RESUMEN
Tyrosine hydroxylase (TH) is the key enzyme that controls the rate of synthesis of the catecholamines. SH-SY5Y cells with stable transfections of either human tyrosine hydroxylase isoform 1 (hTH1) or human tyrosine hydroxylase isoform 4 (hTH4) were used to determined the subcellular distribution of TH protein and phosphorylated TH, under basal conditions and after muscarine stimulation. Muscarine was previously shown to increase the phosphorylation of only serine 19 and serine 40 in hTH1 cells. Under basal conditions, the hTH1 and hTH4 proteins, their serine 19 phosphorylated forms and hTH1 phosphorylated at serine 40 were all similarly distributed; with ~80% in the cytosolic fraction, ~20% in the membrane fraction, and less than 1%, or not detectable, in the nuclear fraction. However, hTH4 phosphorylated at serine 71 had a significantly different distribution with ~65% cytosolic and ~35% membrane associated. Muscarine stimulation led to hTH1 being redistributed from the cytosol and nuclear fractions to the membrane fraction and hTH4 being redistributed from the cytosol to the nuclear fraction. These muscarine stimulated redistributions were not due to TH phosphorylation at serine 19, serine 40, or serine 71 and were most likely due to TH binding to proteins whose phosphorylation was increased by muscarine. This is the first study to show a difference in subcellular distribution between two human TH isoforms under basal and stimulated conditions.
Asunto(s)
Tirosina 3-Monooxigenasa/metabolismo , Línea Celular , Membrana Celular/enzimología , Citosol/metabolismo , Humanos , Isoenzimas/metabolismo , Muscarina/farmacología , Fosforilación , Serina/metabolismo , Fracciones Subcelulares/enzimología , Tirosina 3-Monooxigenasa/genéticaRESUMEN
Ubiquitous low-dose methylmercury (MeHg) exposure through an increased fish consumption represents a global public health problem, especially among pregnant women. A plethora of micronutrients presented in fish affects MeHg uptake/distribution, but limited data is available. Vitamin A (VitA), another fish micronutrient is used in nutritional supplementation, especially during pregnancy. However, there is no information about the health effects arising from their combined exposure. Therefore, the present study aimed to examine the effects of both MeHg and retinyl palmitate administered on pregnant and lactating rats in metabolic and redox parameters from dams and their offspring. Thirty Wistar female rats were orally supplemented with MeHg (0,5â¯mg/kg/day) and retinyl palmitate (7500⯵g RAE/kg/day) via gavage, either individually or in combination from the gestational day 0 to weaning. For dams (150 days old) and their offspring (31 days old), glycogen accumulation (hepatic and cardiac) and retinoid contents (plasma and liver) were analyzed. Hg deposition in liver tissue was quantified. Redox parameters (liver, kidney, and heart) were evaluated for both animals. Cytogenetic damage was analyzed with micronucleus test. Our results showed no general toxic or metabolic alterations in dams and their offspring by MeHg-VitA co-administration during pregnancy and lactation. However, increased lipoperoxidation in maternal liver and a disrupted pro-oxidant response in the heart of male pups was encountered, with apparently no particular effects in the antioxidant response in female offspring. GST activity in dam kidney was altered leading to possible redox disruption of this tissue with no alterations in offspring. Finally, the genomic damage was exacerbated in both male and female pups. In conclusion, low-dose MeHg exposure and retinyl palmitate supplementation during gestation and lactation produced a potentiated pro-oxidant effect, which was tissue-specific. Although this is a pre-clinical approach, we recommend precaution for pregnant women regarding food consumption, and we encourage more epidemiological studies to assess possible modulations effects of MeHg-VitA co-administration at safe or inadvertently used doses in humans, which may be related to specific pathologies in mothers and their children.
Asunto(s)
Antioxidantes/farmacología , Lactancia , Compuestos de Metilmercurio/toxicidad , Vitamina A/análogos & derivados , Animales , Animales Recién Nacidos , Catalasa/metabolismo , Suplementos Dietéticos , Diterpenos , Femenino , Glutatión Peroxidasa/metabolismo , Glutatión Transferasa/metabolismo , Hígado/efectos de los fármacos , Hígado/metabolismo , Masculino , Compuestos de Metilmercurio/sangre , Oxidación-Reducción/efectos de los fármacos , Embarazo , Efectos Tardíos de la Exposición Prenatal , Ratas , Ratas Wistar , Ésteres de Retinilo , Superóxido Dismutasa/metabolismo , Vitamina A/sangre , Vitamina A/metabolismo , Vitamina A/farmacologíaRESUMEN
Retinoids (vitamin A and derivatives) are recognized as essential factors for central nervous system (CNS) development. Retinol (vitamin A) also was postulated to be a major antioxidant component of diet as it modulates reactive species (RS) production and oxidative stress in biological systems. Oxidative stress plays a major role either in pathogenesis or development of neurodegenerative diseases, or even in both. Here we investigate the role of retinol supplementation to human neuron-derived SH-SY5Y cells over RS production and biochemical markers associated to neurodegenerative diseases expressed at neuronal level in Parkinson's disease and Alzheimer's disease: α-synuclein, ß-amyloid peptide, tau phosphorylation and RAGE. Retinol treatment (24 h) impaired cell viability and increased intracellular RS production at the highest concentrations (7 up to 20 µM). Antioxidant co-treatment (Trolox 100 µM) rescued cell viability and inhibited RS production. Furthermore, retinol (10 µM) increased the levels of α-synuclein, tau phosphorylation at Ser396, ß-amyloid peptide and RAGE. Co-treatment with antioxidant Trolox inhibited the increased in RAGE, but not the effect of retinol on α-synuclein, tau phosphorylation and ß-amyloid peptide accumulation. These data indicate that increased availability of retinol to neurons at levels above the cellular physiological concentrations may induce deleterious effects through diverse mechanisms, which include oxidative stress but also include RS-independent modulation of proteins associated to progression of neuronal cell death during the course of neurodegenerative diseases.
Asunto(s)
Péptidos beta-Amiloides/metabolismo , Vitamina A/farmacología , alfa-Sinucleína/metabolismo , Proteínas tau/metabolismo , Antioxidantes/farmacología , Línea Celular , Supervivencia Celular/efectos de los fármacos , Humanos , Neuronas/metabolismo , Fosforilación , Vitamina A/metabolismoRESUMEN
BACKGROUND: Leishmaniasis is a parasitosis caused by several species of the genus Leishmania. These parasites present high resistance against oxidative stress generated by inflammatory cells. OBJECTIVES: To investigate oxidative stress and molecular inflammatory markers in BALB/c mice infected with L. amazonensis and the effect of antioxidant treatment on these parameters. METHODS: Four months after infection, oxidative and inflammatory parameters of liver, kidneys, spleen, heart and lungs from BALB/c mice were assessed. FINDINGS: In liver, L. amazonensis caused thiol oxidation and nitrotyrosine formation; SOD activity and SOD2 protein content were increased while SOD1 protein content decreased. The content of the cytokines IL-1ß, IL-6, TNF-α, and the receptor of advanced glycation endproducts (RAGE) increased in liver. Treatment with the antioxidant N-acetyl-cysteine (20 mg/kg b.w) for five days inhibited oxidative stress parameters. MAIN CONCLUSIONS: L. amazonensis induces significant alterations in the redox status of liver but not in other organs. Acute antioxidant treatment alleviates oxidative stress in liver, but it had no effect on pro-inflammatory markers. These results indicate that the pathobiology of leishmaniasis is not restricted to the cutaneous manifestations and open perspectives for the development of new therapeutic approaches to the disease, especially for liver function.
Asunto(s)
Acetilcisteína/farmacología , Depuradores de Radicales Libres/farmacología , Leishmania mexicana , Leishmaniasis Cutánea/metabolismo , Hígado/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Animales , Leishmaniasis Cutánea/patología , Hígado/enzimología , Ratones , Ratones Endogámicos BALB C , Estrés Oxidativo/fisiologíaRESUMEN
Leishmaniasis is a parasitosis caused by several species of the genus Leishmania, an obligate intramacrophagic parasite. Although neurologic symptoms have been observed in human cases of leishmaniasis, the manifestation of neurodegenerative processes is poorly studied. The aim of the present work was to investigate if peripheral infection of BALB/c mice with Leishmania amazonensis affects tau phosphorylation and RAGE protein content in the brain, which represent biochemical markers of neurodegenerative processes observed in diseases with a pro-inflammatory component, including Alzheimer's disease and Down syndrome. Four months after a single right hind footpad subcutaneous injection of L. amazonensis, the brain cortex of BALB/c mice was isolated. Western blot analysis indicated an increase in tau phosphorylation (Ser(396)) and RAGE immunocontent in infected animals. Brain tissue TNF-α, IL-1ß, and IL-6 levels were not different from control animals; however, increased protein carbonylation, decreased IFN-γ levels and impairment in antioxidant defenses were detected. Systemic antioxidant treatment (NAC 20mg/kg, i.p.) inhibited tau phosphorylation and recovered IFN-γ levels. These data, altogether, indicate an association between impaired redox state, tau phosphorylation and RAGE up-regulation in the brain cortex of animals infected with L. amazonensis. In this context, it is possible that neurologic symptoms associated to chronic leishmaniasis are associated to disruptions in the homeostasis of CNS proteins, such as tau and RAGE, as consequence of oxidative stress. This is the first demonstration of alterations in biochemical parameters of neurodegeneration in an experimental model of Leishmania infection.
Asunto(s)
Encéfalo/parasitología , Leishmania mexicana , Leishmaniasis/metabolismo , Receptores Inmunológicos/metabolismo , Proteínas tau/metabolismo , Animales , Encéfalo/metabolismo , Citocinas/metabolismo , Ratones , Ratones Endogámicos BALB C , Estrés Oxidativo/fisiología , Fosforilación , Receptor para Productos Finales de Glicación Avanzada , Regulación hacia ArribaRESUMEN
Carvacrol (CARV) presents valuable biological properties such as anti-inflammatory and antioxidant activities. However, pharmacological uses of CARV are largely limited due to disadvantages related to solubility, bioavailability, preparation and storage processes. The complexation of monoterpenes with ß-cyclodextrin (ß-CD) increases their stability, solubility and oral bioavailability. Here, the protective effect of oral treatment with CARV/ß-CD complex (25⯵g/kg/day) against dopaminergic (DA) denervation induced by unilateral intranigral injection of 6-hydroxydopamine (6-OHDA - 10⯵g per rat) was analyzed, in order to evaluate a putative application in the development of neuroprotective therapies for Parkinson's disease (PD). Pretreatment with CARV/ß-CD for 15 days prevented the loss of DA neurons induced by 6-OHDA in adult Wistar rats. This effect may occur through CARV anti-inflammatory and antioxidant properties, as the pretreatment with CARV/ß-CD inhibited the release of IL-1ß and TNF-α; besides, CARV prevented the increase of mitochondrial superoxide production induced by 6-OHDA in cultured SH-SY5Y cells. Importantly, hepatotoxicity or alterations in blood cell profile were not observed with oral administration of CARV/ß-CD. Therefore, this study showed a potential pharmacological application of CARV/ß-CD in PD using a non-invasive route of drug delivery, i.e., oral administration.
Asunto(s)
Cimenos/administración & dosificación , Desnervación/efectos adversos , Neuronas Dopaminérgicas/efectos de los fármacos , Fármacos Neuroprotectores/administración & dosificación , Oxidopamina/toxicidad , beta-Ciclodextrinas/administración & dosificación , Administración Oral , Animales , Neuronas Dopaminérgicas/metabolismo , Neuronas Dopaminérgicas/patología , Combinación de Medicamentos , Masculino , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/fisiología , Ratas , Ratas WistarRESUMEN
Retinoic acid (RA) promotes differentiation in multiple neurogenic cell types by promoting gene reprogramming through retinoid receptors and also by inducing cytosolic signaling events. The nuclear RXR receptors are one of the main mediators of RA cellular effects, classically by joining the direct receptors of RA, the nuclear RAR receptors, in RAR/RXR dimers which act as transcription factors. Distinct RXR genes lead to RXRα, RXRß and RXRγ subtypes, but their specific roles in neuronal differentiation remain unclear. We firstly investigated both RXRs and RARs expression profiles during RA-mediated neuronal differentiation of human neuroblastoma cell line SH-SY5Y, and found varying levels of retinoid receptors transcript and protein contents along the process. In order to understand the roles of the expression of distinct RXR subtypes to RA signal transduction, we performed siRNA-mediated silencing of RXRα and RXRß during the first stages of SH-SY5Y differentiation. Our results showed that RXRα is required for RA-induced neuronal differentiation of SH-SY5Y cells, since its silencing compromised cell cycle arrest and prevented the upregulation of neuronal markers and the adoption of neuronal morphology. Besides, silencing of RXRα affected the phosphorylation of ERK1/2. By contrast, silencing of RXRß improved neurite extension and led to increased expression of tau and synaptophysin, suggesting that RXRß may negatively regulate neuronal parameters related to neurite outgrowth and function. Our results indicate distinct functions for RXR subtypes during RA-dependent neuronal differentiation and reveal new perspectives for studying such receptors as clinical targets in therapies aiming at restoring neuronal function.
Asunto(s)
Neuritas/metabolismo , Receptor alfa X Retinoide/fisiología , Receptor beta X Retinoide/fisiología , Animales , Puntos de Control del Ciclo Celular/fisiología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Línea Celular Tumoral , Núcleo Celular/metabolismo , Proliferación Celular/efectos de los fármacos , Neuronas Dopaminérgicas/fisiología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Sistema de Señalización de MAP Quinasas/fisiología , Neuroblastoma/genética , Neuroblastoma/metabolismo , Ratas , Receptores Citoplasmáticos y Nucleares/genética , Receptores de Ácido Retinoico/metabolismo , Receptores de Ácido Retinoico/fisiología , Receptor alfa X Retinoide/metabolismo , Receptor beta X Retinoide/metabolismo , Receptores X Retinoide , Transducción de Señal/efectos de los fármacos , Activación Transcripcional , Tretinoina/metabolismo , Tretinoina/farmacología , Células Tumorales CultivadasRESUMEN
Vitamin A (retinol) is involved in signaling pathways regulating gene expression and was postulated to be a major antioxidant and anti-inflammatory compound of the diet. Parkinson's disease (PD) is a progressive neurodegenerative disorder, characterized by loss of nigral dopaminergic neurons, involving oxidative stress and pro-inflammatory activation. The aim of the present study was to evaluate the neuroprotective effects of retinol oral supplementation against 6-hydroxydopamine (6-OHDA, 12⯵g per rat) nigrostriatal dopaminergic denervation in Wistar rats. Animals supplemented with retinol (retinyl palmitate, 3000 IU/kg/day) during 28 days exhibited increased retinol content in liver, although circulating retinol levels (serum) were unaltered. Retinol supplementation did not protect against the loss of dopaminergic neurons (assessed through tyrosine hydroxylase immunofluorescence and Western blot). Retinol supplementation prevented the effect of 6-OHDA on Iba-1 levels but had no effect on 6-OHDA-induced GFAP increase. Moreover, GFAP levels were increased by retinol supplementation alone. Rats pre-treated with retinol did not present oxidative damage or thiol redox modifications in liver, and the circulating levels of TNF-α, IL-1ß, IL-6 and IL-10 were unaltered by retinol supplementation, demonstrating that the protocol used here did not cause systemic toxicity to animals. Our results indicate that oral retinol supplementation is not able to protect against 6-OHDA-induced dopaminergic denervation, and it may actually stimulate astrocyte reactivity without altering parameters of systemic toxicity.
Asunto(s)
Modelos Animales de Enfermedad , Neuronas Dopaminérgicas/efectos de los fármacos , Degeneración Nerviosa/inducido químicamente , Degeneración Nerviosa/tratamiento farmacológico , Simpatectomía Química/métodos , Vitamina A/administración & dosificación , Administración Oral , Animales , Neuronas Dopaminérgicas/metabolismo , Masculino , Degeneración Nerviosa/metabolismo , Técnicas de Cultivo de Órganos , Ratas , Ratas Wistar , Resultado del TratamientoRESUMEN
The use of dietary supplements to enhance the benefit of exercise training is a common practice. The liver is the organ where all substances are metabolized, and certain supplements have been associated with liver injury. Vitamin A (VA), a liposoluble vitamin stored in the liver, is commonly used as an antioxidant supplement. Here, we evaluated the effect of chronic VA supplementation on oxidative damage and stress parameters in trained rats. Animals were divided into the following groups: sedentary (SE), sedentary/VA (SE+VA), exercise training (ET), and exercise training/VA (ET+VA). During 8 weeks, animals were subjected to swimming (0%, 2%, 4%, 6% body weight) for 5 days/week and a VA daily intake of 450 retinol equivalents/day. Parameters were evaluated by enzymatic activity analysis, ELISA, and Western blotting. VA caused liver lipid peroxidation and protein damage in exercised rats and inhibited the increase in HSP70 expression acquired with exercise alone. The ET group showed higher levels of antioxidant enzyme activity, and VA inhibited this adaptation. Expression of the pro-inflammatory cytokines, interleukin (IL)-1ß, and tumor necrosis factor-α was reduced in the ET+VA group, while the anti-inflammatory cytokine, IL-10, was increased. Western blotting showed that both exercised groups had lower levels of the receptor for advanced glycation end products, suggesting that VA did not affect this receptor. Our study demonstrated that, although VA caused oxidative damage, a controlled administration might exert anti-inflammatory effects. Further studies with higher VA doses and longer ET interventions would elucidate more the effects of the supplementation and exercise on liver parameters.
Asunto(s)
Suplementos Dietéticos , Hígado/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Condicionamiento Físico Animal , Vitamina A/administración & dosificación , Administración Oral , Alanina Transaminasa/sangre , Animales , Antioxidantes , Aspartato Aminotransferasas/sangre , Citocinas/sangre , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Peroxidación de Lípido/efectos de los fármacos , Hígado/metabolismo , Masculino , Ratas , Ratas Wistar , Especies Reactivas de Oxígeno/metabolismo , Superóxido Dismutasa/sangre , Natación , Sustancias Reactivas al Ácido Tiobarbitúrico/metabolismoRESUMEN
Exercise training intensity is the major variant that influences the relationship between exercise, redox balance, and immune response. Supplement intake is a common practice for oxidative stress prevention; the effects of vitamin A (VA) on exercise training are not yet described, even though this molecule exhibits antioxidant properties. We investigated the role of VA supplementation on redox and immune responses of adult Wistar rats subjected to swimming training. Animals were divided into four groups: sedentary, sedentary + VA, exercise training, and exercise training + VA. Over eight weeks, animals were submitted to intense swimming 5 times/week and a VA daily intake of 450 retinol equivalents/day. VA impaired the total serum antioxidant capacity acquired by exercise, with no change in interleukin-1ß and tumor necrosis factor-α levels. In skeletal muscle, VA caused lipid peroxidation and protein damage without differences in antioxidant enzyme activities; however, Western blot analysis showed that expression of superoxide dismutase-1 was downregulated, and upregulation of superoxide dismutase-2 induced by exercise was blunted by VA. Furthermore, VA supplementation decreased anti-inflammatory interleukin-10 and heat shock protein 70 expression, important factors for positive exercise adaptations and tissue damage prevention. Our data showed that VA supplementation did not confer any antioxidative and/or protective effects, attenuating exercise-acquired benefits in the skeletal muscle.
Asunto(s)
Suplementos Dietéticos/efectos adversos , Proteínas HSP70 de Choque Térmico/antagonistas & inhibidores , Interleucina-10/antagonistas & inhibidores , Músculo Esquelético/metabolismo , Miositis/etiología , Estrés Oxidativo , Vitamina A/efectos adversos , Animales , Biomarcadores/sangre , Biomarcadores/metabolismo , Western Blotting , Proteínas HSP70 de Choque Térmico/metabolismo , Mediadores de Inflamación/sangre , Mediadores de Inflamación/metabolismo , Interleucina-10/metabolismo , Peroxidación de Lípido , Masculino , Músculo Esquelético/enzimología , Músculo Esquelético/inmunología , Miositis/sangre , Miositis/inmunología , Miositis/metabolismo , Oxidorreductasas/antagonistas & inhibidores , Oxidorreductasas/química , Oxidorreductasas/metabolismo , Capacidad de Absorbancia de Radicales de Oxígeno , Condicionamiento Físico Animal/efectos adversos , Distribución Aleatoria , Ratas WistarRESUMEN
Most scientific studies are too long to be conducted in a single day or even in a few days. Thus, there is a need to store samples for subsequent investigations. There is sparse information about specific sample storage protocols that minimize analytical error and variability in evaluations of redox parameters. Therefore, the effects of storage temperature and freezing time on enzymatic activities, protein oxidative damage, and CAT (catalase) and SOD1 (superoxide dismutase) immunocontent of blood, liver, and brain from rats were determined for two different sample forms (frozen homogenized tissue or frozen intact tissue). Superoxide dismutase activity was drastically decreased in blood and liver with an increase in freezing time, but not in brain. Catalase activity showed a decrease only in intact liver at -20 and -80°C. In contrast, in blood it showed an increase in intact tissue at -20 and -80°C. Reduced thiol groups generally decreased with freezing time, but showed an increase in intact blood at -20 and -80°C, probably because of color interference. Carbonyl groups in homogenized liver and brain, and in intact blood (except at 80°C) drastically increased with freezing time. Freezing time did not modulate the immunocontent of CAT and SOD1 levels in any tissue. In conclusion, our results indicate that storage at -20°C affects redox parameters more than storage at -80°C. Storage for a long time may compromise the samples, leading to changing parameters due to oxidative stress. Thus, we suggest processing the samples as soon as possible. However, if this is not possible, then material can be aliquoted into different tubes to prevent the effect of refreezing of samples.
Asunto(s)
Encéfalo/enzimología , Congelación , Hígado/enzimología , Manejo de Especímenes/normas , Animales , Encéfalo/inmunología , Catalasa/sangre , Catalasa/inmunología , Catalasa/metabolismo , Calefacción , Hígado/inmunología , Oxidación-Reducción , Ratas , Superóxido Dismutasa/sangre , Superóxido Dismutasa/inmunología , Superóxido Dismutasa/metabolismoRESUMEN
The receptor for advanced glycation endproducts (RAGE) is a pattern-recognition receptor associated with inflammation in most cell types. RAGE up-regulates the expression of proinflammatory mediators and its own expression via activation of NF-kB. Recent works have proposed a role for RAGE in Parkinson's disease (PD). In this study, we used the multimodal blocker of RAGE FPS-ZM1, which has become available recently, to selectively inhibit RAGE in the substantia nigra (SN) of rats intracranially injected with 6-hydroxydopamine (6-OHDA). FPS-ZM1 (40 µg per rat), injected concomitantly with 6-OHDA (10 µg per rat) into the SN, inhibited the increase in RAGE, activation of ERK1/2, Src and nuclear translocation of NF-kB p65 subunit in the SN. RAGE inhibition blocked glial fibrillary acidic protein and Iba-1 upregulation as well as associated astrocyte and microglia activation. Circulating cytokines in serum and CSF were also decreased by FPS-ZM1 injection. The loss of tyrosine hydroxylase and NeuN-positive neurons was significantly inhibited by RAGE blocking. Finally, FPS-ZM1 attenuated locomotory and exploratory deficits induced by 6-OHDA. Our results demonstrate that RAGE is an essential component in the neuroinflammation and dopaminergic denervation induced by 6-OHDA in the SN. Selective inhibition of RAGE may offer perspectives for therapeutic approaches.
Asunto(s)
Dopamina/metabolismo , Neuronas Dopaminérgicas/efectos de los fármacos , Neuronas Dopaminérgicas/metabolismo , Oxidopamina/efectos adversos , Receptor para Productos Finales de Glicación Avanzada/antagonistas & inhibidores , Sustancia Negra/metabolismo , Animales , Citocinas/metabolismo , Modelos Animales de Enfermedad , Neuronas Dopaminérgicas/patología , Mediadores de Inflamación/metabolismo , Masculino , FN-kappa B/metabolismo , Enfermedad de Parkinson/etiología , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/patología , Ratas , Sustancia Negra/efectos de los fármacos , Sustancia Negra/patología , Familia-src Quinasas/metabolismoRESUMEN
Human neuroblastoma SH-SY5Y cells have been used as an in vitro model for neurodegenerative disorders such as Parkinson's disease and can be induced to a mature neuronal phenotype through retinoic acid (RA) differentiation. However, mechanisms of RA-induced differentiation remain unclear. Here, we investigate the role of reactive species (RS) on SH-SY5Y neuroblastoma cells under RA differentiation, using the antioxidant Trolox® as co-treatment. We found that RA treatment for 7 days reduced the cell number and proliferative capacity and induced the expression of adult catecholaminergic/neuronal markers such as tyrosine hydroxylase (TH), ß-III tubulin, and enolase-2. Evaluation of intracellular RS production by DCFH oxidation assay and quantification of cell non-enzymatic antioxidant activity by TRAP demonstrated that RA increases RS production. Furthermore, mitochondrial NADH oxidation showed to be inhibited under differentiation with RA. Cells subjected to co-treatment with antioxidant Trolox® demonstrated a remaining proliferative capacity and a decrease in the pro-oxidant state and RS production. Besides, antioxidant treatment restores the mitochondrial NADH oxidation. Importantly, Trolox® co-treatment inhibited the appearance of morphological characteristics such as neurite extension and branching, and decreased the expression of TH, ß-III tubulin, and enolase-2 after a seven-day differentiation with RA, indicating that RS production is a necessary step in this process. Trolox® also inhibited the phosphorylation of Akt and ERK1/2, which are involved in differentiation and survival, respectively, of these cells. Altogether, these data indicate the presence of a redox-dependent mechanism in SH-SY5Y RA-differentiation process and can be a useful insight to improve understanding of neuronal differentiation signaling.
Asunto(s)
Biomarcadores/metabolismo , Ciclo Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Neuronas/citología , Estrés Oxidativo/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Tretinoina/farmacología , Regulación hacia Arriba/efectos de los fármacos , Antioxidantes/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Forma de la Célula/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Fenotipo , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismoRESUMEN
Glioblastoma multiforme (GBM) is the worst form of primary brain tumor, which has a high rate of infiltration and resistance to radiation and chemotherapy, resulting in poor prognosis for patients. Recent studies show that thiazolidinones have a wide range of pharmacological properties including antimicrobial, anti-inflammatory, anti-oxidant and anti-tumor. Here, we investigate the effect antiglioma in vitro of a panel of sixteen synthetic 2-aryl-3-((piperidin-1-yl)ethyl)thiazolidin-4-ones where 13 of these decreased the viability of glioma cells 30-65% (100 µM) compared with controls. The most promising compounds such as 4d, 4l, 4m and 4p promoted glioma reduction of viability greater than 50%, were further tested at lower concentrations (12.5, 25, 50 and 100 µM). Also, the data showed that the compounds 4d, 4l, 4m and 4p induced cell death primarily through necrosis and late apoptosis mechanisms. Interestingly, none of these 2-aryl-3-((piperidin-1-yl)ethyl)thiazolidin-4-ones were cytotoxic for primary astrocytes, which were used as a non-transformed cell model, indicating selectivity. Our results also show that the treatment with sub-therapeutic doses of 2-aryl-3-((piperidin-1-yl)ethyl)thiazolidin-4-ones (4d, 4l and 4p) reduced in vivo glioma growth as well as malignant characteristics of implanted tumors such as intratumoral hemorrhage and peripheral pseudopalisading. Importantly, 2-aryl-3-((piperidin-1-yl)ethyl)thiazolidin-4-ones treatment did not induce mortality or peripheral damage to animals. Finally, 2-aryl-3-((piperidin-1-yl)ethyl)thiazolidin-4-ones also changed the nitric oxide metabolism which may be associated with reduced growth and malignity characteristics of gliomas. These data indicates for the first time the therapeutic potential of synthetic 2-aryl-3-((piperidin-1-yl)ethyl)thiazolidin-4-ones to GBM treatment.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias Encefálicas/patología , Proliferación Celular/efectos de los fármacos , Glioblastoma/patología , Modelos Biológicos , Tiazolidinas/farmacología , Animales , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Ratas , Ratas WistarRESUMEN
SH-SY5Y cells, a neuroblastoma cell line that is a well-established model system to study the initial phases of neuronal differentiation, have been used in studies to elucidate the mechanisms of neuronal differentiation. In the present study, we investigated alterations of gene expression in SH-SY5Y cells during neuronal differentiation mediated by retinoic acid (RA) treatment. We evaluated important pathways involving nuclear factor kappa B (NF-κB), nuclear E2-related factor 2 (Nrf2), glycolytic, and p53 during neuronal differentiation. We also investigated the involvement of reactive oxygen species (ROS) in modulating the gene expression profile of those pathways by antioxidant co-treatment with Trolox®, a hydrophilic analogue of α-tocopherol. We found that RA treatment increases levels of gene expression of NF-κB, glycolytic, and antioxidant pathway genes during neuronal differentiation of SH-SY5Y cells. We also found that ROS production induced by RA treatment in SH-SY5Y cells is involved in gene expression profile alterations, chiefly in NF-κB, and glycolytic pathways. Antioxidant co-treatment with Trolox® reversed the effects mediated by RA NF-κB, and glycolytic pathways gene expression. Interestingly, co-treatment with Trolox® did not reverse the effects in antioxidant gene expression mediated by RA in SH-SY5Y. To confirm neuronal differentiation, we quantified endogenous levels of tyrosine hydroxylase, a recognized marker of neuronal differentiation. Our data suggest that during neuronal differentiation mediated by RA, changes in profile gene expression of important pathways occur. These alterations are in part mediated by ROS production. Therefore, our results reinforce the importance in understanding the mechanism by which RA induces neuronal differentiation in SH-SY5Y cells, principally due this model being commonly used as a neuronal cell model in studies of neuronal pathologies.
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Diferenciación Celular/efectos de los fármacos , Perfilación de la Expresión Génica , Glucólisis/genética , Factor 2 Relacionado con NF-E2/genética , FN-kappa B/genética , Neuronas/citología , Tretinoina/farmacología , Proteína p53 Supresora de Tumor/genética , Diferenciación Celular/genética , Línea Celular Tumoral , Regulación de la Expresión Génica/efectos de los fármacos , Redes Reguladoras de Genes/efectos de los fármacos , Glucólisis/efectos de los fármacos , Humanos , Factor 2 Relacionado con NF-E2/metabolismo , FN-kappa B/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Thiazolidinones, synthesized from multicomponent reactions of 2-heteroarylmethylamine, arenealdehydes and mercaptoacetic acid, have been tested against six yeasts, namely Candida albicans, C. parapsilosis, C. guilliermondii, Cryptococcus laurentii, Trichosporon asahii and Rhodotorula spp. The activities were expressed as minimum inhibitory concentrations (MIC) and the minimum fungicidal concentrations (MFC). The most affected yeasts were Rhodotorula spp and T. asahii. The cytotoxicities of the thiazolidinones against the fibroblast 3T3/NIH cell line are also described. The antifungal results and the low cytotoxicity of the compounds in this work provide good guides for the further development of active compounds.
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Antifúngicos/farmacología , Candida/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Rhodotorula/efectos de los fármacos , Tiazolidinas/farmacología , Trichosporon/efectos de los fármacos , Animales , Antifúngicos/química , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Ratones , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Células 3T3 NIH , Relación Estructura-Actividad , Tiazolidinas/químicaRESUMEN
Cellular and molecular mechanisms related to lung cancer have been extensively studied in recent years, but the availability of effective treatments is still scarce. Hecogenin acetate, a natural saponin presenting a wide spectrum of reported pharmacological activities, has been previously evaluated for its anticancer/antiproliferative activity in some in vivo and in vitro models. Here, we investigated the effects of hecogenin acetate in a human lung cancer cell line. A549 non-small lung cancer cells were exposed to different concentrations of hecogenin acetate and reactive species production, ERK1/2 activation, matrix metalloproteinase expression, cell cycle arrest and cell senescence parameters were evaluated. Hecogenin acetate significantly inhibited increase in intracellular reactive species production induced by H2O2. In addition, hecogenin acetate blocked ERK1/2 phosphorylation and inhibited the increase in MMP-2 caused by H2O2. Treatment with hecogenin acetate induced G0/G1-phase arrest at two concentrations (75 and 100 µM, 74% and 84.3% respectively), and increased the staining of senescence-associated ß -galactosidase positive cells. These data indicate that hecogenin acetate is able to exert anti-cancer effects by modulating reactive species production, inducing cell cycle arrest and senescence and also modulating ERK1/2 phosphorylation and MMP-2 production.
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Antineoplásicos/farmacología , Carcinoma de Pulmón de Células no Pequeñas/patología , Senescencia Celular/efectos de los fármacos , Puntos de Control de la Fase G1 del Ciclo Celular/efectos de los fármacos , Neoplasias Pulmonares/patología , Especies Reactivas de Oxígeno/metabolismo , Compuestos de Espiro/farmacología , Esteroides/farmacología , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Neoplasias Pulmonares/metabolismo , Metaloproteinasa 2 de la Matriz/metabolismo , Invasividad Neoplásica/patología , Estrés Oxidativo/efectos de los fármacos , FosforilaciónRESUMEN
The present study was elaborated to comparatively evaluate the preventive effect of different peach-derived products obtained from preserved fruits (Syrup and Preserve Pulp Peach [PPP]) and from fresh peels and pulps (Peel and Fresh Pulp Peach [FPP]) in a model of liver/renal toxicity and inflammation induced by carbon tetrachloride (CCl4) in rats. Tissue damage (carbonyl, thiobarbituric acid reactive species and sulfhydril), antioxidant enzymes activity (catalase and superoxide dismutase) and inflammatory parameters [tumor necrosis factor (TNF)-α and interleukin (IL)-1ß levels, and receptor for advanced glycation end-products (RAGE) and nuclear factor (NF)κB-p65 immunocontent] were investigated. Our findings demonstrated that Peel, PPP and FPP (200 or 400 mg/kg) daily administration by oral gavage for 30 days conferred a significant protection against CCl4-induced antioxidant enzymes activation and, most importantly, oxidative damage to lipids and proteins as well as blocked induction of inflammatory mediators such as TNF-α, IL-1ß, RAGE and NFκB. This antioxidant/anti-inflammatory effect seems to be associated with the abundance of carotenoids and polyphenols present in peach-derived products, which are enriched in fresh-fruit-derived preparations (Peel and FPP) but are also present in PPP. The Syrup - which was the least enriched in antioxidants - displayed no protective effect in our experiments. These effects cumulated in decreased levels of transaminases and lactate dehydrogenase leakage into serum and maintenance of organ architecture. Therefore, the herein presented results show evidence that supplementation with peach products may be protective against organ damage caused by oxidative stress, being interesting candidates for production of antioxidant-enriched functional foods.
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Tetracloruro de Carbono/efectos adversos , Frutas/química , Estrés Oxidativo/efectos de los fármacos , Preparaciones de Plantas/farmacología , Prunus/química , Alanina Transaminasa/sangre , Animales , Antioxidantes/farmacología , Aspartato Aminotransferasas/sangre , Bilirrubina/sangre , Glucemia/metabolismo , Carotenoides/análisis , Suplementos Dietéticos , Inflamación/inducido químicamente , Inflamación/tratamiento farmacológico , Interleucina-1beta/sangre , Riñón/efectos de los fármacos , Riñón/metabolismo , Hígado/efectos de los fármacos , Hígado/metabolismo , Masculino , FN-kappa B/sangre , Fitoterapia/métodos , Polifenoles/análisis , Ratas , Ratas Wistar , Receptor para Productos Finales de Glicación Avanzada , Receptores Inmunológicos/sangre , Superóxido Dismutasa/sangre , Sustancias Reactivas al Ácido Tiobarbitúrico/metabolismo , Factor de Necrosis Tumoral alfa/sangreRESUMEN
A series of sixteen novel thiazolidinone derivatives were synthesized from the efficient one-pot reaction of 2-(piperidin-1-yl)ethylamine, arenealdehydes and mercaptoacetic acid in good yields. Identification and characterization of products were achieved by NMR and GC-MS techniques. The in vitro antifungal activities of all synthesized compounds were evaluated against seven fungi: Candida albicans, Candida parapsilosis, Candida guilliermondii, Cryptococcus laurentii, Geotrichum sp, Trichosporon asahii and Rhodotorula sp. The results are expressed as the Minimum Inhibitory Concentration (MIC) and Minimum Fungicidal Concentration (MFC) and the best results were found against the Rhodotorula sp yeast. Two thiazolidinones (4h and 4l), MIC and MFC (16.5 µg/mL) proved to be 1.6 times more active than fluconazole and four of them (4b, 4e, 4g and 4k (MIC and MFC 25 µg/mL)) showed similar activity of standard drug to Rhodotorula sp. In addition, the cytotoxicity of thiazolidinones 4a-p was evaluated on cultured Vero cells and most of them displayed low toxicity (above 98 µg/mL). These preliminary and important results could be considered a starting point for the development of new antifungal agents.
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Antifúngicos/farmacología , Hongos/efectos de los fármacos , Piperidinas/farmacología , Tiazolidinas/farmacología , Animales , Antifúngicos/síntesis química , Antifúngicos/química , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Chlorocebus aethiops , Relación Dosis-Respuesta a Droga , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Piperidinas/síntesis química , Piperidinas/química , Relación Estructura-Actividad , Tiazolidinas/síntesis química , Tiazolidinas/química , Células VeroRESUMEN
BACKGROUND Leishmaniasis is a parasitosis caused by several species of the genus Leishmania. These parasites present high resistance against oxidative stress generated by inflammatory cells. OBJECTIVES To investigate oxidative stress and molecular inflammatory markers in BALB/c mice infected with L. amazonensis and the effect of antioxidant treatment on these parameters. METHODS Four months after infection, oxidative and inflammatory parameters of liver, kidneys, spleen, heart and lungs from BALB/c mice were assessed. FINDINGS In liver, L. amazonensis caused thiol oxidation and nitrotyrosine formation; SOD activity and SOD2 protein content were increased while SOD1 protein content decreased. The content of the cytokines IL-1β, IL-6, TNF-α, and the receptor of advanced glycation endproducts (RAGE) increased in liver. Treatment with the antioxidant N-acetyl-cysteine (20 mg/kg b.w) for five days inhibited oxidative stress parameters. MAIN CONCLUSIONS L. amazonensis induces significant alterations in the redox status of liver but not in other organs. Acute antioxidant treatment alleviates oxidative stress in liver, but it had no effect on pro-inflammatory markers. These results indicate that the pathobiology of leishmaniasis is not restricted to the cutaneous manifestations and open perspectives for the development of new therapeutic approaches to the disease, especially for liver function.