RESUMO
Severe poisoning has recently been diagnosed in humans having hip implants composed of cobalt-chrome alloys due to the release of particulate wear debris on polyethylene and ceramic implants which stimulates macrophagic infiltration and destroys bone and soft tissue, leading to neurological, sensorial and muscular impairments. Consistent with this premise, in this study, we focused on the mechanisms underlying the toxicity of Co(II) ions on skeletal muscle using mouse skeletal C2C12 myotubes as an in vitro model. As detected using propidium iodide incorporation, increasing CoCl2 doses (from 5 to 200µM) affected the viability of C2C12 myotubes, mainly by cell necrosis, which was attenuated by necrostatin-1, an inhibitor of the necroptotic branch of the death domain receptor signaling pathway. On the other hand, apoptosis was hardly detectable as supported by the lack of caspase-3 and -8 activation, the latter resulting in only faint activation after exposure to higher CoCl2 doses for prolonged time points. Furthermore, CoCl2 treatment resulted in atrophy of the C2C12 myotubes which was characterized by the increased expression of HSP25 and GRP94 stress proteins and other typical `pro-atrophic molecular hallmarks, such as early activation of the NF-kB pathway and down-regulation of AKT phosphorylation, followed by the activation of the proteasome and autophagy systems. Overall, these results suggested that cobalt may impact skeletal muscle homeostasis as an inducer of cell necrosis and myofiber atrophy.
Assuntos
Morte Celular/efeitos dos fármacos , Cobalto/toxicidade , Fibras Musculares Esqueléticas/patologia , Atrofia , Autofagia , Western Blotting , Linhagem Celular , Corantes , Fluorometria , Imuno-Histoquímica , Indicadores e Reagentes , Fibras Musculares Esqueléticas/efeitos dos fármacos , Mioblastos/efeitos dos fármacos , NF-kappa B/metabolismo , Necrose , Proteína Oncogênica v-akt/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Próteses e Implantes/efeitos adversos , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais/efeitos dos fármacos , Sais de Tetrazólio , TiazóisRESUMO
Cisplatin (cisPt) use in chemotherapy is limited by the occurrence of a severe nephrotoxicity. Both autophagy and apoptosis seem to contribute in kidney response to cisPt, however their cross-talk is still controversial, since the role played by autophagy (cytoprotective or harmful) and the cellular site driving their switch, are still unclear. Here, we used a multidisciplinary approach to study the correlation between autophagy and apoptosis in renal NRK-52E cells exposed to cisPt. We showed two "sensitivity-thresholds" to cisPt, stating whether apoptosis or autophagy would develop: 10 µM dose of cisPt activated autophagy that preserved cell homeostasis; however 3-methyladenine co-administration affected cell viability and induced apoptosis. In contrast, 50 µM cisPt determined cell death by apoptosis, whereas the pre-conditioning with taurine contributed to cell rescue, delaying apoptosis and maintaining autophagy. Hence, autophagy protects NRK-52E cells from cisPt injury. By studying the expression of ER specific hallmarks, such as GRP78, GRP94 and GADD153/CHOP, we found a possible pivotal role of ER signaling modulation in the crosstalk between autophagy and apoptosis induced by cisPt. To the best of our knowledge, this is the first demonstration that taurine enhances autophagic protection against apoptosis by reducing ER stress, thus making it possible to develop new strategies to reduce severe cisPt-induced side-effects such as nephrotoxicity.
Assuntos
Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Cisplatino/farmacologia , Retículo Endoplasmático/fisiologia , Rim/efeitos dos fármacos , Animais , Antineoplásicos/farmacologia , Apoptose/genética , Apoptose/fisiologia , Autofagia/genética , Autofagia/fisiologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Citoproteção/efeitos dos fármacos , Citoproteção/genética , Citoproteção/fisiologia , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Estresse do Retículo Endoplasmático/genética , Estresse do Retículo Endoplasmático/fisiologia , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Rim/citologia , Rim/metabolismo , Rim/ultraestrutura , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Ratos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genéticaRESUMO
Cisplatin (cisPt) is an antineoplastic drug which causes an array of adverse effects on different organs and tissues, including skeletal muscle. In this work we show that cisPt behaves as a potent trigger to activate protein hypercatabolism in skeletal C2C12 myotubes. Within 24h of 50 µM cisPt administration, C2C12 myotubes displayed unchanged cell viability but showed a subset of hallmark signs typically recognized during atrophy, including severe reduction in body size, repression of Akt phosphorylation, transcriptional up-regulation of atrophy-related genes, such as atrogin-1, gabarap, beclin-1 and bnip-3, and loss of myogenic markers. As a consequence, proteasomal activity and formation of autophagosomes were remarkably increased in cisPt-treated myotubes, but forced stimulation of Akt pathway, as obtained through insulin administration or delivery of a constitutively activated Akt form, was sufficient to counter the cisPt-induced protein breakdown, leading to rescue of atrophic size. Overall, these results indicate that cisPt induces atrophy of C2C12 myotubes via activation of proteasome and autophagy systems, suggesting that the Akt pathway represents one sensitive target of cisPt molecular action in skeletal muscle.
Assuntos
Antineoplásicos/toxicidade , Cisplatino/toxicidade , Fibras Musculares Esqueléticas/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Proto-Oncogênicas c-akt/fisiologia , Animais , Antineoplásicos/administração & dosagem , Apoptose/efeitos dos fármacos , Atrofia , Autofagia/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Cisplatino/administração & dosagem , Relação Dose-Resposta a Droga , Camundongos , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/patologia , Mioblastos/efeitos dos fármacos , Mioblastos/metabolismo , Necrose , Fosforilação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Transcrição GênicaRESUMO
Extremely low-frequency electromagnetic fields (ELF-EMFs) can interact with biological systems. Although they are successfully used as therapeutic agents in physiatrics and rehabilitative practice, they might represent environmental pollutants and pose a risk to human health. Due to the lack of evidence of their mechanism of action, the effects of ELF-EMFs on differentiation processes in skeletal muscle were investigated. C2C12 myoblasts were exposed to ELF-EMFs generated by a solenoid. The effects of ELF-EMFs on cell viability and on growth and differentiation rates were studied using colorimetric and vital dye assays, cytomorphology, and molecular analysis of MyoD and myogenin expression, respectively. The establishment of functional gap junctions was investigated analyzing connexin 43 expression levels and measuring cell permeability, using microinjection/dye-transfer assays. The ELF-EMFs did not affect C2C12 myoblast viability or proliferation rate. Conversely, at ELF-EMF intensity in the mT range, the myogenic process was accelerated, through increased expression of MyoD, myogenin, and connexin 43. The increase in gap-junction function suggests promoting cell fusion and myotube differentiation. These data provide the first evidence of the mechanism through which ELF-EMFs may provide therapeutic benefits and can resolve, at least in part, some conditions of muscle dysfunction.
Assuntos
Conexina 43/genética , Campos Eletromagnéticos , Proteína MyoD/genética , Miogenina/genética , Animais , Comunicação Celular/efeitos da radiação , Técnicas de Cultura de Células , Diferenciação Celular/efeitos da radiação , Proliferação de Células/efeitos da radiação , Sobrevivência Celular/efeitos da radiação , Regulação da Expressão Gênica no Desenvolvimento/efeitos da radiação , Camundongos , Desenvolvimento Muscular/efeitos da radiação , Mioblastos/efeitos da radiaçãoRESUMO
Tubular epithelium represents the primary target of mercuric ions (Hg(2+)) nephrotoxicity. Although widely investigated, the mechanisms of Hg(2+) cell uptake, accumulation and excretion all along the nephron remain largely unknown. In the present study, native distal tubular-derived Madin-Darby canine kidney (MDCK) cells exposed to subcytotoxic (micromolar) HgCl(2) concentrations were used for investigating specific mechanisms involved in the tubular response to toxic metals. Inductively coupled plasma-mass spectrometry (ICP-MS) was firstly used for assessing HgCl(2) solubility and then for quantifying Hg(2+) cell uptake. Exposed to HgCl(2), MDCK cells showed a rapid, but transient, Hg(2+) accumulation. The metallic cation was found to affect cell density and morphology, being these effects related to the dose and the time of exposure. In parallel, an Hg(2+)-induced up-regulation of endogenous MRP1 and MRP2 export pumps, a significant HgCl(2)-dependent induction of protective cellular thiols and an increase in the glutathione conjugates metabolism were also observed. The functional suppression of MRPs activity, obtained by MK-571 treatment, increased the Hg(2+) cell content and the sensitivity of MDCK cells to HgCl(2). Our results demonstrate that, in MDCK cells, inorganic Hg(2+) promotes the activation of specific detoxifying pathways that may, at least partly, depend on the activity of MRP transporters.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/biossíntese , Proteínas de Membrana Transportadoras/biossíntese , Cloreto de Mercúrio/toxicidade , Mercúrio/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/biossíntese , Compostos de Sulfidrila/metabolismo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/antagonistas & inibidores , Animais , Northern Blotting , Cátions Bivalentes/metabolismo , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cães , Glutationa/metabolismo , Glutationa Transferase/metabolismo , Humanos , Immunoblotting , Túbulos Renais/citologia , Túbulos Renais/efeitos dos fármacos , Túbulos Renais/metabolismo , Moduladores de Transporte de Membrana , Proteínas de Membrana Transportadoras/antagonistas & inibidores , Cloreto de Mercúrio/metabolismo , Proteína 2 Associada à Farmacorresistência Múltipla , Proteínas Associadas à Resistência a Múltiplos Medicamentos/antagonistas & inibidores , Propionatos/farmacologia , Quinolinas/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Regulação para CimaRESUMO
We analysed the expression of the hsp70 gene, the phosphorylation status of different members of the mitogen-activated protein kinase (MAPK) family, the behaviour of the Akt-GSK3 pathway, as well as the DNA-binding activity of several transcription factors, potential targets of these kinases, in the brain of rats exposed to a fever-like increase in body temperature. Two different brain regions, the cerebellum and the hippocampus, were studied. Hyperthermia caused HSF activation and the induction of hsp70 mRNA and protein to a greater extent in the cerebellum than in the hippocampus. In the cerebellum, ERK1/2 and p38 MAPK phosphorylation were increased by hyperthermia and returned to basal levels during the recovery from heat stress, whereas JNK3 phosphorylation decreased and recovered to above control levels within 60 min of recovery. JNK1 phosphorylation was never modified. In the hippocampus, ERK phosphorylation did not increase but rather decreased, whereas the behaviour of p38 MAPK and JNK was similar to that observed in the cerebellum. Akt phosphorylation increased after hyperthermia and was accompanied by an increased phosphorylation of two substrates, GSK3 and FKHRL1, in both brain areas, with a major effect in the cerebellum. DNA-binding activities of AP-1, NF-kappaB, and MEF2 were activated by heat shock in the cerebellum, whereas only MEF2 was activated in the hippocampus. Our data indicate that a physiologically relevant increase in body temperature induces brain injury and survival response to it as demonstrated by induction of hsp70 gene expression and activation of specific signalling pathways. Reprogramming of gene expression, by the specific transcription factors activated, probably plays a central role in cell adaptation and survival to heat stress. The hippocampus shows less responsiveness to hyperthermia than the cerebellum.
Assuntos
Febre/enzimologia , Proteínas de Choque Térmico HSP70/metabolismo , Resposta ao Choque Térmico/fisiologia , Sistema de Sinalização das MAP Quinases/fisiologia , Proteínas Serina-Treonina Quinases , Fatores de Transcrição/fisiologia , Animais , Temperatura Corporal/fisiologia , Cerebelo/enzimologia , Cerebelo/fisiopatologia , Proteínas de Ligação a DNA/metabolismo , Febre/fisiopatologia , Proteína Forkhead Box O3 , Fatores de Transcrição Forkhead , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Hipocampo/enzimologia , Hipocampo/fisiopatologia , Proteínas Quinases JNK Ativadas por Mitógeno , Fatores de Transcrição MEF2 , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fatores de Regulação Miogênica , NF-kappa B/metabolismo , Proteínas do Tecido Nervoso , Fosforilação , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Ratos , Ratos Sprague-Dawley , Fator de Transcrição AP-1/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
Cisplatin (CisPt) is a widely used chemotherapeutic drug whose side effects include muscle weakness and cachexia. Here we analysed CisPt-induced atrophy in C2C12 myotubes by a multidisciplinary morphological approach, focusing on the onset and progression of autophagy, a protective cellular process that, when excessively activated, may trigger protein hypercatabolism and atrophy in skeletal muscle. To visualize autophagy we used confocal and transmission electron microscopy at different times of treatment and doses of CisPt. Moreover we evaluated the effects of taurine, a cytoprotective beta-amino acid able to counteract oxidative stress, apoptosis, and endoplasmic reticulum stress in different tissues and organs. Our microscopic results indicate that autophagy occurs very early in 50 µM CisPt challenged myotubes (4 h-8 h) before overt atrophy but it persists even at 24 h, when several autophagic vesicles, damaged mitochondria, and sarcoplasmic blebbings engulf the sarcoplasm. Differently, 25 mM taurine pretreatment rescues the majority of myotubes size upon 50 µM CisPt at 24 h. Taurine appears to counteract atrophy by restoring regular microtubular apparatus and mitochondria and reducing the overload and the localization of autophagolysosomes. Such a promising taurine action in preventing atrophy needs further molecular and biochemical studies to best define its impact on muscle homeostasis and the maintenance of an adequate skeletal mass in vivo.
Assuntos
Cisplatino/efeitos adversos , Atrofia Muscular/induzido quimicamente , Atrofia Muscular/patologia , Taurina/farmacologia , Adenina/análogos & derivados , Adenina/farmacologia , Animais , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Linhagem Celular , Camundongos , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/ultraestrutura , Necrose , Fatores de Tempo , Tubulina (Proteína)/metabolismoRESUMO
The biological effects of electric and magnetic fields, which are ubiquitous in modern society, remain poorly understood. Here, we applied a single-cell approach to study the effects of short-term exposure to extremely low frequency electromagnetic fields (ELF-EMFs) on muscle cell differentiation and function using C2C12 cells as an in vitro model of the skeletal muscle phenotype. Our focus was on markers of oxidative stress and calcium (Ca(2+)) handling, two interrelated cellular processes previously shown to be affected by such radiation in other cell models. Collectively, our data reveal that ELF-EMFs (1) induced reactive oxygen species production in myoblasts and myotubes with a concomitant decrease in mitochondrial membrane potential; (2) activated the cellular detoxification system, increasing catalase and glutathione peroxidase activities; and (3) altered intracellular Ca(2+)homeostasis, increasing the spontaneous activity of myotubes and enhancing cellular reactivity to a depolarizing agent (KCl) or an agonist (caffeine) of intracellular store Ca(2+)channels. In conclusion, our data support a possible link between exposure to ELF-EMFs and modification of the cellular redox state, which could, in turn, increase the level of intracellular Ca(2+)and thus modulate the metabolic activity of C2C12 cells.