Chemopreventive effect of JTE-522, a selective cyclooxygenase-2 inhibitor, on 1, 2-dimethylhydrazine-induced rat colon carcinogenesis

Cancer Letters 202 (2003) 11–16 www.elsevier.com/locate/canlet

Chemopreventive effect of JTE-522, a selective cyclooxygenase-2 inhibitor, on 1, 2-dimethylhydrazine-induced rat colon carcinogenesis Min Wei, Keiichirou Morimura, Hideki Wanibuchi, Jun Shen, Kenichiro Doi, Makoto Mitsuhashi, Masaharu Moku, Elsayed I. Salim, Shoji Fukushima* Department of Pathology, Osaka City University Medical School, 1-4-3 Asahi-machi, Abeno-ku, Osaka 545-8585, Japan Received 12 June 2003; received in revised form 15 July 2003; accepted 18 July 2003

Abstract Selective COX-2 inhibitors have been suggested to be an effective strategy in the prevention of colon cancer without the adverse side effects of non-selective, nonsteroid anti-inflammatory drugs. The present experiment was designed to assess the potential chemopreventive properties of JTE-522, a new selective cyclooxygenase-2 inhibitor, on the induction of 1,2dimethylhydrazine (DMH)-induced colonic aberrant crypt foci (ACF), a marker of rat colon carcinogenesis. A total of 80 male F344 rats were treated with 3 or 10 mg/kg of body weight JTE-522 or vehicle by oral gavage five times weekly from the start of the experiment. One week later, rats received s.c. injections of saline or 20 mg/kg of body weight DMH once weekly for four successive weeks. At the end of 12 weeks after the start of experiment, all rats were sacrificed and colons were evaluated for ACF. 10 mg/kg JTE522 significantly suppressed the total ACF/colon. No inhibitory effect was observed in the 3 mg/kg JTE522 treatment group. This result suggests that JTE-522 possesses chemopreventive activity against colon carcinogenesis. q 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: JTE-522; Selective cyclooxygenase-2 inhibitor; Colon carcinogenesis; Chemoprevention

1. Introduction The protective effect of nonsteroid anti-inflammatory drugs (NSAIDs), such as aspirin and sulindac, for colon cancer has been well documented in epidemiological and animal studies [1]. However, use of traditional NSAIDs for chemoprevention of colon cancer has been hindered by their potential * Corresponding author. Tel.: þ81-6-6645-3737; fax: þ 81-66646-3093. E-mail address: [email protected] (S. Fukushima).

gastrointestinal toxicity that occurs with long-term administration. It is well known that NSAID’s inhibition of colon carcinogenesis is related to their inhibition of prostaglandin production by cyclooxygenase (COX), the constitutively expressed COX-1 and the inducible COX-2 [2,3]. Non-selective NSAIDs can inhibit the activities of both COX-1 and COX-2 [4]. Inhibition of the production of COX1-derived prostaglandins needed for maintenance of gut, kidney, and platelet functions has been suggested as the cause of adverse side effects of nonselective NSAIDs [5]. In contrast, in addition to

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the involvement of COX-2 in many inflammatory processes, it is also overexpressed in approximately 85% of colon cancers and 40% or more of adenomas, indicating that COX-2 may play a key role in colon carcinogenesis [6,7]. Furthermore, selective COX-2 inhibitors such as celecoxib have been shown to suppress colon carcinogenesis in experimental animals [8 – 10] and humans [11]. Therefore, COX-2 may be an important target for both cancer prevention and therapy, and selective targeting of COX-2 may afford protection against colorectal cancer without the gastrointestinal toxicity associated with depletion of COX-1 dependent mucosal cytoprotective prostaglandins. JTE-522 (4-[4-cyclohexyl-2-methyloxazol-5-yl]2-fluorobenzensulfonamide), a novel selective COX2 inhibitor, has been demonstrated to be a highly selective and irreversible inhibitor of both rat and human COX-2 [12]. Three different animal models of intestinal cancer cell growth have been used to test the antitumorigenic properties of non-selective and COX2 selective NSAIDs: mouse with adenomatous polyposis coli (APC) mutations, nude mouse xenograft model, and chemical carcinogen-induced colon carcinogenesis in rodents. JTE-522 has been shown to significantly reduce the number and growth of polyps in APCD474 knockout mice [13,14]. However, the APC mouse model is somewhat limited as a model of colon carcinogenesis in that most of the tumors are small bowel adenomas rather that colorectal cancers. In the nude mouse xenograft model, JTE-522 suppresses tumor growth of human head and neck squamous carcinoma cells [15] and human lung cancer cells [16]. JTE-522 also inhibited liver and lung metastases of colon cancer that showed COX-2 expression but lacked of effect on those lacking COX2 expression [17,18]. In chemically induced carcinogenesis, JTE-522 showed significant inhibitory effects on N-nitrosomethylbenzylamine-induced esophageal tumorigenesis in F344 rats [19]. Given the limited data on the chemoprotective efficacy of JTE522 on chemically induced colon carcinogenesis, the present study was designed to determine the potential inhibitory effect of it on formation of colonic aberrant crypt foci (ACF) in rats, which are preneoplastic lesions of colon carcinogenesis [20,21]. In light of increased cell proliferation, which has been suggested as one of the carcinogenic

properties of COX-2, we evaluated the cell proliferative activity in colonic epithelium using 5-bromo20 -deoxyuridine (BrdU) uptake assessed by immunohistochemistry.

2. Materials and methods 2.1. Chemicals JTE-522 (purity, 100%) was kindly provided by Japan Tobacco Inc., Osaka, Japan. 1, 2-dimethylhydrazine (DMH) (purity, 99%) was purchased from Tokyo Kasei Co., Tokyo, Japan. Carboxymethyl cellulose sodium salt (CMC) was purchased from Wako Life Science Reagent, Osaka, Japan. JTE-522 was prepared by suspension in vehicle of 0.5% CMC just before treatment. The dosing volume was kept constant to 1 ml/rat, and the concentration was adjusted once weekly based on body weight. 2.2. Animals A total of 80 male F344 rats, 5 weeks old, were purchased from Charles River Japan, Inc., Hino, Japan. The rats were housed in polycarbonate cages (5/cage) on wood chip bedding in an animal room targeted under controlled conditions of a 12 h light/12 h dark cycle, 55 ^ 5% humidity and 23 ^ 2 8C room temperature. They were fed powdered CE-2 basal diet (Clea Japan Inc., Tokyo, Japan). Diet and tap water were available ad libitum throughout the experiment. 2.3. Experimental procedures The following protocol was approved by the Institutional Animal Care and Use Committee of the Osaka City University Medical School. Rats at 6 weeks of age after 1 week of acclimatization were divided randomly into five groups. The rats in groups 1– 3 (20 rats each) were injected s.c. with DMH (20 mg/kg body wt) from 1 week after the start of the experiment, once weekly for four successive weeks. Those in groups 4 and 5 (10 rats each) were injected s.c. with 0.9% saline at the same time. Group 2 was treated with JTE-522 at a dose of 3 mg/kg body wt by oral gavage, five times weekly from the start of

M. Wei et al. / Cancer Letters 202 (2003) 11–16

the experiment to the end of the experiment. Groups 3 and 5 were treated with JTE-522 at the dose of 10 mg/kg body wt in the same manner as group 2. Groups 1 and 4 were treated with 0.5% CMC alone, without JTE-522. Body weight, water and food consumption were measured weekly during the experiment. All rats were sacrificed under ether anaesthesia at 12 weeks after the start of the experiment. One hour prior to sacrifice, 10 rats from each group received an i.p. injection of 100 mg/kg body weight BrdU (Sigma Chemical Co., St Louis, MO). The colons were quickly excised, distended in 10% phosphate buffered formalin solution, opened from cecum to anus, and fixed flat between pieces of filter paper in 10% phosphate buffered formalin solution for examination of ACF. The liver and kidney were removed from all animals, weighed, and fixed in 10% phosphatebuffered formalin. After adequate fixation, they were cut and processed for paraffin embedding and routinely stained with hematoxylin and eosin for histological examination. 2.4. ACF analysis After fixation for 24 h at 4 8C, each colon was cut into proximal, middle, and distal portions of equal length. Each portion of colon was further stained using 0.2% methylene blue and examined for ACF by light microscopy. ACF were counted as described previously [22]. The parameters used to assess the aberrant crypts were their occurrence and multiplicity. Crypt multiplicity was determined as the number of crypts in each focus and categorized as containing up to three and four or more aberrant crypts/focus. After ACF counting, samples collected from the proximal, middle, and distal colons were processed for paraffin embedding for histological examination in all rats, and immunohistochemistry in rats that received BrdU i.p. injection. 2.5. BrdU immunohistochemistry BrdU incorporation in colonic epithelium was examined using the avidin – biotin – peroxidase complex (ABC) method. Mouse monoclonal anti-BrdU antibody (M0744, Dako. A/S, Denmark) was used at 1:500 dilutions. The number of BrdU-labeled cells in

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10 complete crypts from proximal, middle, and distal portions of colon, respectively, were counted to determine a labeling index. 2.6. Statistical analysis All data were expressed as the mean ^ SD. Group means for body and tissue weights, BrdU labeling index and ACF were evaluated using ANOVA followed by Dunnett analysis. Differences were considered as statistically significant at p , 0:05: All statistical analyses were performed using StatView J-5.0 software (Abacus Concepts, Berkeley, CA) for the Macintosh computer.

3. Results The body weight gain of rats treated with saline or DMH and given JTE-522 or vehicle were comparable throughout the study period. Water consumption during the experiment was slightly higher in the high dose JTE-522 treated group compared to the control group but the increase was not statistically significant (data not shown). Treatment with JTE-522, DMH or their combination had no effect on the food intake. Final body and organ weights are shown in Table 1. Slightly reduced final body weights were observed in the groups treated with JTE-522 but the decrease was not statistically significant and not dosedependent. There were no significant differences in absolute or relative weights of liver and kidney between groups. No abnormal macroscopic or histological findings in intestine, liver or kidneys were observed in JTE-522-treated rats. The numbers and crypt multiplicities of ACF in the treatment groups are summarized in Table 2. ACF were observed in all of rats treated with DMH. In the rats administered vehicle, DMH induced an average of 409 ACF/colon. Administration of 10 mg/kg JTE-522 significantly suppressed ACF formation with a 30% reduction in total ACF/colon ðp , 0:01Þ: Furthermore, the data on crypt multiplicity show that 10 mg/kg JTE-522 significantly reduced the formation of foci containing 1– 3 crypts but not foci containing four crypts or more. Administration of the low dose of JTE-522 (3 mg/kg) had no inhibitory effects on either the total ACF or crypt multiplicity.

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Table 1 Average final body, liver and kidney weights Group

DMH þ 1 2 3 Saline þ 4 5 a

Treatment

No. of rats

Final body weight (g)

Kidney weighta

Liver weight Absolute (g)

Relative (%)

Absolute (g)

Relative (%)

Vehicle 3 mg/kg JTE-522 10 mg/kg JTE-522

20 20 20

300 ^ 17 293 ^ 16 294 ^ 12

10.4 ^ 1.1 9.9 ^ 0.9 10.1 ^ 1.2

3.5 ^ 0.3 3.4 ^ 0.3 3.4 ^ 0.4

2.1 ^ 0.2 2.1 ^ 0.2 2.1 ^ 0.3

0.7 ^ 0.1 0.7 ^ 0.1 0.7 ^ 0.1

Vehicle 10 mg/kg JTE-522

10 10

310 ^ 17 299 ^ 11

10.7 ^ 1.1 9.9 ^ 0.8

3.5 ^ 0.3 3.3 ^ 0.2

2.1 ^ 0.2 2.0 ^ 0.1

0.7 ^ 0.1 0.7 ^ 0.0

Combined weight of the two kidneys.

Table 2 Effects of JTE-522 on DMH-induced ACF formation in rat colon Group

Treatment

DMH þ vehicle DMH þ 3 mg/kg JTE-522 DMH þ 10 mg/kg JTE-522

1 2 3 a

No. of rats

20 20 20

Total no. of ACF/rat

409 ^ 135 458 ^ 105 285 ^ 65a

Crypt(s)/focus 1

2

3

$4

124 ^ 48 126 ^ 41 87 ^ 29a

137 ^ 42 157 ^ 33 87 ^ 23a

94 ^ 40 110 ^ 24 67 ^ 17a

55 ^ 28 66 ^ 27 44 ^ 13

Significantly different from Group 1.

The control rats treated with saline and given JTE-522 alone showed no evidence of ACF formation in the colon (data not shown). The rate of cell proliferation in colonic epithelium was determined by BrdU incorporation. As shown in Fig. 1, the BrdU labeling index was significantly increased in the DMH alone treatment group. JTE-522 has no significant effect on the cell proliferating activity.

carcinomas based on studies in experimentally induced colon carcinogenesis in animals [20]. They have also been detected in human colorectal epithelium in patients with colorectal cancer [21]. The number, size, and severity of ACF have been shown to correlate with the number of colon tumors in both

4. Discussion The present study demonstrated that administration of the selective COX-2 inhibitor JTE-522 significantly suppressed DMH-induced colonic ACF formation in rats. This result provides the first evidence that JTE-522 possesses chemopreventive activity against colon carcinogenesis in rats. The inhibition of ACF formation was used to define efficacy in this study. ACF have been implicated as precursors to colorectal adenomas and

Fig. 1. BrdU labeling indices in colonic epithelium of rats. *Significantly different from Group 4 value.

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experimental animal [23,24] and patients with colorectal cancer [25], suggesting that they are either precursor lesions for these tumors or indicators of susceptibility to tumor formation. In this study, at 12 weeks 87% of ACF in rats treated with DMH alone were of small size (less than 4 crypts/foci). Moreover, ACF are recognized as early preneoplastic lesions in colon carcinogenesis, and those foci containing four or more crypts may correspond to the promotion step of colon carcinogenesis [20,23,24,26]. Therefore, the findings that inhibitory effects of JTE-522 were virtually restricted to the development of the foci containing one to three aberrant crypts per foci suggest that JTE-522 may possess greater impact on the induction of preneoplastic lesions than growth of preneoplastic lesions in DMH-induced colon carcinogenesis. A long-term experiment using colon tumors as the endpoint is ongoing to assess the chemopreventive activity of JTE-522 on different stages of DMH-induced colon carcinogenesis in rats. To date, various molecular mechanisms have been proposed to be responsible for the antitumor effects of NSAIDs, including inhibition of cell proliferation, angiogenesis, metastasis, induction of apoptosis, and enhancement of immunosurveillance [27,28]. However, the precise mechanism of NSAIDs for their antitumorigenic effects remains unclear. Lack of a significant effect of JTE-522 on cell proliferation activity in colonic epithelium suggests that other effects are involved. Recent studies in APCD474 knockout mice show that JTE-522 did not affect the proliferating potential of adenomas, but it reduced the growth of adenomas mainly by inhibition of angiogenesis and induction of apoptosis [14]. Therefore, although the mechanism of DMH-induced carcinogenesis inhibition by JTE-522 was not determined in the present study, it is possible that induction of apoptosis plays a more important role in the chemopreventive potential of JTE-522. Given diverse activities and interactions with other genetic factors of COX-2 in carcinogenesis [29 –31], the planned molecular analysis of both preneoplastic lesions and tumors in our ongoing long-term bioassay will help understand the COX-related mechanism of carcinogenesis and the mechanism of prevention of JTE-522. In summary, the present study demonstrated the inhibitory effect of JTE-522 on DMH-induced rat

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colon carcinogenesis. Further experiments, including studies elucidating the mechanism involved in its antitumorigenic effects, are warranted to fully evaluate this compound for its chemopreventive properties.

Acknowledgements We thank Japan Tobacco Inc. for providing the JTE-522. We also thank Kaori Touma for technical assistance, and Mari Dokoh and Yuko Onishi for assistance with the preparation of the manuscript (Department of Pathology, Osaka City University Medical School).

References [1] P.A. Janne, R.J. Mayer, Chemoprevention of colorectal cancer, N. Engl. J. Med. 342 (2000) 1960–1968. [2] J.R. Vane, Inhibition of prostaglandin synthesis as a mechanism of action for aspirin-like drugs, Nat. N. Biol. 231 (1971) 232–235. [3] W.L. Xie, J.G. Chipman, D.L. Robertson, R.L. Erikson, D.L. Simmons, Expression of a mitogen-responsive gene encoding prostaglandin synthase is regulated by mRNA splicing, Proc. Natl. Acad. Sci. USA 88 (1991) 2692–2696. [4] M.M. Wolfe, D.R. Lichtenstein, G. Singh, Gastrointestinal toxicity of nonsteroidal antiinflammatory drugs, N. Engl. J. Med. 340 (1999) 1888–1899. [5] P. Needleman, P.C. Isakson, The discovery and function of COX-2, J. Rheumatol. 49 (24 Suppl.) (1997) 6–8. [6] C.E. Eberhart, R.J. Coffey, A. Radhika, F.M. Giardiello, S. Ferrenbach, R.N. DuBois, Up-regulation of cyclooxygenase 2 gene expression in human colorectal adenomas and adenocarcinomas, Gastroenterology 107 (1994) 1183–1188. [7] W. Kutchera, D.A. Jones, N. Matsunami, J. Groden, T.M. McIntyre, G.A. Zimmerman, R.L. White, S.M. Prescott, Prostaglandin H synthase 2 is expressed abnormally in human colon cancer: evidence for a transcriptional effect, Proc. Natl. Acad. Sci. USA 93 (1996) 4816– 4820. [8] T. Kawamori, C.V. Rao, K. Seibert, B.S. Reddy, Chemopreventive activity of celecoxib, a specific cyclooxygenase-2 inhibitor, against colon carcinogenesis, Cancer Res. 58 (1998) 409 –412. [9] B.S. Reddy, Y. Hirose, R. Lubet, V. Steele, G. Kelloff, S. Paulson, K. Seibert, C.V. Rao, Chemoprevention of colon cancer by specific cyclooxygenase-2 inhibitor, celecoxib, administered during different stages of carcinogenesis, Cancer Res. 60 (2000) 293– 297.

16

M. Wei et al. / Cancer Letters 202 (2003) 11–16

[10] C.V. Rao, C. Indranie, B. Simi, P.T. Manning, J.R. Connor, B.S. Reddy, Chemopreventive properties of a selective inducible nitric oxide synthase inhibitor in colon carcinogenesis, administered alone or in combination with celecoxib, a selective cyclooxygenase-2 inhibitor, Cancer Res. 62 (2002) 165–170. [11] G. Steinbach, P.M. Lynch, R.K. Phillips, M.H. Wallace, E. Hawk, G.B. Gordon, N. Wakabayashi, B. Saunders, Y. Shen, T. Fujimura, L.K. Su, B. Levin, The effect of celecoxib, a cyclooxygenase-2 inhibitor, in familial adenomatous polyposis, N. Engl. J. Med. 342 (2000) 1946–1952. [12] M. Matsushita, M. Masaki, Y. Yagi, T. Tanaka, K. Wakitani, Pharmacological profile of JTE-522, a novel prostaglandin H synthase-2 inhibitor, in rats, Inflamm. Res. 46 (1997) 461–466. [13] H. Sasai, M. Masaki, K. Wakitani, Suppression of polypogenesis in a new mouse strain with a truncated Apc(Delta474) by a novel COX-2 inhibitor, JTE-522, Carcinogenesis 21 (2000) 953–958. [14] K. Sunayama, H. Konno, T. Nakamura, H. Kashiwabara, T. Shoji, T. Tsuneyoshi, S. Nakamura, The role of cyclooxygenase-2 (COX-2) in two different morphological stages of intestinal polyps in APC(Delta474) knockout mice, Carcinogenesis 23 (2002) 1351–1359. [15] G. Nishimura, S. Yanoma, H. Mizuno, K. Kawakami, M. Tsukuda, A selective cyclooxygenase-2 inhibitor suppresses tumor growth in nude mouse xenografted with human head and neck squamous carcinoma cells, Jpn. J. Cancer Res. 90 (1999) 1152–1162. [16] T. Hida, K. Kozaki, H. Ito, O. Miyaishi, Y. Tatematsu, T. Suzuki, K. Matsuo, T. Sugiura, M. Ogawa, T. Takahashi, Significant growth inhibition of human lung cancer cells both in vitro and in vivo by the combined use of a selective cyclooxygenase 2 inhibitor, JTE-522, and conventional anticancer agents, Clin. Cancer Res. 8 (2002) 2443–2447. [17] S. Tomozawa, H. Nagawa, N. Tsuno, K. Hatano, T. Osada, J. Kitayama, E. Sunami, M.E. Nita, S. Ishihara, H. Yano, T. Tsuruo, Y. Shibata, T. Muto, Inhibition of haematogenous metastasis of colon cancer in mice by a selective COX-2 inhibitor, JTE-522, Br. J. Cancer 81 (1999) 1274– 1279. [18] I. Nagatsuka, N. Yamada, S. Shimizu, M. Ohira, H. Nishino, S. Seki, K. Hirakawa, Inhibitory effect of a selective cyclooxygenase-2 inhibitor on liver metastasis of colon cancer, Int. J. Cancer 100 (2002) 515 –519.

[19] Z. Li, Y. Shimada, A. Kawabe, F. Sato, M. Maeda, I. Komoto, T. Hong, Y. Ding, J. Kaganoi, M. Imamura, Suppression of Nnitrosomethylbenzylamine (NMBA)-induced esophageal tumorigenesis in F344 rats by JTE-522, a selective COX-2 inhibitor, Carcinogenesis 22 (2001) 547 –551. [20] C.M. Fenoglio-Preiser, A. Noffsinger, Aberrant crypt foci: a review, Toxicol. Pathol. 27 (1999) 632– 642. [21] T.P. Pretlow, B.J. Barrow, W.S. Ashton, M.A. O’Riordan, T.G. Pretlow, J.A. Jurcisek, T.A. Stellato, Aberrant crypts: putative preneoplastic foci in human colonic mucosa, Cancer Res. 51 (1991) 1564– 1567. [22] E.I. Salim, H. Wanibuchi, T. Taniyama, Y. Yano, K. Morimura, S. Yamamoto, S. Otani, Y. Nishizawa, H. Morii, S. Fukushima, Inhibition of development of N; N 0 -dimethylhydrazine-induced rat colonic aberrant crypt foci by pre, post and simultaneous treatments with 24R,25-dihydroxyvitamin D3, Jpn. J. Cancer Res. 88 (1997) 1052–1062. [23] X.M. Zhang, D. Stamp, S. Minkin, A. Medline, D.E. Corpet, W.R. Bruce, M.C. Archer, Promotion of aberrant crypt foci and cancer in rat colon by thermolyzed protein, J. Natl. Cancer Inst. 84 (1992) 1026– 1030. [24] B.A. Magnuson, I. Carr, R.P. Bird, Ability of aberrant crypt foci characteristics to predict colonic tumor incidence in rats fed cholic acid, Cancer Res. 53 (1993) 4499–4504. [25] T. Takayama, S. Katsuki, Y. Takahashi, M. Ohi, S. Nojiri, S. Sakamaki, J. Kato, K. Kogawa, H. Miyake, Y. Niitsu, Aberrant crypt foci of the colon as precursors of adenoma and cancer, N. Engl. J. Med. 339 (1998) 1277–1284. [26] R.P. Bird, Role of aberrant crypt foci in understanding the pathogenesis of colon cancer, Cancer Lett. 93 (1995) 55– 71. [27] S.J. Shiff, B. Rigas, The role of cyclooxygenase inhibition in the antineoplastic effects of nonsteroidal antiinflammatory drugs (NSAIDs), J. Exp. Med. 190 (1999) 445–450. [28] L.R. Howe, K. Subbaramaiah, A.M. Brown, A.J. Dannenberg, Cyclooxygenase-2: a target for the prevention and treatment of breast cancer, Endocr. Relat. Cancer 8 (2001) 97–114. [29] M.M. Taketo, Cyclooxygenase-2 inhibitors in tumorigenesis (part I), J. Natl. Cancer Inst. 90 (1998) 1529–1536. [30] M.M. Taketo, Cyclooxygenase-2 inhibitors in tumorigenesis (part II), J. Natl. Cancer Inst. 90 (1998) 1609– 1620. [31] S.M. Prescott, F.A. Fitzpatrick, Cyclooxygenase-2 and carcinogenesis, Biochim. Biophys. Acta 1470 (2000) M69–M78.