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Cell Type–Specific, Topoisomerase II–Dependent Inhibition of Hypoxia-Inducible Factor-1 Protein Accumulation by NSC 644221

Authors' Affiliations: ¹ Screening Technologies Branch, Developmental Therapeutics Program and ² Developmental Therapeutics Program, SAIC-Frederick, Inc., National Cancer Institute at Frederick, Frederick, Maryland and ³ Auckland Cancer Society Research Centre, Faculty of Medical and Health Sciences, The University of Auckland, Auckland, New Zealand

Requests for reprints: Giovanni Melillo, Developmental Therapeutics Program-Tumor Hypoxia Laboratory, National Cancer Institute at Frederick, Building 432, Room 218, Frederick, MD 21702. Phone: 301-846-5050; Fax: 301-846-6081; E-mail: melillog{at}ncifcrf.gov.

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, NSC 644221.

Experimental Design: We investigated the mechanism by which the novel compound NSC 644221 inhibited HIF-1.

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, but not HIF-1ß, protein expression was inhibited by NSC 644221 in a time- and dose-dependent fashion. Interestingly, NSC 644221 was unable to inhibit HIF-1 protein accumulation in the presence of the proteasome inhibitors MG132 or PS341, yet it did not directly affect the degradation of HIF-1 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-1 translation relative to untreated controls. Silencing of topoisomerase (topo) II, but not topo I, by specific small interfering RNA completely blocked the ability of NSC 644221 to inhibit HIF-1. The data presented show that topo II is required for the inhibition of HIF-1 by NSC 644221. Furthermore, although NSC 644221 induced p21 expression, H2A.X, and G₂-M arrest in the majority of cell lines tested, it only inhibited HIF-1 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.