Experimental Study On Impact Of Ginsenoside Rg3, Sorafenib And Their Joint On C-Met/MAPK Pathway Of Hepatoma Cell Line

Purpose:To investigate the effect of ginsenoside Rg3 and sorafenib singly and combinedly on human hepatoma cell line HepG2 in vitro, recovering the molecular mechanism through c-Met/MAPK pathway, so as to guide the clinical practice.Method:To treat the human hepatoma cell line HepG2 with different concentrations of ginsenoside Rg3 and sorafenib singly and combinedly. Use various methods to detect the growth condition of human hepatoma cell line HepG2:(1) Estimating the inhibition of proliferation by MTT test; (2) Estimating the apoptosis rate by flow cytometry; (3) Using ICC to analyse the expression of proteins c-Met、p-c-Met、MEK、p-MEK、ERK and p-ERK, which are most relevant to c-Met/MAPK pathway; (4) Detecting the expression of proteins c-Met、p-c-Met、MEK、 p-MEK、ERK and p-ERK by Western blotting assay.Result:(1) The inhibition of cell proliferation of HepG2 was significant about administrating the ginsenoside Rg3 and sorafenib alone and combined by MTT test. The effect depends on time and does. The combination of ginsenoside Rg3 and sorafenib shows accumulative or synergistic inhibitory effect. Treating the cells with low does of HGF which has no significant effect on cell proliferation still reduces inhibition ratio of sorafenib. Combining sorafenib with low does of ginsenoside Rg3 can again increase the inhibition ratio, reversing the effect of HGF. (2) The inhibition of cell proliferation of HepG2 was significant about administrating the ginsenoside Rg3 and sorafenib alone and combined by flow cytometry analysis. The effect depends on time and concentration. The combination of ginsenoside Rg3 and sorafenib shows accumulative or synergistic inhibitory effect. HGF of a certain range of concentration can reduce the inhibition ratio of HepG2, while adopting low does of HGF which has no significant effect on cell proliferation still can reduce the inhibition ratio of sorafenib. Combing sorafenib with low does of ginsenoside Rg3 can again increase the inhibition ratio, reversing the effect of HGF. (3) Immunocytochemistry shows that the expression of proteins p-c-Met, MEK,p-MEK, ERK and p-ERK, which are relevant to c-Met/MAPK pathway, can be reduced by ginsenoside Rg3 and sorafenib compared with the control group, with the combination group appearing more effect, while the change of expression of c-Met are not significant. After the activation of c-Met pathway by HGF, the expression of c-Met stays unchanged, while the expression of p-c-Met, MEK, p-MEK, ERK and p-ERK are changed accordantly:the level of HGF group is higher than that of blank control group, the level of sorafenib combing HGF group is higher than that of sorafenib group, and the level of sorafenib plus HGF and ginsenoside Rg3 group is lower than that of sorafenib combing HGF group. (4)Western blottig assay shows that under the treatment of ginsenoside Rg3, sorafenib and the combination of them, the expression of proteins p-c-Met, MEK, p-MEK, ERK and p-ERK can be significantly reduced, and the combing group shows more notable reduction, resulting in synergistic effect. After the activation of c-Met pathway by HGF, the expression of c-Met stays unchanged, while the expression of p-c-Met, MEK, p-MEK, ERK and p-ERK are changed accordantly:the level of HGF group is higher than that of blank control group, the level of sorafenib combing HGF group is higher than that of sorafenib group, and the level of sorafenib plus HGF and ginsenoside Rg3 group is lower than that of sorafenib combing HGF group.reduced expression of proteins p-c-Met, MEK, p-MEK, ERK and p-ERK by ginsenoside Rg3 and sorafenib compared with the control group.Conclusion:Ginsenoside Rg3 and sorafenib can inhibit the proliferation of human hepatoma cell line HepG2, and the combination of them showed additive or synergistic effect. The two of them can reduce the expression of p-c-Met, MEK, p-MEK, ERK and p-ERK singly and combined, and so is the expression of these proteins promoted by HGF. To sum up, the inhibition of proliferation of human hepatoma cell line HepG2 by ginsenoside Rg3 and sorafenib may be related with the reduction of p-c-Met, MEK, p-MEK, ERK and p-ERK.