RESUMO
BACKGROUND: The primary cause of treatment failure in medulloblastomas (MB) is the development of leptomeningeal dissemination (seeding). For translational research on MB seeding, one of the major challenges is the development of reliable experimental models that simulate the seeding and growth characteristics of MBs. To overcome this obstacle, we improved an experimental mouse model by intracisternal inoculation of human MB cells and monitoring with in vivo live images. METHODS: Human MB cells (UW426, D283 and MED8A) were transfected with a firefly luciferase gene and a Thy1.1 (CD90.1) marker linked with IRES under the control of the CMV promoter in a retroviral DNA backbone (effLuc). The MB-effLuc cells were injected into the cisterna magna using an intrathecal catheter, and bioluminescence images were captured. We performed histopathological analysis to confirm the extent of tumor seeding. RESULTS: The luciferase activity of MB-effLuc cells displayed a gradually increasing pattern, which correlated with a quantitative luminometric assay. Live imaging showed that the MB-effLuc cells were diffusely distributed in the cervical spinal cord and the lumbosacral area. All mice injected with UW426-effLuc, D283-effLuc and MED8A-effLuc died within 51 days. The median survival was 22, 41 and 12 days after injection of 1.2 × 10(6) UW426-effLuc, D283-effLuc and MED8A-effLuc cells, respectively. The histopathological studies revealed that the MB-effLuc cells spread extensively and diffusely along the leptomeninges of the brain and spinal cord, forming tumor cell-coated layers. The tumor cells in the subarachnoid space expressed a human nuclei marker and Ki-67. Compared with the intracerebellar injection method in which the subfrontal area and distal spinal cord were spared by tumor cell seeding in some mice, the intracisternal injection model more closely resembled the widespread leptomeningeal seeding observed in MB patients. CONCLUSION: The results and described method are valuable resources for further translational research to overcome MB seeding.
Assuntos
Neoplasias Cerebelares/metabolismo , Meduloblastoma/metabolismo , Neoplasias Meníngeas/secundário , Animais , Linhagem Celular Tumoral , Neoplasias Cerebelares/patologia , Feminino , Genes Reporter , Humanos , Luminescência , Meduloblastoma/patologia , Neoplasias Meníngeas/metabolismo , Camundongos , Microscopia Eletrônica de Varredura , Transplante de NeoplasiasRESUMO
Upon severe head injury (HI), blood vessels of the meninges and brain parenchyma are inevitably damaged. While limited vascular regeneration of the injured brain has been studied extensively, our understanding of meningeal vascular regeneration following head injury is quite limited. Here, we identify key pathways governing meningeal vascular regeneration following HI. Rapid and complete vascular regeneration in the meninges is predominantly driven by VEGFR2 signaling. Substantial increase of VEGFR2 is observed in both human patients and mouse models of HI, and endothelial cell-specific deletion of Vegfr2 in the latter inhibits meningeal vascular regeneration. We further identify the facilitating, stabilizing and arresting roles of Tie2, PDGFRß and Dll4 signaling, respectively, in meningeal vascular regeneration. Prolonged inhibition of this angiogenic process following HI compromises immunological and stromal integrity of the injured meninges. These findings establish a molecular framework for meningeal vascular regeneration after HI, and may guide development of wound healing therapeutics.
Assuntos
Traumatismos Craniocerebrais/genética , Células Endoteliais/metabolismo , Neovascularização Fisiológica/genética , Regeneração/genética , Transdução de Sinais/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Vasos Sanguíneos/metabolismo , Vasos Sanguíneos/patologia , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Circulação Cerebrovascular , Traumatismos Craniocerebrais/metabolismo , Traumatismos Craniocerebrais/patologia , Modelos Animais de Doenças , Células Endoteliais/patologia , Regulação da Expressão Gênica/genética , Humanos , Macrófagos/metabolismo , Macrófagos/patologia , Meninges/lesões , Meninges/metabolismo , Camundongos , Camundongos Knockout , Receptor beta de Fator de Crescimento Derivado de Plaquetas/genética , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Receptor TIE-2/genética , Receptor TIE-2/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Cicatrização/genéticaRESUMO
Human adipose tissue-derived mesenchymal stem cells expressing the secreted form of the tumor necrosis factor-related apoptosis-inducing ligand (hAT-MSC.sTRAIL) have demonstrated therapeutic activity against various tumors in preclinical studies. However, the limited expression of TRAIL death receptors remains a challenge. We evaluated the therapeutic efficacy of panobinostat in enhancing the sensitivity of hAT-MSC.sTRAIL-mediated apoptosis in malignant glioma. Panobinostat effectively inhibited all malignant glioma cells (IC50, 0.03-0.23⯵M), enhancing the expression of DRs, but not in hAT-MSCs. Combined treatment with hAT-MSC.sTRAIL and panobinostat significantly suppressed cell viability and enhanced apoptosis. In a diffuse intrinsic pontine glioma (DIPG) mouse model, the combined treatment induced decreases in tumor volume and prolonged survival. Our study demonstrates that panobinostat enhances the expression of TRAIL DRs and potentiates the anti-cancer effects of hAT-MSC.sTRAIL.
Assuntos
Tecido Adiposo/citologia , Antineoplásicos/farmacologia , Neoplasias Encefálicas/tratamento farmacológico , Terapia Genética/métodos , Glioma/tratamento farmacológico , Inibidores de Histona Desacetilases/farmacologia , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais , Panobinostat/farmacologia , Ligante Indutor de Apoptose Relacionado a TNF/genética , Animais , Apoptose/efeitos dos fármacos , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Sobrevivência Celular/efeitos dos fármacos , Quimioterapia Adjuvante , Técnicas de Cocultura , Feminino , Glioma/genética , Glioma/metabolismo , Glioma/patologia , Humanos , Masculino , Células-Tronco Mesenquimais/metabolismo , Camundongos Endogâmicos BALB C , Camundongos Nus , Pessoa de Meia-Idade , Fenótipo , Transdução de Sinais , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Carga Tumoral/efeitos dos fármacos , Células Tumorais Cultivadas , Receptores Chamariz do Fator de Necrose Tumoral/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
OBJECTIVEEndothelial colony-forming cells (ECFCs) isolated from pediatric patients with moyamoya disease (MMD) have demonstrated decreased numbers and defective functioning in in vitro experiments. However, the function of ECFCs has not been evaluated using in vivo animal models. In this study, the authors compared normal and MMD ECFCs using a chronic cerebral hypoperfusion (CCH) rat model.METHODSA CCH rat model was made via ligation of the bilateral common carotid arteries (2-vessel occlusion [2-VO]). The rats were divided into three experimental groups: vehicle-treated (n = 8), normal ECFC-treated (n = 8), and MMD ECFC-treated (n = 8). ECFCs were injected into the cisterna magna. A laser Doppler flowmeter was used to evaluate cerebral blood flow, and a radial arm maze test was used to examine cognitive function. Neuropathological examinations of the hippocampus and agranular cortex were performed using hematoxylin and eosin and Luxol fast blue staining in addition to immunofluorescence with CD31, von Willebrand factor, NeuN, myelin basic protein, glial fibrillary acidic protein, and cleaved caspase-3 antibodies.RESULTSThe normal ECFC-treated group exhibited improvement in the restoration of cerebral perfusion and in behavior compared with the vehicle-treated and MMD ECFC-treated groups at the 12-week follow-up after the 2-VO surgery. The normal ECFC-treated group showed a greater amount of neovasculogenesis and neurogenesis, with less apoptosis, than the other groups.CONCLUSIONSThese results support the impaired functional recovery of MMD ECFCs compared with normal ECFCs in a CCH rat model. This in vivo study suggests the functional role of ECFCs in the pathogenesis of MMD.
Assuntos
Estenose das Carótidas/terapia , Circulação Cerebrovascular , Células Progenitoras Endoteliais/fisiologia , Células Progenitoras Endoteliais/transplante , Doença de Moyamoya/patologia , Neovascularização Fisiológica , Neurogênese , Adolescente , Animais , Apoptose , Estenose das Carótidas/etiologia , Criança , Pré-Escolar , Doença Crônica , Cognição , Modelos Animais de Doenças , Feminino , Voluntários Saudáveis , Humanos , Fluxometria por Laser-Doppler , Ligadura , Masculino , Aprendizagem em Labirinto , Doença de Moyamoya/etiologia , Ratos , Recuperação de Função FisiológicaRESUMO
There have been extensive efforts to improve the outcome of glioblastoma, but the prognosis of this disease has not been significantly altered to date. Histone deacetylase inhibitors (HDACIs) have been evaluated as promising anti-cancer drugs and regulate cell growth, cell cycle arrest and apoptosis in glioblastoma. Here, we demonstrated the therapeutic efficacy of a novel pan-HDACI, 7-ureido-N-hydroxyheptanamide derivative (CKD5), compared with traditional pan-HDACIs, such as suberoylanilide hydroxamic acid (SAHA) and trichostatin A (TSA), in vitro and in vivo. Compared with SAHA and TSA, CKD5 had improved cytotoxic effects and induced apoptosis, anti-proliferative activity and cell cycle arrest at G2/M phase. Furthermore, CKD5 significantly reduced tumor volume and prolonged the survival in vivo compared with TSA, suggesting improved anti-cancer efficacy among HDACIs. Our results demonstrate that the novel HDACI CKD5 is a promising therapeutic candidate for glioblastoma.
Assuntos
Neoplasias Encefálicas/tratamento farmacológico , Glioblastoma/tratamento farmacológico , Inibidores de Histona Desacetilases/farmacologia , Ureia/análogos & derivados , Animais , Apoptose/efeitos dos fármacos , Neoplasias Encefálicas/enzimologia , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Feminino , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Glioblastoma/enzimologia , Glioblastoma/patologia , Humanos , Ácidos Hidroxâmicos/farmacologia , Camundongos Endogâmicos BALB C , Camundongos Nus , Fatores de Tempo , Carga Tumoral/efeitos dos fármacos , Ureia/farmacologia , Vorinostat , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Leptomeningeal seeding is a strong negative prognostic factor for medulloblastoma (MB). The mechanism of leptomeningeal seeding is unclear but may involve epigenetic regulation. In this study, we evaluated the feasibility of a histone deacetylase (HDAC) inhibitor, panobinostat, in the suppression of MB leptomeningeal seeding. Panobinostat decreased the cell viability and proliferation, inducing cell cycle arrest and apoptosis in MB cell lines. The migration and adhesion capabilities were significantly decreased. Panobinostat effectively down-regulated protein expression of CCND1 and ID3 which has been associated with leptomeningeal seeding of MB. After panobinostat treatment, neurophil-like cellular processes developed and expression of synaptophysin and NeuroD1 was increased, indicating neuronal differentiation. In MB leptomeningeal seeding in vivo model, the panobinostat-treated group showed significantly decreased spinal leptomeningeal seeding and a survival benefit. The findings demonstrate that panobinostat suppresses MB leptomeningeal seeding through the down-regulation of ID3 and the induction of neuronal differentiation. An HDAC inhibitor might be a potent treatment option for the treatment of MB patients with leptomeningeal seeding.
RESUMO
Stem-cell based gene therapy is a promising novel therapeutic approach for inoperable invasive tumors, including brainstem glioma. Previously, we demonstrated the therapeutic potential of human adipose tissue-derived mesenchymal stem cells (hAT-MSC) genetically engineered to express a secreted form of tumor necrosis factor-related apoptosis-inducing ligand (sTRAIL) against brainstem glioma. However, safety concerns should be comprehensively investigated before clinical applications of hAT-MSC.sTRAIL. At first, we injected stereotactically low (1.2 × 10(5) cells/18 µL), medium (2.4 × 10(5)/18 µL), or high dose (3.6 × 10(5)/18 µL) of hAT-MSC.sTRAIL into the brainstems of immunodeficient mice reflecting the plan of the future clinical trial. Local toxicity, systemic toxicity, secondary tumor formation, and biodistribution of hAT-MSC.sTRAIL were investigated. Next, presence of hAT-MSC.sTRAIL was confirmed in the brain and major organs at 4, 9, and 14 weeks in brainstem glioma-bearing mice. In the 15-week subchronic toxicity test, no serious adverse events in terms of body weight, food consumption, clinical symptom, urinalysis, hematology, clinical chemistry, organ weight, and histopathology were observed. In the 26-week tumorigenicity test, hAT-MSC.sTRAIL made no detectable tumors, whereas positive control U-87 MG cells made huge tumors in the brainstem. No remaining hAT-MSC.sTRAIL was observed in any organs examined, including the brainstem at 15 or 26 weeks. In brainstem glioma-bearing mice, injected hAT-MSC.sTRAIL was observed, but gradually decreased over time in the brain. The mRNA of human specific GAPDH and TRAIL was not detected in all major organs. These results indicate that the hAT-MSC.sTRAIL could be applicable to the future clinical trials in terms of biosafety.
Assuntos
Tecido Adiposo/citologia , Neoplasias do Tronco Encefálico/terapia , Terapia Genética , Glioma/terapia , Transplante de Células-Tronco Mesenquimais/efeitos adversos , Células-Tronco Mesenquimais/metabolismo , Adolescente , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Neoplasias do Tronco Encefálico/patologia , Linhagem Celular Tumoral , Criança , Glioma/patologia , Humanos , Masculino , Células-Tronco Mesenquimais/citologia , Camundongos Endogâmicos BALB C , Camundongos Nus , Tamanho do Órgão , Solubilidade , Análise de Sobrevida , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Distribuição TecidualRESUMO
BACKGROUND AND PURPOSE: White-matter (WM) lesions are known to potentiate cognitive impairment in poststroke patients. The present study was designed to assess whether Ginkgo biloba extract (GB) and cilostazol, which were evaluated alone and in a combination formula (Renexin), can attenuate the WM lesions and cognitive decline caused by chronic hypoperfusion in the rat. METHODS: ANIMALS WERE DIVIDED INTO FIVE TREATMENT GROUPS: cilostazol (25 mg/kg/day), GB (20 mg/kg/day), Renexin (25 mg/kg/day cilostazol + 20 mg/kg/day GB), vehicle, and sham. The animals received the treatments orally 1 day after bilateral common carotid artery occlusion [two-vessel occlusion (2VO); except for the sham group, which underwent the surgery but the arteries were not occluded], and then the same dose every day for 21 days thereafter. Prior to sacrificing the rats, repetitive eight-arm radial maze testing was performed to examine their cognitive abilities. After drug administration and cognitive testing, brain tissues were isolated for Klüver-Barrera and terminal deoxynucleotidyl transferase-mediated biotin-dUTP nick end-labeling (TUNEL) staining, immunohistochemical assessment of glial fibrillary acidic protein (GFAP) and CD11b (OX-42), and to assay free-radical scavenging activity. RESULTS: We found that the significant WM lesions induced by 2VO was ameliorated significantly by treatment with cilostazol, GB, and Renexin, in association with increased TUNEL-positive cells. In addition, chronic cerebral hypoperfusion caused a large increase in the degree of GFAP and OX-42 immunoreactivity and free-radical activity in the optic tract. These abnormalities were significantly reversed by the three drugs, but most prominently by Renexin, suggesting a markedly enhanced or supra-additive effect of cilostazol and GB when administered together. CONCLUSIONS: Significant attenuation of cytoarchitectural damage and apoptotic cell death was found with GB and cilostazol, but a markedly enhanced effect was seen for treatment with their combination in the WM of rat brains after bilateral occlusion of the common carotid arteries. We suggest that combination therapy with GB and cilostazol provides enhanced neuroprotective effects and induces subsequent cognitive improvement in patients with chronic ischemic conditions.