Execution Of Ferroptosis Research Articles

Superoxide produced by mitochondrial complex III plays a pivotal role in the execution of ferroptosis induced by cysteine starvation

Ferroptosis is a type of iron-dependent, non-apoptotic cell death, which is typically induced by cysteine starvation or by the inhibition of glutathione peroxidase 4 (GPX4) activity with the accompanying elevation in lipid peroxidation product levels. Despite the central role of mitochondria in oxidative metabolism and hence, as main sources of superoxide, the issue of whether mitochondrial superoxide participates in the execution of ferroptosis remains unclear. To gain additional insights into this issue, we employed suppressors of the site IQ electron leak (S1QEL) and suppressors of the site IIIQo electron leak (S3QEL), small molecules that suppress mitochondrial superoxide production from complex I and III, respectively. The findings indicate that S3QEL, but not S1QEL, significantly protected mouse hepatoma Hepa 1–6 cells from lipid peroxidation and the subsequent ferroptosis induced by cysteine (Cys) starvation (cystine deprivation from culture media or xCT inhibition by erastin). The intracellular levels of Cys and GSH remained low irrespective of life or death. Moreover, S3QEL also suppressed ferroptosis in xCT-knockout mouse-derived embryonic fibroblasts, which usually die under conventional cultivating conditions due to the absence of intracellular Cys and GSH. Although it has been reported that erastin induces the hyperpolarization of the mitochondrial membrane potential, no correlation was observed between hyperpolarization and cell death in xCT-knockout cells. Collectively, these results indicate that superoxide production from complex III plays a pivotal role in the ferroptosis that is induced by Cys starvation, suggesting that protecting mitochondria is a promising therapeutic strategy for the treatment of multiple diseases featuring ferroptosis.

Ferroptosis as a novel therapeutic target for cardiovascular disease.

Cell death is an important component of the pathophysiology of cardiovascular disease. An understanding of how cardiomyocytes die, and why regeneration of cells in the heart is limited, is a critical area of study. Ferroptosis is a form of regulated cell death that is characterized by iron overload, leading to accumulation of lethal levels of lipid hydroperoxides. The metabolism of iron, lipids, amino acids and glutathione tightly controls the initiation and execution of ferroptosis. Emerging evidence shows that ferroptosis is closely associated with the occurrence and progression of various diseases. In recent years, ferroptosis has been found to play critical roles in cardiomyopathy, myocardial infarction, ischemia/reperfusion injury, and heart failure. This article reviews the mechanisms by which ferroptosis is initiated and controlled and discusses ferroptosis as a novel therapeutic target for various cardiovascular diseases.

Ferroptosis in Different Pathological Contexts Seen through the Eyes of Mitochondria

Ferroptosis is a recently described form of regulated cell death characterized by intracellular iron accumulation and severe lipid peroxidation due to an impaired cysteine-glutathione-glutathione peroxidase 4 antioxidant defence axis. One of the hallmarks of ferroptosis is a specific morphological phenotype characterized by extensive ultrastructural changes of mitochondria. Increasing evidence suggests that mitochondria play a significant role in the induction and execution of ferroptosis. The present review summarizes existing knowledge about the mitochondrial impact on ferroptosis in different pathological states, primarily cancer, cardiovascular diseases, and neurodegenerative diseases. Additionally, we highlight pathologies in which the ferroptosis/mitochondria relation remains to be investigated, where the process of ferroptosis has been confirmed (such as liver- and kidney-related pathologies) and those in which ferroptosis has not been studied yet, such as diabetes. We will bring attention to avenues that could be followed in future research, based on the use of mitochondria-targeted approaches as anti- and proferroptotic strategies and directed to the improvement of existing and the development of novel therapeutic strategies.

Acid sphingomyelinase-dependent autophagic degradation of GPX4 is critical for the execution of ferroptosis

Ferroptosis is a type of regulated cell death characterized by ROS accumulation and devastating lipid peroxidation (LPO). The role of acid sphingomyelinase (ASM), a key enzyme in sphingolipid metabolism, in the induction of apoptosis has been studied; however, to date its role in ferroptosis is unclear. In this study, we report that ASM plays a hitherto unanticipated role in promoting ferroptosis. Mechanistically, Erastin (Era) treatment results in the activation of ASM and generation of ceramide, which are required for the Era-induced reactive oxygen species (ROS) generation and LPO. Inhibition of nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase) or removal of intracellular ROS, significantly reduced Era-induced ASM activation, suggesting that NADPH oxidase-derived ROS regulated ASM-initiated redox signaling in a positive feedback manner. Moreover, ASM-mediated activation of autophagy plays a critical role in ferroptosis inducers (FINs)-induced glutathione peroxidase 4 (GPX4) degradation and ferroptosis activation. Genetic or pharmacological inhibition of ASM diminishes Era-induced features of autophagy, GPX4 degradation, LPO, and subsequent ferroptosis. Importantly, genetic activation of ASM increases ferroptosis in cancer cells induced by various FINs. Collectively, these findings reveal that ASM plays a novel role in ferroptosis that could be exploited to improve pathological conditions that link to ferroptosis.

Ferroptosis Resistance in Cancer: An Emerging Crisis of New Hope

Abstract Ferroptosis, a new mode of nonapoptotic cell death, is increasingly recognized as a new hope in overcoming resistance to chemotherapy in cancer. Both canonical and noncanonical pathways can trigger ferroptosis execution via an iron-dependent lethal lipid peroxidation manner. However, growing evidence has shown that some cancer cells can survive ferroptotic stress through metabolic remodeling as regards iron metabolism, anti-oxidative systems, and lipid metabolism. In addition to the well-known roles of the XC−/glutathione/glutathione peroxidase 4 (XC–/GSH/GPX4) axis in blocking ferroptosis, several recently identified pathways, including the Mevalonate-ferroptosis suppressor protein 1 (MVA-FSP1) axis, the GTP cyclohydrolase 1-Tetrahydrobiopterin (GCH1-BH4) axis, the peroxisome-ether-phospholipid axis, the acyl-CoA synthetase long-chain family member 3-monounsaturated fatty acids (ACSL3-MUFA) axis, and the Liver kinase B1-AMP-activated protein kinase (LKB1-AMPK) axis, can negatively regulate susceptibility to ferroptosis. Prominin-2, a newly identified ferroptosis-modulating protein, also drives cancer cells to escape from ferroptosis induction. These findings collectively led to major challenges and opportunities in the development of novel therapies that target the ferroptosis resistance of cancer cells. Significance Ferroptosis is a lethal consequence of accumulated lipid peroxidation catalyzed by ferrous iron and oxygen. This unique cell death process appears to involve many diseases, such as neurodegeneration, ischemia/reperfusion injury, kidney disease, and a druggable target in therapy-resistant cancers. There is great expectation of being able to exploit ferroptosis for the treatment of as yet incurable diseases. However, the state of ferroptosis susceptibility is linked to various regulation pathways. This perspective aims to integrate the current understanding of signaling mechanisms for ferroptotic defenses and facilitates movement toward novel cancer therapeutic strategies.

NCOA4-mediated ferritinophagy promotes ferroptosis induced by erastin, but not by RSL3 in HeLa cells

Ferroptosis is a regulated cell death characterized by a lethal accumulation of lipid peroxides due to an increase of intracellular iron and a decrease of antioxidant capacity. The reduction of antioxidant activity is obtained by using chemical agents, such as erastin and RSL3, the first one inhibiting the transmembrane cystine-glutamate antiporter causing a cysteine and glutathione depletion and the second one inactivating directly the glutathione peroxidase 4 (GPX4) respectively. The role of iron and its related proteins in supporting the formation of lipid peroxides, is not completely understood hence to try to shed light on it we generated HeLa clones with altered ferritinophagy, the ferritin degradation process, by knocking-out or overexpressing Nuclear Receptor Coactivator 4 (NCOA4), the ferritin autophagic cargo-receptor. NCOA4 deficiency abolished ferritinophagy increasing ferritin level and making the cells more resistant to erastin, but unexpectedly more sensitive to RSL3. Interestingly, we found that erastin promoted ferritinophagy in HeLa cells expressing NCOA4, increasing the free iron, lipid peroxidation and the sensitivity to ferroptosis. In contrast, RSL3 did not modulate ferritinophagy, while NCOA4 overexpression delayed RSL3-induced cell death suggesting that RSL3 mechanism of action is independent of ferritin degradation process. Therefore, the ferritin-iron release in the execution of ferroptosis seems to depend on the inducing compound, its target and downstream pathway of cell death activation.

Monitoring autophagy-dependent ferroptosis.

Ferroptosis is an iron-dependent form of regulated cell death, driven by the accumulation of lipid peroxidation. Autophagy is a lysosome-dependent degradation process that can be used to remove and recover intracellular components, such as dysfunctional proteins and damaged organelles. By regulating iron storage and oxidative stress, excessive autophagy is involved in the induction and execution of ferroptosis. In particular, several types of selective autophagy (e.g., ferritinophagy, lipophagy, clockophagy, and chaperone-mediated autophagy) increase the susceptibility to ferroptotic cell death by degrading anti-ferroptotic regulators (e.g., ferritin, GPX4, ARNTL, and lipid droplets). These two integrated biological processes play a pathological role in the occurrence and development of human diseases, such as cancer, neurodegenerative disorders, ischemia and reperfusion injury. Therefore, it is important to develop reliable methods to evaluate the kinetics of autophagosome formation, iron accumulation, and lipid peroxidation. Here, we introduce some protocols (such as western blotting, lipid peroxidation assay kits and probes, and iron probes) to monitor the process of autophagy-dependent ferroptosis.

Dynasore Blocks Ferroptosis through Combined Modulation of Iron Uptake and Inhibition of Mitochondrial Respiration.

Ferroptosis is a form of regulated necrosis characterized by a chain-reaction of detrimental membrane lipid peroxidation following collapse of glutathione peroxidase 4 (Gpx4) activity. This lipid peroxidation is catalyzed by labile ferric iron. Therefore, iron import mediated via transferrin receptors and both, enzymatic and non-enzymatic iron-dependent radical formation are crucial prerequisites for the execution of ferroptosis. Intriguingly, the dynamin inhibitor dynasore, which has been shown to block transferrin receptor endocytosis, can protect from ischemia/reperfusion injury as well as neuronal cell death following spinal cord injury. Yet, it is unknown how dynasore exerts these cell death-protective effects. Using small interfering RNA suppression, lipid reactive oxygen species (ROS), iron tracers and bona fide inducers of ferroptosis, we find that dynasore treatment in lung adenocarcinoma and neuronal cell lines strongly protects these from ferroptosis. Surprisingly, while the dynasore targets dynamin 1 and 2 promote extracellular iron uptake, their silencing was not sufficient to block ferroptosis suggesting that this route of extracellular iron uptake is dispensable for acute induction of ferroptosis and dynasore must have an additional off-target activity mediating full ferroptosis protection. Instead, in intact cells, dynasore inhibited mitochondrial respiration and thereby mitochondrial ROS production which can feed into detrimental lipid peroxidation and ferroptotic cell death in the presence of labile iron. In addition, in cell free systems, dynasore showed radical scavenger properties and acted as a broadly active antioxidant which is superior to N-acetylcysteine (NAC) in blocking ferroptosis. Thus, dynasore can function as a highly active inhibitor of ROS-driven types of cell death via combined modulation of the iron pool and inhibition of general ROS by simultaneously blocking two routes required for ROS and lipid-ROS driven cell death, respectively. These data have important implications for the interpretation of studies observing tissue-protective effects of this dynamin inhibitor as well as raise awareness that off-target ROS scavenging activities of small molecules used to interrogate the ferroptosis pathway should be taken into consideration.

Excessive phospholipid peroxidation distinguishes ferroptosis from other cell death modes including pyroptosis

Lipid peroxidation (LPO) drives ferroptosis execution. However, LPO has been shown to contribute also to other modes of regulated cell death (RCD). To clarify the role of LPO in different modes of RCD, we studied in a comprehensive approach the differential involvement of reactive oxygen species (ROS), phospholipid peroxidation products, and lipid ROS flux in the major prototype modes of RCD viz. apoptosis, necroptosis, ferroptosis, and pyroptosis. LC-MS oxidative lipidomics revealed robust peroxidation of three classes of phospholipids during ferroptosis with quantitative predominance of phosphatidylethanolamine species. Incomparably lower amounts of phospholipid peroxidation products were found in any of the other modes of RCD. Nonetheless, a strong increase in lipid ROS levels was detected in non-canonical pyroptosis, but only during cell membrane rupture. In contrast to ferroptosis, lipid ROS apparently was not involved in non-canonical pyroptosis execution nor in the release of IL-1β and IL-18, while clear dependency on CASP11 and GSDMD was observed. Our data demonstrate that ferroptosis is the only mode of RCD that depends on excessive phospholipid peroxidation for its cytotoxicity. In addition, our results also highlight the importance of performing kinetics and using different methods to monitor the occurrence of LPO. This should open the discussion on the implication of particular LPO events in relation to different modes of RCD.

Ferroptosis occurs through an osmotic mechanism and propagates independently of cell rupture.

Ferroptosis is a regulated form of necrotic cell death that is caused by the accumulation of oxidized phospholipids, leading to membrane damage and cell lysis1, 2. While other types of necrotic death such as pyroptosis and necroptosis are mediated by active mechanisms of execution3–6, ferroptosis is thought to result from the accumulation of unrepaired cell damage1. Previous studies suggested that ferroptosis has the ability to spread through cell populations in a wave-like manner, resulting in a distinct spatiotemporal pattern of cell death7, 8. Here we investigate the mechanism of ferroptosis execution and discover that ferroptotic cell rupture is mediated by plasma membrane pores, similarly to cell lysis in pyroptosis and necroptosis3, 4. We further find that intercellular propagation of death occurs following treatment with some ferroptosis-inducing agents, including erastin2, 9 and C’ dot nanoparticles8, but not upon direct inhibition of the ferroptosis-inhibiting enzyme Glutathione Peroxidase 4 (GPX4)10. Propagation of a ferroptosis-inducing signal occurs upstream of cell rupture, and involves the spreading of a cell swelling effect through cell populations in a lipid peroxide- and iron-dependent manner.

The Latest View on the Mechanism of Ferroptosis and Its Research Progress in Spinal Cord Injury.

Ferroptosis is a recently identified nonapoptotic form of cell death whose major markers are iron dependence and accumulation of lipid reactive oxygen species, accompanied by morphological changes such as shrunken mitochondria and increased membrane density. It appears to contribute to the death of tumors, ischemia-reperfusion, acute renal failure, and nervous system diseases, among others. The generative mechanism of ferroptosis includes iron overloading, lipid peroxidation, and downstream execution, while the regulatory mechanism involves the glutathione/glutathione peroxidase 4 pathway, as well as the mevalonate pathway and the transsulfuration pathway. In-depth research has continuously developed and enriched knowledge on the mechanism by which ferroptosis occurs. In recent years, reports of the noninterchangeable role played by selenium in glutathione peroxidase 4 and its function in suppressing ferroptosis and the discovery of ferroptosis suppressor protein 1, identified as a ferroptosis resistance factor parallel to the glutathione peroxidase 4 pathway, have expanded and deepened our understanding of the mechanism by which ferroptosis works. Ferroptosis has been reported in spinal cord injury animal model experiments, and the inhibition of ferroptosis could promote the recovery of neurological function. Here, we review the latest studies on mechanism by which ferroptosis occurs, focusing on the ferroptosis execution and the contents related to selenium and ferroptosis suppressor protein 1. In addition, we summarize the current research status of ferroptosis in spinal cord injury. The aim of this review is to better understand the mechanisms by which ferroptosis occurs and its role in the pathophysiological process of spinal cord injury, so as to provide a new idea and frame of reference for further exploration.

Aldo-keto reductases protect metastatic melanoma from ER stress-independent ferroptosis

The incidence of melanoma is increasing over the years with a still poor prognosis and the lack of a cure able to guarantee an adequate survival of patients. Although the new immuno-based coupled to target therapeutic strategy is encouraging, the appearance of targeted/cross-resistance and/or side effects such as autoimmune disorders could limit its clinical use. Alternative therapeutic strategies are therefore urgently needed to efficiently kill melanoma cells. Ferroptosis induction and execution were evaluated in metastasis-derived wild-type and oncogenic BRAF melanoma cells, and the process responsible for the resistance has been dissected at molecular level. Although efficiently induced in all cells, in an oncogenic BRAF- and ER stress-independent way, most cells were resistant to ferroptosis execution. At molecular level we found that: resistant cells efficiently activate NRF2 which in turn upregulates the early ferroptotic marker CHAC1, in an ER stress-independent manner, and the aldo-keto reductases AKR1C1 ÷ 3 which degrades the 12/15-LOX-generated lipid peroxides thus resulting in ferroptotic cell death resistance. However, inhibiting AKRs activity/expression completely resensitizes resistant melanoma cells to ferroptosis execution. Finally, we found that the ferroptotic susceptibility associated with the differentiation of melanoma cells cannot be applied to metastatic-derived cells, due to the EMT-associated gene expression reprogramming process. However, we identified SCL7A11 as a valuable marker to predict the susceptibility of metastatic melanoma cells to ferroptosis. Our results identify the use of pro-ferroptotic drugs coupled to AKRs inhibitors as a new valuable strategy to efficiently kill human skin melanoma cells.

The chemical basis of ferroptosis.

Lipid peroxidation underlies the mechanism of oxidative cell death now known as ferroptosis. This modality, distinct from other forms of cell death, has been intensely researched in recent years owing to its relevance in both degenerative disease and cancer. The demonstration that it can be modulated by small molecules in multiple pathophysiological contexts offers exciting opportunities for novel pharmacological interventions. Herein, we introduce the salient features of lipid peroxidation, how it can be modulated by small molecules and what principal aspects require urgent investigation by researchers in the field. The central role of non-enzymatic reactions in the execution of ferroptosis will be emphasized, as these processes have hitherto not been generally considered 'druggable'. Moreover, we provide a critical perspective on the biochemical mechanisms that contribute to cell vulnerability to ferroptosis and discuss how they can be exploited in the design of novel therapeutics.

Arsenic induces pancreatic dysfunction and ferroptosis via mitochondrial ROS-autophagy-lysosomal pathway

Chronic arsenic exposure is a significantly risk factor for pancreatic dysfunction and type 2 diabetes (T2D). Ferroptosis is a newly identified iron-dependent form of oxidative cell death that relies on lipid peroxidation. Previous data have indicated that ferroptosis is involved in various diseases, including cancers, neurodegenerative diseases, and T2D. However, the concrete effect and mechanism of ferroptosis on pancreatic dysfunction triggered by arsenic remains unknown. In this study, we verified that ferroptosis occurred in animal models of arsenic-induced pancreatic dysfunction through assessing proferroptotic markers and morphological changes in mitochondria. In vitro, arsenic caused execution of ferroptosis in a dose-dependent manner, which could be significantly reduced by ferrostatin-1. Additionally, arsenic damaged mitochondria manifested as diminishing of mitochondrial membrane potential, reduced cytochrome c level and production of mitochondrial reactive oxygen species (MtROS) in MIN6 cells. Using the Mito-TEMPO, we found the autophagy level and subsequent ferroptotic cell death induced by arsenic were both alleviated. With autophagy inhibitor chloroquine, we further revealed that ferritin regulated ferroptosis through the MtROS-autophagy pathway. Collectively, NaAsO2-induced ferroptotic cell death is relied on the MtROS-dependent autophagy by regulating the iron homeostasis. Ferroptosis is involved in pancreatic dysfunction triggered by arsenic, and arsenic-induced ferroptosis involves MtROS, autophagy, ferritin.

Quercetin Protects Hepatocyte from Ferroptosis by Depressing Mitochondria-reticulum Interaction Through PERK Downregulation in Alcoholic Liver (P06-056-19)

ObjectivesConsidering the disorder of lipid metabolism and iron deposition in alcoholic liver (ALD), ferroptosis may play a vital role in its pathogenesis. In the present research, we sought to determine whether ferroptosis occurred in ALD and is associated with increased mitochondria-endoplasmic reticulum interaction (MAMs) through RNA-dependent protein kinase like ER kinase (PERK) upregulation and whether quercetin plays a role in ferroptosis. MethodsOur preliminary results temporarily focused on animals model. Adult male C57BL/6 J mice were isocalorically pair-fed either a regular or ethanol-containing Lieber De Carli liquid diets (28% energy replacement) supplemented with quercetin (100 mg/kg.bw) or not for 12 weeks. Results•Serum alanine aminotransferase(ALT) and aspartate aminotransferase (AST) were indicators of liver damage. Compared with regular diet group, alcohol consumption resulted in increased ALT and AST, quercetin treatment reversed the abnormalities induced by ethanol.•Oil Red Ostain, depleted GSH and increased MDA in liver suggested disrupted lipid metabolism and abnormal redox system in E group. Together with the increased liver total iron, liable iron pool iron and mRNA level of prostaglandin endoperoxide synthase 2 (ptgs2), these data indicated the occurrence of ferroptosis in ALD which reversed by quercetin.•Electron microscope pictures showed increased MAMs in E group. Moreover, as a junction protein to maintain MAMs stability and a key sensor of the unfolded protein response, the level of PERKis unchanged in liver tissue but increased in MAMs in E group which decreased in the supplement of quercetin, which suggested upregulated PERK contributed to the increased MAMs in alcohol consumption. Increased Acyl-CoA synthetase long-chain family member 4 (ASCL4) also observed in MAMs in E. Since ACSL4 mainly enriched in MAMs and is also a key protein involved in the execution of ferroptosis, it suggested increased MAMs in E group and correlation between ferroptosis and MAMs. ConclusionsOur results are preliminarily illustrated enhanced ferroptosis regulated by increased MAMs through PERK upregulation in ALD. And quercetin protected hepatocyte from ethanol toxicity by reversing above changes. Funding SourcesNational Natural Science Foundation of China No. 81673164. Supporting Tables, Images and/or Graphs▪▪▪

Ischemia-induced ACSL4 activation contributes to ferroptosis-mediated tissue injury in intestinal ischemia/reperfusion

Ferroptosis is a recently identified form of regulated cell death defined by the iron-dependent accumulation of lipid reactive oxygen species. Ferroptosis has been studied in various diseases such as cancer, Parkinson’s disease, and stroke. However, the exact function and mechanism of ferroptosis in ischemia/reperfusion (I/R) injury, especially in the intestine, remains unknown. Considering the unique conditions required for ferroptosis, we hypothesize that ischemia promotes ferroptosis immediately after intestinal reperfusion. In contrast to conventional strategies employed in I/R studies, we focused on the ischemic phase. Here we verified ferroptosis by assessing proferroptotic changes after ischemia along with protein and lipid peroxidation levels during reperfusion. The inhibition of ferroptosis by liproxstatin-1 ameliorated I/R-induced intestinal injury. Acyl-CoA synthetase long-chain family member 4 (ACSL4), which is a key enzyme that regulates lipid composition, has been shown to contribute to the execution of ferroptosis, but its role in I/R needs clarification. In the present study, we used rosiglitazone (ROSI) and siRNA to inhibit ischemia/hypoxia-induced ACSL4 in vivo and in vitro. The results demonstrated that ACSL4 inhibition before reperfusion protected against ferroptosis and cell death. Further investigation revealed that special protein 1 (Sp1) was a crucial transcription factor that increased ACSL4 transcription by binding to the ACSL4 promoter region. Collectively, this study demonstrates that ferroptosis is closely associated with intestinal I/R injury, and that ACSL4 has a critical role in this lethal process. Sp1 is an important factor in promoting ACSL4 expression. These results suggest a unique and effective mechanistic approach for intestinal I/R injury prevention and treatment.

Chaperone-mediated autophagy is involved in the execution of ferroptosis

Necroptosis and ferroptosis are two distinct necrotic cell death modalities with no known common molecular mechanisms. Necroptosis is activated by ligands of death receptors such as tumor necrosis factor-α (TNF-α) under caspase-deficient conditions, whereas ferroptosis is mediated by the accumulation of lipid peroxides upon the depletion/or inhibition of glutathione peroxidase 4 (GPX4). The molecular mechanism that mediates the execution of ferroptosis remains unclear. In this study, we identified 2-amino-5-chloro-N,3-dimethylbenzamide (CDDO), a compound known to inhibit heat shock protein 90 (HSP90), as an inhibitor of necroptosis that could also inhibit ferroptosis. We found that HSP90 defined a common regulatory nodal between necroptosis and ferroptosis. We showed that inhibition of HSP90 by CDDO blocked necroptosis by inhibiting the activation of RIPK1 kinase. Furthermore, we showed that the activation of ferroptosis by erastin increased the levels of lysosome-associated membrane protein 2a to promote chaperone-mediated autophagy (CMA), which, in turn, promoted the degradation of GPX4. Importantly, inhibition of CMA stabilized GPX4 and reduced ferroptosis. Our results suggest that activation of CMA is involved in the execution of ferroptosis.

Ferroptosis: The Greasy Side of Cell Death.

Ferroptosis is a form of cell death that requires phospholipid peroxidation and has attracted increased attention, both as a means to eradicate tumors resistant to standard chemotherapy and for its potential contribution to tissue damage such as in ischemia/reperfusion. The center stage taken by phospholipid peroxidation in ferroptosis is highlighted by recent discoveries that demonstrate an intricate regulation of both the metabolism of polyunsaturated fatty acids as well as mechanisms leading to their oxidation. These metabolic steps converge at the point of ferroptosis execution through mechanisms that are now only starting to be understood. In this short review, we provide an appraisal of some of the recent advances in the understanding of the ferroptosis process and also provide some perspectives of where this knowledge could take us.

Resolving the Role of Lipoxygenases in the Initiation and Execution of Ferroptosis.

Lipoxygenases (LOXs) have been implicated as central players in ferroptosis, a recently characterized cell death modality associated with the accumulation of lipid hydroperoxides: the products of LOX catalysis. To provide insight on their role, human embryonic kidney cells were transfected to overexpress each of the human isoforms associated with disease, 5-LOX, p12-LOX, and 15-LOX-1, which yielded stable cell lines that were demonstrably sensitized to ferroptosis. Interestingly, the cells could be rescued by less than half of a diverse collection of known LOX inhibitors. Furthermore, the cytoprotective compounds were similarly potent in each of the cell lines even though some were clearly isoform-selective LOX inhibitors. The cytoprotective compounds were subsequently demonstrated to be effective radical-trapping antioxidants, which protect lipids from autoxidation, the autocatalytic radical chain reaction that produces lipid hydroperoxides. From these data (and others reported herein), a picture emerges wherein LOX activity may contribute to the cellular pool of lipid hydroperoxides that initiate ferroptosis, but lipid autoxidation drives the cell death process.

The Potency of Diarylamine Radical-Trapping Antioxidants as Inhibitors of Ferroptosis Underscores the Role of Autoxidation in the Mechanism of Cell Death.

Two aromatic amines (ferrostatin-1 and liproxstatin-1) were recently identified from high-throughput screening efforts to uncover potent inhibitors of ferroptosis, the necrotic-like cell death induced by inhibition of glutathione peroxidase 4 (GPX4), deletion of the corresponding gpx4 gene, or starvation of GPX4 of its reducing cosubstrate, glutathione (GSH). We have since demonstrated that these two aromatic amines are highly effective radical-trapping antioxidants (RTAs) in lipid bilayers, suggesting that they subvert ferroptosis by inhibiting lipid peroxidation (autoxidation) and, thus, that this process drives the execution of ferroptosis. Herein, we show that diarylamine RTAs used to protect petroleum-derived products from autoxidation can be potent inhibitors of ferroptosis. The diarylamines investigated include representative examples of additives to engine oils, greases and rubber (4,4'-dialkyldiphenylamines), core structures of dyes and pharmaceuticals (phenoxazines and phenothiazines), and aza-analogues of these three classes of compounds that we have recently shown can be modified to achieve much greater reactivity. We find that regardless of how ferroptosis is induced (GPX4 inhibition, gpx4 deletion or GSH depletion), compounds which possess good RTA activity in organic solution (kinh > 105 M-1 s-1) and lipid bilayers (kinh > 104 M-1 s-1) are generally potent inhibitors of ferroptosis (in mouse embryonic fibroblasts). Likewise, structural analogs that do not possess RTA activity are devoid of antiferroptotic activity. These results further support the argument that lipid peroxidation (autoxidation) plays a major role in the mechanism of cell death induced by either GPX4 inhibition, gpx4 deletion, or GSH depletion. Moreover, it offers clear direction that ongoing medicinal chemistry efforts on liproxstatin and ferrostatin derivatives, which have been proposed as lead compounds for the treatment and/or prevention of ischemia/reperfusion injury, renal failure, and neurodegeneration, can be widened to include other aminic RTAs. To aid in these efforts, some relevant structure-reactivity relationships are discussed.