Analysis of apoptotic and antiapoptotic signalling pathways induced by Helicobacter pylori.
OBJECTIVE
Although it is reported that Helicobacter pylori induces apoptosis on gastric epithelial cells, the mechanism remains unknown. Antiapoptotic effects generated by H pylori have not yet been evaluated.


METHODS
(1) H pylori strains (type 1 wild, TN2-DeltacagE, TN2-DeltavacA) were cocultured with MKN45, TMK1, and HeLa cells, and cell viability and apoptosis were assessed by trypan blue exclusion and DNA laddering, respectively. (2) Activation of caspases-3, 7, and 8, cytochrome c release from the mitochondria, and Fas, Fas associated death domain protein (FADD), Bax, Bak, and Bcl-X expression were evaluated by immunoblot analysis. (3) To investigate whether nuclear factor kappa B (NFkappaB) activation induced by cag pathogenicity island (PAI) positive H pylori affects antiapoptosis, MKN45 cells stably expressing super-repressor Ikappabetaalpha were cocultured with H pylori, and cell viability and caspase activation were evaluated. NFkappaB regulated gene expression was also evaluated by ribonuclease protection assay.


RESULTS
(1) Wild-type and DeltavacA mutant H pylori induced apoptosis more potently than the DeltacagE mutant. Inhibition of cell contact between H pylori and cancer cells and heat killing H pylori diminished cell death. (2) Caspases-3, 7, and 8 were activated time dependently by H pylori as well as by the agonist anti-Fas. Cytochrome c release from mitochondria was observed and was not inhibited by caspase-8 inhibitor. Although protein expression of Fas, FADD, Bax, Bak, and Bcl-X in the whole cell lysates was not changed by H pylori, Bax was decreased from mitochondria free cytosol suggesting that Bax was translocated into mitochondria. (3) Cell death and the activities of caspases-3 and 8 were promoted in MKN45 cells stably expressing super-repressor Ikappabetaalpha that inhibits NFkappaB activation. Antiapoptotic proteins c-IAP1 and c-IAP2 were upregulated by the wild-type strains.


CONCLUSIONS
cag PAI positive H pylori is capable of inducing apoptotic effects mainly through the mitochondrial pathway. Antiapoptotic effects mediated by NFkappaB activation were also observed.
Maeda
S
S
Department of Gastroenterology, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan. MAEDA-2IM@h.u-tokyo.ac.jp
Yoshida
H
H
Mitsuno
Y
Y
Hirata
Y
Y
Ogura
K
K
Shiratori
Y
Y
Omata
M
M
D048868:Adaptor Proteins, Signal Transducing; D017209:Apoptosis / Q000502:physiology*; D002352:Carrier Proteins / Q000378:metabolism; D053181:Caspase 8; D053453:Caspase 9; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism; D002450:Cell Communication / Q000502:physiology; D002470:Cell Survival / Q000502:physiology; D003574:Cytochrome c Group / Q000378:metabolism*; D053938:DNA Fragmentation / Q000502:physiology; D053200:Fas-Associated Death Domain Protein; D016481:Helicobacter Infections / Q000473:pathology*; D016480:Helicobacter pylori / Q000472:pathogenicity / Q000502:physiology*; D006801:Humans; D008928:Mitochondria / Q000378:metabolism*; D016328:NF-kappa B / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D013274:Stomach Neoplasms / Q000382:microbiology / Q000473:pathology; D014407:Tumor Cells, Cultured; D051028:bcl-2-Associated X Protein; D051020:bcl-X Protein; D019014:fas Receptor / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; C494826:BAX protein, human; C494811:BCL2L1 protein, human; D002352:Carrier Proteins; D061945:Caspase Inhibitors; D003574:Cytochrome c Group; C505533:FADD protein, human; D053200:Fas-Associated Death Domain Protein; D016328:NF-kappa B; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D051028:bcl-2-Associated X Protein; D051020:bcl-X Protein; D019014:fas Receptor; C505530:CASP8 protein, human; C506433:CASP9 protein, human; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases