System Xc−

System Xc− is a heterodimeric cystine/glutamate antiporter and consists of two key members, including the catalytic subunit solute carrier family 7 member 11 (SLC7A11) and solute carrier family 3 member 2 (SLC3A2). System Xc− mainly transports extracellular cystine to cells. Intracellular cystine is rapidly converted to cysteine and then functions as the precursor for glutathione biosynthesis [12] . In terms of regulating system Xc− activity in ferroptosis, SLC7A11 could interact with BECN1 and further induce ferroptosis, as well as being regulated at the transcriptional level, and a decrease in SLC7A11 could consequently induce ferroptosis [13] . The function of System Xc− is very critical, and many mechanisms affecting ferroptosis operate through this system. Erastin, a ferroptosis inducer, can cause strong and persistent inhibition of system Xc− when exposed to cells for a very short duration at low erastin concentrations [14] .

GSH

Cystine input from System Xc− is reduced intracellularly to cysteine, which then acts as a precursor for glutathione (GSH) synthesis. GSH is a tripeptide, γ-L-glutamyl-L-cysteinyl-glycine, found in all mammalian tissues and especially highly concentrated in the liver. Glutathione exists in the thiol-reduced (GSH) (99%) and disulfide-oxidized (GSSG) forms (less than 1%). GSH is synthesized in cytosol in virtually all cells involving two ATP-requiring enzymatic steps: In the first step, L-glutamate acid and L-cysteine are catalyzed by glutamate cysteine ligase (GCL) to produce γ-glutamyl-L-cysteine, and in the second step, γ-glutamyl-L-cysteine and L-glycine are catalyzed by GSH synthase (GS) to produce GSH. GSH serves several vital functions including 1) detoxifying electrophiles; 2) scavenging free radicals; 3) maintaining the essential thiol status of proteins; 4) providing a reservoir for cysteine; and 5) modulating critical cellular processes such as DNA synthesis, microtubular-related processes, and immune function. As a ferroptosis inhibitor, GSH reduces the endogenously produced hydrogen peroxide in the presence of selenium-dependent GSH peroxidase. In the process, GSH is oxidized to GSSG, which in turn is reduced back to GSH by GSSG reductase at the expense of NADPH, forming a redox cycle [15] .

GPX4

GPX4 is a key enzyme in lipid peroxidation. The GPX family comprises many members, including glutathione peroxidase 1 - 8 (GPX1 - 8). Among these enzymes, GPX4 plays an important role in ferroptosis, protecting against cell death associated with lipid peroxidation and oxidative stress [9] [16] . As the co-factor of GPX4 in catalysing peroxides into alcohols, the depletion of GSH caused by cysteine deprivation directly inactivates GPX4 and leads to subsequent induction of ferroptosis. Knockout of GPX4 in mice leads to embryonic lethality and mass lipid peroxides accumulation [17] [18] . RSL3 can inhibit the activity of GPX4 by covalent bonding with GPX4 and leads to lipid peroxide accumulation [8] . As an active-site selenocysteine, selenium is needed for the biosynthesis of GPX4. Thus, selenium deficiency promotes sensitivity to ferroptosis, while selenium supplementation promotes ferroptosis resistance. GPX4 can transform potentially toxic lipid hydroperoxides (L-OOH) into non-toxic lipid alcohols (L-OH) [19] . In the process of ferroptosis resistance, GPX4, converting PL-PUFA-OOH to PL-PUFA-OH, plays a role in eliminating Lipid peroxide (LPO) products. Although oxidizing can take place in various cell membrane lipids, peroxidation of PUFAs in phospholipids by ALOXs appears to be significant for ferroptosis [20] .

Fatty acids include saturated and unsaturated fatty acids, the latter divided into monounsaturated fatty acids (MUFA, one double bond) and polyunsaturated fatty acids (PUFA, multiple double bonds) according to the number of double bonds. The current study found that PUFA present in membrane lipids such as phospholipids (PL-PUFA), rather than free PUFA itself, is the driver of ferroptosis. The synthesis of PL-PUFA consists of two steps catalyzed by two essential enzymes, acylcoenzyme A (CoA) synthetase long-chain family member 4 (ACSL4) and lysophosphatidylcholine acyltransferase 3 (LPCAT3). Long-chain acyl-CoA synthetases (ACSLs), converting long-chain fatty acids into their acyl-CoA, including five ACSL isoenzymes identified in mammals: ACSL1, ACSL3, ACSL4, ACSL5, and ACSL6 [21] . ACSL4 has a clear preference for activating PUFAs, clearance of ACSL4 prevents the entry of polyunsaturated fatty acids into PLs membranes, and ACSL3-dependent MUFA metabolism is a key regulator of ferroptosis [22] . Limiting PUFA biosynthesis through control of acetyl-coenzyme A carboxylase (ACC), energy stress drives resistance to ferroptosis [23] . PUFA in lipid droplet first binds to Acetyl-CoA via ACSL4. Subsequently, they form PUFA-PL under the action of LPCAT3 [24] . The lipid peroxidation process, a key step in iron pyrodeath, is the oxidation of PL-PUFA to PL-PUFA-OOH, which is driven by both the labile iron pool facilitating the Fenton reaction and iron-dependent enzymes, such as lipoxygenases. LPO triggers ferroptosis [24] [25] . As mentioned above, GPX4 mediates the clearance of PL-PUFA-OOH, as known as LPO. Exogenous MUFA, mainly oleic acid (OA) and palmitoleic acid (POA), effectively inhibit ferroptosis by inhibiting the accumulation of toxic lipid reactive oxygen species (ROS), especially on the plasma membrane, and reducing the level of phospholipids containing oxidizable polyunsaturated fatty acids. The free MUFA is activated by ACSL3 into MUFA-CoA, which is incorporated into glycerophospholipids to form PL-MUFA [22] .

Iron is necessary for the accumulation of LPO and the formation of ROS. The Fenton reaction, which plays a significant role in inducing ferroptosis, catalyzes the formation of ROS in the presence of Fe²⁺. Therefore, the sensitivity of ferroptosis can be adjusted by affecting the transport, storage and turnover of iron [19] . Iron contains two oxidation states, Fe²⁺ and Fe³⁺. The serum Fe³⁺ bound to transferrin (TF) is recognized by the transferrin receptor (TFRC) on the cell membrane and transferred into the intracellular endosome, where the STEAP3 metalloreductase reduces Fe³⁺ to Fe²⁺, which is released into the cytoplasm through solute carrier family 11 member 2 (SLC11A2/DMT1) [20] .

Iron storage proteins, including ferritin light chain and ferritin heavy chain 1, can be degraded by lysosomes to increase free iron levels. This process is achieved by nuclear receptor coactivator 4 (NCOA4) mediated ferritin autophagy (a selective autophagy that degrades ferritin through lysosomes), increasing Fe²⁺ and inducing ferripyrosis through the production of reactive oxygen species mediated by the Fenton reaction [26] [27] . NCOA4-induced ferritin autophagy was strongly associated with glioblastoma [28] , pancreatic cancer [29] , ovarian cancer [30] and liver cancer [31] . Such as C-MYC, a regulatory gene closely related to proliferation, down-regulates the expression of NCOA4 through directly targeted binding, and then promotes the immune escape of ovarian cancer cells by inhibiting ferritin autophagy on ferriptosis [30] . Polypyrimidine tract-binding protein 1 (PTBP1) mediates ferroptosis in liver cancer cells by regulating NCOA4 translation [31] .

In contrast to TFRC and ferritin autophagy, which increase iron, ferritransporter (FPN) induces ferroptosis resistance by exporting iron outside the cell. Independent of TFRC, divalent metal transporter 1 (DMT1) and ferritin, siramesine (a lysosome disrupting agent) and lapatinib (a tyrosine kinase inhibitor) increase ROS in breast cancer cells by decreasing FPN expression [32] . Transported into the serum, Fe²⁺ is oxidized to Fe³⁺ by ferroxidases, such as ceruloplasmin (CP) or hephaestin (HEPH).

In summary, the System Xc−, GSH and GPX4, which act as inhibition pathways for ferroptosis, may cause a reduction in the activity or content of GPX4 and PL-PUFA-OOH scavenging suppression, when any substance in the pathway is inhibited. Then the characteristic ferroptosis occurs. Free PUFA produces PL-PUFA-OOH in a series of enzymatic reactions, and MUFA inhibits this process. Iron overload can increase the production of ROS and enhance the sensitivity of ferroptosis.

Previous studies have shown that stanniocalcin 2 (STC2), up-regulated in various tumors such as ESCC, colon cancer, liver cancer, cervical cancer and nasopharyngeal cancer, is involved in cell proliferation, migration, invasion, immune response, and drug resistance biological processes. STC2 interacts with protein methyltransferase 5 (PRMT5) and activates PRMT5. Subsequently, the inhibition of SLC7A11 expression by activating transcription factor 4 (ATF4) induces lipid peroxidation and ferroptosis, and this process can be reversed by GSK, a PRMT5 inhibitor [6] . Moreover, Circ-STC2 induces ferroptosis of nucleus pulposus cells by promoting miR-486-3p/TFR2 axis [38] . Circ-PRMT5 stimulates lymph node metastasis and distant metastasis of EC by binding to miR-203 [39] . PRMT5 inhibitors have been shown to inhibit a variety of tumors, with six oral preparations showing few side effects in mouse xenotransplantation models, and several drugs showing promising results in clinical trials of solid tumors and hematologic malignancies [40] . However, there are few articles about PRMT5 regulating iron coke death. ATF4 inhibition induces ferroptosis in hepatocellular carcinoma [41] , colorectal cancer [42] and pancreatic cancer [43] .

Gliotoxin, a compound derived from marine fungus with well-documented immunosuppressive and anticancer properties, can inhibit the growth of colon and lung cancer through apoptosis. Caspase-9 and caspase-8 are key enzymes in intrinsic and extrinsic pathways, respectively. Deficiency of SUV39H1, a gliotoxin target gene, induces ferroptosis in clear cell renal cell carcinoma. An online tool was used to identify SUV39H1 as a target gene for gliotoxin-induced ESCC ferroptosis. Inhibition of ESCC cells by gliotoxin can be reversed by ferroptosis inhibitors fer-1 and DFO, but caspase-9 and caspase-8 protein levels are not affected. Gliotoxin treatment down-regulates the expression of SLC7A11 and GPX4, but does not affect the expression of FTH1. The reducing of GSH level and increasing of MDA accumulation, ROS and Fe²⁺ levels demonstrate the induction of ferroptosis. The increase in Fe²⁺ may be modulated by other means. In addition, nude mice xenograft tumor model proved that gliotoxin has a strong ability to inhibit ESCC growth [44] .

Transcription factor NRF2, when activated, regulates cellular antioxidant response by transcriptionally activating downstream antioxidant factors such as SLC7A11, ferritin, heme oxygenase 1 (HO-1) and UDP-glucuronosyltransferase (UGT). Under normal circumstances, the rapid degradation of KEEP1-dependent proteasome keeps NRF2 in a low concentration of low activity, but under oxidative stress conditions of ROS or under electrophilic stimulation, the ubiquitination and degradation of NRF2 disappear and maintain the active state [45] . NRF2 inhibition induces ferroptosis in colorectal cancer [46] . In pancreatic cancer, aspartate aminotransferase 1 (GOT1) inhibits ferroptosis by promoting NRF2 expression [47] . Over-expressed NRF2, increasing SLC7A11 expression by binding to its promoter, inhibits ROS production and lipid peroxidation levels, and thus inhibits ESCC ferroptosis. This effect can be restored by fer-1 or SLC7A11 knockout [48] . Pizotifen malate (PZM), an NRF2 inhibitor, induces ESCC ferroptosis by increasing ROS and MDA levels and decreasing the GSH/GSSG ratio in a dose-dependent manner. The induced cell death can be saved by the lipid ROS scavengers fer-1 and Trolox. In terms of molecular mechanism, PZM plays an induction role by binding to the Neh1 domain of NRF2, preventing NRF2 protein from binding to the ARE motif of the target gene, and inhibiting NRF2 transcriptional activity [49] . There is no evidence that PZM induces ferroptosis in other cancers.

ADAM23, a receptor for neuropeptide LGI1, down-regulates SCLC7A11, SLC3A2, and GPX4 protein levels to induce ESCC ferroptosis when overexpressed. MiR-372-3p, an upstream regulator of ADAM23, inhibits ferroptosis by inhibiting ADAM23 expression. LncRNA ARHGEF26-AS1 up-regulates the expression of ADAM23 and induces ferropyrodeath through competitive binding with miR372-3p [50] .

Ferulic acid (FA), a phenolic acid substance, induces ESCC ferroptosis through down-regulates SLC7A11, GSH and GPX4, as well as increases MDA, ROS content and iron load. Induction of ferroptosis could be reversed by DFO. In addition, the increased activity of ACSL4 suggests that lipid metabolism may be involved in FA-induced ferroptosis [51] . However, FA up-regulates Nrf2 signaling in murine MIN6 cells to inhibit ferroptosis [52] . Therefore, the effect of FA on ferroptosis needs further study.

Oridonin (Ori), a natural tetracyclic diterpenoid active compound, induces ESCC ferroptosis by decreasing the GSH/GSSG ratio and GPX4 activity. This can be achieved through two ways: reducing the activity of gamma-glutamyl transpeptidase 1 (GGT1) to inhibit the gamma-glutamyl cycle, and covalently binding with cysteine to form the conjugated oridonin-cysteine (Ori-Cys) to inhibit the synthesis of GSH [33] .

DnaJ/Hsp40 homolog, subfamily B, member 6 (DNAJB6), a member of the heat shock protein 40 (Hsp40) family, negatively correlated with ESCC proliferation, migration, invasion and lymph node metastasis. It plays an anti-tumor role in inducing ESCC ferroptosis by reducing GSH, GPX4 as well as increasing MDA content [53] [54] . At present, the mechanism of DNAJB6 in ESCC is unclear, and we speculate that it directly acts on GSH, but it may also act on cysteine, cystine, or system Xc−. However, DNAJB6 can promote the invasion of colorectal cancer and play a carcinogenic role [55] . This may be due to tissue specificity. Therefore, the role of DNAJB6 in cancer needs further study.

5-aminolevulinic acid (5-ALA) can increase lipid peroxidation by inhibiting GPX4 expression. In the subcutaneous transplanted mice model, 5-ALA treatment demonstrated ESCC growth inhibition while immunohistochemistry (IHC) from tumor sections showed GPX4 expression was down-regulated [56] .

Stearoyl-CoA desaturase (SCD1), a lipase that converts saturated fatty acids to MUFA, inhibits ferroptosis by down-regulating lipid peroxidation levels in pancreatic cancer [57] , gastric cancer stem cells (GCSCs) [58] , ovarian cancer [59] and colorectal cancer [60] . BTB and CNC homology 1 (BACH1) reduces MUFA (especially OA) content by inhibiting SCD1 expression. Subsequently, reduced OA induces ESCC ferroptosis by decreasing the promoted effect of lipid membranes PUFA on lipid peroxidation [61] . MF-438, an inhibitor of SCD1, can also inhibit SCD1 expression and induce ESCC ferroptosis [62] .

Scavenger receptor protein 5 (SCARA5) plays an inhibitory role in pancreatic cancer [63] , oral squamous cell carcinoma [64] , bladder cancer [65] and nasopharyngeal carcinoma [66] by inhibiting proliferation, migration and invasion. However, its effects on ESCC proliferation and migration are contradictory and need to be confirmed by further studies [67] [68] . By increasing intracellular ROS, MDA, and Fe²⁺, SCARA5 induces ESCC ferroptosis and the characteristic cellular changes of ferroptosis with reduced mitochondrial volume, reduced mitochondrial cristae, and increased mitochondrial membrane density are observed in transmission electron microscopy. Mechanologically, SCARA5 increases intracellular ferritin light chain (FTL) concentration by binding to FTL, which can increase Fe²⁺ through NCOA4-mediated ferritinophagy and increase ROS through Fenton reaction. The simultaneous observed reduction in GSH suggests SCARA5 may affect System Xc− or directly affect GSH synthesis [68] .