RESUMO
Patients with EGFR-mutated non-small cell lung cancer (NSCLC) benefit from treatment with tyrosine kinase inhibitors (TKI) targeting EGFR. Despite improvements in patient care, especially with the 3rd generation TKI osimertinib, disease relapse is observed in all patients. Among the various processes involved in TKI resistance, epithelial-to-mesenchymal transition (EMT) is far from being fully characterized. We hypothesized that the cellular prion protein PrPC could be involved in EMT and EGFR-TKI resistance in NSCLC. Using 5 independent lung adenocarcinoma datasets, including our own cohort, we document that the expression of the PRNP gene encoding PrPC is associated with EMT. By manipulating the levels of PrPC in different EGFR-mutated NSCLC cell lines, we firmly establish that the expression of PrPC is mandatory for cells to maintain or acquire a mesenchymal phenotype. Mechanistically, we show that PrPC operates through an ILK-RBPJ cascade, which also controls the expression of EGFR. Our data further demonstrate that PrPC levels are elevated in EGFR-mutated versus wild-type tumours or upon EGFR activation in vitro. In addition, we provide evidence that PRNP levels increase with TKI resistance and that reducing PRNP expression sensitizes cells to osimertinib. Finally, we found that plasma PrPC levels are increased in EGFR-mutated NSCLC patients from 2 independent cohorts and that their longitudinal evolution mirrors that of disease. Altogether, these findings define PrPC as a candidate driver of EMT-dependent resistance to EGFR-TKI in NSCLC. They further suggest that monitoring plasma PrPC levels may represent a valuable non-invasive strategy for patient follow-up and warrant considering PrPC-targeted therapies for EGFR-mutated NSCLC patients with TKI failure.
RESUMO
BACKGROUND: The mesenchymal subtype of colorectal cancer (CRC), associated with poor prognosis, is characterized by abundant expression of the cellular prion protein PrPC, which represents a candidate therapeutic target. How PrPC is induced in CRC remains elusive. This study aims to elucidate the signaling pathways governing PrPC expression and to shed light on the gene regulatory networks linked to PrPC. METHODS: We performed in silico analyses on diverse datasets of in vitro, ex vivo and in vivo models of mouse CRC and patient cohorts. We mined ChIPseq studies and performed promoter analysis. CRC cell lines were manipulated through genetic and pharmacological approaches. We created mice combining conditional inactivation of Apc in intestinal epithelial cells and overexpression of the human prion protein gene PRNP. Bio-informatic analyses were carried out in two randomized control trials totalizing over 3000 CRC patients. RESULTS: In silico analyses combined with cell-based assays identified the Wnt-ß-catenin and glucocorticoid pathways as upstream regulators of PRNP expression, with subtle differences between mouse and human. We uncover multiple feedback loops between PrPC and these two pathways, which translate into an aggravation of CRC pathogenesis in mouse. In stage III CRC patients, the signature defined by PRNP-CTNNB1-NR3C1, encoding PrPC, ß-catenin and the glucocorticoid receptor respectively, is overrepresented in the poor-prognosis, mesenchymal subtype and associates with reduced time to recurrence. CONCLUSIONS: An unleashed PrPC-dependent vicious circle is pathognomonic of poor prognosis, mesenchymal CRC. Patients from this aggressive subtype of CRC may benefit from therapies targeting the PRNP-CTNNB1-NR3C1 axis.
Assuntos
Neoplasias do Colo , Neoplasias Colorretais , Humanos , Camundongos , Animais , Proteínas Priônicas/genética , Proteínas Priônicas/metabolismo , beta Catenina/metabolismo , Glucocorticoides , Neoplasias do Colo/genética , Neoplasias Colorretais/genética , Fenótipo , Prognóstico , Via de Sinalização Wnt , Regulação Neoplásica da Expressão Gênica , Linhagem Celular TumoralRESUMO
BACKGROUND: Colorectal cancer (CRC) can be classified into four molecular subtypes (CMS) among which CMS1 is associated with the best prognosis, while CMS4, the mesenchymal subtype, has the worst outcome. Although mitochondria are considered to be hubs of numerous signaling pathways, the study of mitochondrial metabolism has been neglected for many years. Mitochondrial Complex I (CI) plays a dual role, both in energy and reactive oxygen species (ROS) production. However, the possible contribution of CI to tumorigenesis in cancer remains unclear. The purpose of this study was to investigate the CI under the prism of the CMS classification of CRC in ex vivo models. METHODS: Biochemical dosages, bioenergetics analysis and western-blot were used to characterize CI expression, function and redox balance in LoVo and MDST8 cell lines, belonging to CMS1 and CMS4 subgroups, respectively. Cell proliferation and migration were assessed by xCELLigence technology. Overproduction or scavenging of mitochondrial ROS (mtROS) were performed to analyze the effect of mtROS on proliferation, migration, and mesenchymal markers. Focal adhesion kinase (FAK) and its activation were analyzed by immunofluorescence. We assessed the distribution of two CI scores in CRC cohorts according to CMS classification and their relevance for patient survival. RESULTS: We found that CI is downregulated in CMS4 cells and is associated with elevated mtROS. We establish for the first time that in these migrating cells, mtROS production is maintained at optimal levels not only through changes in CI activity but also by inactivation/acetylation of superoxide dismutase 2 (SOD2), a major mitochondrial antioxidant enzyme. We show that promoting or scavenging mtROS both mitigate CMS4 cells' migration. Our results also point to a mtROS-mediated focal adhesion kinase (FAK) activation, which likely sustains their migratory phenotype. Using cohorts of CRC patients, we document that the expression of CI is downregulated in the CMS4 subgroup, and that low CI expression is associated with poor prognosis. Patients' datasets reveal an inverse correlation between CI and the epithelial-mesenchymal transition (EMT) pathway. CONCLUSION: We showed that inhibition of CI contributes to heighten mtROS, which likely foster MDST8 migration and might account for the specific EMT signature of CMS4 tumors. These data reveal a novel role of mitochondrial CI in CRC, with biological consequences that may be targeted with anti- or pro-oxidant drugs in clinical practice.
Assuntos
Neoplasias Colorretais , Humanos , Neoplasias Colorretais/genética , Espécies Reativas de Oxigênio/metabolismo , Regulação para Baixo , Transdução de Sinais , Proteína-Tirosina Quinases de Adesão Focal/genética , Proteína-Tirosina Quinases de Adesão Focal/metabolismoRESUMO
The cellular prion protein PrPC partners with caveolin-1 (CAV1) in neurodegenerative diseases but whether this interplay occurs in cancer has never been investigated. By leveraging patient and cell line datasets, we uncover a molecular link between PrPC and CAV1 across cancer. Using cell-based assays, we show that PrPC regulates the expression of and interacts with CAV1. PrPC additionally controls the expression of the amyloid precursor protein APP and of the Aß generating enzyme BACE1, and regulates the levels of Aß, whose accumulation is a central event in Alzheimer's disease. We further identify DKK1 and DKK3, involved in both Alzheimer's disease and cancer progression, as targets of the PrPC-dependent axis. Finally, we establish that antibody-mediated blocking of the Aß-PrPC interaction delays the growth of prostate cancer cell line-derived xenografts and prevents the development of metastases. Our data additionally support an enrichment of the Aß-PrPC-dependent pathway in the basal subtype of prostate cancer, associated with anti-hormonal therapy resistance, and in mesenchymal colon cancer, associated with poor prognosis. Thus, based on a parallel with neurodegenerative diseases, our results bring to light an Aß-PrPC axis and support the potential of targeting this pathway in patients with selected subtypes of prostate and colon cancer.
Assuntos
Doença de Alzheimer , Neoplasias do Colo , Neoplasias da Próstata , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Ácido Aspártico Endopeptidases/metabolismo , Caveolina 1/genética , Caveolina 1/metabolismo , Neoplasias do Colo/genética , Humanos , Masculino , Proteínas Priônicas/genética , Proteínas Priônicas/metabolismo , Neoplasias da Próstata/genéticaRESUMO
The CMS4 mesenchymal subtype of colorectal cancer (CRC) is associated with poor prognosis and resistance to treatment. The cellular prion protein PrPC is overexpressed in CMS4 tumors and controls the expression of a panel of CMS4-specific genes in CRC cell lines. Here, we sought to investigate PrPC downstream pathways that may underlie its role in CMS4 CRC. By combining gene set enrichment analyses and gain and loss of function approaches in CRC cell lines, we identify the integrin-linked kinase ILK as a proximal effector of PrPC that mediates its control on the CMS4 phenotype. We further leveraged three independent large CRC cohorts to assess correlations in gene expression pattern with patient outcomes and found that ILK is overexpressed in CMS4 mesenchymal tumors and confers a poor prognosis, especially when combined with high expression of the PrPC encoding gene PRNP. Of note, we discovered that the PrPC-ILK signaling axis controls the expression and activity of the tryptophan metabolizing enzyme indoleamine 2,3 dioxygenase IDO1, a key player in immune tolerance. In addition, we monitored alterations in the levels of tryptophan and its metabolites of the kynurenine pathway in the plasma of metastatic CRC patients (n = 325) and we highlight their prognostic value in combination with plasma PrPC levels. Thus, the PrPC-ILK-IDO1 axis plays a key role in the mesenchymal subtype of CRC. PrPC and IDO1-targeted strategies may represent new avenues for patient stratification and treatment in CRC.
Assuntos
Neoplasias Colorretais , Neoplasias Colorretais/diagnóstico , Humanos , Indolamina-Pirrol 2,3,-Dioxigenase , Proteínas Priônicas , Prognóstico , Proteínas Serina-Treonina QuinasesRESUMO
Endoplasmic Reticulum (ER) stress underlies the pathogenesis of numerous kidney diseases. A better care of patients with kidney disease involves the identification and validation of ER stress biomarkers in the early stages of kidney disease. For the first time to our knowledge, we demonstrate that the prion protein PrPC is secreted in a conventional manner by ER-stressed renal epithelial cell under the control of the transcription factor x-box binding protein 1 (XBP1) and can serve as a sensitive urinary biomarker for detecting tubular ER stress. Urinary PrPC elevation occurs in patients with chronic kidney disease. In addition, in patients undergoing cardiac surgery, detectable urine levels of PrPC significantly increase after cardiopulmonary bypass, a condition associated with activation of the IRE1-XBP1 pathway in the kidney. In conclusion, our study has identified PrPC as a novel urinary ER stress biomarker with potential utility in early diagnosis of ongoing acute or chronic kidney injury.
Assuntos
Biomarcadores/urina , Estresse do Retículo Endoplasmático/fisiologia , Nefropatias/metabolismo , Nefropatias/urina , Rim/lesões , Proteínas Priônicas/metabolismo , Animais , Proliferação de Células , Humanos , Nefropatias/patologia , Masculino , CamundongosRESUMO
Dysfunctions of mitochondrial fatty acid ß-oxidation (ß-FAO) in various tissues represent a hallmark of many common disorders, and are acknowledged to play an essential role in the pathogenesis of diabetes, obesity, and cardiac diseases. Moreover, inborn defects in ß-FAO form a large family of rare diseases with variable phenotypes, ranging from fatal multi-organ failure in the newborn to isolated adult onset myopathy. These pathologies highlight the critical role of ß-FAO in many tissues with high-energy demand (heart, muscle, liver, kidney). Furthermore, and unexpectedly, very recent data unveiled the possible involvement of ß-FAO in instructing complex non energy-related functions, such as chromatin modification, control of neural stem cell activity, or survival and fate of cancer cells. Pharmacological targeting of ß-FAO by small molecules might therefore open new avenues for the treatment of various rare or common diseases.
TITLE: Anomalies de la ß-oxydation mitochondriale des acides gras - Maladies rares et maladies communes. ABSTRACT: Certaines anomalies de la ß-oxydation mitochondriale des acides gras (ß-OAG) apparaissent jouer un rôle majeur dans la pathogenèse de plusieurs maladies communes (diabète, obésité, maladies cardiaques). Des déficits génétiques touchant la ß-OAG sont également à l'origine d'un ensemble de maladies rares de phénotypes très variables, allant de défaillances cardio-hépatiques fatales chez le nourrisson à des myopathies chez l'adulte. Ces différentes pathologies sont révélatrices du rôle clé de la ß-OAG dans plusieurs organes à forts besoins en ATP (cÅur, muscle, foie, rein). Des données récentes suggèrent que la ß-OAG participerait également à d'autres fonctions complexes (modifications de la chromatine, contrôle de l'activité de cellules souches, devenir de cellules cancéreuses).
Assuntos
Ácidos Graxos/metabolismo , Mitocôndrias/metabolismo , Doenças Mitocondriais/metabolismo , Doenças Raras/metabolismo , Humanos , OxirreduçãoRESUMO
BACKGROUND: Comprehensive transcriptomic analyses have shown that colorectal cancer (CRC) is heterogeneous and have led to the definition of molecular subtypes among which the stem-cell, mesenchymal-like group is associated with poor prognosis. The molecular pathways orchestrating the emergence of this subtype are incompletely understood. In line with the contribution of the cellular prion protein PrPC to stemness, we hypothesize that deregulation of this protein could lead to a stem-cell, mesenchymal-like phenotype in CRC. METHODS: We assessed the distribution of the PrPC-encoding PRNP mRNA in two large CRC cohorts according to molecular classification and its association with patient survival. We developed cell-based assays to explore the impact of gain and loss of PrPC function on markers of the mesenchymal subtype and to delineate the signalling pathways recruited by PrPC. We measured soluble PrPC in the plasmas of 325 patients with metastatic CRC and probed associations with disease outcome. FINDINGS: We found that PRNP gene expression is enriched in tumours of the mesenchymal subtype and is associated with poor survival. Our in vitro analyses revealed that PrPC controls the expression of genes that specify the mesenchymal subtype through the recruitment of the Hippo pathway effectors YAP and TAZ and the TGFß pathway. We showed that plasma levels of PrPC are elevated in metastatic CRC and are associated with poor disease control. INTERPRETATION: Our findings define PrPC as a candidate driver of the poor-prognosis mesenchymal subtype of CRC. They suggest that PrPC may serve as a potential biomarker for patient stratification in CRC. FUNDING: Grant support was provided by the following: Cancéropôle Ile de France (grant number 2016-1-EMERG-36-UP 5-1), Association pour la Recherche sur le Cancer (grant number PJA 20171206220), SATT Ile de France Innov (grant number 415) as well as INSERM.
Assuntos
Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Transição Epitelial-Mesenquimal/genética , Proteínas Priônicas/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Biomarcadores , Linhagem Celular Tumoral , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/mortalidade , Feminino , Expressão Gênica , Via de Sinalização Hippo , Humanos , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Proteínas Priônicas/metabolismo , Prognóstico , Proteínas Serina-Treonina Quinases/metabolismo , RNA Mensageiro/genética , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismoRESUMO
Resveratrol (RSV) is a small compound first identified as an activator of sirtuin 1 (SIRT1), a key factor in mediating the effects of caloric restriction. Since then, RSV received great attention for its widespread beneficial effects on health and in connection to many diseases. RSV improves the metabolism and the mitochondrial function, and more recently it was shown to restore fatty acid ß-oxidation (FAO) capacities in patient fibroblasts harboring mutations with residual enzyme activity. Many of RSV's beneficial effects are mediated by the transcriptional coactivator PGC-1α, a direct target of SIRT1 and a master regulator of the mitochondrial fatty acid oxidation. Despite numerous studies RSV's mechanism of action is still not completely elucidated. Our aim was to investigate the effects of RSV on gene regulation on a wide scale, possibly to detect novel genes whose up-regulation by RSV may be of interest with respect to disease treatment. We performed Next Generation Sequencing of RNA on normal fibroblasts treated with RSV. To investigate whether the effects of RSV are mediated through SIRT1 we expanded the analysis to include SIRT1-knockdown fibroblasts. We identified the aspartoacylase (ASPA) gene, mutated in Canavan disease, to be strongly up-regulated by RSV in several cell lines, including Canavan disease fibroblasts. We further link RSV to the up-regulation of other genes involved in myelination including the glial specific transcription factors POU3F1, POU3F2, and myelin basic protein (MBP). We also observe a strong up-regulation by RSV of the riboflavin transporter gene SLC52a1. Mutations in SLC52a1 cause transient multiple acyl-CoA dehydrogenase deficiency (MADD). Our analysis of alternative splicing identified novel metabolically important genes affected by RSV, among which is particularly interesting the α subunit of the stimulatory G protein (Gsα), which regulates the cellular levels of cAMP through adenylyl cyclase. We conclude that in fibroblasts RSV stimulates the PGC-1α and p53 pathways, and up-regulates genes affecting the glucose metabolism, mitochondrial ß-oxidation, and mitochondrial biogenesis. We further confirm that RSV might be a relevant treatment in the correction of FAO deficiencies and we suggest that treatment in other metabolic disorders including Canavan disease and MADD might be also beneficial.
Assuntos
Doença de Canavan/diagnóstico , Fibroblastos/efeitos dos fármacos , Sequenciamento de Nucleotídeos em Larga Escala , Resveratrol/farmacologia , Amidoidrolases/genética , Doença de Canavan/tratamento farmacológico , Linhagem Celular , Células Cultivadas , Regulação da Expressão Gênica , Genes p53 , Glucose/metabolismo , Humanos , Metabolismo dos Lipídeos , Terapia de Alvo Molecular , Proteína Básica da Mielina/genética , Oxirredução , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Receptores Acoplados a Proteínas G/genética , Análise de Sequência de RNA , Sirtuína 1/genética , Fatores de Transcrição/genética , Regulação para CimaRESUMO
Carnitine palmitoyl transferase 2 (CPT2) deficiency is one of the most common inherited fatty acid oxidation (FAO) defects and represents a prototypical mitochondrial metabolic myopathy. Recent studies have suggested a pivotal role of adenosine monophosphate-activated protein kinase (AMPK) in skeletal muscle plasticity and mitochondrial homeostasis. Thus, we tested the potential of GSK773, a novel direct AMPK activator, to improve or correct FAO capacities in muscle cells from patients harboring various mutations. We used controls' and patients' myotubes and studied the parameters of FAO metabolism, of mitochondrial quantity and quality and of differentiation. We found that AMPK is constitutively activated in patients' myotubes, which exhibit both reduced FAO and impaired differentiation. GSK773 improves or corrects several metabolic hallmarks of CPT2 deficiency (deficient FAO flux and C16-acylcarnitine accumulation) by upregulating the expression of CPT2 protein. Beneficial effects of GSK773 are also likely due to stimulation of mitochondrial biogenesis and induction of mitochondrial fusion, by decreasing dynamin-related protein 1 and increasing mitofusin 2. GSK773 also induces a shift in myosin heavy chain isoforms toward the slow oxidative type and, therefore, fully corrects the differentiation process. We establish, through small interfering RNA knockdowns and pharmacological approaches, that these GSK773 effects are mediated through peroxisome proliferator-activated receptor gamma co-activator 1-alpha, reactive oxygen species and p38 mitogen-activated protein kinase, all key players of skeletal muscle plasticity. GSK773 recapitulates several important features of skeletal muscle adaptation to exercise. The results show that AMPK activation by GSK773 evokes the slow, oxidative myogenic program and triggers beneficial phenotypic adaptations in FAO-deficient myotubes. Thus, GSK773 might have therapeutic potential for correction of CPT2 deficiency.
Assuntos
Carnitina O-Palmitoiltransferase/deficiência , Carnitina O-Palmitoiltransferase/genética , Metabolismo dos Lipídeos/genética , Erros Inatos do Metabolismo/genética , Proteínas Quinases/genética , Quinolonas/farmacologia , Quinases Proteína-Quinases Ativadas por AMP , Carnitina O-Palmitoiltransferase/efeitos dos fármacos , Ácidos Graxos/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , Erros Inatos do Metabolismo/fisiopatologia , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/patologia , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Mutação , Cadeias Pesadas de Miosina/genética , PPAR alfa/genética , RNA Interferente Pequeno/genética , Espécies Reativas de Oxigênio/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/genéticaRESUMO
Resveratrol is a natural polyphenolic compound produced by plants under various stress conditions. Resveratrol has been reported to exhibit antioxidant, anti-inflammatory, and anti-proliferative properties in mammalian cells and animal models, and might therefore exert pleiotropic beneficial effects in different pathophysiological states. More recently, resveratrol has also been shown to potentially target many mitochondrial metabolic pathways, including fatty acid ß-oxidation or oxidative phosphorylation, leading to the up-regulation of the energy metabolism via signaling pathways involving PGC-1α, SIRT1, and/or AMP-kinase, which are not yet fully delineated. Some of resveratrol beneficial effects likely arise from its cellular effects in the skeletal muscle, which, surprisingly, has been given relatively little attention, compared to other target tissues. Here, we review the potential for resveratrol to ameliorate or correct mitochondrial metabolic deficiencies responsible for myopathies, due to inherited fatty acid ß-oxidation or to respiratory chain defects, for which no treatment exists to date. We also review recent data supporting therapeutic effects of resveratrol in the Duchenne Muscular Dystrophy, a fatal genetic disease affecting the production of muscle dystrophin, associated to a variety of mitochondrial dysfunctions, which likely contribute to disease pathogenesis.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Doenças Musculares/tratamento farmacológico , Estilbenos/farmacologia , Metabolismo Energético/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , ResveratrolRESUMO
The pathophysiological mechanisms underlying Complex I (CI) deficiencies are understood only partially which severely limits the treatment of this common, devastating, mitochondrial disorder. Recently, we have shown that resveratrol (RSV), a natural polyphenol, has beneficial effects on CI deficiency of nuclear origin. Here, we demonstrate that RSV is able to correct the biochemical defect in oxygen consumption in five of thirteen CI-deficient patient cell lines. Other beneficial effects of RSV include a decrease of total intracellular ROS and the up-regulation of the expression of mitochondrial superoxide dismutase (SOD2) protein, a key antioxidant defense enzyme. The molecular mechanisms leading to the up-regulation of SOD2 protein expression by RSV require the estrogen receptor (ER) and the estrogen-related receptor alpha (ERRα). Although RSV increases the level of SOD2 protein in patients' fibroblasts, the enzyme activity is not increased, in contrast to normal fibroblasts. This led us to hypothesize that SOD2 enzyme activity is regulated post-translationally. This regulation involves SIRT3, a mitochondrial NAD(+)-dependent deacetylase and is critically dependent on NAD(+) levels. Taken together, our data show that the metabolic effects of RSV combined with its antioxidant capacities makes RSV particularly interesting as a candidate molecule for the therapy of CI deficiencies.
Assuntos
Complexo I de Transporte de Elétrons/deficiência , Receptor alfa de Estrogênio/genética , Doenças Mitocondriais/tratamento farmacológico , Receptores de Estrogênio/genética , Sirtuína 3/genética , Estilbenos/administração & dosagem , Superóxido Dismutase/genética , Antioxidantes/metabolismo , Células Cultivadas , Complexo I de Transporte de Elétrons/genética , Complexo I de Transporte de Elétrons/metabolismo , Fibroblastos/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/genética , Doenças Mitocondriais/genética , Doenças Mitocondriais/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/genética , Consumo de Oxigênio/genética , Espécies Reativas de Oxigênio/metabolismo , Resveratrol , Transdução de Sinais/efeitos dos fármacos , Receptor ERRalfa Relacionado ao EstrogênioRESUMO
This review presents recent evidence implicating microRNAs (miRNAs) in the beneficial effects of resveratrol (trihydroxystilbene), a nonflavonoid plant polyphenol, with emphasis on its anti-inflammatory effects. Many diseases and pathologies have been linked, directly or indirectly, to inflammation. These include infections, injuries, atherosclerosis, diabetes mellitus, obesity, cancer, osteoarthritis, age-related macular degeneration, demyelination, and neurodegenerative diseases. Resveratrol can both decrease the secretion of proinflammatory cytokines (e.g., IL-6, IL-8, and TNF-α) and increase the production of anti-inflammatory cytokines; it also decreases the expression of adhesion proteins (e.g., ICAM-1) and leukocyte chemoattractants (e.g., MCP-1). Resveratrol's primary targets appear to be the transcription factors AP-1 and NF-κB, as well as the gene COX2. Although no mechanistic link between any particular miRNA and resveratrol has been identified, resveratrol effects depend at least in part upon the modification of the expression of a variety of miRNAs that can be anti-inflammatory (e.g., miR-663), proinflammatory (e.g., miR-155), tumor suppressing (e.g., miR-663), or oncogenic (e.g., miR-21).
Assuntos
Anti-Inflamatórios/farmacologia , MicroRNAs/fisiologia , Estilbenos/farmacologia , Animais , Anti-Inflamatórios/uso terapêutico , Ácidos Araquidônicos/metabolismo , Expressão Gênica/efeitos dos fármacos , Humanos , Imunidade Inata , Inflamação/tratamento farmacológico , Inflamação/genética , Inflamação/metabolismo , Mediadores da Inflamação/metabolismo , Metabolismo dos Lipídeos/efeitos dos fármacos , Interferência de RNA , Resveratrol , Estilbenos/uso terapêuticoAssuntos
Acil-CoA Desidrogenase de Cadeia Longa/deficiência , Bezafibrato/uso terapêutico , Carnitina O-Palmitoiltransferase/deficiência , Ácidos Graxos/metabolismo , Erros Inatos do Metabolismo Lipídico/tratamento farmacológico , Lipólise/efeitos dos fármacos , Erros Inatos do Metabolismo/tratamento farmacológico , Doenças Mitocondriais/tratamento farmacológico , Doenças Musculares/tratamento farmacológico , Acil-CoA Desidrogenase de Cadeia Longa/genética , Bezafibrato/efeitos adversos , Carnitina O-Palmitoiltransferase/genética , Síndrome Congênita de Insuficiência da Medula Óssea , Frequência Cardíaca/efeitos dos fármacos , Humanos , Erros Inatos do Metabolismo Lipídico/diagnóstico , Erros Inatos do Metabolismo Lipídico/enzimologia , Erros Inatos do Metabolismo Lipídico/genética , Erros Inatos do Metabolismo Lipídico/fisiopatologia , Erros Inatos do Metabolismo/diagnóstico , Erros Inatos do Metabolismo/enzimologia , Erros Inatos do Metabolismo/genética , Erros Inatos do Metabolismo/fisiopatologia , Doenças Mitocondriais/diagnóstico , Doenças Mitocondriais/enzimologia , Doenças Mitocondriais/genética , Doenças Mitocondriais/fisiopatologia , Doenças Musculares/diagnóstico , Doenças Musculares/enzimologia , Doenças Musculares/genética , Doenças Musculares/fisiopatologia , Oxirredução , Resultado do TratamentoRESUMO
BACKGROUND: Inborn enzyme defects of mitochondrial fatty acid beta-oxidation (FAO) form a large group of genetic disorders associated to variable clinical presentations ranging from life-threatening pediatric manifestations up to milder late onset phenotypes, including myopathy. Very few candidate drugs have been identified in this group of disorders. Resveratrol (RSV) is a natural polyphenol with anti-oxidant and anti-inflammatory effects, recently shown to have beneficial metabolic properties in mice models. Our study explores its possible effects on FAO and mitochondrial energy metabolism in human cells, which are still very little documented. METHODS: Using cells from controls and from patients with Carnitine Palmitoyl Transferase 2 (CPT2) or Very Long Chain AcylCoA Dehydrogenase (VLCAD) deficiency we characterized the metabolic effects of RSV, RSV metabolites, and other stilbenes. We also focused on analysis of RSV uptake, and on the effects of low RSV concentrations, considering the limited bioavailability of RSV in vivo. RESULTS: Time course of RSV accumulation in fibroblasts over 48 h of treatment were consistent with the resulting stimulation or correction of FAO capacities. At 48 h, half maximal and maximal FAO stimulations were respectively achieved for 37,5 microM (EC50) and 75 microM RSV, but we found that serum content of culture medium negatively modulated RSV uptake and FAO induction. Indeed, decreasing serum from 12% to 3% led to shift EC50 from 37,5 to 13 microM, and a 2.6-3.6-fold FAO stimulation was reached with 20 microM RSV at 3% serum, that was absent at 12% serum. Two other stilbenes often found associated with RSV, i.e. cis- RSV and piceid, also triggered significant FAO up-regulation. Resveratrol glucuro- or sulfo- conjugates had modest or no effects. In contrast, dihydro-RSV, one of the most abundant circulating RSV metabolites in human significantly stimulated FAO (1.3-2.3-fold). CONCLUSIONS: This study provides the first compared data on mitochondrial effects of resveratrol, its metabolites, and other natural compounds of the stilbene family in human cells. The results clearly indicate that several of these compounds can improve mitochondrial FAO capacities in human FAO-deficient cells.
Assuntos
Ácidos Graxos/metabolismo , Mitocôndrias/metabolismo , Estilbenos/metabolismo , Western Blotting , Fibroblastos/metabolismo , Humanos , Oxirredução , ResveratrolRESUMO
Mitochondrial respiratory chain (RC) disorders are the most prevalent inborn metabolic diseases and remain without effective treatment to date. Up-regulation of residual enzyme activity has been proposed as a possible therapeutic approach in this group of disorders. As resveratrol (RSV), a natural compound, was proposed to stimulate mitochondrial metabolism in rodents, we tested the effect of this compound on mitochondrial functions in control or in Complex I (CI)- or Complex IV (CIV)-deficient patients' fibroblasts. We show that RSV stimulates the expression of a panel of proteins representing structural subunits or assembly factors of the five RC complexes, in control fibroblasts. In moderate RC-deficient patients' cells, RSV treatment increases the amount of mutated proteins and stimulates residual enzyme activities. In these patients' cells, we establish that up-regulation of RC enzyme activities induced by RSV translates into increased cellular O2 consumption rates and results in the correction of RC deficiencies. Importantly, RSV also prevents the accumulation of lactate that occurred in RC-deficient fibroblasts. Different complementary approaches demonstrate that RSV induces a mitochondrial biogenesis that might underlie the increase in mitochondrial capacities. Finally, we showed that, in human fibroblasts, RSV stimulated mitochondrial functions mainly in a SIRT1- and AMPK-independent manner and that its effects rather involved the estrogen receptor (ER) and estrogen-related receptor alpha (ERRα) signaling pathways. These results represent the first demonstration that RSV could have a beneficial effect on inborn CI and CIV deficiencies from nuclear origin, in human fibroblasts and might be clinically relevant for the treatment of some RC deficiencies.
Assuntos
Deficiência de Citocromo-c Oxidase/tratamento farmacológico , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Receptor alfa de Estrogênio/metabolismo , Fibroblastos/efeitos dos fármacos , Receptores de Estrogênio/metabolismo , Pele/efeitos dos fármacos , Estilbenos/farmacologia , Anticarcinógenos/farmacologia , Western Blotting , Células Cultivadas , Deficiência de Citocromo-c Oxidase/metabolismo , Deficiência de Citocromo-c Oxidase/patologia , Transporte de Elétrons/efeitos dos fármacos , Complexo I de Transporte de Elétrons/metabolismo , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , Lactatos , Membranas Mitocondriais/metabolismo , Consumo de Oxigênio/efeitos dos fármacos , Piruvatos , RNA Interferente Pequeno/genética , Resveratrol , Transdução de Sinais/efeitos dos fármacos , Sirtuína 1/antagonistas & inibidores , Sirtuína 1/genética , Sirtuína 1/metabolismo , Pele/metabolismo , Pele/patologia , Receptor ERRalfa Relacionado ao EstrogênioRESUMO
Lipin-1 deficiency is associated with massive rhabdomyolysis episodes in humans, precipitated by febrile illnesses. Despite well-known roles of lipin-1 in lipid biosynthesis and transcriptional regulation, the pathogenic mechanisms leading to rhabdomyolysis remain unknown. Here we show that primary myoblasts from lipin-1-deficient patients exhibit a dramatic decrease in LPIN1 expression and phosphatidic acid phosphatase 1 activity, and a significant accumulation of lipid droplets (LD). The expression levels of LPIN1-target genes [peroxisome proliferator-activated receptors delta and alpha (PPARδ, PPARα), peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), acyl-coenzyme A dehydrogenase, very long (ACADVL), carnitine palmitoyltransferase IB and 2 (CPT1B and CPT2)] were not affected while lipin-2 protein level, a closely related member of the family, was increased. Microarray analysis of patients' myotubes identified 19 down-regulated and 51 up-regulated genes, indicating pleiotropic effects of lipin-1 deficiency. Special attention was paid to the up-regulated ACACB (acetyl-CoA carboxylase beta), a key enzyme in the fatty acid synthesis/oxidation balance. We demonstrated that overexpression of ACACB was associated with free fatty acid accumulation in patients' myoblasts whereas malonyl-carnitine (as a measure of malonyl-CoA) and CPT1 activity were in the normal range in basal conditions accordingly to the normal daily activity reported by the patients. Remarkably ACACB invalidation in patients' myoblasts decreased LD number and size while LPIN1 invalidation in controls induced LD accumulation. Further, pro-inflammatory treatments tumor necrosis factor alpha+Interleukin-1beta(TNF1α+IL-1ß) designed to mimic febrile illness, resulted in increased malonyl-carnitine levels, reduced CPT1 activity and enhanced LD accumulation, a phenomenon reversed by dexamethasone and TNFα or IL-1ß inhibitors. Our data suggest that the pathogenic mechanism of rhabdomyolysis in lipin-1-deficient patients combines the predisposing constitutive impairment of lipid metabolism and its exacerbation by pro-inflammatory cytokines.
Assuntos
Citocinas/farmacologia , Mediadores da Inflamação/farmacologia , Transtornos do Metabolismo dos Lipídeos/etiologia , Lipídeos , Fibras Musculares Esqueléticas/patologia , Mioblastos/patologia , Fosfatidato Fosfatase/genética , Biomarcadores/metabolismo , Western Blotting , Estudos de Casos e Controles , Ciclo Celular , Proliferação de Células , Criança , Pré-Escolar , Estresse do Retículo Endoplasmático , Feminino , Perfilação da Expressão Gênica , Humanos , Transtornos do Metabolismo dos Lipídeos/metabolismo , Transtornos do Metabolismo dos Lipídeos/patologia , Masculino , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Mutação/genética , Mioblastos/efeitos dos fármacos , Mioblastos/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Associadas a Pancreatite , Fosfatidato Fosfatase/metabolismo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Rabdomiólise/etiologia , Rabdomiólise/metabolismo , Rabdomiólise/patologiaRESUMO
Cystic fibrosis (CF), a multisystem disease caused by CFTR (cystic fibrosis transmembrane conductance regulator) gene mutations, is associated with an abnormal inflammatory response and compromised redox homeostasis in the airways. Recent evidence suggests that dysfunctional CFTR leads to redox imbalance and to mitochondrial reduced glutathione (mtGSH) depletion in CF models. This study was designed to investigate the consequences of mtGSH depletion on mitochondrial function and inflammatory response. mtGSH depletion was confirmed in colonic epithelium of CFTR-null mice and in CFTR-mutated human epithelial cells. GSH uptake experiments performed on isolated mitochondria suggest that mtGSH depletion is not due to a defective GSH transport capacity by CF mitochondria, despite the decreased expression of two mtGSH carriers, oxoglutarate carrier and dicarboxylate carrier. CM-H(2)DCFDA [5 (and 6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate, acetyl ester] fluorescence and aconitase activity showed an increase in reactive oxygen species levels in CFTR-defective cells and a pro-oxidative environment within CF mitochondria. The activities of respiratory chain complexes were further examined. Results showed a selective loss of Complex I (CI) function in CF models associated with an altered mitochondrial membrane potential (Δψ(m)). CI analysis showed normal expression but an overoxidation of its NADH-ubiquinone oxidoreductase Fe-S protein 1 subunit. GSH monoethyl ester (GSH-EE) significantly enhanced mtGSH levels in the IB3-1/C38 model and reversed CI inhibition, suggesting that mtGSH depletion is responsible for the loss of CI activity. Furthermore, GSH-EE attenuated Δψ(m) depolarization and restored normal IL-8 secretion by CFTR-defective cells. These studies provide evidence for a critical role of a mtGSH defect in mitochondrial dysfunction and abnormal IL-8 secretion in CF cells and reveal the therapeutic potential of mitochondria-targeted antioxidants in CF.