| Purpose: Determining the cell proliferating activity and the expression level of AFP and Survivin of HepG2 before and after treated with low concentration of ethanol, further investigate the molecular mechanism of apoptosis induced in human hepatocellular carcinoma cell by ethanol, and provide more theoretical knowledge for the clinical application (use) of ethanol in the chemotherapeutic of human hepatocellular carcinoma. Methods: MTT trial was used to detect the effect of low concentration of ethanol on the proliferation ability of HepG2 hepatocellular carcinoma cell. Three kinds of dye, Giemsa, AO, and Hoechst33342 were used to observe the morphological change of HepG2 treated with ethanol. Based on the important role of AFP, Survivin protein in the occurrence and development of human heaptocellular carcinoma, the expression level of AFP and Survivin protein in HepG2 before and after ethanol treatment were detected using immunocytochemistry.Results: The MTT trial showed that the absorbency of HepG2 cell declines as the concentration of ethanol increasing, indicating the decreasing proliferation activity of HepG2. And the proliferation activity differ among different experiment time-point of 6h, 12h, 24h and 48h,with the lowest cell activity at 12h and 24h , the highest cell activity at 6h, the modest at 48h.Observation with light microscopy found that cell untreated with ethanol have a good cell spreading ability and can adhere to the bottom of cell culture flask completely. Most of Cell treated with 2% concentration ethanol adheres to the bottom of flask completely, while few of the treated cells lose its adherence ability. More cell lose its adherence ability after treated with 2% ethanol for 24h. More than half of the cultured cells can' t adhere to the flask bottom completely after treated with 4% ethanol and shrink. Characteristic of cell apoptosis morphology occur after treated with 4% ethanol for 24h following with vesicular phenomenon of cytoplasmic membrane and fragmentation of whole cell into disconnected apoptotic corpuscles encysted with membrane. All the hepatocellular carcinoma cell lose the adherence ability and shrink after treated with ethanol of the concentration from 6% to 10% for 6h, with a more strong shrinkage under 10% ethanol, but no characteristic ofapoptosis was found.Dyeing with Giemsa, it was found that cell treated with 2% ethanolfor 24h has a similar characteristic with the untreated cell, homogeneous cytoplasm, no shrinkage, no condensation of chromatin and no chromatin fragment. Typical characteristic of apoptosis morphology was found among the cell treated with 4% ethanol up to 24h, following with cell shrinkage, condensation of chromatin, cleavage of nuclei which were aggregating along the brim of nuclear membrane, cell membrane blebbing and apoptosome formation. Cell treated with ethanol of concentration from 6% to 10% undergoing a process similar to necrosis, with cell shrinkage, early break of membrane.Control cell and cell treated with 4% ethanol for 24h was dyed with Acridine Orange (AO) and Hoechst 33342, and then viewed by fluorescence microscopy. It was found that the control cell nuclei possess homogeneous circular or elliptical fluorescene, while the treated cell nuclei was deeply dyed with annular, fractional and lunate fluorescene.After secondary antibody and BCIP/NBT were added, positive blue-purple dots were found in the cytoplasm of HepG2 cell incubated with anti-AFP antibody and anti-Survivin antibody by immunocytochemistry. And the blue-purple dots are more in the cell untreated with ethanol than in the cell treated with 4% ethanol. The same treated cell incubated with PBS other than the primary antibody showed negative immunocytochemistry results. Conclusion: Low concentration of ethanol can inhibit the proliferation in a time and dose dependent manner. Cell apoptosis occurrence and decreasing of AFP and Survivin protein in the cell cytoplasm of HepG2 hepatocellular carcinoma cell treated with 4% ethanol. Maybe low concentration of ethanol can inhibit the hepatocellular carcinoma cell proliferation by decreasing the AFP and Survivin protein expression in the hepatocellular carcinoma cell, and induce cell apoptosis under the interaction with other signal transduction pathway, the concrete mechanism deserve further investigation. |
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