Vol. 10, 8105–8113, December 1, 2004 Clinical Cancer Research 8105 The Selective Cyclooxygenase-2 Inhibitor Nimesulide Prevents Helicobacter pylori-Associated Gastric Cancer Development in a Mouse Model Ki Taek Nam,1 Ki-Baik Hahm,2 Sang-Yeon Oh,1 Results: Gastric tumors developed in 68.8% of mice Marie Yeo,2 Sang-Uk Han,2 Byeongwoo Ahn,3 that were given both MNU and H. pylori, whereas less than 10% developed gastric tumors when given either MNU or H. Young-Bae Kim,2 Jin Seok Kang,1 1 1 pylori alone. These findings indicate that H. pylori promotes Dong Deuk Jang, Ki-Hwa Yang, and carcinogen-induced gastric tumorigenesis. In mice treated Dae-Yong Kim4 with both MNU and H. pylori, nimesulide administration 1Department of General Toxicology, National Institute of substantially reduced H. pylori-associated gastric tumori- Toxicological Research, Korea Food and Drug Administration, Seoul; genesis, whereas substantial inductions of apoptosis were 2Genome Center for Gastroenterology, Ajou University School of 3 observed. In vitro studies demonstrated that nimesulide and Medicine, Suwon; Department of Veterinary Pathology, College of H. pylori when combined acted synergistically to induce Veterinary Medicine, Chungbuk National University, Cheongju; and 4Department of Veterinary Pathology, College of Veterinary Medicine more apoptosis than either alone. and School of Agricultural Biotechnology, Seoul National University, Conclusions: Our data show that nimesulide prevents Seoul, Korea H. pylori-associated gastric carcinogenesis, and suggest that COX-2 may be a target for chemoprevention of gastric cancer. ABSTRACT Purpose: Helicobacter pylori infection can lead to gastric INTRODUCTION cancer, and cyclooxygenase-2 (COX-2) is overexpressed in Helicobacter pylori causes chronic active gastritis and pep- the stomach during H. pylori infection. Therefore, we inves- tic ulcer disease, and is linked with gastric adenocarcinomas, tigated whether nonsteroidal anti-inflammatory drugs including gastric mucosa-associated lymphoid tissue lymphoma might protect against this form of cancer. Specifically, we (1). On the basis of epidemiologic data, WHO/IARC classified examined the chemopreventive effect of the COX-2 inhibitor H. pylori as a group 1 carcinogen (2). Generally, gastric ade- nimesulide on H. pylori-associated gastric carcinogenesis in nocarcinoma develops through a multistep process from normal mice. gastric mucosa to chronic active gastritis, to gastric atrophy and Experimental Design: C57BL/6 mice were treated with intestinal metaplasia, and finally to dysplasia and neoplasia (3), the carcinogen N-methyl-N-nitrosourea (MNU) and/or H. and it has been postulated that H. pylori plays a causative role pylori. To determine the effect of COX-2 inhibition, nime- in the early phases of this malignant progression (4, 5). How- sulide was mixed with feed pellets and administered for the ever, debate still exists as to whether H. pylori is really a duration of the experiment. All of the mice were sacrificed carcinogen or a cancer promoter, and whether eradication of 50 weeks after the start of the experiment. Histopathology, H. pylori is beneficial to people free of gastric tumors (6–9). immunohistochemistry, and Western blotting for COX-2, A possible explanation for the link between H. pylori Bax and Bcl-2 were performed in stomach tissues. In vitro infection and gastric carcinogenesis is that H. pylori infection experiments with the human gastric cancer cell line AGS raised cyclooxygenase-2 (COX-2) mRNA/protein levels, and were also performed to identify mechanisms underlying can- stimulated release of prostaglandin E2 in H. pylori-associated cer chemoprevention by nimesulide. premalignant and malignant gastric lesions (10–16). There is strong evidence that COX-2 is causally involved in gastrointes- tinal cancer (17–22). In addition, growth of colon polyps was retarded or blocked by either administration of nonsteroidal anti-inflammatory drugs (NSAIDs; refs. 23–25) or targeted de- Received 5/7/04; revised 8/30/04; accepted 9/9/04. Grant support: Supported by a grant of the Korean Health 21 R&D letion of the COX-2 gene (26). Because induction of COX-2 Project, Ministry of Health & Welfare, Republic of Korea (02-PJ1-PG3- expression has been shown to play an important role in neo- 20802-0014, 01-PJ10-PG6-01GN14-0007) and by Brain Korea 21 Pro- plastic transformation in the large intestine, it is our hypothesis ject; and supported in part by Korea Food and Drug Administration. that COX-2 is involved in H. pylori-associated gastric cancer The costs of publication of this article were defrayed in part by the development, and that NSAID administration can prevent or payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to retard this process. indicate this fact. It has been shown that H. pylori increased release of Requests for reprints: Dae-Yong Kim, Department of Veterinary Pa- prostaglandin E2 and COX-2, which were overexpressed in most thology, College of Veterinary Medicine and School of Agricultural metaplastic and adenomatous tissues, as well as in gastric ade- Biotechnology, Seoul National University, San 56-1, Shillim-dong, Kwanak-gu, Seoul 151-742, Korea. Phone: 82-2-880-1249; Fax: 82-2- nocarcinoma (27). Despite these observations, it remains un- 388-6451; E-mail: [email protected]. known as to whether COX-2 may be a target for chemopreven- ©2004 American Association for Cancer Research. tion of H. pylori-associated gastric carcinogenesis. In the Downloaded from clincancerres.aacrjournals.org on September 27, 2021. © 2004 American Association for Cancer Research. 8106 Chemoprevention of H. pylori-Gastric Cancer by Nimesulide present study, we used an H. pylori-associated gastric cancer CRF-1 diets throughout the experiment. Mice were sacrificed 38 mouse model (28, 29) to investigate the preventive effects of the weeks after infection, making a total of 50 weeks of treatment. selective COX-2 inhibitor nimesulide, and we studied the mech- Histopathologic Examination. Immediately after sacri- anisms underlying nimesulide-induced chemoprevention with fice, mouse stomachs were opened along the greater curvature. AGS human gastric cancer cell line. The number, as well as the long diameter, of tumors in the stomach was measured. A record was kept of the size and MATERIALS AND METHODS number of tumors counted, with a diagnosis made after the final Mice. Male C57BL/6 mice were obtained from the Jack- histopathologic examination. One half of the excised stomachs, son Laboratory (Bar Harbor, ME) at 5 weeks of age. The mice including neoplastic nodules, were fixed in neutral-buffered were maintained in an accredited Korea FDA animal facility in 10% formalin and were cut into approximately six strips, which accordance with the AAALAC International Animal Care pol- were processed by standard methods, embedded in paraffin, icies (Accredited Unit-Korea Food and Drug Administration: sectioned at 4 ␮m, and stained with hematoxylin and eosin Unit Number-000996). All of the mice were given a standard (H&E). The remaining portions were quickly frozen in liquid pellet chow diet (CRF-1, Oriental Yeast Co. Ltd., Tokyo, Japan) nitrogen and stored at Ϫ70°C until analysis. Histologic classi- ad libitum and were maintained in specific pathogen-free con- fication was based on histopathologic and cytologic criteria ditions. proposed by Leininger and Jokinen (30). After histopathologic Chemicals and Bacteria. N-methyl-N-nitrosourea classification was done, tumor incidence and multiplicity was (MNU; Sigma Chemical Co., St. Louis, MO) solutions were calculated. freshly prepared twice a week by dissolving 200 ppm MNU in Identification of H. pylori in Gastric Mucosa. To con- distilled water. When indicated, mice were given the 200-ppm firm H. pylori infection, we transferred samples (ϳ3-mm2)of MNU solution ad libitum in light-shielded bottles in place of stomach mucosa from the greater curvature containing both drinking water. Mouse-adapted H. pylori (SS1) were inoculated fundic and pyloric glands to 1.0 mL of sterile 0.1 mol/L PBS; on Brucella agar plates (Becton Dickinson, Cockeysville, MD) these were homogenized and plated on selective trypticase soy containing 10% heat-inactivated fetal bovine serum and Skirrow agar/5% sheep blood plates containing vancomycin (20 mg/ medium (Difco, Detroit, MI). They were kept at 37°C under mL), nalidixic acid (10 mg/mL), bacitracin (30 mg/mL) and microaerobic conditions with GasPak jars (Difco) and Campy- amphotericin B (2 mg/mL) from Sigma Chemical Co., and Paks (Becton Dickinson). After 24 hours of fasting, a 0.1-mL grown for 3 to 5 days. Colonies were identified by characteristic suspension of H. pylori containing 1 ϫ 109 colony-forming Gram’s stain morphology, and by urease, catalase, and oxidase units (CFU)/mL was administered by intragastric intubation. activity. Another 3-mm2 sample from the antrum was placed Study Design. The experimental design is illustrated in into the gel of a rapid urease test kit (CLO test, Ballard Medical Fig. 1. Mice were randomized into five groups. Animals of Products, Draper, VT) and was left for 6 hours at room temper- groups 2, 3, and 5 were given MNU. One week after completion ature to test for urease activity. The presence of H. pylori in the of MNU administration, mice in groups 3 and 5 were inoculated gastric pit was further confirmed by Warthin–Starry staining. with H. pylori three times every other day. Mice in groups 1 and Immunohistochemistry for COX-2. Immunohisto- 4 were inoculated with H. pylori three times every other day (no chemical identification of COX-2 expression was performed on MNU treatment). Animals of groups 4 and 5 were then given a replicate sections of stomach tissues. The sections were CRF-1 diet (Oriental Yeast Co. Ltd.) containing 200 ppm nime- mounted on silanized slides (Dako, Glostrup, Denmark) and sulide (Choongwae Pharmaceutical Research Institute, Suwon, were dewaxed and rehydrated; endogenous peroxidase activity Korea).