RESUMEN
Polyurethane copolymers based on α,ω-dihydroxypropyl poly(dimethylsiloxane) (PDMS) with a range of soft segment contents were prepared by two-stage polymerization, and their microstructures, thermal, thermomechanical, and surface properties, as well as in vitro hemo- and cytocompatibility were evaluated. All utilized characterization methods confirmed the existence of moderately microphase separated structures with the appearance of some microphase mixing between segments as the PDMS (i.e., soft segment) content increased. Copolymers showed higher crystallinity, storage moduli, surface roughness, and surface free energy, but less hydrophobicity with decreasing PDMS content. Biocompatibility of copolymers was evaluated using an endothelial EA.hy926 cell line by direct contact, an extraction method and after pretreatment of copolymers with multicomponent protein mixture, as well as by a competitive protein adsorption assay. Copolymers showed no toxic effect to endothelial cells and all copolymers, except that with the lowest PDMS content, exhibited resistance to endothelial cell adhesion, suggesting their unsuitability for long-term biomedical devices which particularly require re-endothelialization. All copolymers exhibited excellent resistance to fibrinogen adsorption and adsorbed more albumin than fibrinogen in the competitive adsorption assay, suggesting their good hemocompatibility. The noncytotoxic chemistry of these synthesized materials, combined with their nonadherent properties which are inhospitable to cell attachment and growth, underlie the need for further investigations to clarify their potential for use in short-term biomedical devices.
Asunto(s)
Dimetilpolisiloxanos/toxicidad , Células Endoteliales/citología , Adsorción , Animales , Rastreo Diferencial de Calorimetría , Espectroscopía de Resonancia Magnética con Carbono-13 , Bovinos , Adhesión Celular/efectos de los fármacos , Línea Celular , Proliferación Celular/efectos de los fármacos , Cristalización , Elastómeros/farmacología , Células Endoteliales/efectos de los fármacos , Humanos , Microscopía de Fuerza Atómica , Polimerizacion , Poliuretanos/química , Poliuretanos/toxicidad , Proteínas/aislamiento & purificación , Espectroscopía Infrarroja por Transformada de Fourier , Temperatura , AguaRESUMEN
Properties and biocompatibility of a series of thermoplastic poly(urethane-siloxane)s (TPUSs) based on α,ω-dihydroxy ethoxy propyl poly(dimethylsiloxane) (PDMS) for potential biomedical application were studied. Thin films of TPUSs with a different PDMS soft segment content were characterized by (1) H NMR, quantitative (13) C NMR, Fourier transform infrared spectroscopy (FTIR), atomic force microscopy (AFM), differential scanning calorimetry (DSC), dynamic mechanical analysis (DMA), contact angle, and water absorption measurements. Different techniques (FTIR, AFM, and DMA) showed that decrease of PDMS content promotes microphase separation in TPUSs. Samples with a higher PDMS content have more hydrophobic surface and better waterproof performances, but lower degree of crystallinity. Biocompatibility of TPUSs was examined after attachment of endothelial cells to the untreated copolymer surface or surface pretreated with multicomponent protein mixture, and by using competitive protein adsorption assay. TPUSs did not exhibit any cytotoxicity toward endothelial cells, as measured by lactate dehydrogenase and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide assays. The untreated and proteins preadsorbed TPUS samples favored endothelial cells adhesion and growth, indicating good biocompatibility. All TPUSs adsorbed more albumin than fibrinogen in competitive protein adsorption experiment, which is feature regarded as beneficial for biocompatibility. The results indicate that TPUSs have good surface, thermo-mechanical, and biocompatible properties, which can be tailored for biomedical application requirements by adequate selection of the soft/hard segments ratio of the copolymers.
Asunto(s)
Dimetilpolisiloxanos/química , Células Endoteliales/enzimología , L-Lactato Deshidrogenasa/biosíntesis , Ensayo de Materiales , Membranas Artificiales , Poliuretanos/química , Adsorción , Adhesión Celular , Línea Celular , Células Endoteliales/citología , Fibrinógeno/química , Humanos , Albúmina Sérica Bovina/química , Relación Estructura-ActividadRESUMEN
Macroautophagy (hereafter referred to as autophagy) is an evolutionary conserved catabolic process in which the cytoplasmic content is sequestered and degraded by the lysosomal machinery in order to maintain cellular homeostasis or provide energy during metabolic and hypoxic stress. It also represents an important component of the host response against infectious agents, performing surveillance and effector functions involved in detection and clearance of pathogens, including viruses. Moreover, it appears that autophagy plays a major role in determining the fate of both virally infected and uninfected cells by blocking or promoting their death in a virus- and cell-type-dependent manner. We here review the current knowledge on the complex involvement of autophagy in survival and death of the host cells during viral infection, focusing on the molecular mechanisms underlying viral modulation of autophagic response and its interference with the cell death pathways. We also discuss a possible significance of the autophagy-dependent modulation of cell death for the outcome and therapy of viral infections, emphasizing the need for a time- and cell-type-dependent fine-tuning of the autophagic response in achieving an optimal balance between beneficial and adverse effects.
Asunto(s)
Autofagia , Virosis/patología , Virosis/terapia , Virus/metabolismo , Animales , HumanosRESUMEN
The ratio of affinities toward the dopamine D2 and the 5-hydroxytryptamine 5-HT(1A) receptors is one of the important parameters that determine the efficiency of antipsychotic drugs. Here, we present the synthesis of ortho-, meta-, and para-N-{[2-(4-phenyl-piperazin-1-yl)-ethyl]-phenyl}-arylamides and their structure-activity relationship studies on dopamine D2 and 5-hydroxytryptamine 5-HT(1A) receptors. It was shown that the biological activity of the described ligands strongly depends on their topology as well as on the nature of the heteroaryl group in the head of the molecules. Docking simulations together with conformational analysis revealed a rational explanation for the ligands' behavior. The molecular model of receptor-ligand interactions described herein provided us with a tool for the rational design of new compounds with a favorable D2/5-HT(1A) profile.
Asunto(s)
Amidas/metabolismo , Modelos Moleculares , Receptor de Serotonina 5-HT1A/metabolismo , Receptores de Dopamina D2/metabolismo , Amidas/química , Diseño de Fármacos , Humanos , Ligandos , Simulación del Acoplamiento Molecular , Relación Estructura-ActividadRESUMEN
We investigated the role of autophagy, a stress-inducible lysosomal self-digestion of cellular components, in modulation of herpes simplex virus type 1 (HSV-1)-triggered death of U251 human glioma cells. HSV-1 caused apoptotic death in U251 cells, characterized by phosphatidylserine externalization, caspase activation and DNA fragmentation. HSV-1-induced apoptosis was associated with the induction of autophagic response, as confirmed by the conversion of cytosolic LC3-I to autophagosome-associated LC3-II, increase in intracellular acidification, presence of autophagic vesicles, and increase in proteolysis of the selective autophagic target p62. HSV-1-triggered autophagy was not associated with the significant increase in the expression of proautophagic protein beclin-1 or downregulation of the major autophagy suppressor mammalian target of rapamycin (mTOR). Moreover, the phosphorylation of mTOR and its direct substrate p70 S6 kinase was augmented by HSV-1 infection, while the mTOR stimulator Akt and inhibitor AMPK-activated protein kinase (AMPK) were accordingly activated and suppressed, respectively. An shRNA-mediated knockdown of the autophagy-essential LC3ß, as well as pharmacological inhibition of autophagy with bafilomycin A1 or 3-methyladenine, markedly accelerated apoptotic changes and ensuing cell death in HSV-1-infected glioma cells. These data indicate that AMPK/Akt/mTOR-independent autophagy could prolong survival of HSV-1-infected U251 glioma cells by counteracting the coinciding apoptotic response.
Asunto(s)
Apoptosis , Autofagia , Herpesvirus Humano 1/inmunología , Interacciones Huésped-Patógeno , Neuroglía/inmunología , Serina-Treonina Quinasas TOR/metabolismo , Línea Celular , Supervivencia Celular , Regulación de la Expresión Génica , Herpesvirus Humano 1/patogenicidad , Humanos , Neuroglía/virologíaRESUMEN
We investigated the ability of 19 recently synthesized arylpiperazine compounds to protect human SH-SY5Y neuroblastoma cells from the neurotoxin 6-hydroxydopamine (6-OHDA). The compound with the most potent neuroprotective action was N-{3-[2-(4-phenyl-piperazin-1-yl)-ethyl]-phenyl}-picolinamide (6b), which reduced 6-OHDA-induced apoptotic death through stabilization of mitochondrial membrane and subsequent prevention of superoxide production, caspase activation and DNA fragmentation. 6-OHDA-triggered autophagic response was also reduced by 6b, which prevented inactivation of the main autophagy repressor mTOR, upregulation of proautophagic beclin-1, conversion of microtubule-associated protein 1 light chain 3 (LC3)-I to autophagosome-associated LC3-II, as well as intracytoplasmic acidification induced by 6-OHDA. The inhibition of autophagy using LC3ß gene silencing or pharmacological autophagy blockers 3-methyladenine or bafilomycin A1, mimicked the cytoprotective effect of 6b. While the treatment with 6b had no effect on the phosphorylation of proapoptotic MAP kinases ERK and JNK, it markedly increased the phosphorylation of the prosurvival kinase Akt in 6-OHDA-treated cells. Akt inhibitor DEBC or RNA interference-mediated Akt silencing reduced the ability of 6b to block 6-OHDA-triggered apoptotic and autophagic responses, thus confirming their dependency on Akt activation. The cytoprotective effect of 6b was also observed in 6-OHDA-treated neuronal PC12 cells, but not in SH-SY5Y or PC12 cells exposed to 1-methyl-4-phenylpyridinium, indicating that the observed neuroprotection was dependent on the cytotoxic stimulus. Because of the ability to prevent 6-OHDA induced apoptotic/autophagic cell death through activation of Akt, the investigated arylpiperazines could be potential candidates for treatment of neurodegenerative diseases.
Asunto(s)
Adrenérgicos/farmacología , Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Oxidopamina/farmacología , Piperazinas/farmacología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Adenina/análogos & derivados , Adenina/farmacología , Análisis de Varianza , Supervivencia Celular/efectos de los fármacos , Citoplasma/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Humanos , Macrólidos/farmacología , Neuroblastoma/patología , Fármacos Neuroprotectores/farmacología , Piperazinas/química , Piridinas/farmacología , ARN Interferente Pequeño/farmacología , Transducción de Señal/efectos de los fármacos , Superóxidos/metabolismo , Factores de TiempoRESUMEN
The aim of the study was to investigate serum concentrations of interleukin (IL)-17 and IL-17-inducing cytokines IL-23 and transforming growth factor (TGF)-ß, as well as IL-17 single nucleotide polymorphism (SNP) rs2275913 in patients with primary antiphospholipid syndrome (PAPS). We studied fifty patients with PAPS and fifty age- and sex-matched healthy controls. The cytokine levels were measured by ELISA, while the rs2275913 SNP located in promoter region of IL-17 gene was genotyped using real-time PCR. The significantly higher levels of IL-17 (p=0.002), IL-23 (p<0.001) and TGF-ß (p=0.042) were found in PAPS patients (median 13.1, 9.4, and 125.6 pg/ml, respectively) compared to the control group (6.8, 4.9 and 44.4 pg/ml). There was a significant positive correlation between concentrations of IL-17 and IL-23 (r=0.540, p<0.001), but not between those of IL-17 and TGF-ß. No statistically significant differences were observed in the distribution of genotypes and alleles of the IL-17 rs2275913 variants in patients with PAPS compared to healthy subjects. The blood concentrations of IL-17 did not differ in subjects with different rs2275913 genotypes or patients with or without antiphospholipid antibodies. Finally, a trend toward higher IL-17 levels (p=0.063) and the significantly higher IL-17 concentrations (p=0.012) were observed in PAPS patients with deep vein thrombosis and thrombocytopenia, respectively. These data demonstrate that IL-23/IL-17 axis, stimulated independently of TGF-ß increase IL-17A gene polymorphism and antiphospholipid antibody production, might contribute to vascular manifestations of PAPS.
Asunto(s)
Síndrome Antifosfolípido/inmunología , Interleucina-17/inmunología , Interleucina-23/inmunología , Células Th17/inmunología , Adulto , Síndrome Antifosfolípido/diagnóstico , Síndrome Antifosfolípido/genética , Células Cultivadas , Análisis Mutacional de ADN , Ensayo de Inmunoadsorción Enzimática , Femenino , Estudios de Asociación Genética , Genotipo , Humanos , Interleucina-17/sangre , Interleucina-17/genética , Interleucina-23/sangre , Activación de Linfocitos/genética , Activación de Linfocitos/inmunología , Masculino , Persona de Mediana Edad , Polimorfismo de Nucleótido Simple , Regiones Promotoras Genéticas/genética , Factor de Crecimiento Transformador beta/sangre , Factor de Crecimiento Transformador beta/inmunologíaRESUMEN
The role of the main intracellular energy sensor adenosine monophosphate (AMP)-activated protein kinase (AMPK) in the induction of autophagic response and cell death was investigated in SH-SY5Y human neuroblastoma cells exposed to the dopaminergic neurotoxin 6-hydroxydopamine (6-OHDA). The induction of autophagy in SH-SY5Y cells was demonstrated by acridine orange staining of intracellular acidic vesicles, the presence of autophagosome- and autophagolysosome-like vesicles confirmed by transmission electron microscopy, as well as by microtubule-associated protein 1 light-chain 3 (LC3) conversion and p62 degradation detected by immunoblotting. 6-OHDA induced phosphorylation of AMPK and its target Raptor, followed by the dephosphorylation of the major autophagy inhibitor mammalian target of rapamycin (mTOR) and its substrate p70S6 kinase (S6K). 6-OHDA treatment failed to suppress mTOR/S6K phosphorylation and to increase LC3 conversion, p62 degradation and cytoplasmatic acidification in neuroblastoma cells in which AMPK expression was downregulated by RNA interference. Transfection of SH-SY5Y cells with AMPK or LC3ß shRNA, as well as treatment with pharmacological autophagy inhibitors suppressed, while mTOR inhibitor rapamycin potentiated 6-OHDA-induced oxidative stress and apoptotic cell death. 6-OHDA induced phosphorylation of p38 mitogen-activated protein (MAP) kinase in an AMPK-dependent manner, and pharmacological inhibition of p38 MAP kinase reduced neurotoxicity, but not AMPK activation and autophagy triggered by 6-OHDA. Finally, the antioxidant N-acetyl cysteine antagonized 6-OHDA-induced activation of AMPK, p38 and autophagy. These data suggest that oxidative stress-mediated AMPK/mTOR-dependent autophagy and AMPK/p38-dependent apoptosis could be valid therapeutic targets for neuroprotection.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Neuroblastoma/metabolismo , Oxidopamina/farmacología , Proteínas Quinasas Activadas por AMP/genética , Acetilcisteína/farmacología , Proteínas Adaptadoras Transductoras de Señales , Autofagia/genética , Línea Celular , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Microscopía Electrónica de Transmisión , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Fosforilación , ARN Interferente Pequeño , Proteínas Quinasas S6 Ribosómicas 70-kDa/metabolismo , Proteína Sequestosoma-1 , Sirolimus/farmacología , Serina-Treonina Quinasas TOR/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
The design, synthesis and biological evaluation of a novel C,D-spirolactone analogue of paclitaxel is described. This is the first paclitaxel analogue without an oxetane D-ring that shows a significant cytotoxic effect (activity one order of magnitude lower than paclitaxel). More importantly, its cytotoxicity is a result of a different mechanism of action, involving mTOR inhibition-dependent autophagy instead of G(2)/M cell cycle arrest-dependent apoptosis.
Asunto(s)
Autofagia/efectos de los fármacos , Paclitaxel/química , Espironolactona/análogos & derivados , Línea Celular Tumoral , Humanos , Estructura Molecular , Paclitaxel/farmacologíaRESUMEN
It is suggested that the ratio of dopamine D(2) to 5-hydroxytryptamine 5-HT(1A) activity is an important parameter that determines the efficiency of antipsychotic drugs. Here we present the synthesis of N-{[2-(4-phenyl-piperazin-1-yl)-ethyl]-phenyl}-2-aryl-2-yl-acetamides and 1-{[2-(4-phenyl-piperazin-1-yl)-ethyl]-phenyl}-3-aryl-2-yl-ureas and their structure-activity relationship studies on dopamine D(2) and 5-hydrohytryptamine 5-HT(1A) receptors. It was shown that ligand selectivity and affinity strongly depends on their topology and the presence of a pyridyl group in the head of molecules. Molecular modeling studies using homology modeling and docking simulation revealed a rational explanation for the ligand behavior. The observed binding modes and receptor-ligand interactions provided us with a clue for optimizing the optimal selectivity towards 5-HT(1A) receptors.
Asunto(s)
Acetamidas/síntesis química , Antipsicóticos/síntesis química , Piperazinas/síntesis química , Receptores de Dopamina D2/química , Receptores de Serotonina/química , Urea/análogos & derivados , Urea/síntesis química , Acetamidas/farmacología , Animales , Antipsicóticos/farmacología , Sitios de Unión , Células CHO , Simulación por Computador , Cricetinae , Humanos , Ligandos , Modelos Moleculares , Piperazinas/farmacología , Unión Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Ensayo de Unión Radioligante , Receptores de Dopamina D2/metabolismo , Receptores de Serotonina/metabolismo , Proteínas Recombinantes/química , Relación Estructura-Actividad , Urea/farmacologíaRESUMEN
PURPOSE: To investigate the ability of chloroquine, a lysosomotropic autophagy inhibitor, to enhance the anticancer effect of nutrient deprivation. METHODS: Serum-deprived U251 glioma, B16 melanoma and L929 fibrosarcoma cells were treated with chloroquine in vitro. Cell viability was measured by crystal violet and MTT assay. Oxidative stress, apoptosis/necrosis and intracellular acidification were analyzed by flow cytometry. Cell morphology was examined by light and electron microscopy. Activation of AMP-activated protein kinase (AMPK) and autophagy were monitored by immunoblotting. RNA interference was used for AMPK and LC3b knockdown. The anticancer efficiency of intraperitoneal chloroquine in calorie-restricted mice was assessed using a B16 mouse melanoma model. RESULTS: Chloroquine rapidly killed serum-starved cancer cells in vitro. This effect was not mimicked by autophagy inhibitors or LC3b shRNA, indicating autophagy-independent mechanism. Chloroquine-induced lysosomal accumulation and oxidative stress, leading to mitochondrial depolarization, caspase activation and mixed apoptotic/necrotic cell death, were prevented by lysosomal acidification inhibitor bafilomycin. AMPK downregulation participated in chloroquine action, as AMPK activation reduced, and AMPK shRNA mimicked chloroquine toxicity. Chloroquine inhibited melanoma growth in calorie-restricted mice, causing lysosomal accumulation, mitochondrial disintegration and selective necrosis of tumor cells. CONCLUSION: Combined treatment with chloroquine and calorie restriction might be useful in cancer therapy.
Asunto(s)
Antimaláricos/uso terapéutico , Restricción Calórica , Cloroquina/uso terapéutico , Lisosomas/efectos de los fármacos , Neoplasias/dietoterapia , Neoplasias/tratamiento farmacológico , Animales , Antimaláricos/farmacología , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Cloroquina/farmacología , Femenino , Humanos , Lisosomas/metabolismo , Lisosomas/patología , Melanoma/dietoterapia , Melanoma/tratamiento farmacológico , Melanoma/metabolismo , Melanoma/patología , Ratones , Ratones Endogámicos C57BL , Neoplasias/metabolismo , Neoplasias/patología , Estrés Oxidativo/efectos de los fármacosRESUMEN
The protective ability of novel arylpiperazine-based dopaminergic ligands against nitric oxide (NO)-mediated neurotoxicity is investigated. The most potent neuroprotective arylpiperazine identified during the study was N-{4-[2-(4-phenyl-piperazin-1-yl)ethyl]-phenyl}picolinamide, which protected SH-SY5Y human neuron-like cells from the proapoptotic effect of NO donor sodium nitroprusside (SNP) by decreasing oxidative stress, mitochondrial membrane depolarization, caspase activation and subsequent phosphatydilserine externalization/DNA fragmentation. The protective effect was associated with the inhibition of proapoptotic (JNK, ERK, AMPK) and activation of antiapoptotic (Akt) signaling pathways, in the absence of interference with intracellular NO accumulation. The neuroprotective action of arylpiperazines was shown to be independent of dopamine receptor binding, as it was not affected by the high-affinity D1/D2 receptor blocker butaclamol. These results reported support the further study of arylpiperazines as potential neuroprotective agents.
Asunto(s)
Apoptosis/efectos de los fármacos , Dopaminérgicos/síntesis química , Mitocondrias/efectos de los fármacos , Fármacos Neuroprotectores/síntesis química , Piperazinas/síntesis química , Proteínas Reguladoras de la Apoptosis/antagonistas & inhibidores , Proteínas Reguladoras de la Apoptosis/genética , Línea Celular Tumoral , Fragmentación del ADN/efectos de los fármacos , Dopaminérgicos/farmacología , Expresión Génica/efectos de los fármacos , Humanos , Mitocondrias/metabolismo , Neuroblastoma , Fármacos Neuroprotectores/farmacología , Óxido Nítrico/farmacología , Donantes de Óxido Nítrico/farmacología , Nitroprusiato/farmacología , Fosfatidilserinas/metabolismo , Piperazinas/farmacología , Especies Reactivas de Oxígeno/antagonistas & inhibidores , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
The role of autophagy, a process in which the cell self-digests its own components, was investigated in glioma cell death induced by the hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase-inhibiting drug simvastatin. Induction of autophagy and activation of autophagy-regulating signalling pathways were analyzed by immunoblotting. Flow cytometry/fluorescent microscopy was used to assess autophagy-associated intracellular acidification and apoptotic markers (phosphatidylserine exposure, DNA fragmentation and caspase activation). Cell viability was determined by crystal violet, MTT or LDH release assay. Simvastatin treatment of U251 and C6 glioma cell lines caused the appearance of autophagolysosome-like intracytoplasmic acidic vesicles. The induction of autophagy in U251 cells was confirmed by the upregulation of autophagosome-associated LC3-II and pro-autophagic beclin-1, as well as by the downregulation of the selective autophagic target p62. Simvastatin induced the activation of AMP-activated protein kinase (AMPK) and its target Raptor, while simultaneously downregulating activation of Akt. Mammalian target of rapamycin (mTOR), a major AMPK/Akt downstream target and a major negative autophagy regulator, and its substrate p70 S6 kinase 1 were also inhibited by simvastatin. Mevalonate, the product of HMG-CoA reductase enzymatic activity, AMPK siRNA or pharmacological inactivation of AMPK with compound C suppressed, while the inhibitors of Akt (10-DEBC hydrochloride) and mTOR (rapamycin) mimicked autophagy induction by simvastatin. Inhibition of autophagy with bafilomycin A1, 3-methyladenine and LC3ß shRNA, as well as AMPK inhibition with compound C or AMPK siRNA, markedly increased apoptotic death of simvastatin-treated U251 cells. These data suggest that inhibition of AMPK-dependent autophagic response might sensitize glioma cells to statin-induced apoptotic death.
Asunto(s)
Proteínas Quinasas Activadas por AMP/antagonistas & inhibidores , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Autofagia/efectos de los fármacos , Neoplasias Encefálicas/enzimología , Glioma/enzimología , Proteínas Quinasas Activadas por AMP/genética , Proteínas Quinasas Activadas por AMP/metabolismo , Animales , Apoptosis/efectos de los fármacos , Proteínas Reguladoras de la Apoptosis/metabolismo , Beclina-1 , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Glioma/genética , Glioma/patología , Humanos , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Proteínas de la Membrana/metabolismo , Ratones , Proteínas Asociadas a Microtúbulos/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Interferencia de ARN , Ratas , Transducción de Señal/efectos de los fármacos , Simvastatina/farmacología , Serina-Treonina Quinasas TOR/metabolismo , TransfecciónRESUMEN
Novel polyurethane co-polymers (TPUs), based on poly(ϵ-caprolactone)-block-poly(dimethylsiloxane)-block-poly(ϵ-caprolactone) (PCL-PDMS-PCL) as soft segment and 4,4'-methylenediphenyl diisocyanate (MDI) and 1,4-butanediol (BD) as hard segment, were synthesized and evaluated for biomedical applications. The content of hard segments (HS) in the polymer chains was varied from 9 to 63 wt%. The influence of the content and length of the HS on the thermal, surface, mechanical properties and biocompatibility was investigated. The structure, composition and HS length were examined using (1)H- and quantitative (13)C-NMR spectroscopy. DSC results implied that the synthesized TPUs were semicrystalline polymers in which both the hard MDI/BD and soft PCL-PDMS-PCL segments participated. It was found that an increase in the average HS length (from 1.2 to 14.4 MDI/BD units) was accompanied by an increase in the crystallinity of the hard segments, storage moduli, hydrophilicity and degree of microphase separation of the co-polymers. Depending on the HS content, a gradual variation in surface properties of co-polymers was revealed by FT-IR, AFM and static water contact angle measurements. The in vitro biocompatibility of co-polymers was evaluated using the endothelial EA.hy926 cell line and protein adsorption on the polyurethane films. All synthesized TPUs adsorbed more albumin than fibrinogen from multicomponent protein mixture, which may indicate biocompatibility. The polyurethane films with high HS content and/or high roughness coefficient exhibit good surface properties and biocompatible behavior, which was confirmed by non-toxic effects to cells and good cell adhesion. Therefore, the non-cytotoxic chemistry of the co-polymers makes them good candidates for further development as biomedical implants.
Asunto(s)
Caproatos , Lactonas , Poliuretanos , Siloxanos , Adsorción , Albúminas/química , Animales , Caproatos/química , Espectroscopía de Resonancia Magnética con Carbono-13 , Bovinos , Adhesión Celular , Línea Celular , Supervivencia Celular , Células Endoteliales/fisiología , Fibrinógeno/química , Humanos , Enlace de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Lactonas/química , Ensayo de Materiales , Microscopía de Fuerza Atómica , Poliuretanos/química , Espectroscopía de Protones por Resonancia Magnética , Siloxanos/química , Espectroscopía Infrarroja por Transformada de Fourier , Propiedades de Superficie , Temperatura , Agua/químicaRESUMEN
Five groups of previously synthesized and initially screened non-substituted and 4-halogenated arylpiperazin-1-yl-ethyl-benzimidazoles were estimated for their in-vitro binding affinities at the rat D(2) , 5-HT(2A) , and α(1) -adrenergic receptors. Among all these compounds, 2-methoxyphenyl and 2-chlorophenyl piperazines demonstrate the highest affinities for the tested receptors. The effects of 4-halogenation of benzimidazoles reveal that substitution with bromine may greatly increase the affinity of the compounds for the studied receptors, while the effect of substitution with chlorine is less remarkable. Most of the tested components show 5-HT(2A)/D(2) pK(i) binding ratios slightly above or less than 1, while only 4-chloro-6-(2-{4-[3-(trifluoromethyl)phenyl]piperazin-1-yl}ethyl)-1H-benzimidazole expresses an appropriate higher binding ratio (1.14), which was indicated for atypical neuroleptics. This compound exhibits a non-cataleptic action in rats and prevents d-amphetamine-induced hyperlocomotion in mice, which suggest its atypical antipsychotic potency.
Asunto(s)
Antipsicóticos/metabolismo , Antipsicóticos/farmacología , Bencimidazoles/metabolismo , Bencimidazoles/farmacología , Receptor de Serotonina 5-HT2A/metabolismo , Receptores de Dopamina D2/metabolismo , Animales , Antipsicóticos/química , Conducta Animal/efectos de los fármacos , Bencimidazoles/química , Sitios de Unión , Dextroanfetamina/farmacología , Inhibidores de Captación de Dopamina/farmacología , Evaluación Preclínica de Medicamentos , Halogenación , Ligandos , Ratones , Ratas , Receptores Adrenérgicos alfa 1/metabolismoRESUMEN
Metformin is an antidiabetic drug with anticancer properties, which mainly acts through induction of AMP-activated protein kinase (AMPK). In the present study we investigated the influence of metformin on the in vitro anticancer activity of the well-known chemotherapeutic agent cisplatin. Cell viability was determined by MTT and LDH release assay, oxidative stress and apoptosis (caspase activation, DNA fragmentation, and phosphatidylserine exposure) were assessed by flow cytometry, while activation of AMPK and Akt was analyzed by immunoblotting. Although metformin reduced the number of tumour cells when applied alone, it surprisingly antagonized the cytotoxicity of cisplatin towards U251 human glioma, C6 rat glioma, SHSY5Y human neuroblastoma, L929 mouse fibrosarcoma and HL-60 human leukemia cell lines. Only in B16 mouse melanoma cells metformin augmented the cytotoxicity of cisplatin. In U251 glioma cells metformin suppressed cisplatin-induced apoptotic cell death through inhibition of oxidative stress and caspase activation. The observed cytoprotection was apparently AMPK-independent, as metformin did not further increase cisplatin-induced AMPK activation in U251 cells and other pharmacological AMPK activators failed to block cisplatin-mediated apoptosis. On the other hand, metformin induced Akt activation in cisplatin-treated cells and Akt inhibitor 10-DEBC hydrochloride or phosphoinositide 3-kinase/Akt inhibitor LY294002 abolished metformin-mediated antioxidant and antiapoptotic effects. In conclusion, the antidiabetic drug metformin reduces cisplatin in vitro anticancer activity through AMPK-independent upregulation of Akt survival pathway. These data warrant caution when considering metformin for treatment of diabetic cancer patients receiving cisplatin or as a potential adjuvant in cisplatin-based chemotherapeutic regimens.
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Apoptosis/efectos de los fármacos , Cisplatino/antagonistas & inhibidores , Cisplatino/farmacología , Hipoglucemiantes/farmacología , Metformina/farmacología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Adenilato Quinasa/metabolismo , Animales , Antineoplásicos/antagonistas & inhibidores , Antineoplásicos/farmacología , Autofagia/efectos de los fármacos , Caspasas/metabolismo , Línea Celular Tumoral , Citoprotección/efectos de los fármacos , Antagonismo de Drogas , Activación Enzimática/efectos de los fármacos , Humanos , Ratones , Estrés Oxidativo/efectos de los fármacos , RatasRESUMEN
External magnetic fields (MFs) have the ability to modify motor activity of animals, complex type of behaviour connected with dopaminergic and serotonergic neurotransmissions in the brain. Thus, the purpose of this study was to examine MF-induced changes in the activity of serotonin 5-HT(2A) receptors in the prefrontal cortex, as well as dopamine D(1) and D(2) receptors in the striatum of adult Wistar rats, considering their involvement in motor behavior regulation. Experimental animals were continuously exposed to extremely low frequency MF (ELF-MF, 50 Hz, 0.5 mT) for 1, 3, and 7 days. Subsequently, binding properties (K(d) and B(max)) of receptors were determined by in vitro radioligand receptor binding assays. It was shown that the affinity of serotonin 5-HT(2A) receptors decreased and their density increased in the prefrontal cortex of rats after ELF-MF exposure. Regarding affinity, this effect was duration-dependent and most prominent after 7-day of ELF-MF exposure. In contrast to serotonin 5-HT(2A) receptors in the prefrontal cortex, ELF-MF had no significant effect on the affinity and density of dopamine D(1) and D(2) receptors in the striatum. We can conclude that continuous exposure to ELF-MF up to 7 days affects cortical serotonergic neurotransmission, whereby intensity of these changes depends on ELF-MF exposure duration.
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Encéfalo/metabolismo , Magnetismo , Receptor de Serotonina 5-HT2A/metabolismo , Receptores de Dopamina D1/metabolismo , Receptores de Dopamina D2/metabolismo , Animales , Masculino , Neostriado/metabolismo , Corteza Prefrontal/metabolismo , Ratas , Ratas Wistar , Factores de TiempoRESUMEN
Two new series of substituted arylpiperazines with heterocyclic 3-propoxy-benzimidazole or 3-propoxy-benzimidazole-2-thione groups were synthesized and their in vitro binding affinities for the D(2), 5-HT(1A), 5-HT(2A), and alpha(1)-adrenergic receptors determined. Among them, only two compounds with phenyl aryl-constituent (8a and 9a) showed 5-HT(2A)/D(2) pK(i) binding ratios proposed for atypical neuroleptics. As to their behavioral screening on rodents, both compounds exhibited a non-cataleptic action in rats and antagonized D-amphetamine-induced hyperlocomotion in mice, suggesting their possible atypical antipsychotic potency.
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Antipsicóticos/farmacología , Piperazinas/farmacología , Animales , Antipsicóticos/química , Células CHO , Cricetinae , Cricetulus , Piperazinas/química , RatasRESUMEN
Diethylether extract of aerial parts of Gentiana kochiana mostly consists of two tetraoxygenated xanthones: gentiacaulein (1,7-dihidroxy-3,8-dimethoxyxanthone; 76.1%) and gentiakochianin (1,7,8-trihidroxy-3-methoxyxanthone; 14.2%). The extract and these xanthones were evaluated for the CNS pharmacological activity in rodents. In vitro assays on rat brain preparations revealed insignificant interaction of the compounds with the specific dopamine and serotonin receptors or synaptosomal uptake of serotonin. However, the extract and gentiacaulein strongly inhibited rat microsomal MAO A (IC50=0.22 microg/ml and 0.49 microM, respectively). Their effects on MAO B and a gentiakochianin blocking potential on both MAO enzymes were moderate. Behavioral examinations on mice showed that 10 day s.c. administration of the extract (20 mg/kg) significantly decreased immobility score in a forced swimming test and strongly inhibited ambulation and stereotypy in an open-field test. These effects resembled those induced by 10 mg/kg imipramine. The ex vivo MAO A activity in crude brain mitochondrial fraction of mice treated with 20 mg/kg of the extract was significantly elevated, whilst that outside brain nerve terminals was declined. This study suggests some antidepressant therapeutic potential of G. kochiana, particularly of gentiacaulein, with an ambiguity whether pharmacological mechanism could be related only to the central inhibition of MAO A.