RESUMEN

Familial Alzheimer's disease (FAD) is a complex and multifactorial neurodegenerative disorder for which no curative therapies are yet available. Indeed, no single medication or intervention has proven fully effective thus far. Therefore, the combination of multitarget agents has been appealing as a potential therapeutic approach against FAD. Here, we investigated the potential of combining tramiprosate (TM), curcumin (CU), and the JNK inhibitor SP600125 (SP) as a treatment for FAD. The study analyzed the individual and combined effects of these two natural agents and this pharmacological inhibitor on the accumulation of intracellular amyloid beta iAß; hyperphosphorylated protein TAU at Ser202/Thr205; mitochondrial membrane potential (ΔΨm); generation of reactive oxygen species (ROS); oxidized protein DJ-1; proapoptosis proteins p-c-JUN at Ser63/Ser73, TP53, and cleaved caspase 3 (CC3); and deficiency in acetylcholine (ACh)-induced transient Ca2+ influx response in cholinergic-like neurons (ChLNs) bearing the mutation I416T in presenilin 1 (PSEN1 I416T). We found that single doses of TM (50 µM), CU (10 µM), or SP (1 µM) were efficient at reducing some, but not all, pathological markers in PSEN 1 I416T ChLNs, whereas a combination of TM, CU, and SP at a high (50, 10, 1 µM) concentration was efficient in diminishing the iAß, p-TAU Ser202/Thr205, DJ-1Cys106-SO3, and CC3 markers by -50%, -75%, -86%, and -100%, respectively, in PSEN1 I417T ChLNs. Although combinations at middle (10, 2, 0.2) and low (5, 1, 0.1) concentrations significantly diminished p-TAU Ser202/Thr205, DJ-1Cys106-SO3, and CC3 by -69% and -38%, -100% and -62%, -100% and -62%, respectively, these combinations did not alter the iAß compared to untreated mutant ChLNs. Moreover, a combination of reagents at H concentration was able to restore the dysfunctional ACh-induced Ca2+ influx response in PSEN 1 I416T. Our data suggest that the use of multitarget agents in combination with anti-amyloid (TM, CU), antioxidant (e.g., CU), and antiapoptotic (TM, CU, SP) actions might be beneficial for reducing iAß-induced ChLN damage in FAD.

Asunto(s)

Enfermedad de Alzheimer , Antracenos , Curcumina , Presenilina-1 , Taurina/análogos & derivados , Curcumina/farmacología , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Presenilina-1/genética , Presenilina-1/metabolismo , Antracenos/farmacología , Animales , Especies Reactivas de Oxígeno/metabolismo , Ratones , Péptidos beta-Amiloides/metabolismo , Humanos , Proteínas tau/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neuronas/patología , Potencial de la Membrana Mitocondrial/efectos de los fármacos

RESUMEN

Hypericin (Hy) is a hydrophobic photosensitizer used in photodynamic therapy for cancer therapeutic. In this study, Hy-loaded oil-in-water (O/W) nanoemulsions (NEs) were produced by the ultrasonication method combing different biocompatible oils and surfactants to enhance Hy aqueous solubility and bioavailability. Experimental parameters were optimized by the characterization of droplet size, zeta potential, and physicochemical properties. In vitro studies based on the release profile, cytotoxicity, cell morphology, and Hy intracellular accumulation were assayed. Hy at 100 mg L-1 was incorporated into the low viscosity (~0.005 Pa s) NEs with spherical droplets averaging 20-40 nm in size and polydispersity index <0.02. Hy release from the NE was significantly higher (4-fold) than its suspension (p < 0.001). The NEs demonstrated good physical stability during storage at 5 °C for at least six months. The Hy-loaded NEs exhibited an IC50 value 6-fold lower than Hy suspension during PDT against breast cancer cell lines (MCF-7). Cell microscopy imaging confirmed the increased cytotoxic effects of Hy-loaded NEs, showing damaged and apoptotic cells. Confocal laser scanning microscopy evidenced greater Hy delivery through NE into MCF-7 cells followed by improved intracellular ROS generation. Our results suggest that the Hy-loaded NEs can improve hypericin efficacy and assist Hy-PDT's preclinical development as a cancer treatment.

Asunto(s)

Antracenos/química , Emulsiones/química , Nanoestructuras/química , Perileno/análogos & derivados , Fotoquimioterapia/métodos , Fármacos Sensibilizantes a Radiaciones/química , Antracenos/metabolismo , Antracenos/farmacología , Apoptosis/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Liberación de Fármacos/efectos de la radiación , Estabilidad de Medicamentos , Humanos , Luz , Células MCF-7 , Aceites/química , Perileno/química , Perileno/metabolismo , Perileno/farmacología , Fármacos Sensibilizantes a Radiaciones/metabolismo , Fármacos Sensibilizantes a Radiaciones/farmacología , Especies Reactivas de Oxígeno/metabolismo , Sonicación , Temperatura , Agua/química

RESUMEN

In order to analyze whether the marine macroalga Ulva lactuca can absorb and metabolize anthracene (ANT), the alga was cultivated with 5 µM ANT for 0-72 h, and the level of ANT was detected in the culture medium, and in the alga. The level of ANT rapidly decreased in the culture medium reaching a minimal level at 6 h, and rapidly increased in the alga reaching a maximal level at 12 h and then decreased to reach a minimal level at 48 h of culture. In addition, ANT induced an increase in hydrogen peroxide that remained until 72 h and a higher increase in superoxide anions that reach a maximal level at 24 h and remained unchanged until 72 h, indicating that ANT induced an oxidative stress condition. ANT induced an increase in lipoperoxides that reached a maximal level at 24 h and decreased at 48 h indicating that oxidative stress caused membrane damage. The activity of antioxidant enzymes SOD, CAT, AP, GR and GP increased in the alga treated with ANT whereas DHAR remained unchanged. The level of transcripts encoding these antioxidant enzymes increased and those encoding DHAR did not change. Inhibitors of monooxygenases, dioxygenases, polyphenol oxidases, glutathione-S-transferases and sulfotransferases induced an increase in the level of ANT in the alga cultivated for 24 h. These results strongly suggest that ANT is rapidly absorbed and metabolized in U. lactuca and the latter involves Phase I and II metabolizing enzymes.

Asunto(s)

Antracenos/farmacología , Antioxidantes/farmacología , Enzimas/metabolismo , Estrés Oxidativo/efectos de los fármacos , Ulva/metabolismo , Activación Enzimática , Peróxido de Hidrógeno/farmacología , Ulva/enzimología

RESUMEN

Karwinskia genus consists of shrubs and small trees. Four toxic compounds have been isolated from Karwinskia plants, which were typified as dimeric anthracenones and named T496, T514, T516, and T544. Moreover, several related compounds have been isolated and characterized. Here we review the toxicity of the fruit of Karwinskia plants when ingested (accidentally or experimentally), as well as the toxicity of its isolated compounds. Additionally, we analyze the probable antineoplastic effect of T514. Toxins cause damage mainly to nervous system, liver, lung, and kidney. The pathophysiological mechanism has not been fully understood but includes metabolic and structural alterations that can lead cells to apoptosis or necrosis. T514 has shown selective toxicity in vitro against human cancer cells. T514 causes selective and irreversible damage to peroxisomes; for this reason, it was renamed peroxisomicine A1 (PA1). Since a significant number of malignant cell types contain fewer peroxisomes than normal cells, tumor cells would be more easily destroyed by PA1 than healthy cells. Inhibition of topoisomerase II has also been suggested to play a role in the effect of PA1 on malignant cells. More research is needed, but the evidence obtained so far indicates that PA1 could be an effective anticancer agent.

Asunto(s)

Antracenos/farmacología , Antineoplásicos Fitogénicos/farmacología , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos/etiología , Karwinskia/química , Neoplasias/tratamiento farmacológico , Antracenos/efectos adversos , Antineoplásicos Fitogénicos/efectos adversos , Humanos , Neoplasias/patología

RESUMEN

Peroxisomicine A1 (PA1) is a potential antineoplastic agent with high and selective toxicity toward peroxisomes of tumor cells. Pexophagy is a selective autophagy process that degrades damaged peroxisomes; this process has been studied mainly in methylotrophic yeasts. There are two main modes of pexophagy in yeast: macropexophagy and micropexophagy. Previous studies showed that peroxisomes damaged by a prolonged exposition to PA1 are eliminated by macropexophagy. In this work, Candida boidinii was grown in methanol-containing media, and PA1 was added to the cultures at 2 µg/mL after they reached the mid-exponential growth phase. Samples were taken at 5, 10, 15, 20, and 25 min after the addition of PA1 and processed for ultrastructural analysis. Typical morphological characteristics of micropexophagy were observed: the direct engulfment of peroxisomes by the vacuolar membrane and the presence of the micropexophagic membrane apparatus (MIPA), which mediates the fusion between the opposing tips of the vacuole to complete sequestration of peroxisomes from the cytosol. In conclusion, here we report that, in addition to macropexophagy, peroxisomes damaged by PA1 can be eliminated by micropexophagy. This information is useful to deepen the knowledge of the mechanism of action of PA1 and of that of pexophagy per se.

Asunto(s)

Antracenos/farmacología , Antineoplásicos/farmacología , Candida/efectos de los fármacos , Macroautofagia/efectos de los fármacos , Microautofagia/efectos de los fármacos , Peroxisomas/efectos de los fármacos , Proteínas Fúngicas/metabolismo

RESUMEN

Polycyclic aromatic hydrocarbons (PAHs) were identified as hazardous contaminants that are ubiquitous and persistent in aquatic environments, where bryophytes sensu lato (mosses, liverworts and hornworts) are frequently present. Marchantia polymorpha (Class Hepaticae; thalloid liverwort) is known to respond fast to changes in the environment; it accumulates toxic substances in its tissues due to the lack of vascular and radicular systems and a reduced or absent cuticle. The objective of the present study was to quantify the effects of increasing concentrations of anthracene (0, 50 100, 280 µM) on the germination of propagules, plant morphology and chlorophyll content index (CCI) in M. polymorpha under in vitro cultures. The results show that anthracene had no statistical effect on germination or propagula formation. However, plants exposed to anthracene for 30 days showed significantly lowered the content of chlorophyll (measured as CCI), irregular growth patterns and the induction of thalli asexual reproduction as evidenced by the production of multicellular viable propagules in gemmae cups. Results of epifluorescence microscopy also showed concomitant accumulation of anthracene in the cell walls. All of these distinctive morphological and physiological adaptive responses indicators, clearly suggest that M. polymorpha are capable of resisting high (coal tar) anthracene concentrations.

Asunto(s)

Antracenos/farmacología , Clorofila/metabolismo , Marchantia/efectos de los fármacos , Pared Celular/efectos de los fármacos , Marchantia/anatomía & histología , Marchantia/crecimiento & desarrollo , Marchantia/metabolismo , Microscopía Fluorescente

RESUMEN

Karwinskia parvifolia possesses the highest concentration levels of the anthracenone T-514 (PA1). Studies have demonstrated the induction of apoptosis by PA1 in cancer cell lines. The aim was to investigate the effects of PA1 on the apoptosis of the mouse liver in vivo and its underlying pathway. Sixty CD-1 mice were divided into three groups: untreated, vehicle, and treated with PA1. The animals were euthanized at 4, 8, 12, and 24 h post-treatment. To confirm the toxic effect of PA1 we determined the activity of catalase. Liver sections were prepared for morphological examination and for immunohistochemical evaluation of anti and pro-apoptotic markers. DNA fragmentation was detected by TUNEL assay and electrophoresis. Pre-apoptotic mitochondrial alterations and cytochrome c oxidase activity were analyzed by transmission electron microscopy. PA1 induced pre-apoptotic mitochondrial alterations, a high activity of the cytochrome oxidase, and apoptosis in hepatocytes. PA1 caused p53 over-expression and down regulation of PCNA. PA1 also increased the expression levels of the pro-apoptotic markers Bax and Bak, whereas the anti-apoptotic molecule Bcl-2 was decreased. PA1 induces apoptosis by activating the intrinsic mitochondrial apoptotic pathway. These results will be useful for studies regarding the use of PA1 as an antineoplastic agent.

Asunto(s)

Antracenos/farmacología , Apoptosis/efectos de los fármacos , Hepatocitos/efectos de los fármacos , Animales , Antracenos/aislamiento & purificación , Antracenos/toxicidad , Proteínas Reguladoras de la Apoptosis/metabolismo , Caspasa 3/metabolismo , Catalasa/metabolismo , Citocromos c/metabolismo , Hepatocitos/metabolismo , Hepatocitos/patología , Ratones , Mitocondrias/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Transducción de Señal/efectos de los fármacos , Proteína X Asociada a bcl-2/metabolismo

RESUMEN

In this work, we report on the synthesis of two new mono-alkylated tetrandrine derivatives with acridine and anthracene units, MAcT and MAnT. The compounds were fully characterized by physicochemical techniques and single-crystal X-ray diffraction analysis. In addition, both derivatives were studied as nucleotide receptors and double-stranded DNA binders in aqueous phosphate buffer at pH = 7.2 using UV-vis and fluorescence spectroscopy. According to the molecular recognition studies, MAcT and MAnT exhibit high affinity (K ∼ 105 M-1) and selectivity for ds-DNA, presumably in an intercalation mode. Finally, the anti-proliferative effects of the tetrandrine derivatives on different cancer cell lines were explored, revealing promising activities. Particularly, the mono-anthracene tetrandrine derivative MAnT showed an IC50 of 2.74 µg/mL on the HeLa cervical cancer cell line, representing a value 3.3 times smaller than that obtained for unsubstituted tetrandrine. Examination of the cytotoxic effects on the HeLa cell line by inverted microscopy suggests that the cell death mechanism consists basically in apoptosis. The molecular modelling of three ds-DNA-MAcT complexes, suggested that the macrocycles may use an intercalation binding mode towards DNA. MAcT is predicted to bind into the major groove of the ds-DNA providing non-covalent interactions such as electrostatic, van der Waals and hydrophobic interactions that lead to selectivity. Overall experimental data supports the mode of action of MAnT and MAcT as cytotoxic compounds against cancer cell lines via a DNA interaction mechanism.

Asunto(s)

Acridinas/química , Antracenos/química , Bencilisoquinolinas/química , Compuestos Macrocíclicos/síntesis química , Células A549 , Acridinas/síntesis química , Acridinas/farmacología , Antracenos/síntesis química , Antracenos/farmacología , Apoptosis/efectos de los fármacos , Bencilisoquinolinas/síntesis química , Bencilisoquinolinas/farmacología , Sitios de Unión , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , ADN/química , ADN/metabolismo , Células HeLa , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Sustancias Intercalantes/síntesis química , Sustancias Intercalantes/química , Sustancias Intercalantes/farmacología , Compuestos Macrocíclicos/química , Compuestos Macrocíclicos/farmacología , Simulación del Acoplamiento Molecular , Conformación de Ácido Nucleico , Electricidad Estática

RESUMEN

In Cystic Fibrosis (CF), the impairment of the CFTR channel activity leads to a variety of alterations, including differential gene expression. However, the CFTR signaling mechanisms remain unclear. Recently, culturing IB3-1 CF cells under different intracellular Cl- concentrations ([Cl-]i), we observed several Cl--dependent genes and further characterized one of them as RPS27. Thus, we hypothesized that Cl- might act as a signaling effector for CFTR signaling. Here, to test this idea, we study RPS27 expression in T84 cells modulating the CFTR activity by using CFTR inhibitors. First, we observed that incubation of T84 cells with increasing concentrations of the CFTR inhibitors CFTR(inh)-172 or GlyH-101 determined a progressive increase in the relative [Cl-]i (using the Cl- fluorescent probe SPQ). The [Cl-]i rise was concomitant with a dose-dependent down-regulation of RPS27. These results imply that CFTR inhibition produce Cl- accumulation and that RPS27 expression can be modulated by CFTR inhibition. Therefore, Cl- behaves as a signaling effector for CFTR in the modulation of RPS27 expression. In addition, the IL-1ß receptor antagonist IL1RN or the JNK inhibitor SP600125, both restored the down-regulation of RPS27 induced by CFTRinh-172, implying a role of autocrine IL-1ß and JNK signaling downstream of Cl- in RPS27 modulation.

Asunto(s)

Cloruros/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Células Epiteliales/metabolismo , Metaloproteínas/genética , Proteínas Nucleares/genética , Proteínas de Unión al ARN/genética , Proteínas Ribosómicas/genética , Transducción de Señal , Antracenos/farmacología , Comunicación Autocrina , Benzoatos/farmacología , Línea Celular Tumoral , Regulador de Conductancia de Transmembrana de Fibrosis Quística/antagonistas & inhibidores , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Colorantes Fluorescentes/metabolismo , Regulación de la Expresión Génica , Glicina/análogos & derivados , Glicina/farmacología , Humanos , Hidrazinas/farmacología , Proteína Antagonista del Receptor de Interleucina 1/farmacología , Interleucina-1beta/antagonistas & inhibidores , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Transporte Iónico/efectos de los fármacos , MAP Quinasa Quinasa 4/antagonistas & inhibidores , MAP Quinasa Quinasa 4/genética , MAP Quinasa Quinasa 4/metabolismo , Metaloproteínas/metabolismo , Proteínas Nucleares/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Proteínas de Unión al ARN/metabolismo , Proteínas Ribosómicas/metabolismo , Tiazolidinas/farmacología

RESUMEN

Cystic fibrosis (CF) is caused by mutations in the CFTR gene, which encodes a cAMP-regulated chloride channel. Several cellular functions are altered in CF cells. However, it is not clear how the CFTR failure induces those alterations. We have found previously several genes differentially expressed in CF cells, including c-Src, MUC1, MTND4, and CISD1 (CFTR-dependent genes). Recently, we also reported the existence of several chloride-dependent genes, among them GLRX5 and RPS27. Here, varying the intracellular chloride concentration [Cl- ]i of IB3-1 CF bronchial epithelial cells, we show that IL-1ß mRNA expression and secretion are also under Cl- modulation. The response to Cl- is biphasic, with maximal effects at 75 mM Cl- . The regulation of the IL-1ß mRNA expression involves an IL-1ß autocrine effect, since in the presence of the IL-1ß receptor antagonist IL1RN or anti-IL-1ß blocking antibody, the mRNA response to Cl- disappeared. Similar effects were obtained with the JNK inhibitor SP600125, the c-Src inhibitor PP2 and the IKK inhibitor III (BMS-345541). On the other hand, the IL-1ß secretion is still modulated by Cl- in the presence of IL-1RN, IL-1ß blocking antibody, or cycloheximide, suggesting that Cl- is affecting the IL-1ß maturation/secretion, which in turn starts an autocrine positive feedback loop. In conclusion, the Cl- anion acts as a second messenger for CFTR, modulating the IL-1ß maturation/secretion. The results also imply that, depending on its intracellular concentration, Cl- could be a pro-inflammatory mediator. J. Cell. Biochem. 118: 2131-2140, 2017. © 2016 Wiley Periodicals, Inc.

Asunto(s)

Bronquios/citología , Cloruros/farmacología , Células Epiteliales/metabolismo , Interleucina-1beta/metabolismo , Antracenos/farmacología , Western Blotting , Línea Celular , Cicloheximida/farmacología , Fibrosis Quística/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Células Epiteliales/efectos de los fármacos , Humanos , Proteína Antagonista del Receptor de Interleucina 1/metabolismo , Interleucina-1beta/antagonistas & inhibidores , Interleucina-1beta/genética , Interleucina-6/metabolismo , Pirimidinas/farmacología , ARN Mensajero/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa

RESUMEN

Mixing soil or adding earthworms (Eisenia fetida (Savigny, 1826)) accelerated the removal of anthracene, a polycyclic aromatic hydrocarbon, from a pasture and an arable soil, while a non-ionic surfactant (Surfynol® 485) inhibited the removal of the contaminant compared to the untreated soil. It was unclear if the treatments affected the soil bacterial community and consequently the removal of anthracene. Therefore, the bacterial community structure was monitored by means of 454 pyrosequencing of the 16S rRNA gene in the pasture and arable soil mixed weekly, amended with Surfynol® 485, E. fetida or organic material that served as food for the earthworms for 56 days. In both soils, the removal of anthracene was in the order: mixing soil weekly (100%) > earthworms applied (92%) > organic material applied (77%) > untreated soil (57%) > surfactant applied (34%) after 56 days. There was no clear link between removal of anthracene from soil and changes in the bacterial community structure. On the one hand, application of earthworms removed most of the contaminant from the arable soil and had a strong effect on the bacterial community structure, i.e. a decrease in the relative abundance of the Acidobacteria, Chloroflexi and Gemmatimonadetes, and an increase in that of the Proteobacteria compared to the unamended soil. Mixing the soil weekly removed all anthracene from the arable soil, but had little or no effect on the bacterial community structure. On the other hand, application of the surfactant inhibited the removal of anthracene from the arable soil compared to the untreated soil, but had a strong effect on the bacterial community structure, i.e. a decrease in the relative abundance of Cytophagia (Bacteroidetes), Chloroflexi, Gemmatimonadetes and Planctomycetes and an increase in that of the Flavobacteria (Bacteroidetes) and Proteobacteria. Additionally, the removal of anthracene was similar in the different treatments of both the arable and pasture soil, but the effect of application of carrot residue, earthworms or the surfactant on the bacterial community structure was more accentuated in the arable soil than in the pasture soil. It was found that removal of anthracene was not linked to changes in the bacterial community structure.

Asunto(s)

Antracenos/metabolismo , Bacterias/efectos de los fármacos , Microbiología del Suelo , Acidobacteria/efectos de los fármacos , Acidobacteria/genética , Acidobacteria/crecimiento & desarrollo , Animales , Antracenos/farmacología , Bacterias/genética , Bacterias/crecimiento & desarrollo , Bacteroidetes/efectos de los fármacos , Bacteroidetes/genética , Bacteroidetes/crecimiento & desarrollo , Chloroflexi/efectos de los fármacos , Chloroflexi/genética , Chloroflexi/crecimiento & desarrollo , ADN Bacteriano/química , ADN Bacteriano/aislamiento & purificación , ADN Bacteriano/metabolismo , Oligoquetos/metabolismo , Análisis de Componente Principal , Proteobacteria/efectos de los fármacos , Proteobacteria/genética , Proteobacteria/crecimiento & desarrollo , ARN Ribosómico 16S/química , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADN , Contaminantes del Suelo/química , Contaminantes del Suelo/metabolismo , Contaminantes del Suelo/toxicidad , Tensoactivos/toxicidad

RESUMEN

Hepatocyte growth factor (HGF) is a protective factor in myocardial injury, but its mechanisms of action have not yet been fully elucidated. Nexilin, which locates specifically to the Z-disc, is a novel Z-disc protein that enables the Z-discs to persistently withstand the extreme mechanical forces generated during muscle contraction. Therefore, we investigated the role of HGF in modulating nexilin expression in hypoxia-reoxygenation (H/R)-treated cardiomyocytes. We cultured neonatal cardiomyocytes and treated them with HGF. The mRNA and protein levels of nexilin were determined by RT-PCR and Western blotting. H/R treatment decreased nexilin mRNA expression and nexilin protein levels in cardiomyocytes. Furthermore, treatment with HGF upregulated nexilin expression and the JNK inhibitor SP600125 partly inhibited HGF-induced nexilin upregulation. In conclusion, our results suggest that ischemia-reperfusion injury may downregulate nexilin expression in cardiomyocytes, and HGF may exert its protective role during myocardial ischemic injury through upregulation of nexilin expression in cardiomyocytes.

Asunto(s)

Factor de Crecimiento de Hepatocito/farmacología , Proteínas Quinasas JNK Activadas por Mitógenos/genética , Proteínas de Microfilamentos/genética , Miocitos Cardíacos/efectos de los fármacos , Oxígeno/farmacología , ARN Mensajero/genética , Animales , Animales Recién Nacidos , Antracenos/farmacología , Butadienos/farmacología , Hipoxia de la Célula , Flavonoides/farmacología , Regulación de la Expresión Génica , Ventrículos Cardíacos/citología , Ventrículos Cardíacos/efectos de los fármacos , Ventrículos Cardíacos/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Proteínas de Microfilamentos/agonistas , Proteínas de Microfilamentos/antagonistas & inhibidores , Proteínas de Microfilamentos/metabolismo , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Nitrilos/farmacología , Cultivo Primario de Células , Inhibidores de Proteínas Quinasas/farmacología , ARN Mensajero/metabolismo , Ratas , Transducción de Señal

RESUMEN

Mitoxantrone is an anthracenedione antineoplastic and immunosuppressive agent approved for multiple sclerosis treatment. Novel mono- and disubstituted anthraquinone derivatives, analogues of mitoxantrone, were synthesized through the addition of lipophilic amino alcohols and evaluated for their effect on IL-1ß, TNF-α and nitric oxide production by LPS/IFN-γ-stimulated RAW264.7 cells. The disubstituted 1,4-anthracene-9,10-dione 10 showed significant inhibition of nitric oxide, TNF-α and IL-1ß production at the concentration of 5 µg/mL, with a much lower cytotoxicity than mitoxantrone. The monosubstituted 3, 4, 11, 12 and 13 also displayed a moderate to good inhibitory capacity on IL-1ß production. However, the methylated compounds 11, 12 and 13 failed to inhibit the TNF-α production, and compound 13 was the only one to decrease the production of nitric oxide. None of these derivatives was toxic at the tested concentrations. Compounds 10 and 13 had better inhibitory capacity of the inflammatory mediators analyzed, with reliable viability of the cells.

Asunto(s)

Antracenos/farmacología , Interleucina-1beta/metabolismo , Macrófagos/efectos de los fármacos , Óxido Nítrico/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Línea Celular , Macrófagos/metabolismo , Ratones

RESUMEN

The acetic acid and phenyl-p-benzoquinone are easy and fast screening models to access the activity of novel candidates as analgesic drugs and their mechanisms. These models induce a characteristic and quantifiable overt pain-like behavior described as writhing response or abdominal contortions. The knowledge of the mechanisms involved in the chosen model is a crucial step forward demonstrating the mechanisms that the candidate drug would inhibit because the mechanisms triggered in that model will be addressed. Herein, it was investigated the role of spinal mitogen-activated protein (MAP) kinases ERK (extracellular signal-regulated kinase), JNK (Jun N-terminal Kinase) and p38, PI(3)K (phosphatidylinositol 3-kinase) and microglia in the writhing response induced by acetic acid and phenyl-p-benzoquinone, and flinch induced by formalin in mice. Acetic acid and phenyl-p-benzoquinone induced significant writhing response over 20 min. The nociceptive response in these models were significantly and in a dose-dependent manner reduced by intrathecal pre-treatment with ERK (PD98059), JNK (SB600125), p38 (SB202190) or PI(3)K (wortmannin) inhibitors. Furthermore, the co-treatment with MAP kinase and PI(3)K inhibitors, at doses that were ineffective as single treatment, significantly inhibited acetic acid- and phenyl-p-benzoquinone-induced nociception. The treatment with microglia inhibitors minocycline and fluorocitrate also diminished the nociceptive response. Similar results were obtained in the formalin test. Concluding, MAP kinases and PI(3)K are important spinal signaling kinases in acetic acid and phenyl-p-benzoquinone models of overt pain-like behavior and there is also activation of spinal microglia indicating that it is also important to determine whether drugs tested in these models also modulate such spinal mechanisms.

Asunto(s)

Ácido Acético/toxicidad , Benzoquinonas/toxicidad , Dolor/inducido químicamente , Dolor/fisiopatología , Androstadienos/farmacología , Animales , Antracenos/farmacología , Modelos Animales de Enfermedad , Flavonoides/farmacología , Imidazoles/farmacología , Inyecciones Espinales , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Masculino , Ratones , Microglía/efectos de los fármacos , Microglía/fisiología , Nocicepción/efectos de los fármacos , Nocicepción/fisiología , Dolor Nociceptivo/inducido químicamente , Dolor Nociceptivo/fisiopatología , Dimensión del Dolor , Fosfatidilinositol 3-Quinasas/fisiología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Piridinas/farmacología , Médula Espinal/efectos de los fármacos , Médula Espinal/fisiopatología , Wortmanina

RESUMEN

Gain-of-function mutations in FGFR2 cause Apert syndrome (AS), a disease characterized by craniosynostosis and limb bone defects both due to abnormalities in bone differentiation and remodeling. Although the periosteum is an important cell source for bone remodeling, its role in craniosynostosis remains poorly characterized. We hypothesized that periosteal mesenchymal stem cells (MSCs) and fibroblasts from AS patients have abnormal cell phenotypes that contribute to the recurrent fusion of the coronal sutures. MSCs and fibroblasts were obtained from the periostea of 3 AS patients (S252W) and 3 control individuals (WT). We evaluated the proliferation, migration, and osteogenic differentiation of these cells. Interestingly, S252W mutation had opposite effects on different cell types: S252W MSCs proliferated less than WT MSCs, while S252W fibroblasts proliferated more than WT fibroblasts. Under restrictive media conditions, only S252W fibroblasts showed enhanced migration. The presence of S252W mutation increased in vitro and in vivo osteogenic differentiation in both studied cell types, though the difference compared to WT cells was more pronounced in S252W fibroblasts. This osteogenic differentiation was reversed through inhibition of JNK. We demonstrated that S252W fibroblasts can induce osteogenic differentiation in periosteal MSCs but not in MSCs from another tissue. MSCs and fibroblasts responded differently to the pathogenic effects of the FGFR2(S252W) mutation. We propose that cells from the periosteum have a more important role in the premature fusion of cranial sutures than previously thought and that molecules in JNK pathway are strong candidates for the treatment of AS patients.

Asunto(s)

Fibroblastos/metabolismo , Células Madre Mesenquimatosas/metabolismo , Mutación Missense , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/genética , Acrocefalosindactilia/genética , Acrocefalosindactilia/patología , Animales , Antracenos/farmacología , Antígenos CD/metabolismo , Regeneración Ósea , Estudios de Casos y Controles , Comunicación Celular , Diferenciación Celular , Movimiento Celular , Proliferación Celular , Fibroblastos/trasplante , Estudios de Asociación Genética , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Masculino , Osteogénesis , Periostio/patología , Fenotipo , Ratas , Ratas Wistar , Cráneo/patología , Cráneo/fisiopatología

RESUMEN

The pharmacological inhibitor SP600125 [anthra(1,9-cd)pyrazol-6(2H)-one 1,9-pyrazoloanthrone] has been largely employed as a c-JUN N-terminal kinase (JNK1/2) inhibitor. In this study, we evaluated whether pretreatment with SP600125 was able to prevent Orthopoxviruses Vaccinia virus (VACV), Cowpox virus (CPXV) and modified Vaccinia virus Ankara (MVA) replication. We found that incubation with SP600125 not only blocked virus-stimulated JNK phosphorylation, but also, significantly reduced virus production. We observed 1-3 log decline in viral yield depending on the cell line infected (A31, BSC-40 or BHK-21). The reduction in viral yield correlated with a dramatic impact on virus morphogenesis progress, intracellular mature viruses (IMV) were barely detected. Despite the fact that SP600125 can act as an efficient anti-orthopoxviral compound, we also provide evidence that this antiviral effect is not specifically exerted through JNK1/2 inhibition. This conclusion is supported by the fact that viral titers measured after infections of JNK1/2 knockout cells were not altered as compared to those of wild-type cells. In contrast, a decline in viral titers was verified when the infection of KO cells was carried out in the presence of the pharmacological inhibitor. SP600125 has been the focus of recent studies that have evaluated its action on diverse viral infections including DNA viruses. Our data support the notion that SP600125 can be regarded as a potential antipoxviral compound.

Asunto(s)

Antracenos/farmacología , Antivirales/farmacología , Proteína Quinasa 8 Activada por Mitógenos/metabolismo , Proteína Quinasa 9 Activada por Mitógenos/metabolismo , Orthopoxvirus/efectos de los fármacos , Replicación Viral/efectos de los fármacos , Animales , Línea Celular , Chlorocebus aethiops , Cricetinae , Humanos , Ratones , Ratones Endogámicos BALB C , Células 3T3 NIH , Orthopoxvirus/ultraestructura , Fosforilación , Infecciones por Poxviridae/metabolismo , Células Vero

RESUMEN

Parathyroid hormone (PTH) functions as a major mediator of bone remodeling and as an essential regulator of calcium homeostasis. In this study, we investigated the role of PTH in the regulation of the cell cycle in human colon adenocarcinoma Caco-2 cells. Flow cytometry analysis revealed that PTH (10(-8)M, 12-24h) treatment increases the number of cells in the G0/G1 phase and diminishes the number in both phases S and G2/M. In addition, analysis by Western blot showed that the hormone increases the expression of the inhibitory protein p27Kip1 and diminishes the expression of cyclin D1, cyclin D3 and CDK6. However, the amounts of CDK4, p21Cip1, p15INK4B and p16INK4A were not different in the absence or presence of PTH. Inhibitors of PKC (Ro-318220, bisindolylmaleimide and chelerythine), but not JNK (SP600125) and PP2A (okadaic acid and calyculin A), reversed PTH response in Caco-2 cells. Taken together, our results suggest that PTH induces G0/G1 phase arrest of Caco-2 intestinal cells and changes the expression of proteins involved in cell cycle regulation via the PKC signaling pathway.

Asunto(s)

Adenocarcinoma/patología , Ciclo Celular/efectos de los fármacos , Neoplasias del Colon/patología , Hormona Paratiroidea/farmacología , Adenocarcinoma/metabolismo , Antracenos/farmacología , Antineoplásicos/farmacología , Benzofenantridinas/farmacología , Células CACO-2 , Ciclo Celular/fisiología , Neoplasias del Colon/metabolismo , Quinasas Ciclina-Dependientes/metabolismo , Evaluación Preclínica de Medicamentos , Fase G1/efectos de los fármacos , Fase G1/fisiología , Humanos , Indoles/farmacología , Proteínas Quinasas JNK Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos/fisiología , Hormona Paratiroidea/fisiología , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/metabolismo , Proteína Quinasa C/fisiología , Inhibidores de Proteínas Quinasas/farmacología , Transducción de Señal/efectos de los fármacos

RESUMEN

BACKGROUND: Previous reports suggest that Brazilian propolis has multiple biological functions and may help to restore adiponectin expression and insulin sensitivity. However, little is known about the molecular mechanisms by which these compounds inhibit the downregulation of adiponectin. METHODS: The effect of various Brazilian propolis-derived components on inhibition of tumor necrosis factor-α (TNF-α)-mediated downregulation of adiponectin expression in 3T3-L1 adipocytes and molecular mechanism was investigated. RESULTS AND CONCLUSIONS: Pretreatment with either artepillin C (C3) or its derivative (C4) significantly inhibited TNF-α-mediated downregulation of adiponectin expression in 3T3-L1 adipocytes. Interestingly, C3 strongly activated peroxisome proliferator-activated receptor γ (PPARγ) transcriptional activity. Treatment of adipocytes with C3 resulted in the upregulation of adiponectin and fatty acid-binding protein 4 expression, but C4 did not significantly induce PPARγ transactivation. C4 did, however, inhibit the TNF-α-induced c-Jun-NH(2)-terminal kinase (JNK) signaling that is involved in adiponectin expression. Molecular docking studies based on hPPARγ with C3 and JNK1 with C4 clearly supported our experimental results. These data demonstrate that 1) both C3 and C4 significantly inhibit the TNF-α-mediated downregulation of adiponectin in adipocytes, 2) C3 functions as a PPARγ agonist, and its inhibition of the effect of TNF-α is due to this PPARγ transactivation, and 3) C4 is an effective inhibitor of JNK activation, thus inhibiting the TNF-α-mediated downregulation of adiponectin. GENERAL SIGNIFICANCE: Brazilian propolis-derived components (C3 and C4) can significantly inhibit TNF-α-mediated downregulation of adiponectin in adipocytes, although they do so via different mechanisms.

Asunto(s)

Adipocitos/efectos de los fármacos , Adiponectina/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Própolis/farmacología , Factor de Necrosis Tumoral alfa/farmacología , Células 3T3-L1 , Adipocitos/metabolismo , Anilidas/farmacología , Animales , Antracenos/química , Antracenos/farmacología , Brasil , Ácidos Cumáricos/química , Ácidos Cumáricos/farmacología , Cristalografía por Rayos X , Activación Enzimática/efectos de los fármacos , Immunoblotting , Proteínas Quinasas JNK Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas JNK Activadas por Mitógenos/química , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Ratones , Modelos Moleculares , Estructura Molecular , PPAR gamma/agonistas , PPAR gamma/química , PPAR gamma/metabolismo , Fenilpropionatos/química , Fenilpropionatos/farmacología , Própolis/química , Unión Proteica/efectos de los fármacos

RESUMEN

Striatal cholinergic interneurons show tonic spiking activity in the intact and sliced brain, which stems from intrinsic mechanisms. Because of it, they are also known as "tonically active neurons" (TANs). Another hallmark of TAN electrophysiology is a pause response to appetitive and aversive events and to environmental cues that have predicted these events during learning. Notably, the pause response is lost after the degeneration of dopaminergic neurons in animal models of Parkinson's disease. Moreover, Parkinson's disease patients are in a hypercholinergic state and find some clinical benefit in anticholinergic drugs. Current theories propose that excitatory thalamic inputs conveying information about salient sensory stimuli trigger an intrinsic hyperpolarizing response in the striatal cholinergic interneurons. Moreover, it has been postulated that the loss of the pause response in Parkinson's disease is related to a diminution of I(sAHP), a slow outward current that mediates an afterhyperpolarization following a train of action potentials. Here we report that I(sAHP) induces a marked spike-frequency adaptation in adult rat striatal cholinergic interneurons, inducing an abrupt end of firing during sustained excitation. Chronic loss of dopaminergic neurons markedly reduces I(sAHP) and spike-frequency adaptation in cholinergic interneurons, allowing them to fire continuously and at higher rates during sustained excitation. These findings provide a plausible explanation for the hypercholinergic state in Parkinson's disease. Moreover, a reduction of I(sAHP) may alter synchronization of cholinergic interneurons with afferent inputs, thus contributing to the loss of the pause response in Parkinson's disease.

Asunto(s)

Acetilcolina/metabolismo , Potenciales de Acción/fisiología , Cuerpo Estriado/patología , Interneuronas/fisiología , Trastornos Parkinsonianos/patología , Análisis de Varianza , Animales , Antracenos/farmacología , Apamina/farmacología , Ácido Ascórbico/efectos adversos , Bario/farmacología , Simulación por Computador , Cuerpo Estriado/lesiones , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Estimulación Eléctrica/métodos , Técnicas In Vitro , Indoles/farmacología , Masculino , Modelos Neurológicos , Fármacos Neuroprotectores/farmacología , Oxidopamina/efectos adversos , Trastornos Parkinsonianos/inducido químicamente , Piridinas/farmacología , Ratas , Ratas Sprague-Dawley , Sustancia Negra/lesiones , Sustancia Negra/fisiología

RESUMEN

Growing evidence indicates that the activity of infralimbic prefrontal cortex (IL) is critical for inhibiting inappropriate fear responses following extinction learning. Recently, we showed that fear conditioning and extinction alter the intrinsic excitability and bursting of IL pyramidal neurons in brain slices. IL neurons from Sprague Dawley rats expressing high fear had lower intrinsic excitability and bursting than those from rats expressing low fear, suggesting that regulating the intrinsic excitability and bursting of IL neurons would modulate fear expression. To test this, we combined patch-clamp electrophysiology, auditory fear conditioning, and IL infusions of M-type K(+) channel modulators. Patch-clamp recordings from IL neurons showed that the M-type K(+) channel blocker, XE-991, increased the number of spikes evoked by a depolarizing pulse and reduced the first interspike interval indicating enhanced bursting. To test whether pharmacological enhancement of IL excitability and bursting reduces fear expression and facilitates extinction, fear-conditioned rats were infused with XE-991 into IL before extinction training. XE-infused rats showed reduced freezing and facilitated extinction compared to vehicle-infused rats. The following day, recall of extinction memory was enhanced. Reducing IL excitability and bursting with the M-type K(+) channel agonist, flupirtine, had the opposite effect. Flupirtine reduced IL spike count and bursting in brain slices. Fear-conditioned rats infused with flupirtine into IL before extinction showed significantly higher levels of freezing, indicating that stimulation of M-channels enhanced fear expression. Our findings suggest that the intrinsic excitability and bursting of IL neurons regulate fear expression even before extinction.

Asunto(s)

Potenciales de Acción/fisiología , Extinción Psicológica/fisiología , Miedo/fisiología , Canales de Potasio KCNQ/fisiología , Neuronas/fisiología , Corteza Prefrontal/fisiología , Estimulación Acústica , Potenciales de Acción/efectos de los fármacos , Aminopiridinas/farmacología , Analgésicos/farmacología , Animales , Antracenos/farmacología , Condicionamiento Psicológico/efectos de los fármacos , Condicionamiento Psicológico/fisiología , Extinción Psicológica/efectos de los fármacos , Miedo/efectos de los fármacos , Canales de Potasio KCNQ/agonistas , Canales de Potasio KCNQ/antagonistas & inhibidores , Masculino , Memoria/efectos de los fármacos , Memoria/fisiología , Neuronas/efectos de los fármacos , Técnicas de Cultivo de Órganos , Técnicas de Placa-Clamp , Bloqueadores de los Canales de Potasio/farmacología , Corteza Prefrontal/citología , Ratas , Ratas Sprague-Dawley