Inhibitory Effect And Mechanisms Of DATS On Human Hepatocellular Carcinoma HepG2 Cell Line

Objectives:To investigate the inhibitory effect of Diallyl Trisulfide(DATS),the main component of allicin,on human hepatocellular carcinoma HepG2 cells and its possible mechanism.Methods:1.HepG2 cells in logarithmic phase were randomly divided into blank control group,and DATS group(10 M,20 M,40 M,80 M,160 M,320 M,640 M).CCK-8 was used to detect the activity of cells in each control group,and clone formation test was used to verify the inhibitory effect of DATS on the growth of HepG2 cells.Meanwhile,Hoechst staining cytometry was used to detect the effect of DATS on HepG2 cells.2.HepG2 cells were cultured for 48 hours in blank control group,DATS 40M group and DATS 80M group.The expression of Bax and Bcl-2 was detected by Western blot,the expression of p-ERK was detected by Western blot,and the expression of AMPK and SIRT1 was also detected.The experimental data were analyzed by SPSS 19.0 statistical software.Prism 7.0a and R were used to draw the experimental data.The difference was significant when P<0.05.Results:1.In the inhibition experiment of DATS on HepG2 cells,CCK-8 assay showed that the concentration of DATS at 10 M and 20 M had no significant effect on inhibiting the growth of HepG2 cells(P>0.05).With the increase of DATS concentration,the inhibiting effect of DATS on the growth of HepG2 cells was more obvious,and a large number of hepatocellular carcinoma cells died at the concentration of 32M and 64M.In the time relationship,the inhibitory effect of DATS concentration 40 M,80 M and 160 M for 48 hours was more obvious than that for 24 hours(P<0.05).Plate cloning assay showed that DATS inhibited HepG2 cells,and the number of cells decreased with the increase of DATS concentration.When DATS concentration reached 80 M,no cells formed.Hoechst staining cytometry was used to detect the effect of DATS on HepG2 cell line.It was found that apoptotic rate of HepG2 cells changed significantly.Especially,the apoptotic rate of HepG2 cells treated with 160 M DATS for 24 hours was significantly higher than that of blank control group.2.In the mechanism experiment of DATS on HepG2 cells,the results of Western blot showed that with the increase of DATS concentration,the expression of Bax in HepG2 cells gradually increased,while the expression of Bcl-2 was inhibited,and the ratio of Bax to Bel-2 was increased.The p-ERK/beta-actin ratio of HepG2 hepatocellular carcinoma cell blank control group and HepG2 hepatocellular carcinoma cell+DATS 40M group had no significant change(P>0.05).The p-ERK/beta-actin ratio of HepG2 hepatocellular carcinoma cell+DATS 80M group was signifieantly higher than that of HepG2 hepatocellular earcinoma cell+DATS 40M group(P<0.05).The p-ERK/beta-actin ratio of HepG2 hepatocellular carcinoma cell+DATS 80M group and HepG2 hepatocellular carcinoma cell+40M group was found to be relatively higher than that of HepG2 There was also statistical significance(P<0.05).The results of AMPK/SIRT1 showed that with the increase of DATS concentration,the expression level of p-AMPK increased,the expression level of AMPK decreased,and the expression level of SIRT1 increased.Conclusions:1.DATS can inhibit HepG2 cells in a dose-and time-dependent manner.2.The inhibition of DATS on HepG2 cells may be mediated by Bax/Bcl-2 pathway,ERK pathway and AMPK/SIRT1 pathway.