The Study On Anti-angiogenesis Activity Of Novel 2-methoxy-estradiol Derivatives

PurpuseIt is proposed to study the anti-angiogenic effects of 2-ME and its derivatives,and to screen out new compounds which have good antiangiogenic activity and low toxicity from the derivatives,To provide some reference,for the clinical treatment of cancer combined with adjuvant therapy.Method1.Using Chicken chorioallantoic membranes assay which is the model in vivo anti-angiogenesis,to screen out 2-ME derivatives.2.Using MTTassay to detect the cytotoxicity of the selected compounds GHY on a variety of cells.3.Using Wound-healing assay to test the effect of GHY on the migration of Human HUVEC and MCF-7 cellsin vitro.4.By using Transwell chamber migrationassay to test the effect of compound GHY on the migration and invasion of HUVEC and MCF-7 cells.5.Western blot was used to investigate the molecular mechanism of compound GHY inhibition of migration and invasion.6.By using Nude mice model to study the effect ofthecompounds on anti-tumor activity.The enzymes related to liver function and the HE staining of viscera were measured to evaluatethe toxicity of the compounds.Result1.The Chicken chorioallantoic membranes assay shows that the derivatives GHY and other compounds in the number of blood vessels and vascular volume compared with the control group was statistically significant(P <0.05).2.2-ME and GHY were used in HUVEC cells,GES cells,MCF-7 cells,MGC-803 cells and PC-3 cells for 48 h and 72 h.MTT results show that 2-ME has a higher cytotoxic effect on HUVEC cells,whereas compound GHY showed lower cytotoxicity.The72 h IC50 of the compound GHY of HUVEC cells is 66 ?M,then2-ME is 2.11 ?M.3.The results of HUVEC wound-healing assay and Transwell chamber migration show that GHY could significantly inhibit the migration of endothelial cells,and the results were statistically significant(P <0.05).4.Western blot was used to detect the expression of related proteins in HUVEC,such as VEGF-R,FAK,p-FAK,and SHC.The expression of VEGF-R was down-regulatedand the corresponding SHC protein was also decreased,while the FAK total protein remained unchanged,but the expression of p-FAK in the active form of FAK was down-regulated and eventually inhibited the transfer of endothelial cells.The protein expression was statistically analyzed,Compared with the control group,the compound group had statistical difference.5.The results of MCF-7 cell wound-healing assay and Transwell chambermigration show that GHY could significantly inhibit the migration of tumor cells in a concentration-dependent manner,and the results were statistically significant(P<0.05).6.Western blot was used to detect the expression of EMT pathway marker protein in MCF-7 cells.The results show that GHY can up-regulate the expression ofepithelial phenotype markers ZO-1 and E-cadherin,and down-regulate the expression of N-cadherin And the expression of Vimentin protein.Statistical analysis showed that the expression of protein was statistically different(P <0.05).It is speculated that the mechanism by which GHY inhibits the migration of MCF-7 cells may be interfering with the EMT process.7.The MCF-7 Nude mice results show that the high-dose of compound GHY group in the tumor volume and tumor weight compared with the control group were statistically significant.Indicating that the compound GHY has a good role in inhibiting tumor growth in vivo.8.The results ofbiochemical experiments showed that the GHY had no significant effect on the values of ALT and ALP,while the positive control 5-FU group showed some hepatotoxicity.The value of AST,compared with the control group,there are some effects,but all belong to the normal range.The pathology of visceral HE staining showed that GHY had no visible damage to visceral tissue.Thus,it indicated that compound GHY does not show significant toxicity in vivo.ConclusionIn conclusion,the 2-ME derivative GHY can significantly inhibit the angiogenesis of Chicken chorioallantoic membranes.It also has a low cytotoxic effect,but can significantly inhibit the migration of HUVEC cells.The mechanism of inhibit the migration of HUVEC may be achieved by inhibiting the expression of the relevant transfer protein VEGFR /p-FAK.For the role of tumor cells,the compound GHY can inhibit the MCF-7 cell metastasis and invasion,the mechanism may be by inhibiting the expression of EMT-related protein to achieve.And in vivo nude mice assay,GHY can significantly inhibit the growth of tumor tissue,and no liver toxicity ang less side effects.