RESUMO
Besides the standard parameters used for colorectal cancer (CRC) management, new features are needed in clinical practice to improve progression-free and overall survival. In some cancers, the microenvironment mechanical properties can contribute to cancer progression and metastasis formation, or constitute a physical barrier for drug penetration or immune cell infiltration. These mechanical properties remain poorly known for colon tissues. Using a multidisciplinary approach including clinical data, physics and geostatistics, we characterized the stiffness of healthy and malignant colon specimens. For this purpose, we analyzed a prospective cohort of 18 patients with untreated colon adenocarcinoma using atomic force microscopy to generate micrometer-scale mechanical maps. We characterized the stiffness of normal epithelium samples taken far away or close to the tumor area and selected tumor tissue areas. These data showed that normal epithelium was softer than tumors. In tumors, stroma areas were stiffer than malignant epithelial cell areas. Among the clinical parameters, tumor left location, higher stage, and RAS mutations were associated with increased tissue stiffness. Thus, in patients with CRC, measuring tumor tissue rigidity may have a translational value and an impact on patient care.
Assuntos
Adenocarcinoma , Neoplasias do Colo , Neoplasias Colorretais , Adenocarcinoma/genética , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Humanos , Estudos Prospectivos , Microambiente TumoralRESUMO
Gamma-L-glutamyl-L-glutamate (γ-Glu-Glu) was synthetized and further characterized for its activity on cultured neurons. We observed that γ-Glu-Glu elicited excitatory effects on neurons likely by activating mainly the N-methyl-D-aspartate (NMDA) receptors. These effects were dependent on the integrity of synaptic transmission as they were blocked by tetrodotoxin (TTX). We next evaluated its activity on NMDA receptors by testing it on cells expressing these receptors. We observed that γ-Glu-Glu partially activated NMDA receptors and exhibited better efficacy for NMDA receptors containing the GluN2B subunit. Moreover, at low concentration, γ-Glu-Glu potentiated the responses of glutamate on NMDA receptors. Finally, the endogenous production of γ-Glu-Glu was measured by LC-MS on the extracellular medium of C6 rat astroglioma cells. We found that extracellular γ-Glu-Glu concentration was, to some extent, directly linked to GSH metabolism as γ-Glu-Glu can be a by-product of glutathione (GSH) breakdown after γ-glutamyl transferase action. Therefore, γ-Glu-Glu could exert excitatory effects by activating neuronal NMDA receptors when GSH production is enhanced.
RESUMO
The regulation of the redox status involves the activation of intracellular pathways as Nrf2 which provides hormetic adaptations against oxidative stress in response to environmental stimuli. In the brain, Nrf2 activation upregulates the formation of glutathione (GSH) which is the primary antioxidant system mainly produced by astrocytes. Astrocytes have also been shown to be themselves the target of oxidative stress. However, how changes in the redox status itself could impact the intracellular Ca2+ homeostasis in astrocytes is not known, although this could be of great help to understand the neuronal damage caused by oxidative stress. Indeed, intracellular Ca2+ changes in astrocytes are crucial for their regulatory actions on neuronal networks. We have manipulated GSH concentration in astroglioma cells with selective inhibitors and activators of the enzymes involved in the GSH cycle and analyzed how this could modify Ca2+ homeostasis. IP3-mediated store-operated calcium entry (SOCE), obtained after store depletion elicited by Gq-linked purinergic P2Y receptors activation, are either sensitized or desensitized, following GSH depletion or increase, respectively. The desensitization may involve decreased expression of the proteins STIM2, Orai1, and Orai3 which support SOCE mechanism. The sensitization process revealed by exposing cells to oxidative stress likely involves the increase in the activity of Calcium Release-Activated Channels (CRAC) and/or in their membrane expression. In addition, we observe that GSH depletion drastically impacts P2Y receptor-mediated changes in membrane currents, as evidenced by large increases in Ca2+-dependent K+ currents. We conclude that changes in the redox status of astrocytes could dramatically modify Ca2+ responses to Gq-linked GPCR activation in both directions, by impacting store-dependent Ca2+-channels, and thus modify cellular excitability under purinergic stimulation.
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Acute effects of ghrelin on excitatory synaptic transmission were evaluated on hippocampal CA1 synapses. Ghrelin triggered an enduring enhancement of synaptic transmission independently of NMDA receptor activation and probably via postsynaptic modifications. This ghrelin-mediated potentiation resulted from the activation of GHS-R1a receptors as it was mimicked by the selective agonist JMV1843 and blocked by the selective antagonist JMV2959. This potentiation also required the activation of PKA and ERK pathways to occur as it was inhibited by KT5720 and U0126, respectively. Moreover it most probably involved Ca(2+) influxes as both ghrelin and JMV1843 elicited intracellular Ca(2+) increases, which were dependent on the presence of extracellular Ca(2+) and mediated by L-type Ca(2+) channels opening. In addition, ghrelin potentiated AMPA receptor-mediated [Ca(2+) ]i increases while decreasing NMDA receptor-mediated ones. Thus the potentiation of synaptic transmission by GHS-R1a at hippocampal CA1 excitatory synapses probably results from postsynaptic mechanisms involving PKA and ERK activation, which are producing long-lasting enhancement of AMPA receptor-mediated responses.
Assuntos
Região CA1 Hipocampal/fisiologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Grelina/metabolismo , Potenciação de Longa Duração/fisiologia , Sistema de Sinalização das MAP Quinases/fisiologia , Transmissão Sináptica/fisiologia , Animais , Região CA1 Hipocampal/efeitos dos fármacos , Cálcio/metabolismo , Canais de Cálcio Tipo L/metabolismo , Células Cultivadas , Fármacos do Sistema Nervoso Central/administração & dosagem , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Grelina/administração & dosagem , Potenciação de Longa Duração/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Ratos Sprague-Dawley , Receptores de AMPA/metabolismo , Receptores de Grelina/agonistas , Receptores de Grelina/antagonistas & inibidores , Receptores de Grelina/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Transmissão Sináptica/efeitos dos fármacos , Técnicas de Cultura de TecidosRESUMO
BACKGROUND: Prenatal infection is a major risk factor for the occurrence of neuropsychiatric disorders. These have been associated with hippocampal neuroanatomical and functional abnormalities. In the present study, we evaluated the occurrence of pyramidal cell disarray and reelin neuronal deficit in the hippocampus, and the protective role of N-acetyl-cysteine (NAC) in a rodent experimental model of prenatal immune challenge. METHODS: Sprague-Dawley rats received either 500 µg/kg of endotoxin (lipopolysaccharide, LPS) or 2 ml/kg of isotonic saline by i.p. injection on day 19 of gestation. After LPS injection, rats were or were not maintained on a preventive treatment of NAC (5 g/l in tap water), up to delivery. The pyramidal cell orientation and the number and type of reelin-expressing neurons were determined in male offspring. RESULTS: Prenatal LPS challenge led to permanent pyramidal cell disarray and to an early and transient decreased density of reelin-immunoreactive neurons. These disorders, more pronounced in the CA3 area, were prevented by NAC. CONCLUSION: Hippocampal cytoarchitectural alterations and reelin deficiency may be involved in the development of remote cognitive impairments in this model. The antioxidant NAC is an efficient neuroprotective drug that underlines the role of oxidative stress in prenatal infection and associated neurodevelopmental damage.
Assuntos
Acetilcisteína/farmacologia , Região CA3 Hipocampal/efeitos dos fármacos , Moléculas de Adesão Celular Neuronais/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Exposição Materna , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Células Piramidais/efeitos dos fármacos , Serina Endopeptidases/metabolismo , Animais , Região CA3 Hipocampal/citologia , Feminino , Masculino , Gravidez , Ratos , Ratos Sprague-Dawley , Proteína ReelinaRESUMO
Prenatal infection is a major stressful experience leading to enhanced susceptibility for mental illnesses in humans. We recently reported in rats, that oxidative stress and glutathione (GSH) shortage occurred in fetal male brain after lipopolysaccharide (LPS) to the dams and that these responses might be involved in the neurodevelopmental deficits observed in adolescent offspring. Furthermore, pretreatment with N-acetylcysteine (NAC) before LPS avoided both delayed synaptic plasticity and mnesic performance deficits. Since NAC is one of the few medications permitted in pregnant women, this study evaluated the ability of NAC to serve as a protective therapy even after the LPS challenge. Pregnant rats received a single ip injection of E. coli LPS, two days before delivery, and were given NAC in their tap water after the LPS. GSH was evaluated at the time of its expected drop in the hippocampus of male fetuses, whereas long-term potentiation (LTP) in the CA1 area of the hippocampus and spatial memory in the water-maze were recorded in 28-day-old male offspring. Post-treatment with NAC, four hours after the LPS challenge fully prevented the drop in the GSH hippocampal content. LTP, as well as spatial learning were completely protected. NAC administration at delivery also partially restored the LTP whereas post-treatment two days later was inefficient. Another set of dams were supplemented with alpha-tocopherol prior to LPS exposure, enhancing the alpha-tocopherol levels in fetal hippocampus. This treatment did not prevent the LPS-induced synaptic plasticity impairment. These results point to fetal hippocampal GSH as a major target of the detrimental effects of in utero LPS challenge. The therapeutic window of NAC extends up to birth, suggesting that this drug might be clinically useful even after an immuno-inflammatory episode.
Assuntos
Acetilcisteína/administração & dosagem , Endotoxemia/tratamento farmacológico , Potenciação de Longa Duração , Exposição Materna , Transtornos da Memória/prevenção & controle , Fármacos Neuroprotetores/administração & dosagem , Complicações na Gravidez/tratamento farmacológico , Efeitos Tardios da Exposição Pré-Natal/prevenção & controle , Acetilcisteína/farmacologia , Acetilcisteína/uso terapêutico , Animais , Antioxidantes/administração & dosagem , Antioxidantes/farmacologia , Antioxidantes/uso terapêutico , Esquema de Medicação , Avaliação Pré-Clínica de Medicamentos , Endotoxemia/imunologia , Endotoxemia/fisiopatologia , Feminino , Glutationa/análise , Glutationa/deficiência , Hipocampo/química , Hipocampo/embriologia , Hipocampo/patologia , Lipopolissacarídeos/toxicidade , Potenciação de Longa Duração/efeitos dos fármacos , Potenciação de Longa Duração/fisiologia , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Aprendizagem em Labirinto/fisiologia , Transtornos da Memória/etiologia , Transtornos da Memória/fisiopatologia , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/uso terapêutico , Gravidez , Complicações na Gravidez/imunologia , Efeitos Tardios da Exposição Pré-Natal/fisiopatologia , Ratos , Ratos Sprague-Dawley , alfa-Tocoferol/administração & dosagem , alfa-Tocoferol/análise , alfa-Tocoferol/uso terapêuticoRESUMO
Prenatal infection is a major risk responsible for the occurrence of psychiatric conditions in infants. Mimicking maternal infection by exposing pregnant rodents to bacterial endotoxin lipopolysaccharide (LPS) also leads to major brain disorders in the offspring. The mechanisms of LPS action remain, however, unknown. Here, we show that LPS injection during pregnancy in rats, 2 days before delivery, triggered an oxidative stress in the hippocampus of male fetuses, evidenced by a rapid rise in protein carbonylation and by decreases in alpha-tocopherol levels and in the ratio of reduced/oxidized forms of glutathione (GSH/GSSG). Neither protein carbonylation increase nor decreases in alpha-tocopherol levels and GSH/GSSG ratio were observed in female fetuses. NMDA synaptic currents and long-term potentiation in CA1, as well as spatial recognition in the water maze, were also impaired in male but not in female 28-day-old offspring. Pretreatment with the antioxidant N-acetylcysteine prevented the LPS-induced changes in the biochemical markers of oxidative stress in male fetuses, and the delayed detrimental effects in male 28-day-old offspring, completely restoring both long-term potentiation in the hippocampus and spatial recognition performance. Oxidative stress in the hippocampus of male fetuses may thus participate in the neurodevelopmental damage induced by a prenatal LPS challenge.
Assuntos
Encéfalo/embriologia , Infecções/embriologia , Estresse Oxidativo , Animais , Encefalopatias/induzido quimicamente , Encefalopatias/embriologia , Encefalopatias/etiologia , Cromatografia Líquida de Alta Pressão , Feminino , Glutationa/metabolismo , Dissulfeto de Glutationa/metabolismo , Hipocampo/embriologia , Hipocampo/fisiopatologia , Lipopolissacarídeos/toxicidade , Técnicas de Patch-Clamp , Gravidez , Ratos , Ratos Sprague-Dawley , alfa-Tocoferol/metabolismoRESUMO
Naturally occurring polyphenols are potent antioxidants. Some of these compounds are also ligands for the GABA(A) receptor benzodiazepine site. This feature endows them with sedative properties. Here, the anxiolytic activity of the green tea polyphenol (-)-epigallocatechin gallate (EGCG) was investigated after acute administration in mice, using behavioral tests (elevated plus-maze and passive avoidance tests) and by electrophysiology on cultured hippocampal neurons. Patch-clamp experiments revealed that EGCG (1-10 muM) had no effect on GABA currents. However, EGCG reversed GABA(A) receptor negative modulator methyl beta-carboline-3-carboxylate (beta-CCM) inhibition on GABA currents in a concentration dependent manner. This was also observed at the level of synaptic GABA(A) receptors by recording spontaneous inhibitory synaptic transmission. In addition, EGCG consistently inhibited spontaneous excitatory synaptic transmission. Behavioral tests indicated that EGCG exerted both anxiolytic and amnesic effects just like the benzodiazepine drug, chlordiazepoxide. Indeed, EGCG in a dose-dependent manner both increased the time spent in open arms of the plus-maze and decreased the step-down latency in the passive avoidance test. GABA(A) negative modulator beta-CCM antagonized EGCG-induced amnesia. Finally, state-dependent learning was observable after chlordiazepoxide and EGCG administration using a modified passive avoidance procedure. Optimal retention was observed only when animals were trained and tested in the same state (veh-veh or drug-drug) and significant retrieval alteration was observed in different states (veh-drug or drug-veh). Moreover, EGCG and chlordiazepoxide fully generalized in substitution studies, indicating that they induced indistinguishable chemical states for the brain. Therefore, our data support that EGCG can induce anxiolytic activity which could result from an interaction with GABA(A) receptors.
Assuntos
Ansiolíticos/uso terapêutico , Ansiedade/tratamento farmacológico , Catequina/análogos & derivados , Animais , Ansiedade/etiologia , Aprendizagem da Esquiva/efeitos dos fármacos , Comportamento Animal/efeitos dos fármacos , Carbolinas/farmacologia , Catequina/uso terapêutico , Células Cultivadas , Clordiazepóxido/farmacologia , Convulsivantes/farmacologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Interações Medicamentosas , Estimulação Elétrica/métodos , Eletrochoque/efeitos adversos , Embrião de Mamíferos , Hipocampo/citologia , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Camundongos , Neurônios/efeitos dos fármacos , Técnicas de Patch-Clamp/métodos , Tempo de Reação/efeitos dos fármacos , Ácido gama-Aminobutírico/farmacologiaRESUMO
Low frequency-induced short-term synaptic plasticity was investigated in hippocampal slices with 60-electrode recording array. Remarkably, the application of low-frequency stimulation (1 Hz) for a short duration (3-5 min) resulted in the induction of a slow-onset long-term potentiation (LTP) in the immediate vicinity of the stimulated electrode. This phenomenon was observed exclusively in the CA1 subfield, neither in the CA3 area nor in the dentate gyrus. The induction of this slow-onset LTP required neither N-methyl-D-aspartate (NMDA) nor non-NMDA ionotropic receptor activation but was strongly dependent on metabotropic glutamate mGlu(5) receptor stimulation and [Ca(2+)]i increase. In addition, this form of synaptic plasticity was associated with an increase in cAMP concentration and required protein kinase A activation. Paired-pulse facilitation ratio and presynaptic fiber volley amplitude were unaffected when this LTP was triggered, suggesting the involvement of postsynaptic modifications. Although mitogen activated protein kinase pathway was stimulated after the application of low frequency, the induction and maintenance of this slow-onset LTP were not dependent on the activation of this intracellular pathway. The direct activation of adenylyl cyclase with forskolin also induced a synaptic enhancement displaying similar features. This new form of LTP could represent the mnesic engram of mild and repetitive stimulation involved in latent learning.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Hipocampo/metabolismo , Potenciação de Longa Duração/fisiologia , Receptores de Glutamato Metabotrópico/metabolismo , Sinapses/metabolismo , Transmissão Sináptica/fisiologia , Adenilil Ciclases/efeitos dos fármacos , Adenilil Ciclases/metabolismo , Animais , Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/fisiologia , Colforsina/farmacologia , AMP Cíclico/metabolismo , Estimulação Elétrica/métodos , Inibidores Enzimáticos/farmacologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/fisiologia , Feminino , Hipocampo/efeitos dos fármacos , Aprendizagem/fisiologia , Potenciação de Longa Duração/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/fisiologia , Masculino , Memória/fisiologia , Vias Neurais/efeitos dos fármacos , Vias Neurais/metabolismo , Técnicas de Cultura de Órgãos , Terminações Pré-Sinápticas/efeitos dos fármacos , Terminações Pré-Sinápticas/metabolismo , Ratos , Ratos Sprague-Dawley , Receptor de Glutamato Metabotrópico 5 , Receptores de Glutamato Metabotrópico/efeitos dos fármacos , Sinapses/efeitos dos fármacos , Transmissão Sináptica/efeitos dos fármacosRESUMO
Neuroprotection exerted by alpha-tocopherol against oxidative stress was investigated in cultured rat hippocampal neurons. In addition to its direct action as a radical scavenger revealed at concentrations above 10 microM, a transient application of 1 microM alpha-tocopherol phosphate (alpha-TP) to neurons induced a complete delayed long-lasting protection against oxidative insult elicited by exposure to Fe2+ ions, but not against excitotoxicity. A minimal 16-h application of alpha-TP was required to observe the protection against subsequent oxidative stress. This delayed protection could last up to a week after the application of alpha-TP, even when medium was changed after the alpha-TP treatment. Cycloheximide, added either 2 h before or together with alpha-TP, prevented the delayed neuroprotection, but not the acute. However, cycloheximide applied after the 16-h alpha-TP pretreatment did not alter the delayed neuroprotection. Neither Trolox, a cell-permeant analogue of alpha-tocopherol, nor other antioxidants, such as epigallocatechin-gallate and N-acetyl-L-cysteine, elicited a similar long-lasting protection. Only tert-butylhydroquinone could mimic the alpha-TP effect. Depletion of glutathione (GSH) by L-buthionine sulfoximine did not affect the delayed alpha-TP protection. Thus, in addition to its acute anti-radical action, alpha-TP induces a long-lasting protection of neurons against oxidative damage, via a genomic action on antioxidant defenses apparently unrelated to GSH biosynthesis.