Only live Helicobacter pylori is capable of caspase-3 dependent apoptosis induction in gastric mucosa epithelial cells (original) (raw)
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Inflammation Research, 2012
Objective Apoptosis plays an important role in the regulation of gastric epithelial cell number and gastrointestinal disorders induced by Helicobacter pylori (Hp). Heat shock proteins (HSPs) are involved in cell integrity, cell growth and in gastric mucosa colonized by Hp. COX-2 was implicated in Hp-induced carcinogenesis but the effects of this germ and CagA cytotoxin on HSP70, COX-2, Bax and Bcl-2 in gastric cancer epithelial cells have been little studied. Material and methods We determined the expression for HSP70, Bax and Bcl-2 in human gastric epithelial MKN7 cells incubated with live strain Hp (cagA ? vacA?) with or without co-incubation with exogenous CagA and NS-398, the selective COX-2 inhibitor. After 3-48 h of incubation, the expression of HSP70, COX-2, Bax and Bcl-2 mRNA and proteins were determined by RT-PCR and immunoprecipitation. Results Hp inhibited expression for HSP70 and this was significantly potentiated by exogenous CagA. Co-incubation of epithelial cells with Hp, without or with CagA increased Bax expression and simultaneously decreased expression for Bcl-2. The increase in COX-2 mRNA and Bax expression were significantly inhibited by NS-398. We conclude that Hp promotes apoptosis in adenocarcinoma gastric epithelial cells in vitro and this is associated with activation of COX-2 and inhibition of HSP70.
Effect of Helicobacter pylori on apoptosis and apoptosis related genes in gastric cancer cells
Molecular Pathology, 2003
Background/Aims: Helicobacter pylori induces the apoptosis of gastric epithelial cells in vivo and in vitro. However, the molecular mechanism has not been clarified. The aim of this study was to investigate the effect of H pylori on the apoptosis of gastric epithelial cells and the expression of apoptosis related genes in vitro. Methods: Human gastric adenocarcinoma SGC-7901 cells were co-cultured with a cytotoxic H pylori strain, NCTC 11637, at various densities ranging from 3.2 × 10 4 to 1.0 × 10 8 colony forming units (CFU)/ml for 48 hours. Apoptosis in gastric cells was determined by transmission electron microscopy, Hoechst 33258 fluorochrome staining, and flow cytometry. The expression of apoptosis related proteins, Bcl-2, Bax, and c-Myc, was measured by an immunohistochemical method, and c-Myc mRNA expression was determined by the reverse transcription-polymerase chain reaction. Results: Helicobacter pylori induces morphological changes typical of apoptosis. Both fluorochrome staining and flow cytometry showed that the apoptotic index began to increase when H pylori were at a density of > 1.6 × 10 4 CFU/ml, and in a density dependent manner (p < 0.01; one way ANOVA). The expression of the Bax and c-Myc proteins and of c-Myc mRNA was increased, whereas Bcl-2 expression was decreased after co-culture for 48 hours. Conclusions: Helicobacter pylori induced apoptosis in gastric epithelial cells is mediated by altered expression of the products of the Bcl-2, Bax, and c-Myc genes.
Induction of gastric epithelial apoptosis by Helicobacter pylori
Gut, 1996
Background-Helicobacter pyloni may promote gastric carcinogenesis through increasing gastric epithelial cell proliferation. How H pylon does so is unknown. Programmed, non-necrotic, cell death (apoptosis) occurs throughout the gut and is linked to proliferation. It was hypothesised that H pylori may induce hyperproliferation through increasing apoptosis. Aim-To measure the effect of H pyloni infection on gastric epithelial apoptosis in situ. Patients-Patients with duodenal ulcers treated to eradicate H pylori and patients with H pylori negative non-ulcer dyspepsia. Methods-Retrospective quantification of apoptotic epithelial cells in situ from formalin fixed biopsy specimens, counted after staining by terminal uridine deoxynucleotidyl nick end-labelling. Results-In the uninfected stomach, apoptotic cells were rare and situated in the most superficial portion of gastric glands (mean 2.9% of epithelial cells). In H pyloni infection, they were more numerous and were located throughout the depth of gastric glands, comprising 16.8% of epithelial cells, falling to 3.1% after H pyloni eradication, p=0017.
JNCI Journal of the National Cancer Institute, 1997
Background: Infection with Helicobacter pylori induces chronic gastritis in virtually all infected persons, and such gastritis has been associated with an increased risk of developing gastric cancer. This risk is further enhanced with cagA + (positive for cytotoxin-associated gene A) H. pylori strains and may be a consequence of induced gastric cell proliferation and/or alteration in apoptosis (programmed cell death) in the gastric epithelium. Purpose: To determine whether the H. pylori cagA genotype and another virulence-related characteristic, the vacA (vacuolating cytotoxin A) s1a genotype, differentially affect epithelial cell proliferation, apoptosis, and the histologic parameters of inflammation and injury, we quantitated these characteristics in infected and uninfected persons. Methods: Fifty patients underwent upper gastrointestinal endoscopy, and biopsy specimens were taken. Apoptotic cells in the specimens were quantitated after terminal deoxynucleotidyl transferase labeling of DNA fragments with digoxigenin-deoxyuridine triphosphate; epithelial cell proliferation was scored by immunohistochemical analysis of the proliferation-associated antigen Ki-67. Antibodies directed against H. pylori and CagA protein were measured in the serum of patients by means of enzyme-linked immunosorbent assays. Analysis of H. pylori genomic DNA, by use of the polymerase chain reaction, was performed to determine the cagA and vacA genotypes. Acute and chronic inflammation, epithelial cell degeneration, mucin depletion, intestinal meta
The Journal of Infectious Diseases, 2005
Background. Infection of the gastric mucosa with Helicobacter pylori leads to increased apoptosis. Cytokines and receptors of the tumor necrosis factor (TNF) family are known to be involved in this process. The role that the death-inducing TNF-a-related apoptosis-inducing ligand (TRAIL) and its receptors play, in the context of H. pylori infection, is unknown. Methods. In 74 H. pylori-infected and 51 H. pylori-uninfected gastric antral biopsy specimens, levels of TRAIL mRNA and TRAIL receptor mRNA were determined quantitatively by TaqMan reverse-transcriptase polymerase chain reaction. Recombinant TRAIL-induced apoptosis was measured in human and rat gastric epithelial cells by end-labeling of DNA with fluorescein-dTUP and by fluorescence-activated cell sorter analysis. Results. In patients infected with cagA + /vacAs1 + H. pylori strains, expression of TRAIL and the proapoptotic receptors TRAIL-R1 and-R2 was down-regulated, whereas expression of the antiapoptotic receptors TRAIL-R3 and-R4 was up-regulated. Furthermore, expression of TRAIL and TRAIL-R1 and-R2 correlated inversely with the severity of gastric inflammation. Significant apoptosis of isolated human gastric epithelial cells and highly enriched rat parietal and chief cells was induced by 100 ng/mL TRAIL. Conclusions. Down-regulation of the TRAIL system, in the context of H. pylori infection, may limit exaggerated apoptosis of gastric epithelial cells and destruction of tissue and, therefore, may enable H. pylori to maintain its niche.
Differential Protein Expresion of Helicobacter pylori During Apoptosis Gastric Cells
International Journal of Infectious Diseases, 2008
Background: The major virulence factor of H. pylori is the cag pathogenicity island divided into two parts: the upstream cagII region and the downstream cagI region. The downstream region includes cagA, cagE, and the most important gene introduced in cagII is cagT. The aim of this study was to investigate the important markers of cagI and cagII regions in Helicobacter pylori isolated from Iranian peptic ulcer and non ulcer disease patients. Methods and materials: 80 clinical isolates of H. pyloriwere collected from 120 patients with gastric disorders. The genomic DNA was extracted from biopsy samples by the QIAgen kit. The PCR was performed for detection of cagA, cagE and cagT in cagPAI. Results: Among 80 H. pylori strains, 20 (25%) and 60 (75%) were isolated from PUD and NUD patients respectively. In PUD patients with rate of 14 (70%), all of isolates were cagA-positive and distribution of cagE was 7 (35%) and cagT was 5 (25%). Also the prevalence of cagA, cagE and cagT in NUD patients was 48 (80%), 21 (35%) and 22 (36%) respectively. Discussion: Our results indicated that presence of cag PAI is very common in Iranian strains of H. pylori. The structural variety of the cag PAI might be related to the virulence of H. pylori. Present study showed that the prevalence of cagT, a marker for cagII, in PUD patients is less than NUD patients. In cagI region the presence of cagA in PUD patient is higher than cagE, so it may have an important role in peptic ulcer disease. According to our results it seems that it is not absolutely necessary to coexist of cagII and cagI in peptic ulcer patients.
World journal of gastroenterology : WJG, 2004
To investigate the relations between tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and Helicobacter pylori (H pylori) infection in apoptosis of gastric epithelial cells and to assess the expression of TRAIL on the surface of infiltrating T-cells in H pylori-infected gastric mucosa. Human gastric epithelial cell lines and primary gastric epithelial cells were co-cultured with H pylori in vitro, then recombinant TRAIL proteins were added to the culture. Apoptosis of gastric epithelial cells was determined by a specific ELISA for cell death. Infiltrating lymphocytes were isolated from H pylori-infected gastric mucosa, and expression of TRAIL in T cells was analyzed by flow cytometry. The apoptosis of gastric epithelial cell lines and primary human gastric epithelial cells was mildly increased by interaction with either TRAIL or H pylori alone. Interestingly, the apoptotic indices were markedly elevated when gastric epithelial cells were incubated with both TRAIL and H...
Helicobacter pyloriinduces apoptosis of rat gastric parietal cells
American Journal of Physiology-Gastrointestinal and Liver Physiology, 2002
Gastric Helicobacter pylori infection may lead to multifocal atrophic corpus gastritis associated with loss of epithelial cells as well as glandular structures. The current work investigated H. pylori effects on cell death of isolated, nontransformed rat parietal cells (PC). Highly enriched rat PC (>97%) were isolated from gastric mucosa and cultured in serum-free medium over 24 h. The cells were cocultured over 8 h with cytotoxin-associated immunodominant protein (cagA)+/vacuolating toxin (vacA)+or with cagA−/vacA−H. pylori laboratory strains and also with H. pylori mutants deleted in several genes of the cag pathogenicity island. Staphylococcus aureus or Campylobacter jejuni were used as controls. Apoptosis was determined by terminal deoxynucleotidyl transferase dUTP nick-end labeling staining and electron microscopy. Interleukin (IL)-8 and cytokine-induced neutrophil chemoattractant (CINC)-1 secretion was measured by ELISA. Activation of nuclear factor-κB (NF-κB) was studied i...
2006
Heat shock proteins (HSP) are crucial for the maintenance of cell integrity under normal cell growth and at pathophysiological conditions such as colonization of gastric mucosa by Helicobacter pylori (Hp). The effect of Hp on mRNA expression for HSP70 in the gastric epithelial cells in vitro has been little studied and remains inconclusive. In this study we attempted to determine the alterations in gene expression for HSP70 induced by two live strains of Hp in the epithelial MKN7 cells. The following Hp strains were employed; 1) Hp strain expressing cagA and vacA, and 2) cagA and vacA negative Hp strain without or with addition of exogenous recombinant protein CagA. MKN7 cells were incubated in a standard medium RPMI 1640 supplemented with 10% fetal bovine serum at 37°C with 5% CO 2 and humidified atmosphere under basal condition or in a presence of Hp (1x10 9 CFU per dish) without or with the recombinant CagA (10 µg/ml of RPMI 1640 medium). After 3h, 24h and 48 h of incubation with Hp and in some experiments with the prolonged incubation time up to 72 h, the cells were harvested, the total cellular RNA was isolated and the expression of mRNA for HSP70 was determined by RT-PCR. The incubation of the MKN cells with CagA protein alone failed to affect significantly the expression of HSP70. In contrast, the strain Hp (cagA+, vacA+) inhibited in time-dependent manner the expression of mRNA for HSP70. When the MKN7 cells were coincubated with Hp (cagA+, vacA+) and exogenous CagA, the significant inhibition of the signal intensity for HSP70 mRNA was observed at 3 h and 24 h of incubation and these effects were followed by complete disappearance of the signal for HSP70 mRNA at 48 h. The incubation of MKN7 with Hp (cagA-, vacA-) also significantly attenuated the expression of HSP70 mRNA with the most pronounced inhibitory effect observed at 72 h of incubation with this Hp strain. Addition of the recombinant CagA to Hp (cagA-, vacA-) completely suppressed the expression of HSP70 at 48 h and 72 h after the end of incubation periods. We conclude that 1) both, Hp (cagA+, vacA+) and Hp (cagA-, vacA-) inhibit expression of HSP70 in MKN7 human gastric epithelial cells independently of the presence or absence of cagA gene, and that 2) recombinant CagA protein may exert biological activity in vitro via acceleration of inhibitory effect of Hp negative for Cag A and VacA on HSP70 expression in epithelial cells infected with this bacteria.