EO9 [3-hydroxymethyl-5-aziridinyl-1-methyl-2-(H-
indole-4, 7-indione)-propenol] is a novel indoloquinone structurally related to
mitomycin C, a
quinone anticancer
drug that requires reductive bioactivation.
NAD(P)H: (quinone-acceptor) oxidoreductase (
quinone reductase,
DT-diaphorase, EC 1.6.99.2) is an obligate 2-electron donating
enzyme that can reduce a variety of
quinones resulting either in bioactivation or bioprotection. Using
quinone reductase (QR) preparations from rat Walker 256 mammary
tumor cells and human HT29 colon
carcinoma cells, we have characterized the role of this
enzyme in
EO9 reductive metabolism. QR activity was assayed under optimal conditions by following
cytochrome c reduction at 550 nm in the presence of
enzyme,
quinone substrate,
NADH, and
bovine albumin, and confirmed by loss of
EO9 absorbance at 550 nm. Both the rat and human
tumor cell
enzymes catalyzed reduction of the benchmark
quinone menadione with a similar Km of 1.4-3.1 microM, although the Vmax was 7 to 8-fold lower for the human preparation.
EO9 was readily reduced by the rat Walker QR. The mean Km was about 5-fold higher than for
menadione at around 15 microM and the Vmax was 6-fold lower at around 2.5 mumol of
cytochrome c reduced mg-1 of
protein.
EO9 was also metabolized by QR from HT29 human colon
carcinoma cells but rather less efficiently than by the rat
tumor enzyme. For example, the rate was 6-fold lower than that for the Walker
tumor enzyme at 100 microM substrate concentration after correcting for the 7- to 8-fold difference in specific activity for the two preparations.(ABSTRACT TRUNCATED AT 250 WORDS)