Pylori-infected Gastric Epithelial Cells Research Articles

GKN1–miR-185–DNMT1 Axis Suppresses Gastric Carcinogenesis through Regulation of Epigenetic Alteration and Cell Cycle

Gastrokine 1 (GKN1) functions to protect the gastric antral mucosa and promotes healing by facilitating restoration and proliferation after injury. GKN1 is downregulated in Helicobacter pylori-infected gastric epithelial cells and loss of GKN1 expression is closely associated with gastric carcinogenesis, but underlying mechanisms of the tumor-suppressing effects of GKN1 remain largely unknown. AGS, MKN1, MKN28 gastric cancer cells and HFE-145 immortalized non-neoplastic gastric mucosal cells were transfected with GKN1 or shGKN1. We conducted molecular and functional studies of GKN1 and miR-185 and investigated the mechanisms of alteration. We also analyzed epigenetic alterations in 80 gastric cancer tissues. Restoration of GKN1 protein suppressed gastric cancer cell growth by inducing endogenous miR-185 that directly targets epigenetic effectors DNMT1 and EZH2 in gastric cancer cells. In addition, ectopic expression of GKN1 upregulated Tip60 and downregulated HDAC1 in an miR-185-independent manner, thereby inducing cell-cycle arrest by regulating cell-cycle proteins in gastric cancer cells. Notably, GKN1 expression was inversely correlated with DNMT1 and EZH2 expression in a subset of 80 gastric cancer tissues and various gastric cancer cell lines. Interestingly, it was found that GKN1 exerted a synergistic anti-cancerous effect with 5-fluorouracil on tumor cell growth, which suggests a possible therapeutic intervention method for gastric cancer. Our results show that GKN1 has an miR-185-dependent and -independent mechanism for chromatic and DNA epigenetic modification, thereby regulating the cell cycle. Thus, the loss of GKN1 function contributes to malignant transformation and proliferation of gastric epithelial cells in gastric carcinogenesis.

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.

The phenyl-thiophenyl propenone RK-I-123 reduces the levels of reactive oxygen species and suppresses the activation of NF-κB and AP-1 and IL-8 expression in Helicobacter pylori-infected gastric epithelial AGS cells

To investigate whether the phenyl-thiophenyl propenone RK-I-123 suppresses interleukin-8 (IL-8) expression and activation of mitogen-activated protein kinases (MAPKs) and transcription factors (nuclear factor-κB [NF-κB] and activator protein-1 [AP-1]) by reducing reactive oxygen species (ROS) levels in Helicobacter pylori-infected gastric epithelial cells. Helicobacter pylori in Korean isolates, human gastric epithelial AGS cells. AGS cells pretreated with or without RK-I-123 were cultured in the presence of H. pylori at a bacterium/cell ratio of 300:1. Reactive oxygen species and IL-8 levels were determined by dichlorofluorescein fluorescence and enzyme-linked immunosorbent assay. The IL-8 mRNA expression was analyzed by the real-time reverse transcription-polymerase chain reaction (RT-PCR). The MAPK and IκBα levels were determined by western blotting. The activation of NF-κB and AP-1 was determined by the electrophoretic mobility shift assay. Helicobacter pylori induced an increase in ROS and IL-8 expression and activation of MAPKs and transcription factors (NF-κB and AP-1) together with the degradation of IκBα in AGS cells, all of which were inhibited by RK-I-123. The RK-I-123 suppressed the H. pylori-induced IL-8 expression and activation of MAPKs, NF-κB, and AP-1 by reducing ROS levels in AGS cells. The RK-I-123 may be a potential candidate for the treatment of H. pylori-induced gastric inflammation.

Anti-Helicobacter pylori activity and inhibition of Helicobacter pylori-induced release of IL-8 in AGS cells by plant extracts

Plants used in popular diet were studied for anti-Helicobacter pylori activity and their effect on the expression of interleukin-8 (IL-8) from H. pylori infected gastric epithelial cells. Extracts were prepared of Allium sativum (A. sativum), Cuminum cyminum (C. cyminum),Piper nigrum (P. nigrum) and their mix in two different dilutions. AGS cell line and H. pyloristrains were used for co-culture experiments. Extracts bactericidal activity was determined by a viable colony count. ELISA (Enzyme linked immunosorbent assay) was used to determine IL-8 expression. DNA extracted from bacterial cells was used for polymerase chain reaction (PCR) of cytotoxin-associated gene (cagA) and E (cagE). Results revealedH. pylori strains sensitivity to A. sativum (5.5 mg/ml) was 57% (39/69) (p=0.06), and to 11 mg/ml was 65% (45/69) (p=0.02) compared to amoxicillin, respectively. CagE positive H. pylori, 37% (11/30) (p=0.02) were sensitive to plant mixture (23.5 mg/ml), 60% (18/30) (p<0.001) to plant mix (47 mg/ml), respectively. The expression of IL-8 by AGS cells treated with plant mix, (P=0.0037), C. cyminum (P<0.0001), P. nigrum (P=0.0046) and A. sativum(P=0.0021), respectively compared to positive and negative controls. Thus, dietary plants demonstrated an anti-H. pylori effect. They reduced IL-8 expression from the H. pyloriinfected AGS cells. Key words: Helicobacter pylori; Allium sativum, Cuminum cyminum, Piper nigrum, gastric epithelial cells, interleukin-8, ELISA, cagA, cagE.

Abstract 1995: GKN1-miR-185-DNMT1 axis suppresses gastric carcinogenesis through regulation of epigenetic alteration and cell cycle.

Abstract Gastrokine 1 (GKN1) functions to protect the gastric antral mucosa and promotes healing by facilitating restitution and proliferation after injury. GKN1 is down-regulated in Helicobacter pylori-infected gastric epithelial cells and that loss of GKN1 expression is tightly associated with gastric carcinogenesis, but underlying mechanisms as tumor suppressor are largely unknown. In the present study, we demonstrated that restoration of GKN1 protein suppressed gastric cancer cell growth by inducing endogenous miR-185 that directly targets epigenetic effectors, DNMT1 and EZH2 in gastric cancer cells. Notably, GKN1 expression was inversely correlated with DNMT1 and EZH2 expression in a subset of gastric cancer tissues and various gastric cancer cell lines. In addition, ectopic expression of GKN1 suppressed histone deacetylase 1 and thereby induced cell cycle arrest via regulating cell cycle proteins in gastric cancer cells. Interestingly, it was found that regulatory domains of the GKN1, pGKN1D13N, pGKN1▵68-199 and pGKN1▵1-67, 165-199 recapitulated GKN1 functions, and GKN1 exerted synergistic anti-cancer effect with 5-FU on tumor cell growth implying that the amino-terminal regulatory motif and brichos domain of GKN1 are sufficient for its tumor suppressor activities and thereby suggesting therapeutic intervention of gastric cancer. Collectively, our results demonstrated that loss of GKN1 functions occur and that cause aberrant expression of DMNT1 and EZH2 by suppression of endogenous miR-185 contributing malignant gastric epithelial cell proliferation and transformation in gastric carcinogenesis. Citation Format: Jung Hwan Yoon, Won Suk Choi, Won Sang Park, Olga Kim. GKN1-miR-185-DNMT1 axis suppresses gastric carcinogenesis through regulation of epigenetic alteration and cell cycle. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1995. doi:10.1158/1538-7445.AM2013-1995

Helicobacter pylori Initiates a Mesenchymal Transition through ZEB1 in Gastric Epithelial Cells

Chronic Helicobacter pylori infection provokes an inflammation of the gastric mucosa, at high risk for ulcer and cancer development. The most virulent strains harbor the cag pathogenicity island (cagPAI) encoding a type 4 secretion system, which allows delivery of bacterial effectors into gastric epithelial cells, inducing pro-inflammatory responses and phenotypic alterations reminiscent of an epithelial-to-mesenchymal transition (EMT). This study characterizes EMT features in H. pylori-infected gastric epithelial cells, and investigates their relationship with NF-κB activation. Cultured human gastric epithelial cell lines were challenged with a cagPAI+ H. pylori strain or cag isogenic mutants. Morphological changes, epithelial and mesenchymal gene expression and EMT-related microRNAs were studied. H. pylori up-regulates mesenchymal markers, including ZEB1. This transcription factor is prominently involved in the mesenchymal transition of infected cells and its up-regulation depends on cagPAI and NF-κB activation. ZEB1 expression and NF-κB activation were confirmed by immunohistochemistry in gastric mucosa from cagPAI+ H. pylori-infected patients. Gastric epithelial cell lines express high miR-200 levels, which are linked to ZEB1 in a reciprocal negative feedback loop and maintain their epithelial phenotype in non-infected conditions. However, miR-200b/c were increased upon infection, despite ZEB1 up-regulation and mesenchymal morphology. In the miR-200b-200a-429 cluster promoter, we identified a functional NF-κB binding site, recruiting NF-κB upon infection and trans-activating the microRNA cluster transcription. In conclusion, in gastric epithelial cells, cagPAI+ H. pylori activates NF-κB, which transactivates ZEB1, subsequently promoting mesenchymal transition. The unexpected N-FκB-dependent increase of miR-200 levels likely thwarts the irreversible loss of epithelial identity in that critical situation.

Haem oxygenase-1 inhibits phosphorylation of theHelicobacter pylorioncoprotein 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.

Helicobacter pylori decreases p27 expression through the delta opioid receptor-mediated inhibition of histone acetylation within the p27 promoter

Chronic Helicobacter pylori infection is associated with the decreased expression of the gastric tumour suppressor protein p27. Because transcription of the gene p27 may be regulated epigenetically through histone acetylation, which is mediated by G-protein coupled delta opioid receptor (DOR) stimulation, we examined whether H. pylori regulates the DOR/histone acetylation/p27 promoter pathway. The levels of acetylated histone and p300, a gene-specific histone acetyltransferase within the p27 promoter, were measured using ChIP assays. The expression of phospho-DOR was evaluated by Western blot and immunohistochemical analyses. Growth curves were constructed, and cell proliferation was assessed after BrdU incorporation. Low p27 expression in acutely H. pylori-infected AGS gastric epithelial cells and in chronically H. pylori-infected AGS-derived HS3C cells was associated with approximate 20% and 40% decreases in p27 mRNA expression, respectively, when compared to p27 mRNA levels in uninfected AGS parental cells. The low p27 mRNA levels following H. pylori infection were associated with a 15–60% reduction in p27 promoter histone H4 acetylation. The recruitment of p300 to the p27 promoter was also markedly decreased by H. pylori infection. The expression of phospho-DOR was decreased by H. pylori infection in cell lines in vitro and in H. pylori-infected human gastric mucosa in vivo. The level of cellular p27 inversely correlated with cell proliferation in HS3C cells. These results demonstrate that H. pylori decreases p27 expression by modulating the DOR and thereby inhibiting histone acetylation of the p27 promoter. These findings link low gastric p27 expression levels with increased instances of gastric carcinogenesis associated with H. pylori infection.

Helicobacter pylori에 감염된 위상피세포에서 Skp2의 변화

It has been suggested that Helicobacter pylori(H. pylori) infections can promote the development and progression of gastric cancer through the modulation of cell cycle regulators such as <TEX>$p27^{Kip1}$</TEX> and Skp2. <TEX>$p27^{Kip1}$</TEX> is a cyclin-dependent kinase (CDK) inhibitor that blocks the G1/S transition necessary for cell cycle progression. Skp2 is a component of the ubiquitin ligase complex called <TEX>$SCF^{Skp2}$</TEX>(SKP1-Cullin-F-box), which specifically binds and promotes the degradation of <TEX>$p27^{Kip1}$</TEX>. A low level of <TEX>$p27^{Kip1}$</TEX> and a high level of Skp2 have been reported in many types of cancers, including gastric cancer. In addition, a decrease in <TEX>$p27^{Kip1}$</TEX> has been reported in H. pylori-infected specimens. However, data on Skp2 in H. pylori infections are limited. This study examines the changes in the status of Skp2 in H. pylori-infected gastric epithelial AGS cells. For this, we stimulated AGS cells with H. pylori(NCTC 11637) at the ratio of 300:1(bacterium:cell) for 6 hours. The results of an immunoprecipitation analysis, followed by a western blot, indicate that the interaction between Skp2 and 14-3-3 was elevated 3 hours after the H. pylori treatment. In addition, there was an increase in cytoplasmic Skp2 after 3 hours, whereas there was no change in the nuclear level. Since it has been reported that interaction with 14-3-3 and the subsequent cytoplasmic translocation of Skp2 can increase its protein stability, increases in the interaction with 14-3-3 and the cytoplasmic Skp2 after the H. pylori treatment can increase the level of Skp2 in AGS cells. This phenomenon may explain, at least to some extent, the mechanism underlying the relationship between H. pylori infections and gastric carcinogenesis.

Anti-inflammatory and cytoprotective effects of selected Pakistani medicinal plants in Helicobacter pylori-infected gastric epithelial cells

Ethnopharmacological relevanceHelicobacter pylori infection is associated with gastritis, peptic ulcer, and gastric cancer. Due to its high global prevalence and uprising resistance to available antibiotics, efforts are now directed to identify alternative source to treat and prevent associated disorders. In the present study, effect of selected indigenous medicinal plants of Pakistan was evaluated on the secretion of interleukin-8 (IL-8) and generation of reactive oxygen species (ROS) in a bid to rationalize their medicinal use and to examine the anti-inflammatory and cytoprotective effects in gastric epithelial cells. Materials and methodsAGS cells and clinically isolated Helicobacter pylori strain (193C) were employed for co-culture experiments. Anti-Helicobacter pylori activity and cytotoxic effects of the selected plants were determined by serial dilution method and DNA fragmentation assay respectively. ELISA and flow cytometry were performed to evaluate the effect on IL-8 secretion and ROS generation in Helicobacter pylori-infected cells. ResultsAt 100μg/ml, extracts of Alpinia galangal, Cinnamomum cassia, Cinnamomum tamala, Mentha arvensis, Myrtus communis, Oligochaeta ramose, Polygonum bistorta, Rosa damascena, Ruta graveolens, Syzygium aromaticum, Tamarix dioica, and Terminalia chebula exhibited strong inhibitory activity against IL-8 secretion. Of these, four extracts of Cinnamomum cassia, Myrtus communis, Syzygium aromaticum, and Terminalia chebula markedly inhibited IL-8 secretion at both 50 and 100μg/ml. Cinnamomum cassia was further assessed at different concentrations against Helicobacter pylori and TNF-α stimulated IL-8 secretion, which displayed significant suppression of IL-8 in a concentration-dependent-manner. Among the plants examined against ROS generation, Achillea millefolium, Berberis aristata, Coriandrum sativum, Foeniculum vulgare, Matricaria chamomilla and Prunus domestica demonstrated significant suppression of ROS from Helicobacter pylori-infected cells (p<0.01). ConclusionResults of the study revealed anti-inflammatory and cytoprotective effects of selected medicinal plants which could partially validate the traditional use of these plants in GI disorders particularly associated with Helicobacter pylori. Furthermore, results obtained may lead to possible future candidates of chemoprevention against peptic ulcer or gastric cancer.

Upregulation of progranulin byHelicobacter pyloriin human gastric epithelial cells via p38MAPK and MEK1/2 signaling pathway: role in epithelial cell proliferation and migration

Helicobacter pylori is a major human pathogen associated with gastric diseases such as chronic active gastritis, peptic ulcer, and gastric carcinoma. The growth factor progranulin (PGRN) is a secreted glycoprotein that functions as an important regulator of cell growth, migration, and transformation. We aimed to determine the molecular mechanisms by which H. pylori upregulates the expression of PGRN and the relationship between H. pylori infection and production of PGRN in controlling cell proliferation and migration. Levels of PGRN were examined in gastric tissues from patients and in vitro in gastric epithelial cells. Cell proliferation was measured by colony formation assay. Cell migration was monitored by wound healing migration assay. PGRN protein levels were increased in patients with gastritis and gastric cancer tissue. Infection of gastric epithelial cells with H. pylori significantly increased PGRN expression in a time-dependent manner. Blockade of the p38 and MEK1/2 pathway by inhibitor inhibited H. pylori-mediated PGRN upregulation. Activation of p38 and MEK1/2 pathway by H. pylori was also identified. Knockdown of PGRN attenuated the H. pylori-induced proliferative activity and migration of cancer cells. These findings suggest that the upregulation of PGRN in H. pylori-infected gastric epithelial cells may contribute to the carcinogenic process.

A Mechanism for the Action of the Compound DA‐6034 on NF‐κB Pathway Activation in Helicobacter pylori‐Infected Gastric Epithelial Cells

DA-6034 is a synthetic derivative of eupatilin, a flavonoid with anti-inflammatory effects. The aim of this study was to investigate the effects of DA-6034 on the interactions between IκB kinase (IKK) and heat shock protein 90 (Hsp90), and activation of the nuclear factor-kappaB (NF-κB) signalling pathway in human gastric epithelial cells infected with Helicobacter pylori. MKN-45 gastric epithelial cell line was treated with DA-6034 and H. pylori. DA-6034 significantly inhibited NF-κB activation and upregulated the expressions of interleukin-8 (IL-8) and monocyte chemoattractant protein-1 in MKN-45 cells infected with H. pylori. However, DA-6034 did not influence activator protein-1 DNA binding activity in H. pylori-infected gastric epithelial cells. Pretreatment with DA-6034 attenuated the H. pylori-induced increase in IKK activity, and Hsp90 was associated with IKK-α and IKK-γ in MKN-45 cells. Treatment with DA-6034 dissociated the Hsp90 and IKK-γ complex in H. pylori-infected cells, leading to the inhibition of IL-8 expression. These results suggest that the eupatilin derivative 7-carboxymethyloxy-3',4',5-trimethoxy flavone has anti-inflammatory activity in gastric epithelial cells infected with H. pylori through the promotion of the dissociation of the IKK-γ-Hsp90 complex and suppression of NF-κB signalling.

Loss of DNA Repair Protein and Cell Death in Helicobacter pylori‐Infected Gastric Epithelial Cells

Cell death linked to DNA damage has been implicated in Helicobacter pylori (H. pylori) ‐ infected gastric diseases. During DNA damage, DNA repair proteins, Ku70 and Ku80, prevent cell death but severe DNA damage beyond the capacity of the DNA repair proteins triggers cell death. It has been hypothesized that the decrease in Ku70 and Ku80 may be related to cell death in H. pylori‐infected gastric epithelial cells. In this study, it was found that H. pylori induced cell death with increasing time and amounts of H. pylori added to the cells, which was determined by decreased in viable cell numbers and poly(ADP‐ribose) polymerase (PARP) cleavage. H. pylori resulted in significant decrease in both cytoplasmic and nuclear Ku protein levels as well as nuclear Ku DNA end‐binding with time and amounts of H. pylori added to the cells. H. pylori‐induced decrease in Ku proteins in cytoplasm and nucleus was in parallel with increase in ubiquitinated Ku70 and Ku80 in the whole cell extracts. H. pylori‐induced alterations in Ku expression, cell death and DNA fragmentation were inhibited by a calpain inhibitor, calpeptin and a proteasome inhibitor, lactacystin. In conclusion, loss of Ku proteins may cause the loss of defense against DNA damage and thus, underlie the mechanism of cell death in gastric epithelial cells after H. pylori infection.

Regulation of the actin cytoskeleton in Helicobacter pylori-induced migration and invasive growth of gastric epithelial cells

Dynamic rearrangement of the actin cytoskeleton is a significant hallmark of Helicobacter pylori (H. pylori) infected gastric epithelial cells leading to cell migration and invasive growth. Considering the cellular mechanisms, the type IV secretion system (T4SS) and the effector protein cytotoxin-associated gene A (CagA) of H. pylori are well-studied initiators of distinct signal transduction pathways in host cells targeting kinases, adaptor proteins, GTPases, actin binding and other proteins involved in the regulation of the actin lattice. In this review, we summarize recent findings of how H. pylori functionally interacts with the complex signaling network that controls the actin cytoskeleton of motile and invasive gastric epithelial cells.

Diphenyleneiodonium inhibits the activation of mitogen-activated protein kinases and the expression of monocyte chemoattractant protein-1 in Helicobacter pylori-infected gastric epithelial AGS cells

To investigate whether NADPH oxidase induces MCP-1 expression and the activation of mitogen-activated protein kinases (MAPKs) in H. pylori-infected gastric epithelial cells. H. pylori in Korean isolates, human gastric epithelial AGS cells AGS cells pretreated with or without an NADPH oxidase inhibitor diphenyleneiodonium (DPI) are cultured in the presence of H. pylori at a bacterium/cell ratio of 300:1. Reactive oxygen species (ROS) and MCP-1 were determined by confocal microscopy and enzyme-linked immonosorbent assay. NADPH oxidase activity was measured by lucigenin assay. mRNA expression of MCP-1 was analyzed by reverse transcription-polymerase chain reaction. Levels of MAPKs were assessed by Western blot analysis. H. pylori induced increase in ROS, NADPH oxidase activity, MCP-1 expression, and the activation of MAPKs including extracellular signal-regulated kinases, p38, and jun N-terminal kinases in AGS cells, which was inhibited by DPI. Inhibiting NADPH oxidase by DPI suppresses H. pylori-induced activation of MAPKs and MCP-1 expression in AGS cells.

Down-regulation of Bcl-2 is mediated by NF-κB activation in Helicobacter pylori-induced apoptosis of gastric epithelial cells

Objective. Bcl-2 family is involved in the regulation of apoptosis. NF-κB activation is associated with either the expression of Bcl-2 or down-regulation of Bcl-2 depending on cell types and stimuli. Previously, we showed NF-κB activation, decrease in the level of Bcl-2, and apoptosis in Helicobacter pylori (H. pylori)-infected gastric epithelial cells. The present study aims to investigate the relation of Bcl-2 expression and NF-κB activation in H. pylori-induced apoptotic cell death of AGS (gastric adenocarcinoma) cells. Material and methods. AGS cells were transfected with mutant IκBα to suppress NF-κB activation or Bcl-2 gene to induce overexpression of Bcl-2. mRNA expression of Bcl-2, p53 and Bax, DNA fragmentation, cell viability, and the numbers of apoptotic cells were determined. Results. H. pylori induced decrease in Bcl-2, but increase in p53 and Bax at the levels of mRNA and protein in AGS cells. H. pylori-induced increment of apoptotic cells and decrease in Bcl-2 level were inhibited in the cells transfected with mutant IκBα gene as compared with the cells transfected with control vector. H. pylori-induced apoptosis determined by apoptotic cells, DNA fragmentation, and cell viability was inhibited in the cells transfected with Bcl-2 gene. Conclusion. Down-regulation of Bcl-2 is mediated by NF-κB activation, which may be the underlying mechanism of apoptosis in H. pylori-infected gastric epithelial cells.

Role of protease-activated receptor-2 on cell death and DNA fragmentation in Helicobacter pylori-infected gastric epithelial cells

BackgroundHelicobacter pylori (H. pylori) infection is associated with chronic gastritis, peptic ulceration and gastric carcinoma. Protease-activated receptor-2 (PAR-2), which is activated by trypsin, induced the activation of mitogen-activated protein kinases (MAPK), cell proliferation and apoptosis in several cells. Previously, we found that H. pylori induces the expression of PAR-2, which mediates the expression of adhesion molecules integrins in gastric epithelial cells. In the present study, the role of PAR-2 on H. pylori-induced cell death was investigated by determining cell viability, DNA fragmentation, and the activation of MAPK in gastric epithelial AGS cells.MethodsAGS cells were cultured in the presence of H. pylori transfected with PAR-2 antisense (AS) oligonucleotide (ODN) or treated with a soybean trypsin inhibitor (SBTI). Viable cells and DNA fragmentation were determined by trypan blue exclusion assay and the amount of oligonucleosome-bound DNA, respectively. The activation of MAPK such as extracellular signal-regulated kinases (ERK), p38, and c-Jun N-terminal kinases (JNK), was assessed by Western blotting for phospho-specific forms of MAPK.ResultsH. pylori-induced cell death and DNA fragmentation augmented in the cells transfected with PAR-2 AS ODN or treated with SBTI. The activation of MAPK, induced by H. pylori, were suppressed by transfection with PAR-2 AS ODN or treatment with SBTI.ConclusionPAR-2, whose expression is induced by H. pylori, may prevent cell death and DNA fragmentation with the activation of MAPK in gastric epithelial cells.

HSP90β interacts with Rac1 to activate NADPH oxidase in Helicobacter pylori-infected gastric epithelial cells

Reactive oxygen species (ROS) are involved in the pathogenesis of Helicobacter pylori ( H. pylori)-induced gastric disorders including inflammation. NADPH oxidase has been considered as a major source of ROS in phagocytic and non-phagocytic cells. Small G protein Rac1 is one components of NADPH oxidase complex. Heat shock protein 90 kDa (HSP90) modulates cytokine production in H. pylori-infected gastric epithelial cells. The present study aims to investigate the relation of HSP90β and Rac1 on the activation of NADPH oxidase in H. pylori-infected gastric epithelial cells. As a result, H. pylori-induced translocation of HSP90β from the cytosol to the membrane and activated Rac1 in gastric epithelial AGS cells. HSP90β physically interacted with Rac1, which resulted in the activation of NADPH oxidase in H. pylori-infected AGS cells. Down-regulation of HSP90β by transfection of HSP90β siRNA suppressed the activation of Rac1, activity of NADPH oxidase and the production of H 2O 2 in H. pylori-infected AGS cells. In conclusion, H. pylori induces the translocation of HSP90β from the cytosol to the membrane and interaction of HSP90β and Rac1, which leads to the activation of NADPH oxidase and production of ROS in gastric epithelial cells. HSP90β may be the target molecule for treatment of H. pylori-induced gastric injury.

M1958 Regulation of GLI1 and Snail in Helicobacter pylori Infected Gastric Epithelial Cells

M1958 Regulation of GLI1 and Snail in Helicobacter pylori Infected Gastric Epithelial Cells

651 Apurinic/Apyrimidinic Endonuclease (APE-1) Regulates Oxidative Signaling Pathways Including RAC1 and NADPH Oxidase in H. pylori (HP) Infected Gastric Epithelial Cells (GEC)

Mcl-1 expression in gastric cells co-cultured with strain 7.13, consistent with the marked downregulation of miR-320. To define the effect of the H. pylori oncoprotein CagA on these events, cells were also co-cultured with a 7.13 cagAisogenic mutant. Loss of cagA attenuated both the reduction of miR-320 and the subsequent increase in Mcl-1 expression observed in response to wild-type strain 7.13. In summary,H. pylori strain 7.13 specifically downregulates miR-320 in a CagA-dependent manner, which relieves repression of Mcl-1. Upregulation of Mcl-1 by this carcinogenic strain ofH. pylori likely promotes cell survival through inhibition of apoptosis, which may heighten retention of mutagenized cells and increase the risk of subsequent gastric carcinogenesis.