| Objective:This experiment is aimed to investigate the effect of Vitamin E Succinate on proliferation , apoptosis and expression of associated protein of human esophageal carcinoma cell line Eca-109 in vitro in order to further investigate possible mechanism of Vitamin E Succinate inhibiting esophageal carcinoma cell line. This will provide the theoritic basis for esophageal carcinoma therapy and chemical prevention.Methods:1 Esophageal carcinoma cells were incubated in culture medium in vitro. MTT colorimetric method was used to detect the change of growth rate of different concentration VES on Eca-109 cells in different time (24,48,72h). Morphological changes of Eca-109 cells were observed by light microscopy and transmission electron microscopy. Apoptotic rate of Eca-109 among different concentrition group was examined by flow cytometry.2 The expression of Survivin, Caspase-3 protein was qualitationly studied by immunocytochemistry staining after treated with VES for 48h. 3The expression of Survivin, Caspase-3 protein was semi-quantitately examined by flow cytometry after treated with VES for 48h.Results:1 MTT colorimetric method showed that Vitamin E Succinate(5,10,20,40,80μg/ml)could inhibit the proliferation of Eca-109 cells. After treated with VES for 24h to 72h, compared with control group, the OD values of Vitamin E Succinate -treated groups decreased, and there was statistically significant difference between control group and every treatment group (p<0.05). Among every treatment group, there was also significant difference(p<0.05). Furthermore, with the increasing concentration of Vitamin E Succinate and prolonging of treatment time, the OD values decreased gradually, in other words, VES inhibited the proliferation of Eca-109 cells significantly in a dose and time-dependent manner and the highest inhibition ratio was 68.73% in Vitamin E Succinate 80μg/ml for 72h. The cells untreated with Vitamin E Succinate were round, elliptic or polygonal, and they were diaphanous, well-stacked and in a good condition. But the cells treated with Vitamin E Succinate were shrinked and broken, and became irregular in shape. Furthermore, with the increasing concentration of Vitamin E Succinate, the populationg of apoptotic cell incrased gradually. Vacuolus could be observed in the kytoplasm of some cells. The cells cast-off increased. Morphological changes of Eca-109 cells of Giemsa staining were further observed by light microscopy, Eca-109 cells untreated with Vitamin E Succinate were diamond or polygon, and they were satiation, furthermore, the cell nucleus staining were uniform and clear, no apoptotic cell. But Eca-109 cells treated with Vitamin E Succinate became rounding, the cell kytoplasm was concentrated, the staining cell nucleus was uneven, there were also caryocinesis phenomenon, showing the typical apoptotic morphological changes. The cell ultramicrostructure constitution was observed by transmission electron microscope: the normal Eca-109 cells were big morphous, and cell membranes were integrity with a lot of bloom ecphyma. Cell nucleus were big morphous with rich euchromatins that were uniformly dispered and density coherent. In cytoplasm there were affluent cell organs, for example endocytoplasmic reticulum, bioblast and cytolysosome. The ultramicrostructures of cells treated with 20μg/ml Vitamin E Succinate were changed in different degree. The cell bodies grew downwards, microvilli decreased, nuclear membrane shrinked, chromatin condensed highly, electron density raised up and they became crescent below the nuclear membrane.2 When cells were harvested for the analysis on apoptosis by flow cytometry, the results were: After treated with apoptotic peak which enhanced gradually with the increasing concentration of Vitamin E Succinate was observed and the apoptotic percentage was 14.147%±0.147%, 21.167%±0.702%, 36.087%±0.559%, seperately. 3The expressiong of Survivin and Caspase-3 protein of Eca-109 cells analyzed by immunocytochemical method, and scored in HIS value manner. In the control group, the staining of Survivin was positive in Eca-109 cells and the buffy grains could be seen in endochylema, and the buffy grains was very thick;With the increase of concentration of VES, Eca-109 cells gradually weakened the stain degree of survivin in the Eca-109 cells,the stain and the buffy grains became thinner and the positive cell population was less than control group. After scoring and statistics, expressive values of Survivin (HISvalue)decreased with the increasing concentration of VES. In the control group, the staining of Caspase-3 was weakly positive in Eca-109 cells, the buffy grains was very thin;Vitamin E Succinate strengthened the stain degree of Caspase-3 in the Eca-109 cells,With the increase of concentration of VES, the stain and the buffy grains became thicker and the positive cell population was more than control group.After scoring and statistics , expressive values of Caspase-3 (HIS value) increased with the increasing concentration of VES.To the expressive values of both proteins, there was statistically significant difference between the negative control group and every VES-treated group(p <0.01).4 FCM assay results:The expression of Survivin and Caspase-3 protein had been examined in Eca-109 cells being treated with 0, 10, 20, 40μg/ml Vitamin E Succinate for 48h, the FI-value of Survivin in Eca-109 was 1.000±0.000, 0.855±0.009, 0.689±0.017, 0.313±0.034, respectively. The difference was statistically significant(p<0.05), and the level of Survivin was hightest in 40μg/ml Vitamin E Succinate. The FI-value of Caspase-3 was 1.000±0.000, 1.354±0.092, 1.604±0.078, 1.757±0.084, respectively.The difference was statistically significant(p<0.05), and the level of Caspase-3 was hightest in 40μg/ml Vitamin E SuccinateConclusions: 1 Vitamin E Succinate has the effect of anti-varian cancer cell line Eca-109 in vitro in a dose and time-dependent manner.2 The growth inhibitory effects of Vitamin E Succinate on Eca-109 cells are involved in induction of apoptosis in a dose and time -dependent manner.3 Within a certain drug concentration, Vitamin E Succinate can increase the expression of Caspase-3 and decrease the expression of Survivin in Eca-109 cells. We infer that VES's effects of inhibiting proliferation and inducing apoptosis in Eca-109 cells seem to due to up-regulation of the expression of Caspase-3 protein and down-regulation of the expression of Survivin.
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