Ferroptosis As Ultimate Target of Cancer Therapy

Abstract

Significance:

The significance of ferroptosis in cancer therapeutics has now been unveiled. Specific ferroptosis inducers are expected as a promising strategy for cancer treatment, especially in cancers with epithelial mesenchymal transition and possibly in cancers with activated Hippo signaling pathways, both of which cause resistance to traditional chemotherapy but tend to show ferroptosis susceptibility.

Recent Advances:

Ferroptosis is a new form of regulated non-apoptotic cell death, which is characterized by iron-dependent lipid peroxidation, leading eventually to plasma membrane rupture. Its core mechanisms have been elucidated, consisting of a driving force as catalytic Fe(II)-dependent Fenton reaction and an incorporation of polyunsaturated fatty acids to membrane phospholipids via peroxisome-dependent and -independent pathways, and suppressing factors as prevention of lipid peroxidation with glutathione peroxidase 4 and direct membrane repair via coenzyme Q10 and ESCRT-III pathways.

Critical Issues:

Developments of ferroptosis inducers are in progress by nanotechnology-based drugs or by innovative engineering devices. Especially, low-temperature (non-thermal) plasma is a novel technology at the preclinical stage. The exposure can induce ferroptosis selectively in cancer cells rich in catalytic Fe(II).

Future Directions:

We also summarize and discuss the recently uncovered responsible molecular mechanisms in association with iron metabolism, ferroptosis and cancer therapeutics. Targeting ferroptosis in addition to the current therapeutic modalities would be important to cure advanced-stage cancer. Antioxid. Redox Signal. 39, 206–223.

Introduction

Despite the recent rapid development of precision cancer therapeutics, cancer remains the leading cause of death, accounting for ∼10 million deaths worldwide in 2020 (Sung et al, 2021). Anticancer drugs, mostly inducers of apoptosis, are one of the major approaches of cancer treatment. However, the effects of anticancer drugs are limited, owing to the intrinsic or acquired resistance to apoptosis-inducing drugs (Hassannia et al, 2019; Holohan et al, 2013; Okada and Mak, 2004; Su et al, 2016). Accordingly, establishing therapeutic strategies to induce nonapoptotic cell death is expected to open a new avenue by overcoming chemoresistance.

Recently, a growing number of research efforts suggest ferroptosis, a novel mode of regulated necrosis, as an ultimate candidate for therapeutic target of cancer (Conrad et al, 2016; Hassannia et al, 2019; Vanden Berghe et al, 2014; Yang and Stockwell, 2016). In this review article, we first describe the importance of iron metabolism in cancer. We then explain the molecular mechanisms and involvement of ferroptosis in cancer therapy. Finally, we provide a perspective on this expanding research area.

Classification of Cancer Therapy

Cancer therapy is classified into local therapies (e.g., surgical therapy, radiation therapy, and ablation therapy), systemic therapies (e.g., chemotherapy, targeted therapy, hormone therapy, and immune therapy), and their combination (Baskar et al, 2012; Ramos et al, 2021; Wang et al, 2019b). Local therapies are effective for the local control by eliminating tumor cells from the targeted area, achieving complete cure of cancer at an early stage or symptom relief at the advanced stage. In contrast, systemic therapy plays a pivotal role in controlling advanced or recurrent cancer to extend survival.

Among the systemic therapies, chemotherapy with apoptosis-inducing agents is still a major scheme of the systemic therapy. Thus, intrinsic or acquired resistance to chemotherapeutic agents is a serious problem that causes recurrence and poor survival outcomes in patients (Ramos et al, 2021). Hence, it is expected to develop novel therapeutic options to induce cell death other than apoptosis to overcome the chemoresistance and improve the prognosis of cancer patients.

Chemoresistance

Resistance to chemotherapeutic agents has been observed in nearly all sorts of anticancer drugs (Galluzzi et al, 2014; Murray et al, 2012; Sladek et al, 2002; Smith et al, 2006). Multiple mechanisms are involved in the resistance. One of the molecular mechanisms is the modification of pharmacokinetics in cancer patients, such as via acquired mutations in the genes metabolizing direct target molecules, absorption, and excretion of the drug or its metabolites (Alfarouk et al, 2015). Another mechanism is the mutation or activation of certain signaling pathways, such as the Kelch-like ECH-associated protein 1 (KEAP1)/nuclear factor-erythroid 2-related factor 2 (NRF2, or also known as NFE2L2)/Cullin3 (CUL3) pathway or phosphoinositide 3-kinase (PI3K)/AKT/mitogen-activated protein kinase (MAPK)/nuclear factor-κB (NF-κB)/notch pathway.

KEAP1/NRF2/CUL3 pathway is a master regulator of transcription in the genes associated with glutathione (GSH) synthesis/reduction, reactive oxygen species (ROS)/xenobiotics detoxification, thioredoxin-based antioxidant system, and heme/iron metabolism to gain chemoresistance (Barrera-Rodriguez, 2018; Jeong et al, 2020). PI3K/AKT/MAPK/NF-κB/notch pathway regulates angiogenesis, cellular proliferation, and drug concentration/distribution, which eventually contribute to chemotherapy resistance (Rajabpour et al, 2017).

In addition, tumor microenvironments, including hypoxia, acidity, collagen, hyaluronan, and fibronectin, and the interaction between tumor cells and the surrounding cells, such as cancer-associated fibroblasts, cancer-associated macrophages, or cytotoxic T lymphocytes (CTLs), mediate resistance to the chemotherapeutic drugs (Baghban et al, 2020). Furthermore, characteristics of each cancer of the patients, such as fraction of cancer stem cells and the degree of epithelial–mesenchymal transition (EMT), also determine the chemoresistance. Here we stress that cancer cells even with high chemoresistance inevitably require iron to survive.

Overview of the Role of Iron in Carcinogenesis and Cancer Metabolism

Iron in life

Iron is the most fundamental metal for life. No independent cells, including cancer cells, can live without iron because iron is adopted in the functional catalytic portion of various enzymes for its redox activity, in DNA replication (ribonucleotide reductase), adenosine triphosphate (ATP) synthesis (cytochrome oxidases), and antioxidant activity (catalase).

Iron is the most abundant element by mass inside the Earth, occupying 80% of the core and 6.3% of the surface (Frey and Reed, 2012). Furthermore, 3.8 billion years ago, when the first life was born on the Earth, the atmospheric environment was in a reducing condition and a high concentration of Fe(II) was dissolved in the primordial sea (Gutteridge and Halliwell, 2018). In addition to the abundance, iron holds an affinity to bind a broad range of ligands, thus contributing to redox reaction or electron transfer by changing status mainly between Fe(II) and Fe(III) (Frey and Reed, 2012).

Therefore, iron has been selected in many important catalytic reactions for electron transfer or acid–base reactions in life (Hosseinzadeh and Lu, 2016; Sanchez et al, 2017). After the origination of Cyanobacteria in ∼2.2 billion years ago, Fe(II) was rapidly oxidized to Fe(III) by oxygen released with photosynthesis, resulting in precipitation in the sea by forming insoluble complexes (Gutteridge and Halliwell, 2018). Consequently, as low as 2–3 × 10⁻⁹ kg/L of iron is contained in the sea water today.

Iron metabolism

Iron mainly takes two valences in life, catalytic soluble Fe(II) and noncatalytic nearly insoluble Fe(III) (Sanchez et al, 2017). Iron is stored in the form of Fe(III) to reduce cytotoxicity, bound to transferrin (Tf) or lactoferrin extracellularly, and encapsulated in ferritin core intracellularly to prevent precipitation. When iron goes through iron transporters to cross any biomembrane, Fe(III) needs to be reduced to Fe(II) by the corresponding specific reductase (Fig. 1).

FIG. 1.

Overview of iron metabolism. TBI is the major form of iron in the blood. TBI is received by the TfR1 on the plasma membrane of cells and internalized within the cells by receptor-mediated endocytosis. In the endosome, Fe(III) is released from Tf by decreasing pH, reduced by STEAP3 to soluble Fe(II) and transported across the endosomal membrane into cytosol by DMT1. Fe(II) released from DMT1 can be chaperoned by PCBP2. PCBP2 also can pick up Fe(II) generated by heme degeneration by HO-1/POR. Under iron overload, NTBI is taken up via DMT1 located on cytoplasmic membrane. A major fraction of Fe(II) in cytoplasm is escorted by PCBP1 or PCBP2. In addition, PCBP1 transports Fe(II) to ferritin for iron loading, whereas PCBP2 conveys Fe(II) to FPT1. Under cellular iron deficiency, ferritin is degraded via NCOA4-mediated autophagy called ferritinophagy to retrieve stored Fe(III). Iron sufficiency in cells induces CD63 expression and increases secretion of CD63⁺ EVs containing iron-loaded ferritin. DMT1, divalent metal transporter 1; EVs, extracellular vesicles; FPT1, ferroportin 1; HO-1, heme oxygenase 1; NCOA4, nuclear receptor coactivator 4; NTBI, nontransferrin-bound iron; PCBP, poly r(C)-binding protein; POR, NADPH cytochrome P450 reductase; TBI, transferrin-bound iron; Tf, transferrin; TfR1, transferrin receptor 1.

Cellular iron uptake

There are two major pathways for cellular iron uptake. One is through Tf receptor-mediated uptake of transferrin-bound iron (TBI) and the other is nontransferrin-bound iron (NTBI) uptake (Canonne-Hergaux and Gros, 2002; Dunn et al, 2007; Gunshin et al, 1997; Kautz et al, 2014). Major types of cells, including erythroblasts and cancer cells, take up iron in the form of TBI via binding to and internalization of the transferrin receptor 1 (TfR1) on the plasma membrane (Fillebeen et al, 2019).

After the endocytosis of TfR1, STEAP3 metalloreductase on the endosomal luminal membrane reduces released Fe(III) to soluble Fe(II) (Mancias et al, 2014) and transports Fe(II) into the cytosol through divalent metal transporter 1 (DMT1; solute carrier family 11 member 2, SLC11A2), which is a pH-dependent proton-coupled Fe(II) transporter (Gunshin et al, 1997). In contrast, uptake of NTBI is physiologically observed in duodenum or kidney during the absorptive process of NTBI from foods or reabsorption of NTBI from raw urine, respectively.

Furthermore, NTBI uptake is observed under iron overloaded conditions, where serum iron has exceeded the capacity of Tf, and direct uptake of NTBI into cytosol is mediated by DMT1, ZIP8 (zinc transporter 8, SLC39A8) or ZIP14 (SLC39A14) (Knutson, 2019). ZIP8 and ZIP14 have been originally identified as zinc transporters on the plasma membrane.

Intracellular iron delivery

The understanding of molecular mechanisms on cytoplasmic iron delivery has been revolutionized in the past decade. In the cytoplasm, most of Fe(II) is coordinated by reduced GSH to form Fe(II)-GS, possibly to reduce the oxidation and reactivity of Fe(II) (Philpott et al, 2020). In addition, significant population of Fe(II) or Fe(II)-GS is physiologically escorted by iron chaperones, poly r(C)-binding protein 1–4 (PCBP1–4), which are originally reported as RNA/DNA binding molecules (Swanson and Dreyfuss, 1988). Among the PCBPs, PCBP1 and PCBP2 are most abundantly expressed in a wide range of cells and tissues, and are present in the cytoplasm and nucleus.

PCBP3 and PCBP4 are expressed at low levels only in some tissues and at some periods of development (Leidgens et al, 2013; Yanatori et al, 2020). Though each PCBP escorts Fe(II), they function differently as chaperones. Regarding PCBP1 and PCBP2, major types of PCBPs, both of them deliver Fe(II) to ferritin, a major iron storage protein (Leidgens et al, 2013; Philpott et al, 2020; Shi et al, 2008) and to mononuclear iron enzymes prolyl hydroxylase (Nandal et al, 2011) and dinuclear iron enzyme deoxyhypusine hydroxylase (Frey et al, 2014).

Furthermore, PCBP2 can receive Fe(II) directly from DMT1 (Yanatori et al, 2014), can recover Fe(II) after disassembly of heme via heme oxygenase 1 (HO-1) with NADPH cytochrome P450 reductase (POR) (Yanatori et al, 2017), and can transfer Fe(II) to ferroportin 1 (FPT1, SLC40A1), the only iron exporter (Yanatori et al, 2016). In contrast, PCBP1 does not bind to DMT1 nor FPT1 (Frey et al, 2014; Yanatori et al, 2020; Yanatori et al, 2017; Yanatori et al, 2016), but appears as the only molecule to be able to upload excess cytosolic iron to ferritin cores with the help of ferritin heavy chain subunit (FTH1) (Philpott et al, 2020; Shi et al, 2008). The activity of PCBP1 and PCBP2 may be affected by cell type, expression of iron transporters or intracellular iron level, which requires further investigation.

Iron storage

Ferritin is a universal cellular protein for iron storage. Ferritin makes a hollow complex called apoferritin in cytosol by the assembly of 24 polypeptide units to store iron as Fe(III) in the core and reduce cytotoxity (Lopez-Castro et al, 2012). Ferritin consists of two subunits, H subunit to store iron safely by oxidizing Fe(II) with ferroxidase center and L subunit to mineralize for long-term storage of iron (Lopez-Castro et al, 2012). Under iron overload, due to overwhelming the capacity of ferritin, excess iron deposits as hemosiderin (Hino et al, 2021). In contrast, under cellular iron deficiency, degradation of ferritin is induced via nuclear receptor coactivator 4 (NCOA4)-mediated autophagy, named ferritinophagy.

Cellular iron export via transporter

Ferroportin (FPT1, SLC40A1) is the only known cellular Fe(II) exporter in higher species, mainly expressed in intestinal, splenic, and hepatic cells to transport iron from diets, senescent erythrocytes, and hepatic storage to serum, respectively (Ganz and Nemeth, 2012). FPT1 is post-transcriptionally regulated by two specific mechanisms, hepcidin and iron-responsible element (IRE)–iron regulatory protein (IRP) system.

Hepcidin, a 25 amino acid peptide hormone and a ligand for FPT1, is secreted mainly from hepatocytes, which promotes, after binding to FPT1 as a receptor on the plasma membrane, internalization and lysosomal degeneration of FPT1 to inhibit cellular export of iron (Nemeth et al, 2004; Park et al, 2001; Pigeon et al, 2001). IRE–IRP system as a post-transcriptional regulation plays an important role to maintain cellular iron homeostasis, which is described hereunder.

Cellular iron export via extracellular vesicles

As reported recently, extracellular vesicles (EVs) are implicated in iron export. Prominin2 (PROM2), a member of the prominin family of pentaspan membrane glycoproteins, stimulates the formation of ferritin-containing multivesicular bodies and exosomes to transport iron out of the cells (Brown et al, 2019). In addition, iron loading to cells induces the expression of CD63 and increases secretion of CD63⁺ EVs containing iron-loaded ferritin via the IRE–IRP systems, which indicates that secretion of CD63⁺ EVs is another strategy to export excess iron from iron-sufficient cells to iron-deficient cells within the same individual (Yanatori et al, 2021). Moreover, NCOA4 is also involved in iron-loaded ferritin export via CD63⁺ EVs by mediating the loading of ferritin to CD63⁺ EVs (Yanatori et al, 2021).

Iron regulation via IRE–IRP system

Cellular iron homeostasis is maintained by balanced expression of proteins involved in iron uptake, chaperoning, storage, and export. This balance is post-transcriptionally regulated by the binding of IRP1 and IRP2 to IRE within the messenger RNAs (mRNAs) of genes associated with iron metabolism. IRP1 and IRP2 are RNA-binding proteins, the amounts of which are controlled via iron-dependent regulation (Wang et al, 2007). In iron-starved cells, IRE–IRP interactions stabilize mRNA of genes for iron uptake (TfR1 and DMT1) and simultaneously inhibit translation of genes for iron storage (FTH1 and ferritin light chain subunit [FTL]) and export (FPT1 and CD63) (Kim et al, 1996; Yanatori et al, 2021).

In contrast, in iron-sufficient cells, inactivation of IRPs promotes mRNA degradation of genes for iron uptake (TfR1 and DMT1) and activates translation via releasing block at the 5′ untranslated region of genes for iron storage (FTH and FTL) and export (FPT1 and CD63) (Kim et al, 1996; Yanatori et al, 2021) (Fig. 2).

FIG. 2.

IRE–IRP system. IRP1 and IRP2 bind to IRE in iron-deficient conditions, mediating the repression of the translation of mRNA with 5′-UTR IRE (FTL, FTH1, FPT1, and CD63) and stabilization of mRNA with 3′-UTR (TfR1 and DMT1). In iron overloaded conditions, IRP1 obtains Fe-S cluster, whereas IRP2 undergoes proteasomal degradation after oxidation. Thus, both IRPs are released from the IRE of the corresponding mRNA. Simultaneously, IRP1 gains function of aconitase. In both cases, once dissociated from the IRPs, the target mRNAs will be either translated (5′-UTR IRE) or targeted for faster degradation (3′UTR). FTH1, ferritin heavy chain subunit; FTL, ferritin light chain subunit; IRE, iron-responsible element; IRP, iron regulatory protein; mRNA, messenger RNA; UTR, untranslated region.

Systemic iron regulation

The total amount of iron in normal adult humans is 2.5–4 g, which is the highest among heavy metals in the body. Approximately 60% of iron is involved in erythropoiesis, utilized as the heme of hemoglobin in red blood cells and recycled by destruction of senescent red blood cells (Andrews, 1999). Most of the remaining iron is stored in the liver or spleen as ferritin or hemosiderin. With no active excretion, the body iron metabolism is a semiclosed system, and the amount of body iron is regulated by absorption. Since as low as 1 mg of iron is lost from peeling off or death of surface/intestinal epithelial cells, 1 mg is absorbed daily from duodenum to serum in the portal system under the regulation of hepcidin to stop dietary iron uptake via ferroportin (Harigae, 2018; Toyokuni et al, 2020).

Whereas the amount of iron is regulated through the ways already described, some pathological conditions may induce iron overload. Iron overload can be classified into hereditary hemochromatosis and secondary iron overload. Hereditary hemochromatosis is a disease that causes excess absorption and systemic deposition of iron via the mutations of HFE (Feder et al, 1996), hemojuvelin, HAMP (FPT1), or transferrin receptor 2 (Franchini, 2006).

Secondary iron overload is caused by chronic inflammation (e.g., chronic bacterial infection and asbestos exposure), increased erythroid cell death, including ineffective erythropoiesis (e.g., thalassemia, sideroblastic anemia, and myelodysplastic syndrome), excess iron administration (e.g., iron-rich diet, supplemental drugs, and repeated whole blood transfusion), and liver dysfunction (chronic viral hepatitis, alcoholic liver, nonalcoholic steatohepatitis, etc.) (Kohgo et al, 2008; Yanatori et al, 2020). In addition, local iron overload is developed by ovarian endometriosis (Yamaguchi et al, 2008).

Iron in carcinogenesis

Though iron is essential for every cell and the life, iron behaves as a double-edged sword. Excess iron generates ROS via Fenton reaction (Fe²⁺ + H₂O₂ → Fe³⁺ + ^•OH + OH⁻), leading to oxidative damage in various organs, including liver, heart, kidney, pancreas, and the central nervous system (Andersen et al, 2014; Toyokuni, 2002). Moreover, excess iron is also implicated in carcinogenesis by ROS generation to cause oxidative DNA damage resulting in genomic alterations (Poetsch, 2020). The human epidemiological and clinical observations have been described in detail in other publications.

For example, a significantly high incidence of hepatocellular carcinoma develops in the patients of genetic hemochromatosis (Kowdley, 2004), β-thalassemia (Finianos et al, 2018) or chronic HCV hepatitis (Miyanishi et al, 2019); malignant mesothelioma is induced long after asbestos exposure (Toyokuni, 2019); and ovarian clear cell carcinoma is frequently associated with ovarian endometriosis (Kajiyama et al, 2019; Pearce et al, 2012; Toyokuni, 2016; Toyokuni, 2002; Yamaguchi et al, 2008).

Overview of Ferroptosis, Catalytic Fe(II)-Dependent Regulated Necrosis Accompanied by Lipid Peroxidation

Ferroptosis

After intensive research on cellular death from various aspects, a novel iron-dependent programed necrosis called ferroptosis was coined in 2012. Ferroptosis is characterized by excessive accumulation of catalytic Fe(II) followed by lipid peroxidation, which is prevented by redox-inactive iron chelators (Dixon et al, 2012). Ferroptotic cells are morphologically characterized by shrinking mitochondria with normal or increased membrane density, mitochondrial membrane disruption, or reduced mitochondrial cristae (Dixon et al, 2012; Yagoda et al, 2007), and incomplete plasma membrane with release of intracellular contents, including damage-associated molecular patterns (DAMPs) (Wen et al, 2019), whereas apoptotic cells show membrane blebbing, nuclear/cytoplasmic fragmentation, chromatin condensation, cellular shrinkage, and swollen or ruptured mitochondria (Elmore, 2007).

Ferroptosis was first described on the process of cell death induced by erastin, which inhibits cysteine/glutamate antiporter, SLC7A11 (xCT, system X_c ⁻), resulting in GSH depletion to inactivate glutathione peroxidase 4 (GPX4) (Dixon et al, 2012; Stockwell et al, 2017). Recent research revealed that ferroptosis is regulated by multiple systems, including iron metabolism, sulfur-dependent antioxidants, as well as peroxisome-dependent and peroxisome-independent pathways. Peroxidized phospholipids (PLs) in plasma membrane, especially PLs with polyunsaturated fatty acids (PUFAs) (Hadian and Stockwell, 2020; Jiang et al, 2021b; Li and Li, 2020), are recognized as the possible common end-stage effector.

The incorporation of PUFA onto membrane PLs is mediated by multiple enzymes. PUFAs, such as arachidonic acid (AA) and adrenic acid (AdA), are esterified at first by acyl-CoA synthetase long-chain family member-4 (ACSL4) to PUFA-CoA and are further processed by lysophosphatidylcholine acyltransferase-3 (LPCAT3) before the integration to the cell membrane (Doll et al, 2019). PLs containing PUFA (PL-PUFA) can be oxidized to PUFA-containing-phospholipid hydroperoxide (PL-PUFA-OOH) nonenzymatically by ROS generated via catalytic Fe(II)-dependent Fenton reaction, or enzymatically by lipoxygenases (LOXs), a nonheme iron containing enzyme or by POR (Stockwell et al, 2017; Tang and Kroemer, 2020a; Tang and Kroemer, 2020b; Zou et al, 2020b).

In addition, it was recently revealed that biosynthesis of plasmalogen via peroxisome is another mechanism for lipid peroxidation. Plasmalogen is produced by the collaboration of peroxisomes, a membrane-bound oxidative organelle, and endoplasmic reticulum (ER). Peroxisomal enzymes, such as fatty acyl-CoA reductase 1 (FAR1), glycerone-phosphate O-acyltransferase (GNPAT), and alkylglycerone phosphate synthase (AGPS), contribute to the synthesis of plasmalogens. Distinct from common PLs, plasmalogen uses an ether bond, instead of an ester bond, to connect the glycerophospholipid. Their precursor 1-O-alkyl-glycerol-3-phosphate (AGP) is synthesized by FAR1 and AGPS, and then transported to ER for further biosynthetic reactions involving 1-acylglycerol-3-phosphate O-acyltransferase 3 (AGPAT3).

Finally, the ER-resident enzyme plasmanylethanolamine desaturase 1 (PEDS1) mediates the production of PUFA plasmalogen, which is susceptible to lipid peroxidation (Zou et al, 2020a). Moreover, it was recently suggested that some kinds of selective autophagy are involved in triggering ferroptosis, including ferritinophagy retrieving iron from ferritin, and lipophagy and clockophagy, defined as “the selective degradation of the core circadian clock protein, aryl hydrocarbon receptor nuclear translocator-like (ARNTL), by autophagy,” forming lipid peroxidation (Jiang et al, 2021a; Liu et al, 2020).

Such lipid peroxidation of PUFAs produces a wide variety of oxidation products. Lipid hydroperoxides (LOOHs) are the initial products of peroxidation. Secondary products are aldehydes, among which malondialdehyde and 4-hydroxy-2-nonenal (HNE) are the most abundant and thought to inactivate proteins through crosslinking (Feng and Stockwell, 2018). Recently, our laboratory established that a mouse monoclonal antibody (HNEJ-1) to recognize equally His/Lys/Cys Michael adducts of HNE modification is ideal for the detection of ferroptotic cells in formalin-fixed paraffin-embedded (FFPE) pathological specimens, which demonstrated that fetal erythropoiesis and aging exploit ferroptotic process as physiological phenomena (Zheng et al, 2021).

In contrast, xCT (SLC7A11)-Cys/GSH-GPX4 axis is widely accepted as intracellular suppressive mechanism of ferroptosis, which repairs phospholipid hydroperoxide (PL-OOH) by the use of GSH (Brigelius-Flohe and Maiorino, 2013; Ursini et al, 1982). In addition, it was revealed that ferroptosis suppressor protein 1 (FSP1)/ubiquinone (CoQ10)/NAD(P)H pathway suppresses ferroptosis by repairing cell membrane directly and works as a standalone parallel system in harmony with the GPX4 system (Doll et al, 2019). Endosomal sorting complexes required for transport-III (ESCRT-III) pathway also work in membrane repair for ferroptosis suppression (Dai et al, 2020b) (Fig. 3).

FIG. 3.

Molecular mechanisms of ferroptosis. Incorporation of PUFA-PLs-peroxide into the plasma membrane is a major contributing factor of ferroptosis. Lipid peroxidation can take place either nonenzymatically by ROS generated via catalytic Fe(II)-dependent Fenton reaction, enzymatically by oxidation of PUFAs with LOXs and POR with modification by ACSL4 and LPCAT3, or by biosynthesis of PUFA plasmalogen in peroxisome and ER. PUFA-PL-peroxide formation is counteracted by SLC7A11/GSH/GPX4 axis to reduce peroxided lipids, or by FSP1/CoQ10/NADP(H) pathway or ESCRT-III complex to repair the damaged membrane. AA, arachidonic acid; AA-PE, arachidonic acid–phosphatidylethanolamine; ACSL4, acyl-CoA synthetase long-chain family member-4; AdA, adrenic acid; AdA-PE, adrenic acid–phosphatidylethanolamine; AGP, 1-O-alkyl-glycerol-3-phosphate; AGPAT3, 1-acylglycerol-3-phosphate O-acyltransferase 3; AGPS, alkylglycerone phosphate synthase; CoQ10, coenzyme Q10; DHAP, dihydroxyacetone phosphate; ER, endoplasmic reticulum; ESCRT-III, endosomal sorting complexes required for transport-III; FAR1, fatty acyl-CoA reductase 1; FPP, farnesyl pyrophosphate; FSP1, ferroptosis suppressor protein 1; GNPAT, glycerone-phosphate O-acyltransferase; GPX4, glutathione peroxidase 4; GSH, glutathione; HMG-CoA, 3-hydroxy-3-methylglutaryl coenzyme A; HMGCR, HMG-CoA reductase; IPP, isopentenyl pyrophosphate; LOOH, lipid hydroperoxides; LOX, lipoxygenase; LPCAT3, lysophosphatidylcholine acyltransferase 3; PEDS1, plasmanylethanolamine desaturase 1; PL, phospholipid; PUFA, polyunsaturated fatty acid; ROS, reactive oxygen species.

As already described, molecular mechanisms of ferroptosis are starting to be unveiled. However, the factor that makes cancer cells sensitive to ferroptosis is still elusive. One of the factors that determine the ferroptosis susceptibility is the origin and type of cancer. A sensitivity profiling of 177 cancer cell lines has revealed that cell lines of renal cell carcinoma and diffuse large B cell lymphoma are more sensitive to erastin-induced ferroptosis than cell lines from the other types of cancer (Yang et al, 2014).

In addition, intertumoral heterogeneity is another possible factor to determine ferroptosis sensitivity in cancer cells. In three-dimensional culture of lung cancer cells, the inner cells exhibited vulnerability to ferroptosis than surface cells, which is rescued by hyperactivation of NRF2 (Takahashi et al, 2020). Some transcriptional factors, including NRF2 and Hippo signaling pathway, or tumor differentiation, such as EMT, are reported as described hereunder to affect ferroptosis susceptivity in cancer cells.

NRF2 and ferroptosis

NRF2, which is regulated by KEAP1 by ubiquitination-proteasomal degradation, is a key transcriptional factor in response to oxidative stress and ferroptosis resistance (Ma, 2013; Mohammadzadeh et al, 2012). NRF2 decreases oxidative stress by the expression of proteins involved in the antioxidant systems, such as glutamate-cysteine ligase catalytic subunit (GCLC), HO-1, SLC7A11, and GPX4, and the expression of proteins implicated in the decrease of cellular iron, such as FTH1, FTL, and FPT1 (Anandhan et al, 2020). Thus, either KEAP1 knockdown or NRF2 activation makes the cells ferroptosis resistant (Fan et al, 2017; Sun et al, 2016), whereas NRF2 suppression enhances susceptibility to ferroptosis (Shin et al, 2018).

NRF2 is also modulated by ARF, which is an alternative reading-frame product of the CDKN2A locus. ARF suppresses the cAMP-responsive element binding protein (CBP)-dependent acetylation of NRF2, which is critical for sequence-specific DNA binding and expression of downstream target genes, including SLC7A11 (Chen et al, 2017; Sun and Brodsky, 2017). Therefore, ARF deletion leads to ferroptosis resistance.

EMT and ferroptosis

EMT is a process in which carcinoma cells lose their cellular polarity or adhesion, and acquire mesenchymal properties. During this process, tumor cells achieve more invasive nature and chemoresistance (Brabletz et al, 2018). Conversely, cells with EMT are reported to be vulnerable to ferroptosis inducers (FINs) (Bi et al, 2019; Daher et al, 2019; Hangauer et al, 2017; Viswanathan et al, 2017). Multiple factors for EMT are implicated in ferroptosis susceptibility. Zinc finger E-box-binding homeobox 1 (ZEB1) is a transcriptional factor, which is a major inducer of EMT.

ZEB1 modifies lipid metabolism to increase PUFAs, leading to ferroptosis susceptibility (Gubelmann et al, 2014). As another example, overexpression of ACSLs and stearoyl-CoA desaturase-1, which are associated with EMT of colon cancer, is also known to enhance ferroptosis sensitivity (Sanchez-Martinez et al, 2015). In addition, metadherin (MTDH), also known as Lyric, is also one of the factors to induce EMT, which increases the susceptibility to ferroptosis by inhibiting expressions of GPX4 and SLC3A2 (a system X_c ⁻ heterodimerization partner) (Bi et al, 2019). In contrast, integrin α6β4, which is predominantly expressed in epithelial cells, induces ferroptosis resistance by repression of ACSL4 via activation of the Src-STAT3 pathway (Brown et al, 2017).

Hippo-Yes-associated protein 1/transcriptional coactivator with PDZ-binding motif signaling pathway and ferroptosis

Hippo signaling pathway is an evolutionally conserved pathway that regulates organ size by modulating cellular proliferation and apoptosis (Lai et al, 2005; Tao et al, 1999; Zhao et al, 2011). The core of Hippo signaling pathway is the kinase cascade of mammalian sterile 20-like kinase 1 (MST1)/MST2, Salvador1 (SAV1), large tumor suppressor kinase 1 (LATS1)/LATS2, and MOB kinase activator 1A (MOB1A)/MOB1B. Hippo pathway negatively regulates Yes-associated protein 1 (YAP)/transcriptional coactivator with PDZ-binding motif (TAZ), which forms a complex with TEA domain family member (TEAD) transcription factors (TEAD1–4) to express target genes, resulting in cell proliferation and migration (Goulev et al, 2008; Huang et al, 2005).

Hippo signaling pathway is activated by cell–cell junctions or cell-density sensing via E-cadherin/α-catenin or mechanical signals via integrins to activate MST1/MST2 or LATS1/LATS2, resulting in phosphorylation of YAP/TAZ, followed by proteolytic degradation of YAP/TAZ to suppress expression of the target genes (Kim et al, 2011; Schlegelmilch et al, 2011). In addition, various scaffolding proteins, including Moesin-Ezrin-Radixin like (Merlin) tumor suppressor encoded by neurofibromin type 2 (NF2), are associated with the regulation by activation of some core complexes or suppression of nuclear localization of YAP/TAZ (Boopathy and Hong, 2019; Furukawa et al, 2017; Kim et al, 2011; Moya and Halder, 2019).

Because YAP/TAZ targets several promoters of ferroptosis-associated genes, including TfR1, ACSL4, and NOX2 via ANGPTL4 expression and NOX4 via EMP1 expression (Wu et al, 2019; Yang and Chi, 2020; Yang et al, 2020; Yang et al, 2019), susceptibility to ferroptosis would inversely depend on the activity of Hippo pathway (Fig. 4). Exceptionally, in hepatocellular carcinoma, YAP/TAZ upregulates SLC7A11, suppressor of ferroptosis, to induce ferroptosis resistance (Gao et al, 2021). Collectively, the interaction between ferroptosis and Hippo signaling pathway is component dependent and further studies are necessary on the relationship between Hippo signaling pathway components and ferroptosis sensitivity.

FIG. 4.

Overview of Hippo-YAP/TAZ signaling pathway. E-cadherin, α-catenin, and integrins work as sensors for cellular attachment, cellular density, and mechanical forces to the cell, respectively, which lead to Hippo pathway activation to limit cell/organ size, with E-cadherin, α-catenin, and merlin as initiators. The core of Hippo signaling pathway consists of kinase cascade with MST1/MST2, SAV1, LATS1/LATS2 and MOB1A/MOB1B, which phosphorylates YAP/TAZ. Phosphorylation of YAP/TAZ results in proteolytic degradation of YAP/TAZ, resulting in downregulation of target genes. When Hippo signaling pathway is inactive, unphosphorylated YAP/TAZ localize into the nucleus, to form complex with TEADs, inducing expression of target genes including TfR1 and ACSL4, eventually lading to ferroptosis sensitivity. In contrast, hepatocellular carcinoma cells showed increased expression of SLC7A11 by YAP/TAZ/TEAD complex to form ferroptosis resistance. LATS1, large tumor suppressor kinase 1; Merlin, moesin-ezrin-radixin like; MOB1A, MOB kinase activator 1A; MST1, mammalian sterile 20-like kinase 1; SAV1, Salvador1; TAZ, transcriptional coactivator with PDZ-binding motif; TEAD, TEA domain family member; YAP, Yes-associated protein 1.

Immunological Interaction and Ferroptosis

In considering ferroptosis as a novel target of cancer therapy, we need to understand how ferroptosis of cancer cells eventually affects the tumor microenvironment. Whereas most studies about ferroptosis are performed in single-layer cultured cells and the role of ferroptosis in vivo remains largely elusive, some experimental progress indicates that ferroptosis can be an immunogenic cell death (ICD) (Efimova et al, 2020).

This is based on the facts that ferroptotic cells show common features of ICD, such as release of DAMPs, including high-mobility group box 1 (HMGB1) and ATP and translocation of calreticulin to the cell surface to activate immune responses, though the mechanisms for the releasing DAMPs are unknown and possibly differ from those of necroptotic or pyroptotic cells (Galluzzi et al, 2014; Kepp et al, 2019; Kolbrink et al, 2020; Medina and Ravichandran, 2016).

HMGB1 is released from ferroptotic cells in an autophagy-dependent manner (Wen et al, 2019). It causes inflammatory responses of bone marrow-derived macrophages by activating advanced glycosylation end products (AGE)/receptor for AGE (RAGE) pathway, which is a proinflammatory signaling cascade (Logan and Storey, 2018; Wen et al, 2019). In addition to HMGB1, ATP is also released from early ferroptotic cancer cells, which induce maturation of bone marrow-derived dendric cells (Efimova et al, 2020). Calreticulin (also known as calregulin, CRP55, or endoplasmic reticulum resident protein 60 [ERp60]) is a protein that binds to misfolded proteins to prevent export from ER to Golgi apparatus.

The translocation of calreticulin to cell membrane is known as “eat me signal” to cause engulfment of the cell by phagocytes or dendric cells. Though these processes have been observed during apoptosis, studies reported similar phenomena in ferroptotic tumor cells, inducing maturation of dendritic cells and infiltration of CTLs (Turubanova et al, 2019; Yu et al, 2020).

Whereas many studies suggest that ferroptosis is an ICD, ferroptotic pancreatic ductal adenocarcinoma cells harboring mutant KRAS ^G12D release KRAS^G12D protein via exosomes, which are taken up by macrophages, causing M2-like protumor polarization (Dai et al, 2020a). Prostaglandin E2 (PGE2), a major immunosuppressive factor, negatively affects anticancer immunity by blocking classical type-1 dendritic cell accumulation to tumor, thereby inhibiting CTLs (Wang and DuBois, 2015).

Ferroptotic tumor cells were reported to increase the release of PGE2 (Yang et al, 2014) though the effect is not elucidated yet. In contrast, under immune check point inhibitors such as anti-PD-L1, activated CD8⁺ T cells can secrete interferon-γ (IFN-γ), which induces downregulation of the expression of system Xc⁻ (SLC7A11 and SLC3A2) and contributes to induce ferroptosis (Wang et al, 2019a) (Fig. 5).

FIG. 5.

Ferroptosis from the viewpoint of the association between cancer cells and immune cells. (A) CD8⁺ T cells activated during immunotherapy secrete IFN-γ to activate the STAT1 in cancer cells, which downregulates the expression of SLC7A11 to increase the susceptibility to ferroptosis. (B) Ferroptosis in cancer cells induces the translocation of calreticulin, which is known as “eat-me signal” to trigger engulfment of the cells by phagocytes or DCs. Release of DAMPs including HMGB1 and ATP from ferroptotic cancer cells can induce maturation of DCs and inflammatory responses of macrophages. In case of carcinoma with KRAS ^G12D mutation, ferroptosis in the cancer cell induces release of KRAS^G12D via exosomes, which causes M2-like polarization of macrophages. ATP, adenosine triphosphate; DAMPs, damage-associated molecular patterns; DC, dendritic cell; HMGB1, high-mobility group box 1; IFN-γ, interferon-γ.

Taking together, ferroptosis can induce proinflammatory response by the release of DAMPs and the translocation of calreticulin. However, in some cases, ferroptosis may cause anti-inflammatory responses with KRAS^G12D and PGE2. Further studies on the association of immunological interaction with ferroptosis are necessary to develop precise therapy for each patient.

Ferroptosis Inducers

As already described, ferroptosis is a promising therapeutic endpoint for cancers. Various strategies were developed for ferroptosis induction. FINs are mainly classified into four types (Hassannia et al, 2019). Class I FINs act to reduce GSH, Class II FINs directly inactivate GPX4, Class III FINs deplete GPX4 and CoQ10 in association with squalene synthase (SQS)-mevalonate pathway, and Class IV FINs cause lipid peroxidation by an increase in labile iron pool via blocking iron metabolism or actively degrading heme. These drugs are expected to improve cancer treatment not only by increasing therapeutic choice but also by enhancing the effects of established anticancer drugs in combinational use (Liu and Wang, 2019) (Table 1).

Table 1.

Ferroptosis Inducers

Target point	Drug	Molecular mechanism	References
Class I		GSH depletion
SLC7A11	Erastin	Inhibit SLC7A11, VDAC2/3	Dixon et al (2012); Yagoda et al (2007)
	Piperazine erastin	Inhibit SLC7A11	Yang et al (2014)
	Imidazole ketone erastin	Inhibit SLC7A11	Yang et al (2016)
	Sulfasalazine	Inhibit SLC7A11	Dixon et al (2012); Zhang et al (2019)
	Sorafenib	Inhibit SLC7A11	Dixon et al (2014)
	Glutamate	Inhibit SLC7A11	Dixon et al (2012); Stockwell et al (2017)
	Olaparib	p53-dependent downregulation of SLC7A11	Hong et al (2021)
GCL	Buthionine sulfoximine	Inhibit GCL	Xie et al (2017); Yang et al (2014)
GSH	Cystinase	Degrade cysteine, cystine, and GSH	Badgley et al (2020); Cramer et al (2017)
	Cisplatin	Combine GSH to inactivate GPX4	Guo et al (2018); Liu and Wang (2019); Lou et al (2021); Ma et al (2017)
Class II		Direct GPX4 inactivation
GPX4	RSL3	Inhibit GPX4	Geng et al (2018); Yang et al (2014)
	DPI7/ML162, DPI 10/ML210, DPI12-13, DPI17-19	Inhibit GPX4	Yang et al (2014)
	Altretamine	Inhibit GPX4	Woo et al (2015)
	Withaferin A (high dose)	Upregulate HMOX1 and increase LIP, and inhibit GPX4 in high dose	Hassannia et al (2018); Xiong et al (2019)
Class III		Indirect GPX4 inactivation
GPX4 Squalene synthase	FIN56	Promote degradation of GPX4, reduce CoQ10	Shimada et al (2016); Woo et al (2015)
HMGCR	Statins	Decrease synthesis of CoQ10, selenoproteins including GPX4	Jiang et al (2020); Nagpal et al (2019)
	FINO2	Oxidate ferroptosis-relevant substances directly, inactivate GPX4 indirect	Abrams et al (2016); Yang et al (2014)
Class IV		Increase catalytic Fe(II)-dependent lipid peroxidation
	Ferric ammonium citrate	Increase LIP	Fang et al (2018)
	Ferrous ammonium sulfate	Increase LIP	Hassannia et al (2018)
	Heme	Upregulate HO-1 and increase LIP	Patel et al (2019); Sun et al (2016)
	Withaferin A	Upregulate HO-1 and increase LIP	Hassannia et al (2018); Xiong et al (2019)
	BAY 11-7085	Upregulate HO-1 and increase LIP	Chang et al (2018)
	Neratinib	Upregulate HO-1 and increase LIP	Nagpal et al (2019)
	Lapatinib+siramesine	Increase Tf and LIP, decrease FPT1	Ma et al (2016)
	Artesunate	Ferritinophagy	Du et al (2019); Lin et al (2016)
	Salinomycin	Increase IREB2/IRP2 and TFRC along with a rapid degeneration of the iron storage protein	Mai et al (2017)
	Nonthermal plasma	Iron-dependent lipid peroxidation, ferritin degradation	Jiang et al (2021a); Sato et al (2019); Shi et al (2017)
Others
ROS	BAY 87-2243	Induce mitophagy and increase ROS	Basit et al (2017)
FSP1	iFSP1	Inhibit reduction of CoQ10 by FPS1	Bersuker et al (2019); Doll et al (2019)
NrF2	Trigoneline, brusatol	Inhibit NrF2	Arlt et al (2013); Sun et al (2016)

CoQ10, coenzyme Q10; FPT1, ferroportin 1; FSP1, ferroptosis suppressor protein 1; GCL, glutamate-cysteine ligase; GSH, glutathione; HMGCR, HMG-CoA reductase; HO-1, heme oxygenase 1; GPX4, glutathione peroxidase 4; LIP, labile iron pool; NrF2, nuclear factor-erythroid 2-related factor 2; ROS, reactive oxygen species; Tf, transferrin; VDAC, voltage-dependent anion channel.

Among the Class I FINs, erastin is the first drug recognized as a FIN (Dixon et al, 2012). Erastin reduces GSH by inhibition of system Xc⁻ and also inhibits mitochondrial voltage-dependent anion channel (VDAC) (Yagoda et al, 2007). Moreover, it was revealed that the RAF/MEK/ERK signaling pathway is important in erastin-induced ferroptosis in RAS-mutated carcinoma (Xie et al, 2016). Though erastin induces tumoricidal effects in preclinical studies in various cancer cells, actual use for living body is limited due to its poor solubility and instability. Therefore, more soluble and stable derivatives, piperazine erastin and imidazole ketone erastin, have been developed (Yang et al, 2014; Zhang et al, 2019).

Furthermore, among drugs approved by Food and Drug Administration (FDA), sulfasalazine, cisplatin, and sorafenib are recognized as Class I FINs. Sulfasalazine is currently used in the treatment of rheumatoid arthritis, ulcerative colitis, or Crohn's disease as an immunosuppressant, which inhibits System Xc⁻ to induce ferroptosis. However, the effect of sulfasalazine is weaker than erastin (Dixon et al, 2012; Zhang et al, 2019). Cisplatin is a major antitumor drug, which is revealed to work partially as FIN by direct reduction of GSH (Guo et al, 2018; Ma et al, 2017). Sorafenib is a multitarget kinase inhibitor for the treatment of hepatocellular carcinoma, renal cell carcinoma, and thyroid cancer.

Sorafenib inhibits System Xc⁻ as well to induce ferroptosis (Hou et al, 2019; Louandre et al, 2015; Louandre et al, 2013). Moreover, Olaparib, which is a poly (ADP-ribose) polymerase (PARP) inhibitor used for cancers exhibiting homologous recombination disorder, is also known to induce ferroptosis by p53-dependent downregulation of SLC7A11 (Hong et al, 2021).

Class II FINs directly inactivate GPX4 to induce ferroptosis. RSL3 is a well-studied direct inhibitor of GPX4 (Yang et al, 2014). Among the FDA-approved drugs, altretamine, which is an anticancer drug for ovarian cancer, is reported to inhibit GPX4 as Class II FINs (Woo et al, 2015).

Class III FINs are indirect inactivators of GPX4. FIN56 promotes autophagy-mediated degradation of GPX4 to induce ferroptosis (Shimada et al, 2016; Sun et al, 2021). Among the FDA-approved drugs, statins are reported as Class III FINs. Statins are inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (HMGCR), which are commonly used to reduce serum cholesterol levels. Fluvastatin, lovastatin, and simvastatin inhibit HMGCR, leading to a decreased production of CoQ10 and selenoproteins, including GPX4, resulting in ferroptosis (Viswanathan et al, 2017).

Class IV FINs cause lipid peroxidation by iron overload or enhancement of catalytic reaction of iron. Withaferin A activates NRF2 to upregulate HMOX1, leading to degradation of heme and an increase in catalytic Fe(II) (Hassannia et al, 2018). BAY11-7085 also increases intracellular iron via upregulation of HMOX1 (Chang et al, 2018). Siramesine downregulates FPT1, and a combination of lapatinib and siramesine disrupts iron transport to induce ferroptosis by iron overload (Ma et al, 2016).

Nanomaterials-Based Cancer Therapy to Induce Ferroptosis

In addition to traditional drugs, nanoparticle-based drugs have been developed in the past two decades. Materials sized in nanoscales, generally in 1–100 nm at least in one dimension, possess many unique properties such as high surface/volume ratio and characteristic magnetic/electrical properties, which are distinct from micromaterials. For medical use, they are undertaken to design for efficient drug transportation and controlled release (Cheng et al, 2021). With a high surface/volume ratio, nanomaterials are expected to enhance the specificity of targeted therapy by assembling specified biomolecules on surface to specifically recognize target cells, leading to increase efficacy of the drugs with reduced cytotoxicity to untargeted normal cells.

Various forms of nanomaterials, such as nanoparticles, liposomes, solid lipid nanoparticles, nanostructured lipid, nanoemulsions, dendrimers, graphene, and metallic nanoparticles, have been intensively studied for cancer treatments. Such nanomaterials are already demonstrated to induce ferroptosis in cancer cells (Cheng et al, 2021; Zaffaroni and Beretta, 2021). Iron oxide nanoparticles (IONs) (Zanganeh et al, 2016), LOOH-treated IONs (Zhou et al, 2017), amorphous iron nanoparticles (AFeNPs) (Zhang et al, 2016), and FePt nanoparticles (Yue et al, 2017) can induce ferroptosis via iron overload. Polyethylene glycol-coated ultrasmall nanoparticles called Coined Cornel dots (C'dots) were approved by FDA, which induce iron overload by adsorption of extracellular iron and transporting them into the cells (Kim et al, 2016).

Arginine-capped manganese silicate nanobubbles (AMSNs) deplete GSH and inactivate GPX4 (Wang et al, 2018). Low-density lipoprotein nanoparticles, composed of natural ω-3 PUFA and docosahexaenoic acid, induce GSH depletion and accumulation of ROS to induce ferroptosis in liver cells (Ou et al, 2017). Furthermore, some photodynamic drugs have been designed to induce ferroptosis, such as a disulfide-bearing imidazole ligand coordinated with zinc to form an all-active metal organic framework nanocarrier to deplete GSH (Meng et al, 2019).

Nonthermal Plasma Therapy

Recently, nonthermal plasma (NTP; also as low-temperature plasma, LTP) therapy is one of the novel and promising options for cancer treatment at the preclinical stage. Plasma is the fourth state of physical matter, which is ionized gas, consisting basically of free electrons and positive ions (Rehman et al, 2016). Plasma is common across the space. However, states of solid, liquid, or gas are dominant on the Earth for the pressure and the temperature. Even on the Earth, plasma can be produced by supplying energy to gas for ionization, commonly with electrical discharge. Plasma is classified into two groups according to their temperature, “hot,” thermal plasma, and “cold,” NTP/LTP.

Thermal plasma can reach several thousand degrees Celsius, which has been used in manufacturing applications such as in metallurgy, casting, or chemical synthesis. In contrast, NTP (also known as LTP, cold atmospheric plasma, and nonequilibrium atmospheric pressure plasma) is on the temperature close to the ambient temperature, which can be irradiated to the live body. NTP is currently under intensive investigation in medicine, especially in the areas of wound healing, disinfection, endometriosis (Hayashi et al, 2020; Ishida et al, 2016), and cancer treatment (Kajiyama et al, 2017; Laroussi, 2020; Sato et al, 2019).

NTP is formed by ionized gas flow of Ar or He. Various reactive species, including ^•OH, H₂O₂, O₂ ⁻, and NO, are generated from the surrounding atmosphere. Thus, NTP works to load oxidative stress to the irradiated targets. In addition to direct NTP exposure, NTP can be handled in the forms of plasma-activated medium (PAM) or plasma-activated Ringer's lactate (PAL) as indirect strategies. Both direct and indirect NTP exposures induce ferroptosis specifically in cancer cells where abundant catalytic Fe(II) is responsible to induce Fenton reaction and ferroptosis (Furuta et al, 2018; Jiang et al, 2021b; Nakamura et al, 2021; Okazaki et al, 2014; Shi et al, 2017; Shi et al, 2016; Tanaka and Hori, 2017; Tanaka et al, 2015).

Whereas the responsible chemical reactions have not been completely identified yet, degradation of ferritin and simultaneous increase in catalytic Fe(II) and an increase in NO through inducible NO synthase through NF-κB activation and autophagy induction play important roles (Furuta et al, 2018; Jiang et al, 2021a). We would add here that NTP and PAM/PAL may cause apoptosis as well in certain cancers, such as glioblastoma (Tanaka et al, 2019; Utsumi et al, 2016).

This method is promising for the treatment of cancer with chemoresistance because the therapeutic mechanism is completely different from the other drugs and can target higher amounts of catalytic Fe(II) in cancer cells, which is fundamental for cancer proliferation. The limitation of NTP exposure is the short permeation, which can affect only a few millimeters in depth (Okazaki et al, 2014). Thus, it would be more appropriate for cancers on the epidermal or serosal surface, such as skin cancer, oral cancer, peritoneal disseminations, and the margins of operational resection (Fig. 6).

FIG. 6.

NTP as a handy ferroptosis inducer. (A) NTP is generated by electric power through the ionized gas flow of Ar or He, which produces ^•OH, H₂O₂, O⁻, and NO, by the use of the surrounding atmosphere. (B) NTP can be exposed directly or indirectly as PAM or PAL. (C) NTP exposure is able to load oxidative stress to the target sites, which can be applied into multiple medical use, including promotion of wound healing, disinfection, and cancer therapy. NTP, nonthermal plasma; PAL, plasma-activated Ringer's lactate; PAM, plasma-activated medium.

Conclusion

Ferroptosis is a novel form of nonapoptotic regulatory cell death, characterized by catalytic Fe(II)-dependent lipid peroxidation. Ferroptosis is driven by iron overload, incorporation of PUFAs onto membrane PLs via peroxisome-dependent and peroxisome-independent pathway, and is regulated by GPX4, CoQ10, or ESCRT-III.

Ferroptosis induction therapy is anticipated to innovate cancer therapy because ferroptosis induction is ideal to overcome problems to make cancers resistant to chemotherapy, such as EMT and possibly activated Hippo signaling pathway. Therefore, intensive research is in progress worldwide to establish ferroptosis inducing therapeutic methods of distinct modalities. Among the FINs, NTP therapy is one of the promising methods for the unique anticancer effect as a selective handy FIN for catalytic Fe(II)-rich cancer cells.

Footnotes

Authors' Contributions

Y.M. and S.T. conceived, wrote, and organized the article; prepared the figures; and contributed to the discussion.

Author Disclosure Statement

All authors declare no conflict of interest to present.

Funding Information

This study was supported, in part, by JST CREST (Grant Number JPMJCR19H4), JSPS KAKENHI (Grant Numbers JP16H06276 [AdAMS], JP19H05462, and JP20H05502), and Research Grant of the Princess Takamatsu Cancer Research Fund (19-25126).

Abbreviations Used

References

Abrams

, Carroll

, Woerpel

. Five-membered ring peroxide selectively initiates ferroptosis in cancer cells. ACS Chem Biol, 2016; 11(5):1305–1312; doi: 10.1021/acschembio.5b00900.

Alfarouk

, Stock

, Taylor

, et al. Resistance to cancer chemotherapy: Failure in drug response from ADME to P-gp. Cancer Cell Int, 2015; 15:71; doi: 10.1186/s12935-015-0221-1.

Anandhan

, Dodson

, Schmidlin

, et al. Breakdown of an ironclad defense system: The critical role of NRF2 in mediating ferroptosis. Cell Chem Biol, 2020; 27(4):436–447; doi: 10.1016/j.chembiol.2020.03.011.

Andersen

, Johnsen

, Moos

. Iron deposits in the chronically inflamed central nervous system and contributes to neurodegeneration. Cell Mol Life Sci, 2014; 71(9):1607–1622; doi: 10.1007/s00018-013-1509-8.

Andrews

. Disorders of iron metabolism. N Engl J Med, 1999; 341(26):1986–1995; doi: 10.1056/NEJM199912233412607.

Arlt

, Sebens

, Krebs

, et al. Inhibition of the Nrf2 transcription factor by the alkaloid trigonelline renders pancreatic cancer cells more susceptible to apoptosis through decreased proteasomal gene expression and proteasome activity. Oncogene, 2013; 32(40):4825–4835; doi: 10.1038/onc.2012.493.

Badgley

, Kremer

, Maurer

, et al. Cysteine depletion induces pancreatic tumor ferroptosis in mice. Science, 2020; 368(6486):85–89; doi: 10.1126/science.aaw9872.

Baghban

, Roshangar

, Jahanban-Esfahlan

, et al. Tumor microenvironment complexity and therapeutic implications at a glance. Cell Commun Signal, 2020; 18(1):59; doi: 10.1186/s12964-020-0530-4.

Barrera-Rodriguez

. Importance of the Keap1-Nrf2 pathway in NSCLC: Is it a possible biomarker?. Biomed Rep, 2018; 9(5):375–382; doi: 10.3892/br.2018.1143.

10.

Basit

, van Oppen

, Schockel

, et al. Mitochondrial complex I inhibition triggers a mitophagy-dependent ROS increase leading to necroptosis and ferroptosis in melanoma cells. Cell Death Dis, 2017; 8(3):e2716; doi: 10.1038/cddis.2017.133.

11.

Baskar

, Lee

, Yeo

, et al. Cancer and radiation therapy: Current advances and future directions. Int J Med Sci, 2012; 9(3):193–199; doi: 10.7150/ijms.3635.

12.

Bersuker

, Hendricks

, Li

, et al. The CoQ oxidoreductase FSP1 acts parallel to GPX4 to inhibit ferroptosis. Nature, 2019; 575(7784):688–692; doi: 10.1038/s41586-019-1705-2.

13.

, Yang

, Li

, et al. Metadherin enhances vulnerability of cancer cells to ferroptosis. Cell Death Dis, 2019; 10(10):682; doi: 10.1038/s41419-019-1897-2.

14.

Boopathy

GTK

, Hong

. Role of Hippo pathway-YAP/TAZ signaling in angiogenesis. Front Cell Dev Biol, 2019; 7:49; doi: 10.3389/fcell.2019.00049.

15.

Brabletz

, Kalluri

, Nieto

, et al. EMT in cancer. Nat Rev Cancer, 2018; 18(2):128–134; doi: 10.1038/nrc.2017.118.

16.

Brigelius-Flohe

, Maiorino

. Glutathione peroxidases. Biochim Biophys Acta, 2013; 1830(5):3289–3303; doi: 10.1016/j.bbagen.2012.11.020.

17.

Brown

, Amante

, Chhoy

, et al. Prominin2 drives ferroptosis resistance by stimulating iron export. Dev Cell, 2019; 51(5):575.e4–586.e4; doi: 10.1016/j.devcel.2019.10.007.

18.

Brown

, Amante

, Goel

, et al. The alpha6beta4 integrin promotes resistance to ferroptosis. J Cell Biol, 2017; 216(12):4287–4297; doi: 10.1083/jcb.201701136.

19.

Canonne-Hergaux

, Gros

. Expression of the iron transporter DMT1 in kidney from normal and anemic mk mice. Kidney Int, 2002; 62(1):147–156; doi: 10.1046/j.1523-1755.2002.00405.x.

20.

Chang

, Chiang

, Chen

, et al. Heme oxygenase-1 mediates BAY 11-7085 induced ferroptosis. Cancer Lett, 2018; 416:124–137; doi: 10.1016/j.canlet.2017.12.025.

21.

Chen

, Tavana

, Chu

, et al. NRF2 is a major target of ARF in p53-independent tumor suppression. Mol Cell, 2017; 68(1):224.e4–232.e4; doi: 10.1016/j.molcel.2017.09.009.

22.

Cheng

, Li

, Dey

, et al. Nanomaterials for cancer therapy: Current progress and perspectives. J Hematol Oncol, 2021; 14(1):85; doi: 10.1186/s13045-021-01096-0.

23.

Conrad

, Angeli

, Vandenabeele

, et al. Regulated necrosis: Disease relevance and therapeutic opportunities. Nat Rev Drug Discov, 2016; 15(5):348–366; doi: 10.1038/nrd.2015.6.

24.

Cramer

, Saha

, Liu

, et al. Systemic depletion of L-cyst(e)ine with cyst(e)inase increases reactive oxygen species and suppresses tumor growth. Nat Med, 2017; 23(1):120–127; doi: 10.1038/nm.4232.

25.

Daher

, Parks

, Durivault

, et al. Genetic ablation of the cystine transporter xCT in PDAC cells inhibits mTORC1, growth, survival, and tumor formation via nutrient and oxidative stresses. Cancer Res, 2019; 79(15):3877–3890; doi: 10.1158/0008-5472.CAN-18-3855.

26.

Dai

, Han

, Liu

, et al. Autophagy-dependent ferroptosis drives tumor-associated macrophage polarization via release and uptake of oncogenic KRAS protein. Autophagy, 2020a;16(11):2069–2083; doi: 10.1080/15548627.2020.1714209.

27.

Dai

, Meng

, Kang

, et al. ESCRT-III-dependent membrane repair blocks ferroptosis. Biochem Biophys Res Commun, 2020b;522(2):415–421; doi: 10.1016/j.bbrc.2019.11.110.

28.

Dixon

, Lemberg

, Lamprecht

, et al. Ferroptosis: An iron-dependent form of nonapoptotic cell death. Cell, 2012; 149(5):1060–1072; doi: 10.1016/j.cell.2012.03.042.

29.

Dixon

, Patel

, Welsch

, et al. Pharmacological inhibition of cystine-glutamate exchange induces endoplasmic reticulum stress and ferroptosis. Elife, 2014; 3:e02523; doi: 10.7554/eLife.02523.

30.

Doll

, Freitas

, Shah

, et al. FSP1 is a glutathione-independent ferroptosis suppressor. Nature, 2019; 575(7784):693–698; doi: 10.1038/s41586-019-1707-0.

31.

, Wang

, Li

, et al. DHA inhibits proliferation and induces ferroptosis of leukemia cells through autophagy dependent degradation of ferritin. Free Radic Biol Med, 2019; 131:356–369; doi: 10.1016/j.freeradbiomed.2018.12.011.

32.

Dunn

, Suryo Rahmanto

, Richardson

. Iron uptake and metabolism in the new millennium. Trends Cell Biol, 2007; 17(2):93–100; doi: 10.1016/j.tcb.2006.12.003.

33.

Efimova

, Catanzaro

, Van der Meeren

, et al. Vaccination with early ferroptotic cancer cells induces efficient antitumor immunity. J Immunother Cancer, 2020; 8(2):e001369; doi: 10.1136/jitc-2020-001369.

34.

Elmore

. Apoptosis: A review of programmed cell death. Toxicol Pathol, 2007; 35(4):495–516; doi: 10.1080/01926230701320337.

35.

Fan

, Wirth

, Chen

, et al. Nrf2-Keap1 pathway promotes cell proliferation and diminishes ferroptosis. Oncogenesis, 2017; 6(8):e371; doi: 10.1038/oncsis.2017.65.

36.

Fang

, Yu

, Ding

, et al. Effects of intracellular iron overload on cell death and identification of potent cell death inhibitors. Biochem Biophys Res Commun, 2018; 503(1):297–303; doi: 10.1016/j.bbrc.2018.06.019.

37.

Feder

, Gnirke

, Thomas

, et al. A novel MHC class I-like gene is mutated in patients with hereditary haemochromatosis. Nat Genet, 1996; 13(4):399–408; doi: 10.1038/ng0896-399.

38.

Feng

, Stockwell

. Unsolved mysteries: How does lipid peroxidation cause ferroptosis?. PLoS Biol, 2018; 16(5):e2006203; doi: 10.1371/journal.pbio.2006203.

39.

Fillebeen

, Charlebois

, Wagner

, et al. Transferrin receptor 1 controls systemic iron homeostasis by fine-tuning hepcidin expression to hepatocellular iron load. Blood, 2019; 133(4):344–355; doi: 10.1182/blood-2018-05-850404.

40.

Finianos

, Matar

, Taher

. Hepatocellular carcinoma in beta-thalassemia patients: Review of the literature with molecular insight into liver carcinogenesis. Int J Mol Sci, 2018; 19(12):4070; doi: 10.3390/ijms19124070.

41.

Franchini

. Hereditary iron overload: Update on pathophysiology, diagnosis, and treatment. Am J Hematol, 2006; 81(3):202–209; doi: 10.1002/ajh.20493.

42.

Frey

, Nandal

, Park

, et al. Iron chaperones PCBP1 and PCBP2 mediate the metallation of the dinuclear iron enzyme deoxyhypusine hydroxylase. Proc Natl Acad Sci U S A, 2014; 111(22):8031–8036; doi: 10.1073/pnas.1402732111.

43.

Frey

, Reed

. The ubiquity of iron. ACS Chem Biol, 2012; 7(9):1477–1481; doi: 10.1021/cb300323q.

44.

Furukawa

, Yamashita

, Sakurai

, et al. The epithelial circumferential actin belt regulates YAP/TAZ through nucleocytoplasmic shuttling of Merlin. Cell Rep, 2017; 20(6):1435–1447; doi: 10.1016/j.celrep.2017.07.032.

45.

Furuta

, Shi

, Toyokuni

. Non-thermal plasma as a simple ferroptosis inducer in cancer cells: A possible role of ferritin. Pathol Int, 2018; 68(7):442–443; doi: 10.1111/pin.12665.

46.

Galluzzi

, Vitale

, Michels

, et al. Systems biology of cisplatin resistance: Past, present and future. Cell Death Dis, 2014; 5(5):e1257; doi: 10.1038/cddis.2013.428.

47.

Ganz

, Nemeth

. Hepcidin and iron homeostasis. Biochim Biophys Acta, 2012; 1823(9):1434–1443; doi: 10.1016/j.bbamcr.2012.01.014.

48.

Gao

, Kalathur

RKR

, Coto-Llerena

, et al. YAP/TAZ and ATF4 drive resistance to Sorafenib in hepatocellular carcinoma by preventing ferroptosis. EMBO Mol Med, 2021; 13(12):e14351; doi: 10.15252/emmm.202114351.

49.

Geng

, Shi

, Li

, et al. Knockdown of ferroportin accelerates erastin-induced ferroptosis in neuroblastoma cells. Eur Rev Med Pharmacol Sci, 2018; 22(12):3826–3836; doi: 10.26355/eurrev_201806_15267.

50.

Goulev

, Fauny

, Gonzalez-Marti

, et al. SCALLOPED interacts with YORKIE, the nuclear effector of the hippo tumor-suppressor pathway in Drosophila. Curr Biol, 2008; 18(6):435–441; doi: 10.1016/j.cub.2008.02.034.

51.

Gubelmann

, Schwalie

, Raghav

, et al. Identification of the transcription factor ZEB1 as a central component of the adipogenic gene regulatory network. Elife, 2014; 3:e03346; doi: 10.7554/eLife.03346.

52.

Gunshin

, Mackenzie

, Berger

, et al. Cloning and characterization of a mammalian proton-coupled metal-ion transporter. Nature, 1997; 388(6641):482–488; doi: 10.1038/41343.

53.

Guo

, Xu

, Han

, et al. Ferroptosis: A novel anti-tumor action for cisplatin. Cancer Res Treat, 2018; 50(2):445–460; doi: 10.4143/crt.2016.572.

54.

Gutteridge

JMC

, Halliwell

. Mini-review: Oxidative stress, redox stress or redox success?. Biochem Biophys Res Commun, 2018; 502(2):183–186; doi: 10.1016/j.bbrc.2018.05.045.

55.

Hadian

, Stockwell

. SnapShot: Ferroptosis. Cell, 2020; 181(5):1188–1188.e1; doi: 10.1016/j.cell.2020.04.039.

56.

Hangauer

, Viswanathan

, Ryan

, et al. Drug-tolerant persister cancer cells are vulnerable to GPX4 inhibition. Nature, 2017; 551(7679):247–250; doi: 10.1038/nature24297.

57.

Harigae

. Iron metabolism and related diseases: An overview. Int J Hematol, 2018; 107(1):5–6; doi: 10.1007/s12185-017-2384-0.

58.

Hassannia

, Vandenabeele

, Vanden Berghe

. Targeting ferroptosis to iron out cancer. Cancer Cell, 2019; 35(6):830–849; doi: 10.1016/j.ccell.2019.04.002.

59.

Hassannia

, Wiernicki

, Ingold

, et al. Nano-targeted induction of dual ferroptotic mechanisms eradicates high-risk neuroblastoma. J Clin Invest, 2018; 128(8):3341–3355; doi: 10.1172/JCI99032.

60.

Hayashi

, Nakamura

, Motooka

, et al. Novel ovarian endometriosis model causes infertility via iron-mediated oxidative stress in mice. Redox Biol, 2020; 37:101726; doi: 10.1016/j.redox.2020.101726.

61.

Hino

, Yanatori

, Hara

, et al. Iron and liver cancer: An inseparable connection. FEBS J, 2022; 289(24):7810–7829; doi: 10.1111/febs.16208.

62.

Holohan

, Van Schaeybroeck

, Longley

, et al. Cancer drug resistance: An evolving paradigm. Nat Rev Cancer, 2013; 13(10):714–726; doi: 10.1038/nrc3599.

63.

Hong

, Lei

, Chen

, et al. PARP inhibition promotes ferroptosis via repressing SLC7A11 and synergizes with ferroptosis inducers in BRCA-proficient ovarian cancer. Redox Biol, 2021; 42:101928; doi: 10.1016/j.redox.2021.101928.

64.

Hosseinzadeh

, Lu

. Design and fine-tuning redox potentials of metalloproteins involved in electron transfer in bioenergetics. Biochim Biophys Acta, 2016; 1857(5):557–581; doi: 10.1016/j.bbabio.2015.08.006.

65.

Hou

, Xia

, Zhou

, et al. The MEK inhibitors enhance the efficacy of sorafenib against hepatocellular carcinoma cells through reducing p-ERK rebound. Transl Cancer Res, 2019; 8(4):1224–1232; doi: 10.21037/tcr.2019.07.11.

66.

Huang

, Wu

, Barrera

, et al. The Hippo signaling pathway coordinately regulates cell proliferation and apoptosis by inactivating Yorkie, the Drosophila Homolog of YAP. Cell, 2005; 122(3):421–434; doi: 10.1016/j.cell.2005.06.007.

67.

Ishida

, Mori

, Nakamura

, et al. Non-thermal plasma prevents progression of endometriosis in mice. Free Radic Res, 2016; 50(10):1131–1139; doi: 10.1080/10715762.2016.1211273.

68.

Jeong

, Hellyer

, Stehr

, et al. Role of KEAP1/NFE2L2 mutations in the chemotherapeutic response of patients with non-small cell lung cancer. Clin Cancer Res, 2020; 26(1):274–281; doi: 10.1158/1078-0432.CCR-19-1237.

69.

Jiang

, Zheng

, Lyu

, et al. Lysosomal nitric oxide determines transition from autophagy to ferroptosis after exposure to plasma-activated Ringer's lactate. Redox Biol, 2021a;43:101989; doi: 10.1016/j.redox.2021.101989.

70.

Jiang

, Qiao

, Zhao

, et al. Targeting ferroptosis for cancer therapy: Exploring novel strategies from its mechanisms and role in cancers. Transl Lung Cancer Res, 2020; 9(4):1569–1584; doi: 10.21037/tlcr-20-341.

71.

Jiang

, Stockwell

, Conrad

. Ferroptosis: Mechanisms, biology and role in disease. Nat Rev Mol Cell Biol, 2021b;22(4):266–282; doi: 10.1038/s41580-020-00324-8.

72.

Kajiyama

, Suzuki

, Yoshihara

, et al. Endometriosis and cancer. Free Radic Biol Med, 2019; 133:186–192; doi: 10.1016/j.freeradbiomed.2018.12.015.

73.

Kajiyama

, Utsumi

, Nakamura

, et al. Future perspective of strategic non-thermal plasma therapy for cancer treatment. J Clin Biochem Nutr, 2017; 60(1):33–38; doi: 10.3164/jcbn.16-65.

74.

Kautz

, Jung

, Valore

, et al. Identification of erythroferrone as an erythroid regulator of iron metabolism. Nat Genet, 2014; 46(7):678–684; doi: 10.1038/ng.2996.

75.

Kepp

, Zitvogel

, Kroemer

. Clinical evidence that immunogenic cell death sensitizes to PD-1/PD-L1 blockade. Oncoimmunology, 2019; 8(10):e1637188; doi: 10.1080/2162402X.2019.1637188.

76.

Kim

, LaVaute

, Iwai

, et al. Identification of a conserved and functional iron-responsive element in the 5'-untranslated region of mammalian mitochondrial aconitase. J Biol Chem, 1996; 271(39):24226–24230; doi: 10.1074/jbc.271.39.24226.

77.

Kim

, Koh

, Chen

, et al. E-cadherin mediates contact inhibition of proliferation through Hippo signaling-pathway components. Proc Natl Acad Sci U S A, 2011; 108(29):11930–11935; doi: 10.1073/pnas.1103345108.

78.

Kim

, Zhang

, Ma

, et al. Ultrasmall nanoparticles induce ferroptosis in nutrient-deprived cancer cells and suppress tumour growth. Nat Nanotechnol, 2016; 11(11):977–985; doi: 10.1038/nnano.2016.164.

79.

Knutson

. Non-transferrin-bound iron transporters. Free Radic Biol Med, 2019; 133:101–111; doi: 10.1016/j.freeradbiomed.2018.10.413.

80.

Kohgo

, Ikuta

, Ohtake

, et al. Body iron metabolism and pathophysiology of iron overload. Int J Hematol, 2008; 88(1):7–15; doi: 10.1007/s12185-008-0120-5.

81.

Kolbrink

, Riebeling

, Kunzendorf

, et al. Plasma membrane pores drive inflammatory cell death. Front Cell Dev Biol, 2020; 8:817; doi: 10.3389/fcell.2020.00817.

82.

Kowdley

. Iron, hemochromatosis, and hepatocellular carcinoma. Gastroenterology, 2004; 127(5 Suppl 1):S79–S86; doi: 10.1016/j.gastro.2004.09.019.

83.

Lai

, Wei

, Shimizu

, et al. Control of cell proliferation and apoptosis by mob as tumor suppressor, mats. Cell, 2005; 120(5):675–685; doi: 10.1016/j.cell.2004.12.036.

84.

Laroussi

. Cold plasma in medicine and healthcare: The new frontier in low temperature plasma applications. Front Phys, 2020; 8:74; doi: 10.3389/fphy.2020.00074.

85.

Leidgens

, Bullough

, Shi

, et al. Each member of the poly-r(C)-binding protein 1 (PCBP) family exhibits iron chaperone activity toward ferritin. J Biol Chem, 2013; 288(24):17791–17802; doi: 10.1074/jbc.M113.460253.

86.

, Li

. The interaction between ferroptosis and lipid metabolism in cancer. Signal Transduct Target Ther, 2020; 5(1):108; doi: 10.1038/s41392-020-00216-5.

87.

Lin

, Zhang

, Chen

, et al. Dihydroartemisinin (DHA) induces ferroptosis and causes cell cycle arrest in head and neck carcinoma cells. Cancer Lett, 2016; 381(1):165–175; doi: 10.1016/j.canlet.2016.07.033.

88.

Liu

, Kuang

, Kroemer

, et al. Autophagy-dependent ferroptosis: Machinery and regulation. Cell Chem Biol, 2020; 27(4):420–435; doi: 10.1016/j.chembiol.2020.02.005.

89.

Liu

, Wang

. The induction of ferroptosis by impairing STAT3/Nrf2/GPx4 signaling enhances the sensitivity of osteosarcoma cells to cisplatin. Cell Biol Int, 2019; 43(11):1245–1256; doi: 10.1002/cbin.11121.

90.

Logan

, Storey

. Pro-inflammatory AGE-RAGE signaling is activated during arousal from hibernation in ground squirrel adipose. PeerJ, 2018; 6:e4911; doi: 10.7717/peerj.4911.

91.

Lopez-Castro

, Delgado

, Perez-Omil

, et al. A new approach to the ferritin iron core growth: Influence of the H/L ratio on the core shape. Dalton Trans, 2012; 41(4):1320–1324; doi: 10.1039/c1dt11205h.

92.

Lou

, Zhao

, Huang

, et al. Ginkgetin derived from Ginkgo biloba leaves enhances the therapeutic effect of cisplatin via ferroptosis-mediated disruption of the Nrf2/HO-1 axis in EGFR wild-type non-small-cell lung cancer. Phytomedicine, 2021; 80:153370; doi: 10.1016/j.phymed.2020.153370.

93.

Louandre

, Ezzoukhry

, Godin

, et al. Iron-dependent cell death of hepatocellular carcinoma cells exposed to sorafenib. Int J Cancer, 2013; 133(7):1732–1742; doi: 10.1002/ijc.28159.

94.

Louandre

, Marcq

, Bouhlal

, et al. The retinoblastoma (Rb) protein regulates ferroptosis induced by sorafenib in human hepatocellular carcinoma cells. Cancer Lett, 2015; 356(2 Pt B):971–977; doi: 10.1016/j.canlet.2014.11.014.

95.

, Xiao

, Yu

, et al. Enhanced cisplatin chemotherapy by iron oxide nanocarrier-mediated generation of highly toxic reactive oxygen species. Nano Lett, 2017; 17(2):928–937; doi: 10.1021/acs.nanolett.6b04269.

96.

. Role of nrf2 in oxidative stress and toxicity. Annu Rev Pharmacol Toxicol, 2013; 53:401–426; doi: 10.1146/annurev-pharmtox-011112-140320.

97.

, Henson

, Chen

, et al. Ferroptosis is induced following siramesine and lapatinib treatment of breast cancer cells. Cell Death Dis, 2016; 7(7):e2307; doi: 10.1038/cddis.2016.208.

98.

Mai

, Hamai

, Hienzsch

, et al. Salinomycin kills cancer stem cells by sequestering iron in lysosomes. Nat Chem, 2017; 9(10):1025–1033; doi: 10.1038/nchem.2778.

99.

Mancias

, Wang

, Gygi

, et al. Quantitative proteomics identifies NCOA4 as the cargo receptor mediating ferritinophagy. Nature, 2014; 509(7498):105–109; doi: 10.1038/nature13148.

100.

Medina

, Ravichandran

. Do not let death do us part: ‘Find-me’ signals in communication between dying cells and the phagocytes. Cell Death Differ, 2016; 23(6):979–989; doi: 10.1038/cdd.2016.13.

101.

Meng

, Deng

, Liu

, et al. Triggered all-active metal organic framework: Ferroptosis machinery contributes to the apoptotic photodynamic antitumor therapy. Nano Lett, 2019; 19(11):7866–7876; doi: 10.1021/acs.nanolett.9b02904.

102.

Miyanishi

, Tanaka

, Sakamoto

, et al. The role of iron in hepatic inflammation and hepatocellular carcinoma. Free Radic Biol Med, 2019; 133:200–205; doi: 10.1016/j.freeradbiomed.2018.07.006.

103.

Mohammadzadeh

, Halabian

, Gharehbaghian

, et al. Nrf-2 overexpression in mesenchymal stem cells reduces oxidative stress-induced apoptosis and cytotoxicity. Cell Stress Chaperones, 2012; 17(5):553–565; doi: 10.1007/s12192-012-0331-9.

104.

Moya

, Halder

. Hippo-YAP/TAZ signalling in organ regeneration and regenerative medicine. Nat Rev Mol Cell Biol, 2019; 20(4):211–226; doi: 10.1038/s41580-018-0086-y.

105.

Murray

, Briasoulis

, Linardou

, et al. Taxane resistance in breast cancer: Mechanisms, predictive biomarkers and circumvention strategies. Cancer Treat Rev, 2012; 38(7):890–903; doi: 10.1016/j.ctrv.2012.02.011.

106.

Nagpal

, Redvers

, Ling

, et al. Neoadjuvant neratinib promotes ferroptosis and inhibits brain metastasis in a novel syngeneic model of spontaneous HER2(+ve) breast cancer metastasis. Breast Cancer Res, 2019; 21(1):94; doi: 10.1186/s13058-019-1177-1.

107.

Nakamura

, Yoshikawa

, Mizuno

, et al. Preclinical verification of the efficacy and safety of aqueous plasma for ovarian cancer therapy. Cancers (Basel), 2021; 13(5):1141; doi: 10.3390/cancers13051141.

108.

Nandal

, Ruiz

, Subramanian

, et al. Activation of the HIF prolyl hydroxylase by the iron chaperones PCBP1 and PCBP2. Cell Metab, 2011; 14(5):647–657; doi: 10.1016/j.cmet.2011.08.015.

109.

Nemeth

, Tuttle

, Powelson

, et al. Hepcidin regulates cellular iron efflux by binding to ferroportin and inducing its internalization. Science, 2004; 306(5704):2090–2093; doi: 10.1126/science.1104742.

110.

Okada

, Mak

. Pathways of apoptotic and non-apoptotic death in tumour cells. Nat Rev Cancer, 2004; 4(8):592–603; doi: 10.1038/nrc1412.

111.

Okazaki

, Wang

, Tanaka

, et al. Direct exposure of non-equilibrium atmospheric pressure plasma confers simultaneous oxidative and ultraviolet modifications in biomolecules. J Clin Biochem Nutr, 2014; 55(3):207–215; doi: 10.3164/jcbn.14-40.

112.

, Mulik

, Anwar

, et al. Low-density lipoprotein docosahexaenoic acid nanoparticles induce ferroptotic cell death in hepatocellular carcinoma. Free Radic Biol Med, 2017; 112:597–607; doi: 10.1016/j.freeradbiomed.2017.09.002.

113.

Park

, Valore

, Waring

, et al. Hepcidin, a urinary antimicrobial peptide synthesized in the liver. J Biol Chem, 2001; 276(11):7806–7810; doi: 10.1074/jbc.M008922200.

114.

Patel

, Frey

, Palenchar

, et al. A PCBP1-BolA2 chaperone complex delivers iron for cytosolic [2Fe-2S] cluster assembly. Nat Chem Biol, 2019; 15(9):872–881; doi: 10.1038/s41589-019-0330-6.

115.

Pearce

, Templeman

, Rossing

, et al. Association between endometriosis and risk of histological subtypes of ovarian cancer: A pooled analysis of case–control studies. Lancet Oncol, 2012; 13(4):385–394; doi: 10.1016/s1470-s2045(11)70404-1.

116.

Philpott

, Patel

, Protchenko

. Management versus miscues in the cytosolic labile iron pool: The varied functions of iron chaperones. Biochim Biophys Acta Mol Cell Res, 2020; 1867(11):118830; doi: 10.1016/j.bbamcr.2020.118830.

117.

Pigeon

, Ilyin

, Courselaud

, et al. A new mouse liver-specific gene, encoding a protein homologous to human antimicrobial peptide hepcidin, is overexpressed during iron overload. J Biol Chem, 2001; 276(11):7811–7819; doi: 10.1074/jbc.M008923200.

118.

Poetsch

. The genomics of oxidative DNA damage, repair, and resulting mutagenesis. Comput Struct Biotechnol J, 2020; 18:207–219; doi: 10.1016/j.csbj.2019.12.013.

119.

Rajabpour

, Rajaei

, Teimoori-Toolabi

. Molecular alterations contributing to pancreatic cancer chemoresistance. Pancreatology, 2017; 17(2):310–320; doi: 10.1016/j.pan.2016.12.013.

120.

Ramos

, Sadeghi

, Tabatabaeian

. Battling chemoresistance in cancer: Root causes and strategies to uproot them. Int J Mol Sci, 2021; 22(17):9451; doi: 10.3390/ijms22179451.

121.

Rehman

, Jawaid

, Uchiyama

, et al. Comparison of free radicals formation induced by cold atmospheric plasma, ultrasound, and ionizing radiation. Arch Biochem Biophys, 2016; 605:19–25; doi: 10.1016/j.abb.2016.04.005.

122.

Sanchez

, Sabio

, Galvez

, et al. Iron chemistry at the service of life. IUBMB Life, 2017; 69(6):382–388; doi: 10.1002/iub.1602.

123.

Sanchez-Martinez

, Cruz-Gil

, de Cedron

, et al. A link between lipid metabolism and epithelial-mesenchymal transition provides a target for colon cancer therapy. Oncotarget, 2015; 6(36):38719–38736; doi: 10.18632/oncotarget.5340.

124.

Sato

, Shi

, Ito

, et al. Non-thermal plasma specifically kills oral squamous cell carcinoma cells in a catalytic Fe(II)-dependent manner. J Clin Biochem Nutr, 2019; 65(1):8–15; doi: 10.3164/jcbn.18-91.

125.

Schlegelmilch

, Mohseni

, Kirak

, et al. Yap1 acts downstream of alpha-catenin to control epidermal proliferation. Cell, 2011; 144(5):782–795; doi: 10.1016/j.cell.2011.02.031.

126.

Shi

, Bencze

, Stemmler

, et al. A cytosolic iron chaperone that delivers iron to ferritin. Science, 2008; 320(5880):1207–1210; doi: 10.1126/science.1157643.

127.

Shi

, Ito

, Wang

, et al. Non-thermal plasma induces a stress response in mesothelioma cells resulting in increased endocytosis, lysosome biogenesis and autophagy. Free Radic Biol Med, 2017; 108:904–917; doi: 10.1016/j.freeradbiomed.2017.04.368.

128.

Shi

, Wang

, Ito

, et al. Biphasic effects of l-ascorbate on the tumoricidal activity of non-thermal plasma against malignant mesothelioma cells. Arch Biochem Biophys, 2016; 605:109–116; doi: 10.1016/j.abb.2016.05.016.

129.

Shimada

, Skouta

, Kaplan

, et al. Global survey of cell death mechanisms reveals metabolic regulation of ferroptosis. Nat Chem Biol, 2016; 12(7):497–503; doi: 10.1038/nchembio.2079.

130.

Shin

, Kim

, Lee

, et al. Nrf2 inhibition reverses resistance to GPX4 inhibitor-induced ferroptosis in head and neck cancer. Free Radic Biol Med, 2018; 129:454–462; doi: 10.1016/j.freeradbiomed.2018.10.426.

131.

Sladek

, Kollander

, Sreerama

, et al. Cellular levels of aldehyde dehydrogenases (ALDH1A1 and ALDH3A1) as predictors of therapeutic responses to cyclophosphamide-based chemotherapy of breast cancer: A retrospective study. Rational individualization of oxazaphosphorine-based cancer chemotherapeutic regimens. Cancer Chemother Pharmacol, 2002; 49(4):309–321; doi: 10.1007/s00280-001-0412-4.

132.

Smith

, Watson

, O'Kane

, et al. The analysis of doxorubicin resistance in human breast cancer cells using antibody microarrays. Mol Cancer Ther, 2006; 5(8):2115–2120; doi: 10.1158/1535-7163.MCT-06-0190.

133.

Stockwell

, Friedmann Angeli

, Bayir

, et al. Ferroptosis: A regulated cell death nexus linking metabolism, redox biology, and disease. Cell, 2017; 171(2):273–285; doi: 10.1016/j.cell.2017.09.021.

134.

, Yang

, Xie

, et al. Cancer therapy in the necroptosis era. Cell Death Differ, 2016; 23(5):748–756; doi: 10.1038/cdd.2016.8.

135.

Sun

, Ou

, Chen

, et al. Activation of the p62-Keap1-NRF2 pathway protects against ferroptosis in hepatocellular carcinoma cells. Hepatology, 2016; 63(1):173–184; doi: 10.1002/hep.28251.

136.

Sun

, Berleth

, Wu

, et al. Fin56-induced ferroptosis is supported by autophagy-mediated GPX4 degradation and functions synergistically with mTOR inhibition to kill bladder cancer cells. Cell Death Dis, 2021; 12(11):1028; doi: 10.1038/s41419-021-04306-2.

137.

Sun

, Brodsky

. Guardians of the ERAD Galaxy. Cell, 2017; 171(2):267–268; doi: 10.1016/j.cell.2017.09.023.

138.

Sung

, Ferlay

, Siegel

, et al. Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin, 2021; 71(3):209–249; doi: 10.3322/caac.21660.

139.

Swanson

, Dreyfuss

. Classification and purification of proteins of heterogeneous nuclear ribonucleoprotein particles by RNA-binding specificities. Mol Cell Biol, 1988; 8(5):2237–2241; doi: 10.1128/mcb.8.5.2237-2241.1988.

140.

Takahashi

, Cho

, Selfors

, et al. 3D culture models with CRISPR screens reveal hyperactive NRF2 as a prerequisite for spheroid formation via regulation of proliferation and ferroptosis. Mol Cell, 2020; 80(5):828.e6–844.e6; doi: 10.1016/j.molcel.2020.10.010.

141.

Tanaka

, Hori

. Medical applications of non-thermal atmospheric pressure plasma. J Clin Biochem Nutr, 2017; 60(1):29–32; doi: 10.3164/jcbn.16-67.

142.

Tanaka

, Mizuno

, Katsumata

, et al. Oxidative stress-dependent and -independent death of glioblastoma cells induced by non-thermal plasma-exposed solutions. Sci Rep, 2019; 9(1):13657; doi: 10.1038/s41598-019-50136-w.

143.

Tanaka

, Mizuno

, Toyokuni

, et al. Cancer therapy using non-thermal atmospheric pressure plasma with ultra-high electron density. Phys Plasmas, 2015; 22:122004; doi: 10.1063/1.4933402.

144.

Tang

, Kroemer

. Ferroptosis. Curr Biol, 2020a;30(21):R1292–R1297; doi: 10.1016/j.cub.2020.09.068.

145.

Tang

, Kroemer

. Peroxisome: The new player in ferroptosis. Signal Transduct Target Ther, 2020b;5(1):273; doi: 10.1038/s41392-020-00404-3.

146.

Tao

, Zhang

, Turenchalk

, et al. Human homologue of the Drosophila melanogaster lats tumour suppressor modulates CDC2 activity. Nat Genet, 1999; 21(2):177–181; doi: 10.1038/5960.

147.

Toyokuni

. Iron and carcinogenesis: From Fenton reaction to target genes. Redox Rep, 2002; 7(4):189–197; doi: 10.1179/135100002125000596.

148.

Toyokuni

. Oxidative stress as an iceberg in carcinogenesis and cancer biology. Arch Biochem Biophys, 2016; 595:46–49; doi: 10.1016/j.abb.2015.11.025.

149.

Toyokuni

. Iron addiction with ferroptosis-resistance in asbestos-induced mesothelial carcinogenesis: Toward the era of mesothelioma prevention. Free Radic Biol Med, 2019; 133:206–215; doi: 10.1016/j.freeradbiomed.2018.10.401.

150.

Toyokuni

, Kong

, Cheng

, et al. Carcinogenesis as side effects of iron and oxygen utilization: From the unveiled truth toward ultimate bioengineering. Cancers (Basel), 2020; 12(11):3320; doi: 10.3390/cancers12113320.

151.

Turubanova

, Balalaeva

, Mishchenko

, et al. Immunogenic cell death induced by a new photodynamic therapy based on photosens and photodithazine. J Immunother Cancer, 2019; 7(1):350; doi: 10.1186/s40425-019-0826-3.

152.

Ursini

, Maiorino

, Valente

, et al. Purification from pig-liver of a protein which protects liposomes and biomembranes from peroxidative degradation and exhibits glutathione-peroxidase activity on phosphatidylcholine hydroperoxides. Biochim Biophys Acta, 1982; 710(2):197–211; doi: 10.1016/0005-2760(82)90150-3.

153.

Utsumi

, Kajiyama

, Nakamura

, et al. Variable susceptibility of ovarian cancer cells to non-thermal plasma-activated medium. Oncol Rep, 2016; 35(6):3169–3177; doi: 10.3892/or.2016.4726.

154.

Vanden Berghe

, Linkermann

, Jouan-Lanhouet

, et al. Regulated necrosis: The expanding network of non-apoptotic cell death pathways. Nat Rev Mol Cell Biol, 2014; 15(2):135–147; doi: 10.1038/nrm3737.

155.

Viswanathan

, Ryan

, Dhruv

, et al. Dependency of a therapy-resistant state of cancer cells on a lipid peroxidase pathway. Nature, 2017; 547(7664):453–457; doi: 10.1038/nature23007.

156.

Wang

, DuBois

. Immunosuppression associated with chronic inflammation in the tumor microenvironment. Carcinogenesis, 2015; 36(10):1085–1093; doi: 10.1093/carcin/bgv123.

157.

Wang

, Fillebeen

, Chen

, et al. Iron-dependent degradation of apo-IRP1 by the ubiquitin-proteasome pathway. Mol Cell Biol, 2007; 27(7):2423–2430; doi: 10.1128/MCB.01111-06.

158.

Wang

, Li

, Qiao

, et al. Arginine-rich manganese silicate nanobubbles as a ferroptosis-inducing agent for tumor-targeted theranostics. ACS Nano, 2018; 12(12):12380–12392; doi: 10.1021/acsnano.8b06399.

159.

Wang

, Green

, Choi

, et al. CD8(+) T cells regulate tumour ferroptosis during cancer immunotherapy. Nature, 2019a;569(7755):270–274; doi: 10.1038/s41586-019-1170-y.

160.

Wang

, Zhang

, Chen

. Drug resistance and combating drug resistance in cancer. Cancer Drug Resist, 2019b;2(2):141–160; doi: 10.20517/cdr.2019.10.

161.

Wen

, Liu

, Kang

, et al. The release and activity of HMGB1 in ferroptosis. Biochem Biophys Res Commun, 2019; 510(2):278–283; doi: 10.1016/j.bbrc.2019.01.090.

162.

Woo

, Shimoni

, Yang

, et al. Elucidating compound mechanism of action by network perturbation analysis. Cell, 2015; 162(2):441–451; doi: 10.1016/j.cell.2015.05.056.

163.

, Minikes

, Gao

, et al. Intercellular interaction dictates cancer cell ferroptosis via NF2-YAP signalling. Nature, 2019; 572(7769):402–406; doi: 10.1038/s41586-019-1426-6.

164.

Xie

, Hou

, Song

, et al. Ferroptosis: Process and function. Cell Death Differ, 2016; 23(3):369–379; doi: 10.1038/cdd.2015.158.

165.

Xie

, Zhu

, Song

, et al. The tumor suppressor p53 limits ferroptosis by blocking DPP4 activity. Cell Rep, 2017; 20(7):1692–1704; doi: 10.1016/j.celrep.2017.07.055.

166.

Xiong

, Wang

, et al. Intracellular cascade activated nanosystem for improving ER+ breast cancer therapy through attacking GSH-mediated metabolic vulnerability. J Control Release, 2019; 309:145–157; doi: 10.1016/j.jconrel.2019.07.029.

167.

Yagoda

, von Rechenberg

, Zaganjor

, et al. RAS-RAF-MEK-dependent oxidative cell death involving voltage-dependent anion channels. Nature, 2007; 447(7146):864–868; doi: 10.1038/nature05859.

168.

Yamaguchi

, Mandai

, Toyokuni

, et al. Contents of endometriotic cysts, especially the high concentration of free iron, are a possible cause of carcinogenesis in the cysts through the iron-induced persistent oxidative stress. Clin Cancer Res, 2008; 14(1):32–40; doi: 10.1158/1078-0432.CCR-07-1614.

169.

Yanatori

, Richardson

, Dhekne

, et al. CD63 is regulated by iron via the IRE-IRP system and is important for ferritin secretion by extracellular vesicles. Blood, 2021; 138(16):1490–1503; doi: 10.1182/blood.2021010995.

170.

Yanatori

, Richardson

, Imada

, et al. Iron export through the transporter ferroportin 1 is modulated by the iron chaperone PCBP2. J Biol Chem, 2016; 291(33):17303–17318; doi: 10.1074/jbc.M116.721936.

171.

Yanatori

, Richardson

, Toyokuni

, et al. The iron chaperone poly(rC)-binding protein 2 forms a metabolon with the heme oxygenase 1/cytochrome P450 reductase complex for heme catabolism and iron transfer. J Biol Chem, 2017; 292(32):13205–13229; doi: 10.1074/jbc.M117.776021.

172.

Yanatori

, Richardson

, Toyokuni

, et al. The new role of poly (rC)-binding proteins as iron transport chaperones: Proteins that could couple with inter-organelle interactions to safely traffic iron. Biochim Biophys Acta Gen Subj, 2020; 1864(11):129685; doi: 10.1016/j.bbagen.2020.129685.

173.

Yanatori

, Yasui

, Tabuchi

, et al. Chaperone protein involved in transmembrane transport of iron. Biochem J, 2014; 462(1):25–37; doi: 10.1042/BJ20140225.

174.

Yang

, Chi

. Hippo pathway effectors YAP/TAZ as novel determinants of ferroptosis. Mol Cell Oncol, 2020; 7(1):1699375; doi: 10.1080/23723556.2019.1699375.

175.

Yang

, Ding

, Sun

, et al. The Hippo pathway effector TAZ regulates ferroptosis in renal cell carcinoma. Cell Rep, 2019; 28(10):2501.e4–2508.e4; doi: 10.1016/j.celrep.2019.07.107.

176.

Yang

, Huang

, Wu

, et al. A TAZ-ANGPTL4-NOX2 axis regulates ferroptotic cell death and chemoresistance in epithelial ovarian cancer. Mol Cancer Res, 2020; 18(1):79–90; doi: 10.1158/1541-7786.MCR-19-0691.

177.

Yang

, Kim

, Gaschler

, et al. Peroxidation of polyunsaturated fatty acids by lipoxygenases drives ferroptosis. Proc Natl Acad Sci U S A, 2016; 113(34):E4966–E4975; doi: 10.1073/pnas.1603244113.

178.

Yang

, SriRamaratnam

, Welsch

, et al. Regulation of ferroptotic cancer cell death by GPX4. Cell, 2014; 156(1–2):317–331; doi: 10.1016/j.cell.2013.12.010.

179.

Yang

, Stockwell

. Ferroptosis: Death by lipid peroxidation. Trends Cell Biol, 2016; 26(3):165–176; doi: 10.1016/j.tcb.2015.10.014.

180.

, Choi

, Li

, et al. Magnetic field boosted ferroptosis-like cell death and responsive MRI using hybrid vesicles for cancer immunotherapy. Nat Commun, 2020; 11(1):3637; doi: 10.1038/s41467-020-17380-5.

181.

Yue

, Wang

, Dai

, et al. pH-Responsive, self-sacrificial nanotheranostic agent for potential in vivo and in vitro dual modal MRI/CT imaging, real-time, and in situ monitoring of cancer therapy. Bioconjug Chem, 2017; 28(2):400–409; doi: 10.1021/acs.bioconjchem.6b00562.

182.

Zaffaroni

, Beretta

. Nanoparticles for ferroptosis therapy in cancer. Pharmaceutics, 2021; 13(11):1785; doi: 10.3390/pharmaceutics13111785.

183.

Zanganeh

, Hutter

, Spitler

, et al. Iron oxide nanoparticles inhibit tumour growth by inducing pro-inflammatory macrophage polarization in tumour tissues. Nat Nanotechnol, 2016; 11(11):986–994; doi: 10.1038/nnano.2016.168.

184.

Zhang

, Bu

, Ni

, et al. Synthesis of iron nanometallic glasses and their application in cancer therapy by a localized fenton reaction. Angew Chem Int Ed Engl, 2016; 55(6):2101–2106; doi: 10.1002/anie.201510031.

185.

Zhang

, Tan

, Daniels

, et al. Imidazole ketone erastin induces ferroptosis and slows tumor growth in a mouse lymphoma model. Cell Chem Biol, 2019; 26(5):623.e9–633.e9; doi: 10.1016/j.chembiol.2019.01.008.

186.

Zhao

, Tumaneng

, Guan

. The Hippo pathway in organ size control, tissue regeneration and stem cell self-renewal. Nat Cell Biol, 2011; 13(8):877–883; doi: 10.1038/ncb2303.

187.

Zheng

, Jiang

, Tsuduki

, et al. Embryonal erythropoiesis and aging exploit ferroptosis. Redox Biol, 2021; 48:102175; doi: 10.1016/j.redox.2021.102175.

188.

Zhou

, Song

, Tian

, et al. Activatable singlet oxygen generation from lipid hydroperoxide nanoparticles for cancer therapy. Angew Chem Int Ed Engl, 2017; 56(23):6492–6496; doi: 10.1002/anie.201701181.

189.

Zou

, Henry

, Ricq

, et al. Plasticity of ether lipids promotes ferroptosis susceptibility and evasion. Nature, 2020a;585(7826):603–608; doi: 10.1038/s41586-020-2732-8.

190.

Zou

, Li

, Graham

, et al. Cytochrome P450 oxidoreductase contributes to phospholipid peroxidation in ferroptosis. Nat Chem Biol, 2020b;16(3):302–309; doi: 10.1038/s41589-020-0472-6.