Rh2 weaken effects ofβ-catenin on HepG2 hepatocellular carcinoma through ac-tivating Gsk-3β / 中国免疫学杂志

ABSTRACT

Objective: To investigate the inhibitory effect of Rh2 on HepG2 cells and explore the underlying mechanism.Methods: We used lentivirus carrying β-catenin to infect HepG2 cell, and detected expression of β-catenin using fluorescence microscopy.The effect of Rh2 on proliferation of HepG2-β-catenin and HepG2 cells was measured by CKK-8 assay,and flow cytometry was used to detect cell cycle and apoptosis.The activity of Gsk-3βwas checked by ELISA kit.The expression of Gsk-3β,β-catenin,Bax,Bcl2,CyclinD1,MMP3 genes were measured by qRT-PCR.In order to checked the relationship between β-catenin and TCF4,CHIP assay kit was used,the expression of Bax,Bcl2,CyclinD1,MMP3 genes were measured by PCR.The expressions of Gsk-3β,β-catenin,Bax,Bcl2,CyclinD1,MMP3 proteins were examined by Western blot.Results:HepG2 cells were successfully infected by pLOV-EF1a-MCS-3FLAG-β-catenin lentivirus,named HepG2-β-catenin.CCK-8 showed that ginsenoside Rh2 could effectively inhibit the proliferation of HepG2 and HepG2-β-catenin cells in vitro,which exhibits a dose-dependent manner at range of 10-160 μmol/L Rh2.The IC50 of Rh2 exposure on HepG2 cell for 48,72 h were 100 μmol/L and 58.12 μmol/L,but the IC50 of Rh2 exposure on HepG2-β-catenin for 48,72 h were 129.2 μmol/L,83.33 μmol/L,respectively.The IC50 of Rh2 exposure on HepG2-β-catenin cell was higher than HepG2 cell, compared with HepG2 group the differences was statistically significant ( P<0.01 ).Flow cytometry indicated that Rh2 could arrest HepG2 and HepG2-β-catenin cells in G0/G1 phase;the cell population in G0/G1 phase of HepG2+Rh2 group was(64.57±0.65)%,HepG2-β-catenin+Rh2 group was(58.61±2.01)%.Flow cytometry indicated that Rh2 could induced early apoptosis in HepG2 and HepG2-β-catenin cells.The apoptosis rate of HepG2 +Rh2 group was (17.27 ±2.77)%,HepG2-β-catenin +Rh2 group(9.02 ±1.76)%.The ELISA results indicated that HepG2 cells was induced by Rh2 for 12,24,48,72 h,the activity of Gsk-3βgradually increased,peak in 48 h,then decreased.Compared with control group,Rh2 induced HepG2 and HepG2-β-catenin cells for 48 hours, Gsk-3βactivity were increased, and their activity reduced after adding Bio, there were no significant differences between HepG2+Rh2 and HepG2-β-catenin+Rh2 groups.The PCR,CHIP and WB results showed that the expression of Gsk-3β,Bax gene and proteins increased,while theβ-catenin,CyclinD1,Bcl2,MMP3 gene and proteins down-regulation in HepG2 and HepG2-β-catenin cell induced by Rh2.Compared with HepG2-β-catenin +Rh2 group, the expression of other gene and proteins changed significantly,however,Gsk-3βwas no significant difference.Conclusion:Over-expression of β-catenin may weaken the phar-macological effects of ginsenoside Rh2 on HepG2 cells.The activity of Gsk-3βwas increased by ginsenoside Rh2 to degradeβ-catenin, affecting the expression of downstream genes,promoting apoptosis of liver cancer cells and inhibiting metastasis.