Apoptosis In Gastric Epithelial Cells Research Articles

SOX2 is frequently downregulated in gastric cancers and inhibits cell growth through cell-cycle arrest and apoptosis

SOX transcription factors are essential for embryonic development and play critical roles in cell fate determination, differentiation and proliferation. We previously reported that the SOX2 protein is expressed in normal gastric mucosae but downregulated in some human gastric carcinomas. To clarify the roles of SOX2 in gastric carcinogenesis, we carried out functional characterisation of SOX2 in gastric epithelial cell lines. Exogenous expression of SOX2 suppressed cell proliferation in gastric epithelial cell lines. Flow cytometry analysis revealed that SOX2-overexpressing cells exhibited cell-cycle arrest and apoptosis. We found that SOX2-mediated cell-cycle arrest was associated with decreased levels of cyclin D1 and phosphorylated Rb, and an increased p27Kip1 level. These cells exhibited further characteristics of apoptosis, such as DNA laddering and caspase-3 activation. SOX2 hypermethylation signals were observed in some cultured and primary gastric cancers with no or weak SOX2 expression. Among the 52 patients with advanced gastric cancers, those with cancers showing SOX2 methylation had a significantly shorter survival time than those without this methylation (P=0.0062). Hence, SOX2 plays important roles in growth inhibition through cell-cycle arrest and apoptosis in gastric epithelial cells, and the loss of SOX2 expression may be related to gastric carcinogenesis and poor prognosis.

Differential regulation of ERK1/2 and p38 MAP kinases in VacA-induced apoptosis of gastric epithelial cells

Helicobacter pylori vacuolating cytotoxin A (VacA) has been considered as an apoptosis-inducing factor. Here, we investigated the mechanism of VacA-induced apoptosis in relation to the defense mechanism and MAP kinases pathway in gastric epithelial cells. AGS cells exposed to enriched VacA extracts affected the level of SOD-1 and villin. We further investigated the role of VacA in those inductions using a functional recombinant VacA (rVacA). Activation of p38 MAPK and Bax dimerization by rVacA were increased in a dose-dependent manner. rVacA-induced ERK1/2 MAPK activation was maximal at 30 min and 4 h and 1-4 microg/ml of rVacA. rVacA-induced SOD-1 expression was considerably diminished by inhibiting ERK1/2 MAPK and it was slightly increased by inhibiting p38 MAPK. rVacA increased or decreased villin expression depending on dose and exposure time and its expression was mainly appeared in the contractile actin ring of the dividing cells. Despite its cytoprotective effect, SB-203580, a p38 inhibitor, was unlikely to reduce VacA-induced Bax dimerization and rather inhibited villin and Bcl2 expression, indicating that p38 may also play a role in cell proliferation or differentiation for survival after VacA intoxication. Furthermore, p38 inhibitor accelerated rVacA-induced cell death after exposure of AGS cells to H(2)O(2) but ERK1/2 inhibitor protected cells from H(2)O(2) insult. These results suggest that SOD-1 and villin are expressed differentially upon VacA insult depending on dose and exposure time via ERK and p38 MAP kinases; decrease in SOD-1 and villin expression coupled with Bax dimerization leads to apoptosis of gastric epithelial cells.

In vivo effects of TGFβ1 on the growth of gastric epithelium in suckling rats

As the content of Transforming Growth Factor-β (TGFβ) wanes in the milk of lactating rat, an increase in TGFβ is observed in the gastric epithelia concomitant with differentiation of the glands upon weaning. Whereas TGFβ has been shown to inhibit the proliferation of gastrointestinal cells in vitro, its functional significance and mechanisms of action have not been studied in vivo. Therefore, we administered TGFβ1 (1 ng/g body wt.) to 14-day-old rats in which the gastric epithelium was induced to proliferate by fasting, and determined the involvement of signaling through Smads and the impact on epithelial cell proliferation and apoptosis. After the gavage, we observed the progressive increase of active TGFβ1 while TβRII-receptor remained constant in the gastric mucosa. By immunohistochemistry, we showed Smad2/3 increase at 60 min ( p < 0.05) and Smad2 phosphorylation/activation and translocation to the nucleus most prominently between 0 and 30 min after treatment ( p < 0.05). Importantly, TGFβ1 inhibited cell proliferation ( p < 0.05), which was estimated by BrDU pulse-labeling 12 h after gavage. Lower proliferation was reflected by increased p27 kip1 at 2 h ( p < 0.05). Also, TGFβ1 increased apoptosis as measured by M30 labeling at 60 and 180 min ( p < 0.001), and by morphological features at 12 h ( p < 0.05). In addition, we observed higher levels of activated caspase 3 (17 kDa) from 0 to 30 min. Altogether, these data indicate a direct effect of TGFβ1 signaling through Smads on both inhibiting proliferation, through alteration of cycle proteins, and inducing apoptosis of gastric epithelial cells in vivo. Further, the studies suggest a potential role for both milk and tissue-expressed TGFβ1 in gastric growth during postnatal development.

The Effect of the cag Pathogenicity Island on Binding of Helicobacter pylori to Gastric Epithelial Cells and the Subsequent Induction of Apoptosis

Helicobacter pylori infection leads to gastritis, peptic ulcer, and gastric cancer, in part due to epithelial damage following bacteria binding to the epithelium. Infection with cag pathogenicity island (PAI) bearing strains of H. pylori is associated with increased gastric inflammation and a higher incidence of gastroduodenal diseases. It is now known that various effector molecules are injected into host epithelial cells via a type IV secretion apparatus, resulting in cytoskeletal changes and chemokine secretion. Whether binding of bacteria and subsequent apoptosis of gastric epithelial cells are altered by cag PAI status was examined in this study. AGS, Kato III, and N87 human gastric epithelial cell lines were incubated with cag PAI-positive or cag PAI-negative strains of H. pylori in the presence or absence of clarithromycin. Binding was evaluated by flow cytometry and scanning electron microscopy. Apoptosis was assessed by detection of DNA degradation and ELISA detection of exposed histone residues. cag PAI-negative strains bound to gastric epithelial cells to the same extent as cag PAI-positive strains. Both cag PAI-positive and cag PAI-negative strains induced apoptosis. However, cag PAI-positive strains induced higher levels of DNA degradation. Incubation with clarithromycin inactivated H. pylori but did not affect binding. However, pretreatment with clarithromycin decreased infection-induced apoptosis. cag PAI status did not affect binding of bacteria to gastric epithelial cells but cag PAI-positive H. pylori induced apoptosis more rapidly than cag PAI-negative mutant strains, suggesting that H. pylori binding and subsequent apoptosis are differentially regulated with regard to bacterial properties.

The Helicobacter pylori's protein VacA has direct effects on the regulation of cell cycle and apoptosis in gastric epithelial cells

In this study, we have evaluated the effects on cell cycle regulation of VacA alone and in combination with other two Helicobacter pylori proteins, cytotoxin-associated protein (CagA) and HspB, using the human gastric epithelial cells (AGS). Our results indicate that VacA alone was able to inhibit the G1 to S progression of the cell cycle. The VacA capacity of inhibiting cell progression from G1 to S phase was also observed when cells were co-transfected with CagA or HspB. Moreover, VacA over-expression caused apoptosis in AGS cells through activation of caspase 8 and even more of caspase 9, thus indicating an involvement of both the receptor-mediated and the mitochondrial pathways of apoptosis. Indeed, the two pathways probably can co-operate to execute cell death with a prevalence of the mitochondrial pathways. Our data taken together provide additional information to further enhance our understanding of the molecular mechanism by which H. pylori proteins alter the growth status of human gastric epithelial cells.

NSAIDs enhance proteasomic degradation of survivin, a mechanism of gastric epithelial cell injury and apoptosis

NSAIDs cause severe gastrointestinal injury, in part by suppressing survivin, an inhibitor of apoptosis protein, both in cultured gastric epithelial cells and in human and rat gastric mucosa. The mechanism(s) of survivin down-regulation by NSAIDs is unclear. In this study, we examined whether NSAID treatment decreases survivin mRNA expression and/or enhances degradation of survivin protein via ubiquitin proteasome system in rat gastric mucosal, RGM-1 cells, and whether survivin overexpression prevents indomethacin-induced cell injury and apoptosis. Effects of indomethacin on survivin mRNA expression, survivin protein half-life and ubiquitination were examined in RGM-1 cells. Proteasome inhibitors were utilized to prevent indomethacin-induced survivin protein degradation in RGM-1 cells. The effects of stable overexpression of survivin on indomethacin-induced RGM-1 cell injury and apoptosis were examined. Results showed: (1) Indomethacin treatment did not alter survivin mRNA expression, but significantly reduced survivin protein half-life from 1.5 h to approximately 1 h and increased survivin ubiquitination. (2) Inhibition of ubiquitin proteasome prolonged survivin protein half-life to over 2 h and inhibited indomethacin-induced survivin degradation. (3) Overexpression of survivin significantly reduced indomethacin-induced cell injury and apoptosis. In conclusion, indomethacin treatment enhances degradation of survivin via the ubiquitin proteasome machinery in RGM-1 cells, and maintenance of survivin levels is important for prevention of gastric epithelial cell injury and apoptosis.

Prevention of Deoxycholate-Induced Gastric Apoptosis by Aspirin: Roles of NF-κB and PKC Signaling

Apoptosis is a major mechanism of gastric cell death induced by deoxycholate (DC) and aspirin (ASA), and the caspase cascade and protein kinase C (PKC) signaling play key roles in this process. The transcription factor kappa B (NF-kappaB) has been shown to modulate apoptosis by regulating the transcription of numerous pro- and anti-apoptotic genes. The aim of this study was to investigate the effect of DC and ASA on NF-kappaB signaling, and determine its role in programmed cell death in a human gastric carcinoma cell line.Cells were incubated with DC in the presence or absence of ASA or proteasome inhibitors (PI- I, lactacystin, and MG-132). Cell lysates were evaluated by Western blotting. NF-kappaB (p65) was measured in the cytosol and nuclear fractions.DC induced a translocation of NF-kappaB into the nuclear compartment that was completely blocked by proteasome inhibitors. Although, ASA itself had no effect on the NF-kappaB pathway, nor did it reduce DC-induced NF-kappaB translocation, it did prevent DC-induced caspase-3, -6 and -9 activation, poly (ADP-ribose) polymerase and lamin A processing, DNA degradation, and PKC signaling, all indices of apoptosis. In contrast, proteasome inhibitors had no effect on DC-induced apoptosis.Deoxycholate activates NF-kappaB at the same time that it induces apoptosis in gastric epithelial cells. Prevention of NF-kappaB activation does not alter DC-induced apoptosis, indicating that in our experimental conditions, NF-kappaB is not essential for apoptosis to proceed. In contrast, the ability of aspirin to restore the alterations in PKC isoforms induced by DC and at the same time prevent caspase cascade activation suggests the importance of the PKC signaling system in this process.

Helicobacter pylori infection induces apoptosis in gastric cancer cells through the mitochondrial pathway

To clarify the role of the mitochondrial pathway in apoptosis induced by H. pylori infection in gastric epithelial cells. Cells of a gastric adenocarcinoma cell line SGC-7,901 were co-cultured with H. pylori NCTC 11,637, with or without preincubation with the inhibitors of caspases -3, -8, and -9. Apoptosis was determined by flow cytometry. RT-PCR was used to determine the expression of Bid, Bax, and Bcl-2 mRNA, and Western blotting was used to determine the expression of Bid, Bax, and Bcl-2 proteins, and the activation of caspases -3 and -9. H. pylori directly induced apoptosis in SGC-7,901 cells. Apoptotic indices (AIs) were 6.30 +/- 0.40%, 11.57 +/- 0.78%, 8.63 +/- 0.67%, and 7.22 +/- 0.97%, respectively, at 6, 12, 24, and 48 h after SGC-7,901 cells were co-cultured with H. pylori. H. pylori up-regulated the expression of Bid and Bax at both protein and mRNA levels, and induced a time-dependent activation of caspases -3 and -9. Apoptosis was inhibited significantly by the preincubation of SGC-7,901 cells with the inhibitors of caspase-3 (AIs were 1.72 +/- 0.59%, 2.97 +/- 0.55%, 4.38 +/- 1.56%, and 3.29 +/- 0.83%, respectively, at 6, 12, 24, and 48 h), and caspase -9 (AIs were 2.47 +/- 0.53%, 6.68 +/- 0.47%, 5.97 +/- 0.46%, and 5.43 +/- 0.15%, respectively, at 6, 12, 24, and 48 h). The caspase-8 inhibitor also reduced H. pylori-induced apoptosis by 20%. H. pylori infection induces apoptosis and the activation of caspases -3 and -9 in gastric cancer cells. Moreover, the caspase inhibitors significantly suppress H. pylori-induced apoptosis. These findings suggest that the mitochondrial pathway may be the major pathway in H. pylori-induced apoptosis in gastric epithelial cells.

Aspirin-Induced Apoptosis in Human Gastric Cancer Epithelial Cells: Relationship with Protein Kinase C Signaling

This study examined the relationship of protein kinase C (PKC) signaling with apoptosis induced by aspirin (ASA) in gastric surface cancer cells (AGS cell line). We found increased expression of two PKC isoforms (alpha and betaII) that translocated from the cytosol into the cell membrane fraction after ASA (40 mM) stimulation. PKC betaI expression markedly decreased in response to ASA treatment. This process was independent of caspase activation because no caspase inhibitors used (i.e., inhibitors to caspase 3, 6, 7, 8, and total caspase activity) significantly changed PKC processing, although inhibition of caspase cascade activity markedly attenuated the apoptosis induced by ASA as measured by DNA-histone complex formation. Upstream PKC signaling induced by ASA seems to play an important role in the regulation of apoptosis because PKC inhibitors significantly reduced the magnitude of DNA-histone complex formation. We conclude that ASA-induced apoptosis in gastric cancer cells is mediated, at least in part, through a PKC mechanism involving the (alpha) and (beta) isoforms and that PKC signaling operates upstream of the caspase cascade, which when activated elicits its downstream effects on DNA degradation.

Green Tea Polyphenols Reduce Gastric Epithelial Cell Proliferation and Apoptosis Stimulated by Helicobacter pylori Infection.

Recently the finding of gastric cancer in Helicobacter pylori (H. pylori)-infected mouse models was reported. Studies of humans and animal models have shown that H. pylori infection stimulates gastric epithelial cell proliferation and apoptosis. Polyphenols contained in green tea and related compounds were reported to have a variety anti-tumor effects and bactericidal properties. We studied the effect of green tea polyphenols on gastric cell proliferation and apoptosis in an H. pylori-infected mouse model. This model was prepared by inoculating Balb/c mice with 108 cfu of H. pylori (NCTC 11637 strain) by gavage. Beginning 18 weeks after inoculation, 0.5% polyphenols were given in drinking water every day for 2 weeks. Mice were sacrificed 1 h after bromodeoxyuridine (BrdU) was given i.p. for preparation of paraffin-embedded specimens. Cell proliferation and apoptosis were examined by the avidin-biotin complex method using anti-BrdU antibody and the TUNEL method, respectively. H. pylori infection resulted in increased BrdU-labeled cells in both the antrum and the bodies. Administration of polyphenols suppressed this increased proliferation. H. pylori infection increased apoptotic cells in both the antrum and the corpus in comparison with controls. This increase was not seen in H. pylori-infected mice given polyphenols. We conclude the administration with polyphenols might suppress gastric carcinogenesis that is in part related to H. pylori infection.

Induction of G2/M arrest and apoptosis in human gastric epithelial AGS cells by aqueous extract of Agaricus blazei

Agaricus blazei is well known as a traditional medicinal mushroom and it has been shown to exhibit immunostimulatory and anti-cancer activity. However, the cellular and molecular mechanism of cell cycle arrest and apoptosis of cancer cells is poorly understood. In this study, we have investigated whether A. blazei extract (ABE) exerts anti-proliferative and apoptotic effects on human gastric epithelial AGS cells. It was found that ABE could inhibit cell growth in a dose-dependent manner, which was associated with the arrest of G2/M phase and the induction of apoptotic cell death. Flow cytometric analysis showed that ABE could cause an arrest at the G2/M phase of the cell cycle, which is closely correlated to decreased cyclin B1 and cdc2 levels. Furthermore, this compound induced apoptosis through up-regulation of Bax and the activation of caspases with down-regulation of XIAP and cIAP-1, but not cIAP-2, and a capase-3 inhibitor could block cell death and apoptotic body formation. These data clearly indicate that ABE-induced apoptosis is associated with caspase-3 activation. In summary, the growth inhibition of ABE is highly related to cell cycle arrest at the G2/M phase and the induction of caspase-3-dependent apoptosis in human gastric epithelial AGS cells.

Gastrin-induced apoptosis contributes to carcinogenesis in the stomach

Hypergastrinemia in INS-GAS mice leads to accelerated carcinogenesis of the stomach, but the mechanisms have not been well defined. We investigated the possible role of gastrin-induced gastric cell apoptosis in the development of gastric cancer. We examined apoptosis and the expression of Bcl-2 family proteins in INS-GAS mice of different ages, as well as in gastrin-deficient (GAS-KO) mice after gastrin-17 (G-17) infusion. In addition, we studied the effects of the gastrin/cholecystokinin-2 (CCK-2) receptor antagonist YF476 and/or histamine H2 (H-2) receptor antagonist loxtidine on apoptosis and atrophy in INS-GAS mice with or without Helicobacter felis (H. felis) infection. INS-GAS mice had age-associated increases in Bax protein expression and decreases in Bcl-2 protein expression, along with increased glandular and epithelial cell apoptosis. At 8-week gastrin infusions in GAS-KO mice resulted in a similar pattern of altered Bax and Bcl-2 expression, followed by gastric cell apoptosis. H. felis infection of INS-GAS mice led to increased apoptosis and the development of atrophy, whereas treatment with either YF476 and/or loxtidine strongly inhibited both apoptosis and atrophy. In vitro studies with Fas-expressing RGM1 cells showed that gastrin stimulation alone directly induced apoptosis via gastrin/CCK-2 receptor and synergized with FasL stimulation. These results indicate that gastrin can induce apoptosis in gastric epithelial cells and contribute to the development of gastric carcinogenesis.

Apoptosis in Helicobacter pylori Gastritis is Related to cagA Status

Helicobacter pylori infection increases gastric epithelial cell apoptosis; however, the influence of cagA status is still controversial. We aimed to investigate if cagA status is related to apoptosis in H. pylori gastritis at different anatomic sites of the gastric mucosa. We studied by immunohistochemistry (streptavidin-biotin method) pro-apoptotic (Bax and Bak) and antiapoptotic (Bcl-2 and Bcl-x) proteins expression, scored from 0 to 4, in gastric biopsies, at the antrum (lesser and greater curvatures), incisura, and corpus (greater curvature) from 50 patients with H. pylori gastritis (22 males, 28 females, median age 40 years) and eight non-infected patients (6 males, median age 39.6 years). H. pylori and cagA status were determined by polymerase chain reaction. Apoptotic proteins were expressed in a granular pattern, in the cytoplasm of foveolar cells; Bax and Bak expression was higher than Bcl-2 and Bcl-x in most cases and was significantly higher in patients infected by cagA-positive strains than in those infected by cagA-negative strains (p = .001). Bak expression was higher at the lesser curvature (antrum and incisura) than in the other regions (p = .002) and was correlated with atrophy. Anti-apoptotic proteins were significantly more expressed at the antral lesser curvature than in the other regions of the stomach (Bcl-2: p = .02; Bcl-x: p < .001). Infection with cagA-positive strains is significantly associated with overexpression of pro-apoptotic proteins in the gastric mucosa, mainly at the antral lesser curvature, which may have a role on atrophy development. Anti-apoptotic proteins were also overexpressed at the lesser curvature, which may occur to keep epithelial cell turnover or might be related to malignant transformation.

Uncoupling the Enzymatic and Autoprocessing Activities of Helicobacter pylori γ-Glutamyltranspeptidase

Gamma-glutamyltranspeptidase (gammaGT), a member of the N-terminal nucleophile hydrolase superfamily, initiates extracellular glutathione reclamation by cleaving the gamma-glutamyl amide bond of the tripeptide. This protein is translated as an inactive proenzyme that undergoes autoprocessing to become an active enzyme. The resultant N terminus of the cleaved proenzyme serves as a nucleophile in amide bond hydrolysis. Helicobacter pylori gamma-glutamyltranspeptidase (HpGT) was selected as a model system to study the mechanistic details of autoprocessing and amide bond hydrolysis. In contrast to previously reported gammaGT, large quantities of HpGT were expressed solubly in the inactive precursor form. The 60-kDa proenzyme was kinetically competent to form the mature 40- and 20-kDa subunits and exhibited maximal autoprocessing activity at neutral pH. The activated enzyme hydrolyzed the gamma-glutamyl amide bond of several substrates with comparable rates, but exhibited limited transpeptidase activity relative to mammalian gammaGT. As with autoprocessing, maximal enzymatic activity was observed at neutral pH, with hydrolysis of the acyl-enzyme intermediate as the rate-limiting step. Coexpression of the 20- and 40-kDa subunits of HpGT uncoupled autoprocessing from enzymatic activity and resulted in a fully active heterotetramer with kinetic constants similar to those of the wild-type enzyme. The specific contributions of a conserved threonine residue (Thr380) to autoprocessing and hydrolase activities were examined by mutagenesis using both the standard and coexpression systems. The results of these studies indicate that the gamma-methyl group of Thr380 orients the hydroxyl group of this conserved residue, which is required for both the processing and hydrolase reactions.

External membrane vesicles from Helicobacter pylori induce apoptosis in gastric epithelial cells

The Helicobacter pylori infection of gastric mucosa is one of the most common infectious diseases and is associated with a variety of clinical outcomes, including peptic ulcer disease and gastric cancer. Helicobacter pylori-induced damage to gastric mucosal cells is controlled by bacterial virulence factors, which include VacA and CagA. Outer membrane vesicles are constantly shed by the bacteria and can provide an additional mechanism for pathogenicity by releasing non-secretable factors which can then interact with epithelial cells. The present report shows that external membrane vesicles are able to induce apoptosis not mediated by mitochondrial pathway in gastric (AGS) epithelial cells, as demonstrated by the lack of cytochrome c release with an activation of caspase 8 and 3. Apoptosis induced by these vesicles does not require a classic VacA+ phenotype, as a negative strain with a truncated and therefore non-secretable form of this protein can also induce cell death. These results should be taken into account in future studies of H. pylori pathogenicity in strains apparently VacA-.

Helicobacter pylori infection, histopathological gastritis and gastric epithelial cell apoptosis in children

Abstract Aim: Features of gastritis and gastric epithelial cell apoptosis in children infected with Helicobacter pylori genotypes are seldom studied. Therefore, we investigated the relationship between vacA genotypes and the severity of gastritis, and gastric epithelial cell apoptosis in H. pylori‐infected children. Methods: Antral biopsies from 52 children infected with H. pylori vacA genotypes (s1a/m1 = 17, s1a/m2 = 21 and s2/m2 = 14) were analysed for severity of gastritis on histopathology. Fifteen biopsies infected with different vacA genotypes were studied for gastric epithelial cell apoptosis by terminal uridine deoxynucleotidyl nick‐end labelling. Results: Children infected with the s1a/m1 and s1a/m2 vacA genotypes had higher severity of chronic inflammation than the s2/m2 genotype (s1 a/m1 vs s2/m2, p=0.05; s1a/m2 vs s2/m2, p=0.01). The vacA s1a allele was more independently associated with severe chronic inflammation than the s2 allele (p=0.02). Children infected with the s1a/m1 and s1a/m2 strains had higher gastric epithelial cell apoptosis than the s2/m2 strain (s1a/m1 or s1a/m2 vs s2/m2, p&lt;0.0001). Conclusion: The s1a/m1 and s1a/m2 H. pylori vacA genotypes have significantly higher association with severe chronic gastritis and gastric epithelial cell apoptosis than the s2/m2 genotype in children. The role of H. pylori vacA genotypes and their allelic subtypes in relation to pathogenicity and disease potential in children needs further studies.

Helicobacter pylori infection, histopathological gastritis and gastric epithelial cell apoptosis in children

Features of gastritis and gastric epithelial cell apoptosis in children infected with Helicobacter pylori genotypes are seldom studied. Therefore, we investigated the relationship between vacA genotypes and the severity of gastritis, and gastric epithelial cell apoptosis in H. pylori-infected children. Antral biopsies from 52 children infected with H. pylorivacA genotypes (s1a/m1 = 17, s1a/m2 = 21 and s2/m2 = 14) were analysed for severity of gastritis on histopathology. Fifteen biopsies infected with different vacA genotypes were studied for gastric epithelial cell apoptosis by terminal uridine deoxynucleotidyl nick-end labelling. Children infected with the s1a/m1 and s1a/m2 vacA genotypes had higher severity of chronic inflammation than the s2/m2 genotype (s1a/m1 vs s2/m2, p=0.05; s1a/m2 vs s2/m2, p=0.01). The vacA s1a allele was more independently associated with severe chronic inflammation than the s2 allele (p=0.02). Children infected with the s1a/m1 and s1a/m2 strains had higher gastric epithelial cell apoptosis than the s2/m2 strain (s1a/m1 or s1a/m2 vs s2/m2, p<0.0001). The s1a/m1 and s1a/m2 H. pylorivacA genotypes have significantly higher association with severe chronic gastritis and gastric epithelial cell apoptosis than the s2/m2 genotype in children. The role of H. pylorivacA genotypes and their allelic subtypes in relation to pathogenicity and disease potential in children needs further studies.

Protein Kinase C Involvement in Deoxycholate-Induced Apoptosis in Human Gastric Cells

Bile acids, such as deoxycholic acid (DC), are known to mediate some of their actions by differentially activating various protein kinase C (PKC) isoforms. This study confirms that DC induces apoptosis in gastric epithelial cells through PARP and caspase cascade activation, and examined the role of PKC in DC-induced apoptosis. We found increased activation of PKC in membrane fractions in response to DC that was concentration and time related. The PKC (beta(I)) isoform expression increased with translocation into the cell membrane fraction after DC (300 microM) stimulation. In contrast, PKCepsilon expression markedly decreased in response to DC treatment in a time- and concentration-dependent manner. In addition, this process was regulated by caspases, since the pan-caspase inhibitor z-VAD-fmk and caspase-3-, -6-, and -9-specific inhibitors prevented PKC (beta(I)) and (epsilon epsilon processing induced by DC. Treatment with the caspase-8-specific inhibitor, however, did not affect expression of either PKC isoform. No significant differences in the apoptotic response were observed when PKC (epsilon) overexpressed cells were exposed to DC in the presence of calcium-dependent conventional PKC inhibitors (Gö 6850 or Gö 6976). Our findings demonstrate that PKC is activated in gastric epithelial cells treated with DC with the PKC (beta(I)) and PKC (epsilon) isoforms being particularly involved in this process. The processing of PKC (beta(I) and epsilon) was shown to be closely regulated by caspases; however, modulations in PKC isoform concentrations by themselves have no effect on the apoptotic death of gastric mucosal cells induced by DC.

RUNX3 Cooperates with FoxO3a to Induce Apoptosis in Gastric Cancer Cells

The transcription factor RUNX3, which mediates apoptosis and cell growth inhibition in gastric epithelial cells, is a candidate tumor suppressor that is frequently lost in gastric cancer cells. Here, we found that restoration of RUNX3 expression in the cell line not expressing RUNX3 induced apoptosis and that it physically interacted with the Forkhead transcription factor FoxO3a/FKHRL1, known to be an important regulator of apoptosis and the cell cycle. Active unphosphorylated FoxO3a/FKHRL1 was expressed in the gastric cancer cell lines. RUNX3-induced apoptosis depended on the expression of Bim, a proapoptotic BH3-only protein, and both RUNX3 and FoxO3a/FKHRL1 were required for induction of Bim expression. Furthermore, we showed that interaction of RUNX3 and FoxO3a/FKHRL1 was also indispensable for Bim expression and apoptosis in mouse embryonic fibroblasts. In the Bim promoter, RUNX3 bound to two conserved RUNX-binding elements (RBE1 and RBE2), with RBE1 being immediately downstream of a FoxO-binding element. The physical interaction of RUNX3 and FoxO3a/FKHRL1 on the Bim promoter activated transcription of Bim. These findings show that RUNX3 cooperates with FoxO3a/FKHRL1 to participate in the induction of apoptosis by activating Bim and may play an important role in tumor suppression in gastric cancer.

H pylorireceptor MHC class II contributes to the dynamic gastric epithelial apoptotic response

To investigate the role of MHC class II in the modulation of gastric epithelial cell apoptosis induced by H pylori infection. After stimulating a human gastric epithelial cell line with bacteria or agonist antibodies specific for MHC class II and CD95, the quantitation of apoptotic and anti-apoptotic events, including caspase activation, BCL-2 activation, and FADD recruitment, was performed with a fluorometric assay, a cytometric bead array, and confocal microscopy, respectively. Pretreatment of N87 cells with the anti-MHC class II IgM antibody RFD1 resulted in a reduction in global caspase activation at 24 h of H pylori infection. When caspase 3 activation was specifically measured, crosslinking of MHC class II resulted in a marked reduced caspase activation, while simple ligation of MHC class II did not. Crosslinking of MHC class II also resulted in an increased activation of the anti-apoptosis molecule BCL-2 compared to simple ligation. Confocal microscope analysis demonstrated that the pretreatment of gastric epithelial cells with a crosslinking anti-MHC class II IgM blocked the recruitment of FADD to the cell surface. The results presented here demonstrate that the ability of MHC class II to modulate gastric epithelial apoptosis is at least partially dependent on its crosslinking. Furthermore, while previous research has demonstrated that MHC class II signaling can be pro-apoptotic during extended ligation, we have shown that the crosslinking of this molecule has anti-apoptotic effects during the earlier time points of H pylori infection. This effect is possibly mediated by the ability of MHC class II to modulate the activation of the pro-apoptotic receptor Fas by blocking the recruitment of the accessory molecule FADD, and this delay in apoptosis induction could allow for prolonged cytokine secretion by H pylori-infected gastric epithelial cells.