased ROS levels as miR-153 targets Nrf2 thereby enhances radiosensitivity, triggers apoptosis, and decreases stemness of GSCs. In glioma tissues, there was a low-level expression of miR-9-3p (Yang et al. 2017). Homocysteine inducible ER protein with ubiquitinlike domain 1 (Herpud1) – a mammalian ubiquitin domain protein is the target for miR-9-3p. Herpud1 offers protection against oxidative stress in glioma cells. H2O2 elevated the expression of miR-9-3p, which targets Herpud1 and inhibits its expression. This reduction in the levels of Herpud1 resulted in increased oxidative stress and thus apoptosis in glioma. The low levels of miR-9-3p with elevated levels of Herpud1 might resist apoptotic cell death in glioma cells. In human glioma cells and tissues, miR-34a levels are low (Li et al. 2014). The overexpression of miR-34a using miR-34a mimics triggered apoptosis in A172 glioma cells. NOX2 is the primary ROS generator in different cell types. miR-34a targets NOX2 and reduces ROS production. In glioma cells, miR-34a acts as a tumor suppressor.

Hepatocellular Carcinoma In the neoplastic HCC, miR-92a was downregulated which is a part of the miR 17-92 cluster. The association between miR-92 expression and oxidative damage was

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evidenced from 8-hydroxydeoxyguanosine (8-OHdG) levels (Cardin et al. 2012). The generation of ROS might have inhibited the expression of miR-92. Such induction of ROS led to oxidative DNA damage in HCV-induced liver damage. In Ehrlich acid solid tumor, miR-206b-3p levels were elevated which led to increased oxidative stress and necrosis in liver tissue (Meral et al. 2018). In contrast, thymoquinone (TQ) – a bioactive compound from Nigella sativa oil downregulated miR-206b-3p expression and protected liver tissues from oxidative stress which led to the regeneration of liver tissue evading necrosis.

Lung Cancer In lung cancer, increased expression of mucin-1 (MUC1) confers resistance against apoptosis and chemotherapy (Xu et al. 2014). MUC1 expression suppressed catalase expression thereby increases ROS accumulation. miR-551b targets and inhibits catalase due to MUC1 overexpression. This catalase suppression led to the generation of ROS, which increased MUC1 transcription, stabilized MUC1 preventing the lysosomal degradation. MUC1 favored cell survival pathway through EGR/Akt/ COX-2 signaling pathway and eventually evades drug-induced cell death in lung cancer. miR-200 family of miRs plays key roles in many types of cancer, miR-200s loss promotes cancer cell proliferation and metastasis whereas increased levels prevent cell growth. In lung cancer, overexpression of miR-200c enhanced radiotherapy responses (Cortez et al. 2014). As most miR-200s, miR-200c is also negatively regulated by ZEB1 (Park et al. 2008). An increased miR-200c expression intensifies the generation of intracellular ROS and enhances p21 expression. miR200c interferes redox homeostasis through direct regulation of oxidative stress genes – GABP/Nrf2, PRDX2, and SESN1. In the xenograft model, systemic administration of miR-200c sensitizes radiation treatment in lung cancer. This anticancer efficacy of miR-200c is primarily through the regulation of redox homeostasis in lung cancer.

Medulloblastoma In medulloblastoma, miRs specific for the brain is differentially expressed. Of these, miR-128a targets Bmi-1 – an oncogene and exhibits growth inhibition activity in medulloblastoma (Venkataraman et al. 2010). The suppression of Bmi-1 by miR128a increases ROS in medulloblastoma. This increased ROS due to miR-128a expression triggered the senescence-signaling pathway and decreased cell growth.

Neuroblastoma The response to oxidative stress was dealt with in two different ways in neuroblastoma (NB) cells. Undifferentiated NB cells evade oxidative stress through heme

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oxygenase-1 (HO-1) induction, whereas differentiated NB cells are vulnerable to oxidative stress due to the low levels of HO-1 (Piras et al. 2018). miR-494 regulates the expression of HO-1 in NB cells. On exposure to H2O2, undifferentiated NB cells were restricted due to miR-494 inhibition. Hence, miR-494 favors the induction of HO-1 and cell survival against oxidative stress.

Oral Carcinoma miR-31 is established as an oncogenic miRNA, which perturbs multiple neoplastic processes in many cancers (Zheng et al. 2015). In oral squamous cell carcinoma (OSCC), miR-31 induces metabolic change and consequent progression of OSCC. Silent information regulator 3 (SIRT3), being a NAD-dependent deacetylase, controls the oxidative metabolic process in mitochondria (Hirschey et al. 2010). SIRT3 is a ROS homeostasis regulator and functions as a tumor suppressor or oncogene depending on the type of cancers (Torrens-Mas et al. 2017). miR-31 targets SIRT3 and regulates ROS levels in OSCC cells (Kao et al. 2019). The repression of SIRT3 by miR-31 increases ROS in OSCC cells through the disruption of mitochondrial structure and function eventually switching the energy metabolism from aerobic to glycolysis. Besides this metabolic shift, the SIRT3-miR-31 axis regulates tumor growth, cell migration, and invasion in OSCC cell lines. The miR-125 family participates in multiple cellular processes ranging from proliferation to apoptosis in many different cancer types (Sun et al. 2013). A low level of miR-125b was reported in OSCC tumors. Peroxiredoxin like 2a (PRXL2A) protects the cells from oxidative insult. miR-125b targets PRXL2A thereby increases ROS and suppresses tumorigenesis in OSCC cells (Chen et al. 2019a). But the low levels of miR-125b can no longer target PRXL2A and hence PRXL2A persists with antioxidant defense and checks ROS production in OSCC cells. The overexpression of miR-125b sensitizes cisplatin against OSCC through the induction of ROS. The PRXL2A-miR-125b axis prevents the OSCC cells from oxidative stress and confers resistance against cisplatin.

Ovarian Cancer Oxidative stress stimulates the expression of miR-200s. miR-200 family (miR-200a and miR-141) plays a signature dual role in ovarian cancer (Mateescu et al. 2011). With high sequence homology, both miR-141 and miR-200a target and regulate P38α mitogen-activated protein kinases (p38α MAPK) expression. In aggressive human ovarian adenocarcinomas, these miR-200s were found to accumulate at higher levels and the p38α MAPK regulation was miR-200a-dependent (Bendoraite et al. 2010). This inactivation of p38α MAPK resulted in the accumulation of ROS due to miR-200a overexpression. These miRNAs promote tumor growth in the absence of chemotherapeutic agents, but in contrast to treatment with paclitaxel, these miRNAs sensitize tumor cells to apoptosis. Though miR-141 and mir-200a

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expression favor tumorigenesis, these miRNAs enhance the sensitivity of ovarian cancer cells to paclitaxel in a ROS-dependent manner. MiR-200 family regulation of oxidative stress signature has the potential to be a predictive marker to survival and treatment in ovarian cancer. In ovarian cancer, ROS suppresses miR-199a and miR125b and inhibited angiogenesis with decreased HIF-1a and VEGF (He et al. 2013). In ovarian cancer, ROS influence the expression of miRNAs through epigenetic modifications such as DNA methylation and histone modification. In addition, ROS downregulates miR-199a and miR-125b through hypermethylation of their promoters by DNA methyltransferase 1 (DNMT1) (He et al. 2012). These two miRNAs target ERBB2 and ERBB3 in ovarian cancer cells.

p53 Wild-Type Cancers In p53 wild-type cancers, metformin downregulates Sirt1 expression through the induction of miR-34a in a p53-dependent fashion (Do et al. 2014). This reduction of Sirt1 inhibits Sirt1/Pgc-1α/Nrf2 axis and thereby enhances the susceptibility of wildtype p53 cancer cells to oxidative stress. Metformin inhibited PPARγ transcriptional activity and thereby reduced the binding of PPARγ to PPRE in the promoter of Nrf2 eventually suppressed the expression of Nrf2 thus increased oxidative stress in p53 wild-type cancers. Such metformin suppression of Sirt1 and expression of DR5 triggered TRAIL-induced apoptosis in p53 wild-type cancer cells.

Pancreatic Cancer In pancreatic cancer, the doxycycline-inducible system revealed the upregulation of miR-155 as a result of constitutive K-Ras activation (Wang et al. 2015). K-Ras induced the expression of miR-155 through the MAPK and NF-kB pathway. This increased expression of miR-155 enhanced the production of ROS by inhibiting Foxo3a which resulted in decreased antioxidants, SOD2, and catalase. The generation of ROS by miR-155 increased the proliferation of pancreatic cancer cells.

lncRNAs Regulation of Redox Homeostasis in Cancer lncRNAs are 200 nucleotides long RNA molecules that regulate repertoire of processes – assembly of the multi-subunit protein complex, chromatin dynamics, transcription, splicing, and translation. The errant expression of lncRNA is linked to modulating redox homeostasis, which influences the progression of cancer. Research in the past decade explored the role of various lncRNAs in different cancers and their association with oxidative stress (Table 2). Smoke and cancer-associated lncRNA-1 (SCAL1) is transcriptionally regulated by NRF2 on exposure to cigarette smoke extract in manifold lung cancer cell lines (Thai et al. 2013). SCAL1 offers protection against cytotoxicity elicited by cigarette

Human cholangiocarcinoma – QBC939

HULC

Human malignant melanoma tissue and Human malignant melanoma – A375 and SK-Mel-28 ACA11 CD138- positive bone (Orphan marrow cells of patients snoRNA) with multiple myeloma

Human cholangiocarcinoma – QBC939

H19

GAS5

HCC – CD133+ cancer stem cells

H19

lncRNAs/ Cancer type (cell line/ snoRNAs in vivo model) SCAL1 Lung cancer – CL1–0 and CL1–5

Induces tumorigenesis

Promotes CCA pathogenesis and progression Promotes CCA pathogenesis and progression Inhibits tumorigenesis

Promotes tumorigenesis

Tumorigenesis Associated with cancer

Inhibits oxidative stress

Inhibits SO anion

HULC upregulated by oxidative stress

Inhibits oxidative stress H19 upregulated by oxidative stress

Oxidative stress status Inhibits oxidative stress

ACA11 is highly expressed in t (4;14)-positive MM cancers

HULC sponges miR-372/miR373 and activates CXCR4 GAS5 regulates redox homeostasis through G6PD

H19 sponges let-7a/let-7b and activates IL-6

Regulation Expression of SCAL1 is regulated by NRF2 –

Redox homeostasis and apoptotic signaling pathway –

Chronic inflammation response

MAPK/ERK signaling pathway Chronic inflammation response

Mechanisms/ pathway involved Nrf2-Keap1ARE pathway

Table 2 Summary of long noncoding and small nucleolar RNAs that modulate redox homeostasis in cancer

Downregulation of ACA11 is a functionally important component of the t(4:14) translocation.in MM

Reduced GAS5 expression contributes to disease progression in MM patients

Overexpression of HULC promotes cell migration and invasion

Key findings SCAL1 is induced by cigarette smoke in the airways and is regulated by NRF2 and offers protection Downregulation of H19 elicits oxidative stress and sensitizes chemotherapy Overexpression of H19 promotes cell migration and invasion

Chu et al. (2012)

Chen et al. (2019b)

Wang et al. (2016)

Wang et al. (2016)

Ding et al. (2018)

References Thai et al. (2013)

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smoke in airway epithelial cells. Only a few of the ncRNAs are demonstrated to be transcriptionally regulated by Nrf2 due to the presence of antioxidant response elements in the promoters of these ncRNAs. Though SCAL1 offers protection against oxidative stress, the mechanism remains unclear. The functional characterization of SCAL1 soon will reveal whether it acts as an oncogene or tumor suppressor. H19, a lncRNA, protect cancer cells against oxidative damage and is upregulated in HCC (Ding et al. 2018). Increased expression of H19 activates chemoresistance and survival genes and suppresses apoptotic genes in breast and liver cancer. The inhibition of H19 exacerbates oxidative stress through increased ROS in CD133+ liver cancer stem cells and eventually reversed chemoresistance. In addition, H19 inhibition suppresses the multidrug resistance 1 gene and the MAPK/ERK signaling pathway in HCC. These features of H19 make them an ideal therapeutic target for liver cancer. Growth arrest-specific transcript 5 (GAS5) expression was reduced in MM as well as in A375 (MM cell line) thereby inhibited apoptosis (Chen et al. 2019b). The cell viability of A375 cells increased with reduced GAS5 expression. The low expression of GAS5 increased intracellular ROS levels through NOX4 and altered redox balance eventually contributed to MM progression. GAS5 interacts with G6PD and thus modulates redox homeostasis. Oxidative stress triggers the expression of H19 and HULC lncRNAs in cholangiocarcinoma (CCA) cell lines favoring migration and invasion (Wang et al. 2016). H19 and HULC act as sponges for a set of miRNAs – let-7a, let-7b, miR-372, and miR-373. This sponge activity of H19 and HULC activated inflammatory markers – IL-6 and CXCR4 and promoted migration and invasion in CCA. H19 and HULC could serve as therapeutic targets in CCA.

snoRNA Regulation of Redox Homeostasis in Cancer snoRNA, a noncoding RNA with 60–300 nucleotides, plays major roles in sitespecific modifications in rRNAs and U6 snRNA and also involves in the pre-rRNA folding and endonucleolytic cleavage (Liang et al. 2019). Based on the structure, function, and the presence of conserved motifs, snoRNAs are classified into two groups namely, C/D box (SNORD) and H/ACA box (SNORA). The expression levels of both groups are directly correlated with the growth and invasion of tumor cells thus making a major impact in carcinogenesis and serve as reliable targets for diagnostic approaches (Mei et al. 2012). Though the roles of different snoRNAs on carcinogenesis are studied extensively, their regulation of redox homeostasis in cancer is scarce. ACA11, belongs to the orphan box H/ACA class snoRNA, resides within 18–19 intron of Wolf-Hirschhorn syndrome candidate 1 gene. Besides t(4;14)-positive MM, ACA11 is overexpressed in multiple cancer cell types suggesting its oncogenic role (Chu et al. 2012). ACA11 accelerates the proliferation of cells through the increased ROS levels and phosphorylation of extracellular signal-regulated protein

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kinases 1 and 2 (ERK1/2) (Mahajan et al. 2017). ACA11 sustains the increased ROS levels by inhibiting the NRF2 transcriptional activity. The activation of the Nrf2 antioxidant pathway due to elevated ROS levels is comprised of ACA11 overexpression thereby affords resistance against chemotherapy. Hence, ACA11 could serve as a valuable target for therapeutics against various cancers.

circRNA Regulation of Redox Homeostasis in Cancer Circular RNAs (circRNA), a type of single-stranded and covalently linked noncoding RNAs, are one of the potential cancer biomarkers for diagnosis and prognosis. Many experimental and RNA-sequencing studies reported the presence and differential expression patterns of tumor-specific circRNAs in cancer samples. With the computational algorithm analysis of ENCODE RNA-seq data, the presence of more diverse intronic and exonic circRNA in cancer cells was discovered (Gao et al. 2015). The distinct function of circRNAs is to act as miRNA sponges, which either suppress or induce tumor growth by preventing or promoting cancer-related miRNAs. Reports are scant on their regulation of redox homeostasis in cancer, as these circRNAs act mainly as miR sponges, it is speculated that the sponged miRs may modulate levels of ROS in cancer. Research works are warranted in this area in the near future.

Conclusion A sustainable redox balance is an essential feature for the maintenance of cell health and to dissuade the malignancy transformation of cells. The ROS-miRNA axis regulates tumorigenesis either adversely or beneficially through redox signaling pathways. Cancer therapy resistance is mediated by miRNAs regulation of oxidative stress response. Research endeavors on the interplay between miRNAs and ROS in cancer opened rich avenues that will pave way for the efficacious preventive and therapeutic approaches for cancer in the near future. The ncRNAs-mediated treatment is still in its infancy due to the dearth of knowledge on their distinct dynamic interactions in cancer. The deep understanding is guaranteed on the ncRNAs regulation of redox homeostasis in cancer, which presumably increases exponentially over the next few years.

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A New Mechanism for Regulation of Gene Expression in Cancer Suravi Pramanik, Shrabasti Roychoudhury, and Kishor K. Bhakat

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Oxidative DNA Base Damage Shaping the Mutation Burden in Cancer . . . . . . . . . . . . . . . . . . . . . Oxidative DNA Base Damage Repair . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Interplay Between Posttranslational Modification of BER Proteins and Transcriptional Regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-OxoG and OGG1 as a Novel Epigenetic Regulator for Controlling Gene Expression . . . . . BER in Hypoxia-Induced VEGF mRNA Expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Role of 8-OxoG and OGG1 in the Regulation of Inflammatory Response Genes . . . . . . . . Histone Demethylation, Oxidation of DNA, BER, and Gene Expression . . . . . . . . . . . . . . . . Regulatory Roles of G Oxidation and BER Proteins in G4 Structure Formation in the Genome to Regulate Gene Expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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The DNA bases in the genome are susceptible to oxidation by reactive oxygen species (ROS). The oxidized DNA base lesions such as 8-oxoguanine (8-oxoG) and thymine glycol are primarily repaired by the base excision repair (BER) pathway. Increasing pieces of evidence suggest that oxidative stress-induced base damages in the gene promoter serve as epigenetics marks to regulate gene expression by recruitment of BER proteins. To shed light on this novel role of oxidative DNA base modifications and BER proteins, in this chapter, we focus on how controlled guanine oxidation in gene promoters and BER proteins 8-oxoguanine DNA glycosylase (OGG1) and AP-endonuclease 1 (APE1) regulate expression of multiple genes that drive cancer progression and metastases. S. Pramanik · S. Roychoudhury · K. K. Bhakat (*) Department of Genetics, Cell Biology and Anatomy, University of Nebraska Medical Center, Omaha, NE, USA e-mail: [email protected]; [email protected]; [email protected] © Springer Nature Singapore Pte Ltd. 2022 S. Chakraborti et al. (eds.), Handbook of Oxidative Stress in Cancer: Mechanistic Aspects, https://doi.org/10.1007/978-981-15-9411-3_156

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Further, we highlight the current studies suggesting a novel role of 8-oxoG and BER in regulating the formation of noncanonical DNA secondary structures such as G-quadruplexes (G4s) to regulate gene expression. Since high oxidative stress in tumor cells creates selective pressure on DNA damage repair pathways to maintain sustained growth, invasion, and metastases via modulating gene expression, such efforts to address the intertwined roles of DNA base modification, BER pathway, and gene expression could broaden cancer-therapeutic strategies. Keywords

Oxidized DNA base · 8-Oxoguanine · Base excision repair · OGG1 · APE1 · AP site · Histone demethylation · G-quadruplex structure · Gene expression

Introduction The genome is inherently unstable, and chemical modifications and damaging agents continuously challenge its integrity. Persistent exposure to exogenous and endogenous reactive oxygen species (ROS) generated by metabolic and inflammatory processes can cause DNA base oxidation, one of the most frequent insults to the genome (Winterbourn 2008). ROS comprise of a group of highly reactive molecules such as superoxide anion (O2˙
), and hydroxyl radical (OH•) – some potent oxidants which can directly react with DNA and produce oxidized bases (Schieber and Chandel 2014). Oxidation of purines and pyrimidines can generate multiple modified DNA bases (Bauer et al. 2015). ROS can also directly generate single-strand breaks (SSBs) with nonligatable termini. Deoxyribose in DNA is also prone to oxidation, which usually results in base hydrolysis to form an abasic site and/or SSB (Bauer et al. 2015). Among the four canonical nucleotides in DNA, G base has the lowest redox potential, resulting in G being the most susceptible to oxidative modification (Cadet et al. 2014; Fleming and Burrows 2017). Products of G oxidation by ROS include two-electron oxidation product 8-oxo-7-8-dihydroguanine (8-oxoG), which can pair with A during replication and leading to G to T transversion mutation (Grollman and Moriya 1993). Thus, oxidative damage to DNA may lead to mutations accumulation in the genome, and this eventually results in pathological consequences such as cancer (Cooke et al. 2003). The base excision repair (BER) pathway is the primary mechanism for repairing oxidative, alkylated base lesions, and SSBs in the genome (Lindahl and Barnes 2000; Mitra et al. 2002). BER is an evolutionarily conserved DNA damage repair pathway that is involved in maintaining genomic integrity by removing mutagenic lesions induced due to oxidative stress. BER relies on sequential recruitment and coordinated actions of multiple enzymes, which include DNA glycosylases (such as OGG1), AP-endonucleases (APE1), DNA polymerases, and DNA ligases (Scott et al. 2014). Several studies have demonstrated that in the absence of BER enzymes, cells accumulate mutations and are susceptible to various DNA-damaging agents. Thus, one can envision that BER plays a crucial role in cancer prevention.

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Overexpression of BER proteins confers protection of the cell genome from oxidative stress and toxicity to DNA-damaging drugs. Studies have shown elevated levels of APE1 in diverse cancer tissues and its association with chemo and radiation resistance (Abbotts and Madhusudan 2010; Sengupta et al. 2016). Given the unavoidable exposure to ROS, cells have evolved strategies to utilize ROS as a biological stimulus or signaling molecule to regulate multiple cellular processes, including cell signaling and transcription. An emerging view in transcriptional regulation is that base oxidation in DNA, which is conventionally thought to be mutagenic and detrimental for cells, has an essential regulatory role in controlling transcription and gene expression. Despite high mutagenic potential of 8-oxoG, GC content in the promoter regions of genes in the vertebrate genome is high (Deaton and Bird 2011). This evolutionarily conserved high GC content has been linked with gene expression (Ba and Boldogh 2018). Indeed, low-resolution mapping of 8-oxoG in mammalian cells by several laboratories found that 8-oxoG is enriched in gene promoters and within potential G-quadruplex-forming sequences (PQSs) (Amente et al. 2019; Fang and Zou 2020). The promoter region of many proto-oncogenes, including c-MYC, KRAS, and VEGF, contains PQS that can fold to noncanonical DNA G-quadruplex (G4) structures to regulate their expression (Fleming et al. 2017a; Pastukh et al. 2015). Over the years, the intertwined roles of the 8-oxoG, BER pathway, and transcriptional regulation have merged as a novel epigenetic mechanism for regulating gene expression. The initial cellular observation of controlled G oxidation in VEGF gene promoter upon hypoxia driving the production of Hif-1 protein was reported by Gillespie’s laboratory (Pastukh et al. 2015, 2007). Later, several other laboratories, including ours, have provided evidence that oxidation of bases in these G-rich sequences may serve as critical sensors through which the cells sense the ROS signals, and BER proteins OGG1 and APE1 regulate the transcription of oxidative stress-responsive genes (Fleming et al. 2017a; Pan et al. 2016; Roychoudhury et al. 2020). In this chapter, the roles of oxidative base 8-oxoG and BER enzymes – OGG1 and APE1 in transcriptional regulation – are discussed in detail with an emphasis on how they facilitate the assembly of the transcriptional activators at gene promoters to promote gene expression. Further, we shed light on our current understanding of how oxidative base modification and BER proteins regulate the formation of higher-order DNA secondary structures such as G4s to regulate expression of many oncogenes and promote tumorigenesis.

Oxidative DNA Base Damage Shaping the Mutation Burden in Cancer Cancer cells are characterized by persistent oxidative stress and high levels of ROS. Out of the several different DNA base derivatives that can arise due to ROS, 8-oxoG is one of the major oxidized bases in DNA that pairs with adenine (A) as well as cytosine (C) during DNA replication, potentially leading to G:C to T:A transversion mutations (Grollman and Moriya 1993) (Fig. 1). Left unrepaired, 8-oxoG can compromise transcription, DNA replication, and telomere maintenance, and being

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Fig. 1 A schematic representation of how unrepaired oxidative base damage (8-oxoG) in the DNA can lead to mutation in the genome

a highly mutagenic lesion, it has been associated with cellular transformation and cancer initiation. Elevated levels of 8-oxoG and other oxidative lesions have been found in the urine and tumor tissue DNA from patients with various malignancies (Guo et al. 2017). The levels of 8-oxoG are considered as a factor in risk assessment in cancer (Loft et al. 2012). Apart from 8-oxoG, oxidation of G can also result in the formation of 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyG), a major oxidative lesion (Cadet and Wagner 2013). 8-Oxo-7,8-dihydro-20 -deoxyadenosine (8-oxoA) and 4,6-diamino-5-formamidopyrimidine (FapyA) are the two major products that can arise from the oxidation of A. C can also be oxidized to form 5-hydroxy-20 -deoxycytidine (OH5C), spontaneously on the DNA, or can arise after exposure to ROS generating chemicals (Bjelland and Seeberg 2003; Cadet and Wagner 2013). T can be attacked by free radicals resulting in the generation of different oxidative products. Thymine glycol (Tg) can inhibit replicative polymerases and is highly mutagenic (McCulloch and Kunkel 2008). The 5-methyl group of T can be oxidized to form 5-hydroxymethyluracil (5hmU), which induces transition mutations (Maiti and Drohat 2011). All these mutagenic events pose a serious threat to the DNA and might lead to genetic instability and malignant transformation. Defective DNA maintenance pathways have been associated with distinct signatures that arise due to these mutations (Alexandrov and Stratton 2014). Though the entire course of malignancy is associated with different mutational processes, there can be specific mutational types that are dominant in a particular type of cancer. For instance, a high proportion of T to G and C to T base mutations was found in the immunoglobulin genes of chronic lymphocytic leukemia and melanoma patients (Alexandrov et al. 2013; Puente et al. 2015; Hodis et al. 2012).

Oxidative DNA Base Damage Repair Considering the high frequency at which DNA damaging events occur, and the broad spectrum of damages incurred by the cells, it is remarkable that a majority of these DNA lesions are repaired with impressive precision and efficiency by a host of DNA damage repair pathways.

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As mentioned before, the BER pathway is the primary mechanism that protects the genome from the deleterious effects of exposure to ROS. Being the most versatile among the excision repair pathways, BER repairs multiple types of modified (oxidized or alkylated) DNA bases as well as DNA single-strand breaks (SSBs) (Hegde et al. 2008; Lindahl and Barnes 2000; Robertson et al. 2009). BER is a highly coordinated multistep cellular process that includes (i) damaged base removal, (ii) 30 - or 5-end cleaning, (iii) DNA synthesis, and (iv) DNA nick-sealing (Scott et al. 2014). It is generally initiated by a lesion-specific DNA glycosylase, which removes the damaged base. Among 11 human DNA glycosylases, 8-oxoguanine DNA glycosylase 1 (OGG1), NTH1, endonuclease VII-like (NEIL) 1, NEIL2, and NEIL3 are primarily involved in reaping the oxidized base damages from DNA. Once the base lesions are removed, the product apurinic/apyrimidinic (AP) sites or SSBs with nonligatable termini are a substrate for the next enzyme, AP endonuclease 1 (APE1). APE1 cleaves AP site generating 30 -hydroxyl and 50 -deoxyribose-phosphate (50 -dRP) end which is recognized by DNA polymerase β (pol β). Pol β fills the single nucleotide gap by incorporating a complementary base, and finally, DNA ligase III (Lig III) seals the nick (Fig. 2). Of note is that each of these reactions consequently leaves a new intermediate lesion in the DNA, which is harmful if the entire repair process is not completed through nick-sealing by the DNA ligases (Wilson and Kunkel 2000). Thus, BER is dependent on sequential recruitment and coordinated activities of multiple proteins via the formation of a series of transient repair complexes that assemble at the site of the DNA lesion (Hegde et al. 2012). Apart from the core proteins mentioned above, poly (ADP-ribose) polymerase (PARP) and X-ray repair complementing defective repair in Chinese hamster cells 1 (XRCC1) do not directly participate in the DNA processing but form a scaffold and interact with BER proteins such as APE1 and Lig III which are critical for BER efficiency (El-Khamisy et al. 2003). Furthermore, several other non-BER proteins that are known to be involved in transcription or RNA metabolism have been demonstrated to regulate the activity of BER enzymes (Das et al. 2007). Such auxiliary proteins interact with BER proteins to enhance the overall DNA repair efficiency and coordinate repair and transcription.

The Interplay Between Posttranslational Modification of BER Proteins and Transcriptional Regulation Reversible acetylation of histones plays a profound role in epigenetic regulation of gene expression. Many studies have shown the role of acetylation of BER proteins in the regulation of the BER pathway and gene expression (Bhakat et al. 2009; Carter and Parsons 2016). We discovered that the major human glycosylases, OGG1, NEIL1, and NEIL2, which cleave most oxidatively damaged bases, are acetylated at specific lysine (K) residues in cells (Bhakat et al. 2004, 2006; Sengupta et al. 2018). Acetylation of OGG1 at K338 and K341 increased the activity of OGG1 (Bhakat et al. 2006). OGG1 acetylation was enhanced in cells after oxidative stress, suggesting a DNA damage-dependent activation of OGG1 acetylation due to its acetylation. We also showed that NEIL1 is acetylated at multiple K residues (K296,

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Fig. 2 A schematic representation of the steps involved in the BER pathway

K297, and K298) located in the intrinsically unstructured C-terminal domain, and acetylation of NEIL1 enhanced its glycosylase activity in vitro (Sengupta et al. 2018). We discovered that human APE1 is also acetylated at multiple K residues located in the unstructured N-terminal region (Bhakat et al. 2003; Lirussi et al. 2012). APE1 is acetylated in chromatin, and acetylation of APE1 enhances its AP-endonuclease activity in vitro and modulates its transcriptional coregulatory functions (Roychoudhury et al. 2017). Notably, while unmodified OGG1 and APE1 are present in cytosol and nucleus, acetylated OGG1 and APE1 are exclusively bound to chromatin (Bhakat et al. 2006; Roychoudhury et al. 2017). Using unbiased genome-wide mapping of AP sites, a common intermediate in the BER pathway, and binding of acetylated APE1 and acetylated OGG1, we have documented that endogenous oxidative base damages are not randomly located (Roychoudhury et al. 2020). There is a distinct distribution of oxidative damage and enrichment of acetylated OGG1 or acetylated APE1 predominately in the promoter/enhancer regions of the transcribed genes suggesting a connection of

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DNA base damage or assembly of repair complexes with the regulation of gene expression. Acetylated APE1-bound regions significantly overlap the regions that bear active enhancer and promoter histone marks (Roychoudhury et al. 2020). Studies have shown the interaction of APE1 with transcription activator or repressor factors such as STAT, HIF-1α, AP-1, NF-κB, and HDAC1 in multiple gene promoters (Bhakat et al. 2009; Evans et al. 2000; Xanthoudakis and Curran 1992). Acetylated APE1 was found to be present in the protein complexes that bind to negative calcium response element in gene promoter (Bhakat et al. 2003). Binding of Y-box-binding protein 1 (YB-1) to multidrug resistance gene MDR1 was shown to be dependent on APE1’s acetylation (Sengupta et al. 2011, Chattopadhyay et al. 2008). Further, we demonstrated elevated levels of AcAPE1 in diverse cancer types. The loss of APE1 acetylation resulted in altering the expression of hundreds of genes and enhanced tumor cells’ sensitivity to chemotherapeutic agents (Sengupta et al. 2016).

8-OxoG and OGG1 as a Novel Epigenetic Regulator for Controlling Gene Expression Both transcription and DNA repair have intimate transactions with the DNA, and these two processes are often coupled and are perhaps also interdependent and crossfunctional. A growing list of DNA damage repair proteins that were thought to function in repair pathways solely is increasingly becoming important in transcriptional regulation as well. 8-oxoG is the most prevalent oxidized DNA base found in the genome. Studies have shown that a controlled or localized oxidation of G in a specific region in the genome, such as promoter/enhancer, can occur in a signaldependent manner through localized histone demethylation by LSD1, a flavindependent monoamine oxidase that generates H2O2 (Shi et al. 2005). The locally formed H2O2 drives G oxidation in the discrete regions leading to the recruitment of OGG1 and activation BER pathway to promote induction of transcription (Perillo et al. 2008). Furthermore, supporting this, several laboratories found that 8-oxoG is enriched in gene promoters and within G-rich PQS or G4 sequences (see details below). Lastly, the genome-wide mapping of OGG1 (the primary enzyme responsible for removing 8-oxoG from DNA) further supports that oxidative base damage is not random; they are enriched in promoter regions in the mammalian genome (Ba and Boldogh 2018; Roychoudhury et al. 2020).

BER in Hypoxia-Induced VEGF mRNA Expression The first evidence for a direct connection between oxidized DNA bases, BER pathway, and gene regulation came from the studies by Gillespie’s group. A study by Pastukh et al. demonstrated the hypoxia-mediated formation of 8-oxoG in hypoxic response elements (HREs) in the VEGF gene promoter (Pastukh et al. 2007). Hypoxia-induced oxidative stress was shown to promote the assembly of

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hypoxia-inducible factor-1 (Hif-1α) and BER enzymes OGG1 and APE1. This recruitment of BER machinery was essential for the assembly of transcription complex on the VEGF HRE and for driving VEGF mRNA expression. Inhibition of the BER pathway resulted in hypoxia-induced accumulation of 8-oxoG and attenuated Hif-1α and APE1 binding to VEGF HRE sequences and blunted VEGF mRNA expression. Moreover, compared to wild-type HRE luciferase reporter, luciferase activity increased with AP-site containing HRE element in reporter plasmid (Pastukh et al. 2015, 2007). As described above, the AP site is a common intermediate generated after the removal of oxidized bases in the BER pathway. As the BER pathway repairs AP sites, these results suggested that oxidative base lesions in promoters of hypoxia-inducible genes are linked with transcriptional activation. More, recent studies have demonstrated that the signal-induced formation of localized oxidized bases in the promoter regions in many oncogene promoters such as KRAS, c-MYC, etc., modulates their expression (Fleming et al. 2017b); however, the molecular basis of G oxidation, AP sites formation, and modulation of their gene expression is different in each case (see below).

Role of 8-OxoG and OGG1 in the Regulation of Inflammatory Response Genes It is well-known that inflammation induces ROS production and that ROS-mediated signaling regulates transcription proinflammatory genes. Previous studies have shown ROS’s role in posttranslational modification of TFs such as NF- κB, and AP-1(Ba and Boldogh 2018). In a study by Ba et al., it was shown that TNF-α-induced ROS production results in elevating 8-oxoG levels in the genome, including the promoter regions (Ba and Boldogh 2018). Simultaneously, the binding of OGG1 also increased Cxcl2 expression by facilitating the loading of TFIID, NF- κB/RelA, Sp1, and p-RNA Pol II (Fig. 3). Depletion of OGG1 reduced transcription factor recruitment and the subsequent TNF-α-mediated innate immune response (Ba et al. 2014). A master regulator of gene expression, nuclear factor κB (NF- κB), is a transcription factor (TF) necessary for the inflammatory response, cell proliferation, and differentiation (Oeckinghaus and Ghosh 2009). Constitutive NF- κB activity due to the inflammatory microenvironment and the various oncogenic mutations is found in several human cancers. DNA sequences that contain three or more Gs (5’-GGGRNYYYCC-30 where R is a purine, Y is pyrimidine, and N is any nucleotide) are signatures where NF- κB classically binds. As mentioned before, oxidation of these Gs can lead to the formation of 8-oxoG lesions, which can either change the sequence context of the TF-binding site due to its mutagenicity or directly modulate DNA-protein interactions and thus gene expression. A study by Pan et al. demonstrated that NF- κB-driven gene expression requires 8-oxoG and OGG1 enrichment in the promoter sequences after TNF-α exposure to the cells. OGG1 bound to 8-oxoG facilitates NF- κB’s binding to the DNA (Pan et al. 2016). Hence, binding of OGG1 to its substrate was shown to function in epigenetic regulation of gene

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Fig. 3 Binding of OGG1 to 8-oxoG in promoter sequences bends the DNA and augments proinflammatory gene transcription by facilitating the recruitment of site-specific transcription factors

expression in cells that are under oxidative stress. Additionally, the Tell laboratory proposed that oxidative stress-induced 8-oxoG in the negative calcium responsive elements (nCaRE) in the promoter of sirtuin-1 (SIRT-1) gene recruits OGG1 (Antoniali et al. 2014). The Ras pathway is often activated in human cancers. Boldogh et al. demonstrated that 8-oxoG is bound by OGG1 at a nonsubstrate site with high affinity (Boldogh et al. 2012). The study was one of the first to document the DNA repairindependent function of OGG1 in modulating cellular signaling.

Histone Demethylation, Oxidation of DNA, BER, and Gene Expression Unlike histone acetylation, methylation of histone was considered a permanent and irreversible modification. In 2004, Shi et al. demonstrated that LSD1-mediated removal of the methyl group from both H3K4me2 and H3K4me1 in vitro via Flavin adenine dinucleotide oxidation reaction, generates H2O2 during demethylation

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(Shi et al. 2004). This demethylation resulted in local oxidation of DNA base around promoter or gene body regions and produced 8-oxoG, which is recognized by OGG1. OGG1 initiates the BER pathway, which can regulate transcription and gene expression (Fig. 4). The first direct evidence of histone demethylation and BER’s involvement in transcriptional regulation came from the study by Perillo et al. (Perillo et al. 2008). The authors showed that treatment with 17-beta-estradiol induced estrogen receptor recruitment to the estrogen-responsive elements in the promoter and enhancer of Bcl-2 and pS2 genes and formed a chromatin loop between the promoter and enhancer to activate the gene expression. Demethylation of H3K9me2 occurred at the promoter and enhancer sites of these genes, and 8-oxoG could be detected after treatment with estradiol. Importantly, siRNA-mediated knockdown of LSD1 abolished the accumulation of 8-oxoG lesions. Removal of 8-oxoG lesions by OGG1 followed by recruitment of APE1 generates an SSB facilitating chromatin looping, which provides a local signal for the assembly of transcriptional initiation complex to activate gene expression. OGG1 appeared to be required to remove 8-oxoG, and APE1 was necessary to complete the repair process. However, no additional downstream BER proteins were evaluated in this initial study. Downregulation of OGG1 and APE1 was used to confirm the functional role Fig. 4 LSD1-mediated histone demethylation in gene promoters gives rise to H2O2, which causes local oxidation of DNA base (8-oxoG), leading to the initiation of BER and assembly of transcription factors (TFs) to drive target gene expression

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of BER in transcriptional control, and it dramatically reduced the promoter-enhancer loop formation and gene expression. The same group also demonstrated that DNA oxidation drives Myc-mediated transcription (Amente et al. 2010b). Myc, a transcription factor that is activated in many cancers, contributes to a wide variety of gene regulation involved in cell proliferation, differentiation, and apoptosis (Amente et al. 2010b). They provided a mechanistic framework that links histone demethylation to oxidation of DNA and repairs underlying Myc-activated transcription. They demonstrated that local demethylation of H3K4me2 promotes the recruitment of the OGG1 and APE1 following Myc binding, which produces nicks on the Myc target gene. This subsequently facilitates chromatin loops that bring together RNA polymerase II and Myc. Silencing of OGG1 or APE1 inhibits Myc-mediated transcription (Amente et al. 2010a).

Regulatory Roles of G Oxidation and BER Proteins in G4 Structure Formation in the Genome to Regulate Gene Expression G-quadruplexes (G4s) are DNA secondary structures that can form when four Gs associate through Hoogsteen hydrogen bonds to form a planar tetrad. Stacking of two or more tetrads forms a G4 structure (Bochman et al. 2012; Burge et al. 2006). G4 DNA structures in key regulatory regions in the genome, such as promoters, replication origins, etc., regulate multiple biological processes, including transcription, replication, and telomere maintenance (Hansel-Hertsch et al. 2016, 2017). There is a growing consensus among Cynthia Burrows’ laboratory, ours suggesting that 8-oxoG and BER pathway plays a pivotal role in G4 structure formation in the genome to regulate gene expression (Fleming et al. 2017a, b; Roychoudhury et al. 2020). G4s are overrepresented in regulatory regions such as gene promoters, 50 and 30 untranslated regions, replication initiation sites, and telomeres (Fleming et al. 2019; Hansel-Hertsch et al. 2016). The promoter region of many proto-oncogenes, including c-MYC, KRAS, and VEGF, which are activated by oxidative stress, contains PQS that can fold to G4 to regulate their expression. Through biochemical, cellular, and genetic approaches, Burrows’ laboratory showed that 8-oxoG or AP sites in the PQS of VEGF and endonuclease III-like protein 1(NTHL1) promoter activate transcription of a reporter gene expression (Fleming et al. 2017a). OGG1 and APE1 are required for the induction of transcription. Further, it was found that oxidative modified PQS close to the transcription start site (TSS) can activate transcription when the damaged base and repair chemistry occur in the coding strand (Fleming et al. 2019). On the other hand, modification of PQS in the template strand turned off transcription. Thus formation of 8-oxoG under oxidative stress conditions functions as a signaling or epigenetic mark to promote G4 fold, thus leading to transcriptional activation. This also implies that G oxidation is an epigenetic modification, and G4-forming sequences serve as sensors for oxidative stress. Similarly, in a separate study, Redstone et al. showed that oxidative modification of PQS in the proliferating cell nuclear antigen (PCNA) gene promoter could

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turn on its transcription (Redstone et al. 2019). PCNA overexpression has been detected in malignancies ranging from colorectal cancer to breast cancer. The study showed the possible mechanism of PCNA gene activation during oxidative stress conditions. Briefly, the G-rich PQS promoter element of the PCNA gene can undergo oxidation to produce 8-oxoG. This event directs DNA repair via the BER pathway toward the promoter for initiating events that ultimately induce transcription of the gene. In an independent study by Cogoi et al., it was found that 8-oxoG formation in the PQS of the KRAS oncogene promoter positioned from 148 to 116 relative to the TSS, turned on the transcription (Cogoi et al. 2018). In this study, the authors demonstrated that 8-oxoG in the G4 region of KRAS promoter recruits OGG1. OGG1 removes 8-oxoG from the G4 motif in duplex, but when folded, it binds to the G4 in a nonproductive way. Recruitment of MAZ and hnRNP A1, the two nuclear factors that are essential for KRAS transcription to the KRAS promoter, is enhanced by 8-oxoG (Cogoi et al. 2018). Thus, this oxidative lesion in the G4 region was demonstrated to be a novel player in oncogene expression regulation, adding to other pieces of evidence of the epigenetic potential of oxidative base damage and its associated repair pathway in controlling gene expression. Although these studies mentioned above have established that an oxidized G or AP site in PQSs of many oncogene promoters such as KRAS, MYC, and VEGF modulates their expression, the requirement of G oxidation, AP sites, and APE1 in the spatiotemporal regulation of gene expression in cells was mostly unknown. We have recently demonstrated a mechanistic framework for the role of oxidized DNA-base-derived AP sites and APE1 in the formation and stability of G4 structures (Roychoudhury et al. 2020). We showed that endogenous oxidized DNA-base-derived AP sites in G4 sequences and subsequent recruitment of APE1 drive the spatiotemporal formation and stability of G4 structures to regulate KRAS and MYC oncogene expression. Mapping of the genome-wide APE1 binding and G4 structures revealed that the binding of APE1 is predominant in PQS sequences and is nonrandom. Furthermore, it was shown that APE1 supports the formation of G4 structures and G4-mediated gene expression. Binding of APE1 to G4 sequences promotes G4 folding, and the acetylation of APE1 stabilizes G4 structures by enhancing its residence time. APE1 subsequently facilitates transcription factor loading to the promoter, leading to G4-mediated gene expression. We postulated that cellular oxidants could oxidize G bases within a PQS into 8-oxoG, which is excised by OGG1 to generate an AP site. Unlike 8-oxoG paired with C, an AP site significantly impacts the thermal stability of duplex DNA (Fleming et al. 2017a). We proposed that the generation of an AP site in a PQS destabilizes the duplex and shifts the equilibrium to form an unstable G4. Subsequent binding of APE1 results in the stabilization of a G4 structure, and acetylation of APE1 by p300 enhances its affinity/interaction to stabilize G4 structure and facilitates the loading of TFs to activate gene expression (Fig. 5). This study highlighted a novel role of oxidized DNA bases and APE1 in controlling the formation of G4 structures to regulate transcription (Fleming et al. 2017a; Roychoudhury et al. 2020).

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Fig. 5 Schematic representation linking the role of endogenous oxidized DNA base (8-oxoG) in gene regulatory elements and APE1 in controlling the formation of G4 structures to modulate transcription of target genes

Thus, all these studies collectively demonstrated that oxidative base modifications such as 8-oxoG in the gene promoters that contain PQS might serve as epigenetic marks and serve as a common ground to coordinate the initial steps of DNA repair and the assembly of transcriptional machinery to launch adequate alteration of expression of the gene.

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Conclusion Over the years, studies from several laboratories have supported the view that oxidative DNA modifications such as 8-oxoG in the promoter/enhancer regions could serve as an epigenetic mark to regulate gene expression. Additionally, the BER pathway has been deemed necessary for modulating DNA G4 structures and histone demethylation-mediated epigenetic regulation, which are distinct from its well-established role in maintaining genome stability. Defects in BER may affect the formation of G4 structures and reduce promoter-enhancer formation affecting many oncogene gene expressions. Although several studies discussed above support the concept that action of BER in gene promoters guided by oxidative modification of G regulates transcription of several genes, some obvious questions warranting answers are (a) How a nonselective oxidant such as H2O2 generated during LSD1-mediated histone demethylation induces specific G oxidation in promoter/enhancer sequences to regulate gene expression? (b) Do oxidative base modifications other than 8-oxoG also function as an epigenetic regulator, and are they often required for transcription of oxidative stress-dependent genes? In the context of G4s, future studies are required to understand whether promoter PQSs serve as sensors of oxidative stress to modulate transcription of downstream genes through direct oxidation of DNA bases in the promoters under stressed conditions. Given the fact that tumor cells are highly transcriptionally active and have elevated levels of oxidative stress and BER enzymes, addressing these questions would help illuminate the complex interplay between oxidative DNA damage, BER, and transcriptional regulation in cancer.

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Epigenetic Instability Caused by Oxidative Stress Triggers Tumorigenesis Raman Preet Kaur, Prabhsimran Kaur, and Anjana Munshi

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Oxidative Stress-Induced Epigenetic Changes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Role of Oxidative Stress in Regional Hypermethylation in the Promoter Region of TSGs . . . Oxidative Stress-Induced Global Hypomethylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Therapeutic Strategies to Regulate Epigenetic Alterations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Polyphenols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . HDAC or DNMT Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Histone Deacetylase Inhibitors (HDi) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Nuclear Factor Erythroid 2-Related Factor 2 Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion and Future Directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cross-References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract

Cancer is a multifactorial disease that is caused by various modifiable as well as non-modifiable factors. Among non-modifiable factors, genetic and epigenetic factors have been reported to play a critical role in the progression of cancer. Epigenetics refers to the change in gene expression without any change in the genome. The major epigenome targets include modification of histones by either methylation or acetylation and methylation of DNA at CpG islands. Change in acetylation and methylation pattern leads to not only inhibition of tumor suppressor genes but also the activation of oncogenes. Oxidative stress is a significant phenomenon observed in human body as a result of various intracellular as well as extracellular factors. This stress interferes with the function of histone R. P. Kaur Department of Otolaryngology, School of Medicine, Johns Hopkins University, Baltimore, MD, USA P. Kaur · A. Munshi (*) Department of Human Genetics and Molecular Medicine, School of Health Sciences, Central University of Punjab, Bathinda, India © Springer Nature Singapore Pte Ltd. 2022 S. Chakraborti et al. (eds.), Handbook of Oxidative Stress in Cancer: Mechanistic Aspects, https://doi.org/10.1007/978-981-15-9411-3_184

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methyltransferases and histone deacetylases, resulting in modifications of the epigenome. Further, these oxidative stress-induced epigenetic modifications may occur at various regions, including the promoters of tumor suppressor genes. These result in silencing of genes, leading to increased cell proliferation. In addition, reactive oxygen species have also been found to be involved in regulating the various steps of tumor development, including transformation, survival, proliferation, invasion, metastasis, and angiogenesis. Therefore, oxidative stress can be used as a target for developing cancer-related treatment modalities that reduce oxidative stress levels and thereby help in the resumption of the normal activity of epigenetic enzymes preserving epigenetic integrity. Keywords

Epigenetic instability · Oxidative stress · Tumorigenesis · Regional hypermethylation · Global hypomethylation

Introduction Cancer is a complex disease that is variable in its growth, evolution, presentation, and consequences from one patient to the other. The similar kind of nonuniformity exists at cellular as well as molecular level even in one patient, leading to tumor heterogeneity within the tumor, resulting in variations in response to the treatment among patients. However, the only similarity in this disease from patient to patient is the ability of uncontrolled cell proliferation and failure of the normal cell kinetics regulators (Klaunig and Wang 2018). The concept of carcinogenesis came into light when it was found that the exposure to chemicals is associated with cancer in chimney sweepers and that painting coal tar on rabbit ears resulted in papillomas (Loeb and Harris 2008). Carcinogenesis, a multistep process, involves a profound metabolome and behavioral changes, followed by failure of cell cycle checkpoints in a normal cell that enable cells to proliferate rapidly. This is followed by the development of clusters of cells and tissues, which escape the surveillance by the immune system and ultimately invade distant tissues referred as distant metastases (Merlo et al. 2006). Mostly, the inactivation of apoptotic mechanisms and the uncontrolled cell cycle progression occur either due to deactivation of tumor suppressor genes or due to activation of oncogenes. Further cancer metastasis results from upregulation and downregulation of receptors, which have been found to affect cell motility and tissue-specific cellcell attachment. In addition, other factor contributing to metastatic cancer includes the activation of membrane metalloproteases. Cancer has been found to occur in any organ or tissue and in any part of the body. Although old age has been reported to be associated with cancer, it can still occur at any age since no age group is immune to it. Oxidative stress: Oxidative stress is a process that leads to an imbalance between the production of free radicals and reactive metabolites that are called as oxidants or

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reactive oxygen species (ROS). The elimination of oxidants is caused by protective mechanisms termed as antioxidant pathways. Low levels of ROS are useful, but excessive production of ROS can lead to malignancies. The dysregulation of oxidant and antioxidant levels causes damage to the important biomolecules and cells, causing a potential impact on the whole organism (Reuter et al. 2010). ROS are also produced in response to exogenous sources, including pollutants, tobacco, smoke, drugs, and xenobiotic (Prasad et al. 2017; Niu et al. 2015). Oxidative stress and ROS play an important role in the development of cancer. Exogenous as well as endogenous sources of ROS increase the oxidative. ROS are required to drive regulatory pathways. But excess of ROS can result in damage or modification of the genome, resulting in mutations (Klaunig 2018). Oxidative damage due to oxidative stress promotes the development as well as progression of cancer. A substantial body of evidence suggests that ROS not only promotes but also suppresses the survival of cancer cells (Aggarwal et al. 2019). ROS tunes almost every step of tumor development. It regulates not only metastasis and angiogenesis but also the initial steps, including transformation, survival, proliferation, and invasion. In addition, chronic inflammation, which is reported to mediate cancer majorly, is also regulated by ROS. ROS modulate the various signaling molecules required for cell cycle progression as well as expression of various tumor suppressor genes. Besides, a high level of ROS can suppress cancer proliferation efficiently via cell cycle inhibitor activation. High levels of ROS and oxidative stress not only impair various cell signaling pathways but also lead to the accumulation of damaged biomolecules. In addition, the repair mechanisms are also affected by increasing oxidative stress. Increased damaged biomolecules affect the response of normal cells to oxidants. Oxidative stress has been found to regulate the gene expression of downstream targets, which play a role in cell proliferation, antioxidants, and DNA repair. The altered response might be tumor suppressing, leading to apoptosis or potentially oncogenic. Oncogenic response includes damage accumulation, conserving the potential not only for inflammation but also for proliferation. This set of occurrences results in tumor-initiating cells, which further leads to preneoplastic focal lesions and in the end neoplasia (Sesti et al. 2012). For the same reason, increasing ROS stress is the underlying mechanism used in the currently available chemotherapeutic and radiotherapeutic agents to kill cancer cells. Therefore, emerging cancer therapies include ROS-elevating as well as ROS-eliminating strategies (Prasad et al. 2016). Oxidative stress has been found to induce epigenetic changes that result in tumorigenesis. Besides oxidative stress, different mechanisms by which the genetic and cellular changes lead to carcinogenesis include mutation, chromosomal translocation or deletion, and dysregulated expression or activity of signaling pathways. The genetic and epigenetic alterations lead to the inactivation of the tumor suppressor genes and activation of proto-oncogenes, further promoting dysregulated cell cycling, inactivating apoptotic pathways, and enabling rapid cell proliferation (Vogelstein and Kinzler 2004). In addition to these factors, environmental pollutants, including chemicals, mutagenic agents, and industrial effluents, also contribute to cancer. Other factors such as some therapeutic drugs and ionizing radiation also possess

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the potential to increase the incidence of cancer (Devi 2004). The factors that have been found to contribute to tumorigenesis include family history, radiation, lifestyle, and genetic and epigenetic factor. Cancer genetics has discovered many specific loci playing important roles in cancer progression. Many of these loci are involved in cell cycle control, DNA repair, and cell-death pathways (Frank 2004). Evidences have suggested the key role of several genes with high-penetrance mutations in hereditary cancer, including colorectal cancer (De la Chapelle 2004). According to Benafif and Eeles (2016), rare to moderate to high-risk gene loci, identified by linkage and genetic sequencing studies, predispose to prostate cancer development when altered by mutation (Benafif and Eeles 2016). Epigenetics: Epigenetic modifications have emerged as significant risk factors in the development of carcinogenesis. The most fascinating mechanism of epigenetics is DNA methylation that is a highly governed mechanism, occurring at the cytosine residues of the CpG dinucleotide. CpG islands are present in the promoter regions as well as non-promoter regions. Within the genome, various expression studies have revealed that in normal cells the CpG islands in the promoters of transcriptionally active genes, including TSGs (tumor suppressor genes), remain unmethylated. On the other hand, the CpG islands in non-promoter regions such as repetitive sequences exhibit dense methylation (Felsenfeld 2014). Also known as anti-oncogenes, TSGs function mainly by suppressing the cancer growth. As the name suggests, they must be inactivated or lost for the development of the tumorigenesis. The deletion or mutational gene inactivation of these genes leads to loss of function of the proteins encoded by these genes. This is known to affect cell growth, resulting in malignant transformation of normal cell (Coran et al. 2012). Various cellular activities, including DNA damage repair, cell cycle arrest, and mitogenic signaling, have been found to be modulated by the TSGs such as p53 (tumor protein p53), RB1 (retinoblastoma-associated gene 1), CDKN2A (cyclin dependent kinase inhibitor 2A), and PTEN (phosphatase and tensin homolog). In addition TSGs also regulate cell differentiation, migration, as well as programmed cell death. Besides, TSGs have also been reported to synchronize metabolism; the epigenetic landscape, immune surveillance, and others have also emerged recently. Therefore, TSGs play a crucial role in maintaining not only the progression but also the cellular homeostasis to suppress cancer initiation (Acosta et al. 2018). A number of evidences suggest the antioxidant role of TSGs since they have been found to modulate various antioxidant genes in response to oxidative stress (Vurusaner et al. 2012).

Oxidative Stress-Induced Epigenetic Changes Recent studies suggest a dialogue between ROS and various epigenetic events, including histone modifications, DNA methylation, and miRNAs, not only in normal physiology but also in human pathologies including cancer. Studies have established an association between oxidative stress and epigenetics, resulting in various human malignancies. ROS has been reported to regulate major epigenetic mechanisms such as DNA methylation and histone acetylation. In cancer cells, it has

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been observed that ROS are associated with enhanced DNA methylation in the promoter region of TSGs that leads to silencing of these genes, resulting in increased proliferation of cancer cells under oxidative stress (Ziech et al. 2011; Prasad et al. 2017). ROS-induced oxidative stress has been reported to be related with global hypomethylation and aberrant hypermethylation of TSG promoter regions. 8-hydroxy-20 -deoxyguanosine (8-OHdG), the product of DNA oxidation, has been found to suppress DNA methylation at cytosine bases, resulting in induction of DNA hypomethylation. On the other hand, another product of DNA oxidation, 5-hydroxymethylcytosine (5hmC), may also undergo DNA hypomethylation on account of DNA demethylation processes. Besides, ROS has also been found to play a role as catalyst of DNA methylation, which is responsible for TSG promoter hypermethylation. In addition, ROS might lead to the formation of a new DNMTcontaining complex or upregulation of expression of DNA methyltransferases (DNMTs). This also contributes to the induction of site-specific hypermethylation. Further, DNA methylation pattern alterations induced by ROS have been found to be implicated with malignant transformation as well as the progression of numerous tumors (Wu and Ni 2015). The mechanism of DNA methylation is found to be catalyzed by cytosine-5DNMT, which is involved in the attack on cytosine C5 by the enzyme nucleophile with the subsequent formation of DNA-SAM complex, a transient covalent complex. Nucleophilic insertion of methyl group at the C5 carbon atom of cytosine molecule takes place only after the attachment of negatively charged cysteine residue to C6 carbon in order to supply nucleophilic properties to the C5 position (Vilkaitis et al. 2001). The attachment of cysteine residue to the C6 carbon atom makes possible a nucleophilic attack by positively charged S-adenosyl-L-methionine because it converts cytosine molecule into a negatively charged one. Therefore, the formation of the DNA-SAM complex containing cysteine residue is the first step of DMNT-catalyzed DNA methylation. Another mechanism involves the superoxide, which deprotonates the C5 position of cytosine molecules and accelerates the reaction of DNA with positively charged intermediate, S-adenosyl-L-methionine. In addition, histones also deprotonate lysine residues of histones at the N-terminal domain. This process is similar to DNA methylation and proceeds by a nucleophilic mechanism and consists of the stages of protonation, formation of the (lysine NH3+)-AcCoA-HAT complex, and its dissociation to lysine-NHAc (Jiang et al. 2012; Afanas’ev 2014). Higher levels of ROS produced during mitochondrial respiration also damage mitochondrial DNA. Further, these alterations downregulate the activity of methyltransferases and modulate the epigenetic mechanisms of nuclear DNA (Szumiel 2015).

Role of Oxidative Stress in Regional Hypermethylation in the Promoter Region of TSGs DNA methylation under the oxidative stress in the promoter region of TSGs contributes significantly to tumorigenesis (Fig. 1). Caudal type homeobox-1 (CDX1) is the intestine-specific transcription factor. CDX1 has been reported to

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Fig. 1 Oxidative stress-induced regional hypermethylation. ROS leads to promoter methylation of RUNX 2 and CDX1. ROS also upregulates Snail and recruits oxidative stress-induced upregulated DNMT1 and HDAC1, resulting in methylation at E-cadherin promoter. On the other hand, ROS also recruits polycomb repressive complex, consisting of DNMT1, HDAC, and HMT. Further, ROS leads to inactivation of TSGs as well as their abnormal promoter methylation under the effect of ROS-induced inflammatory cytokines

regulate intestinal epithelial proliferation as well as differentiation. It is decreased in colon cancer-derived cell lines as well as in colorectal cancers. It has been observed that, in colorectal cancer cells, oxidative stress alters the expression of CDX1. Treatment of cells with hydrogen peroxide leads to increased promoter methylation in the CDX1 gene. Besides, oxidative stress also leads to increased expression and activity of DNMTs 1 and HDAC 1 (Zhang et al. 2013). Runt domain transcription factor 3 (RUNX3) is a tumor suppressor that is found to be unexpressed in cancer by way of hypermethylation of its promoter. ROS also silenced the RUNX3 gene by inducing epigenetic alterations as increased promoter methylation of RUNX3 gene has been observed after treatment with hydrogen peroxide. This effect abolishes by treatment with N-acetylcysteine, a ROS scavenger. These findings are suggestive of the fact that ROS silences the TSG RUNX3 expression by an epigenetic mechanism that may further result in the progression of colorectal cancer (Kang et al. 2012). In hepatocellular carcinoma, oxidative stress enhances hypermethylation of the promoter region of E-cadherin protein by stimulating expression of Snail. Snail expression is upregulated by ROS by activating PI3K/Akt/GSK3 pathway and/or other pathways. Snail binds to E boxes of the E-cadherin promoter, repressing transcription. Further, Snail recruits HDAC and DNMT1 for increased methylation at the E-cadherin promoter region (Lim et al. 2008).

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Another study has reported that oxidative stress leads to the enrolment of the polycomb repressive complex that includes DNMT1 and HDAC and histone methyltransferases to the site of damaged chromatin. This phenomenon was observed in the colon cancer cell lines that were treated with H2O2 to induce chromatin damage that leads to delocalization of the polycomb complex from a non-CpG-rich region to CpG island-rich promoter regions carrying 8-oxo-20 -deoxyguanosine (8-oxide) (O’Hagan et al. 2011). The authors of this study suggested that H2O2-induced oxidative stress hires histone modulators to the promoter regions of active genes, where DNA oxidation is observed. This leads to the inactivation of TSGs, such as MBD4 (methyl-CpG-binding domain 4), TDG (thymine-DNA glycosylase), OGG1 (8-oxoguanine DNA glycosylase), MGMT (O-6-methylguanine-DNA methyltransferase), XPC (XPC complex submit), RAD23A (RAD53 homolog A), ERCC1 (ERCC excision repair 1), MLH1 (MutL homolog 1), MSH2 (MutS homolog 2), MSH3 (MutS homolog 3), MSH6 (MutS homolog 6), etc., through an epigenetic mechanism. In addition to the expression of Snail and polycomb complex, inflammatory cytokines also induce the transcription of DNMTs and HDACs. These events have been found to develop an abnormal methylation pattern of the TSG promoters. On the other hand, activities of DNA repair pathways and activation-induced cytidine deaminase (AID) affect 5-methylcytosine that results in a yield of thymine, which is replaced by an unmethylated cytosine through the process of base excision repair. Hence, affecting the epigenome. Association between inflammation and oxidative stress has been documented in various studies. There is a considerable amount of epidemiological data that supports a cross talk between these two processes. ROS induces activation of transcription factors and pro-inflammatory genes, leading to the onset of inflammation. The release of inflammatory markers in turn leads to alteration of epigenetic status. Increased DNA methylation in the gastric mucosa has been observed in response to enhanced inflammation in H. pylori-related gastric cancer in comparison to the patients that were not infected with H. pylori (Maekita et al. 2006; Chan et al. 2003). Similar patterns in DNA methylation were also observed in colonic mucosa in a model of colon cancer, where inflammation was induced by dextran sulfate sodium (Rosenberg et al. 2009). As per the results of various in vitro studies, it has been documented that various cytokines as well as ROS/RNS contribute to the modulation of the activity of the epigenetic proteins. It has been observed that cell line treatment using nitric oxide/cytokines (IL-1β) leads to various epigenetic changes, such as increased methylation and increased DNMT activity. Not only this, decreased expression of several CpG island-containing genes was also found to result from the same. Another study revealed S-nitrosylation of HDAC2, leading to histone modifications and alterations in gene transcription under the effect of RNS nitric oxide in neuronal cells (Nott et al. 2008). Enhanced secretion of IL-6 increased promoter activity of DNMTs, resulting in increased transcription of the gene. Further, IL-6 negatively impacted microRNA that targets DNMT1 (Chiba et al. 2012; Hmadcha et al. 1999). Therefore, inflammation resulting from oxidative stress could result in epigenetic alterations by abnormal methylation in TSG promoters by

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way of inflammation-mediated signaling pathways, leading to the development of tumorigenesis.

Oxidative Stress-Induced Global Hypomethylation DNA hypomethylation is a ubiquitous feature of various cancers. It was the first epigenetic abnormality observed in human tumors. However, most of the times, it was ignored as an unwelcome complication, and all the focus was on the hypermethylation of TSGs because it was believed that global hypomethylation in cancer is observed in repetitive DNA elements only. Therefore, cancer-linked DNA hypomethylation received little attention. The high-throughput genome-wide studies confirmed the importance of genome-wide hypomethylation in cancer (Ehrlich 2009). DNA demethylation during the process of tumorigenesis might include the formation of hemi-methylation dyads, followed by loss of methylation on both the DNA strands. It was observed that metastasis was more linked to hypomethylation than primary tumors (Bedford and Van Helden 1987). DNA hypomethylation may contribute to tumorigenesis by various mechanisms (Fig. 2). Firstly, the chromosomal instability induced by the demethylation of repetitive elements or satellite DNA might lead to the development of tumorigenesis (Wong et al. 2006; Vera et al. 2008). Secondly, it might also result in allowing transcriptional activation of transposition induced due to hypomethylation of mobile repetitive elements (Howard et al. 2008). Thirdly, and most importantly, hypomethylation in the promoter region of oncogenes could induce expression of these genes, leading to carcinogenesis (Wainfan et al. 1992; Stefanska et al. 2011). Hypoxia, an important hallmark of cancer, is the result of excessive cellular proliferation. It has been observed that hypoxia causes oxidative stress due to increased production of ROS during hypoxic conditions (Goudar and Vlahovic 2008; Lawless et al. 2009; Ruan et al. 2009). Generation of ROS is found to be positively correlated with upregulation of VEGFA, DNA oxidation, and hypoxiainducible factor-1α (HIF-1α) (Pialoux et al. 2009). Epigenetic modulators such as lysine demethylases are regulated by HIF-1α. It regulates the gene expression by functionally associating with HDACs under hypoxic conditions (Carrero et al. 2000; Ruas et al. 2002; Xenaki et al. 2008). Increased production of ROS interferes with the binding of a methyl-CpGbinding protein (MBP) 2, a critical epigenetic regulator of DNA. MBP-2 is responsible for recruiting DNMTs and histone HDAC to DNA to regulate gene expression (Valinluck et al. 2004). The presence of 8-oxodG in CpG islands is reported to be associated with the hypomethylation of the CpG site. The ROS converts guanine into 8-oxodG that transforms N7 position of guanine into hydrogen donor, instead of being hydrogen bond acceptor under normal conditions. Under normal conditions, N7 of guanine has been found to accept hydrogen bond in the course of formation of the MBP-DNA complex. The substitution of guanine by 8-oxodG abolishes binding of MBP, when 8-oxodG is adjacent to the 5-methylcytosine. This might result in abnormal gene expression (Turk et al. 1995). In addition, hydroxymethylcytosine,

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Fig. 2 Oxidative stress-induced global hypomethylation. ROS upregulates VEGFA and HIF-α, which associate with HDACs under hypoxic conditions. ROS also modulates MBP2, which recruits DNMTs and HDACs. Further, ROS converts guanine into 8-oxodG, which hinders the MBP-DNA complex. 5-methylcytosine also hinders MBP binding to DNA. On the other hand, superoxide dismutase 1 leads to increased 8-oxodG levels, contributing to DNA hypomethylation

the oxidation product of 5-methylcytosine, also reduces the MBP binding, which leads to DNA hypomethylation (Donkena et al. 2010). Alterations in the superoxide dismutase 1 gene has been reported to elevate 8-oxodG levels in the mice liver, with the progression of global DNA hypomethylation (Bhusari et al. 2010). The presence of 8-oxodG in CpG islands obstructs the methylation of the adjacent cytosine, leading to hypomethylation. It has been observed in hepatocellular carcinoma that the enhanced methyl group requirement reduces the availability of methyl group donors such as SAM. On the other hand, elevated levels of the methylation inhibitor S-adenosylhomocysteine (SAH) result in the global hypomethylation. The oxidative stress and hypomethylation of LINE-1 sequences have been implicated in the pathogenesis of bladder cancer patients (Patchsung et al. 2012). Another phenomenon that leads to the emergence of global hypomethylation is reduced levels of SAM (S-adenosylmethionine) that is necessary for maintaining DNA methylation. SAM is consumed in the synthesis of homocysteine that is required in the replenishing GSH (glutathione) levels during the phase of oxidative stress as there is increased depletion of GSH redox buffer to balance the ROS (Hitchler and Domann 2007).

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Therapeutic Strategies to Regulate Epigenetic Alterations Epigenetic therapy rephrases the abnormal epigenetic codes and modulates the epigenetic machinery by inducing the alterations in cancer phenotypes. Since resistance to conventional chemotherapy as well as to other molecular targeting agents is common in cancer cells, the epigenetic therapy is different from the conventional cancer therapies, peculiar as an anticancer treatment (Snykers et al. 2009). It can also be an effective treatment when used in combination with other chemotherapeutic agents. Also, it has proved to be effective even after administration of the active agent that has been discontinued. Many treatment therapies, which actively target oxidative stress through inhibition of epigenetic enzymes including HDACs, have been proposed. Further, cancer chemoprevention through diet is another field that explores the incorporation of agents targeting HDACs into dietary requirements, which may prove to reduce tumor burden (Lawless et al. 2010). Various treatment strategies that can target oxidative stress and epigenetic changes (Table 1) and result in the improved prognosis of cancer patients have been discussed in the following section.

Polyphenols Natural polyphenols have been identified to possess a promising potential for cancer treatment as well as prevention apart from standard anticancer treatments (Link et al. 2010; Fantini et al. 2015). Besides preventing the oxidative damage caused by oxidative stress, polyphenols have been reported to exert their biological action by way of chromatin remodeling in addition to other epigenetic modifications (Rahman and Chung 2010). Many polyphenols possess the ability to modulate HDAC activity associated with anti-inflammatory properties of polyphenols (Biswas and Rahman 2008). In addition, evidence suggests that polyphenols can also modulate HAT. Oxidative stress leads to the activation of intrinsic HAT activity, which further induces NF-κB pathway that further leads to the expression of pro-inflammatory mediators and can also inhibit HDAC function. For instance, curcumin regulates both acetylation and deacetylation by modulating oxidative stress (Rahman et al. 2004). One of the known modes of action of curcumin involves inhibition of NF-κBmediated responses. This is mediated either by inhibiting the translocation of NF-κB, or might occur on account of suppression of the degradation of IkBa, or by preventing its phosphorylation. Also, it decreases the activity of KATs (lysine acetyltransferases). The inhibition of KAT activity further affects the NF-κB activation as well as translocation and NF-κB-mediated chromatin remodeling, particularly in light of the important roles of KATs/HDACs in regulating NF-κB. Curcumin-induced NF-κB inhibition is associated with upregulation of regulatory genes, which are reported to control pro-inflammatory genes, such as mitogen-activated protein kinase phosphatase-5 (MKP5), as well as reduce pro-inflammatory cytokines, including CXCL1 and CXCL2. Besides, curcumin has also been found to be effective in improving the

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Table 1 Treatment strategies that can target oxidative stress and epigenetic changes Mode of action Regulation of the NF-κB pathway

S. no. 1.

Therapies Polyphenols

Targets HDACs and HATs

2.

HDAC or DNMT inhibitors

HDACs and DNMTs

Reverse the unusual epigenetic changes

3.

Histone deacetylase inhibitors (HDi)

Histone deacetylases

4.

Nuclear factor erythroid 2-related factor 2 inhibitors

Nrf2

Induce apoptosis of cancer cells via generation of oxidative stress Inhibition of Nrf2 pathways

Drugs used Artichoke polyphenols, chlorogenic acid, curcumin, daidzein, epigallocatechin-3gallate, genistein, ginsenoside Rg3, lycopene, phenethyl isothiocyanate, pterostilbene, resveratrol, sulforaphane, and quercetin 5-azacytidine and 5-aza-20 -deoxycytidine

Zolinza (suberoylanilide hydroxamic acid)

Brusatol

References (Mileo and Miccadei 2016)

(Lawless et al. 2009; Nishida and Kudo 2014) (Lawless et al. 2009)

(Kang and Hyun 2017)

oxidative stress conditions associated with a mouse model of steatohepatitis through inhibition of NF-κB activation (Leclercq et al. 2004; Mileo and Miccadei 2016).

HDAC or DNMT Inhibitors There are many pathways involved in the cellular response to oxidative stress, which have been shown to either be linked with or regulated via HDACs. The KEAP1NRF2-ARE pathway involves DNA methylation and lysine acetylation and is reported to act as oxidative stress sensor. HIF-1a pathway regulates responses to oxidative stress by histone methylation, histone acetylation, lysine acetyltransferases, and HDAC. PGC-1a pathway incorporates HDAC and histone acetyltransferases and regulates antioxidant genes along with protection of cells

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from oxidative stress. On the other hand, glucocorticoids regulate expression of protective factors in oxidative stress by recruiting HDAC and lysine acetyltransferases (Lawless et al. 2010). A range of TSGs show unusual DNA methylation in cancer including HCC (Hepatocellular Carcinoma). In addition to DNA methylation, other epigenetic changes could be reversed by HDAC or DNMT inhibitors. 5-azacytidine and 5-aza-20 -deoxycytidine (decitabine) are examples of DNMT inhibitors that induce the expression of TSGs by reversing the protein methylation. These antitumor drugs are used for the treatment of myelodysplastic syndrome and acute myelocytic leukemia (Kantarjian et al. 2006; Silverman et al. 2002). Since overexpression of HDACs and transcriptional inactivation of growthinhibitory as well as apoptosis-related genes are associated with several kinds of cancers, several clinical trials involving HDAC inhibitors, such as the use of vorinostat in the treatment of cutaneous T cell lymphoma, are currently ongoing (Dokmanovic et al. 2007). HDAC has been reported to be overexpressed in HCC (Wu et al. 2010). A study suggested the effectiveness of the HDAC inhibitor panobinostat in a mouse xenograft model of HCC in combination with sorafenib (Lachenmayer et al. 2012). Thus, modulation of the epigenetic machinery should prove a promising approach for treating this type of malignancy (Nishida and Kudo 2014).

Histone Deacetylase Inhibitors (HDi) HDi are reported to be the first-generation inhibitors and have shown welltolerated safety profiles. However, issues about bioavailability and clearance exist. A recent study found that there was a high clearance of a synthetic HDi in the lung due to oxidation (Fonsi et al. 2009). They instigate apoptosis in cancer cells by giving rise to oxidative stress. A lot of evidence has suggested that they might prove a promising candidate to target oxidative stress pathways but may reactivate Brahma (BRM) gene, inhibiting its function, and this functional activity can only be restored after the removal of HDi (Lawless et al. 2009). Therefore, keeping in focus the cellular function of reactivated genes, staggered therapeutic interventions with HDi can be designed. Therefore, following these interventions, the problem of targeting oxidative stress-induced diseases including cancer can be addressed (Lawless et al. 2010).

Nuclear Factor Erythroid 2-Related Factor 2 Inhibitors Nuclear factor E2-related factor 2 (Nrf2) is a transcription factor that plays a significant role in controlling the expression of genes encoding cytoprotective proteins, including antioxidant enzymes that combat oxidative and electrophilic stress to maintain redox homeostasis. However, the Nrf2-ARE pathway is found upregulated in cancer cells via epigenetic mutations to take advantage of the protective power of the Nrf2 pathway for growth and survival. Since KEAP1-

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NRF2-ARE pathway is an important antioxidative stress pathway, this has been found to be linking direct epigenetic regulatory mechanisms to the regulation of this cellular response pathway to oxidative stress. NRF2 has been shown to be directly acetylated by KAT3A/KAT3B in response to cellular stress. Besides, NRF2 promotes the direct chromatin remodeling of its cognate target genes. In addition, the induction of genes is mediated selectively by the SWI/SNF complex component BRG1 interacting with NRF2 under oxidative stress (Lawless et al. 2010). These characteristics can be exploited for anticancer therapy against certain tumors. High-throughput screening compounds inhibiting Nrf2 can prove useful for the designing of Nrf2 inhibitors since few specific inhibitors of Nrf2 are currently available such as brusatol. A study by Wang et al. (2008) suggests that hypermethylation of CpG islands in KEAP1 promoter suppressed KEAP1 expression in human lung adenoma cell lines and tumor tissues. This suggests that epigenetic silencing of KEAP1 can subsequently activate Nrf2 (Ma and He 2012). This established the new insights into pulmonary carcinogenesis as well as chemoprevention, thereby providing a potential target for the treatment of human lung cancer and a powerful tool for early diagnosis (Wang et al. 2008). Also, high Nrf2 activity is reported to contribute to cancer cell resistance to various therapies. Since Nrf2 is exposed to epigenetic regulation by DNA demethylation and histone methylation, the epigenetic modification in Nrf2 expression will provide new candidate therapeutic targets for anticancer drug resistance (Kang and Hyun 2017).

Conclusion and Future Directions Not only genetics but epigenetics also plays a crucial role in the development of carcinogenesis as well as response to various therapeutic strategies used for cancer patients. In combination with other factors, oxidative stress plays a pivotal role in the generation as well as progression of this deadly disease since it has the potential to hit the integrity of the epigenome. It has been found to alter the methylation status of TSGs and oncogenes. These alterations result in loss of function of TSGs and gain of function in oncogenes, resulting in tumorigenesis. Since epigenetic modifications, including DNA methylation, and elevated levels of ROS are two common characteristics of cancer cells, novel drugs aiming to reduce the oxidative stress in patients can be developed to increase the life span of the patients. Furthermore, development of treatment modalities that can reverse the methylation pattern, resulting in activation of TSGs and inactivation of oncogenes, will be of great use to halt the deadly occurrence, recurrence, and distant metastasis of cancer.

Cross-References ▶ ROS Induced by Chemo- and Targeted Therapy Promote Apoptosis in Cancer Cells ▶ ROS-Mediated Apoptosis in Cancer

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Implications of ROS in Cancer Stem Cells Mechanism of Action

Cornelia Amalinei, Raluca Anca Balan, Adriana Grigoras, Ludmila Lozneanu, Elena Roxana Avadanei, Simona Eliza Giusca, and Irina Draga Caruntu

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CSCs Inside Stem Cells Spectrum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CSCs Heterogeneity and Metabolic Landscape . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Plasticity, Quiescence, and Embryonic Signature of CSCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Tumor Microenvironment: CSC Relationship . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Signaling Pathways Involved in CSCs Maintenance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ROS, Carcinogenesis Theories, and CSCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Oxidative Stress in Cancer Initiation and Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Direct Damage of Structural Components and Metabolism with Induction of Gene Mutations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Modulation of Signal Transduction Pathways or Gene Transcription Factors with Alterations of Oncogene Expression and Tumor Progression . . . . . . . . . . . . . . . . . . . . . . . . The Association Between Oxidative Stress and CSCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Single Nucleotide Polymorphisms Results in Cancer Risk Increase . . . . . . . . . . . . . . . . . . . . . . Pathologic Conditions Related to Oxidative Stress and Carcinogenesis Promoters . . . . . . . . . . ROS and Tumor Status . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CSCs and Metastasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract

Cancer stem cells (CSCs) are self-renewal cells involved in tumor progression and recurrence. Cell transformation and reprogramming in CSCs are the results of complex processes, including metabolic changes from glycolytic to oxidative phosphorylation or vice versa. CSCs have a large spectrum, displaying a quiescent status and an embryonic signature. Their activity is C. Amalinei (*) · R. A. Balan · A. Grigoras · L. Lozneanu · E. R. Avadanei · S. E. Giusca · I. D. Caruntu Department of Morphofunctional Sciences I, “Grigore T. Popa” University of Medicine and Pharmacy, Iasi, Romania e-mail: cornelia.amalinei@umfiasi.ro © Springer Nature Singapore Pte Ltd. 2022 S. Chakraborti et al. (eds.), Handbook of Oxidative Stress in Cancer: Mechanistic Aspects, https://doi.org/10.1007/978-981-15-9411-3_113

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influenced by the tumor microenvironment and they are using different signaling pathways for maintenance. Reactive oxygen species (ROS) induce mutations, being involved in the modulation of signal transduction pathways or gene transcription factors which lead to alterations of oncogene expression and tumor progression. CSCs gain an increased resistance in case of increased ROS level, manifested by a reduced oxidative injury and a longer survival. Epigenetic changes induced by chronic inflammation, excessive redox stress, and hypoxic environment stimulate the plasticity of the transition between CSCs and non-CSCs. Oxidative stress may influence tumor development and progression, as recent evidences show a persistent prooxidant state in tumor cells. As a consequence, multiple sequences of tumor progression, such as alteration of cell adhesion, stimulation of tumor angiogenesis, increased matrix metalloproteinases (MMPs) activity, and epithelial-mesenchymal transition (EMT), are associated with the oxidative stress and ROS. The exploitation of ROS dual abilities in the occurrence, development, and evolution of CSCs may be useful for the development of new therapeutic strategies targeting redox regulatory mechanisms. The adaptive resistance of CSCs emphasizes the importance of simultaneous blockage of multiple signaling pathways as a possible therapeutic approach which may overcome CSCs resistance, opening encouraging perspectives. Keywords

Cancer stem cells (CSCs) · Reactive oxygen species (ROS) · Carcinogenesis · Tumor microenvironment · Signal transduction pathways

Introduction Mentioned in literature over 150 years ago, cancer stem cells (CSCs) are now regarded as “tumor-initiating” cells in malignant proliferation, as a self-renewal population, which differentiates into heterogeneous lineages, according to the American Association for Cancer Research (Rich 2016). Taking into consideration their role in tumor progression and recurrence, the main focus of research has been the identification of CSCs characteristic phenotypic markers (Toh et al. 2017), due to mutations, abnormal multiplications, high degree of biological autonomy, loss of normal division control associated to alterations of genes involved in regulation of cell growth, and intercellular interactions (Frank 2007; Sonnenschein and Soto 2018). During the last years, several hypotheses of the correlation between oxidative stress and neoplastic changes have been proposed and tested, aiming to demonstrate reactive oxygen species (ROS) role by different mechanisms of action, with direct damage of cells and indirect changes of cell metabolism (Waris and Ahsan 2006; Klaunig et al. 2010; Gupta et al. 2012; Hatem and Azzi 2015; Prasad et al. 2016; Saha et al. 2017; Bokhari and Sharma 2019).

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CSCs recent theory is based on the correlation between carcinogenesis and a subpopulation of highly potent malignant cells, which may originate from selfrenewal stem cells or other progenitors, tumor heterogeneity being correlated with developing phenotype of the cells of origin as well as genetic and nongenetic influences (Talukdar et al. 2016). Their high aggressiveness is responsible for neoplastic growth, tumor spread, recurrence, and therapy resistance (De Macedo and Machado 2017), as demonstrated by CSCs implantation which can develop a tumor (Nimmakayala et al. 2019). Moreover, recent studies revealed that CSCs are immunoprivileged cells (Romano et al. 2020). CSCs have been isolated from many tumors, showing to be valuable targets for oncotherapy (De Macedo and Machado 2017). Current evidences suggest that the cell transformation and reprogramming in CSCs are the result of complex processes and newly gained mutations, supplemented with metabolic changes from glycolytic to oxidative phosphorylation or vice versa (Nimmakayala et al. 2019). In this context, the role of the modified redox status and high ROS involvement in the occurrence, development, and evolution of CSCs is an objective of research (Leone et al. 2017).

CSCs Inside Stem Cells Spectrum The accumulated knowledge regarding tumor biology, in the 1970s, suggesting that CSCs represent a particular tumor cell population has been demonstrated two decades later, by the identification of “leukemic-initiating cells” in acute myeloid leukemia (AML) (Rich 2016). Following their definition and intrinsic potential in tumor progression and recurrence, the subject has been the focus of research in recent years (Toh et al. 2017). The tumor microenvironment consists of cancer cells as well as CSCs (Rich 2016; Toh et al. 2017) associated with immune cells and stromal cells. The intratumoral heterogeneity is known to develop with asymmetric multiplication and differentiation of CSCs, being reflected in tumor classifications, and having prognosis significance. Moreover, due to CSCs potential to divide uncontrollably, they result in phenotypes resistant to treatment, involved in tumor progression, and metastasis (Rich 2016). Normal stem cells (NSCs) are two distinct types of undifferentiated cells with self-renewal capacity, located in the bone marrow, in adults, surrounded by a complex microenvironment, as reservoirs to replenish cells lost because of damage or aging (Rich 2016; Bhartiya et al. 2016) (Fig. 1). Unlike NSCs, CSCs are capable to generate malignant cells with high proliferation potential and increased plasticity (Badrinath and Yoo 2019). CSCs have lost their “social control,” due to alterations the tumor microenvironment possesses added features such as self-renewal, migratory, and metastatic capacities, angiogenesis induction, apoptotic resistance, telomerase expression, increased membrane transport activity, and long life spans (Rich 2016).

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Fig. 1 The potential of differentiation of NSCs. Normal stem cells (NSCs) are undifferentiated cells with self-renewal capacity, along with the capacity to proliferate, to further undergo clonal expansion, and differentiate into specialized cells. According to their differentiation potential, there are two distinct types of NSCs: pluripotent stem cells, as embryonic stem cells, which have the ability to differentiate into any type of cell and multipotent stem cells which give rise to many cell types, but within a particular lineage, such as: mesenchymal stem cells (MSCs), ovarian stem cells (OSCs), spermatogonial stem cells (SSCs), and hematopoietic stem cells (HSCs)

Another population of early-development stem cells has been identified as very small embryonic-like stem cells (VSELs), being involved in cancer initiation of gonadal tumors and in radiotherapy resistance (Bhartiya et al. 2016; Badrinath and Yoo 2019). They have been identified in mouse bone marrow, human cord blood, and adult organs, showing a weak expression of pluripotency markers, with a morphology characterized by a spherical shape, a minimal cytoplasm, and euchromatic nucleus (Bhartiya et al. 2016). Furthermore, VSELs are actively mobilized into the peripheral blood under stress conditions such as burn, stroke, myocardial infarction, and post-cytotoxic treatments (Bhartiya et al. 2016). CSCs are mainly located in the invasive front or close to blood vessels and depend on the interactions with components of the tumor microenvironment for their survival. CSCs are slow proliferating cells with self-renewal ability and generate progenitor tumor cells (Vander Linden and Corbet 2019). CSCs are identified using several methods as follows: xenotransplantation assays (gold-standard method), anti-CD44 immunohistochemistry (the most well-recognized CSC marker, a glycoprotein involved in cell adhesion and migration), or by identification of aldehyde dehydrogenase (ALDH) cell activity (Bhartiya et al. 2016). CSCs express surface markers associated with stem cells, a low expression of major histocompatibility molecules class I (MHC I) and natural killer (NK) cells receptors, which increase their ability to escape from elimination by T lymphocytes and NK cells and provide therapy resistance, using complex mechanisms (Fig. 2), in

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Fig. 2 The mechanism of therapy resistance of CSCs. CSCs therapy resistance can be attributed to the intervention of EGF, IL-4 enhanced production, ALDH activity, and drug efflux proteins. CSCs pro-survival strategies involve chemotherapy evasion associated to upregulated ABC transporter expression and to anti-PD-1/PD-L1-mediated acquired resistance, dependent on CD38-generated adenosine. This latter mechanism leads to IFN-β and ATRA production that result in increased expression of CD38 by RARα that may catalyze NAD+ to immunosuppressive adenosine, via CD38/CD203a/CD73 pathway. Adenosine activates adenosine receptors of CD8+T cells (A2AR and A2BR) to inhibit their antitumor function. Thus, anti-CD38 monoclonal antibody associated to PD-1/PD-L1 blockade may enhance antitumor immune responses [21]. ABC, ABC transporter; ATRA, all-trans retinoic acid; ALDH, aldehyde dehydrogenases; CSC, cancer stem cell; CD203a, ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (NPP1, also known as CD203a or PC-1); CD38, cluster of differentiation 38 or cyclic ADP ribose hydrolase; CD73, cluster of differentiation 73 or ecto-50 -nucleotidase; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; EMT, epithelial-mesenchymal transition; IFN-β, interferon β; IL-4, interleukin-4; NAD+, nicotinamide adenine dinucleotide; PD-1, programmed cell death protein 1; PD-L1, programmed deathligand 1; RARα, retinoic acid receptor α; ROS, reactive oxygen species

association to ROS production, lymphocytes autophagy activation, and epithelialmesenchymal transition (EMT) (Badrinath and Yoo 2019; Ayob and Ramasamy 2018; Mittal et al. 2018). Development of CSCs is preceded by stem cells influences performed by a microenvironment rich in reactive nitrogen species, ROS, inflammatory chemokines, and cytokines. Non-stem cancer cells can also give rise to CSCs (Badrinath and Yoo 2019), by intervention of IL-6, which mediates the maintenance of tumor heterogeneity and a balance between non-stem cancer cells and CSCs, a conversion demonstrated in

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breast and prostate cancer cells lines. This transformation is mediated by hypoxia, which prevents differentiation of goblet cells and enterocytes, by downregulating CDX1 and Notch1, in colorectal cancer (Badrinath and Yoo 2019). Hypoxia is an important factor of the “niche” that induces CSCs preservation and leads to increased aggressiveness and metastatic potential, as well as cell resistance to conventional treatment. In hypoxic microenvironment, malignant transformation is increased by reduced genomic stability and by diminished expression of DNA repair genes (Ayob and Ramasamy 2018). It is considered that CSCs are mainly able to generate a heterogeneous cells population by an uncontrolled differentiation process rather than a process of dedifferentiation. A high number of asymmetric divisions of CSCs that give rise to the tumor cells population are early events, followed by suppression of asymmetric division, which promotes symmetric self-renewal, in the late tumor stages (Badrinath and Yoo 2019; Ayob and Ramasamy 2018). There are several probable mechanisms involved in CSCs transformation and reprogramming. Current evidence shows that a possible mechanism of CSCs origin is cell fusion, wherein cells get high aneuploidy hybrid forms, with tumorigenic properties and multidrug resistance (Nimmakayala et al. 2019). Another mechanism incriminated to contribute to the CSCs origin is horizontal gene transfer (characteristic for bacteria and fungi in acquiring resistance to antibiotics), with DNA transfer from donor cells to recipient cells, between apoptotic cells and somatic or tumor cells, by endocytosis or phagocytosis, forming more aggressive phenotypes (Nimmakayala et al. 2019). Different external influences like toxin exposure, variable tissue injuries, and radiation therapy can induce gene mutations in embryonic or normal adult stem cells (ASCs), leading to CSCs populations, due to ASCs accumulation of mutations and their transformation into CSCs (Nimmakayala et al. 2019). Variable factors, such as stress, hypoxia, wounding, and ionizing radiations, can induce cellular dedifferentiation of a differentiated cancer cell into a CSC, which may lead to the hypothesis that CSCs possess plasticity (Nimmakayala et al. 2019). The most known theory is that of metabolic adaptation of CSCs due to glycolytic quality of cancer cells, as they can produce energy by glucose anaerobic breakdown, even in aerobic conditions (Nimmakayala et al. 2019).

CSCs Heterogeneity and Metabolic Landscape Most evidences indicate CSCs as a heterogeneous cell population, which make them difficult to characterize in different tumor types. First description of CSCs was detailed in different types of leukemia (Table 1). Different CSCs profiles were also described in solid tumors (Table 1), being responsible for their therapy resistance (Nimmakayala et al. 2019; Badrinath and Yoo 2019). Similarly, other studies identified numerous cancer stem cell populations, based on high expression of pluripotent markers, proving once more that tumors of different organs consist of different CSC subpopulations with heterogeneous

CD34+CD38

CD117+ CD133+CXCR4+

Colorectal cancer

Ovarian, colorectal, gastric, pancreatic, lung, liver, prostate, and head and neck cancer Bladder cancer

Breast cancer

Head and neck cancer

Melanoma, head and neck, gastric, pancreatic, lung, liver, prostate, bladder, ovarian, and colorectal cancer Head and neck cancer

CD29+

CD44+

CD44+BCMab1+

CD49f+CD24+

CD98+

CD133+

CD200+

CD24+

Melanoma

CD20+

CD271+

CD44+CD24+EpCAM + CD87+

CD34+CD19CD10 CD44+ALDH+

BMI1+

Bladder cancer

ALDH1A1+

Melanoma

Ovarian, prostate and lung cancer Colorectal and pancreatic cancer

Colorectal and pancreatic cancer Lung cancer

Breast and head and neck cancer

CXCR4+

CD166+

CD123+

CD90+

CD45RA+

CD34+CD38CD123+ CD34+CD38CD71HLADR CD34Lin+CD38+ CD44+CD24

CD26+

CD13+

ALDH1+

(continued)

Leukemia

Colorectal, gastric, head and neck, prostate, and lung cancer

Lung and liver cancer Leukemia

Leukemia

Breast cancer

Leukemia

Gastric cancer

Liver cancer

Melanoma, pancreatic, and gastric cancer

CSCs markers ABCG2+ Ovarian, colorectal, liver, lung and head and neck cancer Lung and head and neck cancer Colorectal and ovarian cancer AML

Table 1 The heterogeneity of CSCs (Nimmakayala et al. 2019; Badrinath and Yoo 2019) ALDH+

Implications of ROS in Cancer Stem Cells Mechanism of Action

Melanoma, prostate, ovarian, and liver cancer

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K19+ OV6+ Trop2+

Prostate cancer

Melanoma Leukemia

Integrins

Nestin+ TIM3+

Liver cancer Prostate cancer

Liver cancer Liver, colorectal, gastric, ovarian, and lung cancer Liver cancer PD1+ Vimentin+EpCAM VimentinEpCAM+

Lgr5+

DLK1+ GRP78+

Colorectal and gastric cancer Melanoma Breast cancer

Liver cancer Head and neck cancer

ABCG2 ATP-binding cassette super family G member 2, ALL acute lymphoid leukemia, AML acute myeloid leukemia, CML chronic myeloid leukemia, CSCs cancer stem cells, EpCAM epithelial cell adhesion molecule, EMT epithelial-mesenchymal transition, MET mesenchymal-epithelial transition, TIM3 T cell immunoglobulin mucin 3

c-kit+ EpCAM+

Melanoma Bladder cancer

CSCs markers CXCR6+ EMA+

Table 1 (continued)

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phenotypes, which support increased tumorigenicity, metastatic potential, and maintenance of stemness capacity for CSCs and cancer, allowing the design of targeted therapy strategies (Nimmakayala et al. 2019; Wong et al. 2019). The heterogeneity of tumor CSCs has been first explained by genetic and/or epigenetic alterations occurring in transformed cells and providing selective growth privilege for tumors. A second theory assumes that the tumor growth is due to a small group of cells with stem cell–like properties, whereas the differentiated daughter cells dye of clonal breakdown (Wong et al. 2019). The third hypothesis is based on cancer stem cell plasticity, which allows the phenotype shift between CSC and non-CSC, the cancer cell stemness being intrinsic or extrinsic, being influenced by tumor microenvironment or external stimuli (Wong et al. 2019). Like normal pluripotent stem cells, CSCs, which share the same self-renewal potential, can switch their metabolic profile during differentiation from glycolytic to oxidative phosphorylation. This metabolic variability has been illustrated on different CSCs populations in leukemia, breast, nasopharyngeal, and colon cancers, supporting the possibility that different cancer stem cell subpopulations have unique metabolic profiles, being glycolytic or oxyolytic in their actions of tumor proliferation and drug resistance (Nimmakayala et al. 2019).

Plasticity, Quiescence, and Embryonic Signature of CSCs Tumor cell heterogeneity is related to CSC plasticity, the bidirectional transformation between non-stem cells and stem cells modulated by cellular interactions and tumor microenvironment (Talukdar et al. 2016), which depends on EMT (Nimmakayala et al. 2019). CSC plasticity has been proved in breast, pancreas, melanoma, or glioblastoma CSCs, involving ZEB1 EMT transcription factor or PTEN silencing mechanism for breast CSCs, metabolic variability for pancreatic CSCs, histone demethylase JARID1B for melanoma CSCs, and pluripotent transcription factors POU3F2+SOX2+SALL2+OLIG2 for transformation of glioblastoma cells into glioma CSCs (Nimmakayala et al. 2019). Another important feature is CSCs’ dormant state within the tumor, their low-cycling quiescence being responsible for cancer aggressiveness and metastasis. Moreover, ABCG2 and other receptors are incriminated in CSCs drug resistance (Nimmakayala et al. 2019). A significant characteristic of CSCs is the embryonic signature, expressed through the recapitulation of specific activated pluripotent pathways and aberrant expression of embryonic genes, which are related to tumor initiation or progression (Nimmakayala et al. 2019). Cancer initiation and progression are based on activation of specific embryonic stem cells (ESCs) genes triggered by the transcription factors Oct3/4, SOX2, and Nanog, which are activated by different pathways responsible for self-renewal and pluripotency maintenance of ESCs (Nimmakayala et al. 2019).

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Tumor Microenvironment: CSC Relationship During tumorigenesis, the microenvironment supports the proliferation of tumor cells and plays a critical role in drug resistance. Concomitantly, CSCs influence their microenvironment, maintaining specific characteristic features, such as hypoxia, low pH, chronic inflammation, and elevated cytokine levels (Badrinath and Yoo 2019). Tumor stroma or “CSCs niche” consists of cellular components, extracellular matrix, along with cytokines, and growth factors complexes released by stromal or tumor cells, which regulate CSCs activity and promote drug resistance (Rich 2016; Badrinath and Yoo 2019) (Fig. 3). Recent data confirmed that the tumor microenvironment is formed by the correlation between mesenchymal cancer cells and adaptive or innate immune system cells (Romano et al. 2020). Thus, EMT creates a specific immune response

Fig. 3 The composition of CSCs niche. Morphologically, the tumor stroma or “cancer stem cells (CSCs)-niche” consists of: (a) cellular components, such as tumor-infiltrating lymphocytes, mast cells, NK cells, T-cells tumor-associated macrophages, myeloid-derived suppressor cells (MDSCs), blood and lymphatic endothelial cells, pericytes, and cancer-associated fibroblasts; (b) extracellular matrix; (c) cytokines and growth factors complexes released by stromal cells or tumor cells, such as IL-6, IL-8, hepatocyte growth factor (HGF), and stromal cell-derived factor-1 (SDF1), all of which regulate CSCs activity and promote treatment resistance

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represented by high levels of CD3 tumor infiltrating T lymphocytes (TILs) or regulatory T cells (Tregs), as well as numerous activation markers like CD80, CD86, OX40 L, 4-1BB, ICOS, CD127, IFN-γ, and IFN-γ-induced protein CXCL10 (Romano et al. 2020). The activity of this immunoprivileged niche of the tumor microenvironment which supports the activity of CSCs was widely studied on breast, prostate, ovarian, pancreas, and liver cancers, their activity promoting tumor aggressiveness and metastasis (Romano et al. 2020). The processes of tumor cells transformation and drug response regulation are deeply related to the complex of cellular and noncellular microenvironment components represented by extracellular pH, extracellular matrix, different signaling molecules, soluble factors, angiogenesis, and stromal, inflammatory, or hematopoietic cells (Talukdar et al. 2016). These tumor niche factors are also responsible for phenotype maintenance, functionality, and plasticity of CSCs. The influence of the tumor microenvironment is considered a crucial event in the bidirectional shift between the CSC and non-CSC populations, disturbance of the specific signaling networks being able to induce tumor progression arrest (Talukdar et al. 2016; Wong et al. 2019). Current evidence supports the interactions between CSCs and tumor microenvironment, showing specific signaling pathways involvement in different cancers, such as activation of transcription factor NF-κB by the inflammatory tumor microenvironment, which accelerates Wnt signaling, causing dedifferentiation of non-stem cells into CSCs, in intestinal tumors (Talukdar et al. 2016). Additionally, CD133+ CSCs avoid NK cytotoxic effects by expressing low levels of MHC1 molecules in glioblastoma. Furthermore, tumor microenvironment hypoxia preferentially preserves breast CSCs, promoting tumor progression and drug resistance (Nimmakayala et al. 2019). Another mechanism is that of angiogenesis induction by microvesicles of mRNA for vascular endothelial growth factor (VEGF) contained in renal CSCs (Nimmakayala et al. 2019).

The Signaling Pathways Involved in CSCs Maintenance The deregulation of controlled signaling networks, which are responsible for normal stem cells homeostasis, causes an elevated activation, proliferation, and activity of CSCs (Nimmakayala et al. 2019). The most important signaling pathways and factors responsible for CSC maintenance are: Wnt, Hedgehog (Hh), Notch, ATP-binding cassette (ABC) transporters, antiapoptotic pathways, NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells), transcription factor hypoxia-inducible factor 1 (HIF-1), Myc oncoproteins, and Nanog transcription factor (Nimmakayala et al. 2019) (Fig. 4).

ROS, Carcinogenesis Theories, and CSCs Cancer is a disease of cytodifferentiation, with all cells directly derived from the malignant cell losing normal division control, with cumulative mutations of genes (Frank 2007; Sonnenschein and Soto 2018). The genome alteration causes an abnormal karyotype of progenitor cell or a clone of cells with a more aggressive

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Fig. 4 The signaling pathways involved in CSCs maintenance. The most important signaling pathways and factors responsible for CSC maintenance are: Wnt signaling molecules, involved in the activation of self-renewal, proliferation, and dedifferentiation properties of CSCs; Hedgehog (Hh) signaling pathway, by the binding of ligand-receptor, represented by Indian, desert, or sonic hedgehog ligands and PTCH1 or PTCH2 receptor; ABC transporters responsible for drug resistance, regulated by multiple signaling pathways, with three ABC transporters, ABCB1 (MDR1), ABCC1, and ABCG2; Myc oncoproteins and Nanog transcription factor which play crucial roles in alteration of metabolic pathways, reprogramming, and pluripotency maintenance of CSCs, leading to a shift or an inhibition of oxidative phosphorylation, HIF-1, which increases glycolysis, decreases oxidative phosphorylation, and may reprogram a differentiated/cancer cell into a CSC; Notch signaling network, leading to the cleavage of the Notch receptor intracellular part which will be translocated in the nucleus, acting as a transcriptional co-activator, by binding to target gene regions and activating the target genes; anti-apoptotic pathways, with upregulation of antiapoptotic proteins Bcl-2, Mcl-1, c-FLIP and downregulation of proapoptotic proteins, like Bid; and NF-κB pathway, which is deeply related to EMT, responsible for metastasis and drug resistance. ABC, ATP-binding cassette; CSC, cancer stem cell; EMT, epithelial-mesenchymal transition; Hh, Hedgehog; HIF-1, transcription factor hypoxia-inducible factor 1; NF-κB, nuclear factor kappa light chain enhancer of activated B cells; PTCH1, Patched1; PTCH2, Patched2

growth. However, by tumor progression, multiple subclones of biochemically different malignant cells occur by disorders of cellular metabolism and antigenicity (Sonnenschein and Soto 2018). The potent factors of transformation are represented by heredity, chemical and physical agents, irradiation, and viruses. One or more initiating mutagen factors that act together or require cofactors may trigger carcinogenesis (Sonnenschein and Soto 2018). Metabolic activation of mutagenic carcinogens confirms the mutation hypothesis through deficient mechanisms of defense (Sonnenschein and Soto 2018; Manda et al. 2015). In the initiation stage, the

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mutagenic initiator is applied in a subcarcinogenic dose, with minor metabolic changes, activating procarcinogens by covalently binding to DNA. When the promoter continues the action, alteration of the genes expression and enzymes occurs on stem cell populations. During the progression stage, additional genetic events continuously occur, leading to mobility, loss of contact inhibition, independent growth, and adherence to surrounding structures, due to alterations of cellular and/or membrane components (Sonnenschein and Soto 2018; Manda et al. 2015). The natural history of most cancers has suggested different theories of carcinogenesis (Sonnenschein and Soto 2016; Manda et al. 2015), such as theories of intercromosomial recombination aberrations and somatic mutation, multifactorial theory of cumulative action of several carcinogens, multistage theory, multicellular origin with independent lines of progression, proliferation of developmental tissue, genetic hypothesis with clonal expansion of a single transformed cell, and accumulation of mutations, with selection of an autonomous and aggressive population, and metabolic disease theory involving mitochondrial and respiratory dysfunction that can initiate the malignant pathway in which cells are oxygen independent (Frank 2007; Manda et al. 2015). There is enough evidence to support the theory that the development of a tumor is conditioned by both the oncogenic agents and by the failure of the immune surveillance (Frank 2007), as supported by the evidence that children with primary immunodeficiency are at a high risk of developing lymphoid malignancies (Sonnenschein and Soto 2018). The large amount of ROS lead to cellular oxidative stress that indirectly damages DNA, by an oxidation phenomenon, resulting in morphological and functional alterations, by genetic mutations, membrane alterations, and activation of signaling networks, with cellular proliferation and tissue degradation (Leone et al. 2017; Manda et al. 2015). Mutations may determine different quantitative and qualitative effects on chromosomal karyotype, resulting in activation of proto-oncogenes and oncogenes, tumor suppressor genes inhibition, or their imbalance (Leone et al. 2017). Proto-oncogenes stimulate cell growth by encoding some factors and growth factor receptors that stimulate cell division or proteins that participate in the transduction of signals for cell division (Leone et al. 2017; Manda et al. 2015). The oncogenic potential of proto-oncogenes can be activated by: point mutations, structural alterations of oncogenic products, gene amplification, and chromosomal rearrangements. Tumor suppressor genes inhibit cellular multiplication, being major targets for carcinogens (Manda et al. 2015). Suppression of tumor suppressor genes or abnormal activation of proto-oncogenes is the promoter of tumor transformation, while the order of their mutations during the multistep carcinogenesis could result in a large spectrum of human cancers.

Oxidative Stress in Cancer Initiation and Development Within the carcinogenesis multifactorial mechanism, the oxidative stress can intervene as a result of an unregulated or overproduction of ROS, from endogenous and/or exogenous sources. During the last years, several hypotheses on the correlation between oxidative stress and neoplastic changes have been proposed and tested,

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aiming to prove the role and level of ROS action in the carcinogenic sequence (Waris and Ahsan 2006; Klaunig et al. 2010; Gupta et al. 2012; Hatem and Azzi 2015; Prasad et al. 2016; Saha et al. 2017; Bokhari and Sharma 2019). Nowadays, it is accepted that ROS are involved in carcinogenesis through direct damage of cell components and metabolism, modulation of signal transduction pathways or gene transcription factors of cell cycle growth and proliferation, and by single-nucleotide polymorphisms.

Direct Damage of Structural Components and Metabolism with Induction of Gene Mutations ROS acts on cellular components, altering DNA structure or cell membrane constituents, inducing mutations that set off genes involved in carcinogenesis (Waris and Ahsan 2006; Hatem and Azzi 2015; Bokhari and Sharma 2019; Noda and Wakasugi 2001). The oxidative damage of DNA involves several tumor suppressor genes related to different types of malignancies. For example, mutations of p53 and ras have been demonstrated in human liver tumors or human and experimental UV-related skin tumors and mutations in p15INK4B and p16INK4A have been reported in experimental renal cell carcinoma (Klaunig et al. 2010; Hatem and Azzi 2015; Leone et al. 2017). On the other hand, it is proved that the mitochondrial DNA is more vulnerable to oxidation, due to the absence of histones and incapacity of DNA repair enzymes. Changes in the mitochondrial DNA were associated with type I endometrial carcinoma, due to mutations of the mitochondrial genes responsible for the synthesis of some proteins involved in oxidative phosphorylation (Waris and Ahsan 2006; Gupta et al. 2012). Tumor cells are characterized by decreased oxidative phosphorylation capacity, cellular metabolism being maintained by aerobic glycolysis (Hatem and Azzi 2015). The transition from the oxidative phosphorylation to aerobic glycolysis is known as the “Warburg effect” (Warburg et al. 1927). While p53 regulates synthesis of cytochrome c oxidase 2 (SCO2) expression, necessary for the generation of cytochrome c oxidase (COX) complex, in normal cells, an abnormal p53 expression leads to low levels of SCO2, low O2 usage, and high synthesis of lactate (Hatem and Azzi 2015). Consequently, low oxygen levels and hypoxia accelerate tumor transformation. The relationship between glycolysis and the presence of HIF-1α (hypoxia-inducible factor-1α), which stimulates the production of pyruvate dehydrogenase kinase 1, as well as the activity of genes responsible for glucose transport, glycolysis, and lactic acid synthesis have also been demonstrated (Hatem and Azzi 2015). The oxidation of the lipid components of the cell membrane can be followed by their reaction with different metals (i.e., free iron and cooper) and the synthesis of different active substances (i.e., epoxides, aldehydes/malondialdehyde) that induce mutations (Waris and Ahsan 2006; Noda and Wakasugi 2001) in esophageal cancer.

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Modulation of Signal Transduction Pathways or Gene Transcription Factors with Alterations of Oncogene Expression and Tumor Progression ROS result either by enzymatic reactions (involving NADPH-oxidase or NOX metabolic enzymes, such as cytochrome P450 enzymes, lipoxygenase, and cyclooxygenase) or by nonenzymatic reactions, which occur during mitochondrial respiratory chain (Leone et al. 2017). Their involvement in carcinogenesis is supported by the highly reactive potential of ROS against biological molecules and the damage of cellular components, by excessive levels of ROS. To counteract these effects, cells activate “anti-ROS adaptive” mechanisms, which involve several antioxidant agents, such as glutathione peroxidase (GPx), thioredoxin (Trx), catalase (CAT), superoxide dismutase (SOD), and nuclear factor erythroid 2 (NRF2) (Leone et al. 2017). Several studies support the relationship between ROS, the activation of signal transduction pathways or gene transcription factors of cell cycle growth and proliferation, and the modification of oncogene expression, which can lead to cancer initiation and progression. It is proved that the following signaling pathways and transcription factors can be influenced by ROS: NF-κB pathway, Wnt/β –catenin pathway, and NF-E2/rf2 or Nrf2, MAP kinase/AP-1, HIF-1α (Waris and Ahsan 2006; Hatem and Azzi 2015; Prasad et al. 2016; Bokhari and Sharma 2019). Moreover, solid evidences hold up the ROS involvement in the development of variable malignancies, such as breast, lung, liver, colon, pancreas, prostate, ovary, and brain cancer, with unknown specific ROS level correlated to a carcinogenic or a protective role (Hatem and Azzi 2015; Prasad et al. 2016). Recent data reveal that decreased ROS levels are procarcinogenic, by a direct activation of the signaling pathways or by triggering of genomic DNA alterations, mirrored in an altered expression of growth factors and proto-oncogenes (Prasad et al. 2016). As a first step in the carcinogenic process, low ROS levels induce cell proliferation by stimulating cyclin D1 activity, or activate several kinases (ERK, JNK, and MAPK). Through the redox-sensitive cysteine residues located at the catalytic spots, ROS block or annihilate the activity of some tumor suppressors (protein tyrosine phosphatases – PTPs and PTEN). On the other hand, high ROS levels are associated with increased mutations, due to cell sensitivity to mutagenic agents, facilitating DNA damage (Hanahan and Weinberg 2011). Thioredoxin, a redox-controlling agent of redox reactions in oxidative stress, stimulates cell growth and has cytokine-like properties, that aid the activation of protein kinases, Fos and Jun oncogenes, and NF-κB transcription factor (Noda and Wakasugi 2001), being expressed in gastric and liver cancer. Protein kinases (PKCs), involved in the control of several cell functions like proliferation, cell cycle, differentiation, cytoskeletal organization, cell migration, and apoptosis, are activated by the calcium released from intracellular domain by ROS or by ROS itself, stimulating the synthesis of new ROS that inhibit the gap junction and block the intercellular communication. Additionally, H2O2 has the capacity to activate other protein kinases, such as ERK1/2 (signal-regulated kinase

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1/2), P13K/Akt (phosphoinositide 3-kinase/serine-threonine kinase), PKB (proteinkinase B), and PTPs (Waris and Ahsan 2006; Klaunig et al. 2010). AP-1 proteins, members of Jun (c-Jun, JunB, and JunD), Fos (FosB, Fra-1, and Fra-2), musculoaponeurotic fibrosarcoma (Maf), and activating transcription factor (ATF) subfamilies, have a role in basal gene expression and their functions are related to phorbol ester (TPA) or cAMP presence. The oxidative stress conditions activate AP-1 which results in cell proliferation stimulation by a high expression of growth-stimulation genes (i.e., cyclin D1) and inhibition of proteins involved in cell cycle progression (p21waf) (Klaunig et al. 2010).

The Association Between Oxidative Stress and CSCs CSCs gain an increased resistance in case of increased ROS level, expressed by a reduced oxidative injury and a longer survival, and undergo numerous changes depending on the tumor microenvironment, constituting the so-called dynamic tumor strain (Leone et al. 2017; Go and Hideyuki 2016). Epigenetic changes induced by chronic inflammation, excessive redox stress, and hypoxic environment have been shown to stimulate the plasticity of the transition between CSCs and non-CSCs (Go and Hideyuki 2016). There is evidence that ROS are an extremely important factor in maintaining the stem cell character of tumor cells by influencing the expression of the transcription factor SOX-2, the activity of multiple enzyme systems, and the action of variable redox-sensitive metabolic signaling pathways, increasing the expression of molecules with antioxidant effect (Nimmakayala et al. 2019; Leone et al. 2017). It has been proven that higher levels of ROS in CSCs could be associated with reduced action of ROS capture systems, such as SOD, CAT, GPx, and TPx, compared to NSCs and that activation of miR-153/NRF2/GPx1 signaling pathway plays an important role in the regulation of glioma stem cells radiosensitivity (Leone et al. 2017). ROS-induced carcinogenesis is characterized by an association of increased HIF (a transcription factor of VEGF) and tumor angiogenesis, along with poor prognosis (Klaunig et al. 2010). Both HIF-1α and HIF-2α promote the stem cell properties of cancer cells. HIF-2α contributes, together with the intracellular domain of CD44, to the acquisition of radioresistance by glioma stem cells, in osteopontin-rich perivascular niche (Nimmakayala et al. 2019) and a selective HIF-2α-dependent mechanism promotes metastasis in hepatocellular carcinoma. HIF can stimulate CSC population growth by modulating Notch signaling pathway, in glioma and ovarian cancer, by IL-6 - HIF-1α, in small cell lung cancer, and by EMT activation, through increased expression of the Snail transcription factor, in CSCs, in gastric cancer (Leone et al. 2017; Wang et al. 2019). An analogous action is supported by the Hippo pathway, which regulates the molecular switch involved in cell differentiation and stem cell regeneration, by directly stabilizing transcriptional coactivator with PDZ-binding motif (TAZ) in breast cancer, by activating Ras-ERK pathway-ELK3 in liver cancer (Leone et al.

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2017), and by activation of Mst1, a serine/threonine kinase, by oxidative stress (Park et al. 2018). NF-κB, a nuclear transcription factor involved in cell survival, differentiation, inflammation, and growth, and its activation by oxidation can block its binding to DNA, stimulating angiogenesis, tumor invasion, and metastasis (Klaunig et al. 2010). NF-κB has been correlated with HIF-1α expression in breast cancer models, while inhibition of NF-κB signaling induces a significant reduction in the hypoxiadependent CD44+CD24 CSC population, leading to increased CD24 expression (Leone et al. 2017). Additionally, by IκBα inhibition, the enzyme factor Aurora A kinase phosphorylates an inhibitor of NF-κB and thus induces the activation of the NF-κB signaling pathway, in ovarian cancer (Tang et al. 2017). Nuclear factor erythroid 2-related factor 2 (Nrf2) is another antioxidant system involved in cell stability maintenance and modulation of the differentiation process in NSCs (Leone et al. 2017). Nrf2 activation leads to a transcriptional expression of enzymes involved in xenobiotic detoxification, antioxidative response, and proteome maintenance (Klaunig et al. 2010; Hatem and Azzi 2015), such as glutathione reductase, peroxiredoxin, thioredoxin and thioredoxin reductase, catalase, copper/zinc superoxide dismutase, and glutathione peroxidase. Low levels of Nrf2 or loss of Nrf2 activity increase ROS production and result in DNA damage, in pancreatic cancer (Klaunig et al. 2010). In oxidative stress condition, ROS dissociates the complex between Nrf2 and Kelch ECH associating protein 1(Nrf2-Keap 1 complex), stimulating Nrf2 proteasomal degradation and Nrf2 translocation to the nucleus (Klaunig et al. 2010). NRF2 levels correlate with CSCs survival and resistance to cancer drug effects in head and neck squamous cell carcinoma (HNSCC), cervical, breast, and ovarian cancers (Leone et al. 2017). Other signaling pathways involved in regulating the effects of redox status on CSCs are c-Jun and/or p53 and FoxO families. Loss of p53 expression may lead to early expansion of mammary stem/progenitor cells and development of triple-negative breast cancer (Leone et al. 2017). Inhibition of JNK1 or JNK2 expression and/or treatment with JNK-IN-8, an adenosine triphosphate with irreversible pan-JNK inhibitory effect, significantly reduces cell proliferation and limits the presence of the CSCs subpopulation ALDH1+ and CD44+/CD24, in experimental models of triple-negative breast cancer (Leone et al. 2017). Transcription factors FoxO1, FoxO3a, and FoxO4 have been shown to be essential mediators of cellular responses to oxidative stress, being involved in numerous processes of modulating ROS effects (Leone et al. 2017). FoxO is known to compete with TCF for the same β-catenin binding site and suppresses the stimulation of cell proliferation, by inhibiting β-catenin binding with TCF and diminishing WNT-mediated signaling activities. FoxO factors also exert an inhibitory action on c-Myc factor, by reducing excess ROS production (Leone et al. 2017). One of the first response mechanisms to the action of oxidative stress is the translation of mRNA that induces, probably as a means of limiting the energy required for protein synthesis, the formation of stress granules (SGs) in cancer

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cells. Thus, SG formation plays a protective role against stress-induced cell injury and cell death and the involvement of SG in cancer biology has been suggested by different researchers (Leone et al. 2017). It has been shown that, under stress conditions, YB1 factor, located in a nuclear situs sensitive for protein 1 binding to the nuclease, facilitates tumor metastasis by two mechanisms: it can bind directly to HIF-1α, by modulating stress adaptation and metastatic capacity in vivo and mediates the formation of cytosolic SGs, by a translational activation of G3BP1 (Leone et al. 2017).

Single Nucleotide Polymorphisms Results in Cancer Risk Increase The occurrence of a variable number of single nucleotide polymorphisms (SNPs) in the genes responsible for oxidative DNA repair, such as hOGG1 (Human oxoguanine glycosylase 1), XRCC1 (X-ray repair cross-complementing group 1 gene), XPD (Xeroderma pigmentosum group D), and MGMT (O6 methylguanine-DNA methyltransferase) and in antioxidant genes, such as MnSOD/SOD2 (Mn superoxide dismutase), MPO (Myeloperoxidase), CAT, GPX (Glutathione peroxidase), GSTM1, GSTT1, GSTP1 (Glutathione S-transferase), EPHX1 (Epoxide hydrolase 1), and NQO1 (NAD(P)H: quinone oxidoreductase 1), can be associated with an increased risk for cancer development (Klaunig et al. 2010). In keeping with this, a relevant example consists in the deficiency of antioxidative activity reflected by the presence of Mn-SOD with amino acid mutation that is associated to a higher risk of breast cancer. Other related genes with SNPs associated with cancer risk are: cytochrome P450 (CYP1A1) and Vitamin D receptor (VDR) (Klaunig et al. 2010).

Pathologic Conditions Related to Oxidative Stress and Carcinogenesis Promoters Chronic inflammation leads to and maintains oxidative stress and ROS production (Klaunig et al. 2010; Hatem and Azzi 2015), which can produce DNA strand breaks, sister chromatin exchange, and mutations. This has been experimentally demonstrated, using cell lines cocultured with activated neutrophils that, like macrophages, have the ability to produce O2, H2O2, and HO. The genomic damage can result in impairment of DNA repair mechanisms, the activation of oncogenes, or the inactivation of tumor suppressor genes (Bokhari and Sharma 2019). This mechanism has been proved in experimental colon chronic inflammation and adenocarcinoma, in human B and C hepatitis and hepatocellular carcinoma, and in human Helicobacter pylori–induced gastritis (Noda and Wakasugi 2001). Furthermore, the carcinogenic role of the superoxide production has been demonstrated in gastric cancer, by synthesis of azo compounds and active substances (i.e., peroxynitrite) with mutagenic potential, nitric monoxide production by macrophages, free radicals, and cytokines synthesis by gastric epithelium (Noda and Wakasugi 2001).

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ROS and Tumor Status Oxidative stress may influence tumor development and progression, as recent evidences show a persistent prooxidant state in tumor cells (Klaunig et al. 2010; Hatem and Azzi 2015). As a consequence, multiple sequences of tumor progression, such as alteration of cell adhesion, stimulation of tumor angiogenesis, changes of the extracellular matrix by an increased MMPs activity, and EMT are associated with the oxidative stress and ROS (Hatem and Azzi 2015). A characteristic feature of tumor cells, integrin configuration, alters mitochondrial metabolism, leading to the activation of oxidative enzymes, including NOX and COX-2. Consecutively, high levels of ROS influence signaling pathways that control endothelial cell adhesion, resulting in increased permeability, with facilitation of migration and angiogenesis (Hatem and Azzi 2015). Studies on ROS – intercellular adhesion, indicate that ROS activity interferes with Src and Pyk2 kinases, leading to phosphorylation of VE-cadherin, β-catenin, and p120-catenin (Hatem and Azzi 2015). Several experimental and clinical evidences show that ROS stimulate MMPs activity in glioblastoma, breast, pancreatic, and prostate cancer cells. The association between ROS and TGF-β was demonstrated in EMT, as well as ROS action in Smad2, p38, and ERK 1/2 phosphorylation, α-SMA and fibronectin stimulation, and E-cadherin inhibition (Hatem and Azzi 2015). Tumor microenvironment–specific cellular hypoxia, a result of ROS, may contribute to EMT initiation, assumption supported by experimental studies showing that HIF-1 activates transcription factors of the Snail pathway, in breast, prostate tumors, and melanoma (Hatem and Azzi 2015). Although an antioxidant supplementation is frequently associated to the conventional cancer treatment, the classic concept of the benefits of antioxidant drugs seems to be invalid in cancer, as recent studies show that this supplementation does not inhibit carcinogenesis but, on the contrary, they play a role in accelerating tumor progression (Saha et al. 2017). The oxidative stress markers can be considered as supplementary tumor markers, a statement supported by high 8-hydroxydeoxyguanosine levels in different malignancies, in humans and experimental studies, high Mn-SOD serum level in ovarian cancer, and high thioredoxin in liver cancer (Waris and Ahsan 2006).

CSCs and Metastasis It is well known that CSCs can mediate tumor metastasis through plasticity, which is essential for EMT. Plasticity is modulated by the immune system and tumor surrounding stroma, which release different factors like IFN-γ and IL6, responsible for tumor transmembrane protein PD-L1 (CD274, B7-H1) expression (Romano et al. 2020). The “seed and soil” theory of metastasis assumes that the primary tumor cells (seed) move to a certain distant organ (soil) where it finds proper environment for colonization which leads to organ-specific metastasis (Nimmakayala et al. 2019). Although different studies showed the involvement of CSCs in tumor aggressiveness

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and metastasis, the role of these cells in organotropic metastasis is poorly understood. Available data demonstrated CSCs capacity in different cancer types using different CSC markers as follows: highly metastatic CD133+ pancreas, potentially metastatic ALDH+ and CD26+CD24 breast, CD110+ and CDCP1+ colorectal cells, and CD44+CD24 or CXCR4+ breast CSCs which metastasize in lung and lymph nodes (Nimmakayala et al. 2019). Recent data focus on the role of exosomes in CSC-mediated metastasis, considering that primary tumor cells release exosomes which influence the inflammatory environment of target organs because of specific integrins (Nimmakayala et al. 2019). Moreover, exosomes represent the link between CSCs and cancer cells, which share their signature proteins and nucleic acids or other regulatory RNAs, thus ensuring the transformation of a non-stem cell into a stem cell and possibly forming a pre-metastatic niche in a distant organ (Nimmakayala et al. 2019).

Conclusions Emerging data suggests that tumors consist of a heterogeneous population of CSCs subtypes which, with specific phenotype, unique clonogenic and metastatic potential, and consequently molecules of the signaling pathways, together with metabolic modulators, display a recognizable pattern, so a specific cancer type can be assigned to a certain subtype of CSCs. Current evidence suggests that cell transformation and reprogramming in CSCs are the result of complex processes induced by tumor microenvironment and other factors. In this context, the role of the modified redox status and ROS remain controversial regarding their double-face involvement in the occurrence, development, and evolution of CSCs. The exploitation of ROS dual abilities may be useful for the development of new therapeutic strategies targeting redox regulatory mechanisms. The knowledge regarding the remarkably wide range of factors involved in the process of CSCs adaptation to oxidative stress reflects the complexity of the molecular mechanisms involved in CSCs dedifferentiation and reprogramming. This phenomenon of adaptive resistance emphasizes the importance of simultaneous blockage of multiple signaling pathways. As a consequence, new therapeutic approaches that overcome CSCs resistance are needed, followed by their implementation into therapeutic algorithms that target both CSCs and non-CSCs.

References Ayob AZ, Ramasamy TS (2018) Cancer stem cells as key drivers of tumour progression. J Biomed Sci 25:20 Badrinath N, Yoo SY (2019) Recent advances in cancer stem – targeted immunotherapy. Cancers (Basel) 11(3):310 Bhartiya D, Shaikh A, Anand S et al (2016) Endogenous, very small embryonic-like stem cells: critical review, therapeutic potential and a look ahead. Hum Reprod Update 23:41–76

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Metabolism-Redox Interplay in Tumor Stem Cell Signaling

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Vanesa Martin, Maria Turos-Cabal, Ana Maria Sanchez-Sanchez, and Carmen Rodríguez

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cancer Stem Cell Signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cancer Stem Cell Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Redox Regulation in Cancer Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Intracellular Signaling – Redox State Crosstalk: Metabolism Interplay . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cross-References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract

Cancer stem cells (CSCs) are defined as a subpopulation of cells within the heterogeneous tumor mass endowed with the ability to self-renew and differentiate into non-CSCs. Over-activation or abnormal functioning of intracellular pathways that control normal stem cells, participate in, or contribute to the origin, survival, and maintenance of CSCs. In addition, expression of genes involved in the stemness also depends on epigenetic processes controlling by intermediary metabolites – mainly derived from glycolysis – or can be achieved by the V. Martin (*) · M. Turos-Cabal · A. M. Sanchez-Sanchez Departamento de Morfología y Biología Celular, Facultad de Medicina, Universidad de Oviedo, Oviedo, Spain Instituto Universitario de Oncología del Principado de Asturias, Oviedo, Spain Instituto de Investigación Sanitaria del Principado de Asturias, Oviedo, Spain e-mail: [email protected] C. Rodríguez Departamento de Morfología y Biología Celular, Universidad de Oviedo, Oviedo, Spain Instituto Universitario de Oncología del Principado de Asturias, Oviedo, Spain Instituto de Investigación Sanitaria del Principado de Asturias, Oviedo, Spain © Springer Nature Singapore Pte Ltd. 2022 S. Chakraborti et al. (eds.), Handbook of Oxidative Stress in Cancer: Mechanistic Aspects, https://doi.org/10.1007/978-981-15-9411-3_114

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action of reactive oxygen species (ROS) – produced by cellular metabolism – establishing a relationship between metabolism and ROS as a central axis in the CSCs control. In fact, metabolic adaptation is one of the hallmarks of cancer, being aerobic glycolysis the main metabolic change in cancer that moves from oxidative phosphorylation toward lactate production as a way of obtaining energy, even under normal oxygen concentrations. In this sense, data form CSCs indicates that some of them preferably use aerobic glycolysis, while others preferentially use mitochondrial oxidative metabolism. This indicates that CSCs are not a fixed population, but that their metabolic phenotype can be modified depending on the needs. Metabolic factors would be the key for transcription and signaling pathways programs necessary so that intrinsic or environmental factors can direct a particular cell toward a CSC state. In this chapter we review the role of redox state in the regulation of intracellular pathways controlling CSCs and the metabolic plasticity in this tumor subpopulation, thus establishing a point of interconnection between stemness, ROS, and metabolism. Keywords

Cancer stem cells · Metabolism · Epigenetics · Reactive oxygen species · Intracellular signaling pathways

Introduction Self-renewal and pluripotency are the two fundamental characteristics that define a normal stem cell. Thus, every stem cell must be able to undergo both symmetric cell division, giving rise to an identical daughter cell, and asymmetric cell division, giving rise to all the differentiated cell linages needed to populate a specific tissue (Cahan and Daley 2013). Due to the high cellular heterogeneity of the tumors and their recurrence after treatments, is has been suggested that tumors could develop from a small subpopulation of cells within the tumor that share these stem cell properties. This was the foundation of the cancer stem cell (CSC) hypothesis. Thus, CSCs have been thoroughly investigated in recent decades and have been proposed to be responsible for the tumor recurrence and the metastases. CSCs have been identified in hematological and solid tumors, including breast, brain, thyroid, melanoma, colon, pancreas, liver, prostate, lung, head and neck, ovary, and stomach cancer (Turdo et al. 2019), establishing a large number of different biomarkers for identification (Table 1). Thus, CSCs are defined as a subpopulation of cells within the heterogeneous tumor mass endowed with the ability to selfrenew and differentiate into non-CSCs, which is reflected by their ability to reproduce the tumor of origin when transplanted into immunocompromised mice. CSCs are also considered responsible for metastatic spread and chemoresistance. In this way, they evade conventional treatments, including radio and chemotherapy, being responsible for minimal residual disease and cancer relapse. In fact, CSCs are characterized by more pronounced levels of drug transporters, improved DNA damage repair mechanisms, and the ability to escape

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Table 1 Biomarkers reported to characterized CSCs Marker CD133+

CD44+

CD24+ CD123+

CD49+ CD34+ CD90+ ALDHhigh

EpCAM ESA ABCG2high

Tumor type Breast cancer; colon cancer; gastric cancer; glioblastoma; head and neck cancer: liver cancer: lung cancer; ovarian cancer; pancreatic cancer; prostate cancer Breast cancer; colon cancer; gastric cancer; head and neck cancer; liver cancer; lung cancer; liver cancer; lung cancer; Breast cancer; colon cancer; gastric cancer; liver cancer; pancreatic cancer Leukemia (AML); Breast cancer; non-small lung cancer; ovarian cancer; pancreatic cancer; prostate cancer Prostate cancer; breast cancer; glioblastoma Leukemia (AML) Liver cancer; lung cancer Breast cancer; colon cancer; gastric cancer; glioblastoma; liver cancer; lung cancer; ovarian cancer; pancreatic cancer; prostate cancer Breast cancer; colon cancer; pancreatic cancer Breast cancer; colon cancer; pancreatic cancer Liver cancer; lung cancer; prostate cancer; pancreatic cancer; melanoma; head and neck cancer; glioblastoma

Relationship with metabolism Decrease hexokinase II expression; promotes hypoxia Promotes glycolysis via PKM2 suppression. Induced by hypoxia Promotes glycolytic enzymes activity Not specified Not specified Not specified Converts acetaldehyde to acetate; maintains low ROS Not specified Not specified Induced by hypoxia

(Snyder et al. 2018; Turdo et al. 2019)

cytotoxic chemotherapy by maintaining a quiescent state (Turdo et al. 2019). Furthermore, in some tumors, chemotherapy has been shown to stimulate the division of CSCs, thus favoring tumor repopulation (Chen et al. 2012). Mechanistic studies have indicated that dysfunction of various developmentalrelated signaling pathways may cooperate in the dysregulation of the self-renewal and differentiation that characterized CSCs (Matsui 2016). In addition to the involvement of these pathways in the regulation of CSCs, the evidence also suggests that stem cell properties can be acquired as a consequence of mutations and metabolic changes occurring in normal stem cells or differentiated cancer cells that move up the cancer cell hierarchy for their expression of pluripotent genes, making them more susceptible to epigenetic reprogramming. In this sense, many of the CSCs biomarkers identified have some role in cellular metabolism (Table 1). These metabolic changes, capable of inducing this reprogramming in CSCs in the context of a premalignant tumor, are collectively called “metabostemness“(Menendez and Alarcon 2014). This epigenetic reprogramming can also be achieved by the action of reactive oxygen species (ROS), mainly produced by cellular metabolism, establishing a relationship between metabolism and ROS as a central axis in the CSCs behavior control. In this chapter we review the relationship between the main intracellular pathways controlling CSCs and the metabolic plasticity in this tumor subpopulation, focusing on the role that cellular redox state plays in the control of both

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aspects, thus establishing a point of interconnection between stemness, ROS, and metabolism.

Cancer Stem Cell Signaling It is well-known that over-activation or abnormal functioning of the intracellular pathways that control normal stem cells, participate in, or contribute to, the survival and maintenance of CSCs. The proper functioning of a normal stem cell requires fine control of these signaling pathways, which mainly include the pathways governed by JAK/STAT, Sonic/Hedgehog, WNT, Notch, PI3K/AKT, and NFkB (Matsui 2016) (Fig. 1). These pathways, highly regulated in normal stem cells, do not actually represent independent and linear intracellular pathways, but rather interlaced networks of signaling mediators that feed each other. The Sonic/Hedgehog pathway (Fig. 1a) includes three ligands that are secreted (Sonic, Desert, and Indian), its Patched receptor, the transmembrane protein Smoothened, and three transcription factors (Gli 1-3). In the absence of ligand, the receptor acts by repressing the activity of the transmembrane protein, thus keeping the transcriptional activity inactive. The binding of the ligand to the receptor inhibits this repressive action, thus allowing the transcription of the target genes. Aberrant expression of members of this signaling pathway has been described for several tumors. In fact, an increased expression of some of its members has been described in the CSCs subpopulation, so that its inhibition results in a loss of stem cells properties (Merchant and Matsui 2010). The WNT pathway (Fig. 1b) is a complex but highly conserved pathway in evolution that includes 19 ligands and more than 15 receptors. It includes two different, although not independent, pathways known as the canonical and the noncanonical pathway, dependent or independent of the transcriptional activity of βcatenin, respectively. In the absence of ligand, β-catenin levels are kept low by the action of a multiprotein complex responsible for its degradation (consisting of the axin, APC, casein 1, and GSK3B proteins). In the canonical pathway, when the ligand binds to the receptor, this complex is sequestered and anchored to the receptor-associated proteins so that the stabilization and thereby the transcriptional activity of β-catenin is allowed. The noncanonical pathway is activated by family receptors but does not involve the participation of β-catenin. Its function is essential to regulate the release of calcium from the endoplasmic reticulum (ER) to control intracellular calcium levels. Mutations in some of the components of the pathway are very frequent in tumors. In fact, depletion of some of its components in glioma cells inhibits their growth, produces their differentiation, and reduces their tumorigenic capacity (Borcherding et al. 2015). The Notch pathway (Fig. 1c) includes several ligands and receptors, all of them transmembrane proteins, so that activation of the pathway takes place when a ligand expressed in one cell binds to a receptor expressed in the adjacent cell leading to the proteolytic excision of the receptor’s cytoplasmic domain and the release of the intracellular domain. This intracellular domain translocates to the nucleus acting as a

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transcription factor. This pathway is essential for the regulation of CSCs, although its role may be different, acting as a tumor promoter or suppressor in different cellular contexts (Ranganathan et al. 2011). The PI3K/AKT pathway (Fig. 1d) activation is triggered after the binding of ligands to tyrosine kinase receptors. This binding triggers phosphorylation and activation of AKT kinase which can then mediate the activation of several effectors including mTOR. It is a highly conserved signaling pathway and is involved in numerous cellular processes such as proliferation or survival. It also plays an important role in the regulation of normal stem cells, participating in the self-renewal of embryonic cells or in the expansion and differentiation to different lineages of hematopoietic stem cells (Xia and Xu 2015). The main inhibitor of the pathway is PTEN phosphatase, which is usually inactivated by mutations in a large number of tumors. In fact, the loss of function of this phosphatase in hematopoietic stem cells is capable of triggering the development of myeloproliferative diseases and leukemia (Xia and Xu 2015). The pathway is over-activated in CSCs, participating both in the maintenance of these cells and in their capacity to stimulate neovascularization, acting as initiators of new vessel formation by promoting the secretion of proangiogenic factors (Xia and Xu 2015). The JAK/STAT pathway (Fig. 1e) is initiated by the binding of several ligands – interleukins, interferon, hormones, and growth factors – to their respective receptors, inducing their oligomerization and the recruitment of JAK family proteins to their intracellular domains where they are phosphorylated and activated. These active proteins, in turn, induce phosphorylation and activation of STAT family proteins that can thus be translocated to the nucleus, acting as transcription factors. The pathway regulates numerous cellular processes in a large number of different tissues, including the maintenance of embryonic stem cells, hematopoiesis, or neurogenesis. Furthermore, aberrant activation has been described in CSCs of several tumors, including breast cancer, glioblastoma, prostate cancer, and hematological tumors (Stine and Matunis 2013). The NFκB family transcription factors (Fig. 1f) are composed of dimers of five different proteins (p65, c-Rel, RelB, p50, and p52), which are normally inactive in the cytosol due to their binding to IkB proteins. NFκB can be activated by two signaling pathways, the classical and the alternative one. Classical activation, which can be triggered by numerous stimuli, is initiated by phosphorylation of IkB proteins, mediated by IKKα/IKKβ heterodimer, which leads to their proteolytic degradation, allowing the transcription factor release and translocation to the nucleus. In the alternative pathway, IKKα/IKKα homodimer phosphorylates protein p100, given as a result the formation of the mature p52 subunit (Karin 1999). It is actually a very complex pathway, extensively studied for its involvement in inflammation and the immune response, although it is also involved in other functions such as proliferation, survival, or cell differentiation. Unlike the other pathways, its role in regulating normal stem cells has not been widely studied, although there are data that indicate that the loss of their activity produces inhibition of self-renewal, as well as a decrease in the number of normal hematopoietic stem cells and abnormal differentiation into different lineages. Although there are some studies that indicate its participation in

Fig. 1 Cancer stem cell signaling pathways. (a), Sonic/HH pathway. Upon ligand (HH) binding to Patched receptor, repression of Smoothened is released allowing the transcriptional activity of Gli transcription factors. (b), WNT pathway (canonical). In the absence of ligand, β-catenin levels are kept low by the

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self-renewal or the tumorigenic capacity of cancer cells – mainly in breast cancer – the relationship with the maintenance of CSCs is not yet entirely clear. Anyway, activating mutations of this pathway have been described for many types of cancer (Rinkenbaugh and Baldwin 2016). As already mentioned, these are not independent linear pathways. There are studies that demonstrate the necessary cooperation of the Notch and WNT pathways in maintaining the undifferentiated state of intestinal tumor stem cells, or the cooperation of the pathways in the development of epidermal or pancreatic tumors and the increase in resistance to treatments in metastatic breast cancer, favoring the survival of tumor stem cells and thus tumor repopulation (Matsui 2016).

Cancer Stem Cell Metabolism Genetic alterations, with mutations in oncogenes and tumor suppressor genes, and environmental modifications, such as hypoxia, converge in one of the traits that define tumor cells and that are in the spotlight for the design of new therapeutic strategies: metabolic reprogramming. In fact, metabolic adaptation is considered one of the hallmarks of cancer (Hanahan and Weinberg 2011). The main metabolic change in cancer is aerobic glycolysis or the Warburg effect, that moves from oxidative phosphorylation (OXPHOS) as a way of obtaining the necessary energy toward lactate production, even when there are normal oxygen concentrations, hence the name aerobic glycolysis. This allows to redirect metabolic intermediaries toward macromolecule biosynthesis pathways (much needed in highly proliferative cells such as tumor cells) (Jang et al. 2013). In this way, some intermediaries are diverted toward the pentose phosphate pathway (PPP), for the production of nucleotides and NADPH (necessary for the correct maintenance of the cellular redox state) and others toward the formation of glycerol 3 phosphate for lipid synthesis and toward the serine ä Fig. 1 (continued) action of a multiprotein complex responsible for its degradation. After ligand binding to the receptor, the complex is anchored to the receptor-associated proteins allowing the release and stabilization of β-catenin and so the transcription of target genes. (c), Notch pathway. Activation of the pathway takes place when a ligand expressed in one cell binds to a receptor expressed in the adjacent cell leading to the proteolytic excision of the receptor’s intracellular domain and its translocation to the nucleus. (d), PI3K/AKT pathway. Upon activation of tyrosine kinase receptors, AKT is phosphorylated and activated. AKT kinase is then able to phosphorylate several effectors such as FOXO, mTOR, or GSK3β. (e), JAK/STAT pathway. After ligand binding to the receptors, JAK family proteins are recruited to their intracellular domains where they are phosphorylated and activated. These active proteins in turn induce phosphorylation and activation of STAT family proteins that can thus be translocated to the nucleus, acting as transcription factors or to the mitochondria. (f), NFκB pathway. Classical activation (canonical) is initiated by phosphorylation of IkB proteins, mediated by IKKα/IKKβ heterodimer, which leads to their proteolytic degradation, allowing the transcription factor release and translocation to the nucleus. In the alternative pathway (noncanonical), IKKα/IKKα homodimer phosphorylates protein p100 given as a result the formation of the mature p52 subunit

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synthesis for protein synthesis as well as nucleic acids, ATP and reducing power. Lactate is finally secreted out of the cell, generating acidification of the microenvironment that can benefit the aggressiveness of the cancer (Jang et al. 2013). Normal stem cells have a greater glycolytic metabolism compared to differentiated cells derived from them. However, there are relatively few studies and quite a few discrepancies in relation to the metabolism used by CSCs. Some of them indicate that they preferably use aerobic glycolysis, while others show that they preferentially use mitochondrial oxidative metabolism (Peiris-Pagès et al. 2016). Thus, there is abundant literature that supports aerobic glycolysis as the main bioenergetic source in CSCs of various tumor types such as breast, colon, ovarian, or glioblastoma. In fact, it has been described that hypoxia in the tumor niche is a key determinant for the glycolytic metabolism in breast CSCs (Peiris-Pagès et al. 2016). Furthermore, the regression of the mitochondria toward a more immature state induces the epithelialmesenchymal transition and the acquisition of stem properties (Guha et al. 2014). Similarly, glioblastoma CSCs generally use aerobic glycolysis as an energy source, showing a preference for hypoxic niches and a decrease in oxidative metabolism (Zhou et al. 2011). Same has also been described in CSCs derived from osteosarcoma, ovarian carcinoma, or colon cancer (Menendez et al. 2013). However, other authors have also found CSCs that preferentially use OXPHOS, such us breast CSCs (Peiris-Pagès et al. 2016; Snyder et al. 2018). In fact, the inhibition of complex I of electron transport chain (ETC) partially inhibits the stemness in breast cancer. A marked oxidative profile in CSCs has been also described for glioblastoma, where CSCs can move from one to the other type of metabolism. Same occurs in ovarian or pancreatic cancer, where the dependence of CSCs on OXPHOS, as well as overexpression of genes that regulate mitochondrial function, have been described. However, the treatment of pancreatic CSCs with a mitochondrial inhibitor such as metformin is not effective against a proportion of the CSCs subpopulation, which can use either oxidative metabolism or aerobic glycolysis, suggesting the existence of cellular subgroups with great metabolic plasticity (Sancho et al. 2015). These different data, even in the same tumor type, suggest that CSCs must have a more complex biochemical, molecular, and metabolic behavior than their non-tumor counterparts, showing great metabolic plasticity. Thus, metabolic type of CSCs would depend on the characteristics of the niche in which they are located (Sancho et al. 2016). At this point, it seems clear that CSCs can use both aerobic glycolysis and OXPHOS, depending on the state of differentiation, tumor microenvironment, or expression of certain oncogenes. There are several possible causes that could explain these divergent results, even within the same tumor type (Snyder et al. 2018). On the one hand, due to this high plasticity, cells can be collected at different metabolic stages depending on the different laboratory protocols or in different niches of the tumor with different microenvironment, i.e., necrotic areas or hypervascularized areas. Thus, hypoxia promotes dedifferentiation and the maintenance of stem properties, increasing surface markers such as CD133 and at the same time, through the stabilization of hypoxia inducible factor (HIF1α), promotes aerobic glycolysis. Unlike what happens in normal stem cells, in which the niche maintains

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the balance between self-renewal and differentiation, the tumor microenvironment necessary for the maintenance of CSCs is altered, with the signals that favor the proliferation (Li and Neaves 2006). The importance of the tumor niche in the formation and maintenance of CSCs has been well documented, demonstrating the influence of fibroblasts and epithelial cells residing in the niche, as well as the hypoxia that prevails within it (Li and Neaves 2006). On the other hand, the possible different origin of CSCs must be taken into account. While this is unclear, it is now believed that CSCs could originate from transformation of differentiated tumor cells that move up in the cancer cell hierarchy or could derive from transformation of normal stem cells. Menendez et al. (Menendez et al. 2013; Menendez and Alarcon 2014) have proposed a very interesting hypothesis about the origin of CSCs, so that the generation of these cells by transformation of normal stem cells would depend on epigenetic processes controlled by intermediary metabolites that would regulate the expression of genes involved in the stemness. Therefore, cellular metabolism and nutrient availability would play a key role in activating enzymes that will modify histones and DNA and that will later lead to the different gene expression that will originate tumor cells with stem properties. This does not imply that the only origin of CSCs occurs always from transformation of normal stem cells since differentiated tumor cells can also be reprogrammed and acquire characteristics of stem cells by activating pathways not yet fully understood. These cells, which can be reprogrammed, would present different cellular states depending on genetic, epigenetic, metabolic, and extrinsic factors (tumor niche). Thus, the reprogramming of the bioenergetic state of the tumor cell is considered as the metabolic change that defines the origin of the cancer (Menendez et al. 2013). Metabolic factors would be the key for transcription and signaling pathways programs necessary so that intrinsic or environmental factors can direct a particular cell toward a CSC state. Indeed, when transcription factors, oncogenes, or oncomiRNA are used to convert differentiated somatic cells to induced stem cells (iPSCs), these cells spontaneously form teratocarcinomas into nude mice (Blum et al. 2009), demonstrating the close relationship between reprogramming toward stem cells and tumorigenicity. These iPSCs recapitulate all the features of metabolic reprogramming that have been observed in tumor cells, including the appearance of immature mitochondria and low levels of oxidative stress. Therefore, the bioenergetic characteristics of the cells change, going from the use of OXPHOS to aerobic glycolysis. When they differentiate again, the cells reacquire the mitochondrial bioenergetic profile. This means that the ability to develop an anabolic or Warburg-like metabotype would represent a crucial early molecular event that would suppose an a priori barrier to the transformation process of differentiated somatic cells to CSCs. In short, factors present in the tumor niche, such as different cell types, hormones, growth factors, oxygen levels, and metabolites, can regulate epigenetic activity and gene transcription, leading to reprogramming that leads to the transformation of differentiated tumor cells in CSCs. On the other hand, subsequent metabolic changes may be responsible for the characteristics of CSCs within a tumor, with tumor metabolism being one of the hallmarks of cancer (Hanahan and Weinberg

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2011). Thus, tumor metabolism has gone from being an important event in the development and progression of cancer, to being probably the determining factor. As mentioned above, epigenetic reprogramming plays a key role in metabolic changes and in the origin and maintenance of CSCs. Metabolic adaptations that occur after tumor transformation cause epigenetic changes that in turn regulate tumor metabolism and contribute to tumor progression. Thus, some glycolytic enzymes such as pyruvate kinase M2, GAPDH, or LDH translocate to the nucleus to perform non-metabolic functions such as regulating gene transcription and epigenetic modifications (Yu et al. 2018). Similarly, enzymes that participate in other metabolic processes (lipid or nucleotide synthesis) such as ACLY or ACSS2 for the synthesis of ACo A for histone acetylation or that participate in the synthesis of SAMs for DNA methylation have been localized to the nucleus in various tumors (Yu et al. 2018). On the other hand, the enzymes that catalyze DNA and histone modifications use metabolites and coenzymes that come from glycolysis, TCA, and other metabolic pathways, for its catalytic reactions (Yu et al. 2018). Because of the epigenetic modifications caused by metabolic adaptations, the methylation state of DNA or the activity of histones are modified, which in turn regulate metabolic plasticity in tumor cells. Thus, increased expression of histone demethylases observed in several tumors are recruited into the promoters of various genes involved in glycolysis, causing demethylation of histones and activation of their transcription. Similarly, mutations in various histone deacetylases (HDACs) increase aerobic glycolysis or glutamine metabolism (Miranda-Gonçalves et al. 2018). Although the role of epigenetic regulation in CSCs is not yet fully resolved, there are important data that indicate a clear interrelation between the epigenetic state and the origin and maintenance of the stem cell properties. Thus, H1.0 linker histone has been shown to be critical in the self-renewal of CSCs of various tumor types. When the gene that encodes it is repressed due to the methylation of the promoter, genes related to stem properties are expressed, which correlates with the aggressiveness of the tumors (Wainwright and Scaffidi 2017; Li et al. 2019). Chromatin remodeling by the EZH2, BMI1, and SUZ12 polycomb-group proteins, which leads to the silencing of genes through modifications of histones, is a specific trigger for stemness (Wainwright and Scaffidi 2017; Li et al. 2019). On the other hand, suppression of gene expression by histone demethylase LSD1 is essential for the proliferation of pluripotent tumor cells, while it is not relevant in the proliferation of non-pluripotent tumor cells or normal somatic cells (Wainwright and Scaffidi 2017; Li et al. 2019). Genes related to pluripotency and self-renewal have also been shown to be hypomethylated in CSCs (Wainwright and Scaffidi 2017). Epigenetic regulation may also be responsible for changes in cellular metabolism that lead to the acquisition of stem properties. As an example, inhibition of fructose 1-6 biphosphatase 1 (FBP1) expression (specific enzyme of gluconeogenesis) by methylation of the promoter, induces aerobic glycolysis, decreased consumption of oxygen and ROS production, which results in an increase in cancer-stem like properties and tumorigenicity in breast cancer cells (Dong et al. 2013). Thus, the interrelation between metabolism and epigenetic status contributes to the plasticity of CSCs and to tumorigenicity.

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As will be discussed later, metabolic adaptations are not only regulated at the epigenetic level but many of the signaling pathways involved in the CSCs regulation also participate in the control of cellular metabolism. For example, the PI3K/AKT pathway, which establishes a point of convergence for many of the essential pathways for CSCs, stimulates aerobic glycolysis, which can ultimately affect intracellular ROS levels and tumorigenesis. Thus, it seems clear that CSCs are not a fixed population, but that their metabolic phenotype can be modified moving from aerobic glycolysis to OXPHOS by the action of many factors of the microenvironment such as growth factors, inflammatory signals, or by interaction with stromal cells, for example.

Redox Regulation in Cancer Stem Cells Reactive oxygen species (ROS) can be endogenously generated by various oxidases and peroxidases in different cellular compartments such as cell membranes, peroxisomes, or the ER, although the main endogenous source is the mitochondria through the ETC. Initially considered as by-products of cellular metabolism that were harmful to cells, it is currently well known that low or moderate levels of ROS promote cell proliferation and survival acting as second messengers, while only high levels can cause cytotoxicity and trigger cell death. Thus, ROS are involved in the physiological regulation of many biological processes related to cell development at different levels, from gene expression, signal transduction to protein–protein interactions (Martin and Barrett 2002). Maintaining a fine balance between production and removal is therefore essential. To do this, cells have powerful and complex antioxidant systems that include the enzyme superoxide dismutase (SOD), catalase, peroxyredoxins (PRX), thioredoxins (TRX), glutathione peroxidase (GPX), and glutathione reductase (GR). The GPX enzyme breaks down hydrogen peroxide into two water molecules using glutathione (GSH), one of the most abundant antioxidant molecule in cells (Martin and Barrett 2002). Tumor cells have higher ROS levels than their normal counterparts. This increased ROS levels favors tumor promotion and progression by increasing proliferation, survival, and metastasis. Similarly, there are increasing evidence suggesting an important role for ROS and redox signaling in the functioning of CSCs (Lee et al. 2019). In acute lymphoblastic leukemia, the population of CD44+ cells with low levels of ROS has been found to be a tumor-initiating cells enriched subpopulation. Furthermore, there is a correlation between the frequency of CSCs and the expression levels of GPx3 (a ROS scavenger enzyme). Thus, ROS-inducing treatments such as disulfiram (an aldehyde dehydrogenase inhibitor) kill the stem cell population by inhibiting nuclear factor erythroid 2-related factor 2 (Nrf2) activity and activating the JNK pathway. Similar results have also been described in hepatocellular carcinoma, where disulfiram reduces the population of CSCs by increasing cellular ROS levels and activating the p38 MAPK pathway (Lee et al. 2019). However, although many CSCs appear to prefer a low ROS environment, this does not occur in all cases. In fact, CD133+ glioblastoma CSCs have higher ROS levels than non-CSC cells (Lee et al.

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2019). Similarly, breast CSCs have been reported to have higher levels of ROS than non-CSCs due to an increase in mitochondrial biogenesis (Lee et al. 2019). In other words, although a preference for low levels of ROS has been described by CSCs, this does not always occur. This divergence agrees with the discordant data regarding the basal metabolism of CSCs, which could indicate a relationship between both aspects. Thus, a preferential glycolytic metabolism would be related to the maintenance of low ROS levels, whereas a preferentially mitochondrial metabolism would be related to an increased production of ROS. In any case, maintenance of low ROS levels does not always correspond to a preference for glycolytic metabolism. In fact, leukemic CSCs have been described to have low levels of ROS but are surprisingly dependent on OXPHOS for survival and maintenance of the quiescent state (Lagadinou et al. 2013). Therefore, the use of the glycolytic pathway or the mitochondrial pathway by CSCs depending on the state they are in, quiescent or proliferative, is critical in order to maintain energy needs and redox balance, establishing a relationship between ROS and metabolic plasticity. As an example, it has been described that quiescent breast CSCs have a high metabolic rate of the PPP, which favors the generation of reducing power (NADPH), essential for the maintenance of the state cellular redox (Debeb et al. 2016). In any case, a fine regulation of the cellular redox state is essential for the maintenance of CSCs, so that these cells have a powerful antioxidant system that is finely controlled by the hypoxic niche in which they develop, as well as by other factors such as transcription factors of the FOXO family or Nrf2, or other oxidative stress sensors such as Ataxia Telangiectasia Mutated kinase (ATM) (Wang et al. 2013). CSCs can promote the synthesis of GSH due to the increased import of cysteine from the extracellular medium. Thus, there is a decrease in ROS that inhibit the activation of the p38/MAPK intracellular pathway, preventing differentiation and apoptosis (Ding et al. 2015). Along with GSH, thioredoxin metabolism is the other main mechanism of elimination of hydroperoxides that also plays a key role in increasing radiation resistance in CSCs (Ding et al. 2015). Regulation of ROS levels can also be done through transcription factors such as NFκB and Nrf2. NFκB pathway participates in maintaining self-renewal in CSCs. In fact, its inhibition causes a decrease in the size of the CSCs population (Ding et al. 2015). In AML stem cells, treatment with partenolide (NFκB inhibitor) produces an increase in ROS, activation of p53 and triggers a cell death process that can be prevented by antioxidant compounds (Rinkenbaugh and Baldwin 2016). On the other hand, Nrf2 is considered the master regulator of the antioxidant response since it controls the expression of many detoxification and antioxidant genes. Maintaining a low oxidative microenvironment through the activation of Nrf2 favors the development of quiescent CSCs. If these cells suffer an increase in oxidative stress, they differentiate into proliferative cells that support higher levels of ROS and that will also have Nrf2 activated, which will allow them to continue growing and invading tissues (Ding et al. 2015). Ataxia Telangiectasia Mutated kinase (ATM), a master regulator of DNA damage, has also been postulated as one of the main modulators of the response to

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oxidative stress and mitochondrial homeostasis. Defects in ATM cause an increase in ROS in hematopoietic stem cells and the loss of self-renewal that can be reversed by treatment with antioxidant compounds (Wang et al. 2013). Furthermore, the intracellular cascade of ATM has been described to be increased in CSCs compared to normal tumor cells, and treatment with ATM inhibitors reverses resistance to radiological treatments, denoting the importance of this kinase in CSCs (Wang et al. 2013). Finally, there is also a relationship between ROS and the epigenetic state of cells at a given time. Thus, SAM synthetase enzymes are dependent on the cellular redox state, so that an oxidizing environment reduces their activity. Furthermore, methionine synthetase, which participates in the methionine cycle for its recycling, is dependent on cobalamin (vit B12), whose oxidation inactivates the enzyme. These data suggest that the oxidative state of the tumor cell environment could lead to hypomethylation and hence the activation of oncogenes. On the other hand, it is known that ROS cause DNA damage by oxidizing guanine and producing 8oxoguanin (8-OG), which has great mutagenic capacity. If 8-OG formation occurs on a CpG island, binding to DNMTs and thus methylation is inhibited, it leads to DNA hypomethylation in those areas (Hitchler and Domann 2012).

Intracellular Signaling – Redox State Crosstalk: Metabolism Interplay Oncogenic transformation, mitochondrial dysfunction, and alterations in cell signaling pathways in tumors cause an increase in ROS, which at low or moderate levels are capable of modulating a wide variety of intracellular signaling pathways, transcription factors, phosphatases, or kinases such as JAK/STAT, MAPKs, PI3K/AKT, NFkB, Nrf2, FOXO, ATM, HIF1α . . . with the consequent stimulation of survival, proliferation, and differentiation (Ding et al. 2015). Furthermore, many of the intracellular pathways and transcription factors implicated in the maintenance of CSCs also participate in the control of the redox state in these cells, thus establishing a positive feedback mechanism (Ding et al. 2015). A further level of complexity should be added since it must be borne in mind that cellular metabolism is the main source of ROS and many of these intracellular pathways are in turn involved in the control of cellular metabolism. Regulation of the PI3K/AKT pathway in CSCs can be mediated by ROS (Fig. 2, #1), so that they are capable of inducing AKT activation or can inhibit the activity of PTEN (main inhibitor of the pathway). In turn, activation of the pathway can regulate ROS levels in CSCs through the regulation of one of its targets, the transcription factor FOXO. This transcription factor has been described to be essential for maintaining of self-renewal in hematopoietic stem cells through the up-regulation of the expression of the antioxidant enzymes catalase and manganeseSOD. Furthermore, FOXO deficiencies increases ROS production and lose of the quiescent status of CSCs (Miyamoto et al. 2007). On the other hand, PI3K/AKT pathway is considered one of the master regulators of aerobic glycolysis. AKT

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Fig. 2 Crosstalk between CSCs signaling pathways, redox state, and metabolism. 1: activation of PI3K/AKT pathway, that can be mediated by ROS, inhibits FOXO activity, and stimulates mTOR and HIF1α, leading to a stimulation of aerobic glycolysis. Moreover, FOXO also participates in the regulation of cellular redox state reducing ROS levels through the upregulation of antioxidant enzymes expression. 2: the activation of PI3K/AKT pathway can also be achieved by WNT noncanonical pathway (β-catenin independent). 3: on the other hand, β-catenin transcriptional activity can be stimulated by ROS, leading to an increase in aerobic glycolysis and an increase in c-Myc expression, which in turns plays an essential role in the metabolic plasticity of CSCs regulating several metabolic pathways such us aerobic glycolysis, glutaminolysis, or lipid synthesis. PI3K/AKT pathway can be also activated by other ROS-stimulated CSCs signaling pathways such us Sonic/HH or Notch. 4: ROS activate transcription factor Nrf2 that induces the expression of HH triggering activation of Sonic/HH pathway. Through the transcription activity of

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increases gene expression of glucose transporters; increases the phosphorylation of key enzymes in glycolysis, such as hexokinase and phosphofructokinase 2; inhibits FOXO transcription factors, which will result in changes in gene expression that favor aerobic glycolysis; and activates mTOR, which promotes the translation of messenger mRNA and synthesis of macromolecules (Elstrom et al. 2004). PI3K/ AKT pathway also induces HIF1α, which is considered the master regulator of aerobic glycolysis (Elstrom et al. 2004). Furthermore, the AKT signaling pathway represents a point of convergence with other important intracellular pathways for CSCs such as the WNT pathway, which can also be regulated by ROS (Fig. 2, #3). Transcriptional activity of β-catenin can be regulated by ROS (Bowerman 2005). It has been described that nucleoredoxin, an antioxidant protein of the thioredoxin family, enhances the activation of the canonical WNT pathway. In the same way, ROS enhances β-catenin-FOXO interaction, inducing a more differentiated state, decreasing tumorigenicity and pluripotency of cells (Bowerman 2005). Recent studies show that the activity of the WNT pathway plays a key role in the regulation of cellular metabolism, although this role will be different depending on the cellular context (Sherwood 2015). WNT pathway can stimulate both mitochondrial metabolism and aerobic glycolysis, at least in normal cells. A large number of genes involved in cellular metabolism have been described to be transcriptional targets of the pathway, including genes that regulate the metabolism of glucose, glutamine, or fatty acids (Sherwood 2015). In tumor cells, canonical WNT signaling stimulates aerobic glycolysis in several tumors, increasing the transcription of genes such as PDK1 or lactate transporters (Sherwood 2015) or inhibiting transcription of genes involved in the ETC, respectively (Sherwood 2015). Moreover, c-Myc is among the transcriptional targets of β-catenin. The protooncogene, usually dysregulated in tumor cells, coordinates various biological processes in CSCs such as cellular metabolism, redox homeostasis, self-renewal, differentiation, and growth. c-Myc has been shown to be highly expressed in CSCs from glioblastoma and necessary for the maintenance of the glycolytic

ä Fig. 2 (continued) Gli1-3, the pathway stimulates aerobic glycolysis. 5: activation of Notch induces a decrease in the expression of PTEN, which in turn activates PI3K/AKT. 6: Notch also establishes connections with other important intracellular regulators of cellular metabolism. Thus, there is a crosstalk between Notch and HIF1α. On the one hand, Notch stimulates HIF1α transcriptional activity leading to an increase in aerobic glycolysis. And on the other hand, HIF1α increases the proteolytic processing of Notch receptor, leading to the activation of the pathway. Notch activation also results in NFκB activation giving rise to positive feedback mechanisms since NFκB transcriptional activity increases Notch ligand expression. On the other hand, NFκB, which is a well-known redox sensitive transcription factor, has been classically described to inhibit aerobic glycolysis and stimulate OXPHOS, although this effect varies depending on tumor type. 7: finally, ROS can stimulate JAK/STAT signaling pathway in a positive feedback loop since activation of the pathway increases intracellular ROS levels. Once stimulated, the pathway can exert different effects on cellular metabolism depending on the subcellular localization of activated STAT3/5. Thus, translocation to the mitochondria leads to upregulation of OXPHOS, while translocation to the nucleus upregulates aerobic glycolysis

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phenotype. It stimulates the transcription of several genes involved in the regulation of glycolysis, in oxidative phosphorylation, glutaminolysis, or lipid synthesis, among others (Dang et al. 2009). This convergence between c-Myc and WNT leads to the overexpression of key proteins that stimulate glycolysis such as glucose transporters, LDH, or the enzyme PKM2 (which catalyzes the last step of glycolysis) (Dang et al. 2009). Noncanonical WNT activation also regulates glucose metabolism in tumor cells, being its crosstalk with the AKT-mTOR signaling pathway the main mechanism implicated (Fig. 2, #2). In this sense, β-catenin independent WNT activation leads to the activation of mTOR, a master regulator of cell metabolism. This regulation of mTOR occurs at the level of GSK3β, which phosphorylates and activates TSC proteins (mTOR inhibitors). Thus, noncanonical activation of WNT leads to phosphorylation and activation of AKT, which in turn phosphorylates and inhibits GSK3β, resulting in stimulation of mTOR and thereby stimulation of glycolytic metabolism (Inoki et al. 2006). This mechanism of stimulation of glycolytic metabolism mediated by noncanonical WNT activation has been described for several tumors such as prostate cancer or esophageal carcinoma. In addition to the crosstalk with the WNT pathway, AKT also established interconnection with other stem cell pathways such as Sonic/Hedgehog and the Notch pathway (Hales et al. 2014; Sun et al. 2017). Sonic/Hedgehog can also be activated by ROS in CSCs by an indirect mechanism (Fig. 2, #4). Thus, in hepatocarcinoma CSCs, ROS activate the transcription factor Nrf2 that directly interacts with the Sonic hedgehog (HH) promoter triggering activation of the pathway (Wing Leung et al. 2020). Through the activity of the Gli, the Sonic/HH pathway induces a metabolic change stimulating aerobic glycolysis. In fact, it has been described that in SHH-medulloblastoma (a type of tumor caused by mutation of the pathway) there is an increased expression of hexokinase 2 and pyruvate kinase M2 and an increased production of lactate, indicators of a Warburg-type metabolism. These changes are responsible, at least in part, for the maintenance of the undifferentiated and self-renewal state of CSCs in this tumor type. Furthermore, treatment with glycolysis inhibitors reduces HH-induced cell proliferation (Di Magno et al. 2014). Activation of Notch produces a decrease in the expression and activity of PTEN and an increase in phosphorylation and activation of AKT (Fig. 2, #5), which is accompanied by an increase in glucose uptake and glycolysis (Hales et al. 2014). The Notch pathway also establishes connections with other important regulators of cellular metabolism (Fig. 2, #6). In this sense, there is a crosstalk between the Notch pathway and HIF1α. On the one hand, it has been described that HIF1α directly interacts with the Notch intracellular domain, participating in the transcription of Notch-dependent genes and thus blocking the differentiation of normal stem cells. In fact, there is a positive correlation between HIF1α and Notch activity levels (Gustafsson et al. 2005). On the other hand, it has also been described that the Notch pathway stimulates the transcriptional activity of HIF1α and thus aerobic glycolysis. Upregulation of HIF1α, that can be triggered by several pathways, has been demonstrated to favor self-renewal and maintain the redox balance in CSCs through the increase of the glycolytic pathway, decreasing the flow into the

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mitochondria, which favors low ROS levels (Soeda et al. 2009). Moreover, Notch pathway is capable of regulating ROS levels in the CSCs population in a feedback mechanism, since ROS are in turn capable of stimulating the activity of the Notch pathway (Qiang et al. 2012). Notch signaling also leads NFκB activation in a crosstalk between both since activated NFκB also upregulate the expression of Notch ligands (Fig. 2, #6), promoting the production of CSCs by activating Notch signaling pathway (Moriyama et al. 2018). On the other hand, the interrelation between NFκB and ROS is a well-described and well-known fact. ROS- dependent NFκB activation plays an important role in inducing the expression of a wide variety of factors that promote cell survival and prevent cell death in tumor cells. NFκB can activate intracellular cascades that culminate in a decrease in ROS and thus favor the development of quiescent CSCs (Rinkenbaugh and Baldwin 2016). Also, the NFκB pathway has been reported to help maintain CSCs self-renewal. Its inhibition causes a decrease in the CSCs population and also in the expression of stem cell markers such us CD44, Nanog, and Sox, among others (Rinkenbaugh and Baldwin 2016). Several cytokines regulate NFκB signaling which in turns controls the expression of a variety of other cytokines that are essential for CSCs function. Moreover, NFκB signaling has been described to play a key role in the interaction of CSCs and the microenvironment. It has to be noticed that CSCs subpopulation occupy certain niches within the tumors and that the interaction with the niche microenvironment are essential for CSCs maintenance (Rinkenbaugh and Baldwin 2016). Furthermore, NFκB plays a key role in the metabolic adaptation of tumor cells. Activation of NFκB increases mitochondrial respiration and inhibit aerobic glycolysis in mouse embryonic cells. Thus, NFκB is controlling the balance between the use of aerobic glycolysis and mitochondrial respiration (Mauro et al. 2011). This regulation can vary depending on the tumor type, so that while in some tumors NFκB acts by activating mitochondrial respiration, in others it has been clearly described that it is capable of enhancing aerobic glycolysis by increasing the expression of glucose transporters or key glycolytic enzymes such as hexokinase 2 or pyruvate kinase M2 (Mauro et al. 2011). These variations are due in part to the pathway involved in each case, so that in general, the classical pathway would act by promoting aerobic glycolysis, while the alternative pathway would act by promoting mitochondrial respiration. On the other hand, aerobic glycolysis is in turn able to stimulate the activation of NFκB by establishing a reciprocal crosstalk. In this regard, it is common for NFκB signaling to work in coordination with other pathways, such as those regulated by p53 or JAK/STAT3. In fact, constitutive activation of NFκB and STAT3 has been reported in glioblastoma CSCs to regulate the expression of a variety of target genes that lead to activation of the Notch pathway, again demonstrating that these intracellular pathways essential for the control of CSCs are not independent but interconnected pathways and that the final effects depend on a fine regulation of them. JAK/STAT signaling pathway is primarily involved in inflammation, survival, and proliferation by activating transcription factors of the STAT family. Of these

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proteins, STAT3 and STAT 5 constitute the most relevant members in tumors, being overexpressed in a large number of tumor types (Rane and Reddy 2000). ROS can regulate the activation of these transcription factors both positively and negatively by a direct or indirect mechanism through the regulation of tyrosine kinases or tyrosine phosphatases pathways (Fig. 2, #7). Furthermore, the cellular redox state may later be regulated as a consequence of activation of the pathway. Thus, it is well known that once activated, STAT3 can migrate to the mitochondria and stimulate the ETC, increasing the production of ROS. On the other hand, STAT3 activation and translocation to the nucleus stimulates aerobic glycolysis. In other words, STAT3 effect on cellular metabolism will depend on its subcellular location, either in the mitochondria or in the nucleus, which is ultimately determined by the residue that is phosphorylated for activation (Linher-Melville and Singh 2017). These different effects according to the subcellular location will also determine a differential action on the cellular redox state. The stimulation of the ETC leads to an increase in the production of ROS, while among the nuclear targets of STAT3 are some antioxidant enzymes such as SOD that determine a decrease in intracellular ROS (LinherMelville and Singh 2017). Contrary to STAT3, the mitochondrial localization of STAT5 marks a shift from metabolism to an aerobic glycolytic one, mediated by overexpression of HIF2α, an isoform of HIF closely related to HIF1α that was identified in hematopoietic stem cells. Thus, HIF2α stimulates the expression of glycolytic genes. In fact, the inhibition of this HIF2α reduces the expansion and frequency of hematopoietic stem cells (Fatrai et al. 2011). In summary, main intracellular pathways controlling CSCs can be regulated by ROS and act in a coordinated way to control key functions in this tumor subpopulation, including metabolic plasticity. This fine control allows them to move from aerobic glycolytic to mitochondrial metabolism and vice versa, in order to cover the needs of every moment (from quiescent status to a proliferative and differentiated one).

Conclusions CSCs are highly resistant to conventional chemotherapy or radiotherapy and are mainly responsible for tumor relapse in patients. CSCs not only have the ability to initiate a tumor, but also have greater aggressiveness and ability to metastasize. Therefore, cancer-targeted treatments must be able to destroy this cell population in addition to the tumor mass. However, plasticity of CSCs represents a problem in the development of therapeutic options since multiple phenotypes within a single tumor may appear. Thus, a single given therapy will always fail to kill some of the CSCs. For that reason, approaches targeting plasticity of CSCs would be more effective (Das et al. 2020). As previously mentioned, there is great heterogeneity in the metabolic phenotype of CSCs, even in the same type of tumor. Thus, CSCs can modify their metabolic phenotype according to their needs (Peiris-Pagès et al. 2016). This metabolic plasticity offers advantages to CSCs, including chemoresistance and ability to metastasize, making it a potential target for CSCs eradication. Oxidative

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metabolism-dependent cells would respond better to mitochondrial respiration inhibitors while cells most dependent on glycolytic metabolism would respond better to glycolysis inhibitors. Several FDA approved drugs have been shown to affect CSCs in both options (Jagust et al. 2019). However, due to the great metabolic heterogeneity and plasticity present in tumors, combined treatment in which a greater variety of metabolic pathways are affected result much more effective (Jagust et al. 2019). Therapeutic strategies against CSCs targeting different pathways such as lipid, amino acid, or ketone metabolism have been also contemplated with mixed results (Jagust et al. 2019). Heterogeneous microenvironment conditions such as hypoxia, glucose deprivation, or low pH constitutes one of the main sources of metabolic adaptations in CSCs and is regulated by several factors including the HIF1-2 master regulator, making it an interesting therapeutic target for which various compounds have been developed (Das et al. 2020). In addition to metabolic plasticity, the cellular redox state (which have a great dependence on cell metabolism), also plays an essential role in CSCs, which usually prefer a low ROS environment. To achieve low levels of ROS, CSCs rely primarily on GSH redox system. Thus, blocking GSH synthesis could be an interesting therapeutic strategy to eliminate the CSC population. Treatments against SOD or GPX have also shown an improvement in CSCs response to conventional therapies (Jagust et al. 2019). In the same way, several compounds against Nrf2, the master regulator of the antioxidant response, have been tested (Jagust et al. 2019; Kahroba et al. 2019). Finally, the dependence of CSCs on epigenetic regulators both for the origin and for the maintenance and plasticity (Menendez et al. 2013) open the door for using epigenetic modulators as therapeutic strategies (Das et al. 2020). In summary, targeting CSCs plasticity seems to be the key to eliminate this population. However, since this plasticity is regulated by different interconnected mechanisms, the therapeutic approach should be oriented to the development of combined therapies that target more than one CSC property at the same time.

Cross-References ▶ Implications of ROS in Cancer Stem Cells Mechanism of Action ▶ Reactive Oxygen Species-Dependent Signaling Pathways in Cancer Stem Cells ▶ Targeting Redox Signaling and ROS Metabolism in Cancer Treatment ▶ Two-Faced Role of ROS in the Regulation of Cancer Cell Signaling

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Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Epithelial-Mesenchymal Transition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . What Is EMT? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Types of EMT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . EMT and Cytoskeleton . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Transcriptional Regulation of EMT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . EMT and Carcinogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Invasion-Metastasis Cascade . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . EMT and Cancer Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Oxidative Stress: Regulator of EMT and CSC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . What Is Oxidative Stress? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ROS and EMT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Association of ROS with EMT and Stemness . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Differential Level of ROS in CSCs and Non-CSCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract

Phenotypic plasticity is one of the crucial factors for carcinogenesis which involves acquisition of mesenchymal morphologies by epithelial cells. This process of transdifferentiation referred as Epithelial-mesenchymal transition (EMT), plays vital role behind the invasion and metastasis phenomenon during neoplastic progression. EMT has also been reported to be associated with the

S. Law (*) Department of Biochemistry and Medical Biotechnology, Calcutta School of Tropical Medicine, Kolkata, West Bengal, India R. Chatterjee School of Medical Science and Technology, Indian Institute of Technology Kharagpur, Kharagpur, West Bengal, India © Springer Nature Singapore Pte Ltd. 2022 S. Chakraborti et al. (eds.), Handbook of Oxidative Stress in Cancer: Mechanistic Aspects, https://doi.org/10.1007/978-981-15-9411-3_115

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development of a quiescent, drug/radiation resistant sub-population within the bulk tumor mass known as cancer stem cells (CSCs). A wide number of studies reported that the EMT associated process of carcinogenesis is often triggered by the elevation of cellular reactive oxygen species (ROS). The current chapter aims in presenting the mechanistic aspect regarding the correlative association of oxidative stress with EMT and EMT mediated neoplastic processes with special reference to the acquisition of stemness. In-depth knowledge in this domain may be helpful in designing successful anti-neoplastic therapeutic strategies. Keywords

Epithelial-mesenchymal transition · Invasion-metastasis cascade · Cancer stem cells · Reactive oxygen species · Oxidative stress

Introduction Epithelial-mesenchymal transition (EMT) is a natural biological phenomenon of transdifferentiation that directs the highly differentiated, polarized and organized epithelial cells to undergo phenotypic plasticity to become mesenchymal-like cells which are undifferentiated and isolated in nature having migratory and invasive properties (Nieto 2013). EMT is the normal physiological process involved in organogenesis, wound healing, tissue remodeling, etc. (Micalizzi et al. 2010). Pathophysiological attribution of EMT is found during oncogenesis (Acloque et al. 2008). Morphological changes, disorganization of cytoskeleton, disruption of cellcell adhesion and altered expression of transcription factors during EMT of cancer cells promote the cellular metastasis from the primary tumor site to different organs and thereby help in cancer progression (Lamouille et al. 2014). Apart from mediating migration and invasion, there are growing evidences in support of the fact that EMT also promotes the acquisition of multipotent characters and therapeutic resistance in a sub-set of cancer cells designated as cancer stem cells (CSCs) (Chatterjee and Chatterjee 2020). Many oncogenic signaling cascades are known to induce EMT in cancer cells. Interplay between extracellular signals and secreted factors viz.: transforming growth factor-β (TGF-β), epidermal growth factor (EGF), hepatocyte growth factor (HGF), fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), as well as different other signaling pathways mediated by integrin, Wnt, Notch, hedgehog, Ras, MMPs, etc. plays vital role behind the induction of EMT (Lamouille et al. 2014). Several microRNAs (miRNAs) are also known to be involved in the process of EMT (Zhang et al. 2010). Experimental evidences are there which showed that the development of hypoxic condition as well as the generation of reactive oxygen species (ROS) plays critical role in EMT induction (Rhyu et al. 2005; Radisky et al. 2005; Zhou et al. 2009). ROS can act as important cellular messenger that promotes the biological process of EMT by regulating the remodeling of extracellular matrix (ECM) and cytoskeleton, cell-cell junctions, cell mobility, etc. (Jiang et al. 2017).

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However, decline of ROS level has been reported in CSC phenotype, which is crucial for the maintenance of stemness (Chatterjee and Chatterjee 2020). Again, as mentioned earlier acquisition of stemness is dependent on EMT and ROS acts as the promoter of EMT. Thus it appears that ROS can play vital role to promote EMT and CSC phenotype but its level declines after the acquisition of stemness. There is much ambiguity in understanding the intricate molecular mechanistic scenario with this regard. The present chapter discussed the phenomenon of EMT and acquisition of stemness in the perspective of redox regulation. The knowledge in this domain can certainly potentiate the development of successful anti-neoplastic therapeutic strategies.

Epithelial-Mesenchymal Transition What Is EMT? Tightly bound adherent epithelial cells within an organized tissue structure can be morphologically converted to independent mesenchymal phenotypes possessing migratory properties having ability of invading the ECM (Fig. 1). EMT refers to the collection of the events that allows this phenotypic conversion (Chatterjee and Chatterjee 2020). EMT has been regarded as a naturally occurring transdifferentiation program. Epithelial cells are generally polygonal in shape exhibiting apical-basal polarity and remains tightly inter-connected via adherens and tight junctions. On the other hand, mesenchymal cells exhibit spindle-like morphologies and do not show apical-basal polarity. These cells remain loosely attached to the Fig. 1 Schematic representation of EMT

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surrounding ECM via focal adhesion which makes them more motile and invasive than their epithelial counterparts.

Types of EMT The process of EMT can be classified into three types as per the discussion of Kalluri and Weinberg (Kalluri and Weinberg 2009) which are as follows:

Type-1 EMT This type of EMT is not associated with fibrosis or cellular metastasis. Type-1 EMT can generate mesenchymal phenotypes which subsequently undergoes mesenchymal-epithelia transition (MET) to form secondary epithelia. These secondary epithelial cells can differentiate to form different epithelial tissue and can also undergo further EMT to generate connective tissues viz.: astrocytes, adipocytes, chondrocytes, osteoblasts, myocytes, etc. Type-1 EMT mainly occurs during the developmental stages viz.: embryonic implantation, gastrulation, and contribute in the formation of mesoderm, endoderm, neural crest cells, etc. Type-2 EMT This type of EMT contributes in wound healing, tissue regeneration and organ fibrosis. This type of EMT occurs as repair associated phenomenon is tissues that undergoes trauma and inflammatory injuries leading to the generation of fibroblasts and related cell types in order to tissue reconstruction. If the primary inflammatory insult is not removed, type-2 EMT persists and leads to the destruction of the affected organ by promoting fibrosis. Type-3 EMT Neoplastic cells that have previously undergone genetic and epigenetic changes exhibit this type of EMT. Type-3 EMT is responsible for the final life threatening manifestation of cancer because many studies have considered oncogenic EMT as the critical mechanism for the acquisition of malignant property. Morphological alteration of epithelial to mesenchymal phenotype promotes the metastasis of cancer cells as well as invasion to different sites thereby facilitates spreading of the cancer.

EMT and Cytoskeleton EMT process is driven by the alterations of structural proteins those are involved in the maintenance of cytoskeleton and strengthening of cell-cell adhesion. Replacement of E-cadherin by N-cadherin is the hallmark of EMT that leads to the weakening of adhesion between adjacent cells (Gheldof and Berx 2013). Besides the changing of adhesive repertoire, cells undergoing EMT also employ the processes for gaining migratory and invasive properties. These involve dramatic reorganization of actin molecules together with the development of

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membrane protrusions viz.: lamellipodia, filopodia, invadopodia, protostomes, etc. required for invasive growth (Yilmaz and Christofori 2009). Work of Peng et al. 2018 showed that actin polymerization results in the accumulation of intracellular hypoxic stress that results in the activation of HIF and NOTCH signaling which promotes EMT (Peng et al. 2018). Epithelial cells possess different types of keratins to constitute the intermediate filaments. Loss of keratin expression is one of the central features of EMT because it weakens cell-cell adhesion due to the loss of cell junctions viz.: desmosomes and tight junctions (Polyak and Weinberg 2009). The intermediate filaments of the mesenchymal cells are largely constituted by vimentin. Expressional elevation of vimentin is one of the canonical markers of EMT (Chatterjee and Chatterjee 2020). Vimentin promotes the transformation of polygonal epithelial shape of the cells to flatter, elongated mesenchymal structures. Interaction of vimentin with microtubules and associated motor proteins facilitate cellular motility (Dráberová and Dráber 1993; Gilles et al. 1999). Phosphorylation of vimentin also acts as an important factor for EMT. Active AKT can phosphorylate vimentin at ser-39 domain leading to the activation of the latter to further enhance motility and invasiveness of cells (Zhu et al. 2011). Along with increasing cellular motility and directional migration, vimentin also regulates mechanical stiffness of the cell (Danielsson et al. 2018). Vimentin also contributes in the enhancement of focal adhesion dynamics during EMT (Danielsson et al. 2018).

Transcriptional Regulation of EMT Cellular transdifferentiation as well as switching of behavior during EMT are regulated by key transcription factors viz.: SNAIL, zinc-finger E-box-binding (ZEB), basic-helix, and loop-helix transcription factors (Lamouille et al. 2014). Activation of these factors in turn activates mesenchymal proteins viz.: fibronectin, N-cadherins, vimentin, MMPs, etc. and downregulates epithelial markers and cell-cell junction proteins viz.: claudins, occludin, desmoplakin, palkophilin, E-cadherin, etc. Involvement of different signaling cascades and cross-talk among them has been reported to be involved in the initiation and progression of EMT. Tumor growth factor-β (Tgf-β) emerged as a potent EMT inducer in both SMAD dependent and independent pathways (Lamouille et al. 2014). Modulations of EMT transcription factors also occurs through integrin mediated signaling, receptor tyrosine kinases, developmentally important signaling cascades viz.: Wnt, Notch, Hedgehog, etc. (Lamouille et al. 2014). The cellular microenvironment also has profound roles in regulating EMT (Zing et al. 2011). IL-6 plays key role in EMT during inflammation and cancer progression (Zhou et al. 2017). Microenvironmental hypoxia can also induce EMT through HIF-1α (Yang et al. 2008). Some of the miRNAs plays vital role in controlling EMT, for example, miR-30a/b, miR-200 family, miR-203, etc. represses SNAIL expression, miR-200, miR-205, etc. represses the translation of ZEB, miR-300, etc. inhibits TWIST (Abba et al. 2016).

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EMT and Carcinogenesis Nearly 80% of the tumors are of epithelial origin which constitutes the cancers of lung, breast, pancreas, prostate, bladder, ovary, Kidney, gastro-intestinal tract, liver, etc. (Sun et al. 2016). Acquisition of malignancy by the benign state of tumor can be explained by EMT associated metastasis and invasion. EMT also promotes the acquisition of stemness in a sub-set of the bulk tumor (Chatterjee and Chatterjee 2020). These aspects of EMT have been vividly discussed here.

Invasion-Metastasis Cascade The concept of “invasion-metastasis cascade” and EMT has emerged more than two decades ago that refers to multiple steps (Chatterjee and Chatterjee 2020) which are as follows:

Step-1: Invasion It involves the passing of cells from primary tumor location to the surrounding stroma where EMT plays vital role by converting the organized epithelial cells to morphologically altered isolated ectomesenchymal cells which is associated with the loss of cell-cell junction, cytoskeletal reorganization, expressional decline of cell-cell contact proteins viz.: E-cadherin, γ-catenin, etc., increased expression of mesenchymal markers viz.: vimentin, fibronectin, α-smooth muscle actin (SMA), etc. and increased activity of matrix MMPs for making the way for cellular migration through microenvironment (Sun et al. 2016). During this step, negative charge density that separates adjacent tumor cell surfaces, increases resulting in electrostatic repulsion between them that facilitates the tumor cells to come to a free state from tumor tissue. Step-2: Intravasation After the detachment from the primary tumor site, the tumor cells enter to the circulatory system where abnormal loose adhesion contact establishes the implantation of tumor cells in the vascular wall (Jiang et al. 2015). Various factors viz.: growth factors, secretion factors, ECM components stimulates the directional migration of tumor cells through the circulation. Step-3: Extravasation This refers to the physical entrapment of the migrated tumor cells into the tissue parenchyma the organ where they have been transported (Jiang et al. 2015). The tumor cells responds to the environmental stress viz.: altered level of oxygen and nutrients, changed pH, occurrence of active oxygen free radicals and inflammation regulatory factor, to settle at the new site (Sun et al. 2016). Step-4: Colonization The metastasized tumor cells in order to colonize at the new site undergo the reversal of EMT by the process of redifferentiation of mesenchymal cells to epithelial phenotypes known as Mesenchymal-epithelial transition (MET) (Nieto 2013).

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The above discussion pointed towards the crucial involvement of EMT as a pre-requisite for malignant conversion.

EMT and Cancer Stem Cells A wide number of studies identified CSCs, a quiescent sub-population of the bulk tumor cells as the main cause of cancer relapse (Ayob and Ramasamy 2018). These cells confer resistance to both chemo and radio therapies (Chatterjee and Chatterjee 2020). CSCs exhibit efficient drug efflux mechanism due to the high expressions of ABC transporters as compared to that of non-CSCs. CSCs are also reported to possess superior DNA damage repairing system. EMT is reported to promote CSC phenotypes as well as the therapeutic resistance by the CSCs (Chatterjee and Chatterjee 2020). The details regarding the association of EMT and stemness particularly during oxidative stress have been vividly discussed in the latter section.

Oxidative Stress: Regulator of EMT and CSC What Is Oxidative Stress? Oxidative stress refers to the imbalance between level of anti-oxidant and free radicals i.e., oxygen containing molecules having single unpaired electron in the outermost electron shell, in a biological system. Due to the high reactive nature of the oxygen containing free radicals, these are referred as reactive oxygen species (ROS). ROS can react with nucleic acids and other cellular macromolecules to bring up irreversible alterations (Liou and Storz 2010).

ROS and EMT Due to the uncontrolled proliferation of cancer cells, the availability of nutrient and oxygen in the tumor microenvironment decreases and thereby the cells are exposed to hypoxic condition which is generally associated with the over-production of mitochondrial ROS. Works of Zhou et al. (2009), Radisky et al. (2005), Rhyu et al. (2005) identified ROS as a facilitator of EMT. ROS promotes EMT by regulating extra cellular matrix (ECM) remodeling, cytoskeletal rearrangements, cell-cell junctions and cellular mobility (Jiang et al. 2017).

Redox Regulation over EMC Remodeling ROS can initiate EMT through oxidative modification of Integrins. Oxidized integrin α7β1 can bind to laminin-111 to activate focal adhesion kinase (FAK) and Src that promotes cellular migration (Jiang et al. 2017; de Rezende et al. 2012; Canel et al. 2013). ROS also regulate urokinase plasminogen activator (uPA) system to facilitate ECM degradation. ROS activates activator protein-1 (AP-1) and NF-κB signaling pathways to promote the transcription of uPA and urokinase plasminogen activator

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receptor (uPAR) (Lee et al. 2009). ROS can also facilitate the binding of Hu antigen R (HuR) with AU reach elements of uPA and uPAR RNAs there by leading to their stabilization (Tran et al. 2003). Upregulations of the components of uPA system subsequently promote the conversion of plasminogen to plasmin that brings about ECM degradation (Jiang et al. 2017).

ROS and Cytoskeletal Rearrangement ROS has positive effect on the formation of lamellipodia and cellular extension due to its inhibitory action over 14-3-3ζ, a Slingshot-1L inhibitory factor. Slingshot-1L promotes the phosphorylation of cofilin that brings about lamellipodia formation (Kim et al. 2009). Oxidative stress associated oxidation of Cys374 residue of β-actin negatively regulates actin polymerization which leads to cytoskeletal remodeling (DalleDonne et al. 1995). ROS also leads to cytoskeletal injury by promoting tetramerization of tubulin (Jiang et al. 2017; Landino et al. 2014). Oxidative Stress and Cell-Cell Junction Regulation EMT initiation is associated with the decrease of cell-cell junction proteins. Decreased expression of occludin and claudin is related with the loss of tight junction and similarly, downregulations of E-cadherin and connexin are associated with the destabilization of adherens junctions and gap junctions respectively (Mayor and Etienne-Manneville 2016; Zihni et al. 2016). EMT factors viz.: Snail, Slug, Twist and ZEB repress these junction proteins (Jiang et al. 2017). Oxidative stress plays crucial role in the transcriptional upregulations of the EMT factors (Jiang et al. 2017). ROS inhibits Prolyl-hydroxylase mediated degradation of HIF-1α which is associated with the induction of snail and twist (Hielscher and Gerecht 2015). ROS promote degradation of IκB by IκB kinase (IKK) and thereby results in the translocation of NF-κB which leads to the transcription of snail, slug, twist and ZEB1/2 (Jiang et al. 2017). ROS has positive role over TGF-β signaling cascade. It promotes dissolution of TGF-β from latency-associated protein (LAP) which then is able to bind to the respective cell surface receptor (Pociask et al. 2004). ROS plays crucial role in the activation of apoptosis signal-regulating kinase 1 (ASK1) by inhibiting its association with thioredoxin (Trx) (Gotoh and Cooper 1998; Liu and Min 2002; Nadeau et al. 2007). ASK-1 activates Smad which is recruited for the downstream signaling by TGF-β-receptor complex (Jiang et al. 2017). ROS mediated phosphorylation of p53 leads to the formation of p53/Smad/p300 complex that ultimately promotes the transcriptional activation of Snail, Twist and MMPs (Jiang et al. 2017; Overstreet et al. 2014). Redox Regulation over Cell Mobility ROS promotes Src activation by inducing disulfide bond formation (Giannoni et al. 2005). Interaction between Src and FAK acts as the facilitator of focal adhesion formation as well as RhoA/ROCK signaling both of the process associated with the enhanced cell mobility (Jiang et al. 2017). The Src-FAK complex can also promote the transcriptional upregulation of EMT factors viz.: Snail, Twist, and MMPs through ERK activation (Jiang et al. 2017). FAK also confers resistance to anoikic by promoting Mdm2 associated degradation of p53 (Lim et al. 2008). ROS-mediated

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PTP inactivation also promotes FAK phosphorylation (31). ROS can also contribute to the PI3K/Akt signaling activation by repressing PTEN which results in Rac1/ CDC42 and the NF-κB signaling upregulations that promotes cellular mobility (Jiang et al. 2017). PI3K/Akt cascade also inhibits GSK-3β resulting in the nuclear translocation of β-catenin which in turn, upregulates Snail, Twist, and MMPs (Jiang et al. 2017).

Association of ROS with EMT and Stemness It is evident from the above discussion that ROS acts as a potential inducer of EMT and this phenotypic plasticity plays crucial role in imparting stemness in the cancer cells. Many studies revealed the association of ROS with EMT and stemness. Giannoni et al. (2011) showed that in case of prostate cancer, cancer-associated fibroblasts (CAFs) acts as in key determinant of malignant progression (Giannoni et al. 2011). CAF through a proinflammatory signature exploit ROS and induces EMT and stemness which drive the aggressiveness and metastasis of the prostate carcinoma. Work of Kim et al. (2013) regarding mesothelial carcinogenesis demonstrated that hydrogen peroxide associated oxidative stress induces EMT in human mesothelial cells (HMCs) as evident from the upregulation of Slug and Twist 1 and decline of E-cadherin expression (Kim et al. 2013). Stemness related genes viz.: OCT4, SOX2 and NANOG were also found to be increased significantly in these cells. The study also established the correlative association between the alterations of the mentioned molecules with the activation of Hif-1α and TGF-β1. Karicheva et al. (2016) demonstrated that in response to TGF-β induced ROS, Poly (ADP-ribose) polymerase 3 (PARP3) plays crucial role in EMT phenomenon of breast cancer cells by upregulating TG2-Snail-E-cadherin axis (Karicheva et al. 2016). The study considered the link between EMT and CSC induction and showed that PARP3 also facilitate CSC phenotypes by inducing SOX2 and OCT4 expression, increasing CD44high/CD24low population, promoting self-renewal of the stem like subset and in vitro spheroid formation. Lee et al. (2017) presented the correlation between the ionizing radiation associated ROS production and the occurrence of EMT and stemness (Lee et al. 2017). Recent findings of Chang and Singh (2019) for the first time revealed that nicotine exposure associated epigenetic modifications were responsible for carcinogenesis of kidney epithelial cells (Chang and Singh 2019). Their study showed that nicotine exposure was associated with the increase of ROS level in the proximal tubule epithelial cell line, HK-2 which was correlated with the EMT changes observed in the cells as well as stem cell like sphere formation.

Differential Level of ROS in CSCs and Non-CSCs The above mentioned studies clearly hinted towards the ROS associated EMT and acquisition of stemness by the cancer cells. A paradoxical phenomenon has been

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reported by wide number of studies which showed that though ROS acts as stemness inducer but CSCs exhibit low ROS level than non-CSCs within a tumor (Chatterjee and Chatterjee 2020). Decline of ROS level has been reported to sustain the CSC properties (Qian et al. 2018). Shi et al. (2012) hypothesized this as a strategy to protect “tumor-seed” from nucleic acid damage for the future progression of the malignancy (Shi et al. 2012). Lowering of ROS in CSCs was associated with the upregulation of anti-oxidant mechanisms (Qian et al. 2018). Detoxifying enzyme ALDH-1 was also reported as a weapon to decrease ROS level (Xu et al. 2015). It is evident that the ROS acts as a facilitator of CSC formation but after the acquisition of stemness, these cells take up the strategy for lowering of the ROS level. The triggering factors behind such switching of phenomenon remain unexplored.

Conclusion Taken together all the facts, it appears that EMT acts as a vital phenomenon behind the progression of malignancy by promoting tumor metastasis as well as acquisition of stemness. Oxidative stress emerged as a crucial factor associated with EMT especially with the development of CSC population. After the acquisition of stemness, CSCs adopt mechanisms for lowering up of the ROS level for sustaining the stemness. There is enough scope of the future research for identifying the triggering factors behind such paradoxical phenomenon regarding ROS level before and after the acquisition of stemness (Fig. 2).

Fig. 2 Schematic representation of the association of ROS with EMT and acquisition of stemness

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Therapeutic Effect of Natural Compounds in Targeting ROS-Induced Cancer

102

Pawan Kumar Raghav, Zoya Mann, Vishnu Krishnakumar, and Sujata Mohanty

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Natural Compounds in Regulation of ROS Generation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Polyphenols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Other Polyphenols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Alkaloids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Terpenoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Quinones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Miscellaneous Natural Compounds and Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1718 1719 1721 1751 1753 1754 1756 1757 1760 1760

Abstract

Every cell within the body maintains homeostasis for the production of energy through mitochondrial oxidation. This homeostasis is disturbed by high metabolic activity, mutations in cellular organelles (mitochondria, lysosomes, and peroxisomes), and crosstalk with infiltrating immune cells. These changes increase the level of reactive oxygen species (ROS) that activates oncogenes or suppresses the tumor suppressor genes through disrupted signaling pathways. The conventional administered therapies against cancer have incomplete efficiency, usually followed by severe repercussions such as drug resistance and tumor relapse. Therefore, an alternative approach has been adopted to replace these therapeutic models in which phytochemicals supplementation increases the chemotherapeutic efficiency in the treatment of ROS-induced cancer. Since these compounds are employed because of their wide-scale bioavailability and their synergism, this demonstrates a gradual decrease in tumorigenesis. In this chapter, we have summarized the plant-derived natural compounds based on their active P. K. Raghav · Z. Mann · V. Krishnakumar · S. Mohanty (*) Stem Cell Facility, DBT-Centre of Excellence for Stem Cell Research, All India Institute of Medical Sciences, New Delhi, India © Springer Nature Singapore Pte Ltd. 2022 S. Chakraborti et al. (eds.), Handbook of Oxidative Stress in Cancer: Mechanistic Aspects, https://doi.org/10.1007/978-981-15-9411-3_116

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role in modulating oxidative stress and cancer. Also, this chapter describes ROS’s impact on inducing oxidative stress and its regulation through the body’s innate defenses. These defense mechanisms include modulation through various genes and proteins, which tend to curb the stress level through their antioxidant activity. Mainly, the chapter provides a specific therapeutic role of the natural compounds to regulate ROS. The natural compounds have been categorized based on their function in the classes such as polyphenols (flavonoids and nonflavonoids), other polyphenols (diarylheptanoid (curcumin), and vitamins), alkaloids, terpenoids, quinones, and miscellaneous compounds like essential oils, isothiocyanates, and minerals. The combinations of these phytochemicals can be used to regulate ROS’s level for cancer treatment. Keywords

Cancer · ROS · Oxidative stress · Polyphenols · Antioxidant · Prooxidant

Introduction A plethora of exhaustive studies and approaches have been conducted and developed to elucidate the mechanisms of cancer progression. Despite this, the mortality rate of cancer is increasing every year worldwide (Manda et al. 2015). The healthy cells produce energy in the form of ATP by the metabolic process of oxidative phosphorylation (OxPhos) (Ralph et al. 2010a), wherein the oxidation of substrates such as pyruvate or lactate, free fatty acids, glutamine or glutamate, and ketone bodies takes place (Ralph et al. 2010b). However, glucose uptake in cancer cells is high and produces lactate in the presence of oxygen. This glycolysis/respiration ratio represents the difference between cancer and healthy cells, known as the Warburg Effect. The metastasis, cancer growth, and recurrence of several types of cancers are mostly due to cancer stem cells (CSCs). These CSCs possess stemness properties, maintain intratumor heterogeneity, and are resistant to radiotherapy and chemotherapy (Phi et al. 2018). Also, the reactive oxygen species-(ROS) induced stress is absent in CSCs, and they possess different machinery of metabolism compared to the non-CSCs. ROS are highly reactive oxygen-containing chemical species such as hydroxyl radical (OH•), singlet oxygen (1O2), and superoxide anion radicals (O2˙
) (Jackson and Loeb 2001). Mechanistically, the organelles like lysosomes and peroxisomes maintain the intracellular ROS level, but mitochondria majorly contribute this regulation through electron transport chain (ETC). The ETC channel is composed of four complexes, namely I, II, III, and IV. Electrons released from these complexes react with oxygen within the intermembrane mitochondrial space and matrix, reducing it to form superoxide radicals (Ralph et al. 2010a). Furthermore, the proapoptotic proteins, Bax, or Bak on mitochondria, induces the mitochondrial outer membrane permeabilization (MOMP) forming pores (Raghav et al. 2012a, 2019). The higher ratio of Bax/Bcl-2 (proapoptotic/antiapoptotic proteins) induces apoptosis and causes the rupture of the membrane (Raghav et al.

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2012b). Subsequently, the superoxide free radicals are then transported into the cytoplasm through these pores, where they are transmuted into hydrogen peroxide H2O2. The H2O2 levels within the cells are now maintained in two ways, either transported out of the cell to maintain the permissible baseline level of H2O2 within the cell or oxidized back into H2O molecules through the Haber-Weiss reaction. This production and maintenance of ROS are due to an imbalanced reduction of oxygen in mitochondria by cellular enzymes like NADPH oxidase, angiotensin II, lipoxygenase, and MPO, or external exposure such as pollution, excess intake of alcohol and tobacco, heavy metals, drugs, UV light or irradiation (Wu et al. 2006). Although low levels of ROS are required by each cell to sustain its homeostasis and maintain the cell cycle, ROS levels higher than the permissible limits can cause massive insult to cellular organelles and DNA. Interestingly, enzymes like superoxide dismutase (SOD), catalase, and glutathione peroxidase (GPX), maintain the redox balance between the reducing and oxidizing species, which regulate cancer cell’s proliferation and signaling pathways (Raj et al. 2011). The upregulation of naturally occurring antioxidants like glutathione (GSH) also neutralizes the mutilating effect of ROS (Hanot et al. 2012), failure in which causes DNA damage, lipid peroxidation, and disrupted cell cycle, all ultimately culminating in tumorigenesis. Thus, it is essential to maintain the ROS homeostasis to overcome the cell’s cytotoxicity (Noh et al. 2015). ROS is involved in the initiation, promotion, and progression of cancer (Goetz and Luch 2008). The initiation of cancer demonstrates the DNA mutation, and the promotion represents the apoptosis inhibition that leads to abnormal proliferation of mutated cells. However, the progression is associated with alteration in functional activity. Aside from ROS’s role in cancer promotion, it effectively regresses cancer (Diehn et al. 2009; Maraldi et al. 2009). For this purpose, radiotherapy, and phototherapy have been observed to increase ROS’s level in cancer cells, which causes sudden death of cells due to high oxidative stress. This elevated ROS level activates cyt c release from the mitochondria that induces apoptotic and necrotic cell death in human carcinoma cells (Rojo et al. 2014). Therefore, it is required to develop new approaches for targeted drug designing, which would deplete high oxidative stress and increase antioxidant levels to eradicate cancer. The present chapter discusses the regulation of ROS-induced cancer and the biological effect of polyphenols like flavonoids and nonflavonoids, alkaloids, terpenoids, quinines, and other miscellaneous antioxidants such as vitamins, minerals, isothiocyanates, and essential oils. The nonenzymatic important players that control this redox loop are vitamins and minerals, which serve as essential antioxidants. Hereby, this chapter encapsulates a detailed account of all the phytochemicals that have been most exploited to study their roles in the treatment of ROS-induced cancer (Fig. 1).

Natural Compounds in Regulation of ROS Generation Even though the body has its defense mechanism to fight off the oxidative stress within the system, yet the stress level might become grueling and out of control. The body will then need exogenous sources of antioxidants/prooxidants to keep the stress levels in check and maintain homeostasis. Through years of studies, the most

P. K. Raghav et al.

Fig. 1 (continued)

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Therapeutic Effect of Natural Compounds in Targeting ROS-Induced Cancer

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commonly occurring phytochemicals have been discovered and associated in not only tumor prevention but also been modified for therapeutic interventions (Pandey and Rizvi 2009). The commonly explored phytochemicals have been summarized as follows (Table 1).

Polyphenols Polyphenols are naturally occurring plant-derived compounds of the ubiquitous family and found abundantly in vegetables, beverages, fruits, and cereals (Firuzi et al. 2004). The polyphenols perform dual functions of antioxidants and prooxidants, and their structures possess multiple aromatic or a phenolic ring. The antioxidant polyphenols scavenge ROS containing hydroxyl groups on aromatic rings in mammals, reducing oxidative stress in cancer cells. In contrast, prooxidant polyphenols, including vitamins, have shown similar or even better effects. These natural polyphenols are broadly categorized into two main classes of compounds, viz., flavonoids and nonflavonoids (Table 1).

Flavonoids Flavonoids are natural dietary antioxidants, and their derivatives have a remarkable contribution to prevent cancer (Ahn-Jarvis et al. 2019). The epidemiological analyses reported that an abundant flavonoids diet helps to prevent cancer (Ahn-Jarvis et al. 2019). According to pharmacokinetics study, the liver and GI are the primary sources of flavonoids metabolism, wherein methylation, sulfation, and glucuronidation abolish their hydroxyl group (Jung et al. 2003). Thus, this section summarizes the effects of ROS on cancer, which is regulated through flavonoids. ä Fig. 1 Overall representation of anticancer effects of phytochemicals. A typical tumor cell is under oxidative stress due to accumulation of ROS (Reactive oxygen species) such as H2O2 (Hydrogen peroxide), O2 (Oxygen molecule), O2˙
(Superoxide anion radical), 1O2 (Singlet oxygen), and OH
(Hydroxyl free radicals) free radicals. Natural compounds released by phytochemicals drive tumor cells toward cell death via their antioxidant or prooxidant activity through different mechanisms. (a) Apoptosis elevates the expression of proapoptotic proteins, Bak, Bax, and suppressing antiapoptotic protein Bcl2 (B-cell lymphoma 2), which activates caspase 3 and caspase 9, leading to programmed cell death. (b) Mitochondrial membrane polarization (MMP) disruption occurs in the mitochondrial membrane that leads to the release of cyt c (cytochrome complex) into cytoplasm that activates caspases inducing cell death. (c) Cell cycle arrest activates tumor suppressor genes (p21 and p53), and suppression of Cyclin D and CDK 4, CDK 6 (Cyclin-dependent kinase) activity that blocks the cell cycle and halts tumor cell proliferation. (d) Oncogene regulation inhibits oncogenes (c Myc, ERK, Ras). (e) Regulation of free radicals through ROS scavenging activity of the compounds controls the oxidative stress within the tumor cell by upregulating antioxidant enzymes CAT (catalase), GPX (Glutathione peroxidase), and SOD (Superoxide dismutase). (f) The angiogenesis inhibition initiated by downregulating Akt levels and inhibiting VEGF (Vascular endothelial growth factor) activity. (g) The proliferation of tumor cells is controlled through the inhibition of ERK (Extracellular signal-regulated kinase), JNK (c-Jun N-terminal kinase), and mTOR (mammalian Target Of Rapamycin) pathways. Image is created using BioRender.com.

S.No. Classification Polyphenols Flavonoids 1 Anthocyanidin

Repairs/protects DNA. Antiangiogenic

C15H11O7 (Kation)

C17H15O7+

C15H11O5+

Delphinidin

Malvidin

Pelargonidin

Anti-angiogenic that causes inhibition of tumor growth

Stimulates apoptosis causing tumor growth regression

Inhibits tumor formation via its anti-angiogenic property

C15H11O6+

Mechanism of action

Cyanidin

Structures

Molecular formula

Compounds

25533011

25533011

25533011

20494645

PubMed ID

Table 1 Natural compounds are having antioxidants and prooxidant properties classified into flavonoids, nonflavonoids, other phenolic compounds, alkaloids, terpenoids, quinones, and miscellaneous compounds with their anticancer mechanism via ROS scavenging, apoptosis and regulating multiple pathway and genes. The Structure and molecular formula of each compound were retrieved from PubChem

1722 P. K. Raghav et al.

Flavan-3-ol

Flavone

2

3

Antioxidant. Acts as ROS scavenger by inhibiting HAT enzyme, p-JNK and p38 activity

C15H14O6

C22H18O11

Epicatechin

Epigallocatechin-3gallate (EGCG)

Proapoptotic, arrests cell cycle at G2/M phase and suppresses Nrf2 activity

Proapoptotic. Arrests cell cycle at G2 phase

C15H10O5

C15H10O6

Apigenin

Luteolin

Proapoptotic. Activates caspase pathway and Nrf2, suppresses STAT3 pathway, and upregulates Bax and Bak levels

Stimulates apoptosis inhibiting tumor growth

C16H13O6+

Peonidin

Therapeutic Effect of Natural Compounds in Targeting ROS-Induced Cancer (continued)

21601631

26180580

19602054, 24910845

26180580

25533011

102 1723

Classification

Flavonol

S.No.

4

Table 1 (continued)

Proapoptotic. Free radicals deplete GSH, causing cyt c release due to MMP. Bax and Bak elevated and Bcl-2 and Bcl-xL suppressed Induces apoptosis through STAT3, p53, and caspases activation

C15H10O7

C27H30O16

Rutin

Induces apoptosis through STAT3, p53, and caspases’ activation

Mechanism of action Inhibits apoptosis through MMD-intrinsic death pathway, and p53 and PUMA activation

Quercetin

Structures

C15H10O6

Molecular formula C15H10O5

Kaempferol

Compounds Wogonin

32823876

26167193

25147152

PubMed ID 24910845

1724 P. K. Raghav et al.

5

Flavonone

Prooxidant. Elevates ROS levels. Causes cytotoxicity by NFkb suppression

C15H12O6

C16H14O6

C27H32O14

Eriodictyol

Hesperetin

Naringin

Prooxidant. Elevates ROS levels. Causes cytotoxicity by NFkb suppression

Prooxidant. Elevates ROS levels. Causes cytotoxicity by NFkb suppression

Proapoptotic. Caspase 3, 9 activation by cyt c release. p53 and BAX levels increase and Bcl2 reduces

C15H10O5

Baicalein

(continued)

32979141

33265939

21824100

22381695

102 Therapeutic Effect of Natural Compounds in Targeting ROS-Induced Cancer 1725

Proanthocyanidins

7

9

Benzoic acid

Nonflavonoids 8 Benzoate

Classification Isoflavones

S.No. 6

Table 1 (continued)

p-Hydroxybenzoic acid

Anacardic acid

Procyanidin B2

Compounds Genistein

C7H6O3

C22H36O3

C30H26O12

Molecular formula C15H10O5

Structures

Suppresses tumor promotion by inhibiting NFkb and Tip50HAT activation Antioxidant. ROS scavenging activity

Prooxidant. Induces apoptosis and inhibits metastasis

Mechanism of action Proapoptotic. Causes cell cycle arrest by suppression of 3a-4 mediated metabolism, inhibiting oxidative metabolism

32245245

23041058

25533011

PubMed ID 26180580, 23680455

1726 P. K. Raghav et al.

Coumarins

Flavonolignans

Guaiacol

10

11

12

Gingerol

C17H26O4

C25H22O10

C19H12O7

Daphnoretin

Silibinin

C19H12O6

Dicumarol

Induces apoptosis by inhibiting NFkB signaling pathway and p38-MAP kinase

Induces apoptosis by MMP disruption that activates caspases. Acts on ERK ½, NF-KB,c-JUN, c-FOS pathway

Scavenges ROS. Suppresses DNA and protein synthesis

Cytotoxic to cancer cells by upregulating ROS levels

(continued)

30009484

22735354

23680455

28617852

102 Therapeutic Effect of Natural Compounds in Targeting ROS-Induced Cancer 1727

Classification Hydroxycinnamic acid

Lignans

Phenolic acid

S.No. 13

14

15

Table 1 (continued)

Caffeic acid

Secoisolariciresinol diglucoside (SDG)

Compounds Rosmarinic acid

C9H8O4

C32H46O16

Molecular formula C18H16O8

Structures

Proapoptotic at high concentration and antiapoptotic at low concentration

Proapoptotic. Arrests cell cycle at G2/M phase and inhibits STAT3 activity

Mechanism of action Antioxidant. Protects the membranes against oxidative damage

25533011

28990504

PubMed ID 19619938

1728 P. K. Raghav et al.

16

Stilbenoid

C14H12O3

C7H6O5

Gallic acid

Resveratrol

C16H18O9

Chlorogenic acid

Both Prooxidant and Antioxidant. Proapoptotic due to disruption of MMP and the release of cyt c. Scavenger of free radicals

Elicits protection against colon carcinogenesis by preventing DNA damage

Induces apoptosis by DNA methylation and activation of p38

(continued)

23041058

25533011

28990504

102 Therapeutic Effect of Natural Compounds in Targeting ROS-Induced Cancer 1729

Proapoptotic. Disrupts ETC system via MMD. Activates intrinsic death pathway

Catechol

19

C18H27NO3

Protects the membranes against oxidative damage

C20H24O5

Rosmadial

Benzofurans

18

Capsaicin

Induces apoptosis by induction of membrane permeability transition, reduction of bcl-2, and cytochrome c

C10H12O2

Mechanism of action

Eugenol

Structures

Molecular formula

Compounds

S.No. Classification Other phenolic compounds 17 Allylbenzene

Table 1 (continued)

24910845

25533011

25003106

PubMed ID

1730 P. K. Raghav et al.

Diarylheptanoid

Vitamin

20

21

Cytotoxic. Produces an anticancer secondary metabolite 3mercaptopropionaldehyde

C22H32O2

C20H30O

DHA

Vitamin A

Antioxidant. Prevents Lipid peroxidation

Both Antioxidant and Prooxidant. Scavenges ROS radicals and upregulates antioxidant enzymes. Cyt c release due to MMP disruption causes apoptosis

C21H20O6

Curcumin

(continued)

24790705

21774786

26167193, 25597786, 23916858

102 Therapeutic Effect of Natural Compounds in Targeting ROS-Induced Cancer 1731

Classification

Alkaloids 22 Allomatrine

S.No.

Table 1 (continued)

C11H8O2

Vitamin K3

C15H24N2O

C29H50O2

Vitamin E

Matrine

Molecular formula C6H8O6

Compounds Vitamin C

Structures

Proapoptotic. Causes decrease in ratio of Bcl-2/Bax, mitochondrial membrane disruption and caspase -3 activation

Cytotoxic agent, that generates ROS through redox reaction

Antioxidant. Proapoptotic. Arrests cell cycle at G1 phase. Protects lipids peroxidation

Mechanism of action Antioxidant. Reduces skin malonaldehyde, glutathione, and protein thiols content

32477114

26961313

24004441, 24790705

PubMed ID 24790705

1732 P. K. Raghav et al.

Amaryllidaceae

Ammothamnine

Benzophenanthridine

Benzylisoquinoline

23

24

25

26

Berberine

Chelerythrine

Oxymatrine

Pancratistatin

C20H18NO4+

Both prooxidant and antioxidant. Scavenges ROS and stabilizes DNA triplexes. Proapoptotic, triggers mitochondrial caspase release

Proapoptotic causes cell cycle arrest in S phase. Disrupts MMP, causes cyt c release and caspase activation

Induces apoptosis by increasing oxidative stress that disrupts mitochondrial membrane

C15H24N2O2

C21H18NO4

Activates the MMD- intrinsic death pathway, which decreases ATP. Activates Bax, P53 and Caspase

C14H15NO8

(continued)

23680455, 23916858

29780252

21774786

24910845

102 Therapeutic Effect of Natural Compounds in Targeting ROS-Induced Cancer 1733

Classification

Copyrine

Harmala

Imidazole

S.No.

27

28

29

Table 1 (continued)

Naamidine-A

Harmine

Sampangine

Compounds Boldine

C23H23N5O4

C13H12N2O

C15H8N2O

Molecular formula C19H21NO4

Structures

Proapoptotic. Downregulates Bcl-2, Mcl-1, and Bcl-xl without affecting Bax. Mitochondrial disruption and caspase 3, 9 activation Induces apoptosis by mitochondrial disruption and activation of caspases 3, 8 and 9

Prooxidant, inhibits telomerase activity which inhibits cell proliferation

Mechanism of action Anti-inflammatory and cytoprotective

19369860

27625151

26637046

PubMed ID 24944509

1734 P. K. Raghav et al.

Indole alkaloid

Indolizidine

Isoquinoline

30

31

32

Induces apoptosis by activation of caspase-3 and 9. Negative regulation of Mcl-1 and degradation of PARP

Proapoptotic. Causes decrease in ratio of Bcl-2/Bax, mitochondrial membrane disruption, and caspase -3 activation Proapoptotic. Enhances Bax expression and reduces Bcl-2 expression, MMP disruption causing cyt c release

C16H17NO4

C8H15NO3

C22H23NO7

Lycorine

Swainsonine

Noscapine

Proapoptotic. Cell cycle arrest when intercalates with DNA and inhibits topoisomerase II. Activates ERK and JNK pathways

C17H14N2

Ellipticine

(continued)

32791146

31781485

23932729

25107543

102 Therapeutic Effect of Natural Compounds in Targeting ROS-Induced Cancer 1735

Classification Oxazole

Oxoaporphine

Quinazoline

Quinoline

S.No. 33

34

35

36

Table 1 (continued)

Camptothecin

Evodiamine

Oxoisoaporphine

Compounds Streptochlorin

Induces apoptosis through Fas activation through ROS

C20H16N2O4

Prooxidant. Induces apoptosis. Disrupts mitochondrial membrane causing release of cyt c

Mechanism of action Induces apoptosis by activation of Bax and FasL, MMP disruption, Caspase3 activation, and degradation of PARP

Proapoptotic. Activates Bax and p53, downregulates Bcl-2 and caspase activation. Cell cycle arrest in G2/M phase

Structures

C19H17N30

C18H11NO4

Molecular formula C11H7ClN2O

21774786

32863934

31892146

PubMed ID 25931814

1736 P. K. Raghav et al.

Quinolizidine

Vinca alkaloid

37

38

CHIn4

C45H54N4O8

Vinorelbine

C16H25NO

SK228

Lycopodine

22285910, 22015944

Proapoptotic. Causes decrease in ratio of Bcl-2/Bax, mitochondrial membrane disruption and caspase -3, 9 activation Prooxidant. Activates JNK regulated DNA damage. Causes Mitochondrial membrane disruption and caspase activation

(continued)

30245856

31852250, 19786013

Prooxidant and proapoptotic. Causes mitochondrial disruption followed by release of cyt c and caspase-3 activation

102 Therapeutic Effect of Natural Compounds in Targeting ROS-Induced Cancer 1737

S.No. Classification Terpenoids 39 Carotenoid

Table 1 (continued) Molecular formula C40H56

C40H56

Compounds

Carotene

Lycopene

Structures

Antioxidant. Scavenges ROS, inhibits lipid peroxidation and DNA damage

Antioxidative. Acts as ROS scavenger

Mechanism of action

21615277

24790705

PubMed ID

1738 P. K. Raghav et al.

Diterpene

Lactone

40

41

Antioxidative. Elevates levels of antioxidant enzymes like catalase, SOD, and glutathione

C28H40O10S

Withaferin A

Prooxidative. Induces apoptosis, cell cycle arrest at G2/M phase. Activates caspase3 and 9

C18H12O3

Tanshinone

Shields the membrane against free radicals-induced oxidative stress

C20H26O4

Carnosol

Protects the membrane against the free radicals generated

C20H28O4

Carnosic acid

(continued)

25949858

23916858

25533011

25533011

102 Therapeutic Effect of Natural Compounds in Targeting ROS-Induced Cancer 1739

Classification Saponin

Sesquiterpene lactone

S.No. 42

43

Table 1 (continued)

C15H20O2

C42H68O13

Saikosaponin

Alantolactone

Molecular formula C42H66O17

Compounds Ginseng

Structures

Prooxidative. Causes GSH depletion and mitochondrial dysfunction

Prooxidant. Induces apoptosis, through oxidative stress

Mechanism of action Proapoptotic. Suppresses Nrf2dependent pathway

25656627

21774786

PubMed ID 21774786

1740 P. K. Raghav et al.

Cytotoxic, by generating carbon-centered free radicals. Proapoptotic by MMP loss and GSH depletion

Prooxidative via ROS generation and PI3K/AKT/ mTOR/S6K1 signalingdependent apoptosis

Induction of apoptosis under ROS generation due to Jnk activation and MMP loss that leads to cyt c release Prooxidant. ROS generation causes MMP disruption

C15H22O5

C15H24

C15H20O2

C22H28O8

Artemisinins

Beta-Caryophyllene

Costunolide

Eupalinin A

(continued)

25656627

25656627

25003106

21774786, 25656627

102 Therapeutic Effect of Natural Compounds in Targeting ROS-Induced Cancer 1741

S.No.

Classification

Table 1 (continued)

Prooxidative. Causes ROS generation that disrupts mitochondrial membrane potential

Prooxidative. ROS generation that causes mitochondrial membrane potential disruption

C15H18O5

C15H20O3

Salograviolide A Iso-secotanapartholide

Telekin

Induces apoptosis under JNK activation. MMP loss leads to cyt C release and GSH depletion

C15H20O3

Mechanism of action Induces apoptosis by disruption of MMP and caspase activation

Parthenolide

Structures

Molecular formula C15H18O4

Compounds Helenalin

25656627

25656627

25656627

PubMed ID 25656627

1742 P. K. Raghav et al.

Toluquinones

Benzoquinone

46

Antioxidant. Scavenges ROS. Inhibit JAK and STAT pathway

C32H46O9

Cucurbitacin

C7H6O2

Both Prooxidant and Antioxidant. Can scavenge ROS and also cause cytotoxicity in tumor cells

Proapoptotic. Causes cell cycle arrest at G2/M phase and ALP activation

Anti-inflammatory and cytotoxic. Decreases cyclin E, D1 levels. Activation of p53, JNK, caspase 3 and 9 and increase in TSG expression

C29H38O4

Celastrol

C15H10O5

Both prooxidant and antioxidant. Decreases tumorigenic miRNAs by ROS production. Also shows ROS scavenging activity

C30H48O3

Betulinic acid

Aloe-emodin

Triterpenoid

Quinones 45 Anthroquinone

44

Therapeutic Effect of Natural Compounds in Targeting ROS-Induced Cancer (continued)

17876050

17257888

23680455

23916858

23680455

102 1743

Classification Isoquinoline

Benzoquinone

Naphthoquinone

S.No. 47

48

49

Table 1 (continued)

C16H16O5

Shikonin

Anticancer. Inactivates NFkb

Proapoptotic. Cell cycle arrest, Inhibition of PI3K/Akt/mTOR pathway and downregulation of Bcl-2

C10H6O2

Mechanism of action Proapoptotic by ROS production that suppresses cancer cells’ proliferation

Naphtho (1,2-b) furan-4,5-diome

Structures

Dual response, proapoptotic in glioblastoma via cyt c release, antiapoptotic in gastric carcinoma by decreasing caspase activity

Molecular formula C15H14N2O3

C10H12O2

Thymoquinone

Compounds Cribrostatin 6

22381695

22381695

27573448

PubMed ID 20169400

1744 P. K. Raghav et al.

Isothiocyanates

52

BITC

C8H7NS

Zn

Mineral

51

Zinc

C17H19NO5

Miscellaneous natural compounds 50 Dioxolane Piperlongumine

28353636

Under deficiency creates a tumor microenvironment. Accumulation is tumorigenic due to overexpression of estrogen receptor BITC inhibits the proliferation of cells via inhibiting the ERK-1/2, JNK, and regulating the focal adhesion kinases

Therapeutic Effect of Natural Compounds in Targeting ROS-Induced Cancer (continued)

28423628

24910845

Induces apoptosis by p53 independent pathway, postoxidative stress caused due to ROS generation

102 1745

Classification

Essential Oils

S.No.

53

Table 1 (continued)

– – – –

– – – –

Artemisia lavandulaefolia Boswellia sacra

Salvia libanotica Zanthoxylum schinifolium

–

–

C6H11NOS2

Sulforaphane

Structures

Aniba rosaeodora

Molecular formula C9H9NS

Compounds PEITC

Induces apoptosis under JNK activation. Disrupts MMP that depletes GSH. Inhibits PI3K and ERK1/I Selectively induces apoptosis, advantageous against cancer cells Causes mitochondrial stress and caspase-activated apoptosis Selectively induces apoptosis in cancer cells Inhibits tumor growth Prooxidative. Induces apoptosis via ROS generation

Mechanism of action Proapoptotic. Activates JNK and depletes GSH due to MMP disruption

25003106 25003106

25003106

25003106

25003106

23999506, 18671201

PubMed ID 28423628

1746 P. K. Raghav et al.

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Therapeutic Effect of Natural Compounds in Targeting ROS-Induced Cancer

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Majorly, these flavonoids are classified into six sub-classes, including proanthocyanidins (procyanidin B2), anthocyanidins or anthocyanins (cyanidin, delphinidin, malvidin, pelargonidin, peonidin), flavones (luteolin, apigenin), isoflavones (genistein), flavonol (kaempferol, quercetin, rutin), flavan-3-ol (epigallocatechin-3-gallate, (
)-epicatechin, catechin), and flavanone (eriodictyol) (Gibellini et al. 2015; Hollman and Katan 1999). Proanthocyanidins The proanthocyanin such as procyanidin B2, is a free-radical scavenger that significantly induces apoptosis and inhibits the proliferation in the 4T1 cell line (mouse breast cancer) (Li et al. 2014). Later, the results of an in vivo study where 4T1 cells were injected subcutaneously in Balb/c mice and treated with proanthocyanins were found corroborating with in vitro findings. These results exhibited the efficacy of proanthocyanins to exterminate breast cancer and also inhibit metastasis. Anthocyanidin or Anthocyanin Anthocyanin is an anti-angiogenic compound rich in berries, which possesses anticarcinogenic properties. This compound inhibits the growth of blood vessels leading to reduced tumor formation. Grape seed proanthocyanidin has been studied to be proapoptotic and even inhibit metastasis in human breast carcinoma cells (Li et al. 2014). Flavones Flavones are glycosylated forms of luteolin and apigenin, present in abundance in celery and parsley (Shay et al. 2015). The flavones are cytostatic compounds having the parent structure, 2-phenyl-4H-1-benzopyran-4-one, which prevent proliferation of and stimulate apoptosis in human colon cancer cell line, HT-29. Apigenin is a flavone commonly found in garlic, cabbage, guava, bilimbi fruit, celery, French peas, bell pepper, and wolfberry leaves. Apigenin causes G2/M phase cell cycle arrest and inhibits tumorigenesis in colon carcinoma cell lines (SW480, Caco-2 and HT-29). Also, it inhibits chemo-drug-induced ROS-mediated drug resistance in lung cancer, and triggers proapoptotic activity by suppressing NF-κB activation as reported in the prostate, liver, and pancreatic cancer. Luteolin is also found in certain spices like oregano and encourages apoptosis through G2 cell cycle arrest (Shay et al. 2015). Isoflavones Genistein is an isoflavone commonly found in legumes and soybean seeds, and has been used in treating breast cancer, since it inhibits estrogen receptor β, 20-fold higher than estrogen receptor α (Chenand Chien 2014). Therefore, isoflavones are structurally similar to estrogen hormone, hence competing with it to bind at the estrogen receptor and called phytoestrogen. Among all the other polyphenols, genistein is a nontoxic isoflavone, proven to be a potential anticancer agent, though inferior in vivo bioavailability, and the low water solubility makes it less efficient for solid tumors. However, at low doses, it leads to more tumor growth and chemo-

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resistance in breast cancer. It has been reported to show proapoptotic activity in liver and lung cancer as well via cell cycle arrest (Shay et al. 2015). Flavonol Kaempferol (3,5,7-trihydroxy-2-(4-hydroxyphenyl)-4H-1-benzopyran-4-one) is an example of flavonol found in various plants (broccoli, tea, grapes, cabbage, kale, beans, strawberries leek, endive, and tomato). This antioxidant has shown an increased inhibitory effect on ROS due to more hydroxyl groups. Kaempferol exhibits anti-proliferative activity and induces apoptosis by modulating the targets such as STAT3 and p53, activation of caspases, and ROS production. The synergistic effect of conventional chemotherapeutic drugs and kaempferol reduces the toxicity caused by chemotherapy (Rajendran et al. 2014). Quercetin (3,30 ,40 ,5,7-pentahydroxyflavone) has shown anticancer activity and performs dual activities (prooxidant and antioxidant). This ubiquitous natural compound is present in tea, vegetables, onions, nuts, fruits, red wine, and seeds. The effect of quercetin on mitochondrial oxidative phosphorylation is the main topic of interest because of its explicit accumulation in the mitochondria and inhibition of ATP synthase (Gibellini et al. 2015). A similar effect as kaempferol has been shown by quercetin, wherein it scavenges the mitochondrial ROS (O2
and H2O2) by a high number of hydroxyl groups and conjugated π orbitals. Subsequently, the reaction between quercetin and O2
generates an unstable product, semiquinone radicals that cause DNA damage, lipid peroxidation, and production of H2O2, later reacting with H2O2 decreases the peroxidase level. Quercetin induces apoptosis by upregulating the expression of the proapoptotic protein Bax and downregulating expression of the antiapoptotic protein Bcl-2. This change in protein expression triggers the loss of mitochondrial membrane potential (MMP), subsequently releasing cytochrome c from mitochondria and activating caspase-3 and caspase-7. Hydroxyethylrutosides (oxerutins) are semi-synthetically derived hydroxyethyl esters prevailing naturally as flavonol rutin (or rutoside). Hydroxyethylrutosides are widely used to treat chronic venous diseases, which decreases hyperpermeability and edema. The commercial drugs, Venoruton, Relvene, and Paroven are the mixtures of hydroxyethylrutosides and have been prescribed for chronic venous disorders (Aziz et al. 2015). Flavan-3-ol Epigallocatechin-3-gallate (EGCG) is abundantly found in green tea and used to treat colon cancer. EGCG upregulates the expression of the antioxidant promoting gene, nuclear factor-erythroid-2-Related Factor 2 (Nrf-2). Also, EGCG activates caspase-3/7 that inhibits the expression of Bcl-2, XIAP, and survivin, and induces apoptosis in CSCs isolated from the human prostate tumor. In nasopharyngeal carcinoma, EGCG inhibits the STAT3 pathway, reducing tumor growth initiation and progression (Shay et al. 2015). (
)-Epicatechin is associated with the group of flavan-3-ol, commonly found in cacao and cacao-based products (green tea and dark chocolate. However, the seaweed like Halimada (Chlorophyceae) contains a high level of polyphenols, such as

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Therapeutic Effect of Natural Compounds in Targeting ROS-Induced Cancer

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(
)-epicatechin and EGCG. Besides, synergistic use of (
)-epicatechin and chemotherapy has shown an improvement in radiotherapy received by MAP kinase mutated patients (Shay et al. 2015). This improvement is due to the 4-hydroxyl groups of (
)-epicatechin, which can scavenge and neutralize the increased ROS in cancer cells. Also, it suppresses the androgen receptor activation and gene transcription that inhibits the proliferation potential of prostate and breast tumors. Mechanistically, inhibiting histone acetyl-transferase (HAT) activity reduces prostate cancer cell viability, while (
)-epicatechin significantly inhibits p38, p-JNK, and cleaved caspase-3 levels when used in combination with radiation treatment. Catechins, on the other hand, are chemo-preventive agents for prostate cancer, though they have poor efficiency when used in chemotherapy against prostate cancer (Shay et al. 2015). Flavanone Flavanones are found abundantly in the fibrous part of citrus fruits compared to their juice. The citrus fruits such as naringin, hesperetin, and eriodictyol are the major source of flavanones. The flavanones suppress NF-κB and increase the ROS level to induce cytotoxicity in cancer cells (Shay et al. 2015).

Nonflavonoids Nonflavonoids include stilbenes or stilbenoids (pterostilbene and resveratrol), phenolic acid (gallic acid, chlorogenic acid, vanillin acid, ellagic acid, ferulic acid, nordihydroguaiar, methyl gallate, salicylic acid, caffeic acid, and sinapic acid), hydroxycinnamic acid (rosmarinic acid, p-hydroxybenzoic acid, protocatechuic acid, and p-coumaric acid), hydroxybenzoic acids, flavonolignans (silibinin), coumarins (dicumarol, daphnoretin, esculetin, fraxetin, aesculetin), and lignans (secoisolariciresinol diglucoside (SDG)) (Table 1) (Tungmunnithum et al. 2018). Stilbenes or Stillbenoid Resveratrol (3,5,40 -trihydroxystilbene) is a phytoalexin, a robust antioxidant abundantly found in red wine, grapes, blueberries, and raspberries. Resveratrol has two isoforms, “cis” and “trans”, and it upregulates the expression of oxidative phosphorylation and mitochondrial biogenesis genes to enhance the mitochondrial function (Lee et al. 2013). Resveratrol effectively scavenges hydrogen peroxide, hydroxyl radical (OH•), and superoxide anion, through hydrogen atom transfer and SPLET (Sequential proton loss electron transfer) mechanisms. It is also a chelator of transition metals like copper and iron, which inhibit lipid peroxidation and increase glutathione. The cancer-preventive and cytoprotective effect of resveratrol have been displayed because of its antioxidant activity in several types of cells such as keratinocytes, cardiomyocytes, neurons, brain tissue, and adipocytes. At high concentration, it overwhelms the expression of Bcl-2, which subsequently releases cytochrome c that triggers caspases to induce apoptosis in human carcinoma cells (D’Archivio et al. 2008). Resveratrol has been considered for evaluation in ongoing clinical trials for lymphoma and colon cancer. The combination of resveratrol and

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5-fluorouracil (5-FU), compared to 5-FU alone, significantly inhibits the tumor in H22 hepatocarcinoma lymphoma, and colon cancer. Phenolic Acid The phenolic acids are present abundantly in red berries like strawberry and cranberry, black radish, mango, tea, coffee, and onion. The derived phenolic acids, including gallic acid, caffeic acid, coumaric acid, ferulic, and gentisic acid are abundantly found in argan oil, olive oil, oats wheat, berries, coffee, and artichokes. Primarily, it has been studied in the in vitro melanoma model that the phenolic acids and their derivatives inhibit NF-κB and ROS enzymes (lipoxygenases and XOD) that suppress ROS production and promote antiapoptosis (D’Archivio et al. 2008). Gallic acid decreases lipid peroxidation and inhibits the DNA damage that prevents colon cancer. Gallic acid significantly increases the number of antioxidants such as SOD, catalase, glutathione reductase and glutathione peroxidase but reduces glutathione in rats. Synergistically, the gallic acid and ECGC chelate the transition metals that decrease toxicity due to ROS, and also activate tumor suppression genes like p21, p53, and Bax. Caffeic acid works in a dose-dependent manner, being proapoptotic at higher concentration and antiapoptotic at a lower concentration, hence making caffeic acid not a very reliable intervention due to its non-reproducible results. Hydroxycinnamic Acids and Hydroxybenzoic Acids Polyphenols such as rosmarinic acid, p-hydroxybenzoic acid, p-coumaric acid, protocatechuic acid have contributed to Lycopus lucidus and tea antioxidant potential measured using DPPH and NO scavenging assays (Lee et al. 2013). Flavonolignans Silibinin (silybin) is a natural bioactive component of flavonolignans and has proved to be a potential agent to suppress angiogenesis and growth in the colon, liver, bladder, prostate, and lung cancers. Silibinin upregulates the association of p27/CDK4 and p21/CDK4 complexes while downregulating the E2F1/DP1complex association inhibited by phosphorylation of retinoblastoma in HuH7 cells. Additionally, silibinin arrests the cell cycle at G1 and G2-M phase in HepG2 cells and Hep3B cells, respectively, and decreases the expression of cyclin-dependent kinase, (CDK) 2, CDK4, cyclin D1, D3, and E, which collectively increase the level of Kip1/p27 (Varghese et al. 2005). Coumarins Coumarin-derived antioxidant, dicumarol, increases the oxidative stress, inducing ROS and cytotoxicity in human pancreatic cancer cells. It mediates oxidative stress within the tumor cell by affecting the ETC in mitochondria, which leads to production of free radicals O2
and H2O2, culminating in apoptosis (Martin-Cordero et al. 2012).

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Lignans Lignans are formed when residues of cinnamic acid form a dimer. Since they are analogous to human estrogen, they are also called phytoestrogens (Sharma et al. 2018). Lignans are found abundantly in flaxseed and sesame, but not so prevalent in fruits and vegetables. Secoisolariciresinol diglucoside (SDG) is an essential lignan and abundantly found in flaxseed that shows antitumor activity by reducing the high estrogen level in breast and prostate cancer. Sesame oil is a rich source of lignans that shows cell cycle arrest at G2/M phase and STAT3 inhibition that drives the tumor cells toward apoptosis.

Other Polyphenols Polyphenols, including diarylheptanoids (curcumin) and vitamins (A, C, E, and K3), have been studied to effectively regress cancer by regulating the oxidative stress levels within tumor cells (Liu et al. 2018). Although these two classes primarily effectuate an antioxidant effect through the mechanism of ROS scavenging, recent studies show that these polyphenols actually act as double-edged sword, showing both prooxidant and antioxidant activity as summarized in Table 1.

Diarylheptanoid Curcumin Curcumin (1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione) is a diarylheptanoid polyphenol that is isolated from rhizomes of Curcuma longa (turmeric) commonly used as a spice. Curcumin effectively scavenges the hydroxyl radical (OH•), O2
, and H2O2 (Borra et al. 2014). As to its direct role in reacting with ROS, it upregulates the expression of cytoprotective and antioxidant proteins indirectly through Nrf-2. Curcumin suppresses cancer growth by inhibiting in vitro and in vivo cell proliferation and was found nontoxic to humans. Furthermore, curcumin increases apoptosis through p53-pathway and the upregulation of Bax expression in human cancer cells. Curcumin inhibits proliferation and induces apoptosis; it also suppresses mammosphere formation in several cancers due to G2/M phase cell cycle arrest. Turmeric-derived compound, diferuloylmethane, has potent anti-inflammatory and antioxidant properties and has been recommended to be included in the diet for the treatment of inflammatory lung disease. Currently, curcumin-based phase-I/II trials are ongoing for the treatment of pancreatic, colorectal cancer, and multiple myeloma (Borra et al. 2014).

Vitamins Vitamins are essential nutrients not synthesized sufficiently in the human body, thus required for intake from exogenous sources. Vitamins are antioxidants, including vitamins A, C, E, and K considered safe and can be administered in larger doses over a longer duration. Vitamins have the advantage of being recycled back into their

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active forms after ROS detoxification, which improves their bioavailability compared to other antioxidants (Liu et al. 2018). Carotenoid (Vitamin A or Retinol, Fucoxanthin) Vitamin A (retinol) is a β-carotene derivative formed in the liver known to prevent lipid peroxidation. This vitamin belongs to the retinoid family and is considered a weaker antioxidant as proved by the obtained non-significant difference of progression-free survival between vitamin A treated and chemotherapy-alone patients. The weight and number of tumors formed are effectively reduced on the administration of natural form of vitamin A (retinyl palmitate), while vitamin A drug (13-cis-retinoic acid) only decreases the weight of the generated skin tumors within mice. Nevertheless, the topical application of 13-cis-retinoic acid and retinyl palmitate has a synergistic effect of inhibiting the growth of skin papillomas and is proved effective against skin cancers (Liu et al. 2018). Vitamin C Vitamin C (ascorbic acid) is a water-soluble potent reducing antioxidant found mostly in citrus fruits as the oxidized form, L-ascorbic acid. It acts primarily by reacting with hydroxyl and lipid peroxyl free radicals, transforming them into H2O and getting self-oxidized into dehydro-L-ascorbic acid (Liu et al. 2018). Vitamin C protects cell membranes peroxidation efficiently and scavenges H2O2, superoxide, 1 O2, peroxyl radicals, and hydroxyl radical. Under the Warburg effect, GLUT isoforms are overexpressed in the cancer cells’ membranes, which increases the glucose uptake by the tumor cells and permits the preferential uptake of dehydro-Lascorbic acid. This oxidized form of vitamin C is an essential iron scavenger that promotes the reduction of ferric ions into ferrous ions through cellular metalloenzymes. Nonetheless, its oral administration does not affect ultraviolet (UV) radiation-induced erythema in patients. Vitamin C also reduces skin malonaldehyde, glutathione, and protein thiols content, though it increases collagen production, improves inflammatory skin conditions, and protects against the damaging effect of UVA and UVB rays (Pandey and Rizvi 2009). Dehydroascorbic Acid (DHA) DHA is the oxidized form of ascorbic acid and is also abundantly present in the human diet. DHA selectively targets the cancer cells to eliminate the growing tumors in the mouse models. In this approach, DHA reacts with homocysteine thiolactone, a chemical present in cancer cells, and converted into a cytotoxic compound, 3-mercaptopropionaldehyde (Song and Kim 2016). Vitamin E Vitamin E is a lipid-soluble vitamin, prevalent either as tocopherols or tocotrienols. Its variants have four isoforms, which differ in the locus and quantity of methyl groups present on the aliphatic chain. The level of vitamin E was found reduced in UVR-induced oxidative stress conditions, whereas its treatment to the cells reduces the amount of sunburn. Likewise, lower levels of vitamin E compared to control

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have been observed in breast cancer. Tocopherols is a lipid-soluble derivative of tyrosine, found in plasma membranes that reacts with peroxide radicals and singlet molecular oxygen (1O2), and protect lipids against peroxidative damage. Vitamin E has both antioxidant and UV absorptive properties, which protect against UV-induced skin photodamage (Liu et al. 2018). Also, Tocotrienol acted as an antioxidant and have antitumor or proapoptotic properties. The chemistry of this antioxidant property involves the donation of hydrogen ions from its side-chain methyl groups to the ROS, which hence terminates the serial peroxidation of lipids and hydroxyl ions. It promotes epigenetic modifications and demonstrates the antitumor activity by G1 phase cell cycle arrest in pancreatic, colon, cervical, and bladder cancer (Liu et al. 2018). The combination of Tocotrienol with antitumor drugs like simvastatin decreases the self-renewal ability through inhibiting STAT3 levels within the CSCs in breast cancer (Liu et al. 2018).

Vitamin K3 The single-electron reduction of vitamin K3 produces semiquinone that again gets oxidized back to vitamin K3 in the presence of oxygen. This reduction of vitamin K, combined with chemotherapeutic drugs, produces ROS that causes oxidative stress and cytotoxicity in cancer cells (Badave et al. 2016).

Alkaloids Alkaloids are a class of nitrogen-containing organic molecules. Most of the alkaloids have been extracted from natural herbs and serve as a rich reservoir for drug discovery, owing to their anti-proliferation and anti-metastasis activity (Habli et al. 2017). Among the alkaloids discovered so far, the extensively studied molecules, sampangine, boldine, quinoline, ellipticine, and berberine, are summarized in Table 1.

Sampangine Sampangine is a class of polycycles copyrine alkaloids, naturally present in the Cananga odorata, Anaxagorea dolichocarpa, and Duguetia hadrantha stem bark. These molecules exert their anticancer activity through the generation of ROS, inhibition of telomerase activity, and DNA interaction to form G-quadruplex (G4) complexes at telomere, where they block telomerase hybridization and catalytically elongate the telomere, and inhibits cell proliferation (Rodriguez-Arce et al. 2020). Boldine Boldine (boldo tree extract) stands out due to its higher polyphenol content and has various characteristics such as anti-inflammatory, hepatoprotective, cryoprotective, and choleretic property. It induces apoptosis by downregulating Bcl-2 and Hsp70,

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while upregualting Bax protein levels. It is also known to activate caspase 3/7 and caspase 9 that leads to cell death ultimately (Paydar et al. 2014).

Quinoline Camptothecin is a quinoline alkaloid that exhibits antitumor and antileukemia effects. It also activates Fas to induce apoptosis through ROS and oxidative stress pathways in highly resistant medulloblastoma (a malignant brain tumor). Its analogs have also been studied to be proapoptotic in myeloid leukemia cells by activating protein kinase Cδ through proteolytic action (Kovacic and Somanathan 2011). Pyridocarbazole Ellipticine is an antineoplastic pyridocarbazole and intercalates within DNA and is an efficacious inhibitor of mammalian topoisomerase II. Ellipticine alkaloid elevates oxidative stress that breakdown mitochondrial transmembrane potential and releases cytochrome c, leading to apoptosis in human melanoma cells. It induces oxidative stress by arresting cells at G2/M phase and disrupting mitochondrial potential that leads to ROS generation, and ultimately apoptosis by AIF release through caspaseindependent pathway (Saeidnia and Abdollahi 2013). Isoquinoline Berberine is a derivative of isoquinoline alkaloid isolated from Chinese medicinal herb Huanglian and shows antineoplastic activities against various cancers. Berberine triggers mitochondrial-dependent apoptosis that activates caspase release and decreases Bcl-XL, Bcl-2, and Bid expression in hepatocellular carcinoma (Saeidnia et al. 2013).

Terpenoids Terpenoids is another class of chemo-preventive natural compound, mostly isolated from traditional Chinese herbs (Huang et al. 2012; Yang et al. 2020). Based on their structure, the terpenoids have been classified into five major classes, namely, triterpenoids (betulinic acid, oleanolic acid, celastrol), lactone sesquiterpenoids (artemisinin, parthenolide), saponins (ginseng), caretonoids (carotene, lycopene), and diterpernoids (tanshinone) (Table 1).

Triterpenoid Triterpenoids such as betulinic acid, its derivatives (methyl ursolate, β-boswellic acid, and celastrol), and synthetic analogs (glycyrrhetinic acid [2-cyano-3,11-dioxo18β-oleana-1,12-dien-30-oc acid (CDODA)] and oleanolic acid [2-cyano-3,12dioxooleana-1,9-dien-28-oic acid (CDDO)]) are effective anticancer agents that exhibit antiproliferative activities. These compounds induce ROS generation, and sequentially decrease miR-27a, miR-20a/miR-17-5p expression in several cancer cell lines (Saeidnia and Abdollahi 2013).

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Celastrol or tripterine is a Chinese herb extract and an antioxidant of quinine methide triterpenoid, which demonstrates anticancer, and anti-inflammatory effect. Celastrol decreases the levels of cyclin E and cyclin D1 while it increases the levels of p21 and p27 that collectively inhibit the hepatic tumor cells’ growth (Hu et al. 2013).

Lactone Sesquiterpenoid Lactone sesquiterpenoid is a plant-derived compound that induces oxidative stress and acts as cytotoxic/anticancer activity. Increased production of ROS promotes the characteristics of cancer development and progression in healthy cells. Oppositely, increased ROS initiates apoptosis through the mitochondrial-dependent pathway and suppresses cancer progression. Generally, Lactone sesquiterpenoid decreases the high level of GSH to induce apoptosis in cancer cells (Gach et al. 2015). Artemisinins is an antimalarial compound known to have cytotoxic effect in tumors. Artemisinins elevate ROS by accommodating an endoperoxide (–C–O–O–C–) bridge that undergoes cleavage to generate carbon-centered free radicals. This culminates in apoptosis by inducing oxidative stress, cell cycle arrest, and autophagy (Kovacic and Somanathan 2011). Parthenolide is a sesquiterpene-derived compound known to exhibit the antiinflammatory and anticancer activity. Parthenolide suppresses proapoptotic genes through inhibition of the transcriptional activity of NF-κB and STAT genes or by direct inhibition of associated kinases (IKK-β) (Takai et al. 2013). Saponins Saponin derivatives are currently being accepted as anticancer agents. Saikosaponin is a derivative known to sensitize cancer cells to cisplatin through oxidative stressmediated apoptosis, making its combination with cisplatin a practical therapeutic approach. Ginseng is another derivative known to function as a cancer preventive agent, especially for human gastric and breast carcinomas. It acts on the Nrf-2 pathway by ameliorating suppressing oxidative stress within the cancer cells (Takai et al. 2013). Carotenoid Carotene is an antioxidant carotenoid that scavenges the oxidative stress-induced ROS, though its oral consumption for skin treatment has some adverse effects. Carotene is used for photoprotection and effective against UV-induced erythema, but it is found futile against severe UV irradiation. The increased dose concentration and duration of carotene are required for the successful treatment of severe skin diseases (Godic et al. 2014). Lycopene is a tomato-derived carotenoid-based antioxidant that scavenges ROS and prevents lipid peroxidation and DNA damage. The combination of lycopene with other antioxidants would likely play a pivotal role in anticancer treatment (Kelkel et al. 2011).

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Diterpenoids Compounds under this classification consist of four isoprene subunits. Tanshinone shows anticancer activity by intercalating with the DNA of the tumor cell and upregulating TNFα, which induces ROS production followed by apoptosis. It has also been observed to inhibit metastasis by reducing MMP2 and MMP9 levels, which synergizes with its prooxidant effect (Hu et al. 2013).

Quinones Quinone (Table 1) moieties are most commonly present in many drugs, such as doxorubicin, anthracyclins, mitoxantrone, daunorubicin, saintopin, and mitomycin used to treat solid cancers (Ziech et al. 2012).

Cribrostatin 6 Cribrostatin 6 generates large amounts of ROS within the treated cells, which potentially induces apoptosis, not followed by any defined cell cycle arrest (Cui et al. 2014). Naphthoquinone Napthoquinones are naphthalene derivatives having two carbonyl oxygen atoms, in which 1,4-naphthoquinone is the most stable naphthoquinone derivative. Additionally, Lawsone, Shikonin, Juglone, Plumbagin, Naphthoquinoidal and Menadione are the most prevalent derivates of naphthoquinone (Ziech et al. 2012). In vivo and in vitro studies have reported that the Plumbagin or 5-hydroxy-2-methyl-1, 4-naphthoquinone (extracted from roots of the plant Plumbago zeylanica L.), a yellow-colored secondary metabolite of the quinone family, has antioxidant and anticancer properties. This compound shows better antitumor activity when administered synergistically with a chemotherapy drug. However, the mechanism of action differs in different cancer subtypes, in lung cancer it arrests the cell cycle at G2/M phase in tumor cells and inhibits PI3K/Akt/mTOR pathway. Likewise, in HER2 positive cells, it also causes cell cycle arrest at G2/M phase and initiates mitochondria-mediated apoptosis, while it potently targets both receptors for positive and negative tumors in breast cancer. Nonetheless, it shows antitumor activity by inhibiting Bcl-2, which leads to apoptosis on its regimen along with taxol in the case of triple-negative cancers. Plumbagin directly suppresses the generated ROS in mitochondria to inhibit tumor progression in cervical cancer, while it indirectly suppresses ROS by inhibiting STAT3 to induce apoptosis in gastric and esophageal cancer. Another quinone of interest is Shikonin, the bioactive component of Zicao, a root derivative of the plant Lithospermum erythrorhizon. Its vigorous anticancer activity has been observed in various cancer subtypes, including leukemia, gastrointestinal, pancreatic, lung, and breast cancer, primarily by a different mode of ROS generation, and by acting on PI3K/Akt/mTOR pathway, that ultimately leads to apoptosis (Ziech et al. 2012).

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Thymoquinone Thymoquinone is a natural bioactive compound obtained from the black seeds of Nigella sativa L, having a monoterpene structure. The protective effect of Thymoquinone is studied in different cancers such as breast, colorectal, prostate, lung, glioblastoma, and fibrosarcoma. All these studies have shown to decrease angiogenesis via modulating the VEGF through Akt and extracellular receptor kinase pathway. Interestingly, this compound exerts both apoptotic and antiapoptotic activity, like in gastric carcinoma, it significantly reduces the activity of caspase-3 and caspase-9, whereas, in glioblastoma cells, it induces apoptosis via elevating Bax and cytochrome c (Zhu et al. 2016). Toluquinones Toluquinones (triprenylated toluquinones and toluhydroquinones) induce apoptosis in esophageal cancer cell lines. These compounds scavenge ROS and are capable of killing cancer cells (Whibley et al. 2007).

Miscellaneous Natural Compounds and Products Essential Oils (EOs) EOs are lipophilic and concentrated hydrophobic liquids of aromatic plants, they easily penetrate inside the cell (Blowman et al. 2018; Gautam et al. 2014). Essential oils and their constituents such as Citral, Carvacrol, Thymol, Myrcene, α-humulene, perillyl alcohol (POH), Geraniols, β-caryophyllene, d-limonene have been considered as antioxidants and used in cancer therapy. The anticancer effect of EOs involves increasing ROS levels and cell cycle arrest, inducing apoptosis, modulating DNA repair, anti-metastasis, and anti-angiogenesis, anti-proliferative capacity in cancer cells. Furthermore, EOs and their constituents have indicated cytotoxic effects in leukemia, breast, colon, liver, lung, prostate, mouth, and brain cancer. Correspondingly, EOs regulate MAPK-pathway, transcription factors (NF-κB and AP-1), tumor suppressor proteins (p53 and Akt), and detoxification enzymes (glutathione peroxidase, glutathione reductase, SOD, and catalase). Table 1 contains important EOs which regulates ROS in cancer cells. Boswellia Carteri (Frankincense Oil) EO The constituents of this EO modulates the expression of apoptosis-related genes, CDKN1A, NUDT2, GAD45B TNFAIP3, SGK, IL6, IER3, and DEDD2 in bladder cancer cells. Also, this EO downregulates Bcl-2 and upregulates Bax genes, which activates caspase-9 and caspase-3 and stimulates apoptosis in human oral epidermoid carcinoma KB cells (Gautam et al. 2014). Artemisia Lavandulaefolia EO This EO's main compound, 1,8-cineole, induces apoptosis, which involves mitochondrial and MAPKs pathways. EO constituents cleave an indicator of apoptosis,

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poly(ADP-ribose) polymerase-1 (PARP) in mouth cancer KB cells (Gautam et al. 2014). Salvia Libanotica EO This EOs’ principal constituent such as linalyl acetate, terpineol, and camphor and their combinations effectively inhibits proliferation through a caspase-dependent pathway in HCT-116, colon cancer cell lines (p53+/+ and p53
/
), though no significant changes have been noted in standard intestinal cell line. Additionally, EOs and their constituent inactivate PARP-1 protein induces the release of caspases and then cancer cell death (Gautam et al. 2014). Boswellia Sacra EO The Boswellia sacra EOs induces PARP cleavage that leads to apoptosis in MDA-MB-231 cells. Also, it influences the Akt protein expression that regulates a tumor suppressor protein (p53) (Gautam et al. 2014). Aniba Rosaeodora (Rosewood) EOs This EO derivative increases ROS generation that induces apoptosis in cancer cells. The ROS accumulation leads to depolarisation of mitochondrial membrane, followed by caspase activation and phosphatidylserine externalization on the tumor cells, which leads to apoptosis (Gautam et al. 2014). Zanthoxylum Schinifolium EO This EO targets the cancer cells, liver (HepG2) to induce apoptosis wherein, it decreases the levels of glutathione, cellular antioxidants, and increased ROS production has been found. The extract of EO from this plant is known to trigger caspase-independent apoptosis, as seen in hepatocellular carcinoma (Gautam et al. 2014).

Minerals Minerals are an essential component for the proper functioning of the antioxidant machinery of the body (Table 1). Most of the minerals act as cofactors of antioxidant enzymes like zinc for SOD and selenium for GPX. As immunoproteins they play an essential role in regulating oxidative stress with cancer cells (Lee 2018; Hariharan and Dharmaraj 2020). Zinc Zinc is a cofactor for antioxidant enzyme SOD, that induces the synthesis of metallothionein and sequesters to reduce hydroxyl radicals within the cytoplasm. It also acts as a suppressor of oxidative stress that inhibits the NOX enzyme, and TNFα-induced NF-κB pathway. Zinc deficiency has been associated with the risk of breast cancer, as the oxidative stress increases macrophage infiltration, creates a pro-tumor microenvironment, and its accumulation within the cell leads to overexpression of estrogen receptor, ultimately causing mutations within the mammary gland and triggering carcinogenesis (Lee 2018).

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Selenium Selenium is an immunonutrient that is a part of selenocysteine, which is a component of GPX and TXR enzymes. These enzymes, with the aid of selenium derivatives called selenoproteins are responsible for maintaining the oxidative state of the cell by scavenging ROS. It has also been observed that organic selenium compounds show better anticancer activity than their inorganic counterparts. Recent studies have reported that organic selenium derivatives comprise nucleophilic molecules that also play an essential role in controlling metastasis, with minimal systemic effects (Hariharan and Dharmaraj 2020).

Isothiocyanates Glucosinolates are naturally occurring secondary metabolites present in the cruciferous plants, which hydrolyzes to form isothiocyanates. The main classes of glucosinolate are Benzyl Isothiocyanates (BITC), which are mostly seen in cabbage, garden cress; Phenethyl Isothiocyanates (PEITC), which are present in watercress, turnip; and sulforaphane present in abundance in green vegetables like broccoli (Table 1) (Lin et al. 2017; Sestili and Fimognari 2015). BITC Benzyl Isothiocyanate (BITC) has various effects on different cancers. In the case of blood cancer, BITC inhibits cell proliferation by inhibiting the c-Jun N-terminal Kinase (JNK), ERK-1/2, and regulating the focal adhesion kinases. In contrast, in the case of breast cancer, BITC targets p53 activation and suppresses the tumor formation, while in brain tumor, BITC downregulates protein kinase C, which plays a significant role in cell signaling and inflammation (Lin et al. 2017). PEITC Phenethyl Isothiocyanate (PEITC) controls oxidative stress in the cell through thiol modification, particularly affecting the GSH antioxidant system. PEITC inhibits GPX by extruding the GSH out of the cell, which leads to overproduction of ROS, consequently damaging mitochondria leading to apoptosis in the cancer cells. Its preferential selection for the cancer cells makes it a suitable candidate for clinical studies. In combination with rapamycin, it is quite effective in eradicating Akt-activated tumor cells, as observed in preclinical studies, but found ineffective in treating Akt-induced chemo-resistant cells (Lin et al. 2017). Sulforaphane This is a bio-compound found in broccoli as L-sulforaphane (SFN) and identified as a chemo-preventive agent by activating phase II antioxidant enzymes. Sulforaphane, an isothiocyanate, has been studied extensively to induce apoptosis through cell cycle arrest, disruption of microtubule polymerization, and by sensitizing cells to TRAIL factor in lung carcinoma tumor cells. Emerging evidence suggests that it also acts as an HDAC inhibitor, as HDAC acts as an oxidative stress sensor and plays an essential role in cancer progression (Sestili and Fimognari 2015).

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Conclusion It is well established that the oxidative status of the cell forms the ground for the overall functioning of a tumor cell (Hanahan and Weinberg 2011). Since the tumor cell is capable of maneuvering its oxidative status by compromising the standard scavenging mechanisms of a non-tumor cell, this mechanism summarizes that the overall ROS production is upregulated in a cancer cell as compared to a normal cell (Liou and Storz 2010). Conventional interventions like chemotherapy and radiation are not full proof due to the significant concern that they might hamper the ROS levels in a normal cell and turn into tumorigenic (Liou and Storz 2010). The irradiated patients can be supplemented with combinations of dietary antioxidants, which would likely minimize collateral damage. These antioxidants function primarily to downregulate the ROS levels in the tumor cells through regulation of ROS-related apoptotic signaling pathways like PI3K/AKT, MAPK/JNK/p38, JAK/STAT, and ER stress pathways. Importantly, phytochemicals like genistein and daidzein act as a double-edged sword and promotes the overproduction of intracellular ROS, which can be toxic to both the healthy and neoplastic cells (Bonomini et al. 2015). Therefore, for the wide-scale application of these phytochemicals in fighting cancer by targeting oxidative stress of the cancer cells, many factors must be taken into consideration, like study model, dose concentration, and exposure period. This approach can mark the future of therapeutics as an alternative intervention model.

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Therapeutics of Oxidative Stress and Stemness in Breast Cancer

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Balraj Singh, Kalpana Mujoo, and Anthony Lucci

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Oxidative Stress, Tumor Heterogeneity, and Cancer Progression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Role of NO in Stem Cells and DNA Damage Response . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Role of ROS in Cancer Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Role of Oxidative Stress in Evolving Cancer Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Role of Oxidative Stress in Microenvironment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Therapeutic Targeting of Oxidative Stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Future Directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cross-References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract

Oxidative stress (OS) is induced by chemical reactions triggered inside our body by generation of reactive oxygen and nitrogen species and externally by environmental insults and other stresses. However, balance exists between oxidative stress and anti-oxidative mechanisms to maintain homeostasis. OS has a role in cancer initiation, promotion and progression. Most chemotherapeutic drugs, radiation and other therapeutic modalities mainly target highly proliferating cancer cells of various organs, while sparing small number of rare dormant low cycling cells with stem-like properties which may further encounter mutations, chromosomal B. Singh (*) · A. Lucci (*) Department of Breast Surgical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Morgan Welch Inflammatory Breast Cancer Research Program and Clinic, The University of Texas MD Anderson Cancer Center, Houston, TX, USA e-mail: [email protected]; [email protected] K. Mujoo Institute of Molecular Medicine, UT Health at Houston, Houston, TX, USA © Springer Nature Singapore Pte Ltd. 2022 S. Chakraborti et al. (eds.), Handbook of Oxidative Stress in Cancer: Mechanistic Aspects, https://doi.org/10.1007/978-981-15-9411-3_117

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aberrations, changes in transcriptome, epigenetic changes and genomic instability. Cancer progression, being evolution-like process, is governed by various selection pressures. Oxidative stress could be a crucial component of selective pressure (s) triggered by a variety of mechanisms such as metabolic stress. Oxidative stress can also change microenvironment, e.g., rendering it pro-inflammatory, and as cancer reaches to advanced stage, highly abnormal and adaptable cancer cells are selected. Hence, in advanced stage, cancer cells can dictate changes in microenvironment to suit them. Targeting oxidative stress as therapeutic intervention is difficult due to tumor heterogeneity, wherein OS and its connected networks may serve different functions in different subpopulations of cancer cells. Strategies for modeling highly adaptable cancer cells that survive high OS and other bottlenecks in driving cancer will aid drug discovery effort at halting cancer progression. Applying this strategy in resistant breast cancer, low-dose 6-mercaptopurine, which is a safe and effective inhibitor of chronic inflammation, appears suitable for inhibiting highly adaptable cancer cells. Keywords

Cancer evolution · Tumor adaptability · Metabolic stress · Triple-negative breast cancer · Minimal residual disease · Cell culture model of therapeutic resistance · 6-Mercaptopurine

Introduction Alterations in cell metabolism play a pivotal role in initiation, promotion and progression of different types of cancers. Preservation of normal cellular function and cell survival depends on the regulation of redox homeostasis (Gorrini et al. 2013). Cancer cells exhibit high levels of oxidative stress and aerobic glycolysis known as Warburg effect (Cairns et al. 2011). Oxidative stress is triggered due to inequality between reactive oxygen species (ROS) generation and elimination; high ROS levels in cancer cells are counteracted by antioxidant mechanisms (Diehn et al. 2009). Among many cellular processes involved in regulating oxidative stress and its consequences, many of which are described in other chapters of this book, we have chosen to emphasize on nitric oxide (NO) signaling. In this chapter, we will discuss the roles of oxidative stress and stemness in the progression of breast cancer. In this regard, besides the studies on cancer stem cells, we also include studies performed with embryonic and induced pluripotent stem cells. The main goal is to highlight the issues related to oxidative stress and stemness in the context of breast cancers that do not respond to current therapies. In cancer cells, oxidative stress in connected to several vital processes and cancer cell microenvironment. We will discuss how oxidative stress may impact cancer progression when it is viewed as an evolution-like process wherein tumor adaptability plays a

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key role. We also describe a strategy to tackle highly adaptable cancer cells in breast cancer patients in a timely manner before disease relapse.

Oxidative Stress, Tumor Heterogeneity, and Cancer Progression Chemical species (oxygen-containing) with reactive properties are defined as reactive oxygen species (ROS) and these include superoxide (O2• ) and hydroxyl (HO•), nitric oxide radical (NO•), singlet oxygen (1O2), ozone (O3), peroxynitrite (ONOO ), and many other free radicals and nonradical molecules including hydrogen peroxide (H2O2). Mitochondria are the major source of ROS production (Finkel 2012; Handy and Loscalzo 2012); however, technologies which allow the measurement of live cell respiration point to additional sources of ROS generation as contributor to oxidative stress (Nohl et al. 2003). ROSs are produced by both enzymatic and non-enzymatic reactions. ROS generation by enzyme catalyzed reaction involve NADPH oxidase, xanthine oxidase, arachidonic acid, and uncoupled endothelial nitric oxide synthase. In addition, other enzymes such as lipoxygenase, cyclooxygenase, and cytochrome P450 are also involved (Gorrini et al. 2013; Sosa et al. 2013). The tight regulation of ROS is needed to maintain cellular homeostasis. Low levels of ROS act as signaling molecules for cell proliferation and differentiation and also in activation of stress responsive survival pathways (Janssen-Heininger et al. 2008). H2O2 has been shown to induce proliferation, differentiation, and migration (Rhee 2006). Similarly, NO• is a diatomic free radical involved in number of physiological processes including smooth muscle relaxation and in pathological conditions such as diabetes and cancer (Murad 2006).

Role of NO in Stem Cells and DNA Damage Response The NO-cyclic GMP pathway plays an important role in proliferation and differentiation of human and murine embryonic stem (ES) and induced pluripotent stem cells into cells of various lineages (Mujoo et al. 2008, 2011). Since the NO-cyclic GMP pathway exhibits diverse roles in cancer, the roles of the NO-receptor soluble guanylyl cyclase (sGC) and other components of the pathway in the regulation of the tumor cell proliferation were evaluated. These studies collectively suggest that the effect of activators/inhibitors of NO–sGC–cGMP in inhibition of tumor cell proliferation is mediated by both cGMP-dependent and independent mechanisms (Mujoo et al. 2010). High concentrations of NO have also been shown to cause DNA damage in stem cells and cancer cells and robust DNA damage response (DDR) is needed to ensure cell survival. Previous studies indicated that differentiation of stem cells increases residual DNA damage with increased S-phase-specific chromosomal aberration after

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exposure to DNA-damaging agents. Treatment of stem cells with NO donor (NOC-18) reduced double-stranded break repair by homologous recombination (HR) suggesting that DNA repair by HR is impaired in differentiated cells (Mujoo et al. 2017).

Role of ROS in Cancer Cells A connection between ROS and cell transformation was identified when it was found that insulin raised intracellular H2O2 and increased tumor cell proliferation (Oberley 1988). At low levels, ROS stimulates mitogen-activated protein kinase and extracellular signal regulated kinase (ERK) phosphorylation, JUN N-terminal kinase (JNK) activation and cyclin D expression and all of these events are linked to cancer growth and survival (Martindale and Holbrook 2002; Ranjan et al. 2006). Further, studies have shown that ROS can induce pro-inflammatory cytokines and NF-κB pathway (Naik and Dixit 2011). In addition, ROS has been reported to reversibly inactivate tumor suppressors such as protein tyrosine phosphatases (PTPs) and phosphatase and tensin homolog (PTEN) (Leslie et al. 2003). PTPs have been shown to reduce ROS levels by increasing antioxidant expression (Harris et al. 2014). Cancer initiation, promotion, and progression are multistep process and studies have suggested that ROS is possibly involved in all three stages (Weitzman and Gordon 1990). Point mutations and chromosomal aberrations causing irreversible changes in DNA occur during tumor initiation (Bakhoum and Compton 2012) and there are implications that increased levels of oxidative DNA lesions are involved in etiology of various cancers (Sosa et al. 2013). Exposure to chemical carcinogens and radiation leads to loss of genomic integrity and both are potential sources of ROS. Although DNA damage exerted by some chemotherapeutic drugs and IR can cause cancer cell death, it can also lead to mutations and chromosomal rearrangements (Ameziane-El-Hassani et al. 2010). Tumor heterogeneity is developmentally regulated, by mutations and by a large number of changes in transcriptome (alternative splicing, other base-modifications, editing, etc.). Tumor heterogeneity in breast cancers (which include inter and intratumor heterogeneity) is prevalent and represents an important feature of malignancy (Fidler and Kripke 1977). In breast cancers, inter-tumor and intra-tumor heterogeneity is characterized by clinical and histopathological grades, biomarker expression, and genetics. Further, studies indicate heterogeneity in circulating tumor cells and epigenetic heterogeneity in breast cancer patients (Turashvili and Brogi 2017). Based on a myriad of previous studies, it is clear that most chemotherapeutic drugs and radiation and other therapeutic modalities mainly target highly proliferating cancer cells of various organs, while sparing low number of dormant low cycling cells with stem-like properties including some non-stem like cells (Dittmer 2018). Both radiation and chemotherapy exert their anti-tumor effect partly due to generation of ROS, resulting in DNA damage and subsequent single- and double-stranded breaks (Kryston et al. 2011). Human and mouse breast cancer stem cells (BCSCs),

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similar to their normal tissue counterparts, have low levels of ROS but high expression of anti-ROS systems which accounts for their radio-resistance. However, pharmacological blockade with ROS scavengers in BCSCs decreases their clonogenicity thus leading to radio sensitization (Guzman et al. 2005).

Role of Oxidative Stress in Evolving Cancer Cells Cancer progression can be viewed as an evolution like process which is formed by various types of selection pressure bottlenecks. Only a small subpopulation of cancer cells among heterogeneous cancer cells can succeed in facing these challenges. In this model, various intrinsic and extrinsic factors, including oxidative stress, are collectively viewed as determinants of cell fate (Fig. 1a). Further, it is postulated that only stem-like cells, not all proliferating cells, are capable in succeeding in cancer evolution (Fig. 1b). From a clinical perspective, although primary breast cancer can be removed by surgery, persistent minimal residual disease (MRD) could have poor prognosis leading to high risk of relapse and metastasis (Giancotti 2013; Janni et al. 2016). Cancer cells that persist after high dose chemotherapy and radiation have stem-like properties and are also referred as cancer initiating cells (CIC). Studies suggest that ROS mediates normal stem cell differentiation and renewal (Shi et al. 2012); however, comparatively little is known about the redox status of CICs. Some studies have shown that due to high expression of ROS-scavenging systems, breast and liver cancer stem cells have low ROS levels (Diehn et al. 2009; Kim et al. 2012). Since treatments such as IR and some chemotherapeutic drugs

Fig. 1 Interrelationships between oxidative stress, stemness, and cancer. (a) Various factors including oxidative stress that influence each other are depicted as combined determinants of cell fate. (b) For simplicity, cancer cells are categorized into stem-like and non-stem-like cells. They respond to a given stress very differently, exemplified by cell survival versus cell death

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induce high ROS levels for elimination of majority of tumors, a very small percentage of cancer cells (CICs) survive under high ROS conditions by upregulation of antioxidant levels. Additional oxidative stress induced by such treatments can induce mutations and DNA damage leading to drug resistance (Gorrini et al. 2013; Diehn et al. 2009). Breast cancer stem cells (BCSCs) transition between proliferative epithelial-like (E) and dormant mesenchymal-like (M) states with elevated aldehyde dehydrogenase activity and CD24-CD44+ expression respectively (Liu et al. 2014). A balance between the E and M states is tightly regulated by oxidation-reduction (redox) states mediated by metabolic stress besides other factors such as tumor microenvironment, cytokine/chemokine signaling and genetic and epigenetic factors regulating transcription factors (Luo et al. 2018). In the context of highly adaptable cancer cells that persist a variety of bottlenecks in the body, an ability to survive under high oxidative stress may be crucial in surviving after bottlenecks. This ability does not operate in isolation; instead it is an integral part of stemness in cancer. Thus it may be possible to exploit an ability to survive high oxidative stress, and its connectivity to epigenome for instance, for modeling “decathlon winner” cancer cells in cell culture. An application of this strategy for modeling highly adaptable triple-negative breast cancer (TNBC) cells in cell culture has recently been described (Singh et al. 2012, 2014, 2016, 2018, 2019). A wider application of this strategy across cancers has a potential of identifying therapeutic strategies for halting cancer evolution in patients who are not helped by current therapies.

Role of Oxidative Stress in Microenvironment Obviously, cancer cells do not evolve in isolation. They are influenced by various physiological elements including immune system, cancer associated fibroblasts (CAFs) and other cells. Tumor niche contains stromal cells (myofibroblasts, CAFs), vascular (erythrocytes) and immune cells such as lymphocytes, natural killer cells, antigen presenting cells and cancer-associated macrophages (CAMs). Studies have shown that CAFs modulate tumor growth by (a) secreting VEGF and angiopoietin, (b) promoting cell motility and metastasis (via CCL2, CCL5 and MMPs), (c) producing anti-apoptotic factors, and (d) by secretion of IL-6, IL-10, and TGF-β to inhibit the immune response (Kayamori et al. 2010; Tsuyada et al. 2012; Sosa et al. 2013). A study has shown that conditioned media from Caveolin-1 deficient stromal fibroblasts that behave like CAFs, promotes invasiveness in normal breast epithelial cells as evidenced by their epithelial to mesenchymal transition (EMT) (Sotgia et al. 2009). Furthermore, other cells of tumor microenvironment such as CAMs and/or senescent fibroblasts either act independently or in synergy to secrete pro-inflammatory cytokines and proteases, which promote cancer cells to become more aggressive (Sosa et al. 2013). Cancer cells produce ROS, which when released in tumor microenvironment initiates stromal oxidative stress, autophagy, mitophagy by activation of transcription factors such as HIF-1α and NFκB and cyclooxygenase 2 (COX-2) which contribute to angiogenesis (Toullec et al. 2010).

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A direct connection between ROS and EMT was established by studying TGF-β pathway. The activation of TGF-β pathway leads to phosphorylation of Smad2, p38-α, and ERK1/2 which induces an increase in intracellular ROS (Rhyu et al. 2005). Furthermore, role of ROS as EMT promoter is substantiated by studying MMPs. MMP-2, 3, and 9 play important role in GTP-binding protein (Rac1b) stimulation to influence ROS (Radisky et al. 2005). Hence, oxidative stress can change microenvironment leading to cancer initiation and progression. By the time cancer reaches to advanced stage, highly abnormal and adaptable cancer cells are selected and abnormal (pro-inflammatory) microenvironment and oxidative stress are part of this mix. Therefore, it appears that in advanced disease cancer cells can dictate changes in microenvironment to suit them.

Therapeutic Targeting of Oxidative Stress Despite significant advances in understanding the involvement of oxidative stress in pathogenesis of cancer, not much has been achieved in terms of being able to therapeutically modulate oxidative stress in the clinic. The main reason may be tumor heterogeneity that thwarts attempts at targeting oxidative stress only in some subpopulations of cancer cells. Further, it is not obvious in most tumor models whether the oxidative stress is a cause or consequence of cancer. As a result, while a variety of potential therapeutic agents may appear promising in the laboratory, they do not succeed in the clinic. Another big reason for a lack of progress in clinical application is related to how therapeutic agents are evaluated. Testing in laboratorybased models provides very little information regarding the efficacy of potential therapeutic agents. If the goal is to develop therapeutic agents that would impact most resistant cancer cells within a heterogeneous cell population in the body (a desirable goal), then we should ask how we can model such cells better. Certain drugs used for other diseases are being used to repurpose for targeting cancer cells. One such drug is metformin which is used to treat diabetes, but based on evidence from epidemiological and preclinical studies, it is being pursued as a preventive and therapeutic agent in several cancers (Thompson 2014). Metformin impacts not only oxidative stress but many other cellular processes as well. A recent study demonstrated that treatment with a low non-cytotoxic dose of metformin prevented the development of doxorubicin (DOX) resistance in breast cancer cell lines by modulating gene expression related to OS and interferon-α signaling (Marinello et al. 2019). Their studies suggested that metformin treatment group showed increased sensitivity to DOX-induced OS and the cells showed lower levels NO, nuclear NF-κB and Nrf2, and increased nuclear p53 suggesting that metformin can be used as a sensitizer to DOX-based therapies. Recently a strategy for modeling highly adaptable TNBC cells has been described, which involves applying a severe metabolic challenge, e.g., a lack of glutamine in culture medium (Singh et al. 2012). This simple approach modeled the cancer cells that have a high capacity to survive in reversible quiescence under a variety of challenges (including chemotherapeutic drugs and hypoxia) and start

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proliferating when conditions are favorable. These adaptable cancer cells have a high oxidative stress as indicated by reduced levels of GSH and GSSG (Singh et al. 2012). Importantly, they also are good at dealing with oxidative stress; these cells are also more resistant to phenethyl isothiocyanate (an inducer of oxidative stress) than parental cells (Singh et al. 2014). They have embryo-like gene expression and they efficiently metastasize to multiple organs in nude mice (Singh et al. 2012, 2014). This model illustrates the interlinked nature of oxidative stress networks with cell fate networks (Fig. 1). To target oxidative stress in clinic, we must model this connectivity as best as we can while testing therapeutic agents.

Future Directions Making big improvements in cancer treatment will require more effective intervention at minimal residual disease (MRD) stage, before cancer evolves to clinical metastasis stage. An important feature of poor-prognosis MRD leading to relapse is their exceptional ability to switch between quiescence and cell proliferation (Fig. 2). A strategy to model such adaptable breast cancer cells

Fig. 2 Nature of poor-prognosis minimal residual disease in breast cancer. In a setting of an evolution like process, rare stem-like breast cancer cells that persist in the body under a variety of challenges, are depicted as MRD. Their ability to mostly survive in quiescence, and their tendency to proliferate often when conditions are favorable, renders them poor-prognosis MRD representing a high risk of relapse

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has been described (Singh et al. 2012, 2014, 2016, 2018, 2019). With the eventual goal of halting poor-prognosis MRD with repurposed drugs, investigations with low-dose 6-mercaptopurine (6-MP) appear promising. This purine analog has an impressive clinical record of normalizing hyperactive inflammation in inflammatory bowel disease (Korelitz 2013). Regarding the mechanisms of 6-MP effect, although the role of DNA structure/function is often emphasized, 6-MP may also have a significant direct effect on RNA structure/function in abnormal slow-cycling progenitor-like cells. 6-MP undergoes very extensive metabolism in the cell (wherein some intermediary metabolites have regulatory roles), thioguanine base ends up being mis-incorporated into RNA and DNA. It is likely that a high potency of low-dose 6-MP in maintaining remission in this difficult-to-treat disease is due to disruption of abnormal transcriptome that may rely on a variety of RNA modifications for its plasticity. The COVID-19 provides a stark reminder of the power of RNA structure-based cell regulation (positivestrand RNA viruses such as SARS-Cov-2 have a long history of evolution); we suspect that highly resistant cancers are good at harnessing this immense power that needs to be tackled for improving therapies. It is well recognized that chronic inflammation component of abnormal immunity, with links to oxidative stress, has a causative role in cancer. The evaluation of 6-MP in adaptable breast cancer cell model shows that a long treatment with low-dose 6-MP affects cancer cell plasticity (Singh et al. 2019). These results suggest that low-dose 6-MP may produce desirable outcomes against both adaptable cancer cells and abnormal immunity mediated by chronic inflammation at MRD stage in breast cancers with a high risk of relapse. Continuation of such studies with low-dose 6-MP and other nucleoside analogs, which may be effective and safe for a long-term use, will likely lead to innovative clinical trials aimed at halting poor-prognosis MRD in breast cancer. Some nucleoside analogs are very successful in the treatment of advanced cancers, but they may not be suitable for treatment at the MRD stage, when considerations for safety versus efficacy are very different from those at the active disease stage.

Conclusion Oxidative stress in a cell is a readout of its response to intracellular biochemical processes as well as to its environment. It also informs the epigenetic state to deal with these challenges. If we try to therapeutically disrupt regulation of oxidative stress, a disruption that would kill proliferating cells may not similarly affect cancer stem cells. For improving clinical outcomes in breast cancer, a strategy for modeling cancer stem cells that can tolerate high oxidative stress and many other insults to drive cancer has been described (Singh et al. 2012, 2014, 2016, 2018, 2019). Evaluation of potential therapeutic agents in such models will speed up drug discovery efforts. This approach has a potential to change how clinical trials are conducted, i.e., at MRD stage rather than after cancer has relapsed.

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Cross-References ▶ Association of ROS with Epithelial-Mesenchymal Transition and Acquisition of Stemness During Carcinogenesis ▶ Impact of ROS on Cancer and Stem cell Growth and Therapeutics ▶ mRNA Stabilizing Factor HuR: A Crucial Player in ROS-Mediated Cancer Progression ▶ Role of ROS in Cancer Stem Cells ▶ Two-Faced Role of ROS in the Regulation of Cancer Cell Signaling

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Reactive Oxygen Species in Stem Cell Proliferation and Cancer

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Yogesh Kumar Verma, Subodh Kumar, Nishant Tyagi, and Gurudutta Gangenahalli

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Stem Cells and Their Classification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Reactive Oxygen Species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Role of ROS in Cell Proliferation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Role of Hypoxia in Induction of Proliferation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cancer Stem Cells (CSCs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Antioxidant Mechanism of ROS Scavenge . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ROS-Associated Diseases Through Lifestyle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract

Reactive oxygen species (ROS) are chemically reactive species derived from oxygen molecule and can easily react with a variety of other molecules present in the cells. ROS are produced during cellular respiration in mitochondria and other organelles such as peroxisome, endoplasmic reticulum (ER), and phagocytes, where they play a prominent role in a variety of cellular activities such as proliferation, cancer development, differentiation, etc. ROS are also produced by fatty acid oxidation and oxidative burst of immune cells. Among all the known pathways of ROS production, mitochondrial oxidative phosphorylation contributes the most to their production. It has been found that the inner mitochondrial membrane contributes about 80% of free radicals, and the remaining 20% are generated in the matrix. ROS regulate signaling pathways through various transcription factors which either up- or down-regulate the

Y. K. Verma (*) · S. Kumar · N. Tyagi · G. Gangenahalli Division of Stem Cells & Gene Therapy Research, Institute of Nuclear Medicine & Allied Sciences (INMAS), Defence Research and Development Organisation (DRDO), New Delhi, India e-mail: [email protected]; [email protected] © Springer Nature Singapore Pte Ltd. 2022 S. Chakraborti et al. (eds.), Handbook of Oxidative Stress in Cancer: Mechanistic Aspects, https://doi.org/10.1007/978-981-15-9411-3_118

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cascade of signaling thus lead to proliferation, differentiation and maintenance of stem cell pluripotency and cancer development. Most of the time, ROS regulate proliferation by controlling cell cycle with pRB and E2F genes. The ROS phosphorylate these transcription factors with the help of cyclindependent kinases (CDKs). Stem cells are cells with the unique ability to selfrenew and differentiate into specialized cell type in the availability of suitable transcription factors. Proliferation of these cells is achieved through tight regulation of genes such as OCT4, SOX2, KLF4, etc. It has been proved that the normal level of ROS is crucial for stem cell proliferation and differentiation, but any change in ROS level may result in cancer progression by inactivation of tumor suppression gene and activation of oncogenes. This chapter focuses on ROS-mediated signaling, stem cells speciation, and cancer development through stem cells. Keywords

Reactive oxygen species · Pluripotency · Hypoxia · Carcinogenesis · Kinases Abbreviations

ADSCs ALP BCL-2 CDKs EMT ESCs FAD FOXO GPX HIF MAPK MEFs MSCs NADH NF-kB NOS NRS OCT4 PI3K pm-TOR PTEN ROS RUNX2 SOD TRX VEGR

Adipose-derived stem cells Alkaline phosphatase B-cell lymphoma 2 Cyclin-dependent kinases Epithelial-mesenchymal transition Embryonic stem cells Flavin adenine dinucleotide Forkhead box protein Glutathione peroxidase Hypoxia-inducible factor Mitogen-activated protein kinase Mouse-embryonic fibroblasts Mesenchymal stem cells Nicotinamide adenine dinucleotide hydrogen Nuclear factor kappa-light-chain-enhancer of activated B cells Nitrous oxide system Nitrogen-reactive species Octamer-binding transcription factor 4 Phosphoinositide 3-kinases Phosphorylated mammalian target of rapamycin Phosphatase and tensin homolog phosphatase Reactive oxygen species Runt-related transcription factor 2 Superoxide dismutase Thioredoxin Vascular endothelial growth factor

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Introduction Stem Cells and Their Classification Stem cells are special undifferentiated cells that are able to develop into specific cells in the presence of suitable transcriptional factors. They also possess the property of self-renewal and differentiation. This property make them the best candidate for regenerative medicine in which these cells can be helpful in repair/regeneration of all types of tissue of the body. The functions of stem cells are strictly controlled by tissue microenvironment known as “niche” (Drummond-Barbosa 2008). Stem cells are defined by their unique capabilities of self-renewal and differentiation into various types of cells depending on their potency (Table 1). The potency of stem cells is determined by several transcriptional factors. On the basis of their potency, stem cells are categorized into various types of cells such as totipotent, pluripotent, multipotent, oligopotent, and unipotent. Totipotent stem cells are cells that have the capability to self-renew and differentiate into embryo and extraembryonic tissues such as placenta. Totipotent stem cells are the best among SCs due to their unlimited power of differentiation. These cells are produced from single cell zygote and can develope into any lineage of cell. Pluripotent stem cells are originated from totipotent stem cells and can differentiate into nearly all the cells. These cells give rise to exoderm, mesoderm, and endoderm but not all types of cells such as totipotent stem cells. Embryonic stem cells (ESCs) are pluripotent stem cells and can also developed into adult cell type. Multipotent stem cells have a little more closely restricted number of cells. These cells can divide to form a particular lineage of cells such as adult stem cells and hematopoietic stem cells (HSCs). Oligopotent stem cells can differentiate into 2–10 types of cells and are more restricted than multipotent stem cells. Lymphoid or myeloid stem cells are oligopotent stem cells. Unipotent cells are highly restricted in nature. They can differentiate into only one cell type. These cells include muscle stem cells, neural stem cells, epithelial cells, etc. (Ota 2008).

Types of Stem Cells During early phase, embryonic stem cells (ESCs) remain in undifferentiated state, but in late phase, these cells convert into multiple lineages (Morgani et al. 2013). ESCs arise from totipotent stem cells from early embryo but, after 7 days, change into pluripotent stem cells. ESCs have a great therapeutic potential in regenerative medicine due to their pluripotent nature and their keep on dividing for long time; Table 1 Classification of stem cells on the basis of their potency

S. no. 1 2 3 4 5

Types of stem cells Totipotent Pluripotent Oligopotent Multipotent Unipotent

Example One cell zygote Embryonic stem cells Myeloid and lymphoid cells Hematopoietic stem cells Cardiac stem cells

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nevertheless, there are many issues regarding their safety, which need to be sorted out before their use in clinics (Englund et al. 2011). Adult stem cells (ASCs) are isolated from a variety of tissues such as umbilical cord, bone marrow, adipose tissue, retina, skeletal muscle, etc. These cells are similar to Mesenchymal stem cells (MSCs) and are found in developed organism. These cells are further classified into “somatic stem cells” and “germ line stem cells” and, on the basis of their origin ASCs, are found in very small amount and are lineage restricted. ASCs occur in almost all the organs, their major tissue sources are bone marrow, blood vessels, brain tissue, skeletal muscles, liver, skin, etc. ASCs remain in a quiescent state until activated by tissue injury or progression of diseases (Kengla et al. 2017). ASCs treatment has been successfully employed in diseases related to bone/blood cancer through bone marrow transplantation (Kengla et al. 2017). Fetal stem cells (FSCs) are derived from fetus and retain the ability of proliferation and differentiation. These cells are primitive in nature and are found in almost all the organs of the fetus. There are three most reliable sources of FSCs including placenta, umbilical cord blood, and amniotic fluid of fetus. Fetus blood is a rich source of HSCs. These cells are more proliferative in nature than those of cord blood and adult bone marrow. These stem cells display many properties that make them superior to other stem cells for therapeutic application in regenerative medicine. These cells exhibit high growth rate and plasticity and can survive in hypoxic condition. FSCs produce high level of angiogenic factors which facilitate faster healing of wound (Ishii 2014). Cord blood stem cells (CBCs) are multipotent stem cells with limited power of differentiation. They are obtained from umbilical cord of newborn and have been used to treat children with certain blood diseases. They contain fewer numbers of HSCs than a bone marrow derived of cells. The therapeutics application of CBCs is similar to that of adult BM-derived stem cells but restricted to fewer diseases and are currently being explored to treat various diseases (McGuckin et al. 2004). Induced pluripotent stem cells (iPSCs) are derived from fibroblast cells that have been reprogrammed back into pluripotent state by incorporating specialized transcription factors viz.OCT3/4, SOX2, KLF4, and c-MYC. Reprogramming of differentiate cells enables the development of unlimited source of human cell types needed for therapeutic purposes. Human iPSCs such as ESCs also possess a variety of similarities such as surface antigen, gene expression profile, proliferation potential, etc. iPSCs such as ESCs can also self-renew and differentiate into early embryonic cells such as endoderm, mesoderm, and ectoderm except extraembryonic (placenta). The application of iPSCs in regenerative medicine has opened a new possibility of therapeutics of several diseases (Takahashi et al. 2007).

Reactive Oxygen Species Role of Reactive Oxygen Species (ROS) Reactive oxygen species are unstable reactive species that contain oxygen molecules. These molecules can easily react with DNA/RNA and fatty acids. They are known to be produced from aerobic metabolism in mitochondria through electron

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transport chain (ETC) (Liu et al. 2002). For a long time, ROS are considered as cellular dysfunction and death-causing molecules via the destructive oxidation of intracellular components. In recent years, advances in scientific research have opened a new dimension of knowledge to understand the role of signaling molecules. Recent research showed that highly unregulated level of ROS is harmful for cells, whereas a regulated basal level of ROS is necessary for a variety of cellular functions, such as cell survival, proliferation, and differentiation into specialized cells. The effect of ROS on stem cells is very complex, and it is known to enhance cell differentiation, reprogramming, development of malignancy, and premature aging. ROS are also known for epigenetic markings of stem cells. There are many possible cascades of molecular events that are induced by ROS, leading to differentiation of stem cells into their specific lineages. For example, p38 and MAPK pathways induce terminal differentiation of neurons, cardiomyocytes, chondrocytes, and vascular keratinocytes (Nugud et al. 2018).

Chemistry of Reactive Oxygen Species ROS are used to describe a number of free radicals species. ROS include peroxide, superoxide, singlet oxygen, alpha oxygen, and hydroxyl radicals. They are derived from molecular oxygen by stepwise reduction to higher energy exposure or electron transport reactions. Oxygen molecule contains six electrons in its outermost orbital and is not very reactive in the ground state due to the presence of two electrons in a chemical bond. These molecules are not reactive unless activated by stepwise reduction of oxygen or absorption of sufficient energy to reverse the spin of oxygen. Superoxide anion is a result of stepwise one-electron reduction of oxygen, and it is the mediator of ROS-induced oxidative reactions in the cells. In cells, Superoxide is dismutase into hydrogen peroxide by superoxide dismutase enzyme. This molecule is further reduced into water or partially into hydroxyl radical. Reduced form of hydrogen peroxide, i.e., hydroxyl radical, is one of the strongest oxidants. The formation of hydroxyl radical is catalyzed by a variety of reduced transition metals. In addition, superoxide may also react with other radicals including nitric oxide in the cell. The oxidants derived from superoxide and nitric oxide are called as reactive nitrogen species (RNS) (Turrens 2003). Cellular Generation of ROS A large percentage of ROS is generated in the mitochondria during aerobic cellular metabolism (oxidative phosphorylation) that contributes about 80% of its production. In this process, the freely released electrons species are directly reacted with oxygen to produce oxidative species such as peroxide, superoxide, singlet oxygen, alpha oxygen, and hydroxyl radicals. Among all the five complexes, the maximum production of ROS takes place in I, II, and III complex. Mitochondria has double membrane structure, and about 80% of free radicals are generated in the inner mitochondrial membrane and remaining 20% in the matrix of the mitochondria (Kumari et al. 2018). Superoxide radical is prominent among them. These radicals enter into the cytoplasm of the cell due to the permeable nature of the outer membrane of mitochondria where they are dismutated to hydrogen peroxide, a

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highly diffusible secondary messenger. Further, these molecules play various cellular functions. In the matrix and membrane of the peroxisome, superoxide radicals and H2O2 are generated through xanthin. Other minor sources of ROS production include prostaglandins, fatty acids drugs, flavorings, coloring agents, antioxidants, etc. In endoplasmic reticulum (ER), these substances are transformed into highly reactive free radicals. Fewer ROS are also produced as a part of immune response in macrophages and leucocytes (Sareila et al. 2011). The elevated level of ACT1, CPT1/2,HADC1,ECH1, and ACAA1 genes was found in fatty acid oxidation process, which significantly correlated the role of fatty acid oxidation with ROS production (Kumari et al. 2018). Prostaglandin D2 (PD2) is a local modulator of ROS in sertoli cells; this increases ROS and hydrogen peroxide levels and activates signaling related to proliferation through PI3K-signaling pathway (Dong 2015). Another mechanism which induces ROS production in high concentration is immune/oxidative burst; it is referred to an increased ROS production through NADPH oxidase 2 (NOD2). NOX2 gene which is expressed in phagocytes is also responsible for oxidative burst. It is a transmembrane enzyme that oxidizes intracellular NADPH/NADH, leading to the transport of free electrons across the membrane where they react with oxygen and change into free radicals (Jacqueline et al. 2017).

Role of ROS in Cell Proliferation Signaling Through Cell Cycle The fate of cell, either to divide or remain in quiescence, is decided by signaling molecules that act at various checkpoints of cell cycle. Cell cycle is controlled at three checkpoints viz. G1 (integrity of DNA is assessed), G2 (duplication of genetic material is assessed), and M phase (attachment of spindle fiber is assessed) phases. There are many factors controlling the progression of cells from one phase of cell cycle to another. ROS also act as triggering molecules through transactivation of the EFGR, which enhances the cell cycle progression by CDKs (Fig. 1) (Verbon et al. 2012). In cell cycle, progression of cells through G1 is controlled by pRB protein which functions to repress the activity of E2F transcription factor in mitotic cells. Phosphorylation of pRB proteins by CDKs results in the release of E2F factors, thus promoting the cellular transition to S phase. CDK is a specific catalytic core, and its function is regulated by regulatory protein known as cyclin which controls activity and specificity. Most of the antiproliferative agents exert their effect through transcriptional regulation of cyclins and CDK inhibitors (Duronio and Xiong 2013).

Role of Hypoxia in Induction of Proliferation Deprivation of adequate oxygen supply in tissue or cells is known as hypoxia. It helps in embryogenesis and many important signaling events in cells. Hypoxia also acts as modulator of cell proliferation and differentiation. During hypoxia, hypoxiainducible factor (HIF) regulates a variety of cellular processes in response to

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CDKs Phosphorylates G0/G1Phase

E2F

pRB

S-Phase

M-Phase

G2-Phase

Fig. 1 Mechanism of cell proliferation at cell cycle level. pRB protein inhibits function of E2F and does not allow cell cycle to progress, whereas phosphorylation of pRB protein by CDKs activates downstream signaling for cell cycle progression

ROS

Hypoxia At complex III of mitochondria

HIFα

HIFβ

VEGR

NOS

NFKB PI3K

Cox, iNOS

pAKT RAS

VEGFR

Vasodilatation

PI3K

Angiogenesis

Proliferation

pmTOR BCL-2, c-MYC, elFGR

ImmuneResponses MAPK

Proliferation

Cell survival

Fig. 2 Signaling of ROS in cell proliferation: Hypoxia mediates proliferation and survival by upregulating signaling molecules such as ROS, through HIFα and β,which mediate proliferation and angiogenesis. Through PI3K/pmTOR and MAPK/NfkB as alternate pathways, ROS induce survival and proliferation respectively

oxidative stress induced by elevated ROS. HIF-α and HIF-1β have been recognized as key factors for high altitude adaptation, especially for humans and animals. HIF-1 consists of α- subunit (oxygen-regulating unit) and constitutively expressed β-subunit. HIF-1α and HIF-1β contain helix-loop-helix motif that binds to DNA and causes subunit dimerization. These transcription factors modulate gene expression profile of cell induced by oxidative stress through binding to DNA molecule (Fig. 2). Hypoxic condition helps in production of ROS in the mitochondrial

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Hypoxia

HIF-1α

HIF-1β NOTCH

Pluripotency

5%O2 OCT4

OCT3/4 SOX2 Pluripotency

Pluripotency

KLF2 c-Myc

Fig. 3 Role of hypoxia in supporting stem cell pluripotency. Hypoxia induces HIFα and HIFβ, these molecules help in maintaining pluripotency through OCT4. A concentration of 5% O2 helps in upregulating other known transcription factors including OCT3/4, SOX2, KLF2, and c-MYC, and NOTCH to induce pluripotency

respiratory chain (complex III) and thus also helps in regulation of proliferative gene expression (Zhou et al. 2016). ROS also participate in homeostatic responses to hypoxia through the action of prolyl hydroxylase. The two subunits of HIF-1 are known to activate OCT4 gene expression and thus regulate pluripotency of human ESCs. In culture condition, Notch activation of ESCs is a key factor of their pluripotency. ESCs maintain their genomic identity through enhanced ROS removal capacity and limited ROS production due to very small number of mitochondria. A recent finding suggested that the production of human iPSCs has been made possible due to effective reduction in the mitochondrial genome copy number and antioxidant defense as seen in ESCs (Zhu et al. 2005). Hypoxic conditions (5% O2) also facilitate iPSCs production from mouse fibroblasts. Hypoxia also increases efficiency of human iPSCs reprogramming from dermal fibroblasts upon transduction of OCT3/4, SOX2, KLF4, and c-MYC genes (Fig. 3) in stem cells (Silver and Erecińska 1998).

Hypoxia/ROS in Stem Cell Differentiation Although hypoxia and low ROS are critical for docking of stem cells in the bone marrow niche, ROS can also stimulate pathways leading to differentiation of stem cells into adult, embryonic, and cardiac cells through transcription factors (Fig. 4). Niche is a place in bone marrow where stem cells reside and receive various fatedeciding signals. Due to hypoxia and low-ROS concentration in niche, stem cells maintain their naiveness. ROS induce ESCs formation by downregulating OCT4, TRA 1–60, NANOG, and SOX genes. ROS restore osteogenic differentiation via

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p38 and MAPK pathway

Cardiomyogenesis Down-regulation of RUNX2 and ALP

Osteogenesis

ROS

Down-regulation of OCT4, TRA1-60, NANOG and SOX

ESCs formation Regulation of G1/S phase of cell cycle

ASCs formation Through DNA Integrity

iPSCs formation Fig. 4 ROS-mediated differentiation of stem cells into cardiac cells (p38 and MAPK), osteogenic cells (RUNX2 and ALP), ESCs (OCT4, TRA 1–60, NANOG, and SOX), ASCs (G1/S), and iPSCs (DNA integration)

downregulation of RUNX2 and ALP genes. Similarly, a role of ROS has also been reported in differentiation of cells through WNT-signaling pathway and generation of iPSCs. Thus the foregoing discussion suggests that the balance between ROS generation and scavenging has correlation with the stem cell functions.

Cancer Stem Cells (CSCs) The characteristics of CSCs are similar to stem cells such as slow cell cycle, quiescency, and low ROS level as per the requirement. In HSCs, the low level of ROS is maintained by FOXO gene where it directly regulates ATM gene responsible for controlling the downstream proliferation and differentiation of cells (Tothova et al. 2007). Recent studies have suggested that epigenetic regulation also plays an important role in preserving low level of ROS in CSCs (Vira et al. 2012). Like other stem cells, the critical low level of ROS is responsible for tumorigenic and metastatic phenotype of CSCs. According to a latest hypothesis, there are six mutations such as self-sufficiency for growth, insensitive to auto growth signals, invasion of apoptosis, unlimited ability to replicate, and tissue invasion and metastasis are characteristics of CSCs.

Development of Cancer from CSCs Epigenetic events, such as chromatin modeling, DNA methylation, noncoding RNA, histone modification, and controlled gene expression, determine the fate of the cell without changing DNA sequence (Guillaumet-Adkins et al. 2017). Disruption of normal epigenetic process leads to cancer development due to hypermethylation and

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by mitigating the action of tumor-suppressing genes (Iorio and Croce 2012). CSCs have the ability to travel from one point of the body to another through a process known as metastasis (Adjiri 2017; Jang and Sharkis 2007). Metastasis involves movement of genetically unstable cancer cells from their primary site to distant organs, causing morbidity and mortality (Norman 1952). Metastasis occurs as a result of intrinsic burden and interaction between malignant and nonmalignant cells (Brooks et al. 2010). Molecular events, during this process, upregulate several transcription factors such as TGF-β, NF-kB, Twist and Snails, AP-1, and Zeb, etc (Guerrini et al. 2018). Several studies have suggested that ROS are the major factor for epithelial–mesenchymal transition, an important transformation for cell metastasis (Liao et al. 2019).

ROS-Dependent Signaling Pathways in CSCs ROS level in CSCs is regulated by various signaling pathways which either directly or indirectly lead to cancer development from normal cells. These pathways include PTEN/PI3K/AKT/pmTOR, Wnt/β-catenin, and ATM, NOTCH, and STAT signaling (Dong 2015). Numerous studies have demonstrated that genes PTEN/PI3K/AKT/ mToR directly help in ROS production and cancer development. The increased intracellular level of these signaling molecules upregulates the glycolysis process, which have regulatory effect on the rate of ROS formation and tumorigenesis. Mutation in PTEN results in accumulation of PI3K, which subsequently activates AKT signaling for cancer development. Ataxia-telangiectasia-mutated (ATM) pathway also plays a critical role in the maintenance of genomic stability of stem cells. It responds to DNA damage, particularly double strand breaks, and prevents cancer development. The NADPH productions are also regulated by ATM, which in turn decreases ROS production in the cells (Diehn et al. 2009). Dysregulation of ATM signaling results in increased ROS production and oncogenesis. Notch pathway, like other pathways, helps in stem cell proliferation, differentiation, and speciation. Recent advances suggested that it has a role in glioblastoma formation through ROS production and activation of PIK3 pathway (Artavanis-Tsakonas et al. 1999; Che et al. 2011). Another gene, STAT3, directly regulates the proliferation and tumorigenesis of CSCs by interacting with PI3K/mToR pathway (Moncharmont et al. 2012).

Antioxidant Mechanism of ROS Scavenge Almost all cells have developed a defense mechanism to cope with the free radicals produced in the system. They have antioxidants such as catalase superoxide dismutase (SOD), peroxiredoxins, thioredoxin (TRX), and glutathione peroxidase (GPX). From the earlier studies in HSCs, it has been confirmed that a low quantity of ROS maintain p21, TERT, p53, p38, and pmTOR transcription factor in low concentration but in high activation state. This balance is responsible for the quiescency of stem cells and hibernation in the Go phase of cell cycle (Jang and Sharkis 2007).

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ROS-Associated Diseases Through Lifestyle Elevated level of ROS in the cells due to lifestyle and malnutrition is a major health concern in developing nations. Dietary deficiency of nutritional and enzymatic antioxidants contributes to the formation of ROS in the cells. The role of ROS has been seen in the development of various ailments (Fig. 5) including cancer, respiratory, cardiovascular, and neurodegenerative diseases (Liu et al. 2018). Direct interaction of plasma lipoprotein with endothelial cells in vasculature and elevated level of ROS cause modification of low-density lipoprotein (LDL). The modified LDL is transported to arterial lumen to induce apoptosis of endothelial cells, resulting in development of fibrous plaque by macrophages and subsequent hypertension (Le 2014). Like other cells, neuron is also vulnerable to ROS-induced damage. Excessive production of ROS can be correlated with the accumulation of Amyloid plaque (Aβ) in Alzheimer disease and dysfunction of mitochondria (Hoogendam et al. 2017). The oxidative stress leading to dysfunction of mitochondria is commonly observed in neurodegenerative disease such as Huntington’s disease. The elevated level of ROS can also inhibit IGF-1 associate signaling pathway, which is linked to neuron regeneration. Moreover, the role of ROS is reported in digestive diseases where elevated level of ROS disrupts the epithelial balance of intestine and increases its permeability, resulting in inflammation.

1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

Pollutants

Carcinogen

Anthocyanin Lipoic acid Lycopene Melatonin Phytochemicals Polyphenols Resveratrol Selenium Theaflavins Vitamins

1. Electron transport chain 2. β-oxidation of fatty acids 3. Oxidative burst during Immune reaction

Act as

Antioxidants

ROS

Other diseases 1. Respiratory disease 2. Cardiovascular disease 3. Neurodegenerative disease

Stem cell Dysregulation of pathways Cancer

Fig. 5 ROS and their association with various diseases. ROS production is tightly regulated by the availability of antioxidants and control mechanism of cell. Pollutants and carcinogenic compounds initiate the production of ROS in the cells, which further facilitate proliferation of stem cells but dysregulation of pathways leading to CSCs development

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Conclusion ROS are molecules that cause damage to regulatory pathways and infringe upon normal physiological and biological responses. The role of ROS in cell growth, tissue regeneration cellular differentiation, and prevention of aging is well-known. ROS exhibit partly good and partly bad effects in the cells; this has been exploited for their potential therapeutic benefits. The level of ROS in cells and their tight regulation is the determining factor of stem cell functions. The low ROS level enables proliferation and differentiation of stem cells through a variety of signaling pathways. Any imbalance in ROS level leads to cancer development through activation of oncogenes and mitochondrial dysfunction. Almost all cells have also developed antioxidant mechanism to control ROS production, but failure to do so initiates oncogenic activity. Therefore, the controlled production of ROS in the cells is likely to result in a healthy state. Acknowledgments We thank Director INMAS for his continuous support. This work was funded by Defence Research Development Organization (DRDO), India. All the supported image were created using Microsoft office

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Eunus S. Ali, David Barua, Subbroto Kumar Saha, Maizbha Uddin Ahmed, Siddhartha Kumar Mishra, and Mohammad S. Mubarak

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Generation, Pathophysiological, and Regulatory Functions of Reactive Oxygen Species . . . Sources of ROS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ROS in the Regulation of Physiological and Pathophysiological Signal Transduction Pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Excess ROS Perturbs the Cellular Homeostasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Reactive Oxygen Species and Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ROS in Cancer Progression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ROS in Cancer Inhibition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Application of ROS Induction for Anticancer Strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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E. S. Ali College of Medicine and Public Health, Flinders University, Bedford Park, Australia Simpson Querrey Institute for Epigenetics, Northwestern University Feinberg School of Medicine, Chicago, IL, USA Department of Biochemistry and Molecular Genetics, Northwestern University Feinberg School of Medicine, Chicago, IL, USA D. Barua Department of Pathology, National University of Ireland Galway, Galway, Ireland S. K. Saha Department of Biochemistry and Molecular Medicine, University of California, Davis, Sacramento, CA, USA M. U. Ahmed Department of Industrial and Physical Pharmacy (IPPH), Purdue University, West Lafayette, IN, USA S. K. Mishra Department of Life Sciences, Chhatrapati Shahu Ji Maharaj University, Kanpur, India M. S. Mubarak (*) Department of Chemistry, The University of Jordan, Amman, Jordan e-mail: [email protected] © Springer Nature Singapore Pte Ltd. 2022 S. Chakraborti et al. (eds.), Handbook of Oxidative Stress in Cancer: Mechanistic Aspects, https://doi.org/10.1007/978-981-15-9411-3_119

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Direct ROS Generation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Antioxidant Process Inhibition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Combination Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Immunotherapies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions and Future Direction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract

Reactive oxygen species (ROS) are reactive chemical and/or biochemical intermediates or fragments containing oxygen as peroxides, superoxide (O2● ), hydrogen peroxide (H2O2), hydroxyl radical (●OH), hydroxyl ion ( OH), singlet oxygen, and nitric oxide (NO). At optimal concentration ROS have been implicated to serve varieties of important physiological functions in different types of cells under normal physiological conditions. On the other hand, excessive amounts of ROS are one of the main determinants in the pathogenesis of different types of diseases including cancer, metabolic syndromes, cardiovascular, and neurodegeneration. The mechanism of the generation of ROS and its concentration decides the fate of cells in different types and conditions; excessive ROS can cause detrimental effects on normal cells, deregulate cellular homeostasis, and induce carcinogenic changes. In order to continue their growth and proliferation, cancer cells increase ROS production rate compared with normal cells, and to maintain their ROS homeostasis, they simultaneously increase their antioxidant capacity. It is now evident that this unique and altered redox environment of cancer cells upturns or increases their vulnerability to ROS-metabolism therapies. This chapter aims to discuss a current scenario of ROS in physiological and pathological contributions with emphasis on cellular and molecular mechanistic ways. In addition, it discusses the role of oxidative stress in initiation and progression of different types of cancers as well as current and new strategies targeting ROS for the development of therapeutic interventions of ROS-induced cancers. Keywords

Oxidative stress · Reactive oxygen species · Lipid peroxidation · Apoptosis · Inflammation, Cancer metabolism, Anticancer effects

Introduction Oxidative stress is a cellular condition that can be caused by accumulation of ROS due to the imbalance between the generation and elimination of free radicals. It is a successive event of increased production of free radicals and/or impaired physiological antioxidant defense systems. ROS are generally produced in limited amount in the body and are associated with several cellular processes, including normal homeostasis maintenance, signal transduction, and receptor activation (Pizzino et al.

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2017; Vasan et al. 2020), and are considered as signaling molecules (Reczek and Chandel 2015). Enzymes through constant input of metabolic energy within the cells tightly maintain the reduced environment. Perturbations of the redox balance can cause harmful effects mediated via generation of ROS. These stress conditions can damage cellular components, and severe oxidative stress can lead to cell death. It can be noted that the extent of oxidative stress could be resolved by a balance between generation and elimination of ROS (Pizzino et al. 2017). In normal physiological conditions, the balance between prooxidant (oxidative stress inducing components) and antioxidant is slightly maintained in favor of prooxidant products, thus keeping mild oxidative stress. Despite having an essential role (at optimal concentration) in a variety of cellular events including gene expression, excessive production of ROS is associated with tumor development, and tumorigenesis is reported to be mediated by DNA damage, hypoxia, genetic mutations, increased metabolism, and altered antioxidative mechanisms (Perillo et al. 2020). Evidences have established that the effect of ROS is a composite phenomenon depending on the amount of ROS produced and the severity of ROS in a particular biological system. When internal antioxidant systems fail to maintain oxidative stress, external antioxidants are required. In this context, excessive supply of antioxidants may lead to prooxidative or antioxidative stress which is associated with minimal ROS-mediated beneficial effects and increased prooxidative detrimental effects. Several studies suggest the complicated ROS-mediated actions in cellular systems which further helps in determining the extent of oxidative stress in an individual before administration of antioxidant supplements (Harris and DeNicola 2020; Perillo et al. 2020). Choosing ROS-mediated targets for the treatment of malignant cancers, therefore, involves a delicate balance and calibration between physiological and pathological ROS. Due to persistence stress, tumor cells are usually more sensitive to oxidative stress compared to normal cells. Many anticancer chemotherapeutic drugs that are used in cancer treatment can induce oxidative stress by altered regulation of free radicals including hydrogen peroxide (H2O2), superoxide anions (O2● ), and hydroxyl radicals (Montero and Jassem 2011). Thus, these metabolic changes are used in developing anticancer drugs that could target cellular redox regulation (Montero and Jassem 2011; Schieber and Chandel 2014). Cellular redox state can be controlled by three systems that are governed by counteracting free radicals and ROS or by reverting the formation of disulfides. Among the three systems, two are dependent on glutathione (a very small sulfur-containing protein); whereas the other one is thioredoxin dependent. Chemotherapeutic agents that target S-glutathionylation exhibit direct anticancer effects through inhibiting DNA repair of cancer cells and a wide range of cellular signaling pathways. NOV-002, canfosfamide, and several others of this category were under phase III clinical trials along with a number of other drugs are in preclinical studies. PARP inhibitor talazoparib was approved recently for patients with metastatic BRCA1/2-positive, HER2-negative breast cancer (Keung et al. 2020). Mechanistically, NOV-002 can mimic the activity of endogenous GSSG and can serve as a substrate for gamma-glutamyl-transpeptidase (GGT). This leads to S-glutathionylation of proteins and can induce endoplasmic

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reticulum stress-mediated apoptosis (Keung et al. 2020). In contrast, thioredoxin is overproduced in a variety of human cancers and it is associated with poor prognosis of aggressive tumors. Therefore, targeting thioredoxin is another attractive approach to drug development; hence several agents targeting thioredoxin have been developed such as PX-12, dimesna, and motexafin gadolinium. Furthermore, arsenic derivatives have been reported to act as antiproliferative and apoptogenic agents against cancer cells. Although these derivatives exert their action as prooxidant, the detailed mechanisms of oxidative stress induction and apoptosis are yet to be elucidated (Montero and Jassem 2011). In this chapter, we have comprehensively discussed the insights of redox signaling in cancer cell metabolism, proliferation, and explored the means of redox signaling regulation in targeted development of anticancer therapeutics.

Generation, Pathophysiological, and Regulatory Functions of Reactive Oxygen Species Sources of ROS In cancer cells, generation of ROS is mediated by mainly two sources, including mitochondria or nicotinamide adenine dinucleotide phosphate (NADP) oxidase (Winterbourn 2008). The main site of ROS generation is the mitochondrial electron transport chain (ETC). However, other ROS generation pathways are also involved especially the respiratory burst in activated phagocytes and ionizing radiation affecting cell membranes composition. This also includes the byproducts of several cellular enzymes such as NOXs, xanthine oxidase, and uncoupled endothelial nitric oxide synthase (eNOS) (Pizzino et al. 2017). As mentioned above, mitochondria are the key source of ROS generation in cells involving its primary metabolic function, oxidation of energy equivalents in the tricarboxylic acid (TCA) cycle along with concomitant reduction of flavin adenine dinucleotide (FADH2) and nicotinamide adenine dinucleotide (NADH) in the ETC. The electrochemical gradient generates proton motif force at the outer mitochondrial membrane and triggers the ATP synthase to synthesize ATP. During this redox process, electrons often leak out of the ETC and lead to the conversion of molecular oxygen into O2● which is subsequently converted into H2O2 and ●OH (Vasan et al. 2020). Thus, mitochondria consistently and significantly produce cellular ROS, which go into the process of elimination by means of antioxidant enzymes. The most important endogenous sources of ROS that contribute to aging are mitochondrial ETC and NOS reaction. The O2● ions produced during the ETC show a high electrostatic attraction toward mitochondrial iron-sulfur (Fe-S) clusters, which upon degradation impairs mitochondrial respiration. At mitochondrial ETC complexes I, II, and III, a small amount of oxygen undergoes sequential reduction to generate O2● which is released in the matrix (Winterbourn 2008). Among the non-mitochondrial sources of ROS are Fenton’s reaction, peroxisomal β-oxidation, microsomal cytochrome P450 enzymes, and respiratory burst of phagocytic cells. On

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the other hand, external sources of ROS generation include exposure to microbial infections, food and drinks, extensive exercise, ionizing and ultraviolet radiations, pesticides, ozone, cigarette smoke, and alcohol (Winterbourn 2008). A detailed overview of ROS production network has been depicted in Fig. 1. The NOXs are a group of cellular membrane-associated flavocytochrome proteins, which are considered as the largest single producer of ROS in some cell types with physiological functions like phagocytosis (Chio and Tuveson 2017). NOXs transfer an electron from NADPH to the FAD cofactor which then transfers the electron to a heme group and lastly to oxygen leading to the generation of O2● . Research showed the involvement of NOXs in several instances in cancer cells to drive ROS generation and to support tumorigenesis. An increase in NOX1 expression, due to KRAS mutational activity, was linked to ROS generation in colon cancer and cellular transformation in fibroblasts. In addition, HRAS increases intracellular O2● production in lung cancer cells through activation of membrane-associated NOXs (Chio and Tuveson 2017).

ROS in the Regulation of Physiological and Pathophysiological Signal Transduction Pathways The physiological role of ROS requires determining the identity of ROS (O2• , H2O2) and their interactions with specific biomolecules especially lipid and protein. ROS are often associated with cytotoxic effects in both cancer and normal cells; however, these have different paradigm in types and conditions of cells (Wang and Yi 2008). ROS, in particular H2O2, stimulated cancer cell proliferation through rewiring of the cell growth signaling pathways especially, PI3K/Akt. A number of studies suggest that antioxidants supplementation and activation of cellular antioxidant system may enhance tumor survival by reducing ROS-dependent cytotoxicity (Wang and Yi 2008). This indicates that the redox status of various cancer cells may affect the metabolic reprogramming especially the mechanisms associated with tumorigenesis, progression, and metastasis (Cairns et al. 2011). In this respect, mitochondrial ROS, specifically O2● and H2O2, cause a shear-stress and lead to the oxidation of cellular membranes and cytoskeletal proteins. Oxidized proteins can then stimulate production of mitochondrial ROS and initiate various signaling pathways and upregulation of a cellular inflammatory response. It is now well established that mitochondria produce ROS for maintenance of cellular signaling with variable degrees of generation in different pathophysiological conditions of the cell (Cairns et al. 2011). Superoxide formed at the mitochondrial ETC is emitted into both the matrix and intermembrane space yet they have different cellular fates. The enzyme superoxide dismutase 2 (SOD2) catalyzes the conversion of matrix superoxide into H2O2 which then permeates into the cytosol by diffusion through the inner and outer mitochondrial membrane. In cytosol, H2O2 converted into H2O by peroxiredoxin or glutathione peroxidase. Cytosolic H2O2 is believed to impart in the primary cellular signaling and catabolic processes including oxidation of protein thiol residues (Sena and Chandel 2012).

Fig. 1 Major pathways involved in mitochondrial ROS production and antioxidant defense mechanisms in cells. ETC leakage is responsible to release mitochondrial superoxides (O2• ), which can be converted by SOD2 to produce H2O2. NADPH oxidases (NOXs), cytochrome p450, xanthine oxidase, and lipoxygenases, are involved in the production of O2, which can be converted by SOD1 into H2O2. In the extracellular space, SOD3 performs a similar reaction. With a Fenton reaction, H2O2 produces OH•. Glutathione peroxidase (GPX), peroxiredoxin (PRX), and catalase (CAT) convert H2O2 into H2O. Glucose-6phosphate dehydrogenase (G6PD) and 6-Phosphogluconate dehydrogenase (PGD), Methylenetetrahydrofolate dehydrogenase (MTHFD1), Isocitrate

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Another important type of free radical is nitric oxide (NO), which is produced by various group of enzymes termed as NOS found in the cells. NO affects mainly the oxidation of iron-containing proteins and activation of iron regulatory factor. Additionally, NO has upregulatory effects on p53, which is further associated with the tumor suppression property through activation of apoptosis. Moreover, it is also linked with DNA damage through interfering with O2●- and H2O2 activity that leads to tumor growth suppression, angiogenesis, and metastasis. The protective oxidoreductase enzyme, thioredoxin (Trx), which works with glutathione system is associated with the maintenance of cellular redox. Peroxiredoxin I (a Trx peroxidase), heme oxygenase-1 (HO-1), the cystine transporter xc2 are the other set of protective antioxidant systems. The transcriptional activation of these stress-responsive genes is dependent on the mutational activation of the nuclear factor-erythroid 2-related factor 2 (Nrf2). Nrf2 expression is actively induced by the oncogenes KRAS, BRAF, and MYC, which then promotes the ROS detoxification and leads to tumor initiation (DeNicola et al. 2011). The process of cellular protein folding is crucially dependent on the availability of energy and the oxidizing environment of endoplasmic reticulum (ER). The load of protein folding in the ER can cause ROS accumulation leading to inflammatory responses. Two ER enzymes, including protein disulfide isomerase and ER oxidoreductase are critical for protein folding, which act through the oxidative folding formation. Unfolded proteins accumulated into the lumen of the ER can be folded into the native state by cellular GSH, which thus can restore the normal ER homeostasis (Tam et al. 2018). From a mechanistic point of view, the activated Nrf2 translocates to the nucleus and increases the rate of expression of a group of antioxidant and oxidant detoxifying genes (Zhang 2006). The ETC semi-ubiquinone compound nonenzymatically forms O2●-. O2●-, which can be converted into H2O2 by the enzyme SOD. Besides, O2●- can be nonenzymatically converted into H2O2 and singlet oxygen. In the presence of reduced transition metals, H2O2 can be converted into highly reactive ● OH; H2O2 can also be converted into H2O and O2 via the enzyme catalase (Montero and Jassem 2011). Excess glucose also increases oxidative stress through glyceraldehyde autoxidation, PKC activation and sorbitol production, and oxidative phosphorylation. Lipid oxidation and lipotoxicity have also been linked to both oxidative and ER stress ä Fig. 1 (continued) dehydrogenase (IDH1 and IDH2), and Malic enzyme (ME1 and ME2/3) generate NADPH as a byproduct, and maintain cellular redox homeostasis. Glutamate-cysteine ligase (GCL) catalyzes production of GSH, and Glutathione reductase performs reduction of GSSG back to the sulfhydryl form GSH. In order to maintain intracellular redox homeostasis, GSSG is exported out of the cell through multidrug resistant proteins. The cystine-glutamate transport system rises intracellular cystine level, which is reduced to cysteine. γ-glutamyl transferase (GGT) hyrolyses extracellular GSH to produce products which can be imported inside of cells. Dotted lines denote H2O2 diffusion. SOD: superoxide dismutase; RH: organic molecule (e.g., lipid); Radicals: HO2● (hydrogen superoxide); ●H (hydrogen); O2●- (superoxide); HO2●- (hydrogen superoxide anion); NO● (nitric oxide); ONOO● (peroxynitrile); ROO● (organic radical such as lipid peroxyl). (The figure has been adapted from Chio and Tuveson (2017))

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(Ali and Petrovsky 2019). ROS generation and NF-κB activation-induced inflammation manifested an upregulation of active inflammatory mediators involved in monocyte adhesion and chemotaxis. NF-κB is a key redox-regulated sensor gene under the influence of oxidative stress, which gets activated even at low dosage of H2O2 (Li and Karin 1999). In normal conditions, NF-κB inhibitor IκB keeps tightly bound to it and sequesters the transcriptional activities in cell. Post-proteosomal degradation of residual NF-κB translocates into the nucleus and acts as a transcription factor for inducing expression of antiapoptotic genes (Li and Karin 1999). For years, ROS has been known to trigger mitochondria to release cytochrome C, possibly by activating pore-stabilizing proteins and by inactivating poredestabilizing proteins, leading to intrinsic apoptosis. On the other hand, oxidative stress is known to activate some death receptors especially Fas/FADD, TNF-R1, and TRAIL receptors, and impart in inducing extrinsic pathway of apoptosis through caspase-8 activation (Velu et al. 2020). Cell necrosis and autophagy are also associated with the excess amount of ROS generation (Qian et al. 2019). These events are indicative that ROS may culminate into cellular senescence and death via multiple mechanisms including intrinsic and extrinsic apoptosis, necrosis, and autophagy depending on the cellular sensitization mechanisms; these molecular steps can be targeted in cancer therapy.

Excess ROS Perturbs the Cellular Homeostasis Production and scavenging of ROS is a rapid radical chain reaction, and a balance is required between both processes for regulating normal cellular homeostasis (Pizzino et al. 2017). Inability of cells to clear the formed ROS knocks down the oxidative balance resulting in oxidative stress. As has been previously mentioned, excess ROS production is linked to damage redox balance, which, in turn, causes inflammation and other pathological conditions in several chronic diseases. During hypoxia, mitochondrial ETC releases excessive ROS, which upregulate hypoxia-inducible factor-1 (HIF-1) gene expression and maintains oxygen homeostasis by stimulating cellular angiogenesis (Schieber and Chandel 2014). This happens at a very low level of ROS production and can be considered as beneficial, as it can be overcome and stabilized by metabolic responses such as angiogenesis. Additionally, medium and higher levels of ROS production may lead to apoptosis by involving the death receptor pathway and/or mitochondrial pathway (Pizzino et al. 2017). The role of ROS has also been implicated in both the innate and adaptive immune responses. The response of innate immunity upon exposure to environmental pathogens causes the production of ROS leading to the activation of phagocytes (Pizzino et al. 2017). ROS can interfere with the recognition between T cell receptor (TCR) and MHC-peptide complex, and thus inhibit T-cell immune responses. Similarly, inhibition of ROS by antioxidant enzyme catalase could impair suppressive effects on T cell proliferation. In addition, generation of ROS by cancer cells and tumorinfiltrating leukocytes such as myeloid-derived suppressor cells (MDSCs), regulatory T cells (Tregs), and tumor-associated macrophages (TAMs), could suppress the

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immune responses. MDSCs, the major immunosuppressive subsets in tumor cells under the influence of ROS, have been shown to play a vital role in tumor progression and contribute to suppressive tumor microenvironment (Pizzino et al. 2017) . During muscle contraction, ROS act as the intermediates and signals in the regulation of glucose uptake in skeletal muscles. Muscle activity regulates cellular antioxidant defense mechanism, and both acute and chronic exercise increase the cellular resistance to oxidative damage in humans and animals (Pizzino et al. 2017). In this respect, excessive production of ROS is associated with varieties of immune responses in several diseases, including cardiovascular disease, e.g., ischemia and heart attack, and in neurodegenerative diseases such as Alzheimer’s disease (Perillo et al. 2020). In general, the harmful effects of ROS include (a) damage of DNA, (b) oxidation of polyunsaturated fatty acids in lipids (lipid peroxidation), (c) oxidations of amino acids in proteins, and (d) oxidative deactivation of specific enzymes by oxidation of cofactors (Pizzino et al. 2017). ROS, as mentioned before, have been reported, to enhance the proliferation of cancer cells through reprogramming and rewiring of several signaling cascades that regulate cellular homeostasis (Pizzino et al. 2017). In a normal cell, the level of ROS is balanced through several detoxification processes which are mainly regulated by antioxidant enzyme systems. This maintains cellular oxidative homeostasis and contributes to the normal physiological functions of the cell. Generation of ROS by the mitochondrial respiratory chain sustains the cellular oxidative balance with involvement of several other resources such as α-ketoglutarate dehydrogenase, monoamine oxidase, sirtuins, Nrf2, and FOXO3. It is evident now that interferences in this homeostasis add to the initiation and progression of cancer, and excessive ROS promote metastasis of primary tumors, causing increased chances of morbidity and mortality (Aggarwal et al. 2019). In addition, ROS could activate various transcription factors, especially the NF-κB, activator protein-1, HIF-1α and STAT3, which could result in inflammation, tumor cell survival and proliferation, angiogenesis, invasion, and metastasis. Several research findings reported that excessive production of ROS is associated with the cell cycle arrest, increased phosphorylation of ATM and Chk1, and decreased levels of cell division cycle 25 homolog C, and thus can inhibit cell proliferation.(Srinivas et al. 2019). In this context, ROS-mediated oxidation of DNA is one of the main causes of mutations and other changes due to DNA damage (Srinivas et al. 2019). Therefore, these regulators of cellular homeostasis under the influence of ROS appear to be as a double-edged sword, and can be used toward the development of novel cancer therapies.

Reactive Oxygen Species and Cancer The relationship between ROS and cancer receives a considerable amount of attention among scientists. ROS has been described as a double-edged sword in the literature to explain its paradoxical role in cancer. As stated earlier, ROS enhance cancer cell proliferation and promote growth of various types of cancer (Pizzino

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et al. 2017). ROS-related oxidation of DNA is one of the main causes of tumorrelated mutations, which can further cause genomic instability and DNA damage. Excess ROS, however, causes reduced cancer cell proliferation by inducing G2/M phase cell cycle arrest through involvement of ATM, Chk1, Chk2, and cdc25 (Srinivas et al. 2019). These dual properties of ROS are paradoxical and the interplay between them in cancer is substantially tricky and intricate. Normal cells and cancer cells simultaneously utilize ROS to support cell growth and survival. However, higher levels of ROS is present in the cancer cells compared to the normal cells as a result of increased metabolism of glucose, mitochondrial dysfunction, and activity of oncogene (Schieber and Chandel 2014). On one hand, this property enables the activation of central pro-tumorigenic signaling pathways, which results in the oxidative stress and exerts potential antitumor effects (Schieber and Chandel 2014). These paradoxical roles of ROS in cancer create a very important question: “ROS in cancer, is it good? or bad?”. To answer this question, we provided a comprehensive overview on how ROS are associated with cancer progression, and how redox regulation can be exploited to develop anticancer therapies.

ROS in Cancer Progression Tumor initiation, development, and progression is substantially driven by a moderately increased levels in ROS levels (Gorrini et al. 2013). The moderate level of ROS is associated with reduced cellular antioxidant systems which facilitates the malignant transformation through the activation of cell proliferating signaling cascades by recruiting ERK, PI3K/Akt, p38, and JNK, which in turn activate MMPs, NF-κB, and VEGF. Moreover, ROS directly cause mutation to DNA in the early stage of tumor formation, resulting in genome instability, mitochondrial DNA damage, and triggering several signaling cascade to malignancy. Additionally, epigenetic changes such as methylation or acetylation can also affect the expression of oncogenes and tumor suppressor genes and promote carcinogenesis, which further promote ROS production and accumulation (Helfinger and Schroeder 2018). ROS affect all essential processes associated with the tumor formation, survival, and poor prognosis of cancers, along with sustained proliferation, elevated inflammation, reduced apoptosis, and enhanced angiogenesis, invasion, and metastasis (Schieber and Chandel 2014; Ali et al. 2020). Cellular proteins and lipids modified by the disulfide bond formation by ROS catalysis can result in unstable, short-lived lipids, and further attract more ROS through secondary messenger pathways. Moreover, ROS can mediate the oxidation of phosphatase and tensin homologue (PTEN), a tumor suppressor protein, it also can cause oxidation of protein tyrosine phosphatase, and MAPK phosphatases (Aggarwal et al. 2019). As a result, ROS modulate these tumor suppressors and fuel the pro-survival PI3K/AKT and MAPK/ERK signaling pathways. In low to moderate concentrations, ROS signal the cancer cells to survive by activating MAPK/ERK1/2, p38, c-Jun N-terminal kinase (JNK/p38), PI3K/Akt. Consequently, ROS, through all of these signaling cascades, activate NF-κB, matrix metalloproteinases (MMPs), and VEGF at different

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concentration (Aggarwal et al. 2019). Indeed, increased levels of ROS can activate several transcription factors including NF-κB, activator protein 1 (AP-1, NRF2, and HIF-1α), which can induce their target genes involved in cellular proliferation (Qian et al. 2019; Villa et al. 2019). In this context, somatic mutation in Nrf2 has been found to be associated with lung cancer, and research findings have shown that chemo-resistance can develop from some antitumor therapy as a result of such mutations (Hayes and McMahon 2009). Chemotherapeutic drugs reduce cellular ROS to a tolerable level, which can be used by cancer cells for angiogenesis signaling and tumor progression leading to relapse (Hayes and McMahon 2009). Neovasculature formation is associated with tumor, and is also known as angiogenesis. It provides a supportive physiological pathway for delivering oxygen and nutrients for the continued growth of cancer cell and leads toward metastasis (Peshavariya et al. 2009). The NOXs-derived ROS especially H2O2, promote endothelial cell (EC) proliferation and survival, and prevent apoptosis (Peshavariya et al. 2009). Furthermore, phosphorylation of cadherin/catenin by ROS leads to the disassembly of EC junctions and promotes cell migration. In addition, ROS activate VEGF signaling involving multiple intricate pathways including induction of the HIF-1α which further increases VEGF and VEGFR expression (Hwang and Lee 2011). This further leads to the induction of PI3K/Akt and MAPK signaling pathways necessary for the promotion of growth and angiogenesis. This angiogenic signaling cascades upregulates the expression of VEGFR and causes poor prognosis in a variety of malignancies in cancers with markedly high levels of VEGF-C/ VEGFRs signaling (Hwang and Lee 2011). Recently, there have been a number of studies to indicate that overexpressed VEGF-B/VEGFR2 signaling can promote cancer metastasis by remodeling the tumor microvasculature (Ceci et al. 2020). Moreover, studies have demonstrated that ROS are involved in the activation of epithelial mesenchymal transition (EMT) and metastasis in breast cancer cells via the mitochondrial repression by activating the Distal-less homeobox-2 (Dlx-2)/Snail signaling. In breast cancer cells, ROS can also activate EMT through the inhibition of oxidative phosphorylation and mitochondrial cytochrome c oxidase. In line with this, Sun et al. (2019) have recently demonstrated that mutational loss of a mitochondrial transmembrane protein 126A (TMEM126A) in breast cancer cells induced mitochondrial dysfunction and caused overproduction of ROS, which subsequently activated ECM remodeling, EMT, and metastasis. Furthermore, elevation of ROS was correlated with activation of the NF-κB pathway followed by modulation of glycolytic metabolism and high glucosemediated reduction of apoptosis and increased angiogenesis in breast cancer cells (Sun et al. 2019). In addition, elevated levels of mitochondrial ROS attributed to fatty acid β-oxidation in colorectal cancer cells caused activation of MAPK cascades and resulted in EMT and metastasis as depicted in Fig. 2. Metastasis is the primary cause of cancer-related mortality involving migration of cancer cells to distant tissues and organs (Aggarwal et al. 2019). ROS, which is a byproduct of aerobic respiration in mitochondrial ETC, play a pivotal role in the migration and invasion of cancer cells. Cancer cells from blood and metastatic sites display a substantially higher level of cytoplasmic and mitochondrial ROS as

Fig. 2 ROS-mediated angiogenesis and tumor metastasis. Endogenous ROS pro-survival signaling pathways in primary tumors primarily via activation of PTEN, PI3K, EGR, and Akt, which leads to overexpression of HIF-α and causes VEGF to induce angiogenesis. ROS-induced pro-survival signals further activate the processes of invasion and migration primarily via overexpression of MMPs and SNAIL and lead to tumor metastasis

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compared to the primary tumors. In contrast, in some context, a reverse phenomenon such as inhibition of metastasis by oxidative stress is also possible (Peiris-Pagès et al. 2015). In addition, involvement of ROS in the transition of tumor cells from epithelial to mesenchymal phenotype is believed to be a significant factor in tumor metastasis. Epithelial cells lose polarity and cell-cell adhesion, and gain migration ability and invasiveness through ROS-dependent mechanism (Aggarwal et al. 2019). ROS elevates hypoxia-induced MMPs, which, in turn, results in an increased migration of tumor cells. Mitochondrial dysfunction also leads to a considerable amount of ROS generation, which stimulates AP-1 signaling pathway and increases cytosolic Ca2+ levels leading to enhanced cell mobility (Kidd et al. 2013). Similarly, ROS generated from NOXs downregulate the activity of natural killer (NK) cells and influence cancer metastasis. HIF pathway associated with oxidative stress significantly upregulates vimentin gene transcription, an important mesenchymal cell protein involved in the EMT during metastatic progression (Kidd et al. 2013). Increased vimentin gene expression also enhances the formation of invadopodium, which is actively formed by metastatic cancer cells, and helps in the invasion and migration (Kidd et al. 2013). In this respect, HIF-1α and Snail are the important transcription factors induced by ROS and play a major role in EMT, which favors metastasis in chemoresistance cancers. ROS-induced elevation of Snail expression causes epigenetic changes such as DNA methylation in E-cadherin and various other tumor suppressor genes (Aggarwal et al. 2019). ROS are also substantially associated with the direct oxidative damage of protein tyrosine kinase Src, and enhance the invasion ability, growth, and survival of Src-transformed cells. Furthermore, H2O2-mediated oxidative stress accelerates cell migration by activating FAK in a PI3K-dependent manner (Basuroy et al. 2010). Thus, high ROS induce cytotoxic effects, whereas low ROS can induce proliferation, cell survival, EMT, and metastasis of cancer cells (Fig. 2); all of these ROS-mediated events could be potential therapeutic targets in cancer therapy.

ROS in Cancer Inhibition Cancer cells exhibit enhanced levels of ROS compared to normal cells, which consequently promote tumorigenesis. Increased ROS production is correlated with cell cycle arrest and other cell death mechanisms, for instance, apoptosis and autophagy. Apoptosis is a highly regulated programmed cell death, which can be induced through mainly two distinct pathways called the intrinsic pathway (mitochondrial) and the extrinsic pathway (via cell death receptor) (Redza-Dutordoir and Averill-Bates 2016). ROS can affect the activation of both types of apoptosis pathways (Redza-Dutordoir and Averill-Bates 2016). Furthermore, ROS activate death receptors like TNF-R1 and their adaptor proteins FADD and procaspase 8 and/or 10. Upon activation, they form death-inducing signaling complexes (DISCs) and trigger the specific caspase (procaspase 8 activates caspase 3) activation and apoptosis (Redza-Dutordoir and Averill-Bates 2016). In a different study, it has been shown that excessive production of ROS activated apoptosis in hepatocellular

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carcinoma cells by affecting MAPKs and AKT signaling pathways and inducing p53-dependent DNA damage (Chakraborty et al. 2019). Increase of ROS and cellular oxidation state promotes cancer cell survival which was interestingly inhibited by treating with activator of c-Met (a receptor tyrosine kinase), which are over-expressed in renal carcinoma cells. Activated c-Met could promote the expression of antiapoptotic proteins (Bcl-2 and Bcl-xL) and inhibited the expression of apoptotic cleaved caspase 3 through a specific mechanism involving Nrf2-HO-1 pathway (Chakraborty et al. 2019). In the intrinsic apoptosis pathway, elevated levels of ROS cause destruction of the mitochondrial membranes and release cytochrome c, which initiates the cascade of apoptosis (Redza-Dutordoir and Averill-Bates 2016). Mitochondrial cytochrome c forms a complex with apoptotic protein activating factor 1 (Apaf-1) and procaspase 9, which leads to the induction of effector caspase-3/7 through the activation of caspase 9 (Redza-Dutordoir and Averill-Bates 2016). The substantial loss of cytochrome c from mitochondria due to disturbed mitochondrial membrane itself causes further increase in the levels of ROS in a loop or cyclic mode (Redza-Dutordoir and Averill-Bates 2016). Moreover, ROS can regulate the functions of Bcl-2 family proteins, including both antiapoptotic (Bcl-2, Bcl-X, Bcl-XL) and proapoptotic (Bad, Bax, Bak, Bim, and Bid) proteins. Notably, these pro- and antiapoptotic members of Bcl-2 family are known to be transcriptionally regulated by NF-κB, which itself is a direct target of ROS (Li and Karin 1999). In addition, ROS may act as upstream signaling molecules through the ER pathway, and connect to the intrinsic apoptosis pathway. Excessive levels of ROS could trigger protein-misfolding leading to UPR and induction of CCAAT-enhancerbinding protein homologous protein (CHOP), thereby initiating apoptosis (Wilson et al. 2015; Ali et al. 2016, 2019). CHOP is induced by ER stress and mediates apoptosis in connection with ER. In this respect, ROS have the ability to stimulate the release of Ca2+ in the ER lumen (Ali et al. 2017). Excessive release of Ca2+ from ER is further absorbed by mitochondria due to its close proximity (Ali et al. 2019), which in turn causes overload of Ca2+ in the mitochondria. Excess Ca2+ in the mitochondria can facilitate the openings of mitochondrial permeability transition pores (MPTPs) leading to the release of ATP and cytochrome c and thus apoptosis induction under influence of increased ROS generation (Qian et al. 2019). In addition, the control of ER stress-mediated apoptosis also requires ASK-1/JNK cascade, which is directly regulated by ROS. Suppression of ROS by aesthetic midazolam induces apoptosis and S phase cell-cycle arrest in human leukemia and colon cancer cells (Mishra et al. 2013). Midazolam scavenged ROS level in leukemia cancer cells through inhibition of NOX-2 enzyme activity, which further led to the inhibition of ERK1/2 signaling. This also causes the suppression of antiapoptotic proteins Bcl-XL and XIAP and the activation of proapoptotic protein BID (Mishra et al. 2013). Autophagy is one of the cell survival mechanisms, which can play an essential role in the maintenance of normal cellular homeostasis. It is a multistep process where double-membrane autophagosomes are formed. In autophagy, degradation of damaged organelles and macromolecules is mediated via the lysosomes, and thereby

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cells are protected from unwanted stress. Therefore, depending on the cellular micromilieu conditions, autophagy can activate cell death mechanisms, and can also function as a tumor suppressor. Several anticancer agents have shown autophagy induction in cancer cells which also included those that induced ROS-dependent autophagic cell death (Poillet-Perez et al. 2015). Under different cellular conditions, ROS can regulate pro-survival or pro-death signaling of autophagy in cancer cells. One recent study demonstrated that inactivation of ATG4 by ROS (H2O2) leads to the elevated generation of LC3-associated autophagosomes. In addition, ROS can influence autophagy induction mechanism by regulating some transcription factors such as NF-κB and result in altered expression of autophagyassociated genes in cancer cells (Poillet-Perez et al. 2015). On the other hand, autophagy can also decrease ROS levels through activation of the P62 and NRF/KEAP1 pathways. In response to ROS, p62 is activated and interacts with KEAP1, and causes suppression of NRF2 degradation, thereby promoting NRF2 activation, which ultimately can trigger antioxidant defense genes such as GPX and SOD (Poillet-Perez et al. 2015). Ferroptosis is a new form of PCD, characterized by the accumulation of iron-dependent ROS. Ferroptosis is primarily caused by an imbalance in the generation and elimination of the intracellular lipidic ROS and can cause iron-dependent oxidative cell death by reducing the antioxidant capacity. Ferroptosis is also considered as type of programmed necrosis stimulated by lipid peroxidation in an iron-dependent ROS production. Several compounds with abilities to induce ferroptosis can kill cancer cells in a manner mainly related to the metabolism of amino acids/GSH, lipids, and iron and the regulation of p53 (Qian et al. 2019). Erastin is a small molecule inhibitor that can induce ferroptotosis by binding and inhibition of VDAC2 and VDAC3. It can additionally inhibit the activity of the cysteine-glutamate antiporter (system XC ), reduce cystine uptake, and lead to the associated depletion of intracellular GSH. Inhibiting glutathione peroxidase 4 (GPX4) can trigger ferroptosis even at regular cellular cysteine and GSH levels. In contrast, the lipophilic antioxidants like Trolox, ferrostatin-1, and liproxstatin-1 can inhibit ferroptosis (Qian et al. 2019). Intracellular iron, which is involved in lipid oxidation and ROS generation, can induce ferroptosis. Iron can cause oxidative damage and cell death through the generation of lipid-free radicals by lipid peroxidation. Iron chelator, for example, deferoxamine, can suppress ferroptotic-mediated cell death. Moreover, PKC-mediated HSPB1 can inhibit ferroptosis via the reduced production of iron-dependent lipid ROS (Qian et al. 2019). Therefore, irondependent lipid ROS generation plays an important role in the promotion of ferroptosis. Other studies have further demonstrated the role of p53 in inducing ferroptotic cell death. p53 led to ROS-induced ferroptosis and tumor suppression by downregulating the expression of SLC7A11 and averted the system XC from absorbing cystine, which resulted in decreased cystine-dependent GPX activity and cellular antioxidant capacity (Chio and Tuveson 2017). These observations are contrary to those of many other reports, which suggest that P53 reduces cellular levels of ROS. Furthermore, at low levels of ROS, P53 may prevent the further accumulation of

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ROS and promote cell survival; whereas at a higher level of ROS, p53 may induce cell death via ferroptosis. With diverse repertoire on the cellular responsibilities of p53 and ROS, it is suggested that this regulation in tumors needs further study (Qian et al. 2019).

Application of ROS Induction for Anticancer Strategies Numerous anticancer therapies have actively utilized the antioxidant supplements as a strategy to prevent or treat cancers. tert-Butylhydroquinone (t-BHQ) is a such agent that can cause ROS generation during metabolic processes and lead to the dissociation of Nrf2 via oxidative modification of the Keap1 cysteine residues (Perillo et al. 2020). Activation of Nrf2 further promotes the regulation of cytoprotective downstream genes that play vital roles in cancer prevention. In this context, selenocompounds have shown requisite interest with anticancer effects by enhancing the antioxidative defense system in cells with ROS-induced damage. It also acts through modification of redox state and cysteine-rich regions of PKC (a receptor for tumor promoters) (Perillo et al. 2020). However, certain contrasting issues remain to address in view of the chemotherapeutic activities of antioxidants, for example, where antioxidants mainly maintain the cellular redox balance and also intervene the inflammatory signaling pathways. Nrf2 activation has indeed been reported to contribute toward chemoresistance in cancer cells (Pizzino et al. 2017). Moreover, a high concentration of t-BHQ has been shown to increase the carcinogenic risk. Likewise, the safety and efficacy of selenium needs considerable discussion due to its toxicity and side effects. Thus, antioxidant-mediated chemotherapeutics may not be sufficient to treat cancer, and future studies are required to understand any undesired side effects. ROS as a double-edged sword relates to its effect on low-dose cell signaling and high-dose cytotoxicity. A mild level of ROS can regulate cell development and homeostasis; whereas a high level of ROS can inflict severe cellular damage. Additionally, cancer cells are more sensitive toward the oxidative imbalance, the presence of prooxidants and the inhibition of antioxidants due to their excessive ROS levels. On the other hand, ROS-inducing approach to kill cancer cells depends on the oxidative stress-dependent cytotoxicity as well as apoptosis, necroptosis, and autophagy. Moderate levels of ROS affect modulation of cellular antioxidant enzyme systems and promote malignant transformation, while a high level of ROS induces apoptosis in cancer cells (Aggarwal et al. 2019). As mentioned previously, cancer cells proliferate in an uncontrolled manner, which is often involved with the alteration of transcription factors that are related to cellular proliferation. Cancer cells are prone to DNA damage, and thus can be targeted by developing therapeutics that can kill cancer cells by inducing DNA damage and oxidative stress. Enhanced endogenous antioxidants levels can be seen in metastatic tumors, which can balance oxidative stress. The presence of decreased GSH/GSSG ratio in circulating melanoma or metastatic cancers indicates that advanced stage cancers have better antioxidative potential compared to early-stage

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cancers. However, it has been documented that NADPH-independent catalase activity becomes reduced along with the cancer progression. Indeed, the antioxidant feature of advanced cancers is one of the critical factors of chemotherapeutic resistance (Gorrini et al. 2013; Mishra et al. 2013). Thus, an approach to induce ROS and/or to suppress antioxidants can be appropriately applied for the treatment of malignant cancer cells. In this regard, oxidative stress-modulating therapeutics for attacking cancer cells have been currently studied in anticancer studies (Gorrini et al. 2013). The cellkilling capacity of ROS has been attributed for anticancer therapies with two major approaches: (i) direct ROS generation and (ii) inhibition of antioxidant process. NOX1 and NOX2 promote oncogenic signaling pathways by directly associating with HIF1-α, leading to activation of NOX-mediated ROS generation and potentiating HIF-1α by inhibiting its proteasomal degradation (Gorrini et al. 2013). Among other NOXs, NOX4 plays a pro-cancer role through upregulation of oncogenic signaling pathways in renal cell carcinoma, glioblastoma multiform, ovarian carcinoma, and pancreatic cancer (Gorrini et al. 2013). Suppression of NOX-2 dependent ROS generation by midazolam limits the growth of human cancer cells (Mishra et al. 2013). Thus, specifically targeting the NOXs moieties through small molecule inhibitors may act as targeted therapy against cancer cells.

Direct ROS Generation Metabolism and respiratory processes lead to production of electrons, which are the sources of ROS in cells. Disruption of mitochondrial respiratory cycles generates superoxide and the same mechanism follows with metexafin gadolinium and anthracyclines to kill cancer cells (Omura 2008). Metexafin gadolinium, which is effective in the treatment of brain tumors and pediatric glioma patients, acts as an electron acceptor and increases therapeutic index of radiation. It is also involved in impairing the repair activities of cancer cells after irradiation. Besides, anticancer drug doxorubicin generates hydroxyl radicals and leads to cell death through induction of intracellular iron chelation. Another anticancer drug cisplatin, which is effective against a wide variety of cancers, especially ovarian cancer, has been shown to perturb DNA replication machinery and induction of cellular oxidative stress (Omura 2008). It also involves damaging the mtDNA and ETC impairments (Marullo et al. 2013). A number of studies documented the association of 2-methoxyestradiol to inhibit cancer cell proliferation individually or when used in combination with other synergistic drugs. Indeed, this drug has been reported to disrupt the ETC complex I via the production of mitochondrial H2O2. It robustly induces c-Jun N-terminal kinase (JNK) signaling, which leads to the release of cytochrome c and caspase-9, and thereby activates intrinsic apoptosis. Additionally, this drug can synergize the activity of other anticancer agents to kill cancer cells (Marullo et al. 2013). Listed in Table 1 are ROS accelerating anticancer agents described above. Although anticancer drugs with direct ROS-accumulating activity may be effective for treating different types of cancer, their effects on normal and para-cancerous

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Table 1 Redox-active drugs at different stages of validation and development Drug or agent 2-Deoxyglucose

2-Methoxyestradiol 5-Fluorouracil

Arsenic trioxide (As2O3)

Artenimol

Auranofin

Cisplatin

Curcumin

Cyclophosphamide

Disulfiram

Doxorubicin

Mechanism of action/ target Induces oxidative stress via accumulation of glutathione disulfide and NADP+ /NADPH Inhibits ETC complex I, cellular ROS production, HIF-1α stabilization Inhibits thymidylate synthase, blocks DNA and RNA synthesis, increases ROS Mitochondrial dysfunction via the inhibition of thioredoxin 1, covalent cross-linking of vicinal thiols Promotes iron metabolism and ROS-mediated ferroptosis Inhibits redox enzyme thioredoxin reductase

Induces mitochondrial ROS, increases the level of superoxide anions, hydroxyl radical, and hydrogen peroxide; damages mtDNA and ETC, enhances cytotoxicity Enhances intracellular ROS by increasing the potential of mitochondrial membrane Induces marked production of oxidants as an adverse effects Inhibits mitochondrial ALDH activity, activates the p38 pathway and ROS Induces chelation of iron to generate hydroxyl radical, promotes oxidative stress, DNA damage, lipid peroxidation

Clinical status/related notes Phase I/II trials pancreatic carcinoma, prostate cancer, cervical carcinoma Phase I trial

References Ben Sahra et al. (2010)

Rajkumar et al. (2007)

FDA-approved colorectal carcinoma, breast cancer

Longley et al. (2003)

FDA approved to treat promyelocytic leukemia

Zheng et al. (2015)

Treat colon cancer, lung carcinoma

Mou et al. (2019)

Phase I/II trial for head and neck cancer, ovarian cancer, leukemia, and lymphoma FDA-approved chemotherapy agent to treat variety of cancers

Kirkpatrick and Powis (2017) Omura (2008), Marullo et al. (2013), Carroll et al. (2016)

Phase II/III, almost all types of cancers

Shanmugam et al. (2015)

Widely used to treat breast, ovarian, and hematologic cancer FDA approved to treat glioblastoma

Jeelani et al. (2017)

Phase III trial as chemotherapy agent

Xu et al. (2017)

Kim et al. (2019)

(continued)

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Table 1 (continued) Drug or agent Enasidenib

Erastin

Fenretinide

Mechanism of action/ target Specific inhibitors for IDH1/2 mutant and targets mROS for the anticancer effect Inhibits VDAC2/VDAC3, blocks GSH synthesis, increases lipid peroxidation and lipid ROS Generates ROS which lead to apoptosis

FIN56

Degrades GPX4 or inhibits the function GPX4

FINO2

Inhibits GPX4

Imexon

Binds to thiol to disrupt GSH activity, induces oxidative stress; depletes the GSH pool for antioxidative activity Specific inhibitors for IDH1/2 mutant and targets mROS for the anticancer effect Inhibition of system Xc-, enhances ROS production

Ivosidenib

Lanperisone

MitoTEMPO

ML-162 ML-210 NOV-002

Oxaliplatin

Activates SOD2 and inhibits mitochondrial superoxide Inhibits GPX4, enhances ROS production Inhibits GPX4, increases ROS production Mimetic of GSSG complex with nontherapeutic concentrations of cisplatin Retain DACH by the formation of platinum– DNA adducts, blocks DNA replication

Clinical status/related notes FDA approved to treat acute myeloid leukemia and glioblastoma

References Stein et al. (2017)

Phase I/II/III Fibrosarcoma, lung carcinoma, prostate cancer, osteosarcoma

Habermann et al. (2017)

Phase III trial to treat breast cancer

Kirkpatrick and Powis (2017) Gaschler et al. (2018)

Fibrosarcoma and transformed human fibroblast cells Fibrosarcoma, renal cell carcinoma Phase I/II trial

Gaschler et al. (2018) Omura (2008), Yang et al. (2018)

FDA approved to treat acute myeloid leukemia and glioblastoma

Stein et al. (2017)

FDA approved lung carcinoma, Kras-mutant mouse embryonic fibroblast Treat renal cell carcinoma

Mai et al. (2017)

Siska et al. (2017)

Treat colon cancer, melanoma Treat lung carcinoma, colon cancer IND in phase II/III trial

Stockwell (2019) (Stockwell 2019) Townsend et al. (2008)

FDA approved Colon carcinoma, ovarian cancer, lung carcinoma

MikulaPietrasik et al. (2019) (continued)

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Table 1 (continued) Drug or agent Paclitaxel

PARP inhibitors (Olaparib, niraparib)

RSL-3 Sorafenib

Sulfasalazine (SSZ)

Telcyta (TLK-286)

Temozolomide (TMZ) Vitamin A Vitamin C

Vorinostat

Salinomycin and ionomycin Vitamin D

Mechanism of action/ target Increased mitochondria ROS that results in activation of STAT3 signaling Inhibit the activity of PARP enzyme, enhance ROS-mediated DNA damage Inhibits GPX4 and deplete GSH to induce ROS Inhibition of system Xc-, depletes GSH leading to accumulation of lipid ROS Inhibition of system Xc-, induction of ferroptosis

Inhibits glutathione S-transferase, GSTP activated prodrug in ovarian cancer, NSCLC, and others Inhibits autophagy, induces cell death via accumulation of lipid ROS Enhances ROS production Attenuated tumor growth in mutant Kras (G12D)/ Apc murine models Suppresses SLC7A11, enhances ROS lead to DNA damage Iron-mediated ROS production Alteration in ratio of GSSG and GSH, regulate thioredoxin-interacting protein

Clinical status/related notes Treat lung carcinoma, breast cancer

FDA and European Medicines Agency approved for breast cancer, pancreatic carcinoma Clinical study for lung carcinoma, colon cancer FDA approved to treat hepatocellular carcinoma FDA approved to treat glioma, pancreatic carcinoma, lung carcinoma Investigational new drug in phase I/II/III trial to treat breast, ovarian cancer

References Ren et al. (2018)

Franzese et al. (2019)

Stockwell (2019) Stockwell (2019) Stockwell (2019)

Kirkpatrick and Powis (2017)

FDA-approved glioblastoma stem cells

Shireman et al. (2020)

Treat ovarian cancer

Wang and He (2020) Yun et al. (2015)

Clinical trials colorectal carcinoma, pancreatic carcinoma FDA approved

FDA approved to treat breast cancer, colon cancer Clinical trials for endometrial cancer, breast cancer

Wang et al. (2018) Mai et al. (2017) Grant (2020)

cells are still controversial. For example, the radiosensitizer motexafin gadolinium interferes with the DNA repair process and causes injuries to the neighboring normal cells. Similarly, cisplatin-mediated ototoxicity can be caused through the binding of DNA and subsequent induction of inflammatory signaling (Waissbluth and Daniel

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2013). Thus, this specification to cancer or normal cells needs further attention and screening in future.

Antioxidant Process Inhibition Treating invasive cancer cells by the direct ROS induction is one of the effective strategies; however, the combination of ROS induction and inhibition of antioxidative activity leads to favorable outcomes to overcome therapeutic resistance in cancer cells. Depletion of intracellular GSH activity or concentration is considered as an alternative way to generate oxidative stress. For example, buthionine sulfoximine (BSO) is a commercially available GSH inhibitor that can bind to glutamate-cysteine ligase site that usually binds to the acceptor amino acid. BSO is a known oxidative stress-causing agent though intervening GSH synthesis pathway. Chemotherapeutic agent imexon, which is generally employed to treat breast cancers, is involved in depleting GSH levels for antioxidative activity by binding to the thiol group of reduced GSH (Sheveleva et al. 2012). Telintra (Ezatiostat HCl), a small molecule peptidomimetic inhibitor of GST P1–1, can act by interfering with the complex formation between signaling protein GSTP and JNK. Myelodysplastic syndrome is a pre-leukemia where bone marrow cannot produce adequate levels of one or more of the three major blood cell types. Either pharmacological or genetic silencing of GSTP increases WBC formation in normal animals and in animals treated with cancer therapy (Sheveleva et al. 2012). As mentioned before, even though anticancer therapy needs to disrupt the redox balance of malignant cancer cells, the redox state of cancer cells and the inhibition of antioxidative enzymes employ damaging effects on neighboring normal cells in that particular tissue and/or organ. For example, BSO-induced oxidative stress has been shown to be linked with heart hypertrophy (Dvorakova et al. 2001). Due to its general cytotoxicity, imexon exerts potential side effects in normal cells (Dvorakova et al. 2001). Further in-depth researches are necessary to identify appropriate ways in order to reduce associated side effects, and concomitantly to enhance the efficiency and specificity to kill cancer cells.

Combination Therapy Combination therapy is best known as a treatment approach that combines two or more therapeutic methods or agents, and is considered important in cancer therapy. Combination therapy needs to address the two major issues: reduce the toxicity of chemotherapeutics to normal cells, and increase the efficacy of antitumor activity. Metabolic alterations either through the generation of ROS or antioxidant inhibition could serve as an ideal candidate for combination therapy. Glutamine metabolism is critical in antioxidant response, and glutamate, a vital precursor of glutamine metabolic pathway, is essential for GSH synthesis. Glutamine metabolism is vital for the survival of cancer cells, and indeed many cancers are addicted to glutamine (Hayes

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and McMahon 2009). An essential enzyme of this metabolic pathway is glutaminase 1 (GLS1) that converts glutamine to glutamate for the entry into the TCA cycle, and inhibition of this enzyme is associated with the suppression of oncogenic transformation (Hayes and McMahon 2009). Moreover, an anti-leukemic agent inhibits GSH synthesis by causing depletion of glutamine levels. Similarly, the drug that targets L-asparaginase reduces the level of asparagine, and thereby exerts anticancer effect through the alteration of glutamine levels (Hayes and McMahon 2009). Aspartate transaminase (GOT1) is a key enzyme, which is involved in the alternative glutamine metabolic pathway. This enzyme is associated with the aspartate-malate shuttle and produces pyruvate and enhances the ratio of NADPH/NADP+, and thus maintains the reduced GSH levels and redox homeostasis. In addition, GOT1 is essential for KRAS-driven growth of pancreatic ductal adenocarcinoma (PDAC) (Xiang et al. 2018). Therefore, inhibition of GOT1 can cause a decrease ratio of reduced-tooxidized GSH and increases ROS levels, and thereby can suppress the growth of PDAC. Thus, agents that disrupt the glutamine metabolic pathway and GSH production are considered potential anticancer therapies (Xiang et al. 2018). In a pathway analysis, Ricker et al. (2004) found that 2-methoxyestradiol is not only a potent inhibitor of HIF1A and VEGFA playing important roles in angiogenesis, but it also activates MAPK8 to trigger apoptosis (Ricker et al. 2004). In addition, 2-methoxyestradiol was found to be closely associated with modulation of cellular processes like apoptosis, cell proliferation, and angiogenesis. This drug could mainly target breast cancer, melanoma, and pancreatic cancer, as well as attenuated atherosclerosis due to its anti-angiogenetic mechanism. However, 2-methoxyestradiol-mediated autophagy was found to promote cancer cell survival and drug resistance (Ricker et al. 2004). Research findings indicated that BSO effectively inhibits GCLC, and thus blocks GSH synthesis (Liu et al. 2016). Notably, GSH depletion is directly associated with decreased expression of GPX1 and increased expression of NFE2L2 and SOD2. Thus, BSO-mediated inhibition of GSH caused oxidative stress, apoptosis, and cell death as major alterations in the cellular processes. This drug could mainly target breast cancer, hepatocellular carcinoma, and lung cancer and could evoke cataract by increased lipid peroxidation in the lens. The mechanism behind effects of BSO included increased NFE2L2 and upregulation of a cell membrane transporter protein ABCC1 (Liu et al. 2016). This upregulation causes increased drug efflux through the transporter and leads to drug resistance. Additionally, BSO-mediated autophagy was negatively associated with drug sensitivity (Liu et al. 2016). Cisplatin notably induces p53 expression as well as proapoptotic genes (TNF, BAX, CASP3, and FAS), while decreasing antiapoptotic genes (BCL2 and XIAP). In addition, cisplatin caused significant induction of several cell processes like DNA damage apoptosis, ROS generation, and mitochondrial damage. Cisplatin mainly targets ovarian, lung, stomach, and breast cancers. Furthermore, cisplatin-induced pro-inflammatory cytokines IL1β, IL6, and TNFα could cause side effects, especially acute kidney injury and renal dysfunction (Carroll et al. 2016). The other well-known chemotherapeutic agent doxorubicin showed similar effects to cisplatin on targeted genes and cell processes. It significantly increased

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the expressions of TP53, BAX, TNF, CASP3, and FAS, and decreased the expressions of BCL2 and XIAP to promote apoptosis. Doxorubicin caused oxidative stress, DNA damage, and lipid peroxidation as plausible mechanisms of cell processes. This drug is mainly used to treat breast, ovarian, and lung cancers; however, it has notable risk including heart failure and neurotoxicity. Doxorubicin resistance is associated with increased autophagy and increased levels of ABCC1 and NFE2L2 (Carroll et al. 2016). On the other hand, imexon positively regulated the activity of caspases-3 and -9 which are critical mediators of apoptosis. Imexon could also induce oxidative stress and cell cycle arrest as plausible mechanisms of therapeutic effects on multiple myeloma and splenomegaly. Motexafin gadolinium induced apoptosis by inhibiting the activity of TXN and HMOX1, possibly by inducing oxidative stress and disrupted the DNA repair mechanism. Motexafin gadolinium is expected to mainly target lung cancer and cerebral neoplasm. However, findings showed that Mn(III) mesotetrakis (N-n-butoxyethylpyridinium-2yl) porphyrin (a SOD mimic), MnBuOE (MnP), can target malignant cell redox states and exert selective cytotoxicity via differential activation of molecular pathways controlling cell growth and proliferation (Carroll et al. 2016).

Immunotherapies Immunotherapy has emerged as an area of cancer treatment that stimulates the inherent ability of immune system to counter tumor cells. In connection with oxidative stress to immunotherapy, a recent study indicated that using uncouplers as ROS generators with immunotherapy has synergistic effects with inhibition of PD-1 and subsequently blocking of tumor growth. A suggested mechanism involves generation of hypoxia by the synergistic effect of the chemotherapeutics combination where one of the uncoupler alone showed no significant effect on the tumor cell growth (Hodny et al. 2016). In this context, IFN-γ being a part of the cellular defense system triggers NOX2 activity which causes overproduction of superoxide ions into phagosomes of immune cells; this has been correlated with IFNγ-mediated cancer immunotherapy. Moreover, IFNγ induces the constitutive expression of NOX4 in tumor cells, which leads to generation of ROS, damage of the DNA content, and eventually leading to premature cell cycle arrest and cellular senescence (Hodny et al. 2016). IFNγ-induced senescence and antitumor effect have been observed in pancreatic β-cancer cell model (Hodny et al. 2016). The mechanism behind these effects is linked to the regulation of IFNγ/STAT and TGFβ/SMAD signaling modules, which manages the proliferation and growth in cancer cells (Hodny et al. 2016). This has further envisaged that targeting this module can be a novel therapeutic approach in cancer prevention. ROS generation by the mitochondria or exogenous sources within tumor cells can affect the immunity and promote rigorous tumorigenic environment. Mitochondrial ROS generated by tert-butyl hydroperoxide synergizes the effect of PD-1 blockade on tumor growth inhibition and modulates the function of immune cells such as tumor reactive cytotoxic T-cell (T-CTLs). Lower levels of

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ROS and thus lower levels of hypoxic tumor microenvironment favor increased efficacy of PD-1 blockage-dependent immunotherapy (Siska et al. 2017). The mitochondrial ROS can affect the function of tumor-infiltrating T-cells in a concentration-dependent manner. Similarly, ROS generated by myeloid-derived suppressor cells (MDSCs), tumor-associated macrophages (TAMs), and regulatory T cell (Tregs) can also affect the function of other immune cells and regulate the function of T-cells in a tumor environment (Weinberg et al. 2019). The immunosuppressive tumor microenvironment favors greater tumor invasion, metastasis, and therapy resistance, while ROS plays a critical role in promoting these events through deregulation of immune cell suppression (Weinberg et al. 2019). Tregs present in the TME provide other sources of immunosuppression by decreasing T-CTLs immunity, and suggest that the balance between immunosuppressive Tregs and cytotoxic T-cells can be a targeted approach in cancer immunotherapy. This therapeutic possibility with targeting ROS in the tumor microenvironment can reduce ROS generation and limit the immunosuppressive activity in tumor cells. This has been further connected to IL-9 in tumor immunity and Chimeric Antigen Receptor T cells (CAR T cells) for treating the relapse and refractory B cell acute lymphoblastic leukemia (Siska et al. 2017), which offers an insight into potential therapeutic strategies. Studies on renal cell carcinoma tumors showed that CD8 TILs are present but they are functionally and metabolically impaired. The generated excess amounts of mitochondrial ROS can downregulate mitochondrial SOD2 (Siska et al. 2017). Another study showed that T cells modified with a bicistronic expression of CAR and co-expression of catalase (CAT) significantly reduce the intracellular oxidative stress and result in the increased ability of CAR-CAT T-cells to lyse tumor cells under extracellular oxidative stress unlike traditional CAR T-cells (Weinberg et al. 2019). Thus, these immune cell medications and regulation of oxidative stress offer an interesting approach in cancer immunotherapy.

Conclusions and Future Direction In summary, the comprehensive discussion suggests that redox homeostasis plays an essential role in the maintenance of a wide variety of cellular processes. Alteration of the cellular oxidative state can be effectively utilized to kill cancer cells by targeting redox homeostasis, which is being investigated for the development of novel chemotherapeutic regimens against different types of cancers. This chapter encompasses an overview of how ROS-induced cellular stress can be harnessed for anticancer therapy using prooxidative agents. This chapter also highlights the understanding of prooxidant drugs-mediated molecular signaling and their relevance as anticancer drugs through modulation of ROS balancing process. In addition, the current report provides insights for better understanding of the toxicological aspects, and for predicting the efficacy of chemotherapeutics using prooxidative anticancer drugs. Although studies have investigated the mechanisms of prooxidant drugs action, the pharmacogenomics, which is essential to determine the efficacy of a

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particular anticancer drug treatment, is poorly understood. This chapter may further provide information to better understanding of the current prooxidant drug-mediated anticancer strategies to limit the potential undesired side effects. Although ROS-induced physiological functions are complicated and essential, it is vital to maintain a balance between the levels of ROS and physiological or external antioxidant defense system. Indeed, this suggests more likely for having antioxidant supply through several sources including daily foods, drinks, and vitamins. However, ROS-mediated activation of cellular processes is essential for normal homeostatic conditions, and the extent of oxidative measurement is crucial before planning for antioxidant therapy.

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Modulatory Role of Adipocytes and Their Stem Nature in the ROS Signaling Within a Tumor Micro-environment

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Salvatore Chirumbolo, Geir Bjørklund, and Antonio Vella

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Lipid and ROS-Signaling in the Adipocyte-Mediated Tissue Dynamics? . . . . . . . . . . . . . . . . . . . . Conjugated Linoleic Acids as Switching Molecules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mechanism of CLA-Induced Delipidation/Dedifferentiation in the Adipocyte . . . . . . . . . . . Role of ROS in the CLAs Switching Mechanism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Role in Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract

The role of adipocytes as modulatory cells in the micro-environment immune cells/stem cells is particularly intriguing to assess how reactive oxygen species (ROS) play a signaling task in the fine tuning of cell-to-cell communication in a tumor environment. This cross talking should have a major meaning in the pathogenesis and development of cancer. This brief comment highlights the most fundamental issues of the topic. Keywords

Adipocyte · Cancer cell · ROS signaling · Stem cell · Pre-adipocyte · CLAs

S. Chirumbolo (*) Department of Neurosciences, Biomedicine and Movement Sciences-University of Verona, Verona, Italy e-mail: [email protected] G. Bjørklund Council for Nutritional and Environmental Medicine (CONEM), Mo i Rana, Norway A. Vella Unit of Immunology-Azienda Ospedaliera Universitaria Integrata, Verona, Italy © Springer Nature Singapore Pte Ltd. 2022 S. Chakraborti et al. (eds.), Handbook of Oxidative Stress in Cancer: Mechanistic Aspects, https://doi.org/10.1007/978-981-15-9411-3_120

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Introduction Adipocytes are intriguing cells whose function is currently in the spotlight because of their fundamental engagement in the fine tuning of the cancer/immunity cross talk (Wu et al. 2019a). So far, the white adipose tissue close to a tumor (peri-tumoral adipose tissue) has not been yet clarified if being a friend or a foe for cancer, so expanding the debate about the actual role of adipocytes in the immune interplay with tumor cells (Duong et al. 2017; Rio et al. 2015; Wright and Simone 2016; Zhang and Scherer 2018; Wang et al. 2019). According to our opinion, which we would like to address in this chapter, the role of the white adipose tissue (WAT) may be allocated at the crossroad between stress response and cell differentiation, which enables adipocytes to exert a fine regulatory task in the immune microenvironment of tumors. Fundamentally, through their ability in controlling tissue bioenergetics, they should have also a powerful role in modulating tissue turn over. This commitment is much probably exerted by fats released from adipocytes alongside with reactive oxygen species (ROS) acting as signaling molecules during a stress response. Its temping to speculate that both lipids and ROS co-act as a master tuner in tissue turnover and remodeling, particularly following traumas or damages and hence may represent a fundamental language to organize and modulate cancer development. Evidence was reported describing that conjugated linoleic acid (CLA), present in the diet, decreases the storage of fats in the adipose tissue. A hypothesis forwarded to elucidate this mechanism includes the possible formation of delipidated adipocytes (Brown et al. 2004). Delipidation might be a leading mechanism to support a certain degree of plasticity in the white adipose tissue, so to be fully used as a rejuvenation and stem tissue.

Lipid and ROS-Signaling in the Adipocyte-Mediated Tissue Dynamics? Conjugated Linoleic Acids as Switching Molecules Two kinds of CLAs, that is, trans-10 and cis-12 CLA, yet excluding cis-9 and trans-11 CLA, upon addition to stromal vascular fractions (SVFs) in in vitro cocultures containing differentiated human adipocytes, are the major causative factors of a time-dependent reduction in the adipocyte triacylglycerol storage, including glucose and fatty acid uptake upon insulin stimulation (Brown et al. 2004). These kinds of CLAS are able to dampen PPAR-γ and further PPAR-γ induced genes, besides to enhance the expression of leptin. In addition, they trigger a marked activation of the MEK/ERK signaling leading to the subsequent gene expression, causing the rapid release of IL-8 and IL-6. Pertussis toxin and UO126 are able to inhibit the signaling activation elicited by tran-10 e cis-12 CLAs, so highlighting that the complete mechanism induces adipocyte delipidation via an autocrine or even paracrine activity of IL-6 and IL-8 (Brown

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et al. 2004). Delipidation is a crucial step relating lipid signaling with the ability of mature adipocytes to revert to a younger/immature or a de-differentiated phenotype (Obsen et al. 2012; Marques et al. 2015; Yeganeh et al. 2017; Kennedy et al. 2008). These conjugated linoleic acids have been also associated with diet issues, as molecules able to trigger caloric restriction, and actually were included in a list of possible anti-obesity prodrugs and compounds able to reduce adipose-tissue fat storage. Actually, these PUFAs activate adipocyte delipidation (Brown et al. 2004). The most striking evidence is that the counterpart isomers cis-9 and trans-11 CLAs act in an opposite way on adipocytes, promoting adipogenesis (Segovia et al. 2017). Our speculation, therefore, is that adipocytes may be endowed with a tuning switcher, able to set “on” or “off,” via different CLAs, by adjusting or modulating the ratio of the [trans-10, cis-12]/[cis-9, trans-11], a mechanism particularly important in immunity (Albers et al. 2003). At the best of our knowledge, no evidence so far is able to support our speculation about the existence of a molecular CLA-switcher but its possibility from a chemical point of view is particularly easy. It is a switch of two conjugated double bonds in a form of PUFA (linoleic acid), that is, two C-C bonds, cis or trans, are shifted of one single position towards the ω-6 terminus alongside with a reciprocal isomerization, though at least 24 isomers of the 18:2 ω-6 linoleic acid were described, with a signaling meaning (Banni 2002). Furthermore, some recent investigations have focused onto the role of CLAs in adipocyte trans-differentiation, de-differentiation, and desmoplastic process (Brandebourg and Hu 2005). The mechanism by which CLAs act on the adipocyte switching toward a dedifferentiated/delipidated cell is summarized in Fig. 1.

Fig. 1 A The antagonists CLAs in adipocyte differentiation; B Low ROS levels from fibroblasts, activated by linoleic acid, induce FADS in adipocyte and their cycle regulation via CLAs. High ROS levels can induce NADPH-4 in adipocytes (NOX4) and hence the activation of dedifferentiating genes such as Notch. FADS (desaturase of adipocytes), CLA (conjugated linoleic acid isomers)

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Mechanism of CLA-Induced Delipidation/Dedifferentiation in the Adipocyte The isomer trans-10, cis-12 CLA is able to reduce the amount of pre-adipocytes, particularly inhibiting the enzyme glycerol-3-phosphate dehydrogenase (EC 1.1.1.8), whereas the quite-isomeric counterpart.cis-9, trans-11 CLAs act as antagonists of the former (Brandebourg and Hu 2005). Moreover, trans-10, cis-12 CLAs, besides to inhibiting PPAR-γ, reduces the activity of the sterol regulatory element binding protein-1c (SREBP-1c) (Brandebourg and Hu 2005), while it cannot exert an effect on the expression of the CCAAT/enhancer binding protein alpha (C/EBPα). Delipidating CLAs, that is, trans-10, cis-12 CLAs, are not merely chemical tuners of adipogenesis but of lipid building in the adipocyte and storage and ultimately are involved in the complex homeostasis pre/post adipocytes and mature adipose cells (Brandebourg and Hu 2005; House RL et al. 2005). Therefore, it is tempting to speculate that these conjugated PUFAs exert a major role in the “plastic” control of adipocytes and adipose tissue precursors. Figure 1A shows that two kinds of switching isomerization cis/trans and trans/cis in CLAs generate two master tuners of adipogenesis and fat storage promotion (adipose-addressed) or lipidolysis and stem precursors commitment (mesenchymal-addressed).

Role of ROS in the CLAs Switching Mechanism Figure 1B shows that ROS modulate the CLAs switching mechanism. First, trans-10, cis-12 CLAs are able to induce an inflammatory process (LaRosa et al. 2006). Furthermore, in animal models, these CLAs dampen adipogenesis in the subcutaneous fat, whereas promote adipogenesis in the intramuscular adipose tissue (Zhou et al. 2017). The biochemical synthesis of CLAs may involve a α9 desaturase. Actually, in the adipose tissue, PUFAs are usually modulated by the fatty acid desaturase FADS1 and FADS2, while linoleic acid is the major regulator of FADS enzyme expression (Ralston et al. 2015). This evidence would suggest that linoleic acid conjugated derivatives, that is, CLAs, are powerful signaling molecules in the adipocyte differentiation/dedifferentiation cycle, leading to the well-known plasticity of adipocytes, an issue that can elucidate many fundamental concerns about cancer. Probably, CLAs are intrinsic regulators in sub-dermal compartments, where most probably keratinocytes and fibroblasts are main sources of linoleic acid. Interestingly, linoleic acid can trigger with β-NADH an immediate and rapid production of reactive oxygen species (ROS) in fibroblasts, so inducing a fibroblastmediated oxidative burst (Hatanaka et al. 2013). This evidence suggests that a mechanism of cross talking ROS/CLAs may be active in the sub-dermal compartments, where adipocytes undergo plastic transformation. In addition, it is well known that ROS are able to induce adipocyte differentiating mechanisms, by increasing PPAR-γ and also activating C/EBPβ (Lee et al. 2009). In experimental in vitro models, differentiation occurs when cells reach the confluence

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and upon ROS-mediated oxidation. In this occurrence, upon hormonal induction, adipose cells activate PPAR-γ and enter the differentiation mechanism towards mature adipocytes, whereas ROS increase cell phase S during the clonal expansion observed in mitosis (Lee et al. 2009). In our opinion, probably this mechanism is the major tuning process able to modulate adiposity in sub-dermal fat tissues. Any detached adipocyte, more likely reported as loosing lipids (delipidation), has a high probability to access a route of de-differentiation toward mesenchymal and/or stem progenitors, whereas the whole cycle of differentiation/dedifferentiation, passing through delipidated and post-adipocyte phenotypes, is regulated by a CLAs/ROS interplay. Actually, ROS may elicit dedifferentiated adipocytes, probably via a Notch/NADPH oxidase 4 cross talk, and in our opinion this should occur when RS levels exceed a certain threshold, which can be monitored by following mitochondria oscillation (Jiao et al. 2019; Chirumbolo and Bjørklund 2017; Castro et al. 2016).

The Role in Cancer A first concerning question is: which is the task of the white adipose tissue (WAT) in the pathogenesis and progress of tumors? Initially, a purported descriptive function was forwarded, with adipocytes depicted on the basis of the activity associated with lipids as a fuel for cancer growth (Wu et al. 2019b; Muller 2013). According to recent interpretations, peri-tumoral WAT should encompass both mature fat-enriched adipocytes, pre-adipocytes, and adipose tissue derived stem cells (ADSCs) (Wu et al. 2019b). Actually, this classification is going to be much more “fluid” in its representation, as the adipocyte should be considered as a “meta-plastic” cell able to change deeply its differentiated phenotype depending on the complex cross talk with the immune microenvironment close the tumor. In this perspective, soluble factors released by either adipocytes, cancer cells, surrounding tissues, and immune cells are absolutely fundamental to drive the role of peri-tumoral adipocytes in either dampening tumor invasiveness or promoting it. ROS are further likewise signaling molecules in this interplay. A first recognized activity of ROS is to promote adipogenesis (de Villiers et al. 2018), yet ROS should exert a much more intriguing and keen role in this context. ROS are signaling molecules, exerting a pro-survival role when acting at a low or moderate concentration (D’Autréaux and Toledano 2007; Schieber and Chandel 2014; Zhang et al. 2016). So far, the role of ROS has been further highlighted in the adipocyte-mediated thermogenesis (Chouchani et al. 2017). Actually, they might also tune the epithelial-mesenchymal transition (EMT) mechanism close to the interplay adipocyte/tumor cell, which has been addressed in previous reports (Chirumbolo and Bjørklund 2016; Zoico et al. 2017; Zoico et al. 2018; Zoico et al. 2016; Jiang et al. 2017a). The subcellular localization of β-catenin is a critical step to regulate the EMT process in cancer cells and therefore should rule the Wnt signaling in those cells, as well in adipocytes (Chirumbolo and Bjørklund 2016; Zoico et al. 2016). Beta-catenin interacts with E-cadherin in the cell membrane

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and dissociates from this cell adhesion molecule in response to the Wnt-mediated signaling, translocating then into the nucleus and binding to TCF/LEF in order to activate the genetic transcription of Snail, MMPs (such as MMP-7), and Twist (Jiang et al. 2017a). In this context, ROS can contribute to the Wnt/β-catenin pathway leading to EMT by modifying adhesion molecules. Moreover, ROS can oxidize the integrin α7β1, which then binds to laminin-111 activating downstream protein pathways, such as Src and FAK, so moving a complex made by talin, kindlin, and α-actinin leading to the actin stress-fibers mediated cell migration (Jiang et al. 2017a). Peri-tumoral adipose tissue may be a major producer of ROS but this mechanism is strictly related to the different hypoxic conditions in the milieu tumor/WAT during the complex interplay between adipocytes and cancer cells. These latter are notoriously great producers of ROS, a release exacerbated in those highly hypoxic areas due to the limited or abnormal angiogenesis characterizing the early developing tumor (Mazumdar et al. 2009; Heddleston et al. 2010; Keith and Simon 2007). Hypoxia may interfere with the mitochondria electron transport chain (ETC), so representing a critical event for the adipocyte function (Boudina and Graham 2014). The interplay ROS-hypoxia-mitochondria ETC might be particularly massive in the role that the adipose tissue exerts in the tumor/WAT cross talk and EMT (Jiang et al. 2017b). Dysfunctional mitochondria are a leading cause in the EMT mechanism in cancer and it cannot be excluded it may exhibit this role also in the process of mature adipocyte de-differentiation to an adipocyte derived fibroblastlike cell (ADFs) (Guerra et al. 2017). Again, a lipidic signaling may be considered in these processes. Circulating free fatty acids (FFAs) are powerful triggers of apoptosis, in particular palmitate induces a huge ROS production, leading to mitochondria dysfunction with dampening of mitochondria number, impaired activity of complex IV with succinyl dehydrogenase, and reduction of the membrane potential in mitochondria (Li et al. 2018). Particularly for macrophages, then also for adipose tissue associated macrophages (ATMs), FFAs play a major role in inducing apoptosis in these innate immune cells (Li et al. 2018). This role is mediated also by components such as the adipocyte fatty acid binding protein (A-FABP) and the endoplasmic reticulum (ER) stress, as palmitate is able to increase ER stress and the expression of A-FABP (Hoo et al. 2017). The action of palmitate is to reduce autophagy in macrophages by increasing the expression of A-FABP, which in turn attenuates the expression of Atg-7, a major component in the autophagic process (Hoo et al. 2017). The inhibition of the autophagic flux, acting also on the JAK2dependent mechanisms, increases ER stress and leads macrophages to be powerful pro-inflammatory innate cells, a circumstance collectively known as lipotoxicity (Hoo et al. 2017). The idea underneath, when dealing with WAT and its involvement in the inflammatory processes, is that the more FFAs circulation the more the exacerbation of the immune response. Fundamentally, FFAs should act, in association with ROS, as signaling molecules to ensure and establish the WAT activity as a tolerogenic third lymphoid organ (Chirumbolo 2015; Frasca and Blomberg 2019). The apparent Janus-like complex behavior of WAT in the WAT/tumor microenvironment is finely tuned by the need to balance both the ability to counteract the growth and invasiveness of cancer cells and

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the microenvironment exacerbation due to the FFAs and ROS activity on resident immune cells. It is not so an easy task. Therefore, our hypothesis is that FFAs represent not simply a bullet gun but a interplay codified language, as exemplified in the first rows of this text. The ability of ROS to elicit a pro-survival, pro-repairing response seems to be associated with their homeostatic levels within the cell. A possibility to explain this mechanism, called eustress and acknowledged in the widest phenomena of hormetic mechanisms, might be related to autophagy. Autophagy is necessary to prevent apoptosis but its role may be much more complex, so representing a master tuner of cell survival and programmed cell death (Nikoletopoulou et al. 2013). The bottleneck of this mechanism is the interplay death associated protein kinase (DAPK)/protein kinase D1 (PKD), toward which both ROS and ER-stress converge. When a huge deal of stressors is incoming the cell, DAPK works as signal mediator usually inducing apoptosis but also activating both PKD and beclin-1 via a phosphorylation mechanism. Phosphorylated PKD may induce cell death or the downstream phosphorylation of VSP-34 so leading to the formation of auto-phagosomes and hence of autophagy (Nikoletopoulou et al. 2013). This complex multistep route is highly suggestive of the fine tuning occurring in the cell as a response to the ROS input.

Conclusions ROS are not simply free soluble molecules. Cell compartmentation of ROS is fundamental to ascertain the different functions associated with ROS as signaling molecules. ROS are usually produced by mitochondria and can travel to the cytoplasm via the voltage dependent anion channel (VDAC) for superoxide or via aquaporins (peroxides). Moreover, ROS are present in the cytoplasm as the waste product of oxidation mechanisms involving proteins and can be present in redoxosomes, that is, vesicles derived from ER-stress and peroxisomes. Therefore, a significant amount of ROS is not freely released as extracellular ROS, sometimes crossing the membrane via aquaporins but are also embedded in exosomes shedding from cell membranes or secreted with granules (Tafani et al. 2016). This should suggest that the release of ROS, likewise FFAs can be finely regulated.

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Hypoxia-Induced Stress Responses in Cancer and Cancer Stem Cells

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Sandhya Chipurupalli, Snehlata Kumari, Vincenzo Desiderio, and Nirmal Robinson

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . HIF-1α a Key Player in Tumor Hypoxia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Metabolic Adaptation to Hypoxia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Antioxidative Responses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ER-Stress Response to Hypoxia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Autophagy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Hypoxia and Cancer Stem Cells (CSCs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Hypoxia and Cancer Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract

Oxygen is highly critical for the survival of complex organisms and lack of oxygen (hypoxia) promotes stress and death of the organism. However, cancer cells have evolved adaptive mechanisms to survive persistent pathological hypoxia experienced in the inner regions of the tumor. Normal cells rely on oxygen to meet the energy demands through mitochondrial oxidative phosphorylation, protein translation, and proper folding in the endoplasmic reticulum and for S. Chipurupalli Department of Pharmacology, JSS College of Pharmacy, JSS Academy of Higher Education & Research, Ooty, India S. Kumari Diamantina Institute, The University of Queensland, Woolloongabba, Australia V. Desiderio Department of Experimental Medicine, University of Campania “L. Vanvitelli”, Naples, Italy N. Robinson (*) Cellular-Stress and Immune Response Laboratory, Center for Cancer Biology, University of South Australia and SA Pathology, Adelaide, Australia e-mail: [email protected] © Springer Nature Singapore Pte Ltd. 2022 S. Chakraborti et al. (eds.), Handbook of Oxidative Stress in Cancer: Mechanistic Aspects, https://doi.org/10.1007/978-981-15-9411-3_121

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maintaining redox homeostasis. Therefore, cellular adaptive mechanisms will be employed by cancer cells through direct mechanisms such as down regulation of mitochondrial ATP production and autophagy to degrade the damage caused by hypoxia induced oxygen radicals. On the other hand, transcription factors such as hypoxia inducible factor-1 (HIF-1) and nuclear factor erythroid 2-related factor 2 (NRF2) enable the transcription of enzymes and stress responsive mechanisms to restore homeostasis indirectly. These stress response mechanisms promote resistance to cancer therapy. Hypoxia also influences other cells such as cancer stem cells (CSC) present in the hypoxic regions of the tumor. The current theory is CSCs are cells that initiate tumor with the ability to regenerate and hypoxia helps in their maintenance, thus contributes to drug resistance and relapse. Therefore, targeting hypoxia triggered adaptive mechanisms is intense area of research for drug development. Keywords

Cancer · Hypoxia · Cellular stress · Cancer stem cells · HIF-1 · Metabolic adaptation · UPR · Autophagy

Introduction Cancer cells possess the propensity to survive in a typical microenvironment termed tumor microenvironment (TME), which impacts tumor progression. TME constitutes of chemical and cellular microenvironments where the former comprises of the pH, pO2, and availability of carbon sources and metabolites such as lactate secreted by the cancer cells. The cellular component of TME is made up of blood vessels, fibroblasts, T-cells, lymphocytes, granulocytes, extracellular matrix (ECM), and stromal cells together they impact the growth and survival of cancer cells and the therapeutic outcome (Fig. 1). The nature of TME contributes to maladapted tumor vasculature, acidosis, hypoxia, and increased interstitial fluid pressure. However, cancer cells respond reciprocally by activating unique cellular pathways that alter cell growth, invasion, and metastasis. Hence, understanding TME is critical for identifying new strategies to control the growth of cancer cells (Ghosh and Dawson 2018). Hypoxia is a key factor in the TME that enables cancer cells to thrive and develop resistance to available chemotherapies (Muz et al. 2015). The imbalance between oxygen (O2) demand and availability within a tumor leads to varying oxygen levels from well oxygenated to poorly oxygenated and the necrotic area where some tumor cells have died due to lack of oxygen termed as hypoxia. O2 tension in normal tissues exceeds 40 mmHg, whereas regions of tumors experience low O2 tension of 0–20 mmHg (Vaupel et al. 1989). Under hypoxia normal cells will undergo cell death. However, in tumor cells, hypoxia activates a genetic program that enables them to survive in a hostile TME which is also depleted of nutrients. Thus, hypoxia selects for a subpopulation of tumor cells that are genetically programmed to become malignant (Vaupel and Harrison 2004). Solid tumors respond by inducing

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Fig. 1 A typical tumor microenvironment (TME) comprised of the various cellular and the chemical components that influence tumor progression and metastasis

angiogenesis to increase the availability of O2 and nutrients which enable them to overcome the limitations imposed by the TME (Hanahan and Weinberg 2011). Hypoxia activated tumor cells stimulate angiogenesis by secreting angiogenic factors, such as vascular endothelial growth factor (VEGF). As the newly developed blood vessels are poorly organized, regions within the tumor may not still receive sufficient O2 (Helmlinger et al. 1997). Under reduced O2 (hypoxic) conditions, cancer cells upregulate adaptive responses to balance metabolic, bioenergetic, and redox demands. For instance, cancer cells temporarily arrest the cell cycle, alter metabolism to reduce ATP consumption, and secrete factors that promote angiogenesis. These events are coordinated by various cellular pathways, such as transcriptional regulation by hypoxia-inducible factors (HIFs), metabolic alterations, antioxidative responses, unfolded protein response (UPR), and autophagy (Fig. 2).

HIF-1α a Key Player in Tumor Hypoxia Hypoxia inducible factors (HIFs) are a family of heterodimeric proteins and mammals possess HIF1, HIF2, and HIF3. HIF1 alpha (HIF-1α) and HIF-1β are subunits which heterodimerize to form HIF1. However, HIF-1α is frequently overexpressed in tumor cells and is pivotal in regulating the transcriptional response to hypoxic stress. Since its discovery and characterization, interest in HIF-1α’s role in cancer has grown exponentially. Wang and Semenza in 1995 were the first to demonstrate

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Fig. 2 Cellular stress responses and adaptive mechanisms utilized by cancer cells to circumvent hypoxia induced stress

the transcriptional program activated upon stabilization of HIF-1α. It is becoming evident that under hypoxic conditions, HIF-1 transcriptionally activates both pro-survival and cell death pathways. The transcriptional function of HIF-1α is independent of oxygen, but under normoxia or in the presence of oxygen, HIF-1α degraded in the proteasomes. Degradation of HIF-1α is a ubiquitin-dependent process, regulated by VHL (von-Hippel-Lindau protein) an E3-ubiquitin ligase. Interaction of HIF-1α with pVHL–elongin BC–CUL2 complex is mediated by the prolyl hydroxylase (PHD)-hydroxylates proline residues Pro-402 and Pro-564 of HIF-1α in an O2-dependent manner (Semenza 2012, 2003). PHD acts on the substrates molecular O2 and 2-oxoglutarate (α-ketoglutarate) to generate prolylhydroxylated HIF-1α and succinate thereby providing an O2-dependent mechanism of HIF-1α regulation. In contrast, the other subunit HIF-1β is not degraded in the presence of O2 and is constitutively expressed. Under hypoxia, VHL is inactive; therefore, HIF-1α is not degraded; hence, it translocates into the nucleus, heterodimerizes with HIF-1β, and forms an active HIF-1 complex (Zimna and Kurpisz 2015) (Fig. 2). Inner regions of solid tumors turn hypoxic during the rapid expansion of cancer cells which favors the stabilization of HIF-1α. HIF-1 binds to hypoxia responsive element (HRE) motif in the promoter region of the target gene and thus regulates over 100 genes (Goda and Kanai 2012). These genes are implicated in cell growth, erythropoiesis, metabolic adaptation, angiogenesis, and metastasis as several genes induced by HIF-1 such as VEGF, Glucose transporter 1 (GLUT1), GLUT 3, insulin-like growth factor binding protein-1 and -3, insulin-like growth factor II, transforming growth factor-β3 and p21 are highly expressed in tumors (Masoud and

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Fig. 3 Mechanism of HIF-1α degradation under normoxia and its stabilization during hypoxia

Li 2015). Sustained stabilization of HIF-1α enables phenotypic diversity in various solid tumors and is also associated with genetic instability, genetic alterations (Zhong et al. 1999; Luoto et al. 2013) (Fig. 3). Benign tumors also express HIF-1α; however, it is further enhanced in malignant tumors and metastatic tumors but absent in surrounding normal healthy tissues (Zhong et al. 1999). Therefore, expression of HIF-1α has been associated with aggressive form of cancers with poor prognosis and therapeutic failure (Giatromanolaki et al. 2001; Birner et al. 2001; Bos et al. 2003). In hematologic malignancies such as Acute Myeloid Leukemia (AML), although evidences support the role of HIF-1α in the adaptation of hematopoietic stem and progenitor cells, its involvement in AML development is less understood. Function of HIF-1 in heterogenous cancers such as hematological malignancies could vary dependent on a number of factors.

Metabolic Adaptation to Hypoxia Hypoxia alters metabolism in tumor cells from oxidative phosphorylation to glycolysis which is termed as “Warburg effect”. This metabolic shift generates ATP by utilizing large amounts of glucose which results in enhanced production of lactate

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causing acidosis. Hypoxia and acidosis in TME combine to activate adaptive stress responses that promote invasion and metastasis of cancer cells (Weljie and Jirik 2011). Although hypoxia reduces cell proliferation through the induction of p21, some cancer cells are known to maintain cell growth. Such conditions require not only ATP but also other building blocks such as amino acids, ribose-pentose phosphate, and one carbon units for nucleotide synthesis (Spill et al. 2016; Gilkes et al. 2014). Therefore, cancer cells alter their metabolism primarily through HIF-1α-dependent transcriptional activation of glycolytic enzymes: lactate dehydrogenase A (LDHA), phosphoglycerate kinase 1 (PGK-1), hexokinase-1 (HK1), and the glucose transporters (Semenza 2010). Alternatively, HIF-1α transcriptionally activates of pyruvate dehydrogenase kinase 1 (PDK1) which selectively inhibits pyruvate dehydrogenase (PDH) thus prevents the availability of pyruvate for the TCA cycle. Inhibition of PDH by PDK1 that is upregulated in most cancer cells prevents the conversion of pyruvate to acetyl coenzyme A. During hypoxia, loss of glucose availability is also compensated by utilizing the stored glycogen by cancer cells and the storage is also replenished. Mitochondria form dynamic networks and their morphology and distribution change to help the cell to adapt metabolically. Mitochondrial fission and fusion mechanisms regulate mitochondrial morphology which is an adaptive response to metabolic changes in the cell. Consistently, hypoxia alters mitochondrial morphology, mass, composition, and distribution as they are the major consumers of oxygen. Reduced oxygen availability affects the mitochondrial redox status resulting in increased nicotinamide adenine dinucleotide (NADH): oxidized NAD+(NADH: NAD+) which affects the cellular function of proteins. For instance, Sirtuin 1 (SIRT1) is a NAD+ dependent deacetylase and is co-regulated with HIF-1α where SIRT1 acts as redox sensor and HIF-1α as an oxygen sensor (Lim et al. 2010). In normoxic cells, SIRT1 prevents acetylation of HIF-1α by p300 rendering it inactive. Under normoxia SIRT1 deacetylates the Lys674 in HIF-1α which hampers the recruitment of the acetylating enzyme p300/CBP-associated factor (PCAF) and resulting in the repression of HIF-1 transcriptional activity. However, during hypoxia, SIRT1 is downregulated leading to the acetylation and HIF-1α (Lim et al. 2010). These findings suggest that interaction between SIRT1 and HIF-1α regulates the crosstalk between O2-sensing and redox-signaling.

Antioxidative Responses Dynamic equilibrium of electron transfer reactions or in other words redox homeostasis is intrinsically linked to free radicals and is highly essential to maintain cellular homeostasis. The superoxide radicals (O2˙ ) and the hydroxyl radical (OH•) are the primary free radicals derived from molecular oxygen. These oxygen radicals are referred as “reactive oxygen species” (ROS) and the related nonradical mediators such as hydrogen peroxide (H2O2) which together orchestrate the redox system in biological processes. Disequilibrium in redox homeostasis resulting in elevated levels of ROS can result in the death of the cell. However, cells employ backup

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adaptive mechanisms to restore redox homeostasis which are highly exploited by cancer cells. Tumor cells adopt these adaptive mechanisms to survive elevated ROS induced by hypoxia in TME. Mitochondria play a critical role in redox signaling because the ROS superoxide O2˙ is generated through the respiratory chain reaction mainly at the electron transport chain complex I and complex II but also by other enzymatic components (Brand 2016; Bedard and Krause 2007; Murphy 2009). However, manganese superoxide dismutase (MnSOD) rapidly converts O2˙ to H2O2 which can then pass through the mitochondrial membrane and acts as the Redox signal from mitochondria. Several cellular factors such as hypoxia alter the generation of mitochondrial ROS (mROS) within TME (Chandel et al. 1998). ROS is considered to be pro-tumorigenic as it is known to activate PI3K/AKT, MAPK/ ERK, and HIF-1α. They activate pro-tumorigenic factors by inactivating the negative regulators PTEN, MAPK phosphatase, and PHD-2, respectively, which leads to cancer cell survival and metastasis. Moreover, tumor hypoxia also abrogates the adaptive antioxidant mechanisms that help in preventing ROS-dependent damage to the cells. For instance, in breast cancer elevated mROS production as a result of the loss of Sirtuin3 (SIRT3) stabilizes HIF-1α (Finley et al. 2011). Nuclear factor erythroid 2-related factor 2 (NRF2)-dependent transcriptional activation of antioxidative responses is a key cytoprotective response to the stress induced by ROS. Under physiological conditions, kelch-like ECH associated protein 1 (KEAP1)-E3 ubiquitin ligase complex ubiquitinates NRF2 and is degraded in the proteasome. In the presence of ROS, NRF2 degradation is prevented, resulting in the stabilization and nuclear translocation. In the nucleus, it heterodimerizes with small Maf proteins bind to antioxidative response element (ARE) and transcriptionally activates cytoprotective response. Several evidences have suggested that NRF2 is stabilized in nucleus and provides cytoprotection in various cancers; therefore, it has been explored as a therapeutic target (Taguchi and Yamamoto 2017).

ER-Stress Response to Hypoxia The ER is an extensive membranous network of tubules that surround the nucleus and expands throughout the cytosol. In eukaryotic cells, all secreted and transmembrane proteins are processed in the ER. The secreted proteins undergo a series of post-translational modifications, such as glycosylation and disulfide bonding regulated by foldases such as disulphide isomerase, which are vital for the proper functioning of the proteins. Molecular oxygen is shown to be the driving force for the enzymatic reactions involved in protein folding. Therefore, unfolded/misfolded proteins accumulate in ER as a consequence of hypoxia which is commonly known as ER stress (Feldman et al. 2005). The cells respond by activating a transcriptional program to mitigate ER stress and the signaling pathways are collectively termed as the unfolded protein response (UPR). UPR activation transiently attenuates protein synthesis thus limiting the stress on ER. Simultaneously, it augments protein trafficking through ER and ER-associated degradation (ERAD) and autophagy dependent degradation of misfolded proteins

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(Wang et al. 2010). Three primary arms of UPR signaling, namely, PKR-like ER kinase (PERK), IRE1α (inositol-requiring enzyme 1 alpha), and ATF6 (activating transcription factor 6), have been described in mammals. Under basal conditions, the ER-chaperone BiP (Binding immunoglobulin Protein) binds to the luminal domains of PERK and ATF6 and becomes inactive. Upon accumulation of misfolded/ unfolded proteins, BiP dislocates from the complex to facilitate the proper folding of proteins (Fig. 4) (Chipurupalli et al. 2019). The dissociated UPR proteins trigger a

Fig. 4 Protein folding is dependent on oxygen; hence, hypoxia causes perturbations in protein folding and results in the accumulation of misfolded/unfolded proteins resulting in the activation of unfolded protein response (UPR)

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transcriptional response that helps cancer cells in the hypoxic environment to survive and promote tumor development.

Autophagy Autophagy is a lysosome-dependent degradation process where the cargo targeted for degradation in taken into LC3 II positive double membrane vesicles which consequently fuse with lysosomes for degradation. It is a key adaptive response in tumor cells under hypoxic stress and enables cancer cells to maintain metabolic homeostasis (Kroemer et al. 2010). Autophagy is a vital cellular process that helps in the turnover of organelles such as mitochondria and ER as it facilitates the removal of damaged organelles and prevents cellular damage. Impaired autophagy results in defective mitophagy and de-regulated ROS hence is associated with tumorinitiation. In contrast, autophagy also promotes cell death signals, in response to stress factors in the TME. For instance, autophagy induces caspase-dependent cell death in cancer cells as cleavage of autophagic proteins has been observed. Also inhibition of autophagy upon the cleavage of autophagic protein 5 (Atg5) by calpain activates cell death inducing caspases (Norman et al. 2010). Hence, the role of autophagy in cancer remains elusive, but accumulating evidences favor the hypothesis that autophagy is a tumor adaptive response which promotes drug resistance in cancer cells (Mele et al. 2020). Mammalian target of rapamycin (mTOR) is a primary metabolic stress sensor in cancer cells and its inhibition induces autophagy (Paquette et al. 2018). During hypoxia, HIF-1 induces BCL2/adenovirus E1B 19 kDa proteininteracting protein 3 (BNIP3) which in turn inhibits mTOR (Li et al. 2007). BNIP3 also promotes mitochondria specific autophagy (mitophagy) which has been reported to enable tumor development (Sofer et al. 2005). Similarly, hypoxia induced ER-stress is also known to regulate autophagy through Regulated in Development and DNA Damage 1 (REDD1) dependent inhibition of mTOR (Wang and Kaufman 2014). However, the molecular mechanisms that specifically link hypoxic stress and autophagy remain to be elucidated.

Hypoxia and Cancer Stem Cells (CSCs) Cancer stem cells (CSCs) are defined as tumor-initiating cells for their unique ability to form a new tumor in a host organism. These cells are responsible for cancer progression, drug resistance, metastases, and relapse. Regarding the CSCs origin, two theories are mostly accepted: CSCs growing from normal stem cells after an oncogenic hit or misplacement and CSCs surging from cancer cells in which pluripotency genes are activated. In both the theories, hypoxic tumor microenvironment plays a decisive role (Lin and Yun 2010). Indeed, evidences in the last 20 years suggested specific role of hypoxic microenvironment in the maintenance of both stem cells and CSCs. Hypoxic regions of human tumors contain poorly differentiated cells, which express stem cell markers.

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Cells grown under hypoxia show increased clonogenic potential and are more tumorigenic in vivo (Yun and Lin 2014).In several brain tumors, CSCs are located in hypoxic regions (Tong et al. 2018). To further clarify this, Kim et al. lately developed a hypoxia-sensing xenograft model in which hypoxic cells will turn fluorescent (EGFP+). They found that EGFP + cells isolated from tumor specimens are enriched in CSCs. interestingly, cells retained their stem cell characteristics even after culturing under regular conditions, and these cells remain completely different from the non-hypoxic cells isolated from the same tumor. The PI3K/AKT pathway is strongly potentiated in hypoxic cells and seems to be necessary for maintaining CSC properties. Surprisingly, only in vivo hypoxic TME seems to impact CSCs subpopulation, while in vitro hypoxia does not trigger the same effect (Peng and Liu 2015). HIF-1α and HIF-2α are upregulated in CSCs/progenitor cells, where HIFs expression induces the activation of the survival pathway and stemness gene products (Mimeault and Batra 2013). Poorly differentiated pancreatic cancer cells overexpress HIFs. Similar findings have been described in neuroblastoma, glioma, AML, ALL, lung, prostate, breast, gastric, and colorectal cancers (Tong et al. 2018; Miao et al. 2014). Moreover, HIFs seems to promote self-renewal and stem-like features in a different context. In gliomas, HIFs increase the number of CSCs marker CD133 and increase the CSC phenotype. In glioblastoma, HIFs inhibit cell differentiation while HIF-1α and HIF-2α silencing reduces neurosphere formation from patient-derived CD133+ cells, induce apoptosis, and reduce tumor-initiating capacity in vivo (Tong et al. 2018; Peng and Liu 2015). In leukemia, HIF-1α is increased in c-Kit+Sca-1+ CSCs, and its silencing reduces CFU and leukemia-initiating ability. Also, the HIF inhibitor echinomycin eliminates CFU with an IC50 in the picomolar range. In a syngeneic model in which mice received a lethal dose of leukemia cells, echinomycin cured all the treated mice. In AML HIF1-α is overexpressed by CD34 + CD38- CSCs. Echinomycin induces apoptosis in this cell subset with a 100–1000-fold more efficiency compared to the blasts bulk. In vivo treatments of AML mice reduced CSCs frequency and more importantly were able to block tumor initiation when cells were transplanted in a new host. In a model of relapsed AML echinomycin cured about 60% of treated mice and again completely blocked the tumor-initiating-ability of AML bone marrow transplanted in a new host. In CML HIF-1α deficient cells lose their tumor-initiating capacity in secondary recipients. Similarly, in breast cancer, HIF-1α conditional deletion reduced tumor growth and metastases together with a decrease in CSCs number (Peng and Liu 2015). The molecular mechanism by which HIFs promote CSCs phenotype seems to be quite broad and involves different HIFs target genes. For instance, the three major CSCs-associated-genes, OCT3/4, Nanog, and SOX-2, known to induce pluripotency, are regulated by HIF-1. Indeed, HIF knockdown reduces CD44 and OCT3/4 expression in colorectal cancer. In prostate cancer, HIFs expression in CSCs is correlated with increased OCT3/4, Nanog, and CD44, which induce drug resistance and antiapoptotic pathways. In breast cancer, HIF-1α induces the Hippo pathway effector TAZ, which is known to induce CSCs phenotype. Moreover, HIF1-α activates transcription of the

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Siah E3 ubiquitin-protein ligase 1 (SIAH1) gene, which promotes the degradation of the large tumor suppressor kinase 2 (LATS2). This allows the nuclear localization of TAZ. Also, HIF-1α controls the interaction between breast CSCs and mesenchymal stem cell (MSCs) through the chemokine (C-X-C motif) ligand 16 (CXCL16). MSCs, in turn, will recruit tumor-associate-macrophages contributing to shaping TME. Besides, EMT related genes, glucose transporter, metabolic enzymes, and drug resistance-associated genes are described to be regulated by HIFs (Tong et al. 2018; Mimeault and Batra 2013). In gastric cancer peritoneal milky spot serves as a hypoxic niche for metastatic cells, and HIF-1α expression correlates with metastases. Hypoxia promotes CSCs self-renewal and survival trough HIF-1α (Miao et al. 2014). Other than HIFs, hypoxia activates other genes involved in CSC biology. Notch pathway has been widely shown to be fundamental in both stem cells and CSCs. Xing et al. found the Notch ligand Jagged2 overexpressed by the hypoxic invasive front in breast cancer specimen. Jagged2/Notch signaling induces EMT and promotes CSCs survival and self-renewal (Xing et al. 2011). Histone 3 lysine 4 (H3K4) demethylase JARID1B is a CSCs-associated marker in melanoma. JARID1B is rapidly upregulated by hypoxia in both melanoma and renal cell carcinoma together with other histone demethylases genes JMJD1A and JMJD2B. Van Den Beucken et al. showed that tumor hypoxia is associated with reduced DICER expression in large cohorts of breast cancer patients. DICER is fundamental for miRNAs biology. In this context, inhibition of oxygen-dependent H3K27me3 demethylases KDM6A/B reduces DICER expression, which decreases the processing of the miR-200 family leading to EMT and CSCs promotion (Van Den Beucken et al. 2014). From these studies, it is vividly evident that hypoxia critically regulates the CSC’s survival and their ability to initiate tumor progression.

Hypoxia and Cancer Therapy Oxygen gradient within the tumor environment often determines efficacy of the antitumor therapies. Hypoxia interferes with the diffusion and bioavailability, and therefore, the prevalence of hypoxic regions within the TME significantly impacts the clinical outcome of antitumor, radio-, chemo-, and immuno-therapies. Hypoxia not only hampers the homogenous accessibility, but also incites therapeutic resistance in cancer cells, and therefore remained a major clinical challenge in successful cancer therapy. Hypoxia-induced molecular changes in cancer-, immune-, and stromal-cells, as well as manipulated immune landscape have been shown to have great impact on the anti-tumor therapies. Hypoxia can downregulate the pro-apoptotic factors, such as Bid and Bax, inhibit DNA damage, modulate p53 levels, promote autophagy, or alter mitochondrial metabolic functions, which allow the cancer cells to better survive before and after the treatments (Jing et al. 2019). Since radiotherapy relies on the irreversible DNA damage, generation of ROS, and cell death, hypoxia has also remained a major limitation to radiotherapy (Rockwell et al. 2009). Hypoxia can also modulate the outcome of immunotherapy by

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regulating checkpoint inhibitors or immune landscapes of the TME, which hampers cytotoxic actions of immune cells, such as T-Cells or NK-Cells. Studies have shown that the hypoxic condition and HIF-1α up-regulate expression of PD-L1, a ligand of PD-1 on T-cell that suppresses T-cell actions, on cancer cells and myeloid-derived suppressor cells in melanoma, prostate, breast, and lung cancer (Noman et al. 2014). Hypoxia-mediated up-regulation of other immune checkpoints, such as Immune Checkpoint V-Domain Ig Suppressor of T Cell Activation (VISTA), CD47 on macrophages, and nonclassical MHC-I on cancer cells further constraint the success of immunotherapies. Hypoxia is a stress-induced condition that uniquely confined to the tumor microenvironment. Therefore, targeting the hypoxia-associated limitations can open up new avenues for combinatorial cancer therapies. Hypoxia reducing drugs, in combination with chemotherapy, radiotherapy, and immunotherapy, have been exploited in various ways. Pharmaceutical inactive pro-drugs, such as SN30000, TH-302, and EO9, which are activated under hypoxic conditions, have been used in the clinic in combination with the cytotoxic drugs like doxorubicin, or immune check point inhibitors, PD-L1/CTLA-4, to enhance the efficacy of the treatment. Interestingly, these hypoxia-reducing drugs have shown promising outcomes, such as increased infiltration of T-cells and therefore hold promises for the future combinatorial therapies (Borad et al. 2015). Other tumor hypoxia alleviation approaches that have been used in the preclinical and clinical settings include increasing the oxygen transporting capacity under hyperbaric conditions or by providing supplemental oxygen. Hyperbaric oxygen breathing has exhibited improvement in preclinical and clinical models when combined with the radiotherapy; however, the associated side effects like enhanced necrosis require significant consideration for further implementation in the clinic (Graham and Unger 2018). Increasing supplemental oxygen has also shown promising outcomes when used in the combination with checkpoint inhibitors by enhancing the tumoricidal activities of immune cells within the tumor microenvironment (Graham and Unger 2018). Other drugs that have been used to reverse the tumor hypoxia are utilizing the capacity of hemoglobin and fluorocarbons to transport oxygen from high to low oxygen area. Fluorocarbons such as oxygent™ (perfluorooctyl bromide (PFOBe), perflubron), Fluosol(R)-DA 20% (perfluorodecalin (PFDe) with perfluorotripropylamine and NVX-108 are being explored to enhance their active oxygen species and promote oxygen transport in order to boost the effect of anti-tumor therapies (Graham and Unger 2018).

Conclusion Hypoxia is a causative for malignant progression of tumors and results in poor survival, as hypoxia enables cancer cells to develop resistance to therapy. Our knowledge of mechanisms that regulate cancer cells’ adaptive mechanisms to hypoxia has improved considerably, yet not entirely. Especially there is a knowledge gap in understanding the hypoxia mediated mechanisms that help the CSCs to maintain its stemness and tumor initiation properties. Therefore, future

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investigations are required to understand the hypoxia regulated precise mechanism that alters cellular metabolism and cellular stress response mechanism. Although the hypoxic-conditions are continuously being explored, a better understanding of the hypoxia-mediated control of tumor microenvironment will, undoubtedly, add to the future promises to develop effective and successful cancer therapies. Moreover, identifying pathways that are usually dispensable under normoxia, but are vital for survival under hypoxia, will most likely provide therapeutic targets that can be targeted with little adverse effects on normal tissue. Such therapeutic strategies may not be beneficial in killing cancer cells in normoxic tumor regions but will tackle drug resistant cancer cells, prevent relapse, and greatly improve the efficacy of interventions such as chemotherapy and radio therapy leading to improvement in the survival of patients.

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Ajit Kumar Rai and Neeraj Kumar Satija

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Oxidative Stress Targeted Therapies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Prooxidant Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Antioxidant Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ROS-Mediated Differentiation Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cross-References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract

Oxidative stress plays an important role in the etiology and development of leukemia. Although acute myeloid leukemia blasts have high reactive oxygen species (ROS) level, the quiescent leukemia stem cells have low ROS. The presence of recurrent mutations like FLT3, IDH1/2, RAS is responsible for different oxidative states of leukemic cells compared to normal cells. Therefore, targeting the ROS levels in leukemic cells represents a potential therapeutic target. Hence, ROS targeted therapies consist of two major approaches: prooxidant and antioxidant, which involve Increasing or decreasing ROS levels using pharmacological agents to eliminate leukemic cells. Alternative strategies to induce differentiation by modulating ROS are also being developed.

A. K. Rai · N. K. Satija (*) Systems Toxicology and Health Risk Assessment Group, CSIR-Indian Institute of Toxicology Research (CSIR-IITR), Lucknow, India Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, India e-mail: [email protected] © Springer Nature Singapore Pte Ltd. 2022 S. Chakraborti et al. (eds.), Handbook of Oxidative Stress in Cancer: Mechanistic Aspects, https://doi.org/10.1007/978-981-15-9411-3_122

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Keywords

Oxidative stress · Reactive oxygen species · Acute myeloid leukemia · Prooxidant therapy · Antioxidant therapy

Introduction The initiation and progression of leukemia is a multistep process, and accumulating evidence suggests involvement of ROS in hematological malignancies (Prieto-Bermejo et al. 2018). Leukemic cells have high ROS level than normal hematopoietic cells, and it regulates cellular functions like proliferation, survival, differentiation, and migration (Udensi and Tchounwou 2014). In case of acute myeloid leukemia (AML), >60% patients exhibit high superoxide production by AML blasts. Although mitochondria is the major ROS producer, NADPH oxidases (NOX) are responsible for elevated ROS in AML (Hole et al. 2013). Approximately 12–27% of AML patients have Ras mutations (Neubauer et al. 2008). These occur during the progression of AML from MDS and result in high mortality (Brian et al. 2019). Activated RAS level also induces ROS by activating NOX2 and promotes growth factor-independent proliferation in myeloid progenitor cells (Hole et al. 2010). NOX2 is also reported to be essential for leukemic stem cells (LSCs) selfrenewal and maintenance of malignant hematopoiesis (Adane et al. 2019). Other common mutations in AML like FLT3 and IDH1/2 are also found regulating ROS level. FLT3 induces ROS via STAT5 signaling and by RAC1 activation and drives genomic instability which is responsible for AML aggressiveness and poor prognosis of FLT3+AML patients (Sallmyr et al. 2008). In addition, it is reported that oxidation of cysteines in FLT3 are responsible for FLT3ITD-mediated cell transformation (Bohmer et al. 2020). Mutations in IDH1/2 gene lead to increased level of 2-hydroxyglutarate and reduced NADPH, α-ketoglutarate, and glutathione levels (Dang et al. 2009) that can alter normal ROS homeostasis in AML cells (Zhang et al. 2014). It is not necessary that all leukemic cells should have high level of ROS. Leukemic stem cells (LSCs), responsible for leukemia initiation and minimal residual disease (MRD), have low ROS levels with overexpressed BCL-2. Inhibition of BCL-2 abrogated mitochondrial oxidative phosphorylation, depleted ATP, and effectively induced apoptosis in LSCs by increasing ROS (Lagadinou et al. 2013). Leukemia initiating cells adapt to the hypoglycemic bone marrow microenvironment by activating AMPK, which reduces oxidative stress and maintains leukemogenic potential (Saito et al. 2015). CXCR4 signaling is found to be essential for LSC maintenance in a CRISPR-Cas9 screen. Deletion of CXCR4 leads to oxidative stress and differentiation in AML cells. Interestingly, this effect is independent of CXCL12 (Ramakrishnan et al. 2020). The bone marrow microenvironment also confers protection to AML cells by NOX2-derived superoxide which facilitates mitochondrial transfer from stromal cells to AML blasts. This transfer is mediated through AML-derived tunneling nanotubes and supports AML cell survival (Marlein et al.

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2017). Therefore, the redox system is an attractive target for the development of efficient and targeted therapies for treating AML.

Oxidative Stress Targeted Therapies Conventional cytotoxic chemotherapy, such as combination of daunorubicin and cytarabine, has off-target negative effects in patients. In addition, conventional chemotherapy faces the challenge of resistance in AML patients (Estey 2018). High treatment efficiency with minimum side effects can be achieved by targeted therapy in AML. OS-targeted approaches, prooxidant therapy, and antioxidant therapy, which rely on increasing or decreasing cellular ROS, respectively, to kill AML cells are being developed. In addition, ROS-based approaches to induce AML cell differentiation are also under development (Fig. 1).

Fig. 1 Prooxidant and antioxidant targets for AML therapy. (Abbreviation: BSO-buthionine sulfoximine, HDACi- histone deacetylase inhibitor, CDKi- cyclin-dependent kinases inhibitors, As2O3- arsenic trioxide, GSH-glutathione, GSSG- glutathione disulfide, SOD-superoxide dismutase, TFs- transcription factors, NOX- NADPH oxidase, ROS- reactive oxygen spices, NK-1R- neurokinin-1 receptor, SP- substrate P, H2R- H2 receptor)

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Prooxidant Therapy Chemotherapeutic drug such as cytarabine (a purine analogue) targets DNA replication to eliminate leukemic cells. However, high level of ROS has been reported to play an important role in cytarabine induced cytotoxicity. Cytarabine increases ROS level by regulating mitochondria and metabolism enzymes like cytochrome P450 reductase, NADPH oxidase, xanthine oxidase (XO), and thioredoxin reductase. This is also evident from the fact that use of antioxidant can diminish cytarabine effects on leukemia. But another fact suggests that the elevated ROS level could increase resistance in leukemic cells to anthracyclines (Prieto-Bermejo et al. 2018). Redox homeostasis is regulated by glutathione/glutathione peroxidase (GSH/GPx) system, and any alteration may trigger severe oxidative stress in cells. The importance of glutathione in tumor initiation, and suppression of glutathione and thioredoxin pathways resulting in cancer cell death has been demonstrated (Harris et al. 2015). Buthionine sulfoximine (BSO), a glutathione depleting agent, increases the therapeutic efficacy of arsenic trioxide (ATO), when used in combination, in acute promyelocytic leukemia by phosphorylating BCL-2, BIMEL, and MCL1 leading to enhanced apoptotic cell death (Tanaka et al. 2014). It also induces apoptosis in ALL cells via Smac mimetic BV6 that antagonizes IAP proteins (Schoeneberger et al. 2015). Recently, JAK2/STAT3 pathway inhibition by ruxolitinib (a JAK2 inhibitor) has been shown to sensitize AML cells to ATO-mediated cell death by increasing ROS and DNA damage (Mesbahi et al. 2018). Primitive CD34+ AML cells have aberrant glutathione metabolism as a result of enhanced expression of glutathione pathway proteins. Thus, glutathione depletion using parthenolide or piperlongumine resulted in AML cell death but exhibited less toxicity toward normal CD34+ cells (Pei et al. 2013). An important antioxidant enzyme superoxide dismutase (SOD) has also been assigned as a target against leukemia. SOD1 inhibitor ATN-224 has shown effectiveness against leukemia (Lee et al. 2013). ATN-224 synergistically increases cytarabine-mediated inhibition of myeloid leukemic cells proliferation by inhibiting SOD (Chen and Kan 2015). Another enzyme myeloperoxidase (MPO), which catalyzes conversion of H2O2 to hypochlorous acid, is found to be overexpressed in residual leukemic cells. AML cells with high MPO have high mitochondrial oxygen consumption and low ROS levels. Inhibition of this enzyme accumulates mitochondrial superoxide impairing oxidative phosphorylation and energy metabolism and selectively induces apoptosis in Ara-C resistant AML cells in vitro and in vivo (Hosseini et al. 2019). Verdiperstat, an oral, irreversible MPO inhibitor is in phase III trial for multiple system atrophy (NCT03952806). Metformin, an antidiabetic drug, has been reported to alter mitochondria ETC and affect ATP production with increased ROS generation. It suppresses mTOR constitutive expression in leukemia, a hallmark aberration in PI3K/AKT/mTOR axis in AML. Further, the drug also interferes with AML cell proliferation with no or little effect on normal CD34+ HSCs (Biondani and Peyron 2018). It is under clinical trial for relapsed/refractory AML (NCT01849276). Antibiotic tigecycline, approved by FDA for skin and intra-abdominal infection, alters mitochondrial homeostasis and

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increases intracellular ROS level (Jia et al. 2016). Interestingly, tigecycline specifically inhibits mitochondrial translation to target CD34+ CD38 leukemic cells with little or no effect on non-leukemic cells (Skrtic et al. 2011). 2-Methoxyestradiol (2-ME), an estrogen metabolite, is an inhibitor of hypoxia inducible factors (HIF). Since LSCs are present in hypoxic environment and HIF-1α activation is associated with poor prognosis, 2-ME–mediated HIF-1α inhibition induced cell death in AML cells by activating mitochondrial apoptotic pathway (Zhe et al. 2016). Endonuclease APE1/Ref-1 (APE1), which is involved in DNA damage repair system, also controls redox homeostasis by inhibiting ROS generation (Tell et al. 2009). APE1 inhibitor E3330 synergistically increases retinoic acid induced differentiation and apoptosis in myeloid leukemic cell line (Fishel et al. 2010). Another target for AML therapy is neurokinin-1 receptor (NK-1R) which is found elevated in AML patients. A NK-1R antagonist induces Ca2+ flux from endoplasmic reticulum to mitochondria leading to an increase in oxidative stress and mitochondrial dysfunction resulting in apoptosis (Ge et al. 2019). Amino acid glutamine is essential for AML cell survival. The use of glutaminase inhibitor results in ROS generation and apoptosis in AML cells by impairing glutathione production (Gregory et al. 2019). AML cells also express low levels of iron exporter ferroportin. Thus, FDA approved nanoparticle drug, ferumoxytol, causes increase in intracellular free ferrous iron resulting in cell death due to increased oxidative stress (Trujillo-Alonso et al. 2019). Histone deacetylase inhibitors (HDACi), cyclin-dependent kinase inhibitors (CDKi), and proteasome inhibitor (bortezomib approved by FDA for multiple myeloma) also partly mediate their effects by increasing ROS (Prieto-Bermejo et al. 2018). Thus, a wide range of targets is available for development of prooxidant therapies for the treatment of leukemia. However, increasing ROS production is likely associated with challenges such as genomic instability and chemoresistance, damage to neighboring cells, and toxicity to other organs (Sillar et al. 2019).

Antioxidant Therapy As already discussed, leukemic cells have high basal ROS level compared to normal cells. This suggests lowering of ROS is likely to perturb AML cell homeostasis. Capitalizing on this opportunity, different strategies employing antioxidants or inhibitors of ROS generating machinery alone or in combination with conventional chemotherapy are being evaluated. NOX, xanthine oxidoreductase (XOD), and IDH1/2 are recognized as important targets in CML/AML, ALL, and AML, respectively (Prieto-Bermejo et al. 2018). BCR-ABL chimeric gene and FLT3-ITD mutation in CML and AML, respectively, increases ROS level by regulating NADPH oxidase. NOX in turn regulates expression of genes like heme oxygenase-1 which is involved in chemoresistance (Sanchez-Sanchez et al. 2014; Singh et al. 2012). Thus, the use of NOX inhibitor along with chemotherapy is likely to have a stronger effect (Sanchez-Sanchez et al. 2014).

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Macrophages in the tumor microenvironment produce NOX-dependent ROS which impairs T cell and NK cell functionality. Histamine is found to inhibit NOX, thereby reducing ROS in tumor microenvironment, and used in combination with IL2 to activate T cell and NK cell against residual leukemic cells as consolidation therapy resulted in leukemia-free survival in a phase III trial (Romero et al. 2009). Although antioxidant therapy has not been demonstrated to have a major impact on leukemia treatment, a well-balanced diet with antioxidants from food reduces the chance of infections and mucositis in ongoing treatment of leukemia (Ladas et al. 2020). It is also suggested that the use of antioxidant could minimize the negative effects caused by drugs (Garcia et al. 2016). But whether antioxidants may reduce chemotherapy efficacy because they induce cell death via ROS generation is also a question to be addressed.

ROS-Mediated Differentiation Therapy Inducing differentiation in leukemic cells for treating leukemia is another approach. Phorbol 12-myristate 13-acetate (PMA) has been reported to induce cell differentiation by regulating intracellular ROS levels by SOD1 downregulation implicating role of SOD1 in cell differentiation (Chen and Kan 2015). Perturbed iron metabolism has been associated with leukemia, and iron chelators induce differentiation of leukemic blasts by increasing ROS (Callens et al. 2010; Wang et al. 2019). Yang et al. identified mda-7/IL-24 and its splice variant IL-24 delE5, which promotes ROS production and AML cells differentiation (Yang et al. 2011). Through a ROS-dependent mechanism, oncoprotein mucin 1-C inhibition also induces a terminally differentiated myeloid phenotype in AML cell lines and primary blasts (Yin et al. 2011). All-trans retinoic acid (ATRA) is clinically used to induce differentiation in acute promyelocytic leukemia. It is used in combination with ATO as this facilitates differentiation and induces apoptosis by altering redox homeostasis. Also ROS induced by ATO encourages PML-RARA fusion protein degradation (Jeanne et al. 2010). ATPR, a synthetic derivative of ATRA, has recently been shown to induce differentiation and inhibit proliferation in leukemic cell by PTEN/PI3K/ AKT pathway via ROS generation (Feng et al. 2019).

Conclusion The role of ROS in AML is well evident. Oncogenic mutations result in high ROS levels in leukemic cells which supports their proliferation and survival. However, chemoresistant LSCs have low ROS. This difference in the basal ROS levels between LSCs, AML blasts, and normal hematopoietic cells provides an opportunity to develop therapy targeting oxidative stress. Conventional chemotherapy, inhibitors of glutathione pathway, 2-ME, glutaminase inhibitor, HDACi, NK-1R antagonist, and ATO induce ROS to trigger apoptosis in AML. Antioxidant-based approaches

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have not been successful. ATRA-ATO combination works perfectly in acute proemyelocytic leukemia highlighting the importance of combinatorial therapy, which should be exploited for other AML subtypes. Certain issues pertaining to oxidative damage as a result of inducing high ROS levels like genetic stability, acquisition of resistance, and bystrander effects need to be considered when developing such approaches. With increasing understanding of oxidative stress signaling in leukemia, newer targets will emerge and will aid in development of more efficient therapeutic strategies.

Cross-References ▶ Helping Leukemia Cells to Die with Natural or Chemical Compounds Through H2O2 Signaling ▶ Role of ROS in Cancer Stem Cells ▶ Scaffold-Based Selective ROS Generation as Viable Therapeutic Strategies Against Cancer ▶ Targeting Reactive Oxygen Species Homeostasis and Metabolism in Cancer Stem Cells ▶ Therapeutics of Oxidative Stress and Stemness in Breast Cancer Acknowledgments Ajit Kumar Rai is a recipient of Senior Research Fellowship from University Grants Commission, New Delhi, India. CSIR-IITR manuscript number is 3683.

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Sharmistha Chatterjee, Abhishek Kumar Das, and Parames C. Sil

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Stem Cells and Their Role in Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cancer Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Biomarkers of Ovarian CSCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ovarian CSC Microenvironment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Effect of ROS on Cancer Stem Cells: Avenues for Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Stem Cells and ROS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Regulation of ROS in Malignancy and CSCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ROS-Dependent Fundamental Transcription Factors in CSCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . ROS-Dependent Fundamental Signal Transduction Pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . Clinical Relevance of CSCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Recent Developments (Drug Candidates) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract

Cancer, when mostly untreatable, is hypothesized to be sustained due to the presence of highly self-propagating, therapy-resistant cancer stem cells. Their proportion within the tumor is not known, nor any exact correlation could be drawn between the proportion of cancer stem cells in a tumor and the aggressiveness of the metastatic disease. However, extensive research into the mechanisms and therapeutics have revealed numerous pathways and mediator molecules that could be working behind the existence and sustenance of the cancer stem cells, and the cancer having a poor prognosis overall. ROS has been found to be a major role player behind the sustenance as well as degradation

S. Chatterjee · A. K. Das · P. C. Sil (*) Division of Molecular Medicine, Bose Institute, Kolkata, India e-mail: [email protected] © Springer Nature Singapore Pte Ltd. 2022 S. Chakraborti et al. (eds.), Handbook of Oxidative Stress in Cancer: Mechanistic Aspects, https://doi.org/10.1007/978-981-15-9411-3_123

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of these cancer stem cells, making it a double-edged sword. These pathways mediated by ROS have been targeted by various blockers, stimulators, and inhibitors, and in many cases, promising results have been found. All these reports have contributed to the cementing of the existence of cancer stem cells and the extensive role they play in cancer and its metastasis. Apart from contemporary cancer therapeutics, therefore, cancer stem cell targeting has been underway since the past few years. Many answers have been found, and many questions still remain in oblivion. It is expected that more research into this field would allow newer and better avenues in the field of cancer research and therapeutics. Keywords

Reactive oxygen species · Cancer stem cells · Signaling pathways · Biomarkers · Eradication · Therapy · Inhibitors Abbreviations

AML ASC CAF CD CSC EMT ESA ESC FGF GPX GSH HIF HMLE LSC MDSC MMP MSC ROS shRNA SOD SP TAM TIC TMZ TRX R1 TSC TXNIP VEGF

Acute myeloid leukemia Adult stem cell Cancer-associated fibroblasts Cluster of differentiation Cancer stem cell Epithelial-to-mesenchymal transition Epithelial-specific antigen Embryonic stem cell Fibroblast growth factor Glutathione peroxidase Glutathione Hypoxia-inducible factor Human mammary epithelial cells Leukemic stem cell Myeloid-derived suppressor cells Matrix metalloproteinase Mesenchymal stem cell Reactive oxygen species Short hairpin ribonucleic acid Superoxide dismutase Side populations Tumor-associated macrophage Tumor-initiating cells Temozolomide Thioredoxin reductase 1 Tissue stem cell Thioredoxin-interacting protein Vascular endothelial growth factor

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Introduction Cancer has become a global disaster on today’s date. Accounting for the death of an estimated 9.6 million people, it was the second largest cause of mortality in 2018, as per WHO statistics. Cancer is a group of diseases, which arise from uncontrollable growth of abnormal cells going beyond their contact inhibition and finally invading other body tissues and organs. It can initiate from any body organ/tissue and might spread to any other. Initiation is driven by multiple oncogenous mutations which pile up to derange the cellular processes, specifically affecting division and growth. The process of spreading of the malignant cells to various other body parts to generate secondary tumors is called metastasis, which is the primary cause of deaths due to cancer. Cancer tissues are also called neoplasms and malignancies. Men are mostly found to suffer from prostate, lung, liver, and stomach cancer, whereas in women, the most prevalent types are breast, colorectal, thyroid, cervical, and lung cancers. The cancer burden is huge; it continues to grow globally while putting tremendous strain on individuals to families, communities, and health systems as a whole, physically, emotionally, and financially. Economically, the low- and middle-income countries suffer from failing health systems, and these are the ones which are least prepared to manage this burden. Apart from this, majority of the cancer patients around the world do not have access to affordable, quality diagnosis and treatment. Cancer survival rates are increasing in countries with strong health care and accessible early detection, quality treatment, and survivorship care. Stem cells are a group of totipotent and pluripotent undifferentiated cells which have self-renewal capacity. They can differentiate, i.e., specialize under specific conditions to give rise to other cells in the body. Until recently, mainly two types of stem cells were used for study – embryonic stem cells (ESCs) and nonembryonic somatic stem cells, called adult stem cells (ASCs). While the pluripotent ESCs derive from embryos and are the result of first five or six divisions of the zygote, adult stem cells (ASCs) are undifferentiated cells found within the differentiated cells of a tissue or organ, having potential of self-renewal and differentiation into all of the major specialized cell types of the tissue or organ. ASCs are mainly sourced from the bone marrow, brain, blood, cornea and retina of the eye, dental pulp, skeletal muscle, skin, liver, gastrointestinal lining, and the pancreas (Serakinci and Keith 2006). ASCs are limited in potential than ESCs, producing cells which differentiate into mature functioning somatic cells; and they are mostly responsible for cell renewal after injury or disease and during repair. ASCs therefore maintain the steady-state functioning of a tissue, i.e., tissue homeostasis. Cancer stem cells (CSCs) are only a small subset of the heterogeneous tumor cell population. They share the basic properties of self-renewal and also resistance to chemotherapy and radiation with normal stem cells. When transplanted into animal host, they show self-renewal and differentiation, as well as induce tumorigenecity. CSCs have a very high potential of generating tumors. They can be distinctly separated from other cells of the tumor by their cell division symmetry and alterations in gene expression. They divide asymmetrically and hence fulfill two tasks, at once retaining the stem cell identity while also undergoing many rounds of cell

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division and finally postmitotic differentiation to give rise to specific cells. Scholars have not been able to fix the criteria for properly classifying CSCs and hence it has not been possible to define the proportion of CSC subpopulation within a tumor tissue, or their relevance to clinical outcomes definitively. It was previously considered that their proportion within the tumor tissue was very little, however, their proportion is now thought to be quite high, with nearly a quarter of cancer cells within a malignant tissue showing stem-cell-like properties (Kelly et al. 2007). Constant heavy mutations due to disrupted checkpoints in the cell division process finally gives rise to a heterogeneous population of cells within a cancer tissue. As the cancer stem cells keep replenishing the cells inside a cancer tissue while displaying a very high drug efflux rate, hence most chemotherapy and radiotherapy failures are attributed to their doing. Superoxide (O2˙ ), hydrogen peroxide (H2O2), and the hydroxyl free radical (HO.) mainly comprise the group called reactive oxygen species or ROS, and are formed due to oxygen atoms capturing electrons. A critical amount of these oxygenic species is crucial for maintenance of cellular homeostasis encompassing their proliferation, differentiation, and overall survival. This critical level of ROS is maintained within the body by a delicate system of check and balance, the ROS generating and scavenging systems. If endogenous ROS is not well scavenged, their intracellular level increases which lead to certain physiological changes when the cells try to adapt to the altered redox levels. This is called oxidative stress, and is one of the driving forces behind the initiation of malignancy inside the cells. Altered redox balance leads to not only tumorigenesis but also metastasis and drug resistance in the altered cells. However, as cells could be killed by excessive generation of endogenous ROS, and given the fact that cancer cells generate more endogenous ROS than normal cells, xenobiotics which induce such redox reactions producing oxidative stress can selectively kill the cancer cells without significantly affecting the normal ones. In CSC lines like colorectal cancer cell line–derived CSCs, the stemness of the cells has been found to be maintained by hypoxia inside the solid tumor microenvironment, by preventing the differentiation of enterocytes and goblet cells. Given the fact ROS can be induced by hypoxia and the subsequent stabilization of HIF-1α, it is evident that the ROS-hypoxia axis plays a big role in induction and maintenance of stemness in cancer cells. Hence, the therapeutic options which target this axis might prove to be very helpful in removing cancer from the core, without significant chances of relapse (Yu et al. 2012).

Stem Cells and Their Role in Cancer In 1994, a study with human acute myeloid leukemia cells gave the first modern documentation for the role of stem cells, if any, in cancer (Lapidot et al. 1994). The unique AML-initiating cell population sorted based on the presence of the cell surface markers CD34+/CD38 was identified by transplanting the cells from AML patients into severe combined immune-deficient (SCID) mice. Subsequently,

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human CSCs were identified in solid tumors, including brain (Al-Hajj et al. 2003), breast (Singh et al. 2003), colon, pancreas, lung, prostate, melanoma, and glioblastoma. Markedly, even very few numbers of CSCs can promote malignancy, as seen in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice (Al-Hajj et al. 2003).

Cancer Stem Cells Cancer stem cells (CSCs) are a minute subpopulation of tumor cells accountable for carcinogenesis. As the name suggests, CSCs bear characteristics of both tumor cells and stem cells. They exhibit a distinctive ability of self-renewal, differentiation, and proliferation responsible for the initiation of cancer, maintenance and progression of the disease, metastasis to different locations, recurrence of cancer, and resistance to radiation therapy and chemotherapy (Islam et al. 2019). CSCs are heterogeneous, meaning a certain solid mass of tumors comprise different types of CSCs having different molecular signatures (Tang 2012). The functional and phenotypic heterogeneity of the CSC population can be explained by three models, the hierarchical model, the coevolution theory, and the plasticity model. The hierarchical model is based on the linear or branched chain system where a long-term CSC converts to short-term CSC and eventually becomes committed progenitor cell by progressively losing its stemness power. The coevolution theory, on the other hand, foretells that tissue stem cells (TSCs) can be converted to CSCs by “independent initiating events.” TSCs undergo different mutations that ultimately give rise to heterogeneous CSC populations. For example, the cell population which is identified as CD44+/CD24 /low (mammary gland progenitor cells) actually does resemble the lineage of cells used to sort breast cancer CSCs from the patients (CD44 +CD24 /low). Finally, the plasticity model predicts the interchangeable stochastic nature of CSCs. CSCs can spontaneously switch to other cell states with or without stemness and can originate de novo from non-stem cell state. For example, EMT is initiated by cancer cells gaining stem-like characteristics. In immortalized HMLEs (human mammary epithelial cells), the induction of EMT resulted in mesenchymal trait acquisition and stem cell marker expression, which are in reality similar to that found on stem-like cells obtained from HMLE. These altered cells have also been found to have an enhanced capability to form mammospheres (Mani et al. 2008). Certain transcription factors are found to drive cells towards EMT, like ZEB1/2, SNAIL1/2, or TWIST1/2, and they eventually increase the ability of epithelial cells to become invasive. Numerous studies have shown that EMT induction causes CSC character acquisition and aggravates self-renewal (Gang et al. 2004; Mani et al. 2008; Morel et al. 2008). Keeping in line with the above fact, many studies also show that tumor cells possessing an epithelial phenotype actually survive in the circulation and go on to form distant metastasis (Tsuji et al. 2008; Floor et al. 2011; Korpal et al. 2011; Celià-Terrassa et al. 2012). In fact, in prostate cancer cell lines, subpopulations with a strong epithelial gene program were enriched in highly metastatic TIC, whereas mesenchymal subpopulations showed reduced TIC

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(tumor-initiating cells). Collectively, these studies illustrate the plasticity and celltype-specific characteristics governing self-renewal and mesenchymal gene interactions (Celià-Terrassa et al. 2012). Different stemness state of CSCs maintains a dynamic heterogeneous population (Boesch et al. 2016). It is not easy to determine which model is the most suitable to answer the origin of CSCs. It is believed that TSCs transit amplifying progenitor cells and terminally differentiated cells convert to CSCs due to the presence of several stress factors. CSCs subsequently change the niche and the microenvironment to sustain and progress the disease (Aponte and Caicedo 2017). Demonstration by Kaplan et al. depicted that a sequence of events initiates tumor metastasis, and was dependent on a type of cellular “bookmarking” by organizing a site-specific delivery of CSCs, eventually going on to form permissive niches within the target organs. The hematopoietic progenitor cells which are derived from bone marrow are habitated to tumor-specific pre-metastatic sites and they are actually found to form cellular array even before the arrival of tumor cells (Kaplan et al. 2005). In the murine bone marrow, metastatic prostate cancer cells were found to contest with the HSCs (hematopoietic stem cells) for occupying the HSC niche. This led to an increase in the niche size–enhanced metastasis; at the same time, decreasing the niche size reduced the distribution (Shiozawa et al. 2011). Evidently, the cancer CSCs not only maintain cancer at primary sites but aid a lot more than expected in metastasis to distant organs. Hence, it is only natural that these CSCs are taken to be the main culprits behind treatment failures and cancer relapse. Isolation and enrichment of CSCs from different tumors are heavily based on the expression of cell surface markers such as CD24, CD29, CD44, CD90, CD133, epithelial-specific antigen (ESA), and aldehyde dehydrogenase 1 (ALDH1) (Al-Hajj et al. 2003; Singh et al. 2003; Ginestier et al. 2007). The CSC expression on cells is tissue specific, and in case of cancer stem cells it is specific to the tumor subtype. Breast cancers CSCs are widely characterized by CD44+CD24 /low lineage and ALDH+; for colon the signature is CD133+ and the same signature is used for lung and brain as well. CSC signature for leukemia is CD34+CD8 , for head and neck cancer it is CD44+, for liver it is CD90+, and for pancreas it is CD44+/CD24+/ESA+ (Zhang et al. 2010). The ability to generate more of self (self-renewal) and at the same time also differentiate into destined precursors defines CSCs. This is termed as asymmetric division, and it achieves a dual target for the stem cells – one progeny retaining the stem cell characteristics, and the other undergoing multiple cycles of cell division to finally get post-mitotically differentiated. The basis of classifying the CSCs is still not clear, and hence, the exact proportion of CSC subpopulation in a tumor is hard to delineate. For the same reason, their relevance to clinical outcomes of therapy is still ambiguous. Although initially the CSCs had been thought to constitute only a small fraction of the total cellular makeup of a solid tumor, it is now thought that the proportion might be somewhere around as high as 25% (Kelly et al. 2007). Having discussed the above general facts, we would now delve a bit deeper into the role and working of CSCs by focusing on one type of cancer – ovarian. Clinical

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characteristics, heterogeneity, and highly aggressive nature of ovarian carcinoma, for example, strongly suggest that it is a CSC-driven disease. Treatments such as debulking surgery and chemotherapy decrease the tumor mass initially but relapse occurs most of the time as the CSCs can endure the therapeutic approaches (Kenda Suster and Virant-Klun 2019). Ovarian CSC was first identified in 2005 by Bapat et al. from the quarantined ascites of an ovarian cancer patient (Bapat et al. 2005). Later, with the help of various biomarkers, ovarian CSCs were identified and classified.

Biomarkers of Ovarian CSCs The ovarian CSCs are characterized based on surface markers and functional markers. Due to the heterogeneous nature, several markers have been identified.

Surface Markers Several clusters of differentiation have been identified and used to isolate putative ovarian CSCs through flow cytometric analysis. CD24, a glycosylphosphatidylinositol-linked surface protein, was successfully identified as a marker of ovarian CSCs. Gao et al. first isolated CD24+ cells from ovarian cancer patients and demonstrated firm tumor-initiating potential (Gao et al. 2010). Tumor cells obtained from peritoneal effusions were reported to have higher CD24 expression than solid tumors due to the acquisition of CSC-like characteristics (Davidson 2016). CD24+ ovarian CSCs displayed an increased level of STAT3-dependent stemness factors such as Nanog and c-Myc (Burgos-Ojeda et al. 2015). Contrary to these findings, several research groups found little or no association of ovarian CSCs and CD24 expression (Meng et al. 2012; Sharrow et al. 2016; Lupia and Cavallaro 2017). CD44, a transmembrane glycoprotein involved in cellular growth and cell surface adhesion, is a biomarker for ovarian CSC (Senbanjo and Chellaiah 2017) and also involved in pathobiology (Cheng et al. 2012). c-Met, a downstream molecule of CD44-mediated signaling (Li et al. 2011), is involved in different types of other CSCs. cMet promotes ovarian cancer development (Mhawech-Fauceglia et al. 2012) through Wnt/beta-catenin-ATP-binding cassette G2 signaling (Chau et al. 2013), suggesting a potential therapeutic target in ovarian carcinoma. Hyaluronic acid, the major ligand of CD44, is positively correlated with metastasis (Bourguignon et al. 2001; Carpenter and Dao 2003). A specific CD44 variant, CD44v6, was shown to be associated with peritoneal metastasis and metastasis-promoting activity (Tjhay et al. 2015). CD117, a receptor tyrosine kinase, is associated with cellular adhesion, differentiation, and proliferation (Miettinen and Lasota 2005). CD117+ ovarian cancer cells have shown to bear self-renewal, differentiation, regeneration power, and chemoresistance in xenograft model (Luo et al. 2011). CD117 is highly tumorigenic as only 1000 cells can develop ovarian cancer in mouse model (Luo et al. 2011). CD44+/CD117+ cells are chemoresistant and associated with higher expression of MMP2 and MMP9 (Zhang et al. 2008; Chen et al. 2014). CD133 or Prominin-1 is a transmembrane glycoprotein which helps in stem cell maintenance (Yin et al. 1997) and reported as a marker in various types of malignancies, including ovarian carcinoma (Grosse-Gehling et al. 2013). CD133+

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cells have been identified as ovarian tumor-initiating cells (Baba et al. 2009; Curley et al. 2009) and help in ovarian cancer progression (Skubitz et al. 2013). CD133+ CSCs exhibit elevated migration and invasion due to activation of chemokine ligand 5 (CCL5) through NFkB-mediated MMP9 upregulation (Long et al. 2012). The activity of CD133 is epigenetically regulated by specific methylation on the promoter region and histone modification (Baba et al. 2009). Epithelial cell adhesion molecule (EpCAM), a transmembrane glycoprotein, was found to be positively expressed on ovarian cancer cells causing tumor relapse and chemoresistance (Tayama et al. 2017). Cells with CD24+/ CD44+/EpCAM+ phenotype were reported to have massive tumorigenicity, high stemness in OVCAR-5, and SKOV-3 cell lines (Wei et al. 2010).

Functional Markers Besides cell surface markers, several stemness-associated functional markers have been identified in ovarian CSCs. ALDH1, an isozyme of alcohol dehydrogenase enzyme, is extensively used to identify ovarian CSC (Meng et al. 2014; Clark and Palle 2016; Sharrow et al. 2016). High expression of ALDH1 has been reported to be associated with chemoresistance, poor prognosis (Wang et al. 2012a, b), cancer progression, and migration (Wang et al. 2018). CD133+/ALDH+ cells show more stemness than single positive cells (Silva et al. 2011) and are found on the top of the ovarian CSC hierarchy (Silva et al. 2011). Cells with high ALDH activity are related to spheroid formation (Ishiguro et al. 2016). The spheroids were found to be highly aggressive, migratory, invasive, and chemoresistant (Liao et al. 2014). Recently, Ishiguro et al. found that ALDH is a better marker than CD133 for the identification of ovarian CSCs (Ishiguro et al. 2016). Besides ALDH activity, ovarian CSCs have been isolated based on the ability to efflux the lipophilic DNA binding dye Hoechst 33342 by ABC transporters (Wei et al. 2016). According to the position in FACS panel, these cells are referred to as side population (SP) (Behbod and Vivanco 2015), which bears stem-like characteristics (Szotek et al. 2006). However, no precise markers are characterized, as cells isolated through SP analysis are found to be heterogeneous (Lupia and Cavallaro 2017).

Ovarian CSC Microenvironment The tumor microenvironment is the cellular milieu that controls cell growth, proliferation, and metastasis. The CSC niche is composed of tumor microenvironment where CSCs inhabit and interact with nontumor cells (known as stroma), extracellular matrix, and soluble factors (Ruiz-Vela et al. 2009). The stroma is made up of endothelium cells, mesenchymal stem cells (MSCs), cancer-associated fibroblasts (CAFs), tumor-associated macrophages (TAM), etc. (Junttila and de Sauvage 2013).

Growth Factor–Mediated Proliferative Signaling One of the hallmarks of cancer is growth factor–mediated proliferative signaling. Various growth signals and secretory cells have been identified in ovarian carcinoma that are discussed below.

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CAFs are a major component of stroma and involved in the formation and preservation of CSC niche and extracellular matrix modeling. CAFs activate IGF1R expression followed by paracrine/endocrine/autocrine interaction with IGF-1 and results in ovarian cancer progression (Brokaw et al. 2007; Tommelein et al. 2018). CAFs also enhance FGF4 expression and FGF4-FGFR2 signaling which increases ovarian tumor-initiating capacity and stemness of ovarian CSCs (Yasuda et al. 2014). VEGF is one of the most significant growth factors that promote angiogenesis. FGF4 was found to be indirectly increasing angiogenesis through the autocrine induction of VEGF secretion (Deroanne et al. 1997). VEGFA, a specific protein of VEGF family, stimulates stemness of ovarian CSCs via Src-DNMT3A-driven miR-128-2 methylation and Bmi1 upregulation (Jang et al. 2017). Lysophosphatidic acid (LPA)-induced secretion of CXCL12 by MSCs results in increased resistance to hyperthermia in ovarian carcinoma (Lis et al. 2011). MSCs also promote angiogenesis by inducing VEGF and HIF-1α signaling cascade (Pasquet et al. 2010). VEGF was found to induce CXCL12 receptor expression in vascular endothelial cells in ovarian carcinoma (Kryczek et al. 2005). Carcinomaassociated MSCs (CA-MSC), a specific subpopulation of ovarian MSCs, was identified in the ovarian tumor. CA-MSCs upregulate TGF-β and BMP pathways leading to ovarian CSC proliferation (McLean et al. 2011).

Cytokines and Inflammatory Network In ovarian carcinoma, inflammation has been acknowledged as a significant threat in terms of disease development, chemoresistance, and metastasis. Several cytokines have been discovered in ovarian CSCs activating inflammatory network (VarasGodoy et al. 2017). IL-17, one of the critical cytokines present in ovarian CSC niche, is produced by CD68+ macrophages, CD4+ T cells, and tumor-associated macrophages (TAM). CD133+ CSCs express IL-17 receptor and upon IL-17 binding, the self-renewal power of CD133+ cells increases via activation of NFkB and MAPK signaling pathways (Tong et al. 2013). M2 macrophage, a type of TAM, improves tumor progression through TGF-β, VEGF, IL-10, and IL-17 signaling cascade (Varas-Godoy et al. 2017). Recently, it was observed that ovarian CSCs promote M2 macrophage polarization via PPARγ, NFkB, and Wnt signaling pathways resulting in maintenance of stemness, invasiveness, and drug resistance of ovarian CSCs (Deng et al. 2015; Raghavan et al. 2019). Ovarian CSCs produce another cytokine known as CCL-5 which acts in autocrine mode. Binding of CCL-5 to its receptors (CCR1 and CCR3) promotes invasiveness and metastasis of CD133+ CSCs via NFkB-mediated upregulation of MMP9 (Long et al. 2012). miRNA Mediated Stemness Maintenance Ovarian tumor niche contains myeloid-derived suppressor cells (MDSCs) that stimulate miRNA-101 synthesis. miRNA-101 in turn represses the corepressor gene C-terminal binding protein-2 (CtBP-2) and thereby increases the stemness. The exact mechanism of miRNA transfer is still unclear, but it is postulated that the transfer is mediated by extracellular vesicles (Cui et al. 2013; Varas-Godoy et al. 2017).

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Effect of ROS on Cancer Stem Cells: Avenues for Therapy Stem Cells and ROS Reactive Oxygen Species in Cancer and Cancer Stem Cells Reactive oxygen species are generated due to the partial reduction of molecular oxygen by various metabolic reactions. The major members of the ROS family include superoxide anion, hydroxyl radical, peroxides, and singlet oxygen. Under normal physiological conditions, ROS is produced as a by-product of different oxidase and peroxidase reactions. Besides, ROS is generated on the inner mitochondrial membrane due to leakage of electrons from complex I and III during the oxidative phosphorylation. At physiological concentrations, ROS helps in signal transduction and cellular longevity. However, when the concentration exceeds the critical level, ROS reacts with biomolecules like nucleic acids, proteins, lipids, and alters their function. These modifications are a threat to cell survival (Gorrini et al. 2013; Reczek and Chandel 2017). Generally ROS is detoxified by various enzymatic (superoxide dismutase, glutathione peroxidase, catalase, etc.) and nonenzymatic antioxidants (beta carotene, ascorbic acid, glutathione, etc.), maintaining a fine balance in the system. Excessive production of ROS overwhelms the scavenging power of antioxidants, leading to disruption of the redox equilibrium. This phenomenon is referred to as oxidative stress and increase the risk of cellular toxicity (Mukherjee et al. 2015) (Fig.1).

Fig. 1 ROS in cancer stem cells

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Regulation of ROS in Malignancy and CSCs The elevated level of ROS plays a pivotal role in cancer initiation, development, and metastasis. The oxidative stress is much higher in cancer cells than normal cells due to impaired mitochondrial dysfunction. Cancer cells deliberately increase ROS production to enhance its pro-tumorigenic properties and signaling cascade (Moore and Lyle 2011). However, excess ROS level induces apoptosis and necrosis of cancer cells. So, cancer cells have increased antioxidant capacity to combat with the excess buildup of ROS. Thus, cancer cells have a unique redox balance and are highly sensitive to alteration of ROS levels (Thanee et al. 2016). Although in CSCs elevated ROS level is not observed, rather low intracellular ROS level is characteristic of CSCs. For example, cancer-instating cells from head and neck squamous cell carcinoma (HNSCC) maintain low intracellular ROS level to sustain stemness, chemoresistance, quiescence, and tumorigenicity (Emmink et al. 2013). CSCs maintain lower oxidative stress by either lowering the production of ROS or increasing antioxidant capacity. Furthermore, the slow division of stem cells is associated with lower ROS production. It was observed that the asymmetric division of rapidly proliferating breast cancer cells produces slowly proliferating G0-like progeny. This can be referred to as the quiescent stage of the cell and remains outside the cell cycle for a long time (Ding et al. 2015). These quiescent cells with slow proliferative potential are identified as CSCs (Weljie and Jirik 2011). The upregulation of the biosynthesis of antioxidants like glutathione helps to maintain low ROS level in CD44v+ CSCs (Weljie and Jirik 2011). Several epigenetic and microRNA-dependent regulation were also responsible for enhanced antioxidant load in CSCs (Carnero and Lleonart 2016) (Fig. 2).

Fig. 2 Molecules affecting ROS and maintenance of stemness in CSCs

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ROS-Dependent Fundamental Transcription Factors in CSCs HIF-1α Hypoxia, the oxygen-deprived state, is a characteristic feature of cancer cells and CSCs. Due to the lack of oxygen and impaired mitochondrial physiology, the metabolism shifts from aerobic oxidative phosphorylation to anaerobic glycolysis in CSCs, known as the Warburg effect (Tanimoto et al. 2000). This causes the accumulation of lactic acid inside the cell which is eventually released into the tumor microenvironment (Tong et al. 2018). This lactate flux makes the CSC niche acidic and helps in tumor metastasis (Shi et al. 2015). Besides, hypoxia causes the upregulation of hypoxia-inducible factors (HIF) family of transcription factors. HIF is a heterodimeric protein with oxygenresponsive HIF-α subunit and constitutive HIF-α subunit. Under normoxia, HIF-1 α is targeted by Von Hippel-Lindau (VHL), leading to oxygen-dependent proteasomal degradation. However, under hypoxic conditions, the interaction between HIF-1 α and VHL is prohibited and results in nuclear translocation HIF1 α. This facilitates the dimerization of HIF-1 α with HIF-1 β and induction of transcription of the target genes (Zhang et al. 2012). HIFs were found to modulate the CSC population in malignancy (Wei et al. 2018). In lung cancer cells, excess ROS upregulates HIF-1 α expression which shifts glucose metabolism from oxidative phosphorylation to anaerobic glycolysis. This metabolic reprogramming leads to reduced ROS level, which is characteristic of CSCs (Ding et al. 2015). HIF-1 α is also responsible for increasing the number of CD133+ glioma cells and induces self-renewal and inhibits differentiation (Iida et al. 2012; Vadde et al. 2017). TAZ, a Hippo pathway effector, regulates breast CSC activity by interacting with nuclear HIF-1α in hypoxic conditions (Thomas et al. 2012). In lung cancer, HIF-1 α/HIF-2 α induces OCT4 and SOX2, resulting in CD133 expression (Bhagat et al. 2016). Besides, HIF-1 α knockdown leads to OCT4 and CD44 expression in colorectal cancer, leading to decreased stemness (Zhao et al. 2014). On the other hand, CD24 and HIF-1 α expressions are associated with the hypoxic condition in urothelial cancer cells (Jin et al. 2010). The invasiveness and stemness of cancer cells are also upregulated by HIF-2 α via enhancing OCT4 and c-Myc activity in glioma cells (Myant et al. 2013). NF-kB Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) is responsible for cell survival and proliferation. In acute myelogenous leukemia stem cells, inactivation of NF-kB enhances intracellular ROS level leading to apoptosis (Shen et al. 2013). Inactivation of NF-kB also reduces stem cell markers and the CSC population in mammary epithelial cells (Chang et al. 2014). ROS production and NF-kB activation play a vital role in colorectal cancer initiation and intestinal stem cell (Lgr5+ cells) proliferation via Wnt signaling (Gandhi et al. 2014).

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p53 p53 is considered as the most important transcription factor that protects normal cells from developing cancer. Almost all cancer cells have a characteristic feature of p53 mutation. Activation of p53 by resveratrol is found to reduce stemness and epithermalto-mesenchymal transition in nasopharyngeal carcinoma (Wu et al. 2018). The ROS level is increased by p53 activation in CSCs. Selenium was found to be suppressing endogenous antioxidant production and activating oxidative phosphorylation–dependent ROS production leading to apoptosis of leukemia stem cells (Wu et al. 2019). Recently, ginsenoside Rh4 (a saponin) was found to induce apoptosis by activating ROS-dependent JNK/p53 signaling (Wu et al. 2015). NRF2 The nuclear factor erythroid 2 (NFE2)-related factor 2 (NRF2), the master regulator of antioxidant genes, is generally upregulated in CSCs to enhance the antioxidant potential and reduce the oxidative stress. In normal cells, NRF2 binds with its inhibitor protein KEAP1 and is targeted to proteasomal degradation. But in cancer cells, KEAP1 is inactivated by cysteine residue modification and subsequently NRF2 dissociates and translocates to the nucleus (Kim et al. 2018). In colon carcinoma, the NRF2-dependent antioxidant signaling pathways were found to protect CSCs from oxidative stress (Ryoo et al. 2018). Low intracellular ROS level is maintained by abnormal upregulation of NRF2 in mammosphere CSCs derived from breast cancer MCF-7 and MDA-MB-231 cell lines (Shi et al. 2012). Recently, high NRF2 expression level was found to maintain stemness of ALDH-high ovarian CSCs (Cosentino et al. 2011). On the other hand, high CD44 expression induces activation of NRF2 in breast CSCs, causing aggressiveness and drug resistance (Yin and Glass 2011).

ROS-Dependent Fundamental Signal Transduction Pathways ATM Pathway ATM (ataxia telangiectasia mutated) protein is essential for genome stability as it regulates DNA damage such as double-strand breaks (Ping et al. 2011). ATM is involved in reducing ROS level by activating the rate-limiting enzyme of pentose phosphate pathway, glucose-6-phosphate dehydrogenase (G6PD). G6PD subsequently promotes the production of NADPH, an important antioxidant cofactor (Zhou et al. 2007). In CD44+/CD24 breast cancer stem-like cells, the resistance to radiation therapy was due to upregulated ATM pathway. Consistent with the result, when ATM inhibitors were introduced to the cells, the radiation resistance was decreased (Chang et al. 2013). PI3K/AKT Pathway The growth factor–mediated PI3K/AKT pathway is upregulated in CSCs. Activation of this pathway induces VEGF in CD133+ glioma stem-like cells leading to angiogenesis and transdifferentiation-mediated vascularization (Chang et al. 2013).

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Higher intracellular H2O2 level activates AKT by phosphorylation in glioma-initiating cells (Dubrovska et al. 2009). PI3K/AKT pathway, along with STAT3 pathways, was found to be responsible for maintaining stemness and in vitro colony formation of breast cancer cells (Deng et al. 2019). Insulin-like growth factor 1 receptor (IGF-1R) induced PI3K/AKT pathway which helped to maintain stemness of CD44+/CD24-/ALDH+ breast CSCs (Xia et al. 2013). Recently, it was found that activation of PI3K/AKT signaling triggers EMT and CSC marker expression in epithelial ovarian cancer. Combinatorial effect of cisplatin and BEZ235 increases the ROS level and decreases colony formation leading to reduced CSC marker expression (Lee et al. 2002). Two major regulators of this pathway are PTEN and FOXOs. PTEN Phosphatase and tensin homolog (PTEN) is a negative regulator of the PI3K/AKT pathway and its mutation or deletion leads to the development of malignancy. Higher intracellular H2O2 level irreversibly inactivates PTEN by inducing the formation of a disulfide bond between Cys71 and Cys124 in the active site (Chen et al. 2016). Targeting PTEN by upregulating miR-26a/217 activates PI3K/AKT pathway and promotes EMT and stem-like properties in hepatocellular carcinoma (Klotz et al. 2015). Moreover, knockdown of PTEN by shRNA results in increased sphere formation which further results in the enrichment of stem cells in prostate cancer (Dey-Guha et al. 2011). FOXOs Class O of forkhead box transcription factors (FOXOs) are transcriptional factors that are tightly regulated by PI3K/AKT pathway. Specific phosphorylation of serine/ threonine residue by AKT deactivates and inhibits nuclear translocation of FOXOs (Dey-Guha et al. 2011). FOXOs elevate antioxidant enzymes including SOD2, catalase, and extracellular antioxidant proteins like selenoprotein P and ceruloplasmin (Wang et al. 2012a, b). In the MCF-7 breast cancer cell line, ROS-low cells downregulate the AKT pathway. These cells belong to a G0-like stage with higher nuclear localization of FOXO1. Later, these quiescent cells were found to be CSC (McAuliffe et al. 2012; Qiang et al. 2012). Contradictory to these findings, deletion and inactivation of FOXO3a results in enhancement of self-renewal, clonogenicity, and tumorigenicity of prostate CSCs (Dey-Guha et al. 2011).

Notch Pathway The direct cell-cell communication of Notch signaling is essential for maintaining stemness, survival, and self-renewal in neural stem cells and CSCs (Wang et al. 2012a, b). In the presence of oxidative stress, HIF-1α activates Notch signaling pathway for the hypoxia-mediated maintenance of glioblastoma stem cells (Qiang et al. 2012). NOTCH3 was found to be critical for drug resistance of CSCs in ovarian carcinoma (McAuliffe et al. 2012). Isolated CD133+/CD24+ stem cells from renal cell carcinoma ACHN and Caki-1 cell lines were found to have elevated Notch receptors (NOTCH1, NOTCH2) and Notch ligands (JAGGED1, JAGGED2, DLL1,

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and DLL4) (McAuliffe et al. 2012). Consistent with the results, Notch inhibition by shRNA and γ-secretase inhibitor 18 depleted stemness and induced apoptosis of CSCs (McAuliffe et al. 2012).

Wnt Pathway Upregulation of Wnt/β-catenin signaling is prevalent in CSCs to maintain its stemness and tumorigenicity. Upon activation of Wnt signaling, β - catenin translocates to the nucleus, interacts with TCF, and activates stemness-enhancing genes like CD44, OCT4, NANOG, and SOX2 (Li et al. 2019). In basal-like breast carcinoma, loss of function of fructose-1,6-bis phosphatase results in lower ROS level and increased glycolysis under hypoxia. The metabolic shift induces the interaction between β - catenin and TCF leading to the increased CSC-like properties (Dong et al. 2013). In hepatocellular carcinoma, targeting Glutaminase 1 (GLS-1) by GLS-specific inhibitor 968 or silencing GLS-1 by CRISPR/Cas-9-mediated knockdown inactivates β - catenin. It was also found that targeting GLS-1 enhances the ROS level and loss of stemness of CSCs (Li et al. 2019). Contradictorily, activated RAC1 was found to promote ROS production and NF-kB activation that leads to activate Wnt signaling (Myant et al. 2013).

Clinical Relevance of CSCs Evidently, cancer stem cells are novel cancer therapy targets. Antibodies were developed by Hoey et al. against DLL4 (delta-like 4 ligand), a component of Notch, the embryological signaling pathway highly active in stem cells. It worked fine in a human colon cancer murine xenograft where the anti-DLL4 inhibited Notch target genes expression and reduced tumor cells proliferation. Also, anti-DLL4 reduced CSC function alone as well as in combination with irinotecan, a chemotherapeutic agent (Hoey et al. 2009). Jin et al. attempted treating leukemia stem cells in a murine model of AML with 7G3, a monoclonal antibody to CD123, the IL-3 receptor alpha chain. As a result, it was found that 7G3 inhibited the IL-3-mediated intracellular signaling as well as self-renewal of CD34+CD38 leukemia stem cells, and finally was able to reduce leukemia stem cell engraftment (Jin et al. 2009). Ginestier et al. strategically targeted breast cancer stem cells using an anti-CXCR1 antibody as well as a small-molecule inhibitor of the IL-8 receptor CXCR1, repertaxin. Indeed, the anti-CXCR1 treatment reduced the breast CSC population. Moreover, the repertaxin treatment resulted in retardation of tumor growth, inhibition of metastasis, and reduction of the breast cancer stem cell population in xenografts of human breast cancer cells (Ginestier et al. 2010). Wu et al. had earlier conducted immunotherapy studies in mice, and the above results were consistent with it. Wu and his group had demonstrated in vivo an antibody immunoneutralizing the IL-8 receptor blocked breast cancer metastasis (Wu et al. 2008). It even was able to overexpress Dachshund (DACH1), the cell fate determination factor (Chen et al. 2013), whose expression is lost in patients with a poor prognosis of the disease. Not surprisingly, the endogenous DACH1 had a

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reduced expression in breast cancer cell lines which had a high expression of cancer stem cell markers and in general, the patient samples of basal breast cancer phenotype. The antibody-induced overexpression of DACH1 in breast cancer cells resulted in reduction of formation of tumor in vivo as well as reduction of in vitro mammosphere formation (Chen et al. 2013). If a tumor from which CSCs are extracted regenerates to give rise to a tumor that resembles its original form, then that is considered to be the gold standard for CSCs. However, there remains a restraint which questions the direct translational application value of targeting CSCs, and that is a lack of correlation between proportion of CSCs and the clinical outcome of therapy. It is evident that the cancer stem cells are able to resist current traditional cancer therapies, hence aiding in the metastasis and recurrence of cancer. Not surprisingly then, it has also been hypothesized that the CSC proportion within a tumor might correlate with how severe the cancer is (Diehn and Clarke 2006). Accordingly, more research and clinical evidence is needed to define the correlation between tumor aggression and the proportion of therapy-resistant CSC in a tumor. To redirect the therapeutic applications of studies targeting CSCs towards the clinic, we need further progress in CSC-specific marker identification, understanding and chalking out the regulations of CSC and tumorigenic capacity, and finally making successful progress in linking the CSC and its proportions to clinical outcomes.

Recent Developments (Drug Candidates) Ishimoto et al. (56) showed that in gastrointestinal CSCs, CD44v could increase intracellular GSH levels via stabilization of xCT, which is a part of a glutamatecystine exchange transporter and situated at the plasma membrane. The xCT increases the uptake of cystine, a substrate for GSH synthesis at the cell surface. It is hence critical in controlling intracellular redox status. Hence, ROS defense impairing CD44v-targeted therapy provided a potent approach of CSC destruction. Sulfasalazine could be a promising molecule in this regard (Seishima et al. 2016). Kim et al. (2012) demonstrated the effect of increased CD13 expression in liver cancer. It reduced ROS and promoted survival of liver CSCs via a TGF-β-induced EMT-like process. CD13 is actually involved in ROS scavenging in human liver CSCs. Gclm (encoding glutamate-cysteine ligase) catalyzing the rate-limiting step in GSH synthesis was found to be overexpressed in CD13+/CD90- and the CD13+ fractions of cells (HuH7 and PLC/PRF/5). All of these cells have lower ROS concentrations than CD13- fraction. CD13 was suppressed by the CD13-neutralizing antibody ubenimex (clone WM15), and eventually, the CD13+ cells had increased concentration of ROS; in fact, it actually was comparable to the level of ROS in the CD13- cells (Hills et al. 2009). Ubenimex administration inhibited self-renewal and tumor-initiation capacity of CD13+ cells in CD13+ cell-enriched mice xenografts (Haraguchi et al. 2010). Hence, targeting CD13 is also a viable option for CSC elimination. A potent agent increasing ROS, Niclosamide, selectively kills CD34+/ CD38- AML stem cells while conferring minimal cytotoxicity against normal bone marrow progenitor cells in healthy individuals (Jin et al. 2010). A natural product,

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parthenolide, was shown by Guzman et al. to induce CSC apoptosis in patients with AML and CML, partially via ROS increase (Guzman et al. 2005). PML (promyelocytic leukemia), the tumor suppressor gene involved in (Carew et al. 2003; Brandon et al. 2006) chromosomal translocation of acute promyelocytic leukemia, was shown to be a crucial factor to maintain CML CSCs quiescence (Ito et al. 2008). Arsenic trioxide (AS2O3), which is potent for PML degradation and ROS increase, has been pitted to eliminate CML stem cells (Ito et al. 2008). Lagadinou et al. found that LSCs with low ROS overexpressed BCL-2. BCL-2 inhibition decreased GSH levels, which increased the oxidative state and selectively eradicated quiescent LSCs (Lagadinou et al. 2013). Inhibitors of GSH synthesis potentiated TMZ or temozolomide (DNA alkylating agent) induced bystander effect when treating glioblastoma multiforme (Kohsaka et al. 2013). Brusatol has been found to sensitize mammospheres to taxol. It is a Nrf2 pathway inhibitor and downregulates the Nrf2 protein levels as well as that of its target genes (Wu et al. 2015). miRNA deregulation related to ROS is also a recent therapeutic approach in the treatment of cancer (Bao et al. 2014). ROS induces the expression of miR-200 family and further downregulates ZEB1, hence plays a key role in ROS-induced apoptosis and senescence (Magenta et al. 2011). ROS induction and Nrf2 and HIF1α pathways inhibition can also decrease the ability to form colonies in LSC-like cells and apoptosis (Liu et al. 2013a, b). A new drug has been recently developed to directly target AML stem cells, called fenretinide. The drug can augment the deaths of AML stem cells by rapidly generating ROS, upregulating stress responses and apoptosis-related genes, as well as downregulating genes in NF-kB and Wnt signaling (Zhang et al. 2013). Recent studies have also showed that shikonin (TrxR1 inhibitor) induced ROS-mediated apoptosis in human PML HL-60 cells. It was able to break the ROS balance by targeting the TrxR1 and blocked physiological functions in cancer stem cells (Duan et al. 2014). 3-deazaneplanocin A could reactivate TXNIP, which eventually inhibits Trx activity and increases the ROS levels. As a result of the above, the AML cell lines are driven towards apoptosis in AML cell lines, primary cells as well as CD34+CD38 LSCs (Zhou et al. 2011). Other than these, targeting mitochondrial metabolism is another effective way to selectively kill CSCs via ROS-mediated ways. Menadione acts dually by inhibiting complex I of Oxphos pathway and at the same time inducing ROS, hence has been shown to prevent the development of resistance (Sancho et al. 2016). It has been discussed earlier that CSCs do have a finely curated regulation of redox metabolism, ROS is a double-edged sword in the context, and glutathione there plays a critical role in maintaining the stemness characteristics (Chang et al. 2008; Le Belle et al. 2011), hence blocking the synthesis of glutathione and thereby increasing oxidative stress appears to be a promising therapeutic option for CSC population elimination and also restricting tumor growth (Diehn et al. 2009; Rodman et al. 2016). A glutathione biosysnthesis inhibitor called buthionine sulfoximine (BSO) has been proven to be quite effective in sensitizing the CSCs to radiotherapy and decreasing their clonogenicity, both in vitro and in vivo (Diehn et al. 2009; Rodman et al. 2016). Glutamine biosynthesis is essential to cancer cells and CSCs, and hence they are addicted to glutamine. Hence glutamine deprivation is also a strategy to be looked

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into. Glutamine deprivation has already been proven to be effective in reducing the metastatic potential of cancer cells, thus hinting to be effectively working against the CSCs (Gameiro et al. 2013). Apart from glutathione, targeting other cellular antioxidants in view of increasing oxidative stress to kill CSCs has also been under focus. For example, a thioredoxin reductase inhibitor auranofin was able to increase human breast CSC sensitivity to radiotherapy (Rodman et al. 2016). The same molecule synergistically reduced CD44v9+ cell population when applied alongside glutathione-S-transferase inhibition in PDX (patient derived xenograft) models of CRC (Tanaka et al. 2016). Arsenic trioxide (ATO) is FDA approved for treatment of PML (promyelocytic leukemia) as discussed earlier. It works by depleting SOD and GPX, thereby increasing ROS; this has been proved to be effective in reducing CSC population in various cancer types (Li et al. 2006; Ding et al. 2014; Ally et al. 2016; Zhang et al. 2016; Bell et al. 2018). Given the effectiveness of both these therapies, ATO along with glutathione inhibition synergism might prove to be deadly for cancers unresponsive to ATO. Apart from these, disulfiram, the anti-alcohol addiction drug, has been pitted as an anticancer agent as well, as it could block SOD activation (Calderon-Aparicio et al. 2015), inhibit Nrf2 (Xu et al. 2017), and thereby increase oxidative stress. Disulfiram could diminish mammosphere formation in breast cancer cell lines, both in vitro and in vivo (Yip et al. 2011; Kim et al. 2017a, b). It also could reduce CD49f+CD24+ and CD44+CD24 subpopulations of CSCs as well as reversed cisplatin and paclitaxel resistance in TNBCs (Liu et al. 2013a, b). It also reduced ALDH1+ subpopulations in NSCLCs, and CD34+CD38+ populations in AML as well as primary cell lines (Huang et al. 2016; Xu et al. 2017). Disulfiram has been in phase III clinical trials and presented as a potent adjuvant for cancer therapy. One shortcoming of it is that it is not much stable in the blood; for this purpose, nanoparticles based on disulfiram have also been developed to increase their stability. They did increase disulfiram stability (Song et al. 2016), but even then, more research is required to understand the full spectrum of its in vivo working, biological and antioxidant properties. As discussed above, Nrf2 is another potential target for CSC elimination owing to its role in CSC chemoresistance and survival (Kwak and Kensler 2010; Zhu et al. 2013; Ryoo et al. 2016). ATRA (all-trans retinoic acid) was found to be able to block Nrf2 activation, thereby being able to diminish the tumorigenicity and self-renewal of ALDH1+ lung cancer and ovarian cancer cell lines (Hayes and McMahon 2006; Kim et al. 2018). Brusatol has already been discussed above to decrease Nrf2 levels, which was found to inhibit formation of mammospheres and increase human breast CSC sensitivity to Taxol (Wu et al. 2015). Natural inhibitors of Nrf2 like apigenin, a natural flavonoid, and trigonelline, an alkaloid, inhibit Nrf2 at both transcriptional and translational levels, while sensitizing CSCs towards chemotherapeutic drugs (Arlt et al. 2013; Kim and Keum 2016; Gao et al. 2017; Kim et al. 2017a, b). Synergistic ROS induction and Nrf2 inhibition is a potent strategy for elimination of CSC effectively (Ishimoto et al. 2011; Kubben et al. 2016; Ryoo et al. 2016; Choi et al. 2017; Kim et al. 2018; Luo et al. 2018; Dong et al. 2019). On the contrary, it is paradoxical that a few natural antioxidants which potentially increase Nrf2 levels have also been shown to have therapeutic value in this regard. Sulforaphane is a Nrf2

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inducer and is found in broccoli; it was shown to inhibit self-renewal in CD44+ LDH1+ pancreatic and ALDH1+ breast cancer cells both in vitro and in vivo (Rausch et al. 2010; Burnett et al. 2017). The widely researched active ingredient of turmeric, curcumin, was found to effectively reduce self-renewal of CD44+ EpCAM+ pancreatic cancer cells both in vitro and in vivo, as well as reduce mammosphere formation and proliferation of breast cancer cells that were ALDH1+ (Kakarala et al. 2010; Bao et al. 2012). Other widely known natural molecules like resveratrol, carnosol, and oleanane triterpenoids also were found to increase Nrf2 levels which might be used for diminishing the population of CSCs (Probst et al. 2015; Giacomelli et al. 2017). Other natural antioxidants like phenethylisothiocyanates (PEITCs) are found in Brussel sprouts and broccoli, or the widely known Vitamin C have been shown to reduce clonogenicity and self-renewal tendency of human colon cancer and NCCIT human embryonic carcinoma cell lines, diminished OV6+, EpCAM+ and CD133+ cells, while also restricting formation of tumorspheres, diminishing growth of hepatocellular carcinoma cells, as well as in vivo PDX models (Lv et al. 2018; Ngo et al. 2019). An overview of the drug candidates is given in Table 1. The natural antioxidants do provide exciting avenues for anticancer therapies. However, clinical trials did not show any significant positive effects on patient survival. All published data indicate towards the drawback of antioxidant treatments owing to lack of specificity. There is a possibility that the antioxidants might actually aid in maintenance of stemness and aggravate the development of cancer, thus weakening this approach’s potential (Figs. 2 and 3, Table 1).

Table 1 Targeting ROS in cancer stem cells Mechanism Targeting CD-44v Targeting CD-13 Bcl2 inhibition Complex I inhibition Glutathione inhibition Nrf2 inhibition and downregulation Nrf2 upregulation Wnt inhibition and downregulation ROS upregulation Thioredoxin reductase 1 downregulation Synergystic downregulation of glutathione and thioredoxin 1 Synergystic downregulation of superoxide dismutase and glutathione Other antioxidants

Candidates Sulfasalazine Ubenimex Venetoclax Menadione Buthionine sulfoximine Brusatol, all-trans retinoic acid (ARA), fenretinide, apigenin, and trigonelline Sulforaphane Chloroquine and emodin Niclosamide, parthenolide, arsenic trioxide, miR200 upregulation Shikonin, auranofin, 3-deazaneplanocin A Auranofin Arsenic trioxide and disulfiram Curcumin, resveratrol, carnosol, oleanane triterpenoids, phenethyl isothiocyanate, and Vitamin C

Fig. 3 Interaction of various factors and signaling molecules to uphold the maintenance of CSCs. These provide avenues for successful cancer therapy with minimal chances of relapse, as they directly target the self-sustaining rogue stem cell populations thriving within malignant tissues

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Conclusion Exogenously induced and increased ROS imposing a state of oxidative stress have been known to be effective in selectively killing cancer cells. Stemness of CSCs has been associated with low ROS levels. Unregulated redox states might sustain or kill the cancer cells and CSCs; however, the mechanism which induces such a reduced ROS state has not been completely understood. Oxidative stress already exists in cancer cells and any external hit might tip the redox balance towards selective propagation or selective killing of the cancer cells and CSCs. While there exists no wholesome information regarding the complete regulation of ROS in the CSCs, there exist evidences which are fast emerging that indicate the major role of ROS in the differentiation and self-renewal ability of CSCs. Transcriptional activities and signaling pathways which are ROS dependent, control and regulate the intracellular redox balance and ROS levels in CSCs. Evidently, targeting the cancer stem cells via regulation of ROS and various antioxidant molecules hold a great potential in improving current cancer therapy. Therefore, intensive research is needed to understand the entire map of ROS role in CSCs. Complete eradication of CSCs might just be the magic cure for the deadly disease called cancer.

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Implications in Resistance to Therapy Vijay Kumar Kutala and Shaik Mohammad Naushad

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cancer Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ROS-Mediated Signaling in CSCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ataxia Telangiectasia Mutation (ATM) Pathway in CSCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . PI3K/AKT Pathway in CSCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Notch Pathway in CSCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Wnt Pathways in CSCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . STAT Pathway in CSCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ROS-Dependent Transcription Factors in CSCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Role of ROS in Epithelial-Mesenchymal Transition in CSCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CSCs and Drug Resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract

Cancer stem cells (CSC) are a divergent subpopulation of cells that resides within a tumor. CSC possesses similar properties to normal stem cells, for instance, selfrenewal and quiescence. These cells have shown to involve in cancer recurrence, metastasis, heterogeneity, and multidrug and radiation resistance. The presence of low levels of reactive oxygen species (ROS) is necessary for stem cells and CSCs to retain quiescence and self-renewal. The ROS-dependent transcription and signaling pathways are the vital mechanisms to maintain ROSlow in CSCs. An increase in ROS production has shown to be involved in stem cell proliferation/differentiation, V. K. Kutala (*) Department of Clinical Pharmacology and Therapeutics, Nizam’s Institute of Medical Sciences, Hyderabad, India S. M. Naushad Department of Biochemical Genetics and Pharmacogenomics, Sandor Speciality Diagnostics Pvt Ltd, Hyderabad, India e-mail: [email protected] © Springer Nature Singapore Pte Ltd. 2022 S. Chakraborti et al. (eds.), Handbook of Oxidative Stress in Cancer: Mechanistic Aspects, https://doi.org/10.1007/978-981-15-9411-3_124

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senescence, and apoptosis leading to cell death. Hence, the ROS production in cancer stem cells be firmly regulated to maintain stemness and plays a vital role in cancer recurrence, metastasis, and resistance to cancer therapy. Recent studies have established that ataxia telangiectasia mutated (ATM), forkhead box O3 (FoxO3), nuclear factor-erythroid 2-related factor 2 (NRF2), Notch, PI3K/AKT/mTOR, Wnt β-Catenin, nuclear factor-κB (NF-κB), and hypoxia inducible factor (HIF) are implicated in the survival, quiescence, and oxidative stress-mediated resistance of CSCs. In the present chapter, though there are several factors that are involved for the maintaining of the stemness of CSCs, we highlight the role of enzymes and transcription factors implicated in redox signaling on stemness, recurrence, metastasis, and chemo- and radioresistance and possible application of newer strategies to sensitize the CSCs is discussed. Keywords

Stem cells · Cancer stem cells · ROS · Resistance · Oxidant · Antioxidant signaling

Introduction Increased metabolic rate, mitochondrial dysfunction, activation of proto-oncogenes, and inactivation of tumor suppressor genes contribute to increased production of reactive oxygen species (ROS) in cancer cells compared to normal cells (Trachootham et al. 2009; Galadari et al. 2017). Through the ROS signaling pathways, cancer cells might obtain proliferative, invasive, and metastatic properties and are also involved in every phase of cancer development, such as initiation, promotion, progression and metastasis, and poor prognosis (Trachootham et al. 2009). Increased ROS levels facilitate the activation of downstream targets, that is, Keap1/ Nrf2 pathway thus contributing to the proliferation of cancer cells (Ohta et al. 2008). The other important downstream target activated by ROS is AKT/mTOR pathway, which stimulates pro-survival signals via the activation of nuclear factor kB (NF-kB) (Lee et al. 2019). Further, activation of PI3K/AKT/mTOR signaling pathway contributes to enhanced cellular metabolism and glycolysis, thus enhancing the intracellular ROS level and facilitating tumorigenesis (Elstrom et al. 2004). In addition, it has been reported that cancer cells may acquire adaptive mechanisms by inducing the redox-sensitive transcription factors that activate SODs and glutathione synthase, survival factors, and as well as the inhibition of apoptosis that can drive to malignant transformation, metastasis, and chemo-resistance (Trachootham et al. 2009). The majority of the tumors have a subpopulation of extremely tumorigenic cells, known as cancer stem cells (CSCs). CSCs, an intrinsic part of tumor mass, which are the sub-population of quiescent cells enrich with the properties of self-renewal and differentiation and highly influence the tumor propagation, cancer metastasis, and multidrug resistance (Lee et al. 2019). CSCs share identical characteristics like normal stem cells or progenitor cells such as self-renewal capability and multi-

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lineage differentiation (O’Brien et al. 2010). CSCs have been shown to promote cell cycle arrest that provides the evidence about their capability to become chemo- and radioresistance (Lee et al. 2019). Similar to stem cells, CSCs also maintain low ROS cells for stemness, which is modulated by the various antioxidant signaling mechanisms, which are the key mediator for chemo- or radiation therapy-induced resistance (Lee et al. 2019). In this chapter, we emphasize the role of redox sensors in CSCs in maintaining stemness, tumor propagation, metastasis, and resistance to both radiation and chemotherapy and newer strategies to overcome the resistance.

Cancer Stem Cells A subpopulation of cancer cells in various cancers (leukemia, breast cancer, glioblastoma, liver cancer, and pancreatic cancer) exhibit stem cell-like properties and hence were denoted as cancer stem cells (CSCs) (Lee et al. 2019). These cells resemble normal stem cells with self-renewal and differentiation capacity and are shown to be the cause of radio- and chemo resistance and tend to survive after either of the therapies, and these survived cells can generate new tumors, recurrence, and metastasis (Lee et al. 2019). Hence, newer therapies, apart from cancer cells, must target to CSCs. Since cancer cells share the unique redox systems, conventional therapies such as radiation or chemotherapy and targeting redox systems can eradicate the majority of the cancer cells together with the CSCs (Lee et al. 2019) (Tables 1 and 2). Recent investigations have found that the redox status of cancer stem cells have some similar characteristics of normal stem cells (Lee et al. 2019). Recent studies have implicated that low levels of ROS are required for the CSCs survival and also involved in resistance to therapy (Lee et al. 2019). The mechanism of adaptation to low ROS levels in CSCs is tumor-specific. High tumorigenicity and radiotherapy resistance was observed in the CD24/low/CD4+ breast cancer stem cells, which have ROSlow levels (Phillips et al. 2006). High degree of antioxidant defense was observed in the human gastrointestinal cancer stem cells with an enhanced expression of CD44 due to increased synthesis of glutathione (Ishimoto et al. 2011). Similarly, compared to non-stem breast cancer cells, low ROS levels have been reported in human and murine breast cancer stem cells (Diehn et al. 2009). The possible mechanism for the low ROS levels in breast cancer stem cells is the upregulation of antioxidant enzymes contributing to tumor radioresistance. In another study, it was observed that because of efficient DNA repair mechanism, the CD133+ glioblastoma stem cells (GSCs) show higher survival rates when exposed to ionizing radiation as compared to normal counterpart (Gilbertson and Rich 2007). The aldehyde dehydrogenases (ALDH), a marker in many CSCs, reduce the free radicals and enhance the resistance to chemotherapeutic drugs, such as taxanes, oxazolidine, and platinum drugs (Singh et al. 2013). The chemotherapy- or radiotherapy-induced DNA damage and apoptosis are the possible mechanisms involved in cancer therapy; however, CSCs might protect the cancer cells from apoptosis by inducing DNA repair mechanisms (Diehn et al. 2009). On treatment

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Table 1 Cancer stem cell surface markers and metabolic phenotypes in human cancers Type of CSCs Leukemia CSCs Breast CSCs

CSC marker CD34 CD44, CD133

Liver CSCs

CD90, CD45

Ovarian CSCs

CD44,CD117, CD133 CD133, nestin, and A2B5

Gliobastoma CSCs

Pancreatic CSCs

CD44, CD133

Colon CSCs

CD24,CD29,CD44 CD51, CD133, CD166

Effect on ROS through metabolic process Low ROS CSCs due to dormant cell cycle status CSC population is maintained by low ROS due to suppressing oxidative phosphorylation and enhancing glycolysis and induction of antioxidant system CSC population is maintained by low ROS due to suppressing oxidative phosphorylation and enhancing glycolysis and induction of antioxidant system CD44+/CD117+ CSCs possess higher ROS than CD44+/CD117+ cells Reduced NADPH by inhibition of fatty acid oxidation and increase in ROS levels CSCs are less glycolytic. Induction of ROS in glioma CSCs through electron transport chain Upregulation of glycolysis, low ROS promotes stemness, EMT and chemoresistance, low ROS CSCs depend on non-canonical glutamine metabolic pathway Maintain low ROS levels by enhanced antioxidant systems and contribute for drug resistance. CSCs secretome is enriched with proteins implicated in glycolysis and gluconeogenesis

Ref: Lee et al. (2019), Yang et al. (2020)

with parthenolide (PTL), apoptosis was induced in leukemic stem cells (LSCs) due to increased ROS levels, activation of p53, and inhibition of NF-κB (Guzman et al. 2005). The promyelocytic leukemia protein (PML), a tumor suppressor, was reported to play a pivotal role in the maintenance of the dormant CML stem cells. Inhibition of PML with arsenic trioxide was shown to prevent CML stem cell proliferation via generation of ROS (Ito et al. 2008). In another study, it was demonstrated that PML inhibits the mTOR and HIF-1α under hypoxic conditions, suggesting the possible crucial targets in LSCs (Bernardi et al. 2006). The ROS-mediated activation of AKT/mTOR signaling pathway in mutated hematopoetic progenitors was shown to cause a myeloproliferative syndrome via loss of FoxO3 activity (Yalcin et al. 2010).

ROS-Mediated Signaling in CSCs Several ROS-dependent signaling pathways in regulating CSCs have been explored, which include ATM/p53, PTEN/PI3K/AKT/mTOR, Notch, JAK/STAT, Wnt, and NF-kB cascades that have been implicated in the modulation of CSCs and redox

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Table 2 Regulation of redox sensor molecules and its effect on cancer stem cells Redox sensor molecules AKT kinases

Detrimental effects of ROS Akt phosphorylation affected due to cysteine oxidation

Effect on ROS generation Modulates ROS through transcription factors such as FOXO ATM upregulates the glucose-6phosphate dehydrogenase to promote NADPH production, and thus reduces the ROS level Stem cell selfrenewal mediates through ROS

ATM

Cysteine oxidation in ATM by ROS, activates ATM

p38 MAPK

Activated by cysteine oxidation

mTOR

Activated by cysteine oxidation

Mitochondrial biogenesis and ROS production

PTEN

Inactivated due to cysteine oxidation by ROS

Modulation of PI3K/AKT pathway

Thioredoxins

ROS induces TRX disassociation from ASK1, ASK1 then activates p38MAPK and JNK and induces apoptosis

Reduction state of biomolecules is maintained

Influence of ROS on cancer stem cells High H2O2 can induce the phosphorylation of AKT in gliomainitiating cells Involved in radiation resistance as ATM inhibitor reversed the radiation resistance of CD44+/CD24– cells In glioma-initiating cells, H2O2induced ROS can increase p38 MAPK. The upregulated p38MAPK will induce BMI1 protein degradation and FOXO3 activation, leading to differentiation In CSCs, mTOR controls the cellular ROS levels through regulation of nuclear-localized FOXOs PTEN deletion contributes to the depletion of normal HSCs, but increases the generation of leukemia-initiating cells Histone methyltransferase inhibitor showed toxicity against CD34+CD38– leukemia stem cells by reactivating TXNIP and also by inhibiting Trx activity

References Sato et al. (2014)

Shi et al. (2012), Yin and Glass (2011)

Sato et al. (2014)

Diehn et al. (2009)

Yilmaz et al. (2006)

Zhou et al. (2011)

(continued)

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Table 2 (continued) Redox sensor molecules HIFs

FOXOs

p53

NRF2

Detrimental effects of ROS Either stabilized (normoxia or inhibited (hypoxia) by ROS

Effect on ROS generation Reduces ROS levels by modulating cell metabolism

Contains 5–10 cysteine residues, switch from cell cycle arrest to apoptosis mediated by ROS. ROS modulates PTEN or AKT effect on FOXOs DNA repair binding is impaired by oxidation or maintained by interaction with oxidized APE/REF1 Modulated by APE/REF1 and KEAP1, the oxidation results in NRF2 activation/ inactivation

Regulate the antioxidant enzymes (SOD2, catalase, and GPx1)

Regulates of pro-oxidants and antioxidants

Regulates of transcription of antioxidant enzymes

Influence of ROS on cancer stem cells Mediate the EGF-induced prostate cancer cell EMT phenotype and STAT3 downstream of ROS is implicated in EGF-induced HIF-1a transcription and protein expression ER+/HER2 human breast cancer MCF7 cell line, the ROSlow cancer cells had higher levels of nuclear localized FOXO1 Resveratrol suppressed the CSC properties that include resistance to therapy and selfrenewal, tumor initiation, and metastatic potential Mediator of CSC resistance, increased drug efflux

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Sablina et al. (2005), Muller et al. (2009)

Alam et al. (1999), Ryoo et al. (2016)

Akt, protein kinase B; APE/REF1, apurinic/apyrimidinic endonuclease; ASK1, apoptosis signalregulating kinase 1; MAP3K5, mitogen-activated protein kinase 5; BID, BH3 interacting domain death agonist; CSCs, cancer stem cells, FOXOS, Forkhead homebox type O proteins; GPX1, glutathione peroxidase 1; JNK, Janus kinase; KEAP1, Kelch-like ECH-associated protein; NRF2, nuclear factor erythroid 2; MAPK, mitogen-activated protein kinase; p53, transformation related protein 53; mTOR, mammalian target of rapamycin; PTEN, phosphate and tension homolog

homeostasis (Lee et al. 2019). Recent studies have demonstrated the crucial role of ROS-induced HIF-1α and Nrf2, two redox-sensitive transcription factors, in the regulation of breast cancer stem cell (BCSC) plasticity during metabolic stress (Luo et al. 2018). It was found that the low level of ROS in CSCs is attributable to the overexpression of antioxidant systems, such as glutathione (GSH) (Diehn et al.

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2009). Furthermore, in a study, it was observed that increased expression CD44v, the variant isoform of CD44+ in CSCs, contributes to increased synthesis of GSH (Nagano et al. 2013). The main antioxidant enzymes which are highly activated in CSCs are superoxide dismutase-2 (SOD2), glutathione peroxidase (GPX), hemeoxygenase-1 (HO-1), NAD(P)H quinone oxidoreductase1 (NQO-1), and peroxiredoxin II (Chandimali et al. 2018; Kim et al. 2018).

Ataxia Telangiectasia Mutation (ATM) Pathway in CSCs The ATM gene plays a crucial role in the ionizing irradiation-induced impairment in DNA double-strand breaks (DSB) (Shi et al. 2012). In a study, it was demonstrated that ATM exhibits an enhanced expression in glioma stem cells (GSCs) and contributes to radioresistance, and this observation was substantiated by silencing the ATM gene in GSCs which was shown to promote the therapeutic affect by sensitizing GSCs to radiation therapy (Li et al. 2017). The expression of ATM was significantly enhanced in CD44+/CD24 stem-like cells in breast tumors and breast cancer cell lines (Yin & Glass 2011). In a study, the radiation-induced resistance of CD44+/CD24 cells is shown to be reversed by ATM inhibitor, indicating the critical role of ATM signaling in CSCs (Yin and Glass 2011). The low levels of ROS and CSCs stemness is possibly due to increased ATM activity, subsequently leading to the upregulation of antioxidant enzymes and downregulation of genes of proliferation and differentiation (Shi et al. 2012). The other mechanism that reduces ROS levels by ATM is by enhancing NADPH production through the upregulation of glucose-6-phosphate (Cosentino et al. 2011).

PI3K/AKT Pathway in CSCs The adaptive inhibition of AKT in cancer stem cells is shown to be crucial for stemness and resistance (Arasanz et al. 2020; Yang et al. 2020). The regulation of PTEN/PI3K/AKT/mTOR signaling pathway in CSCs is dependent on ROS (Lee et al. 2019), and it is mediated by nuclear-localized FOXOs (Diehn et al. 2009), and then the FOXOs regulate the activation of MnSOD and catalase to scavenge ROS (Kops et al. 2002). In a study, it was shown that ROSlow in MCF7 human breast cancer cells possesses enhanced nuclear localized FOXO1 (Dey-Guha et al. 2011). The higher concentrations of H2O2 treatment can induce the phosphorylation of AKT in glioma-initiating cells (Sato et al. 2014). In CSCs, for the regulation of catalytic activity of PTEN, ROS-dependent oxidized cellular milieu is crucial, as evidenced by the treatment of H2O2 which has shown to diminish the activity of PTEN by the disulfide bond formation between the Cys124 and Cys71 active sites (Lee et al. 2002). In another study, it was demonstrated that abolishing PTEN activity through PTEN deletion resulted in the reduction of normal HSCs and enhanced the formation of leukemia-initiating cells, indicating the crucial role of PTEN regulation in the normal stem cell maintenance (Yilmaz et al. 2006).

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Notch Pathway in CSCs Notch signaling pathway has shown to be implicated in preserving the self-renewal and maintenance of CSCs, also involved in the formation, spread, and cancer recurrence (Wang et al. 2010; Meisel et al. 2020). In a study, it was observed that the Notch3 signaling pathway is crucial for the survival and resistance of CSCs (McAuliffe et al. 2012). Similarly, Notch1 or Notch2 deletion has shown that glioma stem-like cells sensitize to radiation (Wang et al. 2010). The Notch signaling pathway is also important for the maintenance of ROS levels in CSCs by targeting the PI3K/AKT pathway. In a study, it was demonstrated that the activation of Notch pathway induced by HIF-1α is important for hypoxia-mediated maintenance of glioblastoma stem cells (Qiang et al. 2012). In glioma stem cells, the AKT is induced by Notch resulting in the upregulation of the ROS scavenging enzymes (Qiang et al. 2012). Nevertheless, ROS is able to activate the Notch signaling pathway in order to retain the CSCs stemness.

Wnt Pathways in CSCs Wnt/β-catenin signaling pathway plays a key role in determining the cell fate, facilitating the cell migration, in dictating the cell polarity and neural patterning and in the development of organs during the embryonic development. The high ROS levels have shown to inhibit the activation of Wnt/β-catenin signaling (Korswagen 2006). The exogenously activated Wnt pathway has shown to enhance the proliferation of colon CSCs (Vermeulen et al. 2010). The radiation therapy-induced resistance of breast cancer cells displays CSC-like properties with the increase of Wnt/β-catenin, whereas the treatment of breast cancer cells with a cyclooxygenase inhibitor, NS398, sensitizes the cells to radiation, and this observed effect is due to the decreased expression of Wnt/β-catenin (Che et al. 2011). In a recent finding, the Wnt/β-catenin signaling pathway is involved in stemness maintenance of irradiated hepatocellular carcinoma stem cells (Hou et al. 2020).

STAT Pathway in CSCs The continual activation of STAT3 has been shown to promote cell survival and the maintenance of stemness in breast CSCs (Yang et al. 2020). In non-small lung cancer, high p-STAT3 level was shown in CD133+ stem-like cells as compared to CD133 cells. This is supported by further observation that the inhibition of STAT3 by cucurbitacin 1 decreases the CD133+ cells by inducing the apoptosis (Hsu et al. 2011). Breast cancer cells treated with H2O2 exhibit decreased STAT3 binding with the inhibition of cell proliferation (Li et al. 2010). In breast cancer stem cells, the STAT3 pathway has shown to be positively regulated by mTOR signaling (Zhou et al. 2007). Both the STAT3 and mTOR are negatively regulated by PTEN, hence

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the effect of ROS on CSCs by STAT3 signaling might be modulated by the PTEN/ PI3K/ATK/mTOR signaling pathway.

ROS-Dependent Transcription Factors in CSCs Hypoxia Inducing Factor (HIF) in CSCs Cells in response to hypoxia have shown to be regulated by the HIF-1 and are generally seen in tumor hypoxic environment, stem cells, and CSCs. An increase in the expression of HIF-1 inside the tumors has been well associated with the radioresistance, progression, and metastasis (Olivares-Urbano et al. 2020). Cells can adapt to low oxygen milieu by the activation of HIFs under hypoxia condition. The HIF activation was shown to be enhanced by ROS scavengers and triggered pro-survival signaling factors, leading to radioresistance to tumor cells, and this process is mediated by Notch, Wnt, and Hedgehog signaling pathways (OlivaresUrbano et al. 2020). Studies have shown that the HIF-α expression is regulated by ROS. HIF-1α is shown to modulate the epidermal growth factor (EGF)-induced prostate cancer cell EMT phenotype, whereas STAT3 is shown to involve in EGF-induced HIF-1α transcription and protein expression (Yang et al. 2020). HIF-1α is shown to regulate the proliferation and outcome of CSCs in medulloblastoma and glioblastoma by activating the NF-κB pathway to enhance the CSC survival. Thus, CSCs tend to adapt under hypoxic condition through cell energy metabolism and preventing the apoptosis (Yang et al. 2020). The possible role of low-oxygen tension, ROS production, and EMT was also established (Yang et al. 2020). The higher ROS levels can promote EMT, but the antioxidant treatment can mitigate hypoxia-induced EMT and metastasis propagation in cancer cells; hence, maintaining the low ROS levels is critical to preserve the CSCs (Yang et al. 2020). NF-kB in CSCs The transcription factor, NF-κB plays a vital role in cell survival, proliferation, inflammation, and immunity (Yang et al. 2020). Studies have shown that there is a link between the ROS and NF-κB signaling. ROS can regulate NF-κB activation to induce antioxidant genes code for CuZn-SOD, MnSOD, catalase, and Trx and protect the cells from ROS-induced cytotoxicity (Morgan and Liu 2011). The low ROS levels as seen in CSCs are possibly due to the enhanced expression of NF-κB that might involve in redox balance. The NF-κB suppresses the anticancer agentinduced ROS-JNK-mediated cytotoxicity (Bubici et al. 2006). Treatment of acute myelogenous leukemic stem cells (LSCs) with niclosamide (an antineoplastic) might inhibit the TNFα-induced NF-κB activation by increasing the intracellular ROS levels (Jin et al. 2010), whereas suppresses the ROS by the N-acetylcysteine and has shown to attenuate the niclosamide-induced apoptosis. AML progenitor and stem cell population treated with Parthenolide, a sesquiterpene lactone, promote cell death and decrease of engraftment in vivo by blocking NF-κB and increased ROS generation (Myant et al. 2013). The Rac1 activation and ROS induced activation of NF-κB is crucial for the colorectal cancers initiation (Morgan and Liu 2011).

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Tumor Suppressor p53 in CSCs The tumor suppressor p53 plays an important role in protecting the normal cells from cancer development. It can also regulate genes which produce or quench the ROS and might exert either pro-oxidant or antioxidant effects depending on its expression level and cellular statuses (Yang et al. 2020). It is well documented that all human cancers lose the p53 activity (Muller et al. 2009). The treatment of resveratrol to CSCs of nasopharyngeal carcinoma cells has found to abolish the CSC properties that include self-renewal, metastatic potential, and resistance to therapy (Shen et al. 2013), possibly due to the activation of p53, as the knockdown of p53 reverses this effect. In addition, resveratrol utilizes p53 to suppress stemness and EMT (Shen et al. 2013). In addition, treatment of leukemia CSCs with selenium has been shown to enhance the ROS levels and induces apoptosis, and this process is mediated by the activation of the ATM-p53 (Gandhi et al. 2014). CSCs treated with parthenolide have shown to block NF-κB, activate p53, and increase ROS levels thereby inducing the apoptosis of LSCs in AML (Guzman et al. 2005). The other possible mechanism by which p53 is regulated by ROS is by inactivating p53 through the oxidation of p53 cysteine residues, thus demonstrating the crucial role of p53 and ROS signaling in the regulation of cell cycle and apoptosis. The Nuclear Factor Erythroid 2-Related Factor (Nrf2) in CSCs Nuclear factor-erythroid 2-related factor 2 (Nrf2) encoded by the NFE2L2 gene is a basic leucine zipper that increases the expression of antioxidant proteins (glutathione peroxidase, glutathione reductase, thioredoxin reductase, ferritin, NADPH: quinone oxidoreductase, etc.) by recognizing the cellular oxidative stress thus maintaining the cellular redox status (Emmink et al. 2013). In normal cells, Nrf2 is in inactive state by binding to Keap1. However, in many cancer cells, loss of Keap1 function due to several factors activates the Nrf2 and induces cancer growth (Ohta et al. 2008). In CSCs, Nrf2 maintains the low ROS levels by regulating the expression of antioxidant genes in response to oxidative stress, and thus leading to CSC resistance and enhanced drug efflux (Ryoo et al. 2016). In a study, the differentiation of glioma stem-like cells was inhibited by Nrf2, as the knockout of Nrf2 induces the differentiation process (Alam et al. 1999). The antioxidant genes such as GSH reductase, GSH synthetase and peroxidases are regulated by Nrf2 (Alam et al. 1999). In a study, it was observed that the colon CSCs are protected from oxidative stress by the Nrf2antioxidant pathway (Emmink et al. 2013).

Role of ROS in Epithelial-Mesenchymal Transition in CSCs Recent findings have shown that ROS signaling mechanisms are involved in promoting the epithelial-mesenchymal transition (EMT)-like phenotype of CSCs, which plays a critical role in tumor progression. Mesenchymal CSCs in breast cancer with ROSlow are found to be greatly effective in inhibiting glycolysis than the ROShigh epithelial CSCs and this is meditated through Nrf2 antioxidant response

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(Luo et al. 2018). The oxidative/metabolic stress induced by hypoxia, H2O2, or 2-Deoxy D-glucose has shown to stimulate the shift of breast cancer stem cells (BCSCs) from a dormant, ROSlow M-like state to a ROShigh proliferative E-state which is mediated by the enhanced Nrf2 expression through the AMPK-dependent HIF-1α stabilization (Luo et al. 2018). In a recent study, it was shown the elevated mitochondrial ROS in CSCs can potentiate the cancer metastasis (Wang et al. 2019). The mesenchymal as well as epithelial CSCs can be targeted by inhibiting glycolysis and antioxidant pathways simultaneously. The inhibition of glycolysis by 2-deoxy-2-D-glucose in gemicitabine-resistance cell line was found to inhibit CSC and EMT processes. N-acetyl cysteine treatment restored CSC and EMT phenotype, while H2O2 treatment showed the effects similar to glycolysis inhibition (Zhao et al. 2017). Dual Oxidase 1 (DUOX1) expression was shown to be lower in CD24low CSC compared to the CD24 high non-CSC in lung cancer (Little et al. 2016). Silencing of DUOX1 may result in the decrease in H2O2 levels, enhancing the CSC cell number, tumor invasiveness, and resistance to therapy (Little et al. 2016). These findings suggest that the association of ROSlow with the EMT phenotype of CSCs

CSCs and Drug Resistance There is compelling evidence that the CSC population is extremely resistant to current chemo- and radiation therapies (Phillips et al. 2006; Lee et al. 2019). This is most probably due to some of the characteristics which are very specific to CSCs. Hence, it is possible that CSCs can survive even after both the therapy regimens. The survival of these CSCs may cause recurrences, metastasis, and poor prognosis. The role of ROSlow in CSCs resistance to therapy is well documented in several studies. In AML, the ROSlow, dormant leukemic cells have shown to exhibit CSC properties and increased expression of the anti-apoptotic protein, Bcl-2 (Lagadinou et al. 2014). The inhibition of Bcl-2 increases the mitochondrial ROS levels and decreases the GSH levels, thus preferentially eliminating the therapy-resistant and therapydormant CSCs. In hepatocellular carcinoma, anticancer drugs or radiation therapy can result in the enhancement of CSCs with a ROSlow form accompanied by the reduction in oxidative DNA damage or increased glutathione or facilitate the activation of MAPK/PI3K pathway (Song et al. 2017). Sulfasalazine, a cystineglutamate antiporter inhibitor, was shown to combat resistance to chemo-/ radiotherapy in CD133+ CSCs by increasing the production of ROS (Song et al. 2017; Zhang et al. 2018). This is further substantiated by the observed radioresistance in ROSlow CSCs in pancreatic adenocarcinoma (Li et al. 2015; Wang et al. 2017). The susceptibility of CSCs toward Sorafenib was shown to increase through the inhibition of fatty acid oxidation and restoration of oxidative phosphorylation in a mice model of hepatocellular carcinoma (HCC) thus emphasizing the importance of modulating the redox environment in overcoming the chemoresistance of HCC (Chen et al. 2016).

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In a study, glutamine deficit or the inhibition of inducible glutamine metabolism has shown to sensitize the pancreatic CSCs to radiation treatment in vitro and also in in vivo tumor xenograft models through the accumulation of intracellular ROS (Li et al. 2015). Higher resistance of CSCs was observed for both chemotherapy and radiotherapy. In a study, the survived glioblastoma cells after radiation treatment became highly resistant to irradiation, indicating that CSCs are not eliminated and thus contribute to self-renewal after the radiation therapy (Bao et al. 2006). In another study, the mechanism of higher resistance to radiotherapy in glioblastoma CSCs was demonstrated (Carruthers et al. 2018). The mechanistic basis of resistance of CSCs to DNA damage can be attributed to induction of antioxidant defense system, increased DNA repair potential or induction of crucial signaling pathways, such as PI3K/Akt, WNT/b-catenin, Notch, etc. (Peitzsch et al. 2013). The stimulation of ATM signaling was shown to increase the resistance to radiotherapy in the CD44+/CD24 /low CSC subset in breast cancer (Yin and Glass 2011). The isolated CD133+ cells of glioma tumors were found to have higher efficiency than CD133- tumor cells in repairing the radiation-induced DNA damage due to enhanced stimulation of the pathways of DNA damage and repair (Bao et al. 2006). The role of Notch signaling pathway in the radioresistance of glioma stem cells was demonstrated in a study wherein the inhibition of Notch pathway by gamma-secretase inhibitors (GSIs) sensitized the glioma stem cells to radiotherapy (Wang et al. 2010). In a recent finding, the nanoparticle-based encapsulation of a manganese(II)-3,4,7,8-tetramethyl-1,10-phenanthroline complex, 1 bearing a diclofenac showed cytotoxicity to CSCs and further showed the possible mechanism through ROS generation and COX-2 inhibition (Eskandari and Suntharalingam 2019).

Conclusion CSCs have stem cell-like properties and are found in all types of cancers and play a vital role for chemo- and radiation resistance and cancer recurrence. In order to survive in tumor milieu, CSCs adapt to themselves to low ROS levels through the modulation of antioxidant defenses. In addition, CSCs are able to demonstrate the numerous effects such as EMT, stimulation of signaling pathways which can regulate self-renewal or impact on the tumor environments. Recent study showed the epithelial (E)- and mesenchymal (M)-like CSCs extensively differ in its metabolic pathways, redox states, and sensitivities to metabolic/oxidant stress (Yang et al. 2020). As increased oxidant stress might potentiate the transition of quiescent M-CSCs to an E-like state through the over-expression of HIF1α and Nrf2 pathways that are further prone to the inhibition of redox metabolic pathways, particularly Nrf2-mediated antioxidant pathways; hence, the pro-oxidant-based therapeutic strategies along with the conventional cancer therapies may be a useful way in reducing the resistance associated with current cancer therapies. Also, the current therapeutic approach of combining drugs that are in particularly targeting the CSCs with traditional anticancer agents might be a better approach for anticancer therapy and

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PTEN

AKT NOTCH

mTOR ATM

FOXO

Chemotherapy Radiation therapy ROS

AKT

Antioxidant system

FOXO3 p53 HIF1 ATM Hypoxia

NRf 2 HIF-1

High survival

EGF

Quiescence

Increased Drug eff lux

Tumor

EMT

Cancer stem cell resistance

Enhanced DNA repair mechanism and anti-apoptotic pathway

Tumor recurrence Metastasis

Fig. 1 Reactive oxygen species’ (ROS) effects on cancer stem cells (CSCs). CSCs display low ROS levels due to increased expression of antioxidant systems. CSCs resistance to chemo- or radiation therapy is due to strong antioxidant defense system (through activation of Nrf2, upregulation of FOXO3, enhanced DNA repair mechanism, and activation of anti-apoptotic pathway). Prosurvival factor AKT is upregulated by Notch signaling. Tumor metastasis is due to HIF-1α induced EMT which is mediated by EGF

outcome. In addition, as normal stem cells and CSCs share the similar cell surface markers and signaling pathways, it is crucial to develop novel therapeutic agents targeting only CSCs without harming normal stem cells (Fig. 1).

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Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Relation Between Hypoxia and ROS in Development and Progression of Cancer . . . . . . . . . . . Computational Systems Biology in Revealing Complex Molecular Interconnectivity Network in Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . In Silico Approaches in Oxidative Stress-Induced Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Construction of Disease-Specific Interaction Network . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Identification of IIP and Bottlenecks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Construction of Signaling to Metabolic Crosstalk Pathway and Connectivity Analysis . . . Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cross-References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract

Oxidative stress refers to the pathophysiological condition when production of reactive oxygen species (ROS) saturates the intrinsic antioxidant defense mechanism. ROS have been shown to modulate diverse physiological processes including cellular signaling by acting as second messengers, hypoxic response pathways, inflammation, and immune response in mammalian cells. However, defects in antioxidant defense machinery contribute to elevated levels of ROS resulting in cytotoxicity and impaired cellular functions. In mitochondria, ROS are produced as an inescapable byproduct of oxidative phosphorylation, and hypoxia also promotes amplification of cellular levels of ROS. Hypoxia and oxidative stress-induced generation of ROS contribute to each step of carcinogenesis, starting from tumor formation to malignant transformation. This transS. Bose · K. Kumar · S. Chakrabarti (*) Structural Biology and Bioinformatics Division, Indian Institute of Chemical Biology, Kolkata, India e-mail: [email protected] © Springer Nature Singapore Pte Ltd. 2022 S. Chakraborti et al. (eds.), Handbook of Oxidative Stress in Cancer: Mechanistic Aspects, https://doi.org/10.1007/978-981-15-9411-3_158

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formation involves dysregulation of an enormous number of genes and proteins, involved in various pathways including the conventional hypoxia-inducible factor (HIF) pathway. Understanding the interconnectivity between signaling and metabolic pathways is indispensable for early disease detection and therapeutics to develop. System biology aids in this process by analyzing disease dynamics as an integrated system of genes, networks, and pathways to gain important biological insights. Network-based comprehensive analysis integrates multifaceted high throughput data to identify novel cancer biomarkers. Integration of network and pathway analysis can effectively detect molecules and pathways that get perturbed during tumorigenesis. Eventually, dynamic analysis of biological networks using systems biology approaches may provide useful information to gain better understanding of a multifactorial disease like cancer. Keywords

Hypoxia · Oxidative stress · ROS · Systems biology · Interaction network · Pathway analysis

Introduction Cancer is a heterogeneous disease having high morbidity and mortality rate with poor prognosis. Cancer cells display a broad range of metabolic phenotypes that are essential for them to survive and proliferate in highly selective microenvironment. Reactive oxygen species (ROS) are suggested as prime determinant of cancer’s metabolic phenotype (Kumari et al. 2017; Rodic and Vincent 2017). Oxidative stress is characterized by elevated levels of ROS that damage DNA, proteins, and lipids and cause genetic instability. Reactive oxygen species (ROS) are free radicals, ions, and molecules having a single unpaired electron conferring high reactivity. ROS are formed as natural by-products of normal cellular activity and take part in various intracellular signaling pathways to maintain cellular homeostasis. In cancer cells, high levels of ROS result from enhanced metabolic activity, cellular receptor signaling, oncogene activation, mitochondrial dysfunction, peroxisome activity, and increased activity of oxidases or through crosstalk with impregnating immune cells (Liou and Storz 2010; Szatrowski and Nathan 1991). Oxidative damage also occurs due to the noxious effect of prolonged systemic hypoxia which is characterized by low oxygen concentration and pressure in the cellular environment and caused due to low level of oxygen content and pressure in the blood, a condition known as hypoxemia, reduced in oxygen delivery and cellular oxygen uptake (MacIntyr 2014). During the initiation of a tumor, insufficient angiogenesis generates hypoxic areas of different intensity in which ROS increase, thereby satisfying an environment in which tumor cell survives and develops. The underlying mechanism of hypoxia inducing ROS has remained unclear, but from previous studies, it is evident that hypoxia contributes to ROS production through mitochondrial electron transport chain (ETC) (Tafani et al. 2016).

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Cancer is a multiprocess aberration where genetic, epigenetic, transcriptional, and protein deregulation occurs simultaneously. To detect these changes, we need to develop a combinatorial systemic approach by which the key mechanisms/regulators could be identified. It is rather difficult to identify and observe all those crossconnective pathways experimentally. Thus, analyzing multiomics data, such as genomic, transcriptomics, and proteomics data, may retrieve important genes, proteins, and regulatory pathways that are involved in cellular transformation, proliferation, and survival. Construction of disease-specific protein-protein interaction network may contribute toward identification of functionally significant proteins involved in that specific cancer scenario (Yu et al. 2008; Guruharsha et al. 2011). These biological networks follow the principles of graph and information theory (Lesne 2006). Thus, based on the graph theory certain proteins have been assigned as important interacting proteins (IIPs) that could be crucial in the particular disease condition. Nodes with maximum connectivity, higher centrality score, and higher global and local perturbation score are considered as important in a biological network. A node satisfying more than one of these criteria is termed as IIP (Bhattacharyya et al. 2015). Application of network biological approach and studying the interconnection between regulatory, signaling, and metabolic pathways may provide new insights in cancer research. It has been widely studied and accepted that cancer cells reprogram their metabolic status to achieve a more aggressive state that is considered as one of the new “hallmarks of cancer” that broadens the existing set of hallmarks (Hanahan et al. 2011). Understanding the coordination and interlinks between diverse cellular pathways, such as signaling, gene-regulatory and metabolic pathways are essential and may provide insights into the molecular machinery of metabolic transformations that are simultaneously occurring within cancer and associated cells. In order to understand the adaptation machinery, it is important to first investigate the underlying mechanism by which impact of gene-regulatory and signaling alterations is transgressed to metabolic reprogramming. To cope with the complexity of interconnected cellular pathways, efficient systems biological techniques need to be developed. Mathematical model-based system biology approaches have been successful for signaling and metabolic network analysis (Puniya et al. 2016; Samaga et al. 2013; Albert et al. 2014; Le Novère et al. 2015; Naldi et al. 2015). Signaling pathwayspecific mathematical models have been developed based on logical models (SaezRodriguez et al. 2011; Giacomantonio and Goodhill 2010), kinetic models (Schoeberl et al. 2002; Huang et al. 2010), decision tree (Hautaniemi et al. 2005), and differential equation-based models (Hughey et al. 2010). The different omics data viz., gene expressions, protein levels, and metabolomes in different cancer are integrated to study metabolic regulation in cancer (Hernández Patiño et al. 2013). Oxidative stress encompasses a variety of factors that interplay with diverse set of biological molecules and pathways, and standard approaches of studying a single gene or protein could not help to decipher the broader scenario of disease progression. Topological analysis of protein-protein interaction networks may help to identify the key genes/proteins and their molecular connections with central regulatory pathways. Pathway analysis in contrast can expand the current knowledge of hypoxia-induced metabolic rearrangements during cancer development.

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Relation Between Hypoxia and ROS in Development and Progression of Cancer In response to depleted levels of oxygen, cells up-regulate a highly conserved transcription factor HIF or hypoxia-inducible factor that regulates physiological and pathophysiological processes such as cancer. HIF is a highly conserved transcription factor that consists of a regulatory α subunit and a constitutively stable β subunit. The regulatory HIF-α subunit has three isoforms in humans, i.e., HIF-1α, HIF-2α, and HIF-3α, whereas two isoforms exist for HIF-β subunit (often referred to as ARNTs or Aryl hydrocarbon receptor nuclear translocators), e.g., HIF-1β and HIF-2β. HIF-1 and HIF-2 are regulators of oxygen homeostasis, although from previous studies it has been reported that HIF-3α suppresses hypoxia-induced gene expression in von Hippel-Lindau disease. During hypoxia, either HIF-1α or HIF-2α heterodimerizes with any of the HIF-β subunits to form functional HIF complexes (Graham and Presnell 2017; Raymaekers et al. 2015). In normoxic state, the proline residues of oxygen-dependent degradation domain (ODDD) in HIF-1α get hydroxylated by a family of prolyl hydroxylase enzymes (PHD). These hydroxylated prolines are recognized by an E3 ubiquitin ligase, Von Hippel-Lindau tumor suppressor protein (pVHL). The recognition component of pVHL instantly targets α-subunit of HIF1 for ubiquitin-mediated proteasomal degradation. But in case of hypoxia, ROS level increases via transfer of an electron from ubisemiquinone to molecular oxygen at the Q0 site of mitochondrial complex III. Mitochondrial ROS stabilizes HIF-1α by preventing its hydroxylation, which allows it to translocate to the nucleus followed by dimerization with HIF-1β. This dimerization promotes initiation of transcription of several downstream target genes of HIF1 (Fig. 1) (Bell et al. 2007a, b). Although mitochondrial ROS do not activate DNA damage response, they act as signaling molecule to stabilize HIF-1α and activate HIF-dependent increase in telomerase activity, thereby positively regulating replicative lifespan in human cells. Telomerase, the enzyme involved in maintaining telomere integrity, plays a crucial role in regulating replicative lifespan of human cells. hTERT or human telomerase reverse transcriptase, the rate-limiting catalytic subunit of telomerase, is one of the target genes of HIF. Thus, during hypoxia, stabilization of HIF-α increases hTERT and telomerase activity; this in turn provides growth advantage in cancer cells. Vascular endothelial growth factor (VEGF) and its receptor VEGFR1 are target genes of HIF, and VEGF has been shown to promote malignant cell proliferation. Thus, under hypoxic conditions, cells have the ability to accelerate their rate of cell division and initiate angiogenesis. The inherent resistance to apoptosis in malignant cells results due to the expression of an antiapoptotic protein known as survivin, which blocks caspase activation. In cervical cancer cells, survivin has shown to be a downstream target gene of HIF-α, illustrating another mechanism by which hypoxia induces tumor growth through the regulation of HIF-α (Macklin et al. 2017). HIF-1 acts as a transcriptional activator for a wide range of glycolytic enzymes; as a result, it interconnects an extensive range of metabolic and signaling pathways (Masson and Ratcliffe 2014; Seeza et al. 1994). Reactive oxygen species (ROS) are known to modulate several signal transduction pathways,

Fig. 1 A schematic diagram depicting the interconnectivity between hypoxia and ROS leading to cancer. Under normoxia, HIF-1α is hydroxylated by prolyl hydroxylase enzyme (PHD) and undergoes Von Hippel-Lindau tumor suppressor protein (pVHL)-mediated proteosomal degradation. During hypoxia, ROS generation is amplified within mitochondria via transfer of electrons at the Qo site of complex III, from ubisemiquinone to molecular oxygen. Elevated levels of ROS prevent PHD-mediated hydroxylation of HIF-1α, thereby inhibiting degradation of the same and resulting in heterodimerization with HIF-1β to form the functional HIF complex. The HIF complex interacts with p300/CBP coactivators and then binds to HREs (hypoxia-response element) leading to transcription of its downstream genes that are responsible for cell survival, growth, and metastasis

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including adaptation to hypoxia, and promote cellular survival through pathways such as nuclear factor-κB (NF-κB) (Gorrini et al. 2013; Waypa et al. 2010). With the enormous number of pathways associated with development and progression of cancer induced by various oxidative stresses, it becomes too obscure to distinguish the principal pathways or regulators that are expressing predominantly. Hence, integrative systems biology approach may be beneficial in recognizing key regulators from the plethora of pathways and molecules. This approach combines largescale data collection and employs mathematical and statistical analysis, followed by experimental validation. Systems biology approaches may lead to identification of novel players and regulators which could be utilized as therapeutic targets involved in ROS-mediated progression of cancer.

Computational Systems Biology in Revealing Complex Molecular Interconnectivity Network in Cancer Oxidative stress pathways that are involved in cancer have been extensively discussed earlier. But it is still unclear how they are associated with poor prognosis of the disease and if their regulation is cancer-type specific. Computational biology combines systems biology along with network biology approaches leading to better understanding of the complex interconnectivity between various genes, proteins, and regulatory elements such as noncoding RNAs. Application of systems biology in cancer research has been helpful to identify various novel marker genes or proteins and regulatory pathways that are involved in disease advancement. In this section, we will discuss some of the existing works that involved application of bioinformatics and systems biology to study oxidative stress inducing cancer in different tissues or organs and will adjoin some newer approaches that are not being discussed yet. As described by Leone et al. (2017) for addressing the two-faced effects of ROS, bioinformatics and systemic approach could be used to figure out the connection between oxidative stress and cancer stem cells so as to define druggable targets (Leone et al. 2017). In this study, they have compared overall survival of cancer patients and mRNA levels of 73 oxidative stress signature genes including glutathione peroxidases (GPx), genes associated with ROS metabolism (i.e., DUSP1, FoxM1, and HMOX1), and genes that encompasses in superoxide metabolism, such as superoxide dismutase (SOD). RNA expression profiles and DNA mutational status of genes involved in different type of solid tumors were extracted from The Cancer Genome Atlas (TCGA) datasets and were analyzed using SynTarget and PPISURV programs. SynTarget is an online tool that is able to test the cumulative effect of two genes on survival outcome, and therefore, it can identify gene pairs with synergistic effects (Amelio et al. 2016). On the contrary, PPISURV (URL: http:// www.bioprofiling.de/PPISURV) is a data-mining tool that employs several publicly available databases including protein–protein interactions, regulatory and signaling pathways, and protein posttranslational modifications to extract the query genespecific interactome (Antonov et al. 2014). Both SynTarget and PPISURV are

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incorporated into BioProfiling.de, an analytical web portal for retrieving highthroughput cell biology data (URL: http://bioprofiling.de/). PPISURV derives interactome of a gene and correlates its expression levels with survival outcomes from publicly available clinical expression datasets. Thereby it evaluates deregulation of a particular gene and is either associated with positive or negative survival outcome. In this study, the authors established that a significant number of oxidative stress genes are correlated negatively with survival in solid carcinomas, such as breast, lung, and HNSCC, emphasizing the concept that oxidative stress plays a pivotal role in cancer progression. From this analysis, two ROS metabolism genes, FoxM1 and TXNRD1, are appeared to be significantly high for poor prognosis patients in most of the tumor types. Further, in order to identify more admissible oxidative stress markers, an additional analysis was carried out by mapping all modulated genes, irrespective of their expression profiles, on the protein-protein interaction database STRING (Szklarczyk et al. 2019). This revealed an enriched cellular network, including GPx, SOD, and Trx pathways (including TXNRD1), which are highly correlated. TXNRD1 encodes thioredoxin reductase 1(TrxR1), which is a component of thioredoxin system that recruits several redox enzymes and signaling proteins to modulate the redox status by scavenging ROS, in response to oxidative stress (Arnér and Holmgren 2006). Another component of thioredoxin system Trx1 (Thioredoxin) is known to be a serum marker for breast cancer and also known to modulate transcription of cyclooxygenase-2 (COX-2) via HIF-1α in nonsmall cell lung cancer (Park et al. 2014; Csiki et al. 2006). Collectively, by synchronizing some bioinformatics tools and databases, a detailed idea about involvement and interconnectivity between pathways could be established more comprehensively.

In Silico Approaches in Oxidative Stress-Induced Cancer System biology focuses on the broader scenario of the dynamic interactions among various biological molecules. Implication of systems biology approaches using large-scale “omics” data will facilitate our understanding of complex biological or cellular activity networks. In these networks, the nodes represent biomolecules and edges denote different type of physical interactions, starting from protein-protein interaction, transcriptional regulation, microRNA-mRNA regulation, and enzymesubstrate reactions. Network representation is carried out to simplify the complexity of interactome data and to focus precisely on the biomolecules and their interacting partners. In this study, we performed a systemic analysis approach to showcase the power and utility of the approach in discerning complex molecular relationship pattern that emphasizes in pathophysiological condition (Fig. 2). Accumulating evidences suggest that dysregulation of hypoxia-regulated transcriptional mechanisms is crucial for adaptation of cancer cells to the hypoxic microenvironment which leads to modifications in its cellular bioenergetics machinery, an event known as metabolic reprogramming. This phenomenon contributes to tumor development as well as malignant transformation. Metabolic and genetic heterogeneity has also

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Fig. 2 Schematic representation of the steps involved in network and pathway analysis. The left panel describes workflow of constructing disease-specific protein-protein interaction network and important interacting protein (IIP) analysis resulting in IIP (Important interacting protein), BN (Bottleneck), CP (Central protein), GNPP (Global network perturbation protein), and LNPP (Local network perturbation protein). The right segment refers to the signaling (S) and metabolic (M) crosstalk pathway connection construction and analysis protocol using HMM (Hidden Markov model)

been observed within and across a particular type of cancer (Yoshida 2015). Development of therapeutics from individual knowledge of signaling and metabolic pathways is difficult. To investigate the heterogeneity and pathway interconnectivity within a particular cancer type, we have performed a comparative genome-wide expression profile analysis of three different breast cancer transcriptomics datasets (GSE37340, GSE47533, and GSE89891, respectively) collected from different

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geographic locations. Breast cancer is the most frequent and lethal type of cancer in women in more developed regions, although mortality rates are increasing globally. In the dataset GSE37340, MDA-MB-231 breast-cancer cells were exposed to oxygen and glucose-deprived condition for a prolonged period (5 weeks), whereas in the other two datasets, GSE47533 and GSE89891, MCF7 cell line was kept in the hypoxic microenvironment for several hours in increasing order. For GSE47533, cells were kept under hypoxia for 16 h, 32 h, and 48 h, while in case of GSE89891 cells were exposed to 1% O2 for 4 hours and 24 hours (https://www.ncbi.nlm.nih. gov/geo/query/acc.cgi?acc¼GSE37340; Camps et al. 2014; Ho et al. 2017). Gene expression profiles were extracted using an R-package-based analysis tool. Moreover, systems-based context-specific network study was carried out to reveal the key interactors and pathways that are involved in disease development under hypoxic stress response. Network and pathway analysis leads to identification of novel marker genes that could have major footprints contributing to malignancy. The detailed method of network construction and analysis is discussed in the following.

Construction of Disease-Specific Interaction Network Construction of disease-specific network was carried out for the three different datasets where cells were treated with low O2 concentration (1% O2) representing hypoxic environment. Raw transcriptomic data was normalized to obtain differential gene expression using an R-based dataset analysis tool, GEO2R analyzer (https:// www.ncbi.nlm.nih.gov/geo/geo2r/). For retrieving the normalized gene expression values of GSE37340, MDA-MB-231 WT normoxia samples were treated as control groups, and MDA-MB-231 WT hypoxia samples were assigned as diseased group. Likewise, for GSE47533, four separate time point-specific comparative studies were performed. MCF7 cells treated with 21% O2 for 48 hours were assigned as control group, and four disease groups were assigned to samples treated with 1% O2 for 16, 32, and 48 hours considering all of them as a single disease group. Similarly, for GSE89891, MCF7 exposed to 24 hours of normoxia was considered as control, and exposures of 4 hours and 24 hours of hypoxia were assigned as two different disease groups, respectively. The normalized data was further filtered using a cutoff P value 0.05. Genes having log fold change (Log FC) value 1 were considered as upregulated, genes having LogFC value  1 were considered as downregulated, and those having LogFC from < +1 to > 1 were considered to be expressed. Upand downregulated genes were collectively called as deregulated genes. Human protein-protein interaction (HPPIN) data with confidence score  700 was extracted from STRING interactome database. The disease-specific protein-protein interaction network was generated by mapping the deregulated and expressed genes onto HPPIN. Network construction up to second level interaction and topological analysis was carried out using in-house computational programs. Network visualization and representation was done using Cytoscape (version 3.7.1) (Fig. 3a) (Shannon et al. 2003).

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Fig. 3 (a) Representation of protein-protein interaction network of GSE47533 (16, 32, and 48 hours of hypoxic exposure compared to 48 hours of normoxia). (b) Second-level interaction network of HIF1A

Identification of IIP and Bottlenecks Important interacting protein (IIP) is broadly classified into four categories, i.e., Hub, CP, GNPP, and LNPP. Nodes, i.e., genes/proteins satisfying more than one category, are termed as IIP. Hubs are the nodes that have maximum degree or connectivity and thus may play critical role in the regulation of network. For identifying hubs, all the degrees were normalized to z-score, and the fraction of nodes having z-score  1 was considered to have significantly higher degree than others in the respective network. Bottlenecks (BN) are characterized based on their high betweenness values that connect different functional clusters within a network and also maintain dynamics of the network. The proportion of number of shortest paths passing along a node to the number of total shortest paths in the network is termed as betweenness of a node. Betweenness calculates the frequency of a node to act as a connector along the

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shortest paths between two other nodes or clusters. Thus, bottlenecks have great influence on the information flow of the network, and removing a bottleneck can cause disruption of the network. CP or central proteins are selected based on several centrality parameters like high betweenness (the number of shortest paths going through a certain node), high clustering coefficient (the degree of nodes in a network tends to congregate together), high closeness, high stress, and high eccentricity along with low radiality. An in silico perturbation analysis was carried out at the subgraph level to investigate important nodes that are required to conserve network integrity. By considering each node and its second level interactor as an independent network, a transient local subgraph was created. Perturbation potential of each node was estimated by calculating the change in network centrality before and after perturbation of a node from the network, which is termed as global network perturbation score (GNPS). Similarly, the same scoring criterion is followed for the local networks to calculate local network perturbation score (LNPS). Nodes having higher GNPS than others were calculated by statistical z-score analysis and are termed as GNPP or global network perturbation protein. Likewise, nodes having higher LNPS than others were termed as LNPP or local network perturbation protein. Incorporation of network parameters, e.g., hubness, centrality, and perturbation potential led to recognition of such nodes that are critical for overall integrity of the PPIN. Here, in the comparative analysis of three different breast cancer datasets, we have found five topologically important genes that are being expressed in two of all three datasets (Table 1). Two of them are associated with protein metabolism (NUP155 and IKBKG), two correspond to nucleic acid binding (SLU7 and RBPMS), and one is involved in DNA-repair machinery (GPS1). Enolase 2 or Gamma-Enolase (ENO2) is found to be an IIP (including properties such as CP, GNPP, and LNPP) in GSE37340, which actively participates in carbohydrate metabolism. Time point-dependent analyses in GSE47533 have identified 68 hubs, 25 CP, 4 GNPP, 2 LNPP, 5 IIP, and 7 BNs that are getting expressed throughout 16–48 hours of hypoxic treatment. HIF1A is found to physically interact with CREBBP (CREB Binding Protein) that appears to be a BN and is involved in the transcriptional coactivation of many different transcription factors that are Hub, BN, and GNPPs in the respective PPIN (Fig. 3b). Genes involved in ERK pathway and lung cancer development (RALBP1), transcriptional regulation of androgen receptor (HMG20B), Wnt signaling (VPS35), and thyroid cancer development (TRIM33) along with mTOR, BCL7, MCM7, CASP8, and CUL2 are expressed as bottleneck in the PPIN. Another time point-dependent study of GSE89891 revealed 1 hub (PPP2CA), 2 CP (MOB4, HLA-DRA), 1 GNPP Table 1 List of genes common in three different datasets and their network property Gene name SLU7 NUP155 GPS1 RBPMS IKBKG

Network property Hub GNPP GNPP LNPP BN

GSE37340 Expressed – – – –

GSE47533 Expressed Expressed Expressed Expressed Expressed

GSE89891 – Expressed Expressed Expressed Expressed

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(GAPDH), and 2 bottlenecks (PPP2CA, UBC) to be expressed thoroughly in 4 and 24 hours of hypoxic treatment. ENO1 and ENO2 were identified as CN and LNPP, respectively, for the 4 hour treatment PPI, whereas mTOR and GAPDH appeared to be crucial (IIP) in 24 hours of treatment group.

Construction of Signaling to Metabolic Crosstalk Pathway and Connectivity Analysis In search of promising drug targets and to develop more precise therapeutics, we need to understand the highly dynamic interconnectivity between different oncogenic signaling and metabolic pathways. To understand the interconnectivity between signaling pathways and metabolic pathways, signaling-metabolic pathway cross-connectivity was established by using all the genes/proteins of 23 signaling pathways (cancer-specific) and all the metabolic pathways available at KEGG database (Kanehisa and Goto 2000). All possible unique connections (maximum three proteins involved in between) to a metabolic pathway protein (M) were established using PPIs (up to second level), considering a signaling pathway protein (S) as a starting point in the HPPIN. Four different types of linking paths were established where signaling proteins were connected to metabolic pathway proteins either directly (S-M) or via one (S-P-M), two (S-P-P-M), or three (S-P-P-P-M) PPIs, respectively. The resulting signaling-metabolic cross-connecting paths/connections/ links were consisted of 210 direct (S-M) connections, 2669 via one PPI (S-P-M), 40266 via two PPIs (S-P-P-M), and 735395 via three PPIs (S-P-P-P-M) interconnections. These interconnections/paths/links were formed between 210, 1697, 7965, and 28920 signaling metabolic pathway protein (S-M) pairs, respectively. These paths/connections/links were converted into network to construct signaling-metabolic interaction network (SMIN) (Table 2). Further differentially expressed genes (i.e., upregulated, downregulated, and simply expressed) identified from transcriptomics data (GSE47533) of hypoxia-induced cancer cell line were mapped to extract context-specific cross-connected paths/links. The paths/connections/links having deregulated (upregulated and downregulated) genes/proteins at the terminals and deregulated or expressed genes/proteins in middle were filtered out and considered for additional analysis. The resulting paths/connections/links were consisted of 4 (S-M), 11 (S-P-M), 33 (S-P-P-M), and 89 (S-P-P-P-M) paths formed between 4, 9, 16, and 26 S-M pairs, respectively. The filtered paths were converted into network to construct the cervical cancer-specific SMIN (C-SMIN) network. The C-SMIN consisted of 108 interactions forming among 72 genes/proteins (Table 2 and Fig. 4a). To evaluate the biological and functional relevance of signaling to metabolic pathway interconnections, weights based on biological properties, differential expression, and network topological properties were assigned on each nodes and edges in the context-specific paths/links. Edge weight was assigned in terms of normalized gene expression value, and node weight was assigned based on their biological properties including signaling crosstalk genes (SC), rate-limiting enzyme (RLE), deregulated genes (up- and downregulated), and network topological properties

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including Hub, CP, GNPP, and LNPP. Node weights and edge weights were incorporated into a Hidden Markov model (HMM) based mathematical models to calculate the score of each path/link (Bag et al. 2019). Two separate models viz. model1 and model 2 were used to identify the significant S-M pairs and S-M paths, respectively. Model 1 was applied to identify the S-M pairs. Model 2 was applied to identify the S-M interconnecting paths between the S-M pairs selected after model 1. The path score of each S-to-M linking path calculated by model 1 and model 2 was converted into statistical z-score to identify paths deviating from the mean. A z-score  1 filter was applied to select the significant S-M pairs. Paths having path score  80% of the highest path score for every S-M pair were considered as significant S-M interconnecting paths from model 2. Using the above-mentioned threshold, a total of nine S-M pairs and nine S-to-M interconnecting paths/links were identified. The resulted S-M interconnecting paths were converted into network to form cancer-specific significant signaling metabolic interaction network (C-SMIN) (Table 2 and Fig. 4b). The significant paths/links obtained consisted of two paths/ links, where signaling pathway proteins were connected to metabolic pathways proteins directly (S-M type), four paths/links via one PPI (S-P-M type), one via two PPIs (S-P-P-M type), and two via three PPIs (S-P-P-M type), respectively. Seven signaling pathways proteins (ALDOA, GYS1, ENO2, VCL, TNF, REL8, and RUVBL1) were found to be linked with eight metabolic pathways proteins/ enzymes (ALDOC, GBE1, GAPDH, PGK1, PGAM1, CKB, MPI, and POLR3B). Three upregulated signaling pathway proteins (ALDOA, GYS1, and ENO2) were linked to five upregulated metabolic enzymes (ALDOC, GBE1, GAPDH, PGK1, and PGAM1) forming a total of five paths (two direct path/links and three linked via one PPI). Two paths/links were identified, where both the signaling pathway proteins (REL8 and RUVBL1) and metabolic enzyme (POLR3B) were downregulated and two paths were identified where signaling pathways proteins (VCL

Table 2 Signaling to metabolic interconnecting paths and pairs Pairs and paths

Connection types S-M S-P-M S-P-P-M S-P-P-P-M Total Interactions Total genes/ proteins

Signalingmetabolic interaction network (SM1N)

Cancerspecific SMIN (C-SMIN)

Significant C-SMIN

Pairs 210 1697 7964 28920 29179 11242 2603

Pairs 4 9 16 26 28 108 72

Z  1 pairs 1 1 1 7 9 19 27

Paths 210 2649 40266 735395 778540

Paths 4 11 33 89 137

Mode1 pair selection

Model2 paths selection Z  1 or Score  80% paths 2 4 1 2 9

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Fig. 4 Signaling to metabolic pathways interconnecting paths/links. Panel A represents the signaling metabolic interaction network in hypoxia-induced breast cancer cell line. Panel B shows the significant (path score  80%) signaling to metabolic proteins interconnecting paths. Nodes represented as blue, orange, and purple are signaling proteins, protein-protein interactors, and metabolic proteins, respectively. Panel C circos plot represents the connections/links between signaling pathways (purple font) and metabolic pathways (black font)

and TNF) were downregulated and metabolic enzymes (CKB and MPI) were upregulated (Fig. 4b). In order to identify which signaling pathways made maximum connections with which type of metabolic pathways in hypoxia-induced cancer cell line, the pathways information was mapped to the terminal nodes (source signaling protein and

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destination metabolic enzyme) of significant S-M paths/links. A total of ten signaling pathways and three metabolic pathways were mapped to the identified paths/ links in hypoxia-induced cancer cell line. The signaling pathways mapped were HIF1 (hypoxia-inducible factor 1), NFκB (nuclear factor κ B), MAPK (mitogenactivated protein kinase), Wnt (wingless-type), TGFβ (transforming growth factor β), mTOR (mechanistic target of rapamycin kinase), Cytokine receptor pathway, apoptosis, AKT (protein kinase B) pathway, and Adh. (adherence junction) pathways. However, carbohydrate metabolism, nucleotide metabolism, and amino acid metabolism were metabolic pathways mapped to the identified S-M paths. HIF-1 signaling pathway had maximum connections (7 links) to carbohydrate metabolic pathways (6 links) and amino acid metabolism (1 link). However, among metabolic pathways, carbohydrate metabolism had maximum connections (11 links) with signaling pathways, followed by nucleotide metabolism (3 links) and amino acid (2 links). 1:1 interconnection between signaling and metabolic pathways showed that the HIF-1 signaling pathways had maximum connections with carbohydrate metabolism (6 connections) (Fig. 4c).

Discussion In order to understand the cumulative effects of oxidative stress and hypoxia to transform a healthy cell into carcinogenic one, evaluation of complex network of signaling and metabolic pathways and mutations leading to alterations in network dynamics are indispensable. Since these networks are typically complex involving a diverse range of signaling molecules and pathways in different stages of the disease, it is essential to represent them in the form of a computational model that can be used for a meticulous study. Tissue, cell, and stage-specific high-throughput data collection drives better understanding of biological networks and also opens the possibility of discovery-oriented approach. Effective combinatorial drug prediction could also be achieved by integrating mathematical models to measure the modulations in oncogenic pathways. Comparative analysis of IIPs within three different breast cancer datasets revealed certain topologically important genes that are associated with protein metabolism. Cancer cells need a constant supply of amino acids to maintain the proliferative drive, and protein metabolism serves in this aspect as a source of amino acid production. Genes/proteins involved in carbohydrate metabolism, such as GAPDH, ENO1, and ENO2, also emerged to be critical in terms of network properties. HIF-1 showed connectivity with different hub and bottlenecks along with maximum pathway connections with carbohydrate metabolism.

Conclusion This chapter discusses about construction of disease-specific human protein-protein interaction networks (HPPI) and graph-theory-based topological analysis of those to reveal key regulators that are involved in disease progression. Mathematical model-

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dependent signaling to metabolic crosstalk pathway analysis further facilitates detection of a number of pathways that are associated with oxidative stress-induced cancer formation and development. To summarize, cancer is a heterogeneous disease integrating different gene/proteins and pathways across different stages and tissue types that requires identification of a finite number of genes. These genes are central to the proper biological processes that are involved in disease development and which, if altered, can lead to pathological conditions. To distinguish these genes from a complex disease network, we have proposed a systemic approach that combines information of context-specific gene-gene (protein-protein) interactions and the functional pathways that can interpret the driver genes/proteins and pathways involved in disease progression.

Cross-References ▶ Analytical and Omics Approaches in the Identification of Oxidative StressInduced Cancer Biomarkers ▶ Proteomics and Metabolomics in Cancer Diagnosis and Therapy ▶ System Biology and Network Analysis Approaches on Oxidative Stress in Cancer ▶ Systems Biology and Bioinformatics Insights into the Role of Free RadicalMediated Oxidative Damage in the Pathophysiology of Cancer ▶ Systems Biology Resources and Their Applications to Understand the Cancer

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Amelioration by Botanicals N. A. Chugh and A. Koul

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Involvement of ROS in Progression and Metastasis of Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sources of ROS in Cancer Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mechanisms Involved in ROS-Mediated Cancer Progression and Metastasis . . . . . . . . . . . . Botanicals Inhibit Cancer Progression and Metastasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract

Reactive oxygen species (ROS) are involved in all stages of cancer and therefore their levels have a profound effect on the outcome of this disease. ROS levels during cancer are enhanced by both cancer-associated and environmental factors. The sources of ROS in cancer cells include: increased metabolic activity, altered mitochondrial energetics, chronic inflammation, altered signaling, hypoxia, oncogene activation, etc. It has been observed that cancer cells survive by manipulating ROS production, i.e. by enhanced production of antioxidants or by boosting the ROS production in order to maintain ROS levels that provide sustenance for tumor cell proliferation and survival. ROS facilitates cancer progression and metastasis via several ways including activation of transcription factors, triggering of epithelial to mesenchymal transition, modification of extra cellular matrix, inducing resistance to anoikis, promotion of angiogenesis, development of a conducive tumor microenvironment to enable field cancerization, etc. Progression and metastatic spread of this disease has been linked to poor prognosis and morbidity. Botanicals have been widely used for their curative properties since eternity and are still used popularly. Their beneficial effects in cancer have been observed in epidemiological, animal, and human studies. Owing to the presence N. A. Chugh · A. Koul (*) Department of Biophysics, Panjab University, Chandigarh, India e-mail: [email protected] © Springer Nature Singapore Pte Ltd. 2022 S. Chakraborti et al. (eds.), Handbook of Oxidative Stress in Cancer: Mechanistic Aspects, https://doi.org/10.1007/978-981-15-9411-3_160

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of multiple components, botanicals are able to affect all stages of cancer development. Inhibition of ROS production is considered as one of the important strategies to inhibit spread of cancer. This chapter illustrates the role of ROS in cancer progression and metastasis and the potential of botanicals in inhibiting it. Keywords

Reactive oxygen species · Cancer · Botanicals · Phytochemicals · Progression · Metastasis

Introduction Tumor development is a complex process which has been divided into three stages. The first is the initiation stage which is followed by the promotion and progression stages. The stages of cancer have been studied by using animal models of chemical carcinogenesis. These models have been reviewed extensively in literature (Kemp 2005; Oliveira et al. 2007). The initiation stage involves irreversible genetic changes that have the propensity to direct the cells toward an oncogenic fate. Although genetic alterations exist in the initiated cells, the phenotype remains unaltered. DNA damage kick-starts the initiation of the carcinogenic cascade. Several endogenous and exogenous agents can cause oncogenic alterations, especially if the damage occurs in oncogenes, tumor suppressor genes, and DNA repair genes. If the genetic alterations persist in these cancer-related genes and the cells undergo division, they can produce daughter cells with the same altered (damaged) genetic makeup. The initiated cells harboring potentially oncogenic mutations can remain dormant for weeks, months, or years or they can continue to divide and proliferate. The clonal expansion of the initiated cells is an effect of the ensuing mitosis and apoptosis inhibition. However, it is of importance to understand that not all cells exposed to initiating agents/events will be initiated even if they have mutations in cancer-associated genes. The promotion stage is responsive to the presence of physiological and environmental conditions. Promotion is considered a reversible stage because disappearance of a promoting agent can regress cell proliferation, thereby limiting cancer development. The promoting agents mainly act by enhancing cell proliferation in the susceptible (initiated) cells. They do so by fixing the mutations and modifying gene expression of the initiated cells. Therefore, promoting actions allow for increased cellular growth and survival of the initiated clone. Unlike the initiating agents, promoters do not have genotoxical and mutational activity. The mitogenic activity of the promoters serves to decrease the latent period by activating the initiated quiescent cells into mitogenically active state. Exposure of cells to promoters does not ascertain their involvement in promotion stage because only those cells that have been mitogenically stimulated to divide, are undifferentiated, and have evaded apoptosis can contribute to the imbalance between cell division and cell death, thereby leading to the appearance of pre-neoplastic lesions/benign neoplasia.

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The lesions appearing between initiation and promotion stages are considered as pre-neoplastic lesions (benign neoplasias). Their transformation into malign forms is the progression stage and is the most extended stage of the carcinogenic cascade. Several genetic and epigenetic mechanisms are involved in the acquisition of the malignant/neoplastic phenotype. During this stage the aberrant cell proliferation is free of any control exerted by the presence of mitogenic stimuli. This stage is characterized by irreversibility, genetic instability, dysplasia, hyperproliferation, local and distant spread, and a range of biochemical, metabolic, and morphological changes in cells. The progression stage involves the conversion of benign tumors into malignant neoplasms capable of invading adjacent tissues and metastasizing to distant sites. Metastasis has been recognized as the major cause of cancer-associated morbidity and mortality. It occurs through a series of coordinated steps. Cancer cells detach from the primary site, enter into the circulatory and lymphatic system, dodge the immune system, and settle and proliferate at sites in distant organs. These metastatic cells form a favorable microenvironment that enable their proliferation, survival, and angiogenesis which results in formation of secondary tumors (Seyfried and Huysentruyt 2013). Reactive oxygen species (ROS), as the name implies, are oxygen derivatives which are unstable, reactive, and are produced as by-products of normal metabolism and also in conditions of disease and pathology. In normal functioning of the body ROS play important roles in immune defense mechanisms, autophagy, inflammation, stress responses, and several signaling pathways. However, the uncontrolled generation and insufficient scavenging of these reactive species can lead to oxidative stress, cellular toxicity, disturbed cellular functions, and development of diseases like cancer. Cancer cells have elevated levels of ROS which result from defects in ROS production and their removal. ROS are involved in all stages of cancer and therefore their levels have a profound effect on the outcome of this disease (Fig. 1). Inhibition of ROS production is considered as one of the important strategies to inhibit spread of cancer. This chapter illustrates the role of ROS in cancer progression and metastasis and the potential of botanicals in inhibiting it.

Involvement of ROS in Progression and Metastasis of Cancer Sources of ROS in Cancer Cells During cancer development, ROS levels are increased by both cancer-associated and environmental factors (Chio and Tuveson 2017; Yang et al. 2018). Environmental agents linked to cancer etiology such as UV radiation, smoking, and chemical carcinogens are known to increase ROS production in cells. The biochemical processes underlying the activation of carcinogens by carcinogen metabolizing enzymes also involve the production of ROS. Since ROS are obligatory by-products of metabolism, therefore increased metabolism supporting hyper proliferation of cancer cells further contributes to the load of ROS produced. The activation of oncogenes such as C-myc, K-ras, and BRCA1 is also accompanied

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Fig. 1 Involvement of ROS in carcinogenesis. There is an imbalance in ROS production and removal in cancer cells. Several types of ROS species are generated in response to exogenous and endogenous factors. These reactive species affect all stages of cancer development (Kumari et al. 2018)

by an increase in ROS production (Tanaka et al. 2002; Cao et al. 2007). Cancer cells have elevated expression of membrane-associated nitric oxide synthase (NOX) which is a major contributor of extra cellular ROS. Oncogenic K-ras is known to enhance the activity of NOX. Hypoxia in growing tumors is another major source of ROS production. The altered signaling during cancer metastasis is also linked to increased ROS production. Cancer cells mount their defense against elevated ROS by soaring their activity of membrane-associated catalase and superoxide dismutase. Impaired signaling of transforming growth factor-β (TGF-β) suppresses the function of glutathione peroxidase, enhances mitochondria linked intracellular ROS production and NOX-mediated extracellular ROS production. This leads to the enhanced production of H2O2 and its accumulation in the tumor microenvironment (TME). Cancer-associated fibroblasts (CAFs) in comparison to normal fibroblasts exhibit high production of intracellular and extracellular ROS. Tumor-associated macrophages which originate from the circulating blood monocytes also release ROS.

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Mechanisms Involved in ROS-Mediated Cancer Progression and Metastasis During carcinogenesis, ROS produced intracellularly participate in signaling pathways that enable acquisition of cancer-associated features while extracellular ROS assist in propagating, amplifying, and effectively creating an area for the formation of multifoci tumors and spring board for metastatic tumor cells (Liao et al. 2019). ROS has the potential to affect cellular macromolecules with carcinogenic consequences. Additionally, ROS facilitates cancer progression and metastasis via several ways (Fig. 2). They may catalyze the formation of a modified DNA base (8-OHdG) in cancer-associated genes, increasing the likelihood of mutations consequently initiating carcinogenesis (Dizdaroglu and Jaruga 2012). In early stages of cancer, intracellular ROS potentiate cancer initiation by causing oxidative damage to DNA and base pair substitution mutations in tumor suppressor and oncogenes such as p53 and Ras. ROS can attack membrane/cellular lipids leading to the formation of unstable, short-lived lipid intermediates that may act as secondary messengers. This attack on lipids may lead to membrane damage and altered signaling. Aberrant disulfide bond formation in proteins can also occur in response to ROS (Valko et al. 2016). As cancer progresses and ROS continues to be produced, apoptosis can be triggered; however, this is avoided as the cells begin to produce high levels of intracellular antioxidants. In the late stages, extracellular ROS create an environment for the successful dissemination of cancer cells. ROS is also involved in causing tumor promoting inflammation and hyperproliferation. Pro-inflammatory transcription factors such as NF-κB are activated by ROS, thereby resulting in the transcriptional activation of downstream genes involved in cancer spread. NF-kβ regulates the expression of genes which contribute to tumorigenesis, such as inflammatory, anti-apoptotic, and positive regulators of cell

Fig. 2 ROS facilitates cancer progression and metastasis via several ways

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proliferation and angiogenesis (Mitisiades et al. 2002). The high levels of ROS in the primary sites activate the matrix modifying enzymes such as matrix metalloproteinases (MMPs) by triggering signaling pathways involving redox sensitive transcription factors such as mitogen-activated protein kinases (MAPK), activator protein- (AP-1), NF-κB, etc. These activated pathways then initiate the migration processes. Studies using ROS inhibitors also lend support to the view of ROS facilitated metastasis of cancer (Kashyap et al. 2019). In cell lines such as MCF10 and MDAMB-231, the TGF-β induced epithelial to mesenchymal transition (EMT) is dependent upon ROS production by NOX-4 (Zhang et al. 2013). The cross-talk between ROS and NF-κB is crucial in TGF-β1-mediated induction of MMPs and urokinase plasminogen activator during cancer (Tobar et al. 2010; Kashyap et al. 2019). EMT is a complex process that promotes tumor progression. It involves alterations in cell junctions, morphology, cytoskeleton, expression of receptors, fibroblastic markers, etc. that enable the cancer cells to detach from their primary site and invade nearby and distant tissues. Studies have revealed that EMT is under a redox control and pro-oxidant conditions induce EMT (Lamouille et al. 2014; Clark and Vignjevic 2015). ROS production regulates TGF-β which in turn is a vital factor governing EMT (Saitoh 2018). MMPs play an indisputable role in cancer invasion and metastasis. These enzymes degrade collagen in the extracellular matrix (ECM) which helps in cell detachment and invasion at new sites. Increased expression of these enzymes is predictive of poor prognosis, tumor progression, and metastasis. MMPs are also involved in triggering EMT which assists in cancer spread. Activation of IntegrinRac pathway in cancer cells generates ROS resulting in tumor cell migration and invasion. Rac signaling is involved in cytoskeletal rearrangement which helps in cancer cell motility. Increased production of ROS and upregulated expression of Rac1b is involved in MMP3-mediated EMT (Radisky et al. 2005; Kumari et al. 2018). The cross talk between ROS and other pathways mediated by TGF-β1, nuclear factor (erythroid-derived 2)-like 2 (Nrf2) lead to accelerated EMT (Liao et al. 2019). ROS-mediated increase in MMPs and cathepsin expression contributes to tumor migration. The presence of inflammation in the TME affects cellular proliferation, survival, angiogenesis, local and distant spread, response to therapy, etc. Studies suggest that over 25% of cancers in humans result from sustained inflammation. Apart from the cancer cells, a number of other cell types including fibroblasts, myeloid-derived suppressor cells (MDSCs), macrophages, neutrophils, cancer stem cells, and CAFs are present in the TME. These pro-inflammatory cells produce factors such as TNF-α, TGF-β, interleukin-6 (IL-6), and interleukin-1 (IL-1) which activate transcription factors such as NF-κB and signal transducers and activators of transcription-3 (STAT3) which in turn trigger the EMT transdifferentiation program and the activity of matrix modifiers (Wu and Zhou 2009). In healthy cells, apoptosis is induced after the cells lose cell matrix attachment. This process is known as anoikis. However, in executing metastasis, survival of cancer cells in absence of their attachment to the ECM is crucial. ROS promote resistance to anoikis and apoptosis (programmed cell death) inhibition in cancer cells thereby aiding in survival of matrix detached cancer cells (Douma et al. 2004;

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Schafer et al. 2009). In cancer cells, angiopoietin-like 4 (ANGPTL4) in association with integrin stimulates the production of ROS leading to the activation of PI3K/Akt and extracellular signal-regulated kinases (ERK) pathway which confers resistance against anoikis. The upregulation of growth factors mediated by ROS such as epidermal growth factor (EGF) also contributes to anoikis inhibition and cell survival. Angiogenesis is essential for tumor growth and metastasis. The acquisition of the angiogenic phenotype precedes the attainment of attributes that confer characteristics of malignancy/malignant characteristics (Hawighorst et al. 2001). NADPH oxidases are family of ROS-generating enzymes which play significant part in redox signaling between tumor, endothelial, and immune cells to promote formation of blood vessels. MDSCs are a heterogeneous population of cells that regulate immune responses in cancer patients by suppressing T-cell functions. Apart from modifying immune response, these cells promote tumor angiogenesis, invasion, and metastasis. The action of MDSCs has been linked to NOX-mediated upregulation of ROS production (Corzo et al. 2009; OuYang et al. 2015). ROS play a major role in creating conditions that support the growth and survival of cancer cells (seed) along with an immunosuppressive environment appropriate for field cancerization and metastasis (Liao et al. 2019). “Field cancerization” refers to the development of abnormal epithelia and stroma in proximity to a tumorigenic area. If a regional carcinogenic signal persists for a long enough time at areas of cellular abnormalities it can lead to irreversible changes to the cells causing their eventual oncogenic transformation and cancerization of that area. This “field cancerization” results in the development of multifocal primary tumors in proximity with an enhanced likelihood of recurrence even after removal of the malignant tumor. This cancerization of the areas adjoining to the tumor indicates the changes in many areas of the primary tumor including the epithelial cells and surrounding stromal cells that drive the transformation of the cells. ROS is known to initiate and propagate field cancerization (Liao et al. 2019). Chronic exposure to extracellular H2O2 promotes the transformation of normal epithelial cells and fibroblasts. Normal fibroblasts on treatment with H2O2 can transform into CAF like state. These CAFs like cells have impaired TGF-β signaling and also produce higher amounts of H2O2 (Cirri and Chiarugi 2011; Avagliano et al. 2018). Stromal cells such as CAFs are influenced by redox signaling and employ mitogenic signaling to enter into a reciprocal relation with the epithelial tumor, indicating that extracellular oxidative stress can act as an agent that promotes spread of cancer (Liao et al. 2018, 2019). This implies that ROS can promote stroma-mediated cancerization which is different than the typical mode of inducing cancer (Chan et al. 2017). MAPK pathway is an important intracellular signal transduction cascade involved in cell survival, growth, death, and differentiation. It consists of ERK1/2, c-Jun N-terminal kinase (JNK), MAPK-11, and MAPK1. Studies have revealed that ROS can activate growth factors such as EGF and platelet-derived growth factor (PDGF) without their corresponding signals, which in turn can activate Ras and subsequently trigger the ERK pathway (Zhang et al. 2016). This independence from receptor-mediated activation contributes to resistance against antibody-based cancer

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therapies. Additionally, ROS can also activate the MAPK pathway by causing oxidative modification of downstream kinases such as apoptosis signal regulating kinase-1 (ASK-1) (Son et al. 2011). PI3K/PTEN is another pathway involved in “field cancerization” and “metastasis” (Zhang et al. 2016). This pathway involves several redox sensitive signal mediators and ROS affects this pathway by causing oxidative modification of these mediators. Oxidation of the cysteine thiol groups of phosphatases can result in in inactivation of PTEN. Setting up a new site for the successful development of secondary tumor requires a conducive environment. The survival of the disseminated tumor cells at the new secondary tumor site is dependent upon the interactions from the primary tumor and its ability to create a pre-metastatic niche. Additionally, the TME at the new site also determines the fate of the incoming tumor cells. ROS is known to produce a supportive environment for the disseminated cancer cells and help create a suitable soil for their survival (Liao et al. 2019). Studies have demonstrated that metastatic niches have an accumulation of MDSCs in the microenvironment which suppresses the activity of cytotoxic CD8+ T cells, consequently promoting the disseminated cancer cells (Youn and Gabrilovich 2010; Hamilton et al. 2014).

Botanicals Inhibit Cancer Progression and Metastasis Keeping in mind the biological and physical factors that confer complexity to cancer, it has been realized that adopting measures to prevent this disease could prove effective in controlling its morbidity. Considering their easy availability, better acceptability, enhanced positive effects and fewer side effects, the use of botanicals (whole plants/plant parts/plant products) as chemopreventive agents has been regarded useful in controlling this disease. The presence of multiple components in botanicals allows for several dysregulated pathways (multiple targets) to be attacked simultaneously which is crucial to a disease like cancer (Arora and Koul 2014). There is an inexhaustible literature evidence highlighting the anticancer potential of herbs, spices, fruits, vegetables, medicinal plants, etc. The proposed mechanism of action of phytochemicals against cancer include modulation of carcinogen biotransformation, free radical scavenging, suppression of inflammation, inhibition of cell proliferation, induction of apoptosis, promotion of cell differentiation, inhibition of angiogenesis and metastasis, etc. Free radical scavenging is an effective strategy for interfering with carcinogenesis at all the stages. Other options for restricting tumor promotion and progression include: altering the expression of genes involved in cell signaling, particularly those regulating cell proliferation, apoptosis and differentiation, decreasing inflammation etc. The anti-oncogenic activity of botanicals is accompanied by pro-oxidant/antioxidant effects suggesting dual role of ROS in cancer (Fig. 3). Studies from our laboratory have provided evidence that extracts of plants such as Azadirachta indica (neem), Solanum lycopersicum (tomato), Silibum marianum (milk thistle), and Tinospora cordifolia (giloya) can control several dysregulated pathways including carcinogen metabolism, oxidant-antioxidant

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Fig. 3 Amelioration of cancer by botanicals. Botanicals have the potential to mitigate cancer at all stages. The presence of multiple phytochemicals in botanicals allows for several pathways to be targeted simultaneously. The antioncogenic activity is accompanied by pro-oxidant/antioxidant effects suggesting dual role of ROS in cancer

defense, cell proliferation, cell death, angiogenesis, metastasis, etc. during chemically induced cancer in mice (Koul et al. 2006; Gangar and Koul 2008; Arora et al. 2011a, b; Gupta et al. 2013; Bhatia et al. 2015; Sati et al. 2016; Koul et al. 2019). Lycopene enriched tomato extract (LycT) exerted chemopreventive effects against hepatocellular carcinoma (HCC) in mice and this was accompanied by a decrease in elevated ROS levels, along with a mitigation in angiogenic and metastatic markers including vascular endothelial growth factor (VEGF), cluster of differentiation-31, hemoxygenase, hypoxia inducible factor, MMP-2, MMP-9, etc. (Gupta et al. 2013; Bhatia et al. 2015). This extract was also effective against squamous cell carcinoma (SCC) in mice and decreased the expression of angiogenesis-associated genes (VEGF, ANGPT-2, basic fibroblast growth factor), increased the expression of cell-cell communication proteins (connexins), and modulated the ECM components (Koul et al. 2019). Azadirachta indica leaf extract (AAILE) exhibited good radical scavenging activity as revealed by in vitro radical scavenging assays (Koul et al.

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2013). AAILE administration during HCC exhibited chemopreventive effects which were accompanied by a decrease in pro-inflammatory TNF-α levels (Bharati et al. 2014). AAILE-mediated chemoprevention of SCC in mice was accompanied by a decrease in the expression of angiogenesis and metastasis associated transcription factors including NF-κB, AP-1, p53, STAT 1 (Arora et al. 2011a, b). Quercetin, a flavonoid isolated from Azadirachta indica, has been documented to retard carcinogenesis at early and late stages of carcinogenesis by virtue of its radical scavenging properties (Rice-Evans et al. 1996). The major polyphenolic component of green tea, epigallocatechin-3-gallate (EGCG), possesses remarkable cancer chemopreventive properties. As reviewed by Khan et al. (2006), studies in animal models and humans have revealed that EGCG modulated multiple signal transduction pathways including those mediated by NF-κB, AP-1, MAPK, and EGFR. EGCG also modulated cell signaling associated with angiogenesis, metastasis, and migration. ECGC inhibited binding of VEGF to its receptor and diminished activity of MMP-2 and 9, thereby exhibiting antiangiogenic, anti-invasive, and antimetastatic properties. Ng et al. (2018) have reviewed the work of several authors and discussed in detail the anticarcinogenic properties of several phytochemicals including resveratrol, silibinin, luteolin, eugenol, gingerol, curcumin, etc. The anti-oncogenic effects of phytochemicals have been explained on the basis of their antiproliferative, antioxidative, anti-inflammatory, and antiangiogenic effects. Resveratrol is a polyphenol found in fruits such as grapes, mulberries, blueberries, raspberries etc which exhibits inhibitory effects on all stages of cancer. It is a potent scavenger of free radicals like peroxyl and superoxide radicals. It also antagonized inflammation by inhibiting the activity of cyclooxygenases mediated via inhibition of NF-κB, ERK, and p38 MAPK. Silibinin is the main and active component of Silymarin complex, which consists of flavonoids and flavonolignans. The health beneficial effects of silibinin have been attributed to its direct/indirect antioxidative action including ROS scavenging. Silibinin inhibits angiogenesis by targeting VEGF and inducible nitric oxide synthase (iNOS). Topical and dietary administration of silibinin have been effective in inhibiting MAPK (ERK1/2, JNK, p38) which are important for cell migration and invasion. Luteolin is a flavonoid found in carrots, celery, olives, pepper, etc. The antitumor action of luteloin has been attributed to antioxidant, anti-inflammatory, antiangiogenesis, pro-apoptotic, and antiproliferative activities. Eugenol is a phenolic component of cloves, nutmeg, cinnamon, bay leaves, basil etc. Its anticancer action is mediated by its antioxidative, antiproliferative, and anti-inflammatory activities. The antioxidative action of eugenol has been ascribed to its scavenging activity thereby, reducing the formation of superoxides and lipid peroxides. The anti-inflammatory effects are mediated by inhibition of cyclooxygenase (COX), iNOS, cytokines, and NF-κB. Gingerol is a phenolic phytochemical isolated from the roots of Zingiber officinale ginger plant. Its anticancer activity against mouse skin cancer has been attributed to its anti-inflammatory activity mediated by inhibition of ornithine decarboxylase, COX, NF- κB, MAPK, etc. It has also been observed to act as an antioxidant by reducing intracellular ROS.

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Polyphenols affect EMT pathway by increasing the levels of epithelial markers and decreasing the mesenchymal markers. Dietary polyphenols also decrease the formation of ROS that allows for minimizing the oxidative damage to cellular macromolecules. Resveratrol inhibited the EMT in gastric, lung, pancreatic, and breast cancer and also suppressed cancer invasion and metastasis in vitro (Amawi et al. 2017). Reduction of ROS and consequent promotion of apoptosis was considered responsible for the decrease in glioma cell growth in response to sea buck thorn leaf extract. Studies have demonstrated that grape seed extract induced anoikis in LnCaP cells which was accompanied by an inhibition in focal adhesion kinase levels, increase in caspase 3 and 9 levels and poly-ADP ribose levels (Kaur et al. 2006). Curcumin blocked NF-kβ signaling, thereby suppressing cell survival and cell proliferation genes like bcl-2, cyclin D1, IL-6, COX-2, MMP-9 (Thangapazham et al. 2006). Although several evidences support the pro-oncogenic role of ROS; however, ROS inhibition has also been linked with increased tumorigenesis. This is particularly evident during use of antioxidants/antioxidant therapy during cancer treatment, when ROS inhibition was associated with decreased survival (Goodman et al. 2011). Many cancer chemotherapeutic agents exert their action by generating ROS in cancer cells. This additional load of ROS to the already ROS-rich cancer/tumor cells pushes their level above a point that induces cell death (Kong et al. 2000; Conklin 2004; Pelicano et al. 2004; Yang et al. 2018). Doxorubicin, daunorubicin, and epirubicin are among the agents that induce highest amount of ROS. Alkylating agents, arsenic agents, camptothecins, and topoisomerase inhibitors also induce high levels of ROS. Other agents such as taxanes, vinca alkaloids, anti-folates, and nucleotide analogues generate lower levels of ROS (Apel and Hirt 2004; Shi et al. 2004; Yen et al. 2012). Studies from our laboratory and those of several others have revealed that chemoprevention of cancer using plant extracts in animal models was accompanied by an increase in peroxidative damage in target tissues with a concomitant decrease in low molecular weight nonenzymatic antioxidants (Subapriya and Nagini 2003; Gangar and Koul 2008; Arora et al. 2013; Sati et al. 2016). The pro-oxidant effects of several phytochemicals have been observed during cancer treatment and prevention (Chikara et al. 2018). Studies on anticancer agents suggest that it would be reasonable to consider that ROS exhibits dual role in cancer. The detrimental/beneficial effects of ROS depend upon the state/type of tissue and the concentration of the anticancer agent being used. Although increase in ROS is a causative factor for cancer; however, increase in ROS levels beyond a certain level can slow cell cycle progression of cancer cells, cause cell cycle checkpoint arrest, and induce apoptosis thereby limiting cancer.

Conclusion Although evidences are available that support both the pro- and anti-oncogenic action of ROS; however, yet it is difficult to ascertain the role of ROS in cancer “development” and “amelioration.” Even though botanicals have exhibited good anticancer effects, their roles as ROS inhibitors or promoters while executing their action still remain to be exhaustively explored.

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Therapeutic Implications in ROS-Induced Cancer Charles Elias Assmann, Grazielle Castagna Cezimbra Weis, Je´ssica Righi da Rosa, Beatriz da Silva Rosa Bonadiman, Audrei de Oliveira Alves, Felipe Tecchio Borsoi, and Margarete Dulce Bagatini Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Anthocyanins and Anthocyanidins: General Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cyanidin and Cyanidin-3-O-Glicoside . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Delphinidin and Delphinidin-3-O-Glucoside . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Flavonols: General Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Kaempferol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Quercetin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract

Cancer stands among the main causes of death worldwide characterized by a combination of different factors which drive tumor progression and invasiveness. Oxidative stress has been shown to possess a central involvement in cancer development for promoting an imbalanced microenvironment, favoring DNA Charles Elias Assmann and Grazielle Castagna Cezimbra Weis share the first authorship of this chapter. C. E. Assmann (*) · G. C. C. Weis (*) · J. R. da Rosa · A. d. O. Alves Federal University of Santa Maria, Santa Maria, RS, Brazil B. d. S. R. Bonadiman Federal University of Santa Catarina, Florianópolis, SC, Brazil F. T. Borsoi University of the State of Santa Catarina, Pinhalzinho, SC, Brazil M. D. Bagatini (*) Federal University of the South Border, Chapecó, SC, Brazil © Springer Nature Singapore Pte Ltd. 2022 S. Chakraborti et al. (eds.), Handbook of Oxidative Stress in Cancer: Mechanistic Aspects, https://doi.org/10.1007/978-981-15-9411-3_139

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mutations and damage to biological structures. Flavonoids are naturally occurring compounds present in plant-based foods, mainly fruits and vegetables, which exhibit notable antioxidant and radical scavenging activities. Due to their biological activities and their availability in nature, anthocyanins and flavonols, one of the flavonoids subclasses, have been studied as potential anticarcinogenic agents. This chapter summarizes the main therapeutic implications of some of the most efficacious diet-derivate anthocyanidins and flavonols, and its glycosides, on cancer chemotherapy and chemoprevention. Keywords

Flavonoids · Anthocyanidins · Glycosides · Cyanidin · Delphinidin · Quercetin · Kaempferol · Oxidative stress

Introduction Flavonoids are naturally occurring phenolic compounds and ubiquitously present in the plant kingdom. This class of natural molecules is subdivided into flavones, flavonols, isoflavones, flavanones, flavan-3-ols, and anthocyanins. Plant-food flavonoids are known for their remarkable radical scavenging activities, which make them potential compounds for the use against many diseases (George et al. 2017). Anthocyanins are molecules largely encountered in nature responsible for the blue (açai berry, bilberry, blueberry, eggplant, e.g.), purple (purple carrot, purple corn, e.g.), and red (raspeberry, red cabagge, red onion, red potato, red wine, e.g.) colors of foods. The range of reddish-purple color is attributed to cyanidin, delphinidin, malvidin, pelargonidin, peonidin, and petunidin, which stand among the predominant anthocyanidins in food sources (Khoo et al. 2017). Cyanidin, delphinidin, and their glycosides are the most common anthocyanin compounds found in the plant kingdom with well-understood biological functions including neuroprotective, anti-inflammatory, antioxidant, antimicrobial, antidiabetic, antiobesity, and anticancer properties (Khoo et al. 2017; Lee et al. 2017; Chen et al. 2019). Quercetin and kaempferol are examples of often-studied dietary flavonoids, being representatives of the flavonols subclass. These molecules, widely present in the human diet, have displayed antioxidant, neuroprotective, anti-inflammatory, and anticancer activity in many in vitro and in vivo studies (George et al. 2017; D’Andrea 2015; Kashyap et al. 2017). Cancer is a multifactorial disease characterized by the uncontrolled growth of cells, evading of antiproliferative signals, angiogenesis, invading of surrounding tissues, among others; inflammation and genome instability have been presented as underlying hallmarks that promote tumor development and progression (Hanahan and Weinberg 2011). A large body of evidence has shown that especially inflammation and oxidative stress, mainly by the action of reactive oxygen species (ROS) and inflammatory molecules, trigger damage to important biomolecules and consequently to the cell, and thus possess a close link to tumor formation, driving a healthy cell to a tumor cell (Reuter et al. 2010).

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Taking into consideration the healthy benefits of anthocyanins and flavonols, several studies are investigating the potential of these molecules against cancer development (George et al. 2017; Khoo et al. 2017; Chen et al. 2019). In this scenario, this chapter presents some insights on the role in vitro and in vivo of anthocyanidins, especially cyanidin, delphinidin, and their glycosides, and flavonols, quercetin and kaempferol, on cancer.

Anthocyanins and Anthocyanidins: General Overview Anthocyanins are pigments soluble in water which provide blue, red, and purple colors to fruits, vegetables, and beverages (e.g., blueberry, cherry, cranberry, eggplant, red cabbage, and red wine, among others) (Khoo et al. 2017). Approximately 700 structurally different anthocyanins have been identified in nature. The aglycone form, called anthocyanidin, which is less frequent in nature, has approximately 30 identified molecules. Among them, six anthocyanidins are usually encountered in food sources: cyanidin, delphinidin, malvidin, pelargonidin, peonidin, and petunidin. Meanwhile, anthocyanins are defined as glycosylated anthocyanidins with one or more sugar, and frequently present glucose, galactose, and arabinose in their chemical structure (Khoo et al. 2017; Rodriguez-Amaya 2019). The table below (Table 1) shows some examples of food sources of anthocyanins and anthocyanidins, and their major molecules. Literature data has reported that anthocyanins from berries are powerful anticarcinogenic agents mainly due to the protective capacity against genomic instability (Khoo et al. 2017). In the study conducted by Hogan et al. (Hogan et al. 2010), the antioxidant properties and antiproliferative activity of an anthocyanin-rich açai berry extract (Euterpe oleracea Mart.) was investigated against MDA-468 human breast cancer cells and C-6 rat brain glioma cells. The principal anthocyanins identified in the extract were cyanidin-3-glucoside, cyanidin-3-rutinoside, delphinidin 3-(600 -acetoyl) glucoside, and peonidin-3-(600 -malonylglucoside). The açai extract presented effective antioxidant activities and antiproliferative capacity against C-6 brain glioma cells. Besides, DNA damage results suggested that the extract promotes apoptosis in these cells, indicating a correlation between this effect and the antiproliferative capacity. Shi et al. (Shi et al. 2017) evaluated the anticarcinogenic impact of lyophilized black raspberries (BRB) (Rubus occidentalis), which contain anthocyanins as major phenolic components, mainly cyanidin-3-rutinoside, in esophageal squamous cells carcinogenesis in rats. The diet with 5% BRB significantly reduced the rate of esophageal cancer in rats treated with N-nitrosomethylbenzylamine (NMBA) when compared to rats treated with NMBA plus BRB (100% to 81.5%, respectively). Results showed that treatment with NMBA enhanced the levels of hydrogen peroxide (H2O2) and lipid hydroperoxide, decreased the expression and activity of glutathione peroxidase (GPx) and superoxide dismutase 2 (SOD2), and triggered the NFκB/MAPK signaling in rat esophagus. Overall, BRB treatment was shown to

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Table 1 Anthocyanins and anthocyanidins composition of different fruits and vegetables Source Açai berry Purple carrot

Red potato

Red onion

Eggplant

Blueberry

Bilberry

Anthocyanins and anthocyanidins Cyanidin-3-glucoside, cyanidin-3-rutinoside, and peonidin-3-rutinoside Cyanidin-3-(xylosyl)(coumaroyl)glucosidegalactoside, cyanidin-3-(xylosyl)(feruloyl)glucosidegalactoside, cyanidin-3-(xylosyl)(sinapoyl)glucosidegalactoside, and cyanidin-3-(xylosyl)glucosidegalactoside Pelargonidin, pelargonidin-3-(feruloyl)-rutinoside-5glucoside, pelargonidin-3-rutinoside, pelargonidin-3rutinoside-5-glucoside, and peonidin-3-(p-coumaroyl)rutinoside-5-glucoside Cyanidin-3-(600 -malonyl)glucopyranoside, cyanidin3,5-diglucosides, cyanidin-3-glucosides, and delphinidin-3,5-diglucosides Cyanidin-3-rutinoside, delphinidin-3-rutinoside, delphinidin-3-rutinoside-5-glucoside, malvidin-3rutinoside-5-glucoside, and petunidin-3-rutinoside Cyanidin, cyanidin-3-glucoside, delphinidin, delphinidin 3-arabinoside, delphinidin 3-galactoside, malvidin, malvidin 3-arabinoside, petunidin, and petunidin-3-arabinoside Cyanidin-3-O-galactoside, cyanidin-3-O-glucoside, delphinidin-3-O-arabinoside, delphinidin-3-Ogalactoside, delphinidin-3-O-glucoside, malvidin-3-Oglucoside, and petunidin-3-O-glucoside

Reference Hogan et al. (2010) Montilla et al. (2011), Li et al. (2012)

Li et al. (2012), Ieri et al. (2011)

Zhang et al. (2016a)

Ferarsa et al. (2018)

Li et al. (2016)

Benvenuti et al. (2018)

reverse oxidative stress and suppress NFκB/MAPK pathways, and these effects were suggested to promote the chemopreventive activity of BRB in esophageal cancer. Bornsek et al. (Bornsek et al. 2012) tested the cellular antioxidant action of blueberries (Vaccinium corymbosum L.) crude extract and bilberries (Vaccinium myrtillus L.) crude and purified extract in human colon cancer (Caco-2), human endothelial (EA.hy926), human hepatocarcinoma (HepG2), and rat vascular smooth muscle (A7r5) cells. The anthocyanins represented the majority of all phenolics in both extracts. While cyanidin and delphinidin glycosides were the main anthocyanins in the bilberry extracts, malvidin glycosides were more recurrent in the blueberry extract. Data showed that bilberry and blueberry anthocyanins have potent intracellular antioxidant properties at very small concentrations, however, the antioxidant activity of the bilberry extract was greater when compared to the blueberry extract. In a recent study performed by Léon-González et al. (León-González et al. 2018), an anthocyanin-rich bilberry extract was able to promote apoptosis in acute lymphoblastic leukemia cells by diminishing the expression levels of Polycomb Group proteins (PcG), and, thus, the following PcG proteins-dependent pro-survival events via a redox-dependent process, after 24 h of exposition. An investigation performed with anthocyanins from roselle (Hibiscus sabdariffa L.) reported antileukemic action in a rat model of chemical-induced leukemia.

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Animals were allocated into four groups: control, nitrosomethylurea (NMU), and roselle anthocyanin extract orally supplemented in the diet at 0.1% and 0.2% plus NMU treatment. The results displayed that the administration of anthocyanin extract from roselle (0.2%) orally given to rats significantly suppressed the progression of NMU-induced leukemia by 33.3% (Tsai et al. 2014). Besides the conventional sources such as fruits, pigment-rich root vegetables are powerful sources of phytochemical antioxidants, notably anthocyanins. Zhang et al. (Zhang et al. 2016b) investigated the antioxidant and anti-inflammatory activity of purple carrots and potato extracts in H2O2-exposed Caco-2 cells. These anthocyaninrich phenolic extracts, mainly in cyanidin and petunidin, besides presenting strong antioxidant actions by direct radical scavenging activity, also stimulated the expression of antioxidant enzymes such as catalase (CAT) and GPx, and reduced the production of pro-inflammatory interleukins mediated by H2O2 in Caco-2 cells at low doses (50–100 μg/mL). Results indicate that anthocyanin-rich purple types of potatoes and carrots could improve oxidative stress by intestinal inflammatory responses. The proliferation inhibitory potential of anthocyanin-rich extracts of cereals was also reported by Mazewski et al. (Mazewski et al. 2017) that analyzed the antiproliferative action of anthocyanin-rich purple and red corn on human colorectal cancer cells (HT-29 and HCT-116). Both extracts increased apoptosis and suppressed angiogenesis. In a recent work performed by Mazewski et al. (Mazewski et al. 2018), the antiproliferative consequence of 11 anthocyanin-rich extracts: black and purple bean, black lentil, black peanut, black rice, blue wheat, purple carrot, purple sweet potato, red and purple grape, and sorghum was tested against human colon cells. The extracts decreased expression of antiapoptotic proteins, induced apoptosis, and promoted cell cycle arrest at the G1 phase. The black lentil, red grape, and sorghum extract inhibited HCT-116 and HT-29 cell proliferation at concentrations of 0.9–2.0 mg/mL (IC50). Anthocyanins and anthocyanidins act attenuating oxidative stress and inflammation as cellular mechanisms of inhibiting carcinogenesis (Khoo et al. 2017), especially anthocyanins which display more effective anticancer activity than anthocyanidins (Zhou et al. 2018). Among the anthocyanins that have a substantial key role in cancer development are delphinidin, cyanidin and their glycosides, which exhibit potent antioxidant, anti-inflammatory, and anticancer properties (Khoo et al. 2017; Mazewski et al. 2017; Mazewski et al. 2018).

Cyanidin and Cyanidin-3-O-Glicoside Cyanidins and their glycosides are one of the main groups of anthocyanins that occur naturally, being considered one of the most abundant in the plant kingdom (Khoo et al. 2017). They are compounds with well-understood biological properties, being widely researched mainly for their antioxidant and anti-inflammatory properties. Cyanidin rarely occurs in nature, on the other hand, its glycosides (Table 2) are

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Table 2 Names, molecular characteristics, and chemical structures of cyanidin, delphinidin and their O-glycosides Name

Abbr. Substitution pattern R1 R2

Cyanidin and its O-glycosidesa Cyanidin C Cyanidin-3-5-O-diglucoside CDG Cyanidin-3-O-arabinoside CA Cyanidin-3-O-galactoside CGA Cyanidin-3-O-glucoside CG Cyanidin-3-O-rutinoside CR Delphinidin and its O-glycosidesb Delphinidin D Delphinidin-3-O-arabinoside DA Delphinidin-3-O-galactoside DGA Delphinidin-3-O-glucoside DG Delphinidin-3-O-rutinoside DR Delphinidin-3-O-sambubioside DS a

H Glucose Arabinose Galactose Glucose Rutinose

H Glucose H H H H

H Arabinose Galactose Glucose Rutinose Sambubiose

– – – – – –

Structure

Adapted from Cyboran-Mikolajczyk et al. (Cyboran-Mikolajczyk et al. 2019) Adapted from Chen et al. (Chen et al. 2019)

b

widely found in the plant kingdom, among these glycosides is cyanidin-3-glucoside (CG) (Liu et al. 2018; Cyboran-Mikolajczyk et al. 2019). Cyboran-Mikolajczyk et al. (Cyboran-Mikolajczyk et al. 2019), when analyzing the significance of O-glycosylation on the interplay of cyanidin with red blood cells and human microvascular endothelial cells (HMEC-1), reported that the bioactive properties of cyanidin and its glycosides relies on the type and number of sugar substituents and differs according to the cell type and extracellular milieu. Furthermore, the results showed that the compounds did not present cytotoxicity, not induced apoptosis or altered the promotion of the cell cycle in HMEC-1 cells. In addition, despite the compounds having altered the shape of red blood cells, they did not impair their transmembrane potential, also protecting erythrocytes against free radicals and reducing the generation of ROS (Cyboran-Mikolajczyk et al. 2019). Hosseini et al. (Hosseini et al. 2017) analyzed the cytotoxic and apoptotic effect of CG on the U87 glioblastoma cell line at different concentrations showing that treatment with 40 μg/mL of the compound caused the apoptosis of 32% of the cells after 24 h. More recently, Liu et al. (Liu et al. 2018) conducted a study to investigate the effects of cyanidin at different concentrations on the proliferation, invasion, cell cycle, and apoptosis in vitro using renal cell carcinoma lines (786-O and ACHN). Data reported that cyanidin inhibited cell proliferation and migration in a wide range of concentrations, significantly reducing tumorigenesis independent of the concentration. In addition, cyanidin promoted cell apoptosis in treated cells (786-O and ACHN) and induced cell cycle arrest. Furthermore, protocols were also carried out in vivo injecting mice with the suspension of ACHN cells and administering cyanidin at the dose of 6 mg/Kg two times a week. After 4 weeks, tumors were

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collected and evaluated, and results showed that the mice treated with cyanidin significantly reduced tumor growth. In another study performed by Takeuchi et al. (Takeuchi et al. 2011), cyanidin diminished the intracellular levels of ROS in several cell cancer lines such as MCF-7, Huh-7, HepG2, and Caco2, also inhibiting the proliferation of these cells. However, while cyanidin suppressed the growth of cells, CG did not. In the same way, peonidin, peonidin-3-glucoside, and cyanidin-3-rutinoside (CR) were also tested, though the glycosylated structure inhibited the biological effects.

Delphinidin and Delphinidin-3-O-Glucoside Delphinidin, one of the major anthocyanidins found in plants, and its glycosides, have displayed several health benefits, especially antioxidant and anti-inflammatory properties (Lee et al. 2017; Chen et al. 2019). Research has also presented that these compounds have anticarcinogenic activities. The chemical structure of delphinidin affects its stability and bioavailability and the addition of distinct sugar substituents originates different delphinidin glycosides, as shown in Table 2 (Chen et al. 2019). Anthocyanins have been shown to mediate apoptosis in various cancer cells, including non-small-cell lung cancer (NSCLC) (Pal et al. 2013) and colon cancer cells (Shin et al. 2009). Delphinidin was shown to suppress the expression of epidermal growth factor receptor (EGFR) and vascular endothelial growth factor receptor 2 (VEGFR2) in NSCLC cells. The effects of this molecule in vitro and in vivo on NSCLC cells and in athymic nude mice, respectively, were investigated. Data showed that delphinidin significantly inhibited cell growth in vitro without harmful effects on healthy human bronchial epithelial cells. In nude mice, treatment with the compound suppressed tumor growth, inducing apoptosis, and decreasing angiogenesis and markers of cell proliferation compared to control animals (Pal et al. 2013). In human colon cancer cells (HCT-116), anthocyanins isolated from Vitis coignetiae Pulliat were proposed to regulate apoptosis proteins by stimulating p38-MAPK and inhibiting Akt proteins (Shin et al. 2009). In another study, the outcome of different anthocyanins was tested in vitro on the viability of human fibrosarcoma HT1080 cells. Results showed that at the concentration of 100 μM, delphinidin-3-glucoside (DG) significantly decreased cell viability, whereas other anthocyanins (3-glucosides of malvidin, cyanidin, pelargonidin, and peonidin) did not affect this parameter; also, DG only presented a 20% decrease in the viability of normal fibroblast cells (NIH 3T3) (Filipiak et al. 2014). Recent investigation performed in vivo using NMU as a model to induce breast carcinogenesis in rats suggested that delphinidin might possibly inhibit breast tumor formation and present its anticancer effects by modulating the HOTAIR/miR-34a axis (Han et al. 2019). Another recent work addressed the effect on cell viability of DG and delphinidin on human colorectal cancer cells (HT-29 and HCT-116). Overall, results demonstrated that DG and delphinidin promoted apoptosis and inhibited colorectal cancer cell survival according to the dose. Furthermore, DG also was shown to potentially

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suppress immune checkpoints, programmed cell death protein 1 (PD-1), and programmed death-ligand 1 (PD-L1). Overexpressed PD-L1 in cancer cells contributes to escape immune destruction, and its binding to PD-1 on T cells disables these cells to recognize and kill cancer cells. Therefore, these proteins are being used as targets for colon cancer treatment once inhibiting their expression could boost immune response in order to promote cancer cell death (Mazewski et al. 2019). Thus, anthocyanins such as delphinidin and its glycosides appear to be effective in reducing oxidative stress and decreasing proinflammatory factors, as well as positively interfering with some cancers.

Flavonols: General Overview Among flavonoids, flavonols are one of the most abundant classes in nature. Flavonols are present in considerable amounts in the human diet, through edible portions of many food plants, leafy vegetables, tubers and bulbs, several fruits, tea, and wine (Perez-Vizcaino and Duarte 2010). Flavonols show a broad range of biological actions: antioxidant, neuroprotective, anti-inflammatory, and anticancer activity (George et al. 2017; Kashyap et al. 2017). The main representant of the flavonol class is quercetin (Fig. 1B), which is the most abundant and most often studied dietary flavonol, found especially in glucoside forms. Another common flavonol is kaempferol (Fig. 1A), also present in many foods (Perez-Vizcaino and Duarte 2010). Due to its availability in nature, presence in the human diet, and biological activities, quercetin and kaempferol are compounds of interest in research against cancer development.

Kaempferol Kaempferol (Fig. 1A) is a flavonol, belonging to the large group of flavonoids, found in several species of plants around the world. Some examples of food sources that contain this compound are: garlic, chives, broccoli, mustard, turnip, grapefruit,

Fig. 1 Chemical structure of the main flavonols: kaempferol (A) and quercetin (B)

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cucumber, strawberry, sweet potato, lettuce, apple, peach, spinach, tomatoes, grape, raspberry, aloe vera, capers, and saffron, among others (Calderón-Montaño et al. 2011). The interest in the kaempferol compound has increased due to its antioxidant, cardio- and neuroprotective, and anti-inflammatory activities, demonstrated in many in vitro and in vivo studies (Kashyap et al. 2017). Regarding kaempferol antitumor activity, several investigations have addressed and highlighted possible mechanisms of this compound through cellular and in vivo models (Imran et al. 2019). For example, in the case of breast cancer, kaempferol anticancer activity was explored in the cell cycle of MCF-7 cancer cells and verified to inhibit cyclins D and E in the G1 phase, possibly by suppressing estrogen activity (Kim et al. 2016). Another study showed that kaempferol triggered apoptosis of human cervical cancer (HeLa) cells by positively regulating pro-apoptotic genes such as TP53, P21, caspase 3, caspase 9, Bax, and PTEN and negative modulation of survival genes, including AKT, Bcl-2, and PI3K (Kashafi et al. 2017). Han et al. (Han et al. 2018), studying pulmonary adenocarcinoma (A549 cells) found that kaempferol reduced cell viability and proliferation, while inducing autophagy and apoptosis. Additionally, the compound also upregulated PTEN expression besides inactivating the PI3K/AKT pathway. Besides, in another study, Heo et al. (Heo et al. 2018) investigated kaempferol in melanoma cells (A375SM) and verified the inhibition of the Bcl-2 protein and the stimulus of the pro-apoptotic protein Bax, the activation of the P21 gene expression, promoting the arrest of the cell cycle and cell progression at the G1 phase by inhibiting cyclin B and E. In addition, cells treated with kaempferol increased the generation of ROS, which may contribute to apoptosis and suppress cell growth in melanoma cells.

Quercetin Quercetin (Fig. 1B) is a polyphenolic flavonoid that possesses antioxidant properties and is usually found in several fruits and vegetables, including green vegetables, berries, green tea, citrus fruits, legumes, onions, and parsley (Hashemzaei et al. 2017). Researches have shown several health benefits of quercetin such as improvement of endothelial function, antihypertensive, antioxidant, antithrombotic, antiinflammatory, anti-obesity, and anticancer activity (D’Andrea 2015). Due to its wide distribution in nature, quercetin is a flavonol with potential as an anticancer agent against several cancer types and by being a further candidate for anticancer drug design. An investigation addressed the action of natural products such as quercetin and green tea on the efficacy of inoculated androgen-independent prostate PC-3 cancer cells in mice. Results showed that quercetin and green tea in combination with docetaxel (Doc) increased its efficacy and considerably decreased tumor progression.

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Also, blood concentrations of growth factors, such as vascular endothelial growth factor (VEGF) and epidermal growth factor (EGF) were found decreased after the intervention by the mixture of quercetin, green tea, and Doc (Wang et al. 2016a). Zhao and coworkers investigated the action of quercetin against HepG2 human liver cancer cells. Results showed that quercetin was able to trigger apoptosis in these cells which was followed by the decrease in fatty acid synthase (FASN) activity. In this sense, suggesting that apoptosis may be triggered by quercetin via the suppression of FASN and that this compound could help to prevent human liver cancer (Zhao et al. 2014). The effect of quercetin has been also investigated against colorectal lung metastasis. In a study conducted both in vitro and in vivo, quercetin suppressed the viability of colon 26 (CT26) and colon 38 (MC38) cells. Besides, it triggered apoptosis by regulating the MAPKs pathway in CT26 cells and also suppressed the migration and invasion abilities of these cells by the modulation of tissue inhibitor of metalloproteinases (TIMPs) and matrix metalloproteinases (MMPs) expression. Quercetin also reduced lung metastasis of CT26 cells in a mouse model. This result suggested that this compound was able to suppress the ability of CT26 cells to metastasize and it also inhibited lung metastasis in vivo (Kee et al. 2016). In the case of cervical cancer, research showed that quercetin could inhibit the viability and proliferation of HeLa cells by promoting apoptosis through a p53-dependent mechanism and also inducing G2/M phase cell cycle arrest (Priyadarsini et al. 2010; Wang et al. 2016b). Quercetin has been also indicated to enhance the chemosensitivity of breast cancer cells (MCF-7) to the chemotherapeutic drug doxorubicin (Dox) and the mechanism has been suggested to be through enhanced cell apoptosis, suppression of cell invasion and proliferation, downregulation of p-Akt expression, and upregulation of PTEN expression (Li et al. 2015). Besides, in another study using human breast cancer cell lines (MCF-7 and MDA-MB-231), quercetin showed inhibitory effects on cell proliferation, suppressed invasion by the downregulation of the epidermal growth factor receptor (EGFR) expression, promoted apoptosis by caspase-3 activation, and an upregulation in the miR-146a expression (Tao et al. 2015). Balakrishnan et al. (Balakrishnan et al. 2016) investigated the antitumor potential of a gold nanoparticle-conjugated quercetin system against breast cancer cells and also in an induced mammary carcinoma in rats. Results showed that the combination of quercetin and gold nanoparticles suppressed invasion and migration of MDA-MB and MCF-7 cells and formation of blood vessels using in vivo and in vitro angiogenesis assays. Besides, the combination inhibited tumor growth in the rat model. Overall, findings indicated that the conjugation of quercetin with gold nanoparticles was able to inhibit metastasis and angiogenesis of breast cancer cells. Figure 2 summarizes some biological effects and central mechanisms by which anthocyanins and flavonols, presented in this chapter, demonstrate their anticancer properties acting at stages of carcinogenesis, such as angiogenesis, metastasis, migration, inhibition of the cell cycle phases, and promoting apoptosis.

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Fig. 2 Overview of some of the biological activities and general anticancer mechanisms of action induced by the anthocyanins and flavonols discussed in this chapter

Conclusion In summary, literature data using in vitro cellular models and in vivo protocols provides evidence on the anticancer properties of anthocyanins and flavonols, showing that some of the main underlying mechanisms of action are related to the antiproliferative activity, apoptosis induction, suppression of angiogenesis and migration, and improvement of oxidative stress. Furthermore, these molecules did not show or showed reduced negative effects on healthy cellular models. Therefore, isolated anthocyanins and flavonols or derived from extracts could potentially be used as chemopreventive or adjuvant agents in cancer treatment. Acknowledgments The authors are thankful for grants and fellowships provided by the Brazilian Agencies Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES; Finance Code 001). Conflict of Interest None.

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Zhang S-L, Deng P, Xu Y-C et al (2016a) Quantification and analysis of anthocyanin and flavonoids compositions, and antioxidant activities in onions with three different colors. J Integr Agri 15 (9):2175–2181 Zhang H, Liu R, Tsao R (2016b) Anthocyanin-rich phenolic extracts of purple root vegetables inhibit pro-inflammatory cytokines induced by H2O2 and enhance antioxidant enzyme activity in Caco-2 cells. J Funct Food 22:363–375 Zhao P, Mao J-M, Zhang S-Y et al (2014) Quercetin induces HepG2 cell apoptosis by inhibiting fatty acid biosynthesis. Oncol Lett 8(2):765–769 Zhou F, Wang T, Zhang B et al (2018) Addition of sucrose during the blueberry heating process is good or bad? Evaluating the changes of anthocyanins/anthocyanidins and the anticancer ability in HepG-2 cells. Food Res Int 107:509–517

Implication of Nanomedicine in Therapy of Oxidative Stress-Induced Cancer

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Tanweer Haider, Vikas Pandey, Kamalpreet Kaur Sandha, Prem N. Gupta, and Vandana Soni

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ROS and Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ROS and Its Biological Functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Role and Mechanism of ROS in Cancer Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ROS Induced Transcription Factor and Regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ROS Altered Biomolecules Functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Oxidative Stress Adaptation in Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Therapeutics Targeting Oxidative Stress Alteration in Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Nanoparticulate Carriers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Nanovesicular Carriers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract

The disproportion between the concentration of reactive oxygen species (ROS) and the detoxification of ROS by the body results in oxidative stress. ROS like O2˙
, OH•, organic hydroperoxide, peroxynitrite, etc., are formed as byproducts of the normal oxygen metabolism and are involved in cell signaling and homeostasis. In cancer, the cells have been found in oxidative stress condition, which disfigures the cellular proteins, lipids, and eventually leads to oxidation of DNA, thereby causing lethal lesions and contributing to carcinogenesis. The elevated ROS modulates different transcription factors which lead to the cellular T. Haider · V. Pandey · V. Soni (*) Department of Pharmaceutical Sciences, Dr. Harisingh Gour University, Sagar, Madhya Pradesh, India e-mail: [email protected] K. K. Sandha · P. N. Gupta (*) Formulation & Drug Delivery Division, CSIR-Indian Institute of Integrative Medicine, Jammu, India e-mail: [email protected] © Springer Nature Singapore Pte Ltd. 2022 S. Chakraborti et al. (eds.), Handbook of Oxidative Stress in Cancer: Mechanistic Aspects, https://doi.org/10.1007/978-981-15-9411-3_128

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transformation, angiogenesis, proliferation, metastasis, cancer cell survival, as well as control of tumor suppressor genes. In cancer cells, elevated ROS levels are offset by an increase in antioxidant enzyme activity. In this case, increased ROS production and/or reduced antioxidant defense may be one of the therapeutic strategies that inhibit tumor progression. The development of nanotherapeutics approaches, which will overproduce the ROS, may initiate the ROS-induced tumor cells apoptosis. Other chemotherapeutic approaches in which the antioxidant may be delivered to the tumor site at an early stage may inhibit the early events where ROS required for tumor development. In this section, we will discuss the production of ROS and signaling in cancer, therapeutic approaches, and drug delivery system which elevate the ROS induced tumor apoptosis. The modulation of ROS may act as a potential target of chemotherapeutic delivery for tumor treatment. Keywords

Oxidative stress · Biomolecules alteration · Transcription factors · Reactive oxygen species · ROS signaling · Nanotherapeutics Abbreviations

13-HPODE 5-FU Akt AMPK AP-1 ATP Cyt c DNA DOX ER ERK1 ETC FOXO G6P GSH GSSG GTP HIF-1α JNK MAPK MCSCs MEKK1 miR-34a MMP-9

13 hydroperoxyoctadecadenoic acid 5-flourouracil Protein kinase B 50 adenosine monophosphate-activated protein kinase Activated protein 1 Adenosine triphosphate cytochrome c Deoxyribonucleic acid Doxorubicin Endoplasmic reticulum Extracellular signal regulatory kinase 1 Electron transport chain Forkhead box O Glucose 6- phosphate Glutathione Glutathione disulfide Glutathione peroxidase hypoxia-inducible-factor-1α c-Jun N-terminal kinase Mitogen-activated protein kinase P38 Metastatic cancer stem cells Mitogen-activated protein kinase kinase kinase 1 microRNA-34a Matrix metallopeptidase 9

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mTOR NADPH NCs NF-kLB NOX NPs Nrf2 O/W PEG–PLA PI3K PKM2 PLK1 PPP PTEN PTPC RGD ROS SDOs STAT 3 TICSCs TME TNF VDAC W/O

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mammalian target of rapamycin Nicotinamide adenine dinucleotide phosphate Nanocarrier systems Nuclear factor kappa-light-chain-enhancer of activated B cells NADPH oxidase Nanoparticles Nuclear factor-like 2 Oil in water poly(ethylene glycol)–polylactide Phosphoinositide 3-kinase pyruvate kinase embryonic M2 Polo-like kinase 1 pentose phosphate pathway Phosphatase and tensin homolog Permeability transition pore complex Arginylglycylaspartic acid Reactive oxygen species Superoxide dismutases signal transducer and activator of transcription 3 Tumor-initiating cancer stem cells Tumor microenvironment Tumor necrosis factor Voltage-dependent anion channel Water in oil

Introduction The oxidative stress may define as a disproportion of the generation of free radicals and reactive oxygen species (ROS) and their detoxification by protective mechanisms of the body. ROS, a chemically reactive oxygen-containing species (superoxide, peroxide, hydroxyl radicals, etc.), are byproducts of normal oxygen metabolism and play a role in the cell signaling and homeostasis (Haider et al. 2019). ROS is highly reactive with DNA, proteins, lipids, and other biological molecules. The slight increase of ROS may be useful for cellular functions but the excessive increase of ROS may cause irreversible oxidative damage. ROS generation and altered oxidative stress are observed in most cancer cells. The dramatic increase of ROS level that damage and distorted the cell’s proteins, lipids, and DNA oxidation causes the malignant lesion in cells and finally contributed to carcinoma (Haider et al. 2019). The increased ROS production and the altered biochemical status of cancer cells can be used for therapeutic targeting. Large amounts of ROS production in cancer cells might occur either due to the suppression of the antioxidant system or by increased ROS production. Initially, therapeutic antioxidants may protect the cells from damage and prevent tumor development in early stages. However, in later stages, the expression of antioxidant proteins also increased to detoxify the ROS,

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probably maintaining suitable intracellular ROS levels for uninterrupted cancer cell function and ensuring cancer cell survival. Since excessive ROS levels can be toxic to cells, cancer cells that possess high oxidative stress make them more prone to be harmed by further ROS insults induced by exogenous agents. Numerous studies confirmed that the high concentration of peroxide ions is found in most cancer cell lines viz. colon carcinoma, breast carcinoma, pancreatic carcinoma, malignant melanoma, ovarian carcinoma, neuroblastoma, etc. (Halliwell 2007). Alteration in ROS levels results in the activation of the different transcription factors like nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kLB), activator protein-1 (AP-1), signal transducer and activator of transcription 3 (STAT 3), which plays a role in cells transformation, survival, proliferation, metastasis, etc., in cancer (Tabish et al. 2018). Cancer cells have many genetic changes. The oxidative stress regulates the expression of numerous genes like tumor cells suppressor genes, antiapoptotic, proapoptotic genes, etc., as well as modification in gene expression and signaling pathways that overall supports in cancer development and survival (Yang et al. 2016). The targeting of unique biochemical alteration, which causes oxidative alteration, may be an effective therapeutic tool to achieve the anticancer effects. The dramatic changes in ROS overproduction/oxidative stress are cases in which cancer cells are used the ROS for their development, survival, etc. These biochemical aspects can be exploited for treating cancer. Cancer cells require some levels of ROS, up or down, which causes cytotoxicity in cancer cells. There are two strategies either reduce the overproduced ROS or enhance ROS production. The specific signaling pathways may be identified to target cancer cells ROS-sensing signaling pathways, which are involved in various stress-regulated cellular functions. Initially, the level of ROS overgeneration plays an essential part in the cancer development. The reduction of ROS levels may be one of the strategies for tumor treatment. The free radicals scavenging system, that is, superoxide dismutases (SDOs), glutathione peroxidase (GTX), peroxiredoxins, glutaredoxin, thioredoxin, are disrupted in cancer which helps in elevation in the level of ROS. Melatonin (MLT) is a molecule derived from tryptophan, which reduces verities of free radicals that limiting the oxidative damage by enhancing/stimulating several antioxidant enzymes such as GTX, SDOs, G-6-P dehydrogenase, glutathione S-transferase, and glutathione, which inhibit ROS by catalytic activity (Paulsen and Carroll 2013). The studies showed that antioxidants have anticancer activities in different cancers. The antioxidant tempol (4-hydroxy2,2,6,6-tetramethylpiperidine-N-oxyl) acts as a chemo-preventative agent by reducing the ROS, restored mitochondrial membrane potential, reduced tissue oxidative damage, and oxidative stress (Schubert et al. 2004). The antioxidants β-carotene and maesil have anticancer effects in 7,12-dimethylbenz[a]anthracene, 12-O-tetradecanoyl phorbol 13-acetate-induced mouse skin carcinogenesis (Kim et al. 2014). Other strategies involving the elevation of ROS generation in cancer cells can lead to apoptosis via mRNA mutation and defective biosynthesis, which initiates and stimulates caspase activity (Haider et al. 2019). Some of the drugs alteronol, procarbazine, monoclonal antibodies like rituximab, arsenic trioxide, STA-4783 (also known as elesclomol), etc., induce the ROS production.

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The nanocarrier systems (NCs) are nanosized particulate and vesicular systems used to deliver drugs, genetic materials, peptides, etc., to the specific target sites for treating different diseases. The NCs may be used as a targeting tool for different cellular components like the nucleus, mitochondrial, etc., for cancer treatment. Various polymeric, non-polymeric, metallic nanoparticles (NPs), and other NPs are used as the cancer chemotherapeutics (Haider et al. 2019). There are several NCSs that respond to ROS for drug triggers and efficacious cancer therapy (Shanmugapriya et al. 2019; Xin et al. 2019; Qiao et al. 2018). In this chapter, we will discuss the biological functions of ROS, transcriptions factors, the mechanism for cancer development, biomolecules functions, etc., and the NCs explored for ROS induced cytotoxic treatment in different cancers.

ROS and Cancer ROS causes genomic instability, alterations in signaling pathways, and gene expression, thus promoting cancer development. The molecular and cellular makeup is altered by the transformation process which leads cell to become malignant. The overexpressed ROS damaged the DNA and accumulation of incomplete repair or mis-repair may induce the mutagenesis and transformation, leading to the lack of apoptosis. NADPH oxidase (NOX)-4 generated ROS mediates the antiapoptotic effects by inhibiting the protein tyrosine phosphatases and assisting in the sustained activation of kinases (Lee et al. 2007). The overexpressed ROS in cancer also induces the tumor cells epigenetics, inflammatory conditions, metastasis, angiogenesis, invasion, proliferation, etc. ROS also plays a role in autophagy regulation with the help of transcription factor activity such as NFƙB leads to the induction of autophagy genes, that is, the expression of BECLIN1/ATG6 in breast cancer cells (Haider et al. 2019).

ROS and Its Biological Functions ROS are naturally occurring byproducts of oxidative metabolism and play an integral role as signaling molecules in the various biological processes like cell survival, differentiation, death, inflammation-related factors, etc. The production of ROS predominantly takes place in the mitochondria at the electron transport chain (ETC) in the inner mitochondrial membrane during the oxidative phosphorylation as well as external stimuli (Fig. 1) (Abdal Dayem et al. 2017). The endoplasmic reticulum (ER) plays a pivotal role in ROS production. The leakage of electrons from the electron complex1 and electron complex 3 leads to a partial reduction of the oxygen molecule into superoxide (O2•
), a short life-spanned free radical, which rapidly reduced to hydrogen peroxide (H2O2) by SODs, that is, mitochondrial interspace SOD1 and mitochondria matrix SOD2 H2O2, has a long biological lifespan and high stability non-radical ROS. The NOX also generates the O2•
has an activity to initiate the lipid peroxidation and inactivation of enzymes

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Fig. 1 Sources of reactive oxygen species (ROS) generation. (a) Descriptive diagram outlining the extracellular and intracellular sources of ROS generation. The extracellular sources of ROS are represented by environmental pollutants, radiation exposure, microbial infection, and exposure to engineered NPs. Intracellular ROS can be generated from the mitochondria, endoplasmic reticulum (ER) stress, cellular-metabolizing enzymes, and the NOX family; and (b) a schematic diagram summarizing the formation of ROS from nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and mitochondria and the mechanisms involved in ROS scavenging of ROS. CAT: catalase; e
: electron; GR: glutathione reductase; and GSSG: Glutathione disulfide. (Copyrighted image republished under CC4.0 license Ref. Abdal Dayem et al. (2017))

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Fig. 2 In the normal cellular metabolic condition, the antiapoptotic protein, that is, Bcl-2 only protein attaches to voltage-dependent anion channel (VDAC) and is inactive form. The proapoptotic protein like Bax/Bak presents in the inactive form. Hexokinase attaches to VDAC and converts glucose (Glu) to glucose 6-phosphate (G6P). Permeability transition pore complex (PTPC) normally allows the selective entry and the mitochondrial transition potential is high, it is due to the respiratory chain and PTPC conductance is low. But if any pro-apoptotic signal appears, the dissipation in mitochondrial potential takes place which activates the oligomerization of Bax/ Bak, and Bax directly binds to PTPC and BH3-only protein bind to Bcl-2 and detached the from PTPC and finally enhancement in PTPC conductance. The outer mitochondrial membrane permeation increased by which Cyt-c release from intra-mitochondrial membrane space to cytosol which finally activates the caspase 3 and cell go for apoptosis. (Copyrighted image republished with permission Ref. Haider et al. (2019))

(Abdal Dayem et al. 2017). The various detoxifying enzymes of intermediate ROS, that is, catalase, SODs, GTXs, etc., and antioxidants such as ascorbic acids, vitamin A, glutathione (GSH) act as free radicals scavenging agents. There is a wellestablished balance between the ROS generation and its metabolism required for biological signaling and other vital cellular functions. In stress cell conditions, increased ROS leads to increased apoptosis by the released cytochrome c (Cyt c) and leads to activation of caspase 3 (Fig. 2) (Haider et al. 2019), but in moderate ROS elevation regulate survival, differentiation, and proliferation. Increased Ca2+ in cytoplasm activates the mitochondrial ETC, which produces the ROS by complex I and complex III (Figs. 1b, 2). Nitrogen oxides are nonmitochondrial source of ROS generation and play a role in superoxide formation by reducing oxygen by the electronic donor NADPH (Abdal Dayem et al. 2017). Unlike the participation of cellular processes and signals, abnormally high ROS, in the case of oxidative stress

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responsible for damage and deformation of cellular proteins and lipids, eventually lead to oxidation of DNA and, therefore, cause fatal lesions, which contribute to carcinogenesis and also support the survival of cancer cells by activation of different transcription factors as well as by regulation of autophagy, cells transformation, tumor cell survival, tumor cell proliferation, angiogenesis, metastasis (Haider et al. 2019).

Role and Mechanism of ROS in Cancer Development Cancer cells meet their energy requirement in the form of ATP (adenosine triphosphate) mainly produced from aerobic glycolysis rather than the mitochondrial respiration, as in the case of normal cells, and this is known as the Warburg phenomenon. The presence of carcinogenic mutations in highly proliferative cancer cells promotes metabolic and protein translation, increasing ROS production rate. The Warburg effect is useful for modulating the antioxidant system, preventing ROS accumulation and transforming cells. The glycolytic enzyme pyruvate kinase embryonic M2 isoform (PKM2) prevents intracellular ROS accumulation, but the increase in intracellular ROS can inhibit PKM2. The inhibition of PKM2 redirects glucose flux into the pentose phosphate pathway, resulting in the generation of reduction potential for detoxification (Haider et al. 2019). By this, regulatory properties of PKM2 give a protective function to cancer cells against excessive ROS production. Cancer cells produce high ROS levels, reflecting an increase in metabolic rate, which can reach their peak in a constant state of oxidative stress. The continuous increase in ROS production contributes to the incidence of tumors and cancer development by increasing genetic instability, increasing DNA damage, and reducing mismatch repair. Alteration in DNA, such as base modifications and crosslinking of DNA-protein and strand breaks, is strongly involved in the initial stage of carcinogenesis. The hydroxyl radicals cause the DNA double-strand breaks as well as purine and pyrimidine lesions, by which the genomic integrity is distorted in cancer (Ziech et al. 2011). The mitochondrial DNA damage has contributed to human carcinogenesis. Mutation in the mitochondrial DNA occurs due to ROS exposure because of the presence close to the respiratory chain. ROS controls the expression of several genes to suppress the tumor and improves the process of cancer development by modifying gene expression, which causes gene instability and alterations in signaling pathways. The oxidative DNA damage during normal cell proliferation, p53 triggers the mechanisms that eliminate oxidative DNA and leads to programmed cell death. However, during the carcinogenic process, there is an imbalance between cell proliferation and cell death that moves towards uncontrolled cell proliferation (Ziech et al. 2011). The p38 mitogen-activated protein kinase (MAPK) plays a significant part in tumor initiation by oxidative stress. Human tumors are shown to have mutated or overexpressed p38 MAPK regulating genes (Ziech et al. 2011). Some oncogenes such as HRas, NRas, and Neu are induced by the high levels of ROS, and this result is associated with an elevated transformation of p38α-deficient fibroblasts (Dolado et al. 2007). The abnormal elevated ROS

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during the tumorigenesis may transform Ras-transformed fibroblast and overexpressed superoxide-generating oxidase Mox1 transform immortalized NIH3T3 fibroblasts (Myatt et al. 2011). The ionizing radiation-induced oxidative stress has been shown to induce phosphoinositide 3-kinase (PI3K), MAPK, c-Jun N-terminal kinase (JNK), and p38 signaling pathways as well as inhibit protein tyrosine phosphatase activity, both of which play an essential role in controlling cell proliferation and inducing metastasis (Ziech et al. 2011). Besides causing genetic changes, ROS can also give rise to epigenetic alterations; thus, playing a role in cancer development. The elevated ROS specifically induces alterations in the DNA methylation pattern (Ziech et al. 2011). In gliomas, epigenetic changes are shown to be involved in the deregulation of DNA methylation, nucleosome rearrangement, and histone acetylation (Sanchez-Perez et al. 2017). Excessive ROS production promotes these modifications. Oxidative DNA damage causes interference in interaction of methyltransferase with DNA, inducing alteration in methylation of cytosine residues at CpG sites. ROS also reduces catalase expression by stimulating the methylation of CpG Island in liver cancer (Ziech et al. 2011). ROS can modify indulgent and oppressive histone markers. The post-transitional modification in histone has been implicated in chromatin regulation and gene expression. Mitochondrial dysfunction can result in histone hypermethylation by an increased ROS level, succinate, or fumarate, which inhibits histone demethylase (Matilainen et al. 2017). Heterogeneity is the hallmark of cancer and an essential feature of differentiated stem cell populations from tumor cells to normal and causes resistance to chemotherapeutic agents and compromises efficacy. Heterogeneity is overwhelmingly affected by elevated levels of ROS. ROS has an impact on cancer stem cells. Considering ROS as a mutagen can block self-renewal or stimulate stem cell differentiation (de Sá Junior et al. 2017). The ROS signaling network may also influence cancer drug-resistant cells by regulating Forkhead box O (FOXO) transcription factor. FOXO transcription factors can control ROS generation and metabolism and contrarily FOXO activity regulated by ROS, thus play a crucial role in cancer development (Myatt et al. 2011). Mitochondrial overproduction of ROS reduces the amount of ATP available to the efflux pump by oxidizing NADH to NAD+, and this phenomenon is helpful to reduce the cytotoxic drug resistance (Myatt et al. 2011).

ROS Induced Transcription Factor and Regulation Many studies established that ROS can be responsible for modulation of activity of transcription factors directly or indirectly by acting as their activators. Cells’ physiological function requires ROS, but excessive ROS activates JNK, disruption in mitochondrial membrane potential, and/or caspase directly mechanism, leading to cell apoptosis (Fig. 2). In cancer cells, ROS is associated with a variety of transcription factors, such as AP-1, NF-kLB, FOXO, STAT 3, and others, which are involved in cellular transformation, cancer cell survival, cancer cell proliferation,

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angiogenesis, metastasis, etc. (Haider et al. 2019; Myatt et al. 2011). The outcome of ROS formation in the growth factor receptor signal is the activation of downstream effector kinases in the MAPK (also known as modulating transcription factor) family. The other MAPK family members, such as p38 MAPK, extracellular signal regulatory kinase 1 (ERK1), and cJNK, have been shown to have ROS-sensitive kinase activity (Benhar et al. 2002). The functional role of elevated ROS level is associated with the activation of Erk-1/2 and p38 MAP kinase and tumor progression (Ziech et al. 2011). It has been shown that MAPK regulates AP-1 activity in response to various stimuli. The nuclear factor κB (NFκB) is one of the antiapoptotic factors, and its activities are linked with tumorigenesis. The generation of ROS stimulates transcription by AP-1 and NFκB (Dhar et al. 2002). According to the report, NFκB activation was regulated by MEKK1 by JNKs upstream and p38 MAPK (Sakon et al. 2003). The studies suggested that antioxidants can inhibit MAPK activation. NFκB stimulates cell death by downregulation tumor necrosis factor (TNF), which induced JNK activation. TNF stimulation accumulates the ROS, later is essential for the prolonged MAPK activation (Sakon et al. 2003). NFκB activation may activate from oxidizing agents without any physiological stimulation and is inhibited by a wide range of chemically unrelated antioxidants. The ROS accumulation may induce by overexpressed c-Myc or E2F1 (Tanaka et al. 2002). Another transcription factor, nuclear factor erythroid-2-related factor 2 (Nrf2), a bZIP transcription, plays a role in controlling the cellular response to increased oxidation states. It has been established that Nrf2 modulators of antioxidant responses are essential for the survival and development of myeloid hematopoietic stem progenitor cells. Inhibition of NFκB by ikkβ or rela gene deficiency sensitivity to stress response through enhanced or longterm activation of JNK and persistent activation of NFκB inhibits cytokine-induced JNK activation (Tanaka et al. 2002). ROS regulates various transcription factors that play an essential role in cancer development, progression, metastasis, invasion, drug resistance, etc. In addition to it, ROS also gets regulated by various factors like Nrf2, TNF, MAPK, etc.

ROS Altered Biomolecules Functions When the production of free radicals exceeds the ability to destroy cells, high oxidative stress is established, which reduces the redox-sensitive signaling pathways and multicellular biological molecules, that is, DNA, proteins, sugars, and lipids (Sesti et al. 2012). The alteration in DNA due to ROS is already discussed above. In brief, ROS can cause DNA alterations like alteration in DNA sites (apurinic/ apyrimidinic), oxidation in purines and pyrimidines, DNA strands break, DNA base modifications (8-oxo-7,8-dihydroguanine, and 2,6-diamino-4-hydroxy-5formamidopyrimidine), DNA protein cross-linking, non-DSB clustered DNA lesions, etc. (Sesti et al. 2012). Increased oxidative stress affects the proteome destroying proteins via the formation of peroxyl radicals or oxidation of cysteine residues, which causes conformational alteration resulting in alteration or degradation of other proteins or DNA

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and enzymatic inactivation (Sesti et al. 2012). These alterations of protein cause the loss of function, initiation of some functions or switches over the functions. For example, inhibition of phosphatases causes the inability in the regulation kinasemediated transduction pathways. The deregulation in AP-1 protein may affect the signaling pathways and cause oncogenic transformation by activating of some oncogenic genes (Dhar et al. 2002). The level of O2˙
is significantly increased, which may cause the decrease GSH, GSSG, and ratio of GSH/GSSG in cancer (Schieber and Chandel 2014). Glutamine (nonessential amino acid), an essential intermediate and precursor of glutathione synthesis, plays an essential role in tumor metabolism. The enhanced catabolism is a crucial tumor metabolism hallmark through which cancer cells support cell proliferation, signal transduction, and redox homeostasis. A decreased activity and expression of the mitochondrial form of manganese superoxide dismutase has been reported to some cancers like colorectal carcinomas, pancreatic cancer, breast cancer, lung cancer, etc. Other than the DNA, the orientation of tyrosine and collagen is also changed in cervical cancer (Daniel et al. 2016). ROS exerts the physiologic action on the lipids. Mitochondrial ROS production is known to cause covalent modifications in lipids that produce lipid peroxidation. Lipid peroxidation in normal cellular condition proceed in controlled and limited in some cellular compartment, but the uncontrolled lipid peroxidation is harmful. Lipid peroxidation may alter the noncovalent interactions within the membrane bilayer, contributing to local membrane destabilization. Elevated ROS generation can deregulate the equilibrium and cause excess lipid peroxidation. Lipid peroxides cause cell destruction in the breakdown of toxic products that are soluble aldehydes. They exhibit easy reactivity with biomolecules such as proteins, DNA, and phospholipids to form various intra- and inter-molecular toxic covalent additives and cause other cellular abnormality. The main target of lipid oxidation is the cell membrane. The ROS causes alteration in the plasma membrane lipids like linoleic acid and arachidonic acid. Other important reactive aldehydes malondialdehyde are produced by membrane lipid hydroperoxides breakdown. The reduced phospholipid hydroperoxide glutathione expression is found in different cancers, that is, pancreatic, lung cancer, etc. The overexpression of phospholipid hydroperoxide GTX may provide effective targets for cancer therapy.

Oxidative Stress Adaptation in Cancer Cancer cells generate a high ROS level to support tumorigenesis and activate different signaling pathways to encourage proliferation, survival, and metabolic alteration (Fig. 3a). Nevertheless, the high level of ROS has also increased the susceptibility of cancer cells to cell death. The increased level of ROS causes apoptosis (Fig. 2). Cancer cells create some adaptation to maintain the tolerable ROS level to survive and avoid apoptosis (Fig. 3b) (Schieber and Chandel 2014). The enzyme, 50 adenosine monophosphate-activated protein kinase (AMPK) activation, is one of the critical adaptions for the energy stress that later induces oxidative

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Fig. 3 ROS regulation of normal and cancer cell proliferation. (a) H2O2 is required for activation of a number of cellular pathways involved in cellular growth, survival, and proliferation and in metabolism and angiogenesis. (b) Cancer cells generate higher levels of ROS that are essential for tumorigenesis. Genetic alterations leading to activation of oncogenes (PI3K, MAP kinase, HIFs, NF-kB) and loss of tumor suppressors (p53) coordinate an elevated redox state. ROS is also generated by increased oxidative metabolism and hypoxia in rapidly expanding tumors. In addition, cancer cells express elevated levels of cellular antioxidants (SODs, GSH, and PRx), in part through NRF2, to protect against oxidative-stress-induced cell death (Copyrighted image republished with permission Ref. Schieber and Chandel (2014)

stress. In cancer cells, high levels of ROS cause the inhibition of antioxidant gene expression and increases mitochondrial metabolism through the inhibition of FOXO transcription factors, which leads to hyperactivity of Akt (Myatt et al. 2011). The aberrant regulation of carcinogenic signal transduction stages, such as PI3K /Akt pathways, is among the most common unregulated pathways found in cancer. FOXO1 induces Sestrin3 gene expression. FOXO may act as a regulator that maintains a homeostatic balance between Akt and mTOR complexes activities (Myatt et al. 2011). The other is FOXM1 has several prototyping effects but has been shown to reduce ROS levels through transcriptional induction of SOD2, catalases, and mitochondrial thioredoxin-dependent peroxide reductase (Nogueira and Hay 2013). The detoxifying enzymes concentration such as glutathione transferases, NAD(P)H: quinone oxidoreductase 1, etc., are elevated in cancer to maintain the tolerable limits of ROS (Belinsky and Jaiswal 1993). The pentose phosphate

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pathway (PPP) is one of adaptation of cancer cells. PPP is vital for cancer cell metabolism and survival. In the first phase of glucose metabolism, PPP derived from glycolysis is essential for ribonucleotide synthesis and is the main source of NADPH. Thus, PPP plays an essential role in meeting the anabolic needs of these cancer cells and fighting against oxidative stress.

Therapeutics Targeting Oxidative Stress Alteration in Cancer Several promising strategies using the concept of nanotechnology have been used in anticancer therapies. The uses of nanocarriers system have been widely studied from time to time, introducing newer concepts and approaches providing the tremendous potential in anticancer therapies. Various smart nanocarriers (as nanoparticulate and nanovesicular) like nanoparticles (NPs), liposomes, micelles, nanoemulsion, etc., are found to be useful as therapeutics targeting oxidative stress alteration in cancer (Table 1). The extent of ROS production and cytotoxicity caused by NPs relies on their unique physico-chemical properties. Some important parameters are size, shape, electrical charge, stability, and composition of the NPs core and shell (Sukhanova et al. 2018). Small-sized NPs penetrate the cell and organelles easily and cause more cytotoxicity as compared to the larger NPs. Other than the size, chemical composition, and crystal structure are important influencing the cytotoxicity of NPs. 20-nm silicon dioxide NPs alter the DNA structure and zinc oxide NPs cause oxidative stress (Sukhanova et al. 2018). Reports have shown that upon UV-A irradiation, out of four different sizes (800 possible peak identified in CSF Alpha-1-acid glycoprotein, clusterin, transthyretin in serum Exoprotease in serum Serum peptidome, dehydro-Ala(3)fibrinopeptide Transitional cell carcinoma (TCC) from urine Multiple plasma protein Neural-network models

Breast

Uterus

Analytes >100 peptides

Cancer Type Prostate

Table 1 (continued)

Capillary microsampling MS with fluorescence microscopy

H1/ C13 1D and 2D NMR, HR MAS MR spectroscopy UPLC-MS and tandem MS

Detected more than 30 other metabolites Accuracy 100% Sensitivity and specificity 100%

Surface plasmon resonance imaging (SPRi) with MALDITOF mass spectrometry (SUPRAMS)

SELDI-TOF/MS SELDI-MS

SELDI-MS

MALDI-TOF(-TOF)-mass spectrometry MALDI-TOF/MS MALDI-TOF/MS

MALDI-TOF

Platform used MALDI-MS

29 metabolites and 54 lipids in different cell cycle stages from single cell of average 18μm size

78.3% sensitivity and 65.0% specificity 92% sensitivity and 97% specificity Sensitivity 81.8% and specificity 83.3% Limit of detection1 μg/ mL

97.5% sensitivity 95% sensitivity and 95% specificity

Salient observations 71.2% specificity and 67.4% sensitivity Sensitivity of 79% and specificity of 76%

Gaul et al. (2015)

Sitter et al. (2002)

Zhang et al. (2018)

Remy-Martin et al. (2012)

Lin et al. (2006) Rogers et al. (2003)

Villanueva (2005) Villanueva et al. (2006) Munro et al. (2006)

Goufman et al. (2006)

Dekker et al. (2005)

References M’Koma et al. (2007)

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Inositol, changes in Nacetyleaspertate to choline ratio, altered level of lipid and creatine. Amino acids, carnitines, lysophospholipids

Pyruvic acid, glycolic acid, tryptophan, palmitoleic acid, fumaric acid, ornithine, lysine, and 3-hydroxyisovaleric acid.

2,4-hexadienoic acid, 4-methylphenyl dodecanoate and glycerol tributanoate Phosphocholine, glutamate, hypoxanthine, arginine and α-glucose

Brain cancer

Colorectal cancer

Gastric cancer

Prostate cancer

Lung cancer

Renal cancer

Cervical cancer

Pancreatic cancer

Elevated level in AAs, lactate and phosphorylethalonamine Phosphatidylinositol, phosphocholine and glycerophosphocholin High level of Choline and AAs, and low level of glucose in malignant tissues Different Triglyceride and cholesterol

Liver cancer

Differentiate between prostate cancer and benign tissue as well as ERG-positive prostate cancers from the ERG-negative ones.

42 altered metabolites, mostly related to glutamate and glutamine metabolism pathway Detected 64 metabolites in the plasma samples, 29 metabolites differed significantly between healthy and cancer patients. Sensitivity-99.3%, specificity93.8% Detected 16 serum metabolites

As low as 2 mg samples

Accuracy 100%

Sensitivity 98%, Specificity 94% Accuracy 96% High in resolution

High

1H HR MAS NMR and 1H/31P NMR and LC-MS

HPLCESI/Q-TOF-MS

GC/QQQ-MS

DI-ESI-QTOF-MS and GC-MS

1H Magic angle spinning (MAS) NMR, 2D NMR, 2D JRES, 2D TOCSY, HMQC H1, C13, 1D and 2D NMR,

HR MAS MR spectroscopy

H1 NMR

H1 NMR

Proteomics and Metabolomics in Cancer Diagnosis and Therapy (continued)

Dudka et al. (2020)

Wang et al. (2017)

Nishiumi et al. (2017)

Callejón-Leblic et al. (2016)

Tzika et al. (2007)

Moka et al. (1998)

Sitter et al. (2004)

Beger et al. (2006) and Fang et al. (2007)

Yang et al. (2007)

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Bladder cancer

Thyroid cancer

Cancer Type Squamous cell carcinoma

Analytes Choline, p-hydroxyphenylacetic acid, 2-hydroxy-4-methylvaleric acid, valine, 3-phenyllactic acid, leucine, hexanoic acid, octanoic acid, terephthalic acid, γ-butyrobetaine, and 3-(4-hydroxyphenyl)propionic acid, isoleucine, tryptophan, 3-phenylpropionic acid, 2-hydroxyvaleric acid, butyric acid, cadaverine, 2-oxoisovaleric acid, N6,N6,N6-trimethyllysine, taurine, Glycolic acid, 3-hydroxybutyric acid, heptanoic acid, alanine, and urea Cysteine, cystine, glutamic acid, α-ketoglutarate, 3-hydroxybutyric acid, adenosine-5-monophosphate, uracil, and sucrose Dopamine 4-sulfate, aspartylhistidine, tyrosyl-methionine, 3-hydroxy-cis-5tetradecenoylcarnitine, 6-ketoestriol, beta-cortolone, tetrahydrocorticosterone, heptylmalonic acid

Table 1 (continued)

Suggested sucrose as a circulating biomarker for differential diagnosis between malignant and benign thyroid nodules, ROC value 0.92 Sensitivity and specificity were 0.881 and 0.786, respectively

Salient observations Detected25 metabolites as potential markers to discriminate between healthy and cancer patients

LC-HRMS

GC-quadrupole-MS

Platform used CE-MS

Cheng et al. (2018)

Abooshahab et al. (2020)

References Ohshima et al. (2017)

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Fig. 1 General workflow for cancer proteomics

ultra-performance liquid chromatography (UPLC), capillary electrophoresis (CE), one or two-dimensional electrophoresis (1D or 2DE) followed by mass spectrometric (MS) analyses. 3. Mass spectrometry (MS) is the backbone of cutting-edge proteomics analytical platforms. Matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) are the most commonly employed peptide ionization method used for MS analyses. Other less-frequently used ionization methods include but not limited to surface-enhanced laser desorption/ionization (SELDI), nanoelectrospray ionization (nanoESI), atmospheric pressure chemical ionization (APCI), matrix-free LDI mass spectrometry including Desorption/ionization on silicon (DIOS), matrix-free on-chip pulse-heating desorption/ionization (PHDI). Followed by ionization, the analytes need to be separated based up on their mass to charge ratio which is carried out through suitable mass analyzer. Usually, four types of mass analyzers are most commonly used in proteomics research: quadrupole (Q), ion trap (quadrupole ion trap, QIT; linear ion trap, LIT or LTQ), TOF, mass analyzer, and Fourier-transform ion cyclotron resonance (FTICR) mass analyzer. Combination of these mass analyzers, for instance, Q-Q-Q, Q-Q-LIT, Q-TOF, TOF-TOF, LTQ-FTICR, etc., provides enhanced sensitivity. Further, introduction of Orbitrap mass analyzer and its integration with any ion-trap type mass analyzer, such as LTQ-Orbitrap, increases robustness, sensitivity, mass accuracy, and resolution capacity of tandem MS (MS/MS) analyses. After

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sorting, the charged analytes are directed to the detector for generation of mass spectra. The detector recognizes the charge induced or current produced as the analyte ions pass by or hit the detector surface. Finally, protein identification is performed by using bioinformatics software and database search such as Mascot or Sequest, etc. 4. MS-based proteomic analyses for protein identification can follow either topdown or bottom-up approach. The top-down proteomics launches intact ionized proteins to a mass analyzer, while enzymatically or chemically fragmented peptides are introduced for MS analysis in bottom-up approach. Shotgun proteomics exploits bottom-up strategy for protein identification from a complex proteome by peptide mass fingerprinting (PMF). Quantitative proteomics can be carried out using labeling or label-free quantitation method. Stable isotopes such as isotopic coded affinity tags (ICAT), isobaric tags for relative and absolute quantitation (iTRAQ), and stable isotope labeling amino acids in cell culture (SILAC) are the most commonly employed label-based quantitative proteomic techniques. In label-free quantitative proteomics, quantitation is performed based on peptide ion intensity counting and spectral counting. Targeted proteomics is performed for analysis of selective known proteins from the objective sample, whereas untargeted approach is the choice for introspection of unknown protein from any target sample. 5. Non-MS based proteomics mostly rely upon antibody-mediated approaches including protein or antibody microarray, confocal microscopy, fluorescencebased methods, lab-on-chip devices and sensors, etc. 6. Additionally, data validation can be carried out on larger sample for confirmation of initial mining.

Proteomics Platforms MS Based Proteomic Approaches in Cancer Early diagnosis, prognosis, and targeted therapy are the key factors in “fight against cancer.” After coming through its “infancy stage,” MS based proteomic platforms are now mature enough and become a robust and powerful tool in screening of novel cancer biomarkers in advance onco-proteomic research and development. Various combinations of MS based platforms are in practices; some of the most common advance high through-put platforms for analytical purposes are MADLI and SELDI in combination with TOF/MS (MALDI-TOF/MS, SELDI-TOF/MS) or MALDIMSI or SELDI-TOF-MALDI. Another variant is ESI-MS in combination with LC (LC-ESI-MS, LC-MS/MS). All of the MS platforms work on the principle of measuring the mass-to-charge ratio (m/z) of different analytes when separated through a mass analyzer into a gaseous phase ions. Different analytes in their ionized form produce characteristic peak on the basis of their different m/z ratio which has been further recorded through a detector and decoded through existing software based databases using bioinformatics tools (Minakshi et al. 2019a).

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Electrospray Ionization (ESI) and Matrix-Assisted Laser Desorption/ Ionization (MALDI) These are the two most common method of ionization of protein or peptides from liquid and solid crystals, respectively. In MALDI, proteins are mixed with an excess of organic matrix and after ionization, allowed to travel through a vacuum channel where the mass analyzer (TOF) separate ions based on m/z ratio (Minakshi et al. 2019a). High sensitivity and versatility made MALDI-TOF/MS-MS is very useful tool in protein/peptide fingerprinting on novel biomarkers research for cancer diagnosis. MALDI profiling was used as a diagnostic tool in detection of cancer biomarker in various human cancers such as prostate (M’Koma et al. 2007), breast (Dekker et al. 2005), ovarian (Tiss et al. 2010) cancers, etc. SELDI-TOF In contrast to MALDI, SELDI or Surface-Enhanced Laser Desorption-Ionization utilizes protein chips with different physicochemical properties, that is, immobilized metal ions, affinity antibodies, or ion exchange. SELDI-MS is found useful for detection of melanoma (Caputo et al. 2005), cervical (Lin et al. 2006), and renal cancer (Tolson et al. 2004) in human. QUEST-MS Quick Enrichment of Small Targets for Mass Spectrometry is a unique and advance proteomic technology developed to detect low-molecular weight plasma biomarkers associated with cancer. Purification enrichment of low molecular weight proteins in a sequential reversed-phase chromatography followed by analyzing with LC/MS/MS is the working principle of the technique. Specific detection of neurotransmitter polypeptide neuropeptide-Y (NPY) as a marker of prostate cancer was identified and validated through this modality (Ueda et al. 2013). SPR-MS The combination of SPR and MS approach enables to detect analytes at a rangebound concentration. SPR produce a real-time, label-free sensitive platform for interaction of affinity-bound surface analytes with light photons followed by ligand identification using a mass detector alike MS. Quantitation and characterization of LAG 3 protein, a potential biomarker of breast cancer, was performed with SPR-MS platform (Remy-Martin et al. 2012). Chromatography Coupled with MS Precise separation of proteins from a complex biological specimen prior to MS analysis is often required to improve the sensitivity and specificity of the platform. Liquid chromatography (LC) integrated to tandem MS (LC-MS/MS), nano-RPLC-ESI-MS/MS, GC-MS/MS are the platforms widely employed for identification and quantitation of cancer biomarkers. For instance, multidimensional protein identification technology (MudPIT) is an emergent strategy which employs two or more LC columns in association with ESI-MS for protein identification (Palmblad et al. 2009).

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Non-MS Based Proteomic Approaches in Cancer Biology Antibody Based Platform Conventionally antibody is a component of adaptive immune system, produced in a response of any exogenous/altered endogenous signal as a normal adaptive defense mechanism of the body. Antibody specifically binds with its target, that is, “epitope” with its receptor binding domain “paratope.” Being bivalent in nature, antibody binds two different epitopes of multivalent antigen at a single time. Advancement came with the invention of hybridoma technique (Nobel Prize in 1984) for production of monoclonal antibody (mAb) which in contra to conventional antibody, bind single epitope of a multivalent antigen specifically. Down the decade, rDNA technology has achieved the potential to produce different components of antibodies (scFv, bFv) from these conventional as well as unconventional antibodies (dAb from HCAbs). Cancer cells generate many aberrant proteins having altered characteristics due to mutation or overexpression which evokes an immune response within the body. As a result, normal body produce circulating autoantibodies against those altered proteins, known as tumor specific antigens (TSAs). Early detection of either such circulating autoantibodies or TSAs makes antibody based cancer diagnostics platforms a viable and popular option.

Immuno-sensors Based on Lateral flow Assay (LFA) Lateral flow immunoassay is an antibody based “point of care” detection system which provides rapid qualitative response in terms of signaling either presence or absence of marker analytes. Horizontal or lateral (along the x-axis) movement of liquid sample containing specific biomolecules via capillary action of the membrane toward the test zone where conjugate-bound target analyte interacts with the specific antibody attached is the principle of any LFA device. A typical LFA strip/device contains three zones: first, sample zone impregnated with compatible buffer solution; second, conjugate pad zone containing target specific labeled-captured-antibody; and third, test zone. Samples with target analyte interact with conjugated antibody and move towards the test line where the antibody present at the test line interacts with the target analyte producing a visible line which indicates the presence of specific analyte in the sample. Further the sample continues to flow towards the control line where it interacts with antispecies antibody against captured antibody. Appearance of visible line indicates integrity and correctness of the device. LFAbased proteomic platforms have been successfully employed in cancer diagnosis and some of them are already commercially available. “Onsite” range of rapid tests for the prostate specific antigen (PSA) by CTK Biotech’s, OncoE6™ lateral flow device from Arbor Vita for detection of E6 onco-proteins of human papilloma virus (HPV) types 16 and 18 causing cervical cancer, CEA SerumRapid Test for detection of carcino embryonic antigen (CEA) in colorectal, breast and lung cancer by S or P Cortez Diagnostics Inc. with sensitivity 97% and specificity 100% are the few examples to mention.

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Tissue Micro Array (TMA) Tissue microarrays (TMAs) are high-throughput platform, introduced by Kononen and co-workers for automated analysis of protein expression level in multiple tissue samples simultaneously. Principally, the technique is similar to immunohistochemistry (IHC) except that in this, paraffin-embedded tissues with diameter 0.6–2.0 mm from donor re-embedded in an individual recipient block. This TMA block then subjected to re-sectioning, incubation with primary antibody and labeled (enzyme or florescence) secondary antibody. Final image is processed with advanced image analysis software assembled with scanning hardware and automatic IHC scoring system. Fluorescent-based AQUA (HistoRx) platform is an established, validated system available to study cytokeratin expression level in various tumor cells. Enzyme-Linked Immunosorbent Assay (ELISA) Antibody based enzyme-linked immunosorbent assay (ELISA) remains the gold standard test since its discovery by Engvall and Perlmann in 1971 against many diseases (Engvall and Perlmann 1972). The basic principle of ELISA is detection of antigen bound to solid-phase from a liquid sample (serum or tissue fluid) with specific primary antibody (targeted and raised against marker proteins) and antispecies enzyme labeled secondary antibody (Crowther 1995). The technique most commonly employs a chromogenic system to generate information regarding presence or absent of a marker protein in the solution, whereas recently fluorogenic and electrochemiluminescent system has produced higher sensitivity and specificity with plausible multiplexing (Leng et al. 2008). For instance, electrochemiluminescent based ELISA platform is mesoscale Discovery (msD) modality which has been successfully exploited for investigation of the effect of phosphoprotein inhibitor LY294002 on tumor xenograft in human (Gowan et al. 2007). Antibody Array Antibody Micro Array (AMA) is a protein array forward phase quantitative platform which requires two highly specific antibodies for each assay designed. In this technique, the first antibody directed against one of the epitope of the targeted protein coated on a suitable solid surface. The analyte then incubates against the spotted antibody. Specific protein can be detected by a labeled secondary antibody directed against another epitope of the same protein. One of the advantages of this platform is measurement of many analytes in a single sample simultaneously but in every case both the antibodies needed to be validated by western blot for its specificity (Boellner and Becker 2015). DotScan™ CLL antibody microarray for chronic lymphocytic leukemia and DotScan™ CRC microarrays for colorectal cancer manufactured by Medsaic Pty, Antibody Microarray 507 for prostate cancer by Clontech, PEX100 for lung and esophageal squamous cell carcinoma, and phosphorylation-specific antibody microarray for prostate cancer by Fullmoon Biosystems Inc. are few examples of commercially available antibody microarray systems for cancer biology applications (Chen et al. 2018).

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Reverse Phase Protein Array (RPPA) Reverse phase protein array (RPPA) is a high throughput, sensitive platform for analysis of hundreds to thousands of proteins from plethora of biological samples such as direct cells or laser captured microdissected tissues, CSF, urine, saliva. The technique was pioneered by Lance Liotta and Emanuel Petricoin in 2001 (Paweletz et al. 2001). Basically, the technique is a protein microarray system with a dot-blot platform. Processed samples are immobilized on a microarrays platform as a spot which is subsequently incubated with high quality specific antibodies for the measurement of protein expression level in a quantitative manner. This technique has gained popularity in personalized theranostics approach against cancer and implemented against patients with metastatic colorectal and breast cancers. Recently Theranostics Health, Inc. has developed the TheraLink HER Family Assay which targets the expression of marker protein human epidermal growth factor receptor 2 (HER2) for cancer detection (Lu et al. 2016).

Single Cell Proteomics in Cancer Single cell proteomics (SCP) is an advance technology in the field of proteomic research which allows studying all the proteins expressed within a homogeneous population of cells or precisely, a single cell at any specific time or over a period of time frame. Cancerous tumor often comprises of a heterogeneous cell population and few of them often shed and get circulated within different organs via blood stream or lymphatic system. This circulating peripheral tumor cells usually termed as circulating tumor cells or CTCs. These CTCs are often helps in spreading of cancer but a major constrain in detection of such cells includes their low abundance in the circulation (1 in 107 to 108 cell/ml). SCP based platform is very useful to overcome this challenge as the modalities produce ultra-high sensitivity. The isolation of single cell from a heterogeneous cell population often relies upon few techniques such as FACS, MACS, microfluidics, high density microarrays. Following isolation, the cells undergo various processing steps before subjected to analytical platforms for proteome analysis. Analytical platforms for SCM analyses include but not limited to antibody-based assay techniques such as fluorescence flow cytometry, mass cytometry (CyTOF), enzyme-linked immunospot assay (ELISpot assay), SingleMolecule Array (SiMoA), Microfluidic Antibody Capture Chip (MACS Chip), Proximity Extension Assay/Abseq, single-cell western blotting (scWestern), multiplexed profiling of single-cell proteome and transcriptome in a single reaction and MS-based platforms as well as living cell proteomics by mass spectrometry imaging (MSI) (Minakshi et al. 2019a). A FDA approved CTCs detection system is available in the name of CELLSEARCH ® which primarily targets EpCAM molecule expressed on the surface of CTCs via a specific antibody coupled with magnetic beads. Besides SCP, in 2018, a group of scientists from University of Toronto described a more advanced platform for capturing CTCs. They developed a “single cell mRNA cytometry” platform which targets specific mRNA within a single cell. The device is

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designed to detect CTCs originated from prostate cancer and has limit of detection 10 cells/ml of blood approximately.

Cancer Metabolomics Metabolomics is one of the emerging branches of the state-of-the-art “omics” technologies which can be defined as global analysis of small molecules (metabolites) including their identification, characterization, and quantification from a biological specimen such as body fluids, cell, tissue, organ, or organism at a specific point of time under precise genetic and environmental conditions (Oliver et al. 1998; German et al. 2005; Griffin and Vidal-Puig 2008); and the entire set of metabolites from the specific biological sample comprises the metabolome (Querengesser et al. 2007). Metabolites are the small active components generated as the end products or intermediates of many complex metabolic processes related to energy production, cell growth and division, signal transduction, etc. Alike marker proteins, signature metabolites can also be exploited to find out metabolic aberrations leading to disease establishment and progression. Hence, metabolites carry the most nascent information on any underlying biological event; thus, metabolomic introspection is extremely worthy to explore the molecular pathology of numerous diseases including cancer. Metabolomics can also aid in identifying the therapeutic check-points by elucidating the altered metabolic pathways nodes to customize effective drug regime. This can be helpful for precise drug targeting to avoid systemic side effects. Further, monitoring of key disease-specific metabolites can also provide an accountability regarding the effectiveness of undergoing therapeutic regime in due course of disease progression along with the tentative prognosis. Thus metabolomics has enormous potential in the diagnostic as well as therapeutic arena of complex disorders including cancer which has been depicted in Table 1.

Metabolomics Workflow Untargeted and targeted metabolomics are the two different approaches of metabolomics introspection which can be chosen according to the goal of the experiment (Patti et al. 2012). Untargeted metabolomics carry out exhaustive metabolite profiling to include all the metabolites from a given biological sample irrespective of any preference. The approach enables identification and comparison of metabolites from two contrasting groups (control and case) which can be undertaken to extrapolate any disease-associated metabolite signatures. The workflow of untargeted metabolomics includes sample preparation encompassing isolation of metabolites from biological samples and their enrichment to achieve suitable concentration followed by their identification in either qualitative or quantitative manner and subsequent structural as well as functional characterization employing suitable analytical platform such as NMR, CE/LC/GC-MS, fluorescence-based methods, or MSI equipped with advanced bioinformatics programs for database searching and functional

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annotations. A few prominent metabolomic databases include but not limited to XCMS (Smith et al. 2006), Pubchem, Metabolights, KEGG, METLIN (Smith et al. 2005), ChEBI, Chemspider, Massbank, MetaboAnalyst, LIPID MAPS, MS-Dial, mzCloud, HMDB, etc. (Minakshi et al. 2019b) (Fig. 2). Principal Component Analysis (PCA), Analysis of Variance (ANOVA), etc., are the usual methods of statistical analysis for disease-specific metabolite biomarker(s) identification and quantitation which often require further validation to establish their significance in relation to precise cellular or metabolic processes (Gowda et al. 2008; Patti et al. 2012). The Human Metabolome Database (HMDB) is currently the largest and most comprehensive, organism-specific freely available metabolomics database containing 114,223 metabolite entries. Moreover, it is linked with several other databases (KEGG, PubChem, MetaCyc, ChEBI, PDB, UniProt, and GenBank) along with different structure and pathway viewing applets. Further, the repository is associated with additional databases such as DrugBank, T3DB, SMPDB, and FooDB which provide information regarding drug and drug metabolites, toxins and environmental pollutants, pathway maps, food components and food additives, etc., relevant for disease diagnosis and therapy (https://hmdb.ca/). Analyses of specified known metabolite(s) involved in selected pathways are the objective of targeted metabolomics approach (Dudley et al. 2010). It is a quantitative method and usually intended to verify the postulated hypothesis regarding the predefined metabolites information dug out from existing references, novel experiments, and/or clinical findings. Typical targeted metabolomics workflow follows the

Fig. 2 Schematic outline of cancer metabolomics

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path of sample processing for extraction of target metabolite(s) followed by their confirmation and quantitation through suitable analytical platforms such as NMR, MS coupled with apposite chromatographic separation methods, fluorescence-based or other labeling-based imaging modalities. Finally, data processing and interpretation is carried out using specialized programs and bioinformatics tools. Targeted serum metabolomics has already been proved to be an efficient method for identification of several cancers such as ovarian cancer, lung cancer, pancreatic cancer. (Plewa et al. 2019; Ni et al. 2019; Jiao et al. 2019). Moreover, a combination of untargeted and targeted metabolomics can also be applied which has found valuable in cancer biomarker identification (Yang et al. 2020).

Metabolomics Analytical Platforms Most commonly employed analytical platforms for metabolomics introspection are nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS) coupled with different chromatographic separation methods. Liquid chromatography-MS (LC-MS) and gas chromatography-MS (GC-MS) are the most widely used MS-based metabolomics modality (Shao and Le 2019). Every platform has their inherent merits as well as limitations which need to be carefully considered to choose the suitable one that fits into the experimental requirement. For instance, NMR is very robust and reliable technique for routine metabolomic analysis requiring minimum sample processing; however, the sensitivity is less than MS. Thus, for single cell metabolomics study which demands extreme sensitivity, MS is most commonly employed rather than NMR. Hyphenated LC-MS-NMR has put its foot-print in the field of metabolomics achieving super spectral resolution and enhanced metabolite identification (Walker et al. 2016). Integration of these two complementary platforms will definitely strengthen the ability of quantitative metabolomics. Even the scope of LC-UV-Solid-Phase Extraction-NMR-MS based combinatorial approach has been explored with significant sensitivity improvement (Exarchou et al. 2003). Although several state-of-the-art metabolomics analytical platforms have been employed to explore cancer metabolomics with persistent enrichment in the literature by regular introduction of newer or modified ones, however within the limited purview of the current chapter, only some of the major analytical platforms are described hereunder along with suitable examples (Table 1).

NMR NMR is a robust technique for metabolite identification and quantitation from diverse biological samples without requiring prior chromatographic separation. One-dimensional nuclear overhauser enhancement spectroscopy (NOESY) sequence with water suppression is the most widely employed NMR method due to its robustness and generation of flatter baseline. However, sophisticated twodimensional (2D) NMR techniques such as 2D-J spectroscopy, correlation spectroscopy (COSY), total correlation spectroscopy (TOCSY), heteronuclear correlation (CH-COSY or HETCOR) spectroscopy, heteronuclear single quantum coherence

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(HSQC) spectroscopy, heteronuclear multiquantum coherence (HMQC) spectroscopy, exchange spectroscopy (EXSY), heteronuclear overhauser effect spectroscopy (HOESY) are also gaining importance in metabolomics studies including oncology. For instance, 2D HSQC in-cell NMR metabolomics of live HepG2 cancer cells using 13 C6-labeled glucose have successfully unearthed the temporal differences in metabolism between healthy and cancer cells and elucidated large in- and out-flux of pyruvate along with enhanced production of alanine and acetate only in cancer cells. Further, the approach has extended the scope of evaluating anticancerous metabolic effects of galloflavin along with identification of its novel functional targets (Wen et al. 2015). The excellent attributes of NMR include minimal sample processing, short analysis time, robust signal, and absolute quantification of metabolites. However, it can only detect the most abundant metabolites ( 1μM) due to relatively low sensitivity. Moreover, overlapping metabolite signals in absence of a proper separation system render accurate structure identification of metabolites often a tricky task. Recent technological achievements have alleviated some of the shortcomings and continuous pursuit is going on to improve the sensitivity and resolution of the NMR modalities (Gowda et al. 2008; Shao and Le 2019). 2D-COSYwith a microfluidic diamond quantum sensor is such an improved modality to overcome the resolution and concentration sensitivity issues of conventional NMR to achieve spectral resolution of 0.65  0.05 Hz from 40picoliter detection volume (Smits et al. 2019). Further improvements involving nitrogen-vacancy (NV)-doped diamond nanograting chip based 1H and 19F NMR have enabled to obtain high-resolution spectra from even 1-picoliter of fluid (Kehayias et al. 2017). Recently another modified NMR method employing solidstate spin sensor (a magnetometer) comprised of NV centers along with a narrowband synchronized readout protocol has achieved spectral resolution about one hertz from micrometer-scale sample volume of 10-picoliter (Glenn et al. 2018). The instances have paved the way toward potential NMR applications in masslimited metabolite analysis and single-cell biology.

MS MS is another major metabolomics analytical platform which is often preferred over the other modalities due to better sensitivity, high throughput, and its ability to detect wide array of metabolites from complex biological specimens. It can facilitate metabolite measurement at femtomolar to attomolar concentrations producing significantly higher resolution but quantitation is challenging and metabolite identification from complex samples often pose difficulty due to ion suppression. Thus, separation based MS techniques are often preferred where MS is usually coupled with prior separation modalities for analyte segregation from a complex biological cocktail. It can be broadly categorized into either chromatography based gas chromatography-MS (GC-MS) and liquid chromatography-MS (LC-MS) or electrophoresis based capillary electrophoresis-MS (CE-MS) and ion mobility-MS (IM-MS) (Ren et al. 2018).

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GC-MS GC-MS is usually the go to option for volatile metabolite analysis. It produces improved analyte separation, high sensitivity and resolution, reproducibility and stability, precise repeatable fragmentation along with easy and relatively inexpensive operation. Moreover, the availability of universal spectral database (such as NIST) and spectral deconvolution system (AMDIS) facilitates easy metabolite identification through GC-MS modality. However, tedious sample processing and derivatization of nonvolatile analyte counterparts are certain demerits of the technique to mention (Ren et al. 2018). GC-MS based metabolomic introspection has proficiently elucidated several key metabolic signatures from diverse cancer type cells and serum samples such as papillary thyroid carcinoma tissues, HT-29 colon cancer cells, MCF-7 and MDA-MB231 breast cancer cells, colorectal cancers, etc. (Semreen et al. 2020; Ibáñez et al. 2017; Chen et al. 2015; Hadi et al. 2017; Amir Hashim et al. 2019). LC-MS LC-MS is the predominant analytical platform in metabolic profiling due to high sensitivity and compatibility to diverse metabolites eliminating the criteria of analyte volatility and derivatization which is necessary for GC separation. Instead of gas used as mobile phase in GC, certain liquids serve the purpose in LC. Advent of cutting-edge ultra-high-performance LC (UPLC) platform employing sub-2μm stationary phase particles which can withstand enhanced solvent flow rates and high pressures ranged between 6000–19,000 psi have enabled to reduce the peak broadening thus increasing the peak capacity and reducing overlap, curtail the analytical run times, and improve analyte ionization resulting into reliable metabolite identification and improved structural determination even better than the conventional HPLC platforms. Equipped with gradient elution starting from highly aqueous to organic contents, C18 or C8 column based Reversed-phase liquid chromatography (RPLC) system coupled with MS is a well-acquainted analytical platform for diverse metabolites with enormous separation selectivity and detection sensitivity from complex biological samples. Ion-pairing liquid chromatography (IPLC) employing addition of ion-pairing agents into the chromatographic mobile phase is a modified RPLC platform for better retention of highly polar and ionic compounds which is usually difficult in conventional RPLC system. Hydrophilic interaction chromatography (HILIC) is another modified LC method employing polar stationary phase and organic mobile phase with at least 3% water. The water percentage is increased slowly during the elution; thus, lipophilic compounds are eluted early followed by elution of more polar compounds in due course of simultaneous increase in aqueous phase content. Although it generates wider peaks with lower peak capacity and lower resolution than RPLC, HILIC-MS is often used as complementary to RPLC-MS for its high selectivity to increase the coverage of metabolite identification.

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Supercritical Fluid Chromatography Supercritical fluid chromatography (SFC) is another analyte separation platform usually employs carbon dioxide as supercritical fluid to form the primary mobile phase, facilitating higher flow rates and lower pressure drops to be used for separation of polar as well as nonpolar compounds. Thus, SFC-MS has also introduced an additional artillery in the verse arsenal of metabolomics analytical modalities (Ren et al. 2018). These state-of-the-art techniques are already delivering plethora of valuable metabolomic insights towards diagnostic and therapeutic intervention in several patho-physiological conditions including diverse type of cancers. For instance, urinary metabolite analyses from liver cancer patients by both RPLC-MS and HILIC-MS have identified several key metabolites of amino acid metabolism as potential biomarkers to differentiate the cancerous patients from the healthy ones (Chen et al. 2009). Similarly, RPLC-MS and HILIC-MS based metabolite analysis has been depicted as potential method for identification of biomarkers of several cancer types such as renal cell carcinoma, prostate cancer, ovarian cancer (Kim et al. 2008; Zhang et al. 2013; Alonezi et al. 2016). Capillary Electrophoresis Capillary electrophoresis (CE) has gradually matured as a complementary method to chromatography for the electrophoretic separation of polar and charged metabolites under the influence of externally applied uniform electric field. Capillary zone electrophoresis (CZE) facilitates metabolite separation initially based up on charge-to-size ratios and subsequently on their mass-to-charge ratios, thus providing comprehensive separation and has become the mainstay of current CE-MS based metabolite identification. Low separation volume, excellent separation efficiency, sometimes even better than the GC and LC are certain advantages of the platform; however, poor system stability and frequent capillary blockage due to inadequate salt removal from the sample are posing limitations for routine use of this technique (Ren et al. 2018). CE-MS has been successfully employed to elucidate metabolite signatures from several cancer types such as colorectal cancers, colon and stomach cancer, uterine myoma and cervical cancer, hepatocellular carcinoma (Chen et al. 2012; Hirayama et al. 2009; Kim et al. 2001; Zeng et al. 2014). Due to small sample volume requirement, CE-MS can be preferred over other modalities for single-cellmetabolomics (SCM) analyses. For instance, CE-ESI-MS has been effectively employed for metabolite profiling of single Xenopus laevis embryo or single neuron cell of Aplysia californica (Nemes et al. 2012; Onjiko et al. 2017). So, application of the technique for metabolomic introspection in single cell oncology may not be too far away. Ion Mobility Spectrometry Ion mobility spectrometry (IMS) operates as a condensed gas phase electrophoresis analogue to separate gas-phase ions based on their size and shape which can be coupled with MS for metabolite analyses. It can be under taken by three approaches: drift-time IMS (DTIMS), travelling-wave IMS (TWIMS), and field-asymmetric IMS (FAIMS). IMS-MS can be beneficial for detecting isobars and isomers; further,

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increased signal-to-noise ratio, fast separation in milliseconds adds extra advantage to this modality (Lanucara et al. 2014). Recently, FAIMS-based metabolic and lipidomic profiling by paper spray ionization-IMS-MS (PSI-IMS-MS) has been depicted as potential method for rapid breast cancer diagnosis and early detection of cervical cancer (Huang et al. 2019; Mendes et al. 2020). IMS-MS can also be applied in structural proteomics to resolve macromolecular assemblies. For instance, DTIMS-MS has been successfully employed to elucidate the conformational diversity of cancer-induced P53 mutants (Jurneczko et al. 2013). The efficiency of separation-based MS platform depends upon both the competence of separation technique as well as the type of mass analyzer used in the MS modality. Different mass analyzers generally used in MS are ion traps, quadrupoles, triple quads, and time-of-flight. Validation of unknown molecules can be carried out through tandem MS (MS/MS or even MSn) analysis (Zhang et al. 2020). High resolution and a better mass accuracy can be achieved through Fourier-transform ion cyclotron resonance (FT-ICR) Orbitrap mass analyzers (Brown et al. 2005). Different separation-free MS techniques are also observed to be effective for metabolite identification. Commonly employed separation-free MS techniques include but not limited to Direct infusion-MS (DI-MS), MALDI-MS, mass spectrometry imaging (MSI), direct analysis in real time (DART)-MS, etc.

DI-MS DI-MS enables direct sample infusion to MS platform without involving any prior separation methods. Usually ultra-high-resolution MS modalities involving high end mass analyzers like FT-ICR or Orbitrap mass analyzers are employed for this purpose. Significantly shortened analyses time facilitating high-throughput screenings along with relatively simple data processing are certain advantages to the platform. However, ion suppression and spectral overlap due to narrow molecularweight distribution of the metabolomic components may pose bottle-neck to DI-MS analysis. Further, the platform is unsuitable for differentiating isomeric compounds. DI-MS has been recommended as a rapid diagnostic method for kidney cancer due to short analysis time while LC can be preferred for comprehensive biomarker screening (Lin et al. 2010). A novel DI-MS approach, termed as MS/MS-stitch, has successfully elucidated stearoyl CoA desaturase 1 (SCD1) as the therapeutic target of bezafibrate and medroxyprogesterone acetate as an anticancer drug combination against acute myelogenous leukemia (AML) and endemic Burkitt lymphoma (eBL) by introspecting the lipid metabolism (Southam et al. 2015). MALDI-MS MALDI-MS is another potent analytical platform for metabolomic profiling having the capability of rapid analysis, low sample volume requirement in the range of 0.1–1μL, and relatively high salt endurance. It can be carried out with or without prior separation method. In direct MALDI-MS analysis without involving any prior separation method, the sample is mixed with suitable matrix material such as α-cyano-4-hydroxycinnamic acidor 2,5-dihydroxybenzoic acid followed by soft ionization and MS analysis. The technique is mostly suitable for macromolecule

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metabolite analysis, however, development of novel matrices; precisely the nanomaterials have potentiated the modality for small metabolites analysis too. The reproducibility is greatly influenced by the matrix materials and sample processing method. Difficulty in quantitative analysis is a potential limitation of this platform (Ren et al. 2018). Quantitative analysis of serum nonesterified and esterified fatty acids by MALDI-FTICR-MS has revealed significant correlation between increased or decreased concentration of certain fatty acids with lung cancer (Ren et al. 2016). In a similar way, direct MALDI-MS approach has elucidated pronounced differences in lipid metabolism between A549 and H596 human nonsmall-cell lung cancer cell lines with marked variation in eicosapentaenoic acid (EPA) derived prostaglandin E3 production (Pirman et al. 2013).

Direct Analysis in Real Time (DART)-MS Direct analysis in real time (DART)-MS is another cutting edge analytical platform for rapid direct analysis of solids, liquids, and gases at atmospheric pressure without requiring prior analyte extraction and lengthy sample preparation procedure. DART ionization facilitates gas-phase ionization of the sample analytes followed by their delivery to the MS platform for identification and characterization. It is a highthroughput technique, mostly suitable for small analytes, preferably m/z of 50–1200. However, ionization and analysis of polar compounds is relatively difficult in this modality (Ren et al. 2018). A combination of DART-MS and 1H-NMR has been successfully used for metabolomic introspection to detect breast cancer (Gu et al. 2011). DART-MS alone has also been used for serum metabolomic profiling and detection of ovarian cancer with marked accuracy (Zhou et al. 2010). Mass Spectrometry Imaging Mass spectrometry imaging (MSI) is arguably the most promising technique for insitu metabolite analysis precisely for untargeted approach. It is having the potential to generate information regarding diverse metabolites of wide molecular weight range, their in situ distribution within a tissue section or even inside a single target cell. MSI can be performed through different ionization methods and approaches such as MALDI-MSI, secondary ion mass spectrometry (SIMS), matrix-free Desorption Ionization on Silicon (DIOS)-MSI, surface-based matrix-free method Nanostructure-Initiator Mass Spectrometry Imaging (NIMS), Silicon Nanopost Array (NAPA)-MSI, Desorption Electrospray Ionization (DESI)-MSI, Laser Ablation Electrospray Ionization (LAESI)-MSI, Live Single-Cell Video-Mass Spectrometry using Nano-ESI-MSI, Single-Probe Mass Spectrometry, 3D MALDI-MSI, microfluidic chips or microarray system-based MSI, etc. (Minakshi et al. 2019b). MALDI-MSI has been successfully employed in metabolomic introspection of several types of cancers including breast cancer, brain tumor, colorectal cancer, gastric cancer, colon tumor, renal cell carcinoma, leiosarcoma, myxoidliposarcoma, thyroid cancer, prostate cancer, osteosarcoma, glioblastoma, nonsmall cell lung carcinoma, etc. Beyond detection, the technique is also extremely useful in cancer grading, prediction of therapeutic outcome and cancer prognosis (Buck et al. 2017; Kreutzer et al. 2019). Airflow-assisted desorption electrospray ionization

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(AFADESI)-MSI is another such approach that has explored several metabolites along with the underlying enzymes related to altered metabolic pathways in esophageal squamous cell carcinoma (Sun et al. 2018). MSI is also the most promising and powerful technique for single-cell metabolomics (SCM) analyses as evidenced by numerous examples (Minakshi et al. 2019b; Kumar et al. 2020). Although SCM analysis in oncology research is yet to bloom adequately, considering the potential, it will certainly hold key position in near future. NMR and MS are the most prominent analytical platforms for targeted as well as untargeted metabolomics; however, other methods are also available but mostly for targeted analyses with limited multiplexing such as fluorescence spectroscopy, Raman spectroscopy, 18F-fluorodeoxyglucose positron emission tomography (FDG-PET), metabolic sensors, etc. These methods can be very useful for validation purpose.

Challenges and Opportunities Challenges are integral part of any technology, nothing different the case of proteomics and metabolomics. Proper sample processing to achieve adequate analyte concentration to match the sensitivity level of the analytical platforms often becomes tricky. Spatiotemporal analyte dynamicity and heterogeneity also poses complexity to sample handling prior analysis. Integrated omics or multiomics or even individual omics techniques are associated with handling of “big data.” The volume, variety, velocity, and veracity of the “big data” engender several challenges such as data cleaning and normalization, reduction in data dimensionality, data storage, statistical analyses, and biological contextualization. Dearth of available standardized software/website and scanty databases often renders data processing and analysis difficult as different software may produce highly variable result in MS data analyses. Further absolute quantitation is usually vogue due to nonstandard experimental conditions, thus heavily reliant on relative quantitation of the data. As a combinatorial outcome, the obtained data from the high-throughput analytical platforms often face the question of reproducibility and reliability. Thus, with the progress in sample collection and handling methods, it is highly recommended to carry out omics approaches in much standardized way beginning from sampling to data acquisition to interpretation towards unhindered data sharing, multiplatform data integration and their effective use for verification to increase the utility and credibility of these state-of-the-art techniques in clinical applications. Although it seems tricky to deal with these advanced omics techniques and the generated complex large data, it must be remembered that most of the platforms are in naïve stage. It is evident that the techniques are mostly automated, high-throughput, and having the potential to scan through the entire set of analytes expressed in a target sample within a spatio-temporal frame at one go. Here lies the opportunity to extract benefit from these cutting-edge modalities over the traditional methods like histology staining, or immunohistochemistry which are mostly manual, low through-put and

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targeted approach. Thus, novel biomarker discovery, identification of new therapeutic checkpoints, novel drug discovery, or elucidation of therapeutic outcome at molecular monitoring basis are much easier through these omics approaches. Continuous progress in sampling methods, analytical platforms, analytical software and bioinformatics programs, and specialized databases will certainly remove the hurdles and prosper the path for effective “lab to land” transition of these omics approaches towards their routine application in oncology and clinical medicine.

Conclusions The eventual objective of cancer omics technologies is their adaptation for routine use in clinical medicine for the purpose of rapid and early diagnosis, prognostic evaluation of disease states, identification of novel therapeutic targets, as well as evaluation of drug efficacy and toxicity. The information which remains obscure even in genomic and transcriptomic analysis can be elucidated through tumorspecific proteomic and metabolomic profiling. These high-throughput technologies are capable of producing a real-time snap-shot of the regulatory proteins and metabolites from diverse tissue as well as biofluid samples. Further, untargeted quantitative automated omics approaches are of unparalleled utility for conducting exploration on discovery mode which is cumbersome or sometimes beyond the scope of manual conventional approaches. These high-resolution platforms have the penetration capability up to single individual cell or even beyond at the level of subcellular organelles which extends the opportunity to detect any aberration at molecular level much earlier than the traditional methods which mostly rely up on phenotypic manifestation of the disease symbols resulting into inevitable delay. Early detection is extremely crucial for cancer remedial; thus, cutting edge proteomics and metabolomic techniques can be of at most importance in clinical oncology. These techniques are progressively getting matured and standardized by surmounting the bottlenecks for their effective transition from research arena to the field of routine clinical diagnosis and therapy of cancer.

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Systems Biology and Bioinformatics Insights into the Role of Free Radical-Mediated Oxidative Damage in the Pathophysiology of Cancer

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Shaik Mohammad Naushad and Vijay Kumar Kutala

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Computational Approaches in Systems Biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Artificial Intelligence (AI) Predicting Cancer Susceptibility Based on Redox Data . . . . . . Artificial Intelligence (AI) Predicting Cancer Prognosis Based on Redox Data . . . . . . . . . . Artificial Intelligence (AI) Predicting Therapeutic Outcome Based on Redox Data . . . . . . Bayesian Networks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Boolean Networks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Multi-omics Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Network Analyses Showing Association of Redox Proteome with Cancer Susceptibility . . . Network Analyses Showing Association of Redox Proteome with Cancer Prognosis . . . . . . . Network Analyses Showing Association of Redox Proteome with Cancer Therapy . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract

Oxidative stress plays a pivotal role in various physiological and pathological processes. The interplay between pro-oxidants and antioxidants (enzymatic and non-enzymatic) is highly complex requiring the application of systems biology approaches for differentiation between physiological and pathological states. In this review, we have provided a comprehensive overview of the existing literature on oxidative damage and cancer by combining genomics, transcriptomics, proteomics, and metabolomics of redox signaling. Systems biology approaches such as artificial intelligence, Bayesian networks, Boolean networks, and Network analyses were used by various researchers to facilitate prediction, early diagnosis, S. M. Naushad (*) Department of Biochemical Genetics and Pharmacogenomics, Sandor Speciality Diagnostics Pvt Ltd, Hyderabad, India V. K. Kutala Department of Clinical Pharmacology and Therapeutics, Nizam’s Institute of Medical Sciences, Hyderabad, India © Springer Nature Singapore Pte Ltd. 2022 S. Chakraborti et al. (eds.), Handbook of Oxidative Stress in Cancer: Mechanistic Aspects, https://doi.org/10.1007/978-981-15-9411-3_154

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prognosis, and therapeutic outcome of cancers. These studies clearly display the interplay of pro-oxidants and antioxidants in modulating the cellular metabolism and in affecting various carcinogenesis pathways, that is, mutagenicity, cell cycle deregulation, metastasis, resistance to apoptosis, and angiogenesis. Multi-omics approach with systems biology tools has the potential in unraveling the complexities associated with cancer biology thus facilitating effective management of cancer. Consortium-based approaches are essential in pooling up the data from different sources and generate more efficient translational tools. Keywords

Reactive oxygen species · Systems Biology · Pathophysiology of Cancer

Introduction Reactive oxygen species (ROS) are the highly reactive free radicals produced during mitochondrial respiration and cellular metabolism. Environmental factors such as UV radiation, cigarette smoking, and pollution can induce oxidative damage to DNA thus promoting oncogenic mutations that initiate tumorigenesis (Migliore and Coppedè 2002). ROS act as signaling molecules and contribute to abnormal cell growth, metastasis, resistance to apoptosis, and angiogenesis (Schieber and Chandel 2014). In vitro and in vivo studies showed beneficial effects of N-acetylcysteine (NAC) in reducing the tumor growth in many cancers (Aggarwal et al. 2020; Yildiz 2004). As an effect of mitochondrial DNA mutations, mitochondrial dysfunction was observed in several cancers (Hsu et al. 2016). The ROS production in tumor cells was shown to be induced by the oncogenes such as c-Myc, c-Met, or Ras (Maya-Mendoza et al. 2015). K-Ras oncogenic mutation was found to increase superoxide production by activating Rac, the regulatory component of NOX (Calvert et al. 2013). The external stimuli such as UV radiation and smoking were reported to activate tyrosine protein kinases EGFR, PDGFR, and Src by inducing phosphorylation (Yang et al. 2009). The association of oxidative stress is well documented now. However, due to complexity of various strata of information in the form of redox genomics, redox transcriptomics, redox proteomics, and redox metabolomics, big data analytics using systems biology and bioinformatics tools are emerging to translate the available information in clinical decision in terms of predicting the susceptibility to cancer, early diagnosis, evaluation of prognosis, and in personalizing the therapy. In this book chapter, we are providing an overview of recent developments in systems biology in addressing the association of oxidative damage with cancer.

Computational Approaches in Systems Biology Artificial intelligence (AI) is an interdisciplinary science capable of performing complex tasks that typically mimic human intelligence. It involves artificial neural network analysis, machine learning (supervised and unsupervised learning), and

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deep learning. Bayesian networks are probabilistic graphical models built based on the probability distributions and are governed by laws of probability. These models are useful in prediction, anomaly detection, decision making, and time-series predictions. Boolean network consists of a set of Boolean variables whose state is determined by other variables in the network. Usually, the nodes of these networks represent the status of the expression of genes and directed edges show the actions of genes on other genes.

Artificial Intelligence (AI) Predicting Cancer Susceptibility Based on Redox Data An artificial neural network (ANN) model of breast cancer prediction revealed that increased dietary folate can restore the expression of ER and PR and decrease the promoter CpG island methylation of extracellular superoxide dismutase and BRCA1 (Naushad et al. 2016). N-(Biotinyl)-N0-(Iodoacetyl) Ethylenediamine (BIAM), a reagent specific to thiols was used to observe the thiol proteome changes on 2D gel, and ANN analysis was performed to identify potential selenium targets (Lee et al. 2006). Classification and regression tree (CART) of base excision repair pathway genes covering 13SNPs explored high order gene-gene interactions associated with increased susceptibility to bladder cancer (Xie et al. 2015). Whole exome sequencing-based CART model revealed the association of MSH6 and BRIP1 variants with risk for TNBC by inducing oxidative DNA damage and increased susceptibility to breast cancer (Aravind Kumar et al. 2018). Integrative analysis and machine learning on the cancer genomics data obtained from the Cancer Systems Biology Database (CancerSysDB) revealed G > T transversions in lung cancers of tobacco smokers were induced by oxidative stress (Krempel et al. 2018).

Artificial Intelligence (AI) Predicting Cancer Prognosis Based on Redox Data A mathematical model was proposed to deduce the biological age associated with oxidative stress based on serum levels of HDL-cholesterol and magnesium, and total AKT1 and glutathione in liver (Sáez-Freire et al. 2018). Biologically older mice were shown to develop enhanced aggressive breast cancer than the biologically younger mice (Sáez-Freire et al. 2018). The genomic regions on chromosome 2 and 15 were associated with the grade of oxidative aging (Sáez-Freire et al. 2018). Flavoproteins pivotal for the mitochondrial electron transport, fatty acid degradation, and redox regulation were studied for their association with survival and prognosis in esophageal squamous cell carcinoma (ESCC) (Peng et al. 2019). Six-gene signature of flavoproteins (CTNN, GGH, GPD2, PYROXD2, SRC, SYNJ2BP) was shown to have excellent prognostic ability for six-year survival in ESCC patients with AUC of 0.86 (Peng et al. 2019). Clustering-based analysis of 155 ROS/RNS regulating genes and 128 ROS regulated genes for breast cancer patients’ survival revealed the association of GSTK1, PRDX2, PRDX3, and SLC36A1 with LumB breast cancer survival (Luthra et al. 2018).

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Artificial Intelligence (AI) Predicting Therapeutic Outcome Based on Redox Data An ANN-based sequential classification of photosensitizers located in mitochondria and lysosomes was developed to facilitate virtual screening of drug candidates for photodynamic therapy (Tejedor-Estrada et al. 2012). The modulation of cell metabolism and the redox capability using herbal compositions optimized by artificial intelligence are emerging as new therapeutic approaches for the treatment of cancer and autoimmune disorders (Fan et al. 2018). A classification and regression tree (CART) model revealed higher risk for radiotoxicity in women with high BMI (>25 kgm-2) and eNOS GG/MPO GG-genotype compared to those with BMI < 25 kgm2 (Ahn et al. 2006).

Bayesian Networks Semi-Bayesian Hierarchical modeling to explore gene-nutrient interactions modulating breast cancer risk revealed that DNA repair pathway genetic variants increase breast cancer risk by 27% after adjusting for oxidative stress, carcinogen metabolism, and one-carbon metabolism with average flavonoid intake (Khankari et al. 2014). As an indirect method to evaluate PI3K pathway activity, FOXO activity, which is an inverse readout of PI3K pathway activity, was inferred from the mRNA expression profile of 26 FOXO target genes using a knowledge-based Bayesian network computational model (van de Stolpe 2019). In pediatric brain tumors, upregulation of dihydropyrimidinase-related protein 2 (DPYSL2), glial fibrillary acidic protein (GFAP) isoform 2, phosphoserine aminotransferase isoform 1 (PSAT1), Sirt2 histone deacetylase (SirT2), and Twinkle MtDNA helicase (TWNK) was reported (Luna et al. 2015). Further, these tumors showed downregulation of heat shock protein 90 kDa beta (HSP90beta), guanine nucleotide binding protein (subunit beta-2-like 1(GNB2L1), Igg Fab Fragment, histone H2B.1, neurofilament light polypeptide (NEFL), Annexin I, and RAN (Luna et al. 2015).

Boolean Networks Boolean network model based on the information on pathways from the existing literature linked oxidative stress response to Ras-mediated activation of PI3K/Akt pathway resulting in the apoptosis (Sridharan et al. 2012). A systems-based approach called Entropy Minimization and Boolean Parsimony (EMBP) was used to recognize the association with disease directly from the gene expression data and modules of genes (Varadan and Anastassiou 2006). This approach identified association of prostate cancer and cellular damage with oxidative stress together with the inhibition of apoptotic mechanisms (Varadan and Anastassiou 2006).

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Multi-omics Analysis Multi-omics factor analysis (MOFA) of CLL data revealed tagging of Factor 5 with a set of genes enhanced for oxidative stress and senescence pathways (Argelaguet et al. 2018). Systems biology analysis of biomarkers in lung cancer identified miR21, miR155, MALATI, and miR31 as the top non-coding RNA biomarkers; TP53, KRAS, CDKN2A, ENO2, KRT19, RASSF1, GRP, SHOX2, and ERBB2 as the top protein biomarkers in lung cancer (Alanazi et al. 2018). MOFA of non-small cell lung cancer (NSCLC) identified members of hypoxia-inducible factor (HIF) gene family at the center of gene network along with well-recognized lung cancerrelated genes such as EGFR and TERT (Wang et al. 2018). Using multi-omics approach, it was demonstrated that the deficiency of S-adenosylhomocysteine hydrolase (AHCY) might increase susceptibility to late-onset liver diseases by inducing chronic DNA stress (Belužić et al. 2018). These adverse effects can be mitigated via adenosine supplementation (Belužić et al. 2018). Further, the possibility of exploring AHCY inhibitors is an alternative therapeutic modality in certain cancer types that are highly sensitive to increased DNA damage (Belužić et al. 2018). The microRNA and transcription factor gene regulatory network analysis revealed the association of HOXD10, BCL2, and PGR with primary prostate samples, and STAT3, JUN, and JUNB with metastasis (Sadeghi et al. 2016). Five miRs (miR-671-5p, miR-665, miR-663, miR-512-3p, and miR-371-5p) were shown to dysregulate STAT3 in the metastatic tumors (Sadeghi et al. 2016). Protein S-sulfenylation (SOH), a post-translational modification, acts as a redox switch in crucial cellular processes and hence playing a pivotal role in signal transduction, protein folding, and enzymatic analysis (Wang et al. 2016). Reversible SOH is a component of redox homeostasis and involved in cancer and cardiovascular diseases (Wang et al. 2016). SOHPRED, a bioinformatic tool, was able to predict potential SOH sites in 193 S-sulfenylated substrates (Wang et al. 2016).

Network Analyses Showing Association of Redox Proteome with Cancer Susceptibility Data mining, gene ontology, and network analyses identified 98 genes associated with glucose-6-phosphate dehydrogenase (G6PD) and 33miRs that regulate G6PD, suggesting that alteration of G6PD can increase susceptibility to cancer and autoimmune diseases (amyotrophic lateral sclerosis, autoimmune thyroid disease, graftversus-host diseases) by inducing oxidative stress (Chen et al. 2017). Comparison of tumor microenvironment with healthy tissues in hepatocellular carcinoma in terms of differentially expressed mRNAs (n ¼ 1770) and miRs (n ¼ 12) revealed the involvement of RNAs corresponding to Wnt, MAPK, mTOR, Jak-STAT, and VEGF signaling pathways (Gao et al. 2015).

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Redox proteome has emerged as an interface between genome-directed biological structure and functions and the environmental determinants of those structures and functions (Go and Jones 2013). Cys proteome with thiol of Cys serving as a sulfur switch links redox chemistry with structure and function (Go and Jones 2013). The integration of redox proteome data with genomic data facilitated the quantification of the intracellular oxidative stress in cancer cells (Liu et al. 2020). Mutation rate, expression, and association with oxidative stress were integrated to generate this model (Liu et al. 2020). GO enrichment analysis of prostate cancer data from TCGA database suggested an association between the genes that are regulated by an epigenetic phenomenon with redox signaling, ALDH and NADP+ activity (Xu et al. 2019). Bioinformatics analysis of gene expression profiles of GEO database corresponding to hepatocellular carcinoma revealed upregulation of 101 differentially expressed genes and downregulation of 146 genes (Fu et al. 2018). The functional annotation of these differentially expressed genes revealed their possible association with redox signaling, glutathione metabolism, autophagy, cell growth, mitotic cytokinesis, and endome or lysozome organization (Fu et al. 2018).

Network Analyses Showing Association of Redox Proteome with Cancer Prognosis Transcriptome analysis of 19 ALDH genes revealed that each ALDH isoform has a specific differential expression pattern related to the prognosis of human cancer (Chang et al. 2018). A lower expression of ALDH2 was reported in tumors and was linked to poor cancer prognosis (Chang et al. 2018). Differences in the function of redox regulatory systems in the tumor tissue and its surrounding tissues of various histological origin and localization were reported (Surikova et al. 2016). In cancer cells, as a result of inactivation of components of electron transport chain or TCA cycle, there is an increased ratio of NADH/NAD+ in the mitochondrial matrix, which increases NADPH/NADP+ ratio through the catalytic action of nictotinamide nucleotide transhydrogenase (NNT) (Alberghina and Gaglio 2014). The high mitochondrial NADPH/NADP+ ratio facilitates the production of adequate citrate for the reductive carboxylation of glutamine (Alberghina and Gaglio 2014). The supply of NAD(+) precursors reduces tumor aggressiveness (Alberghina and Gaglio 2014). In breast cancer metastases, the activation of specific signaling networks was shown to facilitate the cancer cells to adapt to organs of metastasis, for example, activation of glutathionemediated detoxification, semaphoring signaling in neurons, NRF2-mediated oxidative stress response contributes to brain metastasis; increased acute phase response contributes to lung metastasis; activation of hematopoietic stem cell signaling contributes to bone metastasis; and activation of Notch/orphan nuclear receptor signaling contributes to adrenal metastasis (Burnett et al. 2015). Systems biology-based calculations of metabolic flux states for the NC160 cell lines revealed the correlation between chemotherapeutic resistance and the glucose

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uptake mediated through excess of redox and energy production (Zielinski et al. 2017). Bioinformatics analysis of mRNAs of 73 oxidative stress genes from the Cancer Genome Atlas revealed an inverse correlation between the oxidative stress genes and survival in solid carcinomas with breast, lung, and HNSCC cancers being more vulnerable to oxidative stress (Leone et al. 2017). The statistical significance was high for FOXM1 and TXNRD1 in four out of six tumor types (Leone et al. 2017).

Network Analyses Showing Association of Redox Proteome with Cancer Therapy The autophagy-related protein 4B (ATG4B) cleaves the C-terminal amino acid of ATG8 family proteins to expose C-terminal glycine residue for the conjugation of these proteins with phosphatidylethanolamine leading to their insertion to membranes for autophagy. Hence, small molecular agonists for this protein were designed through systems biology approaches, and the in silico results were validated through experiments on MDA-MB-231 cells that demonstrated the efficacy of Flubendazole in targeting ATG4B and inducing autophagy thus projecting it as a potential drug for TNBC therapy (Zhang et al. 2015). The impact of cellular redox environment on the expression of BRCA1 was elucidated through in vitro experiments wherein decreased ROS production (mitochondrial electron transport deficient cell lines) or reduced NOX activity (with peptide inhibitors) were found to restore BRCA1 expression (Wilson and Yakovlev 2016). This was replicated in TCGA database analysis that showed an inverse association between BRCA1 expression and expression of NOX regulatory subunits (Wilson and Yakovlev 2016). In more than 400 tumor lines, the inhibition of glutaminase (GLS1) and gamma-glutamylcysteine synthetase (GCS) was found to be effective specifically in the mesenchymal subtype, which depends on GLS1-derived glutamate for the de novo synthesis of glutathione and TCA cycle anaplerosis. In the absence of GLS1, these tumors are susceptible to increased oxidative stress (Daemen et al. 2018) (Fig. 1).

Conclusions Integration of OMICS data related to oxidative damage in cancer can open new avenues in translational research in terms of risk prediction, early diagnosis, identification of prognostic markers, and alternative therapeutic modalities. The efficacy of systems biology and bioinformatics approaches in delineating the complexity of cancer biology depends on the availability of data in the public domain and its integration through formation of consortiums. Multi-omics approach with systems biology tools has the potential in unraveling the complexities associated with cancer biology thus facilitating effective management of cancer.

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Prooxidants

Anti-oxidants

RNS

ROS

Enzymatic

Non-Enzymatic

Mutagenicity Activation of Proto-oncogenes c-Myc, c-Met, or Ras

tyrosine protein kinases

Metabolomics 1 Proteomics

Genomics

Data mining

2 3

• Artificial Intelligence

Imbalances in redox systems Increased ROS/RNS production Altered signal transduction Cancer risk Cancer susceptibility

• Bayesian and Boolean Networks • Network analyses

Systems Biology

Prognosis Novel Therapeutics

Fig. 1 Systems biology approaches delineating the association of Free radical-mediated oxidative damage in the pathophysiology of cancer

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Pawan Kumar Raghav, Zoya Mann, Pranav K. Pandey, and Sujata Mohanty

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Systems Biology Databases Used in Cancer Research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Expression and Variation Databases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Immune System and Personalized Medicine Databases Used in Cancer Drug Designing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Databases Used for Interaction and Pathway Analyses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Tumor Databases Used for Drug Designing in Experimental and Clinical Studies . . . . . . . Systems Biology Approaches and Tools to Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Expression and Variation-Based Systems Biology Tools and Approaches for Cancer Prediction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Common Immunoinformatic and Bioinformatics Tools to Cancer Drug Discovery . . . . . . Biomolecular Networks Tools in Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Text Mining Tools Used in Cancer Research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mathematical Modeling and Simulation Tools to Model Cancer Pathways and Networks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Clinical Applications of Systems Biology Tools and Approaches . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract

Tumor development is not an abrupt process, but rather begins as a slow and gradual anomaly in a random cell’s machinery. Such an aberration disturbs the cell’s homeostasis and leads to transformation of its niche into a tumor microenvironment. These changes alter the genetic stability of the surrounding cells through different molecules and associated pathways. Although tumor niche P. K. Raghav · Z. Mann · S. Mohanty (*) Stem Cell Facility, DBT- Centre of Excellence for Stem Cell Research, All India Institute of Medical Sciences, New Delhi, India P. K. Pandey Department of Ophthalmic Sciences, Dr. Rajendra Prasad Centre, All India Institute of Medical Sciences, New Delhi, India © Springer Nature Singapore Pte Ltd. 2022 S. Chakraborti et al. (eds.), Handbook of Oxidative Stress in Cancer: Mechanistic Aspects, https://doi.org/10.1007/978-981-15-9411-3_140

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development is a random and complicated process, most of the conventional therapy modes have only a symptomatic effect. Therefore, targeted therapeutic interventions and a better understanding of tumor complexity are required. This phenomenon can be achieved with systems biology approaches by using highthroughput techniques that assist in the prognosis and diagnosis of cancer. These approaches work primarily in two interdependent ways collating the available data in the form of a database and extracting data from these platforms via tools and software to create networks and prediction models. Such tools are efficient in generating data that can help in personalized medicine through drug discovery. Therefore, in this chapter, we have reported the publicly available systems biology databases, software, and tools used in cancer data analysis. Keywords

Systems biology · Databases · Tools · Software · Mathematical modeling and simulations · Cancer

Introduction Cancer occurs due to the abnormal proliferation of cells regulated predominantly by anti-apoptotic, proapoptotic, and other proteins (Verma et al. 2013; Raghav et al. 2012a, b, 2019). Death from cancer is still prevalent despite the advancement of prevention and treatment. Based on predictions, the deaths from cancer will continue to increase, and 11.4 million are expected to die by 2030 (World Health Organization 2012). The early prognosis and diagnosis are tedious task due to the lack of specific molecular markers for cancer, although regulating genes and their products are being studied to eradicate cancer. The solution to these issues is provided by data integration from the “Omics” data generation technologies to explain the molecular mechanisms of cancer pathogenesis. Currently, cancer is considered as systems biology disease (Hornberg et al. 2006). Systems biology is an integrative approach through which the new molecular events in cancer can be revealed based on high-throughput “Omics” technologies (Chakraborty et al. 2018). The high-throughput research tools belong to “Omics” technologies and include genomics, proteomics, and transcriptomics. The genome sequencing and high-throughput technologies (microarray and next-generation sequences) provide extensive datasets stored in several databases (Kitano 2002). These datasets containing genes and proteins information extracted from samples have been analyzed using systems biology tools (Nagaraj 2009). Furthermore, systems biology explains the complex interactions between genes and networks of all cellular elements (Liu 2005). A comprehensive network of diverse data, such as gene expression, mutation, DNA-protein, and protein-protein interactions, can be constructed to understand molecular processes associated with cancer (Schadt et al. 2009). These complex networks are represented as a systems biology model that can be used to identify specific regulatory molecules or pathways involved in

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cancer (Laubenbacher et al. 2009; Morrow et al. 2010; Mac et al. 2010). This inclusive systems biology approach is necessary to understand the interaction between the adaptive antioxidant response and ROS signaling (Kitano 2002). The scope of the current chapter is to provide an overview of web resources, databases, tools, and software of systems biology used for cancer research (Tables 1 and 2, Fig. 1). This chapter summarizes the advanced bioinformatics tools with systems biology approaches for “Omics” analysis. Such analysis reports multiple facets of protein and gene expression, variations, experimental pathways and interactions, immunoinformatics, drug designing and GWAS (Genome-wide association studies) (Mac et al. 2010).

Systems Biology Databases Used in Cancer Research Expression and Variation Databases The number of bioinformatics data repositories is exponentially growing. The search for biomarkers and novel cancer targets needs various databases and tools (Table 1). The high-throughput data obtained from cancer studies have been integrated into publicly available centralized databases. For cancer therapy, aggregation, analysis, and integration of clinical data are required to identify novel biomarkers of cancer and their targets (Hackl et al. 2010). This biological data includes both gene expression and mutation data available at the international consortium, TCGA (The Cancer Genome Atlas, http://cancergenome.nih.gov), ICGC (International Cancer Genome Consortium, http://icgc.org), cBioPortal (https://www.cbioportal. org/), and GEN2PHEN (Genotype to Phenotype Database, http://www.gen2phen. org). The primary aim of these portals is to generate genetic variation data of cancer patients compared to healthy. However, these databases are used in combination with other tools to identify cancer-associated mutations (Raghav et al. 2019; Forman et al. 2010). The TCGA database is funded by the NIH (National Institutes of Health) and has clinical and high-throughput data for 33 types of cancer (Gao et al. 2019). The extensive resources, GEO (Gene Expression Omnibus) and microarray informatics at EBI (European Bioinformatics Institute), are available for retrieving microarray data, analysis, and storage. The querying abilities of the GEO repository are enhanced and achieved in GEOmetadb. The GEO and ArrayExpress public repositories provide the datasets for microarray gene expression data of cancer types and subtypes acquired on several platforms and tumor samples, while PRIDE (PRoteomics IDentifications database) is a proteomics data repository. Oncomine is another cancer gene expression database that includes cancer genomic profiles in 15 datasets and 86,733 samples. Data integration, combined with data mining tools, identifies correlations, which is essential for systems biology studies. The gene name redundancies and inconsistencies can be reduced using genetic nomenclature in GO (Gene Ontology) and the caBIG (cancer Biomedical Informatics Grid) database. High-throughput analyses pertain to the GWAS method used to identify cancer biomarkers. However, a protein microarray approach has identified a

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Table 1 List of systems biology and related databases used in cancer studies Databases/Webportals Description Expression and variation databases ArrayExpress Archive of the genomics data, based on microarray data, gene expression profiles and their functionality. [https://www.ebi.ac.uk/arrayexpress/] Atlas of Genetics and Genetic mutations related to cancer and Cytogenetics in Oncology respective chromosomal abnormalities. and Hematology [http://atlasgeneticsoncology.org/] BASE Web-based microarray database and analysis platform. [http://base.thep.lu.se/] BMERC Completed Genomes collected resources. (BioMolecular Engineering [https://www.bu.edu/bmerc/] Research Center) caBIG Large multidisciplinary data sets, analysis tools (Cancer Biomedical and other related tools resources for analysis. Informatics [https://orbit.nlm.nih.gov/browse-repository/ Grid) online-community/forum-message/28-cancerbiomedical-informatics-grid-cabig] Cancer GAMAdb Cancer genetic data from integrated knowledge database of cancer with genome-wide association studies and meta-analyses, where specific information can be searched. [https://omictools.com/cancer-gamadb-tool] Cancer Gene Census Catalogue of annotation of genes and their mutations. [https://cancer.sanger.ac.uk/census] caGWAS Integrate, analyze, query, and report plausible (Cancer Genome Wide associations among genetic variations, the Association Scan) respective drug responses, and diseases or other clinical outcomes. [https://www.nitrc.org/projects/cagwas/] cBioPortal for Cancer A platform for analysis, visualization, and Genomics downloading of the enormous genomic data. [https://www.cbioportal.org/] CGAP Genetic expression profiles’ resource of (Cancer Genome Anatomy normal vs. cancer cells. Project) [http://cgap.nci.nih.gov/] CGEMS Identification of commonly inherited genetic (Cancer Genetic Markers of mutations linked with the risk of prostate and Susceptibility) breast cancer. [https://dl.acm.org/doi/10.1145/965106. 965131] COLT-Cancer Collection of all the shRNA-based signatures database profiles, covering approximately16000 human genes. [https://omictools.com/colt-cancer-tool]

PubMed IDs 17132828

18396036

19822003

17604447

22986455

18396036

22986455

22986455

24492837

22986455

22986455

18396036

(continued)

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Table 1 (continued) Databases/Webportals COSMIC (Catalogue of Somatic Mutations in Cancer)

CRC gene Database (Colorectal Cancer)

caSNP (Database for copy number alterations of cancer genome from SNP array data) dbDEPC (Database of Differentially Expressed Proteins in Human Cancers) EBI (European Bioinformatics Institute) EBI- EMBL

Ensembl Genome Browser

FaCD (Familial Cancer Database) GEM (Grid-Enabled Measures)

GEN2PHEN (Genotype to Phenotype Database) caSNP (Gene Expression Omnibus)

Description Contains details of sample aberrations or mutations and literature. Also provides mutational range and frequency statistics for any gene of interest and/or cancer phenotype. [https://cancer.sanger.ac.uk/cosmic] Gathers all gene-based studies on CRC with a precise interpretation of all the possible risk factors. [http://colonatlas.org/] Collection of copy number variations (CNV) from numerous SNP arrays. [https://bioinformaticshome.com/tools/cnv/ descriptions/CaSNP.html] Provides proteomics data for cancer, details about changes in expressions at protein level and exploring the differences in protein profiles among different cancers subtypes. [https://www.scbit.org/dbdepc3/protein.php] Maintains SwissProt and EMBL Nucleotide Sequence Database, Europe’s primary and most reliable nucleotide sequence database. [https://ebi.ac.uk] Europe’s primary sequence data resource that maintains EMBL Nucleotide Sequence Database and SWISS- PROT protein Sequence Database. [https://www.ebi.ac.uk/] Genomic information on Human, archiving all the sequenced genes. [http://www.ensembl.org/] Aids differential diagnosis in cancer patients at genetic level. [https://www.familialcancerdatabase.nl/] A platform by NCI to promote use of standardized measures organized by theoretical constructs and share the harmonized data hence generated. [grids.ucs.indiana.edu/ptliupages/publications/ hpjavaapril04.pdf] This database collates the G2P data from various resources with the facility of data annotation and user feedback. [http://www.gen2phen.org] Stores high-throughput genomic data, comprised of chromatin structure, transcription factor binding information, methylation status, and CNVs. [https://www.ncbi.nlm.nih.gov/geo/]

PubMed IDs 18396036 22986455

18396036

22986455

22986455

7937043

8594602

11752248

18396036

21521586

21438073

22986455

(continued)

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Table 1 (continued) Databases/Webportals GeneCards

GEOmetadb

GO (Gene Ontology)

HGMD (Human Gene Mutation Database) HGMP Resource (Human Genome Mapping Project) HNOCDB (Head and Neck and Oral Cancer) ICGC Data Portal (International Cancer Genome Consortium)

IGDB NSCLC (Integrated Genomic Database of Non-Small Cell Lung Cancer) IMB Jena

Liverome

MethDB

MINT (Molecular INTeraction database)

Description A comprehensive database for all predicted and known human genes. [https://www.genecards.org/] SQLite database that contains the metadata associated with the GEO repository. [https://gbnci-abcc.ncifcrf.gov/geo/] The largest database on function of all the genes. Data has been derived from molecular and genetics experiments. [http://geneontology.org/] Collates all the human gene lesions underlying genetic diseases, most of which are published. [http://www.hgmd.org] Homology information for mouse, human and over 70 other species. [http://www.hgmp.mrc.ac.uk/Genome] Comprehensive information on microRNAs and genes of the head, neck and oral cancer. [https://omictools.com/hnocdb-tool] Catalogues of genetic aberrations (somatic mutations, epigenetic modifications, abnormal expression of genes) among various types of tumors. Project for ‘omics’ data, ICGC is focused in understanding the genomic abnormalities caused in cancer with detailed information on somatic mutations in 50 different cancer types. [https://icgc.org/] Consolidated database of genetic mutations (LOH, CNA, aCGH, SNP) archived on lung cancer. [http://igdb.nsclc.ibms.sinica.edu.tw/] Structural genomics data for human, mouse, and primates. [http://genome.imb-jena.de/] Curated database of liver cancer-related genes retrieved from available open source proteomics and microarray studies. [http://liverome.kobic.re.kr/] Database for environmental epigenetic effects and DNA methylation. [http://www.methdb.net/] Gene expression patterns and DNA methylation in cancers vs. normal cells. [https://mint.bio.uniroma2.it/]

PubMed IDs 24492837

18842599

23161678

28349240

10193186

18396036

18396036 24492837 22986455

18396036

10592237

18396036

11125109

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(continued)

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Table 1 (continued) Databases/Webportals miRCancer

Mitelman Database of Chromosome Aberrations in Cancer NCBI dbGaP (Database of Genotypes and Phenotypes ) Oncomine

Oral Cancer Gene Database Version I and II

PC-GDB (Pancreatic Cancer gene database) PEpiD

Progenetix database

RCDB (Roller Coaster Database)

RefDIC

Roche Cancer Genome Database

SageBio

Description Archived microRNA expression description in different human cancers and subtypes using chi-square sequence and clustering analytical tool. [http://mircancer.ecu.edu/] Chromosomal mutations and tumor characteristics, from either associated or individual cases. [https://mitelmandatabase.isb-cgc.org/] Archives the exposure, genotype, phenotype, and sequence-based data of the individuals and the possible associations among them. [https://www.ncbi.nlm.nih.gov/gap/] Database and data-mining platform built to retrieve microarray data. Collects networks and pathways and gene expression data. [https://www.oncomine.org/resource/login.html] Oral cancer associated genes archived in this database, with two versionsVersion I has 242 genes. Version II has 374 genes. [http://www.actrec.gov.in/OCDB/] Details on pancreatic cancer associated genes. [https://www.bioinformatics.org/pcgdb/] Records prostate cancer epigenetic data of experimental rodents and humans. [https://www.pepid.com/] Aberrations in copy number of genes associated with human cancer. [https://www.progenetix.org/] Manually collated information about 269 microRNA and 240 protein-coding genes, describing pathogenesis and etiology of different renal cancers. [https://rcdb.com/] Immunoinformatic resources for microarray analyses. [http://refdic.rcai.riken.jp/welcome.cgi] Comprehensive information on chromosomal aberrations and SNPs archived from techniques like CGH and FISH. [http://rcgdb.bioinf.uni-sb.de/MutomeWeb/] Provides a platform for data curation, sharing and running solutions on complex biomedical problems. [https://sagebionetworks.org/]

PubMed IDs 18396036

18396036

22986455

18396036 22986455

18396036

18396036

18396036

18396036

18396036

17893089

18396036

24492837

(continued)

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P. K. Raghav et al.

Table 1 (continued) Databases/Webportals Sanger Institute databases

dbSNP (Single Nucleotide Polymorphism Database)

SNP500Cancer

TCGA (The Cancer Genome Atlas)

TUMIR

Tumor Gene Family Databases UCSC Xena (University of California Santa Cruz) Immune system databases dbLRC-KIR

dbMHC

IEDB (Immune epitope database and analysis resource) IMGT (International ImMunoGeneTics information system) International HapMap Project

Description A set of databases within the institute to store and analyze a large scale of data. [https://www.sanger.ac.uk/science/tools/ categories/database-software] Various kinds of databases are categorized based on different mutations like substitutions, deletion, insertion polymorphism, and microsatellite repeats. [https://www.ncbi.nlm.nih.gov/snp/] Main library for sequence verification of SNPs and related assay information. [https://hsls.pitt.edu/obrc/index.php? page¼URL1097241151] Resource designed to understand molecular basis of the cancer, by performing analysis of genetic and miRNA expression, copy number and methylation status of brain, ovarian and lung cancer. [https://www.cancer.gov/about-nci/ organization/ccg/research/structural-genomics/ tcga] Archives manually collected but experimentally backed data for understanding the role of miRNAs in different cancer types. [https://omictools.com/tumir-tool] Molecular and cellular data of genes associated with different cancers subtypes. [http://www.tumor-gene.org/tgdf.html] Visualizes and hosts functional genomic data. [https://xena.ucsc.edu/welcome-to-ucsc-xena/]

PubMed IDs 24492837

22986455

18396036

24492837 22986455

18396036

18396036

18396036

Database of the human Leukocyte Receptor Complex (LRC). [https://www.ebi.ac.uk/ipd/kir/] Experimental and clinical data on MHC. [https://www.ncbi.nlm.nih.gov/Web/Newsltr/ Summer03/dbMHC.html] Contains immune epitope data related to all species. [https://www.iedb.org/] Resource of the IgSF, MHC, MhcSF, RPI, IG, TR. [http://www.imgt.org/]

23193264

Databases and linkage maps of sequence variations. [http://snp.cshl.org/]

16251469

15215374

30357391

15608269

(continued)

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Table 1 (continued) Databases/Webportals IPD (Immuno-polymorphism database)

Description Database containing information on polymorphic genes of the immune system and their respective variations. [http://www.ebi.ac.uk/ipd/] MHC Haplotype Project Studies of the MHC linked-diseases to find and possible associations. [http://www.sanger.ac.uk/HGP/Chr6/MHC/] SNPBinder The database helps in prediction of the tissuespecific minor histocompatibility antigens. [http://www.sipep.org/] SIGMA Immunoinformatic resources for microarray (System for Integrative analyses. Genomic Microarray Analysis) [http://sigma.bccrc.ca] TumorHoPe Collated information on tumor homing peptides and their respective target cells, validated by experimental data. [https://webs.iiitd.edu.in/raghava/tumorhope/ help.php] Drug designing databases used for systems biology analysis BindingDB Web-based open source database for measuring binding affinities of protein-ligand complexes. [https://www.bindingdb.org/bind/index.jsp] ChEMBL Freely available database on bioactive molecules that have properties similar to drugs, curating the chemical, biological and genomic data. Primarily used for the purpose of drug discovery. [https://www.ebi.ac.uk/chembl/] DrugBank Combines information on drug and its details on structure and function with the specific targets, collating bioinformatics and cheminformatics. [https://www.drugbank.ca/] MMDB Archives 3D structures of biomolecular nucleic (Molecular Modeling acids and proteins derived from PDB and Database) determines their biological functions. [https://www.ncbi.nlm.nih.gov/Structure/ MMDB/mmdb.shtml] MOAD Largest archive about maximum of the (Mother of all databases) identified protein-ligand complexes retrieved from PDB. [http://bindingmoad.org/] PDB This repository archives information about (Protein Data Bank) macromolecules, nucleic acids and proteins. [https://www.rcsb.org/] PharmGKB Online database predicting clinical data by investigating effect of genetic variations in respective drug responses. [https://www.pharmgkb.org/]

PubMed IDs 16944494

18193213

16893394

17192189

18396036

26481362

27899562

18048412

24319143

15971202

10592235

23824865

(continued)

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P. K. Raghav et al.

Table 1 (continued) Databases/Webportals PubChem

Description The largest freely accessible chemistry database, with information about the chemicals’ name, molecular formula, structure and biological applications. [https://pubchem.ncbi.nlm.nih.gov/] STITCH Explores and exploits drug-target relationships (Search Tool for Interactions of by integrating data of interaction of proteins and Chemicals) chemicals. [http://stitch.embl.de/] SwissProt Manually annotated section of UniProt with details retrieved from available literature and also curator-evaluated computated analysis. Acts as a protein sequencing database. [https://web.expasy.org/docs/swiss-prot_ guideline.html] UniProt Information on protein sequence and respective (Universal Protein Resource) function applied for drug designing. [http://www.uniprot.org/] ZINC Database of commercially available chemical (Zinc Is Not Commercial) compounds for virtual screening. [https://zinc.docking.org/] Databases for interaction and pathway analyses CancerProView Cancer-related Gene/Protein and Disease Pathway Database. [https://omictools.com/cancerproview-tool] DIP Catalogs protein-protein interactions and (Database of Interacting their application and roles in biological Proteins) networks. [http://dip.doe-mbi.ucla.edu] HPRD Pathways and protein interaction networks. (Human Protein Reference [http://www.hprd.org/] Database) InnateDB Interactions and signaling pathways in the innate immune response. [http://www.innatedb.ca/] JenPep Immunological protein– peptide interactions database. [http://www.jenner.ac.uk/jenpep/] KEGG Collection of databases of genomes, pathways, (Kyoto Encyclopedia of Genes drugs and chemical structures. and Genomes) [https://www.genome.jp/kegg/] LINCS LINCS aims to enhance a network-based (Library of Integrated understanding of biology, cataloguing changes Network-based Cellular in gene expression, and other cellular processes Signatures) in response to perturbations. [http://www.lincsproject.org/]

PubMed IDs 26400175

18084021

10592178

18045787

15667143

18396036

10592249

22159132

23180781

11934742

10592173

24492837

(continued)

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Table 1 (continued) Databases/Webportals OMIM (Online Mendelian Inheritance in Man)

Description Compendium of human genes and related genetic disorders and traits, focusing on genotype- phenotype relationship, to enhance practice of clinical genetics. [https://omim.org/] PIG Host–pathogen, protein– protein interactions (Pathogen Interaction (PPIs) data. Gateway) [http://molvis.vbi.vt.edu/pig/] Pathway Commons Pathway Commons is a portal to access biological pathway information collected from public pathway databases. [https://www.pathwaycommons.org/] Reactome Database for pathways analysis to add information to proteomics data. [www.reactome.org/] VirusMINT Interactions between human and viral proteins are archived here. [http://mint.bio.uniroma2.it/virusmint/ Welcome.do] Databases for experimental and clinical studies caGWAS Integrate, query, report, and analyze significant (Cancer Genome Wide associations between genetic variations and Association Scan) disease, drug response, or other clinical outcomes. [https://www.nitrc.org/projects/cagwas/] CancerDR Provides information about 148 anti-cancer drugs, and their pharmacological profiling across 1000 cancer cell lines. [http://crdd.osdd.net/raghava/cancerdr/] canSAR v 2.0 Brings together biological, chemical, pharmacological, and disease data, distils them and makes them accessible to cancer research scientists from all disciplines to support translational research and drug discovery. [https://cansarblack.icr.ac.uk/] CDISC Standards to support the use of clinical research (Clinical Data Interchange data and metadata. Standards Consortium) [http://www.cdisc.org/] CGAP Resource of gene expression profiles of normal, (Cancer Genome Anatomy precancer, and cancer cells. Project) [http://cgap.nci.nig.gov/] CMAP Available for analysis gene associated with (Cancer Molecular Analysis oncogenesis and cancer profiles, clinical trials Project) and therapies. [https://www.g6g-softwaredirectory.com/bio/ cross-omics/dbs-kbs/20768-CMAP.php]

PubMed IDs 11752252

18984614

24492837

21067998

18974184

22986455

18396036

18396036

29888049

22986455

22986455

(continued)

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P. K. Raghav et al.

Table 1 (continued) Databases/Webportals CPT-4 (Current Procedural Terminology) DICOM (Digital Imaging and Communication in Medicine) eTUMOUR

FaCD (Familial Cancer Database) FuGE (Functional Genomics Experiments) GLIF (GuideLine Interchange Format) HL7 (Health Level Seven) ICD (International Classification of Disease) LGA

LOINC (Logical Observation Identifiers Names and Codes) MammoGrid

METAcancer

MIAPE (Minimum Information About a Proteomics Experiment

Description Describes medical, surgical, and diagnostic services. [https://catalog.ama-assn.org/Catalog/cpt/cpt_ search.jsp] A standard for information in medical imaging. [http://medical.nema.org/]

PubMed IDs 24761332

9147339

This project curates database based on transcriptomic and clinical data from brain tumor patients. [http://www.etumour.net] Assist genetic differential diagnosis in cancer patients. [https://www.familialcancerdatabase.nl/] Enlists standards for high-throughput biological experiments to maintain repositories and a defined set of standards. [https://omictools.com/fuge-tool] For sharing of clinical practice guidelines. [http://www.glif.org/glif_main.html]

23180768

Standards for interoperability of health information technology. [http://www.hl7.org/] Classifications of diseases. [http://www.who.int/classifications/icd/en/]

30353411

Public platform that supports research and analysis of molecular data of leukemias. [http://www.leukemia-gene-atlas.org/LGAtlas/] Universal codes and names to identify laboratory and other clinical observations. [http://loinc.org/] Archives mammograms and related reports of patients. [http://www.cems.uwe.ac.uk/cccs/project.php? name.mammogrid] A consortium of metabolomics, proteomics, and transcriptomic data of breast cancer patients. [http://www.metacancer-fp7.eu] Uses modules for recording use and interpretation of proteomics data derived from protein dependent techniques. [http://www.psidev.info/miape/]

18396036

18396036

17921998

9670133

25879045

12651816

17920862

22546809

18688244

(continued)

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Table 1 (continued) Databases/Webportals MIBBI (Minimum Information for Biological and Biomedical Investigations)

MIFlowCyt (Minimum Information about a Flow Cytometry Experiment)

MINI (Minimum Information about a Neuroscience Investigation) MIAME (Minimum Information About a Microarray Experiment) MMHCC (Mouse Models of Human Cancer Consortium)

NCCN (Oncology Outcomes Database)

NCDB

PRIDE (PRoteomics IDentifications database) SEER (Surveillance, Epidemiology, and End Results Program)

Description Web-based freely accessible tool for creating checklists to facilitate coordination with the aim to build an integrated checklist resource site. [http://www.dcc.ac.uk/resources/metadatastandards/mibbi-minimum-informationbiological-and-biomedical-investigations] Establishes standards for recording and reporting information related to flow cytometer experiments like samples, instrumentation and data analysis. [https://isac-net.org/page/MIFlowCyt] Maintains a checklist that establishes minimum requirements for the use of electrophysiology in a neuroscience study. [http://carmen.org.uk/standards] For the interpretation of microarray experimental results. [http://www.mged.org/Workgroups/MIAME/ miame.html] Resource for mouse cancer models of mouse and associated strains that combines the basic and translational data to derive at genetically engineered mouse models for cancer research. [http://emice.nci.nih.gov/] Network-based data collection, reporting, and analytic system to describe patterns and outcomes of care delivered in the management of patients with cancer. [https://www.nccn.org/clinical_trials/ SharedResource.aspx] A joint program of the CoC of the ACoS and the ACS evaluate and compare cancer care delivered to patients diagnosed and/or treated at state, regional, and national cancer facilities. [https://www.facs.org/quality-programs/cancer/ ncdb] An integrated database for sharing the tremendous proteomics data generated till date among the proteomics community. [http://www.ebi.ac.uk/pride] Collects cancer incidence, prevalence, and survival data, further categorized based on Epidemiological features providing reliable cancer statistics. [https://seer.cancer.gov/data-software/linked_ databases.html]

PubMed IDs 18688244

18752282

18688244

19484163

19259381

18396036

18396036

18592187

30647547

(continued)

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P. K. Raghav et al.

Table 1 (continued) Databases/Webportals SEER (Medicare Linked Database)

SNOMED CT (Systematized Nomenclature of Medicine –Clinical Terms) TRANS-BIG

UMLS (Unified Medical Language System)

Description This database combines clinical information from different cancer registries for health services research. [https://healthcaredelivery.cancer.gov/ seermedicare/] A comprehensive clinical terminology collated. [http://www.nlm.nih.gov/research/umls/ Snomed/snomed_main.html] Consortium of breast cancer patients’ data. [http://www.breastinternationalgroup.org/ Research/TRANSBIG.aspx] Terminology, classification and coding standards. [http://www.nlm.nih.gov/research/umls/]

PubMed IDs 12187163

28566995

28451965

14681409

set of antigens in one particular colon cancer study (Nam et al. 2003), while the mRNA and protein profiles are stored in the RefDIC database. Several systems biology genes and protein expression, and variation databases related to cancer are shown in Table 1 (Expression and Variation Databases). The dbSNP (Single Nucleotide Polymorphism Database) database is primarily used to retrieve detailed genetic sequence and SNPs (Single Nucleotide Polymorphisms) information.

Immune System and Personalized Medicine Databases Used in Cancer Drug Designing Pharmacogenomics analysis and personalized medicine development recognized the relationship between disease and genetic variations (Yan 2008a). This phenomenon shows that different patient subgroups respond to different vaccines or drugs (Wang et al. 2010). The systems biology and immunoinformatic approaches lead to drug and vaccine development required for personalized medicine, treatment, and prevention of cancer. This data is available at an Immunoinformatics Portal (http:// immune.pharmtao.com). Immune epitopes are the most strongly studied areas of the mammalian immune system and play an essential role in designing different cancer vaccines (Gao et al. 2019). Epitopes are the binding domain of antigens that interact with the respective receptors (Yan 2008b). This epitope interaction triggers immune responses in the host immune cells. The immune epitope databases have contributed to the vaccine development, targeted drug design, and interaction analyses (Table 1, Immune System databases). The epitope analysis can be performed using the IEDB (Immune Epitope Database and Analysis Resource). IMGT (International ImMunoGeneTics) is a complete resource database with information about proteins of the human immune system like MHC (Major Histocompatibility Complex), MhcSF (MHC superfamily), Ig (immunoglobulins), and IgSF (Immunoglobulin

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Table 2 Systems biology tools used in cancer studies Tools/Software Description Expression and variation tools ArrayMiner Set of analysis tools for microarray data. Windows and Mac based commercial software. [https://arraymining.net/] ArrayWiki A common sharing platform for collection and storage of microarray experimental data and meta-analysis results. [http://www.bio-miblab.org/arraywiki] caArray Microarray data management system by NCI for analyzing and visualizing gene expression data. [https://array.nci.nih.gov/caarray/home. action] Camelot Outputs a linear regression model that uses (CAusal Modeling with genotype and expression to predict Expression Linkage for phenotype; powered by regularized linear cOmplex Traits) regression. CARMAweb Web-based tool for microarray data analysis (Comprehensive R-based by performing data processing, cluster Microarray Analysis web analysis, and gene oncology term analysis. service) [https://www.genepattern.org/] Cluster For clustering, SOM of microarray data. Windows based commercial software. [http://bonsai.hgc.jp/~mdehoon/software/ cluster/] CNAmet Identification of genes that show simultaneous methylation, copy number, and expression alterations. [omictools.com/cnamet-tool] Consensus clustering Starting from multiple clusterings (each can represent a data type), obtaining a single integrated cluster assignment.[http://code. google.com/p/consensus-cluster] dChip Widely used for the analysis of Affymetrix gene chip data. [http://www.dchip.org/] ECR Browser Publicly available resource for regulatory (Evolutionary Conserved genome data mining for sequence alignment Regions) comparison. [https://ecrbrowser.dcode.org/] Elastic Net Regularized regression method to improve the overall prediction accuracy, using all data as covariates. [https://github.com/ kiwtir/RWEN] Entrez SNP Search tool to look for SNP mutations in dbSNP. [https://www.ncbi.nlm.nih.gov/snp/]

PUBMED IDs 19863798

18541053

19208739

19888205

16845058

31115888

22986455

20141333

18528524

15215395

29688307

23241512

(continued)

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P. K. Raghav et al.

Table 2 (continued) Tools/Software Expression Profiler

GEI (Genes and the Environment Initiative, Exposure Biology Program )

GenePattern

GeneTrailExpress

GEPAS (Gene Expression Profile Analysis Suite) ILOOP

IntegrOmics

iPAC

KOBAS (KEGG Orthology-Based Annotation System ) Lasso

Description Analysis and clustering of gene expression data Web based commercial software. [https://omictools.com/expression-profilertool] A tool that links the genetic and environmental aspects of any tumor type, identifying the environmental exposures and lifestyle factors that make a certain population more susceptible to carcinogenesis. [www.gei.nih.gov/exposurebiology/] Open source software package that provides tools in the form of modules for genomic data analysis. [https://www.genepattern.org/] Analyzes microarray data through standard normalization procedures and statistical analysis. [http://genetrail.bioinf.uni-sb.de] Web-based microarray data analysis tool with algorithms for gene selection, class prediction and functional profiling. [http://www.gepas.org] To analyze two-channel microarray data in different clinical settings. [http://mcbc.usm.edu/iloop] Identification of relationships between two ‘omics’ data sets. [https://omictools.com/integromics-tool] Integration of copy number and gene expression to detect genes and associated pathways or processes that are influenced in trans by copy number. [http://bioconductor.org/packages/release/ bioc/html/iPAC.html] Pathway and disease annotation of gene sets. [http://kobas.cbi.pku.edu.cn/]

PUBMED IDs 15215431

Identification of ‘Omics’ features with predictive ability for a given response (such as survival), using all data as covariates or using some data to decide the penalty of others. [https://github.com?HaohanWang/ thePrecisionLasso]

22986455

21308768

16642009

19099609

18508806

18831776

22986455

22986455

22986455

(continued)

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Table 2 (continued) Tools/Software Lol (Lots of Lasso)

MAGMA

MAPPFinder

MCD (Multiple Concerted Disruption analysis) MGSA (Model-based Gene Set Analysis) Microarray Retriever

Netwalker

omniBioMarker

PatholOgist

PLRS (Piecewise Linear Regression Splines)

RMA Express (Robust Multichip Average)

ScanAlyze

Description Integration of copy number and gene expression to detect in-cis and in-trans regulation of gene expression. [https://rdrr.io/bioc/lol/] A tool for gene analysis and generalized gene-set analysis of genome-wide association studies’ data. [http://ctglab.nl/software/magma] Tool for gene ontology term annotation of differentially expressed genes. [http://www.genmapp.org/] Identification of subsets of genes that are affected on multiple levels by some condition. Identification of active gene sets and their analysis. [https://www.bioconductor.org/packages/ release/bioc/html/mgsa.html] Web-based tool for screening of publicly available microarray data from GEO and ArrayExpress. [http://www.lgtc.nl/MaRe/] Netwalker is a platform to assist in functional analyses of large-scale genomics datasets focused on molecular networks. [http://bioinfo.vanderbilt.edu/netwalker/] Biomarker detection tool that scans through NCI Cancer Gene Index through particular algorithms to improve microarray-based clinical prediction performance. [http://omnibiomarker.bme.gatech.edu/] A consistency score and an activity score is calculated for each pathway. [https://thepathologist.com/inside-the-lab/ bioinformatics] Studying relationships between copy number and mRNA expression; detection of copy number-induced sample subgroupspecific effects. [http://bioconductor.org/packages/release/ bioc/html/plrs.html] Online tool to compute gene expression summary values for qualitative assessment using probe-level metrics. [http://rmaexpress.bmbolstad.com/] Processes fluorescent images of microarrays. Windows based commercial software. [https://omictools.com/scanalyze-tool]

PUBMED IDs 22986455

25885710

22986455

20478067

22986455

18463138

24492837

22893372

22986455

22986455

12925520, 19145252

24298393

(continued)

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P. K. Raghav et al.

Table 2 (continued) Tools/Software SPIA (Signaling Pathway Impact Analysis) SubpathwayMiner

Taverna

TreeView

Tumorscape

VIDA (Visulaization of DAta)

Immunoinformatic tools BLAST (Basic Local Alignment Search Tool)

CLUSTAL W

CTLPred

Cytoscape

IntBioSim

Motif Scan

Description Pathway annotation of differentially expressed genes. [http://bioconductor.org/packages/release/ bioc/html/SPIA.html] Pathway annotation of gene sets. [https://omictools.com/subpathwayminertool] Combines web services and local tools into workflow pipelines for high-throughput ‘Omics’ analyses. [http://www.taverna.org.uk] Graphically browse and analyzes results of clustering. Windows based commercial software. [https://visualcomposer.com/help/interface/ tree-view/] Provide copy number alterations across multiple cancer types. [http://portals.broadinstitute.org/tcga/home] Homologous protein families from virus genomes. [http://www.biochem.ucl.ac.uk/bsm/virus_ database/VIDA.html]

PUBMED IDs 22986455

Most widely used web-based sequence similarity search tool used for comparing nucleotide and protein queries with their respective databases, examining multiple parameters. [https://blast.ncbi.nlm.nih.gov/Blast.cgi] Sequence alignment tool for similarities and differences. [http://www.ebi.ac.uk/clustalw/] Tool for CTL epitopes prediction, for vaccine designing. [http://crdd.osdd.net/raghava/ctlpred/] Cytoscope is an open source platform for visualizing complex networks and integrating the networks with other data types. [https://cytoscape.org/] Integrative multiscale projects relevant to produce biomolecular simulations. [http://intbiosim.org/] Helps finding motifs in a sequence. [http://myhits.isb-sib.ch/cgi-bin/motif_scan]

18440982

22986455

23640334

18792942

22986455

11125070

7984417

30406342

24492837

16766357

19351663 (continued)

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Table 2 (continued) Tools/Software Physiome Project

PredictProtein SiPep

SNeP Biomolecular Network Tools ARACNe (Algorithm for the Reconstruction of Accurate Cellular Networks) ClueGo

DAVID (Database for Annotation, Visualization and Integration Discovery) DREAM (Dialogue for Reverse Engineering Assessments and Methods) Metacore

PSORT

STRING

Text mining tools Anni 2.0

Description Prediction of physiological and functional dynamics of biomolecules. [http://physiomeproject.org//] Protein secondary structure prediction. [http://www.predictprotein. org/] Prediction of tissue-specific minor histocompatibility antigens. [http://www.sipep.org/] Prediction of SNP-derived epitopes. [http://elchtools.de/SNEP/]

PUBMED IDs 12539957

Uses microarray expression profiles to construe functional mechanisms of cellular processes in mammalian cells by analyzing transcriptional interactions. [http://califano.c2b2.columbia.edu/aracne] It is a CytoScape App that improves biological interpretation by integrating GO and KEGG data to create functionally grouped terms with similar associated genes to reduce redundancy. [http://apps.cytoscape.org/apps/cluego] Tool used for functional interpretation of genes’ enlisted and archived on the basis of genomic studies. [https://david.ncifcrf.gov/] DREAM aims to be a catalyzer for the interaction between experiment and theory focused on cellular network inference and quantitative model building. [http://dreamchallenges.org/] For functional analysis of expression and genetic variation data. [https://portal.genego.com/] Prediction tool for protein subcellular localization sites. [https://psort.hgc.jp/] String is a tool for predicting physical and functional protein interactions. [https://string-db.org/]

16723010

Medline interface with retrieved information of genes, drugs, and diseases to conduct more efficient oncological research. [http://biosemantics.org/anni/]

24799431 19513250

25852748

19237447

17576678

24492837

29163640

10087920

24492837

18549479

(continued)

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P. K. Raghav et al.

Table 2 (continued) Tools/Software CAESAR (CAndidatE Search And Rank) CARGO (Cancer And Related Genes Online)

CGMIM

Coag-MDB (Coagulation Serine Protease Mutation Database) COBRA

CoPub

ENDEAVOUR

FACTA+ (Finding Associated Concepts with Text Analysis) G2D

GAPscreener

HuGE Navigator

Description Annotates human genes as tumor associated candidates and ranks them based on a score system. [http://visionlab.bio.unc.edu/caesar] Web-based visualization tool for integrating customized biological information, designed to aid researches with little or no Bioinformatics’ background. [http://cargo-dev.bioinfo.cnio.es/] Text mining of OMIM to detect genetically associated cancer and the related genes. [http://www.bccrc.ca/ccr/CGMIM] Provides data on point mutations with structural analyses of coagulation proteases. [http://www.coagmdb.org/] Text mining tool to construct biochemical networks and enforcing necessary QC measures. [http://opencobra.sourceforge.net] Text mining system that uses Medline abstracts to narrow down keyword co-occurrences. [http://www.copub.org] Tool for prioritization of candidate genes for more complex studies by input of already known genes and integrating the genomic data. [https://endeavour.esat.kuleuven.be/] Text search engine like PubMed for visualizing associations between genes, diseases and chemical compounds. http://www.nactem.ac.uk/facta/] A tool for gene by based on its link to a particular tumor type. Does so by associating various databases like MEDLINE, STRING, GO and RefSeq. [https://omictools.com/g2d-tool] An automatic SVM tool for screening the human genetic associated literature in PubMed. [http://www.hugenavigator.net/download/ GAPscreener_src.zip] .A platform for integrated genetic dataextracted from PubMed using text mining algorithms. [https://phgkb.cdc.gov/ PHGKB/hNHome.action]

PUBMED IDs 20074336

17483515

15796777

18058827

21596791

21622961

18508807

21685059

25392685

18430222

23999671

(continued)

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Table 2 (continued) Tools/Software MarkerInfoFinder

Description For direct retrieval of publications related to a particular or set of genetic markers, making it a more efficient tool for identifying genetic disorders. Can help in delineating genetic causes and mechanism underlying the tissuespecific tumors. [http://brainarray.mbni.med.umich.edu/ brainarray/datamining/MarkerInfoFinder] MedlineR Open source library in R language for Medline biomedical literature data mining for more relevant data scanning. [http://dbsr.duke.edu/pub/MedlineR] MeInfoText Database for information on gene methylation pattern and other epigenetic modifications associated with a tumor type. [http://bws.iis.sinica.edu.tw:8081/ MeInfoText2/] NetCutter Co-occurrence analysis tool to identify coordinately deregulated set of genes in a particular cancer type. [http://bio.ifom-ieo-campus.it/NetCutter/] OSIRISv1.2 Upgraded version of OSIRIS, it is a Named Entity Recognition System to fish out allelic variants of genes from MEDLINE literature to relate SNPs with tissue malignancies. [http://ibi.imim.es/OSIRISv1.2.html] PolySearch Web-based text mining system for correlating cancer types, associated genetic mutations, proteins and metabolites that helps in decoding underlying mechanisms. [http://polysearch.cs.ualberta.ca] Mathematical modeling and simulation tools BioNetTGen Generates physicochemical models of biological systems, where cellular signaling from user-specified rules for biomolecular interactions at the protein domain level is integrated with tools for reaction network simulation and analysis. [http://cellsignaling.lanl.gov/bionetgen/] Bio-SPICE Cellular modelling and simulation of pathways and interaction- network tools for data analysis. [https://biospice.org/index.php] CellDesigner Gene regulatory network modelling, supported by SBML format. [http://celldesigner.org/index.html]

PUBMED IDs 17823133

15284107

18194557

18781200

18251998

18487273

27402907

14683613

24927840

(continued)

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Table 2 (continued) Tools/Software Cellerator

CoExMiner COPASI

Dizzy

Grid Cellware

MCell

MesoRD

PathwayPro

SBTOOLBOX

SIGTRAN

SimBiology

Description For simulation and analysis of signal transduction networks in cells and multicellular tissues. [http://www-aig.jpl.nasa.gov/public/mls/ cellerator/] Used for modeling gene co-expression patterns from transcriptional data. Simulation of network pathways and metabolomic analysis. [http://www.copasi.org/tiki-index.php] Kinetic modelling of integrated large-scale genetic, metabolic, and signaling networks. [http://magnet.systemsbiology.net/software/ Dizzy] Grid-based modeling and simulation tool for biochemical pathways. [http://www.bii.a-star.edu.sg/research/sbg/ cellware] Monte Carlo simulator for investigating cellular physiology, particularly ligand receptor binding, its dynamics and chemical reactions involved. [http://www.mcell.cnl.salk.edu and www. mcell.psc.edu] Open source C++ software for 3D simulation of kinetic reactions based on SBML format. [http://mesord.sourceforge.net] Provides quantitative assessment of gene expression profiles of ligands and their respective receptors. [https://www.accessionhealth.com/digitaldisruption/pathwaypro/] Matlab toolbox for prototyping new algorithms, and building applications for the analysis and simulation of biological systems. [http://www.sbtoolbox.org/] Simulation platform for large- scale reaction networks with Java swing graphical user interface and SBML file support. [https://datatracker.ietf.org/wg/sigtran/ about/] Matlab toolkit for modeling, simulating, and analyzing biochemical pathways. [http://www.mathworks.com/products/ simbiology/description4.html]

PUBMED IDs 12651737

19381544 28655634

15852513

30945248

30945248

15817692

19381544

19381542

19381542

19381542

(continued)

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Table 2 (continued) Tools/Software SmartCell

StochKit

Virtual Cell

Clinical applications of tools AfCS (Alliance for Cellular Signaling)

caBIG ® (Cancer Biomedical Informatics Grid)

caMATCH

caTIES, (Text Information Extraction System)

CERTS (Centers for Education in Research and Therapeutics)

Description C++ platform for the modeling and simulation of diffusion- reaction networks in different subcellular compartments. [http://smartcell.embl.de/introduction.html] C++ simulation tool for intracellular biochemical processes. [http://www.engineering.ucsb.edu/~cse/ StochKit/StochKit.html] Model creation and simulation of cell biological processes, integrating biochemical and electrophysiological data for individual reactions with experimental microscopic image data describing subcellular locations. [http://www.vcell.org/]

PUBMED IDs 19381542

Reconstructs signaling networks by combining the experimental data with genetic and protein annotations, that ultimately leads to quantitative understanding of cellular responses and translational applications. [http://www.signaling-gateway.org/aboutus/ afcs.html] An initiative to develop a system of networks to support translational research in field of cancer. It allows extraction of structured data from pathology reports. [cabig.nci.nih.gov] This subsystem of caBIG is used in recruitment of patients for clinical trials. [cabig.nci.nih.gov/tools/caMATCH] It is an extraction system for generating clinical information based in surgical pathological reports. It is a tool used under caBIG. [cabig.nci.nih.gov/tools/caties] The most widely used online platform operated as a collaborative effort of CERTs is Clinician- Consumer Health Advisory Information Network (CHAIN), which is primarily a resource dissemination program that provides practical information to health care professionals for better patient handling. [www.certs.hhs.gov/]

12539952

19381542

19381542

17911733

20859412

19108734

17632533

(continued)

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Table 2 (continued) Tools/Software CRN (Cancer Research Network)

CTSAs (Clinical and Translational Sciences Awards Programs)

CVRN (Cardiovascular Research Network)

Medi-Class

PROMIS (Patient-Reported Outcomes Measuring System)

STKE (Signal Transduction Knowledge Environment)

Description It is a consortium of 14 different research groups connected by their respective health care delivery sites with information on a wide variety of topics like cancer prevention, detection and treatment. [crn.cancer.gov] This program has been developed to enhance the clinical application of research data collected, to improve the health of the public. [ctsaweb.org] Standardizes data elements like demographics, diagnosis and pathology reports in a virtual data warehouse in conjunction with NCI’s caBIG. [https://cvrn.org/] Decodes both free-text and coded records to detect clinical events in EMRs to optimize record keeping and clinical practice both. [http://www.mediclass.ro/] Patient-centered tool for evaluating physical, mental, and social health. Primarily used to assess symptom burden, or impact of the disease on quality of life through clinical information. [outcomes.cancer.gov/tools/promis] Includes tools to collate information in interdisciplinary fields of signal transduction with a more patient specific approach. [https://libraries.usc.edu/databases/signaltransduction-knowledge-environment-stke]

PUBMED IDs 30972356

21896519

18793105

15905485

30426667

12438188

superfamily). The IPD (Immuno Polymorphism Database) provides an integrated system to study gene polymorphism of the immune system and is further categorized into diverse databases. The IPD-IMGT/HLA (Human Leucocyte Antigen) database incorporates sequences of the human MHC, which is named by the WHO Nomenclature Committee for Factors of the HLA System. Another database, IPD–KIR, contains human killer-cell immunoglobulin-like receptors’ (KIR) allelic sequences, though IPD–MHC database holds MHC information. The dbMHC is an MHC database that provides MHC microsatellite sub-database and interactive alignment visualization for HLA and related genes. Also, database, IPD–HPA includes human platelet antigens (HPA) information. The immunologically characterized tumor cells data is encompassed in the IPD–ESTDAB database. Further, Sanger MHC Haplotype Project comprises data related to MHC-linked-diseases. HapMap is a genomic variation database that provides information on individual genotype data. Moreover, an enormous amount of biological data is required to construct drugtarget networks, which are available in publicly available chemical databases. The

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Fig. 1 Common systems biology databases and tools used in cancer studies

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databases, such as PubChem, MOAD (Mother of All Databases), PDB (Protein Data Bank), ZINC (Zinc Is Not Commercial), and ChEMBL are valuable resources in drug discovery (Table 1, Drug designing databases). Subsequently, these databases can be accessed using STITCH (Search Tool for InTeractions of CHemicals) that integrates relevant information from crystal structures, metabolic pathways, drugtarget relationships, and binding experiments. However, STITCH 2 incorporates BindingDB, PharmGKB, comparative toxicogenomics, and similarity prediction based on text mining, a chemical structure database containing 74,000 compounds and 2200 drugs. Prominently, three-dimensional (3D) modeling databases such as PDB and MMDB (Molecular Modeling Database) have protein 3D structures of antibodies, HLA, and TCRs (T Cell Receptors), required to develop protein interaction network (Wang et al. 2010; Yan 2008b).

Databases Used for Interaction and Pathway Analyses The databases for interaction and pathway analyses are specified in Table 1 (Databases for Interaction and Pathways Analyses). The signaling pathways and molecular interactions associated with the immune systems are provided by InnateDB, PIG (Pathogen Interaction Gateway), and JenPep. Another database, VirusMINT, contains interactions data between human and viral proteins. The typical databases, Reactome, HPRD (Human Protein Reference Database), and DIP (Database of Interacting Proteins), are useful to predict the gene network, protein-protein interactions, and pathways. The linkage between sequence mutation and its function is reported under the OMIM (Online Mendelian Inheritance in Man) database. The miRNAs (miRNA-21, miRNA 125b, miRNA-221, and miRNA-326) play a significant role in the cancer regression by modulating the apoptosis pathway (Hur 2015; Singh and Mo 2013). The miRBase database contains sequences, nomenclature, annotation, and the target prediction of the specific miRNAs. Epigenetic mechanistic studies involving DNA methylation and chromatin modification helps to understand cancer progression and potential cancer therapies (Tomasi et al. 2006). The epigenetic database, MethDB, revealed the methylation patterns and profiles. The central database, KEGG (Kyoto Encyclopedia of Genes and Genomes, http://www.genome.jp/kegg/), is used for studying the biological functions of genes or proteins. KEGG database is comprised of the following sections: KEGG PATHWAY, KEGG BRITE, KEGG GENES, KEGG COMPOUND, KEGG GLYCAN, KEGG REACTIONS, KEGG ENZYMES, KEGG NETWORK, KEGG DISEASE, and KEGG DRUG. The KEGG PATHWAY database is composed of a group of pathway-based maps of molecular reactions and interactions for nucleotide metabolism, metabolic paths, signal transduction paths, and other cell-based processes. KEGG BRITE provides more extensive interaction pathways with diverse types of relationships. The gene catalog database, KEGG GENES, retrieves genomic sequences from NCBI RefSeq. The information for structures of the chemical compound can be retrieved from KEGG COMPOUNDS. The KEGG GLYCAN is a repository of glycan structures. KEGG REACTIONS is used to find the formula of

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chemical reactions, while the nomenclature of enzymes is contained in KEGG ENZYMES. KEGG NETWORK illustrates the networks of gene variations, KEGG DISEASE is used to visualize the details of the molecular network between the type of diseases and therapeutic drugs, KEGG DRUG for chemical structures, the target molecule of drugs and therapeutic categories.

Tumor Databases Used for Drug Designing in Experimental and Clinical Studies Multiple target approaches are used to design drugs against cancer and to identify the interactions between genes and the molecules that target a particular pathway and selectively eradicates cancer (Sharom et al. 2004). Moreover, the unique repositories such as MIBBI (Minimum Information for Biological and Biomedical Investigations), MINI (Minimum Information about a Neuroscience Investigation), MIFlowCyt (Minimum Information about a Flow Cytometry Experiment), and MIAPE (Minimum Information About a Proteomics Experiment) and MIAME (Minimum Information about a Microarray Experiment) are essential for researchers to retrieve the experimental data for analysis. FuGE (Functional Genomics Experiments) repository provides a general description of experimental conditions stored in FuGE as FuGE Object Model (FuGE-OM) or FuGE MarkupLanguage (FuGE-ML). Conversely, the proteomic data is stored in a modified database called PRIDE. Systems biology plays an essential role in identifying crosstalk molecules between pathways and transient behavior of the tumor clinical data. The multicenter project, IOTA, delivers the information of characterized ovarian tumors. Also, the multidisciplinary eTUMOUR project includes clinical “Omics” data, used to develop the brain cancer diagnostics tools. Afterward, this developed model was incorporated into a form of the GUI (Graphical User Interface) to make it user friendly for clinicians. Additionally, breast cancer consortia such as METAcancer, TRANS-BIG, and MammoGrid provide the solution to improve prognosis and breast cancer diagnosis. Besides, the TME.db (Tumor MicroEnvironment database) includes processed, clinical data and R-based statistical tests and approaches for survival analysis.

Systems Biology Approaches and Tools to Cancer Expression and Variation-Based Systems Biology Tools and Approaches for Cancer Prediction All databases perform the meta-analysis (aggregation) of identical data, an essential step of data integration. This approach expands the sample size and consequently advances statistical strength, used to perform the analysis of expression profiling data (Mathew et al. 2007). Data integration indicates numerous issues to be considered for data analysis:

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1. The quantile normalization applied to raw data from any platform (Affymetrix, Agilent, and Illumina.) eliminates the batch-specific effects based on the study and platform analysis (Orlov et al. 2007). 2. Identification of differentially expressed genes and improvement for multiple hypothesis testing (Motakis et al. 2009). 3. Accurate annotation of probe-sets, transcripts, or genes is vital to compare the expression levels of transcript isoform. 4. Consistency should be followed for the clinical sample description and sample nomenclature in all studies. Furthermore, microarray data analysis can be performed based on two different approaches. Either of the separate microarray experiments can be clustered to form one dataset, or each microarray experiment analyzed followed by statistical analysis of all experiments, then ranked according to the aggregation approach (Pihur et al. 2008). Thus, the use of conventional statistical methods for meta-analysis or combining p-values can be implemented to microarray data (Whitehead and Whitehead 1991). However, in the case of meta-regression or stratified groups, a correlation method is used (Mantel and Haenszel 1959), while the developed latent variable approach is the advancement of this method (Mac et al. 2010; Choi et al. 2007). The meta-analysis primarily identifies cancer biomarkers and prognosis signatures (Pihur et al. 2008; Rhodes et al. 2004). In addition to gene expression analysis, the metaanalyses also perform genomics, genetics, and GWAS in cancer (Guerra and Goldstein 2016). The gene expression microarray data at the clinical level has to be analyzed based on either unusual expression levels in one sample, varied expression across all the samples, samples with similar expression patterns, or similar expression patterns over all the samples (Kostka and Spang 2004; Affara 2003). The fundamental evolutionary issues and influence of the amount and quality of available data are addressed based on comparative genome analysis using ECR (Evolutionary Conserved Regions) Browser. Resources and tools for genetic expression and respective functional analysis are listed in Table 2. DNA microarrays can be used for mRNA expression assessment. Nevertheless, protein expression is achieved using 2D-DIGE and MudPit techniques. Two-channel-based microarrays tools, ILOOP (Interwoven Loop) and MAGMA are used to analyze differentially expressed genes at various conditions. Another tool used to perform microarray analysis is GEPAS (Gene Expression Profile Analysis Suite), which includes feature selection, data normalization, unsupervised clustering, and class prediction. Also, a new tool for microarray data analysis, CARMAweb (Comprehensive R-based Microarray Analysis web service), is a Bioconductor module that can be accessed through R programming language. This package includes background correction, normalization, differential gene detection, quality control, visualization, clustering, and dimensionality reduction. GenePattern and caArray are analysis tools that collect differential gene expression data to integrate into the caBIG database (cancer Biomedical Informatics Grid, https://cabig.nci.nih.gov/). The statistical gene function data analysis is performed based on data extracted from the GO database using several available tools or downloadable packages, like GoMiner, AmiGO, GOStat,

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GOEAST, and BiNGO. Furthermore, CoPub tool mines literature and visualizes the searched genes to specific keywords derived from the literature by searching through Medline abstracts (http://services.nbic.nl/cgi-bin/copub/CoPub.pl). Some databases such as GEO and ArrayExpress follow community data standards and established by MIAME, though the community users are allowed to annotate gene expression data using ArrayWiki. Correspondingly, the Microarray Retriever tool retrieves gene expression data from ArrayExpress in addition to GEO databases to increase the sample size in the microarray-based studies. GeneTrailExpress, a web-based application module, is used for implementation, normalization, interpretation, visualization, and statistical analysis based on the standard methods. However, Taverna has been building workflows for web services like caBIG. A web-resource, omniBioMarker (http://omnibiomarker.bme.gatech.edu/) identifies biomarkers based on quality control and normalization, biological interpretation, feature selection, clinical prediction, and validation. There are several existing applications such as RMA Express (Robust Multichip Average), dChip, and caCORRECT that assess microarray data quality and normalizes gene expression. However, some efficient commercial software and source codes are available such as ScanAlyze, Cluster, and TreeView (Table 2, Expression and Variation Tool) (Kostka and Spang 2004; Affara 2003).

Common Immunoinformatic and Bioinformatics Tools to Cancer Drug Discovery Several immune epitope prediction tools are listed in Table 2 (Immunoinformatic Tools). CTLPred predicts the cytotoxic T lymphocyte epitopes, which is an essential tool for designing vaccines. The SNP-derived potential T-cell epitopes for mHAgs (Minor histocompatibility antigens) can be predicted using the SNeP tool. Similarly, SiPep is a mHAgs prediction tool responsible for predicting the chances of rejection of skin grafts transplants and tumors from MHC identical donors, since mHAgs is an antigen in addition to the MHC that determines the functional feasibility of transplantation. Besides, some commonly used bioinformatics tools such as BLAST (Basic Local Alignment Search Tool), Motif Scan and CLUSTAL W are required to compare genetic sequences, building phylogenetic trees, evolutionary relationships, and sequence pattern analysis for immune molecules (Table 2, Expression and Variation Tools) (Affara 2003).

Biomolecular Networks Tools in Cancer Systems approaches have confirmed to be of great advantage for cancer studies, including CSCs (Cancer Stem Cells) diagnosis and characterization (Al-Hajj et al. 2003; Ricci-Vitiani et al. 2007; Singh et al. 2004). A systems approach to cancer identifies crosstalk molecules through global analyses and biological networks (Alberghina et al. 2004; Stilwell et al. 2007). The most widely used systems

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approach is to identify cancer-related gene interaction networks, protein-DNA, and protein-protein interaction networks based on their differential gene expression. These interaction networks are used to visualize large data sets, though they cannot be used to construct drug development and predictive medicine network models (Price et al. 2008). Biomolecular networks integrate clinical data to change highthroughput genomics information into a more comprehensive learning of personalized medicine for the respective disease (Baudot et al. 2009). Network modeling approaches have proved useful to recognize cancer (Wong et al. 2008; Kreeger and Lauffenburger 2009). The network modeling constructs the gene co-expression network, which represents a significant gene correlation map based on their expression profiles at a specific cut-off for tumors samples. These co-expression connections can be weighed with a sigmoid function. The neighboring genes connections can be measured effectively by hierarchical clustering in cancer. Furthermore, the ARACNe (Algorithm for Reconstruction of Accurate Cellular Networks) and Relevance Networks are the two approaches that measure correlation among genes. ClueGo and DAVID build the gene network based on gene ontology terms using the kappa score. The STRING predicts protein or gene relationships. The tools widely used for multiple pathways and interaction analyses in the cancer-immune responses are given in Table 2 (Biomolecular Network Tools). PSORT tool predicts the protein localization sites within cells. A popular software, Cytoscape, has been used to model and visualize cancer biomolecular interactions with options for integration with other data. In addition to Cytoscape, the Metacore is used to visualize biological systems and complex interaction modeling.

Text Mining Tools Used in Cancer Research The text mining tools have proved to be beneficial to identify the relationship between a biological entity and diseases such as cancer. FACTA+ (Finding Associated Concepts with Text Analysis) is a text-mining tool available to analyze the association between genes, proteins, diseases, and chemical compounds. Also, STITCH provides the association links between several drugs and compounds. Nevertheless, NetCutter determines the significant associations between biological entities from the literature. An automatic concept recognition software, Anni 2.0, elicits conceptual profiles from the articles and the ontological relationships among genes. The programming language R has a large-scale archive of statistical packages used for microarray data analysis. An open-source R library, MedlineR, is designed explicitly for Medline literature data mining to build the association matrix and network of query terms, which is further visualized by Pajek. The visualization can also be performed using another literature mining tool CARGO (Cancer And Related Genes Online) that retrieves information of SNPs, genetically inherited diseases, and structural information from iHOP, OMIM, and PDB, respectively. Similarly, other tools like MarkerInfoFinder and OSIRISv1.2 extract SNPs data from the literature, and make suitable connections to cancers and other diseases. However, the programming languages such as Python and Java-based standalone scripts extract mutations

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contained from the literature, whereas R-based script is used to create recursive computational sequential mutations (Raghav et al. 2019). A machine learning-based approach, CRFs algorithm, has been implemented in VTag software to collect mutations information related to cancer at the amino acid and nucleic acid levels from the text. Likewise, a coagulation protein database, Coag-MDB, extracts mutation data from full-text articles and abstracts, further manually inspected and validated. The OMIM collects summaries of Mendelian disorders, described phenotypic and genotypic features of human genes. The OMIM is thoroughly utilized by the CGMIM tool to identify the cancer-associated genes, though the HuGE Navigator tool identifies these genes based on PubMed abstracts using SVM text classifier application, GAP screener. The PolySearch tool extracts gene and disease association information from abstracts, sentences from the literature, and multiple databases (e.g., DrugBank, Entrez SNP, SwissProt, and HGMD). The MeInfoText provides the systemic search from the literature for DNA methylation (hypermethylation and hypomethylation), methods (MSP and COBRA), and gene methylation-related pathway associated with cancer. Similarly, a PubMeth database extracts cancerassociated methylated genes information from abstracts contained in PubMed. These articles are indexed based on gene names from Ensembl and Entrez Gene, incorporated in GeneCard. The text mining tool ENDEAVOU, developed a model for gene ranking, by integrating multiple databases like KEGG (for pathway analysis), BIND (for interaction analysis) and GO (for functional terms assessment). However, the G2D tool prioritizes genes depending on the genetically inherited diseases, and CAESAR (CAndidatE Search And Rank) ranks genes based on retrieved information from OMIM (Kreeger and Lauffenburger 2009; Moding et al. 2013).

Mathematical Modeling and Simulation Tools to Model Cancer Pathways and Networks Three main approaches used for the treatment of cancer are surgery, radiotherapy, and chemotherapy (Mitra et al. 2015). A large proportion of cancer patients receive radiotherapy in combination with other therapies. The proliferative cells within a tumor are primarily more sensitive to DNA mutation or damage caused by radiation. Nonetheless, some tumor cells became quiescent due to hypoxia and are not susceptible to radiation therapy, which is a drawback of the efficacy of radiotherapy (Moding et al. 2013). Therefore, the sensitivity of tumor cells to ionization by radiation depends on the cell cycle phases. Typically, a radiation dose of 2 Gγ/day for 5 days a week, followed by repetition for several weeks, is the standard protocol for radiation therapy given to patients (Moding et al. 2013). The radiation dose’s optimal timing needs to be set to study the cumulative effects of radiation therapies using systems biology, mathematical modeling, and simulation approaches (Ribba et al. 2006). The development of various mathematical models for describing tumor dynamics is increasing enormously. Cancer mathematical model is subdivided into two broad groups, mechanistic and descriptive (Anderson and Quaranta 2008). The

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descriptive models describe the regulation of cancer growth without giving out cellular biological detail (Anderson and Quaranta 2008), while these mechanistic models represent the mechanism of tumor progression, which helps to initiate cancer treatment (Anderson and Quaranta 2008; Araujo and McElwain 2004). The mathematical modeling of biochemical reaction network models demonstrates their utility in constructing predictive models (Price and Shmulevich 2007). The two computational algorithms, PathwayPro and CoExMiner, are used to investigate dynamic behaviors of the gene networks and stable features of gene co-expression. Table 2 (Text Mining Tools) briefly describes some of the existing software tools that can be applied for the stochastic kinetic simulations of various biological systems.

Clinical Applications of Systems Biology Tools and Approaches Natural language–processing services, caBIG’s, caTIES (Cancer Text Information Extraction System), and Medi-Class (Hazlehurst et al. 2005), examine pathology records, chart notes, and other free-text elements measure the frequency of different cancer subtypes for the clinical trials. The programs, caBIG and caMATCH (www. breastcancertrials.org) match patients’ EHR with extensive treatment trials and enlist eligible cancer patients for the clinical trials. The healthcare delivery systems answer the related research questions by patterning with a virtual data warehouse using resources such as CRN (Cancer Research Network), CVRN (Cardiovascular Research Network), and CERTS (Centers for Education in Research and Therapeutics). The SEER-Medicare Linked Database shares knowledge for cancer prevention and research studies related to the treatments (Hazlehurst et al. 2005). Several clinical bioinformatics tools are being employed for risk assessment, early diagnosis, prognosis, and classification of cancer (Kapetanovic et al. 2004). Additionally, opensource software for analyzing and modeling of data has been developed under projects, CGAP (Cancer Genome Anatomy Project), SBML (Systems Biology Markup Language, http://sbml.org/index.psp), the MMHCC (Mouse Models of Human Cancer Consortium), CellML language (http://www.cellml.org/public/ news/index.html), and Systems Biology Workbench (http://www.sbw-sbml.org/ oldindex.html). Besides these, remarkable pathways databases involve the development of the systems biology models, for instance, the KEGG, STKE (Signal Transduction Knowledge Environment) (http://stke.sciencemag.org/), and AfCS (Alliance for Cellular Signaling). In the field of clinical bioinformatics, the applications of systems biology tools and software support the design of future personalized medicines.

Conclusion Cancer is considered to be one of the most lethal diseases in current times (Siegel et al. 2015). The primary concern is the lack of target-specific medicine because it is practically impossible to analyze the big data through conventional study models

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(Stephens et al. 2012; Mossé et al. 2008; Kumar-Sinha et al. 2008; Greenman et al. 2007). The lacunae are now being identified and addressed through Systems Biology approaches. Bioinformatics tools and software are widely used for high-throughput molecular analyses on different data types, involving those generated from the microarray experiments, RNA-seq, WES (Whole-Exome Sequencing), DNA copy number, DNA methylation assays, pathways’ delineation, and protein structure analysis (Kunz et al. 2017). Thorough analysis of these databases and exploiting the mentioned tools and software has helped both types of research and healthcare professionals in understanding cancer statistics and epidemiological studies, diagnosis, biomarker prediction and detection, drug designing, and, most importantly, targeted personalized medicine. Network-based models are being employed and proved to be a good strategy for augmenting the therapeutic efficacy in cancer treatments. These developed networks are being availed to understand inter-tumoral heterogeneity and facilitate data integration among genomic, transcriptomic, and epigenetic alterations. Although more user-friendly tools with simpler interfaces needed to be developed for their full-scale acceptance and application. The present work provides a detailed overview of how bioinformatics algorithms and analytic innovations are being used in the cancer domain to extract maximum biological information.

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Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ROS and Cancer Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cross Talk Between Signaling Pathways and ROS in CSCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . PI3K/AKT/mTOR Pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Notch Pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Wnt Pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . JAK-STAT Pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Metabolic Control of ROS in CSCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Glucose Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Lipid Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Amino Acid Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Therapeutically Targeting ROS Dynamics in CSCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion and Perspective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Both authors designed the literature search strategies and wrote the manuscript. Stacy Grieve and Dipsikha Biswas contributed equally with all other contributors. S. Grieve Department of Biology, University of New Brunswick, Saint John, NB, Canada D. Biswas (*) Department of Biochemistry and Molecular Biology, Dalhousie University, Dalhousie Medicine New Brunswick, Saint John, NB, Canada Novo Nordisk Foundation Center for Basic Metabolic Research, University of Copenhagen, Copenhagen, Denmark e-mail: [email protected]; [email protected] © Springer Nature Singapore Pte Ltd. 2022 S. Chakraborti et al. (eds.), Handbook of Oxidative Stress in Cancer: Mechanistic Aspects, https://doi.org/10.1007/978-981-15-9411-3_150

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Abstract

Cancer stem cells (CSCs) are key tumor-initiating cells residing as a small subpopulation within the tumor mass. Due to their self-renewal and differentiation capacity, CSCs contribute to tumor malignancy and recurrence, metastasis, cancer cell heterogeneity as well as resistance to conventional anticancer therapies. Reactive oxygen species (ROS) acts as an important signaling molecule that tightly regulates the epithelial and mesenchymal states that CSCs exist in. ROS can influence the fate of CSCs by regulating several intersecting intracellular signaling pathways. Here we discuss some of the key signaling pathways that precipitate in changes in ROS levels or are triggered by ROS levels, such as the PI3K/AKT/mTOR (phosphoinositide 3-kinase/AKT/mammalian target of rapamycin), Notch, Wnt, JAK-STAT (Janus kinase/signal transducers and activators of transcription). These intricate signaling cues in response to ROS levels allow CSCs to adapt to the microenvironment by switching their metabolic phenotypes. CSC metabolism is also impacted by the type of tumor and can utilize both aerobic glycolysis and oxidative phosphorylation as energy sources. We discuss some of the important nutrient sources and metabolic pathways and their interaction with ROS that helps sustain CSC stemness. Finally, we summarize different potential strategies targeting the ROS machinery as well as signaling and metabolic pathways operating in CSCs to overcome therapeutic resistance. Keywords

CSCs · Redox · Therapy resistance · Metabolism · Glycolysis · OXPHOS · Signal transduction pathway

Introduction More than a century ago, the idea that tumors may arise from a small subpopulation of cells emerged (Capp 2019). This concept of cancer stem cells (CSCs) took hold in the mid-1990s when acute myeloid leukemia cells possessing specific cell surface markers were shown to reproduce the complete leukemic hierarchy after xenotransplantation in NOD/SCID mice (Capp 2019). Since this discovery, CSCs have been identified not only in hematological malignancies but also in solid tumors such as breast, colon, cervical, and lung cancers, among others (Desai et al. 2019). Understanding how CSCs contribute towards tumor progression, metastasis, relapse, and drug resistance will lead to the development of CSCs-specific therapies that could potentially improve patient outcomes. There are now several defining criteria that distinguish CSCs from the remaining malignant cell population. These characteristics include (1) self-renewal ability; (2) ability to recreate the full phenotypic heterogeneity of the parent tumor; (3) capacity to differentiate into nontumorigenic cells; (4) ability to express distinctive cell

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markers; and (5) ability to exist as a small subpopulation of the total malignant cell pool (Capp 2019). These distinctive features of CSCs render them more resistant to conventional therapeutic strategies, including chemotherapy and radiation (Desai et al. 2019). Recent evidences have highlighted the role of reactive oxygen species (ROS) as a critical driver of metabolic remodeling for sustaining the CSC phenotype, contributing to their therapeutic resistance (Jagust et al. 2019).

ROS and Cancer Stem Cells ROS are chemically reactive species primarily generated during mitochondrial oxidative metabolism. A powerful antioxidant system that includes several antioxidant enzymes including SOD1/SOD2/SOD3, catalase, thioredoxin 1 (TRX1), and the glutathione (GSH) system (Aggarwal et al. 2019) as well as redox-sensitive transcription factors such as NRF2, HIF1α, FOXO, or MYC (Jagust et al. 2019) is necessary to maintain cellular redox homeostasis. Elevated ROS, often seen in malignant cell populations, exerts oxidative stress leading to damage of nucleic acids, proteins, and lipids. Combined with the ability of ROS to affect key cellular signaling pathways, an imbalance of ROS has been shown to contribute towards tumor progression (Aggarwal et al. 2019). While higher levels of ROS are generally thought to contribute to tumor progression, studies also show that low ROS concentrations are critical for CSCs. Diehn et al showed that ROS levels were lower in the CD24medCD49fhigh murine mammary cells, a population enriched for mammary CSCs, when compared to the progenitor cell population. These mammary CSCs with low and intermediate ROS levels were able to engraft and develop new tumors when transplanted into mice mammary fat pads. A similar enrichment of CSCs with low ROS levels was demonstrated in human breast tumors and head and neck tumors (Diehn et al. 2009). Since this observation, lower levels of ROS have been reported in numerous CSC models, including leukemia (Herault et al. 2012) and solid tumors such as breast (Achuthan et al. 2011; Yip et al. 2011; Luo et al. 2018), head and neck (Gammon et al. 2013; Chang et al. 2014, 2018), liver (Muramatsu et al. 2013), nasopharyngeal (Shen et al. 2015), and glioblastoma (Svendsen et al. 2011), among many others. Consistently in these studies, enrichment of CSCs with low ROS correlated with established stem cell markers including Nanog (Chang et al. 2014), OCT4 (Achuthan et al. 2011; Chang et al. 2014; Shen et al. 2015), CD44 (Muramatsu et al. 2013), SOX2 (Achuthan et al. 2011; Hou et al. 2018; Seino et al. 2015), and/or aldehyde dehydrogenase 1 (ALDH 1) (Luo et al. 2018; Chang et al. 2014). There are several mechanisms through which CSCs maintain low ROS levels. Often, one or more antioxidant enzymes are found to be upregulated in CSCs compared to either progenitor cell populations or bulk malignant tumor cell population. For example, ROS scavenger genes such as SOD2, catalase, and peroxiredoxin 1, 2, or 3 (PRDX1,2,3) were upregulated in CSCs derived from head and neck cancer cell lines (Chang et al. 2014), and breast cancer (Achuthan et al. 2011) as well as in tissues from patients with glioblastoma (Svendsen et al.

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2011). Expression of ROS scavenging enzymes is critical for the maintenance of stemness (Herault et al. 2012) and associated with increased resistance to radiotherapy or chemotherapy; enrichment for CSCs can also lead to decreased survival (Herault et al. 2012; Achuthan et al. 2011; Svendsen et al. 2011). Importantly, inhibition of either SOD2 or catalase reduced the number of ROSlow cells in spheroid cultures as well as reduced stemness characteristics, such as the ability of spheroids to grow in anchorage-independent conditions or to form tumors in mouse models (Chang et al. 2014). The upregulation of these antioxidant enzymes has been attributed to altered expression and function of specific transcription factors including the FOXO family and NRF2. The FOXO family of transcription factors were first shown to be critical in maintaining ROS levels in hematopoetic stem cells (HSCs) and later shown to upregulate SOD2 or catalase in CSCs compared to the normal tumor cell population (Diehn et al. 2009). Similarly, increased NRF2 expression has also been reported in CSCs and attributed to the regulation of SOD and other antioxidant genes involved in GSH and TRX anti-ROS defense and redox balance (Achuthan et al. 2011; Kahroba et al. 2019). NRF2 is essential for maintaining low ROS in CSCs even in the absence of oxidative stress and its activation is required for the maintenance of CSC properties (Chang et al. 2018). Apart from regulating the expression of powerful antioxidants, NRF2 also modulates cellular metabolism and influences the switch between anabolic/catabolic glucose metabolism (Jagust et al. 2019). Metabolic switching towards glycolysis is a prominent trait of CSCs, which further contributes towards decreased ROS levels in CSCs. NRF2 is able to maintain low ROS levels by inhibiting the TCA cycle and promoting glycolysis (Chang et al. 2018). NRF2 can also promote the pentose phosphate pathway (PPP) and impede mitochondrial metabolism by activating PDK1 (Chang et al. 2018) and altering expression of several other genes including G6PD, PGD, MTHFD2, and IDH1 (Kahroba et al. 2019). Additionally, the transcription factor HIF1α contributes to metabolic remodeling in CSCs residing in hypoxic lesions and is crucial for maintaining its stemness (Gammon et al. 2013). While originally thought to exist in a hierarchy, stem cells were more recently shown to possess bidirectionality, mediated in part by ROS and highlighting the dynamic interplay between ROS and CSCs. Increasing ROS by inhibiting antioxidants (Diehn et al. 2009; Chang et al. 2014) or by blocking glycolysis (Luo et al. 2018; Shen et al. 2015) have been shown to deplete CSC of its stem cell characteristics. The plasticity of stemness is closely related to the epithelial-mesenchymal transition (EMT). Indeed, CSCs have been shown to exist in two states – epithelial and mesenchymal – that is tightly controlled by ROS (Luo et al. 2018; Chang et al. 2014; Peitzsch et al. 2014). ROS generated during hypoxia regulates EMT, where the transition to a mesenchymal phenotype confers CSC characteristics (Dong et al. 2013). This occurs as a shift in metabolic reprogramming. Specifically, the EMT transcription factor, Snail decreases expression of fructose 1,6 bisphosphatase 1 (FBP1), a rate-limiting enzyme in gluconeogenesis, thereby inhibiting oxygen consumption and suppressing mitochondrial complex I (Dong et al. 2013). HIF1α can also upregulate anaerobic metabolic genes (Gammon et al. 2013), where hypoxia promotes CSC proliferation (Peitzsch et al. 2014). Ultimately, ROS can control

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the stem cell–like state by acting as a signaling molecule and affecting numerous pathways implicated in CSC biology. Thus, not only is ROS a by-product of oxidative imbalance and altered metabolism but is also a critical mediator of CSC plasticity.

Cross Talk Between Signaling Pathways and ROS in CSCs ROS plays an important role in cellular signaling and redox regulation, where it oxidizes proteins to allosterically regulate their conformation and function by targeting the redox-sensitive cysteine and methionine residues (Lee et al. 2019). Among the different ROS agents, H2O2 acts as the second messenger for intracellular signaling through cysteine-based modifications, while the other ROS agents, O2•- and •OH, are associated with cellular damage (Lee et al. 2019). This redox regulation is crucial in maintaining the hallmark features of CSCs and may point to important therapeutic targets for effective elimination of CSCs. A summary of some of the key ROS-dependent signaling pathways important for effective CSCs functioning are provided in Fig. 1.

Fig. 1 ROS-mediated signaling in the CSCs. Wnt, Notch, PI3K/mTOR/AKT, and JAK-STAT are some of the key signaling pathways associated with CSC self-renewal, maintenance of stemness, tumorigenesis, and differentiation. The activation of these signaling pathways is synchronous with the ROS levels and is in turn responsible to modulate ROS levels within the CSCs. The intricate cross talk between the signaling pathways are integral for redox regulation

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PI3K/AKT/mTOR Pathway The PI3K/AKT/mTOR pathway is upregulated in CSCs and is vital for preserving stemness. Activation of this signaling pathway is associated with increased expression of CSC markers as well as EMT in chemoresistant ovarian cancer cells (Deng et al. 2019). Not only does activation of the PI3K/AKT/mTOR pathway result in elevated glycolysis influencing intracellular ROS levels and tumorigenesis (Ding et al. 2015), but modulation of ROS affects PI3K/AKT/mTOR signaling in CSCs. For example, treatment with high concentrations of H2O2 induced AKT phosphorylation and activity in glioma-initiating cells (Sato et al. 2014). While mTOR activation is necessary for the tumorigenic properties of breast cancer stem-like cells, inhibition of mTOR results in decreased expression of stem cell marker ALDH1 in colorectal CSCs and epithelial cell adhesion molecule (EpCAM) in hepatocellular CSCs (Xia and Xu 2015). mTOR activity has been linked with modulating both mitochondrial number and function and overall homeostasis of ROS in HSCs. Indeed, in HSCs lacking tuberous sclerosis complex 1 (TSC1), the upstream activator of mTOR, the expression of mitochondrial oxidative genes was significantly enhanced, resulting in ROS accumulation as well as triggering loss of HSC quiescence and selfrenewal capacities (Finkel 2012). Reduced stem cell–like properties and loss of tumorgenicity due to inhibition of PI3K/AKT/mTOR activity has also been reported across different tumor types (Deng et al. 2019; Xia and Xu 2015; Dubrovska et al. 2009). AKT also signals through the FOXO family of transcription factors to control cellular viability, metastasis, and ROS levels. Since the FOXO family maintain production of redox catalysts such as manganese superoxide dismutase (MnSOD/ SOD2) and catalase, cancer cells with higher nuclear FOXO1 levels have lower ROS levels (Ding et al. 2015). The SOD2 to catalase ratio is considered a potential marker of invasion and metastatic progression in different cancers such as lung, colon, prostrate, and squamous carcinomas (Miar et al. 2015). Moreover, SOD2 overexpression increased mitochondrial ROS and HIF2α activity and increased CSC formation leading to tumor invasiveness and worse outcomes in breast cancer patients (He et al. 2019). In a feed-forward mechanism, FOXOs are also transcriptional targets of HIF1α, further influencing ROS levels and cancer cell stemness (Ding et al. 2015). The tumor suppressor, phosphatase and tensin homolog deleted on chromosome 10 (PTEN), is a major negative regulator of PI3K. PTEN regulation plays a critical role in CSCs and is differentially regulated in CSCs versus normal stem cells. ROSdependent redox regulation has been shown to affect the catalytic activity of PTEN in CSCs, where NADPH oxidase (NOX)-mediated ROS production inactivates PTEN through oxidation of a cysteine residue, thereby affecting PI3K/AKT signaling (Nakanishi et al. 2014). PTEN deletion has also been reported to result in exhaustion of HSCs while increasing the leukemia-initiating cell population and prostrate cancer stem-like cells (Dubrovska et al. 2009). Similarly, downregulation of PTEN by miR-216a/217 cluster evokes EMT and cancer stem-like properties in hepatocellular carcinoma (HCC) (Ding et al. 2015).

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Notch Pathway Signaling through the Notch family of receptors is often deregulated in cancers and is thought to play a critical role in regulating CSC proliferation, renewal, differentiation, and tumorigenesis. The expression and activation of Notch pathway components leads to poor prognosis and resistance to chemotherapy or radiation (Wang et al. 2012). Notch signaling promotes the drug-resistant capacity of CSCs by triggering angiogenesis, EMT, and their ability to self-renew. Aberrant Notch 1 and Notch 4 signaling has been shown to regulate the proliferation and colony formation of CSCs from numerous cancer types. Similarly, ligands such as Jagged 1, Jagged 2, Delta-like ligand 1, and Delta-like ligand 4 that activate Notch signaling in tissue-specific contexts are often overexpressed in CSCs and have also been shown to play critical roles in maintaining stemness (Yang et al. 2020). In part, Notch signaling maintains CSCs by interacting with other key signaling proteins. For example, Notch activates the epidermal growth factor receptor type 2 (HER2) in breast cancer–derived spheroid cells with stem cell–like properties, promoting CSC propagation and self-renewal (Baker et al. 2014). Similarly, Notch 3 activity dictates interleukin-6 (IL-6)-mediated self-renewal of mammary CSCs. The PI3K/AKT pathway is also influenced by Notch signaling where the radioprotective role of Notch signaling in glioma was found to be dependent on AKT (Ding et al. 2015). However, the mechanisms by which AKT is activated by Notch depends on the type of CSCs and can be independent of transcriptional regulation. Similarly, activation of Notch signaling may depend on its interaction with HIF-1α or the Wnt or Hedgehog (Hh) pathways (Ding et al. 2015). The interplay of Notch signaling with these and other pathways is critical for regulating the maintenance of stemness in CSCs and their self-renewal capabilities. The Notch pathway can also maintain CSCs by regulating ROS levels. Notch upregulation of AKT in CSCs enhances expression of ROS scavenging enzymes, leading to a decrease in ROS (Ding et al. 2015). On the other hand, ROS can also stimulate Notch signaling pathway to maintain CSCs. The endoplasmic reticulum cargo protein MAP 17 alters redox balance by increasing ROS levels resulting in increased glucose and mannose metabolism. MAP 17 sequesters the Notch signaling inhibitor, NUMB, by interacting through its PDZ-binding domain and activating the Notch pathway in cervical CSCs (Garcia-Heredia et al. 2017). Moreover, ROS levels increase in response to hypoxia, stabilizing HIF-1α levels, inducing Jagged 2 activation, and increasing the invasive properties of breast and lung CSCs. Both HIF-1α and HIF-2α have been implicated in activating the Notch signaling pathway and subsequent maintenance of solid tumor CSCs (Yan et al. 2018). Inducible nitric oxide synthase (iNOS) can also act as major ROS-generating systems influencing the Notch signaling pathway. iNOS is associated with more aggressive solid tumors and has been found to promote stemness in gliomas, colon cancer, and liver CSCs through TACE/ADAM17 activation of Notch 1 signaling (Wang et al. 2018a). NO released from endothelial cells can also activate Notch signaling, promoting the stemness of PDGF-induced glioma cells, their tumorigenesis, and capacity to form neurospheres. Conversely, loss of NO activity

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suppressed Notch signaling, reduced tumor burden, and prolonged survival in a mouse glioma model (Charles et al. 2010).

Wnt Pathway The Wnt signaling pathway has been associated with CSC stemness, self-renewal, invasion, and differentiation along with their metabolic reprograming. The Wnt pathway is an evolutionarily conserved pathway that can be divided into three branches: β-catenin or canonical Wnt pathway that activates its downstream targets in the nucleus; the noncanonical pathway that constitutes the planar cell polarity pathway involving jun N-terminal kinase (JNK); and the Wnt/Ca2+ pathway (Kahn 2018). Both the canonical and noncanonical pathways have been shown to be critical for the maintenance of CSCs. Activation of the canonical Wnt/β-catenin pathway upregulates numerous genes involved in CSC maintenance including MYC, Snail, Cyclin D1, CD44, and Jagged 1. Collectively, gene expression changes modulated by β-catenin regulate EMT, oxidative stress, lineage commitment, and metabolism (Le et al. 2008). An important attribute of CSCs is a high functioning telomerase reverse transcriptase (TERT) gene, which helps them maintain long telomeres and potentiates their longer survival. TERT is a direct transcriptional target of β-catenin and its expression is enhanced upon canonical Wnt signaling activation (Zhan et al. 2017). Due to the critical role β-catenin plays in stemness, reduction of active β-catenin leads to a decrease in CSCs population. For example, the tumor suppressor cadherin-11 induces apoptosis in colorectal CSCs by inhibiting active β-catenin (Satriyo et al. 2019). Similarly, apoptosis can be stimulated in breast CSCs by the Wnt signaling pathway antagonists DACT-1 or Dickkopf-related protein 2 (Yang et al. 2020). Indeed, β-catenin protects CSCs against oxidative stress by maintaining an equilibrium between the TCF signaling pathway, which regulates CSC proliferation and the FOXO signaling pathway, which in turn regulate ROS scavenging proteins (Zhao et al. 2018). Apart from β-catenin, Wnt signaling can also activate the PI3K/AKT signaling cascade or Hippo pathway components YAP/TAZ. Both the PI3K/AKT and the Hippo pathway promote the survival of CSCs and their resistance to therapeutic drugs (Kahn 2018). Not only does β-catenin regulate ROS levels, but ROS levels also regulate CSC proliferation through the canonical Wnt/β-catenin pathway. During oxidative stress, binding of β-catenin to the FOXO promoter increases while the binding of β-catenin with TCF is restored when ROS levels are controlled (Zhao et al. 2018). In HCC, targeting glutaminolysis through GLS1, an enzyme involved in glutamate conversion and mediating antioxidant defense, was shown to increase ROS levels, inhibit β-catenin translocation to the nucleus, and reduce stemness (Li et al. 2019). Deprivation of glutamine was also shown to enhance ROS production and decrease β-catenin stability in stem cells isolated from a lung cancer cell lines (Liao et al. 2017). Similarly, in breast CSCs, a shift in metabolic programming can increase ROS production and decrease β-catenin signaling (Dong et al. 2013).

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The interplay between ROS and Wnt/β-catenin signaling is likely complex. Other studies have suggested that low levels of oxidative stress can activate β-catenin and enhance proliferation of CSCs. Under homeostatic conditions, the TRX family protein nucleoredoxin binds a redox-sensitive Wnt adaptor protein activating the ubiquitin ligase activity of GSK3β to decrease β-catenin stability. On the contrary, under oxidative stress, this interaction is inhibited and β-catenin signaling through the TCF transcription factor is enhanced (Ding et al. 2015). Oxidative stress also activates Wnt signaling pathway through induction of CD44. Specifically, the splice variant of CD44 (CD44v) stabilizes the cystine/glutamate (xCT) antiporter in order to provide cystine, the rate limiting substrate of GSH synthesis, to the CSCs. This reduces ROS levels within CSCs and influences Wnt signaling in a negative feedback loop. Indeed, cancer cells at the metastatic front are composed of a heterogeneous population of quiescent CSCs which exhibit high CD44v/low MYC and proliferative CSCs exhibiting high CD44v/high MYC. Further, the number of CSCs at the invasive front with enhanced ROS is decreased in response to the drug sulfasalazine, a drug that disrupts the CD44v-xCT axis (Yoshida 2017).

JAK-STAT Pathway The Janus kinase/signal transducers and activators of transcription (JAK-STAT) signaling pathway is another evolutionarily conserved pathway that is involved in cellular stress response, apoptosis, oncogenic transformation, and immune regulation in cancers. This pathway is stimulated by cytokines and growth factors, such as interleukins, growth hormones, epidermal growth factor (EGF), platelet-derived growth factor (PDGF), granulocyte/monocyte colony-stimulating factor (GMCSF), and interferons. Several of these cytokines are integral in regulating CSCs. The cytokines, IL-10 and IL-6, have been documented to induce invasion and migration in non-small-cell lung and breast CSCs, respectively (Yang et al. 2020). IL-6 can convert bulk cancer cells into CSCs in breast and ovarian cancer by activating the transcription factor OCT4 and subsequently activating JAK1/STAT3 signaling. Similarly, IL-6 also activates the JAK1/STAT3 pathway in high ALDH expressing CD126+ endometrial CSCs. JAK2/STAT3 signaling is activated by IL-6, erythropoietin, and retinolbinding protein 4 in CD44+CD24
breast and colorectal CSCs as well as colon CSCs (Yang et al. 2020). Furthermore, chronic activation of STAT3 promotes survival and stemness of breast CSCs. Similarly, cell adhesion, differentiation, invasion, and proliferation of colorectal CSCs are promoted via the JAK1/STAT1 pathway when it is activated by the scaffold protein AJUBA (Yang et al. 2020). The JAK/STAT pathway is activated in CSCs in response to intracellular ROS where STAT1 and STAT3 are activated in response to H2O2.. Alternatively, depleting GSH can also activate the STAT pathway (Simon et al. 1998). Further, ROS acts as a second messenger for PDGF, which in turn activates STAT signaling. Interestingly, STAT activation is dependent on different cancer types and is also oxidant specific. For instance, in squamous cell carcinoma cells, STAT is not activated in response to

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superoxide or NO-generating stimuli (Simon et al. 1998). HIF-1α-mediated activation of the JAK1/STAT3 pathway enhances self-renewal of glioma CSCs (Yang et al. 2020). In non-small-cell lung cancers, increased NOX4 expression results in ROSdependent IL-6 secretion and subsequent activation of STAT3. Conversely, NOX4 depletion reduced H2O2 and inhibited IL-6-dependent STAT3 activity, suggesting a positive feed-forward loop between NOX, ROS, IL6, STAT3 factors (Kirtonia et al. 2020). STAT3 is also involved in the formation of NO that promotes CSCs survival. STAT can trigger the formation of ROS by processing mitochondrial respiratory complexes and import. ROS can, in turn, rapidly activate STAT. Indeed, interferons α/β/γ induced STAT1/3 phosphorylation and import into the mitochondria. This in turn increased the oxygen consumption rate and expression of mitochondrial complexes, resulting in increased ROS accumulation. Alternately, suppression of ROS hindered interferon-mediated STAT activation. This STAT-ROS cycle has been reported in different cancer cell types; however, it may not be exclusive to CSCs (Wang et al. 2018b).

Metabolic Control of ROS in CSCs Metabolism plays a causal role in dictating different phenotypic states exhibited by CSCs. Unlike other multipotent stem cells that are primarily glycolytic and can switch to oxidative phosphorylation (OXPHOS) during differentiation, CSCs can adopt both metabolic phenotypes in a tumor-type-dependent manner, summarized in Fig. 2. Moreover, high functioning and increased number of mitochondria are crucial benefactors for sustaining CSC functionality irrespective of their metabolic states. Mitochondria are crucial for regulating signaling pathways, controlling apoptotic signals, as well as releasing ROS (Sancho et al. 2016).

Glucose Metabolism Although the Warburg effect is considered a common phenomenon in all cancer cells, depending on their origin and the complex interactions with the tumor microenvironment, CSCs can either be more dependent on OXPHOS or anaerobic glycolysis (De Francesco et al. 2018). For instance, breast CSCs depend on distinct metabolic pathways that allow them to transition between the quiescent low ROS mesenchymal (M) and the high ROS proliferative epithelial-like (E) states. The Mtype breast CSCs display augmented glycolysis and gluconeogenesis while E-type breast CSCs have increased mitochondrial OXPHOS and TCA cycle coupled with increased NRF2-mediated antioxidant mechanisms (Luo et al. 2018). Several studies have suggested that high PPP supports the function and survival of CSCs in response to high ROS (Ayob and Ramasamy 2018). Along with ROS levels, the expression of various glycolytic or PPP enzymes can be regulated by the AMPK-HIF1α axis (Luo et al. 2018). This association between cellular redox and CSC state suggest that

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Fig. 2 Redox regulation of CSC metabolism. Tumor cell heterogeneity is determined by not only the different cell population, but also its metabolism. Contrary to other multipotent stem cells, proliferative CSCs exhibit both glycolysis and OXPHOS metabolic phenotypes depending on its tumor origin. Under normoxic conditions, both glycolysis and the pentose phosphate pathway (PPP) are upregulated in the CSCs. CSCs also display increased number of high functioning mitochondria, increased lipid droplet (LD) formation, fat oxidation (FAO) as well as a concomitant increase in fatty acid synthesis and lipid desaturation. The NAPH generated from the PPP as well as the FAO is utilized to maintain the redox balance within the CSCs. Increased ROS mediates HIF1α activation resulting in increased expression of glucose transporters and glycolytic proteins. CSCs also rely on utilization of catabolites such as lactate and amino acids like glutamate and BCAAs

antioxidants might allow metabolic switching of CSCs to the quiescent-like states allowing them to escape therapy and revert back to proliferative states. Lower oxygen availability in the CSC niche is characterized by increased reliance on glucose as an energy substrate. This metabolic remodeling is primarily mediated by the transcriptional regulation of glycolytic enzymes and glucose transporters by the HIFs and NRF2 and can lead to drug resistance. Indeed, chemoresistant pancreatic CSCs present with a strict reliance on low ROS coupled with glycolysis while chemoresistance in lung CSCs correlated with HIF-1-mediated increased expression of GLUT1 (De Francesco et al. 2018). Drug resistance is associated with enhanced oxidative PPP along with high levels of GSH and G6PD, which are regulated by NRF2. Inhibiting G6PD results in increased ROS and ER stress leading to apoptosis (Ayob and Ramasamy 2018). Several important proteins play pivotal roles in regulating the metabolic plasticity of CSCs. For example, CD44, particularly CD44v isotypes, protect CSCs against redox stress by promoting the synthesis of GDH through the interaction of xCT with pyruvate kinase isoform M2 (PKM2), suppressing PKM2 activity. Due to low

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PKM2 activity, glycolytic intermediates flow into PPP instead of being converted to lactate (Ayob and Ramasamy 2018). Conversely, high MYC activity in CSCs correlate with high PKM2/PKM1 ratios that stimulate glycolysis and coordinate with HIF-1 to regulate cellular responses to hypoxia. In combination with HIF, MYC promotes glycolysis by upregulating GLUT1, hexokinase 2 (HK2), and pyruvate dehydrogenase kinase1 (PDK1) (Ayob and Ramasamy 2018). Additionally, basallike breast CSCs silence FBP1 as a mechanism to maintain flux through both glycolysis and other secondary biosynthetic pathways (Dong et al. 2013). Overexpression of FBP1 lead to increased mitochondrial oxygen consumption and reduced flux through the PPP pathway resulting in ROS accumulation and increased tumor suppression, an effect reversed by the antioxidant, N-acetylcysteine (Dong et al. 2013). Finally, some of the energy requirements that drive stemness and ultimately result in poor clinical outcomes may come from the more differentiated glycolytic cancer cells or auxiliary catabolites, such as ketones and lactate (De Francesco et al. 2018).

Lipid Metabolism CSC metabolism is heavily dependent on lipid metabolism. Current evidence suggest that glycolytic intermediates feed de novo lipogenesis to fuel CSC self-renewal and proliferation (Yi et al. 2018). Modulations involving both lipid anabolism and catabolism are associated with stemness of CSCs. Glioma CSCs display an increased reliance on de novo lipogenesis (DNL) due to high fatty acid synthase expression (Yasumoto et al. 2016). Additionally, increased lipid uptake and rates of DNL have been associated with elevated accumulation of lipid droplets (LDs) that are composed of fatty acids, cholesterol, and triacylglycerols, which adds to the tumor initiating capacity of the CSCs. Increased LD content is a distinctive feature of several types of CSCs, particularly colorectal CSCs which display a direct correlation between CD133+ cells and LD content (Begicevic et al. 2019). Interestingly, high glucose triggered ROS accumulation as well as increased LD content in colorectal CRCs. Administering lipid-lowering drugs, such as a diacylglycerol O-acyltransferase 2 (DGAT2) inhibitor, markedly reduced colorectal CRC population (Tirinato et al. 2019). Several factors can contribute to increased LD content. For example, hypoxia induces LD formation via HIF1- and HIF2-mediated suppression of carnitine palmitoyl transferase (CPT1A), a key mitochondrial fatty acid oxidation (FAO) enzyme (Yi et al. 2018). Conversely, the fatty acid transporter CD36 increases lipolysis and FAO. CSCs have a greater reliance on FAO compared to the non-stem cancer cells and inhibiting FAO results in reduced viability and tumorsphere forming capacity of CSCs. Likewise, the HCC CSCs undergo metabolic reprograming from OXPHOS to FAO to promote their enhanced clonogenic potential (Begicevic et al. 2019). Increased LD content has also been associated with increased activity of stearoyl-CoA desaturase 1 (SCD1). SCD1 and LD content can activate the Wnt/β-catenin, NF-κB, and Hippo pathways that dictate CSC fate (Cruz et al. 2020). Thus, LDs and associated increases in fatty acid supply and FAO may

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dictate the fate of CSCs by conferring survival to CSCs under adverse metabolic demands. LDs can also protect lipids from peroxidation due to defective GSH and excessive ROS, shielding the CSCs from a type of nonapoptotic cell death called ferroptosis (Yi et al. 2018). Inducing this process by the synthetic derivative, ironomycin, potentiated selective antitumor activities against breast CSCs (Mai et al. 2017). These studies suggest that CSCs rely on enhanced lipid metabolism to support their energy demands and sustenance as well as protection from high ROS toxicity.

Amino Acid Metabolism Amino acids also play a critical role in the metabolic plasticity of CSCs. Most prominently, the nonessential amino acid glutamine contributes to the energy and metabolic balance during CSC proliferation by modulating the uptake and utilization of other amino acids and maintaining redox potential. Glutamine plays an important role in mediating metabolic remodeling in cells with defective OXPHOS and the severity of the defects determine whether glutamine will undertake oxidative or reductive metabolic routes (Yadav et al. 2020). CSCs display increased expression of the glutamine-metabolizing enzymes GSH, GOT1, and GOT2. In turn, glutamine can also regulate CSC metabolism by modulating expression of the stem cell marker ALDH (Samanta et al. 2016). Additionally, increased plasma membrane localization of the glutamine transporter 2 (ASCT2) is observed in different CSCs, notably prostrate and colorectal CSCs. Inhibiting ASCT2 resulted in reduced mTOR signaling and E2F cell cycle proteins, arresting CSC growth (Yadav et al. 2020). Interestingly, augmenting GSH activity increased CSC proliferation even under glutamine starvation (Samanta et al. 2016). CSCs thus depend on glutamine metabolism for their survival and redox homeostasis. Both mTOR and MYC influences glutamine uptake and catabolism and can in turn be regulated by intracellular glutamine levels. Specifically, MYC mediated conversion of glutamine to α-ketoglutarate which acts as an anaplerotic substrate to replenish TCA cycle pool in response to hypoxia and low pyruvate availability (Yadav et al. 2020). Targeting these pathways may disrupt CSC stemness and proliferation. In recent years, additional amino acids have also been shown to influence CSC metabolism. The serine biosynthesis enzyme, phosphoglycerate dehydrogenase (PHGDH), is highly expressed in breast CSCs which helps in sustaining the CSC population by maintaining redox homeostasis by metabolic rewiring towards increased glycolysis (Samanta et al. 2016). This also helps CSCs to adapt and survive under hypoxic conditions. On the contrary, depleting tryptophan resulted in preserving CSC phenotype by augmenting OCT4 transcription, a key transcription factor for CSC self-renewal (Liu et al. 2019). Moreover, metabolome analysis of leukemia initiating cells (LICs) revealed increased branched chain amino acid (BCAAs) levels that are correlated with high levels of BCAA transporters and increased transport of BCAAs into the cytoplasm, irrespective of their lineage origin. Indeed, mice fed a BCAA-deficient diet demonstrated significantly reduced tumor growth when engrafted with human LICs (Kikushige et al. 2019).

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Therapeutically Targeting ROS Dynamics in CSCs Therapeutically targeting CSCs remains a challenge to date. Many conventional therapies mediate cell death through induction of free radicals. CSCs, characterized in part by their increased ROS defense pathways, may develop less DNA damage after treatment compared to bulk malignant cell population, thus developing resistance to conventional therapies. Indeed, enrichment of CSCs has been observed after irradiation (Diehn et al. 2009; Shen et al. 2015) or cytotoxic chemotherapies (Achuthan et al. 2011; Luo et al. 2018; Chang et al. 2014; Muramatsu et al. 2013). With the understanding of the critical role ROS plays in maintaining CSCs properties, targeting ROS in CSCs offers an alternative approach to enhance efficacy of antitumor therapies. Given the different mechanisms through which ROS levels are regulated in CSCs, various therapeutic approaches (Table 1) have been investigated in the preclinical setting. One approach involves directly targeting signaling pathways known to modulate ROS levels. For example, inhibiting PI3K/AKT/mTOR activity by the dual PI3K/mTOR inhibitor, NVP-BEZ235, decreases CD133+/CD44+ stemlike populations (Dubrovska et al. 2009). Moreover, combining BEZ235 with the chemotherapeutic drug cisplatin induced ROS levels resulting in decreased population of CSCs and increased apoptosis in chemoresistant ovarian cancer cells (Deng et al. 2019). Moreover, CSCs display selective sensitivity to the inhibition of the PI3K/ AKT/mTOR pathway compared to normal stem cells making this pathway an attractive therapeutic target for combination therapy. Similarly, inhibition of Notch signaling through compounds such as psoralidin (Suman et al. 2013) or L685458 (Yan et al. 2018) decreased the number of stem-like cells, limited spheroid growth, and reversed chemotherapy resistance in different cancer types. Additionally, cotreatment of GSI-18, a γ-secretase inhibitor that leads to inhibition of Notch signaling, has been shown in several settings such as medulloblastoma or glioblastoma (Yang et al. 2020) to deplete stem cell populations and reduce tumor growth in mouse models. Because of its success in preclinical studies, other γ-secretase inhibitors have now entered clinical trials for the treatment of both solid and hematological cancers (Yang et al. 2020). Loss of β-catenin has been shown to impede the recovery of CSCs from ionizing radiation by elevating ROS levels and enhancing double-strand DNA breaks (Zhao et al. 2018). Similarly, treatment with the cyclooxygenase 2 inhibitor NS398 resulted in increased sensitivity in radioresistant breast CSCs due to downregulation of β-catenin levels (Ding et al. 2015). Specific targeting of β-catenin with epigallocatechin-3-gallate (EGCG) or the small molecule inhibitor CWP232228 has been shown to induce apoptosis in lung and liver CSCs, respectively (Yang et al. 2020). Finally, inhibiting STAT3 by cucurbitacin 1 reduced tumorigenicity, sphere formation, and resistance to ionizing radiation in head and neck squamous cell carcinoma and reduced CD133+ lung CSC populations (Ding et al. 2015). Targeting the antioxidant system has also been shown to be effective in eradicating CSCs. The combination of disulfuram and copper, which synergistically act as a potent ROS inducer, has been shown to inhibit the proliferation and self-renewal

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Table 1 ROS targeted therapies in CSCs Drug Signaling pathways NVP-BEZ35

Target

Cancer

Reference

PI3K/AKT

Prostrate, ovarian

Psoralidin L685458 GSI-18

Notch Notch Notch

NS398

WNT, Betacatenin WNT, Betacatenin STAT3

Breast Breast Medulloblastoma, glioblastoma Breast

Deng et al. (2019) and Dubrovska et al. (2009) Suman et al. (2013) Yan et al. (2018) Yang et al. (2020)

CWP232228 Cucurbitacin 1 Antioxidant Disulfuram and copper Resveratrol

Ding et al. (2015)

Lung, liver

Yang et al. (2020)

HNSCC, lung

Ding et al. (2015)

" ROS

Breast, glioma

Yip et al. (2011)

" ROS

Emodin PEITC/LBL21

" ROS GSH

Breast, colon, glioma Glioma Colon, lung

Curcumin Sulfasalizaine Auranofin Shikonin

GSH GSH TRX TRX

Liver HNSCC, liver Lung Colon

ATRA

NRF2

Ovarian

Brusatol Hinokitiol Metabolic dichloroacetate Atovaquone

NRF2 NRF2

Breast Glioma

Seino et al. (2015) and Zhang et al. (2018) Kim et al. (2015) Yun et al. (2017) and Wang et al. (2017) Wang et al. (2018c) Okazaki et al. (2019) Hou et al. (2018) Ding et al. (2015) and Liang et al. (2017) Robledinos-Antón et al. (2019) Kahroba et al. (2019) Kahroba et al. (2019)

Pancreatic Breast

Tataranni et al. (2019) Fiorillo et al. (2016)

Glioma

Bijangi-Vishehsaraei et al. (2017) Jagust et al. (2019)

Rapamycin

Glycolysis Oxidative phosphorylation Oxidative phosphorylation Oxidative phosphorylation mTOR

Ketogenic diet

Glycolysis

Glioma

Sulforaphane Metformin

HNSCC, osteosarcoma Pancreatic, breast

Lai et al. (2016) and Kuo et al. (2019) Ji et al. (2020)

capacity of ALDH+ breast CSCs (Yip et al. 2011) or other tumor-specific CSCs, increasing their sensitivity to common chemotherapeutics such as paclitaxel. Resveratrol, a type of natural phenol, is a nonspecific ROS inducer reported to inhibit CSCs proliferation and sphere-forming ability, effects that can be reversed by

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N-acetylcysteine-mediated suppression of ROS production (Seino et al. 2015). Resveratrol or its analogs have been shown to affect CSCs in many different cancers by altering signaling through several pathways including Notch (Zhang et al. 2018). Emodin is another naturally occurring anthraquinone shown to decrease stemness in various CSCs and increase sensitivity to conventional chemotherapeutics, such as cisplatin (Kim et al. 2015). More targeted approaches against the antioxidant machinery include inhibitors of GSH such as PEITC (Yun et al. 2017) or its more potent analog LBL21 (Wang et al. 2017) as well as curcumin (Wang et al. 2018c) and sulfasalazine (Okazaki et al. 2019). Sulfasalazine inhibits the xCT cysteine-glutamate antiporter, thereby reducing GSH-mediated antioxidant properties in stem cells from HNSCC and HCC (Okazaki et al. 2019; Wada et al. 2018). Sulfasalazine increased ROS-mediated apoptosis and reduced tumor growth in a HCC mouse model when used in combination with cisplatin (Wada et al. 2018). Similarly, inhibitors of another powerful antioxidant, TRX, such as auranofin (Hou et al. 2018) or shikonin (Ding et al. 2015), can also affect ROS dynamics in CSCs. Specifically, shikonin has been shown to inhibit TRX to increase ROS-dependent apoptosis in colon CSCs (Liang et al. 2017). Use of auranofin decreases ROS levels in a concentration-dependent manner, reducing the number of cells expressing stem cell markers. Importantly, the expression of these markers returns when auranofin treatment is prematurely halted, showing that auranofin specifically inhibit CSCs (Hou et al. 2018). Inhibition of NRF2 has also been shown to inhibit CSC proliferation and self-renewal ability by suppressing antioxidant protein expression (Kahroba et al. 2019). For example, all-trans retinoic acid (ATRA) inhibits NRF2 transcriptional activity leading to the attenuation of CSC-like properties in ovarian cancer (Robledinos-Antón et al. 2019). Brusatol also inactivates NRF2 to increase ROS and sensitivity to paclitaxel in mammosphere cultures, while hinokitiol decreases NRF2 expression in glioma stem cells (Kahroba et al. 2019). Collectively, these compounds sensitize CSCs to available anticancer therapies by reducing their ROS defense capacity. Targeting metabolic dynamics in CSCs is another approach to modulate ROS levels and render them more susceptible to cell death. For example, dichloroacetate diverts metabolism from glycolysis to oxidative phosphorylation, increasing ROS and decreasing self-renewing capability and spheroid formation while also increasing radiosensitivity (Tataranni et al. 2019). Other compounds such as atovaquone (Fiorillo et al. 2016), sulforaphane (Bijangi-Vishehsaraei et al. 2017), and metformin or rapamycin (Jagust et al. 2019; Lai et al. 2016) can inhibit oxidative phosphorylation. Clinical utility for metformin and rapamycin has been shown through their inhibition of the mTOR complex. Increases in ROS formation after treatment with metformin or rapamycin may result from a decrease in antioxidants or inhibition of oxidative phosphorylation and disintegration of the mitochondrial network. Metformin has been shown to specifically increase apoptosis of pancreatic or breast CSCs (Jagust et al. 2019). Although rapamycin does not affect the proliferation of CSCs, its use does affect the viability of CSCs. As such, low-dose radiation in combination with rapamycin decreases mammosphere efficiency through an upregulation of ROS and asymmetric cell division (Lai et al. 2016). The use of metformin in CSCs is still controversial as some studies report that metformin decreases ROS levels, although

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this still leads to a reduction in cancer stemness (Kuo et al. 2019). Altering metabolic dynamics can also be achieved through diet and recently researchers suggested that a ketogenic diet may reduce CSC pool (Ji et al. 2020). In this study, patient-derived glioma stem–like cells were cultured in glucose-restricted media, which lead to reduced proliferation and increased apoptosis of glioma CSCs through the inhibition of glycolysis as well as morphological and functional disturbances to mitochondria. These studies highlight how hijacking the altered metabolic phenotype of CSCs may be a valuable therapeutic approach for the eradication of CSCs.

Conclusion and Perspective Apart from the established characteristics that define CSCs, ROS are now known to be a critical driver of metabolic remodeling and essential for sustaining CSC stemness. Contrary to the malignant cell population, ROS concentrations are significantly lower in the CSCs as reported in a broad spectrum of both solid and hematopoietic cancers. As we have outlined in this chapter, lower levels of ROS can result from an altered balance within the antioxidant defense system, favoring the expression of key antioxidants, such as SOD and catalase, and the transcription factors that regulate their expression, including NRF2 and HIF1/2. Alterations in key signaling pathways important for both stem cell maintenance and survival, including PI3K/AKT, Wnt/β-catenin, Notch, and JAK/STAT, also contributes to the lower levels of ROS observed in CSCs. Another key feature of CSCs is their ability to reprogram glucose, lipid, and amino acid metabolism in response to changes in the tumorigenic niche and extracellular matrix. In contrast to the conventional notion of multipotent stem cells, proliferative CSCs can exist in both glycolytic and oxidative phenotypes, depending on their origin. Such plasticity contributes to the ability of CSCs to withstand conventional chemotherapeutics. Accumulating evidences of ROS playing an integral role in sustaining CSC stemness have established ROS modulating agents as potential measures of targeting CSCs in order to improve the resilience of current treatment regimens. ROS-mediated signaling and metabolic pathways are instrumental in maintaining CSC phenotypes and targeting regulatory factors of these pathway have been reported to be beneficial in suppressing antioxidants and enhancing CSCs in diverse in vitro and in vivo rodent models. However, developing an effective and safe therapeutic strategy translatable to clinical settings would require careful delineation of specific redox signaling pathways that are unique to the CSCs to prevent damage to the normal tissue stem cells. New combination therapies to target and prolong ROS generation in specific cellular compartments will result in efficient tumor therapy.

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Cancer Cells’ Defensive Weapons Against Oxidative Stress Yezhou Yu, Giovanna Di Trapani, and Kathryn F. Tonissen

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ROS and Redox Control Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Glutathione and Thioredoxin Systems in Cancers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Targeting Antioxidant Systems for Cancer Therapies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cross-References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract

Most cancer cells contain high levels of reactive oxygen species (ROS), which result in higher expression levels of the glutathione and thioredoxin reductase systems. These antioxidant systems convert ROS to nonharmful compounds and they also reduce disulfide bonds in oxidized proteins. They also control the glutathionylation and nitrosylation of proteins, which alters the activity of the proteins. Through these functions the upregulated antioxidant systems act on transcription factors and on signaling pathways, which result in the promotion of cancer cell growth and the prevention of cell apoptosis. High levels of certain members of the glutathione and thioredoxin systems in specific cancers correlate with poor patient prognosis and with drug resistance. Therefore, these antioxidants represent a potential new target for chemotherapy treatments, especially the thioredoxin reductase (TrxR) protein, which can be targeted through its

Y. Yu · K. F. Tonissen (*) School of Environment and Science, Griffith University, Nathan, Australia Griffith Institute for Drug Discovery, Griffith University, Nathan, Australia e-mail: k.tonissen@griffith.edu.au G. Di Trapani School of Environment and Science, Griffith University, Nathan, Australia © Springer Nature Singapore Pte Ltd. 2022 S. Chakraborti et al. (eds.), Handbook of Oxidative Stress in Cancer: Mechanistic Aspects, https://doi.org/10.1007/978-981-15-9411-3_143

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selenocysteine residue. Inhibition of TrxR leads to increased ROS levels in cancer cells to a lethal level, consequently leading to activation of cell death mechanisms. However, ROS levels remain at nonlethal levels in normal cells, thus enabling specific targeting of cancer cells. The design and availability of inhibitors for the specific upregulated antioxidant proteins in each type of cancer may also overcome drug resistance by resensitizing cancers to existing chemotherapy treatments. Keywords

Thioredoxin · Glutathione · Peroxidase · Cell signaling · Cancer · Reactive oxygen species · Chemotherapy

Introduction Free radicals, including reactive oxygen species (ROS) and reactive nitrogen species (RNS), are known to damage cells. ROS are mostly generated in the mitochondria as by-products of cell metabolism (Boveris and Chance 1973). Low levels of ROS can play a positive role in redox signaling and are required for activation of many cell signaling pathways (Schieber and Chandel 2014). However, an excess of ROS results in oxidative stress and can cause damage to cell lipids, proteins, and DNA (Zorov et al. 2014), which results in autophagy or cell apoptosis. To keep the ROS at a harmless level and to maintain the redox homeostasis in cells, cellular antioxidants such as thioredoxin (Trx) and glutathione (GSH) are generated. Antioxidants can either act as redox buffers to neutralize the excess of ROS or as rescuers to reduce the oxidized proteins. These antioxidants are essential for cell survival and growth and it was found that the absence of either Trx or GSH is embryonically lethal in mice (Matsui et al. 1996; Dalton et al. 2000), showing that the two systems are not redundant. As will be discussed, each antioxidant system has their own specific targets (Go et al. 2013), in addition to acting as redox buffers, in both normal and cancer cells. Compared with normal cells, cancer cells exhibit a much faster proliferation rate. Due to this fast metabolism, the ROS production is increased to a potentially dangerous level. In response, antioxidants are upregulated to protect cells from ROS-induced damage, which would otherwise lead to apoptosis. The upregulation of antioxidant expression has been observed in many different types of cancer (Berggren et al. 1996; Nishiyama et al. 2001; Lincoln et al. 2003; Raninga et al. 2015; Leone et al. 2017). The inhibition of these antioxidant systems can decrease cancer cells’ proliferation or lead to cell apoptosis (Saitoh et al. 1998; Pan and Berk 2007). Thus, antioxidants are currently considered to be potential targets for cancer therapy (Desideri et al. 2019; Arner 2020). This chapter discusses how antioxidants protect cancer cells from ROS and the potential of antioxidant systems as effective targets for cancer therapy.

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ROS and Redox Control Systems Various ROS, including the superoxide anion, hydrogen peroxide, and hydroxyl radicals, are generated in the mitochondria. They can directly react with proteins and result in oxidization of thiol groups (Reddie and Carroll 2008; van Bergen et al. 2014). The reaction can be either reversible or irreversible depending on the ROS levels. Thiol groups (
SH) will firstly be oxidized to sulfenic groups (-SOH) (Reddie and Carroll 2008; van Bergen et al. 2014). This step of the reaction is reversible and sulfenic groups (
SOH) can further react with thiol groups (
SH) to form disulfide bonds (
SS-), which can lead to altered functionality of the proteins. With higher ROS levels, sulfenic groups (
SOH) can be converted to sulfinic groups (
SO2H) (Reddie and Carroll 2008; van Bergen et al. 2014). This reaction was previously considered as irreversible, however sulfinic groups (
SO2H) were later shown to be reduced by the enzyme called sulfiredoxin (Chang et al. 2004). If the ROS levels increase even further, sulfonic groups (-SO3H) will eventually be formed. This reaction is irreversible. Proteins with sulfonic groups are regarded as damaged and are degraded (Reddie and Carroll 2008). Although ROS are important and necessary for some signaling pathways (Schieber and Chandel 2014), uncontrolled ROS can lead to protein and other biomolecule damage, potentially culminating in apoptosis. Therefore, antioxidants are necessary to maintain the redox balance in cells. Since glutathione (GSH) and thioredoxin (Trx) are two of the most important thiol-dependent antioxidants and both have been shown to be important in cancers, this chapter will focus on these systems. The glutathione system is the most abundant antioxidant system in cells. The key compound in the system is GSH, which is a tripeptide (γ-L-glutamyl-Lcysteinyl-glycine) (Meister and Anderson 1983). Its concentration in cells is much higher than other antioxidants, normally around 0.5–10 mM (Meister and Anderson 1983). The synthesis of GSH only involves two steps. Glutamate and cysteine are first ligated by the enzyme called glutamate cysteine ligase (GCL). Then the enzyme glutathione synthetase (GS) catalyzes the addition of glycine, resulting in GSH (Meister and Anderson 1983). The synthesis can be suppressed by knocking out the expression of either the GCL or GS enzymes. For example, after disrupting the expression of GCL, mice died during embryogenesis because of insufficient GSH synthesis (Dalton et al. 2000). Glutathione reductase (GR) and glutathione peroxidase (GPX) are also important enzymes required for a functional GSH system (Meister and Anderson 1983) (Fig. 1a). GPX is a family of selenium-containing enzymes (Brigelius-Flohe and Maiorino 2013), of which eight are known to exist in mammals. Each GPX has different tissue distribution and substrate specificity, with only some members containing a selenocysteine residue (Brigelius-Flohe and Maiorino 2013). GPX1, found in both cytoplasm and mitochondria, is the most abundant and widely expressed and in vivo experiments showed that it is essential for counteracting oxidative stress and it cannot be replaced by any other selenoprotein (Cheng et al. 1998). GPX can continuously reduce H2O2 to H2O with the consumption of GSH,

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Fig. 1 The glutathione and thioredoxin systems (a) The glutathione system utilizes GSH as the reducing agent. GPX reduces H2O2 and converts GSH to GSSG. Glutaredoxin (GRX) can reduce oxidized proteins. GR reduces GSSG to GSH with the consumption of NADPH. (b) The thioredoxin (Trx) system contains the 12 kDa Trx protein. Trx can assist in the scavenging of ROS by recycling peroxiredoxin (PRX) and it can also reduce oxidized proteins. TrxR catalyzes the reduction of oxidized Trx by NADPH

resulting in the reduced form GSH being converted to the oxidized form GSSG (two GSH molecules linked by a disulfide bond). Then GR reduces the GSSG back to GSH, with the consumption of NADPH. This process is one of the major pathways to metabolize H2O2 in most cells, however the GPX proteins are also involved in other physiological processes (Brigelius-Flohe and Maiorino 2013). Glutaredoxin (GRX) is another antioxidant protein that utilizes glutathione as a substrate (Lillig et al. 2008). A family of GRX proteins exist in mammalian cells, including dithiol GRXs with a conserved Cys-Pro-Tyr-Cys-active site sequence and monothiol Grxs with a CGFS-active site sequence, but which share similar structural characteristics and using GSH as a substrate. The GRX proteins collectively have a broad substrate range and are able to reduce disulfide bonds in proteins (Fig. 1a) as well as reducing other small molecules, including ascorbic acid (Lillig et al. 2008). The thioredoxin (Trx) system is another important thiol-dependent antioxidant system (Lu and Holmgren 2014). This system consists of thioredoxin (Trx), thioredoxin reductase (TrxR), and NADPH (Holmgren 1985) (Fig. 1b). Trx can act by either reducing oxidized proteins (Holmgren 1985) or by neutralizing ROS by recycling peroxiredoxins (PRX) (Rhee and Kil 2017). After reducing other proteins, an intramolecular disulfide will be formed in Trx and the oxidized Trx can be reduced by TrxR, with the consumption of NADPH. This regenerates an active reduced Trx protein (Holmgren 1985). Thioredoxin was first purified from E. coli and found to have the ability to mediate the enzymatic reduction of disulfide bonds (Holmgren 1985). The active site sequence Cys-Gly-Pro-Cys is conserved between E.coli and human thioredoxin proteins. In mammalian cells Trx1 and Trx2 exist. Trx1 is cytosolic and has five cysteine residues, while Trx2 is mitochondrial and has two cysteine residues. Both Trx proteins have a conserved active site, which includes two Cys, responsible for reducing disulfide bonds, which are situated at positions 32 and 35 in the Trx1 protein. Trx functions as an oxidoreductase by initially forming a bond between

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Cys32 on Trx and the target protein, leading to the break of the intramolecular disulfide bond in the target protein. Then Cys35 reacts with Cys32 to form an intramolecular disulfide bond in Trx1 and the target protein is reduced (Fig. 1b) (Holmgren 1985; Lu and Holmgren 2014). One of the main protein substrates of Trx1 is the peroxiredoxin family of proteins. There are six Prx proteins in mammals of which Prx1-4 contain two conserved cysteine residues required for the catalytic reduction of H2O2, and all are dependent on Trx for recycling to an active form (Fig. 1b) (Rhee and Kil 2017). Prx5, also a substrate for Trx, prefers to reduce alkyl hydroperoxides or peroxynitrate rather than H2O2 (Knoops et al. 2011). Prx6 reduces phospholipid hydroperoxides and functions independently of Trx since it lacks a resolving cysteine and instead uses GSH to complete its catalytic reaction (Arevalo and Vazquez-Medina 2018). TrxR is another important member of the Trx system. TrxR is a selenoprotein and three TrxR proteins have been found in mammalian cells, the cytosolic TrxR1, the mitochondrial TrxR2, and the testis-special thioredoxin glutathione reductase (TGR) (Holmgren 1985; Lu and Holmgren 2014). All TrxRs share a similar structure containing a flavin adenine dinucleotide (FAD) binding domain, an NADPH binding domain, and an interface domain, while TGR has an extra glutaredoxin domain at the N-terminal. There are two active sites in TrxR1, one is the N-terminal active site, -CVNVGC-, and another is the C-terminal active site, -GCUG-, which contains a selenocysteine residue (Zhong et al. 2000; Cheng et al. 2008; Lu and Holmgren 2014). Electrons are first transferred from NADPH to FAD, then FAD reduces the disulfide bond in the N-terminal active site. The N-terminal active site will reduce the C-terminal active site enabling TrxR to reduce its main target, Trx, in addition to a few other substrates, including selenite and lipoic acid (Cheng et al. 2008; Lu and Holmgren 2014). Overall, the GSH and Trx antioxidant systems act like guardians for cells to maintain redox homeostasis and protect cells from excessive levels of ROS generated during cell metabolism. As described above, they can either directly quench ROS or can reduce oxidized proteins. While these systems have some overlapping function, especially in terms of neutralizing ROS, they also have some specific targets. However, whether the antioxidant system plays a role of “good guy” greatly depends on the cells they are protecting. Protecting cancer cells can lead to a worse prognosis for patients through development of drug resistance or by negating the effects of drugs that would otherwise act by increasing ROS levels (Kim et al. 2005; Chandra 2009; Raninga et al. 2015; Leone et al. 2017; Zhu et al. 2019).

Glutathione and Thioredoxin Systems in Cancers The highly metabolic cancer cells often generate a much higher level of ROS than observed in normal cells (Kumari et al. 2018). As a result, antioxidants in cancer cells are also upregulated to maintain redox homeostasis and to avoid cellular damage caused by ROS (Berggren et al. 1996; Bansal and Simon 2018). Upregulation of the Trx system (both Trx and TrxR) has been reported in many

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types of cancer, with the most aggressive types of breast cancer and melanoma tumors having the highest levels, along with a poor prognosis for patients (Lincoln et al. 2003; Bhatia et al. 2016; Leone et al. 2017). Many of the antioxidant enzymes, including Trx1, TrxR1, GPX2, GPX4, GRX1, Prx1, and Prx6, are coordinately upregulated by the transcription factor Nrf-2 (Hayes and Dinkova-Kostova 2014). This transcription factor is usually kept in an inactive state in the cytoplasm by the binding of Keap1. When ROS are present key cysteine residues in Keap 1 are oxidized, resulting in release of Nrf2 and subsequent ubiquitin-mediated degradation of Keap1 (Hayes and Dinkova-Kostova 2014). Nrf2 moves into the nucleus and therefore when ROS levels are increased, such as the case in cancer cells, many of the antioxidant genes become highly expressed (Leone et al. 2017). Not only are the antioxidant systems highly expressed but this upregulation has consequences for patient outcomes. For patients with breast cancer the upregulation of a number of antioxidants relates to a poor prognosis, as shown in Table 1. Notably, patients with higher expression of the genes encoding the Trx1, TrxR1, Prx1, or GPX1 antioxidant proteins display lower overall survival. All of these genes are Nrf2 regulated (Schachtele et al. 2012; Hayes and Dinkova-Kostova 2014) and are likely upregulated due to the high ROS levels present in the cancer cells (Kumari et al. 2018). In turn, both the glutathione and Trx systems are utilized by the cancer cells as defensive weapons to protect from damage that may occur due to these ROS levels. However, while both systems play roles in preventing apoptosis or in promoting cell growth, both systems regulate some specific signaling pathways, and facilitate tumor progression and survival (Hawk et al. 2016), as discussed below. With a very high cytosol concentration, the reduced form of glutathione (GSH) acts as a redox buffer in cells, including cancer cells (Bansal and Simon 2018). While cancer cells exhibit increased oxidative stress, they also display a high GSH/GSSG ratio, which can result from enhanced upregulation of the GSH synthesis enzymes or from the conversion of GSSG back to GSH using GR and NADPH, which is generated through the pentose phosphate pathway (Zhang et al. 2016). The GSH system can directly neutralize the excess ROS by conversion to nontoxic compounds or alternatively the thiol group of GSH can react with the sulfenic groups of oxidized proteins in a reversible process called glutathionylation. The glutathionylation can protect proteins from undergoing further oxidization and thus avoid irreversible oxiditive modifications, and permanent dectivation (Cooper et al. 2011; Xiong et al. 2011). Glutathionylation of key proteins is also a mechanism by which cell signaling is regulated, especially protein kinases, which can result in cancer cell proliferation (Cooper et al. 2011; Xiong et al. 2011). Under oxidative stress conditions, GSH can be added to cysteine residues of specific proteins, while it can be removed through the action of GRX (Lillig et al. 2008). Due to its reversible nature, the gluathionylation is thought to act as a switch, since the additional glutathione moiety can result in a change in protein structure with consequential functional changes. A number of cytoskeletal proteins, with known roles in cancer cell migration and metastasis, can also be glutathionylated, leading to the possibility that this reversible

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Table 1 Antioxidant gene expression and patient survival in breast cancer Antioxidant name *Trx1 Trx2 *TrxR1 TrxR2 TrxR3 *Prx1 *Prx2 Prx3 *Prx4 *Prx5 *Prx6 *GPX1 GPX2 GPX3 GPX4 GPX5 GPX7 *GPX8 GRX1 *GRX2 *GRX3 *GRX5 GR

Hazard ratio (HR) (95% confidence interval) 1.62 (1.45–1.8) 0.87 (0.77–0.98) 1.59 (1.43–1.78) 0.85 (0.76–0.94) 0.79 (0.71–0.88) 1.38 (1.23–1.54) 1.42 (1.26–1.59) 0.76 (0.68–0.85) 1.59 (1.42–1.77) 1.57 (1.33–1.85) 1.24 (1.1–1.39) 1.29 (1.15–1.46) 0.73 (0.65–0.82) 0.86 (0.77–0.97) 0.86 (0.77–0.96) 0.69 (0.61–0.77) 0.89 (0.8–1) 1.25 (1.06–1.48) 0.91 (0.82–1.02) 1.59 (1.42–1.77) 1.39 (1.25–1.55) 1.31 (1.17–1.47) 1.08 (0.91–1.27)

P value