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Digestive Diseases and Sciences

, Volume 57, Issue 10, pp 2615–2624 | Cite as

Effects of Helicobacter pylori γ-Glutamyltranspeptidase on Apoptosis and Inflammation in Human Biliary Cells

  • Wongwarut Boonyanugomol
  • Chariya Chomvarin
  • Jea-Young Song
  • Kyung-Mi Kim
  • Jung-Min Kim
  • Myung-Je Cho
  • Woo-Kon Lee
  • Hyung-Lyun Kang
  • Kwang-Ho Rhee
  • Banchob Sripa
  • Chariya Hahnvajanawong
  • Seung-Chul Baik
Original Article

Abstract

Background

Several studies have reported the presence of H. pylori in individuals with hepatobiliary diseases, but in vitro and in vivo studies are still needed. Here, we determined the effects of H. pylori γ-glutamyltranspeptidase (GGT) on the induction of apoptosis and IL-8 production in a human cholangiocarcinoma cell line (KKU-100 cells).

Methods

Cell viability and DNA synthesis were examined by MTT and BrdU assays, respectively. RT-PCR and western blot analysis were performed to assess gene and protein expression, respectively. IL-8 secretion in KKU-100 cells was measured by ELISA.

Results

Exposure to the H. pylori ggt + strain decreased KKU-100 cell survival and DNA synthesis when compared with cells exposed to the H. pylori ggt mutant strain. Treatment with recombinant H. pylori GGT (rHP-GGT) dramatically decreased cell survival and DNA synthesis, and stimulated apoptosis; these features corresponded to an increased level of iNOS gene expression in KKU-100 cells treated with rHP-GGT. RT-PCR and western blot analyses revealed that rHP-GGT treatment enhanced the expression of pro-apoptotic molecules (Bax, Caspase-9, and Caspase-3) and down-regulated the expression of anti-apoptotic molecules (Bcl-2 and Bcl-xL). The extrinsic-mediated apoptosis molecules, including Fas and activated Caspase-8, were not expressed after treatment with rHP-GGT. Furthermore, rHP-GGT significantly stimulated IL-8 secretion in KKU-100 cells.

Conclusion

Our data indicate that H. pylori GGT might be involved in the development of cancer in hepatobiliary cells by altering cell kinetics and promoting inflammation.

Keywords

H. pylori γ-Glutamyltranspeptidase Apoptosis Hepatobiliary diseases Cholangiocarcinoma 

Notes

Acknowledgments

We would like to thank the Commission on Higher Education, Thailand, for supporting this research with a grant funds under the Strategic Scholarships for Frontier Research Network for the Joint Ph.D. Program Thai Doctoral degree. We would also like to thank Khon Kaen University and the National R&D Program for Cancer Control, Ministry for Health, Welfare and Family affairs, Republic of Korea (0820050) for supporting some parts of this work. We would further like to thank the Liver Fluke and Cholangiocarcinoma Research Center, Khon Kaen University for providing the cell line.

Conflict of interest

We declare that we have no conflicts of interest.

References

  1. 1.
    Shin HR, Oh JK, Masuyer E, et al. Comparison of incidence of intrahepatic and extrahepatic cholangiocarcinoma—focus on East and South-eastern Asia. Asian Pac J Cancer Prev. 2010;11:1159–1166.PubMedGoogle Scholar
  2. 2.
    Sripa B, Kaewkes S, Sithithaworn P, et al. Liver fluke induces cholangiocarcinoma. PLoS Med. 2007;4:e201.PubMedCrossRefGoogle Scholar
  3. 3.
    Pellicano R, Menard A, Rizzetto M, Megraud F. Helicobacter species and liver diseases: association or causation? Lancet Infect Dis. 2008;8:254–260.PubMedCrossRefGoogle Scholar
  4. 4.
    Kusters JG, van Vliet AH, Kuipers EJ. Pathogenesis of Helicobacter pylori infection. Clin Microbiol Rev. 2006;19:449–490.PubMedCrossRefGoogle Scholar
  5. 5.
    Pellicano R, Mazzaferro V, Grigioni WF, et al. Helicobacter species sequences in liver samples from patients with and without hepatocellular carcinoma. World J Gastroenterol. 2004;10:598–601.PubMedGoogle Scholar
  6. 6.
    Xuan SY, Li N, Qiang X, Zhou RR, Shi YX, Jiang WJ. Helicobacter infection in hepatocellular carcinoma tissue. World J Gastroenterol. 2006;12:2335–2340.PubMedGoogle Scholar
  7. 7.
    Hamada T, Yokota K, Ayada K, et al. Detection of Helicobacter hepaticus in human bile samples of patients with biliary disease. Helicobacter. 2009;14:545–551.PubMedCrossRefGoogle Scholar
  8. 8.
    Murata H, Tsuji S, Tsujii M, et al. Helicobacter bilis infection in biliary tract cancer. Aliment Pharmacol Ther. 2004;20:90–94.PubMedCrossRefGoogle Scholar
  9. 9.
    Huang Y, Fan XG, Wang ZM, Zhou JH, Tian XF, Li N. Identification of helicobacter species in human liver samples from patients with primary hepatocellular carcinoma. J Clin Pathol. 2004;57:1273–1277.PubMedCrossRefGoogle Scholar
  10. 10.
    Boonyanugomol W, Chomvarin C, Sripa B, et al. Helicobacter pylori in Thai patients with cholangiocarcinoma and its association with biliary inflammation and proliferation. HPB (Oxford). 2012;14:177–184.CrossRefGoogle Scholar
  11. 11.
    Kawahara T, Teshima S, Kuwano Y, Oka A, Kishi K, Rokutan K. Helicobacter pylori lipopolysaccharide induces apoptosis of cultured guinea pig gastric mucosal cells. Am J Physiol Gastrointest Liver Physiol. 2001;281:G726–G734.PubMedGoogle Scholar
  12. 12.
    Kuck D, Kolmerer B, Iking-Konert C, Krammer PH, Stremmel W, Rudi J. Vacuolating cytotoxin of Helicobacter pylori induces apoptosis in the human gastric epithelial cell line AGS. Infect Immun. 2001;69:5080–5087.PubMedCrossRefGoogle Scholar
  13. 13.
    Fan X, Gunasena H, Cheng Z, et al. Helicobacter pylori urease binds to class II MHC on gastric epithelial cells and induces their apoptosis. J Immunol. 2000;165:1918–1924.PubMedGoogle Scholar
  14. 14.
    Jones NL, Shannon PT, Cutz E, Yeger H, Sherman PM. Increase in proliferation and apoptosis of gastric epithelial cells early in the natural history of Helicobacter pylori infection. Am J Pathol. 1997;151:1695–1703.PubMedGoogle Scholar
  15. 15.
    Shimada T, Terano A. Chemokine expression in Helicobacter pylori-infected gastric mucosa. J Gastroenterol. 1998;33:613–617.PubMedCrossRefGoogle Scholar
  16. 16.
    Shibayama K, Kamachi K, Nagata N, et al. A novel apoptosis-inducing protein from Helicobacter pylori. Mol Microbiol. 2003;47:443–451.Google Scholar
  17. 17.
    Ito K, Yamaoka Y, Yoffe B, Graham DY. Disturbance of apoptosis and DNA synthesis by Helicobacter pylori infection of hepatocytes. Dig Dis Sci. 2008;53:2532–2540.PubMedCrossRefGoogle Scholar
  18. 18.
    Boonyanugomol W, Chomvarin C, Baik SC, et al. Role of cagA-positive Helicobacter pylori on cell proliferation, apoptosis, and inflammation in biliary cells. Dig Dis Sci. 2011;56:1682–1692.PubMedCrossRefGoogle Scholar
  19. 19.
    Kim KM, Lee SG, Park MG, et al. Gamma-glutamyltranspeptidase of Helicobacter pylori induces mitochondria-mediated apoptosis in AGS cells. Biochem Biophys Res Commun. 2007;355:562–567.PubMedCrossRefGoogle Scholar
  20. 20.
    Perfetto B, Buommino E, Canozo N, et al. Interferon-gamma cooperates with Helicobacter pylori to induce iNOS-related apoptosis in AGS gastric adenocarcinoma cells. Res Microbiol. 2004;155:259–266.PubMedCrossRefGoogle Scholar
  21. 21.
    Greenlund LJ, Korsmeyer SJ, Johnson EM Jr. Role of BCL-2 in the survival and function of developing and mature sympathetic neurons. Neuron. 1995;15:649–661.PubMedCrossRefGoogle Scholar
  22. 22.
    Winter RN, Kramer A, Borkowski A, Kyprianou N. Loss of caspase-1 and caspase-3 protein expression in human prostate cancer. Cancer Res. 2001;61:1227–1232.PubMedGoogle Scholar
  23. 23.
    Leelawat K, Suksumek N, Leelawat S, Lek-Uthai U. Detection of vacA gene specific for Helicobactor pylori in hepatocellular carcinoma and cholangiocarcinoma specimens of Thai patients. Southeast Asian J Trop Med Public Health. 2007;38:881–885.PubMedGoogle Scholar
  24. 24.
    Goo MJ, Ki MR, Lee HR, et al. Helicobacter pylori promotes hepatic fibrosis in the animal model. Lab Invest. 2009;89:1291–1303.PubMedCrossRefGoogle Scholar
  25. 25.
    Allison CC, Ferrero RL. Role of virulence factors and host cell signaling in the recognition of Helicobacter pylori and the generation of immune responses. Future Microbiol. 2010;5:1233–1255.PubMedCrossRefGoogle Scholar
  26. 26.
    Tate SS, Meister A. gamma-Glutamyl transpeptidase: catalytic, structural and functional aspects. Mol Cell Biochem. 1981;39:357–368.PubMedCrossRefGoogle Scholar
  27. 27.
    Suzuki H, Kumagai H, Tochikura T. gamma-Glutamyltranspeptidase from Escherichia coli K-12: purification and properties. J Bacteriol. 1986;168:1325–1331.PubMedGoogle Scholar
  28. 28.
    Ishiye M, Yamashita M, Niwa M. Molecular cloning of the gamma-glutamyltranspeptidase gene from a Pseudomonas strain. Biotechnol Prog. 1993;9:323–331.PubMedCrossRefGoogle Scholar
  29. 29.
    Xu K, Strauch MA. Identification, sequence, and expression of the gene encoding gamma-glutamyltranspeptidase in Bacillus subtilis. J Bacteriol. 1996;178:4319–4322.PubMedGoogle Scholar
  30. 30.
    Chevalier C, Thiberge JM, Ferrero RL, Labigne A. Essential role of Helicobacter pylori gamma-glutamyltranspeptidase for the colonization of the gastric mucosa of mice. Mol Microbiol. 1999;31:1359–1372.PubMedCrossRefGoogle Scholar
  31. 31.
    Gong M, Ho B. Prominent role of gamma-glutamyl-transpeptidase on the growth of Helicobacter pylori. World J Gastroenterol. 2004;10:2994–2996.PubMedGoogle Scholar
  32. 32.
    Shibayama K, Wachino J, Arakawa Y, Saidijam M, Rutherford NG, Henderson PJ. Metabolism of glutamine and glutathione via gamma-glutamyltranspeptidase and glutamate transport in Helicobacter pylori: possible significance in the pathophysiology of the organism. Mol Microbiol. 2007;64:396–406.PubMedCrossRefGoogle Scholar
  33. 33.
    Flahou B, Haesebrouck F, Chiers K, et al. Gastric epithelial cell death caused by Helicobacter suis and Helicobacter pylori gamma-glutamyl transpeptidase is mainly glutathione degradation-dependent. Cell Microbiol. 2011;13:1933–1955.PubMedCrossRefGoogle Scholar
  34. 34.
    Kim KM, Lee SG, Kim JM, et al. Helicobacter pylori gamma-glutamyltranspeptidase induces cell cycle arrest at the G1-S phase transition. J Microbiol. 2010;48:372–377.PubMedCrossRefGoogle Scholar
  35. 35.
    Elmore S. Apoptosis: a review of programmed cell death. Toxicol Pathol. 2007;35:495–516.PubMedCrossRefGoogle Scholar
  36. 36.
    Plati J, Bucur O, Khosravi-Far R. Apoptotic cell signaling in cancer progression and therapy. Integr Biol (Camb). 2011;3:279–296.CrossRefGoogle Scholar
  37. 37.
    Bras M, Queenan B, Susin SA. Programmed cell death via mitochondria: different modes of dying. Biochemistry (Mosc). 2005;70:231–239.CrossRefGoogle Scholar
  38. 38.
    Schulte-Hermann R, Bursch W, Grasl-Kraupp B, Torok L, Ellinger A, Mullauer L. Role of active cell death (apoptosis) in multi-stage carcinogenesis. Toxicol Lett. 1995;82–83:143–148.PubMedCrossRefGoogle Scholar
  39. 39.
    Grimm MC, Elsbury SK, Pavli P, Doe WF. Interleukin 8: cells of origin in inflammatory bowel disease. Gut. 1996;38:90–98.PubMedCrossRefGoogle Scholar
  40. 40.
    Yamaoka Y, Kita M, Kodama T, Sawai N, Imanishi J. Helicobacter pylori cagA gene and expression of cytokine messenger RNA in gastric mucosa. Gastroenterology. 1996;110:1744–1752.PubMedCrossRefGoogle Scholar
  41. 41.
    Waugh DJ, Wilson C. The interleukin-8 pathway in cancer. Clin Cancer Res. 2008;14:6735–6741.PubMedCrossRefGoogle Scholar
  42. 42.
    Gong M, Ling SS, Lui SY, Yeoh KG, Ho B. Helicobacter pylori gamma-glutamyl transpeptidase is a pathogenic factor in the development of peptic ulcer disease. Gastroenterology. 2010;139:564–573.PubMedCrossRefGoogle Scholar
  43. 43.
    Seo JY, Yu JH, Lim JW, Mukaida N, Kim H. Nitric oxide-induced IL-8 expression is mediated by NF-kappaB and AP-1 in gastric epithelial AGS cells. J Physiol Pharmacol. 2009;60:101–106.PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Wongwarut Boonyanugomol
    • 1
    • 2
  • Chariya Chomvarin
    • 1
    • 2
  • Jea-Young Song
    • 3
    • 4
  • Kyung-Mi Kim
    • 3
    • 4
  • Jung-Min Kim
    • 3
    • 4
  • Myung-Je Cho
    • 3
    • 4
  • Woo-Kon Lee
    • 3
    • 4
  • Hyung-Lyun Kang
    • 3
    • 4
  • Kwang-Ho Rhee
    • 3
    • 4
  • Banchob Sripa
    • 2
    • 5
  • Chariya Hahnvajanawong
    • 1
    • 2
  • Seung-Chul Baik
    • 3
    • 4
  1. 1.Department of Microbiology, Faculty of MedicineKhon Kaen UniversityKhon KaenThailand
  2. 2.Liver Fluke and Cholangiocarcinoma Research CenterKhon Kaen UniversityKhon KaenThailand
  3. 3.Department of MicrobiologyGyeongsang National University School of MedicineJinjuRepublic of Korea
  4. 4.Research Institute of Life ScienceGyeongsang National University School of MedicineJinjuRepublic of Korea
  5. 5.Department of Pathology, Faculty of MedicineKhon Kaen UniversityKhon KaenThailand

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