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CISD2 protects against Erastin induced hepatocellular carcinoma ferroptosis by upregulating FSP1

CISD2 protects against Erastin induced hepatocellular carcinoma ferroptosis by upregulating FSP1 IntroductionHCC is the most common type of liver cancer with high mortality and high incidence rate. At present, HCC is affected by cirrhosis and other end-stage liver diseases, and the incidence rate is on the rise. HCC has surgery (hepatectomy, liver transplantation), local treatment (image-guided ablation and chemoembolization) and drug treatment [1, 2]. Systemic therapies such as polykinase inhibitors (such as sorafenib) have been used to treat unresectable liver cancer. Although recent progress has been made in HCC management, these drugs have shown limited survival effects [3, 4], which makes it necessary to find new HCC treatment schemes and provide patients with better medical treatment.Ferroptosis, a regulatory cell death (RCD) mode different from apoptosis and necrosis, was first discovered by Dixon team. It can be triggered by the inhibition of cystine-glutamate reverse transporter (known as system xc-, xCT) by Ras-selective lethal compounds (RSLs) and other drugs, RSLs include Erastin and Ras-selective lethal small molecule 3 (RSL3) [5, 6]. Ferroptosis has attracted more and more attention in the development of anti-cancer drugs. Sulfasalazine, sorafenib and other drugs can also induce ferroptosis of cancer cells. Many cancer cell lines are highly sensitive to ferroptosis drugs, and ferroptosis plays a key role in killing cancer cells and inhibiting the growth and proliferation of cancer cells [7]. Therefore, Erastin induced ferroptosis in HCC may be a promising cancer treatment strategy.CISD2 is a member of NEET protein family and has the characteristics of NEET protein family. It is a key protein mainly located in the outer membrane of cell mitochondria that can maintain the integrity of mitochondrial structure and energy metabolism function [8, 9]. CISD2, as a NEET protein, also participates in the process of cell aging and plays a key regulatory role in Fe and ROS homeostasis [10]. Previous studies have shown that CISD2 can inhibit the ferroptosis of head and neck cancer cells induced by sulfasalazine by inhibiting the accumulation of ferrous and lipid ROS in mitochondria [11], and CISD2 can also inhibit the accumulation of ROS in lung adenocarcinoma and promote the increase of mitochondrial membrane potential [12]. FSP1 was once known as Apoptosis-inducing factor mitochondria associated 2 (AIFM2). FSP1 is one of the oxidase of Nicotinamide adenine dinucleotide phosphate (NADPH). Recently, two teams of Doll and Bersuker found that it plays a role in inhibiting ferroptosis in the plasma membrane through the NADPH-FSP1-CoQ10 pathway, and this pathway has a parallel effect with glutathione (GSH)/GPX4 (glutathione peroxidase 4) ferroptosis inhibition axis [13], [14], [15], [16]. In conclusion, CISD2 and FSP1 are promising targets in the anti-cancer treatment of ferroptosis. However, the potential role and mechanism of CISD2 and FSP1 in regulating ferroptosis in HCC have not been determined. This study will explore whether CISD2 and FSP1 participate in the ferroptosis process of HCC induced by Erastin, and clarify their role and mechanism in the ferroptosis process of HCC induced by Erastin.Materials and methodsCell culture and ferroptosis inducing drugs treatmentThe LO2 normal liver cell lines and HepG2, Bel-7404, and SMMC-7721 liver cancer cell lines were donated by the specimen bank (Guangxi, China) and cultured in complete medium containing 10% fetal bovine serum (GEMINI, America, 900-108) in a 37 °C, 5% constant CO2 incubator (SANYO, Japan, 10050098). If the cells were treated with drug, the subsequent experiments were performed after 24 h of treatment with Erastin (TargetMOI, America, T1765, drug dissolved with DMSO, final concentration of DMSO below 0.1%).Cell viability testCells were plated in a 96-well plate (4,000 cells/well) and incubated with the CCK-8 kit (MCE, America, HY-K0301). The absorbance value of the cells at 450 nm was then measured using a multi-function microplate reader (BERTHOLD, Germany, TriStar LB941), and the cell viability was calculated according to the formula in the instruction.Detection of Fe contentThe cells to be tested were collected and processed with the Fe assay kit (BaiOLaiBo, China, GL1920). The absorbance value of cells at 562 nm was then measured using a multi-function microplate reader, and the Fe content was calculated according to the formula in the instruction.Detection of GSH contentThe cells to be tested were collected and processed with a GSH content detection kit (Solarbio, China, BC1175). The absorbance value of the cells at 412 nm was then measured using a multi-function microplate reader, and the GSH content was calculated according to the formula in the instruction.Detection of ROS contentCells were plated in 6-well plates (2.5 × 105 cells/well) and the probe was loaded with the ROS assay kit (Solarbio, China, CA1410). Cells were then visualized with an inverted multifunctional microimager (OLYMPUS, Japan, IX73 + DP80) at an excitation wavelength of 488 nm and emission wavelength of 525 nm (green fluorescence).Western blotThe cells to be tested were collected, lysed into protein with RIPA lysate (RIPA, EpiZyme, China, PC101) and PMSF protease inhibitor (PMSF, Solarbio, China, P0100) and measured by protein concentration with BCA Protein Concentration Measurement kit (Beyotime, China, Cat #P0010). Samples, SDS-PAGE protein loading buffer (Beyotime, China, P0015) and appropriate PBS were mixed and boiled. Proteins were separated by gel electrophoresis using an SDS-PAGE solution (Beyotime, China, P0014B). After electrophoresis, proteins were transferred to the PVDF membranes (Merck Millipore, Germany, Cat# ISEQ00010) using Western membrane transfer fluid (Beyotime, China, P0021B) (Merck Millipore, Germany, Cat# ISEQ00010). Then, the PVDF membranes were blocked with the Western blocking fluid (Beyotime, China, P0252). Next, The PVDF membranes were incubated with CISD2 (1:1000 dilution), FSP1 (1:1,500 dilution), and GAPDH (Proteintech, China, 10494-1-AP, 1:30000 dilution) for incubation, And the PVDF membranes were further incubated with HRP-conjugated Affinipire Goat Anti-Rabbit IgG antibody (Proteintech, China, SA00001-2, 1:10000 dilution). Finally, the PVDF membranes were visualized using ECL ultrasensitive chemiluminescence detection kit (EpiZyme, China, SQ 201) and automatic chemiluminescence image analyzer (Tanon, China, Tanon-5200 multi). Grayscale analysis was performed with the Image J software.RT-qPCRTotal RNA was extracted from the cells with a total RNA extraction kit (TIANGEN, China, DP419). Then, the purity and concentration of the total RNA were determined by a trace UV spectrophotometer (TIANGEN, China, TGEM Plus), and the total RNA was reverse transcribed into cDNA using the cDNA first strand synthesis kit (Beyotime, China, D7168L) and the gradient PCR instrument (eppendorf, Finland, mastercycler gradient). Finally, SYBR Green qPCR Mix (MonDNA, China, 130526), primers (GENEray, China), and fluorescent quantitative PCR instrument (Roche, Switzerland, LightCycle) were used to make the cDNA go through the process of pre-denaturation, denaturation, annealing and extension, and finally obtain the melting curve. Relative mRNA expression was calculated by the 2−ΔΔCt method.The primer sequence used for RT-qPCR is as follows. GAPDH Forward primer sequence: 5′-GGACCTGACCTGCCGTCTAG-3′, GAPDH Reversed primer sequence: 5′-GTAGCCCAGGATGCCCTTGA-3′. CISD2 Forward primer sequence: 5′-GTGGCCCGTATCGTGAAGG-3′, CISD2 Reversed primer sequence: 5′-CTAGCGAACCCGGTAATGCTT-3′. FSP1 Forward primer sequence: 5′-TCTTGGTTTGGTGCTTCTG-3′, FSP1 Reversed primer sequence: 5′-CCCTCTTTGCCCGAGTA-3′.Immunohistochemical stainingThe cancer tissue and adjacent tissue wax blocks from 25 patients were collected and the tissue wax blocks were cut into thin pieces and attached to adhesive glass slides. After the glass slides with tissue were dewaxed, hydrated and repaired by antigen, were incubated with CISD2 antibody (CST, America, 60758S, 1:200 dilution) and FSP1 antibody (Proteintech, China, 2003130425h, 1:350 dilution) at room temperature. Next, the glass slides with tissues were dripped with immune chromogenic reagent (MaxVision, China, 200825S407m) and DAB staining solution (MaxVision, China, 2009270805M) for coloration, and then the processes of contrast staining, differentiation, blue-return, dehydration, and sealing piece were performed. Finally, immunohistochemistry scoring was performed using an upright fluorescence microscope (OLYMPUS, Japanese, BX 53).The immunohistochemical staining results were scored as follows. FSP1 protein in cells was localized in the cytoplasm and cell membrane, and CISD2 protein in the cytoplasm was scored by pathologists according to the positive degree and the proportion of positive cells at high magnification (400×). According to the positive score, negative score is 0 point, pale yellow is one point, brown is two point, brown is three point and dark brown is four point. According to the proportion of positive cells, <5% is 0 point, 5–25% is one point, 26% is two point, 51–75% is three point, and >75% is four point. The total score of multiplying the degree of positive score and the proportion of positive cell score was taken as the final result.Plasmid amplificationEmpty vector plasmid, CISD2 overexpression vector plasmid and FSP1 overexpression vector plasmid were amplified using DH-5α competent cells (Huayueyang, China, 2110 # 09D211009D). Then, plasmid extraction was performed with an endotoxin-free plasmid bulk lift kit (TIANGEN, China, # DP117). Next, DNA concentration was determined using a trace UV spectrophotometer and purity was determined using an agarose horizontal electrophoresis tank (LIUYI, CHYA, DYCP-31DN) and an automated chemiluminescence image analyzer.Cell transfectionThe cells were plated into 6-well plates. When the cell density reached about 90%, the empty vector, CISD2 overexpression vector plasmid and FSP1 were transfected into the cells with lipofectamine 3,000 transfection reagent (invitrogen, America, 2328417), respectively.Statistical methodThe statistical analysis of data results was carried out by using the software of grahpad prism 8.0.2, and the line chart and histogram were drawn. Use the data results of repeated experiments for 3 times to verify whether the difference is statistically significant. The data in the figure is the mean ± standard deviation (x ± s). */**/*** in the figure represents p value<0.05/0.01/0.001, and it is considered that the difference is/is significant/has extremely significant statistical significance. T-test was used to analyze the differences between the two samples. The one-way ANOVA is used to analyze the differences between groups of multiple samples, and the two-way ANOVA is used to analyze the differences between groups of multiple samples with multiple factors.ResultsErastin induced ferroptosis in hepatoma cells, and HepG2 hepatoma cell line was sensitive to ErastinThe chemical structural formula of Erastin is shown (Figure 1A). CCK-8 kit was used to measure the cell viability of HepG2, Bel-7404 and SMMC-7721 hepatoma cells treated with Erastin at different concentrations (0, 2.5, 5, 10 and 20 µM). Compared with the untreated control group, Erastin reduced the cell viability of the three hepatoma cells in a dose-dependent manner (Figure 1B). In addition, HepG2 cells with median lethal dose (LD50) of 10 µM were selected as the hepatoma cell line sensitive to Erastin. In the following experiments, Erastin with a concentration of 10 µM was selected to treat HepG2 cells. Fe accumulation, GSH consumption and lipid ROS production are three key signaling events of ferroptosis. Compared with the control group, the total Fe content increased significantly after Erastin treatment (Figure 1C), while the GSH content decreased significantly (Figure 1D). In addition, ROS levels increased significantly under the influence of Erastin (Figure 1E, 100×). Taken together, these data suggest that Erastin inhibits the cell viability of hepatoma cells and promotes ferroptosis in HepG2 cells.Figure 1:Erastin induced ferroptosis in hepatoma cells, and hepG2 hepatoma cell line was sensitive to Erastin. Chemical structure formula of Erastin from the drug instruction (A). The cell viability of HepG2, Bel-7404 and SMMC-7721 cells treated with Erastin (0, 2.5, 5, 10 and 20 µM) was detected using CCK-8 kit. (B) Erastin treated hepatoma cells sensitive to it, and the relative contents of (C) total Fe and (D) GSH and (E) ROS were detected using Fe detection kit, glutathione content detection kit and ROS detection kit respectively. *p < 0.05, **p< 0.005, ***p < 0.001.The protein and mRNA expression of CISD2 and FSP1 changed significantly during the ferroptosis of HepG2 cellsSubsequently, the protein and mRNA expression of CISD2 and FSP1 after Erastin treatment were detected by Western blot and RT-qPCR. Compared with the control group, Erastin significantly up-regulated the protein and mRNA expression levels of CISD2 (Figure 2A and B), while down regulated the protein and mRNA expression levels of FSP1 (Figure 2C and D). These data showed that the protein and mRNA expression of CISD2 and FSP1 changed significantly during ferroptosis in HepG2 cells.Figure 2:The protein and mRNA expression of CISD2 and FSP1 changed significantly during the ferroptosis of HepG2 cells. The protein and mRNA expression levels of CISD2 in HepG2 cells treated with Erastin were detected by (A) Western blot and (B) RT-qPCR. And the protein and mRNA expression levels of FSP1 in HepG2 cells treated with Erastin were detected by (C) Western blot and (D) RT-qPCR. *p < 0.05, **p< 0.005, ***p < 0.001.Overexpression of CISD2 reversed the effect of Erastin on ferroptosis of HepG2 cells and Partially Restored the Regulatory Effect of FSP1 in ferroptosis of HepG2 cellsCompared with the adjacent tissues, the protein expression level of CISD2 in HCC tissues was significantly increased (Figure 3A, 400×). In order to further study the specific role of CISD2 in HCC cells, we transfected CISD2 overexpression plasmid to increase the expression level of CISD2. After transfection, the protein expression level of CISD2 was significantly up-regulated, and the protein expression level of FSP1 was also increased (Figure 3B). Then, the cell viability was detected by CCK-8 kit. Compared with the OE-NC + 10 µM Erastin group, the up-regulation of CISD2 significantly increased the cell viability of HepG2 cells (Figure 3C). In addition, we also detected the contents of total Fe and GSH in cells to analyze the role of overexpression of CISD2 in ferroptosis of HCC. The effect of Erastin on total Fe and GSH content in HepG2 cells was reversed by overexpression of CISD2 (Figure 3D and E). In addition, compared with HepG2 cells stimulated only by Erastin, the production of ROS in HepG2 cells treated with Erastin after up-regulation of CISD2 was significantly inhibited (Figure 3F, 100×). Taken together, these results suggest that overexpression of CISD2 can reverse the effects of Erastin on cell viability of HepG2 cells and ferroptosis in HCC.Figure 3:Overexpression of CISD2 reversed the effect of Erastin on ferroptosis of HepG2 cells. The protein expression level of CISD2 in HCC tissues and paired adjacent tissues was detected by IHC (A). After transfection of CISD2 overexpression plasmid, the protein expression levels of CISD2 and FSP1 in HepG2 cells were detected by Western blot to verify the transfection efficiency (B). The cell viability of HepG2 cells overexpressing CISD2 and treated with Erastin was detected using CCK-8 kit (C). The relative contents of (D) total Fe and (E) GSH, and (F) ROS levels of HepG2 cells overexpressing CISD2 and treated with Erastin were detected using the Fe detection kit, GSH content detection kit, and ROS detection kit, respectively. *p < 0.05, **p< 0.005, ***p < 0.001.Then, compared with HepG2 cells stimulated only by Erastin, the protein expression level of CISD2 was significantly up-regulated in HepG2 cells treated with Erastin after up-regulation of CISD2, and the protein expression level of FSP1 was also significantly up-regulated (Figure 4A–C). Subsequently, the string database was used to predict the potential proteins interacting with CISD2 (Figure 4D). CISD3, CIAPIN1, MRFAP1, SFXN4, WFS1, SOD1, ITPR1, BECN1, COX4I1, GIMAP5 and so on were the potential proteins of CISD2. FSP1 was not found to be an important protein of CISD2, which was the first time that we found the relationship between CISD2 and FSP1. Based on the above findings, we conclude that overexpression of CISD2 can partially restore the regulatory effect of Erastin on FSP1 in HepG2 cells.Figure 4:Overexpression of CISD2 partially restored the regulatory effect of FSP1 in ferroptosis of HepG2 cells. Western blot was used to detect the protein expression levels of CISD2 and FSP1 in HepG2 cells overexpressing CISD2 and then treated with Erastin (A–C). STRING database (https://string-db.org/) showed (D) the protein-protein interaction network of CISD2. *p < 0.05, **p< 0.005, ***p < 0.001.Overexpression of FSP1 reversed the effect of Erastin on ferroptosis of HepG2 cellsCompared with adjacent tissues, the level of FSP1 in HCC tissues was significantly reduced (Figure 5A, 400×). In addition, we also found that compared with normal liver cell line LO2, the mRNA expression level of FSP1 in HepG2, Bel-7404 and SMMC-7721 liver cancer cell lines was also significantly reduced (Figure 5B). In order to further study the specific role of FSP1 in HCC cells, we transfected FSP1 overexpression plasmid to increase the expression level of FSP1. After transfection, the protein expression level of FSP1 was significantly up-regulated (Figure 5C). Then, the cell viability was detected by CCK-8 kit. Compared with the OE-NC + 10 µM Erastin group, the up regulation of FSP1 significantly increased the cell viability of HepG2 cells (Figure 5D). In addition, we also detected the total Fe content in cells to analyze the role of FSP1 overexpression in ferroptosis of HCC. The effect of Erastin on total Fe content in HepG2 cells was reversed by overexpression of FSP1 (Figure 5E). In addition, compared with HepG2 cells stimulated only by Erastin, ROS production in HepG2 cells treated with Erastin after FSP1 up regulation was significantly inhibited (Figure 5F, 100×). Then, compared with HepG2 cells stimulated only by Erastin, the protein expression level of FSP1 in HepG2 cells treated with Erastin increased significantly after up-regulation of FSP1 (Figure 5G). Taken together, these results suggest that overexpression of FSP1 can reverse the effects of Erastin on cell viability of HepG2 cells and ferroptosis in HCC.Figure 5:Overexpression of FSP1 reversed the effect of Erastin on ferroptosis of HepG2 cells. The expression level of FSP1 protein in HCC tissues and matched adjacent tissues was detected by IHC (A). The mRNA expression level of FSP1 in normal liver cell line LO2, HepG2, Bel-7404 and SMMC-7721 was detected by RT-qPCR (B). After transfection of FSP1 overexpression plasmid, the protein expression level of FSP1 in HepG2 cells was detected by Western blot to verify the transfection efficiency (C). The cell viability of HepG2 cells overexpressing FSP1 and treated with Erastin was detected using CCK-8 kit (D). The relative content of (E) total Fe and (F) ROS in HepG2 cells treated with FSP1 and Erastin were detected using the Fe detection kit and ROS detection kit respectively. Western blot was used to detect the protein expression level of FSP1 in HepG2 cells overexpressing FSP1 and then treated with Erastin (G). *p < 0.05, **p< 0.005, ***p < 0.001.DiscussionA new type of cell death driven by Fe dependent lipid peroxidation was found in 2012. This cell death different from apoptosis and necrosis was induced by drugs such as Erastin, which was called ferroptosis, and such drugs received great attention in anti-cancer [17, 18]. Ferroptosis can occur through two main pathways, namely, exogenous/transporter dependent pathway and endogenous/enzyme regulatory pathway. Ferroptosis is caused by the redox imbalance between the production of oxidants and antioxidants, which is driven by the abnormal expression and activity of a variety of redox active enzymes that produce/remove free radicals and lipid oxidation products [19]. In this study, we found that CISD2 inhibits Erastin induced ferroptosis in HCC by up regulating FSP1, decreased Fe content, increased GSH content, and decreased ROS content. And we provided a proposed model (Figure 6).Figure 6:A proposed model for the role of CISD2 and FSP1 in regulating ferroptosis in HCC cells was provided.Fe metabolism is closely related to tumor biology. Fe promotes the production of oxygen free radicals, which may lead to ferroptosis or mutation and malignant transformation [20]. Lipid peroxidation refers to the process that oxygen combines with lipids to form peroxy free radicals through intermediates to generate lipid hydrogen peroxide. Lipids containing polyunsaturated fatty acids (PUFAs) carrying unstable diallyl hydrogen atoms are most vulnerable to lipid peroxidation. Other lipids may undergo peroxidation under extreme conditions. Lipid peroxidation is necessary for normal ferroptosis [21]. Antioxidant GSH is a tripeptide composed of glutamic acid, cysteine and glycine, and it is synthesized by the continuous action of its constituent amino acids through glutamate-cysteine ligase. The inactivation of GSH-GPX4 dependent antioxidant defense system causes the inactivation of cellular antioxidant system, which leads to the accumulation of lipid peroxides necessary for ferroptosis [22]. Therefore, Fe dependence, accumulation of lipid peroxides such as ROS and consumption of GSH are the chemical basis of ferroptosis, and they are also the key to the occurrence of ferroptosis [23]. In this study, after Erastin intervention, HepG2 cells were selected as the cells sensitive to Erastin in HCC cells. The total Fe content and ROS level in HepG2 cells increased significantly, while the cell viability and GSH content decreased, confirming the occurrence of ferroptosis in HCC cells.CISD2 protein, a new connection between cancer, Fe metabolism and ROS, is located in the endoplasmic reticulum and the outer membrane of mitochondria, supporting cell proliferation and tumor growth. CISD2 is involved in mediating the mobilization of Fe and Fe–S clusters between mitochondria and cytoplasm, thereby regulating mitochondrial Fe content and ROS metabolism [9]. Recently, FSP1 was found to protect tumors from ferroptosis by inhibiting lipid peroxidation. The identification of this FSP1 mediated process shows that drugs that inhibit FSP1 may be developed as anticancer therapy. Doll’s team screened 10,000 compounds, thereby an effective anti-FSP1 drug called FSP1 inhibitor (iFSP1) was identified, which selectively induces tumor ferroptosis [14, 16]. In this study, we observed that CISD2 was significantly up-regulated and FSP1 was significantly down regulated in Erastin treated HepG2 cells, confirming the regulatory effect of Erastin on CISD2 and FSP1 in the ferroptosis process of HCC cells. CISD2 and FSP1 were involved in the ferroptosis process of HCC cells induced by Erastin.It is reported that the abnormal expression of human gene expression regulators (such as CISD2) is crucial for the development of human aging related diseases and tumors. Overexpression of CISD2 seems to promote the occurrence of cancer [24]. CISD2 is highly expressed in many human cancer cells and tissues. For example, CISD2 is highly expressed in neuroblastoma, pancreatic cancer, prostate cancer and laryngeal squamous cell carcinoma and is closely related to disease progression [25], [26], [27], [28]. Our research group studied the expression of CISD2 in HCC and found that CISD2 was highly expressed in HCC tissues and cells, and its expression level was related to the prognosis of patients [29]. Besides, CISD2 was also found to be involved in and regulate ferroptosis in several types of cancer. For example, in the study of Shao’s team, it was found that CISD2 was abnormally overexpressed on non-small cell lung cancer, which was related to the poor overall survival of non-small cell lung cancer. It was also found that inhibiting the expression of CISD2 in non-small cell lung cancer cells promoted the accumulation of ROS in cells and mitochondria, and the morphology of mitochondria changed and function was impaired [30]. Similar results were also obtained in the study of lung adenocarcinoma of non-small cell lung cancer by Li’s team [12]. In the study of breast cancer cells by Sohn’s team, it was found that the level of CISD2 protein increased in human epithelial breast cancer cells, and inhibition of CISD2 expression in human breast cancer cells resulted in an increase in the accumulation of mitochondrial Fe and ROS [8]. At the same time, Holt’s team also studied the regulation of CISD2 on Fe and ROS content in breast cancer, and similar results were also obtained [31]. In addition, Li’s team found that knockdown of CISD2 in HCC promoted resistance to sorafenib induced ferroptosis, and the levels of ROS, MDA and Fe ion increased, but GSH did not change significantly [32]. And our research group has also confirmed through bioinformatics analysis that the high expression level of CISD2 mRNA in HCC is related to the poor prognosis of HCC patients. CISD2 may promote the survival and proliferation of tumor cells by affecting the Fe ion, energy metabolism and tumor immune microenvironment of tumor cells [33]. FSP1 has been proved to be an ferroptosis related protein. Recently, Koppulas’s team identified the CoQ-FSP1 axis as a key downstream effector of the KEAP1 (kelch-like ECH associated protein (1)-NRF2 (nuclear factor erythroid 2-related factor (2) pathway to mediate ferroptosis- and radiation-resistance in KEAP1 deficient lung cancers [34]. Therefore, we investigated whether CISD2 affects ferroptosis in HCC induced by Erastin by regulating FSP1. The results showed that overexpression of CISD2 could reverse the effect of Erastin on ferroptosis in HepG2 cells and partially restore the regulatory effect of Erastin on FSP1 in HepG2 cells. In addition, we also overexpressed FSP1 to confirm the effect of FSP1 on ferroptosis in HCC and found that overexpression of FSP1 could reverse the effect of Erastin on ferroptosis in HepG2 cells. In addition, in order to further confirm whether it is the first time to associate CISD2 with FSP1, we used the STRING database to find potential proteins that interact with CISD2. Among the obtained proteins, CISD3 and others are potential proteins of CISD2, but the interaction between CISD2 and FSP1 has not been explored, and has not been found in the research related to ferroptosis. These findings suggest that CISD2 inhibits the ferroptosis process of HCC cells induced by Erastin by promoting the expression of FSP1.ConclusionsTo sum up, this study found for the first time the relationship between CISD2 and FSP1 in the ferroptosis process of HCC induced by Erastin. In the mechanism, we have proved that CISD2 protects against Erastin induced hepatocellular carcinoma ferroptosis by upregulating FSP1, which provides a new mechanism support for the clinical treatment of HCC with ferroptosis inducing drugs such as Erastin. However, this study has not explored the specific molecular mechanism of CISD2 regulating FSP1 in ferroptosis of HCC. In the future, we will further study to solve the scientific problem of how CISD2 regulates FSP1. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png ONCOLOGIE de Gruyter

CISD2 protects against Erastin induced hepatocellular carcinoma ferroptosis by upregulating FSP1

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de Gruyter
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Abstract

IntroductionHCC is the most common type of liver cancer with high mortality and high incidence rate. At present, HCC is affected by cirrhosis and other end-stage liver diseases, and the incidence rate is on the rise. HCC has surgery (hepatectomy, liver transplantation), local treatment (image-guided ablation and chemoembolization) and drug treatment [1, 2]. Systemic therapies such as polykinase inhibitors (such as sorafenib) have been used to treat unresectable liver cancer. Although recent progress has been made in HCC management, these drugs have shown limited survival effects [3, 4], which makes it necessary to find new HCC treatment schemes and provide patients with better medical treatment.Ferroptosis, a regulatory cell death (RCD) mode different from apoptosis and necrosis, was first discovered by Dixon team. It can be triggered by the inhibition of cystine-glutamate reverse transporter (known as system xc-, xCT) by Ras-selective lethal compounds (RSLs) and other drugs, RSLs include Erastin and Ras-selective lethal small molecule 3 (RSL3) [5, 6]. Ferroptosis has attracted more and more attention in the development of anti-cancer drugs. Sulfasalazine, sorafenib and other drugs can also induce ferroptosis of cancer cells. Many cancer cell lines are highly sensitive to ferroptosis drugs, and ferroptosis plays a key role in killing cancer cells and inhibiting the growth and proliferation of cancer cells [7]. Therefore, Erastin induced ferroptosis in HCC may be a promising cancer treatment strategy.CISD2 is a member of NEET protein family and has the characteristics of NEET protein family. It is a key protein mainly located in the outer membrane of cell mitochondria that can maintain the integrity of mitochondrial structure and energy metabolism function [8, 9]. CISD2, as a NEET protein, also participates in the process of cell aging and plays a key regulatory role in Fe and ROS homeostasis [10]. Previous studies have shown that CISD2 can inhibit the ferroptosis of head and neck cancer cells induced by sulfasalazine by inhibiting the accumulation of ferrous and lipid ROS in mitochondria [11], and CISD2 can also inhibit the accumulation of ROS in lung adenocarcinoma and promote the increase of mitochondrial membrane potential [12]. FSP1 was once known as Apoptosis-inducing factor mitochondria associated 2 (AIFM2). FSP1 is one of the oxidase of Nicotinamide adenine dinucleotide phosphate (NADPH). Recently, two teams of Doll and Bersuker found that it plays a role in inhibiting ferroptosis in the plasma membrane through the NADPH-FSP1-CoQ10 pathway, and this pathway has a parallel effect with glutathione (GSH)/GPX4 (glutathione peroxidase 4) ferroptosis inhibition axis [13], [14], [15], [16]. In conclusion, CISD2 and FSP1 are promising targets in the anti-cancer treatment of ferroptosis. However, the potential role and mechanism of CISD2 and FSP1 in regulating ferroptosis in HCC have not been determined. This study will explore whether CISD2 and FSP1 participate in the ferroptosis process of HCC induced by Erastin, and clarify their role and mechanism in the ferroptosis process of HCC induced by Erastin.Materials and methodsCell culture and ferroptosis inducing drugs treatmentThe LO2 normal liver cell lines and HepG2, Bel-7404, and SMMC-7721 liver cancer cell lines were donated by the specimen bank (Guangxi, China) and cultured in complete medium containing 10% fetal bovine serum (GEMINI, America, 900-108) in a 37 °C, 5% constant CO2 incubator (SANYO, Japan, 10050098). If the cells were treated with drug, the subsequent experiments were performed after 24 h of treatment with Erastin (TargetMOI, America, T1765, drug dissolved with DMSO, final concentration of DMSO below 0.1%).Cell viability testCells were plated in a 96-well plate (4,000 cells/well) and incubated with the CCK-8 kit (MCE, America, HY-K0301). The absorbance value of the cells at 450 nm was then measured using a multi-function microplate reader (BERTHOLD, Germany, TriStar LB941), and the cell viability was calculated according to the formula in the instruction.Detection of Fe contentThe cells to be tested were collected and processed with the Fe assay kit (BaiOLaiBo, China, GL1920). The absorbance value of cells at 562 nm was then measured using a multi-function microplate reader, and the Fe content was calculated according to the formula in the instruction.Detection of GSH contentThe cells to be tested were collected and processed with a GSH content detection kit (Solarbio, China, BC1175). The absorbance value of the cells at 412 nm was then measured using a multi-function microplate reader, and the GSH content was calculated according to the formula in the instruction.Detection of ROS contentCells were plated in 6-well plates (2.5 × 105 cells/well) and the probe was loaded with the ROS assay kit (Solarbio, China, CA1410). Cells were then visualized with an inverted multifunctional microimager (OLYMPUS, Japan, IX73 + DP80) at an excitation wavelength of 488 nm and emission wavelength of 525 nm (green fluorescence).Western blotThe cells to be tested were collected, lysed into protein with RIPA lysate (RIPA, EpiZyme, China, PC101) and PMSF protease inhibitor (PMSF, Solarbio, China, P0100) and measured by protein concentration with BCA Protein Concentration Measurement kit (Beyotime, China, Cat #P0010). Samples, SDS-PAGE protein loading buffer (Beyotime, China, P0015) and appropriate PBS were mixed and boiled. Proteins were separated by gel electrophoresis using an SDS-PAGE solution (Beyotime, China, P0014B). After electrophoresis, proteins were transferred to the PVDF membranes (Merck Millipore, Germany, Cat# ISEQ00010) using Western membrane transfer fluid (Beyotime, China, P0021B) (Merck Millipore, Germany, Cat# ISEQ00010). Then, the PVDF membranes were blocked with the Western blocking fluid (Beyotime, China, P0252). Next, The PVDF membranes were incubated with CISD2 (1:1000 dilution), FSP1 (1:1,500 dilution), and GAPDH (Proteintech, China, 10494-1-AP, 1:30000 dilution) for incubation, And the PVDF membranes were further incubated with HRP-conjugated Affinipire Goat Anti-Rabbit IgG antibody (Proteintech, China, SA00001-2, 1:10000 dilution). Finally, the PVDF membranes were visualized using ECL ultrasensitive chemiluminescence detection kit (EpiZyme, China, SQ 201) and automatic chemiluminescence image analyzer (Tanon, China, Tanon-5200 multi). Grayscale analysis was performed with the Image J software.RT-qPCRTotal RNA was extracted from the cells with a total RNA extraction kit (TIANGEN, China, DP419). Then, the purity and concentration of the total RNA were determined by a trace UV spectrophotometer (TIANGEN, China, TGEM Plus), and the total RNA was reverse transcribed into cDNA using the cDNA first strand synthesis kit (Beyotime, China, D7168L) and the gradient PCR instrument (eppendorf, Finland, mastercycler gradient). Finally, SYBR Green qPCR Mix (MonDNA, China, 130526), primers (GENEray, China), and fluorescent quantitative PCR instrument (Roche, Switzerland, LightCycle) were used to make the cDNA go through the process of pre-denaturation, denaturation, annealing and extension, and finally obtain the melting curve. Relative mRNA expression was calculated by the 2−ΔΔCt method.The primer sequence used for RT-qPCR is as follows. GAPDH Forward primer sequence: 5′-GGACCTGACCTGCCGTCTAG-3′, GAPDH Reversed primer sequence: 5′-GTAGCCCAGGATGCCCTTGA-3′. CISD2 Forward primer sequence: 5′-GTGGCCCGTATCGTGAAGG-3′, CISD2 Reversed primer sequence: 5′-CTAGCGAACCCGGTAATGCTT-3′. FSP1 Forward primer sequence: 5′-TCTTGGTTTGGTGCTTCTG-3′, FSP1 Reversed primer sequence: 5′-CCCTCTTTGCCCGAGTA-3′.Immunohistochemical stainingThe cancer tissue and adjacent tissue wax blocks from 25 patients were collected and the tissue wax blocks were cut into thin pieces and attached to adhesive glass slides. After the glass slides with tissue were dewaxed, hydrated and repaired by antigen, were incubated with CISD2 antibody (CST, America, 60758S, 1:200 dilution) and FSP1 antibody (Proteintech, China, 2003130425h, 1:350 dilution) at room temperature. Next, the glass slides with tissues were dripped with immune chromogenic reagent (MaxVision, China, 200825S407m) and DAB staining solution (MaxVision, China, 2009270805M) for coloration, and then the processes of contrast staining, differentiation, blue-return, dehydration, and sealing piece were performed. Finally, immunohistochemistry scoring was performed using an upright fluorescence microscope (OLYMPUS, Japanese, BX 53).The immunohistochemical staining results were scored as follows. FSP1 protein in cells was localized in the cytoplasm and cell membrane, and CISD2 protein in the cytoplasm was scored by pathologists according to the positive degree and the proportion of positive cells at high magnification (400×). According to the positive score, negative score is 0 point, pale yellow is one point, brown is two point, brown is three point and dark brown is four point. According to the proportion of positive cells, <5% is 0 point, 5–25% is one point, 26% is two point, 51–75% is three point, and >75% is four point. The total score of multiplying the degree of positive score and the proportion of positive cell score was taken as the final result.Plasmid amplificationEmpty vector plasmid, CISD2 overexpression vector plasmid and FSP1 overexpression vector plasmid were amplified using DH-5α competent cells (Huayueyang, China, 2110 # 09D211009D). Then, plasmid extraction was performed with an endotoxin-free plasmid bulk lift kit (TIANGEN, China, # DP117). Next, DNA concentration was determined using a trace UV spectrophotometer and purity was determined using an agarose horizontal electrophoresis tank (LIUYI, CHYA, DYCP-31DN) and an automated chemiluminescence image analyzer.Cell transfectionThe cells were plated into 6-well plates. When the cell density reached about 90%, the empty vector, CISD2 overexpression vector plasmid and FSP1 were transfected into the cells with lipofectamine 3,000 transfection reagent (invitrogen, America, 2328417), respectively.Statistical methodThe statistical analysis of data results was carried out by using the software of grahpad prism 8.0.2, and the line chart and histogram were drawn. Use the data results of repeated experiments for 3 times to verify whether the difference is statistically significant. The data in the figure is the mean ± standard deviation (x ± s). */**/*** in the figure represents p value<0.05/0.01/0.001, and it is considered that the difference is/is significant/has extremely significant statistical significance. T-test was used to analyze the differences between the two samples. The one-way ANOVA is used to analyze the differences between groups of multiple samples, and the two-way ANOVA is used to analyze the differences between groups of multiple samples with multiple factors.ResultsErastin induced ferroptosis in hepatoma cells, and HepG2 hepatoma cell line was sensitive to ErastinThe chemical structural formula of Erastin is shown (Figure 1A). CCK-8 kit was used to measure the cell viability of HepG2, Bel-7404 and SMMC-7721 hepatoma cells treated with Erastin at different concentrations (0, 2.5, 5, 10 and 20 µM). Compared with the untreated control group, Erastin reduced the cell viability of the three hepatoma cells in a dose-dependent manner (Figure 1B). In addition, HepG2 cells with median lethal dose (LD50) of 10 µM were selected as the hepatoma cell line sensitive to Erastin. In the following experiments, Erastin with a concentration of 10 µM was selected to treat HepG2 cells. Fe accumulation, GSH consumption and lipid ROS production are three key signaling events of ferroptosis. Compared with the control group, the total Fe content increased significantly after Erastin treatment (Figure 1C), while the GSH content decreased significantly (Figure 1D). In addition, ROS levels increased significantly under the influence of Erastin (Figure 1E, 100×). Taken together, these data suggest that Erastin inhibits the cell viability of hepatoma cells and promotes ferroptosis in HepG2 cells.Figure 1:Erastin induced ferroptosis in hepatoma cells, and hepG2 hepatoma cell line was sensitive to Erastin. Chemical structure formula of Erastin from the drug instruction (A). The cell viability of HepG2, Bel-7404 and SMMC-7721 cells treated with Erastin (0, 2.5, 5, 10 and 20 µM) was detected using CCK-8 kit. (B) Erastin treated hepatoma cells sensitive to it, and the relative contents of (C) total Fe and (D) GSH and (E) ROS were detected using Fe detection kit, glutathione content detection kit and ROS detection kit respectively. *p < 0.05, **p< 0.005, ***p < 0.001.The protein and mRNA expression of CISD2 and FSP1 changed significantly during the ferroptosis of HepG2 cellsSubsequently, the protein and mRNA expression of CISD2 and FSP1 after Erastin treatment were detected by Western blot and RT-qPCR. Compared with the control group, Erastin significantly up-regulated the protein and mRNA expression levels of CISD2 (Figure 2A and B), while down regulated the protein and mRNA expression levels of FSP1 (Figure 2C and D). These data showed that the protein and mRNA expression of CISD2 and FSP1 changed significantly during ferroptosis in HepG2 cells.Figure 2:The protein and mRNA expression of CISD2 and FSP1 changed significantly during the ferroptosis of HepG2 cells. The protein and mRNA expression levels of CISD2 in HepG2 cells treated with Erastin were detected by (A) Western blot and (B) RT-qPCR. And the protein and mRNA expression levels of FSP1 in HepG2 cells treated with Erastin were detected by (C) Western blot and (D) RT-qPCR. *p < 0.05, **p< 0.005, ***p < 0.001.Overexpression of CISD2 reversed the effect of Erastin on ferroptosis of HepG2 cells and Partially Restored the Regulatory Effect of FSP1 in ferroptosis of HepG2 cellsCompared with the adjacent tissues, the protein expression level of CISD2 in HCC tissues was significantly increased (Figure 3A, 400×). In order to further study the specific role of CISD2 in HCC cells, we transfected CISD2 overexpression plasmid to increase the expression level of CISD2. After transfection, the protein expression level of CISD2 was significantly up-regulated, and the protein expression level of FSP1 was also increased (Figure 3B). Then, the cell viability was detected by CCK-8 kit. Compared with the OE-NC + 10 µM Erastin group, the up-regulation of CISD2 significantly increased the cell viability of HepG2 cells (Figure 3C). In addition, we also detected the contents of total Fe and GSH in cells to analyze the role of overexpression of CISD2 in ferroptosis of HCC. The effect of Erastin on total Fe and GSH content in HepG2 cells was reversed by overexpression of CISD2 (Figure 3D and E). In addition, compared with HepG2 cells stimulated only by Erastin, the production of ROS in HepG2 cells treated with Erastin after up-regulation of CISD2 was significantly inhibited (Figure 3F, 100×). Taken together, these results suggest that overexpression of CISD2 can reverse the effects of Erastin on cell viability of HepG2 cells and ferroptosis in HCC.Figure 3:Overexpression of CISD2 reversed the effect of Erastin on ferroptosis of HepG2 cells. The protein expression level of CISD2 in HCC tissues and paired adjacent tissues was detected by IHC (A). After transfection of CISD2 overexpression plasmid, the protein expression levels of CISD2 and FSP1 in HepG2 cells were detected by Western blot to verify the transfection efficiency (B). The cell viability of HepG2 cells overexpressing CISD2 and treated with Erastin was detected using CCK-8 kit (C). The relative contents of (D) total Fe and (E) GSH, and (F) ROS levels of HepG2 cells overexpressing CISD2 and treated with Erastin were detected using the Fe detection kit, GSH content detection kit, and ROS detection kit, respectively. *p < 0.05, **p< 0.005, ***p < 0.001.Then, compared with HepG2 cells stimulated only by Erastin, the protein expression level of CISD2 was significantly up-regulated in HepG2 cells treated with Erastin after up-regulation of CISD2, and the protein expression level of FSP1 was also significantly up-regulated (Figure 4A–C). Subsequently, the string database was used to predict the potential proteins interacting with CISD2 (Figure 4D). CISD3, CIAPIN1, MRFAP1, SFXN4, WFS1, SOD1, ITPR1, BECN1, COX4I1, GIMAP5 and so on were the potential proteins of CISD2. FSP1 was not found to be an important protein of CISD2, which was the first time that we found the relationship between CISD2 and FSP1. Based on the above findings, we conclude that overexpression of CISD2 can partially restore the regulatory effect of Erastin on FSP1 in HepG2 cells.Figure 4:Overexpression of CISD2 partially restored the regulatory effect of FSP1 in ferroptosis of HepG2 cells. Western blot was used to detect the protein expression levels of CISD2 and FSP1 in HepG2 cells overexpressing CISD2 and then treated with Erastin (A–C). STRING database (https://string-db.org/) showed (D) the protein-protein interaction network of CISD2. *p < 0.05, **p< 0.005, ***p < 0.001.Overexpression of FSP1 reversed the effect of Erastin on ferroptosis of HepG2 cellsCompared with adjacent tissues, the level of FSP1 in HCC tissues was significantly reduced (Figure 5A, 400×). In addition, we also found that compared with normal liver cell line LO2, the mRNA expression level of FSP1 in HepG2, Bel-7404 and SMMC-7721 liver cancer cell lines was also significantly reduced (Figure 5B). In order to further study the specific role of FSP1 in HCC cells, we transfected FSP1 overexpression plasmid to increase the expression level of FSP1. After transfection, the protein expression level of FSP1 was significantly up-regulated (Figure 5C). Then, the cell viability was detected by CCK-8 kit. Compared with the OE-NC + 10 µM Erastin group, the up regulation of FSP1 significantly increased the cell viability of HepG2 cells (Figure 5D). In addition, we also detected the total Fe content in cells to analyze the role of FSP1 overexpression in ferroptosis of HCC. The effect of Erastin on total Fe content in HepG2 cells was reversed by overexpression of FSP1 (Figure 5E). In addition, compared with HepG2 cells stimulated only by Erastin, ROS production in HepG2 cells treated with Erastin after FSP1 up regulation was significantly inhibited (Figure 5F, 100×). Then, compared with HepG2 cells stimulated only by Erastin, the protein expression level of FSP1 in HepG2 cells treated with Erastin increased significantly after up-regulation of FSP1 (Figure 5G). Taken together, these results suggest that overexpression of FSP1 can reverse the effects of Erastin on cell viability of HepG2 cells and ferroptosis in HCC.Figure 5:Overexpression of FSP1 reversed the effect of Erastin on ferroptosis of HepG2 cells. The expression level of FSP1 protein in HCC tissues and matched adjacent tissues was detected by IHC (A). The mRNA expression level of FSP1 in normal liver cell line LO2, HepG2, Bel-7404 and SMMC-7721 was detected by RT-qPCR (B). After transfection of FSP1 overexpression plasmid, the protein expression level of FSP1 in HepG2 cells was detected by Western blot to verify the transfection efficiency (C). The cell viability of HepG2 cells overexpressing FSP1 and treated with Erastin was detected using CCK-8 kit (D). The relative content of (E) total Fe and (F) ROS in HepG2 cells treated with FSP1 and Erastin were detected using the Fe detection kit and ROS detection kit respectively. Western blot was used to detect the protein expression level of FSP1 in HepG2 cells overexpressing FSP1 and then treated with Erastin (G). *p < 0.05, **p< 0.005, ***p < 0.001.DiscussionA new type of cell death driven by Fe dependent lipid peroxidation was found in 2012. This cell death different from apoptosis and necrosis was induced by drugs such as Erastin, which was called ferroptosis, and such drugs received great attention in anti-cancer [17, 18]. Ferroptosis can occur through two main pathways, namely, exogenous/transporter dependent pathway and endogenous/enzyme regulatory pathway. Ferroptosis is caused by the redox imbalance between the production of oxidants and antioxidants, which is driven by the abnormal expression and activity of a variety of redox active enzymes that produce/remove free radicals and lipid oxidation products [19]. In this study, we found that CISD2 inhibits Erastin induced ferroptosis in HCC by up regulating FSP1, decreased Fe content, increased GSH content, and decreased ROS content. And we provided a proposed model (Figure 6).Figure 6:A proposed model for the role of CISD2 and FSP1 in regulating ferroptosis in HCC cells was provided.Fe metabolism is closely related to tumor biology. Fe promotes the production of oxygen free radicals, which may lead to ferroptosis or mutation and malignant transformation [20]. Lipid peroxidation refers to the process that oxygen combines with lipids to form peroxy free radicals through intermediates to generate lipid hydrogen peroxide. Lipids containing polyunsaturated fatty acids (PUFAs) carrying unstable diallyl hydrogen atoms are most vulnerable to lipid peroxidation. Other lipids may undergo peroxidation under extreme conditions. Lipid peroxidation is necessary for normal ferroptosis [21]. Antioxidant GSH is a tripeptide composed of glutamic acid, cysteine and glycine, and it is synthesized by the continuous action of its constituent amino acids through glutamate-cysteine ligase. The inactivation of GSH-GPX4 dependent antioxidant defense system causes the inactivation of cellular antioxidant system, which leads to the accumulation of lipid peroxides necessary for ferroptosis [22]. Therefore, Fe dependence, accumulation of lipid peroxides such as ROS and consumption of GSH are the chemical basis of ferroptosis, and they are also the key to the occurrence of ferroptosis [23]. In this study, after Erastin intervention, HepG2 cells were selected as the cells sensitive to Erastin in HCC cells. The total Fe content and ROS level in HepG2 cells increased significantly, while the cell viability and GSH content decreased, confirming the occurrence of ferroptosis in HCC cells.CISD2 protein, a new connection between cancer, Fe metabolism and ROS, is located in the endoplasmic reticulum and the outer membrane of mitochondria, supporting cell proliferation and tumor growth. CISD2 is involved in mediating the mobilization of Fe and Fe–S clusters between mitochondria and cytoplasm, thereby regulating mitochondrial Fe content and ROS metabolism [9]. Recently, FSP1 was found to protect tumors from ferroptosis by inhibiting lipid peroxidation. The identification of this FSP1 mediated process shows that drugs that inhibit FSP1 may be developed as anticancer therapy. Doll’s team screened 10,000 compounds, thereby an effective anti-FSP1 drug called FSP1 inhibitor (iFSP1) was identified, which selectively induces tumor ferroptosis [14, 16]. In this study, we observed that CISD2 was significantly up-regulated and FSP1 was significantly down regulated in Erastin treated HepG2 cells, confirming the regulatory effect of Erastin on CISD2 and FSP1 in the ferroptosis process of HCC cells. CISD2 and FSP1 were involved in the ferroptosis process of HCC cells induced by Erastin.It is reported that the abnormal expression of human gene expression regulators (such as CISD2) is crucial for the development of human aging related diseases and tumors. Overexpression of CISD2 seems to promote the occurrence of cancer [24]. CISD2 is highly expressed in many human cancer cells and tissues. For example, CISD2 is highly expressed in neuroblastoma, pancreatic cancer, prostate cancer and laryngeal squamous cell carcinoma and is closely related to disease progression [25], [26], [27], [28]. Our research group studied the expression of CISD2 in HCC and found that CISD2 was highly expressed in HCC tissues and cells, and its expression level was related to the prognosis of patients [29]. Besides, CISD2 was also found to be involved in and regulate ferroptosis in several types of cancer. For example, in the study of Shao’s team, it was found that CISD2 was abnormally overexpressed on non-small cell lung cancer, which was related to the poor overall survival of non-small cell lung cancer. It was also found that inhibiting the expression of CISD2 in non-small cell lung cancer cells promoted the accumulation of ROS in cells and mitochondria, and the morphology of mitochondria changed and function was impaired [30]. Similar results were also obtained in the study of lung adenocarcinoma of non-small cell lung cancer by Li’s team [12]. In the study of breast cancer cells by Sohn’s team, it was found that the level of CISD2 protein increased in human epithelial breast cancer cells, and inhibition of CISD2 expression in human breast cancer cells resulted in an increase in the accumulation of mitochondrial Fe and ROS [8]. At the same time, Holt’s team also studied the regulation of CISD2 on Fe and ROS content in breast cancer, and similar results were also obtained [31]. In addition, Li’s team found that knockdown of CISD2 in HCC promoted resistance to sorafenib induced ferroptosis, and the levels of ROS, MDA and Fe ion increased, but GSH did not change significantly [32]. And our research group has also confirmed through bioinformatics analysis that the high expression level of CISD2 mRNA in HCC is related to the poor prognosis of HCC patients. CISD2 may promote the survival and proliferation of tumor cells by affecting the Fe ion, energy metabolism and tumor immune microenvironment of tumor cells [33]. FSP1 has been proved to be an ferroptosis related protein. Recently, Koppulas’s team identified the CoQ-FSP1 axis as a key downstream effector of the KEAP1 (kelch-like ECH associated protein (1)-NRF2 (nuclear factor erythroid 2-related factor (2) pathway to mediate ferroptosis- and radiation-resistance in KEAP1 deficient lung cancers [34]. Therefore, we investigated whether CISD2 affects ferroptosis in HCC induced by Erastin by regulating FSP1. The results showed that overexpression of CISD2 could reverse the effect of Erastin on ferroptosis in HepG2 cells and partially restore the regulatory effect of Erastin on FSP1 in HepG2 cells. In addition, we also overexpressed FSP1 to confirm the effect of FSP1 on ferroptosis in HCC and found that overexpression of FSP1 could reverse the effect of Erastin on ferroptosis in HepG2 cells. In addition, in order to further confirm whether it is the first time to associate CISD2 with FSP1, we used the STRING database to find potential proteins that interact with CISD2. Among the obtained proteins, CISD3 and others are potential proteins of CISD2, but the interaction between CISD2 and FSP1 has not been explored, and has not been found in the research related to ferroptosis. These findings suggest that CISD2 inhibits the ferroptosis process of HCC cells induced by Erastin by promoting the expression of FSP1.ConclusionsTo sum up, this study found for the first time the relationship between CISD2 and FSP1 in the ferroptosis process of HCC induced by Erastin. In the mechanism, we have proved that CISD2 protects against Erastin induced hepatocellular carcinoma ferroptosis by upregulating FSP1, which provides a new mechanism support for the clinical treatment of HCC with ferroptosis inducing drugs such as Erastin. However, this study has not explored the specific molecular mechanism of CISD2 regulating FSP1 in ferroptosis of HCC. In the future, we will further study to solve the scientific problem of how CISD2 regulates FSP1.

Journal

ONCOLOGIE – de Gruyter

Published: May 1, 2023

Keywords: CISD2; Erastin; ferroptosis; FSP1; HCC

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