Reduction of the indoloquinone anticancer drug EO9 by purified DT-diaphorase: a detailed kinetic study and analysis of metabolites.
DT-diaphorase has been implicated in the activation and mechanism of cytotoxicity of the investigational indoloquinone anticancer drug EO9. Here, we have used a highly purified DT-diaphorase isolated from rat Walker tumour cells to provide unambiguous evidence for the ability of this enzyme to catalyze reduction of EO9 and to provide a more detailed characterization of the reaction. Under the conditions used hypoxia had no effect on the initial rate of this reduction but did effect the nature and stability of metabolites formed. Electron spin resonance (ESR) spectrometry studies showed that DT-diaphorase reduced EO9 to a highly oxygen-sensitive metabolite that is probably the hydroquinone. In the presence of air, this metabolite is auto-oxidized to generate both drug- and oxygen-based radicals. Comproportionation:disproportionation reactions may also be involved in the generation of these radical species. The identification of these metabolites may contribute to the understanding of the molecular mechanism of DNA damage and cytotoxicity exerted by EO9.
CRC Department of Medical Oncology, CRC Beatson Laboratories, Bearsden, Glasgow, Scotland.
S M
SM
Bailey
A D
AD
Lewis
R J
RJ
Knox
L H
LH
Patterson
G R
GR
Fisher
P
P
Workman
D000818:Animals; D000970:Antineoplastic Agents / Q000378:metabolism*; D001388:Aziridines / Q000378:metabolism*; D002279:Carcinoma 256, Walker / Q000378:metabolism* / Q000473:pathology; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D003574:Cytochrome c Group / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D005609:Free Radicals; D019073:HT29 Cells; D006801:Humans; D045563:Indolequinones; D007211:Indoles / Q000378:metabolism*; D007700:Kinetics; D016660:NAD(P)H Dehydrogenase (Quinone) / Q000302:isolation & purification / Q000378:metabolism*; D010084:Oxidation-Reduction; D051381:Rats; D014407:Tumor Cells, Cultured
D000970:Antineoplastic Agents; D001388:Aziridines; D003574:Cytochrome c Group; D005609:Free Radicals; D045563:Indolequinones; D007211:Indoles; D016660:NAD(P)H Dehydrogenase (Quinone); C060817:apaziquone