The Effects Of Tetrandrine In Combination With Cisplatin On The Proliferation And Apoptosis In A549 Cells

Objective:Non-small cell lung cancer comprises the majority of lung cancer cases and is insensitive to chemotherapy.Most patients develop drug resistance.Recently,tetrandrine,a bis-benzylisoquinoline alkaloid,was identified as a novel anti-cancer agent.We aimed to identify a possible synergistic effect between tetrandrine(TET)and cisplatin(DDP),besides,to investigate the effects of TET in combination with DDP on apoptosis and proliferation in cisplatin-resistant and cisplatin-sensitive A549 cell lines,and to study the underlying mechanism.Methods:Cisplatin-resistant A549(A549/DDP)cells and A549 cells were cultured in RPMI-1640 medium supplemented with 10%fetal bovine serum,100 U/ml penicillin and 100 mg/L streptomycin,5 ?M cisplatin was added to the medium of A549/DDP cells to metain the resistance.Four groups were arranged for the experiments:control group(Con)?TET group(the concentration of TET were:0.5,1,2,4,8 ?g/ml)?DDP group(the concentration of DDP were:6.25,12.5,25,50,100?M)?TET combined with DDP group(the concentration of TET was:0.25 ?g/ml,the concentration of DDP were:6.25,12.5,25,50,100 ?M),the time of drug treatment was 48 h.Cell viability was confirmed with CCK8 assay,and the IC50 value for each treatment group was calculated.The synergistic interaction of these drugs was evaluated using an isobolographic analysis.Four groups were arranged for next experiments:control group(Con)?TET group(the concentration of TET was:0.25 ?g/ml)?DDP group(the concentration of DDPon A549/DDP cells was 60?M,the concentration of DDP on A549 cells was 25 ?M)?TET combined with DDP group(the concentration of TET was:0.25 ?g/ml,the concentration of DDP on A549/DDP cells was 60 ?M,the concentration of DDP on A549 cells was 25 ?M),the time of drug treatment was 12 h.Hoechst staining and flow cytometry were used to assess apoptosis.Proliferation was assessed by EdU staining.Apoptosis-and autophagy-associated proteins were analyzed by western blot.Transmission electron microscopy was used to detect autophagy.We used 3-methyladenine to inhibit the formation of autophagosomes and thereby verify the role of autophagy.Results:TET and DDP exerted synergistic cytotoxic effects on both A549/DDP cells and A549 cells.From the hoechst staining and the flow cytometry,we could see that the apoptosis rate of combination treatment group was significantly higher than that of control group or DDP group.The EdU staining showed that,compared with control group and DDP group,cell proliferation decreased significantly when TET was combined with DDP.The results of western blot showed that in the combination treatment group,the expression of Bax was upregulated,whereas Bcl-2 and p-Akt were downregulated.Thus,the combination of TET and DDP induced apoptosis and inhibited proliferation in a synergistic manner.The formation of autophagosomes was evident by transmission electron microscopy.In addition,treatment with the autophagy antagonist 3-methyladenine increased cell viability and decreased apoptosis.After adding 3-MA,the cell viability of each group was rising;the apoptosis rate was decreased in hoechst staining and flow cytometry;the proliferation cells of combination treatment were increased in EdU staining;the expression of Bax was downregulated and Bcl-2 was upregulated;the expression of p-Akt was elevated;.Conclusions:TET synergized with DDP to reduce the viability of A549/DDP cells and A549 cells,TET could reverse the resistance of A549/DDP cells and A549 cells to DDP.The drug treatments induced cell death via apoptosis and autophagy.Apoptosis was decreased and proliferation was increased when autophagy was inhibited,indicating that the drugs promoted apoptosis and inhibited proliferation by inducing autophagy.