Study On Apoptosis Of ACC-M Cells Induced By Down-regulating SIRT3 Due To Triptolide And Effects In Combination With Cisplatin

Objective:Initially to investigate the proliferation inhibition and apoptosis effect of triptolide or in combination with cisplatin?DDP?on ACC-M cells through studying on the expression of SIRT3,Cyt-C,and Caspase-9,to provide experimental basis for clinical treatment of salivary adenoid cystic carcinoma with TP alone or in combination with DDP.Methods:After the ACC-M cells were resuscitated and subcultured in RPMI-1640medium mixed with 10%FBS and 1%double antibody,at 37?,5%CO₂ incubator,logarithmic growth cells were used for the experiment.Determine drug concentration gradients through pre-experiments,the negative control group was 0.1%DMSO and the experimental groups were divided into?2.5,5.0,10,20,40,80,160 ng/mL?TP group,?0.25,0.50,1.0,2.0,4.0?g/mL?DDP group and combination group.?1?CCK8 method was used to measure the cell viability of each group after 24h,48h and 72h.The 24h IC50 values of TP and DDP were calculated by SPSS20.0.The combination index?CI?was used in the follow-up experiments,and the combined interaction of the two drugs was judged by Chou talalalay method.?2?Flow cytometry was used to detect the effect of apoptosis by 2.5,40,160 ng/ml TP group,and 1.0?g/mL DDP.Collect apoptosis data for each group at 24h and 48h.The effects of IC50 TP,IC50 DDP and their combination on apoptosis were detected 24 hours later.?3?The expression of SIRT3,Cyt-C and Caspase-9 protein in ACC-M cells were evaluated by Western blot.?4?The transcription of Caspase-9,SIRT3 and Cyt-C genes in ACC-M cells was detected by quantitative PCR.Results:?1?In the experimental groups,the viability of ACC-M was inhibited?P=0.000<0.05?,and exhibits concentration and time-dependent?P=0.000<0.05?.It shows interaction between the time and concentration of TP?F=76.09,P=0.000<0.05?.The inhibitory effect was affected by the different concentrations of TP and the change of action time.The inhibition effect entered the plateau stage when the drug concentration reached 80ng/ml after 48h and72h(P₄₈=0.07>0.05,P₇₂=0.15>0.05);In the range of experimental concentration,the inhibitory effect of DPP and combination group on ACC-M cell viability was proportional to time and concentration?P=0.000<0.05?;The combination was stronger than single drug in inhibition of cell viability?P=0.000<0.05?;The 24h IC50 of TP was 75.67ng/mL and cisplatin was 1.89?g/mL;the CI average of combination was 0.721,that confirmed triptolide has synergistic effect on cisplatin in a certain concentration range.?2?The results of annexin V-FITC/PI double staining showed that 2.5ng/ml TP had no significant apoptosis effect on ACC-M cells(P_24h=0.51>0.05,P_48h=0.26>0.05);Then the TP has a concentration and time-dependent effect on ACC-M cell apoptosis?F=2965.83,P=0.000<0.05,F=234.29,P=0.00<0.05?;Compared with the single drug group,the combined drug group had stronger apoptosis inducing effect on ACC-M cells?P=0.000<0.05?.Compared with the positive control group?1.0?g/mL DDP?,the apoptosis effect of 40ng/mL TP was stronger?P=0.000<0.05?.?3?Western blot showed that Caspase-9 and Cyt-C were up-regulated in three experimental groups,TP could specifically down regulate SIRT3 protein,while DDP had no effect?P=0.48>0.05?.The ability of increasing caspase-9 and Cyt-C in the combined group was stronger than the single group?P=0.000<0.05?,but no significant difference in SIRT3 expression between the combined group and the TP group?P=0.77>0.05?.?4?The results of real time qPCR showed that TP could down regulate SIRT3 mRNA and up regulate the expression of Cyt-C and Caspase-9 mRNA in a concentration and time-dependent manner?P=0.000<0.05?.Conclusion:?1?In vitro,TP has a significant inhibitory viability on ACC-M cells,which is dose and time-dependent.In addition,TP has the synergistic effect combined with DDP.?2?The mechanism of TP-induced ACC-M cell apoptosis is related to the down-regulation of SIRT3 and mediated Cyt-C/Caspase-9 mitochondrial apoptosis pathway.?3?The mechanism of synergistic inhibition of TP combined with DDP on ACC-M cell proliferation is related to the up regulation of Cyt-C and caspase-9 protein expression.