Antiangiogenic activity of genistein in pancreatic carcinoma cells is mediated by the inhibition of hypoxia-inducible factor-1 and the down-regulation of VEGF gene expression.
BACKGROUND
Previous reports indicate that Genistein, a naturally occurring isoflavonoid, exhibits strong antiangiogenic activity. The underlying mechanism of inhibition, however, remains unclear. Among the biologic effects of Genistein are the inhibition of tyrosine kinases and the inhibition of hypoxic activation of hypoxia-inducible factor-1 (HIF-1), one of the main regulators of VEGF gene expression.


METHODS
Hypoxic cell culture was performed in a modular incubator chamber. Vascular endothelial growth factor (VEGF) protein secretion was measured using the enzyme-linked immunosorbent assay, binding of DNA by HIF-1 was measured using the electrophoretic mobility shift assay, and mRNA quantification was performed using Northern blot analysis. Pancreatic carcinoma was studied in an orthotopic murine model. Angiogenesis in vivo was quantified by staining xenograft tumors for endothelial cell markers.


RESULTS
VEGF protein secretion was dose-dependently suppressed with increasing doses of Genistein. Furthermore, treatment of pancreatic carcinoma cells with Genistein led to impaired activation of HIF-1 under hypoxic culture conditions. Northern blot analysis indicated that VEGF mRNA expression decreased upon treatment with Genistein, both under normoxic and hypoxic culture conditions. In vivo, Genistein inhibited tumor growth for xenograft pancreatic carcinoma cells, whereas extensive hypoxia was observed in xenograft tumors and was not influenced by Genistein therapy. Similarly, decreased VEGF mRNA levels were observed in Genistein-treated Capan-1 xenograft tumors.


CONCLUSIONS
The current study indicates that the previously reported antiangiogenic activity of Genistein probably is mediated by the inhibition of HIF-1, an important regulator of VEGF gene homeostasis, particularly under low-oxygen conditions. Therefore, this bioactive compound may well be beneficial to patients with pancreatic carcinoma.
Büchler
Peter
P
Department of Surgery, School of Medicine, University of California at Los Angeles, Los Angeles, California, USA. peter.buechler@med.uni-heidelberg.de
Reber
Howard A
HA
Büchler
Markus W
MW
Friess
Helmut
H
Lavey
Robert S
RS
Hines
Oscar J
OJ
D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D015152:Blotting, Northern; D002277:Carcinoma / Q000473:pathology*; D018076:DNA, Complementary; D004268:DNA-Binding Proteins / Q000096:biosynthesis* / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation; D024202:Electrophoretic Mobility Shift Assay; D015972:Gene Expression Regulation, Neoplastic; D019833:Genistein / Q000494:pharmacology*; D018257:Helix-Loop-Helix Motifs; D006706:Homeostasis; D006801:Humans; D000860:Hypoxia; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D007150:Immunohistochemistry; D051379:Mice; D008819:Mice, Nude; D009389:Neovascularization, Pathologic; D009687:Nuclear Proteins / Q000096:biosynthesis* / Q000494:pharmacology; D010190:Pancreatic Neoplasms / Q000473:pathology*; D012333:RNA, Messenger / Q000032:analysis; D014157:Transcription Factors; D014183:Transplantation, Heterologous; D014407:Tumor Cells, Cultured; D042461:Vascular Endothelial Growth Factor A / Q000096:biosynthesis* / Q000494:pharmacology
D000970:Antineoplastic Agents; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; C497442:HIF1A protein, human; C497443:Hif1a protein, mouse; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D009687:Nuclear Proteins; D012333:RNA, Messenger; D014157:Transcription Factors; D042461:Vascular Endothelial Growth Factor A; D019833:Genistein