？？-Glutamyltranspeptidase (GGT) is a novel protein in the induction of H. pylori-mediated apoptosis; however, the signal pathway involved in GGT-induced apoptosis remains unclear. Using DNA recombination techniques, ggt was cloned into pET117b and tra ...
？？-Glutamyltranspeptidase (GGT) is a novel protein in the induction of H. pylori-mediated apoptosis; however, the signal pathway involved in GGT-induced apoptosis remains unclear. Using DNA recombination techniques, ggt was cloned into pET117b and transformed into E. coli. rGGT was purified using nickel-affinity resin. rGGT induced apoptosis in AGS cells in a time-dependent manner, which was confirmed by TUNEL staining, the MTT assay and immunoblot analysis for caspases-9, -3, Bax, Bcl-2, Bcl-xL and cytochrome c release. Activation of caspase-3, and -9 following exposure to GGT increased in a time-dependent manner. In addition, upregulation of proapoptotic Bax and a downregulation of antiapoptotic Bcl-2 and Bcl-xL were detected.
Apoptotic signals also trigger changes in mitochondria, which lead to a release of cytochrome c into the cytosolic space. The ggt-deficient mutant was not as able to induce apoptosis as the wild-type strain. These results indicated that GGT of H. pylori induces apoptosis via a mitochondria-mediated pathway.
Fallowing study was carried out to demonstrate the role of mitogen-activated protein (MAP) kinase in H. pylori GGT-induced apoptosis in AGS cells. The activation of MAP kinase was confirmed by western blot. In an attempt to elucidate the pathway, the cells were treated with PD98059, SB203580 and SP600125 for the inhibition of ERK, p38 kinase and JNK, respectively, prior to exposure to the rGGT protein. Data showed that ERK was phosphorylated and GGT-induced apoptosis was inhibited by PD98059.
To better understand the mechanism of apoptosis by H. pylori GGT, cell cycle-related events were examined following exposure to the rGGT protein. During co-culture, H. pylori rGGT inhibited cell cycle progression at G1-S in AGS cells which were confirmed by immunoblotting analysis for CDK4, CDK6, cyclin A, cyclin E, p21, and p27. p21and p27 were up-regulated and CDK4, CDK6, cyclin A and cyclin E were down-regulated. These results indicated that rGGT inhibits cell cycle progression at G1-S for apoptosis.
In conclusion, H. pylori rGGT induces apoptosis and inhibits cell cycle progression at G1-S, in association with ERK phosphorylation, caspase-3 activation, cytochrome c release in AGS cells.