RESUMO
Os astrocitomas são neoplasias primárias do Sistema Nervoso Central, graduadas de I a IV com base em critérios clínicos e histológicos. O astrocitoma de grau IV, também denominado glioblastoma, é o tipo mais comum e agressivo dos tumores gliais e apresenta baixa resposta a agentes quimioterápicos. Em glioblastomas, mutações e superexpressão dos receptores de fatores de crescimento podem levar à ativação desregulada da via das proteínas cinases ativadas por mitógenos (MAPK). A via de MAPK é ativadora direta da cinase RSK (do inglês, p90 ribosomal S6 kinase), que está envolvida com diversos processos celulares. Apesar dessa proteína ter sido estudada em diferentes tipos tumorais, sua participação em glioblastomas nunca foi avaliada. Dessa maneira, neste trabalho tivemos como objetivo analisar o envolvimento de RSK na tumorigênese em glioblastoma, e observar a participação dessa proteína em processos como proliferação e senescência celular. Observamos que a regulação dessa proteína ocorre principalmente a nível traducional em linhagens celulares de glioblastoma. Utilizando células nocaute para RSK1 e/ou 2 obtidas pela metodologia CRISPR/Cas9, pudemos constatar o envolvimento dessa proteína na proliferação celular. Além disso, a relação entre RSK e a senescência celular induzida pela perda de PTEN foi demonstrada em células não transformadas, utilizando siRNA e inibidores químicos de RSK. Pudemos também descrever que inibidores de RSK comumente utilizados na literatura possuem importantes efeitos inespecíficos que podem levar a interpretações errôneas sobre as funções de RSK. Adicionalmente, determinamos que o melhor alvo para acessar a atividade de RSK é a proteína TSC2 (Ser1798).
Astrocytomas are primary Central Nervous System tumors graded from I to IV based on histological and clinical criteria. The grade IV astrocytoma, also known as glioblastoma, is the most common and aggressive of glial tumors and presents low response to chemotherapeutic agents. In glioblastomas, mutations and overexpression of growth factor receptors can lead to the upregulated activation of the mitogen-activated protein kinases pathways (MAPK). MAPK pathway is the major activator of the p90 ribosomal S6 kinase (RSK), which is involved in many cellular processes. RSK involvement has been performed in several tumor types; however it has never been studied in glioblastomas. Thus, this work aimed to analyze the involvement of RSK in glioblastoma. Firstily, it was observed that the regulation of this protein occurs mainly at translational level or by pos-tranlstional control in glioblastoma cell lines. Using RSK1 and/or RSK2 knockout cells obtained by the CRISPR/Cas9 methodology, it was found the involvement of this protein on cell proliferation. Furthermore, the possible relationship between RSK and cellular senescence induced by PTEN loss has been demonstrated in untransformed cells by using siRNA and chemical RSK inhibitors. Additionally, it also was observed that RKS inhibitors commonly used in the literature have unspecific effects that can lead to wrongful conclusions about the true functions of RSKs. It was also demonstrated that the best target to access the RSK activity is the TSC2 protein (Ser1798).
Assuntos
Humanos , Neoplasias do Sistema Nervoso Central/genética , Glioblastoma/genética , Proteínas Quinases S6 Ribossômicas/genética , Plasmídeos , Western Blotting , Neoplasias do Sistema Nervoso Central/metabolismo , Senescência Celular , Glioblastoma/metabolismo , Proteínas Quinases S6 Ribossômicas/metabolismo , RNA Interferente Pequeno , Proliferação de Células , Transcrição Reversa , PTEN Fosfo-Hidrolase/metabolismo , Reação em Cadeia da Polimerase em Tempo RealRESUMO
INTRODUCTION: Heart failure (HF) is a progressive myopathy, with clinical signs of fatigue and limb weakness that can damage the nerve-muscle interaction, altering synaptic transmission and nicotinic acetylcholine receptors (nAChR) in neuromuscular junctions (NMJs). The diaphragm is composed of a mixed proportion of muscle fibres, and during HF, this muscle becomes slower and can alter its function. As exercise training is an accepted practice to minimise abnormalities of skeletal muscle during HF, in this study, we evaluated the hypothesis that aerobic training attenuates alterations in the expression of nAChR subunits in NMJs diaphragm during heart failure. OBJECTIVE: The aim of this study was to evaluate the distribution and expression of nAChR subunits in the diaphragm muscle fibres of rats subjected to an aerobic training programme during HF. METHODS: Control (Sham), control training (ShamTR), aortic stenosis (AS) and aortic stenosis training (ASTR) groups were evaluated. The expression of nAChR subunits (γ, α1, ε, ß1 and δ) was determined by qRT-PCR, and NMJs were analysed using confocal microscopy. RESULTS: We observed increased expression of the γ, α1 and ß1 subunits in the AS group compared with the ASTR group. The distribution of NMJs was modulated in these groups. DISCUSSION: HF alters the mRNA expression of nAChR subunits and the structural characteristics of diaphragm NMJs. In addition, aerobic training did not alter NMJs morphology but attenuated the alterations in heart structure and function and in nAChR subunit mRNA expression. Our findings demonstrate the beneficial effects of aerobic exercise training in maintaining the integrity of the neuromuscular system in the diaphragm muscle during HF and may be critical for non-pharmacological therapy to improve the quality of life for patients with this syndrome.