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Carcinogenesis Advance Access originally published online on November 2, 2005
Carcinogenesis 2006 27(4):803-810; doi:10.1093/carcin/bgi262
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© The Author 2005. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

NO-donating aspirin induces phase II enzymes in vitro and in vivo

Jianjun Gao, Khosrow Kashfi 1, Xiaoping Liu and Basil Rigas *

Division of Cancer Prevention, Department of Medicine, SUNY at Stony Brook, NY 11794, USA and 1 Department of Physiology and Pharmacology, City University of New York Medical School, New York, NY 10031, USA

* To whom correspondence should be addressed at: Division of Cancer Prevention, Department of Medicine, Life Sciences Building 06, SUNY at Stony Brook, Stony Brook, NY 11794-5200, USA. Tel: +1 631 632 9035; Fax: +1 631 632 1992; Email: basil.rigas{at}sunysb.edu


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Modulation of drug metabolizing enzymes, leading to facilitated elimination of carcinogens represents a successful strategy for cancer chemoprevention. Nitric oxide-donating aspirin (NO-ASA) is a promising agent for the prevention of colon and other cancers. We studied the effect of NO-ASA on drug metabolizing enzymes in HT-29 human colon adenocarcinoma and Hepa 1c1c7 mouse liver adenocarcinoma cells and in Min mice treated with NO-ASA for 3 weeks. In these cell lines, NO-ASA induced the activity and expression of NAD(P)H:quinone oxireductase (NQO) and glutathione S-transferase (GST). Compared with untreated Min mice, NO-ASA increased in the liver the activity (nmol/min/mg; mean ± SEM for all) of NQO (85 ± 6 versus 128 ± 11, P<0.05) and GST (2560 ± 233 versus 4254 ± 608, P < 0.005) and also in the intestine but not in the kidney; the expression of NQO1 and GST P1-1 was also increased. NO-ASA had only a marginal effect on P450 1A1 and P450 2E1, two phase I enzymes. The release of NO from NO-ASA, determined with a selective microelectrode was paralleled by the induction of NQO1 and abrogated by NO scavengers; an exogenous NO donor also induced the expression of NQO1. NO-ASA induced concentration-dependently the translocation of Nrf2 into the nucleus as documented by immunofluorescence and immunoblotting; this paralleled the induction of NQO1 and GST P1-1. Thus NO-ASA induces phase II enzymes, at least in part, through the action of NO that it releases and by modulating the Keap1–Nrf2 pathway; this effect may be part of its mechanism of action against colon and other cancers.

Abbreviations: ARE, antioxidant response element; CDNB, 1-chloro-2,4-dinitrobenzene; CYP, cytochrome P450; DMSO, dimethyl sulfoxide; DTNB, 5,5'-dithiobis 2-nitrobenzoic acid; GST, glutathione S-transferase; NO-ASA, NO-donating aspirin; NO-NSAIDs, Nitric oxide-donating non-steroidal anti-inflammatory drugs; NQO, NAD(P)H:quinone oxireductase; PTIO, 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide; SNAP, S-nitroso-N-acetyl-penicillamine; UGT, UDP-glucuronyltransferase


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Nitric oxide-donating non-steroidal anti-inflammatory drugs (NO-NSAIDs) are an emerging novel class of compounds with significant potential for the control of cancer (reviewed in (1)). Each consists of a traditional NSAID that bears covalently attached to it an NO-releasing moiety. NO-donating aspirin (NO-ASA), the most extensively studied NO-NSAID, inhibits the growth of various cancer cell lines (2), inhibits colon carcinogenesis in preclinical animal models (3,4) and a clinical trial for colon cancer prevention is underway. Animal and early human studies suggest that NO-ASA is much safer than conventional ASA (5,6). Thus NO-ASA is a promising agent for the prevention of colon and other cancers.

We have been exploring the important question of the mechanism of NO-ASA's apparently strong chemopreventive effect. NO-ASA has pleiotropic effects encompassing a strong cell kinetic effect and modulation of several cell-signaling pathways, including the eicosanoid, nitric oxide, Wnt, NF-{kappa}B and MAPK pathways (7–10). The relative contribution of each of these effects of NO-ASA to its chemopreventive efficacy remains unclear and is the subject of intense study.

The xenobiotic metabolizing enzymes are classified as phase I (oxidation and reduction reactions) and phase II (conjugation reactions) enzymes; the balance between the phase I carcinogen-activating enzymes and the phase II detoxifying enzymes is critical to determining an individual's risk for cancer (reviewed in (11)). Phase I enzymes primarily consist of the cytochrome P450 (CYP) superfamily. In humans, four CYP gene families (CYP1–CYP4) are considered the most important (12). Phase II enzymes comprise many enzyme superfamilies including the NAD(P)H:quinone oxireductase (NQO), glutathione S-transferases (GSTs) and UDP-glucuronyltransferases (UGTs). NQO protects cells against the toxicity of quinones and their metabolic precursors by promoting the obligatory two-electron reduction of quinones to semiquinones. The human GSTs consist of four main classes, {alpha} (A), µ (M), {theta} (T) and {pi} (P), each of which is divided into one or more isoforms (13). GSTs detoxify a number of carcinogenic electrophiles by transforming them into their conjugates with reduced glutathione. A major consequence of the inactivation of chemical carcinogens by GST is thought to be blocking the formation of DNA adducts (reviewed in Ref. 14).

Modulation of drug metabolizing enzymes, leading to facilitated elimination of endogenous and environmental carcinogens represents a successful strategy for cancer chemoprevention (15). This notion is exemplified by dithiolethiones, which induce phase II enzymes. These compounds inhibit tumorigenesis of environmental carcinogens in various animal models and, as shown in human clinical trials, modulate the metabolism of the carcinogen aflatoxin B1 (15). In general, the induction of phase II enzymes is a sufficient strategy for protecting mammals and their cells against carcinogens and other forms of electrophile and oxidant toxicity.

Several chemopreventive agents induce the expression of phase II genes through their effects on the Keap1–Nrf2 complex (16,17). In the nucleus, the transcription factor Nrf2, a member of the NF-E2 family, dimerizes with the small Maf protein and binds to the antioxidant response element (ARE), a cis-acting regulatory element in the promoter region of phase II enzymes. A cytoplasmic actin-binding protein, Keap1, is an inhibitor of Nrf2 that sequesters it in the cytoplasm. Inducers dissociate this complex, allowing Nrf2 to translocate to the nucleus.

In the present studies, we evaluated the effect of NO-ASA on xenobiotic metabolizing enzymes both in vitro and vivo. We studied its effect on phase I and II enzymes in liver and colon cell lines as well as in the liver and colon of Min mice, a mouse model system of intestinal cancer that displays many of the molecular and histological features of human colon cancer. Our data demonstrate that NO-ASA is a significant inducer of phase II enzymes that probably acts through the Keap1–Nrf2 complex.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Reagents
Stock (100 mM) solutions of NO-ASA [NCX4016, 2-(acetyloxy)benzoic acid 3-(nitrooxy methyl)phenyl ester, Nicox, SA, Sophia Antipolis, France] and ASA and DMSO (Sigma Chemical, St Louis, MO) were prepared in dimethyl sulfoxide (DMSO; Fisher Scientific, Fair Lawn, NJ); the final DMSO concentration was adjusted in all media to 1%. S-nitroso-N-acetyl-penicillamine (SNAP), 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO) were from Sigma.

Cell culture
HT-29 human colon adenocarcinoma and Hepa 1c1c7 mouse liver adenocarcinoma cell lines were obtained from American Type Tissue Collection (Rockville, MD). Cells were grown in 5% CO2 at 37°C in a-MEM (Hepa1c1c7) or McCoy 5A (HT-29) medium supplemented with 10% fetal calf serum, 10 000 IU/ml penicillin and 10 mg/ml streptomycin. To assay GST activity and perform immunoblot analyses, cells were seeded in 10 cm cell culture dishes at a density of 5.0 x 105 cells/cm2 in 10 ml of media and allowed to attach for 24 h. Cells were exposed to various concentrations of NO-ASA for 24 h; control cultures were supplemented with DMSO only. Cells were washed twice with ice-cold PBS and harvested using a rubber policeman. The cell pellet was stored dry at –70°C until analyses.

Cells were lysed by incubating them for 30 min on ice in lysis buffer (50 mM Tris–HCl, 0.8% digitonin and 2 mM EDTA, pH 6.8). After centrifugation, the lysate was assayed for GST enzyme activity and subjected to immunoblot analysis.

Animal treatments
Six-week-old female C57BL/6J APCMin/+ mice were purchased from Jackson Laboratories (Bar Harbor, ME). After acclimation, the animals were housed and maintained according to the approved standards of Institutional Animal Care and Use Committee. NO-ASA was suspended (35 mg/ml, wt/v) in a solution of 0.5% carboxy methylcellulose (Sigma Chemical) immediately before treatment. Mice were divided into two groups of four animals each and treated via gavage either with vehicle or with NO-ASA 100 mg/kg/day. After 21 days of treatment, animals were killed. For preparation of mitochondria, microsomes and cytosol, the liver, kidneys and segments of the small intestine were removed rapidly. Liver and kidney were immediately perfused with cold NaCl (0.9 w/v) and homogenized in 3 mM Tris buffer (pH 7.2) containing 0.25 M sucrose and 1 mM EGTA. Hepatic and kidney mitochondria were isolated by the method of Johnson and Lardy (18) with modifications as described previously (19). The post-mitochondrial supernatant was centrifuged at 20 000x g for 20 min to remove light mitochondria and peroxisomes, and the resulting supernatant was centrifuged at 100 000x g for 60 min to collect microsomes and cytosol. Microsomes were further purified and characterized as previously described (20). The small intestine was homogenized in 50 mM Tris–HCl (pH 7.5) containing 1 mM DTT, 1 mM EDTA, 10 µM leupeptin, heparin 25 U/ml and 20% glycerol (v/v) (21). Protein concentration was determined using the Bradford assay.

Assay of NQO
In vitro NQO activity was assayed as described previously (22). Briefly, Hepa 1c1c7 (10 000 cells/well) or HT-29 (15 000 cells/well) were plated into 96 well plates and incubated overnight. Cells were then treated with various concentrations of NO-ASA. Cells were lysed in 50 µl 0.8% digitonin, after which we added to each well 200 µl of reaction mixture [25 mM Tris–HCl buffer (pH 7.4), 0.5% bovine serum albumin, 0.025 % Tween-20, 5 µM FAD, 30 uM NADP, 1 µM glucose-6-phosphate, 2 U/ml glucose-6-phosphate dehydrogenase baker's yeast (Sigma), 0.3 mg/ml 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrozolium bromide (MTT)] containing 25 µM menadion. The reaction was arrested after 5 min by adding 50 µl of solution containing 0.3 mM dicoumarol in 0.5% DMSO and 5 mM potassium phosphate (pH 7.4). Initial reaction rates were measured at 610 nm. Protein concentration was measured using crystal violet staining in an identical 96-well plate treated in parallel (22). Briefly, after treatment, the medium was removed and cells were stained with 0.5% crystal violet red for 10 min. Cells were then dissolved in 0.5% SDS in 50% ETOH for 1 h. The 610 nm absorbance was measured using a plate reader.

In vivo NQO activity was carried out by adding the cytosolic fractions into the above reaction mixture (final volume 1 ml) initiating the reaction. After 5 min, the absorbance at 610 nm was measured.

GST assay
The activity of GST was determined spectrophotometrically at 340 nm at 30°C with 1-chloro-2,4-dinitrobenzene (CDNB) as substrate according to the procedure of Habig et al. (23). One milliliter of 0.1 M sodium phosphate (pH 6.5) reaction mixture contained 50 µl 20 mM GSH, 50 µl of 20 mM CDNB and ~30–50 µg of cell lysate, and animal tissue fractions. The initial reaction rate was measured at 340 nm at 30 s.

GSH determination
Cellular GSH levels were determined using the glutathione reductase-coupled 5,5'-dithiobis 2-nitrobenzoic acid (DTNB) enzyme-linked immunosorbent assay (ELISA) in 96-well plates (24).

Immunoblotting
Immunoblotting was performed following standard protocols. Proteins were probed with primary antibody and visualized using an ECL kit according to the manufacturer's instructions (Amersham Pittsburg, PA). Primary antibodies were from the following sources (the final concentrations used are shown in parentheses): Goat polyclonal antibodies against NQO (1:500), rabbit polyclonal antibodies against Nrf2 (1:1000) and P450 1A1 (1:1000) were from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit polyclonal antibodies to GST P1-1 (1:1000) and GST A1-1 (1:1000) were from BD Transduction Laboratories, Caribbean, CA. The mouse monoclonal antibody against {alpha}-tubulin (1:1500) was from Oncogene Research Products (San Diego, CA).

Immunofluorescence
HT-29 cells were grown on coverslips and allowed to attach overnight. Cells were then exposed to DMSO or NO-ASA for 24 h, washed with PBS and fixed with methanol for 30 min. After blocking in 2% BSA for 15 min, cells were treated for 2 h with anti-Nrf2 antibody (1 mg/ml). After washing, cells were treated with fluorescein isothiocyanate (FITC) second antibody for 1 h at the room temperature. Slides were mounted and examined under a fluorescence microscope at 100 x magnification.

NO measurements
After 24 h treatment of cultured cells with NO-ASA, the medium was collected and stored at –20°C until assayed. Nitrite levels were quantified using an NO selective microelectrode (amiNO-700, Innovative Instruments, Queen Brooks Court, FL) and an amplifier (inNO meter, Innovative Instruments) following the instructions of the manufacturer. Addition of the sample to a 100 mM H2SO4 solution in the presence of NaI converted the nitrite in the medium to NO, which was measured by the electrode using NaNO2 as standard.

Statistics
The results we expressed as mean ± SEM and differences were compared using Student's t-test.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
NO-ASA induces phase II enzyme activity in HT-29 human colon cancer and Hepa 1c1c7 murine hepatoma cells
We examined the effect of NO-ASA on the activity of NQO and GST in mouse hepatoma Hepa 1c1c7 cells, which have been the model cell line for the study of phase II enzymes (25) and in HT-29 human colon cancer cells, which also express both NQO and GST (26,27).

Cells were plated as described in Materials and methods and treated with various concentrations of NO-ASA or equimolar concentrations of conventional ASA for 24 h, when the activity of both NQO and GST was determined. As shown in Figure 1, NO-ASA induces the activity of NQO and GST in these two cell lines in a concentration-dependent manner. In both instances, the induction of NQO is quantitatively more pronounced than that of GST. For example, treatment of Hepa 1c1c7 cells with 100 µM NO-ASA resulted in >2-fold induction of NQO activity but only ~1.3-fold induction of GST activity. Of note, at each concentration the effect of NO-ASA on Hepa 1c1c7 cells was more pronounced than that on HT-29 cells. ASA has no effect on either NQO or GST activity, even at the highest concentration studied (400 µM, not shown) concentration.


Figure 1
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  		Fig. 1. The effect of NO-ASA on the activity of NQO and GST in cultured cells. HT-29 and Hepa 1c1c7 cells were grown in the presence of various NO-ASA and ASA concentrations for 24 h. Cells were harvested and assayed for NQO (A) and GST (B) activity as described under Materials and methods. NQO and GST activities were not determined in Hepa 1c1c7 cells treated with 200 µM NO-ASA, as most cells were dead.

 
Treatment of HT-29 cells with 50 µM NO-ASA for 24 h increased significantly (2-fold) the intracellular GSH levels, but there was no change at NO-ASA concentrations of ≤5 µM. This finding suggests that NO-ASA, at the higher concentration, may have induced {gamma}-glutamylcystein synthetase, the rate-limiting enzyme of GSH biosynthesis (28). Conventional ASA at equimolar concentrations had no effect on GSH levels under our experimental protocol.

Induction of phase II enzyme activity is accompanied by an increase of their protein levels
In order to determine whether changes in NQO and GST levels were responsible for their increased enzymatic activity, we determined by immunoblotting the cellular levels of NQO1 and GST P1-1 in response to NO-ASA. These two isoforms are expressed in both cell lines and are easily assayable with existing antibodies. NO-ASA increased the levels of NQO1 and GST P1-1 in both HT-29 and Hepa1c1c7 cells in a concentration-dependent manner. In contrast, ASA did not affect the levels of these two proteins even at 400 µM (data not shown). It is of interest that the degree of increase in protein expression reflects the respective changes in catalytic activity. For example, NQO1 is induced more in liver than colon cells, whereas the difference in induction of GST between these two cell lines is less pronounced; as already mentioned, NQO (but not GST) is more active in the liver cells compared with colon cells.

Many phase II enzyme inducers also induce phase I enzymes (16,17). Thus we examined the effect of NO-ASA on the levels of P450 1A1, an important phase I enzyme in mammalian livers. As shown in Figure 2, NO-ASA induced the expression of P450 1A1 only slightly in Hepa 1c1c7 cells.


Figure 2
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  		Fig. 2. The effect of NO-ASA on NQO1, GST P1-1 and P450 1A1. Hepa 1c1c7 (A) or HT-29 (B) cells were incubated with various concentrations of NO-ASA for 24 h. NQO1, GST P1-1 and P450 1A1 levels were determined by immunoblotting in cell extracts. {alpha}-tubulin is the loading control.

 
Induction of phase II enzymes by NO-ASA is related to NO release
To evaluate whether induction of phase II enzymes by NO-ASA is dependent on NO, we determined the amount of NO released from NO-ASA. In HT-29 cells treated with NO-ASA for 18 h, the release of NO was paralleled by the induction of NQO1; conventional ASA did not release any NO (as expected) and the expression of NQO1 was not altered (Figure 3A). We next determined whether NO is responsible for NQO1's induction. Pretreatment with PTIO, a specific NO scavenger, essentially abrogated the induction of NQO activity by NO-ASA (Figure 3B). Another NO scavenger, hemoglobin, also decreased the induction of NQO by NO-ASA (data not shown). These results indicate that the NO released from NO-ASA is, at least in part, responsible for the induction of this phase II enzyme.


Figure 3
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  		Fig. 3. Phase II enzyme induction by NO-ASA is dependent on NO release. (A) HT-29 cells were treated with NO-ASA 200 µM or ASA 200 µM for 24 h or with solvent only (Con). Nitrite levels in culture medium were determined using an NO-sensing electrode following conversion of nitrite to NO. NQO1 expression also was determined in lysates of these cells. (B) HT-29 cells, pretreated with and without the NO scavenger PTIO 50 µM, were treated with various concentration of NO-ASA (0, 100 and 200 µM) for 24 h. Cells were harvested and assayed for NQO activity as described under Materials and methods. (C) Hepa 1c1c7 cells were treated for 24 h with the exogenous NO donor SNAP, or SNAP plus ASA 100 µM or NO-ASA 100 µM. NQO was assayed in lysates of these cells as described under Materials and methods.

 
To further explore this observation, we determined the effect of SNAP, a widely used NO donor on NQO1 expression. Treatment of Hepa 1c1c7 cells with various concentrations of SNAP for 24 h increased the activity of NQO in a concentration-dependent manner (Figure 3C), with SNAP 400 µM increasing the activity of NQO >3-fold over control. Induction of phase II enzymes by NO in other cell lines has been recently reported (29). We treated Hepa 1c1c7 cells either with NO-ASA 100 µM or ASA 100 µM, or combinations of 100 µM ASA with SNAP (50–400 µM). As shown in Figure 3C, the presence of ASA did not alter the effect of SNAP, suggesting that the ASA moiety of NO-ASA had very little effect on the induction of phase II enzymes by NO-ASA.

Induction of phase II enzymes by NO-ASA employs the Nrf2-dependent pathway
The Nrf2 transcription factor is normally largely bound in the cytoplasm to Keap1. When inducers disrupt its complex with Keap1, Nrf2 translocates to the nucleus where it accelerates the transcription of phase II genes. To determine whether NO-ASA indeed induces phase II enzymes by modulating Nrf2, we assessed the cellular distribution of Nrf2 after treatment with NO-ASA. Immunofluorescence analysis revealed that control cells showed both cytoplasmic and nuclear staining of Nrf2, whereas NO-ASA-treated cells showed increased nuclear staining, which increased progressively with increasing NO-ASA concentrations (Figure 4A). Immunoblotting also indicated that the nuclear level of Nrf2 was elevated in NO-ASA-treated cells. ASA had no effect on the cellular distribution of Nrf2.


Figure 4
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  		Fig. 4. The effect of NO-ASA on Nrf2 nuclear translocation. HT-29 cells grown on coverslips were treated with NO-ASA for 24 h and the cellular localization of Nrf2 was studied by immunofluorescence. (A) untreated control; (B) 100 µM NO-ASA; (C) 200 µM NO-ASA, causing almost complete nuclear translocation of Nrf2. (D) Immunoblot of nuclear lysates from HT-29 cells treated with various concentrations of NO-ASA or ASA 200 µM for 24 h. NO-ASA induces nuclear accumulation of the Nrf2 protein. Sam 68, a nuclear protein, represents a loading control.

 
NO-ASA induces the activity of NQO and GSTs in Min mice
To determine whether NO-ASA induces phase II enzymes in vivo, we measured its effect on both the activity and expression of NQO and GST in Min mice. Compared with controls, after 3 weeks of treatment NO-ASA significantly induced the activity of GST (66% increase; P < 0.005) and NQO (50% increase; P < 0.05) in the liver (Table I). Similarly, NO-ASA increased significantly (but to a lesser extent) the activity of GST (36% increase; P < 0.01) and NQO (30% increase; P < 0.005) in the small intestine. In contrast, the increases in the activity of these two enzymes in the kidney did not reach statistical significance.


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  		Table I. NQO and GST activity in tissues of Min mice

 
Immunoblot analysis (Figure 5) demonstrated that NO-ASA significantly increased the liver cytosolic GST P1-1 and NQO1 levels. The levels of two phase I enzymes, P450 1A1 and cytochrome P450 2E1, did not differ between the untreated and treated groups. This finding indicates that in vivo NO-ASA is a monofunctional phase II inducer.


Figure 5
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  		Fig. 5. Effect of NO-ASA on the expression of murine hepatic detoxification enzymes. Upper panels: Liver cytosol or microsome fractions from Min mice treated with NO-ASA for 21 days were subjected to immunoblotting for the proteins shown here. Each lane represents one animal. Lower panel: The expression of each protein (fold over control) for each group of animals (mean ± SEM, N = 4). *, P < 0.05; **, P < 0.01; ***, P < 0.005, compared with the corresponding control; all other differences are not statistically significant.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
Our findings establish that NO-ASA induces phase II enzymes both in cultured cells and in an animal model of colon cancer. This induction is probably brought about, at least in part, through its effect on the Keap1–Nrf2 complex and parallels the release of NO by NO-ASA (Figure 6). Conventional ASA at equimolar concentrations failed to generate similar effects. Of the two classes of xenobiotic metabolizing enzymes, the effect of NO-ASA appears restricted to phase II enzymes; in cultured cells and in mice, NO-ASA had only a marginal effect on P450 1A1 and P450 2E1, two phase I enzymes.


Figure 6
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  		Fig. 6. Proposed mechanism for the induction of phase II enzymes by NO-ASA. Biotransformation of NO-ASA by the cell ultimately generates NO (38), which may modify Keap1 (via S-nitrosylation?) thus disrupting the Keap1–Nrf2 complex. This is followed by nuclear translocation of the transcription factor Nrf2, which in turn interacts with the ARE in the promoter region of phase II detoxifying enzymes inducing their expression.

 
We studied the effect of NO-ASA in cultured Hepa 1c1c7 murine hepatoma cells and in HT-29 human colon cancer cells; they have been used extensively to study the biology of drug metabolizing enzymes and of colon cancer, respectively. These two cell lines also represent two organs relevant to the chemoprevention of colon cancer: the liver, the major organ for drug metabolizing activities where carcinogens can be (in)activated; and the colon, the target organ of NO-ASA's effect. Of the many phase II enzymes, we focused on NQO and GST, two biologically significant members of the phase II enzyme superfamily; each is present as several isoforms and both are potentially important in mediating the effects of chemoprevention agents.

Our data establish that NO-ASA has a dual effect on these phase II enzymes: it induces in a concentration-dependent manner both their intracellular levels and also their catalytic activity. Although the effect on these two enzymes in cultured cells was quantitatively more pronounced for NQO, compared with GST, the differences between the two are generally small (and just the reverse in the liver and intestine of Min mice). The degree of induction of protein levels and of enhancement of catalytic activities is similar for both NQO and GST. However, it is difficult to ascribe the enhanced activity exclusively to the increase in the amount of the enzyme, particularly since our antibody was specific for enzyme isoforms and the assay for enzymatic activity was not isoform-specific.

That our findings with cultured cells may be biologically relevant is suggested by the observation that Min mice showed similar changes in NQO and GST. Interestingly, this was not an ‘acute’ effect, as it was observed following a 3 week administration of the drug. Of note, NO-ASA had no effect on the kidneys of these mice, suggesting a degree of tissue specificity, although the size of our study groups precludes a firm conclusion on this.

Three aspects of the mechanism by which NO-ASA induces phase II enzymes merit discussion. First, NO, the molecule that defines this drug, appears involved in this effect. There are two supporting lines of evidence: (i) the release of NO from NO-ASA was paralleled by the induction of NQO1 and this effect was abrogated by NO scavengers; and (ii) an exogenous NO donor (SNAP) also induced the expression of NQO1, consistent with findings by others indicating the ability of NO to induce phase II enzymes (29). Second, NO-ASA appears to induce the expression of phase II genes by modulating the Keap1–Nrf2 complex. This is evidenced by the concentration-dependent translocation of Nrf2 into the nucleus following treatment with NO-ASA. Of note, the translocation of Nrf2 parallels the induction of NQO1 and GST P1-1. Although our data do not conclusively prove that this is the mechanism of action of NO-ASA, they are, nevertheless, consistent with it (15). We can only speculate on the modification of Keap1 that leads to the dissociation of Nrf2; S-nitrosylation of Keap1 by NO-ASA is a distinct possibility (30). And, third, equimolar concentrations of conventional ASA, the prototypical colon cancer chemoprevention agent (31), failed to induce phase II enzymes. Our findings do not, however, preclude such a possibility at high drug concentrations. In general, ASA is known to differ in terms of pharmacological potency from NO-ASA, often requiring much higher concentrations to achieve the same effect as NO-ASA (1).

The biological significance of our findings cannot be deduced with certainty from the present data. In general, there is a dichotomy in the activity of these enzymes; they can mediate cancer chemoprevention, or they can activate carcinogens or even be part of the mechanism of drug resistance (12,15,32). NQO1 detoxifies carcinogenic compounds and prevents the generation of oxygen radicals. For example, it catalyzes two-electron transfer reactions to quinones (33,34), thus bypassing the formation of semiquinones and, subsequently, of superoxide anion radicals (35). In general, NQO enzymes provide a cellular detoxifying system and are considered protective. It should be kept in mind, however, that NQO1 can activate important mutagens and carcinogens (36) and may accelerate the formation of reactive oxygen species through the semiquinone pathway. The end result often depends on biological context, such as tissue type or route of administration. A similar argument can be made for GST (13,14). Given the abundant evidence that NO-ASA is chemopreventive against colon cancer (3,4,37), it is reasonable to speculate that the induction of phase II enzymes by NO-ASA is part of its mechanism of chemoprevention.

In conclusion, our data establish that NO-ASA induces phase II enzymes, probably, at least in part, through the action of the NO that it releases and by modulating the Keap1–Nrf2 pathway. It is possible that this effect is part of its mechanism of action against colon and other cancers. Further work is required to provide additional details on the mechanism of these effects and an assessment of the relative contribution of the individual changes that we have described.


   Acknowledgments
 
This work was supported by the NIH Grant CA92423 and PSC-CUNY Grant 65201-00 34.

Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

  1. Rigas,B. and Kashfi,K. (2004) Nitric-oxide-donating NSAIDs as agents for cancer prevention. Trends Mol. Med., 10, 324–330.[CrossRef][ISI][Medline]
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Received September 14, 2005; revised October 28, 2005; accepted October 28, 2005.


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M.G. Luciani, C. Campregher, and C. Gasche
Aspirin blocks proliferation in colon cells by inducing a G1 arrest and apoptosis through activation of the checkpoint kinase ATM
Carcinogenesis, October 1, 2007; 28(10): 2207 - 2217.
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