RESUMEN
OBJECTIVE: Cytotoxicity of the antirheumatic drug auranofin (Aur) and the non-steroidal anti-inflammatory drug meclofenamic acid (MA) on several cancer cell lines and isolated mitochondria was examined to assess whether these drugs behave as oxidative phosphorylation inhibitors. METHODS: The effect of Aur or MA for 24 h was assayed on metastatic cancer and non-cancer cell proliferation, energy metabolism, mitophagy and metastasis; as well as on oxygen consumption rates of cancer and non-cancer mitochondria. RESULTS: Aur doses in the low micromolar range were required to decrease proliferation of metastatic HeLa and MDA-MB-231 cells, whereas one or two orders of magnitude higher levels were required to affect proliferation of non-cancer cells. MA doses required to affect cancer cell growth were one order of magnitude higher than those of Aur. At the same doses, Aur impaired oxidative phosphorylation in isolated mitochondria and intact cells through mitophagy induction, as well as glycolysis. Consequently, cell migration and invasiveness were severely affected. The combination of Aur with very low cisplatin concentrations promoted that the effects on cellular functions were potentiated. CONCLUSION: Aur surges as a highly promising anticancer drug, suggesting that efforts to establish this drug in the clinical treatment protocols are warranted and worthy to undertake.
Asunto(s)
Antineoplásicos , Auranofina , Proliferación Celular , Reposicionamiento de Medicamentos , Metabolismo Energético , Ácido Meclofenámico , Mitocondrias , Fosforilación Oxidativa , Humanos , Auranofina/farmacología , Antineoplásicos/farmacología , Proliferación Celular/efectos de los fármacos , Metabolismo Energético/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Ácido Meclofenámico/farmacología , Línea Celular Tumoral , Fosforilación Oxidativa/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Células HeLa , Mitofagia/efectos de los fármacos , Glucólisis/efectos de los fármacos , Consumo de Oxígeno/efectos de los fármacosRESUMEN
Fourteen cobalt(II) complexes with the non-steroidal anti-inflammatory drugs sodium meclofenamate, tolfenamic acid, mefenamic acid, naproxen, sodium diclofenac, and diflunisal were prepared in the presence or absence of a series of nitrogen-donors (namely imidazole, pyridine, 3-aminopyridine, neocuproine, 2,2'-bipyridine, 1,10-phenanthroline and 2,2'-bipyridylamine) as co-ligands and were characterised by spectroscopic and physicochemical techniques. Single-crystal X-ray crystallography was employed to determine the crystal structure of eight complexes. The biological profile of the complexes was investigated regarding their interaction with serum albumins and DNA, and their antioxidant potency. The interaction of the compounds with calf-thymus DNA takes place via intercalation. The ability of the complexes to cleave pBR322 plasmid DNA at the concentration of 500 µM is rather low. The complexes demonstrated tight and reversible binding to human and bovine serum albumins and the binding site of bovine serum albumin was also examined. In order to assess the antioxidant activity of the compounds, the in vitro scavenging activity towards free radicals, namely 1,1-diphenyl-picrylhydrazyl and 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid), and their ability to reduce H2O2 were studied.
Asunto(s)
Antiinflamatorios no Esteroideos , Cobalto , Complejos de Coordinación , ADN , Ácido Mefenámico , Antiinflamatorios no Esteroideos/química , Antiinflamatorios no Esteroideos/farmacología , Cobalto/química , Complejos de Coordinación/química , Complejos de Coordinación/farmacología , Complejos de Coordinación/síntesis química , Humanos , ADN/química , ADN/metabolismo , Bovinos , Animales , Ácido Mefenámico/química , Ácido Mefenámico/farmacología , Antioxidantes/química , Antioxidantes/farmacología , Diflunisal/química , Diflunisal/farmacología , Ácido Meclofenámico/química , Ácido Meclofenámico/farmacología , Cristalografía por Rayos X , Albúmina Sérica Bovina/química , Albúmina Sérica Bovina/metabolismo , Diclofenaco/química , Diclofenaco/farmacología , Naproxeno/química , Naproxeno/farmacología , ortoaminobenzoatos/química , ortoaminobenzoatos/farmacologíaRESUMEN
Epigenetics is brought to RNA, introducing a new dimension to gene expression regulation. Among numerous RNA modifications, N6-methyladenosine (m6A) is an abundant internal modification on eukaryote mRNA first identified in the 1970s. However, the significance of m6A modification in mRNA had been long neglected until the fat mass and obesity-associated (FTO) enzyme was identified as the first m6A demethylase almost 40 years later. The m6A modification influences nearly every step of RNA metabolism and thus broadly affects gene expression at multiple levels, playing a critical role in many biological processes, including cancer progression, metastasis, and immune evasion. The m6A level is dynamically regulated by RNA epigenetic machinery comprising methyltransferases such as methyltransferase-like protein 3 (METTL3), demethylases FTO and AlkB human homologue 5 (ALKBH5), and multiple reader proteins. The understanding of the biology of RNA epigenetics and its translational drug discovery is still in its infancy. It is essential to further develop chemical probes and lead compounds for an in-depth investigation into m6A biology and the translational discovery of anticancer drugs targeting m6A modifying oncogenic proteins.In this Account, we present our work on the development of chemical inhibitors to regulate m6A in mRNA by targeting the FTO demethylase, and the elucidation of their mode of action. We reported rhein to be the first substrate competitive FTO inhibitor. Due to rhein's poor selectivity, we identified meclofenamic acid (MA) that selectively inhibits FTO compared with ALKBH5. Based on the structural complex of MA bound with FTO, we designed MA analogs FB23-2 and Dac51, which exhibit significantly improved activities compared with MA. For example, FB23-2 is specific to FTO inhibition in vitro among over 400 other oncogenic proteins, including kinases, proteases, and DNA and histone epigenetic proteins. Mimicking FTO depletion, FB23-2 promotes the differentiation/apoptosis of human acute myeloid leukemia (AML) cells and inhibits the progression of primary cells in xenotransplanted mice. Dac51 treatment impairs the glycolytic activity of tumor cells and restores the function of CD8+ T cells, thereby inhibiting the growth of solid tumors in vivo. These FTO inhibitors were and will continue to be used as probes to promote biological studies of m6A modification and as lead compounds to target FTO in anticancer drug discovery.Toward the end, we also include a brief review of ALKBH5 demethylase inhibitors and METTL3 methyltransferase modulators. Collectively, these small-molecule modulators that selectively target RNA epigenetic proteins will promote in-depth studies on the regulation of gene expression and potentially accelerate anticancer target discovery.
Asunto(s)
Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato , Linfocitos T CD8-positivos , Humanos , Ratones , Animales , Linfocitos T CD8-positivos/metabolismo , Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato/genética , Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato/metabolismo , Proteínas/química , ARN , ARN Mensajero/metabolismo , Ácido Meclofenámico/farmacología , MetiltransferasasRESUMEN
In a companion paper we examined whether combinations of Kv7 channel openers (Retigabine and Diclofenac; RET, DIC) could be effective modifiers of deep tissue nociceptor activity; and whether such combinations could then be optimized for use as safe analgesics for pain-like signs that developed in a rat model of GWI (Gulf War Illness) pain. In the present report, we examined the combinations of Retigabine/Meclofenamate (RET/MEC) and Meclofenamate/Diclofenac (MEC/DIC). Voltage clamp experiments were performed on deep tissue nociceptors isolated from rat DRG (dorsal root ganglion). In voltage clamp studies, a stepped voltage protocol was applied (-55 to -40 mV; Vh=-60 mV; 1500 msec) and Kv7 evoked currents were subsequently isolated by Linopirdine subtraction. MEC greatly enhanced voltage dependent conductance and produced exceptional maximum sustained currents of 6.01 ± 0.26 pA/pF (EC50: 62.2 ± 8.99 µM). Combinations of RET/MEC, and MEC/DIC substantially amplified resting currents at low concentrations. MEC/DIC also greatly improved voltage dependent conductance. In current clamp experiments, a cholinergic challenge test (Oxotremorine-M, 10 µM; OXO), associated with our GWI rat model, produced powerful action potential (AP) bursts (85 APs). Optimized combinations of RET/MEC (5 and 0.5 µM) and MEC/DIC (0.5 and 2.5 µM) significantly reduced AP discharges to 3 and 7 Aps, respectively. Treatment of pain-like ambulatory behavior in our rat model with a RET/MEC combination (5 and 0.5 mg/kg) successfully rescued ambulation deficits, but could not be fully separated from the effect of RET alone. Further development of this approach is recommended.
Asunto(s)
Dolor Crónico , Síndrome del Golfo Pérsico , Animales , Ratas , Diclofenaco/farmacología , Ganglios Espinales , Ácido Meclofenámico/farmacología , Nociceptores , Canales de PotasioRESUMEN
BACKGROUND: Glioblastoma cells assemble to a syncytial communicating network based on tumor microtubes (TMs) as ultra-long membrane protrusions. The relationship between network architecture and transcriptional profile remains poorly investigated. Drugs that interfere with this syncytial connectivity such as meclofenamate (MFA) may be highly attractive for glioblastoma therapy. METHODS: In a human neocortical slice model using glioblastoma cell populations of different transcriptional signatures, three-dimensional tumor networks were reconstructed, and TM-based intercellular connectivity was mapped on the basis of two-photon imaging data. MFA was used to modulate morphological and functional connectivity; downstream effects of MFA treatment were investigated by RNA sequencing and fluorescence-activated cell sorting (FACS) analysis. RESULTS: TM-based network morphology strongly differed between the transcriptional cellular subtypes of glioblastoma and was dependent on axon guidance molecule expression. MFA revealed both a functional and morphological demolishment of glioblastoma network architectures which was reflected by a reduction of TM-mediated intercellular cytosolic traffic as well as a breakdown of TM length. RNA sequencing confirmed a downregulation of NCAM and axon guidance molecule signaling upon MFA treatment. Loss of glioblastoma communicating networks was accompanied by a failure in the upregulation of genes that are required for DNA repair in response to temozolomide (TMZ) treatment and culminated in profound treatment response to TMZ-mediated toxicity. CONCLUSION: The capacity of TM formation reflects transcriptional cellular heterogeneity. MFA effectively demolishes functional and morphological TM-based syncytial network architectures. These findings might pave the way to a clinical implementation of MFA as a TM-targeted therapeutic approach.
Asunto(s)
Neoplasias Encefálicas , Glioblastoma , Ácido Meclofenámico/farmacología , Neoplasias Encefálicas/tratamiento farmacológico , Línea Celular Tumoral , Proliferación Celular , Glioblastoma/tratamiento farmacológico , Humanos , Técnicas In VitroRESUMEN
Gap junctions are ubiquitous within the retina, but in general, it remains to be determined whether gap junction coupling between specific cell types is sufficiently strong to mediate functionally relevant coupling via electrical synapses. From ultrastructural, tracer coupling and immunolabeling studies, there is clear evidence for gap junctions between cone bipolar cells, but it is not known if these gap junctions function as electrical synapses. Here, using whole-cell voltage-clamp recording in rat (male and female) retinal slices, we investigated whether the gap junctions of bipolar cells make a measurable contribution to the membrane properties of these cells. We measured the input resistance (RN) of bipolar cells before and after applying meclofenamic acid (MFA) to block gap junctions. In the presence of MFA, RN of ON-cone bipolar cells displayed a clear increase, paralleled by block of the electrical coupling between these cells and AII amacrine cells in recordings of coupled cell pairs. For OFF-cone and rod bipolar cells, RN did not increase in the presence of MFA. The results for rod bipolar cells are consistent with the lack of gap junctions in these cells. However, for OFF-cone bipolar cells, our results suggest that the morphologically identified gap junctions between these cells do not support a junctional conductance that is sufficient to mediate effective electrical coupling. Instead, these junctions might play a role in chemical and/or metabolic coupling between subcellular compartments.
Asunto(s)
Membrana Celular/metabolismo , Uniones Comunicantes/metabolismo , Células Bipolares de la Retina/metabolismo , Células Amacrinas/efectos de los fármacos , Células Amacrinas/metabolismo , Animales , Membrana Celular/efectos de los fármacos , Fenómenos Electrofisiológicos/efectos de los fármacos , Femenino , Uniones Comunicantes/efectos de los fármacos , Masculino , Ácido Meclofenámico/farmacología , Ratas , Células Bipolares de la Retina/efectos de los fármacos , Células Fotorreceptoras Retinianas Bastones/efectos de los fármacos , Células Fotorreceptoras Retinianas Bastones/metabolismoRESUMEN
In vertebrates, intercellular communication is largely mediated by connexins (Cx), a family of structurally related transmembrane proteins that assemble to form hemichannels (HCs) at the plasma membrane. HCs are upregulated in different brain disorders and represent innovative therapeutic targets. Identifying modulators of Cx-based HCs is of great interest to better understand their function and define new treatments. In this study, we developed automated versions of two different cell-based assays to identify new pharmacological modulators of Cx43-HCs. As HCs remain mostly closed under physiological conditions in cell culture, depletion of extracellular Ca2+ was used to increase the probability of opening of HCs. The first assay follows the incorporation of a fluorescent dye, Yo-Pro, by real-time imaging, while the second is based on the quenching of a fluorescent protein, YFPQL, by iodide after iodide uptake. These assays were then used to screen a collection of 2242 approved drugs and compounds under development. This study led to the identification of 11 candidate hits blocking Cx43-HC, active in the two assays, with 5 drugs active on HC but not on gap junction (GJ) activities. To our knowledge, this is the first screening on HC activity and our results suggest the potential of a new use of already approved drugs in central nervous system disorders with HC impairments.
Asunto(s)
Bioensayo , Conexina 43/genética , Drogas en Investigación/farmacología , Neuroglía/efectos de los fármacos , Medicamentos bajo Prescripción/farmacología , Automatización de Laboratorios , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Benzoxazoles/química , Calcio/metabolismo , Carbenoxolona/farmacología , Línea Celular Tumoral , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Conexina 43/antagonistas & inhibidores , Conexina 43/metabolismo , Colorantes Fluorescentes/química , Expresión Génica , Humanos , Yoduros/farmacología , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Ácido Meclofenámico/farmacología , Neuroglía/citología , Neuroglía/metabolismo , Compuestos de Quinolinio/química , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Imagen de Lapso de TiempoRESUMEN
AIMS: There has been an increasing trend towards the ulcerative colitis (UC) incidence worldwide. The present study aimed to explore novel biomarkers and potential therapeutic agents for UC. MATERIALS AND METHODS: Differentially expressed genes (DEGs) among UC and healthy control samples were identified by GEO2R online tool. Functional analysis was performed and protein-protein interaction networks were constructed. The hub genes were explored by Cytoscape, and quantitative real-time-PCR (qRT-PCR) was used to valid their expression in clinical samples. ImmuCellAI was utilized to analyze the fraction of 24 types of immune cells. The L1000 platform was applied to determine potential agents for UC treatment. The dextran sulfate sodium (DSS)-induced colitis model was used to identify the therapeutic effect of meclofenamic acid. KEY FINDINGS: A total of 270 DEGs were identified among UC and healthy control samples. Functional analysis indicated that the DEGs were primarily enriched in several immune response and digestion pathways. A proportion of 18 immune-cell types was found to be significantly altered between UC and healthy control samples. 10 compounds were predicted to have therapeutic potentials for treating UC. Among them, we selected meclofenamic acid to identify its therapeutic effect on UC treatment by animal experiments. SIGNIFICANCE: The current study comprehensively analyzed the DEGs and immune infiltration in UC, as well as screened for potential agents for UC treatment.
Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Colitis Ulcerosa/tratamiento farmacológico , Colitis Ulcerosa/inmunología , Ácido Meclofenámico/farmacología , Transcriptoma , Animales , Estudios de Casos y Controles , Colitis/inducido químicamente , Colitis/tratamiento farmacológico , Colitis Ulcerosa/genética , Sulfato de Dextran/toxicidad , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Femenino , Marcadores Genéticos/genética , Humanos , Ratones Endogámicos C57BL , Mapas de Interacción de Proteínas/genéticaRESUMEN
Malignant glioma constitutes one of the fatal primary brain tumors in adults. Such poor prognosis calls for a better understanding of cancer-related signaling pathways of this disease. Here we elucidate a MYC-miRNA-MXI1 feedback loop that regulates proliferation and tumorigenesis in glioma. MYC suppressed MXI1 expression via microRNA-155 (miR-155) and the microRNA-23aâ¼27aâ¼24-2 cluster (miR-23a cluster), whereas MXI1, in turn, inhibited MYC expression by binding to its promoter. Overexpression of miR-155 and the miR-23a cluster promoted tumorigenesis in U87 glioma cells. Furthermore, fat mass and obesity-associated protein (FTO), an N6-methyladenosine (m6A) RNA demethylase, regulated the loop by targeting MYC. The ethyl ester form of meclofenamic acid (MA2) inhibited FTO and enhanced the effect of the chemotherapy drug temozolomide on suppressing proliferation of glioma cells and negatively regulated the loop. These data collectively highlight a key regulatory circuit in glioma and provide potential targets for clinical treatment. SIGNIFICANCE: These findings elucidate a novel feedback loop that regulates proliferation in glioma and can be targeted via inhibition of FTO to enhance the efficacy of temozolomide.
Asunto(s)
Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato/antagonistas & inhibidores , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Neoplasias Encefálicas/metabolismo , Glioma/metabolismo , MicroARNs/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato/metabolismo , Animales , Antineoplásicos Alquilantes/farmacología , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Inhibidores de la Ciclooxigenasa/farmacología , Sinergismo Farmacológico , Retroalimentación Fisiológica/efectos de los fármacos , Retroalimentación Fisiológica/fisiología , Femenino , Glioma/tratamiento farmacológico , Glioma/patología , Humanos , Ácido Meclofenámico/farmacología , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Temozolomida/farmacologíaRESUMEN
The role of RNA methylation on the sixth N atom of adenylate (m6A) in acute kidney injury (AKI) is unknown. FTO (fat mass and obesity-associated protein) reverses the m6A modification in cisplatin-induced AKI. Here, we aimed to determine FTO's role in AKI. We induced AKI in c57BL/6 mice by intraperitoneal cisplatin injection and treated the animal with vehicle or an FTO inhibitor meclofenamic acid (MA) for 3 days. Moreover, as an in vitro model, human kidney proximal tubular cells (HK2 cells) were treated with cisplatin. We found that the cisplatin treatment reduces FTO expression and increases m6A levels in vivo and in vitro MA aggravated renal damage and increased apoptosis in cisplatin-treated kidneys, phenotypes that were correlated with reduced FTO expression and increased m6A levels. Moreover, MA promoted apoptosis in cisplatin-treated HK2 cells, which was correlated with the reduced FTO expression and increased m6A in HK2 cells. FTO protein overexpression reduced m6A levels and inhibited apoptosis in cisplatin-treated HK2 cells and also blocked the MA-induced increase in m6A levels and apoptosis rates. In agreement, overexpression of the m6A-generating methyltransferase-like 3 and 14 (METTL3 and METTL14) or siRNA-mediated FTO knockdown promoted apoptosis and enhanced m6A levels in cisplatin-treated HK2 cells. MA increased p53 mRNA and protein levels in AKI both in vitro and in vivo, and FTO overexpression reduced p53 expression and reversed the MA-induced p53 increase in AKI. In conclusion, reduced renal FTO expression in cisplatin-induced AKI increases RNA m6A levels and aggravates renal damages.
Asunto(s)
Lesión Renal Aguda/patología , Tejido Adiposo/efectos de los fármacos , Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato/metabolismo , Cisplatino/farmacología , Ácido Meclofenámico/farmacología , ARN/química , ARN/metabolismo , Lesión Renal Aguda/inducido químicamente , Lesión Renal Aguda/genética , Lesión Renal Aguda/metabolismo , Tejido Adiposo/metabolismo , Tejido Adiposo/patología , Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato/deficiencia , Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato/genética , Animales , Apoptosis/efectos de los fármacos , Línea Celular , Sinergismo Farmacológico , Regulación de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Many functions of glial cells depend on the formation of selective glial networks mediated by gap junctions formed by members of the connexin family. Olfactory ensheathing cells (OECs) are specialized glia associated with olfactory sensory neuron axons. Like other glia, they form selective networks, however, the connexins that support OEC connectivity in vivo have not been identified. We used an in vivo mouse model to selectively delete candidate connexin genes with temporal control from OECs and address the physiological consequences. Using this model, we effectively abolished the expression of connexin 43 (Cx43) in OECs in both juvenile and adult mice. Cx43-deleted OECs exhibited features consistent with the loss of gap junctions including reduced membrane conductance, largely reduced sensitivity to the gap junction blocker meclofenamic acid and loss of dye coupling. This indicates that Cx43, a typically astrocytic connexin, is the main connexin forming functional channels in OECs. Despite these changes in functional properties, the deletion of Cx43 deletion did not alter the density of OECs. The strategy used here may prove useful to delete other candidate genes to better understand the functional roles of OECs in vivo.
Asunto(s)
Conexina 43/fisiología , Uniones Comunicantes/fisiología , Técnicas de Inactivación de Genes , Neuroglía/fisiología , Bulbo Olfatorio/citología , Envejecimiento/metabolismo , Animales , Conexina 43/deficiencia , Conexina 43/genética , Cruzamientos Genéticos , Femenino , Uniones Comunicantes/efectos de los fármacos , Genes Reporteros , Genes Sintéticos , Integrasas/genética , Masculino , Ácido Meclofenámico/farmacología , Ratones , Ratones Noqueados , Proteína Proteolipídica de la Mielina/genética , Bulbo Olfatorio/metabolismo , Técnicas de Placa-Clamp , Tamoxifeno/farmacologíaRESUMEN
Oligodendrocyte precursor cells (OPCs) proliferation and differentiation are essential for remyelination after white matter injury. Astrocytes could promote oligodendrogenesis after white matter damage whereas the underlying mechanisms are unknown. In this study, the role of astrocytic connexin43 (Cx43) hemichannels involved in OPC proliferation and differentiation in chronic hypoxia was evaluated. In an astrocyte-OPC co-culture chronic hypoxia model, OPCs became proliferative but failed to mature into oligodendrocytes. Application of astrocytic Cx43 blockers attenuated astrocyte activation, suppressed Cx43 hemichannel uptake activity and glutamate release induced by hypoxia, as well as improved OPC differentiation. Moreover, AMPA but not NMDA glutamate receptor antagonist rescued OPC differentiation in hypoxia. In conclusion, these findings suggested that astrocytic Cx43 hemichannel inhibition could potentially improve OPC maturation by attenuating AMPAR-mediated glutamate signaling. Astrocytic Cx43 hemichannels could serve as a potential therapeutic target for remyelination after chronic hypoxia.
Asunto(s)
Conexina 43/antagonistas & inhibidores , Neurogénesis , Células Precursoras de Oligodendrocitos/efectos de los fármacos , Oxígeno/metabolismo , Animales , Astrocitos/citología , Astrocitos/metabolismo , Carbenoxolona/farmacología , Hipoxia de la Célula , Proliferación Celular , Células Cultivadas , Ácido Meclofenámico/farmacología , Ratones , Ratones Endogámicos C57BL , Células Precursoras de Oligodendrocitos/citología , Células Precursoras de Oligodendrocitos/metabolismo , Oligodendroglía/citología , Oligodendroglía/metabolismo , Receptores AMPA/antagonistas & inhibidores , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidoresRESUMEN
Coherent spike activity occurs between widely separated retinal ganglion cells (RGCs) in response to a large, contiguous object, but not to disjointed objects. Since the large spatial separation between the RGCs precludes common excitatory inputs from bipolar cells, the mechanism underlying this long-range coherence remains unclear. Here, we show that electrical coupling between RGCs and polyaxonal amacrine cells in mouse retina forms the synaptic mechanism responsible for long-range coherent activity in the retina. Pharmacological blockade of gap junctions or genetic ablation of connexin 36 (Cx36) subunits eliminates the long-range correlated spiking between RGCs. Moreover, we find that blockade of gap junctions or ablation of Cx36 significantly reduces the ability of mice to discriminate large, global objects from small, disjointed stimuli. Our results indicate that synchronous activity of RGCs, derived from electrical coupling with amacrine cells, encodes information critical to global object perception.
Asunto(s)
Células Amacrinas/fisiología , Sinapsis Eléctricas/fisiología , Retina/fisiología , Células Ganglionares de la Retina/fisiología , Percepción Visual/fisiología , Células Amacrinas/citología , Animales , Conexinas/genética , Conexinas/fisiología , Sinapsis Eléctricas/efectos de los fármacos , Sinapsis Eléctricas/genética , Inyecciones Intravítreas , Aprendizaje por Laberinto , Ácido Meclofenámico/farmacología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Animales , Retina/citología , Retina/efectos de los fármacos , Células Ganglionares de la Retina/citología , Percepción Visual/efectos de los fármacos , Proteína delta-6 de Union ComunicanteRESUMEN
PURPOSE: Retinal dystrophy through outer photoreceptor cell death affects 1 in 2,500 people worldwide with severe impairment of vision in advanced stages of the disease. Optogenetic strategies to restore visual function to animal models of retinal degeneration by introducing photopigments to neurons spared degeneration in the inner retina have been explored, with variable degrees of success. It has recently been shown that the non-steroidal anti-inflammatory and non-selective gap-junction blocker meclofenamic acid (MFA) can enhance the visual responses produced by an optogenetic actuator (channelrhodopsin) expressed in retinal ganglion cells (RGCs) in the degenerate retina. Here, we set out to determine whether MFA could also enhance photoreception by another optogenetic strategy in which ectopic human rod opsin is expressed in ON bipolar cells. METHODS: We used in vitro multielectrode array (MEA) recordings to characterize the light responses of RGCs in the rd1 mouse model of advanced retinal degeneration following intravitreal injection of an adenoassociated virus (AAV2) driving the expression of human rod opsin under a minimal grm6 promoter active in ON bipolar cells. RESULTS: We found treated retinas were light responsive over five decades of irradiance (from 1011 to 1015 photons/cm2/s) with individual RGCs covering up to four decades. Application of MFA reduced the spontaneous firing rate of the visually responsive neurons under light- and dark-adapted conditions. The change in the firing rate produced by the 2 s light pulses was increased across all intensities following MFA treatment, and there was a concomitant increase in the signal to noise ratio for the visual response. Restored light responses were abolished by agents inhibiting glutamatergic or gamma-aminobutyric acid (GABA)ergic signaling in the MFA-treated preparation. CONCLUSIONS: These results confirm the potential of MFA to inhibit spontaneous activity and enhance the signal to noise ratio of visual responses in optogenetic therapies to restore sight.
Asunto(s)
Ácido Meclofenámico/farmacología , Opsinas de Bastones/metabolismo , Relación Señal-Ruido , Vías Visuales/efectos de los fármacos , Vías Visuales/fisiología , Potenciales de Acción/efectos de los fármacos , Adaptación Ocular/efectos de los fármacos , Animales , Humanos , Ratones , Células Ganglionares de la Retina/efectos de los fármacos , Células Ganglionares de la Retina/metabolismoRESUMEN
RNA modifications play critical roles in important biological processes. However, the functions of N6-methyladenosine (m6A) mRNA modification in cancer biology and cancer stem cells remain largely unknown. Here, we show that m6A mRNA modification is critical for glioblastoma stem cell (GSC) self-renewal and tumorigenesis. Knockdown of METTL3 or METTL14, key components of the RNA methyltransferase complex, dramatically promotes human GSC growth, self-renewal, and tumorigenesis. In contrast, overexpression of METTL3 or inhibition of the RNA demethylase FTO suppresses GSC growth and self-renewal. Moreover, inhibition of FTO suppresses tumor progression and prolongs lifespan of GSC-grafted mice substantially. m6A sequencing reveals that knockdown of METTL3 or METTL14 induced changes in mRNA m6A enrichment and altered mRNA expression of genes (e.g., ADAM19) with critical biological functions in GSCs. In summary, this study identifies the m6A mRNA methylation machinery as promising therapeutic targets for glioblastoma.
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Adenosina/análogos & derivados , Carcinogénesis/patología , Autorrenovación de las Células , Glioblastoma/patología , Células Madre Neoplásicas/patología , ARN/metabolismo , Adenosina/metabolismo , Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato/antagonistas & inhibidores , Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato/metabolismo , Animales , Secuencia de Bases , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Carcinogénesis/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Proliferación Celular/efectos de los fármacos , Autorrenovación de las Células/efectos de los fármacos , Autorrenovación de las Células/genética , Progresión de la Enfermedad , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Glioblastoma/genética , Humanos , Ácido Meclofenámico/farmacología , Metilación , Metiltransferasas/metabolismo , Ratones , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
A subpopulation of olivary pretectal nucleus (OPN) neurons fire action potentials in a rhythmic manner with an eruption of activity occurring approximately every two minutes. These infra-slow oscillations depend critically on functional retinal input and are subject to modulation by light. Interestingly, the activity of photoreceptors is necessary for the emergence of the rhythm and while classic photoreceptors (rods and cones) are necessary in darkness and dim light, melanopsin photoreceptors are indispensable in bright light. Using pharmacological and electrophysiological approaches in vivo, we show that also blocking retinal gap junctions (GJs), which are expressed by multitude of retinal cells, leads to the disruption of oscillatory activity in the rat OPN. Intravitreal injection of carbenoxolone (CBX) quenched oscillations in a concentration-dependent manner with 1mM being ineffective, 5mM showing partial and 20mM showing complete effectiveness in disrupting oscillations. Moreover, the most effective CBX concentration depressed cone-mediated light-induced responses of oscillatory neurons suggesting that CBX is also acting on targets other than GJs. In contrast, intravitreal injection of meclofenamic acid (MFA, 20mM) led to disruption of the rhythm but did not interfere with cone-mediated light-induced responses of oscillatory neurons, implying that MFA is more specific toward GJs than CBX, as suggested before. We conclude that electrical coupling between various types of retinal cells and resultant synchronous firing of retinal ganglion cells is necessary for the generation of infra-slow oscillations in the rat OPN.
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Uniones Comunicantes/fisiología , Periodicidad , Área Pretectal/fisiología , Retina/fisiología , Animales , Carbenoxolona/farmacología , Relación Dosis-Respuesta a Droga , Uniones Comunicantes/efectos de los fármacos , Inyecciones Intravítreas , Masculino , Ácido Meclofenámico/farmacología , Vías Nerviosas/efectos de los fármacos , Vías Nerviosas/fisiología , Fármacos del Sistema Nervioso Periférico/farmacología , Ratas , Ratas Wistar , Retina/efectos de los fármacos , Visión Ocular/efectos de los fármacos , Visión Ocular/fisiologíaRESUMEN
Brain metastasis represents a substantial source of morbidity and mortality in various cancers, and is characterized by high resistance to chemotherapy. Here we define the role of the most abundant cell type in the brain, the astrocyte, in promoting brain metastasis. We show that human and mouse breast and lung cancer cells express protocadherin 7 (PCDH7), which promotes the assembly of carcinoma-astrocyte gap junctions composed of connexin 43 (Cx43). Once engaged with the astrocyte gap-junctional network, brain metastatic cancer cells use these channels to transfer the second messenger cGAMP to astrocytes, activating the STING pathway and production of inflammatory cytokines such as interferon-α (IFNα) and tumour necrosis factor (TNF). As paracrine signals, these factors activate the STAT1 and NF-κB pathways in brain metastatic cells, thereby supporting tumour growth and chemoresistance. The orally bioavailable modulators of gap junctions meclofenamate and tonabersat break this paracrine loop, and we provide proof-of-principle that these drugs could be used to treat established brain metastasis.
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Astrocitos/metabolismo , Neoplasias Encefálicas/patología , Neoplasias Encefálicas/secundario , Uniones Comunicantes/metabolismo , Nucleótidos Cíclicos/metabolismo , Animales , Astrocitos/citología , Astrocitos/efectos de los fármacos , Benzamidas/farmacología , Benzamidas/uso terapéutico , Benzopiranos/farmacología , Benzopiranos/uso terapéutico , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/metabolismo , Neoplasias de la Mama/patología , Cadherinas/metabolismo , Línea Celular Tumoral , Técnicas de Cocultivo , Conexina 43/metabolismo , Resistencia a Antineoplásicos , Femenino , Uniones Comunicantes/efectos de los fármacos , Humanos , Inmunidad Innata , Interferón-alfa/metabolismo , Neoplasias Pulmonares/patología , Ácido Meclofenámico/farmacología , Ácido Meclofenámico/uso terapéutico , Proteínas de la Membrana/metabolismo , Ratones , FN-kappa B/metabolismo , Comunicación Paracrina/efectos de los fármacos , Protocadherinas , Factor de Transcripción STAT1/metabolismo , Factores de Necrosis Tumoral/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
STUDY OBJECTIVES: Modafinil is a non-amphetaminic wake-promoting compound used as therapy against sleepiness and narcolepsy. Its mode of action is complex, but modafinil has been recently proposed to act as a cellular-coupling enhancer in glial cells, through modulation of gap junctions constituted by connexins. The present study investigated in mice the impact of connexins on the effects of modafinil using connexin inhibitors. METHODS: Modafinil was administered alone or combined with inhibitors of astrocyte connexin, meclofenamic acid, or flecainide, respectively, acting on Cx30 and Cx43. Sleep-wake states were monitored in wild-type and narcoleptic orexin knockout mice. A spontaneous alternation task was used to evaluate working memory in wild-type mice. The effects of the compounds on astroglial intercellular coupling were determined using dye transfer in acute cortical slices. RESULTS: Meclofenamic acid had little modulation on the effects of modafinil, but flecainide enhanced the wake-promoting and pro-cognitive effects of modafinil. Co-administration of modafinil/flecainide resulted in a marked decrease in the number and duration of direct transitions to rapid eye movement sleep, which are characteristic of narcoleptic episodes in orexin knockout mice. Furthermore, modafinil enhanced the connexin-mediated astroglial cell coupling, whereas flecainide reduced it. Finally, this modafinil-induced effect was reversed by co-administration with flecainide. CONCLUSIONS: Our study indicates that flecainide impacts the pharmacological effects of modafinil, likely through the normalization of Cx30-dependent gap junctional coupling in astroglial networks. The enhancement of the wake-promoting, behavioral, and cognitive outcomes of modafinil demonstrated here with flecainide would open new perspectives in the management of sleep disorders such as narcolepsy. COMMENTARY: A commentary on this article appears in this issue on page 1175.
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Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Compuestos de Bencidrilo/farmacología , Conexina 43/metabolismo , Conexinas/metabolismo , Animales , Astrocitos/citología , Compuestos de Bencidrilo/administración & dosificación , Conexina 30 , Conexina 43/antagonistas & inhibidores , Conexinas/antagonistas & inhibidores , Modelos Animales de Enfermedad , Flecainida/farmacología , Masculino , Ácido Meclofenámico/farmacología , Ratones , Ratones Noqueados , Modafinilo , Narcolepsia/tratamiento farmacológico , Narcolepsia/genética , Narcolepsia/patología , Narcolepsia/fisiopatología , Orexinas/deficiencia , Orexinas/genética , Sueño/efectos de los fármacos , Vigilia/efectos de los fármacosRESUMEN
The use of nonsteroidal anti-inflammatory drugs (NSAIDs) like meclofenamate sodium (MS), used to reduce pain, has been associated with an increased risk of cardiovascular disease (CVD). Naproxen (NAP), another NSAID, is not associated with increased risk of CVD. The molecular mechanism(s) by which NSAIDs induce CVD is unknown. We investigated the effects of MS and NAP on protein homeostasis and cardiotoxicity in rat cardiac H9c2 cells and murine neonatal cardiomyocytes. MS, but not NAP, significantly inhibited proteasome activity and reduced cardiac cell viability at pharmacological levels found in humans. Although proteasome subunit gene and protein expression were unaffected by NSAIDs, MS treated cell lysates showed higher 20S proteasome content, while purified proteasomes from MS treated cells had lower proteasome activity and higher levels of oxidized subunits than proteasomes from control cells. Addition of exogenous proteasome to MS treated cells improved cell viability. Both MS and NAP increased ROS production, but the rate of ROS production was greater in MS than in NAP treated cells. The ROS production is likely from mitochondria, as MS inhibited mitochondrial Complexes I and III, major sources of ROS, while NAP inhibited Complex I. MS also impaired mitochondrial membrane potential while NAP did not. Antioxidants were able to prevent the reduced cell viability caused by MS treatment. These results suggest that NSAIDs induce cardiotoxicity by a ROS dependent mechanism involving mitochondrial and proteasome dysfunction and may explain why some NSAIDs should not be given to patients for long periods.
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Antiinflamatorios no Esteroideos/farmacología , Ácido Meclofenámico/farmacología , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Naproxeno/farmacología , Complejo de la Endopetidasa Proteasomal/metabolismo , Animales , Supervivencia Celular/efectos de los fármacos , Complejo II de Transporte de Electrones/metabolismo , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Ratones , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Oxidación-Reducción , Prostaglandina-Endoperóxido Sintasas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Proteínas Ubiquitinadas/metabolismoRESUMEN
Mammalian pituitaries exhibit a high degree of intercellular coordination; this enables them to mount large-scale coordinated responses to various physiological stimuli. This type of communication has not been adequately demonstrated in teleost pituitaries, which exhibit direct hypothalamic innervation and expression of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) in distinct cell types. We found that in two fish species, namely tilapia and zebrafish, LH cells exhibit close cell-cell contacts and form a continuous network throughout the gland. FSH cells were more loosely distributed but maintained some degree of cell-cell contact by virtue of cytoplasmic processes. These anatomical differences also manifest themselves at the functional level as evidenced by the effect of gap-junction uncouplers on gonadotropin release. These substances abolished the LH response to gonadotropin-releasing hormone stimulation but did not affect the FSH response to the same stimuli. Dye transfer between neighboring LH cells provides further evidence for functional coupling. The two gonadotropins were also found to be differently packaged within their corresponding cell types. Our findings highlight the evolutionary origin of pituitary cell networks and demonstrate how the different levels of cell-cell coordination within the LH and FSH cell populations are reflected in their distinct secretion patterns.