Relationship between NAD(P)H:quinone oxidoreductase 1 (NQO1) levels in a series of stably transfected cell lines and susceptibility to antitumor quinones.
To investigate the importance of NAD(P)H:quinone oxidoreductase 1 (or DT-diaphorase; NQO1) in the bioactivation of antitumor quinones, we established a series of stably transfected cell lines derived from BE human colon adenocarcinoma cells. BE cells have no NQO1 activity due to a genetic polymorphism. The new cell lines, BE-NQ, stably express wild-type NQO1. BE-NQ7 cells expressed the highest level of NQO1 and were more susceptible [determined by the thiazolyl blue (MTT) assay] to known antitumor quinones and newer clinical candidates. Inhibition of NQO1 by pretreatment with an irreversible inhibitor, ES936 [5-methoxy-1,2-dimethyl-3-[(4-nitrophenoxy)methyl]indole-4,7-dione], protected BE-NQ7 cells from toxicity induced by streptonigrin, ES921 [5-(aziridin-1-yl)-3-(hydroxymethyl)-1,2-dimethylindole-4,7-dione], and RH1 [2,5-diaziridinyl-3-(hydroxymethyl)-6-methyl-1,4-benzoquinone]. RH1 was evaluated further by clonogenic assay for cytotoxic response and was more cytotoxic to BE-NQ7 cells than to BE cells. Cytotoxicity was abrogated by inhibition of NQO1 with ES936 pretreatment. Using a comet assay to evaluate DNA cross-linking, BE-NQ7 cells demonstrated significantly higher DNA cross-links than did BE cells in response to RH1 treatment. DNA cross-linking in BE-NQ7 cells was observed at very low concentrations of RH1 (5 nM), confirming that NQO1 activates RH1 to a potent cross-linking species. Further studies using streptonigrin, ES921, and RH1 were undertaken to analyze the relationship between NQO1 activity and quinone toxicity. Toxicity of these compounds was measured in a panel of BE-NQ cells expressing a range of NQO1 activity (23-433 nmol/min/mg). Data obtained suggest a threshold for NQO1-induced toxicity above 23 nmol/min/mg and a sharp dose-response curve between the no effect level of NQO1 (23 nmol/min/mg) and the maximal effect level (>77 nmol/min/mg). These data provide evidence that NQO1 can bioactivate antitumor quinones in this system and suggest that a threshold level of NQO1 activity is required to initiate toxic events.
Winski
S L
SL
Department of Pharmaceutical Sciences, School of Pharmacy and Cancer Center, Campus Box C238, University of Colorado Health Sciences Center, Denver, CO 80262, USA.
Swann
E
E
Hargreaves
R H
RH
Dehn
D L
DL
Butler
J
J
Moody
C J
CJ
Ross
D
D
D000903:Antibiotics, Antineoplastic / Q000494:pharmacology; D000970:Antineoplastic Agents / Q000494:pharmacology*; D001388:Aziridines / Q000494:pharmacology; D016227:Benzoquinones / Q000494:pharmacology; D001711:Biotransformation; D002455:Cell Division / Q000187:drug effects; D004347:Drug Interactions; D006801:Humans; D020128:Inhibitory Concentration 50; D016660:NAD(P)H Dehydrogenase (Quinone) / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011809:Quinones / Q000378:metabolism / Q000494:pharmacology*; D013308:Streptonigrin / Q000494:pharmacology; D014162:Transfection; D014407:Tumor Cells, Cultured
C117776:2,5-diaziridinyl-3-(hydroxymethyl)-6-methyl-1,4-benzoquinone; D000903:Antibiotics, Antineoplastic; D000970:Antineoplastic Agents; D001388:Aziridines; D016227:Benzoquinones; D011809:Quinones; D013308:Streptonigrin; D016660:NAD(P)H Dehydrogenase (Quinone)