RESUMEN
A considerable body of evidence exists implicating high levels of free saturated fatty acids in beta pancreatic cell death, although the molecular mechanisms and the signaling pathways involved have not been clearly defined. The membrane protein caveolin-1 has long been implicated in cell death, either by sensitizing to or directly inducing apoptosis and it is normally expressed in beta cells. Here, we tested whether the presence of caveolin-1 modulates free fatty acid-induced beta cell death by reexpressing this protein in MIN6 murine beta cells lacking caveolin-1. Incubation of MIN6 with palmitate, but not oleate, induced apoptotic cell death that was enhanced by the presence of caveolin-1. Moreover, palmitate induced de novo ceramide synthesis, loss of mitochondrial transmembrane potential and reactive oxygen species (ROS) formation in MIN6 cells. ROS generation promoted caveolin-1 phosphorylation on tyrosine-14 that was abrogated by the anti-oxidant N-acetylcysteine or the incubation with the Src-family kinase inhibitor, PP2 (4-amino-5-(4-chlorophenyl)-7(dimethylethyl)pyrazolo[3,4-d]pyrimidine). The expression of a non-phosphorylatable caveolin-1 tyrosine-14 to phenylalanine mutant failed to enhance palmitate-induced apoptosis while for MIN6 cells expressing the phospho-mimetic tyrosine-14 to glutamic acid mutant caveolin-1 palmitate sensitivity was comparable to that observed for MIN6 cells expressing wild type caveolin-1. Thus, caveolin-1 expression promotes palmitate-induced ROS-dependent apoptosis in MIN6 cells in a manner requiring Src family kinase mediated tyrosine-14 phosphorylation.
Asunto(s)
Apoptosis/efectos de los fármacos , Caveolina 1/metabolismo , Células Secretoras de Insulina/efectos de los fármacos , Palmitatos/farmacología , Especies Reactivas de Oxígeno/metabolismo , Acetilcisteína/farmacología , Animales , Western Blotting , Caveolina 1/genética , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Depuradores de Radicales Libres/farmacología , Peróxido de Hidrógeno/farmacología , Células Secretoras de Insulina/metabolismo , Masculino , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Ratones Endogámicos C57BL , Microscopía Confocal , Oxidantes/farmacología , Fosforilación/efectos de los fármacos , Pirimidinas/farmacología , Esfingosina/análogos & derivados , Esfingosina/farmacología , Tirosina/metabolismo , Familia-src Quinasas/antagonistas & inhibidores , Familia-src Quinasas/metabolismoRESUMEN
Helicobacter pylori infection is highly prevalent in Chile (73%). Usually a minority of infected patients develops complications such as ulcers and gastric cancer that have been associated with the presence of virulence factors (cagA, vacA) and host T helper response (Th1/Th2). Our aim was to evaluate the relationship between strain virulence and host immune response, using a multiple regression approach for the development of a model based on data collected from H. pylori infected patients in Chile. We analyzed levels of selected cytokines determined by ELISA (interleukin (IL)-12, IL-10, interferon (IFN)-gamma and IL-4) and the presence of cagA and vacA alleles polymorphisms determined by PCR in antral biopsies of 41 patients referred to endoscopy. By multiple regression analysis we established a correlation between bacterial and host factors using clinical outcome (gastritis and duodenal ulcer) as dependent variables. The selected model was described by: clinical outcome=0.867491 (cagA)+0.0131847 (IL-12/IL-10)+0.0103503 (IFN-gamma/IL-4) and it was able to explain over 90% of clinical outcomes observations (R(2)=96.4). This model considers that clinical outcomes are better explained by the interaction of host immune factors and strain virulence as a complex and interdependent mechanism.
Asunto(s)
Citocinas/inmunología , Infecciones por Helicobacter/inmunología , Infecciones por Helicobacter/virología , Helicobacter pylori/inmunología , Helicobacter pylori/patogenicidad , Factores de Virulencia/genética , Adolescente , Adulto , Alelos , Antígenos Bacterianos/genética , Proteínas Bacterianas/genética , Niño , Preescolar , Úlcera Duodenal/inmunología , Úlcera Duodenal/virología , Femenino , Gastritis/inmunología , Gastritis/virología , Helicobacter pylori/genética , Humanos , Masculino , Persona de Mediana Edad , Células TH1/inmunología , Células Th2/inmunologíaRESUMEN
Caveolin-1 (CAV1) is a scaffolding protein that plays a dual role in cancer. In advanced stages of this disease, CAV1 expression in tumor cells is associated with enhanced metastatic potential, while, at earlier stages, CAV1 functions as a tumor suppressor. We recently implicated CAV1 phosphorylation on tyrosine 14 (Y14) in CAV1-enhanced cell migration. However, the contribution of this modification to the dual role of CAV1 in cancer remained unexplored. Here, we used in vitro [2D and transendothelial cell migration (TEM), invasion] and in vivo (metastasis) assays, as well as genetic and biochemical approaches to address this question in B16F10 murine melanoma cells. CAV1 promoted directional migration on fibronectin or laminin, two abundant lung extracellular matrix (ECM) components, which correlated with enhanced Y14 phosphorylation during spreading. Moreover, CAV1-driven migration, invasion, TEM and metastasis were ablated by expression of the phosphorylation null CAV1(Y14F), but not the phosphorylation mimicking CAV1(Y14E) mutation. Finally, CAV1-enhanced focal adhesion dynamics and surface expression of beta1 integrin were required for CAV1-driven TEM. Importantly, CAV1 function as a tumor suppressor in tumor formation assays was not altered by the Y14F mutation. In conclusion, our results provide critical insight to the mechanisms of CAV1 action during cancer development. Specific ECM-integrin interactions and Y14 phosphorylation are required for CAV1-enhanced melanoma cell migration, invasion and metastasis to the lung. Because Y14F mutation diminishes metastasis without inhibiting the tumor suppressor function of CAV1, Y14 phosphorylation emerges as an attractive therapeutic target to prevent metastasis without altering beneficial traits of CAV1.
Asunto(s)
Caveolina 1/metabolismo , Melanoma/metabolismo , Neoplasias Cutáneas/metabolismo , Tirosina/química , Animales , Carcinogénesis , Caveolina 1/química , Adhesión Celular , Movimiento Celular , Femenino , Fibroblastos/metabolismo , Fibronectinas/química , Humanos , Integrina beta1/metabolismo , Laminina/química , Neoplasias Pulmonares/secundario , Masculino , Melanoma Experimental , Ratones , Ratones Endogámicos C57BL , Invasividad Neoplásica , Metástasis de la Neoplasia , FosforilaciónRESUMEN
AIM: To evaluate how widely Helicobacter pylori (H. pylori) HopE and HopV porins are expressed among Chilean isolates and how seroprevalent they are among infected patients in Chile. METHODS: H. pylori hopE and hopV genes derived from strain CHCTX-1 were cloned by polymerase chain reaction (PCR), sequenced and expressed in Escherichia coli AD494 (DE3). Gel-purified porins were used to prepare polyclonal antibodies. The presence of both genes was tested by PCR in a collection of H. pylori clinical isolates and their expression was detected in lysates by immunoblotting. Immune responses against HopE, HopV and other H. pylori antigens in sera from infected and non-infected patients were tested by Western blotting using these sera as first antibody on recombinant H. pylori antigens. RESULTS: PCR and Western blotting assays revealed that 60 and 82 out of 130 Chilean isolates carried hopE and hopV genes, respectively, but only 16 and 9, respectively, expressed these porins. IgG serum immunoreactivity evaluation of 69 H. pylori-infected patients revealed that HopE and HopV were infrequently recognized (8.7% and 10.1% respectively) compared to H. pylori VacA (68.1%) and CagA (59.5%) antigens. Similar values were detected for IgA serum immunoreactivity against HopE (11.6%) and HopV (10.5%) although lower values for VacA (42%) and CagA (17.4%) were obtained when compared to the IgG response. CONCLUSION: A scarce expression of HopE and HopV among Chilean isolates was found, in agreement with the infrequent seroconversion against these antigens when tested in infected Chilean patients.