title: Diflavin oxidoreductases activate the bioreductive prodrug PR-104A under hypoxia.
abstract: The clinical agent PR-104 is converted systemically to PR-104A, a nitrogen mustard prodrug designed to target tumor hypoxia. Reductive activation of PR-104A is initiated by one-electron oxidoreductases in a process reversed by oxygen. The identity of these oxidoreductases is unknown, with the exception of cytochrome P450 reductase (POR). To identify other hypoxia-selective PR-104A reductases, nine candidate oxidoreductases were expressed in HCT116 cells. Increased PR-104A-cytotoxicity was observed in cells expressing methionine synthase reductase (MTRR), novel diflavin oxidoreductase 1 (NDOR1), and inducible nitric-oxide synthase (NOS2A), in addition to POR. Plasmid-based expression of these diflavin oxidoreductases also enhanced bioreductive metabolism of PR-104A in an anoxia-specific manner. Diflavin oxidoreductase-dependent PR-104A metabolism was suppressed >90% by pan-flavoenzyme inhibition with diphenyliodonium chloride. Antibodies were used to quantify endogenous POR, MTRR, NDOR1, and NOS2A expression in 23 human tumor cell lines; however, only POR protein was detectable and its expression correlated with anoxic PR-104A reduction (r(2) = 0.712). An anti-POR monoclonal antibody was used to probe expression using human tissue microarrays; 13 of 19 cancer types expressed detectable POR with 21% of cores (185 of 874) staining positive; this heterogeneity suggests that POR is a useful biomarker for PR-104A activation. Immunostaining for carbonic anhydrase 9 (CAIX), reportedly an endogenous marker of hypoxia, revealed only moderate coexpression (9.6%) of both CAIX and POR across a subset of five cancer types.
authors: Auckland Cancer Society Research Centre, School of Medical Sciences, the University of Auckland, Auckland, New Zealand.
authors: Christopher P
authors: CP
authors: Guise
authors: Maria R
authors: MR
authors: Abbattista
authors: Smitha R
authors: SR
authors: Tipparaju
authors: Neil K
authors: NK
authors: Lambie
authors: Jiechuang
authors: J
authors: Su
authors: Dan
authors: D
authors: Li
authors: William R
authors: WR
authors: Wilson
authors: Gabi U
authors: GU
authors: Dachs
authors: Adam V
authors: AV
authors: Patterson
mesh_terms: D000818:Animals; D015687:Cell Hypoxia / Q000187:drug effects / Q000502:physiology; D005415:Flavins / Q000502:physiology*; D005420:Flavoproteins / Q000378:metabolism / Q000502:physiology*; D045325:HCT116 Cells; D006801:Humans; D051379:Mice; D008819:Mice, Nude; D009251:NADPH-Ferrihemoprotein Reductase / Q000502:physiology; D009588:Nitrogen Mustard Compounds / Q000378:metabolism* / Q000494:pharmacology; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000378:metabolism / Q000502:physiology*; D011355:Prodrugs / Q000494:pharmacology*
chemical_list: D005415:Flavins; D005420:Flavoproteins; D009588:Nitrogen Mustard Compounds; C524170:PR-104A; D011355:Prodrugs; C478386:NDOR1 protein, human; D010088:Oxidoreductases; D009251:NADPH-Ferrihemoprotein Reductase