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 IkappaBalpha 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 IkappaBalpha 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.
D017209:Apoptosis; D020169:Caspases / Q000378:metabolism; D002470:Cell Survival; D003574:Cytochrome c Group / Q000378:metabolism; D053938:DNA Fragmentation; D004273:DNA, Neoplasm / Q000235:genetics; D006367:HeLa Cells; D016481:Helicobacter Infections / Q000378:metabolism / Q000473:pathology*; D016480:Helicobacter pylori / Q000472:pathogenicity*; D006801:Humans; D008928:Mitochondria / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism; D009363:Neoplasm Proteins / Q000378:metabolism; D015398:Signal Transduction; D013274:Stomach Neoplasms / Q000378:metabolism / Q000382:microbiology* / Q000473:pathology; D014407:Tumor Cells, Cultured; D014774:Virulence
D003574:Cytochrome c Group; D004273:DNA, Neoplasm; D016328:NF-kappa B; D009363:Neoplasm Proteins; D020169:Caspases