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Glioblastoma is the most common and aggressive primary brain tumor, characterized by its high chemoresistance and the presence of a cell subpopulation that persists under hypoxic niches, called glioblastoma stem-like cells (GSCs). The chemoresistance of GSCs is mediated in part by adenosine signaling and ABC transporters, which extrude drugs outside the cell, such as the multidrug resistance-associated proteins (MRPs) subfamily. Adenosine promotes MRP1-dependent chemoresistance under normoxia. However, adenosine/MRPs-dependent chemoresistance under hypoxia has not been studied until now. Transcript and protein levels were determined by RT-qPCR and Western blot, respectively. MRP extrusion capacity was determined by intracellular 5 (6)-Carboxyfluorescein diacetate (CFDA) accumulation. Cell viability was measured by MTS assays. Cell cycle and apoptosis were determined by flow cytometry. Here, we show for the first time that MRP3 expression is induced under hypoxia through the A2B adenosine receptor. Hypoxia enhances MRP-dependent extrusion capacity and the chemoresistance of GSCs. Meanwhile, MRP3 knockdown decreases GSC viability under hypoxia. Downregulation of the A

Glioblastoma is the most common and aggressive primary brain tumor, characterized by its high chemoresistance and the presence of a cell subpopulation that persists under hypoxic niches, called glioblastoma stem-like cells (GSCs). The chemoresistance of GSCs is mediated in part by adenosine signaling and ABC transporters, which extrude drugs outside the cell, such as the multidrug resistance-associated proteins (MRPs) subfamily. Adenosine promotes MRP1-dependent chemoresistance under normoxia. However, adenosine/MRPs-dependent chemoresistance under hypoxia has not been studied until now. Transcript and protein levels were determined by RT-qPCR and Western blot, respectively. MRP extrusion capacity was determined by intracellular 5 (6)-Carboxyfluorescein diacetate (CFDA) accumulation. Cell viability was measured by MTS assays. Cell cycle and apoptosis were determined by flow cytometry. Here, we show for the first time that MRP3 expression is induced under hypoxia through the A2B adenosine receptor. Hypoxia enhances MRP-dependent extrusion capacity and the chemoresistance of GSCs. Meanwhile, MRP3 knockdown decreases GSC viability under hypoxia. Downregulation of the A2B receptor decreases MRP3 expression and chemosensibilizes GSCs treated with teniposide under hypoxia. These data suggest that hypoxia-dependent activation of A2B adenosine receptor promotes survival of GSCs through MRP3 induction.

Immunohistochemistry (IHC) for O6-methylguanine-DNA-methyltransferase (MGMT) has shown heterogeneous results. Cell staining intensity has not been included as a quantifiable variable in IHC analyses. We performed MGMT IHC in 29 patients diagnosed as glioblastoma classifying cells into three categories based on nuclear staining intensity compared with adjacent endothelium. The median proportions of strong-moderate, weak and no staining cells were 10%, 16% and 71%, respectively. The proportion of positive cases for MGMT expression varies from 38% to 52% depending on the classification of weakly stained cells. This letter challenges previous studies that have not included intensity as a variable for IHC analysis.

// Angelo Torres 1 , Yosselyn Vargas 1 , Daniel Uribe 1 , Catherine Jaramillo 1 , Alejandra Gleisner 2 , Flavio Salazar-Onfray 2 , Mercedes N. Lopez 2 , Romulo Melo 3 , Carlos Oyarzun 1 , Rody San Martin 1 , Claudia Quezada 1 1 Laboratorio de Patologia Molecular, Instituto de Bioquimica y Microbiologia, Facultad de Ciencias, Universidad Austral de Chile, Valdivia, Chile 2 Instituto Milenio de Inmunologia e Inmunoterapia, Facultad de Medicina, Universidad de Chile, Santiago, Chile 3 Servicio de Neurocirugia, Instituto de Neurocirugia Dr. Asenjo, Santiago, Chile Correspondence to: Claudia Quezada, email: claudiaquezada@uach.cl Keywords: adenosine receptors, ATP-binding cassette (ABC) transporter superfamily, glioblastoma stem-like cells, multiple drug resistance Received: February 18, 2016 Accepted: August 29, 2016 Published: September 15, 2016 ABSTRACT MRP1 transporter correlates positively with glioma malignancy and the Multiple Drug Resistance (MDR) phenotype in Glioblastoma Multiforme (GBM). Evidence shows that the MRP1 transporter is controlled by the adenosine signalling axis. The aim of this study was to identify the role of adenosine on the MDR phenotype in Glioblastoma Stem-like Cells (GSCs), the cell population responsible for the tumorigenic and chemoresistance capabilities of this tumour. We found that GSCs have increased intrinsic capacity to generate extracellular adenosine, thus controlling MRP1 transporter expression and activity via activation of the adenosine A 3 receptor (A 3 AR). We showed PI3K/Akt and MEK/ERK1/2 signaling pathways downstream A 3 AR to control MRP1 in GSCs. In vitro pharmacological blockade of A 3 AR had a chemosensitizing effect, enhancing the actions of antitumour drugs and decreasing cell viability and proliferation of GSCs. In addition, we produced an in vivo xenograft model by subcutaneous inoculation of human GSCs in NOD/SCID-IL2Rg null mice. Pharmacological blockade of A 3 AR generated a chemosensitizing effect, enhancing the effectiveness of the MRP1 transporter substrate, vincristine, reducing tumour size and the levels of CD44 and Nestin stem cell markers as well as the Ki-67 proliferation indicator. In conclusion, we demonstrated the chemosensitizing effect of A 3 AR blockade on GSCs.

Background: Patients with Glioblastoma multiforme (GBM) have a five years survival of less than 5%, but the response to chemotherapy with alkylating agents can vary depending on the methylation status of O6-methylguanine-DNA-methyltransferase (MGMT). Genetic testing has limitations for routine use, while immunohistochemistry (IHC) offers a fast and affordable technique but with heterogeneous results in the literature. Aim: To evaluate MGMT expression by IHC in tumor tissue of Chilean patients with GBM. Material and Methods: Tumor samples of 29 patients with a pathological diagnosis of GBM were studied. We performed IHC staining and manual analysis of positive and negative cells for MGMT expression. A cut-off of at least 10% of cells expressing MGMT was used. Demographic and clinical features of patients were obtained from clinical records. Results: The median number of cells counted per case was 692 (interquartile range [IQR] 492-928). Fifteen cases (52%) were positive for MGMT expression. Median overall survival was 5.3 months (IQR 3.4-12-8). The effect of MGMT expression on the therapeutic response was not studied since only 3 patients received chemotherapy. Conclusions: Our results are similar to international reports, but we were not able to determine the association between MGMT expression and therapeutic response.

Patients with Glioblastoma multiforme (GBM) have a five years survival of less than 5%, but the response to chemotherapy with alkylating agents can vary depending on the methylation status of O6-methylguanine-DNA-methyltransferase (MGMT). Genetic testing has limitations for routine use, while immunohistochemistry (IHC) offers a fast and affordable technique but with heterogeneous results in the literature.To evaluate MGMT expression by IHC in tumor tissue of Chilean patients with GBM.Tumor samples of 29 patients with a pathological diagnosis of GBM were studied. We performed IHC staining and manual analysis of positive and negative cells for MGMT expression. A cut-off of at least 10% of cells expressing MGMT was used. Demographic and clinical features of patients were obtained from clinical records.The median number of cells counted per case was 692 (interquartile range [IQR] 492-928). Fifteen cases (52%) were positive for MGMT expression. Median overall survival was 5.3 months (IQR 3.4-12-8). The effect of MGMT expression on the therapeutic response was not studied since only 3 patients received chemotherapy.Our results are similar to international reports, but we were not able to determine the association between MGMT expression and therapeutic response.

Traumatic brain injury (TBI) is the most important cause of disability in individuals under the age of 45 years and thus represents a significant social and economic burden. Evidence strongly suggests that oxidative stress is a cornerstone event leading to and propagating secondary injury mechanisms such as excitotoxicity, mitochondrial dysfunction, apoptosis, autophagy, brain edema, and inflammation. TBI has defied conventional approaches to diagnosis and therapy development because of its heterogeneity and complexity. Therefore, it is necessary to explore alternative approaches to therapy development for TBI. The aim of this review is to present a therapeutic approach for TBI, taking into account the evidence supporting the role for oxidative stress in the pathophysiological processes of secondary brain injury. The role of agents such as mitochondria-targeted antioxidants (melatonin and new mitochondria-targeted antioxidants), nicotinamide adenine dinucleotide phosphate (NADPH) inhibitors (antioxidant vitamins and apocynin), and other compounds having mainly antioxidant properties (hydrogen-rich saline, sulforaphane, U-83836E, omega-3, and polyphenols) is covered. The rationale for innovative antioxidant therapies based on current knowledge and particularly the most recent studies regarding this field is discussed. Particular considerations and translational potential of new TBI treatments are examined and a novel therapeutic proposal for TBI is presented.

Glioblastoma multiforme (GBM) is a brain tumour characterised by a remarkably high chemoresistance and infiltrating capability. To date, chemotherapy with temozolomide has contributed only poorly to improved survival rates in patients. One of the most important mechanisms of chemoresistance comes about through the activity of certain proteins from the ATP-binding cassette superfamily that extrudes antitumour drugs, or their metabolites, from cells. We identify an increased expression of the multiple drug resistance-associated protein 1 (Mrp1) in glioblastoma multiforme biopsies and in T98G and G44 cell lines. The activity of this transporter was also confirmed by measuring the extrusion of the fluorescent substrate CFDA. The sensitivity of GBM cells was low upon exposure to temozolomide, vincristine and etoposide, with decreases in cell viability of below 20% seen at therapeutic concentrations of these drugs. However, combined exposure to vincristine or etoposide with an inhibitor of Mrp1 efficiently decreased cell viability by up to 80%. We conclude that chemosensitization of cells with inhibitors of Mrp1 activity might be an efficient tool for the treatment of human GBM.

Background: Mortality rate is dramatically high in high grade brain tumors. The presence of multiple drug resistance transporters in glioblastoma multiforme, has contributed largely to the poor effcacy of targeted therapy against cancer in the central nervous system. Aim: To analyze the percentage of survival and mortality of patients with glioblastoma multiforme in a cohort of patients in Chile and to co-rrelate the chemo-resistance of these cells with the expression level of multiple drug resistance transporters. Materials and Methods: Eighteen biopsies of glioblastoma multiforme were obtained from patients at the Institute of Neurosurgery Dr. Asenjo (INCA). The tumor cells were obtained from primary cultures and the expression and activity of multiple drug resistance transporters was assessed by RT-PCR and immunohistochemistry. Population-based study was performed using the databases of the Department of Neurosurgery of INCA. Results: The number of patients with glioblastoma multiforme increased between 2007 and 2009, from 3.5% to 7.9% of total brain tumors. Mortality of these tumors is 90 % at three years. A high expression and activity of the multiple drugs resistance associated protein 1 (Mrp1) transporter was observed in primary cultures of biopsies. Conclusions: We propose that Mrp1 activity is responsible for the chemo-resistance of the glioblastoma multiforme and inhibition of this transporter could represent a plausible strategy for the treatment.

Glioblastoma multiforme (GBM) cells are characterised by their extreme chemoresistance. The activity of multiple-drug resistance (MDR) transporters that extrude antitumor drugs from cells plays the most important role in this phenomenon. To date, the mechanism controlling the expression and activity of MDR transporters is poorly understood. Activity of the enzyme ecto-5′-nucleotidase (CD73) in tumor cells, which hydrolyses AMP to adenosine, has been linked to immunosuppression and prometastatic effects in breast cancer and to the proliferation of glioma cells. In this study, we identify a high expression of CD73 in surgically resected samples of human GBM. In primary cultures of GBM, inhibition of CD73 activity or knocking down its expression by siRNA reversed the MDR phenotype and cell viability was decreased up to 60% on exposure to the antitumoral drug vincristine. This GBM chemosensitization was caused by a decrease in the expression and activity of the multiple drug associated protein 1 (Mrp1), the most important transporter conferring multiple drug resistance in these cells. Using pharmacological modulators, we have recognized the adenosine A3 receptor subtype in mediation of the chemoresistant phenotype in these cells. In conclusion, we have determined that the activity of CD73 to trigger adenosine signaling sustains chemoresistant phenotype in GBM cells. J. Cell. Physiol. 228: 602–608, 2013. © 2012 Wiley Periodicals, Inc.

Mortality rate is dramatically high in high grade brain tumors. The presence of multiple drug resistance transporters in glioblastoma multiforme, has contributed largely to the poor efficacy of targeted therapy against cancer in the central nervous system.To analyze the percentage of survival and mortality of patients with glioblastoma multiforme in a cohort of patients in Chile and to co-rrelate the chemo-resistance of these cells with the expression level of multiple drug resistance transporters.Eighteen biopsies of glioblastoma multiforme were obtained from patients at the Institute of Neurosurgery Dr. Asenjo (INCA). The tumor cells were obtained from primary cultures and the expression and activity of multiple drug resistance transporters was assessed by RT-PCR and immunohistochemistry. Population-based study was performed using the databases of the Department of Neurosurgery of INCA.The number of patients with glioblastoma multiforme increased between 2007 and 2009, from 3.5% to 7.9% of total brain tumors. Mortality of these tumors is 90 % at three years. A high expression and activity of the multiple drugs resistance associated protein 1 (Mrp1) transporter was observed in primary cultures of biopsies.We propose that Mrp1 activity is responsible for the chemo-resistance of the glioblastoma multiforme and inhibition of this transporter could represent a plausible strategy for the treatment.