RESUMO
The role of P2 receptors for purines/pyrimidines is not well characterized in neuroblastoma, although a variety of purinergic mRNAs/proteins are expressed in these cells. Among these, the P2Y(6) receptor is the only subtype distinguished by UDP-specific activation. In this work, after over-expressing the P2Y(6) protein in human neuroblastoma SH-SY5Y cells, we find that UDP arrests cell cycle and induces apoptosis, by counteracting the pathological functioning of neuroblastoma in vitro. UDP also causes mitochondrial damage through diffusion of cytochrome c in the cytoplasm, and stimulates caspase-3,7,8 activities, with extensive over-expression of manganese superoxide dismutase. Our data establish the direct toxic role and anti-cancer activity of UDP in a neuroblastoma cell line, and identify the P2Y(6) receptor as a novel potential target in anti-tumoural therapies. This constitutes an advancement not only in the knowledge of purinergic signalling, but also in the biological and pathological aspects of neuroblastoma in vitro.
Assuntos
Antineoplásicos , Citostáticos , Receptores Purinérgicos P2/biossíntese , Difosfato de Uridina/farmacologia , Anexina A5 , Apoptose/efeitos dos fármacos , Western Blotting , Cálcio/metabolismo , Caspase 3/metabolismo , Caspase 7/metabolismo , Contagem de Células , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Clonagem Molecular , Eletroforese em Gel de Poliacrilamida , Humanos , Microscopia de Fluorescência , Plasmídeos/genética , RNA Neoplásico/biossíntese , Receptores Purinérgicos P2/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by the selective loss of lower and upper motoneurons. The pathology is imputable in approximately 2% of cases to mutations in the ubiquitous enzyme Cu, Zn superoxide dismutase (SOD1). Common theories to explain the pathogenic mechanisms of ALS include activation of microglia, responsible for the release of proinflammatory factors. However, how mutant SOD1 affects microglial activation and subsequently injures neurons is still unclear. Considering that extracellular ATP, through purinergic P2 receptors, constitutes a well recognized neuron-to-microglia alarm signal, the aim of this study was to investigate how the expression of mutant SOD1 affects P2 receptor-mediated proinflammatory microglial properties. We used primary and immortalized microglial cells from mutant SOD1 mice to explore several aspects of activation by purinergic ligands and to analyze the overall effect of such stimulation on the viability of NSC-34 and SH-SY5Y neuronal cell lines. We observed up-regulation of P2X(4), P2X(7), and P2Y(6) receptors and down-regulation of ATP-hydrolyzing activities in mutant SOD1 microglia. This potentiation of the purinergic machinery reflected into enhanced sensitivity mainly to 2'-3'-O-(benzoyl-benzoyl) ATP, a P2X(7) receptor preferential agonist, and translated into deeper morphological changes, enhancement of TNF-alpha and cyclooxygenase-2 content, and finally into toxic effects exerted on neuronal cell lines by microglia expressing mutant SOD1. All these parameters were prevented by the antagonist Brilliant Blue G. The purinergic activation of microglia may thus constitute a new route involved in the progression of ALS to be exploited to potentially halt the disease.
Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/patologia , Mediadores da Inflamação/fisiologia , Microglia/metabolismo , Microglia/patologia , Receptores Purinérgicos P2/fisiologia , Superóxido Dismutase/fisiologia , Regulação para Cima , Alanina/genética , Substituição de Aminoácidos/genética , Esclerose Lateral Amiotrófica/enzimologia , Esclerose Lateral Amiotrófica/genética , Animais , Linhagem Celular Transformada , Linhagem Celular Tumoral , Células Cultivadas , Técnicas de Cocultura , Modelos Animais de Doenças , Progressão da Doença , Regulação Enzimológica da Expressão Gênica , Glicina/genética , Humanos , Camundongos , Camundongos Transgênicos , Microglia/enzimologia , Fenótipo , Receptores Purinérgicos P2/biossíntese , Receptores Purinérgicos P2/genética , Transdução de Sinais/genética , Superóxido Dismutase/biossíntese , Superóxido Dismutase/genética , Superóxido Dismutase-1 , Regulação para Cima/genéticaRESUMO
ATP is a potent signalling molecule abundantly present in the nervous system, where it exerts physiological actions ranging from short-term responses such as neurotransmission, neuromodulation and glial communication, to long-term effects such as trophic actions. The fast signalling targets of extracellular ATP are represented by the ionotropic P2X receptors, which are broadly and abundantly expressed in neurons and glia in the whole central and peripheral nervous systems. Because massive extracellular release of ATP often occurs by lytic and non-lytic mechanisms, especially after stressful events and pathological conditions, purinergic signalling is correlated to and involved in the aetiopathology and/or progression of many neurodegenerative diseases. In this minireview, we highlight the contribution of the subclass of ionotropic P2X receptors to several diseases of the human nervous system, such as neurodegenerative disorders and immune-mediated neuroinflammatory dysfunctions including ischaemia, Parkinson's, Alzheimer's and Huntington's diseases, amyotrophic lateral sclerosis and multiple sclerosis. The role of P2X receptors as novel and effective targets for the genetic/pharmacological manipulation of purinergic mechanisms in several neuropathological conditions is now well established. Nevertheless, any successful therapeutic intervention against these diseases cannot be restricted to P2X receptors, but should take into consideration the whole and multipart ATP signalling machinery.