Deficient HER3 expression in poorly-differentiated colorectal cancer cells enhances gefitinib sensitivity.

Poorly-differentiated colorectal cancers (PD-CRC) show high metastatic potential and poor prognosis. However, molecular characteristics of PD-CRC remain unknown to date, particularly in molecular targeting therapy for patients with PD-CRC. In this study, we examined the expression of EGFR, HER2 and HER3 in PD-CRC by immunohistochemical analysis of archived clinical specimens of primary tumors and investigated the sensitivity of PD-CRC cell lines to gefitinib, a tyrosine kinase inhibitor for EGFR in vitro. We found that HER3 expression of PD-CRC among members of the HER family was significantly lower than that of well to moderately differentiated CRC (WMD-CRC) and 37% of the PD cases showed a EGFR+/HER2+/HER3- expression pattern. COLM-5 cells, a PD-CRC-derived cell line, which exhibits EGFR+/HER2+/HER3- expression pattern and recapitulates the typical histology of PD-CRC in xenografted tumors, showed high gefitinib sensitivity both in vitro and in vivo, compared with WMD-CRC cell line (COLM-2). Treatment with gefitinib resulted in the upregulation of p27Kip1 expression and induction of G1 cell cycle arrest, concomitantly associated with inactivation of PI3K/Akt signaling in COLM-5 cells and marked inhibition of xenografted tumors in nude mice, but not evident in COLM-2 cells. Treatment with sodium butyrate, an HDAC inhibitor that induces differentiation, upregulated the expression of HER3 associated with enhancement of the PI3K/Akt signaling, attenuated gefitinib-mediated p27Kip1 upregulation and reduced gefitinib sensitivity in COLM-5 cells in vitro. Furthermore, enforced expression of HER3 in COLM-5 cells resulted in significant resistance to gefitinib treatment both in vitro and in vivo. These findings suggest that deficient HER3 expression plays an important role in gefitinib sensitivity and that a malignant subset of PD with EGFR+/HER2+/HER3- phenotype is a potential candidate for a target of anti-EGFR molecular therapy such as gefitinib.

Lapatinib sensitivities of two novel trastuzumab-resistant HER2 gene-amplified gastric cancer cell lines.

BACKGROUND: Trastuzumab (Tmab) resistance is a major clinical problem to be resolved in patients with HER2-positive gastric cancers. However, in contrast to the situation for HER2-positive breast cancer lines, the Tmab-resistant gastric cancer preclinical models that are needed to develop a new therapy to overcome this problem are not yet available. METHODS: We developed three new cell lines from HER2 gene-amplified gastric cancer cell lines (GLM-1, GLM-4, NCI N-87) by a new in vivo selection method consisting of the repeated culture of small residual peritoneal metastasis but not subcutaneous tumor after Tmab treatment. We then evaluated the anti-tumor efficacy of lapatinib for these Tmab-resistant cells. RESULTS: We successfully isolated two Tmab-resistant cell lines (GLM1-HerR2(3), GLM4-HerR2) among the three tested cell lines. These resistant cells differed from the parental cells in their flat morphology and rapid growth in vitro, but HER2, P95HER2 expression, and Tmab binding were essentially the same for the parental and resistant cells. MUC4 expression was up- or downregulated depending on the cell line. These resistant cells were still sensitive to lapatinib, similar to the parental cells, in vitro. This growth inhibition of the Tmab-resistant cells by lapatinib was due to both G1 cell-cycle arrest and apoptosis induction via effective blockade of the PI3K/Akt and MAPK pathways. A preclinical study confirmed that the Tmab-resistant tumors are significantly susceptible to lapatinib. CONCLUSION: These results suggest that lapatinib has antitumor activity against the Tmab-resistant gastric cancer cell lines, and that these cell lines are useful for understanding the mechanism of Tmab resistance and for developing a new molecular therapy for Tmab-resistant HER2-positive gastric cancers.

Establishment of new intraperitoneal paclitaxel-resistant gastric cancer cell lines and comprehensive gene expression analysis.

BACKGROUND: Intraperitoneal (i.p.) chemotherapy with paclitaxel is a potential therapeutic modality for patients with peritoneal metastasis of gastric cancer. To overcome paclitaxel resistance, which is a major clinical problem with this modality, prediction of i.p. paclitaxel resistance is critically important. MATERIALS AND METHODS: We developed three new i.p. paclitaxel-resistant cell lines from parental gastric cancer cell lines by an in vivo selection method using i.p. paclitaxel chemotherapy. With these cell lines, we performed gene expression profiling analysis to select up-regulated genes in i.p. paclitaxel-resistant cells and validated the genes with clinical samples. RESULTS: We successfully isolated nine up-regulated genes in i.p. paclitaxel-resistant cell lines compared with parental cells by microarray analysis, followed by confirmation with quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). Among these, we identified four genes, namely kinesin family member-23 (KIF23), ERBB2 interacting protein (ERBB2IP), ATPase family, AAA domain containing-2 (ATAD2) and PHD finger protein (PHF19) as candidate genes for paclitaxel resistance after validation with clinical samples derived from responders and non-responders to paclitaxel treatment. CONCLUSION: These i.p. paclitaxel-resistant cell lines are ideal models for understanding the mechanism of resistance to i.p. paclitaxel and development of a new therapeutic modality. Four up-regulated genes may be potential new predictive markers for resistance to i.p. paclitaxel in patients with peritoneal metastasis of gastric cancer.

Positive and negative regulation of podoplanin expression by TGF-ß and histone deacetylase inhibitors in oral and pharyngeal squamous cell carcinoma cell lines.

OBJECTIVES: Podoplanin, a transmembrane sialomucin-like glycoprotein, is known to express at high frequency in oral squamous cell carcinomas (OSCC) and possess metastasis-promoting activity such as increased invasion and platelet-aggregating activity. However, the regulatory mechanism of podoplanin expression in OSCC remains unknown. MATERIALS AND METHODS: In the present study, we investigated the podoplanin expression in both clinical specimens from total 80 patients (50 OSCC and 30 pharyngeal SCC) and in 4 OSCC cell lines in vitro. RESULTS: Immunohistochemical analysis of surgically resected specimens of OSCC revealed podoplanin expression in 70% of OSCC cases with localization primarily in the basal layer of squamous cancer nest and the expression was inversely correlated with squamous cell differentiation. In vitro analysis of OSCC cell lines revealed 36 that podoplanin expression was decreased in response to the squamous cell differentiation (Cytokeratin 10 expression as a marker) induced by treatment with histone deacetylase (HDAC) inhibitors such as sodium butyrate and trichostatin. Furthermore, transforming growth factor-ß (TGF-ß) significantly enhanced podoplanin expression in OSCC cell lines in line with increased phosphorylation of Smad2. A TGF-ß type I receptor inhibitor (SB431542) significantly inhibited such induction of podoplanin expression by TGF-ß at both the protein and mRNA level. However, in a subset of OSCC cell line, its expression was only weakly dependent on TGF-ß and squamous differentiation. CONCLUSION: These results suggest that regulation of podoplanin is not simple, but in the majority of OSCC cell lines, its expression is positively and negatively regulated by TGF-ß receptor/Smad signaling pathway and epigenetic mechanism leading to squamous differentiation, respectively.

Establishment and characterization of novel gastric signet-ring cell and non signet-ring cell poorly differentiated adenocarcinoma cell lines with low and high malignant potential.

BACKGROUND: Poorly differentiated signet-ring cell carcinoma (SRCC) and non signet-ring cell carcinoma (NSRCC) are prevalent histological subtypes of gastric cancers with distinct morphological features. To date, however, the molecular basis of their growth, differentiation, and metastasis still remains unclear, because of the limitation of available cell lines. METHODS: In the present study, we established novel SRCC and NSRCC cell lines (designated GPM-2 and GPM-1) derived from the ascites of two individual gastric cancer patients with peritoneal metastasis. RESULTS: Immunohistochemical and quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis revealed that GPM-2 cells showed both gastric and intestinal differentiation phenotypes (E-cadherin+/MUC5AC+/MUC6+/Villin+), and formed xenografted tumors with typical SRCC histology in nude mice. In contrast, GPM-1 cells only weakly expressed differentiation markers, showing a phenotype of E-cadherin(low)+/MUC2-/MUC5AC-/Villin(low)+. Characteristically, GPM-2 cells were found to highly express both membrane-bound mucin (MUC1/MUC4) and secreted mucin glycoproteins (MUC5AC/MUC6), whose expression is regulated by an epigenetic mechanism such as histone acetylation. GPM-2 cells also secreted a large amount of sTn antigen into the culture medium. These mucin profiles of GPM-2 cells are distinct from those of conventional SRCC cell lines (KATO III and HSC-39), which preferentially express intestinal MUC2/MUC4 as well as sLe(x) and sLe(A) antigens. In addition, GPM-2 cells showed a slow growth rate, and a lower metastatic potential than GPM-1 cells. CONCLUSIONS: These results indicate that the cells of the new SRCC line, GPM-2 cells, are more differentiated and less aggressive than NSRCC-type GPM-1 cells, and would thus offer an excellent model for understanding the molecular mechanism underlying the growth, differentiation, and mucin production of an SRCC gastric cancer cell line.

Inflammatory processes triggered by Helicobacter pylori infection cause aberrant DNA methylation in gastric epithelial cells.

Altered patterns of DNA methylation associated with Helicobacter pylori (HP) infection of gastric epithelial cells are thought to contribute to gastric cancer risk. However, it is unclear whether this increased risk reflects an infection-associated inflammatory response or the infection itself. In this study, we sought to clarify mechanisms in a gerbil model of gastric cancer where we showed that HP infection is causally involved in induction of aberrant DNA methylation. By genome-wide screening, CpG islands that were aberrantly methylated in gerbil gastric cancer cell lines were isolated, and 10 islands were shown to be specifically methylated only in gastric mucosae infected with HP. By temporal analysis, methylation levels in gastric epithelial cells started to increase at 5 to 10 weeks after infection and reached high levels by 50 weeks. When HP was eradicated, methylation levels markedly decreased 10 and 20 weeks later, but they remained higher than those in gerbils that were not infected by HP. Expression levels of several inflammation-related genes (CXCL2, IL-1beta, NOS2, and TNF-alpha) paralleled the temporal changes of methylation levels. Significantly suppressing inflammation with the immunosuppressive drug cyclosporin A did not affect colonization by HP but blocked the induction of altered DNA methylation. Our findings argue that DNA methylation alterations that occur in gastric mucosae after HP infection are composed of transient components and permanent components, and that it is the infection-associated inflammatory response, rather than HP itself, which is responsible for inducing the altered DNA methylation.

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-1 beta (IL-1 beta), tumor necrosis factor-alpha (TNF-alpha), 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.

Emergence of spasmolytic polypeptide-expressing metaplasia in Mongolian gerbils infected with Helicobacter pylori.

Spasmolytic polypeptide (TFF2)-expressing metaplasia (SPEM) is observed in mucosa adjacent to human gastric cancer and in fundic glands showing oxyntic atrophy in Helicobacter felis-infected mice. Mongolian gerbils infected with Helicobacter pylori (Hp) develop goblet cell intestinal metaplasia and adenocarcinoma, but the presence of SPEM has not been studied in gerbils. We therefore have sought to examine the development of metaplastic mucosal changes in Hp-infected Mongolian gerbils. Mongolian gerbils were assigned to either uninfected controls or infected with Hp at 17 weeks of age. The animals were killed at 17, 20, 26, 31, 41 and 56 weeks of age. Stomach sections were stained using antibodies for TFF2, intrinsic factor, H/K-ATPase, BrdU and MUC2. Dual immunofluorescence staining for TFF2 with intrinsic factor and for TFF2 with MUC2 was performed. In uninfected animals, no SPEM or intestinal metaplasia was observed. Infected gerbils developed SPEM initially in the intermediate zone along the lesser curvature and subsequently spread out towards the greater curvature. In the earlier stages of infection, SPEM glands demonstrated TFF2 and intrinsic factor double staining cells. However, after 35 weeks of infection, the number of double staining SPEM cells decreased. While early in infection SPEM organized in straight glands, in the later stages of infections, SPEM glands became distorted or dilated along with the development of gastritis cystica profunda that was TFF2 positive. Goblet cell intestinal metaplasia developed only late in the infection. Dual staining for TFF2 and MUC2 showed glands containing both SPEM- and MUC2-positive goblet cell intestinal metaplasia. SPEM develops early in Hp infection in Mongolian gerbils, and alterations in gland morphology arise from SPEM glands during the course of gastric infection with goblet cell intestinal metaplasia developing subsequent to SPEM.

Inhibitory effect of nordihydroguaiaretic acid, a plant lignan, on Helicobacter pylori-associated gastric carcinogenesis in Mongolian gerbils.

Recent epidemiological studies have demonstrated that consumption of certain natural products can lower cancer risk in humans. For example, plant-derived lignans have been shown to exert chemopreventive effects against cancer in vitro and in vivo. In the present study, the effects of three such lignans, termed arctiin, arctigenin, and nordihydroguaiaretic acid (NDGA), on the proliferation of Helicobacter pylori and the prevention of H. pylori-associated gastric cancer were investigated in Mongolian gerbils. To examine the effects of arctigenin and NDGA on stomach carcinogenesis, specific pathogen-free male, 5-week-old gerbils were infected with H. pylori, administered 10 p.p.m. N-methyl-N-nitrosourea in their drinking water and fed diets containing various concentrations of lignans until they were killed after 52 weeks. At a dietary level of 0.25%, NDGA significantly decreased the incidence of gastric adenocarcinomas. Arctigenin, in contrast, failed to attenuate neoplasia at a level of 0.1%. Both NDGA and arctigenin significantly reduced serum 8-hydroxy-2'-deoxyguanosine levels at doses of 0.25 and 0.05% (NDGA), and 0.1% (arctigenin). Administration of 0.25% NDGA significantly suppressed the formation of intestinal metaplasia both in the antrum and the corpus. Although all three lignans dose-dependently inhibited the in vitro proliferation of H. pylori, there were no differences in the titers of anti-H. pylori antibodies or the amount of the H. pylori-specific urease A gene among all H. pylori-infected groups. These results suggest that NDGA might be effective for prevention of gastric carcinogenesis. The possible mechanisms appear to be related to inhibitory effects on progression of gastritis and antioxidative activity rather than direct antimicrobial influence.

Severity of gastritis determines glandular stomach carcinogenesis in Helicobacter pylori-infected Mongolian gerbils.

Helicobacter pylori (H. pylori) infection causes chronic gastritis and is also related to gastric carcinoma. The present study focused on severity of H. pylori-induced gastritis as a determinant of carcinogenesis. Seven-week-old male Mongolian gerbils were inoculated with H. pylori at experimental weeks 0, 12, or 18, then given N-methyl-N-nitorosourea (MNU) from weeks 20-40. At week 70, stomachs were then excised for histological examination 70, 58, or 52 weeks after H. pylori inoculation, respectively (Groups A, B, and C for long-, middle-, and short-term). The respective incidences of glandular stomach adenocarcinomas were 65.0% (13/20), 20.0% (2/10), and 23.0% (3/13) (P<0.05). Higher scores of infiltration of inflammatory cells, hyperplasia, intestinal metaplasia and mucosal bromodeoxyuridine (BrdU) labeling index in antrum and corpus mucosa, were seen in group A than B or C (P<0.05) and serum anti-H. pylori IgG titer and gastrin levels were also significantly higher, along with mRNA levels for mucosal interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha), cyclooxygenase-2 (COX-2), and inducible nitric oxide synthase (iNOS). The results demonstrated the term and severity of H. pylori infection to play important roles in gastric carcinogenesis, with essential involvement of chronic inflammation, especially increased rates of cell proliferation, in H. pylori-associated carcinogenesis.

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.

Inhibition of Helicobacter pylori motility by (+)-Syringaresinol from unripe Japanese apricot.

A methanol extract from unripe Japanese apricot showed inhibitory activity of Helicobacter pylori motility. Inhibitory compound 1 was isolated and identified as (+)-syringaresinol (1) by spectoroscopic means. (+)-Syringaresinol (1) inhibited >90% of the H. pylori motility at a concentration of 500 microg/ml and the IC50 value was 50 microg/ml.

Suppressive effects of fruit-juice concentrate of Prunus mume Sieb. et Zucc. (Japanese apricot, Ume) on Helicobacter pylori-induced glandular stomach lesions in Mongolian gerbils.

Helicobacter pylori (Hp) infection is an important factor in human gastric disorders, including chronic active gastritis, peptic ulcers, intestinal metaplasia and cancer. Since epidemiologic studies overwhelmingly agree on a protective influence of fruits and vegetables in reducing the risk of gastric neoplasia and processed foods made from Prunus mume Sieb. et Zucc. (Japanese apricot or "Ume" in Japanese) are traditionally known for their miscellaneous medical effects, in the present study we investigated the efficacy of a fruit-juice concentrate of Japanese apricot (CJA) in the glandular stomach of Hp-infected Mongolian gerbils. Hp-inoculated gerbils were given CJA in their drinking water at concentrations of 1 and 3% for 10 weeks. The microscopic scores for gastritis and mucosal hyperplasia in the CJA groups were significantly lower than in the Hp-inoculated control group, with dose-dependence. Real-time PCR was performed to quantitate Hp by demonstrating urease A gene amount using gerbils glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene as an internal control. Average relative urease A gene dosage in the glandular stomach in the 1 and 3% CJA and Hp-inoculated control groups was 26.6 +/- 11.6% (average +/- SE), 30.3 +/- 10.5%, 100 +/- 40.9%, respectively, the fruit-juice concentrate causing significant lowering (P<0.01 and P<0.05, respectively, with 1 and 3%). These findings suggest that suppressive effects on gastric cancer development might also be expected as a result of decreased numbers of Hp and improvement of Hp-induced chronic active gastritis on administration of CJA.

Colonic and small-intestinal phenotypes in gastric cancers: relationships with clinicopathological findings.

The clinicopathological significance of colonic and small-intestinal phenotypes has hitherto remained unclear in gastric cancers. The purpose of the present study was therefore to examine 86 gastric carcinomas histologically and phenotypically using several phenotypic markers, including colon-specific carbonic anhydrase 1 (CA1) and sucrase as small-intestine specific marker. Of 86 gastric cancers, sucrase and CA1 expression was observed in 12 (14.0%) and only in two cases (2.3%), respectively, associated with other intestinal markers such as villin and mucin core protein (MUC)2. In the sucrase cases, expression appeared independent of the stage. However, CA1 expression was observed only in two advanced cases. No association was observed between colonic and small-intestinal phenotypes, and lymph node metastasis and postoperative survival in the advanced gastric cancer cases with intestinal phenotypic expression. Cdx2 appeared to be linked to upregulation of both CA1 and sucrase. In conclusion, the data suggest that colonic phenotype occurs rarely in gastric carcinogenesis. Colonic and small-intestinal phenotypes appear with expression of several intestinal phenotypic markers under the control of Cdx2 and presumably other related transcription factors.

Expression of small intestinal and colonic phenotypes in complete intestinal metaplasia of the human stomach.

The incomplete intestinal metaplasia (IM) that is reported to be a risk factor for gastric carcinogenesis in man usually features sulfomucin production and thus is considered of colonic type. To cast light on the underlying mechanisms, we here examined the proportions of colonic and small intestinal phenotypes in IM by immunohistochemistry and real-time reverse transcription-polymerase chain reaction at the single isolated gland level. Carbonic anhydrase 1 (CA1) is a specific marker of colonic epithelial cells, whereas sucrase is specific to absorptive cells of the small intestine. Totals of 139 (23.5%) and 452 (76.5%) IM glands were judged to be CA1 positive and CA1 negative, respectively, in resected pyloric mucosa from cancer patients. The average score for MUC5AC in CA1-positive IMs was significantly lower than in CA1-negative counterpart tissue (P<0.0001), whereas the opposite was the case for sucrase (P<0.0001). High iron diamine-Alcian blue staining revealed CA1 expression to coincide with type I complete IM. The expression of CA1 mRNA strongly correlated with that of sucrase-isomaltase, and inversely with that of MUC5AC in isolated IM glands. In conclusion, CA1 could be colocalized with small intestinal proteins such as sucrase, but only rarely with the gastric mucin, MUC5AC. Its expression warrants further study, with the focus on stimulation and/or suppression mechanisms by gastric and intestinal transcription factors.

Helicobacter pylori-dependent NF-kappa B activation in newly established Mongolian gerbil gastric cancer cell lines.

Mongolian gerbils are an ideal animal model to explore the role of H. pylori on cancer development. However, there have been no established adenocarcinoma cell lines from this model animal. In the present study, we have established cancer cell lines from a primary gastric cancer tissue of a Mongolian gerbil. The derived cells could be stably attached with H. pylori, revealed under a scanning electron microscope, and easily transplanted to the nude mice. Rapid phosphorylation of IkappaB, Erk1/2, and AKT of these cells was observed by Interleukin-1 beta stimulation, and luciferase reporter gene assay on transcriptional activation of Nuclear Factor kappa B after challenging with either H. pylori NCTC11637 or its isogenic cagE-knockout mutant, H. pylori revealed the cagE-dependent NF-kappaB transcriptional activation. The newly established cancer cell lines from the in vivo gastric carcinogenesis model animal, the Mongolian gerbil, can be used to develop effective therapeutic strategies against gastric cancer, especially in exploring the effect of H. pylori, and thus might greatly contribute to gastric cancer prevention and treatment in humans.

Mixed gastric- and intestinal-type metaplasia is formed by cells with dual intestinal and gastric differentiation.

We have proposed to divide intestinal metaplasia (IM) into two categories, i.e., a mixed gastric and intestinal (GI) type, and a solely intestinal (I) type, based on the residual gastric phenotype cells. The GI-mixed-type IM can be identified by the presence of both cells with either gastric or intestinal phenotypes in a single gland. This study is conducted to elucidate whether cells in the GI-mixed-type IM glands can simultaneously present both gastric and intestinal phenotypes. MUC5AC, MUC2, CD10 and villin expressions were investigated in 20 samples from five gastric cancer cases, directly using either AlexaFluor 488- or 568-labeled specific monoclonal antibodies and observed by fluorescent microscopy and confocal laser-scanning microscopy. GI-mixed IM glands comprise a population expressing MUC5AC and MUC2, MUC5AC and villin, and MUC5AC and CD10. MUC2 and villin expressions were reciprocally increased with decreasing MUC5AC expression, while CD10 expression was limited to cells with only a residual MUC5AC expression or no expression. These results suggest that a heterogeneous cell population with both gastric and intestinal phenotypes would develop into a single intestinal phenotype, as reflected in the progression of intestinal metaplasia from GI-mixed-type- to I-type IM-type glands.

Beta-catenin gene alteration in glandular stomach adenocarcinomas in N-methyl-N-nitrosourea-treated and Helicobacter pylori-infected Mongolian gerbils.

The goal of this study was to elucidate whether beta-catenin gene mutations might contribute to glandular stomach carcinogenesis in Helicobacter pylori (H.pylori)-infected Mongolian gerbils. Firstly, exon 3 of gerbil beta-catenin cDNA, a mutation hot spot, was cloned and sequenced and found to have 89.3% homology with the human form and 95.5% with the rat and mouse forms. Peptide sequence in this region was shown to be 100% conserved in these mammals. Then, 45 stomach adenocarcinomas induced with N-methyl-N-nitrosourea (MNU) plus H. pylori infection and 7 induced with MNU alone were examined for beta-catenin expression by immunohistochemistry and for DNA mutations using a combination of microdissection and PCR-single strand conformation polymorphism analysis. One gastric cancer in the MNU + H. pylori group (2.2%) displayed nuclear (N) beta-catenin localization, 3 (6.7%) showed cytoplasmic (C) distribution in local regions, and 41 (91.1%) demonstrated cell membrane (M) localization. Tumors induced by MNU alone showed only membranous beta-catenin localization (7/7). Analysis of exon 3 of the beta-catenin gene dem-onstrated all tumors with membrane or cytoplasmic staining as well as surrounding normal mucosa (S) to feature wild-type beta-catenin. In contrast, the lesion with nuclear staining had a missense mutation at codon 34 [GAC (Gly) --> GAA (Glu)] in exon 3 (1/1 = 100%, N vs. M, P < 0.05; and N vs. S, P < 0.05). In conclusion, these results suggest that beta-catenin may not be a frequent target for mutation in stomach carcinogenesis in MNU + H. pylori-treated gerbils.

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.

Effect of early eradication on Helicobacter pylori-related gastric carcinogenesis in Mongolian gerbils.

Helicobacter pylori (Hp) infection and gastric carcinogenesis are known to have a close relationship, but the effect of eradication of Hp on Hp-related gastric carcinogenesis has not been fully studied experimentally. To evaluate the effect of eradication in gastric carcinogenesis, an experimental model with eradication in the early, middle or late period was studied using Hp-infected and N-methyl-N-nitrosourea (MNU)-treated Mongolian gerbils. The animals were divided into seven groups: group A (MNU + Hp + eradication at 15 w), group B (MNU + Hp + eradication at 35 w), group C (MNU + Hp + eradication at 55 w), group D (MNU + Hp), group E (MNU), group F (Hp) and group G (control). The tumor incidences at week 75 in the early (group A), middle (group B) and late (group C) eradicated groups were 6.7%, 27.3% and 38.2%, respectively. The incidence of 56.3% in the non-eradicated group was the highest (group D). Incidences in group E, group F and group G were 6.3%, 0.0% and 0.0%, respectively. The tumor incidences were related to the period of inflammatory status induced by Hp infection. Hp infection strongly enhanced gastric carcinogenesis initiated with a chemical carcinogen, and following eradication at an early period this enhancing effect was effectively reduced. Eradication at an early stage of inflammation might be effective in preventing Hp-related gastric carcinogenesis.