Impact Of Gamabufotalin Inhibits VEGF-induced Angiogenesis

Objective: Gamabufotalin(CS-6),a majoractive compound isolated from Chinese medicine Chansu,has been shown to strongly inhibit cancer cell growth and inflammatory response.However,its effects on angiogenesis have not been known yet.Here,we sought to determine the biological effects of CS-6 on signaling mechanisms during angiogenesis.Methods: The effects of CS-6 on VEGF induced human umbilical vein endothelial cells(HUVEC)proliferation,migration and invasion were determined by MTT,scratch wound healing and transwell invasion assays.Moreover,the in vitro,ex vivo and in vivo assays including tube formation on Matrigel,spheroid sprouting in collage gel,mouse aortic ring sprouting as well as Matrigel plug were applied to evaluatethe influence of CS-6 on VEGF stimulatingendothelial angiogenesis.Immunofluorescence,Co-immunoprecipitation and Western blotting were employed to explore the underlying mechanisms of CS-6 suppressing VEGFR-2 activation.Results: CS-6 could significantly inhibit VEGF triggered HUVECs proliferation,migration,invasion and tubulogenesis in vitro and blocked vascularization in Matrigel plugs impregnated in C57/BL6 mice as well as reduced vessel density in human lung tumor xenograft implanted in nude mice.Computer simulations revealed that CS-6 interacted with the ATP-binding sites of VEGFR-2 using molecular docking.Furthermore,western blot analysis indicated that CS-6 inhibited VEGF-induced phosphorylation of VEGFR-2 kinase and suppressed the activity of VEGFR-2-mediated signaling cascades.Conclusions: These results demonstrated that CS-6 inhibited tumor angiogenesis by inhibiting the activation of VEGFR-2 signaling pathways and CS-6 could be a potential candidate in angiogenesis-related disease therapy.