ABSTRACT
This study aimed to investigate the effect of Helicobacter pylori (H. pylori) infection on the sonic Hedgehog (Shh) signaling in gastric cancer. Shh, Patched (Ptch), and transcription factor Gli1 were overexpressed in H. pylori-infected gastric cancer cells. The oncoprotein, CagA positive H. pylori resulted in significantly higher Shh expression. Pretreatment with MG-132 or PDTC significantly lowered Shh expression. Significant overexpression of Shh and Gli1 were noted in H. pylori-infected compared to non-infected gastric cancer tissues. Conclusively, H. pylori activated the Shh signaling pathway in CagA-dependent manner partly through the NF-kappaB pathway in gastric cancer cells.
Subject(s)
Hedgehog Proteins/metabolism , Helicobacter Infections/metabolism , Helicobacter pylori/pathogenicity , Receptors, Cell Surface/metabolism , Stomach Neoplasms/metabolism , Transcription Factors/metabolism , Cell Line, Tumor , Hedgehog Proteins/genetics , Helicobacter Infections/microbiology , Helicobacter Infections/pathology , Humans , Immunoblotting , Patched Receptors , Patched-1 Receptor , RNA, Messenger/genetics , Receptors, Cell Surface/genetics , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction , Stomach Neoplasms/microbiology , Stomach Neoplasms/pathology , Transcription Factors/genetics , Zinc Finger Protein GLI1ABSTRACT
The Helicobacter pylori protein CagA may undergo tyrosine phosphorylation following its entry into human gastric epithelial cells with downstream effects on signal transduction. Disruption of the gp130 receptor that modulates the balance of the SHP2/ERK and JAK/STAT pathways enhanced peptic ulceration and gastric cancer in gp130 knock-out mice. In this study, we evaluated the effect of translocated CagA in relation to its tyrosine phosphorylation status on the gp130-mediated signal switch between the SHP2/ERK and JAK/STAT3 pathways. We showed that in the presence of CagA, SHP2 was recruited to gp130. Phosphorylated CagA showed enhanced SHP2 binding activity and ERK1/2 phosphorylation, whereas unphosphorylated CagA showed preferential STAT3 activation. These findings indicate that the phosphorylation status of CagA affects the signal switch between the SHP2/ERK and JAK/STAT3 pathways through gp130, providing a novel mechanism to explain H. pylori signaling.
Subject(s)
Antigens, Bacterial/metabolism , Bacterial Proteins/metabolism , Cytokine Receptor gp130/metabolism , Epithelial Cells/metabolism , Helicobacter pylori/metabolism , Janus Kinases/metabolism , MAP Kinase Signaling System , Mitogen-Activated Protein Kinase 3/metabolism , Protein Tyrosine Phosphatase, Non-Receptor Type 11/metabolism , STAT3 Transcription Factor/metabolism , Animals , Antigens, Bacterial/genetics , Bacterial Proteins/genetics , Cells, Cultured , Cytokine Receptor gp130/genetics , Epithelial Cells/microbiology , Gastric Mucosa/metabolism , Helicobacter pylori/genetics , Humans , Janus Kinases/genetics , Mice , Mice, Knockout , Mitogen-Activated Protein Kinase 3/genetics , Phosphorylation/genetics , Protein Tyrosine Phosphatase, Non-Receptor Type 11/genetics , STAT3 Transcription Factor/genetics , Stomach/microbiologyABSTRACT
BACKGROUND AND AIM: Capsaicin, the main pungent ingredient of hot red and chilli pepper, has been considered as not only a cytoprotective but also a detrimental agent to the gastric mucosa. However, the effect and mechanism of capsaicin that modulate the induction of pro-inflammatory cytokine in Helicobacter pylori-infected epithelial cells have not been investigated previously. Herein, we demonstrated that capsaicin inhibited the release of pro-inflammatory cytokine, interleukin-8 (IL-8) by H. pylori-infected gastric epithelial cells through nuclear factor-kappaB (NF-kappaB) signal pathway. MATERIALS AND METHODS: AGS or MKN45 cells as gastric epithelial cells and Vac A+, CagA+ wild-type H. pylori strain ATCC 49503 were used. Gastric epithelial cells were pre-treated with various concentrations of capsaicin and infected with H. pylori for different periods of time to determine IL-8 concentrations in culture supernatant by an ELISA assay. We measured IL-8 mRNA transcripts in H. pylori-infected gastric epithelial cells co-treated with capsaicin by reverse transcriptase-polymerase chain reaction analysis. We performed electrophoretic mobility shift assay to examine the NF-kappaB DNA binding activity with capsaicin and immunofluorescence microscopy to examine nuclear staining of p65. We also performed immunoblotting for IkappaB, IKK activity with capsaicin. RESULTS: Capsaicin inhibits H. pylori-induced IL-8 production by gastric epithelial cells in dose- and time-dependent manner. Capsaicin as low as 100 micromol/L significantly inhibited IL-8 production in H. pylori-infected MKN45 cells (43.2% of control) at 24 hours incubation, whereas inhibited IL-8 production in H. pylori-infected AGS cells (70% of control). We confirmed that capsaicin inhibited IL-8 mRNA expression after infection of gastric epithelial cells with H. pylori for 6 hours. The addition of capsaicin (100 micromol/L) suppressed H. pylori-induced NF-kappaB activation in gastric epithelial cells at 1 hour post-infection. We also found that the degradation of IkappaB and IKK activation were inhibited by capsaicin. CONCLUSIONS: Nontoxic dose of capsaicin inhibited H. pylori-induced IL-8 production by gastric epithelial cells through the modulation of IkappaB-, NF-kappaB-, and IL-8 pathways. We conclude that capsaicin can be proposed as a potential anti-inflammatory drug by inhibition of the production of IL-8 in H. pylori-infected gastric epithelium.
