ABT737 Increases The Sensitivity Of Human Hepatocellular Carcinoma Cell HepG2 To Cisplatin Through Apoptosis Pathway

ObjectiveTo explore the feasibility of enhancing the sensitivity of tumor cells by using anti-apoptotic protein Bcl-2,Bcl-xl and Bcl-w inhibitor ABT737 as the breakthrough point,and to analyze the mechanism of apoptosis from the perspective of the apoptosis pathway.The aim was to analyze the therapeutic effect of ABT737 combined with cisplatin,and to provide a new direction for the combined medication strategy of cisplatin in clinical practice.Methods1.The differences in the expression of Bcl-2,Bcl-xl(bcl2L1)and Bcl-w(bcl2L2)were analyzed in normal tissues and liver cancer tissues by retrieving TGCA database.2.Cisplatin with different concentrations of 0,1.25,2.5,5,10,20,40 and 80(?g/ml)were used to treat 24h in HepG2 cells respectively.HepG2 cells were treated by 5 ?M ABT737 combined with cisplatin at different concentrations for 24 h;MTT assay detected the activity of HepG2 cells after single treatment of cisplatin or combined application.3.HepG2 cells were treated with 5 ?M ABT737,10 ?g/ml cisplatin,and 5 ?M ABT73 7 combined with 10 ?g/ml cisplatin for 24 h,respectively;Hoechst 33342 was used for stainin..Confocal fluorescence microscopy was utilized to observe chromatin changes in HepG2 cells.4.HepG2 cells were treated with 5 ?M ABT737,10 ?g/ml cisplatin,and 5 ?M ABT737 combined with 10 ?g/ml cisplatin for 24 h,respectively.The cells were stained by Annexin V/PI and detected by flow cytometry.The apoptosis level of HepG2 cells was observed accordingly.5.HepG2 cells were treated with the combination of ?M ABT737 and 10?g/ml cisplatin with the addition of 100 ?M DFO,1 ?M Fer-1,10 ?M Nec-1 and 10?M zVAD,respectively.The activity of HepG2 cells was detected by MTT in each experimental group.6.HepG2 cells were treated with 5 ?M ABT737,10 ?g/ml cisplatin,5 ?M ABT737 and 10 ?g/ml cisplatin for 24 h,respectively.The mRNA expression level in each experimental group(control group,cisplatin group,ABT737 group,cisplain and ABT737 group)was detected by Real-time Quantitative PCR.7.The expression levels of Bax protein and cleaved caspase-3 protein in each experimental group(control group,cisplatin group,ABT737 group,cisplain and ABT737 group)were detected by Western blot.Results1.The expression levels of Bcl-xl gene and Bcl-w gene were obviously higher in HCC tissues than those in normal tissues.2.MTT experiment result showed that the combined treatment group had more obvious killing effect on HepG2 cells compared with cisplatin alone treatment group at the same concentration of cisplatin,and ABT737 increased the sensitivity of HepG2 cells to cisplatin.3.After staining with Hoechst 33342,confocal fluorescence microscopy revealed that there was no significant change in chromatin in HepG2 cells between the control group and ABT737 alone treatment group.In cisplatin alone treatment group,there were nuclear condensation and enhanced chromatin staining.However,combined treatment group of ABT737 and cisplatin showed nuclear condensation,and the number of cells with enhanced staining was increased evidently compared with that of cisplatin alone treatment group.4.The detection of apoptosis by flow cytometry with Annexin V/PI staining indicated that the rate of apoptosis in the combined treatment group of ABT737 and cisplatin was higher than that of the cisplatin along treatment group.5.The survival rate of HepG2 cells in combined treatment group of ABT737 and cisplatin added with DFO,Fer-1 and Nec-1 was undifferentiated,however,it was elevated remarkably after the addition of zVAD,suggesting that ABT737 combined with cisplatin medicated HepG2 cells death was achieved mainly by the apoptotic pathway.6.Cisplatin alone could upregulated the mRNA expression level of Bcl-xl,while AB T737 combined with cisplatin inhibited Bcl-xl mRNA expression.7.Western blot results displayed that the expression levels of Bax and caspase-3 protein were increased significantly in combined treatment group of ABT737 and cisplatin than those in cisplatin alone treatment group.Conclusion The combined application of ABT737 and cisplatin enhances the sensitivity of HepG2 cells to cisplatin in the form of inducing apoptosis in cells.