RESUMEN
Disruption of alternative splicing frequently causes or contributes to human diseases and disorders. Consequently, there is a need for efficient and sensitive reporter assays capable of screening chemical libraries for compounds with efficacy in modulating important splicing events. Here, we describe a screening workflow employing dual Nano and Firefly luciferase alternative splicing reporters that affords efficient, sensitive, and linear detection of small molecule responses. Applying this system to a screen of ~95,000 small molecules identified compounds that stimulate or repress the splicing of neuronal microexons, a class of alternative exons often disrupted in autism and activated in neuroendocrine cancers. One of these compounds rescues the splicing of several analyzed microexons in the cerebral cortex of an autism mouse model haploinsufficient for Srrm4, a major activator of brain microexons. We thus describe a broadly applicable high-throughput screening system for identifying candidate splicing therapeutics, and a resource of small molecule modulators of microexons with potential for further development in correcting aberrant splicing patterns linked to human disorders and disease.
Asunto(s)
Empalme Alternativo , Exones , Genes Reporteros , Ensayos Analíticos de Alto Rendimiento , Luciferasas de Luciérnaga , Bibliotecas de Moléculas Pequeñas , Animales , Empalme Alternativo/efectos de los fármacos , Humanos , Ensayos Analíticos de Alto Rendimiento/métodos , Ratones , Exones/genética , Bibliotecas de Moléculas Pequeñas/farmacología , Luciferasas de Luciérnaga/genética , Luciferasas de Luciérnaga/metabolismo , Células HEK293 , Corteza Cerebral/metabolismo , Corteza Cerebral/efectos de los fármacos , Neuronas/metabolismo , Neuronas/efectos de los fármacosRESUMEN
Anaplastic thyroid carcinoma is arguably the most lethal human malignancy. It often co-occurs with differentiated thyroid cancers, yet the molecular origins of its aggressivity are unknown. We sequenced tumor DNA from 329 regions of thyroid cancer, including 213 from patients with primary anaplastic thyroid carcinomas. We also whole genome sequenced 9 patients using multi-region sequencing of both differentiated and anaplastic thyroid cancer components. Using these data, we demonstrate thatanaplastic thyroid carcinomas have a higher burden of mutations than other thyroid cancers, with distinct mutational signatures and molecular subtypes. Further, different cancer driver genes are mutated in anaplastic and differentiated thyroid carcinomas, even those arising in a single patient. Finally, we unambiguously demonstrate that anaplastic thyroid carcinomas share a genomic origin with co-occurring differentiated carcinomas and emerge from a common malignant field through acquisition of characteristic clonal driver mutations.
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Adenocarcinoma , Carcinoma Anaplásico de Tiroides , Neoplasias de la Tiroides , Humanos , Carcinoma Anaplásico de Tiroides/genética , Carcinoma Anaplásico de Tiroides/patología , Neoplasias de la Tiroides/genética , Neoplasias de la Tiroides/patología , Mutación/genética , GenómicaRESUMEN
BACKGROUND: S100B and Tau are implicated with both brain growth and injury. Their urinary levels in 30-to-40-day-old full-term, preterm, IUGR, and preterm-IUGR subjects were measured to investigate their possible relationship with future delayed neurodevelopment. METHODS: Values were related to the neuro-behavioral outcome at two years of age, as well as to brain volumes and urinary NGF assessed at the same postnatal time point. RESULTS: Using the Griffiths III test, cognitive and motor performances were determined to establish subgroups characterized by either normal or impaired neuro-behavior. The latter included preterm, IUGR, and preterm-IUGR individuals who exhibited significantly higher and lower S100B and Tau levels, respectively, along with markedly reduced cerebral volumes and urinary NGF, as previously demonstrated. Contrary to NGF, however, Tau and S100B displayed a weak correlation with brain volumes. CONCLUSIONS: Delayed cognitive and motor performances observed in two-year-old preterm and IUGR-born individuals were also found to be associated with anomalous urinary levels of S100B and Tau, assessed at 30-40 days of the postnatal period, and their changes did not correlate with brain growth. Thus, our data suggests that, in addition to cerebral volumes and NGF, urinary S100B and Tau can also be considered as valuable parameters for the early detection of future neurodevelopmental abnormalities.
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Encéfalo , Retardo del Crecimiento Fetal , Recién Nacido , Femenino , Humanos , Preescolar , Retardo del Crecimiento Fetal/diagnósticoRESUMEN
During cancer development, tumor cells acquire changes that enable them to invade surrounding tissues and seed metastasis at distant sites. These changes contribute to the aggressiveness of metastatic cancer and interfere with success of therapy. Our comprehensive analysis of "matched" pairs of HNSCC lines derived from primary tumors and corresponding metastatic sites identified several components of Notch3 signaling that are differentially expressed and/or altered in metastatic lines and confer a dependency on this pathway. These components were also shown to be differentially expressed between early and late stages of tumors in a TMA constructed from over 200 HNSCC patients. Finally, we show that suppression of Notch3 improves survival in mice in both subcutaneous and orthotopic models of metastatic HNSCC. Novel treatments targeting components of this pathway may prove effective in targeting metastatic HNSCC cells alone or in combination with conventional therapies.
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Carcinoma de Células Escamosas , Neoplasias de Cabeza y Cuello , Animales , Ratones , Transducción de Señal , Carcinoma de Células Escamosas de Cabeza y Cuello , HumanosRESUMEN
High-throughput screening (HTS) is a vaunted technology in drug discovery, and drug repositioning a celebrated strategy with famous examples of successful stories; however, repositioned drugs have primarily resulted from serendipitous observations, retrospective studies, and pharmacological analyses as opposed to experimental routes. This observation points to a methodological paradox, considering that academic laboratories of the post-genomic era have benefited from unprecedented technological progress, and a facilitated access to powerful resources that, historically, were a prerogative of the pharma industry. This disconnect is exacerbated by financial, practical, and regulatory complexities affecting drug repositioning; however, the pivotal significance of stringent and rigorous data is what unconditionally sits at the crossroad of go/no-go decisions concerning the therapeutic significance, or predictive validity, of selected drugs. Here, I propose a visionary approach, to which I assigned the term labsourcing, to dramatically enhance efficiency and clinical relevance of academic drug screens and, ultimately, generate contextual and reproducible data for correct interpretations and reliable selection of drug candidates. The overall concept implies intra- and intermural aggregation of expertise (e.g., assay development, cell biology, statistics, bioinformatics) to perform multiple bioassays, under multiple conditions and readouts, using a common screening collection. Advantages of high input screens can be manifold: (i) to tackle discrepancies that may arise from the screens of libraries of variable size and content and assay types and conditions too narrow in scope; (ii) the opportunity to generate massive amounts of data applicable for multiple publications and funding requests; (iii) the educational benefits for students and post-docs collegially exposed to long-term programs; and (iv) the opportunity to democratize research and recruit small labs that could not otherwise join screening programs due to costs, timelines, and risks.
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Descubrimiento de Drogas , Reposicionamiento de Medicamentos , Humanos , Estudios Retrospectivos , Descubrimiento de Drogas/métodos , Ensayos Analíticos de Alto Rendimiento , GenómicaRESUMEN
Alternative splicing (AS) is a critical regulatory layer; yet, factors controlling functionally coordinated splicing programs during developmental transitions are poorly understood. Here, we employ a screening strategy to identify factors controlling dynamic splicing events important for mammalian neurogenesis. Among previously unknown regulators, Rbm38 acts widely to negatively control neural AS, in part through interactions mediated by the established repressor of splicing, Ptbp1. Puf60, a ubiquitous factor, is surprisingly found to promote neural splicing patterns. This activity requires a conserved, neural-differential exon that remodels Puf60 co-factor interactions. Ablation of this exon rewires distinct AS networks in embryonic stem cells and at different stages of mouse neurogenesis. Single-cell transcriptome analyses further reveal distinct roles for Rbm38 and Puf60 isoforms in establishing neuronal identity. Our results describe important roles for previously unknown regulators of neurogenesis and establish how an alternative exon in a widely expressed splicing factor orchestrates temporal control over cell differentiation.
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Neurogénesis , Empalme del ARN , Empalme Alternativo , Animales , Exones/genética , Mamíferos , Ratones , Neurogénesis/genética , Neuronas , Proteínas de Unión al ARN/genéticaRESUMEN
OBJECTIVE: This study aimed at improving the discrimination of Prostate Imaging - Reporting and Data System version 2.1 (PI-RADS v2.1) score 3 suspicious prostate cancer lesions using lesion volume evaluation. MATERIAL AND METHODS: Two hundred five PI-RADS v2.1 score 3 lesions were submitted to transperineal MRI/TRUS fusion-targeted biopsy. The lesion volumes were estimated on diffusion-weighted imaging sequence and distributed in PI-RADS 3a (LV < 0.5 mL) and PI-RADS 3b (LV ≥ 0.5 mL) subcategories, using a 0.5 mL cutoff value. Data were retrospectively matched with histopathological findings from the biopsy. Assuming that lesions with LV < or ≥ 0.5 mL were respectively not eligible (benign and indolent PCa lesions) or eligible for biopsy (significant PCa lesions), the diagnostic accuracy of lesion volume in determining clinically significant PCa at biopsy was evaluated using a bi- or multivariate model. RESULTS: About 55.1% and 44.9% of lesions were distributed in subcategories 3a and 3b, respectively. The overall PI-RADS score 3 detection rate was 273%. 3.5% (1.95% of total), and 25% (11.7% of total) significant PCa were found in PI-RADS 3a and 3b subcategory, respectively. The method showed 85.2% sensitivity, 61.2% specificity, 25% positive predictive value, and 96.5% negative predictive value and avoided 55.1% of unnecessary biopsies. The diagnostic accuracy in determining significant PCa at biopsy was 73.2% or 86.5% depending on whether lesion volume was used alone or in combination with prostate volume and patient age in a multivariate model. CONCLUSION: 0.5 mL lesion volume cutoff value significantly discriminates fusion-targeted biopsy need in PI-RADS v2.1 score 3 lesions and its diagnostic accuracy improves when it combines with prostate volume and age in a multivariate model.
RESUMEN
Despite extensive analysis of pRB phosphorylation in vitro, how this modification influences development and homeostasis in vivo is unclear. Here, we show that homozygous Rb∆K4 and Rb∆K7 knock-in mice, in which either four or all seven phosphorylation sites in the C-terminal region of pRb, respectively, have been abolished by Ser/Thr-to-Ala substitutions, undergo normal embryogenesis and early development, notwithstanding suppressed phosphorylation of additional upstream sites. Whereas Rb∆K4 mice exhibit telomere attrition but no other abnormalities, Rb∆K7 mice are smaller and display additional hallmarks of premature aging including infertility, kyphosis, and diabetes, indicating an accumulative effect of blocking pRb phosphorylation. Diabetes in Rb∆K7 mice is insulin-sensitive and associated with failure of quiescent pancreatic ß-cells to re-enter the cell cycle in response to mitogens, resulting in induction of DNA damage response (DDR), senescence-associated secretory phenotype (SASP), and reduced pancreatic islet mass and circulating insulin level. Pre-treatment with the epigenetic regulator vitamin C reduces DDR, increases cell cycle re-entry, improves islet morphology, and attenuates diabetes. These results have direct implications for cell cycle regulation, CDK-inhibitor therapeutics, diabetes, and longevity.
Asunto(s)
Envejecimiento/fisiología , Ácido Ascórbico/farmacología , Diabetes Mellitus Experimental/prevención & control , Proteína de Retinoblastoma/metabolismo , Animales , Senescencia Celular/efectos de los fármacos , Quinasa 2 Dependiente de la Ciclina/antagonistas & inhibidores , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/patología , Factor de Transcripción E2F1/metabolismo , Desarrollo Embrionario/genética , Femenino , Fibroblastos/efectos de los fármacos , Técnicas de Sustitución del Gen , Células Secretoras de Insulina/patología , Ratones , Fosforilación , Embarazo , Proteína de Retinoblastoma/genética , Telómero/genéticaRESUMEN
Astrocytes can support neuronal survival through a range of secreted signals that protect against neurotoxicity, oxidative stress, and apoptotic cascades. Thus, analyzing the effects of the astrocyte secretome may provide valuable insight into these neuroprotective mechanisms. Previously, we characterized a potent neuroprotective activity mediated by retinal astrocyte conditioned media (ACM) on retinal and cortical neurons in metabolic stress models. However, the molecular mechanism underlying this complex activity in neuronal cells has remained unclear. Here, a chemical genetics screen of kinase inhibitors revealed phosphoinositide 3-kinase (PI3K) as a central player transducing ACM-mediated neuroprotection. To identify additional proteins contributing to the protective cascade, endogenous PI3K was immunoprecipitated from neuronal cells exposed to ACM or control media, followed by MS/MS proteomic analyses. These data pointed toward a relatively small number of proteins that coimmunoprecipitated with PI3K, and surprisingly only five were regulated by the ACM signal. These hits included expected PI3K interactors, such as the platelet-derived growth factor receptor A (PDGFRA), as well as novel RNA-binding protein interactors ZC3H14 (zinc finger CCCH-type containing 14) and THOC1 (THO complex protein 1). In particular, ZC3H14 has recently emerged as an important RNA-binding protein with multiple roles in posttranscriptional regulation. In validation studies, we show that PI3K recruitment of ZC3H14 is necessary for PDGF-induced neuroprotection and that this interaction is present in primary retinal ganglion cells. Thus, we identified a novel non-cell autonomous neuroprotective signaling cascade mediated through PI3K that requires recruitment of ZC3H14 and may present a promising strategy to promote astrocyte-secreted prosurvival signals.
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Astrocitos/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas de Unión a Poli(A)/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Inmunoprecipitación , Neuroprotección/fisiología , Fosfatidilinositol 3-Quinasas/química , Proteínas de Unión a Poli(A)/genética , Proteínas de Unión al ARN/genética , Espectrometría de Masas en TándemRESUMEN
Anaplastic thyroid cancer (ATC) is a rare, but nearly uniformly fatal disease that is typically resistant to chemotherapy and radiation. Alternative strategies to target this cancer at a molecular level are necessary in order to improve dismal outcomes for ATC patients. We examined the effects of flavopiridol, a CDK inhibitor, in a panel of ATC cell lines. When cell lines were treated over a ten-point concentration range, CAL62, KMH2 and BHT-101 cell lines had a sub micromolar half-maximal inhibitory concentration, while no effect was seen in the non-cancerous cell line IMR-90. Flavopiridol treatment resulted in decreased levels of the cell cycle proteins CDK9 and MCL1, and induced cell cycle arrest. Flavopiridol also decreased the in vitro ability of ATC cells to form colonies and impeded migration using a transwell migration assay. In vivo, flavopiridol decreased tumor weight and tumor volume over time in a patient-derived xenograft model of ATC. Given the observed in vitro and in vivo activity, flavopiridol warrants further investigation for treatment of ATC.
Asunto(s)
Puntos de Control del Ciclo Celular , Flavonoides/uso terapéutico , Piperidinas/uso terapéutico , Inhibidores de Proteínas Quinasas/uso terapéutico , Carcinoma Anaplásico de Tiroides/tratamiento farmacológico , Neoplasias de la Tiroides/tratamiento farmacológico , Animales , Apoptosis/efectos de los fármacos , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Quinasa 9 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 9 Dependiente de la Ciclina/genética , Quinasa 9 Dependiente de la Ciclina/metabolismo , Flavonoides/farmacología , Humanos , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Piperidinas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Carcinoma Anaplásico de Tiroides/patología , Neoplasias de la Tiroides/patología , Trasplante HeterólogoRESUMEN
RUVBL1 and RUVBL2 are highly conserved ATPases that belong to the AAA+ (ATPases Associated with various cellular Activities) superfamily and are involved in various complexes and cellular processes, several of which are closely linked to oncogenesis. The proteins were implicated in DNA damage signaling and repair, chromatin remodeling, telomerase activity, and in modulating the transcriptional activities of proto-oncogenes such as c-Myc and ß-catenin. Moreover, both proteins were found to be overexpressed in several different types of cancers such as breast, lung, kidney, bladder, and leukemia. Given their various roles and strong involvement in carcinogenesis, the RUVBL proteins are considered to be novel targets for the discovery and development of therapeutic cancer drugs. Here, we describe the identification of sorafenib as a novel inhibitor of the ATPase activity of human RUVBL2. Enzyme kinetics and surface plasmon resonance experiments revealed that sorafenib is a weak, mixed non-competitive inhibitor of the protein's ATPase activity. Size exclusion chromatography and small angle X-ray scattering data indicated that the interaction of sorafenib with RUVBL2 does not cause a significant effect on the solution conformation of the protein; however, the data suggested that the effect of sorafenib on RUVBL2 activity is mediated by the insertion domain in the protein. Sorafenib also inhibited the ATPase activity of the RUVBL1/2 complex. Hence, we propose that sorafenib could be further optimized to be a potent inhibitor of the RUVBL proteins.
Asunto(s)
ATPasas Asociadas con Actividades Celulares Diversas/antagonistas & inhibidores , Proteínas Portadoras/antagonistas & inhibidores , ADN Helicasas/antagonistas & inhibidores , Sorafenib/farmacología , ATPasas Asociadas con Actividades Celulares Diversas/química , ATPasas Asociadas con Actividades Celulares Diversas/metabolismo , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , ADN Helicasas/química , ADN Helicasas/metabolismo , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Humanos , Concentración 50 Inhibidora , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Agregado de Proteínas/efectos de los fármacos , Multimerización de Proteína/efectos de los fármacos , Sorafenib/químicaRESUMEN
Receptor tyrosine kinases (RTKs) are transmembrane receptors of great clinical interest due to their role in disease. Historically, therapeutics targeting RTKs have been identified using in vitro kinase assays. Due to frequent development of drug resistance, however, there is a need to identify more diverse compounds that inhibit mutated but not wild-type RTKs. Here, we describe MaMTH-DS (mammalian membrane two-hybrid drug screening), a live-cell platform for high-throughput identification of small molecules targeting functional protein-protein interactions of RTKs. We applied MaMTH-DS to an oncogenic epidermal growth factor receptor (EGFR) mutant resistant to the latest generation of clinically approved tyrosine kinase inhibitors (TKIs). We identified four mutant-specific compounds, including two that would not have been detected by conventional in vitro kinase assays. One of these targets mutant EGFR via a new mechanism of action, distinct from classical TKI inhibition. Our results demonstrate how MaMTH-DS is a powerful complement to traditional drug screening approaches.
Asunto(s)
Ensayos Analíticos de Alto Rendimiento/métodos , Inhibidores de Proteínas Quinasas/farmacología , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Línea Celular , Línea Celular Tumoral , ADN Nucleotidiltransferasas/genética , Descubrimiento de Drogas , Resistencia a Antineoplásicos/genética , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/genética , Genes Reporteros , Humanos , Luciferasas/genética , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Mutación , Fosforilación/efectos de los fármacos , Reproducibilidad de los Resultados , Bibliotecas de Moléculas Pequeñas/farmacología , Estaurosporina/análogos & derivados , Estaurosporina/farmacologíaRESUMEN
BACKGROUND: Previously, we found that amplification of chromosome 17q24.1-24.2 is associated with lymph node metastasis, tumour size, and lymphovascular invasion in invasive ductal carcinoma. A gene within this amplicon, CACNG4, an L-type voltage-gated calcium channel gamma subunit, is elevated in breast cancers with poor prognosis. Calcium homeostasis is achieved by maintaining low intracellular calcium levels. Altering calcium influx/efflux mechanisms allows tumour cells to maintain homeostasis despite high serum calcium levels often associated with advanced cancer (hypercalcemia) and aberrant calcium signaling. METHODS: In vitro 2-D and 3-D assays, and intracellular calcium influx assays were utilized to measure tumourigenic activity in response to altered CANCG4 levels and calcium channel blockers. A chick-CAM model and mouse model for metastasis confirmed these results in vivo. FINDINGS: CACNG4 alters cell motility in vitro, induces malignant transformation in 3-dimensional culture, and increases lung-specific metastasis in vivo. CACNG4 functions by closing the channel pore, inhibiting calcium influx, and altering calcium signaling events involving key survival and metastatic pathway genes (AKT2, HDAC3, RASA1 and PKCζ). INTERPRETATION: CACNG4 may promote homeostasis, thus increasing the survival and metastatic ability of tumour cells in breast cancer. Our findings suggest an underlying pathway for tumour growth and dissemination regulated by CACNG4 that is significant with respect to developing treatments that target these channels in tumours with aberrant calcium signaling. FUNDING: Canadian Breast Cancer Foundation, Ontario; Canadian Institutes of Health Research.
Asunto(s)
Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Canales de Calcio/genética , Amplificación de Genes , Animales , Neoplasias de la Mama/metabolismo , Calcio/metabolismo , Canales de Calcio/química , Canales de Calcio/metabolismo , Señalización del Calcio , Línea Celular , Movimiento Celular/genética , Proliferación Celular/efectos de los fármacos , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Progresión de la Enfermedad , Femenino , Expresión Génica , Humanos , Inmunohistoquímica , Ratones , Modelos Biológicos , Metástasis de la Neoplasia , Estadificación de Neoplasias , Dominios y Motivos de Interacción de ProteínasRESUMEN
The accumulation of damaged mitochondria causes the death of dopaminergic neurons. The Parkin-mediated mitophagy pathway functions to remove these mitochondria from cells. Targeting this pathway represents a therapeutic strategy for several neurodegenerative diseases, most notably Parkinson's disease. We describe a discovery pipeline to identify small molecules that increase Parkin recruitment to damaged mitochondria and ensuing mitophagy. We show that ROCK inhibitors promote the activity of this pathway by increasing the recruitment of HK2, a positive regulator of Parkin, to mitochondria. This leads to the increased targeting of mitochondria to lysosomes and removal of damaged mitochondria from cells. Furthermore, ROCK inhibitors demonstrate neuroprotective effects in flies subjected to paraquat, a parkinsonian toxin that induces mitochondrial damage. Importantly, parkin and rok are required for these effects, revealing a signaling axis which controls Parkin-mediated mitophagy that may be exploited for the development of Parkinson's disease therapeutics.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Mitocondrias/metabolismo , Mitofagia/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Ubiquitina-Proteína Ligasas/metabolismo , Quinasas Asociadas a rho/antagonistas & inhibidores , Animales , Línea Celular Tumoral , Dípteros , Hexoquinasa/genética , Hexoquinasa/metabolismo , Humanos , Masculino , Mitocondrias/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Ubiquitina-Proteína Ligasas/genética , Quinasas Asociadas a rho/genética , Quinasas Asociadas a rho/metabolismoRESUMEN
OBJECTIVES: Spleen tyrosine kinase (SYK) is a promoter of cell survival in a variety of cell types, including normal and cancerous epithelial cells. We hypothesized that SYK would an important therapeutic target to inhibit for the treatment of HNSCC. MATERIALS AND METHODS: SYK protein abundance in patient tumours was evaluated. SYK protein and mRNA abundance was used to examine patient survival and human papillomavirus (HPV) status. Small-interfering RNAs and gene editing with CRISPR/Cas9 were used to evaluate SYK expression on proliferation in HNSCC cell lines. The potency of SYK inhibitor ER27319 maleate on cellular proliferation was tested using a panel of 28 HNSCC cell lines and in vivo in HNSCC patient-derived xenograft (PDX) models. RESULTS: Moderate to high protein expression of SYK was observed in 24% of patient tumors and high SYK expression was exclusively observed in HPV-positive samples (p < 0.001). SYK inhibition with RNA interference, gene editing or a SYK inhibitor (ER27319) decreased cell proliferation and migration. Treatment of PDXs with ER27319 maleate was observed to reduce tumour burden in vivo in two of three models. CONCLUSIONS: HPV-positive HNSCC harbours high SYK protein levels. We demonstrate that proliferation, migration and overall burden of these tumours can be reduced by genetic or pharmacologic inhibition of SYK. Taken together, these data establish SYK as a therapeutic target for HNSCC.
Asunto(s)
Neoplasias de Cabeza y Cuello/etiología , Infecciones por Papillomavirus/complicaciones , Quinasa Syk/genética , Adulto , Anciano , Animales , Biomarcadores de Tumor , Línea Celular Tumoral , Proliferación Celular , Modelos Animales de Enfermedad , Susceptibilidad a Enfermedades , Femenino , Edición Génica , Regulación Neoplásica de la Expresión Génica , Neoplasias de Cabeza y Cuello/metabolismo , Neoplasias de Cabeza y Cuello/patología , Humanos , Inmunohistoquímica , Masculino , Ratones , Persona de Mediana Edad , Clasificación del Tumor , Estadificación de Neoplasias , Infecciones por Papillomavirus/virología , Interferencia de ARN , ARN Mensajero/genética , ARN Interferente Pequeño , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Selenium (Se) displays antioxidant properties that can be exploited, in plants, to counteract abiotic stresses caused by overly-produced reactive oxygen species (ROS). Here, we show that fertigation of maize crops with sodium selenate effectively protects pollen against oxidative stress. Pollen isolated from Se-treated plants (Se1) and untreated controls (Se0) was incubated in vitro with H2O2 to produce oxidative challenge. Given the impact of ROS on Ca2+ homeostasis and Ca2+-dependent signaling, cytosolic Ca2+ was measured to monitor cellular perturbations. We found that H2O2 disrupted Ca2+ homeostasis in Se0 pollen only, while Se1 samples were preserved. The same trend was observed when Se0 samples were treated with sodium selenate or Se-methionine, which recapitulated in vitro the protective capacity of Se-fertigation. Furthermore, we found that germination rates were much better retained in Se1 as compared to Se0 (46% vs 8%, respectively) after exposure to 20 mM H2O2. The same was observed with Se0 pollen treated with Se-methionine, which is the organic form of Se into which most fertigated sodium selenate converts in the plant. These results, together, show a close correlation between ROS, Ca2+ homeostasis and pollen fertility, and provide strong evidence that Se-fertigation is an excellent approach to preserve or enhance agricultural productivity.
Asunto(s)
Calcio/metabolismo , Selenio/metabolismo , Zea mays/metabolismo , Antioxidantes/farmacología , Citosol/metabolismo , Germinación/efectos de los fármacos , Germinación/fisiología , Homeostasis/fisiología , Peróxido de Hidrógeno/farmacología , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/fisiología , Hojas de la Planta/efectos de los fármacos , Polen/efectos de los fármacos , Especies Reactivas de Oxígeno/farmacología , Ácido Selénico/farmacología , Selenio/farmacología , Zea mays/efectos de los fármacosRESUMEN
Aberrant regulation of programmed cell death (PCD) has been tied to an array of human pathologies ranging from cancers to autoimmune disorders to diverse forms of neurodegeneration. Pharmacologic modulation of PCD signalling is therefore of central interest to a number of clinical and biomedical applications. A key component of PCD signalling involves the modulation of pro- and anti-apoptotic Bcl-2 family members. Among these, Bax translocation represents a critical regulatory phase in PCD. In the present study, we have employed a high-content high-throughput screen to identify small molecules which inhibit the cellular process of Bax re-distribution to the mitochondria following commitment of the cell to die. Screening of 6246 Generally Recognized As Safe compounds from four chemical libraries post-induction of cisplatin-mediated PCD resulted in the identification of 18 compounds which significantly reduced levels of Bax translocation. Further examination revealed protective effects via reduction of executioner caspase activity and enhanced mitochondrial function. Consistent with their effects on Bax translocation, these compounds exhibited significant rescue against in vitro and in vivo cisplatin-induced apoptosis. Altogether, our findings identify a new set of clinically useful small molecules PCD inhibitors and highlight the role which cAMP plays in regulating Bax-mediated PCD.
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Proliferación Celular/efectos de los fármacos , Proteínas Fluorescentes Verdes/antagonistas & inhibidores , Ensayos Analíticos de Alto Rendimiento/métodos , Transporte de Proteínas/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/farmacología , Proteína X Asociada a bcl-2/antagonistas & inhibidores , Animales , Células CHO , Cricetulus , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Proteína X Asociada a bcl-2/metabolismoRESUMEN
Anaplastic thyroid cancer (ATC) is a rare and lethal human malignancy with no known effective therapies in the majority of cases. Despite the use of conventional treatments such as chemotherapy, radiation and surgical resection, this disease remains almost universally fatal. In the present study, we identified the JAK2 inhibitor Lestaurtinib as a potent compound when testing against 13 ATC cell lines. Lestaurtinib demonstrated a potent antiproliferative effect in vitro at nanomolar concentrations. Furthermore, Lestaurtinib impeded cell migration and the ability to form colonies from single cells using scratch-wound and colony formation assays, respectively. Flow cytometry was used for cell cycle analysis following drug treatment and demonstrated arrest at the G2/M phase of the cell cycle, indicative of a cytostatic effect. In vivo studies using the chick chorioallantoic membrane xenograft models demonstrated that treatment with Lestaurtinib resulted in a significant decrease in endpoint tumor volume and vascularity using power Doppler ultrasound imaging. Overall, this study provides evidence that Lestaurtinib is a potent antiproliferative agent with potential antiangiogenic activity that warrants further investigation as a targeted therapy for ATC.
Asunto(s)
Antineoplásicos/farmacología , Carbazoles/farmacología , Carcinoma Anaplásico de Tiroides/tratamiento farmacológico , Neoplasias de la Tiroides/tratamiento farmacológico , Animales , Apoptosis/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Embrión de Pollo , Relación Dosis-Respuesta a Droga , Furanos , Humanos , Janus Quinasa 2/metabolismo , Carcinoma Anaplásico de Tiroides/irrigación sanguínea , Carcinoma Anaplásico de Tiroides/diagnóstico por imagen , Neoplasias de la Tiroides/irrigación sanguínea , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Nonsteroidal anti-inflammatory drugs (NSAIDs) are commonly prescribed for the treatment of pain and inflammation. Although it is well known that NSAIDs can suppress bone growth, remodelling and repair, they are largely used post-operatively and post-traumatically to achieve analgesia and reduce inflammation in bone tissue. AIMS: The impact of two NO-releasing, non-selective NSAIDs, NCX-4016 and HCT-3012 (NO-derivatives of Aspirin and Naproxen, respectively) on osteoblasts were evaluated and compared to the non-selective, parent chemicals and to the COX-2-selective inhibitor Celecoxib. MAIN METHODS: Using MG-63 osteoblast-like cells, we considered proliferation, the early and late stage of differentiation, and the activity of proteinases thought to be involved in osteoid degradation, a preliminary fundamental event of bone remodelling. KEY FINDINGS: Unlike Aspirin, Naproxen and Celecoxib, the two NO-NSAIDs did not alter proliferation and differentiation of osteoblasts. They also reduced the activity of plasminogen activator, metalloproteinases, and cathepsin B. Similar inhibitory effects against these proteinases were recapitulated by the NO-donor sodium nitroprusside, thereby suggesting a NO-mediated mechanism. SIGNIFICANCE: Due to a differential effect on cell proliferation and differentiation, the two NO-NSAIDs exhibit a safer impact on osteoblast metabolism compared to Celecoxib and their parent compounds. This suggests an advantageous option for these drugs in individuals with a need of COX-inhibiting treatment, in general. In addition, their capability of modulating the proteinases involved in osteoid degradation may specifically suggest an additional safer use in comorbidity conditions of inflammation or pain with bone disorders characterized by high rate of remodelling, such as high-turnover osteoporosis in post-menopausal women.