Inhibitory effects of mofezolac, a cyclooxygenase-1 selective inhibitor, on intestinal carcinogenesis
Tomohiro Kitamura1,
Toshihiko Kawamori1,
Naoaki Uchiya1,
Masaki Itoh2,3,
Tetsuo Noda2,4,
Mamoru Matsuura5,
Takashi Sugimura1 and
Keiji Wakabayashi1,6
1 Cancer Prevention Division, National Cancer Center Research Institute, 1-1 Tsukiji 5-chome, Chuo-ku, Tokyo 104-0045, Japan,
2 Cell Biology Department, Cancer Institute, 37-1 Kami-Ikebukuro 1-chome, Toshima-ku, Tokyo 170-8455, Japan,
3 Oncology Department, Jikei University School of Medicine, 25-8 Nishi-shinbashi 3-chome, Minato-ku, Tokyo 105-8461, Japan,
4 The Core Research for Evolutional Science and Technology Program, Japan Science and Technology Corporation, 4-1-8 Motomachi, Kawaguchi 332-0012, Japan and
5 Research Laboratory III, Mitsubishi Pharma Corporation, 1000 Kamoshida-cho, Aoba-ku, Yokohama, Kangawawa 2270033, Japan
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Abstract
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Cyclooxygenase (COX)-2, one enzyme isoform responsible for producing prostanoids from arachidonic acid, contributes to colon carcinogenesis. Recently, genetic disruption of COX-1, the other isoform, was shown to decrease the number of intestinal polyps and prostaglandin E2 levels in intestinal mucosa, like the case with COX-2 gene disruption, in Min mice. We therefore investigated whether a COX-1 selective inhibitor, mofezolac, suppresses intestinal carcinogenesis in rodents. F344 male rats, receiving azoxymethane (AOM, 15 mg/kg body wt) s.c. injections at 5 and 6 weeks of age, were fed a diet containing 600 or 1200 p.p.m. mofezolac for 4 weeks. The number of aberrant crypt foci (ACFs) per rat and the bromodeoxyuridine labeling index of the crypt epithelium were dose-dependently decreased by administration of mofezolac, the value for the former at 1200 p.p.m. being 60% of control value. When Apc gene knockout mice (APC1309 mice) were given 600 or 1200 p.p.m. mofezolac in their diet for 8 weeks, the numbers of intestinal polyps were also dose-dependently decreased, with reduction to 59% of that in the control diet group at the higher dose. Nimesulide, a COX-2 selective inhibitor used as positive control, showed similar suppressive effects on the development of ACFs in AOM-treated rats and polyps in Apc gene knockout mice. The data indicate that both COX-1 and COX-2 can contribute to intestinal tumorigenesis.
Abbreviations: AOM, azoxymethane; ACs, aberrant crypts; ACFs, aberrant crypt foci; Apc, adenomatous polyposis coli; BrdU, bromodeoxyuridine; COX, cyclooxygenase; FAP, familial adenomatous polyposis; NSAIDs, non-steroidal anti-inflammatory drugs; PGE2, prostaglandin E2
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Introduction
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Accumulating evidence indicates that the mortality rate from colorectal cancer in man and the development of chemically induced colon cancers in experimental animals, can be suppressed by non-steroidal anti-inflammatory drugs (NSAIDs) (1). NSAID administration has also been found to decrease the appearance of intestinal polyps in familial adenomatous polyposis (FAP) patients and in an animal model of the disease, mutant mice with a truncated adenomatous polyposis coli (Apc) gene (46). NSAIDs exert inhibitory effects on the activity of cyclooxygenase (COX), which is the rate-limiting enzyme in the conversion of arachidonic acid to prostanoids. Two enzyme isoforms of COX, referred to as COX-1 and COX-2, have been identified. COX-1 is constitutively expressed in most tissues and plays a role in various physiological functions, while COX-2 is transiently inducible by stimuli such as cytokines, growth factors, hormones, etc., and contributes to inflammation and abnormal cell proliferation (2). COX-2 has also been reported to have a significant relevance to carcinogenesis in various organs, including the colon (36).
On the other hand, perhaps reflecting its ubiquitous housekeeping expression, the contribution of the COX-1 isoform to tumorigenesis has yet to be examined extensively. However, there is evidence suggesting possible involvement of COX-1 in colon carcinogenesis. Genetic disruption of the COX-1 gene, as well as the COX-2 gene, decreased the number of intestinal polyps in Min mice by
80% (7). Prostaglandin E2 (PGE2) levels have been reported to be increased in intestinal polyps compared with surrounding normal tissue, and both COX-1 and COX-2 were known to contribute to PGE2 production (7). Moreover, with a genetic and pharmacological approach, PGE2 was shown to be involved in intestinal carcinogenesis through its binding to the PGE2 receptor subtype EP1 (8) and/or EP2 (9).
In the light of these observations, the present study was designed to evaluate the effects of mofezolac, a COX-1 selective inhibitor, on the formation of azoxymethane (AOM)-induced colonic aberrant crypt foci (ACFs), putative pre-neoplastic lesions (10) in F344 rats and also on intestinal polyp development in Apc gene knockout mice. Under the same experimental conditions, the inhibitory effects of a COX-2 selective inhibitor, nimesulide, on intestinal carcinogenesis were examined to allow comparison with those of the COX-1 selective inhibitor.
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Materials and methods
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Animals
Male F344/Du Crj rats, 4 weeks of age, were purchased from Charles River Japan (Atsugi, Japan). Progeny of APC1309 mice, produced by a gene knockout method and bred by artificial insemination, were genotyped by the allele-specific polymerase chain reaction using tail tips (11) and used at 6 weeks of age. The rats and mice were quarantined for 1 week and then randomized into experimental and control groups. The animals were housed two or three to a plastic cage in a holding room controlled at 24 ± 2°C and a 55% relative humidity with a 12/12 h light/dark cycle. Water and basal diet (AIN-76A, Dyets, Bethlehem, PA) or basal diet containing test chemical were provided ad libitum during the experiments. The animals were weighed weekly throughout the experiment.
Chemicals
AOM and bromodeoxyuridine (BrdU) were purchased from Sigma Chemical (St Louis, MO). Mofezolac, [3,4-di(4-methoxyphenyl)-5-isoxazolyl]acetic acid (Mitsubishi Pharma, Chikujo-gun, Fukuoka, Japan), a COX-1 selective inhibitor, has been clinically used to control acute pain and inflammation from operation, injury, or odontectomy. The ratio of the mofezolac IC50 values for ovine COX-1 to COX-2 is reported to be 0.003:1 (12). Nimesulide, 4-nitro-2-phenoxymethanesulfonanilide, a COX-2 selective inhibitor, was kindly provided by Helsinn Healthcare SA (Pazzallo-Lugano, Switzerland). Experimental diets were prepared at least every 2 weeks by thoroughly mixing each compound into the basal diet. The chemicals were confirmed to be stable under the experimental conditions used in the present study.
Experimental methods
The rats received AOM at a dose of 15 mg/kg body wt or the vehicle (saline) subcutaneously once a week for 2 weeks from 5 weeks of age. Starting 1 day before the first dosing of AOM or vehicle, rats were fed control diet or experimental diet containing 600 or 1200 p.p.m. mofezolac throughout the experiment. In the vehicle-treated group, 1200 p.p.m. mofezolac was also given. A separate group of rats was similarly given a diet containing 400 p.p.m. nimesulide for the period of the experiment. Four weeks after the first dosing of AOM, all rats were necropsied 1 h after being injected i.p. with 50 mg BrdU/kg body wt. After laparotomy, the entire large intestine was resected, flushed with saline, slit open longitudinally from the cecum to the anus, placed between two pieces of filter paper and fixed in 10% neutral-buffered formalin. They were then stained with 0.5% methylene blue in saline, and ACFs were scored as described previously (13).
APC1309 heterozygous female mice, starting at 7 weeks of age, were fed control diet or experimental diet containing 600 or 1200 p.p.m. mofezolac throughout the experiment. For comparison, wild-type animals received 1200 p.p.m. mofezolac. Under the same conditions, APC1309 mice were also fed an experimental diet containing 400 p.p.m. nimesulide. After 8 weeks treatment, complete necropsies were performed on all animals. After laparotomy, the entire intestinal tract was resected, filled with 10% neutral-buffered formalin, and divided into the small intestine, cecum, and large intestine. The small intestine was divided into the duodenum (4 cm in length; proximal) and the proximal (middle) and distal halves of the remainder (distal). These segments were opened longitudinally and fixed flat between sheets of filter paper in 10% neutral-buffered formalin. The numbers and sizes of polyps as well as their distribution in the intestine were determined under x5 magnification with a stereoscopic microscope, detectable polyps being >0.2 mm in diameter (14).
BrdU labeling index
After counting ACFs, the large intestines embedded in paraffin were submitted to immunohistochemistry using an anti-BrdU antibody and a Vectastain ABC kit (Vector Laboratories, Burlingame, CA) (15). For determination of BrdU labeling indices, 60 well-oriented crypts, in which the base, lumen, and top of the crypts could be seen, were counted for each animal, 20 in each of the three portions of the large intestine, namely the middle colon, distal colon, and rectum. The numbers and positions of the labeled cells in each crypt column were recorded in terms of serial position counting upward from position 1, at the base of crypt, to the mouth of the crypt. The percentage of labeled cells (labeling index) was determined for the whole crypt by calculating the labeled cells per total number of cells x100.
Statistical analysis
Statistical analysis was performed with the Students or Welchs t tests versus the basal diet group. Results were considered statistically significant when P < 0.05.
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Results
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Body weights of AOM-treated rats were slightly lower than those of counterparts receiving the vehicle, but values for rats fed the control and experimental diets were comparable throughout the study period (Table I
). Food intake by rats fed the experimental diets was
15 g/animal/day and almost equal to that in the control diet group. No neoplastic lesions were found on macroscopic examination of all organs. In vehicle-treated animals, administration of mofezolac did not produce any gross changes in the liver, kidneys, stomach, intestines and lungs that would indicate toxicity in rats.
Data for ACF are shown in Table I
. ACFs were detected in all rats treated with AOM but not in any of the animals receiving the vehicle. They were located mainly in the distal colon, but some were found in the middle colon, and a few in the rectum. In the 600 and 1200 p.p.m. mofezolac groups, the numbers of ACFs per rat were suppressed dose-dependently to 76 and 60% relative to that (182 ± 20) for the AOM alone group, respectively. Mean numbers of aberrant crypts (ACs) per focus did not differ among the three AOM-treated groups (Table I
). The BrdU labeling index for large intestine epithelium in the AOM alone group (10.4%) was higher than in the AOM-treated groups receiving 600 and 1200 p.p.m. mofezolac, which demonstrated to decrease to 7.3 and 6.8%, respectively (Table I
). The length of crypts, estimated by the numbers of epithelial cells, were 46.5 ± 2.9 (mean ± SD) cells per crypt for the AOM alone group. These values were shortened to 42.1 ± 1.3 cells per crypt (P < 0.05) and 38.9 ± 1.2 cells per crypt (P < 0.01) by treatment with 600 and 1200 p.p.m. mofezolac, respectively. In addition, the lengths of crypts in vehicle-treated groups with basal diet and 1200 p.p.m. mofezolac were 34.2 ± 0.4 and 34.6 ± 2.2 cells/crypt, respectively, and these were shorter (P < 0.01) than that of the AOM alone group.
Under the same conditions, the numbers of ACFs per rat were significantly decreased to 111 ± 20 (mean ± SD, P < 0.01) by administration of 400 p.p.m. nimesulide. The mean numbers of ACs/focus were 2.1 ± 0.1. The data are consistent with those in our previous report (1). The average BrdU labeling index was decreased to 6.5 ± 0.9% (P < 0.01) and crypt length was 41.4 ± 1.8 cells/crypt (P < 0.01).
Administration of mofezolac at doses of 600 and 1200 p.p.m. in the diet did not affect feeding, body weights or behavior of the APC1309 mice. The average body weight for the control group was 19.8 ± 3.5 g (mean ± SD), and those for the 600 and 1200 p.p.m. mofezolac groups were 21.8 ± 2.5 and 21.0 ± 2.8 g, respectively, at 15 weeks of age. Food intake by mice fed the experimental diets was
3 g/animal/day and almost equal to that in the control diet group. Administration of mofezolac did not produce any gross changes in the liver, kidneys, stomach, intestines and lungs of the mice. In the stomach, neither bleeding nor gastritis was observed microscopically. Table II
summarizes data for the number and distribution of intestinal polyps in untreated control and test chemical-treated groups of mice. Intestinal polyps were detected in all APC1309 mice but not in any wild-type animals. Most polyps were located in the small intestine, with only a few apparent in the large intestines, and these polyps were identified as adenoma. In the 600 and 1200 p.p.m. mofezolac groups, the numbers of polyps in the small and large intestines were reduced dose-dependently to
90 and 59% relative to the control values, respectively. The same patterns were also observed regarding the number of polyps in the small intestines only, where inhibitory effects were obvious in the middle and distal segments. In the large and proximal small intestines, the suppression was non-significant. Figure 1
shows data for the size distribution of the intestinal polyps developing in control and test chemical-treated mice. It is noteworthy that mofezolac significantly reduced the numbers of polyps >1.5 mm in diameter at 1200 p.p.m. and >2.0 mm in diameter at 600 p.p.m..

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Fig. 1. Effects of mofezolac and nimesulide on the size distribution of intestinal polyps in APC1309 mice. APC1309 mice were fed basal diet (open columns) or diet containing 600 p.p.m. (hatched columns), 1200 p.p.m. (filled columns) mofezolac or 400 p.p.m. nimesulide (shaded columns) for 8 weeks. Polyps were grouped at intervals of 0.5 mm according to their diameters. The number of polyps per mouse in each size group is presented as the mean ± SD. *P < 0.05, **P < 0.01 versus the basal diet group.
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Under the same conditions, the average total number of polyps in the small and large intestines was significantly decreased by administration of 400 p.p.m. nimesulide (Table II
). Moreover, nimesulide also significantly reduced the number of polyps >1.5 mm in diameter (Figure 1
). The data were well consistent with those previously reported for Min mice (14).
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Discussion
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The present study provided clear evidence that a selective COX-1 inhibitor, mofezolac, can suppress both AOM-induced ACF formation in rats and spontaneous intestinal polyp development in Apc knockout mice. The data obtained with a pharmacological approach are in line with the suggestion of an involvement of the COX-1 isoform, as well as COX-2, in colon carcinogenesis, reported on the basis of a genetic approach by Chulada et al. (7). A human colon carcinoma cell line transfected with cDNAs for either COX-1 or COX-2 expresses either isoform with increased growth rates in both cases, the stimulated growth being suppressed by indomethacin treatment (16). These findings also suggest a possible contribution of COX-1 to colon carcinogenesis.
In the present study, reduction in polyp size as well as polyp number in the intestine of APC1309 mice was observed on treatment with mofezolac and nimesulide. Changes in cell proliferation and apoptosis, often together with altered angiogenesis, are closely associated with tumor growth. It has been reported that sulindac decreases the size and number of intestinal polyps in Min mice with increased apoptosis, but concomitant treatment with EP receptor agonists decreases the apoptosis (17), suggesting PGE2 to be a pivotal factor in the growth of intestinal polyps, possibly by affecting cell turnover. Regarding COX-1 and COX-2, both are known to contribute to increased PGE2 production in intestinal polyps (7). Furthermore, they have been shown to be involved in angiogenesis in gastrointestinal cancer xenograft in nude mice (18) and tube formation from endothelial cells co-cultured with colon carcinoma cells (19). In addition, expression of COX-1, as well as COX-2, is enhanced in cervical carcinomas, and artificial overexpression of COX-1 in Hela cells results in induction of expression of COX-2 and prostaglandin E synthase, concomitant with increased PGE2 synthesis and also induction of angiogenic factors (20). From these findings, it is possible that both COX-1 and COX-2 contribute, independently or cooperatively, to growth of intestinal polyps through increased PGE2 levels and angiogenesis, and affect on cell turnover. It is also possible that the COX isoforms are involved in production of prostaglandins and contribute to carcinogenesis through different receptor-mediated pathways, for example, by impacting on EP1 and EP2 receptors (8,9).
Inhibition of COX-1 by traditional NSAIDs is suggested to be a causal factor in their gastrointestinal side effects, such as the development of gastritis, gastric ulcers, and gastrointestinal bleeding. On the other hand, recently, it was indicated that NSAID-induced gastric injury required suppression of both COX-1 and COX-2 in rats (21). Actually, administration of a COX-1 selective inhibitor, mofezolac, did not induce any gastrointestinal side effects in the rats and mice under the conditions in the present study. From these observations, it may be that COX-1 or COX-2 selective inhibitors suppress colon carcinogenesis with lowered side effects, each isoform possibly compensating for the lack of expression of the other (22). Whereas traditional NSAIDs, COX-dual inhibitors, exhibit potent inhibition of colon carcinogenesis, they also cause severe side effects. To establish more effective and safe chemopreventive measures for colon carcinogenesis, further investigation of the effects of combinations of COX-1 and COX-2 dual and selective inhibitors on colon cancer development is clearly warranted.
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Notes
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6 To whom correspondence should be addressed Email: kwakabay{at}gan2.ncc.go.jp 
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Acknowledgments
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This work was supported in part by grants-in-aid for Cancer Research and the Second-Term Comprehensive 10-Year Strategy for Cancer Control from the Ministry of Health, Labor and Welfare, Japan.
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Received November 14, 2001;
revised May 13, 2002;
accepted May 14, 2002.