α-Lipoic acid inhibits Helicobacter pylori-induced oncogene expression and hyperproliferation by suppressing the activation of NADPH oxidase in gastric epithelial cells

Abstract

Hyperproliferation and oncogene expression are observed in the mucosa of Helicobacter pylori- (H. pylori-) infected patients with gastritis or adenocarcinoma. Expression of oncogenes such as β-catenin and c-myc is related to oxidative stress. α-Lipoic acid (α-LA), a naturally occurring thiol compound, acts as an antioxidant and has an anticancer effect. The aim of this study is to investigate the effect of α-LA on H. pylori-induced hyperproliferation and oncogene expression in gastric epithelial AGS cells by determining cell proliferation (viable cell numbers, thymidine incorporation), levels of reactive oxygen species (ROS), NADPH oxidase activation (enzyme activity, subcellular levels of NADPH oxidase subunits), activation of redox-sensitive transcription factors (NF-κB, AP-1), expression of oncogenes (β-catenin, c-myc), and nuclear localization of β-catenin. Furthermore, we examined whether NADPH oxidase mediates oncogene expression and hyperproliferation in H. pylori-infected AGS cells using treatment of diphenyleneiodonium (DPI), an inhibitor of NADPH oxidase. As a result, α-LA inhibited the activation of NADPH oxidase and, thus, reduced ROS production, resulting in inhibition on activation of NF-κB and AP-1, induction of oncogenes, nuclear translocation of β-catenin, and hyperproliferation in H. pylori-infected AGS cells. DPI inhibited H. pylori-induced activation of NF-κB and AP-1, oncogene expression and hyperproliferation by reducing ROS levels in AGS cells. In conclusion, we propose that inhibiting NADPH oxidase by α-LA could prevent oncogene expression and hyperproliferation occurring in H. pylori-infected gastric epithelial cells.

Figure 1

Cell proliferation of AGS cells infected with H. pylori. AGS cells were cultured in the presence or absence of H. pylori (at bacterium/cell ratio of 10, 20, 50 : 1). Cell proliferation was assessed by viable cell numbers and [³H] thymidine incorporation. (a) Viable cell numbers were determined by trypan blue exclusion assay at the indicated time period. (b) The cells were treated with [³H] thymidine at 24 h after H. pylori infection and incubated for 8 h. The amount of [³H] thymidine incorporation of AGS cells cultured in the absence of H. pylori is considered as 100%. *P < 0.05 versus corresponding none (the cells cultured in the absence of H. pylori).

Figure 2

ROS levels, NADPH oxidase activity, and mRNA and protein levels of β-catenin and c-myc in AGS cells infected with H. pylori. (a, b, d, f) The cells were cultured in the presence or absence of H. pylori (at bacterium/cell ratio of 10, 20, 50 : 1) for 24 h. (c, e) AGS cells were cultured in the presence or absence of H. pylori (bacterium/cell ratio of 50 : 1) for the indicated time period. (a) ROS levels were determined by DCF fluorescence after 30 min of H. pylori infection. The levels of ROS trapped in the cells and cultured in the absence of H. pylori (none) are considered as 100%. (b) NADPH oxidase activity was determined by lucigenin assay using membrane extracts of the cells. NADPH oxidase activity was also monitored by addition of cytosolic extracts to the reaction mixture as a negative control. (c–f) mRNA and protein expression were determined by real-time PCR and Western blot analysis, respectively. Actin served as a loading control. *P < 0.05 versus 0 h or none (the cells cultured in the absence of H. pylori).

Figure 3

Effect of α-LA on cell proliferation of H. pylori-infected AGS cells. The cells were pretreated with α-LA for 2 h and cultured in the presence or absence of H. pylori. Cell proliferation was assessed by viable cell numbers and [³H] thymidine incorporation. (a) Viable cell numbers were determined by trypan blue exclusion assay for the indicated time period. (b) The cells were treated with [³H] thymidine at 24 h after H. pylori infection and incubated for 8 h. The amount of [³H] thymidine incorporation of AGS cells cultured in the absence of H. pylori is considered as 100%. *P < 0.05 versus corresponding none (the cells cultured in the absence of H. pylori); ⁺ P < 0.05 versus corresponding H. pylori control (the cells cultured in the presence of H. pylori and treated without α-LA).

Figure 4

Effect of α-LA on ROS levels, NADPH oxidase activity, and levels of NADPH oxidase subunits in H. pylori-infected AGS cells. The cells were pretreated with α-LA for 2 h and cultured in the presence or absence of H. pylori. (a) ROS levels were determined by DCF fluorescence after 30 min of H. pylori infection. The levels of ROS trapped in the cells treated without DPI and cultured in the absence of H. pylori are considered as 100%. (b) NADPH oxidase activity was determined by lucigenin assay using membrane extracts of the cells. NADPH oxidase activity was also monitored by addition of cytosolic extracts to the reaction mixture as a negative control. (c), Protein levels of NADPH oxidase subunits (p47, p67) in cytosolic and membrane extracts were determined by Western blotting. Aldolase A and Nox1 were used as indices for cytosol and membrane, respectively. *P < 0.05 versus none (the cells cultured in the absence of H. pylori); ⁺ P < 0.05 versus control (the cells cultured in the presence of H. pylori and treated without α-LA).

Figure 5

Effect of α-LA on expression of β-catenin and c-myc as well as activation of NF-κB and AP-1 in H. pylori-infected AGS cells. The cells were pretreated with α-LA for 2 h and cultured in the presence or absence of H. pylori for 24 h (mRNA, protein levels) or 1 h (activation of NF-κB and AP-1). (a) mRNA expression of β-catenin and c-myc was measured by real-time PCR analysis. (b) Protein levels of β-catenin and c-myc were determined by Western blot analysis. Actin served as a loading control. (c) EMSA was performed for DNA binding activities of NF-κB and AP-1. *P < 0.05 versus none (the cells cultured in the absence of H. pylori); ⁺ P < 0.05 versus control (the cells cultured in the presence of H. pylori and treated without α-LA).

Figure 6

Effect of α-LA on activation of β-catenin in H. pylori-infected AGS cells. AGS cells were pretreated with α-LA for 2 h and cultured in the presence or absence of H. pylori for 24 h. (a) Protein levels of β-catenin in cytosolic and nuclear extracts were determined by Western blot analysis. Aldolase A and histone H1 were used as indices for cytosolic and nuclear extracts, respectively. (b) Immunofluorescence staining was performed to determine the levels of β-catenin in nucleus and cytosol. β-catenin was visualized using fluorescein isothiocyanate-conjugated anti-mouse IgG antibody (lower panel) with DAPI counter staining (upper panel) of the same field.

Figure 7

Effect of DPI on ROS levels and cell proliferation of H. pylori-infected AGS cells. The cells were pretreated with DPI for 2 h and cultured in the presence or absence of H. pylori. (a) ROS levels were determined by DCF fluorescence after 30 min of H. pylori infection. The levels of ROS trapped in the cells treated without DPI and cultured in the absence of H. pylori are considered as 100%. (b) Viable cell numbers were determined by trypan blue exclusion assay for the indicated time period. (c) The cells were treated with [³H] thymidine at 24 h after H. pylori infection and incubated for 8 h. The amount of [³H] thymidine incorporation of AGS cells cultured in the absence of H. pylori is considered as 100%. *P < 0.05 versus corresponding none (the cells cultured in the absence of H. pylori); ⁺ P < 0.05 versus corresponding H. pylori control (the cells cultured in the presence of H. pylori and treated without DPI).

Figure 8

Effect of DPI on expression of β-catenin and c-myc as well as activation of NF-κB and AP-1 in H. pylori-infected AGS cells. The cells were pretreated with DPI for 2 h and cultured in the presence or absence of H. pylori for 24 h (mRNA, protein levels) or 1 h (activation of NF-κB and AP-1). (a) mRNA expression of β-catenin and c-myc was measured by real-time PCR analysis. (b) Protein levels of β-catenin and c-myc were determined by Western blot analysis. Actin served as a loading control. (c), EMSA was performed for DNA binding activities of NF-κB and AP-1. *P < 0.05 versus corresponding none (the cells cultured in the absence of H. pylori); ⁺ P < 0.05 versus corresponding H. pylori control (the cells cultured in the presence of H. pylori and treated without DPI).