RESUMEN
Biomarkers able to improve the cost/benefit ratio are urgently needed for metastatic colorectal cancer patients that are eligible to receive regorafenib. Here, we measured plasma levels of ten circulating microRNAs (c-miRNAs) and we investigated their early changes during treatment, as well as possible correlation with clinical outcome. Ten literature-selected c-miRNAs were quantified by qRT-PCR on plasma samples collected at baseline (d1) and after 15 days of treatment (d15). C-miRNAs showing significant changes were further analyzed to establish correlations with outcome. A decision tree-based approach was employed to define a c-miRNA signature able to predict the outcome. Results achieved in an exploratory cohort were tested in a validation group. In the exploratory cohort (n = 34), the levels of c-miR-21 (p = 0.06), c-miR-141 (p = 0.04), and c-miR-601 (p = 0.01) increased at d15 compared with d1. A c-miRNA signature involving c-miR-21, c-miR-221, and c-miR-760 predicted response to treatment (p < 0.0001) and was significantly associated to PFS (HR = 10.68; 95% CI 3.2-35.65; p < 0.0001). In the validation cohort (n = 36), the increase in c-miR-21 (p = 0.02) and c-miR-601 (p = 0.02) levels at d15 was confirmed, but the associations with outcome were not. Our data indicate that early changes of c-miRNA levels might be influenced by regorafenib treatment. However, further studies are needed to establish the predictive power of such modifications.
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MicroARN Circulante/sangre , Neoplasias Colorrectales/tratamiento farmacológico , Compuestos de Fenilurea/uso terapéutico , Piridinas/uso terapéutico , Adulto , Anciano , Anciano de 80 o más Años , Estudios de Cohortes , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/mortalidad , Neoplasias Colorrectales/patología , Femenino , Humanos , Masculino , MicroARNs/sangre , Persona de Mediana Edad , Metástasis de la NeoplasiaRESUMEN
Protein acetylation, which is central to transcriptional control as well as other cellular processes, is disrupted in Huntington's disease (HD). Treatments that restore global acetylation levels, such as inhibiting histone deacetylases (HDACs), are effective in suppressing HD pathology in model organisms. However, agents that selectively target the disease-relevant HDACs have not been available. SirT1 (Sir2 in Drosophila melanogaster) deacetylates histones and other proteins including transcription factors. Genetically reducing, but not eliminating, Sir2 has been shown to suppress HD pathology in model organisms. To date, small molecule inhibitors of sirtuins have exhibited low potency and unattractive pharmacological and biopharmaceutical properties. Here, we show that highly selective pharmacological inhibition of Drosophila Sir2 and mammalian SirT1 using the novel inhibitor selisistat (selisistat; 6-chloro-2,3,4,9-tetrahydro-1H-carbazole-1-carboxamide) can suppress HD pathology caused by mutant huntingtin exon 1 fragments in Drosophila, mammalian cells and mice. We have validated Sir2 as the in vivo target of selisistat by showing that genetic elimination of Sir2 eradicates the effect of this inhibitor in Drosophila. The specificity of selisistat is shown by its effect on recombinant sirtuins in mammalian cells. Reduction of HD pathology by selisistat in Drosophila, mammalian cells and mouse models of HD suggests that this inhibitor has potential as an effective therapeutic treatment for human disease and may also serve as a tool to better understand the downstream pathways of SirT1/Sir2 that may be critical for HD.
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Carbazoles/administración & dosificación , Proteínas de Drosophila/antagonistas & inhibidores , Inhibidores Enzimáticos/administración & dosificación , Enfermedad de Huntington/tratamiento farmacológico , Enfermedad de Huntington/enzimología , Sirtuina 1/antagonistas & inhibidores , Sirtuinas/antagonistas & inhibidores , Animales , Modelos Animales de Enfermedad , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/efectos de los fármacos , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Femenino , Histona Desacetilasas/genética , Histona Desacetilasas/metabolismo , Humanos , Enfermedad de Huntington/genética , Enfermedad de Huntington/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Células PC12 , Ratas , Ratas Sprague-Dawley , Sirtuina 1/genética , Sirtuina 1/metabolismo , Sirtuinas/genética , Sirtuinas/metabolismoRESUMEN
AIM: Selisistat (SEN0014196), a first-in-class SirT1 inhibitor, is being developed as a disease-modifying therapy for Huntington's disease. This first-in-human study investigated the safety, pharmacokinetics and pharmacogenomics of single and multiple doses of selisistat in healthy male and female subjects. METHOD: In this double-blind, randomized, placebo-controlled study, seven cohorts of eight subjects received a single dose of selisistat at dose levels of 5, 25, 75, 150, 300 and 600 mg and four cohorts of eight subjects were administered 100, 200 and 300 mg once daily for 7 days. Blood sampling and safety assessments were conducted throughout the study. RESULTS: Selisistat was rapidly absorbed and systemic exposure increased in proportion to dose in the 5-300 mg range. Steady-state plasma concentrations were achieved within 4 days of repeated dosing. The incidence of drug related adverse events showed no correlation with dose level or number of doses received and was comparable with the placebo group. No serious adverse events were reported and no subjects were withdrawn due to adverse events. There were no trends in clinical laboratory parameters or vital signs. No trends in heart rate or ECG parameters, including the QTc interval and T-wave morphology, were observed. There were no findings in physical or neurological examinations or postural control. Transcriptional alteration was observed in peripheral blood. CONCLUSION: Selisistat was safe and well tolerated by healthy male and female subjects after single doses up to 600 mg and multiple doses up to 300 mg day(-1).
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Carbazoles/efectos adversos , Carbazoles/farmacocinética , Electrocardiografía/efectos de los fármacos , Sirtuina 1/antagonistas & inhibidores , Transcriptoma/efectos de los fármacos , Adolescente , Adulto , Área Bajo la Curva , Disponibilidad Biológica , Carbazoles/administración & dosificación , Carbazoles/sangre , Relación Dosis-Respuesta a Droga , Método Doble Ciego , Femenino , Voluntarios Sanos , Frecuencia Cardíaca/efectos de los fármacos , Humanos , Masculino , Persona de Mediana Edad , Adulto JovenRESUMEN
AIMS: Selisistat, a selective SirT1 inhibitor is being developed as a potentially disease-modifying therapeutic for Huntington's disease (HD). This was the first study of selisistat in HD patients and was primarily aimed at development of pharmacodynamic biomarkers. METHODS: This was a randomized, double-blind, placebo-controlled, multicentre exploratory study. Fifty-five male and female patients in early stage HD were randomized to receive 10 mg or 100 mg of selisistat or placebo once daily for 14 days. Blood sampling, clinical and safety assessments were conducted throughout the study. Candidate pharmacodynamic markers included circulating soluble huntingtin and innate immune markers. RESULTS: Selisistat was found to be safe and well tolerated, and systemic exposure parameters showed that the average steady-state plasma concentration achieved at the 10 mg dose level (125 nm) was comparable with the IC50 for SirT1 inhibition. No adverse effects on motor, cognitive or functional readouts were recorded. While circulating levels of soluble huntingtin were not affected by selisistat in this study, the biological samples collected have allowed development of assay technology for use in future studies. No effects on innate immune markers were seen. CONCLUSIONS: Selisistat was found to be safe and well tolerated in early stage HD patients at plasma concentrations within the anticipated therapeutic concentration range.
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Carbazoles/uso terapéutico , Enfermedad de Huntington/tratamiento farmacológico , Sirtuina 1/antagonistas & inhibidores , Administración Oral , Adolescente , Adulto , Anciano , Área Bajo la Curva , Carbazoles/administración & dosificación , Carbazoles/efectos adversos , Carbazoles/sangre , Cognición/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Método Doble Ciego , Esquema de Medicación , Femenino , Humanos , Enfermedad de Huntington/sangre , Enfermedad de Huntington/psicología , Masculino , Persona de Mediana Edad , Pruebas Neuropsicológicas , Índice de Severidad de la Enfermedad , Distribución Tisular , Resultado del Tratamiento , Adulto JovenRESUMEN
BACKGROUND: Huntington's disease (HD) is a monogenic disorder caused by an aberrant expansion of CAG repeats in the huntingtin gene (HTT). Pathogenesis is associated with expression of the mutant (mHTT) protein in the CNS, with its levels most likely related to disease progression and symptom severity. Since non-invasive methods to quantify HTT in the CNS do not exist, measuring amount of soluble HTT in peripheral cells represents an important step in development of disease-modifying interventions in HD. RESULTS: An ELISA assay using commercially available antibodies was developed to quantify HTT levels in complex matrices like mammalian cell cultures lysates and human samples. The immunoassay was optimized using a recombinant full-length HTT protein, and validated both on wild-type and mutant HTT species. The ability of the assay to detect significant variations of soluble HTT levels was evaluated using an HSP90 inhibitor that is known to enhance HTT degradation. Once optimized, the bioassay was applied to peripheral blood mononuclear cells (PBMCs) from HD patients, demonstrating good potential in tracking the disease course. CONCLUSIONS: The method described here represents a validated, simple and rapid bio-molecular assay to evaluate soluble HTT levels in blood cells as useful tool in disease and pharmacodynamic marker identification for observational and clinical trials.
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Análisis Químico de la Sangre/métodos , Ensayo de Inmunoadsorción Enzimática , Leucocitos Mononucleares/metabolismo , Proteínas del Tejido Nervioso/sangre , Anticuerpos/inmunología , Ensayo de Inmunoadsorción Enzimática/normas , Células HEK293 , Humanos , Proteína Huntingtina , Enfermedad de Huntington/metabolismo , Enfermedad de Huntington/patología , Espectrometría de Masas , Mutación , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/normas , Control de Calidad , Proteínas Recombinantes/análisis , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/normasRESUMEN
Glioblastoma multiforme (GBM) is composed of heterogeneous and genetically different cells, which are highly invasive and motile. The standard chemotherapeutic agent, temozolomide, affects GBM cell proliferation but is generally unable to prevent tumor recurrence. Hedgehog pathway activation has been reported to be relevant in GBM and different pharmacological pathway modulators have been identified. We report that by growing a commercially available recurrent GBM cell line (DBTRG-05MG) without serum and in the presence of defined growth factors; we obtained a less differentiated cell population, growing in suspension as neurospheres, in which the Hedgehog pathway is activated. Furthermore, the expression profile of Hedgehog pathway components found in DBTRG-05MG neurospheres is similar to primary stem-like cells derived from recurrent GBM patients. We report the effect of our novel specific Smoothened receptor antagonist (SEN450) on neurosphere growing cells and compared its effect to that of well known benchmark compounds. Finally, we showed that SEN450 is both antiproliferative on its own and further reduces tumor volume after temozolomide pretreatment in a mouse xenograft model using DBTRG-05MG neurosphere cells. Altogether our data indicate that the Hedgehog pathway is not irreversibly switched off in adherent cells but can be reactivated when exposed to well-defined culture conditions, thus restoring the condition observed in primary tumor-derived material, and that pharmacological modulation of this pathway can have profound influences on tumor proliferation. Therefore, pharmacological inhibition of the Hedgehog pathway is a potentially useful therapeutic approach in GBM.
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Glioblastoma/tratamiento farmacológico , Glioblastoma/metabolismo , Proteínas Hedgehog/antagonistas & inhibidores , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Anilidas/farmacología , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Dacarbazina/análogos & derivados , Dacarbazina/farmacología , Perfilación de la Expresión Génica , Glioblastoma/genética , Glioblastoma/patología , Proteínas Hedgehog/metabolismo , Humanos , Ratones , Ratones Desnudos , Piridinas/farmacología , Receptor Smoothened , Temozolomida , Factores de Transcripción/metabolismo , Alcaloides de Veratrum/farmacología , Ensayos Antitumor por Modelo de Xenoinjerto , Proteína con Dedos de Zinc GLI1RESUMEN
BACKGROUND: Quantitative measurements of specific protein phosphorylation sites, as presented here, can be used to investigate signal transduction pathways, which is an important aspect of cell dynamics. The presented method quantitatively compares peptide abundances from experiments using 18O/16O labeling starting from elaborated MS spectra. It was originally developed to study signaling cascades activated by amyloid-beta treatment of neurons used as a cellular model system with relevance to Alzheimer's disease, but is generally applicable. RESULTS: The presented method assesses, in complete cell lysates, the degree of phosphorylation of specific peptide residues from MS spectra using 18O/16O labeling. The abundance of each observed phospho-peptide from two cell states was estimated from three overlapping isotope contours. The influence of peptide-specific labeling efficiency was removed by performing a label swapped experiment and assuming that the labeling efficiency was unchanged upon label swapping. Different degrees of phosphorylation were reported using the fold change measure which was extended with a confidence interval found to reflect the quality of the underlying spectra. Furthermore a new way of method assessment using simulated data is presented. Using simulated data generated in a manner mimicking real data it was possible to show the method's robustness both with increasing noise levels and with decreasing labeling efficiency. CONCLUSION: The fold change error assessable on simulated data was on average 0.16 (median 0.10) with an error-to-signal ratio and labeling efficiency distributions similar to the ones found in the experimentally observed spectra. Applied to experimentally observed spectra a very good match was found to the model (<10% error for 85% of spectra) with a high degree of robustness, as assessed by data removal. This new method can thus be used for quantitative signal cascade analysis of total cell extracts in a high throughput mode.
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Espectrometría de Masas/métodos , Fosfopéptidos/análisis , Biología Computacional/métodos , Isótopos de Oxígeno/química , Fosfopéptidos/química , Proteoma/análisis , Proteómica/métodosRESUMEN
Proteomics studies employing primary neurons are difficult due to the neurons' characteristics. We have developed a detergent-based fractionation method which reduces complexity of the protein extracts, is sufficiently fast to allow differential proteomics analysis after treatments of neurons for short time periods, can be applied to small numbers of cells directly in culture plates, and allows differential extraction of proteins in a compartment-specific manner. The sequential use of detergent-containing buffers on neurons in culture plates yields four extracts enriched in cytosolic, membrane-bound or enclosed, nuclear, and cytoskeletal proteins. Fractionation of neurons was validated by comparison of the distribution of known subcellular marker proteins in the four extracts using Western blotting. Comparison of extracts by DIGE showed a clear difference in protein composition demonstrating significant variations with a fold change (FC) of at least 1.20 for 82% of the detected spots. Using proteins identified in these spots that could be assigned a subcellular localization based on descriptions in the Uniprot database, an extraction efficiency of 85% was calculated for cytosolic proteins in extract 1, 90% for membrane-bound and membrane-enclosed proteins in extract 2, 82% for nuclear proteins in extract 3 and 38% for cytoskeletal and RAFT proteins in extract 4.
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Fraccionamiento Celular/métodos , Detergentes , Neuronas/química , Proteómica , Animales , Técnicas de Cultivo de Célula , Electroforesis en Gel Bidimensional , RatasRESUMEN
In a comprehensive proteomics study aiming at the identification of proteins associated with amyloid-beta (Abeta)-mediated toxicity in cultured cortical neurons, we have identified Thimet oligopeptidase (THOP1). Functional modulation of THOP1 levels in primary cortical neurons demonstrated that its overexpression was neuroprotective against Abeta toxicity, while RNAi knockdown made neurons more vulnerable to amyloid peptide. In the TgCRND8 transgenic mouse model of amyloid plaque deposition, an age-dependent increase of THOP1 expression was found in brain tissue, where it co-localized with Abeta plaques. In accordance with these findings, THOP1 expression was significantly increased in human AD brain tissue as compared to non-demented controls. These results provide compelling evidence for a neuroprotective role of THOP1 against toxic effects of Abeta in the early stages of AD pathology, and suggest that the observed increase in THOP1 expression might be part of a compensatory defense mechanism of the brain against an increased Abeta load.
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Enfermedad de Alzheimer/enzimología , Péptidos beta-Amiloides/toxicidad , Corteza Cerebral/enzimología , Metaloendopeptidasas/biosíntesis , Neuronas/enzimología , Anciano , Anciano de 80 o más Años , Enfermedad de Alzheimer/patología , Animales , Western Blotting , Células Cultivadas , Corteza Cerebral/patología , Femenino , Expresión Génica , Humanos , Inmunohistoquímica , Masculino , Ratones , Ratones Transgénicos , Microscopía Confocal , Persona de Mediana Edad , Neuronas/patología , Placa Amiloide/metabolismo , ARN Interferente Pequeño , Ratas , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , TransfecciónRESUMEN
A parallel synthesis of aryl azoles with neuroprotective activity is described. All compounds obtained were evaluated in an in vitro assay using a NMDA toxicity paradigm showing a neuroprotective activity between 15% and 40%. The potential biological target of the active compounds was investigated by extensive literature searches based around similar scaffolds with reported neuroprotective activity. The most interesting molecules active in the NMDA toxicity assay (3a and 2g) showed moderate but significant activity in the inhibition of the Site 2 Sodium Channel binding assay at 10 microM. To confirm our hypothesis compounds 3a, c, f and 2g were tested in the Veratridine assay which is one of the excitotoxicity assays of relevance to NaV channels. The compounds tested showed an activity between 40% and 70%. The identification of neuroprotective small molecules and the identification of NaV channels as the potential site of action were the most important goals of this work.
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Azoles/farmacología , Fármacos Neuroprotectores/síntesis química , Animales , Azoles/síntesis química , Humanos , N-Metilaspartato/toxicidad , Fármacos Neuroprotectores/farmacología , Bloqueadores de los Canales de Sodio/síntesis química , Bloqueadores de los Canales de Sodio/farmacología , Canales de Sodio/efectos de los fármacosRESUMEN
Preclinical imaging modalities represent an essential tool to develop a modern and translational biomedical research. To date, Optical Imaging (OI) and Magnetic Resonance Imaging (MRI) are used principally in separate studies for molecular imaging studies. We decided to combine OI and MRI together through the development of a lentiviral vector to monitor the Wnt pathway response to Lithium Chloride (LiCl) treatment. The construct was stably infected in glioblastoma cells and, after intracranial transplantation in mice, serial MRI and OI imaging sessions were performed to detect human ferritin heavy chain protein (hFTH) and firefly luciferase enzyme (FLuc) respectively. The system allowed also ex vivo analysis using a constitutive fluorescence protein expression. In mice, LiCl administration has shown significantly increment of luminescence signal and a lower signal of T2 values (P<0.05), recorded noninvasively with OI and a 7 Tesla MRI scanner. This study indicates that OI and MRI can be performed in a single in vivo experiment, providing an in vivo proof-of-concept for drug discovery projects in preclinical phase.
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Genes Reporteros/genética , Imagen Molecular , Animales , Apoferritinas/genética , Apoferritinas/metabolismo , Encéfalo/metabolismo , Línea Celular Tumoral , Femenino , Expresión Génica , Humanos , Cloruro de Litio/farmacología , Luciferasas de Luciérnaga/genética , Imagen por Resonancia Magnética , Ratones Desnudos , Imagen Óptica , Vía de Señalización WntRESUMEN
The aspartic protease beta-site amyloid precursor protein cleaving enzyme 1 (BACE1) mediates the production of the neurotoxic amyloid beta peptide and is therefore considered an important drug target for treatment of Alzheimer's disease. We describe a new homogeneous time-resolved fluorescence quenching assay for the identification of BACE1 inhibitors that is characterized by minimal compound interference and allows both kinetic and end-point measurements. A fluorescent Eu-chelate as fluorescent donor, coupled to the N-terminus of a peptide containing the amyloid precursor protein Swedish mutation with a quenching molecule at the C-terminus as acceptor, is used as substrate. Upon peptide cleavage by BACE1, the energy transfer between donor and acceptor molecules is interrupted, leading to increased fluorescence emission of the donor. Compound interference, a common problem in fluorescence assays, is minimized with this technology because of the large Stoke's shift and the time-resolved fluorescence emission of the Eu-chelate. The assay reproduced IC50 values of known inhibitors and detected them also as hits in a screening campaign. A high signal-to-noise ratio of 289 and a Z' factor of 0.76 make this assay suitable for high-throughput screening.
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Endopeptidasas/metabolismo , Fluorescencia , Inhibidores de Proteasas/análisis , Secretasas de la Proteína Precursora del Amiloide , Precursor de Proteína beta-Amiloide/análisis , Precursor de Proteína beta-Amiloide/metabolismo , Cinética , Inhibidores de Proteasas/farmacología , Reproducibilidad de los Resultados , Especificidad por Sustrato , Tecnología Farmacéutica/métodos , Factores de TiempoRESUMEN
Cystic fibrosis (CF) is a lethal genetic disease caused by mutations of the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) with a prevalence of the ΔF508 mutation. Whereas the detailed mechanisms underlying disease have yet to be fully elucidated, recent breakthroughs in clinical trials have demonstrated that CFTR dysfunction can be corrected by drug-like molecules. On the basis of this success, a screening campaign was carried out, seeking new drug-like compounds able to rescue ΔF508-CFTR that led to the discovery of a novel series of correctors based on a tetrahydropyrido[4,3-d]pyrimidine core. These molecules proved to be soluble, cell-permeable, and active in a disease relevant functional-assay. The series was then further optimized with emphasis on biological data from multiple cell systems while keeping physicochemical properties under strict control. The pharmacological and ADME profile of this corrector series hold promise for the development of more efficacious compounds to be explored for therapeutic use in CF.
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Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Piridinas/química , Pirimidinas/química , Animales , Línea Celular , Permeabilidad de la Membrana Celular , Fibrosis Quística/tratamiento farmacológico , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Perros , Humanos , Membranas Artificiales , Microsomas Hepáticos/metabolismo , Mutación , Piridinas/síntesis química , Piridinas/metabolismo , Pirimidinas/síntesis química , Pirimidinas/metabolismo , Mucosa Respiratoria/citología , Mucosa Respiratoria/metabolismo , Solubilidad , Relación Estructura-ActividadRESUMEN
Enhancement of cellular senescence in tumours triggers a stable cell growth arrest and activation of an antitumour immune response that can be exploited for cancer therapy. Currently, there are only a limited number of targeted therapies that act by increasing senescence in cancers, but the majority of them are not selective and also target healthy cells. Here we developed a chemogenomic screening to identify compounds that enhance senescence in PTEN-deficient cells without affecting normal cells. By using this approach, we identified casein kinase 2 (CK2) as a pro-senescent target. Mechanistically, we show that Pten loss increases CK2 levels by activating STAT3. CK2 upregulation in Pten null tumours affects the stability of Pml, an essential regulator of senescence. However, CK2 inhibition stabilizes Pml levels enhancing senescence in Pten null tumours. Taken together, our screening strategy has identified a novel STAT3-CK2-PML network that can be targeted for pro-senescence therapy for cancer.
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Quinasa de la Caseína II/antagonistas & inhibidores , Senescencia Celular/efectos de los fármacos , Terapia Molecular Dirigida , Naftiridinas/uso terapéutico , Fosfohidrolasa PTEN/deficiencia , Neoplasias de la Próstata/tratamiento farmacológico , Animales , Quinasa de la Caseína II/metabolismo , Evaluación Preclínica de Medicamentos , Femenino , Células HCT116 , Humanos , Masculino , Ratones Transgénicos , Naftiridinas/farmacología , Proteínas Nucleares/metabolismo , Fenazinas , Proteína de la Leucemia Promielocítica , ARN Interferente Pequeño , Factor de Transcripción STAT3/metabolismo , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/metabolismoRESUMEN
BACKGROUND: Huntington Disease (HD) is a progressive neurological disorder, with pathological manifestations in brain areas and in periphery caused by the ubiquitous expression of mutant Huntingtin protein. Transcriptional dysregulation is considered a key molecular mechanism responsible of HD pathogenesis but, although numerous studies investigated mRNA alterations in HD, so far none evaluated a whole gene expression profile in blood of R6/2 mouse model. FINDINGS: To discover novel pathogenic mechanisms and potential peripheral biomarkers useful to monitor disease progression or drug efficacy, a microarray study was performed in blood of R6/2 at manifest stage and wild type littermate mice. This approach allowed to propose new peripheral molecular processes involved in HD and to suggest different panels of candidate biomarkers. Among the discovered deregulated processes, we focused on specific ones: complement and coagulation cascades, PPAR signaling, cardiac muscle contraction, and dilated cardiomyopathy pathways. Selected genes derived from these pathways were additionally investigated in other accessible tissues to validate these matrices as source of biomarkers, and in brain, to link central and peripheral disease manifestations. CONCLUSIONS: Our findings validated the skeletal muscle as suitable source to investigate peripheral transcriptional alterations in HD and supported the hypothesis that immunological alteration may contribute to neurological degeneration. Moreover, the identification of altered signaling in mouse blood enforce R6/2 transgenic mouse as a powerful HD model while suggesting novel disease biomarkers for pre-clinical investigation.
RESUMEN
BACKGROUND: Huntington's disease is a neurodegenerative disorder characterized by transcriptional alterations both in central and peripheral tissues. Therefore, the identification of a transcriptional signature in an accessible tissue can meaningfully complement current efforts in clinical biomarker development. Gene expression normalization represents an essential step in transcriptional signatures identification, and since many reference genes show altered expressions in several pathologies, the definition of stable genes in the desired tissue is required to allow correct result interpretations. OBJECTIVE: The present work aimed at identifying a set of suitable reference genes for expression normalization in blood of HD patients and R6/2 mice. METHODS: By crossing literature investigation and analysis of microarrays performed on blood of HD patients and healthy subjects, a set of genes was selected and tested by RT-qPCR. Employment of statistical algorithms allowed the identification of the most stable genes in human samples that were than confirmed in R6/2. RESULTS: PPIB, PGK1, ACTB and YWHAZ represent the best possible genes combination, useful to normalize blood transcriptional analysis. To link clinical and preclinical studies, the identified genes were investigated also in blood of R6/2 and wild type mice, confirming that Ppib, Actb and Ywhaz were appropriate for expression normalization. Selected references were subsequently applied to evaluate expression of genes known to be involved in Huntington's pathological progression. CONCLUSIONS: This work highlights the importance for correct data normalization to avoid misinterpretation of results, while providing a suitable method to support quantitative gene expression analysis in preclinical and clinical investigations.
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Perfilación de la Expresión Génica/métodos , Expresión Génica/genética , Enfermedad de Huntington/genética , Animales , Estudios de Casos y Controles , Femenino , Humanos , Masculino , Ratones , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Estándares de Referencia , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Huntington's Disease is a rare neurodegenerative disease caused by an abnormal expansion of CAG repeats encoding polyglutamine in the first exon of the huntingtin gene. N-terminal fragments containing polyglutamine (polyQ) sequences aggregate and can bind to cellular proteins, resulting in several pathophysiological consequences for affected neurons such as changes in gene transcription. One transcriptional pathway that has been implicated in HD pathogenesis is the CREB binding protein (CBP)/cAMP responsive element binding (CREB) pathway. We developed a phenotypic assay to screen for compounds that can reverse the transcriptional dysregulation of the pathway caused by induced mutated huntingtin protein (µHtt). 293/T-REx cells were stably co-transfected with an inducible full-length mutated huntingtin gene containing 138 glutamine repeats and with a reporter gene under control of the cAMP responsive element (CRE). One clone, which showed reversible inhibition of µHtt-induced reporter activity upon treatment with the neuroprotective Rho kinase inhibitor Y27632, was used for the development of a high-throughput phenotypic assay suitable for a primary screening campaign, which was performed on a library of 24,000 compounds. Several hit compounds were identified and validated further in a cell viability adenosine triphosphate assay. The assay has the potential for finding new drug candidates for the treatment of HD.
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Bioensayo , Proteínas del Tejido Nervioso/genética , Neuronas/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Bibliotecas de Moléculas Pequeñas/farmacología , Amidas/química , Amidas/farmacología , Proteína de Unión a CREB/genética , Proteína de Unión a CREB/metabolismo , Línea Celular Tumoral , Regulación de la Expresión Génica/efectos de los fármacos , Genes Reporteros , Humanos , Proteína Huntingtina , Enfermedad de Huntington/tratamiento farmacológico , Enfermedad de Huntington/genética , Enfermedad de Huntington/metabolismo , Enfermedad de Huntington/patología , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Neuronas/patología , Fármacos Neuroprotectores/química , Péptidos/química , Péptidos/metabolismo , Unión Proteica/efectos de los fármacos , Piridinas/química , Piridinas/farmacología , Elementos de Respuesta , Transducción de Señal , Bibliotecas de Moléculas Pequeñas/química , Transcripción Genética/efectos de los fármacos , Quinasas Asociadas a rho/antagonistas & inhibidores , Quinasas Asociadas a rho/genética , Quinasas Asociadas a rho/metabolismoRESUMEN
Here, we describe the selection and optimization of a chemical series active in both a full-length and a fragment-based Huntington's disease (HD) assay. Twenty-four thousand small molecules were screened in a phenotypic HD assay, identifying a series of compounds bearing a 3-hydroxy-3-trifluoromethylpyrazole moiety as able to revert the toxicity induced by full-length mutant Htt by up to 50%. A chemical exploration around the series led to the identification of compound 4f, which demonstrated to be active in a Htt171-82Q rat primary striatal neuron assay and a PC12-Exon-1 based assay. This compound was selected for testing in R6/2 mice, in which it was well-tolerated and showed a positive effect on body weight and a positive trend in preventing ventricular volume enlargment. These studies provide strong rationale for further testing the potential benefits of 3-hydroxy-3-trifluoromethylpyrazoles in treating HD.
RESUMEN
Glioblastoma multiforme (GBM) is among the most deadly cancers. A number of studies suggest that a fraction of tumor cells with stem cell features (Glioma Stem-like Cells, GSC) might be responsible for GBM recurrence and aggressiveness. GSC similarly to normal neural stem cells, can form neurospheres (NS) in vitro, and seem to mirror the genetic features of the original tumor better than glioma cells growing adherently in the presence of serum. Using cDNA microarray analysis we identified a number of relevant genes for glioma biology that are differentially expressed in adherent cells and neurospheres derived from the same tumor. Fatty acid-binding protein 7 (FABP7) was identified as one of the most highly expressed genes in NS compared to their adherent counterpart. We found that down-regulation of FABP7 expression in NS by small interfering RNAs significantly reduced cell proliferation and migration. We also evaluated the potential involvement of FABP7 in response to radiotherapy, as this treatment may cause increased tumor infiltration. Migration of irradiated NS was associated to increased expression of FABP7. In agreement with this, in vivo reduced tumorigenicity of GBM cells with down-regulated expression of FABP7 was associated to decreased expression of the migration marker doublecortin. Notably, we observed that PPAR antagonists affect FABP7 expression and decrease the migration capability of NS after irradiation. As a whole, the data emphasize the role of FABP7 expression in GBM migration and provide translational hints on the timing of treatment with anti-FABP7 agents like PPAR antagonists during GBM evolution.
Asunto(s)
Proteínas Portadoras/genética , Glioblastoma/genética , Glioblastoma/patología , Proteínas Supresoras de Tumor/genética , Región de Flanqueo 5' , Anilidas/farmacología , Animales , Secuencia de Bases , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Línea Celular Tumoral , Movimiento Celular/genética , Movimiento Celular/efectos de la radiación , Proliferación Celular/efectos de la radiación , Análisis por Conglomerados , Proteína de Unión a los Ácidos Grasos 7 , Expresión Génica , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de la radiación , Redes Reguladoras de Genes , Silenciador del Gen , Glioblastoma/metabolismo , Humanos , Ratones , Datos de Secuencia Molecular , Invasividad Neoplásica , Receptores Activados del Proliferador del Peroxisoma/antagonistas & inhibidores , Transducción de Señal , Proteínas Supresoras de Tumor/química , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Glioblastomas (GBMs), the most common primary malignancies of the central nervous system, are highly aggressive and heterogeneous, and remain a dramatic therapeutic challenge. Markers mirroring the complex molecular profile of GBMs that are predictive of patient outcomes are needed to define novel multi-targeted treatment strategies. Resistance to current GBM therapies is partly due to a subpopulation of stem-like and other self-renewing cells (hereafter called glioma stem-like cancer cells, GSCC), which are therefore of key interest as therapeutic entry points. Wnt and Hedgehog are among the main pathways involved in GSCC renewal. ß-catenin and Gli1 are markers of Wnt and Hedgehog activation respectively and both pathways are known to be altered in gliomas. To date, there are no investigations of Gli1 protein expression in GBM tissue, and recently a high expression of ß-catenin has been found to have a poor prognostic impact in GBM patients in a study. We have therefore quantified the positivity for ß-catenin, Gli1, as well as Ki-67, p53, and EGFR proteins on immunohistochemically-stained GBM sections from 106 patients in an investigation for potential predictive biomarkers. Correlation between these markers and survival was evaluated by pair-wise Pearson correlation coefficient and by bi-dimensional hierarchical clustering, followed by survival estimations using linear regression models and classification trees. We demonstrated that both ß-catenin and, for the first time, Gli1 proteins are highly predictive markers of short survival, being found in 75 and 90% of the highly predictive trees, respectively, whereas Ki-67, p53 and EGFR were under 30% and thus, not considered as predictive. Our results indicate a role of ß-catenin and Gli1 in GBM malignant behaviour, and suggest that inhibiting members of Wnt and Hedgehog pathways could be a valuable therapeutic strategy for GBM patients.