Study On Effects Of Nitidine Chloride On Apoptosis And Cell Cycle In Human Lung Cancer A549 Cells

Background and Objective: The incidence of lung cancer increased with each passing year due to the change of environment and lifestyle.The problem has become the world’s urgent need to overcome.Most patients are in the advanced stage when they are diagnosed,which result in a low level in the five year survival rate of lung cancer.Therefore,we are eager to find more effective drugs for the treatment of lung cancer.In recent years,the natural products nitidine chloride has been considered to have antitumor activity.It has been proved by many experiments that nitidine chloride can inhibit the proliferation of breast cancer,liver cancer,colon cancer and prostate cancer cells.But there’s no study on the effect of lung cancer cells.In this study,we used the human lung cancer A549 cells as the research object,to observe the inhibitory effect of nitidine chloride and its mechanism.1.To observe the effects of nitidine chloride on the growth and proliferation of human lung cancer cells.2.To observe the effect of nitidine chloride on apoptosis and cell cycle of human lung cancer cells,and discuss its mechanism.Methods: 1.After being treated with different concentrations of nitidine chloride and cultured for 48 h in vitro,the growth and proliferation of A549 cells was detected by MTT method.2.After being treated with different concentrations of nitidine chloride,the morphologic alterations of the apoptosis of A549 cells were observed by Hoechst33258 staining;The apoptosis rate of each group was detected by Annexin V-FITC/PI double staining flow cytometry;The proportion of cell cycle in each group was detected by PI single staining method;The expression of the apoptotic pathway,the Wnt pathway and cell cycle related molecules in each group was detected by Western blot method.Results: 1.The MTT results showed that nitidine chloride significantly inhibited the proliferation of A549 cells,the inhibitory effect was time and dose dependent.The statistical analysis was performed with single factor analysis of variance.Compared with the control group,the difference was statistically significant.**P<0.05,***P<0.01.2.Hoechst33258 staining method showed,in the nucleus or cytoplasm which was treated with nitidine chloride,dense granular and massive fluorescence was observed,different sizes of apoptotic body which contained organelles and nucleus formed.3.A n n e x i n V-F I T C / P I d o u b l e s t a i n i n g fl o w c y t o m e t r y s h o w e d,d i ff e r e n t concentrations of nitidine chloride induced apoptosis in A549 cells.Compared wit h the control group,the difference was statistically significant.**P<0.05,***P<0.01.4.PI single staining showed,G2/M stage cells increased significantly among the A549 cells treated with nitidine chloride,which is dose dependent.The results suggest that nitidine chloride can block cell cycle.5.The Western blot result showed,With the increase of nitidine chloride concentrat ion,the expression level of Bcl-xl,Bid,PARP,p-β-catenin in the apoptotic pathway of A549 cells was down regulated;the expression level of Cyto-C and β-catenin was up regulated;The activity of Caspase-9,Caspase-3,Caspase-8 enhanced.It was suggested that nitidine chloride induced apoptosis of A549 cells in a dose-dependent manner.The expression of cell cycle related protein Cyclin D1 was gradually down regulated.The results suggest that nitidine chloride block the cell cycle in a dose-dependent manner.Conclusions: 1.Nitidine chloride could inhibit the growth and proliferation of lung cancer A549 cells.2.Nitidine chloride could induce apoptosis of lung cancer A549 cells through the mitochondrial pathway and the death receptor pathway,Whose process may be involved in the Wnt/β-catenin signaling pathway.This process is regulated by the Bcl-2 protein family.3.Nitidine chloride blocked lung cancer A549 cells in G2/M phase.