Effects Of Rosiglitazone On Proliferation Of Human Colorectal Cancer Cell SW480 And Its Mechanisms

Objective: To study the effects of rosiglitazone on the proliferation of human colorectal cancer cell SW480 and its mechanisms. Meanwhile, to examine the effects of rosiglitazone combining with anti-cancer agents 5-FU on humancolirectal cancer cell SW480, and to investigate possible mechanism of inhibiting cell proliferation and inducing apoptosis. To investigate the mechanisms of rosiglitazone on colon carcinoma, and provide the theoretic basis for colon carcinoma therapy.Methods: 1 SW480 cells were cultured in vitro. Effect of rosiglitazone on SW480 cells was measured by MTT colorimetric method, and choose suitable dose. Distribution of cell cycle and apoptosis were measured by flow cytometry. Morphological changes of SW480 cells were observed by light microscopy and transmission electron microscopy. The expression of PTEN,VEGF protein were examined by semi-quantitativly flow cytometry. 2 MTT colorimetric method was performed to evaluate the potential cytosatic effect of combining rosiglitazone and 5-FU on human colorectal cancer cell SW480. To determine whether the combination rosiglitazone and 5-FU results in asynergistic cytostatic effect or not, 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.Results: 1.1 The effects of rosiglitazone on the proliferation of SW480 cells: MTT colorimetric method showed that rosiglitazone (12.5,25,50,100μmol/L) could inhibit the proliferation of SW480 cells. After treated with rosiglitazone for 24h to 72h, the OD values of rosiglitazone groups decreased, compared with control group, there was statistically significant difference between control group and every treatment group(P<0.01). Moreover, with the increasing concentration of rosiglitazone and prolonging of treatment time, the OD values decreased gradually, in other words, rosiglitazone inhibited the proliferation of SW480 cells significantly in a dose-dependent and time-dependent manner and the highest inhibition ratio was 66.7% in rosiglitazone 100μmol/L for 72h.1.2 Distribution of cell cycle was measured by flow cytometry, the results were: after SW480 cells were treated with 0,12.5,25,50μmol/L rosiglitazone for 48h, with the increasing concentration of rosiglitazone, the number of cells in G0/G1 phase increased gradually, while the number of cells in S phase decreased grudually. It showed that rosiglitazone could induce an arrest of cell cycle in G2/M G0/G1 in a dose-dependent manner. In addition, after treated with 0,12.5,25,50μmol/L rosiglitazone for 48h, the apoptotic percentage were 3.76%,7.55%,20.54%,31.85% respectively. The typical apoptotic peak which enhanced gradually with the increasing concentration of rosiglitazone was observed, compared with control group, there was statistically significant difference between control group and every treatment group(P<0.01).1.3 Morphological changes of SW480 cells were observed by light microscopy: morphouses of cells untreated with rosiglitazone were multiple. They were round,fusiform or polygon, and they were satiation. While the cells treated with rosiglitazone crimpled and fractured. Some cells stretched out long and thin pseudopodia in each end. Vacuolus could be found in endochylema of some cells. The cells fell off increased.1.4 The ultramicrostructure changes of the cells were observed by transmission electron microscopy: 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.1.5 Analysis on the expression of PTEN,VEGF protein by FCM showed that: after SW480 cells were treated with 0,12.5,25,50μmol/L rosiglitazone for 48h, the FI values of VEGF decreased with the increasing concentration of rosiglitazone, and the FI values of PTEN increased with the increasing concentration of rosiglitazone. There was statistically significant difference between control group and every treatment group for the FI values of every protein (P<0.05 or P<0.01).1.6 The results of immunohistochemistry showed that: PPARγ,PTEN protein were expressed in cell kytoplasm, and VEGF protein were expressed in kytoplasm and vascular endothelial cell. We could observed brown colouring. Immunohistochemical score was carried by IHS. The IHS values of VEGF protein decreased with the increasing concentration of rosiglitazone, the IHS values of PPARγ,PTEN protein increased with the increasing concentration of rosiglitazone. To the IHS values of every protein, there was statistically significant difference between the negative control group and every treatment group (P<0.05 or P<0.01).2 MTT colorimetric method was performed to evaluate the potential cytostaic effect of combining rosiglitazone with anti-cancer agents 5-FU in SW480 cells. The results showed: 12.5,25,50μmol/L rosiglitazone excerted a synergistic cytostatic effect in SW480 cells when combined with 5-FU .The synergistic effect combining with 5-FU was enhanced while concentration of rosiglitazone increased.The analysis of Jin's formula showed that the interaction index for 12.5,25,50μmol/L rosiglitazone and 5-FU used in combination on SW480 cells was more than 0.85. It proved that rosiglitazone indeed had a synergistic effect on inhibiting the proliferation of SW480 cells combined with 5-FU.Conclusions:1 Rosiglitazone could inhibit proliferation of colon carcinoma in a dose-dependent manner within the certain concentration.2 The growth inhibitory effects of rosiglitazone on colon carcinoma were involved in induction of apoptosis and arrest of cell cycle in G0/G1 phase, and it was in a dose-dependent manner.3 Rosiglitazone could bring into effect of anti-colon carcinoma. The mechanisms might be concerned with down-regulating the expression levels of VEGF and up-regulating the expression levels of PTEN.4 The experiment showed that: rosiglitazone had the effect of inhibiting proliferation and inducing apoptosis of colon carcinoma, which provided a new theoretical foundation for appropriate clinical treatment of colon carcinoma.5 With MTT colorimetric method, we confirmed that rosiglitazone combined with 5-FU had a synergistic effect on inhibiting the proliferation of SW480 cells.