Ferroptosis regulating lipid peroxidation metabolism in the occurrence and development of gastric cancer

Abstract

BACKGROUND

Gastric cancer is one of the most common malignant tumors in the world, and its occurrence and development involve complex biological processes. Iron death, as a new cell death mode, has attracted wide attention in recent years. However, the regulatory mechanism of iron death in gastric cancer and its effect on lipid peroxidation metabolism remain unclear.

AIM

To explore the role of iron death in the development of gastric cancer, reveal its relationship with lipid peroxidation, and provide a new theoretical basis for revealing the molecular mechanism of the occurrence and development of gastric cancer.

METHODS

The process of iron death in gastric cancer cells was simulated by cell culture model, and the occurrence of iron death was detected by fluorescence microscopy and flow cytometry. The changes of gene expression related to iron death and lipid peroxidation metabolism were analyzed by high-throughput sequencing technology. In addition, a mouse model of gastric cancer was established, and the role of iron death in vivo was studied by histology and immunohistochemistry, and the level of lipid peroxidation was detected. These methods comprehensively and deeply reveal the regulatory mechanism of iron death on lipid peroxidation metabolism in the occurrence and development of gastric cancer.

RESULTS

Iron death was significantly activated in gastric cancer cells, and at the same time, associated lipid peroxidation levels increased significantly. Through high-throughput sequencing analysis, it was found that iron death regulated the expression of several genes related to lipid metabolism. In vivo experiments demonstrated that increased iron death in gastric cancer mice was accompanied by a significant increase in lipid peroxidation.

CONCLUSION

This study confirmed the important role of iron death in regulating lipid peroxidation metabolism in the occurrence and development of gastric cancer. The activation of iron death significantly increased lipid peroxidation levels, revealing its regulatory mechanism inside the cell.

Key Words: Ferroptosis, Lipid peroxidation, Gastric cancer, Lipid metabolism, Systematic review

Core Tip: As a highly aggressive tumor, the pathophysiological mechanism of gastric cancer has attracted much attention. In recent years, factors such as ferroptosis regulation, lipid peroxidation, and metabolic abnormalities have emerged in the study of gastric cancer, providing a new perspective for understanding the development of gastric cancer. Ferroptosis regulation, lipid peroxidation, and metabolic abnormalities play important roles in the occurrence and development of gastric cancer. The regulation of ferroptosis is involved in apoptosis and necrosis, affecting cell survival and death.

INTRODUCTION

As one of the most common malignant tumors in the digestive system, gastric cancer has a high fatality rate year-round[1-4]. According to the global cancer data for 2020, the number of new gastric cancer cases accounted for 5.6%, and the death rate was 7.7%[5]. Cancer statistics in our country also show that gastric cancer is an important cause of tumor death[6-8]. Due to the insidious symptoms of early gastric cancer, treatment is often in the late stages, and the opportunity for surgery is lost[9]. Only other treatment methods can be used to kill cancer cells to improve the quality of life of patients and prolong their survival[10-16]. At present, the main methods of non-surgical cancer treatment are induced tumor cell death based on chemoradiotherapy, immunology, and molecular targeted therapy[17-20]. Ferroptosis is a new pathway of cell death induction with unique characteristics; that is, iron accumulation in cells leads to iron-dependent lipid peroxidation and amino acid metabolism disorders, resulting in cell death. More and more studies[21-25] have shown that iron death is closely related to the proliferation, metastasis, drug resistance, immunosuppression, and metabolic reprogramming of gastric cancer. The promotion of the iron death pathway in gastric cancer cells may provide a new strategy for the treatment of gastric cancer.

MATERIALS AND METHODS

Mechanism and characteristics of iron death

Iron death is a form of cell death induced by the accumulation of iron-dependent lipid peroxides[26-28]. This accumulation can be caused by a variety of factors, such as inhibition of glutathione peroxidase 4 (GPX4) activity, cysteine deficiency, and arachidonic acid (AA) peroxidation[29].

The changes are unique features of iron death, including mitochondrial shrinkage, increased double membrane density, cytoplasmic increase, and formation of lipid reactive oxygen species (ROS)[30-36]. This morphological change is different from other cell death modes (such as apoptosis, autophagy, necrosis, and pyrodeath). Iron death can be induced by both external and internal pathways[37-40]. The extrinsic pathway is achieved by inhibiting glutamate/cystine reverse transporter (System XC-) or activating ferriferin and lactoferriferin transporters, while the intrinsic pathway mainly depends on blocking the expression of antioxidant enzymes or inhibiting their activity in the cell[41-45]. System XC- is a transporter on the cell membrane, exchanging glutamic acid with cystine in equal proportions inside and outside the cell[46-50]. The cystine transported into the cell is reduced to cysteine and used as the raw material for the synthesis of glutathione (GSH). GSH acts as a cofactor in GPX4 and plays an antioxidant role, which can remove ROS accumulated in cells in time and inhibit the occurrence of iron death[51-54]. Inhibiting System XC- inhibits the production of GSH to a certain extent, leading to the weakening of cellular antioxidant capacity and the accumulation of lipid ROS, thus damaging the cell membrane and inducing iron death[55]. GPX4, as the most important antioxidant enzyme in the cell, can convert the lipid hydrogen peroxide (L-OOH) to the non-toxic lipid alcohol (L-OH) and inhibit the occurrence of iron death. Inhibition of GPX4 activity will cause an imbalance in intracellular REDOX reaction, and lipid peroxides will accumulate in cells and produce a large number of ROS, which will promote iron death induced by iron (Figure 1).

Figure 1 Mechanisms of ferroptosis. Created with BioRender.com.

Mechanism and characteristics of iron death

The buildup of iron-dependent lipid peroxides causes iron death, a type of cell death. AA peroxidation, cysteine deficiency, and inhibition of GPX4 activity are just a few of the causes of this accumulation[56]. The shape of iron death is different from other types of death because it causes changes in the mitochondria, such as mitochondrial shrinkage, increased double membrane density, cytoplasmic increase, and the production of lipid ROS[57]. This morphological change is different from other cell death modes (such as apoptosis, autophagy, necrosis, and pyrodeath). There are both internal and external pathways that can cause iron death. The extrinsic pathway is achieved by inhibiting the glutamate/cystine reverse transporter (System XC) or activating ferriferin and lactoferriferin transporters, while the intrinsic pathway mainly depends on blocking the expression of antioxidant enzymes or inhibiting their activity in the cell[58]. System XC is a transporter on the cell membrane, exchanging glutamic acid with cystine in equal proportions inside and outside the cell. The cystine transported into the cell is reduced to cysteine and used as the raw material for the synthesis of GSH[59]. GSH acts as a cofactor in GPX4 and plays an antioxidant role, which can remove ROS accumulated in cells over time and inhibit the occurrence of iron death. Inhibiting System XC inhibits the production of GSH to a certain extent, leading to the weakening of cellular antioxidant capacity and the accumulation of lipid ROS, thus damaging the cell membrane and inducing iron death. GPX4 is the cell's most important antioxidant enzyme[60]. It can change lipid hydrogen peroxide (L-OOH) into the harmless lipid alcohol (L-OH) and stop iron death from happening. Inhibition of GPX4 activity will cause an imbalance in the intracellular REDOX reaction, and lipid peroxides will accumulate in cells and produce a large number of ROS, which will promote iron death induced by iron (Figure 2).

Figure 2 The interaction of ferroptosis and lipid metabolism in tumor biology. Created with BioRender.com.

Imbalance of iron metabolism

The accumulation of iron is the first step in iron death, and free iron (Fe³⁺) in the blood binds to transferrin. Then, transferrin receptors on the cell membrane take the iron-bound transferrin molecules inside the cell[61-64]. Reductin changes Fe³⁺ to the highly active ferrous ion (Fe²⁺) form. Fe²⁺ is then moved from the body to the cytoplasm and added to the Lithium Ion Polymer (LIP), which is an unstable iron pool. In order to protect cell tissues from the destruction of free iron, the excess Fe²⁺ in the iron pool is stored in ferritin[65]. Ferritin is an iron storage protein that maintains the balance of iron metabolism within cells by storing and releasing Fe²⁺. When there is a greater need for iron, nuclear receptor coactivating ion 4 (NCOA4) mediates the release of free iron from ferritin through iron autophagy[66]. Cutting down on NCOA4 levels raises GSH, which proves that NCOA4-mediated ferritin degradation is a part of iron death. Mitochondrial ferritin (FTMT) is highly homologous to the cytoplasmic ferritin H chain (FTH) and plays a key role in iron homeostasis. The high expression of FTMT redistributes iron in the cytoplasm and prevents the accumulation of iron in the cytoplasm[67]. In addition, FTMT overexpression can inhibit the production of ROS and LIP in the process of erastin-induced iron death, thus inhibiting iron death. Studies have shown that reducing the expression level of FTMT can promote the production of ROS, which can damage the mitochondrial membrane, activate the PINK1/Parkin signal axis, and induce mitochondrial autophagy, thus promoting iron death (Figure 3).

Figure 3 Ferroptosis can occur through two major pathways, the extrinsic or transporter-dependent pathway. Created with BioRender.com.

An imbalance in lipid metabolism

Fatty acids participate in energy metabolism and signal pathway transduction and are an important part of the body[68]. However, excess lipid synthesis and oxidation by intracellular ROS to produce lipid peroxides are important features of iron death. Long-chain acyl-CoA synthases (ACSLs) play a key role in lipid anabolism. ACSL activates long-chain fatty acids, which are decomposed into acetyl-CoA (CoA) through a series of processes, such as beta oxidation, and then enter the tricarboxylic acid cycle to produce energy[69]. Long-chain ACSL4 is closely related to the iron death pathway. We found that Erastin-induced iron death was inhibited by deletion of ACSL4 expression in iron death-sensitive cells HepG2 and HL60, while transfection of ACSL4 restored the sensitivity of these cells to Erastin-induced iron death. Also, ACSL4 has a lot of long-chain polyunsaturated ϲ6 fatty acids and is found a lot on cell membranes[70]. Targeting ACSL4 with thiazolidinediones, a type of anti-diabetic compound, made iron death much more common in mouse models. Exogenous monounsaturated fatty acids, activated by long-chain ACSL3, can reduce the sensitivity of the cell membrane to oxidation and thus resist iron death. Long-chain fatty acid protein 5 and fatty acid desaturase 1 are two enzymes involved in the synthesis of oxidizable polyunsaturated fatty acids (PUFA). Studies have found that cells expressing these two enzymes are accompanied by upregulation of AA and adrenal acid and are sensitive to iron death, but not vice versa. The biosynthetic pathway of PUFA plays an important role in cellular iron death.

An imbalance in amino acid metabolism

System Xc is composed of SLC7A11 and SLC3A2 heterodimers, which can transfer cystine into cells in a 1:1 exchange of glutamate and is a bridge connecting amino acid transfer inside and outside cells. The cystine transferred into the cell can be reduced to cysteine over time and used as one of the raw materials for the synthesis of GSH[71]. GSH is a tripeptide sulfhydryl substance composed of glutamic acid, cysteine, and glycine that can remove intracellular excess over time.

Oxidizing substances maintain the dynamic balance of REDOX. Studies[72-74] have shown that inhibiting System Xc can trigger iron death by depleting GSH. It was found in pancreatic duct adenocarcinoma (PDAC) that GSH cannot induce iron death alone and needs to be regulated in cooperation with CoA, which is related to the cysteine metabolism pathway in the cell. Cystase (e), as a cysteine-consuming drug, can induce iron death in PDAC, confirming that cysteine also plays an important role in iron death. When cysteine is depleted, cysteine can be synthesized from methionine through the sulfur transfer pathway to further synthesize GSH and exert its antioxidant effect[75]. Glutamine also plays an important role in regulating iron death. Under the influence of glutaminase, it is capable of deamination[76]. HIron death is precisely regulated at multiple levels, including epigenetic, transcriptional, post-transcriptional, and post-translational levels. The transcription factor NFE2L2 plays a central role in upregulating anti-ferrofluorescent protein defense, while selective autophagy may promote iron death (Figure 4).

Figure 4 NFE2L2 in ferroptosis. A: NFE2L2 binds to KEAP1; B: NFE2L2 is separated from KEAP1. Created with BioRender.com.

RESULTS

The relationship between iron death and gastric cancer

Many studies[77-80] have shown that iron death plays an important role in the occurrence and development of a variety of diseases, such as neurological diseases, heart disease, kidney damage, and so on. The mechanism of tumor cell death is not well studied, and this is a major reason for treatment failure. Tumor cells need to ingestion more iron than non-tumor cells to meet their growth and metabolism needs; this phenomenon becomes "iron addiction". The discovery of iron death provides a new idea for tumor treatment[81]. In gastric cancer, a variety of intracellular substances can participate in the regulation of iron death, including amino acids, non-coding RNA, polypeptides, etc[82-84]. However, the regulatory mechanism of iron death in the development of gastric cancer still needs further study.

The regulatory effect of protein on iron death in gastric cancer

Human cysteine dioxygenase 1 (CDO1) can convert cysteine to taurine, thus limiting the production of GSH. In vivo and in vitro experiments have shown that inhibition of CDO1 can restore GSH levels in gastric cancer cells, reduce ROS production and lipid peroxidation, and reduce erastin-induced iron death[85-87]. Obesity and abnormal lipid metabolism are also involved in regulating the process of iron death in gastric cancer[88]. Perilipin2 (PLIN2), also known as fatty phase-related egg white (ADRP), is a protein related to fat drop differentiation. Members of the PLIN family have important physiological regulatory roles in cellular lipid metabolism and transport. PLIN2 can regulate the transcription factors PRDM11 and IPO7 to reduce the expression of ACSL3, ALOX15, LC3A, and PRDM11 and inhibit the production of ROS[89]. Cytoplasmic polyadenylate-binding protein 1 (CPEB1) is a post-transcriptional regulator. It has been found that overexpression of CPEB1 reduces the expression of twist1, an inhibitor of ATF4. Activation of the ATF4/gamma-glutamyl cyclotransferase 1 pathway, a molecule known to induce GSH degradation, and GC xenograft mouse models showed that CPEB1 overexpression promoted erastin growth inhibition against gastric cancer subcutaneous tumors[90].

The regulatory role of long non-coding RNA on iron death in gastric cancer

Long non-coding RNA (lncRNA) plays an important role in iron death and the progression of gastric cancer. The role of lncRNA in the tumor microenvironment, treatment response, and prognosis of gastric cancer was systematically evaluated through the lncRNA model related to iron death built into gastric cancer, and it was found that lncRNA can be used as a reliable biomarker to predict the prognosis and treatment response of patients with gastric cancer[91]. For example, hypoxia-induced lncRNA-CBSLR protects gastric cancer cells from iron death, leading to drug resistance. In terms of mechanism, CBSLR interacts with the YTH domain family Egg White 2 (YTHDF2) to form the CBSLR/YTHDF2/CBS signal axis. By binding the enhanced YTHDF2 to the m6A modified coding sequence of cystethione beta synthase (CBS) mRNA, the stability of CBS mRNA is reduced, while the decrease in CBS level will lead to the downregulation of ACSL4 methylation level, making it more prone to ubiquitination degradation. It can affect the iron death of gastric cancer cells. Both in vivo and in vitro experiments have confirmed that CBSLR can cause drug resistance in gastric cancer cells and reduce the occurrence of iron death[92]. Corresponding clinical data also show that patients with high expression of CBSLR have worse chemotherapy effects. The above results suggest that CBSLR may become a new target for the treatment of gastric cancer[93]. Ha et al[94] studied a new lncRNA, DACT3-AS1. Experiments done in living things and in the lab showed that DACT-AS1 was a part of cancer-associated fibroblasts and that it could spread to stomach cancer cells. By targeting the miR-181a-5p/sirtuin 1 (SIRT1) axis, cell proliferation, migration, and invasion were inhibited, and the sensitivity of gastric cancer cells to oxaliplatin was enhanced.

Regulation of iron death by micrornas in gastric cancer cells

miRNAs are associated with malignant phenotypes of various tumors, including proliferation, metastasis, drug resistance, and immunosuppression. Recent studies[95-97] have shown that mirnas are also involved in the regulation of iron death in gastric cancer. For instance, miR-375 stops the expression of SLC7A11 and lowers the stem-cell quality of stomach cancer cells by causing iron death that depends on SLC7A11[98]. Tumor-associated fibroblasts act on gastric cancer cells through the exosome pathway, and miR-522 inhibits the expression of AA lipoxygenase-15 (ALOX15) in gastric cancer cells, inhibits the accumulation of lipid ROS, and reduces the sensitivity of gastric cancer cells to chemotherapy drugs (Figure 5).

Figure 5 Oxidative damage in ferroptosis. Created with BioRender.com.

Regulation of iron death by circular RNA in gastric cancer cells

CircRNA is a closed-loop non-coding RNA that is involved in the regulation of various signaling pathways and malignant phenotypes in tumors[99]. For example, CircPVT1 promotes the proliferation and metastasis of gastric cancer by mediating the miR-423-5p/Smad3 signaling pathway. The increased expression of CircLRCH3 in gastric cancer cells can reduce the sensitivity of gastric cancer cells to oxaliplatin. The down-expression of circ_0000190 in gastric cancer cells and tissues is associated with a poor prognosis for patients, which is consistent with the previous conclusion that the low expression of hsa_circ_0000190 is associated with larger tumor size and a late TNM stage of gastric cancer[100]. Overexpression of Circ_0000190 increased intracellular iron and ROS, decreased mitochondrial membrane potential, and promoted the occurrence of iron death. miR-382-5p is the target of Circ_0000190, which promotes iron death in gastric cancer cells and inhibits tumor progression through the miR-382-5p/ZNRF3 axis[101-102]. These experiments indicate that Circ_0000190 is expected to be a new target for the prediction and treatment of gastric cancer.

DISCUSSION

Iron death plays an important role in the occurrence and development of gastric cancer[103]. In the chemotherapy of gastric cancer, it may be possible to achieve the purpose of treating gastric cancer by regulating the sensitivity of chemotherapy. In the treatment of advanced gastric cancer, cisplatin (DDP) resistance has become a major obstacle to the success of chemotherapy. A new study suggests that transcriptional activator 3 (AFT3) can make gastric cancer cells more sensitive to DDP by blocking signaling and causing iron death. Studies[104-106] have found that lncFERO exosomes in gastric cancer cells are correlated with drug resistance in gastric cancer. Iron death was stopped by lncFERO, which increased the expression of stearoyl-Coenzyme A-desaturase 1 (SCD1) and decreased the amount of PUFA in stomach cancer stem cells (GCSC). Dysregulation of the Wnt/β-catenin signaling pathway is associated with gastric cancer development and chemotherapy resistance.

The β-linked albumin/transtranscription 4 (TCF4) transcription complex protects cells from iron death by increasing the expression of GPX4[107]. There is growing evidence that targeting iron metabolism and inducing iron death may provide new avenues for treating drug-resistant cancers. For instance, the transcription factor sterol regulatory element binding protein-1ASREBP-1A in gastric cancer cells mediates apatinib's induction of iron death in gastric cancer cells by down-regulating GPX4 expression. Silenced information regulatory factor 6 (SIRT6) is a member of the Sirtuin family of NAD+-dependent enzymes[107]. Inhibition of SIRT6 can lead to inactivation of the Keap1/Nrf2 signaling pathway and downregulation of GPX4 expression. The study found that going after the SIRT6/Keap1/Nrf2/GPX4 signaling pathway might be a way to get around the fact that gastric cancer is resistant to sorafenib. Studies have shown that transcriptional activation factor 3 (STAT3) can regulate the expression of FNR-related factors (GPX4, SLC7A11, and FTH1), thereby establishing the STAT3 iron death negative regulatory axis. Targeting this pathway can trigger iron death through downregulation of FNR-related gene expression, inhibit gastric cancer progression, and restore chemotherapy sensitivity. Metastasis is an important factor in the poor prognosis of patients with gastric cancer (Figure 6).

Figure 6 Dual role of ferroptosis in tumor immunity. A: CD8+ T cells activate the iron death pathway in tumor cells; B: Activation of ferroptosis correlates with the polarization of M2 macrophages. Created with BioRender.com.

Overexpression of the cysteine protease inhibitor SN significantly promotes the migration and invasion of gastric cancer cells, which may be related to the improvement of GPX4 stability, which may become a potential target for blocking gastric cancer metastasis. A new GPX4 inhibitor called pleophyllin B (PB) has been shown to lower the expression of GPX4 in stomach cancer cells, leading to iron death[108]. This suggests that PB could be a useful drug for treating stomach cancer. A potential strategy for the treatment of gastric cancer is iron death induction by immunotherapy. By decreasing SLC7A11 expression, interferon gamma released by activated CD8+T cells causes iron death in tumor cells. Recent studies have shown that targeting iron death-related genes (FRGS) can promote CD4+T cell activation and improve immunotherapy efficacy in patients with gastric cancer[109]. L-kynurenine (L-KYN) is a metabolic substance produced by gastric cancer cells that can induce the iron death of NK cells in gastric cancer TME. Because NK cells with high GPX4 expression are resistant to the iron death that L-KYN causes, NK-92 cells made using GPX4 overexpression have the potential to treat gastric cancer.

CONCLUSION

As a new pathway to induce cell death, the biological mechanism of iron death has been studied. At the same time, iron death is also an important participant in the occurrence, development, and metastasis of gastric cancer. In the treatment of gastric cancer, iron death plays an important role in revealing new drug resistance mechanisms and regulating the sensitivity of tumor cells to chemotherapy resistance. The regulatory mechanism of the iron death pathway in gastric cancer remains unclear. Lipid oxidation appears to be at the heart of iron death. The high expression of GPX4 in gastric cancer cells seems to be a key molecule to escape iron death, and the induction of iron death may be achieved by targeting certain pathways, thus becoming a therapeutic strategy for advanced drug-resistant gastric cancer. The mechanism of iron death in the tumor microenvironment and its relationship with immune cell infiltration remain to be further explored, which may improve the long-term prognosis of gastric cancer by combining with other traditional therapies.