N-substituted Benzylidene Pyrrolidine Diketone Compounds Screeing Activity And Mechanism Of Anti-angiogensis Study

Tumor angiogenesis in tumor occurrence,development and metastasis plays a very important role.Especially the growth of solid tumors is dependent on intricate and complex network of blood vessels.Blood vessels supply nutrition and oxygen and exclude toxic wastes of the cells metabolism.Therefore,inhibition of angiogenesis can effectively block the nutrient supply so as to restrain the tumor growth.Dynamic equilibrium of pro-angiogenic and anti-angiogenic factors lose regulation during tumor growth,which rapidly generate abnormal angiogenesis.Vascular endothelial growth factor plays a key regulatory role in the process of neovascularization.Especially VEGF and its receptor-mediated signaling pathways is major regulator of angiogenesis.Therefore,targeting VEGF and VEGFR receptor-specific tyrosine kinase inhibitors have become a researchful hot.Some small molecule tyrosine kinase inhibitor have significant efficacy in clinical anti-tumor therapy.This paper based on the mechanism of angiogenesis,with natural antineoplastic drugs for nucleus independent synthesis novel the tyrosine kinase inhibitor for the purpose,which screen anti-angiogenesis activity compounds through molecular,cellular and animal models.After a series of experiments to screen the active better N-substituted benzylidene pyrrolidine diketones XCF-37b/43b and Urea based benzo imidazoles compounds ZD-21,then for further study the compounds mechanism of anti-angiogenic active.First,using of chorioallantoic membrane model in vivo to screen chemical synthesis of compounds(targeting the receptor tyrosine kinases)with anti-angiogenic active.Subsequently,MTT assay of HUVEC cells were further screened with a specific inhibit the proliferation of endothelial cells,which found the XCF-37b/43b compound IC50 values with VEGF-induced HUVEC proliferation is three times the normal HUVEC.ZD-21 has significantly inhibited HUVEC proliferation,and its IC50 value is 5.6?M.Next,the scratch assay found 2.5?M XCF-37b/43b and ZD-21 within HUVEC after 24h,and inhibit migration of endothelial cells at 70%.Meanwhile 2.5?M of XCF-37b/43b significantly inhibite forming microtubules ability of endothelial cells.In Western blotting detection of VEGF and mTOR signaling pathway;5?M XCF-37b suppressed expression P-VEGFR2,P-FAK,P-Src,P-Erk,P-AKT,P-mTOR protein and P-P70S6K etc.However,XCF-37b inhibite endothelial cell proliferation independent-apoptosis,but dependent on the induction of VEGF.In addition,5?M XCF-37b significantly inhibite vascular sprouting process and invasion of tumor cells.Scratch assay show ZD-21 not only inhibit endothelial cell migration but also becomes larger the gap of cells and reduce intercellular adhesion between endothelial cells.By immunofluorescence staining clearly see the changes cell morphology and cell adhesion decreases,with the concentration increases result in cell apoptosis.In summary,XCF-37b has a specific inhibition of endothelial cell proliferation and migration,and inhibits VEGF and mTOR signaling pathway and its downstream activity of the protein to produce an anti-angiogenic effect.However,XCF-3 7b inhibit endothelial cell proliferation is dependent on VEGF induced vascular sprouting,microtubule formation and cell invasion.In addition,ZD-21 also shows a certain degree of anti-angiogenic activity and may destructe cytoskeleton of endothelial cells.In short,we screened tyrosine kinase inhibitors XCF-3 7b with a better anti-angiogenesis activity,which has a potential value against tumor angiogenesis.