Preliminary Study On Apoptosis Of Human Hepatoma Cell Strain MMC7721 Induced By Water-soluble Extract Components Of Toad

Objective:To observe the proliferation and apotosis of human hepatoma cell strain SMMC7721 induced by Water-soluble extract components of Toad and detection the effect of cell cycle on SMMC7721 cells by Water-soluble extract components of Toad and preliminaryly investigate the related mechanism of the apoptosis.Methods:The SMMC7721 cells were treated with Water-soluble extract components of Toad of different concentrations. The growth inhibition and cytotoxicity of Water-soluble extract components of Toad on SMMC7721 cells was measured by MTT reduction law assay; The change of morphologic of SMMC7721 cells which were intervented by Water-soluble extract components of Toad was observed by the inverted microscopy. Flow cytometry and Annexin-V/PI was used to study the apoptosis and Necrosis; Cell cycle distribution was analyzed with flowcytometry.Results:(1) The water-soluble extract components of Toad could remarkably inhibited the cell growth, the IC50 value of Water-soluble extract components of Toad for cells was 0.065mg/mL and the effect enhanced with it's concentration.(2) Water-soluble extract components of Toad can significantly inhibit the growth of SMMC7721 cells, the growth curve shows that the proliferation of the cells which were dealed with the water-soluble extract components of Toad was far slower than the control group. Different time slots cells also significantly increased inhibition rate(P﹤0.01﹚ After the intervention, SMMC7721 cells performance a typical pathological changes and the morphological changes with time-dependent. Cell changed from polygon into round. Chromatin looseness and karylysis were observed. (3) The Apoptosis have dependency relation to the drug density and drug action time, the percentage of apoptosis increase along with the density increase and the time extension. There was a significant difference(P﹤0.01)between drug group and control group in apoptosis rate. (4) There is a remarkably change of cell cycle distribution which was analyzed with flow cytometry , Treatment of cells with Water-soluble extract components of Toad induced arrest in S phase of cell cycle progression.Conclusion:1. The Water-soluble extract components of Toad at concentration of 0.004mg/ml or higher could have a remarkable cytotoxic effect on human hepatoma cells SMMC7721. Water-soluble extract components of Toad could efficiently induce apoptosis and necrosis in SMMC 7721 cells. 2. Water-soluble extract components of Toad suppresses the multiplication of SMMC7721 cells in dose and time - dependent manner. The IC50 value of Water-soluble extract components of Toad for cells was 0.214mg/mL,0.065mg/mL and 0.023 mg/mL which was treated distinctive for 24,48 and 72 hours.3. Water-soluble extract components of Toad induced apoptosis of SMMC7721 cells. And it assumes the time and the density dependence. 4. Water-soluble extract components of Toad could inhibit SMMC7721 cell growth and arrest cells in S phase of cell cycle,which is possibly one of the mechanisms for anticancer of Water-soluble extract components of Toad.