Pylori-infected Cells Research Articles

The role of C1q in recognition of apoptotic epithelial cells and inflammatory cytokine production by phagocytes during Helicobacter pylori infection.

BackgroundGastric epithelial cells (GECs) undergo apoptosis during H. pylori infection and phagocytes within the mucosa engulf these cells. The recognition and clearance of apoptotic cells is a multifactorial process, enhanced by the presence of various bridging molecules and opsonins which are abundant in serum. However, it is not clear how recognition or clearance may differ in the context of H. pylori infection induced apoptosis. In addition, efferocytosis of sterile apoptotic cells is known to confer anti-inflammatory properties in the engulfing phagocyte, however it is unknown if this is maintained when phagocytes encounter H. pylori-infected cells. Thus, the ability of macrophages to bind and engulf gastric epithelial cells rendered apoptotic by H. pylori infection and the association of these interactions to the modulation of phagocyte inflammatory responses was investigated in the absence and presence of serum with a particular focus on the role of serum protein C1q.MethodsControl (uninfected) or H. pylori-infected AGS cells were co-cultured with THP-1 macrophages in the presence or absence of serum or serum free conditions + C1q protein (40–80 μg/mL). Binding of AGS cells to THP-1 macrophages was assessed by microscopy and cytokine (IL-6 and TNF-α) release from LPS stimulated THP-1 macrophages was quantified by ELISA.ResultsWe show that macrophages bound preferentially to cells undergoing apoptosis subsequent to infection with H. pylori. Binding of apoptotic AGS to THP-1 macrophages was significantly inhibited when studied in the absence of serum and reconstitution of serum-free medium with purified human C1q restored binding of macrophages to apoptotic cells. Co-culture of sterile apoptotic and H. pylori-infected AGS cells both attenuated LPS-stimulated cytokine production by THP-1 macrophages. Further, direct treatment of THP-1 macrophages with C1q attenuated LPS stimulated TNF-α production.ConclusionsThese studies suggest that C1q opsonizes GECs rendered apoptotic by H. pylori. No differences existed in the ability of infected or sterile apoptotic cells to attenuate macrophage cytokine production, however, there may be a direct role for C1q in modulating macrophage inflammatory cytokine production to infectious stimuli.Electronic supplementary materialThe online version of this article (doi:10.1186/s12950-015-0098-8) contains supplementary material, which is available to authorized users.

PP8 - Crosstalk of JAK1/STAT3 and WNT/β-catenin in Helicobacter pylori-induced hyper-proliferation in gastric epithelial cells: Effect of antioxidant lycopene

Helicobacter pylori (H. pylori) infection, as a risk factor for gastric cancer, is associated with hyper-proliferation and oncogene expression in gastric epithelial cells. Previously, we showed that reactive oxygen species (ROS) mediate activation of JAK/STAT signaling and hyper-proliferation in H. pylori–infected gastric epithelial cells. Since JAK signaling is known to mediate Wnt/β-catenin pathway in various cells, we investigated the relation of ROS, JAK/STAT activation, and Wnt/β-catenin pathway for hyper-proliferation in H. pylori-infected gastric epithelial AGS cells using an antioxidant lycopene. Lycopene, a bright red carotenoid pigment in fruits and vegetables, has anticancer activity in many types of cancers. As a result, H. pylori induced hyper-proliferation in parallel with induction and nuclear translocation of β-catenin. For Wnt/β-catenin signaling, H. pylori decreased the level of adenomatous polyposis coli (APC), a component of the destruction complex targeting β-catenin, and induced β-catenin target genes (cyclin-E, c-myc) in AGS cells. H. pylori activated GSK3β, but reduced APC and Axin in AGS cells. For JAK/STAT signaling, Jak1 and Stat3 were activated in H. pylori-infected AGS cells. Jak1 inhibitor AG490 decreased levels of β-catenin and its target genes (cyclin-E, c-myc) and increased the level of APC in H. pylori-infected cells. Lycopene inhibited H. pylori-induced ROS production, activation of JAK1/STAT3, reduction of APC/AXIN, and induction of β-catenin and its target genes (cyclin-E, c-myc), and hyper-proliferation in AGS cells. In conclusion, lycopene inhibits H. pylori-induced hyper-proliferation by suppressing ROS-mediated activation of JAK/STAT and WNT/β-catenin signaling in gastric epithelial cells.

Dietary prevention of Helicobacter pylori-associated gastric cancer with kimchi.

To prove whether dietary intervention can prevent Helicobacter pylori-induced atrophic gastritis and gastric cancer, we developed cancer preventive kimchi (cpKimchi) through special recipe and administered to chronic H. pylori-initiated, high salt diet-promoted, gastric tumorigenesis mice model. H. pylori-infected C57BL/6 mice were administered with cpKimchi mixed in drinking water up to 36 weeks. Gross and pathological gastric lesions were evaluated after 24 and 36 weeks, respectively and explored underlying molecular changes to explain efficacies. Cancer preventive actions of anti-inflammation and anti-mutagenesis were compared between standard recipe kimchi (sKimchi) and special recipe cpKimchi in in vitro H. pylori-infected cell model. The erythematous and nodular changes, mucosal ulcerative and erosive lesions in the stomach were noted at 24th weeks, but cpKimchi administration significantly ameliorated. After 36th weeks, scattered nodular masses, some ulcers, and thin nodular gastric mucosa were noted in H. pylori-infected mice, whereas these gross lesions were significantly attenuated in cpKimchi group. On molecular analysis, significant expressions of COX-2 and IL-6, activated NF-κB and STAT3, increased apoptosis, and marked oxidative stresses were noted in H. pylori-infected group relevant to tumorigenesis, but these were all significantly attenuated in cpKimchi group. cpKimchi extracts imparted significant selective induction of apoptosis only in cancer cells, led to inhibition of H. pylori-induced proliferation, while no cytotoxicity through significant HO-1 induction in non-transformed gastric cells. In conclusion, daily dietary intake of cpKimchi can be an effective way either to rejuvenate H. pylori-atrophic gastritis or to prevent tumorigenesis supported with the concerted actions of anti-oxidative, anti-inflammatory, and anti-mutagenic mechanisms.

GATA-3 augmentation down-regulates Connexin43 in Helicobacter pylori associated gastric carcinogenesis

Helicobacter pylori (H. pylori) is a risk factor of gastric carcinoma, and inflammation with H.pylori infection has widely been suggested to trigger gastric carcinogenesis through “inflammation-carcinoma chain” (non-atrophic gastritis (NAG) → chronic atrophic gastritis (CAG) → intestinal metaplasia (IM) → dysplasia (DYS) and gastric carcinoma (GC)). Connexin43 (Cx43) is a major constituent of gap junction in normal gastric mucosa (NGM) and it is continuously down-regulated from normal gastric mucosa to precancerous lesions or ultimate gastric carcinoma, which shows novel target against gastric carcinoma by preventing the Cx43 decline. Our previous studies demonstrated that H. pylori infection in gastric mucosa down-regulates Cx43 expression, but its mechanism remains unknown. The transcriptional factor, GATA binding protein 3 (GATA-3) is the key to regulate adaptive immune response, which possibly relates to inflammation toward malignant transformation. Here the substantial rising of GATA-3 was screened by transcriptional factor microarray along the developmental stages of H. pylori associated gastric carcinoma. Moreover, the increased GATA-3 and inhibited Cx43 were confirmed in clinical specimens, Mongolian gerbils and normal gastric epithelial cell line GES-1 with H. pylori infection. GATA-3 silencing generated the Cx43 restoration both in intermediate differentiation gastric cancer cells BGC-803 and in H. pylori infected GES-1 cells. Dual-luciferase reporter assay further revealed the GATA-3 as one of Cx43 down-regulators by directly binding to its promoters. Together, the incremental GATA-3 is found in H. pylori associated gastric carcinogenesis, which is responsible for Cx43 inhibition as well.

Su1826 Pseudokinase Tribbles 3 Enhances SHP2-Dependent ERK Signaling in Helicobacter pylori-Induced Gastric Carcinogenesis

Background & Aim: Helicobacter pylori (H. pylori) infection is known to induce gastric inflammation, gastric intestinal metaplasia and gastric carcinogenesis, but the details are still ambiguous. In DDW2012, we reported that H. pylori infection induced the expression of pseudokinase Tribbles 3 (Trib3) both in vitro and in vivo, and overexpression of this pesudokinase led to augmented phosphorylation of tyrosine phosphatase SHP2. In this study, we aimed to identify the downstream signaling pathway of SHP2 affected by Trib3, which contributes to gastric carcinogenesis.Methods & Results: First, we generated Trib3 overexpressing cell line Trib3GSM06 from normal murine gastric epithelial cell line GSM06. We then infected these cells with H. pylori and investigated the signaling pathway that is affected by the overexpression of Trib3. Western blot analysis of these cells showed that although neither β-catenin nor STAT3 was affected by over-expression of Trib3, the expression of phosphorylated ERK1/2 (p44/42 MAPK) was augmented up to 2.4 folds in Trib3GSM06 compared to GSM06 cells. To verify if the phosphorylation of ERK1/2 was the result of SHP2 activation, we assessed the effect of SHP2 inhibitor (II-B08) on ERK1/2 phosphorylation by western blot. As a result, the addition of SHP2 inhibitor diminished the expression of phosphorylated ERK1/2. In addition, MTS assay of Trib3GSM06 and GSM06 cells showed that over-expression of Trib3 enhanced cell proliferation, and this enhancement was suppressed by the addition of SHP2 inhibitor. Moreover, expression of c-Myc was enhanced up to 2.0 folds by overexpression of Trib3, and this enhancement was abolished by the addition of SHP2 inhibitor. Finally, we investigated the physical interaction between Trib3 and SHP2 in H. pylori-infected GSM06 cells by immunoprecipitation and western blotting, which clearly showed the binding of these two molecules. These findings suggested that over-expression of Trib3 activates ERK1/2 by directly binding to and activating SHP2. Conclusion: Our study demonstrated that H. pylori-induced Trib3 directly interacts with SHP2 and activates ERK1/2 signaling, leading to gastric carcinogenesis. This finding suggests Trib3 as a possible candidate of therapeutic target for gastric cancers.

MiR-223 promotes the cisplatin resistance of human gastric cancer cells via regulating cell cycle by targeting FBXW7.

BackgroundIncreasing evidence showed that miRNAs serve as modulators of human cancer, either as oncogene or tumor suppressors. Cisplatin resistance is the most common cause of chemotherapy failure in gastric cancer (GC). However, the roles of miRNAs in cisplatin resistance of GC remain largely unknown. The aim of the study was to identify a novel miRNA/gene pathway that regulates the sensitivity of GC cells to cisplatin.MethodsIn this study, we chose miR-223 by qRT-PCR analysis, the most significantly up-regulated miRNA in GC, to investigate its formation of DDP-resistant phenotype of GC cells and possible molecular mechanisms.ResultsWe found that miR-223 was most significantly up-regulated miRNA in DDP-resistant GC cells compared with parental GC cells. Besides, its expression was also significantly up-regulated in GC tissues. FBXW7 was identified as the direct and functional target gene of miR-223. Overexpression of FBXW7 could mimic the effect of miR-223 down-regulation and silencing of FBXW7 could partially reverse the effect of miR-223 down-regulation on DDP resistance of DDP-resistant GC cells. Besides, miR-223 and FBXW7 could affect the G1/S transition of cell cycle by altering some certain cell cycle regulators. Furthermore, miR-223 was found to be significantly up-regulated in H. pylori infected tissues and cells, suggesting that H. pylori infection may lead to GC development and DDP resistance.ConclusionsOur findings revealed the roles of miR-223/FBXW7 signaling in the DDP resistance of GC cells and targeting it will be a potential strategic approach for reversing the DDP resistance in human GC.Electronic supplementary materialThe online version of this article (doi:10.1186/s13046-015-0145-6) contains supplementary material, which is available to authorized users.

Helicobacter pylori Induces Serine Phosphorylation of EGFR via Novel TAK1-p38 Activation Pathway in an HB-EGF-Independent Manner.

The interaction of Helicobacter pylori with gastric epithelial cells can result in the activation of transcription factor NF-κB via TGF-β-activated kinase 1 (TAK1). In this study, we have demonstrated the role of H. pylori in the activation of EGFR via TAK1-mediated phosphorylation of p38. Gastric epithelial AGS or MKN-45 cells were co-cultured with wild-type or cagA(-) H. pylori strains. H. pylori was added to the cells, and the activation of EGFR, p65 (NF-κB) subunit, p38, ERK, and TAK1 was examined by Western blotting. Infected cells were pretreated with or without ligands, chemical inhibitors, anti-HB-EGF antibody, and siRNAs to evaluate the effects on phosphorylation of various EGFR residues. Fluorescence microscopy and flow cytometry were performed to detect the internalization of EGFR. Incubating cells with wild-type and CagA(-) H. pylori strains resulted in the rapid and transient phosphorylation of serine residues of EGFR. RNAi experiments using siRNA against TAK1 and p38 pathways blocked the phosphorylation of serine residue. Immunofluorescence and flow cytometry revealed that EGFR was internalized in H. pylori-infected cells after EGFR phosphorylation in a p38-dependent manner. In contrast, pretreatment with gefitinib and anti-HB-EGF antibody did not block both the phosphorylation and internalization of EGFR. Helicobacter pylori induces internalization of EGFR via novel TAK1-p38-serine activation pathway which is independent of HB-EGF. The interaction between TAK1 and EGFR in H. pylori-infected cells might open new dimensions in understanding H. pylori-associated gastric carcinogenesis.

Helicobacter pylori bacteria alter the p53 stress response via ERK-HDM2 pathway.

H. pylori infection is the strongest known risk factor for gastric cancer. Inhibition of host tumor suppressor mechanisms by the bacteria underlies the development of this disease. Among the tumor suppressors affected by H. pylori are p53 and E-cadherin, which inhibition has been shown to increase the risk of gastric cancer. In this report, we investigated the interaction between E-cadherin and p53 in H. pylori-infected cells. We found that downregulation of E-cadherin leads to cellular stress and activation of p53. In the setting of H. pylori infection, this mechanism, however, is disrupted. We found that although co-culture of gastric epithelial cells with H. pylori led to downregulation of E-cadherin and cellular stress, it resulted in inhibition of p53, which is mediated by intracellular Erk kinases and HDM2 protein induced by H. pylori. Experimental inhibition of HDM2/p53 interactions restored p53 activity, and decreased survival of infected cells. Collectively, our results revealed that regulation of p53 and E-cadherin is tightly linked through the p53 stress response mechanism that is inhibited by H. pylori via activation of Erk1/2-HDM2-p53 pathway leading to survival of damaged cells. This might be advantageous to the bacteria but may increase the cancer risk.

Helicobacter pylori induces IL-1β and IL-18 production in human monocytic cell line through activation of NLRP3 inflammasome via ROS signaling pathway.

This study investigated whether Helicobacter pylori could activate the nucleotide-binding oligomerization domain-like receptor (NLR) family, pyrin domain-containing 3 (NLRP3) inflammasome in human macrophages and the involvement of reactive oxygen species (ROS) in inflammasome activation. Phorbol-12-myristate-13-acetate (PMA)-differentiated human acute monocytic leukemia cell line THP-1 was infected with H. pylori. The levels of pro-inflammatory cytokines interleukin (IL)-1β and IL-18 in supernatant were measured by ELISA. Intracellular ROS level was analyzed by flow cytometry. Quantitative real-time PCR and western blot analysis were employed to determine the mRNA and protein expression levels of NLRP3 and caspase-1 in THP-1 cells, respectively. Our results showed that H. pylori infection could induce IL-1β and IL-18 production in PMA-differentiated THP-1 cells in a dose- and time-dependent manner. Moreover, secretion of IL-1β and IL-18 in THP-1 cells following H. pylori infection was remarkably reduced by NLRP3-specific small interfering RNA treatment. In addition, the intracellular ROS level was elevated by H. pylori infection, which could be eliminated by the ROS scavenger N-acetylcysteine (NAC). Furthermore, NAC treatment could inhibit NLRP3 inflammasome formation and caspase-1 activation and suppress the release of IL-1β and IL-18 from H. pylori-infected THP-1 cells. These findings provide novel insights into the innate immune response against H. pylori infection, which could potentially be used for the prevention and treatment of H. pylori-related diseases.

Anti-inflammatory effect of cinnamaldehyde in Helicobacter pylori induced gastric inflammation.

Cinnamomum cassia is widely employed for gastrointestinal complaints such as dyspepsia, flatulence, diarrhea, and vomiting. Studies report cinnamaldehyde (CM) as a major active constituent of cinnamon. The aim of this study was to evaluate the anti-inflammatory mechanism of CM on Helicobacter (H.) pylori-infected gastric epithelial cells in order to validate cinnamon traditional use in gastrointestinal (GI)-related disorders. AGS/MKN-45 cells and H. pylori (193C) were employed for co-culture experiments. Anti-H. pylori cytotoxic and anti-adhesion activity of CM were determined. Enzyme linked immunosorbent assay, real time polymerase chain reaction analysis and immunoblotting were used to measure the effect on interleukin-8 (IL-8) secretion/expression. The effect on activation of nuclear factor kappa B (NF-κB) was determined by immunoblot analysis. The non-cytotoxic CM (≤125 µM) was also non-bactericidal at the given time, suggesting the effect in H. pylori/cell co-culture system was not due to alteration in H. pylori viability or the toxicity to the cells. Also, CM did not show any anti-adhesion effect against H. pylori/cell co-culture. However, pre-incubation of the cells with CM significantly inhibited the IL-8 secretion/expression from H. pylori-infected cells (p<0.01). In addition, CM suppressed H. pylori-induced NF-κB activation and prevented degradation of inhibitor (I)-κB This study provides evidence that the anti-inflammatory effect of C. cassia on H. pylori-infected gastric cells is due to blockage of the NF-κB pathway by cinnamaldehyde. This agent can be considered as a potential candidate for in vivo and clinical studies against various H. pylori related gastric pathogenic processes.

Jak1/Stat3 is an upstream signaling of NF-κB activation in Helicobacter pylori-induced IL-8 production in gastric epithelial AGS cells.

Helicobacter pylori (H. pylori) induces the activation of nuclear factor-kB (NF-κB) and cytokine expression in gastric epithelial cells. The Janus kinase/signal transducers and activators of transcription (Jak/Stat) cascade is the inflammatory signaling in various cells. The purpose of the present study is to determine whether H. pylori-induced activation of NF-κB and the expression of interleukin-8 (IL-8) are mediated by the activation of Jak1/Stat3 in gastric epithelial (AGS) cells. Thus, gastric epithelial AGS cells were infected with H. pylori in Korean isolates (HP99) at bacterium/cell ratio of 300:1, and the level of IL-8 in the medium was determined by enzyme-linked immonosorbent assay. Phospho-specific and total forms of Jak1/Stat3 and IκBα were assessed by Western blot analysis, and NF-κB activation was determined by electrophoretic mobility shift assay. The results showed that H. pylori induced the activation of Jak1/Stat3 and IL-8 production, which was inhibited by a Jak/Stat3 specific inhibitor AG490 in AGS cells in a dose-dependent manner. H. pylori-induced activation of NF-κB, determined by phosphorylation of IκBα and NF-κB-DNA binding activity, were inhibited by AG490. In conclusion, Jak1/Stat3 activation may mediate the activation of NF-κB and the expression of IL-8 in H. pylori-infected AGS cells. Inhibition of Jak1/Stat3 may be beneficial for the treatment of H. pylori-induced gastric inflammation, since the activation of NF-κB is inhibited and inflammatory cytokine expression is suppressed.

Measurement of total iron in Helicobacter pylori-infected gastric epithelial cells.

Despite the evidence suggesting a role for Helicobacter pylori in the induction of systemic iron deficiency anaemia, little is known about the possibility of infection-associated changes in cellular iron homeostasis at the gastric epithelium. In this study we compared four different techniques for measuring iron in AGS cells, a gastric epithelial cell line that is widely used to model to H. pylori infection in vitro. Inductively coupled plasma-mass spectrometry proved to be an efficient method, but only when large numbers of cells were used. Two colorimetric assays that included the use of concentrated hydrochloric acid with or without potassium ferrocyanide detected iron in the micromolar but not the nanomolar range in cell-free standards. However, the third colorimetric assay that incorporated ferrozine proved to be highly accurate at detecting iron in the nanomolar range, and was able to detect iron in AGS cells, Moreover, using this assay, we were able to show that the level of iron in H. pylori-infected AGS cells is significantly increased when compared to uninfected cells.

Helicobacter pylori promotes VEGF expression via the p38 MAPK‑mediated COX‑2‑PGE2 pathway in MKN45 cells.

Helicobacter pylori has been suggested to be the major cause of gastric malignancy. However, the pathogenesis and molecular mechanisms of gastric tumorigenesis induced by H. pylori infection are yet to be elucidated. In the present study, the expression levels of vascular endothelial growth factor (VEGF), which has been suggested to promote angiogenesis in gastric cancer, were found to be elevated in H. pylori-infected MKN45 cells. Furthermore, it was demonstrated that the expression of VEGF was modulated by the p38 mitogen-activated protein kinases (MAPK) pathway via regulation of the cyclooxygenase (COX)-2 pathway. It was also found that prostaglandin E2 (PGE2) and its receptor EP2/EP4 may mediate the upregulation of VEGF in gastric cells exposed to H. pylori. In combination, these results suggest that VEGF expression is regulated by the p38 MAPK COX‑2-PGE2-EP2/EP4 pathway in gastric cancer cells induced by H. pylori. This provides a theoretical basis for the investigation of the pathogenesis of H. pylori‑induced gastric cancer.

MicroRNA-146a Enhances Helicobacter pylori Induced Cell Apoptosis in Human Gastric Cancer Epithelial Cells

Helicobacter pylori (H. pylori) infection induces apoptosis in gastric epithelial cells, and this occurrence may link to gastric carcinogenesis. However, the regulatory mechanism of H. pylori-induced apoptosis is not clear. MicroRNA-146a has been implicated as a key regulator of the immune system. This report describes our discovery of molecular mechanisms of microRNA-146a regulation of apoptosis in human gastric cancer cells. We found that overexpression of microRNA-146a by transfecting microRNA-146a mimics could significantly enhance apoptosis, and this up-regulation was triggered by COX-2 inhibition. Furthermore, we found that microRNA-146a density was positively correlated with apoptosis rates in H. pylori-positive gastric cancer tissues and intratumoral microRNA-146a density was negatively correlated with lymph node metastasis among H. pylori-positive gastric cancer patients. Understanding the important roles of microRNA-146a in regulating cell apoptosis in H. pylori infected human gastric cancer cells will contribute to the development of microRNA targeted therapy in the future.

Nucleotide Binding Oligomerization Domain 1 Is an Essential Signal Transducer in Human Epithelial Cells Infected with Helicobacter pylori That Induces the Transepithelial Migration of Neutrophils.

Background/AimsThe cytosolic host protein nucleotide binding oligomerization domain 1 (Nod1) has emerged as a key pathogen recognition molecule for innate immune responses in epithelial cells. The purpose of the study was to elucidate the mechanism by which Helicobacter pylori infection leads to transepithelial neutrophil migration in a Nod1-mediated manner.MethodsHuman epithelial cell lines AGS and Caco-2 were grown and infected with H. pylori. Interleukin (IL)-8 mRNA expression and IL-8 secretion were assessed, and nuclear factor κB (NF-κB) activation was determined. Stable transfections of AGS and Caco-2 cells with dominant negative Nod1 were generated. Neutrophil migration across the monolayer was quantified.ResultsCytotoxin-associated gene pathogenicity island (cagPAI)(+) H. pylori infection upregulated IL-8 mRNA expression and IL-8 secretion in AGS and Caco-2 cells compared with controls. NF-κB activation, IL-8 mRNA expression and IL-8 secretion by cagPAI knockdown strains were reduced compared with those infected with the wild-type strain. NF-κB activation, IL-8 mRNA expression and IL-8 secretion in dominant-negative (DN)-Nod1 stably transfected cells were reduced compared with the controls. The transepithelial migration of neutrophils in DN-Nod1 stably transfected cells was reduced compared with that in controls.ConclusionsSignaling through Nod1 plays an essential role in neutrophil migration induced by the upregulated NF-κB activation and IL-8 expression in H. pylori-infected human epithelial cells.

Helicobacter pylori cytotoxin-associated gene A protein upregulates α-enolase expression via Src/MEK/ERK pathway: implication for progression of gastric cancer.

Persistent infection with Helicobacter pylori confers an increased risk for the development of gastric cancer. In our previous investigations, we found that ENO1 was overexpression in cagA-positive H. pylori-infected gastric epithelial AGS cells by proteomic method, in contrast to the isogenic cagA knock out mutant H. pylori-infected cells. ENO1 is a newly identified oncoprotein overexpressed in some cancer. However, the relationship between H. pylori infection and ENO1 expression still remains undefined. The AGS gastric cancer cells were transfected with WT-cagA plasmid and PR-cagA plasmids. Expression of ENO1 mRNA and protein were measured by real-time quantitative PCR and western blot analysis. Signal protein inhibitor treatment was used to investigate the signal pathways. It was found that the ENO1 mRNA and protein overexpression levels were dependent on cagA gene expression and CagA protein phosphorylation. Further analysis revealed that the Src, MEK and ERK pathway was involved in this upregulation effect. Our data suggest that ENO1 was upregulated by CagA protein through activating the Src and MEK/ERK signal pathways, thereby providing a novel mechanism underlying H. pylori-mediated gastric diseases.

The inhibition of Helicobacter pylori infected cells by Origanum minutiflorum

This study aimed to investigate the potential use of Origanum minutiflorum for the eradication of Helicobacter pylori by determining antimicrobial activity, minimum bactericidal concentration (MBC), and chemical composition of its essential oil (EO). Furthermore, cell cytotoxicity and anti-H. pylori action of EO on AGS/H. pylori infected cells were also determined. Essential oil extracts displayed a strong antimicrobial activity against H. pylori. Carvacrol (29.22%), a known antimicrobial agent, was observed as the predominant compound. The MBC (1/20,000 v/v) of EO was nontoxic to AGS cells, revealing a >80% reduction. Moreover, EO at the same concentration displayed the ability to kill H. pylori in/on infected cells. The findings of this study demonstrated the positive antimicrobial and anti-adherence/anti-invasion effect of O. minutiflorum EO extract for the eradication of H. pylori and H. pylori infected AGS cells.

Elevated Interleukin-32 Expression Is Associated with Helicobacter pylori-Related Gastritis

BackgroundInterleukin-32 (IL-32) is a recently discovered proinflammatory cytokine involved in inflammatory diseases. We investigated the expression of IL-32 and its regulation mechanism in the inflammatory response of patients with Helicobacter pylori (H. pylori) infection.Design and MethodsIL-32 mRNA and protein expression in gastric tissues was detected by quantitative real-time PCR and immunohistochemistry. The regulation of IL-32 in human gastric epithelia cell line AGS was investigated by different cytokine stimulation and different H. pylori strain infection.ResultsGastric IL-32 mRNA and protein expression were elevated in patients with H. pylori infection and positively correlated with gastritis. In H. pylori-infected patients, the mRNA level of IL-32 was also correlated with that of proinflammatory cytokines IL-1β and TNF-α. In vitro IL-1β and TNF-α could upregulate IL-32 mRNA and protein level in AGS cells, which was dependent on NF-κB signal pathway. The regulation of IL-32 expression in response to H. pylori-infection could be weakened by using neutralizing antibodies to block IL-1β and TNF-α. Moreover, H. pylori-infected AGS cells also induced IL-32 mRNA and protein expression, which was dependent on CagA.ConclusionsIL-32 level is elevated in patients with H. pylori infection and its expression is regulated by proinflammatory stimuli, suggesting that IL-32 may play a role in the pathogenesis of H. pylori-related gastritis.

Anti-Inflammatory Action of Isorhamnetin

Dear Editor, A recent paper from Kynhwa Seo and colleagues in the latest issue of this journal reported that isorhamnetin, n ame l y ( 3 , 5 , 7 t r i h y d r o x y 2 ( 4 h y d r o x y 3 methoxyphenyl)chromen-4-one or quercetin-3methylether), a 3′-O-methylated metabolite of quercetin, reduced inducible nitric oxide synthase (iNOS) expression and NO production [1]. Isorhamnetin is able to increase the nuclear translocation of Nrf2 in a doseand time-dependent manner, hence the anti-oxidant response element (ARE) reporter gene activity, the expression and synthesis of proteins such as heme-oxygenase (HO-1) and cysteine lygase, and the increase in intracellular glutathione (GSH) level [2]. The expression of HO-1, via ERK/Nrf2 signaling pathway, is induced also by quercetin (2-(3,4dihydroxyphenyl)-3,5,7-trihydroxy-4H-chromen-4-one) [3], a fact that would suggest a major role of OH-flavone ring, compared to the methoxyl group, in the Nrf2-mediated anti-oxidant and anti-inflammatory activities of isorhamnetin. In a recent paper of mine, I suggested that the O-methylated group should increase plasma bioavailability of a flavonoid, and –OCH3 lacking flavone-sharing molecules are rapidly metabolized as they undergo glucuronidation and sulfation [4, 5]. In this context, isorhamnetin should deserve higher attention than quercetin. The authors suggested a role for isorhamnetin in the inhibition of cyclooxygenase-2 (COX-2) expression, in order to explain the anti-inflammatory action of the O-3′methylated flavonoid [1]. Other flavonoids, such as wogonin (5,7-dihydroxy-8-methoxy-2-phenyl-4H-chromen-4-one) and acacetin (5,7-dihydroxy-2-(4methoxyphenyl)chromen-4-one), two flavones bearing a methoxyl group, showed an inhibitory activity towards COX-2 expression [6–8]. The inhibitory effect on COX-2 by flavone-ring bearing phytochemicals has been commonly described elsewhere. Flavones without an – OCH3 group are able to affect COX-2 or iNOS expression, and reports demonstrating that flavonoids inhibit COX-2 activity were previously published [9]. A past report showed that isorhamnetin and quercetin 3′-O-sulphate are major plasma metabolites from the more widely diffused quercetin and that these metabolites, unlike quercetin, are enzymatic inhibitors of COX-2 activity [10]. Interestingly, many COX-2 synthetic inhibitors possess a flavone-like backbone [11]. Targeting COX-2 by flavonoids should represent a common hallmark of most polyphenols bearing a flavone backbone, such as flavones. Apigenin showed an anti-inflammatory action against Helicobacter pylori-infected gastric adenocarcinoma cells by affecting NF-κB and COX-2 [12]; luteolin and chrysin inhibited LPS-induced inflammation by inhibiting COX-2 and iNOS expression [13, 14]; scutellarein in Siam weed extract (Chromolaena odorata L.) exhibited an anti-inflammatory action by targeting COX-2 activity [15]. This evidence strongly suggests that many flavone-bearing phytochemicals, such as flavonoids, widely affect COX-2 function, often through ancillary mechanisms involving NF-κB [16]. Flavonoids related to quercetin, such as isorhamnetin or rutin, exhibit the potential to affect the expression of NFE2-related factor 2 (Nrf2) and HO-1 [17]. Isorhamnetin showed an anti-inflammatory action through an anti-oxidant activity [1]. In my opinion, this may represent an interpretation of a more complex pattern, in which isorhamnetin plays a fundamental role, by targeting different pathways. For example, isorhamnetin is able to reduce the inflammatory response by targeting and inhibiting high mobility group box 1 (HMGB1) receptors [17]. In the pulmonary system, binding of HMGB1 to its receptor triggers the production of pro-inflammatory cytokines, chemokines, adhesion molecules, and reactive oxygen species, promoting the development of acute lung injury and acute respiratory distress syndrome. Recent findings have shown that induction of HO-1 protects tissues and cells from extracellular stress by reducing HMGB1 production and HO-1 may mediate the protective effects of peroxisome proliferator-activated 1 Laboratory of Physiopathology of Obesity, Department of Medicine, University of Verona, Italy, LURM est Policlinico GB Rossi, Piazzale AL Scuro 10, 37134 Verona, Italy 2 To whom correspondence should be addressed at Laboratory of Physiopathology of Obesity, Department of Medicine, University of Verona, Italy, LURM est Policlinico GB Rossi, Piazzale AL Scuro 10, 37134 Verona, Italy. E-mail: salvatore.chirumbolo@univr.it

Activation of EGFR and ERBB2 by Helicobacter pylori Results in Survival of Gastric Epithelial Cells With DNA Damage

The gastric cancer-causing pathogen Helicobacter pylori up-regulates spermine oxidase (SMOX) in gastric epithelial cells, causing oxidative stress-induced apoptosis and DNA damage. A subpopulation of SMOX(high) cells are resistant to apoptosis, despite their high levels of DNA damage. Because epidermal growth factor receptor (EGFR) activation can regulate apoptosis, we determined its role in SMOX-mediated effects. SMOX, apoptosis, and DNA damage were measured in gastric epithelial cells from H. pylori-infected Egfr(wa5) mice (which have attenuated EGFR activity), Egfr wild-type mice, or in infected cells incubated with EGFR inhibitors or deficient in EGFR. A phosphoproteomic analysis was performed. Two independent tissue microarrays containing each stage of disease, from gastritis to carcinoma, and gastric biopsy specimens from Colombian and Honduran cohorts were analyzed by immunohistochemistry. SMOX expression and DNA damage were decreased, and apoptosis increased in H. pylori-infected Egfr(wa5) mice. H. pylori-infected cells with deletion or inhibition of EGFR had reduced levels of SMOX, DNA damage, and DNA damage(high) apoptosis(low) cells. Phosphoproteomic analysis showed increased EGFR and erythroblastic leukemia-associated viral oncogene B (ERBB)2 signaling. Immunoblot analysis showed the presence of a phosphorylated (p)EGFR-ERBB2 heterodimer and pERBB2; knockdown of ErbB2 facilitated apoptosis of DNA damage(high) apoptosis(low) cells. SMOX was increased in all stages of gastric disease, peaking in tissues with intestinal metaplasia, whereas pEGFR, pEGFR-ERBB2, and pERBB2 were increased predominantly in tissues showing gastritis or atrophic gastritis. Principal component analysis separated gastritis tissues from patients with cancer vs those without cancer. pEGFR, pEGFR-ERBB2, pERBB2, and SMOX were increased in gastric samples from patients whose disease progressed to intestinal metaplasia or dysplasia, compared with patients whose disease did not progress. In an analysis of gastric tissues from mice and patients, we identified a molecular signature (based on levels of pEGFR, pERBB2, and SMOX) for the initiation of gastric carcinogenesis.