Mechanisms Of Inhibitory Effect Of Diosgenin On Human Hepatic Carcinoma Cell SMMC-7721

Research backgroundHepatocelluLar carcinoma (HCC) is one of the most common human fatal malignancy that is greatly harmfuL to human health. In China, HCC is one of the major malignant cancer which is second only to lung cancer. Currently, the incidence of HCC shows a slowly upward trend, and the new oneset cases will continue to increase in the near future. Therefore, HCC will be one of the most important disease prevention and control. At present, there is no effective treatment for intermediate and advanced liver cancer, traditional Chinese medicine treatment as one of the most important composition of comprehensive treatment has certain curative effect and unique advantage. The antitumor effect of the active ingredients of traditional Chinese medicine with modern molecuLar biology techniques has been the focus of domestic research. Professor Ling Leading his research team developed a granuLe named Ganfujian, they have confirmed that Ganfujian granuLe have a certain role in preventing and delaying the formation of liver cancer. Rhizoma Dioscoreae as a monarch drug in this prescription can tonify qi and nourish yin, invigorate spleen and kidney. It is a clinical common drug which have strengthening body resistance function. Therefore, in order to clear the antitumor mechanism of the granuLe, we chose Rhizoma Dioscoreae to start our study, the antitumor effect of diosgenin which is one active ingredient of the Dioscoreae have been the key point of this research.Research ObjectivesTo study the effect of diosgenin on cell proliferation of human HepatocelluLar carcinoma cell line SMMC-7721in vitro, preliminary study the antitumor effect mechanisms with cycle arrest, apoptosis and autophagy. Open avenues for further study of diosgenin.Method1. Study the anti-proliferation effects of different concentrations diosgenin (10,20,30,40,50μM) on human hepatocelluLar carcinoma cell line SMMC-7721in vitro using MTT assay, and obvese the morphological changes of SMMC-7721cells by inverted microscope.2. Apply flow cytometry to detect the impact on the cell cycle of SMMC-7721cells that have been intervened with different concentrations diosgenin24h.3. SMMC-7721cells were treated with different concentrations diosgenin24h, with the application of flow cytometry, the apoptosis of SMMC-7721cells were determinated by Annexin-V/PI double staining.4. Using transmission electron microscope (LEM) and MDC staining to detect the autophagy of SMMC-7721cells which were treated with different concentrations diosgenin24h.5. After SMMC-7721cells were treated with diosgenin24h, the expression of cell autophagy related protein light chain3(LC3), p-Akt and caspase family were investigated by Western Blot method.6. After SMMC-7721cells were treated with diosgenin24h, the expressions of p21and p27gene were investigated by RT-PCR.Result1. Diosgenin had an anti-proliferation effect on human hepatocelluLar carcinoma cell line SMMC-7721cells in a dose-and time-dependent manner. In addition, SMMC-7721cells shrinked,becoming smaller and rounder compared with normal cells.2. After SMMC-7721cells were treated with diosgenin24h, the number of cells in the G0/G1phase was decreased, while the number of cells in the G2/M phase was increased. With the concentration of diosgenin increased, G2/M phase cells were significantly increased compared with control group (P<0.05). The resuLts of Western Blot and RT-PCR show that p21and p27are over expression, so diosgenin induced SMMC-7721cell cycle arrest in G2/M phase are associated with the overexpression of p21and p27.3. SMMC-7721cells were induced to apoptosis when treated with diosgenin, with the concentration of diosgenin gradually increased, cell viability was gradually decreased, while the rate of early apoptosis and late apoptosis were significantly increased with the control group (P<0.05) respectively.4. SMMC-7721cells were induced to autophagy by diosgenin the number of autophagic vacuole which marked with green fluorescence spot was gradually increased with different diosgenin except for40μM and50μM group, the expression of LC3was increased, the proportion of LC3-Ⅰ/LC3-Ⅱ was decreased. Autophagy bubble structure observed by LEM confirmed the diosgenin has the role of induced autophagy in SMMC-7721cells5. After cells were treated with diosgenin24h, with the concentration of diosgenin gradually increased, the expression of caspase-3, caspase-7and caspase-8were gradually decreased, cleaved caspase-8and cleaved caspase-3were increased, the expression of p-Akt were decreased.Conclusion1. G2/M phase arrest induced by diosgenin are associated with the inhibition of p-Akt and overepression of p21and p27in SMMC-7721cells, it indicate that the antitumor of diosgenin probably through PI3K-Akt signal pathway.2. Caspase-8might be activated by diosgenin through the extrinsic apoptotic pathway to induce SMMC-7721cell apoptosis.3. Diosgenin couLd induce autophagy in SMMC-7721cells.