Helicobacter pylori infection‐induced H3Ser10 phosphorylation in stepwise gastric carcinogenesis and its clinical implications

Background

Our previous works have demonstrated that Helicobacter pylori (Hp) infection can alter histone H3 serine 10 phosphorylation status in gastric epithelial cells. However, whether Helicobacter pylori‐induced histone H3 serine 10 phosphorylation participates in gastric carcinogenesis is unknown. We investigate the expression of histone H3 serine 10 phosphorylation in various stages of gastric disease and explore its clinical implication.

Materials and Methods

Stomach biopsy samples from 129 patients were collected and stained with histone H3 serine 10 phosphorylation, Ki67, and Helicobacter pylori by immunohistochemistry staining, expressed as labeling index. They were categorized into nonatrophic gastritis, chronic atrophic gastritis, intestinal metaplasia, low‐grade intraepithelial neoplasia, high‐grade intraepithelial neoplasia, and intestinal‐type gastric cancer groups. Helicobacter pylori infection was determined by either ¹³C‐urea breath test or immunohistochemistry staining.

Results

In Helicobacter pylori‐negative patients, labeling index of histone H3 serine 10 phosphorylation was gradually increased in nonatrophic gastritis, chronic atrophic gastritis, intestinal metaplasia groups, peaked at low‐grade intraepithelial neoplasia, and declined in high‐grade intraepithelial neoplasia and gastric cancer groups. In Helicobacter pylori‐infected patients, labeling index of histone H3 serine 10 phosphorylation followed the similar pattern as above, with increased expression over the corresponding Helicobacter pylori‐negative controls except in nonatrophic gastritis patient whose labeling index was decreased when compared with Helicobacter pylori‐negative control. Labeling index of Ki67 in Helicobacter pylori‐negative groups was higher in gastric cancer than chronic atrophic gastritis and low‐grade intraepithelial neoplasia groups, and higher in intestinal metaplasia group compared with chronic atrophic gastritis group. In Helicobacter pylori‐positive groups, Ki67 labeling index was increased stepwise from nonatrophic gastritis to gastric cancer except slightly decrease in chronic atrophic gastritis group. In addition, we noted that histone H3 serine 10 phosphorylation staining is accompanied with its location changes from gastric gland bottom expanded to whole gland as disease stage progress.

Conclusions

These results indicate that stepwise gastric carcinogenesis is associated with altered histone H3 serine 10 phosphorylation, Helicobacter pylori infection enhances histone H3 serine 10 phosphorylation expression in these processes; it is also accompanied with histone H3 serine 10 phosphorylation location change from gland bottom staining expand to whole gland expression. The results suggest that epigenetic dysregulation may play important roles in Helicobacter pylori‐induced gastric cancer.     

Helicobacter pylori infection up-regulates gland mucous cell-type mucins in gastric pyloric mucosa

Background. Two types of mucous cell are present in gastric mucosa: surface mucous cells (SMCs) and gland mucous cells (GMCs), which consist of cardiac gland cells, mucous neck cells, and pyloric gland cells. We have previously reported that the patterns of glycosylation of SMC mucins are reversibly altered by Helicobacter pylori infection. In this study, we evaluated the effects of H. pylori infection on the expression of GMC mucins in pyloric gland cells. Methods. Gastric biopsy specimens from the antrums of 30 H. pylori‐infected patients before and after eradication of H. pylori and 10 normal uninfected volunteers were examined by immunostaining for MUC6 (a core protein of GMC mucins), α1,4‐N‐acetyl‐glucosaminyl transferase (α4GnT) (the glycosyltransferase which forms GlcNAcα1‐4Galβ‐R), and GlcNAcα1‐4Galβ‐R (a GMC mucin‐specific glycan). Results. MUC6, α4GnT, and HIK1083‐reactive glycan were expressed in the cytoplasm, supranuclear region, and secretory granules in pyloric gland cells, respectively. The immunoreactivity of MUC6 and α4GnT, but not of GlcNAcα1‐4Galβ‐R, in the pyloric gland increased in H. pylori‐associated gastritis, and after the eradication of H. pylori, the increased expression of MUC6 and α4GnT in the gastric mucosa of H. pylori‐infected patients decreased to almost normal levels. This up‐regulation was correlated with the degree of inflammation. Conclusions. In addition to the synthesis of GMC mucins increasing reversibly, their metabolism or release may also increase reversibly in H. pylori‐associated gastritis. The up‐regulation of the expression of gastric GMC mucins may be involved in defense against H. pylori infection in the gastric surface mucous gel layer and on the gastric mucosa.     

Anthocyanins from black soybean inhibit Helicobacter pylori‐induced inflammation in human gastric epithelial AGS cells

Infection with Helicobacter pylori leads to gastritis, peptic ulcers and gastric cancer. Moreover, when the gastric mucosa is exposed to H. pylori, gastric mucosal inflammatory cytokine interleukin‐8 (Il‐8) and reactive oxygen species increase. Anthocyanins have anti‐oxidative, antibacterial and anti‐inflammatory properties. However, the effect of anthocyanins in H. pylori‐infected cells is not yet clear. In this study, therefore, the effect of anthocyanins on H. pylori‐infected human gastric epithelial cells was examined. AGS cells were pretreated with anthocyanins for 24 hrs followed by H. pylori 26695 infection for up to 24 hrs. Cell viability and ROS production were examined by 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide and 2′,7′–dichlorofluorescein diacetate assay, respectively. Western blot analyses and RT‐PCR were performed to assess gene and protein expression, respectively. IL‐8 secretion in AGS cells was measured by ELISA. It was found that anthocyanins decrease H. pylori‐induced ROS enhancement. Anthocyanins also inhibited phosphorylation of mitogen‐activated protein kinases, translocation of nuclear factor‐kappa B and Iκβα degradation. Furthermore anthocyanins inhibited H. pylori‐induced inducible nitric oxide synthases and cyclooxygenase‐2 mRNA expression and inhibited IL‐8 production by 45.8%. Based on the above findings, anthocyanins might have an anti‐inflammatory effect in H. pylori‐infected gastric epithelial cells.     

Protein changes in gastric epithelial cells RGM-1 in response to Helicobacter pylori infection

Helicobacter pylori-induced inflammation significantly increases the risk of gastric cancer. To investigate the role of H. pylori infection in gastric epithelial cell carcinogenesis, flow cytometry was used to analyze the apoptosis of gastric epithelial cells infected by H. pylori. Next, LTQ MS mass spectrometry (MS) was applied to identify protein changes in gastric epithelial cells infected with H. pylori, and then bioinformatics was adopted to analyze the cellular localization and biological function of differential proteins. LTQ MS/MS successfully identified identified 22 differential proteins successfully, including 20 host-cell proteins and two H. pylori bacterial proteins. Also, human proteins were located in all areas of cells and involved in various cell biological functions. The oncogene proteins p53, p16, and C-erbB-2 proteins in H. pylori-infected RGM-1 cells were remarkably increased from the analysis by Western blot analysis. H. pylori infection of gastric epithelial cells leads to changes in various protein components in the cell, and enhances the expression of oncogene proteins, thereby increasing the possibility of possibility of carcinogenesis of H. pylori infection.     

Teprenone, but not H2-receptor blocker or sucralfate, suppresses corpus Helicobacter pylori colonization and gastritis in humans: teprenone inhibition of H. pylori-induced interleukin-8 in MKN28 gastric epithelial cell lines

Background. The role of teprenone in Helicobacter pylori‐associated gastritis has yet to be determined. To investigate the effect of teprenone on inflammatory cell infiltration, and on H. pylori colonization of the gastric mucosa in H. pylori‐infected patients, we first compared the effect of teprenone with that of both histamine H2 receptor antagonists (H2‐RA) and sucralfate on the histological scores of H. pylori gastritis. We then examined its in vitro effect on H. pylori‐induced interleukin (IL)‐8 production in MKN28 gastric epithelial cells. Materials and Methods. A total of 68 patients were divided into three groups, each group undergoing a 3‐month treatment with either teprenone (150 mg/day), H2‐RA (nizatidine, 300 mg/day), or sucralfate (3 g/day). All subjects underwent endoscopic examination of the stomach before and after treatment. IL‐8 production in MKN28 gastric epithelial cells was measured by enzyme‐linked immunosorbent assay (ELISA). Results. Following treatment, the teprenone group showed a significant decrease in both neutrophil infiltration and H. pylori density of the corpus (before vs. after: 2.49 ± 0.22 vs. 2.15 ± 0.23, p = .009; 2.36 ± 0.25 vs. 2.00 ± 0.24, p = .035, respectively), with no significant differences seen in either the sucralfate or H2‐RA groups. Teprenone inhibited H. pylori‐enhanced IL‐8 production in MKN28 gastric epithelial cells in vitro, in a dose‐dependent manner. Conclusions. Teprenone may modify corpus H. pylori‐associated gastritis through its effect on neutrophil infiltration and H. pylori density, in part by its inhibition of IL‐8 production in the gastric mucosa.     

Interleukin-6 genetic polymorphisms are not related to Helicobacter pylori-associated gastroduodenal diseases

Background. Polymorphisms in the promoter region of the proinflammatory cytokine, interleukin (IL)‐6 have been related to several chronic inflammatory diseases. Inter‐individual variation in the severity of gastric inflammation may be important in determining the clinical outcome of an Helicobacter pylori infection and relate to polymorphisms in this region. Materials and Methods. We studied H. pylori‐infected patients with duodenal ulcer or gastric cancer. In addition six gastric cancer cell lines, AGS, SNU‐668, MKN‐1, MKN‐7, MKN28 and KATOIII, were cocultured with both cag pathogenicity island‐positive and ‐negative H. pylori. Single nucleotide polymorphisms at positions ?174, ?572, and ?597 in the IL‐6 promoter region were identified by PCR‐RFLP. The IL‐6 production from the cancer cells was determined by ELISA. Results. Sixty patients with gastric cancer and 60 with duodenal ulcer were studied. The alleles at positions ?174 and ?597 were closely linked (?174G/?597G or ?174C/?597A) regardless of the ethnic group or disease presentation. There was no difference in the allele frequency at any of the sites among patient groups. H. pylori‐induced IL‐6 production from the gastric cancer cell lines was also independent of the IL‐6 polymorphisms or the presence of the cag pathogenicity island. Conclusions. The genetic polymorphisms in IL‐6 can be attributable to ethnicity and appear to be independent of the clinical outcome of an H. pylori infection.     

Modulation of Activation-Induced Cytidine Deaminase by Curcumin in Helicobacter pylori-Infected Gastric Epithelial Cells

Background: Anomalous expression of activation‐induced cytidine deaminase (AID) in Helicobacter pylori‐infected gastric epithelial cells has been postulated as one of the key mechanisms in the development of gastric cancer. AID is overexpressed in the cells through nuclear factor (NF)‐κB activation by H. pylori and hence, inhibition of NF‐κB pathway can downregulate the expression of AID. Curcumin, a spice‐derived polyphenol, is known for its anti‐inflammatory activity via NF‐κB inhibition. Therefore, it was hypothesized that curcumin might suppress AID overexpression via NF‐κB inhibitory activity in H. pylori‐infected gastric epithelial cells. Materials and Methods: MKN‐28 or MKN‐45 cells and H. pylori strain 193C isolated from gastric cancer patient were used for co‐culture experiments. Cells were pretreated with or without nonbactericidal concentrations of curcumin. Apoptosis was determined by DNA fragmentation assay. Enzyme‐linked immunosorbent assay was performed to evaluate the anti‐adhesion activity of curcumin. Real‐time polymerase chain reaction was employed to evaluate the expression of AID mRNA. Immunoblot assay was performed for the analysis of AID, NF‐κB, inhibitors of NF‐κB (IκB), and IκB kinase (IKK) complex regulation with or without curcumin. Results: The adhesion of H. pylori to gastric epithelial cells was not inhibited by curcumin pretreatment at nonbactericidal concentrations (≤10 μmol/L). Pretreatment with nonbactericidal concentration of curcumin downregulated the expression of AID induced by H. pylori. Similarly, NF‐κB activation inhibitor (SN‐50) and proteasome inhibitor (MG‐132) also downregulated the mRNA expression of AID. Moreover, curcumin (≤10 μmol/L) has suppressed H. pylori‐induced NF‐κB activation via inhibition of IKK activation and IκB degradation. Conclusion: Nonbactericidal concentrations of curcumin downregulated H. pylori‐induced AID expression in gastric epithelial cells, probably via the inhibition of NF‐κB pathway. Hence, curcumin can be considered as a potential chemopreventive candidate against H. pylori‐related gastric carcinogenesis.     

Role of γ‐glutamyltranspeptidase in the pathogenesis of Helicobacter pylori infection

γ‐Glutamyltranspeptidase and asparaginase have been shown to play important roles in Helicobacter pylori colonization and cell death induced by H. pylori infection. In this study, the association of γ‐glutamyltranspeptidase and asparaginase was elucidated by comparing activities of both deamidases in H. pylori strains from patients with chronic gastritis, gastric and duodenal ulcers, and gastric cancer. γ‐Glutamyltranspeptidase activities in H. pylori strains from patients with gastric cancer were significantly higher than in those from patients with chronic gastritis or gastric ulcers. There was a wide range of asparaginase activities in H. pylori strains from patients with gastric cancer and these were not significantly than those from patients with other diseases. To identify the contributions of γ‐glutamyltranspeptidase and asparaginase to gastric cell inflammation, human gastric epithelial cells (AGS line) were infected with H. pylori wild‐type and knockout strains and inflammatory responses evaluated by induction of interleukin‐8 (IL‐8). IL‐8 response was significantly decreased by knockout of the γ‐glutamyltranspeptidase‐encoding gene but not by knockout of the asparaginase‐encoding gene. Additionally, IL‐8 induction by infection with the H. pylori wild‐type strain was significantly decreased by adding glutamine during infection. These findings indicate that IL‐8 induction caused by γ‐glutamyltranspeptidase activity in H. pylori is mainly attributable to depletion of glutamine. These data suggest that γ‐glutamyltranspeptidase plays a significant role in the chronic inflammation caused by H. pylori infection.     

Haem oxygenase‐1 inhibits phosphorylation of the Helicobacter pylori oncoprotein CagA in gastric epithelial cells

The cytotoxin‐associated gene A protein (CagA) plays a pivotal role in the aetiology of Helicobacter pylori‐associated gastric diseases. CagA is injected into the cytoplasm of host cells by a type IV secretion system, and is phosphorylated on tyrosine residues by the host enzyme c‐Src. We previously reported that the enzyme haem oxygenase‐1 (HO‐1) inhibits IL‐8 secretion by H. pylori‐infected cells. However, the cellular mechanism by which HO‐1 regulates the innate immune function of infected cells remains unknown. We now show that nitric oxide and haemin, two inducers of HO‐1, decrease the level of phosphorylated CagA (p‐CagA) in H. pylori‐infected gastric epithelial cells and this is blocked by either pharmacological inhibition of HO‐1 or siRNA knockdown of hmox‐1. Moreover, forced expression of HO‐1 by transfection of a plasmid expressing hmox‐1 also results in a strong attenuation of CagA phosphorylation. This occurs through the inhibition of H. pylori‐induced c‐Src phosphorylation/activation by HO‐1.Consequently, H. pylori‐induced cytoskeletal rearrangements and activation of the pro‐inflammatory response mediated by p‐CagA are inhibited in HO‐1‐expressing cells. These data highlight a mechanism by which the innate immune response of the host can restrict the pathogenicity of H. pylori by attenuating CagA phosphorylation in gastric epithelial cells.     

Increased gastric osteopontin expression by Helicobacter pylori Infection can correlate with more severe gastric inflammation and intestinal metaplasia

Background: Osteopontin (OPN) is involved in the gastric cancer progression. The study validated whether OPN expressions correlate with Helicobacter pylori‐related chronic gastric inflammation and the precancerous change as intestinal metaplasia (IM). Methods: This study included 105 H. pylori‐infected patients (63 without and 42 with IM) and 29 H. pylori‐negative controls. In each subject, the gastric OPN expression intensity was evaluated by immunohistochemistry, and graded from 0 to 4 for the epithelium, lamina propria, and areas with IM, respectively. For the H. pylori‐infected subjects, the gastric inflammation was assessed by the Updated Sydney System. Forty‐nine patients received follow‐up endoscopy to assess OPN change on gastric mucosa after H. pylori eradication. The in vitro cell‐H. pylori coculture were performed to test the cell origin of OPN. Results: The H. pylori‐infected patients had higher gastric OPN expression than the noninfected controls (p < .001). For the H. pylori‐infected patients, an increased OPN expression correlated with more severe chronic gastric inflammation (p < .001) and the presence of IM (OR: 2.6, 95% CI: 1.15–5.94, p = .02). Within the same gastric bits, lamina propria expressed OPN stronger than epithelium (p < .001), suggesting OPN predominantly originates from inflammatory cells. The in vitro assay confirmed H. pylori stimulate OPN expression in the monocytes, but not in the gastric epithelial cells. After H. pylori eradication, the gastric OPN expression could be decreased only in areas without IM (p < .05). Conclusions: Increased gastric OPN expression by H. pylori infection can correlate with a more severe gastric inflammation and the presence of IM.     

Helicobacter pylori strain and the pattern of gastritis among first-degree relatives of patients with gastric carcinoma

Background. Relatives of gastric cancer patients have an increased risk of gastric cancer, possibly related to genetically‐related strains of Helicobacter pylori or a common environment. Methods. The pattern of gastritis and H. pylori from gastric cancer patients and their first‐degree relatives were compared using detailed DNA fingerprints and vacA, cagA, and iceA genotyping. Results. Sixteen index cases from Korea, the US, or Colombia and their 38 first‐degree relatives (brothers, sisters, sons and daughters) were studied. No definite, or consistent, relationship between the pattern of gastritis and the relatedness of the H. pylori strain was observed (i.e. relatives could have an identical or a totally different pattern of gastritis regardless if they were infected with identical or highly similar organisms). For example, three elderly siblings of an index case with atrophic pangastritis had identical H. pylori isolates and environments in childhood and yet two had antral predominant nonatrophic gastritis, which is typically associated with duodenal ulcer instead of gastric cancer. Conclusions. The results of this study are not consistent with the hypothesis that specific virulence factors or similar H. pylori strains correlate with a specific histologic pattern or outcome even among those sharing the same environment in childhood.     

CagA tyrosine phosphorylation in gastric epithelial cells caused by Helicobacter pylori in patients with gastric adenocarcinoma

Background. Tyrosine phosphorylation of Helicobacter pylori cytotoxin‐associated protein of in gastric epithelial cells is reported. The goals of this study are first to examine the occurrence of CagA tyrosine phosphorylation in H. pylori strains isolated from patients with gastric adenocarcinoma and gastritis, and second to clarify the relationship between the diversity of tyrosine phosphorylation motifs and the presence of CagA tyrosine phosphorylation. Methods. Fifty‐eight clinical isolates of H. pylori from patients with gastric adenocarcinoma (29 cases) and gastritis (29 cases) were studied for CagA tyrosine phosphorylation by Western blotting. Sequence diversity of tyrosine phosphorylation motifs was analysed among positive‐ or negative‐CagA tyrosine phosphorylation isolates. Results. Positive CagA tyrosine phosphorylation was found in 93.1% (27 of 29) of strains from gastric adenocarcinoma patients and 51.7% (15 of 29) of strains from gastritis patients (p < 0.001). Intact motifs were found in H. pylori isolates with CagA tyrosine phosphorylation. Of the 16 negative CagA tyrosine phosphorylation isolates, intact tyrosine phosphorylation motifs were found in 15 isolates. Conclusions. CagA tyrosine phosphorylation, which is significantly greater in strains from gastric adenocarcinoma patients, may play a role in gastric carcinogenesis, and could be a better marker of more virulent strains than the cag pathogenicity island in Asia, where the cag pathogenicity island is present in nearly all H. pylori strains. Sequence diversity of tyrosine phosphorylation motifs on CagA was not related to the presence of tyrosine phosphorylation. The absence of tyrosine phosphorylation motif might result in negative tyrosine phosphorylation phenotypes, but such motifs are not the sole factors associated with CagA tyrosine phosphorylation.     

The effect of Helicobacter pylori infection on levels of DNA damage in gastric epithelial cells

Background. Helicobacter pylori infection leads to an increased risk of developing gastric cancer. The mechanism through which this occurs is not known. We aimed to determine the effect of H. pylori and gastritis on levels of DNA damage in gastric epithelial cells. Methods. Epithelial cells were isolated from antral biopsies from 111 patients. DNA damage was determined using single cell gel electrophoresis and the proportion of cells with damage calculated before and 6 weeks after eradication of H. pylori. Cell suspensions generated by sequential digestions of the same biopsies were assayed to determine the effect of cell position within the gastric pit on DNA damage. Results. DNA damage was significantly higher in normal gastric mucosa than in H. pylori gastritis [median (interquartile range) 65% (58.5–75.8), n = 18 and 21% (11.9–29.8), n = 65, respectively, p < .001]. Intermediate levels were found in reactive gastritis [55.5% (41.3–71.7), n = 13] and H. pylori negative chronic gastritis [50.5% (36.3–60.0), n = 15]. DNA damage rose 6 weeks after successful eradication of H. pylori[to 39.5% (26.3–51.0), p = .007] but was still lower than in normal mucosa. Chronic inflammation was the most important histological factor that determined DNA damage. DNA damage fell with increasing digestion times (r = –.92 and –.88 for normal mucosa and H. pylori gastritis, respectively). Conclusions. Lower levels of DNA damage in cells isolated from H. pylori infected gastric biopsies may be a reflection of increased cell turnover in H. pylori gastritis. The investigation of mature gastric epithelial cells for DNA damage is unlikely to elucidate the mechanisms underlying gastric carcinogenesis.     

Recruitment of CCR6+ Foxp3+ regulatory gastric infiltrating lymphocytes in Helicobacter pylori gastritis

Helicobacter pylori (H. pylori) infection is associated with an inflammatory response in the gastric mucosa, leading to chronic gastritis, peptic ulcers, and gastric cancer. Increased T‐cell infiltration is found at sites of H. pylori infection. The CCR6⁺ subset of CD4⁺ regulatory T cells (Tregs), a newly characterized subset of Tregs, has been reported to contribute to local immune inhibition. However, whether CCR6⁺ Tregs are present in H. pylori gastritis, and what their relationship is to disease prognosis, remains to be elucidated. In this study, gastric infiltrating lymphocytes were isolated from endoscopic biopsy specimens of H. pylori gastritis patients and analyzed. We found that in gastric infiltrating lymphocytes, CCR6⁺ CD4⁺ CD25^high Tregs, which express high levels of CD45RO, are positively associated with more severe inflammation in gastric mucosa during H. pylori infection. Furthermore, the frequency of CCR6⁺ Tregs in gastric infiltrating lymphocytes, but not CCR6^? Tregs, is significantly increased in inflamed gastric tissues, which is inversely correlated with significantly lower expression of IFN‐γ⁺ CD8⁺ T cells. We also found that the frequency of CCR6⁺ Tregs is positively correlated with the frequency of CD4⁺ IFN‐γ⁺ T cells. In addition, the frequency of CCR6⁺ Tregs, but not that of CCR6^? Tregs, is significantly correlated with increased inflammation in H. pylori gastritis. This study demonstrates that immunosuppression in H. pylori gastritis might be related to the activity of CCR6⁺ Tregs, which could influence disease prognosis.