Effect Of 20(S)-Ginsenoside Rg3 On Epithelial Mesenchymal Transformation Mediated The Sensitivity Of Hepatocellular Carcinoma Cells To Sorafenib

Objective:We induce hepatocellular carcinoma cell HepG2 become epithelial-mesenchymal transition(HepG2/EMT).In order to find the effect of 20(S)-Ginsenoside Rg3(Rg3)and sorafenib sensitivity of HepG2/EMT cells and to research the mechanism in vitro.Methods:TGF?1 induce the hepatocellular carcinoma cell HepG2 become epithelial-mesenchymal transition(EMT)and creat HepG2/EMT cells.In this process the cell morphology change was observed and test the expression of marker protein.The sensitivity of HepG2/EMT and HepG2 cells to sorafenib and Rg3 by MTT method,respectivly.The proliferation inhibition of sorafenib plus Rg3 in HepG2/EMT and HepG2 cells were detected by MTT method too.The effection of apoptosis and cycle by sorafenib and Rg3 in the two kinds of cells were measured by flow cytometry.The effect of the two medicine and EMT on the cell signal transduction pathway Ras/Raf/MEK/ERK in HepG2/EMT and HepG2 cells were detected by western blotting method.Result:1.In different time(Oh,24h,48h,72h)the epithelial cell marker protein expression decreased and mesenchymal cell marker protein expression increased(P<0.01,P<0.01)when establishment the HepG2/EMT cells,2.Sorafenib and Rg3 can inhibit HepG2/EMT and HepG2 cell proliferation showing time-effect and dose-effect relationship.The sensitive of HepG2/EMT cell to low concentration sorafenib were reduced(P<0.05).The sensitive of HepG2/EMT cell to Rg3 were not significant and irregular.The union of sorafenib and Rg3 can enhance the inhibition effect of HepG2 and HepG2/EMT(P<0.05).Sorafenib plus different concentrations Rg3 also can enhance the depressant effects(P<0.05)and the inhibition effect of HepG2/EMT cells were lower than it in HepG2 cells(P<0.05).3.The cell cycle do not change when HepG2 cells become EMT.Sorafenib,Rg3 and the drug combination arrest HepG2 cell cycle at G1 phase.Sorafenib and drug combination arrest HepG2/EMT cell cycle at G1 phase.Rg3 arrest HepG2/EMT cell cycle at S phase.The natural apoptosia rete of HepG2 cells decreased after EMT.The apoptosis rate of HepG2/EMT cells induced by sorafenib and Rg3 were reduced(P<0.01,P<0.01).The apoptosis rate of HepG2/EMT cells induced by drug combination were increased(P<0.01).4.Sorafenib,Rg3 and drug combination can inhibit cell signal transduction pathway Ras/Raf/MEK/ERK(P<0.05).The Ras,Raf,pMEK and pERK1/2 proteins were up-regulated(P<0.05)and the expressions of MEK and ERK1/2 were down regulated(P<0.05)in HepG2 cells after EMT.The inhibition of Sorafenib,Rg3 alone or in combination to the.expression of Ras,Raf,pMEK and pERK 1/2 protein in HepG2/EMT cells were decreased(P<0.05,P<0.05,P<0.05,P<0.05).Conclusion:The sensitve of HepG2 cells to low concentrations sorafenib were reduced after theHepG2 cells induced EMT.EMT has little effect on Rg3.Rg3 can improve the sensitivity of cells to sorafenib and these two drugs combination is synergistic effect.Expression of Ras/Raf/MEK/ERK signaling pathway in HepG2/EMT cells influence sorafenib and two drug combination effects.