intracellular recording techniques were used to investigate the effects of the endogenous CB1 receptor agonist anandamide, the synthetic CB1 receptor agonist WIN 55,212-2 and the CBI specific antagonist SR141716 on smooth muscle membrane potentials following electrical neuronal stimulation. Experiments were performed in wild-type as well as in CB1- deficient mice. Results: Focal electrical stimulation of the myenteric plexus induced a fast excitatory junction potential (EJP; abolished by atropine) followed by a fast inhibitory junction potential (fliP; reduced by apamin) and a slow inhibitory junction potential (slJP; abolished by L-NNA). None of the used drugs had an influence on the resting membrane potential. Addition of anandamide (10-6-10-5M) in the wildtype abolished the EJP while leaving the flJP and the slJP unchanged. The more potent CB1 agonist WIN 55,212-2 reduced the EJP and the flJP but did not change the sUP. In the CB1 deficient mice anandamide and WIN 55,212-2 had no effect. SR141716 when given alone increased the EJP in wildtype mice while leaving the IJPs in the wildtype and the EJPs and IJPs in the knockout mice unchanged. 5R141716 reversed all anandamide and WIN 55,212-2 effects in the wildtype mice. In the CB 1 defficient mice, the EJFs are significantly increased compared to wildtype, the UPs remein unaltered. Conclusions: We conclude that the CB1 eannabinoi- dreceptor modulates excitatory and inhibitory neurotranamission in the mouse colon. Since in the CB1 deficient mice EJPs are sigmficantly increased these findings strongly suggesting a physiologic involvement of CB1 receptors in cholinergic neurotransmission. Finally, our study supports that no other receptors than CB1 are involved in the anandamide action on mouse distal colon since the endogenous agonist anandamide had no effect in CB1- deficient mice.

846

Host and Microbial Determinants Influence Helicobacter pylorMnduced Gastric Carcinogenesis in a Murine Model of Hypergastrinemia James Fox, Timothy Wang, Arlin Rogers, Theofflos Poutahidis, Zhongming Ge, Nancy Taylor, Charles Dangler, Dawn Israel, Urea Krishua, Kristen Gaus, Richard M. Peek Jr.

Hdicobacter pylori LS a strong risk factor for intestinal-type gastric adenocarcinoma, a malig- nancy that predominates in males, and strains possessing the tag pathogenicity island further augment cancer risk. We examined the effects of an H. pylori cancer-associated deterrmuant (cagE) and host gender on tbe development of pre-maliguant and malignant lesions in a transgenic hypergasmnemic (INS-GAS) munne model of gastric carcinogenesis. Methods: Male (n = 70) and female (n = 22) INS-GAS mice infected with H. pylori wild-type strain Bi28, its isogenic cagE mutant, or H. Jelis, were sacrificed 2-24 weeks post-challenge. Colonization efficiency was determined by immunohistocbeimstry, fluorescent in situ hybnd- Lzation, real-time PCR, and quantitative culture; inflammation and injury were scored from 0-4 Results: All mice challenged with H. pylori were successfully colonized. Male INS-GAS mice infected with H. pylori strain B128 developed glandular atrophy, intestinal metaplasia, and dysplasia by 6 weeks, and carcinoma was evident in all colonized males by 24 weeks. Inactivation of cagE delayed the progression to carcinoma, but neoplasia ultanately developed in all male mice challenged with the H. pylori mutant strain. In contrast to the malignant phenotype displayed by male mice, none of the H. pylor~-infected females developed cancer, and injury scores were significantly higher in males compared to females at each time-point. Conclusions: H. pylor~ infection engenders the development of gastric adenocarcinoma in an experimental mouse model of disease. Cancer is restricted to males, which parallels the epidemiologic features of intestinal-type gastric cancer in humans, and loss of cage temporally retards, but does not abrogate pathologic progression. The INS-GAS model is effective for investigating discrete host-microbial interactions that lead to gastric cancer within the context of biological conditions, such as inflammation and bypergastrinemia, induced by H. pylori col- onization.

847

Chemoprevention of Mnng-Induced Gastric Cancer in Rat by Celecoxih Is Independent of COX-2 and PGE2 Suppression Jun Yu, Baodong Tang, Wai K. Leung, Pinjin Hu, Ka-Fai To, Alfa Bai, Po-Ki Ma, Minnie Y Go, Zhirong Zeng, Joseph J. Sung

Chemopreventive effects of nonsteroidal anti-intlammatory drugs (NSAIDs) have been sug- gested to be mediated by inhibition of cyclooxygenase (COX). Although overexpression of COX2 was frequently demonstrated in gastric cancer, the role of NSAIDs or specific COX2 inhibitors in prevention of gastric carcinogenesis and the underlying mechanism remains unknown. Aim: This study characterizes the expressions of COX1, COX2, prostaglandin E2 (PGE2) and monitor changes in cell kinetics in MNNG induced gastric cancer with celecoxib or indomethacin treatment. Methods:Wistar rats were gnven gastric carcinogen MNNG alone or MNNG with celecoxib (10 mg/kg/d) or MNNG with nidomethacin (3 mg/kg/d) for 40 weeks. The levels of COX1 and COX2 mRNA and protein expresstons in gastric mucosa were determined by real time PCR and immunohistochemistry. PGE2 levels were measured by an ELISA. Apoptosis (AI) was quantified by apoptouc nuclei counting and proliferation (P1) by Ki67 immunostaining. Results: Treatment with celecoxib, but not indomethacin, significantly inhibited MNNG induced gastric cancer in rats (Hu et al. DDW 2003). There was no stguificant alteration in COX1 expression with MNNG or COX inhibitors treatment. MNNG induced expression of COX2 (8.5-+ 2.0 vs 0.5-+ 0.2, p <0.0001) and PGE2 (541+_271 vs 178+_54, p =0.018) in tumor when compared to control rats. Although treatment with celecoxib and indomethacin only marginally reduced the COX-2 and PGE2 production in MNNG induced gastric tumor (p = 0.32 and 0.21 ), celecoxib induced apoptosis (p = 0.043) and inhibited proliferation (p = 0.012) of tumor cells. Indomethacin only caused suppression of cell proliferation (p = 0.0002) but did not increase apoptotic activity (p = 0.5). Conclusion: Our results demonstrate: (a) upregulation of COX2 in MNNG induced gastric cancer in rats, (b) the anti-neoplastic effect of celecoxib is via induction of apoptosis and inhibition of gastric cancer cell proliferation. These effects are independent of COX2 and PGE2 suppression

COX2 6`5T0 ~1" 2.9N0.5 T NT L2~2,4 L7'O,8 6,01'3.2 12N0.4 3.90.8 130.9 0.9~0.4 P ~ 541~Z/t 23473 20~96 397186 ~l'O 152 164107 296 196 217131 ~02~ 139 AI 0./,19 ~ 6.24 003'0.08 0.03~0.02 L0t '0,37 056'015 a02,O.Ol a6?~o 21 0.42'013 001'0.01 I:t 22.1 ~7.6 16`1113 1.5,07 2.9~2.0 6`2L0 0.80.7 6.9,3.9 1078.7 r r=ttwo', I ~ = ~ t ~ N=r, umal from ,o~mcer ra/~, Al=apoplolr i naex, Pl=pr~lllera~ index

848

Treatment of Human Gastric Cancer by Adenoviral-Vector Mediated Expression of NK4 - in Vitro and in Vivo Experiments Danielle Heideman, Victor V. Bensechem, Efisabeth Bloemena, Peter Snijders, Mikael Craanen, Johan Offerhaus, Patrick Derksen, Michiel D. Bruin, Adhiambo Witlox, Bonnie Molenaar, Chris Meijer, Winald Gerritsen

Hepatocyte Growth Factor (HGF) is a pleiotropic factor that binds to and activates its cellular receptor c-met. Stimulation of c-met triggers mitogenic and motogenic responses of cells and, in the context of cancer, may induce tumor growth, metastasis and angiogenesis. In this study, we demonstrated that normal gastric epithelial cells did not express HGF and c-met, whereas the majority of gastr c cancers, i.e. 76%, overexpressed c-met and in all cancers HGF was produced by either tumor or stromal cells. Therefore, we investzgated if blocking the HGF-c-met signaling pathway would affect the biological behavior of gastric adenocarcinoma. To this end, we employed adenoviral vector-mediated expression of NK4, a competitive antagonist of HGF and effective inhibitor of angiogenesis. We demonstrated efficient production and secretion of NK4 by gastric cancer cells transduced with the replica- tion-deficient adenoviral vector AdCMV.NK4. This resulted in significant inhibition of prolif- eration, mtgration and invasion of gastric cancer cells. Gastric carcinoma cells produced biologically significant amounts of angiogenic factors such as VEGF, and induced mitogenic responses of endothelial cells. Transduction of tumor cells with AdCMV.NK4 significantly reduced the proliferation of vascular endothelial cells and microvessel sprouting from aortic rings. Finally, treatment of established human gastric tumor xenografts in nude mice with AdCMV.NK4 resulted in a significant tumor growth delay. In conclusion, the present study shows, in vitro and in vivo, that adenoviral vector-mediated expression of NK4 is a promising strategy to treat human gastr c cancer by simultaneously inhibiting gastn c tumor cell motility and proliferation as well as tumor angiogenesis.

849

A Pro-lnflammatory Genetic Profile Increases the Risk of Chronic Atrophic Gastritis and Gastric Carcinoma Jose C. Machado, Ceu Figueiredo, Paulo Canedo, Serglo Nabais, Catarina C. Alves, Maria L. Campos, Raquel Seruca, Fatima Cameiro, Manuel Sobrinho-Simoes

It was demonstrated that proinflammatory polymorphisms within the genes interteukin (IL)IB and IL1RN are associated with risk of gastric carcinoma in Helicobacter pylon- infected individuals. We aimed to determine the assoctation between variation of the tumor necrosis factor alpha (TNFA) gene and risk of chronic atrophic gastritis and gastric carcinoma, and to investigate the extent to which the combined effect of proinflammatory genetic polymorphisms influences such risk. In a case-control study including 306 controls, 221 individuals with chrome gastritis and 287 gastric carcinoma patients, the TNFA-308 and 1L1B-511 bi-aflehc polymorphisms, and the IL1RN variable number of tandem repeats were genotyped. Odds ratios (OR) were estimated by logistic regression analysis. We found a significant association between the TNFA-308 polymorphism and increased risk of gastric carcinoma development with an OR of 1.9 (95% CI 1.3-2.7). Both for chronic atrophic gastritis and gastric carcinoma the odds of developing disease increased with the number of high-risk genotypes. Individuals carrying the three high-risk genotypes have an increased risk of chronic atrophic gastritis and gastric carcinoma with an OR of 5.8 (95% CI 1.1- 31.0) and 9.7 (95% CI 2.6-36.0), respectively. These findings demonstrate that it is possible to define a specific genetic profile associated with highest risk of chronic atrophic gastritis and gastric carcinoma. Taking into account the high prevalence of H. pylon infection in many countries, current costs of therapy and antibiotic resistance, host and bacterial genotyping are likely to constitute a reasonable basis for identifynig individuals who are at greatest risk of developing gastric carcinoma.

850

Adenocarcinomas of the Gastro-Esophageal Junction: A Comparative Study of the Gastric Cardia and the Esophagus with Respect to Cyclooxygenase-2 Expression Christiartne Buskens, Anna Sivula, Marinke Westerterp, Johan Offerhaus, Ari Ristimaki, Jan Van Lansehot

Adenocarcinomas of the gastric cardia and distal esophagus are nowadays often considered as one clinical entity due to their comparable increasing incidence, prognosis and optimal treatment options. However, it is not yet known if these malignancies have the same etiology and genotype. In a previous study, we demonstrated that elevated expression of the cyclooxygenase-2 (COX-2) protein is an independent prognostic factor in a Barrett carcinoma. The aim of this study was to analyze the role of COX-2 in the development of cardia carcinoma, to correlate the COX-2 expression to clinicopathological parameters and survival, and to compare these resuks to the prognostic value of COX-2 found in esophageal adenocarcinomas. Tumor sections of 134 consecutive patients undergoing potentially curative surgery for an adenocarcinoma arising from the gastric cardia and substantially invading the distal esophagus were immunohistochemicany stained using a COX-2 monoclonal anti- body. The specimens were blindly scored based on the intensity and extent of COX-2 immunopnsitivity. COX-2 immunoreactivity was negative to weak in 59% (COX-2 Low) and moderate to strong in 41% (COX-2 High) of the tumors. This was significantly different when compared to the Barrett carcinomas of which 79% had a COX-2 High expression (p<0.0001). COX-2 expression was not significantly correlated with any clinicopathological

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