RESUMO
Grape pomace is the source of bioactive compounds (anthocyanins, flavonols, flavan-3-ols, and stilbenes) which exhibit antiproliferative actions on cell cultures. We have investigated the antitumoral effects of grape pomace and grape seed extracts on colon cancer cells (Caco-2, HT-29) and fibroblasts. Crude extracts prepared from white and red pomace, and grape seeds, reduced the viability and proliferation of Caco-2. HT-29 cells were resistant to these actions. Purified extracts were then prepared from the same sources and compared with the LDH test; again, all three extracts were active and purified extract from grape seed was the most potent and specific on Caco-2 cells. HT-29 cells were more sensitive to these purified extracts. The biological activity resided almost exclusively in the flavonol and flavan-3-ols subfractions, rather than the anthocyanin subfraction. Preliminary results on the mechanisms involved in these effects revealed downregulation of Myc gene expression in HT-29 and upregulation of Ptg2 in Caco-2 cells.
RESUMO
In this study, the role of CB2r on aversive memory consolidation was further evaluated. Mice lacking CB2r (CB2KO) and their corresponding littermates (WT) were exposed to the step-down inhibitory avoidance test (SDIA). MAP2, NF200 and synaptophysin (SYN)-immunoreactive fibers were studied in the hippocampus (HIP) of both genotypes. The number of synapses, postsynaptic density thickness and the relation between the synaptic length across the synaptic cleft and the distance between the synaptic ends were evaluated in the HIP (dentate gyrus (DG) and CA1 fields) by electron microscopy. Brain-derived neurotrophic factor (BDNF), glucocorticoid receptor (NR3C1) gene expressions and mTOR/p70S6K signaling cascade were evaluated in the HIP and prefrontal cortex (PFC). Finally, the effects of acute administration of CB2r-agonist JWH133 or CB2r-antagonist AM630 on memory consolidation were evaluated in WT mice by using the SDIA. The lack of CB2r impaired aversive memory consolidation, reduced MAP2, NF200 and SYN-immunoreactive fibers and also reduced the number of synapses in DG of CB2KO mice. BDNF and NR3C1 gene expression were reduced in the HIP of CB2KO mice. An increase of p-p70S6K (T389 and S424) and p-AKT protein expression was observed in the HIP and PFC of CB2KO mice. Interestingly, administration of AM630 impaired aversive memory consolidation, whereas JWH133 enhanced it. Further functional and molecular assessments would have been helpful to further support our conclusions. These results revealed that CB2r are involved in memory consolidation, suggesting that this receptor could be a promising target for developing novel treatments for different cognitive impairment-related disorders.
Assuntos
Transtornos da Memória/fisiopatologia , Plasticidade Neuronal/fisiologia , Receptor CB2 de Canabinoide/deficiência , Receptor CB2 de Canabinoide/fisiologia , Sinapses/ultraestrutura , Animais , Aprendizagem da Esquiva/fisiologia , Fator Neurotrófico Derivado do Encéfalo/biossíntese , Agonistas de Receptores de Canabinoides/farmacologia , Antagonistas de Receptores de Canabinoides/farmacologia , Canabinoides/farmacologia , Expressão Gênica/fisiologia , Hipocampo/metabolismo , Hipocampo/fisiopatologia , Indóis/farmacologia , Memória/efeitos dos fármacos , Memória/fisiologia , Transtornos da Memória/genética , Camundongos , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas de Neurofilamentos/metabolismo , Plasticidade Neuronal/genética , Córtex Pré-Frontal/metabolismo , Receptor CB2 de Canabinoide/genética , Receptores de Glucocorticoides/biossíntese , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Sinaptofisina/metabolismo , Serina-Treonina Quinases TOR/metabolismoRESUMO
Recent reports suggest that FKBP5 gene and its corresponding FKBP5 protein play a relevant role in the regulation of anxiety and depression in animal models and human stress-related disorders. In the present study, FKBP5 and glucocorticoid receptor (GR) gene and protein expression were analyzed in the amygdala (AMY) of suicide victims (n=13 males, without clinical psychiatric history and non-treated with anxiolytic or antidepressant drugs) and its corresponding controls (n=13 males) by real-time PCR and Western blotting. The results revealed that FKBP5 and GR gene expression were significantly reduced in the AMY (-38% and -48%, respectively) of suicide victims compared with controls. Interestingly, FKBP5 and GR protein expression were also significantly decreased (-41% and -42%, respectively) in the AMY of suicide victims compared with controls. These results suggest that the FKBP5 plays a relevant role in human emotional responses and suggest this receptor as a new promising target in the treatment of suicide behavior.
Assuntos
Tonsila do Cerebelo/metabolismo , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo , Suicídio , Proteínas de Ligação a Tacrolimo/genética , Proteínas de Ligação a Tacrolimo/metabolismo , Adolescente , Adulto , Idoso , Estudos de Casos e Controles , Expressão Gênica/genética , Humanos , Masculino , Pessoa de Meia-Idade , RNA/metabolismo , Receptores de Glucocorticoides/biossíntese , Proteínas de Ligação a Tacrolimo/biossínteseRESUMO
BACKGROUND AND PURPOSE: Impulsivity is a core symptom in many neuropsychiatric disorders. The main objective of this study was to evaluate the effects of topiramate and pregabalin on the modulation of different impulsivity dimensions in DBA/2 mice. EXPERIMENTAL APPROACH: The effects of acute and chronic administration of pregabalin (10, 20 and 40 mg·kg(-1) ) and topiramate (12.5, 25 and 50 mg·kg(-1) ) were evaluated in the light-dark box (LDB), hole board test (HBT) and delayed reinforcement task (DRT). α(2A) -Adrenoceptor, D(2) -receptor and TH gene expression were evaluated by real-time PCR in the prefrontal cortex (PFC), accumbens (ACC) and ventral tegmental area (VTA), respectively. KEY RESULTS: Acute pregabalin administration showed a clear anxiolytic-like effect (LDB) but did not modify novelty-seeking behaviour (HBT). In contrast, topiramate produced an anxiolytic effect only at the highest dose, whereas it reduced novelty seeking at all doses tested. In the DRT, acute pregabalin had no effect, whereas topiramate only reduced motor impulsivity. Chronically, pregabalin significantly increased motor impulsivity and topiramate diminished cognitive impulsivity. Pregabalin decreased α(2A) -adrenoceptor and D(2) -receptor gene expression in the PFC and ACC, respectively, and increased TH in the VTA. In contrast, chronic administration of topiramate increased α(2A) -adrenoceptor and D(2) -receptor gene expression in the PFC and ACC, respectively, and also increased TH in the VTA. CONCLUSIONS AND IMPLICATIONS: These results suggest that the usefulness of pregabalin in impulsivity-related disorders is related to its anxiolytic properties, whereas topiramate modulates impulsivity. These differences could be linked to their opposite effects on α(2A) -adrenoceptor and D(2) -receptor gene expression in the PFC and ACC, respectively.
Assuntos
Anticonvulsivantes/uso terapêutico , Ansiedade/tratamento farmacológico , Frutose/análogos & derivados , Comportamento Impulsivo/tratamento farmacológico , Ácido gama-Aminobutírico/análogos & derivados , Animais , Anticonvulsivantes/farmacologia , Ansiedade/fisiopatologia , Comportamento Animal/efeitos dos fármacos , Cognição/efeitos dos fármacos , Frutose/farmacologia , Frutose/uso terapêutico , Comportamento Impulsivo/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos DBA , Atividade Motora/efeitos dos fármacos , Núcleo Accumbens/metabolismo , Córtex Pré-Frontal/metabolismo , Pregabalina , Receptores Adrenérgicos alfa 2/genética , Receptores de Dopamina D2/genética , Topiramato , Área Tegmentar Ventral/metabolismo , Ácido gama-Aminobutírico/farmacologia , Ácido gama-Aminobutírico/uso terapêuticoRESUMO
BACKGROUND AND PURPOSE: This study evaluated gene expression differences between two mouse strains, characterized by opposite impulsivity-like traits and the involvement of the cannabinoid CB(2) receptor in the modulation of impulsivity. EXPERIMENTAL APPROACH: Behavioural tests were conducted to compare motor activity, exploration and novelty seeking, attention and cognitive and motor impulsivity (delayed reinforcement task: session duration 30 min; timeout 30 s) between A/J and DBA/2 mice. Expression of genes for dopamine D(2) receptors, CB(1) and CB(2) receptors were measured in the cingulate cortex (CgCtx), caudate-putamen (CPu), accumbens (Acc), amygdala (Amy) and hippocampus (Hipp). Involvement of CB(2) receptors in impulsivity was evaluated in DBA/2 mice with a CB(2) receptor agonist (JWH133) and an antagonist (AM630). KEY RESULTS: DBA/2 mice presented higher motor and exploratory activity, pre-pulse inhibition impairment and higher cognitive and motor impulsivity level than A/J mice. In addition, DBA/2 mice showed lower (CgCtx, Acc, CPu) D(2) receptor, lower (Amy) and higher (CgCtx, Acc, CPu, Hipp) CB(1) receptor and higher (CgCtx, Acc, Amy) and similar (CPu, Hipp) CB(2) receptor gene expressions. Treatment with JWH133 (0.5, 1, 3 mg·kg(-1), i.p.) reduced cognitive and motor impulsivity level, accompanied by CB(2) receptor down-regulation (CgCtx, Acc, Amy) but did not modify other behaviours. In contrast, AM630 (1, 2, 3 mg·kg(-1), i.p.) improved pre-pulse inhibition and reduced novelty seeking behaviour in DBA/2 mice. CONCLUSIONS AND IMPLICATIONS: CB(2) receptors might play an important role in regulating impulsive behaviours and should be considered a promising therapeutic target in the treatment of impulsivity-related disorders.
Assuntos
Canabinoides/farmacologia , Comportamento Impulsivo/tratamento farmacológico , Indóis/farmacologia , Receptor CB2 de Canabinoide/metabolismo , Animais , Relação Dose-Resposta a Droga , Regulação da Expressão Gênica/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos DBA , Receptor CB2 de Canabinoide/genéticaRESUMO
The role of CB2 cannabinoid receptors in the behavioral and neurochemical changes induced by intracaudate administration of 6-hydroxydopamine (6-OHDA) was evaluated. 6-OHDA (12 µg/4 µL) or its vehicle was injected in the caudate-putamen (CPu) of mice overexpressing the CB2 cannabinoid receptor (CB2xP) and wild type (WT) mice. Motor impairment, emotional behavior, and cognitive alterations were evaluated. Tyrosine hydroxylase (TH), glial fibrillary acidic protein (GFAP), and ionized calcium-binding adapter molecule 1 (Iba-1) were measured by immunocytochemistry in the CPu and/or substantia nigra (SN) of CB2xP mice and WT mice. Oxidative/nitrosative and neuroinflammatory parameters were also measured in the CPu and cortex of 6-OHDA-treated and sham-treated mice. 6-OHDA-treated CB2xP mice presented significantly less motor deterioration than 6-OHDA-treated WT mice. Immunocytochemical analysis of tyrosine hydroxylase in the SN and CPu revealed significantly fewer lesions in CB2xP mice than in WT mice. GFAP and Iba-1 immunostaining revealed less astrocyte and microglia recruitment to the treated area of the CPu in CB2xP mice. Malonyldialdehyde (MDA) concentrations were lower in the striatum and cerebral cortex of sham-treated CB2xP mice than in sham-treated WT mice. The administration of 6-OHDA increased MDA levels in both WT mice and CB2xP mice; it increased the oxidized (GSSG)/reduced (GSH) glutathione ratio in the striatum in WT mice alone compared with matched sham-treated controls. The results revealed that overexpression of CB2 cannabinoid receptors decreased the extent of motor impairment and dopaminergic neuronal loss, reduced the recruitment of astrocytes and microglia to the lesion, and decreased the level of various oxidative parameters. These results suggest that CB2 receptors offer neuroprotection against dopaminergic injury.
Assuntos
Comportamento Animal/efeitos dos fármacos , Comportamento Animal/fisiologia , Núcleo Caudado/fisiologia , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Oxidopamina/farmacologia , Receptor CB2 de Canabinoide/metabolismo , Adrenérgicos/farmacologia , Animais , Núcleo Caudado/efeitos dos fármacos , Quimiocinas/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Fármacos Neuroprotetores/farmacologia , Regulação para Cima/efeitos dos fármacosRESUMO
Ca2+-dependent calcineurin is upregulated in reactive astrocytes in neuroinflammatory models. Therefore, the fact that the nuclear factor of activated T cells (NFAT) is activated in response to calcineurin qualifies this family of transcription factors with immune functions as candidates to mediate astrogliosis. Brain trauma induces a neuroinflammatory state in which ATP is released from astrocytes, stimulating calcium signalling. Our goal here is to characterize NFATc1 and NFATc2 in mouse primary astrocyte cultures, also exploring the implication of NFAT in astrocyte activation by mechanical lesion. Quantitative reverse transcriptase-polymerase chain reaction, Western blot analysis and immunofluorescence microscopy identified NFATc1 in astrocytes, but not NFATc2. Moreover, NFATc1 was expressed in the cytosol of resting astrocytes, whereas activation of the Ca2+-calcineurin pathway by ionomycin translocated NFATc1 to the nucleus, which is a requirement for activation. The implication of astrocytic NFAT in brain trauma was analysed using an in vitro scratch lesion model. Mechanical lesion caused a rapid NFATc1 translocation that progressed throughout the culture as a gradient and was maintained for at least 4 h. We also demonstrate that ATP, released by lesion, is a potent inducer of NFATc1 translocation and activation. Moreover, the use of P2Y receptor modulators showed that such ATP action is mediated by stimulation of several G(q)-protein-coupled P2Y purinergic receptors, among which P2Y(1) and P2Y(6) are included. In conclusion, this work provides evidence that newly identified NFATc1 is translocated in astrocytes in response to lesion following a pathway that involves ATP release and activation of metabotropic purinergic receptors.
Assuntos
Trifosfato de Adenosina/metabolismo , Astrócitos/metabolismo , Fatores de Transcrição NFATC/metabolismo , Receptores Purinérgicos/metabolismo , Animais , Astrócitos/patologia , Western Blotting , Células Cultivadas , Citoplasma/química , Citoplasma/metabolismo , Ativação Enzimática/fisiologia , Imunofluorescência , Camundongos , Camundongos Endogâmicos C57BL , Fatores de Transcrição NFATC/genética , Transporte Proteico/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição GênicaRESUMO
The glitazones (or thiazolidinediones) are synthetic compounds used in type-2 diabetes, but they also have broad antiproliferative and anti-inflammatory properties still not well understood. We described previously the apoptotic effects of glitazones on astroglioma cells ( J Biol Chem 279: 8976-8985, 2004 ). At certain concentrations, we found a selective lethality on glioma cells versus astrocytes that was dependent on a rapid production of reactive oxygen species (ROS) and seemed unrelated to the receptor peroxisome proliferator activated receptor-gamma. The present study was aimed at characterizing the oxygen derivatives induced by ciglitazone, rosiglitazone, and pioglitazone in C6 glioma cells and to investigate their intracellular source. We examined the interaction of ROS with nitric oxide (NO) and its consequences for glioma cell survival. Fluorescence microscopy and flow cytometry showed that glitazones induced superoxide anion, peroxynitrite, and hydrogen peroxide, with ciglitazone being the most active. ROS production was completely prevented by uncoupling of the electron transport chain and by removal of glucose as an energy substrate, whereas it was unaffected by inhibition of NADPH-oxidase and xanthine-oxidase. Moreover, glitazones inhibited state 3 respiration in permeabilized cells, and experiments with mitochondrial inhibitors suggested that complex I was the likely target of glitazones. Therefore, these results point to the mitochondrial electron transport chain as the source of glitazone-induced ROS in C6 cells. Glitazones also depolarized mitochondria and reduced mitochondrial pH. NO synthase inhibitors revealed that superoxide anion combines with NO to yield peroxynitrite and that the latter contributes to the cytotoxicity of glitazones in astroglioma cells. Future antitumoral strategies may take advantage of these findings.
Assuntos
Astrocitoma/tratamento farmacológico , Mitocôndrias/efeitos dos fármacos , Óxido Nítrico/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Tiazolidinedionas/farmacologia , Animais , Astrocitoma/metabolismo , Astrocitoma/patologia , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/farmacologia , Linhagem Celular Tumoral , Transporte de Elétrons/efeitos dos fármacos , Concentração de Íons de Hidrogênio , Mitocôndrias/metabolismo , NADPH Oxidases/fisiologia , NG-Nitroarginina Metil Éster/farmacologia , Ratos , Superóxidos/metabolismoRESUMO
The glitazones or thiazolidinediones are ligands of the peroxisome proliferator-activated receptor gamma (PPARgamma). The glitazones are used in the treatment of diabetes, regulate adipogenesis, inflammation, cell proliferation, and induce apoptosis in several cancer cell types. High grade astrocytomas are rapidly growing tumors derived from astrocytes, for which new treatments are needed. We determined the effects of two glitazones, ciglitazone and the therapeutic rosiglitazone, on the survival of serum-deprived primary rat astrocytes and glioma cell lines C6 and U251, which were assessed by the methylthiazolyl tetrazolium assay and lactate dehydrogenase release. Rosiglitazone (5-20 microM) decreased survival of glioma cells without affecting primary astrocytes, whereas ciglitazone at 20 microM was toxic for both cell types. Ciglitazone at 10 microM was cytoprotective for primary astrocytes but toxic to glioma cells. Cell death induced by ciglitazone, but not rosiglitazone, presented apoptotic features (Hoechst staining and externalization of phosphatidylserine). Two mechanisms to explain cytotoxicity were investigated: activation of PPARgamma and production of reactive oxygen species (ROS). PPARgamma does not seem to be the main mechanism involved, because the order of efficacy for cytotoxicity, ciglitazone > rosiglitazone, was inverse of their reported affinities for activating PPARgamma. In addition, GW9662, an inhibitor of PPARgamma, only slightly attenuated cytotoxicity. However, the rapid increase in ROS production and the marked reduction of cell death with the antioxidants ebselen and N-acetylcysteine, indicate that ROS have a key role in glitazone cytotoxicity. Ciglitazone caused a dose-dependent and rapid loss (in minutes) of mitochondrial membrane potential in glioma cells. Therefore, mitochondria are a likely source of ROS and early targets of glitazone cytotoxicity. Our results highlight the potential of rosiglitazone and related compounds for the treatment of astrogliomas.