The Investigation On The Induced Differentiation Of Human Hepatoma Cells By Ginsenoside Rh₂ And Its Mechanisms

There has been an increasing interest in compounds that inhibit the proliferation of cancer cells and induce neoplastic cells to differentiate into normal cells, and these compounds are expected to be a new type of anticancer agent. It is a key to search for non-toxic and natural origin substances that induce the differentiation of cancer cells. Ginseng, the root of Panax ginseng C.A.Meyer, is one of the most widely used natural tonics in oriental countries for thousands of years and has been reported to have various biological effects. Ginsenosides are thought to be the major effective ingredients in ginseng. Among them, ginsenoside Rh₂ (G-Rh₂) has been suggested to have a cell-growth suppressive effect on various cancer cells, but the mechanism is not clear. In this study, the induced differentiation effects of G-Rh₂ on SMMC-7721 hepatocarcinoma cells and the differentiation mechanism from different aspects were investigated.1. Study on the differentiation of SMMC-7721 cells induced by G-Rh₂. (1) MTT assay was used to investigate the inhibitory effect of G-Rh₂ on cultured SMMC-7721 cell proliferation. It was shown that G-Rh₂ inhibited the growth of the cells in time- and dose-dependent manners. (2) Morphological observation: It was demonstrated that 20μg/ml G-Rh₂ induced the mature and normality of morphology and ultra-structure in SMMC-7721 cells under light-microscope and electronic microscope. (3) Biochemistrical investigation: 10μg/ml, 20μg/ml G-Rh₂ significantly increased the secretory amount of albumin (Alb) and alkaline phosphatase (ALP) activity, which were specific markers of hepatocyte differentiation, while significantly decreased the production and secretory amount of alpha-fetoprotein (AFP),the activity ofγ-glutamyltranspeptidase(γ-GT) and heat-resistant ALP, which were specific markers of hepatocarcinoma. It is suggested that G-Rh₂ could directly inhibit the proliferation of SMMC-7721 cells and induce the cells to differentiate into normal cells.2. Study on differentiation mechanism from the regulation of cell cycle. The change of the cell cycle was examined by flow cytometry assay; the protein expressions of cyclin D1,cyclin E,p16 were detected with flow cytometry assay; The mRNA expression of p21 was determined with reverse transcription-polymerase chain reaction (RT-PCR). It was indicated that 20μg/ml G-Rh₂ arrested SMMC-7721 cells at the G1/G0 phase; weakened the expression of positive-regulating factor:cyclin D1, cyclin E; increased the expressions of negative-regulating factors: p16, p21 in SMMC-7721 cells. It is suggested that G-Rh₂ could down-regulate or up-regulate the mentioned factors of cell cycles and eventually inhibit the cell cycle progression, lead to the differentiation of the cell.3. Study on differentiation mechanism from the regulation of signal transduction. (1) The level of intracellular calcium ion , measured with Fura-2/AM fluorescent probe, remarkably decreased after cells treated with 20μg/ml G-Rh₂. It was implied that 20μg/ml G-Rh₂ could inhibit the protein expressions of protein kinase C (PKC)α, insulin-like growth factor â…  receptor (IGFâ… R)β measured with flow cytometry and immunocytochemistry assays . (2) It was found that 20μg/ml G-Rh₂ induced the mRNA expression of glucocorticoid receptor (GR) detected with RT-PCR. (3) After SMMC-7721 cells treated with 20μg/ml G-Rh₂ and 5μg/ml RU486, a glucocorticoid antagonist with a high affinity for the GR, the cellular proliferation and the protein expressions of cyclin D1, cyclin E, p16, PKCα, IGFâ… Rβwere not changed, different from the effects induced by G-Rh₂ alone. The results showed RU486 could block the induced differentiation effects of G-Rh₂. It is suggested that the effects of G-Rh₂ might be exerted via binding with GR or its analogous nuclear receptor and PKCα, IGFâ… Rβ might play an important role in this process.4. Study on differentiation mechanism from the regulation of genes. It was shown that 20μg/ml G-Rh₂ could suppress the mRNA expressions of onco...