Synergistic Inhibitory Effects of Gastrin and Histamine Receptor Antagonists on Helicobacter-Induced Gastric Cancer

Transcription

1 GASTROENTEROLOGY 2005;128: Synergistic Inhibitory Effects of Gastrin and Histamine Receptor Antagonists on Helicobacter-Induced Gastric Cancer SHIGEO TAKAISHI,* GUANGLIN CUI, DANA M. FREDERICK, JANE E. CARLSON, JEANMARIE HOUGHTON, ANDREA VARRO, GRAHAM J. DOCKRAY, ZHONGMING GE, MARK T. WHARY, ARLIN B. ROGERS, JAMES G. FOX, and TIMOTHY C. WANG* *Division of Digestive and Liver Disease, Department of Medicine, Columbia University, College of Physicians and Surgeons, New York, New York; Gastroenterology Division, Department of Medicine, University of Massachusetts Medical School, Worcester, Massachusetts; Department of Physiology, School of Medicine, University of Liverpool, Liverpool, United Kingdom; and Division of Comparative Medicine, Massachusetts Institute of Technology, Cambridge, Massachusetts Background & Aims: Apart from its importance as an acid secretogogue, the role of histamine as a downstream target of gastrin has not been fully explored. Previous studies have shown that the combination of hypergastrinemia and Helicobacter infection resulted in accelerated gastric cancer in mice. We used this model to examine the role of cholecystokinin 2 (CCK2)/gastrin receptor and histamine H 2 -receptor signaling in the development of gastric atrophy and cancer. Methods: Male hypergastrinemic mice (INS-GAS mice) were infected with Helicobacter felis and given the CCK2/gastrin receptor antagonist YF476 and/or the histamine H 2 -receptor antagonist loxtidine for 3 or 6 months. In addition, mice were treated with omeprazole alone or in combination with either YF476 or loxtidine for 3 months. Results: Mice treated with YF476 or loxtidine alone showed partial suppression of both gastric acid secretion and progression to neoplasia. The combination of YF476 plus loxtidine treatment resulted in nearly complete inhibition of both parameters. YF476 and/or loxtidine treatment did not alter the overall level of H felis colonization but did result in significant down-regulation of the growth factors regenerating gene I and amphiregulin. Loxtidine treatment, with or without YF476, induced a mild shift in T-helper cell polarization. In contrast, omeprazole treatment resulted in mild progression of gastric hyperplasia/dysplasia, which was ameliorated by the addition of YF476 or loxtidine. Conclusions: The combination of CCK2/gastrin- and histamine H 2 -receptor antagonists has synergistic inhibitory effects on development of gastric atrophy and cancer in H felis/ins-gas mice, while the proton pump inhibitor showed no such effects. These results support an important role for the gastrin-histamine axis in Helicobacter-induced gastric carcinogenesis. Recent studies have confirmed that Helicobacter pylori infection represents the primary environmental risk factor for noncardia gastric cancer. 1,2 Based on accumulated epidemiologic evidence, a Working Group of the International Agency for Research on Cancer, a branch of the World Health Organization, classified H pylori as a group I carcinogen in Nevertheless, many questions remain regarding the possible mechanisms by which this infectious agent is able to promote neoplasia of the stomach. Data derived from both animal models and human studies have pointed to host immune responses, particularly strong Th1 immune responses, as critical to the development of gastric atrophy and intestinal metaplasia, preneoplastic conditions strongly associated with progression to cancer. 4,5 However, more recent studies in mice have also suggested a role for hypergastrinemia in the pathogenesis of gastric cancer. In the absence of Helicobacter infection, the hypergastrinemic (insulin-gastrin [INS-GAS]) mouse at early time points (age, 1 4 months) shows moderate hypergastrinemia and a 2- to 3-fold increase in stimulated acid secretion consistent with increased parietal cell mass. However, this is followed at later time points (5 months and older) by a marked decline in acid secretion with the spontaneous development of gastric atrophy, metaplasia, and invasive cancer that can be markedly accelerated by concurrent Helicobacter infection. 6,7 These data suggested the notion that elevations in circulating amidated gastrin levels might directly promote gastric atrophy and preneoplasia as well as neopla- Abbreviations used in this paper: ECL, enterochromaffin-like; EGF, epidermal growth factor; ELISA, enzyme-linked immunosorbent assay; GERD, gastroesophageal reflux disease; HB-EGF, heparin-binding epidermal growth factor like growth factor; HDC, histidine decarboxylase; IFN, interferon; IL, interleukin; INS-GAS, insulin-gastrin; PPI, proton pump inhibitor; Reg I, regenerating gene I; RT-PCR, reverse-transcription polymerase chain reaction; TGF, transforming growth factor; TNF, tumor necrosis factor by the American Gastroenterological Association /05/$30.00 doi: /j.gastro

2 1966 TAKAISHI ET AL GASTROENTEROLOGY Vol. 128, No. 7 sia of the stomach in a susceptible host. Nevertheless, the precise mechanism of action of gastrin and the critical downstream cellular targets of gastrin in this model have not been defined. The decrease in parietal cells observed over time in hypergastrinemic mice could be due to either decreased production or increased turnover of parietal cells. In addition, the effects of gastrin on the gastric mucosa could be either a direct response of cells expressing the cholecystokinin 2 (CCK2)/gastrin receptor or a response by other cells responding to growth factors released by gastrin. The effects of amidated gastrin are clearly transduced by the CCK2/gastrin receptor, a member of the larger G-protein coupled receptor family, and expression of the CCK2 receptor on both parietal and enterochromaffin-like (ECL) cells of the stomach has clearly been demonstrated. 8,9 Thus, gastrin likely exerts its proliferative effects at least partly indirectly through up-regulation of paracrine growth factors such as heparin-binding epidermal growth factor like growth factor (HB-EGF) (expressed in parietal cells) and regenerating gene I (Reg I) (expressed in ECL cells). The possibility that gastrin exerts direct effects on a progenitor cell population in the oxyntic glands has also been suggested. 10 However, one of the main targets of gastrin in the gastric mucosa is certainly histamine production, given that histamine is the final common mediator of acid secretion. Hypergastrinemia acting on CCK2 receptors on ECL cells leads to both increased histamine release and increased histamine production through upregulation of histidine decarboxylase (HDC), which stimulate acid secretion through histamine H 2 receptors on parietal cells. Nevertheless, the relative importance of histamine in the long-term mucosal response to hypergastrinemia in the INS-GAS mouse has not been directly addressed. The role of hypergastrinemia in the development of gastric atrophy, particularly in human patients with or without H pylori infection, has been somewhat more controversial. It is generally accepted that infection with H pylori results in a 1.5- to 2.0-fold elevation in serum amidated gastrin levels that occurs early in the course of infection, precedes the development of atrophic gastritis, and typically resolves after eradication of infection. In addition, the majority of clinical studies have accepted that proton pump inhibitors (PPIs), which typically induce hypergastrinemia, accelerate the onset of atrophic gastritis in H pylori positive patients In general, the assumption has been that the potential promotion of atrophic gastritis by PPIs may primarily be due to the decrease in gastric acid secretion, which is believed to lead to changes in the distribution of H pylori colonization and worsening of corpus gastritis. Indeed, acid suppression in mice has been shown to produce similar changes in Helicobacter felis colonization within the murine stomach. 17 However, in most of the studies performed primarily in patients with gastroesophageal reflux disease (GERD), there was a strong correlation between the degree of atrophic gastritis and serum gastrin levels. In one study, H pylori infected patients with the highest serum gastrin levels before PPI therapy showed the most marked progression in gastric atrophy during PPI therapy. 18 To explore the relative importance of achlorhydria versus hypergastrinemia in the pathogenesis of Helicobacter-mediated gastric atrophy, we examined in our model the effects of 2 acid-suppressive reagents: the CCK2/ gastrin receptor antagonist YF and the irreversible histamine H 2 -receptor antagonist loxtidine. 20 Short-term studies with the PPI omeprazole were also undertaken for purposes of comparison. The 3 drugs act on different targets, but all 3 inhibit gastric acid secretion and also induce hypergastrinemia. Importantly, the first 2 drugs inhibited progression of gastric preneoplasia while the third appeared to accelerate it. The results of this study point to the importance of the gastrin-histamine axis in the pathophysiology of Helicobacter-induced gastric atrophy and carcinogenesis and may also have clinical implications for the development of safer therapeutic approaches that result in chronic acid suppression. Materials and Methods Animals The INS-GAS transgenic mice (FVB/N background) have been described previously 7,21 and were free of specific murine pathogens. Animals were housed in microisolator, solid-bottomed polycarbonate cages, fed a commercially prepared pelleted diet, and given water ad libitum. A total of 124 male INS-GAS mice at 2 or 3 months of age were inoculated with H felis (ATCC 49179) 3 times every other day in 1 week with a dose of about 1 billion colony-forming units per mouse each time. 7 Infection status was confirmed at 14 weeks and 26 weeks after inoculation by enzyme-linked immunosorbent assay (ELISA) to measure immunoglobulin (Ig) G antibody to H felis and by quantitative real-time polymerase chain reaction (PCR) assays of gastric corpus tissue at necropsy as described in the following text. All experiments were approved by the Institutional Animal Care and Use Committee of University of Massachusetts Medical School. Drugs and Experimental Design The CCK2/gastrin receptor antagonist YF476 was a kind gift of Dr Keiji Miyata and Dr Hidenobu Yuki (Yamanouchi Pharmaceutical Co Ltd, Tsukuba, Japan). 19 The drug was dissolved in polyethylene glycol (PEG) 300 at a concentration of 12 mg/ml and subcutaneously injected every week

3 June 2005 GASTRIN/HISTAMINE AND GASTRIC CANCER 1967 at a dose of 40 mg/kg (80 mol/kg). 22 The irreversible histamine H 2 -receptor antagonist loxtidine was manufactured by GlaxoSmithKline (Research Triangle Park, NC) and a kind gift of Prof Duan Chen and Prof Rolf Hakanson. The drug was dissolved in sterilized drinking water at a concentration of 0.5 g/l and given to the mice ad libitum as previously described. 23 Loxtidine-containing water bottles were changed weekly, and consumed water volume was measured for each bottle. The PPI omeprazole was purchased from Sigma Chemical Co (St Louis, MO) and dissolved in dimethyl sulfoxide/peg 300, a 1:1 mixture at a concentration of 40 mg/ml, and injected intraperitoneally daily at a dose of 4 mg/mouse (350 mol/kg). 24 One hundred male INS-GAS mice approximately 3 months old with H felis infection were divided into 4 groups (25 mice per group) and treated with YF476 and loxtidine for 3 or 6 months. Mice in the first group were treated with vehicle only as controls, whereas mice in the second group were subcutaneously injected with the CCK2/gastrin receptor antagonist YF476 at a dose of 80 mol/kg once per week. Mice in the third group were treated orally with the histamine H 2 -receptor antagonist loxtidine (in drinking water) at a dose of 0.5 g/l, and mice in the fourth group received both drugs. Ten mice in each group were assigned to 3 months of drug study, and 15 mice in each group were assigned to 6 months of drug study. An additional 24 mice, approximately 2 months old with H felis infection, were divided into 4 groups (6 mice per group). Mice in the first group were treated with vehicle only as controls, and mice in the second group were treated with omeprazole alone for 3 months. Mice in the third and fourth groups were treated with a combination of omeprazole and YF476 or loxtidine for 3 months, respectively. Histologic Evaluation and Immunohistochemical Studies At necropsy, linear strips extending from the squamocolumnar junction through the proximal duodenum were fixed in 10% neutral-buffered formalin, paraffin embedded, cut at 5 m, and stained with H&E. Inflammation, atrophy, hyperplasia, and dysplasia in the gastric corpus were each scored on an ordinal scale from 0 to 4 in increments of 0.5 as previously described 6,25 by a single veterinary pathologist (A.B.R.) blinded to treatment groups. Paraffin-embedded sections were also stained with the Warthin Starry silver staining method for detection of H felis as previously described. 25 Immunohistochemical studies were performed with avidinbiotin-peroxidase complex kits (Vector Laboratories, Burlingame, CA) according to the manufacturer s instructions. The following primary antibodies were used: against chromogranin A to stain ECL cells (working dilution 1:500, rabbit antiporcine; Immunostar, Hudson, WI) and against H,K adenosine triphosphatase subunit to stain parietal cells (1:2000, mouse anti-porcine; Affinity Bioreagents, Golden, CO). Primary antibodies were incubated at room temperature for 1 hour in a humidified chamber. Diaminobenzidine (Vector Laboratories) was used as the chromogen, and slides were counterstained with Mayer s hematoxylin. With respect to cell density counting, immunoreactive epithelial cells with appropriate morphology and location in well-oriented sections were counted using routine microscopy (objective, 20 ; eyepiece, 10 ; visual field diameter, 1.85 mm; CX31, Olympus America, Inc, Woodbury, NY). The immunoreactive epithelial cell densities were expressed as number per gland for parietal cells (10 glands were selected per slide) and number per field for ECL cells (10 visual fields were selected per slide) as previously reported. 26 Measurement of Gastric Acid Output The pyloric ligation method was used to measure gastric acid output as previously described. 27 Briefly, mice were fasted overnight and anesthetized by isoflurane inhalation. The abdomen was incised by midline celiotomy, the pylorus ligated firmly, and the abdomen closed with surgical sutures. After 4 hours, the mice were killed and the gastric juice was collected and then diluted in 100 ml distilled water. The acidity of diluted gastric juice was measured with a ph meter (AR 25; Fisher Scientific, Houston, TX) by 0.01N NaOH titration and results expressed as microequivalents of protons. Evaluation of Serum Antibody Responses to H felis Serum was prepared from mice blood obtained by cutting the left brachial artery just before necropsy at 3 and 6 months after H felis infection and evaluated by ELISA for serum IgG, IgG2a, and IgG1 using an outer membrane antigen preparation of H felis as previously described. 5,6 Antigen was coated overnight at 4 C on Immulon II plates (Thermo Labsystems, Franklin, MA) at a concentration of 1 or 10 g/ml (IgG and subclasses, respectively), and sera were diluted 1:400. Biotinylated secondary antibodies included polyclonal goat anti-mouse IgG (Southern Biotechnology Associates, Birmingham, AL) and monoclonal anti-mouse antibodies produced by clones G1-6.5 and R (PharMingen, San Diego, CA) for detecting IgG1 and IgG2a, respectively. Incubation with ExtrAvidin peroxidase (Sigma Chemical Co, St. Louis, MO) was followed by ABTS substrate (Kirkegaard and Perry Laboratories, Gaithersburg, MD) for color development. Optical density development at 405 nm was recorded by an ELISA plate reader (Dynatech MR7000; Dynatech Laboratories, Inc, Chantilly, VA). Gastrin Radioimmunoassay Plasma gastrin levels (COOH-terminally amidated gastrin) were determined by radioimmunoassay using rabbit antiserum L2 that reacts similarly with G17 and G Real-Time PCR Assay of H felis Infection in Mouse Stomachs H felis DNA present in infected mouse stomachs was quantified using a modification of a previously described realtime PCR assay that accurately quantified H pylori. 28 Two

4 1968 TAKAISHI ET AL GASTROENTEROLOGY Vol. 128, No. 7 primers (forward, 5=-ttcgattggtcctacaggctcaga-3=; reverse, 5=-ttcttgttgatgacattgaccaacgca-3=) were designed to hybridize within the conserved region of the single copy H felis flab gene locus. No products were amplified from DNA isolated from H pylori, Helicobacter mustelae, orhelicobacter bizzozeroni, confirming the specificity of these oligonucleotides for H felis. DNA from plate-grown H felis and from mouse gastric corpus was prepared using a high pure PCR kit (Roche Molecular Biochemicals, Indianapolis, IN). Real-time PCR was performed using SmartCycler (Cepheid, Sunnyvale, CA) and Quantitect SYBR Green PCR kit (Qiagen Inc, Valencia, CA) following the manufacturer s instructions. Briefly, the PCR assay was performed under the following conditions: 95 C for 15 minutes followed by 45 cycles at 95 C for 15 seconds, 55 C for 30 seconds, and 72 C for 30 seconds. Ten-fold dilutions ( to copies) of DNA from H felis were used to generate a standard curve, and serially diluted standards were simultaneously amplified with in vivo samples of mice gastric corpus DNA (100 ng per sample) and H felis mucosal DNA copy numbers were then normalized per stomach DNA (copies per microgram). 25,28 Real-Time Reverse-Transcription PCR Assay of Growth Factors and Cytokine Expression Profiles Total RNAs were extracted from a sample of whole stomach from each animal with TRIzol reagent (Invitrogen, Carlsbad, CA), and 5 g of total RNA was used for first-strand complementary DNA synthesis using the Superscript II cdna Amplification System (Invitrogen) following the manufacturer s instructions. Real-time PCR was performed as previously described using reverse-transcription (RT)-PCR primers for each gene as listed in the following text. All primers were designed using Lasergene version 5.0 software (DNASTAR Inc, Madison, WI). Results were calculated by the Ct method. 29 Briefly, the threshold cycle Ct 1 of each sample reaction was deducted from the threshold cycle Ct 2 of glyceraldehyde-3-phosphate dehydrogenase reaction for normalization and then deducted from the threshold cycle Ct 3 of calibration control (45 cycles in this experiment), that is, the final result was represented by the formula Ct 3 (Ct 1 Ct 2 ). Sequences of RT-PCR primers are as follows. Reg I: forward, 5=-aaggagagtggcactacagacg-3=; reverse 5=-gtattgggcatcacagttgtca-3=; HB-EGF: forward, 5=-gacccatgcctcaggaaataca-3=; reverse, 5=-tacagccaccacagccaagact-3=; amphiregulin: forward, 5=-ggcaaaaatggaaaaggcagaa-3=; reverse, 5=-cgaggatgatggcagagacaaa-3=; transforming growth factor (TGF)- : forward, 5=- gccggtttttggtgcaggaaga-3=; reverse, 5=ttgcggagctgacagcagtgga-3=; interferon (IFN)- : forward, 5=-catggctgtttctggctgttactg-3=; reverse, 5=-gttgctgatggcctgattgtcttt-3=; tumor necrosis factor (TNF)- : forward, 5=-tggcccagaccctcacactcag-3=; reverse, 5=- acccatcggctggcaccact-3=; interleukin (IL)-4: forward, 5=-atcggcattttgaacgaggtca-3=; reverse, 5=-catcgaaaagcccgaaag-3=; somatostatin: forward, 5=-gtcctggctttgggcggtgtca-3=; reverse, 5=-tgcagctccagcctcatctcgt-3=; glyceraldehyde-3-phosphate dehydrogenase: Figure 1. YF476 and/or loxtidine treatment for 3 months strongly inhibited gastric acid output in H felis infected INS-GAS mice. Gastric acid output measured by the pyloric ligation method; acid secretion over 4 hours was determined by the pyloric ligation method and expressed as microequivalent protons. H felis infected INS-GAS mice treated with YF476 and/or loxtidine for 3 months showed a significant decrease of gastric acid output compared with mice receiving no drug. (*P.05; n 4 per each group). LOX, loxtidine; YF LOX, YF476 plus loxtidine; FVB, untreated FVB/N control mice. forward, 5=-gacatcaagaaggtggtgaagc-3=; reverse, 5=-gtccaccaccctgttgctgtag-3=. Statistical Analysis Results are expressed as mean SD unless otherwise stated. Student t test or the Mann Whitney test was used to evaluate statistical significance. Values of P.01 or P.05 were considered statistically significant. Results YF476 and/or Loxtidine Treatment for 3 Months Resulted in Synergistic Inhibition of Gastric Acid Output and Gastric Atrophy, Hyperplasia, and Dysplasia in H felis Infected INS-GAS Mice While the highly specific CCK2/gastrin receptor antagonist YF476 and the irreversible histamine H 2 - receptor antagonist loxtidine have previously been shown to inhibit acid secretion in mice, they have not been studied in models of chronic Helicobacter infection. In addition, they have not previously been examined in a mouse model of gastric cancer such as the hypergastrinemic INS-GAS mouse model. Previous studies have shown that young ( 6 months of age) INS-GAS mice have substantially elevated gastric acid secretion but that Helicobacter infection leads over time to a rapid reduction in gastric acid secretion. 7 Other groups have also shown an inhibitory effect of Helicobacter infection on gastric acid secretion. 30,31 Consequently, we first examined gastric acid output by using the pyloric ligation method in INS-GAS mice infected for 3 months with H felis. As shown in Figure 1, YF476 or loxtidine treatment for 3 months significantly inhibited gastric acid output in H

5 June 2005 GASTRIN/HISTAMINE AND GASTRIC CANCER 1969 Table 1. Gastric Mucosa Histologic Scores in H felis Infected INS-GAS Mice Treated With YF476 and/or Loxtidine for 3 Months Corpus No drug YF476 Loxtidine YF476 loxtidine Inflammation Atrophy a Hyperplasia b Dysplasia b NOTE. n 4 for each group. a P.05; YF476-plus loxtidine-treated mice compared with mice receiving no drug. b P.01; YF476-plus loxtidine-treated mice compared with mice receiving no drug. felis infected INS-GAS mice, and mice treated with both drugs exhibited essentially no acid output. The study also confirms that 3-month H felis infection resulted in a level of acid secretion that was lower than in uninfected FVB/N control mice. H felis infection of INS-GAS mice results in a rapid parietal cell loss and progression to gastric atrophy. 7 While YF476 and loxtidine are known acid inhibitors, an additional explanation for the profound inhibition of acid secretion observed in Figure 1 might be that the drugs potentiated Helicobacter-mediated parietal cell loss. Thus, we examined the gastric histology of the 3-month H felis infected INS-GAS mice in each group. As shown in Table 1 and Figure 2, treatment with either YF476 alone or loxtidine alone showed no significant effect but a slight tendency toward improvement in Helicobactermediated gastric atrophy and reduction of foveolar hyperplasia compared with the untreated, infected INS- GAS mice. However, the mice treated with the Figure 2. YF476 and/or loxtidine treatment for 3 months synergistically inhibited gastric atrophy, hyperplasia, and dysplasia in H felis infected INS-GAS mice. Representative H&E stains are shown (original magnification 60 ; scale bar 250 m). (A) No drug, (B) YF476, (C) loxtidine, and (D) YF476 plus loxtidine. Treatment with (B) YF476 alone or (C) loxtidine alone for 3 months resulted in partial inhibition of gastric atrophy and foveolar hyperplasia noted in H felis infected INS-GAS mice receiving (A) no drug. In addition, the mice treated with (D) YF476 plus loxtidine for 3 months showed almost complete inhibition of gastric atrophy, hyperplasia, and dysplasia.

6 1970 TAKAISHI ET AL GASTROENTEROLOGY Vol. 128, No. 7 Figure 3. Serum amidated gastrin levels of H felis infected INS-GAS mice treated with YF476 and/or loxtidine for 6 months. Serum amidated gastrin levels of H felis infected INS-GAS mice treated with YF476 alone or YF476 plus loxtidine for 6 months were significantly higher than mice receiving no drug (*P.05; **P.01), whereas those of mice treated with loxtidine alone were not significant, but the values tended to be higher when compared with serum amidated gastrin levels of mice receiving no drug ( # P.054). LOX, loxtidine; YF LOX, YF476 plus loxtidine (n 10 for each group). combination of YF476 and loxtidine showed almost complete inhibition of atrophy, gastric hyperplasia, and dysplasia. Thus, YF476 and loxtidine appeared to have a synergistic inhibitory effect on both gastric acid output and the development of gastric preneoplasia in H felis infected INS-GAS mice. YF476 and Loxtidine Treatment for 6 Months Resulted in Synergistic Inhibition of Gastric Carcinogenesis in H felis Infected INS-GAS Mice To determine the effect of the CCK2/gastrin receptor and the histamine H 2 -receptor antagonist on the progression to gastric cancer, we undertook longer-term (6 months) drug treatment studies in the H felis infected INS-GAS mice. Treatment with YF476 and loxtidine, through their inhibitory effects on acid production, have previously been shown to induce hypergastrinemia in nontransgenic rodents. 32,33 Furthermore, H felis infection of the hypergastrinemic INS-GAS mice has been shown to lead to worsening of the hypergastrinemia, primarily due to progression of gastric atrophy. 7 Thus, not surprisingly, all 4 groups of H felis infected INS-GAS mice showed serum gastrin levels 500 pmol/l. In the case of YF476 alone or YF476 plus loxtidine double-treated mice, the serum gastrin levels were significantly higher than those of the untreated mice (P.01 for YF476 alone, P.05 for YF476 plus loxtidine; Figure 3). In the mice treated with loxtidine alone, there was a tendency toward higher amidated gastrin levels but it was not significant (P.054; Figure 3). As previously reported, 7 H felis infection of INS-GAS mice for more than 6 months resulted in a high prevalence of gastric tumors in the corpus area that was associated with a marked gastric thickening. Consequently, we examined the wet-tissue weights of stomachs from the mice treated with YF476 and/or loxtidine for 6 months. Both the gross presentation (Figure 4A E) and the actual weights of stomachs from the mice treated with YF476 and/or loxtidine were considerably reduced in comparison with untreated H felis infected INS-GAS mice (Figure 4F). In particular, the stomachs from mice treated with YF476 plus loxtidine closely resembled those from uninfected nontransgenic normal FVB/N mice. The body weights of YF476- and/or loxtidine-treated mice showed a significant increase from untreated mice, suggesting overall improved health conditions (Figure 4G). The H felis infected INS-GAS mice developed progressive cachexia after 6 months of infection, and this was largely ameliorated by treatment with YF476 and/or loxtidine. The ratios of stomach weight to body weight in the mice treated with YF476 and/or loxtidine were significantly lower than in untreated H felis infected INS-GAS mice (Figure 4H). Histologic examination and scoring of stomachs from YF476- and/or loxtidine-treated mice confirmed an inhibitory effect by the drugs on progression to gastric neoplasia. The mice treated with either YF476 or loxtidine alone showed a significant decrease in overall mucosal thickness and the elimination of submucosal invasion observed in untreated mice (Figure 5A C), that is, either YF476 or loxtidine alone seemed to have a partial inhibitory effect on progression to neoplasia (P.05 in Table 2). In addition, treatment with the combination of YF476 plus loxtidine significantly improved histologic scoring and resulted in nearly complete inhibition of neoplasia and normalization of histology with only mild inflammation and edema (P.01 in Table 2; Figure 5D). Thus, the combination of YF476 plus loxtidine appeared to have a synergistic inhibitory effect on gastric carcinogenesis in H felis infected INS-GAS mice. We also performed an immunohistochemical study to determine whether YF476 and/or loxtidine treatment would affect specific gastric cell lineages such as ECL cells and parietal cells. Our previous study showed that uninfected INS-GAS mice showed an early increase followed by a later, sustained reduction in the cell densities of ECL cells and parietal cells. 7 As shown in Table 3 and Figure 6A D, treatment with either YF476 or loxtidine alone for 6 months, as well as with both agents together, significantly increased ECL cell density, whereas parietal cell loss was signif-

7 June 2005 GASTRIN/HISTAMINE AND GASTRIC CANCER 1971 Figure 4. YF476 and/or loxtidine treatment for 6 months synergistically inhibited gastric tumors in H felis infected INS-GAS mice. (A E) Gross presentation of the stomachs of H felis infected INS-GAS mice treated with YF476 and/or loxtidine for 6 months. (A) No drug, (B) YF476, (C) loxtidine, (D) YF476 plus loxtidine, and (E) untreated FVB/N control mice. The stomachs of H felis infected INS-GAS mice treated with (B) YF476 alone or (C) loxtidine alone for 6 months were smaller than those of mice receiving (A) no drug, and the size of the stomachs of mice treated with (D) YF476 plus loxtidine was almost same as those of (E) untreated FVB/N control mice. Each distance between 2 adjoining bars in the scale represents 1 mm. (F H) Stomach wet weight, body weight, and the ratio of stomach wet weight over body weight of H felis infected INS-GAS mice treated with YF476 and/or loxtidine for 6 months. (F) Stomach wet weight and (H) the ratio of stomach wet weight over body weight of mice treated with YF476 and/or loxtidine for 6 months were significantly smaller than those of mice receiving no drug (*P.01; n 6 per each group), whereas (G) body weight of those mice was significantly larger than that of mice receiving no drug. (*P.01; n 6 per each group). LOX, loxtidine; YF LOX, YF476 plus loxtidine; FVB, untreated FVB/N control mice.

8 1972 TAKAISHI ET AL GASTROENTEROLOGY Vol. 128, No. 7 Figure 5. YF476 and/or loxtidine treatment for 6 months synergistically inhibited gastric carcinogenesis in H felis infected INS-GAS mice. Representative H&E stains are shown (original magnification 60 ; scale bar 250 m). (A) No drug, (B) YF476, (C) loxtidine, and (D) YF476 plus loxtidine. Treatment with (B) YF476 alone or (C) loxtidine alone for 6 months resulted in a significant decrease in overall mucosal thickness, a partial inhibition of neoplasia, and the elimination of submucosal invasion observed in H felis infected INS-GAS mice receiving (A) no drug. In addition, treatment with (D) YF476 plus loxtidine for 6 months resulted in nearly complete inhibition of neoplasia and normalization of histology, with only mild inflammation and edema as noted in mice treated with (B) YF476 alone or (C) loxtidine alone. Table 2. Gastric Mucosa Histologic Scores in H felis Infected INS-GAS Mice Treated With YF476 and/or Loxtidine for 6 Months Corpus No drug YF476 Loxtidine YF476 loxtidine Inflammation a a Atrophy a Hyperplasia b a Dysplasia b b a NOTE. n 5 for each group. a P.01; YF476- and/or loxtidine-treated mice compared with mice receiving no drug. b P.05; YF476- and/or loxtidine-treated mice compared with mice receiving no drug. icantly prevented only by the YF476 plus loxtidine combination treatment. The increase in ECL cell density seen with treatment with YF476 (either with or without loxtidine) was unexpected in light of earlier studies showing that histamine-immunoreactive ECL cells are largely absent in CCK2/gastrin receptor knockout mice. 34 This discrepancy may arise from the different time points at which the CCK2/gastrin receptor was inhibited (ie, either at the embryonic stage in the knockout case mice or later in adulthood in the current study). Alternatively, the dose of YF476 used in this study may only partially inhibit the CCK2/ gastrin receptor in ECL cells. Finally, we would note that the inbred murine strain (FVB/N) used in our

9 June 2005 GASTRIN/HISTAMINE AND GASTRIC CANCER 1973 Table 3. Cell Densities of ECL Cells and Parietal Cells in H felis Infected INS-GAS Mice Treated With YF476 and/or Loxtidine for 6 Months No drug YF476 Loxtidine YF476 loxtidine INS-GAS FVB ECL cells a a a a Parietal cells b b NOTE. Cell densities are expressed as number per field for ECL cells (10 visual fields of 200 magnification were selected per slide) and number per gland for parietal cells (10 glands were selected per slide). FVB, untreated FVB/N control mice. a P.05; comparison with mice receiving no drug. b P.01; comparison with mice receiving no drug. study differed from that used in early CCK2/gastrin receptor deletion studies. 34 Treatment With YF476 or Loxtidine Does Not Reduce H felis Colonization of Infected INS-GAS Mice To elucidate the possible mechanisms for the synergistic inhibitory effect of YF476 and loxtidine on gastric atrophy and carcinogenesis in H felis infected INS-GAS mice, we first examined the possibility that drug treatment could block the ability of H felis to colonize the stomachs of INS-GAS mice. We addressed this question using 3 separate approaches. First, we performed Warthin Starry silver staining to clarify whether H felis organisms might in fact be eradicated by these acid-suppressive reagents. These studies confirmed that H felis could still be detected in the stomachs of Figure 6. Immunohistochemical studies of ECL cells and parietal cells in H felis infected INS-GAS mice treated with YF476 and/or loxtidine for 6 months. (A and B) Immunohistochemical staining of ECL cells: (A) no drug and (B) YF476 plus loxtidine treatment for 6 months (anti chromogranin A antibody; original magnification 100 ). (C and D) Immunohistochemical staining of parietal cells: (C) no drug and (D) YF476 plus loxtidine treatment for 6 months (anti H,K adenosine triphosphatase subunit antibody; original magnification 100 ).

10 1974 TAKAISHI ET AL GASTROENTEROLOGY Vol. 128, No. 7 YF476- and/or loxtidine-treated mice at the same frequency as untreated mice and that the shape of these bacteria appeared to be the spiral form, not the coccoid form (Figure 7A). 35 Next, we performed ELISA assays of serum from the treated and untreated mice for H felis specific antibodies, and the results showed a very similar IgG titer against H felis among the 4 groups of mice (Figure 7B). Finally, we analyzed DNA from mice gastric corpus for H felis DNA using quantitative real-time PCR assay. YF476- and/or loxtidine-treated mice showed a slight increase of H felis DNA per stomach DNA, although this difference was not statistically significant (P.05 in Figure 7C). These data suggest that the inhibitory effect of YF476 and loxtidine was not due to a significant reduction in H felis colonization. Growth Factor Expression Analysis in YF476- and/or Loxtidine-Treated H felis Infected INS-GAS Mice Gastrin appears to modulate growth and differentiation of the gastric mucosa through both direct and indirect actions. 8 Two important classes of downstream growth factor targets that have been identified in recent years have included Reg I and the epidermal growth factor (EGF) family members such as HB-EGF, amphiregulin, and TGF-. Thus, to investigate the further possible mechanisms for the inhibitory effects of YF476 and loxtidine, we analyzed the expression levels of various growth factors by quantitative real-time RT-PCR. Among the 4 growth factors analyzed, Reg I showed the highest ratio of up-regulation in untreated H felis infected INS-GAS mice compared with uninfected nontransgenic FVB/N control mice. In response to treatment with YF476 and/or loxtidine, the level of expression was markedly reduced (Figure 8A). Similarly, amphiregulin was also highly expressed in H felis infected INS-GAS mice, and YF476 and/or loxtidine treatment significantly down-regulated the expression of this gene (Figure 8B). In contrast, HB-EGF was mildly down-regulated by YF476 and/or loxtidine treatment, but this change was not statistically significant (Figure 8C). In addition, another EGF family gene, TGF-, did not show any significant changes in response to YF476 and/or loxtidine treatment (Figure 8D). Loxtidine Treatment of H felis Infected INS- GAS Mice Resulted in a Mild Shift of Th1 to Th2 Polarization Given the importance of cytokine response in the pathogenesis of Helicobacter-induced gastric carcinogenesis, we analyzed cytokine expression profiles by quantitative real-time RT-PCR in the 4 groups of mice. Th1- polarized cytokines such as IFN- and TNF- were significantly down-regulated in H felis infected INS- GAS mice treated with loxtidine alone or YF476 plus loxtidine for 6 months (Figure 9A and B). Treatment with YF476 alone resulted in a small but not statistically significant decrease in IFN- and TNF- gene expression. In contrast, the cytokine IL-4 showed a slight but not statistically significant increase in YF476-treated mice, whereas a large and significant increase was shown in mice treated with loxtidine alone or YF476 plus loxtidine (Figure 9C). In addition, given the previous report by Zavros et al, 36 we examined somatostatin expression in YF476- and/or loxtidine-treated mice. As shown in Figure 9D, loxtidine alone or in combination with YF476 showed significant down-regulation of somatostatin expression. These findings were confirmed with the investigation of serum immunoglobulin IgG1 versus IgG2a subclass titers for H felis infection. As shown in Figure 8E, YF476- or loxtidine-treated mice showed significant up-regulation of the ratio of IgG1 versus IgG2a titers, although the double-treated mice showed a slight increase in this ratio that was not significant. Taken together, loxtidine treatment, with or without YF476, resulted in a mild shift toward a Th2- polarized response to H felis infection. Omeprazole Treatment for 3 Months Resulted in Mild Progression of Gastric Hyperplasia and Dysplasia in H felis Infected INS-GAS Mice We also examined the effect of another type of acid-suppressive reagent, the PPI omeprazole. In contrast to our findings with YF476 and/or loxtidine treatment, H felis infected INS-GAS mice treated with omeprazole for 3 months did not show a reduction in atrophy, but instead appeared to manifest a more rapid progression of gastric foveolar hyperplasia and dysplasia compared with the control, untreated, H felis infected INS-GAS mice (Table 4 and Figure 10A and B). The combination of omeprazole with YF476 or loxtidine resulted in some improvement of histologic features compared with treatment with omeprazole alone. In fact, treatment of H felis infected INS-GAS mice with the combination of omeprazole and YF476 led to significant suppression of gastric hyperplasia and dysplasia compared with mice treated with omeprazole alone (Table 4 and Figure 10C). Furthermore, treatment with the combination of omeprazole and loxtidine resulted in remarkable suppression of gastric atrophy, hyperplasia, and dysplasia compared with mice treated with omeprazole alone (Table 4 and Figure 10D). Of note, the control H felis infected INS- GAS mice in this experiment showed lower histologic

11 June 2005 GASTRIN/HISTAMINE AND GASTRIC CANCER 1975 scores for inflammation and hyperplasia/dysplasia (Table 4) compared with mice in the earlier experiment (Table 1), although these differences were not inconsistent with the experimental variability typically observed in the H felis model. Some of the variability may be attributed to the older age (3 months) of the mice in the first experiment compared with the second experiment (2 months) or to the difference in the injected vehicles used (daily dimethyl sulfoxide/peg 300 mixture in the second experiment versus weekly PEG 300 alone in the first) or to a different batch of H felis used, although there was not a significant difference in H felis infection status in the 2 experiments. As was the case for the other acid-suppressive drugs, serum gastrin levels were significantly higher in mice treated with omeprazole alone or combination with YF476 or loxtidine than in untreated mice (Figure 11A). Consequently, we also analyzed gastric cell lineage alterations induced by omeprazole treatment alone or by omeprazole with YF476 or loxtidine. Treatment with omeprazole alone or in combination with YF476 or loxtidine significantly increased ECL cell density compared with the non drug-treated control mice (Table 5 and Figure 11B and C). In contrast, omeprazole alone did not change parietal cell density, whereas omeprazole plus YF476 or loxtidine treatment significantly increased the density of parietal cells (Table 5 and Figure 11D and E). The increase in the cellular density of ECL and parietal cells, found in mice treated with omeprazole in combination with YF476, was unexpected in light of earlier studies. 34 The possible explanations have already been discussed. Figure 7. H felis infection status in the stomachs of YF476- and/or loxtidine-treated H felis infected INS-GAS mice. (A) Warthin Starry silver staining of H felis in the antrum of stomach of H felis infected INS-GAS mice treated with YF476 plus loxtidine for 6 months; H felis were detected as spiral-formed bacteria (original magnification 1000 ). (B) ELISA assay for mice measuring serum H felis specific IgG antibody. H felis IgG titers indicated minor changes among mice treated with YF476 and/or loxtidine for 6 months and mice receiving no drug (n 6 per each group). (C) Quantitative real-time PCR analysis for H felis DNA from the gastric corpus DNA of H felis infected INS-GAS mice. Increased H felis DNA copy numbers per gastric corpus DNA (copy per microgram) were not significant but were slightly increased in mice treated with YF476 and/or loxtidine for 6 months compared with mice receiving no drug (n 6 per each group). LOX, loxtidine; YF LOX, YF476 plus loxtidine; FVB, uninfected FVB/N control mice. Discussion In this study, we have investigated in our H felis infected INS-GAS mouse model the effect of 3 distinct acid-suppressive reagents: the CCK2/gastrin receptor antagonist YF476, the histamine H 2 -receptor antagonist loxtidine, and the PPI omeprazole. All 3 drugs strongly inhibit gastric acid secretion and were initially developed as antiulcer or anti gastroesophageal reflux disease drugs. Our results strongly suggest that the gastrin-histamine axis contributes to the development of gastric atrophy and neoplasia in this mouse model. While YF476 and loxtidine both inhibited the atrophic and proliferative response in H felis infected hypergastrinemic mice, omeprazole appeared to worsen disease progression. Because all 3 regimens inhibit acid secretion and induce hypergastrinemia, the different responses seen with the 3 drugs support a possible pathogenic role for CCK2/gastrin receptor and histamine H 2 -receptor

12 1976 TAKAISHI ET AL GASTROENTEROLOGY Vol. 128, No. 7 Figure 8. Growth factor expression analysis in YF476- and/or loxtidine-treated H felis infected INS-GAS mice. Quantitative real-time RT-PCR analysis of growth factor expression level: (A) Reg I, (B) amphiregulin, (C) HB-EGF, and (D) TGF-. All 3 groups of mice treated with YF476 and/or loxtidine for 6 months showed a significantly lower expression level of (A) Reg I and (B) amphiregulin than mice receiving no drug, whereas the expression level of (C) HB-EGF was not significant but showed a tendency for lower values. (D) TGF- expression did not show a significant change in response to YF476 and/or loxtidine treatment (*P.05, **P.01, n 6 for each group). LOX, loxtidine; YF LOX, YF476 plus loxtidine; INSGAS, INS-GAS mice without H felis infection; FVB, untreated FVB/N control mice. signaling in the INS-GAS/H felis mouse model of gastric cancer. The finding that YF476 could block disease progression was not surprising, given the hypergastrinemic nature of the INS-GAS mouse model. YF476 is a potent and highly selective CCK2/gastrin receptor antagonist that can also induce long-lasting suppression of acid secretion after a single injection. Previous in vivo studies have shown that it can inhibit many of the pathologic and proliferative effects induced by hypergastrinemia. Martinsen et al reported that administration of YF476 for 6 months into cotton rats markedly reduced the development of spontaneous gastric carcinomas in this hypergastrinemic rodent model. 37 The inhibitory effect of YF476 on the development of gastric carcinogenesis in our study does provide further proof that the gastric cancer observed in the INS-GAS mice is mediated at least in part by amidated gastrin. The inhibitory effect of loxtidine was, on the other hand, somewhat unexpected. In fact, we initially postulated that treatment with a histamine H 2 -receptor antagonist would, through acid suppression and induction of hypergastrinemia, accelerate gastric carcinogenesis. 38 Loxtidine is a well-studied irreversible and highly potent histamine H 2 -receptor antagonist that has been extensively used as a model of hypergastrinemia. For example, it has been shown to induce gastric hyperplasia and ECL cell carcinoids in rodents, 39 similar to that observed with omeprazole. 40 Poynter et al reported that loxtidinetreated rats showed ECL cell hyperplasia after 39 days of administration, while similar lesions were observed after 28 days of treatment with omeprazole. 41 However, in contrast to the effects of hypergastrinemia seen in the INS-GAS mouse model, long-term treatment of rats and mice with loxtidine did not result in loss of parietal cells but instead appeared to result in increased parietal cells. 42 Similar observations have been noted in histamine-deficient mice; histidine decarboxylase knockout (HDC / ) mice kept on a low-histamine diet showed an expanded parietal cell pool despite exhibiting marked hypergastrinemia. 43 Thus, these earlier observations are consistent with our current results, which suggest that loxtidine treatment of H felis infected INS-GAS mice inhibits progression to atrophy. Taken together, these observations suggest that excessive histamine produced in response to gastrin stimulation may contribute to the

13 June 2005 GASTRIN/HISTAMINE AND GASTRIC CANCER 1977 Figure 9. Loxtidine treatment of H felis infected INS-GAS mice showed mild shifts of cytokine expression profiles from Th1 to Th2 polarization. (A D) Quantitative real-time RT-PCR analysis of Th1, Th2 cytokines and somatostatin expression level: (A) IFN-, (B) TNF-, (C) IL-4, and (D) somatostatin (*P.05; **P.01; N.D., not detected; n 6 for each group). Mice treated with loxtidine alone or YF476 plus loxtidine for 6 months showed a significantly lower expression level of (A) IFN- and (B) TNF- compared with mice receiving no drug (*P.05), whereas mice treated with YF476 alone for 6 months did not show a significant change. Similarly, mice treated with loxtidine alone or YF476 plus loxtidine for 6 months showed significantly higher expression levels of (C) IL-4 and (D) somatostatin compared with mice receiving no drug (*P.05), whereas mice treated with YF476 alone for 6 months did not show a significant change. (E) Serum H felis IgG1/IgG2a ratio (n 6 for each group). Serum H felis IgG1/IgG2a ratio of mice treated with YF476 alone or loxtidine alone for 6 months was significantly lower than of mice receiving no drug (*P.05, **P.01), whereas mice treated with YF476 plus loxtidine for 6 months did not show a significant change, although there was a tendency for higher values ( # P.054). LOX, loxtidine; YF LOX, YF476 plus loxtidine; INSGAS, INS-GAS mice without H felis infection; FVB, untreated FVB/N control mice. gradual down-regulation of parietal cell number (gastric atrophy) and eventual progression to gastric cancer. The inhibition of gastric disease progression in this study occurred without significant change of H felis colonization. Danon et al reported that the localization of H felis in the stomach was affected by gastric acid suppressive reagents, that is, acid suppression caused the shift of H felis colonization from antrum to corpus. 16 Thus, our observation of a slight (but not significant) increase of H felis DNA in the gastric corpus treated with YF476 and/or loxtidine would be consistent with this shift. Nevertheless, treatment with YF476 and/or loxtidine did not lead to a measurable change in H felis infection status overall, that is, there was no decrease of serum H felis specific IgG titer and there was no change in bacterial shape from spiral form to coccoid form.

14 1978 TAKAISHI ET AL GASTROENTEROLOGY Vol. 128, No. 7 Table 4. Gastric Mucosa Histologic Scores in H felis Infected INS-GAS Mice Treated With Omeprazole Alone or Omeprazole With YF476 or Loxtidine for 3 Months Corpus No drug Omeprazole alone Omeprazole YF476 Omeprazole loxtidine Inflammation a b Atrophy c Hyperplasia a c c Dysplasia b b NOTE. n 4 for each group. a P.05; mice treated with omeprazole alone compared with mice receiving no drug. b P.05; mice treated with omeprazole plus YF476 or loxtidine compared with mice treated with omeprazole alone. c P.01; mice treated with omeprazole plus YF476 or loxtidine compared with mice treated with omeprazole alone. Figure 10. Omeprazole treatment for 3 months resulted in the mild progression of gastric hyperplasia and dysplasia in H felis infected INS-GAS mice. Representative H&E stains are shown (original magnification 60 ; scale bar 250 m). (A) No drug, (B) omeprazole alone, (C) omeprazole plus YF476, and (D) omeprazole plus loxtidine. Treatment with (B) omeprazole alone for 3 months did not show a reduction in atrophy but instead appeared to manifest a more rapid progression of gastric foveolar hyperplasia and dysplasia than H felis infected INS-GAS mice receiving (A) no drug, whereas the combination of omeprazole with (C) YF476 or (D) loxtidine for 3 months resulted in a significant suppression of gastric hyperplasia and dysplasia compared with mice treated with omeprazole alone.

15 June 2005 GASTRIN/HISTAMINE AND GASTRIC CANCER 1979 Figure 11. Serum gastrin levels and immunohistochemical studies of ECL cells and parietal cells in H felis infected INS-GAS mice treated with omeprazole alone or in combination with YF476 or loxtidine for 3 months. (A) Serum amidated gastrin levels of H felis infected INS-GAS mice treated with omeprazole alone or in combination with YF476 and/or loxtidine for 3 months. Serum amidated gastrin levels of H felis infected INS-GAS mice treated with omeprazole alone or in combination with YF476 or loxtidine for 3 months were significantly higher than mice receiving no drug (*P.05). OMP, omeprazole alone; OMP YF, omeprazole plus YF476; OMP LOX, omeprazole plus loxtidine; n 5 for each group. (B and C) Immunohistochemical staining of ECL cells: (B) no drug and (C) treated with omeprazole alone for 3 months (anti chromogranin A antibody; original magnification 100 ). (D and E) Immunohistochemical staining of parietal cells: (D) no drug and (E) treated with omeprazole alone for 3 months (anti H,K adenosine triphosphatase subunit antibody; original magnification 100 ). However, treatment with YF476 and/or loxtidine did result in significant down-regulation in the expression of Reg I. Previous reports have shown that the Reg I gene and protein, primarily expressed in gastric ECL cells, are up-regulated by amidated gastrin. 44,45 In addition, transgenic overexpression of Reg I can result in a gastric phenotype that closely mirrors the hypergastrinemic phenotype. 46 Nevertheless, while Reg I is considered a gastrin-regulated gene, our results with loxtidine suggest that the Reg I gene may also be regulated by