The Effects Of Tetrandrine Combined With Cisplatin On The Proliferation And Apoptosis In Human Small Cell Lung Cancer Cell Line NCI-H446 Cells

Objective To investigate the effects of tetrandrine(TET)combined with cisplatin(DDP)on proliferation and apoptosis in the human small cell lung cancer cell line NCI-H446,to study the potential mechanism of TET enhancing the sensitivities of DDP on NCI-H446 cell line.Methods Human small cell lung cancer cell line NCI-H446 cells were routinely cultured in RPMI-1640 medium supplemented with 10%fetal bovine serum,100 U/ml penicillin and 100 mg/L streptomycin;Firstly,The Inhibitory Concentration 50(IC50)of TET and DDP were calculated according to the cell viability test;secondly,a low concentration of TET was chosen for combination with DDP concentration gradient,Using the isobologram analysis method to determine the synergistic effects between TET and DDP;Four groups were arranged for next experiments:control group(con)?TET 1 ?g/ml group(TET)?DDP 2.5 ?g/ml group(DDP)?TET 1 jig/ml combined with DDP 2.5?g/ml(DT);Four groups were treated with or without N-acetyl-cysteine(NAC)for 48 hours.Cell viability of each group were analyzed with CCK-8 assay after treated with different drugs for 48 hours;Hoechst staining and flow cytometry(FCM)were used to evaluate the apoptosis rate of NCI-H446;The proliferation rate were determined by EdU staining;The intracellular levels of reactive oxygen species(ROS)in indicated times which induced by TET.DDP and DT were determined by FCM.The expression of the related proteins such as P-Akt/Akt?Bax?Bcl-2?cleaved-caspase-3/pro-caspase-3 were detected by western blot.Results(1)Both TET and DDP could inhibit the cell viability of NCI-H446 cells in a time dependent manner,TET combined with DDP could further enhance the inhibitory effects;The IC50 of combination group was signifcantly lower than DDP group;EdU staining result shown that the proliferation cells were decreased notably in combination group when compared with TET group or DDP group;On the contrary,the amount of apoptosis cells was increased with Hoechst staining in combination group;moreover,the combination of TET and DDP could trigger large amount production of ROS in a time dependent manner until 16 hours,and then began to decrease;after treated with each drugs for 16 hours,the combination of TET and DDP could triggered the most amount production of ROS compared with other groups;As shown in western blot,the phosphorylation levels of Akt was decreased,and the protein level of Bax?cleaved-caspase-3 were increased,the expression of anti-apoptosis protein Bcl-2 was decreased in the combination group;(2)The synergistic effect of apoptosis induction and proliferation inhibiting were partially attenuated when each group were co-treatment with ROS-scavenger NAC.Conclusion The current study reported that TET could enhance the cytotoxicity of DDP on NCI-H446 cell line,the combination of TET and DDP could exert synergistic apoptosis induction and proliferation inhibiting effects on NCI-H446 cells;the potential mechanism might be targeting the intracellular redox system and then triggering the downstream signal pathway such as PI3K-Akt pathway and mitochondrial apoptosis pathway.