cyclin-d1 and Glioma

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

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

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

Glioblastoma (GBM) is the most common primary brain malignancy in adults. Despite multimodal treatment that involves maximal safe resection, concurrent chemoradiotherapy, and tumour treatment for supratentorial lesions, the prognosis remains poor. The current median overall survival is only <2 years, and the 5-year survival is only 7.2%. Thioredoxin domain-containing protein 11 (TXNDC11), also known as EF-hand binding protein 1, was reported as an endoplasmic reticulum stress-induced protein. The present study aimed to elucidate the prognostic role of TXNDC11 in GBM. We evaluated the clinical parameters and TXNDC11 scores in gliomas from hospitals. Additionally, proliferation, invasion, migration assays, apoptosis, and temozolomide (TMZ)-sensitivity assays of GBM cells were conducted to evaluate the effects of short interfering RNA (siRNA) on these processes. In addition, these cells were subjected to Western blotting to detect the expression levels of N-cadherin, E-cadherin, and Cyclin D1. High levels of TXNDC11 protein expression were significantly associated with World Health Organization (WHO) high-grade tumour classification and poor prognosis. Multivariate analysis revealed that in addition to the WHO grade, TXNDC11 protein expression was also an independent prognostic factor of glioma. In addition, TXNDC11 silencing inhibited proliferation, migration, and invasion and led to apoptosis of GBM cells. However, over-expression of TXNDC11 enhanced proliferation, migration, and invasion. Further, TXNDC11 knockdown downregulated N-cadherin and cyclin D1 expression and upregulated E-cadherin expression in GBM cells. Knock-in TXNDC11 return these. Finally, in vivo, orthotopic xenotransplantation of TXNDC11-silenced GBM cells into nude rats promoted slower tumour growth and prolonged survival time. TXNDC11 is a potential oncogene in GBMs and may be an emerging therapeutic target.

Topics: Animals; Cadherins; Cyclin D1; Glioblastoma; Glioma; Humans; Rats; Thioredoxins

Leukocyte-associated immunoglobulin-like receptor-1 (LAIR1), an immune receptor containing immunoreceptor tyrosine-based inhibiory motifs (ITIMs), has emerged as an attractive target for cancer therapy. However, the intrinsic function of LAIR1 in gliomas remains unclear. In this study, the poor prognosis of glioma patients and the malignant proliferation of glioma cells in vitro and in vivo were found to be closely correlated with LAIR1. LAIR1 facilitates focal adhesion kinase (FAK) nuclear localization, resulting in increased transcription of cyclin D1 and chemokines/cytokines (CCL5, TGFβ2, and IL33). LAIR1 specifically supports in the immunosuppressive glioma microenvironment via CCL5-mediated microglia/macrophage polarization. SHP2

Topics: Chemokines; Cyclin D1; Cytokines; Focal Adhesion Kinase 1; Focal Adhesion Protein-Tyrosine Kinases; Glioma; Humans; Tumor Microenvironment

To explore the functions of circRNA cyclin B1 (circCCNB1) in glioma and its possible mechanisms. The expression of circCCNB1, eukaryotic translation initiation factor 4A3 (EIF4A3), cyclin D1 (CCND1) and miR-516b-5p was determined by qRT-PCR, western blot or immunohistochemistry (IHC) assay. The feature of circCCNB1 was analyzed by Actinomycin D (ActD), RNase R and subcellular fraction assays. The molecule relationships were analyzed by RIP, dual-luciferase reporter and RNA pull-down assays. CCK-8, EdU and colony formation assays were performed to analyze cell proliferation. Flow cytometry analysis was executed to estimate the cell cycle. Murine xenograft model assay was used for the role of circCCNB1 in vivo. CircCCNB1 was overexpressed in glioma tissues and cells. EIF4A3 positively regulated circCCNB1 expression. CircCCNB1 knockdown repressed glioma cell proliferation and cell cycle process in vitro and blocked tumor growth in vivo. CircCCNB1 knockdown reduced CCND1 expression in glioma cells and CCND1 overexpression bated the effect of circCCNB1 knockdown on glioma cell growth. CircCCNB1 interacted with HuR to elevate CCND1 expression. miR-516b-5p could interact with circCCNB1 and CCND1. CircCCNB1 regulated glioma cell progression and CCND1 expression by miR-516b-5p and HuR. CircCCNB1 aggravated glioma cell growth by elevating CCND1 through targeting miR-516b-5p and HuR.

Topics: Animals; Cell Line, Tumor; Cell Proliferation; Cyclin B1; Cyclin D1; DEAD-box RNA Helicases; ELAV-Like Protein 1; Eukaryotic Initiation Factor-4A; Glioma; Humans; Mice; MicroRNAs; RNA, Circular

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

Dysregulation of microRNAs has been frequently implicated in the progression of human diseases, including glioma. This study aims to explore the interaction between E2F transcription factor 1 (E2F1) and miR-107 in the progression of glioma.. Expression of miR-107 in glioma tissues and cells was examined. Putative binding sites between E2F1 and the promoter region of miR-107, and between miR-107 and cyclin D1 (CCND1) mRNA were predicted via bioinformatic systems and validated via chromatin immunoprecipitation and luciferase reporter gene assays. Altered expression of miR-107, E2F1, and CCND1 was introduced in A172 and T98G cells to examine their roles in cell growth and the activity of the Wnt/β-catenin signaling. In vivo experiments were performed by injecting cells in nude mice.. miR-107 was poorly expressed, whereas E2F1 and CCND1 were highly expressed in glioma tissues and cells. E2F1 bound to the promoter region of miR-107 to induce transcriptional repression, and miR-107 directly bound to CCND1 mRNA to reduce its expression. Overexpression of miR-107 reduced proliferation, migration and invasion, and augmented apoptosis of glioma cells, and it reduced activity of the Wnt/β-catenin pathway. The anti-tumorigenic roles of miR-107 were blocked by E2F1 or CCND1 overexpression. Similar results were reproduced in vivo where miR-107 overexpression or E2F1 inhibition blocked tumor growth in nude mice.. This study suggested that E2F1 reduces miR-107 transcription to induce CCND1 upregulation, which leads to progression of glioma via Wnt/β-catenin signaling activation.

Topics: Animals; Apoptosis; Cell Line, Tumor; Cell Proliferation; Cyclin D1; E2F Transcription Factors; Glioma; Humans; Mice; Mice, Nude; MicroRNAs

Topics: Cell Proliferation; Cisplatin; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase 6; Drug Resistance, Neoplasm; Glioma; Humans; Immunohistochemistry; Intercellular Signaling Peptides and Proteins; Nucleosome Assembly Protein 1; Oncogenes; Prognosis; Up-Regulation

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

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

As the key factor of the polarity protein complex, Par6 not only regulates polarization processes, but also plays important roles in tumor metastasis and progression in many epithelium malignancy tumors. Here, we showed that Par6 is an essential component in glioma tumorigenesis. Our results indicated the aberrant expression of Par6 in malignant glioma tissues and cell lines. We found that the regulation of Par6 expression induces cell proliferation and tumor growth in vivo and in vitro. Additionally, RNA-seq revealed the effects of Par6 were associated with cyclin D1-regulated cell cycle progression in glioma cells. Moreover, our results demonstrated that the regulation of Par6 can enhance the activation of Akt/PI3K signaling pathway, and subsequently upregulate the expression level of GSK-3β protein, which then regulate cyclin D1-mediated cell cycle regulation. Furthermore, we found that TGF-β-induced the upregulation of Par6 expression may be involved in this process. The pathological analysis confirmed the correlation between Par6 expression and the prognosis in human glioma tissues, suggesting the regulation of Par6 expression regulates glioma tumorigenesis and progression. Thus, our findings showed that Par6 might be a potential biomarker for the diagnosis and providing a therapeutic strategy for the treatment of malignant glioma.

Topics: Adaptor Proteins, Signal Transducing; Animals; Biomarkers, Tumor; Cell Cycle; Cell Line, Tumor; Cyclin D1; Gene Expression Regulation, Neoplastic; Glioma; Glycogen Synthase Kinase 3 beta; Humans; Male; Mice; Mice, Nude; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Signal Transduction

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

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

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

The current study investigated the effect of plasmid small interfering RNA (psiRNA)‑mediated silencing of signal transducer and activator of transcription‑3 (STAT3) on the invasion, apoptosis, and expression levels of cyclin D1, caspase‑3 and B‑cell lymphoma‑2 (Bcl‑2) in C6 glioma cells. Cell invasion was determined using a Transwell assay, while the apoptosis rate and cell cycle distribution of cells were assessed using Annexin V‑FITC/PI double staining. The expression levels of cyclin D1, caspase‑3 and Bcl‑2 proteins were measured by western blotting. Transfection with psiRNA‑STAT3 was observed to significantly decrease the number of transmembrane cells compared with the control groups (P<0.05). In addition, the proportion of cells at G0/G1 phase was increased in the psiRNA‑STAT3 group compared with the controls. Western blotting indicated that psiRNA‑STAT3 decreased the expression of cyclin D1, caspase‑3 and Bcl‑2 proteins. Taken together, psiRNA‑STAT3 inhibited the migration and invasive abilities, and induced the apoptosis of C6 glioma cells, possibly through regulation of the expression of cyclin D1, caspase‑3 and Bcl‑2 proteins.

Topics: Animals; Apoptosis; Caspase 3; Cell Cycle; Cell Line, Tumor; Cyclin D1; Gene Expression Regulation, Neoplastic; Glioma; Neoplasm Invasiveness; Plasmids; Rats; RNA Interference; RNA, Small Interfering; STAT3 Transcription Factor

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 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

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

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

MiR-638 is constantly downregulated and serves as a tumor suppressor in various cancers. Its role in gliomas remains unclear. This study is designed to investigate the clinical significance and the pathogenic role of miR-638 in human gliomas.. Quantitative Real-time PCR was performed to analyze the expression of miR-638 in the tumor and adjacent tissues of 24 glioma patients. The association between the expression of miR-638 and clinical features were examined. Survival of patients was studied by Kaplan-Meier curves. The impact of miR-638 on cell growth and apoptosis was determined by CCK-8 assay, colony formation assay, cell cycle analysis and Annexin V-FITC-PI apoptosis assay. The effect of miR-638 on HOXA9 was determined by luciferase assay and Western blot. The effect of miR-638 and HOXA9 on expression of oncogenes, Cyclin D1 and C-MYC was determined by Western blot.. MiR-638 expression was constantly downregulated in glioma tumor tissue, which is negatively correlated with the WHO grade. MiR-638 expression was associated with clinical features such as tumor size, KPS score and WHO grade. Patients with low miR-638 had a worse overall survival than those with high expression. Experimentally, miR-638 directly targeted HOXA9 to suppress its expression, leading to attenuations of cell proliferation, colony formation and cell cycle progression and enhanced basal apoptosis level. MiR-638/HOXA9 axis also suppressed the expression of Wnt/beta-catenin-regulated oncogenes, Cyclin D1 and C-MYC.. MiR-638 is a constantly downregulated microRNA in gliomas and is associated with its prognosis. MiR-638 regulates cellular malignancy of gliomas through targeting HOXA9. Thus, miR-638/HOXA9 signaling axis may have therapeutic potential in gliomas.

Topics: Apoptosis; beta Catenin; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Down-Regulation; Female; Genes, Tumor Suppressor; Glioma; Homeodomain Proteins; Humans; Male; MicroRNAs; Middle Aged; Prognosis; Signal Transduction

To investigate the impact of CCND1 and EFEMP1 gene polymorphism, and additional their gene-gene interactions and haplotype within EFEMP1 gene on glioma risk based on Chinese population.. Logistic regression was performed to investigate association between single-nucleotide polymorphisms (SNP) and glioma risk and generalized multifactor dimensionality reduction (GMDR) was used to analyze the gene-gene interaction.. Glioma risks were higher in carriers of homozygous mutant of rs603965 within CCND1 gene, rs1346787 and rs3791679 in EFEMP1 gene than those with wild-type homozygotes, OR (95%CI) were 1.67 (1.23-2.02), 1.59 (1.25-2.01) and 1.42 (1.15-1.82), respectively. GMDR analysis indicated a significant two-locus model (p=0.0010) involving rs603965 within CCND1 gene and rs1346787 within EFEMP1 gene. Overall, the cross-validation consistency of the two- locus models was 10\\ 10, and the testing accuracy is 60.17%. Participants with rs603965 - GA or AA and rs1346787- AG or GG genotype have the highest glioma risk, compared to participants with rs603965 - GG and rs1346787- AA genotype, OR (95%CI) was 3.65 (1.81-5.22). We conducted haplotype analysis for rs1346787 and rs3791679, because D' value between rs1346787 and rs3791679 was more than 0.8. The most common haplotype was rs1346787 - A and rs3791679- G haplotype, the frequency of which was 0.4905 and 0.4428 in case and control group.. Polymorphism in rs603965 within CCND1 gene and rs1346787 within EFEMP1 gene and its gene- gene interaction were associated with increased glioma risk.

Topics: Adult; Asian People; Case-Control Studies; China; Cyclin D1; Extracellular Matrix Proteins; Female; Gene Frequency; Genetic Predisposition to Disease; Genotype; Glioma; Haplotypes; Humans; Logistic Models; Male; Middle Aged; Polymorphism, Single Nucleotide; Risk Factors

Accumulating evidence demonstrates that dysregulated microRNAs (miRNAs) play a critical role in tumorigenesis and progression of various cancers. miR-320b, a member of miR‑320 family, was revealed downregulated in numerous human cancers, including nasopharyngeal carcinoma and colorectal cancer. However, the function of miR‑320b in human glioma remained poorly defined. In this study, we report that miR‑320b was lowly expressed in glioma tissues and cell lines in contrast with controls, being closely correlated with histological malignancy of glioma. Furthermore, patients with low expression of miR‑320b were associated with poor prognostic outcomes. In vitro functional assays indicated that overexpression of miR‑320b could markedly enhance cell apoptosis rate and suppress cell proliferation, migration and invasion. miR-320b mimic impaired cell cycle and metastasis through inhibiting the expression of G1/S transition key regulator Cyclin D1 as well as decreasing the expression level of MMP2 and MMP9. Additionally, upregulation of miR‑320b could markedly promote apoptosis by increasing the level of Bax and reducing Bcl-2 expression in glioma. Taken together, our data suggested that miR‑320b might serve as a novel prognostic marker and potential therapeutic target for glioma.

Topics: Adult; Aged; Apoptosis; bcl-2-Associated X Protein; Biomarkers, Tumor; Carcinogenesis; Cell Movement; Cell Proliferation; Cyclin D1; Female; Gene Expression Regulation, Neoplastic; Glioma; Humans; Male; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; MicroRNAs; Middle Aged; Prognosis; Proto-Oncogene Proteins c-bcl-2

Angelica sinensis (Oliv) Diels (Apiaceae) is a traditional medicine that has been used for more than 2000 years in China. It exhibits various therapeutic effects including neuroprotective, anti-oxidant, anti-inflammatory, and immunomodulatory activities. Angelica polysaccharides (APs), bioactive constituents of Angelica have been shown to be responsible for these effects; however, the utility of APs for the treatment of glioma and their mechanism of action remain to be elucidated.. In this study, we investigated the inhibitory effects of APs on a glioma cell line and their molecular mechanism of action.. U251 cells were utilized to confirm the effects of APs on glioma.. The human glioblastoma cell line U251 was utilized for both in vitro and in vivo models, in which we tested the effects of APs. Flow cytometry, gene expression analysis, western blotting, and MTT assays were used to elucidate the effects of APs on cell proliferation, cell cycle, and apoptosis.. The results demonstrated that APs significantly inhibited the growth and proliferation of U251 cells and induced their apoptosis. Furthermore, APs effectively reduced the expression of several cell cycle regulators: cyclins D1, B, and E. The apoptosis suppressor protein Bcl-2 was also downregulated, and the expression of pro-apoptotic proteins Bax and cleaved-caspase-3 increased. Additionally, APs inhibited the transforming growth factor (TGF)-β signaling pathway and stimulated the expression of E-cadherin, thus prohibiting cell growth.. In conclusion, the results indicate that APs attenuate the tumorigenicity of glioma cells and promote their apoptosis by suppressing the TGF-β signaling pathway. The present study therefore provides evidence of the inhibitory effects of APs against glioma progression, and proposes their potential application as alternative therapeutic agents for glioma.

Topics: Angelica sinensis; Antigens, CD; Apoptosis; Apoptosis Regulatory Proteins; Cadherins; Caspase 3; Cell Cycle; Cell Line, Tumor; Cell Proliferation; China; Cyclin D1; Glioma; Humans; Polysaccharides; Transforming Growth Factor beta

Experimental and clinical evidence points to a critical role of Upregulator of cell proliferation (URGCP/URG4) in controlling the progression of multiple tumors. However, the oncogenic role of URGCP in glioma still remains elusive. In this study we tried to investigate the oncogenic roles and molecular mechanisms of URGCP in glioma. We found that the levels of URGCP were upregulated in glioma, and that the high-levels of URGCP indicated a worse prognosis in glioma patients. URGCP and miR-16 are critical for glioma growth: silencing URGCP (shURGCP) inhibited glioma growth, while, the shURGCP-mediated proliferative inhibition could be recovered by antagonizing miR-16 (anta-miR-16) in vivo and in vitro. Mechanically, URGCP repressed miR-16 expression via activating NF-κB/c-myc pathway in glioma; Cyclins D1 and Cyclin E1 were identified as the direct targets of miR-16, thus, URGCP-mediated miR-16 downregulation accelerated cell proliferation by upregulating Cyclin D1 and Cyclin E1 expression. All these results suggested that URGCP accelerates glioma growth through the NF-κB/c-myc/miR-16/Cyclin D1/E1 pathway, and both URGCP and miR-16 function as a novel cell cycle regulators in glioma and could be considered as potential targets for glioma therapy.

Topics: Animals; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin E; Disease Models, Animal; Flow Cytometry; Gene Expression; Gene Expression Regulation, Neoplastic; Glioma; Heterografts; Humans; Immunohistochemistry; Male; Mice; MicroRNAs; Neoplasm Proteins; NF-kappa B; Oncogene Proteins; Polymerase Chain Reaction; Signal Transduction

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

The metastasis-associated lung adenocarcinoma transcription 1 (MALAT1) is a highly conserved long non-coding RNA (lncRNA) gene. However, little is known about the pathological role of lncRNA MALAT1 in glioma. In the present study, we explored the expression level of lncRNA MALAT1 in primary glioma tissues as well as in U87 and U251 glioma cell lines. Using qRT-PCR, we found that the expression of lncRNA MALAT1 was significantly increased in glioma tissues compared with that of paracancerous tissues. Meanwhile, the expression of MALAT1 was highly expressed in U98 and U251 cells. In order to explore the function of MALAT1, the expression of MALAT1 was greatly reduced in U87 and U251 cells transfected with siRNA specifically targeting MALAT1. Consequently, cell viability of U87 and U251 cells were drastically decreased after the knockdown of MALAT1. Concomitantly, the apoptosis rate of the two cell lines was dramatically increased. Furthermore, the expression levels of some tumor markers were reduced after the knockdown of MALAT1, such as CCND1 and MYC. In summary, the current study indicated a promoting role of MALAT1 in the development of glioma cell.

Topics: Apoptosis; Biomarkers, Tumor; Blotting, Western; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Down-Regulation; Flow Cytometry; Glioma; Humans; Proto-Oncogene Proteins c-myc; Real-Time Polymerase Chain Reaction; RNA Interference; RNA, Long Noncoding; RNA, Small Interfering

Glioma differentiation therapy is a novel strategy that has been used to induce glioma cells to differentiate into glia-like cells. Although some advances in experimental methods for exploring the molecular mechanisms involved in differentiation therapy have been made, a model-based comprehensive analysis is still needed to understand these differentiation mechanisms and improve the effects of anti-cancer therapeutics. This type of analysis becomes necessary in stochastic cases for two main reasons: stochastic noise inherently exists in signal transduction and phenotypic regulation during targeted therapy and chemotherapy, and the relationship between this noise and drug efficacy in differentiation therapy is largely unknown.. In this study, we developed both an additive noise model and a Chemical-Langenvin-Equation model for the signaling pathways involved in glioma differentiation therapy to investigate the functional role of noise in the drug response. Our model analysis revealed an ultrasensitive mechanism of cyclin D1 degradation that controls the glioma differentiation induced by the cAMP inducer cholera toxin (CT). The role of cyclin D1 degradation in human glioblastoma cell differentiation was then experimentally verified. Our stochastic simulation demonstrated that noise not only renders some glioma cells insensitive to cyclin D1 degradation during drug treatment but also induce heterogeneous differentiation responses among individual glioma cells by modulating the ultrasensitive response of cyclin D1. As such, the noise can reduce the differentiation efficiency in drug-treated glioma cells, which was verified by the decreased evolution of differentiation potential, which quantified the impact of noise on the dynamics of the drug-treated glioma cell population.. Our results demonstrated that targeting the noise-induced dynamics of cyclin D1 during glioma differentiation therapy can increase anti-glioma effects, implying that noise is a considerable factor in assessing and optimizing anti-cancer drug interventions.

Topics: Cell Differentiation; Cyclin D1; Feedback, Physiological; Glioma; Models, Biological; Molecular Targeted Therapy; Phenotype; Signal Transduction; Stochastic Processes; Treatment Outcome

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

Aberrant expression of oncogenes and/or tumor suppressors play fundamental roles in the pathogenesis of glioma. B-cell CLL/lymphoma 3 (BCL3) was previously found to be a putative proto-oncogene in human cancers and the decoy receptor DcR1 is induced in a p50/Bcl3-dependent manner and attenuates the efficacy of temozolomide in glioblastoma cells. However, its expression status, clinical significance and biological functions in glioma remain largely unknown. In the present study, the levels of BCL3 were overexpressed in glioma compared to normal brain tissues. Furthermore, high expression of BCL3 protein was confirmed by immunoblotting in glioma cells as compared with normal human astrocyte cell line. The positive expression of BCL3 was correlated with adverse prognostic features and reduced overall survival rate of glioma patients. BCL3 silencing resulted in prominent decreased proliferation, cell cycle arrest in G1 phase and increased apoptosis in U251 cells. In contrast, BCL3 overexpression in U87 cells remarkably facilitated proliferative ability and cell cycle progression and induced apoptosis. In vivo studies showed that BCL3 knockdown inhibited the tumor growth of U251 cells in a mouse xenograft model. Mechanistically, BCL3 positively regulated the abundance of STAT3, p-STAT3 and the downstream targets of STAT3 pathway including BCL2, MCL-1 and cyclin D1 in glioma cells. Furthermore, a positive correlation between BCL3 and STAT3 expression was observed in glioma specimens. Notably, we confirmed that STAT3 knockdown abolished the oncogenic roles of BCL3 in glioma. In conclusion, we suggest that BCL3 serves as an oncogene in glioma by modulating proliferation, cell cycle progression and apoptosis, and its oncogenic effects are mediated by the STAT3 signaling pathway.

Topics: Apoptosis; Astrocytes; B-Cell Lymphoma 3 Protein; Brain; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Disease Progression; Female; G1 Phase Cell Cycle Checkpoints; Gene Expression Regulation, Neoplastic; Glioma; Humans; Male; Middle Aged; Myeloid Cell Leukemia Sequence 1 Protein; Proto-Oncogene Mas; Proto-Oncogene Proteins; RNA Interference; RNA, Small Interfering; STAT3 Transcription Factor; Survival Rate; Transcription Factors; Xenograft Model Antitumor Assays

Topics: Apoptosis; Cell Movement; Cell Proliferation; Cyclin D1; Epithelial-Mesenchymal Transition; Glioma; Humans; Kaplan-Meier Estimate; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Peptides; Prognosis; Receptors for Activated C Kinase; RNA, Messenger; Up-Regulation

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

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

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

Signal transducer and activator of transcription 5b (STAT5b) is likely the relevant STAT5 isoform with respect to the process of malignant progression in gliomas. STAT5b is a latent cytoplasmic protein involved in cell signaling through the modulation of growth factors, apoptosis, and angiogenesis. Previous in vitro studies have shown increased STAT5b expression in glioblastomas relative to low-grade tumors and normal brain. We recently demonstrated that phosphorylated STAT5b associates with delta epidermal growth factor receptor in the nucleus and subsequently binds the promoters of downstream effector molecules, including aurora kinase A. Analysis of TCGA dataset reveals that STAT5b is predominantly expressed in proneural (PN) gliomas relative to mesenchymal and neural gliomas. Here, we modeled ectopic expression of STAT5b in vivo using a platelet-derived growth factor subunit B (PDGFB)-dependent mouse model of PN glioma to determine its effect on tumor formation and progression. We showed that coexpression of STAT5b and PDGFB in mice yielded a significantly higher rate of high-grade gliomas than PDGFB expression alone. We also observed shorter survival in the combined expression set. High-grade tumors from the STAT5b + PDGFB expression set were found to have a lower rate of apoptosis than those from PDGFB alone. Furthermore, we showed that increased expression of STAT5b + PDGFB led to increased expression of downstream STAT5b targets, including Bcl-xL, cyclin D1 and aurora kinase A in high-grade tumors when compared to tumors derived from PDGFB alone. Our findings show that STAT5b promotes the malignant transformation of gliomas, particularly the PN subtype, and is a potential therapeutic target.

Topics: Animals; Apoptosis; Aurora Kinase A; Cell Proliferation; Cell Transformation, Neoplastic; Cyclin D1; Disease Progression; Disease-Free Survival; Glioma; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Transgenic; Proto-Oncogene Proteins c-sis; STAT5 Transcription Factor

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

To investigate the effect of nitric oxide on tumor development, we established a rat tumor xenograft model in zebrafish embryos. The injected tumor cells formed masses in which nitric oxide production could be detected by the use of the cell-permeant DAF-FM-DA (diaminofluorophore 4-amino-5-methylamino-2'-7'-difluorofluorescein diacetate) and DAR-4M-AM (diaminorhodamine-4M). This method revealed that nitric oxide production could be co-localized with the tumor xenograft in 46% of the embryos. In 85% of these embryos, tumors were vascularized and blood vessels were observed on day 4 post injection. Furthermore, we demonstrated by qRT-PCR that the transplanted glioma cells highly expressed Nos2, Vegfa and Cyclin D1 mRNA. In the xenografted embryos we also found increased zebrafish vegfa expression. Glioma and zebrafish derived Vegfa and tumor Cyclin D1 expression could be down regulated by the nitric oxide scavenger 2-(4-Carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide or CPTIO. We conclude that even if there is a heterogeneous nitric oxide production by the xenografted glioma cells that impacts Vegfa and Cyclin D1 expression levels, our results suggest that reduction of nitric oxide levels by nitric oxide scavenging could be an efficient approach to treat glioma.

Topics: Analysis of Variance; Animals; Benzoates; Cyclin D1; Flow Cytometry; Gene Expression Regulation, Neoplastic; Glioma; Heterografts; Histological Techniques; Imidazoles; Nitric Oxide; Rats; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; Vascular Endothelial Growth Factor A; Zebrafish

Integrin-linked kinase (ILK) is a highly conserved serine-threonine protein kinase that interacts with cytoplasmic domains of integrin subunits in tumor tissues. However, the relationship between gliomas and ILK is elusive. The present study aimed to investigate the role of ILK in a human glioma cell line (U251). ILK stable expressing vector, U251ILK-PGFP-V-RS-shRNA, was established and named as U251-si. The empty-PGFP-V-RS-shRNA (U251-N) was employed as the control. Quantitative real-time PCR and western blot analysis were used to detect ILK and E-cadherin mRNA and protein expression, respectively. Cell cycle analysis was employed to examine the cell cycle distribution. Cell migration was detected using a wound healing assay, and cell invasion was detected using a Transwell invasion assay. Tumor size and weight were also examined. The results indicated that ILK was expressed at a lower level at both the mRNA and protein levels in the U251-si group compared with the U251-N group (p<0.01). ILK knockdown suppressed cell proliferation of the glioma cells. Knockdown of ILK reduced the migratory and invasive potentials of the glioma cells. Inhibition of ILK expression upregulated E-cadherin and downregulated cyclin D1 in the glioma cells compared to the U251-N group (p<0.05). Knockdown of ILK in the U251 cells attenuated the ability of U251 cells to form tumors in nude mice and impaired glioma cell in vivo tumorigenicity. In conclusion, knockdown of ILK inhibits glioma cell migration, invasion and proliferation through upregulation of E-cadherin and downregulation of cyclin D1. Our results suggest that ILK may serve as a promising therapeutic target for glioma.

Topics: Animals; Cadherins; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Gene Expression Regulation, Neoplastic; Glioma; Humans; Mice; Neoplasm Invasiveness; Protein Serine-Threonine Kinases; Xenograft Model Antitumor Assays

Glioma differentiation therapy is a novel modality to increase anti-glioma effects using specific drugs to induce glioma cell differentiation to glia-like cells. However, the molecular mechanisms underlying glioma differentiation remain poorly understood. In this study, we built an experiment-integrated mathematical model for glioma differentiation signaling pathways. Our modeling and experimental analysis revealed that a "one-way-switch" bifurcation of cyclin D1 dynamics was critical for controlling the phenotypic transition of glioma cells. We also quantitatively evaluated drug combinations toward a synergistic therapeutic effect. These results provide insights into the molecular mechanisms underlying glioma differentiation and implications for the design of novel therapeutic targets in anti-cancer therapy.

Topics: Animals; Antineoplastic Agents; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Drug Synergism; Glioma; Models, Biological; Phenotype; Rats; Signal Transduction

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

The present study is to investigate the effect of the combination of small-interfering RNA (siRNA) treatment with bis-chloroethylnitrosourea (BCNU) on the proliferation and apoptosis of glioma cells.. According to different treatments, glioma U251 cells were randomly divided into blank group, Lipofectamine group, siRNA-Gli1 group, BCNU group and combination group. After treatments, the morphology of U251 cells was visualized under the microscope. Afterwards, semi-quantitative real-time polymerase chain reaction and Western blotting were used to determine Gli1, Bcl-2, Bax and cyclin D1 mRNA levels and protein expression, respectively. MTT assay was used detect the proliferation of U251 cells, while flow cytometry was performed to determine cell apoptosis and cell cycle.. The combination of siRNA-Gli1 and BCNU caused more severe damages to U251 cell shapes compared with siRNA-Gli1 or BCNU alone. The combination of BCNU and siRNA-Gli1 altered mRNA level and protein expression of Bcl-2 and Bax, but not those of Gli1 and cyclin D1. The combination of siRNA-Gli1 and BCNU promoted U251 cell apoptosis. The combination of siRNA-Gli1 and BCNU enhanced the arrestment of U251 cells in G0/G1 phase. The combination of siRNA-Gli1 and BCNU significantly inhibited U251 cell proliferation.. The present study demonstrates that combined treatment with siRNA-Gli1 and BCNU significantly inhibits the proliferation and promotes the apoptosis of glioma U251 cells, possibly by the up-regulation of Bax and the down-regulation of Bcl-2. The combination of siRNA-Gli1 and BCNU enhances the inhibition of cell cycles, but does not down-regulate the expression of cell cycle protein cyclin D1.

Topics: Antineoplastic Agents, Alkylating; Apoptosis; Carmustine; Cell Cycle; Cell Division; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Down-Regulation; Gene Knockdown Techniques; Glioma; Humans; RNA, Messenger; RNA, Small Interfering; Up-Regulation; Zinc Finger Protein GLI1

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

PCDH9, a member of the protocadherin superfamily, is frequently lost in many different cancer types. This study aimed to detect PCDH9 expression in glioma tissues. This study also assessed the effects of PCDH9 expression in two different glioma cell lines. This was accomplished by manipulating PCDH9 expression in these glioma cell lines. The data showed that the expression of PCDH9 mRNA and protein was significantly decreased in gliomas compared to normal brain tissues. Lentivirus carrying PCDH9 cDNA restored PCDH9 expression in the U87 and U251 glioma cell lines. PCDH9 restoration in these cell lines reduced tumor cell viability, induced apoptosis, and caused G0/G1 cell cycle arrest. PCDH9 expression also suppressed the colony formation ability and invasion capacity of U87 and U251 cells. Molecularly, the restoration of PCDH9 expression upregulated Bax protein expression, but downregulated Bcl-2 and cyclin D1 expression. These data from the current study suggest that the loss of PCDH9 expression could contribute to glioma development and/or progression. Further studies will evaluate PCDH9 expression as a biomarker for the early detection of gliomas and as a prognostic indicator for this cancer type.

Topics: Apoptosis; bcl-2-Associated X Protein; Cadherins; Cell Cycle Checkpoints; Cell Line, Tumor; Cyclin D1; Glioma; Humans; Neoplasm Invasiveness; Proto-Oncogene Proteins c-bcl-2; Protocadherins; RNA, Messenger

Special AT-rich sequence-binding protein-1 (SATB1) has been reported to be over-expressed in many human tumors and knockdown of SATB1 can inhibit tumor growth. The present study was designed to determine the role of SATB1 in the growth of human glioma U251 cells using the plasmid-based SATB1 short hairpin RNA (shRNA) delivered by hydroxyapatite nanoparticles in vitro and in vivo. The in vitro growth, invasion and angiogenesis assays of human glioma U251 cells were done. U251 cells tumor blocks were transplanted into the nude mice. CaCl2-modified hydroxyapatite nanoparticles carrying shRNA-SATB1 plasmids were injected into the tumors. The apoptosis of the tumor U251 cells was examined with TUNEL assay and flow cytometer (FCM). The tumor growth and immunohistochemistry were measured. The expression level of SATB1 mRNA was investigated by RT-PCR. The expression levels of SATB1, Cyclin D1, MMP-2, VEGF, Bax and Caspase-9 protein were determined by western blot analysis. The results showed that hydroxyapatite nanoparticles-delivered shRNA-SATB1 could significantly inhibit the growth, invasion and angiogenesis of U251 cells in vitro and the growth of U251 cells in vivo. FCM results showed that Nano HAP-shRNA-SATB1-induced apoptosis (up to 67.8 %). SATB1 expression was strongly down-regulated in the tumor U251 cells. Cyclin D1, MMP-2 and VEGF were also down-regulated in the tumor tissues that also displayed significant increased in Bax expression and Caspase-9 activity. These results show that Nano HAP-shRNA-SATB1 can inhibit the growth of human glioma U251 cells in vitro and in vivo, and hydroxyapatite nanoparticles can be used for the in vitro and in vivo delivery of plasmid-based shRNAs into U251 cells.

Topics: Animals; bcl-2-Associated X Protein; Caspase 9; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Durapatite; Gene Expression Regulation, Neoplastic; Glioma; Humans; Matrix Attachment Region Binding Proteins; Matrix Metalloproteinase 2; Mice; Nanoparticles; RNA, Small Interfering

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

Recently, mutations of the isocitrate dehydrogenase (IDH) 1 gene, which specifically occur in the majority of low-grade and secondary high-grade gliomas, have drawn particular attention of neuro-oncologists. Mutations of the IDH1 gene have been proposed to have significant roles in the tumorigenesis, progression and prognosis of gliomas. However, the molecular mechanism of the role of IDH1 mutants in gliomagenesis remains to be elucidated. The present study, showed that forced expression of an IDH1 mutant, of which the 132th amino acid residue arginine is substituted by histidine (IDH1R132H), promoted cell proliferation in cultured cells, while wild-type IDH1 overexpression had no effect on cell proliferation. Consistent with previous studies, it was also observed that expression of hypoxia-inducible factor 1-α (HIF1-α) was upregulated in IDH1R132H expressing cells with the induction of vascular endothelial growth factor (VEGF) expression. However, knockdown of VEGF via small RNA interference had no significant influence on the cell proliferation induced by overexpression of IDH1R132H, implying that another signaling pathway may be involved. Next, forced expression of IDH1R132H was found to activate nuclear factor-κB (NF-κB), since the inhibitory IκB protein (IκBα) was highly phosphorylated and the NF-κB p65 subunit was translocated into the nucleus. Notably, knockdown of HIF1-α significantly blocked NF-κB activation, which was induced by the overexpression of IDH1 mutants. In addition, expression of IDH1 mutants markedly induced the NF-κB target gene expression, including cyclin D1 and E and c-myc, which were involved in the regulation of cell proliferation. In conclusion, it was demonstrated that the IDH1 mutant activated NF-κB in a HIF1-α‑dependent manner and was involved in the regulation of cell proliferation.

Topics: Active Transport, Cell Nucleus; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin E; Enzyme Activation; Gene Expression; Gene Knockdown Techniques; Glioma; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; I-kappa B Proteins; Isocitrate Dehydrogenase; Mutation; NF-kappa B; NF-KappaB Inhibitor alpha; Phosphorylation; Protein Transport; Proto-Oncogene Proteins c-myc; Vascular Endothelial Growth Factor A

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

Viability, cell cycle distribution, and expressions of eukaryotic translation initiation factor-2α (eIF-2α), cyclin D1, poly(ADP-ribose) polymerase 1 (PARP-1), and apoptosis-inducing factor (AIF) of RT-2 glioma cells were assayed under treatment of tetrandrine and caffeine for 48 h. The results showed that cell viability decreased significantly under treatment with tetrandrine (5 μM) alone or under combined treatment with tetrandrine (5 μM) and caffeine (0.5 or 1 mM). The ratio of RT-2 cells at sub G1 and G0/G1 stages increased significantly during combined treatment of tetrandrine (5 μM) and caffeine (0.5, 1 mM). The ratio of phospharylated eIF-2α to dephospharylated eIF-2α increased, whereas cyclin D1 decreased significantly under combined treatment of tetrandrine (5 μM) and caffeine (1 mM). The cleaved PARP-1 to PARP-1 ratio was elevated significantly under treatment of 5 μM tetrandrine alone, and combined treatment of 5 μM tetrandrine and caffeine (0.5, 1 mM). The expression levels of AIF increased significantly under treatment of 5 μM tetrandrine alone or 1 mM caffeine alone, and combined treatment of 5 μM tetrandrine and caffeine (0.5, 1 mM). In conclusion, tetrandrine and caffeine could induce glioma cell death possibly via increasing eIF-2α phospharylation, decreasing cyclin-D1 expression, and increasing caspase-dependent and -independent apoptosis pathways.

Topics: Animals; Apoptosis; Apoptosis Inducing Factor; Apoptotic Protease-Activating Factor 1; Benzylisoquinolines; Caffeine; Caspase 3; Cell Cycle; Cell Line, Tumor; Cell Survival; Cyclin D1; Eukaryotic Initiation Factor-2; Glioma; Phosphorylation; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerases; Rats; Rats, Inbred F344; Signal Transduction

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

Germacrone is one of the major bioactive components of the traditional Chinese Medicinal plant Curcuma aromatica Salisb. and has been shown to possess anti‑tumor properties. In the present study, the anti‑proliferative effect of germacrone on human glioma cells and the molecular mechanism underlying its cytotoxicity were investigated. Treatment of the U87 and U251 human glioma cell lines with germacrone inhibited the cell proliferation in a dose‑ and time‑dependent manner as assessed by MTT assay, while significantly lower effects were observed on normal human astrocytes. Flow cytometric analysis and DNA fragmentation revealed that germacrone promoted apoptosis of glioma cells, associated with an increased expression of p53 and bax and decreased expression of bcl‑2. Furthermore, flow cytometric cell cycle analysis revealed that germacrone induced G1 phase arrest, associated with an obvious decrease in the expression of cyclin D1 and CDK2 and an increased expression of p21. In conclusion, the present study suggested that germacrone may be a novel potent chemopreventive drug candidate for gliomas via regulating the expression of proteins associated with apoptosis and G1 cell cycle arrest.

Topics: Apoptosis; bcl-2-Associated X Protein; Cell Line, Tumor; Cell Proliferation; Curcuma; Cyclin D1; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase Inhibitor p21; DNA Fragmentation; G1 Phase Cell Cycle Checkpoints; Glioma; Humans; Proto-Oncogene Proteins c-bcl-2; Sesquiterpenes, Germacrane; Tumor Suppressor Protein p53

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

To investigate the role and mechanism of miR-15b in the proliferation and apoptosis of glioma.. The miR-15b mimics were transfected into human glioma cells to upregulate the miR-15b expression. Cyclin D1 was determined by both western blotting analysis and luciferase reporter assay. Methylthiazol tetrazolium (MTT) and flow cytometry were employed to detect the cell proliferation, cell cycle, and apoptosis.. Overexpression of miR-15b inhibits proliferation by arrested cell cycle progression and induces apoptosis, possibly by directly targeting Cyclin D1. Both luciferase assay and bioinformatics search revealed a putative target site of miR-15b binding to the 3'-UTR of Cyclin D1. Moreover, expression of miR-15b in glioma tissues was found to be inversely correlated with Cyclin D1 expression. Enforced Cyclin D1 could abrogate the miR-15b-mediated cell cycle arrest and apoptosis.. Our findings identified that miR-15b may function as a glioma suppressor by targeting the Cyclin D1, which may provide a novel therapeutic strategy for treatment of glioma.

Topics: Apoptosis; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Gene Expression Regulation, Neoplastic; Glioma; Humans; MicroRNAs

The HIWI gene is one of the members of the PIWI gene family that is important for stem cell self‑renewal and expressed highly in certain human tumors. Some studies have demonstrated that HIWI plays a key role in the development of tumors in cervical, colon and liver cancer. Previous studies have demonstrated that HIWI is associated with prognosis of patients with glioma. However, there is no report on the analysis of HIWI in the biological characteristics of glioma cells. The aim of the study was to investigate whether HIWI plays an important role in the progress of glioma. Silencing HIWI inhibited cell proliferation by promoting apoptosis and increased cell cycle arrest. The expression of proteins related to apoptosis and the cell cycle, including p21, cyclin D1, Bcl-2, and Bax was significantly altered. Moreover, knockdown of HIWI inhibited the migration and invasion of glioma cells by reducing the expression of MMP-2 and MMP‑9. Furthermore, we found that reduction of HIWI inhibited tumor growth in vivo. These findings suggest that HIWI is an oncogene involved in the progression of glioma.

Topics: Apoptosis; Argonaute Proteins; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Gene Expression Regulation, Neoplastic; Glioma; Humans; Matrix Metalloproteinase 2; Neoplasm Invasiveness; Prognosis

Notch signaling has been reported to be oncogenic or tumor suppressive, depending on the tissue context. To investigate the effects of Notch2 knockdown on U87 human glioma cell proliferation in vitro and in vivo, and the associated mechanisms, U87 cells were stably transfected with p green fluorescent protein (GFP)‑V‑RS Notch2 short hairpin (sh) RNA plasmid and pGFP‑V‑RS scramble‑shRNA plasmid. The former was referred to as the Notch2‑shRNA group and the latter as the negative‑shRNA group. mRNA and protein expression, cell proliferation, cell cycle and apoptosis were measured by reverse transcription‑polymerase chain reaction, western blot analysis, 3‑(4,5‑dimethylthiazol‑2‑yl)‑2,5‑diphenyltetrazolium bromide analysis and flow cytometry using propidium iodide, respectively. Tumor volume, tumor weight and cumulative survival rate were determined in a nude mouse xenograft tumor model. Notch2 mRNA and protein expression in the Notch2‑shRNA group were reduced by 87.6 and 94.5% compared with the negative‑shRNA group (P<0.001). Notch2 knockdown significantly inhibited U87 cell proliferation after three days of culture (P<0.05). Notch2 silencing induced cell cycle arrest at G0/G1 phase by upregulation of p21 protein expression and downregulation of mini chromosome maintenance complex 2 and cyclin‑D1 protein expression. Furthermore, knockdown of Notch2 also induced U87 cell apoptosis. On day 50 after inoculation, tumor weight in the Notch2‑shRNA group was significantly lower than that in the negative‑shRNA group (0.55±0.10 vs. 1.23±0.52 g; P<0.01). The cumulative survival rate was significantly longer in the Notch2‑shRNA group compared with the negative‑shRNA group (log rank test P=0.01). In conclusion, Notch2 silencing inhibited U87 glioma cell proliferation by inducing cell cycle arrest and apoptosis in vitro and in vivo. Thus, Notch2 may be a key therapeutic target for the treatment of glioma.

Topics: Animals; Apoptosis; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Down-Regulation; Female; G1 Phase; Gene Expression Regulation, Neoplastic; Glioma; Humans; In Vitro Techniques; Mice; Mice, Inbred BALB C; Mice, Nude; Receptor, Notch2; Resting Phase, Cell Cycle; RNA Interference; RNA, Messenger; RNA, Small Interfering

In recent years, microRNAs (miRNAs) have been proved to be closely related to the tumorigenesis and progression. An increasing number of researches have shown that microRNAs function as oncogenes or tumor suppressor genes in human malignant tumors. This study aims to explore the effects of microRNA-383 (miR-383) on malignant biological function of human gliomas. We detected the expression of miR-383 in glioma tissues and normal brain tissues by quantitative real-time PCR. Anchorage-independent growth assays, and flow cytometry were used to evaluate the functions of miR-383 that involves in cell growth and cell cycle. Western blotting assay was used to examine protein expression levels of Cyclin D1 (CCND1), a cell cycle-associated oncogene which has a predicted binding site of miR-383 within its 3'-untranslated region (3'-UTR), and luciferase activity assay was used to evaluate the 3'-UTR activity of CCND1. In this study, we found that miR-383 expression level was lower in gliomas than normal brain tissues. Overexpression of miR-383 in U251 and U87 cells showed a significant inhibitory effect on cell growth, which accompanied with cell cycle G0/G1 arrest as well as downregulation of CCND1 expression. Moreover, CCND1 was verified to be one of the direct targets of miR-383. In summary, this study suggested that miR-383 plays the role of tumor suppressor by targeting CCND1 in glioma cells, and may be useful for developing a new therapeutic strategy for gliomas.

Topics: Apoptosis; Cell Adhesion; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Glioma; Humans; MicroRNAs

Rosette-forming glioneuronal tumor of the fourth ventricle (RGNT) is a new entity in the WHO 2007 Classification of Tumors of the Central Nervous System. RGNT has two components: neurocytic rosettes and low-grade gliomas. Neurocytic rosettes are conventionally described as consisting of uniform neurocytes. However, some studies have reported rosette-forming tumor cells that expressed glial markers such as Olig2. We indicated the expression of glial markers including Olig2, cyclinD1, glial fibrillary acidic protein (GFAP), and platelet-derived growth factor receptor alpha (PDGFRα) in the neurocytic rosettes in our previous study, and we suggested that these tumor cells had a heterogeneous nature. In this study, we used double and triple immunostaining to demonstrate that these tumor cells have both glial and neuronal characteristics. We found that rosette-forming tumor cells coexpressed Olig2/cyclinD1 and synaptophysin. Furthermore, the cores of the rosettes coexpressed GFAP/PDGFRα in the peripheral zone and synaptophysin in the central zone. These findings imply that rosette-forming tumor cells have a similar nature to neuronal-glial progenitor cells, and we believe that the nomination "neurocytic rosette" may be unsuitable given their heterogeneous nature. Our study appears to clarify some of the properties of RGNT tumor cells and may help elucidate the histogenesis of RGNT.

Topics: Adolescent; Adult; Basic Helix-Loop-Helix Transcription Factors; Cerebral Ventricle Neoplasms; Cyclin D1; Female; Fourth Ventricle; Gene Expression; Glial Fibrillary Acidic Protein; Glioma; Humans; Immunohistochemistry; Male; Nerve Tissue Proteins; Oligodendrocyte Transcription Factor 2; Receptor, Platelet-Derived Growth Factor alpha; Rosette Formation; Young Adult

Malignant gliomas persist as a major disease responsible for high morbidity and mortality rates in adults. Differentiation therapy has emerged as a promising treatment modality. Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) gene function is commonly lost in primary gliomas, particularly in glioblastomas, and this is associated with tumor differentiation. PTEN gene deletion is one of the main molecular events in gliomas. In this study, we aimed to explore the effect and mechanisms of PTEN on cholera toxin (CT)‑induced SWO-38 glioma cell differentiation. It has been shown that transfection of the exogenous PTEN gene induces glioma cell differentiation; however, the underlying mechanism remains to be elucidated. Results of the present study showed that CT-induced SWO-38 glioma cell differentiation was characterized by morphological changes, the increased expression of glial fibrillary acidic protein (GFAP), an accumulation of cells in the G0/G1 phase of the cell cycle, the decreased expression of cyclin D1 and a decreased invasion and migration capacity. Silencing of the PTEN protein using RNA interference resulted in suppressed cell differentiation. Furthermore, inhibition of the PI3K/AKT pathway by the inhibitor LY294002 led to attenuated differentiation, while differentiation remained stable with the inhibition of the MAPK/ERK pathway by PD0325901. Thus, PTEN may be important in glioma cell differentiation.

Topics: Cell Differentiation; Cell Line, Tumor; Cell Movement; Cholera Toxin; Chromones; Cyclin D1; G1 Phase Cell Cycle Checkpoints; Glial Fibrillary Acidic Protein; Glioma; Humans; Morpholines; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; RNA Interference; RNA, Small Interfering; Signal Transduction; Transfection

To study the expression of FOS protein in human glioma tissues and its effect on tumor growth.. FOS protein expression in glioma tissues was determined with immunohistochemical (IHC) staining. Subsequently, 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-diphenytetrazoliumromide (MTT) assay, flow cytometry, transwell invasion and Western blotting were used to assay U87 and U251 cells with reduced FOS expression.. The expression of FOS in glioma was increased and strongly correlated with its pathological grade. Abrogating expression of FOS has suppressed proliferation and invasion, and delayed cell cycle at G1 phrase for both U87 and U251 cells.. The expression of FOS protein in human glioma was strong. FOS protein probably plays a critical role in the progression of gliomas.

Topics: Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Gene Expression; Glioma; Humans; Matrix Metalloproteinase 9; Neoplasm Grading; Proto-Oncogene Proteins c-fos; RNA Interference

Emerging evidence have suggested that calorie restriction (CR) is a reliable method to decrease cancer development since it produces changes in tumor microenvironment that interfere with cell proliferation, tissue invasion, and formation of metastases. Studies on the role of pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) in cancer cells indicate that their influence on cell growth is either cell type specific or dependent on culture conditions. Evidence showing the effect of PACAP and VIP in glioma cells grown under conditions mimicking CR are currently unavailable. Therefore, we explored the effects of both PACAP and VIP in C6 glioma cells either grown in a normal growth medium or exposed to serum starvation, to resemble an acute condition of CR. Cell viability, expression of proteins related to cell proliferation (cyclin D1), apoptosis (Bcl2, p53, and cleaved caspase-3), and cell malignancy (GFAP and nestin) were assessed by MTT assay, immunoblot, and immunolocalization, respectively. Results demonstrated that CR significantly decreased cell proliferation, reduced levels of cyclin D1 and Bcl2, and increased the expression of p53 and cleaved caspase-3. Surprisingly, all of these CR-driven effects were further exacerbated by PACAP or VIP treatment. We also found that PACAP or VIP prevented GFAP decrease caused by CR and further reduced the expression of nestin, a prognostic marker of malignancy. In conclusion, these data demonstrate that PACAP and VIP possess antiproliferative properties against glioma cells that depend on the specific culture settings, further supporting the idea that CR might offer new avenues to improve peptide-oriented glioma cancer treatment.

Topics: Animals; Apoptosis Regulatory Proteins; Cell Line, Tumor; Cell Proliferation; Culture Media, Serum-Free; Cyclin D1; Glial Fibrillary Acidic Protein; Glioma; Nestin; Pituitary Adenylate Cyclase-Activating Polypeptide; Rats; Transcription, Genetic; Tumor Suppressor Protein p53; Vasoactive Intestinal Peptide

Using a GWA analysis of a comprehensive glioma specimen population, we identified whole gain of chromosome 19 as one of the major chromosomal aberrations that correlates to patients' outcomes. Our analysis of significant loci revealed for the first time NOTCH3 as one of the most significant amplification. NOTCH3 amplification is associated with worse outcome compared to tumors with non-amplified locus. NOTCH receptors (NOTCH1-4) are key positive regulators of cell-cell interactions, angiogenesis, cell adhesion and stem cell niche development which have been shown to play critical roles in several human cancers. Our objective is to determine the molecular roles of NOTCH3 in glioma pathogenesis and aggressiveness. Here we show for the first time that NOTCH3 plays a major role in glioma cell proliferation, cell migration, invasion and apoptosis. Therefore, our study uncovers the prognostic value and the oncogenic function of NOTCH3 in gliomagenesis and supports NOTCH3 as a promising target of therapy in high grade glioma. Our studies allowed the identification of a subset of population that may benefit from GSI- or anti-NOTCH3- based therapies. This may lead to the design of novel strategies to improve therapeutic outcome of patients with glioma by establishing medical and scientific basis for personalized chemotherapies.

Topics: Apoptosis; Cell Cycle; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; ErbB Receptors; Gene Knockdown Techniques; Glioma; Humans; Neoplasm Grading; Neoplasm Invasiveness; Oncogenes; Prognosis; Receptor, Notch3; Receptors, Notch

Previous studies have shown that miR-137 functions as a tumor suppressor in various cancers, but its role in the initiation and development of gliomas is still unknown. Currently, we found that miR-137 exhibited the most significant increase in normal brain tissues compared with glioma specimens, and the miR-137 expression was greatly decreased with the ascending of tumor pathological grades. Furthermore, overexpression of miR-137 in vitro by chemically synthesized miR-137 mimics suppressed the proliferation, inhibited cell cycle arrest in the G1/G0 phase, and induced cell apoptosis. The tumor-suppressive effects of miR-137 were indeed induced by Rac1, which was verified as a direct target of miR-137. These findings indicate that miR-137 inhibits the growth of gliomas cells by directly targeting Rac1, suggesting that miR-137 could be a new important therapeutic strategy for glioma treatment and warrants further investigation.

Topics: Apoptosis; Brain Chemistry; Cell Line, Tumor; Cell Proliferation; Central Nervous System Neoplasms; Cyclin D1; G1 Phase Cell Cycle Checkpoints; Glioma; Humans; Matrix Metalloproteinase 2; MicroRNAs; Neoplasm Grading; Neoplasm Invasiveness; Proto-Oncogene Proteins c-bcl-2; rac1 GTP-Binding Protein; RNA, Small Interfering; Signal Transduction; Transfection

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

5'-Βenzylglycinyl-amiloride (UCD38B) and glycinyl-amiloride (UCD74A) are cell-permeant and cell-impermeant derivatives of amiloride, respectively, and used here to identify the cellular mechanisms of action underlying their antiglioma effects. UCD38B comparably kills proliferating and nonproliferating gliomas cells when cell cycle progression is arrested either by cyclin D1 siRNA or by acidification. Cell impermeant UCD74A inhibits plasmalemmal urokinase plasminogen activator (uPA) and the type 1 sodium-proton exchanger with potencies analogous to UCD38B, but is cytostatic. In contrast, UCD38B targets intracellular uPA causing mistrafficking of uPA into perinuclear mitochondria, reducing the mitochondrial membrane potential, and followed by the release of apoptotic inducible factor (AIF). AIF nuclear translocation is followed by a caspase-independent necroptotic cell death. Reduction in AIF expression by siRNA reduces the antiglioma cytotoxic effects of UCD38B, while not activating the caspase pathway. Ultrastructural changes shortly following treatment with UCD38B demonstrate dilation of endoplasmic reticulum (ER) and mitochondrial swelling followed by nuclear condensation within hours consistent with a necroptotic cell death differing from apoptosis and from autophagy. These drug mechanism of action studies demonstrate that UCD38B induces a cell cycle-independent, caspase-independent necroptotic glioma cell death that is mediated by AIF and independent of poly (ADP-ribose) polymerase and H2AX activation.

Topics: Amiloride; Apoptosis; Apoptosis Inducing Factor; Autophagy; Caspases; Cell Cycle; Cell Cycle Checkpoints; Cell Death; Cell Line, Tumor; Cell Nucleus; Cell Proliferation; Cyclin D1; Endoplasmic Reticulum; Glioma; Histones; Humans; Membrane Potential, Mitochondrial; Mitochondria; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerases; Protein Transport; RNA, Small Interfering; Sodium-Hydrogen Exchangers; Urokinase-Type Plasminogen Activator

RLIP76, a GTPase-activating protein, is a central regulator in multiple pathways that respond to redox states and control cell growth, motility, division, and apoptosis in many malignant cancer cells. In this study, human glioblastoma cell lines U87 and U251 were stably transfected with a lentivirus vector expressing a short hairpin RNA (shRNA) targeting RLIP76. shRNA knockdown of RLIP76 induced cell cycle arrest in U87 and U251 cells and inhibited their invasiveness. Quantitative Western blot analysis revealed that cells stably underexpressing RLIP76 showed lower expression of cyclin D1 and decreased expression and activity of matrix metalloproteinase 2 compared to cells stably transfected with a control vector. Furthermore, RLIP76 expression levels were correlated with IC(50) values for the antitumor drug temozolomide (TMZ). Compared with TMZ alone (17.19 ± 1.78 and 22.18 ± 1.99 μg/mL in U87 and U251 cells, respectively) or combined shGFP and TMZ (18.04 ± 1.07 and 23.040 ± 1.77 μg/mL in U87 and U251 cells, respectively), combined shRNA and TMZ therapy resulted in a significant decrease in IC(50) value (7.61 ± 2.99 and 6.91 ± 2.59 μg/mL in U87 and U251 cells, respectively). Combined RLIP76 knockdown and TMZ treatment inhibited cell proliferation in vitro more effectively than either treatment alone. Furthermore, RLIP76 downregulation enhanced chemosensitivity to TMZ without affecting protein expression of MDR1 and MRP1. The results indicate that inhibition of RLIP76 expression may be an effective means for overcoming RLIP76-associated chemoresistance in human malignant glioma cells and may represent a potential gene-targeting approach for glioma treatment.

Topics: Antineoplastic Agents, Alkylating; ATP Binding Cassette Transporter, Subfamily B; ATP Binding Cassette Transporter, Subfamily B, Member 1; ATP-Binding Cassette Transporters; Cell Cycle Checkpoints; Cell Line, Tumor; Cyclin D1; Dacarbazine; Flow Cytometry; Gene Expression Regulation, Neoplastic; Glioma; Green Fluorescent Proteins; GTPase-Activating Proteins; Humans; Multidrug Resistance-Associated Proteins; Neoplasm Invasiveness; RNA Interference; RNA, Messenger; RNA, Small Interfering; Temozolomide; Time Factors; Transfection

Tumor necrosis factor receptor-associated factor 6 (TRAF6), which plays an important role in inflammation and immune response, is an essential adaptor protein for the NF-κB (nuclear factor κB) signaling pathway. Recent studies have shown that TRAF6 played an important role in tumorigenesis and invasion by suppressing NF-κB activation. However, up to now, the biologic role of TRAF6 in glioma has still remained unknown. To address the expression of TRAF6 in glioma cells, four glioma cell lines (U251, U-87MG, LN-18, and U373) and a non-cancerous human glial cell line SVG p12 were used to explore the protein expression of TRAF6 by Western blot. Our results indicated that TRAF6 expression was upregulated in human glioma cell lines, especially in metastatic cell lines. To investigate the role of TRAF6 in cell proliferation, apoptosis, invasion, and migration of glioma, we generated human glioma U-87MG cell lines in which TRAF6 was either overexpressed or depleted. Subsequently, the effects of TRAF6 on cell viability, cell cycle distribution, apoptosis, invasion, and migration in U-87MG cells were determined with 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyl tetrazolium bromide (MTT) assay, flow cytometry analysis, transwell invasion assay, and wound-healing assay. The results showed that knockdown of TRAF6 could decrease cell viability, suppress cell proliferation, invasion and migration, and promote cell apoptosis, whereas overexpression of TRAF6 displayed the opposite effects. In addition, the effects of TRAF6 on the expression of phosphor-NF-κB (p-p65), cyclin D1, caspase 3, and MMP-9 were also probed. Knockdown of TRAF6 could lower the expression of p-p65, cyclin D1, and MMP-9, and raise the expression of caspase 3. All these results suggested that TRAF6 might be involved in the potentiation of growth, proliferation, invasion, and migration of U-87MG cell, as well as inhibition of apoptosis of U-87MG cell by abrogating activation of NF-κB.

Topics: Apoptosis; Caspase 3; Cell Cycle; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Survival; Cyclin D1; Gene Expression; Glioma; Humans; Matrix Metalloproteinase 9; Phosphorylation; Protein Processing, Post-Translational; TNF Receptor-Associated Factor 6; Transcription Factor RelA

Glioma proliferation is a multistep process during which a sequence of genetic and epigenetic alterations randomly occur to affect the genes controlling cell proliferation, cell death and genetic stability. microRNAs are emerging as important epigenetic modulators of multiple target genes, leading to abnormal cellular signaling involving cellular proliferation in cancers.In the present study, we found that expression of miR-195 was markedly downregulated in glioma cell lines and human primary glioma tissues, compared to normal human astrocytes and matched non-tumor associated tissues. Upregulation of miR-195 dramatically reduced the proliferation of glioma cells. Flow cytometry analysis showed that ectopic expression of miR-195 significantly decreased the percentage of S phase cells and increased the percentage of G1/G0 phase cells. Overexpression of miR-195 dramatically reduced the anchorage-independent growth ability of glioma cells. Furthermore, overexpression of miR-195 downregulated the levels of phosphorylated retinoblastoma (pRb) and proliferating cell nuclear antigen (PCNA) in glioma cells. Conversely, inhibition of miR-195 promoted cell proliferation, increased the percentage of S phase cells, reduced the percentage of G1/G0 phase cells, enhanced anchorage-independent growth ability, upregulated the phosphorylation of pRb and PCNA in glioma cells. Moreover, we show that miR-195 inhibited glioma cell proliferation by downregulating expression of cyclin D1 and cyclin E1, via directly targeting the 3'-untranslated regions (3'-UTR) of cyclin D1 and cyclin E1 mRNA. Taken together, our results suggest that miR-195 plays an important role to inhibit the proliferation of glioma cells, and present a novel mechanism for direct miRNA-mediated suppression of cyclin D1 and cyclin E1 in glioma.

Topics: 3' Untranslated Regions; Animals; Astrocytes; Base Sequence; Biomarkers, Tumor; Cell Line, Tumor; Cell Proliferation; Cell Transformation, Neoplastic; Cyclin D1; Cyclin E; Down-Regulation; Glioma; Humans; Mice; Mice, Inbred BALB C; MicroRNAs; Oncogene Proteins; Proliferating Cell Nuclear Antigen

Glioma is the most common type of brain tumor, and has the worst prognosis in human malignancy. Experimental evidence suggests that the use of high concentrations of various amino acids may perturb neoplastic cell growth. Thus, the aim of this study was to investigate whether essential amino acids can alter the growth and proliferation of glioma cells. Studies were performed using C6 rat glioma cell lines. High concentration of L-leucine induced growth arrest of glioma cell lines. Terminal transferase uridyl nick end labeling assay and cell cycle analysis showed that the effect of L-leucine on glioma cells growth was not cytotoxic, but rather cytostatic. Additionally, the extracellular signal-regulated protein kinase was activated in L-leucine-treated glioma cells, and inhibition of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1 (MEK) enhanced the effect of L-leucine on glioma cell growth. These data suggest that high concentration L-leucine combined with inhibition of MEK is a potential strategy for glioma cell growth arrest.

Topics: Amino Acids, Cyclic; Animals; Antineoplastic Agents; Apoptosis; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cell Shape; Cell Survival; Cyclin D1; Drug Synergism; Enzyme Activation; Extracellular Signal-Regulated MAP Kinases; Flavonoids; Glioma; JNK Mitogen-Activated Protein Kinases; Large Neutral Amino Acid-Transporter 1; Leucine; MAP Kinase Signaling System; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Protein Processing, Post-Translational; Rats

A common polymorphism (G870A) in the exon 4/intron 4 boundary of CCND1 gene has been linked to an alternate transcript, cyclin D1b, which preferentially encodes a protein with an increased transforming capability compared with the full-length D1a. Therefore, the CCND1 G870A polymorphism may influence an individual's susceptibility to the development of certain tumors. In the present study, we investigated the association of CCND1 G870A polymorphism with glioma cancer risk in a northern Chinese population. By polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis, CCND1 G870A genotyping was carried out among 170 glioma patients and 170 age, gender-matched healthy control subjects. The CCND1 870 A allele was more frequently observed in patients than in controls (0.57 vs. 0.48, p=0.03), and an increased risk of glioma cancer was observed for the AA genotype compared with the GG and AG genotypes (odds ratio [OR]=1.828; 95% confidence interval [CI]: 1.150-2.908, p=0.01), particularly among female groups, or ages ≤45 groups (OR=2.204, 95% CI: 1.220-3.981, p=0.008). Significant associations were also observed between the AA genotype and glioma risk in the subgroups of World Health Organization (WHO) grade II gliomas and grade III gliomas. Further reverse transcriptase-polymerase chain reaction analysis (RT-PCR) also revealed a stronger positive correlation between the AA genotype and higher cyclin D1b expression among glioma patients either in the mean quantitative value, or the cyclin D1b/cyclin D1a ratio in the same tumor tissue (p=0.008, p=0.004, respectively). In conclusion, our data indicate that the CCND1 G870A polymorphism modulated oncogenic cyclin D1b expression in glioma tissues and may be associated with an increased risk of gliomas in Chinese population.

Topics: Adult; Age of Onset; Asian People; Cyclin D1; Female; Gene Expression Regulation, Neoplastic; Genetic Predisposition to Disease; Glioma; Humans; Male; Polymorphism, Single Nucleotide; RNA, Messenger

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

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

Phosphorylation of Tyr-88/Tyr-89 in the 3(10) helix of p27 reduces its cyclin-dependent kinase (CDK) inhibitory activity. This modification does not affect the interaction of p27 with cyclin-CDK complexes but does interfere with van der Waals and hydrogen bond contacts between p27 and amino acids in the catalytic cleft of the CDK. Thus, it had been suggested that phosphorylation of this site could switch the tumor-suppressive CDK inhibitory activity to an oncogenic activity. Here, we examined this hypothesis in the RCAS-PDGF-HA/nestin-TvA proneural glioma mouse model, in which p21 facilitates accumulation of nuclear cyclin D1-CDK4 and promotes tumor development. In these tumor cells, approximately one-third of the p21 is phosphorylated at Tyr-76 in the 3(10) helix. Mutation of this residue to glutamate reduced inhibitory activity in vitro. Mutation of this residue to phenylalanine reduced the tumor-promoting activity of p21 in the animal model, whereas glutamate or alanine substitution allowed tumor formation. Consequently, we conclude that tyrosine phosphorylation contributes to the conversion of CDK inhibitors from tumor-suppressive roles to oncogenic roles.

Topics: Amino Acid Sequence; Animals; Cell Proliferation; Central Nervous System Neoplasms; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Disease Models, Animal; Gene Expression Regulation, Neoplastic; Glioblastoma; Glioma; HEK293 Cells; Humans; Mice; Molecular Sequence Data; Mutation; Phosphorylation; Tyrosine

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

Gliomas are the most common primary intracranial tumors. Understanding the molecular basis of gliomas' progression is required to develop more effective therapies. The Wnt/β-catenin signaling cascade is an important signal transduction pathway in human cancers. Although, overactivation of this pathway is a hallmark of several forms of cancer, little is known about its role in human gliomas. Here, we aimed to determine the clinical significance of Wnt/β-catenin pathway components in gliomas. Immunohistochemical staining was performed to detect the expression patterns of Wnt1, β-catenin and Cyclin D1 in the biopsies from 96 patients with primary gliomas. Kaplan-Meier survival and Cox regression analyses were performed to evaluate the prognosis of patients. Cytoplasmic staining pattern of Wnt1, membranous, cytoplasmic and nuclear accumulation of β-catenin, and nuclear localization of Cyclin D1 were demonstrated by immunohistochemical staining. The Wnt1 expression significantly correlated with the expression of Cyclin D1 (P < 0.0001). The ratio of tumors with a cytoplasmic-nuclear pattern or a cytoplasmic pattern of β-catenin was significantly higher in Wnt1-positive (P < 0.01) and Cyclin D1-positive (P < 0.01) tumors than in Wnt1-negative and Cyclin D1-negative tumors, respectively. The protein expression levels of Wnt1, β-catenin and Cyclin D1 were all positively correlated with the Karnofsky performance scale (KPS) score and World Health Organization (WHO) grades of patients with gliomas. Furthermore, Wnt1, cytoplasmic-nuclear β-catenin and Cyclin D1 status were all the independent prognostic factors for glioma patients (P = 0.01, 0.007 and 0.005, respectively). These results provide convincing evidence that the Wnt/β-catenin pathway correlated closely with the progression of gliomas and might be a novel prognostic marker for this neoplasm.

Topics: Adult; Aged; Aged, 80 and over; beta Catenin; Biopsy; Cyclin D1; Gene Expression Profiling; Glioma; Humans; Immunohistochemistry; Male; Middle Aged; Prognosis; Severity of Illness Index; Wnt1 Protein

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

Glioma stem cells (GSCs), which are originated from transformed neural stem cells, are tumor-initiating cells of glioma, the most common primary malignant neoplasm of the central nervous system. Extensive studies have shown that bone morphogenetic protein 4 (BMP4) plays an important role in the differentiation and proliferation of neural stem cells. To seek the functions and mechanisms of BMP4 in GSCs, GSCs isolated from U87 human glioma cells by using vincristine were exposed to BMP4 protein. This study shows that BMP4 inhibited U87 GSC proliferation (p < 0.01) via downregulation of cyclin D1 level and promoted GSC apoptosis through induction of Bax expression and inhibition of Bcl-2 and Bcl-xL levels. Thus, these results indicate a new approach of GSC-based glioma treatment.

Topics: Apoptosis; bcl-2-Associated X Protein; bcl-X Protein; Bone Morphogenetic Protein 4; Cell Differentiation; Cell Growth Processes; Cell Line, Tumor; Cyclin D1; Down-Regulation; Glioma; Humans; Neoplastic Stem Cells; Proto-Oncogene Proteins c-bcl-2; Vincristine

Malignant gliomas are the most common and lethal intracranial tumors, and differentiation therapy shows great potential to be a promising candidate for their treatment. Here, we have elaborated that a PKA activator, forskolin, represses cell growth via cell cycle arrest in the G0/G1 phase and induces cell differentiation characteristic with elongated processes and restoration of GFAP expression. In mechanisms, we verified that forskolin significantly diminishes the mRNA and protein level of a key cell cycle regulator cyclin D1, and maintenance of low cyclin D1 expression level was required for forskolin-induced proliferation inhibition and differentiation by gain and loss of function approaches. In addition, that forskolin down-regulated the cyclin D1 by proteolytic (post-transcriptional) mechanisms was dependent on GSK-3β activation at Ser9. The pro-differentiation activity of forskolin and related molecular mechanisms imply that forskolin can be developed into a candidate for the future in differentiation therapy of glioma, and cyclin D1 is a promising target for pro-differentiation strategy.

Topics: Animals; Cell Cycle Checkpoints; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Colforsin; Cyclin D1; Down-Regulation; Enzyme Activators; Gene Expression Regulation; Glioma; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Phosphorylation; Proteolysis; Rats; Transcription, Genetic

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

Signal transducer and activator of transcription 3 (STAT3) is usually constitutively activated in a variety of malignancies. Thus, STAT3 may be a promising target for treatment of tumor cells. Recently, Tanshinone IIA (Tan IIA), a major active constituent from the root of Salvia miltiorrhiza Bunge, was reported to have apoptosis inducing effects on a large variety of cancer cells. In this study, we evaluate the anti-proliferation and apoptosis inducing effects of Tan IIA on C6 glioma cells. Cell growth and proliferation were measured by MTT assay, cell apoptosis was observed by flow cytometry and DNA-fragmentation analysis. Further more, we investigated inhibitory effects of Tan IIA on STAT3 activity and its downstream targets: Bcl-XL, cyclin D1. Alteration of STAT3 activity was examined by measuring their DNA binding activity and tyrosine phosphorylation. Changes in the expression levels of Bcl-XL and cyclin D1 were examined by Western blot analysis. We found that the cellular growth were inhibited and cell apoptosis were observed after the treatment with Tan IIA. The STAT3 activity was significantly reduced by Tan IIA parallel with a significant attenuation of expression of Bcl-XL and cyclin D1. These results suggest that Tan IIA may serve as an effective adjunctive reagent in the treatment of glioma for its targeting of constitutive STAT3 signaling.

Topics: Abietanes; Animals; Antineoplastic Agents, Phytogenic; Apoptosis; bcl-X Protein; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cyclin D1; DNA Fragmentation; Dose-Response Relationship, Drug; Glioma; Phenanthrenes; Rats; STAT3 Transcription Factor; Time Factors

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

Cathepsin B and urokinase plasminogen activator receptor (uPAR) are both known to be overexpressed in gliomas. Our previous work and that of others strongly suggest a relationship between the infiltrative phenotype of glioma and the expression of cathepsin B and uPAR. Though their role in migration and adhesion are well studied the effect of these molecules on cell cycle progression has not been thoroughly examined.. Cathepsin B and uPAR single and bicistronic siRNA plasmids were used to downregulate these molecules in SNB19 and U251 glioma cells. FACS analysis and BrdU incorporation assay demonstrated G0/G1 arrest and decreased proliferation with the treatments, respectively. Immunoblot and immunocyto analysis demonstrated increased expression of p27(Kip1) and its nuclear localization with the knockdown of cathepsin B and uPAR. These effects could be mediated by alphaVbeta3/PI3K/AKT/FOXO pathway as observed by the decreased alphaVbeta3 expression, PI3K and AKT phosphorylation accompanied by elevated FOXO3a levels. These results were further confirmed with the increased expression of p27(Kip1) and FOXO3a when treated with Ly294002 (10 microM) and increased luciferase expression with the siRNA and Ly294002 treatments when the FOXO binding promoter region of p27(Kip1) was used. Our treatment also reduced the expression of cyclin D1, cyclin D2, p-Rb and cyclin E while the expression of Cdk2 was unaffected. Of note, the Cdk2-cyclin E complex formation was reduced significantly.. Our study indicates that cathepsin B and uPAR knockdown induces G0/G1 arrest by modulating the PI3K/AKT signaling pathway and further increases expression of p27(Kip1) accompanied by the binding of FOXO3a to its promoter. Taken together, our findings provide molecular mechanism for the G0/G1 arrest induced by the downregulation of cathepsin B and uPAR in SNB19 and U251 glioma cells.

Topics: Animals; Blotting, Western; Cathepsin B; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Chromones; Cyclin D1; Cyclin D2; Cyclin E; Cyclin-Dependent Kinase Inhibitor p27; Enzyme-Linked Immunosorbent Assay; Fluorescent Antibody Technique; Forkhead Transcription Factors; Glioma; Humans; Immunoprecipitation; Mice; Mice, Nude; Models, Biological; Morpholines; Promoter Regions, Genetic; Receptors, Urokinase Plasminogen Activator; Reverse Transcriptase Polymerase Chain Reaction; RNA, Small Interfering

To investigate the role of bone morphogenetic protein 4 (BMP4) on the proliferation and apoptosis in glioma stem cells.. Stem cells were isolated from a human glioma cell line U87 by using vincristine and characterized by immunofluorescence assay. Proliferation and apoptosis were determined by soft agar colony assay and flow cytometry; Cyclin D1, Bcl-2 and Bax were detected by Western blot analysis.. BMP4 inhibited cell proliferation and promoted apoptosis in U87 glioma stem cells. Moreover, Bcl-2 and Cyclin D1 expression were decreased by BMP4, while Bax level was elevated.. BMP4 can inhibit U87 glioma stem cells proliferation through downregulating Cyclin D1 level, and promote apoptosis through induction of Bax expression and inhibition of Bcl-2 level. It suggests that BMP4 plays an important role in human glioma stem cell biology.

Topics: Apoptosis; Bone Morphogenetic Protein 4; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Gene Expression Regulation, Neoplastic; Glioma; Humans; Neoplastic Stem Cells; Proto-Oncogene Proteins c-bcl-2

The detection of cell cycle proteins in Alzheimer's disease (AD) brains may represent an early event leading to neurodegeneration. To identify cell cycle modifiers with anti-Abeta properties, we assessed the effect of Differentiation-Inducing Factor-1 (DIF-1), a unique, small-molecule from Dictyostelium discoideum, on the proteolysis of the amyloid beta-protein precursor (APP) in a variety of different cell types. We show that DIF-1 slows cell cycle progression through G0/G1 that correlates with a reduction in cyclin D1 protein levels. Western blot analysis of DIF-treated cells and conditioned medium revealed decreases in the levels of secreted APP, mature APP, and C-terminal fragments. Assessment of conditioned media by sandwich ELISA showed reduced levels of Abeta40 and Abeta42, also demonstrating that treatment with DIF-1 effectively decreases the ratio of Abeta42 to Abeta40. In addition, DIF-1 significantly diminished APP phosphorylation at residue T668. Interestingly, site-directed mutagenesis of APP residue Thr668 to alanine or glutamic acid abolished the effect of DIF-1 on APP proteolysis and restored secreted levels of Abeta. Finally, DIF-1 prevented the accumulation of APP C-terminal fragments induced by the proteasome inhibitor lactacystin, and calpain inhibitor N-acetyl-leucyl-leucyl-norleucinal (ALLN). Our findings suggest that DIF-1 affects G0/G1-associated amyloidogenic processing of APP by a gamma-secretase-, proteasome- and calpain-insensitive pathway, and that this effect requires the presence of residue Thr668.

Topics: Acetylcysteine; Amyloid beta-Peptides; Amyloid beta-Protein Precursor; Animals; Benzazepines; Cell Line; Cell Line, Tumor; CHO Cells; Cricetinae; Cricetulus; Cyclin D1; Fibroblasts; Glioma; Hexanones; Humans; Hydrocarbons, Chlorinated; Indoles; Leupeptins; Mice; Peptide Fragments; Proteasome Inhibitors; Purines; Recombinant Fusion Proteins; Roscovitine; Threonine

Gamma-linolenic acid is a known inhibitor of tumour cell proliferation and migration in both in vitro and in vivo conditions. The aim of the present study was to determine the mechanisms by which gamma-linolenic acid (GLA) osmotic pump infusion alters glioma cell proliferation, and whether it affects cell cycle control and angiogenesis in the C6 glioma in vivo.. Established C6 rat gliomas were treated for 14 days with 5 mM GLA in CSF or CSF alone. Tumour size was estimated, microvessel density (MVD) counted and protein and mRNA expression measured by immunohistochemistry, western blotting and RT-PCR.. GLA caused a significant decrease in tumour size (75 +/- 8.8%) and reduced MVD by 44 +/- 5.4%. These changes were associated with reduced expression of vascular endothelial growth factor (VEGF) (71 +/- 16%) and the VEGF receptor Flt1 (57 +/- 5.8%) but not Flk1. Expression of ERK1/2 was also reduced by 27 +/- 7.7% and 31 +/- 8.7% respectively. mRNA expression of matrix metalloproteinase-2 (MMP2) was reduced by 35 +/- 6.8% and zymography showed MMP2 proteolytic activity was reduced by 32 +/- 8.5%. GLA altered the expression of several proteins involved in cell cycle control. pRb protein expression was decreased (62 +/- 18%) while E2F1 remained unchanged. Cyclin D1 protein expression was increased by 42 +/- 12% in the presence of GLA. The cyclin dependent kinase inhibitors p21 and p27 responded differently to GLA, p27 expression was increased (27 +/- 7.3%) while p21 remained unchanged. The expression of p53 was increased (44 +/- 16%) by GLA. Finally, the BrdU incorporation studies found a significant inhibition (32 +/- 11%) of BrdU incorporation into the tumour in vivo.. Overall the findings reported in the present study lend further support to the potential of GLA as an inhibitor of glioma cell proliferation in vivo and show it has direct effects upon cell cycle control and angiogenesis. These effects involve changes in protein expression of VEGF, Flt1, ERK1, ERK2, MMP2, Cyclin D1, pRb, p53 and p27. Combination therapy using drugs with other, complementary targets and GLA could lead to gains in treatment efficacy in this notoriously difficult to treat tumour.

Topics: Animals; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin D1; gamma-Linolenic Acid; Glioma; Matrix Metalloproteinase 2; Mitogen-Activated Protein Kinase 3; Neovascularization, Pathologic; Proliferating Cell Nuclear Antigen; Rats; Retinoblastoma Protein; Tumor Suppressor Protein p53; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factor Receptor-1

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

To investigate the suppressive effect of resveratrol on growth of U251 human glioma cells and its correlated mechanism.. U251 human glioma cells were treated with resveratrol at various concentrations, MTT assay was used to determine the inhibitory rate of cell proliferation, FCM to detect the cell apoptosis, the expressions of Bcl-2, Bcl-XL, STAT3 and CyclinD1 were analysed by immunohistochemistry and Western blot to examine the expression of Bcl-2, Bcl-XL, STAT3, CyclinD1, Caspase-3 and Bax.. After treatment with resveratrol, MTT assay showed the growth of U251 cells was inhibited in dose-dependent and time-dependent manners, apoptosis of cells advanced stage was built up, immunohistochemical staining displayed decreased the expression of Bcl-2, Bcl-XL, STAT3 and CyclinD1 and Western blot showed that resveratrol decreased the expression of Bcl-2, Bcl-XL, STAT3 and CyclinD1, and built up Bax and Caspase-3.. It is possible that downregulated the expression of Bcl-2, Bcl-XL, but upregulated Bax and Caspase-3, and the indication was obviously in dose-dependent and time-dependent manners.

Topics: Apoptosis; bcl-2-Associated X Protein; bcl-X Protein; Blotting, Western; Caspase 3; Cell Line, Tumor; Cyclin D1; Gene Expression Regulation, Neoplastic; Glioma; Humans; Immunohistochemistry; Proto-Oncogene Proteins c-bcl-2; Resveratrol; STAT3 Transcription Factor; Stilbenes

Resveratrol (trans-3,4', 5-trihydroxystilbene) is a naturally occurring polyphenolic compound that has antiinflammatory, antioxidant, neuroprotective properties and acts as a chemopreventive agent. Resveratrol causes cell cycle arrest and induces apoptotic cell death in various types of cancer cells. In the current studies, the effect of resveratrol on phosphoinositide kinase-3 (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling pathway was examined in human U251 glioma cells. Resveratrol decreased both the expression and phosphorylation of Akt. Inhibitors of PI3K (LY294002) and Akt (SH-6) enhanced resveratrol-induced LDH release and caspase-3 activation. Resveratrol reduced phosphorylation of ribosomal protein S6 and the mTOR inhibitor rapamycin further enhanced resveratrol-induced cell death. These results suggest that the downregulation of PI3K/Akt/mTOR signaling pathways may be an important mediator in resveratrol-induced apoptosis in glioma cells.

Topics: Antineoplastic Agents, Phytogenic; Caspase 3; Cell Line, Tumor; Cyclin D1; Dose-Response Relationship, Drug; Down-Regulation; Glioma; Humans; Phosphoinositide-3 Kinase Inhibitors; Protein Kinases; Proto-Oncogene Proteins c-akt; Resveratrol; Signal Transduction; Stilbenes; TOR Serine-Threonine Kinases

To investigate the expression and the significance of beta-catenin and Cyclin D1 in gliomas.. The SABC immunohistochemistry were used to detect the expression of beta-catenin and Cyclin D1 in 49 brain gliomas and 5 normal brain tissues.. The beta-catenin positive ratio in normal brain tissues and glioma samples are 2/5 and 38/49(P<0.01) respectively, and the Cyclin D1 positive ratio in normal brain tissues and gliomas are 1/5 and 42/49(P<0.01); Compared with the normal brain tissue, both the immunohistochemical expression of beta-catenin and Cyclin D1 were up-regulated(P<0.05), and the expression of beta-catenin and Cyclin D1 between the normal brain tissues and gliomas exhibited significantly difference (P<0.01).. beta-catenin and Cyclin D1 increased in gliomas may be precipitate the tumorigenesis of glioma.

Topics: Adolescent; Adult; Aged; beta Catenin; Cell Nucleus; Child; Cyclin D1; Female; Gene Expression Regulation, Neoplastic; Glioma; Humans; Immunohistochemistry; Male; Middle Aged; Up-Regulation; Young Adult

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

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

To explore the effect of LRRC4, a glioma suppressive gene, on blocking U251 cells in G0/G1 by MAPK signaling pathway.. LRRC4 was transfected into U251 cells, and at 24 hour of post-transfection, cells were split at a 1:3 dilution, challenged with 500 microg /mL G418 and formed a stable transfected clone pool. RT-PCR, Northern blot and Western blot were used to identify the stable transfectants. ERK, JNK and P38 expression changes were analyzed by Western blot. FACS analysis, Luciferase reporter gene assay and Western blot were used to detect the cell cycle and cyclin D1.. LRRC4 down-regulated the expression of phosphorylated ERK2 and up-regulated the expression of total protein JNK2 (a key molecule of MAPK signaling pathway) and phosphorylated c-Jun. LRRC4 decreased the expression of mutation P53, cyclin D1 activation and its expression. U251 cells were blocked in G0/G1 by LRRC4.. LRRC4 can decrease JNK2, up-regulate the phosphoralated c-Jun, down-regulate mutant P53 and cyclin D1, and therefore block U251 cells in G0/G1.

Topics: Blotting, Northern; Blotting, Western; Cell Line, Tumor; Cyclin D1; Flow Cytometry; G1 Phase; Glioma; Humans; Luciferases; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase Kinases; Nerve Tissue Proteins; Recombinant Fusion Proteins; Resting Phase, Cell Cycle; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transfection

Brain tumorigenesis is a complex process involving multiple genetic alterations. Cyclin D1 and BAX genes are two of the most important regulators in controlling the normal proliferation and apoptosis of cells, respectively. In this study, we analysed the possibilities of involvement of cyclin D1 and BAX genes in the gliomagenesis.. In determining gene alterations of exon 4 of cyclin D1 gene and exon 6 of BAX gene, all samples were amplified by polymerase chain reaction (PCR) and subsequently by direct sequencing. Our results showed a frameshift mutation (G base deletion) at nucleotide 82 of codon 28 in exon 4 of the cyclin D1 gene and another frameshift mutation with a deletion of C base at nucleotide 153 of exon 6 of the BAX gene in two separate cases of a glioblastoma multiform (WHO Grade IV) sample.. These findings suggest that both cyclin D1 and BAX genes alteration are rarely found in brain tumors. However, the alteration might cause a significant effect of the normal protein production and this might contribute to the development of brain tumorigenesis in Malaysian patients.

Topics: bcl-2-Associated X Protein; Central Nervous System Neoplasms; Cyclin D1; DNA Mutational Analysis; Exons; Glioma; Humans; Mutation; Retrospective Studies; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

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

In a variety of tumors, the susceptibility of the tumor cells to apoptotic cell death following chemotherapy is a major determinant of therapeutic outcome. Gliomas are resistant to most chemotherapeutic agents, and its mechanism is not known in detail. In an attempt to understand the mechanism of chemo-resistance, we investigated the roles of insulin-like growth factor-I (IGF-I), IGF-I receptors (IGF-IR), and their relationship with the apoptotic response of two glioma cell lines to etoposide, a chemotherapeutic agent for malignant gliomas.. Two human glioma cell lines, U-87MG and KNS-42, were used. Etoposide-induced cell growth inhibition was quantified using a modified MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrasodium bromide), colorimetric assay. Hoechst 33258 staining, DNA fragmentation assay, and western blot were used for the evaluation of apoptosis. ApoAlert caspase assay was used for measuring the activity of caspase-3 (CPP32) and interleukin-1 beta -converting enzyme (ICE) protease. In addition, the effect of IGF-IR antisense was tested in U-87MG and KNS-42 glioma cell lines.. Etoposide inhibited the growth of U-87MG and KNS-42 cells in a concentration-dependent manner. Etoposide increased the expression of wild-type p53, activated CPP32 (but not ICE) activity, and induced apoptosis in these cells. IGF-I prevented etoposide-induced apoptosis by increasing the expression of bcl-2 and decreasing the activity of CPP32. IGF-IR antisense enhanced the apoptotic effect of etoposide.. IGF-I decreased etoposide-induced apoptosis in glioma cells by increasing the expression of bcl-2 and decreasing the activity of CPP32. The antisense of IGF-IR increased etoposide-induced apoptosis. The anti-apoptotic effect of IGF-I and IGF-IR might be related to the chemo-resistance of glioma to chemotherapeutic agents.

Topics: Apoptosis; Benzimidazoles; Blotting, Southern; Blotting, Western; Caspase 3; Caspases; Cell Count; Cell Line, Tumor; Cell Proliferation; Colorimetry; Cyclin D1; DNA Fragmentation; Dose-Response Relationship, Drug; Drug Interactions; Endopeptidases; Etoposide; Gene Expression Regulation, Neoplastic; Glioma; Humans; Insulin-Like Growth Factor I; Nerve Tissue Proteins; Nucleic Acid Synthesis Inhibitors; Oligodeoxyribonucleotides, Antisense; Receptor, IGF Type 1; Tetrazolium Salts; Thiazoles; Time Factors; Transfection

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

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

The early growth response gene-1 (Egr-1) is a tumor suppressor which plays an important role in cell growth, differentiation and apoptosis. Egr-1 has been shown to be down-regulated in many types of tumor tissues. Trifluoperazine (TFP), a phenothiazine class of antipsychotics, restored serum-induced Egr-1 expression in several cancer cell lines. We investigated the effect of Egr-1 expression on the TFP-induced inhibition of cell growth. Ectopic expression of Egr-1 enhanced the TFP-induced antiproliferative activity and downregulated cyclin D1 level in U87MG glioma cells. Our results suggest that antipsychotics TFP exhibits antiproliferative activity through up-regulation of Egr-1.

Topics: Cell Cycle; Cell Proliferation; Cyclin D1; DNA-Binding Proteins; Early Growth Response Protein 1; Gene Expression; Glioma; Humans; Immediate-Early Proteins; Promoter Regions, Genetic; Transcription Factors; Trifluoperazine; Tumor Cells, Cultured

Glial cell proliferation in culture is under the control of metabotropic glutamate (mGlu) receptors. We have examined whether this control extends to human glioma cells. Primary cultures were prepared from surgically removed human glioblastomas. RT-PCR combined with western blot analysis showed that most of the cultures (eight out of 11) expressed group-II mGlu receptors. In two selected cultures (MZC-12 and FCN-9), the mGlu2/3 receptor antagonist, LY341495, slowed cell proliferation when applied to the growth medium from the second day after plating. This effect was reversible because linear cell growth was restored after washing out the drug. LY341495 reduced glioma cell proliferation at concentrations lower than 100 nm, which are considered as selective for mGlu2/3 receptors. In addition, its action was mimicked by the putative mGlu2/3 receptor antagonist (2S)-alpha-ethylglutamate. The anti-proliferative effect of LY341495 was confirmed by measuring [methyl-3H]-thymidine incorporation in cultures arrested in G0 phase of the cell cycle and then stimulated to proliferate by the addition of 10% fetal calf serum or 100 ng/mL of epidermal growth factor (EGF). In cultures treated with EGF, LY341495 was also able to reduce the stimulation of the mitogen-activated protein kinase (MAPK) pathway, as well as the induction of cyclin D1. Both effects, as well as decreased [methyl-3H]-thymidine incorporation, were partially reduced by co-addition of the potent mGlu2/3 receptor agonist, LY379268. We conclude that activation of group-II mGlu receptors supports the growth of human glioma cells in culture and that antagonists of these receptors should be tested for their ability to reduce tumour growth in vivo.

Topics: Amino Acids; Bridged Bicyclo Compounds, Heterocyclic; Cell Division; Cells, Cultured; Cyclin D1; Cyclin D2; Cyclins; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Glioma; Humans; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Receptors, Metabotropic Glutamate; Thymidine; Tritium; Xanthenes

The progression of mammalian cells through G1 phase of the cell cycle is governed by the D-type cyclins (D1, D2, D3). These proteins are induced at the beginning of the G1 phase and associate with serine/threonine cyclin-dependent kinases to form holoenzymes. Overexpression of cyclin D1 in human cancers as well as in several cancer cell lines has been reported. Here, we employed mitotic selection to synchronize the C6 glioma cell cycle at the start of the G1 phase and assessed the effects of neomycin on cyclin D1 protein detection by immunocytochemical analysis. Cyclin D1 activation as well as cell proliferation were already significantly reduced after 3 h of incubation of the cells with neomycin. These findings suggested that the antiproliferative effects of neomycin in gliomas could be mediated by inhibition of the expression of cyclin D1 gene and support further consideration of therapeutic use of neomycin in a Phase I clinical study for patients with recurrent glioblastoma.

Topics: Animals; Cell Division; Cell Line, Tumor; Cyclin D1; G1 Phase; Glioma; Neomycin; Rats

Cell cycle progression is tightly regulated by cyclins, cyclin-dependent kinases (cdks) and related inhibitory phophatases. Here, we employed mitotic selection to synchronize the C6 glioma cell cycle at the start of the G1 phase and mapped the temporal regulation of selected cyclins, cdks and inhibitory proteins throughout the 12 h of G1 by immunoblot analysis. The D-type cyclins, D3 and D1, were differentially expressed during the C6 glioma G1 phase. Cyclin D1 was up-regulated in the mid-G1 phase (4-6 h) while cyclin D3 expression emerged only in late G1 (9-12 h). The influence of the anticonvulsant agent valproic acid (VPA) on expression of cyclins and related proteins was determined, since its teratogenic potency has been linked to cell cycle arrest in the mid-G1 phase. Exposure of C6 glioma to VPA induced a marked up-regulation of cyclin D3 and decreased expression of the proliferating cell nuclear antigen. In synchronized cell populations, increased expression of cyclin D3 by VPA was detected in the mid-G1 phase (3-5 h). Immunocytochemical localization demonstrated rapid intracellular translocation of cyclin D3 to the nucleus following VPA exposure, suggesting that VPA-induced cell cycle arrest may be mediated by precocious activation of cyclin D3 in the G1 phase.

Topics: Active Transport, Cell Nucleus; Animals; Anticonvulsants; Cell Cycle; Cell Cycle Proteins; Cell Division; Cell Line; Cell Nucleus; Cyclin D1; Cyclin D3; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase Inhibitor p27; Cyclin-Dependent Kinases; Cyclins; Dose-Response Relationship, Drug; G1 Phase; Glioma; Immunoblotting; Immunohistochemistry; Proliferating Cell Nuclear Antigen; Proto-Oncogene Proteins; Rats; Tumor Suppressor Proteins; Valproic Acid

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

Analysis of tumor-derived genetic lesions has provided insights into molecular pathogenesis of human gliomas. Because these changes represent only one of several mechanisms that alter gene expression during tumorigenesis, it is likely that further information will be obtained from a careful analysis of important regulatory proteins present in these tumors.. We have quantified the levels of key cell cycle/signaling proteins in 94 prospectively collected, meticulously preserved, "snap frozen" glioma specimens and have compared these levels with histopathological data and patient outcome.. The results of these experiments confirm that the levels of wild-type tumor suppressor proteins, such as p53, pRB, PTEN, p14(ARF), and p16(INK4), are lost or severely reduced in most gliomas, and that epidermal growth factor receptor, 2human telomerase reverse transcriptase, and cyclin-dependent kinase 4 are overexpressed frequently and with a few exceptions, almost exclusively, in glioblastomas. In addition, we report frequent underexpression of E2F-1 (in 55% of gliomas) and cyclin E overexpression (in 26% of gliomas), which have not yet been reported on the genomic level. Several of these markers significantly correlated with histopathological grade, and the levels of five proteins showed significant association with patient outcome. In particular, overexpression of epidermal growth factor receptor, human telomerase reverse transcriptase, cyclin-dependent kinase 4, and cyclin E was largely restricted to glioblastomas and was significantly associated with reduced patient survivals.. We conclude that the quantitation of cell cycle/signaling proteins from meticulously preserved glioma specimens provides further insights into the molecular pathogenesis of human gliomas and yields valuable prognostic information.

Topics: Blotting, Western; Cell Cycle Proteins; Cyclin D1; Cyclin E; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinases; DNA-Binding Proteins; ErbB Receptors; Glioma; Humans; Phosphoric Monoester Hydrolases; Prognosis; Proteins; Proto-Oncogene Proteins; PTEN Phosphohydrolase; Retinoblastoma Protein; Telomerase; Tumor Suppressor Protein p14ARF; Tumor Suppressor Protein p53; Tumor Suppressor Proteins

Protein kinase C (PKC) plays an important role in the regulation of glioma growth; however, the identity of the specific isoform and mechanism by which PKC fulfills this function remain unknown. In this study, we demonstrate that PKC activation in glioma cells increased their progression through the cell cycle. Of the six PKC isoforms that were present in glioma cells, PKC alpha was both necessary and sufficient to promote cell cycle progression when stimulated with phorbol 12-myristate 13-acetate. Also, decreased PKC alpha expression resulted in a marked decrease in cell proliferation. The only cell cycle-regulatory molecule whose expression was rapidly altered and increased by PKC alpha activity was the cyclin-cyclin-dependent kinase (CDK) inhibitor p21(Waf1/Cip1). Coimmunoprecipitation studies revealed that p21(Waf1/Cip1) upregulation was accompanied by an incorporation of p21(Waf1/Cip1) into various cyclin-CDK complexes and that the kinase activity of these complexes was increased, thus resulting in cell cycle progression. Furthermore, depletion of p21(Waf1/Cip1) by antisense strategy attenuated the PKC-induced cell cycle progression. These results suggest that PKC alpha activity controls glioma cell cycle progression through the upregulation of p21(Waf1/Cip1), which facilitates active cyclin-CDK complex formation.

Topics: Cell Cycle; Cell Division; Central Nervous System Neoplasms; Cyclin A; Cyclin B; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinases; Cyclins; Enzyme Activation; Glioma; Humans; Isoenzymes; Oligonucleotides, Antisense; Phorbol Esters; Protein Kinase C; Protein Kinase C-delta; Protein Kinase C-epsilon; Protein Kinase C-theta; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured; Up-Regulation

The genes encoding the cyclin-dependent kinase inhibitors p16INK4A (CDKN2A) and p15INK4B (CDKN2B) are frequently homozygously deleted in a variety of tumor cell lines and primary tumors, including glioblastomas in which 40-50% of primary tumors display homozygous deletions of these two loci. Although the role of p16 as a tumor suppressor has been well documented, it has remained less well studied whether p15 plays a similar growth-suppressing role. Here, we have used replication-defective recombinant adenoviruses to compare the effects of expressing wild-type p16 and p15 in glioma cell lines. After infection, high levels of p16 and p15 were observed in two human glioma cell lines (U251 MG and U373 MG). Both inhibitors were found in complex with CDK4 and CDK6. Expression of p16 and p15 had indistinguishable effects on U251 MG, which has homozygous deletion of CDKN2A and CDKN2B, but a wild-type retinoblastoma (RB) gene. Cells were growth-arrested, showed no increased apoptosis, and displayed a markedly altered cellular morphology and repression of telomerase activity. Transduced cells became enlarged and flattened and expressed senescence-associated beta-galactosidase, thus fulfilling criteria for replicative senescence. In contrast, the growth and morphology of U373 MG, which expresses p16 and p15 endogenously, but undetectable levels of RB protein, were not affected by exogenous overexpression of either inhibitor. Thus, we conclude that overexpression of p15 has a similar ability to inhibit cell proliferation, to cause replicative senescence, and to inhibit telomerase activity as p16 in glioma cells with an intact RB protein pathway.

Topics: Adenoviridae; Carrier Proteins; Cell Cycle Proteins; Cell Division; Cell Line; Cell Size; Cellular Senescence; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase 6; Cyclin-Dependent Kinase Inhibitor p15; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinases; Enzyme Induction; Glioma; Humans; Immunoblotting; Mutation; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Recombinant Proteins; Retinoblastoma Protein; Telomerase; Transfection; Tumor Cells, Cultured; Tumor Suppressor Protein p53; Tumor Suppressor Proteins

The tumour suppressor gene p16/INK4a encodes a specific inhibitor of the cyclin D-dependent kinases CDK4 and CDK6. p16/INK4a prevents the association of CDK4 with cyclin D1, and subsequently inhibits phosphorylation of retinoblastoma tumour suppressor protein (pRb), thus preventing exit from the G1 phase. In human cancers, the estimated frequency of genetic alteration involving the p16/INK4a locus is believed to be second only to alteration of p53. A high frequency (greater than 50%) of homozygous p16/INK4a gene deletion has been demonstrated in glioblastoma tissues and p16/INK4a is altered in 80% of glioma cell lines. Therefore, restoration of p16/INK4a would suppress cell proliferation and induce cell growth arrest. We showed here that restoration of p16/INK4a expression in p16 negative U87MG, U251MG and partially deleted U373MG by Ad-CMV-p16/INK4a induced growth suppression in vitro and in vivo. Expression of p16 transferred by Ad-CMV-p16/INK4a in glioma cells was highly efficient and maintained for more than seven days. In addition, we found that the endogenous status of p16 and Rb might affect the expression of exogenous p16/INK4a gene and inhibitory effect of cell proliferation. Even though, there were several factors affecting the efficiency of Ad-CMV-p16/INK4 gene transfer, our results suggest that Ad-CMV-p16 gene therapy strategy is potentially useful and warrants further clinical investigation for the treatment of gliomas.

Topics: Adenoviridae; Animals; Carrier Proteins; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinases; Genetic Therapy; Genetic Vectors; Glioma; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Transplantation; Proto-Oncogene Proteins; Retinoblastoma Protein; Transfection; Tumor Cells, Cultured

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

In order to define the role of cyclin D1 in the progression of malignant glioma, cells over-expressing cyclin D1 were constructed (a-1 cells). They exhibited significantly increased invasiveness as compared with mock-transfected cells. Since cellular invasion is thought to depend on extracellular-matrix degradation, we determined whether cyclin-D1 expression modifies the activity of matrix metalloproteinases (MMPs). Increased gelatinolytic activity of latent type MMP-2 (proMMP-2) and active MMP-2 was observed in a-1 cells. Moreover, cyclin-D1 expression was associated with increased activation of proMMP-9 through MMP-3. Wound assays showed an increase of cell motility in a-1 cells. Cyclin-D1 expression was found to be associated with up-regulation of Rac1, which modulates the formation of ruffling membranes and cell motility. Our results show that cyclin D1 may modulate invasive ability by increasing MMP activity and cell motility, and suggests a novel function of cyclin D1 in the progression of malignant gliomas.

Topics: Actins; Calcium-Calmodulin-Dependent Protein Kinases; Cell Movement; Collagen; Collagenases; Cyclin D1; Drug Combinations; Gelatinases; Glioma; Humans; Laminin; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Metalloendopeptidases; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Neoplasm Invasiveness; Proteoglycans; Transfection; Tumor Cells, Cultured

C6 glioma - Ca2+ depletion - proliferation arrest morphology change - CDK inhibitor In this study, we investigated the role of the intracellular calcium store in modulating the cellular proliferation and the expression of cell cycle regulatory proteins in cultured C6 glioma cells. By means of microspectrofluorimetry and Ca(2+)-sensitive indicator fura-2, we found that the intracellular Ca2+ pump inhibitors, thapsigargin (TG) irreversibly and 2,5-ditert-butyl-hydroquinone (DBHQ) reversibly depleted the Ca(2+)-store accompanied with the induction of G0/G1 arrest, an increase in glial fibrillary acidic protein (GFAP) expression and morphological changes from a round flat shape to a differentiated spindle-shaped cell. The machinery underlying these changes induced by Ca(2+)-store depletion was investigated. The results indicated that Ca(2+)-store depletion caused an increased expression of p21 and p27 proteins (cyclin-dependent kinase inhibitors), with unchanged mutant p53 protein of C6 cells but reduced amounts of the cell cycle regulators: cyclin-dependent kinase 2 (CDK2), cdc2, cyclin C, cyclin D1, cyclin D3 and proliferating cell nuclear antigen (PCNA) in a time-dependent manner. These findings indicate a new function of the endoplasmic reticulum (ER) Ca2+ store in regulating cellular proliferation rate through altering the expression of p21 and p27 proteins. Moreover, cellular differentiation as revealed by spindle-shaped morphology and induced GFAP expression were also modulated by the ER Ca2+ store. The implication of this finding is that the abnormal growth of cancer cells such as C6 glioma cells may be derived from a signalling of the ER which can be manipulated by depleting the Ca2+ store.

Topics: Blotting, Western; Calcium-Transporting ATPases; CDC2 Protein Kinase; CDC2-CDC28 Kinases; Cell Cycle; Cell Differentiation; Cell Division; Cyclin D1; Cyclin D3; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinases; Cyclins; Cytosol; Glial Fibrillary Acidic Protein; Glioma; Humans; Hydroquinones; Microfilament Proteins; Muscle Proteins; Protein Serine-Threonine Kinases; Thapsigargin; Tumor Cells, Cultured; Tumor Suppressor Protein p53

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

Alternative splicing of cyclin D1 gene mRNA has recently been demonstrated. The novel transcript shows no splicing at the downstream exon 4 boundary and encodes a protein with an altered carboxyl-terminal domain that is a cyclin D1 variant; exon 5 is not included in the coding sequence which terminates downstream of exon 4. We here produced cells that exogenously express each form of cyclin D1 and analysed their cell cycle regulation. We found that (1) alternative splicing forms of cyclin D1 modulated entry into the cell cycle in an inverse manner; (2) both splicing forms suppressed cell growth; and (3) cells overexpressing form [a] were inhibited from entry into and completion of the S phase, although form [b]-expressing cells showed no reduction of G1- to S transition. We also found that overexpression of either cyclin D1 form upregulated Rb gene products, suggesting that this upregulation may be one of the causes of growth suppression in cyclin D1 overexpressing cells.

Topics: Alternative Splicing; Bromodeoxyuridine; Cell Cycle; Cell Cycle Proteins; Cell Division; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Cyclin-Dependent Kinases; Cyclins; Flow Cytometry; G1 Phase; Gene Expression Regulation; Genes, p53; Glioma; Humans; Immunoblotting; Microtubule-Associated Proteins; Proliferating Cell Nuclear Antigen; Proto-Oncogene Proteins; Resting Phase, Cell Cycle; Retinoblastoma Protein; Transfection; Tumor Cells, Cultured; Tumor Suppressor Proteins

Although the cyclopentenone prostaglandin A1 (PGA1) is known to arrest the cell cycle at the G1 phase in vitro and to suppress tumor growth in vivo, its relatively weak activity limits its usefulness in cancer chemotherapy. In an attempt to develop antitumor drugs of greater potency and conspicuous biological specificity, we synthesized novel analogs based on the structure of PGA1. Of the newly synthesized analogs, 15-epi-delta7-PGA1 methyl ester (NAG-0092), 12-iso-delta7-PGA1 methyl ester (NAG-0093), and ent-delta7-PGA1 methyl ester (NAG-0022) possess a cross-conjugated dienone structure around the five-member ring with unnatural configurations at C(12) and/or C(15) and were found to be far more potent than native PGA1 in inhibiting cell growth and causing G1 arrest in A172 human glioma cells. These three analogs induced the expression of p21 at both RNA and protein levels in a time- and dose-dependent fashion. Kinase assays with A172 cells treated with these analogs revealed that both cyclin A- and E-dependent kinase activities were markedly reduced, although cyclin D1-dependent kinase activity was unaffected. Immunoprecipitation-Western blot analysis showed that the decrease in cyclin A-dependent kinase activity was due to an increased association of p21 with cyclin A-cyclin-dependent kinase 2 complexes, whereas the decrease in cyclin E-dependent activity was due to a combined mechanism involving reduction in cyclin E protein itself and increased association of p21. Thus, these newly synthesized PGA1 analogs may prove to be powerful tools in cancer chemotherapy as well as in investigations of the structural basis of the antiproliferative activity of A series prostaglandins.

Topics: Antineoplastic Agents; Cell Division; Cyclin A; Cyclin D1; Cyclin E; G1 Phase; Glioma; Humans; Prostaglandins A; Protein Kinases; Proto-Oncogene Proteins p21(ras); Tumor Cells, Cultured

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

Cyclin D1 plays a key regulatory role during the G1 phase of the cell cycle and its gene is amplified and overexpressed in many cancers. To address the relationship between cyclin D1 and other cell cycle regulatory proteins, we established human glioma and rodent fibroblast cell lines in which cyclin D1 expression could be regulated ectopically with tetracycline. In both of these cell lines, we found that ectopic expression of cyclin D1 in asynchronously growing cells was accompanied by increased levels of the p53 tumor suppressor protein and the cyclin/cdk inhibitor p21. Despite the induction of these cell cycle inhibitory proteins, cyclin D1-associated cdk kinase remained activated and the cells grew essentially like that of the parent cells. Although growth parameters were unchanged in these cells, morphological changes were clearly identifiable and anchorage independent growth was observed in NIH3T3 cells. In a first step toward elaborating the mechanism for cyclin D1-mediated induction of p21 gene expression we show that co-expression of E2F-1 and DP-1 can specifically transactivate the p21 promoter. In support of these findings and a direct effect of E2F on induction of p21 gene expression a putative E2F binding site was identified within the p21 promoter. In summary, our results demonstrate that ectopic expression of cyclin D1 can induce gene expression of the cdk inhibitor p21 through an E2F mechanism the consequences of which are not to growth arrest cells but possibly to stabilize cyclin D1/cdk function.

Topics: 3T3 Cells; Animals; Base Sequence; Blotting, Western; Carrier Proteins; Cell Cycle; Cell Cycle Proteins; Cell Division; Cell Size; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinases; Cyclins; DNA-Binding Proteins; E2F Transcription Factors; E2F1 Transcription Factor; Enzyme Inhibitors; Fibroblasts; Gene Expression Regulation; Glioma; Humans; Immunohistochemistry; Mice; Models, Biological; Oncogene Proteins; Proto-Oncogene Proteins; Retinoblastoma-Binding Protein 1; Tetracycline; Time Factors; Transcription Factor DP1; Transcription Factors; Transcriptional Activation; Tumor Suppressor Protein p53