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Nitric Oxide-releasing Nonsteroidal Anti-inflammatory Drugs (NSAIDs) Alter the Kinetics of Human Colon Cancer Cell Lines More Effectively than Traditional NSAIDs

Implications for Colon Cancer Chemoprevention¹

American Health Foundation, Valhalla, New York 06595 [J. L. W., B. R.]; Division of Gastroenterology [J. L. W., I. H., E. C., B. R.] and Brader Cancer Canter [F. T.], New York Medical College, Valhalla, New York 06595; and Strang Cancer Prevention Center [S. B.] and Rockefeller University [B. R.], New York, New York 10021

	ABSTRACT

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Nitric oxide-releasing nonsteroidal anti-inflammatory drugs (NO-NSAIDs), consisting of a known nonsteroidal anti-inflammatory drug (NSAID) and a nitric oxide (NO)-releasing group, are reported safer than NSAIDs. To assess their potential in colon cancer chemoprevention, we studied in vitro the effect of NO-aspirin, NO-sulindac, and NO-ibuprofen on colonocyte kinetics. These three NO-NSAIDs reduced the growth of cultured HT-29 colon adenocarcinoma cells much more effectively than the corresponding NSAIDs; e.g., at 24 h, their IC₅₀ values were as follows: (a) aspirin, >5000 µM; (b) NO-aspirin, 1 µM; (c) sulindac, 750 µM; (d) NO-sulindac, 150 µM; (e) ibuprofen, >1000 µM; and (f) NO-ibuprofen, 42 µM. This effect was due to inhibition of proliferation and induction of apoptosis and perhaps to the induction of novel cell changes, characterized by extensive DNA degradation. NO-NSAIDs also blocked the G₀-G₁ to S cell cycle transition. Their superior effectiveness compared with traditional NSAIDs, combined with their reported safety, makes them promising candidates for chemopreventive agents against colon cancer.

	Introduction

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Ample evidence supports the notion that chemoprevention has the potential to be a major component of the control of colorectal cancer, one of the most common human malignancies in the Western world (1) . NSAIDs³ are prototypical chemopreventive agents against colorectal cancer (reviewed in Refs. 2 and 3 ), but side effects are a major obstacle to their large-scale application to prevent colorectal cancer. NSAID toxicity includes (a) GI side effects, which range from dyspepsia to GI bleeding, obstruction, and perforation; (b) renal side effects, including a wide range of tubular, interstitial, glomerular, and vascular lesions; and (c) a large number of additional side effects, some of them serious, ranging from hypersensitivity reactions to the distinct salicylate intoxication (summarized in Ref. 4 ). Among patients using NSAIDs, up to 4% per year suffer serious GI complications; in 1998, the number of deaths in the United States from NSAID-induced GI complications was 16,550, virtually identical to the 16,685 deaths from AIDS (5 , 6) . The synthesis of NO-NSAIDs represents a novel approach to reduce the side effects of NSAIDs, especially their gastric toxicity (7, 8, 9) . NO-NSAIDs consist of a known NSAID molecule and a NO-releasing group (typically -NO₂) linked to it via a chemical spacer (Fig. 1) . The rationale for their development was based on the observation that NO possesses some of the same properties as PGs within the gastric mucosa. NO increases mucosal blood flow, mucous release, and repair of the mucosa, whereas it inhibits neutrophil activation and adherence. These effects can theoretically compensate for gastric PG reduction. Coupling a NO-releasing moiety to a NSAID might deliver NO to the site of NSAID-induced damage and thus decrease gastric toxicity. Existing data, mostly from animal studies, indicate that this prediction may be true (7, 8, 9) .

View larger version (12K): [in this window] [in a new window] [Download PPT slide]   Fig. 1. The chemical structure of NO-aspirin. The three structural components of this novel derivative of aspirin (designated NCX 4040) are indicated: the spacer molecule links the traditional NSAID to -NO2, which can release NO. The structure of traditional aspirin is also shown for comparison.

	Materials and Methods

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Cell Lines.
HT-29 and HCT-15 human colon adenocarcinoma cell lines (American Type Culture Collection, Manassas, VA) were grown as monolayers in McCoy 5A medium and RPMI 1640, respectively, and supplemented with 10% FCS (Mediatech, Herndon, VA), penicillin (50 units/ml), and streptomycin (50 µg/ml; Life Technologies, Inc., Grand Island, NY). Cells were seeded at a density of 1.5 x 10⁶ cells/100-cm² culture dish and incubated at 37°C in 5% CO₂ and 90% relative humidity. Single-cell suspensions were obtained by trypsinization (0.05% trypsin/EDTA), and cells were counted using a hemacytometer. Viability was determined by the trypan blue dye exclusion method.

Reagents.
NO-sulindac (NCX1102; (Z)-5-fluoro-2-methyl-1-{[4-(methylsulfinyl)phenyl]methylene}-1H-indene-3-acetic acid 4-(nitrooxy)butyl ester), NO-ibuprofen (NCX2210; trans-3-{4-[-methyl-4-(2-methylpropyl)benzeneacetylpxy]-3-methoxyphenyl}-2-propenoic acid 4-nitrooxy)butyl ester), and NO-aspirin (NCX4040; 2-(acetyloxy)benzoic acid 4-(nitrooxy methyl)phenyl ester) were gifts of Dr. P. Del Soldato (Nicox, SA, France). Stock (100 mM) solutions of NO-NSAIDs and NSAIDs were prepared in DMSO (Fisher Scientific, Fair Lawn, NJ). All compounds were added to the culture medium immediately before plating. Final DMSO concentration was adjusted in all media to 1%.

Flow Cytometry.
Cell cycle phase distributions of control and treated colon cancer cell lines were obtained using a Coulter Profile XL equipped with a single argon ion laser. For each subset, we analyzed 10,000 events. All parameters were collected in listmode files. Data were analyzed on an XL Elite Work station (Coulter) using the software programs Multigraph and Multicycle.

Cell Proliferation and Cell Cycle Analysis.
Cells (0.5 x 10⁶) were fixed in 100% methanol for 10 min at -20°C, pelleted (5000 rpm for 10 min at 4°C), resuspended, and incubated in PBS containing 1% FBS/0.5% NP40 on ice for 5 min. Cells were washed twice in PBS/1% FBS, pelleted, and resuspended in 50 µl of a 1:10 dilution of the anti-PCNA primary antibody (PC-10; all antibodies were from Santa Cruz Biotechnology, Santa Cruz, CA) in PBS/1% FBS for 60 min at room temperature. Nonspecific IgG1/IgG2 was used as an isotypic control. Cells were then washed and incubated with goat antimouse phycoerythrin antibody (diluted 1:50) for 60 min at room temperature in the dark. Cells were washed again in 500 µl of PBS/1% FBS containing 40 µg/ml propidium iodide and 200 µg/ml RNase type IIA and analyzed within 30 min by flow cytometry. The percentage of cells in G₀-G₁, G₂-M, and S phases was determined from DNA content histograms.

Assays for Apoptosis.
The induction of apoptosis was determined by the presence of a subdiploid (sub-G₀-G₁) peak in DNA content histograms obtained by flow cytometry as described above and by fluorescence microscopy of cells stained with DAPI (Accurate Chemical, Westbury, NY). For each sample, at least five fields were examined. The morphological criteria used to identify apoptosis included cytoplasmic and nuclear shrinkage, chromatin condensation, and cytoplasmic blebbing with maintenance of the integrity of the cell membrane.

	Results

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NO-NSAIDs Reduce the Number of Cultured Colon Cancer Cells More Effectively than Their Parent Compounds.
Compared with their parent NSAIDs, all three NO-NSAIDs reduced the number of HT-29 cells in culture at lower concentrations and after shorter incubation periods (Table 1 ; Fig. 2 ). Remarkably, even at the maximal concentrations used, ASA and ibuprofen failed to reduce the number of cells by half, whereas their NO-derivatives reduced the cell number to near zero by 24 h. Indeed, the ratio of the IC₅₀ for ASA compared with that of NO-ASA (ASA:NO-ASA) ranged between 1270 and >5000 over the 72 h of observation; for ibuprofen:NO-ibuprofen, it was >42, and for sulindac:NO-sulindac, it ranged between 5 and 20. Similar ratios were obtained with HCT-15 cells (data not shown).

View larger version (31K): [in this window] [in a new window] [Download PPT slide]   Fig. 2. The effect of NO-NSAIDs on HT-29 cell growth. HT-29 cells were treated with various concentrations of NO-NSAIDs or their corresponding traditional NSAIDs as described in "Materials and Methods." Cell number was determined at 24, 48, and 72 h.

Microscopic examination of DAPI-stained cells revealed that, compared with controls, cultures treated with NO-NSAIDs had an increased number of apoptotic cells, as defined by standard morphological criteria such as cytoplasmic and nuclear shrinkage or chromatin condensation (Table 2 ; Fig. 3 ). For example, exposure of cells to 100 µM NO-ASA for 48 h increased the frequency of apoptotic cells from 2% in controls to 46%; 1 µM NO-ASA had no such effect. The other two compounds had a similar but less pronounced effect on apoptosis: 48 h of exposure to 100 µM NO-ibuprofen resulted in 19% apoptotic cells, whereas NO-sulindac under identical culture conditions increased apoptosis to 34%.

View larger version (45K): [in this window] [in a new window] [Download PPT slide]   Fig. 3. The effect of NO-aspirin on apoptosis in HT-29 cells. A, control samples (no NO-NSAID treatment) showed rare apoptotic cells. Samples treated for 72 h with 200 µM NO-sulindac (B), 10 µM NO-aspirin (C), or 100 µM NO-ibuprofen (D) show classical apoptotic cells (cytoplasmic and nuclear shrinkage; chromatin condensation). In addition, especially in D, there are cells that maintain their basic structure but show diminished to no detectable amounts of DNA without DNA condensation or cytoplasmic shrinkage (atypical cells in Table 2 ).

As shown in Table 2 , all three NO-NSAIDs also induced apoptosis in HT-29 cells when apoptosis was determined by assaying the proportion of cells with a subdiploid DNA content. For example, exposure to 10 µM NO-ASA resulted in 20% apoptotic cells. Indeed, at that concentration, the effect already appeared maximal, being around 10-fold higher than control. The relative efficacy of these compounds in inducing apoptosis can be assessed by determining the frequency of apoptotic cells after treatment of HT-29 cells with equimolar concentrations of these compounds for the same period of time. Thus, after exposure to 100 µM of each NO-NSAID for 48 h, the increase in the percentage of apoptotic cells over control was 8.7-fold for NO-ASA, 4.6-fold for NO-ibuprofen, and 1.9-fold for NO-sulindac. As expected (10 , 11) , the percentage of apoptotic cells was higher when apoptosis was determined by morphological criteria rather than by cell DNA content, although the changes in apoptosis detected by these two methods were always concordant.

NO-NSAIDs Block Cell Cycle Transitions.
All three NO-NSAIDs significantly altered the cell cycle distribution of HT-29 cells, inducing a block in the G₀-G₁ to S transition. This was evident by the increased percentage of cells in the G₀-G₁ phase that was accompanied by corresponding reductions of the proportion of cells in S and G₂-M phases. This effect was concentration dependent. NO-ASA was the most effective of the three in inducing these changes. This is evident, for example, when equimolar concentrations of the three compounds are compared, e.g., exposure to 100 µM for 48 h. NO-ASA increased the G₀-G₁ fraction by 57% over control versus no change or a 31% increase after exposure to NO-ibuprofen and NO-sulindac, respectively. The corresponding changes for the S-phase were a 61% reduction for NO-ASA, no change for NO-ibuprofen, and a 60% reduction for NO-sulindac. For G₂-M, there was a 78% reduction for NO-ASA and virtually no change for the other two.

	Discussion

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Our data demonstrate that three members of the family of the novel NO-NSAIDs inhibit cell growth much more effectively than their parent NSAIDs; their superior effectiveness reached a very high level in the case of NO-ASA.

NO-NSAIDs inhibited cell growth through an effect on cell proliferation, apoptosis, or both. Although it is difficult to accurately determine their individual contribution to these complex kinetic changes, our data provide some indication of the relative magnitude of these effects. The case of NO-ASA illustrates this point. Compared with controls, exposure to 1 µM NO-ASA for 48 h reduced the cell number by 11% and PCNA expression by 17%, whereas there was no detectable induction of apoptosis and no change in the cell cycle phase distribution of these cells. Thus, it appears that at this concentration of NO-ASA, there is only a minor antiproliferative effect. In contrast, exposure of colonic tumor cells to 10 µM NO-ASA reduced the number of cells by 83% and PCNA expression by 25% and increased apoptosis at least 10-fold, not taking into account the atypical cells. This treatment also increased the percentage of cells that accumulated in the G₀-G₁ phase. Therefore, at this concentration, the predominant effect of NO-ASA is to induce apoptosis.

The induction of the atypical cells in response to NO-NSAIDs was intriguing. This phenomenon, which is currently under investigation, requires further assessment. It is important to note, however, that traditional NSAIDs, even at high concentrations, failed to induce this morphologically unique cell type. It is reasonable to speculate that cellular forms devoid of apparent DNA, akin to platelets, have a shortened life span, which may contribute in a unique way to the dramatic effect of NO-NSAIDs on cell kinetics.

All three NO-NSAIDs were superior to their parent NSAIDs in affecting the parameters we studied. However, they differed substantially among themselves when compared on an equimolar basis; NO-ASA was clearly the most effective in all respects. Given the structural complexity of these molecules and the paucity of information on the biological role of each of their structural components (traditional NSAID, spacer, -NO₂), it is difficult to deduce plausible structure-activity correlates that could account for such pronounced differences. Remarkably, of the three parent NSAIDs, ASA has the highest IC₅₀, followed by ibuprofen and sulindac. However, of the NO derivatives, NO-ASA has by far the lowest IC₅₀.

It is unclear at this point what accounts for the enhanced effectiveness of NO-NSAIDs compared with their NSAID counterparts. The magnitude of their enhanced activity, especially that seen with NO-ASA, is difficult to ascribe to simple changes in the physical properties of each NSAID. The spacer part of the molecule and the -NO₂ group, which releases NO, must contribute to this effect. Our preliminary data indicate that these compounds do not block PG synthesis, at least at concentrations that inhibit cell growth. Combined with their effect on HCT-15 cells that lack both COX isozymes (12) , this suggests that the effect of NO-NSAIDs does not necessarily involve inhibition of COX, the classical target of traditional NSAIDs and of the selective COX-2 inhibitors. Rather, it appears likely that NO-NSAIDs act on targets beyond COX and may use novel modes of action. Such a notion has been considered for traditional NSAIDs as well (13) .

An important biological question is whether NO-NSAIDs maintain their superior performance in vivo. Although it is difficult to extrapolate results from cultured cells to animal systems and humans, a recent study suggested that NO-NSAIDs may be more effective in vivo than traditional NSAIDs. The chemopreventive effect of aspirin versus a NO-ASA derivative (NCX 4016; a positional isomer of the one reported here) was assessed using a rat azoxymethane model of colon cancer (14) . Whereas ASA reduced the number of aberrant crypt foci, the putative preneoplastic lesions of the colon, by 64%; NO-ASA reduced them even more (85%). Further work, some of it already in progress, will assess this critical question. Of interest, it was recently reported that low-dose ASA (<150 mg) used for cardiac prophylaxis failed to protect patients from colon cancer (15) . NO-NSAIDs, with their potential for enhanced effectiveness against colon cancer, may provide double protection against coronary artery disease and colon cancer.

In conclusion, our data demonstrate that a novel class of NSAID derivatives, the NO-NSAIDs, which promise to be less toxic than traditional NSAIDs, affects colon cancer cell kinetics in vitro in a way consistent with a chemopreventive effect. In this respect, NO-NSAIDS are severalfold more powerful than traditional NSAIDs. Their potential role in human colon cancer prevention remains to be established.

	ACKNOWLEDGMENTS

 
We thank Dr. Z. Darzynkiewicz for helpful discussions, Jan Kunicki for assistance with flow cytometry, and Dr. P. Del Soldato (Nicox, SA, Nice, France) for providing the NO-NSAIDs and for helpful discussions.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Supported by a grant from the I. Weinstein Foundation.

² To whom requests for reprints should be addressed, at American Health Foundation, One Dana Road, Valhalla, NY 06595. Phone: (914) 789-7295; Fax: (914) 592-6317.

³ The abbreviations used are: NSAID, nonsteroidal anti-inflammatory drug; NO, nitric oxide; NO-NSAID, NO-releasing NSAID; DAPI, 4',6-diamidino-2-phenylindole; GI, gastrointestinal; FBS, fetal bovine serum; PCNA, proliferating cell nuclear antigen; PG, prostaglandin; COX, cyclooxygenase; ASA, aspirin.

Received 2/12/01. Accepted 2/23/01.

	REFERENCES

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