Assuntos
Acetamidas/uso terapêutico , Anemia Hemolítica Congênita/sangue , Eritrócitos/metabolismo , Mutação com Ganho de Função , Hidropisia Fetal/sangue , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/antagonistas & inibidores , Transporte de Íons/efeitos dos fármacos , Mutação de Sentido Incorreto , Bloqueadores dos Canais de Potássio/uso terapêutico , Potássio/sangue , Compostos de Tritil/uso terapêutico , Acetamidas/farmacologia , Anemia Hemolítica Congênita/genética , Contagem de Células , Avaliação Pré-Clínica de Medicamentos , Deformação Eritrocítica , Eritrócitos/efeitos dos fármacos , Heterozigoto , Humanos , Hidropisia Fetal/genética , Técnicas In Vitro , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/genética , Fragilidade Osmótica , Técnicas de Patch-Clamp , Bloqueadores dos Canais de Potássio/farmacologia , Compostos de Tritil/farmacologiaRESUMO
Subtype-specific human cardiomyocytes (CMs) are valuable for basic and applied research. Induction of cardiomyogenesis and enrichment of nodal-like CMs was described for mouse pluripotent stem cells (mPSCs) in response to 1-ethyl-2-benzimidazolinone (EBIO), a chemical modulator of small-/intermediate-conductance Ca2+-activated potassium channels (SKs 1-4). Investigating EBIO in human pluripotent stem cells (PSCs), we have applied three independent differentiation protocols of low to high cardiomyogenic efficiency. Equivalent to mPSCs, timed EBIO supplementation during hPSC differentiation resulted in dose-dependent enrichment of up to 80% CMs, including an increase in nodal- and atrial-like phenotypes. However, our study revealed extensive EBIO-triggered cell loss favoring cardiac progenitor preservation and, subsequently, CMs with shortened action potentials. Proliferative cells were generally more sensitive to EBIO, presumably via an SK-independent mechanism. Together, EBIO did not promote cardiogenic differentiation of PSCs, opposing previous findings, but triggered lineage-selective survival at a cardiac progenitor stage, which we propose as a pharmacological strategy to modulate CM subtype composition.
Assuntos
Benzimidazóis/farmacologia , Agonistas dos Canais de Cálcio/farmacologia , Diferenciação Celular/efeitos dos fármacos , Miócitos Cardíacos/citologia , Miócitos Cardíacos/efeitos dos fármacos , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/efeitos dos fármacos , Biomarcadores , Diferenciação Celular/genética , Linhagem Celular , Linhagem da Célula , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/genética , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/metabolismo , Mesoderma/citologia , Mesoderma/efeitos dos fármacos , Mesoderma/embriologia , Miócitos Cardíacos/metabolismo , Células-Tronco Pluripotentes/metabolismoRESUMO
Angiosarcoma is a rare malignant mesenchymal tumor with poor prognosis. We aimed to identify malignancy-associated miRNAs and their target genes, and explore biological functions of miRNA and its target in angiosarcoma. By miRNA microarrays and reverse transcription polymerase chain reaction, we identified 1 up-regulated miRNA (miR-222-3p) and 3 down-regulated miRNAs (miR-497-5p, miR-378-3p and miR-483-5p) in human angiosarcomas compared with human capillary hemangiomas. The intermediate-conductance calcium activated potassium channel KCa3.1 was one of the putative target genes of miR-497-5p, and marked up-regulation of KCa3.1 was detected in angiosarcoma biopsy specimens by immunohistochemistry. The inverse correlation of miR-497-5p and KCa3.1 also was observed in the ISO-HAS angiosarcoma cell line at the mRNA and protein levels. The direct targeting of KCa3.1 by miR-497-5p was evidenced by reduced luciferase activity due to complementary binding of miR-497-5p to KCa3.1 mRNA 3' untranslated region. For the functional role of miR-497-5p/KCa3.1 pair, we showed that application of TRAM-34, a specific KCa3.1 channel blocker, or transfection of ISO-HAS cells with KCa3.1 siRNA or miR-497-5p mimics inhibited cell proliferation, cell cycle progression, and invasion by down-regulating cell-cycle related proteins including cyclin D1, surviving and P53 and down-regulating matrix metallopeptidase 9. In an in vivo angiosarcoma xenograft model, TRAM-34 or miR-497-5p mimics both inhibited tumor growth. In conclusion, the tumor suppressor miR-497-5p down-regulates KCa3.1 expression and contributes to the inhibition of angiosarcoma malignancy development. The miR-497-5p or KCa3.1 might be potential new targets for angiosarcoma treatment.
Assuntos
Regulação Neoplásica da Expressão Gênica , Genes Supressores de Tumor , Hemangiossarcoma/genética , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/genética , MicroRNAs/metabolismo , Regiões 3' não Traduzidas , Animais , Carcinogênese/genética , Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Regulação para Baixo , Feminino , Hemangiossarcoma/patologia , Humanos , Imuno-Histoquímica , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , MicroRNAs/genética , Análise em Microsséries , Invasividade Neoplásica/genética , Pirazóis/farmacologia , Interferência de RNA , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Transfecção , Regulação para CimaRESUMO
Malignant gliomas are among the most frequent and aggressive cerebral tumors, characterized by high proliferative and invasive indexes. Standard therapy for patients, after surgery and radiotherapy, consists of temozolomide (TMZ), a methylating agent that blocks tumor cell proliferation. Currently, there are no therapies aimed at reducing tumor cell invasion. Ion channels are candidate molecular targets involved in glioma cell migration and infiltration into the brain parenchyma. In this paper we demonstrate that: i) blockade of the calcium-activated potassium channel KCa3.1 with TRAM-34 has co-adjuvant effects with TMZ, reducing GL261 glioma cell migration, invasion and colony forming activity, increasing apoptosis, and forcing cells to pass the G2/M cell cycle phase, likely through cdc2 de-phosphorylation; ii) KCa3.1 silencing potentiates the inhibitory effect of TMZ on glioma cell viability; iii) the combination of TMZ/TRAM-34 attenuates the toxic effects of glioma conditioned medium on neuronal cultures, through a microglia dependent mechanism since the effect is abolished by clodronate-induced microglia killing; iv) TMZ/TRAM-34 co-treatment increases the number of apoptotic tumor cells, and the mean survival time in a syngeneic mouse glioma model (C57BL6 mice implanted with GL261 cells); v) TMZ/TRAM-34 co-treatment reduces cell viability of GBM cells and cancer stem cells (CSC) freshly isolated from patients.Taken together, these data suggest a new therapeutic approach for malignant glioma, targeting both glioma cell proliferating and migration, and demonstrate that TMZ/TRAM-34 co-treatment affects both glioma cells and infiltrating microglia, resulting in an overall reduction of tumor cell progression.
Assuntos
Antineoplásicos Alquilantes/uso terapêutico , Neoplasias Encefálicas/tratamento farmacológico , Dacarbazina/análogos & derivados , Glioma/tratamento farmacológico , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/antagonistas & inibidores , Pirazóis/farmacologia , Animais , Apoptose/efeitos dos fármacos , Neoplasias Encefálicas/mortalidade , Proteína Quinase CDC2/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Dacarbazina/farmacologia , Dacarbazina/uso terapêutico , Sinergismo Farmacológico , Quimioterapia Combinada , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Glioma/mortalidade , Humanos , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/genética , Estimativa de Kaplan-Meier , Camundongos , Camundongos Endogâmicos C57BL , Microglia/efeitos dos fármacos , Microglia/metabolismo , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/mortalidade , Células-Tronco Neoplásicas/efeitos dos fármacos , Fosforilação , Cultura Primária de Células , Pirazóis/uso terapêutico , TemozolomidaRESUMO
BACKGROUND: Extensive alveolar epithelial injury and remodelling is a common feature of acute lung injury and acute respiratory distress syndrome (ARDS) and it has been established that epithelial regeneration, and secondary lung oedema resorption, is crucial for ARDS resolution. Much evidence indicates that K(+) channels are regulating epithelial repair processes; however, involvement of the KCa3.1 channels in alveolar repair has never been investigated before. RESULTS: Wound-healing assays demonstrated that the repair rates were increased in primary rat alveolar cell monolayers grown on a fibronectin matrix compared to non-coated supports, whereas an anti-ß1-integrin antibody reduced it. KCa3.1 inhibition/silencing impaired the fibronectin-stimulated wound-healing rates, as well as cell migration and proliferation, but had no effect in the absence of coating. We then evaluated a putative relationship between KCa3.1 channel and the migratory machinery protein ß1-integrin, which is activated by fibronectin. Co-immunoprecipitation and immunofluorescence experiments indicated a link between the two proteins and revealed their cellular co-distribution. In addition, we demonstrated that KCa3.1 channel and ß1-integrin membrane expressions were increased on a fibronectin matrix. We also showed increased intracellular calcium concentrations as well as enhanced expression of TRPC4, a voltage-independent calcium channel belonging to the large TRP channel family, on a fibronectin matrix. Finally, wound-healing assays showed additive effects of KCa3.1 and TRPC4 inhibitors on alveolar epithelial repair. CONCLUSION: Taken together, our data demonstrate for the first time complementary roles of KCa3.1 and TRPC4 channels with extracellular matrix and ß1-integrin in the regulation of alveolar repair processes.
Assuntos
Células Epiteliais Alveolares/metabolismo , Integrina beta1/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/metabolismo , Alvéolos Pulmonares/metabolismo , Cicatrização , Células Epiteliais Alveolares/efeitos dos fármacos , Células Epiteliais Alveolares/patologia , Animais , Movimento Celular , Proliferação de Células , Células Cultivadas , Relação Dose-Resposta a Droga , Fibronectinas/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/antagonistas & inibidores , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/genética , Masculino , Bloqueadores dos Canais de Potássio/farmacologia , Alvéolos Pulmonares/efeitos dos fármacos , Alvéolos Pulmonares/patologia , Interferência de RNA , Ratos Sprague-Dawley , Transdução de Sinais , Canais de Cátion TRPC/antagonistas & inibidores , Canais de Cátion TRPC/genética , Canais de Cátion TRPC/metabolismo , Fatores de Tempo , Transfecção , Cicatrização/efeitos dos fármacosRESUMO
We performed genomic mapping of a family with autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) and intellectual and psychiatric problems, identifying a disease-associated region on chromosome 9q34.3. Whole-exome sequencing identified a mutation in KCNT1, encoding a sodium-gated potassium channel subunit. KCNT1 mutations were identified in two additional families and a sporadic case with severe ADNFLE and psychiatric features. These findings implicate the sodium-gated potassium channel complex in ADNFLE and, more broadly, in the pathogenesis of focal epilepsies.