Effect of inhibition of extracellular signal-regulated kinase 1 and 2 pathway on apoptosis and bcl-2 expression in Helicobacter pylori-infected AGS cells

Abstract

Helicobacter pylori induces activation of mitogen-activated protein kinases (MAPKs). However, its effect on H. pylori-induced apoptosis has not been evaluated. Thus, we examined whether H. pylori-induced extracellular signal-regulated kinase 1 and 2 (ERK1/2) and p38 MAPK activation affects gastric epithelial cell apoptosis and bcl-2 family gene expression, especially in relation to the cagA status of an H. pylori strain. In flow cytometric and oligonucleosome-bound DNA enzyme-linked immunosorbent assay analyses, infection with cagA(+) H. pylori strains induced gastric cancer cell apoptosis in AGS cells more prominently than infection with cagA mutants. Activation of ERK1/2 and p38 MAPKs was also more prominent in cagA(+) strains. Pretreatment with a MEK inhibitor (PD98059) inhibited ERK1/2 activation and increased H. pylori-induced apoptosis significantly. This increased apoptosis was accompanied by decreased antiapoptotic bcl-2 mRNA expression among bcl-2-related genes (bcl-2, bax, bak, mcl-1, and bcl-X(L/S)), and the effect was also more prominent in the cagA(+) strains. However, the alteration of bcl-2 gene expression was not accompanied by protein level changes. Inhibition of p38 using specific inhibitor SB203580 decreased H. pylori-induced apoptosis but resulted in little alteration of bcl-2-related gene expression. In conclusion, H. pylori-induced ERK1/2 activation, especially by the cagA(+) H. pylori strain, may play a protective role against gastric epithelial cell apoptosis partially through maintenance of bcl-2 gene expression.

FIG. 1.

H. pylori-induced ERK1/2 and p38 MAPK activation. AGS gastric epithelial cells were stimulated by H. pylori at a cell-to-bacterium ratio of 1:100. (A) Cell lysates were prepared at the designated time points after H. pylori strain 99 (cagA⁺, cytotoxin positive) infection. Western blot analysis was performed for phosphorylated ERK1/2 (P-ERK1/2), total ERK1/2, phosphorylated p38 (P-p38), and total p38. (B) AGS gastric epithelial cells were stimulated with H. pylori cagA mutants (strains 28 and 45) and cagA⁺ strains (strains 92 and 99) with or without pretreatment by MEK1/2 inhibitor PD98050 (20 μM) for 1 h. Cell lysates were prepared at 90 min after H. pylori infection. Western blot analysis was performed for phosphorylated ERK1/2 (upper panel) and total ERK1/2 (lower panel). (C) AGS gastric epithelial cells were stimulated with H. pylori cagA mutants (strains 28 and 45) and cagA⁺ strains (strains 92 and 99) with or without pretreatment by p38 inhibitor SB203580 (10 μM) for 1 h. Western blot analysis was performed for phosphorylated p38 (upper panel) and total p38 (lower panel). Representative data of three independent experiments are shown.

FIG. 2.

H. pylori-induced apoptosis in AGS gastric epithelial cells. Subconfluent monolayers of AGS gastric epithelial cells were cultured without or with H. pylori strain 99 (cagA⁺, cytotoxin positive). AGS cells were harvested at 16 h after H. pylori infection and incubated with FITC-conjugated annexin V (AV) and propidium iodide (PI) double staining. Flow cytometric analysis was performed, and the data shown are representative of three separate experiments. The lower right quadrants represent early apoptotic cells that were stained by annexin V but not by propidium iodide. The upper right quadrants represent late apoptotic cells that were stained by both annexin V and propidium iodide.

FIG. 3.

Augmentation of H. pylori-induced apoptosis by inhibition of ERK1/2 pathway. (A) H. pylori strain 99 (cagA⁺, cytotoxin positive) and a subconfluent monolayer of AGS cells were cocultured for 16 h with or without pretreatment of PD98059 (20 μM) or SB203580 (10 μM). Data represent early and total apoptotic cell proportions measured by flow cytometric analysis after staining with FITC-conjugated annexin V and propidium iodide. (B) Apoptosis of AGS cells was assessed by a cell death detection ELISA. Values refer to DNA fragmentation as measured by the enrichment factor. Values are expressed as means ± standard deviations of a triplicate experiment and are representative of three separate experiments. *, P < 0.05 (compared with results for H. pylori infection alone).

FIG. 4.

Decreased bcl-2 gene expression by inhibition of the ERK1/2 pathway. AGS epithelial cells were cocultured with H. pylori strain 99 (cagA⁺, cytotoxin positive) with or without pretreatment by MEK1/2 inhibitor PD98050 (20 μM) or p38 inhibitor SB203580 (10 μM) for 1 h. After 6 h of coculture, total cellular RNA was extracted and RT-PCR was performed for bcl-2 family genes. PCR products were separated on a 2% NuSieve agarose gel and stained with ethidium bromide.

FIG. 5.

Increased apoptosis by cagA⁺ H. pylori strains. AGS gastric epithelial cells were infected with H. pylori cagA mutants (strains 28 and 45) or cagA⁺ strains (strains 44, 92, and 99). H. pylori and a subconfluent monolayer of AGS cells were cocultured for 16 h with or without pretreatment by PD98059 (20 μM). (A) Early apoptotic cell proportions measured by flow cytometric analysis after staining with FITC-conjugated annexin V and propidium iodide. (B) Apoptosis of AGS cells was assessed by means of cell death detection ELISA. Values refer to DNA fragmentation as measured by the enrichment factor. Values are means ± standard deviations of triplicate experiments, and representative data of three independent experiments were shown. *, P < 0.05 (compared with each result for cells without PD98059 treatment).

FIG. 6.

Decreased bcl-2 gene expression by the inhibition of the ERK1/2 pathway, especially in cagA⁺ H. pylori strains. AGS gastric epithelial cells were infected with H. pylori cagA mutants (strains 28 and 45) or cagA⁺ strains (strains 44, 92, and 99) with or without pretreatment by PD98059 (20 μM). After 6 h of coculturing, total cellular RNA was extracted and RT-PCR was performed for bcl-2-related genes. PCR products were separated on a 2% NuSieve agarose gel and stained with ethidium bromide. Representative data of three separate experiments are shown.

FIG. 7.

cagA⁺ H. pylori strain-induced Bcl-2 protein expression. AGS gastric epithelial cells were stimulated with H. pylori cagA mutants (strains 28 and 45) and cagA⁺ strains (strains 92 and 99) at a cell-to-bacterium ratio of 1:100, with or without pretreatment by PD98059 (20 μM). Cell lysates were prepared 16 h after H. pylori infection. Western blot analysis was performed for Bcl-2 family proteins. These experiments were performed on three occasions, and representative blots are shown.