Generation of an inducible expression system to perform molecular pharmacology studies with mu opioid receptors

Máster en Biología Molecular y Biomedicin

Potentiation of morphine-induced antinociception and locomotion by citalopram is accompanied by anxiolytic-like effects

Morphine and related opioids are the mainstay of analgesic treatment, especially in patients suffering chronic pain. Besides their antinociceptive effects they may also exhibit anxiolytic-like properties that could contribute to pain relief. The pharmacological manipulation of the serotonergic system may not only modulate pain transmission and processing but also other behavioral effects of opioids. The present study aimed to analyze the effect of the concurrent treatment with citalopram, a selective serotonin reuptake inhibitor, on the antinociceptive, locomotor and anxiety-related effects induced by acute and subchronic administration of morphine in mice. Citalopram (15 mg/kg) enhanced the acute antinociceptive effects of morphine when concurrently administered as evidenced by a two-fold increase in the ED50 for the antinociceptive effect of morphine in the hot-plate test. Chronic studies also revealed that concurrent citalopram treatment (15 mg/kg) delayed the development of tolerance to the thermal antinociceptive effects of morphine. Additionally, morphine-induced hyperlocomotion was potentiated by citalopram as assessed in the open-field test and in the spontaneous activity recording in the home cage, a behavioral outcome to which tolerance or desensitization was not developed. Interestingly, chronic administration of both drugs promoted an anxiolytic effect as evidenced by the increased central activity in the open field test. Future investigations on this pharmacological interaction, such as the possible translational research in clinics, might have consequences in future strategies for the therapeutic management of pain.This research was supported by the grant SAF2010-15663 from the Spanish Government (MINECO). We thank David Ramos and Raquel Lanza for their technical assistance in handling living animals. We kindly appreciate Belen Palacio support for video footage and editing and Dr. Albert Adell for the critical reading of the manuscript

Role of SOX family of transcription factors in central nervous system tumors

SOX genes are developmental regulators with functions in the instruction of cell fate and maintenance of progenitor’s identity during embryogenesis. They play additional roles during tissue homeostasis and regeneration in adults particularly in the Central Nervous System (CNS). In the last years a growing number of evidences has shown that mutations and dysfunction of SOX factors are implicated in several human diseases, including a variety of cancers. In this review, we will summarize the current knowledge about SOX family in CNS tumors and their role in the origin and maintenance of the subpopulation of cancer stem cells in these tumors

Haplotipo del gen del factor VIII en el diagnóstico molecular de la hemofilia A. Estudio de una familia.

La hemofilia A se caracteriza por ser una enfermedad congénita del trastorno de la coagulación y constituye un desorden recesivo ligado al cromosoma X. El estudio molecular se realiza por estudio indirecto, por ser causada por mutaciones heterogéneas en el gen del factor VIII. Se estudió una familia afectada, para la detección de portadora de la gestante y posteriormente se realizó el diagnóstico prenatal. La extracción de ADN se hizo por el método de precipitación salina Salting Out a tres muestras de sangre y una de líquido amniótico. Se efectuó el análisis de los polimorfismos St14, Bcl I y Hind III. Para la determinación de sexo fetal, se estudió el gen AMXY. La técnica empleada fue la reacción en cadena de la polimerasa.  El análisis del marcador Bcl I arrojó que la gestante era portadora de hemofilia A, pero al ser homocigótica no era informativa; el polimorfismo St14 por sí solo no brindaba la información de la condición de la gestante, pero al ser heterocigótica para el mismo y conociendo de antemano la información de ser portadora, se pudo realizar diagnóstico prenatal, gracias al análisis conjunto de los marcadores. El polimorfismo Hind III no fue informativo. El feto resultó ser varón sano.  Palabras clave: Hemofilia A, Bcl I, St14, Hind III, factor VIII

Characterization of the Antiglioma Effect of the Oncolytic Adenovirus VCN-01

Despite the recent advances in the development of antitumor therapies, the prognosis for patients with malignant gliomas remains dismal. Therapy with tumor-selective viruses is emerging as a treatment option for this devastating disease. In this study we characterize the anti-glioma effect of VCN-01, an improved hyaluronidase-armed pRB-pathway-selective oncolytic adenovirus that has proven safe and effective in the treatment of several solid tumors. VCN-01 displayed a significant cytotoxic effect on glioma cells in vitro. In vivo, in two different orthotopic glioma models, a single intra-tumoral administration of VCN-01 increased overall survival significantly and led to long-term survivors free of disease

Endocytosis as a biological response in receptor pharmacology: evaluation by fluorescence microscopy

The activation of G-protein coupled receptors by agonist compounds results in diverse biological responses in cells, such as the endocytosis process consisting in the translocation of receptors from the plasma membrane to the cytoplasm within internalizing vesicles or endosomes. In order to functionally evaluate endocytosis events resulted from pharmacological responses, we have developed an image analysis method -the Q-Endosomes algorithm- that specifically discriminates the fluorescent signal originated at endosomes from that one observed at the plasma membrane in images obtained from living cells by fluorescence microscopy. Mu opioid (MOP) receptor tagged at the carboxy-terminus with yellow fluorescent protein (YFP) and permanently expressed in HEK293 cells was used as experimental model to validate this methodology. Time-course experiments performed with several agonists resulted in different sigmoid curves depending on the drug used to initiate MOP receptor endocytosis. Thus, endocytosis resulting from the simultaneous activation of co-expressed MOP and serotonin 5-HT2C receptors by morphine plus serotonin was significantly different, in kinetics as well as in maximal response parameters, from the one caused by DAMGO, sufentanyl or methadone. Therefore, this analytical tool permits the pharmacological characterization of receptor endocytosis in living cells with functional and temporal resolution

Transcriptional networks controlled by SOX2 in glioblastoma stem cells

Glioblastoma is the most common and aggressive primary malignant brain tumor in adults. It constitutes 45,2% of all malignant central nervous system (CNS) tumors and 80% of all primary malignant CNS tumors. The current standard of care for glioblastoma includes maximal surgical resection, radiation and chemotherapy. Despite steady advances in new treatments to improve survival rates, the overall survival of patients with glioblastoma has not improved over the last decades, remaining at 12 to 18 months from diagnosis. At the cellular level glioblastoma is composed by heterogeneous cell populations, among which the glioma stem-like cells (GSCs) exhibits self-renewal potential and the ability to reconstitute the original tumor upon orthotopic implantation. GSCs are the culprit of glioma chemo- and radio-resistance ultimately leading to relapse. The elucidation of the transcriptome and the molecular pathways involved in the generation and maintenance of GSCs is critical to understand the molecular underpinnings of glioblastoma malignancy and could allow the identification of relevant and novel therapeutic targets. SOX2, a critical transcription regulator of embryonic and neural stem cell function, is deregulated in GSCs and is highly expressed in glioblastoma. However the precise molecular pathways regulated by SOX2 in GSCs remain poorly understood. We hypothesized that SOX2 plays a prominent role in driving the growth, treatment resistance and recurrence of glioblastoma cells, through the orchestration of different transcriptional pathways. In this work we performed a genome-wide analysis of SOX2-regulated coding and non-coding transcripts in GSCs. We identified 2048 differentially expressed coding transcripts regulated by SOX2. These genes are involved in different biological process, such as cell adhesion and cell-cell signaling, and in canonical pathways related with intracellular signaling cascades and amino-acid metabolism pathways associated with GSC propagation. SOX2 regulates 261 non-coding transcripts differentially expressed in GSCs, including miRNAs and lncRNAs amongst other. We identified 2 interesting miRNAs regulated by SOX2. miR-301a-3p is over-expressed in glioblastoma tissues, positively correlates with SOX2 expression and participates in the invasive properties of GSCs, acting as an onco-miR. miR-425-5p is significantly overexpressed in glioblastoma tissue and also correlates with SOX2 expression. Moreover, SOX2 activates its expression by directly binding to its promoter. Inhibition of miR-425-5p expression results in inhibition of neurosphere formation, cell proliferation and finally apoptotic cell death. Inhibition of mir-425-5p in vivo leads to a significant increase in overall median survival time of mice bearing orthotopic glioma xenografts. In summary this work integrates for the first time the coding and non-coding transcriptome controlled by SOX2 in GSCs, defining miR-301 and miR-425 as novel onco-miRs in GSCs and gaining new insights about the molecular circuitries governing glioblastoma biology

Role of SOX family of transcription factors in central nervous system tumors

SOX genes are developmental regulators with functions in the instruction of cell fate and maintenance of progenitor’s identity during embryogenesis. They play additional roles during tissue homeostasis and regeneration in adults particularly in the Central Nervous System (CNS). In the last years a growing number of evidences has shown that mutations and dysfunction of SOX factors are implicated in several human diseases, including a variety of cancers. In this review, we will summarize the current knowledge about SOX family in CNS tumors and their role in the origin and maintenance of the subpopulation of cancer stem cells in these tumors

Tyrosyl-DNA Phosphodiesterase 1 and Topoisomerase I Activities as Predictive Indicators for Glioblastoma Susceptibility to Genotoxic Agents

Glioblastoma (GBM) patients have an estimated survival of ~15 months with treatment, and the standard of care only modestly enhances patient survival. Identifying biomarkers representing vulnerabilities may allow for the selection of efficacious chemotherapy options to address personalized variations in GBM tumors. Irinotecan targets topoisomerase I (TOP1) by forming a ternary DNA-TOP1 cleavage complex (TOP1cc), inducing apoptosis. Tyrosyl-DNA phosphodiesterase 1 (TDP1) is a crucial repair enzyme that may reduce the effectiveness of irinotecan. We treated GBM cell lines with increasing concentrations of irinotecan and compared the IC values. We found that the TDP1/TOP1 activity ratio had the strongest correlation (Pearson correlation coefficient R = 0.972, based on the average from three sets of experiments) with IC values following irinotecan treatment. Increasing the TDP1/TOP1 activity ratio by the ectopic expression of wild-type TDP1 increased in irinotecan IC, while the expression of the TDP1 catalytic-null mutant did not alter the susceptibility to irinotecan. The TDP1/TOP1 activity ratio may be a new predictive indicator for GBM vulnerability to irinotecan, allowing for the selection of individual patients for irinotecan treatment based on risk-benefit. Moreover, TDP1 inhibitors may be a novel combination treatment with irinotecan to improve GBM patient responsiveness to genotoxic chemotherapies