title: Chlorogenic acid inhibits hypoxia-induced angiogenesis via down-regulation of the HIF-1α/AKT pathway.
abstract: BACKGROUND
  The hypoxia-inducible factor-1 (HIF-1) is known to play an important role in cellular responses to hypoxia, including the transcriptional activation of a number of genes involved in tumor angiogenesis. Chlorogenic acid (CGA), one of the most abundant polyphenols in the human diet, has been reported to inhibit cancer cell growth. The effect of CGA on tumor angiogenesis and its underlying mechanisms are, as yet, unknown.
  
  
  METHODS
  The effect of CGA on HIF-1α expression was assessed by Western blot and reverse transcriptase-polymerase chain reaction (RT-PCR) assays in A549 lung cancer cells. The transcriptional activity of the HIF-1 complex was confirmed using a luciferase assay. To assess whether angiogenic factors are increased under hypoxic conditions in these cells, vascular endothelial growth factor (VEGF) expression levels were measured by RT-PCR and Western blotting. The direct effect of CGA on human vascular endothelial cells (HUVEC) under hypoxic conditions was analyzed using in vitro assays, including tube-formation, wound healing and Transwell invasion assays. To investigate the effect of CGA on angiogenesis in vivo, we performed a Matrigel plug assay in a mouse model. Finally, the effect of CGA on AKT and ERK activation (phosphorylation) as a putative mechanism underlying the effect of CGA on VEGF-mediated angiogenesis inhibition was assessed using Western blotting.
  
  
  RESULTS
  We found that CGA significantly decreases the hypoxia-induced HIF-1α protein level in A549 cells, without changing its mRNA level. CGA was, however, found to suppress the transcriptional activity of HIF-1α under hypoxic conditions, leading to a decrease in the expression of its downstream target VEGF. We also found that CGA can block hypoxia-stimulated angiogenesis in vitro and VEGF-stimulated angiogenesis in vivo using HUVEC cells. In addition, we found that CGA can inhibit the HIF-1α/AKT signaling pathway, which plays an important role in VEGF activation and angiogenesis.
  
  
  CONCLUSIONS
  Our data indicate that CGA plays a role in the suppression of angiogenesis via inhibition of the HIF-1α/AKT pathway. CGA may represent a novel therapeutic option for the treatment of (lung) cancer.
authors.affiliation: College of Pharmacy, Inje University, 197 Inje-ro, Gimhae, Gyeongnam, 621-749, Republic of Korea.
authors.forename: Jin Ju
authors.initials: JJ
authors.lastname: Park
authors.forename: Su Jung
authors.initials: SJ
authors.lastname: Hwang
authors.forename: Ji-Hyeon
authors.initials: JH
authors.lastname: Park
authors.forename: Hyo-Jong
authors.initials: HJ
authors.lastname: Lee
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D015153:Blotting, Western; D015687:Cell Hypoxia / Q000502:physiology; D045744:Cell Line, Tumor; D002726:Chlorogenic Acid / Q000494:pharmacology*; D015536:Down-Regulation; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000378:metabolism; D007150:Immunohistochemistry; D051379:Mice; D008810:Mice, Inbred C57BL; D009389:Neovascularization, Pathologic / Q000503:physiopathology*; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000187:drug effects*
chemical_list: D000970:Antineoplastic Agents; C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D002726:Chlorogenic Acid; D051057:Proto-Oncogene Proteins c-akt