cyclin-d1 and Brain-Neoplasms

Some studies already tried to assess the associations between cyclin D1 (CCND1) polymorphisms and brain tumor. However, the results of these studies were not consistent. Thus, we performed the present meta-analysis to explore the relationship between CCND1 polymorphisms and brain tumor in a larger pooled population.. PubMed, Web of Science, Embase, and CNKI were searched for related articles. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated to assess the potential associations.. Our findings suggested that CCND1 rs603965 polymorphism may serve as a potential genetic biomarker of brain tumor, especially for glioma.

Topics: Asian People; Biomarkers, Tumor; Brain Neoplasms; Cyclin D1; Glioma; Humans; Polymorphism, Single Nucleotide; White People

Topics: Antineoplastic Combined Chemotherapy Protocols; Biomarkers, Tumor; Biopsy; Brain Neoplasms; Cyclin D1; Diagnosis, Differential; Fatal Outcome; Flow Cytometry; Humans; Lymphoma, B-Cell; Magnetic Resonance Imaging; Male; Middle Aged; Proto-Oncogene Proteins c-myc; Tomography, X-Ray Computed; Translocation, Genetic

Previous studies have shown the association of the Cyclin D1 (CCND1) G870A polymorphism with glioma risk, but the findings are inconsistent and inconclusive. To shed some light on the findings across individual studies and acquire a quantitative assessment of this association, we conducted a meta-analysis of all published case-control studies thus far. Four independent studies with a total of 690 cases and 1,014 controls were identified after a systematic search of the PubMed, Embase, Web of Science, and Wanfang databases. The strength of the association between the CCND1 G870A polymorphism and glioma risk was estimated by the pooled odds ratios (ORs) with 95 % confidence intervals (95 %CIs). Subgroup analysis by ethnicity was also performed. Overall, a statistically significant association was found between the CCND1 G870A polymorphism and glioma risk in three genetic models (ORA vs. G = 1.178, 95 %CI 1.025-1.354, P OR = 0.021; ORAA vs. GG = 1.328, 95 %CI 1.007-1.750, P OR = 0.045; ORAA + AG vs. GG = 1.253, 95 %CI 1.006-1.516, P OR = 0.044). In subgroup analysis, the pooled ORs suggested that the CCND1 G870A polymorphism was associated with an increased risk of glioma in Caucasians under the heterozygote and dominant genetic models (ORAG vs. GG = 1.329, 95 %CI 1.001-1.766, P OR = 0.049; ORAA + AG vs. GG = 1.332, 95 %CI 1.019-1.740, P OR = 0.036). The meta-analysis suggests that the CCND1 G870A polymorphism is a risk factor for the development of glioma.

Topics: Brain Neoplasms; Cyclin D1; Genetic Predisposition to Disease; Glioma; Humans; Polymorphism, Genetic; Publication Bias; Risk

In recent years, numerous studies have been performed to investigate the CCND1 G870A gene polymorphism impact on brain tumors susceptibility. Unfortunately, the results of previous studies were inconsistent. Therefore, we performed a meta-analysis to derive a more precise estimation of any association.. We conducted a search in PubMed, Embase and CNKI covering all published papers up to November, 2013. Odds ratios (ORs) and their 95% confidence intervals (95%CIs) were applied to assess associations.. A total of 6 publications including 9 case-control studies met the inclusion criteria. The pooled ORs for the total included studies showed significant association among comparison A vs G (OR= 1.246, 95%CI= 1.092-1.423, p= 0.001), homozygote comparison AA vs GG (OR= 1.566, 95%CI= 1.194-2.054, p= 0.001), heterozygote comparison AG vs GG (OR= 1.290, 95%CI= 0.934-1.782, p= 0.122), dominant model AA/GA vs GG (OR= 1.381, 95%CI= 1.048-1.821, p= 0.022) and recessive model AA vs GA/GG (OR= 1.323, 95%CI= 1.057- 1.657, p= 0.015) especially in glioma.. CCND1 G870A polymorphism may increase brain tumor risk, especially for gliomas. However, more primary large scale and well-designed studies are still required to evaluate the interaction of CCND1 G870A polymorphism with brain tumor risk.

Topics: Adenoma; Brain Neoplasms; Cyclin D1; Genetic Predisposition to Disease; Glioma; Humans; Meningioma; Neuroma, Acoustic; Odds Ratio; Pituitary Neoplasms; Polymorphism, Single Nucleotide

miR-135a-5p has been reported as a tumor suppressor in several extracranial tumors. However, its exact roles in gliomagenesis and relevance to the patients' prognoses are largely unknown. Herein, we detected the miR-135a-5p and tumor necrosis factor receptor-associated factor 5 (TRAF5) levels in 120 human glioma specimens and 20 nontumoral brain tissues; we found the miR-135a-5p level decreased, whereas the TRAF5 level increased, with the elevation of glioma grade. Their labeling indexes were inversely correlated with each other and showed strong negative (miR-135a-5p) and positive (TRAF5) correlation with the Ki-67 index. Cox regression demonstrated that both of their expression levels were independent survival predictors, whereas Kaplan-Meier analysis showed that subgrouping the glioma patients according to their levels could perfectly reflect the patients' prognoses regardless of the similarities in pathologic, molecular, and clinical features. In the following in vitro and in vivo studies, it was demonstrated that miR-135a-5p induced G

Topics: Animals; Brain Neoplasms; Cell Line, Tumor; Cyclin D1; Genes, Tumor Suppressor; Glioblastoma; Humans; Kaplan-Meier Estimate; Mice; Mice, Inbred BALB C; Mice, Nude; MicroRNAs; Neoplasm Staging; Neoplasm Transplantation; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-myc; RNA, Neoplasm; TNF Receptor-Associated Factor 5; Transplantation, Heterologous

Alterations of cyclin D1, one of the main regulators of the cell cycle, are known to be involved in various cancers. The CCDN1 G870A polymorphism causes production of a truncated variant with a shorter half-life and thus thought to impact the regulatory effect of CCDN1. The aim of the present study was to contribute to existing results to help to determine the prognostic value of this specific gene variant and evaluate the role of CCDN1 G870A polymorphism in brain cancer susceptibility. A Turkish study group including 99 patients with primary brain tumors and 155 healthy controls were examined. Genotypes were determined by polymerase chain reaction-restriction fragment length polymorphism analysis. The CCDN1 genotype frequencies in meningioma, glioma and control cases were not significantly different (p>0.05). No significant association was detected according to clinical parameters or tumor characteristics; however, a higher frequency of AG genotype was recorded within patients with astrocytic or oligoastrocytic tumors. A significant association between AG genotype and gliobilastoma multiforme (GBM) was recorded within the patients with glial tumors (p value=0.048 OR: 1.87 CI% 1.010-3.463). According to tumor characteristics, no statistically significant difference was detected within astrocytic, oligoasltrocytic tumors and oligodentrioglias. However, patients with astrocytic astrocytic or oligoastrocytic tumors showed a higher frequency of AG genotype (50%) when compared to those with oligodendrioglial tumors (27.3%). Our results indicate a possible relation between GBM formation and CCDN1 genotype.

Topics: Adult; Brain Neoplasms; Case-Control Studies; Cyclin D1; Female; Follow-Up Studies; Genetic Predisposition to Disease; Genotype; Glioma; Humans; Male; Meningeal Neoplasms; Meningioma; Middle Aged; Neoplasm Staging; Polymerase Chain Reaction; Polymorphism, Genetic; Polymorphism, Restriction Fragment Length; Prognosis; Risk Factors

Gliomas are the most common brain malignancies characterized by high degree of aggressiveness and high mortality. However, the underlying mechanism of glioma progression remains unclear. Here, we probed the role of CDC42EP3 (CDC42 effector protein 3) played in glioma development and its potential downstream mechanism. The expression of CDC42EP3 in tumor and normal brain tissues were examined through immunohistochemistry and we found the likelihood of CDC42EP3 overexpression was positively correlated with pathological grading. Patients with higher expression of CDC42EP3 were more likely to suffer from recurrence as well. Through constructing CDC42EP3-knockdown cell models, we discovered that silencing CDC42EP3 significantly restricted cell proliferation and migration but facilitated cell apoptosis in vitro. Inhibition on tumor growth mediated by CDC42EP3 depletion was further verified in vivo. Regarding downstream target of CDC42EP3, we found that it may positively regulate the expression of CCND1 through c-Myc-mediated transcription. Furthermore, our findings affirmed that effects of CDC42EP3 overexpression on cell proliferation, migration and apoptosis could be confined by depleting CCND1. In a word, this study reported the tumor-promoting role of CDC42EP3 in glioma progression which probably functioned through targeting CCND1.

Topics: Apoptosis; Brain Neoplasms; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Gene Expression Regulation, Neoplastic; Glioma; GTP-Binding Protein Regulators; Humans

This study aimed to investigate FAM84B expression in glioma tissues and explore the role of FAM84B in promoting the proliferation of glioma cells and the mechanism of regulating the cell cycle pathways.. The TCGA database was adopted to analyze FAM84B expression in glioma tissues. The FAM84B expression was detected by qRT-PCR in patients with glioma, especially that in glioma cells, U251, LN-229, U98, and U87. Two glioma cell lines U87 and T98 were selected for siRNA transfection, which were divided into si-NC si-FAM84B-1 and si-FAM84B-2 groups. The effect of FAM84B on the proliferation of glioma cells was detected with the MTT experiment and that on the glioma cell cycle was detected with the flow cytometry. The signaling pathways potentially regulated by FAM84B in glioma were analyzed through the bioinformatics analysis. The expression of proteins, Cyclin D1, CDK4, Cdk6, and p21, in the cell cycle-related pathways in cells of each group was detected by the Western blot.. TCGA database results showed a significantly higher FAM84B expression in glioma tissues than that in paracancerous tissues. According to the detection of qRT-PCR, FAM84B expressed the highest in the glioma cell line U87 (P < 0.05). Compared with the serum of healthy controls, FAM84B mRNA expression significantly increased in patients with gliomas. And compared with the si-NC group, the proliferation ability of U87 and T98 cells decreased and the cell cycle was blocked in the G0/G1 phase in both si-FAM84B transfection groups (P < 0.05). According to the bioinformatics analysis, FAM84B regulated the cell cycle pathways in glioma. FAM84B siRNA inhibited the expression of key proteins, Cyclin D1, CDK2, CDK4, and Cdk6, of the cell cycle pathways in glioma cells and promoted the expression of P53 and P21 proteins.. In conclusion, FAM84B may inhibit the proliferation of glioma cells by regulating the cell cycle pathways.

Topics: Brain Neoplasms; Cell Cycle; Cell Proliferation; Cyclin D1; Gene Expression Regulation, Neoplastic; Glioma; Humans; RNA, Small Interfering

This study aims to explore the impact of LINC00887 on the malignant progression of glioma via upregulating CCND1.. LINC00887 and CCND1 levels in glioma patients in different tumor grades or metastasis statuses were detected by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). Kaplan-Meier curves were depicted for analyzing the prognostic potential of LINC00887 in glioma patients. Meanwhile, Pearson correlation test was conducted to assess the expression correlation between LINC00887 and CCND1 in glioma tissues. After knockdown of LINC00887 in LN229 and U251 cells, proliferative abilities were examined by cell counting kit-8 (CCK-8) and 5-Ethynyl-2'- deoxyuridine (EdU) assays. Subcellular distribution of LINC00887 was determined. Thereafter, RNA Binding Protein Immunoprecipitation (RIP) was performed to uncover the interaction between LINC00887 and CCND1. After α-amanitin induction in glioma cells overexpressing LINC00887, RNA degradation of CCND1 was examined at 0, 6, 12 and 24 h, respectively. Finally, the synergistic regulation of both LINC00887 and CCND1 on glioma proliferation was explored by CCK-8 assay.. It was found that LINC00887 was upregulated in glioma tissues, especially in stage III+IV or metastatic glioma cases. Overall survival was remarkably worse in glioma patients expressing a high level of LINC00887 than those with a low level. CCND1 was upregulated in glioma tissues as well, showing a positive correlation to LINC00887. In addition, LINC00887 was mainly distributed in the cytoplasm and interacted with CCND1, and it shortened the half-life of CCND1. Moreover, the knockdown of LINC00887 inhibited glioma cell proliferation, and this inhibitory effect was abolished by overexpression of CCND1.. LINC00887 is upregulated in glioma tissues, and it aggravates the malignant progression of glioma by upregulating CCND1.

Topics: Brain Neoplasms; Cell Proliferation; Cells, Cultured; Cyclin D1; Glioma; Humans; RNA, Long Noncoding; Up-Regulation

Glioma, the most common primary brain tumor, account Preparing figures for 30 to 40% of all intracranial tumors. Herein, we aimed to study the effects of M2 macrophage-derived exosomal microRNAs (miRNAs) on glioma cells.. First, we identified seven differentially expressed miRNAs in infiltrating macrophages and detected the expression of these seven miRNAs in M2 macrophages. We then selected hsa-miR-15a-5p (miR-15a) and hsa-miR-92a-3p (miR-92a) for follow-up studies, and confirmed that miR-15a and miR-92a were under-expressed in M2 macrophage exosomes. Subsequently, we demonstrated that M2 macrophage-derived exosomes promoted migration and invasion of glioma cells, while exosomal miR-15a and miR-92a had the opposite effects on glioma cells. Next, we performed the target gene prediction in four databases and conducted target gene validation by qRT-PCR, western blot and dual luciferase reporter gene assays.. The results revealed that miR-15a and miR-92a were bound to CCND1 and RAP1B, respectively. Western blot assays demonstrated that interference with the expression of CCND1 or RAP1B reduced the phosphorylation level of AKT and mTOR, indicating that both CCND1 and RAP1B can activate the PI3K/AKT/mTOR signaling pathway.. Collectively, these findings indicate that M2 macrophage-derived exosomal miR-15a and miR-92a inhibit cell migration and invasion of glioma cells through PI3K/AKT/mTOR signaling pathway.

Topics: Brain Neoplasms; Cell Line, Tumor; Cell Movement; Computational Biology; Cyclin D1; Exosomes; Glioma; Humans; Macrophages; MicroRNAs; Microscopy, Electron, Transmission; Nanoparticles; Neoplasm Invasiveness; Phosphorylation; Proto-Oncogene Proteins c-akt; rap GTP-Binding Proteins; Real-Time Polymerase Chain Reaction; Signal Transduction; THP-1 Cells; TOR Serine-Threonine Kinases

5-Aminolevulinic acid (5-ALA) is a naturally occurring non-proteinogenic amino acid, which contributes to the diagnosis and therapeutic approaches of various cancers, including glioblastoma (GBM). In the present study, we aimed to investigate whether 5-ALA exerted cytotoxic effects on GBM cells. We assessed cell viability, apoptosis rate, mRNA expressions of various apoptosis-related genes, generation of reactive oxygen species (ROS), and migration ability of the human U-87 malignant GBM cell line (U87MG) treated with 5-ALA at different doses. The half-maximal inhibitory concentration of 5-ALA on U87MG cells was 500 μg/mL after 7 days; 5-ALA was not toxic for human optic cells and NIH-3T3 cells at this concentration. The application of 5-ALA led to a significant increase in apoptotic cells, enhancement of Bax and p53 expressions, reduction in Bcl-2 expression, and an increase in ROS generation. Furthermore, the application of 5-ALA increased the accumulation of U87MG cells in the SUB-G1 population, decreased the expression of cyclin D1, and reduced the migration ability of U87MG cells. Our data indicate the potential cytotoxic effects of 5-ALA on U87MG cells. Further studies are required to determine the spectrum of the antitumor activity of 5-ALA on GBM.

Topics: Aminolevulinic Acid; Animals; Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; Brain Neoplasms; Cell Line, Tumor; Cell Movement; Cell Survival; Cyclin D1; G1 Phase Cell Cycle Checkpoints; Gene Expression Regulation, Neoplastic; Glioblastoma; Humans; Mice; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Tumor Suppressor Protein p53

Curcumin is a natural polyphenol extracted from the rhizome of Curcuma that has an important antitumour effect, but its effect on adverse psychological stress-induced tumour proliferation and invasion has not been reported to date. Here, we found that curcumin not only inhibited the growth of xenografts in chronically stressed nude mice, but also decreased the expression of matrix metalloproteinase (MMP)-2/9 and CD147 in tumour tissues. Exogenous norepinephrine (NE) was used to stimulate glioma cells to simulate the stress environment in vitro, and it was found that curcumin inhibited the NE-induced proliferation and invasion of glioma cells in a dose-dependent manner. Further research found that the effects of NE on glioma cells could lead to the activation of the mitogen-activated protein kinase (MAPK) signalling pathway through β-adrenergic receptor, while curcumin suppressed the level of extracellular signal-regulated kinase (ERK)1/2 phosphorylation. In addition, blocking ERK1/2 expression with U0126 resulted in the down-regulated expression of CD147, which further led to the decreased expression of MMP-2 and MMP-9. Curcumin could also inhibit the expression of cyclin D1/CDK4/6 and anti-apoptotic protein Bcl-2/Bcl-XL induced by NE, and induced cell cycle changes and increased apoptosis. Therefore, curcumin may be a potential candidate drug for preventing and treating the progression of glioma induced by adverse psychological stress.

Topics: Animals; Apoptosis; Brain Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; Curcumin; Cyclin D1; Down-Regulation; Female; Glioma; Humans; MAP Kinase Signaling System; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Mice; Mice, Inbred BALB C; Mice, Nude; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Norepinephrine; Stress, Psychological

A maximal surgical resection followed by radiotherapy and chemotherapy with temozolomide (TMZ) as the representative agent is the standard therapy for gliomas. However, tumor cell resistance to radiotherapy and chemotherapy leads to poor prognosis and high mortality in patients with glioma. In the present study, we demonstrated that JARID2 was downregulated and CCND1 was upregulated within glioma tissues of different grades and glioma cells. In tissue samples, JARID2 was negatively correlated with CCND1. JARID2 overexpression significantly inhibited glioma cell viability, promoted glioma cell apoptosis upon TMZ treatment, and increased p21, cleaved-PARP, and cleaved-caspase3 in TMZ-treated glioma cells. JASPAR tool predicted the possible binding sites between JARID2 and CCND1 promoter regions; through direct binding to CCND1 promoter region, JARID2 negatively regulated CCND1 expression. Under TMZ treatment, JARID2 overexpression inhibited CCND1 expression, promoted glioma cell apoptosis, and increased p21, cleaved-PARP, and cleaved-caspase3 in glioma cells treated with TMZ; meanwhile, CCND1 overexpression exerted opposite effects on glioma cells treated with TMZ and partially reversed the effects of JARID2 overexpression. In conclusion, JARID2 targets and inhibits CCND1. The JARID2/CCND1 axis modulates glioma cell growth and glioma cell sensitivity to TMZ.

Topics: Brain Neoplasms; Cell Line, Tumor; Cyclin D1; Drug Resistance, Neoplasm; Glioma; Humans; MicroRNAs; Polycomb Repressive Complex 2; Temozolomide

Glioblastoma and Alzheimer's disease (AD) are the most common and devastating diseases in the central nervous system. The dysfunction of Presenilin1 is the main reason for AD pathogenesis. However, the molecular function of Presenilin1 and its relative mechanism in glioblastoma remain unclear.. Expression of presenilin1 in glioma was determined by IHC. CCK-8, colony formation, Flow cytometry, Edu staining were utilized to evaluate functions of presenilin1 on glioblastoma proliferation. The mechanism of above process was assessed by Western blotting and cell immunofluorescence. Mouse transplanting glioblastoma model and micro-MRI detection were used to verified presenilin1 function in vivo.. In this study, we found that all grades of glioma maintained relatively low Presenilin1 expression and that the expression of Presenilin1 in high-grade glioma was significantly lower than that in low-grade glioma. Moreover, the Presenilin1 level had a positive correlation with glioma and glioblastoma patient prognosis. Next, we determined that Presenilin1 inhibited the growth and proliferation of glioblastoma cells by downregulating CDK6, C-myc and Cyclin D1 to arrest the cell cycle at the G1/S phase. Mechanistically, Presenilin1 promoted the direct phosphorylation of β-catenin at the 45 site and indirect phosphorylation at the 33/37/41 site, then decreased the stabilized part of β-catenin and hindered its translocation from the cytoplasm to the nucleus. Furthermore, we found that Presenilin1 downregulation clearly accelerated the growth of subcutaneous glioblastoma, and Presenilin1 overexpression significantly repressed the subcutaneous and intracranial transplantation of glioblastoma by hindering β-catenin-dependent cell proliferation.. Our data implicate the antiproliferative effect of Presenilin1 in glioblastoma by suppressing Wnt/β-catenin signaling, which may provide a novel therapeutic agent for glioblastoma. Video Abstract.

Topics: Animals; beta Catenin; Brain Neoplasms; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Down-Regulation; G1 Phase; Glioblastoma; Humans; Mice, Nude; Phosphorylation; Presenilin-1; Prognosis; Proteolysis; Proto-Oncogene Proteins c-myc; S Phase; Wnt Signaling Pathway; Xenograft Model Antitumor Assays

MicroRNAs (miRNA/miRs) serve an important function in the regulation of gene expression, and have been indicated to mediate a number of cellular biological processes, including cell proliferation, the cell cycle, cell apoptosis and cell differentiation. The altered expression of miRNAs has been revealed to result in a variety of human diseases, including glioblastoma multiforme (GBM). The present study indicated an increase in miR‑296‑3p in glioma tumor types compared with normal brain, particularly in the samples from patients with high grade GBM. Antagonizing miR‑296‑3p was demonstrated to induce cell growth arrest and cell cycle redistribution in U251 cells. The miR‑296‑3p antagonist altered the expression of a number of key genes that are involved in cell cycle control, including cyclin D1 and p21. Additionally, the decrease of miR‑296‑3p increased inhibitor of β‑catenin and T cell factor (ICAT) expression, and increased miR‑296‑3p‑inhibited ICAT expression in U251 cells. Bioinformatics analysis indicated that ICAT is a target gene of miR‑296‑3p, which was further validated using a dual‑luciferase reporter assay. Through the regulation of ICAT, the miR‑296‑3p antagonist decreased β‑catenin protein expression and increased the expression of its target genes. Silencing ICAT was indicated to reverse the miR‑296‑3p downregulation‑induced inactivation of Wnt signaling and cell growth arrest in glioma cells. The present study also indicated a negative correlation between ICAT mRNA levels and miR‑296‑3p levels in glioma tumor types. In conclusion, the present study identified an oncogenic function of miR‑296‑3p in glioblastoma via the direct regulation of ICAT.

Topics: 3' Untranslated Regions; Adaptor Proteins, Signal Transducing; Apoptosis; Brain Neoplasms; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Down-Regulation; G1 Phase Cell Cycle Checkpoints; Gene Expression Regulation, Neoplastic; Gene Silencing; Glioblastoma; Humans; MicroRNAs; Neoplasm Grading; Up-Regulation; Wnt Signaling Pathway

The present study was designed to determine the association between the genetic polymorphisms/expression variations of RB1 and CCND1 genes and brain tumor risk. For this purpose, 250 blood samples of brain tumor patients along with 250 controls (cohort I) and 96 brain tumor tissues (cohort II) with adjacent control section were collected. Mutation analysis of RB1 (rs137853294, rs121913300) and CCND1 (rs614367, rs498136) genes was performed using ARMS-PCR followed by sequencing, and expression analysis was performed using real-time PCR and immunohistochemistry. The results showed homozygous mutant genotype of RB1 gene polymorphism, rs121913300 (P=0.003) and CCND1 gene polymorphism rs614367 (P=0.01) were associated significantly with brain tumor risk. Moreover, significant down-regulation of RB1 (P=0.005) and up-regulation of CCND1 (P=0.0001) gene was observed in brain tumor sections vs controls. Spearman correlation showed significant negative correlation between RB1 vs proliferation marker, Ki-67 (r = -0.291*, P<0.05) in brain tumors. Expression levels of selected genes were also assessed at protein level using immunohistochemical analysis (IHC) and signification down-regulation of RB1 (P=0.0001) and up-regulation of CCND1 (P=0.0001) was observed in brain tumor compared with control sections. In conclusion, it is suggested that polymorphisms/expression variations of RB1 and CCND1 genes may be associated with increased risk of brain tumor.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Brain; Brain Neoplasms; Cell Cycle; Child; Cyclin D1; Down-Regulation; Female; Gene Expression Regulation, Neoplastic; Gene Frequency; Genetic Predisposition to Disease; Humans; Male; Middle Aged; Polymorphism, Single Nucleotide; Retinoblastoma Binding Proteins; Retrospective Studies; Ubiquitin-Protein Ligases; Up-Regulation; Young Adult

The KDEL receptor is a seven-transmembrane-domain protein, which plays a key role in ER quality control and in the ER stress response, KDELR2 involved in regulation of cellular functions, including cell proliferation, survival, promotes glioblastoma tumorigenesis. The aim of this study was to investigate the clinicpathological value and biological role of KDELR2 in glioma.. We studied the expression of KEDLR2 and its association with the prognosis through the TCGA, CGGA, and GSE16011 database. To explore the role of KDELR2 in glioma, KDELR2 siRNA was constructed and transfected into U87 glioma cells. CCK-8, colony formation and Transwell assays were used to investigate the roles of KDELR2 on GBM cell proliferation. We further studied the effect of KDELR2 on tumorigenesis in animal model. Additionally, flow cytometry was used to monitor the changes in the cell cycle and apoptosis following transfection with KDELR2 siRNA. We applied GeneChip primeview expression array to analysis the differential gene expression profiling. Ingenuity Pathway Analysis to show that KDELR2 has a significant impact in canonical pathway in cell cycle regulation and participate in multiple pathways. And we detected the cell cycle proteins CCND1 expression by Western blot analysis.. Our results showed that KDELR2 was up-regulated in glioma tissue and cell lines. Knockdown KDELR2 was able to reduce cell viability, promote cell cycle arrest at the G1 phase, and induce apoptotic cell death. Moreover, our results suggested that KDELR2 regulated the cellular functions of U87 cells by targeting CCND1. Therefore, we demonstrated that KDELR2 is a novel biomarker in glioma.. KDELR2 is highly expressed in human glioma tissues and cell lines, a higher expression of KDELR2 is associated with a poor prognosis of glioma patients. Moreover, KDELR2 regulated the cellular functions of U87 cells by targeting CCND1. The KDELR2/CCND1 axis may provide a new therapeutic target for the treatment of glioma and deepen our understanding of glioma mechanisms.

Topics: Animals; Biomarkers, Tumor; Brain Neoplasms; Carcinogenesis; Cyclin D1; Disease Progression; Female; Gene Expression Regulation, Neoplastic; Glioma; Heterografts; Humans; Male; Mice; Mice, Nude; Prognosis; Vesicular Transport Proteins

Background: Glioma, most common primary malignant brain tumor in adults, is highly aggressive and associated\ with a poor prognosis. Evaluate the association of polymorphisms related of to the cell cycle, integrity and DNA repair\ with gliomas, as well as lifestyle habits, comorbidities, survival and response to treatment. Methods: Were studied\ 303 individuals distributed into: Study Group - 100 patients with gliomas, regardless of the degree of malignancy, and\ Control Group - 203 individuals without clinical signs of the disease. These polymorphisms were genotyped by TaqMan®\ SNP Genotyping Assay. Significance level was set at 5%. Results: Smoking, alcohol consumption, systemic arterial\ hypertension (SAH) and diabetes mellitus (DM) prevailed in patients, compared to controls (P=0.0088, P=0.0001,\ P=0.0001, P=0.0011, respectively). In the logistic regression analysis, alcohol consumption and SAH were identified\ as independent risk factors for gliomas (P=0.0001, P=0.0027, respectively). Patients with low-grade gliomas showed\ survival in one year (92.0±6.8%), compared to patients with high-grade gliomas (24.0±5.3; P=0.011). Conclusion:\ Polymorphisms involved in cell cycle, telomere protection and stability and DNA repair are not associated with gliomas.\ On the other hand, alcohol consumption and SAH stand out as independent risk factors for the disease. Low-grade\ gliomas, response to treatment and the combination of chemotherapy with Temozolomide and radiation therapy show\ increased survival of patients.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Brain Neoplasms; Case-Control Studies; Child; Child, Preschool; Cyclin D1; DNA Helicases; Female; Follow-Up Studies; Gene Expression Regulation, Neoplastic; Genotype; Glioma; Humans; Infant; Male; Middle Aged; Neoplasm Grading; Polymorphism, Genetic; Survival Rate; Telomere; X-ray Repair Cross Complementing Protein 1; Young Adult

Ganoderic acid A (GA-A), recognized as a lanostanetriterpene isolated from Ganoderma lucidum, demonstrates an efficient antitumor activity in multiple cancers. To date, it is unclear whether and how GA-A functions on human glioblastoma (GBM). To unravel the functional significance of GA-A on human glioblastoma (GBM), the cell-counting kit-8 and transwell assays were used to detect proliferation, migration, and invasion of human GBM cell after GA-A treatment. Then, we utilized the flow cytometry and western blot to further evaluate the effect of GA-A on GBM cell. Further, activities of autophagy and PI3K/AKT signaling were assessed by Western blot assay. We found that GA-A significantly inhibited proliferation, migration, and invasion of GBM cell. Additionally, GA-A markedly triggered cell apoptosis, which incarnated an elevation trend in apoptotic percentage, simultaneously, an increased level of proapoptosis protein (Bax and active caspase-3) and a decreased level of antiapoptosis protein (Bcl-2), induced by GA-A treatment. Meanwhile, levels of two well-known autophagy markers (beclin 1 and LC3 II) increased while another autophagic substrate (P-62) was reduced. Moreover, the expressions levels of phosphorylated AKT, mTOR, p-P70S6K, and cyclin D1 in the PI3K/AKT pathway were significantly reduced, which revealed GA-A repressed the activation of PI3K/AKT signaling pathway. Collectively, these results indicate that GA-A may encourage U251 cell growth and invasion/migration inhibition, apoptosis, and autophagy through the inactivation of PI3K/AKT signaling pathway in human GBM. Hence, GA-A may be a potent antitumorigenic agent for human GBM in future clinical practice.

Topics: Apoptosis; Autophagy; Brain Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Cytotoxins; Glioblastoma; Heptanoic Acids; Humans; Lanosterol; Neoplasm Invasiveness; Phosphatidylinositol 3-Kinases; Phosphorylation; Proto-Oncogene Proteins c-akt; Reishi; Ribosomal Protein S6 Kinases, 70-kDa; Signal Transduction; TOR Serine-Threonine Kinases

Topics: Adult; Animals; Apoptosis; beta Catenin; Brain Neoplasms; Cell Cycle; Cell Proliferation; Cyclin D1; Disease Progression; Female; Gene Expression Regulation, Neoplastic; Glioma; Glycogen Synthase Kinase 3 beta; Humans; Male; Mice; MicroRNAs; Middle Aged; Neuroglia; Proto-Oncogene Proteins c-myc; RNA, Long Noncoding; RNA, Small Interfering; Wnt Proteins; Wnt Signaling Pathway; Xenograft Model Antitumor Assays

The PAX3 (paired box 3) gene plays an important role in embryonic development, diseases, and cancer formation. Our preliminary studies have shown that PAX3 gene is upregulated in glioma cells, which is associated with a worse prognosis. Moreover, PAX3, by facilitating cell proliferation and invasion and inhibiting cell apoptosis, plays an oncogenic role in glioma. However, the specific molecular mechanism of PAX3 acting as an oncogene in glioma remains unclarified. In the present study, we have found that PAX3 overexpression was observed in high grade glioma and predicted a worse prognosis. PAX3 overexpression did not correlate significantly to IDH1 mutation and MGMT methylation. Moreover, the expression of PAX3 was positively correlated with that of β-catenin. In U87 glioma cells, PAX3 interacted with β-catenin, as was confirmed by CO-IP. Besides, PAX3 overexpression promoted cell proliferation and cell cycle progression, while it inhibited cell apoptosis by altering the expressions of important molecules associated with the Wnt signaling pathway, including β-catenin, Myc, VEGF, cyclinD1, MMP7, and Wnt1. In the meantime, it was also proved that PAX3 correlated to β-catenin through a negative regulatory mechanism with respect to the promotion of U87 glioma cell proliferation and cell cycle progression and inhibition of the cell apoptosis. Our experiment demonstrated the role of PAX3 in promoting glioma growth and development, possibly by interacting directly with β-catenin and regulating the Wnt signaling pathway.

Topics: Adult; Aged; Apoptosis; beta Catenin; Brain Neoplasms; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Female; Glioma; Humans; Male; Matrix Metalloproteinase 7; Middle Aged; PAX3 Transcription Factor; Protein Binding; Vascular Endothelial Growth Factor A; Wnt Signaling Pathway; Wnt1 Protein

Glioblastoma (GBM) is a highly invasive brain neoplasia with an elevated recurrence rate after surgical resection. The cyclin D1 (Ccnd1)/Cdk4-retinoblastoma 1 (RB1) axis is frequently altered in GBM, leading to overproliferation by RB1 deletion or by Ccnd1-Cdk4 overactivation. High levels of Ccnd1-Cdk4 also promote GBM cell invasion by mechanisms that are not so well understood. The purpose of this work is to elucidate the in vivo role of cytoplasmic Ccnd1-Cdk4 activity in the dissemination of GBM. We show that Ccnd1 activates the invasion of primary human GBM cells through cytoplasmic RB1-independent mechanisms. By using GBM mouse models, we observed that evaded GBM cells showed cytoplasmic Ccnd1 colocalizing with regulators of cell invasion such as RalA and paxillin. Our genetic data strongly suggest that, in GBM cells, the Ccnd1-Cdk4 complex is acting upstream of those regulators. Accordingly, expression of Ccnd1 induces focal adhesion kinase, RalA and Rac1 activities. Finally, in vivo experiments demonstrated increased GBM dissemination after expression of membrane-targeted Ccnd1. We conclude that Ccnd1-Cdk4 activity promotes GBM dissemination through cytoplasmic and RB1-independent mechanisms. Therefore, inhibition of Ccnd1-Cdk4 activity may be useful to hinder the dissemination of recurrent GBM. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Topics: Animals; Biomarkers, Tumor; Brain Neoplasms; Cell Line, Tumor; Cell Movement; Cyclin D1; Cytoplasm; Gene Expression Regulation, Neoplastic; Glioblastoma; Humans; Male; Mice; Mice, SCID; Neoplasm Invasiveness

Annexin A2 (ANXA2) acts as a calcium‑dependent phospholipid‑binding protein that is widely expressed in vertebrate cells and has abnormally high expression in various tumor cells. However, the detailed molecular mechanism underlying the effects of ANXA2 on glioma cells remains unclear. The present study aimed to investigate the role and underlying molecular mechanisms of ANXA2 in glioma cell proliferation. The results revealed that knockdown of ANXA2 inhibited the proliferation of U251 and U87 glioma cell lines and decreased phosphorylated (p) signal transducer and activator of transcription 3 (STAT3)(Y705) and cyclin D1 expression, leading to impedance of the G1‑to‑S phase transition. Furthermore, it was suggested that ANXA2 may regulate pSTAT3(Y705) levels through direct binding with STAT3, thereby affecting STAT3‑cyclin D1 pathway‑mediated cell proliferation. When ANXA2 was re‑expressed in ANXA2‑knockdown cells, the expression of pSTAT3(Y705) and cyclin D1 was restored. Furthermore, overexpression of ANXA2 significantly promoted the proliferation of U251 cells, as determined by an MTT assay and a tumor formation assay in nude mice, but had no statistically significant effect on colony formation rate, cell cycle progression or the STAT3‑cyclin D1 pathway, suggesting that endogenous ANXA2 may be redundant. Additionally, the present study provided evidence that the overexpression of ANXA2 enhanced the expression of pSTAT3(Y705) in the presence of epidermal growth factor (EGF), indicating that the proliferation‑promoting effect of ANXA2 may be due to the accumulation and synergistic effect of paracrine EGF. Taken together, the present results indicated that ANXA2 may affect the proliferation of human glioma cells through the STAT3‑cyclin D1 pathway via direct interaction with STAT3 in U251 and U87 glioma cells. ANXA2 was redundant in this pathway, but positive synergy was revealed to exist between ANXA2 and EGF.

Topics: Animals; Annexin A2; Brain Neoplasms; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Epidermal Growth Factor; G1 Phase Cell Cycle Checkpoints; Gene Knockdown Techniques; Glioma; Humans; Mice; Mice, Nude; Neoplasm Transplantation; Phosphorylation; Signal Transduction; STAT3 Transcription Factor

Cell division cycle associated 7 like (CDCA7L) belongs to the JPO protein family, recently identified as a target gene of c‑Myc and is frequently dysregulated in multiple cancers. However, to the best of our knowledge, no studies to date have been carried out to investigate the functions of CDCA7L in glioma. Thus, in this study, the expression level of CDCA7L and its association with the prognosis in glioma were detected through the TCGA database. The mRNA expression levels of CDCA7L in glioblastoma (GBM) tissues and normal brain tissues were detected by RT‑qPCR and western blot analysis. To explore the role of CDCA7L in glioma, CDCA7L siRNA was constructed and transfected into U87 glioma cells. The expression levels of CDCA7L and cyclin D1 (CCND1) in glioma U87 cells following transfection with CDCA7L siRNA were measured by RT‑qPCR and western blot analysis. CCK‑8, colony formation, EdU and Transwell assays were used to measure the effects of CDCA7L on U87 cell proliferation, and flow cytometry was used to monitor the changes in the cell cycle following transfection with CDCA7L siRNA. Xenograft tumors were examined in vivo for the carcinogenic effects, as well as the mechanisms and prognostic value of CDCA7L in glioma tissues. The results revealed that CDCA7L was highly expressed in human GBM tissues, and a high expression of CDCA7L was associated with a poor prognosis of glioma patients through the TCGA database. We demonstrated that CDCA7L was highly expressed in human GBM tissues and 3 glioma cell lines. The downregulation CDCA7L expression significantly inhibited the proliferation and colony formation ability of U87 cells by blocking cell cycle progression in the G0/G1 phase. In addition, we found that the mRNA and protein levels of CCND1 were markedly decreased following transfection with CDCA7L siRNA compared with NC siRNA in vitro. The downregulation CDCA7L expression reduced the number of invading cells. Consistent with the results of the in vitro assays, the xenograft assay, immunohistochemistry (IHC) assay and western blot analysis demonstrated that, in response to CDCA7L inhibition, tumor growth was inhibited, Ki‑67 and CCND1 expression levels were decreased in vivo. On the whole, the results of the current study indicate that CDCA7L is highly expressed in human glioma tissues and that a high CDCA7L expression predicts a poor prognosis of glioma patients. CDCA7L promotes glioma U87 cell growth through CCND1.

Topics: Animals; Brain Neoplasms; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Gene Expression Regulation, Neoplastic; Glioblastoma; Humans; Male; Mice, Inbred BALB C; Mice, Nude; Prognosis; Repressor Proteins; Xenograft Model Antitumor Assays

The GATAD1 gene overexpression induced by GATAD1 amplification upregulation is detected in different human tumors. To date, the relationship between GATAD1 amplification and glioma oncogenesis and malignancy is still unknown.. GATAD1 gene amplification and expression were analyzed in 187 gliomas using qPCR and immunostaining. The relation of GATAD1 to patients' prognoses was assessed via the Kaplan-Meier method. The MTT and orthotopic tumor transplantation assays were used to identify the function of GATAD1 in glioma proliferation. cDNA microarray, ChIP qPCR, EMSA and 3C were used to screen the downstream mechanism of GATAD1 regulating glioma proliferation.. Our results indicated that GATAD1 gene amplification and GATAD1 gene expression are novel independent diagnosis biomarkers to indicate poor outcome of glioma patients. GATAD1 knockdown can remarkably suppress GBM cell proliferation both in vitro and in vivo. GATAD1 could promote CCND1 gene transcription by inducing long range chromatin architectural interaction on the CCND1 promoter. Then GATAD1 sequentially accelerates GBM cell cycle transition and proliferation via regulating CCND1.. We identify GATAD1 as a novel potential diagnosis biomarker and promising prognosis predictor in glioma patients. Functionally, we confirm GATAD1 as an epigenetic chromatin topological regulator that promotes glioma proliferation by targeting CCND1.

Topics: Biomarkers, Tumor; Brain Neoplasms; Cell Cycle; Cell Line, Tumor; Cyclin D1; DNA Copy Number Variations; Eye Proteins; Gene Amplification; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Glioblastoma; Histones; Humans; Kaplan-Meier Estimate; Mutation; Prognosis; Promoter Regions, Genetic

Patients with brain metastases (BMs) have a poor prognosis and limited therapeutic options. Lung cancer is the most common primary malignancy giving rise to BMs; thus, understanding the molecular mechanisms behind increased BM risk is essential for identifying therapeutic targets and developing effective interventions.. Sixty-one patients who underwent surgical resection of primary non-small cell lung cancer (NSCLC) and BMs were retrospectively studied. Comprehensive genomic profiling of primary NSCLC and matched BMs was performed with next-generation sequencing targeting 416 cancer-relevant genes.. Mutations of major drivers, including EGFR, KRAS, TP53, and ALK, were highly concordant between primary NSCLC and matched BMs (>80%), whereas discordance suggested the unique genomic evolution and oncogenic mechanisms of NSCLC BMs. BMs also demonstrated higher levels of copy number variations in comparison with primary NSCLC. Furthermore, the alterations of genes encoding CDK4/CCND1, CDKN2A/2B, and PI3K signaling pathways were enriched in BMs, and this suggested their correlation with increased metastatic risk. Indeed, patients with activated PI3K signaling in their primary NSCLC had significantly shorter BM-free survival (hazard ratio, 8.49; P = .0005). In addition, mutated TP53 or an activated WNT pathway via CTNNB1, APC, and AXIN2 mutations trended toward shorter BM-free intervals but not significantly so.. These findings yield detailed insights into the genomic complexity and heterogeneity of primary NSCLC and matched BMs. This study highlights the significant correlation of PI3K signaling with increased metastatic risk in patients with NSCLC and identifies genomic alterations enriched in NSCLC BMs that could serve as prognostic markers and potential therapeutic targets for treating patients with NSCLC BMs.

Topics: Adult; Aged; Brain Neoplasms; Carcinoma, Non-Small-Cell Lung; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase Inhibitor p15; Cyclin-Dependent Kinase Inhibitor p16; DNA Copy Number Variations; Female; Gene Expression Regulation, Neoplastic; Genomic Instability; Genomics; High-Throughput Nucleotide Sequencing; Humans; Male; Middle Aged; Mutation; Neoplasm Metastasis; Neoplasm Proteins; Retrospective Studies; Transcriptome

Ependymal tumors are pathologically defined intrinsic neoplasms originating in the intracranial compartments or the spinal cord that affect both children and adults. The recently integrated classification of ependymomas based on both histological and molecular characteristics is capable of subgrouping patients with various prognoses. However, the application of histological and molecular markers in Chinese patients with ependymomas has rarely been reported. We aimed to demonstrate the significance of histological characteristics, the v-relavian reticuloendotheliosis viral oncogene homolog A (RELA) fusions and other molecular features in ependymal tumors.. We reviewed the histological characteristics of ependymal tumors using conventional pathological slides and investigate the RELA fusions and Cylclin D1 (CCND1) amplification by Fluorescence in situ hybridization (FISH) and trimethylation of histone 3 lysine 27 (H3K27me3) expression by immunohistochemistry (IHC) methods. SPSS software was used to analyze the data.. We demonstrated that hypercellularity, atypia, microvascular proliferation, necrosis, mitosis, and an elevated Ki-67 index, were tightly associated with an advanced tumor grade. Tumor location, necrosis, mitosis and the Ki-67 index were related to the survival of the ependymomas, but Ki67 was the only independent prognostic factor. Additionally, RELA fusions, mostly presented in pediatric grade III intracranial ependymomas, indicated decreased survival times of patients, and closely related to the patients' age, tumor grade, cellularity, cellular atypia, necrosis and Ki67 index in the intracranial ependymal tumors, whereas reduction of H3K27me3 predicted the worse prognosis in ependymal tumors.. Histological and molecular features facilitate tumor grading and prognostic predictions for ependymal tumors in Chinese patients.

Topics: Adolescent; Adult; Biomarkers, Tumor; Brain Neoplasms; Child; China; Cyclin D1; Ependymoma; Female; Follow-Up Studies; Histones; Humans; Immunohistochemistry; In Situ Hybridization, Fluorescence; Kaplan-Meier Estimate; Ki-67 Antigen; Male; Necrosis; Neoplasm Grading; Prognosis; Spinal Cord Neoplasms; Transcription Factor RelA; Young Adult

Glioblastoma with primitive neuroectodermal tumor-like component (GBM-PNET) is a rare variant of glioblastoma, which was renamed as glioblastoma with a primitive neuronal component (GBM-PN) in new WHO classification of tumours of the central nervous system in 2016. There are few publications on the investigation of GBM-PN. In this study, PCR mRNA arrays on 6 cases of conventional GBM and 10 cases of GBM-PN showed high mRNA level of CDK4 in GBM-PN and low mRNA level of EGFR in GBM-PN. Immunohistochemical stains on tissue microarrays with 28 cases of conventional GBM and 13 cases of GBM-PN demonstrated that CDK4 was selectively expressed in the primitive neuronal component of all GBM-PN cases while EGFR was positive in conventional GBM and glial component of GBM-PN, but was negative in the primitive neuronal component of all GBM-PN cases. Immunohistochemical stains with antibodies against proteins that interact with CDK4 in cell cycle regulation, such as CDK6, cyclin D1 and p16(INK4a), were performed on these GBM-PN and GBM cases. CDK6 was patchily positive in rare cases of GBM-PN and cyclin D1 was negative in GBM-PN cases. p16(INK4a) is traditionally known as an inhibitor of CDK4 and CDK6. p16(INK4a) might not be the inhibitor of CDK4 in GBM-PN cases because seven GBM-PN cases were positive for both CDK4 and p16(INK4a). It indicates that CDK4 and p16(INK4a) might play a crucial role in GBM-PN pathogenesis. Since CDK4 and EGFR are highly expressed in the primitive neuronal component and in the glial component of GBM-PN respectively, the combination of CDK4/6 inhibitor and targeted therapy against EGFR might be potential effective therapeutic regimen for GBM-PN. CDK4 and EGFR immuohistochemical stain patterns make the diagnosis of GBM-PN much easier.

Topics: Adult; Aged; Aged, 80 and over; Brain; Brain Neoplasms; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase 6; Cyclin-Dependent Kinase Inhibitor p16; ErbB Receptors; Female; Gene Expression Regulation, Neoplastic; Glioblastoma; Humans; Male; Middle Aged; Neuroectodermal Tumors, Primitive; RNA, Messenger

CXCR4 expression in neuroblastoma tumors correlates with disease severity. In this study, we describe mechanisms by which CXCR4 signaling controls neuroblastoma tumor growth and response to therapy. We found that overexpression of CXCR4 or stimulation with CXCL12 supports neuroblastoma tumorigenesis. Moreover, CXCR4 inhibition with the high-affinity CXCR4 antagonist BL-8040 prevented tumor growth and reduced survival of tumor cells. These effects were mediated by the upregulation of miR-15a/16-1, which resulted in downregulation of their target genes BCL-2 and cyclin D1, as well as inhibition of ERK. Overexpression of miR-15a/16-1 in cells increased cell death, whereas antagomirs to miR-15a/16-1 abolished the proapoptotic effects of BL-8040. CXCR4 overexpression also increased miR-15a/16-1, shifting their oncogenic dependency from the BCL-2 to the ERK signaling pathway. Overall, our results demonstrate the therapeutic potential of CXCR4 inhibition in neuroblastoma treatment and provide a rationale to test combination therapies employing CXCR4 and BCL-2 inhibitors to increase the efficacy of these agents.

Topics: Animals; Brain Neoplasms; Cell Line, Tumor; Cyclin D1; Drug Resistance, Neoplasm; Gene Expression Regulation, Neoplastic; Humans; MAP Kinase Signaling System; Mice; MicroRNAs; Neuroblastoma; Peptides; Proto-Oncogene Proteins c-bcl-2; Receptors, CXCR4; Xenograft Model Antitumor Assays

The activating mutations of micro RNA (miR)-17 have been revealed in tumors such as human non-Hodgkin's lymphoma and T cell leukemia. However, it is unclear about the role of miR-17 in glioma cells. The current study aimed to investigate effects of miR-17 mimics or inhibitor on the viability and migration of rat glioma C6 cells, and explore possible mechanisms.. The expression of miR-17 in rat glioma C6 cells and normal brain tissue was detected by quantitative PCR. Protein expression of Cyclin D1 in rat glioma C6 cells and normal brain tissue was measured by Western Blot. Glioma C6 cells were transfected with MiR-17 mimics or inhibitor. Cells that were not transfected (Lipofectamine only) and cells that were transfected with nonsense RNA negative control served as control. MTT assay was utilized to detect cell viability, and cell wound scratch assay was utilized to examine the migration index. In addition, protein expression of Cyclin D1, p-Akt and Akt in MiR-17 mimics or inhibitor-transfected glioma C6 cells was detected by Western Blot. This study had been approved by the Medical Ethics Committee of the First Affiliated Hospital of Soochow University. All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.. The expression of miR-17 was significantly lower, whereas the expression of Cyclin D1 was significantly higher in glioma C6 cells compared to normal brain tissue. MiR-17 mimics decreased the viability and migration of glioma C6 cells markedly at 48 h. In addition, MiR-17 inhibitor increased the viability and migration of glioma C6 cells at 24 and 48 h. The protein expression of Cyclin D1, p-Akt and Akt in glioma C6 cells decreased after transfection with miR-17 mimics for 72 h, and increased after transfection with miR-17 inhibitor for 72 h.. The reduced miR-17 levels in glioma cells increased cell viability and migration, which correlates with increased expression of Cyclin D1, p-Akt and Akt.

Topics: Animals; Brain Neoplasms; Cell Line, Tumor; Cell Movement; Cell Survival; Cyclin D1; Glioma; Male; MicroRNAs; Proto-Oncogene Proteins c-akt; Rats; Rats, Wistar

Emerging studies show that dysregulated miRNAs are implicated in tumorigenesis and progression of various cancers. MiRNA-320c, an important member of miRNA-320 family, was characterized as a new candidate miRNA that suppressed the development of colorectal cancer and bladder cancer. However, the function of miRNA-320c in human glioma remained unclear. Here, we found that miRNA-320c was significantly down-regulated in glioma tissues in contrast with normal brain tissues, being tightly related to clinical stage of glioma by qRT-PCR. Moreover, Kaplan-Meier analysis demonstrated that patients with low miRNA-320c expression had a shorter survival. Multivariate Cox regression analysis indicated that miRNA-320c could serve as an independent poor prognostic factor for patients with glioma. Functionally, overexpression of miRNA-320c could dramatically inhibit glioma cell proliferation, migration and invasion, as well as promote apoptosis. Further analysis indicated that overexpression of miRNA-320c dramatically led to the G0/G1 phase arrest and correspondingly decreased the percentage of S phase cells by suppressing the expression of G1/S transition key regulators, such as Cyclin D1 and CDK6. Additionally, up-regulation of miRNA-320c could significantly impair migration and invasion of glioma cells via reducing the expression of MMP2, MMP9, N-cadherin and Integrin β1. Collectively, our data revealed that miRNA-320c played a crucial role in the carcinoma processes of glioma and might serve as a new prognosis biomarker and therapeutic target of glioma.

Topics: Adult; Apoptosis; Brain Neoplasms; Cell Cycle; Cell Line, Tumor; Collagenases; Colony-Forming Units Assay; Cyclin D1; Cyclin-Dependent Kinase 6; Female; Gene Expression Regulation, Neoplastic; Glioma; Humans; Male; MicroRNAs; Middle Aged; Neoplasm Invasiveness; Retrospective Studies; Time Factors; Wound Healing

To study the presence of 9p deletion and p16, cyclin D1 and Myc expression and their respective diagnostic and prognostic interest in oligodendrogliomas.. We analyzed a retrospective series of 40 consecutive anaplastic oligodendrogliomas (OIII) from a single institution and compared them to a control series of 10 low grade oligodendrogliomas (OII). Automated FISH analysis of chromosome 9p status and immunohistochemistry for p16, cyclin D1 and Myc was performed for all cases and correlated with clinical and histological data, event free survival (EFS) and overall survival (OS).. Chromosome 9p deletion was observed in 55% of OIII (22/40) but not in OII. Deletion was highly correlated to EFS (median = 29 versus 53 months, p<0.0001) and OS (median = 48 versus 83 months, p<0.0001) in both the total cohort and the OIII population. In 9p non-deleted oligodendrogliomas, p16 hyperexpression correlated with a shorter OS (p = 0.02 in OII and p = 0.0001 in OIII) whereas lack of p16 expression was correlated to a shorter EFS and OS in 9p deleted OIII (p = 0.001 and p = 0.0002 respectively). Expression of Cyclin D1 was significantly higher in OIII (median expression 45% versus 14% for OII, p = 0.0006) and was correlated with MIB-1 expression (p<0.0001), vascular proliferation (p = 0.002), tumor necrosis (p = 0.04) and a shorter EFS in the total cohort (p = 0.05). Hyperexpression of Myc was correlated to grade (median expression 27% in OII versus 35% in OIII, p = 0.03), and to a shorter EFS in 9p non-deleted OIII (p = 0.01).. Chromosome 9p deletion identifies a subset of OIII with significantly worse prognosis. The combination of 9p status and p16 expression level identifies two distinct OIII populations with divergent prognosis. Hyperexpression of Bcl1 and Myc appears highly linked to anaplasia but the prognostic value is unclear and should be investigated further.

Topics: Brain Neoplasms; Chromosome Deletion; Chromosomes, Human, Pair 9; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; Disease-Free Survival; Female; Gene Expression Regulation, Neoplastic; Humans; Male; Middle Aged; Oligodendroglioma; Proto-Oncogene Proteins c-myc; Survival Rate

To determine whether erythropoietin (EPO) promotes rapid proliferation of glioma through Akt pathway.. We detected the expression of EPO in human glioma tissues using immunohistochemistry. A nude mouse model bearing human glioma U87 cell xenograft was established and given intraperitoneal injection of EPO or saline every other day, and the tumor growth was observed. In the in vitro experiment, U87 cells were treated with PBS (control), EPO, or EPO with Akt inhibitor, and the expression of p-Akt and cyclin D1 was detected using Western blotting; the cell proliferation rate was determined using cell counting kit-8 and clone formation assay, and the cell cycle changes were analyzed with flow cytometry.. Compared with low-grade glioma tissues, high-grade glioma tissues exhibited a significantly increased EPO expression (P=0.0002). In the tumor-bearing mice, EPO treatment significantly increased the expression of EPO (P=0.0006) and p-Akt (P=0.0003) in the tumor and obviously increased the tumor volume (P<0.0001) and weight (P=0.0003). In U87 cells cultured in vitro, EPO treatment obviously accelerated the cell proliferation (P=0.020 on day 3 and 0.028 on day 5), promoted clone formation (P=0.0010), and increased proliferation index (P=0.0028); EPO significantly enhanced the protein expression of p-Akt (P=0.0020) and cyclin D1 (P=0.0022). The application of Akt inhibitor significantly suppressed the effect of EPO in enhancing cyclin D1 and p-Akt expression (both P<0.0001) and promoting cell proliferation.. EPO can significantly accelerate the proliferation of glioma through Akt pathway.

Topics: Animals; Brain Neoplasms; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Erythropoietin; Glioma; Humans; Mice; Mice, Nude; Neoplasm Transplantation; Proto-Oncogene Proteins c-akt; Signal Transduction

Aggressive cancers such as glioblastoma (GBM) contain intermingled apoptotic cells adjacent to proliferating tumor cells. Nonetheless, intercellular signaling between apoptotic and surviving cancer cells remain elusive. In this study, we demonstrate that apoptotic GBM cells paradoxically promote proliferation and therapy resistance of surviving tumor cells by secreting apoptotic extracellular vesicles (apoEVs) enriched with various components of spliceosomes. apoEVs alter RNA splicing in recipient cells, thereby promoting their therapy resistance and aggressive migratory phenotype. Mechanistically, we identified RBM11 as a representative splicing factor that is upregulated in tumors after therapy and shed in extracellular vesicles upon induction of apoptosis. Once internalized in recipient cells, exogenous RBM11 switches splicing of MDM4 and Cyclin D1 toward the expression of more oncogenic isoforms.

Topics: Animals; Apoptosis; Brain Neoplasms; Cell Communication; Cell Cycle Proteins; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Drug Resistance, Neoplasm; Extracellular Vesicles; Female; Glioblastoma; Humans; Mice, Inbred NOD; Mice, SCID; Nuclear Proteins; Phenotype; Proto-Oncogene Proteins; RNA Splicing; RNA-Binding Proteins; Signal Transduction; Spliceosomes; Tumor Burden

Adrenocortical dysplasia (ACD) is a shelterin protein involved in the maintenance of telomere length and in cancer radioresistance. This study investigated the expression profile of ACD in human gliomas and its role in radioresistance of glioma cells. The expression of ACD was analyzed in 62 different grades of glioma tissues and correlated with prognosis. A radioresistant cell line was generated from U87MG cells. For mechanistic studies, ACD was inhibited by small interfering RNA-targeting ACD and the effect on cell radioresistance, telomerase activity, cyclinD1, caspase-3, hTERT, and BIRC1 was evaluated. Clonogenic assay was performed after irradiation, to investigate the effect of ACD silencing on radiation sensitivity. ACD expression appeared strongly upregulated in higher grade gliomas, and its expression was significantly correlated to grading and poor prognosis. In glioma cell lines, ACD expression pattern was similar to those observed in glioma tissues. In irradiated cells, ACD expression was increased in an ionizing radiation dose-dependent manner. A higher expression of ACD was observed in the radioresistant clones than parental cells. Silencing of ACD led to the enhanced radiation sensitivity, decreased telomerase activity and cyclin D1 expression, reduced expression of BIRC1, and finally to the upregulation of caspase-3. This study represents the first report, which demonstrated the expression pattern of ACD in gliomas and its prognostic value. Our results suggested that ACD is involved in glioblastoma radioresistance, likely through the modulation of telomerase activity, proliferation, and apoptosis. ACD might represent a potential molecular biomarker and a novel therapeutic target in glioblastoma.

Topics: Brain Neoplasms; Caspase 3; Cell Line, Tumor; Cell Survival; Cyclin D1; Gene Silencing; Glioblastoma; Humans; Neoplasm Grading; Neuronal Apoptosis-Inhibitory Protein; Radiation Tolerance; Shelterin Complex; Telomerase; Telomere-Binding Proteins

Glioma is a type of tumor that starts in the glial cells of brain and spine. However, the underlying molecular mechanisms of miRNAs dysregulation in glioma initiation and progression is largely unclear.. To further understand the molecular mechanism of miR-490-5P functions and how miR-490 regulated CCND1 function.. The expression of miR-490-5P in glioma tissues and cells was measured by qRT-PCR and ISH. Cell transfection is responsible for miR-490-5P overexpression and knockdown. CCK-8 and clone formation assay are applicable to examine the capacity of glioma cells proliferation. Cell cycle analysis is used to test glioma cells cycle distribution with miR-490-5P overexpression or downregulation. Further, in vivo tumor exnograft studies are used to examine the effects of miR- 490-5P on glioma malignancy in vivo.. We found overexpression of miR-490 lead to glioma cells cycle arrest at G1 phase and decreased proliferation. Next-step functional assays showed miR-490 regulated CCND1 expression and manipulated giloma cells proliferation. Finally, negative regulation of miR-490 in CCND1 function was validated through in vivo nude mice tumorigenesis assay and IHC examination in glioma tissue.. Overall, these results showed that epigenetic regulation of CCND1 via miR-490 was essential to glioma and provide a new insight into glioma diagnosis, treatment, prognosis and further translational investigations.

Topics: Animals; Brain Neoplasms; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Colony-Forming Units Assay; Cyclin D1; Female; Gene Expression Regulation, Neoplastic; Glioma; Humans; Male; Mice, Nude; MicroRNAs; Transfection; Xenograft Model Antitumor Assays

Glioblastoma (GB) is the most lethal, aggressive, and diffuse brain tumor. The main challenge for successful treatment is targeting the cancer stem cell (CSC) subpopulation responsible for tumor origin, progression, and recurrence. Chloride Intracellular Channel 1 (CLIC1), highly expressed in CSCs, is constitutively present in the plasma membrane where it is associated with chloride ion permeability.

Topics: Brain Neoplasms; Cell Line, Tumor; Cell Membrane; Cell Proliferation; Chloride Channels; Cyclin D1; G1 Phase; Glioblastoma; Humans; Hydrogen-Ion Concentration; Middle Aged; NADPH Oxidases; Neoplastic Stem Cells; Reactive Oxygen Species; S Phase; Sodium-Hydrogen Exchanger 1; Time Factors

Topics: Animals; Apoptosis; beta Catenin; Brain; Brain Neoplasms; Cell Cycle; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Gene Silencing; Glioma; Humans; Mice; Neoplasm Invasiveness; Neuroglia; Real-Time Polymerase Chain Reaction; Receptors, Formyl Peptide; Receptors, Lipoxin; RNA, Messenger; RNA, Small Interfering; Signal Transduction

Cyclin D1 (CCND1) is frequently overexpressed in malignant gliomas. We have previously shown ectopic overexpression of CCND1 in human malignant gliomas cell lines.. Quantitative reverse transcriptase PCR (qRT-PCR) and Western Blot (WB) was performed to investigate the expression of CCND1 in glioma tissues and cell lines. The biological function of CCND1 was also investigated through knockdown and overexpression of BCYRN1 in vitro.. Here we reported that CCND1 expression was positively associated with the pathological grade and proliferative activity of astrocytomas, as the lowest expression was found in normal brain tissue (N = 3) whereas the highest expression was in high-grade glioma tissue (N = 25). Additionally, we found that the expression level of CCND1 was associated with IC50 values in malignant glioma cell lines. Forced inhibition of CCND1 increased temozolomide efficacy in U251 and SHG-44 cells. After CCND1 overexpression, the temozolomide efficacy decreased in U251 and SHG-44 cells. Colony survival assay and apoptosis analysis confirmed that CCND1 inhibition renders cells more sensitive to temozolomide treatment and temozolomide-induced apoptosis in U251 and SHG-44 cells. Inhibition of P-gp (MDR1) by Tariquidar overcomes the effects of CCND1 overexpression on inhibiting temozolomide-induced apoptosis. Inhibition of CCND1 inhibited cell growth in vitro and in vivo significantly more effectively after temozolomide treatments than single temozolomide treatments. Finally, inhibition of CCND1 in glioma cells reduced tumor volume in a murine model.. Taken together, these data indicate that CCND1 overexpression upregulate P-gp and induces chemoresistance in human malignant gliomas cells and that inhibition of CCND1 may be an effective means of overcoming CCND1 associated chemoresistance in human malignant glioma cells.

Topics: Adult; Antineoplastic Agents, Alkylating; Apoptosis; Brain Neoplasms; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Down-Regulation; Female; Gene Expression Regulation, Neoplastic; Glioblastoma; Humans; Male; Middle Aged; Temozolomide

The function of miR16 in multiforme glioblastoma multiforme (GBM) and its stem cells (GSCs) remains elusive. To this end, we investigated the patterns of miR16 expression in these cells and their correlation with malignant behaviors and clinical outcomes. The levels of miR16 and its targeted genes in tumor tissue of GBM and GBM SGH44, U87, U251 cells as well as their stem cell counterparts were measured by qRT-PCR or western blot or immunohistochemistry. Luciferase reporter assay was used to confirm the binding of miR16 to 3'-UTR of its target genes. The effects of miR16 on malignant behaviors were investigated, including tumor cell viability, soft-agar colony formation, GSCs Matrigel colony forming and migration and invasion as well as nude mice xenograft model. Differentially expression patterns of miR16 in glioblastoma cells and GSCs cells were found in this study. Changes of miR16 targeted genes, Bcl2 (B cell lymphoma 2), CDK6 (Cyclin-dependent kinase 6), CCND1 (cyclin D1), CCNE1 (cyclin E1) and SOX5 were confirmed in glioblastoma cell lines and tissue specimens. In vitro and in vivo studies showed that tumor cell proliferation was inhibited by miR16 mimic, but enhanced by miR16 inhibitor. The expression level of miR16 positively correlates with GSCs differentiation, but negatively with the abilities of migration, motility, invasion and colony formation in glioblastoma cells. The inhibitory effects of miR16 on its target genes were also found in nude mice xenograft model. Our findings revealed that the miR16 functions as a tumor suppressor in GSCs and its association with prognosis in GBM.

Topics: Adolescent; Adult; Aged; Animals; Biomarkers, Tumor; Brain; Brain Neoplasms; Case-Control Studies; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Child; Cyclin D1; Cyclin E; Cyclin-Dependent Kinase 6; Female; Gene Expression Regulation, Neoplastic; Glioblastoma; Humans; Male; Mice; Mice, Nude; MicroRNAs; Middle Aged; Neoplastic Stem Cells; Oncogene Proteins; Prognosis; Proto-Oncogene Proteins c-bcl-2; SOXD Transcription Factors; Survival Rate; Xenograft Model Antitumor Assays; Young Adult

Gliomas are the most common primary tumors in central nervous system. The prognosis of the patients with glioma is poor regardless of the development of therapeutic strategies. Its aggressive behavior mainly depends on the potent ability of proliferation. The transcription factor EGR1 (early growth response 1) is a member of a zinc finger transcription factor family which plays an essential role in cell growth and proliferation.. EGR1 expression levels in 39 glioma tissues and 10 normal brain tissues were tested by RT-qPCR and Western-blotting. The effects of EGR1 on U251 cells, U251 stem-like cells (GSCs), and U87 cells proliferation were assessed using in vitro and in vivo cell proliferation assays. The specific binding between EGR1 and CCND1 promoter was confirmed by CHIP assay. EGF was used to improve EGR1 expression in this assay.. EGR1 expression levels in human gliomas are decreased compared with normal brain tissues, however, the patients with low EGR1 expression level showed significantly enhanced patient survival in all glioma patients. EGR1 silencing inhibited proliferation and induced G1 phase arrest in glioma cells. EGR1 contributed to proliferation by directly raising CCND1. Meanwhile, EGR1 overexpression induced by EGF was able to promote the proliferation of glioma cells.. Our results show that stable knockdown EGR1 would inhibit glioma proliferation. The results suggest EGR1 showing lower expression in cancer tissues compared with normal tissues maybe still play an important role in tumor proliferation.

Topics: Animals; Brain Neoplasms; Cell Proliferation; Cyclin D1; Early Growth Response Protein 1; Gene Expression Regulation, Neoplastic; Glioma; Humans; Mice; Mice, SCID; Promoter Regions, Genetic

Acting as a proto-oncogene, long noncoding RNAs (lncRNAs) urothelial carcinoembryonic antigen 1 (UCA1) plays a key role in the occurrence and development of several human tumors. However, the expression and biological functions of UCA1 in glioma are less known. This study discussed the expression of UCA1 in glioma and its effect on the proliferation and cell cycle of glioma cells.. LncRNA UCA1 expressions in 64 glioma samples (Grade I-II in 22 cases and Grade III-IV in 42 cases, according to WHO criteria) and 10 normal brain samples were detected using real-time fluorescence quantitative PCR. On this basis, the correlations of UCA1 to clinicopathological characteristics and prognosis of glioma were assessed. Then, using qPCR, the lncRNA UCA1 expressions in glioma cell lines and astrocytes were detected. UCA1-overexpressing glioma cell lines U87 and U251 were further detected after siRNA transfection of these two cell lines, and the impact on cell proliferation and cell cycle was assessed with CCK-8 (cell counting kit-8) assay and flow cytometry method (FCM), respectively. The expression of cyclin D1, a cell cycle-related protein, was detected using Western Blot.. LncRNA UCA1 expression in the glioma samples was obviously higher as compared with the normal brain samples (P < 0.001), and the expression was correlated significantly with grading of the tumors (P < 0.05). However, lncRNA UCA1 expression was not correlated with age, gender, tumor size and KPS score (P > 0.05). After interference of UCA1 expression by siRNA transfection, the proliferation of both U251 and SHG-44 cells was inhibited (P < 0.05), with more cells arrested in G0/G1 (P < 0.05). Moreover, cyclin D1 expression was also downregulated considerably.. LncRNA UCA1 can promote the proliferation and cell cycle progression of glioma cells by upregulating cyclin D1 transcription. So UCA1 may serve as an independent prognostic indicator and a novel therapeutic target for glioma.

Topics: Adult; Biomarkers, Tumor; Brain Neoplasms; Cell Proliferation; Cyclin D1; Female; Gene Expression Regulation, Neoplastic; Glioma; Humans; Kaplan-Meier Estimate; Male; Middle Aged; Prognosis; Proto-Oncogene Mas; RNA, Long Noncoding; Up-Regulation

Emerging evidence has suggested that aberrantly expressed microRNAs (miRNAs) are associated with glioma development and progression. The aberrant expression of miR-409-3p has been reported in several human cancers. However, little is known about the function of miR-409-3p in gliomas. The aim of this study was to investigate the specific role and molecular mechanism of miR-409-3p in gliomas. In the present study, we found that miR-409-3p was downregulated in glioma tissue and cell lines. Overexpression of miR-409-3p inhibited glioma cell invasion and proliferation, whereas suppression of miR-409-3p promoted glioma cell invasion and proliferation. High-mobility group nucleosome-binding domain 5 (HMGN5), a well-known oncogene in gliomas, was identified as a functional target of miR-409-3p using bioinformatics, dual-luciferase reporter assay, real-time quantitative polymerase chain reaction, and Western blot analysis. Furthermore, miR-409-3p was found to regulate the expression of matrix metalloproteinase 2 and cyclin D1. Restoration of HMGN5 expression significantly reversed the inhibitory effects of miR-409-3p overexpression on glioma cell invasion and proliferation. Taken together, our results suggest that miR-409-3p inhibits glioma cell invasion and proliferation by targeting HMGN5, representing a potential therapeutic target for glioma.

Topics: 3' Untranslated Regions; Brain Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Gene Expression Regulation, Neoplastic; Genes, Reporter; Glioma; HMGN Proteins; Humans; Matrix Metalloproteinase 2; MicroRNAs; RNA Interference; Trans-Activators

Gliomas are the most frequent type of primary brain tumor in adults. Their highly proliferative nature, complex cellular composition, and ability to escape therapies have confronted investigators for years, hindering the advancement toward an effective treatment. Agents that are safe and can be administered as dietary supplements have always remained priority to be most feasible for cancer therapy. Withania somnifera (ashwagandha) is an essential ingredient of Ayurvedic preparations and is known to eliminate cancer cells derived from a variety of peripheral tissues. Although our previous studies have addressed the in vitro anti-proliferative and differentiation-inducing properties of ashwagandha on neuronal cell lines, in vivo studies validating the same are lacking. While exploring the mechanism of its action in vitro, we observed that the ashwagandha water extract (ASH-WEX) induced the G2/M phase blockade and caused the activation of multiple pro-apoptotic pathways, leading to suppression of cyclin D1, bcl-xl, and p-Akt, and reduced the expression of polysialylated form of neural cell adhesion molecule (PSA-NCAM) as well as the activity of matrix metalloproteinases. ASH-WEX reduced the intracranial tumor volumes in vivo and suppressed the tumor-promoting proteins p-nuclear factor kappa B (NF-κB), p-Akt, vascular endothelial growth factor (VEGF), heat shock protein 70 (HSP70), PSA-NCAM, and cyclin D1 in the rat model of orthotopic glioma allograft. Reduction in glial fibrillary acidic protein (GFAP) and upregulation of mortalin and neural cell adhesion molecule (NCAM) expression specifically in tumor-bearing tissue further indicated the anti-glioma efficacy of ASH-WEX in vivo. Combining this enhanced understanding of the molecular mechanisms of ASH-WEX in glioma with in vivo model system offers new opportunities to develop therapeutic strategy for safe, specific, and effective formulations for treating brain tumors.

Topics: Allografts; Animals; Apoptosis; bcl-X Protein; Biomarkers, Tumor; Brain Neoplasms; Cell Adhesion; Cell Cycle; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cyclin D1; Glioma; Male; Neoplasm Invasiveness; Phosphorylation; Plant Extracts; Proto-Oncogene Proteins c-akt; Rats, Wistar; Signal Transduction; Vascular Endothelial Growth Factor A; Withania

Glioblastoma (GBM) is the most common malignant brain tumor, and glioma stem cells (GSCs) are considered a major source of treatment resistance for glioblastoma. Identifying new compounds that inhibit the growth of GSCs and understanding their underlying molecular mechanisms are therefore important for developing novel therapy for GBM.. We investigated the potential inhibitory effect of isorhapontigenin (ISO), an anticancer compound identified in our recent investigations, on anchorage-independent growth of patient-derived glioblastoma spheres (PDGS) and its mechanism of action.. ISO treatment resulted in significant anchorage-independent growth inhibition, accompanied with cell cycle G0-G1 arrest and cyclin D1 protein downregulation in PDGS. Further studies established that cyclin D1 was downregulated by ISO at transcription levels in a SOX2-dependent manner. In addition, ISO attenuated SOX2 expression by specific induction of miR-145, which in turn suppressed 3'UTR activity of SOX2 mRNA without affecting its mRNA stability. Moreover, ectopic expression of exogenous SOX2 rendered D456 cells resistant to induction of cell cycle G0-G1 arrest and anchorage-independent growth inhibition upon ISO treatment, whereas inhibition of miR-145 resulted in D456 cells resistant to ISO inhibition of SOX2 and cyclin D1 expression. In addition, overexpression of miR-145 mimicked ISO treatment in D456 cells.. ISO induces miR-145 expression, which binds to the SOX2 mRNA 3'UTR region and inhibits SOX2 protein translation. Inhibition of SOX2 leads to cyclin D1 downregulation and PDGS anchorage-independent growth inhibition. The elucidation of the miR-145/SOX2/cyclin D1 axis in PDGS provides a significant insight into understanding the anti-GBM effect of ISO compound.

Topics: Brain Neoplasms; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Gene Expression Regulation, Neoplastic; Glioblastoma; Humans; MicroRNAs; SOXB1 Transcription Factors; Stilbenes

Given that glioblastoma multiforme (GBM) is associated with poor prognosis, new agents are urgently needed. We developed phospho-glycerol-ibuprofen-amide (PGIA), a novel ibuprofen derivative, and evaluated its safety and efficacy in preclinical models of GBM, and its mechanism of action using human GBM cells and animal tumor models. Furthermore, we explored whether formulating PGIA in polymeric nanoparticles could enhance its levels in the brain. PGIA was 3.7- to 5.1-fold more potent than ibuprofen in suppressing the growth of human GBM cell lines. PGIA 0.75× IC50 inhibited cell proliferation by 91 and 87% in human LN-229 and U87-MG GBM cells, respectively, and induced strong G1/S arrest.In vivo, compared with control, PGIA reduced U118-MG and U87-MG xenograft growth by 77 and 56%, respectively (P< 0.05), and was >2-fold more efficacious than ibuprofen. Normal human astrocytes were resistant to PGIA, indicating selectivity. Mechanistically, PGIA reduced cyclin D1 levels in a time- and concentration-dependent manner in GBM cells and in xenografts. PGIA induced cyclin D1 degradation via the proteasome pathway and induced dephosphorylation of GSK3β, which was required for cyclin D1 turnover. Furthermore, cyclin D1 overexpression rescued GBM cells from the cell growth inhibition by PGIA. Moreover, the formulation of PGIA in poly-(L)-lactic acid poly(ethylene glycol) polymeric nanoparticles improved its pharmacokinetics in mice, delivering PGIA to the brain. PGIA displays strong efficacy against GBM, crosses the blood-brain barrier when properly formulated, reaching the target tissue, and establishes cyclin D1 as an important molecular target. Thus, PGIA merits further evaluation as a potential therapeutic option for GBM.

Topics: Animals; Brain Neoplasms; Cell Line, Tumor; Cyclin D1; Glioblastoma; Ibuprofen; Mice

Our previous work has demonstrated an intrinsic mRNA-specific protein synthesis salvage pathway operative in glioblastoma (GBM) tumor cells that is resistant to mechanistic target of rapamycin (mTOR) inhibitors. The activation of this internal ribosome entry site (IRES)-dependent mRNA translation initiation pathway results in continued translation of critical transcripts involved in cell cycle progression in the face of global eIF-4E-mediated translation inhibition. Recently we identified compound 11 (C11), a small molecule capable of inhibiting c-MYC IRES translation as a consequence of blocking the interaction of a requisite c-MYC IRES trans-acting factor, heterogeneous nuclear ribonucleoprotein A1, with its IRES. Here we demonstrate that C11 also blocks cyclin D1 IRES-dependent initiation and demonstrates synergistic anti-GBM properties when combined with the mechanistic target of rapamycin kinase inhibitor PP242. The structure-activity relationship of C11 was investigated and resulted in the identification of IRES-J007, which displayed improved IRES-dependent initiation blockade and synergistic anti-GBM effects with PP242. Mechanistic studies with C11 and IRES-J007 revealed binding of the inhibitors within the UP1 fragment of heterogeneous nuclear ribonucleoprotein A1, and docking analysis suggested a small pocket within close proximity to RRM2 as the potential binding site. We further demonstrate that co-therapy with IRES-J007 and PP242 significantly reduces tumor growth of GBM xenografts in mice and that combined inhibitor treatments markedly reduce the mRNA translational state of cyclin D1 and c-MYC transcripts in these tumors. These data support the combined use of IRES-J007 and PP242 to achieve synergistic antitumor responses in GBM.

Topics: Animals; Brain Neoplasms; Cell Line, Tumor; Cyclin D1; Female; Genes, myc; Glioblastoma; Heterografts; Humans; Internal Ribosome Entry Sites; Mice; Protein Biosynthesis; RNA, Messenger; TOR Serine-Threonine Kinases

Low-grade gliomas (LGGs) account for about a third of all brain tumours in children. We conducted a detailed study of DNA methylation and gene expression to improve our understanding of the biology of pilocytic and diffuse astrocytomas. Pilocytic astrocytomas were found to have a distinctive signature at 315 CpG sites, of which 312 were hypomethylated and 3 were hypermethylated. Genomic analysis revealed that 182 of these sites are within annotated enhancers. The signature was not present in diffuse astrocytomas, or in published profiles of other brain tumours and normal brain tissue. The AP-1 transcription factor was predicted to bind within 200 bp of a subset of the 315 differentially methylated CpG sites; the AP-1 factors, FOS and FOSL1 were found to be up-regulated in pilocytic astrocytomas. We also analysed splice variants of the AP-1 target gene, CCND1, which encodes cell cycle regulator cyclin D1. CCND1a was found to be highly expressed in both pilocytic and diffuse astrocytomas, but diffuse astrocytomas have far higher expression of the oncogenic variant, CCND1b. These findings highlight novel genetic and epigenetic differences between pilocytic and diffuse astrocytoma, in addition to well-described alterations involving BRAF, MYB and FGFR1.

Topics: Adolescent; Adult; Astrocytoma; Biomarkers, Tumor; Brain; Brain Neoplasms; Child; Child, Preschool; CpG Islands; Cyclin D1; DNA Methylation; Female; Humans; Infant; Infant, Newborn; Male; Middle Aged; Neoplasm Grading; Promoter Regions, Genetic; Transcription Factor AP-1; Young Adult

Emerging studies show that long noncoding RNAs (lncRNAs) have important roles in carcinogenesis. lncRNA ZEB1 antisense 1 (ZEB1-AS1) is a novel lncRNA, whose clinical significance, biological function, and underlying mechanism remains unclear in glioma. Here, we found that ZEB1-AS1 was highly expressed in glioma tissues, being closely related to clinical stage of glioma. Moreover, patients with high ZEB1-AS1 levels had poor prognoses, with the evidence provided by multivariate Cox regression analysis indicating that ZEB1-AS1 expression could serve as an independent prognostic factor in glioma patients. Functionally, silencing of ZEB1-AS1 could significantly inhibit cell proliferation, migration, and invasion, as well as promote apoptosis. Knockdown of ZEB1-AS1 significantly induced the G0/G1 phase arrest and correspondingly decreased the percentage of S phase cells. Further analysis indicated that ZEB1-AS1 could regulate the cell cycle by inhibiting the expression of G1/S transition key regulators, such as Cyclin D1 and CDK2. Furthermore, ZEB1-AS1 functioned as an important regulator of migration and invasion via activating epithelial to mesenchymal transition (EMT) through up-regulating the expression of ZEB1, MMP2, MMP9, N-cadherin, and Integrin-β1 as well as decreasing E-cadherin levels in the metastatic progression of glioma. Additionally, forced down-regulation of ZEB1-AS1 could dramatically promote apoptosis by increasing the expression level of Bax and reducing Bcl-2 expression in glioma. Taken together, our data suggest that ZEB1-AS1 may serve as a new prognostic biomarker and therapeutic target of glioma.

Topics: Adult; Apoptosis; Biomarkers, Tumor; Brain Neoplasms; Cadherins; Carcinogenesis; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase 2; Epithelial-Mesenchymal Transition; Female; G1 Phase Cell Cycle Checkpoints; Glioma; Humans; Male; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Middle Aged; RNA, Long Noncoding; Zinc Finger E-box-Binding Homeobox 1

Thrombin activates its G-coupled seven transmembrane protease-activated receptor (PAR-1) by cleaving the receptor's N-terminal end. In several human cancers, PAR1 expression and activation correlates with tumor progression and metastatization. This provides compelling evidence for the effectiveness of an appropriate antithrombin agent for the adjuvant treatment of patients with cancer. Dabigatran is a selective direct thrombin inhibitor that reversibly binds to thrombin. In this study, we aimed to explore if dabigatran may affect mechanisms favoring tumor growth by interfering with thrombin-induced PAR-1 activation. We confirmed that exposure of tumor cells to thrombin significantly increased cell proliferation and this was coupled with downregulation of p27 and concomitant induction of cyclin D1. Dabigatran was consistently effective in antagonizing thrombin-induced proliferation as well as it restored the baseline pattern of cell cycle protein expression. Thrombin significantly upregulated the expression of proangiogenetic proteins like Twist and GRO-α in human umbilical vascular endothelial cells (HUVEC) cells and their expression was significantly brought down to control levels when dabigatran was added to culture. We also found that the chemoattractant effect of thrombin on tumor cells was lost in the presence of dabigatran, and that the thrombin antagonist was effective in dampening vascular tube formation induced by thrombin. Our data support a role of thrombin in inducing the proliferation, migration, and proangiogenetic effects of tumor cells in vitro. Dabigatran has activity in antagonizing all these effects, thereby impairing tumor growth and progression. In vivo models may help to understand the relevance of this pathway.

Topics: Antithrombins; Brain Neoplasms; Breast Neoplasms; Cell Cycle; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p27; Dabigatran; Female; Gene Expression Regulation, Neoplastic; Glioblastoma; Humans; Poly (ADP-Ribose) Polymerase-1; Thrombin

The transcriptional coactivator with PDZ-binding motif (TAZ) is one of the important downstream effectors of Hippo pathway. In this study, the potential implication of TAZ in gliomagenesis was explored. TAZ expression was identified to be upregulated in glioma specimens and positively correlated with tumor grade. Meanwhile, its expression in nucleus was increased more significantly with the ascending order of tumor grade. Knocking down TAZ inhibited glioma cell proliferation, invasion and promoted apoptosis. Conversely, enforced upregulation of TAZ promoted proliferation, invasion of glioma cells, and suppressed apoptosis in vitro. When orthotopic glioblastoma mouse model implanted with TAZ knocked down cells, glioma growth was inhibited and survival period was prolonged. Expression of Ki67, MMP-9, Cyclin D1, Bcl-2 and C-myc was varied in accordance with the level of TAZ in glioma cell. The biomarkers of EMT (epithelial-mesenchymal transition), vimentin and N-cadherin, were downregulated when TAZ was suppressed. Using Co-immunoprecipitation TAZ was identified to bind to TEAD4. Therefore, our findings indicate that TAZ is overexpressed in glioma and translocated more into nucleus in high grade glioma. TAZ is involved in gliomagenesis by promoting glioma growth and may benefit to EMT progression. This result suggests that TAZ serves as a potential target for the treatment of glioma.

Topics: Animals; Apoptosis; Biomarkers, Tumor; Brain Neoplasms; Cell Line, Tumor; Cell Movement; Cell Nucleus; Cell Proliferation; Cyclin D1; DNA-Binding Proteins; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Glioma; Humans; Intracellular Signaling Peptides and Proteins; Ki-67 Antigen; Male; Matrix Metalloproteinase 9; Mice, Inbred BALB C; Mice, Nude; Muscle Proteins; Neoplasm Grading; Neoplasm Invasiveness; Proto-Oncogene Proteins c-bcl-2; Proto-Oncogene Proteins c-myc; RNA Interference; Signal Transduction; TEA Domain Transcription Factors; Time Factors; Trans-Activators; Transcription Factors; Transcriptional Coactivator with PDZ-Binding Motif Proteins; Tumor Burden; Up-Regulation

Aberrant DNA methylation has been shown to inactivate tumor suppressor genes during carcinogenesis. MicroRNA-149 (miR-149) was recently demonstrated to function as a tumor suppressor gene in glioblastoma multiforme (GBM). However, the potential linkage of miR-149 levels and the underlying epigenetic regulatory mechanism in human GBM has not been studied. We used quantitative real-time polymerase chain reaction to investigate the levels of miR-149 in GBM tissues, their matched adjacent normal tissues, and glioblastoma U87MG cell line. Using bisulfite genomic sequencing technology, DNA methylation status of upstream region of miR-149 was evaluated in study population groups and the U87MG cell line. After treatment of cells with 5-aza-2'-deoxycitidine (5-aza-dC), the DNA methylation status, gene expression, and target protein levels of miR-149 were investigated. Our studies revealed that methylation and expression levels of miR-149 were significantly increased and decreased, respectively in GBM patients relative to the adjacent normal tissues (P < 0.01). MiR-149 suppressed the expression of AKT1 and cyclin D1 and reduced the proliferative activities of the U87MG cell line. Treatment of U87MG cells with 5-aza-dC reversed the hypermethylation status of miR-149, enhanced the expression of its gene, and decreased target mRNA and proteins levels (P < 0.01). These findings suggest that the methylation mechanism is associated with decreased expression levels of miR-149, which may in turn lead to the increased levels of its oncogenic target proteins.

Topics: Antimetabolites, Antineoplastic; Apoptosis; Azacitidine; Base Sequence; Brain; Brain Neoplasms; Cell Proliferation; Cyclin D1; DNA Methylation; Epigenesis, Genetic; Gene Expression Regulation, Neoplastic; Glioblastoma; Humans; MicroRNAs; Promoter Regions, Genetic; Proto-Oncogene Proteins c-akt; Real-Time Polymerase Chain Reaction; Tumor Cells, Cultured

β-catenin is a crucial signal transduction molecule in the Wnt/β-catenin signal pathway, and increased β-catenin expression has consistently been found in high grade gliomas. However, the mechanisms responsible for β-catenin overexpression have remained elusive.Here we show that the deubiquitinase USP9X stabilizes β-catenin and thereby promotes high grade glioma cell growth. USP9X binds β-catenin and removes the Lys 48-linked polyubiquitin chains that normally mark β-catenin for proteasomal degradation. Increased USP9X expression correlates with increased β-catenin protein in high grade glioma tissues. Moreover, patients with high grade glioma overexpressing USP9X have a poor prognosis. Knockdown of USP9X suppresses cell proliferation, inhibits G1/S phase conversion, and induces apoptosis in U251 and A172 cells. Interestingly, c-Myc and cyclinD1, which are important downstream target genes in the Wnt/β-catenin signal pathway, also show decreased expression in cells with siRNA-mediated down-regulation of USP9X. Down-regulation of USP9X also consistently inhibits the tumorigenicity of primary glioma cells in vivo.In summary, these results indicate that USP9X stabilizes β-catenin and activates Wnt/β-catenin signal pathway to promote glioma cell proliferation and survival. USP9X could also potentially be a novel therapeutic target for high grade gliomas.

Topics: Adolescent; Adult; Aged; Animals; Apoptosis; beta Catenin; Brain Neoplasms; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cyclin D1; Enzyme Stability; Female; G1 Phase Cell Cycle Checkpoints; Gene Expression Regulation, Neoplastic; Glioma; Humans; Kaplan-Meier Estimate; Male; Mice, Inbred BALB C; Mice, Nude; Middle Aged; Neoplasm Grading; Proportional Hazards Models; Proteasome Endopeptidase Complex; Proteolysis; Proto-Oncogene Proteins c-myc; RNA Interference; Time Factors; Transfection; Ubiquitin Thiolesterase; Ubiquitination; Wnt Signaling Pathway; Young Adult

Glioblastoma is the most common and aggressive primary brain tumor in adults, with a poor prognosis because of its resistance to radiotherapy and chemotherapy. Merlin/NF2 (moesin-ezrin-radixin-like protein/neurofibromatosis type 2) is a tumor suppressor found to be mutated in most nervous system tumors; however, it is not mutated in glioblastomas. Merlin associates with several transmembrane receptors and intracellular proteins serving as an anchoring molecule. Additionally, it acts as a key component of cell motility. By selecting sub-populations of U251 glioblastoma cells, we observed that high expression of phosphorylated Merlin at serine 518 (S518-Merlin), NOTCH1 and epidermal growth factor receptor (EGFR) correlated with increased cell proliferation and tumorigenesis. These cells were defective in cell-contact inhibition with changes in Merlin phosphorylation directly affecting NOTCH1 and EGFR expression, as well as downstream targets HES1 (hairy and enhancer of split-1) and CCND1 (cyclin D1). Of note, we identified a function for S518-Merlin, which is distinct from what has been reported when the expression of Merlin is diminished in relation to EGFR and NOTCH1 expression, providing first-time evidence that demonstrates that the phosphorylation of S518-Merlin in glioblastoma promotes oncogenic properties that are not only the result of inactivation of the tumor suppressor role of Merlin but also an independent process implicating a Merlin-driven regulation of NOTCH1 and EGFR.

Topics: Animals; Basic Helix-Loop-Helix Transcription Factors; Brain Neoplasms; Cell Line, Tumor; Cyclin D1; ErbB Receptors; Female; Gene Expression Regulation, Neoplastic; Glioblastoma; Homeodomain Proteins; Humans; Mice; Mice, Nude; Neurofibromin 2; Phosphorylation; Receptor, Notch1; Transcription Factor HES-1

Rho-associated protein kinase 1 (ROCK1), a serine/threonine protein kinase, affects cell invasion and migration by changing the status of the cytoskeleton. In recent years, ROCK1 was found to be overexpressed in a variety of tumors. However, the information of ROCK1 in glioma still remains elusive. In our study, the expression of ROCK1 in glioma tissues was examined by real-time PCR and the relationship between ROCK1 expression and clinical characteristics of patients with glioma was also analyzed. With the inhibition of ROCK1 expression by RNAi, the effects of ROCK1 on biological behaviors of glioma cells including cell viability, cell cycle, and cell invasion were probed in the U251 cell line by methyl thiazolyl tetrazolium (MTT) assay, flow cytometer analysis, and Transwell invasion experiment. In addition, the effects of ROCK1 on the regulation of Ki67, cyclin D1, matrix metalloproteinases 9 (MMP9), and E-cadherin were also investigated. The results indicated that ROCK1 messenger RNA (mRNA) was increased significantly compared to that in the adjacent normal tissue (P < 0.05) and the expression level of ROCK1 mRNA in high-grade malignant glioma tissue was significantly higher than that in low-grade malignant glioma tissue (P < 0.05). MTT assay and flow cytometer analysis revealed that the cell viability and cell proliferation in the ROCK1 small interfering RNA (siRNA) transfection group were markedly lower than those in the blank or negative control group (P < 0.05), and no obvious differences were found between the blank group and negative control group. The Transwell invasion experiments showed that the invasive ability of U251 cells in the ROCK1 siRNA transfection group was obviously lower than that in the blank or negative control group (P < 0.05), and there were no visible differences between the blank group and negative control group. Western blot demonstrated that the protein levels of Ki67, cyclin D1, and MMP9 in the ROCK1 siRNA transfection group were distinctly lower than those in the blank or negative control group (P < 0.05) and that the protein level of E-cadherin displayed an opposite variation (P < 0.05). In summary, the expressions of ROCK1 in glioma tissue were visibly upregulated and the increase of ROCK1 had a positive correlation with the malignant grade of glioma. The results implied that the proliferation and metastasis of the glioma cell could be inhibited by suppressing the expression of ROCK1, and our findings would provide a n

Topics: Adult; Antigens, CD; Brain Neoplasms; Cadherins; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Female; Gene Expression; Gene Knockdown Techniques; Glioma; Humans; Ki-67 Antigen; Male; Matrix Metalloproteinase 9; Neoplasm Grading; Neoplasm Invasiveness; rho-Associated Kinases

Cathepsin L, a lysosomal cysteine proteinase, is exclusively elevated in a variety of malignancies, including gliomas. In this study we investigated the relationship between cathepsin L and NF-κB, two radiation-responsive elements, in regulating the sensitivity of human glioma cells ionizing radiation (IR) in vitro.. Human glioma U251 cells were exposed to IR (10 Gy), and the expression of cathepsin L and NF-κB was measured using Western blotting. The nuclear translocation of NF-κB p65 and p50 was analyzed with immunofluorescence assays. Cell apoptosis was examined with clonogenic assays. NF-κB transcription and NF-κB-dependent cyclin D1 and ATM transactivation were monitored using luciferase reporter and ChIP assays, respectively. DNA damage repair was investigated using the comet assay.. IR significantly increased expression of cathepsin L and NF-κB p65 and p50 in the cells. Furthermore, IR significantly increased the nuclear translocation of NF-κB, and NF-κB-dependent cyclin D1 and ATM transactivation in the cells. Knockdown of p65 did not change the expression of cathepsin L in IR-treated cells. Pretreatment with Z-FY-CHO (a selective cathepsin L inhibitor), or knockdown of cathepsin L significantly attenuated IR-induced nuclear translocation of NF-κB and cyclin D1 and ATM transactivation, and sensitized the cells to IR. Pretreatment with Z-FY-CHO, or knockdown of p65 also decreased IR-induced DNA damage repair and clonogenic cell survival, and sensitized the cells to IR.. Cathepsin L acts as an upstream regulator of NF-κB activation in human glioma cells and contributes to their sensitivity to IR in vitro. Inhibition of cathepsin L can sensitize the cells to IR.

Topics: Active Transport, Cell Nucleus; Ataxia Telangiectasia Mutated Proteins; Brain Neoplasms; Cathepsin L; Cell Line, Tumor; Cyclin D1; Cysteine Proteinase Inhibitors; DNA Damage; Dose-Response Relationship, Radiation; Glioma; Humans; Neurons; NF-kappa B; NF-kappa B p50 Subunit; Radiation-Sensitizing Agents; RNA Interference; Signal Transduction; Time Factors; Transcription Factor RelA; Transfection

Glioblastoma development is often associated with alteration in the activity and expression of cell cycle regulators, such as cyclin-dependent kinases (CKDs) and cyclins, resulting in aberrant cell proliferation. Recent studies have highlighted the pivotal roles of miRNAs in controlling the development and growth of glioblastoma. Here, we provide evidence for a function of miR-340 in the inhibition of glioblastoma cell proliferation. We found that miR-340 is downregulated in human glioblastoma tissue samples and several established glioblastoma cell lines. Proliferation and neurosphere formation assays revealed that miR-340 plays an oncosuppressive role in glioblastoma, and that its ectopic expression causes significant defect in glioblastoma cell growth. Further, using bioinformatics, luciferase assay and western blot, we found that miR-340 specifically targets the 3'UTRs of CDK6, cyclin-D1 and cyclin-D2, leading to the arrest of glioblastoma cells in the G0/G1 cell cycle phase. Confirming these results, we found that re-introducing CDK6, cyclin-D1 or cyclin-D2 expression partially, but significantly, rescues cells from the suppression of cell proliferation and cell cycle arrest mediated by miR-340. Collectively, our results demonstrate that miR-340 plays a tumor-suppressive role in glioblastoma and may be useful as a diagnostic biomarker and/or a therapeutic avenue for glioblastoma.

Topics: Base Sequence; Brain Neoplasms; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin D2; Cyclin-Dependent Kinase 6; DNA Primers; Glioblastoma; Humans; MicroRNAs; Real-Time Polymerase Chain Reaction

Despite great efforts to improve survival rates, the prognosis of lung cancer patients is still very poor, mainly due to high invasiveness. We developed brain metastatic PC14PE6/LvBr4 cells through intracardiac injection of lung adenocarcinoma PC14PE6 cells. Western blot and RT-qPCR analyses revealed that PC14PE6/LvBr4 cells had mesenchymal characteristics and higher invasiveness than PC14PE6 cells. We found that cyclin D1 was upregulated, miR-95-3p was inversely downregulated, and pri-miR-95 and its host gene, ABLIM2, were consistently decreased in PC14PE6/LvBr4 cells. MiR-95-3p suppressed cyclin D1 expression through direct binding to the 3' UTR of cyclin D1 mRNA and suppressed invasiveness, proliferation, and clonogenicity of PC14PE6/LvBr4 cells. Ectopic cyclin D1 reversed miR-95-3p-mediated inhibition of invasiveness and clonogenicity, demonstrating cyclin D1 downregulation is involved in function of miR-95-3p. Using bioluminescence imaging, we found that miR-95-3p suppressed orthotopic tumorigenicity and brain metastasis in vivo and increased overall survival and brain metastasis-free survival. Consistent with in vitro metastatic cells, the levels of miR-95-3p, pri-miR-95, and ABLIM2 mRNA were decreased in brain metastatic tissues compared with lung cancer tissues and higher cyclin D1 expression was involved in poor prognosis. Taken together, our results demonstrate that miR-95-3p is a potential therapeutic target for brain metastasis of lung adenocarcinoma cells.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Animals; Brain Neoplasms; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Down-Regulation; Female; Heterografts; Humans; Lung Neoplasms; Mice; Mice, Inbred BALB C; Mice, Nude; MicroRNAs; Neoplasm Metastasis; Prognosis; Transfection

To define the DNA methylation landscape of neuroblastoma and its clinicopathological impact.. Microarray DNA methylation data were analyzed and associated with functional/regulatory genome annotation data, transcriptional profiles and clinicobiological parameters.. DNA methylation changes in neuroblastoma affect not only promoters but also intragenic and intergenic regions at cytosine-phosphate-guanine (CpG) and non-CpG sites, and target functional chromatin domains of development and cancer-related genes such as CCND1. Tumors with diverse clinical risk showed differences affecting CpG and, remarkably, non-CpG sites. Non-CpG methylation observed essentially in clinically favorable cases was associated with the differentiation status of neuroblastoma and expression of key genes such as ALK.. This epigenetic fingerprint of neuroblastoma provides new insights into the pathogenesis and clinical behavior of this pediatric tumor.

Topics: Anaplastic Lymphoma Kinase; Brain Neoplasms; Cell Line, Tumor; Child; Child, Preschool; Chromatin; CpG Islands; Cyclin D1; DNA Fingerprinting; DNA Methylation; DNA, Intergenic; Epigenesis, Genetic; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Genome, Human; Humans; Infant; Male; Neuroblastoma; Oligonucleotide Array Sequence Analysis; Prognosis; Promoter Regions, Genetic; Receptor Protein-Tyrosine Kinases; Survival Analysis

Oligodendroglial tumors with neuronal differentiation cases have been reported in recent studies. Oligodendrocyte precursor cells (OPCs) give rise to both oligodendrocytes and neurons; however, little is known about the association between OPCs and oligodendroglial tumors with neuronal differentiation. Previously, we observed the coexpression of cyclin D1, one of the OPC markers, and alpha-internexin (INA) in oligodendroglial tumor cells. INA is a neuronal marker, and has been indicated as an immunohistochemical surrogate of chromosome 1p/19q co-deletion in oligodendroglial tumors. In this study, we investigated the expression status in 83 gliomas immunohistochemically, and found that cyclin D1-positive cells were commonly detected in gliomas. There was no correlation between the cyclin D1 and Ki-67 labeling indices, suggesting an unrecognized role of cyclin D1 other than a cell cycle regulator in gliomas. Cyclin D1/INA double-positive cells were consistently observed in oligodendroglial tumors regardless of histological grade. In 2 cases of oligodendroglioma with neuronal differentiation, the tumor cells of neuronal morphology showed higher expression of INA, suggesting INA expression may be associated with a bona fide neuronal phenotype. The prevalence of cyclin D1/INA double-positive cells is a distinct feature of oligodendroglial tumors. This new characteristic finding may have practical utility in glioma classification.

Topics: Brain Neoplasms; Cyclin D1; Gene Expression; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Intermediate Filament Proteins; Oligodendroglioma

It has been well documented that aberrant expression of microRNAs is associated with carcinogenesis of glioblastoma (GBM), however the underlying mechanisms are not clear. In this present study, we aimed to clarify the biological function of miR-454 in GBM. MiR-454 was identified to be significantly down-regulated in GBM primary tumors and cell lines. Overexpression of miR-454 in GBM cells resulted in arresting cells at G0/G1 phase and thus inhibiting cell proliferation. Bioinformatic analysis predicted 3-phosphoinositide-dependent protein kinase-1 (PDK1) as a target of miR-454 which acted as a tumor promoter gene. Increased miR-454 significantly repressed PDK1 expression, and then regulating cell proliferation and cell cycle regulators, down-regulation of Cyclin D1 and p-pRb and p21 was up-regulated. Taken together, our study has revealed miR-454 as a tumor suppressor in GBM.

Topics: 3-Phosphoinositide-Dependent Protein Kinases; 3' Untranslated Regions; Binding Sites; Brain Neoplasms; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Computational Biology; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Glioma; Humans; MicroRNAs; Phosphorylation; Retinoblastoma Protein; Signal Transduction; Time Factors; Transfection

Astrocytic gliomas, which are derived from glial cells, are considered the most common primary neoplasias of the central nervous system (CNS) and are histologically classified as low grade (I and II) or high grade (III and IV). Recent studies have shown that astrocytoma formation is the result of the deregulation of several pathways, including the RB/E2F pathway, which is commonly deregulated in various human cancers via genetic or epigenetic mechanisms. On the basis of the assumption that the study of the mechanisms controlling the INK4/ARF locus can help elucidate the molecular pathogenesis of astrocytic tumors, identify diagnostic and prognostic markers, and help select appropriate clinical treatments, the present study aimed to evaluate and compare methylation patterns using bisulfite sequencing PCR and evaluate the gene expression profile using real-time PCR in the genes CDKN2A, CDKN2B, CDC6, Bmi-1, CCND1, and RB1 in astrocytic tumors. Our results indicate that all the evaluated genes are not methylated independent of the tumor grade. However, the real-time PCR results indicate that these genes undergo progressive deregulation as a function of the tumor grade. In addition, the genes CDKN2A, CDKN2B, and RB1 were underexpressed, whereas CDC6, Bmi-1, and CCND1 were overexpressed; the increase in gene expression was significantly associated with decreased patient survival. Therefore, we propose that the evaluation of the expression levels of the genes involved in the RB/E2F pathway can be used in the monitoring of patients with astrocytomas in clinical practice and for the prognostic indication of disease progression.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Astrocytoma; Brain Neoplasms; Cell Cycle Proteins; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p15; Cyclin-Dependent Kinase Inhibitor p16; DNA Methylation; E2F Transcription Factors; Female; Gene Expression Regulation, Neoplastic; Humans; Male; Middle Aged; Nuclear Proteins; Polycomb Repressive Complex 1; Prognosis; Retinoblastoma Protein; Signal Transduction; Young Adult

Topics: Brain Neoplasms; Cyclin D1; Genetic Predisposition to Disease; Glioma; Humans; Polymorphism, Genetic

Deregulation of SOX9 expression has been detected in various human cancer tissues; however, the functional role of SOX9 expression has not been fully elucidated in glioma. SOX9 expression in glioma tissues was analyzed using public tumor datasets and quantitative reverse transcription polymerase chain reaction. The association of SOX9 expression with clinical prognosis in glioma patients was analyzed by examining publically available microarray profiling datasets. The functional roles of SOX9 in glioma were examined using gene set enrichment analysis (GSEA). Cell growth was measured using soft agar colony formation assay, and the cell cycle was analyzed using flow cytometry. Our data showed that SOX9 expression was commonly upregulated in glioma tissues, and patients with high SOX9 levels had shorter survival times. GSEA identified that the gene sets regulating cell proliferation and cell cycle progression were significantly enriched in glioma cells with high SOX9 expression. SOX9 downregulation decreased cyclin D1, CDK4 expression and Rb phosphorylation, which correlated with a reduced population of cells in the S phase and suppressed growth. SOX9, as an oncogene, is highly expressed in gliomas and may be potential indicators of a poor prognosis in glioma patients. SOX9 knockdown may suppress cancer cell growth by inducing cell cycle arrest, which suggests that SOX9 is a potential therapeutic target in glioma.

Topics: Adult; Aged; Brain Neoplasms; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase 4; Down-Regulation; Female; Gene Expression Regulation, Neoplastic; Glioma; Humans; Male; Middle Aged; Phosphorylation; Prognosis; Retinoblastoma Protein; SOX9 Transcription Factor; Survival Rate; Up-Regulation

Vitamin D and its analogs have been shown to display anti-proliferative effects in a wide variety of cancer types including glioblastoma multiforme (GBM). These anticancer effects are mediated by its active metabolite, 1α, 25-dihydroxyvitamin D3 (calcitriol) acting mainly through vitamin D receptor (VDR) signaling. In addition to its involvement in calcitriol action, VDR has also been demonstrated to be useful as a prognostic factor for some types of cancer. However, to our knowledge, there are no studies evaluating the expression of VDR protein and its association with outcome in gliomas. Therefore, we investigated VDR expression by using immunohistochemical analysis in human glioma tissue microarrays, and analyzed the association between VDR expression and clinico-pathological parameters. We further investigated the effects of genetic and pharmacologic modulation of VDR on survival and migration of glioma cell lines. Our data demonstrate that VDR is increased in tumor tissues when compared with VDR in non-malignant brains, and that VDR expression is associated with an improved outcome in patients with GBM. We also show that both genetic and pharmacologic modulation of VDR modulates GBM cellular migration and survival and that VDR is necessary for calcitriol-mediated effects on migration. Altogether these results provide some limited evidence supporting a role for VDR in glioma progression.

Topics: Adult; Age Factors; Brain Neoplasms; Calcitriol; Calcium Channel Agonists; Cell Line, Tumor; Cell Movement; Cell Survival; Cyclin D1; Female; Gene Expression Regulation, Neoplastic; Glioblastoma; Humans; Male; Middle Aged; Oncogene Proteins; Receptors, Calcitriol; RNA, Small Interfering; Sex Factors; Time Factors; Tissue Array Analysis

To investigate the expression of cyclinD1 and Ki-67 proteins in gliomas and its significance.. The immunohistochemistry was used to detect the expression of cyclinD1 and Ki-67 proteins in 18 cases of normal brain tissues, 32 cases of low-grade gliomas, and 24 cases of high-grade gliomas.. The cyclinD1 positive ratio in normal brain tissues, low-grade gliomas, and high-grade gliomas were 4/18, 15/32, and 18/24, respectively, with statistically significant difference (p < 0.05). Differences were significant by pairwise comparison between normal brain tissue with high-grade gliomas and low-grade gliomas with high-grade glioma groups (p < 0.01). However, there was no significant differences between normal brain tissue with low-grade gliomas. The Ki-67 positive ratio in normal brain tissues, low-grade gliomas, and high-grade gliomas were 5/18, 21/32, and 20/24, respectively. The difference among three tissues was statistically significant (p < 0.05). Differences were significant by pairwise comparison between normal brain tissue with low-grade gliomas and normal brain tissue with high-grade glioma group (p < 0.01). There is no difference between low-grade gliomas and high-grade gliomas (p > 0.05). Spearman's rank correlation confirmed that cyclinD1 and Ki-67 was positively correlated in low-grade gliomas and high-level brain tumor (p < 0.05), but no correlation in the normal brain tissue (p > 0.05).. The expression of CyclinD1 and Ki-67 increased in gliomas, suggesting that both may play an important role in the occurrence of gliomas.

Topics: Adolescent; Adult; Aged; Brain Neoplasms; Chi-Square Distribution; Child; Cyclin D1; Female; Glioma; Humans; Immunohistochemistry; Ki-67 Antigen; Male; Middle Aged; Neoplasm Grading; Predictive Value of Tests; Up-Regulation; Young Adult

Signal transducer and activator of transcription 3 (Stat3) is a transcription factor that is involved in cell survival and proliferation and has been found to be persistently activated in most human cancers mainly through its phosphorylation at Tyr-705. However, the role and regulation of Stat3 Ser-727 phosphorylation in cancer cells have not been clearly evaluated. In our findings, correlation studies on the expression of CK2 and Stat3 Ser-727 phosphorylation levels in human glioma patient samples as well as rat orthotopic tumor model show a degree of negative correlation. Moreover, brain tumor cell lines were treated with various pharmacological inhibitors to inactivate the CK2 pathway. Here, increased Stat3 Ser-727 phosphorylation upon CK2 inhibition was observed. Overexpression of CK2 (α, α' or β subunits) by transient transfection resulted in decreased Stat3 Ser-727 phosphorylation. Stat3 Tyr-705 residue was conversely phosphorylated in similar situations. Interestingly, we found PP2A, a protein phosphatase, to be a mediator in the negative regulation of Stat3 Ser-727 phosphorylation by CK2. In vitro assays prove that Ser-727 phosphorylation of Stat3 affects the transcriptional activity of its downstream targets like SOCS3, bcl-xl and Cyclin D1. Stable cell lines constitutively expressing Stat3 S727A mutant showed increased survival, proliferation and invasion which are characteristics of a cancer cell. Rat tumor models generated with the Stat3 S727A mutant cell line formed more aggressive tumors when compared to the Stat3 WT expressing stable cell line. Thus, in glioma, reduced Stat3 Ser-727 phosphorylation enhances tumorigenicity which may be regulated in part by CK2-PP2A pathway.

Topics: Animals; bcl-X Protein; Brain Neoplasms; Casein Kinase II; Cell Line, Tumor; Cell Movement; Cell Transformation, Neoplastic; Cyclin D1; Glioma; HEK293 Cells; Humans; Okadaic Acid; Phosphorylation; Protein Phosphatase 2; Rats; Rats, Sprague-Dawley; Serine; STAT3 Transcription Factor; Suppressor of Cytokine Signaling 3 Protein; Suppressor of Cytokine Signaling Proteins; Transplantation, Heterologous

The Fyn related kinase (FRK) is a noteworthy member of the Src non-receptor tyrosine kinase family for its distinctive tumor suppressive function. Recently, we have shown that FRK plays a protective role against the progression of glioma by suppressing cell migration and invasion. However, it is unclear whether the cell growth of glioma is also regulated by FRK and by which mechanism FRK alters its specific biological functions. In the current study, we found that FRK over-expression significantly suppressed the proliferation of glioma cells. In contrast, FRK knockdown by siRNA promoted glioma cell growth. In addition, FRK over-expression caused G1 phase arrest as well as apoptosis of glioma cells. Further investigation disclosed that FRK-induced G1 arrest was accompanied by down-regulation of hyperphosphorylated retinoblastoma protein (pRb), which led to the consequent suppression of E2F1. More importantly, we found that over-expression of FRK inhibited proper cyclin D1 accumulation in the nucleus of proliferating cells. Taken together, our results demonstrate a combined mechanism for the anti-proliferative effects of FRK by inhibiting cyclin D1 nucleus accumulation and pRb phosphorylation in glioma cells.

Topics: Brain Neoplasms; Cell Line, Tumor; Cell Nucleus; Cell Proliferation; Cyclin D1; Down-Regulation; G1 Phase; Glioma; Humans; Neoplasm Proteins; Phosphorylation; Protein-Tyrosine Kinases; Retinoblastoma Protein; Signal Transduction

The Rab protein family is composed of small GTP-binding proteins involved in intracellular vesicle trafficking. In particular, Rab3a which is one of four Rab3 proteins (a, b, c, and d isoforms) is associated with synaptic vesicle trafficking in normal brain. However, despite the elevated level of Rab3a in tumors, its role remains unclear. Here we report a tumorigenic role of Rab3a in brain tumors. Elevated level of Rab3a expression in human was confirmed in both glioma cell lines and glioblastoma multiforme patient specimens. Ectopic Rab3a expression in glioma cell lines and primary astrocytes promoted cell proliferation by increasing cyclin D1 expression, induced resistance to anti-cancer drug and irradiation, and accelerated foci formation in soft agar and tumor formation in nude mice. The overexpression of Rab3a augmented the tumorsphere-forming ability of glioma cells and p53(-/-) astrocytes and increased expression levels of various stem cell markers. Taken together, our results indicate that Rab3a is a novel oncogene involved in glioma initiation and progression.

Topics: Animals; Astrocytes; Brain Neoplasms; Carcinogenesis; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Transformation, Neoplastic; Cyclin D1; Disease Progression; Drug Resistance, Neoplasm; Gene Expression Regulation, Neoplastic; Glioblastoma; Humans; Mice; Mice, Knockout; Mice, Nude; rab3A GTP-Binding Protein

SYF2 is thought to be a cell cycle regulator at the G1/S transition, which encodes a nuclear protein that interacts with cyclin D-type binding-protein 1. In the present study, we investigated the role of SYF2 in human glioma progression. Immunohistochemical and Western blot analyses were performed in human glioma tissues. High SYF2 expression (located in cell nuclei) was observed in 80 samples, and its level was correlated with the grade of malignancy. A strongly positive correlation was observed between SYF2 and Ki-67 expression (P < 0.01). More importantly, high expression of SYF2 was associated with a poor outcome. In vitro, after the release of U87 cell lines from serum starvation, the expression of SYF2 was upregulated, as well as PCNA and cyclin D1. In addition, knockdown of SYF2 by small interfering RNA transfection diminished the expression of PCNA, cyclin D1 and arrested cell growth at G1 phase. These results indicate that SYF2 in glioma is essential for cell proliferation; thus, targeting SYF2 or its downstream targets may lead to novel therapies for glioblastomas.

Topics: Adult; Biomarkers, Tumor; Brain Neoplasms; Cell Cycle; Cell Line, Tumor; Cell Movement; Cyclin D1; Female; Gene Knockdown Techniques; Glioma; Humans; Male; Middle Aged; Nuclear Proteins; Reference Values; RNA-Binding Proteins; RNA, Small Interfering

Oxysterols possess anti-proliferative properties that may be used with much effect in the treatment of cancer. We have demonstrated previously that 7 beta-hydroxycholesterol (7b-HC) provokes both metabolic stress, as witnessed by AMPK activation, and changes in lipid raft composition in C6 glioblastoma cells. These observations suggested that glycolysis might have been changed. Here we will show that 7b-HC increases cell cycle time and that it changes the affinity of pyruvate kinase to its substrate, phosphoenol pyruvate. The latter effect is mimicked by glutamine withdrawal.

Topics: AMP-Activated Protein Kinases; Animals; Brain Neoplasms; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin E; Enzyme Activation; Glioblastoma; Glutamine; Glycolysis; Hydroxycholesterols; Phosphoenolpyruvate; Pyruvate Kinase; Rats; Stress, Physiological

Glioblastoma (GBM) is one of the most lethal forms of human cancer, and new clinical biomarkers and therapeutic targets are urgently required. microRNAs (miRNAs) are small, non-coding RNAs that negatively regulate gene expression at the post-transcriptional and/or translational level by binding the 3' untranslated regions (3' UTRs) of target mRNAs. The dysregulated expression of several miRNAs has been reported to modulate glioma progression. In the present study, we defined the expression and function of miR-708, which, based on real-time PCR analysis, were downregulated in GBM cells. The overexpression of miR-708 inhibited cell proliferation and invasion and induced apoptosis in the human GBM cell lines A172 and T98G. Furthermore, the overexpression of miR-708 reduced the expression of Akt1, CCND1, MMP2, EZH2, Parp-1 and Bcl2 in A172 and T98G cells. Taken together, our study suggests that miR-708 affects GBM cell proliferation and invasion, and induces apoptosis. It is suggested that miR-708 may play an important role as a tumor suppressor in GBM and it may be an attractive target for therapeutic intervention in GBM.

Topics: Apoptosis; bcl-2-Associated X Protein; Brain Neoplasms; Cell Growth Processes; Cell Line, Tumor; Cyclin D1; Down-Regulation; Enhancer of Zeste Homolog 2 Protein; Gene Expression Regulation, Neoplastic; Genes, Tumor Suppressor; Glioblastoma; Humans; Matrix Metalloproteinase 2; MicroRNAs; Neoplasm Invasiveness; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerases; Polycomb Repressive Complex 2; Proto-Oncogene Proteins c-akt

Glioblastoma is the most common type of primary brain tumors. Cisplatin is a commonly used chemotherapeutic agent for Glioblastoma patients. Despite a consistent rate of initial responses, cisplatin treatment often develops chemoresistance, leading to therapeutic failure. Cellular resistance to cisplatin is of great concern and understanding the molecular mechanisms is an utter need.. Glioblastoma cell line U251 cells were exposed to increasing doses of cisplatin for 6 months to establish cisplatin-resistant cell line U251R. The differential miRNA expression profiles in U251 and U251R cell lines were identified by microarray analysis and confirmed by Q-PCR. MiRNA mimics were transfected into U251R cells, and cellular response to cisplatin-induced apoptosis and cell cycle distribution were examined by FACS analysis.. U251R cells showed 3.1-fold increase in cisplatin resistance compared to its parental U251 cells. Microarray analysis identified Let-7b and other miRNAs significantly down-regulated in U251R cells compared to U251 cells. Transfection of Let-7b mimics greatly re-sensitized U251R cells to cisplatin, while transfection of other miRNAs has no effect or slightly effect. Cyclin D1 is predicted as a target of Let-7b through bioinformatics analysis. Over-expression of Let-7b mimics suppressed cyclin D1 protein expression and inhibited cyclin D1-3'-UTR luciferase activity. Knockdown of cyclin D1 expression significantly increased cisplatin-induced G1 arrest and apoptosis.. Collectively, our results indicated that cisplatin treatment leads to Let-7b suppression, which in turn up-regulates cyclin D1 expression. Let-7b may serve as a marker of cisplatin resistance, and can enhance the therapeutic benefit of cisplatin in glioblastoma cells.

Topics: Antineoplastic Agents; Apoptosis; Brain Neoplasms; Cell Line, Tumor; Cisplatin; Cyclin D1; Drug Resistance, Neoplasm; G1 Phase Cell Cycle Checkpoints; Gene Expression; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Glioblastoma; Humans; MicroRNAs; Transfection

Dysregulation of cell cycle genes such as Cyclin D1 and Chk1 contributes to the undifferentiated phenotype of neuroblastoma (NB). CASZ1 functions as a tumor suppressor in NB; here we sought to determine how loss of CASZ1 contributes to cell cycle dysregulation in NB. CASZ1 restoration in NB cells delays NB cell cycle progression. The earliest changes occur within 8 h of CASZ1 restoration in SY5Y cells with a 2.8-fold increase in the level of p21, an inhibitor of Cdk2/4. By 16 h, there is a 40% decrease in the steady-state levels of Cdk6. Restoration of CASZ1 decreases Cdk2-dependent cyclins A and E protein levels and Cdk4/6-dependent Cyclin D1 protein levels. The restoration of CASZ1 resulted in a decrease in pRb phosphorylation and a significant reduction of E2F transcriptional activity. Subsequent to the changes in the G 1/S transition, induction of CASZ1 results in a decrease in Cyclin B levels and Cdc25c phosphatase levels, an upstream activator of the G 2/M regulator CyclinB:Cdk1. In addition, induction of CASZ1 results in a decrease in the levels of phospho-Chk1, a key M-phase regulatory kinase. Similar results were found in a NB cell line with MYCN amplification. Taken together, this study indicates that restoration of CASZ1 activates pRb in G 1 and inhibits the G 2/M regulators Cyclin B1 and Chk1, leading to a lengthening of NB cell cycle progression and a subsequent decrease in cell proliferation.

Topics: Brain Neoplasms; Cell Cycle Checkpoints; Cell Line, Tumor; Checkpoint Kinase 1; Cyclin A; Cyclin B1; Cyclin D1; Cyclin E; Cyclin-Dependent Kinases; DNA-Binding Proteins; Gene Expression Regulation, Neoplastic; Humans; Neuroblastoma; p21-Activated Kinases; Protein Kinases; Retinoblastoma Protein; Signal Transduction; Transcription Factors

Currently, there are few effective adjuvant therapies for pediatric ependymoma outside confocal radiation, and prognosis remains poor. The phosphoinositide 3-kinase (PI3K) pathway is one of the most commonly activated pathways in cancer. PI3Ks transduce signals from growth factors and cytokines, resulting in the phosphorylation and activation of AKT, which in turn induces changes in cell growth, proliferation, and apoptosis.. PI3K pathway status was analyzed in ependymoma using gene expression data and immunohistochemical analysis of phosphorylated AKT (P-AKT). The effect of the PI3K pathway on cell proliferation was investigated by immunohistochemical analysis of cyclin D1 and Ki67, plus in vitro functional analysis. To identify a potential mechanism of PI3K pathway activation, PTEN protein expression and the mutation status of PI3K catalytic subunit α-isoform gene (PIK3CA) was investigated.. Genes in the pathway displayed significantly higher expression in supratentorial than in posterior fossa and spinal ependymomas. P-AKT protein expression, indicating pathway activation, was seen in 72% of tumors (n = 169) and P-AKT expression was found to be an independent marker of a poorer progression-free survival. A significant association between PI3K pathway activation and cell proliferation was identified, suggesting that pathway activation was influencing this process. PTEN protein loss was not associated with P-AKT staining and no mutations were identified in PIK3CA.. Our results suggest that the PI3K pathway could act as a biomarker, not only identifying patients with a worse prognosis but also those that could be treated with therapies targeted against the pathway, a strategy potentially effective in a high percentage of ependymoma patients.

Topics: Biomarkers, Tumor; Brain Neoplasms; Cell Line, Tumor; Cell Proliferation; Child; Child, Preschool; Class I Phosphatidylinositol 3-Kinases; Cyclin D1; Disease-Free Survival; Ependymoma; Female; Humans; Kaplan-Meier Estimate; Male; Molecular Targeted Therapy; Phosphatidylinositol 3-Kinases; Prognosis; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; Signal Transduction; Transcriptome

TRIM11 (tripartite motif-containing protein 11), an E3 ubiquitin ligase, is known to be involved in the development of the central nervous system. However, very little is known regarding the role of TRIM11 in cancer biology. Here, we examined the expression profile of TRIM11, along with two stem cell markers CD133 and nestin, in multiple glioma patient specimens, glioma primary cultures derived from tumors taken at surgery and normal neural stem/progenitor cells (NSCs). The oncogenic function of TRIM11 in glioma biology was investigated by knockdown and/or overexpression in vitro and in vivo experiments. Our results showed that TRIM11 expression levels were upregulated in malignant glioma specimens and in high-grade glioma-derived primary cultures, whereas remaining low in glioblastoma multiforme (GBM) stable cell lines, low-grade glioma-derived primary cultures and NSCs. The expression pattern of TRIM11 strongly correlated with that of CD133 and nestin and differentiation status of malignant glioma cells. Knock down of TRIM11 inhibited proliferation, migration and invasion of GBM cells, significantly decreased epidermal growth factor receptor (EGFR) levels and mitogen-activated protein kinase activity, and downregulated HB-EGF (heparin-binding EGF-like growth factor) mRNA levels. Meanwhile, TRIM11 overexpression promoted a stem-like phenotype in vitro (tumorsphere formation) and enhanced glial tumor growth in immunocompromised mice. These findings suggest that TRIM11 might be an indicator of glioma malignancy and has an oncogenic function mediated through the EGFR signaling pathway. TRIM11 overexpression potentially leads to a more aggressive glioma phenotype, along with increased malignant tumor growth and poor survival. Taken together, clarification of the biological function of TRIM11 and pathways it affects may provide novel therapeutic strategies for treating malignant glioma patients.

Topics: AC133 Antigen; Adult; Aged; Animals; Antigens, CD; Brain Neoplasms; Cell Differentiation; Cell Movement; Cell Proliferation; Cyclin D1; ErbB Receptors; Female; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Glioblastoma; Glioma; Glycoproteins; Heparin-binding EGF-like Growth Factor; Humans; Intercellular Signaling Peptides and Proteins; Male; Mice; Mice, Inbred BALB C; Middle Aged; Mitogen-Activated Protein Kinases; Nestin; Oncogenes; Peptides; Signal Transduction; Tripartite Motif Proteins; Tumor Cells, Cultured; Ubiquitin-Protein Ligases; Xenograft Model Antitumor Assays

Estradiol (E2) regulates several cellular functions through the interaction with estrogen receptor subtypes, ERα and ERβ, which present different functional and regulation properties. ER subtypes have been identified in human astrocytomas, the most common and aggressive primary brain tumors. We studied the role of ER subtypes in cell growth of two human astrocytoma cell lines derived from tumors of different evolution grades: U373 and D54 (grades III and IV, respectively). E2 significantly increased the number of cells in both lines and the co-administration with an ER antagonist (ICI 182, 780) significantly blocked E2 effects. ERα was the predominant subtype in both cell lines. E2 and ICI 182, 780 down-regulated ERα expression. The number of U373 and D54 cells significantly increased after PPT (ERα agonist) treatment but not after DPN (ERβ agonist) one. To determine the role of SRC-1 and SRC-3 coactivators in ERα induced cell growth, we silenced them with RNA interference. Coactivator silencing blocked the increase in cell number induced by PPT. The content of proteins involved in proliferation and metastasis was also determined after PPT treatment. Western blot analysis showed that in U373 cells the content of PR isoforms (PR-A and PR-B), EGFR, VEGF and cyclin D1 increased after PPT treatment while in D54 cells only the content of EGFR was increased. Our results demonstrate that E2 induces cell growth of human astrocytoma cell lines through ERα and its interaction with SRC-1 and SRC-3 and also suggest differential roles of ERα on cell growth depending on astrocytoma grade.

Topics: Astrocytoma; Brain Neoplasms; Cell Line, Tumor; Cyclin D1; ErbB Receptors; Estradiol; Estrogen Receptor alpha; Humans; Nuclear Receptor Coactivator 1; Nuclear Receptor Coactivator 3; Protein Isoforms; Receptors, Progesterone; Receptors, Vascular Endothelial Growth Factor; RNA Interference

The purposes of our study were to elucidate the role of BRG1 in the development of human glioma and to determine the effect of BRG1 on glioma cell growth, migration and invasion.. Using tissue microarray and immunohistochemistry, we evaluated BRG1 staining in 190 glioma tissues, 8 normal brain tissues and 8 tumor adjacent normal brain tissues. We studied glioma cell proliferative ability with reduced BRG1 expression by siRNA using CCK-8 cell proliferation assay and cell cycle analysis. We studied the role of BRG1 in glioma cell migration and invasion by cell migration assay and matrigel invasion assay. We performed western blot to detect cyclin D1, cyclin B1 and MMP-2 protein expression. We also detected MMP-2 enzyme activity by gelatin zymography.. Our results showed that BRG1 expression was increased in benign tumor and malignant tumor compared with tumor adjacent normal brain tissue (P < 0.01 for both). We did not find any correlation between BRG1 expression and clinicopathological parameters. In addition, we found that knockdown of BRG1 in glioma cell lines inhibits cell growth due to the G1 phase arrest by downregulating cyclin D1. We further demonstrated that silencing of BRG1 in glioma cells inhibited the cell migration and invasion abilities, and downregulation of MMP-2 expression greatly contributed to the reduced cell invasion and migration abilities.. Our data indicated that BRG1 expression is significantly increased in human glioma and it may be involved in the process of glioma cell proliferation, migration and invasion.

Topics: Brain Neoplasms; Cell Cycle; Cell Growth Processes; Cell Line, Tumor; Cell Movement; Cyclin B1; Cyclin D1; DNA Helicases; Female; G1 Phase Cell Cycle Checkpoints; Gene Knockdown Techniques; Gene Silencing; Glioma; Humans; Immunohistochemistry; Male; Matrix Metalloproteinase 2; Middle Aged; Neoplasm Invasiveness; Nuclear Proteins; RNA, Small Interfering; Sincalide; Transcription Factors

Although sEH inhibitors are well studied in inflammatory and cardiovascular diseases, their effects on gliomas are unclear. In this study, we investigated the effects of t-AUCB, a more potent and selective sEH inhibitor, on U251 and U87 human glioblastoma cell lines and the HepG2 human hepatocellular carcinoma cell line. Our results showed that t-AUCB efficiently inhibited sEH activities in all three cell lines (the inhibition rate was more than 80% in each) and suppressed U251 and U87 cell growth in a dose-dependent manner, but exhibited no cell growth inhibition on HepG2. We detected high levels of phosphorylated NF-κB-p65 (Ser536) in t-AUCB-treated U251 and U87 cells, and then found that the NF-κB inhibitor PDTC can completely abolish t-AUCB-induced growth inhibition. This indicated that t-AUCB suppresses U251 and U87 cell growth by activating NF-κB-p65. Moreover, we found that t-AUCB induces cell-cycle G0/G1 phase arrest by regulating Cyclin D1 mRNA and protein levels and CDC2 (Thr161) phosphorylation level. We propose to further test this promising reagent for its anti-glioma activity in clinical relevant orthotopic brain glioma models.

Topics: Apoptosis; Benzoates; Blotting, Western; Brain Neoplasms; Carcinoma, Hepatocellular; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Epoxide Hydrolases; Flow Cytometry; Glioblastoma; Humans; Liver Neoplasms; NF-kappa B; Phosphorylation; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Urea

Mutations in the parkin gene, which encodes a ubiquitin ligase, are a major genetic cause of parkinsonism. Interestingly, parkin also plays a role in cancer as a putative tumor suppressor, and the gene is frequently targeted by deletion and inactivation in human malignant tumors. Here, we investigated a potential tumor suppressor role for parkin in gliomas. We found that parkin expression was dramatically reduced in glioma cells. Restoration of parkin expression promoted G(1) phase cell-cycle arrest and mitigated the proliferation rate of glioma cells in vitro and in vivo. Notably, parkin-expressing glioma cells showed a reduction in levels of cyclin D1, but not cyclin E, and a selective downregulation of Akt serine-473 phosphorylation and VEGF receptor levels. In accordance, cells derived from a parkin-null mouse model exhibited increased levels of cyclin D1, VEGF receptor, and Akt phosphorylation, and divided significantly faster when compared with wild-type cells, with suppression of these changes following parkin reintroduction. Clinically, analysis of parkin pathway activation was predictive for the survival outcome of patients with glioma. Taken together, our study provides mechanistic insight into the tumor suppressor function of parkin in brain tumors and suggests that measurement of parkin pathway activation may be used clinically as a prognostic tool in patients with brain tumor.

Topics: Animals; Brain Neoplasms; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Down-Regulation; Genes, Tumor Suppressor; Glioma; Humans; Mice; Mice, Inbred NOD; Mice, SCID; Prognosis; Ubiquitin-Protein Ligases; Vascular Endothelial Growth Factor Receptor-2

The purpose of this study was to investigate the impact of SAM- and SH3-domain containing 1 (SASH1) on the biological behavior of glioma cells, including its effects on cellular growth, proliferation, apoptosis, invasion, and metastasis, and thereby to provide an experimental basis for future therapeutic treatments. A pcDNA3.1-SASH1 eukaryotic expression vector was constructed and transfected into the U251 human glioma cell line. Using the tetrazolium-based colorimetric (MTT) assay, flow cytometry analyses, transwell invasion chamber experiments, and other methods, we examined the impact of SASH1 on the biological behaviors of U251 cells, including effects on viability, cell cycle, apoptosis, and invasion. Furthermore, the effect of SASH1 on the expression of cyclin D1, caspase-3, matrix metalloproteinase (MMP)-2, MMP-9, and other proteins was observed. Compared to the empty vector and blank control groups, the pcDNA3.1-SASH1 group of U251 cells exhibited significantly reduced cell viability, proliferation, and invasion (p < 0.05), although there was no difference between the empty vector and blank control groups. The pcDNA3.1-SASH1 group demonstrated a significantly higher apoptotic index than did the empty vector and blank control groups (p < 0.05), and the percentage of apoptotic cells was similar between the empty vector and blank control groups. In addition, the pcDNA3.1-SASH1 group expressed significantly lower protein levels of cyclin D1 and MMP-2/9 compared to the control and empty vector groups (p < 0.05) and significantly higher protein levels of caspase-3 than the other two groups (p < 0.05). Cyclin D1, caspase-3, and MMP-2/9 expression was unchanged between the empty vector and blank control groups. SASH1 gene expression might be related to the inhibition of the growth, proliferation, and invasion of U251 cells and the promotion of U251 cells apoptosis.

Topics: Apoptosis; Blotting, Western; Brain Neoplasms; Caspase 3; Cell Cycle; Cell Movement; Cell Proliferation; Cyclin D1; Flow Cytometry; Glioma; Humans; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Neoplasm Invasiveness; Tumor Cells, Cultured; Tumor Suppressor Proteins

Glioma is a common malignant tumor of the human neural system, and Wnt signaling activation is closely connected with glioma malignancy. Pygopus 2 (Pygo2) was recently discovered as a component of the Wnt signaling pathway regulating β-catenin/Tcf dependent transcription. However, the role of Pygo2 in glioma cells has not yet been defined. In the current study, we investigated the role of Pygo2 in rat glioma C6 cells for the first time. Our results showed that over-expression of Pygo2 promoted cell proliferation as well as enhanced cell cycle progression from G₁ to S phase associated with an increase in the expression of the Wnt target gene cyclin D1. In contrast, knockdown of Pygo2 suppressed cell proliferation with cell cycle block from G₁ to S phase and down-regulation of cyclin D1. In addition, the expression of Pygo2 and cyclin D1 in 67 glioma tissue samples was quantified by real-time reverse transcription polymerase chain reaction (RT-PCR) and immunochemistry. The data indicated that tumor grade was significantly associated with over-expression of Pygo2 and cyclin D1. We conclude that Pygo2 is highly expressed in and promotes the growth of glioma cells by an increase in the expression of cyclin D1 to improve G₁/S transition.

Topics: Animals; Blotting, Western; Brain Neoplasms; Cell Cycle; Cell Proliferation; Cyclin D1; Fluorescent Antibody Technique; Gene Expression; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Glioma; Humans; Immunohistochemistry; Intracellular Signaling Peptides and Proteins; Rats; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Wnt Proteins

Tumor cells are able to survive and proliferate in spite of their increased oxidative stress. This was taken as a hint for the implication of oxidants/antioxidants in the proliferation of glial-tumor cells. In the present study, an anti-proliferative effect of Naringenin, an antioxidant against cerebrally implanted C6 glioma cells in rats has been investigated. The status of lipid peroxidation/antioxidants, expressions of protein kinase C, nuclear factor κB, cyclin D1, cyclin dependent kinase 4, proliferating cell nuclear antigen, vascular endothelial growth factor, argyophillic nucleolar organizing regions and histopathology of brain tissues of control and experimental rats were analyzed. On supplementation of naringenin (50mg/kg BW for 30 days) to glioma induced rats, there was a reduction in lipid peroxidation with an increased antioxidant status. There was a significant decrease in the expressions of protein kinase C, nuclear factor κB, cyclin D1 and cyclin dependent kinase 4 on naringenin treatment. Further, the drug could modulate the glial-tumor cell proliferation as evidenced from the histopathological findings, argyophillic nucleolar organizing regions staining, proliferating cell nuclear antigen and vascular endothelial growth factor immunostaining. The findings suggest that naringenin could underlie the inhibition of glial tumor cell proliferation in C6 glioma models of rat.

Topics: Animals; Antioxidants; Ascorbic Acid; Brain Neoplasms; Cell Growth Processes; Cell Line, Tumor; Cyclin D1; Cyclin-Dependent Kinase 4; Flavanones; Glioma; Glutathione; Glutathione Peroxidase; Immunohistochemistry; Male; NF-kappa B; Proliferating Cell Nuclear Antigen; Protein Kinase C; Rats; Receptors, Peptide; Thiobarbituric Acid Reactive Substances; Vascular Endothelial Growth Factor A; Vitamin E

It is necessary to understand mechanisms by which differentiating agents influence tumor-initiating cancer stem cells. Toward this end, we investigated the cellular and molecular responses of glioblastoma stem-like cells (GBM-SCs) to all-trans retinoic acid (RA). GBM-SCs were grown as non-adherent neurospheres in growth factor supplemented serum-free medium. RA treatment rapidly induced morphology changes, induced growth arrest at G1/G0 to S transition, decreased cyclin D1 expression and increased p27 expression. Immunofluorescence and western blot analysis indicated that RA induced the expression of lineage-specific differentiation markers Tuj1 and GFAP and reduced the expression of neural stem cell markers such as CD133, Msi-1, nestin and Sox-2. RA treatment dramatically decreased neurosphere-forming capacity, inhibited the ability of neurospheres to form colonies in soft agar and inhibited their capacity to propagate subcutaneous and intracranial xenografts. Expression microarray analysis identified ∼350 genes that were altered within 48 h of RA treatment. Affected pathways included retinoid signaling and metabolism, cell-cycle regulation, lineage determination, cell adhesion, cell-matrix interaction and cytoskeleton remodeling. Notch signaling was the most prominent of these RA-responsive pathways. Notch pathway downregulation was confirmed based on the downregulation of HES and HEY family members. Constitutive activation of Notch signaling with the Notch intracellular domain rescued GBM neurospheres from the RA-induced differentiation and stem cell depletion. Our findings identify mechanisms by which RA targets GBM-derived stem-like tumor-initiating cells and novel targets applicable to differentiation therapies for glioblastoma.

Topics: AC133 Antigen; Animals; Antigens, CD; Antineoplastic Agents; Brain Neoplasms; Cell Differentiation; Cell Line, Tumor; Cyclin D1; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Glial Fibrillary Acidic Protein; Glioblastoma; Glycoproteins; Humans; Intermediate Filament Proteins; Mice; Mice, Nude; Neoplastic Stem Cells; Nerve Tissue Proteins; Nestin; Peptides; Proliferating Cell Nuclear Antigen; Receptors, Notch; RNA-Binding Proteins; SOXB1 Transcription Factors; Tretinoin; Tubulin

Glioblastoma Multiforme (GBM), the most common and lethal primary human brain tumor, exhibits multiple molecular aberrations. We report that loss of the transcription factor GATA4, a negative regulator of normal astrocyte proliferation, is a driver in glioma formation and fulfills the hallmarks of a tumor suppressor gene (TSG). Although GATA4 was expressed in normal brain, loss of GATA4 was observed in 94/163 GBM operative samples and was a negative survival prognostic marker. GATA4 loss occurred through promoter hypermethylation or novel somatic mutations. Loss of GATA4 in normal human astrocytes promoted high-grade astrocytoma formation, in cooperation with other relevant genetic alterations such as activated Ras or loss of TP53. Loss of GATA4 with activated Ras in normal astrocytes promoted a progenitor-like phenotype, formation of neurospheres, and the ability to differentiate into astrocytes, neurons, and oligodendrocytes. Re-expression of GATA4 in human GBM cell lines, primary cultures, and brain tumor-initiating cells suppressed tumor growth in vitro and in vivo through direct activation of the cell cycle inhibitor P21(CIP1), independent of TP53. Re-expression of GATA4 also conferred sensitivity of GBM cells to temozolomide, a DNA alkylating agent currently used in GBM therapy. This sensitivity was independent of MGMT (O-6-methylguanine-DNA-methyltransferase), the DNA repair enzyme which is often implicated in temozolomide resistance. Instead, GATA4 reduced expression of APNG (alkylpurine-DNA-N-glycosylase), a DNA repair enzyme which is poorly characterized in GBM-mediated temozolomide resistance. Identification and validation of GATA4 as a TSG and its downstream targets in GBM may yield promising novel therapeutic strategies.

Topics: Animals; Brain Neoplasms; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Dacarbazine; DNA Methylation; DNA Modification Methylases; DNA Repair Enzymes; GATA4 Transcription Factor; Glioblastoma; Humans; Mice; Promoter Regions, Genetic; Temozolomide; Tumor Suppressor Proteins

Glioblastoma multiforme, a highly aggressive tumor of the central nervous system, has a dismal prognosis that is due in part to its resistance to radio- and chemotherapy. The protein kinase C (PKC) family of serine threonine kinases has been implicated in the formation and proliferation of glioblastoma multiforme. Members of the protein kinase D (PKD) family, which consists of PKD1, -2 and, -3, are prominent downstream targets of PKCs and could play a major role in glioblastoma growth. PKD2 was highly expressed in both low-grade and high-grade human gliomas. The number of PKD2-positive tumor cells increased with glioma grading (P < .001). PKD2 was also expressed in CD133-positive glioblastoma stem cells and various glioblastoma cell lines in which the kinase was found to be constitutively active. Inhibition of PKDs by pharmacological inhibitors resulted in substantial inhibition of glioblastoma proliferation. Furthermore, specific depletion of PKD2 by siRNA resulted in a marked inhibition of anchorage-dependent and -independent proliferation and an accumulation of glioblastoma cells in G0/G1, accompanied by a down-regulation of cyclin D1 expression. In addition, PKD2-depleted glioblastoma cells exhibited substantially reduced tumor formation in vivo on chicken chorioallantoic membranes. These findings identify PKD2 as a novel mediator of glioblastoma cell growth in vitro and in vivo and thereby as a potential therapeutic target for this devastating disease.

Topics: Animals; Apoptosis; Blotting, Western; Brain; Brain Neoplasms; Cell Cycle; Cell Proliferation; Chickens; Chorioallantoic Membrane; Cyclin D1; Glioblastoma; Humans; Immunoenzyme Techniques; RNA, Small Interfering; TRPP Cation Channels

Malignant gliomas are the most common in central nervous system cancer. Genome-wide association study identifies that CDKN2A was a susceptibility loci for glioma. The CDKN2A/cyclin-dependent kinase 4, 6/Retinoblastoma protein (Rb) pathway is thought to play a crucial role in malignant gliomas pathogenesis. We have investigated the expression of CDKN2A for potential correlations with malignant gliomas grade and potential role of CDKN2A on malignant gliomas pathogenesis.. Tumour tissue samples from 61 patients suffering from malignant gliomas were investigated. The expression levels of CDKN2A were detected using immunohistochemical staining and western blot. Overexpression and knockdown of CDKN2A were performed in human glioma cell lines. Subsequently, colony formation, growth curves and CDKN2A-Cyclin-Rb pathway were analyzed.. Here we show that a lower expression of CDKN2A and a higher expression of cyclin D1 in the patients with high-grade malignant gliomas than low-grade gliomas, respectively. Moreover, overexpression of CDKN2A inhibits growth of glioma cell lines by suppression of cyclin D1 gene expression.. Our study suggests that CDKN2A as a malignant gliomas suppressor gene, appears to be useful for predicting behaviour of high-grade malignant gliomas. CDKN2A-Cyclin-Rb pathway plays a key role on malignant gliomas formation and that therapeutic targeting of this pathway may be useful in malignant gliomas treatment.

Topics: Adult; Blotting, Western; Brain Neoplasms; Cell Proliferation; Colony-Forming Units Assay; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; Down-Regulation; Female; Glioma; Humans; Immunoenzyme Techniques; Male; Middle Aged; Prognosis; RNA, Small Interfering

Our previous miRNAs profiling study showed that miR-27b was up-regulated in glioma cells compared with H4 low grade astrocytoma cells. However, the main function of miR-27b in glioma in not known yet. The aim of this study was to investigate the expression and function of miR-27b in the pathogenesis of glioma. Real-time PCR showed that miR-27b was up-regulated in glioma samples and glioma cells. Down-regulation of miR-27b triggered growth inhibition, induced apoptosis and inhibited invasion in glioma cells. Furthermore, TOPflash luciferase activity was decreased significantly, while FOPflash luciferase did not change significantly. In addition, Western blot assay showed that STAT3, c-myc and cyclin D1 were knocked down after treatment with miR-27b inhibitor. These findings suggest that aberrantly up-regulated miR-27b may be one of the critical factors that contribute to malignancy in human gliomas.

Topics: Apoptosis; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; beta Catenin; Brain Neoplasms; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Gene Expression; Gene Knockdown Techniques; Genes, Reporter; Glioma; Humans; Luciferases, Renilla; MicroRNAs; Neoplasm Invasiveness; Proto-Oncogene Proteins c-myc; STAT3 Transcription Factor; Transcription Factor 4; Transcription Factors; Up-Regulation

In our laboratory, a novel therapeutic probe, T11TS, a membrane glycoprotein, was isolated which had antineoplastic activity against experimental glioma. Development of a novel therapeutic strategy with T11TS has unearthed a newer dimension of its mechanism of action: modulation of the cell cycle. In this study, we have presented evidence to support the finding that T11TS induces G1 cell cycle arrest of rat glioma cells. Results of flow cytometric studies showed that the treatment produced a marked increase in the proportion of cells in the G1 phase. Flow cytometry, immunoblotting, immunoprecipitation, and kinase assays were performed for investigating the involvement of G1 cell cycle regulators. T11TS induces downregulation of the cyclin-D (1 and 3) expression with the concurrent upregulation of p21 and p27 and their concomitant association with cyclin-dependent kinase 4, proliferating cell nuclear antigen and cyclin E respectively leading to a decrease in cyclin-dependent kinase 4 kinase activity. A transient rise in retinoblastoma protein level and coordinated binding of retinoblastoma protein with E2F coincided with the accumulation of cells in G1 phase. Thus, our observations have uncovered an antiproliferative pathway for T11TS, causing retardation of glioma cell cycle.

Topics: Animals; Animals, Newborn; Antineoplastic Agents; Brain Neoplasms; Cyclin D; Cyclin D1; Cyclin D3; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Down-Regulation; Erythrocyte Membrane; Ethylnitrosourea; Female; Flow Cytometry; G1 Phase; Glioma; Glycoproteins; Male; Rats; Rats, Inbred Strains; Sheep; Tumor Cells, Cultured; Up-Regulation

Our recent studies have shown that the FoxM1B transcription factor is overexpressed in human glioma tissues and that the level of its expression correlates directly with glioma grade. However, whether FoxM1B plays a role in the early development of glioma (i.e., in transformation) is unknown. In this study, we found that the FoxM1B molecule causes cellular transformation and tumor formation in normal human astrocytes (NHA) immortalized by p53 and pRB inhibition. Moreover, brain tumors that arose from intracranial injection of FoxM1B-expressing immortalized NHAs displayed glioblastoma multiforme (GBM) phenotypes, suggesting that FoxM1B overexpression in immortalized NHAs not only transforms the cells but also leads to GBM formation. Mechanistically, our results showed that overexpression of FoxM1B upregulated NEDD4-1, an E3 ligase that mediates the degradation and downregulation of phosphatase and tensin homologue (PTEN) in multiple cell lines. Decreased PTEN in turn resulted in the hyperactivation of Akt, which led to phosphorylation and cytoplasmic retention of FoxO3a. Blocking Akt activation with phosphoinositide 3-kinase/Akt inhibitors inhibited the FoxM1B-induced transformation of immortalized NHAs. Furthermore, overexpression of FoxM1B in immortalized NHAs increased the expression of survivin, cyclin D1, and cyclin E, which are important molecules for tumor growth. Collectively, these results indicate that overexpression of FoxM1B, in cooperation with p53 and pRB inhibition in NHA cells, promotes astrocyte transformation and GBM formation through multiple mechanisms.

Topics: Animals; Astrocytes; Brain Neoplasms; Cell Line, Tumor; Cell Transformation, Neoplastic; Cyclin D1; Cyclin E; Endosomal Sorting Complexes Required for Transport; Enzyme Activation; Forkhead Box Protein M1; Forkhead Transcription Factors; Glioma; Humans; Inhibitor of Apoptosis Proteins; Mice; Mice, Nude; Microtubule-Associated Proteins; Nedd4 Ubiquitin Protein Ligases; Oncogene Protein v-akt; Phenotype; PTEN Phosphohydrolase; Survivin; Ubiquitin-Protein Ligases

In humans, the targeting protein for Xenopus kinesin-like protein 2 (TPX2) is a cell cycle-associated protein, and altered TPX2 expression has been found in various malignancies. However, the contribution of TPX2 expression to astrocytoma progression is unclear. The aim of this study was to investigate TPX2 expression in human astrocytoma samples and cell lines. TPX2 protein expression was detected in the nucleus of astrocytoma tissues by immunohistochemistry and immunofluorescence staining. Real-time PCR and Western blot analysis showed that the expression levels of TPX2 were higher in high-grade astrocytoma tissues and cell lines than that in low-grade astrocytoma tissues and normal cell lines. Immunohistochemical analysis of tumor tissues from 52 patients with astrocytoma showed that TPX2 over-expression was significantly associated with decreased patient survival. In addition, down-regulation of the TPX2 gene by RNA interference inhibited proliferation of U87 cells. TPX2 gene silencing also increased early-stage apoptosis in U87 cells. Western blotting and real-time PCR showed changes in the protein and mRNA expression of Aurora A, Ran, p53, c-Myc and cyclin B1 in U87 cells that had been transfected with pSUPER/TPX2/siRNA. These data suggest that TPX2 expression is associated with the progression of malignant astrocytoma.

Topics: Apoptosis; Astrocytoma; Aurora Kinases; Brain; Brain Neoplasms; Cell Cycle Proteins; Cell Division; Cell Line, Tumor; Cyclin B1; Cyclin D1; Disease Progression; Down-Regulation; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Microtubule-Associated Proteins; Nuclear Proteins; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-myc; Survival Rate; Tumor Suppressor Protein p53

The retinoblastoma (RB) tumor suppressor pathway is likely important in primitive neuroectodermal tumors (PNET) of the brain. In fact, 10% to 15% of children born with RB mutations develop brain PNETs, commonly in the pineal gland. Cyclin D1, which in association with cyclin-dependent kinase (Cdk) 4 and Cdk6 phosphorylates and inactivates the RB protein, is expressed in 40% of sporadic medulloblastoma, a PNET of the cerebellum. To understand tumorigenic events cooperating with RB pathway disruption in brain PNET, we generated a transgenic mouse where cyclin D1 was expressed in pineal cells. Cyclin D1 enhanced pinealocyte proliferation, causing pineal gland enlargement. However, proliferation ceased beyond 2 weeks of age with reversal of Cdk4-mediated Rb phosphorylation despite continued expression of the transgene, and the pineal cells showed heterochromatin foci suggestive of a senescent-like state. In the absence of the p53 tumor suppressor, cell proliferation continued, resulting in pineal PNET that limited mouse survival to approximately 4 months. Interestingly, the Cdk inhibitor p18(Ink4c) was induced in the transgenic pineal glands independently of p53, and transgenic mice that lacked Ink4c developed invasive PNET, although at an older age than those lacking p53. Analogous to our mouse model, we found that children with heritable RB often had asymptomatic pineal gland enlargement that only rarely progressed to PNET. Our finding that the Cdk4 inhibitor p18(Ink4c) is a tumor suppressor in cyclin D1-driven PNET suggests that pharmacologic interventions to inhibit Cdk4 activity may be a useful chemoprevention or therapeutic strategy in cancer driven by primary RB pathway disruption.

Topics: Animals; Brain Neoplasms; Cell Growth Processes; Child, Preschool; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinase Inhibitor p18; Cyclin-Dependent Kinase Inhibitor p21; Disease Progression; Eye Proteins; Genes, bcl-1; Genes, p53; Humans; Hyperplasia; Infant; Mice; Mice, Inbred C57BL; Mice, Transgenic; Neuroectodermal Tumors, Primitive; Phosphorylation; Pineal Gland; Retinoblastoma; Retinoblastoma Protein; Retinol-Binding Proteins

Increased protein synthesis is regulated, in part, by two eukaryotic translation initiation factors (eIFs): eIF4E and eIF2alpha. One or both of these factors are often overexpressed in several types of cancer cells; however, no data are available at present regarding eIF4E and eIF2alpha levels in brain tumors. In this study, we analyzed the expression, subcellular localization and phosphorylation states of eIF4E and eIF2alpha in 64 brain tumors (26 meningiomas, 16 oligodendroglial tumors, and 22 astrocytomas) and investigated the correlation with the expression of MIB-1, p53, and cyclin D1 proteins as well. There are significant differences in the phosphorylated eIF4E levels between the tumors studied, being the highest in meningiomas and the lowest in the oligodendroglial tumors. Relative to subcellular localization, eIF4E is frequently found in the nucleus of the oligodendroglial tumors and rarely in the same compartment of the meningiomas, whereas eIF2alpha showed an inverse pattern. Finally, cyclin D1 levels directly correlate with the phosphorylation status of both factors. The different expression, phosphorylation, or/and subcellular distribution of eIF2alpha and eIF4E within the brain types of tumors studied could indicate that different pathways are activated for promoting cell cycle proliferation, for instance, leading to increased cyclin D1 expression.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Brain Neoplasms; Cell Proliferation; Cyclin D1; Eukaryotic Initiation Factor-2; Eukaryotic Initiation Factor-4E; Female; Humans; Ki-67 Antigen; Male; Middle Aged; Phosphorylation; Tumor Suppressor Protein p53; Young Adult

Astrocytic tumors are the most common brain tumors with various genetic defects. As a tumor suppressor gene, Axin could control cell death and growth. Axin possesses a separate domain that directly interacts with p53 and regulates the activity of p53 pathway. Our aims were to elucidate the effects of Axin on the progression of astrocytoma. We examined the expression of Axin in 96 cases of astrocytoma using immunohistochemistry. The apoptotic index, proliferactive acitivity and the expression levels of p53 and its downstream genes, p21 and Cyclin D1, were evaluated in the C6 astrocytoma cells with overexpression or silencing of Axin. The results showed the levels of Axin correlated significantly inversely with the grades of astrocytoma (R=-0.286, P<0.05) and correlated negatively with Ki-67 labeling index (R=-0.227, P<0.05). Overexpression of Axin in C6 cells induces cell death, and reduces the cell proliferation, up-regulates the expression of p53. Silencing of Axin reduces p53 expression. The p53 inhibitor, pifithrin-alpha, was able to counteract the effect of Axin in C6 cells. Our data demonstrate that the expression of Axin is associated negatively with the progression of astrocytoma. In conclusion, Axin induces cell death and reduces cell proliferation, partially by activating the p53 pathway in astrocytoma cells. This knowledge is helpful in understanding the role of Axin in the progression of astrocytoma.

Topics: Adolescent; Adult; Aged; Animals; Apoptosis; Astrocytoma; Axin Protein; Benzothiazoles; Brain Neoplasms; Cell Line, Tumor; Cell Proliferation; Child; Child, Preschool; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Female; Glioblastoma; Humans; Immunohistochemistry; Male; Middle Aged; Neoplasm Staging; Rats; Repressor Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; Time Factors; Toluene; Transfection; Tumor Suppressor Protein p53; Young Adult

Gliomas are the most common adult primary brain tumors, and the most malignant form, glioblastoma multiforme, is invariably fatal. The phosphatidylinositol 3-kinase (PI3K)-Akt signaling pathway is altered in most glioblastoma multiforme. PTEN, an important negative regulator of the PI3K-Akt pathway, is also commonly mutated in glioma, leading to constitutive activation of Akt. One ultimate consequence is phosphorylation and inactivation of FOXO forkhead transcription factors that regulate genes involved in apoptosis, cell cycle arrest, nutrient availability, DNA repair, stress, and angiogenesis. We tested the ability of a mutant FOXO1 factor that is not subject to Akt phosphorylation to overcome dysregulated PI3K-Akt signaling in two PTEN-null glioma cell lines, U87 and U251. Adenovirus-mediated gene transfer of the mutant FOXO1 successfully restored cell cycle arrest and induced cell death in vitro and prolonged survival in vivo in xenograft models of human glioma (33% survival at 1 year of animals bearing U251 tumors). However, U87 were much more resistant than U251 to mutant FOXO1-induced death, showing evidence of increased nuclear export and Akt-independent phosphorylation of FOXO1 at S249. A cyclin-dependent kinase 2 inhibitor decreased phosphorylation of S249 and rendered U87 cells significantly more susceptible to mutant FOXO1-induced death. Our results indicate that targeting FOXO1, which is at the convergence point of several growth factor receptor tyrosine kinase pathways, can effectively induce glioma cell death and inhibit tumor growth. They also highlight the importance of Akt-independent phosphorylation events in the nuclear export of FOXO1.

Topics: Adenoviridae; Animals; Brain Neoplasms; Cell Cycle; Cell Line, Tumor; Cyclin D1; Cyclin D2; Cyclin-Dependent Kinase 2; Cyclins; Disease Models, Animal; DNA Repair; Forkhead Box Protein O1; Forkhead Transcription Factors; Gene Expression Regulation, Neoplastic; Glioma; Humans; Mice; Neovascularization, Pathologic; Phosphorylation

Glioblastomas, the most aggressive primary brain tumors, occur almost exclusively in adult patients. Neural precursor cells (NPCs) are antitumorigenic in mice, as they can migrate to glioblastomas and induce tumor cell death. Here, we show that the antitumor effect of NPCs is age-dependently controlled by cell proliferation in the subventricular zone (SVZ) and that NPCs accumulating at a glioblastoma are diverted from their normal migratory path to the olfactory bulb. Experimentally induced cortical glioblastomas resulted in decreased subventricular proliferation in adult (postnatal day 90) but not in young (postnatal day 30) mice. Adult mice supplied fewer NPCs to glioblastomas and had larger tumors than young mice. Apart from the difference in proliferation, there was neither a change in cell number and death rate in the SVZ nor a change in angiogenesis and immune cell density in the tumors. The ability to kill glioblastomas was similar in NPCs isolated from young and adult mice. The proliferative response of NPCs to glioblastomas depended on the expression of D-type cyclins. In young mice, NPCs express the cyclins D1 and D2, but the expression of cyclin D1 is lost during aging, and in adult NPCs only cyclin D2 remains. In young and adult cyclin D2-deficient mice we observed a reduced supply of NPCs to glioblastomas and the generation of larger tumors compared with wild-type mice. We conclude that cyclin D1 and D2 are nonredundant for the antitumor response of subventricular NPCs. Loss of a single D-type cyclin results in a smaller pool of proliferating NPCs, lower number of NPCs migrating to the tumor, and reduced antitumor activity. Disclosure of potential conflicts of interest is found at the end of this article.

Topics: Age Factors; Animals; Brain Neoplasms; Cell Proliferation; Cells, Cultured; Cyclin D1; Cyclin D2; Cyclins; Glioblastoma; Mice; Mice, Inbred C57BL; Neurons; Stem Cell Transplantation; Stem Cells

Deregulated signaling through the epidermal growth factor receptor (EGFR) is involved in chemoresistance. To identify the molecular determinants of sensitivity to the EGFR inhibitor gefitinib (Iressa, ZD1839) in chemoresistance, we compared the response of matched chemosensitive and chemoresistant glioma and ovarian cancer cell lines. We found that chemoresistant cell lines were 2- to 3-fold more sensitive to gefitinib growth-inhibitory effects, because of decreased proliferation rather than survival. Sensitivity to gefitinib correlated with overexpression and constitutive phosphorylation of HER2 and HER3, but not EGFR, altered HER ligand expression, and enhanced activation of EGF-triggered EGFR pathway. No activating mutations were found in EGFR. Gefitinib fully inhibited EGF-induced and constitutive Akt activation only in chemoresistant cells. In parallel, gefitinib downregulated constitutively phosphorylated HER2 and HER3, and activated GSK3beta with a concomitant degradation of cyclin D1. Ectopically overexpressed HER2 on its own was insufficient to sensitize chemonaive cells to gefitinib. pHER3 coimmunoprecipitated with p85-PI3K in chemoresistant cells and gefitinib dissociated these complexes. siRNA-mediated inhibition of HER3 decreased constitutive activation of Akt and sensitivity to gefitinib in chemoresistant cells. Our study indicates that in chemoresistant cells gefitinib inhibits both an enhanced EGF-triggered pathway and a constitutive HER3-mediated Akt activation, indicating that inhibition of HER3 together with that of EGFR could be relevant in chemorefractory tumors. Furthermore, in combination experiments gefitinib enhanced the effects of coadministered drugs more in chemoresistant than chemosensitive ovarian cancer cells. Combined treatment might be therapeutically beneficial in chemoresistant tumors from ovary and likely from other tissues.

Topics: Antineoplastic Agents; Apoptosis; Brain Neoplasms; Cell Cycle; Cell Line, Tumor; Cisplatin; Cyclin D1; DNA, Complementary; Down-Regulation; Drug Resistance, Neoplasm; ErbB Receptors; Female; Gefitinib; Gene Expression Regulation, Neoplastic; Glioma; Humans; Immunoblotting; Immunoprecipitation; Neoplasms; Ovarian Neoplasms; Phosphorylation; Polymerase Chain Reaction; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Quinazolines; Receptor, ErbB-2; Receptor, ErbB-3; Repressor Proteins; RNA, Small Interfering; Sequence Analysis, DNA; Signal Transduction

Inappropriate Hedgehog (Hh) signaling underlies development of a subset of medulloblastomas, and tumors with elevated HH signaling activity express the stem cell self-renewal gene BMI1. To test whether Bmi1 is required for Hh-driven medulloblastoma development, we varied Bmi1 gene dosage in transgenic mice expressing an oncogenic Hh effector, SmoA1, driven by a glial fibrillary acidic protein (GFAP) promoter. Whereas 100% of SmoA1; Bmi1(+/+) or SmoA1;Bmi1(+/-) mice examined between postnatal (P) days 14 and 26 had typical medulloblastomas (N = 29), tumors were not detected in any of the SmoA1;Bmi1(-/-) animals examined (N = 6). Instead, small ectopic collections of cells were present in the region of greatest tumor load in SmoA1 animals, suggesting that medulloblastomas were initiated but failed to undergo expansion into frank tumors. Cells within these Bmi1(-/-) lesions expressed SmoA1 but were largely nonproliferative, in contrast to cells in Bmi1(+/+) tumors (6.2% vs 81.9% PCNA-positive, respectively). Ectopic cells were negative for the progenitor marker nestin, strongly GFAP-positive, and highly apoptotic, relative to Bmi1(+/+) tumor cells (29.6% vs 6.3% TUNEL-positive). The alterations in proliferation and apoptosis in SmoA1;Bmi1(-/-) ectopic cells are associated with reduced levels of Cyclin D1 and elevated expression of cyclin-dependent kinase inhibitor p19(Arf), two inversely regulated downstream targets of Bmi1. These data provide the first demonstration that Bmi1 is required for spontaneous de novo development of a solid tumor arising in the brain, suggest a crucial role for Bmi1-dependent, nestin-expressing progenitor cells in medulloblastoma expansion, and implicate Bmi1 as a key factor required for Hh pathway-driven tumorigenesis.

Topics: Animals; Brain Neoplasms; Cell Proliferation; Cerebellum; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; Gene Expression Regulation, Neoplastic; Genotype; Glial Fibrillary Acidic Protein; Hedgehog Proteins; Medulloblastoma; Mice; Mice, Transgenic; Nerve Tissue Proteins; Nuclear Proteins; Polycomb Repressive Complex 1; Promoter Regions, Genetic; Proto-Oncogene Proteins; Repressor Proteins

The human glioma cell lines, U87 and T98G, were evaluated for their ability to survive and form colonies in an acidic environment of pH(ext) 6.0. In contrast to U87, which showed an 80-90% survival rate, only 40% of T98G cells survived 6 days at pH(ext) 6.0 and lost their colony forming ability when returned to a normocidic environment. Although both U87 and T98G cells maintain an intracellular pH (pH(i)) of 7.0 at pH(ext) 6.0 and arrest mostly in G1 phase of the cell cycle, only T98G demonstrated a major loss of cyclin D1 that was prevented by the proteasome inhibitor MG132. Colony forming ability was restored by stably transfecting T98G cells with a cyclin D1-expressing plasmid. Both U87 and T98G cells demonstrated increased cytoplasmic localization of cyclin D1 during exposure at pH(ext) 6.0. Upon prolonged (24 h) incubation at pH(ext) 6.0, nuclear cyclin D1 was nearly absent in T98G in contrast to U87 cells. Thus, an acidic environment triggers cytoplasmic localization and proteasomal degradation of cyclin D1.

Topics: Acids; Active Transport, Cell Nucleus; Brain Neoplasms; Cell Line, Tumor; Cell Nucleus; Cell Proliferation; Cell Survival; Cyclin D1; Cytoplasm; Down-Regulation; Enzyme Inhibitors; Extracellular Space; Glioma; Humans; Hydrogen-Ion Concentration; Proteasome Endopeptidase Complex; Proteasome Inhibitors; RNA, Small Interfering; Tumor Stem Cell Assay

Gliomas are the most common type of primary tumor in the human central nervous system. STAT3, a signal transducer and activator of transcription 3, is over expressed in gliomas. Its involvement in tumorgenesis can be attributed to its ability to induce cell proliferation and inhibit apoptosis. Double-stranded decoy oligodeoxynucleotides (ODNs) which correspond closely to the STAT3 response element within the c-fos promoter are a potential tool for inhibiting a variety of tumor cell growth. To investigate its therapeutic potential in malignant gliomas, a 15-mer double-stranded decoy ODN mimicking STAT3-specific cis-elements was transfected into two glioma cell lines, U251 and A172. The STAT3 decoy ODN treatment specifically blocked STAT3 signaling and subsequently inhibited U251 and A172 cell proliferation by inducing apoptosis and cell-cycle arrest. The ODN treatment also decreased transcription and translation of downstream STAT3 target genes including c-myc, cyclin D1 and bcl-xl in both cell lines. Thus, targeted blockade of the STAT3 signaling pathway with a decoy ODN is a potential anti-glioma therapeutic approach.

Topics: Antineoplastic Agents; Apoptosis; bcl-X Protein; Brain Neoplasms; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Gene Expression Regulation, Neoplastic; Genes, cdc; Glioma; Growth Inhibitors; Humans; Oligonucleotides; Proto-Oncogene Proteins c-myc; Signal Transduction; STAT3 Transcription Factor; Transfection

Tuberous sclerosis (TS), autosomal dominant disorder manifested by the formation of usually benign tumors in the brain, heart, kidneys and skin, results from an inactivating mutation in one of two tumor suppressor genes TSC1 or TSC2. Protein products of these genes, hamartin and tuberin, respectively, have been shown to participate in the mTOR pathway controlling translation of approx. 10-15% of all proteins. In the current paper, we aimed at verifying whether hamartin and tuberin may also be implicated in the control of gene transcription. Very recently it has been hypothesized that the pathway triggered by WNT, one of embryonic growth factors involved in cell differentiation and migration, could be disturbed in TS. In order to test this hypothesis we evaluated samples of four subependymal giant cell astrocytomas (SEGAs), brain tumors developing in the progress of TS. We found that beta-catenin, transcription factor and mediator of WNT pathway activity is indeed present and active in SEGAs. mRNA transcripts for c-Myc and N-Myc, proteins whose transcription is regulated by beta-catenin, were upregulated in two of four SEGAs, while cyclin D1 mRNA was significantly higher in three SEGAs. At the same time, c-Myc and N-Myc proteins were detected in the same two samples. Thus, we show for the first time that aberrant WNT signaling may contribute to the pathogenesis of TS-associated SEGAs.

Topics: Astrocytoma; beta Catenin; Blotting, Western; Brain Neoplasms; Cell Nucleus; Cyclin D1; Genes, myc; Humans; Immunohistochemistry; Nerve Tissue Proteins; Phosphorylation; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Transcription, Genetic; Translocation, Genetic; Tuberous Sclerosis; Up-Regulation; Wnt Proteins

Forty-four evaluable patients with intracranial meningiomas were assessed for the expression of the cell-cycle regulator cyclin D1 and of proteins involved in proliferation and apoptosis such as PCNA, MIB-1, p53 and bcl-2. Analyses were carried out by western blot and immunohistochemistry after immediate processing of fresh tumor specimens. By western blot, expression of cyclin D1 significantly correlated with p53 (p=0.02) and with proliferative activity, as assessed by PCNA expression (p=0.0009). By immunohistochemistry, a significant relationship between cyclin D1 and the proliferation marker MIB-1 was confirmed (p=0.05), whereas significance with bcl-2 expression was not found (p=0.01). Moreover, although the association with tumor grade appeared of borderline statistical significance (p=0.07), all the grade II/III meningiomas showed increased expression of cyclin D1 and high proliferative activity. In conclusion, data from this preliminary study seem to suggest a potential value of the combined expression of cyclin D1 and proliferation indicators in defining subgroups of meningiomas with a more aggressive biological behavior.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Brain Neoplasms; Cell Proliferation; Cyclin D1; Female; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Ki-67 Antigen; Male; Meningioma; Middle Aged; Proliferating Cell Nuclear Antigen; Proto-Oncogene Proteins c-bcl-2; Severity of Illness Index; Tumor Suppressor Protein p53

Because fibroblast growth factor (FGF) causes the intracellular accumulation of activated signal transducer and activator of transcription 3 (STAT3), we assessed whether dobesilate, a synthetic FGF inhibitor that has been reported to show antiproliferative and proapoptotic activities in glioma cell cultures, down-regulates the STAT3 signaling pathway in growing cultures of those cells. Because STAT3 signaling pathway plays pleiotropic roles in tumor proliferation, maintenance of STAT3 in its inactive state may prevent glioma growth and spreading.. Rat glioma C6 cells were treated with dobesilate and cultures were evaluated immunocytochemically for STAT3 activation and enhancement of the expression rate of cyclin D1 and bcl-XL.. Dobesilate abrogates the accumulation of activated STAT3 in glioma cells. The decrease in the intracellular levels of activated STAT3 by the dobesilate treatment runs parallel with a significant attenuation of cyclin D1 and bcl-XL expression.. Treatment with inhibitors of FGF down-regulates the STAT3 signaling pathway. These alterations could be correlated to the already observed inhibition of cell proliferation and promotion of apoptosis in glioma cell cultures by dobesilate. The reported results may open new avenues for developing new treatments against these tumors.

Topics: Animals; Apoptosis; bcl-X Protein; Brain Neoplasms; Calcium Dobesilate; Cell Division; Cell Line, Tumor; Cell Proliferation; Cell Transformation, Neoplastic; Cyclin D1; Down-Regulation; Fibroblast Growth Factors; Gene Expression Regulation, Neoplastic; Glioma; Growth Inhibitors; Hemostatics; Rats; Signal Transduction; STAT3 Transcription Factor; Transcriptional Activation

The transcription factor Forkhead box M1 (FoxM1) is overexpressed in malignant glioma. However, the functional importance of this factor in human glioma is not known. In the present study, we found that FoxM1B was the predominant FoxM1 isoform expressed in human glioma but not in normal brain tissue. The level of FoxM1 protein expression in human glioma tissues was directly correlated with the glioma grade. The level of FoxM1 protein expression in human glioblastoma tissues was inversely correlated with patient survival. Enforced FoxM1B expression caused SW1783 and Hs683 glioma cells, which do not form tumor xenografts, to regain tumorigenicity in nude mouse model systems. Moreover, gliomas that arose from FoxM1B-transfected anaplastic astrocytoma SW1783 cells displayed glioblastoma multiforme phenotypes. Inhibition of FoxM1 expression in glioblastoma U-87MG cells suppressed their anchorage-independent growth in vitro and tumorigenicity in vivo. Furthermore, we found that FoxM1 regulates the expression of Skp2 protein, which is known to promote degradation of the cell cycle regulator p27(Kip1). These results showed that FoxM1 is overexpressed in human glioblastomas and contributes to glioma tumorigenicity. Therefore, FoxM1 might be a new potential target of therapy for human malignant gliomas.

Topics: Animals; Astrocytoma; Brain Neoplasms; Cell Line, Tumor; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p27; Forkhead Box Protein M1; Forkhead Transcription Factors; Glioblastoma; Humans; Mice; Mice, Nude; Protein Isoforms; RNA, Messenger; S-Phase Kinase-Associated Proteins; Transplantation, Heterologous

U87MG human glioma cells in cultures expressed metabotropic glutamate (mGlu) receptors mGlu2 and mGlu3. Addition of the mGlu2/3 receptor antagonist LY341495 to the cultures reduced cell growth, expression of cyclin D1/2, and activation of the MAP kinase and phosphatidylinositol-3-kinase pathways. This is in line with the evidence that activation of mGlu2/3 receptors sustains glioma cell proliferation. U87MG cells were either implanted under the skin (1x10(6) cells/0.5 ml) or infused into the caudate nucleus (0.5x10(6) cells/5 microl) of nude mice. Animals were treated for 28 days with mGlu receptor antagonists by means of subcutaneous osmotic minipumps. Treatments with LY341495 or (2S)-alpha-ethylglutamate (both infused at a rate of 1 mg/kg per day) reduced the size of tumors growing under the skin. Infusion of LY341495 (10 mg/kg per day) also reduced the growth of brain tumors, as assessed by magnetic resonance imaging analysis carried out every seven days. The effect of drug treatment was particularly evident during the exponential phase of tumor growth, that is, between the third and the fourth week following cell implantation. Immunohistochemical analysis showed that U87MG cells retained the expression of mGlu2/3 receptors when implanted into the brain of nude mice. These data suggest that mGlu2/3 receptor antagonists are of potential use in the experimental treatment of malignant gliomas.

Topics: Amino Acids; Animals; Blotting, Western; Brain Neoplasms; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin D2; Cyclins; Enzyme Activation; Excitatory Amino Acid Antagonists; Glioma; Humans; Immunohistochemistry; Ki-67 Antigen; Magnetic Resonance Imaging; MAP Kinase Signaling System; Mice; Mice, Nude; Phosphatidylinositol 3-Kinases; Receptors, Metabotropic Glutamate; Reverse Transcriptase Polymerase Chain Reaction; Xanthenes

An immunohistochemical method for assessing cell cycle phase distribution in neurosurgical biopsies would enable such data to be incorporated into diagnostic algorithms for the estimation of prognosis and response to adjuvant chemotherapy in glial neoplasms, without the requirement for flow cytometric analysis. Paraffin-embedded sections of intracerebral gliomas (n = 48), consisting of diffuse astrocytoma (n = 9), anaplastic astrocytoma (n = 8) and glioblastoma (n = 31), were analysed by immunohistochemistry using markers of cell cycle entry, Mcm-2 and Ki67, and putative markers of cell cycle phase, cyclins D1 (G1-phase), cyclin A (S-phase), cyclin B1 (G2-phase) and phosphohistone H3 (Mitosis). Double labelling confocal microscopy confirmed that the phase markers were infrequently coexpressed. Cell cycle estimations by immunohistochemistry were corroborated by flow cytometric analysis. There was a significant increase in Mcm-2 (P < 0.0001), Ki67 (P < 0.0001), cyclin A (P < 0.0001) and cyclin B1 (P = 0.002) expression with increasing grade from diffuse astrocytoma through anaplastic astrocytoma to glioblastoma, suggesting that any of these four markers has potential as a marker of tumour grade. In a subset of glioblastomas (n = 16) for which accurate clinical follow-up data were available, there was a suggestion that the cyclin A:Mcm-2 labelling fraction might predict a relatively favourable response to radical radiotherapy. These provisional findings, however, require confirmation by a larger study. We conclude that it is feasible to obtain detailed cell cycle data by immunohistochemical analysis of tissue biopsies. Such information may facilitate tumour grading and may enable information of prognostic value to be obtained in the routine diagnostic laboratory.

Topics: Adult; Astrocytoma; Biomarkers, Tumor; Brain Neoplasms; Cell Cycle; Cell Cycle Proteins; Cyclin A; Cyclin B; Cyclin B1; Cyclin D1; Flow Cytometry; Histones; Humans; Immunohistochemistry; Microscopy, Confocal; Prognosis; Reproducibility of Results

Overexpression of cyclin D1 was shown in many tumors. The excessive expression of cyclin D1 is an important cause of many tumors. But there are still some controversies of whether the overexpression of cyclin D1 exists in brain gliomas. This study was to determine the expression level of cyclin D1 in glioma tissues of human brain, and to analyze the relationship of cyclin D1 with the malignancy and prognosis of gliomas.. The expression levels of cyclin D1 in 84 specimens were determined by SP immunohistochemical assay. The correlation of expression intensity of cyclin D1, positive cell ratio of glioma tissues with the tumors malignancy, and the prognosis of the patients was analyzed.. (1) The average percentages of cyclin D1 positive cells were (9.82+/-9.75)% and (27.45+/-21.03)% in the low grade gliomas and the high grade gliomas, respectively. There was significant difference between two groups (P< 0.01). (2) The cyclin D1 positive ratios were 31.25% (10/32) and 61.53% (32/52) in the low grade gliomas and the high grade gliomas, respectively. There was significant difference between two groups (P< 0.01). (3) The cyclin D1 positive ratios were 76.19% (16/21) and 24.00% (6/25) in recurrence group and non-recurrence group,respectively. There was significant difference between two groups (P< 0.01). (4) The cyclin D1 positive ratios were 66.67% (14/21) and 32.00%(8/25) in dead group and survival group, respectively. There was significant difference between two groups (P< 0.05). In dead group, the cyclin D1 positive ratios were 86.66%(13/15) and 16.66%(1/6) in the high grade gliomas and the low grade gliomas, respectively. There was significant difference between two groups (P< 0.05).. (1) The expression of cyclin D1 increased with the increased grade of glioma. (2) The higher cyclin D1 expressed, the worse prognosis the patients had. (3) The expression of cyclin D1 can act as a biological marker in evaluating malignancy of gliomas and prognosis of patients.

Topics: Adolescent; Adult; Aged; Brain Neoplasms; Child; Cyclin D1; Female; Glioma; Humans; Male; Middle Aged; Prognosis

Penta-acetyl geniposide, (Ac)5-GP, the acetylated compound of geniposide, is able to inhibit the growth of rat C6 glioma cells in culture and in the bearing rats. Our recent data indicated that the induction of cell apoptosis and cell cycle arrest at G0/gap phase 1 (G1) by (Ac)5-GP might be associated with the induction of p53 and c-Myc, and mediated via the apoptosis-related bcl-2 family proteins. In this report, we further investigated the mechanism involved in the cell cycle arrest induced by (Ac)5-GP in C6 glioma cells. The inhibitory effect of (Ac)5-GP on the cell cycle progression of C6 glioma cells which arrested cells at the G0/G1 phase was associated with a marked decrease in the protein expression of cyclin D1, and an induction in the content of cyclin-dependent kinase (cdk) inhibitor p21 protein. This effect was correlated with the elevation in p53 levels. Further immunoprecipitation studies found that, in response to the treatment, the formation of cyclin D1/cdk 4 complex declined, preventing the phosphorylation of retinoblastoma (Rb) and the subsequent dissociation of Rb/E2F complex. These results illustrated that the apoptotic effect of (Ac)5-GP, arresting cells at the G0/G1 phase, was exerted by inducing the expression of p21 that, in turn, repressed the activity of cyclin D1/cdk 4 and the phosphorylation of Rb.

Topics: Animals; Antineoplastic Agents; Brain Neoplasms; Cell Cycle; Cell Line, Tumor; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinases; Cyclins; Drugs, Chinese Herbal; Glioma; Glucosides; Iridoid Glucosides; Iridoids; Phosphorylation; Plant Extracts; Pyrans; Rats; Retinoblastoma Protein; Tumor Suppressor Protein p53

This study was designed to investigate immunoexpression of cyclin A and D1, and topoisomerase IIalpha in oligodendrogliomas and to evaluate the correlation with MIB-1 (Ki67), tumour grade, and survival of the patients.. Forty cases of oligodendrogliomas (20 high- and 20 low-grade) were studied immunohistochemically with the above-mentioned monoclonal antibodies.. Normal brain tissues included in tumour sections did not express any of cyclin A, MIB-1 and topoisomerase IIalpha except cyclin D1, which was shown in perineuronal and interfascicular normal oligodendroglial cells. In low-grade and high-grade oligodendrogliomas, the mean cyclin A labelling index (LI) was 1.18 +/- 0.98% versus 4.65 +/- 1.99%, respectively; the mean topoisomerase IIalpha LI was 1.32 +/- 1.04% versus 6.63 +/- 4.31%, respectively; and the mean MIB-1 LI was 1.69 +/- 1.55% versus 9.46 +/- 4.66%, respectively. Interestingly, cyclin D1 was not expressed in any oligodendrogliomas. Both cyclin A and topoisomerase IIalpha LI showed a significant positive correlation with MIB-1 LI and histological grade of oligodendrogliomas (P < 0.01) and an inverse correlation with overall survival (P < 0.01). Univariate analysis showed that cyclin A and topoisomerase IIalpha LIs with a cut-off point at 3% were a significant prognostic factor (P: cyclin A = 0.0040, topoisomerase IIalpha = 0.0033).. Cyclin A and topoisomerase IIalpha expression are closely correlated with anaplastic oligodendrogliomas and worse clinical outcomes. Cyclin D1 seems not to be involved in the tumorigenesis of oligodendrogliomas.

Topics: Adult; Antigens, Neoplasm; Brain Neoplasms; Cyclin A; Cyclin D1; DNA Topoisomerases, Type II; DNA-Binding Proteins; Female; Fluorescent Antibody Technique, Indirect; Humans; Immunoenzyme Techniques; Ki-67 Antigen; Male; Middle Aged; Oligodendroglioma; Survival Rate

Recent clinical studies have demonstrated that As2O3 is an effective drug in the treatment of acute promyelocytic leukemia (APL) by inducing apoptosis and inhibiting the proliferation of leukemia cells both in vitro and in vivo. As a novel anticancer agent for the treatment of solid cancer, As2O3 is promising, but no experimental investigations of its efficacy on glioblastoma have been conducted at concentrations that may be achieved clinically. In addition, the cell proliferation and cell cycle regulating mechanism of As2O3 has not yet to be clarified, especially in solid cancers. We investigated the effect of As2O3 on proliferation and cell cycle regulation with change in cyclins in two human glioblastoma cell lines differing in p53 status (U87MG-wt; T98G-mutated). Sensitivity to As2O3 varied depending on the dose with the IC50 of the U87MG and T98G cells being 1.78 and 3.55 microM, respectively. Analysis by laser scanning cytometry (LSC) indicated that As2O3 inhibited the proliferation of the two cell lines via cell cycle arrest both at the G1 and G2 phases. To address the mechanism of the antiproliferative effect of As2O3, we examined its effect on cell cycle-related proteins by means of LSC, confocal microscopy and Western blot analysis. As2O3 induced an increase in p53 level and a decrease in level of cyclin B1 combined with cell arrest at G2/M in both cell lines. Cell arrest in G1, however, was associated with a decline in cyclin D1 expression only in the wt U87MG cells. As2O3 also induced apoptosis of U87MG cells as evidenced by the presence of cells with fractional DNA content ( cell populations). The present evidence that As2O3 at relatively low concentration effectively inhibited proliferation of U87MG and T98G cells in vitro, suggests that the drug may be considered for in vivo testing on animal models and possibly clinical trials on glioma patients.

Topics: Arsenic Trioxide; Arsenicals; Blotting, Western; Brain Neoplasms; Cell Cycle; Cell Division; Cyclin B; Cyclin B1; Cyclin D1; Dose-Response Relationship, Drug; Flow Cytometry; Glioblastoma; Humans; Oxides; Tumor Cells, Cultured; Tumor Suppressor Protein p53

This study was designed to investigate the mechanisms by which mutant versican constructs play a dominant-negative effect on astrocytoma cell proliferation. Although a mini-versican or a versican G3 construct promoted growth of U87 astrocytoma cells, a mini-versican lacking epidermal growth factor (EGF) motifs (versicanDeltaEGF) and a G3 mutant (G3DeltaEGF) exerted a dominant-negative effect on cell proliferation. G3DeltaEGF-transfected cells formed smaller colonies, arrested cell cycle at G(1) phase, inhibited expression of cell cycle proteins cdk4 and cyclin D1, and contained multiple nucleoli. In cell surface binding assays, G3 products expressed in COS-7 cells and bacteria bound to U87 cell surface. G3DeltaEGF products exhibited decreased binding activity, but higher levels of G3DeltaEGF products were able to inhibit the binding of G3 to the cell surface. G3DeltaEGF expression inhibited secretion of endogenous versican in astrocytoma cells and also inhibited the secretion of mini-versican in COS-7 cells co-transfected with the mini-versican and G3DeltaEGF constructs. The effect seems to depend on the expression efficiency of G3DeltaEGF, and it occurred via the carbohydrate recognition domain.

Topics: Amino Acid Motifs; Animals; Astrocytoma; Binding, Competitive; Blotting, Western; Brain Neoplasms; Cell Cycle; Cell Division; Cell Nucleolus; Cells, Cultured; Chondroitin Sulfate Proteoglycans; COS Cells; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinases; DNA, Complementary; Gene Deletion; Genes, Dominant; Genetic Vectors; Glioma; Lectins, C-Type; Mutation; Protein Binding; Protein Structure, Tertiary; Proteoglycans; Proto-Oncogene Proteins; Recombinant Proteins; Reverse Transcriptase Polymerase Chain Reaction; Transfection; Tumor Cells, Cultured; Versicans

Malignant gliomas frequently show genetic aberrations of genes coding for cell cycle regulatory proteins involved in the control of G1/S phase transition. These include mutation and/or deletion of the retinoblastoma (RB1) gene, homozygous deletion of the CDKN2A and CDKN2B genes, as well as amplification and overexpression of the CDK4 and CDK6 genes. The D-type cyclins (cyclin D1, D2, and D3) promote cell cycle progression from G1 to S phase by binding to and activating the cyclin dependent kinases Cdk4 and Cdk6. Here, we have investigated a series of 110 primary malignant gliomas and 8 glioma cell lines for amplification and expression of the D-type cyclin genes CCND1 (11q13), CCND2 (12p13), and CCND3 (6p21). We found the CCND1 gene amplified and overexpressed in one anaplastic astrocytoma of our tumor series. Two glioblastomas and one anaplastic astrocytoma showed CCND2 gene amplification, but lacked significant overexpression of CCND2 transcripts. Amplification and overexpression of the CCND3 gene was detected in the glioblastoma cell line CCF-STTG1, as well as in one primary glioblastoma and in the sarcomatous component of one gliosarcoma. Our data thus suggest that amplification and increased expression of CCND1 and CCND3 contribute to the loss of cell cycle control in a small fraction of human malignant gliomas.

Topics: Blotting, Southern; Brain Neoplasms; Cell Cycle; Cyclin D; Cyclin D1; Cyclin D2; Cyclin D3; Cyclins; Gene Amplification; Glioblastoma; Glioma; Gliosarcoma; Humans; Immunohistochemistry; Nucleic Acid Hybridization; Polymerase Chain Reaction

Cyclin D1 (cycD1) expression was defined immunohistochemically using monoclonal antibody DCS-6 and polyclonal antiserum H-295 in 50 glioma biopsies. The number of positive nuclei was higher for H-295 than for DCS-6, with a ratio of 3:1. The labelling index (LI) was compared to the grade of histological malignancy and to Ki-67 MIB-1 LI. The LI for cycD1 increased with histological malignancy, in parallel with the increase in MIB-1 LI. In most tumours, the maximum LI for cycD1 and MIB-1 were found in the same areas. The mean MIB-1 LI: mean cycD1 LI ratio does not vary in the three grades of astrocytic tumours. However, in this study the correlation between the two LIs was not statistically significant. Staining for cycD1 antigen does not necessarily imply that the gene is overexpressed since other molecular mechanisms can also be responsible for cell cycle deregulation. In invasive areas, the cycD1 LI is frequently higher than in solid tumour, either because more tumour cells are positive or because reactive astrocytes and activated microglia express cycD1. The relative contribution of neoplastic and reactive cells remains to be defined.

Topics: Astrocytes; Astrocytoma; Brain Neoplasms; Cell Cycle; Cyclin D1; Glioblastoma; Glioma; Humans; Immunohistochemistry; Mitotic Index; Neoplasm Invasiveness; Oligodendroglioma

Cyclins are key components of the cell cycle progression machinery. Immunohistochemical detection of cyclins in relation to cell position, DNA content, by multiparameter flow cytometry has provided new cell cycle studies. T98G, U373MG, and U251MG were scheduled expression of cyclin A, B1 which are maximal in late S and G2M phase cells. T98G was scheduled expression of cyclin E. being maximal in G1 and S phase cells, but U251MG, U373MG were unscheduled expression of cyclin E. The expression of cyclin D1 were not expressed a pattern. These result may mean that cyclin E in an important protein of cell cycle machinery of malignant brain tumor.

Topics: Brain Neoplasms; Cell Cycle; Cyclin A; Cyclin B; Cyclin B1; Cyclin D1; Cyclin E; Cyclins; Flow Cytometry; Glioma; Humans; Tumor Cells, Cultured

The molecular genetic events involved in the development of ependymal neoplasms are not well understood. This study retrospectively examines 41 ependymomas; in all tumors the cyclin D1 labeling index (LI) (percent of positive immunostaining tumor cells) was correlated with MIB-1 LI and outcome. The study included 41 patients (25 males and 16 females) ranging in age from 1.5 to 70 years (mean, 30.7 years). Twenty-five patients underwent a subtotal resection or biopsy, and 16 underwent a gross total resection. Thirty-two patents had an ordinary or tanycytic ependymoma, and 9 had anaplastic/malignant tumors. The cyclin D1 LI ranged from 0 to 8.2 (mean, 0.7); 21 tumors had an LI of 0, and 8 had an LI of >1.0. The MIB-1 LI ranged from 0.1 to 34 (mean, 4.6); 16 tumors had an LI >2.0. All 8 patients with a cyclin D1 LI of >1.0 had an MIB-1 LI of >2.0. The tumors in the 6 of 8 patients with a cyclin D1 LI of >1.0 were classified as anaplastic/malignant tumors. The findings at the most recent follow-up were as follows: alive with no evidence of tumor (n = 11; mean, 64.1 months); died with evidence of tumor (n = 11; mean, 45.9 months); alive with tumor (n = 10; mean, 39.0 months). Two patients were alive with evidence of residual disease at follow-up intervals of 7 and 16 months but were lost to further follow-up. The remaining 6 patients either were lost to follow-up or else died in the immediate postoperative period. Cyclin D1 and MIB-1 LI did not reliably correlate with clinical outcome or recurrence. All tumors with an elevated cyclin D1 LI also had an elevated MIB-1 LI; however, the converse was not true. An elevated cyclin D1 LI (>1.0 in this study) appeared to be associated with anaplastic/malignant histology; however, cyclin D1 LI along with MIB-1 LI and histology did not always reliably correlate with clinical outcome.

Topics: Adolescent; Adult; Aged; Antigens, Nuclear; Biomarkers, Tumor; Brain Neoplasms; Child; Child, Preschool; Cyclin D1; Ependymoma; Female; Humans; Immunohistochemistry; Infant; Ki-67 Antigen; Male; Middle Aged; Nuclear Proteins; Spinal Cord Neoplasms

Pilocytic astrocytoma is an infrequently encountered, generally low-grade neoplasm. No study has extensively looked at both cyclin D1 and MIB-1 labeling indices in pilocytic astrocytoma and their relation to clinical outcome. This study retrospectively examines the clinicopathologic features of 48 patients with pilocytic astrocytoma including MIB-1 (cell proliferation marker) and cyclin D1 (protein that regulates progression from G1 to S phase of the cell cycle) immunohistochemistry. Of 48 patients (27 females and 21 males; mean age, 12.7 years; age range, 2 to 57 years), 26 initially underwent gross total resection; 17, subtotal resection; four, biopsy alone; in one patient, the extent of tumor resection was unknown. Histological features observed included Rosenthal fibers (83.3%), granular bodies (75%), vascular sclerosis (56.2%), vascular proliferation (56.2%), prominent nuclear pleomorphism (14.6%), necrosis (10.4%), and identifiable mitotic figures (2.1%). MIB-1 labeling indices (n=45) (positive staining tumor nuclei per 1,000 nuclei evaluated) ranged from 0 to 3.5% (mean, 0.6%); seven tumors had a labeling index greater than 1.0%. Cyclin D1 labeling indices (n=45) ranged from 0 to 0.8% (mean, 0.1%). Most tumors (N=29, 66.7%) had no immunostaining. At last known follow-up, 27 patients were alive with no evidence of disease (mean, 49.2 months), 17 patients were alive with evidence of disease (mean, 36.8 months), three died with tumor at 2, 22, and 156 months, and one patient was lost to follow-up. Eight patients had at least one tumor recurrence requiring additional surgery; seven of these patients had an initial subtotal resection. In summary, MIB-1 labeling indices were generally low (mean, 0.6%) and are reflective of the slow growth of the tumors. Cyclin D1 immunostaining does not appear to be significantly increased in pilocytic astrocytoma. Adverse outcome in patients with pilocytic astrocytoma may be related to extent of surgical resection and does not seem to correlate with histology, MIB-1 labeling indices, or cyclin D1 immunoreactivity.

Topics: Adolescent; Adult; Antigens, Nuclear; Astrocytoma; Brain Neoplasms; Cell Nucleus; Child; Child, Preschool; Cyclin D1; Female; Humans; Immunoenzyme Techniques; Ki-67 Antigen; Male; Middle Aged; Neoplasm Recurrence, Local; Nuclear Proteins; Retrospective Studies

Well-documented cases of malignant degeneration in pleomorphic xanthoastrocytoma and of anaplastic pleomorphic xanthoastrocytoma are rare in the literature. We report 2 cases of pleomorphic xanthoastrocytoma, 1 of which demonstrated clear evidence of malignant degeneration in the absence of prior radiation therapy over an 18-year period. Both anaplastic tumors were characterized by foci of necrosis and increased mitotic activity (3 and 2 mitotic figures/10 high-power fields). Both tumors demonstrated focal positive staining for glial fibrillary acidic protein and showed marked reticulin deposition. An MIB-1 labeling index (marker of cell proliferation) in the initial low-grade-appearing tumor in case 1 was 0.1%. The recurrent tumor in case 1 had an MIB-1 labeling index of 4.9%, and the anaplastic tumor in case 2 had an MIB-1 labeling index of 5.4%. Significant cyclin D1 immunoreactivity was not observed in either anaplastic tumor. Two percent to 3% of tumor cells stained positive with p53 protein antibody in the recurrent anaplastic tumor in case 1. Although histology may not reliably predict aggressive behavior in pleomorphic xanthoastrocytomas, the presence of increased mitosis, necrosis, and increased cell proliferation labeling indices may be indicative of a higher grade tumor.

Topics: Adolescent; Anaplasia; Antigens, Nuclear; Astrocytoma; Brain Neoplasms; Child; Cyclin D1; Female; Humans; Immunohistochemistry; Ki-67 Antigen; Neoplasm Recurrence, Local; Nuclear Proteins; Tumor Suppressor Protein p53

Alterations of CDKN2A, RB, and cyclin D1 genes and expression of their products in astrocytic tumors were studied using a combination of molecular genetic and immunohistochemical assays. In addition, the association of gene status with clinical outcome was evaluated. Alterations of CDKN2A and RB gene in 30 lesions were analyzed by single-strand conformation polymorphism of polymerase chain reaction (PCR-SSCP), direct sequencing, and Western blotting. Methylation of the CDKN2A promoter was detected by methylation-specific PCR. Immunohistochemistry was applied to determine the expression of gene products in tumors from 94 patients for whom clinical outcome was also evaluated. Analyses of the CDKN2A gene revealed 12 homozygous or hemizygous deletions, one mutation in exon 1, and three methylations in the promoter. Expression of p 16 protein was not detected in 18 of 30 cases. RB mutations leading to loss of expression of the pRb were found in four (13%) cases, and six were immunohistochemically negative for this protein. Overexpression of cyclin D1 was obtained in 51 (54%) of 94 cases. Patients with pRb-negative tumors had a significantly greater risk of earlier death than those with p16 and cyclin D1 alterations, Both p16 and pRb immunohistochemistry provides useful complementary information and may provide valuable predictive information in screening. The biological consequences of deregulating individual components along cell control pathways are unequal, perhaps reflecting their hierarchical roles in the G1 checkpoint.

Topics: Adolescent; Adult; Aged; Astrocytoma; Blotting, Western; Brain Neoplasms; Cell Cycle Proteins; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; Humans; Immunohistochemistry; Middle Aged; Polymerase Chain Reaction; Polymorphism, Single-Stranded Conformational; Predictive Value of Tests; Retinoblastoma Protein; Survival Rate

The difference in biological features between recurrent and nonrecurrent intracranial chordomas has not been studied. In this study, proliferative potentials of chordomas were studied with an immunohistochemical staining method, mainly using anti-Ki-67 antibody, MIB-1, which is known to be available for archival paraffin sections, together with immunohistochemical studies on the expression of cell cycle or apoptosis-related proteins, including p53, cyclin D1, and bcl-2 proteins. The correlation among MIB-1 staining indices, the immunoreactivities of these proteins, and clinical courses of intracranial chordomas were analyzed retrospectively, and the statistically significant correlation between MIB-1 staining index (SI) and recurrence has been clarified. The mean MIB-1 SI of recurrent tumors was 10.2%, being shown to be higher than that of nonrecurrent tumors (2.8%). The immunohistochemically positive staining of cell cycle-related protein, especially p53 and cyclin D1 proteins, correlated well with recurrence and high MIB-1 SI. In conclusion, both the examination of proliferative potentials of chordomas using MIB-1 SI and the study of the immunoreactivity of p53 and cyclin D1 proteins are important for their biological and histopathological analyses and the prediction of future recurrence.

Topics: Adolescent; Adult; Antigens, Nuclear; Biomarkers; Brain Neoplasms; Cell Division; Chordoma; Cyclin D1; Cyclins; Female; Histones; Humans; Immunohistochemistry; In Situ Hybridization; Ki-67 Antigen; Male; Middle Aged; Neoplasm Recurrence, Local; Nuclear Proteins; Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Retrospective Studies; Sex Factors; Tumor Suppressor Protein p53

While several genetic alterations associated with the evolution of the astrocytomas have been identified, the molecular basis of non-astrocytic brain tumors has remained largely unknown. In this study, p15, p16, CDK4 and cyclin D1 genes were analyzed in 69 nonastrocytic human brain tumors, including 17 oligodendrogliomas, 16 medulloblastomas/primitive neuroectodermal tumors (PNETs), 14 ependymomas and 22 meningiomas. Southern blot analysis of DNA from frozen samples showed no homozygous deletions in p15 or p16 genes in any of these tumors. No mobility shift was found by PCR-single-strand conformation polymorphism (PCR-SSCP) analysis in exons 1 and 2 of the p15 gene and exons 1 and 2 of the p16 genes, except for one oligodendroglioma. Direct sequencing of DNA from this tumor showed a G --> A transition at nucleotide 436 (codon 140) in exon 2 of the p16 gene, which is a common polymorphism. Southern blot analyses revealed no amplification of CDK4 and cyclin D1 genes in any of the neoplasms analyzed. In contrast to astrocytic brain tumors, which show frequent loss of the p16 gene and amplification of the CDK4 gene, alteration of these genes appears to be rare in other neoplasms of the human nervous system.

Topics: Astrocytoma; Base Sequence; Blotting, Southern; Brain Neoplasms; Cerebellar Neoplasms; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinases; Cyclins; DNA Primers; Ependymoma; Humans; Medulloblastoma; Meningeal Neoplasms; Meningioma; Molecular Sequence Data; Neuroectodermal Tumors; Oligodendroglioma; Oligonucleotide Probes; Oncogene Proteins; Point Mutation; Polymerase Chain Reaction; Polymorphism, Single-Stranded Conformational; Proto-Oncogene Proteins; Reference Values

Forty-eight astrocytic tumours were stained immunohistochemically with antibodies to the cell cycle-regulating protein, cyclin D1, and to the proliferation marker MIB1 (Ki-67) using formalin fixed paraffin embedded tissue and a microwave antigen retrieval system. Cases were classified by the WHO system (1993). The labelling indices (LI) for both antibodies were compared with each other and with the tumour type. The mean labelling indices for both antibodies increased with the degree of malignancy, and a significant difference was seen between the pilocytic astrocytoma and diffuse astrocytoma together vs anaplastic astrocytoma and glioblastoma together. However, within each tumour type there was considerable variation in the labelling indices and a clear cut off value could not be demonstrated. There was a strong positive correlation between labelling indices for cyclin D1 and MIB1 in diffuse astrocytoma, but this correlation broke down increasingly in anaplastic astrocytoma and glioblastoma. There was poor correlation between cyclin D1 and MIB1 in pilocytic astrocytoma, a feature which appeared to separate them from the diffuse astrocytoma. Average labelling indices for cyclin D1 were higher than those of MIB1, which suggests that cyclin D1 positive cells represent a pool of cells from which proliferation and hence MIB1 expression can take place. In conclusion, cyclin D1 is overexpressed in astrocytic tumours, more so with increasing grade of malignancy and in a way which approximately correlates with MIB1 expression.

Topics: Antigens, Neoplasm; Astrocytoma; Brain Neoplasms; Cell Nucleus; Cyclin D1; Cyclins; Glioblastoma; Humans; Immunohistochemistry; Ki-67 Antigen; Oncogene Proteins