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Department of Pharmaceutical Sciences, School of Pharmacy and Cancer Center, University of Colorado Health Sciences Center, Denver, Colorado, and School of Chemistry, University of Exeter, Exeter, United Kingdom
Received February 5, 2003; accepted May 22, 2003
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Abstract |
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 Top Abstract Materials and MethodsResultsDiscussionReferences |
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; Malkinson et al.,
1992; Mikami et al.,
1998). NQO1 is generally considered to be a detoxification enzyme
(Ernster, 1967;
Lind et al., 1982;
Thor et al., 1982) that
reduces quinones directly to hydroquinones, eliminating the formation of
reactive oxygen species generated by redox cycling. NQO1 may also act as an
antioxidant enzyme by regenerating antioxidant forms of ubiquinone and vitamin
E quinone (Beyer et al., 1996;
Siegel et al., 1997).
Alternatively, NQO1 may also bioactivate quinones to reactive DNA-alkylating
agents. It is in this capacity that NQO1 may be useful in the treatment of
cancer, and there has been considerable interest in the development of
chemotherapeutic prodrugs that are specifically bioactivated by NQO1.
The involvement of NQO1 in biochemical reactions has historically been
determined by inhibiting its enzymatic activity with dicoumarol, a nonspecific
competitive inhibitor that binds to the pyridine nucleotide binding site in
NQO1 (Hosoda et al., 1974;
Hollander and Ernster, 1975).
Dicoumarol has been shown to inhibit a variety of enzymes
(Wang et al., 1982;
Segura-Aguilar et al., 1986;
Ross et al., 1993), including
glutathione (GSH) transferases and GSH peroxidase II
(Mays and Benson, 1992;
Karczewski et al., 1999), as
well as NADH-ubiquinone reductase (Tampo
and Yonaha, 1996). It may also affect mitochondrial oxidative
phosphorylation (Karczewski et al.,
1999), potentiate tumor necrosis factor-
–induced
apoptosis in HeLa cells, and inhibit stress-activated protein kinase and
nuclear factor-
B activation (Cross
et al., 1999). Many of the ancillary effects of dicoumarol in
cells are probably caused by the relatively high micromolar concentrations
necessary to inhibit NQO1 activity. Even at micromolar concentrations of
dicoumarol, only partial inhibition of NQO1 may occur, depending on the
quinone substrate (Preusch et al.,
1991; Nakamura and Hayashi,
1994). Therefore, the data obtained with the use of dicoumarol
cannot solely be interpreted in terms of NQO1 inhibition.
We reported recently the chemical and biochemical properties of ES936, a
mechanism-based inhibitor of NQO1, in cell-free systems
(Winski et al., 2001a). ES936
undergoes reduction by NQO1 followed by the loss of para-nitrophenol
to generate a reactive iminium species that alkylates one of two tyrosine
residues in the active site, resulting in irreversible inhibition of the
enzyme. Our previous work used purified recombinant human wild-type NQO1 and
allowed for the investigation of the specific mechanism of ES936 interaction
with the human wild-type NQO1*1 protein. However, the biological consequences
of the use of ES936 in cells are not known and need to be defined before
beginning routine use of this agent to inhibit NQO1 in cellular systems.
ES936, although a potent and specific NQO1 inhibitor, is a quinone and therefore may have deleterious effects in cellular systems. This study examines the biochemical, cytotoxic, and genotoxic effects of ES936 in cells that express a range of NQO1 activities. To measure the effectiveness of ES936 as an inhibitor of NQO1, we evaluated the ability of ES936 to abrogate the toxicity of an antitumor quinone bioactivated by NQO1. ES936, as a mechanism-based inhibitor of NQO1, provides a potentially useful tool to clarify the precise in vivo role of NQO1 without the ancillary effects of the NQO1 inhibitor, dicoumarol.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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). Human and rat liver microsomes were obtained from BD
Gentest (Woburn, MA).
Cell Culture. HCT116 and HT-29, human colon carcinoma cell lines,
and the human breast cancer cell line MDA-MB-468 (MDA468) were obtained from
the American Type Culture Collection and were grown as monolayers in either
minimal essential medium or RPMI 1640 medium supplemented with 10% fetal
bovine serum, 2 mM l-glutamine, 100 U/ml of penicillin, and 100
µg/ml streptomycin (complete media) at 37°C in a humidified atmosphere
with 5% CO2. The MDA468 NQ16 cell line is a NQO1 stable
transfectant cell line generated from the human breast cancer cell line,
MDA468. The parental MDA468 cell line is homozygous for a polymorphism in NQO1
(NQO1*2) that results in extremely low NQO1 activity (<5 nmol DCPIP/min/mg
protein). The cytomegalovirus-driven mammalian expression vector pcDNA3.0
containing human wild-type NQO1 cDNA (construction described by
Gustafson et al., 1996) was
used to transfect MD468 cells as described previously
(Winski et al., 1998). The
stable expression of NQO1 has been monitored by repeated measurement of NQO1
activity as well as by NQO1 immunoblot analysis, as described previously
(Gustafson et al., 1996).
Enzyme Activity Assay. NQO1 activity was measured as the rate of
dicoumarol-inhibitable DCPIP reduction in cell cytosolic samples as described
previously (Ernster, 1967)
with modifications (Benson et al.,
1980) and normalized to total cytosolic protein
(Lowry et al., 1951). Briefly,
cells were lifted with trypsin/EDTA, neutralized with complete media, and
centrifuged at 1500 rpm for 5 min (4°C); the supernatant was then
aspirated, and the pellet was resuspended in 200 to 500 µl of buffer A (25
mM Tris-HCl, 250 µM sucrose, 5 µM FAD). The cell suspension was
probe-sonicated on ice for 5 s and then centrifuged at 13,000 rpm for 15 min
with the supernatant removed to a clean tube and kept on ice for analysis.
NQO1 activity was measured in 1-ml reactions (27°C) containing 25 mM
Tris-HCl, pH 7.4, 0.7 mg/ml bovine serum albumin, 200 µM NADH, and 40 µM
DCPIP. Reactions were performed in the presence and absence of 20 µM
dicoumarol. Cytochrome P450 reductase was measured in cellular samples as the
NAD(P)H-dependent reduction of cytochrome c according to the method
used by Vermilion and Coon
(1978). The activity of
cytochrome b5 reductase was measured as the NADH-dependent
reduction of cytochrome c according to the method used by Mihara and
Sato (1978).
NQO1 Inhibition by ES936. The inhibition of NQO1 activity by ES936 was monitored over a time and concentration range to determine the lowest concentration of ES936 that would inhibit >95% of NQO1 activity over a given duration of exposure. Cells were treated with varying concentrations of ES936 in complete media for 30, 60, or 120 min, at which time cell cytosols were prepared and assayed for NQO1 activity.
ES936 Biostability. To determine the biostability of ES936 in tissue culture media, the inhibitor was added to complete media at a final concentration of 100 nM and maintained at 37°C in a humidified atmosphere with 5% CO2. At various times afterward, cells that were 60 to 70% confluent were dosed with an aliquot of the media containing ES936 for 30 min, at which time the NQO1 activity was measured as described above.
Growth Inhibition Assays. Toxicity was determined by growth inhibition (MTT) assays. HCT116 cells and MDA468 NQ16 cells were seeded at 1000 to 3000 cells/well, respectively, in 96-well plates and were allowed to attach for 16 to 20 h. Plates (in triplicate) were treated with ES936 in complete media or with streptonigrin in serum-free media for 2 h. Additional plates were pretreated with ES936 (100 nM) in complete media for 30 min before exposure to streptonigrin in serum-free media for 2 h. After drug treatment, the media were removed and replaced with complete media. Cells were then allowed to grow for 3 to 5 days. Cell viability was assessed by measuring the NADH-dependent reduction of MTT to a formazan product that was extracted from cells with dimethyl sulfoxide. The optical density of the extract was determined at 550 nm with a microplate reader. IC50 values, defined as the concentration that results in a 50% reduction in cell density (from untreated controls), were calculated from a minimum of three separate experiments.
Soluble Thiols. Cells were seeded 24 h before treatment, and all
cells used were approximately 60 to 70% confluent. Soluble thiols were
determined according to a modified method used by Sedlak and Lindsay
(1968) after treatment with
100 nM ES936 in complete media for the indicated times. After dosing, media
were aspirated, and the cells were rinsed with phosphate-buffered saline,
lifted with trypsin/EDTA, and neutralized with complete media. The cell
suspension was vortexed, and 1-ml aliquots were distributed into two tubes on
ice. Cells were centrifuged at 1,500 rpm for 5 min (4°C), and the
supernatant was aspirated. The cell pellet in one tube was rinsed with
phosphate-buffered saline, vortexed lightly, recentrifuged, and aspirated, and
the pellet was brought up in buffer A for determination of protein
concentration according to the methods used by Lowry et al.
(1951). The cell pellet of the
second tube was lysed in 5% trichloroacetic acid, vortexed immediately for 10
s, and then centrifuged at 5,000 rpm for 10 min to pellet cellular protein.
The supernatant was removed to a glass tube containing 2 ml of 0.4 M Tris-HCl,
pH 8.9, and 5,5'-dithio-bis(2-nitrobenzoid acid) was added to a final
concentration of 100 µM. Samples were vortexed and incubated for 5 min at
room temperature, and the absorbance at 412 nm was determined. Results were
expressed as µM acid-soluble thiols/mg protein calculated from a reduced
glutathione calibration curve.
NAD(P)H Oxidation. Quinone-stimulated microsomal-dependent NAD(P)H
oxidation was measured spectrophotometrically at 340 nm in 1-ml reactions at
32°C. Reactions were performed in 100 mM potassium phosphate buffer, pH
7.4, containing 200 µM NAD(P)H, 330 mg rat microsomal protein, and 50 µM
quinone (ES936, menadione, and 
-lapachone). Reactions were started with
the addition of quinone, and the decrease in absorbance at 340 nm was
monitored for 1 min.
Comet Assay. DNA damage was evaluated by the single-cell gel
electrophoresis method, commonly known as the alkaline comet assay. The comet
assay is a sensitive method used to evaluate DNA strand breaks in single
cells. Briefly, cells are treated with drug before embedding in a thin coating
of agarose on a microscope slide. The slides are submerged in an alkaline
buffer and subjected to horizontal electrophoresis. Single- or double-strand
breaks cause a relaxation of supercoiled DNA, allowing migration toward the
anode, forming a "comet". The percentage of DNA in the comet tail
provides an indication of the degree of DNA damage. For this study, cells were
incubated in complete media with 100, 250, or 500 nM ES936. After a 2-h
incubation period, the cells were lifted, immediately placed on ice, and
processed as described previously (Winski
et al., 2001b). Comet slides were examined on a Nikon Eclipse
TE300 (Nikon, Tokyo, Japan) equipped with epifluorescence capabilities. An
excitation filter of 530 nm and a magnification of 400x was used to
visually score a minimum of 200 comets on each slide. Each comet was visually
identified as belonging to one of four classes according to the degree of DNA
damage observed, in a fashion similar to that of Collins et al.
(1997). Slides were scored
blinded. Comet classes are as follows and are shown in
Fig. 1: class 0, intact nuclei,
no DNA damage present; class 1, well-defined nucleus with light tail
formation, <20% DNA in the tail; class 2, defined nucleus with 20 and 75%
DNA in the tail; class 3: nucleus is no longer well-defined and tail consists
of more than 75% of the DNA.
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To verify the visual classification system, random fields of comet images from all treatment groups were captured with a Nikon Coolpix 990 digital camera mounted to the microscope. All single-comet images (no image overlap) in each photo were copied into individual files with the use of Adobe Photoshop 6.0 (Adobe Systems, Mountain View, CA). The single images were visually scored and placed into one of the four classes. The percentage of DNA in the tail was then determined for each image using Euclid Comet Analysis software (St. Louis, MO). The mean percentage of DNA in the tail was calculated and assigned to each comet class.
A score for each slide was derived by multiplying the comet class (0–3) by the percentage of the comets in that class and then summing all scores to obtain a total for the slide. Scores for each slide could range between 0 (100% of comets in class 0) and 300 (100% of comets in class 3) (units are arbitrary) and represent the overall DNA damage of the cell population for a specific treatment.
Statistical Analysis. Statistical analysis was performed using NCSS software (Kaysville, UT). The two-sample t test was used to evaluate differences between two independent groups, and P values <0.05 were considered significant. Differences between more than two independent groups were evaluated with analysis of variance followed by Scheffé F test for multiple comparisons. Data are presented as means ± S.E.M.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Biostability of ES936. The biostability of 100 nM ES936 in complete media was measured over time (Fig. 3). In these experiments, ES936 was preincubated in complete media (minus cells) for the indicated times, after which the ES936-containing media was added to cells, and NQO1 activity was measured after 30 min. In experiments with the HCT116 cell line, more than 95% inhibition of NQO1 activity was observed at all time points. In experiments with the very high NQO1 activity cell line MDA468 NQ16, a reduction in the ability of ES936 to inhibit NQO1 was observed between 2 and 4 h of incubation in media.
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Growth-Inhibitory Activity of ES936. The growth-inhibitory activity
of ES936 was measured by use of the MTT assay in the MDA468 NQ16 and HCT116
cell lines because these two lines represent the upper and lower limits of
NQO1 activity, respectively, examined in this study. IC50 values
are shown in Table 1. The
IC50 values for the cell lines used in this study are 8 to 20 times
higher than the concentration of ES936 needed for effective inhibition of
NQO1. The ability of ES936 to affect the toxicity of compounds that are
metabolized by NQO1 was also investigated. ES936 pretreatment significantly
increased the IC50 value for streptonigrin by approximately 6-fold
for the MDA468 NQ16 cell line and 2-fold for the HCT116 cell line, which is
consistent with a bioactivation role of NQO1 in streptonigrin metabolism, as
described previously (Beall et al.,
1996).
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Other Reductases. We examined the effects of ES936 on cytochrome P450 reductase and cytochrome b5 reductase in cell lines and purified microsomal preparations to ascertain whether other cellular reductases in addition to NQO1 were inhibited by this compound. As shown in Table 2, treatment with 100 nM ES936 for 30 min did not inhibit the activity of cytochrome P450 reductase or cytochrome b5 reductase in the cell lines tested, although more than 95% of the NQO1 activity was inhibited. Although the activities of these two reductases were low in these cell lines, there was no significant difference between treated and untreated cells, regardless of initial NQO1 activity. Furthermore, we evaluated the effects of ES936 at 100 nM in purified human and rat liver microsomes supplemented with NADH/NAD(P)H (data also shown in Table 2). As seen in the cell lines tested, the activities of cytochrome P450 reductase or cytochrome b5 reductase were not significantly different between treated and control human or rat microsomes. In these experiments, we also observed no effect of ES936 at concentrations up to 10 µM on the activities of either cytochrome P450 reductase or cytochrome b5 reductase (data not shown).
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Cellular Nonprotein Thiols. To determine the effect of ES936 on cellular redox homeostasis, total cellular thiols were measured in vehicle-treated (control) cells and cells treated with 100 nM ES936 for various times (Fig. 4). Statistical analysis revealed no difference between treated and control cells for either cell line at any time point.
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Microsomal-Dependent NAD(P)H Oxidation. The ability of ES936 to
stimulate microsomal NAD(P)H oxidation was measured in purified rat liver
microsomes. ES936 did stimulate low levels of NAD(P)H oxidation, but compared
with other quinones (menadione, -lapachone) under identical conditions,
the rate of NAD(P)H oxidation with ES936 was very low
(Fig. 5).
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DNA Damage. We used the comet assay to investigate the potential for the induction of DNA strand breaks after ES936 exposure. A dose-dependent increase in the degree of DNA damage with increasing concentration of ES936 can be seen in a comparison of the overall comet scores (see Materials and Methods) for each treatment (Fig. 6A). For each ES936 concentration evaluated, the contribution of each degree of DNA damage is presented in Fig. 6B. In both cell lines studied (open and closed symbols in Fig. 6B), ES936 at a concentration of 100 nM (triangles) induced a similar number of comets in each class, and the level of damage was up to twice that seen in the controls. At doses of ES936 greater than 100 nM in the MDA468 NQ16 cells, more severely damaged nuclei are seen, as indicated by increased numbers of class 3 comets. In HCT116 cells, doses greater than 100 nM ES936 resulted in more comets formed in classes 1 and 2 and fewer in class 3, indicating less severe damage to the nuclei. This is reflected in the lower total comet score (Fig. 6A) for HCT116 cells compared with scores for MDA468 NQ16 cells at each increasing dose of ES936.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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To measure of the effectiveness of ES936, we evaluated its ability to
abrogate the toxicity of streptonigrin, an aminoquinone antitumor antibiotic
efficiently bioactivated by NQO1 (Beall et
al., 1996). Streptonigrin was chosen as a test compound because it
showed the best correlation between NQO1 activity and toxicity of the more
than 31,000 compounds tested in the National Cancer Institute's 60-tumor cell
line panel (Paull et al.,
1994). Streptonigrin is 6-fold less toxic to MDA468 NQ16 cells and
approximately 2-fold less toxic to HCT116 cells when pretreated with 100 nM
ES936. Although NQO1 is clearly inhibited by ES936, it does not inhibit other
cellular reductases such as NAD(P)H cytochrome P450 reductase or NADH
cytochrome b5 reductase. We have reported previously a
similar result in the BE cell line (Winski
et al., 1998). The activities of xanthine oxidase and xanthine
dehydrogenase were not measured because they are consistently below the levels
of detection in these cell lines (Beall et
al., 1996; Winski et al.,
1998).
Electrophilic and redox cycling reactions of quinones may lead to decreases
in cellular thiol content. Levels of glutathione, the most abundant
intracellular thiol (>99.5% of total glutathione is GSH)
(Anderson, 1985) and an
important detoxicant for free radicals, would be expected to decrease if ES936
were generating reactive oxygen species through one-electron reduction and
redox cycling. The lack of change in acid-soluble thiol levels, regardless of
the duration of exposure, indicates that ES936 is not generating substantial
levels of reactive oxygen species. These data correlate with the relatively
low degree of NAD(P)H oxidation detected in rat liver microsomes.
Some DNA damage may occur with the concentrations of ES936 (
100 nM)
used to inhibit NQO1 activity. A small increase in the DNA content of the
comet tail after treatment with ES936 is consistent with the induction of
single-strand breaks. To place the extent of DNA damage resulting from 100 nM
ES936 exposure in perspective, exposure to streptonigrin at a dose 1000-fold
lower results in a higher overall comet score (data not shown). The low amount
of rat liver microsome-dependent NAD(P)H oxidation seen with ES936 treatment
could relate to the DNA damage observed using the comet assay. No data are
available to determine whether the degree of DNA damage observed at 100 nM
ES936 might result in cellular toxicity, because DNA repair was not evaluated
in this study. ES936, at all concentrations used in this study, had no effect
on p53 status in HCT116 cells (wild-type p53), indicating that downstream
markers of DNA damage were not activated
(Anwar et al., 2003).
In summary, ES936 is a potent and specific inhibitor of NQO1 in cellular systems at concentrations of more than 1000-fold lower than the nonspecific inhibitor dicoumarol. The resumption of NQO1 catalytic activity depends on the synthesis of new protein, which is cell line-dependent, and therefore the duration of inactivation induced by ES936 varies between cell lines. The inhibition of other reductases by ES936 was not observed in two different cell lines. Using streptonigrin, a compound that is bioactivated by NQO1, we validated the use of ES936 as an inhibitor of NQO1 in cells. One caveat in the use of ES936 is the induction of a relatively small amount of DNA damage in the form of strand breaks. The degree of damage was relatively minor compared with that observed with other genotoxic quinones, such as streptonigrin, and was in agreement with a limited amount of redox cycling observed in rat liver microsomal systems. However, care should be taken if ES936 is used as an inhibitor of NQO1 in studies in which DNA damage is used as an endpoint. From the findings in our studies, we propose the use of ES936 (100 nM) as a mechanism-based inhibitor of NQO1 in cellular systems and for use as a component of the routine activity assay for NQO1.
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Footnotes |
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ABBREVIATIONS: NQO1, NAD(P)H:quinone oxidoreductase 1; ES936, 5-methoxy-1,2-dimethyl-3-[(4-nitrophenoxy)methyl]indole-4,7-dione; GSH, glutathione; DCPIP, 2,6-dichlorophenol-indophenol; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium.
Address correspondence to: Dr. David Ross, Department of Pharmaceutical Sciences, Campus Box C238, University of Colorado Health Sciences Center, 4200 East Ninth Avenue, Denver, CO 80262. E-mail: david.ross{at}uchsc.edu
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Winski SL, Swann E, Hargreaves RH, Dehn DL, Butler J, Moody CJ, and Ross D (2001b) Relationship between NAD(P)H:quinone oxidoreductase 1 (NQO1) levels in a series of stably transfected cell lines and susceptibility to antitumor quinones. Biochem Pharmacol 61: 1509–1516.[CrossRef][Medline]
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), 60 min
(
), or 120 min (
) of exposure at each ES936 concentration. Values
given are the means ± S.E.M. of a minimum of three independent
experiments. Error bars are not visible in most cases.
) by ES936 after incubation of 100 nM ES936 in media at 37°C for
the durations indicated. NQO1 activity was measured in cell sonicates after a
30-min dosing period. Results are the mean ± S.E.M. of a minimum of
three independent experiments.
, respectively). Values represent the means ±
S.E.M. of a minimum of three independent experiments. There was no statistical
difference (
