Introduction
In 2012, Dixony et al. proposed a type of unique programmed cell death type, ferroptosis, which is mainly characterized by high intracellular lipid peroxidation and iron disorders.1 Morphologically, ferroptosis is mainly manifested by decreased mitochondrial volume or disappeared mitochondrial crest, increased mitochondrial membrane potential and membrane density.2 In terms of biochemistry, it is mainly involved the increase of reactive oxygen species (ROS), decreased activity of glutathione peroxidase 4 (GPX4), iron disorder and accumulation of lipid peroxidation.3 Ferroptosis is a newly discovered programmed cell death, many signaling pathways and proteins involved in ferroptosis are continuously discovered and reported (Fig. 1). It is well known that Solute Carrier Family 7 Member 11 (SLC7A11)/GPX4 inhibits ferroptosis by eliminating the ROS and lipid peroxidation. While iron metabolism and lipid metabolism participate in ferroptosis by supplying ferrous iron and unsaturated lipid.4,5 In addition, ferroptosis can also be regulated by transsulfuration pathway and mevalonate pathway through regulating cysteine level and coenzyme Q10 (CoQ10) content, respectively.6,7
Ferroptosis is associated with various diseases, including (neuro)degenerative diseases, ischemia-reperfusion injury, acute kidney injury, and cancer.8 Evading ferroptosis is one of manners that tumor cells bypass cell death, inhibiting different key molecules of ferroptosis can re-sensitive tumor cells to ferroptosis. In addition, the higher the level of ferroptosis in cancer patients receiving radiotherapy, the better the radiation response and the longer the survival of patient.9 Evading ferroptosis plays key role in resistance of cancer to chemotherapy or radiotherapy, inducing ferroptosis might overcome chemotherapy or radiotherapy resistance.10 Recently reported ferroptosis inducers can be divided into four categories according to their mechanisms. The first and second categories are the ferroptosis inducers that directly targeting SLC7A11 and GPX4, respectively.11–13 The third type of ferroptosis inducers is ferroptosis inducing 56 (FIN56) inducers that consume CoQ10 by activating squalene synthase, promoting cellular lipid peroxidation, and ultimately inducing ferroptosis.7,12 The fourth type is 1,2-dioxolane FINO2 inducers that indirectly inhibit GPX4 and directly oxidize iron to induce lipid peroxidation.14
Ferroptosis signaling pathways
SLC7A11/GPX4 axis
SLC7A11, also known as xCT, together with heavy chain subunit Solute Carrier Family 3 Member 2 (SLC3A2) form cystine-glutamate acid reverse transporter (System Xc-), mediating the exchange of intracellular glutamate with extracellular cystine at 1:1. Once transported into the cell, cystine is rapidly converted into cysteine (L-cysteine), which is then utilized to synthesize glutathione (GSH).15 In the heterodimer of System Xc-, SLC7A11 executes the function of transporting amino acids, while SLC3A2 is required for the stability and trafficking of SLC7A11 to the cell membrane.16 mTORC2, as a growth factor integrating variety of signaling pathway, phosphorylates the 26th serine of SLC7A11 to reduce its activity, thereby inhibiting cystine intake and glutathione metabolism.17 The regulation of SLC7A11 has been well reviewed by Boyi Gan group.18 GPX4 plays a pivotal role in ferroptosis by reducing lipid hydroperoxides to lipid alcohols.19 GPX4, as a selenase, requires the participation of GSH acts as a cofactor.20,21 Inhibition of SLC7A11 or GPX4 activity can induce the production of lipid ROS, which in turn induces ferroptosis.19,22
Transsulfuration pathway
Transsulfuration pathway, as the backup supply of cysteine, plays important role in the growth of cancer cells. Transsulfuration pathway negatively regulates ferroptosis by de novo synthesizing cysteine (Fig. 2), which is a rate-limiting precursor for the synthesis of GSH.23 Intracellular cysteine is indirectly supplied with cystine by System Xc- or directly produced by the transsulfuration pathway.15 Once intracellular cysteine is deficient, the cell will initiate transsulfuration pathway to synthesize cysteine from methionine.6 In transsulfuration pathway, methionine reacts with ATP to form S-adenosine methionine (SAM) under the catalysis of methionine adenosine transferase Iα/IIα (MAT1A/MAT2A).24,25 Then SAM is converted to s-adenosine homocysteine (SAH) by losing a methyl group in the presence of methyltransferase (MTs).25 SAH is then hydrolyzed to form homocysteine in the presence of adenosine homocysteine 1 (AHCYL1) or S-adenosine homocysteine hydrolase (SAHH).25,26 Finally, Under the action of the enzyme cystathionine-β-synthase (CBS), serine is coupled into homocysteine to form cystathionine, which can then be cleaved by cystathionine c-lyase (CTH) to release cysteine.6,27
Several investigations have reported that upregulation of transsulfuration pathway prevented ferroptosis induced by SLC7A11 inhibition. Knockdown of cysteinyl- tRNA synthetase (CARS) could upregulate the transsulfuration pathway by accumulation of cystathionine, thus suppressing erastin induced ferroptosis.28 It has been reported that transformation of SAM to SAH determined the transsulfuration pathway activity, inducible of which contributed to cellular cysteine pool and promoted cancer cell growth upon cysteine restriction.27 Depletion of parkinsonism-associated deglycase DJ-1, an oncogene located at 1P36.12-13 of human genome, reduced the activity of SAHH, thus leading to inhibition of transsulfuration pathway, enhancing the sensitivity of cancer cells to SLC7A11 inhibitor.29,30
Iron metabolism
Iron is an indispensable trace element in the human body and is widely involved in metabolism and biology process as a cofactor or component of the enzymes.31 Excess irons will damage cells by catalyzing Fenton reaction, thus inducing ferroptosis.32–35 Normally, absorbed irons in blood vessel are ferric irons (Fe3+) bound by transferrin (Tf). Tf containing one or two ferric ions binds to transferrin receptor 1 (TfR1), forming a complex that enters the cell via receptor-mediated endocytosis and then enters the non-lysosomal acid compartment.36 Ferric ions are then released from the transferrin complex by endosomal acidification and reduced to ferrous ions (Fe2+) by six-transmembrane epithelial antigen of prostate 3 (STEAP3), Tf will separate from its receptor TfR1 and re-enter the bloodstream to capture ferric ions.37,38 Meanwhile, divalent metal transporter protein 1 (DMT1) accounts for the transfer of ferrous ions to the labile iron pool, in which ferrous ions are easily transported to different cell machinery to participate in the synthesis of DNA synthase, heme and iron-sulfur cluster enzymes.38,39 The excess ferrous ions are then stored in ferritin as the type of ferric irons.39 Nuclear receptor coactivator 4 (NCOA4), as the selective cargo receptor of ferritin, participating in the autophagy of ferritin, thus releasing the stored ferrous irons.40 Ferroportin 1 (FPN1) is a transmembrane protein responsible for iron output and iron balance in cells, degradation of which is regulated by hepcidin, a circulatory regulatory hormone peptide produced by liver cells.41,42
Accumulation of ferrous irons in tumor cells affected by various factors will induce ferroptosis by participating in the production of lipid ROS. Knock-down of NCOA4 could abolish degradation of ferritin and reduce the intracellular Fe2+ level, thus inhibiting ferroptosis induced by erastin.43 New studies have shown that upregulated FPN1 can also inhibit ferroptosis, while silencing or downregulating FPN1 enhances the sensitivity of tumor cells to ferroptosis inducer.44,45
IRE (iron response element) /IRP (iron regulatory protein) axis plays critical role in maintaining iron homeostasis through posttranscriptional regulation of iron-related genes. IRP1 and IRP2 specifically recognize the IREs and regulate cell iron homeostasis by blocking or promoting the translation of target mRNAs bound by IRE, which contains conserved stem-loop.46,47 Binding of IREs with IRPs is well regulated by iron concentration.48 When intracellular irons are deficient, IRP1 binds to the IRE of TfR1 and ferritin, thus increasing the iron uptake and reducing iron storage.49 On the contrary, high iron content in cells will reduce iron intake and increase iron storage by releasing of IRP1/2 from IRE of TfR1 and ferritin.38,50 It has been found dihydroartemisinin made cells sensitive to ferroptosis by impinging IRP-IRE system and lysosomal mediated ferritin degradation, both of which increased the intracellular free iron content.51
Lipid metabolism pathway
Lipid peroxidation is an important manifestation of ferroptosis and unsaturated fatty acids are the substrates of lipid peroxidation, thus disorder of lipid metabolism is closely related with ferroptosis. Identifying the lipid substances and pivotal enzymes for lipid peroxidation will supply more information for ferroptosis. Among the various lipids, phospholipid-related polyunsaturated fatty acids (PUFAs) such as phosphatidylethanolamine (PE) and phosphatidylcholine (PC) play key role in ferroptosis as the substrates for lipid peroxidation.52 Genome-wide CRISPR-based genetic screen uncovers that Acyl-CoA synthase long chain member 4 (ACSL4) is essential for ferroptosis execution.53 ACSL4 connects CoA to arachidonic acid (AA) and adrenal acid (AdA) to form fatty acyl-coA esters.53 Then, under the catalysis of lysophosphatidylcholine transferase 3 (LPCAT3), AA-CoA and AdA-CoA react with PC or PE to form PE-AA and PE-AdA.54 Lipoxygenases (LOXs) are dioxygenases that catalyze the peroxidation of PUFAs to produce various lipid peroxides and bioactive lipids in the presence of molecular oxygen.55,56 Genetic or pharmacological inhibition of ACSL4 inhibited ferroptosis by preventing the esterification of AA or AdA into PE, which was the only lipid oxidized in endoplasmic-reticulum-associated compartments during ferroptosis.57 Arachidonate 12-Lipoxygenase (ALOX12) was indispensable for p53 induced ferroptosis and dispensable for erastin induced ferroptosis.58 In addition, Genome-wide CRISPR/Cas9-mediated screen uncovered that cytochrome P450 oxidoreductase promoted ferroptosis across a wide range of lineages and cell-states by peroxidation of membrane polyunsaturated phospholipids.59 The relationship between ferroptosis and lipid metabolism is complex, and its specific mechanism still needs to be further explored.
Mevalonate pathway
The mevalonate pathway occurs in the cytoplasm, where Acetoacetyl-CoA thiolase (AACT) produces acetoacetyl-CoA by condensation of two units of acetyl-coA, and then 3-hydroxy-3-methylglutaryl CoA synthase (HMGS) condensates acetoacetyl-CoA and acetyl-CoA to form 3-hydroxy-3-methylglutaryl CoA (HMG-CoA), which is then catalyzed by hydroxymethylglutaryl-CoA reductase (HMGR) to generate mevalonic acid (MVA).60 MVA is then phosphorylated by MVA kinase and converted to isopentenyl pyrophosphate (IPP), which contributes to the synthesis of farnesyl pyrophosphate (FPP). FPP is converted to squalene, and finally squalene is converted to cholesterol.61 In addition, FPP can also participate in the production of CoQ10.62 Thus, the mevalonate pathway finally leads to the production of CoQ10, cholesterol, and IPP (Fig. 3).7,52,63 CoQ10 is a fat-soluble antioxidant with the function of scavenging oxygen free radicals, protecting lipids in golgi and plasma membranes from oxidation.7,64 Ferroptosis suppressor protein 1 (FSP1) with myristic acylation is recruited to the plasma membrane to reduce CoQ10 by using NAD(P)H, preventing the reproduction of lipid peroxides, thus inhibiting ferroptosis.65–68 FSP1-CoQ10-NAD(P)H pathway is the stand-alone parallel system with GSH-GPX4 pathway to inhibit phospholipid peroxidation and ferroptosis.69
In addition, the mevalonate pathway participates in ferroptosis by supplying IPP for production of selenoproteins, including GPX4. As an intermediate product of the mevalonate pathway, IPP is required for isoprenylation of selenocysteine tRNA, which is indispensable for synthesizing selenoprotein GPX4.70 HMGR inhibitor statins reduced GPX4 expression in cancer cells, leading to elevated lipid peroxides and making cancer cells sensitive to ferroptosis.71
p53
p53 is a well-known tumor suppressor that induces cell cycle arrest and cell apoptosis. Recently, it has been reported that p53 induces ferroptosis through pleiotropic effect.72 Mutant p533KR, which is defective for the conventional p53 functions, still be able to repress SLC7A11 and induce ferroptosis upon ROS-induced stress.73,74 In addition, p53 induced ferroptosis required enzymatic activity of ALOX12, which is inhibited by SLC7A11, but independent of GPX4.58 Recently, scientists found that p53-driven ferroptosis under ROS stress can also suppressed by calcium-independent phospholipase iPLA2β, which mediated detoxification of peroxidized lipids.75
Moreover, p53 induced ferroptosis by directly activating its target gene spermidine/spermine N1-acetyltransferase 1 (SAT1), which induced lipid peroxidation.76 p53 suppresses tumor through regulating its target gene, glutamine synthase 2 (GLS2), which exerts antioxidant defense function.77 Another article reported that GLS2 knockdown inhibits ferroptosis in mouse embryonic fibroblasts.78 Therefore, whether p53 induces ferroptosis by promoting GLS2 expression is worth further investigation.
However, wild-type p53 could delay induction of ferroptosis by regulating its transcriptional target CDKN1A (encoding p21).79 p53 can also inhibit ferroptosis in a transcription-independent manner. For example, the deletion of p53 prevents the nuclear accumulation of dipeptidyl peptidase-4 (DPP4), thus facilitates plasma-membrane-associated DPP4-dependent lipid peroxidation, which finally results in ferroptosis.80
Targeting ferroptosis in cancer
In recent years, more and more researchers are committed to discovering the development process of cancer, and found that ferroptosis is closely related to the occurrence and development of cancers. The ferroptosis inducers are attracting increasing attention in cancer treatment. In addition, ferroptosis inducers can enhance the effect of chemotherapy by inducing ferroptosis and reducing chemotherapy resistance, thereby improving the treatment effect. In this part, we elucidate the rational of targeting ferroptosis and the reported ferroptosis inducers (Table 1)1,7,13,22,81–115.
Table 1Ferroptosis inducers
| Ferroptosis inducers | Target | Basic mechanism | Tumor type | Reference |
|---|
| Erastin, IKE, Sorafenib, Sulfasalazine, 6-Thioguanine | SLC7A11 | Inhibit System Xc- | Fibrosarcoma, Colorectal cancer, Diffuse large B cell lymphoma, Astrocytoma, Lung carcinoma cancer, Lymphoma, Non-Hodgkin’s lymphoma,Gastric cancer | 1,86,22,82,83,84 |
| Tirapazamine, T-2 Toxin,Tanshinone IIA, Talaroconvolutin A | SLC7A11 | Inhibit the expression of SLC7A11 | Osteosarcoma, Lung cancer,Gastric cancer,Colorectal cancer | 85,86,88,89 |
| Metformin | SLC7A11 | Reduces the protein stability of SLC7A11 | Breast cancer | 87 |
| Actinidia chinensis Planch, Capsaicin | SLC7A11 | Inhibit the expression of SLC7A11 and GPX4 | Gastric cancer, Non-small cell lung cancer | 90,91 |
| RSL3, QD394, Compound 26a, Honokiol, FINO2, | GPX4 | Inhibit GPX4 enzymatic activity | Renal carcinoma, Colon cancer, B-lymphoblastic cell leukemia line, Pancreatic cancer, Breast cancer, Breast adenocarcinoma, Fibrosarcoma | 13,92,93,103,110 |
| Jiyuan oridonin A derivative a2 Compound 21, Tetrahydro citrate, Bufortaline, Dihydroisotanshinone I Solasonine, Cucurbitin B, Gambogenic acid, Thiostrepton, Red ginseng polysaccharide, Apatinib, Simvastatin, Atorvastatin | GPX4 | Downregulate GPX4 expression | Gastric cancer, Hepatocellular carcinoma, Nasopharyngeal carcinoma, Melanoma, Human cardiomyocytes, Murine skeletal muscle cells, Breast cancer, Non-small cell lung cancer,
Lung cancer, Gastric cancer, Triple-negative breast cancer, Pancreatic cancer | 94–109,102,104–109 |
| DMOCPTL, FIN56 | GPX4 | Promote degradation of GPX4, | Triple-negative breast cancer, Fibrosarcoma | 7,101 |
| Curcumin analogues EF24, Cadmium | HMOX1 | Directly up-regulating HMOX1 expression | Osteosarcoma, Hepatic stellate cells | 111,112 |
| Artesunate, Saponin Formosanin C | Ferritin | Promote ferritin phagocytosis | Hepatic stellate cells, Hepatocellular carcinoma | 113,114 |
| Baicalin | Ferritin | Down-regulate ferritin | Bladder cancer | 115 |
SLC7A11 inhibitors
SLC7A11 mediates uptake of extracellular cystine, which guarantees glutathione synthesis and maintains GPX4 enzyme activity to inhibit ferroptosis.15,20 SLC7A11 overexpression has been observed in hepatocellular carcinoma and is associated with poor prognosis.116,117 SLC7A11 is regulated by multiple enzymes and pathways. It has been reported that mutant p53 fail to induce cell cycle arrest and cell apoptosis, but still be able to repress SLC7A11 and induce ferroptosis upon ROS-induced stress.73,74 Tumor suppressor BRCA1-associated protein 1 (BAP1) frequently mutated or deleted in sporadic human cancers, inactivation of BAP1 inhibited ferroptosis by releasing SLC7A11 inhibition, thus BAP1 mutated cancer cells were sensitive to SLC7A11 inhibitor erastin.118 OTUB1 is overexpressed in a variety of human cancers, and it can inhibit ferroptosis by stabilizing SLC7A11.119 In short, SLC7A11 is an important target for the treatment of cancer and more and more SLC7A11 inhibitors have been discovered and reported.
In 2012, researchers firstly proposed that erastin induced ferroptosis by inhibiting System Xc- activity.1 Later, more reports proved that erastin induce ferroptosis by inhibiting SLC7A11 activity in variety of cancer cells.81,120 Given the fact that poor water solubility and unstability of erastin, a metabolically stable erastin analogue, imidazole ketone erastin (IKE), has been developed.22,82 Sorafenib, as a multi-target kinase inhibitor, is clinically used for the treatment of unresectable hepatocellular carcinoma.121 In 2014, it was firstly reported that sorafenib can induce ferroptosis by inhibiting System Xc-.122 However, it was recently reported that Sorafenib could not induce ferroptosis by inhibiting System Xc-, thus it might not be a true ferroptosis inducer.123 Sulfasalazine is clinically used for treating ulcerative colitis.124 Recent study found that sulfasalazine is also a novel potent inhibitor of the System Xc-.83 However, all SLC7A11 inhibitors currently identified, including sulfasalazine and erastin, have off-target effects, which limits their clinical use as SLC7A11 specific inhibitors.125
In addition, a variety of compounds have been reported to be able to indirectly inhibit SLC7A11, such as the newly discovered small molecule compound 6-Thioguanine,84 the drug Tirapazamine,85 and T-2 Toxin.86 The oral anti-diabetic drug metformin can also reduce the stability of SLC7A11 by inhibiting UFMylation of SLC7A11, and its combination with sulfasalazine can synergistically induce ferroptosis in breast cancer cells.87 Some natural products, including Talaroconvolutin A,88 Tanshinone IIA,89 Actinidia chinensis Planch and Capsaicin,90,91 can also induce ferroptosis by partially inhibiting SLC7A11.
GPX4 inhibitors
GPX4 is a selenase that utilizes GSH as a cofactor to reduce membrane phospholipid peroxide to maintain cellular redox homeostasis, thus negatively regulating ferroptosis.20 Pharmacological inhibition or genetic absence of GPX4 induce ferroptosis in mouse tumor xenografts.19 Analyzing the data from TCGA, scientists found that GPX4 expression in various cancer tissues is higher than that in normal tissues, and it is negatively correlated with the prognosis of cancer patients.126 High expression of GPX4 promotes tumor recurrence in melanoma xenograft mice models.71 In addition, GPX4 plays important role in the survival of drug resistant cancer cells.127 Therefore, GPX4 serves as the potential target to treat cancer by inducing ferroptosis.
The first reported GPX4 inhibitor RSL3 covalently interacts with selenocysteine, the active site of GPX4, to inhibit enzymatic activity of GPX4, thus inducing ferroptosis.13 Poor pharmacokinetic property of RSL3 restricted its application in vivo. Recent studies reported that small-molecule compound QD394 and compound 26a induce ferroptosis by directly targeting GPX4.92,93 In addition, many small-molecule compounds induce ferroptosis mainly through GPX4 inhibition, including Jiyuan oridonin A derivative a2,94 compound 21,95 tetrahydrocitrate,96 and Bufortaline.97 It has long been reported that many natural products inhibited cancer cells through multiple targets. Accompany with the report of ferroptosis, researchers found that many natural compounds, including dihydroisotanshinone I,98,99 solasonine,100 DMOCPTL,101 cucurbitin B,102 and honokiol103 could induce ferroptosis by inhibiting GPX4 activity or reducing GPX4 protein level. Natural products gambogenic acid,104 thiostrepton,105 and red ginseng polysaccharide106 could suppress GPX4 expression by regulating upstream transcriptional factors, thus inducing ferroptosis. In addition, small molecule compounds atorvastatin, apatinib, and simvastatin induce ferroptosis by indirectly downregulating GPX4.107–109 The discovery of GPX4 inhibitors also supplies the information for the regulation of GPX4 in cancer cells.
FIN56
As a type 3 ferroptosis inducer, FIN56 induces ferroptosis by promoting GPX4 degradation.7 Except degrading GPX4, FIN56 can also reduce intracellular antioxidants (like CoQ10) by activating squalene synthase, thus promoting cellular lipid peroxidation and ferroptosis.7 In addition, FIN56-induced ferroptosis is related to autophagy, and inhibition of autophagy at different stages could weaken FIN56-induced lipid peroxidation and GPX4 degradation.128
FINO2
FINO2 triggers ferroptosis through mechanisms different from that of SLC7A11 inhibitors or GPX4 inhibitors.14 FINO2, serves as an endoperoxide, induces ferroptosis by directly oxidizing iron and indirectly inhibiting GPX4, which effect can be reversed by iron chelating agents.110
Others ferroptosis inducers
In addition to above mentioned ferroptosis inducers, researchers have developed many ferroptosis inducers by targeting other key ferroptosis regulators, like heme oxygenase-1 (HMOX1), ferrous ions or ACSL4 etc. HMOX1 protects cell through its byproduct bilirubin, and its overexpression reduces ROS levels and lipid peroxidation.129 Combination of lapatinib and cilacine co-induces ferroptosis by reducing heme oxygenase-1 protein expression.129 However, it has also been reported that HMOX1 can induce cell death by promoting intracellular ferrous accumulation.130 Newly discovered ferroptosis inducers such as curcumin analogues EF24111 and heavy metal cadmium112 can induce ferroptosis by directly up-regulating HMOX1 expression.
Triggering ferritin phagocytosis, a lysosome-mediated autophagy process that degrading ferritin and inducing ferroptosis, is a new strategy to induce ferroptosis.131 It had been reported that natural compounds Artesunate,113 Saponin Formosanin C,114 and Arsenite132 can induce ferroptosis by promoting ferritin phagocytosis. The natural product Baicalin can also induce ferroptosis by down-regulating ferritin heavy chain 1.115
In addition, Yiqi Huayu Decoction, created by Professor Liu Shenlin, can cause ferroptosis through multiple targets, such as reducing the content of GSH in cells and up-regulating the expression of ACSL4.133