Helicobacter pylori—Induced Loss of the Inhibitor-of-Apoptosis Protein Survivin Is Linked to Gastritis and Death of Human Gastric Cells

Manuel Valenzuela 1 2 3 Guillermo Pe rez-Pe rez 2 4 Alejandro H. Corvala n 0 2 Gonzalo Carrasco 0 2 Hery Urra 2 3 Denisse Bravo 2 3 H ector Toledo (.) 1 2 Andrew F. G. Quest () 2 3 0 Pathology and Molecular Epidemiology Laboratory, Centro Investigaciones Me dicas, Pontificia Universidad Cato lica de Chile , Santiago , Chile 1 Laboratory of Molecular Microbiology, Programa de Biolog a Celular y Molecular, Instituto de Ciencias Biome dicas, Facultad de Medicina, Universidad de Chile 2 Received 13 November 2009; accepted 27 April 2010; electronically published 24 August 2010. Potential conflicts of interest: none reported. Presented in part: Reunio n Annual de la Sociedad de Biolog a de Chile , Puco n, Chile, 26-29 November 2008 (abstract in Biol Res 2008; 41:R-57). Financial support: Fondo de Investigacio n Avanzada en A reas Prioritarias (grant 1501006 to A.F.G.Q.); Fondo Nacional de Investigacio n Cient fica y Tecnolo gica (grants 1080563 to A.H.C., 1085193 to H.T., and 1090071 to A.F.G.Q.). Studies of the Cell, Facultad de Medicina, Universidad de Chile , Av Independencia 1027, Santiago , Chile 3 Laboratory of Cellular Communication, Fondo de Investigacio n Avanzada en A reas Prioritarias Center for Molecular Studies of the Cell, Programa de Biolog a Celular y Molecular, Facultad de Medicina 4 Departments of Medicine and Microbiology, New York University , New York , New York Helicobacter pylori infects the human stomach and modifies signaling pathways that affect gastric epithelial cell proliferation and viability. Chronic exposure to this pathogen contributes to the onset of gastric atrophy, an early event in the genesis of gastric cancer associated with H. pylori infection. Susceptibility to H. pyloriinduced cell death ultimately depends on the presence of protective host cell factors. Although expression of the inhibitor-of-apoptosis protein survivin in adults is frequently linked to the development of cancer, evidence indicating that the protein is present in normal gastric mucosa is also available. Thus, we investigated in human gastric tissue samples and cell lines whether H. pylori infection is linked to loss of survivin and increased cell death. Our results show that infection with H. pylori decreased survivin protein levels in the mucosa of patients with gastritis. Furthermore, survivin down-regulation correlated with apoptosis and loss of cell viability in gastrointestinal cells cocultured with different H. pylori strains. Finally, overexpression of survivin in human gastric cells was sufficient to reduce cell death after infection. Taken together, these findings implicate survivin as an important survival factor in the gastric mucosa of humans. - sequence of histological changes in the mucosa, which initially involves nonatrophic gastritis, followed by multifocal atrophic gastritis, intestinal metaplasia, and, finally, intestinal-type gastric cancer [1, 2]. This progression has also been recapitulated in animal models, for instance, after inoculation of Mongolian gerbils with H. pylori [3]. Enhanced apoptosis after infection of the gastric epithelium is thought to represent an important contributing factor to the development of this cancer [4, 5] that correlates directly with the loss of specialized secretory gland cells of the mucosa, a precursor lesion referred to as gastric atrophy [1]. Consistent with this view, the gland cells lost in an animal model after Helicobacter infection were subsequently replaced by an influx of bone marrowderived cells, which then differentiated and contributed to the development of metaplasia, dysplasia, and gastric cancer [6, 7]. Two major events have been associated with apoptosis of gastric epithelial cellsparticularly in the proliferative neck zone of the glandsafter H. pylori infection of the mucosa [8, 9]. On the one hand, an inflammatory response is mounted against the bacteria that results in the production of reactive oxygen species and reactive nitrogen species as well as the release of proapoptotic molecules, such as tumor necrosis factor a, interferon g, and FasL [4, 5, 10, 11]. Alternatively, secreted (VacA and urease), injected (CagA and peptidoglycan), or surface (lipopolysaccharide) components of the bacteria modulate signal transduction pathways and reduce the viability of gastric epithelial cells [5, 1214]. Thus, while H. pylori infection is considered to be a contributing factor to epithelial cell damage, the mechanisms underlying H. pyloriinduced apoptosis in gastric epithelial cells remain unclear [15]. Survivin is an inhibitor-of-apoptosis protein that reportedly modulates apoptosis by a variety of mechanisms [1618]. Survivin is essential for embryonic development [19] but is generally not expressed in adult tissues, although expression is maintained in some proliferating cell populations [20]. Despite being poorly expressed in adult tissues, survivin plays an important physiological role in the repair of human tissues and organs [20] and after damage [21, 22]. Furthermore, survivin expression is dramatically elevated in the majority of human cancers, including gastric cancer [2326], and down-regulation of survivin in cancer cells reduces their viability [27, 28]. Available studies have focused predominantly on investigating the relationship between survivin expression and gastric cancer. However, whether survivin is present in normal tissue and might change in the presence of H. pylori were not considered, although published data indicate that survivin is present in normal human gastric tissue [29] and may be important for the maintenance of stomach mucosa integrity in rats [30]. Such issues were addressed in the present study. Tissue samples. Biopsy specimens from the corpus and antrum of 37 patients who underwent upper endoscopy between 1998 and 2005 at the Clinical Hospital and the San Carlos Clinic of the Pontificia Universidad Cato lica de Chile were analyzed. For this group of patients, the absence of tumors was the only inclusion criteria. Written informed consent approved by the Ethics Committee of the Pontificia Universidad Cato lica de Chile was obtained from each patient. Gastric tissue samples were subsequently classified as either normal or H. pyloripositive gastritis by evaluating the following histological variables: location (corpus or antrum), chronic inflammation, polymorphonuclear activity, atrophy, intestinal metaplasia, and H. pylori status (in agreement with the criteria of the updated Sydney system [31]). All samples were evaluated by 2 independent observers who were unaware of the clinical data and the objectives of the study (A.H.C. and G.C.). According to these criteria, all H. pyloripositive cases included in this study displayed only mild or moderate chronic gastritis, without any evidence of intestinal metaplasia. In samples from control individuals, neither H. pylori presence or evidence of gastritis was detected. Tissue microarrays. Tissue microarrays were pe (...truncated)