The antioxidant system Xc- consists of transporters located on cell membranes. The primary system maintains homeostasis of glutamate and cystine on cell membranes, where it pumps out glutamate molecules and pumps in cystine molecules (68). The functional subunits of the system Xc- are SLC7A11 and SLC3A2 (68). GSH is synthesized by reduction of cystine to cysteine by system Xc-. Glutamyl-L-cysteine-L-glycine (GSH) is an endogenous antioxidant consisting of glutamic acid, cysteine and glycine that scavenges free radicals. Its production is dependent on activity of glutamate-cysteine ligase and inhibition of system Xc- attenuates its activity, resulting in GSH depletion and leading to ferroptosis (69). Trans-sulfuration is another mechanism by which methionine synthesizes cysteine. Cysteine is still synthesized when intracellular system Xc- is inhibited (70). Thus, ferroptotic inducers that negatively regulate system Xc- cannot effectively kill cells. The trans-sulfuration pathway is activated by interfering with RNA dynamics to decrease expression of cysteine transfer RNA synthetase. As a result, cells become less sensitive to ferroptosis-inducing agents (71). Cells that contain GPX4 decrease lipid peroxidation, which promotes their survival. Aberrant expression of GPX4 is linked to a range of diseases (breast cancer) in humans and depletion of GPX4 increases H₂O₂-containing phospholipids (72), promotes lipoxygenase-mediated lipid peroxidation and induces ferroptosis (73). GPX4 is highly expressed in tumor tissue, while histone H3 is trimethylated at lysine 4 (H3K4me3) and acetylated at lysine 27 (H3K27ac) at the GPX4 transcriptional start site. Patients with cancer have a poor prognosis when epigenetic modifications of GPX4 are present; thus, elevated expression of GPX4 in tumor tissue may be due to epigenetic modifications (74). In addition, the activation of autophagy degrades intracellular ferritin and directly induces ferroptosis in tumor cells. Autophagy is a lysosome-dependent process that promotes ferroptosis by generating lysosomal ROS. Promoting autophagy effectively enhances ferroptosis in tumor cells (75). The autophagy-mediated nuclear receptor coactivator (NCOA4) signaling pathway promotes ferritin degradation and enhances ferroptosis when NCOA4 is overexpressed (76), which demonstrates the association between ferroptosis and apoptosis.

Although GPX4 is a key regulator of ferroptosis, inhibition of GPX4 does not induce ferroptosis in certain cell lines. A previous study identified potential factors that regulate ferroptosis independently of the GPX4 signaling pathway (72). Cells were treated with a ferroptosis initiator [RAS selective lethal 3 (RSL3)], and it was observed that Glutathione Independent Iron Death Inhibitor Protein (FSP1) induced apoptosis. The inhibition of GPX4 leads to ferroptosis when FSP1 is overexpressed. Therefore, FSP1 may act as a ferroptosis inhibitor (77). However, AIFM1, although homologous to FSP1, does not inhibit ferroptosis (78). FSP1 may inhibit ferroptosis because its N-terminal myristoylated mediates FSP1 localization to the plasma membrane. Furthermore, myristoylation of the N-terminus of FSP1 promotes binding of target proteins to the cell membrane. In the plasma membrane, COQ10 serves as a lipophilic radical scavenger (79), while FSP1 serves as a NADPH-dependent COQ oxidoreductase that regulates COQ10 in vitro. FSP1 is a component of the traditional mitochondrial respiratory chain that catalyzes the same reactions as complex I. Extra-mitochondrial ubiquinone is reduced from COQ10 by FSP1, which either directly captures lipid free radicals or indirectly serves as an antioxidant by recycling α-tocopherol (77). Idebenone, a soluble analog of COQ10, inhibits ferroptosis and lipid peroxidation by catalyzing the first step in the biosynthesis of COQ2 (80). The aforementioned findings showed that FSP1, like GPX4, inhibits ferroptosis by regulating the non-mitochondrial COQ10 antioxidant system (80).

The redox-active cofactor BH4 serves a role in production of neurotransmitters, carbon monoxide and aromatic amino acids. In vitro, BH4 exhibits antioxidant properties and GCH1 limits BH4 synthesis (82). In tumor cells, GCH1 overexpression clears lipid peroxidation and protects cells from ferroptosis. Overexpression of GCH1 protects against RSL3-induced ferroptosis when cells were treated with RSL3 and apoptosis-inducing agents. GCH1 protects cells from ferroptosis (83). Treatment of RSL3-exposed cells with BH4 completely prevents ferroptosis. It has been previously shown that GCH1 acts independently of the ferroptosis and GSH pathways to prevent ferroptosis (84). Notably, BH4 as a cofactor for biosynthetic enzymes does not serve a role in ferroptosis protection (85). In addition, BH4 converts phenylalanine to tyrosine to promote synthesis of COQ10, thereby exerting an antioxidant effect (86).

There are numerous roles for DNA methylation in cancer, including suppression of DNA methylation at transcriptional start sites of key gene regulatory elements, such as enhancers and promoters (88). In DNA methylation, a methyl group is typically inserted at the carbon 5 position of the cytosine base (5mC) via DNA methylation mediated by CpG transferase DNMTs (89). DNA methylation primarily regulates mitotic gene expression, centromere stability and chromatin segregation. In addition, 5-hydroxymethylcytosine (5hmC) has been demonstrated to be produced by 5mC oxidation. 5hmC has been widely studied in relation to its potential role in modifying methylation landscapes by ten-eleven translocation (TET) enzymes (90,91). The ability of TET proteins to oxidize 5hmC to 5-formylcytosine and 5-carboxycytosine indicates utilization of the base excision repair pathway, while thymine DNA glycosylase excises cytosine and replaces it with an unmodified cytosine (92). CpG sites are found at ~28 million sites in the human genome. The distribution of CpG sites in somatic cells is uneven and ~70% of CpG sites are oxidized in normal somatic cells (93). Clusters of CpG sites are known as CGIs; CpG sites in CGIs tend not to be methylated in somatic cells. Chromosomes are their primary medium. Unmethylated CpG sites are found in promoter CGIs, where transcription factors bind to control gene expression. The CpG coast is ~2 kb pairs away from the CGI and has a lower density of CpGs than the CGI (94). Normal cells also contain these unmethylated regions that regulate gene activity (95). During tumorigenesis, normal epigenetic processes such as DNA methylation are disrupted. Tumor development is primarily accompanied by genome-wide hypomethylation and DNA hypermethylation in the promoter regions of CGIs (96). Cell proliferation and tumor suppressor genes and downstream signaling pathways are primarily silenced by the hypermethylation of CGIs (97). Moreover, tumors also abnormally express DNMT enzymes, resulting in aberrant DNA methylation across the genome, potentially causing mutations in genomic sequences (98). Aneuploidy and genomic instability are associated with genome-wide hypomethylation in cancer (99). The abnormal expression of transposable elements and oncogenes is also associated with genome-wide DNA hypomethylation, which disrupts cellular pathways and changes chromatin structure, as chromatin structure and DNA methylation are associated and a nucleosome is required before 5mC can be obtained (100). To anchor the DNMT enzyme, abnormal DNA methylation must disrupt chromatin structure (101). The most studied (102) epigenetic modification is DNA methylation (103). However, the mechanism of this process in unclear and the number of cancer-associated genes that are silenced and targeted is unknown. A recent study on the association between DNA methylation, ferroptosis and cancer has led to novel targeted therapy for cancer (104). Hydricase, lymphoid specific (HELLS/LSH) is a chromatin remodeling enzyme that inhibits ferroptosis by activating metabolic genes such as stearoyl-coenzyme A desaturase (SCD). HELLS induces epigenetic silencing of the long non-coding RNA (lncRNA) LINC00472. LINC00472, as a tumor suppressor, is downregulated in tumors and can inhibit ferroptosis (105). Egl nine homolog 1 and c-Myc in tumor cells activate LSH expression by inhibiting hypoxia inducible factor 1. LSH inhibits iron dynamics by activating GLUT1, SCD1 and fatty acid desaturase 2, which are involved in lipid metabolism, by interacting with WD repeat domain 76. Several types of cancer tissue (pancreatic and lung cancer) express high levels of SCD1, an enzyme that catalyzes fatty acid synthesis at the rate-limiting step. Through inhibition of SCD1, COQ10 activity is decreased, resulting in lipid oxidation and cell death (106). GPX4 DNA methylation in the nucleus pulposus is induced by homocysteine treatment, resulting in ferroptosis (107). Furthermore, DNA hypermethylation of the cadherin 1 (CDH1) gene promoter increases ferroptosis by suppressing expression of E-cadherin, which is encoded by CDH1 (108). DNA methylation contributes to ferroptosis-associated gene silencing, as demonstrated by the aforementioned studies. There is, however, a need for further research to determine whether DNA methylation also affects other ferroptosis-associated genes. Notably, TET proteins also catalyze DNA demethylation by oxidizing 5mC (109).

RNA is a key biological macromolecule in cells and it can be classified into two categories according to its function, namely, coding RNA and ncRNA. RNA is responsible for gene regulation and information transmission and is involved in disease. For example, nc-RNAs encode tumor peptides or proteins (110,111). The following subsections review ncRNAs in the context of microRNAs (miRNAs or miRs), lncRNAs and circular RNAs (circRNAs) involved in ferroptosis.

miRNAs are the most well-studied (112) ncRNAs in tumor biology (113). Endogenous genes encode a class of RNA molecules known as miRNAs, which are single-stranded ncRNA molecules with a length of 20–24 nucleotides. miRNAs regulate translational and transcriptional activities that control metabolic pathways, tumor growth and cell migration and invasion and induce resistance to chemotherapeutic agents (113). During post-transcriptional processing, miRNAs bind to the 3′ untranslated region of target mRNAs and regulate gene expression. miRNAs that bind to specific mRNAs directly inhibit translation by suppressing translation or decreasing mRNA stability post-transcriptionally (114). In addition to controlling cell differentiation, proliferation, drug resistance and lipid metabolism, miRNAs also participate in several other biological processes. For example, mirnas prevent translation by inactivating or inducing mRNA degradation, which is a key step in post-transcriptional gene regulation (115). Cancer cells are genetically or epigenetically altered as a result. Furthermore, miRNAs inhibit tumor cell proliferation by altering ferroptosis (116). For example, miR-9 interferes with the catalytic efficacy of glutamic-oxaloacetic transaminase 1 in melanoma cells, thus inhibiting its ability to transaminate α-ketoglutarate. By contrast, miR-9 inhibition causes lipid ROS accumulation, thereby increasing ferroptosis (117). Upregulation of miR-137 protects melanoma cells against ferroptosis by inhibiting the glutamine transporter SLC1A5 (118). By decreasing ferrous iron levels in the cytoplasm, miR-7-5p also decreases tumor cell migration and invasion (119). By decreasing tumor cell susceptibility to ferroptosis induced by treatment with RSL3 and erastin, activating transcription factor 4 knockout increases inhibition of tumor cell proliferation (120). In addition, GPX4 is inhibited by miR-4715-3p and cisplatin sensitivity is enhanced by miR-4715-3p. As a result of inhibition of the intracellular aurora kinase A, a key serine-threonine kinase in mitosis, the expression of miR-4715-3p is suppressed (121). In addition, inhibition of miR-4715-3p results in decreased levels of intracellular GSH, thereby preventing ferroptosis (122). Human colon adenocarcinoma cells are protected from oxidative damage by miR-185, which increases GPX2 levels in cells (123). Furthermore, specific transcriptional targets of miRNAs are involved in GSH metabolism, which is associated with ferroptosis and tumorigenesis (124,125). miRNAs serve both tumor-suppressive and -promotive functions in ferroptosis. Additionally, miRNAs modulate gene transcription at the mRNA level to decrease tumorigenesis, ferroptosis or degraded exon production (126). To determine the roles of miRNAs in tumor resistance mechanisms, it is imperative to understand the molecular mechanisms of miRNAs.

RNA molecules >200 nucleotides in length are known as lncRNAs and influence gene expression at different levels, including transcription and post-transcriptional translation. lncRNAs have also been reported to be key for a variety of biological processes, such as cell differentiation, regulation of the cell cycle and maintenance of stem cell pluripotency (127). Via downregulation of membrane receptors and inhibition of necroptosis-associated proteins, overexpression of lncRNAs inhibits the extrinsic apoptotic pathway in tumor cells (128). lncRNAs are important regulators of ferroptosis. A number of them affect miRNAs, while others bind to specific enzymes. Wang et al (129) reported that the lncRNAs LINC00336 and embryonic lethal, abnormal vision, Drosophila-like 1 (ELAVL1) inhibit ferroptosis by interacting with each other. In addition, ELAVL1 increases LINC00336 expression by stabilizing its post-transcriptional levels when RSL3 is used to activate ferroptosis in lung cancer cell lines. Overexpression of LINC00336 inhibits ferroptosis caused by RSL3 treatment, as well as protein kinase-induced ferroptosis. Furthermore, overexpression of LINC00336 decreases intracellular Fe²⁺ and mitochondrial superoxide concentrations, as well as ROS production, demonstrating that LINC00336 overexpression inhibits ferroptosis (129). Urothelial cancer associated 1 (UCA1) sponges miR-16 and increases expression of glutaminase 2 (GLS2) in bladder cancer cells. There are two mechanisms by which lncRNAs affect ferroptosis (130). GLS2 enzyme catalyzes conversion of glutamine to glutamate and subsequent synthesis of GSH. NADPH is produced when glutamine enters the tricarboxylic acid cycle. Glutathione is reduced to GSH in the presence of NADPH (131). Therefore, UCA1 regulates GSH and NADPH expression, thereby enhancing the antioxidant effects of GPX4 and inhibiting ferroptosis in tumor cells by decreasing ROS production (132). The transcription factor NRF2 also has antioxidant properties and can activate downstream antioxidant factors. NRF2 can promote the production of NADPH via the pentose phosphate pathway, thereby enhancing antioxidant activity. Additionally, NRF2 activates transcription of GSH and GPX family genes, while GPX4 is activated to exert antioxidant effects during ferroptosis (133). The balance of intracellular iron levels is key for cell survival. The co-function of transferrin and transferrin receptor is required for the absorption of Fe³⁺ by cells. Subsequently, Fe³⁺ becomes Fe²⁺ through a redox reaction and it is then stored in the iron pool (134). Ferroptosis occurs when Fe²⁺ donates electrons to oxygen in the Fenton reaction to form ROS, which catalyzes generation of lipid free oxygen radicals (135). In HCC, for example, expression of the ncRNA plasmacytoma variant translocation 1 (PVT1) is increased; PVT1 binds to miR-150 and regulates the target hypoxia-inducible gene (HIG2) (136). HIG2 is involved in ferroptosis and iron uptake via hypoxia-induced protein expression. Decreased transferrin receptor and increased ferritin light chain expression are observed when PVT1 is silenced, thereby decreasing iron uptake and affecting ferroptosis (137). The induction of ferroptosis by erastin results in upregulation of the lncRNA GA binding protein subunit β1 (GABPB1)-antisense RNA 1, blocking of the transcription and translation of GABPB1 and the inhibition of peroxidase 5 (PRDX5) expression. PRDX5 is less effective than non tumor tissue at preventing ROS production from H₂O₂, which ultimately impairs the antioxidant capacity of tumor cells, resulting in increased cell death (138). Melanoma glucose-6-phosphate dehydrogenase (G6PD) may stimulate NADPH production, thereby activating NADPH oxidase 4 (NOX4) (139). Superoxide and ROS are produced by NOX4, a transmembrane protein. When growth arrest specific 5 is silenced, G6PD and NOX4 activity is increased (140). Furthermore, ferroptosis is induced by G6PD and NOX4 in tumor cells (141). Inhibition of iron-induced cell death by targeting the miR-106B-5p/acyl-coenzyme A synthetase long chain family member 4 axis is a mechanism by which H19 promotes cancer progression (142). Iron-induced death is also regulated by PVT1 via activation of TFR1 and TP53 by miR-214. The PVT1/miR-214-3p/GPX4 signaling pathway activates ketamine and inhibits the proliferation of HCC cells both in vitro and in vivo, leading to iron-induced cell death (143). In addition, lncRNA ZNFX antisense RNA 1 acts as a competing endogenous RNA for iron-induced death via the miR-150-5p/SLC38A1 axis (144). Multiple other lncRNAs are involved in iron-induced tumor cell death. Further studies are needed to investigate the specific mechanism by which lncRNA regulates iron-related cell death.

circRNAs are formed by the 3′ and 5′ ends of mRNAs. According to their structural features, they are primarily classified into three categories, namely, exonic, circular intronic and exonic intronic circRNAs, and they function as miRNA sponges and protein scaffolds (145). Compared with miRNAs, the abundance, conservation and tissue specificity of circRNAs make them better biomarkers for certain pathological states. For example, circrNA-002178 is abnormally expressed in lung cancer, which is of great help in the diagnosis of lung cancer (146). The majority of circRNAs are highly stable and specific. Cancer cells express abnormal levels of circRNAs, which are involved in cell proliferation, autophagy and apoptosis, among other aspects of programmed cell death (147). circRNAs affect the post-transcriptional stability of miRNAs by altering their target gene expression. In tumor cells, circRNAs serve as sponges and regulate ferroptosis (148). Furthermore, they enhance tumor cell proliferation and invasion by sponging miR-761 and activating integrin β8 (ITGB8) in glioma, inhibit ferroptosis by activating ITGB8 and inhibit apoptosis by targeting MET, while knockdown of circ-tubulin kinase 2 promotes erastin-induced ferroptosis (149). In addition, 526 circRNAs are dysregulated in cervical cancer cells according to a previous high-throughput microarray-based study (150). circRNAs are predominantly involved in GSH metabolism according to a previous bioinformatics analysis (151). However, associated downstream factors and miRNAs have not been screened (152). Therefore, further studies on regulation of circRNAs in ferroptosis are needed (Fig. 2).

The histone H3 lysine 9 demethylase that regulates SLC7A11 expression in response to erastin-induced ferroptosis is lysine demethylase 3B (156). E1A binding protein p300-CREB binding protein (CREBBP), which is involved in transactivation of genes, acetylates nuclear factor erythroid-derived 2-like 2 (NFE2L2) (157). Mouse double minute 2 homolog is a proto-oncogene associated with TP53 stability and activation, while cyclin-dependent kinase inhibitor 2A/ARF promotes its degradation. Ferroptosis is promoted by ARF independently of TP53 (158). Cancer is frequently caused by TP53 inactivation or mutations. A number of genes controlled by TP53 serve key roles in various cellular processes such as cell proliferation, cell cycle progression, cell death and response to DNA damage. Cancer cells, when activated by TP53, not only promote tumor growth but also inhibit ferroptosis (159). ARF protein disrupts CREBBP-NFE2L2 interaction, resulting in attenuated acetylation of NFE2L2 and the decreased expression of SLC7A11 mediated by NFE2L2 (160). Furthermore, members of the bromodomain (BRD) family are epigenetic regulators that recognize acetylated lysine residues on histones. JQ1, an inhibitor of the BRD4 complex, induces ferroptosis in breast and lung cancer cells by decreasing expression of GPX4, SLC7A11 and SLC3A2. Other anti-ferroptotic genes are also regulated by BRD4 (161).

Cancer cells overexpress deubiquitinase OUT deubiquitinase ubiquitin aldehyde binding 1 (OTUB1), which stabilizes cystine transporter SLC7A11, inhibits ferroptosis and promotes tumor growth (162). BRCA1-associated protein 1 (BAP1) decreases SLC7A11 promoter H2A ubiquitination via an H2A deubiquitinase that functions as a tumor suppressor. As a result, ferroptosis is regulated and SLC7A11 expression is inhibited. The H2A ubiquitin ligases BAP1 and protein regulator of cytokinesis 1 inhibit expression of SLC7A11. The tumor suppressor deubiquitinating enzymes (DUB) inactivates P53 and BAP1 by downregulating SLC7A11 expression or enhancing OTUB1 or CD44 expression to stabilize SLC7A11 (153). As a result, tumor growth is suppressed and proteasomal activity and apoptosis are inhibited. The DUB inhibitor ubiquitin-specific protease (USP7) enhances caspase-dependent apoptosis during ferroptosis while degrading GPX4, which results in ubiquitinated protein accumulation and cell death (163). The expression of H2Bub1, which binds to the regulatory region of SLC7A11, is decreased by P53, thereby inhibiting SLC7A11 expression (164). Furthermore, ferritin degradation occurs via lysosomal or proteasomal mechanisms. Via ferritin heavy chain 1, NCOA4 binds to iron-rich ferritin on autophagosomes, thus transporting it to lysosomes for iron release. The ubiquitin ligase HERC2, which affects stability of proteins, ubiquitinates and degrades NCOA4 in environments with high iron concentrations. Therefore, ferroptosis is suppressed by inhibition of NCOA4 (165).

The four aforementioned histones are acetylated on specific lysine residues, which is associated not only with gene transcription but also with DNA replication and repair (166). During transcription, histone acetylation induces HAT to acetylate lysine residues in histones, while histone deacetylation inhibits HDAC (167). HAT and HDAC catalyze histone acetylation and deacetylation, respectively (168). Patients with cancer have a poor prognosis when GPX4 expression is higher in tumor than in normal tissue (169). In tumor cells, high GPX4 levels may be due to epigenetic regulation such as methylation of DNA and histones and acetylation of histones, as reported in a previous study that analyzed upstream regulation of GPX4 (170). Mutant P533KR causes lipid peroxidation and ferroptosis via decreased acetylation and cysteine uptake (171). As a result of hydrolysis of cystinase, GSH is synthesized and GSH metabolism is affected. Cysteinase depletion also cause ferroptosis (Table I) (172).