Inhibition Of Autophagy Enhances Cisplatin-induced Cell Apoptosis And Its Preliminary Mechanical Research In Breast Cancer

Objective: To investigate the effect of cisplatin on autophagy in human breast cancer cells,clarify the role of autophagy inhibition in apoptosis regulated by cisplatin,and illuminate the molecular mechanism of cisplatin in mediating autophagy.Methods: 1.After the treatment of cisplatin on human breast cancer MCF-7 and MDA-MB-231 cells at different concentrations,the half maximal inhibitory concentration of cisplatin on MCF-7 and MDA-MB-231 cells were measured by CCK-8 assay,the expression of Cleaved-caspase3,LC3,p62 and Cleaved-PARP were measured by western blotting,and the nuclear morphological was detected by hochest staining.The LC3 punctate dots were detected by immunofluorescence,the cytoplasmic vacuolation,mitochondrion swelling,chromatin condensation,the formation of apoptosis body and autophagosome in cisplatin-treated MCF-7 cells were observed by transmission electron microscopy.2.MDA-MB-231 cells were treated with cisplatin at different concentrations,and western blotting was used to detect the expression of relevant molecules in MAPK signaling pathway.Cells in one group were treated with ERK inhibitor PD98059,p38 inhibitor PD169316 or JNK inhibitor SP600126 respectively,and the expression of LC3 was detected by western blotting.3.After the treatment of cisplatin on human breast cancer MCF-7 and MDA-MB-231 cells at different concentrations,the expression of p-YAP and the cellular distribution of YAP were determined by western blotting.Cells in one group were treated with autophagy inhibitor hydroxychloroquine,then the expression of LC3,p-YAP,YAP,CTGF and Cleaved-caspase3 were measured by western blotting,and the cell apoptosis rates were calculated by flow cytometry.4.YAP knockdown MCF-7 and MDA-MB-231 stable cell lines were established by shRNA lentivirus infection and puromycin selection.The interference effect of YAP protein expression was detected by western blotting.The IC50 values were detected by CCK-8 assay with different concentrations of cisplatin administration,and the expression of Cleaved-caspase3 was measured by western blotting.Results: 1.Results of CCK-8 assay showed that cisplatin could significantly inhibit the proliferation of human breast cancer cells,and the IC50 of MCF-7 and MDA-MB-231 cells were 6μg/ml and 8μg/ml respectively.Results of western blotting indicated that the levels of Cleaved-PARP,Cleaved-caspase3 and autophagy associated markers LC3 were increased,and p62 was decreased after cisplatin treatment in a dose-dependent manner.Results of Hochest staining revealed the chromatin condensation and aggregation of cells.Immunofluorescence showed the increase of LC3 punctate dots.Transmission electron microscopy observed the cytoplasmic vacuolation,mitochondrial swelling,chromatin condensation,apoptosis and autophagosome formation.2.Results of western blotting showed that cisplatin activated the ERK,p38 and JNK signaling pathways,and the ERK inhibitor,but not the p38 or JNK inhibitor decreased the level of LC3 in MDA-MB-231 cells.3.Results of western blotting showed that p-YAP was decreased after cisplatin treatment in a dose-dependent manner,and the accumulation of YAP in the nucleus was increased.Compared with cisplatin treatment,the expression of LC3,CTGF were decreased and cell apoptosis,p-YAP were increased when treated by cisplatin and HCQ.4.CCK-8 experimental results showed that YAP knockdown enhanced the sensibility of breast cancer cells to cisplatin.Meanwhile,the apoptosis rates and the expression of Cleaved-caspase3 were up-regulated together with cisplatin treatment in YAP-knockdown cells..Conclusion: 1.Cisplatin promotes apoptosis and induces autophagy in human breast cancer cells MCF-7 and MDA-MB-231.2.Cisplatin induces the autophagy of breast cancer cells by activating the MAPK/ERK signaling pathway.3.Cisplatin induces autophagy and regulates the nuclear import of YAP in human breast cancer cells.4.YAP depletion potentiates the sensitivity of human breast cancer cells to cisplatin.5.Inhibition of autophagy enhances the apoptosis induced by cisplatin in breast cancer cells.