Hypoxia, a ubiquitous feature of
tumors, can be exploited by
hypoxia-activated
prodrugs (HAP) that are substrates for one-electron reduction in the absence of
oxygen.
NADPH:cytochrome P450 oxidoreductase (POR) is considered one of the major
enzymes responsible, based on studies using purified
enzyme or forced overexpression in cell lines. To examine the role of POR in HAP activation at endogenous levels of expression, POR knock-outs were generated in HCT116 and SiHa cells by targeted mutation of exon 8 using
zinc finger nucleases. Absolute quantitation by proteotypic
peptide mass spectrometry of DNA sequence-confirmed multiallelic mutants demonstrated expression of
proteins with residual one-electron
reductase activity in some clones and identified two (Hko2 from HCT116 and S2ko1 from SiHa) that were functionally null by multiple criteria. Sensitivities of the clones to 11 HAP (six nitroaromatics, three benzotriazine N-
oxides, and two
quinones) were compared with wild-type and POR-overexpressing cells. All except the
quinones were potentiated by POR overexpression. Knocking out POR had a marked effect on antiproliferative activity of the
5-nitroquinoline SN24349 in both genetic backgrounds after anoxic exposure but little or no effect on activity of most other HAP, including the clinical stage
2-nitroimidazole mustard
TH-302, dinitrobenzamide mustard
PR-104A, and benzotriazine N-
oxide SN30000. Clonogenic cell killing and reductive metabolism of
PR-104A and
SN30000 under
anoxia also showed little change in the POR knock-outs. Thus, although POR expression is a potential
biomarker of sensitivity to some HAP, identification of other one-electron
reductases responsible for HAP activation is needed for their rational clinical development.