The peroxidation of lipids is an advantageous feedback chain reaction initiated by free radicals; these ROS, including superoxide (O2), peroxide (H2O2 and ROOH), and free radicals (HO and RO), catalyze polyunsaturated fatty acid oxidation (10). Mitochondria are the main producer of intracellular ROS, i.e., partial reduction inside the respiratory chain of the mitochondria (11, 12). Additionally, ROS are produced by enzymes in the endoplasmatic reticulum, peroxisomes, and the plasma membrane (such as NOX and NADPH oxidases), and additionally non-enzymatically via the Fenton reaction (13, 14). Under the catalyst effect of Fe²⁺, hydrogen peroxide creates hydroxyl radicals (HO), which is the foundation of free radical lipid peroxidation, known as the Fenton reaction. The two most common ROS that impact lipids are hydroxyl radicals (HO) and hydroperoxides (ROO) (15).

The GSH/GPX 4 axis prevents iron-catalyzed apoptosis, whereas cysteine (cys), an inhibitory amino acid in GSH synthesis that blocks cys input via SLC7A11, causes ferroptosis by lowering GSH levels.

Lipid peroxidation is a biomarker of ferroptosis (48). By changing the lipid composition of cellular membranes, abnormal lipid metabolism increases lipid peroxidation and promotes ferroptosis (49). During ferroptosis, acyl-CoA synthetase long-chain family member 4 (ACSL 4), lysophosphatidylcholine acyltransferase 3 (LPCAT 3), and arachidonic acid 15-lipoxygenase (ALOX 15) are required for lipid peroxidation. Arachidonic acid (AA), epinephrine (AdA), and phosphatidylethanolamines (PE), which are necessary for ferroptosis, have been found by quantitative lipidomics (50). ACSL4 encodes for the formation of proteins that link acetyl coenzyme A to AA during lipid synthesis to generate long-chain polyunsaturated lipoyl coenzyme A (AA-CoA). In the presence of LPCAT3, AA-CoA then interacts with PE to generate PE-AA, which then enters the lipid oxidation pathway. In the presence of LOX, PE-AA produces phospholipid peroxides. The process described above happens when ACSL4 and LPCAT3 dope PUFA (for example, arachidonic acid, AA) into membrane-localized lipids, which must be present in a membrane-bound milieu for PUFA to be lethal following peroxidation (51). Phospholipid peroxide accumulation depletes intracellular COQ10 and GSH on the one hand while increasing cellular vulnerability to ferroptosis on the other. Thus, it is clear that lipid anabolism works as a precursor to and regulator of lipid oxidation metabolism (52).

Iron is a trace element that exists in two forms in the human body: divalent iron (Fe²⁺, absorbable) and trivalent iron (Fe³⁺, non-absorbable) (68, 69). The liver is the primary organ in charge of iron metabolism, delivering and regulating the iron required for metabolism throughout the body, and there are two pathways for iron uptake: transferrin-bound iron (TBI) and non-transferrin-bound iron (NTBI). Under normal conditions, Fe²⁺ is largely imported by membrane iron transport protein (FPN) on the basolateral membrane of the duodenum, where it is oxidized to Fe³⁺ and then collected by transferrin (Tf) in plasma (70). Transferrin is a plasma glycoprotein generated by hepatocytes and transported to organs throughout the body, where it chelates circulating Fe³⁺ safely (70). The iron-loaded transferrin is then recognized by the cell membrane receptor TfR1 and translocated into endosomes via the receptor-mediated endocytosis pathway, where ferric ions are released from the transferrin in acidic endosomes and then reduced to Fe²⁺ by the enzyme prostate 3 metal reductase (STEAP 3) (71). But in conditions of iron overload, plasma transferrin’s iron-binding capability is exceeded, resulting in NTBI. Through the presence of divalent metal transporter protein 1 (DMT1, also known as soluble protein carrier [SLC11A2]) and ZRT,IRT-related protein 14 (ZIP14, also known as SLC39A14), Fe²⁺ can be transferred into the cytoplasm as an unstable iron pool (LIP) (72). To balance the redox state and intracellular iron homeostasis, excess intracellular Fe²⁺ can be translocated out of the cell via the carrier SLC40A1 (73, 74). The remainder of the iron leaves the cell via membrane iron transport protein (FPN). Ferritin is a cytosolic protein with two subunits (FTL and FTH1) that serves as the primary protein for iron storage (as Fe³⁺) (75). Iron metabolism can regulate ferroptosis via a variety of methods. Excess iron is retained in ferritin during iron overload, where it binds nuclear receptor coactivator 4 (NOCA4). NOCA4 attaches to ferritin and is destroyed by ferritin phagocytosis (76), eventually releasing free iron to participate in the Fenton reaction, which generates ROS and increases the accumulation of intracellular lipid peroxides (LPOs) and ferroptosis (77) ( Figure 2 ).

Several studies in recent years have shown that silencing TRFC (the gene encoding TFR1) inhibits erastin-induced iron apoptosis and prevents LIP accumulation, whereas the heme oxygenase HO-1 increases intracellular free iron levels and promotes heme degradation, which speeds up the ferroptosis process (78, 79). Furthermore, inhibiting iron response element binding protein 2 (IREB2) using shRNA changes the expression of various iron genes (e.g., TRFC, FTH1, and FTL), affecting iron absorption, metabolism, and storage (3). LIP is coordinated in the cytoplasm by a complex buffer of both small and large molecules, the main proteins of which are poly-r(C)-binding protein 1 (PCBP1) and PCBP2. They are required for iron chaperone action and are in charge of iron transport to ferritin. PCBP2 has also been demonstrated to preferentially interact with the iron transporter proteins divalent metallotransporter1 (DMT1) and membrane iron transporter protein to increase their iron import and export activities (80, 81). Sun et al. (78) previously established that heat shock protein beta-1 (HSPB1) is a negative regulator of ferroptosis in cancer cells. In cancer cells, erastin, a particular iron apoptosis-inducing chemical, increases heat shock factor 1 (HSF1)-dependent HSPB1 expression. HSF1 and HSPB1 knockdown promotes erastin-induced ferroptosis, but heat shock preconditioning and HSPB1 overexpression prevent erastin-induced ferroptosis. The activation of HSPB1 by protein kinase C decreased ferroptosis by lowering iron-mediated generation of lipid reactive oxygen species. Iron can also be exported by prominin-2 (prom 2)-mediated multivesicular bodies (MVB) of iron-containing proteins, and exosomes improve resistance to ferroptosis by depleting cells of iron and their potential to trigger lipid peroxidation (82). Under oxidative stress conditions, NRF2 separates from Keap1 and activates the expression of downstream target genes containing antioxidant response elements (ARE), such as iron heavy chain protein (FTH1) and iron light chain protein (FTL), promoting GSH synthesis and regenerating NADPH to exceed ROS production (36, 83). NRF2 stimulates the production of heme oxygenase-1 (HO-1) and the accumulation of Fe²⁺ in LIP as well as the release of iron from hemoglobin, hence inducing ferroptosis.

Among the numerous mechanisms that regulate ferroptosis, NRF2 is a significant regulator of the antioxidant system, and its regulation of SLC7A11 has already been discussed. ( Figure 3 ) Higher levels of oxidative stress are caused by excessive metabolic and proliferative loads in cancer cells (85). A transcription factor called NFE2-related factor 2 (NRF2) combines information from stressed cells and controls different transcriptional elements (86). NRF2, however, is no longer ubiquitinated and degraded in response to elevated oxidative stress or mutations in KEAP1, CUL3, or NRF2; this enables translated NRF2 to be translocated to the nucleus and stimulate transcription of genes with antioxidant response elements (AREs) (87, 88). NRF 2 transcriptionally induces the expression of several antioxidant defense proteins involved in iron and ROS metabolism, such as SOD1, GPX4, glutathione synthetase (GCL and GSS), HO-1, and FTH 1 (86–88). Under conditions of oxidative stress, NRF2 associates with Keap1 and activates the expression of downstream target genes containing AREs, such as ferric heavy chain proteins (FTH1) and ferric light chain proteins (FTL), which encourages GSH synthesis and regenerates NADPH to compete with ROS production (36, 83). A NRF2’s downstream antioxidant enzyme, NAD(P)H-quinone oxidoreductase 1 (NQO1), catalyzes the conversion of quinone to hydroquinone while lowering the formation of semi-hydroquinone and ROS. Meanwhile, NRF2 activated heme oxygenase-1 (HO-1) expression, which encouraged Fe²⁺ buildup in LIP and iron release from hemoglobin, as well as iron apoptosis development. A study of the drug resistance mechanism of sorafenib in hepatocellular carcinoma (HCC) cells discovered the first evidence that NFE2L2 inhibits ferroptosis (87). Sorafenib and erastin can activate the NFE2L2 pathway, resulting in the expression of antioxidant genes such as quinone oxidoreductase 1 (NQO1) and metallothionein 1G (MT1G) in HCC cells (87, 89). ABCC5, an ATP-binding cassette (ABC) protein, is a member of the ATP-dependent transporter family (90). ABCC5’s primary biological role is to expel xenobiotics and certain endogenous metabolites from cells. Sorafenib upregulates ABCC5 via the PI3K/Akt/Nrf2 pathway, which inhibits lipid peroxidation-mediated ferroptosis and promotes cancer growth, resulting in acquired sorafenib resistance in human HCC cells, according to Huang et al (91). Furthermore, ABCC5 overexpression increased SLC7A11 expression, decreased lipid peroxidation, and increased intracellular GSH levels, resulting in reduced ferroptosis (91). NRF2 activation increases the expression of the metallothionein-1 (MT1) family’s MT1G mRNA. MT1G knockdown enhances glutathione (GSH) depletion and lipid peroxidation, resulting in iron apoptosis (92). The cytosolic pro-oxidant quiescent sulfhydryl oxidase 1 (QSOX1) also enhanced ferroptosis in HCC cells by inhibiting NRF2. GSTZ1 loss in HCC can activate NRF2-associated pathways, according to recent research. GSTZ1 is thought to act as a tumor suppressor in HCC due to its reported downregulation, which results in a worse prognosis for patients and increased carcinogenesis (93, 94). Li et al. demonstrated that GSTZ1 downregulation resulted in decreased GSH and elevated ROS levels, which activated NRF2-associated pathways (94). CDGSH iron sulfur domain 2 (CISD2) is a protein that contains iron and sulfur. Sorafenib-induced ferroptosis in resistant cells was boosted by inhibiting CISD2 via ferritinophagy or the p62-Keap1-NRF2 pathway (95). Sorafenib-induced ferroptosis and cell death is aided by the downregulation of complexin II (CPLX2) and haloperidol (a sigma receptor 1 antagonist) (96, 97).

When the System Xc-GSH-GPX4 pathway is blocked, the transsulfuration pathway acts as an alternative antioxidant process to limit lipid peroxidation. The transsulfuration (TSS) route promotes ferroptosis resistance by generating cys from methionine, which is negatively controlled by cysteyl-tRNA synthetase 1 (CARS1) (84, 98). Methionine is converted to S-adenosylmethionine (SAM) by methionine adenosyltransferase (MAT), which then creates S-adenosylhomocysteine (SAH) via TSS.SAH is converted to homocysteine (HCY), a precursor of cysteine, by S-adenosylhomocysteine hydrolase (SAHH) (99, 100). Homocysteine, a metabolite of the transsulfur pathway, has been found to promote iron apoptosis rather than promote iron resistance (101). As opposed to serving as a source of cysteine, this shows that homocysteine can act independently (84). In hepatocellular carcinoma (HCC) cells, post-translational activation of cystathionine -synthase (CBS) under TNF-induced oxidative stress increases TSS activity through a proteolytically cleaved and highly active form of CBS, increasing cystathionine and GSH production (102), which in turn promotes tumor progression and inhibits ferroptosis. Using either CBS knockdown or the cystathionase (CTH) inhibitor propargylglycine (PAG) to block the TSS pathway significantly increases ferroptosis in tumor cells (98).

While mTORC2 primarily promotes cell proliferation and survival through the phosphorylation of AKT, many tumors need mTORC1 to maintain their ability to control protein synthesis and cell growth, as well as to coordinate nutrient and energy supply to ensure that they undergo unrestricted cell division (103). Mammalian target of rapamycin complex 2 (mTORC2; also known as mechanotarget of rapamycin complex 2) is a serine/threonine kinase made up of several protein components, including mTOR, and has the ability to phosphorylate a number of downstream targets to integrate upstream growth factor stimulation with cellular processes (such as cell survival) (104). By modifying SLC7A11, the mammalian target of rapamycin (mTOR) complex family of proteins controls iron apoptosis (46). High cell density, on the other hand, inhibits mTORC1 and encourages SLC7A11 lysosomal degradation while improving SLC7A11 protein stability by lowering lysosomal degradation (46). Additionally, via phosphorylating SLC7A11 at serine 26, mTORC2 reduces the functioning of the SLC7A11 transporter (105). According to Zhang et al. (106), the mTOR pathway increases GPX4 protein synthesis and sterol response element binding protein (SREBP)-mediated lipogenesis to encourage resistance to ferroptosis (107). Hepcidin transcription regulation is linked to the pathways that react to mitogen stimulation and nutritional status through components of Ras/RAF MAPK and mTOR signaling. Additionally, mTOR is crucial for iron metabolism. In murine primary hepatocytes, mTOR and low doses of RAF inhibitors promoted ferredoxin mRNA expression (108).

Particularly, when ACSL4 was overexpressed, mTOR phosphorylation greatly increased, and when ACSL4 was downregulated, mTOR phosphorylation significantly decreased. Rapamycin therapy also preserved the function of ACSL4 overexpression in increasing cell proliferation and preventing cell death (109). Additionally, according to Chen et al. (110), curcumin promoted iron apoptosis in colorectal cancer by controlling the expression of vital iron apoptosis indicators Fer 1, SLC7A11, GSH, MDA, and ROS through the PI3K/mTOR pathway. Recent studies have shown that the mTOR-related signaling system can either promote or inhibit ferroptosis in hepatocellular carcinoma cells (111, 112). For instance, HU et al. (113) found that miR-21-5p controlled the AKT/mTOR signaling pathway through MELK in vivo, reducing iron apoptosis in hepatocellular carcinoma cells. They did this by using a subcutaneous tumorigenic model in nude mice. Additionally, by phosphorylating p62, mTORC1 facilitates the binding of p62 and Keap1, which causes Keap1 to degrade and NRF2 to accumulate (107).

HCC is difficult to detect in the early stages of clinical work due to examination methods and clinical manifestations limitations. Once diagnosed, it progresses into advanced hepatocellular carcinoma that is extremely resistant to treatment and has unsatisfactory therapeutic results. Therefore, the need for innovative therapeutic approaches is urgent, and in recent years, targeted or chemotherapeutic drug delivery approaches using nanoparticles have started to emerge. Tang et al. (124) discovered that iron-catalyzed apoptosis might be induced by the degradation of manganese-silica nanoparticles themselves (through breakage of Mn-Obond). Manganese-silica nanoparticles loaded with sorafenib were created and combined with the drug’s effects. Significant anti-tumor effects were produced when applied to HCC cells. Similar to the above, Liu et al. (125) created an iron-apoptosis-associated therapy for hepatocellular cancer using a sorafenib-loaded MIL-101 (Fe) nanomedicine that was delivered in conjunction with iRGD. The neurociliin-1 (NRP-1) receptors are largely found in tumor and vascular tissues. The iRGD peptide preferentially binds to these receptors, activating the endocytosis transport route and facilitating drug penetration into tumor tissues (125). This procedure dramatically raised lipid peroxidation and malondialdehyde levels, increased auto-induced iron apoptosis in HepG 2 cells, and lowered glutathione and glutathione peroxidase 4 (GPX4) levels. This combination (125) have several promising characteristics including drug-loading, controllable release, peroxidase activity, biocompatibility, and T2 magnetic resonance imaging ability. One of the primary palliative treatment options for advanced hepatocellular carcinoma is transcatheter arterial chemoembolization (TACE). However, TACE creates a hypoxic microenvironment in the tumor that reduces the sensitivity of chemotherapy and plays a significant role in the unpredictable treatment outcome (126). Gelatin was used as the wall material for the microspheres during the preparation of adriamycin/triiron tetraoxide composite gelatin microspheres (ADM/Fe3O4-MS), and it was confirmed (127) that it is possible to use uniformly sized gelatin microspheres coloaded with-Fe3O4 nanoparticles and adriamycin to increase the effectiveness of TACE in the treatment of HCC.

Active substances derived from plants are referred to as phytochemicals. Examples include polysaccharides, polyphenols, and alkaloids. Numerous studies have demonstrated the exceptional anti-tumor efficacies of numerous phytochemicals, including baicalin and curcumin, with less adverse effects when compared to other chemotherapy medicines (128). Numerous phytochemicals have been employed as ferroptosis modulators in HCC cells to far. By encouraging iron apoptosis through the increase of ER stress and the production of PEBP1/15-LO, DHA can limit the progression of HCC (129, 130). Additionally, Heteronemin and Artesunate increased ROS and promoted ferritin degradation to induce ferroptosis, respectively, to enhance the anticancer activity of sorafenib against HCC (131). An isolated substance from Lonergania japonica called lonerganine has anti-infective properties. Lonerganine was originally used to treat HCC by Jin et al. (132). Metabolomics and a mouse xenograft model both supported the findings that lobelia alkaloids reduced tumor volume and weight and prevented hepatocyte invasion and migration. By inhibiting GPX4 and GSS, solasonine increased the amounts of lipid ROS in HepG2 cells to produce these effects (132).

Despite the advent of novel and rising HCC therapeutic techniques during the last two decades, chemotherapy remains the anticancer treatment pioneer. It is employed as a first-line therapy method for the majority of advanced cancers (133), but drug resistance poses a significant obstacle to tumor treatment. Sorafenib blocks systemic XC-mediated cystine input, causing endoplasmic reticulum stress, GSH depletion, and iron-dependent lipid ROS buildup (114). Nrf2 and metallothionein-1G (MT-1G) are two critical variables in sorafenib-resistant HCC (87). Sorafenib causes the production of P62, which inhibits NRF2 degradation and promotes the inactivation of KEAP1, followed by the activation of heme oxygenase-1 (HO-1), quinone oxidoreductase-1, and ferritin heavy chain-1, which enhances NRF2 nuclear localization. Sorafenib stimulates NRF2 expression in HCC cells, which boosts MT-1G expression, which prevents ferroptosis by inhibiting GSH depletion, which is one of the pathways associated in sorafenib resistance (87). Inhibiting any of the aforementioned pathways might thus theoretically accelerate ferroptosis in HCC cells. An iron-sulfur protein is CDGSH iron-sulfur structural domain 2 (CISD 2). CISD 2 inhibition improved sorafenib-induced iron apoptosis in drug-resistant cells via inhibiting ferritin phagocytosis or the p62-Keap1-NRF2 pathway (95).

Metformin is commonly used to treat diabetes, and its involvement in malignancies has been studied extensively. Metformin has been shown (134) to induce iron apoptosis and increase ROS levels in hepatocellular carcinoma cells via ATF4/STAT3, thereby decreasing hepatocellular carcinoma cell resistance to sorafenib. Meanwhile, metformin used with other medications to treat malignancies has produced impressive effects. Tang et al. (135) demonstrated that metformin mixed with sorafenib promoted iron apoptosis via the p62-Keap1-Nrf2/HO1 signaling pathway, inhibiting the value-added of HCC cells.

Unfortunately, chemotherapy alone is often unsatisfactory, with intrinsic and acquired resistance to conventional chemotherapeutic agents and targeted drugs developing in a short period of time. This largely limits the efficacy of the drugs and the survival of the patients. Therefore, the use of combination therapy is a viable path. A meta-analysis by Li et al. (136) demonstrated that the efficacy of combination therapy was superior to that of monotherapy. Interventional therapy, which promotes ischemic necrosis of tumor tissues by blocking the blood-supplying arteries of the tumor, has become the optimal choice for patients with advanced unresectable hepatocellular carcinoma. Drug-eluting microspheres have been widely used in the treatment of hepatocellular carcinoma for the simultaneous effects of embolization of blood vessels supplying tumors and sustained release of chemotherapeutic agents (137). However, in a hypoxic environment, vascular endothelial growth factor and HIF-1α are activated in tumor cells, increasing tumor neovascularization and the likelihood of metastasis. Ionizing radiation from radiation therapy also induced HIF-1α accumulation, which resulted in nuclear translocation of SLC7A11 and inhibited ferroptosis in HCC (138). In this situation, combination therapy shows unique advantages, CPT (an NRF2 inhibitor) (139), anti-angiogenic drugs (140), immune drugs (PD-1 inhibitors), etc. (141) in conjunction with chemotherapeutic drugs can reduce the resistance of tumor tissues.

Noncoding RNA (ncRNA) studies have revealed that ncRNAs such as microRNAs, long noncoding RNAs (lncRNAs), and circular RNAs play important roles in the carcinogenesis and progression of HCC. These non-coding RNAs are involved in tumorigenesis and progression and affect ferroptosis (142). Two homeoboxes Through activities on the Fork head Box M1 (FoxM1) protein, A pseudogene 8 (DUXAP8), a pseudogenederived lncRNA, may promote pancreatic cancer, non-small-cell lung cancer, and HCC. LncRNA DUXAP 8 is overexpressed in hepatocellular carcinoma and is associated with a poor prognosis, possibly contributing to sorafenib resistance by suppressing ferroptosis (143). Shi and colleagues (143) explored whether DUXAP 8 reduced HCC vulnerability to sorafenib-induced ferroptosis by acting on SLC7A11. Through binding to SLC7A11, DUXAP 8 decreases membrane translocation and facilitates sorting of depalmitoylated SLC7A11 to lysosomes. Thus, suppressing DUXAP8 accelerates iron mortality in HCC, and combining it with sorafenib improves treatment efficacy in patients with advanced HCC. The above results were demonstrated in CDX models at the same time. According to a recent study (144), the lncRNA HEPFAL acts on SLC7A11 and reduces its expression, resulting in a decrease in the amount of cystine transported into the cell and, as a result, a decrease in the intracellular GSH level, which effectively inhibits the iron apoptosis function of GPX4 and, ultimately, iron apoptosis in tumor cells. In the meantime, the PI3K-AKT-mTOR signaling pathway is involved in this process. Mechanistically, it could be because PI3K activates AKT via downstream molecules, and AKT activates mTOR by phosphorylation. mTORC1 is activated by AKT and governs ferroptosis through modulating a number of molecules, including SLC7A11, GPX4, and ferroportin, primarily via NRF2. In conclusion, these should be investigated further as additional research uncover the significance of RNA in ferroptosis in HCC.