Tsutomu Mizoshita

Down-regulation of a gastric transcription factor, Sox2, and ectopic expression of intestinal homeobox genes, Cdx1 and Cdx2: inverse correlation during progression from gastric/intestinal-mixed to complete intestinal metaplasia

Purpose The molecular mechanisms underlying the development of intestinal metaplasia (IM) of the human stomach have yet to be clarified in detail. Besides ectopic expression of intestinal transcription factors, Cdx1 and Cdx2, little information is available regarding other regulatory factors. Hence, we here analyzed Sox2, a human homolog of a chicken gastric transcription factor, with reference to our new classification for gastric/intestinal (GI)-mixed type IM.Methods Twenty specimens of surgically resected antral mucosa were subjected to a gland isolation technique. Isolated glands were classified into gastric (G), GI-mixed, and solely intestinal (I) types according to Alcian blue and paradoxical concanavalin A staining and were quantified for mRNA levels of gastrointestinal markers.Results MUC5AC and MUC6 transcripts decreased with the progression of IM, while MUC2 and villin-1 were inversely correlated. Sox2 showed a gradual decrease from G, through GI, to the I type (G vs GI and GI vs I, P<0.01 and P<0.005, respectively). On the other hand, Cdx1 (G vs GI and GI vs I, P<0.0001 and P=0.337, respectively) and Cdx2 (G vs GI and GI vs I, P<0.0001 and P<0.05, respectively) appeared in IM. Immunohistochemical study confirmed decreased expression of Sox2 and ectopic emergence of Cdx2 protein in IM glands.Conclusion Down-regulation of Sox2, besides ectopic expression of Cdx genes, may be important factors for the development of IM.

Expression of Cdx2 and the phenotype of advanced gastric cancers: relationship with prognosis

PurposeThe clinicopathologic significance of the phenotype and Cdx2 expression has hitherto remained unclear in gastric cancers. In the present study, we therefore examined the correlation between prognosis, phenotype, and Cdx2 expression in advanced cases.MethodsWe evaluated 177 advanced gastric carcinomas histologically and phenotypically. The expression of Cdx2 was also assessed by immunohistochemistry.ResultsThe lesions were phenotypically divided into 32 gastric (G type), 36 gastric and intestinal mixed (GI type), 53 intestinal (I type), and 56 null (N type) types, independent of the histological classification. Cdx2 nuclear staining demonstrated a close relation to intestinal phenotypic expression, not with the histological classification. Kaplan-Meier analysis of Cdx2 expression and the phenotype showed that the Cdx2 positive groups had a significantly better outcome than the negative ones (P =0.0013), and the patients with GI type cancers had significant better survival than those with N type (P =0.0052).ConclusionsOur results suggest that Cdx2 is a useful prognostic marker. In addition, advanced gastric cancers with both intestinal and gastric phenotypic expression have a relatively good prognosis. Combined evaluation of gastric and intestinal epithelial cell markers, including Cdx2, is clinically useful to predict outcome in patients with advanced neoplasm of the stomach.

Sox2 expression in human stomach adenocarcinomas with gastric and gastric‐and‐intestinal‐mixed phenotypes

Aims:  Other than ectopic expression of intestinal transcription factors, Cdx1 and Cdx2, the molecular mechanisms underlying gastric and intestinal phenotypes of human stomach adenocarcinomas have yet to be clarified in detail. We have reported that Sox2, an HMG‐box gastric transcription factor, is expressed in normal gastric mucosa and down‐regulated in intestinal metaplasia.

Expression of Cdx1 and Cdx2 mRNAs and relevance of this expression to differentiation in human gastrointestinal mucosa--with special emphasis on participation in intestinal metaplasia of the human stomach.

Background. The caudal-type homeobox genes, Cdx1 and Cdx2, are candidates for directing intestinal development, differentiation, and maintenance of the intestinal phenotype. The aims of this study were: (1) to assess the normal tissue expression patterns of Cdx1 and Cdx2 in the human gastrointestinal tract and (2) to ascertain levels in intestinal metaplasia (IM) of the stomach associated with gastritis. Methods. Fresh human tissues were collected by surgical resection from 39 patients after informed consent had been received. RNAs were extracted from 11 distinct sites in the gastrointestinal mucosa (gastric body, gastric antrum, duodenum, jejunum, ileum, cecum, ascending colon, transverse colon, descending colon, sigmoid colon, and rectum), and Northern hybridization was performed for Cdx1 and Cdx2 mRNAs. In addition, RNAs were also extracted from normal gastric mucosa, and gastric mucosa with mild to severe IM, confirmed histopathologically. Reverse-transcriptase polymerase chain reaction (RT-PCR) was then carried out for Cdx1 and Cdx2. Results. The expression of Cdx1 mRNA increased gradually from the duodenum to the distal colon, with no expression detected in the stomach. Compared with the distribution of Cdx1 mRNA in the mouse gastrointestinal tract, the expression of Cdx1 mRNA in the human gastrointestinal tract showed greater predominance in the jejunum and ileum. The expression of Cdx2 mRNA increased gradually from the duodenum to the proximal colon and decreased from the ascending colon to the rectum. Compared with the expression pattern of Cdx2 mRNA in the mouse gastrointestinal tract, the expression of Cdx2 mRNA in the human gastrointestinal tract showed greater predominance in the ileum. By RT-PCR, both Cdx1 and Cdx2 mRNAs were detected in the mild and severe types of IM. However, neither of these mRNAs was identified in normal gastric mucosa without IM. Conclusions.Cdx1 and Cdx2 mRNAs are widely present in the human intestinal and colonic mucosae, but not in the gastric mucosa, suggesting that their expression may contribute to the intestinal phenotype. The high levels of these mRNAs in IM mucosa associated with chronic atrophic gastritis point to an association with this phenotypic shift in the gastric mucosa.

High salt diets dose-dependently promote gastric chemical carcinogenesis in Helicobacter pylori-infected Mongolian gerbils associated with a shift in mucin production from glandular to surface mucous cells.

Intake of salt and salty food is known as a risk factor for gastric carcinogenesis. To examine the dose‐dependence and the mechanisms underlying enhancing effects, Mongolian gerbils were treated with N‐methyl‐N‐nitrosourea (MNU), Helicobacter pylori and food containing various concentrations of salt, and were sacrificed after 50 weeks. Among gerbils treated with MNU and H. pylori, the incidences of glandular stomach cancers were 15% in the normal diet group and 33%, 36% and 63% in the 2.5%, 5% and 10% NaCl diet groups, showing dose‐dependent increase (p < 0.01). Intermittent intragastric injection of saturated NaCl solution, in contrast, did not promote gastric carcinogenesis. In gerbils infected with H. pylori, a high salt diet was associated with elevation of anti‐H. pylori antibody titers, serum gastrin levels and inflammatory cell infiltration in a dose‐dependent fashion. Ten percent NaCl diet upregulated the amount of surface mucous cell mucin (p < 0.05), suitable for H. pylori colonization, despite no increment of MUC5AC mRNA, while H. pylori infection itself had an opposing effect, stimulating transcription of MUC6 and increasing the amount of gland mucous cell mucin (GMCM). High salt diet, in turn, decreased the amount of GMCM, which acts against H. pylori infection. In conclusion, the present study demonstrated dose‐dependent enhancing effects of salt in gastric chemical carcinogenesis in H. pylori‐infected Mongolian gerbils associated with alteration of the mucous microenvironment. Reduction of salt intake could thus be one of the most important chemopreventive methods for human gastric carcinogenesis.

Changes in 12-Year First-Line Eradication Rate of Helicobacter pylori Based on Triple Therapy with Proton Pump Inhibitor, Amoxicillin and Clarithromycin

A triple therapy based on a proton pump inhibitor (PPI), amoxicillin (AMPC), and clarithromycin (CAM) is recommended as a first-line therapy for Helicobacter pylori (H. pylori) eradication and is widely used in Japan. However, a decline in eradication rate associated with an increase in prevalence of CAM resistance is viewed as a problem. We investigated CAM resistance and eradication rates over time retrospectively in 750 patients who had undergone the triple therapy as first-line eradication therapy at Nagoya City University Hospital from 1995 to 2008, divided into four terms (Term 1: 1997–2000, Term 2: 2001–2003, Term 3: 2004–2006, Term 4: 2007–2008). Primary resistance to CAM rose significantly over time from 8.7% to 23.5%, 26.7% and 34.5% while the eradication rate decreased significantly from 90.6% to 80.2%, 76.0% and 74.8%. Based on the PPI type, significant declines in eradication rates were observed with omeprazole or lansoprazole, but not with rabeprazole. A decrease in the H. pylori eradication rate after triple therapy using a PPI + AMPC + CAM has been acknowledged, and an increase in CAM resistance is considered to be a factor. From now on, a first-line eradication regimen that results in a higher eradication rate ought to be investigated.

Helicobacter pylori infection-negative gastric cancer in Japanese hospital patients: Incidence and pathological characteristics

We used Helicobacter pylori sero‐positivity and mucosal atrophy as detected by the serum pepsinogen method to identify H. pylori infection‐negative gastric cancer patients with or without atrophy. One hundred and six of 748 (14.2%) primary gastric cancer patients were infection‐negative by a serum antibody detection system. Further, 121 (16.2%) of the 748 were negative for gastric mucosal atrophy by the pepsinogen method, of whom 15/748 (2.0%) were H. pylori‐negative by pepsinogen I level (>70 ng/mL) and pepsinogen I/II ratio (>3.0). Twenty‐seven of 782 (3.6%) gastric cancer patients were H. pylori‐negative by antibodies and severe atrophy as determined by pepsinogen I level (<30 ng/mL) and pepsinogen I/II ratio (<2.0). H. pylori‐negative gastric cancer patients with severe atrophy likely had a previous infection. These results indicate that the actual number of H. pylori‐negative patients is 2.0% at minimum and 10.6% (14.2% minus 3.6%) at maximum in the general Japanese population. Five of 15 (33%) cases displaying neither anti‐H. pylori antibodies nor atrophy were intestinal‐type and 10 (67%) were diffuse‐type adenocarcinomas. Thirteen surgical patients with primary gastric cancer displaying neither antibodies nor mucosal atrophy were further analyzed for pathological and phenotypic characteristics. The mucin phenotype was divided into four gastric, five gastric and intestinal, two intestinal and two null types, independent of histological classification. Intestinal phenotype elements were detected by Cdx2 immunohistochemical methods in nine of 13 (70%) cases examined. We conclude that a small fraction of gastric cancer patients displayed multifactorial carcinogenesis without H. pylori infection, indicating that gastric cancer risk still exists in the absence of H. pylori infection, at an incidence of 2.0% at minimum and 10.6% at maximum in the general Japanese population. (Cancer Sci 2007; 98: 790–794)

4-Vinyl-2,6-dimethoxyphenol (canolol) suppresses oxidative stress and gastric carcinogenesis in Helicobacter pylori-infected carcinogen-treated Mongolian gerbils.

Oxidative stress is linked to gastric carcinogenesis because of its ability to damage DNA. Here we examined antioxidative and anti‐inflammatory effects of 4‐vinyl‐2,6‐dimethoxyphenol (canolol), a recently identified potent antioxidative compound obtained from crude canola oil, on Helicobacter (H.) pylori‐induced gastritis and gastric carcinogenesis using a Mongolian gerbil model. The animals were allocated to H. pylori‐infection alone (12 weeks) or H.pylori + N‐methyl‐N‐nitrosourea (MNU) administration (52 weeks). After oral inoculation of H. pylori, they were fed for 10 and 44 weeks with or without 0.1% canolol. H. pylori‐induced gastritis, 5′‐bromo‐2′‐deoxyuridine (BrdU) labeling and scores for cyclooxygenase‐2 (COX‐2) and inducible nitric oxide synthase (iNOS) immunohistochemistry were attenuated in the canolol‐treated groups. Expression of interleukin‐1β (IL‐1β), tumor necrosis factor‐α (TNF‐α), COX‐2 and iNOS mRNA in the gastric mucosa, and serum 8‐hydroxy‐2′‐deoxyguanosine (8‐OHdG), anti‐H. pylori IgG and gastrin levels were also significantly lower in canolol‐treated groups. Furthermore, the incidence of gastric adenocarcinomas was markedly reduced in the H. pylori + MNU + canolol‐treated group [15.0% (6/40)] compared to the control group [39.4% (13/33)] (p < 0.05). These data indicate canolol to be effective for suppressing inflammation, gastric epithelial cell proliferation and gastric carcinogenesis in H. pylori‐infected Mongolian gerbils. Interestingly, the viable H. pylori count was not changed by the canolol containing diet. Thus, the data point to the level of inflammation because of H. pylori rather than the existence of the bacteria as the determining factor. Importantly, canolol appears to suppress induction of mRNAs for inflammatory cytokines.

Animal Models of Stomach Carcinogenesis

Although incidences of stomach cancer have decreased over the past several decades, the disease remains an important public health problem. To identify pathological and molecular biochemical mechanisms, various experimental animal models have been established in rats and mice with chemical carcinogens including N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) and N-methyl-N-nitrosourea (MNU). Helicobacter pylori (H. pylori) is one of the most important factors for human stomach disorders, including neoplasia, and the H. pylori-infected and carcinogen-treated Mongolian gerbil (MG) has proven very useful for analyses of underlying processes. The findings with this model support the hypothesis that intestinal metaplasia is important not as a precancerous lesion but rather as a paracancerous condition and that intestinalization of stomach cancer progresses with chronic inflammation. Furthermore, dose-dependent enhancing effects of salt on stomach carcinogenesis could be demonstrated in MGs treated with MNU and H. pylori modifying surface mucous gel layer. H. pylori itself only causes chronic inflammation and acts as a promoter of stomach carcinogenesis in experimental models. Based on the precise pathological diagnosis of stomach lesions such as noncancerous heterotopic proliferative glands (HPG) and adenocarcinomas, a basis for understanding mechanisms of carcinogenesis has been established on which chemoprevention can be modeled.

Role of helicobacter pylori in gastric carcinogenesis: The origin of gastric cancers and heterotopic proliferative glands in mongolian gerbils

Helicobacter pylori infection is well accepted to be a very important factor for the development of gastric carcinogenesis in the human stomach. In Mongolian gerbils treated with chemical carcinogens, H. pylori infection enhances glandular stomach carcinogenesis, and eradication of infection and results in curtailment of enhancing effects, particularly at early stages of associated inflammation. A high‐salt diet exacerbates the effects of H. pylori infection on gastric carcinogenesis, and these two factors act synergistically to promote the development of gastric cancers in this animal model. However, the bacterium exerts the greater effects. Early acquisition significantly increases gastric chemical carcinogenesis in Mongolian gerbils, as compared to later infection. While heterotopic proliferative glands, hyperplastic and dilated glands localized beneath the muscularis mucosae, frequently develop with H. pylori infection alone in this animal model, they obviously regress on eradication, suggesting a relation to severe gastritis, rather than a malignant character. Furthermore, endocrine cells, positive for chromogranin A, are observed in the heterotopic proliferative glands, in contrast to cancerous lesions which lack endocrine elements. In conclusion, H. pylori is not an initiator, but rather a strong promoter of gastric carcinogenesis, whose eradication, together with reduction in salt intake, might effectively prevent gastric cancer development.