title: Anti-angiogenic activity and mechanism of kaurane diterpenoids from Wedelia chinensis.
abstract: BACKGROUND
  Wedelia chinensis is a traditional medicinal herb used in Asia and it has been reported to possess various bioactivities including anti-inflammatory and anticancer effects. However, its anti-angiogenic activity has never been reported.
  
  
  OBJECTIVE
  To determine the most potent anti-angiogenic component in W. chinensis and its molecular mechanism of action.
  
  
  METHODS
  Initially, the active fraction of the plant was studied. Then, we determined the active components of the fraction and explored the mechanism of the most active compound.
  
  
  METHODS
  The ethanol extract of W. chinensis and its four fractions with different polarities were evaluated for their anti-angiogenic activity in the Zebrafish model using quantitative endogenous alkaline phosphatase (EAP) assay. The molecular mechanism of the most active compound from the active fraction was studied using the real-time polymerase chain reaction (PCR) assay on Zebrafish embryos. The inhibitory effect of the most active compound on the proliferation, invasion and tube formation steps of angiogenesis was evaluated using the vascular endothelial growth factor (VEGF)-induced human umbilical vein endothelial cells (HUVECs) model, and the influences of the active compound on tyrosine phosphorylation of VEGF receptor (VEGFR-2) and its downstream signal pathway were evaluated by western blotting assay. Moreover, its anti-angiogenic effect was further evaluated by the VEGF-induced sprouts formation on aortic ring assay and the VEGF-induced vessel formation of mice on matrigel plug assay, respectively.
  
  
  RESULTS
  Petroleum ether (PE) fraction of the plant displayed potent anti-angiogenic activity. Twelve kaurane diterpenoids (1-12) isolated from this fraction showed quite different effects. Compounds 9-12 could dose-dependently inhibit vessel formation in the Zebrafish embryos while the others showed little inhibitory effect. Among the active diterpenoids, compound 10, 3α-cinnamoyloxy-9ß-hydroxy-ent-kaura-16-en-19-oic acid (CHKA), possessed the strongest effect, and it affected multiple molecular targets related to angiogenesis including VEGF and angiopoietin in Zerbrafish. Moreover, CHKA significantly inhibited a series of VEGF-induced angiogenesis processes including proliferation, invasion, and tube formation of endothelial cells. Besides, it directly inhibited VEGFR-2 tyrosine kinase activity and its downstream signaling pathways in HUVECs. CHKA also obviously inhibited sprouts formation of aortic ring, and block vessel formation in mice.
  
  
  CONCLUSIONS
  Our findings demonstrate that kaurane diterpenoids is one of anti-angiogenic components in W. chinensis, and CHKA may become a promising candidate for the development of anti-angiogenic agent.
authors.affiliation: Institute of Traditional Chinese Medicine and Natural Products, College of Pharmacy, Jinan University, 601 Huangpu Avenue West, Guangzhou 510632, P. R. China.
authors.forename: Weihuan
authors.initials: W
authors.lastname: Huang
authors.affiliation: Institute of Traditional Chinese Medicine and Natural Products, College of Pharmacy, Jinan University, 601 Huangpu Avenue West, Guangzhou 510632, P. R. China.
authors.forename: Yeyin
authors.initials: Y
authors.lastname: Liang
authors.affiliation: Institute of Traditional Chinese Medicine and Natural Products, College of Pharmacy, Jinan University, 601 Huangpu Avenue West, Guangzhou 510632, P. R. China.
authors.forename: Jiajian
authors.initials: J
authors.lastname: Wang
authors.affiliation: Institute of Traditional Chinese Medicine and Natural Products, College of Pharmacy, Jinan University, 601 Huangpu Avenue West, Guangzhou 510632, P. R. China.
authors.forename: Guoqiang
authors.initials: G
authors.lastname: Li
authors.affiliation: Institute of Traditional Chinese Medicine and Natural Products, College of Pharmacy, Jinan University, 601 Huangpu Avenue West, Guangzhou 510632, P. R. China.
authors.forename: Guocai
authors.initials: G
authors.lastname: Wang
authors.affiliation: Institute of Traditional Chinese Medicine and Natural Products, College of Pharmacy, Jinan University, 601 Huangpu Avenue West, Guangzhou 510632, P. R. China. Electronic address: tliyl@jnu.edu.cn.
authors.forename: Yaolan
authors.initials: Y
authors.lastname: Li
authors.affiliation: Food and Nutritional Sciences Programme, School of Life Sciences, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong SAR, China. Electronic address: anthonychung@cuhk.edu.hk.
authors.forename: Hau Yin
authors.initials: HY
authors.lastname: Chung
mesh_terms: D020533:Angiogenesis Inhibitors / Q000494:pharmacology*; D000818:Animals; D001011:Aorta / Q000187:drug effects; D002465:Cell Movement / Q000187:drug effects; D045786:Diterpenes, Kaurane / Q000494:pharmacology*; D004625:Embryo, Nonmammalian / Q000187:drug effects; D061307:Human Umbilical Vein Endothelial Cells / Q000187:drug effects*; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D015394:Molecular Structure; D009389:Neovascularization, Pathologic / Q000188:drug therapy; D010766:Phosphorylation; D010936:Plant Extracts / Q000494:pharmacology; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction / Q000187:drug effects; D042461:Vascular Endothelial Growth Factor A / Q000494:pharmacology; D040281:Vascular Endothelial Growth Factor Receptor-1 / Q000378:metabolism; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000378:metabolism; D031209:Wedelia / Q000737:chemistry*; D015027:Zebrafish
chemical_list: D020533:Angiogenesis Inhibitors; D045786:Diterpenes, Kaurane; D010936:Plant Extracts; D042461:Vascular Endothelial Growth Factor A; C501162:FLT1 protein, human; D040281:Vascular Endothelial Growth Factor Receptor-1; D040301:Vascular Endothelial Growth Factor Receptor-2
keywords: Anti-angiogenesis; Kaurane diterpenoid; Mechanism; Wedelia chinensis; Zerbrafish