Chaetomugilin J Enhances Apoptosis In Human Ovarian Cancer A2780 Cells Induced By Cisplatin Through Inhibiting PINK1/Parkin Mediated Mitophagy

The tumor resistance of traditional chemotherapy drugs and their toxicity to normal cells have become obstacles to their anti-tumor effects in ovarian cancer.Therefore,it is particularly important to develop new anti-cancer drugs to improve the sensitivity of chemotherapy and reduce the toxicity of chemotherapy drugs.As a key organelle,mitochondria play an important role in chemotherapy resistance.Cells develop resistance to drugs by maintaining the homeostasis of mitochondria.In recent years,endophytes are considered to be an important source of secondary metabolites with novel structures with anti-cancer,antibacterial and other biological activities.Chaetomugilin J is an endophytic Chaetomium globosum strain from Polygonatum sibiricum.According to reports,these compounds exhibited cell proliferation inhibitory effects on 36 human cell lines.In this study,we aimed to explore the relationship between apoptosis and mitochondrial dysfunction and mitophagy caused by Chaetomugilin J combined with cisplatin in ovarian cancer A2780 cells.Experimental method:1.The effect of endophytic metabolite Chaetomugilin J on the viability and cisplatin sensitivity of human ovarian cancer A2780 cells.A2780 cells were cultured,and the experiment was set as the control(Con)group,the cisplatin(CDDP)group,the Chaetomugilin J group,and the Chaetomugilin J+CDDP group.MTT determines the optimal concentration of Chaetomugilin J combined with cisplatin.The clone formation test detects the proliferation ability of A2780 cells.Western blot was used to detect the expression of apoptosis-related protein Cleaved caspase-3.Flow cytometry was used to detect the rate of apoptosis.2.The effect of Chaetomugilin J combined with cisplatin on the mitochondrial function of human ovarian cancer A2780 cells.DCFH-DA and JC-1 stained the cells,flow cytometry was used to detect the total active oxygen level and mitochondrial membrane potential;Mitosox stained the cells,and fluorescence microscope was used to observe the mitochondrial active oxygen level.3.The effect of Chaetomugilin J combined with cisplatin on mitochondrial apoptosis of human ovarian cancer A2780 cells.Western blot was used to detect the expression of Bcl-2 family proteins Bak,Mcl-1,Bax and Bcl-2.4.The effect of Chaetomugilin J combined with cisplatin on mitophagy in human ovarian cancer A2780 cells.Western blot was used to detect the expression of mitochondrial autophagy-related proteins PINK1 and Parkin.5.The role of mitophagy in the apoptosis induced by Chaetomugilin J combined with cisplatin.Taking wild-type HEK293 T cells and HEK293 T cells overexpressing Parkin as the research objects,clone formation was used to detect cell proliferation ability,and Western blot was used to detect the expression of apoptosis-related proteins Bcl-2 and Bax.And then the cells were treated with Mdivi-1,an inhibitor of mitochondrial autophagy,cell viability was measured by MTT,and apoptosis rate was measured by flow cytometry.Experimental results:1.The endophytic metabolite Chaetomugilin J enhances the chemosensitivity of human ovarian cancer A2780 cells to cisplatin.MTT results show that Chaetomugilin J has a significant inhibitory effect on cell viability,and when combined with low dose(1μg/m L)cisplatin,cell viability is significantly reduced.The clone formation experiment showed that the cell proliferation ability of Chaetomugilin J+CDDP group was significantly inhibited.Western blot results showed that the Cleaved caspase-3 protein expression level in the Chaetomugilin J+CDDP group was significantly increased.Flow cytometry detected cell apoptosis and found that the apoptotic rate of Chaetomugilin J+CDDP group increased significantly.2.Chaetomugilin J combined with cisplatin induces mitochondrial dysfunction in human ovarian cancer A2780 cells.Flow cytometry detected the total cell ROS level and mitochondrial membrane potential.It was found that the total cellular ROS level in the Chaetomugilin J+CDDP group increased significantly and the mitochondrial membrane potential decreased significantly in the Chaetomugilin J+CDDP group.Mitosox staining was used to detect the level of mitochondrial ROS,and we found that the mitochondrial ROS level of Chaetomugilin J+CDDP group increased significantly.3.Chaetomugilin J combined with cisplatin induces human ovarian cancer A2780 cells to induce mitochondrial apoptosis.Western blot detection of Bcl-2 family proteins Bak,Mcl-1,Bax,Bcl-2 found that the ratio of Bax/Bcl-2 in the Chaetomugilin J group and the Chaetomugilin J+CDDP group was significantly increased,and the ratio of Bak/Mcl-1 in the Chaetomugilin J+CDDP group increased significantly.4.Chaetomugilin J combined with cisplatin inhibits the occurrence of mitophagy in human ovarian cancer A2780 cells.Western blot detection of the expression of mitophagy-related proteins PINK1 and Parkin showed that the expression of PINK1 and Parkin proteins in the Chaetomugilin J group and the Chaetomugilin J+CDDP group were significantly decreased.5.Mitophagy plays a protective role in Chaetomugilin J combined with cisplatin to induce apoptosis.The results of clone formation,Western blot,MTT and flow cytometry showed that overexpression of Parkin can attenuate the inhibition of Chaetomugilin J combined with cisplatin on cell proliferation and reduce the expression of Bax/Bcl-2 protein;after treatment with mitophagy inhibitor Mdivi-1,cell viability decreases and apoptosis rate increases.Research conclusion:1.Chaetomugilin J,a metabolite of endophytes,enhances the chemosensitivity of human ovarian cancer A2780 cells to cisplatin.2.Chaetomugilin J combined with cisplatin induces mitochondrial dysfunction and mitochondrial apoptosis in human ovarian cancer A2780 cells.3.Chaetomugilin J combined with cisplatin promotes apoptosis by inhibiting the occurrence of mitophagy in human ovarian cancer A2780 cells.