NS398 reduces hypoxia‐inducible factor (HIF)‐1α 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‐1α 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‐1α. 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‐1α 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‐1α mRNA and reduces HIF‐1α synthesis in a COX‐2/PGE₂ dependent way, which can be restored by addition of exogenous PGE₂ that activates the phosphatidylinositol 3‐kinase/AKT/p70^s6k signaling pathway. On the other hand, NS398 accelerates HIF‐1α 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‐1α 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‐1α and HIF‐1 transcriptional activity but also demonstrate that such an effect occurs at multiple levels involving both COX‐2 dependent and independent mechanisms.