ABSTRACT
RESUMEN La quinasa dependiente de ciclina 5 (CDK5) regula diversas funciones en neuronas, células endoteliales y epiteliales, entre ellas la dinámica del citoesqueleto. Así mismo, se ha reportado que componentes del citoesqueleto, tales como, filamentos de actina y microtúbulos juegan un rol importante durante la infección por el virus dengue (DENV). El objetivo del presente trabajo fue evaluar por dos métodos, inhibición química y silenciamiento génico, la participación de CDK5 durante la infección por DENV-2. La actividad antiviral de roscovitina fue evaluada usando ensayos de Unidades Formadoras de Placa (PFU). La eficiencia de transfección y el silenciamiento de CDK5, empleando miARNs artificiales, se determinó por citometría de flujo. El efecto sobre la proteína de envoltura viral y elementos del citoesqueleto se evidenció mediante microscopia avanzada de fluorescencia y análisis de imágenes. Roscovitina mostró actividad antiviral en etapas pre y post-infectivas en una forma dependiente de la dosis. El tratamiento con roscovitina y miRCDK5 mostró ser efectivo reduciendo la cantidad de CDK5 en células no infectadas. En células infectadas y transfectadas con miRCDK5, así como tratadas con el inhibidor, se observó una reducción significativa de la proteína de envoltura viral; sin embargo, no se encontró reducción significativa de CDK5. Además, el tratamiento con roscovitina indujo cambios celulares morfológicos evidentes en células infectadas. Los resultados indican la potencial participación de CDK5 durante la infección por DENV-2, posiblemente mediando la traducción proteica o la replicación del genoma viral a través de la regulación de la dinámica del citoesqueleto. Se requieren datos adicionales para esclarecer la mecanística del fenómeno usando métodos alternativos.
ABSTRACT Cyclin-Dependent Kinase 5 (CDK5) regulates several functions in neurons, endothelial, and epithelial cells, including the cytoskeleton dynamics. Likewise, it has been reported that some cytoskeleton elements, such as actin filaments and microtubules, play an essential role during Dengue virus (DENV) infection. This work aimed to evaluate the role of CDK5 during DENV-2 infection by two methods, chemical inhibition, and gene silencing. The antiviral activity of roscovitine was evaluated using Plaque Forming Units (PFU) assay. The transfection efficiency and knockdown of CDK5, using artificial miRNAs, was carried out by flow cytometry. The effect on the viral envelope protein and cytoskeleton elements was evidenced by advanced fluorescence microscopy and image analysis. Roscovitine showed antiviral activity in pre and post-infection stages in a dose-dependent manner. Treatment with roscovitine and miRCDK5 decrease the amount of CDK5 in uninfected cells. In cells infected and transfected with miRCDK5, as well as treated with the inhibitor, a significant reduction of the viral envelope protein was observed; however, no significant reduction of CDK5 was found. Also, evident morphological cellular changes were observed during the treatment with roscovitine in infected cells. The results indicate the potential participation of CDK5 during DENV-2 infection, possibly mediating protein translation or replication of the viral genome through the cytoskeletal dynamics regulation. Additional data are required to clarify the mechanistic of these phenomena using alternative methods.
ABSTRACT
OBJECTIVE: To analyze the associated expression of STMN1, MELK and FOXM1 in search of alternative drugable target in glioblastoma (GBM) and to review relevant functional roles of STMN1 in cancer biology. METHOD: STMN1, MELK and FOXM1 expressions were studied by quantitative PCR and their coexpressions were analyzed in two independent glioblastoma cohorts. A review of articles in indexed journals that addressed the multiple functional aspects of STMN1 was conducted, focusing on the most recent reports discussing its role in cancer, in chemoresistance and in upstream pathways involving MELK and FOXM1. RESULTS: Significant associated expressions of MELK and FOXM1 were observed with STMN1 in GBM. Additionally, the literature review highlighted the relevance of STMN1 in cancer progression. CONCLUSION: STMN1 is very important to induce events in cancer development and progression, as cellular proliferation, migration, and drug resistance. Therefore, STMN1 can be an important therapeutic target for a large number of human cancers. In glioblastoma, the most aggressive brain tumor, the MELK/FOXM1/STMN1 presented significant associated expressions, thus pointing MELK and FOXM1 as alternative targets for therapy instead of STMN1, which is highly expressed in normal brain tissue. Continuous functional research to understand the STMN1 signaling pathway is worthwhile to improve the therapeutic approaches in cancer.
OBJETIVO: Analisar as expressões associadas de STMN1, MELK e FOXM1 na procura de alvos alternativos de tratamento em glioblastoma (GBM) e revisar os papeis funcionais relevantes de STMN1 na biologia do câncer. MÉTODO: As expressões de STMN1, MELK e FOXM1 foram estudadas por PCR quantitativo e suas coexpressões foram analisadas em dois coortes independentes de GBM. A revisão dos artigos publicados em revistas indexadas na procura dos aspectos funcionais múltiplos de STMN1 foi conduzida focando-se nos estudos mais recentes discutindo o seu papel em câncer, quimiorresistência e vias de sinalização envolvendo MELK e FOXM1. RESULTADOS: Observou-se expressões associadas significantes de MELK e FOXM1 com STMN1. Adicionalmente, a revisão da literatura salientou a relevância do STMN1 na progressão do câncer. CONCLUSÃO: STMN1 é muito importante nos eventos relacionados ao desenvolvimento e progressão do câncer, como proliferação celular, migração e resistência ao tratamento. Desta forma, STMN1 pode ser um forte alvo terapêutico em um grande número de cânceres humanos. Em GBM, o tumor cerebral mais agressivo, MELK/FOXM1/STMN1 apresentaram significativa associação em suas expressões gênicas, indicando, portanto, MELK e FOXM1 como alvos alternativos para terapia em substituição ao STMN1, que apresenta alta expressão no tecido cerebral normal. Perseverar nos estudos funcionais para o entendimento da via de sinalização do STMN1 é relevante para melhorar os esquemas terapêuticos para câncer.
Subject(s)
Humans , Glioblastoma/therapy , Stathmin/analysis , Forkhead Box Protein M1/analysis , Cytoskeleton , MicrotubulesABSTRACT
Objetivos: Estudiar el efecto de la infección con rabia sobre la ultraestructura dendrítica de las neuronas piramidales de la corteza cerebral en ratones inoculados con el virus por vía intramuscular. Métodos: Ratones adultos inoculados con el virus de la rabia y ratones inoculados con solución vehículo sin el virus (controles) fueron fijados por perfusión intracardiaca, con una solución que contenía paraformaldehído al 4% y glutaraldehído al 2%, cuando los animales infectados manifestaron signos avanzados de la enfermedad. Los encéfalos fueron extraídos y cortados en plano coronal en un vibrátomo. Fragmentos pequeños y delgados de estos cortes, que contenían el área de la corteza cerebral motora, fueron procesados para microscopía electrónica de transmisión. Resultados: En las dendritas distales de las neuronas piramidales de los animales controles se observaron mitocondrias largas y estrechas, así como abundantes microtúbulos organizados en paralelo con la membrana celular. En las dendritas distales de las neuronas piramidales de los ratones infectados con el virus se observaron unas estructuras electrodensas de forma irregular semejantes a figuras de mielina, pero no se observaron las mitocondrias alargadas y los microtúbulos fueron escasos. Algunas dendritas también exhibieron la formación de vacuolas que interrumpían la continuidad del citoplasma y los microtúbulos. Conclusión: La infección con virus de la rabia generó cambios ultraestructurales en las dendritas de las neuronas piramidales corticales que aparentemente no se conocían. Estos resultados son coherentes con hallazgos previos, usando otras técnicas y modelos experimentales, en donde se ha demostrado patología dendrítica inducida por la infección con rabia.
Objectives: To study the effect of rabies infection on the dendritic ultrastructure of pyramidal neurons in the cerebral cortex of mice intramuscularly inoculated with rabies virus. Methods: Adult mice inoculated with rabies virus and mice inoculated with vehicle solution without the virus (controls) were fixed by intracardiac perfusion with a solution containing 4% paraformaldehyde and 2% glutaraldehyde. When infected animals showed advanced signs of disease, their brains were extracted and cut into the coronal plane on a vibratome. Small and thin fragments of these cuts containing motor cortex area were processed for transmission electron microscopy. Results: Distal dendrites of pyramidal neurons of control animals showed long and narrow mitochondria and abundant microtubules arranged in parallel with the cell membrane. In distal dendrites of pyramidal neurons of rabiesinfected mice some irregular shape electrondense structures similar to myelin figures were observed but elongated mitochondria were not observed, and microtubules were scarce. Some dendrites also exhibited vacuole formation interrupting the continuity of cytoplasm and microtubules. Conclusion: Infection with rabies virus produced ultrastructural changes within dendrites of the cortical pyramidal neurons that apparently were not known. These results are consistent with previous findings using other techniques and experimental models where it has been shown dendritic pathology induced by infection with rabies.
ABSTRACT
Circadian rhythms are oscillations of physiological functions. The period of their oscillation is about 24 h, and can be synchronized by environmental periodic signals as night-day cycle. The endogenous periodical changes depend on various structural elements of the circadian system which consists of the effectors, the secondary oscillators, the synchronizers and the circadian pacemaker. In mammalian species, the physiological function better understood respect their oscillation pattern are the synthesis and release of several hormones (i.e. cortisol and melatonin), the body temperature, the sleep-awake cycle, the locomotive activity, cell proliferation, neuronal activity among other rhythms. The Suprachiasmatic nucleus is the main circadian pacemarker in mammals; its oscillation keeps the circadian system synchronized particularly with respect to the environment photo period. When light reaches the pigment melanopsin in ganglionar neurons in the retina, the photoperiod signal is sent to Suprachiasmatic nucleus, and its postsinaptic neurons distributes the temporal signal to pheripheral oscillators by nervous or humoral pathways. Among the oscillators, the pineal gland is a peripheral one modulated by Suprachiasmatic nucleus. At night, the indolamine melatonin is synthesized and released from pinealocytes, and reaches other peripheral oscillators. Melatonin interacts with membrane receptors on Suprachiasmatic nucleus pacemarker neurons, reinforcing the signal of the photoperiod. In mammals, exogenous melatonin synchronizes several circadian rhythms including locomotive activity and melatonin release. When this indolamine is applied directly into the Suprachiasmatic nucleus, it produces a phase advance of the endogenous melatonin peak and increases the amplitude of the oscillation. In humans, melatonin effect on the circadian system is evident because it changes the circadian rhythms phase in subjects with advanced sleep-phase syndrome, night workers or blind people. Also it reduces jet lag symptoms enhancing sleep quality and reseting the circadian system to local time. Melatonin effects on circadian rhythms indicate their role as a chronobiotic, since decreased daily melatonin levels that occur with age and in neuropsychiatric disorders are associated with disturbances in the sleep-awake cycle. In particular, it has been described that Alzheimer's disease patients have disturbed sleep-awake cycle and have decreased serum melatonin levels. Sleep disorders in Alzheimer's disease patients decrease when they are treated with melatonin. Moreover, sleep disturbances have been observed in bipolar disorder patients and often precede relapses of insomnia-associated mania and hypersomnia-associated depression. These disturbances are linked to delayed- and advanced- phases of circadian rhythms or arrhythmia; therefore, it has been suggested that bipolar disorder patients could be treated with light and dark therapy. In depressed patients, the levels of melatonin are low throughout the 24 hour period and have a delayed onset of the indolamine concentration and showed an advance of its peak. Schizophrenic patients have decreased levels in the plasmatic melatonin in both phases of the light-dark cycle. Melatonin administration to these patients increases their sleep efficiency. In addition, melatonin acts as a neuroprotector because of its potent antioxidant action and through its cytoskeletal modulation properties. In neurodegenerative animal models, its protector effect has been observed using okadaic acid. This neurotoxin is employed for reproducing cytoskeletal damage in neurons and increased oxidative stress levels, which are molecular events similar to those that occur in Alzheimer's disease. In N1E-115 cell cultures incubated with okadaic acid, the administration of melatonin diminishes hyperphosphorylated tau and oxidative stress levels, and prevents the neurocytoskeletal damage caused by the neurotoxin. Although it is known that melatonin plays a key role in the circadian rhythms entrainment, little is known about its synchronizing effects at molecular and structural level. In algae, it has been observed a link between morphological changes and the light-dark cycle and it is known that shape is determinated by the cytoskeletal structure. In particular, the alga Euglena gracilis changes its shape two times per day under the effect of a daily light-dark cycle. This alga has a long shape when there is a higher photosynthetic capacity at the half period of the day; on the contrary, it showed a rounded shape at the end of 24 h cycle. Also, the influence of the cell shape changes on the photosynthetic reactions was investigated by altering them with drugs that disrupt the cytoskeletal structure as cytochalasin B and colchicine. Both inhibitors blocked the rhythmic shape changes and the photo-synthetic rhythm. Moreover, there are some reports about cytoskeletal changes in plants targeted by circadian rhythms. Guarda cells of Vicia faba L. showed a diurnal cycle on the alpha and beta tubulin levels. In addition, it has been proposed that melatonin synchronizes different body rhythms through cytoskeletal rearrangements. In culture cells, nanomolar melatonin concentrations cause an increase in both the polimerization rate and microtubule formation through calmodulin antagonism. A cyclic pattern produced by melatonin in the actin microfilament organization has been demonstrated in canine kidney cells. Cyclic incubation of MDCK cells with nanomolar concentrations of melatonin, resembling the cyclic pattern of secretion and release to plasma produces a microfilament reorganization and the formation of domes. Studies in animals are controvertial regarding if the amount of microtubules in different tissues varies cyclically. In rats and baboons, melatonin administration or exposure of rats to darkness induced an increased number of microtubules in the pineal gland. However, in the hypothalamus, the exposure of rats to light resulted in an increase in the microtubular protein content. Similarly, (X-tubulin mRNA was augmented during the light phase in the hypothalamus, hippocampus and cortex. By contrast, in rats maintained in constant darkness, a decreased level in the tubulin content was observed in the visual cortex. Additional information on cycle variations observed in cytoskeletal molecules indicated that beta actin mRNA levels are lower during the day in the hippocampus and cortex. But no change was observed in actin protein levels in the cerebral cortex. However, increased levels of actin and its mRNA were observed in the hypothalamus. Exogenous melatonin administration at onset of night decreased the amount of actin in the hypothalamus, while the actin mRNA levels decreased when the administration was realized in the morning. In this review we will describe the synchronizer role of melatonin in the sleep-awake cycle and in the organization of cytoskeletal proteins and their mRNAs. Also, we will describe alterations in the melatonin secretion rhythm associated with a neuronal cytoskeleton disorganization in the neuropsychiatric diseases such as Alzheimer, depression, bipolar disorder and schizophrenia.
Los ritmos circadianos son patrones de oscilación con un periodo cercano a 24h que se observan en los procesos fisiológicos. En los mamíferos se han descrito funciones biológicas con regulación circádica tal como el ciclo sueño-vigilia. La administración de la melatonina, una indolamina secretada por la glándula pineal, sincroniza los ritmos circadianos. En los humanos, este efecto se ha estudiado en sujetos con síndrome de <
ABSTRACT
The main objective of anti-carcinogenic chemotherapy is to stop uncontrolled cellular proliferation. This has prompted us to begin a systematic survey of new effective inhibitors with ability to react with cytoskeletal components and arrest living, dividing cells. Even for traditional populations herbs-consuming, encouraging the use of species with chemopreventive actions could be helpful as part of life expectancy improvement strategies. Herbal products have significantly lower costs, exhibit little or no toxicity during long-term oral administration and are relatively available at large scale. Current work involved the screening of 85 extracts from Cuban medicinal plants, selected on the basis of traditional use, ethnobotanics and pharmacological information (antiparasitic, antitumour, abortive, etc.). Antitubulinic activity in the hydroalcoholics extracts was evaluated by using a modified version of the conventional turbidity assay of tubulin assembly/ disassembly. The activity limits of the news isolated antitubulin agents were thoroughly investigated. According to the presented results, the extracts displaying the highest antitubulinic activity were Tamarindus indica L., Lawsonia inermes L and Xanthium strumarium L.
Detener la proliferación celular es el principal propósito de la quimioterapia anticarcinogénica. Para ello se ha realizado una búsqueda a partir de fuentes naturales de nuevos inhibidores efectivos que reaccionen con los componentes del citoesqueleto y puedan detener la división celular. En poblaciones que tradicionalmente utilizan plantas medicinales se estimula el uso de aquellas especies con acción quimiopreventivas como parte de una estrategia que contribuya a la calidad de vida. Los productos herbarios tienen costos significativamente más bajos, exhiben poca o ninguna toxicidad durante la administración oral a largo plazo y están al alcance de todos. Nuestro trabajo consistió en realizar un tamizaje de 85 extractos de plantas medicinales cubanas seleccionadas en base al uso tradicional, en las encuestas etnobotánicas e información farmacológica (actividad antiparasitaria, antitumoral, abortiva, etc). La actividad antitubulínica fue evaluada mediante una versión modificada del ensayo turbimétrico del ensamblaje/desensamblaje de la tubulina. Se determinó la actividad límite de los nuevos agentes antitubulínicos siendo los extractos de Tamarindus indica L., Lawsonia inermes L and Xanthium strumarium L. los de mejor actividad antitubulínica según las condiciones ensayadas.
Subject(s)
Antimitotic Agents/pharmacology , Plant Extracts/pharmacology , Plants/chemistry , Antineoplastic Agents/pharmacology , Cuba , Flora , Lawsonia Plant/chemistry , Microtubules , Plant Preparations/pharmacology , Tamarindus/chemistry , Xanthium/chemistryABSTRACT
INTRODUÇÃO: O citoesqueleto é uma complexa rede de proteínas que determina a forma da célula. Ele é fundamental para que ocorra a movimentação celular; proporciona o suporte estrutural e mobilidade de organelas intracelulares e a estrutura para movimentação e separação de cromossomos durante a divisão celular. Os componentes principais do citoesqueleto são os microfilamentos, os filamentos intermediários e os microtúbulos. Os microtúbulos são formados por dímeros de α e β tubulina que se associam à proteínas específicas, as proteínas asssociadas aos microtúbulos (MAPs). A associação diferencial entre estas proteínas possibilita ampla variedade na modulação de função dos componentes do citoesqueleto no meio celular. As MAPs expressas no sistema nervoso central (SNC), MAP2 e tau, possuem diferentes isoformas geradas por processamento alternativo. O objetivo da presente revisão é de descrever e discutir as principais funções das proteínas do citoesqueleto em condições normais e patológicas, com destaque na fisiopatologia das epilepsias. RESULTADOS: As MAPs possuem funções essenciais nas células neuronais, agem principalmente na formação estrutural destas células, garantindo sua morfologia e regulando funções específicas. Alterações nos níveis de expressão de proteínas estruturais estão envolvidas em diversas patologias do SNC como a esquizofrenia, a epilepsia do lobo temporal, as displasias corticais e as desordens do desenvolvimento. Estudos com modelos animais de epilepsia e tecido humano proveniente de pacientes epilépticos têm mostrado que as crises epilépticas podem modificar a expressão das proteínas do citoesqueleto. CONCLUSÕES: Apesar do significativo conhecimento existente sobre o citoesqueleto e proteínas associadas aos microtúbulos, não se sabe exatamente os mecanismos responsáveis pelas modificações estruturais encontradas em algumas patologias. Além do papel bem estabelecido do citoesqueleto como componente estrutural e citoarquitetônico, sua participação como facilitador do tráfico intracelular de neurotransmissores e outras macromoléculas é função ainda a ser melhor explorada e compreendida.
INTRODUCTION: The cytoskeleton is a complex network of protein fibers that determines the shape of cells. It is essential for the movements of cells and provides the structural support and movements of organelles within the cells, α and β the framework for moving and separating chromosomes during the cell division. The main components of the cytoskeleton are microfilaments, intermediate filaments and microtubules. Microtubules are assembled dimers of a and b tubulin that bind to specific proteins, the microtubules associated proteins (MAPs). Differential association between these proteins enables a wide variety in functional modulation of the cytoskeleton components in the cellular environment. The MAPs expressed in the central nervous system (CNS), MAP2 and tau, have different isoforms generated by alternative splicing. The aim of the present short review is to describe and discuss the key functions of cytoskeleton proteins in physiological and pathological states, mainly in the epileptic condition. RESULTS: The MAPs have critical roles in neurons, they act mainly on the structural formation of these cells, ensuring their morphology and regulating specific functions. Changes in the expression levels of structural proteins are involved in various CNS pathologies such as schizophrenia, temporal lobe epilepsy, cortical dysplasia and developmental disorders. Studies with animal models of epilepsy and human tissue from epileptic patients have shown that seizures can change the expression of cytoskeletal proteins. CONCLUSIONS: Despite the significant amount of knowledge on cytoskeleton and microtubules associated proteins, the precise mechanisms responsible for structural changes found in some pathological conditions are still not known. Besides the well-established role of the cytoskeleton as a structural and cytoarchitectural component, its participation in the facilitation of intracellular trafficking of neurotransmitters and other macromolecules is a function to be further explored and understood.
Subject(s)
Humans , Cytoskeleton , Epilepsy, Temporal Lobe , Neurodevelopmental Disorders , Neuropathology , Microtubule-Associated ProteinsABSTRACT
Usnic acid, a lichen metabolite, is known to exert antimitotic and antiproliferative activities against normal and malignant human cells. Many chemotherapy agents exert their activities by blocking cell cycle progression, inducing cell death through apoptosis. Microtubules, protein structure involved in the segregation of chromosomes during mitosis, serve as chemotherapeutical targets due to their key role in cellular division as well as apoptosis. The aim of this work was to investigate whether usnic acid affects the formation and/or stabilisation of microtubules by visualising microtubules and determining mitotic indices after treatment. The breast cancer cell line MCF7 and the lung cancer cell line H1299 were treated with usnic acid 29 µM for 24 hours and two positive controls: vincristine (which prevents the formation of microtubules) or taxol (which stabilizes microtubules). Treatment of MCF7 and H1299 cells with usnic acid did not result in any morphological changes in microtubules or increase in the mitotic index. These results suggest that the antineoplastic activity of usnic acid is not related to alterations in the formation and/or stabilisation of microtubules.
O ácido úsnico, um metabólito de liquens, é conhecido por sua atividade antimitótica e antiproliferativa em células humanas normais e malignas. Muitos quimioterápicos exercem suas atividades bloqueando a progressão do ciclo celular e induzindo morte celular por apoptose. Os microtúbulos, estruturas protéicas envolvidas na segregação dos cromossomos durante a mitose, servem como alvo quimioterapêutico devido ao seu importante papel tanto na divisão celular quanto nos mecanismos de morte celular por apoptose. O objetivo deste trabalho foi investigar se o ácido úsnico afeta a formação e/ou estabilização dos microtúbulos, a partir da visualização de microtúbulos e determinação de índices mitóticos após o tratamento. Células de câncer de mama MCF7 e de câncer de pulmão H1299 foram tratadas por 24 horas com 29 µM de ácido úsnico e dois controles positivos: vincristina (que impede a formação de microtúbulos) e taxol (que estabiliza microtúbulos). O tratamento das células MCF7 e H1299 com o ácido úsnico não resultou em aumento do índice mitótico. Os resultados sugerem que a atividade antineoplásica do ácido úsnico não está relacionada a alterações na formação e/ou estabilização de microtúbulos.