The synergistic effect of radiation and inhibition of SPAK/OSR1 in the reduction of cell proliferation and clonogenic potential in patient-derived GBM cells in vitro

Glioblastoma (GBM) is the most aggressive brain tumor in adults. Cell invasion, migration and proliferation into the heathy brain parenchyma it’s one of the most important challenges in the treatment of this deadly tumor. One potential mechanism that GBM cells can utilize to enhance cell migration and evade pro-apoptotic signals is the tight regulation of cell volume by the STE20/SPS1-Related Proline-Alanine-Rich Protein Kinase (SPAK) and (Oxidative Stress Responsive Kinase 1) OSR1 kinases. Dynamic changes in cell volume can be used by GBM cells to disseminate through the narrow perivascular spaces of the brain. In addition, cancer cells could counteract pro-apoptotic reduction of cell volume by increasing the activity of these kinases. The objective of this project is to test the efficacy of SPAK and OSR1 inhibition alone or in combination with radiotherapy. For this purpose we evaluated the impact of this novel therapy on the proliferation, clonogenicity and apoptosis of primary patient-derived GBM cells in vitro. To achieve our goal we tested a novel SPAK/OSR1 inhibitor (a small molecule called YU566) in two patient derived GBM lines. Cell proliferation and colony formation were determined after treatment using 1uM YU566 alone or in combination with radiotherapy (at different doses 2, 4 Gray (Gy)). We found that radiation and inhibition of SPAK/OSR1 could act in a synergistic fashion, decreasing cell proliferation and clonogenic potential. The next steps in our research will be to determine the mechanisms of cell death and the implications of this therapy in vivo

A Human iPSC-derived 3D platform using primary brain cancer cells to study drug development and personalized medicine

Abstract A high throughput histology (microTMA) platform was applied for testing drugs against tumors in a novel 3D heterotypic glioblastoma brain sphere (gBS) model consisting of glioblastoma tumor cells, iPSC-derived neurons, glial cells and astrocytes grown in a spheroid. The differential responses of gBS tumors and normal neuronal cells to sustained treatments with anti-cancer drugs temozolomide (TMZ) and doxorubicin (DOX) were investigated. gBS were exposed to TMZ or DOX over a 7-day period. Untreated gBS tumors increased in size over a 4-week culture period, however, there was no increase in the number of normal neuronal cells. TMZ (100 uM) and DOX (0.3 uM) treatments caused ~30% (P~0.07) and ~80% (P < 0.001) decreases in the size of the tumors, respectively. Neither treatment altered the number of normal neuronal cells in the model. The anti-tumor effects of TMZ and DOX were mediated in part by selective induction of apoptosis. This platform provides a novel approach for screening new anti-glioblastoma agents and evaluating different treatment options for a given patient

De usuario competente del sistema monetario al dominio de la escritura de los números

En este texto nos interesa discutir algunos presupuestos sobre los saberes previos de los sujetos adultos, para quienes se elaboran propuestas de enseñanza de la numeración en materiales para la modalidad. Particularmente, nos interrogaremos qué aprendizajes se presuponen asociados al uso cotidiano del dinero por parte de adultos no escolarizados. Para ello, analizaremos el uso del dinero como recurso de enseñanza en dichos materiales y lo compararemos con el uso en libros de textos para niños para, finalmente, narrar una secuencia de enseñanza en la que se sugiere un uso diverso de este recurso, "El juego del cajero". Los resultados que presentaremos son fruto del trabajo colectivo en un taller semanal realizado entre docentes de primarias de adultos e investigadores de la FFyH y de la Facultad de Matemática, Astronomía y Física (FAMAF), UNC, constituido inicialmente para la realización del trabajo de campo de la tesis de doctorado de Delprato y en el que seguimos estudiando propuestas para llevar a cabo en el aula y poder comunicarlas. El espacio en el que se implementó esta secuencia es un Centro de Nivel Primario de Adultos (CENPA) de la ciudad de Córdoba, es decir, un espacio de enseñanza formal de matemática cuyo propósito es la atención del rezago educativo de jóvenes y adultos en el Nivel Primario. Este CENPA funciona por la tarde en un rincón del comedor de una escuela de niños. Las docentes participantes de la indagación iniciaron su ejercicio en la modalidad de la Educación de Jóvenes y Adultos (EDJA), lo que posibilita priorizar la discusión sobre los sujetos adultos y la especificidad de una propuesta que recupere, interprete, potencie y desarrolle los conocimientos y saberes de estos sujetos. Los alumnos asistentes son veinte como máximo, la mayoría de ellos mujeres de entre 20 y 30 años, migrantes (provenientes sobre todo de Bolivia), aunque también hay algunos jóvenes. Esta composición del alumnado incide en que haya una presencia continua de niños (los hijos de las alumnas, alrededor de ocho niños de 0 a 4 años) y genera que el horario y la asistencia estén sujetos a las "obligaciones" domésticas. Las alumnas están distribuidas en dos ciclos. Además, hay un grupo de alfabetización que concurre dos veces por semana, en un horario diferenciado. Proyectos "Educación básica rural y de jóvenes y adultos. Políticas, instituciones y actores", CIFFyH, UNC. Agencia Nacional de Investigación Científica y Tecnológica-FONCYT, Convocatoria Proyectos Bicentenario, Temas Abiertos-PICT-2010-0890, SECYT, Res. 214/10; "Indagaciones sobre la formación de docentes en Matemática. Perspectivas, tendencias y desafíos" FaMAF, UNC,SECYT, Res. 159/09; MCyT Res. 210/2007. "Condiciones de la enseñanza matemática a adultos con baja escolaridad", tesis en proceso bajo la dirección de Fregona, Doctorado en Educación de la FFyH, UNC.Fil: Delprato, María Fernanda. Universidad Nacional de Córdoba. Facultad de Filosofía y Humanidades. Escuela de Ciencias de la Educación; Argentina.Fil: Fregona, Dilma. Universidad Nacional de Córdoba. Facultad de Filosofía y Humanidades. Escuela de Ciencias de la Educación; Argentina.Fil: Gerez Cuevas, José Nicolás. Universidad Nacional de Córdoba. Facultad de Filosofía y Humanidades. Escuela de Ciencias de la Educación; Argentina.Educación General (incluye capacitación, pedagogía y didáctica

Supramolecular Crafting of Self-Assembling Camptothecin Prodrugs with Enhanced Efficacy against Primary Cancer Cells

Abstract Chemical modification of small molecule hydrophobic drugs is a clinically proven strategy to devise prodrugs with enhanced treatment efficacy. While this prodrug strategy improves the parent drug&apos;s water solubility and pharmacokinetic profile, it typically compromises the drug&apos;s potency against cancer cells due to the retarded drug release rate and reduced cellular uptake efficiency. Here we report on the supramolecular design of self-assembling prodrugs (SAPD) with much improved water solubility while maintaining high potency against cancer cells. We found that camptothecin (CPT) prodrugs created by conjugating two CPT molecules onto a hydrophilic segment can associate into filamentous nanostructures in water. Our results suggest that these SAPD exhibit much greater efficacy against primary brain cancer cells relative to that of irinotecan, a clinically used CPT prodrug. We believe these findings open a new avenue for rational design of supramolecular prodrugs for cancer treatment

Regulation of Glioblastoma Tumor-Propagating Cells by the Integrin Partner Tetraspanin CD151

Glioblastoma (GBM) stem cells (GSCs) represent tumor-propagating cells with stem-like characteristics (stemness) that contribute disproportionately to GBM drug resistance and tumor recurrence. Understanding the mechanisms supporting GSC stemness is important for developing therapeutic strategies for targeting GSC-dependent oncogenic mechanisms. Using GBM-derived neurospheres, we identified the cell surface tetraspanin family member CD151 as a novel regulator of glioma cell stemness, GSC self-renewal capacity, migration, and tumor growth. CD151 was found to be overexpressed in GBM tumors and GBM neurospheres enriched in GSCs. Silencing CD151 inhibited neurosphere forming capacity, neurosphere cell proliferation, and migration and attenuated the expression of markers and transcriptional drivers of the GSC phenotype. Conversely, forced CD151 expression promoted neurosphere self-renewal, cell migration, and expression of stemness-associated transcription factors. CD151 was found to complex with integrins α3, α6, and β1 in neurosphere cells, and blocking CD151 interactions with integrins α3 and α6 inhibited AKT phosphorylation, a downstream effector of integrin signaling, and impaired sphere formation and neurosphere cell migration. Additionally, targeting CD151 in vivo inhibited the growth of GBM neurosphere-derived xenografts. These findings identify CD151 and its interactions with integrins α3 and α6 as potential therapeutic targets for inhibiting stemness-driving mechanisms and stem cell populations in GBM

Expression and epigenetic modulation of sonic hedgehog-GLI1 pathway genes in neuroblastoma cell lines and tumors

It is well known that sonic hedgehog signaling pathway plays a vital role during early embryonic development. It is also responsible for stem cell renewal and development of several cancers like colorectal and breast carcinoma and major brain tumors as medulloblastoma and glioblastoma. The role of sonic hedgehog signaling in the development of neuroblastoma has not been thoroughly investigated. In this study, we attempted to determine the expression of Bmi-1 stem cell marker and of Shh pathway downstream target genes glioma-associated oncogene homolog 1 (GLI1), protein patched homolog 1 (PTCH1), Cyclin D2, plakoglobin (γ catenin), NK2 homeobox 2 (NKX2.2), paired box gene 6 (PAX6), secreted frizzled-related protein 1 (SFRP1), and hedgehog interacting protein (HHIP) in 11 neuroblastoma cell lines and 41 neuroblastoma samples. Also, inhibition of the pathway was performed genetically by GLI1 knockdown siRNA or chemically by cyclopamine. After inhibition, low transcript expression was detected in downstream target genes like PTCH1, in the cell lines. We further preformed promoter methylation studies of Cyclin D2, PTCH1, HHIP, and SFRP1 genes by melting curve analysis-based methylation assay (MCA-Meth) and methylation-specific PCR (MSP). Results revealed no methylation in Cyclin D2 gene promoter in neuroblastoma samples or in cell lines; one cell line (MHH-NB-11) showed PTCH1 methylation; 3/11 (27%) cell lines and 9/41 (22%) neuroblastoma samples showed HHIP methylation; and 3/11 (27%) cell lines and 11/41 (27%) samples showed SFRP1 methylation. Taken together, our results suggest the possibility of two levels of control of the sonic hedgehog signaling pathway: transcriptional and epigenetic, which might offer new therapeutic possibilities to modulate the pathway and try to suppress tumor growth

Brain-on-a-chip model enables analysis of human neuronal differentiation and chemotaxis

Migration of neural progenitors in the complex tissue environment of the central nervous system is not well understood. Progress in this area has the potential to drive breakthroughs in neuroregenerative therapies, brain cancer treatments, and neurodevelopmental studies. To a large extent, advances have been limited due to a lack of controlled environments recapitulating characteristics of the central nervous system milieu. Reductionist cell culture models are frequently too simplistic, and physiologically more relevant approaches such as ex vivo brain slices or in situ experiments provide little control and make information extraction difficult. Here, we present a brain-on-chip model that bridges the gap between cell culture and ex vivo/in vivo conditions through recapitulation of self-organized neural differentiation. We use a new multi-layer silicone elastomer device, over the course of four weeks to differentiate pluripotent human (NTERA2) cells into neuronal clusters interconnected with thick axonal bundles and interspersed with astrocytes, resembling the brain parenchyma. Neurons within the device express the neurofilament heavy (NF200) mature axonal marker and the microtubule-associated protein (MAP2ab) mature dendritic marker, demonstrating that the devices are sufficiently biocompatible to allow neuronal maturation. This neuronal-glial environment is interfaced with a layer of human brain microvascular endothelial cells showing characteristics of the blood-brain barrier including the expression of zonula occludens (ZO1) tight junctions and increased trans-endothelial electrical resistance. We used this device to model migration of human neural progenitors in response to chemotactic cues within a brain-tissue setting. We show that in the presence of an environment mimicking brain conditions, neural progenitor cells show a significantly enhanced chemotactic response towards shallow gradients of CXCL12, a key chemokine expressed during embryonic brain development and in pathological tissue regions of the central nervous system. Our brain-on-chip model thus provides a convenient and scalable model of neural differentiation and maturation extensible to analysis of complex cell and tissue behaviors.publishe

NKCC1 Regulates Migration Ability of Glioblastoma Cells by Modulation of Actin Dynamics and Interacting with Cofilin

Glioblastoma (GBM) is the most aggressive primary brain tumor in adults. The mechanisms that confer GBM cells their invasive behavior are poorly understood. The electroneutral Na+-K+-2Cl− co-transporter 1 (NKCC1) is an important cell volume regulator that participates in cell migration. We have shown that inhibition of NKCC1 in GBM cells leads to decreased cell migration, in vitro and in vivo. We now report on the role of NKCC1 on cytoskeletal dynamics. We show that GBM cells display a significant decrease in F-actin content upon NKCC1 knockdown (NKCC1-KD). To determine the potential actin-regulatory mechanisms affected by NKCC1 inhibition, we studied NKCC1 protein interactions. We found that NKCC1 interacts with the actin-regulating protein Cofilin-1 and can regulate its membrane localization. Finally, we analyzed whether NKCC1 could regulate the activity of the small Rho-GTPases RhoA and Rac1. We observed that the active forms of RhoA and Rac1 were decreased in NKCC1-KD cells. In summary, we report that NKCC1 regulates GBM cell migration by modulating the cytoskeleton through multiple targets including F-actin regulation through Cofilin-1 and RhoGTPase activity. Due to its essential role in cell migration NKCC1 may serve as a specific therapeutic target to decrease cell invasion in patients with primary brain cancer