A novel small-molecule HIF-1 inhibitor that increases HIF-1 alpha degradation Free

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The hypoxia-inducible factor (HIF) is a transcription factor that regulates a variety of genes enabling cells to adapt to a low-oxygen environment. In recent years HIFs have become a very attractive target for cancer therapy, since HIF-1 activates the transcription of genes that are involved in critical aspects of cancer biology, including but not limited to angiogenesis, cell survival, glucose metabolism, invasion and metastasis. A cell-based high-throughput screen of the National Cancer Institute library of synthetic compounds sought to uncover inhibitors of the aforementioned pathway by utilizing a genetically engineered U251 human glioma cell line to stably express a recombinant vector in which the luciferase reporter gene is under the control of three copies of a canonical hypoxia-responsive element (U251-HRE) (Rapisarda et al Cancer Res. 2002). This screen yielded many novel and distinct small-molecule inhibitors of HIF-1 transactivation, one such small molecule inhibitor was a tricyclic carboxamide: NSC 644221. In addition to inhibiting HRE-luciferase activity, a subsequent decrease in vascular endothelial growth factor (VEGF) and glucose transporter-3 (GLUT-3) mRNA expression was also observed in U251 cells. Further studies using an effective and non-cytotoxic dose of NSC 644221, between 1-5 microM, demonstrated that NSC 644221 had the capacity to accelerate the degradation of HIF-1 alpha when cells were exposed to hypoxia. Analysis of protein half-life, using cyclohexamide, demonstrated that the half-life of HIF-1 alpha was decreased in the presence of NSC 644221. However, this inhibition was not observed in the presence of the proteosome inhibitors MG132, ALLNL and PS-341. NSC 644221 appears to be a specific and potent inhibitor of HIF-1 alpha in several different cancer cell lines and has no effect on the expression of a variety of other short-lived proteins. Interestingly, the mode of action of NSC 644221 was found to be independent of the Von-Hippel Lindau (VHL) tumor suppressor protein, which is used by cells for the endogenous degradation of HIF-1 alpha in the presence of oxygen. The inhibition of HIF-1 alpha expression by NSC644221 was not mediated by heat-shock protein-90 (HSP90), AKT or histone deacetylation. Unlike other previously described non-selective HIF-1 inhibitors, NSC 644221 is a compound that targets the cellular degradative pathway of HIF-1 alpha in what appears to be a novel and selective manner. This compound has potential both as a useful analytical tool and as a potential anticancer therapy. Contract No. NO1-CO-12400