| Objective:Ovarian cancer is one of the common malignant tumors in gynecology.Due to the lack of typical symptoms and signs of the disease,most patients are diagnosed as advanced when they are found.Ovarian cancer has the characteristics of high recurrence rate and high mortality rate,which brings great challenges to clinical treatment.Surgery combined with chemotherapy is now the main method for treating ovarian cancer.Platinum combined with paclitaxel is the first-line chemotherapy program in China.However,in the course of clinical treatment,we found that most patients will inevitably develop platinum resistance,which has a great impact on the effect of chemotherapy and prognosis of patients.Therefore,domestic and foreign scholars have put a lot of effort into exploring and developing new drugs.Due to its pharmacological effects such as anti-inflammatory,hemostatic and analgesic,regulating immunity,and small side effects,polyphyllin ? has been widely used in clinical practice.In recent years,studies have found that polyphyllin ? has obvious anti-cancer activity on ovarian cancer,prostate cancer,gastric cancer,nasopharyngeal cancer and colon cancer.Some studies have found that saponins can regulate the expression of Bcl-2 family proteins and induce apoptosis of mitochondrial pathway of cells by causing a decrease in mitochondrial membrane potential.Other studies have found that heavy cells can increase intracellular ROS production and decrease mitochondrial membrane potential,with the activation of the caspase pathway and the release of cytochrome c from the cytoplasm,which induces mitochondrial apoptosis.It is suggested that saponin may cause mitochondrial dysfunction of tumor cells and induce apoptosis,but the specific mechanism of regulating mitochondrial function is not clear.Therefore,investigating the specific regulation mechanism of polyphyllin ? on mitochondrial function will provide some targeted evidence for clinical treatment of ovarian cancer.Mitochondria are important organelles that generate energy in cells,and play important roles in signal transduction,cell proliferation,differentiation,autophagy,and cellular immunity.Mitochondria are dynamic organelles,whose imbalance in division and fusion often leads to changes in mitochondrial structure and dysfunction.Although mitochondrial division often occurs inapoptosis,some scholars believe that division is an essential step in the process of apoptosis,but the specific regulatory mechanism of mitochondrial division and apoptosis is unclear.Mitochondrial fission is a multi-step process in which the recruitment of GTPase DRP1 on mitochondria plays a key role.Studies have shown that Bax / Bak activation co-localizes Bax and DRP1 at the mitochondrial site where division occurs.DRP1 and the outer membrane of mitochondria stably bind,leading to mitochondrial fragmentation.Therefore,the localization of DRP1 on mitochondria may mediate mitochondrial dynamics and mitochondrial pathway apoptosis,and this process is regulated by its phosphorylation level.Protein phosphatase 2A(PP2A)is an important and ubiquitous serine threonine phosphatase and a tumor suppressor.PP2 A is a key negative regulator of tumorigenesis,affecting protein synthesis,cell proliferation,cell survival,cell migration,and invasiveness.PP2 A is inactivated in a variety of malignancies,including breast,ovarian,cervical,and lung cancer.Studies have shown that activating PP2 A is an effective method to fight cancer.It was found that the saponin glycosides could inhibit the proliferation and invasion of cisplatin-resistant cells by regulating the PP2 A / AKT signal axis.Drp1 may be a direct substrate of AKT,and its activity is regulated by AKT.The interaction between them induces phosphorylation of Drp1,leading to mitochondrial division.Therefore,we conclude that saponins can phosphorylate DRP1 and regulate its mitochondrial localization by regulating PP2 A / AKT.Here,we focus on the changes of Drp1 phosphorylation and use different concentrations of diosgenin to treat human ovarian cancer cells,and explore the relationship between changes in mitochondrial localization of Drp1 and apoptosis,and provide a new method for clinical treatment of ovarian cancer with heavy Ideas.In this study,human ovarian cancer cells A2780 and SKOV3 were treated with diosgenin at different concentrations,and the relationship between changes in DRP1 mitochondrial localization and apoptosis was explored through changes in DRP1 phosphorylation levels.It was clarified that the polyphyllin ? regulated the mitochondrial division through the PP2 A / AKT / DRP1 axis,and then promoted cell apoptosis,which provided a new idea for the clinical application of the saponin glycosides treated with ovarian cancer.Experimental method:(1)MTT method was used to detect the effect of Polyphyllin ? on the viability of human ovarian cancer cells A2780 and SKOV3 cells;apoptosis was detected by flow cytometry after Annexin V-PI staining.(2)To observe the effect of Polyphyllin ? on mitochondrial function of ovarian cancer cells.DCFH-DA,JC-1 staining flow cytometry was used to detect changes in ROS and mitochondrial membrane potentials of cells.Mitochondria were extracted using a mitochondrial extraction kit.Western Blot was used to detect changes in the expression of apoptosis-related proteins Bcl-2 and Bax on mitochondria and the activation of Capase3.(3)To observe the effect of Polyphyllin ? on mitochondrial dynamics of ovarian cancer cells.After Mito Tracker staining,the morphology of mitochondria was observed under a microscope.Western Blot was used to detect changes in mitochondrial localization of Drp1 and changes in p-AKT and p-Drp1.The PP2 A ELISA kit was used to detect changes in PP2 A activity.(4)To explore the regulatory relationship between Polyphyllin ? and PP2 A / AKT / Drp1 signal axis,and the role played by Drp1.PP2 A inhibitor LB-100 was used as a tool for ovarian cancer cells.Western Blot was used to detect changes in mitochondrial localization of Drp1,release of Cyto C,and changes in p-AKT and p-Drp1.The PP2 A ELISA kit was used to detect changes in PP2 A activity.After Mito Tracker staining,the fluorescent secondary antibody stained Drp1,and the co-localization of the two was observed with a fluorescence microscope.Result:(1)Polyphyllin ? acts on A2780 and SKOV3 cells,which significantly inhibits cell viability,promotes its apoptosis in a dose-dependent manner,and Polyphyllin ? 3mM can significantly inhibit cell viability and significantly promote apoptosis.(2)Polyphyllin ? can obviously induce ROS generation in A2780 and SKOV3 cells,leading to a decrease in mitochondrial membrane potential.Western Blot detected the expression level of apoptosis-related proteins,the expression of pro-apoptotic protein Bcl-2 increased,the expression of anti-apoptotic protein Bax decreased,and the expression of activated cleved-caspase3 increased.(3)Polyphyllin ? can affect the mitochondrial dynamics of A2780 and SKOV3,and break the original mitochondrial fusion and division balance.After Mito Tracker staining,the mitochondria were observed to be fragmented under a microscope.Western Blot detected that Drp1 increased mitochondrial localization,while p-Drp1 and p-AKT expression decreased.PP2 A enzymelinked immunosorbent kit detected increased PP2 A activity.(4)After inhibiting PP2 A,it can obviously reverse the translocation of Drp1 to mitochondria,and can inhibit the decrease of p-Drp1 and p-AKT expression.Experimental results:1.Polyphyllin ? can inhibit ovarian cancer A2780 and SKOV3 cells,and promote mitochondrial apoptosis in ovarian cancer cells.2.Polyphyllin ? treated human ovarian cancer cells A2780 and SKOV3,and activated PP2 A / AKT to dephosphorylate p-Drp1(ser637)to induce mitochondrial translocation of Drp1,and then induce mitochondrial division,mitochondrial Bcl-2 family expression changes and cytochromes C is released into the cytoplasm and eventually leads to apoptosis.3.Polyphyllin ? regulate the PP2A/AKT/Drp1 axis and thereby the mitochondrial translocation of Drp1 may provide a new way to treat cancer. |
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