RESUMEN
The oxidative phosphorylation (OXPHOS) system couples the transfer of electrons to oxygen with pumping of protons across the inner mitochondrial membrane, ensuring the ATP production. Evidence suggests that respiratory chain complexes may also assemble into supramolecular structures, called supercomplexes (SCs). The SCs appear to increase the efficiency/capacity of OXPHOS and reduce the reactive oxygen species (ROS) production, especially that which is produced by complex I. Studies suggest a mutual regulation between complex I and SCs, while SCs organization is important for complex I assembly/stability, complex I is involved in the supercomplex formation. Complex I is a pacemaker of the OXPHOS system, and it has been shown that the PKA-dependent phosphorylation of some of its subunits increases the activity of the complex, reducing the ROS production. In this work, using in ex vivo and in vitro models, we show that the activation of cAMP/PKA cascade resulted in an increase in SCs formation associated with an enhanced capacity of electron flux and ATP production rate. This is also associated with the phosphorylation of the NDUFS4 subunit of complex I. This aspect highlights the key role of complex I in cellular energy production.
Asunto(s)
Membranas Mitocondriales , Fosforilación Oxidativa , Adenosina Trifosfato/metabolismo , Complejo I de Transporte de Electrón/metabolismo , Membranas Mitocondriales/metabolismo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Red onion, a species of great economic importance rich in phytochemicals (bioactive compounds) known for its medicinal properties, was fertilized with sulphur-bentonite enriched with orange residue or olive pomace, with the aim of producing onion enriched in health beneficial compounds. There is a worldwide great demand of minimally processed food or food ingredients with functional properties because of a new awareness of how important healthy functional nutrition is in life. Phytochemicals have the capacity to regulate most of the metabolic processes resulting in health benefits. Red onion bioactive compound quantity and quality can vary according to cultivation practices. The main aims of the current research were to determine the chemical characteristics of the crude extracts from red onion bulbs differently fertilized and to evaluate their biological activity in normal and oxidative stress conditions. The lyophilized onion bulbs have been tested in vitro on two cellular models, i.e., the H9c2 rat cardiomyoblast cell line and primary human dermal fibroblasts, in terms of viability and oxygen radical homeostasis. The results evidenced different phytochemical compositions and antioxidant activities of the extracts obtained from red onions differently fertilized. Sulphur-bentonite fertilizers containing orange waste and olive pomace positively affected the red onion quality with respect to the red onion control, evidencing that sulphur-bentonite-organic fertilization was able to stimulate plant a secondary metabolism inducing the production of phytochemicals with healthy functions. A positive effect of the extracts from red onions treated with fertilizers-in particular, with those containing orange waste, such as the reduction of oxidative stress and induction of cell viability of H9c2 and human fibroblasts-was observed, showing a concentration- and time-dependent profile. The results evidenced that the positive effects were related to the phenols and, in particular, to chlorogenic and p-coumaric acids and to the flavonol kaempferol, which were more present in red onion treated with low orange residue than in the other treated ones.
Asunto(s)
Ingredientes Alimentarios , Olea , Animales , Antioxidantes/metabolismo , Antioxidantes/farmacología , Bentonita , Ácidos Cumáricos/farmacología , Fertilizantes , Humanos , Quempferoles/farmacología , Mamíferos/metabolismo , Olea/metabolismo , Cebollas/química , Estrés Oxidativo , Fenoles/farmacología , Fitoquímicos/metabolismo , Fitoquímicos/farmacología , Extractos Vegetales/química , Extractos Vegetales/farmacología , Ratas , Especies Reactivas de Oxígeno/metabolismo , Azufre/farmacologíaRESUMEN
Parkinson's disease is one of the most common neurodegenerative disorders worldwide, characterized by a progressive loss of dopaminergic neurons mainly localized in the substantia nigra pars compacta. In recent years, the detailed analyses of both genetic and idiopathic forms of the disease have led to a better understanding of the molecular and cellular pathways involved in PD, pointing to the centrality of mitochondrial dysfunctions in the pathogenic process. Failure of mitochondrial quality control is now considered a hallmark of the disease. The peroxisome proliferator-activated receptor gamma coactivator 1 (PGC-1) family acts as a master regulator of mitochondrial biogenesis. Therefore, keeping PGC-1 level in a proper range is fundamental to guarantee functional neurons. Here we review the major findings that tightly bond PD and PGC-1s, raising important points that might lead to future investigations.
Asunto(s)
Neuronas/metabolismo , Enfermedad de Parkinson/metabolismo , Porción Compacta de la Sustancia Negra/metabolismo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/genética , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/fisiología , Animales , ADN Mitocondrial/metabolismo , Proteínas de Unión al ADN/metabolismo , Neuronas Dopaminérgicas/metabolismo , Estudio de Asociación del Genoma Completo , Humanos , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/metabolismo , Ratones , Mitocondrias/metabolismo , Enfermedades Neurodegenerativas/metabolismo , Biogénesis de Organelos , Estrés Oxidativo , Fosforilación , Proteína Desglicasa DJ-1/metabolismo , Proteínas Quinasas/metabolismo , Factores de Transcripción/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas de Transporte Vesicular/metabolismo , alfa-Sinucleína/metabolismoRESUMEN
Vimentin, a member of cytoskeleton intermediate filaments proteins, plays a critical role in cell structure and dynamics. The present proteomic study reveals reduced amount of six different lengths, N-terminal truncated proteolytic products of vimentin, in the primary skin fibroblasts from two unrelated PD patients, as compared to control fibroblasts. The decreased amount of N-terminal truncated forms of vimentin in parkin-mutant fibroblasts, could contribute to impairment of cellular function, potentially contributing to the pathogenesis of Parkinson disease.
Asunto(s)
Fibroblastos/metabolismo , Enfermedad de Parkinson/metabolismo , Ubiquitina-Proteína Ligasas/genética , Vimentina/metabolismo , Adulto , Células Cultivadas , Femenino , Fibroblastos/patología , Humanos , Persona de Mediana Edad , Mutación , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/patología , Isoformas de Proteínas/análisis , Isoformas de Proteínas/metabolismo , Proteolisis , Proteómica , Piel/metabolismo , Piel/patología , Vimentina/análisisRESUMEN
The scientific community continuously strives to get new disease models, to discover early markers or novel therapeutic approaches, improving the diagnosis and prognosis of several human pathologies. Parkinson's Disease (PD) is characterized by a long asymptomatic phase, characterized by a selective loss of dopaminergic neurons. Recently, the human Periapical Cyst-Mesenchymal Stem Cells (hPCy-MSCs) have been differentiated in functional dopaminergic neurons: such oral-derived MSCs and the hPCy-MSCs-derived exosomes may represent a strategic and useful in vitro study-model, as well as intriguing therapeutic carriers. Circadian rhythm (CR) alteration variously impacts on PD pathways: an interesting research target is represented by the analysis of the exosomes released by dopaminergic neurons, derived from neural-differentiated hPCy-MSCs, after having reproduced in-vitro PD-like conditions. This review aims to describe the crosstalk among some aspects of circadian rhythm related to the onset of PD and the exosomes released by cells of PD patients. More in detail: the first part of this article will describe the main characteristics of circadian rhythm and the involvement of the exosomes found to be effective in the pathogenesis of PD. Finally, the authors will suggest how those exosomes derived from dopaminergic neurons, obtained by oral-derived stem cells (hPCy-MSCs) may represent a smart model for the in vitro research on PD, to find new biomarkers, to test new drugs or, fatally, to find new pathways applicable in future therapeutic approaches.
Asunto(s)
Ritmo Circadiano , Exosomas , Células Madre Mesenquimatosas , Enfermedad de Parkinson , Quiste Radicular , Humanos , Modelos Biológicos , Medicina de PrecisiónRESUMEN
There is a growing interest in the cytotoxic effects of bioactive glycoalkaloids, such as α-tomatine on tumor cells. Here, for the first time, we determine the antitumor potential of tomatine, a mixture of α-tomatine and dehydrotomatine, in metastatic melanoma (MM) cell lines harboring different BRAF and MC1R variants. We performed cytotoxicity experiments and annexin-V/propidium iodide staining to assess the apoptotic/necrotic status of the cells. ER stress and autophagy markers were revealed by Western Blot, whereas antiangiogenic and vascular-disrupting effects were evaluated through a capillary tube formation assay on matrigel and by ELISA kit for VEGF release determination. Cell invasion was determined by a Boyden chamber matrigel assay. Tomatine reduced 50% of cell viability and induced a concentration-dependent increase of apoptotic cells in the range of 0.5-1 µM in terms of α-tomatine. The extent of apoptosis was more than two-fold higher in V600BRAF-D184H/D184H MC1R cells than in BRAF wild-type cells and V600BRAF-MC1R wild-type cell lines. Additionally, tomatine increased the LC3I/II autophagy marker, p-eIF2α, and p-Erk1/2 levels in BRAF wild-type cells. Notably, tomatine strongly reduced cell invasion and melanoma-dependent angiogenesis by reducing VEGF release and tumor-stimulating effects on capillary tube formation. Collectively, our findings support tomatine as a potential antitumor agent in MM.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis , Sistema de Señalización de MAP Quinasas , Melanoma , Tomatina/farmacología , Sustitución de Aminoácidos , Apoptosis/efectos de los fármacos , Apoptosis/genética , Línea Celular Tumoral , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/genética , Melanoma/tratamiento farmacológico , Melanoma/genética , Melanoma/metabolismo , Melanoma/patología , Mutación Missense , Necrosis , Invasividad Neoplásica , Metástasis de la Neoplasia , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas B-raf/metabolismoRESUMEN
BACKGROUND: Melanoma cells develop adaptive responses in order to cope with particular conditions of tumor microenvironment, characterized by stress conditions and deregulated proliferation. Recently, the interplay between the stress response and the gene expression programs leading to metastatic spread has been reported. METHODS: We evaluated levels and localization of eIF2α/peIF2α in V600BRAF and wtBRAF metastatic melanoma cell lines by means of western blot and confocal microscopy analyses. Furthermore, we performed a sequence analyses and structure and dynamics studies of eIF2α protein to reveal the role of eIF2α and its correlations in different pathways involved in the invasive phase of melanoma. RESULTS: We found peIF2α both in cytoplasm and nucleus. Nuclear localization was more represented in V600BRAF melanoma cell lines. Our studies on eIF2α protein sequence indicated the presence of a predicted bipartite NLS as well as a nuclear export signal NES and an S1 domain, typical of RNA interacting proteins. Furthermore, we found high levels of transcription factor EB (TFEB), a component of the MiT/TFE family, and low ß-catenin levels in V600BRAF cells. CONCLUSIONS: Based on our results, we suggest that peIF2α nuclear localization can be crucial in ER stress response and in driving the metastatic spread of melanoma, through lysosomal signaling and Wnt/ß-catenin pathway. In conclusion, this is the first evidence of nuclear localization of peIF2α, representing a possible target for future therapeutic approaches for metastatic melanoma.
Asunto(s)
Factor 2 Eucariótico de Iniciación/metabolismo , Melanoma/metabolismo , Biosíntesis de Proteínas , Neoplasias Cutáneas/metabolismo , Secuencia de Aminoácidos , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Línea Celular Tumoral , Factor 2 Eucariótico de Iniciación/química , Humanos , Fosforilación , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , beta Catenina/metabolismo , Melanoma Cutáneo MalignoRESUMEN
Growing evidence highlights a tight connection between circadian rhythms, molecular clockworks, and mitochondrial function. In particular, mitochondrial quality control and bioenergetics have been proven to undergo circadian oscillations driven by core clock genes. Parkinson's disease (PD) is a chronic neurodegenerative disease characterized by a selective loss of dopaminergic neurons. Almost half of the autosomal recessive forms of juvenile parkinsonism have been associated with mutations in the PARK2 gene coding for parkin, shown to be involved in mitophagy-mediated mitochondrial quality control. The aim of this study was to investigate, in fibroblasts from genetic PD patients carrying parkin mutations, the interplay between mitochondrial bioenergetics and the cell autonomous circadian clock. Using two different in vitro synchronization protocols, we demonstrated that normal fibroblasts displayed rhythmic oscillations of both mitochondrial respiration and glycolytic activity. Conversely, in fibroblasts obtained from PD patients, a severe damping of the bioenergetic oscillatory patterns was observed. Analysis of the core clock genes showed deregulation of their expression patterns in PD fibroblasts, which was confirmed in induced pluripotent stem cells (iPSCs) and induced neural stem cells (iNSCs) derived thereof. The results from this study support a reciprocal interplay between the clockwork machinery and mitochondrial energy metabolism, point to a parkin-dependent mechanism of regulation, and unveil a hitherto unappreciated level of complexity in the pathophysiology of PD and eventually other neurodegenerative diseases.
Asunto(s)
Proteínas CLOCK/genética , Metabolismo Energético/genética , Mutación/genética , Ubiquitina-Proteína Ligasas/genética , Animales , Proteínas CLOCK/metabolismo , Respiración de la Célula , Ritmo Circadiano/genética , Fibroblastos/metabolismo , Regulación de la Expresión Génica , Glucólisis , Humanos , Ratones Desnudos , Mitocondrias/metabolismo , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/patología , Transcripción GenéticaRESUMEN
This study explores the V600BRAF-MITF-PGC-1α axis and compares metabolic and functional changes occurring in primary and metastatic V600BRAF melanoma cell lines. V600BRAF mutations in homo/heterozygosis were found to be correlated to high levels of pERK, to downregulate PGC-1α/ß, MITF and tyrosinase activity, resulting in a reduced melanin synthesis as compared to BRAFwt melanoma cells. In this scenario, V600BRAF switches on a metabolic reprogramming in melanoma, leading to a decreased OXPHOS activity and increased glycolytic ATP, lactate, HIF-1α and MCT4 levels. Furthermore, the induction of autophagy and the presence of ER stress markers in V600BRAF metastatic melanoma cells suggest that metabolic adaptations of these cells occur as compensatory survival mechanisms. For the first time, we underline the role of peIF2α as an important marker of metastatic behaviour in melanoma. Our results suggest the hypothesis that inhibition of the glycolytic pathway, inactivation of peIF2α and a reduction of basal autophagy could be suitable targets for novel combination therapies in a specific subgroup of metastatic melanoma.
Asunto(s)
Metabolismo Energético , Melanoma/enzimología , Melanoma/genética , Mutación , Proteínas Proto-Oncogénicas B-raf/genética , Neoplasias Cutáneas/enzimología , Neoplasias Cutáneas/genética , Autofagia , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Línea Celular , AMP Cíclico/metabolismo , Estrés del Retículo Endoplásmico , Factor 2 Eucariótico de Iniciación/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Glucólisis , Heterocigoto , Homocigoto , Humanos , Melanoma/secundario , Factor de Transcripción Asociado a Microftalmía/genética , Factor de Transcripción Asociado a Microftalmía/metabolismo , Monofenol Monooxigenasa/metabolismo , Metástasis de la Neoplasia , Fosforilación Oxidativa , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/genética , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/metabolismo , Fenotipo , Fosforilación , Proteínas Proto-Oncogénicas B-raf/metabolismo , Proteínas de Unión al ARN , Neoplasias Cutáneas/patologíaRESUMEN
Parkin mutations are a major cause of early-onset Parkinson's disease (PD). The impairment of protein quality control system together with defects in mitochondria and autophagy process are consequences of the lack of parkin, which leads to neurodegeneration. Little is known about the role of lipids in these alterations of cell functions. In the present study, parkin-mutant human skin primary fibroblasts have been considered as cellular model of PD to investigate on possible lipid alterations associated with the lack of parkin protein. Dermal fibroblasts were obtained from two unrelated PD patients with different parkin mutations and their lipid compositions were compared with that of two control fibroblasts. The lipid extracts of fibroblasts have been analyzed by combined matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF/MS) and thin-layer chromatography (TLC). In parallel, we have performed direct MALDI-TOF/MS lipid analyses of intact fibroblasts by skipping lipid extraction steps. Results show that the proportions of some phospholipids and glycosphingolipids were altered in the lipid profiles of parkin-mutant fibroblasts. The detected higher level of gangliosides, phosphatidylinositol, and phosphatidylserine could be linked to dysfunction of autophagy and mitochondrial turnover; in addition, the lysophosphatidylcholine increase could represent the marker of neuroinflammatory state, a well-known component of PD.
Asunto(s)
Fibroblastos/metabolismo , Glicoesfingolípidos/metabolismo , Mutación , Enfermedad de Parkinson/metabolismo , Fosfolípidos/metabolismo , Piel/metabolismo , Ubiquitina-Proteína Ligasas/genética , Biomarcadores/metabolismo , Células Cultivadas , Cromatografía en Capa Delgada , Fibroblastos/patología , Predisposición Genética a la Enfermedad , Humanos , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/patología , Fenotipo , Cultivo Primario de Células , Piel/patología , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Parkinson's disease (PD) is the most common neurodegenerative movement disorder caused primarily by selective degeneration of the dopaminergic neurons in substantia nigra. In this work the proteomes extracted from primary fibroblasts of two unrelated, hereditary cases of PD patients, with different parkin mutations, were compared with the proteomes extracted from commercial adult normal human dermal fibroblasts (NHDF) and primary fibroblasts from the healthy mother of one of the two patients. The results show that the fibroblasts from the two different cases of parkin-mutant patients display analogous alterations in the expression level of proteins involved in different cellular functions, like cytoskeleton structure-dynamics, calcium homeostasis, oxidative stress response, protein and RNA processing.
RESUMEN
Mitochondrial dysfunction and oxidative stress occur in Parkinson's disease (PD), but the molecular mechanisms controlling these events are not completely understood. Peroxisome proliferator-activated receptor-gamma coactivator-1α (PGC-1α) is a transcriptional coactivator known as master regulator of mitochondrial functions and oxidative metabolism. Recent studies, including one from our group, have highlighted altered PGC-1α activity and transcriptional deregulation of its target genes in PD pathogenesis suggesting it as a new potential therapeutic target. Resveratrol, a natural polyphenolic compound proved to improve mitochondrial activity through the activation of several metabolic sensors resulting in PGC-1α activation. Here we have tested in vitro the effect of resveratrol treatment on primary fibroblast cultures from two patients with early-onset PD linked to different Park2 mutations. We show that resveratrol regulates energy homeostasis through activation of AMP-activated protein kinase (AMPK) and sirtuin 1 (SIRT1) and raise of mRNA expression of a number of PGC-1α's target genes resulting in enhanced mitochondrial oxidative function, likely related to a decrease of oxidative stress and to an increase of mitochondrial biogenesis. The functional impact of resveratrol treatment encompassed an increase of complex I and citrate synthase activities, basal oxygen consumption, and mitochondrial ATP production and a decrease in lactate content, thus supporting a switch from glycolytic to oxidative metabolism. Moreover, resveratrol treatment caused an enhanced macro-autophagic flux through activation of an LC3-independent pathway. Our results, obtained in early-onset PD fibroblasts, suggest that resveratrol may have potential clinical application in selected cases of PD-affected patients.
Asunto(s)
Mitocondrias/efectos de los fármacos , Enfermedad de Parkinson/tratamiento farmacológico , Enfermedad de Parkinson/fisiopatología , Estilbenos/farmacología , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas Quinasas Activadas por AMP/genética , Proteínas Quinasas Activadas por AMP/metabolismo , Adenosina Trifosfato/genética , Adenosina Trifosfato/metabolismo , Células Cultivadas , Femenino , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Predisposición Genética a la Enfermedad , Humanos , Persona de Mediana Edad , Mitocondrias/genética , Mitocondrias/metabolismo , NAD/genética , NAD/metabolismo , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/genética , Consumo de Oxígeno/efectos de los fármacos , Consumo de Oxígeno/genética , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/metabolismo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma , Resveratrol , Sirtuina 1/genética , Sirtuina 1/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
The p66Shc protein mediates oxidative stress-related injury in multiple tissues. Steatohepatitis is characterized by enhanced oxidative stress-mediated cell damage. The role of p66Shc in redox signaling was investigated in human liver cells and alcoholic steatohepatitis. HepG2 cells with overexpression of wild-type or mutant p66Shc, with Ser36 replacement by Ala, were obtained through infection with recombinant adenoviruses. Reactive oxygen species and oxidation-dependent DNA damage were assessed by measuring dihydroethidium oxidation and 8-hydroxy-2'-deoxyguanosine accumulation into DNA, respectively. mRNA and protein levels of signaling intermediates were evaluated in HepG2 cells and liver biopsies from control and alcoholic steatohepatitis subjects. Exposure to H2O2 increased reactive oxygen species and phosphorylation of p66Shc on Ser36 in HepG2 cells. Overexpression of p66Shc promoted reactive oxygen species synthesis and oxidation-dependent DNA damage, which were further enhanced by H2O2. p66Shc activation also resulted in increased Erk-1/2, Akt, and FoxO3a phosphorylation. Blocking of Erk-1/2 activation inhibited p66Shc phosphorylation on Ser36. Increased p66Shc expression was associated with reduced mRNA levels of antioxidant molecules, such as NF-E2-related factor 2 and its target genes. In contrast, overexpression of the phosphorylation defective p66Shc Ala36 mutant inhibited p66Shc signaling, enhanced antioxidant genes, and suppressed reactive oxygen species and oxidation-dependent DNA damage. Increased p66Shc protein levels and Akt phosphorylation were observed in liver biopsies from alcoholic steatohepatitis compared with control subjects. In human alcoholic steatohepatitis, increased hepatocyte p66Shc protein levels may enhance susceptibility to DNA damage by oxidative stress by promoting reactive oxygen species synthesis and repressing antioxidant pathways.
Asunto(s)
Daño del ADN , Hígado Graso Alcohólico/metabolismo , Hepatocitos/metabolismo , Oxidación-Reducción , Estrés Oxidativo , Proteínas Adaptadoras de la Señalización Shc , Técnicas de Cultivo de Célula , Proteína Forkhead Box O3 , Factores de Transcripción Forkhead/metabolismo , Humanos , Proteína Oncogénica v-akt/metabolismo , Fosforilación , Especies Reactivas de Oxígeno/metabolismo , Proteínas Adaptadoras de la Señalización Shc/genética , Proteínas Adaptadoras de la Señalización Shc/metabolismo , Transducción de Señal , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de SrcRESUMEN
BACKGROUND: The poor response to chemotherapy and the brief response to vemurafenib in metastatic melanoma patients, make the identification of new therapeutic approaches an urgent need. Interestingly the increased expression and activity of the Aurora kinase B during melanoma progression suggests it as a promising therapeutic target. METHODS: The efficacy of the Aurora B kinase inhibitor barasertib-HQPA was evaluated in BRAF mutated cells, sensitive and made resistant to vemurafenib after chronic exposure to the drug, and in BRAF wild type cells. The drug effectiveness has been evaluated as cell growth inhibition, cell cycle progression and cell migration. In addition, cellular effectors of drug resistance and response were investigated. RESULTS: The characterization of the effectors responsible for the resistance to vemurafenib evidenced the increased expression of MITF or the activation of Erk1/2 and p-38 kinases in the newly established cell lines with a phenotype resistant to vemurafenib. The sensitivity of cells to barasertib-HQPA was irrespective of BRAF mutational status. Barasertib-HQPA induced the mitotic catastrophe, ultimately causing apoptosis and necrosis of cells, inhibited cell migration and strongly affected the glycolytic metabolism of cells inducing the release of lactate. In association i) with vemurafenib the gain in effectiveness was found only in BRAF(V600K) cells while ii) with nab-paclitaxel, the combination was more effective than each drug alone in all cells. CONCLUSIONS: These findings suggest barasertib as a new therapeutic agent and as enhancer of chemotherapy in metastatic melanoma treatment.
Asunto(s)
Aurora Quinasa B/antagonistas & inhibidores , Melanoma/tratamiento farmacológico , Melanoma/patología , Inhibidores de Proteínas Quinasas/uso terapéutico , Albúminas/farmacología , Albúminas/uso terapéutico , Apoptosis/efectos de los fármacos , Aurora Quinasa B/metabolismo , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Forma del Núcleo Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Forma de la Célula/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Espacio Extracelular/metabolismo , Humanos , Ácido Láctico/metabolismo , Melanoma/enzimología , Mitosis/efectos de los fármacos , Necrosis , Metástasis de la Neoplasia , Organofosfatos/farmacología , Organofosfatos/uso terapéutico , Paclitaxel/farmacología , Paclitaxel/uso terapéutico , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas B-raf/genética , Quinazolinas/farmacología , Quinazolinas/uso terapéuticoRESUMEN
Neurons are highly dependent on mitochondria to meet their bioenergetic needs and understanding the metabolic changes during the differentiation process is crucial in the neurodegeneration context. Several in vitro approaches have been developed to study neuronal differentiation and bioenergetic changes. The human SH-SY5Y cell line is a widely used cellular model and several differentiation protocols have been developed to induce a neuron-like phenotype including retinoic acid (RA) treatment. In this work we obtained a homogeneous functional population of neuron-like cells by a two-step differentiation protocol in which SH-SY5Y cells were treated with RA plus the mitotic inhibitor 2-deoxy-5-fluorouridine (FUdr). RA-FUdr treatment induced a neuronal phenotype characterized by increased expression of neuronal markers and electrical properties specific to excitable cells. In addition, the RA-FUdr differentiated cells showed an enrichment of long chain and unsaturated fatty acids (FA) in the acyl chain composition of cardiolipin (CL) and the bioenergetic analysis evidences a high coupled and maximal respiration associated with high mitochondrial ATP levels. Our results suggest that the observed high oxidative phosphorylation (OXPHOS) capacity may be related to the activation of the cyclic adenosine monophosphate (cAMP) pathway and the assembly of respiratory supercomplexes (SCs), highlighting the change in mitochondrial phenotype during neuronal differentiation.
Asunto(s)
Neuroblastoma , Tretinoina , Humanos , Tretinoina/farmacología , Tretinoina/metabolismo , Floxuridina , Fosforilación Oxidativa , Línea Celular Tumoral , Neuroblastoma/metabolismo , Diferenciación CelularRESUMEN
A simple and fast method of lipid analysis of isolated intact mitochondria by means of MALDI-TOF mass spectrometry is described. Mitochondria isolated from bovine heart and yeast have been employed to set up and validate the new method of lipid analysis. The mitochondrial suspension is directly applied over the target and, after drying, covered by a thin layer of the 9-aminoacridine matrix solution. The lipid profiles acquired with this procedure contain all peaks previously obtained by analyzing the lipid extracts of isolated mitochondria by TLC and/or mass spectrometry. The novel procedure allows the quick, simple, precise, and accurate analysis of membrane lipids, utilizing only a tiny amount of isolated organelle; it has also been tested with intact membranes of the bacterium Paracoccus denitrificans for its evolutionary link to present-day mitochondria. The method is of general validity for the lipid analysis of other cell fractions and isolated organelles.
Asunto(s)
Lípidos/análisis , Mitocondrias/química , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/citología , Animales , Bovinos , Mitocondrias Cardíacas/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Mutations in the parkin gene are expected to play an essential role in autosomal recessive Parkinson's disease. Recent studies have established an impact of parkin mutations on mitochondrial function and autophagy. In primary skin fibroblasts from two patients affected by an early onset Parkinson's disease, we identified a hitherto unreported compound heterozygous mutation del exon2-3/del exon3 in the parkin gene, leading to the complete loss of the full-length protein. In both patients, but not in their heterozygous parental control, we observed severe ultrastructural abnormalities, mainly in mitochondria. This was associated with impaired energy metabolism, deregulated reactive oxygen species (ROS) production, resulting in lipid oxidation, and peroxisomal alteration. In view of the involvement of parkin in the mitochondrial quality control system, we have investigated upstream events in the organelles' biogenesis. The expression of the peroxisome proliferator-activated receptor gamma-coactivator 1-alpha (PGC-1α), a strong stimulator of mitochondrial biogenesis, was remarkably upregulated in both patients. However, the function of PGC-1α was blocked, as revealed by the lack of its downstream target gene induction. In conclusion, our data confirm the role of parkin in mitochondrial homeostasis and suggest a potential involvement of the PGC-1α pathway in the pathogenesis of Parkinson's disease. This article is part of a Special Issue entitled: Translating nuclear receptors from health to disease.
Asunto(s)
Proteínas de Choque Térmico/fisiología , Mitocondrias/fisiología , Enfermedad de Parkinson/fisiopatología , Factores de Transcripción/fisiología , Ubiquitina-Proteína Ligasas/fisiología , Adulto , Secuencia de Bases , Cartilla de ADN , Metabolismo Energético , Femenino , Fibroblastos/ultraestructura , Humanos , Masculino , Mutación , Estrés Oxidativo , Enfermedad de Parkinson/genética , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Parkin plays an important role in ensuring efficient mitochondrial function and calcium homeostasis. Parkin-mutant human fibroblasts, with defective oxidative phosphorylation activity, showed high basal cAMP level likely ascribed to increased activity/expression of soluble adenylyl cyclase and/or low expression/activity of the phosphodiesterase isoform 4 and to a higher Ca2+ level. Overall, these findings support the existence, in parkin-mutant fibroblasts, of an abnormal Ca2+ and cAMP homeostasis in mitochondria. In our previous studies resveratrol treatment of parkin-mutant fibroblasts induced a partial rescue of mitochondrial functions associated with stimulation of the AMPK/SIRT1/PGC-1α pathway. In this study we provide additional evidence of the potential beneficial effects of resveratrol inducing an increase in the pre-existing high Ca2+ level and remodulation of the cAMP homeostasis in parkin-mutant fibroblasts. Consistently, we report in these fibroblasts higher expression of proteins implicated in the tethering of ER and mitochondrial contact sites along with their renormalization after resveratrol treatment. On this basis we hypothesize that resveratrol-mediated enhancement of the Ca2+ level, fine-tuned by the ER-mitochondria Ca2+ crosstalk, might modulate the pAMPK/AMPK pathway in parkin-mutant fibroblasts.
Asunto(s)
Quinasas de la Proteína-Quinasa Activada por el AMP/genética , Calcio/metabolismo , Enfermedad de Parkinson/tratamiento farmacológico , Resveratrol/farmacología , Ubiquitina-Proteína Ligasas/genética , Señalización del Calcio/efectos de los fármacos , AMP Cíclico/genética , Retículo Endoplásmico/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Humanos , Mitocondrias/efectos de los fármacos , Mitofagia/efectos de los fármacos , Mitofagia/genética , Proteínas Mutantes/genética , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/patología , Sirtuina 1/genéticaRESUMEN
Dietary choline deprivation (CD) is associated with behavioral changes, but mechanisms underlying these detrimental effects are not well characterized. For instance, no literature data are available concerning the CD effects on brain mitochondrial function related to impairment in cognition. Therefore, we investigated brain mitochondrial function and redox status in male Wistar rats fed a CD diet for 28 d. Moreover, the CD behavioral phenotype was characterized. Compared with rats fed a control diet (CTRL), CD rats showed lower NAD-dependent mitochondrial state III and state IV respiration, 40% lower complex I activity, and significantly higher reactive oxygen species production. Total glutathione was oxidatively consumed more in CD than in CTRL rats and the rate of protein oxidation was 40% higher in CD than in CTRL rats, reflecting an oxidative stress condition. The mitochondrial concentrations of cardiolipin, a phospholipid required for optimal activity of complex I, was 20% lower in CD rats than in CTRL rats. Compared with CTRL rats, the behavioral phenotype of CD rats was characterized by impairment in motor coordination and motor learning assessed with the rotarod/accelerod test. Furthermore, compared with CTRL rats, CD rats were less capable of learning the active avoidance task and the number of attempts they made to avoid foot shock was fewer. The results suggest that CD-induced dysfunction in brain mitochondria may be responsible for impairment in cognition and underline that, similar to the liver, the brain also needs an adequate choline supply for its normal functioning.
Asunto(s)
Conducta Animal , Encéfalo/metabolismo , Colina/metabolismo , Colina/farmacología , Dieta , Mitocondrias/metabolismo , Animales , Masculino , Oxidación-Reducción , RatasRESUMEN
Crosstalk between lysosomes and mitochondria plays a central role in Parkinson's Disease (PD). Lysosomal function may be influenced by mitochondrial quality control, dynamics and/or respiration, but whether dysfunction of endocytic or autophagic pathway is associated with mitochondrial impairment determining accumulation of defective mitochondria, is not yet understood. Here, we performed live imaging, western blotting analysis, sequencing of mitochondrial DNA (mtDNA) and senescence-associated beta-galactosidase activity assay on primary fibroblasts from a young patient affected by PD, her mother and a healthy control to analyze the occurrence of mtDNA mutations, lysosomal abundance, acidification and function, mitochondrial biogenesis activation and senescence. We showed synergistic alterations in lysosomal functions and mitochondrial biogenesis, likely associated with a mitochondrial genetic defect, with a consequent block of mitochondrial turnover and occurrence of premature cellular senescence in PARK2-PD fibroblasts, suggesting that these alterations represent potential mechanisms contributing to the loss of dopaminergic neurons.