Journal of Pharmacology And Experimental Therapeutics Fast Forward
First published on March 20, 2003; DOI: 10.1124/jpet.103.050070
0022-3565/03/3053-1079-1086$20.00
JPET 305:1079-1086, 2003
CHEMOTHERAPY, ANTIBIOTICS, AND GENE THERAPY
Kinetics of NAD(P)H:Quinone Oxidoreductase I (NQO1) Inhibition by Mitomycin C in Vitro and in Vivo
Daniel L. Gustafson,
David Siegel,
Jeffrey C. Rastatter,
Andrea L. Merz,
Jacqueline C. Parpal,
Jadwiga K. Kepa,
David Ross, and
Michael E. Long
Department of Pharmaceutical Sciences, School of Pharmacy and the Cancer
Center, University of Colorado Health Sciences Center, Denver, Colorado
Received for publication
February 6, 2003
Accepted
March 11, 2003.
 |
Abstract
|
|---|
The bioreductive activation of the antitumor quinone mitomycin C (MMC) by
NAD(P)H: quinone oxidoreductase 1 (NQO1) is complicated by the ability of MMC
to also act as a mechanism-based inhibitor of NQO1 in a pH dependent manner.
Inhibition of NQO1 by MMC has been studied in purified enzyme preparations and
in cultured cells but has not determined in vivo. In the studies presented
here, NQO1 activity was measured in mouse tissues following treatment with MMC
or the potent mechanism-based human NQO1 inhibitor
5-methoxy-1,2-dimethyl-[(4-nitrophenoxy)methyl]indole-4,7-dione (ES936). NQO1
activity was significantly decreased at 1, 2, and 4 h following MMC (10 or 20
mg/kg) treatment in kidney and lung but was unchanged in brain, heart, liver,
and bladder. ES936 (1 mg/kg) treatment led to a significant and much more
potent inhibition of NQO1 in all murine tissues analyzed except for bladder.
To extrapolate these in vivo results from mice to humans, the species-specific
kinetics of NQO1 inactivation by MMC was determined in vitro using mouse, rat,
and human recombinant NQO1 proteins. Results showed the inactivation kinetics
of mouse and human proteins by MMC were similar. Treatment of human and murine
endothelial cells with MMC or ES936 showed similar inhibition of NQO1
activity. The aforementioned results clearly demonstrate that MMC can serve as
a substrate for NQO1 in vivo; however, the metabolism resulting in enzyme
inactivation is possibly tissue-specific. Furthermore, the kinetic
similarities for inactivation between murine and human forms of NQO1 show
these results are apropos to clinical use of MMC.
Although it is clear that NAD(P)H:quinone oxidoreductase 1 (NQO1; EC
1.6.99.2
[EC]
) is capable of reducing the antineoplastic antibiotic mitomycin C
(MMC) in vitro (Siegel et al.,
1990b
,
1992
), the role of this enzyme
in determining the toxicity of MMC remains controversial. A correlative
relationship has been shown between NQO1 activity levels and sensitivity to
MMC in the NCI tumor cell line panel
(Fitzsimmons et al., 1996
).
Nevertheless, transfection and expression of NQO1 in cell lines has produced
varied results (Powis et al.,
1995
; Belcourt et al.,
1996
; Gustafson et al.,
1996
; Winski et al.,
1998
), as have xenograft studies
(Malkinson et al., 1992
;
Nishiyama et al., 1995
;
Phillips et al., 2001
). The
lack of an apparent relationship between NQO1 levels and MMC toxicity is not
surprising for a number of reasons. MMC can be metabolically activated by
multiple enzyme systems including NADPH:cytochrome P450 reductase
(Keyes et al., 1984
;
Pan et al., 1984
), cytochrome
b5 reductase (Hodnick
and Sartorelli, 1993
), xanthine oxidase/dehydrogenase
(Pan et al., 1984
;
Gustafson and Pritsos, 1992
),
and probably others not yet identified. MMC bioactivation by NQO1 is pH
dependent in vitro (Siegel et al.,
1990b
), and this pH dependence seems to be due to inactivation of
NQO1 by MMC at more neutral pH. Furthermore, evidence suggests that the
relationship between NQO1 levels and the toxicity of bioreductive substrates
is best described by a threshold relationship, and thus, a simple correlation
between activity and toxicity may not exist
(Gustafson et al., 1996
;
Winski et al., 1998
).
The role of NQO1 in the selective toxicity of antitumor quinones is an
important unknown since this enzyme is a target for the recent development of
bioreductive antitumor agents (Riley and
Workman, 1992
; Winski et al.,
1998
), and MMC is the prototype for this class of agents. The use
of NQO1 as a target for drug activation is precipitated by the elevation of
NQO1 enzyme activity in many human tumors
(Schlager and Powis, 1990
)
including breast, colon, and lung and that this increase in activity is
associated with enhanced expression (Marin
et al., 1997
; Siegel et al.,
1998
). Furthermore, the presence of a polymorphism in the human
NQO1 gene rendering a fraction of the population null for enzyme activity
(Traver et al., 1997
;
Siegel et al., 1999
) makes the
role of this enzyme in drug action an important question for efficacy in this
subpopulation (Kelsey et al.,
1997
).
Previous studies in enzyme preparations have determined MMC to be an
inhibitor of NQO1 (Schlager and Powis,
1988
) and that the characteristics of this inhibition are
consistent with mechanism-based inactivation of the enzyme
(Siegel et al., 1993
).
Inhibition of NQO1 by MMC is pH dependent, with enzyme inactivation occurring
more readily at neutral pH (6.57.4) compared with a more acidic pH
(5.8) (Siegel et al., 1993
).
Consistent with this data, other metabolic studies of MMC with NQO1 have shown
MMC reduction was much more pronounced at an acidic pH (5.8) as opposed to
more neutral or basic pH values (7.07.8)
(Siegel et al., 1990b
). Taken
together, such data are consistent with the idea that the release of reduced
MMC is more favored at an acidic pH as opposed to more neutral or basic pH
values and that if NQO1 does not release the reduced form of MMC, the result
is alkylation and inactivation of the enzyme
(Siegel et al., 1993
). The
interplay between drug activation and enzyme inactivation mechanisms involving
MMC and NQO1 and the potential added effect of the cellular pool of other
metabolizing enzymes suggests there will be wide variability in the
contribution of each of these processes in various tissues.
The previous studies that determined the kinetics and mechanism of MMC
inactivation of NQO1 were carried out using rat liver enzyme
(Siegel et al., 1993
). Studies
have shown MMC acts as an inhibitor of the human form of the enzyme in both
cells and purified preparations (Schlager
and Powis, 1988
; Siegel et
al., 1993
), although the inactivation kinetics of the human form
have not been determined. Comparative studies have determined the kinetics of
antitumor substrates using both rat and human recombinant NQO1, and the
results from these studies showed rat NQO1, in general, carried out substrate
reduction more rapidly than human NQO1
(Beall et al., 1994
) and that
for MMC the rate is 5-times higher for rat than human. The kinetic differences
between rat and human NQO1 reveal the importance of carrying out
species-specific studies. In the studies presented here, we have determined
the kinetics of NQO1 inactivation by MMC in vitro using mouse, rat, and human
recombinant NQO1. We have also measured the dose dependence of NQO1 inhibition
by MMC and another mechanism-based NQO1 inhibitor ES936
(Winski et al., 2001
) in mouse
and human endothelial cells and determined the kinetics and tissue specificity
of NQO1 inhibition by MMC and ES936 in mice
(Fig. 1). The results from
these in vitro and in vivo studies will allow for the extrapolation of in vivo
results in mice to humans using species-specific kinetic parameters.
 |
Materials and Methods
|
|---|
Materials. Bovine serum albumin (fraction V),
2,6-dichlorophenolindophenol, dicumarol, FAD, mitomycin C, NADH, and Tween 20
were purchased from Sigma-Aldrich (St. Louis, MO). Dulbecco's modified Eagle's
medium, Hank's balanced salt solution, and trypsin were purchased from
Mediatech, Inc. (Herndon, VA). The murine endothelial cell line bEnd.3 was
purchased from the American Type Culture Collection (Manassas, VA). Human
umbilical vein endothelial cells (HUVEC), endothelial cell growth medium, and
reagent kits were purchased from Cell Applications, Inc. (San Diego, CA).
Penicillin-streptomycin and L-glutamine were purchased from
Invitrogen Corp. (Carlsbad, CA). Fetal bovine serum was purchased from Summit
Biotechnology (Fort Collins, CO). SDS-polyacrylamide gel electrophoresis
reagents were purchased from Bio-Rad Laboratories (Hercules, CA).
Bicinchoninic acid protein assay reagents and SuperSignal chemiluminescent
substrate were purchased from Pierce Chemical Co. (Rockford, IL). All other
reagents were of analytical grade.
Mouse NQO1 Escherichia coli Expression. The coding region
for mouse NQO1 was kindly provided by Dr. Vasilis Vasiliou (School of
Pharmacy, University of Colorado Health Sciences Center, Denver, CO)
(Vasiliou et al., 1994
) and
was amplified using primers that spanned the entire coding region with linkers
attached to allow for cloning into the NcoI/HindIII site of
the pKK2332 vector. The resulting plasmid was used to express murine
NQO1 in transformed E. coli when induced with 1 mM isopropyl
-D-thiogalactopyranoside.
Human, Rat, and Mouse NQO1 E. coli Expression and
Preparation. The E. coli containing expression plasmids for rat
and human NQO1 have been previously described
(Beall et al., 1994
). Cultures
of E. coli (50 ml) containing the human, rat, or mouse NQO1
expression plasmids were grown in Luria-Bertani (LB) broth to an optical
density at 660 nm of 0.44, and
-D-thiogalactopyranoside was
added to a final concentration of 1 mM. The cultures were then allowed to grow
for another 2 h before harvesting by centrifugation at 2000g for 1 h.
The resulting pellets were resuspended in 500 µl of 25 mM Tris (pH 7.4)
containing 250 mM sucrose and 5 µM FAD+. The suspensions were
sonicated with three 5-s bursts at 30% power, and the resulting sonicate was
centrifuged at 15,000g for 15 min. The resulting supernatants were
diluted to an appropriate enzyme activity concentration in the resuspension
buffer, aliquoted, and stored at 80°C.
NQO1 Activity Measurements. For studies with recombinant enzyme
preparations, NQO1 activity was determined essentially as previously described
(Hollander and Ernster, 1975
).
The reaction mix contained 25 mM Tris (pH 7.4), 200 µM NADH, 1% Tween 20
(w/v), and varying concentrations of DCPIP (10120 µM). The presence
of 20 µM dicumarol completely inhibited enzyme-dependent DCPIP reduction in
the enzyme preparations and was therefore not included in all activity
determinations. Nevertheless, complete dicumarol inhibition was verified in
each enzyme preparation before assays being carried out. Enzyme activity was
determined by monitoring the enzyme-dependent decrease in absorbance at 600
nm, and activity is expressed as nanomoles of DCPIP reduced per minute using
an extinction coefficient of 21 mM1
cm1.
For NQO1 measurements in tissues and cells, the assay was performed as
described by Ernster (1967
) and
modified by Benson et al.
(1980
) using DCPIP as a
substrate. The reaction mix contained 25 mM Tris (pH 7.4), 0.07% bovine serum
albumin (w/v), 200 µM NADH, and 40 µM DCPIP, and assays were carried out
in the presence and absence of 20 µM dicumarol. NQO1 activity is described
as the dicumarol inhibitable decrease in absorbance at 600 nm with DCPIP as a
substrate and is expressed in nanomoles of DCPIP reduced per minute per
milligram of protein. Total protein in tissue and cell culture preparations
was determined by the bicinchoninic acid assay using bovine serum albumin as a
standard.
Tissue Culture. The murine endothelial cell line bEnd.3 was grown in
Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum
and penicillin-streptomycin. HUVECs were grown in complete endothelial cell
growth medium as supplied by the manufacturer. Both cell lines were grown in a
humidified atmosphere of 95% air and 5% CO2 at 37°C. HUVEC and
bEnd.3 cells were plated out in 60-mm tissue culture dishes and were grown to
approximately 80% confluency before being treated with graded concentrations
of MMC or ES936 for 1 h. Following treatment, cells were washed twice with
Hank's balanced salt solution and scraped into 100 µl of 25 mM Tris (pH
7.4) containing 250 mM sucrose and 5 µM FAD+. The cells were
then disrupted by sonication using three 2-second bursts at 30% power; the
resulting sonicates were centrifuged at 15,000g, and the supernatant
was collected and stored at 80°C until assayed for NQO1 activity
and total protein.
Animals and Animal Treatments. Female Balb/C mice 6 to 8 weeks of
age were purchased from Charles River Laboratories, Inc. (Wilmington, MA) and
were acclimated for at least 1 week before being used for experiments. All
studies were conducted in accordance with NIH guidelines for the care and use
of laboratory animals, and animals were housed in a facility accredited by the
American Association of Laboratory Animal Care. Following acclimation, animals
were randomly assigned to one of four treatment groups. These included a
control group (saline-treated), an ES936 group (1 mg/kg i.p. ES936), and two
MMC-treated groups, MMC 10 and MMC 20 (10 and 20 mg/kg mitomycin C i.v.).
Following drug administration by intravenous tail vein injection of MMC
dissolved in 0.9% NaCl or intraperitoneal injection of ES936 dissolved in
DMSO, animals were killed by cardiac stick exsanguinations under isoflurane
anesthesia at 1, 2, 4, and 24 h post-treatment, and tissues were removed.
Tissues were immediately rinsed in ice-cold 25 mM Tris (pH 7.4) containing 250
mM sucrose and 5 µM FAD+. Tissues were then frozen in liquid
nitrogen and stored until needed. Tissue homogenates were prepared by
homogenization of frozen tissue in 25 mM Tris (pH 7.4) containing 250 mM
sucrose and 5 µM FAD+. The resulting homogenates were
centrifuged at 15,000g, and the supernatant collected and stored at
80°C until assayed for NQO1 activity and total protein.
Western Blotting. Lung tissue was homogenized in 50 mM Tris-HCl (pH
8) buffer containing 150 mM NaCl, 1 mM EDTA, 50 µg/ml phenylmethylsulfonyl
fluoride, 1 µg/ml aprotinin, and 1% Triton X-100. The resulting homogenate
was then centrifuged for 10 min at 15,000g and the supernatant
collected. Total protein was determined in the samples, and 2.5 µg of total
protein was then separated on a 15% SDS-polyacrylamide gel electrophoresis gel
and transferred to nitrocellulose membranes by electrophoretic transfer.
Filters were then blocked overnight at 4°C with TBST (10 mM Tris-HCL, pH
7.5, 100 mM NaCl, 0.1% Tween 20) containing 5% nonfat dried milk (NFDM). The
membrane was then incubated with a 1:250 dilution in TBST with 5% NFDM of a
rabbit polyclonal antibody to human NQO1 that was kindly provided by Dr.
Su-Shu Pan (University of Pittsburgh Cancer Center, Pittsburgh, PA). After the
addition of primary antibody, the filter was washed three times with TBST. The
secondary antibody (anti-rabbit HRP conjugate; Amersham Biosciences, Inc.,
Piscataway, NJ) was then added at a 1:2,500 dilution in TBST with 5% NFDM,
followed by three washes in TBST. The blot was developed using
chemiluminescent detection.
Data Analysis. Kinetic data were analyzed using SigmaPlot 2001 and
its' associated Enzyme Kinetics Module software (SPSS, Inc., Chicago, IL) and
a PC-based computer. Determination of significant differences between groups
was done by one-way ANOVA analysis and Tukey post-tests using SigmaStat
software (SPSS, Inc.).
 |
Results
|
|---|
Species-Specific Kinetics of NQO1 with DCPIP as a Substrate. Kinetic
constants for mouse, rat, and human recombinant NQO1-mediated reduction of
DCPIP were measured, and the results are shown in
Fig. 2 for the mouse enzyme and
species-specific kinetic constants summarized in
Table 1.
Figure 2 also shows that NQO1
kinetics are best fit using an uncompetitive substrate inhibition model as
opposed to the classical Michaelis-Menten equation. The ability of MMC to
serve as a competitive inhibitor of mouse, rat, and human recombinant NQO1
with DCPIP as a substrate was also determined using MMC at concentrations of
25, 50, and 100 µM. MMC behaved as a competitive inhibitor of DCPIP
reduction by mouse NQO1 only and did not show competitive inhibition of rat or
human NQO1, as determined by kinetic analysis. The Ki
calculated for MMC as a competitive inhibitor of mouse NQO1 was 1140
µM.
View this table:
[in this window]
[in a new window]
|
TABLE 1 Species specific kinetic parameters for NQO1 with DCPIP as a substrate
using Michaelis-Menten models with no inhibition and uncompetitive substrate
inhibition
|
|
Species-Specific Inactivation Kinetics of NQO1 by MMC. The time
course of NQO1 inactivation by MMC was determined using mouse, rat, and human
recombinant NQO1, and the resulting curves at MMC concentrations ranging from
2.5 to 20 µM are shown in Fig.
3. The slopes of the linear regression lines were used to
calculate half-lives of inactivation and are plotted versus the inverse of
inhibitor concentration in a Kitz and Wilson plot shown in
Fig. 4.
Table 2 summarizes the kinetic
constants for inactivation of mouse, rat, and human NQO1, as calculated from
the Kitz and Wilson plots. The half-lives of inactivation for mouse, rat, and
human NQO1 by MMC are calculated to be 4.9, 22.9, and 4.1 min,
respectively.

View larger version (12K):
[in this window]
[in a new window]
|
Fig. 3. Time- and concentration-dependent inhibition of recombinant mouse, rat, and
human NQO1 by MMC. Values represent the mean ± S.D. of three
independent determinations. Lines represent the linear regression line of log
percent remaining activity versus time. Incubations were performed at 37°C
in 25 mM Tris (pH 7.4) with 500 µM NADH, 1% (w/v) Tween 20 and 0 µM MMC
(circles), 2.5 µM MMC (squares), 5 µM MMC (triangles), 10 µM MMC
(inverted triangles), or 20 µM MMC (diamonds) µM MMC. NQO1 activity
measurements were done using a DCPIP concentration of 40 µM.
|
|

View larger version (15K):
[in this window]
[in a new window]
|
Fig. 4. Kitz and Wilson plots for MMC-dependent inactivation of mouse, rat, and
human recombinant NQO1 proteins. Concentration-dependent inactivation
half-lives were calculated from the slope of the regression line of
time-dependent inactivation (Fig.
3).
|
|
Inhibition of NQO1 by MMC and ES936 in Mouse and Human Endothelial
Cells. Inhibition of NQO1 in mouse bEnd.3 endothelial cells and HUVECs
following a 1-h incubation with MMC at concentrations ranging from 1 to 100
µM are shown in Fig. 5. MMC
treatment led to a significant (P < 0.05) decrease in NQO1
activity in bEnd.3 cells at MMC concentrations above 5 µM and in HUVECs
above 10 µM. NQO1 activity in murine cells was inhibited by 61 ± 8%
at 100 µM MMC, and HUVEC NQO1 activity was inhibited by 94 ± 42% at
this same dose. ES936, a novel mechanism-based inhibitor of NQO1, also
inhibits NQO1 in endothelial cells but is much more potent an inhibitor than
MMC (Fig. 6), inhibiting
>95% of NQO1 activity at concentrations as low as 25 nM.

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 5. Inhibition of NQO1 activity in bEnd.3 cells and HUVECs treated with MMC.
Values represent the mean ± S.D. of three independent determinations.
Cells were treated with graded concentrations of MMC for 1 h, followed by the
measurement of NQO1 activity. Significant inhibition (*, p < 0.05)
compared with untreated cells was determined by one-way ANOVA with Tukey
post-tests for between group comparisons.
|
|

View larger version (13K):
[in this window]
[in a new window]
|
Fig. 6. Inhibition of NQO1 activity in bEnd.3 cells and HUVECs treated with ES936.
Values represent the mean ± S.D. of three independent determinations.
Cells were treated with graded concentrations of ES936 for 1 h, followed by
the measurement of NQO1 activity. Significant inhibition (*, p <
0.05) compared with untreated cells was determined by one-way ANOVA with Tukey
post-tests for between group comparisons.
|
|
Inhibition of NQO1 by MMC and ES936 in Mouse Tissues Following Drug
Treatment. The effect of MMC treatment at doses of 10 and 20 mg/kg and
ES936 at 1 mg/kg was measured on NQO1 activity in mouse tissues at 1, 2, 4,
and 24 h post-treatment, and the results for MMC and ES936 are shown in Figs.
7 and
8, respectively. MMC treatment
at both 10 and 20 mg/kg lead to a significant (P < 0.05) decrease
in NQO1 activity in kidney and lung tissues at early timepoints but had no
effect on NQO1 activity in bladder, brain, and heart tissues. There was no
indication of any dose responsiveness with MMC treatment, as the 10- and
20-mg/kg doses showed similar results as the maximum percent inhibition
measured in kidney and lung tissues were similar as was the time course of
inhibition (Table 3). In no
tissue at any timepoint was the degree of inhibition of NQO1 activity
>50%.

View larger version (47K):
[in this window]
[in a new window]
|
Fig. 7. NQO1 Activity in mouse tissues following treatment with (A) 10 mg/kg or (B)
20 mg/kg MMC. Values represent the mean ± S.D. of three independent
determinations in each tissue at each timepoint. Values are expressed as a
percentage of control tissues that were collected from saline treated animals
killed at the same timepoints. Significant inhibition (*, p <
0.05) compared with timepoint matched control tissues was determined by
one-way ANOVA.
|
|

View larger version (33K):
[in this window]
[in a new window]
|
Fig. 8. NQO1 Activity in mouse tissues following treatment with 1 mg/kg ES936.
Values represent the mean ± S.D. of three independent determinations in
each tissue at each timepoint. Values are expressed as a percentage of control
tissues that were collected from saline treated animals killed at the same
timepoints. Significant inhibition (*, p < 0.05) compared with
timepoint matched control tissues was determined by one-way ANOVA.
|
|
ES936 treatment led to a significant decrease in mouse NQO1 activity at 1,
2, and 4 h post-treatment in all tissues measured except bladder
(Fig. 8). The maximum percent
inhibition measured in murine tissues ranged from 61 to 96%, and the maximum
percent inhibition was seen at 1 or 2 h post-treatment
(Table 3). In all tissues that
showed significant inhibition at 1, 2, or 4 h, NQO1 activity was not
significantly inhibited at 24 h post-treatment. NQO1 protein levels in lung
tissue from MMC and ES936 treated animals remained essentially unchanged
relative to control animals (Fig.
9), whereas the NQO1 activity was significantly decreased in these
tissues at various times after dosing. These results show that the measured
decreases in NQO1 activity determined in the tissues is due to a functional
inactivation in the protein and not due to a decrease in protein expression.
For example, NQO1 activity in the lung tissue of an ES936-treated animal at 1
h after dosing is inhibited by 96% (Table
3), but NQO1 protein levels, as shown by immunoblot in
Fig. 9, are roughly equivalent
to the untreated lung tissue sample.

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 9. Immunoblot of NQO1 in mouse lung tissues following treatment with MMC or
ES936. Representative lung samples at 1, 2, 4, and 24 h post-treatment. Murine
NQO1 was identified by comigration with recombinant murine NQO1 standards. U,
untreated sample.
|
|
 |
Discussion
|
|---|
NQO1 has received a great deal of attention as a target for bioreductive
drug therapy and a number of studies have been done attempting to correlate
NQO1 levels with differential toxicity of agents that are bioreductive
substrates for this enzyme. Antitumor agents that have been investigated as
NQO1 substrates include MMC (Siegel et
al., 1990b
),
3-hydroxymethyl-5-aziridinyl-1-methyl-2-(1H-indole-4,7-dione)propenol
(Walton et al., 1991
),
streptonigrin (Beall et al.,
1994
), diaziquone (Siegel et
al., 1990a
), 2,5-dimethyl-3,6-diaziridinyl-1,4-benzoquinone
(Lee et al., 1992
),
5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB1954)
(Boland et al., 1991
), and
others. Metabolism studies carried out with purified rat and human NQO1 for
these substrates have shown that, in general, rat NQO1 reduces antitumor
substrates at a significantly greater rate than human NQO1
(Beall et al., 1994
). The
impact of these enzyme rate differentials can be large differences in the
cytotoxicity of agents in rat versus human cells
(Boland et al., 1991
).
Therefore, the species-specific kinetics of NQO1 with respect to various
agents is an important component of preclinical screening.
The kinetics of NQO1 inhibition are complicated by the fact that NQO1 is
substrate-inhibited at high substrate concentrations for a number of
substrates, including DCPIP. That NQO1 kinetics cannot follow classical
Michaelis-Menten kinetics makes it difficult to assess the ability of
substrates to competitively inhibit NQO1. Previous studies have shown that MMC
acts as a competitive inhibitor of NQO1 activity, with a
KI of 319 µM at pH 7.8 using NQO1 purified from human
kidney (Schlager and Powis,
1988
). When we measured the ability of MMC to act as a competitive
inhibitor of mouse rat and human NQO1, only mouse NQO1 was competitively
inhibited, with a Ki (1140 µM) calculated to be less
than 400 mM. These results were calculated at DCPIP concentrations of 10 to 60
µM, in which NQO1 activity fit the classical Michaelis-Menten equation and
thus could be analyzed for competitive inhibition. These DCPIP concentration
ranges are similar to those used in the previous report; thus, kinetic issues
having to do with substrate inhibition cannot be invoked as playing a role in
the discrepancy between the two studies.
For MMC, the issue of rate of metabolic activation is complicated by the
mechanism-based inactivation of NQO1 by MMC upon reduction in the active site
of the enzyme. Previous studies have determined the kinetics of MMC and
porfiromycin inactivation of rat liver NQO1 as well as the pH dependence of
this process (Siegel et al.,
1993
). The pH dependence of NQO1 inactivation by MMC is inversely
related to the pH dependence of MMC activation by NQO1
(Siegel et al., 1990b
). The
mechanism by which pH influences release of or alkylation by activated MMC has
been discussed elsewhere (Siegel et al.,
1993
). From these studies, a schematic summarizing the mechanism
by which the interaction of MMC with NQO1 can result in either drug activation
or enzyme inactivation can be proposed
(Fig. 10) that is based on
kinetics that have been proposed for mechanism-based enzyme inactivation
(Silverman, 1988
).

View larger version (6K):
[in this window]
[in a new window]
|
Fig. 10. Kinetic scheme of NQO1 mediated metabolism of MMC with subsequent release
of activated MMC or alkylation and mechanism-based inactivation of NQO1.
|
|
The studies presented here expand on previous studies of NQO1 inhibition by
MMC in that they determine the kinetics of this inactivation in mouse, rat,
and human recombinant NQO1 enzyme preparations and extend these findings to
mouse and human endothelial cells and in vivo studies with mice. The fact that
the inactivation kinetics are similar with the mouse and the human enzyme
suggests that studies carried out in mice will be more reflective of what
happens in humans than studies using rat models. This is supported by the
results using the mouse bEnd.3 and the HUVEC cell lines that show similar
inhibition of NQO1 following MMC treatment. Furthermore, these studies support
the previous findings that the kinetics of MMC activation by rat NQO1 are
greater than that of human NQO1 and that part of the increased activation
observed could be due in part to the slower inactivation rate exhibited by the
rat enzyme.
The tissue-specific, in vivo inhibition of NQO1 by MMC provides some data
showing that the role of NQO1 in the metabolism of MMC is tissue-specific and
that the presence of other enzymes that are capable of MMC bioreduction may
decrease the role that NQO1 plays in specific tissues. For example, NQO1
activity was inhibited in kidney and lung tissue where the abundance of other
metabolizing enzymes such as NADPH:cytochrome c reductase, xanthine
dehydrogenase, and NADH:cytochrome b5 reductase can be
presumed to be lower compared with liver tissue, where NQO1 levels were not
affected by MMC treatment. For ES936, which is much more specific for NQO1
mediated metabolism, inhibition was significant in all tissues examined except
for bladder.
The species-dependent differences in activation and inactivation kinetics
are presumably the reason that stable Chinese hamster ovary cell lines that
express human NQO1 are not more sensitive to MMC than the non-NQO1 expressing
parental cell line, whereas Chinese hamster ovary cell lines stably
transfected with rat NQO1 are more sensitive to MMC than the parental line
(Baumann et al., 2001
). MMC is
activated by rat NQO1 at a rate approximately 4.5-times that of human NQO1
(Beall et al., 1994
) and, as
shown here, is inactivated at a rate of approximately 5.5-times less than
human NQO1. These factors, increased activation and decreased inactivation,
dramatically increase the discrepancy between the abilities of rat and human
NQO1 to activate MMC and must be taken into account when comparing studies
that use the human or rat form of the enzyme for metabolic or transfection
studies.
In summary, the studies presented here determined the species-specific
kinetics of NQO1 inactivation by MMC in recombinant enzyme preparations and
show that similar results are obtained in cell culture as well as in mice
treated with MMC. Furthermore, we show that a potent mechanism-based inhibitor
of NQO1, ES936, can inhibit NQO1 enzyme activity in vivo in various tissues.
The in vitro studies in mouse, rat, and human recombinant enzyme preparations
show that the mouse and human enzymes show similar kinetics of inactivation by
MMC. In vivo studies with MMC show that treatment at doses (10 and 20 mg/kg)
that correspond to appreciably higher plasma and tissue levels than those seen
in humans presently undergoing MMC therapy
(van Hazel et al., 1983
;
Buice et al., 1984
) results in
significant inhibition in lung and kidney tissue but not in other tissues
measured. Whether inhibition of NQO1 occurs following exposure of patients
with therapeutic doses of MMC remains to be established.
 |
Acknowledgements
|
|---|
We thank Drs. Elizabeth Swann and Christopher J. Moody at the University of
Exeter (Exeter, UK) for providing ES936 for these studies. We also thank
Patrick J. Kerzic for assistance with the in vivo studies.
 |
Footnotes
|
|---|
This work was supported by CA75955 (D.L.G.) and CA51210 (D.R.).
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
DOI: 10.1124/jpet.103.050070.
ABBREVIATIONS: NQO1, NAD(P)H:quinone oxidoreductase 1; MMC,
mitomycin C; HUVEC, human umbilical vein endothelial cells; DCPIP,
2,6-dichlorophenolindophenol; ES936,
5-methoxy-1,2-dimethyl-[(4-nitrophenoxy)methyl]indole-4,7-dione; TBST,
Tris-buffered saline/Tween 20; NFDM, nonfat dried milk; ANOVA, analysis of
variance.
Address correspondence to: Dr. Daniel L. Gustafson, School of Pharmacy,
C-238, University of Colorado Health Sciences Center, 4200 E. 9th Ave.,
Denver, CO 80220. E-mail:
daniel.gustafson{at}uchsc.edu
 |
References
|
|---|
Baumann RP, Hodnick WF, Seow HA, Belcourt MF, Rockwell S, Sherman
DH, and Sartorelli AC (2001) Reversal of mitomycin C resistance
by overexpression of bioreductive enzymes in chinese hamster ovary cells.
Cancer Res 61:
77707776.[Abstract/Free Full Text]
Beall HD, Mulcahy RT, Siegel D, Traver RD, Gibson NW, and Ross D
(1994) Metabolism of bioreductive antitumor compounds by purified
rat and human DT-diaphorase. Cancer Res
54:
31963201.[Abstract/Free Full Text]
Belcourt MF, Hodnick WF, Rockwell S, and Sartorelli AC
(1996) Bioactivation of mitomycin antibiotics by aerobic and
hypoxic Chinese hamster ovary cells overexpressing DT-diaphorase.
Biochem Pharmacol 51:
16691678.[CrossRef][Medline]
Benson AM, Hunkler MJ, and Talalay P (1980) Increase
of NAD(P)H:quinone reductase by dietary antioxidants: possible role in
protection against carcinogenesis and toxicity. Proc Natl Acad Sci
USA 77:
52165220.[Abstract/Free Full Text]
Boland MP, Knox RJ, and Roberts JJ (1991) The
differences in kinetics of rat and human DT diaphorase result in a
differential sensitivity of derived cell lines to CB 1954
(5-(aziridinyl-1-yl)-2,4-dinitrobenzamide). Biochem
Pharmacol 41:
867875.[CrossRef][Medline]
Buice RG, Niell HB, Sidhu P, and Gurley BJ (1984)
Pharmacokinetics of mitomycin C in non-oat cell carcinoma of the lung.
Cancer Chemother Pharmacol
13:
14.[CrossRef][Medline]
Ernster L (1967) DT-diaphorase. Meth
Enzymol 10:
309317.[CrossRef]
Fitzsimmons SA, Workman P, Grever M, Paull K, Camalier R, and Lewis
AD (1996) Reductase enzyme expression across the National Cancer
Institute tumor cell line panel: correlation with sensitivity to mitomycin C
and EO9. J Natl Cancer Inst
88:
259269.[Abstract/Free Full Text]
Gustafson DL, Beall HD, Bolton EM, Ross D, and Waldren CA
(1996) Expression of NAD(P)H:quinone oxidoreductase
(DT-diaphorase) in Chinese hamster ovary cells: effect on the toxicity of
antitumor quinones. Mol Pharmacol
50:
728735.[Abstract]
Gustafson DL and Pritsos CA (1992) Bioactivation of
mitomycin C by xanthine dehydrogenase from EMT6 mouse mammary carcinoma
tumors. J Natl Cancer Inst
84:
11801185.[Abstract/Free Full Text]
Hodnick WF and Sartorelli AC (1993) Reductive
activation of mitomycin C by NADH:cytochrome b5 reductase.
Cancer Res 53:
49074912.[Abstract/Free Full Text]
Hollander PM and Ernster L (1975) Studies on the
reaction mechanism of DT-diaphorase: action of dead-end inhibitors and effects
of phospholipids. Arch Biochem Biophys
169:
560567.[CrossRef][Medline]
Kelsey KT, Ross D, Traver RD, Christiani DC, Zuo Z-F, Spitz MR,
Wang M, Xu X, Lee B-K, Schwartz BS, and Wiencke JK (1997) Ethnic
variation in the prevalence of a common NAD(P)H:quinone oxidoreductase
polymorphism and its implications for anti-cancer chemotherapy. Br
J Cancer 76:
852854.[Medline]
Keyes SR, Fracasso PM, Heimbrook DC, Rockwell S, Sligar SG, and
Sartorelli AC (1984) Role of NADPH:cytochrome c
reductase and DT-diaphorase in the biotransformation of mitomycin C.
Cancer Res 44:
56385643.[Abstract/Free Full Text]
Lee CS, Hartley JA, Berardini MD, Butler J, Siegel D, Ross D, and
Gibson NW (1992) Alteration in DNA cross-linking and sequence
selectivity of a series of aziridinylbenzoquinones after enzymatic reduction
by DT-diaphorase. Biochem
31:
30193025.[CrossRef][Medline]
Malkinson AM, Siegel D, Forrest GL, Gazdar AF, Oie HK, Chan DC,
Bunn PA, Mabry M, Dykes DJ, Harrison SD Jr, and Ross D (1992)
Elevated DT-diaphorase activity and messenger RNA content in human non-small
lung cell carcinoma: relationship to the response of lung tumor xenografts to
mitomycin C. Cancer Res
52:
47524757.[Abstract/Free Full Text]
Marin A, Lopez de Cerain A, Hamilton E, Lewis AD, Martinez-Penuela
JM, Idoate MA, and Bello J (1997) DT-diaphorase and cytochrome
b5 reductase in human lung and breast tumours.
Br J Cancer 76:
923929.[Medline]
Nishiyama M, Saeki S, Aogi K, Hirabayashi N, and Toge T
(1995) Relevance of DT-diaphorase activity to mitomycin C (MMC)
efficacy on human cancer cells: differences in in vitro and in vivo systems.
Int J Cancer 53:
10131016.
Pan S-S, Andrews PA, and Glover CJ (1984) Reductive
activation of mitomycin C and mitomycin C metabolites catalyzed by
NADPH-cytochrome P-450 reductase and xanthine oxidase. J Biol
Chem 259:
959966.[Abstract/Free Full Text]
Phillips RM, Burger AM, Loadman PM, Jarrett CM, Swaine DJ, and
Fiebig H-H (2001) Predicting tumor responses to mitomycin C on
the basis of DT-diaphorase activity of drug metabolism by tumor homogenates:
implications for enzyme-directed bioreductive drug development.
Cancer Res 60:
63846390.
Powis G, Gasdaska PY, Gallegos A, Sherrill K, and Goodman D
(1995) Overexpression of DT-diaphorase in transfected NIH 3T3
Cells does not lead to increased anticancer quinone drug sensitivity: a
questionable role for the enzyme as a target for bioreductively activated
anticancer drugs. Anticancer Res
15:
11411146.[Medline]
Riley RJ and Workman P (1992) DT-diaphorase and cancer
chemotherapy. Biochem Pharmacol
43:
16571669.[CrossRef][Medline]
Schlager JJ and Powis G (1988) Mitomycin C is not
metabolized by but is an inhibitor of human kidney
NAD(P)H:(quinone-acceptor)oxidoreductase. Cancer Chemother
Pharmacol 22:
126130.[Medline]
Schlager JJ and Powis G (1990) NAD(P)H:quinone
acceptor oxidoreductase in human normal and tumor tissue: effects of cigarette
smoking and alcohol. Int J Cancer
45:
403409.[Medline]
Siegel D, Beall H, Kasai M, Arai H, Gibson NW, and Ross D
(1993) pH dependent inactivation of DT-diaphorase by mitomycin C
and porfiromycin. Mol Pharmacol
44:
11281134.[Abstract]
Siegel D, Beall H, Senekowitsch C, Kasai M, Arai H, Gibson NW, and
Ross D (1992) Bioreductive activation of mitomycin C by
DT-diaphorase. Biochem
31:
78797885.[CrossRef][Medline]
Siegel D, Franklin WA, and Ross D (1998)
Immunohistochemical detection of NAD-(P)H:quinone oxidoreductase in human lung
and lung tumors. Clin Cancer Res
4:
20652070.[Abstract]
Siegel D, Gibson NW, Preusch PC, and Ross D (1990a)
Metabolism of diaziquone by NAD(P)H:(quinone acceptor) oxidoreductase
(DT-diaphorase): role in diaziquoneinduced DNA damage and cytotoxicity in
human colon carcinoma cells. Cancer Res
50:
72937300.[Abstract/Free Full Text]
Siegel D, Gibson NW, Preusch PC, and Ross D (1990b)
Metabolism of mitomycin C by DT-diaphorase: role in mitomycin C-induced DNA
damage and cytotoxicity in human colon carcinoma cells. Cancer
Res 50:
74837489.[Abstract/Free Full Text]
Siegel D, McGuinness SM, Winski SL, and Ross D (1999)
Genotype-phenotype relationships in studies of a polymorphism in
NAD(P)H:quinone oxidoreductase 1. Pharmacogenetics
9:
113121.[Medline]
Silverman RB (1988) Mechanism-Based Enzyme
Inactivation: Chemistry and Enzymology, vol
1, CRC Press, Inc., Boca Raton, FL.
Traver RD, Siegel D, Beall HD, Phillips RM, Gibson NW, Franklin WA,
and Ross D (1997) Characterization of a polymorphism in
NAD(P)H:quinone oxidoreductase (DT-diaphorase). Br J
Cancer 75:
6975.[Medline]
van Hazel GA, Scott M, Rubin J, Moertel CG, Eagan RT, O'Connell MJ,
and Kovach JS (1983) Pharmacokinetics of mitomycin C in patients
receiving the drug alone or in combination. Cancer Treat
Rep 67:
805810.[Medline]
Vasiliou V, Theurer MJ, Puga A, Reuter SF, and Nebert DW
(1994) Mouse dioxin-inducible NAD(P)H:menadione oxidoreductase:
NMO1 cDNA sequence and genetic differences in mRNA levels.
Pharmacogenetics 4:
341348.[Medline]
Walton MI, Smith PJ, and Workman P (1991) The role of
NAD(P)H:quinone reductase (EC 1.6.99.2
[EC]
, DT-diaphorase) in the reductive
bioactivation of the novel indolquinone antitumor agent EO9. Cancer
Commun 3:
199206.[Medline]
Winski SL, Faig M, Bianchet MA, Siegel D, Swann E, Fung K, Duncan
MW, Moody CJ, Amzel LM, and Ross D (2001) Characterization of a
mechanism-based inhibitor of NAD(P)H:quinone oxidoreductase 1 by biochemical,
X-ray crystallographic and mass spectrometric approaches.
Biochem 40:
1513515142.[CrossRef][Medline]
Winski SL, Hargreaves RHJ, Butler J, and Ross D (1998)
A new screening system for NAD(P)H:quinone oxidoreductase (NQO1)-directed
antitumor quinones: identification of a new aziridinylbenzoquinone, RH1, as a
NQO1-directed antitumor agent. Clin Cancer Res
4:
30833088.[Abstract]