Role of bacterial strain diversity of Helicobacter pylori in gastric carcinogenesis induced by N-methyl-N-nitrosourea in Mongolian gerbils

AIM: Helicobacter pylori is known to enhance gastric carcinogenesis induced by chemical carcinogens. We previously demonstrated that infection with H. pylori strain SS1 did not enhance such carcinogenesis in C57BL/6 mice. Whether this result was due to the bacterial strain SS1 or to the experimental host, C57BL/6 mice, should be addressed. Therefore, we examined whether H. pylori strains introduced to the same host (Mongolian gerbils) differed in carcinogenicity. MATERIALS AND METHODS: H. pylori TN2GF4 strain (CagA(+), VacA(+)) and SS1 strain (CagA functionally(-), VacA(-)) were infected to Mongolian gerbils (n = 126). In the first experiment (induction of gastritis), histologic change in gastric mucosa of gerbils infected by H. pylori (TN2GF4, SS1, vehicle) without N-methyl-N-nitrosourea (MNU) at 1 month or 6 months was assessed. In the second experiment (experimental carcinogenesis), H. pylori (TN2GF4, SS1, vehicle) was inoculated to the gerbils after administration of MNU for 10 weeks, and the number of cancers and histopathologic changes at week 54 were assessed. RESULTS: In the first experiment, activity and inflammation in the TN2GF4 group were significantly greater than in the SS1 group at 1 month, while no significant difference was noted at 6 months. On the other hand, intestinal metaplasia and atrophy were significantly greater with TN2GF4 than with SS1 at 6 months but not at 1 month. In studies on experimental carcinogenesis, microscopically, 47.8% (11/23), 26% (7/26), and 0% (0/26), of animals had gastric adenocarcinoma in the MNU + TN2GF4 group, MNU + SS1 group, and MNU alone group, respectively. CONCLUSION: Both H. pylori strains, TN2GF4 and SS1, promoted carcinogenesis in Mongolian gerbils. The severity of gastritis and destruction and restoration of gastric mucosa may be related to gastric carcinogenesis. That the SS1 strain significantly accelerated carcinogenesis only in Mongolian gerbils and not in C57BL/6 mice suggests the crucial role of host factors in carcinogenesis by H. pylori infection.     

Helicobacter pylori inhibits gastric cell cycle progression

Helicobacter pylori infection of the gastric mucosa is associated with changes in gastric epithelial cell proliferation. In vitro studies have shown that exposure to H. pylori inhibits proliferation of gastric cells. This study sought to investigate the cell cycle progression of gastric epithelial cell lines in the presence and absence of H. pylori. Unsynchronized and synchronized gastric epithelial cell lines AGS and KatoIII were exposed to H. pylori over a 24-h period. Cell cycle progression was determined by flow cytometry using propidium iodide (PI), and by analysis of cyclin E, p21, and p53 protein expression using Western blots. In the absence of H. pylori 40, 45, and 15% of unsynchronized AGS cells were in G(0)-G(1), S, and G(2)-M phases, respectively, by flow cytometry analysis. When AGS cells were cultured in the presence of H. pylori, the S phase decreased 10% and the G(0)-G(1) phase increased 17% after 24 h compared with the controls. KatoIII cells, which have a deleted p53 gene, showed little or no response to H. pylori. When G1/S synchronized AGS cells were incubated with media containing H. pylori, the G(1) phase increased significantly (25%, P < 0.05) compared with controls after 24 h. In contrast, the control cells were able to pass through S phase. The inhibitory effects of H. pylori on the cell cycle of AGS cells were associated with a significant increase in p53 and p21 expression after 24 h. The expression of cyclin E was downregulated in AGS cells following exposure of AGS cells to H. pylori for 24 h. This study shows that H. pylori-induced growth inhibition in vitro is predominantly at the G(0)-G(1) checkpoint. Our results suggest that p53 may be important in H. pylori-induced cell cycle arrest. These results support a role for cyclin-dependent kinase inhibitors in the G(1) cell cycle arrest exerted by H. pylori and its involvement in changing the regulatory proteins, p53, p21, and cyclin E in the cell cycle.     

Influence of vitamin E on gastric mucosal injury induced by Helicobacter pylori infection

We investigated the effect of vitamin E on gastric mucosal injury induced by Helicobacter pylori (H. pylori) infection. Male Mongolian gerbils were divided into 4 groups (normal group without H. pylori infection, vitamin E-deficient, -sufficient and -supplemented groups with H. pylori infection). Following oral inoculation with H. pylori (ATCC43504 2 x 10(8) CFU), animals were fed diets alpha-tocopherol 2 mg/100 g diet in the normal and vitamin E-sufficient groups and alpha-tocopherol 0.1 mg/100 g and 50 mg/100 g in the vitamin E-deficient and -supplemented groups, respectively, for 24 weeks. Chronic gastritis was detected in all gerbils inoculated H. pylori. Gastric ulcer was detected in 2 of 7 gerbils only in the vitamin E-deficient group. In the vitamin E-deficient group, myeloperoxidase activity and mouse keratinocyte derived chemokine (KC) in gastric mucosa was significantly higher than in the vitamin E supplemented group. Subsequently, in an in vitro study expression of CD11b/CD18 on neutrophils was enhanced by H. pylori water extract. This effect was suppressed in a dose dependent manner by the addition of alpha-tocopherol. These results suggest that vitamin E has a protective effect on gastric mucosal injury induced by H. pylori infection in gerbils, through the inhibition of accumulation of activated neutrophils.     

Helicobacter pylori encoding the pathogenicity island activates matrix metalloproteinase 1 in gastric epithelial cells via JNK and ERK

Helicobacter pylori colonizes the human gastric epithelium and induces an inflammatory response that is a trigger for gastric carcinogenesis. Matrix metalloproteinases (MMPs) have recently been shown to be up-regulated in gastric epithelial cells infected with H. pylori and might contribute to the pathogenesis of peptic ulcer. The aim of this study was to extend the knowledge about the effect of H. pylori infection on MMP-1 expression by gastric epithelial cells, the kinetics of induction, the pathogenetic properties of the bacterium, and the intracellular signaling pathways required for MMP-1 up-regulation. Expression of MMP-1 was induced more than 10-fold by co-culture of AGS+cells with H. pylori strains carrying the pathogenicity island (PAI). H. pylori strains with mutations in the PAI and a defective type IV secretion system had no effect on MMP-1. Double immunofluorescence revealed strong MMP-1 staining in epithelial cells of gastric biopsies at sites of bacterial attachment. In vitro, MMP-1 is up-regulated by interleukin-1beta and tumor necrosis factor-alpha, but these regulatory mechanisms are not operating in H. pylori infection as shown by inhibitory antibodies. Specific inhibitors of JNK kinase and ERK1/2 kinase were found to suppress the H. pylori-induced MMP-1 expression and activity. AGS cells treated with antisense MMP-1 showed a significantly reduced potential to degrade reconstituted basement membrane. Our results suggest that in gastric epithelial cells, H. pylori up-regulates MMP-1 in a type IV secretion system-dependent manner via JNK and ERK1/2. Induction of MMP-1 is further implicated in complex processes induced by H. pylori, resulting in tissue degradation and remodeling of the gastric mucosa.     

Urokinase plasminogen activator receptor is upregulated by Helicobacter pylori in human gastric cancer AGS cells via ERK, JNK, and AP-1

The gastric pathogen Helicobacter pylori (H. pylori) is suggested to be associated with gastric cancer progression. In this study, we investigated the effect of H. pylori on urokinase plasminogen activator receptor (uPAR) expression which has been known to correlate closely with gastric cancer invasion. H. pylori induced the uPAR expression in a time- and concentration-dependent manner. Specific inhibitors and inactive mutants of MEK-1 and JNK were found to suppress the H. pylori-induced uPAR expression and the uPAR promoter activity. Electrophoretic mobility shift assay and transient transfection study using an AP-1 decoy oligonucleotide confirmed that the activation of AP-1 is involved in the H. pylori-induced uPAR upregulation. The AGS cells treated with H. pylori showed a remarkably enhanced invasiveness, and this effect was partially abrogated by uPAR-neutralizing antibodies. These results suggest that H. pylori induces uPAR expression via Erk-1/2, JNK, and AP-1 signaling pathways and, in turn, stimulates the cell invasiveness in human gastric cancer AGS cells.     

Effect of probiotics and triple eradication therapy on the cyclooxygenase (COX)-2 expression, apoptosis, and functional gastric mucosal impairment in Helicobacter pylori-infected Mongolian gerbils

BACKGROUND: Helicobacter pylori infection in Mongolian gerbils is an established experimental model of gastric carcinogenesis that mimics H. pylori-positive patients developing gastric ulcer and gastric cancer, but the effect of probiotic therapy on functional aspects of this infection remains unknown. METHODS: We compared the effects of intragastric inoculation of gerbils with H. pylori strain (cagA+ vacA+, 5 x 10(6) colony forming units/ml) with or without triple therapy including omeprazole, amoxicillin, and tinidazol or probiotic bacteria Lacidofil. Histology of glandular mucosa, the viable H. pylori, and density of H. pylori colonization were evaluated. The gastric blood flow was measured by H2-gas clearance method; the plasma gastrin and gastric luminal somatostatin were determined by RIA and expression of cyclooxygenase (COX)-2 and apoptotic Bax and Bcl-2 proteins were evaluated by Western blot. RESULTS: The gastric H. pylori infection was detected in all animals by histology and H. pylori culture. Basal gastric acid was significantly reduced in H. pylori-infected animals but not in those with triple therapy or Lacidofil. Early lesions were seen already 4 weeks upon H. pylori inoculation and consisted of chronic gastritis and glandular atypia associated with typical regenerative hyperplasia and increased mitotic activity and formation of apoptotic bodies. The H. pylori infection was accompanied by the fall in gastric blood flow, the marked increase in plasma gastrin, the significant fall in gastric somatostatin levels and Bcl-2 protein expression, and the rise in expression of COX-2 and Bax proteins. These mucosal changes were counteracted by the triple therapy and Lacidofil. CONCLUSIONS: H. pylori infection in gerbils, associated with regenerative hyperplasia of glandular structure, results in the suppression of gastric secretion, overexpression of COX-2, and enhancement in apoptosis and impairment of both, gastric blood flow and gastrin-somatostatin link that were reversed by anti-H. pylori triple therapy and attenuated by probiotics.     

A role for Id in the regulation of TGF-beta-induced epithelial-mesenchymal transdifferentiation

Epithelial-mesenchymal transdifferentiation (EMT) is a critical morphogenic event that occurs during embryonic development and during the progression of various epithelial tumors. EMT can be induced by transforming growth factor (TGF)-beta in mouse NMuMG mammary epithelial cells. Here, we demonstrate a central role of helix-loop-helix factors, E2A and inhibitor of differentiation (Id) proteins, in TGF-beta-induced EMT. Epithelial cells ectopically expressing E2A adopt a fibroblastic phenotype and acquire migratory/invasive properties, concomitant with the suppression of E-cadherin expression. Id proteins interacted with E2A proteins and antagonized E2A-dependent suppression of the E-cadherin promoter. Levels of Id proteins were dramatically decreased by TGF-beta. Moreover, NMuMG cells overexpressed Id2 showed partial resistance to TGF-beta-induced EMT. Id proteins thus inhibit the action of E2A proteins on the expression of E-cadherin, but after TGF-beta stimulation, E2A proteins are present in molar excess of the Id proteins, thus over-riding their inhibitory function and leading to EMT.     

Helicobacter pylori induces gastric epithelial cell invasion in a c-Met and type IV secretion system-dependent manner

Helicobacter pylori interacts with gastric epithelial cells, activating signaling pathways important for carcinogenesis. In this study we examined the role of H. pylori on cell invasion and the molecular mechanisms underlying this process. The relevance of H. pylori cag pathogenicity island-encoded type IV secretion system (T4SS), CagA, and VacA for cell invasion was also investigated. We found that H. pylori induces AGS cell invasion in collagen type I and in Matrigel invasion assays. H. pylori-induced cell invasion requires the direct contact between bacteria and cancer cells. H. pylori-mediated cell invasion was dependent on the activation of the c-Met receptor and on increased MMP-2 and MMP-9 activity. The abrogation of the c-Met receptor using the specific NK4 inhibitor or the silencing of c-Met expression with small interference RNA suppressed both cell invasion and MMP activity. Studies with different H. pylori strains revealed that cell invasion, c-Met tyrosine phosphorylation, and increased MMP-2 and MMP-9 activity were all dependent on the presence of a functional bacterial T4SS, but not on VacA cytotoxicity. Our findings demonstrate that H. pylori strains with a functional T4SS stimulate gastric epithelial cell invasion through a c-Met-dependent signaling pathway that comprises an increase in MMP-2 and MMP-9 activity.     

Cellular stress-related protein expression in Helicobacter pylori-infected gastric epithelial AGS cells

Helicobacter pylori infection leads to gastroduodenal inflammation, peptic ulceration, and gastric carcinoma. Moreover, H. pylori may induce disease-specific protein expression in gastric epithelial cells. The present study was aimed at determining differentially expressed proteins in H. pylori-infected gastric epithelial AGS cells. AGS cells were treated with H. pylori at a bacterium/cell ratio of 300:1 for 12 h. Altered protein patterns as separated by two-dimensional electrophoresis using pH gradients of 4-7 were conclusively identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis of the peptide digests. Four differentially expressed proteins, whose expression levels were increased by more than two-fold in H. pylori-infected cells, were analyzed. These proteins (14-3-3 protein alpha/beta, cullin homolog 3, alpha-enolase, ezrin) are known to be related to cell proliferation, cell adhesion, and carcinogenesis, and may be mediated by cellular stress, such as reactive oxygen species. In conclusion, the identification of these differentially expressed proteins provide valuable information for the understanding of the pathophysiologic mechanisms of H. pylori-induced gastric diseases, and may be useful as prognostic indices of H. pylori-related gastric disorders.