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Polo-like kinase 1 (PLK1) is an important cell cycle kinase and an attractive target for anticancer treatments. An ATP-competitive small molecular PLK1 inhibitor, volasertib, has reached phase III in clinical trials in patients with refractory acute myeloid leukemia as a combination treatment with cytarabine. However, severe side effects limited its use. The origin of the side effects is unclear and might be due to insufficient specificity of the drug. Thus, identifying potential off-targets to volasertib is important for future clinical trials and for the development of more specific drugs. In this study, we used thermal proteome profiling (TPP) to identify proteome-wide targets of volasertib. Apart from PLK1 and proteins regulated by PLK1, we identified about 200 potential volasertib off-targets. Comparison of this result with the mass-spectrometry analysis of volasertib-treated cells showed that phosphatidylinositol phosphate and prostaglandin metabolism pathways are affected by volasertib. We confirmed that PIP4K2A and ZADH2-marker proteins for these pathways-are, indeed, stabilized by volasertib. PIP4K2A, however, was not affected by another PLK1 inhibitor onvansertib, suggesting that PIP4K2A is a true off-target of volasertib. Inhibition of these proteins is known to impact both the immune response and fatty acid metabolism and could explain some of the side effects seen in volasertib-treated patients.
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Antígenos de Superficie/metabolismo , Proteínas de Ciclo Celular/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Pteridinas/farmacología , Citarabina/farmacología , Ácidos Grasos/metabolismo , Células HL-60 , Humanos , Inmunidad/efectos de los fármacos , Células Jurkat , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/metabolismo , Piperazinas/farmacología , Proteoma/metabolismo , Pirazoles/farmacología , Quinazolinas/farmacología , Quinasa Tipo Polo 1RESUMEN
Host proteins incorporated into virus particles have been reported to contribute to infectivity and tissue-tropism. This incorporation of host proteins is expected to be variable among viral particles, however, protein analysis at single-virus levels has been challenging. We have developed a method to detect host proteins incorporated on the surface of virions using the in situ proximity ligation assay (isPLA) with rolling circle amplification (RCA), employing oligonucleotide-conjugated antibody pairs. The technique allows highly selective and sensitive antibody-based detection of viral and host proteins on the surface of individual virions. We detected recombinant noninfectious sub-viral particles (SVPs) of tick-borne encephalitis virus (TBEV) immobilized in microtiter wells as fluorescent particles detected by regular fluorescence microscopy. Counting the particles in the images enabled us to estimate individual TBEV-SVP counts in different samples. Using isPLA we detected individual calnexin-, CD9-, CD81-, CD29- and CD59-positive SVPs among the viral particles. Our data suggests that a diversity of host proteins may be incorporated into TEBV, illustrating that isPLA with digital counting enables single-virus analysis of host protein incorporation.
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Virus de la Encefalitis Transmitidos por Garrapatas/metabolismo , Técnicas de Amplificación de Ácido Nucleico/métodos , Proteínas/metabolismo , Línea Celular , Humanos , Proteínas/ultraestructura , Virión/metabolismo , Virión/ultraestructuraRESUMEN
Novel therapies are undergoing clinical trials, for example, the Hsp90 inhibitor, XL888, in combination with BRAF inhibitors for the treatment of therapy-resistant melanomas. Unfortunately, our data show that this combination elicits a heterogeneous response in a panel of melanoma cell lines including PDX-derived models. We sought to understand the mechanisms underlying the differential responses and suggest a patient stratification strategy. Thermal proteome profiling (TPP) identified the protein targets of XL888 in a pair of sensitive and unresponsive cell lines. Unbiased proteomics and phosphoproteomics analyses identified CDK2 as a driver of resistance to both BRAF and Hsp90 inhibitors and its expression is regulated by the transcription factor MITF upon XL888 treatment. The CDK2 inhibitor, dinaciclib, attenuated resistance to both classes of inhibitors and combinations thereof. Notably, we found that MITF expression correlates with CDK2 upregulation in patients; thus, dinaciclib would warrant consideration for treatment of patients unresponsive to BRAF-MEK and/or Hsp90 inhibitors and/or harboring MITF amplification/overexpression.
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Compuestos de Azabiciclo/farmacología , Quinasa 2 Dependiente de la Ciclina/metabolismo , Resistencia a Antineoplásicos , Imidazoles/farmacología , Melanoma/metabolismo , Factor de Transcripción Asociado a Microftalmía/metabolismo , Oximas/farmacología , Ácidos Ftálicos/farmacología , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Óxidos N-Cíclicos , Quinasa 2 Dependiente de la Ciclina/genética , Resistencia a Antineoplásicos/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Humanos , Indolizinas , Melanoma/tratamiento farmacológico , Melanoma/genética , Factor de Transcripción Asociado a Microftalmía/genética , Fosfoproteínas/metabolismo , Proteómica , Compuestos de Piridinio/farmacología , Regulación hacia ArribaRESUMEN
INTRODUCTION: Amantadine, as a dopamine receptor agonist, may stimulate and help the recovery of the nervous system after traumatic brain injury (TBI). METHODS: We performed this study as a double-blind, randomized, controlled clinical trial with target population including all patients with TBI who scored nine or lower on the Glasgow Coma Scale (GCS), admitted to our hospital between January 2013 and April 2014. The protocol included administration of the drug (placebo or amantadine) for 6 weeks and patient evaluation using the GCS and FOUR score on the first, third and seventh days after the drug was started. After 6 months from starting study drug, the patients were evaluated on the Mini-Mental State Examination, Glasgow Outcome Study, Disability Rating Scale and Karnofsky Performance Scale. RESULTS: We included 40 patients in the study. The mean age of the patients was 36.77 ± 18.21. As an only important finding, the amantadine group registered an important rise between the first and the seventh day of study drug (∆GCS7-GCS1) with p-value = 0.044. CONCLUSION: Based on our findings during the first week and the 6 months (since starting drug) follow-ups, prescribing amantadine did not lead to reportable effects on the patients' level of consciousness, memory, disability, cognition, mortality and performance.
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Amantadina/uso terapéutico , Lesiones Traumáticas del Encéfalo/tratamiento farmacológico , Dopaminérgicos/uso terapéutico , Resultado del Tratamiento , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Evaluación de la Discapacidad , Método Doble Ciego , Femenino , Escala de Coma de Glasgow , Humanos , Estado de Ejecución de Karnofsky , Masculino , Pruebas de Estado Mental y Demencia , Persona de Mediana Edad , Recuperación de la Función/efectos de los fármacos , Estudios Retrospectivos , Adulto JovenRESUMEN
The potential of cell therapy is promising in nerve regeneration, but is limited by ethical considerations about the proper and technically safe source of stem cells. We report the successful differentiation of human EnSCs (endometrial stem cells) as a rich source of renewable and safe progenitors into high-efficiency cholinergic neurons. The extracellular signals of NGF (nerve growth factor) and bFGF (basic fibroblast growth factor) could induce cholinergic neuron differentiation. ChAT (choline acetyltransferase), MAP2 (microtubule associated protein 2) and NF-l (neurofilament L) increased after administration of bFGF and NGF to the EnSC cultures. trkC and FGFR2 (fibroblast growth factor receptor 2), which belong to the NGF and bFGF receptors respectively, were determined in populations of EnSCs. NGF, bFGF and their combination differentially influenced human EnSCs high efficiency differentiation. By inducing cholinergic neurons from EnSCs in a chemically defined medium, we could produce human neural cells without resorting to primary culture of neurons. This in vitro method provides an unlimited source of human neural cells and facilitates clinical applications of EnSCs for neurological diseases.
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Endometrio/citología , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Factores de Crecimiento Nervioso/metabolismo , Neurogénesis , Neuronas/citología , Células Madre/citología , Adulto , Antígeno CD146/análisis , Separación Celular , Supervivencia Celular , Células Cultivadas , Colina O-Acetiltransferasa/análisis , Femenino , Humanos , Proteínas Asociadas a Microtúbulos/análisis , Proteínas de Neurofilamentos/análisis , Neuronas/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/análisis , Antígenos Thy-1/análisis , Molécula 1 de Adhesión Celular Vascular/análisisRESUMEN
Extracellular vesicles (EVs) are mediators of intercellular communication and a promising class of biomarkers. Surface proteins of EVs play decisive roles in establishing a connection with recipient cells, and they are putative targets for diagnostic assays. Analysis of the surface proteins can thus both illuminate the biological functions of EVs and help identify potential biomarkers. We developed a strategy combining high-resolution mass spectrometry (HRMS) and proximity ligation assays (PLA) to first identify and then validate surface proteins discovered on EVs. We applied our workflow to investigate surface proteins of small EVs found in seminal fluid (SF-sEV). We identified 1,014 surface proteins and verified the presence of a subset of these on the surface of SF-sEVs. Our work demonstrates a general strategy for deep analysis of EVs' surface proteins across patients and pathological conditions, proceeding from unbiased screening by HRMS to ultra-sensitive targeted analyses via PLA.
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Vesículas Extracelulares , Próstata , Masculino , Humanos , Vesículas Extracelulares/metabolismo , Biomarcadores/metabolismo , Espectrometría de Masas/métodos , Proteínas de la Membrana/metabolismoRESUMEN
BRAF inhibitors (BRAFi) selectively target oncogenic BRAFV600E/K and are effective in 80% of advanced cutaneous malignant melanoma cases carrying the V600 mutation. However, the development of drug resistance limits their clinical efficacy. Better characterization of the underlying molecular processes is needed to further improve treatments. We previously demonstrated that transcription of PTEN is negatively regulated by the PTEN pseudogene antisense RNA, PTENP1-AS, and here we investigated the impact of this transcript on clinical outcome and BRAFi resistance in melanoma. We observed that increased expression levels of PTENP1-AS in BRAFi resistant cells associated with enrichment of EZH2 and H3K27me3 at the PTEN promoter, consequently reducing the expression levels of PTEN. Further, we showed that targeting of the PTENP1-AS transcript sensitized resistant cells to BRAFi treatment and that high expression of PTENP1-AS in stage III melanoma correlated with poor survival. Collectively, the data presented here show that PTENP1-AS is a promising target for re-sensitizing cells to BRAFi and also a possible prognostic marker for clinical outcome in stage III melanoma.
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Melanoma , Proteínas Proto-Oncogénicas B-raf , Neoplasias Cutáneas , Línea Celular Tumoral , Resistencia a Antineoplásicos/efectos de los fármacos , Humanos , Inhibidores de Proteínas Quinasas/farmacología , Transducción de Señal/efectos de los fármacos , Vemurafenib/farmacología , Melanoma Cutáneo MalignoRESUMEN
Introduction of targeted therapy in the treatment of metastatic cutaneous malignant melanoma (CMM) has improved clinical outcome during the last years. However, only in a subset of the CMM patients, this will lead to long-term effects. CEBPB is a transcription factor that has been implicated in various physiological and pathological processes, including cancer development. We have investigated its prognostic impact on CMM and unexpectedly found that higher CEBPB mRNA levels correlated with a longer overall survival. Furthermore, in a small cohort of patients with metastatic CMM treated with BRAF-inhibitors, higher levels of CEBPB mRNA expression in the tumor cells prior treatment correlated to a longer progression-free survival. We have characterized an overlapping antisense transcript, CEBPB-AS1, with the aim to investigate the regulation of CEBPB expression in CMM and its impact on BRAF-inhibitor sensitivity. We demonstrated that silencing of CEBPB-AS1 resulted in epigenetic modifications in the CEBPB promoter and in increased CEBPB mRNA and protein levels, inhibited proliferation and partially resensitized BRAF-inhibitor resistant CMM cells to this drug-induced apoptosis. Our data suggest that targeting CEBPB-AS1 may represent a valuable tool to sensitize CMM cells to the BRAF-inhibitor-based therapies.
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Proteína beta Potenciadora de Unión a CCAAT/metabolismo , Melanoma/tratamiento farmacológico , ARN sin Sentido/genética , Vemurafenib/farmacología , Adulto , Anciano , Anciano de 80 o más Años , Proteína beta Potenciadora de Unión a CCAAT/biosíntesis , Proteína beta Potenciadora de Unión a CCAAT/genética , Línea Celular Tumoral , Resistencia a Antineoplásicos , Femenino , Humanos , Masculino , Melanoma/genética , Melanoma/metabolismo , Persona de Mediana Edad , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , ARN sin Sentido/metabolismo , ARN Mensajero/biosíntesis , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Aneurysmal bone cyst (ABC) is defined as a benign lesion of bone containing blood-filled channels divided by soft-tissue septa and mostly diagnosed in children and adolescents. In the present study, the case of a 7-year-old girl with spastic paraparesia and left foot drop was investigated. Spinal imaging revealed a large lytic mass in the posterior elements of the T2 vertebral body, which resulted in the partial collapse of the T2 vertebral body, a T1 to T3 dislocation, and a spinal sagittal imbalance. The patient underwent a two-stage surgical procedure including an anterior T2 corpectomy, T1/T2 and T2/T3 discectomy, anterior instrumentation, posterior total tumor resection, and posterior fixation with laminar hooks. Postoperative imaging demonstrated cord decompression and good spinal alignment. Although a spinal ABC is a rare entity, it can occur in any segment of the spinal column and cause cord compression and spinal instability. Thus, proper treatment planning is crucial for total tumor resection to avoid tumor recurrence and correct spinal imbalance.
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Acute leukemia is a common malignancy among children and adults worldwide and many patients suffer from chronic health issues using current therapeutic approaches. Therefore, there is a great need for the development of novel and more specific therapies with fewer side effects. The family of Polo-like kinases (Plks) is a group of five serine/threonine kinases that play an important role in cell cycle regulation and are critical targets for therapeutic invention. Plk1 and Plk4 are novel targets for cancer therapy as leukemic cells often express higher levels than normal cells. In contrast, Plk2 and Plk3 are considered to be tumor suppressors. Several small molecule inhibitors have been developed for targeting Plk1 inhibition. Despite reaching phase III clinical trials, one of the ATP-competitive Plk1 inhibitor, volasertib, did not induce an objective clinical response and even caused lethal side effects in some patients. In order to improve the specificity of the Plk1 inhibitors and reduce off-target side effects, novel RNA interference (RNAi)-based therapies have been developed. In this review, we summarize the mechanisms of action of the Plk family members in acute leukemia, describe preclinical studies and clinical trials involving Plk-targeting drugs and discuss novel approaches in Plk targeting.
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Leucemia/enzimología , Terapia Molecular Dirigida , Proteínas de Neoplasias/fisiología , Proteínas Serina-Treonina Quinasas/fisiología , Proteínas Supresoras de Tumor/fisiología , Enfermedad Aguda , Adulto , Animales , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/fisiología , Niño , Ensayos Clínicos como Asunto , Terapia Combinada , Ensayos de Selección de Medicamentos Antitumorales , Regulación Enzimológica de la Expresión Génica , Regulación Leucémica de la Expresión Génica , Trasplante de Células Madre Hematopoyéticas , Humanos , Leucemia/tratamiento farmacológico , Leucemia/terapia , Ratones , Familia de Multigenes , Proteínas de Neoplasias/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Interferencia de ARN , Tasa de Supervivencia , Proteínas Supresoras de Tumor/genética , Quinasa Tipo Polo 1RESUMEN
Kynurenine pathway (KP) activation by the enzymatic activity of indoleamine 2,3-dioxygenase1 (IDO1) and kynurenine (KYN) production represents an attractive target for reducing tumour progression and improving anti-tumour immunity in multiple cancers. However, immunomodulatory properties of other KP metabolites such as 3-hydroxy kynurenine (3-HK) and kynurenic acid (KYNA) are poorly understood. The association of the kynurenine metabolic pathway with T-cell status in the tumour microenvironment were characterized, using gene expression data of 368 cutaneous skin melanoma (SKCM) patients from the TCGA cohort. Based on the identified correlations, we characterized the production of KYN, 3-HK, and KYNA in vitro using melanoma-derived cell lines and primary CD4+ CD25- T-cells. Activation of the CD4+ T-cells produced IFNγ, which yielded increased levels of KYN and KYNA. Concurrently, kynurenine 3-monooxygenase (KMO) expression and proliferation of CD4+ T-cells were reduced, whereas exhaustion markers such as PD-L1, AHR, FOXP3, and CTLA4 were increased. Additionally, an analysis of the correlation network reconstructed using TCGA-SKCM emphasized KMO and KYNU with high variability among BRAF wild-type compared with V600E, which underscored their role in distinct CD4+ T-cell behavior in tumour immunity. Our results suggest that, in addition to IDO1, there is an alternative immune regulatory mechanism associated with the lower KMO expression and the higher KYNA production, which contributes to dysfunctional effector CD4+ T-cell response.
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Quinurenina/metabolismo , Melanoma/patología , Neoplasias Cutáneas/patología , Antígeno B7-H1/genética , Antígeno B7-H1/metabolismo , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/metabolismo , Línea Celular , Proliferación Celular/efectos de los fármacos , Técnicas de Cocultivo , Medios de Cultivo Condicionados/farmacología , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Regulación de la Expresión Génica , Humanos , Indolamina-Pirrol 2,3,-Dioxigenasa/metabolismo , Interferón gamma/farmacología , Ácido Quinurénico/análisis , Ácido Quinurénico/metabolismo , Melanoma/inmunología , Melanoma/metabolismo , Redes y Vías Metabólicas , Metabolómica , Proteínas Proto-Oncogénicas B-raf/genética , Neoplasias Cutáneas/inmunología , Neoplasias Cutáneas/metabolismo , Triptófano/análisis , Triptófano/metabolismo , Microambiente Tumoral , Regulación hacia Arriba , Melanoma Cutáneo MalignoRESUMEN
OBJECTIVES: Tourette syndrome (TS) is a neuropsychiatric disorder characterized by childhood onset motor and phonic tics. In refractory cases, deep brain stimulation (DBS) with different targets including anteromedial Globus pallidus (AM-GPi) looks promising. PATIENTS AND METHODS: Patients with TS diagnosed according to DSM-IV TR criteria with severe medication-recalcitrant disease referred to our DBS clinic, were recruited for this study. They underwent bilateral AM-GPi DBS with Model 3389, Medtronic electrodes. Patients were assessed using Yale Global Tic Severity Scale (YGTSS) and Gilles de la Touretts syndrome-quality of life (GTS-QOL) questionnaire before and one year after DBS. RESULTS: Six patients (four men and two women) with severe medication-recalcitrant TS, mean age of 26.33⯱â¯7.25 years fulfilled the follow up visits. All patients revealed significant improvement in tics severity one year after surgery. Based on YGTSS, total tic severity score decreased from 75.66⯱â¯16.54 to 28.33⯱â¯13.95, P-value:0.005. Quality of life improved significantly after DBS (26.66⯱â¯20.65 before and 70.00⯱â¯17.88 one year after surgery, P-value:0.02). CONCLUSIONS: Results of our study in accordance to previous ones suggest AM-GPi DBS as an effective and well-tolerated therapeutic modality for patients with medication refractory TS.
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Estimulación Encefálica Profunda , Globo Pálido/cirugía , Síndrome de Tourette/terapia , Adulto , Estimulación Encefálica Profunda/métodos , Manual Diagnóstico y Estadístico de los Trastornos Mentales , Femenino , Humanos , Irán , Masculino , Calidad de Vida , Índice de Severidad de la Enfermedad , Resultado del TratamientoRESUMEN
There is a need for standardized validation methods for antibody specificity and selectivity. Recently, five alternative validation pillars were proposed to explore the specificity of research antibodies using methods with no need for prior knowledge about the protein target. Here, we show that these principles can be used in a streamlined manner for enhanced validation of research antibodies in Western blot applications. More than 6,000 antibodies were validated with at least one of these strategies involving orthogonal methods, genetic knockdown, recombinant expression, independent antibodies, and capture mass spectrometry analysis. The results show a path forward for efforts to validate antibodies in an application-specific manner suitable for both providers and users.
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Anticuerpos/inmunología , Especificidad de Anticuerpos/inmunología , Ensayos Analíticos de Alto Rendimiento/métodos , Estudios de Validación como Asunto , Animales , Western Blotting/métodos , Western Blotting/normas , Ensayos Analíticos de Alto Rendimiento/normas , Humanos , Estándares de Referencia , Reproducibilidad de los ResultadosRESUMEN
A majority of patients with BRAF-mutated metastatic melanoma respond to therapy with BRAF inhibitors (BRAFi), but relapses are common owing to acquired resistance. To unravel BRAFi resistance mechanisms we have performed gene expression and mass spectrometry based proteome profiling of the sensitive parental A375 BRAF V600E-mutated human melanoma cell line and of daughter cell lines with induced BRAFi resistance. Increased expression of two novel resistance candidates, aminopeptidase-N (CD13/ANPEP) and ETS transcription factor FLI1 was observed in the BRAFi-resistant daughter cell lines. In addition, increased levels of the previously reported resistance mediators, receptor tyrosine kinase ephrine receptor A2 (EPHA2) and the hepatocyte growth factor receptor MET were also identified. The expression of these proteins was assessed in matched tumor samples from melanoma patients obtained before BRAFi and after disease progression. MET was overexpressed in all progression samples while the expression of the other candidates varied between the individual patients. Targeting CD13/ANPEP by a blocking antibody induced apoptosis in both parental A375- and BRAFi-resistant daughter cells as well as in melanoma cells with intrinsic BRAFi resistance and led to dephosphorylation of EPHA2 on S897, previously demonstrated to cause inhibition of the migratory capacity. AKT and RSK, both reported to induce EPHA2 S897 phosphorylation, were also dephosphorylated after inhibition of CD13/ANPEP. FLI1 silencing also caused decreases in EPHA2 S897 phosphorylation and in total MET protein expression. In addition, silencing of FLI1 sensitized the resistant cells to BRAFi. Furthermore, we show that BRAFi in combination with the multi kinase inhibitor dasatinib can abrogate BRAFi resistance and decrease both EPHA2 S897 phosphorylation and total FLI1 protein expression. This is the first report presenting CD13/ANPEP and FLI1 as important mediators of resistance to BRAF inhibition with potential as drug targets in BRAFi refractory melanoma.
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Antígenos CD13/genética , Efrina-A2/genética , Regulación Neoplásica de la Expresión Génica , Melanoma/genética , Proteínas de Microfilamentos/antagonistas & inhibidores , Proteínas Proto-Oncogénicas B-raf/genética , Receptores Citoplasmáticos y Nucleares/antagonistas & inhibidores , Neoplasias Cutáneas/genética , Antineoplásicos/uso terapéutico , Apoptosis/efectos de los fármacos , Apoptosis/genética , Antígenos CD13/antagonistas & inhibidores , Antígenos CD13/metabolismo , Puntos de Control del Ciclo Celular/efectos de los fármacos , Puntos de Control del Ciclo Celular/genética , Línea Celular Tumoral , Dasatinib/uso terapéutico , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Efrina-A2/metabolismo , Humanos , Indoles/uso terapéutico , Melanoma/tratamiento farmacológico , Melanoma/metabolismo , Melanoma/patología , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Fosforilación , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Proteínas Proto-Oncogénicas B-raf/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-met/genética , Proteínas Proto-Oncogénicas c-met/metabolismo , Piridonas/uso terapéutico , Pirimidinonas/uso terapéutico , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Receptor EphA2 , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Proteínas Quinasas S6 Ribosómicas 90-kDa/genética , Proteínas Quinasas S6 Ribosómicas 90-kDa/metabolismo , Transducción de Señal , Neoplasias Cutáneas/tratamiento farmacológico , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/patología , Sulfonamidas/uso terapéutico , Transactivadores , VemurafenibRESUMEN
Docetaxel is a cornerstone treatment for metastatic, castration resistant prostate cancer (CRPC) which remains a leading cause of cancer-related deaths, worldwide. The clinical usage of docetaxel has resulted in modest gains in survival, primarily due to the development of resistance. There are currently no clinical biomarkers available that predict whether a CRPC patient will respond or acquire resistance to this therapy. Comparative proteomics analysis of exosomes secreted from DU145 prostate cancer cells that are sensitive (DU145 Tax-Sen) or have acquired resistance (DU145 Tax-Res) to docetaxel, demonstrated significant differences in the amount of exosomes secreted and in their molecular composition. A panel of proteins was identified by proteomics to be differentially enriched in DU145 Tax-Res compared to DU145 Tax-Sen exosomes and was validated by western blotting. Importantly, we identified MDR-1, MDR-3, Endophilin-A2 and PABP4 that were enriched only in DU145 Tax-Res exosomes. We validated the presence of these proteins in the serum of a small cohort of patients. DU145 cells that have uptaken DU145 Tax-Res exosomes show properties of increased matrix degradation. In summary, exosomes derived from DU145 Tax-Res cells may be a valuable source of biomarkers for response to therapy.
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Antineoplásicos/química , Resistencia a Antineoplásicos , Exosomas/efectos de los fármacos , Neoplasias de la Próstata Resistentes a la Castración/tratamiento farmacológico , Neoplasias de la Próstata/metabolismo , Taxoides/química , Taxoides/uso terapéutico , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Biomarcadores de Tumor/metabolismo , Proteínas Sanguíneas/metabolismo , Muerte Celular , Línea Celular Tumoral/efectos de los fármacos , Estudios de Cohortes , Biología Computacional , Docetaxel , Exosomas/metabolismo , Matriz Extracelular , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Masculino , Nanopartículas/química , Proteínas de Unión a Poli(A)/metabolismo , Neoplasias de la Próstata Resistentes a la Castración/metabolismo , ProteomaRESUMEN
Reconstruction of the bladder wall via in vitro differentiated stem cells on an appropriate scaffold could be used in such conditions as cancer and neurogenic urinary bladder. This study aimed to examine the potential of human endometrial stem cells (EnSCs) to form urinary bladder epithelial cells (urothelium) on nanofibrous silk-collagen scaffolds, for construction of the urinary bladder wall. After passage 4, EnSCs were induced by keratinocyte growth factor (KGF) and epidermal growth factor (EGF) and seeded on electrospun collagen-V, silk and silk-collagen nanofibres. Later we tested urothelium-specific genes and proteins (uroplakin-Ia, uroplakin-Ib, uroplakin-II, uroplakin-III and cytokeratin 20) by immunocytochemistry, RT-PCR and western blot analyses. Scanning electron microscopy (SEM) and histology were used to detect cell-matrix interactions. DMEM/F12 supplemented by KGF and EGF induced EnSCs to express urothelial cell-specific genes and proteins. Either collagen, silk or silk-collagen scaffolds promoted cell proliferation. The nanofibrous silk-collagen scaffolds provided a three-dimensional (3D) structure to maximize cell-matrix penetration and increase differentiation of the EnSCs. Human EnSCs seeded on 3D nanofibrous silk-collagen scaffolds and differentiated to urothelial cells provide a suitable source for potential use in bladder wall reconstruction in women.
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Endometrio/citología , Células Madre/citología , Ingeniería de Tejidos/métodos , Andamios del Tejido/química , Vejiga Urinaria/patología , Urotelio/patología , Adulto , Materiales Biocompatibles/farmacología , Técnicas de Cultivo de Célula , Diferenciación Celular , Células Cultivadas , Colágeno/química , Ensayo de Inmunoadsorción Enzimática , Factor de Crecimiento Epidérmico/metabolismo , Femenino , Factor 7 de Crecimiento de Fibroblastos/metabolismo , Humanos , Inmunohistoquímica , Queratinocitos/citología , Microscopía Electrónica de Rastreo , Nanofibras/química , Fenotipo , Seda/química , Urotelio/citologíaRESUMEN
Previous studies in cell lines have suggested a role for melanosomes and related protein trafficking pathways in melanoma drug response. We have investigated the expression of six proteins related to melanosomes and melanogenesis (MITF, GPR143, gp100/PMEL, MLANA, TYRP1, and RAB27A) in pretreatment metastases from melanoma patients (n = 52) with different response to dacarbazine/temozolomide. Microphthalmia-associated transcription factor (MITF) and G-protein coupled receptor 143 (GPR143) showed significantly higher expression in nonresponders compared with responders. The premelanosome protein (gp100/PMEL) has been indicated previously in resistance to cisplatin in melanoma cells, but the expression levels of gp100/PMEL showed no association with response to dacarbazine/temozolomide in our clinical material. We also investigated the effects on chemosensitivity of siRNA inhibition of gp100/PMEL in the MNT-1 melanoma cell line. As expected from the study of the tumor material, no effect was detected with respect to response to temozolomide. However, knockdown of gp100/PMEL sensitized the cells to both paclitaxel and cisplatin. Overall, our results suggest that MITF, and several MITF-regulated factors, are associated with resistance to chemotherapy in melanoma and that different MITF targets can be of importance for different drugs.
Asunto(s)
Antineoplásicos/uso terapéutico , Biomarcadores de Tumor/metabolismo , Resistencia a Antineoplásicos , Melanoma/metabolismo , Melanosomas/metabolismo , Factor de Transcripción Asociado a Microftalmía/metabolismo , Neoplasias Cutáneas/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/genética , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Resistencia a Antineoplásicos/genética , Proteínas del Ojo/genética , Proteínas del Ojo/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Concentración 50 Inhibidora , Masculino , Melanoma/tratamiento farmacológico , Melanoma/genética , Melanoma/patología , Melanosomas/genética , Melanosomas/patología , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Factor de Transcripción Asociado a Microftalmía/genética , Persona de Mediana Edad , Interferencia de ARN , Neoplasias Cutáneas/tratamiento farmacológico , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/patología , Transcripción Genética , Transfección , Resultado del Tratamiento , Antígeno gp100 del Melanoma/genética , Antígeno gp100 del Melanoma/metabolismoRESUMEN
Male Zucker diabetic fatty (mZDF) rats spontaneously develop type 2 diabetes, whereas females only become diabetic when fed a diabetogenic high-fat diet (high-fat-fed female ZDF rat, HF-fZDF). The aim of this study was to investigate if differences in liver functions could provide clues to this sex difference. Non-diabetic obese fZDF rats were compared with either mZDF or HF-fZDF regarding hepatic molecular profiles, to single out those components that might be protective in the females. High-fat feeding in fZDF led to enhanced weight gain, increased blood glucose and insulin levels, reduced insulin sensitivity and a trend towards reduced glucose tolerance, indicative of a prediabetic state. mZDF rats were diabetic, with low levels of insulin, high levels of glucose, reduced insulin sensitivity and impaired glucose tolerance. Transcript profiling and capillary electrophoresis time-of-flight mass spectrometry were used to indentify hepatic transcripts and metabolites that might be related to this. Many diet-induced alterations in transcript and metabolite levels in female rats were towards a 'male-like' phenotype, including reduced lipogenesis, increased fatty acid (FA) oxidation and increased oxidative stress responses. Alterations detected at the level of hepatic metabolites, indicated lower capacity for glutathione (GSH) production in male rats, and higher GSH turnover in females. Taken together, this could be interpreted as if anabolic pathways involving lipogenesis and lipid output might limit the degree of FA oxidation and oxidative stress in female rats. Together with a greater capacity to produce GSH, these hepatic sex differences might contribute to the sex-different development of diabetes in ZDF rats.