Studies Of The Anti-tumor Effect And Mechanism Of Nobiletin On Ovarian Cancer

Background:Ovarian cancer is a serious threat to women's health and life with high morbidity and mortality.Chemotherapy of ovarian cancer is combination chemotherapy base on platinum.However,side effects and drug resistance of chemotherapy drugs are scientific problems that need to be solved urgently.Natural plant and fruit extract with safety and low toxicity have wide application prospect with biological activities of antioxidant,anti-inflammatory and anti-tumor.Nobiletin,an active ingredient extracted from tangerine,has significant antitumor activity,but its antitumor mechanism remains unclear.In this study,the effects of nobiletin on apoptosis,pyroptosis and autophagy of ovarian cancer cells are analyzed,and the anti-tumor effect and mechanism of nobiletin on ovarian cancer are elucidated,which lay a foundation for the treatment of ovarian cancer with nobiletin.Objectives:1.Evaluate the effect of nobiletin on apoptosis of ovarian cancer cells.2.Evaluate the effect of nobiletin on pyroptosis of ovarian cancer cells.3.Evaluate the effect of nobiletin on autophagy and mitochondrial autophagy of ovarian cancer cells.4.Explore the correlation mechanism of ROS,autophagy and pyroptosis.Methods:1.CCK-8 and Live/Dead studies were applied to evaluate the effects of nobiletin on the growth,proliferation,and death of ovarian cancer cells.ROS and JC-1 staining were used to evaluate the effect of nobiletin on ROS production and mitochondrial membrane potential in ovarian cancer cells.The effects of nobiletin on apoptosis of ovarian cancer cells were detected by flow analysis,Hoechst staining and western blotting.2.The effects of nobiletin on pyroptosis of ovarian cancer cells were evaluated by electron microscopy,qPCR and western blotting.3.The effects of nobiletin on autophagy and mitochondrial autophagy of ovarian cancer cells were studied by transmission electron microscopy,MDC staining,western blotting and immunofluorescence.4.Associations among ROS,autophagy and pyroptosis were investigated using 3-MA,NAC and NSA.Results:1.CCK-8 and Live/Dead results showed that nobiletin inhibited the growth and proliferation of ovarian cancer cells and promoted cell death.ROS and JC-1 staining showed that nobiletin induced ROS production and decreased mitochondrial membrane potential.Hoechst staining,western blotting and apoptotic flow analysis showed that nobiletin induced dense staining of cell nucleus,protein expression of c-PARP and yH2AX and cell apoptosis in ovarian cancer.2.The results showed that the phagocytes were spherical and swollen.The protein expression of c-GSDMD and c-GSDME were gradually increased,and the mRNA expression of IL-1? and ASC were also increased.3.Autophagosomes and mitochondrial autophagosomes were observed by transmission electron microscopy.Immunofluorescence results showed that the expression of LC3-II was increased.The morphology of mitochondria was swollen,and the co-localization of lysosomes and mitochondria were increased.MDC staining showed increased eosinophilic autophagosomes,and western blotting showed increased expression of autophagy-related proteins LC3-? and p62.4.ROS production was reduced with NAC,and protein expressions of c-GSDMD,c-GSDME and LC3-? were also inhibited.LC3-? was inhibited by using 3-MA,c-GSDMD and c-GSDME were also inhibited.The protein expression of c-GSDMD was inhibited with NSA,as well as LC3-?.Conclusions:1.Nobiletin inhibited cell growth and proliferation and promote cell death.Nobiletin can promote ROS production and reduce mitochondrial membrane potential,and then induce the apoptosis of ovarian cancer cells.2.Nobiletin induced pyroptosis in ovarian cancer cells.3.Nobiletin induced autophagy and mitochondrial autophagy in ovarian cancer cells.4.Nobiletin induced autophagy and pyroptosis in ovarian cancer cells by regulating ROS...