| Objective: To study the effects of simvastatin on prolif- eration of human colorectal cancer cell SW480 and its mechanisms. Meanwhile, to examine the effects of simvastatin combined with anti-cancer agents Oxaliplation on human colorectal cancer cell SW480 to investigate possible mechanism of inhibiting cell proliferation and inducing apoptosis, and feasibility therapy and chemoprevention of colorectal cancer.Methods: 1 SW480 cells were incubated in culture med- ium in vitro. Effect of simvastatin on the proliferation of SW480 cells was measured by MTT colorimetric method. Apoptosis and distribution of cell cycle were examined by flow cytometry. Morphological changes of SW480 cells were observed by light microscopy and transmission electron microscopy. The expression of bcl-2,cyclind1 protein was analyzed by flow cytometric indirect immunofluorescent technigue. 2 MTT colorimetric method was performed to evaluate the potential cytostatic effect of combining simvastatin and Oxaliplation on human colorectal cancer cell SW480. To determine whether the combination of simvastatin and Oxaliplation results in a synergistic cytostatic effect, Jin's formula was performed. In the analysis, there is synergy when the interaction index is more than 0.85; antagonism when the interaction index is less than 0.85. Apoptosis induced by combining simvastatin and Oxaliplation was examined by flow cytometry.Results: 1 MTT colorimetric method showed that simvas- tatin(2,5,10,20μmol/L) could inhibit the proliferation of SW480 cells. After treated with simvastatin for 24h to 72h, compared with control group, the OD values of simvastatin-treated groups decreased, and there was statistically significant difference between control group and every treatment group (P<0.01). Among every treatment group, there was also statistically significant difference (P<0.01). Furthermore, with the increasing concentration of simvastatin and prolonging of treatment time, the OD values decreased gradually, that was, and simvastatin inhibited the proliferation of SW480 cells significantly in a dose-and time-dependent manner.When cells were harvested for the analysis on distribution of cell cycle and apoptosis by flow cytometry, the results were: After treated with 0,2,5,10,20μmol/L simvastatin for 48h, the number of cells in G0/G1 phase increased gradually, while the number of cells in S phase decreased grudually. But G2/M phase were not changed obviously. That was, simvastatin could induce an arrest of cell cycle in G1 phase by a dose-dependent manner. In addition, after treated with 0,5,10,20μmol/L simvastatin for 48h, the typical apoptotic peak which enhanced gradually with the increasing concentration of simvastatin was observed and the apoptotic percentage was 1.97%,11.71%,26.66%,34.07% seperately. When treated with simvastatin, SW480 cells had significant morphological changes which were observed by light microscopy: The cells untreated with simvastatin were diamond or fusiform, and they were satiation. But the cells treated with simvastatin shrinked and broken, became irregular in shape, spreaded a thin and long pseudopodium in each end. Vacuolus could be observed in the kytoplasm of some cells. The cells cast-off increased. The cell ultramicrostructure changes were observed by transmission electron microscope: nucelus overshoot in acute angle outwards, chromatin concentratede highly, electron density raised and they were enriched as crescent below the nuclear membrane. We also observed karyopycnosis. Analysis on expression of bcl-2 and cyclind1 by FCM showed that: Treating SW480 cells with 0,5,10,20μmol/L simvastatin for 48h resulted in the FI values of bcl-2 and cyclind1 decreasing with the increasing concentration of simvastatin. To the FI values of each kind of protein, there was statistically significant difference between every simvastatin-treated group and control group (P<0.05). 2 MTT colorimetric method was performed to evaluate the potential cytostatic effect of combining simvastatin with anti-cancer agents Oxaliplation in SW480 cells. The results showed: 2,5,10μmol/L simvastatin excerted a synergistic cytostatic effect in SW480 cells when combined with Oxaliplation. The synergistic effect combined with Oxaliplation was enhanced while concentration of simvastatin increased. 3 The analysis of Jin's formula showed that the interaction index for 2,5,10μmol/L simvastatin and Oxaliplation used in combination on SW480 cells was more than 0.85. It proved that simvastatin indeed had a synergistic effect on inhibiting the proliferation of SW480 cells combined with Oxaliplation. Apoptosis induced by combined simvastatin and Oxaliplation was examined by flow cytometry, the result showed: the apoptotic percentage increased by combining 2μmol/L simvastatin with Oxaliplation of different concentration, and there was statistically significant difference between the group of treating with Oxaliplation alone and the group of treating with combining simvastatin and Oxaliplation (P<0.01).Conclusions: 1 Simvastatin, an inhibitor of 3-hydroxy-3- methylglutaryl-CoA, had the effect of anti-colorectal cancer cell line SW480, which provided a new theoretical foundation for appropriate clinical treatment and chemoprevention of colorectal cancer. 2 Simvastatin had the capability of inhibiting the proliferation of SW480 cells in vitro in a dose-and time-dependent way. 3 Simvastatin induced an arrest of cell cycle in G1 phase as well as apoptosis in SW480 cells. 4 Simvastatin's effects of inhibiting proliferation and inducing apoptosis in SW480 cells is due to down-regulating the expression of bcl-2,cyclind1. 5 With MTT colorimetric method, we confirmed that simvastatin combined with Oxaliplation had a synergistic effect on inhibiting the proliferation of SW480 cells. 6 By FCM, we confirmed that simvastatin combined with Oxaliplation had a synergistic effect on inducing the apoptosis of SW480 cells, thus, we assumed that simvastatin could have increased the sensitivity to anti-cancer agents of SW480 cells.
|
PDF Full Text Request