The Role And Mechanism Of BCAT2 In Cancer Ferroptosis

Objective Ferroptosis is a non-apoptotic and non-necrotic programmed cell death pattern discovered in recent years.The regulatory mechanism of ferroptosis is significantly different from the traditional apoptotic or necrotic and is still in the exploratory stage.This study was to investigate the role of branched chain amino acid transaminase 2(BCAT2)in drug-induced ferroptosis in human tumor cells and to explore its potential molecular mechanism.Methods(1)CCK-8 assay was used to screen the proper concentration of Erastin,sorafenib and sulfasalazine to induce human pancreatic cancer cell(Aspc-1)and human hepatoma cancer cell(Hep G2)cell death.(2)CCK-8 assay was used to access the ability of ferroptosis inhibitor(ferrostatin-1),the apoptosis inhibitor(ZVAD-FMK),and the necrosis inhibitor(necrosulfonamide)to rescue Erastin,sorafenib and sulfasalazine induced cell death in Aspc-1 and Hep G2 cancer cells.(3)Western blot was used to access the protein expression levels of SREBP1,p-AMPK,BCAT2 and BCAT1 in Aspc-1 and Hep G2 cancer cells following Erastin,sorafenib,and sulfasalazine treatment,respectively.(4)qRT-PCR was used to access the m RNA expression levels of BCAT2 and BCAT1 in Aspc-1 and Hep G2 cancer cells following Erastin,sorafenib,and sulfasalazine treatment,respectively.(5)Chromatin immunoprecipitation(Ch IP)was used to detect the binding of SREBP1 to BCAT2 promoter region in Hep G2 cells treated with ferroptosis inducers(6)AMPK activator(AICAR)and inhibitor(Compound C)were used to treat Aspc-1and Hep G2 cells with Erastin,sorafenib and sulfasalazine respectively.The expression of BCAT2 protein in each group were detected by Western blot.(7)BCAT2 overexpressed Aspc-1 and Hep G2 cancer cells were treated with Erastin,sorafenib,and sulfasalazine.CCK-8 assay kit was used to access the cell viability;Iron Assay Kit,Malondialdehyde Assay Kit and Glutathione Assay Kit were used to detect the levels of Fe2+,malondialdehyde,and reduced glutathione,respectively;Glutamate Assay Kit was used to detect the intracellular glutamate level.(8)BCAT2 silenced Aspc-1 and HepG2 cancer cells were treated with Erastin,sorafenib,and sulfasalazine.CCK-8 assay kit was used to access the cell viability;Iron Assay Kit,Malondialdehyde Assay Kit and Glutathione Assay Kit were used to detect the levels of Fe2+,malondialdehyde,and reduced glutathione,respectively;Glutamate Assay Kit was used to detect the intracellular glutamate level;Western blot was used to detect the protein levels of BCAT2 in Aspc-1 and Hep G2 cancer cells.(9)Aspc-1 and Hep G2 cells were divided into six groups: DMSO(control group),sorafenib group,sulfasalazine group,sorafenib + BCAT2 sh RNA1 group,sulfasalazine + BCAT2 sh RNA1 group,sorafenib + sulfasalazine group.CCK-8assay was used to access the cell viability;Malondialdehyde Assay Kit was used to detect the levels of malondialdehyde in each group.(10)Aspc-1 and HepG2 cells were divided into five groups: DMSO(control group),sorafenib group,sulfasalazine group,sorafenib + sulfasalazine group,sorafenib+Sulfasalazine + ferrostatin-1 group.Western blot was used to detect the protein levels of BCAT2 in each group.(11)The Panc02 subcutaneous tumor model were treated with the following agents:DMSO(control),sorafenib,sulfasalazine,sorafenib + sulfasalazine.The tumor growth rate and BCAT2 m RNA levels were measured.Results(1)The half lethal doses of Erastin,sorafenib and sulfasalazine for Aspc-1 were 10?mol/L,5 ?mol/L,1 mmol/L,whlie for Hep G2 were 20 ?mol/L,10 ?mol/L,2mmol/L,respectively.(2)The cytotoxic effects of Erastin,sorafenib and sulfasalazine on cancer cells could be reversed by ferrostatin-1(ferroptosis inhibitor),rather than by ZVAD-FMK(apoptosis inhibitor)or necrosulfonamide(necrosis inhibitor).(3)Aspc-1 and Hep G2 cells were treated with Erastin,sorafenib,sulfasalazine,SREBP1 and BCAT2 protein expression levels were down-regulated,AMPK phosphorylation was activated,whereas BCAT1 protein levels were unchanged.(4)Aspc-1 and HepG2 cells were treated with Erastin,sorafenib,sulfasalazine,BCAT2 m RNA expression levels in human cancer cells were down-regulated,while the BCAT1 m RNA levels were not significantly changed.(5)Chromatin immunoprecipitation(ChIP)showed that the binding of SREBP1 to the BCAT2 promoter region was reduced in Hep G2 cancer cells treated with Erastin,sorafenib,sulfasalazine.(6)Compound C could reverse Erastin,sorafenib,sulfasalazine induced BCAT2 decrease.(7)BCAT2 overexpred Aspc-1 and HepG2 cancer cells were treated with Erastin,sorafenib and sulfasalazine,the levels of Fe2+ were not changed,malondialdehyde levels were decreased,while the release of glutamate,the intracellular glutamate levels and the reduced glutathione levels were increased.(8)BCAT2 silenced Aspc-1 and Hep G2 cancer cells were treated with Erastin,sorafenib and sulfasalazine,the Fe2+ levels were not changed,the malondialdehyde levels were increased,the intracellular glutamate levels and the reduced glutathione levels were decreased(9)The cell viability and the malondialdehyde levels in sorafenib + sulfasalazine treated groups were significantly lower than that in the sorafenib group,which could be reversed by ferrostatin-1(ferroptosis inhibitor).(10)The protein levels of BCAT2 in sorafenib + sulfasalazine treated groups were significantly lower than that in the sorafenib groups,which could be reversed by ferrostatin-1(ferroptosis inhibitor).(11)The tumor growth rate and BCAT2 m RNA levels in sorafenib + sulfasalazine group were significantly lower than that in the singal sorafenib or sulfasalazine group in Panc02 subcutaneous tumors.Conclusions(1)Erastin,sorafenib and sulfasalazine could induce ferroptosis in human pancreatic cancer cells and human hepatoma cancer cells.AMPK/SREBP1/BCAT2 pathway might play an important role in this process.(2)BCAT2 inhibits Erastin,sorafenib,and sulfasalazine induced ferroptosis depending on promoting intracellular glutamate production.(3)Combining sulfasalazine and sorafenib can significantly promote ferroptosis in cancer,and the down-regulation of BCAT2 might play an important role in this process.