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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. [ABSTRACT FROM AUTHOR]

Background: Gastric cancer stem cells (CSCs), which require activation of Wnt signaling to maintain their self-renewal and tumorigenicity, are proposed to be critical targets for effective therapy of gastric carcinomas. Gene therapies that are delivered by adenovirus of serotype 5 (Ad5) or chimeric 5/35(Ad5/35) adenovirus have shown promise for treating various cancers. Here we aimed to develop a gene therapy strategy that targeted gastric CSCs (CD44 cells). Methods: CD44 cells were isolated by fluorescence activated cell sorting from both primary gastric cancer cells and cell lines. Expression of adenovirus receptors was examined in CD44 and CD44 cells. A potent Wnt antagonist Dickkopf-1 (DKK1) was delivered into CD44 cells using Ad5/35 (Ad5/35-DKK1). The therapeutic outcomes were evaluated. Results: Expression of Coxsakievirus adenovirus receptor for Ad5 was significantly reduced, while abundance of CD46, the receptor for Ad5/35, was slightly higher in CD44 cells. Accordingly, CD44 cells were sensitive to Ad5/35 infection, but not to Ad5. Ad5/35-DKK1 introduced DKK1 into CD44 cells and deactivated endogenous Wnt/ β-catenin signaling efficiently. Overexpression of DKK1 inhibited survival, anchorage-independent colony formation, and invasion of CD44 cells, which were restored by a GSK-3 specific inhibitor BIO-acetoxime. More importantly, introduction of DKK1 abrogated the tumorigenicity of CD44 cells in vivo. However, Ad5/35-DKK1 only showed minimal cytotoxicity to normal tissue-derived cells, L-02 and GES-1. Conclusions: We developed, for the first time, a novel Ad5/35-DKK1-based approach to abrogate Wnt signaling in CSCs and demonstrated that gastric CSC-targeting gene therapy was effective in preclinical experiments. [ABSTRACT FROM AUTHOR]