Development of a New Isogenic Cell-Xenograft System for Evaluation of NAD(P)H:Quinone Oxidoreductase-Directed Antitumor Quinones

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Abstract
Purpose: The purpose of our study was to develop and validate an isogenic cell line pair that differs only in the expression of NAD(P)H:quinone oxidoreductase (NQO1) that can be used to examine the in vitro and in vivo role of NQO1 in the bioactivation of the antitumor quinone RH1 (2,5-diaziridinyl-3-(hydroxymethyl)-6-methyl-1,4-benzoquinone), a compound currently in Phase I clinical trials. Experimental Design: MDA-MB-468 (MDA468) human breast adenocarcinoma cells, homozygous for a polymorphism in NQO1 (NQO1*2/*2) and with low levels of NQO1 activity, were stably transfected with human NQO1 to generate a clone (NQ16) expressing very high NQO1 activity. We examined levels of other reductases and looked at biochemical systems that might influence response to antitumor quinones to validate that the isogenic cell line pair differed only in the expression of NQO1. The 3-(4,5-dimethylthiazol-2,5-diphenyl)tetrazolium (MTT) assay was used to determine the differential toxicity of various quinones, including the most recent NQO1-directed antitumor quinone, RH1, between the two cell lines. Human tumor xenografts were established from both MDA468 and NQ16 cells, and the antitumor activity of RH1 was evaluated. Results: Levels of cytochrome P450 reductase, cytochrome b5 reductase, soluble thiols, and superoxide dismutase in the NQ16 line were unchanged from the parental line. The functional significance of wild-type NQO1 expression was confirmed by measurement of the differential toxicity of compounds activated or deactivated by NQO1 in the two cell lines. The toxicity of the NQO1-directed antitumor quinones RH1 and streptonigrin were markedly greater and the toxicity of menadione, which is detoxified by NQO1, was ameliorated in the NQ16 line. High levels of NQO1 expression were observed throughout xenograft tumors established from the NQ16 cell line. RH1 treatment was effective at statistically reducing tumor volume in NQ16 xenografts at all of the doses tested [0.1, 0.2, 0.4 mg/kg every day for 5 days), whereas only the highest dose of RH1 resulted in a significant reduction in tumor volume in MDA468 xenografts. Conclusions: The MDA468/NQ16 isogenic cell line pair is a useful model system for evaluating the role of NQO1 in the bioactivation of antitumor quinones in both cell lines and xenografts. In addition, our data demonstrate that the novel antitumor quinone RH1, is effectively activated by NQO1 both in vitro and in vivo.