Ferroptosis in Health and Disease [1st ed. 2019] 978-3-030-26779-7, 978-3-030-26780-3

Table of contents :
Front Matter ....Pages i-vi
Lipid Metabolism and Ferroptosis (Amy Tarangelo, Scott J. Dixon)....Pages 1-26
Iron Metabolism and Ferroptosis (Shinya Toyokuni, Izumi Yanatori)....Pages 27-41
Regulation and Function of Autophagy During Ferroptosis (Daolin Tang, Rui Kang)....Pages 43-59
Heat Shock Proteins: Endogenous Modulators of Ferroptosis (Rui Kang, Daolin Tang)....Pages 61-81
Biological Aspects of Endoplasmic Reticulum Stress in Ferroptosis (Young-Sun Lee, Yong J. Lee)....Pages 83-98
Gpx4 and Ferroptosis (Qitao Ran, Paulina Mozolewska)....Pages 99-109
ACSL4 as the First Reliable Biomarker of Ferroptosis Under Pathophysiological Conditions (Caroline Moerke, Franziska Theilig, Ulrich Kunzendorf, Stefan Krautwald)....Pages 111-123
Regulation of Ferroptosis by MicroRNAs (Yongfei Yang)....Pages 125-145
Ferroptosis in Cardiovascular Disease (Jason K. Higa, Nicholas K. Kawasaki, Takashi Matsui)....Pages 147-172
Ferroptosis in Nervous System Diseases (Jieru Wan, Xiuli Yang, Jian Wang)....Pages 173-195
Regulation of Ferroptosis Through the Cysteine-Glutathione Redox Axis (Junichi Fujii, Sho Kobayashi, Takujiro Homma)....Pages 197-213
Iron–Sulfur Cluster Metabolism Impacts Iron Homeostasis, Ferroptosis Sensitivity, and Human Disease (Vladislav O. Sviderskiy, Erdem M. Terzi, Richard Possemato)....Pages 215-237
Ferroptosis in Liver Disease (Antoine Galmiche)....Pages 239-248
p53 and Ferroptosis (Shun Jiang, Yangchun Xie)....Pages 249-256
Ferroptosis in Hemolytic Disorders (Lyla A. Youssef, Steven L. Spitalnik)....Pages 257-272
Lipoxygenase in Ferroptosis (Xiaoyuan Mao)....Pages 273-284
Ferroptosis in Cancer Disease (Behrouz Hassannia, Tom Vanden Berghe)....Pages 285-301
Ferroptosis in Cancer Therapy (Xiao Zhang, Susu Guo, Yueyue Yang, Xiangfei Xue, Jiayi Wang)....Pages 303-324

Citation preview

Daolin Tang Editor

Ferroptosis in Health and Disease

Ferroptosis in Health and Disease

Daolin Tang Editor

Ferroptosis in Health and Disease

Editor Daolin Tang Department of Surgery UT Southwestern Medical Center Dallas, Texas, USA

ISBN 978-3-030-26779-7 ISBN 978-3-030-26780-3 https://doi.org/10.1007/978-3-030-26780-3

(eBook)

© Springer Nature Switzerland AG 2019 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, express 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 Switzerland AG. The registered company address is: Gewerbestrasse 11, 6330 Cham, Switzerland

Contents

1

Lipid Metabolism and Ferroptosis . . . . . . . . . . . . . . . . . . . . . . . . . . Amy Tarangelo and Scott J. Dixon

1

2

Iron Metabolism and Ferroptosis . . . . . . . . . . . . . . . . . . . . . . . . . . Shinya Toyokuni and Izumi Yanatori

27

3

Regulation and Function of Autophagy During Ferroptosis . . . . . . Daolin Tang and Rui Kang

43

4

Heat Shock Proteins: Endogenous Modulators of Ferroptosis . . . . . Rui Kang and Daolin Tang

61

5

Biological Aspects of Endoplasmic Reticulum Stress in Ferroptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Young-Sun Lee and Yong J. Lee

83

6

Gpx4 and Ferroptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Qitao Ran and Paulina Mozolewska

99

7

ACSL4 as the First Reliable Biomarker of Ferroptosis Under Pathophysiological Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111 Caroline Moerke, Franziska Theilig, Ulrich Kunzendorf, and Stefan Krautwald

8

Regulation of Ferroptosis by MicroRNAs . . . . . . . . . . . . . . . . . . . . 125 Yongfei Yang

9

Ferroptosis in Cardiovascular Disease . . . . . . . . . . . . . . . . . . . . . . . 147 Jason K. Higa, Nicholas K. Kawasaki, and Takashi Matsui

10

Ferroptosis in Nervous System Diseases . . . . . . . . . . . . . . . . . . . . . 173 Jieru Wan, Xiuli Yang, and Jian Wang

v

vi

Contents

11

Regulation of Ferroptosis Through the Cysteine-Glutathione Redox Axis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197 Junichi Fujii, Sho Kobayashi, and Takujiro Homma

12

Iron–Sulfur Cluster Metabolism Impacts Iron Homeostasis, Ferroptosis Sensitivity, and Human Disease . . . . . . . . . . . . . . . . . . 215 Vladislav O. Sviderskiy, Erdem M. Terzi, and Richard Possemato

13

Ferroptosis in Liver Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 239 Antoine Galmiche

14

p53 and Ferroptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 249 Shun Jiang and Yangchun Xie

15

Ferroptosis in Hemolytic Disorders . . . . . . . . . . . . . . . . . . . . . . . . . 257 Lyla A. Youssef and Steven L. Spitalnik

16

Lipoxygenase in Ferroptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 273 Xiaoyuan Mao

17

Ferroptosis in Cancer Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 285 Behrouz Hassannia and Tom Vanden Berghe

18

Ferroptosis in Cancer Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . 303 Xiao Zhang, Susu Guo, Yueyue Yang, Xiangfei Xue, and Jiayi Wang

Chapter 1

Lipid Metabolism and Ferroptosis Amy Tarangelo and Scott J. Dixon

Abstract Ferroptosis is an iron-dependent form of non-apoptotic cell death that is characterized by the accumulation of toxic lipid reactive oxygen species (ROS). The execution of ferroptosis does not appear to rely on particular protein effectors. Rather, the initiation and execution of this process depends upon modulation of intracellular metabolism. In particular, the process of lipid peroxidation is essential for ferroptosis. While the central role of lipid peroxidation in ferroptosis is clear, the precise lipid peroxide species formed during ferroptosis, as well as how and where they are generated, remain poorly understood. This chapter explores these questions and describes the role of lipids and lipid metabolism in the initiation, execution, and suppression of ferroptosis.

1.1

Introduction

Lipids are a ubiquitous class of hydrophobic molecules with exceptional structural diversity that are indispensable for life. In mammalian cells, lipids perform a variety of critical functions in membrane architecture and function (van Meer et al. 2008), signaling (Shimizu 2009), bioenergetics (Nakamura et al. 2014), proliferation (Storck et al. 2018), and stress response (Okazaki and Saito 2014). As the major components of cell membranes, lipids compartmentalize the biochemical processes of the cell interior from the extracellular environment. Disrupting membrane integrity is central to the execution of all forms of regulated cell death (RCD), including apoptosis, necroptosis, pyroptosis, and ferroptosis (Magtanong et al. 2016). In this chapter, we focus on ferroptosis, a non-apoptotic, iron-dependent, oxidative process characterized by membrane lipid peroxidation (Dixon et al. 2012; Cao and Dixon 2016; Gaschler and Stockwell 2017). While a central role for lipid peroxidation in ferroptosis is well established, the precise lipid peroxide species formed during ferroptosis, as well as how and where they are generated, remain poorly understood. A. Tarangelo · S. J. Dixon (*) Cancer Biology Graduate Program and Department of Biology, Stanford University, Stanford, CA, USA e-mail: [email protected] © Springer Nature Switzerland AG 2019 D. Tang (ed.), Ferroptosis in Health and Disease, https://doi.org/10.1007/978-3-030-26780-3_1

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A. Tarangelo and S. J. Dixon

This chapter explores these questions and describes the role of lipids and lipid metabolism in the initiation, execution, and suppression of ferroptosis.

1.2

Lipids Are a Diverse Class of Molecules that Mediate Cellular Life and Death

Lipids are a heterogeneous class of molecules that are soluble in organic solvents, but insoluble or sparingly soluble in water (Cammack et al. 2006). Six major classes of lipids are found in mammalian cells, each with distinct structural and chemical properties: fatty acids (FAs), glycerolipids, glycerophospholipids, sphingolipids, sterol lipids, and prenol lipids (Fahy et al. 2011). Lipids demonstrate incredible structural diversity owing to factors such as variable hydrocarbon chain lengths, biochemical transformations, and modifications with sugar residues or other functional groups. Due to the technical challenges of elucidating the full extent of this structural diversity, the exact number of discrete lipid species present in the mammalian cell remains unclear (Fahy et al. 2011). However, some estimates place the size of the mammalian lipidome on the order of tens of thousands of different species (Han 2016).

1.2.1

The Composition and Function of Lipids in Cell Membranes

Lipids are the main component of cell membranes. The precise composition of lipids in membranes varies between organism, cell type, organelle, and membrane leaflets (Harayama and Riezman 2018). The cellular repertoire of membrane lipids is diverse but composed mainly of glycerophospholipids (hereafter, phospholipids, or PLs), sphingolipids, and sterols. Phospholipids are comprised of a hydrophobic diacylglycerol region containing saturated or unsaturated fatty acyl chains of varying lengths, and a hydrophilic head group comprised of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), or phosphatidic acid (PA) (van Meer et al. 2008). Sphingolipids are a distinct class of structural lipids with a hydrophobic ceramide backbone and saturated or trans-unsaturated tail groups. Like phospholipids, sphingolipids display extensive structural diversity and serve as essential components of membranes and as signaling molecules (Hannun and Obeid 2018). Sterols include non-polar lipids critical for membrane architecture and function. Cholesterol is the main sterol of vertebrates and can be equimolar with phospholipids in cell membranes (Dufourc 2008).

1 Lipid Metabolism and Ferroptosis

1.2.2

3

Lipid Peroxidation: A Balance Between Signaling and Survival

Oxidative damage to lipids, as occurs during ferroptosis, produces potentially toxic lipid hydroperoxides (hereafter lipid peroxides, or LOOH). Lipid peroxidation preferentially occurs on polyunsaturated fatty acids (PUFAs), a class of long chain fatty acids that contain at least two carbon-carbon double bonds. These include linoleic, arachidonic, and docosahexaenoic acids. The bisallylic hydrogen of PUFAs with a (1Z, 4Z) pentadiene moiety is most susceptible to oxidation (Gaschler and Stockwell 2017). Other unsaturated lipids such as cholesterol can also be oxidized to hydroperoxides, albeit less readily than PUFAs (Smith 1981). Peroxidation can occur on both PUFA free fatty acids and PUFA-containing membrane phospholipids (i.e., PUFA-PLs). Peroxidation of PUFA-PLs can alter lipid bilayer structure and geometry, potentially disrupting the structure and dynamics of cell membranes with toxic effects to the cell. Clusters of oxidized lipids in membranes can also form hydrophilic pores that disrupt the barrier function of membranes (Boonnoy et al. 2017). Additionally, LOOHs can react with redox active transition metals such as iron (i.e., Fe2+) to generate lipid reactive oxygen species (ROS) that are highly reactive and can propagate further lipid peroxidation reactions or decompose into harmful reactive products that can damage essential proteins or DNA (Gaschler and Stockwell 2017). LOOHs can be formed through controlled, enzymatic reactions or through spontaneous, non-enzymatic processes. The major enzymes that generate LOOHs include lipoxygenases, cyclooxygenases, and P450 (CYP) enzymes. Each of these enzyme groups catalyzes the formation of a complex array of bioactive LOOHs that regulate cell signaling. In particular, the lipoxygenase (LOX) family of enzymes are a major contributor to the synthesis of LOOHs and have been implicated in a variety of pathological states including neurodegeneration, ischemia-reperfusion injury, infection, inflammation, and diabetes (Kuhn et al. 2015). The non-enzymatic production of LOOHs occurs through a three-step reaction composed of an initiation, propagation, and termination phase. In the initiation phase, radicals are produced from non-radical molecules, often through redox reactions catalyzed by “labile,” or loosely ligated, iron. The term “Fenton chemistry” describes a set of reactions in which the labile iron pool catalyzes the production of oxygen-centered radicals from hydrogen peroxide. Radicals produced through Fenton chemistry can abstract a hydrogen from an allylic carbon on a non-radical lipid to form a resonancestabilized, carbon-centered lipid radical (L•). The reaction is propagated when the lipid radical reacts with molecular oxygen to form a lipid peroxyl radical (LOO•). Lipid peroxyl radicals can then abstract a hydrogen from another lipid to generate a lipid hydroperoxide (LOOH) and a new LOO• that propagates the chain reaction. The reaction is terminated when the concentration of lipid radicals becomes high enough that two radicals can bond and form a non-radical molecule, or when an antioxidant donates a hydrogen to a lipid peroxyl radical (Ayala et al. 2014; Gaschler

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A. Tarangelo and S. J. Dixon

and Stockwell 2017). Ferroptosis, in particular, is characterized as a form of cell death caused by an excess of lipid peroxidation.

1.3

Ferroptosis Is a Lipid-Dependent Form of Cell Death

Oxidative damage to membrane lipids is central to the execution of ferroptosis (Dixon and Stockwell 2019). The selenoprotein glutathione peroxidase 4 (GPX4) normally guards membranes from oxidative damage and thereby prevents the onset of this process (Conrad and Friedmann Angeli 2015). GPX4 uses two molecules of the thiol-containing tripeptide glutathione (GSH) as cofactors to reduce LOOH species to non-toxic lipid alcohol (LOH) species. Ferroptosis can be instigated by depletion of GSH or direct inactivation of GPX4, which lead to loss of GPX4 activity and the accumulation of LOOHs (Yang et al. 2014). A number of ferroptosis-induced compounds have been identified, including those that degrade the rate-limiting GSH biosynthetic precursor cysteine (e.g., cyst(e)inase), inhibit plasma membrane cysteine/glutamate antiporter system xc activity (e.g., erastin, sorafenib), inhibit the rate-limiting glutathione biosynthetic enzyme glutamatecysteine ligase (e.g., buthionine sulfoximine), disrupt pathways that modulate GPX4 synthesis or stability (e.g., statins, FIN56, FINO2), or covalently inhibit GPX4 itself (e.g., 1S,3R-RSL3, ML162, ML210) (Weïwer et al. 2012; Dixon et al. 2012, 2014; Yang et al. 2014, 2016; Lachaier et al. 2014; Gao et al. 2015; Shimada et al. 2016; Cramer et al. 2017; Gaschler et al. 2018a; Zhang et al. 2019). Upon GPX4 inactivation, the majority of LOOHs formed in ferroptosis originate in phospholipids containing PUFA acyl chains, though additional oxidized lipid species, such as cholesterol and sphingolipids, may be generated. Ferroptosis can be suppressed through pharmacologic or genetic perturbations. The major classes of pharmacological suppressors of ferroptosis are iron chelators and lipophilic antioxidants. Though the exact role of iron in ferroptosis has yet to be determined, iron chelators likely inhibit death by sequestering redox active, labile iron to inhibit ironcatalyzed lipid ROS formation. Alternatively or additionally, iron chelators may suppress death by removing iron from the active site of iron-containing enzymes that catalyze membrane lipid oxidation (Cao and Dixon 2016). A diverse class of endogenous and synthetic lipophilic antioxidants including α-tocopherol (i.e., vitamin E), trolox, ferrostatin-1 (Fer-1), and liproxstatin-1 (Lip-1) can also suppress ferroptosis by preventing lipid ROS accumulation, which occurs as a consequence of LOOH interaction with iron (see below)(Stockwell et al. 2017).

1 Lipid Metabolism and Ferroptosis

1.4

5

The Role of Lipids and Lipid Metabolism in Ferroptosis

Ferroptosis was initially identified as a novel form of cell death caused by the toxic accumulation of LOOHs (Dixon et al. 2012; Yang et al. 2014; Skouta et al. 2014; Friedmann Angeli et al. 2014; Gaschler et al. 2018a). The necessity for LOOH formation in ferroptosis was elegantly demonstrated using deuterated PUFAs (D-PUFAs), in which the replacement of bisallylic hydrogens with deuterium slows the rate of hydrogen abstraction, thereby suppressing lipid radical generation. In G-401 cells, replacing natural PUFAs with D-PUFAs strongly suppresses ferroptosis induced by erastin or RSL3 (Yang et al. 2016). Further, the oxidative destruction of PUFAs during ferroptosis was directly demonstrated using alkynelabeled linoleic acid (LA, C18:2) followed by copper-catalyzed cycloaddition (Click)-labeling. In cells treated with alkyne-LA and erastin, Click reactions revealed the accumulation of oxidative breakdown products of LA, which was suppressible with Fer-1 (Skouta et al. 2014). Additional evidence for the requirement of PUFA activation and incorporation into PLs in the execution of ferroptosis is provided by results showing that exogenous administration of the monounsaturated fatty acid (MUFA) oleic acid (OA, C18:1) strongly suppresses erastin-induced ferroptosis by competing with PUFAs for incorporation into PLs. In HT-1080 fibrosarcoma cells, OA treatment decreases the abundance of PUFA-containing PLs and the accumulation of LOOHs at the plasma membrane. The ability of exogenous OA to suppress lipid peroxidation and ferroptosis is dependent on the acyl-CoA synthetase long chain family member 3 (ACSL3), which activates MUFA free fatty acids to MUFA-CoAs, a step necessary for their incorporation into membrane PLs (Magtanong et al. 2019). Thus, reducing the number of oxidizable PUFA acyl chains on membrane PLs may be sufficient to suppress ferroptosis.

1.4.1

Lipid Signatures of Cells Undergoing Ferroptosis

Determining which lipid species are modulated during ferroptosis provides insights into the underlying lethal mechanism. Specific lipid signatures of ferroptotic cells have been identified using various analytic techniques (summarized in Table 1.1). Erastin-treated cancer cells are depleted of PUFA free fatty acids (Skouta et al. 2014) as well as PUFA-PCs (Yang et al. 2016). Ferroptosis induced by the deletion of Gpx4 in mouse kidneys leads to the accumulation of oxygenated PUFA-PCs and PUFA-PEs, as well as oxygenated derivatives of free PUFAs (Friedmann Angeli et al. 2014). RSL3-treated mouse embryonic fibroblasts (MEFs) specifically accumulate four oxygenated PUFA-PE species (Kagan et al. 2017). Human cancer cells treated with FINO2, a ferroptosis-inducing compound that inhibits GPX4 activity, accumulate a wide array of oxidized PLs containing PE, PS, phosphatidylinositol (PI), and cardiolipin (CL) head groups. However, only the increase in oxidized PEs and PIs is suppressed by the lipophilic antioxidant Fer-1, suggesting that oxidized PS

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Table 1.1 Modifications of the lipidome observed in cells undergoing ferroptosis

Model system HT-1080 human fibrosarcoma cell line

Inducible Gpx4 deletion in mouse kidney

Assay type LC-MS/MS for polar and lipid metabolites Oxidative lipidomics with LC-MS/ MS

Method of induction Erastin

Gpx4 deletion

HT-1080 human fibrosarcoma cell line

GC/MS and LC-MS/MS

Erastin +/ Ferrostatin1

HT-1080 human fibrosarcoma cell line

LC-MS/MS for lipid metabolites

Piperazine erastin

Wild-type and Acsl4 knockout cells derived from Gpx4fl/fl mouse embryonic fibroblasts from GPX4fl/fl mice

LC-MS/MS

RSL3 +/ Lip-1

Lipid signatures (ferroptotic cells vs controls) Increased lyso-PCs

Increased di-oxygenated PCs and PEs at 10 days postdeletion Increased di-oxygenated CL and mono-lyso-CL Increased hydroperoxy species of free linoleic (C18:2-OOH), arachidonic (C20:4OOH), and docosahexaenoic acid (C22:6-OOH) Increased lyso-PC and lyso-PE at 10 days postdeletion Depletion of free PUFAs and PUFAderivatives, including eicosapentaenoate (20:5n3), linoleate (18:2n6), docosahexaenoate (22:6n3), PUFA depletion reversible with Fer-1 treatment Decreased phosphatidylcholines with PUFA acyl chains Increased ceramide Increased lysophosphatidylcholine In WT cells, RSL3 caused increased accumulation of oxygenated esterified PUFAs, while ACSL4 KO cells preferentially accumulated oxygenated free PUFAs RSL3 treatment led to increased levels of

References Yang et al. (2014) Cell

FriedmannAngeli et al. (2014) Nat Cell Biol

Skouta et al. (2014) JACS

Yang et al. (2016) PNAS

Kagan et al. (2017) Nat Chem Biol

(continued)

1 Lipid Metabolism and Ferroptosis

7

Table 1.1 (continued)

Model system

Assay type

Method of induction

HT-1080 human fibrosarcoma cell line

LC-MS for oxidized and esterified phospholipids

FINO2, erastin, +/ Ferrostatin1

Diffuse large B-cell lymphoma cell lines and tumor xenografts

LC-MS untargeted lipidomics

Imidazoleketo erastin (IKE) +/ Fer-1, β-Me, DFO

Lipid signatures (ferroptotic cells vs controls) oxygenated PLs containing PC, PE, PS, PG, and PI Deletion of GPX4 in murine kidney cells led to increased levels of oxygenated PEs containing PUFA acyl chains, which was suppressible with Lip-1 Erastin caused an increased in 1 oxidized PE species FINO2 caused an increase in 21 oxidized PE species, as well as PS, PI, and CL species with PUFA acyl chains Accumulation of oxidized PE and PI species upon FINO2 treatment was suppressed with Fer-1 Increases in oxidized PS and CL species upon FINO2 treatment were not responsive to Fer-1 treatment In vitro: Decreased PCs and PE species Decrease in triacylglycerols (TAGs), reversible with Fer-1 In vivo: Increased free fatty acids, phospholipids, and diacylglycerols (DAGs) Identified lipids enriched in linoleic or arachidonic acid metabolism pathways

References

Gaschler et al. (2018a) Nat Chem Biol

Zhang et al. (2019) Cell Chem Biol

Abbreviations: PC phosphatidylcholine, PE phosphatidylethanolamine, PS phosphatidylserine, PI phosphatidylinositol, CL cardiolipin, Lyso-PC lysolipid phosphatidylcholine, PG phosphatidylglycerol, Lyso-PE lysolipid phosphatidylethanolamine, PUFA polyunsaturated fatty acid, Fer-1 ferrostatin-1, Lip-1 liproxstatin-1, β-Me 2-mercaptoethanol, DFO deferoxamine

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and CL may not contribute to ferroptosis. These results suggest that different methods of ferroptosis induction may produce qualitatively different types of oxidized PL species, and that only some of these oxidized species contribute directly to cell death (Gaschler et al. 2018a). As noted above, the strongest current evidence suggests a key role for oxidation of PUFA-containing PEs in ferroptosis. The preferential oxidation of PEs may be explained by their molecular geometry. In contrast to PCs, which are largely cylindrical in structure, PEs assume a more conical shape due to the smaller size of the PE head-group. The consequence of this geometry is that PEs can assume non-bilayer arrangements in membranes, including hexagonal phases that form transient, local regions of negative curvature (van den Brink-van der Laan et al. 2004). The formation of non-bilayer structures may cause PUFA acyl chains on individual PEs to be more accessible to oxidants and/or oxygenating enzymes. Indeed, computational modeling suggests that hexagonal phase lipids are preferential substrates for 15-lipoxygenase (15-LOX) enzymes, which have been implicated in ferroptosis (Kagan et al. 2017). In vitro experiments validate these in silico predictions and show that a hexagonal-phase forming PL is a far better 15-LOX substrate as compared with a PL that forms bilayer arrangements (Kagan et al. 2017). Importantly, PEs are specifically enriched on the inner leaflet of the plasma membrane in eukaryotes, suggesting that they may also be more accessible to cytosolic oxidizing enzymes or ROS that promote autoxidation of membrane lipids (Zwall et al. 1973; Verkleij et al. 1973). A second lipid signature of cells undergoing ferroptosis is the accumulation of lysophospholipids (hereafter, lysolipids). Lysolipids containing PC (i.e., lyso-PCs) accumulate in human cancer cells treated with erastin-analogs (Yang et al. 2014, 2016; Zhang et al. 2019) and in mouse kidneys following inducible deletion of Gpx4 (Friedmann Angeli et al. 2014). Lysolipids are produced when one acyl chain of a phospholipid is hydrolyzed. Under physiological conditions, lysolipids generally comprise a small proportion of membrane lipids and are rapidly reacylated or metabolized (Robertson and Lands 1964). Lysolipids accumulation can result from the selective cleavage of PUFA acyl chains from phospholipids by phospholipases (Parthasarathy et al. 1985). This process might slow lipid peroxidation during ferroptosis by decreasing the number of oxidizable PUFAs in the membrane. However, treatment with lyso-PCs has also been shown to produce ROS and disrupt plasma membrane integrity in human fibroblasts (Colles and Chisolm 2000), so a positive role for lysolipids in promoting ferroptosis cannot be ruled out. In one study, the exogenous administration of lyso-PC was not sufficient to modulate the lethality of RSL3 in human cancer cells, suggesting that lysolipids with PC head-groups do not promote ferroptosis (Yang et al. 2016). It remains unclear whether exogenous administration of other lysolipid types, such as lyso-PEs, contributes to ferroptosis susceptibility. Overall, these lipid signatures suggest that ferroptotic stimuli lead to the peroxidation of PEs containing PUFA acyl chains, thus depleting PUFA-PEs from the cell and inducing the accumulation of oxygenated PUFA-PE derivatives (Fig. 1.1). Simultaneously, cells may respond to redox imbalance by increasing

1 Lipid Metabolism and Ferroptosis

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A Glutamate

Cystine

System XcOH-PUFA-PE

sma Pla

OOH-PUFA-PE

OH

brane Mem

Extracellular Space

OOH PUFA-PE

Glutamate Cysteine

Lyso-PE

ROS GPX4

LPCAT3

γ-Glutamylcysteine Glycine

PUFA-CoA CoA

Reduced glutathione

ACSL4

PUFA

Oxidized glutathione

B Erastin Glutamate System Xc-

sma Pla

Cystine

Ferroptosis

OOH-PUFA-PE Accumulation OOH

brane Mem

OOH

OOH

Extracellular Space OOH

PUFA-PE

Cystei teine Glutamate Cys

Lyso-PE

RSL3

ROS GPX4

γ-Glu γ-G lutamylcysteine Glycine

LPCAT3

PUFA-CoA CoA

Reduced glutathione

Oxidized glutathione

ACSL4

PUFA

Fig. 1.1 Overview of the ferroptosis pathway. Outline of key proteins and metabolites involved in the regulation of ferroptosis. (a) The (reduced) glutathione (GSH)-dependent lipid hydroperoxidase glutathione peroxidase 4 (GPX4) prevents the accumulation of potentially toxic lipid peroxides, such as polyunsaturated phosphatidylethanolamines (OOH-PUFA-PE), by conversion to lipid alcohols. (b) In response to direct or indirect inhibition of GPX4, lipid peroxide acccumulation leads to membrane damage and cell death due to membrane permeabilization and/or damage to intracellular proteins by reactive lipid fragments. The role of other proteins shown here is described in the main text.

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phospholipase-mediated cleavage of oxidizable acyl chains from PLs, driving the accumulation of lyso-PLs.

1.4.2

Specific PUFA-Modifying Enzymes Are Required for Ferroptosis

The central requirement for PUFA oxidation in ferroptosis is supported by genetic evidence linking specific lipid metabolic genes to the execution of ferroptosis. AcylCoA synthetase long chain family member 4 (ACSL4) is an acyl CoA synthetase enzyme that preferentially activates PUFAs (Soupene and Kuypers 2008). ACSL4 expression promotes sensitivity to ferroptosis-inducing compounds (Dixon et al. 2015; Doll et al. 2017; Yuan et al. 2016). Overexpression of ACSL4 is sufficient to increase the ferroptosis susceptibility of resistant cell lines, while knockdown or deletion of ACSL4 can reduce ferroptosis sensitivity (Yuan et al. 2016; Doll et al. 2017; Kagan et al. 2017; Garcia-Bermudez et al. 2019). Deletion of Acsl4 in Gpx4 fl/fl MEFs suppresses cell death upon Gpx4 deletion. Inhibition of ACSL4 with triacsin C or thiazolidinedione compounds is also sufficient to inhibit ferroptosis in response to GPX4 inhibitors (Doll et al. 2017; Kagan et al. 2017), but not cystine deprivation (Magtanong et al. 2019). However, the accumulation of oxygenated PUFAcontaining PEs that accumulate following RSL3 treatment was strongly suppressed with pharmacological inhibition or genetic inactivation of Acsl4 compared with controls, clearly linking Acsl4 function to ferroptosis-specific changes in lipid oxidation in some contexts (Doll et al. 2017). It is possible that ferroptosis induced by GPX4 inhibition versus cystine deprivation yield different patterns of lipid oxidation that render ACSL4 more or less important for the execution of ferroptosis. LPCAT3 preferentially catalyzes the insertion of activated AA into membrane PLs or the reacylation of lyso-PLs, with a preference for PCs and PEs (Shindou and Shimizu 2009). While LPCAT3 was initially identified as necessary for RSL3induced death (Dixon et al. 2015), a follow-up study in mouse Gpx4 fl/fl MEFs found that knocking out Lpcat3 only slightly protected cells from ferroptosis induced by RSL3 or Gpx4 deletion (Kagan et al. 2017). Meanwhile, knockdown of Lpcat3 partially protected mouse lung epithelial cells and MEFs from RSL3induced ferroptosis (Doll et al. 2017). These results suggest that the requirement of LPCAT3 for ferroptosis may be species or cell-type dependent. Further studies in diverse cell types are necessary to clarify the role of ACSL and LPCAT enzymes in ferroptosis.

1 Lipid Metabolism and Ferroptosis

1.4.3

11

The Role of Other Lipids and Lipid Metabolic Enzymes in Ferroptosis

In addition to the roles of MUFAs and PUFAs in opposing or promoting ferroptosis through effects on lipid peroxidation, other classes of lipids may also be involved in this process directly or more indirectly. Along with PUFAs, sterol lipids including cholesterols can be oxidized in membranes or in low-density lipoprotein particles (Girotti and Korytowski 2017), and cholesterol esters can be oxygenated by lipoxygenase enzymes (Belkner et al. 1991). Oxidized cholesterol is also a substrate for the peroxidase activity of GPX4 (Thomas et al. 1990). It is therefore possible that cholesterol oxidation may be involved in ferroptosis; however, the exogenous administration of cholesterol was found to be insufficient to modulate the lethality of RSL3 in human cancer cell lines, leaving the role of this lipid in ferroptosis presently unclear (Yang et al. 2016). Cholesterol is a terminal product of the mevalonate pathway. Other products of this lipid metabolic pathway may regulate ferroptosis—most notably the cholesterol precursor squalene. The accumulation of squalene in cholesterol in certain lymphoma cells is correlated with protection from ferroptosis induced by GPX4 inhibitors (Garcia-Bermudez et al. 2019). While the mechanism of squalene’s protective effect is unclear, prior studies suggest that squalene is an efficient oxygen scavenging agent that can prevent the propagation of free radical reactions (Kohno et al. 1995). Squalene may therefore protect membrane lipids from peroxidation during ferroptosis. In Archea, squalene can influence the spatial organization of lipids in the membranes to facilitate tighter packing of bilayers (Gilmore et al. 2013). One possibility is that high levels of squalene in mammalian cells may help maintain membrane architecture and circumvent the changes in membrane fluidity or permeability associated with LOOH accumulation during ferroptosis. Of note, squalene is generally undetectable in human cell lines, and it is possible that squalene only exerts its protective effects at high concentrations found in certain cells or contexts. Squalene accumulation is also observed in cells treated with the ferroptosis inducer FIN56, which induces ferroptosis through a partially characterized mechanism that involves activation of squalene synthetase (SQS) and simultaneous inactivation of GPX4 (Shimada et al. 2016). SQS synthesizes squalene by coupling two molecules of farnesyl pyrophosphate (FPP). FPP is also a precursor to the endogenous antioxidant coenzyme Q10 (CoQ10). SQS activation therefore leads to CoQ10 depletion, which may contribute to the lethality of FIN56. Inhibitors of upstream enzymes in the mevalonate pathway enhance the lethality of FIN56, perhaps by further depleting CoQ10, and/or by preventing squalene synthesis. Intriguingly, inhibitors of squalene monooxygenase (SQLE), the enzyme directly downstream of SQS, suppresses the lethality of FIN56. Inhibition of SQLE likely promotes the accumulation of squalene in cells with active SQS (Shimada et al. 2016), further supporting the role for squalene as an endogenous inhibitor of ferroptosis, at least at high concentrations.

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A recent study found that ferroptosis caused by glutamate-induced suppression of system xc activity led to increased levels of ceramide and sphingosine (Novgorodov et al. 2018). This upregulation was dependent on the activity of acid sphingomyelinase (ASM), an enzyme that generates ceramide from sphingomyelin. Downregulation of ASM or treatment with ASM inhibitors could prolong survival upon glutamate-induced ferroptosis, suggesting a possible role for sphingosine and ceramide in ferroptosis progression. Further, in HT-1080 cells, erastin treatment leads to elevated levels of ceramides (Yang et al. 2016). However, addition of exogenous ceramide does not modulate the lethality of RSL3 in human cancer cells. Intriguingly, the addition of C2-ceramide to mitochondria may increase the production of hydrogen peroxide. While C2-ceramide treatment alone was not sufficient to induce lipid peroxidation, C2-ceramide treatment combined with glutathione depletion in the mitochondria enhances hydrogen peroxide production and induces lipid peroxidation (García-Ruiz et al. 1997). Thus, glutathione depletion may cause an increase in the ASM-dependent accumulation of ceramides and production of hydrogen peroxide. This elevation in the pool of ROS may increase free radicals through iron-catalyzed Fenton chemistry and accelerate lipid peroxidation in other cellular compartments. However, sphingolipid accumulation itself is not sufficient to induce lipid peroxidation in the absence of glutathione depletion. Thus, while sphingolipids may potentiate ferroptosis, they are likely neither necessary nor sufficient for its execution.

1.5

Lipid Peroxidation: Mechanism, Location, and Consequences

Previous studies have broadly characterized the lipid oxidation signature of ferroptosis and defined the specific lipid species necessary for ferroptosis induction. However, a great deal remains unknown about the role of lipid peroxidation in ferroptosis, including how LOOHs form, where they are localized, and how they initiate the terminal lethal events of ferroptosis.

1.5.1

How Do LOOHs Form in Ferroptosis?

The essential role of oxidized PUFA-PLs in ferroptosis is now well established. In addition to the ACSL4 genetic evidence cited above, administration of deoxygenated AA esterified onto PE (PE-AA-OOH) can enhance the lethality of RSL3 in Gpx4 fl/fl MEFs (Kagan et al. 2017). This suggests that lethal PUFA-PL oxidation occurs on PUFAs esterified onto PLs. The next important question concerns the process that generates oxidized PUFA-PL species in cells. One model is that lipid peroxidation occurs through the regulated activity of enzymes—the lipoxygenase

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(LOX) enzymes—that can selectively oxidize PUFAs. The second model holds that the majority of lipid peroxidation in ferroptosis arises due to the spontaneous, non-catalytic process of autoxidation. Below, we consider the evidence for each of these models.

1.5.1.1

The Role of Lipoxygenase Enzymes

LOX enzymes are a family of non-heme iron containing dioxygenases that oxidize PUFAs in a regio-, stereo-, and enantio-specific manner (Noguchi et al. 2002). LOX enzymes can catalyze the oxygenation of PUFAs to their corresponding hydroperoxy derivatives, which can be further converted to a diverse series of bioactive lipid mediators (i.e., eicosanoids) (Kuhn et al. 1990; Kuhn et al. 2015). Some genetic evidence supports the notion that LOX enzymes are important for ferroptosis. For example, MEFs deficient for murine 12/15-Lox are resistant to the lethal effects of buthionine sulfoximine (BSO)-mediated glutathione depletion (Seiler et al. 2008). Likewise, in a model of developing rat oligodendrocytes, 12/ 15-Lox suppressed glutamate-induced neurotoxicity, which likely involves ferroptosis due to glutathione depletion (Li et al. 2009). Moreover, genetic knockdown of all six human ALOX genes (encoding human LOX enzymes) rendered G-401 human cancer cells partially resistant to erastin analogs, but not to RSL3 (Yang et al. 2016). Knockdown of ALOX15B or ALOXE3 in two additional human cancer cell lines strongly suppressed erastin-induced ferroptosis (Yang et al. 2016). The production of HpETE and other oxygenation products of AA by 15-LOX has been hypothesized as a critical death signal in ferroptosis and may be important in initiating or accelerating lipid peroxidation (Anthonymuthu et al. 2018). Intriguingly, LOX-mediated oxygenation products of AA are observed in MEFs following Gpx4 deletion, and the addition of exogenous LOX products 5-, 12-, and 15-hydroperoxyeicosatetraenoic acid (HpETE) is sufficient to promote ferroptosis (Friedmann Angeli et al. 2014). Finally, LOX-mediated induction of ferroptosis in host cells appears to be a mechanism of pathogenesis. Pseudomonas aeruginosa, a gram-negative bacteria that forms biofilms, can secrete vesicles containing LOX enzymes, thereby initiating ferroptosis in mammalian lung epithelium and facilitating infection (Dar et al. 2018). These data suggest that LOX enzymes are sufficient to induce ferroptosis in this context and support the overall contention that LOX activity is pro-ferroptotic. Because the preferred substrates for LOX enzymes are free PUFAs, the activity of phospholipases to cleave PUFA acyl chains from PLs is thought to be required for LOX function (Kuhn et al. 2015). Supporting this, a phospholipase inhibitor prevented ferroptosis induced by Gpx4 deletion in Gpx4 fl/fl MEFs (Friedmann Angeli et al. 2014). However, this model is inconsistent with the aforementioned findings that lipid peroxidation in ferroptosis appears to occur on esterified PUFAPLs rather than on free PUFAs (Kagan et al. 2017). This discrepancy is potentially reconciled by a study showing that the expression of PEBP1, a scaffold protein, complexes with 15-LOX to alter its specificity from free PUFAs to PUFA-PEs

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(Wenzel et al. 2017). Further, 15-LOX and PEBP1 were proposed to co-localize with and inhibit GPX4. Over-expression of PEBP1 sensitized cells to RSL3-induced death, while PEBP1 knockdown marginally suppressed death. As a caveat, in this study, PEBP1 expression itself induced some cell death that was not suppressible with Fer-1, suggesting that PEBP1 may also induce non-ferroptotic cell death (Wenzel et al. 2017).

1.5.1.2

Evidence for Non-enzymatic Lipid Autoxidation

A second model hypothesizes that LOOHs in ferroptosis are formed by spontaneous free radical chain reactions, otherwise known as autoxidation. Evidence for autoxidation occurring in ferroptosis comes from studies of the mechanism by which ferroptosis inhibitors block lipid peroxidation and death. Generally, compounds that are good radical trapping antioxidants (RTAs) in organic solutions and lipid bilayers are potent inhibitors of ferroptosis. For instance, aromatic amines that inhibit ferroptosis (i.e., Fer-1 and Lip-1) are highly effective RTAs in lipid bilayers, but are poor inhibitors of 15-LOX enzyme (Zilka et al. 2017). Further, a set of rationally designed lipophilic RTAs were as effective as Fer-1 and Lip-1 at suppressing ferroptosis induced by GPX4 inhibition or glutathione depletion in hippocampal cells but could not inhibit 15-LOX (Zilka et al. 2017). Likewise, many small molecule LOX inhibitors are potent radical trapping antioxidants (RTAs) that can inhibit non-enzymatic lipid autoxidation reactions, independent of on-target LOX inhibition (Shah et al. 2018). For example, the LOX inhibitors zileuton, PD146176, and NDGA are comparable with Fer-1 and Lip-1 in their ability to suppress ferroptosis through their reactivity as radical-trapping antioxidants (Shah et al. 2018). This result suggests that it is the radical-trapping ability of these inhibitors to block autoxidation that explains ferroptosis suppression. It is possible that other LOX enzymes contribute to ferroptosis and are suppressed by lipophilic antioxidants, but this remains untested. A second line of evidence in support of the autoxidation model comes from a deeper consideration of the role of LOX enzyme activity in ferroptosis. For example, the mechanism of LOX activity appears inconsistent with the ability for lipophilic antioxidants to suppress ferroptosis. LOX-catalyzed oxygenation does involve the formation of radical intermediates, but these are bound and stabilized by the enzyme active site in most cases (Noguchi et al. 2002). Accordingly, in one study, antioxidants including ascorbate, alpha-tocopherol, and Trolox did not suppress the stereospecific oxygenation activity of LOX enzymes. These inhibitors did, however, suppress non-stereospecific side reactions that can occur due to LOX activity (Noguchi et al. 2002). Indeed, while LOX enzymes are generally characterized by a high degree of specificity in their reaction products, when PUFA levels are high, stereo-random side reaction products can occur (Kuhn et al. 1990; Noguchi et al. 2002). These data suggest that the activity of lipophilic antioxidants to suppress ferroptosis is not due to inhibition of the stereospecific LOX reactions and production of specific oxygenation products, but rather due to inhibition of non-specific

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oxidation products. These data suggest that LOX enzymes and their oxidation products are not universally required for ferroptosis. Indeed, several additional lines of evidence argue against an essential role for LOX enzymes in ferroptosis. 12/15-LOX deletion cannot rescue the embryonic lethality of Gpx4 knockout mice or abrogate cell death following whole-body Gpx4 deletion in adult mice (Friedmann Angeli et al. 2014; Brütsch et al. 2015). Furthermore, some cell lines sensitive to ferroptosis do not detectably express any of the major LOX enzymes (Shah et al. 2018), and HT-1080 cells, which are a classic model of ferroptosis, express only ALOX15B and ALOXE3 (Yang et al. 2016). The role of individual LOX enzymes in ferroptosis was tested by overexpressing each LOX enzyme in a cell line deficient for LOX expression, then feeding cells AA deuterated at each (or all) of the bisallylic position from which each LOX enzyme can regiospecifically abstract a hydrogen. In this model, only AA deuterated at all bisallylic positions strongly suppressed RSL3-induced death (Shah et al. 2018). These results support earlier work showing that deuterated linoleate (D-Lin) strongly protected human cancer cell lines from both RSL3 and erastin-induced ferroptosis (Yang et al. 2016). Altogether, these data suggest that LOX enzymes are not universally required for ferroptosis and thus by default favor an autoxidation model of lipid peroxidation. However, it is likely that a mixture of both LOX-catalyzed and autoxidation-dependent processes can contribute to lipid peroxidation in ferroptosis in a context and cell type-specific manner.

1.5.2

Where Are LOOHs Localized in Ferroptosis?

During ferroptosis lipid oxidation occurs within the plasma membrane and in membranes of the mitochondria, ER, lysosomes, and lipid droplets (Kagan et al. 2017; Gaschler et al. 2018a). Where LOOHs accumulate in ferroptosis and whether accumulation is necessary or sufficient for membrane permeabilization during ferroptosis remains to be fully clarified, although progress has been made. This section explores the potential role of LOOH accumulation at different membranes in the cell.

1.5.2.1

The Role of the Plasma Membrane

LOOH accumulation at the plasma membrane may be necessary for ferroptosis. Experiments using the LOOH-sensitive dye BODIPY C11 581/591 and confocal microscopy showed that the execution of ferroptosis correlates with the formation of a distinct “ring” of lipid oxidation around the plasma membrane (Tarangelo et al. 2018; Magtanong et al. 2019). These results suggest that the PM lipid ROS accumulation is necessary for the execution of ferroptosis, at least in the cell types examined. In support of this hypothesis, treatment with the monounsaturated fatty acid (MUFA) oleic acid (C18:1) suppresses erastin-induced ferroptosis in human

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cancer cells specifically by inhibiting lipid peroxidation at the plasma membrane (Magtanong et al. 2019). Erastin-induced lipid peroxidation is also associated with “blisters” in the PM that form rapidly just prior to membrane permeabilization, and this blistering process could conceivably be related to biophysical changes that occur in the lipid bilayer in response to plasma membrane lipid oxidation.

1.5.2.2

The Role of Mitochondria

Human cancer cells treated with erastin undergo morphological changes including mitochondrial shrinkage, cristae enlargement (Yagoda et al. 2007; Dixon et al. 2012), and outer membrane rupture (Friedmann Angeli et al. 2014). The mitochondria itself is a major source of endogenous ROS production and is susceptible to lipid peroxidation. To counter-balance this oxidative stress, one isoform of GPX4 is specifically localized to the mitochondria, and mitochondria contain high levels of reduced glutathione (~10–14 mM) (Marí et al. 2009). Thus, it is plausible that inhibitors of GPX4 activity or conditions that deplete glutathione might also induce lipid peroxidation in the mitochondria. Oxidized cardiolipin and phosphatidylglycerol—phospholipids mostly localized at the mitochondrial inner membrane—are observed in ferroptotic cells (Friedmann Angeli et al. 2014; Kagan et al. 2017; Gaschler et al. 2018a). However, the accumulation of oxidized cardiolipin was not inhibited by the lipophilic antioxidant Fer-1, which otherwise suppresses ferroptosis, suggesting that cardiolipin oxidation is not sufficient for ferroptosis to proceed. Mitochondria also contain high levels of PE and are a major source of PE synthesis (Voelker 1984). The accumulation of oxidized PEs in ferroptosis is well established, yet their localization remains unknown. Thus, it is possible that PE oxidation may initially occur in mitochondria, but then spreads elsewhere. ROS arising from the mitochondria may potentiate lipid peroxidation in the mitochondria and/or in other compartments. As previously discussed, inhibition of cystine import can activate acid sphingomyelinase (ASM), an enzyme that increases ceramide synthesis in the mitochondria and upregulates ROS production. Inhibition of ASM partially protected cells from ferroptosis, suggesting that mitochondriaderived ROS may promote ferroptosis (Novgorodov et al. 2018). Furthermore, a mitochondria-targeted nitroxide antioxidant, XJB-5-131, suppresses ferroptosis better than non-targeted nitroxides (Krainz et al. 2016). Further supporting a role for mitochondria, a recent report suggests that mitochondria and mitochondrial metabolism play an essential role in ferroptosis induced by cysteine deprivation. Gao and colleagues show that depleting cells of mitochondria through the induction of mitophagy partially suppressed erastin-induced ferroptosis and lipid peroxidation in HT-1080 cells. This study also showed that following cysteine deprivation, the earliest LOOHs co-localize with mitochondrial markers. Further, the authors find that the mitochondrial TCA cycle and electron transport chain modulate ferroptosis (Gao et al. 2018). These results are consistent with a prior report showing that

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glutaminolysis, which provides intermediates for the TCA cycle, is necessary for ferroptosis induced by cysteine deprivation (Gao et al. 2015). However, other lines of evidence argue against a central role for core mitochondrial metabolic functions in ferroptosis. Cells depleted of mitochondrial DNA (ρ0) lack mitochondrial respiration, mitochondrial ROS generation, or TCA function (King and Attardi 1989; Martínez-Reyes et al. 2016), yet show no alterations in their sensitivity to ferroptosis induced by erastin, RSL3, or FIN56 (Dixon et al. 2012; Shimada et al. 2016). In contrast to the results obtained by Gao and colleagues, the Stockwell lab found that elimination of mitochondria by inducing mitophagy did not inhibit ferroptosis induced by erastin, RSL3, or FIN56 (Gaschler et al. 2018b). This study also found that ferrostatin-1 and iron chelators were equally effective in preventing erastin-induced ferroptosis in cells with and without mitochondria. Moreover, a lipophilic antioxidant was approximately 100-fold more efficient at preventing ferroptosis induced by erastin, Gpx4 deletion, or RSL3 compared with an analog that is specifically targeted to the mitochondria (Friedmann Angeli et al. 2014). Thus, while mitochondria might help promote ferroptosis under some conditions, they do not appear to be essential for the execution of this process. It is possible that mitochondrial respiration contributes to the overall pool of ROS in the cell, thus potentiating lipid peroxidation that occurs upon glutathione depletion or GPX4 inactivation in other compartments. Mitochondrial membranes may also be oxidized during ferroptosis, although this oxidation does not appear required for death. The accumulation of LOOHs in the mitochondrial outer membrane might lead to increased membrane permeability, thus explaining the mitochondrial swelling (Dixon et al. 2012) and possible outer membrane rupture (Friedmann Angeli et al. 2014) that is observed in ferroptosis.

1.5.2.3

The Role of the Lysosome

Lysosomes are a major site of lipid catabolism and recycling of membrane lipids (Jaishy and Abel 2016). High levels of ROS are continuously produced at the lysosomes and LOOHs accumulate in the perinuclear region early in ferroptosis (Torii et al. 2016). The lysosome is also implicated in ferroptosis through its role in autophagy. The autophagic degradation of iron-loaded ferritin and release of labile iron can promote ferroptosis, and inhibition of autophagy can suppress erastininduced ferroptosis in human cancer cells and MEFs (Gao et al. 2015, 2016; Hou et al. 2016). Inhibitors of lysosomal acidification and proteolytic function, including bafilomycin A1, ammonium chloride, and PepA-Me, decrease lysosomal ROS and can suppress ferroptosis induced by erastin or RSL3 (Torii et al. 2016). On the other hand, no evidence of lysosomal bursting is observed in erastin-treated cells (Skouta et al. 2014). Moreover, while ferrostatins can localize to the lysosome, modifications to the structure of ferrostatins that reduce their trapping in the lysosome increased, rather than decreased, their efficacy in suppressing ferroptosis (Gaschler et al. 2018b). These results suggest that lipid peroxidation at the lysosome is not be

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essential for ferroptosis, though more studies are needed to directly investigate this question.

1.5.2.4

The Role of the ER

The endoplasmic reticulum (ER) is the main intracellular site of de novo lipid synthesis and contains a high density of intracellular membranes (Flis and Daum 2013). Stimulated Raman scattering microscopy coupled with small vibrational tags showed that ferrostatins accumulate in the ER, lysosomes, and mitochondria. Selective depletion of mitochondria failed to suppress the inhibitory effects of ferrostatins on ferroptosis, suggesting that localization of ferrostatins to the ER might instead be required for ferroptosis suppression in some contexts (Gaschler et al. 2018b). Additional studies are needed to further examine whether lipid peroxidation in the ER is required for ferroptosis.

1.5.2.5

The Role of Lipid Droplets

Lipid droplets (LDs) are evolutionarily conserved, highly dynamic organelles composed of a phospholipid monolayer surrounding a hydrophobic core of neutral lipids. LDs can store lipids for energy and membrane synthesis but also regulate cellular stress responses by sequestering certain toxic lipid species (Olzmann and Carvalho 2019). LDs have been proposed to suppress lipid peroxidation by sequestering PUFAs away from membrane PLs (Bailey et al. 2015; Li et al. 2018). Interestingly, the autophagic degradation of lipid droplets (lipolysis) can promote ferroptosis induced by RSL3 in murine hepatocytes (Bai et al. 2019). The knockdown of proteins required for neutral lipid storage results in decreased LD accumulation and enhanced RSL3-induced death. Conversely, inhibition of proteins required for lipophagy modestly inhibits RSL3-induced death (Bai et al. 2019). These data suggest that LD degradation by lipophagy could serve as a source of PUFAs that can be activated and incorporated into membrane phospholipids that are susceptible to lipid peroxidation during ferroptosis. This effect may, however, be cell-type or stimulus specific as the treatment of cells with inhibitors of diglyceride acyltransferase (DGAT) 1/2—enzymes required for the synthesis of TAGs and lipid droplet formation—has no effect on erastin-induced ferroptosis in HT-1080 cells (Magtanong et al. 2019).

1.5.3

How Does Lipid Peroxidation Kill Cells?

While the role of lipid peroxidation in ferroptosis is undisputed, it remains unclear exactly how LOOHs exert their toxic effects on the cell. At least three processes may contribute to lethal effects of lipid peroxidation: (1) LOOH accumulation may alter

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the biophysical properties of membranes and disrupt their integrity; (2) compositional changes in membranes may alter membrane-embedded proteins and disrupt functions essential for survival; or (3) the degradation of LOOHs into highly reactive byproducts may impair other essential cellular processes (Feng and Stockwell 2018). In this section, we will explore the evidence for each of these models.

1.5.3.1

The Effect of Lipid Peroxidation on Membrane Dynamics

The accumulation of LOOHs can alter the physical properties of lipid bilayers by decreasing membrane fluidity, slowing the lateral diffusion of lipids, and increasing membrane permeability (Wong-ekkabut et al. 2007; Catalá and Díaz 2016; Feng and Stockwell 2018). Additionally, the clustering of LOOHs can lead to the formation of membrane pores (Paal et al. 2015) that can be suppressed with the addition of alpha tocopherol (Boonnoy et al. 2017). However, whether lipid-based pores form locally during ferroptosis, or whether a decrease in membrane thickness or structure throughout the membrane result in permeabilization, remains unclear. Computational modeling of lipid peroxidation during ferroptosis using molecular dynamics simulations suggests that changes in the lipid composition of membranes may be sufficient to explain ferroptosis (Agmon et al. 2018). An increase in ferroptosis-competent lipids in membranes increases loacl membrane curvature and the accessibility of oxidants into the membrane interior. These results were validated in vitro using giant unilamellar vesicles and optical microscopy, demonstrating that lipid peroxidation results in local regions of high membrane curvature. Additional molecular dynamics simulations suggest that acyl tails of LOOHs may bend toward the water phase, disrupting the bilayer structure and leading to increased permeabilization (Wong-ekkabut et al. 2007). In vivo, high-resolution phase-contrast imaging of HT-1080 cells revealed that erastin treatment produced “blister”-like deformations with positive curvature on the plasma membrane (Magtanong et al. 2019). The formation of these “blisters” appeared to directly precede membrane permeabilization and cell death in cells undergoing ferroptosis. Together, these data suggest that LOOH accumulation may alter the properties of membranes to induce permeabilization. The effect of specific LOOH species on membrane properties may provide further insights into the lethal mechanism of ferroptosis. In particular, the distinct geometries of membrane PLs can influence membrane function and dynamics (van den Brink-van der Laan et al. 2004). PEs are conical-shaped, non-bilayer lipids that are preferentially oxidized during ferroptosis. An attractive hypothesis is that PE oxidation disrupts non-bilayer lipid phases, thereby altering membrane properties. In fact, molecular dynamics simulations suggest that the incorporation of PE into membranes plays a critical structural role by increasing membrane thickness and decreasing curvature, counterbalancing the effect of PUFAs to decrease thickness and increase membrane curvature (Agmon et al. 2018). This buffering function of PE against the deleterious effects of PUFAs may be critical for membrane integrity. Thus, oxidizing PUFA-containing PLs such as PEs might be a final, necessary step

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to fully disrupting membrane integrity in ferroptosis (Agmon et al. 2018). Like PUFAs, lysolipids can also impose curvature stress on membranes and modulate membrane dynamics (Fuller and Rand 2001). Since the accumulation of lysolipids is characteristic of the lipid signature of ferroptosis (Friedmann Angeli et al. 2014; Yang et al. 2016; Zhang et al. 2019), it is possible that lysolipid accumulation also contributes to curvature stress and membrane disruption.

1.5.3.2

The Effect of Lipid Peroxidation on Membrane-Embedded Protein Function

LOOHs may exert their lethal effects by disrupting the localization or function of membrane-associated proteins. For example, while it is conceivable that the accumulation of LOOHs may recruit pore-forming proteins that disrupt membrane permeability, there is presently no evidence supporting a role for pore-forming proteins in ferroptosis. Alternatively, changes in membrane properties might disrupt the localization or function of essential membrane-associated proteins, such as nutrient transporters or cell signaling machinery. Models using bacterial proteins suggest that non-bilayer membrane regions (such as those formed by PE) may form “insertion sites” where transmembrane proteins can be more easily inserted (van den Brink-van der Laan et al. 2004). Thus, PE oxidation may disrupt non-bilayer phases and prevent the insertion and function of essential transmembrane proteins.

1.5.3.3

The Effect of LOOH Breakdown Products on Essential Cellular Processes

The breakdown of LOOHs into reactive end products during ferroptosis may help physically permeabilize the membrane and also be directly toxic to cells (Gaschler and Stockwell 2017). Reactive compounds derived from lipid peroxidation have been implicated in the cytotoxicity and pathogenicity of many degenerative disorders (Zielinski and Pratt 2017). The best-described and most abundant of these are malondialdehyde (MDA) and 4-hydroxy-nonenol (4-HNE), which are formed through the enzymatic and non-enzymatic decomposition of AA and other PUFAs (Feng and Stockwell 2018). Both MDA and 4-HNE are toxic and mutagenic due to their ability to react with DNA bases, proteins, and other nucleophilic molecules (Esterbauer et al. 1991; Marnett 1999; Ayala et al. 2014). The accumulation of MDA or 4-HNE in cells undergoing ferroptosis has been demonstrated in vitro and in vivo (Gaschler et al. 2018a; Zhang et al. 2019). The formation of adducts by toxic end products has been hypothesized to inhibit essential cellular processes. Supporting this, cell lines cultured in low levels of erastin acquired resistance to ferroptosis through upregulation of aldoketoreductases that detoxify aldehydes like 4-HNE (Dixon et al. 2014). Further, a recent study found that over 400 proteins were endogenously modified by 4-HNE following RSL3 treatment (Chen et al. 2018). Further validation is necessary to determine whether any of these covalent modifications promote the initiation or execution of ferroptosis. Ultimately, it is

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conceivable that multiple mechanisms contribute to the toxicity associated with the lipid peroxidation process.

1.6

Conclusions and Future Perspectives

While many forms of cell death are executed by specific pro-death proteins, ferroptosis is instead executed by metabolites, namely, LOOH. At this time, the role of lipid peroxidation in ferroptosis is firmly established but, as discussed throughout this chapter, the roles of specific lipids, enzymes, and organelles is likely to vary in a context or cell type-specific manner. For example, LOX enzyme may be essential for ferroptosis in some cells but not others. Likewise, mitochondrial lipid oxidation may be essential to promote ferroptosis in response to cysteine deprivation but not in response to different pro-ferroptotic stimuli. Moving forward, advances in untargeted lipidomics, in silico modeling, and chemical probes may proffer further insights into the mechanisms of ferroptosis initiation and execution. It will be important to bear in mind the potential context-dependency of various lipids and lipid metabolic enzymes in ferroptosis. Further studies using in vivo models will also certainly help clarify which of the pathways identified in genetic and small molecule screens are most central to oxidative lipid damage and ferroptotic cell death. We anticipate that these investigations will provide critical insights into the mechanisms and consequences of ferroptosis in biology and ultimately aid in the development of therapeutics to target ferroptosis in human disease. Acknowledgments This work is supported by grants from the NIH to A.T. (1F99CA234650) and S.J.D. (1R01GM122923). Conflict of Interest S.J.D. is on the scientific advisory board of Ferro Therapeutics.

References Agmon E, Solon J, Bassereau P, Stockwell BR (2018) Modeling the effects of lipid peroxidation during ferroptosis on membrane properties. Sci Rep 8:5155. https://doi.org/10.1038/s41598018-23408-0 Anthonymuthu TS, Kenny EM, Shrivastava IH, Tyurina YY, Hier ZE, Ting H-C, Dar H, Tyurin VA, Nesterova A, Amoscato AA, Mikulska-Ruminska K, Rosenbaum JC, Mao G, Jinming Z, Conrad M, Kellum JA, Wenzel SE, VanDemark AP, Bahar I, Kagan VE, Bayir H (2018) Empowerment of 15-lipoxygenase catalytic competence in selective oxidation of membrane ETE-PE to ferroptotic death signals, HpETE-PE. J Am Chem Soc. https://doi.org/10.1021/jacs. 8b09913 Ayala A, Muñoz MF, Argüelles S (2014) Lipid peroxidation: production, metabolism, and signaling mechanisms of malondialdehyde and 4-hydroxy-2-nonenal. Oxid Med Cell Longev. https:// www.hindawi.com/journals/omcl/2014/360438/. Accessed 24 Mar 2019

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Chapter 2

Iron Metabolism and Ferroptosis Shinya Toyokuni and Izumi Yanatori

Abstract No life on earth can survive without iron. Iron absorption, transfer, storage, and its retrieval are precisely regulated by numerous interacting mechanisms, including peptide hormones and transcriptional/posttranscriptional mechanisms. Mammals have no physiological pathway to excrete iron out of the body except for hemorrhage. Iron deficiency may cause anemia and muscle weakness. However, excess iron is a risk for cancer with augmented oxidative stress. The word “ferroptosis” consists of Fe(II) and “falling down,” respecting the Fenton reaction. Cancer cells accommodate higher catalytic Fe(II) in general but with more resistance to oxidative stress in comparison to the non-tumorous counterpart cells. Therefore, carcinogenesis may be a genetic evolutionary process to obtain this ferroptosisresistance, which small molecules, such as erastin, can demolish. Recently, non-thermal plasma became available with the development of engineering, which can precisely load oxidative stress to preferred surface locations. This novel strategy may specifically kill certain cancer cells or even germs by targeting higher catalytic Fe(II) to result in ferroptosis and is expected to work as an additional medical intervention.

2.1

Introduction

Iron is abundant in space because many meteors, in which the major component is iron, have hit the earth thus far (Ruzicka et al. 2017). The earth also contains huge amounts of iron (Ohta et al. 2016), and it is known by geological studies that the ancient sea of a few billion years ago harbored a high concentration of ferrous iron (Olson and Straub 2016), when the first life appeared on earth. Thus, it is not surprising that no independent life on earth can live without iron. A great oxygen event occurred approximately 2.5 billion years ago with the crystallization of iron ore from ferrous iron. Then there was a period of relatively high concentration of S. Toyokuni (*) · I. Yanatori Department of Pathology and Biological Responses, Nagoya University Graduate School of Medicine, Nagoya, Japan e-mail: [email protected] © Springer Nature Switzerland AG 2019 D. Tang (ed.), Ferroptosis in Health and Disease, https://doi.org/10.1007/978-3-030-26780-3_2

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hydrogen sulfur, and the age of oxygen started 0.6 billion years ago (Olson and Straub 2016). Mammals have chosen iron to transport oxygen in the body during the evolutionary process. Molecular oxygen, abundant over the present earth, is versatile in the transfer of 1–4 electrons, thus ensuring constant electron flow. Oxygen is transported in mammals via heme in hemoglobin in red blood cells (Wriggleworth and Baum 1980).

2.2

Iron Metabolism in Circulation

Iron is the most abundant heavy metal in humans and the males have ~4 g and the females have ~2.5 g, and as much as 60% of iron is in the hemoglobin (Wriggleworth and Baum 1980). Iron is a transition metal and the solubility is much higher as ferrous iron [Fe(II)] and ferric iron [Fe(III)] is almost insoluble at neutral pH (Toyokuni 1996; Koppenol and Hider 2018). Apparently, iron has been extremely precious as a nutrient to all lives, so each species has a tactic to obtain iron efficiently. For example, bacteria secrete siderophore, a low molecular weight chemical with high iron affinity, to adsorb iron from other molecules or outer environments (Miethke and Marahiel 2007). Mammals lack any pathway to discharge iron from the body except for hemorrhage or phlebotomy (Toyokuni 2009b). Iron solubility is greatly increased after acidification, which is the reason why iron is absorbed from duodenal villous epithelial cells from the diet because pH of normal gastric juice is 1–2. Fe(III) is always reduced to Fe(II) when passing through the double-layered lipid membrane, including plasma membrane. Two distinct iron transporters, DMT1 (Slc11A2) (Andrews 1999) and ferroportin (Slc40A1) (Donovan et al. 2000), are essential to absorb iron from the duodenum, namely, DMT1 to take Fe(II) into cytoplasm and ferroportin to take Fe(II) from the cytoplasm to the portal vein circulation (Toyokuni 2009b). Secreted Fe(II) is immediately oxidized to Fe(III) by hephaestin (Vulpe et al. 1999) and captured by serum transferrin (Fig. 2.1). Portal vein pours into the liver, which is the major iron reservoir (hepatocytes) in our body. Curiously enough, hepatocytes secrete a peptide hormone called hepcidin (Ganz and Nemeth 2012), which as a ligand for ferroportin decreases intestinal iron absorption as well as available iron in circulation by the degradation of ferroportin through the ubiquitin proteasome system (Fig. 2.2). As already described, erythropoiesis is the largest consumer of iron. The major erythroid regulator of hepcidin is erythroferrone, which is synthesized and secreted by erythroblasts in the bone marrow (Ganz 2018). Erythropoietin produced by the renal interstitial cells induces the production of erythroferrone (Fig. 2.2).

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Fig. 2.1 Summary of iron acquisition by mammals. How iron is absorbed from the diet in the duodenum after solubilization with low pH in the stomach and delivered to each cell is schematically shown. Blue filled circles indicate Fe(III) (ferric iron) and red filled circles correspond to Fe (II) (ferrous iron). Note that iron ions are always reduced to Fe(II) from Fe(III) when they cross the double-layered lipid membrane through the iron transporters (DMT1 and ferroportin). Duodenal iron reductase is duodenal cytochrome b (DCYTB; also as CYBRD1) and duodenal iron oxidase is membrane-bound hephaestin. As soon as iron as Fe(III) is released to circulation (portal vein) from the basolateral membrane of duodenal enterocytes, transferrin captures Fe(III), which is transported to all sorts of cells via the blood stream. Cellular iron metabolism would be described in Fig. 2.2. There is no active pathway to excrete iron

2.3

Iron Metabolism in Cells

Iron is a major cofactor for a variety of enzymes either as Fe(II), heme, or Fe-S cluster. These enzymes include ribonucleotide reductase for DNA synthesis (Bollinger et al. 1991), cytochrome oxidases for energy production and metabolizing xenobiotics (Powers et al. 1981; de Ungria et al. 2000), and nitric oxide synthases for modulating vascular tones and epithelial secretion (McMillan et al. 1992). Therefore, every cell in our body requires iron, which is delivered mainly by serum transferrin (Figs. 2.1 and 2.2). Of note, humans have possible siderophores (Devireddy et al. 2010) and siderophore-binding proteins including lipocalin 2 (Flo et al. 2004;

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Fig. 2.2 Cellular regulation of iron metabolism. Major iron receiver in cells is transferrin receptor whereas certain cells may have DMT1 on the plasma membrane. Transferrin receptor after binding with transferrin is sorted to endosomes, where the binding of transferrin and transferrin receptors as well as that of iron and transferrin are dissolved in acidic environment. Here again Fe(III) in endosomes is reduced by six-transmembrane epithelial antigens of prostate 3 (STEAP3) prior to release to cytosol via DMT1, where Fe(II) is captured by PCBP1/2 (iron chaperone) and sorted to its destination for functional use or storage in ferritin. Ferritin heavy chain works as oxidase to accommodate iron as Fe(III). Stored Fe(III) in ferritin can be retrieved via NCOA4-mediated ferritinophagy, and heme can be degraded by heme oxygenases (HOs) to recover Fe(II). Surplus iron may be secreted from the cell to circulation via ferroportin, which can transport Fe(II) through plasma membrane from Fe(II) bound to PCBP1/2. Cellular iron status is finely regulated through multiple mechanisms, both transcriptional and post-transcriptional mechanisms. Iron regulatory proteins 1 and 2 (IRP1/IRP2), of which IRP2 can be degraded by FBXL5 through ubiquitinproteasome system. Prolyl hydroxylase (PDH), a sensor for iron and oxygen, is the key enzyme for the degradation of hypoxia-inducible factor (HIF)-2α, which works as a transcription factor for DMT1 and CYBRD1. Furthermore, hepcidin, a peptide hormone secreted by hepatocytes, is a ligand for ferroportin, which is degraded after binding via proteasome. Hepcidin transcription is inhibited by erythroferrone secreted by erythroblasts. Blue filled circle, Fe(III) (ferric iron); red filled circle, Fe(II) (ferrous iron)

Rodvold et al. 2012), and some of the cells other than duodenal villous epithelia have DMT1 on the plasma membrane, which may be released via membrane budding as extracellular vesicles (Mackenzie et al. 2016). However, the human siderophore system has not been well established as yet.

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Iron metabolism is precisely regulated via iron regulatory proteins (IRPs; IRP1 and IRP2) and iron responsive element (IRE) (Hentze and Kuhn 1996). In the case of iron deficiency, the number of transferrin receptors per cell is increased via increasing the lifespan of message of transferrin receptor with the binding of IRP to IREs present at the 30 end of the message whereas ferritin, an iron storage protein, is reduced by the translation block with the binding of IRP to IREs present at the 50 end of the message. Indeed, these are the posttranscriptional regulation mechanism. Furthermore, F-box and leucine-rich repeat protein 5 (FBXL5) can target IRP2 for its degradation under iron repletion (Moroishi et al. 2011; Iwai 2018) (Fig. 2.2). After the binding of transferrin with transferrin receptor, this complex is sorted to endosomes and lysosomes, where Fe(III) is released at acidic pH. Here again, DMT1 is used at the endosomal and lysosomal membranes after reduction to transport Fe (II) to a cytosolic fraction for use. Recently, poly(rC)-binding protein 1 and 2 (PCBP1/2) were identified as major Fe(II) chaperones in the cytosol (Shi et al. 2008; Yanatori et al. 2014, 2017). Some of the Fe(II) is carried into mitochondria via mitoferrin for the synthesis of heme and Fe-S cluster (Paradkar et al. 2009). Surplus Fe(II) is stored in ferritin core, consisting of ferritin heavy chain with oxidase activity of Fe(II) and light chain. Unnecessary Fe(II) may be excreted into extracellular circulation via the only iron exporter, ferroportin, in each cell (Yanatori et al. 2016; Yanatori and Kishi 2018) (Fig. 2.2).

2.4

Excess Iron and Cancer

Iron is essential for every kind of life on earth as described in the previous sections. Thus, iron deficiency due to food intake insufficiency or iron loss due to continued hemorrhage causes anemia and muscle dysfunction. However, iron excess is not good for health either but is closely associated with carcinogenesis (Toyokuni 2002, 2009b, 2014; Toyokuni et al. 2017). There are three distinct categories of data supporting this fact: patient information on specific diseases, human population data based on epidemiological analyses, and the abundant data on animal experiments. Table 2.1 summarizes human-specific diseases associated with local iron overload and the carcinogenesis of the corresponding cells. Phlebotomy or iron chelation therapy are the only ways to reduce iron from the body. Of note, there is an epidemiological study that regular phlebotomy (500-ml whole blood) twice a year for 5 years reduced not only the incidence but also the mortality of many representative cancers (Zacharski et al. 2008). On the other hand, there are many animal models established on the association of iron excess and carcinogenesis. In 1959, Richmond reported that intramuscular injection of iron dextran complex produced spindle-cell sarcoma to highly pleomorphic sarcoma at the site of injection (Richmond 1959). In 1982, Okada and Midorikawa reported that repeated intraperitoneal injections of an iron chelate, ferric nitrilotriacetate (Fe-NTA), induced renal adenocarcinoma with a high incidence in rats (Okada and Midorikawa 1982; Ebina et al. 1986; Li et al. 1987; Toyokuni et al.

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Table 2.1 Epidemiological association between local excess iron and cancer in humans Disease Genetic (hereditary) hemochromatosis

Target cell Hepatocytes

Cancer Hepatocellular carcinoma

Viral hepatitis (hepatitis B and C virus)

Hepatocytes

Hepatocellular carcinoma

Environmental exposure to asbestos

Mesothelial cells; alveolar/ bronchial cells

Malignant mesothelioma, lung cancer

Ovarian endometriosis

Ovarian surface epithelial cells

Clear cell carcinoma, endometrioid adenocarcinoma

Molecular mechanism(s) Genetic; type 1–5 (six different types) affecting different single genes (directly associated with iron metabolism), ultimately leading to uncontrolled systemic iron loading Infection; hepatocytes are chronically damaged by autoimmune process, which decreases secretion of hepcidin, leading to unregulated iron absorption from duodenum Environmental exposure; asbestos fibers reach pulmonary alveoli after inhalation; macrophages cannot cope with long and thin fibers, which have high affinity for hemoglobin and histones; fibers accumulate endogenous iron and provide oxidative damage to pulmonary epithelial cells and mesothelial cells Ectopia/regurgitation of menstruation; monthly hemorrhage induces severe iron deposits in the ovary

1998). This model was found with serendipity and I, one of the authors (ST), have been studying this model for over 30 years (Toyokuni 2016a). Fe-NTA is soluble at neutral pH and still works as a catalyst (Toyokuni and Sagripanti 1992, 1993). The molecular mechanisms of excess iron-associated carcinogenesis are in two ways: iron is an inorganic essential nutrient for cell proliferation and excess iron generates catalytic Fe(II) in the body, which causes the Fenton reaction [Fe (II) + H2O2 ! Fe(III) + •OH + OH] (Fenton 1894; Koppenol and Hider 2018). The generated hydroxyl radical (•OH) is indeed the most reactive species in the biological system, inducing strand breaks, modifications, and cross-links in all sorts of biomolecules, including DNA (Toyokuni and Sagripanti 1996). These covalent molecular alterations are the causes of somatic mutations, leading to carcinogenesis. In the Fe-NTA model, it was a mystery why intraperitoneal injections of Fe-NTA caused renal adenocarcinoma. We have elucidated that absorbed Fe-NTA into the blood flow is filtered through the glomeruli of the kidney into the lumina of the proximal tubules of the nephron system, where the Fenton reaction is induced presumably by an abundant local reductant L-cysteine (Hamazaki et al. 1985; Hamazaki et al. 1986, 1988, 1989; Toyokuni et al. 1990; Okada et al. 1993). It is interesting that the genetic alterations observed in Fe-NTA-induced renal adenocarcinoma reveal massive chromosomal change, including homozygous deletion of p16/p15 tumor suppressor genes and amplification of c-Met oncogene (Tanaka et al. 1999; Toyokuni 2011; Akatsuka et al. 2012). Notably, Fe-NTA-induced

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carcinogenesis occurs in wild-type animals in a high incidence (~90% in rats) (Ebina et al. 1986; Toyokuni 2016a). This is the reason why we believe that iron excess is one of the major causes of human carcinogenesis (Toyokuni 2016b). Iron gradually becomes excessive in our body with aging with a decrease in metabolic rates (Toyokuni 2018). Excess iron is also associated with foreign body-induced carcinogenesis. Typically, this occurs in asbestos-induced mesothelial carcinogenesis in humans (Toyokuni 2018). Humans always try to use new materials to improve the quality of our life especially after the industrial revolution, which sometimes has caused unexpected diseases thus far. Asbestos is just one of them. Asbestos is a natural fibrous mineral, so is resistant to heat, acid, and friction and is used for various industrial purposes to increase the quality of the products, such as masks, cloths, brakes, ships, locomotives, and houses. These extremely thin mineral fibers could be mined and processed economically and with versatility, and so they were used in huge amounts worldwide. In the 1960s, it became clear that asbestos causes various pulmonary diseases because it flies in the air and reaches human pulmonary alveoli due to its physical characteristics (IARC 2012; Oury et al. 2014). Within the lung, macrophages and histiocytes try to dispose of these thin fibrous foreign materials. However, they cannot digest asbestos because they are mineral, which was generated on earth, consuming billions of years. Indeed, thin (