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CHEMOTHERAPY, ANTIBIOTICS, AND GENE THERAPY
Department of Pharmaceutical Sciences, School of Pharmacy and the Cancer Center, University of Colorado Health Sciences Center, Denver, Colorado
Received February 6, 2003; accepted March 11, 2003.
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Abstract |
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 Top Abstract Materials and MethodsResultsDiscussionReferences |
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,
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 (6.5–7.4) 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 (7.0–7.8)
(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.
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Materials and Methods |
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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 pKK233–2 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 (10–120 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 mM–1
cm–1.
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.).
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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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.
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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.
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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%.
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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.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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),
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).
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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.
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Acknowledgements |
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Footnotes |
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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
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=
Vmax · [S]/KM +
[S]), and the solid line represents data fit to the uncompetitive
substrate inhibition model (
