Cell type-specific, topoisomerase II-dependent inhibition of hypoxia-inducible factor-1alpha 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-1alpha, NSC 644221.

EXPERIMENTAL DESIGN

We investigated the mechanism by which the novel compound NSC 644221 inhibited HIF-1alpha.

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-1alpha, but not HIF-1beta, protein expression was inhibited by NSC 644221 in a time- and dose-dependent fashion. Interestingly, NSC 644221 was unable to inhibit HIF-1alpha protein accumulation in the presence of the proteasome inhibitors MG132 or PS341, yet it did not directly affect the degradation of HIF-1alpha as shown by experiments done in the presence of cyclohexamide or pulse-chase labeling using [35S]methionine. In contrast, NSC 644221 decreased the rate of HIF-1alpha translation relative to untreated controls. Silencing of topoisomerase (topo) IIalpha, but not topo I, by specific small interfering RNA completely blocked the ability of NSC 644221 to inhibit HIF-1alpha. The data presented show that topo II is required for the inhibition of HIF-1alpha by NSC 644221. Furthermore, although NSC 644221 induced p21 expression, gammaH2A.X, and G2-M arrest in the majority of cell lines tested, it only inhibited HIF-1alpha 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.