Hui Chang1, Dongfeng Chen1, Bosheng Ni2, Qianfei Zuo3, Chunhua Wang3, Ran Han4, Chunhui Lan5. 1. Department of Gastroenterology, Daping Hospital, Third Military Medical University, 10 Changjiang Branch Road, Chongqing, 400042, China. 2. Department of Microbiology and Biochemical Pharmacy, College of Pharmacy, The Third Military Medical University, Chongqing, China. 3. Cadre's Ward of Bethune International Peace Hospital, PLA, Shijiazhuang City, Hebei Province, China. 4. Department of Gastroenterology, Guizhou Aerospace Hospital, Zunyi City, Guizhou Province, China. 5. Department of Gastroenterology, Daping Hospital, Third Military Medical University, 10 Changjiang Branch Road, Chongqing, 400042, China. tiandaochouqin99@hotmail.com.
Abstract
BACKGROUND: Vacuolating cytotoxin antigen (VacA) is one of the major virulence factors in Helicobacter pylori (H. pylori), which is responsible for cell vacuolar degeneration and apoptotic cell death. A candidate host factor which mediates this process is cortactin, a protein associated with the processes of colonization and adhesion of H. pylori in gastric epithelium. AIM: To investigate the role of cortactin in VacA-induced apoptosis of gastric epithelial cells. METHODS: Cortactin expression and shRNA lentiviral constructs were developed and transduced into the human gastric cancer cell line, AGS. VacA protein was purified from H. pylori cultures, acid-activated, and co-incubated with the transduced cell populations. Apoptosis was detected by flow cytometry, and the levels of the pro- and anti-apoptotic proteins Bax and Bcl-2 were determined by Western blot. RESULTS: Acid-activated purified VacA induced apoptosis in the parental AGS cells. Increased expression of cortactin (AGS/cortactin) led to a greater percentage of cells undergoing apoptosis. In contrast, knockdown of cortactin with shRNA (AGS/cortactin-shRNA) decreased the percentage of apoptotic cells. The protein levels of pro- and anti-apoptotic proteins Bax and Bcl-2 were increased and decreased in AGS/cortactin cells relative to the parental AGS cells. In the AGS/cortactin-shRNA cells, Bax protein levels were decreased, while Bcl-2 protein was increased. CONCLUSIONS: The results indicate that cortactin is involved in the regulation of apoptosis induced by VacA in gastric cells.
BACKGROUND: Vacuolating cytotoxin antigen (VacA) is one of the major virulence factors in Helicobacter pylori (H. pylori), which is responsible for cell vacuolar degeneration and apoptotic cell death. A candidate host factor which mediates this process is cortactin, a protein associated with the processes of colonization and adhesion of H. pylori in gastric epithelium. AIM: To investigate the role of cortactin in VacA-induced apoptosis of gastric epithelial cells. METHODS:Cortactin expression and shRNA lentiviral constructs were developed and transduced into the humangastric cancer cell line, AGS. VacA protein was purified from H. pylori cultures, acid-activated, and co-incubated with the transduced cell populations. Apoptosis was detected by flow cytometry, and the levels of the pro- and anti-apoptotic proteins Bax and Bcl-2 were determined by Western blot. RESULTS: Acid-activated purified VacA induced apoptosis in the parental AGS cells. Increased expression of cortactin (AGS/cortactin) led to a greater percentage of cells undergoing apoptosis. In contrast, knockdown of cortactin with shRNA (AGS/cortactin-shRNA) decreased the percentage of apoptotic cells. The protein levels of pro- and anti-apoptotic proteins Bax and Bcl-2 were increased and decreased in AGS/cortactin cells relative to the parental AGS cells. In the AGS/cortactin-shRNA cells, Bax protein levels were decreased, while Bcl-2 protein was increased. CONCLUSIONS: The results indicate that cortactin is involved in the regulation of apoptosis induced by VacA in gastric cells.
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