Approximately 65% of the body’s iron is found in the form of haemoglobin iron, with 25% stored in ferritin and ferrous haemoflavin, and only about 0.1% bound to transferrin (Tf). Metabolic processes such as absorption, storage, excretion and recycling are responsible for regulating iron levels in the body [ 42 , 43 ]. Iron cations (Fe³⁺) are absorbed from food by intestinal epithelial cells, mainly in the duodenum and upper part of the jejunum [ 44 ]. Duodenal epithelial cytochrome b reductase 1 (DCYTB) converts Fe³⁺ to ferrous iron (Fe²⁺) in the presence of the reducing agent vitamin C or gastric acid [ 45 , 46 ], and then transports it into the intestinal epithelial cells via the divalent metal transporter 1 (DMT1) [ 47 ]. Some of the iron is stored as ferritin, so supplementation with Fe and vitamin C, as well as overexpression of DMT1, can promote iron uptake and sensitivity to iron-induced cell death [ 48 ]. The remaining reduced Fe²⁺ enters the plasma via endocytosis by binding to the iron transporter (FPN) in intestinal epithelial cells. Hepcidin—the dominant regulator of systemic iron homeostasis—binds to FPN to modulate cellular iron export, thereby influencing iron availability for ferroptosis in tumor cells. Subsequently, exported iron is oxidised by ceruloplasmin to Fe³⁺ and binds to transferrin (Tf) [ 49 ]. Transferrin enters cells after passing through the transferrin receptor 1 (TFR1) on the cell surface [ 50 ] and is reduced by prostate transmembrane epithelial antigen 3 (STEAP3) to Fe²⁺, which is then stored as ferritin. Some free Fe²⁺ is also stored in the labile iron pool (LIP) [ 51 ]. Degradation or autophagy of ferritin in response to other regulators such as autophagy-associated protein (Atg) and Nuclear receptor coactivator 4 can result in release of Fe²⁺ from ferritin into the LIP [ 52 ]. Excess labile iron is cytotoxic and can lead to lipid peroxidation and the generation of large amounts of ROS via the Fenton reaction or auxiliary lipid oxidases (LOXs) [ 53 - 55 ]. Therefore, depleting iron stores and increasing ferritin autophagy to increase unstable iron levels may promote iron-induced cell death [ 56 ].

Lipid peroxidation is a crucial process in ferroptosis, yet the mechanism of the oxidative system involved remains elusive and controversial. What is clear, however, is that polyunsaturated fatty acids (PUFAs) play a central role in ferroptosis by undergoing peroxidation. This leads to the accumulation of oxidized phospholipids on cell and organelle membranes, resulting in the formation of lipid peroxides and ultimately, ferroptosis [ 57 , 58 ]. Specifically, arachidonic acid (AA) and adrenoic acid (AdA) are dienophiles that are susceptible to oxidation by lipoxygenase (LOX), generating AA-PE-OOH or AdA-PE-OOH [ 59 , 60 ]. These oxidized lipids disrupt membrane integrity [ 61 ], leading to ferroptosis. LOX-12 and LOX-15 also contribute to oxidative stress and depletion of glutathione (GSH), further increasing lipid peroxides. Acyl coenzyme A synthase long-chain family member 4 (ACSL4) and lysophosphatidylcholine transferase (LPCAT3) are enzymes that mediate the biosynthesis, remodelling [ 62 ], and transmembrane properties of PUFAs, promoting their oxidation to phosphatidylethanolamine (PE) [ 63 ]. Additionally, the oxidative degradation of PUFAs generates two secondary lipid peroxides, 4-hydroxy-2-nonenal (4-HNE) and malondialdehyde (MDA), which are highly expressed during the onset of ferroptosis and serve as markers of this process. Interestingly, 4-HNE and MDA can activate the autophagic lysosomal pathway and promote ferritin degradation, further promoting ferroptosis [ 64 , 65 ].

The metabolic pathways of amino acids involved in ferroptosis are centered around the molecules glutathione (GSH), glutathione peroxidase 4(GPX4), and system Xc⁻ [ 2 ]. GPX4, an enzyme, plays a crucial role in the cellular antioxidant defense system by catalytically neutralizing lipid peroxides, converting them into non-toxic alcohols [ 66 ]. GSH, a non-protein molecule, influences the activity of GPX4 by serving as a recyclable substrate for the enzyme’s thiol group. Inactivation of GPX4 can occur in the presence of the inhibitor Ras-selective Lethal 3 (RSL3) or by binding to cisplatin, leading to the accumulation of ROS and ultimately promoting ferroptosis [ 67 ]. Furthermore, resbufogenin has been found to inhibit oxidative stress and promote the eradication of colorectal cancer cells [ 68 ].

System Xc⁻ is a heterodimer composed of SLC7A11 and SLC3A2, also known as the cysteine/glutamate transport system [ 66 ]. Its primary function is to regulate the levels of intracellular cysteine, which is essential for GSH synthesis. Inhibiting system Xc⁻ reduces GSH production, thereby diminishing the activity of GPX4 and leading to ROS accumulation, ultimately driving ferroptosis in CRC cells [ 61 , 69 - 72 ]. Additionally, the expression of SLC7A11 can be downregulated by the tumor suppressor protein P53 [ 73 ]. Erastin, in conjunction with SLC7A5 and the SLC7A5/SLC3A2 complex, also downregulates the expression of SLC3A2 and SLC7A11, thereby influencing the occurrence of ferroptosis [ 12 , 74 , 75 ].

The thioredoxin reductase (TXN) system, alongside system Xc⁻ GPX4-GSH, plays a critical role in antioxidant defence [ 76 , 77 ]. Comprised of TXN, thioredoxin reductase (TrxR), and nicotinamide adenine dinucleotide phosphate (NADPH), this system facilitates the reduction of oxidative GSH to its active form in response to NADPH [ 78 ], thereby supporting the removal of cytotoxic GSH by GPX4 [ 79 , 80 ]. This process helps to combat toxic peroxides, maintain the intracellular reduced state, and prevent DNA and lipid peroxidation. In addition, TXN acts as an antioxidant by facilitating the reduction of oxidised proteins through cysteine-thiol-disulfide bond exchange [ 81 , 82 ]. Consequently, inhibition of TXN, TrxR and NADPH can lead to inhibited glutathione uptake, reduced GPX4 levels, increased levels of peroxides and oxidised proteins, and ultimately to iron-mediated cell death.

Ferroptosis also involves mitochondrial activity [ 83 , 84 ]. Mitochondria are important sites for ferroptosis as they undergo oxidative exergy via the electron transfer chain (ETC) and the tricarboxylic acid (TCA) cycle, generating ROS [ 83 , 85 ]. Glutamine is a protein that provides energy for the TCA cycle by metabolically degrading glutamine and generating ROS. Carbonyl cyanide m-chlorophenyl hydrazine renders the inner mitochondrial membrane permeable to H+, leading to membrane potential destabilisation on both sides of the inner mitochondrial membrane, and then, via the Pink1-Parkin pathway, which is specific for membrane potential destabilisation, the mitochondria undergo further membrane potential (MMP) hyperpolarisation, which generates ROS. MMP hyperpolarisation, leading to mitochondrial autophagy and mitochondrial deletion. Cysteine deprivation (CC) leads to mitochondrial membrane potential hyperpolarisation and accumulation of lipid peroxides. Mitochondrial TCA cycle, MMP hyperpolarisation and ETC have all been shown to be involved in CC starvation-induced ferroptosis [ 84 ]. However, mitochondrial function is not essential for GPX4 inhibition-induced ferroptosis. This means that when mitochondrial activity is disrupted, as is the case with w inhibitors, low levels of GPX4 can still trigger ferroptosis.

In addition, mitochondria-associated stress releases mtDNA from the mitochondria into the cytoplasm, which activates a variety of immune responses and induces ferroptosis [ 86 ]. mtDNA is required for a large amount of Fe²⁺, and iron ions in the mitochondria are mainly used for synthesis of haemoglobin and the Fe-S family, or are stored in mitochondrial ferritin [ 37 , 87 ]. Haem can directly induce ferroptosis under the regulation of mitochondrial haem oxygenase 1 and cytoplasmic haem oxygenase. Ferroptosis can also be directly induced when mitochondria are destroyed and ferritin is degraded [ 86 , 88 ] ( Figure 1 and Figure 2 ).

Selenium primarily regulates ferroptosis by influencing selenoprotein levels. Selenocysteine is crucial for maintaining GPX4 activity, so a deficiency in selenium leads to reduced GPX4 activity and increased sensitivity to ferroptosis [ 90 , 91 ]. Coenzyme Q10 (CoQ10), also known as ubiquinone, is a fat-soluble vitamin analog produced in the inner mitochondrial membrane through the mevalonate pathway [ 92 ]. It has an antioxidant effect and protects cells from ferroptosis [ 93 , 94 ]. Its inhibitors, such as statin drugs, reduce levels of free radical scavenging and trigger ferroptosis. Sugar metabolism, including glycolysis, the pentose phosphate pathway, and the TCA pathway, plays a crucial role in the generation of ferroptosis-related compounds like NADPH, GSH, and ROS, which indirectly contribute to ferroptosis. Research indicates that glucose deprivation leads to an increase in the AMP/ATP ratio, activation of the LKB1-AMPK pathway, inhibition of PUFA synthesis, and suppression of ferroptosis. Consequently, AMP inhibitors or glucose supplementation can be utilized to promote ferroptosis [ 95 , 96 ].

Nevertheless, it has also been demonstrated that glucose deficiency, activation of the system Xc⁻ system, high expression of SLC7A11, elevated NADP+/NADPH ratio, extensive NADPH consumption, cystine accumulation, and decreased GSH levels collectively drive ferroptosis [ 97 ].

Hippo is a protein kinase, and yes-associated protein (YAP) is a transcription factor. When iron ion accumulation reaches a certain level, oxidative stress activates the Hippo pathway, which includes the oncogene NF2 and kinases [ 98 , 99 ]. These kinases phosphorylate YAP, reducing its transcriptional activity level [ 98 ]. At the same time, Hippo inhibits the G1/S transition point in the cell cycle, thereby inhibiting cell proliferation and differentiation. Additionally, the Hippo-YAP pathway promotes the G2/M transition to promote apoptosis [ 100 ].

Numerous studies have demonstrated that intestinal epithelial cells are more vulnerable to ferroptosis in a low-density environment. The mechanism is mediated by E-cadherin-mediated cell-to-cell contact, activation of the Hippo-YAP pathway via the NF2/Merlin tumor suppressor protein, and inhibition of Hippo with increased levels of YAP activity. YAP targets and regulates ACSL4 and TFRC (transferrin), which increases susceptibility to ferroptosis [ 61 , 101 ]. TAZ, an analogue of YAP, is also involved in ferroptosis in a density-regulated manner, as per the research [ 102 ].

The AMP-activated protein kinase (AMPK) is a heterotrimeric complex that regulates ATP levels via serine/threonine kinase activity. AMPK is involved in several pathways that induce ferroptosis, including AMPK-mTOR-SLC7A11, AMPK-BECN1-SLC7A11, and TIGAR-AMPK-SCD1 [ 103 - 105 ]. SLC7A11 is a key regulator of autophagy, while mTOR is a serine/threonine kinase involved in cell proliferation. Glucose starvation activates AMPK, which promotes phosphorylation of BECN1 at the S90 and S93 sites, leading to increased binding of BEC7A11 to SLC7A11 and decreased SLC7A11 binding. AMPK also downregulates SLC7A11 activity, inhibits system Xc⁻, and decreases cystine transport and GSH levels, ultimately leading to ROS accumulation and the onset of ferroptosis [ 104 , 106 ].

High levels of ROS can lead to TIGAR overexpression, which increases antioxidant capacity by reducing the NADP+/NADPH ratio and increasing GSH [ 105 , 107 ]. However, a TIGAR inhibitor can inhibit stearoyl-CoA desaturase 1 (SCD1), which induces ROS and promotes ferroptosis [ 105 , 108 ].

The nuclear factor erythrocyte 2-associated factor 2 (Nrf2) pathway is a major intracellular antioxidant stress pathway that plays an important role in oxidative stress response, cytoprotection and anti-inflammation in cells. It helps cells to resist and repair free radicals and other harmful substances [ 109 - 111 ]. Nrf2-related pathways are mainly AKT-GSK3β-Nrf2-ARE, MKLP2-APK5/PP1β-GSK3β-Nrf2, KEAP1-Nrf2.

Both protein kinase B (AKT) and glycogen synthase kinase 3 β (GSK3β) are serine/threonine kinases. They are involved in cell proliferation, signalling, etc. A certain level of ROS phosphorylates AKT at the Ser9 site and downregulates GSK3β, promoting the nuclear translocation of Nrf2 and AKT [ 112 , 113 ]. Nrf2 reduces ferroptosis by regulating the expression of genes related to iron metabolism and activating the expression of antioxidant enzymes in the cell [ 114 ]. The mitotic kinesin-like protein 2 (MKlp2) is a kinesin involved in mitosis [ 115 ]. AMPK-related protein kinase 5 (ARK5) is a kinase that has the ability to enable p53 binding activity and regulates the activity of protein phosphatase 1β (PP1β) [ 116 , 117 ]. While PP1β induces GSK3β activation and decreases nuclear Nrf2 levels, thereby promoting ferroptosis, the presence of ROS upregulates MKLP2, increases GPX4 and GSH levels, and decreases ferroptosis [ 118 , 119 ]. At the same time, MKLP2 activates ARK5 or ARK5 inhibits PP1β activity, thereby decreasing GSK3β activity, increasing nuclear translocation of Nrf2 and inhibiting ferroptosis [ 120 ]. Therefore, ARK5 inhibitors such as WZ4003 and HTH-01-015 can be used to reduce Nrf2 levels in the nucleus and drive ferroptosis [ 121 , 122 ]. In addition, Kelch-like ECH-associated protein 1 (KEAP1) binds to Nrf2, mediates its ubiquitination and degradation [ 123 ], and inhibits its nuclear translocation activity [ 124 ]. when ROS accumulate, they can be released from Nrf2 bound to KEAP1 by an oxidative reaction to increase antioxidant capacity [ 124 , 125 ].

In addition, research has shown that the absence of Nrf2 leads to the downregulation of vesicle-associated membrane protein 8, causing ferritin autophagy and the accumulation of nuclear receptor coactivator 4/apoferritin autophagosomes, which regulate ferritin synthesis and degradation, increasing LIP and thereby increasing susceptibility to ferroptosis [ 126 ].

The stress-activated protein kinase/c-Jun N-terminal kinase pathway (SAPK/JNK) plays a crucial role in regulating cell growth, apoptosis, and inflammatory response [ 127 , 128 ]. It primarily influences ferroptosis through several pathways. In an iron-deficient or hypoxic environment, activation of the SAPK/JNK pathway can enhance transferrin synthesis and iron ion uptake, leading to elevated intracellular iron levels. Simultaneously, the SAPK/JNK pathway can suppress ferroportin expression, limiting the efflux of intracellular iron and further contributing to iron accumulation, thus promoting ferroptosis [ 129 , 130 ]. Furthermore, activation of the SAPK/JNK pathway can stimulate the release of inflammatory factors, such as NF-κB, a transcription factor involved in regulating inflammation and apoptosis. SAPK/JNK activation inhibits NF-κB activity, reducing its anti-apoptotic effects and weakening its protective role against oxidative stress and apoptosis [ 131 , 132 ]. The mitogen-activated protein kinase (MAPK) signaling pathway is also involved in regulating cell growth, differentiation, and apoptosis [ 133 ]. SAPK/JNK activation can influence the activity of the MAPK pathway, impacting cell survival and death. Additionally, when activated, the SAPK/JNK pathway can trigger the P53-YAP1 pathway, decrease GPX4 levels, promote ROS accumulation, and increase susceptibility to ferroptosis [ 134 - 136 ].

Signal transduction and transcription activator 3 (STAT3) can be activated by phosphorylation of extracellular signaling molecules and then enter the nucleus, bind to DNA, regulate the transcription of ferroptosis-related genes (e.g., GPX4, superoxide dismutase, etc.) and affect the production of ROS, thereby inhibiting ferroptosis [ 137 ]. In addition, STAT3 can regulate the membrane expression of a variety of mitochondrial and cell membrane-related genes, thereby affecting mitochondrial function and cell membrane integrity, thereby affecting cell survival and death. STAT3 is highly expressed in various tumor cells [ 138 , 139 ]. Therefore, STAT3 inhibitors (e.g., isoproterenol, thiostreptozotocin, etc.) can be used to induce ferroptosis. It is worth noting that thiostreptozotocin promotes ferroptosis by downregulating GPX4 mRNA and GPX levels mainly by decreasing intracellular STAT3 and p-STAT3 expression rather than affecting SLC7A11 protein expression [ 140 ].

In addition to the main mechanism of ferroptosis mentioned above, other pathways can also regulate ferroptosis. Voltage-dependent anion channels (VDAC) are a transmembrane pathway found in the cell membrane and mitochondria. It can interfere with the expression of VDAC, affecting the transport of -related substances and mitochondrial function, leading to the accumulation of oxidant substances [ 141 ]. Fatty acid elongase 6 is an endoplasmic reticulum enzyme that inhibits GPX4 and GSH expression and ferritin heavy chain 1 (FTH1) activity by interacting with ACSL4, leading to ROS accumulation [ 142 , 143 ]. In addition, circular RNA inhibits ferroptosis by inhibiting miR-874-3p and regulating gene expression of related transmembrane proteins such as glycerophosphodiester phosphodiesterase 2 [ 144 ].

Cysteine plays a crucial role in the synthesis of GSH. Apart from being produced through the exchange of cystine and glutamate, it can also be obtained via the transsulfur pathway (TSP). Cysteine-b-synthase and cysteine gamma-lyase synthesize cysteine from the intermediate homocysteine and cysteine thioether in the presence of the sulfur donor methionine [ 145 ]. Consequently, when system Xc⁻ is inhibited, the diversion pathway can counteract ferroptosis resulting from system Xc⁻ inhibition [ 146 ]. Furthermore, research indicates that knocking out cysteine-TRNA synthetase can enhance GSH synthesis by inducing the sulfur transfer pathway, thereby inhibiting ferroptosis [ 147 ].

The mevalonate (MVA) pathway is a metabolic pathway for synthesizing isoprenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate from acetyl coenzyme A. IPP can impact the production of selenocysteine tRNA, which in turn incorporates selenocysteine into GPX4, thereby affecting its activity [ 148 ]. As a result, the MVA pathway can stimulate GPX4 synthesis and impede ferroptosis by increasing IPP levels. Squalene, CoQ10, and cholesterol generated by the MVA pathway are also implicated in ferroptosis. Squalene, an isoprenoid compound with multiple unsaturated hydrocarbons, binds various free radicals, boosts the expression of SLC7A11 and GPX4, and exerts an antioxidant effect, reducing susceptibility to ferroptosis [ 149 , 150 ]. However, depletion of CoQ10 leads to squalene synthase SQS triggering ferroptosis in response to the agonist FIN56 [ 76 , 151 ]. Cholesterol is inherently sensitive to oxidants such as hydroxyl radicals, and exogenous hydroperoxycholesterol contributes to ferroptosis in a dose-dependent manner. Specifically, 7-dehydrocholesterol, a cholesterol precursor, exhibits significantly greater oxidizing activity than cholesterol and arachidonic acid. However, there is no direct evidence that it promotes ferroptosis [ 151 - 153 ].

GCH1/ BH4 represents a lipid antioxidant pathway that operates independently of GPX4. Guanosine triphosphate cyclohydrolase (GCH1) has the ability to limit the production of tetrahydrobiopterin (BH4), which possesses lipophilic antioxidant properties [ 76 , 154 ]. Moreover, GCH1 can facilitate the restructuring of the lipid membrane environment, leading to an increase in the presence of CoQ10. Furthermore, BH4 can facilitate the conversion of phenylalanine to tyrosine, thereby promoting the synthesis of CoQ10 [ 155 ]. The combined depletion of GPX4 and the inducer of ferroptosis, PUFAPL, works synergistically to decrease the susceptibility of cancer cells to ferroptosis.

Some enzymes also play a critical role in regulating ferroptosis, including GPX4, FSP1, DHODH, iNOS, IL4i1, and others. GPX4, which was mentioned earlier, will be further elaborated on, while the following four will be the main focus.

Ferroptosis Suppressor Protein 1 (FSP1) is a glutathione-independent NADPH-ubiquinone reductase that inhibits ferroptosis by aggregating on cell membranes through myristoylation and catalyzing ubiquitin ketone to produce the antioxidant dihydroubiquinone (CoQH2). Dihydroorotate dehydrogenase (DHODH), located in the inner mitochondrial membrane, catalyzes the pyrimidine nucleotide pathway to produce CoQH2, thereby increasing antioxidant capacity [ 156 ]. Nitric oxide synthase (iNOS) can reduce lipid peroxidation and inhibit ferroptosis by producing nitric oxide [ 157 ]. Interleukin-4 inducible protein 1 (IL4i1) is a metabolic enzyme found in high levels in tumor cells, and In3Py is its metabolite. IL4i1/In3Py regulates ferroptosis by scavenging free radicals and regulating ferroptosis-related suppressor genes [ 158 ].

In the presence of FSP1, vitamin K is converted to vitamin K hydroquinone, VKH₂, a powerful lipophilic antioxidant that can neutralize lipid peroxides in the cell membrane. Vitamin K epoxide reductase complex subunit 1 contributes to the generation of VKH₂ [ 159 ]. Additionally, FSP1 also reduces CoQ10 to hydroquinone, further suppressing ferroptosis. Membrane-bound O-acyltransferase domain-containing 2 (MBOAT2), a ferroptosis inhibitor, operates independently of GPX4 or FSP1 [ 160 ]. When activated by sex hormone signaling transcription, specifically estrogen receptor and androgen receptor, MBOAT1 and MBOAT2 facilitate the restructuring of cytophospholipid composition to impede ferroptosis in cancer cells. Lipocalin-2 is a protein that carries iron and is highly expressed in the tumor microenvironment. By disrupting iron balance, it stimulates the expression of GPX4 and xCT to prevent ferroptosis [ 161 ]. B7H3 is a transmembrane protein belonging to the B7 immunostimulatory and co-inhibitory family. Research indicates that B7H3 can activate the AKT pathway and decrease sterol regulatory element binding protein 2-mediated cholesterol metabolism, thus inhibiting RSL3-mediated ferroptosis [ 162 ].