RESUMO
Accumulating studies have investigated the efficacy of receptor-mediated delivery of hydrophobic drugs in glioma chemotherapy. Here, a delivery vehicle comprising polyethylene glycol (PEG) and oxidized nanocrystalline mesoporous carbon particles (OMCN) linked to the Pep22 polypeptide targeting the low-density lipoprotein receptor (LDLR) is designed to generate a novel drug-loaded system, designated as OMCN-PEG-Pep22/DOX (OPPD). This system effectively targets glioma cells and the blood-brain barrier and exerts therapeutic efficacy through both near-infrared (NIR) photothermal and chemotherapeutic effects of loaded doxycycline (DOX). Pathological tissue microarrays show an association of LDLR overexpression in human glioma tissue with patient survival.NIR irradiation treatment and magnetic resonance imaging results show that OPPD reaches the effective glioma-killing temperature in a glioma-bearing rat with a skull bone removal model and considerably reduces glioma sizes relative to the drug-loaded system without the Pep22 peptide modification and the control respectively. Thus, OPPD not only effectively targets LDLR-overexpressing glioma but also exerts a dual therapeutic effect by transporting DOX into the glioma and generating thermal effects with near-infrared irradiation to kill tumor cells. These collective findings support the utility of the novel OPPD drug-loaded system as a promising drug delivery vehicle for clinical application in glioma therapy.
Assuntos
Carbono/química , Doxiciclina/química , Glioma/tratamento farmacológico , Nanopartículas/química , Peptídeos/química , Polietilenoglicóis/química , Animais , Doxiciclina/uso terapêutico , Sistemas de Liberação de Medicamentos/métodos , HumanosRESUMO
Gliomas are the most malignant and aggressive primary brain tumor in adults. Despite concerted efforts to improve therapies, their prognosis remains very poor. Isocitrate dehydrogenase 1 (IDH1) mutations have been discovered frequently in glioma patients and are strongly correlated with improved survival. However, the effect of IDH1 mutations on the chemosensitivity of gliomas remains unclear. In this study, we generated clonal U87 and U251 glioma cell lines overexpressing the R132H mutant protein (IDH1-R132H). Compared with control cells and cells overexpressing IDH wild type (IDH1-WT), both types of IDH1-R132H cells were more sensitive to temozolomide (TMZ) and cis-diamminedichloroplatinum (CDDP) in a time- and dose-dependent manner. The IDH1-R132H-induced higher chemosensitivity was associated with nicotine adenine disphosphonucleotide (NADPH), glutathione (GSH) depletion, and reactive oxygen species (ROS) generation. Accordingly, this IDH1-R132H-induced growth inhibition was effectively abrogated by GSH in vitro and in vivo. Our study provides direct evidence that the improved survival in patients with IDH1-R132H tumors may partly result from the effects of the IDH1-R132H protein on chemosensitivity. The primary cellular events associated with improved survival are the GSH depletion and increased ROS generation.
Assuntos
Cisplatino/administração & dosagem , Dacarbazina/análogos & derivados , Glioma/tratamento farmacológico , Isocitrato Desidrogenase/genética , Proliferação de Células , Dacarbazina/administração & dosagem , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glioma/genética , Glioma/patologia , Humanos , Isocitrato Desidrogenase/biossíntese , Mutação , Prognóstico , Espécies Reativas de Oxigênio/metabolismo , TemozolomidaRESUMO
The isocitrate dehydrogenase 1 (IDH1) gene mutation occurs frequently in glioma. While some studies have demonstrated that IDH1 mutations are associated with prolonged survival, the mechanism remains unclear. In this study, we found that growth was significantly inhibited in glioma cells overexpressing the mutated IDH1 gene. Furthermore, these cells were characterized by decreased intracellular NADPH levels accompanied by glutathione (GSH) depletion and reactive oxygen species (ROS) generation. Moreover, the increased apoptosis and the decreased proliferation were found in the glioma cells overexpressing the mutant IDH1 gene. Accordingly, our study demonstrates that using H2O2-regulated mutant IDH1 glioma cells could obviously increase the inhibition of cell growth; nevertheless, GSH had the opposite result. Our study provides direct evidence that mutation of IDH1 profoundly inhibits the growth of glioma cells, and we speculate that this is the major factor behind its association with prolonged survival in glioma. Finally, our study indicates that depletion of GSH and generation of ROS are the primary cellular events associated with this mutation.
Assuntos
Glioblastoma/genética , Glioblastoma/metabolismo , Glutationa/metabolismo , Isocitrato Desidrogenase/genética , Espécies Reativas de Oxigênio/metabolismo , Apoptose/genética , Neoplasias Encefálicas/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Humanos , Mutação , NADP/metabolismoRESUMO
TRAF6 (TNF receptor-associated factor 6), a member of tumor necrosis factor receptor-associated factors family was identified as a molecule that binds to the cytoplasmic domain of CD40. TRAF6 functions as an adaptor, positively regulating the NF-κB, JNK pathway. Additionally, some studies have reported that TRAF6 is required for apoptosis within the developing CNS and regulates cell fate decisions by inducing caspase 8-dependent apoptosis. However, its distribution and function in the central nervous system (CNS) lesion are not well understood. In this study, we performed an acute traumatic brain injury model in adult rats. And we mainly examined protein expression and cellular localization of TRAF6 during rat traumatic brain injury (TBI). Western blot analysis showed TRAF6 level significantly improved at 7 days after injury, and then declined during the following days. The protein expression of TRAF6 was further analysed by immunohistochemistry. In comparison to contralateral cerebral cortex, we observed a highly significant accumulation of TRAF6 at the ipsilateral brain. Immunofluorescence double-labeling showed that TRAF6 was co-expressed with NeuN and GFAP. Besides, co-localization of TRAF6/active caspase 3 and TRAF6/proliferating cell nuclear antigen (PCNA) were detected in NeuN and GFAP, respectively. We also examined the expression profiles of proliferating cell nuclear antigen (PCNA) and active caspase 3 whose changes were correlated with the expression profiles of TRAF6. In conclusion, this is the first description of TRAF6 expression in traumatic brains. Our data suggested that TRAF6 might play important roles in CNS pathophysiology after TBI.
Assuntos
Lesões Encefálicas/metabolismo , Fator 6 Associado a Receptor de TNF/metabolismo , Animais , Apoptose , Biomarcadores/metabolismo , Western Blotting , Lesões Encefálicas/patologia , Proliferação de Células , Córtex Cerebral/metabolismo , Córtex Cerebral/patologia , Imunofluorescência , Immunoblotting , Masculino , Neurônios/metabolismo , Neurônios/patologia , Transporte Proteico , Ratos , Ratos Sprague-DawleyRESUMO
HS1-associated protein X-1 (Hax-1) is an intracellular protein with anti-apoptotic properties that, in addition to suppressing cell death by inhibiting the activation of initiator caspase-9 and death caspase-3, is involved in an increasing number of signaling cascades. However, its expression and function in the central nervous system lesion are still unclear. In this study, we performed a traumatic brain injury (TBI) model in adult rats and investigated the dynamic changes of Hax-1 expression in the brain cortex. Western blot and immunohistochemistry analysis revealed that Hax-1 was present in normal brain. It gradually increased, reached a peak at day 3 after TBI, and then declined during the following days. Double immunofluorescence staining showed that Hax-1 immunoreactivity (IR) was found in neurons, but not astrocytes and microglia. Moreover, the 3rd day post injury was the apoptotic peak implied by the alteration of caspase-3, Bcl-2 and TUNEL. All these results suggested that Hax-1 may be involved in the pathophysiology of TBI and further research is needed to have a good understanding of its function and mechanism.
Assuntos
Lesões Encefálicas/metabolismo , Proteínas de Transporte/metabolismo , Córtex Cerebral/lesões , Córtex Cerebral/metabolismo , Animais , Biomarcadores/metabolismo , Lesões Encefálicas/patologia , Lesões Encefálicas/fisiopatologia , Proteínas de Transporte/genética , Caspase 3/metabolismo , Córtex Cerebral/patologia , Peptídeos e Proteínas de Sinalização Intracelular , Masculino , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ratos , Ratos Sprague-Dawley , Regulação para CimaRESUMO
BACKGROUND: Both the pterygopalatine fossa (PPF) and the infratemporal fossa (ITF) lie outside the midline of the skull base. Lesions in the PPF or ITF include trigeminal schwannoma (trigeminal schwannoma, TS), which originates from the second or third branch of the trigeminal nerve (maxillary nerve or mandibular nerve). Due to their typically deep anatomic location, lesions in the PPF or ITF can be difficult to treat using traditional surgical approaches. In recent years, because of their advantages, which include the fact that they allow the problem to be observed close up, neuroendoscopic techniques are increasingly being applied in skull base surgery, especially in treatment of lesions around the midline of the base of the skull. This study aims to 1) evaluate the neuroendoscopic treatment of lesions in PPF or ITF via the transnasal palate bone perpendicular plate or transnasal maxillary sinus approach and 2) analyze the clinical significance of this approach. METHODS: We retrospectively analyzed 3 cases of PPF TSs and 1 case of ITF TS treated between January 2015 and May 2017. All of the cases underwent neuroendoscopic resection of TSs located in the PPF via the nasal perpendicular plate palatine bone (or nasal maxillary sinus) approach. RESULTS: Two cases of PPF TSs were characterized by a thin palate bone perpendicular plate due to oppressed absorption of the tumor. Therefore the endoscopic transnasal palate bone perpendicular plate approach was employed. Additionally, 1 case of PPF TSs and 1 case of ITF TS were resected via the transnasal maxillary sinus approach. All 4 patients received total resection under endoscopy and recovered well after their respective operations without cerebrospinal fluid leakage, although 1 patient experienced postoperative dry eye symptoms and 1 other patient showed no improvement in facial numbness before and after the operation. CONCLUSIONS: Neuroendoscopic surgery performed via the transnasal perpendicular plate palatine bone or transnasal maxillary sinus approach has its own unique advantages in removing TSs in PPF and in ITF: Notably, the tumor can be exposed and dealt with under direct vision, which prevents damage to important structures, such as the internal carotid and maxillary nerves, while at the same time helping to achieve total removal of TSs. Furthermore, by adopting this approach versus traditional skull base surgery, postoperative trauma can be reduced significantly, which should be advocated for in this time of minimal invasive surgery.
Assuntos
Neoplasias dos Nervos Cranianos/cirurgia , Neurilemoma/cirurgia , Neuroendoscopia/métodos , Doenças do Nervo Trigêmeo/cirurgia , Idoso , Neoplasias dos Nervos Cranianos/diagnóstico por imagem , Feminino , Seguimentos , Humanos , Masculino , Seio Maxilar , Pessoa de Meia-Idade , Cirurgia Endoscópica por Orifício Natural/métodos , Neurilemoma/diagnóstico por imagem , Palato Duro , Fossa Pterigopalatina , Estudos Retrospectivos , Doenças do Nervo Trigêmeo/diagnóstico por imagemRESUMO
The present study aimed to investigate the role of pituitary tumor-transforming gene 1 (PTTG1) in the proliferation, invasion and apoptosis of human malignant glioma U251 cells. Firstly, 2 microRNAs (miRNAs) targeting PTTG1 messenger (m)RNA were ligated into a pcDNA6.2-GW/EmGFP-miR expression vector. The recombinant plasmids, miRNA-1 and miRNA-2 (miR-2), were transfected into U251 cells using the liposome method. PTTG1 mRNA and protein levels were evaluated using quantitative polymerase chain reaction and western blot analysis. The proliferation and invasion abilities of U251 cells were determined using methylthiazol tetrazolium and Matrigel assays. Flow cytometry analysis with Annexin V/propidium iodide double staining was used to determine the percentage of apoptotic cells. PTTG1 expression was effectively suppressed by miR-2. U251 cell growth was inhibited between 10.7 and 34.7% in the miR-2 group compared with the blank group. The Matrigel assay demonstrated that the percentage of infiltrating U251 cells was significantly lower in the miR-2 group (12.3±1.0%) compared to the blank group (24.7±1.4%; P<0.001) and the negative control group (24.0±2.0%; P<0.05). A higher percentage of apoptotic U251 cells were observed in the miR-2 group compared with the blank group (53.6 vs. 32.4%) using flow cytometry due to cycle arrests at the G2/M phase. The miR-2-transfected U251 cells were subcutaneously injected into nude mice, and these mice possessed a decreased tumor tissue growth rate and higher percentage of apoptotic cells compared with the blank and negative control groups. In conclusion, PTTG1 gene expression in human malignant glioma U251 cells was effectively suppressed by exogenous miR-2. The downregulation of PTTG1 induced glioma cell apoptosis and cell cycle arrest at the G2/M phase, which inhibited cell proliferation, reverse invasion and infiltration of glioma cells.
RESUMO
Gliomas are the most common type of brain tumor in the central nervous system of adults, and are highly aggressive, resistant to treatment, and prone to recurrence. Brain tumor stem cells (BTSCs) are implicated in tumor initiation and recurrence. Cluster of differentiation (CD)133 is currently the most widely used BTSC marker; however, its role in glioma development and progression is largely unknown. In this study, we evaluated CD133 expression in pairs of primary and recurrent human glioma specimens from 24 patients. We found that recurrent gliomas have aberrantly upregulated CD133 levels. To clarify the mechanism underlying this observation, we assessed CD133 promoter (P)2 methylation status by bisulfite sequencing and found that P2 hypomethylation was associated with the increase in CD133 expression and glioma recurrence. These results suggest that CD133 overexpression in BTSCs due to P2 hypomethylation underlies glioma recurrence, which may provide insight into the mechanism of glioma recurrence and provide a basis for novel therapies for glioma treatment.
Assuntos
Antígeno AC133/genética , Neoplasias Encefálicas/genética , Metilação de DNA/genética , Glioma/genética , Recidiva Local de Neoplasia/genética , Regiões Promotoras Genéticas/genética , Biomarcadores Tumorais/genética , Linhagem Celular Tumoral , Glioma/patologia , Humanos , Recidiva Local de Neoplasia/patologia , Células-Tronco Neoplásicas/patologia , Regulação para Cima/genéticaRESUMO
Glioblastomas are understood to evolve from brain glioma stem cells (BGSCs), and yet the biology underlying this model of tumorigenesis is largely unknown. Paired box 3 protein (Pax3) is a member of the paired box (Pax) family of transcription factors that is normally expressed during embryonic development, but has recently been implicated in tumorigenesis. The present study demonstrated that Pax3 is differentially expressed in U87MG human glioma cell, BGSC and normal 1800 human astrocyte lines. Herein, we identified that the glial fibrillary acidic protein (GFAP), a major intermediate filament protein of mature astrocytes, is directly downregulated during the differentiation of BGSCs via the binding of Pax3 to the promoter region of GFAP. Moreover, siRNA silencing of Pax3 arrested BGSC differentiation, while overexpression of Pax3 promoted the differentiation in BGSCs. Furthermore, we studied the cell proliferation, invasion, apoptosis, differentiation and expression of Pax3 and GFAP in Pax3 siRNA-knockdown and Pax3-overexpressing BGSC models by CCK-8, Transwell migration, flow cytometry and western blot assays. The results indicate that Pax3 regulates GFAP expression, and that Pax3 may contribute to the evolution of BGSCs towards malignancy.
Assuntos
Regulação Neoplásica da Expressão Gênica/genética , Proteína Glial Fibrilar Ácida/genética , Glioma/genética , Células-Tronco Neoplásicas/metabolismo , Fator de Transcrição PAX3/genética , Apoptose/genética , Astrócitos/metabolismo , Astrócitos/patologia , Diferenciação Celular/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Regulação para Baixo/genética , Glioma/patologia , Humanos , Regiões Promotoras Genéticas/genética , RNA Interferente Pequeno/genéticaRESUMO
Tentorial dural arteriovenous fistula (DAVF) as a cause of trigeminal neuralgia is extremely rare. Although less than 10 cases have been reported in the literature, all cases presented with ipsilateral trigeminal neuralgia. Here we report a unique case of tentorial DAVF manifesting as contralateral trigeminal neuralgia. A 58-year-old man presented with right-sided trigeminal neuralgia. Cerebral angiography revealed a left tentorial DAVF and the MR imaging demonstrated a variceal venous dilatation occupying the left lateral pontine cistern and multiple venous flow voids adjacent to the right trigeminal nerve root entry zone. Transarterial Onyx embolization resulted in near complete obliteration of the fistula with immediate resolution of facial pain. The patient remains symptom free and without evidence of recurrence during 9â months of follow-up. Transarterial Onyx embolization may be an effective treatment modality for such an usual case.
Assuntos
Malformações Vasculares do Sistema Nervoso Central/complicações , Malformações Vasculares do Sistema Nervoso Central/terapia , Embolização Terapêutica/métodos , Polivinil/uso terapêutico , Tantálio/uso terapêutico , Neuralgia do Trigêmeo/etiologia , Malformações Vasculares do Sistema Nervoso Central/diagnóstico , Angiografia Cerebral , Combinação de Medicamentos , Humanos , Angiografia por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Resultado do Tratamento , Nervo Trigêmeo/patologiaRESUMO
OBJECTIVE: Transarterial treatment of direct carotid cavernous fistulas (DCCF) via embolic materials has been well documented. This study reports, validates, and compares with existing literature our experience treating DCCFs via endovascular approaches by using detachable balloons, coils, and covered stents. METHODS: Between June 2006 to October 2011, 32 patients (21 male, 11 female) with 32 DCCFs (30 traumatic, 2 spontaneous cavernous ICA aneurysms) were embolized endovascularly. Followup was performed for at least 6 months. RESULTS: Among the 32 DCCFs, 21 (65.6%) were embolized using detachable balloons, eight (25.0%) with coils, one (3.1%) with balloons and coils, and two (6.3%) with covered stents. Complete DCCF obliteration was achieved in 31 (96.9%) cases. One fistula failed to respond due to premature balloon detachment. Intracranial bruit in 31 (100%) chemosis and exophthalmos in 28 (100%) cases resolved after embolization. Visual acuity and oculomotor palsy improved in 18 (90%) and 18 (69.2%) cases, respectively. There was no evidence of DCCF recurrence. Thirteen DCCFs were followed up by MRI and five by DSA. In these cases, four (4/13, 30.8%) balloon-embolized DCCFs showed pseudoaneurysms. Three patients were asymptomatic; one had minor left oculomotor palsy. CONCLUSIONS: Our results correlate and reinforce literature regarding endovascular treatment of DCCFs. Application of Transarterial embolization with detachable balloons, despite extensive use has been decreasing. Coil embolization is an effective and safe alternative for treatment, especially when balloon embolization fails. Covered stent placement may be used as another alternative for selected cases.
RESUMO
Tentorial dural arteriovenous fistula (DAVF) as a cause of trigeminal neuralgia is extremely rare. Although less than 10 cases have been reported in the literature, all cases presented with ipsilateral trigeminal neuralgia. Here we report a unique case of tentorial DAVF manifesting as contralateral trigeminal neuralgia. A 58-year-old man presented with right-sided trigeminal neuralgia. Cerebral angiography revealed a left tentorial DAVF and the MR imaging demonstrated a variceal venous dilatation occupying the left lateral pontine cistern and multiple venous flow voids adjacent to the right trigeminal nerve root entry zone. Transarterial Onyx embolization resulted in near complete obliteration of the fistula with immediate resolution of facial pain. The patient remains symptom free and without evidence of recurrence during 9 months of follow-up. Transarterial Onyx embolization may be an effective treatment modality for such an usual case.
Assuntos
Fístula Arteriovenosa/complicações , Fístula Arteriovenosa/terapia , Malformações Vasculares do Sistema Nervoso Central/complicações , Malformações Vasculares do Sistema Nervoso Central/terapia , Dimetil Sulfóxido/uso terapêutico , Dura-Máter/irrigação sanguínea , Embolização Terapêutica/métodos , Polivinil/uso terapêutico , Neuralgia do Trigêmeo/etiologia , Neuralgia do Trigêmeo/terapia , Fístula Arteriovenosa/diagnóstico , Malformações Vasculares do Sistema Nervoso Central/diagnóstico , Angiografia Cerebral , Diagnóstico Diferencial , Humanos , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Neuralgia do Trigêmeo/diagnósticoRESUMO
SKIP (Ski-interacting protein), is part of nuclear regulatory complexes and interacts with factors involved in preinitiation, splicing and polyadenylation, potentiates the activity of important transcription factors, involved in an increasing number of signaling cascades. However, its distribution and function in the central nervous system remains poorly understood. In this study, western blot analysis, RT-PCR and immunohistochemistry showed a significant up-regulation of SKIP in ipsilateral peritrauma cortex compared with the sham group. Immunofluorescent labeling indicated that SKIP was localized striking in the neurons, but not astrocytes and oligodendrocytes; co-localization of SKIP and active caspase-3 and PCNA in the ipsilateral cortex. In addition, the expression patterns of active caspase-3 and PCNA were parallel with that of SKIP. Based on our data, we speculated that SKIP might play an important role in neuronal apoptosis following TBI; and might provide a basis for the further study on its role in cell cycle re-entry in traumatic brain injury.
Assuntos
Lesões Encefálicas/metabolismo , Córtex Cerebral/metabolismo , Fatores de Transcrição/metabolismo , Regulação para Cima , Animais , Lesões Encefálicas/genética , Caspase 3/metabolismo , Ciclo Celular/genética , Córtex Cerebral/patologia , Modelos Animais de Doenças , Masculino , Neurônios/metabolismo , Ratos , Fatores de Tempo , Fatores de Transcrição/genética , Regulação para Cima/genéticaRESUMO
Septins are a novel group of GTPases, which are first identified in yeast and more recently found in a wide range of animal cells. Septin-9, a novel septin family member, is expressed ubiquitously and involved in an increasing number of signaling cascades. However, information regarding its distribution and possible function in the central nervous system (CNS) is limited. In this study, western blot analysis and immunohistochemistry showed a significant upregulation of Septin-9 in the ipsilateral peritrauma cortex compared with the sham group. Immunofluorescent labeling indicated that Septin-9 was localized strikingly in the cytoplasm of neurons, but not astrocytes and oligodendrocytes. The co-localization of Septin-9 and active caspase-3 was also examined in the ipsilateral cortex. In addition, the expression patterns of active caspase-3 were parallel with that of Septin-9. Quantitative real-time RT-PCR also was used to detect Seption-9 mRNA level. Based on our data, we speculated that traumatic brain injury may be a stimulus to induce the Septin-9 expression, and Septin-9 might play an important role in the pathophysiology process in the CNS after traumatic brain injury. Taken together, this is the first description of Septin-9 expression changes during the central nervous system lesion and repair, but further studies are needed to understand the cell signaling pathway which can direct the exact role of Septin-9 following traumatic brain injury.
Assuntos
Lesões Encefálicas/metabolismo , Córtex Cerebral/metabolismo , Septinas/metabolismo , Animais , Apoptose , Astrócitos/metabolismo , Caspase 3/genética , Caspase 3/metabolismo , Córtex Cerebral/patologia , Citoplasma/metabolismo , Masculino , Neurônios/metabolismo , Oligodendroglia/metabolismo , Especificidade de Órgãos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Septinas/genéticaRESUMO
Glioma is the leading cause of tumor-related mortality in the central nervous system. There is increasing evidence that the self-renewal capacity of cancer cells is critical for the initiation, growth and recurrence of tumors. OCT4 is a transcription factor that plays a key role in regulating the self-renewal ability of embryonic stem cells. DNA methylation is involved in the regulation of OCT4 expression during the development and differentiation of embryonic stem cells and neural stem cells. In the present study, we reported that OCT4 was highly expressed in primary gliomas and its expression levels increased in parallel with pathological grades. BSP analysis showed that the methylation levels of OCT4 gene promoter and exon were significantly reduced in comparison with the normal group and were negatively correlated with OCT4 gene expression in primary gliomas. In vitro, OCT4 gene expression was upregulated following treatment by a demethylation reagent in glioma cell lines. Our findings suggest that OCT4 is epigenetically regulated by DNA hypomethylation in primary gliomas, which may provide evidence for the role of DNA methylation in tumor and may present a new direction for developing more powerful strategies to treat glioma in the clinic.
Assuntos
Glioma/genética , Fator 3 de Transcrição de Octâmero/genética , Regiões Promotoras Genéticas/genética , Diferenciação Celular , Linhagem Celular Tumoral , Metilação de DNA , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Glioma/metabolismo , Humanos , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Fator 3 de Transcrição de Octâmero/metabolismo , Sequências Reguladoras de Ácido NucleicoRESUMO
PURPOSE: It is generally accepted that inflammation has a role in the progression of many central nervous system (CNS) diseases, although the mechanisms through which this occurs remain unclear. Among mitogen-activated protein kinase (MAPK) targets, mitogen- and stress-activated protein kinase (MSK1) has been thought to be involved in the pathology of inflammatory gene expression. In this study, the roles of MSK1 activation in neuroinflammation were investigated. METHODS: The bacterial lipopolysaccharide (LPS)-induced brain injury model was performed on Sprague-Dawley rats. The dynamic expression changes and the cellular location of p-MSK1 in the brain cortex were detected by Western blot and immunofluorescence staining. The synthesis of inflammatory cytokines in astrocytes was detected by enzyme-linked immunosorbent assay (ELISA). RESULTS: Phosphorylated MSK1 (p-MSK1 Thr-581) was induced significantly after intracerebral injection of LPS into the lateral ventricles of the rat brain. Specific upregulation of p-MSK1 in astrocytes was also observed in inflamed cerebral cortex. At 1 day after LPS stimulation, iNOS, TNFα expression, and the astrocyte marker glial fibrillary acidic protein (GFAP) were increased significantly. Also, in vitro studies indicated that the upregulation of p-MSK1 (Thr-581) may be involved in the subsequent astrocyte inflammatory process, following LPS challenge. Using an enzyme-linked immunosorbent assay (ELISA), it was confirmed that treatment with LPS in primary astrocytes stimulated the synthesis of inflammatory cytokines, through MAPKs signaling pathways. In cultured primary astrocytes, both knock-down of total MSK1 by small interfering RNAs (siRNA) or specific mutation of Thr-581 resulted in higher production of certain cytokines, such as TNFα and IL-6. CONCLUSIONS: Collectively, these results suggest that MSK1 phosphorylation is associated with the regulation of LPS-induced brain injury and possibly acts as a negative regulator of inflammation.
Assuntos
Astrócitos/metabolismo , Lesões Encefálicas/genética , Córtex Cerebral/metabolismo , Proteínas Quinases S6 Ribossômicas 90-kDa/genética , Animais , Astrócitos/patologia , Lesões Encefálicas/induzido quimicamente , Lesões Encefálicas/metabolismo , Lesões Encefálicas/patologia , Córtex Cerebral/patologia , Regulação da Expressão Gênica , Proteína Glial Fibrilar Ácida/genética , Proteína Glial Fibrilar Ácida/metabolismo , Inflamação/induzido quimicamente , Inflamação/genética , Inflamação/metabolismo , Inflamação/patologia , Injeções Intraventriculares , Interleucina-6/genética , Interleucina-6/metabolismo , Lipopolissacarídeos , Masculino , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Fosforilação , Cultura Primária de Células , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ratos , Ratos Sprague-Dawley , Proteínas Quinases S6 Ribossômicas 90-kDa/antagonistas & inibidores , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Transdução de Sinais , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Traumatic brain injury (TBI) triggers a complex series of neurochemical and signaling changes that lead to neuronal dysfunction and overreactive astrocytes. In the current study, we showed that interactions between SCYL1-bp1 and Pirh2 are involved in central nervous system (CNS) injury and repair. Western blot and immunohistochemical analysis of an acute traumatic brain injury model in adult rats revealed significantly increased levels of SCYL1-bp1 and Pirh2 in the ipsilateral brain cortex, compared to contralateral cerebral cortex. Immunofluorescence double-labeling analyses further revealed that SCYL1-bp1 is mainly co-expressed with NeuN. Terminal deoxynucleotidyl transferase-mediated biotinylated-dUTP nick-end labeling staining data supported the involvement of SCYL1-bp1 and Pirh2 in neuronal apoptosis after brain injury. We additionally examined the expression profiles of active caspase-3, which were altered in correlation with the levels of SCYL1-bp1 and Pirh2. Notably, both SCYL1-bp1 and Pirh2 were colocalized with active caspase-3, and all three proteins participated in neuronal apoptosis. Immunoprecipitation experiments further revealed interactions of these proteins with each other in the pathophysiology process. To our knowledge, this is the first study to report interactions between SCYL1-bp1 and Pirh2 in traumatic brain. Our data collectively indicate that SCYL1-bp1 and Pirh2 play important roles in CNS pathophysiology after TBI.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Lesões Encefálicas/metabolismo , Doenças do Sistema Nervoso Central/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/genética , Animais , Antígenos Nucleares/genética , Antígenos Nucleares/metabolismo , Apoptose/genética , Astrócitos/metabolismo , Biomarcadores/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Caspase 3/genética , Caspase 3/metabolismo , Doenças do Sistema Nervoso Central/genética , Córtex Cerebral/metabolismo , Masculino , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Domínios e Motivos de Interação entre Proteínas , Ratos , Ratos Sprague-Dawley , Proteína Supressora de Tumor p53/genética , Ubiquitina-Proteína Ligases/genética , Regulação para CimaRESUMO
MLTK (mixed-lineage kinase-like mitogen-activated protein triple kinase) is a member of the mitogen-activated protein kinase family and functioned as a mitogen activated kinase kinase kinase. MLTKα, one of the alternatively spliced forms of MLTK, could activate the c-Jun N-terminal kinase pathway, which involved in cellular stress responses and apoptosis. But the role of MLTKα in neural apoptosis was still unclear. Here, we performed a transient global cerebral ischemia model (TGCI) in adult rats and detected the dynamic changes of MLTKα in hippocampal CA1 neurons and brain cortex. We found the MLTKα expression was increased shortly after TGCI and peaked after 8 h. In spatial distribution, MLTKα was widely located in neurons rather than astrocytes and microglia. Moreover, there was a concomitant up-regulation of active caspase-3. Taken together, we hypothesized the up-regulation of MLTKα played an essential role in the apoptosis of hippocampal CA1 neurons.
Assuntos
Astrócitos/metabolismo , Região CA1 Hipocampal/metabolismo , Ataque Isquêmico Transitório/genética , MAP Quinase Quinase Quinases/genética , Microglia/metabolismo , Neurônios/metabolismo , Animais , Apoptose , Astrócitos/patologia , Região CA1 Hipocampal/patologia , Caspase 3/genética , Caspase 3/metabolismo , Expressão Gênica , Ataque Isquêmico Transitório/enzimologia , Ataque Isquêmico Transitório/patologia , Isoenzimas/genética , Isoenzimas/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/genética , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , MAP Quinase Quinase Quinases/metabolismo , Masculino , Microglia/patologia , Neurônios/patologia , Ratos , Ratos Wistar , Transdução de Sinais , Regulação para CimaRESUMO
Mammalian ecto ADP-ribosyltransferases (ARTs) can regulate the biological functions of various types of cells by catalyzing the transfer of single ADP-ribose moiety from NAD+ to a specific amino acid in a target protein. ART3 is a member of the known ART family which is involved in cell division, DNA-repair and the regulation of the inflammatory response. To elucidate the expression, cellular localization and possible functions of ART3 in central nervous system (CNS) lesion and repair, we performed an acute traumatic brain injury model in adult rats. Western blot analysis showed that the expression of ART3 in ipsilateral brain cortex increased, then reached a peak at day 3 after traumatic brain injury (TBI), and gradually declined during the following days. But in the contralateral brain cortex, no obvious alterations were observed. Immunohistochemistry revealed the highly significant accumulation of ART3 at the ipsilateral brain in comparison to contralateral cerebral cortex. Double immunofluorescence labeling suggested that ART3 was localized mainly in the plasmalemma of neurons, but not in astrocytes or microglias within 3 mm from the lesion site at day 3 post-injury. In addition, we detected the expression profiles of caspase-3 and growth associated protein 43 (GAP-43) whose changes were correlated with the expression profiles of ART3 in this TBI model. Besides, co-localization of ART3/active caspase-3 and ART3/GAP43 were detected in NeuN-positive cells, respectively. Moreover, Pheochromocytoma (PC12) cells were treated with H2O2 to establish an apoptosis model. The results showed that the expression of ART3 was increased in the concentration and time dependence way. To further examine the involvement of ART3 in apoptosis of PC12, 3-Methoxybenzamide was used in flow cytometry analysis of apoptotic cells stained with Annexin V and PI. The experimental group in which 3-Methoxybenzamide used had a relative low level of apoptotic index compared with the untreated group. Together with previous reports, we hypothesize that ART3 may play important roles in CNS pathophysiology after TBI and further research is needed to have a good understanding of its function and mechanism.
Assuntos
ADP Ribose Transferases/metabolismo , Lesões Encefálicas/enzimologia , ADP Ribose Transferases/genética , Animais , Apoptose/genética , Biomarcadores/metabolismo , Western Blotting , Lesões Encefálicas/genética , Lesões Encefálicas/patologia , Linhagem Celular , Córtex Cerebral/metabolismo , Córtex Cerebral/patologia , Modelos Animais de Doenças , Proteína GAP-43/metabolismo , Expressão Gênica , Perfilação da Expressão Gênica , Masculino , Neurônios/metabolismo , Ligação Proteica , Ratos , Ratos Sprague-Dawley , Fatores de TempoRESUMO
The paired box 3 (PAX3), a crucial transcription factor, is normally expressed during embryonic development and is absent in normal adult human tissues. Deregulated expression of PAX3 has been observed in tumors like rhabdomyosarcoma and melanomas. To assess deregulated PAX3 expression in patients with gliomas, these samples from 57 glioma patients (13 grade I, 16 grade II, 14 grade III, and 14 grade IV tumors) and 10 normal brain specimens acquired from 10 patients undergoing surgery for epilepsy as control were obtained. PAX3 expression was measured by RT-PCR, Western blot, and immunohistochemistry. Survival analyses were performed using the Kaplan-Meier method. Association between PAX3 expression, clinicopathological characteristics, and patients' survival were analyzed by using SPSS 17.0. We found that the expression of PAX3 was upregulated in high-grade glioma tissues compared with that in low-grade and normal brain tissues, and increased with ascending tumor World Health Organization (WHO) grades (P = 0.001). The increased PAX3 expression in gliomas was significantly associated with higher WHO grade (P = 0.021) and poorer disease-specific survival of patients (P = 0.001). Our results suggested that PAX3 might be an intrinsic regulator of progression in glioma cells and it might serve as a prognostic factor for this dismal disease.