Cell type-specific, topoisomerase II-dependent inhibition of hypoxia-inducible factor-1α protein accumulation by NSC 644221

Purpose: The discovery and development of small-molecule inhibitors of hypoxia-inducible factor-1 (HIF-1) is an attractive, yet challenging, strategy for the development of new cancer therapeutic agents. Here, we report on a novel tricyclic carboxamide inhibitor of HIF-1 alpha, NSC 644221. Experimental Design: We investigated the mechanism by which the novel compound NSC 644221 inhibited HIF-1 alpha. Results: NSC 644221 inhibited HIF-1 -dependent, but not constitutive, luciferase expression in U251-HRE and U251-pGL3 cells, respectively, as well as hypoxic induction of vascular endothelial growth factor mRNA expression in U251 cells. HIF-1 alpha, but not HIF-1 beta, protein expression was inhibited by NSC 644221 in a time- and dose-dependent fashion. Interestingly, NSC 644221 was unable to inhibit HIF-1 alpha protein accumulation in the presence of the proteasome inhibitors MG132 or PS341, yet it did not directly affect the degradation of HIF-1 alpha as shown by experiments done in the presence of cyclohexamide or pulse-chase labeling using [S-35] methionine. In contrast, NSC 644221 decreased the rate of HIF-1 alpha translation relative to untreated controls. Silencing of topoisomerase (topo) II alpha, but not topo I, by specific small interfering RNA completely blocked the ability of NSC 644221 to inhibit HIF-1 alpha. The data presented show that topo II is required for the inhibition of HIF-1 alpha by NSC 644221. Furthermore, although NSC 644221 induced p21 expression, gamma H2A.X, and G(2)-M arrest in the majority of cell lines tested, it only inhibited HIF-1 alpha in a distinct subset of cells, raising the possibility of pathway-specific "resistance" to HIF-1 inhibition in cancer cells. Conclusions: NSC 644221 is a novel HIF-1 inhibitor with potential for use as both an analytic tool and a therapeutic agent. Our data provide a strong rationale for pursuing the preclinical development of NSC 644221 as a HIF-1 inhibitor.