| Objective:To observe the effect of medicine monomer ginsenosides Rg3, molecular targeted drugs sorafenib,chemotherapy drug oxaliplatin combined in different ways in HepG2human hepatoma cell lines in vitro and in mice transfer model (LCI-D20) tumor,and to investigate the impact of cell cycle and related protein,providing the basis for traditional Chinese medicine,chemotherapy drugs and molecularly targeted drugs to treat HCC patients.Method:(1) observed by MTT method ginsenosides Rg3, sorafenib, oxaliplatin monotherapy and combined in different ways (two-drug combination, the three-drug combination) in vitro proliferation of HepG2cells, and calculate the50%inhibitory concentration (IC50).(2)using flow cytometry to detect cell apoptosis and cell cycle distribution.(3) using western boltting method to detect human hepatocellular carcinoma xenografted in nude mice tumor tissue cell cycle-related proteins p53, p21, p27, PTEN expression.Results:(1)Compared with the control group,except of sorafenib,low concentrations of ginsenoside Rg3, oxaliplatin in24h couldn’t inhibit HepG2cells significantly (P>0.05), but in48h and72h the three drugs all could significantly inhibit cell proliferation (P<0.01); along with the increasing concentration of the three monotherapy group effecting on cells24h,48h,72h, cell inhibition rate showed an upward trend;the same concentration of the same drugs compared with different time,they all had statistically significant (P<0.05).(2) combination therapy: compared with monotherapy group of oxaliplatin,oxaliplatin+ginsenoside Rg3group (P<0.05) and oxaliplatin+sorafenib both inhibition rate increased (P<0.01); compared with the monotherapy group of Rg3,oxaliplatin+Rg3group and ginsenoside Rg3+sorafenib group inhibition rate increased (all P<0.01); compared with the monotherapy group of sorafenib, oxaliplatin+sorafenib group and ginsenoside Rg3+sorafenib inhibition rate increased (P <0.01).Compared with two-drug group and the monotherapy group,the three-drug combination group had significantly higher inhibition (P<0.01).Compared with the monotherapy group,oxaliplatin+ginsenoside Rg3combined performance additive effect (q=0.92), ginsenoside Rg3+sorafenib (q=1.39) and oxaliplatin+sorafenib (q=1.23) showed synergistic joint.Compared with the two drugs combined, on the basis of ginsenosides Rg3+oxaliplatin plus sorafenib (q=1.14)and on the basis of oxaliplatin+sorafenib plus ginsenosides Rg3(q=0.86) showed additive effect.On the basis of ginsenosides Rg3+sorafenib plus oxaliplatin (q=1.17) showed synergistic effect.(3) flow cytometry cycle conditions found cells of Rg3group were arrested in G0/G1phase, oxaliplatin group of cells were arrested in S phase, in sorafenib group cells were arrested in G0/G1phase.Combination therapy:ginsenoside Rg3+oxaliplatin group of cells were arrested in G0/G1phase, S phase; cells in ginsenoside Rg3+sorafenib group arrested in G0/G1phase and S phase; cells in oxaliplatin+sorafenib group arrested in S phase;cells in ginsenoside Rg3+oxaliplatin combined with sorafenib group arrested in G0/G1phase, S phase,G2/M phase.(4) flow cytometry apoptosis found that ginsenoside Rg3, oxaliplatin, sorafenib either monotherapy or in combination induced HepG2cells apoptosis. compared with the negative control group, all the experimental groups were significantly higher rate of apoptosis (P<0.01); compared with the monotherapy groups, with the addition of ginsenoside Rg3group compared with ginsenoside Rg3+sorafenib group, the two-drug combination groups induction of apoptosis were enhanced (P<0.05);compared with the monotherapy groups and the two drugs combined group, the three-drug combination group had significant induction of apoptosis (P<0.01).(5) western bolting found that the three-drug monotherapy and combination inhibited cell proliferation of human hepatocellular carcinoma xenografted in nude mice may be associated with reduced mutant p53, increased p21, p27and PTEN expression.Conclusion:(1) ginsenoside Rg3, sorafenib, oxaliplatin either alone or in combination on the proliferation of HepG2cells were significantly reduced,and the inhibition showed time and dose-dependent manner;two drugs and three-drug combination in each group showed an additive or synergistic effect.(2) the experimental groups major arrest cells in G0/G1phase and S phase and the blacking effect of combination therapy is more wide.(3) flow cytometry found that the experimental group had influence on cell apoptosis.The three-drug group had higher apoptosis influence than the two-drug group, the two-drug group higher than the monotherapy group.(4) further confirmed by research in vivo that these drugs could reduced mutant p53and increased the expression of cyclin-related protein to suppress the tumor growth,and the role of combination therapy compared to monotherapy is more obvious. |
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