title: Bear bile powder inhibits angiogenesis in vivo and in vitro.
abstract: OBJECTIVE
  To evaluate the effect of bear bile powder (BBP) on angiogenesis, and investigate the underlying molecular mechanisms.
  
  
  METHODS
  A chick embryo chorioallantoic membrane (CAM) assay was used to evaluate the angiogensis in vivo. Human umbilical vein endothelial cells (HUVECs) were treated with 0, 0.25, 0.5, 0.75, and 1.0 mg/mL of BBP for 24, 48 and 72 h, respectively. The 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was performed to determine the viability of HUVECs. Cell cycle progression of HUVECs was examined by fluorescence-activated cell sorting (FACS) analysis with propidium iodide staining. HUVEC migration was determined by wound healing method. An ECMatrix gel system was used to evaluate the tube formation of HUVECs. The mRNA and protein expression of vascular endothelial growth factor (VEGF)-A in both HUVECs and HepG2 human cells were examined by reverse transcription-polymerase chain reaction and enzyme linked immunosorbent assay, respectively.
  
  
  RESULTS
  Compared with the untreated group, BBP inhibited angiogenesis in vivo in the CAM model (P< 0.01). In addition, treatment with 0.25-1 mg/mL of BBP for 24, 48, or 72 h respectively reduced cell viability by 14%-27%, 29%-69% and 33%-91%, compared with the untreated control cells (P< 0.01). Additionally, BBP inhibited the proliferation of HUVECs via blocking the cell cycle G to S progression, compared with the S phase of untreated cells 48.05%± 5.00%, 0.25-0.75 mg/mL BBP reduced S phase to 40.38%± 5.30%, 36.54± 4.50% and 32.13± 3.50%, respectively (Pglt; 0.05). Moreover, BBP inhibited the migration and tube formation of HUVECs, compared with the tube length of untreated cells 100%± 12%, 0.25-0.75 mg/mL BBP reduced the tube length to 62%± 9%, 43%± 5% and 17%± 3%, respectively (p< 0.01). Furthermore, BBP treatment down-regulated the mRNA and protein expression levels of VEGF-A in both HepG2 cells and HUVECs.
  
  
  CONCLUSIONS
  BBP could inhibit the angiogenesis by reducing VEGF-A expression, which may, in part, explain its anti-tumor activity.
authors.affiliation: Fujian Academy of Integrative Medicine, Fuzhou, 350122, China.
authors.forename: Jin-yan
authors.initials: JY
authors.lastname: Zhao
authors.forename: Wei
authors.initials: W
authors.lastname: Lin
authors.forename: Qun-chuan
authors.initials: QC
authors.lastname: Zhuang
authors.forename: Xiao-yong
authors.initials: XY
authors.lastname: Zhong
authors.forename: Jun
authors.initials: J
authors.lastname: Peng
authors.forename: Zhen-feng
authors.initials: ZF
authors.lastname: Hong
mesh_terms: D000818:Animals; D001646:Bile / Q000737:chemistry*; D002453:Cell Cycle; D002465:Cell Movement; D049109:Cell Proliferation; D002642:Chick Embryo; D049033:Chorioallantoic Membrane / Q000098:blood supply; D005786:Gene Expression Regulation; D056945:Hep G2 Cells; D061307:Human Umbilical Vein Endothelial Cells / Q000166:cytology; D006801:Humans; D018919:Neovascularization, Physiologic; D011208:Powders; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D001503:Ursidae; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics / Q000378:metabolism
chemical_list: D011208:Powders; D012333:RNA, Messenger; D042461:Vascular Endothelial Growth Factor A