Cultivated Orostachys japonicus extract inhibits VEGF-induced angiogenesis via regulation of VEGFR2 signaling pathway in vitro and in vivo

Ethnopharmacological relevance: Orostachys japonicus A. Berger (O. japonicus), so-called Wa-song in Korea, a traditional food and medicine that grows on mountain rocks and roof files. Wa-song containing various phenolic compounds have been reported as a medicinal plant for prevention of fibrosis, cancer, inflammation, and oxidative damage. Aim of the study: The present study was designed to examine the anti-angiogenic effects of cultivated Orostachys japonicus 70% ethanol extract (CE) in vascular endothelial growth factor (VEGF)-stimulated human umbilical vein endothelial cells (HUVECs). Materials and methods: CE was prepared with 70% ethanol. HUVECs, rat aortic rings, and matrigel plug in mice were treated with CE (10-20 mu g/mL) and VEGF (20-50 ng/mL), and the anti-angiogenic activities of CE were analyzed by SRB, wound healing, trans-well invasion, capillary-like tubule formation, rat aortas, Western blot, and matrigel plug assay. Phenolic compounds in CE were analyzed using a high-performance liquid chromatography (HPLC)-PDA system. Results: Treatment of CE (10-20 mu g/mL) markedly suppressed proliferation of HUVECs in the presence (from 136.5% to 112.2%) or absence of VEGF (from 100.0% to 92.1%). The proliferation inhibitory effect of CE was caused by G0/G1 cell cycle arrest, and the decrease of CDK-2, CDK-4, Cyclin D1 and Cyclin E1. Furthermore, CE treatment showed significant angiogenesis inhibitory effects on motility, invasion and micro-vessel formation of HUVECs, rat aortic rings and subcutaneous matrigels under VEGF-stimulation condition. In HUVECs, CE-induced anti-angiogenic effect was regulated by inhibition of the PI3K/AKT/mTOR, MAPK/p38, MAPK/ERK, FAK-Src, and VEGF-VEGFR2 signaling pathways. Conclusion: This study demonstrated that CE might be used as a potential natural substance, multi-targeted angiogenesis inhibitor, functional food material.