RESUMEN
Gliomas are the most common primary brain tumours, and glioblastomas (GBMs) are subgrouped into four distinct molecular subtypes. This study aimed to identify the potential gene related to glioma progression. Weighted gene co-expression network analysis (WGCNA) was used to explore the related gene. Correlation, ROC, survival and Cox regression analyses were performed. Blue module was strongly associated with WHO grade (r = .65, P = 1e-19). GNG5 in gliomas was overexpressed compared with normal samples and associated with clinicopathologic characteristics. GNG5 was frequent in Mesenchymal subtype and lowly expressed in Proneural subtype of GBMs. Survival and Cox regression analyses showed that glioma patients with GNG5 overexpression had shorter survival time, and GNG5 was an independent prognostic indicator of overall survival. Overall, GNG5 expression is closely associated with clinicopathologic characteristics and is an independent prognostic indicator for glioma patients, as well as a promising subtype-associated biomarker in molecular classification of gliomas.
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Neoplasias Encefálicas/genética , Subunidades gamma de la Proteína de Unión al GTP/metabolismo , Glioma/genética , Neoplasias Encefálicas/patología , Subunidades gamma de la Proteína de Unión al GTP/genética , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Redes Reguladoras de Genes , Glioma/patología , Humanos , PronósticoRESUMEN
Meningiomas are the most common primary intracranial tumor in adults. However, to date, systemic coexpression analyses for meningiomas fail to explain its pathogenesis. The aim of the present study was to construct coexpression modules and identify potential biomarkers associated with meningioma progression. Weighted gene coexpression network analysis (WGCNA) was performed based on GSE43290, and module preservation was tested by GSE74385. Functional annotations were performed to analyze biological significance. Hub genes were selected for efficacy evaluations and correlation analyses using two independent cohorts. A total of 14 coexpression modules were identified, and module lightcyan was significantly associated with WHO grades. Functional enrichment analyses of module lightcyan were associated with tumor pathogenesis. The top 10 hub genes were extracted. Ten biomarkers, particularly AHCYL2, FGL2, and KCNMA1, were significantly related to grades and prognosis of meningioma. These findings not only construct coexpression modules leading to the better understanding of its pathogenesis but also provide potential biomarkers that represent specific on tumor grades and identify recurrence, predicting prognosis and progression of meningiomas.
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Biología Computacional/métodos , Perfilación de la Expresión Génica/métodos , Neoplasias Meníngeas , Meningioma , Transcriptoma/genética , Bases de Datos Genéticas , Humanos , Neoplasias Meníngeas/genética , Neoplasias Meníngeas/metabolismo , Meningioma/genética , Meningioma/metabolismo , Mapas de Interacción de Proteínas/genéticaRESUMEN
Gliomas are the most prevalent malignant primary brain tumors with poor outcome, and four different molecular subtypes (Mesenchymal, Proneural, Neural, and Classical) are popularly applied in scientific researches and clinics of gliomas. Public databases contain an abundant genome-wide resource to explore the potential biomarker and molecular mechanisms using the informatics analysis. The aim of this study was to discover the potential biomarker and investigate its effect in gliomas. Weighted gene co-expression network analysis (WGCNA) was used to construct the co-expression modules and explore the biomarker among the dataset CGGA mRNAseq_693 carrying 693 glioma samples. Functional annotations, ROC, correlation, survival, univariate, and multivariate Cox regression analyses were implemented to investigate the functional effect in gliomas, and molecular experiments in vitro were performed to study the biological effect on glioma pathogenesis. The brown module was found to be strongly related to WHO grade of gliomas, and KEGG pathway analysis demonstrated that TNFRSF1A was enriched in MAPK signaling pathway and TNF signaling pathway. Overexpressed TNFRSF1A was strongly related to clinical features such as WHO grade, and functioned as an independent poor prognostic predictor of glioma patients. Notably, TNFRSF1A was preferentially upregulated in the Mesenchymal subtype gliomas (Mesenchymal-associated). Knockdown of TNFRSF1A inhibited proliferation and migration of glioma cell lines in vitro. Our findings provide a further understanding of the progression of gliomas, and Mesenchymal-associated TNFRSF1A might be a promising target of diagnosis, therapy, and prognosis of gliomas.
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Ependymomas (EPNs) are one of the most common types of malignant neuroepithelial tumors. In an effort to identify potential biomarkers involved in the pathogenesis of EPN, the mRNA expression profiles of the GSE25604, GSE50161, GSE66354, GSE74195 and GSE86574 datasets, in addition to the microRNA (miRNA/miR) expression profiles of GSE42657 were downloaded from the Gene Expression Omnibus (GEO) database. Differentially expressed genes (DEGs) and differentially expressed miRNAs (DEMs) between EPN and normal brain tissue samples were identified using the Limma package in R and GEO2R, respectively. Functional and pathway enrichment analyses were conducted using the Database for Annotation, Visualization and Integrated Discovery. A protein-protein interaction network was constructed using the Search Tool for Retrieval of Interacting Genes database, which was visualized using Cytoscape. The targeted genes of DEMs were predicted using miRWalk2.0 and a miRNA-mRNA regulatory network was constructed. Following analysis, a total of 948 DEGs and 129 DEMs were identified. Functional enrichment analysis revealed that 609 upregulated DEGs were significantly enriched in 'PI3K-Akt signaling pathway', while 339 downregulated DEGs were primarily involved in 'cell junction' and 'retrograde endocannabinoid signaling'. In addition, 6 hub genes [cyclin dependent kinase 1, CD44 molecule (Indian blood group) (CD44), proliferating cell nuclear antigen (PCNA), MYC, synaptotagmin 1 (SYT1) and kinesin family member 4A] and 6 crucial miRNAs [homo sapiens (hsa)-miR-34a-5p, hsa-miR-449a, hsa-miR-106a-5p, hsa-miR-124-3p, hsa-miR-128-3p and hsa-miR-330-3p] were identified as biomarkers and potential therapeutic targets for EPN. Furthermore, a microRNA-mRNA regulatory network was constructed to highlight the interactions between DEMs and their target DEGs; this included the hsa-miR-449a-SYT1, hsa-miR-34a-5p-SYT1, hsa-miR-330-3p-CD44 and hsa-miR-124-3p-PCNA pairs, whose expression levels were confirmed using reverse transcription-quantitative polymerase chain reaction. In conclusion, the present study may provide important data for the investigation of the molecular mechanisms of EPN pathogenesis.
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Medulloblastoma (MB) is the most common malignant brain tumor in children. The aim of the present study was to predict biomarkers and reveal their potential molecular mechanisms in MB. The gene expression profiles of GSE35493, GSE50161, GSE74195 and GSE86574 were downloaded from the Gene Expression Omnibus (GEO) database. Using the Limma package in R, a total of 1,006 overlapped differentially expressed genes (DEGs) with the cut-off criteria of P<0.05 and |log2fold-change (FC)|>1 were identified between MB and normal samples, including 540 upregulated and 466 downregulated genes. Furthermore, the Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were also performed using the Database for Annotation, Visualization and Integrated Discovery (DAVID) online tool to analyze functional and pathway enrichment. The Search Tool for Retrieval of Interacting Genes database was subsequently used to construct a protein-protein interaction (PPI) network and the network was visualized in Cytoscape. The top 11 hub genes, including CDK1, CCNB1, CCNB2, PLK1, CDC20, MAD2L1, AURKB, CENPE, TOP2A, KIF2C and PCNA, were identified from the PPI network. The survival curves for hub genes in the dataset GSE85217 predicted the association between the genes and survival of patients with MB. The top 3 modules were identified by the Molecular Complex Detection plugin. The results indicated that the pathways of DEGs in module 1 were primarily enriched in cell cycle, progesterone-mediated oocyte maturation and oocyte meiosis; and the most significant functional pathways in modules 2 and 3 were primarily enriched in mismatch repair and ubiquitin-mediated proteolysis, respectively. These results may help elucidate the pathogenesis and design novel treatments for MB.
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Glioblastoma multiforme (GBM) is the most common malignant tumor of the central nervous system and has a very poor prognosis. Currently, patients were treated by resection followed by radiotherapy plus concurrent temozolomide (TMZ) chemotherapy. However, many patients are resistant to TMZ-induced DNA damage because of upregulated expression of the DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT). In this study, upregulation of SATB1 and MGMT, and downregulation of SLC22A18 resulted in acquisition of TMZ resistance in GBM U87 cells. Inactivation of special AT-rich sequence-binding protein 1 (SATB1) using short hairpin RNA (shRNA) downregulated MGMT expression and upregulated solute carrier family 22 member 18 (SLC22A18) expression in GBM cells. This suggested SATB1-mediated posttranscriptional regulation of the MGMT and SLC22A18 protein levels. Immunohistochemical analysis of malignant glioma specimens demonstrated a significant positive correlation between the levels of MGMT and SATB1, and a negative correlation between the levels of SLC22A18 and SATB1. Importantly, in recurrent, compared with the primary, lesions in 15 paired identical tumors, the SATB1 and MGMT protein levels were increased and the SLC22A18 levels were decreased. Finally, in TMZ-resistant GBM, SATB1 knockdown enhanced TMZ efficacy. Consequently, SATB1 inhibition might be a promising strategy combined with TMZ chemotherapy to treat TMZ-resistant GBM.
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Antineoplásicos Alquilantes/uso terapéutico , Metilasas de Modificación del ADN/metabolismo , Enzimas Reparadoras del ADN/metabolismo , Glioblastoma/tratamiento farmacológico , Proteínas de Unión a la Región de Fijación a la Matriz/metabolismo , Proteínas de Transporte de Catión Orgánico/metabolismo , Temozolomida/uso terapéutico , Proteínas Supresoras de Tumor/metabolismo , Antineoplásicos Alquilantes/farmacología , Movimiento Celular , Regulación hacia Abajo , Resistencia a Antineoplásicos , Glioblastoma/genética , Glioblastoma/patología , Humanos , Temozolomida/farmacología , Regulación hacia ArribaRESUMEN
BACKGROUND: Anti-angiogenic therapy inhibits tumor growth and is considered as a potential clinical therapy for malignant glioma. However, inevitable recurrences and unexpected tumor resistance, particularly increased invasion ability of glioma cell, were observed after anti-angiogenic treatment. The underlying mechanism remains undetermined. Focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2 (Pyk2) are closely associated with cell migration; therefore, we investigated the possible role of these kinases in rat C6 glioma cell invasion induced by bevacizumab, a recombinant monoclonal antibody against vascular endothelial growth factor (VEGF). METHODS: The effects of bevacizumab on migration and invasion of C6 glioma cells were investigated in vitro and in vivo. The cells proliferation, migration, and invasion were determined by MTT assay, wound healing, and transwell assay, respectively. Invasive potential of glioma cells in vivo was assessed by counting vimentin-positive cells crossing the solid tumor rim by immunohistochemical staining. The total and phosphorylated protein levels of FAK and Pyk2 were detected by Western blotting. RESULTS: Bevacizumab exposure increased migration and invasion of cultured C6 cells in a concentration-dependent manner. In addition, the continuous bevacizumab treatment also promoted tumor invasion in rat C6 intracranial glioma models. Bevacizumab treatment enhanced Pyk2 phosphorylation at Tyr402, but no effect on FAK phosphorylation at Tyr397 both in vitro and in vivo. Knockdown of Pyk2 by siRNA or inhibition of Pyk2 phosphorylation by Src kinase specific inhibitor PP1 partially inhibited bevacizumab-induced cell invasion in cultured C6 glioma cells. Furthermore, the combined administration of bevacizumab and PP1 significantly suppressed glioma cell invasion into surrounding brain tissues compared to bevacizumab treatment alone in experimental rats. CONCLUSIONS: These results suggest that anti-VEGF treatment promotes glioma cell invasion via activation of Pyk2. Inhibition of Pyk2 phosphorylation might be a potential target to ameliorate the therapeutic efficiency of anti-VEGF treatment.
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Anticuerpos Monoclonales Humanizados/uso terapéutico , Neoplasias Encefálicas/patología , Quinasa 2 de Adhesión Focal/metabolismo , Glioma/patología , Invasividad Neoplásica , Animales , Bevacizumab , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/enzimología , Línea Celular Tumoral , Activación Enzimática , Inducción Enzimática , Glioma/tratamiento farmacológico , Glioma/enzimología , Metástasis de la Neoplasia , RatasRESUMEN
Special AT-rich sequence-binding protein-1 (SATB1) has been reported to be over-expressed in many human tumors and knockdown of SATB1 can inhibit tumor growth. The present study was designed to determine the role of SATB1 in the growth of human glioma U251 cells using the plasmid-based SATB1 short hairpin RNA (shRNA) delivered by hydroxyapatite nanoparticles in vitro and in vivo. The in vitro growth, invasion and angiogenesis assays of human glioma U251 cells were done. U251 cells tumor blocks were transplanted into the nude mice. CaCl2-modified hydroxyapatite nanoparticles carrying shRNA-SATB1 plasmids were injected into the tumors. The apoptosis of the tumor U251 cells was examined with TUNEL assay and flow cytometer (FCM). The tumor growth and immunohistochemistry were measured. The expression level of SATB1 mRNA was investigated by RT-PCR. The expression levels of SATB1, Cyclin D1, MMP-2, VEGF, Bax and Caspase-9 protein were determined by western blot analysis. The results showed that hydroxyapatite nanoparticles-delivered shRNA-SATB1 could significantly inhibit the growth, invasion and angiogenesis of U251 cells in vitro and the growth of U251 cells in vivo. FCM results showed that Nano HAP-shRNA-SATB1-induced apoptosis (up to 67.8 %). SATB1 expression was strongly down-regulated in the tumor U251 cells. Cyclin D1, MMP-2 and VEGF were also down-regulated in the tumor tissues that also displayed significant increased in Bax expression and Caspase-9 activity. These results show that Nano HAP-shRNA-SATB1 can inhibit the growth of human glioma U251 cells in vitro and in vivo, and hydroxyapatite nanoparticles can be used for the in vitro and in vivo delivery of plasmid-based shRNAs into U251 cells.
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Durapatita/administración & dosificación , Glioma/genética , Proteínas de Unión a la Región de Fijación a la Matriz/genética , Nanopartículas/administración & dosificación , Animales , Caspasa 9/biosíntesis , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Ciclina D1/biosíntesis , Durapatita/química , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Glioma/tratamiento farmacológico , Glioma/patología , Humanos , Metaloproteinasa 2 de la Matriz/biosíntesis , Ratones , Nanopartículas/química , ARN Interferente Pequeño/efectos de los fármacos , Proteína X Asociada a bcl-2/biosíntesisRESUMEN
BACKGROUND: Previous study showed that hydroxyapatite nanoparticles (nano-HAPs) inhibited glioma growth in vitro and in vivo; and in a drug combination, they could reduce adverse reactions. We investigated the possible enhancement of radiosensitivity induced by nano-HAPs. METHODS: In vitro radiosensitization of nano-HAPs was measured using a clonogenic survival assay in human glioblastoma U251 and breast tumor brain metastatic tumor MDA-MB-231BR cells. DNA damage and repair were measured using γH2AX foci, and mitotic catastrophe was determined by immunostaining. The effect of nano-HAPs on in vivo tumor radiosensitivity was investigated in a subcutaneous and an orthotopic model. RESULTS: Nano-HAPs enhanced each cell line's radiosensitivity when the exposure was 1 h before irradiation, and they had no significant effect on irradiation-induced apoptosis or on the activation of the G2 cell cycle checkpoint. The number of γH2AX foci per cell was significantly large at 24 h after the combination modality of nano-HAPs + irradiation compared with single treatments. Mitotic catastrophe was also significantly increased at an interval of 72 h in tumor cells receiving the combined modality compared with the individual treatments. In a subcutaneous model, nano-HAPs caused a larger than additive increase in tumor growth delay. In an orthotopic model, nano-HAPs significantly reduced tumor growth and extended the prolongation of survival induced by irradiation. CONCLUSIONS: These results show that nano-HAPs can enhance the radiosensitivity of tumor cells in vitro and in vivo through the inhibition of DNA repair, resulting in an increase in mitotic catastrophe.
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Neoplasias Encefálicas/prevención & control , Neoplasias de la Mama/prevención & control , Durapatita/farmacología , Glioblastoma/prevención & control , Nanopartículas , Tolerancia a Radiación/efectos de los fármacos , Fármacos Sensibilizantes a Radiaciones/farmacología , Animales , Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , Materiales Biocompatibles/farmacología , Western Blotting , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/secundario , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Ciclo Celular/efectos de los fármacos , Ciclo Celular/efectos de la radiación , Proliferación Celular/efectos de los fármacos , Proliferación Celular/efectos de la radiación , Femenino , Citometría de Flujo , Glioblastoma/metabolismo , Glioblastoma/patología , Histonas/metabolismo , Humanos , Técnicas para Inmunoenzimas , Técnicas In Vitro , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Células Tumorales Cultivadas , Rayos XRESUMEN
BACKGROUND: Our previous study showed that SLC22A18 downregulation and promoter methylation were associated with the development and progression of glioma and the elevated expression of SLC22A18 was found to increase the sensitivity of glioma U251 cells to the anticancer drug 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). In this study, we investigated the predictive value of SLC22A18 promoter methylation and protein expression in glioblastoma multiforme (GBM) patients receiving temozolomide (TMZ) therapy. PATIENTS AND METHODS: SLC22A18 promoter methylation and protein expression were examined by methylation-specific polymerase chain reaction (MSP) and Western blotting respectively, then we compared SLC22A18 promoter methylation and protein expression in tumor cell explants in regard to prediction of TMZ response and survival time of 86 GBM patients. RESULTS: SLC22A18 promoter methylation was detected in 61 of 86 (71%) samples, whereas 36 of 86 (42%) cases were scored positive for SLC22A18 protein expression. Overall SLC22A18 promoter methylation was significantly related to SLC22A18 protein expression, but a subgroup of cases did not follow this association. Multivariate Cox regression analysis indicated that SLC22A18 protein expression, but not promoter methylation, was significantly correlated with TMZ therapy. SLC22A18 protein expression predicted a significantly shorter overall survival in 51 patients receiving TMZ therapy, whereas no differences in overall survival were observed in 35 patients without TMZ therapy. CONCLUSIONS: These results show that lack of SLC22A18 protein expression is superior to promoter methylation as a predictive tumor biomarker in GBM patients receiving temozolomide therapy.
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Antineoplásicos/uso terapéutico , Neoplasias Encefálicas/metabolismo , Dacarbazina/análogos & derivados , Glioblastoma/metabolismo , Proteínas de Transporte de Catión Orgánico/metabolismo , Western Blotting , Neoplasias Encefálicas/tratamiento farmacológico , Metilación de ADN , Dacarbazina/uso terapéutico , Femenino , Glioblastoma/tratamiento farmacológico , Humanos , Masculino , Proteínas de Transporte de Catión Orgánico/genética , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas , TemozolomidaRESUMEN
Special AT-rich-sequence-binding protein 1 (SATB1), a new type of gene regulator, has been reported to be expressed in various human cancers and may be associated with malignancy. The aim of this study was to investigate the expression of SATB1 in astrocytoma and to determine its prognostic value for the overall survival of patients with astrocytoma. The expression of SATB1 protein and messenger RNA (mRNA) in human astrocytoma specimens was examined using immunohistochemistry and semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). The relationship between SATB1 expression and O-6-methylguanine-DNA methyltransferase (MGMT) promoter methylation status was also investigated. Spearman's correlation coefficient was used to describe the association between SATB1 expression and the clinical parameters of astrocytoma patients. SATB1 protein and mRNA were expressed at significant levels in astrocytoma specimens. SATB1 expression was positively correlated with astrocytoma pathological grade but negatively correlated with the life span of astrocytoma patients. SATB1 expression was also significantly lower in astrocytoma specimens with MGMT promoter methylation than in those without MGMT promoter methylation. Our findings suggest that SATB1 may have an important role as a positive regulator of astrocytoma development and progression and that SATB1 might be a useful molecular marker for predicting the prognosis of patients with astrocytoma and could be a novel target for treating astrocytoma.
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Astrocitoma/genética , Astrocitoma/patología , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Proteínas de Unión a la Región de Fijación a la Matriz/genética , Adulto , Anciano , Astrocitoma/cirugía , Biomarcadores de Tumor/genética , Neoplasias Encefálicas/cirugía , Carcinógenos , Metilación de ADN , Femenino , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Análisis Multivariante , Procedimientos Neuroquirúrgicos , O(6)-Metilguanina-ADN Metiltransferasa/genética , Pronóstico , ARN/biosíntesis , ARN/aislamiento & purificación , ARN Mensajero/biosíntesis , ARN Mensajero/genética , ARN Neoplásico/biosíntesis , ARN Neoplásico/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Análisis de Regresión , Sobrevida , Análisis de SupervivenciaRESUMEN
Special AT-rich sequence-binding protein-1 (SATB1) has been reported to be overexpressed in numerous human tumors. The aim of the present study was to determine the correlation and clinical significance between the expression of SATB1 and B-cell lymphoma 2 (Bcl-2) in human glioblastoma multiforme (GBM). Samples from 70 patients with GBMs were analyzed and 10 normal brain tissues were used as the control group. In situ hybridization was used to detect SATB1 mRNA expression and immunohistochemistry was used to detect Bcl-2 and proliferating cell nuclear antigen (PCNA) protein expression. Apoptosis was detected with flow cytometry. The SATB1 mRNA and Bcl-2 protein levels were found to be significantly higher in GBM tissues than in normal brain and their levels were associated with patient survival, but not associated with patient gender, age and tumor size and site. A positive correlation was observed between SATB1 mRNA and Bcl-2 protein and between SATB1 mRNA and PCNA. A negative correlation was observed between SATB1 mRNA and apoptosis and between Bcl-2 and apoptosis. A positive correlation existed between Bcl-2 and PCNA. Patients with GBM identified as SATB1 mRNA (+) and Bcl-2 (+) were associated with a poor prognosis. Therefore, assessment of SATB1 and Bcl-2 co-expression may provide important information for the diagnosis, therapy and prognosis of GBM.
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Neoplasias Encefálicas/genética , Regulación Neoplásica de la Expresión Génica , Glioblastoma/genética , Proteínas de Unión a la Región de Fijación a la Matriz/genética , Proteínas Proto-Oncogénicas c-bcl-2/genética , Adulto , Anciano , Apoptosis/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/mortalidad , Femenino , Citometría de Flujo , Glioblastoma/metabolismo , Glioblastoma/mortalidad , Humanos , Inmunohistoquímica , Hibridación Fluorescente in Situ , Masculino , Proteínas de Unión a la Región de Fijación a la Matriz/metabolismo , Persona de Mediana Edad , Pronóstico , Antígeno Nuclear de Célula en Proliferación/genética , Antígeno Nuclear de Célula en Proliferación/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismoRESUMEN
Hydroxyapatite nanoparticles (nano-HAPs) have been reported to exhibit antitumor effects on various human cancers, but the effects of nano-HAPs on human glioma cells remain unclear. The aim of this study was to explore the inhibitory effect of nano-HAPs on the growth of human glioma U251 and SHG44 cells in vitro and in vivo. Nano-HAPs could inhibit the growth of U251 and SHG44 cells in a dose- and time-dependent manner, according to methyl thiazoletetrazolium assay and flow cytometry. Treated with 120 mg/L and 240 mg/L nano-HAPs for 48 hours, typical apoptotic morphological changes were noted under Hoechst staining and transmission electron microscopy. The tumor growth of cells was inhibited after the injection in vivo, and the related side effects significantly decreased in the nano-HAP-and-drug combination group. Because of the function of nano-HAPs, the expression of c-Met, SATB1, Ki-67, and bcl-2 protein decreased, and the expression of SLC22A18 and caspase-3 protein decreased noticeably. The findings indicate that nano-HAPs have an evident inhibitory action and induce apoptosis of human glioma cells in vitro and in vivo. In a drug combination, they can significantly reduce the adverse reaction related to the chemotherapeutic drug 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU).
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Durapatita/farmacología , Glioma/tratamiento farmacológico , Nanopartículas/química , Animales , Antineoplásicos/química , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Caspasa 3/metabolismo , Procesos de Crecimiento Celular/efectos de los fármacos , Línea Celular Tumoral , Forma de la Célula/efectos de los fármacos , Durapatita/química , Glioma/metabolismo , Glioma/patología , Humanos , Etiquetado Corte-Fin in Situ , Masculino , Proteínas de Unión a la Región de Fijación a la Matriz/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Nanopartículas/administración & dosificación , Proteínas de Transporte de Catión Orgánico/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteínas Proto-Oncogénicas c-met/metabolismoRESUMEN
BACKGROUND: Special AT-rich sequence-binding protein-1 (SATB1) has been reported to be expressed in several human cancers and may have malignant potential. This study was aimed at investigating the expression and potential role of SATB1 in human glioma. METHOD: The relationship between SATB1 expression, clinicopathological parameters, Ki67 expression and MGMT promoter methylation status was evaluated, and the prognostic value of SATB1 expression in patients with gliomas was analyzed. SATB1-specific shRNA sequences were synthesized, and U251 cells were transfected with SATB1 RNAi plasmids. Expression of SATB1 mRNA and protein was investigated by RT-PCR and immunofluoresence staining and western blotting. The expression of c-Met, SLC22A18, caspase-3 and bcl-2 protein was determined by western blotting. U251 cell growth and adherence was detected by methyl thiazole tetrazolium assay. The apoptosis of U251 cells was examined with a flow cytometer. The adherence, invasion, and in vitro angiogenesis assays of U251 cells were done. The growth and angiogenesis of SATB1 low expressing U251 cells was measured in an in vivo xenograft model. RESULTS: Of 70 tumors, 44 (62.9%) were positive for SATB1 expression. SATB1 expression was significantly associated with a high histological grade and with poor survival in univariate and multivariate analyses. SATB1 expression was also positively correlated with Ki67 expression but negatively with MGMT promoter methylation in glioma tissues. SATB1 shRNA expression vectors could efficiently induce the expression of SLC22A18 protein, increase the caspase-3 protein, inhibit the expression of SATB1, c-Met and bcl-2 protein, the growth, invasion, metastasis and angiogenesis of U251 cells, and induce apoptosis in vitro. Furthermore, the tumor growth of U251 cells expressing SATB1 shRNA were inhibited in vivo, and immunohistochemical analyses of tumor sections revealed a decreased vessel density in the animals where shRNA against SATB1 were expressed. CONCLUSIONS: SATB1 may have an important role as a positive regulator of glioma development and progression, and that SATB1 might be a useful molecular marker for predicting the prognosis of glioma.
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Neoplasias Encefálicas/metabolismo , Glioma/metabolismo , Proteínas de Unión a la Región de Fijación a la Matriz/metabolismo , Regulación hacia Arriba , Animales , Western Blotting , Neoplasias Encefálicas/patología , Adhesión Celular , Línea Celular Tumoral , Metilación de ADN , Progresión de la Enfermedad , Técnica del Anticuerpo Fluorescente , Glioma/patología , Humanos , Inmunohistoquímica , Proteínas de Unión a la Región de Fijación a la Matriz/genética , Ratones , Invasividad Neoplásica , Neovascularización Patológica , Regiones Promotoras Genéticas , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
SLC22A18 [solute carrier family 22 (organic cation transporter) member 18] is located within the 11p15.5 cluster, and may be a new tumor suppressor gene; evidence of SLC22A18 hypermethylation is documented in several types of human cancers. In order to determine whether SLC22A18 hypermethylation is involved in glioma, we determined the SLC22A18 gene protein expression, mRNA expression and methylation status in glioma U251 cells before and after treatment with 5-Aza-2'deoxycytidine (5-Aza-CdR), and observed the change in growth. Glioma U251 cells treated with 5-Aza-CdR were analyzed by flow cytometry to identify any change in their cell cycle profiles. Tumors induced via the injection of untreated U251 cells were measured. Immunohistochemistry, reverse transcriptase-polymerase chain reaction (RT-PCR) and PCR-based methylation assay were carried out to determine SLC22A18 gene protein expression, mRNA expression and methylation status in glioma U251 cells before and after treatment with 5-AzaCdR. The treated cells showed an increase in their proportion in G1, from 79.2 to 83.5%, and a decrease in S phase, from 12.4 to 5.8%. The apoptotic rate increased from 6.4 to 15.8%. Tumors induced via the injection of untreated U251 cells were approximately 1.46 cm³ in size, whereas the tumors induced by U251 cells treated with 5-Aza-CdR averaged 0.88 cm³ in size. The expression levels of SLC22A18 protein and mRNA in U251 cells were increased following treatment with 5x10â»7 M 5-AzaCdR. Prior to 5-Aza-CdR treatment, the SLC22A18 gene demonstrated hypermethylation and therefore could not be cleaved by HpaII and MspI. It is known that only the DNA digested with HpaII or MspI can be amplified. Following treatment with 5-AzaCdR, the SLC22A18 gene became demethylated, and could then be cleaved by both of the enzymes, and this failed to be amplified. 5-Aza-cdR may induce glioma U251 cell division and apoptosis and enhance demethylation and protein and mRNA expression of SLC22A18. The hypermethylation of SLC22A18 may be related to the transcriptional silencing of this gene. The growth inhibitory effects of 5-Aza-CdR treatment in vivo remain recognizable.
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Antimetabolitos Antineoplásicos/farmacología , Azacitidina/análogos & derivados , Proteínas de Transporte de Catión Orgánico/metabolismo , Apoptosis/efectos de los fármacos , Azacitidina/farmacología , Puntos de Control del Ciclo Celular , Línea Celular Tumoral , Metilación de ADN/efectos de los fármacos , Decitabina , Fase G1 , Glioma , Humanos , Proteínas de Transporte de Catión Orgánico/genética , ARN Mensajero/metabolismo , Fase SRESUMEN
We investigated the expression of the putative tumor suppressor SLC22A18 to evaluate it as a prognostic marker in glioma patients. Immunohistochemical and Western blot analyses of clinical tissue samples obtained from 120 patients with glioma were performed. Low expression of SLC22A18 was observed in 71.7% of patients. Loss of SLC22A18 expression in glioma was significantly related to pathological grade (p = 0.003). High pathological grade (World Health Organization III-IV) was correlated with negative (low or absent) expression of SLC22A18, which was correlated with a significantly shorter overall patient survival than in those with positive (high) expression (p = 0.007). Multivariate Cox regression analysis indicated that SLC22A18 expression level is an independent survival prognostic factor for patients with glioma (p = 0.011). Western blotting analysis confirmed decreased expression of SLC22A18 in glioma tissues compared with adjacent brain tissues. This study suggests that SLC22A18 functions as a tumor suppressor in glioma and represents a candidate biomarker for long-term survival in this disease.
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Biomarcadores de Tumor/deficiencia , Neoplasias Encefálicas/diagnóstico , Neoplasias Encefálicas/metabolismo , Regulación hacia Abajo/fisiología , Glioma/diagnóstico , Glioma/metabolismo , Proteínas de Transporte de Catión Orgánico/deficiencia , Adulto , Anciano , Biomarcadores de Tumor/biosíntesis , Biomarcadores de Tumor/fisiología , Neoplasias Encefálicas/mortalidad , Femenino , Glioma/mortalidad , Humanos , Masculino , Persona de Mediana Edad , Proteínas de Transporte de Catión Orgánico/biosíntesis , Proteínas de Transporte de Catión Orgánico/fisiología , Pronóstico , Tasa de Supervivencia , Proteínas Supresoras de Tumor/deficiencia , Adulto JovenRESUMEN
BACKGROUND: Downregulation of the putative tumor suppressor gene SLC22A18 has been reported in a number of human cancers. The aim of this study was to investigate the relationship between SLC22A18 downregulation, promoter methylation and the development and progression of human glioma. METHOD: SLC22A18 expression and promoter methylation was examined in human gliomas and the adjacent normal tissues. U251 glioma cells stably overexpressing SLC22A18 were generated to investigate the effect of SLC22A18 on cell growth and adherence in vitro using the methyl thiazole tetrazolium assay. Apoptosis was quantified using flow cytometry and the growth of SLC22A18 overexpressing U251 cells was measured in an in vivo xenograft model. RESULTS: SLC22A18 protein expression is significantly decreased in human gliomas compared to the adjacent normal brain tissues. SLC22A18 protein expression is significantly lower in gliomas which recurred within six months after surgery than gliomas which did not recur within six months. SLC22A18 promoter methylation was detected in 50% of the gliomas, but not in the adjacent normal tissues of any patient. SLC22A18 expression was significantly decreased in gliomas with SLC22A18 promoter methylation, compared to gliomas without methylation. The SLC22A18 promoter is methylated in U251 cells and treatment with the demethylating agent 5-aza-2-deoxycytidine increased SLC22A18 expression and reduced cell proliferation. Stable overexpression of SLC22A18 inhibited growth and adherence, induced apoptosis in vitro and reduced in vivo tumor growth of U251 cells. CONCLUSION: SLC22A18 downregulation via promoter methylation is associated with the development and progression of glioma, suggesting that SLC22A18 is an important tumor suppressor in glioma.
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Metilación de ADN/genética , Progresión de la Enfermedad , Regulación hacia Abajo/genética , Glioma/genética , Glioma/patología , Proteínas de Transporte de Catión Orgánico/genética , Regiones Promotoras Genéticas , Animales , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Azacitidina/farmacología , Western Blotting , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Adhesión Celular/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Metilación de ADN/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Ratones , Clasificación del Tumor , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Oligodendroglía/efectos de los fármacos , Oligodendroglía/metabolismo , Proteínas de Transporte de Catión Orgánico/metabolismo , Reacción en Cadena de la Polimerasa , ARN Mensajero/genética , ARN Mensajero/metabolismo , RecurrenciaRESUMEN
The hepatocyte growth factor (HGF) and vascular endothelial growth factor (VEGF) are important cytokines with modulatory actions in the nervous system. The present study aimed to investigate the role and expression of HGF and VEGF in the cerebral tissue of adult rats with chronic hydrocephalus after subarachnoid hemorrhage. Adult female Wistar rats were randomly divided into 4 groups: a control group (n=20) and 3 experimental subgroups (n=60). Subarachnoid hemorrhage was induced by the injection of 0.4 ml of non-heparinized autologous arterial blood into the cisterna magna of experimental animals on day 0 with a second injection 2 days later. The rats were sacrificed within 24 h of magnetic resonance imaging (MRI) examination at 2, 4, or 6 weeks. The excised brains were studied by RT-PCR, immunohistochemical and Western blot analyses as we examined HGF and VEGF mRNA and protein expression. Chronic hydrocephalus was induced in 21 rats after subarachnoid hemorrhage. After 2 weeks, the expression of HGF and VEGF in the cerebral tissue was significantly increased in the experimental group compared to the controls, especially in periventricular white matter. Our results indicate that HGF and VEGF participate in the pathological injury and repair of cerebral tissue in rats with chronic hydrocephalus after subarachnoid hemorrhage.
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Envejecimiento/metabolismo , Cerebro/metabolismo , Cerebro/patología , Factor de Crecimiento de Hepatocito/metabolismo , Hidrocefalia/etiología , Hemorragia Subaracnoidea/complicaciones , Factor A de Crecimiento Endotelial Vascular/metabolismo , Envejecimiento/patología , Animales , Western Blotting , Ventrículos Cerebrales/metabolismo , Ventrículos Cerebrales/patología , Enfermedad Crónica , Densitometría , Femenino , Regulación de la Expresión Génica , Factor de Crecimiento de Hepatocito/genética , Hidrocefalia/genética , Inmunohistoquímica , Imagen por Resonancia Magnética , Tamaño de los Órganos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Coloración y Etiquetado , Hemorragia Subaracnoidea/genética , Factor A de Crecimiento Endotelial Vascular/genéticaRESUMEN
Previous studies showed that solute carrier family 22 member 18 (SLC22A18) is involved in tumorigenesis. The aim of this study was to examine the role of SLC22A18 in glioma cells. Glioma U251 cells were transfected with the human SLC22A18 gene. Transfection of the empty vector pcDNA3.1 was used as a negative control. Sensitivity to BCNU was measured by Annexin V staining. The expression of caspase-3 and bcl-2 was determined by immunohistochemistry. The transfection was confirmed by PCR, RT-PCR and Western blotting. Augmented apoptotic cell death was observed in the SLC22A18-transfected cells, compared to the non-transfected cells or cells with the empty vector. Caspase-3 expression increased in U251-SLC22A18 cells, whereas the bcl-2 expression decreased. These results indicated that SLC22A18 has a pro-apoptotic function in glioma cells.
RESUMEN
Cell culture, tissue chemistry and flow cytometry were used to determine whether antisense c-Met oligodeoxynucleotides enhanced the sensitivity of human glioma cells to paclitaxel. A combination of paclitaxel with antisense c-Met oligodeoxynucleotides inhibited cell growth, induced apoptosis and induced c-Met protein expression in U251 and SHG44 human glioma cells more significantly than either paclitaxel or the oligodeoxynucleotides on their own (P<0.01). Thus, c-Met antisense oligodeoxynucleotides increase the sensitivity of human glioma cells to paclitaxel. Combined use of the two agents could be a novel and attractive strategy in human glioma treatment.