title: NS398 reduces hypoxia-inducible factor (HIF)-1alpha and HIF-1 activity: multiple-level effects involving cyclooxygenase-2 dependent and independent mechanisms.
abstract: Tissue hypoxia is a common feature in solid tumors. Hypoxia-inducible factor 1 (HIF-1) is a critical transcription factor that regulates the expression of genes encoding factors that influence tumor growth including vascular endothelial growth factor. Previous studies have demonstrated that post-transcriptional modification events are important for regulation of HIF-1alpha protein expression and HIF-1 transcriptional activity. Prostaglandin E2 (PGE2), a major end product of the cyclooxygenase-2 (COX-2) enzyme, induces the basal and hypoxia-induced nuclear relocalization of HIF-1alpha. This is suppressed by NS398, a COX-2 selective inhibitor. NS398 also inhibits hypoxia-induced angiogenesis, which may be mediated by the inhibition of HIF-1 function in a COX-2-dependent manner. Here, we show that NS398 reduces HIF-1alpha and HIF-1 transcriptional function in both COX-2 positive PC-3 cells and COX-2 negative HCT116 cells under normoxic and hypoxic conditions. On the one hand, NS398 decreases the expression of HIF-1alpha mRNA and reduces HIF-1alpha synthesis in a COX-2/PGE2 dependent way, which can be restored by addition of exogenous PGE2 that activates the phosphatidylinositol 3-kinase/AKT/p70s6k signaling pathway. On the other hand, NS398 accelerates HIF-1alpha degradation by moderately increasing ubiquitination and remarkably promoting the clearance of ubiquitylated protein, an effect most likely independent of COX-2/PGE2 since exogenous PGE2 fails to reverse it. Finally, NS398 decreases hypoxia-induced shifted form of HIF-1alpha and attenuates HIF-1 activation in greater extent under hypoxic than normoxic conditions. These data not only confirm the inhibitory effect of NS398 on HIF-1alpha and HIF-1 transcriptional activity but also demonstrate that such an effect occurs at multiple levels involving both COX-2 dependent and independent mechanisms.
authors.affiliation: Winship Cancer Institute, Department of Hematology and Oncology, Emory University School of Medicine, Atlanta, GA 30322, USA. hua_zhong@emoryhealthcare.org
authors.forename: Hua
authors.initials: H
authors.lastname: Zhong
authors.forename: Margaret
authors.initials: M
authors.lastname: Willard
authors.forename: Jonathan
authors.initials: J
authors.lastname: Simons
mesh_terms: D015687:Cell Hypoxia; D051546:Cyclooxygenase 2; D052246:Cyclooxygenase 2 Inhibitors; D016861:Cyclooxygenase Inhibitors / Q000494:pharmacology*; D004268:DNA-Binding Proteins / Q000096:biosynthesis* / Q000494:pharmacology*; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D007527:Isoenzymes / Q000494:pharmacology*; D008565:Membrane Proteins; D009369:Neoplasms / Q000503:physiopathology; D009389:Neovascularization, Pathologic; D009578:Nitrobenzenes / Q000494:pharmacology*; D009687:Nuclear Proteins / Q000096:biosynthesis* / Q000494:pharmacology*; D011451:Prostaglandin-Endoperoxide Synthases / Q000494:pharmacology*; D013449:Sulfonamides / Q000494:pharmacology*; D014157:Transcription Factors / Q000096:biosynthesis* / Q000494:pharmacology*; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
chemical_list: D052246:Cyclooxygenase 2 Inhibitors; D016861:Cyclooxygenase Inhibitors; D004268:DNA-Binding Proteins; C497442:HIF1A protein, human; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D007527:Isoenzymes; D008565:Membrane Proteins; D009578:Nitrobenzenes; D009687:Nuclear Proteins; D013449:Sulfonamides; D014157:Transcription Factors; C080955:N-(2-cyclohexyloxy-4-nitrophenyl)methanesulfonamide; D051546:Cyclooxygenase 2; C496540:PTGS2 protein, human; D011451:Prostaglandin-Endoperoxide Synthases