Experimental Study On The Impact Of Hepatocelluar Carcinoma Cells PI3K/Akt Signaling Pathway Of Ginsenoside Rg3, Sorafenib Monotherapy And Combination

Purpose:Cell experiment in vitro to observe Chinese medicine monomer ginsenoside Rg3, molecular targeted drug sorafenib monotherapy and combination on human hepatocellular carcinoma SMMC-7721 cells proliferation, apoptosis, cell cycle, and expression of PI3K/Akt signaling pathway associated proteins. Explore the mechanism of drug action from molecular biology to provide a theoretical basis for the future joint use of traditional Chinese medicine and molecular targeted drugs.Method:We choose human hepatocellular carcinoma SMMC-7721 cell line for the study and PI3K/Akt pathway inhibitor LY294002 as positive controls. Specific experimental methods are as follows:(1)Using MTT assay to observe ginsenoside Rg3, sorafenib, LY294002 monotherapy and combined with different ways of SMMC-7721 cell proliferation; (2) Flow cytometry to detect apoptosis and cycle distribution of each drug group; (3) Immunofluorescence cytochemistry and Western blotting to detect the expression PI3K/Akt signal pathway associated proteins (PI3K, p-PI3K, Akt, p-Akt) on each group SMMC-7721 cells.Results:(1) MTT assay showed:PI3K/Akt pathway inhibitor LY294002 can inhibit the proliferation of SMMC-7721 cells, and this inhibition has a time and dose dependent. Appropriate concentration of ginsenosides Rg3, sorafenib, LY294002 monotherapy and combination can inhibit the proliferation of human hepatoma SMMC-7721 cells. The two-drug group is better than monotherapy group, and any combination of two drugs shows an additive or synergistic effects. But there is no significant difference between the three-drug group and sorafenib plus LY294002 group(P<0.05).(2) Flow cytometry showed that:ginsenoside Rg3, sorafenib, LY294002 monotherapy and combination can induce apoptosis of SMMC-7721 cells. And the two-drug group is better than monotherapy group, the three-drug group is better than two-drug groups (P<0.05). Ginsenosides Rg3 and LY294002 primarily arrest SMMC-7721 cells in G1 phase.Cells in groups containing sorafenib are mainly arrested in S phase.(3) Immunofluorescence method and Western blotting assay showed that:ginsenoside Rg3, sorafenib, LY294002 can down-regulate expression of p-PI3K, p-Akt and inhibit the phosphorylation of PI3K/Akt pathway respectively. Ginsenoside Rg3 plus sorafenib group and sorafenib plus LY294002 group both significantly inhibit PI3K/Akt pathway, significantly better than monotherapy. The three-drug group showed no significant difference with sorafenib plus LY294002 group in the inhibiting effect on PI3K/Akt pathway(P> 0.05).Conclusion:We confirmed by this experiment that ginsenoside Rg3, sorafenib monotherapy and combination can effectively inhibit the proliferation of human hepatocellular carcinoma SMMC-7721 cells, induce apoptosis and block cell cycle. And the two-drug combination adds the effect. The mechanism may be related to inhibition of phosphorylation of PI3K/Akt signaling pathway and down regulation of p-PI3K and p-Akt protein expression. This experiment provides some experimental basis to hepatocellular carcinoma comprehensive treatment program of combination of molecular targeted drug with traditional Chinese medicine or signaling pathway inhibitor, and it has a certain reference value for the improvement of the clinical efficacy of hepatocellular carcinoma.