Helicobacter pylori-Mediated Oxidative Stress and Gastric Diseases: A Review

Variants in Autophagy Genes Affect Susceptibility to Both Crohn's Disease and Helicobacter pylori Infection

Objective To detect the methylation status of the promoter region of cytokine signal transduction inhibitor 1 (SOCS-1) in gastric cancer tissues and to explore the relationship between the methylation status and Helicobacter pylori (Hp) infection in gastric cancer. Methods From September 2016 to September 2018, sixty-three patients undergoing gastric cancer surgery (gastric cancer group) and sixty-seven patients undergoing gastric mucosal biopsy for gastric mucosal polyps (control group) were selected. The methylation status of SOCS-1 gene was detected in gastric mucosa tissues of the two groups. The expression of SOCS-1 gene and protein in pathological tissues were detected by RT-PCR and Western blotting. Rapid urease test was used to detect Hp infection and the correlation between SOCS-1methylation and Hp infection in gastric cancer was analyzed. Results Compared with the control group, the positive rate of Hp infection in gastric cancer patients increased (74.60% vs 11.94%, χ2=52.234, P<0.001). Compared with the control group, the abnormal methylation rate of CpG island in the promoter region of SOCS-1 in gastric cancer group increased (80.95% vs 5.97%, χ2=74.491, P<0.001). Compared with negative Hp infection, the proportion of SOCS-1 methylation in Hp-positive gastric cancer patients increased significantly (95.74% vs 4.26%, χ2=26.261, P<0.001). Positive Hp infection was a risk factor for SOCS-1 methylation (OR=1.576, 95% CI: 1.126-2.205, P=0.035). SOCS-1 methylation was associated with low differentiation of gastric cancer and lymph node metastasis in TNM stage (χ2=11.530, 9.380, 11.581, all P<0.01). Compared with the negative Hp infection group, the expressions of SOCS-1 mRNA and protein in the positive group decreased significantly (P<0.05). Conclusion Hp infection may be closely related to SOCS-1 methylation and promote the occurrence and development of gastric cancer by influencing SOCS-1 methylation. Key words: Stomach neoplasms; Helicobacter pylori; DNA methylation; Signal transduction inhibitor

Helicobacter pylori in childhood: new insights into the immunopathogenesis of gastric disease and implications for managing infection in children.

Objective To investigate the interaction between known genetic variation of gastric cancer and Helicobacter pylori (H. pylori) in the occurrence of gastric cancer, and its effect on tumor site and the age at onset of gastric cancer. Methods With a case-only design, the interaction between genetic variation and H. pylori was analyzed by binary Logistic regression analysis in 2 426 patients with gastric cancer. Results After adjusting confounding factors, the interactions of genetic variation NSUN 3 rs7624041 and DEFB rs 2376549 with H. pylori infection in gastric cancer were statistically significant (OR=1.257, 95% CI: 1.006-1.571, P=0.044; OR=0.845, 95% CI: 0.715-0.999, P=0.048). Based on the hierarchical analysis of tumor location, there was no interaction between genetic variation and H. pylori infection. Hierarchical analysis based on tumor stage showed that the interaction between lnc-POLR3G-4 rs7712641 and H. pylori infection was statistically significant in patient with early gastric cancer (OR=1.757, 95% CI: 1.060-2.915, P=0.029). The age-based stratified analysis results showed that the interactions between ASHIL rs80142782, NSUN 3 rs7624041, DEFB rs2376549 and H. pylori infection were statistically significant in people aged Conclusions The occurrence and development of gastric cancer is complex. It is helpful to prevent the occurrence and development of gastric cancer by integrating genetic factors and environmental factors and taking targeted H. pylori eradication measures for susceptible and high-risk groups.

Persistent gastritis induced by Helicobacter pylori is the strongest known risk factor for peptic ulcer disease and distal gastric adenocarcinoma, a process for which adherence of H. pylori to gastric epithelial cells is critical. Decay-accelerating factor (DAF), a protein that protects epithelial cells from complement-mediated lysis, also functions as a receptor for several microbial pathogens. In this study, we investigated whether H. pylori utilizes DAF as a receptor and the role of DAF within H. pylori-infected gastric mucosa. In vitro studies showed that H. pylori adhered avidly to Chinese hamster ovary cells expressing human DAF but not to vector controls. In H. pylori, disruption of the virulence factors vacA, cagA, and cagE did not alter adherence, but deletion of DAF complement control protein (CCP) domains 1-4 or the heavily O-glycosylated serine-threonine-rich COOH-terminal domain reduced binding. In cultured gastric epithelial cells, H. pylori induced transcriptional up-regulation of DAF, and genetic deficiency of DAF attenuated the development of inflammation among H. pylori-infected mice. These results indicate that DAF may regulate H. pylori-epithelial cell interactions relevant to pathogenesis.

ABSTRACTWe investigated the methylation status of multidrug resistance 1 (MDR1) gene promoter in noncancerous gastric mucosa in relation to gastric cancer (GC) occurrence. Methylation of the MDR1 promoter was estimated in 127 GC and 82 non-GC patients. A significant association was found between higher methylation ratio and occurrence of GC. Higher methylation ratio was also associated with occurrence of GC in both Helicobacter pylori–positive and Helicobacter pylori–negative subjects. Higher MR was also associated with Helicobacter pylori infection. Methylation status of MDR1 gene in noncancerous gastric mucosa would be useful for predicting GC occurrence.

Helicobacter pylori is an important risk factor of gastric cancer (GC). Although many H. pylori virulence factors have been reported, the pathogenic mechanism by which H. pylori infection causes GC remains unclear. The aims of this study were to identify GC-related antigens from H. pylori and characterize their roles in the development of GC. As GC and duodenal ulcer (DU) are considered clinically divergent, we compared two-dimensional immunoblots of an acid-glycine extract of H. pylori probed with serum samples from 15 patients with GC and 15 with DU to find GC-related antigens, which were subsequently identified by mass spectrometry. Many protein spots were recognized by more than one serum, and 24 of these were better recognized by GC sera. The proteins showing higher frequency of recognition in GC group are threonine synthase, rod shape-determining protein, S-adenosylmethionine synthetase, peptide chain release factor 1, DNA-directed RNA polymerase alpha subunit, co-chaperonin GroES (monomeric and dimeric forms), response regulator OmpR, and membrane fusion protein. Of these proteins, GroES was identified as a dominant GC-related antigen with a much higher seropositivity of GC samples (64.2%, n = 95) compared with 30.9% for gastritis (n = 94) and 35.5% for DU (n = 124). GroES seropositivity was more commonly associated with antral GC than with non-antral GC (odds ratio = 2.7; 95% confidence interval, 1.1-6.7). In peripheral blood mononuclear cells, GroES stimulated production of interleukin (IL)-8, IL-6, granulocyte macrophage colony-stimulating factor, IL-1beta, tumor necrosis factor-alpha, cyclooxygenase-2, and prostaglandin E(2). Moreover when incubated with gastric epithelial cells, GroES induced expression of IL-8, cell proliferation, and up-regulation of c-jun, c-fos, and cyclin D1 but caused down-regulation of p27(Kip1). We conclude that GroES of H. pylori is a novel GC-associated virulence factor and may contribute to gastric carcinogenesis via induction of inflammation and promotion of cell proliferation.

Regulation of Gastric Carcinogenesis by InflammatoryCytokines.

Objective To investigate the expressions of large tumor suppressor kinase 1 (LATS1) and large tumor suppressor kinase 2 (LATS2) proteins in gastric cancer tissues, and to explore the correlation between expressions of LATS1 and LATS2 proteins and the occurrence and development of gastric cancer. Methods A total of 93 gastric cancer paraffin tissues and the corresponding adjacent gastric normal mucosa in the Department of Pathology in Baotou Cancer Hospital from September 2008 to June 2010 were collected. The immunohistochemistry was used to detect the expressions of LATS1 and LATS2 proteins in gastric cancer and adjacent normal tissues. The differences of the expressions of LATS1 and LATS2 proteins in gastric cancer and adjacent normal tissues were compared by using χ2 test. The relationship between the expressions of LATS1 and LATS2 proteins and the clinicopathological features was also analyzed. Results In gastric cancer tissues, LATS1 was negatively expressed in 54 cases (58.1%), weakly positive expressed in 15 cases (16.1%), moderately positive expressed in 16 cases (17.2%), and strongly positive expressed in 8 cases (8.6%); in adjacent normal tissues, LATS1 was negatively expressed in 17 cases (18.3%), weakly positive expressed in 16 cases (17.2%), moderately positive expressed in 31 cases (33.3%), and strongly positive expressed in 29 cases (31.2%). The positive expression rate of LATS1 in gastric cancer tissues was lower than that in adjacent normal tissues, and the difference was statistically significant (χ2= 37.460, P 0.05). Conclusion LATS1 and LATS2 proteins may be involved in the occurrence of gastric cancer and have the inhibiting effect on the occurrence and development of gastric cancer. Key words: Gastric neoplasms; Large tumor suppressor kinase 1; Large tumor suppressor kinase 2

Helicobacter pylori and MALT Lymphoma

Helicobacter pylori (Hp) has been classified as a class I carcinogen in gastric cancer, and one of the important mechanisms by which it affects the gastric environment and promotes cancerogenesis is by triggering inflammation.Inflammatory responses caused by Hp’s ‘crosstalk’ with various types of cells in the microenvironment of the gastric tumour play a key role in cancer progression, but the exact mechanism of its ‘crosstalk’ is still unclear. This paper focuses on the regulatory impact of Hp infection on the tumor microenvironment, systematically explores the activation process of immune stromal cells such as tumor-associated macrophages (TAMs), cancer-associated fibroblasts (CAFs), mesenchymal stem cells (MSCs), and myeloid-derived suppressor cells by directly or indirectly promoting the secretion of inflammatory factors by Hp, and analyzes the molecular mechanism of these cells that influence the occurrence and development of gastric cancer through multiple signal pathways, in order to provide innovative theoretical basis for the development of accurate prevention and treatment strategies for Hp infection-related gastric cancer.

Backgrounds: We previously revealed that CD44, cancer stem-like marker, interacts with xCT, a glutamate-cystine transporter, and controls the intracellular level of reduced glutathione. Helicobacter pylori (HP) infection is known to be a major cause of gastric cancer and be associated with increased expression of CD44 on gastric epithelial cells, but precise mechanism of CD44 up-regulation still has uncertain. On the other hand, microRNAs (miRNAs) are noncoding RNAs that regulate molecular pathways in cancer by targeting various genes. The aim of the current study was to identify the miRNA regulating CD44 expression in H. pylori-infected gastric mucosa during the development of preneoplastic lesions. Methods and Results: To investigate the regulation of CD44 through miRNA, we performed 384 miRNA quantitative RT-PCR array analyses in six human gastrointestinal cancer cell lines that differ in CD44 expression status. We identified miR-328 which is predicted as CD44-targeted and established the functional relationship between miR-328 and CD44. MiR-328 overexpression decreased CD44 expression and led to inhibition of cancer cell growth and impairing chemo- and ROS (reactive oxygen species) - resistance. We performed luciferase assay using vectors containing CD44 3′- UTR, which might be directly bound by miR-328, using the miRanda algorithm, and the results indicated that miR-328 regulated CD44 expression by directly targeting its 3′-UTR. We polarized THP-1 that human monocytic leukemia cell line to M1- and M2 macrophages, and detected ROS production of its by using 2′,7′-dichlorofluorescein diacetate, and ROS induced miR-328 down-regulation and CD44 up-regulation by co-culture gastric cancer cell lines with M1- and M2-polarized THP-1 macrophages. We further analyzed the association between miR-328-CD44 pathway and HP infection in human gastric mucosa. We identified that HP infection correlated with miR-328 down-regulation and CD44 up-regulation. Lastly, we examined the DNA methylation of the miR-328 5′-flanking region by bisulfite pyrosequencing in human gastric mucosa, but HP infection were not related with the DNA methylation level in this region. Conclusions: We show that inflammatory response with HP infection causes CD44 expression via miR-328 suppression, resulting in cancer initiation and progression, but HP infection was unrelated to the DNA methylation in the miR-328 5′-flanking region. Citation Format: Keisuke Miyake, Takatsugu Ishimoto, Hidetaka Sugihara, Kojiro Eto, Daisuke Izumi, Junji Kurashige, Yukiharu Hiyoshi, Shiro Iwagami, Yoshifumi Baba, Yuji Miyamoto, Naoya Yoshida, Hideo Baba. Helicobacter pylori infection via miR-328 suppression and CD44 expression in gastric mucosa causes gastric cancer initiation and progression. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 198. doi:10.1158/1538-7445.AM2015-198

A Functional Polymorphism of Toll-Like Receptor 4 Gene Increases Risk of Gastric Carcinoma and Its Precursors

Aberrant DNA methylation accumulates in non-malignant gastric mucosa after exposure to environmental pathogens such as H. pylori (HP). To understand how environmental factors and DNA methylation interplay to influence primary gastric cancer (GC) risk, we performed an integrated analysis of clinical factors and DNA methylation data of gastric tissues from a longitudinally monitored cohort in Japan, with validation in a separate cohort from Singapore. The Japanese check-up cohort included 4,234 healthy subjects who underwent gastric mucosal biopsy. The median observation period was 4.2 years, and 77 subjects developed GC. GC incidence correlated with age, drinking, smoking, GC family history, and HP status in the multivariable Cox model. Next, we conducted comprehensive DNA methylation analysis using Infinium MethylationEPIC arrays on gastric tissues (n=164), including (1) mucosal biopsies from subjects who later developed GC (“future GC patients”), (2) mucosal biopsies from HP(+) subjects who did not later develop GC (“future non-GC subjects”), (3) mucosal biopsies from future non-GC HP(−) subjects (“control mucosae”), and (4) GCs and surrounding mucosae. Infinium data of gastric mucosae (n=137) collected in the GCEP cohort (Singapore) were also analyzed. DNA methylation of promoter region observed in GCs, accumulated not only in mucosae adjacent to GCs but also in the biopsy mucosae of future GC patients. Mucosae of future non-GC subjects were more methylated than control mucosae but less methylated than mucosae of future GC patients. Similar findings were observed in the GCEP cohort. DNA methylation levels were associated with clinical factors and histopathological alterations - however, in multivariable analyses, DNA methylation remained an independent GC risk factor. Methylation levels were predictive of not only higher GC risk but also a shorter period to GC incidence. We then focused on the associations between environmental factors and methylation. Heavy drinking and smoking were associated with the accumulation of DNA methylation only in HP(+) subjects. The increases in methylation over time in subjects who quit smoking were significantly attenuated compared to continuous smokers. These results suggest that pro-carcinogenic epigenetic alterations initiated by HP exposure are amplified by unfavorable but modifiable lifestyle choices. Furthermore, target genes methylated by each environmental factor may overlap in part; however, they were not necessarily methylated similarly, suggesting that the best markers for stratifying GC risk may differ for each subgroup classified by exposure to environmental factors. Indeed, candidate markers for stratifying GC risk overlapped among subgroups, whereas markers unique to each subgroup were also identified, highlighting the potential of integrating environmental, lifestyle, and epigenetic information to inform GC precision prevention. Citation Format: Genki Usui, Keisuke Matsusaka, Kie K. Huang, Feng Zhu, Tomohiro Shinozaki, Masaki Fukuyo, Bahityar Rahmutulla, Norikazu Yogi, Tomoka Okada, Motoaki Seki, Eiji Sakai, Kazutoshi Fujibayashi, Stephen K. Tsao, Christopher Khor, Tiing L. Ang, Hiroyuki Abe, Hisahiro Matsubara, Masashi Fukayama, Toshiaki Gunji, Nobuyuki Matsuhashi, Teppei Morikawa, Tetsuo Ushiku, Khay G. Yeoh, Patrick Tan, Atsushi Kaneda. Integrated environmental, lifestyle, and epigenetic risk prediction of primary gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 761.

DNA Hypermethylation Contributes to Incomplete Synthesis of Carbohydrate Determinants in Gastrointestinal Cancer