Suppression of N-nitrosomethylbenzylamine (NMBA)-induced esophageal tumorigenesis in F344 rats by JTE-522, a selective COX-2 inhibitor

Zhigang Li, Yutaka Shimada,1, Atsushi Kawabe, Fumiaki Sato, Masato Maeda, Izumi Komoto, Tao Hong, Yongzeng Ding, Junichi Kaganoi and Masayuki Imamura

Department of Surgery and Surgical Basic Science, Graduate School of Medicine, Kyoto University, 54-Shogoin Kawahra-cho, Sakyoku Kyoto 606-8507, Japan


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Recent studies have demonstrated that overexpression of cyclooxygenase-2 (COX-2) and elevation of COX-2-mediated synthesis of prostaglandin E2 (PGE2) were observed in various cancers including esophageal cancer, but their roles in carcinogenesis of the esophagi still remain unclear. To address the issue, we observed the reduction of N-nitrosomethylbenzylamine (NMBA)-induced tumorigenesis in rat esophagi via JTE-522 (4-[4-cyclohexyl-2-methyloxazol-5-yl]-2-fluorobenzenesulfonamide), a selective COX-2 inhibitor. In this study, 54 F344 male rats were divided into nine groups; JTE-522 (3, 9 and 30 mg/kg) was administered orally. We also examined the effects of JTE-522 on COX-2 mRNA and synthesis of PGE2. In the group in which JTE-522 was administered intermittently at a daily dose of 30 mg/kg, the number of NMBA-induced esophageal tumors per rat significantly reduced, to 62% (P < 0.05), but the size of the tumors was not significantly inhibited. In the group in which JTE-522 was administered continuously five times weekly for 24 weeks at a daily dose of 9 mg/kg, both the number and size of tumors significantly reduced, to 29 and 44%, respectively (P < 0.05). Furthermore, JTE-522 suppressed not only tumor formation but also developing carcinomas (P < 0.0001). In this study, treatment with NMBA alone resulted in an ~5-fold rise in expression of COX-2 mRNA detected by semi-quantitative RT–PCR analysis and an ~7-fold increase in the production of PGE2 measured by ELISA compared with the normal esophageal mucosa. The up-regulated COX-2 expression did not decrease with the treatment of JTE-522 at the 3, 9 and 30 mg/kg doses; however, the increased levels of PGE2 synthesis were significantly decreased by administering JTE-522 (P < 0.01). Our study suggests that COX-2-mediated PGE2 is important in NMBA-induced esophageal tumorigenesis in rats, and therefore may be a promising chemotherapeutic target for the prevention and treatment of esophageal cancer, especially with selective COX-2 inhibitors.

Abbreviations: CMC-Na, carboxymethyl celluose sodium; COX, cyclooxygenase; JTE-522, 4-[4-cyclohexyl-2-methyloxazol-5-yl]-2-fluorobenzenesulfonamide; NSAIDs, non-steroidal anti-inflammatory drugs; NMBA, N-nitrosomethylbenzylamine; PGE2, prostaglandin E2.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
It is known that cyclooxygenase (COX), or prostaglandin endoperoxidase, is the rate limiting enzyme involved in the conversion of arachidonic acid to prostaglandin H2 (PGH2), the unstable bicycloendoperoxide intermediate, which undergoes further metabolism to the parent eicosanoids PGD2, PGE2, PGF2{alpha}, PGI2 and thromboxane 2. Of these PGE2 was thought to be a chief factor associated with several kinds of human carcinogenesis including lung, prostatic and esophageal carcinoma (13). Other evidence reported that exogenous PGE2 stimulated the proliferation of both human colon cancer cell lines and normal mouse colonocytes (4), modulated apoptosis and Bcl-2 expression in human colon cancer cell (5) and enhanced angiogenesis in vivo. (6). It was suggested that one of the mechanisms by which PGE2 supports tumor growth is by inducing the angiogenesis necessary to supply oxygen and nutrients to tumors >2 mm in diameter (7).

Recently, two isoforms of COX have been identified which share 60% homology, COX-1 and COX-2 (8). COX-1 is considered primarily to be a housekeeping form, constitutively expressed in most cells and tissues (9), which produces the prostanoids involved in a range of normal physiologic functions. In contrast, COX-2 was originally found to be increased in inflammatory cells and sites of inflammation. COX-2 expression is highly regulated and induced by a variety of agents including cytokins, growth factors and tumor promoters (10). Multiple lines of evidence suggest that COX-2 expression is up-regulated in several types of human cancers, including the colon, lung, breast, stomach and pancreatic cancer (1115), and it was also reported that overexpression of COX-2 is observed in human esophageal cancer (16). In addition, in the N-nitrosomethylbenzylamine (NMBA)-induced rat esophageal tumorigenesis model, the results of semi-quantitative RT–PCR and northern blot analysis indicated that expression of COX-2 was significantly higher in preneoplastic tissues at 16 weeks, and in preneoplastic tissues and papillomas at 26 weeks (the end of experiment), compared with normal tissues (17).

Much evidence has demonstrated that non-steroidal anti-inflammatory drugs (NSAIDs) inhibited both COX-1 and COX-2 (18). Several recent studies have reported a 40–50% lower colorectal cancer risk in people who are continuously taking aspirin or other NSAIDs (19), and the inhibitive effects of NSAIDs on tumorigenesis of the colon were observed in many animal experimental models (20). In another investigation, intake of aspirin was associated with up to a 90% decreased risk of developing esophageal cancer (21). These findings indicated a possibility that NSAIDs also reduced esophageal carcinogenesis by inhibition of COX. Because NSAIDs inhibit COX-1 so that a range of normal physiologic functions are affected, which frequently results in untoward gastrointestinal side effects such as ulceration and bleeding (22), this limits their clinical use and has led to the development of more selective COX inhibitors. Selective COX-2 inhibitors may be more effective and safer cancer chemopreventive agents than classical NSAIDs. It has been reported that selective inhibitors of COX-2 decrease tumor formation in experimental animals (23) and these compounds induce apoptosis and inhibit growth in several types of cancer cells (2426).

JTE-522 (4-[4-cyclohexyl-2-methyloxazol-5-yl]-2-fluorobenzenesulfonamide) was determined to be a novel selective COX-2 inhibitor. In vitro COX activity assays using isolated COX-1 and COX-2 enzymes from sheep seminal vesicle microsome and placenta, respectively, have confirmed that JTE-522 selectively inhibits COX-2 without affecting COX-1 even at 100 µM. JTE-522 has anti-inflammatory effects and does not cause severe gastric lesions at oral doses up to 300 mg/kg (27,28). These properties of JTE-522 may allow it to be administered as a prolonged preventative agent. Recently, it has also been reported that selective inhibition of COX-2 by JTE-522 in mice reduced hematogenous metastasis of a human colorectal cancer cell that had high levels of COX-2 expression, but JTE-522 did not have a significant inhibitory effect on another colorectal cancer cell lacking COX-2 protein (29). It was furthermore reported that JTE-522 suppresses tumor growth in nude mouse xenografted with human head and neck squamous carcinoma cells (30).

In this study, we examined whether JTE-522 can inhibit NMBA-induced tumorigenesis in rats and whether sensitivity to JTE-522 is related to COX-2 expression levels and/or to COX-2-mediated PGE2 production. Based on the results of these studies, it will be important to establish whether selective inhibitors of COX-2 are useful for the prevention and treatment of esophageal carcinomas.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Animals
A total of 54 F344 male rats (10 weeks of age) were purchased from Japan SLC (Haruno, Shizuoka, Japan). Each cage contained three animals, which were kept in our animal center for 2 weeks before use. Rats were given water and food freely and kept on a 12 h light/12 h dark cycle.

Chemical carcinogen and the selective inhibitor
NMBA was purchased from NARD (Osaka, Japan). Purified JTE-522 was obtained from Japan Tobacco Co. (Tokyo, Japan). NMBA was dissolved in saline solution and given at a dose of 1 mg/kg, subcutaneously, while JTE-522 was suspended in a vehicle of 0.5% carboxymethyl celluose sodium (CMC-Na) and administered orally at a volume of 5 ml/kg body weight (28). To ensure the dose was exact, JTE-522 was administered by means of esophageal intubation.

Tumorigenesis and tumorisuppression protocol
In this experiment, 54 F344 rats were randomly divided into nine experimental groups according to the different regimens they were submitted to. This consisted of the following groups: group 1, five rats were injected subcutaneously with saline 1 ml/kg; group 2, five rats were orally administered with 0.5% CMC-Na 5 ml/kg; group 3, five rats orally received JTE-522 9 mg/kg; group 4, eight rats were injected subcutaneously with NMBA 1 mg/kg; group 5, five rats received NMBA 1 mg/kg plus 0.5% CMC-Na 5 ml/kg; group 6, five rats received NMBA 1 mg/kg plus JTE-522 3 mg/kg; group 7, five rats received NMBA 1 mg/kg plus JTE-522 9 mg/kg; group 8, eight rats received NMBA 1 mg/kg plus JTE-522 30 mg/kg; group 9, eight rats received NMBA 1 mg/kg plus JTE-522 9 mg/kg (continuous administration). Groups 1, 2 and 3 were negative controls; group 4 was a positive control.

The administration of drugs in groups 1–8 was scheduled as follows: five times weekly for 5 weeks followed by the same dose once per week for another 10 weeks; weight was then only measured once or twice per week for 9 weeks. The administration of JTE-522 in group 9 was performed continuously five times per week until the end of the experiment (illustrated in Figure 1Go).



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Fig. 1. Schematic representation of the experimental protocol. The downward arrows indicate injection of NMBA at 1 mg/kg body weight; upward arrows indicate injection of JTE-522 at 3 (group 6), 9 (groups 3, 7 and 9) or 30 mg/kg body weight (group 8). The hatched box indicates injection of saline at 1 ml/kg body weight or CMC-Na at 5 ml/kg body weight.

 
At the end of 24 weeks, all surviving animals were killed using diethyl ether in a glass container according to institutional protocols. Esophagi were excised, the total number of tumors >2 mm in diameter were counted and the volume of tumors in each rat esophagus was calculated as lengthxwidthxheightx0.52 (31). One part of each tissue sample was inflated, fixed in 10% phosphate-buffer formalin solution and routinely embedded in paraffin for H&E staining; the remaining sample was immediately frozen in liquid nitrogen for semi-quantitative RT–PCR assay and PGE2 analysis.

Semi-quantitative RT-PCR for COX-2 expression
Total RNA was extracted from tumor and non-tumor tissues of esophagi and cDNA was synthesized from 1 µg total RNA using a First Strand cDNA Synthesis Kit (Pharmacia). The primers for COX-2 were designed as: 5'-GGTCTGGTGCCGGGTCT GATGATG-3' (sense) and 5'-GGCCTTTCAAGGAGAATGGAGC-3' (antisense). Aliquots of 5 µl of the reserve-transcribed cDNA samples were added to 50 µl of a reaction mixture that contained: 5 µl of 10x buffer, 5 µl of 2 mM dNTP mix, 3 µl of 2.5 mM MgCl2, 0.2 µl of Ex Taq polymerase (TaKaRa) and 1 µl of each primer. Samples were co-amplified for 25 cycles: denaturation at 94°C for 20 s, annealing at 65°C for 20 s, extension at 72°C for 30 s and final extension at 72°C for 10 min. A constitutively expressed gene, GAPDH, was used as an internal control, generating a 230 bp PCR product. The primers for GAPDH were 5'-AGATGGTGAAG G T C G G T GTG-3' (sense) and 5'-CTGGAAGATGGTGATGGGTT-3' (antisense). The PCR conditions for GAPDH were identical to those for COX-2. The 12 µl of PCR products were applied to a 2% agarose gel and electrophoresed. The gel was then stained with ethidium bromide and illuminated on a UV table. Electrophoresed PCR products were scanned using a computer densitometer (NIH image software package) to determine the density of the bands, and the relative value of the COX-2 band to GAPDH was calculated in each sample (32).

Measurement of PGE2 production
To determine basal PGE2 levels, frozen samples were homogenized at ice temperature in 0.5 ml of 0.1 M Tris–HCl buffer containing 5.6 µM indomethacin (pH 7.4) with a microtube pestel and vortexed thoroughly for 2 min. The quantity of PGE2 in supernatants was immediately determined with the PGE2 Monoclonnal Enzyme Immunoassay Kit (Caymen Chemical), according to the manufacturer's instructions. Results were measured using a Dynatech MR5000 microplate reader and normalized to micrograms of protein.

Statistical analysis
Tumor multiplicity and PGE2 production expressed as mean ± standard deviation (SD). Comparisons between groups were made using the Tukey–Kramer test. Comparisons of the incidence of esophageal tumors in rats treated with NMBA or a combination of NMBA and JTE-522 were made using the Kruskal–Wallis test. Software used in this study was StatView version 5.0 (SAS. Co.). Differences were considered statistically significant at P < 0.05.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
General observation and histopathological examination
Two rats in group 9 died during weeks 13 and 15, respectively, due to misadministration of the drug into the airway; the rest survived to the end of the experiment. The body weights of rats treated with saline, CMC-Na, NMBA and different doses of JTE-522 were recorded and compared throughout the study. The initial and final average body weights of rats in each group are summarized in Table IGo. The average body weights of rats in NMBA-untreated groups (groups 1–3) were similar, and administration of JTE-522 alone (group 3) did not produce any toxic effects. However, the final average body weights of rats that received NMBA treatment (groups 4–9) were significantly reduced in comparison to the saline-treated animals (P < 0.05). Of these, the reduction in body weight of JTE-522-treated rats (groups 6–9) was less than of JTE-522-untreated rats (group 4 and 5). In preliminary experiments, NMBA was demonstrated to first induce tumor formation in esophagi in F344 rats at 12 weeks. Rats that were treated with only NMBA had an esophageal tumor incidence of 100% at the end of 24 weeks, while those that did not receive NMBA had no tumors. The tumor incidence of groups 6, 7, 8 and 9 (treated with JTE-522) was lower than group 4 (treated with NMBA alone). Of these, we compared the results of histopathological examination of esophageal tumors in rats treated with NMBA (group 4) and NMBA plus JTE-522 9 mg/kg (group 7), these results are summarized in Table IIGo. Our data clearly showed that COX-2 inhibitor suppressed not only tumor formation but also developing carcinomas (P < 0.0001).


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Table I. Change of body weights of treated rats
 

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Table II. Incidence of esophageal tumors in rats treated with NMBA and a combination of NMBA and JTE-522
 
Effects of JTE-522 on tumor multiplicity
The data for the effects of JTE-522 on NMBA-induced esophageal tumor multiplicity (number and size of tumors in each rat) in each group are summarized in Table IIIGo. JTE-522 showed significant inhibitory effects on the mean number of tumors per rat in groups 8 and 9 (62 and 29%, respectively; P < 0.05). However, the mean volume of tumors was significantly reduced only in group 9 (44%; P < 0.05).


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Table III. Inhibitory effects of JTE-522 on NMBA-induced esophageal tumorigenesis in rats
 
Effects of JTE-522 on COX-2 mRNA levels
Semi-quantitative analysis for COX-2 mRNA levels was performed using a multiplex RT–PCR technique. We compared levels of COX-2 mRNA in paired samples of esophageal tumors and the adjacent apparently normal tissue from groups 4, 8 and 9 with normal esophageal mucosa from group 1. The data are shown in Figure 2Go.There was nearly a 5-fold increase in the amount of COX-2 mRNA in esophageal tumors that received NMBA treatment alone as compared with normal mucosa; levels of COX-2 mRNA also increased in non-tumor tissues compared with normal mucosa. However, up-regulated COX-2 mRNA expression was not affected by administering JTE-522 at different doses (3–30 mg/kg) or intervals.



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Fig. 2. Semi-quantitative RT–PCR assay for COX-2 mRNA in relation to JTE-522 treatment. Expression levels of COX-2 in different tissue samples are not affected by administering JTE-522 at different doses or intervals. Results for groups 1 (normal mucosa), 4 (NMBA alone), 8 (NMBA plus JTE-522 30 mg/kg) and 9 (NMBA plus JTE-522 9mg/kg, continuous administration) are shown. N, normal tissue; T, tumor tissue. GAPDH was used as the internal standard in normal esophageal epithelium and tumor tissues. The relative value of the COX-2 band to GAPDH was calculated for each tissue sample and noted beneath each lane. PCR product sizes: COX-2, 702 bp; GAPDH, 230 bp. M, 100 bp molecular marker.

 
Effects of JTE-522 on PGE2 synthesis
Induction of COX-2 by NMBA can be assessed in a variety of ways, including changes in the synthesis of PGE2. In the present study, PGE2 levels increased in both normal tissues and tumor tissues in esophagi that received NMBA. The 7-fold increase in production of PGE2 in tumor tissues that received NMBA alone, compared with the normal esophageal mucosa, was significant (P < 0.01). Moreover, the elevated levels of PGE2 synthesis were significantly inhibited by JTE-522 at each of the 3–30 mg/kg doses; in particular, a 3-fold decrease was observed in the rats continuously treated with JTE-522 9 mg/kg (P < 0.01) (shown in Figure 3Go).



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Fig. 3. Effect of JTE-522 on NMBA-induced PGE2 production. Group 1 is the normal esophageal mucosa; group 4 received NMBA alone; group 5 was given NMBA plus solvent; groups 6, 7 and 8 were given JTE-522 at different doses (3, 9 and 30 mg/kg); group 9 also received JTE-522 9 mg/kg, while administration was continued for 24 weeks. N, normal tissue; T, tumor tissue. Data are the means ± SD of pg/µg protein accumulation. *P < 0.01, group 4 (tumor tissue) versus group 1 (normal tissue); **P < 0.01, groups 6, 7, 8 and 9 (tumor tissue) versus group 4 (tumor tissue) (significance determined using the Tukey–Kramer test).

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Since esophageal carcinogenesis is a complicated and multistep process, and treatment by curative surgical resection is difficult, prevention of carcinogenesis is thought to be an important strategy in reducing the incidence and mortality rates of esophageal cancer. Consistent with recent epidemiological investigation, intake of aspirin was associated with an up to 90% decrease in the risk of developing esophageal cancer (21). This result may imply that it is possible NSAIDs reduce esophageal carcinogenesis by inhibiting COX. However, due to the non-selective inhibition of NSAIDs on both COX-1 and COX-2, which results in many untoward gastrointestinal side effects (e.g. ulceration and bleeding), their clinical use is limited. As such, development of more selective COX inhibitors, such as JTE-522, for the prevention and treatment of esophageal carcinogenesis is thought to be an important strategy.

In the present study, we were interested in a comparison of the tumor multiplicity (number and size of tumors per rat) in each group (summarized in Table IIIGo) in order to understand the effects of JTE-522 against the formation and growth of tumors. Oral administration of the selective COX-2 inhibitor JTE-522 potently reduced NMBA-induced esophageal tumor multiplicity. Our results indicated that JTE-522 dose-dependently reduced the formation of NMBA-induced esophageal tumors in rats and time-dependently inhibited the formation and growth of tumors. This finding is perhaps important for future clinical use of JTE-522. Our data clearly showed that JTE-522 suppressed not only tumor formation but also developing carcinomas. The same results were also observed in colon cancer and other esophageal carcinogenesis animal models (33,34).

With regard to the mechanism of the inhibitory effects of JTE-522 on NMBA-induced esophageal tumorigenesis in rats, our study revealed that COX-2 expression in tumor tissues from rats that received NMBA alone was elevated 5-fold, while the level of PGE2 production increased 7-fold, compared with normal tissue. Treatment with JTE-522 did not change the increased COX-2 mRNA expression in every group; however, the level of PGE2 in the continuously administered group was significantly reduced. These findings indicate that JTE-522 cannot suppress COX-2 mRNA expression, but it can inhibit one activity of COX-2: the synthesis of COX-2-mediated PGE2. Increased PGE2 levels due to increased COX-2 levels have not been shown definitively according to our data; however, COX-2 is the rate limiting enzyme of PGE2 production (35), therefore, a strong correlation may exist between PGE2 and COX-2 levels. Zimmermann et al. (16) also reported that, in esophageal cancer cells producing a large amount of PGE2, COX-2 inhibitors induced apoptic cell death and reduced proliferation through the inhibition of PG synthesis. In another study, PGE2-stimulated human prostatic carcinoma cell growth and inhibition of PGE2 synthesis by COX-2 inhibitors was thought to be one of the chemopreventative mechanisms (36). Thus, excessively synthesized PGE2 mediated by overexpression of COX-2 is believed to play an important role in neoplasma formation in NMBA-induced esophageal tumorigenesis models. Inhibition of COX-2 activity may at least partly explain the chemopreventative effect of aspirin against esophageal cancer in humans (21). The investigation of the potential for COX-2 inhibitors in preventing adenocarcinoma is a major issue in western countries. However, our NMBA model did not develop adenocarcinomas. In order to evaluate the effect of COX-2 inhibitors on carcinogenesis of esophageal adenocarcinoma, other carcinogenesis models, such as the reflux esophagitis model (37) or prophylactic administration of COX-2 inhibitor for patients with Barrett's esophagus, may be useful.

In conclusion, our data suggest that COX-2 and COX-2-mediated PGE2 synthesis may play an important role in rat NMBA-induced esophageal tumorigenesis, which is partly inhibited by selective COX-2 inhibition. This well-defined animal model is valuable for studying modulation of COX-2 expression in esophageal carcinogenesis, and COX-2 therefore may be a promising chemotherapeutic target for the treatment of human esophageal cancer.


    Notes
 
1 To whom correspondence should be addressed. Email: shimada{at}kuhp.kyoto-u.ac.jp Back


    Acknowledgments
 
We thank Japan Tobacco Co. (Tokyo, Japan) for kindly donating the selective COX-2 inhibitor, JTE-522. We thank Dr T.Sakurai (the Department of Clinical Pathology, Kyoto University (Kyoto, Japan) for valuable advice and teaching about the histological diagnosis of tumor tissues in rat esophagi. We thank Dr H. Chikuma for statistical analysis suggestions. We also thank Drs A.J.Dannenberg and N.K.Artorki for useful suggestions.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received September 4, 2000; revised December 4, 2000; accepted December 5, 2000.