| Apoptosis is the programmed death of a cell,an autonomous and orderly death of a cell controlled by genes.Apoptosis is a process strictly controlled by multiple genes,and the Bcl-2 family is one of the important families involved in it.According to function,Bcl-2 family proteins can be divided into anti-apoptotic proteins,pro-apoptotic proteins and BH3-only proteins.These molecules influence and interact with each other to form a molecular network regulating apoptosis.Therefore,targeting the anti-apoptotic proteins of Bcl-2 family and inducing apoptosis of tumor cells has become an alternative strategy for cancer therapy and chemotherapy drug development.ABT737 is a kind of synthetic molecule that mimic BH3 domain,and it can induces apoptosis by targeting the anti-apoptotic proteins like Bcl-2,Bcl-xl and Mcl-1,which provides an useful tool for us to investigate the apoptosis resistance of cancer cells.SIRT3 is a member of the sirtuin family which is a kind of nicotinamide adenine NAD+-dependent deacetylases mainly locating in the mitochondria and regulates the level of mitochondrial proteins acetylation.SIRT3 participates in many cellular processes through its function of deacetylation,including metabolism,electron transport,oxidative stress,and apoptosis.SIRT3 has a dual role in cancer,showing tumor suppression in some occasions,and promoting cancer cell survival in others.Our previous study found that SIRT3 can promote apoptosis induced by Bcl-2 inhibitor S1 in ovarian cancer cell SKOV-3,which indicated that Sirt3 is involved in the apoptosis process regulated by Bcl-2 family protein.The purpose of this study was to investigate the role of SIRT3 in apoptosis induced by Bcl-2 anti-apoptosis protein inhibitor ABT737 in ovarian cancer cell SKOV-3 and its mechanism,providing the basis for clinical selection of chemotherapy drugs.Methods:(1)The MTT assay was performed to test the effect of ABT737 on the cell viability of NC and over-expression SIRT3 groups.And test the effect of ABT737 on the growth rate of NC and SIRT3 over-expression groups by Real Time Cellular Analysis(RTCA).(2)Test the rate of cell apoptosis and mitochondrial membrane potential changes by Flow Cytometry.(3)BH3 analysis test the level of mitochondrial priming.(4)Western Blot analyze the expression level of Bcl-2 family protein and HK? on mitochondrial membrane.(5)Test the acetylation level of CyclophilinD by immunoprecipitation.Results:(1)The cells in SIRT3 over-expression group are more sensitive to the cytotoxicity of ABT737,and both of the cell viability and rate of cell growth it are are lower than those in NC group.(2)The apoptosis rate of SIRT3 over-expression group was not significantly increased compared with NC group,but there was a significant increase in SIRT3 over-expression and ABT737 treatment group compared to ABT737 treatment group.(3)The mitochondrial membrane potential in the SIRT3 over-expression group was not significantly lower than that in the NC group,but the decrease in the mitochondrial membrane potential of the SIRT3 over-expression and ABT737 treatment group was significantly greater than that of the ABT737 treatment group.(4)Compared with NC group,the cells in SIRT3 over-expression group are more sensitive to BH3 peptide treatment and the rate of depolarization is higher.(5)Over express SIRT3 can up-regulate the expression of Bcl-2 pro-apoptotic proteins,including BAX,BAK,BIM and BID on mitochondria(6)Over express SIRT3 resulted in decreased acetylation level of Cyclophilin D and decreased expression of HKII in mitochondria.Conclusions:(1)SIRT3 can increase the pro-apoptotic effect of Bcl-2 anti-apoptosis protein inhibitor ABT737 on ovarian cancer cells.(2)High level of SIRT3 increase mitochondrial priming in SKOV-3 cells.(3)SIRT3 mediates the dissociation of HKII from the mitochondrial outer membrane by decreasing the acetylation level of CyclophilinD,and promotes the localization of anti-apoptotic proteins on the mitochondria,resulting in a high level of mitochondrial priming of the cells. |
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