Effects Of Silent Information Regulation 3(SIRT3) On The Sensitivity Of HCC Cells To Chemotherapeutic Agents And Its Mechanisms

ObjectiveTo determine the mechanism of SIRT3 regualting the sensitivity of HCC cells to chemotherapeutic agentsMethods1 SIRT3 expression in three HCC cell lines(SMMC-7721,Huh-7 and PLC/PRF/5)and one immortalized liver cell line(MIHA)treated with chemotherapeutic agents(doxorubicin,cisplatin,epirubicin)were detected by Western blot and q PCR.2 The effect of SIRT3 overexpression on cell viability of SMMC-7721,Huh-7 and PLC/PRF/5 cells treated with various concentrations of chemotherapeutic agents(doxorubicin,cisplatin,epirubicin)was dectected by MTS.3 The effect of SIRT3 overexpression on apoptotic rates in HCC cells treated with or without different chemotherapeutic agents(doxorubicin,cisplatin,epirubicin)were detected by Apoptosis Flow Cytometry.And the level of PARP cleavage,Caspase cleavage and Bax was detected by Western blot.4 The effect of SIRT3 silencing on cell viability and apoptotic rates in SMMC-7721 and Huh-7 cells treated with different chemotherapeutic agents(doxorubicin,cisplatin,epirubicin)were detected by MTS and Apoptosis Flow Cytometry,respectively.5 The expression of SIRT3 in HCC cells treated with various concentrations of sorafenib was examined by western blot.Effects of SIRT3 overexpression on cell viability and apoptotic rates were detected by MTS and Appotosis Flow Cytometry,respectively.6 Futhermore,effects of SIRT3 overexpression on the expression of GSTP1 with or without different chemotherapeutic agents(doxorubicin,cisplatin,epirubicin)in HCC cells were detected by Western blot and qPCR.And protein expression level of phosphorylated-JNK,total JNK,phosphorylated-c-Jun and total c-Jun treated with chemotherapeutic agents was detected by Western blot.Then,effects of SIRT3 overexpression on the combination of GSTP1 and JNK in SMMC-7721 cells treated with doxorubicin were detected by Co-immunoprecipitation.7 Effects of GSTP1 overexpression on apoptotic rates and PARP cleavage with or without different chemotherapeutic agents(doxorubicin,cisplatin,epirubicin)in HCC cells were detected by Appotosis Flow Cytometry and Western blot,respectively.And JNK inhibitor SP600125 inducing apoptosis in HCC cells was detected by Appotosis Flow Cytometry.Result:1 SIRT3 mRNA and protein expression in three HCC cell lines(SMMC-7721,Huh-7 and PLC/PRF/5)were less than immortalized liver cell line(MIHA).The chemotherapeutic agents significantly inhibited SIRT3 expression in HCC cells in a dose-dependent manner.2 SIRT3-overexpression significantly enhanced cellular sensitivity to three chemotherapeutic agents in SMMC-7721,Huh-7 and PLC/PRF/5.3 SIRT3-overexpression increased the apoptotic rates in HCC cells(SMMC-7721,Huh-7 and PLC/PRF/5)with the treatment of three different chemotherapeutic agents.Furthermore,SIRT3 overexpression also resulted in increased level of cleaved-PARP and cleaved-caspase 9.4 SIRT3 silencing reduced cellular sensitivity to three chemotherapeutic agents in SMMC-7721 and Huh-7 cells.5 Sorafenib inhibited SIRT3 expression in HCC cells in a dose-dependent manner.SIRT3 overexpression could enhance cellular sensitivity to sorafenib in SMMC-7721 and Huh-7 cells.6 SIRT3-overexpression reduced the mRNA and protein level of GSTP1 under different chemotherapeutic agents.And SIRT3 overexpression promoted the phosphorylation status of JNK and c-Jun.Futhermore,immunoprecipitation assay showed that SIRT3 overexpression decreased the combination of GSTP1 and JNK.7 GSTP1 overexpression or JNK inhibitor SP600125 abolished SIRT3-induced apoptosis in HCC cells exposed to three different chemotherapeutic agents.ConclusionSIRT3 overexpression enhanced the sensitivity of human hepatoma cells to chemotherapeutic agents through Glutathione S-transferase pi 1/JNK signaling pathway.