Effects And Its Mechanisms Of Genistein On Colon Carcinoma In Vivo And Vitro

Objective: To study the effects of genistein on the proliferation,inducing apoptosis and expression of correlative protein of colon carcinoma in vivo and vitro. To investigate the mechanisms of genistein on colon carcinoma, and provide the theoretic basis for colon carcinoma therapy.Methods: 1 In vitro: SW480 cells were cultured in vitro. Using MTT colorimetric method to study the effects of genistein on the proliferation of SW480 cells, and to 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 PCNA,P21,VEGF protein was semi-quantitativly examined by flow cytometry. 2 In vivo: sixty healthy male BALB/c mice were divided randomly into five groups(twelve in each): the control group(physiological saline); the low-dose genistein group(1mg/kg); the middle-dose genistein group(10mg/kg); the high-dose genistein group(20mg/kg), and the 5-Fu group. Murine colonic adenocarcinoma cells (colon26) were inoculated subcutaneously into each mouse to establish model of subcutaneous transplantation of colon carcinoma. Beginning to administer and receive intraperitoneal injection respectively when tumors were touched on most mice. The mice were treated with Sodium Chloride (0.2ml/d, DMSO<1%),low-dose genistein (1mg/kg/d),middle-dose genistein (10mg/kg/d),high-dose genistein(20mg/kg/d) for consecutive fourteen days; 5-Fu(0.2ml per mouse, 16.5mg/ml) once per week for twice. Observe animal action, record weight of animals and the size of tumor. Fourteen days later all mice were weighed before being killed and then the tumors were taken and weighed. The volume of tumor and the inhibition ratio of tumor growth were calculated. Distribution of cell cycle and apoptosis were measured by flow cytometry. The expression of PCNA,P21,VEGF protein was studied by immunohistochemistry.Results1 the results in vitro1.1 The effects of genistein on the proliferation of SW480 cells: MTT colorimetric method showed that genistein(10,20,40,80μg/ml) could inhibit the proliferation of SW480 cells. After being treated with genistein for 24h to 72h, the OD values of genistein 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 genistein and prolonging of treatment time, the OD values decreased gradually, in other words, genistein inhibited the proliferation of SW480 cells significantly in a dose-dependent and time-dependent manner and the highest inhibition ratio was 60.2% in genistein 80μg/ml for 72h.1.2 Distribution of cell cycle was measured by flow cytometry, the results were: after SW480 cells were treated with 0,20,40,80μg/ml genistein for 48h, with the increasing concentration of genistein, the number of cells in G2/M phase increased gradually, while the number of cells in G0/G1 phase decreased grudually. It showed that genistein could induce an arrest of cell cycle in G2/M phase in a dose-dependent manner. In addition, after being treated with 0,20,40,80μg/ml genistein for 48h, the apoptotic percentage were 1.40%,8.01%,24.22%,47.01% respectively.The typical apoptotic peak which enhanced gradually with the increasing concentration of genistein 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 genistein were multiple. They were round,fusiform or polygon, and they were satiation. While the cells treated with genistein 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 falling 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 PCNA,P21,VEGF protein by FCM showed that: after SW480 cells were treated with 0,20,40,80μg/ml genistein for 48h, the FI values of PCNA,VEGF decreased with the increasing concentration of genistein, the FI values of P21 increased with the increasing concentration of genistein. To the FI values of every protein, there was statistically significant difference between control group and every treatment group (P<0.05 or P<0.01).2 the results in vivo2.1 Genistein inhibited obviously the growth of tumor. After all mice were killed, the volume of tumor of the low-dose genistein group,the middle-dose genistein group,the high-dose genistein group and the 5-Fu group were lower than the negative control group. There was statistically significant difference between control group and every treatment group (P<0.01). Moreover, the volume of tumor of three treatment groups decreased with the increasing dose of genistein. The inhibition ratio of tumor of the low-dose genistein group,the middle-dose genistein group,the high-dose genistein group and the 5-Fu group were 11.7%,28.8%,40.8%,62.0% respectively. The inhibition ratio of three treatment groups increased with the increasing dose of genistein.2.2 Distribution of cell cycle was measured by flow cytometry, the results were: comparing the low-dose genistein group,the middle-dose genistein group,the high-dose genistein group and control group, the number of cells in G2/M phase increased gradually, while the number of cells in G0/G1 phase decreased grudually. That was, genistein could induce an arrest of cell cycle in G2/M phase in a dose-dependent manner. Furthermore, the apoptotic percentage of control group,the low-dose genistein group,the middle-dose genistein group,the high-dose genistein group and the 5-Fu group were 3.16%,10.26%,19.35%,27.27%,49.64% respectively. The typical apoptotic peak which enhanced gradually with the increasing concentration of genistein was observed, and 5-Fu group was also observed, compared with control group, there was statistically significant difference between control group and every treatment group(P<0.01).2.3 The results of immunohistochemistry showed that: PCNA,P21 protein were expressed in cell nucleus, 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 PCNA,VEGF protein decreased with the increasing concentration of genistein, the IHS values of P21 protein increased with the increasing concentration of genistein. To the IHS values of every protein, there was statistically significant difference between control group and every treatment group (P<0.05 or P<0.01).Conclusions1 Genistein could inhibit proliferation of colon carcinoma in a dose-dependent manner within a certain concentration. 2 The growth inhibitory effects of genistein on colon carcinoma were involved in induction of apoptosis and arrest of cell cycle in G2/M phase, and it was in a dose-dependent manner.3 Genistein could bring into effect of anti-colon carcinoma. The mechanisms might be concerned with downregulating the expression levels of VEGF,PCNA and up-regulating the expression levels of P21.4 The experiment showed that: genistein had the effect of inhibiting proliferation and inducing apoptosis of colon carcinoma, which provided a new theoretical foundation for appropriate treatment and chemoprevention of colon carcinoma.