Oleanolic Acid Inhibits Proliferation And Induces Apoptosis By Which Wnt/β-catenin Signaling Pathway In Lung Adenocarcinoma A549 Cells

Objective:To study the effect of oleanolic acid(OA)on the proliferation and apoptosis of lung adenocarcinoma A549 cells,and to explore its mechanism.Methods:1.Group:Control group,OA group(0.625,1.25,2.5,5,10 μmol/L),positive control group(cisplatin,0.004g/L);2.MTT and RTCA were used to detect the effect of OA on A549 cell proliferation;3.The effect of OA on the cell cycle distribution of A549 was detected by fluorescence double staining flow cytometry;4.Hoechst33342 staining,JC-1 and FCM were used to detect the effect of OA on A549 cell apoptosis;5.Western Blot method to detect the expression levels of cell proliferation related proteins PCNA,cell cycle related proteins cyclinD1,cyclinD3,CDK2,CDK6,P18,apoptosis related proteins XIAP,Survivin,Bcl-2,Bax,apoptotic pathway related proteins cleaved caspase-3,7,8,9,1 2,and the expression levels of Wnt5a/b and β-catenin,key proteins of the Wnt/β-catenin signaling pathway.Results:1.OA inhibits the proliferation of A549 cells:MTT and RTCA results show that different concentrations of OA treated A549 cells can significantly inhibit the proliferation of A549 cells compared with the control group for 24,48and 72 hours(P<0.01).A549 cells were treated with OA(1.25,5 μmol/L)for 48h for subsequent experiments.Western Blot results showed that the expression of PCNA could be reduced after 48h of drug treatment;2.OA blocks A549 cell cycle:Fluorescence double staining flow cytometry results show that compared with the control group,the proportion of Sub G1 phase in OA group is increased,and the A549 cell cycle is blocked in Sub G1 phase(P<0.01).Western Blot results showed that the expression of cycle-related proteins cyclinDl,cyclinD3,CDK2,and CDK6 decreased,and the expression of P18 increased;3.OA induces apoptosis of A549 cells:Hoe chst3 3 3 42 staining method,mitochondrial membrane potential detection kit(JC-1),and fluorescent double-staining flow cytometry results show that after 48 hours of drug treatment,different concentrations of OA were compared with the control group can significantly induce apoptosis of A549 cells(P<0.0 1,P<0.0 5).Western Blot results showed that the expressions of anti-apoptotic proteins XIAP,Survivin,and Bcl-2 decreased,and the expressions of pro-apoptotic proteins Bax increased.Increased expression of apoptotic pathway-related proteins cleaved caspase-3,7,8,and9;4.OA inhibits Wnt/β-catenin signaling pathway:Western Blot results showed that the expression levels of Wnt5a/b and β-catenin were significantly reduced after 48 hours of drug treatment;5.OA inhibits the proliferation and promotes apoptosis of A549 cells via the Wnt/β-catenin signaling pathway:Add Wnt/β-catenin signaling pathway activator HLY78 for 48h,Western Blot results showed that compared with the control group(Control),the expression levels of Wnt5a/b and β-catenin were increased.Compared with OA group,HLY78 combined with OA group the expression of proliferation-related protein PCNA,cycle-related protein cyclinD1,cyclinD3,CDK2,CDK6 was increased,P18 was decreased,and the proportion of cells in Sub G1 phase was decreased.The difference was significant.And the expression of apoptosis proteins XIAP,Survivin and Bcl-2 was increased,the expression of pro-apoptotic protein Bax was decreased,and the apoptosis rate was decreased.The difference was significant.Conclusion:1.Oleanolic acid can block the cell cycle and inhibit the proliferation of A549 cells;2.Oleanolic acid can induce apoptosis in A549 cells,which may induce apoptosis through the death receptor or mitochondrial pathway;3.Oleanolic acid can inhibit the proliferation and promote apoptosis of A549 cells by inhibiting the activition of Wnt/β-catenin signaling pathway.