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2-arachidonoylglycerol (2-AG) is the most abundant endocannabinoid (EC), acting as a full agonist at both CB1 and CB2 cannabinoid receptors. It is synthesized on demand in postsynaptic membranes through the sequential action of phosphoinositide-specific phospholipase Cß1 (PLCß1) and diacylglycerol lipase α (DAGLα), contributing to retrograde signaling upon interaction with presynaptic CB1. However, 2-AG production might also involve various combinations of PLC and DAGL isoforms, as well as additional intracellular pathways implying other enzymes and substrates. Three other alternative pathways of 2-AG synthesis rest on the extracellular cleavage of 2-arachidonoyl-lysophospholipids by three different hydrolases: glycerophosphodiesterase 3 (GDE3), lipid phosphate phosphatases (LPPs), and two members of ecto-nucleotide pyrophosphatase/phosphodiesterases (ENPP6-7). We propose the names of AlterAG-1, -2, and -3 for three pathways sharing an ectocellular localization, allowing them to convert extracellular lysophospholipid mediators into 2-AG, thus inducing typical signaling switches between various G-protein-coupled receptors (GPCRs). This implies the critical importance of the regioisomerism of both lysophospholipid (LPLs) and 2-AG, which is the object of deep analysis within this review. The precise functional roles of AlterAGs are still poorly understood and will require gene invalidation approaches, knowing that both 2-AG and its related lysophospholipids are involved in numerous aspects of physiology and pathology, including cancer, inflammation, immune defenses, obesity, bone development, neurodegeneration, or psychiatric disorders.
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Ácidos Araquidônicos , Endocanabinoides , Glicerídeos , Lisofosfolipídeos , Transdução de Sinais , Endocanabinoides/metabolismo , Glicerídeos/metabolismo , Lisofosfolipídeos/metabolismo , Humanos , Ácidos Araquidônicos/metabolismo , Animais , Diester Fosfórico Hidrolases/metabolismoRESUMO
Neuroinflammation and iron accumulation are hallmarks of a variety of adult neurodegenerative diseases. In Sanfilippo syndrome (mucopolysaccharidosis type III, MPSIII, a pediatric neurodegenerative disease that shares some features with adult neurodegenerative diseases), the progressive accumulation of heparan sulfate oligosaccharides (HSOs) induces microglia and astrocytes to produce pro-inflammatory cytokines leading to severe neuroinflammation. The objectives of the present study were (1) to measure the local iron concentration and to assess iron metabolism in the brain of a MPSIIIB murine model and (2) to identify the brain cells involved in this accumulation. We found that iron accumulation in MPSIIIB mice primarily affected the cerebral cortex where hepcidin levels were higher than in wild-type mice, and increased with aging. This increase was correlated with low expression of ferroportin 1 (FPN1), and thus brain iron retention. Moreover, we showed in vitro that HSOs are directly responsible for the production of hepcidin and the relative decrease in FPN1 expression when added to cultures of microglia and, to a lesser extent, to cultures of astrocytes. In contrast, no significant differences were observed in neurons. Hepcidin induction results from activation of the TLR4 pathway and STAT3 signaling, and leads to iron retention within microglia. Our results show that microglia have a key role in cerebral hepcidin overexpression and thus in the brain iron accumulation observed in the MPSIIIB model.
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Encéfalo/metabolismo , Ferro/metabolismo , Microglia/metabolismo , Mucopolissacaridose III/metabolismo , Animais , Astrócitos/metabolismo , Camundongos Knockout , Doenças Neurodegenerativas/metabolismo , Neurônios/metabolismoRESUMO
BACKGROUND: Epidermal growth factor receptor (EGFR) gene alterations and amplification are frequently reported in cases of glioblastoma (GBM). However, EGFR-activating mutations that confer proven sensitivity to tyrosine kinase inhibitors (TKIs) in lung cancer have not yet been reported in GBM. CASE PRESENTATION: Using next-generation sequencing, array comparative genomic hybridization and droplet digital PCR, we identified the p.L861Q EGFR mutation in a case of GBM for the first time. The mutation was associated with gene amplification. L861Q may be a clinically valuable mutation because it is known to sensitize non-small-cell lung cancers to treatment with the second-generation EGFR TKI afatinib in particular. Furthermore, we used slice culture of the patient's GBM explant to evaluate the tumour's sensitivity to various EGFR-targeting drugs. Our results suggested that the tumour was not intrinsically sensitive to these drugs. CONCLUSIONS: Our results highlight (i) the value of comprehensive genomic analyses for identifying patient-specific, targetable alterations, and (ii) the need to combine genomic analyses with functional assays, such as tumour-derived slice cultures.
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Neoplasias Encefálicas , Receptores ErbB/genética , Glioblastoma , Mutação , Idoso , Neoplasias Encefálicas/enzimologia , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/terapia , Hibridização Genômica Comparativa , Ativação Enzimática/genética , Receptores ErbB/antagonistas & inibidores , Feminino , Glioblastoma/enzimologia , Glioblastoma/genética , Glioblastoma/terapia , Humanos , Reação em Cadeia da Polimerase , Inibidores de Proteínas Quinases/farmacologia , Análise de Sequência de DNA , Células Tumorais CultivadasAssuntos
COVID-19 , Antidepressivos/uso terapêutico , Humanos , Prescrições , Rimonabanto/uso terapêutico , SARS-CoV-2RESUMO
Mucopolysaccharidosis (MPS) type IIIB is a genetic deficiency of α-N-acetylglucosaminidase, inducing accumulation of partially degraded heparan sulfate (HS) oligosaccharides in tissues. In the central nervous system, this accumulation is associated with microglial activation, neurodegeneration, and oxidative stress. We have already shown that HS activates microglial cells through toll-like receptor 4 (TLR4) and triggers neuroinflammation. The present study investigates whether oxidative stress is a direct consequence of inflammation or is an independent event directly caused by HS accumulation. The present study addresses causative links between oxidative stress and inflammation by analyzing the corresponding markers in the cortex of control mice, MPSIIIB mice (with neuroinflammation), and double mutant TLR4 knockout MPSIIIB mice (without neuroinflammation at early stages). Results showed that, although inflammation was not present in the cortex of 10-day-old double mutant MPSIIIB/TLR4(-/-) mice, the enzymatic activity of total superoxide dismutase (SOD) was already greater than in control animals. Moreover, at 3 and 8 months of age, the total enzymatic activities of glutathione peroxidase, SOD, and carbonyl protein levels in the cortex of MPSIIIB/TLR4(-/-) mice were similar to those measured in MPSIIIB mice and were higher than those in controls. The results indicate that the oxidative stress present at a very early stage in the brain of MPSIIIB mice is not the consequence of neuroinflammation. Insofar as it has an impact on the development of neurological disease, reducing oxidative stress might prevent or slow the progression of MPSIIIB.
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Encéfalo/metabolismo , Inflamação/metabolismo , Mucopolissacaridose III/metabolismo , Estresse Oxidativo/fisiologia , Animais , Encéfalo/patologia , Modelos Animais de Doenças , Progressão da Doença , Glutationa Peroxidase/metabolismo , Inflamação/patologia , Camundongos , Camundongos Knockout , Mucopolissacaridose III/patologia , NADPH Oxidases/metabolismo , Superóxido Dismutase/metabolismo , Receptor 4 Toll-Like/genéticaRESUMO
In B cells, B-cell receptor (BCR) immunoglobulin revision is a common route for modifying unwanted antibody specificities via a mechanism called VH replacement. This in vivo process, mostly affecting heavy-chain rearrangement, involves the replacement of all or part of a previously rearranged IGHV gene with another germline IGHV gene located upstream. Two different mechanisms of IGHV replacement have been reported: type 1, involving the recombination activating genes complex and requiring a framework region 3 internal recombination signal; and type 2, involving an unidentified mechanism different from that of type 1. In the case of light-chain loci, BCR immunoglobulin editing ensures that a second V-J rearrangement occurs. This helps to maintain tolerance, by generating a novel BCR with a new antigenic specificity. We report that human B cells can, surprisingly, undergo type 2 replacement associated with κ light-chain rearrangements. The de novo IGKV-IGKJ products result from the partial replacement of a previously rearranged IGKV gene by a new germline IGKV gene, in-frame and without deletion or addition of nucleotides. There are wrcy/rgyw motifs at the 'IGKV donor-IGKV recipient chimera junction' as described for type 2 IGHV replacement, but activation-induced cytidine deaminase (AID) expression was not detected. This unusual mechanism of homologous recombination seems to be a variant of gene conversion-like recombination, which does not require AID. The recombination phenomenon described here provides new insight into immunoglobulin locus recombination and BCR immunoglobulin repertoire diversity.
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Citidina Desaminase/fisiologia , Recombinação Homóloga , Região Variável de Imunoglobulina/genética , Receptores de Antígenos de Linfócitos B/genética , Sequência de Aminoácidos , Sequência de Bases , Linhagem Celular , Rearranjo Gênico , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias kappa de Imunoglobulina/genética , Interleucina-1/farmacologia , Lipopolissacarídeos/farmacologia , Dados de Sequência MolecularRESUMO
We have previously reported an increased expression of cytokeratins 8/18 (K8/K18) in cells expressing the F508del mutation of cystic fibrosis transmembrane conductance regulator (CFTR). This is associated with increased colocalization of CFTR and K18 in the vicinity of the endoplasmic reticulum, although this is reversed by treating cells with curcumin, resulting in the rescue of F508del-CFTR. In the present work, we hypothesized that (i) the K8/K18 network may interact physically with CFTR, and that (ii) this interaction may modify CFTR function. CFTR was immunoprecipitated from HeLa cells transfected with either wild-type (WT) CFTR or F508del-CFTR. Precipitates were subjected to 2D-gel electrophoresis and differential spots identified by mass spectrometry. K8 and K18 were found significantly increased in F508del-CFTR precipitates. Using surface plasmon resonance, we demonstrate that K8, but not K18, binds directly and preferentially to the F508del over the WT human NBD1 (nucleotide-binding domain-1). In vivo K8 interaction with F508del-CFTR was confirmed by proximity ligation assay in HeLa cells and in primary cultures of human respiratory epithelial cells. Ablation of K8 expression by siRNA in F508del-expressing HeLa cells led to the recovery of CFTR-dependent iodide efflux. Moreover, F508del-expressing mice topically treated with K8-siRNA showed restored nasal potential difference, equivalent to that of WT mice. These results show that disruption of F508del-CFTR and K8 interaction leads to the correction of the F508del-CFTR processing defect, suggesting a novel potential therapeutic target in CF.
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Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Queratina-8/metabolismo , Animais , Regulador de Condutância Transmembrana em Fibrose Cística/química , Células Epiteliais/metabolismo , Feminino , Inativação Gênica , Células HeLa , Humanos , Queratina-18/metabolismo , Queratina-8/antagonistas & inibidores , Queratina-8/genética , Masculino , Camundongos , Mutação , Nariz/citologia , Domínios e Motivos de Interação entre ProteínasRESUMO
In this study, we investigated the capacity of chronic lymphocytic leukemia (CLL) B cells to undergo terminal differentiation into Ig-secreting plasma cells in T cell-independent and T cell-dependent responses. We used a two-step model involving stimulation with phorbol myristate acetate (PMA) and CD40L, together with cytokines (PMA/c and CD40L/c), for 7 days. We describe immunophenotypic modifications, changes in the levels of mRNA and protein for transcription factors and morphological and functional events occurring during the differentiation of CLL B cells into antibody-secreting cells (ASCs). The induction of differentiation differed significantly between the CD40L/c and PMA/c culture systems. The PMA/c culture system allowed CLL B cells to differentiate into IgM-secreting cells with an immunophenotype and molecular profile resembling those of preplasmablasts. By contrast, CD40L/c-stimulated cells had a phenotype and morphology similar to those of activated B cells and resembling those of the CLL B cells residing in the lymph node and bone marrow. These data suggest that the CLL B cells are not frozen permanently at a stage of differentiation and are able to differentiate into ASCs as appropriate stimulation are provided. The data presented here raise questions about the molecular processes and stimulation required for CLL B-cell differentiation and about the inability of CD40 ligand to induce differentiation of the CLL B cells.
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Formação de Anticorpos/imunologia , Linfócitos B/imunologia , Linfócitos B/metabolismo , Ligante de CD40/imunologia , Leucemia Linfocítica Crônica de Células B/imunologia , Leucemia Linfocítica Crônica de Células B/metabolismo , Acetato de Tetradecanoilforbol/imunologia , Idoso , Idoso de 80 Anos ou mais , Antígenos de Superfície/metabolismo , Linfócitos B/patologia , Diferenciação Celular , Citocinas/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Imunoglobulina M/biossíntese , Imunofenotipagem , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/patologia , Masculino , Pessoa de Meia-Idade , Fenótipo , Transcrição GênicaRESUMO
BACKGROUND: Mutations in the activation segment of the v-raf murine sarcoma viral oncogene homolog B (BRAF) gene are present in approximately 50% of melanomas. The selective BRAF inhibitor vemurafenib has demonstrated significant clinical benefits in patients with melanomas harboring the most common mutations (V600E, V600K and V600R). However, the clinical activity of BRAF inhibitors in patients with rare mutations of codon 600 and the surrounding codons has not been documented. CASE PRESENTATION: We used the BRAF inhibitor vemurafenib to treat a patient presenting a rare p.V600_K601delinsD-mutated melanoma. An objective response was evidenced by two months of progression-free survival. By cloning and sequencing BRAF exon 15, we confirmed that a dual mutation was present on a single allele and thus resulted in a BRAFV(600DK601del) mutant protein. We also performed an in silico crystal structure analysis of the mutated protein, in order to characterize the nature of the putative interaction between vemurafenib and the mutant protein. CONCLUSION: This clinical experience suggests that (i) patients with BRAFV(600DK601del)-mutation-positive melanoma can be treated successfully with the oral BRAF inhibitor vemurafenib and (ii) molecular screening in this context should encompass rare and complex mutations.
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Antineoplásicos/administração & dosagem , Indóis/administração & dosagem , Melanoma/tratamento farmacológico , Melanoma/genética , Proteínas Proto-Oncogênicas B-raf/genética , Sulfonamidas/administração & dosagem , Idoso , Antineoplásicos/uso terapêutico , Feminino , Humanos , Indóis/uso terapêutico , Modelos Moleculares , Metástase Neoplásica/tratamento farmacológico , Mutação Puntual , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas B-raf/química , Deleção de Sequência , Sulfonamidas/uso terapêutico , Resultado do Tratamento , VemurafenibRESUMO
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.
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Fibrose Cística/tratamento farmacológico , Glutationa/análogos & derivados , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/metabolismo , Protetores contra Radiação/farmacologia , Animais , Linhagem Celular , Fibrose Cística/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Transportadores de Ácidos Dicarboxílicos/genética , Transportadores de Ácidos Dicarboxílicos/metabolismo , Complexo I de Transporte de Elétrons/genética , Complexo I de Transporte de Elétrons/metabolismo , Glutationa/farmacologia , Interleucina-8/metabolismo , Masculino , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Camundongos , Camundongos Endogâmicos CFTR , Camundongos Knockout , Mitocôndrias/genética , Mitocôndrias/patologia , Mutação , Recuperação de Função Fisiológica/efeitos dos fármacosRESUMO
Background: Embryonal rhabdomyosarcomas (ERMS) of the uterine cervix and corpus are rare pediatric tumors usually associated with a late age of onset and frequent somatic DICER1 mutation. It may also develop in the context of a familial predisposition such as DICER1 syndrome requiring specific medical care for children and young adults at risk for a broad range of tumors. Case presentation: This is a case of a prepubescent 9-year-old girl who was presented to our department for metrorrhagias due to a vaginal cervical mass, initially classified as a müllerian endocervical polyp on negative myogenin immunostaining. The patient subsequently manifested growth retardation (-2DS) and learning disabilities leading to genetic explorations and the identification of a germline pathogenic DICER1 variant. The family history revealed thyroid diseases in the father, aunt and paternal grandmother before the age of 20. Conclusion: Rare tumors such as cervical ERMS associated with a family history of thyroid disease during infancy could be related to DICER1 syndrome. Identifying at-risk relatives is challenging but necessary to detect early DICER1 spectrum tumors in young patients.
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Cystic fibrosis (CF) is associated with abnormal lipid metabolism. We have recently shown variations in plasma levels of several phosphatidylcholine (PC) and lysophopshatidylcholine (LPC) species related to disease severity in CF patients. Here our goal was to search for blood plasma lipid signatures characteristic of CF patients bearing the same mutation (F508del) and different phenotypes, and to study their correlation with forced expiratory volume in 1 s (FEV1) and Pseudomonas aeruginosa chronic infection, evaluated at the time of testing (t = 0) and three years later (t = 3). Samples from 44 F508del homozygotes were subjected to a lipidomic approach based on LC-ESI-MS. Twelve free fatty acids were positively correlated with FEV1 at t = 0 (n = 29). Four of them (C20:3n-9, C20:5n-3, C22:5n-3, and C22:6n-3) were also positively correlated with FEV1 three years later, along with PC(32:2) and PC(36:4) (n = 31). Oleoylethanolamide (OEA) was negatively correlated with FEV1 progression (n = 17). Chronically infected patients at t = 0 showed lower PC(32:2), PC(38:5), and C18:3n-3 and higher cholesterol, cholesterol esters, and triacylglycerols (TAG). Chronically infected patients at t = 3 showed significantly lower levels of LPC(18:0). These results suggest a potential prognostic value for some lipid signatures in, to our knowledge, the first longitudinal study aimed at identifying lipid biomarkers for CF.
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Fibrose Cística/sangue , Fibrose Cística/patologia , Lipídeos/sangue , Adolescente , Adulto , Criança , Pré-Escolar , Colesterol/sangue , Ésteres do Colesterol/sangue , Cromatografia Líquida , Endocanabinoides , Feminino , Volume Expiratório Forçado/fisiologia , Humanos , Lactente , Lisofosfatidilcolinas/sangue , Masculino , Espectrometria de Massas , Ácidos Oleicos/sangue , Fosfatidilcolinas/sangue , Infecções por Pseudomonas/fisiopatologia , Triglicerídeos/sangue , Adulto JovemRESUMO
Mucopolysaccharidosis type I (MPS I) is caused by a deficiency of the lysosomal hydroxylase alpha-l-iduronidase (IDUA). The resulting accumulation of dermatan and heparan sulfate induces intellectual disabilities and pre-mature death, and only a few treatment options are available. In a previous study, we demonstrated the feasibility, safety, and efficacy of gene therapy by injecting recombinant adeno-associated viral vector serotype (AAV)2/5-IDUA into the brain of a canine model of MPS I. We report on a quantitative proteomic analysis of control dogs and untreated dogs with MPS I cerebrospinal fluid (CSF) that had been collected throughout the study in the MPS I dogs. Mass spectrometry (MS) analysis identified numerous proteins present at altered levels in MPS I CSF samples. Quantitative immunoblotting, performed on CSF from healthy controls, untreated MPS I dogs, and MPS I dogs early treated and late treated by gene therapy, confirmed the MS data for a subset of proteins with higher abundance (neuronal pentraxin 1, chitinase 3-like 1, monocyte differentiation antigen CD14, and insulin-like growth factor-binding protein 2). Scoring of the results shows that the expression levels of these proteins are close to those of the control group for dogs that underwent gene therapy early in life but not for older treated animals. Our results disclose four novel predictive biomarker candidates that might be valuable in monitoring the course of the neurological disease in MPS patients at diagnosis, during clinical follow-up, and after treatment.
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Integrin α5ß1 was suggested to be involved in glioblastoma (GBM) aggressiveness and treatment resistance through preclinical studies and genomic analysis in patients. However, further protein expression data are still required to confirm this hypothesis. In the present study, we investigated by immunofluorescence the expression of integrin α5 and its prognostic impact in a glioblastoma series of patients scheduled to undergo the Stupp protocol as first-line treatment for GBM. The integrin α5 protein expression level was estimated in each tumor by the mean fluorescence intensity (MFI) and allowed us to identify two subpopulations showing either a high or low expression level. The distribution of patients in both subpopulations was not significantly different according to age, gender, recursive partitioning analysis (RPA) prognostic score, molecular markers or surgical and medical treatment. A high integrin α5 protein expression level was associated with a high risk of recurrence (HR = 1.696, 95% CI 1.031-2.792, p = 0.0377) and reduced overall survival (OS), even more significant in patients who completed the Stupp protocol (median OS: 15.6 vs. 22.8 months; HR = 2.324; 95% CI 1.168-4.621, p = 0.0162). In multivariate analysis, a high integrin α5 protein expression level was confirmed as an independent prognostic factor in the subpopulation of patients who completed the temozolomide-based first-line treatment for predicting OS over age, extent of surgery, RPA score and O-6-methylguanine-DNA methyltransferase (MGMT) promoter methylation (p = 0.029). In summary, for the first time, our study validates that a high integrin α5 protein expression level is associated with poor prognosis in GBM and confirms its potential as a therapeutic target implicated in the Stupp protocol resistance.
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Two highly potent and selective cystic fibrosis (CF) transmembrane regulator (CFTR) inhibitors have been identified by high-throughput screening: the thiazolidinone CFTR(inh)-172 [3-[(3-trifluoromethyl)phenyl]-5-[(4-carboxyphenyl)methylene]- 2-thioxo-4-thiazolidinone] and the glycine hydrazide GlyH-101 [N-(2-naphthalenyl)-((3,5-dibromo-2,4-dihydroxyphenyl)methylene)glycine hydrazide]. Inhibition of the CFTR chloride channel by these compounds has been suggested to be of pharmacological interest in the treatment of secretory diarrheas and polycystic kidney disease. In addition, functional inhibition of CFTR by CFTR(inh)-172 has been proposed to be sufficient to mimic the CF inflammatory profile. In the present study, we investigated the effects of the two compounds on reactive oxygen species (ROS) production and mitochondrial membrane potential in several cell lines: the CFTR-deficient human lung epithelial IB3-1 (expressing the heterozygous F508del/W1282X mutation), the isogenic CFTR-corrected C38, and HeLa and A549 as non-CFTR-expressing controls. Both inhibitors were able to induce a rapid increase in ROS levels and depolarize mitochondria in the four cell types, suggesting that these effects are independent of CFTR inhibition. In HeLa cells, these events were associated with a decrease in the rate of oxygen consumption, with GlyH-101 demonstrating a higher potency than CFTR(inh)-172. The impact of CFTR inhibitors on inflammatory parameters was also tested in HeLa cells. CFTR(inh)-172, but not GlyH-101, induced nuclear translocation of nuclear factor-kappaB (NF-kappaB). CFTR(inh)-172 slightly decreased interleukin-8 secretion, whereas GlyH-101 induced a slight increase. These results support the conclusion that CFTR inhibitors may exert nonspecific effects regarding ROS production, mitochondrial failure, and activation of the NF-kappaB signaling pathway, independently of CFTR inhibition.
Assuntos
Benzoatos/farmacologia , Canais de Cloreto/antagonistas & inibidores , Regulador de Condutância Transmembrana em Fibrose Cística/antagonistas & inibidores , Glicina/análogos & derivados , Hidrazinas/farmacologia , Mitocôndrias/efeitos dos fármacos , Tiazolidinas/farmacologia , Aconitato Hidratase/metabolismo , Linhagem Celular , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Glicina/farmacologia , Humanos , Interleucina-8/biossíntese , Potencial da Membrana Mitocondrial , Mitocôndrias/fisiologia , Mutação , NF-kappa B/metabolismo , Fosforilação Oxidativa , Consumo de Oxigênio , Espécies Reativas de Oxigênio/metabolismoRESUMO
INTRODUCTION: Adult granulosa cell tumors (aGCTs) are extremely rare tumors characterized by the presence of the single missense mutation (c.402 C>G, p. C134W) in the FOXL2 gene. These tumors are frequently associated with a slow, indolent disease progression and a high probability of aggressive tumor recurrence. Hence, the identification of molecular markers that are predictive of recurrence and/or aggressive behavior would be a great asset in the management of aGCT. The present study focused on the influence of the FOXL2 genotype (heterozygous or homozygous) and copy number variations (CNVs) in recurrence by comparing the primary tumor with recurrent lesions in the same patient. We performed array comparative genomic hybridization (CGH) experiments and FOXL2 genotyping by allelic discrimination on 40 tumor samples. RESULTS AND DISCUSSION: In array CGH results of recurrent tumors, few samples presented the multiple chromosome losses and gains characteristic of chromosome instability (CIN). We also observed that three recurrent tumors and one primary tumor appeared to be homozygous for the FOXL2 c.402C>G mutation. Interestingly, the homozygous FOXL2 genotype was correlated with a shorter time to relapse. A change in the FOXL2 genotype in cases of recurrence was correlated with the appearance of CIN. CONCLUSION: Despite the small number of matching primary and recurrent tumors analyzed here, the present study is the first to have shown that the FOXL2 homozygous genotype and CIN are prevalent in recurrent aGCTs. The two mechanisms are probably linked, and both almost certainly have a role in the molecular transformation of aGCTs.
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Background. New approaches are needed for patients newly diagnosed with bulky glioblastoma (GB) and/or with severe neurological impairment that cannot benefit from first line temozolomide (TMZ)-based chemoradiotherapy. Bevacizumab (BEV), an antiangiogenic anti-VEGF-R monoclonal antibody, has a rapid impact on tumor-related brain edema in recurrent GB. The present study reports the feasibility and efficacy of an induction treatment with TMZ and BEV to alleviate the initial neurological impairment and/or to reduce the tumor volume before a delayed chemoradiotherapy. Methods. We retrospectively analyzed tumor and target volumes and clinical neurological status in 39 patients with bulky GB and/or with severe neurological impairment after an induction treatment combining TMZ and BEV. Neurological and radiological responses were assessed according to RANO criteria. Calculating gross tumor and clinical target volumes (GTV and CTV) was done at diagnosis and before radiotherapy. Progression-free survival (PFS) and overall survival (OS) were determined by Kaplan Meier methods. Safety was reported according to NCTCAE. Results. A cohort of 39 patients was analyzed between December 2010 and April 2014. Upfront standard TMZ-based chemoradiotherapy was recused due either to tumor volume or impairment of neurological status and/or performance status. After TMZ/BEV induction (median time of 3 months), 6 (15%) patients achieved a partial response (PR), and 17 (44%) had a stable disease. 24 patients (62%) received a radical-intent chemoradiotherapy. TMZ-BEV induced median reduction of the clinical target volume (CTV) was 25.9% [-84.4%; - 4.8%]. The median PFS and OS were 8.4 months [95% CI: (6.6 - 9.9)] and 11.0 months [95% CI: (9.3 - 13.7)], respectively in the whole cohort and 10.8 [95% CI: (9.3 - 12.9)] and 15.0 [95% CI: (13.2 - 17.8)] for irradiated patients. Induction treatment led to corticosteroid dose reduction or cessation in 21 patients (54%). KPS improvement was observed in 38% of patients. Toxicity was mild with only 7/39 (18%) grade III-IV toxicity, including 1 digestive bleeding and 1 epistaxis. Conclusion. TMZ-BEV induction led to CTV reduction allowing for optimal chemoradiotherapy in a majority (62%) of patients for which radiotherapy was initially recused. A clinical benefit was obtained with improved KPS and a decrease in steroid dose.
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Chronic lymphocytic leukemia (CLL) and multiple myeloma (MM) are hematological disorders that occur at different stages of B-cell development. It has been shown that CLL B-cells can differentiate into plasma cells in vitro and in vivo. CLL is the most frequent adult leukemia in the western world. It is a heterogeneous disease, characterized by clonal proliferation and the accumulation of mature CD5+ B lymphocytes (1). MM is a clonal plasma cell malignancy that accounts for more than 10% of all hematologic cancers (2). Although secondary cancers [particularly solid tumors (3-5)] can occur with CLL and MM, the concomitant occurrence of these two disorders in the same patient is rare [for a review of the few reported cases, see Ref. (6)]. The clonal relationship between these diseases has not always been clarified but is important in terms of understanding the pathogenesis and optimizing treatment. The clonal relationship between CLL and MM can be evaluated by (i) analyzing immunoglobulin (Ig) heavy chain and light chain (Ig kappa light chain and Ig lambda light chain) gene rearrangement, (ii) identifying and comparing somatic mutations, and (iii) studying chromosomic aberrations. Nevertheless, Ig rearrangements must always be interpreted in the light of specific phenomena such as allelic exclusion, B-cell receptor (BCR) revision (VH and DH gene replacement), BCR editing, and somatic mutations-events that were not considered in previous studies. These issues can be addressed by sequencing the rearranged Ig genes from sorted populations and interpreting the generated data. In the present study, we evaluated the putative clonal relationship between the two diseases by combining DNA copy number analysis with an assessment of Ig gene rearrangements [clonality assessment, V(D)J sequencing, and somatic hypermutation analysis] in highly enriched CD19+ CD5+ (CLL) and CD38+ CD138+ (MM) cell populations. Array comparative genomic hybridization data suggested a possible phylogenic progression from CLL to MM. Moreover, V(D)J sequencing indicated that both CLL and MM cells used the same VH and JH genes but different DH genes. However, in-depth analysis and interpretation of Ig gene rearrangements ultimately suggested that the two diseases had distinct clonal origins.
RESUMO
Recent research has shown that chronic lymphocytic leukemia (CLL) B-cells display a strong tendency to differentiate into antibody-secreting cells (ASCs) and thus may be amenable to differentiation therapy. However, the effect of this differentiation on factors associated with CLL pathogenesis has not been reported. In the present study, purified CLL B-cells were stimulated to differentiate into ASCs by phorbol myristate acetate or CpG oligodeoxynucleotide, in combination with CD40 ligand and cytokines in a two-step, seven-day culture system. We investigated (i) changes in the immunophenotypic, molecular, functional, morphological features associated with terminal differentiation into ASCs, (ii) the expression of factors involved in CLL pathogenesis, and (iii) the expression of pro- and anti-apoptotic proteins in the differentiated cells. Our results show that differentiated CLL B-cells are able to display the transcriptional program of ASCs. Differentiation leads to depletion of the malignant program and deregulation of the apoptosis/survival balance. Analysis of apoptosis and the cell cycle showed that differentiation is associated with low cell viability and a low rate of cell cycle entry. Our findings shed new light on the potential for differentiation therapy as a part of treatment strategies for CLL.