The epithelial cytokine response, associated with reactive oxygen species (ROS), is important in Helicobacter pylori (H. pylori)-induced inflammation. H. pylori induces the production of ROS, which may be involved in the activation of mitogen-activated protein kinases (MAPK), janus kinase/signal transducers and activators of transcription (Jak/Stat), and oxidant-sensitive transcription factor, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kappaB), and thus, expression of interleukin-8 (IL-8) in gastric epithelial cells. alpha-lipoic acid, a naturally occurring thiol compound, is a potential antioxidant. It shows beneficial effects in treatment of oxidant-associated diseases including diabetes. The present study is purposed to investigate whether alpha-lipoic acid inhibits expression of inflammatory cytokine IL-8 by suppressing activation of MAPK, Jak/Stat, and NF-kappaB in H. pylori-infected gastric epithelial cells. Gastric epithelial AGS cells were pretreated with or without alpha-lipoic acid for 2 h and infected with H. pylori in a Korean isolate (HP99) at a ratio of 300:1. IL-8 mRNA expression was analyzed by RT-PCR analysis. IL-8 levels in the medium were determined by enzyme-linked immunosorbent assay. NF-kappaB-DNA binding activity was determined by electrophoretic mobility shift assay. Phospho-specific and total forms of MAPK and Jak/Stat were assessed by Western blot analysis. ROS levels were determined using dichlorofluorescein fluorescence. As a result, H. pylori induced increases in ROS levels, mRNA, and protein levels of IL-8, as well as the activation of MAPK [extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun NH2-terminal kinase 1/2 (JNK1/2), p38], Jak/Stat (Jak1/2, Stat3), and NF-kappaB in AGS cells, which was inhibited by alpha-lipoic acid. In conclusion, alpha-lipoic acid may be beneficial for prevention and/or treatment of H. pylori infection-associated gastric inflammation.
Enzyme-Linked Immunosorbent Assay ; Epithelial Cells/metabolism ; Gastric Mucosa/*drug effects/metabolism/microbiology ; Gene Expression Regulation, Bacterial ; Helicobacter Infections/immunology/*metabolism ; Helicobacter pylori/drug effects/*pathogenicity ; Humans ; Interleukin-8/genetics/*metabolism ; JNK Mitogen-Activated Protein Kinases ; Janus Kinase 1 ; Mitogen-Activated Protein Kinases/*biosynthesis ; NF-kappa B/*metabolism ; RNA, Messenger/isolation & purification/metabolism ; Reactive Oxygen Species/metabolism ; STAT3 Transcription Factor ; Stomach/metabolism/*microbiology ; Thioctic Acid/*pharmacology

NADPH oxidase produces a large amount of reactive oxygen species (ROS) in Helicobacter pylori (H. pylori)-induced gastric epithelial cells. Even though ROS mediate apoptotic cell death, direct involvement of NADPH oxidase on H. pylori-induced apoptosis remains unclear. Besides, H. pylori isolates show a high degree of genetic variability. The predominant genotype of H. pylori in Korea has been reported as cagA+, vacA s1b, m2, iceA genotype. Present study aims to investigate whether NADPH oxidase-generated ROS mediate apoptosis in human gastric epithelial AGS cells infected with H. pylori in a Korean isolate. AGS cells were pretreated with or without an NADPH oxidase inhibitor diphenyleneiodonium (DPI) and cultured in the presence of H. pylori at a bacterium/cell ratio of 300:1. Cell viability, hydrogen peroxide level, DNA fragmentation, and protein levels of p53, Bcl-2, and Bax were determined. Results showed that H. pylori inhibited cell viability with the density of H. pylori added to the cells. Inhibition of NADPH oxidase by DPI suppressed H. pylori-induced cell death, increased hydrogen peroxide, DNA fragmentation, and the ratio of Bax/Bcl-2, and p53 induction in AGS cells dose-dependently. The results suggest that targeting NADPH oxidase may prevent the development of gastric inflammation associated with H. pylori infection by suppressing abnormal apoptotic cell death of gastric epithelial cells.
Apoptosis ; Apoptosis Regulatory Proteins/metabolism ; Cell Survival ; Epithelial Cells/metabolism/microbiology ; Gastric Mucosa/metabolism ; Helicobacter Infections/*metabolism/microbiology ; Helicobacter pylori/drug effects/genetics/*isolation & purification ; Humans ; NADPH Oxidase/metabolism ; Onium Compounds/*antagonists & inhibitors/pharmacology ; Oxidative Stress/drug effects ; Reactive Oxygen Species/metabolism ; Republic of Korea ; Stomach/cytology/*metabolism/microbiology

BACKGROUND/AIMS: Helicobacter pylori cytotoxin-associated gene A (CagA) has been suggested to be involved in the inactivation of Runt-related transcription factor 3 (RUNX3), a known gastric carcinoma tumor suppressor gene. It remains unclear how H. pylori CagA initiates or maintains RUNX3 promoter methylation and inactivates its protein expression in gastric carcinoma. METHODS: RUNX3 promoter methylation status, RUNX3 expression, and H. pylori CagA were investigated in 76 sample pairs of gastric carcinoma tissue. The patients' medical records were reviewed. The association between RUNX3 methylation or loss of RUNX3 expression and clinicopathologic variables according to H. pylori CagA status were investigated. RESULTS: In gastric carcinoma patients with H. pylori CagA-positive infection, RUNX3 methylation did not show association with lymphatic invasion, venous invasion, and TNM stages. However RUNX3 methylation was observed more frequently in poorly differentiated adenocarcinoma and signet ring cell carcinoma (77.8% vs. 20.0%, p=0.023) in early stage. In gastric carcinoma patients with H. pylori CagA-positive infection, loss of RUNX3 expression did not show association with lymphatic invasion, venous invasion, and TNM stages. However loss of RUNX3 expression was observed more frequently in early gastric carcinoma than in advanced gastric carcinoma (84.2% vs. 75.0%, p=0.51), but this difference was not significant. CONCLUSIONS: In gastric carcinoma patients with H. pylori CagA-positive infection, RUNX3 methylation or loss of RUNX3 expression did not show correlation with lymphovascular invasion and TNM stages. In early gastric carcinoma patients with H. pylori CagA-positive infection, RUNX3 methylation was observed more in poorly differentiated adenocarcinoma and signet ring cell carcinoma.
Adult ; Aged ; Aged, 80 and over ; Antigens, Bacterial/*metabolism ; Bacterial Proteins/*metabolism ; Cell Line, Tumor ; Core Binding Factor Alpha 3 Subunit/genetics/*metabolism ; Female ; *Gene Expression Regulation, Neoplastic ; Helicobacter Infections/complications/microbiology ; Helicobacter pylori/isolation & purification/*metabolism ; Humans ; Immunohistochemistry ; Lymphatic Metastasis ; Male ; Methylation ; Middle Aged ; Neoplasm Staging ; Promoter Regions, Genetic ; Retrospective Studies ; Stomach Neoplasms/complications/microbiology/*pathology

OBJECTIVETo establish an animal model of gastric cancer by long-term infection of Helicobacter pylori (H.pylori) and to elucidate the pathogenesis by proteomics analysis.

METHODSFifty male Mongolian gerbils (4-5 week-old and weighted 60-100 g) were infected with H.pylori and the gastric tissues were obtained after the infection at 3, 6, 12 and 24 months. Histological changes were evaluated by H-E staining of the gastric tissue sections. Detection of H.pylori was performed by in-vitro culture of fresh gastric tissue samples, PCR amplification of H.pylori 16s rRNA and localization by silver staining. In addition, proteins extracted from gastric tissue samples were subjected to two-dimensional electrophoresis (2-DE) at various infection time points. Protein spots with increased quantity over the course of H.pylori infection were selected and analyzed by LC-MS/MS. Finally, differentially expressed proteins between human gastric cancer tissue samples and lymph nodes were analyzed by real-time RT-PCR.

RESULTSColonization of H.pylori was observed in gastric tissue of gerbils as early as 3 months after H.pylori infection, and persisted till 24 months. Pathological examination of infected animals showed various histological changes including acute gastritis, atrophic gastritis, intestinal metaplasia and gastric carcinoma. Seventy-eight differentially expressed proteins were identified by proteomics analysis, among which 36 proteins were up-regulated and 42 were down-regulated. Analyzed by LC-MS/MS, ten proteins were identified, including lactate dehydrogenase, ATP synthase, fatty acid-binding protein, COX5B, peroxiredoxin-4, peroxide reductase, transgelin, succinyl-CoA ligase, keratin and protein disulfide-isomerase A2, among which transgelin, ATP synthase and lactate dehydrogenase were highly expressed in human gastric carcinoma and lymph nodes.

CONCLUSIONSH.pylori infection induces the expression of transgelin, ATP synthase and lactate dehydrogenase, implying possible roles in the pathogenesis of gastric diseases including cancer.

Animals ; Disease Models, Animal ; Gastritis ; microbiology ; pathology ; Gerbillinae ; Helicobacter Infections ; complications ; metabolism ; Helicobacter pylori ; genetics ; Humans ; L-Lactate Dehydrogenase ; metabolism ; Male ; Metaplasia ; Microfilament Proteins ; metabolism ; Muscle Proteins ; metabolism ; Proteomics ; Proton-Translocating ATPases ; metabolism ; RNA, Ribosomal, 16S ; analysis ; Stomach Neoplasms ; metabolism ; microbiology ; Tandem Mass Spectrometry

OBJECTIVETo investigate the correlation between infection with L-form of Helicobacter pylori (Hp-L) and the expressions of macrophage migration inhibition factor (MIF), matrix metalloproteinase 9 (MMP9), and vascular endothelial growth factor (VEGF) in gastric cancer.

METHODSHp-L was examined in 80 gastric carcinoma and 50 adjacent normal tissues by Gram staining and immunohistochemical staining, and the expressions of MIF, MMP9 and VEGF were detected by immunohistochemical staining; the expression of MIF mRNA was detected by RT-PCR and the expression of MIF, MMP9 and VEGF proteins were detected by Western blotting in 30 fresh gastric cancer tissues and the corresponding adjacent tissues.

RESULTSOf the 80 gastric carcinoma tissues, 57 (71.25%) showed Hp-L positivity detected by both Gram staining and immunohistochemical staining, as compared with a rate of only 14% in the adjacent normal tissues (P<0.05). The gastric carcinoma tissues showed higher expression levels of MIF, MMP9 and VEGF proteins than the corresponding adjacent normal mucosa; the positivity MIF, MMP-9 and VEGF proteins were significantly higher in Hp-L-positive gastric carcinoma than in Hp-L-negative cases (P<0.05). Positive correlations were found between Hp-L positivity and the expressions of MIF, MMP-9 and VEGF (r=0.598, 0.292, 0.341, respectively, P<0.05). The 30 fresh gastric cancer tissues showed also significantly higher MIF mRNA expression and MIF, MMP-9 and VEGF protein expressions than the adjacent tissues (t=3.729, P<0.01). The expressions of MIF and MMP-9 were also related to the clinicopathological factors including lymph node metastasis and depth of invasion (P<0.05).

CONCLUSIONInfection with L-form of Hp-L can be an important factor that contributes to the invasion and metastasis of gastric carcinoma, the mechanism of which involves up-regulated expressions of MIF, MMP-9 and VEGF.

Adult ; Aged ; Aged, 80 and over ; Female ; Helicobacter Infections ; metabolism ; pathology ; Helicobacter pylori ; Humans ; L Forms ; Lymphatic Metastasis ; Macrophage Migration-Inhibitory Factors ; metabolism ; Male ; Matrix Metalloproteinase 9 ; metabolism ; Middle Aged ; Stomach Neoplasms ; metabolism ; microbiology ; pathology ; Vascular Endothelial Growth Factor A ; metabolism

Gastric cancer is one of the most common malignancies and a leading cause of cancer mortality worldwide. The pathogenesis mechanisms of gastric cancer are still not fully clear. Inactivation of tumor suppressor genes and activation of oncogenes caused by genetic and epigenetic alterations are known to play significant roles in carcinogenesis. Accumulating evidence has shown that epigenetic silencing of the tumor suppressor genes, particularly caused by hypermethylation of CpG islands in promoters, is critical to carcinogenesis and metastasis. Here, we review the recent progress in the study of methylations of tumor suppressor genes involved in the pathogenesis of gastric cancer. We also briefly describe the mechanisms that induce tumor suppressor gene methylation and the status of translating these molecular mechanisms into clinical applications.
Apoptosis ; Cell Adhesion ; Cell Cycle ; CpG Islands ; genetics ; DNA Methylation ; DNA Repair ; Epigenesis, Genetic ; Genes, Tumor Suppressor ; Helicobacter Infections ; genetics ; Helicobacter pylori ; Humans ; Neoplasm Invasiveness ; Promoter Regions, Genetic ; genetics ; Stomach Neoplasms ; genetics ; metabolism ; microbiology ; pathology ; Tumor Suppressor Proteins ; genetics ; metabolism

OBJECTIVETo explore the possible angiogenesis mechanism of Huazhuo Jiedu Hewei Recipe (HJHR) in preventing and treating precancerous lesions of gastric cancer (PLGC).

METHODSTotally 66 Wistar rats were randomly divided into 6 groups, i.e., the normal control group, the model group, the retinoic acid (RA) group, the high dose HJHR group, the middle dose HJHR group, the low dose HJHR group, 11 in each group. PLGC model was duplicated by inserting a spring with Helicobacter. Corresponding medicines were administered to rats in each medicated group once daily by gastrogavage, 2 mL each time for 12 successive weeks. The effect of HJHR on hypoxia induced factor (HIF-1alpha) and vascular endothelial growth factor (VEGF) of PLGC in chronic atrophic gastritis (CAG) rats' gastric mucosa was observed by immunohistochemical assay and Western blot method.

RESULTSCompared with the normal control group, the expression of VEGF and HIF-1alpha increased in the model group (P < 0.05). Compared with the model group, the expression of VEGF and HIF-1alpha decreased in each medicated group (P < 0.05). Besides, they were lower in the high and middle dose HJHR groups than in the RA group and the low dose HJHR group (P < 0. 05). There was no statistical difference between the low dose HJHR group and the RA group (P > 0.05).

CONCLUSIONHJHR could prevent and treat PLGC of CAG rats possibly through decreasing the expression of HIF-1alpha and VEGF in a dose-dependent manner.

Animals ; Drugs, Chinese Herbal ; therapeutic use ; Gastric Mucosa ; metabolism ; Gastritis ; metabolism ; microbiology ; Helicobacter ; Hypoxia-Inducible Factor 1, alpha Subunit ; metabolism ; Male ; Neovascularization, Pathologic ; Precancerous Conditions ; blood supply ; drug therapy ; metabolism ; Rats ; Rats, Wistar ; Stomach Neoplasms ; blood supply ; drug therapy ; metabolism ; Vascular Endothelial Growth Factor A ; metabolism

OBJECTIVETo evaluate the H. pylori and Epstein-Barr virus infection in cardiac and distal gastric adenocarcinoma tissues in residents in Cixian county, a high risk area of esophageal cancer in Hebei province, and to explore the putative role of H. pylori and Epstein-Barr virus infection in the carcinogenesis of adenocarcinoma at different subsites of stomach.

METHODSH. pylori and Epstein-Barr virus latent membrane protein 1 (EBV-LMP1) immunopositivities were determined by Elivision(TM) plus immunohistochemical staining in 190 gastric adenocarcinoma tissues including 144 cases of cardiac adenocarcinoma and 46 cases of distal gastric adenocarcinoma. The relationship between H. pylori and Epstein-Barr virus infection and the subsite, Laurén type as well as other clinicopathological features of gastric adenocarcinoma were analyzed.

RESULTSNo significant difference was found between the H. pylori detection rates in cardiac and distal gastric adenocarcinomas(56.9% vs. 65.2%, P > 0.05). The detection rate of H. pylori in intestinal type was significantly higher than that in the diffuse type distal gastric adenocarcinomas (71.8% vs. 28.6%, P < 0.05). No positive expression of EBV-LMP1 was found in the gastric adenocarcinomas in this study.

CONCLUSIONSNo significant differences in H. pylori and EBV-LMP1 infections were found between cardiac and distal gastric adenocarcinomas in Cixian county. H. pylori infection is related with the intestinal type of distal gastric adenocarcinoma.

Adenocarcinoma ; microbiology ; pathology ; virology ; Aged ; Cardia ; China ; Epstein-Barr Virus Infections ; pathology ; Female ; Helicobacter Infections ; pathology ; Helicobacter pylori ; isolation & purification ; Humans ; Male ; Middle Aged ; Stomach Neoplasms ; microbiology ; pathology ; virology ; Viral Matrix Proteins ; metabolism

OBJECTIVETo study the effect of Jianpi Jiedu Recipe (JJR) on the expression of cyclooxygenase (COX-2) in Helicobacter pylori (Hp) infected gastric cancer cell line MKN 45, and its regulatory mechanism of p38MAPK signal transduction.

METHODSThe expressions of COX-2 mRNA and protein in human gastric cancer cell line MKN 45 infected by Hp type strain NCTC 11637 and the regulatory effect of JJR containing serum were detected using Real-time fluorescent quantitative polymerase chain reaction (RFQ-PCR) and Western blot. The effects of Hp on COX-2 mRNA and protein expressions in human gastric cancer cell line MKN 45 were observed using blocking p38MAPK signal transduction pathway by p38MAPK specific inhibitor SB203580. The effects of JJR on Hp-infection activated p38MAPK signal transduction pathway and its downstream activating transcription factor 2 (ATF-2) were observed.

RESULTSCOX-2 mRNA and protein expressions were obviously higher after human gastric cancer cell line MKN 45 were infected by Hp (P<0.01). After blocking p38MAPK signal transduction pathway, COX-2 mRNA and protein expressions in Hp-induced MKN 45 cell line were obviously down-regulated (P<0.01). JJR containing serum down-regulated Hp-induced COX-2 mRNA and protein expressions in MKN 45 cell line in a dose dependent manner. Besides, it could inhibit the activation of Hp-induced p38MAPK signal pathway. It also showed obvious inhibition on the activity of its downstream transcription factor ATF-2.

CONCLUSIONSHp infection induced COX-2 expressions of gastric cancer cells via p38MAPK signal transduction pathway. JJR inhibited Hp-induced the expression of COX-2 through regulating p38MAPK/ATF-2 signal transduction pathway, which may be one of its mechanisms in prevention and treatment of Hp-induced gastric cancer.

Activating Transcription Factor 2 ; metabolism ; Animals ; Cell Line, Tumor ; Cyclooxygenase 2 ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; Gene Expression Regulation, Neoplastic ; Helicobacter pylori ; drug effects ; Humans ; Imidazoles ; pharmacology ; MAP Kinase Signaling System ; drug effects ; Pyridines ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Serum ; Signal Transduction ; drug effects ; Stomach Neoplasms ; metabolism ; microbiology ; p38 Mitogen-Activated Protein Kinases ; antagonists & inhibitors

OBJECTIVETo investigate the regulatory effect of jianpi jiedu Recipe (JJR) on the microvessel density (MVD) and cyclooxygenase-2 (COX-2) in long-term infection of Helicobacter pylori induced gastric cancer of C57BL/6 mice, thus providing experimental bases for its treatment of the H. pylori correlated gastropathy.

METHODSC57BL/6 mouse gastric cancer model induced by H. pylori infection was established by gastrogavage of H. pylori standard strain SS1. Mice were divided into the control group, the model group, low dose JJR group, and the high dose JJR group, 40 in each group. Mice were sacrificed after 72-week medication. Changes of the gastric mucosa MVD of mice in each group were detected by immunohistochemical method. Expressions of COX-2 mRNA and protein were detected by Real-time fluorescent quantitative polymerase chain reaction and immunohistochemical method.

RESULTSThe occurrence rate of gastric cancer in the control group, the model group, the low dose JJR group, and the high dose JJR group was 0, 22.2%, 11.1%, and 10.0%, respectively. The gastric mucosa MVD (number/cm2) of mice in each group was 2.50 +/- 1.54, 18.56 +/- 2.62, 14.61 +/- 3.60, and 7.39 +/- 1.75, respectively. The gastric mucosa MVD in the model group increased more obviously than that in the control group (P < 0.01). The gastric mucosa MVD significantly decreased in the low dose JJR group and the high dose JJR group (P < 0.01). Expressions of COX-2 mRNA and protein in the model group increased more obviously than those in the control group (P < 0.01). Low dose JJR and high dose JJR could decrease their expressions in a dose dependent manner.

CONCLUSIONSH. pylori infection could increase the gastric mucosa MVD of C57BL/6 mice and promote COX-2 expressions, which might play a promoting effect in the incidence of H. pylori induced gastric cancer. JJR could decrease the gastric mucosa MVD and inhibit COX-2 expressions, which might be one of its important mechanisms of preventing and treating gastric cancer.

Animals ; Cyclooxygenase 2 ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; Gastric Mucosa ; blood supply ; drug effects ; metabolism ; Helicobacter Infections ; metabolism ; Helicobacter pylori ; Mice ; Mice, Inbred C57BL ; Microvessels ; pathology ; Stomach Neoplasms ; blood supply ; metabolism ; microbiology