Anti-tumor Molecular Targets Of Intestinal Metabolite IH-901 Of Ginseng Saponin In Human Hepatocellular Carcinoma Cells

Hepatocellular carcinoma (HCC) is a common malignant tumor which threatens seriously human life and health. As HCC has a high degree of malignancy and is easy to metastasize and invade, there are still no effective anti-hepatoma agents so far. Panax ginseng C. A. Meyer, a rare Chinese traditional medicine, has an obvious action to anti-tumor, and its main effective component is ginsenoside. The latest pharmacology study indicates that the final active anti-tumor substance is mainly the series of metabolites produced by intestinal flora rather than the natural ginsenoside. And the ginsenoside intestinal metabolite IH-901 (20-O-β-D-glucopyranosyl-20(S)-protopanaxadiol; Ml or Compound K) which is the ultimate-metabolic products of intestinal flora of natural panaxadiol saponins is the ultimate main active form of ginsenoside anti-tumor action. But the molecular mechanisms and function targets of IH-901 resistance to tumor remain poorly understood up to now.According to the phytochemistry method, the nature protopanaxadiol ginsenoside was isolated from Panax notoginseng subordinating to Araliaceae. Then in virtue of Enzyme Engineering, six glycosidases were used to screen their ability of enzymatic transformation for preparation of the metabolites ginsenoside IH-901 in vitro referring to the intestine metabolites mechanism of ginsenoside. The study result indicates that snailase has the best ability of enzymatic transformation. Under the condition of Orthogonal Test, the affecting factors of enzymatic transformation were studied and optimized, and the best optimal condition was obtained as following: 6/1 for material ratio, 9 h for reaction time, 45℃as reaction temperature and 3 as pH value, the optimum rate of crude hydrolysate add to 54.24%. The crude hydrolyaste was purify by silica gel column chromatogram to obtain the high purified ginsenoside IH-901. The chemical structures were elucidated by spectroscopic methods including IR and NMR and tested the purity by TLC and HPLC, and the purified compound is 20-O-β-D-glucopyranosyl-20(S)-protopanaxadiol, that is ginsenoside IH-901, its purity adding to about 98%. In order to research the functional effect of pharmacodynamics of the ginsenoside intestinal metabolite IH-901, we found that IH-901 had an obvious inhibitory effect on the proliferation of three common hepatoma cell lines SMMC7721,Bel7402,HepG2 and a highly metastatic hepatoma cell line MHCC97-H in time and concentration dependent manners by MTT method. We also found that IH-901 could obviously induce the apoptosis of SMMC7721 and MHCC97-H, and both the cell cycles were arrested at G0/G1 phase through the morphological observation, DNA agarose electrophoresis, Hoechst 33258 and AO/EB nucleus staining, and cell cycle analysis by flow cytometry. IH-901 has higher apoptosis rate and in vitro cytotoxicity against hepatoma cell line than that of normal humanhepatic cell Chang-liver.To understand the molecular mechanisms and function targets of apoptosis induced by IH-901 on human hepatocellular carcinoma cells, firstly we explored the mitochondrium apoptosis signal pathway activated by IH-901 in human hepatocellular carcinoma cell line SMMC7721. The investigation demonstrated that the mitochondrium transmembrane potential (Δψm) obviously breakdown, cytochrome c (Cyt c) released from mitochondria to cytoplasm, at the same time, the Caspase-3 and Caspase-9 were activated by IH-901. Fas/FasL pathway is another significant cell apoptotic signal pathway. Here, we found that IH-901 also could up-regulated the expression of FasL and Fas. These data suggests that the IH-901 can induce human hepatocellular carcinoma SMMC7721 cell line apoptotic via mitochondrium apoptosis signal pathway, and the Fas/FasL signal pathway is intimate correlation with IH-901-induced apoptosis. We presume the two signal pathway are reciprocal chiasmata in IH-901-induced apoptotic, and together execute the network regulation to enhance the apoptotic signal. Moreover, to study the apoptotic associated protein, we observed that IH-901 up-regulated the expression of pro-apoptotic protein Bax and P53 protein in a dose-dependent manner. At the same time, we also noticed that Bcl-2 and BcI-X_L did not change in IH-901-induced apoptosis in SMMC7721 cells. Our study demonstrates that, in this network apoptotic regulation, P53 protein could activate downstream Caspase proteins, and lead to cell DNA reparation or cell apoptosis by cell cycle blockage. As the P53 protein is transcription factor of pro-apoptotic protein Bax, the multiplication of P53 protein expression can lead to the the multiplication of Bax protein, and Bax protein promote Cyt c liberation. Thereby, the apoptotic proceeding is promoted.In order to extremely analyze the function and molecular targets of IH-901 on angiogenesis, and on human hepatocellular carcinoma cell migration and invasion. Firstly, the outcome of this study showed that IH-901 not only inhibited the cell growth of human endothelial ECV304 cell line in a time- and concentration-dependent manner, but also induced the cell apoptosis in vitro. Meanwhile, the IH-901-induced cell cycle was arrested at the G0/G1 phase in ECV304 cells. In addition, our study demonstrated that IH-901 inhibited ECV304 cells adhesion and migration in vitro. In this study, we also observed that IH-901 obviously inhibited high metabasis human hepatocellular carcinoma MHCC97-H cells migration, invasion and adhesion in vitro. In the molecular level, our study demonstrated that IH-901 down-regulated MMP-9 and VEGF proteins expression in a dose-dependent manner in MHCC97-H cells in vitro. Furthermore, we found that, IH-901 inhibited tumor metastasis in the lung and liver by subcutaneously injection as xenografts in nude mice. Moreover, this outcome demonstrated that IH-901 inhibited MMP-9, VEGF and MVD, and the correlations existed between the expression of MVD and MMP-9, and between VEGF and MVD in nude mice subcutaneous tumors by immunohistochemistry analysis. All these results suggest that IH-901 can not only inhibit human hepatocellular carcinoma cell migration and invasion, but also plays an important role in angiogenesis, MMP-9 and VEGF are the significance molecular targets in this process.