The Inhibitory Effect Of Genistein On Xenografted Tumor Of Human Ovarian Cancinoma Cell In Nude Mice And Its Effect On CyclinB1, CyclinD1 Protein Expression

IntroductionOvarian cancer has the highest mortality rate of all gynecologic malignancies. In recent years, despite the chemotherapy has made important progress, but the 5-year survival rate still has not improved markedly. Looking for the treatment of ovarian cancer, the role of new drugs and new targets has become a pressing need.Genistein was a plant estrogen extracted from the soybeans originally, as a specific inhibitor of protein tyrosine kinase. In recent years many studies at home and abroad have shown that it can inhibit breast cancer, bladder cancer, stomach cancer, liver cancer, ovarian cancer and other tumor growth. Our previous studies confirmed, genistein has inhibitory effect on ovarian epithelial cancer cell line CAOV3. WangXin and others's research on model of tumor induced by ovarian cancer in nude mice through gastrogavage show that, genistein can play an inhibitory role in tumor by inhibit ovarian cancer cell proliferation and angiogenesis. This study used the clinical use of the intraperitoneal injection route to discuss genistein's inhibitory effect on xenografted tumor of ovarian cancinoma cell in nude mice and its effect on cyclinB1, cyclinD1 protein expression.Materials and methods1. Materials(1) Experimental objectsHuman ovarian cancer cell line HO-8910 acquired from Cell Resource Center, Shanghai Institutes for Biological Sciences.Female BALB/c genetic background nu/nu nude mice, aged from 4 to 6 weeks, weighing 18 to 22g, were used as experimental animals. The mice were purchased from Shanghai SLRC Experimental Animal Center, and bred in the SPF-class laminar flow cabinet of Laboratory Animal Center of China Medical University.(2) ApparatusUltra clean cabinet, carbon dioxide incubator, refrigerator, inverted microscope and camera system, centrifugal sedimentation machine, constant temperature oscillators, calipers,electronic analytical balance, the level of shaking, electrophoresis, transfer electrophoresis tank, vertical electrophoresis unit, UV spectrophotometer, gel electrophoresis image analysis system.(3) Main reagentGenistein, DMEM medium, DMSO, PBS buffer, trypsin, calf serum and penicillin, streptomycin, cyclinB1 monoclonal mouse anti-human antibody, mouse anti-human cyclinD1 monoclonal antibody, alkaline phosphatase labeled goat anti-mouse secondary antibody, alkaline phosphatase color kit, O-dianidine,β-naphthyl acid phosphate, cellulose nitrate membrane, sodium dodecyl sulfate (SDS), three-aminomethane (Tris), acrylamide (Acr), methanol.2. Methods(1) Drug ConfigurationReserve liquid was composed of Genistein and dimethyl sulfoxide (DMSO), and saved in-20℃. When experiments are to be made, it is diluted to the required concentration with DMEM medium.(2) Cell CultureHuman ovarian cancer cell line HO-8910 cultured adherent from DMEM medium with 10% fetal calf serum, 100U/ml penicillin, 100μg/ml streptomycin, and subcultured at 37℃in 5%carbon dioxide incubator. (3) Model built on xenografted tumor of ovarian cancinoma cell in nude miceThe cell line of human ovarian cancer named HO-8910, was cultured from DMEM with 10% calf serum, and suspended in serum-free culture medium by 1×107/mL. Tumors were established by injection of 0.2mL tumors cells respectively into the right axilla of each nude mouse. Visible tumors block formed in about 7 to 10 days, and intraperitoneal began when the tumor blocks Grew to a diameter of about 4mm.(4) Animal experimentsThrough the establishment of model on xenografted tumor of ovarian cancinoma cell in nude mice, to discuss the Inhibitory Effect of Genistein on Ovarian Cancer.(5) Western blot methodWith the method of western blot to measure cyclinB1, cyclinD1 protein expression in xenografted tumor of ovarian cancinoma cell in nude mice.(6) Statistical MethodsStatistical software SPSS 13.0 was used for t test of differences between the two groups, the difference of P<0.05 was significant.Results1. Research on establishment of model on subcutaneously implanted tumor induced by ovarian cancer in nude mice and if Genistein has inhibitory effect on Ovarian Cancer:Model on subcutaneously implanted subcutaneously tumor induced by ovarian cancer in nude mice was erected successfully, experiment such as intraperitoneal injection was conducted smoothly. Will each dose group of Genistein compared with the control group, low-dose group, Middle and high dose group significantly inhibited tumor growth (P<0.05).In this study, tumor-bearing nude mice were given different doses of Genistein treatment for 21 days. During and after the course of treatment, observation on the rat's diet, growth and activity showed no obvious abnormality, while no significant difference in body weight.2. Genistein's effect on cyclinB1, cyclinD1 protein expression on xenografted tumor of ovarian cancinoma cell in nude mice:Western blot showed that compared with control group, genistein low, medium and high dose group after treatment decreased protein expression cyclinB1, cyclinD1 protein gradually increased, there were a dose-response relationship and it was statistically significant (P<0.05).Conclusions1. Genistein has inhibitory effect of on xenografted tumor of ovarian cancinoma cell in nude mice and in a dose-effect of positive correlation;2. Genistein has inhibitory effect of on xenografted tumor of ovarian cancinoma cell in nude mice and has dose-effect correlation,and it can decline cyclinB1's protein expression and raise cyclinD1's protein expression.