The Effect Of GDF15 On Erastin-induced Ferroptosis In Gastric Cancer Cells And Its Mechanisms

Objective:Ferroptosis,a new form of regulatory cell death,is characterized by iron-dependent lipid reactive oxygen species?lipid ROS?that accumulates to lethal level.Ferroptosis can't be inhibited by apoptotic inhibitors such as necrostatin-1 or autophagy inhibitors such as chloroquine,whereas can be inhibited by iron chelators such as deferoxamine or synthetic antioxidants such as ferrostatin-1.Ferroptosis mainly involves iron metabolism,lipid peroxidation and system Xc^--GSH-GPX4 axis.Intracellular iron homeostasis is related to the import and export as well as the storage of iron.Disorder of iron regulatory proteins leads to excessive accumulation of Fe²⁺which promotes lipid ROS production by Fenton reaction.Mitochondrial fatty acid metabolism provides specific lipid precursors for ferroptosis.ROS reacts with polyunsaturated fatty acids on the lipid membrane produces lipid ROS.System Xc^-is an amino acid transporter and widely exists in phospholipid bilayer.The extracellular cystine is uptaken by system Xc^-into cells to synthesize GSH?Glutathione?.GPX4?Glutathione peroxidase 4?converts reduced GSH to oxidized glutathione,meanwhile removing lipid ROS.The light chain encoded by SLC7A11?Solute carrier family 7 member 11?is a unique subunit of system Xc^-.The expression level of SLC7A11 is usually positively correlated with the activity of system Xc^-.Ferroptosis can be regulated by pharmacology or genetics,killing cancer cells by inducing ferroptosis is a new antitumor treatment strategy.Small molecule compounds,such as erastin,sorafenib can deplete GSH through directly inhibiting the activity of system Xc^-,which increases lipid ROS accumulation and induces ferroptosis.Some studies have shown that changes of some related genes or molecules affect the process of drug-induced ferroptosis in cancer cells,for example,ATF3?Activating transcription factor 3?binds to SLC7A11 promoter inhibits system Xc^-activity and promotes erastin-induced ferroptosis.GDF15?Growth differentiation factor 15?is a member of the TGF-?superfamily.It has been reported that GDF15 level is increased in serums or cancer tissues of patients with gastric cancer or other cancers,moreover,high level of GDF15 is associated with poor prognosis,suggesting that GDF15 may be a reliable predictor of cancer progression.Cell biology research shows that GDF15 has effects on apoptosis,invasion and metastasis of cancer cells.In previous study,we found that GDF15 promoted the stem cell-like property of gastric cancer cell MGC803.The above results suggest that GDF15plays a very important role in gastric cancer and other cancers,but its mechanism is still unclear.It has been reported that GDF15 is related to hepcidin level and regulates its expression.Hepcidin leads to ferroportin degradation.GDF15 mRNA is positively correlated with the mRNA of ferroportin and FTH1?Ferritin heavy chain 1?in the blood cells of children with erythrocytic lymphocytosis.Above results indicate that GDF15may be involved in the regulation of intracellular iron metabolism,moreover,iron is a key regulator of ferroptosis.We therefore speculate that GDF15 may play a role in ferroptosis.In order to confirm our speculation,we first investigate whether erastin induce ferroptosis in human gastric cancer cell lines MGC803 and SGC7901.Then,we study the effect of GDF15 knockdown on erastin-induced ferroptosis in vitro and vivo.Last,we further study the mechanism of GDF15 in ferroptosis.We hope to understand the role and molecular mechanism of GDF15 in erastin-induced ferroptosis in human gastric cancer cells and provide a new target for the treatment of gastric cancer with ferroptosis through this study.Methods:1.Treating with erastin and ferrostatin-1 to confirm whether erastin induces ferroptosis in human gastric cancer cell lines MGC803 and SGC7901.2.MGC803 and SGC7901 cells were transfected with GDF15 specific shRNA,the expressions of GDF15 and ferroptosis related genes were examined by real-time quantitative PCR.The protein level of GDF15 and SLC7A11 were examined by Western blotting.3.MGC803 cells were transfected with ATF3 specific siRNA,mRNA and protein levels of ATF3 and SLC7A11 were examined by real-time quantitative PCR and Western blotting.4.The interaction between ATF3 and GDF15 was detected by Co-IP assay.5.After being stably transfected with GDF15 specific RNAi vector,MGC803 cells were transplanted into BALB/c mice.After tumor formation,the mice were intraperitoneal injected with erastin and the tumor size was measured,recorded and pictured.6.CCK 8 assay was used to detect the cell viability.7.Glutamate assay was used to detect the glutamate amount in the culture medium.8.Glutathione assay was used to detect the glutathione amount in cells.9.Flow cytometry was used to detect the lipid ROS level in cells.10.Inverted fluorescence microscopy was used to observe the cell status.Results:1.The viability of MGC803 and SGC7901 cells was decreased along with the erastin increase?P<0.05?.Ferrostatin-1 rescued the decreased cell viability of MGC803and SGC7901 cells induced by erastin.2.Treating MGC803 and SGC7901 cells with erastin,the cell viability of GDF15knockdown group was significantly lower than that of control group?P<0.01?.3.After erastin treatment,the tumor volume of GDF15 knockdown group was significantly lower than that of control group in BALB/c mice?P<0.05?.4.In GDF15 knockdown MGC803 cells,mRNA and protein levels of SLC7A11were significantly reduced?P<0.001?.5.In MGC803 cells,mRNA and protein levels of GDF15 and SLC7A11 were increased along with erastin increase?P<0.05?.6.In MGC803 cells,GDF15 knockdown inhibited erastin-induced protein levels increase of GDF15 and SLC7A11?P<0.05?.7.GDF15 knockdown reduced the amount of glutmate in the culture medium and glutathione in cells,whereas increased the accumulation of lipid ROS in cells?P<0.05?.8.GDF15 knockdown promoted erastin-induced the reduction of glutmate in the culture medium,the decrease of glutathione amount and the increase of lipid ROS accumulation in cells?P<0.01?.9.The levels of SLC7A11 mRNA and protein were significantly decreased after ATF3 knockdown?P<0.001?.10.GDF15 and ATF3 proteins interacted with each other.Conclusion:1.GDF15 knockdown promotes erstin-induced ferroptosis in MGC803 and SGC7901 cells as well as the antitumor activity of erastin in nude mice.2.GDF15 knockdown promotes erastin-induced ferroptosis in MGC803 cells by inhibiting SLC7A11 expression and system Xc^-function.3.ATF3 regulates SLC7A11 expression and interacts with GDF15.