2-5-diaziridinyl-3-(hydroxymethyl)-6-methyl-1-4-benzoquinone and Colonic-Neoplasms

RH1 (2,5-diaziridinyl-3-(hydroxymethyl)-6-methyl-1,4-benzoquinone), which is currently in clinical trials, is a diaziridinyl benzoquinone bioreductive anticancer drug that was designed to be activated by the obligate two-electron reductive enzyme NAD(P)H quinone oxidoreductase 1 (NQO1). In this electron paramagnetic resonance (EPR) study we showed that RH1 was reductively activated by the one-electron reductive enzyme NADPH cytochrome P450 reductase and by a suspension of HCT116 human colon cancer cells to yield a semiquinone free radical. As shown by EPR spin trapping experiments RH1 was reductively activated by cytochrome P450 reductase and underwent redox cycling to produce damaging hydroxyl radicals in reactions that were both H2O2- and iron-dependent. Thus, reductive activation by cytochrome P450 reductase or other reductases to produce a semiquinone that can redox cycle to produce damaging hydroxyl radicals and/or DNA-reactive alkylating species may contribute to the potent cell growth inhibitory effects of RH1. These results also suggest that selection of patients for treatment with RH1 based on their expression levels of NQO1 may be problematic.

Topics: Antineoplastic Agents; Aziridines; Benzoquinones; Cell Line, Tumor; Cell Proliferation; Colonic Neoplasms; Electron Spin Resonance Spectroscopy; Humans; Hydrogen Peroxide; Hydroxyl Radical; Iron; Molecular Structure; NADPH-Ferrihemoprotein Reductase; Oxidation-Reduction; Quinones

NAD(P)H:quinone oxidoreductase (NQO1; DT-diaphorase) is elevated in certain tumors, such as non-small cell lung cancer (NSCLC). Compounds such as mitomycin C and streptonigrin are efficiently bioactivated by NQO1 and have been used in an enzyme-directed approach to chemotherapy. Previously, 2,5-diaziridinyl-3,6-dimethyl-1,4-benzoquinone (MeDZQ) was identified as a potential antitumor agent based on its high rate of bioactivation by human NQO1 and its selective cytotoxicity to cells containing elevated NQO1. RH1, a water-soluble analogue of MeDZQ synthesized in this work, was a better substrate for recombinant human NQO1 than the parent compound. RH1 was, correspondingly, more cytotoxic to human tumor cells expressing elevated NQO1 activity (H460 NSCLC and HT29 human colon carcinoma), as measured by 3-(4,5-dimethylthiazol-2,5-diphenyl)tetrazolium assay, than it was to cells deficient in NQO1 activity (H596 NSCLC and BE human colon carcinoma). RH1 exhibited a greater selective toxicity (ratio of IC50s in H596:H460 and BE:HT29) to cells with elevated NQO1 activity relative to MeDZQ. Additionally, we report the establishment of a stable line of BE human colon carcinoma cells transfected with wild-type human NQO1 (BE-NQ7). BE cells are devoid of NQO1 activity due to a homozygous point mutation in the NQO1 gene. In comparison to the parental cell line, RH1, MeDZQ, and mitomycin C were significantly more cytotoxic to BE-NQ7 cells (17-, 7-, and 3-fold, respectively), confirming that the presence of NQO1 is sufficient to increase cytotoxicity of these antitumor quinones. These data suggest that RH1 may be an effective NQO1-directed antitumor agent for the therapy of tumors with elevated NQO1 activity, such as NSCLC.

Topics: Antineoplastic Agents; Aziridines; Benzoquinones; Biotransformation; Cell Division; Colonic Neoplasms; Drug Screening Assays, Antitumor; Gene Targeting; Humans; Hydroquinones; NAD(P)H Dehydrogenase (Quinone); Transfection; Tumor Cells, Cultured