title: RH1 induces cellular damage in an NAD(P)H:quinone oxidoreductase 1-dependent manner: relationship between DNA cross-linking, cell cycle perturbations, and apoptosis.
abstract: Structure-based development of NAD(P)H:quinone oxidoreductase (NQO1)-directed antitumor quinones resulted in development of RH1 [2,5-diaziridinyl-3-(hydroxymethyl)-6-methyl-1,4-benzoquinone], a methyl-substituted diaziridinyl quinone. We conducted experiments to evaluate the mechanism of RH1-induced cytotoxicity and the inter-relationship between DNA cross-linking, cell cycle changes, and apoptosis using an isogenic cell line pair developed from the human breast cancer cell line MDA-MB-468 differing only in expression of wtNQO1 (NQ16 cells). Statistically significant DNA cross-linking was detected using a modified comet assay in cells with wtNQO1 within 1 h of dosing, whereas in parental cells, only marginal DNA cross-linking was observed and required a concentration up to 50 times higher. Cross-linking in NQ16 cells could be abrogated with 5-methoxy-1,2-dimethyl-3-[(4-nitrophenoxy)methyl]indole-4,7-dione, a mechanism-based inhibitor of NQO1. RH1 prolonged S phase and caused a G(2)/M block. Cell cycle changes were observed up to 10-fold lower in RH1 concentrations in NQ16 cells relative to parental cells. Apoptosis was similarly observed morphologically in both cell lines after RH1 treatment but was induced preferentially in NQ16 cells at lower concentrations and earlier time points. Marked cleavage of caspase-3 was observed in NQ16 cells relative to parental cells using lower concentrations of RH1. Temporally, low doses of RH1-induced rapid DNA cross-linking in NQ16 cells followed by induction of apoptosis at times when a G(2)/M block was not observed. This suggests that cell cycle arrest is not required for RH1-induced apoptosis and that DNA damage may directly initiate apoptotic events. In summary, RH1-induced preferential DNA cross-linking, cell cycle changes, and apoptosis in an NQO1-dependent manner.
authors.affiliation: Department of Pharmaceutical Sciences, University of Colorado Health Sciences Center, Denver, 80262, USA.
authors.forename: Donna L
authors.initials: DL
authors.lastname: Dehn
authors.forename: Salmaan H
authors.initials: SH
authors.lastname: Inayat-Hussain
authors.forename: David
authors.initials: D
authors.lastname: Ross
mesh_terms: D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D001388:Aziridines / Q000633:toxicity*; D016227:Benzoquinones / Q000633:toxicity*; D002453:Cell Cycle / Q000187:drug effects* / Q000502:physiology; D045744:Cell Line, Tumor; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D004247:DNA / Q000378:metabolism*; D006801:Humans; D016660:NAD(P)H Dehydrogenase (Quinone) / Q000502:physiology*
chemical_list: C117776:2,5-diaziridinyl-3-(hydroxymethyl)-6-methyl-1,4-benzoquinone; D001388:Aziridines; D016227:Benzoquinones; D003432:Cross-Linking Reagents; D004247:DNA; D016660:NAD(P)H Dehydrogenase (Quinone); C447479:NQO1 protein, human