| Objective: to study the effects of VES on the proliferati- on,apoptosis,distribution of cell cycle and expression of correlative protein of colon carcinoma in vivo and vitro. To investigate the mechanisms of VES on colon carcinoma, then to provide evidence for VES on the therapy and chemoprevention of colon carcinoma.Methods: 1 in vitro: SW480 cells were incubated in culture medium in vitro. Effect of different dose of VES on the prolifera- tion of SW480 cells was measured by MTT colorimetric method, after 24h,48h,72h. Distribution of cell cycle and apoptotic rate of SW480 cells treated by VES after 48h were examined by flow cytometry(FCM). Morphological changes of SW480 cells were observed by light microscopy and transmission electron microscopy. The expression of Fas,bcl-2 and PCNA protein of SW480 cells were analyzed by flow cytometry. 2 in vivo: sixty healthy male BALB/c mice were randomized into 5 groups, twelve mice in each group. A suspension of 106 murine colonic adenocarcinoma cells (colon26) was inoculated into right armpit of each mouse to establish murine colonic adenocarcinoma metastatic tumor model. The control group received intraperit- oneal injection of seasame oil (0.05ml per mouse) once per day; the mice in VES groups received intraperitoneal injection of low-dose VES (25mg/kg),middle-dose VES (50mg/kg),high-dose VES (100mg/kg) once per day respectively; 5-Fu group received intraperitoneal injection of 5-Fu (3.3mg/0.2ml per mouse) once per week. Fourteen days later, all mice were killed, the tumor and spleen were taken and weighed, then the inhibitory rate of tumor growth and the spleen index could be calculated; at the same time, distribution of cell cycle and apoptotic rate of tumor tissue cells of every group were examined by FCM; the expression of Fas,bcl-2 and PCNA protein of tumor tissue cells was analyzed by immunohistoche- mistry.Results1 the results in vitro1.1 VES inhibited the proliferation of SW480 cells: MTT colorimetric method showed that: after the administration of VES(5,10,20,40,80μg/ml) for 24h to 72h, the OD values of VES-treated groups decreased, compared with control group, there was statistically significant difference between control group and every treatment group (P<0.01). Among every treatment group, there was also statistically significant different- ce(P<0.01). Furthermore, with the increasing concentration of VES and prolonging of treatment time, the OD values decreased gradually, the inhibitory rate increased, that was, VES inhibited the proliferation of SW480 cells significantly in a dose- dependent and time-dependent manner. 1.2 VES could induce the arrestment of cell cycle and apoptosis of SW480 cells: when cells were harvested for the analysis on distribution of cell cycle and apoptosis by flow cytometry, the results were: after the administration of VES at the concentration of 10,20,40μg/ml for 48h, with the increasing concentration of VES, the number of cells in G0/G1 phase increased gradually,while the number of cells in S phase decreased gradually,the proliferation index(PI) significantly decreased (P<0.01). That was, VES could induce an arrestment of cell cycle in G0/G1 phase in a dose-dependent manner. In addition, after the administration of VES, the apoptotic rate of every VES-treated group were 8.50%,14.30%,35.76% respectively, and the control group was 1.39%,there was statistically significant difference between control group and every VES-treated group (P<0.01).1.3 VES could cause morphological changes of SW480 cells: the morphological changes of SW480 cells were observed by light microscopy: the cells of normal SW480 cells were diamond or fusiform, and they were satiation, after treating with VES(20μg/ml), cellular density was rare extremely, and cellular volume grew small, the cells shrinked and broke, became irregular in shape, spreaded a thin and long pseudopodium in each end, vacuolus could be observed in the cytoplasm of some cells, the cells cast-off increased. The ultramicrostructure changes of SW480 cells were observed by transmission electron microscope: after treating with VES, the cells presented apoptotic changes of different levels: cellular volume was smaller, vacuolus could be observed in the kytoplasm of some cells, nuclear chromatin concentrated highly, electron density raised and they were enriched as crescent below the nuclear membrane. We also observed karyopycnosis and karyorrhexis. 1.4 Analysis on expressions of Fas,bcl-2 and PCNA by FCM showed that: after the administration of VES at the concentrations of 10,20,40μg/ml for 48h, the FI values of Fas increased with the increasing concentration of VES, and the FI values of bcl-2 and PCNA decreased with the increasing concentration of VES. To the FI values of every protein, there was statistically significant difference between control group and every VES-treated group (P<0.05 or P<0.01).2 the results in vivo2.1 VES could inhibit the growth of mice tumors: the inhibitory rate of colon26 metastatic tumor treated with low-dose,middle-dose and high-dose VES were 14.55%,34.79%,50.9%6respectively, and it was in a dose-dependent manner. The inhibitory rate of 5-Fu group was 62.93%, higher than every VES-treated group. Comparing murine weight of every group before being killed: there was no statistically difference between the control group and every VES-treated group (P>0.05). The weight of 5-Fu group was obviously lower than the control group, there was statistically significant difference(P<0.01).2.2 The spleen indexes of mice treated with VES in low-dose,middle-dose,high-dose and 5-Fu were 12.403±1.749,12.967 ±1.212,14.868±1.607,5.058±1.816 respectively,there was significant difference between every VES-treated group and 5-Fu group(P<0.01).2.3 VES could arrest cell cycle and induce apoptosis of mice tumor cells: the distribution of cell cycle and apoptosis of colon26 metastatic tumor cell treated with low-dose,middle- dose and high-dose VES were analyzed by flow cytometry: the number of cells in G0/G1 phase increased,while the number of cells in S phase decreased,there was significant difference comparing to the control group(P<0.05 or P<0.01). The apoptotic rate of every VES-treated group were 15.99%,20.8%,39.71% respectively,the control group was 5.38%,there was statistically significant difference between the control group and every VES-treated group(P<0.01). The apoptotic rate of 5-Fu group was 51.65%, which was higher than high-dose VES group(P<0.01).2.4 The expressions of Fas,bcl-2 and PCNA protein of tumor cells were analyzed by immunohistochemistry,and scored in IHS value manner. Expressive values of Fas (IHS value) increased with the increasing concentration of VES, bcl-2 and PCNA decreased with the increasing concentration of VES. To the expressive values of every protein, there was statistically significant difference between the control group and every VES- treated group(P<0.05 or P<0.01).Conclusions1 VES could inhibit proliferation of SW480 cells in a dose-dependent and time-dependent manner within a certain concentration.2 VES could inhibit growth of murine colonic adenocarcin- oma metastatic tumor in a dose-dependent manner within a certain concentration, VES could elevate the spleen index of the tumor-bearing mouse.3 VES could induce apoptosis, arrest cell cycle in G0/G1 phase of colon carcinoma, in a dose-dependent manner.4 VES could bring into effect of anti-colon carcinoma, the mechanisms might be concerned with up-regulating the expression levels of Fas and downregulating the expression levels of bcl-2 and PCNA.5 The experiment showed that: VES had the effect of inhibiting proliferation,inducing apoptosis and arresting cell cycle, which provided a new theoretical foundation for approp- riate treatment and chemoprevention of colon carcinoma.
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