Hypoxia-inducible factor HIF-1α modulates drugs resistance in colon cancer cells / Factor inducible por hipoxia HIF-1α modula la resistencia a drogas en células de cáncer de colon

Abstract Introduction: Drug resistance mechanisms may be associated with decreased cell death and its induction may depend on the response to oxidative stress caused by hypoxia. The correlation between hypoxia-inducible factor HIF-1α, the number of reactive oxygen species and their effect on cell survival has not yet been evaluated. Objective: The purpose of this study was to evaluate the effect of HIF-1α activity and reactive oxygen species (ROS) accumulation in apoptosis of colon cancer cells. Materials and methods: HT29 colon cancer cells were treated with Cobalt(II) chloride (CoCl2) or doxorubicin and the activity of HIF-1α was determined by ELISA assay. ROS were determined using fluorescence probe carboxy-H2DFFDA. Apoptosis was assessed by caspase-3 activation analysis, and PUMA and BAX mRNA levels by qRT-PCR. The reduction of the antiapoptotic effect due to hypoxia was attenuated by use of the endonuclease APE-1 (E3330) inhibitor. The endonuclease E3330 APE-1 inhibitor allowed evaluating the effect of ROS generated by doxorubicin and CoCl2 on apoptosis. Results: Chemical hypoxia in combination with doxorubicin is an oxidative stressor in HT29 cells and induces a reduction in the apoptotic process in a time-dependent manner. Conclusion: Resistance to hypoxia and doxorubicin-mediated cell death could be controlled by a mechanism related to the activity of HIF-1α and the amount of reactive oxygen species generated.

Resumen Introducción. Los mecanismos de resistencia a drogas podrían asociarse con disminución en la muerte celular y su inducción podría depender de la respuesta al estrés oxidativo que origina la hipoxia. La correlación entre factor inducible por hipoxia HIF-1α, cantidad de especies reactivas de oxígeno y su efecto sobre la supervivencia celular aún no ha sido evaluada. Objetivo. Evaluar el efecto de la inducción de la actividad de HIF-1α y la cantidad de especies reactivas de oxígeno sobre la apoptosis en células de cáncer de colon. Materiales y métodos. Células de cáncer de colon HT29 fueron tratadas con cloruro de cobalto (CoCl2) o doxorrubicina; la actividad de HIF-1α se evaluó por ELISA. Las especies reactivas de oxígeno fueron determinadas con sonda fluorescente carboxi-H2DFFDA. La apoptosis fue evaluada por la actividad de caspasa-3 y los niveles de mRNA de los genes proapoptóticos PUMA y BAX por qRT-PCR. El inhibidor de la endonucleasa APE-1 E3330 permitió evaluar el efecto de las especies reactivas de oxígeno generadas por doxorubicina y CoCl2 sobre la apoptosis. Resultados. La hipoxia química combinada + doxorubicina es estresor oxidativo en células HT29 e induce una reducción en el proceso apoptótico de manera tiempo dependiente. Conclusión. La resistencia a la muerte celular mediada por hipoxia y doxorubicina podría estar controlada por un mecanismo relacionado con la actividad de HIF-1α y la cantidad de especies reactivas de oxígeno generadas.

[STAT3 activation by hypoxia in in vitro models of cervix cancer and endothelial cells].

INTRODUCTION: The biological behavior of cancer cells is influenced by the tumor microenvironment in which they develop. In this context, stressor stimuli such as hypoxia are considered critical for tumor development and therapeutic management. Cellular response to various stimuli is evidenced in the activation of intracellular signaling pathways such as JAK/STAT, which is one of the most important for its effects in differentiation and cell proliferation. OBJECTIVE: To evaluate the condition of the JAK/STAT pathway through the expression/activation of the STAT3 protein in cervix cancer cells (HeLa) and endothelial cells (EA.hy926) subjected to hypoxia. MATERIAL AND METHODS: Cell lines were subjected to physical (1% O2) or chemical (deferoxamine, DFO, 100 µM) hypoxia for 2, 6 and 24 hours. Changes in the expression and activation of STAT3, and its subcellular localization by indirect immunofluorescence, were determined by western blot. RESULTS: Hypoxia was evidenced by the activation and translocation to the nucleus of HIF-1. Neither physical nor chemical hypoxia altered STAT3 expression, but it did affect its activation, as seen in its phosphorylation and translocation to the nucleus in the two models under study. CONCLUSIONS: The present study highlights the importance of hypoxia as a stimulus that modifies the activation of the STAT3 protein in HeLa and EA.hy926 cells, which makes it an important factor in the design of therapeutic strategies against cancer.

Oxidative Stress Promotes Doxorubicin-Induced Pgp and BCRP Expression in Colon Cancer Cells Under Hypoxic Conditions.

P-glycoprotein (Pgp) and breast cancer resistance protein (BCRP) are ATP binding cassette (ABC) transporters that are overexpressed in different drug-resistant cancer cell lines. In this study, we investigated whether doxorubicin promotes Pgp and/or BCRP expression to induce drug resistance in colon cancer cells under hypoxic conditions. We analyzed HIF-1α activity via ELISA, Pgp, and BCRP expression by qRT-PCR and the relationship between doxorubicin uptake and ABC transporter expression via confocal microscopy in HT-29WT and HT-29 doxorubicin-resistant colon cancer cells (HT-29DxR). These cells were treated with doxorubicin and/or CoCl2 (chemical hypoxia), and reactive oxygen species inductors. We found that the combination of chemically induced hypoxia and doxorubicin promoted Pgp mRNA expression within 24 h in HT-29WT and HT-29DxR cells. Both doxorubicin and CoCl2 alone or in combination induced Pgp and BCRP expression, as demonstrated via confocal microscopy in each of the above two cell lines. Thus, we surmised that Pgp and BCRP expression may result from synergistic effects exerted by the combination of doxorubicin-induced ROS production and HIF-1α activity under hypoxic conditions. However, HIF-1α activity disruption via the administration of E3330, an APE-1 inhibitor, downregulated Pgp expression and increased doxorubicin delivery to HT-29 cells, where it served as a substrate for Pgp, indicating the existence of an indirect relationship between Pgp expression and doxorubicin accumulation. Thus, we concluded that Pgp and BCRP expression can be regulated via cross-talk between doxorubicin and hypoxia, promoting drug resistance in HT-29 WT, and HT-29DxR cells and that this process may be ROS dependent. J. Cell. Biochem. 118: 1868-1878, 2017. © 2017 Wiley Periodicals, Inc.

Activación de STAT3 por hipoxia en modelos in vitro de cáncer de cuello uterino y en células endoteliales / STAT3 activation by hypoxia in in vitro models of cervix cancer and endothelial cells

resumen Introducción: El microambiente tumoral influye en el comportamiento de las células cancerosas. Especialmente, el estímulo de agentes estresantes, como la hipoxia, se convierte en un factor crítico para la evolución y el tratamiento del cáncer. La reacción celular frente a diversos estímulos se manifiesta en la activación de vías de señalización como la JAK/STAT, una de las más importantes por sus efectos en la diferenciación y proliferación celular. Objetivo: Evaluar el estado de la vía JAK/STAT mediante la expresión o activación de la proteína STAT3 en células de cáncer de cuello uterino (HeLa) y en células endoteliales (EA.hy926) sometidas a hipoxia. Materiales y métodos: Las líneas celulares se sometieron a condiciones de hipoxia física (1 % de O2) o química (100 μM de deferoxamina, DFO) durante dos, seis y 24 horas. Mediante Western blot se determinó el cambio en la expresión y activación de STAT3, y mediante inmunofluorescencia indirecta su localización subcelular. Resultados:. La hipoxia se evidenció por la activación y translocación al núcleo del HIF-1. Ni la hipoxia física ni la química alteraron la expresión de STAT3, pero sí la activación, según se comprobó por su fosforilación y su translocación al núcleo en los dos modelos bajo estudio. Conclusiones: Se evidenció la importancia de la hipoxia como un estímulo que modifica la activación de la proteína STAT3 en las células HeLa y EA.hy926, lo cual la convierte en un elemento importante en el diseño de estrategias terapéuticas contra el cáncer.

Abstract Introduction: The biological behavior of cancer cells is influenced by the tumor microenvironment in which they develop. In this context, stressor stimuli such as hypoxia are considered critical for tumor development and therapeutic management. Cellular response to various stimuli is evidenced in the activation of intracellular signaling pathways such as JAK/STAT, which is one of the most important for its effects in differentiation and cell proliferation. Objective: To evaluate the condition of the JAK/STAT pathway through the expression/activation of the STAT3 protein in cervix cancer cells (HeLa) and endothelial cells (EA.hy926) subjected to ypoxia. Material and methods: Cell lines were subjected to physical (1% O2) or chemical (deferoxamine, DFO, 100 μM) hypoxia for 2, 6 and 24 hours. Changes in the expression and activation of STAT3, and its subcellular localization by indirect immunofluorescence, were determined by western blot. Results: Hypoxia was evidenced by the activation and translocation to the nucleus of HIF-1. Neither physical nor chemical hypoxia altered STAT3 expression, but it did affect its activation, as seen in its phosphorylation and translocation to the nucleus in the two models under study. Conclusions: The present study highlights the importance of hypoxia as a stimulus that modifies the activation of the STAT3 protein in HeLa and EA.hy926 cells, which makes it an important factor in the design of therapeutic strategies against cancer.

The cross-talk between canonical and non-canonical Wnt-dependent pathways regulates P-glycoprotein expression in human blood-brain barrier cells.

In this work, we investigate if and how transducers of the 'canonical' Wnt pathway, i.e., Wnt/glycogen synthase kinase 3 (GSK3)/ß-catenin, and transducers of the 'non-canonical' Wnt pathway, i.e., Wnt/RhoA/RhoA kinase (RhoAK), cooperate to control the expression of P-glycoprotein (Pgp) in blood-brain barrier (BBB) cells. By analyzing human primary brain microvascular endothelial cells constitutively activated for RhoA, silenced for RhoA or treated with the RhoAK inhibitor Y27632, we found that RhoAK phosphorylated and activated the protein tyrosine phosphatase 1B (PTP1B), which dephosphorylated tyrosine 216 of GSK3, decreasing the GSK3-mediated inhibition of ß-catenin. By contrast, the inhibition of RhoA/RhoAK axis prevented the activation of PTP1B, enhanced the GSK3-induced phosphorylation and ubiquitination of ß-catenin, and reduced the ß-catenin-driven transcription of Pgp. The RhoAK inhibition increased the delivery of Pgp substrates like doxorubicin across the BBB and improved the doxorubicin efficacy against glioblastoma cells co-cultured under a BBB monolayer. Our data demonstrate that in human BBB cells the expression of Pgp is controlled by a cross-talk between canonical and non-canonical Wnt pathways. The disruption of this cross-talk, e.g., by inhibiting RhoAK, downregulates Pgp and increases the delivery of Pgp substrates across the BBB.

Nanoparticle- and liposome-carried drugs: new strategies for active targeting and drug delivery across blood-brain barrier.

The blood-brain barrier (BBB), the unusual microvascular endothelial interface between the central nervous system (CNS) and the circulatory system, is a major hindrance to drug delivery in the brain parenchyma. Besides the absence of fenestrations and the abundance of tight junctions, ATP-binding cassette (ABC) transporters critically reduce drug entry within the CNS, as they carry many drugs back into the bloodstream. Nanoparticle- and liposome-carried drugs, because of their increased cellular uptake and reduced efflux through ABC transporters, have been developed in recent times to circumvent the low drug permeability of the BBB. This review discusses the role of ABC transporters in controlling drug penetration into the brain parenchyma, the rationale for using nanoparticle- and liposome-based strategies to increase drug delivery across the BBB and new therapeutic strategies for using nanoparticle- and liposome-carried drugs in different conditions, ranging from CNS tumors and neurodegenerative diseases to viral infections and epilepsy.

¿Continuamos informando o asumimos el cambio? / May We Continue Informing or May We Assume the Change?

Los sistemas educativos tradicionales han convertido a los estudiantes en entidades pasivas que guardan información transitoriamente, sin mayores capacidades para procesarla y aplicarla a la solución de situaciones de la vida diaria. La discusión generalizada entre los investigadores de la educación plantea la necesidad de cambiar las estrategias pedagógicas que han favorecido esas situaciones, para permitir una formación integral del individuo como ser capaz de razonar, crear, innovar y desarrollarse autónomamente. Las exigencias gubernamentales, institucionales e incluso las políticas de la globalización y el desarrollo del ser humano basado en la economía del conocimiento reclaman ese cambio de estrategias en el sistema pedagógico de enseñanza-aprendizaje; pues aunque éste se encuentre amenazado por la resistencia natural que ofrece el enfrentamiento con una cultura establecida, debe darse desde el aula de una manera inmediata. Como respuesta a esa necesidad, la Unidad de Bioquímica del Instituto de Ciencias Básicas de la Facultad de Medicina (Universidad del Rosario) plantea una estrategia no tradicional en pos del cambio.

Traditional educative systems, had convert students in passive recipient, who are keeping information without any possibility to process and apply it for the solution of daily problems. General discussion between educative researchers, had been establish the necessity to change the educative strategies that have been supporting those situations, looking for an integral education of the individual in terms of the capacity to argue, to create, to innovate, to develop by him self with independence. Governments, institutions and the politics requirements, that handle the globalization and the human development based in the knowledge economy, demand changes in the pedagogical system strategies in spite of the barriers from the traditional educative system. Responding to these necessity, the Biochemistry Department belong to the Basic Sciences Institute from the Medicine Faculty in the University of Rosario, is applying a different pedagogical strategy.

CXCR4-mediated suppressor of cytokine signaling up-regulation inactivates growth hormone function.

Coordinated action between cytokines and chemokines is required for effective endocrine and immune responses. Proteins of both families promote receptor oligomerization, activation of the Janus kinase (JAK)/STAT pathway, and transcription of many genes, including the suppressor of cytokine signaling (SOCS) family. In this study, we show that chemokine-mediated SOCS1 and SOCS3 up-regulation modulates the signaling and function associated to a cytokine receptor, both in vitro and in vivo. The effect is mediated by SOCS binding to JAK2 and to the cytokine receptor, which blocks subsequent signaling events. The data reinforce the premise of cytokine-chemokine cross-talk, which helps contribute to modulating individual responses and in defining the functional plasticity of the immune system.

Functional inactivation of CXC chemokine receptor 4-mediated responses through SOCS3 up-regulation.

Hematopoietic cell growth, differentiation, and chemotactic responses require coordinated action between cytokines and chemokines. Cytokines promote receptor oligomerization, followed by Janus kinase (JAK) kinase activation, signal transducers and transactivators of transcription (STAT) nuclear translocation, and transcription of cytokine-responsive genes. These include genes that encode a family of negative regulators of cytokine signaling, the suppressors of cytokine signaling (SOCS) proteins. After binding their specific receptors, chemokines trigger receptor dimerization and activate the JAK/STAT pathway. We show that SOCS3 overexpression or up-regulation, stimulated by a cytokine such as growth hormone, impairs the response to CXCL12, measured by Ca(2+) flux and chemotaxis in vitro and in vivo. This effect is mediated by SOCS3 binding to the CXC chemokine receptor 4 receptor, blocking JAK/STAT and Galpha(i) pathways, without interfering with cell surface chemokine receptor expression. The data provide clear evidence for signaling cross-talk between cytokine and chemokine responses in building a functional immune system.

Producción de la proteína recombinante JAK2 de ratón / Production of mouse recombinant JAK2 protein

El presente trabajo describe la producción de la proteína recombinante JAK2, empleando un sistema de vectores de expresión baculovirales. La infección de células Sf9, derivadas del tejido de ovario de Spodoptera frugiperda, permitió establecer las condiciones óptimas de dilución del virus en 10 µl/ml de medio y de tiempo de recolección en 72 horas post-infección. Se recolectaron y ultrafiltraron extractos correspondientes a la infección de 5 x 10 a la 7 células, aproximadamente, y se sometieron a cromatografía de intercambio iónico (DEAE-Sephacel), lográndose purificar parcialmente la proteína JAK2 por medio de un gradiente continuo de fuerza iónica (NaCl 10-500 mM). La presencia de JAK2 se verificó por Western blot, empleando un anticuerpo policlonal producido en conejo contra los residuos 758-776 de JAK2 de ratón. Los resultados indicaron que la proteína obtenida por este sistema de expresión se sintetiza en forma fosforilada/activada