Inhibitive Efficacy And Associated Mechanisms Of Apigenin On Human Ovarian Carcinoma Cell Line CAOV3

Inhibitive efficacy and associated mechanisms of apigenin on human ovarian carcinoma cell line CAOV3ObjectiveThe mortality of ovarian carcinoma is the highest among that of the obstetric malignant disease. Ovarian cancer is fast growing and insidious in that it is late to cause symptoms. Chemotherapy has proved curative in advanced ovarian cancers.Because serious side effects and chemotherapy resistance are major problems, it is our important aim to seek new agents that have relatively high response rates and low side effects and non-cross-resistance properties.The tumor is caused by multifactor, multistep carcinogenesis and multigene mutation. Cell cycle regulation, anti-apoptosis and angiogenesis was known necessary for the tumor development. Survivin is a kind of protein works on both cell cycle regulation and anti-apoptosis, and is the most effective anti-apoptotic factor. Survivin and p21 could competitively bind to CDK4, form survivn/CDK4 complex, release p21 from the complex, stop its suppression on cell cycle, and induce cell proliferation. Survivin could bind to caspase-9, active the mitochondrial pathway, and its antiapoptotic effect was achieved through death receptor pathway. Caspase-3 is the crosslink between mitochondrial pathway and death receptor pathway, and the crucial factor of apoptosis. Survivin could bind to caspase-3, suppress its activity, then induce apoptosis. Expression of VEGF and survivin are positively related, VEGF could up regulate positively the expression of survivin in HUVEC. Survivin expression was observed in almost all the tumors, but not in the already differentiated normal tissues. Inhibition the expression of survivin will induce apoptosis and cell division suppression in tumor cells, while the normal cells were not affected. So survivin is an attractive target for new anti-cancer interventions.Natural drugs were well studied recently as its low toxicity, and high activity. Apigenin(4',5,7,-trihydroxyflavone) is a common dietary flavonoid. It has low toxicity, is nonmutagenic, and is widely distributed in many fruits and vegetables. It has the effects of sedative, antibiosis, depressurization, et al. And it has recently been shown to possess anti-tumor properties. It was reported that apigenin could inhibit cell proliferation and induce apoptosis in tumor cells. But the mechanism is still unclear, it was suggested that apigenin may execute its anticancer effect through various pathway: suppressing cell proliferation, inducing apoptosis, inhibiting tumor angiogenesis, invasion and metastasis. But there is no report yet on whether apigenin could inhibit the growth of human ovarian carcinoma cell line CAOV3, and its mechanism.In this study, based on the great pharmaceutical effect of apigenin on inhibition of cell proliferation and blockage on cell cycle, we provided theoretical evidence for apigenin as chemotherapeutic drug, so that to kill tumor cells and assist ovarian cancer therapy. And for further understanding the relation between diet and ovarian cancer. Through choosing diet, it may be possible to find a proper prevention strategy for ovarian cancerMethods1,Using inverted phase constract microscopy, methyl thiazolyl tetrazolium(MTT) method and Flow cytometry analysis(FCM) to investigate the effect of apigenin on CAOV3 cell proliferation. Cell morphological changes of CAOV3 under the 48h treatment of 10,20,40,80,160μM apigenin were observed with inverted phase contrast microscopy, cells without treatment were taken as controls. MTT assay was performed to determine CAOV3 cell proliferation with or without apigenin treatment. Cells treated with 10,20,40,80,160μM apigenin for 48h, or 80μM apigenin for 24h, 48h and 72h were studied. Cell cycle changes of CAVO3 under the treatment of 10,20, 40,80μM apigenin were determined by Flow cytometry analysis(FCM).2,To analysis the mechanism of inhibitive effect of Apigenin on CAOV3 cell proliferation, immunohistochemistry, RT-PCR, and western blot were performed. Expression of survivin protein in CAOV3 cells under the treatment of 80μM apigenin for 72h was determined by immunohistochemistry(SP method). Effect of apigenin on the expression of survivin, p21, caspase-3 and VEGF mRNA were studied by RT-PCR; CAOV3 cells were treated with 10,20,40,80,160μM apigenin for 48h, or with 80μM apigenin for 24h, 48h and 72h. Effect of apigenin on the expression of survivin, p21 and caspase-3 protein were studied by using western blot.3,Expression of survivin and VEGF in ovarian cancer tissues and normal ovarian tissues were determined by using immunohistochemistry and RT-PCR. Expression of survivin and VEGF protein in 38 ovarian cancer tissues and 8 normal ovarian tissues were determined by using immunohistochemistry. Expression of survivin and VEGF mRNA in 20 ovarian cancer tissues and 4 normal ovarian tissues were determined by using RT-PCR.4,Statistic analysis: experimental data were shown as mean±s, and analyzed by SPSS10.0 statistic software. Student's t test, chi-square test, Pearson and Spearman correlation coefficients was used, size of testα=0.05.Results1,To observe the effect of Apigenin on CAOV3 cell proliferation. Morphological changes of cell: under inverted phase contrast microscopy, in the cells treated with 10,20,40,80,160μM apigenin for 48h, the cell density and cellular volume of experimental group were decreasing gradually, intercellular space and intracellular cells granules were increasing accompany with the increasing of agents. In 160μM apigenin treating group, great amount of cell debris were observed. The cells in control group were observed to be no obvious morphological changes, cells were spindle shape, cell membrane are intact, chromatin distributed evenly. MTT assay shows that after inoculation 48 hours, the suppress rates of CAOV3 cell proliferation at 10, 20, 40, 80, 160μM apigenin were 2.7％, 9.0％, 24.5％, 42.8％, 63.8％respectively. (P＜0.01).After treated with 80μM apigenin for 24h, 48h and 72h, the suppress rates of CAOV3 cell proliferation were 13％, 43％, 68％respectively(P＜0.01). It suggests that accompany with the increasing of apigenin and the longer inoculation time, the inhibition of CAOV3 cell proliferation was increasing and showed perfect time-effect relationship and dose-effect relationship.Results of cytometry analysis show that after inoculation of apigenin 24 hours at different concentrations, the ratio of cell on G2/M phase was increased from 6.45％to 43.17％, and the ratio on G0/G1 and S phase was decreased, showing G2/M phase blockage. After 48h apigenin treatment, G2/M ratio increased from 0.49％to 63.2％, the extent of decrease in the ratio of G0/G1 and S phase, G2/M phase blockage was remarkble when compared with 24h treatment. And in the group treated with 80μM apigenin, peak of SubG1 cell(apoptotic cells) were observed after 24h and 48h treatment.2,To analysis the mechanism of inhibitive effect of Apigenin on CAOV3 cell proliferation. Results of immunohistochemistry show that in control group, brown particles covered almost all cytoplasmic area, which indicates positive expression of survivin. After treatment with 80μM apigenin for 72h, expression level of survivin protein greatly decreased. Results of RT-PCR and Western blot show that in CAOV3 cells, with the apigenin amount increasing, expression level of survivin mRNA and protein and VEGF mRNA decreased while expression of p21 and caspase-3 mRNA and protein increased.3,Expression of survivin and VEGF in ovarian cancer tissues and normal ovarian tissues. Immunohistochemistry results show that expression of survivin and VEGF in ovarian cancer tissues were increased. Andin 27 cases of survivin positive ovarian cancers, 19 cases positively expressed VEGF, coexpression ratio of the two protein was 50％. In 11 survivin negative cases, 8 cases lacked the expression of VEGF, 21.05％showed negative expression of the two protein. Statistic analysis indicates that expression of Survivin and VEGF are positively related(P＜0.05). RT-PCR results indicate that ovarian cancer tissues showed higher expression level of survivin and VEGF mRNA comparing with normal ovarian tissues(P＜0.05).Conclusions1,Apigenin could block CAOV3 at G2/M phase, induce apoptosis, thus execute inhibit CAOV3 cell proliferation. And the inhibition of CAOV3 cell proliferation showed perfect time and dose dependence.2,Apigenin down regulated expression of survivin mRNA and protein, while expression of p21 and caspase-3 mRNA and protein were upregulated. The mechanism for inhibition effect of apigenin on CAOV3 cells is speculated to be as follows: Downregulation of survivin expression by apigenin blocked CAOV3 cells at G2/M phase, and induced apoptosis. Besides the inhibition of CAOV3 cell proliferation by apigenin, expression of VEGFmRNA was also inhibited by apigenin, so that tumor angiogenesis was inhibited. Our results suggest that apigenin may be a new agent of treatment ovarian caner.3,Expression of survivin and VEGF were increased in ovarian cancer tissues. And expression of survivin and VEGF are positively related. Our research in vitro had proved that apigenin could down regulated expression of survivin and VEGF, which suggests apigenin as a new agent of treatment ovarian caner could have the good future in clinical therapy.