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


METHODS
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.
Creighton-Gutteridge
Mark
M
Screening Technologies Branch, Developmental Therapeutics Program, SAIC-Frederick, Inc., National Cancer Institute at Frederick, Frederick, Maryland 21702, USA.
Cardellina
John H
JH
Stephen
Andrew G
AG
Rapisarda
Annamaria
A
Uranchimeg
Badarch
B
Hite
Karen
K
Denny
William A
WA
Shoemaker
Robert H
RH
Melillo
Giovanni
G
D000577:Amides / Q000494:pharmacology*; D000970:Antineoplastic Agents / Q000494:pharmacology*; D045744:Cell Line, Tumor; D004250:DNA Topoisomerases, Type II / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000037:antagonists & inhibitors*; D008156:Luciferases / Q000378:metabolism; D008956:Models, Chemical; D009369:Neoplasms / Q000188:drug therapy*; D011083:Polycyclic Compounds / Q000494:pharmacology*; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D013696:Temperature; D013997:Time Factors; D014162:Transfection
D000577:Amides; D000970:Antineoplastic Agents; D004791:Enzyme Inhibitors; C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; C519238:NSC 644221; D011083:Polycyclic Compounds; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D008156:Luciferases; D004250:DNA Topoisomerases, Type II