Bcl-2 Inhibition Enhances Ovarian Cancer Cells Sensitivity To Cisplatin Through Regulation Of Mitochondrial Fission Via SIRT3 Activation

Background:Ovarian cancer is a common malignancy that seriously threatens the health of women in the world.Due to the lack of effective early diagnostic method,ovarian cancer has often progressed to advanced stage and the prognosis is usually poor.The five-year survival rate is less than 40%.At present,the main treatment methods for ovarian cancer include surgical treatment and treatment with platinum drugs as the core,while the long-term application of chemotherapy drugs has severely restricted the treatment effect.Therefore,it is urgent to study the resistance mechanism of ovarian cancer cells to platinum drugs,and to find an effective method to improve the sensitivity of ovarian cancer cells to chemotherapy drugs.It has been found that cisplatin can produce cell killing by targeting mitochondrial function of damaged tumor cells.However,when cisplatin is applied,tumor cells will produce a series of mitochondrial quality controls to counteract the cytotoxicity of cisplatin.Recently,there is increasing evidence that the Bcl-2 family is closely related to mitochondrial function regulation,and it is involved in the regulation of mitochondrial autophagy,apoptosis and mitochondrial division and fusion,and is a key factor in mitochondrial quality control.In the process of apoptosis,Bcl-2 family pro-apoptotic proteins can cause mitochondrial division by inhibiting Bcl-2 family anti-apoptotic protein-mediated mitochondrial fusion.In addition,overexpression of BAX and BAK proteins can also promote mitochondrial division.However,it is unclear how the Bcl-2family of proteins affects the mitochondrial dynamics at the molecular level.The Bcl-2 family regulates mitochondrial quality through a number of pathways,including the SIRT3 protein pathway involved in the regulation of mitochondrial function.SIRT3 is a member of the NAD+-dependent histone deacetylase family.In recent years,ithas attracted much attention due to its role in tumor genetics,aging,and neurodegenerative diseases.It is located in the nucleus and mitochondria under physiological conditions.When stress or SIRT3 expression is increased,SIRT3 is transferred from the nucleus to the mitochondria.According to reports in the literature,SIRT3 is indispensable in silencing Bcl-2-mediated apoptosis.In other words,SIRT3 is an important mediator of Bcl-2regulation of apoptosis.Given that SIRT3 and Bcl-2 play important regulatory roles in regulating mitochondrial function,we sought to investigate whether SIRT3 is involved in the regulation of mitochondrial dysfunction associated with Bcl-2 function.Objective:The poor prognosis of ovarian cancer is mainly caused by chemotherapy resistance.Studies show that the Bcl-2 inhibitor ABT737 can significantly improve the effect of cisplatin and induce mitochondrial pathway apoptosis.However,the mechanism of ABT737 increases sensitivity to cisplatin by regulating mitochondrial function remains unclear in ovarian cancer cells.SIRT3,as a histone deacetylase,is involved in the regulation of mitochondrial function in cancers.In this study,we intend to explore the mechanistic link between SIRT3 and mitochondrial dysfunction induced by ABT737 and cisplatin in ovarian cancer cells.Methods:Apoptosis was examined by flow cytometry following Annexin V and PI staining.SIRT3 activity was assessed using SIRT3 deacetylase fluorometric assay.The mitochondrial membrane potential was examined by flow cytometry following JC-1 staining.Overexpression and knock-down of SIRT3 were confirmed by western blot analysis.Mitochondrial fission/fusion dynamics were detected by immunofluorescence staining or western blot analysis.Results:It was determined that the Bcl-2 inhibitor ABT737 can regulate the expression level of Bcl-2 family proteins,cause the depolarization of mitochondrial membrane potential,and eventually lead to mitochondrial apoptosis in ovarian cancer cells;Excessive activation of SIRT3 promotes SKOV3 cell apoptosis through the mitochondrial pathway.The specific knockdown of SIRT3 can partially reverse the effect of cisplatin combined with ABT737 on the mitochondrial membrane potential.SIRT3 is a key molecule that ABT737 enhances the sensitivity of SKOV3 ovarian cancer cells to cisplatin,and the expression and activity of SIRT3 have an effect on this process;Platinum combined with ABT737 had no significant effect on mitochondrial fusion.ABT737 combined with cisplatin promoted the translocation of DRP1 from the cytoplasm into mitochondria,leading to mitochondrial division.The activation of SIRT3 increased the localization of DRP1 in mitochondria,while the localization of DRP1 in cytoplasm decreased,suggesting that activation of SIRT3 caused DRP1 to translocate from cytoplasm into mitochondria,which may promote mitochondrial division.Conclusion:Using ABT737,the small molecule inhibitor Bcl-2,in combination with cisplatin,the expression of SIRT3 is up-regulated and its activity is increased,which may promote mitochondrial fission of SKOV3 ovarian cancer cells and eventually lead to mitochondrial apoptosis,thereby increasing its sensitivity to cisplatin.This study provides a new method for reversing cisplatin resistance in tumor cells.