Role of activation of the protein tyrosine kinase, Src, in colorectal cancer chemoresistance

Advances in therapy for colorectal cancer have been hampered by development of resistance to chemotherapy. The Src family of protein tyrosine kinases has been associated with colorectal cancer development and progression. Activation of the prototypic member of the family, Src, occurs in advanced colorectal cancer and is associated with a worse outcome. This work tests the hypotheses that Src activation contributes to chemoresistance in some colon tumors and that this resistance can be overcome by use of Src inhibitors. The aims of the proposal were to (1) determine if constitutive Src activation is sufficient to induce oxaliplatin resistance; (2) evaluate the role of reactive oxygen species (ROS) in the activation of Src after oxaliplatin treatment; (3) determine the frequency of Src activation in liver metastases after oxaliplatin treatment; and (4) evaluate the safety, preliminary efficacy, and pharmacodynamics of the combination of dasatinib with oxaliplatin-based therapy in patients with metastatic colorectal cancer. ^ Using a panel of colon cancer cell lines and murine models, I demonstrate that administration of oxaliplatin, a commonly utilized chemotherapy for colorectal cancer, results in an increased activation of Src. The activation occurs acutely in some, but not all, colorectal carcinoma cell lines. Cell lines selected for oxaliplatin resistance are further increased in Src activity. Treatment of cell lines with dasatinib, a non-selective pharmacologic inhibitor of the Src family kinases synergistically killed some, but not all cell lines. Cell lines with the highest acute activation of Src after oxaliplatin administration were the most sensitive to the combination therapy. Previous work demonstrated that siRNA to Src increased sensitivity to oxaliplatin, suggesting that the effects of dasatinib are primarily due to its ability to inhibit Src in these cell lines. ^ To examine the mechanism underlying these results, I examined the effects of reactive oxygen species (ROS), as previous studies have demonstrated that platinum chemotherapeutics result in intracellular oxidative stress. I demonstrated that oxaliplatin-induced reactive oxygen species were higher in the cell lines with Src activation, relative to those in which Src was not activated. This oxaliplatin-induced Src activation was blocked by the administration of anti-oxidants, thereby demonstrating that synergistic killing between dasatinib and oxaliplatin was associated with the ability of the latter to generate ROS. ^ In a murine model of colorectal cancer metastasis to the liver, the combination of dasatinib and oxaliplatin was more effective in reducing tumor volume than either agent alone. However, when oxaliplatin resistant cell lines were treated with a combination of oxaliplatin and AZD0530, an inhibitor in the clinic with increased specificity for Src, no additional benefit was seen, although Src was activated by oxaliplatin and Src substrates were inhibited. The indolent growth of oxaliplatin-resistant cells, unlike the growth of oxaliplatin resistant tumors in patients, precludes definitive interpretation of these results. ^ To further explore Src activation in patients with oxaliplatin exposure and resistance, an immunohistochemistry analysis of tumor tissue from resected liver metastases of colorectal cancer was performed. Utilizing a tissue microarray, staining for phosphorylated Src and FAK demonstrated strong staining of tumor relative to stromal and normal liver. In patients recently exposed to oxaliplatin, there was increased FAK activation, supporting the clinical relevance of the prior preclinical studies. ^ To pursue the potential clinical benefit of the combination of Src inhibition with oxaliplatin, a phase IB clinical trial was completed. Thirty patients with refractory metastatic colorectal cancer were treated with a combination of 5-FU, oxaliplatin, an epidermal-growth factor receptor monoclonal antibody, and dasatinib. The recommended phase II dose of dasatinib was established, and toxicities were quantified. Pharmacodynamic studies demonstrated increased phosphorylation of the Src substrate paxillin after dasatinib therapy. Tumor biopsies were obtained and Src expression levels were quantitated. Clinical benefit was seen with the combination, including a response rate of 20% and disease control rate of 56%, prompting a larger clinical study. ^ In summary, although Src is constitutively activated in metastatic colorectal cancer, administration of oxaliplatin chemotherapy can further increase its activity, through a reactive oxygen species dependent manner. Inhibition of Src in combination with oxaliplatin provides additional benefit in vitro, in preclinical animal models, and in the clinic. Further study of Src inhibition in the clinic and identification of predictive biomarkers of response will be required to further advance this promising therapeutic target. ^

Syntaxin 6- and microtubule- mediated intracellular trafficking contributes to Golgi and nuclear translocation of EGFR

Receptor-mediated endocytosis is well known for its degradation and recycling trafficking. Recent evidence shows that these cell surface receptors translocate from cell surface to different cellular compartments, including the Golgi, mitochondria, endoplasmic reticulum (ER), and the nucleus to regulate physiological and pathological functions. Although some trafficking mechanisms have been resolved, the mechanism of intracellular trafficking from cell surface to the Golgi is not yet completed understood. Here we report a mechanism of Golgi translocation of EGFR in which EGF-induced EGFR travels to the Golgi via microtubule (MT)-dependent movement by interacting with dynein and fuses with the Golgi through syntaxin 6 (Syn6)-mediated membrane fusion. We also demonstrate that the Golgi translocation of EGFR is necessary for its consequent nuclear translocation and transcriptional activity. Interestingly, foreign protein such as bacterial cholera toxin, which is known to activate its pathological function through the Golgi/ER retrograde pathway, also utilizes the MT/Syn6 pathway. Thus, the MT, and syntaxin 6 mediated trafficking pathway from cell surface to the Golgi and ER defines a comprehensive retrograde trafficking route for both cellular and foreign molecules to travel from cell surface to the Golgi and the nucleus.

OXIDATIVE STRESS BASED STRATEGIES FOR ENHANCING THE EFFICACY OF HISTONE DEACETYLASE INHIBITORS (HDACi)

Histone deacetylase inhibitors (HDACi) are anti-cancer drugs that primarily act upon acetylation of histones, however they also increase levels of intracellular reactive oxygen species (ROS). We hypothesized that agents that cause oxidative stress might enhance the efficacy of HDACi. To test this hypothesis, we treated acute lymphocytic leukemia cells (ALL) with HDACi and adaphostin (ROS generating agent). The combination of two different HDACi (vorinostat or entinostat) with adaphostin synergistically induced apoptosis in ALL. This synergistic effect was blocked when cells were pre-treated with the caspase-9 inhibitor, LEHD. In addition, we showed that loss of the mitochondrial membrane potential is the earliest event observed starting at 12 h. Following this event, we observed increased levels of superoxide at 16 h, and ultimately caspase-3 activation. Pre-treatment with the antioxidant N-acetylcysteine (NAC) blocked ROS generation and reversed the loss of mitochondrial membrane potential for both combinations. Interestingly, DNA fragmentation and caspase-3 activity was only blocked by NAC in cells treated with vorinostat-adaphostin; but not with entinostat-adaphostin. These results suggest that different redox mechanisms are involved in the induction of ROS-mediated apoptosis. To further understand these events, we studied the role of the antioxidants glutathione (GSH) and thioredoxin (Trx). We found that the combination of entinostat-adaphostin induced acetylation of the antioxidant thioredoxin (Trx) and decreased intracellular levels of GSH. However, no effect on Trx activity was observed in either combination. In addition, pre-treatment with GSH ethyl ester, a soluble form of GSH, did not block DNA fragmentation. Together these results suggested that GSH and Trx are not major players in the induction of oxidative stress. Array data examining the expression of genes involved in oxidative stress demonstrated a differential regulation between cells treated with vorinostat-adaphostin and entinostat-adaphostin. Some of the genes differentially expressed between the combinations include aldehyde oxidase 1, glutathione peroxidase-5, -6, peroxiredoxin 6 and myeloperoxidase. Taken together, these experimental results indicate that the synergistic activity of two different HDACi with adaphostin is mediated by distinct redox mechanisms in ALL cells. Understanding the mechanism involved in these combinations will advance scientific knowledge of how the action of HDACi could be augmented in leukemia models. Moreover, this information could be used for the development of effective clinical trials combining HDACi with other anticancer agents.

Calmodulin -regulated processes and intracellular calcium in the heat stress response of mammalian cells

Three approaches were used to examine the role of Ca$\sp{2+}$- and/or calmodulin (CaM)-regulated processes in the mammalian heat stress response. The focus of the first approach was on the major Ca$\sp{2+}$-binding protein, CaM, and involved the use of CaM antagonists that perturbed CaM-regulated processes during heat stress. The second approach involved the use of a cell line and its BPV-1 transformants that express increased basal levels of CaM, or parvalbumin--a Ca$\sp{2+}$-binding protein not normally found in these cells. The last approach used Ca$\sp{2+}$ chelators to buffer Ca$\sp{2+}$-transients.^ The principle conclusions resulting from these three experimental approaches are: (1) CaM antagonists cause a temperature-dependent potentiation of heat killing, but do not inhibit the triggering and development of thermotolerance suggesting some targets for heat killing are different from those that lead to thermotolerance; (2) Members of major HSP families (especially HSP70) can bind to CaM in a Ca$\sp{2+}$-dependent manner in vitro, and HSP have been associated with events leading to thermotolerance. But, because thermotolerance is not affected by CaM antagonists, and antagonists should interfere with HSP binding to CaM, the events leading to triggering or developing thermotolerance were not strongly dependent on HSP binding to CaM; (3) CaM antagonists can also bind to HSP70 (and possibly other HSP) suggesting an alternative mechanism for the action of these agents in heat killing may involve direct binding to other proteins, like HSP70, whose function is important for survival following heating and inhibiting their activity; and (4) The signal governing the rate of synthesis of another major HSP group, the HSP26 family, can be largely abrogated by elevated Ca$\sp{2+}$-binding proteins or Ca$\sp{2+}$ chelators without significantly reducing survival or thermotolerance suggesting if the HSP26 family is involved in either end point, it may function in (Ca$\sp{2+}$) $\sb{\rm i}$ homeostasis. ^

The role of the intracellular second messenger cAMP in the induction of long-term morphological changes in sensory neurons of {\it Aplysia\/}

The present work examines the role of cAMP in the induction of the type of long-term morphological changes that have been shown to be correlated with long-term sensitization in Aplysia.^ To examine this issue, cAMP was injected into individual tail sensory neurons in the pleural ganglion to mimic, at the single cell level, the effects of behavioral training. After a 22 hr incubation period, the same cells were filled with horseradish peroxidase and 2 hours later the tissue was fixed and processed. Morphological analysis revealed that cAMP induced an increase in two morphological features of the neurons, varicosities and branch points. These structural alterations, which are similar to those seen in siphon sensory neurons of the abdominal ganglion following long-term sensitization training of the siphon-gill withdrawal reflex, could subserve the altered behavioral response of the animal. These results expose another role played by cAMP in the induction of learning, the initiation of a structural substrate, which, in concert with other correlates, underlies learning.^ cAMP was injected into sensory neurons in the presence of the reversible protein synthesis inhibitor, anisomycin. The presence of anisomycin during and immediately following the nucleotide injection completely blocked the structural remodeling. These results indicate that the induction of morphological changes by cAMP is a process dependent on protein synthesis.^ To further examine the temporal requirement for protein synthesis in the induction of these changes, the time of anisomycin exposure was varied. The results indicate that the cellular processes triggered by cAMP are sensitive to the inhibition of protein synthesis for at least 7 hours after the nucleotide injection. This is a longer period of sensitivity than that for the induction of another correlate of long-term sensitization, facilitation of the sensory to motor neuron synaptic connection. Thus, these findings demonstrate that the period of sensitivity to protein synthesis inhibition is not identical for all correlates of learning. In addition, since the induction of the morphological changes can be blocked by anisomycin pulses administered at different times during and following the cAMP injection, this suggests that cAMP is triggering a cascade of protein synthesis, with successive rounds of synthesis being dependent on successful completion of preceding rounds. Inhibition at any time during this cascade can block the entire process and so prevent the development of the structural changes.^ The extent to which cAMP can mimic the structural remodeling induced by long-term training was also examined. Animals were subjected to unilateral sensitization training and the morphology of the sensory neurons was examined twenty-four hours later. Both cAMP injection and long-term training produced a twofold increase in varicosities and approximately a fifty percent increase in the number of branch points in the sensory neuron arborization within the pleural ganglion. (Abstract shortened by UMI.) ^

Intrinsic oxidative stress in cancer cells: A biochemical basis for therapeutic selectivity using ROS -generating agents

A major goal of chemotherapy is to selectively kill cancer cells while minimizing toxicity to normal cells. Identifying biological differences between cancer and normal cells is essential in designing new strategies to improve therapeutic selectivity. Superoxide dismutases (SOD) are crucial antioxidant enzymes required for the elimination of superoxide (O2·− ), a free radical produced during normal cellular metabolism. Previous studies in our laboratory demonstrated that 2-methoxyestradiol (2-ME), an estradiol derivative, inhibits the function of SOD and selectively kills human leukemia cells without exhibiting significant cytotoxicity in normal lymphocytes. The present work was initiated to examine the biochemical basis for the selective anticancer activity of 2-ME. Investigations using two-parameter flow cytometric analyses and ROS scavengers established that O2·− is a primary and essential mediator of 2-ME-induced apoptosis in cancer cells. In addition, experiments using SOD overexpression vectors and SOD knockout cells found that SOD is a critical target of 2-ME. Importantly, the administration of 2-ME resulted in the selective accumulation of O 2·− and apoptosis in leukemia and ovarian cancer cells. The preferential activity of 2-ME was found to be due to increased intrinsic oxidative stress in these cancer cells versus their normal counterparts. This intrinsic oxidative stress was associated with the upregulation of the antioxidant enzymes SOD and catalase as a mechanism to cope with the increase in ROS. Furthermore, oxygen consumption experiments revealed that normal lymphocytes decrease their respiration rate in response to 2-ME-induced oxidative stress, while human leukemia cells seem to lack this regulatory mechanism. This leads to an uncontrolled production of O2·−, severe accumulation of ROS, and ultimately ROS-mediated apoptosis in leukemia cells treated with 2-ME. The biochemical differences between cancer and normal cells identified here provide a basis for the development of drug combination strategies using 2-ME with other ROS-generating agents to enhance anticancer activity. The effectiveness of such a combination strategy in killing cancer cells was demonstrated by the use of 2-ME with agents/modalities such as ionizing radiation and doxorubicin. Collectively, the data presented here strongly suggests that 2-ME may have important clinical implications for the selective killing of cancer cells. ^

El modelo 1 a 1 en las Escuelas Secundarias. Un estudio a partir de la experiencia de las provincias de Entre Ríos, Formosa y Misiones. 16H362

En el Marco del Convenio de Cooperación, N° 375/12, suscripto entre la Universidad Nacional de Misiones, el Ministerio de Educación de la Nación y la Organización de Estados Iberoamericanos (OEI), el presente proyecto pretende conocer y analizar los cambios y/o continuidades que se estarían produciendo en las aulas, instituciones, sujetos y comunidades de las provincias de Entre Ríos, Formosa y Misiones, a partir de la implementación del Programa Conectar Igualdad. Programa que resulta ser uno de los principales pilares y referentes significativos de un nuevo perfil de política educativa, por parte de un estado nacional que asume su centralidad y responsabilidad como garante del derecho a la educación y la inclusión social, educativa y digital. Cuyo fin, en última instancia, es la revalorización de la escuela pública, a partir de la promoción de la inclusión digital y el mejoramiento de la calidad de la educación, garantizando el acceso y uso de las TIC’s en los procesos de enseñanza-aprendizaje.

Césped deportivo : comportamiento del cultivar AZ-1 de Pennisetum clandestinum Hochst. ex Chiov. (kikuyo). Paraná, Entre Ríos, Argentina

A fines de 2001 se incorporó al mercado argentino el cultivar AZ-1 de Pennisetum clandestinum Hochst. ex Chiov. (kikuyo) para ser utilizado en campos deportivos o con fines ornamentales. Con el propósito de caracterizar esta variedad comercial en el departamento Paraná (provincia de Entre Ríos, Argentina) se evaluó su comportamiento y aptitud para césped ornamental y/o deportivo en condiciones de mantenimiento para césped de calidad, bajo dos condiciones de drenaje. Se evaluaron cobertura, color, textura, distancia de entrenudos, grosor de estolones y período de dormición. El ensayo se realizó en Oro Verde, departamento Paraná, en un suelo Molisol y consistió en 2 tratamientos: con y sin drenaje, con 4 repeticiones cada uno. El tamaño de la parcela fue de 2,5 por 5,0 m. El diseño experimental utilizado fue parcelas apareadas y las mediciones se realizaron desde junio hasta noviembre de 2005. Las características climáticas locales son: temperatura media anual de 18,1°C y un régimen isohigro de 947,6 mm de precipitación anual. El cultivar se comportó como apto para césped en las características y condiciones de manejo evaluadas, sin diferencias entre los tratamientos con y sin drenaje. Presentó rápida implantación, niveles altos de cobertura, color claro y uniforme, textura media y estolones gruesos a medianos a través del tiempo. En invierno no perdió cobertura ni color. Se caracterizó como un césped no apto para campos deportivos de alta exigencia, pero recomendable para clubes de bajo presupuesto y mantenimiento.

Ríos de agua y tiempo

Fil: Badui de Zogbi, María Banura. Universidad Nacional de Cuyo

Variaciones numéricas de Diaphorina citri Kuwayama (Sternorrhyncha: Psyllidae) y del Ectoparasitoide Tamarixia radiata (Waterston) (Hymenoptera: Eulophidae) en una plantación de naranjos de Entre Ríos, Argentina

Diaphorina citri es vector de la bacteria que produce la enfermedad HLB en cítricos, una de las más destructivas. En lotes comerciales de naranja dulce en Entre Ríos se analizó la abundancia espacio-temporal de adultos de D. citri y del ectoparasitoide Tamarixia radiata. Quincenalmente y durante tres años se colectaron adultos en 10 trampas cromotrópicas y los datos obtenidos fueron relacionados con el porcentaje medio de brotación de otra plantación. El número de D. citri/trampa/quincena se analizó mediante Kruskal-wallis y prueba de Mantel y la respuesta de agregación de T. radiata mediante correlación. Ambas poblaciones exhibieron la mayor abundancia el primer año: las frecuentes aplicaciones de abamectina en el segundo y tercero provocaron una marcada reducción. Espacialmente, la diferencia numérica de D. citri entre árboles no estuvo asociada a su cercanía pero la correlación entre ellos fue significativa. Temporalmente se evidenciaron cuatro picos de abundancia: tres asociados a brotación (invernal, primaveral y estival), y un cuarto no asociado a brotación. La mayor abundancia de T. radiata ocurrió en los árboles con mayor abundancia de D. citri y hubo una significativa correlación espacial entre ambas especies. El enrollamiento anti-horario del gráfico entre D. citri - T. radiata en árboles individuales sugiere una interacción huésped- parasitoide, estructurada como poblaciones locales.

Alejandro Bekes. Breviario filológico. Términos usuales en Lingüística y Teoría Literaria. Segunda edición ampliada y corregida. Entre Ríos, EDUNER, 2013, 424 pp.

Fil: Zubiría, María Isabel. Universidad Nacional de Cuyo

El sauce criollo (Salix Humboldtiana Willd.) en los ríos San Juan y Desagüadero

Fil: Roig, Fidel Antonio. Universidad Nacional de Cuyo. Facultad de Ciencias Agrarias

Antecedentes del rol mediador del Éros en los discursos de Erixímaco y Aristófanes

En el Banquete Platón pone en práctica como en ningún otro diálogo un tipo de escritura proléptica, en el sentido de que cada encomio contiene anticipos fragmentarios de tópicos que, a través de un sutil juego de rectificación y complementación, serán retomados en los discursos posteriores. Esta escritura proléptica entronca con la lectura perspectivista del diálogo, ya que cada posición discursiva no se hace sola sino en el contrapunto dialógico-filósofico que mantiene con la asumida por los otros oradores, contrapunto cuyo despliegue permite dar cuenta de las infinitas armonías invisibles que subyacen en el texto. De los múltiples contrapuntos dialógico-filosóficos que pueden trazarse en el Banquete, en este trabajo me interesa limitarme a los pueden advertirse entre los discursos de Erixímaco, Aristófanes y Sócrates-Diotima, a fin de destacar en qué medida los dos primeros discursos operan, desde distintos marcos conceptuales, como un antecedente del tercero en lo que respecta a la función mediadora del Eros

Nuevo enlace ferrovial interprovincial Santa Fe-Entre Ríos : Una oportunidad de escala regional

La provincia de Santa Fe, Argentina, se encuentra en una localización estratégica. La potencialidad de la Hidrovía Paraná-Paraguay, los corredores bioceánicos viales y la red existente de trazados ferroviarios le confieren gran dinamismo a su integración económica, social, cultural y política, no sólo hacia el interior del propio territorio, sino también en relación a las demás provincias y más allá de los confines nacionales. La región capital, cuyo núcleo es la ciudad de Santa Fe, se encuentra caracterizada por factores realmente dinámicos: el riesgo hídrico que es intrínseco del área, la intensificación de los flujos económicos pasantes, los procesos de concentración demográfica y la creciente interdependencia entre ciudades, como es el casode Santa Fe y Paraná (capital de la vecina provincia de Entre Ríos), bajo un progresivo proceso de metropolización binuclear. Estos factores, sumados a la escasa cantidad de conexiones físicas sobre el sistema fluvial del río Paraná, han instalado la creciente necesidad de contar con un nuevo enlace interprovincial, adaptado a una hipótesis de reactivación ferroviaria. El proyecto se encuentra en fase preliminar. La cuestión principal gira en torno a la decisión de su localización específica, que deberá considerar el profundo efecto transformador propio de una obra civil de gran calibre, tanto en relación a la plataforma natural como al sistema de asentamientos humanos. También sus alcances territoriales y el impacto potencial en la micro, meso y macroescala. El propósito de la investigación reside en profundizar sobre las dimensiones involucradas por el proyecto (técnica, social, económica, ambiental, de movilidad), en la búsqueda de una toma de posición que permita echar luz sobre los escenarios más beneficiosos y/o menos desfavorables, en relación a las numerosas propuestas de localización que se encuentran actualmente en discusión. El resultado es una matriz analítica basada en variables cuantitativas y cualitativas, que permite una evaluación integral de las propuestas en función de considerar, en síntesis, el grado de impacto sobre la plataforma natural sustentante, sus capacidades para revertir las problemáticas territoriales actuales, y finalmente sus posibilidades para generar nuevos ejes de desarrollo en la región o bien potenciar los existentes. Se concluye que análisis preliminares de tipo pluridimensional son necesarios para someter a discusión, como instancia previa a estudios específicos de factibilidad y viabilidad, puesto que permiten una visualización integral de las variables intervinientes, marcando el camino hacia su adecuada ponderación. Palabras clave: enlace, multimodalidad, región, transformaciones