Yonsei Med J.  2015 Jul;56(4):1150-1154. 10.3349/ymj.2015.56.4.1150.

Diphenyleneiodonium Inhibits Apoptotic Cell Death of Gastric Epithelial Cells Infected with Helicobacter pylori in a Korean Isolate

• Cho SO
• Lim JW
• Kim H

• KMID: 2366360
• DOI: http://doi.org/10.3349/ymj.2015.56.4.1150

Abstract

NADPH oxidase produces a large amount of reactive oxygen species (ROS) in Helicobacter pylori (H. pylori)-induced gastric epithelial cells. Even though ROS mediate apoptotic cell death, direct involvement of NADPH oxidase on H. pylori-induced apoptosis remains unclear. Besides, H. pylori isolates show a high degree of genetic variability. The predominant genotype of H. pylori in Korea has been reported as cagA+, vacA s1b, m2, iceA genotype. Present study aims to investigate whether NADPH oxidase-generated ROS mediate apoptosis in human gastric epithelial AGS cells infected with H. pylori in a Korean isolate. AGS cells were pretreated with or without an NADPH oxidase inhibitor diphenyleneiodonium (DPI) and cultured in the presence of H. pylori at a bacterium/cell ratio of 300:1. Cell viability, hydrogen peroxide level, DNA fragmentation, and protein levels of p53, Bcl-2, and Bax were determined. Results showed that H. pylori inhibited cell viability with the density of H. pylori added to the cells. Inhibition of NADPH oxidase by DPI suppressed H. pylori-induced cell death, increased hydrogen peroxide, DNA fragmentation, and the ratio of Bax/Bcl-2, and p53 induction in AGS cells dose-dependently. The results suggest that targeting NADPH oxidase may prevent the development of gastric inflammation associated with H. pylori infection by suppressing abnormal apoptotic cell death of gastric epithelial cells.

Keyword

Helicobacter pylori; diphenyleneiodonium; NADPH oxidase; apoptosis; gastric epithelial cells

MeSH Terms

Apoptosis
Apoptosis Regulatory Proteins/metabolism
Cell Survival
Epithelial Cells/metabolism/microbiology
Gastric Mucosa/metabolism
Helicobacter Infections/*metabolism/microbiology
Helicobacter pylori/drug effects/genetics/*isolation & purification
Humans
NADPH Oxidase/metabolism
Onium Compounds/*antagonists & inhibitors/pharmacology
Oxidative Stress/drug effects
Reactive Oxygen Species/metabolism
Republic of Korea
Stomach/cytology/*metabolism/microbiology
Apoptosis Regulatory Proteins
NADPH Oxidase
Onium Compounds
Reactive Oxygen Species

Figure

• 

  Fig. 1 Cell viability, hydrogen peroxide level, DNA fragmentation, and apoptotic indices of H. pylori-infected AGS cells treated with or without DPI. Prior to the experiment, the cells (1×105/mL/well) were cultured in the presence of H. pylori at a bacterium/cell ratio of 100:1, 300:1, and 500:1 for 24 h (A). For the following study on DPI, the cells were pre-treated with DPI (2.5 or 5 μM) for 2 h and infected with H. pylori at a bacterium/cell ratio of 300:1 for 2 h (hydrogen peroxide level) (B), 12 h (protein levels of p53, Bcl-2, and Bax) (E and F), and 24 h [DNA fragmentation (D) and cell viability (C)]. The values are expressed as means±SE of four different experiments. *p<0.05 versus none control (the cells without DPI and cultured in the absence of H. pylori), †p<0.05 versus H. pylori control (the cells without DPI and cultured in the presence of H. pylori). AGS, gastric adenocarcinoma; DPI, diphenyleneiodonium; H. pylori, Helicobacter pylori.

Reference

1. Clément MV, Pervaiz S. Reactive oxygen intermediates regulate cellular response to apoptotic stimuli: an hypothesis. Free Radic Res. 1999; 30:247–252.

2. Davies GR, Simmonds NJ, Stevens TR, Sheaff MT, Banatvala N, Laurenson IF, et al. Helicobacter pylori stimulates antral mucosal reactive oxygen metabolite production in vivo. Gut. 1994; 35:179–185.

3. Nagata K, Yu H, Nishikawa M, Kashiba M, Nakamura A, Sato EF, et al. Helicobacter pylori generates superoxide radicals and modulates nitric oxide metabolism. J Biol Chem. 1998; 273:14071–14073.

4. Bagchi D, Bhattacharya G, Stohs SJ. Production of reactive oxygen species by gastric cells in association with Helicobacter pylori. Free Radic Res. 1996; 24:439–450.

5. Kawahara T, Kohjima M, Kuwano Y, Mino H, Teshima-Kondo S, Takeya R, et al. Helicobacter pylori lipopolysaccharide activates Rac1 and transcription of NADPH oxidase Nox1 and its organizer NOXO1 in guinea pig gastric mucosal cells. Am J Physiol Cell Physiol. 2005; 288:C450–C457.

6. Cha B, Lim JW, Kim KH, Kim H. HSP90beta interacts with Rac1 to activate NADPH oxidase in Helicobacter pylori-infected gastric epithelial cells. Int J Biochem Cell Biol. 2010; 42:1455–1461.

7. Cho SO, Lim JW, Kim KH, Kim H. Diphenyleneiodonium inhibits the activation of mitogen-activated protein kinases and the expression of monocyte chemoattractant protein-1 in Helicobacter pylori-infected gastric epithelial AGS cells. Inflamm Res. 2011; 60:501–507.

8. Jang SH, Lim JW, Morio T, Kim H. Lycopene inhibits Helicobacter pylori-induced ATM/ATR-dependent DNA damage response in gastric epithelial AGS cells. Free Radic Biol Med. 2012; 52:607–615.

9. Labigne A, de Reuse H. Determinants of Helicobacter pylori pathogenicity. Infect Agents Dis. 1996; 5:191–202.

10. Mobley HL. Defining Helicobacter pylori as a pathogen: strain heterogeneity and virulence. Am J Med. 1996; 100(5A):2S–9S.

11. Seo JH, Lim JW, Kim H, Kim KH. Helicobacter pylori in a Korean isolate activates mitogen-activated protein kinases, AP-1, and NF-kappaB and induces chemokine expression in gastric epithelial AGS cells. Lab Invest. 2004; 84:49–62.

12. Kim MH, Kim A, Yu JH, Lim JW, Kim H. Glutamine deprivation induces interleukin-8 expression in ataxia telangiectasia fibroblasts. Inflamm Res. 2014; 63:347–356.

13. Cho SO, Kim KH, Yoon JH, Kim H. Signaling for integrin alpha5/beta1 expression in Helicobacter pylori-infected gastric epithelial AGS cells. Ann N Y Acad Sci. 2006; 1090:298–304.

14. Tominaga K, Kawahara T, Sano T, Toida K, Kuwano Y, Sasaki H, et al. Evidence for cancer-associated expression of NADPH oxidase 1 (Nox1)-based oxidase system in the human stomach. Free Radic Biol Med. 2007; 43:1627–1638.

15. Keenan JI, Peterson RA 2nd, Hampton MB. NADPH oxidase involvement in the pathology of Helicobacter pylori infection. Free Radic Biol Med. 2005; 38:1188–1196.

16. Jones RD, Hancock JT, Morice AH. NADPH oxidase: a universal oxygen sensor? Free Radic Biol Med. 2000; 29:416–424.

17. Mori M, Suzuki H, Suzuki M, Kai A, Miura S, Ishii H. Catalase and superoxide dismutase secreted from Helicobacter pylori. Helicobacter. 1997; 2:100–105.

18. Arslan D, Kose K, Patiroglu TE. Is there an oxidative stress in children with Helicobacter pylori infection? Saudi Med J. 2007; 28:1222–1226.

19. Morishita K, Takeuchi H, Morimoto N, Shimamura T, Kadota Y, Tsuda M, et al. Superoxide dismutase activity of Helicobacter pylori per se from 158 clinical isolates and the characteristics. Microbiol Immunol. 2012; 56:262–272.

20. Yuan X, Zhou Y, Wang W, Li J, Xie G, Zhao Y, et al. Activation of TLR4 signaling promotes gastric cancer progression by inducing mitochondrial ROS production. Cell Death Dis. 2013; 4:e794.

21. Bauer G, Bereswill S, Aichele P, Glocker E. Helicobacter pylori protects oncogenically transformed cells from reactive oxygen species-mediated intercellular induction of apoptosis. Carcinogenesis. 2014; 35:1582–1591.