Screening Of Molecular Mechanisms Underlying The Activity Of Ginsenoside Rg3in Esophagus Cancer

Ginsenoside Rg3is a major active component of ginseng with multiple pharmacological activities. Rg3has been reported to exhibit in vitro and in vivo anti-carcinogenic and anti-metastatic effects. The molecular mechanism on anti-tumor effects of Ginsenoside Rg3is now one of the attracting fields.The study includes:(1) The effect of Rg3on the proliferation and the effect on the cell cycle of human esophageal cancer cells EC9706were analyzed by MTT analysis and flow cytometry. The expression of cell cycle related factors such as p21, p27, c-Myc, PCNA and Cyclin D1of EC9706cell were detected by immunohistochemical assay, RT-PCR and western-blot in order to study the molecular mechanism of Rg3in anti-proliferation.(2)The apoptosis analysis was performed to evaluate the role of anti-tumor of Rg3in EC9706cell by MTT assay, AO/EB fluorescence double-dye technology and flow cytometry. The expression of apoptosis related factors such as Bcl-2, Bax, Bcl-xL, p53, Fas and caspase-3were analyzed by immunohistochemical assay, RT-PCR and western-blot in order to explore the molecular mechanism of Rg3in apoptosis.(3) The human umbilical vein endothelial cells were isolated, cultured and identified in this study. The effects of proliferation limitation of Rg3on HUVECs were determined by the way of MTT and flow cytometry. The effects of Rg3on HUVECs migration and invasion of Rg3migration were studied by the way of Transwell little room, tube forming methods in order to explore the anti-angiogenesis effects in vitro.(4) The anti-angiogenesis related molecular such as VEGF, MMP-2and MMP-9were analyzed by IHC, RT-PCR and western-blot in order to explain the anti-angiogenesis mechanism induced by Rg3.The results showed that:(1)The inhibition rates on EC9706cells in each Rg3treatment group were significantly higher than those in control group (P<0.05), and the inhibition rates were increased in a concentration and time dependent manner. Rg3can affect EC9706cell cycle phase distribution. The number of G0/G1phase cells was increased and the number of the S and G2/M phase cells was gradually reduced when treated with Rg3. The expression levels of p21and p27protein were up-regulated, while the Cyclin-D1and PCNA expression levels were down-regulated.(2) Compared with control group, the apoptosis rates of EC9706cells of Rg3treatment group were significantly increased in a concentration and time dependent manner. The results of RT-PCR, western-blot and IHC showed that the expressions of Bax, Caspase-3, p53, Fas, Caspase-3gene and protein were up-regulated with increased Rg3concentration, but Bcl-2remained unchanged.(3) MTT assay displayed that Rg3inhibited the growth HUVECs. Rg3could reduce the migration activity of HUVECs and inhibit the capillary-like tube formation of HUVECs with a dose-dependently manner.(4)The result of immunohistochemical assay displayed that the expression levels of VEGF in EC9706cells were weakly positive and negative in100μmol/L and200μmol/L Rg3treated group, respectively. Rg3could decrease the expression of VEGF and MMP-2in EC9706cell with a dose-response relationship.The study showed that:(1) Rg3could restrain the proliferation of esophageal cancer cells EC9706, mainly block esophageal cancer cells in G0/G1phase with up-regulation of p21expression and down-regulation c-Myc and Cyclin-D1protein expression level. (2) Rg3can induce esophageal EC9706cells apoptosis, via increased Bax, p53and Caspase3levels to promote tumor cell apoptosis.(3) Rg3can significantly inhibit the proliferation of HUVEC cells, migration and tubules formation, which suggests that Rg3can inhibit neovascularization. Specific mechanism remains to be further study. Rg3is expected to become a new type of anti-angiogenesis herbs monomer composition.(4) Rg3could inhibit inhibit tumor angiogenes by down-regulation the VEGF, MMP-2and MMP-9expression level in esophageal cancer cells EC9706.These studies subject to explore the effects of Rg3on cell cycle, apoptosis and anti-tumor angiogenesis in EC9706cell and their antitumor molecular mechanisms. This will help to provide the basis for the clinical reasonable application of Rg3.