H pylori stimulates proliferation of gastric cancer cells through activating mitogen-activated protein kinase cascade

Abstract

Aim: To explore the mechanism by which H pylori causes activation of gastric epithelial cells.

Methods: A VacA (+) and CagA (+) standard H pylori line NCTC 11637 and a human gastric adenocarcinoma derived gastric epithelial cell line BGC-823 were applied in the study. MTT assay and (3)H-TdR incorporation test were used to detect the proliferation of BGC-823 cells and Western blotting was used to detect the activity and existence of related proteins.

Results: Incubation with H pylori extract increased the proliferation of gastric epithelial cells, reflected by both live cell number and DNA synthesis rate. The activity of extracellular signal-regulated protein kinase (ERK) signal transduction cascade increased within 20 min after incubation with H pylori extract and appeared to be a sustained event. MAPK/ERK kinase (MEK) inhibitor PD98059 abolished the action of H pylori extract on both ERK activity and cell proliferation. Incubation with H pylori extract increased c-Fos expression and SRE-dependent gene expression. H pylori extract caused phosphorylation of several proteins including a protein with molecular size of 97.4 kDa and tyrosine kinase inhibitor genistein inhibited the activation of ERK and the proliferation of cells caused by H pylori extract.

Conclusion: Biologically active elements in H pylori extract cause proliferation of gastric epithelial cells through activating tyrosine kinase and ERK signal transduction cascade.

Figure 1

MTT assay (A) and ³H-TdR-incorporation test (B) showing H pylori extract- stimulated proliferation of BGC-823 cells. ^bP < 0.01 vs control.

Figure 2

Western blotting showing phosphorylated ERK (A) and total ERK (B) in serum-starved BGC-823 cells incubated with H pylori extract. M: Protein molecular marker; 0: Control; 1-7: Incubation with 50 mg/L H pylori extract for 20, 40, 60 min and 3, 6, 12, 24 h.

Figure 3

Western blotting showing PD98059-blocked stimulating effect of H pylori extract on ERK activation in BGC-823 cells. M: Molecular marker; C: Control; Hp: 50 mg/L H pylori extract; PD1: 25 μmol/L PD98059 + 50 mg/L H pylori extract; PD2: 50 μmol/L PD98059 + 50 mg/L H pylori extract.

Figure 4

MTT assay showing PD98059-blocked proliferation-stimulating effect of H pylori extract. ^aP < 0.05, ^cP < 0.05, ^dP < 0.01 vs control only; ^bP < 0.01 vs Hp only.

Figure 5

Western blotting showing genistein-prevented ERK activation by H pylori extract. M: Molecular marker; C: Control; Hp: H pylori extract; Hp + G: Genistein and H pylori extract.

Figure 6

Western blotting showing H pylori extract-caused tyrosine pho-sphorylation of cell lysate of BGC-823. M: Molecular marker; 1: control; 2-7: H pylori extract incubated for 20, 40 min and 1, 3, 6, 12 h.

Figure 7

Western blotting showing H pylori extract- increased expression of c-Fos. 1: Control; 2-8: H pylori extract incubated for 20, 40 min and 1, 3, 6, 12 and 24 h.

Figure 8

H pylori extract-stimulated SRE-dependent reporter gene expression. ^aP < 0.05 vs control.