Effects Of Genistein On Invasion Of Methotrexate-resistant Human Choriocarcinoma JAR/MTX Cells And Associated Mechanism

Choriocarcinoma is a highly malignant neoplasm of placenta characterized by high potential to metastasize. The choriocarcinoma cell has high tendency to invade into blood vessel and metastasize even in the early stage. It has got satisfied prognosis with the advancement of chemotherapy. However, the chemotherapy was limited for its evident side-effects. Furthermore, the emergency of drug resistance also leads to final treatment failure. Metastasis is proved to be the major cause of death.Genistein (4,5,7-trihydroxyisoflavone) is a phytoestrogen coming from soy diets. Epidemiological study indicated that genistein was evidently correlated with reduced incidence of cancer including breast, prostate and colon cancer. Many studies of in vivo and in vitro have indicated that genistein could inhibit the activity of DNA topoisomerase and tyrosine protein kinase,as well as inhibit proliferation and induce differentiation of cancer cells. Several studies have shown that genistein inhibits invasion of cancer cells including breast, Melanoma and fibrosarcoma cells in in vitro assay. The exact mechanism of genistein action has not been determined. In this study, we have investigated the effect of genistein on the biological behavior of choriocarcimoma and the effect on the invasiveness of methotrexate - resistant choriocarcinoma cell JAR/MTX and the associated mechenism.There are three parts in the study: part I Effects of Genistein on the choriocarcinoma cell line JAR; part II Effects of Genistein on proliferation, apoptosis and invasiveness of methotrexate-resistant human choriocarcinoma JAR/MTX cells and associated mechanism; part III Invasiveness inhibition induced by Genistein in methotrexate-resistant human choriocarcinoma JAR/MTX cells is mediated by ER-beta.Part I Effects of Genistein on the choriocarcinoma cell line JARObjective To study the effects of Genistein on morphology , proliferation , differentiation , cell cycle and invasion of choriocarcinoma cell line JAR . Methods The Genistein was incubated in different concentrations with choriocarcinoma cells JAR. Every 24 hours, aliquots were removed and tested for human chorionic gonadotrop(h) in (hCG). In addition we tested the effects of Genistein on cell proliferation by MTT assay. To study the effects of Genistein on morphology, ultrastructure, cell cycle, immigration and invasion by HE stain, electron microscope, flowcytometry(FCM) and invasion assay. In vivo , in order to reveal the tumor-suppressive effect of Genistein, JAR cells incubated in Genistein were hypodermic injected in nude mice. Results With this study we could show that Genistein can reduce the proliferation of trophoblast tumour cell JAR, in addition it has correlation with times and doses. 100nmol/ml Genistein can increase the secretion of hCG, In addition it has correlation with times. In Comparison of a vursus control group, 10 , 25 , 50 and 100 nmol/ml Genistein downregulated invasion of JAR by -14. 1% ( P>0. 05 ), 75. 6 % ( P<0. 05 ) , 100% ( P<0. 05 ) and 100% (P<0. 05 ) . In Comparison of a vursus control group ( 17. 44 ±6. 16) , and Genistein can block cell cycle on G2-M stage (67. 14 ±1.02) .10 and 100nmol/ml Genistein downregulated migration of JAR by 42. 3%(P = 0. 013) and 41.3% ( P = 0. 014). In vivo, tumor generation rate of control group was 100% and Genistein group was 16. 7%. Tumor weight were 1. 10 ± 0. 95g and 0. 008 ± 0. 020g. Conclusions Genistein can suppresses proliferation, cell cycle, migration , invasion and increase secretion of HCG of choriocarcinoma cell line JAR.Part II Effects of Genistein on proliferation, apoptosis and invasiveness of methotrexate-resistant human choriocarcinoma JAR/MTX cells andassociated mechanismObjective To study the effects of Genistein on JAR/MTX cells proliferation, apoptosis and invasion and associated mechanism in vitro. Methods MTT assay, Annxin-Vand propidium iodide label analysis and invasion assay were used to determine the effects of Genistein on proliferation, apoptosis and invasiveness of JAR/MTX methotrexate -resistant human choriocarcinoma cells . RT-PCR was used to estimate the relative mRNA amounts of estrogen receptor(ER), MTA3 and snail in the cells. Western blot and Gelatin zymography assay were used to estimate the relative protein amounts of MMP-2. MMP-9 and E-cd in the cells. Results After treated by Genistein, the proliferation and invasiveness of JAR/MTX cells were decreased significantly in dose-dependent manner. 10nmol/ml Genistein induced apoptosis (16.85%) whereas 25,50, 100nmol/ml Genistein induced apoptosis (54.84%, 54.67%, 47.50% ) and necrosis (28.59%, 21.60%, 44. 12% ) significantly. Through exposure to Genistein led to a increase in ERG, MTA3 mRNA and E-cd protein expression. Decreases of the amounts for snail mRNA and MMP-2 and MMP-9 protein expression of JAR/MTX cells were observed. Conclusions Genistein can inhibit the proliferation by induced apoptosis and necrosis. Genistein also could inhibit JAR/MTX cells invasion in part through the upregulation of E-cadherin and downregulation of MMP-2 and MMP-9. Genistein may be suppresses invasion of JAR/MTX cells by one of signalway of MTA3→snail→ E-cd.Part III Invasiveness inhibition induced by Genistein in methotrexate-resistant human choriocarcinoma JAR/MTX cells is mediatedby estrogen receptor betaSubjective In this study , we investigated the effects of estrogen receptor β (ER-β) in invasion inhibited by Genistein. Methods After ER-β have bene interferenced , invasion assay, western blot and Gelatin zymography assay were used to determine the effects of Genistein on invasion, to estimate the relative protein amounts of MMP-2, MMP-9 and E-cadherine of JAR/MTX methotrexate - resistant human choriocarcinoma cells . RT-PCR was used to estimate the relative mRNA amounts of MTA3 and snail in the cells. Results After RNA interference in ER- β , the effects of 10 and 25 nmol/ml Genistein-induced invasiveness inhibition (62%和 72%) were inhibited ( -19. 9%和 6. 3 % ) completly . 50 和 100 nmol/ml Genistein-induced invasiveness inhibition ( 94% 和 96% )were also inhibited ( 68. 7% 和 81.7 % ) partly. In Comparison of a vursus control group, Genistein-induced upregulation of E-cadherine and downregulation of MMP-2 and MMP-9 were suppressed. Comparing with Oh, upregulation of MTA3 and downregulation of Snail induced by Genistein were suppressed. Conclusion we demonstrate that invasiveness inhibition of methotrexate-resistant human choriocarcinoma JAR/MTX cells induced by Genistein is mediated by ER-β .