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Accumulating evidence indicates time-of-day variations in ischemic neuronal injury. Under ischemic conditions, Zn2+ is massively released from hippocampal glutamatergic neurons, and intracellular Zn2+ accumulation results in neuron death. Notably, excitatory amino acid carrier 1 (EAAC1), known as a cysteine transporter, is involved in Zn2+ homeostasis, and its expressions exhibit a diurnal fluctuation. This study aimed to investigate whether time of day of an ischemic insult affects Zn2+ accumulation and neuronal injury and determine whether altered Zn2+ accumulation is modulated by EAAC1 diurnal fluctuation in the hippocampus in a mouse model of ischemic stroke. Mice subjected to transient global ischemia for 40 min at Zeitgeber time 18 (ZT18) (23:00) exhibited reduced Zn2+ accumulation and neuronal death in the hilar region of the hippocampus compared to those at ZT4 (09:00). The EAAC1 protein expression in the hippocampus was increased at ZT18 relative to ZT4. Intracerebroventricular injection of a non-selective excitatory amino acid transporter inhibitor, DL-threo-ß-benzyloxyaspartate, or a selective EAAC1 inhibitor, L-aspartic acid ß-hydroxamate, increased ischemia-induced Zn2+ accumulation and neuronal death in the hilus at ZT18. These findings suggest that ischemia-induced Zn2+ accumulation displays circadian fluctuations through diurnal variations in EAAC1 expressions and affects susceptibility to ischemic neuronal injury in the hippocampal hilar region.
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Isquemia Encefálica/metabolismo , Ritmo Circadiano/fisiología , Transportador 3 de Aminoácidos Excitadores/metabolismo , Hipocampo/metabolismo , Zinc/metabolismo , Animales , Muerte Celular , Transportador 3 de Aminoácidos Excitadores/antagonistas & inhibidores , Glutatión/metabolismo , Inyecciones Intraventriculares , Masculino , Ratones , Ratones Endogámicos C57BL , Degeneración Nerviosa/patología , Neuronas/patologíaRESUMEN
OBJECTIVE: Glioma stem cells (GSCs) are responsible for tumor initiation, therapeutic resistance, and recurrence. CD146 is mainly expressed in dividing GSCs and regulates cell cycle progression. However, the evaluation of the efficacy of targeted therapy against CD146 in vivo remains to be investigated. In this study, the authors aimed to develop gene therapy targeting GSCs using chitosan oligosaccharide lactate (COL) nanoparticles (NPs) conjugated with folic acid-polyethylene glycol (FA-PEG-COL NPs) for in vitro and in vivo delivery of CD146 small-interfering RNA (siCD146) and to determine the effect of CD146 knockdown on tumor growth. METHODS: To examine the uptake of NPs by tumor cells, immunofluorescence staining, flow cytometry, and in vivo imaging were performed. The knockdown effect of siCD146 was measured by western blot and water-soluble tetrazolium salt-8 assay in mouse glioma cells. The efficacy of siRNA therapy-targeted GSCs was evaluated by monitoring tumor growth through in vivo imaging and histological analysis. RESULTS: In vivo accumulation of the FA-PEG-COL NPs in subcutaneous and intracranial gliomas following NP administration via a mouse tail vein was observed. Additionally, in vitro delivery of siCD146 ionically cross-linked NPs, reduced CD146 levels, and suppressed growth in the glioma tumor sphere. Evaluation of the in vivo therapeutic effects of siCD146-cross-linked NPs in a mouse glioma model revealed significant suppression of intracranial tumor growth, with complete removal of the tumor observed in some mice on histological examination. Furthermore, delivery of siCD146 significantly reduced the Ki-67 index in residual tumor tissues relative to that in control mice. CONCLUSIONS: CD146 is a potential therapeutic target, and folic acid-conjugated NPs delivering siRNA may facilitate gene therapy in malignant gliomas.
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Neoplasias Encefálicas/terapia , Ácido Fólico/administración & dosificación , Glioma/terapia , Nanopartículas/administración & dosificación , ARN Interferente Pequeño/administración & dosificación , Animales , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Antígeno CD146/antagonistas & inhibidores , Antígeno CD146/genética , Línea Celular Tumoral , Pollos , Marcación de Gen/métodos , Terapia Genética/métodos , Glioma/genética , Glioma/patología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Ratones Desnudos , ARN Interferente Pequeño/genéticaRESUMEN
BACKGROUND: Glial cells such as astrocytes and microglia play an important role in the central nervous system via communication between these glial cells. Activated microglia can exhibit either the inflammatory M1 phenotype or the anti-inflammatory M2 phenotype, which influences astrocytic neuroprotective functions, including engulfment of cell debris. Recently, extracellular zinc has been shown to promote the inflammatory M1 phenotype in microglia through intracellular zinc accumulation and reactive oxygen species (ROS) generation. PURPOSE: Here, we investigated whether the zinc-enhanced inflammatory M1 phenotype of microglia affects the astrocytic engulfing activity. METHODS: Engulfing activity was assessed in astrocytes treated with microglial-conditioned medium (MCM) from lipopolysaccharide (LPS)-activated or from ZnCl2-pretreated LPS-activated M1 microglia. The effect of zinc on microglia phenotype was also validated using the zinc chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) and the ROS scavenger Trolox. RESULTS: Although treatment of astrocytes with LPS showed no significant effect on the engulfing activity, MCM from LPS-induced M1 microglia increased the beads uptake by astrocytes. This increased uptake activity was suppressed when MCM from LPS-induced M1 microglia pretreated with ZnCl2 was applied to astrocytes, which was further abolished by the intracellular zinc chelator TPEN and the ROS scavenger Trolox. In addition, expression of P2×7 receptors (P2×7R) was increased in astrocytes treated with MCM derived from M1 microglia but not in the M1 microglia pretreated with ZnCl2. CONCLUSION: These findings suggest that zinc pre-treatment abolishes the ability of LPS-induced M1 microglia to increase the engulfing activity in astrocytes via alteration of astrocytic P2×7R.
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INTRODUCTION: CD146 is highly expressed in various malignant tumors and contributes to their malignancy phenotype, which involves metastatic and tumorigenic activity. However, studies on the expression and function of CD146 in brain tumors are limited. METHODS: We over-expressed or knocked-down CD146 in both conventionally cultured glioma cells and tumor spheres (TS). The distribution of glioma cells and their stem cells in different cell cycle phases was analyzed by flow cytometry using the stem cell marker CD133 and the glial precursor marker A2B5. CD146 expression was immunohistochemically examined in glioma tissues. RESULTS: The majority of glioma stem cells (GSCs) expressing CD133 were also CD146-positive. CD146 knockdown in GSCs significantly compromised cell growth. Cell cycle analysis revealed that most of the CD146 and CD133 double-positive cells were in the G2/M phase. Ectopic expression of CD146 in parental glioma cells resulted in cell cycle arrest of most differentiated cells in G0/G1 phase. In contrast, ectopic expression of CD146 in GSCs resulted in an increase in the number of CD133-positive cells in the G2/M phase. Furthermore, CD146 knockdown reduced the number of CD133-positive cells in the G2/M phase, which was consistent with effects of cell growth inhibition. Immunohistochemical analysis revealed that CD146 expression was significantly upregulated in World Health Organization (WHO) Grade III and IV glioma and positively correlated with CD133 expression. CONCLUSIONS: CD146 is mainly expressed in dividing GSCs and may be a potential target for eradicating glioma stem cells.
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Neoplasias Encefálicas/metabolismo , Ciclo Celular , Glioma/metabolismo , Células Madre Neoplásicas/metabolismo , Apoptosis , Neoplasias Encefálicas/patología , Antígeno CD146/metabolismo , Diferenciación Celular , Proliferación Celular , Glioma/patología , Humanos , Células Madre Neoplásicas/patología , Pronóstico , Células Tumorales CultivadasRESUMEN
We report the use of small interfering RNAs (siRNAs) against ARHGEF4, CCDC88A, LAMTOR2, mTOR, NUP85, and WASF2 and folic acid (FA)-modified polyethylene glycol (PEG)-chitosan oligosaccharide lactate (COL) nanoparticles for targeting, imaging, delivery, gene silencing, and inhibition of invasiveness and metastasis in an orthotopic xenograft model. In vitro assays revealed that these siRNA-FA-PEG-COL nanoparticles were specifically inserted into pancreatic cancer cells compared to immortalized normal pancreatic epithelial cells and knocked down expression of the corresponding targets in pancreatic cancer cells. Cell motility and invasion were significantly inhibited by adding target siRNA-FA-PEG-COL nanoparticles into the culture medium. In vivo mouse experiments confirmed that when intravenously delivered, these siRNA-FA-PEG-COL nanoparticles became incorporated into human pancreatic cancer cells in mouse pancreatic tumors. Little accumulation was seen in the normal pancreas and vital organs. All target siRNA-FA-PEG-COL nanoparticles significantly inhibited retroperitoneal invasion. The siRNA-FA-PEG-COL nanoparticles against LAMTOR2, mTOR, and NUP85, which strongly inhibited retroperitoneal invasion and significantly inhibited peritoneal dissemination compared to the other nanoparticles, improved prognosis of the mice. Our results imply that siRNA-FA-PEG-COL nanoparticles against these six targets could have great potential as biodegradable drug carriers. In particular, siRNA nanoparticles against LAMTOR2, mTOR, and NUP85 may hold significant clinical promise.
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Activated microglia exhibit two opposite activation states, the inflammatory M1 and the anti-inflammatory M2 activation states. In the mammalian brain, ischemia elicits a massive release of zinc from hippocampal neurons, and the extracellular zinc primes M1 microglia-by inducing reactive oxygen species (ROS) generation-to enhance their production of proinflammatory cytokines, which ultimately results in short-term spatial memory impairment. Here, we examined how peridinin, a carotenoid in dinoflagellates, affects the zinc-enhanced inflammatory M1 phenotype of microglia. Treatment of microglia with 30-300â¯ng/mL peridinin caused a dose-dependent attenuation of zinc-enhanced interleukin (IL)-1ß, IL-6, and tumor necrosis factor-α (TNFα) secretion when M1 activation was induced by lipopolysaccharide exposure. Moreover, peridinin inhibited the increase in ROS levels in zinc-treated microglia without directly interacting with zinc. Notably, when mice were administrated peridinin (20-200 ng/animal) intracerebroventricularly 5â¯min before cerebral ischemia-reperfusion, the peridinin treatment not only suppressed the increase in expression of IL-1ß, IL-6, TNFα, and the microglial M1 surface marker CD16/32, but also protected the mice against ischemia-induced short-term spatial-memory impairment. Our findings suggest that peridinin prevents extracellular zinc-enhanced proinflammatory cytokine secretion from M1 microglia by inhibiting the increase in microglial ROS levels, and that this anti-inflammatory effect of peridinin might result in protection against deficits in short-term spatial memory.
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Isquemia Encefálica/tratamiento farmacológico , Isquemia Encefálica/fisiopatología , Carotenoides/uso terapéutico , Inflamación/patología , Microglía/patología , Sustancias Protectoras/farmacología , Memoria Espacial/efectos de los fármacos , Zinc/efectos adversos , Animales , Carotenoides/química , Carotenoides/farmacología , Quelantes/farmacología , Cognición/efectos de los fármacos , Citocinas/metabolismo , Hipocampo/patología , Mediadores de Inflamación/metabolismo , Lipopolisacáridos/farmacología , Ratones Endogámicos C57BL , Microglía/efectos de los fármacos , Fenotipo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Microglia, the resident immune cells of the central nervous system, can display a pro-inflammatory M1 phenotype or an anti-inflammatory M2 phenotype. Arginase (Arg)-1 expressed in interleukin-4 (IL-4)-induced M2 microglia reduces nitric oxide (NO) production by competing with inducible NO synthase for l-arginine, which contributes to the attenuation of brain inflammation. Although previous studies have indicated that brain zinc promotes M1 activation, the effect of zinc on M2 microglial activation remains to be determined. In the present study, murine primary microglia treated with 10 ng mL-1 IL-4 exhibited increased Arg-1 mRNA expression and levels of intracellular free zinc. Chelation of this increased intracellular free zinc by the cell permeable zinc chelator N,N,N',N'-tetrakis-(2-pyridylmethyl)ethylenediamine (TPEN) aggravated the IL-4-induced mRNA expression and enzymatic activity of Arg-1. However, the cell impermeable zinc chelator CaEDTA had no effect on Arg-1 expression or cytosolic levels of free zinc in IL-4-induced M2-polarized microglia. Furthermore, treatment with IL-4 resulted in upregulation of phagocytic activity in microglia, while administration of TPEN abolished IL-4-induced phagocytic activity. Moreover, this effect was reversed vial-arginine supplementation. These findings suggest that IL-4 induces an increase in intracellular free zinc in microglia, which may act as a negative regulator of IL-4-induced Arg-1 expression, and that such negative regulation is essential for microglial phagocytic activity.
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Arginasa/metabolismo , Regulación de la Expresión Génica , Interleucina-4/metabolismo , Microglía/efectos de los fármacos , Zinc/farmacología , Animales , Arginasa/genética , Células Cultivadas , Ratones , Ratones Endogámicos C57BL , Microglía/metabolismoRESUMEN
BACKGROUND AND PURPOSE: We have demonstrated that i.c.v.-administered (±)-epibatidine, a nicotinic ACh receptor (nAChR) agonist, induced secretion of noradrenaline and adrenaline (catecholamines) from the rat adrenal medulla with dihydro-ß-erythroidin (an α4ß2 nAChR antagonist)-sensitive brain mechanisms. Here, we examined central mechanisms for the (±)-epibatidine-induced responses, focusing on brain NOS and NO-mediated mechanisms, soluble GC (sGC) and protein S-nitrosylation (a posttranslational modification of protein cysteine thiol groups), in urethane-anaesthetized (1.0 g·kg-1 , i.p.) male Wistar rats. EXPERIMENTAL APPROACH: (±)-Epibatidine was i.c.v. treated after i.c.v. pretreatment with each inhibitor described below. Then, plasma catecholamines were measured electrochemically after HPLC. Immunoreactivity of S-nitrosylated cysteine (SNO-Cys) in α4 nAChR subunit (α4)-positive spinally projecting neurones in the rat hypothalamic paraventricular nucleus (PVN, a regulatory centre of adrenomedullary outflow) after i.c.v. (±)-epibatidine administration was also investigated. KEY RESULTS: (±)-Epibatidine-induced elevation of plasma catecholamines was significantly attenuated by L-NAME (non-selective NOS inhibitor), carboxy-PTIO (NO scavenger), BYK191023 [selective inducible NOS (iNOS) inhibitor] and dithiothreitol (thiol-reducing reagent), but not by 3-bromo-7-nitroindazole (selective neuronal NOS inhibitor) or ODQ (sGC inhibitor). (±)-Epibatidine increased the number of spinally projecting PVN neurones with α4- and SNO-Cys-immunoreactivities, and this increment was reduced by BYK191023. CONCLUSIONS AND IMPLICATIONS: Stimulation of brain nAChRs can induce elevation of plasma catecholamines through brain iNOS-derived NO-mediated protein S-nitrosylation in rats. Therefore, brain nAChRs (at least α4ß2 subtype) and NO might be useful targets for alleviation of catecholamines overflow induced by smoking.
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Médula Suprarrenal/efectos de los fármacos , Encéfalo/efectos de los fármacos , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Óxido Nítrico Sintasa de Tipo II/antagonistas & inhibidores , Óxido Nítrico/antagonistas & inhibidores , Piridinas/farmacología , Receptores Nicotínicos/metabolismo , Médula Suprarrenal/metabolismo , Animales , Encéfalo/metabolismo , Compuestos Bicíclicos Heterocíclicos con Puentes/administración & dosificación , Catecolaminas/sangre , Catecolaminas/metabolismo , Infusiones Intraventriculares , Masculino , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo II/metabolismo , Piridinas/administración & dosificación , Ratas , Ratas WistarRESUMEN
Extracellular zinc, which is released from hippocampal neurons in response to brain ischaemia, triggers morphological changes in microglia. Under ischaemic conditions, microglia exhibit two opposite activation states (M1 and M2 activation), which may be further regulated by the microenvironment. We examined the role of extracellular zinc on M1 activation of microglia. Pre-treatment of microglia with 30-60 µM ZnCl2 resulted in dose-dependent increases in interleukin-1 beta (IL-1ß), interleukin-6 (IL-6), and tumour necrosis factor-alpha (TNFα) secretion when M1 activation was induced by lipopolysaccharide administration. In contrast, the cell-permeable zinc chelator TPEN, the radical scavenger Trolox, and the P2X7 receptor antagonist A438079 suppressed the effects of zinc pre-treatment on microglia. Furthermore, endogenous zinc release was induced by cerebral ischaemia-reperfusion, resulting in increased expression of IL-1ß, IL-6, TNFα, and the microglial M1 surface marker CD16/32, without hippocampal neuronal cell loss, in addition to impairments in object recognition memory. However, these effects were suppressed by the zinc chelator CaEDTA. These findings suggest that extracellular zinc may prime microglia to enhance production of pro-inflammatory cytokines via P2X7 receptor activation followed by reactive oxygen species generation in response to stimuli that trigger M1 activation, and that these inflammatory processes may result in deficits in object recognition memory.
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Cloruros/farmacología , Citocinas/metabolismo , Microglía/efectos de los fármacos , Compuestos de Zinc/farmacología , Animales , Animales Recién Nacidos , Isquemia Encefálica/metabolismo , Línea Celular , Línea Celular Tumoral , Células Cultivadas , Humanos , Lipopolisacáridos/farmacología , Masculino , Ratones Endogámicos C57BL , Microglía/clasificación , Microglía/metabolismo , Neuronas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Zinc/metabolismoRESUMEN
Previously, we reported that central administration of bombesin, a stress-related peptide, elevated plasma levels of catecholamines (noradrenaline and adrenaline) in the rat. The sympatho-adrenomedullary system, which is an important component of stress responses, can be regulated by the central opioid system. In the present study, therefore, we examined the roles of brain opioid receptor subtypes (µ, δ, and κ) and nociceptin receptors, originally identified as opioid-like orphan receptors, in the bombesin-induced activation of central sympatho-adrenomedullary outflow using anesthetized male Wistar rats. Intracerebroventricularly (i.c.v.) administered bombesin-(1 nmol/animal) induced elevation of plasma catecholamines was significantly potentiated by pretreatment with naloxone (300 and 1000 µg/animal, i.c.v.), a non-selective antagonist for µ-, δ-, and κ-opioid receptors. Pretreatment with cyprodime (100 µg/animal, i.c.v.), a selective antagonist for µ-opioid receptors, also potentiated the bombesin-induced responses. In contrast, pretreatment with naltrindole (100 µg/animal, i.c.v.) or nor-binaltorphimine (100 µg/animal, i.c.v.), a selective antagonist for δ- or κ-opioid receptors, significantly reduced the elevation of bombesin-induced catecholamines. In addition, pretreatment with JTC-801 (30 and 100 µg/animal, i.c.v.) or J-113397 (100 µg/animal, i.c.v.), which are selective antagonists for nociceptin receptors, also reduced the bombesin-induced responses. These results suggest that brain µ-opioid receptors play a suppressive role and that brain δ-, κ-opioid, and nociceptin receptors play a facilitative role in the bombesin-induced elevation of plasma catecholamines in the rat. Thus, in the brain, these receptors could play differential roles in regulating the activation of central sympatho-adrenomedullary outflow.
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Médula Suprarrenal/fisiología , Bombesina/farmacología , Encéfalo/metabolismo , Receptores Opioides/fisiología , Sistema Nervioso Simpático/fisiología , Animales , Catecolaminas/sangre , Morfinanos/administración & dosificación , Naloxona/administración & dosificación , Naltrexona/administración & dosificación , Naltrexona/análogos & derivados , Ratas , Receptor de NociceptinaRESUMEN
We previously reported that intracerebroventricularly (i.c.v.) administered (±)-epibatidine (1, 5 or 10 nmol/animal), a nicotinic acetylcholine receptor agonist, dose-dependently induced secretion of noradrenaline and adrenaline (catecholamines) from the rat adrenal medulla by brain diacylglycerol lipase- (DGL), monoacylglycerol lipase- (MGL) and cyclooxygenase-mediated mechanisms. Diacylglycerol is hydrolyzed by DGL into 2-arachidonoylglycerol (2-AG), which is further hydrolyzed by MGL to arachidonic acid (AA), a cyclooxygenase substrate. These findings suggest that brain 2-AG-derived AA is involved in the (±)-epibatidine-induced response. This AA precursor 2-AG is also a major brain endocannabinoid, which inhibits synaptic transmission through presynaptic cannabinoid CB1 receptors. Released 2-AG into the synaptic cleft is rapidly inactivated by cellular uptake. Here, we examined a role of brain 2-AG as an endocannabinoid in the (±)-epibatidine-induced activation of central adrenomedullary outflow using anesthetized male Wistar rats. In central presence of AM251 (CB1 antagonist) (90 and 180 nmol/animal, i.c.v.), (±)-epibatidine elevated plasma catecholamines even at an ineffective dose (1 nmol/animal, i.c.v.). Central pretreatment with ACEA (CB1 agonist) (0.7 and 1.4 µmol/animal, i.c.v.), 2-AG ether (stable 2-AG analog for MGL) (0.5 and 1.0 µmol/animal, i.c.v.) or AM404 (endocannabinoid uptake inhibitor) (80 and 250 nmol/animal, i.c.v.) significantly reduced an effective dose of (±)-epibatidine- (5 nmol/animal, i.c.v.) induced elevation of plasma catecholamines, and AM251 (90 and 180 nmol/animal, i.c.v.) centrally abolished the reduction induced by 2-AG ether (1.0 µmol/animal, i.c.v.) or AM404 (250 nmol/animal, i.c.v.). Immunohistochemical studies demonstrated that (±)-epibatidine (10 nmol/animal, i.c.v.) activated DGLα-positive spinally projecting neurons in the hypothalamic paraventricular nucleus, a control center of central adrenomedullary system. These results suggest a possibility that a brain endocannabinoid, probably 2-AG, plays an inhibitory role in (±)-epibatidine-induced activation of central adrenomedullary outflow through brain CB1 receptors in the rat.
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Médula Suprarrenal/efectos de los fármacos , Médula Suprarrenal/metabolismo , Ácidos Araquidónicos/metabolismo , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Endocannabinoides/metabolismo , Glicéridos/metabolismo , Agonistas Nicotínicos/farmacología , Piridinas/farmacología , Médula Suprarrenal/citología , Animales , Ácidos Araquidónicos/administración & dosificación , Ácidos Araquidónicos/farmacología , Catecolaminas/sangre , Relación Dosis-Respuesta a Droga , Endocannabinoides/administración & dosificación , Glicéridos/administración & dosificación , Inmunohistoquímica , Masculino , Neuronas/citología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neurotransmisores/administración & dosificación , Neurotransmisores/farmacología , Núcleo Hipotalámico Paraventricular/citología , Núcleo Hipotalámico Paraventricular/efectos de los fármacos , Núcleo Hipotalámico Paraventricular/metabolismo , Piperidinas/farmacología , Pirazoles/farmacología , Ratas Wistar , Receptor Cannabinoide CB1/agonistas , Receptor Cannabinoide CB1/antagonistas & inhibidores , Receptor Cannabinoide CB1/metabolismoRESUMEN
Cancer stem cells (CSCs) are the sole population possessing high self-renewal activity in tumors, with their existence affecting tumor recurrence. However, the invasive activity of CSCs has yet to be fully understood. In this article, we established a tumor sphere culture of RSV-M mouse glioma cells (RSV-M-TS) and evaluated their migration and invasion activities. Histological analysis of a tumor formed by cranial injection of the RSV-M-TS cells showed highly invasive properties and similarities with human malignant glioma tissues. When the migration activity of both RSV-M and RSV-M-TS cells were compared by intracranial injection, rapid migration of RSV-M-TS cells was observed. To confirm the invasive capabilities of RSV-M-TS cells, a three-dimensional collagen invasion assay was performed in vitro using RSV-M, RSV-M-TS, and RSV-M-TS cells cultured with medium containing serum. RSV-M and RSV-M-TS cultured with medium containing serum for 8 days indicated low migration activity, while moderate invasion activity was observed in RSV-M-TS cells. This activity was further enhanced by incubation with medium containing serum overnight. To identify the genes involved in this invasion activity, we performed quantitative polymerase chain reaction (PCR) array analysis of RSV-M and RSV-M-TS cells. Of 84 cancer metastasis-related genes, up-regulation was observed in 24 genes, while 4 genes appeared to be down-regulated in RSV-M-TS cells. These results suggest that the enhanced invasive activity of glioma sphere cells correlates with a number of tumor metastasis-related genes and plays a role in the dissemination and invasion of glioma cells.
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Glioma/patología , Animales , Línea Celular Tumoral , Movimiento Celular , Regulación Neoplásica de la Expresión Génica , Ratones , Ratones Endogámicos C3H , Invasividad NeoplásicaRESUMEN
BACKGROUND: The association between human cytomegalovirus (HCMV) and glioblastoma multiforme (GBM) is becoming a new concept. However, information on the geographic variability of HCMV prevalence in GBM remains scarce. Moreover, the potential roles of various viruses, such as polyomaviruses and oncogenic viruses, in gliomagenesis remain unclear. Our aim was to investigate the prevalence of HCMV in GBM among Japanese patients. Furthermore, this was the first study that evaluated infection with four new human polyomaviruses in GBMs. This study also provided the first data on the detection of human papillomavirus (HPV) in GBM in the Eastern world. METHODS: We measured the number of various viral genomes in GBM samples from 39 Japanese patients using real-time quantitative PCR. The tested viruses included HCMV, Merkel cell polyomavirus, human polyomavirus (HPyV) 6, HPyV7, HPyV9, Epstein-Barr virus, human herpesvirus 8, and HPV. Our quantitative PCR analysis led to the detection of eight copies of the HCMV DNA mixed with DNA extracted from 10(4) HCMV-negative cells. The presence of HCMV and HPV genomes was also assessed by nested PCR. Immunohistochemical study was also carried out to detect HPV-derived protein in GBM tissues. RESULTS: The viral DNAs were not detectable, with the exception of HPV, which was present in eight out of 39 (21%) GBMs. All HPV-positive cases harbored high-risk-type HPV (HPV16 and HPV18). Moreover, the HPV major capsid protein was detected in GBM tumor cells. CONCLUSIONS: In contrast with previous reports from Caucasian patients, we did not obtain direct evidence in support of the association between HCMV and GBM. However, high-risk-type HPV infection may play a potential etiological role in gliomagenesis in a subset of patients. These findings should prompt further worldwide epidemiological studies aimed at defining the pathogenicity of virus-associated GBM.
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Angiotensin II (AngII) plays important roles in the regulation of cardiovascular function. Both peripheral and central actions of AngII are involved in this regulation, but mechanisms of the latter actions as a neurotransmitter/neuromodulator within the brain are still unclear. Here we show that (1) intracerebroventricularly (i.c.v.) administered AngII in urethane-anesthetized male rats elevates plasma adrenaline derived from the adrenal medulla but not noradrenaline with valsartan- (AT1 receptor blocker) sensitive brain mechanisms, (2) peripheral AT1 receptors are not involved in the AngII-induced elevation of plasma adrenaline, although AngII induces both noradrenaline and adrenaline secretion from bovine adrenal medulla cells, and (3) i.c.v. administered AngII elevates blood pressure but not heart rate with the valsartan-sensitive mechanisms. From these results, i.c.v. administered AngII acts on brain AT1 receptors, thereby inducing the secretion of adrenaline and pressor responses. We propose that the central angiotensinergic system can activate central adrenomedullary outflow and modulate blood pressure.
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Angiotensina II/farmacología , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Epinefrina/metabolismo , Receptor de Angiotensina Tipo 1/metabolismo , Médula Suprarrenal/efectos de los fármacos , Médula Suprarrenal/metabolismo , Bloqueadores del Receptor Tipo 1 de Angiotensina II/farmacología , Animales , Presión Sanguínea/efectos de los fármacos , Inyecciones Intraventriculares/métodos , Masculino , Norepinefrina/metabolismo , Ratas , Ratas Wistar , Tetrazoles/farmacología , Valina/análogos & derivados , Valina/farmacología , ValsartánRESUMEN
Brain concussions are a serious public concern and are associated with neuropsychiatric disorders, such as depression. Patients with concussion who suffer from depression often experience distress. Nevertheless, few pre-clinical studies have examined concussion-induced depression, and there is little information regarding its pharmacological management. Edaravone, a free radical scavenger, can exert neuroprotective effects in several animal models of neurological disorders. However, the effectiveness of edaravone in animal models of concussion-induced depression remains unclear. In this study, we examined whether edaravone could prevent concussion-induced depression. Mice were subjected to a weight-drop injury and intravenously administered edaravone (3.0 mg/kg) or vehicle immediately after impact. Serial magnetic resonance imaging showed no abnormalities of the cerebrum on diffusion T1- and T2-weighted images. We found that edaravone suppressed concussion-induced depressive-like behavior in the forced swim test, which was accompanied by inhibition of increased hippocampal and cortical oxidative stress (OS) and suppression of 5-lipoxygenase (5-LOX) translocation to the nuclear envelope in hippocampal astrocytes. Hippocampal OS in concussed mice was also prevented by the nicotinamide adenine dinucleotide phosphate oxidase inhibitor, apocynin, and administration of BWB70C, a 5-LOX inhibitor, immediately and 24 h after injury prevented depressive-like behaviors in concussed mice. Further, antidepressant effects of edaravone were observed in mice receiving 1.0 or 3.0 mg/kg of edaravone immediately after impact, but not at a lower dose of 0.1 mg/kg. This antidepressant effect persisted up to 1 h after impact, whereas edaravone treatment at 3 h after impact had no effect on concussion-induced depressive-like behavior. These results suggest that edaravone protects against concussion-induced depression, and this protection is mediated by suppression of OS and 5-LOX translocation.
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Antipirina/análogos & derivados , Araquidonato 5-Lipooxigenasa/metabolismo , Conmoción Encefálica/complicaciones , Conmoción Encefálica/psicología , Trastorno Depresivo/tratamiento farmacológico , Trastorno Depresivo/psicología , Inhibidores de la Lipooxigenasa/uso terapéutico , Fármacos Neuroprotectores/uso terapéutico , Estrés Oxidativo/efectos de los fármacos , Animales , Antipirina/farmacología , Antipirina/uso terapéutico , Encéfalo/patología , Química Encefálica/efectos de los fármacos , Conmoción Encefálica/patología , Trastorno Depresivo/etiología , Relación Dosis-Respuesta a Droga , Edaravona , Activación Enzimática/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Fármacos Neuroprotectores/farmacología , Desempeño Psicomotor/efectos de los fármacos , Natación/psicologíaRESUMEN
We recently reported that intracerebroventricularly administered 2-arachidonoylglycerol elevated plasma noradrenaline and adrenaline by brain monoacylglycerol lipase- (MGL) and cyclooxygenase-mediated mechanisms in the rat. These results suggest that 2-arachidonoylglycerol is hydrolyzed by MGL to free arachidonic acid, which is further metabolized to prostaglandins (PGs) by cyclooxygenase in the brain, thereby elevating plasma noradrenaline and adrenaline. On the other hand, 2-arachidonoylglycerol can be also metabolized by cyclooxygenase to PG glycerol esters (PG-Gs), which seems to be hydrolyzed by MGL to free PGs. Here, we examined the involvement of brain PG-Gs in the elevation of plasma noradrenaline and adrenaline regarding PGE2-G and prostanoid EP receptors using anesthetized male Wistar rats. Intracerebroventricularly administered PGE2-G (1.5 and 3 nmol/animal) dose-dependently elevated plasma noradrenaline but not adrenaline. PGE2-G also elevated systolic, mean and diastolic blood pressure and heart rate. The PGE2-G-induced elevation of plasma noradrenaline was attenuated by JZL184 (MGL inhibitor). Intracerebroventricularly administered PGE2 (0.3 and 1.5 nmol/animal) and sulprostone (0.1 and 0.3 nmol/animal) (EP1/EP3 agonist) also elevated plasma noradrenaline but not adrenaline in a dose-dependent manner. The sulprostone-induced elevation was attenuated by L-798,106 (EP3 antagonist), but not by SC-51322 (EP1 antagonist). L-798,106 also attenuated the PGE2-G- and PGE2-induced elevation of plasma noradrenaline, while PF-04418948 (EP2 antagonist) and L-161,982 (EP4 antagonist) had no effect on the PGE2-G-induced response. These results suggest a possibility that brain PGE2-G produced from 2-arachidonoylglycerol can be hydrolyzed to free PGE2, thereby activating central sympathetic outflow by brain prostanoid EP3 receptor-mediated mechanisms in the rat.
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Presión Sanguínea/fisiología , Encéfalo/fisiología , Catecolaminas/sangre , Dinoprostona/análogos & derivados , Frecuencia Cardíaca/fisiología , Subtipo EP3 de Receptores de Prostaglandina E/metabolismo , Animales , Benzodioxoles/farmacología , Presión Sanguínea/efectos de los fármacos , Encéfalo/efectos de los fármacos , Fármacos del Sistema Nervioso Central/farmacología , Dinoprostona/metabolismo , Dinoprostona/farmacología , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Epinefrina/sangre , Frecuencia Cardíaca/efectos de los fármacos , Masculino , Monoacilglicerol Lipasas/antagonistas & inhibidores , Monoacilglicerol Lipasas/metabolismo , Norepinefrina/sangre , Piperidinas/farmacología , Ratas Wistar , Subtipo EP1 de Receptores de Prostaglandina E/agonistas , Subtipo EP1 de Receptores de Prostaglandina E/antagonistas & inhibidores , Subtipo EP1 de Receptores de Prostaglandina E/metabolismo , Subtipo EP2 de Receptores de Prostaglandina E/antagonistas & inhibidores , Subtipo EP2 de Receptores de Prostaglandina E/metabolismo , Subtipo EP3 de Receptores de Prostaglandina E/agonistas , Subtipo EP3 de Receptores de Prostaglandina E/antagonistas & inhibidores , Subtipo EP4 de Receptores de Prostaglandina E/antagonistas & inhibidores , Subtipo EP4 de Receptores de Prostaglandina E/metabolismoRESUMEN
For effective ovarian cancer gene therapy, systemic administrated tumor-targeting siRNA/folic acid-poly(ethylene glycol)-chitosan oligosaccharide lactate (FA-PEG-COL) nanoparticles is vital for delivery to cancer site(s). siRNA/FA-PEG-COL nanoparticles were prepared by ionic gelation for effective FA receptor-expressing ovarian cancer cells transfection and in vivo accumulation. The chemical structure of FA-PEG-COL conjugate was characterized by MALDI-TOF-MS, FT-IR and (1)H NMR. The average size of siRNA/FA-PEG-COL nanoparticles was approximately 200 nm, and the surface charge was +8.4 mV compared to +30.5 mV with siRNA/COL nanoparticles. FA-PEG-COL nanoparticles demonstrated superior compatibility with erythrocytes in terms of degree of aggregation and haemolytic activity and also effects on cell viability was lower when compared with COL nanoparticles. FA grafting significantly facilitated the uptake of nanoparticles via receptor mediated endocytosis as demonstrated by flow cytometry. The in vitro transfection and gene knockdown efficiency of HIF-1α were superior to COL nanoparticles (76-62%, respectively) and was comparable to Lipofectamine 2000 (79%) as demonstrated by RT-qPCR and Western blot. Gene knockdown at the molecular level translated into effective inhibition of proliferation in vitro. Accumulation efficiency of FA-PEG-COL nanoparticles was investigated in BALB/c mice bearing OVK18 #2 tumor xenograft using in vivo imaging. The active targeting FA-PEG-COL nanoparticles showed significantly greater accumulation than the passive targeting COL nanoparticles. Based on the results obtained, siRNA/FA-PEG-COL nanoparticles show much potential for effective ovarian cancer treatment via gene therapy.
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Quitosano/análogos & derivados , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Nanopartículas/administración & dosificación , Neoplasias Ováricas/terapia , ARN Interferente Pequeño/administración & dosificación , Animales , Carbocianinas/administración & dosificación , Carbocianinas/química , Línea Celular , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Quitosano/administración & dosificación , Quitosano/química , Femenino , Colorantes Fluorescentes/administración & dosificación , Colorantes Fluorescentes/química , Ácido Fólico/química , Técnicas de Silenciamiento del Gen , Terapia Genética/métodos , Hemólisis , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Ratones , Ratones Desnudos , Nanopartículas/química , Neoplasias Ováricas/genética , Polietilenglicoles/química , ARN Interferente Pequeño/químicaRESUMEN
Traumatic axonal injury (TAI), a feature of traumatic brain injury (TBI), progressively evolves over hours through impaired axonal transport and is thought to be a major contributor to cognitive dysfunction. In spite of various studies suggesting that pharmacologic or physiologic interventions might reduce TAI, clinical neuroprotective treatments are still unavailable. Edaravone, a free radical scavenger, has been shown to exert neuroprotective effects in animal models of several brain disorders. In this study, to evaluate whether edaravone suppresses TAI following TBI, mice were subjected to weight drop injury and had either edaravone (3.0mg/kg) or saline administered intravenously immediately after impact. Axonal injury and oxidative stress were assessed using immunohistochemistry with antibodies against amyloid precursor protein, a marker of impaired axonal transport, and with 8-hydroxy-2'-deoxyguanosine, a marker of oxidative DNA damage. Edaravone significantly suppressed axonal injury and oxidative stress in the cortex, corpus callosum, and hippocampus 24h after injury. The neuroprotective effects of edaravone were observed in mice receiving 1.0, 3.0, or 10mg/kg of edaravone immediately after impact, but not after 0.3mg/kg of edaravone. With treatment 1h after impact, axonal injury was also significantly suppressed and this therapeutic effect persisted up to 6h after impact. Furthermore, behavioral tests performed 9 days after injury showed memory deficits in saline-treated traumatized mice, which were not evident in the edaravone-treated group. These results suggest that edaravone protects against memory deficits following TBI and that this protection is mediated by suppression of TAI and oxidative stress.
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Antipirina/análogos & derivados , Lesiones Encefálicas/tratamiento farmacológico , Lesiones Encefálicas/psicología , Trastornos del Conocimiento/prevención & control , Trastornos del Conocimiento/psicología , Lesión Axonal Difusa/tratamiento farmacológico , Depuradores de Radicales Libres/uso terapéutico , Fármacos Neuroprotectores/uso terapéutico , Estrés Oxidativo/efectos de los fármacos , 8-Hidroxi-2'-Desoxicoguanosina , Animales , Antipirina/uso terapéutico , Lesiones Encefálicas/complicaciones , Cognición/efectos de los fármacos , Trastornos del Conocimiento/etiología , Daño del ADN , Desoxiguanosina/análogos & derivados , Desoxiguanosina/sangre , Lesión Axonal Difusa/patología , Relación Dosis-Respuesta a Droga , Edaravona , Conducta Exploratoria/efectos de los fármacos , Inmunohistoquímica , Masculino , Trastornos de la Memoria/etiología , Trastornos de la Memoria/psicología , Ratones , Ratones Endogámicos C57BL , Reconocimiento en Psicología/efectos de los fármacosRESUMEN
This study is the first to report that Spirulina complex polysaccharides (CPS) suppress glioma growth by down-regulating angiogenesis via a Toll-like receptor 4 signal. Murine RSV-M glioma cells were implanted s.c. into C3H/HeN mice and TLR4 mutant C3H/HeJ mice. Treatment with either Spirulina CPS or Escherichia coli (E. coli) lipopolysaccharides (LPS) strongly suppressed RSV-M glioma cell growth in C3H/HeN, but not C3H/HeJ, mice. Glioma cells stimulated production of interleukin (IL)-17 in both C3H/HeN and C3H/HeJ tumor-bearing mice. Treatment with E. coli LPS induced much greater IL-17 production in tumor-bearing C3H/HeN mice than in tumor-bearing C3H/HeJ mice. In C3H/HeN mice, treatment with Spirulina CPS suppressed growth of re-transplanted glioma; however, treatment with E. coli LPS did not, suggesting that Spirulina CPS enhance the immune response. Administration of anti-cluster of differentiation (CD)8, anti-CD4, anti-CD8 antibodies, and anti-asialo GM1 antibodies enhanced tumor growth, suggesting that T cells and natural killer cells or macrophages are involved in suppression of tumor growth by Spirulina CPS. Although anti-interferon-γ antibodies had no effect on glioma cell growth, anti-IL-17 antibodies administered four days after tumor transplantation suppressed growth similarly to treatment with Spirulina CPS. Less angiogenesis was observed in gliomas from Spirulina CPS-treated mice than in those from saline- or E. coli LPS-treated mice. These findings suggest that, in C3H/HeN mice, Spirulina CPS antagonize glioma cell growth by down-regulating angiogenesis, and that this down-regulation is mediated in part by regulating IL-17 production.