Iron plays a crucial role in regulating ferroptosis, not only by triggering the non-enzymatic Fenton reaction that directly oxidizes polyunsaturated fatty acid-phospholipids (PUFA-PLs), but also by serving as a vital cofactor for enzymes involved in lipid peroxidation, such as ALOX and POR (42–45). Under normal circumstances, cells maintain a relatively stable level of labile iron through a finely tuned balance of iron absorption, use, storage, and export. However, disruptions in these iron metabolism processes can either accelerate or inhibit ferroptosis, depending on whether they increase or decrease the intracellular labile iron pool. For instance, most intracellular iron is stored as inert iron within ferritin. The autophagic breakdown of ferritin, known as ferritinophagy, releases iron from ferritin into the labile iron pool. Blocking NCOA4-mediated ferritinophagy lowers the labile iron pool and thus inhibits ferroptosis (46, 47). On the other hand, promoting ferritinophagy, such as by inhibiting cytosolic glutamate oxaloacetate transaminase 1 (GOT1), increases the labile iron pool and encourages ferroptosis. The exact mechanism by which GOT1 inhibition facilitates the release of labile iron through ferritinophagy is still under investigation (48–50). For a more comprehensive discussion on iron metabolism in the context of ferroptosis, interested readers are directed to recent detailed reviews on the subject (51).

Iron absorption and metabolism in the body can be summarized as following. Firstly, iron is a crucial trace element necessary for various biological functions in cells. Dietary iron is mainly categorized into non-heme and heme iron, with the latter being Fe2+ combined with protoporphyrin IX. Non-heme iron primarily exists as ferric salts in the diet, which are converted to Fe2+ by intestinal iron reductase. This Fe2+ is then absorbed by intestinal epithelial cells (IECs) through the DMT1 transporter and exits these cells via ferroportin 1 (FPN1) (52, 53). Once in the bloodstream, Fe2+ is oxidized to Fe3+ by ceruloplasmin (CP) and hephaestin (HP), and then binds to transferrin (Tf) for transport (54). Tf-Fe3+ attaches to cell membranes via the transferrin receptor (TfR), internalizes into cells as endosomes, and is reduced back to Fe2+ by STEAP3 in various cells before entering the cytoplasm through DMT1 on the endosomal membrane (55) (see Figure 3 ).

Heme iron, a component of hemoglobin and myoglobin, has a less clear absorption mechanism. It’s believed that heme iron ingested is broken down by heme oxygenase in intestinal cells, releasing free ferric iron. Subsequently, a significant amount of Fe2+ accumulates in the cytoplasm, forming a labile iron pool crucial for processes like ferroptosis (56, 57). Excessive intracellular iron, in the presence of H2O2, triggers the Fenton reaction, leading to ROS formation such as •OH, which induces lipid peroxidation and hence ferroptosis (58–60). Iron responsive element binding protein 2 (IREB2) is an important regulator of iron metabolism and may influence sensitivity to ferroptosis (61–63). Autophagy also plays a role in iron regulation, affecting the recruitment of ferritin to autophagosomes for lysosomal degradation, thus releasing free iron (64–66). For example, ferritinophagy, facilitated by the cargo receptor NCOA4, specifically targets the ferritin heavy chain 1 (FTH1), releasing iron into autophagosomes for degradation (67). Conversely, a decrease in intracellular Fe2+ levels can hinder ferroptosis, as seen when erastin-induced ferroptosis is reduced following the knockout of autophagy-related genes ATG5 or ATG7 (68, 69). Therefore, iron metabolism is integral to the process of ferroptosis.

Lipid peroxidation stands out as a key characteristic and the primary mechanism that drives ferroptosis. Reactive oxygen species (ROS) generated through the Fenton reaction interact with polyunsaturated fatty acids (PUFAs) in cell or organelle membranes, resulting in the formation of toxic phospholipid hydroperoxides (PLOOHs) and triggering ferroptosis (70–72). Various factors contribute to the process of lipid peroxidation, including enzymes such as acyl–coenzyme A synthetase long-chain family member 4 (ACSL4), lysophosphatidylcholine acyltransferase 3 (LPCAT3), and lipoxygenases (LOXs) (73–75). ACSL4, vital for lipid metabolism, and LPCAT3, key in catalyzing the reacylation of lysophospholipids to phospholipids, play crucial roles (76–78). They activate free long-chain PUFAs, facilitate the conversion of lysophosphatidylcholine (LPC) to lecithin, mediate the synthesis of oxidized phospholipids in cell membranes, and thus influence ferroptosis. ACSL4 specifically esterifies arachidonic acid (AA) into acyl-coenzyme A (acyl-CoA), essential for PUFA biosynthesis, which is pivotal in lipid peroxidation and ferroptosis. LOXs are critical regulators in this process. They significantly impact the onset of ferroptosis by promoting lipid autoxidation and are indicative of a cell’s sensitivity to ferroptosis (74, 79). Therefore, in the context of ferroptosis, lipid peroxidation leads to cell death by disrupting the lipid bilayer structure of cellular and organelle membranes (see Figure 4 ).

Mitochondrial metabolic processes play a crucial role in initiating ferroptosis (80). One key aspect is the generation of mitochondrial reactive oxygen species (ROS), essential for lipid peroxidation and the onset of ferroptosis. Mitochondria are significant cellular sources of ROS, where electron leakage from complexes I and III of the electron transport chain leads to the formation of superoxides. These superoxides are then converted into hydrogen peroxide (H2O2) by superoxide dismutase (81). The H2O2 reacts with labile iron through the Fenton reaction to produce hydroxyl radicals, which in turn drive the peroxidation of polyunsaturated fatty acid-phospholipids (PUFA-PLs) (81, 82). Additionally, mitochondrial electron transport and proton pumping, crucial for ATP production, also play a role in promoting ferroptosis (83–86). Under conditions where ATP is depleted, AMP-activated protein kinase (AMPK) phosphorylates and inhibits acetyl-CoA carboxylase (ACC), leading to a suppression of PUFA-PL synthesis and thus blocking ferroptosis. Conversely, when ATP levels are sufficient, AMPK activation is inefficient, resulting in the activation of ACC and promotion of PUFA-PL synthesis and ferroptosis (85, 86). Furthermore, mitochondria’s role in biosynthetic pathways, such as the tricarboxylic acid (TCA) cycle and anaplerotic reactions (like glutaminolysis), contributes to ferroptosis (83). These processes likely drive ferroptosis through the promotion of ROS, ATP, and/or PUFA-PL generation (87–89). Hence, the multifaceted functions of mitochondria in bioenergetics, biosynthesis, and ROS generation are central to driving mitochondrial lipid peroxidation and ferroptosis (80).

Environmental stresses like high temperature and hypoxia can trigger iron reactions, necessitating cells to establish defense mechanisms against ferroptosis. The most classic defense against ferroptosis involves the antioxidant axis consisting of xCT, glutathione (GSH), and GPX4. xCT, a transmembrane protein, comprises the light-chain solute carrier family 7 member 11 (SLC7A11) and the heavy-chain solute carrier family 3 member 2 (SLC3A2, also known as CD98hc or 4F2hc) (90, 91). SLC7A11, the primary functional unit of xCT, regulates the intake of extracellular cysteine (Cys) into cells and the export of intracellular glutamic acid (Glu). SLC3A2 helps maintain xCT’s stability by anchoring and stabilizing SLC7A11 (92).

Cysteine is converted into reduced GSH alongside Glu and glycine (Gly) under the influence of glutamate cysteine ligase (GCL) and glutamylcysteine synthetase (GCS) (93, 94). Beclin 1 can suppress xCT activity, thereby promoting ferroptosis, by directly binding to SLC7A11 (95). GPX4, a crucial enzyme in preventing ferroptosis, reduces toxic phospholipid hydroperoxides (PLOOH) to non-toxic phospholipid alcohols (PLOH) in membranes, using GSH (96). The inhibitor 2-cyano-3,12-dioxooleana-1,9 (11)-dien-28-oic acid (CDDO) helps prevent the specific degradation of GPX4 through chaperone-mediated autophagy (CMA). It does so by affecting the interaction between heat shock protein 90 (HSP90) and lysosomes, thus inhibiting cell ferroptosis (13). However, this inhibition can be counteracted by suppressing the mammalian target of rapamycin (MTOR) pathway, which may lead to GPX4 degradation and promote ferroptosis (97). Consequently, xCT, GPX4, and GSH are key regulators and have a significant impact on the control of ferroptosis (see Figure 4 ).

The FSP1-CoQH2 system has challenged the earlier belief that GPX4 was the sole defense mechanism against ferroptosis. Recent studies have shown that ferroptosis suppressor protein 1 (FSP1, also known as AIFM2) works independently of GPX4 to protect against ferroptosis (98, 99). FSP1, located on the plasma membrane and other subcellular compartments, is crucial in this role. Its presence on the plasma membrane is both necessary and sufficient for its function in ferroptosis suppression (98, 99). FSP1 acts as an NAD(P)H-dependent oxidoreductase, reducing ubiquinone (also known as coenzyme Q or CoQ) to ubiquinol (CoQH2). CoQH2, apart from its known role in mitochondrial electron transport, can intercept lipid peroxyl radicals, thus inhibiting lipid peroxidation and ferroptosis (98, 99). The generation of a non-mitochondrial CoQH2 pool by FSP1 as radical-trapping antioxidants plays a key role in its anti-ferroptosis activity (98, 99). Though CoQ is mainly produced in mitochondria, it is also found in non-mitochondrial membranes, including the plasma membrane (100–102). The exact sources of non-mitochondrial CoQ used by FSP1 in ferroptosis defense are yet to be determined.

Additionally, the DHODH-CoQH2 system has been identified as another mitochondria-localized defense mechanism. This system, mediated by dihydroorotate dehydrogenase (DHODH), can compensate for the loss of GPX4 in detoxifying mitochondrial lipid peroxidation (36). DHODH, an enzyme in pyrimidine synthesis, reduces CoQ to CoQH2 in the inner mitochondrial membrane (36). When GPX4 is acutely inactivated, DHODH activity increases, leading to more CoQH2 production which neutralizes lipid peroxidation and protects against mitochondrial ferroptosis. Inactivating both mitochondrial GPX4 and DHODH leads to significant mitochondrial lipid peroxidation and ferroptosis (36). Notably, while mitochondrial GPX4 and DHODH can substitute for each other in reducing mitochondrial lipid peroxidation, cytosolic GPX4 and FSP1 cannot, likely due to their non-mitochondrial localization.

These findings suggest a model where ferroptosis defense systems are divided into two main categories: the GPX4 system and the CoQH2 system, each further divided into non-mitochondrial and mitochondrial components. For instance, cytosolic and mitochondrial GPX4 are part of the GPX4 system, while non-mitochondrial FSP1 and mitochondrial DHODH belong to the CoQH2 system. This division is presumably due to the need to mitigate lipid peroxides in mitochondria and the unique double-membrane structure of mitochondria, which limits the effectiveness of defense systems located in other compartments (103). However, further research is required to fully understand this compartmentalization model in ferroptosis regulation and reconcile it with some conflicting findings, such as the significant localization of cytosolic GPX4 in the intermembrane space of mitochondria (104). The role of cytosolic GPX4 in mitochondria and its potential impact on suppressing mitochondrial lipid peroxidation needs further investigation.

Recent research has highlighted the role of GTP cyclohydrolase 1 (GCH1) as a key player in regulating ferroptosis (105, 106). GCH1 is involved in the rate-limiting step of the biosynthesis pathway for tetrahydrobiopterin (BH4), a cofactor for aromatic amino acid hydroxylases and other enzymes (107). BH4, aside from its role as a cofactor, is a radical-trapping antioxidant capable of capturing lipid peroxyl radicals, thus contributing to the inhibition of ferroptosis independently of its cofactor function (106). It is suggested that GCH1 helps prevent ferroptosis in two ways: firstly, through the generation of BH4 as a radical-trapping antioxidant, and secondly, via the GCH1-mediated production of coenzyme QH2 (CoQH2) and phospholipids (PLs) containing two polyunsaturated fatty acid (PUFA) tails. This is notable because most PUFA-PLs typically exhibit an asymmetric structure, with a PUFA tail at the sn-2 position and a saturated fatty acid tail at the sn-1 position (105, 106). The specific subcellular location where the GCH1-BH4 system functions, however, is still to be determined. This discovery adds another layer to the complex regulatory network of ferroptosis and underscores the multifaceted nature of cellular defense mechanisms against this form of cell death.

System Xc− plays a significant role in the survival and growth of many tumor cells, making it a potential target for cancer treatment. Erastin, known for inhibiting system Xc−, reduces glutathione (GSH) levels and induces ferroptosis. A notable discovery in Dixon’s 2012 study was that erastin led to ROS accumulation in NRAS-mutant HT-1080 fibrosarcoma cells, and this cell death was hindered by the iron chelator deferoxamine, indicating erastin’s role in inducing ferroptosis (4, 96, 126, 127). Further research highlighted the importance of the RAF/MEK/ERK signaling pathway in erastin-triggered ferroptosis in RAS-mutated cancers (128). Derivatives of erastin, like piperazine erastin and imidazole ketone erastin (IKE), have been developed to address erastin’s limitations, such as poor water solubility and unstable in-vivo metabolism. For instance, IKE has been effectively used in treating diffuse large B cell lymphoma (DLBCL) in the SUDHL6 xenograft animal model (129).

Sorafenib, a multi-kinase inhibitor used in treating advanced renal cell carcinoma, thyroid carcinoma, and hepatocellular carcinoma, is another inducer of ferroptosis. Its cytotoxicity to hepatocellular carcinoma was negated when treated with an iron chelator (130). However, some cancer cell lines have developed resistance to sorafenib, as seen in hepatocellular carcinoma cells with retinoblastoma (Rb) protein, where sorafenib-induced ferroptosis was inhibited (131). Additionally, the anti-inflammatory drug sulfasalazine (SAS) can induce ferroptosis in glioma cells by inhibiting system Xc− (132).

Some cancer cells induce ferroptosis through the transsulfuration pathway instead of system Xc−. GPX4 inactivation can eliminate these tumor cells, as seen with (1S, 3R)-RSL, which induces ferroptosis through direct GPX4 inhibition, and FIN56, which promotes ferroptosis by degrading GPX4 (133). Ferroptosis can also be induced by increasing the labile iron pool (LIP). Compounds like BAY 11-7085 can induce ferroptosis through the Nrf2-SLC7A11-HO-1 pathway, and overexpression of heme oxygenase-1 (HO-1), encoded by HMOX1, has been observed in MDA-MB-231 breast cancer and DBTRG-05MG glioblastoma cells (134). Increased TF expression and decreased FPN-1 expression, mediated by compounds like siramesine and lapatinib, can also induce ferroptosis. Autophagy contributes to this process by degrading ferritin in cancer cells. The cargo receptor nuclear receptor coactivator 4 (NCOA4) is significant in the autophagic turnover of ferritin in ferroptosis. In pancreatic cancer cells, NCOA4 overexpression inhibited FIH1 expression and promoted erastin-induced ferroptosis (47). Further research is necessary to discover novel molecules targeting NCOA4 for cancer treatment through ferroptosis. Besides these, more small molecules are being studied for their potential to induce ferroptosis in cancer therapy (see Figure 6 ).

Nanotechnology applications, particularly due to their unique physicochemical properties, have garnered significant interest in recent years. Many nanomaterials, including iron-containing nanoparticles, leverage the Fenton reaction for their functionality. For instance, Chen and colleagues developed a tumor-targeted nanoparticle called α‐enolase targeting peptide modified Pt-prodrug loaded Fe3O4 nanoparticles (ETP-PtFeNP). Treatment of tumor cells with ETP-PtFeNP resulted in increased ROS generation, enhanced immunogenicity, and a robust anti-tumor immune response (135). Additionally, a novel nanoparticle named SRF@FeIIITA (SFT) has been shown to be effective in inhibiting tumor progression. This nanoparticle combines photodynamic therapy (PDT) and ferroptosis by loading methylene blue (MB) into SFT. This is achieved by depositing tannic acid (TA) and Fe3+ onto SRF nanocrystals, enabling a dual-therapy approach (136).

Nanomaterials can also trigger ferroptosis through the manipulation of GSH metabolism. Arginine-capped manganese silicate nanobubbles (AMSNs), for example, have been developed to efficiently deplete GSH (137). This efficiency is attributed to their high surface area to volume ratio. In-vivo studies have demonstrated that AMSN treatment can suppress the growth of Huh7 xenograft tumors by downregulating GPX4. The effectiveness of AMSN-induced ferroptosis can be inhibited by the ferroptosis inhibitor liproxstatin-1, highlighting the potential of these nanomaterials in targeted cancer therapy through the induction of ferroptosis.

Drug resistance presents a significant hurdle in chemotherapy treatment, but the use of ferroptosis inducers might offer a way to surmount this challenge. Incorporating ferroptosis agonists with chemotherapy drugs could emerge as an innovative approach to cancer therapy (137). Persister cells, which are cancer cells that survive after multiple rounds of chemotherapy, exhibit downregulated Nrf2-targeted genes (138). Accelerating ferroptosis in these cells can be achieved by inhibiting intracellular NF2 and the hippo signaling pathway (139). Moreover, persister cells typically show reduced levels of glutathione (GSH) and nicotinamide adenine dinucleotide phosphate (NADPH), making them more susceptible to lipid peroxidation. GPX4 inhibitors have been found to be particularly effective against these cells. Therefore, inducing ferroptosis in persister cells may be a promising strategy to overcome their drug resistance, offering a new avenue in the fight against cancer.

There have been limited studies on combining ferroptosis inducers with other anti-tumor therapies for clinical treatment, with many investigations still in the experimental phase. An important aspect in cancer cells, especially when the oxygen concentration is above 3–8% as found in most tissues, is the iron-sulfur cluster biosynthetic enzyme NFS1. Its high expression is often observed in well-differentiated adenocarcinomas. Research by Alvarez SW’s group suggested that inhibiting NFS1, along with suppressing cysteine transport, could induce ferroptosis in tumor cells (140). Interestingly, some tumors that are resistant to certain chemotherapy drugs show a heightened sensitivity to ferroptosis inducers. For instance, pancreatic cancer cells, known for their resistance to chemotherapy-induced apoptosis, demonstrate considerable sensitivity to artemisinin-induced ferroptosis. Thus, ferroptosis inducers emerge as a promising strategy for cancer therapies, particularly for types of cancer that are resistant to conventional treatments.

In terms of preventing tumor metastasis, targeting ferroptosis could be a potential approach. Clinical treatment of tumor metastasis is complex due to factors like tumor heterogeneity, oncogene activity, epithelial-mesenchymal transition (EMT), and the microenvironment of metastatic sites (141). Since cancer metastasis can be inhibited by high intracellular oxidative stress, ferroptosis, which involves the accumulation of such stress, may be an effective strategy. Nanoparticles present a substantial advantage in treating cancer metastasis due to their relatively low risk compared to locally injected agents (142). For example, a metal–organic network encapsulating the p53 plasmid (MON-p53) was developed using coordination between ferric iron (Fe3+) and tannic acid (TA). Treatment with MON-p53 showed the potential to suppress cancer cell migration in in-vitro wound healing assays, suggesting its possible role in inhibiting tumor metastasis (143, 144). Moreover, mesenchymal cancer cells, known for their metastatic potential and resistance to anti-cancer treatments, could be rendered more sensitive to chemotherapy and thus reduce metastasis. Antagonizing the NF2-YAP pathway, which promotes ferroptosis by up-regulating modulators like ACSL4 and transferrin receptor (TFRC), offers a new perspective on treating mesenchymal or metastatic cancer cells, which are highly sensitive to ferroptosis (139).