cyclin-d1 and Glioblastoma

To review the evidence implicating the deregulation of cyclin D1 in the pathogenesis of non-small cell lung cancer (NSCLC), and to discuss the opportunities for targeted clinical intervention.. Data published until June 2006 are summarized, and previously unpublished results from our own research are included.. In normal cells, cyclin D1 complexes with and activates cyclin-dependent kinases (CDK) and acts as a transcriptional regulator. The protein is frequently overexpressed in a wide range of cancers, sometimes coincident with CCND1 (cyclin D1) gene amplification (5-20% of tumours). A low level of somatic mutations have been seen in certain tumours. CCND1 is amplified in NSCLC and cyclin D1 is frequently overexpressed in tumours and pre-invasive bronchial lesions, generally from one parental allele. Mutation analyses revealed a frequent CCND1 gene polymorphism (A870G) that modulates alternative splicing and allows expression of an alternative cyclin D1 transcript (transcript cyclin D1b). The encoded cyclin D1b protein lacks a specific phosphorylation site required for nuclear export. Genotype has been correlated with the risk and/or severity of disease or drug response across a range of malignancies, including lung cancer. Together, these findings suggest a strong pathological role for cyclin D1 deregulation in bronchial neoplasia.. Current data indicate that cyclin D1 overexpression is not a consequence of, but rather a pivotal element in the process of malignant transformation in the lung and other tissues. This understanding may open new avenues for lung cancer diagnosis, treatment and prevention.

Topics: Adenoma; Carcinoma, Non-Small-Cell Lung; Cell Transformation, Neoplastic; Cyclin D1; Gene Expression Regulation, Neoplastic; Glioblastoma; Humans; Lung Neoplasms; Polymorphism, Genetic

Temsirolimus (CCI779), an intravenous analog of rapamycin, presents immunosuppressive properties and also antiproliferative activity. Its principal target is the mTOR serine/threonin kinase which controls the initiation of the transcription of many ARNm implicated in carcinogenesis. Breast cancers, glioblastoma and renal cell carcinoma were particularly studied with response rates from 10 to 20 %. In haematology, mantle-cell lymphoma is of particular interest because of constitutional activation of cyclin D1 (response rate of 40 %). As a whole these data define temsirolimus as a promising new drug. Current and further developments are based on its association with chemotherapy in a concomitant or sequential way.

Topics: Antineoplastic Agents; Breast Neoplasms; Cyclin D1; Glioblastoma; Humans; Kidney Neoplasms; Lymphoma, Mantle-Cell; Protein Kinase Inhibitors; Protein Kinases; Sirolimus; TOR Serine-Threonine Kinases

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

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

The high-grade astrocytoma, glioblastoma multiforme (GBM), is the most common primary tumour of the brain, known for being aggressive and developing drug resistance. The non-coding RNAs (ncRNAs), such as microRNAs (miRNAs) and Piwi-interacting RNAs (piRNAs), have critical functions in tumorigenesis and cancer drug resistance. Hence, we profiled miRNAs, piRNAs, and genes in U-87 MG GBM cells by next-generation sequencing and performed target prediction, pathway enrichment, protein-protein interaction, co-expression studies, and qRT-PCR validations to predict their possible roles in the malignancy. The study identified 335 miRNAs, 665 piRNAs, and 4286 genes differentially expressed (DE) in GBM. Among them 128 DE genes (DEGs) were targeted by both miRNAs and piRNAs, while 1817 and 192 were targeted solely by miRNAs or piRNAs, respectively. Interestingly, all the DEG targets enriched in cancer processes were overexpressed in GBM. Among these, BRAF was solely targeted by two piRNAs and this was found to be co-expressed with 19 sole targets of 5 miRNAs, including CCND1, and both were found to regulate cell proliferation in cancer. We conjectured that upregulated HRH1 and ATXN3 were targeted by both piRNAs and miRNAs, and along with BRAF and CCND1 might induce cell proliferation in GBM through G-protein-coupled receptor or Akt signalling pathways due to downregulation of the respective targeting small RNAs. These targets were also linked to the progression and overall survival of GBM patients, suggesting that they could be used as biomarkers. Overall, this study has identified a few novel ncRNA targets, which might aid in a better understanding of GBM pathogenesis.

Topics: Ataxin-3; Carcinogenesis; Cell Transformation, Neoplastic; Cyclin D1; Glioblastoma; Humans; MicroRNAs; Piwi-Interacting RNA; Proto-Oncogene Proteins B-raf; Repressor Proteins; RNA, Untranslated

A major mechanism conferring resistance to mTOR inhibitors is activation of a salvage pathway stimulating internal ribosome entry site (IRES)-mediated mRNA translation, driving the synthesis of proteins promoting resistance of glioblastoma (GBM). Previously, we found this pathway is stimulated by the requisite IRES-trans-acting factor (ITAF) hnRNP A1, which itself is subject to phosphorylation and methylation events regulating cyclin D1 and c-myc IRES activity. Here we describe the requirement for m

Topics: Cyclin D1; Genes, myc; Glioblastoma; Heterogeneous Nuclear Ribonucleoprotein A1; Humans; Internal Ribosome Entry Sites; Methyltransferases; Protein Biosynthesis; TOR Serine-Threonine Kinases

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

LncRNA RUNX1-IT1 has been characterized as a tumor suppressive lncRNA in several cancers, while its role in glioblastoma (GBM) is unknown. This study aimed to investigate the potential involvement of RUNX1-IT1 in GBM.. Expression of RUNX1-IT1 in GBM tissues and paired non-tumor tissues was determined by RT-qPCR. The interaction between RUNX1-IT1 and miR-195 was analyzed by dual luciferase activity assay. Overexpression of RUNX1-IT1 and miR-195 was achieved in GBM cells to explore the interaction between them. The effects of RUNX1-IT1 and miR-195 overexpression on the expression of cyclin D1 were analyzed by RT-qPCR and Western blot. Cell proliferation was analyzed by CCK-8 assay.. RUNX1-IT1 was upregulated in GBM. RUNX1-IT1 and miR-195 interacted with each other, but failed to regulate the expression of each other. Overexpression of RUNX1-IT1 resulted in the upregulation of cyclin D1, and also reduced the effects of miR-195 overexpression on cyclin D1 expression. RUNX1-IT1 and cyclin overexpression increased cell proliferation, while miR-195 overexpression decreased cell proliferation. In addition, RUNX1-IT1 overexpression reduced the effects of miR-195 overexpression on cell proliferation.. RUNX1-IT1 may sponge miR-195 to upregulate cyclin D1, thereby increasing the proliferation of glioblastoma cells.

Topics: Cell Line, Tumor; Cell Proliferation; Core Binding Factor Alpha 2 Subunit; Cyclin D1; Gene Expression Regulation, Neoplastic; Glioblastoma; Humans; MicroRNAs; RNA, Long Noncoding

Understanding human genome alterations is necessary to optimize genome-based cancer therapeutics. However, some newly discovered mutations remain as variants of unknown significance (VUS). Here, the mutation c.1403A > G in exon 10 of the platelet-derived growth factor receptor-alpha (PDGFRA) gene, a VUS found in adult glioblastoma multiforme (GBM), was introduced in human embryonal kidney 293 T (HEK293T) cells using genome editing to investigate its potential oncogenic functions. Genome editing was performed using CRISPR/Cas9; the proliferation, drug sensitivity, and carcinogenic potential of genome-edited cells were investigated. We also investigated the mechanism underlying the observed phenotypes. Three GBM patients carrying the c.1403A > G mutation were studied to validate the in vitro results. The c.1403A > G mutation led to a splice variant (p.K455_N468delinsN) because of the generation of a 3'-acceptor splice site in exon 10. PDGFRA-mutated HEK293T cells exhibited a higher proliferative activity via PDGFRα and the cyclin-dependent kinase (CDK)4/CDK6-cyclin D1 signaling pathway in a ligand-independent manner. They showed higher sensitivity to multi-kinase, receptor tyrosine kinase, and CDK4/CDK6 inhibitors. Of the three GBM patients studied, two harbored the p.K455_N468delinsN splice variant. The splicing mutation c.1403A > G in PDGFRA is oncogenic in nature. Kinase inhibitors targeting PDGFRα and CDK4/CDK6 signaling should be evaluated for treating GBM patients harboring this mutation.

Topics: Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase 6; Gene Editing; Glioblastoma; HEK293 Cells; Humans; Molecular Targeted Therapy; Receptor, Platelet-Derived Growth Factor alpha; RNA Splice Sites; Signal Transduction

Long non-coding RNA (lncRNA) SEMA3B antisense RNA 1 (head to head) (SEMA3B-AS1) is a recently identified tumor suppressor in gastric cancer. However, its role in glioblastoma (GBM) is unclear. This study was conducted to explore the role of SEMA3B-AS1 in GBM. In this study, the expression of SEMA3B-AS1, cyclin D1 and miR-195 were determined by RT-qPCR. Gene interactions were evaluated by dual-luciferase assay and overexpression experiments. BrdU assay was performed to monitor cell proliferation. We observed downregulation of SEMA3B-AS1 in GBM. The expression of SEMA3B-AS1 was inversely correlated with the expression of cyclin D1 but positively correlated with the expression of miR-195. In GBM cells, overexpression of SEMA3B-AS1 and miR-195 caused reduced expression levels of cyclin D1. MiR-195 inhibitor reduced the effects of overexpression of SEMA3B-AS1 on the expression of cyclin D1. Moreover, overexpression of SEMA3B-AS1 increased the expression levels of miR-195. Cell proliferation data showed that, SEMA3B-AS1 and miR-195 decreased cell proliferation, while overexpression of cyclin D1 increased GBM cell proliferation. In addition, miR-195 inhibitor inhibited the role of overexpression of SEMA3B-AS1 in cancer cell proliferation. Moreover, miR-195 interacted with cyclin D1, but not SEMA3B-AS1. Furthermore, SEMA3B-AS1 decreased the methylation of the promoter region of miR-195. Therefore, we concluded that miR-195 links lncRNA SEMA3B-AS1 and cyclin D1 to regulate the proliferation of GBM cells.

Topics: Cell Line, Tumor; Cell Proliferation; Cyclin D1; Gene Expression Regulation, Neoplastic; Glioblastoma; Humans; Membrane Glycoproteins; MicroRNAs; RNA, Antisense; RNA, Long Noncoding; Semaphorins

The dual-specificity tyrosine-regulated kinases DYRK1A and DYRK1B play a key role in controlling the quiescence-proliferation switch in cancer cells. Serum reduction of U87MG 2D cultures or multi-cellular tumour spheroids induced a quiescent like state characterized by increased DYRK1B and p27, and decreased pRb and cyclin D1. VER-239353 is a potent, selective inhibitor of the DYRK1A and DYRK1B kinases identified through fragment and structure-guided drug discovery. Inhibition of DYRK1A/B by VER-239353 in quiescent U87MG cells increased pRb, DYRK1B and cyclin D1 but also increased the cell cycle inhibitors p21 and p27. This resulted in exit from G0 but subsequent arrest in G1. DYRK1A/B inhibition reduced the proliferation of U87MG cells in 2D and 3D culture with greater effects observed under reduced serum conditions. Paradoxically, the induced re-expression of cell cycle proteins by DYRK1A/B inhibition further inhibited cell proliferation. Cell growth arrest induced in quiescent cells by DYRK1A/B inhibition was reversible through the addition of growth-promoting factors. DYRK inhibition-induced DNA damage and synergized with a CHK1 inhibitor in the U87MG spheroids. In vivo, DYRK1A/B inhibition-induced tumour stasis in a U87MG tumour xenograft model. These results suggest that further evaluation of VER-239353 as a treatment for glioblastoma is therefore warranted.

Topics: Animals; Cell Cycle; Cell Line, Tumor; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Disease Models, Animal; Dyrk Kinases; Female; Glioblastoma; Humans; Mice; Protein Kinase Inhibitors; Protein Serine-Threonine Kinases; Protein-Tyrosine Kinases; Signal Transduction

Recent studies show that the expression of CCND1, a key factor in cell cycle control, is increased following the progress and deteriotation of glioma and predicts poor outcomes. On the other hand, dysregulated deubiquitinase USP10 also predicts poor prognosis for patients with glioblastoma (GBM). In the present study, we investigated the interplay between CCND1 protein and USP10 in GBM cells. We showed that the expression of CCND1 was significantly higher in both GBM tissues and GBM-derived stem cells. USP10 interacted with CCND1 and prevented its K48- but not K63-linked polyubiquitination in GBM U251 and HS683 cells, which led to increased CCND1 stability. Consistent with the action of USP10 on CCND1, knockdown of USP10 by single-guided RNA downregulated CCND1 and caused GBM cell cycle arrest at the G1 phase and induced GBM cell apoptosis. To implement this finding in the treatment of GBMs, we screened a natural product library and found that acevaltrate (AVT), an active component derived from the herbal plant Valeriana jatamansi Jones was strikingly potent to induce GBM cell apoptosis, which was confirmed by the Annexin V staining and activation of the apoptotic signals. Furthermore, we revealed that AVT concentration-dependently suppressed USP10-mediated deubiquitination on CCND1 therefore inducing CCND1 protein degradation. Collectively, the present study demonstrates that the USP10/CCND1 axis could be a promising therapeutic target for patients with GBMs.

Topics: Apoptosis; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Glioblastoma; HEK293 Cells; Humans; Iridoids; Ubiquitin Thiolesterase; Ubiquitination

Annotation of structural variations (SVs) and base-level karyotyping in cancer cells remains challenging. Here, we present Integrative Framework for Genome Reconstruction (InfoGenomeR)-a graph-based framework that can reconstruct individual SVs into karyotypes based on whole-genome sequencing data, by integrating SVs, total copy number alterations, allele-specific copy numbers, and haplotype information. Using whole-genome sequencing data sets of patients with breast cancer, glioblastoma multiforme, and ovarian cancer, we demonstrate the analytical potential of InfoGenomeR. We identify recurrent derivative chromosomes derived from chromosomes 11 and 17 in breast cancer samples, with homogeneously staining regions for CCND1 and ERBB2, and double minutes and breakage-fusion-bridge cycles in glioblastoma multiforme and ovarian cancer samples, respectively. Moreover, we show that InfoGenomeR can discriminate private and shared SVs between primary and metastatic cancer sites that could contribute to tumour evolution. These findings indicate that InfoGenomeR can guide targeted therapies by unravelling cancer-specific SVs on a genome-wide scale.

Topics: A549 Cells; Breast Neoplasms; Cell Line, Tumor; Chromosome Aberrations; Cyclin D1; DNA Copy Number Variations; Female; Genome, Human; Genomic Structural Variation; Glioblastoma; HeLa Cells; High-Throughput Nucleotide Sequencing; Humans; Karyotyping; Ovarian Neoplasms; Polyploidy; Receptor, ErbB-2; Sequence Analysis, DNA; Whole Genome Sequencing

Ganglioglioma, pleomorphic xanthoastrocytoma (PXA) and pilocytic astrocytoma are rare brain neoplasms with frequent activation of mitogen-activated protein (MAP) kinase pathway. A downstream marker of MAP-kinase pathway activation is cyclin D1. However, the expression of cyclin D1 has not been studied in the differential diagnosis between these brain tumors. The aim of this work is to compare the expression of cyclin D1 in ganglioglioma, PXA, pilocytic astrocytoma. We also compared cyclin D1 expression in giant cell glioblastoma and in IDH wild type glioblastoma. Our work shows that roughly half of gangliogliomas have ganglion cells stained by cyclin D1 while two third of PXA have pleormophic cells stained by cyclin D1 and 15% of giant cell glioblastoma have pleomorphic cells stained by cyclin D1 (p < 0.001). Cyclin D1 never stains normal neurons either in the adjacent cortex of circumscribed tumor, or in entrapped neurons in IDH wild type glioblastomas. The expression of cyclin D1 is correlated to the presence of BRAF V600E mutation in ganglioglioma and PXA (p = 0.002). To conclude, cyclin D1 positivity might be used to confirm the neoplastic nature of ganglion cells. Cyclin D1 is expressed in most cases of BRAF V600E mutated gangliogliomas but also in cases without BRAF mutations suggesting an activation of MAP-kinase pathway through another way. Cyclin D1 immunohistochemistry has currently no or little role in the differential diagnosis of pilocytic astrocytoma. Its role in the differential diagnosis between PXA and giant cell glioblastoma needs to be further investigated on external series.

Topics: Adolescent; Adult; Astrocytoma; Cyclin D1; Diagnosis, Differential; Female; Ganglioglioma; Glioblastoma; Humans; Male; Middle Aged; Mutation; Prognosis; Proto-Oncogene Proteins B-raf; Young Adult

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

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

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

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

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

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

Clinical trials directed at mechanistic target of rapamycin (mTOR) inhibition have yielded disappointing results in glioblastoma (GBM). A major mechanism of resistance involves the activation of a salvage pathway stimulating internal ribosome entry site (IRES)-mediated protein synthesis. PRMT5 activity has been implicated in the enhancement of IRES activity.. We analyzed the expression and activity of PRMT5 in response to mTOR inhibition in GBM cell lines and short-term patient cultures. To determine whether PRMT5 conferred resistance we used genetic and pharmacological approaches to ablate PRMT5 activity and assessed the effects on in vitro and in vivo sensitivity. Mutational analyses of the requisite IRES-trans-acting factor (ITAF), hnRNP A1 determined whether PRMT5-mediated methylation was necessary for ITAF RNA binding and IRES activity.. PRMT5 activity is stimulated in response to mTOR inhibitors. Knockdown or treatment with a PRMT5 inhibitor blocked IRES activity and sensitizes GBM cells. Ectopic expression of non-methylatable hnRNP A1 mutants demonstrated that methylation of either arginine residues 218 or 225 was sufficient to maintain IRES binding and hnRNP A1-dependent cyclin D1 or c-MYC IRES activity, however a double R218K/R225K mutant was unable to do so. The PRMT5 inhibitor EPZ015666 displayed synergistic anti-GBM effects in vitro and in a xenograft mouse model in combination with PP242.. These results demonstrate that PRMT5 activity is stimulated upon mTOR inhibition in GBM. Our data further support a signaling cascade in which PRMT5-mediated methylation of hnRNP A1 promotes IRES RNA binding and activation of IRES-mediated protein synthesis and resultant mTOR inhibitor resistance.

Topics: Animals; Apoptosis; Cell Proliferation; Cyclin D1; DNA Methylation; Drug Resistance, Neoplasm; Gene Expression Regulation, Neoplastic; Glioblastoma; Heterogeneous Nuclear Ribonucleoprotein A1; Humans; Indoles; Internal Ribosome Entry Sites; Isoquinolines; Mice; Protein-Arginine N-Methyltransferases; Proto-Oncogene Proteins c-myc; Purines; Pyrimidines; Signal Transduction; TOR Serine-Threonine Kinases; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

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

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

Topics: Animals; beta Catenin; Calcium; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Gene Expression Regulation, Neoplastic; Glioblastoma; Heterografts; Humans; Mice; Neurocalcin; Proto-Oncogene Proteins c-myc; Wnt Signaling Pathway

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

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

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

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

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

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

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

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

Glioblastoma (GBM) ranks among the most challenging cancers to treat and there is an urgent need for clinically relevant prognostic and diagnostic biomarkers. Here, we set out to investigate the expression of eight proteins (bcl-2, cyclin D1, p16, p21, p27, p53, Sox11 and WT1) in GBM with the specific aim to establish immunohistochemistry cut-off points with clinical relevance.. Immunohistochemistry (IHC) was used to examine protein expression in 55 surgical GBM specimens using H-scores, and IHC cut-off points were established using the Cutoff Finder web platform. Protein co-expression and its correlation with histopathological features were assessed, and cases were classified according to IDH1 mutation status. Survival curves were determined using Kaplan-Meier analyses.. Clinical and molecular parameters found to be correlated with overall survival (OS) were tumor size (r = -0.278; p = 0.048), p53 (r = -0.452; p = 0.001), p16 (r = 0.351; p = 0.012) and Sox11 (r = 0.324; p = 0.020). In addition, we found that tumor size correlated with cyclin D1 (r = -0.282; p = 0.037), p53 (r = 0.269; p = 0.041), Sox11 (r = -0.309; p = 0.022) and WT1 (r = -0.372; p = 0.003). Variables found to be significantly associated with IDH1 mutation status were OS (p < 0.01), age (p < 0.01), cyclin D1 (p = 0.046), p16 (p = 0.019) and Sox11 (p = 0.012). Variables found to be significantly associated with a poor survival were tumor size >5 cm (p < 0.001), bcl-2 score > 40 (p = 0.034), cyclin D1 score ≤ 70 (p = 0.004), p16 score ≤ 130 (p = 0.005), p53 score > 20 (p = 0.003), Sox11 score ≤ 40 (p < 0.001) and WT1 score ≤ 270 (p = 0.02).. Correlations between protein biomarkers and main clinical GBM variables were identified. The establishment of distinct biomarker cut-off points may enable clinicians and pathologists to better weigh their prognostic value.

Topics: Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinase Inhibitor p21; Female; Glioblastoma; Humans; Immunohistochemistry; Male; Middle Aged; SOXC Transcription Factors; Tissue Array Analysis; Tumor Suppressor Protein p53; WT1 Proteins

Topics: Antineoplastic Agents; Apoptosis; Artesunate; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cyclin B1; Cyclin D1; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase 4; Glioblastoma; Humans

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

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

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

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

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

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

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

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

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

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

The impact of extremely low-frequency pulsed electromagnetic fields (ELF-PEMFs) at various frequencies and amplitudes was investigated on cell cycle, apoptosis and viability of the Glioblastoma Multiforme (GBM) cell line (U87), in vitro. The GBM is a malignant brain tumor with high mortality in humans and poorly responsive to the most common type of cancer treatments, such as surgery, chemotherapy and radiation therapy. U87 cells with five experimental groups (I-V) were exposed to various ELF-PEMFs for 2, 4 and 24 h, as follows: (I) no exposure, control; (II) 50 Hz 100 ± 15 G; (III) 100 Hz 100 ± 15 G; (IV) 10 Hz 50 ± 10 G; (V) 50 Hz 50 ± 10 G. The morphology properties, cell viability and gene expression of proteins involved in cell cycle regulation (Cyclin-D1 and P53) and apoptosis (Caspase-3) were investigated. After 24 h, the cell viability and Cyclin-D1 expression increased in Group II (30%, 45%), whereas they decreased in Groups III (29%, 31%) and IV (21%, 34%); P53 and Caspase-3 elevated only in Group III; and no significant difference was observed in Group V, respectively, compared with the control (p < 0.05). The data suggest that the proliferation and apoptosis of human GBM are influenced by exposure to ELF-PEMFs in different time-dependent frequencies and amplitudes. The fact that some of the ELF-PEMFs frequencies and amplitudes favor U87 cells proliferation indicates precaution for the use of medical devices related to the MFs on cancer patients. On the other hand, some other ELF-PEMFs frequencies and intensities arresting U87 cells growth could open the way to develop novel therapeutic approaches.

Topics: Apoptosis; Caspase 3; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Survival; Cyclin D1; Electromagnetic Fields; Glioblastoma; Humans

Allopregnanolone (3α-THP) is one of the main reduced progesterone (P

Topics: Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Cholestenone 5 alpha-Reductase; Cyclin D1; ErbB Receptors; Glioblastoma; Humans; Pregnanolone; Progesterone; Transforming Growth Factor beta1; Vascular Endothelial Growth Factor A

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

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

Glioblastoma (GBM) represents one of most common tumors in humans. However, the biological processes and molecular mechanisms of GBM are still unclear. It is known that microRNA-520b (miR-520b) participates in the development of various tumor progressions. The present study was to evaluate the level of miR-520b in GBM tissues and cells. We further investigated the molecular mechanisms of miR-520b in U87 and U251 cell lines. Here, our data showed that the expression levels of miR-520b were significantly reduced in clinical GBM tissues and cell lines. Accordingly, the expression levels of cyclin D1 were significantly increased in clinical GBM tissues and cell lines. Ectopic expression of miR-520b in U87 and U251 cells resulted in decreased cell proliferation and enhanced cell apoptosis. Further study characterized the 3' untranslated region (3'-UTR) of cyclin D1 gene as a direct target of miR-520b in U87 and U251 cells as determined by luciferase reporter assays. In addition, ectopic expression of miR-520b led to the down-regulation of phosphorylated retinoblastoma (p-Rb, a downstream effector of cyclin D1), while the overexpression of cyclin D1 reversed the miR-520b-induced inhibition of p-Rb expression. In conclusion, this study highlights the importance of miR-520b in regulating the proliferation and apoptosis of GBM by directly targeting cyclin D1, and miR-520b may represent a potential therapeutic strategy for GBM.

Topics: Apoptosis; Blotting, Western; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Gene Expression Regulation, Neoplastic; Glioblastoma; Humans; MicroRNAs; Reverse Transcriptase Polymerase Chain Reaction

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

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

Glioblastoma (GBM) is the most common primary brain tumor, with poor prognosis and a lack of effective therapeutic options. The aberrant expression of transcription factor REST (repressor element 1-silencing transcription factor) had been reported in different kinds of tumors. However, the function of REST and its mechanisms in GBM remain elusive. Here, REST expression was inhibited by siRNA silencing in U-87 and U-251 GBM cells. Then CCK-8 assay showed significantly decreased cell proliferation, and the inhibition of migration was verified by scratch wound healing assay and transwell assay. Using cell cycle analysis and Annexin V/PI straining assay, G1 phase cell cycle arrest was found to be a reason for the suppression of cell proliferation and migration upon REST silencing, while apoptosis was not affected by REST silencing. Further, the detection of REST-downstream genes involved in cytostasis and migration inhibition demonstrated that CCND1 and CCNE1 were reduced; CDK5R1, BBC3, EGR1, SLC25A4, PDCD7, MAPK11, MAPK12, FADD and DAXX were enhanced, among which BBC3 and DAXX were direct targets of REST, as verified by ChIP (chromatin immunoprecipitation) and Western blotting. These data suggested that REST is a master regulator that maintains GBM cells proliferation and migration, partly through regulating cell cycle by repressing downstream genes, which might represent a potential target for GBM therapy.

Topics: Adaptor Proteins, Signal Transducing; Apoptosis Regulatory Proteins; Carrier Proteins; Cell Line, Tumor; Cell Movement; Cell Proliferation; Co-Repressor Proteins; Cyclin D1; Cyclin E; Early Growth Response Protein 1; Fas-Associated Death Domain Protein; G1 Phase; Glioblastoma; Humans; Membrane Transport Proteins; Mitogen-Activated Protein Kinases; Molecular Chaperones; Nerve Tissue Proteins; Nuclear Proteins; Oncogene Proteins; Proto-Oncogene Proteins; Repressor Proteins

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

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

Glioblastoma (GB) recurrences are rarely removed, therefore, tissue modifications induced by radiotherapy, and temozolomide chemotherapy are scarcely known. Nuclear cyclin D1 is associated with GB progression and resistance to therapy. We previously found that the expression of autophagic protein beclin-1 is a major determinant of prognosis in GB.. In 31 patients with primary GB and their recurrences, we investigated the protein expression of cyclin D1 and beclin-1, before and after radiotherapy-temozolomide therapy by immunohistochemistry.. Most (20/31) primary GBs were negative for nuclear cyclin D1, and highly expressed beclin-1. In their recurrences, cytoplasmic cyclin D1 positivity was observable, which co-localized with beclin-1. Eleven primary GBs instead exhibited low beclin-1 expression and were positive for nuclear cyclin D1; three of their recurrences exhibited an increase of beclin-1, which co-localized with cyclin D1 in the cytoplasm.. Our results suggest therapy-induced degradation of cyclin D1 via autophagy.

Topics: Adult; Apoptosis; Autophagy; Beclin-1; Cell Line, Tumor; Cyclin D1; Dacarbazine; Drug Resistance, Neoplasm; Female; Gene Expression Regulation, Neoplastic; Glioblastoma; Humans; Male; Middle Aged; Neoplasm Recurrence, Local; Prognosis; Temozolomide

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

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

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

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

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

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

Aberrant PDGF-PDGFR signaling and its effects on downstream effectors have been implicated in glioma development. A crucial AKT regulator, ACK1 (TNK2) has been shown to be a downstream mediator of PDGF signaling; however, the exact underlying mechanisms in gliomas remain elusive. Here, we report that in glioma cells, PDGFR-β activation enhanced the interaction between ACK1 and AKT, resulting in AKT activation. PDGF treatment consistently promoted the formation of complexes containing PDGFR-β and ACK1. Mutational analysis suggested that Y635 of ACK1 is a PDGFR-β phosphorylation site and that the ACK1 Y635F mutant abrogated the sequential activation of AKT. Moreover, PDK1 interacted with ACK1 during PDGF stimulation, which is required for the binding of ACK1 to PDGFR-β. Further mutational analysis showed that T325 of ACK1 was crucial for the ACK1 and PDK1 interaction. ACK1 Y635F or T325A mutants abolished PDGFR-β-induced AKT activation, the subsequent nuclear translocation of β-catenin and the expression of cyclin D1. Glioma cell cycle progression, proliferation and tumorigenesis were accordingly blocked by ACK1 Y635F or T325A. In glioblastoma multiforme samples from 51 patients, increased ACK1 tyrosine phosphorylation correlated with upregulated PDGFR-β activity and AKT activation. Taken together, our data demonstrate that ACK1 plays a pivotal role in PDGF-PDGFR-induced AKT signaling in glioma tumorigenesis. This knowledge contributes to our understanding of glioma progression and may facilitate the identification of novel therapeutic targets for future glioma treatment.

Topics: beta Catenin; Carcinogenesis; Cell Cycle; Cell Line; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Glioblastoma; HEK293 Cells; Humans; Phosphorylation; Protein-Tyrosine Kinases; Proto-Oncogene Proteins c-akt; Receptor, Platelet-Derived Growth Factor beta; Signal Transduction; Up-Regulation

Glioblastoma multiforme (GBM) displays high resistance to radiation and chemotherapy, due to the presence of a fraction of GBM stem-like cells (GSLCs), which are thus representing the target for GBM elimination. Since mesenchymal stem cells (MSCs) display high tumor tropism, we examined possible antitumor effects of the secreted factors from human MSCs on four GSLC lines (NCH421k, NCH644, NIB26, and NIB50). We found that conditioned media from bone marrow and umbilical cord-derived MSCs (MSC-CM) mediated cell cycle arrest of GSLCs by downregulating cyclin D1. PCR arrays revealed significantly deregulated expression of 13 genes associated with senescence in NCH421k cells exposed to MSC-CM. Among these, ATM, CD44, COL1A1, MORC3, NOX4, CDKN1A, IGFBP5, and SERPINE1 genes were upregulated, whereas IGFBP3, CDKN2A, CITED2, FN1, and PRKCD genes were found to be downregulated. Pathway analyses in GO and KEGG revealed their association with p53 signaling, which can trigger senescence via cell cycle inhibitors p21 or p16. For both, upregulated expression was proven in all four GSLC lines exhibiting senescence after MSC-CM exposure. Moreover, MSC paracrine signals were shown to increase the sensitivity of NCH421k and NCH644 cells toward temozolomide, possibly by altering them toward more differentiated cell types, as evidenced by vimentin and GFAP upregulation, and Sox-2 and Notch-1 downregulation. Our findings support the notion that MSCs posses an intrinsic ability to inhibit cell cycle and induce senescence and differentiation of GSLCs.

Topics: Aged; Aged, 80 and over; Apoptosis; Bone Marrow Cells; Cell Differentiation; Cellular Senescence; Culture Media, Conditioned; Cyclin D1; Female; G1 Phase Cell Cycle Checkpoints; Gene Expression Regulation; Glioblastoma; Humans; Male; Membrane Potential, Mitochondrial; Mesenchymal Stem Cells; Paracrine Communication; Tumor Cells, Cultured; Umbilical Cord

Products of the Pim (the proviral integration site for the Moloney murine leukemia virus) family of proto—oncogenes possess serine/threonine kinase activity and belong to the Ca2+/calmodulin—dependent protein kinase group. Pim—3, a member of the Pim family is closely linked to the development of a variety of tumors. However, the role of Pim—3 in human glioblastoma remains unknown. In this study, we elucidated the role of Pim—3 in the growth and apoptosis of glioblastoma cells. Western blotting was used for determination of protein levels, and shRNA was used for Pim—3 knockdown. The MTT assay was used to evaluate cell proliferation and flow cytometry was used to determine cell cycle status and the number of apoptotic cells. A mouse xenograft model was established by injecting nude mice with Pim—3—depleted glioblastoma cells in order to determine tumor growth in vivo. We demonstrated that Pim—3 was highly expressed in human glioblastoma cell lines. We also found that knockdown of Pim—3 by specific shRNA slowed decreased proliferation, induced cell cycle arrest in the G0/G1 phase, and increased apoptosis in glioblastoma cells. Pim—3 knockdown potently inhibited the growth of subcutaneously implanted glioblastoma cells in vivo. We further revealed that Pim—3 knockdown induced growth inhibition by reducing the levels of the anti—apoptotic protein Bcl—xl and cell cycle regulatory proteins, including cyclin D1 and Cdc25C, and increasing the levels of the pro—apoptotic protein Bax.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; bcl-X Protein; Carcinogenesis; cdc25 Phosphatases; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Central Nervous System Neoplasms; Cyclin D1; Disease Models, Animal; Down-Regulation; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Glioblastoma; Heterografts; Humans; Mice, Nude; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; RNA, Small Interfering

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

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

Glioblastoma, the most common type of primary malignant brain tumor, is a devastating disease associated with a median survival of approximately 12 months. Here, we have tested E804, the commercially available indirubin derivatives, against U251 and U87 glioblastoma cells. Treatment with E804 significantly inhibits the growth of human glioblastoma cells lines via induction of differentiation and apoptosis. Differentiation induction is coupled with increased expression of glial fibriliary acidic protein, a marker for mature astrocytes. Apoptosis is associated with activation of Caspase 3 and reduction of Bcl-xL and Mcl-1. Furthermore, we demonstrate that E804 reduces signal transducer and activator of transcription-3 (Stat3) signaling to a remarkable extent, suggesting that inactivation of Stat3, at least in part, mediates the effects of this indirubin derivative. Consistently, reduction of Stat3 activity promotes E804-mediated anti-tumor effects, whereas overexpression of Stat3C mutant recues cell apoptosis induced by E804. Taken together, our results indicate that E804 can effectively suppress tumor cell growth, induce tumor cell differentiation and apoptosis mediated partially by Stat3 signaling pathway, suggesting that E804 could be useful for a potential anti-glioblastoma therapeutic approach.

Topics: Animals; Annexin A5; Apoptosis; bcl-X Protein; Cell Cycle; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Colony-Forming Units Assay; Cyclin D1; Dose-Response Relationship, Drug; Gene Expression Regulation, Neoplastic; Glioblastoma; Humans; Indoles; Inhibitor of Apoptosis Proteins; Myeloid Cell Leukemia Sequence 1 Protein; Oximes; Rats; RNA, Small Interfering; Signal Transduction; STAT3 Transcription Factor; Survivin; Time Factors

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

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

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

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

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

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

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

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

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

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

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

The human Distal-less Homeobox (DLX) gene family encodes homeobox transcription factors involved in the control of morphogenesis and tissue homeostasis, which is primarily expressed in embryonic development. Recently, DLX gene family was reported to have essential roles in carcinogenesis. We have profiled whole genome expressed genes in 83 glioblastoma multiforme (GBM) patients from the Chinese Glioma Genome Atlas (CGGA) Group. Two major groups of samples were identified in mRNA expression profiles (referred to as Cluster 1 (C1) and Cluster 2 (C2)). We identified 7 out of the top 10 Gene Ontology terms in the C1 group were associated with differentiation and development of neuronal cell. The most significant prognostic gene was DLX2 (P < 0.001, OR = 1.744); overexpression of DLX2 indicated poor survival in the 83 GBM patients (low DLX2 vs high DLX2, 77.6 vs 44.7 weeks, P < 0.001). Annotation of mRNA profiling data on GBM from The Cancer Genome Atlas and MD Anderson Cancer Center showed the proneural and neural subtypes highly correlated with low and high DLX2 expression, respectively. Knocking down of DLX2 in GBM cell line-LN229 results in decreased cyclin D1 expression and cell proliferation. Collectively, these data identified high expression of DLX2 as a poor prognostic marker to GBM patients.

Topics: Adolescent; Adult; Aged; Biomarkers, Tumor; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Glioblastoma; Homeodomain Proteins; Humans; Male; Middle Aged; Neurons; Prognosis; RNA Interference; RNA, Messenger; RNA, Small Interfering; Survival; Transcription Factors; Young Adult

Elevated cyclin D1 (CCND1) in human glioblastoma correlates with poor clinical prognosis. In this study, the human glioblastoma cell lines SHG-44 and U251 were stably transfected with short hairpin RNA (shRNA) targeting cyclin D1 or with ectogenic cyclin D1 by lentivirus-mediated transfection. Glioblastoma cells overexpressing or underexpressing cyclin D1 were then examined by in vitro growth assays, apoptosis assays, cell cycle analysis, and invasion assays. Cyclin D1 knockdown in SHG-44 cells inhibited cell proliferation, induced apoptosis, and attenuated migration across Matrigel, a model of invasive capacity. Western blot analysis and quantitative reverse-transcription polymerase chain reaction (RT-PCR) revealed that cells underexpressing CCND1 exhibited decreased multidrug resistance protein 1 (MDR1) and B-cell lymphoma-2 (Bcl-2) expression, but enhanced apoptosis effector caspase-3 expression. In contrast, cyclin D1 overexpression promoted cell proliferation, attenuated apoptosis, and enhanced invasive capacity. Furthermore, cyclin D1 overexpression was associated with increased expression of MDR1 and Bcl-2, and decreased caspase-3 expression. Results using the U251 cell line confirmed the effects of CCND1-targeted shRNA and lentivirus-mediated overexpression on proliferation and apoptosis of glioblastoma cells. Overexpression of cyclin D1 enhanced the proliferation and invasive potential of human glioblastoma cells, while reducing apoptosis. The ability to suppress the malignant phenotype by downregulating cyclin D1 expression may provide a new gene therapy approach for patients with malignant glioma.

Topics: Analysis of Variance; Annexin A5; Apoptosis; ATP Binding Cassette Transporter, Subfamily B, Member 1; Caspase 3; Cell Cycle; Cell Line, Transformed; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Flow Cytometry; Gene Expression Regulation, Neoplastic; Glioblastoma; Green Fluorescent Proteins; Humans; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Neoplasm Invasiveness; Proto-Oncogene Proteins c-bcl-2; RNA, Small Interfering; Time Factors; Transfection

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

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

Evidence has suggested that STAT3 functions as an oncogene in gliomagenesis. As a consequence, changes in the inflammatory microenvironment are thought to promote tumor development. Regardless of its origin, cancer-related inflammation has many tumor-promoting effects, such as the promotion of cell cycle progression, cell proliferation, cell migration and cell survival. Given that IL-6, a major cancer-related inflammatory cytokine, regulates STAT3 activation and is upregulated in glioblastoma, we sought to investigate the inhibitory effects of the chemopreventive flavonoid quercetin on glioblastoma cell proliferation and migration triggered by IL-6, and to determine the underlying mechanisms of action. In this study, we show that quercetin is a potent inhibitor of the IL-6-induced STAT3 signaling pathway in T98G and U87 glioblastoma cells. Exposure to quercetin resulted in the reduction of GP130, JAK1 and STAT3 activation by IL-6, as well as a marked decrease of the proliferative and migratory properties of glioblastoma cells induced by IL-6. Interestingly, quercetin also modulated the expression of two target genes regulated by STAT3, i.e. cyclin D1 and matrix metalloproteinase-2 (MMP-2). Moreover, quercetin reduced the recruitment of STAT3 at the cyclin D1 promoter and inhibited Rb phosphorylation in the presence of IL-6. Overall, these results provide new insight into the role of quercetin as a blocker of the STAT3 activation pathway stimulated by IL-6, with a potential role in the prevention and treatment of glioblastoma.

Topics: Cell Cycle; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Cytokine Receptor gp130; Glioblastoma; Humans; Interleukin-6; Janus Kinase 1; Matrix Metalloproteinase 2; Phosphorylation; Quercetin; Retinoblastoma Protein; Signal Transduction; STAT3 Transcription Factor

Tumor-specific pyruvate kinase M2 (PKM2) is essential for the Warburg effect. In addition to its well-established role in aerobic glycolysis, PKM2 directly regulates gene transcription. However, the mechanism underlying this nonmetabolic function of PKM2 remains elusive. We show here that PKM2 directly binds to histone H3 and phosphorylates histone H3 at T11 upon EGF receptor activation. This phosphorylation is required for the dissociation of HDAC3 from the CCND1 and MYC promoter regions and subsequent acetylation of histone H3 at K9. PKM2-dependent histone H3 modifications are instrumental in EGF-induced expression of cyclin D1 and c-Myc, tumor cell proliferation, cell-cycle progression, and brain tumorigenesis. In addition, levels of histone H3 T11 phosphorylation correlate with nuclear PKM2 expression levels, glioma malignancy grades, and prognosis. These findings highlight the role of PKM2 as a protein kinase in its nonmetabolic functions of histone modification, which is essential for its epigenetic regulation of gene expression and tumorigenesis.

Topics: Animals; Astrocytoma; Carrier Proteins; Cell Line; Cell Line, Tumor; Cell Transformation, Neoplastic; Cyclin D1; Epidermal Growth Factor; Epigenesis, Genetic; Female; Gene Expression Regulation, Neoplastic; Glioblastoma; Histones; Humans; Membrane Proteins; Mice; Mice, Nude; Neoplasm Transplantation; Proto-Oncogene Proteins c-myc; Thyroid Hormone-Binding Proteins; Thyroid Hormones; Transcription, Genetic; Transplantation, Heterologous

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

Moesin-ezrin-radixin-like protein (merlin) has long been considered a unique tumour suppressor that inhibits mitogenic signalling only at the membrane-cytoskeleton interface. However, the nucleocytoplasmic shuttling of merlin in a cell cycle-dependent manner has recently been observed, indicating that merlin may also exert its tumour-suppressive activity by interacting with specific nuclear protein partners. We have identified protein interacting with carboxyl terminus 1 (PICT-1) as a novel merlin-binding partner. Although the detailed mechanisms are not fully understood, several lines of evidence have previously implicated PICT-1 as a candidate tumour suppressor, including its phosphatase and tensin homolog deleted on chromosome 10 (PTEN)-dependent growth-suppression and cell-killing activities. We show here that PICT-1 is localised to the nucleolus, and Ser518-dephosphorylated merlin (the growth-inhibitory form of merlin) can interact with PICT-1 in the nucleolus. Ectopic expression of PICT-1, both in PTEN-positive HeLa cells and in PTEN-deficient U251 cells, effectively represses cyclin D1 expression, arrests the cell cycle at G0/G1, and promotes cell apoptosis. PICT-1 (1-356), a carboxyl-terminus truncated mutant that has lost the ability to bind merlin, has a markedly reduced inhibitory effect on the cell cycle and proliferation. Knockdown of merlin expression by siRNA attenuates the inhibitory effects induced by PICT-1 over-expression. We propose that merlin mediates PICT-1-induced growth inhibition by translocating to the nucleolus and binding PICT-1.

Topics: Active Transport, Cell Nucleus; Animals; Apoptosis; Cell Line; Cell Nucleus; Cell Proliferation; Cyclin D1; G1 Phase; Gene Expression Regulation, Neoplastic; Glioblastoma; Humans; Male; Mice; Neurofibromin 2; Phosphorylation; Protein Binding; Resting Phase, Cell Cycle; Serine; Tumor Suppressor Proteins

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

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

Glioblastoma multiforme is one of the most devastating cancers and presents unique challenges to therapy because of its aggressive behavior. Cancer-initiating or progenitor cells have been described to be the only cell population with tumorigenic capacity in glioblastoma. Therefore, effective therapeutic strategies targeting these cells or the early precursors may be beneficial. We have established different cultures of glioblastoma-initiating cells (GICs) derived from surgical specimens and found that, after induction of differentiation, the NFκB transcriptional pathway was activated, as determined by analyzing key proteins such as p65 and IκB and the upregulation of a number of target genes. We also showed that blockade of nuclear factor (NF)κB signaling in differentiating GICs by different genetic strategies or treatment with small-molecule inhibitors, promoted replication arrest and senescence. This effect was partly mediated by reduced levels of the NFκB target gene cyclin D1, because its downregulation by RNA interference reproduced a similar phenotype. Furthermore, these results were confirmed in a xenograft model. Intravenous treatment of immunodeficient mice bearing human GIC-derived tumors with a novel small-molecule inhibitor of the NFκB pathway induced senescence of tumor cells but no ultrastructural alterations of the brain parenchyma were detected. These findings reveal that activation of NFκB may keep differentiating GICs from acquiring a mature postmitotic phenotype, thus allowing cell proliferation, and support the rationale for therapeutic strategies aimed to promote premature senescence of differentiating GICs by blocking key factors within the NFκB pathway.

Topics: Animals; Blotting, Western; Carbazoles; Cell Differentiation; Cell Proliferation; Cellular Senescence; Cyclin D1; Female; Gene Expression Profiling; Glioblastoma; Glycosides; Humans; I-kappa B Kinase; I-kappa B Proteins; Mice; Mice, Inbred BALB C; Mice, Knockout; Neural Stem Cells; NF-kappa B; Nitriles; Oligonucleotide Array Sequence Analysis; Protein Kinase Inhibitors; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; Signal Transduction; Sulfones; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

The normal progression of the cell cycle requires sequential expression of cyclins. Rapid induction of cyclin D1 and its associated binding with cyclin-dependent kinases, in the presence or absence of mitogenic signals, often is considered a rate-limiting step during cell cycle progression through the G(1) phase.. In the present study, human umbilical cord blood stem cells (hUCBSC) in co-cultures with glioblastoma cells (U251 and 5310) not only induced G(0)-G(1) phase arrest, but also reduced the number of cells at S and G(2)-M phases of cell cycle. Cell cycle regulatory proteins showed decreased expression levels upon treatment with hUCBSC as revealed by Western and FACS analyses. Inhibition of cyclin D1 activity by hUCBSC treatment is sufficient to abolish the expression levels of Cdk 4, Cdk 6, cyclin B1, β-Catenin levels. Our immuno precipitation experiments present evidence that, treatment of glioma cells with hUCBSC leads to the arrest of cell-cycle progression through inactivation of both cyclin D1/Cdk 4 and cyclin D1/Cdk 6 complexes. It is observed that hUCBSC, when co-cultured with glioma cells, caused an increased G(0)-G(1) phase despite the reduction of G(0)-G(1) regulatory proteins cyclin D1 and Cdk 4. We found that this reduction of G(0)-G(1) regulatory proteins, cyclin D1 and Cdk 4 may be in part compensated by the expression of cyclin E1, when co-cultured with hUCBSC. Co-localization experiments under in vivo conditions in nude mice brain xenografts with cyclin D1 and CD81 antibodies demonstrated, decreased expression of cyclin D1 in the presence of hUCBSC.. This paper elucidates a model to regulate glioma cell cycle progression in which hUCBSC acts to control cyclin D1 induction and in concert its partner kinases, Cdk 4 and Cdk 6 by mediating cell cycle arrest at G(0)-G(1) phase.

Topics: Animals; Cell Cycle; Cell Line, Tumor; Cells, Cultured; Coculture Techniques; Cyclin B1; Cyclin D1; Cyclin E; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase 6; Fetal Blood; Flow Cytometry; Glioblastoma; Humans; Immunoprecipitation; Mice; Mice, Nude; Oligonucleotide Array Sequence Analysis; Oncogene Proteins; Polymerase Chain Reaction; Protein Binding; Stem Cells

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

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

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

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

Constitutive activation of signal transducer and activator of transcription 3 (STAT3) signaling is frequently detected in cancer, promoting its emergence as a promising target for cancer treatment. Inhibiting constitutive STAT3 signaling represents a potential therapeutic approach. We used structure-based design to develop a nonpeptide, cell-permeable, small molecule, termed as LLL12, which targets STAT3. LLL12 was found to inhibit STAT3 phosphorylation (tyrosine 705) and induce apoptosis as indicated by the increases of cleaved caspase-3 and poly (ADP-ribose) polymerase in various breast, pancreatic, and glioblastoma cancer cell lines expressing elevated levels of STAT3 phosphorylation. LLL12 could also inhibit STAT3 phosphorylation induced by interleukin-6 in MDA-MB-453 breast cancer cells. The inhibition of STAT3 by LLL12 was confirmed by the inhibition of STAT3 DNA binding activity and STAT3-dependent transcriptional luciferase activity. Downstream targets of STAT3, cyclin D1, Bcl-2, and survivin were also downregulated by LLL12 at both protein and messenger RNA levels. LLL12 is a potent inhibitor of cell viability, with half-maximal inhibitory concentrations values ranging between 0.16 and 3.09 microM, which are lower than the reported JAK2 inhibitor WP1066 and STAT3 inhibitor S3I-201 in six cancer cell lines expressing elevated levels of STAT3 phosphorylation. In addition, LLL12 inhibits colony formation and cell migration and works synergistically with doxorubicin and gemcitabine. Furthermore, LLL12 demonstrated a potent inhibitory activity on breast and glioblastoma tumor growth in a mouse xenograft model. Our results indicate that LLL12 may be a potential therapeutic agent for human cancer cells expressing constitutive STAT3 signaling.

Topics: Animals; Anthraquinones; Antineoplastic Agents; Apoptosis; Blotting, Western; Breast Neoplasms; Caspase 3; Cell Line; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Female; Glioblastoma; Humans; Inhibitor of Apoptosis Proteins; Male; Mice; Mice, Nude; Microtubule-Associated Proteins; Neoplasms; Pancreatic Neoplasms; Phosphorylation; Poly(ADP-ribose) Polymerases; Protein Binding; Proto-Oncogene Proteins c-bcl-2; Reverse Transcriptase Polymerase Chain Reaction; STAT3 Transcription Factor; Sulfonamides; Survivin; Xenograft Model Antitumor Assays

Glioblastoma is the most common type of primary brain tumor and is rapidly progressive with few treatment options. Here, we report that sorafenib (< or =10 micromol/L) inhibited cell proliferation and induced apoptosis in two established cell lines (U87 and U251) and two primary cultures (PBT015 and PBT022) from human glioblastomas. The effects of sorafenib on these tumor cells were associated with inhibiting phosphorylated signal transducers and activators of transcription 3 (STAT3; Tyr705). Expression of a constitutively activated STAT3 mutant partially blocked the effects of sorafenib, consistent with a role for STAT3 inhibition in the response to sorafenib. Phosphorylated Janus-activated kinase (JAK)1 was inhibited in U87 and U251 cells, whereas phosphorylated JAK2 was inhibited in primary cultures. Sodium vanadate, a general inhibitor of protein tyrosine phosphatases, blocked the inhibition of phosphorylation of STAT3 (Tyr705) induced by sorafenib. These data indicate that the inhibition of STAT3 activity by sorafenib involves both the inhibition of upstream kinases (JAK1 and JAK2) of STAT3 and increased phosphatase activity. Phosphorylation of AKT was also reduced by sorafenib. In contrast, mitogen-activated protein kinases were not consistently inhibited by sorafenib in these cells. Two key cyclins (D and E) and the antiapoptotic protein Mcl-1 were downregulated by sorafenib in both cell lines and primary cultures. Our data suggest that inhibition of STAT3 signaling by sorafenib contributes to growth arrest and induction of apoptosis in glioblastoma cells. These findings provide a rationale for potential treatment of malignant gliomas with sorafenib. Mol Cancer Ther; 9(4); 953-62. (c)2010 AACR.

Topics: Apoptosis; Benzenesulfonates; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cyclin D1; Cyclin E; DNA, Neoplasm; Glioblastoma; Humans; Interleukin-6; Janus Kinase 1; Janus Kinase 2; Mutant Proteins; Myeloid Cell Leukemia Sequence 1 Protein; Niacinamide; Oncogene Proteins; Phenylurea Compounds; Phosphorylation; Phosphotyrosine; Protein Binding; Protein Tyrosine Phosphatases; Proto-Oncogene Proteins c-bcl-2; Pyridines; Signal Transduction; Sorafenib; src-Family Kinases; STAT3 Transcription Factor; Tumor Cells, Cultured; Vanadates

Adrenomedullin is a secreted peptide hormone with multiple functions. Although a number of reports have indicated that adrenomedullin may be involved in tumor progression, its mechanism of action remains obscure. In this study, we have analysed the signal transduction pathway activated by adrenomedullin in human glioma cells. Our results revealed that adrenomedullin induced the phosphorylation of both c-Jun and JNK in glioblastoma cells. Silencing JNK expression with siRNA reversed the phosphorylation of c-Jun induced by adrenomedullin, indicating that JNK is responsible of c-Jun activation. In addition, electrophoretic mobility-shift assays showed that the increase in phosphorylation of c-Jun was associated with increased AP-1 DNA binding activity. Supershift assays and co-immunoprecipitation demonstrated that c-Jun and JunD are part of the AP-1 complex, indicating that activated c-Jun is dimerized with JunD in response to adrenomedullin. Furthermore, adrenomedullin was shown to promote cell transit beyond cell cycle phases with a concomittant increase in cyclin D1 protein level, suggesting that adrenomedullin effects cell proliferation through up-regulation of cyclin D1. The inhibition of JNK activation or the suppression of c-Jun or JunD expression with siRNA impaired the effects of adrenomedullin on cell proliferation and on cyclin D1. Taken together, these data demonstrate that activation of cJun/JNK pathway is involved in the growth regulatory activity of adrenomedullin in glioblastoma cells.

Topics: Adrenomedullin; Apoptosis Regulatory Proteins; Cell Cycle; Cell Line, Tumor; Cyclic AMP; Cyclin D1; Dimerization; Enzyme Activation; Gene Expression Regulation, Neoplastic; Glioblastoma; Humans; JNK Mitogen-Activated Protein Kinases; Neoplasm Proteins; Protease Inhibitors; Proto-Oncogene Proteins c-jun; RNA, Small Interfering; Signal Transduction; Transcription Factor AP-1

Isoquercitrin isolated from the aerial parts of Hyptis fasciculata was evaluated according to its capacity to interfere with glioblastoma (Gbm) cell growth. Gbm cells were incubated with isoquercitrin, quercetin, or rutin at concentrations of 25, 50, and 100 mumol/l for 24, 48, and 72 h. Quercetin and rutin affected Gbm cell proliferation after treatment times of longer than 24 h. However, increasing concentrations of isoquercitrin inhibited 50% of Gbm cell proliferation at 24 h and further reached nearly 90% inhibition at 72 h. This effect did not affect cell morphology, cell viability, or cleaved capase-3 levels, indicating that isoquercitrin did not induce Gbm cell death. A marked reduction in cyclin D1 levels and an increase in p27 levels were observed when 100 micromol/l of isoquercitrin was added to Gbm cells. Interestingly, nuclear beta-catenin staining observed in a subpopulation of untreated Gbm cells was found in the cytoplasm after 100-micromol/l isoquercitrin treatment. Collectively, these data show that isoquercitrin reduces Gbm cell growth without inducing apoptosis, possibly by modulating the control of the cell cycle. Our data also suggest that beta-catenin-mediated signaling may be involved on the antiproliferative activity of isoquercitrin.

Topics: Antineoplastic Agents, Phytogenic; beta Catenin; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p27; Dose-Response Relationship, Drug; Glioblastoma; Humans; Hyptis; Quercetin; Rutin; Time Factors

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

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

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

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

Rho GTPases are important regulators of actin cytoskeleton, but they are also involved in cell proliferation, transformation and oncogenesis. One of this proteins, RhoE, inhibits cell proliferation, however the mechanism that regulates this effect remains poorly understood. Therefore, we undertook the present study to determine the role of RhoE in the regulation of cell proliferation. For this purpose we generated an adenovirus system to overexpress RhoE in U87 glioblastoma cells. Our results show that RhoE disrupts actin cytoskeleton organization and inhibits U87 glioblastoma cell proliferation. Importantly, RhoE expressing cells show a reduction in Rb phosphorylation and in cyclin D1 expression. Furthermore, RhoE inhibits ERK activation following serum stimulation of quiescent cells. Based in these findings, we propose that RhoE inhibits ERK activation, thereby decreasing cyclin D1 expression and leading to a reduction in Rb inactivation, and that this mechanism is involved in the RhoE-induced cell growth inhibition. Moreover, we also demonstrate that RhoE induces apoptosis in U87 cells and also in colon carcinoma and melanoma cells. These results indicate that RhoE plays an important role in the regulation of cell proliferation and survival, and suggest that this protein may be considered as an oncosupressor since it is capable to induce apoptosis in several tumor cell lines.

Topics: Actin Cytoskeleton; Apoptosis; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cyclin D1; Cytoskeleton; Glioblastoma; Humans; Retinoblastoma Protein; rho GTP-Binding Proteins; Transfection

Active Ras contributes to the malignant phenotype of glioblastoma multiforme. Recent studies showed that the Ras inhibitor farnesyl thiosalicylic acid downregulates the transcription factor hypoxia-inducible factor-1alpha, causing shutdown of glycolysis in U87 glioblastoma cells. Farnesyl thiosalicylic acid also inhibited the growth of U87 cells. The way in which Ras inhibition affects U87 cell proliferation was not clear. Here we applied a computational method in which gene expression profile clustering is combined with promoter sequence analysis to obtain global dissection of the transcriptional response to farnesyl thiosalicylic acid in U87 cells. The analysis revealed a prominent Ras-dependent cell-cycle arrest response, in which a major component is highly enriched for the binding-site signature of the transcription factor E2F1. Electrophoretic mobility shift assays together with E2F-luciferase reporter assays showed that E2F1 was inactivated by the Ras inhibitor. Inhibition of Ras by farnesyl thiosalicylic acid promoted proteasomal degradation of cyclin D1, with a concomitant decrease in phosphorylated retinoblastoma protein accompanied by downregulation of E2F1 and decreased expression of key E2F1-regulated genes critical for cell-cycle progression. U87 cell growth arrest induced by farnesyl thiosalicylic acid was overridden by constitutive expression of E2F1. Thus, downregulation of E2F1 and of hypoxia-inducible factor-1alpha represents 2 distinct arms of the antioncogenic effect of Ras inhibitors in glioblastoma.

Topics: Cell Cycle; Cell Line, Tumor; Cell Survival; Cyclin D1; Dose-Response Relationship, Drug; E2F1 Transcription Factor; Electrophoretic Mobility Shift Assay; Enzyme Inhibitors; Farnesol; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Glioblastoma; Green Fluorescent Proteins; Humans; Immunoblotting; Immunohistochemistry; Luciferases; Phosphorylation; Promoter Regions, Genetic; Proteasome Endopeptidase Complex; ras Proteins; Recombinant Fusion Proteins; Retinoblastoma Protein; Salicylates

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

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

We have reported previously that the expression of focal adhesion kinase (FAK) is elevated in glioblastomas and that expression of FAK promotes the proliferation of glioblastoma cells propagated in either soft agar or in the C.B.17 severe combined immunodeficiency (scid) mouse brain. We therefore determined the effect of FAK on cell cycle progression in these cells. We found that overexpression of wild-type FAK promoted exit from G(1) in monolayer cultures of glioblastoma cells, enhanced the expression of cyclins D1 and E while reducing the expression of p27(Kip1) and p21(Waf1), and enhanced the kinase activity of the cyclin D1-cyclin-dependent kinase-4 (cdk4) complex. Transfection of the monolayers with a FAK molecule in which the autophosphorylation site is mutated (FAK397F) inhibited exit from G(1) and reduced the expression of cyclins D1 and E while enhancing the expression of p27(Kip1) and p21(Waf1). Small interfering RNA (siRNA)-mediated down-regulation of cyclin D1 inhibited the enhancement of cell cycle progression observed on expression of wild-type FAK, whereas siRNA-mediated down-regulation of cyclin E had no effect. siRNA-mediated down-regulation of p27(Kip1) overcame the inhibition of cell cycle progression observed on expression of FAK397F, whereas down-regulation of p21(Waf1) had no effect. These results were confirmed in vivo in the scid mouse brain xenograft model in which propagation of glioblastoma cells expressing FAK397F resulted in a 50% inhibition of tumor growth and inhibited exit from G(1). Taken together, our results indicate that FAK promotes proliferation of glioblastoma cells by enhancing exit from G(1) through a mechanism that involves cyclin D1 and p27(Kip1).

Topics: Animals; Brain; Cell Cycle; Cell Cycle Proteins; Cell Line, Tumor; Cyclin D1; Cyclin E; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Focal Adhesion Kinase 1; Focal Adhesion Protein-Tyrosine Kinases; G1 Phase; Gene Expression Regulation, Neoplastic; Glioblastoma; Humans; Mice; Mice, SCID; Mutation; Neoplasms, Experimental; Protein-Tyrosine Kinases; Transplantation, Heterologous; Tumor Suppressor Proteins

The objectives of this study were to investigate a nuclear factor-kappa B (NFkappaB) decoy oligonucleotide (ODN) strategy on the inhibition of glioblastoma (GBM) cell line growth and to evaluate a poly(DL-lactic-co-glycolic acid) (PLGA) microparticle delivery system for the NFKB decoy ODNs in vitro. We have demonstrated that NFkappaB activation is important in regulating GBM cell line growth. Aberrant nuclear expression of NFkappaB was found in a panel of GBM cell lines, while untransformed glial cells did not display NFkappaB activity. Nuclear translocation of NFkappaB was inhibited by using a 'decoy" ODN strategy. NFkappaB decoy ODNs designed to inhibit NFkappaB resulted in a significant reduction in cell number (up to 45%) compared to control cultures after 2 days. The reduction in cell number correlated with a decrease in cyclin D1 protein expression and a commensurate decrease in Cdk-4 activity. These results provide evidence suggesting that NFkappaB mediates cell cycle progression and demonstrates a mechanism linking increased NFkappaB activity with GBM cell growth and cell cycle disregulation. Decoy ODNs were encapsulated at a yield of 66% in PLGA microparticles and released in a controlled manner in phosphate buffered saline for up to 28 days. Approximately 83% of entrapped ODNs were released by day 28. During 3 days of GBM cell line culture, the released decoy ODNs retained their biologic activity and led to significantly reduced cell number as compared to control cultures. These findings offer a potential therapeutic strategy in the control of human GBM cell line growth in vitro and suggest that PLGA microparticles may be appropriate as delivery vehicles for the "decoy" ODN strategy.

Topics: Active Transport, Cell Nucleus; Biocompatible Materials; Biodegradation, Environmental; Blotting, Western; Cell Division; Cell Line; Cells, Cultured; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinases; Glioblastoma; Humans; Immunoblotting; Kinetics; Lactic Acid; Neuroglia; NF-kappa B; Oligonucleotides; Polyglycolic Acid; Polylactic Acid-Polyglycolic Acid Copolymer; Polymers; Proto-Oncogene Proteins; Time Factors; Tumor Cells, Cultured

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

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

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

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

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

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

The p16INK4a gene product acts as a negative regulator of the cell cycle by binding to cyclin-dependent kinases (CDKs) 4 and 6, thereby inhibiting the formation of an active CDK/cyclin D complex. Deletion of the p16 locus has been observed in tumor cell lines and, less frequently, in primary human neoplasms. We analyzed 31 glioblastomas and identified 6 cases with hemizygous and 6 with homozygous deletions of the p16 locus. Eight of these cases showed a concurrent amplification of the EGFR gene (epidermal growth factor receptor) while the overall frequency was 35%. This close correlation suggests that deletion of the p16 chromosomal region constitutes another genetic hallmark of the primary glioblastoma, which rapidly develops de novo, without a less malignant precursor lesion and for which EGFR amplification is a characteristic genetic change. The p16 protein was not detectable in 15 of 22 glioblastomas but only 4 of these showed homozygous deletion of the gene. The alternative transcript p16 beta, for which a growth-suppressing function has been suggested, was co-expressed with p16 alpha mRNA in most cases. Hypermethylation of CpG islands in the 5' region of the p16 gene was identified in only 1 case, suggesting that this alternative mechanism of gene silencing is rarely responsible for loss of p16 expression in glioblastomas. Likewise, only 1 glioblastoma carried a p16 mutation and in addition, unexpectedly, a homozygous deletion of p16 in approximately 80% of tumor cells. This mutation, Arg24Pro, has previously been identified in a melanoma kindred.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; DNA Methylation; ErbB Receptors; Female; Gene Deletion; Glioblastoma; Humans; Male; Middle Aged; Mutation; Polymerase Chain Reaction; RNA, Messenger

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

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

The mammalian nuclear protein E2F-1 has recently been cloned based on its ability to bind the retinoblastoma protein. To determine whether E2F-1 plays a role in the control of the cell proliferation, we introduced an inducible construct expressing an E2F-1 antisense RNA into the human glioblastoma T98G cell line and assessed DNA synthesis during the cell cycle. Expression of the antisense transcripts during the G1-S transition resulted in a marked delay in the completion of DNA synthesis. Band-shift analysis of bacterially produced E2F-1 showed that this protein bound to the promoters of human DNA polymerase-alpha, cyclin D1, and c-myb but not to the cdc2 gene promoter. E2F-1 also transactivated the bound promoters in transient transfection assays. These results suggest a major role for E2F-1 in the control of cell cycle progression via transcriptional regulation of proliferation-associated genes.

Topics: Base Sequence; Carrier Proteins; CDC2 Protein Kinase; Cell Cycle; Cell Cycle Proteins; Cyclin D1; Cyclins; DNA Polymerase II; DNA-Binding Proteins; DNA, Antisense; DNA, Complementary; DNA, Neoplasm; E2F Transcription Factors; E2F1 Transcription Factor; Glioblastoma; Humans; Molecular Sequence Data; Oncogene Protein p55(v-myc); Oncogene Proteins; Promoter Regions, Genetic; Retinoblastoma-Binding Protein 1; S Phase; Transcription Factor DP1; Transcription Factors; Transcription, Genetic; Transcriptional Activation; Tumor Cells, Cultured