transforming-growth-factor-beta and Glioma

Transforming growth factor beta (TGF-β) signaling is involved in the regulation of proliferation, differentiation and survival/or apoptosis of many cells, including glioma cells. TGF-β acts via specific receptors activating multiple intracellular pathways resulting in phosphorylation of receptor-regulated Smad2/3 proteins that associate with the common mediator, Smad4. Such complex translocates to the nucleus, binds to DNA and regulates transcription of many genes. Furthermore, TGF-β-activated kinase-1 (TAK1) is a component of TGF-β signaling and activates mitogen-activated protein kinase (MAPK) cascades. Negative regulation of TGF-β/Smad signaling may occur through the inhibitory Smad6/7. While genetic alterations in genes related to TGF-β signaling are relatively rare in gliomas, the altered expression of those genes is a frequent event. The increased expression of TGF-β1-3 correlates with a degree of malignancy of human gliomas. TGF-β may contribute to tumor pathogenesis in many ways: by direct support of tumor growth, by maintaining self-renewal of glioma initiating stem cells and inhibiting anti-tumor immunity. Glioma initiating cells are dedifferentiated cells that retain many stem cell-like properties, play a role in tumor initiation and contribute to its recurrence. TGF-β1,2 stimulate expression of the vascular endothelial growth factor as well as the plasminogen activator inhibitor and some metalloproteinases that are involved in vascular remodeling, angiogenesis and degradation of the extracellular matrix. Inhibitors of TGF-β signaling reduce viability and invasion of gliomas in animal models and show a great promise as novel, potential anti-tumor therapeutics.

Topics: Animals; Carcinogenesis; Glioma; Humans; Phosphorylation; Receptors, Transforming Growth Factor beta; Signal Transduction; Transforming Growth Factor beta

Malignant gliomas are the most common type of primary malignant brain tumors. They are characterized by enhanced growing capabilities, neoangiogenic proliferation, and extensive infiltration of the brain parenchyma, which make their complete surgical resection impossible. Together with transient and refractory responses to standard therapy, these aggressive neoplasms are incurable and present a median survival of 12 to 14 months. Transforming growth factor-beta (TGF-β) is a pleiotropic cytokine of which two of the three isoforms expressed in humans have been shown to be overexpressed proportionally to the histologic grade of glioma malignancy. The increase of chromosomal aberrations and genetic mutations observed in glioma cells turns TGF-β into an oncogene. For that reason, it plays critical roles in glioma progression through induction of several genes implicated in many carcinogenic processes such as proliferation, angiogenesis, and invasion. Consequently, investigators have begun developing innovative therapeutics targeting this growth factor or its signaling pathway in an attempt to hinder TGF-β's appalling effects in order to refine the treatment of malignant gliomas and improve their prognosis. In this paper, we extensively review the TGF-β-induced oncogenic pathways and discuss the diverse new molecules targeting this growth factor.

Topics: Brain Neoplasms; Glioma; Humans; Transforming Growth Factor beta

The transforming growth factor beta (TGF-β) pathway plays a key role in oncogenesis of advanced cancers. However, the effects of TGF-β pathway on gliomas are still controversial. So, it is essential to conduct a meta-analysis to determine their correlations. Eligible studies were included, and then odds ratios (ORs), standard mean differences (SMDs), and hazard ratios (HRs) with 95 % confidence intervals (95% CIs) were estimated. Funnel plots were available for evaluation of publication bias. In this meta-analysis, all 14 eligible studies involving 875 patients were included and conducted in China. Six studies with dichotomous data revealed altered TGF-β expression in glioma tissues was closely associated with high WHO grade (III + IV) (OR 4.39, 95% CI 2.90-6.63; p = 0.000), meanwhile, seven studies with continuous data also demonstrated TGF-β expression intensity extremely related to high grade (SMD -2.44, 95% CI -2.71, -2.16; p = 0.000). To our interest, TGF-β expression was associated with old age (OR 0.59, 95% CI 0.36-0.93; p = 0.025) rather than gender (OR 1.04, 95% CI 0.64-1.67; p = 0.884). Besides, TGF-β expression significantly correlated to 3-year-OS (n = 2; HR 2.53, 95% CI 1.18-5.41; p = 0.017) rather than 5-year-OS (n = 1; HR 1.04, 95% CI 0.66-1.64; p = 0.872) in glioma patients. No heterogeneity and publication bias were observed across all studies. Taken together, the present meta-analysis testifies TGF-β is potently associated with high grade and poor 3 years prognosis, and TGF-β test combined with survivin [1 Mol Neurobiol] and MMP9 [2 Mol Neurobiol] in glioma tissues should be clinically recommended as criteria of glioma grade in department of pathology.

Topics: Adult; Brain Neoplasms; Confidence Intervals; Female; Glioma; Humans; Male; Middle Aged; Neoplasm Grading; Odds Ratio; Prognosis; Publication Bias; Transforming Growth Factor beta

Glioma, a neuroglia originated malignancy, consists of one of the most aggressive primary tumors of the central nervous system with poor prognosis and lack of efficient treatment strategy. Cytokines have been implicated in several stages of glioma progression, participating in tumor onset, growth enhancement, angiogenesis and aggressiveness. Interestingly, cytokines have also the ability to inhibit glioma growth upon specific regulation or interplay with other molecules. This review addresses the dual role of major cytokines implicated in glioma pathology, pointing toward promising therapeutic approaches.

Topics: Animals; Apoptosis; Biomarkers; Brain Neoplasms; Cytokines; Disease Progression; Epigenesis, Genetic; Gene Expression Regulation; Gene Expression Regulation, Neoplastic; Genetic Therapy; Glioma; Humans; Interleukin-18; Interleukin-1beta; Interleukin-6; Interleukin-8; Microglia; Neovascularization, Pathologic; Prognosis; Transforming Growth Factor beta

Despite recent advances in treatment, the prognosis for glioblastoma multiforme (GBM) remains poor. The lack of response to treatment in GBM patients may be attributed to the immunosuppressed microenvironment that is characteristic of invasive glioma. Regulatory T-cells (Tregs) are immunosuppressive T-cells that normally prevent autoimmunity when the human immune response is evoked; however, there have been strong correlations between glioma-induced immunosuppression and Tregs. In fact, induction of Treg activity has been correlated with glioma development in both murine models and patients. While the exact mechanisms by which regulatory T-cells function require further elucidation, various cytokines such as interleukin-10 (IL-10) and transforming growth factor-β (TFG-β) have been implicated in these processes and are currently under investigation. In addition, hypoxia is characteristic of tumor development and is also correlated with downstream induction of Tregs. Due to the poor prognosis associated with immunosuppression in glioma patients, Tregs remain a promising area for immunotherapeutic research.

Topics: Animals; Brain Neoplasms; Glioblastoma; Glioma; Humans; Immune Tolerance; Interleukin-10; T-Lymphocytes, Regulatory; Transforming Growth Factor beta

With recent advances in microarray technology, including genomics, proteomics, and metabolomics, it brings a great challenge for integrating this "-omics" data to analysis complex disease. Glioma is an extremely aggressive and lethal form of brain tumor, and thus the study of the molecule mechanism underlying glioma remains very important. To date, most studies focus on detecting the differentially expressed genes in glioma. However, the meta-analysis for pathway analysis based on multiple microarray datasets has not been systematically pursued.. In this study, we therefore developed a systems biology based approach by integrating three types of omics data to identify common pathways in glioma. Firstly, the meta-analysis has been performed to study the overlapping of signatures at different levels based on the microarray gene expression data of glioma. Among these gene expression datasets, 12 pathways were found in GeneGO database that shared by four stages. Then, microRNA expression profiles and ChIP-seq data were integrated for the further pathway enrichment analysis. As a result, we suggest 5 of these pathways could be served as putative pathways in glioma. Among them, the pathway of TGF-beta-dependent induction of EMT via SMAD is of particular importance.. Our results demonstrate that the meta-analysis based on systems biology level provide a more useful approach to study the molecule mechanism of complex disease. The integration of different types of omics data, including gene expression microarrays, microRNA and ChIP-seq data, suggest some common pathways correlated with glioma. These findings will offer useful potential candidates for targeted therapeutic intervention of glioma.

Topics: Databases, Genetic; Epithelial-Mesenchymal Transition; Gene Ontology; Glioma; Humans; Oligonucleotide Array Sequence Analysis; Systems Biology; Transcriptome; Transforming Growth Factor beta

Transforming growth factor beta (TGF-β) signaling is involved in the regulation of proliferation, differentiation and survival/or apoptosis of many cells, including glioma cells. TGF-β acts via specific receptors activating multiple intracellular pathways resulting in phosphorylation of receptor-regulated Smad2/3 proteins that associate with the common mediator, Smad4. Such complex translocates to the nucleus, binds to DNA and regulates transcription of many genes. Furthermore, TGF-β-activated kinase-1 (TAK1) is a component of TGF-β signaling and activates mitogen-activated protein kinase cascades. Negative regulation of TGF-β/Smad signaling may occur through the inhibitory Smad6/7. Increased expression of TGF-β1-3 correlates with a degree of malignancy of human gliomas. TGF-β may contribute to tumor pathogenesis by direct support of tumor growth, self-renewal of glioma initiating stem cells and inhibiting of anti-tumor immunity. TGF-β1,2 stimulate expression of the vascular endothelial growth factor as well as the plasminogen activator inhibitor and some metalloproteinases that are involved in vascular remodeling, angiogenesis and degradation of the extracellular matrix. Inhibitors of TGF-β signaling reduce viability and invasion of gliomas in animal models and show promises as novel, potential anti-tumor therapeutics.

Topics: Animals; Brain Neoplasms; Disease Progression; Glioma; Humans; Signal Transduction; Transforming Growth Factor beta

Transforming growth factor-β (TGF-β) is a cytokine with a key role in tissue homeostasis and cancer. TGF-β elicits both tumor suppressive and tumor promoting functions during cancer progression, in a wide range of cancers. Here, we review the tumor promoting function of TGF-β and its possible promise as a therapeutic target in high grade gliomas, including glioblastoma multiforme (GBM), a disease with very poor prognosis. TGF-β signaling is highly active in high grade gliomas and elevated TGF-β activity has been associated with poor clinical outcome in this deadly disease. Common features of GBMs include fast cell proliferation, invasion into normal brain parenchyma, hypoxia, high angiogenic - and immunosuppressive activity, characteristics that all have been linked to activation of the TGF-β pathway. TGF-β signaling has also been connected with the cancer stem cell (CSC) phenotype in GBM. CSCs represent a subset of GBM cells thought to be responsible for tumor initiation, progression and relapse of disease. Following the description of these different properties of TGF-β signaling and the underlying mechanisms identified thus far, the promise of TGF-β targeted therapy in malignant gliomas is discussed. Several drugs targeting TGF-β signaling have been developed that showed potent antitumor activity in preclinical models. A number of agents are currently evaluated in early clinical studies in glioma patients. Available results of these studies are highlighted and a perspective on the promise of TGF-β-targeted therapy is given.

Topics: Antineoplastic Agents; Brain Neoplasms; Glioma; Humans; Signal Transduction; Transforming Growth Factor beta

A major contributing factor to glioma development and progression is its ability to evade the immune system. This chapter will explore the mechanisms utilized by glioma to mediate immunosuppression and immune evasion. These include intrinsic mechanisms linked to its location within the brain and interactions between glioma cells and immune cells. Lack of recruitment of naïve effector immune cells perhaps accounts for most of the immune suppression mediated by these tumor cells. This is enhanced by increased recruitment of microglia which resemble immature antigen presenting cells that are unable to support T-cell mediated immunity. Furthermore, secreted factors like TGF-β, COX-2 and IL-10, altered costimulatory molecules and inhibition of STAT-3 all contribute to the recruitment and expansion of regulatory T cells, which further modulate the immunosuppressive environment of glioma. In light of these findings, multiple immunotherapeutic treatment modalities are currently being explored.

Topics: Antigen-Presenting Cells; Brain Neoplasms; Cyclooxygenase 2; Glioma; Humans; Immune Tolerance; Interleukin-10; T-Lymphocytes, Regulatory; Transforming Growth Factor beta; Tumor Escape

Treatment of malignant gliomas represents one of the most formidable challenges in oncology. Despite treatment with surgery, radiation therapy, and chemotherapy, the prognosis remains poor, particularly for glioblastoma, which has a median survival of 12 to 15 months. An important impediment to finding effective treatments for malignant gliomas is the presence of the blood brain barrier, which serves to prevent delivery of potentially active therapeutic compounds. Multiple efforts are focused on developing strategies to effectively deliver active drugs to brain tumor cells. Blood brain barrier disruption and convection-enhanced delivery have emerged as leading investigational delivery techniques for the treatment of malignant brain tumors. Clinical trials using these methods have been completed, with mixed results, and several more are being initiated. In this review, we describe the clinically available methods used to circumvent the blood brain barrier and summarize the results to date of ongoing and completed clinical trials.

Topics: Animals; Antineoplastic Agents; Blood-Brain Barrier; Brain; Brain Neoplasms; Catheterization; Drug Delivery Systems; Drug Implants; Exotoxins; Genetic Vectors; Glioblastoma; Glioma; Humans; Immunotoxins; Interleukins; Transferrin; Transforming Growth Factor alpha; Transforming Growth Factor beta; Ultrasonic Therapy

We developed human mesenchymal stem cell (MSC) lines that could differentiate into various tissue cells including bone, neural cells, bone marrow (BM) stromal cells supporting the growth of hematopoietic stem cell (HSC), and so-called 'tumor stromal cells' mixing with tumor cells. We investigated the applicability of MSC as therapeutic cell transplanting reagents (cytoreagents). Telomerized human BM derived stromal cells exhibited a prolonged lifespan and supported the growth of hematopoietic clonogenic cells. The gene transfer of Indian hedgehog (Ihh) remarkably enhanced the HSC expansion supported by the human BM stromal cells. Gene-modified MSC are useful as therapeutic tools for brain tissue damage (e.g. brain infarction) and malignant brain neoplasms. MSC transplantation protected the brain tissue from acute ischemic damage in the midcerebral artery occlusion (MCAO) animal model. Brain-derived neurotrophic factor (BDNF)-gene transduction further enhanced the protective efficacy against the ischemic damage. MSC possessed excellent migratory ability and exerted inhibitory effects on the proliferation of glioma cells. Gene-modification of MSC with therapeutic cytokines clearly augmented the antitumor effect and prolonged the survival of tumor-bearing animals. Gene therapy employing MSC as a tissue-protecting and targeting cytoreagent would be a promising approach.

Topics: Animals; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Brain Ischemia; Brain Neoplasms; Cell Differentiation; Cell Survival; Disease Models, Animal; DNA-Binding Proteins; Gene Transfer Techniques; Genetic Therapy; Glioma; Hedgehog Proteins; Hematopoietic Stem Cells; Humans; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Telomerase; Trans-Activators; Transforming Growth Factor beta

Topics: Animals; Brain Neoplasms; Gene Expression Regulation, Neoplastic; Genes, p53; Glioma; Humans; Mutation; Neoplasm Invasiveness; Neural Cell Adhesion Molecule L1; Signal Transduction; Transforming Growth Factor beta

Topics: Antigens, Neoplasm; Brain Neoplasms; Cell Adhesion Molecules; Clinical Trials as Topic; Dendritic Cells; Fas Ligand Protein; Glioma; Humans; Immune Tolerance; Immunotherapy; Immunotherapy, Adoptive; Interferon-gamma; Interleukin-2; Killer Cells, Lymphokine-Activated; Membrane Glycoproteins; Receptors, Antigen, T-Cell; Signal Transduction; T-Lymphocytes; T-Lymphocytes, Cytotoxic; Transforming Growth Factor beta; Tumor Necrosis Factors

Brain tumors arise at a rate of nearly 5/100,000 in the general population, with over 17,000 U.S. residents being diagnosed each year. Approximately 60% of all brain tumors are gliomas, which are derived from interstitial tissue of the brain, such as astrocytic or ependymal tissue, or oligodendrocytes. The traditional protocols for treatment of malignant gliomas include diagnostic surgery, followed by regimens of radio- and chemotherapies. In the case of chemotherapy, the treatment protocols have remained nearly unchanged for over 30 years despite high mortality rates, and with little to no improvement in outcome. New advances in the fields of molecular biology and immunology have resulted in new possibilities for treating malignant gliomas by targeting cellular and molecular mechanisms of tumor cells, and stand in contrast to traditional forms of treatment. In the field of gene therapy, the possibility of using oncolytic viruses, such as HSV-1, for glioma therapy--specifically, of high grade astrocytomas--is being explored, and trials have begun using a replication-selective mutant strain known as G207. An increased understanding of the role of the cytokine TGF-beta2 has led to developments of anti-sense immunotherapy targeting this factor. The two examples mentioned here are discussed in this review and cited as possible improvements in the treatment of high grade astrocytomas.

Topics: Brain Neoplasms; Genetic Therapy; Genetic Vectors; Glioma; Humans; Immunotherapy; Transforming Growth Factor beta; Transforming Growth Factor beta2; Viruses

Numerous growth factors have been implicated in glioma angiogenesis. This chapter focuses on the role of scatter factor/hepatocyte growth factor, fibroblast growth factor, platelet-derived growth factor and transforming growth factor beta. We review the expression pattern of these factors in gliomas, their functional contribution to tumor angiogenesis - also in relation to vascular endothelial growth factor, and the effects resulting from their inhibition or overexpression in gliomas in vivo.

Topics: Animals; Brain Neoplasms; Cell Division; Disease Progression; Fibroblast Growth Factors; Glioma; Growth Substances; Hepatocyte Growth Factor; Humans; Ligands; Neovascularization, Pathologic; Platelet-Derived Growth Factor; Transforming Growth Factor beta

Characteristics of human malignant glioma are excessive proliferation, infiltrative growth, angiogenesis and suppression of anti-tumor immune surveillance. Transforming growth factor-beta (TGF-beta), a versatile cytokine, is intimately involved in the regulation of these processes. Here, we discuss the interactions of TGF-beta with growth factors, such as basic fibroblast growth factor (bFGF), epidermal growth factor (EGF) and platelet derived growth factor (PDGF), metalloproteinases (MMP-2, MMP-9) and their inhibitor, plasmin activator inhibitor-1 (PAI-1), and immune cells, like natural killer cells, T-cells and microglia. The differential effects of TGF-beta in glioma biology are outlined with emphasis on the induction of a survival advantage for glioma cells by enforced cell growth, migration, invasion, angiogenesis and immune paralysis. By virtue of its growth regulatory and immunomodulatory properties, TGF-beta promises to become a novel target for the experimental therapy of human malignant glioma.

Topics: Brain Neoplasms; Glioma; Humans; Neovascularization, Pathologic; Transforming Growth Factor beta; Tumor Escape

Matrix metalloproteinases (MMPs) are a family of extracellular endopeptidases that selectively degrade components of the extracellular matrix. MMPs are implicated in tumor cell invasion because they mediate the breakdown of the basal membrane. In addition, they seem to be important for the creation and maintenance of a microenvironment that facilitates tumor cell survival. Among the essential characteristics of human malignant gliomas are infiltrative growth, angiogenesis and suppression of antitumor immune surveillance. Transforming growth factor-beta (TGF-beta) is intimately involved in the regulation of these processes. We have previously demonstrated that TGF-beta promotes the migration of LN- 18 and LN-229 glioma cells via a process that may involve the upregulation of alphaVbeta3 integrin expression. Furthermore, we have defined a novel pathway for hepatocyte growth factor (HGF)-induced glioma cell migration and invasion which requires the induction of TGF-beta2 expression. Here, we demonstrate that TGF-beta2 induces MMP-2 expression and suppresses tissue inhibitor of metalloproteinases (TIMP)-2 expression and that concentration-dependently promotes the invasion of U87MG and LN-229 glioma cells in a matrigel invasion assay. Similarly, ectopic expression of the anti-apoptotic BCL-x, protein leads to enhanced matrigel invasion by LN-18 and LN-229 glioma cells. We outline the possible interrelations of TGF-beta, proteins of the BCL-2 family, integrins and metalloprotease activity. By virtue of its promotion of glioma invasion and its growth regulatory and immunomodulatory properties. TGF-beta continues to be one of the most promising targets for the experimental therapy of human malignant glioma.

Topics: 3T3 Cells; Animals; bcl-X Protein; Brain Neoplasms; Cell Movement; Collagen; Cytoskeletal Proteins; Drug Combinations; Enzyme Induction; Extracellular Matrix; Glioma; Humans; Laminin; Matrix Metalloproteinase 2; Mice; Neoplasm Invasiveness; Neoplasm Proteins; Phosphoproteins; Proteoglycans; Proto-Oncogene Proteins c-bcl-2; Receptors, Vitronectin; Recombinant Proteins; Tissue Inhibitor of Metalloproteinase-2; Transfection; Transforming Growth Factor beta; Transforming Growth Factor beta1; Transforming Growth Factor beta2; Tumor Cells, Cultured

Malignant brain tumors are notoriously invasive. Although surgical debulking can relieve the patient of the main mass of tumor, adjuvant treatments are needed to target the glioma cells that infiltrate through normal parenchyma as single cells or pockets of tumor cells from which recurrent tumors arise. Successful adjuvant cellular therapy of brain tumors, or activation of endogenous immune cells, requires that either cell effectors make direct contact with tumor cells or come within close proximity to them and exert an indirect effect. This review examines current clinical trials aimed at direct lysis of glioma cells and trials making gliomas more visible to the endogenous immune system.

Topics: Animals; Brain Neoplasms; Clinical Trials as Topic; Combined Modality Therapy; Cytokines; DNA, Antisense; Forecasting; Genetic Therapy; Glioma; Humans; Immune Tolerance; Immunization, Passive; Immunologic Factors; Immunotherapy; Immunotherapy, Adoptive; Injections, Intralesional; Interferon-gamma; Killer Cells, Natural; Lymphocytes, Tumor-Infiltrating; Neoplasm Proteins; T-Lymphocytes, Cytotoxic; Transforming Growth Factor beta; Transforming Growth Factor beta2; Tumor Cells, Cultured; Vaccination

Malignant glioblastomas (gliomas) account for approximately one third of all diagnosed brain tumors. Yet, a decade of research has made little progress in advancing the treatment of these tumors. In part this lack of progress is linked to the challenge of discovering how glial tumors are capable of both modulating host immune function and neutralizing immune-based therapies. Patients with gliomas exhibit a broad suppression of cell-mediated immunity. The impaired cell-mediated immunity observed in patients with gliomas appears to result from immunosuppressive factor(s) secreted by the tumor. This article reviews what has been elucidated about the immune defects of patients harboring glioma and the glioma-derived factors which mediate this immunosuppression. A model involving systemic cytokine dysregulation is presented to suggest how the immune defects arise in these individuals.

Topics: Apoptosis; Brain Neoplasms; Dinoprostone; Glioblastoma; Glioma; Humans; Interleukin-10; Killer Cells, Natural; Monocytes; Receptors, Interleukin-2; Signal Transduction; T-Lymphocytes; Transforming Growth Factor beta

We review the role of peptide growth factors in angiogenesis and progression of low grade glial tumors to higher grade glioblastoma multiforme (GBM).. Vascular pathology is a key feature of glioblastoma multiforme characterized by hypervascularity, vascular permeability, and hypercoagulability.. Vascular endothelial growth factor (VEGF) can mediate all of these effects, but by itself does not promote malignant growth. Epidermal growth factor (EGF), platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), and transforming growth factor beta (TGF-beta) are implicated in the angiogenesis of a number of tumors including those of glial origin.. These growth factors are suggested to play a role in autocrine and/or paracrine mediated tumorogenesis of astrocytic tumors. VEGF secretion might be the product of induction by physiologic concentrations of other growth factors with VEGF being the common pathway of neovascularization and progression to GBM.

Topics: Animals; Disease Progression; Endothelial Growth Factors; Epidermal Growth Factor; Fibroblast Growth Factors; Glioma; Growth Substances; Humans; Lymphokines; Neovascularization, Pathologic; Platelet-Derived Growth Factor; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

This review examines the apparently paradoxical conversion of transforming growth factor beta's (TGFbeta) regulatory role as a growth inhibitor among normal glial cells to that of a progression factor among glioblastomas (GM). In vitro, TGFbeta functions as an autocrine growth inhibitor of near-diploid gliomas of any grade. In contrast, hyperdiploid glioblastoma multiforme (HD-GM) cultures proliferate in response to TGFbeta, which is mediated by induction of platelet-derived growth factor B chain (PDGF-BB). The dominant hypothesis of TGFbeta's pathogenetic association with malignant transformation has been predicated upon acquisition of resistance to its growth inhibitory effects. However, the lack of obvious correlation with TGFbeta receptor (TbetaR) expression (or loss) between the HD-GM and the TGFbeta-inhibited GM cultures suggests the existence of intrinsically opposed regulatory mechanisms influenced by TGFbeta. The mechanism of conversion might be explained either by the loss of a putative tumor suppressor gene (TSG) which mediates TGFbeta's inhibition of growth or by enhancement of an active oncogenic pathway among the HD-GM. The frequency of mutations within glioma-associated TSG, such as TP53 and RB, suggests that defects in TGFbeta's inhibitory signaling pathway may have analogous effects in the progression to HD-GM, and TGFbeta's conversion to a mitogen. Alternative sites of inactivation which might explain the loss of TGFbeta's inhibitory effect include inactivating mutation/loss of the TbetaR type II, alterations in post-receptor signal transmission or the cyclin/cyclin dependent kinase system which regulates the phosphorylation of pRB. Loss or inactivation of a glial TSG with a consequent failure of inhibition appears to allow TGFbeta's other constitutive effects, such as induction of c-sis, to become functionally dominant. Mechanistically, TGFbeta's conversion from autocrine inhibitor to mitogen promotes 'clonal dominance' by conferring a Darwinian advantage to the hyperdiploid subpopulations through qualitative and quantitative differences in its modulation of PDGF-A and c-sis, with concomitant paracrine inhibition of competing, near-diploid elements.

Topics: Animals; Brain Neoplasms; Cell Transformation, Neoplastic; Disease Progression; Glioma; Humans; Transforming Growth Factor beta

Human malignant gliomas are rather resistant to all current therapeutic approaches including surgery, radiotherapy and chemotherapy as well as antibody-guided or cellular immunotherapy. The immunotherapy of malignant glioma has attracted interest because of the immunosuppressed state of malignant glioma patients which resides mainly in the T-cell compartment. This T-cell suppression has been attributed to the release by the glioma cells of immunosuppressive factors like transforming growth factor-beta (TGF-beta) and prostaglandins. TGF-beta has multiple effects in the immune system, most of which are inhibitory. TGF-beta appears to control downstream elements of various cellular activation cascades and regulates the expression of genes that are essential for cell cycle progression and mitosis. Since TGF-beta-mediated growth arrest of T-cell lines results in their apoptosis in vitro, glioma-derived TGF-beta may prevent immune-mediated glioma cell elimination by inducing apoptosis of tumor-infiltrating lymphocytes in vivo. T-cell apoptosis in the brain may be augmented by the absence of professional antigen-presenting cells and of appropriate costimulating signals. Numerous in vitro studies predict that tumor-derived TGF-beta will incapacitate in vitro-expanded and locally administered lymphokine-activated killer cells (LAK-cells) or tumor-infiltrating lymphocytes. Thus, TGF-beta may be partly responsible for the failure of current adoptive cellular immunotherapy of malignant glioma. Recent experimental in vivo studies on non-glial tumors have corroborated that neutralization of tumor-derived TGF-beta activity may facilitate immune-mediated tumor rejection. Current efforts to improve the efficacy of immunotherapy for malignant glioma include various strategies to enhance the immunogenicity of glioma cells and the cytotoxic activity of immune effector cells, e.g., by cytokine gene transfer. Future strategies of cellular immunotherapy for malignant glioma will have to focus on rendering glioma cell-targeting immune cells resistent to local inactivation and apoptosis which may be induced by TGF-beta and other immunosuppressive molecules at the site of neoplastic growth. Cytotoxic effectors targeting Fas/APO-1, the receptor protein for perforin-independent cytotoxic T-cell killing, might be promising, since Fas/APO-1 is expressed by glioma cells but not by untransformed brain cells, and since Fas/APO-1-mediated killing in vitro is not inhibited by TGF-beta.

Topics: Apoptosis; Brain; Glioma; Humans; Immunotherapy; T-Lymphocytes; Transforming Growth Factor beta; Treatment Failure

Patients with primary intracranial tumors (gliomas) exhibit a profound decrease in immunity, the mechanism of which has, until recently, remained obscure. Here Thomas Roszman, Lucinda Elliott and William Brooks reveal that T cells obtained from these patients exhibit defects in interleukin 2 secretion and in expression of the high-affinity IL-2 receptor and they discuss the role played by immunosuppressive factors produced by gliomas in inducing these defects.

Topics: Brain Neoplasms; Glioma; Humans; Interleukin-2; Lymphocyte Activation; Receptors, Interleukin-2; T-Lymphocytes; Transforming Growth Factor beta

Transforming growth factor-β (TGF-β) signaling is involved in glioma development. The monoclonal antibody fresolimumab (GC1008) can neutralize all mammalian isoforms of TGF-β, and tumor uptake can be visualized and quantified with (89)Zr-fresolimumab PET in mice. The aim of this study was to investigate the fresolimumab uptake in recurrent high-grade gliomas using (89)Zr-fresolimumab PET and to assess treatment outcome in patients with recurrent high-grade glioma treated with fresolimumab.. Patients with recurrent glioma were eligible. After intravenous administration of 37 MBq (5 mg) of (89)Zr-fresolimumab, PET scans were acquired on day 2 or day 4 after tracer injection. Thereafter, patients were treated with 5 mg of fresolimumab per kilogram intravenously every 3 wk. (89)Zr-fresolimumab tumor uptake was quantified as maximum standardized uptake value (SUVmax). MR imaging for response evaluation was performed after 3 infusions or as clinically indicated.. Twelve patients with recurrent high-grade glioma were included: 10 glioblastomas, 1 anaplastic oligodendroglioma, and 1 anaplastic astrocytoma. All patients underwent (89)Zr-fresolimumab PET 4 d after injection. In 4 patients, an additional PET scan was obtained on day 2 after injection. SUVmax on day 4 in tumor lesions was 4.6 (range, 1.5-13.9) versus a median SUVmean of 0.3 (range, 0.2-0.5) in normal brain tissue. All patients showed clinical or radiologic progression after 1-3 infusions of fresolimumab. Median progression-free survival was 61 d (range, 25-80 d), and median overall survival was 106 d (range, 37-417 d).. (89)Zr-fresolimumab penetrated recurrent high-grade gliomas very well but did not result in clinical benefit.

Topics: Adult; Aged; Animals; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Brain Neoplasms; Cell Line, Tumor; Female; Glioma; Humans; Male; Metabolic Clearance Rate; Middle Aged; Neoplasm Recurrence, Local; Positron-Emission Tomography; Radioisotopes; Radiopharmaceuticals; Tissue Distribution; Transforming Growth Factor beta; Zirconium

Activation of the p21-ras signaling pathway from aberrantly expressed receptors promotes the growth of malignant human astrocytomas. Perillyl alcohol has shown to have both chemopreventive and chemotherapeutic activities in preclinical studies. The underlying action mechanism(s) of POH has yet to be delineated but may involve effects on the TGF-beta and/or the Ras signaling pathways. The intranasal delivery allows drugs that do not cross the BBB to enter the CNS; moreover, it eliminates the need for systemic delivery, thereby reducing unwanted systemic side effects.. We are conducting a phase I/II study to evaluate the antitumoral activity of POH intranasal delivery in a 4x daily schedule in patients with recurrent MG. The objective was to determine PFS at 6 months and the safety for POH in adult patients who failed conventional treatment. Assessments were performed every 27 days. Thirty-seven patients with progressive disease after prior surgery, radiotherapy, and at least temozolomide-based chemotherapy were enrolled, 29 of whom had GBM, 5 who had anaplastic astrocytoma, and 3 had AO.. One patient (3.4%) with GBM and 1 patient (33.3%) with AO achieved partial response; 13 patients (44.8%) with GBM, 3 patients (60%) with AA, and 1 (33.3%) with AO achieved stable disease; 15 (51.7%) patients with GBM, 2 (40%) patients with AA, and 1 (33.3%) with AO showed progressive disease. Progression-free survival (partial response and stable disease) was 48.2% for patients with GBM, 60% for patients with AA, and 66.6% for patients with AO.. There were no toxicity events. Perillyl alcohol is well tolerated and regression of tumor size in some patients is suggestive of antitumor activity. This work discusses POH intranasal delivery as a potential adjuvant therapeutic strategy for patients with malignant gliomas.

Topics: Administration, Intranasal; Antineoplastic Agents; Antineoplastic Agents, Alkylating; Blood-Brain Barrier; Brain Neoplasms; Dacarbazine; Disease Progression; Drug Administration Schedule; Enzyme Inhibitors; Female; Glioma; Humans; Magnetic Resonance Imaging; Male; Monoterpenes; Neoplasm Recurrence, Local; Oncogene Protein p21(ras); Signal Transduction; Survival Rate; Temozolomide; Transforming Growth Factor beta; Treatment Outcome

To elucidate the role of tumor necrosis factor alpha (TNF alpha) as a biological response modifier, we studied cellular and cytokine responses of the central nervous system to TNF alpha administered intracranially in a phase I clinical trial for patients with malignant gliomas. Six patients received injections of TNF alpha (1.25 x 10(3)-10 x 10(3) U/injection) into the tumor cavities, and regional fluids (RF) and lumbar cerebrospinal fluids (CF) were serially sampled before and after the injections. Recruitment of neutrophils occurred, mostly peaking 8 h after TNF alpha injection, and fewer numbers of CD4+ T cells and monocytes/macrophages migrated, subsequently peaking at 24 h. The CF leukocytosis persisted for 48 h and was associated with an increased level of neutrophil chemotactic activity in the CF. This neutrophil chemotactic activity was attributed to interleukin-8 (IL-8) by HPLC. The level of IL-6 activity in the CF and RF consistently increased; beginning 2 h after TNF alpha injection and reaching the maximum between 8 h and 12 h. It returned to the basal level within 48 h. IL-1 beta was detected in the CF of three patients, its level peaking at 8 h. Prostaglandin E2 also increased after injection of TNF alpha, peaking between 4 h and 12 h and then gradually decreasing. Transforming growth factor beta was found in all cases tested and one patient showed a significant change after TNF alpha injection. IL-2 activity, interferon alpha (INF alpha) activity, IFN beta, and granulocyte/macrophage-colony-stimulating factor were not detected in the CF or RF. In conclusion, TNF alpha is biologically effective in inducing migration of immune cells and generating multiple cytokine responses in the human central nervous system.

Topics: Adult; Chemotaxis, Leukocyte; Dinoprostone; Glioma; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Interferons; Interleukin-1; Interleukin-2; Interleukin-6; Middle Aged; Neutrophils; Time Factors; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Adult-type diffuse gliomas, CNS WHO grade 4 are the most aggressive primary brain tumors and represent a particular challenge for therapeutic intervention.. In a single-center retrospective study of matched pairs of initial and post-therapeutic glioma cases with a recurrence period greater than 1 year, we performed whole exome sequencing combined with mRNA and microRNA expression profiling to identify processes that are altered in recurrent gliomas.. Mutational analysis of recurrent gliomas revealed early branching evolution in 75% of the patients. High plasticity was confirmed at the mRNA and miRNA levels. SBS1 signature was reduced and SBS11 was elevated, demonstrating the effect of alkylating agent therapy on the mutational landscape. There was no evidence for secondary genomic alterations driving therapy resistance. ALK7/ACVR1C and LTBP1 were upregulated, whereas LEFTY2 was downregulated, pointing towards enhanced Tumor Growth Factor β (TGF-β) signaling in recurrent gliomas. Consistently, altered microRNA expression profiles pointed towards enhanced Nuclear Factor Kappa B and Wnt signaling that, cooperatively with TGF-β, induces epithelial to mesenchymal transition (EMT), migration, and stemness. TGF-β-induced expression of pro-apoptotic proteins and repression of antiapoptotic proteins were uncoupled in the recurrent tumor.. Our results suggest an important role of TGF-β signaling in recurrent gliomas. This may have clinical implications since TGF-β inhibitors have entered clinical phase studies and may potentially be used in combination therapy to interfere with chemoradiation resistance. Recurrent gliomas show high incidence of early branching evolution. High tumor plasticity is confirmed at the level of microRNA and mRNA expression profiles.

Topics: Activin Receptors, Type I; Adult; Brain Neoplasms; Cell Line, Tumor; Epithelial-Mesenchymal Transition; Glioma; Humans; MicroRNAs; Recurrence; Retrospective Studies; RNA, Messenger; Transforming Growth Factor beta; Up-Regulation

Gliomas are a group of primary intracranial tumors with high morbidity and mortality. The previous researches indicated a crucial role of CKS2 (cyclin-dependent kinases regulatory subunit 2) in hepatocellular carcinoma and breast cancer; however, little is known about the molecular mechanism of CKS2 in the tumorigenesis and epithelial-mesenchymal transition-like (EMT) process in glioma.. Datasets for bioinformatics analysis were obtained from the GEO, TCGA and CGGA databases. qRT-PCR, western blotting (WB), and immunohistochemistry (IHC) assays were used to investigate the expression patterns of CKS2 among glioma and brain tissues. Glioma cells were transfected with small interfering RNA/overexpression plasmid against CKS2, then clone formation assay, CCK-8, wound healing, Transwell assay, and flow cytometry were performed to detect changes in cell viability, invasiveness, and the apoptosis rate. Markers of cell invasion, apoptosis, EMT and TGFβ/SMAD signaling were evaluated by WB and immunofluorescence (IF) assays.. We found that CKS2 overexpression correlates with poor prognosis in human glioma and knockdown of CKS2 could inhibit cell proliferation, migration, invasion, and induced apoptosis in glioma cells. Besides, we also found that knockdown of CKS2 could reverse the EMT process via modulating EMT-related molecules. Glioma cells with overexpression of CKS2 were constructed to confirmed the fact that CKS2 induced nucleocytoplasmic translocation of SMAD2/3 and activated TGFβ/SMAD pathway, then upregulated its downstream targets expression, while inhibition of TGFβ/SMAD (by TGFβ inhibitor LY2157299 or SMAD4 siRNA) could reverse the tumor-promoting effects and malignant phenotype caused by CKS2 overexpression.. We identified CKS2 as a critical contributor to the gliomagenesis, which might provide a novel therapeutic target for inhibiting the spread and infiltration of glioma.

Topics: CDC2-CDC28 Kinases; Cell Cycle Proteins; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin-Dependent Kinases; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Glioma; Humans; Liver Neoplasms; Phenotype; RNA, Small Interfering; Transforming Growth Factor beta

GBM is the central nervous system's most aggressive and malignant tumor. TGF-β expression is elevated in GBM, and it promotes invasion and EMT. TGF-β regulates the expression of several lncRNAs, which promote glioma pathogenesis. Here we characterize the role of TGF-β-induced lncRNA- LINC01711 in glioma pathogenesis. We show that LINC01711 expression is significantly upregulated in GBM tissues and is associated with poor overall survival of GBM patients. Loss-of-function studies illustrate that LINC01711 promotes proliferation, migration, and invasion in GBM. In addition, LINC01711 depletion sensitizes glioma cells to Temozolomide (TMZ) induced apoptosis by inhibiting ZEB1 expression. LINC01711 functions as a competing endogenous RNA for miR-34a and promotes ZEB1 expression to regulate invasion. Our findings suggest that LINC01711 is an attractive therapeutic target for GBM.

Topics: Brain Neoplasms; Cell Line, Tumor; Cell Proliferation; Glioblastoma; Glioma; Humans; MicroRNAs; RNA, Long Noncoding; Transforming Growth Factor beta; Transforming Growth Factors; Zinc Finger E-box-Binding Homeobox 1

Gliomas are aggressive brain tumors characterized by uncontrolled cell proliferation. FAM64A, a cell cycle-related gene, has been found to promote cell proliferation in various tumors, including gliomas. However, the regulatory mechanism and clinical significance of FAM64A in gliomas remain unclear. In this study, we investigated FAM64A expression in gliomas with different grades and constructed FAM64A silenced cell lines to study its functions. Our results demonstrated that FAM64A was highly expressed in glioblastoma (P < 0.001) and associated with a poor prognosis (P < 0.001). Expression profiles at the single-cell resolution indicated FAM64A could play a role in a cell-cycle-dependent way to promote glioma cell proliferation. We further observed that FAM64A silencing in glioma cells resulted in disrupted proliferation and migration ability, and increased cell accumulation in the G2/M phase (P = 0.034). Additionally, TGF-β signaling upregulates FAM64A expression, and SMAD4 and FAM64A co-localize in high-grade glioma tissues. We found FAM64A knockdown inhibited TGF-β-induced epithelial-mesenchymal transition in glioma. Our findings suggest that FAM64A could serve as a diagnostic and therapeutic target in gliomas.

Topics: Brain Neoplasms; Cell Cycle; Cell Division; Cell Line, Tumor; Cell Movement; Cell Proliferation; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Glioma; Humans; Transforming Growth Factor beta

Microtubes (MTs), cytoplasmic extensions of glioma cells, are important cell communication structures promoting invasion and treatment resistance through network formation. MTs are abundant in chemoresistant gliomas, in particular, glioblastomas (GBMs), while they are uncommon in chemosensitive IDH-mutant and 1p/19q co-deleted oligodendrogliomas. The aim of this study was to identify potential signaling pathways involved in MT formation.. Bioinformatics analysis of TCGA was performed to analyze differences between GBM and oligodendroglioma. Patient-derived GBM stem cell lines were used to investigate MT formation under transforming growth factor-beta (TGF-β) stimulation and inhibition in vitro and in vivo in an orthotopic xenograft model. RNA sequencing and proteomics were performed to detect commonalities and differences between GBM cell lines stimulated with TGF-β.. Analysis of TCGA data showed that the TGF-β pathway is highly activated in GBMs compared to oligodendroglial tumors. We demonstrated that TGF-β1 stimulation of GBM cell lines promotes enhanced MT formation and communication via calcium signaling. Inhibition of the TGF-β pathway significantly reduced MT formation and its associated invasion in vitro and in vivo. Downstream of TGF-β, we identified thrombospondin 1 (TSP1) as a potential mediator of MT formation in GBM through SMAD activation. TSP1 was upregulated upon TGF-β stimulation and enhanced MT formation, which was inhibited by TSP1 shRNAs in vitro and in vivo.. TGF-β and its downstream mediator TSP1 are important mediators of the MT network in GBM and blocking this pathway could potentially help to break the complex MT-driven invasion/resistance network.

Topics: Glioblastoma; Glioma; Humans; Oligodendroglioma; Thrombospondin 1; Transforming Growth Factor beta

Topics: Animals; Cell Line, Tumor; Cell Movement; Cell Proliferation; Gene Expression Regulation, Neoplastic; Glioma; Humans; Mice; MicroRNAs; RNA, Long Noncoding; RNA, Small Interfering; Transforming Growth Factor beta; Tumor Suppressor Protein p53

Glioblastoma (GBM) is the most common and aggressive primary malignant brain tumor. The unregulated expression of Claudin-4 (CLDN4) plays an important role in tumor progression. However, the biological role of CLDN4 in GBM is still unknown. This study aimed to determine whether CLDN4 mediates glioma malignant progression, if so, it would further explore the molecular mechanisms of carcinogenesis. Our results revealed that CLDN4 was significantly upregulated in glioma specimens and cells. The inhibition of CLND4 expression could inhibit mesenchymal transformation, cell invasion, cell migration and tumor growth in vitro and in vivo. Moreover, combined with in vitro analysis, we found that CLDN4 can modulate tumor necrosis factor-α (TNF-α) signal pathway. Meanwhile, we also validated that the transforming growth factor-β (TGF-β) signal pathway can upregulate the expression of CLDN4, and promote the invasion ability of GBM cells. Conversely, TGF-β signal pathway inhibitor ITD-1 can downregulate the expression of CLDN4, and inhibit the invasion ability of GBM cells. Furthermore, we found that TGF-β can promote the nuclear translocation of CLDN4. In summary, our findings indicated that the TGF-β/CLDN4/TNF-α/NF-κB signal axis plays a key role in the biological progression of glioma. Disrupting the function of this signal axis may represent a new treatment strategy for patients with GBM.

Topics: Cell Line, Tumor; Claudin-4; Glioblastoma; Glioma; Humans; NF-kappa B; Signal Transduction; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha; Up-Regulation

Topics: Brain Neoplasms; Cell Line, Tumor; Glioblastoma; Glioma; Humans; Neoplastic Stem Cells; Receptors, Vitronectin; STAT3 Transcription Factor; Transforming Growth Factor beta; Tumor Microenvironment; Tumor-Associated Macrophages

DNA damage response (DDR) proficiency is the principal mechanism of temozolomide (TMZ) resistance in glioma. Accumulating evidence has also suggested the determining role of DDR in anticancer immunity. We propose that a comprehensive investigation of the DDR landscape can optimize glioma treatment.. We identified the pronounced enrichment of DDR in TMZ-resistant glioma cells by RNA sequencing. Nine differentially expressed genes between TMZ-sensitive/resistant glioma cells were selected to construct the DDR score through lasso regression analysis. Two glioma cohorts from TCGA and CGGA were interrogated to evaluate the predictive ability of DDR score. Multiple algorithms were applied to estimate the immunotherapeutic responses of two DDR phenotypes. Immunohistochemistry was used to determine the protein levels of PD-L1 and TGFβ in glioma specimens. The oncoPredict package was employed to predict the candidate chemotherapy agents.. DDR score exhibited a robust prognostic capability in TCGA and CGGA cohorts and served as an independent predictive biomarker in glioma patients. Functional enrichment analyses revealed that high and low DDR score groups were characterized by distinct immune activity and metabolic processes. Elevated levels of infiltrating immune cells (including CD8+ T cells, CD4+ T cells, and dendritic cells) were observed in the high DDR score glioma. Further, high DDR scores correlated with increased mutation burden, up-regulated immune checkpoints, and tumor immunity activation, indicating a profound interplay between DDR score and glioma immunogenicity. In addition, PD-L1 and TGFβ were overexpressed in recurrent glioma specimens compared with primary ones. Finally, we estimated that PI3K inhibitors may serve as latent regimens for high DDR score patients.. Our study highlighted the promising prognostic role of DDR score in glioma. Individual assessment of DDR status for patients with glioma may provide new clues for developing immunotherapeutic strategies.

Topics: B7-H1 Antigen; DNA Damage; Glioma; Humans; Immunotherapy; Neoplasm Recurrence, Local; Phosphatidylinositol 3-Kinases; Transforming Growth Factor beta

Glioblastoma multiforme (GBM) is the most lethal type of craniocerebral gliomas. Glioma stem cells (GSCs) are fundamental reasons for the malignancy and recurrence of GBM. Revealing the critical mechanism within GSCs' self-renewal ability is essential. Our study found a novel circular RNA (circRPPH1) that was up-regulated in GSCs and correlated with poor survival. The effect of circRPPH1 on the malignant phenotype and self-renewal of GSCs was detected in vitro and in vivo. Mechanistically, UPF1 can bind to circRPPH1 and maintain its stability. Therefore, more existing circRPPH1 can interact with transcription factor ATF3 to further transcribe UPF1 and Nestin expression. It formed a feedback loop to keep a stable stream for stemness biomarker Nestin to strengthen tumorigenesis of GSCs continually. Besides, ATF3 can activate the TGF-β signaling to drive GSCs for tumorigenesis. Knocking down the expression of circRPPH1 significantly inhibited the proliferation and clonogenicity of GSCs both in vitro and in vivo. The overexpression of circRPPH1 enhanced the self-renewal of GSCs. Our findings suggest that UPF1/circRPPH1/ATF3 maintains the potential self-renewal of GSCs through interacting with RNA-binding protein and activating the TGF-β signal pathway. Breaking the feedback loop against self-renewing GSCs may represent a novel therapeutic target in GBM treatment.

Topics: Activating Transcription Factor 3; Brain Neoplasms; Carcinogenesis; Cell Line, Tumor; Cell Proliferation; Cell Transformation, Neoplastic; Feedback; Glioblastoma; Glioma; Humans; Neoplastic Stem Cells; Nestin; Phenotype; RNA Helicases; Trans-Activators; Transforming Growth Factor beta

Microglia is the major cellular component of glioma mass that promotes glioma growth, invasion, and chemoresistance by releasing inflammatory factors. Sterile alpha and HEAT/Armadillo motif (SARM), a member of the Toll-interleukin-1 receptor (TIR) domain-containing adaptor family, is primarily expressed in the central nervous system. However, the role of SARM in glioma is still undefined. In the present work, we examined the function of SARM in microglial polarization and glioma progression. Our results showed that forced the expression of SARM in GL261 glioma cells inhibited tumor growth, and reduced interleukin (IL)-6 secretion in conditioned media. Silencing of SARM in microglia cells inhibited IL-4-induced M2 polarization, enhanced lipopolysaccharide -induced M1 microglial polarization. Furthermore, overexpression of SARM increased the migration of microglia cells upon TGFβ stimulation. These data suggested that SARM is involved in neuro-inflammation and microglia activation. In summary, this study provides novel insight into the mechanisms of microglial polarization.

Topics: Culture Media, Conditioned; Glioma; Humans; Interleukin-4; Lipopolysaccharides; Microglia; Receptors, Interleukin-1; Transforming Growth Factor beta

Synergies of transcription factors, chromatin modifiers and their target genes are vital for cell fate determination in human cancer. Although the importance of numerous epigenetic machinery for regulating gliomagenesis has been previously recognized, how chromatin modifiers collaborate with specific transcription factors remains largely elusive. Herein we report that Pontin chromatin remodelling factor acts as a coactivator for LEF1 to activate TGFβ/SMAD signalling, thereby contributing to gliomagenesis. Pontin is highly expressed in gliomas, and its overexpression paralleled the grade elevation and poor prognosis of patients. Functional studies verified its oncogenic roles in GBM cells by facilitating cell proliferation, survival and invasion both in vitro and in vivo. RNA sequencing results revealed that Pontin regulated multiple target genes involved in TGFβ/SMAD signalling. Intriguingly, we found that Pontin amplified TGFβR2 gene transcription by recruiting LEF1, thereby activating TGFβ/SMAD signalling and facilitating gliomagenesis. Furthermore, higher TGFβR2 expression conferred worse patient outcomes in glioma. To conclude, our study revealed that the Pontin-LEF1 module plays a crucial role in driving TGFβR2 gene transcription, which could be exploited to target TGFβ/SMAD signalling for anti-glioma therapy.

Topics: ATPases Associated with Diverse Cellular Activities; Carrier Proteins; Cell Proliferation; Chromatin; DNA Helicases; Glioma; Humans; Lymphoid Enhancer-Binding Factor 1; Receptor, Transforming Growth Factor-beta Type II; Transcription Factors; Transforming Growth Factor beta

Topics: Animals; Antibodies, Neutralizing; Biomarkers; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Endothelial Cells; Glioma; Humans; Liver Neoplasms; Mice; Mice, Nude; Neovascularization, Pathologic; Nogo Proteins; Receptors, Transforming Growth Factor beta; Smad Proteins; Transforming Growth Factor beta; Tumor Microenvironment

Under the influence of transforming growth factor-beta (TGFβ), glioma-associated microglia produce molecules that promote glioma growth and invasion. Olfactomedin-like 3 (

Topics: Animals; Brain Neoplasms; Cell Line, Tumor; Cell Movement; Chemotaxis; Gene Expression Regulation, Neoplastic; Glioma; Glycoproteins; Humans; Intercellular Signaling Peptides and Proteins; Mice, Knockout; Microglia; Phagocytosis; Transforming Growth Factor beta; Tumor Microenvironment

Glioma stem cells (GSCs) contribute to the malignant growth of glioma, but little is known about the interaction between GSCs and tumor microenvironment. Here, we found that intense infiltration of regulatory T cells (Tregs) facilitated the qualities of GSCs through TGF-β secretion that helped coordinately tumor growth. Mechanistic investigations indicated that TGF-β acted on cancer cells to induce the core cancer stem cell-related genes CD133, SOX2, NESTIN, MUSASHI1 and ALDH1A expression and spheres formation via NF-κB-IL6-STAT3 signaling pathway, resulting in the increased cancer stemness and tumorigenic potential. Furthermore, Tregs promoted glioma tumor growth, and this effect could be abrogated with blockade of IL6 receptor by tocilizumab which also demonstrated certain level of therapeutic efficacy in xenograft model. Additionally, expression levels of CD133, IL6 and TGF-β were found to serve as prognosis markers of glioma patients. Collectively, our findings reveal a new immune-associated mechanism underlying Tregs-induced GSCs. Moreover, efforts to target this network may be an effective strategy for treating glioma.

Topics: Animals; Antibodies, Monoclonal, Humanized; Biomarkers; Cell Self Renewal; Cytokines; Disease Models, Animal; Female; Glioma; Humans; Immunophenotyping; Interleukin-6; Mice; Neoplastic Stem Cells; NF-kappa B; Prognosis; Signal Transduction; STAT3 Transcription Factor; T-Lymphocytes, Regulatory; Transforming Growth Factor beta

In primary central nervous system tumours, epithelial-to-mesenchymal transition (EMT) gene expression is associated with increased malignancy. However, it has also been shown that EMT factors in gliomas are almost exclusively expressed by glioma vessel-associated pericytes (GA-Peris). In this study, we aimed to identify the mechanism of EMT in GA-Peris and its impact on angiogenic processes.. In glioma patients, vascular density and the expression of the pericytic markers platelet derived growth factor receptor (PDGFR)-β and smooth muscle actin (αSMA) were examined in relation to the expression of the EMT transcription factor SLUG and were correlated with survival of patients with glioblastoma (GBM). Functional mechanisms of SLUG regulation and the effects on primary human brain vascular pericytes (HBVP) were studied in vitro by measuring proliferation, cell motility and growth characteristics.. The number of PDGFR-β- and αSMA-positive pericytes did not change with increased malignancy nor showed an association with the survival of GBM patients. However, SLUG-expressing pericytes displayed considerable morphological changes in GBM-associated vessels, and TGF-β induced SLUG upregulation led to enhanced proliferation, motility and altered growth patterns in HBVP. Downregulation of SLUG or addition of a TGF-β antagonising antibody abolished these effects.. We provide evidence that in GA-Peris, elevated SLUG expression is mediated by TGF-β, a cytokine secreted by most glioma cells, indicating that the latter actively modulate neovascularisation not only by modulating endothelial cells, but also by influencing pericytes. This process might be responsible for the formation of an unstructured tumour vasculature as well as for the breakdown of the blood-brain barrier in GBM.

Topics: Brain Neoplasms; Cell Movement; Endothelial Cells; Gene Expression Regulation, Neoplastic; Glioblastoma; Glioma; Humans; Pericytes; Receptor, Platelet-Derived Growth Factor beta; Snail Family Transcription Factors; Transforming Growth Factor beta

MDM2 proto‑oncogene, E3 ubiquitin protein ligase (MDM2) is a well‑known oncogene and has been reported to be closely associated with epithelial‑to‑mesenchymal transition (EMT). The present study first demonstrated that the expression levels of MDM2 were markedly increased in TGF‑β‑induced EMT using quantitative PCR and western blotting. In addition, MDM2 was demonstrated to be associated with pathological grade in clinical glioma samples by immunohistochemical staining. Furthermore, overexpression of MDM2 promoted EMT in glioma, lung cancer and breast cancer cell lines using a scratch wound migration assay. Subsequently, the present study explored the mechanism by which MDM2 promoted EMT and revealed that MDM2 induced EMT by upregulating EMT‑related transcription factors via activation of the B‑Raf signaling pathway through tyrosine 3‑monooxygenase activation protein ε using RNA sequencing and western blotting. This mechanism depended on the p53 gene. Furthermore,

Topics: 14-3-3 Proteins; A549 Cells; Adult; Animals; Brain Neoplasms; Cell Line, Tumor; Epithelial-Mesenchymal Transition; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Glioma; HEK293 Cells; High-Throughput Nucleotide Sequencing; Humans; Male; MCF-7 Cells; Mice; Middle Aged; Neoplasm Grading; Neoplasm Transplantation; Proto-Oncogene Proteins B-raf; Proto-Oncogene Proteins c-mdm2; Sequence Analysis, RNA; Signal Transduction; Transforming Growth Factor beta; Up-Regulation; Young Adult

Topics: Animals; Cell Line, Tumor; Cell Movement; Cell Proliferation; F-Box Proteins; Gene Expression Regulation, Neoplastic; Glioma; Jumonji Domain-Containing Histone Demethylases; Mice; Mice, Nude; MicroRNAs; Neoplastic Stem Cells; Signal Transduction; Transforming Growth Factor beta; Up-Regulation

Glioma is the most aggressive primary brain tumor. We have previously provided evidence that IFITM3 promoted glioma cells migration. However, the mechanism of how IFITM3 regulates glioma cells invasion and whether IFITM3 participates in TGF-β-mediated glioma invasion are still unknown. In this paper, we proved that IFITM3 was notably up-regulated in glioma tissues. Knockdown of IFITM3 suppressed STAT3 phosphorylation in vitro, and a specific STAT3 inhibitor AG490 reversed IFITM3-induced invasion of glioma cells. Furthermore, IFITM3 expression was induced by TGF-β in glioma and IFITM3 knockdown abolished TGF-β-mediated glioma cells invasion. Collectively, the results indicate that IFITM3/STAT3 axis may promote TGF-β-induced glioma cells invasion. This study provided some suggestions for the clinical treatment of the brain tumor.

Topics: Adult; Brain Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; Female; Gene Expression Regulation, Neoplastic; Glioma; Humans; Male; Membrane Proteins; Middle Aged; Phosphorylation; RNA-Binding Proteins; STAT3 Transcription Factor; Transcriptional Activation; Transforming Growth Factor beta; Tyrphostins

BRCA1-associated protein (BRAP) was first identified by its ability to bind to the nuclear localization signalling motif of BRCA1 and other proteins. Subsequently, human BRAP has been found to exert multiple functions, many of which are related to cancer development. Up till now, however, the role of BRAP in glioma development has remained obscure. Here, we report a role for BRAP in mediating the proliferation and migration of glioma cells both in vitro and in vivo.. The expression of BRAP in 98 glioma patient samples was determined by immunohistochemistry, after which associations between BRAP expression and patient prognosis were assessed. A short hairpin RNA (shRNA) was used to knock down BRAP and an expression vector was used to exogenously overexpress BRAP in glioma cells. The effects of BRAP expression on tumour cell behaviour in vitro and in an in vivo xenograft mouse model were examined.. We found that in glioma patients BRAP expression was associated with a favourable prognosis. We also found that shRNA-mediated knockdown of BRAP facilitated the proliferation and migration of glioma cells and the self-renewal of glioma stem cells. In parallel, we found that BRAP knockdown increased tumour growth and invasion and decreased survival in an in vivo glioma xenograft mouse model. Mechanistically, we found that BRAP inhibited glioma cell proliferation and migration, as well as glioma stem cell self-renewal via the TGF-β/PI3K/AKT/mTOR signalling pathway.. Together, our findings identify BRAP as a mediator of glioma cell proliferation, migration and glioma stem cell self-renewal.

Topics: Animals; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Self Renewal; Female; Gene Expression Regulation, Neoplastic; Glioma; Humans; Kaplan-Meier Estimate; Male; Mice, Inbred BALB C; Mice, Nude; Middle Aged; Neoplastic Stem Cells; Phosphatidylinositol 3-Kinases; Proteins; Proto-Oncogene Proteins c-akt; RNA Interference; Signal Transduction; TOR Serine-Threonine Kinases; Transforming Growth Factor beta; Ubiquitin-Protein Ligases; Xenograft Model Antitumor Assays

Topics: Apoptosis; Cadherins; Cell Line, Tumor; Cell Movement; Cell Proliferation; Flavonoids; Glioma; Humans; Lamiaceae; Signal Transduction; Transforming Growth Factor beta

Diffuse intrinsic pontine glioma (DIPG) bears a dismal prognosis. A genetically engineered brainstem glioma model harboring the recurrent DIPG mutation, Activin A receptor type I (ACVR1)-G328V (mACVR1), was developed for testing an immune-stimulatory gene therapy.. We utilized the Sleeping Beauty transposase system to generate an endogenous mouse model of mACVR1 brainstem glioma. Histology was used to characterize and validate the model. We performed RNA-sequencing analysis on neurospheres harboring mACVR1. mACVR1 neurospheres were implanted into the pons of immune-competent mice to test the therapeutic efficacy and toxicity of immune-stimulatory gene therapy using adenoviruses expressing thymidine kinase (TK) and fms-like tyrosine kinase 3 ligand (Flt3L). mACVR1 neurospheres expressing the surrogate tumor antigen ovalbumin were generated to investigate whether TK/Flt3L treatment induces the recruitment of tumor antigen-specific T cells.. Histologic analysis of mACVR1 tumors indicates that they are localized in the brainstem and have increased downstream signaling of bone morphogenetic pathway as demonstrated by increased phospho-smad1/5 and Id1 levels. Transcriptome analysis of mACVR1 neurosphere identified an increase in the TGFβ signaling pathway and the regulation of cell differentiation. Adenoviral delivery of TK/Flt3L in mice bearing brainstem gliomas resulted in antitumor immunity, recruitment of antitumor-specific T cells, and increased median survival (MS).. This study provides insights into the phenotype and function of the tumor immune microenvironment in a mouse model of brainstem glioma harboring mACVR1. Immune-stimulatory gene therapy targeting the hosts' antitumor immune response inhibits tumor progression and increases MS of mice bearing mACVR1 tumors.

Topics: Activin Receptors, Type I; Animals; Brain Stem Neoplasms; Disease Models, Animal; Female; Genetic Therapy; Genetic Vectors; Glioma; Humans; Immunotherapy; Male; Membrane Proteins; Mice; Mice, Transgenic; Mutation; Pons; Primary Cell Culture; RNA-Seq; Signal Transduction; Spheroids, Cellular; Thymidine Kinase; Transforming Growth Factor beta; Tumor Cells, Cultured; Tumor Microenvironment

Glioma stem cells (GSC) play a major role in drug resistance and tumor recurrence. Using a genetic screen with a set of shRNAs that can target chromatin regulators in a GSC model, we have HDAC3 as a major negative regulator of GSC differentiation. Inhibition of HDAC3 using a pharmacological inhibitor or a siRNA led to the induction of GSC differentiation into astrocytes. Consequently, HDAC3-inhibition also caused a strong reduction of tumor-promoting and self-renewal capabilities of GSCs. These phenotypes were highly associated with an increased acetylation of SMAD7, which protected its ubiquitination. SMAD7 inhibits a TGF-β signaling axis that is required for maintaining stemness. These results demonstrate that HDAC3 appears to be a proper target in anti-glioma therapy.

Topics: Acrylamides; Animals; Brain Neoplasms; Cell Differentiation; Cell Line, Tumor; Cell Survival; Cell Transformation, Neoplastic; Glioma; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplastic Stem Cells; Phenylenediamines; RNA, Small Interfering; Signal Transduction; Smad7 Protein; Transforming Growth Factor beta; Xenograft Model Antitumor Assays

Gliomas are brain and spinal cord malignancies characterized by high malignancy, high recurrence and poor prognosis, the underlying mechanisms of which remain largely elusive. Here, we found that the Sry-related high mobility group box (Sox) family transcription factor, Sox9, was upregulated and correlated with poor prognosis of clinical gliomas. Sox9 promotes migration and invasion of glioma cells and

Topics: Animals; Brain Neoplasms; Cell Line, Tumor; Disease Models, Animal; Disease Progression; Disease Susceptibility; Gene Expression; Gene Knockdown Techniques; Glioma; Humans; Immunohistochemistry; Mice; Protein Binding; Protein Stability; Signal Transduction; SOX9 Transcription Factor; Transforming Growth Factor beta; Tumor Cells, Cultured

Cannabinoid receptors have been detected in human gliomas and cannabinoids have been proposed as novel drug candidates in the treatment of brain tumors.. To test the in vitro antitumor activity of COR167, a novel cannabinoid CB2-selective agonist displaying a high binding affinity for human CB2 receptors, on tumor cells isolated from human glioblastoma multiforme and anaplastic astrocytoma.. Glioma cell cultures were established from two glioblastoma multiforme and two anaplastic astrocytomas. Proliferation was measured in the presence or absence of COR167 with a bromodeoxyuridine (BrdU) cell proliferation ELISA assay. CB2 receptor expression was detected by western blotting. Apoptosis was assessed with phycoerythrin (PE) annexin V flow cytometry kit. TGF-beta 1 and 2 levels were analyzed in culture supernatants with commercial ELISAs.. COR167 was found to significantly reduce the proliferation of both glioblastoma and anaplastic astrocytoma in a dose-dependent manner at lower doses than other known, less specific CB2 agonists. This activity is independent of apoptosis and is associated with a significant reduction of TGF-beta 1 and 2 levels in supernatants of glioma cell cultures.. These findings add to the role of cannabinoid CB2 receptor as a possible pharmacological target to counteract glial tumor growth and encourage further work to explore any other pharmacological effect of this novel CB2 agonist useful in the treatment of human gliomas.

Topics: Adamantane; Apoptosis; Cannabinoid Receptor Agonists; Cannabinoids; Cell Proliferation; Dose-Response Relationship, Drug; Glioma; Humans; Quinolones; Receptor, Cannabinoid, CB2; Transforming Growth Factor beta; Tumor Cells, Cultured

Long non-coding RNAs (lncRNAs) are critical regulators in cancer. However, the involvement of lncRNAs in TGF-β-regulated tumorigenicity is still unclear. Here, we identify TGF-β-activated lncRNA LINC00115 as a critical regulator of glioma stem-like cell (GSC) self-renewal and tumorigenicity. LINC00115 is upregulated by TGF-β, acts as a miRNA sponge, and upregulates ZEB1 by competitively binding of miR-200s, thereby enhancing ZEB1 signaling and GSC self-renewal. LINC00115 also promotes ZNF596 transcription by preventing binding of miR-200s to the 5'-UTR of ZNF596, resulting in augmented ZNF596/EZH2/STAT3 signaling and GBM tumor growth. Inhibition of EZH2 by genetic approaches or a small molecular inhibitor markedly suppresses LINC00115-driven GSC self-renewal and tumorigenicity. Moreover, LINC00115 is highly expressed in GBM, and LINC00115 expression or correlated co-expression with ZEB1 or ZNF596 is prognostic for clinical GBM survival. Our work defines a critical role of LINC00115 in GSC self-renewal and tumorigenicity, and suggests LINC00115 as a potential target for GBM treatment.

Topics: Animals; Brain Neoplasms; Carcinogenesis; Cell Line, Tumor; Cells, Cultured; Enhancer of Zeste Homolog 2 Protein; Female; Gene Expression Regulation, Neoplastic; Glioma; Humans; Mice; MicroRNAs; Neoplastic Stem Cells; RNA, Long Noncoding; Transforming Growth Factor beta; Zinc Finger E-box-Binding Homeobox 1

Despite advances in therapy, glioblastoma remains an incurable disease with a dismal prognosis. Recent studies have implicated cancer stem cells within glioblastoma (glioma stem cells, GSCs) as mediators of therapeutic resistance and tumor progression. In this study, we investigated the role of the transforming growth factor-β (TGF-β) superfamily, which has been found to play an integral role in the maintenance of stem cell homeostasis within multiple stem cell systems, as a mediator of stem-like cells in glioblastoma. We find that BMP and TGF-β signaling define divergent molecular and functional identities in glioblastoma, and mark relatively quiescent and proliferative GSCs, respectively. Treatment of GSCs with BMP inhibits cell proliferation, but does not abrogate their stem-ness, as measured by self-renewal and tumorigencity. Further, BMP pathway activation confers relative resistance to radiation and temozolomide chemotherapy. Our findings define a quiescent cancer stem cell population in glioblastoma that may be a cellular reservoir for tumor recurrence following cytotoxic therapy.

Topics: Animals; Antineoplastic Agents; Bone Morphogenetic Protein 4; Bone Morphogenetic Proteins; Brain Neoplasms; Cell Division; Cell Line, Tumor; Cell Proliferation; Disease Progression; Drug Resistance, Neoplasm; Glioblastoma; Glioma; Homeostasis; Humans; Mice; Mice, Inbred NOD; Neoplasm Recurrence, Local; Neoplasm Transplantation; Neoplastic Stem Cells; Phenotype; RNA, Small Interfering; Sequence Analysis, RNA; Signal Transduction; Temozolomide; Transforming Growth Factor beta; Transforming Growth Factor beta1

Glioma is the most common primary malignant tumor in the central nervous system. Because of the resistance of glioma to chemoradiotherapy and its aggressive growth, the survival rate of patients with glioma has not improved. This study aimed to disclose the effect of retinol dehydrogenase 10 (RDH10) on the migration and invasion of glioma cells, and to explore the potential mechanism.. Reverse transcription-polymerase chain reaction (RT-PCR) was used to determine the expression levels of RDH10 in healthy glial cells and glioma cells. Human glioma cell strains, U87 and U251, were infected with negative control or RDH10-interfering lentiviruses. RT-PCR and Western blotting were performed to determine the knockdown efficiency. Scratch and transwell assays were used to assess cell migration and invasion after RDH10 knockdown. Finally, changes in transforming growth factor-β (TGF-β)/SMAD signaling pathway-related expression were examined by Western blotting. Differences between groups were analyzed by one-way analysis of variance.. RDH10 was highly expressed in glioma cells. Compared with the control group, RDH10 knockdown significantly reduced RDH10 messenger RNA and protein expression levels in U87 and U251 glioma cells (U87: 1.00 ± 0.08 vs. 0.22 ± 0.02, t = 16.55, P < 0.001; U251: 1.00 ± 0.17 vs. 0.39 ± 0.01, t = 6.30, P < 0.001). The scratch assay indicated that compared with the control group, RDH10 knockdown significantly inhibited the migration of glioma cells (U87: 1.00% ± 0.04% vs. 2.00% ± 0.25%, t = 6.08, P < 0.01; U251: 1.00% ± 0.11% vs. 2.48% ± 0.31%, t = 5.79, P < 0.01). Furthermore, RDH10 knockdown significantly inhibited the invasive capacity of glioma cells (U87: 97.30 ± 7.01 vs. 13.70 ± 0.58, t = 20.36, P < 0.001; U251: 96.20 ± 7.10 vs. 18.30 ± 2.08, t = 18.51, P < 0.001). Finally, Western blotting demonstrated that compared with the control group, downregulation of RDH10 significantly inhibited TGF-β expression, phosphorylated SMAD2, and phosphorylated SMAD3 (TGF-β: 1.00 ± 0.10 vs. 0.53 ± 0.06, t = 7.05, P < 0.01; phosphorylated SMAD2: 1.00 ± 0.20 vs. 0.42 ± 0.17, t = 4.01, P < 0.01; phosphorylated SMAD3: 1.00 ± 0.18 vs. 0.41 ± 0.12, t = 4.12, P < 0.01).. RDH10 knockdown might inhibit metastasis of glioma cells via the TGF-β/SMAD signaling pathway.

Topics: Alcohol Oxidoreductases; Brain Neoplasms; Cell Line, Tumor; Cell Movement; Glioma; Humans; Neoplasm Invasiveness; RNA Interference; Signal Transduction; Smad Proteins; Transforming Growth Factor beta

Glioma invasion is a main cause of a poor prognosis and relapse in patients suffering from the disease. However, the molecular mechanisms responsible for glioma cell invasion remain poorly understood. In this study, the characteristics of exosomes were identified using electron microscope (TEM), and western blot analysis. The potential mechanism of long non‑coding RNA (lncRNA) activated by TGF‑β (lncRNA‑ATB) was demonstrated using luciferase reporter assays and RNA immunoprecipitation. We found that glioma cell‑derived exosomes promoted the activation of astrocytes and had the ability to shuttle long non‑coding RNA (lncRNA) activated by TGF‑β (lncRNA‑ATB) to astrocytes. More importantly, lncRNA‑ATB activated astrocytes through the suppression of microRNA (miRNA or miR)‑204‑3p in an Argonaute 2 (Ago2)‑dependent manner. Furthermore, astrocytes activated by lncRNA‑ATB in turn promoted the migration and invasion of glioma cells. Taken together, the findings of this study suggest that lncRNA‑ATB may play an important role in modulating glioma microenvironment through exosomes. Thus, a better understanding of this process may provide implications for the prevention of highly invasive glioma.

Topics: Astrocytes; Cell Line, Tumor; Cell Movement; Cell Proliferation; Exosomes; Gene Expression Regulation, Neoplastic; Glioma; Humans; MicroRNAs; Microscopy, Electron; Neoplasm Invasiveness; Neoplasm Recurrence, Local; RNA, Long Noncoding; Transforming Growth Factor beta; Tumor Microenvironment

Glioma constitutes the most aggressive primary intracranial malignancy in adults. We previously showed that long noncoding RNA activated by TGF-β (lncRNA-ATB) promoted the glioma cells invasion. However, whether lncRNA-ATB is involved in TGF-β-mediated invasion of glioma cells remains unknown. In this study, quantitative real-time polymerase chain reaction and western blot analysis were used for detecting the mRNA and protein expression of related genes, respectively. Transwell assay was performed to assess the impact of lncRNA-ATB on TGF-β-induced glioma cells migration and invasion. Immunofluorescence staining was utilized to characterize related protein distribution. Results showed that TGF-β upregulated lncRNA-ATB expression in glioma LN-18 and U251 cells. Overexpression of lncRNA-ATB activated nuclear factor-κB (NF-κB) pathway and promoted P65 translocation into the nucleus, thus facilitated glioma cells invasion stimulated by TGF-β. Similarly, lncRNA-ATB markedly enhanced TGF-β-mediated invasion of glioma cells through activation P38 mitogen-activated protein kinase (P38/MAPK) pathway. Moreover, both the NF-κB selected inhibitor pyrrolidinedithiocarbamate ammonium and P38/MAPK specific inhibitor SB203580 partly reversed lncRNA-ATB induced glioma cells invasion mediated by TGF-β. Collectively, this study revealed that lncRNA-ATB promotes TGF-β-induced glioma cell invasion through NF-κB and P38/MAPK pathway and established a detailed framework for understanding the way how lncRNA-ATB performs its function in TGF-β-mediated glioma invasion.

Topics: Brain Neoplasms; Cell Line, Tumor; Gene Expression Regulation, Neoplastic; Glioma; Humans; MAP Kinase Signaling System; Neoplasm Invasiveness; NF-kappa B; p38 Mitogen-Activated Protein Kinases; RNA, Long Noncoding; Transforming Growth Factor beta

Tumor-associated macrophages (TAMs) are pivotal effector cells in angiogenesis. Here, we tested whether CYP4X1 inhibition in TAMs by flavonoid CH625 prolongs survival and normalizes glioma vasculature. CH625 was selected against the CYP4X1 3D model by virtual screening and showed inhibitory activity on the CYP4X1 catalytic production of 14,15-EET-EA in the M2-polarized human peripheral blood mononuclear cells (IC

Topics: Cell Line, Tumor; Cell Movement; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System; Down-Regulation; ErbB Receptors; Flavonoids; Glioma; Humans; Macrophages; Neovascularization, Pathologic; Receptor, Cannabinoid, CB2; STAT3 Transcription Factor; T-Lymphocytes; Transforming Growth Factor beta

BACKGROUND Malignant glioma is intractable primary brain carcinoma that has a poor survival rate. Natural diterpenoid isoferritin A (IsoA) presents antitumor effects by regulating signal pathways in tumor cells. In the present study we investigated the inhibitory effects of IsoA on glioma cells. MATERIAL AND METHODS The potential molecular mechanism of IsoA-mediated glioma cell growth and metastasis were investigated using Western blot, gene knockdown, immunofluorescence, and immunohistochemistry. RESULTS Results showed that IsoA significantly inhibits growth and metastasis of glioma cells in multiple preclinical settings. In vitro assay showed that IsoA (4 mg/ml) treatment significantly induced apoptosis of glioma cells. Mechanism analysis demonstrated that IsoA (4 mg/ml) treatment decreased TGFβ and regulated EMT markers expression in glioma cells. Reduced expression of TGFβ in glioma cells was closely correlated with inhibitory effects of IsoA on growth and metastasis of glioma cells. TGFβ overexpression promoted glioma cell growth and invasion. Results also showed that IsoA treatment significantly decreased Fibronectin and Vimentin and increased E-cadherin, while TGFβ overexpression abolished the regulation mediated by IsoA in glioma cells. In vivo assay showed that IsoA treatment inhibited tumor growth in a glioma-bearing mouse model. CONCLUSIONS Results indicate that IsoA could be regarded as a potential anti-cancer agent by regulating TGFβ-induced EMT signal pathway.

Topics: Animals; Brain Neoplasms; Cell Cycle; Cell Line, Tumor; Cell Movement; Cell Proliferation; Diterpenes; Epithelial-Mesenchymal Transition; Ferritins; Gene Expression Regulation, Neoplastic; Glioma; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Invasiveness; Neoplasm Metastasis; Signal Transduction; Transforming Growth Factor beta; Xenograft Model Antitumor Assays

Glioblastoma multiforme (GBM) is the most common primary malignant tumor affecting the human brain. Despite improvements in therapeutic technologies, patients with GBM have a poor clinical result and the molecular mechanisms responsible for the development of GBM have not yet been fully elucidated. 3-phosphoinositide dependent protein kinase 1 (PDK1) is upregulated in various tumors and promotes tumor invasion. In glioma, transforming growth factor-β (TGF‑β) promotes cell invasion; however, whether TGF‑β directly regulates PDK1 protein and promotes proliferation and invasion is not yet clear. In this study, PDK1 levels were measured in glioma tissues using tissue microarray (TMA) by immunohistochemistry (IHC) and RT‑qPCR. Kaplan-Meier analyses were used to calculate the survival rate of patients with glioma. In vitro, U251 and U87 glioma cell lines were used for functional analyses. Cell proliferation and invasion were analyzed using siRNA transfection, MTT assay, RT‑qPCR, western blot analysis, flow cytometry and invasion assay. In vivo, U251 glioma cell xenografts were established. The results revealed that PDK1 protein was significantly upregulated in glioma tissues compared with non-tumorous tissues. Furthermore, the higher PDK1 levels were associated with a large tumor size (>5.0 cm), a higher WHO grade and a shorter survival of patients with GBM. Univariate and multivariate analyses indicated that PDK1 was an independent prognostic factor. In vivo, PDK1 promoted glioma tumor xenograft growth. In vitro, functional analyses confirmed that TGF‑β upregulated PDK1 protein expression and PDK1 promoted cell migration and invasion, and functioned as an oncogene in GBM, by upregulating c‑Jun protein and inducing epithelial-mesenchymal transition (EMT). c‑Jun protein were overexpressed in glioma tissues and positively correlated with PDK1 levels. Moreover, our findings were further validated by the online Oncomine database. On the whole, the findings of this study indicate that in GBM, PDK1 functions as an oncogene, promoting proliferation and invasion.

Topics: 3-Phosphoinositide-Dependent Protein Kinases; Adult; Aged; Animals; Brain Neoplasms; Cell Proliferation; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Glioblastoma; Glioma; Humans; Male; Mice, Nude; Middle Aged; Oncogenes; Proto-Oncogene Proteins c-jun; Survival Rate; Transforming Growth Factor beta; Xenograft Model Antitumor Assays

To understand the molecular mechanism underlying the causal relationship between aberrant upregulation of transforming growth factor beta (TGF-β) and radio-resistance in glioma. The mouse glioma cell GL261 was irradiated, and relative expression of TGF-β/Smad signaling genes was determined by real-time PCR and western blotting. The DNA repair response on exogenous TGF-β or LY2109761 was evaluated by quantification of diverse genes by real-time PCR and western blotting. Xenograft mice were employed for in vivo investigation to assess the response to irradiation and LY2109761 either alone or in combination. The expression of DNA repair genes was further determined in the xenograft tumor. The TGF-β/Smad signaling pathway was activated by radiation in the GL261 cell line. The exogenous complement of TGF-β significantly stimulated DNA repair response. Administration of LY2109761 suppressed DNA repair genes. Simultaneous treatment with LY2109761 abrogated the upregulation of DNA repair genes in GL261. In the xenograft tumor model, LY2109761 synergistically improved the therapeutic effect of radiation via improvement of sensitivity. Our data suggested that LY2109761 treatment re-sensitized glioma to radiation via antagonizing TGF-β/Smad-induced DNA repair.

Topics: Acid Anhydride Hydrolases; Animals; ATP-Binding Cassette Transporters; Brain Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; DNA Damage; DNA Glycosylases; DNA Modification Methylases; DNA Repair; DNA Repair Enzymes; DNA-Binding Proteins; Gamma Rays; Gene Expression Regulation, Neoplastic; Glioma; Humans; Mice; MutL Protein Homolog 1; MutS Homolog 2 Protein; Neuroglia; Pyrazoles; Pyrroles; RNA, Small Interfering; Signal Transduction; Smad Proteins; Survival Analysis; Transforming Growth Factor beta; Tumor Suppressor Proteins; Xenograft Model Antitumor Assays

Tumors are capable of coopting hematopoietic cells to create a suitable microenvironment to support malignant growth. Here, we have demonstrated that upregulation of kinase insert domain receptor (KDR), also known as VEGFR2, in a myeloid cell sublineage is necessary for malignant progression of gliomas in transgenic murine models and is associated with high-grade tumors in patients. KDR expression increased in myeloid cells as myeloid-derived suppressor cells (MDSCs) accumulated, which was associated with the transformation and progression of low-grade fibrillary astrocytoma to high-grade anaplastic gliomas. KDR deficiency in murine BM-derived cells (BMDCs) suppressed the differentiation of myeloid lineages and reduced granulocytic/monocytic populations. The depletion of myeloid-derived KDR compromised its proangiogenic function, which inhibited the angiogenic switch necessary for malignant progression of low-grade to high-grade tumors. We also identified inhibitor of DNA binding protein 2 (ID2) as a key upstream regulator of KDR activation during myeloid differentiation. Deficiency of ID2 in BMDCs led to downregulation of KDR, suppression of proangiogenic myeloid cells, and prevention of low-grade to high-grade transition. Tumor-secreted TGF-β and granulocyte-macrophage CSF (GM-CSF) enhanced the KDR/ID2 signaling axis in BMDCs. Our results suggest that modulation of KDR/ID2 signaling may restrict tumor-associated myeloid cells and could potentially be a therapeutic strategy for preventing transformation of premalignant gliomas.

Topics: Animals; Bone Marrow Cells; Cell Line, Tumor; Glioma; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Inhibitor of Differentiation Protein 2; Mice; Mice, Transgenic; Myeloid Cells; Neoplasm Proteins; Neovascularization, Pathologic; Signal Transduction; Transforming Growth Factor beta; Vascular Endothelial Growth Factor Receptor-2

The paper describes a mathematical model with synergistic interaction between the malignant glioma cells and the immune system, namely, macrophages, activated Cytotoxic T-Lymphocytes (CTLs), the immunosuppressive cytokine Transforming Growth Factor - β (TGF-β) and the immuno-stimulatory cytokine Interferon - γ (IFN-γ), using a system of coupled non-linear ordinary differential equations (ODEs). We have introduced a new immunotherapeutic drug T11 Target structure (T11TS) into the model, which boosts the macrophages and CTLs to kill the glioma cells. In our analysis, we have established a criteria for the threshold level of immunotherapeutic drug T11TS for which the system will be gliomas free or tumor free. The analytical findings are supported by numerical simulations using parameters estimated from experimental data.

Topics: Brain Neoplasms; Computer Simulation; Disease Progression; Glioma; Humans; Interferon-gamma; Macrophages; Models, Immunological; Reproducibility of Results; T-Lymphocytes, Cytotoxic; Transforming Growth Factor beta

Glioblastomas rapidly become refractory to anti-VEGF therapies. We previously showed that cytochrome P450 (CYP) 4A-derived 20-hydroxyeicosatetraenoic acid (20-HETE) promotes angiogenesis. Here, we tested whether a novel flavonoid (FLA-16) prolongs survival and normalizes tumor vasculature in glioma through CYP4A inhibition. FLA-16 improved survival, reduced tumor burden, and normalized vasculature, accompanied with the decreased secretion of 20-HETE, VEGF and TGF-β in tumor-associated macrophages (TAMs) and endothelial progenitor cells (EPCs) in C6 and U87 gliomas. FLA-16 attenuated vascular abnormalization induced by co-implantation of GL261 glioma cells with CYP4A10

Topics: Angiogenesis Inhibitors; Animals; Brain Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; Chalcones; Culture Media, Conditioned; Cytochrome P-450 CYP4A; Cytochrome P-450 Enzyme Inhibitors; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; Endothelial Progenitor Cells; Flavonoids; Glioma; Humans; Hydroxyeicosatetraenoic Acids; Macrophages; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Nude; Neovascularization, Pathologic; Paracrine Communication; Pericytes; Rats, Wistar; Time Factors; Transforming Growth Factor beta; Tumor Burden; Tumor Microenvironment; Vascular Endothelial Growth Factor A; Xenograft Model Antitumor Assays

Glioblastoma (GBM) is the most common and aggressive tumor of the central nervous system. Unfortunately, patients affected by this disease have a very poor prognosis, due to high level of invasiveness and resistance to standard therapies. Although the molecular profile of GBM has been extensively investigated, the events responsible for its pathogenesis and progression remain largely unknown. Histone Deacetylases (HDAC) dependent epigenetic modifications and transforming growth factor (TGF)-β/Smad pathway seem to play an important role in GBM tumorigenesis, resistance to common therapies and poor clinical outcome.  The aim of this study was to evaluate the involvement and the possible interaction between these two molecular cascades in the pathogenesis and prognosis of GBM. Immunohistochemistry (IHC) was performed on microdissected GBM samples, collected from 14 patients (6 men and 8 women) ranging in age from 43 to 74 years. The patients were previously divided, on the basis of their overall survival (OS), into two groups: short and long OS. Patients with poor prognosis showed hyperexpression of HDAC4 and HDAC6, an activation of the TGF-β/Smad pathway, with high levels of IL-13, Smad2, PDGF and MMP3 expression, compared to the long survivors. The short OS group exhibits a decrease in Smad 7 expression and also low levels of p21 immunostaining, which represents a common target of the two pathways. The IHC data was confirmed by quantitative analysis and Immunoblotting. Our preliminary results suggest that both HDAC4 and HDAC6 together with the TGF-β/Smad pathway may be involved in progression of GBM and this cross talking could be a useful prognostic marker in this deadly disease.

Topics: Adult; Aged; Chemoradiotherapy; Female; Glioma; Histone Deacetylases; Humans; Male; Middle Aged; Prognosis; Signal Transduction; Smad2 Protein; Staining and Labeling; Transcriptome; Transforming Growth Factor beta

BACKGROUND Glioblastoma multiforme (GBM) evades immune surveillance by inducing immunosuppression via receptor-ligand interactions between immune checkpoint molecules. T cell immunoglobulin and mucin domain 3 (Tim-3) is a key checkpoint receptor responsible for exhaustion and dysfunction of T cells and plays a critical role in immunosuppression. Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) has been recently identified as a heterophilic ligand for Tim-3. MATERIAL AND METHODS We established an intracranial GBM model using C57BL/6 mice and GL261 cells, and treated the mice with single or combined monoclonal antibodies (mAbs) against Tim-3/CEACAM1. The CD4+, CD8+, and regulatory T cells in brain-infiltrating lymphocytes were analyzed using flow cytometry, and the effector function of T cells was assessed using ELISA. We performed a rechallenge by subcutaneous injection of GL261 cells in the "cured" (>90 days post-orthotopic tumor implantation) and naïve mice. RESULTS The mean survival time in the control, anti-Tim-3, anti-CEACAM1, and combined treatment groups was 29.8, 43.4, 42.3, and 86.0 days, respectively, with 80% of the mice in the combined group becoming long-term survivors showing immune memory against glioma cells. Infiltrating CD4+ and CD8+ T cells increased and immunosuppressive Tregs decreased with the combined therapy, which resulted in a markedly elevated ratio of CD4+ and CD8+ cells to Tregs. Additionally, plasma IFN-γ and TGF-β levels were upregulated and downregulated, respectively. CONCLUSIONS Our data indicate that combined blockade of Tim-3 and CEACAM1 generates robust therapeutic efficacy in mice with intracranial tumors, and provides a promising option for GBM immunotherapy.

Topics: Animals; Antibodies, Monoclonal; Antilymphocyte Serum; Brain Neoplasms; Carcinoembryonic Antigen; CD8-Positive T-Lymphocytes; Disease Models, Animal; Glioblastoma; Glioma; Hepatitis A Virus Cellular Receptor 2; Immune Tolerance; Immunotherapy; Mice; Mice, Inbred C57BL; Receptors, Virus; Transforming Growth Factor beta; Treatment Outcome

The epidermal growth factor receptor (EGFR) frequently undergoes high-level genomic amplification and variant III (vIII) deletion in adult glioblastoma. MicroRNAs (miRNAs) are recognized to participate in gene expression regulation. We found that miR-524-3p and miR-524-5p were suppressed in the classical molecular subtype of glioblastoma (GBM) from Chinese Glioma Genome Atlas (CGGA) data, and the suppression was associated with EGFR overexpression and EGFRvIII mutation. These two miRNAs improved overall survival time of patients with glioma, and their overexpression could restrain glioma cell migration, proliferation, and cell cycle, and control tumor formation in vivo. Interestingly, both of the miRNAs had a synergistic inhibitory effect on glioma cells. Furthermore, we confirmed that EGFR amplification/EGFRvIII mutation can repress the expression of Pri-miR-524 by histone modification. MiR-524-3p and miR-524-5p inhibited TGF/β, Notch and the Hippo pathway by targeting Smad2, Hes1 and Tead1, respectively; these pathways repressed their common downstream transcription factor, C-myc. More interestingly, C-myc bound to the promoter region of EGFR/EGFRvIII and activated its expression. These findings indicate that miR-524 mediates the EGFR/EGFRvIII stimulating effect. It may serve as a potential therapeutic agent and classical-specific biomarker for the development of glioma.

Topics: Adult; Animals; Epigenesis, Genetic; ErbB Receptors; Female; Gene Amplification; Gene Expression Regulation, Neoplastic; Gene Silencing; Glioma; Hippo Signaling Pathway; Humans; Male; Mice; Mice, Nude; MicroRNAs; Mutation; Neoplasm Staging; Prognosis; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-myc; Receptors, Notch; Survival Rate; Transforming Growth Factor beta; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

Human gliomas are related to high rates of morbidity and mortality. TGF-β promotes the growth of glioma cells, and correlate with the degree of malignancy of human gliomas. However, the molecular mechanisms involved in the malignant function of TGF-β are not fully elucidated. Here, we showed that TGF-β induced the downregulation of MST1 expression in U87 and U251 glioma cells. Treatment of glioma cells with the DNA methylation inhibitor 5-aza-2'-deoxycytidine (5-AzadC) prevented the loss of MST1 expression. Addition of 5-AzadC also reduced the TGF-β-stimulated proliferation, migration and invasiveness of glioma cells. Furthermore, Knockdown of DNMT1 upregulated MST1 expression in gliomas cells. In addition, the inhibition of DNMT1 blocked TGF-β-induced proliferation, migration and invasiveness in glioma cells. These results suggest that TGF-β promotes glioma malignancy through DNMT1-mediated loss of MST1 expression.

Topics: Antimetabolites, Antineoplastic; Azacitidine; Brain Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; Decitabine; DNA (Cytosine-5-)-Methyltransferase 1; Down-Regulation; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Glioma; Hepatocyte Growth Factor; Humans; Neoplasm Invasiveness; Proto-Oncogene Proteins; Transforming Growth Factor beta; Up-Regulation

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

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

This study aimed to deliver gellan sulfate core platinum coil with tenascin-C (GSCC-TNC) into rabbit side-wall aneurysms endovascularly and to evaluate the organization effects in a simulated clinical setting.. Elastase-induced rabbit side-wall aneurysms were randomly coiled via a transfemoral route like clinical settings with platinum coils (PCs), gellan sulfate core platinum coils (GSCCs), or GSCC-TNCs (n = 5, respectively). Aneurysm-occlusion status was evaluated angiographically and histologically at 2 weeks post coiling. As each rabbit coiled aneurysm provided only 2-3 tissue slices due to technical limitations and prevented immunohistochemical evaluations, a PC, GSCC, or GSCC-TNC was randomly implanted in a rat blind-ended model (n = 3, respectively) and the organization effects were immunohistochemically evaluated for expressions of tenascin-C (TNC), transforming growth factor-beta (TGF-β), and matrix metalloproteinase-9 (MMP-9) 2 weeks later.. Coil handling was similar among the 3 kinds of coils. GSCCs showed a significantly higher ratio of organized area to the aneurysmal cavity than PCs, but GSCC-TNCs had the greatest organization-promoting effects on aneurysms (the ratio of organized area/aneurysmal luminal area: PC, 17.9 ± 7.1%; GSCC, 54.2 ± 18.3%; GSCC-TNC, 82.5 ± 5.8%). GSCC-TNCs had intense immunoreactivities for TNC, TGF-β, and MMP-9 in the organized thrombosis and tunica media. GSCCs also showed intense immunoreactivities for TNC, TGF-β, and MMP-9, although the extent was less than GSCC-TNCs. The immunoreactivities were hardly found in unorganized thrombus and the tunica media of aneurysm wall in the PC group.. This study first showed that GSCC-TNCs promote intra-aneurysmal clot organization in simulated clinical settings using rabbits possibly through the TGF-β and MMP-9 upregulation.

Topics: Angiography; Animals; Cell Line, Tumor; Disease Models, Animal; Embolization, Therapeutic; Female; Glioma; Intracranial Aneurysm; Male; Matrix Metalloproteinase 9; Platinum; Polysaccharides; Rabbits; Rats; Rats, Sprague-Dawley; Sulfuric Acid Esters; Tenascin; Transforming Growth Factor beta

Periostin (POSTN) interacts with multiple integrins to coordinate a variety of cellular processes, including epithelial-to-mesenchymal transition (EMT) and cell migration. In our previous study, anti-VEGF-A therapy was associated with resistance and EMT. This study sought to determine the role of POSTN in the resistance of glioma stem cells (GSC) to antiangiogenic therapy. In mouse xenograft models of human glioma, POSTN expression was associated with acquired resistance to anti-VEGF-A therapy and had a synergistic effect with bevacizumab in prolonging survival and decreasing tumor volume. Resistance to anti-VEGF-A therapy regulated by POSTN was associated with increased expression of TGFβ1 and hypoxia-inducible factor-1α (HIF1α) in GSCs. At the molecular level, POSTN regulated invasion and expression of EMT (caveolin-1) and angiogenesis-related genes (HIF1α and VEGF-A) through activation of STAT3. Moreover, recombinant POSTN increased GSC invasion. Collectively, our findings suggest that POSTN plays an important role in glioma invasion and resistance to antiangiogenic therapy. Mol Cancer Ther; 15(9); 2187-97. ©2016 AACR.

Topics: Angiogenesis Inhibitors; Animals; Antineoplastic Agents; Brain Neoplasms; Cell Adhesion Molecules; Cell Line, Tumor; Disease Models, Animal; Drug Resistance, Neoplasm; Female; Gene Expression; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Glioma; Humans; Immunohistochemistry; Macrophages; Mice; Signal Transduction; Transforming Growth Factor beta; Xenograft Model Antitumor Assays

Transforming growth factor-β (TGF-β) proteins are important cytokines in the occurrence and development of tumors. However, its neural functions in glioma are still not understood. In the present study, we evaluated the effects of TGF-β1 on glioma cell line U87. miR-205 and miR-195 were involved in TGF-β1 signaling pathway. Quantitative real-time PCR was used to detect miR-205 and miR-195 levels in human glioma tissue samples and U87 cells treated with different concentrations of TGF-β1. Enzyme-linked immunosorbent assay (ELISA) was performed to determine TGF-β1 in the glioma patients peripheral blood. In vitro, U87 cells were transfected with mimics or inhibitors of miR-205 and miR-195. SMAD proteins were assayed by western blotting. Luciferase assay and co-immunoprecipitation (Co-IP)were used to determine the relationships between miR-205 and SMAD2, miR-195 and SMAD7. Effects of miR-205 and miR-195 on glioma cell proliferation and invasion using colony forming and cell migration assays. It was shown that miR-205 was decreased in glioma tissue, but miR-195 and TGF-β1 was increased. In addition, TGF-β1 concentration was negatively correlated with miR-205 mRNA level, but positively correlated with miR-195 mRNA. In addition, miR-205 was downregulated and miR-195 was upregulated by TGF-β1 in a dose-dependent manner. miR-205 and miR-195 targeted and inhibited SMAD2 and SMAD7 expression, respectively, in U87. High expression of miR-205 but not miR-195 reduced SMAD2 and SMAD4 heteromer formation. In addition, it was also shown that miR-205 overexpression inhibited U87 proliferation and invasion efficiently. All the results suggested that miR-205 and miR-195 participated in the TGF-β1 signaling pathway and showed opposite effects in glioma. These findings contribute to the understanding of TGF-β1 function in glioma.

Topics: Blotting, Western; Brain Neoplasms; Cell Line, Tumor; Enzyme-Linked Immunosorbent Assay; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Glioma; Humans; Immunoprecipitation; MicroRNAs; Real-Time Polymerase Chain Reaction; Signal Transduction; Smad2 Protein; Smad7 Protein; Transforming Growth Factor beta

Tumor-associated macrophages (TAMs) are enriched in gliomas and help create a tumor-immunosuppressive microenvironment. A distinct M2-skewed type of macrophages makes up the majority of glioma TAMs, and these cells exhibit pro-tumor functions. Gliomas contain large hypoxic areas, and the presence of a correlation between the density of M2-polarized TAMs and hypoxic areas suggests that hypoxia plays a supportive role during TAM recruitment and induction. Here, we investigated the effects of hypoxia on human macrophage recruitment and M2 polarization. We also investigated the influence of the HIF inhibitor acriflavine (ACF) on M2 TAM infiltration and tumor progression in vivo. We found that hypoxia increased periostin (POSTN) expression in glioma cells and promoted the recruitment of macrophages. Hypoxia-inducible POSTN expression was increased by TGF-α via the RTK/PI3K pathway, and this effect was blocked by treating hypoxic cells with ACF. We also demonstrated that both a hypoxic environment and hypoxia-treated glioma cell supernatants were capable of polarizing macrophages toward a M2 phenotype. ACF partially reversed the M2 polarization of macrophages by inhibiting the upregulation of M-CSFR in macrophages and TGF-β in glioma cells under hypoxic conditions. Administering ACF also ablated tumor progression in vivo. Our findings reveal a mechanism that underlies hypoxia-induced TAM enrichment and M2 polarization and suggest that pharmacologically inhibiting HIFs may reduce M2-polarized TAM infiltration and glioma progression.

Topics: Acriflavine; Animals; Antineoplastic Agents; Brain Neoplasms; Cell Adhesion Molecules; Cell Communication; Cell Plasticity; Cell Proliferation; Chemotaxis; Cytokines; ErbB Receptors; Glioma; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Macrophages; Male; Mice, Inbred BALB C; Mice, Nude; Phosphatidylinositol 3-Kinase; Receptors, Granulocyte-Macrophage Colony-Stimulating Factor; RNA Interference; Signal Transduction; THP-1 Cells; Time Factors; Transfection; Transforming Growth Factor alpha; Transforming Growth Factor beta; Tumor Burden; Tumor Hypoxia; Tumor Microenvironment; Up-Regulation

TGF-β-induced epithelial-mesenchymal transition (EMT) plays an important role in tumor progression. We assessed whether the TGF-β-induced EMT contributed to vasculogenic mimicry (VM) formation in glioma, we established an SHG44 cell line stably transfected with TGF-β cDNA loaded lentivirus. SB203580 was employed to inhibit the TGF-β-induced EMT. The results showed that the VM forming ability of cells could be improved by TGF-β over-expression. The migration and invasion capabilities of cells were also enhanced due to EMT. SB203580 was able to weaken cell migration, invasion and VM forming abilities via blocking p38/MAPK signaling pathways, but it had tiny influence on MMP/LAMC2 chain. Consequently, we concluded that EMT inhibition via p38/MAPK signaling pathways would partly impair TGF-β-induced VM formation in glioma.

Topics: Angiogenesis Inhibitors; Animals; Cell Line, Tumor; Cell Movement; Cell Plasticity; Cell Proliferation; Epithelial-Mesenchymal Transition; Glioma; Humans; Imidazoles; Lentivirus; MAP Kinase Signaling System; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Invasiveness; Neoplasm Transplantation; p38 Mitogen-Activated Protein Kinases; Pyridines; Transfection; Transforming Growth Factor beta; Transplantation, Heterologous

High-grade glioma (HGG), a deadly primary brain malignancy, manifests radioresistance mediated by cell-intrinsic and microenvironmental mechanisms. High levels of the cytokine transforming growth factor-β (TGF-β) in HGG promote radioresistance by enforcing an effective DNA damage response and supporting glioma stem cell self-renewal. Our analysis of HGG TCGA data and immunohistochemical staining of phosphorylated Smad2, which is the main transducer of canonical TGF-β signaling, indicated variable levels of TGF-β pathway activation across HGG tumors. These data suggest that evaluating the putative benefit of inhibiting TGF-β during radiotherapy requires personalized screening. Thus, we used explant cultures of seven HGG specimens as a rapid, patient-specific ex vivo platform to test the hypothesis that LY364947, a small molecule inhibitor of the TGF-β type I receptor, acts as a radiosensitizer in HGG. Immunofluorescence detection and image analysis of γ-H2AX foci, a marker of cellular recognition of radiation-induced DNA damage, and Sox2, a stem cell marker that increases post-radiation, indicated that LY364947 blocked these radiation responses in five of seven specimens. Collectively, our findings suggest that TGF-β signaling increases radioresistance in most, but not all, HGGs. We propose that short-term culture of HGG explants provides a flexible and rapid platform for screening context-dependent efficacy of radiosensitizing agents in patient-specific fashion. This time- and cost-effective approach could be used to personalize treatment plans in HGG patients.

Topics: Antineoplastic Agents; Databases, Genetic; DNA Damage; Gene Expression Profiling; Glioma; Humans; Immunohistochemistry; In Vitro Techniques; Neoplasm Grading; Precision Medicine; Radiation Tolerance; Radiation-Sensitizing Agents; Signal Transduction; Smad2 Protein; SOXB1 Transcription Factors; Transforming Growth Factor beta; X-Rays

In a new report, Bayin et al. described an ex vivo explant model to test the patient-specific response to transforming growth factor-β inhibition in high-grade gliomas.

Topics: Brain Neoplasms; Glioma; Humans; Precision Medicine; Receptors, Transforming Growth Factor beta; Signal Transduction; Transforming Growth Factor beta

Thrombospondin-1 (TSP1) plays a role in the immune tolerance, and is involved in the pathogenesis of glioma. This study aims to investigate the role of the glioma-derived TSP1 in the induction of the tumor immune tolerance. The results showed that the primary human glioma cells expressed high levels of TSP1. Glioma cells enhanced the expression of transforming growth factor (TGF)-β in CD4⁺ CD16⁻ naïve monocytes (Mos). The TGF-β⁺ Mos showed inhibitory effect on CD8⁺ T cell proliferation. We conclude that glioma cell-derived TSP1 facilitates the induction of TGF-β in Mos. The TSP1 may be a potential therapeutic target of glioma.

Topics: Adult; Female; Glioma; Humans; Immune Tolerance; Lipopolysaccharide Receptors; Male; Middle Aged; Monocytes; Thrombospondin 1; Transforming Growth Factor beta

T11 Target structure (T11TS), a membrane glycoprotein isolated from sheep erythrocytes, reverses the immune suppressed state of brain tumor induced animals by boosting the functional status of the immune cells. This study aims at aiding in the design of more efficacious brain tumor therapies with T11 target structure. We propose a mathematical model for brain tumor (glioma) and the immune system interactions, which aims in designing efficacious brain tumor therapy. The model encompasses considerations of the interactive dynamics of glioma cells, macrophages, cytotoxic T-lymphocytes (CD8(+) T-cells), TGF-β, IFN-γ and the T11TS. The system undergoes sensitivity analysis, that determines which state variables are sensitive to the given parameters and the parameters are estimated from the published data. Computer simulations were used for model verification and validation, which highlight the importance of T11 target structure in brain tumor therapy.

Topics: Animals; Animals, Newborn; Brain Neoplasms; CD8-Positive T-Lymphocytes; Female; Glioma; Humans; Immunotherapy; Interferon-gamma; Least-Squares Analysis; Macrophages; Male; Membrane Glycoproteins; Models, Biological; Numerical Analysis, Computer-Assisted; Rats; Sheep; Transforming Growth Factor beta

Transforming growth factors β (TGF-β) pathway has been proven to play important roles in oncogenesis and angiogenesis of gliomas. MiR-132 might be related to TGF-β signaling pathway and high miR-132 expression was reported to be a biomarker of poor prognosis in patients diagnosed with glioma. However, the expression regulation way involved in TGF-β pathway and clinical significance of miR-132 have not been investigated in glioma cells. Here we reported that the mRNA level of miR-132 and TGF-β concentration were both increased in patients with brain glioma. Correlation analysis revealed that TGF-β concentration was positively correlated with mRNA level of miR-132. In addition, the mRNA level of miR-132 was up-regulated by TGF-β in a concentration-dependent and time-dependent manner. Furthermore, we found that miR-132 was involved in modulation of the TGF-β signaling pathway and down-regulation of SMAD7 expression by directly targeting the SMAD7 3'-UTR. MiR-132 was negatively correlated with SMAD7 in patients with brain glioma. Taken together, our results suggest that miR-132 could be stimulated by TGF-β and might enhance the activation of TGF-β signaling through inhibiting SMAD7 expression in glioma cells. These findings contribute to a better understanding of the mechanism of the activation of TGF-β signaling by miR-132.

Topics: Brain; Brain Neoplasms; Cell Line; Female; Gene Expression Regulation, Neoplastic; Glioma; Humans; Male; MicroRNAs; Signal Transduction; Smad7 Protein; Transforming Growth Factor beta

Studies have shown that the abnormal expression of leucine-rich α2 glycoprotein 1 (LRG1) is associated with multiple malignancies, yet its role in glioma pathology remains to be elucidated. In this study, we investigated the role of LRG1 in regulating proliferation, migration and invasion of glioma cells by establishing glioma cell strains with constitutively silenced or elevated LRG1 expression. LRG1 overexpression and silenced cell lines demonstrated modulation of glioma cellular proliferation, migration and invasion through MTT, cell scratching and Transwell assays. Furthermore, overexpression of LRG1 led to augmented activation of transforming growth factor-β (TGF-β) signaling pathway as well as downregulation of E-cadherin and resultant enhanced invasiveness, which was reversed by TGF-β signaling pathway inhibitor SB431542. In summary, our findings suggest that LRG1 promotes invasion and migration of glioma cells through TGF-β signaling pathway.

Topics: Benzamides; Cell Line, Tumor; Cell Movement; Dioxoles; Glioma; Glycoproteins; Humans; Neoplasm Invasiveness; Receptors, Transforming Growth Factor beta; Signal Transduction; Transforming Growth Factor beta

While there were certain studies focusing on the mechanism of TGF-β promoting the growth of glioma cells, the present work revealed another novel mechanism that TGF-β may promote glioma cell growth via enhancing Nodal expression. Our results showed that Nodal expression was significantly upregulated in glioma cells when TGF-β was added, whereas the TGF-β-induced Nodal expression was evidently inhibited by transfection Smad2 or Smad3 siRNAs, and the suppression was especially significant when the Smad3 was downregulated. Another, the attenuation of TGF-β-induced Nodal expression was observed with blockade of the ERK1/2 pathway also. Further detection of the proliferation, apoptosis, and invasion of glioma cells indicated that Nodal overexpression promoted the proliferation and invasion of tumor cells and inhibited their apoptosis, resembling the effect of TGF-β addition. Downregulation of Nodal expression via transfection Nodal-specific siRNA in the presence of TGF-β weakened the promoting effect of the latter on glioma cells growth, and transfecting Nodal siRNA alone in the absence of exogenous TGF-β more profoundly inhibited the growth of glioma cells. These results demonstrated that while both TGF-β and Nodal promoted glioma cells growth, the former might exert such effect by enhancing Nodal expression, which may form a new target for glioma therapy.

Topics: Apoptosis; Brain Neoplasms; Cell Line, Tumor; Cell Proliferation; Extracellular Signal-Regulated MAP Kinases; Gene Expression Regulation, Neoplastic; Glioma; Humans; MAP Kinase Signaling System; Neoplasm Invasiveness; Nodal Protein; Smad2 Protein; Smad3 Protein; Transforming Growth Factor beta

Idiopathic pulmonary fibrosis has been associated with the reactivation of developmental pathways, notably the Hedgehog-Glioma-associated oncogene homolog (GLI) pathway. In this study, we determined whether the Hedgehog pathway was activated in bleomycin-induced lung injury in mice, and whether targeting the Hedgehog-Gli pathway could decrease bleomycin-induced lung fibrosis. After intratracheal injection of bleomycin on Day 0, C57Bl6 mice received GDC-0449 (an inhibitor of Smoothened, the transducer of the pathway), or 2,2'-[[Dihydro-2-(4-pyridinyl)-1,3(2H,4H)-pyrimidinediyl]bis(methylene)]bis[N,N dimethylbenzenamine (GANT61; an inhibitor of GLI transcription factors in the nucleus), from Day 7 to Day 13. At Day 14, whole-lung homogenates were obtained for morphological analysis, assessment of cell apoptosis and proliferation, collagen quantification, and evaluation of profibrotic (transforming growth factor-β, connective tissue growth factor, plasminogen activator inhibitor 1, vascular endothelial growth factor-A) and proinflammatory mediators (IL-1β) expression. We showed that the Hedgehog pathway was activated in bleomycin-induced lung fibrosis on Day 14 after injury, with an increased lung expression of the ligand, Sonic Hedgehog, and with increased messenger RNA expression and nuclear localization of GLI1 and GLI2. Inhibition of Smoothened with GDC-0449 did not influence the development of bleomycin-induced lung fibrosis. By contrast, the inhibition of GLI activity with GANT61 decreased lung fibrosis and lung collagen accumulation, and promoted an antifibrotic and anti-inflammatory environment. Our results identify the hedgehog-Gli pathway as a profibrotic pathway in experimental fibrosis. Inhibition of the Hedgehog-Gli pathway at the level of GLI transcriptional activity could be a therapeutic option in fibrotic lung diseases.

Topics: Anilides; Animals; Antibiotics, Antineoplastic; Apoptosis; Bleomycin; Blotting, Western; Cell Proliferation; Collagen; Fluorescent Antibody Technique; Glioma; Hedgehog Proteins; Immunoenzyme Techniques; Kruppel-Like Transcription Factors; Male; Mice; Mice, Inbred C57BL; Pulmonary Fibrosis; Pyridines; Pyrimidines; Real-Time Polymerase Chain Reaction; Receptors, G-Protein-Coupled; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Smoothened Receptor; Transforming Growth Factor beta; Zinc Finger Protein GLI1

Integrins regulate cellular adhesion and transmit signals important for cell survival, proliferation and motility. They are expressed by glioma cells and may contribute to their malignant phenotype. Integrin inhibition may therefore represent a promising therapeutic strategy. GL-261 and SMA-560 glioma cells grown under standard conditions uniformly detached and formed large cell clusters after integrin gene silencing or pharmacological inhibition using EMD-121974, a synthetic Arg-Gly-Asp-motif peptide, or GLPG0187, a nonpeptidic integrin inhibitor. After 120 h, the clusters induced by integrin inhibition decayed and cells died. In contrast, when cells were cultured under stem cell (sphere) conditions, no disaggregation became apparent upon integrin inhibition, and cell death was not observed. As poly-HEMA-mediated detachment had similar effects on cell viability as integrin inhibition, we postulated that cell death may result from detachment alone, which was confirmed using various permissive and nonpermissive substrates. No surrogate markers of apoptosis were detected and electron microscopy confirmed that necrosis represents the dominant morphology of detachment-induced cell death. In addition, integrin inhibition resulted in the induction of autophagy that represents a survival signal. When integrins were inhibited in nonsphere glioma cells, the TGF-β pathway was strongly impaired, whereas no such effect was observed in glioma cells cultured under sphere conditions. Cell death induced by integrin inhibition was rescued by the addition of recombinant transforming growth factor-β (TGF-β) and accelerated by exposure to the TGF-β receptor inhibitor, SD-208. In summary, cell death following integrin inhibition is detachment mediated, represents an atypical form of anoikis involving necrosis as well as autophagy, and is modulated by TGF-β pathway activity.

Topics: Animals; Anoikis; Apoptosis; Cell Adhesion; Cell Line, Tumor; Down-Regulation; Glioma; Humans; Integrins; Mice; Peptides, Cyclic; Snake Venoms; Transforming Growth Factor beta

Studies have shown that several miRNAs play important roles in regulating a variety of cellular processes in gliomas. In these reports, upregulation of miR-193b has been found to be associated with a poor prognosis for glioma, but its functional mechanism in glioma remains unclear. This study investigates the roles of miR-193b in glioma tumor growth. We first showed that the expression of miR-193b was elevated in both glioma samples and glioma cells. Furthermore, downregulation of miR-193b by inhibitors was statistically correlated with a decrease in cell growth and a restored G1 accumulation. Luciferase assay and Western blot analysis revealed that Smad3 is a direct target of miR-193b. To prove that miR-193b regulated cell growth through the transforming growth factor-β (TGF-β) pathway in glioma cells by regulating Smad3, we tested endogenous targets of the TGF-β pathway by measuring the accumulation of p21 mRNAs after downregulation of miR-193b. The results confirmed that induction of p21 was promoted by miR-193b inhibitors in glioma cells, although this induction disappeared when Smad3 was knocked down with siRNA. Moreover, downregulation of Smad3 mitigates the miR-193b suppression of glioma proliferation. In conclusion, these results suggest that miR-193b regulated cell growth in glioma through the TGF-β pathway by regulating Smad3. Thus, our study indicates that miR-193b promotes cell proliferation by targeting Smad3 in human glioma, which may serve as a potentially useful target for development of miRNA-based therapies in the future.

Topics: Adult; Brain Neoplasms; Cell Line, Tumor; Cell Proliferation; Female; Gene Expression Regulation, Neoplastic; Glioma; HEK293 Cells; Humans; Male; MicroRNAs; Oligonucleotides; RNA, Small Interfering; Sincalide; Smad3 Protein; Transfection; Transforming Growth Factor beta; Tumor Cells, Cultured; Young Adult

HDGF is overexpressed in gliomas as compared to normal brain. We therefore analyzed the molecular mechanisms of HDGF action in gliomas. HDGF was downregulated in normal brain tissue as compared to glioma specimens at both the mRNA and the protein levels. In glioma samples, increased HDGF expression was associated with disease progression. Knocking down HDGF expression not only significantly decreased cellular proliferation, migration, invasion, and tumorigenesis, but also markedly enhanced TMZ-induced cytotoxicity and apoptosis in glioma cells. Mechanistic analyses revealed that CCND1, c-myc, and TGF-β were downregulated after stable HDGF knockdown in the U251 and U87 glioma cells. HDGF knockdown restored E-cadherin expression and suppressed mesenchymal cell markers such as vimentin, β-catenin, and N-cadherin. The expression of cleaved caspase-3 increased, while Bcl-2 decreased in each cell line following treatment with shHDGF and TMZ, as compared to TMZ alone. Furthermore, RNAi-based knockdown study revealed that HDGF is probably involved in the activation of both the PI3K/Akt and the TGF-β signaling pathways. Together, our data suggested that HDGF regulates glioma cell growth, apoptosis and epithelial-mesenchymal transition (EMT) probably through the Akt and the TGF-β signaling pathways. These results provide evidence that targeting HDGF or its downstream targets may lead to novel therapies for gliomas.

Topics: Adolescent; Adult; Aged; Antineoplastic Agents, Alkylating; Apoptosis; Brain Neoplasms; Cadherins; Carcinogenesis; Cell Line, Tumor; Cell Movement; Child; Dacarbazine; Epithelial-Mesenchymal Transition; Female; Glioma; Humans; Intercellular Signaling Peptides and Proteins; Male; Middle Aged; Proto-Oncogene Proteins c-akt; Signal Transduction; Temozolomide; Transforming Growth Factor beta; Young Adult

Glioma is one of the most prevalent types of brain tumor and is associated with the highest mortality rate of all CNS cancers. Epithelial‑mesenchymal transition (EMT) has been recognized as an important factor in tumor metastasis. Previously, it has been demonstrated that microRNA-16 (miR-16) has an important role in tumor metastasis in human cancer cell lines. However, the role of miR-16 in epithelial‑mesenchymal transition of human glioma cells remains unclear. In the present study, U87 and U251 glioma cell lines overexpressing miR-16 were established and it was identified that miR-16 suppressed invasion, adhesion, cell cycle, production of interleukin (IL)-6, IL-8 and transforming growth factor-β, and EMT-related gene expression, including vimentin, β-catenin and E-cadherin in miR-16 overexpressing U87 and U251 glioma cells. Furthermore, miR-16 suppressed EMT mainly through the downregulation of p-FAK and p-Akt expression, and nuclear factor-κB and Slug transcriptional activity. Therefore, miR-16 may be an important therapeutic target and predictor for glioma therapy.

Topics: beta Catenin; Cadherins; Cell Line, Tumor; Cell Movement; Cell Proliferation; Down-Regulation; Epithelial-Mesenchymal Transition; Focal Adhesion Kinase 1; Gene Expression; Gene Expression Regulation, Neoplastic; Glioma; Humans; Interleukin-6; Interleukin-8; MicroRNAs; NF-kappa B; Proto-Oncogene Proteins c-akt; Snail Family Transcription Factors; Transcription Factors; Transforming Growth Factor beta; Vimentin

Tumor specific immune regulatory cells play an important role in the pathogenesis of glioma. The mechanisms have not been fully understood yet. It is suggested that placenta growth factor (PlGF) is involved in the generation of immune regulatory cells. This study aims to investigate the role of glioma cell-derived PlGF in the generation of regulatory B cells (Breg). Glioma cells were isolated from surgically removed glioma tissue. Cytokines were measured by enzyme-linked immunosorbent assay, quantitative real time RT-PCR and Western blotting. Immune suppressor functions of Bregs were assessed by T cell proliferation assay. The results showed that glioma cells expressed PlGF, which was increased after a non-specific activation. Naïve B cells captured the PlGF to differentiate into transforming growth factor-β positive Bregs. The Bregs were activated upon exposure to protein extracts of glioma tissue to suppress the CD8(+) T cell proliferation and the release of perforin and granzyme B. We conclude that glioma cell-released PlGF can induce Bregs to suppress CD8(+) T cell activities.

Topics: Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; B-Lymphocytes, Regulatory; CD8-Positive T-Lymphocytes; Cell Growth Processes; Cell Line, Tumor; Epitopes, B-Lymphocyte; Exosomes; Female; Glioma; Humans; Male; Membrane Proteins; Transforming Growth Factor beta; Tumor Cells, Cultured

Human gliomas are associated with high rates of morbidity and mortality. In the brain, increased mRNA levels of transforming growth factor β (TGF-β) correlate with the degree of malignancy of human gliomas. miR10a/10b expression has been demonstrated to be associated with TGF-β expression in brain tumors, and it is reported that TGF-β induces miR10 expression. Therefore, miR10a/10b expression may be induced by TGF-β expression and may be involved in the TGF-β-induced migration of brain tumor cells. The present study examined the expression of TGF-β and miR10a/10b in the tissues of 10 patients with brain tumors using quantitative PCR (qPCR), and the correlation between TGF-β and miR10a or miR10b expression was analyzed. Additionally, U251 and SHG-44 cells were treated with TGF-β and the expression of miR10a/10b was examined. Further, cell migration was analyzed following transfection of U251 cells with miR10a/10b and the association between miR10a/10b and phosphatase and tensin homolog deleted on chromosome 10 (PTEN) was investigated. U251 cells were transfected with miR10a/10b inhibitors and a PTEN expression plasmid prior to TGF-β treatment and then cell migration was assessed. A significant correlation was identified between TGF-β and miR10a expression (r2=0.6936, P=0.007) and between TGF-β and miR10b expression (r2=0.5876, P=0.02) in the tissues of patients with brain tumors. The results also showed that TGF-β induces miR10a/10b expression and that TGF-β-induced miR10a/10b expression promotes cell migration through the suppression of PTEN. In conclusion, TGF-β-induced miR10a/10b promotes brain tumor migration. This study may provide a number of suggestions for the clinical treatment of brain tumors.

Topics: 3' Untranslated Regions; Base Sequence; Brain Neoplasms; Cell Line, Tumor; Cell Movement; Gene Expression Regulation, Neoplastic; Glioma; Humans; MicroRNAs; Molecular Sequence Data; PTEN Phosphohydrolase; Transforming Growth Factor beta

Although studies have suggested that bone marrow human mesenchymal stem cells (BM-hMSC) may be used as delivery vehicles for cancer therapy, it remains unclear whether BM-hMSCs are capable of targeting cancer stem cells, including glioma stem cells (GSC), which are the tumor-initiating cells responsible for treatment failures. Using standard glioma models, we identify TGF-β as a tumor factor that attracts BM-hMSCs via TGF-β receptors (TGFβR) on BM-hMSCs. Using human and rat GSCs, we then show for the first time that intravascularly administered BM-hMSCs home to GSC-xenografts that express TGF-β. In therapeutic studies, we show that BM-hMSCs carrying the oncolytic adenovirus Delta-24-RGD prolonged the survival of TGF-β-secreting GSC xenografts and that the efficacy of this strategy can be abrogated by inhibition of TGFβR on BM-hMSCs. These findings reveal the TGF-β/TGFβR axis as a mediator of the tropism of BM-hMSCs for GSCs and suggest that TGF-β predicts patients in whom BM-hMSC delivery will be effective.

Topics: Adenoviridae; Animals; Apoptosis; Blotting, Western; Bone Marrow; Brain Neoplasms; Cell Differentiation; Cell Proliferation; Cells, Cultured; Enzyme-Linked Immunosorbent Assay; Flow Cytometry; Glioma; Humans; Mesenchymal Stem Cells; Neoplastic Stem Cells; Rats; Receptors, Transforming Growth Factor beta; Transforming Growth Factor beta; Xenograft Model Antitumor Assays

A major concern in targeted drug therapy is that the inhibition of receptors and signaling molecules in tumor cells may also affect similar components in the tumor microenvironment or in the immune system, with undefined consequences for inhibition of tumor growth. One example is given by the Ras inhibitor salirasib (Farnesythiosalycilic acid, FTS), which in addition to its antitumor activity in mice and humans also exhibits anti-inflammatory activity. Here we show three major effects through which Ras inhibition by FTS provides a favorable antitumor environment in immune-competent mice with subcutaneous or intracranial tumors. First, FTS exhibited antitumor activity in intracranial immune-competent tumor-bearing mice and increased their survival relative to tumor-bearing immune-compromised mice. Second, FTS induced an increase in regulatory T cells in mouse splenocytes, in which Foxp3+ T cells did not interfere with the tumor growth inhibitory effects of FTS. Third, FTS induced an increase in antitumor cytotoxic T-cell reactivity in glioma cells by downregulating their own expression of Foxp3. This downregulation induced a TGF-β-associated mechanism in glioma cells altering the tumor microenvironment and causing reduced resistance of the tumor to the immune system. These results are important as they might explain some of the major beneficial effects of Ras inhibitors. They may provide an experimental framework for examination of the impact of other anticancer drugs on cancer and the immune system.

Topics: Animals; Antineoplastic Agents; Brain Neoplasms; CD8-Positive T-Lymphocytes; Cell Line, Tumor; Cell Proliferation; Extracellular Signal-Regulated MAP Kinases; Farnesol; Forkhead Transcription Factors; Glioma; Male; Mice; Mice, Inbred C57BL; Mice, Nude; Molecular Targeted Therapy; Oncogene Protein v-akt; Proto-Oncogene Proteins p21(ras); ras Proteins; RNA, Messenger; Salicylates; T-Lymphocytes, Cytotoxic; Transforming Growth Factor beta

Glioma stem cells (GSCs) have the property of self-renewal and appear to be a driving force for the initiation and recurrence of gliomas. We recently found that the human tumorigenic LN-229 glioma cell line failed to form neurospheres in serum-free conditions and generated mostly small tumors in vivo, suggesting that either LN-229 GSCs are not active in these conditions or GSCs are absent in the LN-229 cell line.. Using self-renewal assay, soft-agar colony assay, cell proliferation assay, invasion assay, real time PCR analysis, ELISA and in vivo tumorigenic assay, we investigated the effects of interleukin (IL)-1β and transforming growth factor (TGF)-β on the development of GSCs from LN-229 cells.. Here, we demonstrate that the combination of IL-1β and TGF-β can induce LN-229 cells to form neurospheres in serum-free medium. IL-1β/TGF-β-induced neurospheres display up-regulated expression of stemness factor genes (nestin, Bmi-1, Notch-2 and LIF), and increased invasiveness, drug resistance and tumor growth in vivo: hallmarks of GSCs. These results indicate that IL-1β and TGF-β cooperate to induce a GSC phenotype in the LN-229 cell line. Induction of nestin, LIF and Notch-2 by IL-1β/TGF-β can be reverted after cytokine withdrawal. Remarkably, however, up-regulated Bmi-1 levels remained unchanged after cytokine withdrawal; and the cytokine-withdrawn cells maintained strong clonogenicity, suggesting that Bmi-1 may play a crucial role in tumorigenesis.. Our finding indicates that glioma cells without self-renewal capability in standard conditions could also contribute to glioma malignancy when cytokines, such as IL-1β and TGF-β, are present in the tumor environment. Targeting GSC-promoting cytokines that are highly expressed in glioblastomas may contribute to the development of more effective glioma therapies.

Topics: Animals; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Transformation, Neoplastic; Glioma; Humans; Interleukin-1beta; Intermediate Filament Proteins; Leukemia Inhibitory Factor; Mice; Mice, Nude; Neoplastic Stem Cells; Nerve Tissue Proteins; Nestin; Nuclear Proteins; Polycomb Repressive Complex 1; Proto-Oncogene Proteins; Receptor, Notch2; Repressor Proteins; Transforming Growth Factor beta

Glioma is one of the most malignant tumors in the central nervous system. As a peroxisome proliferator-activated receptor γ (PPAR-γ) activator, the thiazolidinediones (TZDs) induce growth arrest and cell death in a broad spectrum of tumor cells. In this study, we investigated the role of rosiglitazone in glioma cells. We found that rosiglitazone, a member of TZDs, suppresses growth of human glioma cell lines U87 and U251. Rosiglitazone also induces cell cycle arrest and apoptosis, which may be the mechanism of its anti-proliferation effect. Next, we found that rosiglitazone suppresses the expression of TGF-beta and its receptor TGF-betaR2, and suppresses phosphorylation of Smad3. Rosiglitazone also inhibits formation of the Smad3/Smad4 complex. Furthermore, Rosiglitazone affects the expression of Smad3/Smad4 associated regulators of gene expression, including p21 and c-Myc. These results suggest that rosiglitazone suppresses growth and cell cycle of human glioma cells by blocking the TGF-beta mediated pathway.

Topics: Blotting, Western; Cell Cycle; Cell Division; Cell Line, Tumor; Enzyme-Linked Immunosorbent Assay; Flow Cytometry; Glioma; Humans; Phosphorylation; Rosiglitazone; Smad3 Protein; Thiazolidinediones; Transforming Growth Factor beta

The strength and duration of NF-κB signaling are tightly controlled by multiple negative feedback mechanisms. However, in cancer cells, these feedback loops are overridden through unclear mechanisms to sustain oncogenic activation of NF-κB signaling. Previously, we demonstrated that overexpression of miR-30e* directly represses IκBα expression and leads to hyperactivation of NF-κB. Here, we report that miR-182 was overexpressed in a different set of gliomas with relatively lower miR-30e* expression and that miR-182 directly suppressed cylindromatosis (CYLD), an NF-κB negative regulator. This suppression of CYLD promoted ubiquitin conjugation of NF-κB signaling pathway components and induction of an aggressive phenotype of glioma cells both in vitro and in vivo. Furthermore, we found that TGF-β induced miR-182 expression, leading to prolonged NF-κB activation. Importantly, the results of these experiments were consistent with an identified significant correlation between miR-182 levels with TGF-β hyperactivation and activated NF-κB in a cohort of human glioma specimens. These findings uncover a plausible mechanism for sustained NF-κB activation in malignant gliomas and may suggest a new target for clinical intervention in human cancer.

Topics: Animals; Brain Neoplasms; Cell Line, Tumor; Deubiquitinating Enzyme CYLD; Genes, Reporter; Glioma; Humans; I-kappa B Kinase; I-kappa B Proteins; Mice; Mice, Nude; MicroRNAs; Neoplasm Invasiveness; Neoplasm Proteins; Neovascularization, Pathologic; NF-kappa B; NF-KappaB Inhibitor alpha; Phosphorylation; Protein Processing, Post-Translational; RNA; RNA, Neoplasm; Signal Transduction; Smad Proteins; Transcription, Genetic; Transforming Growth Factor beta; Tumor Suppressor Proteins; Ubiquitination

Even with current standard-of-care therapies, the prognosis for patients with malignant gliomas is very poor and several new treatment modalities for glioblastomas are currently under investigation. Given the role of TGF-β in gliomas, anti-TGF-β strategies against gliomas are currently being investigated. Biodistribution of intravenously injected AF680-labeled 1D11, a pan-neutralizing TGF-β antibody, was monitored in mice bearing either subcutaneous or orthotopic gliomas using in vivo imaging and fluorescence microscopy. AF680-labeled 1D11 entered both the subcutaneous and intracranial tumors and the antibody was detected within the tumor tissue for several days whereas only low fluorescence was found in organs. The effects of 1D11 on subcutaneous versus orthotopic U87MG and GL261 gliomas in immunocompetent C57BL/6J versus immunodeficient CD1-Foxn1nu mice were observed by direct tumor size measurement, H&E staining and immunohistochemistry. Treatment of immunocompetent mice bearing subcutaneous GL261 tumors with 1D11 resulted in complete remission. In immune deficient mice, the growth of subcutaneous GL261 tumors was increased following treatment with 1D11. Intracranially implanted gliomas in C57Bl/6J mice showed no size reduction after 1D11 treatment but there was reduced invasion of the glioma cells into the adjacent normal brain. Together these data demonstrate that TGF-β plays different roles in combating the tumor depending on subcutaneous versus orthotopic implantation site.

Topics: Animals; Antibodies, Monoclonal; Antibody Specificity; Brain Neoplasms; Cell Line, Tumor; Glioma; Humans; Immunocompetence; Immunohistochemistry; Mice; Mice, Inbred C57BL; Mice, Nude; Microscopy, Fluorescence; Neoplasm Invasiveness; Spectroscopy, Near-Infrared; Tissue Distribution; Transforming Growth Factor beta; Xenograft Model Antitumor Assays

Bone morphogenetic protein 7 (BMP-7) belongs to the superfamily of transforming growth factor β-like cytokines, which can act either as tumor suppressors or as tumor promoters depending on cell type and differentiation. Our investigations focused on analyzing the effects of BMP-7 during glioma cell proliferation in vitro and in vivo. BMP-7 treatment decreased the proliferation of Gli36ΔEGFR-LITG glioma cells up to 50%through a cell cycle arrest in the G(1) phase but not by induction of apoptosis. This effect was mediated by the modulation of the expression and phosphorylation of cyclin-dependent kinase 2, cyclin-dependent kinase inhibitor p21, and downstream retinoblastoma protein. Furthermore, in vivo optical imaging of luciferase activity of Gli36ΔEGFR-LITG cells implanted intracranially into nude mice in the presence or absence of BMP-7 treatment corroborated the antiproliferative effects of this cytokine. This report clearly underlines the tumor-suppressive role of BMP-7 in glioma-derived cells. Taken together, our results indicate that manipulating the BMP/transforming growth factor β signaling cascade may serve as a new strategy for imaging-guided molecular-targeted therapy of malignant gliomas.

Topics: Animals; Blotting, Western; Bone Morphogenetic Protein 7; Cell Differentiation; Cell Proliferation; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase Inhibitor p21; Fluorescent Antibody Technique; G1 Phase; Glioma; Luciferases; Luminescence; Mice; Mice, Inbred BALB C; Molecular Targeted Therapy; Phosphorylation; Retinoblastoma Protein; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transforming Growth Factor beta

The Th17 cell subset is involved in many autoimmune and infectious pathologies. It is also associated with the tumorigenesis process and poor prognosis of certain tumors. However, its expression and function in glioma cases remain unclear. We measured the percentage of Th17 cells in peripheral blood mononuclear cells (PBMCs) and compared the concentrations of relevant cytokines in the serum of 35 glioma patients and 20 healthy donors. Protein, mRNA, and levels of Th17-relevant cytokines in 24 glioma tissues and 5 cerebral trauma tissues were also assessed. We evaluated whether Th17-relevant cytokines were associated with the clinical stages of glioma. The results showed that there were no significant differences in the volume of Th17 cells in PBMCs and serum concentrations of interferon (IFN)-γ, interleukin (IL)-17, IL-6, IL-23, and TGF-β between glioma patients and healthy donors nor did these differences exist in patients with different clinical stages of glioma. Different expression patterns of Th17-relevant cytokines were observed in glioma tissues when compared to trauma tissues. High mRNA-positive ratios of IL-17 (19/24) and retinoid-related orphan receptor (RORC) (18/24) were observed in glioma tissues, but not in trauma tissues. Positive ratios of transforming growth factor (TGF)-β were higher in trauma tissues and glioma grade II than in glioma grade IV. IL-6, IFN-γ, and IL-1β showed high positive ratios, but showed no significant differences between trauma tissues or grades of glioma. None of the glioma and trauma tissues was positive for IL-23. High positive ratios of IL-17 in glioma tissue were confirmed via analysis of immunohistochemical staining. The results demonstrated that IL-17 and other Th17-relevant cytokines could be expressed in glioma tissues. IL-17 expression, the hallmark of Th17 cell subset, may play an important role in glioma tumorigenesis and progression.

Topics: Adult; CD4 Antigens; Female; Glioma; Humans; Interferon-gamma; Interleukins; Leukocytes, Mononuclear; Male; Neoplasm Staging; Nuclear Receptor Subfamily 1, Group F, Member 3; RNA, Messenger; Th17 Cells; Transforming Growth Factor beta

Glioma cells are exposed to elevated interstitial fluid flow during the onset of angiogenesis, at the tumor periphery while invading normal parenchyma, within white matter tracts, and during vascular normalization therapy. Glioma cell lines that have been exposed to fluid flow forces in vivo have much lower invasive potentials than in vitro cell motility assays without flow would indicate.. A 3D Modified Boyden chamber (Darcy flow through collagen/cell suspension) model was designed to mimic the fluid dynamic microenvironment to study the effects of fluid shear stress on the migratory activity of glioma cells. Novel methods for gel compaction and isolation of chemotactic migration from flow stimulation were utilized for three glioma cell lines: U87, CNS-1, and U251. All physiologic levels of fluid shear stress suppressed the migratory activity of U87 and CNS-1 cell lines. U251 motility remained unaltered within the 3D interstitial flow model. Matrix Metalloproteinase (MMP) inhibition experiments and assays demonstrated that the glioma cells depended on MMP activity to invade, and suppression in motility correlated with downregulation of MMP-1 and MMP-2 levels. This was confirmed by RT-PCR and with the aid of MMP-1 and MMP-2 shRNA constructs.. Fluid shear stress in the tumor microenvironment may explain reduced glioma invasion through modulation of cell motility and MMP levels. The flow-induced migration trends were consistent with reported invasive potentials of implanted gliomas. The models developed for this study imply that flow-modulated motility involves mechanotransduction of fluid shear stress affecting MMP activation and expression. These models should be useful for the continued study of interstitial flow effects on processes that affect tumor progression.

Topics: Animals; Apoptosis; Cell Line, Tumor; Cell Movement; Cell Survival; Chemotactic Factors; Chemotaxis; Down-Regulation; Fluorescence; Gels; Glioma; Humans; Hydrodynamics; Matrix Metalloproteinase 1; Matrix Metalloproteinase 2; Neoplasm Invasiveness; Permeability; Rats; Reproducibility of Results; Rheology; Stress, Mechanical; Transforming Growth Factor beta

A human glioblastoma multiforme cell line (U87) and its derived-spheroids were irradiated either using a conventional irradiation (CIR) or a CK-like irradiation (IIR) in which the 8 Gy was delivered intermittently over a period of 40 minutes. The ability of glioma cells to migrate into a matrigel matrix was evaluated on days 1-8 from irradiation. Irradiation with CK-driven IIR significantly increased the invasion potential of U87 cells in a matrigel-based assay. In contrast to CIR, IIR was associated with increased levels of TGF-β at four days (Real time PCR), β1-integrin at 4-5 days (real-time PCR and western blot) and no elevation in phosphorylated AKT at days 4 and 5 (western blot). Our data suggests that glioma cell invasion as well as elevations of TGF-β and β1-integrin are associated with IIR and not CIR.

Topics: Cell Line, Tumor; Cell Movement; Central Nervous System Neoplasms; Collagen; Drug Combinations; Glioblastoma; Glioma; Humans; Integrin beta1; Laminin; Neoplasm Invasiveness; Oncogene Protein v-akt; Phosphorylation; Proteoglycans; Radiosurgery; Transforming Growth Factor beta; Up-Regulation

Bevacizumab has promising activity against glioma, although reasons for poor efficacy and variable response rates in certain patients are unclear. Vascular endothelial growth factor receptor 2 (VEGFR2) is heterogeneously expressed within the microvasculature of various malignancies. Moreover, transforming growth factor β (TGF-β), a negative prognostic factor for glioma, is intimately involved in angiogenesis including VEGFR2 regulation. Our objective was to associate expression of VEGFR2 and TGF-β activity with clinicopathological features of human glioma.. Expression patterns determined by immunohistochemistry for VEGFR2 and phosphorylated Smad2 in human gliomas were compared to overall survival, progression-free survival (PFS), initial versus recurrent tumors and tumor grade.. Endothelial VEGFR2 expression was low or undetectable in normal tissue but the proportion of VEGFR2-positive vessels increased with tumor grade. Decreased PFS was associated with tumors whose vessels had increased proportions of VEGFR2 at recurrence. Neither parenchymal nor endothelial cell p-Smad2 was associated with tumor grade; however, the former was negatively correlated with overall survival in glioblastoma multiforme.. The molecular phenotype of the vasculature based on the status of VEGFR2 but not p-Smad2 is related to aspects of glioma progression and patient response. Changes in VEGFR2-positive vessels may account for variable therapeutic efficacy of anti-angiogenic agents.

Topics: Adult; Aged; Brain Neoplasms; Disease-Free Survival; Female; Glioma; Humans; Male; Middle Aged; Neoplasm Grading; Neoplasm Recurrence, Local; Recurrence; Signal Transduction; Smad2 Protein; Transforming Growth Factor beta; Vascular Endothelial Growth Factor Receptor-2

Both vasculogenic mimicry (VM) and transforming growth factor-β (TGFβ) are positively correlated with malignancy in glioma. Accordingly, we supposed that TGFβ might be related with VM, and aimed to detect whether TGFβ could influence VM formation in two glioma cell lines U251MG and SHG44, which were different in malignancy. We found that the VM-positive U251MG had a significantly higher TGFβ expression than the VM-negative SHG44. Downregulating TGFβ in U251MG by RNAi technology resulted in a significantly impaired VM formation, which could be rescued by rhTGFβ. However, adding rhTGFβ could not induce VM in SHG44. To investigate the possible mechanism, we detected the changes of some VM-related genes including EphA2, VE-cadherin, MMP-2, MMP-9, MT1-MMP and LAMC2 by RT-PCR and found that MT1-MMP transcript was affected by TGFβ expression. Gelatin zymography showed a declined MMP-2 activity in the TGFβ-inhibited cells. Further studies showed that MT1-MMP inhibition impaired VM formation in U251MG. Moreover, TGFβ induced MT1-MMP expression and VM formation in a dose-dependent manner. These findings indicated us that TGFβ was required for VM formation in U251MG. MT1-MMP was correlated with TGFβ-induced VM formation. Thus, TGFβ might be a potential target for VM inhibition in glioma.

Topics: Cell Line, Tumor; Gene Expression; Gene Expression Regulation, Neoplastic; Gene Silencing; Glioma; Humans; Matrix Metalloproteinase 14; Matrix Metalloproteinase Inhibitors; Matrix Metalloproteinases; Neovascularization, Pathologic; Transforming Growth Factor beta

The activating receptor NKG2D, expressed by natural killer (NK) cells and CD8(+) T cells, has a role in the specific killing of transformed cells. We examined NKG2D expression in patients with glioblastoma multiforme and found that NKG2D was downregulated on NK cells and CD8(+) T cells. Expression of NKG2D on lymphocytes significantly increased following tumor resection and correlated with an increased ability to kill NKG2D ligand-positive tumor targets. Despite the presence of soluble NKG2D ligands in the sera of glioblastoma patients, NKG2D downregulation was primarily caused by tumor-derived tumor growth factor-beta, suggesting that blocking of this cytokine may have therapeutic benefit.

Topics: Brain Neoplasms; CD8-Positive T-Lymphocytes; Cell Separation; Cytotoxicity, Immunologic; Down-Regulation; Enzyme-Linked Immunosorbent Assay; Flow Cytometry; Fluorescent Antibody Technique; Gene Expression Regulation, Neoplastic; Glioma; Humans; Immune Tolerance; Killer Cells, Natural; NK Cell Lectin-Like Receptor Subfamily K; Reverse Transcriptase Polymerase Chain Reaction; Transforming Growth Factor beta

Glioblastoma multiforme (GBM, WHO grade IV) is the most common and most malignant of astrocytic brain tumors, and is associated with rapid invasion into neighboring tissue. In other tumor types it is well established that such invasion involves a complex interaction between tumor cells and locally produced extracellular matrix. In GBMs, surprisingly little is known about the associated matrix components, in particular the fibrillar proteins such as collagens that are known to play a key role in the invasion of other tumor types.. In this study we have used both the Masson's trichrome staining and a high resolution multiple immunofluorescence labeling method to demonstrate that intratumoral fibrillar collagens are an integral part of the extracellular matrix in a subset of GBMs. Correlated with this collagen deposition we observed high level expression of the collagen-binding receptor Endo180 (CD280) in the tumor cells. Further, interrogation of multiple expression array datasets identified Endo180 as one of the most highly upregulated transcripts in grade IV GBMs compared to grade III gliomas. Using promoter analysis studies we show that this increased expression is, in part, mediated via TGF-beta signaling. Functionally, we demonstrate that Endo180 serves as the major collagen internalization receptor in GBM cell lines and provide the first evidence that this activity is critical for the invasion of GBM cells through fibrillar collagen matrices.. This study demonstrates, for the first time, that fibrillar collagens are extensively deposited in GBMs and that the collagen internalization receptor Endo180 is both highly expressed in these tumors and that it serves to mediate the invasion of tumor cells through collagen-containing matrices. Together these data provide important insights into the mechanism of GBM invasion and identify Endo180 as a potential target to limit matrix turnover by glioma cells and thereby restrict tumor progression.

Topics: Adult; Aged; Aged, 80 and over; Collagen; Female; Gene Expression Regulation, Neoplastic; Glioblastoma; Glioma; Humans; Male; Middle Aged; Neoplasm Invasiveness; Receptors, Mitogen; Signal Transduction; Transforming Growth Factor beta

Gliomas localized within the CNS are generally not rejected by the immune system despite being immunogenic. This failure of the immune system has been associated both with glioma-derived immunosuppressive molecules and the immune-privileged state of the CNS. However, the relative contribution of tumor location to the glioma-mediated immunosuppression, as well as the immune mechanisms involved in the failure of glioma rejection are not fully defined. We report here that syngeneic GL261 gliomas growing either intracranially or subcutaneously in mice are infiltrated by DC and T cells. However, only subcutaneous gliomas elicit an effective anti-tumor immune response. In contrast to DC infiltrating subcutaneously grown GL261 gliomas, tumor-infiltrating DC from intracranial gliomas do not activate antigen-dependent T-cell proliferation in vitro. In addition, brain-localized GL261 gliomas are characterized by significantly higher numbers of Foxp3(+) Treg and higher levels of TGF-beta1 mRNA and protein expression when compared with GL261 gliomas in the skin. Our data show that gliomas in the CNS, but not in the skin, give rise to TGF-beta production and accumulation of both Treg and functionally impaired DC. Thus, not the tumor itself, but its location dictates the efficiency of the anti-tumor immune response.

Topics: Animals; Cell Line, Tumor; Central Nervous System Neoplasms; Dendritic Cells; Flow Cytometry; Forkhead Transcription Factors; Glioma; Histocytochemistry; Kaplan-Meier Estimate; Lymphocyte Activation; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Knockout; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Skin Neoplasms; T-Lymphocytes, Regulatory; Transforming Growth Factor beta

A growing body of evidence suggests that only a small subpopulation of malignant glioma cells have true tumorigenic potential. A study by Peñuelas et al. in this issue of Cancer Cell demonstrates that TGF-beta can stimulate self-renewal and inhibit differentiation in a proportion of these glioma-initiating cells.

Topics: Animals; Brain Neoplasms; Cell Differentiation; Glioma; Humans; Neoplastic Stem Cells; Transforming Growth Factor beta

High biological activity of the transforming growth factor (TGF)-beta-Smad pathway characterizes the malignant phenotype of malignant gliomas and confers poor prognosis to glioma patients. Accordingly, TGF-beta has become a novel target for the experimental treatment of these tumors. TGF-beta is processed by furin-like proteases (FLP) and secreted from cells in a latent complex with its processed propeptide, the latency-associated peptide (LAP). Latent TGF-beta-binding protein 1 (LTBP-1) covalently binds to this small latent TGF-beta complex (SLC) and regulates its function, presumably via interaction with the extracellular matrix (ECM). We report here that the levels of LTBP-1 protein in vivo increase with the grade of malignancy in gliomas. LTBP-1 is associated with the ECM as well as secreted into the medium in cultured malignant glioma cells. The release of LTBP-1 into the medium is decreased by the inhibition of FLP activity. Gene-transfer mediated overexpression of LTBP-1 in glioma cell lines results in an increase inTGF-beta activity. Accordingly, Smad2 phosphorylation as an intracellular marker of TGF-beta activity is enhanced. Conversely, LTBP-1 gene silencing reduces TGF-beta activity and Smad2 phosphorylation without affecting TGF-beta protein levels. Collectively, we identify LTBP-1 as an important modulator of TGF-beta activation in glioma cells, which may contribute to the malignant phenotype of these tumors.

Topics: Astrocytoma; Brain Neoplasms; Cell Line, Tumor; Cell Proliferation; Extracellular Matrix; Gene Expression Regulation, Neoplastic; Glioblastoma; Glioma; Humans; Immunoblotting; Immunohistochemistry; Latent TGF-beta Binding Proteins; Oligonucleotide Array Sequence Analysis; Phenotype; Phosphorylation; Polymerase Chain Reaction; Signal Transduction; Smad2 Protein; Transforming Growth Factor beta; Up-Regulation

A variety of cancers, including malignant gliomas, overexpress transforming growth factor-beta (TGF-beta), which helps tumors evade effective immune surveillance through a variety of mechanisms, including inhibition of CD8(+) CTLs and enhancing the generation of regulatory T (T(reg)) cells. We hypothesized that inhibition of TGF-beta would improve the efficacy of vaccines targeting glioma-associated antigen (GAA)-derived CTL epitopes by reversal of immunosuppression.. Mice bearing orthotopic GL261 gliomas were treated systemically with a TGF-beta-neutralizing monoclonal antibody, 1D11, with or without s.c. vaccinations of synthetic peptides for GAA-derived CTL epitopes, GARC-1 (77-85) and EphA2 (671-679), emulsified in incomplete Freund's adjuvant.. Mice receiving the combination regimen exhibited significantly prolonged survival compared with mice receiving either 1D11 alone, GAA vaccines alone, or mock treatments alone. TGF-beta neutralization enhanced the systemic induction of antigen-specific CTLs in glioma-bearing mice. Flow cytometric analyses of brain-infiltrating lymphocytes revealed that 1D11 treatment suppressed phosphorylation of Smad2, increased GAA-reactive/IFN-gamma-producing CD8(+) T cells, and reduced CD4(+)/FoxP3(+) T(reg) cells in the glioma microenvironment. Neutralization of TGF-beta also upregulated plasma levels of interleukin-12, macrophage inflammatory protein-1 alpha, and IFN-inducible protein-10, suggesting a systemic promotion of type-1 cytokine/chemokine production. Furthermore, 1D11 treatment upregulated plasma interleukin-15 levels and promoted the persistence of GAA-reactive CD8(+) T cells in glioma-bearing mice.. These data suggest that systemic inhibition of TGF-beta by 1D11 can reverse the suppressive immunologic environment of orthotopic tumor-bearing mice both systemically and locally, thereby enhancing the therapeutic efficacy of GAA vaccines.

Topics: Animals; Antibodies, Monoclonal; Antibodies, Neutralizing; Antigens, Neoplasm; Brain Neoplasms; Cancer Vaccines; Cell Line, Tumor; Epitopes, T-Lymphocyte; Glioma; Male; Mice; Mice, Inbred BALB C; Neoplasm Transplantation; T-Lymphocytes, Cytotoxic; T-Lymphocytes, Regulatory; Transforming Growth Factor beta; Vaccines, Subunit

Haematopoietic progenitor cells (HPC) are attracted by experimental gliomas in vivo. This attraction is further enhanced by irradiation or hypoxic preconditioning of the glioma cells. Adhesive interactions might be critical to the preferential accumulation of HPC within the glioma tissue. Here, we studied the interactions of HPC with endothelial cells. Exposure of human cerebral endothelial cells (SV-HCEC), human microvascular endothelial cells (HMEC) and brain tumour endothelial cells derived from human glioblastomas (BTEC) to supernatants of glioma cells and primary glioma cells (SN-G) induced the expression of E-selectin (CD62E). CD62E expression was further enhanced when the glioma cells had been exposed to irradiation or hypoxia prior to the collection of supernatants, as well as by irradiation or exposure to hypoxia of the endothelial cells. Vascular cell adhesion molecule 1 (VCAM-1) was constitutively expressed on SV-HCEC, HMEC and BTEC, but was not modulated by SN-G, irradiation or hypoxia. Transendothelial HPC migration was enhanced after CD62E induction in vitro. Neutralizing antibodies to CD62E strongly reduced the homing of lin(-)Sca-1(+)c-kit(+) cells to orthotopic SMA-560 gliomas in vivo. Tissue microarray sampling normal brain tissue and astrocytomas of WHO grades II-IV revealed a selective expression of CD62E on endothelial cells of tumour vessels. SN-G-induced CD62E expression on endothelial cells in vitro required transforming growth factor (TGF)-beta signalling in glioma cells and vascular endothelial growth factor (VEGF)/VEGF receptor 2 (VEGF-R2) signalling in endothelial cells. Further, we observed a nuclear factor kappa B-dependent activation of the CD62E promoter peaking at 12 h after VEGF-R2 activation by glioma-derived VEGF. Taken together, we identify glioma cell-induced CD62E expression on endothelial cells as one mediator of the glioma tropism of HPC.

Topics: Adult Stem Cells; Animals; Autoantibodies; Cell Hypoxia; Cell Line, Tumor; Cell Migration Inhibition; Cell Movement; Coculture Techniques; E-Selectin; Endothelial Cells; Gene Expression; Glioma; Hematopoietic Stem Cells; Humans; Mice; Mice, Mutant Strains; Neoplasm Transplantation; NF-kappa B; Signal Transduction; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factor Receptor-2

Transforming growth factor-beta (TGF-beta) is abundantly expressed in malignant gliomas and is crucial for the tumor micromilieu. TGF-beta not only enhances migration and invasion of glioma cells but also inhibits an effective anti-glioma immune response. TGF-beta mediates its biologic effects through interactions with TGF-beta receptors (TbetaR)-I to -III. Binding of TGF-beta leads to the activation of an intracellular signaling cascade and subsequent phosphorylation of Sma and MAD-related proteins (SMAD). Soluble TGF-beta receptors (TbetaRs) abrogate the TGF-beta effect by competing for the binding of the ligand to its receptor. Here we used adenoviral gene transfer to express TbetaR-IIs and -IIIs in human glioma cell lines. TbetaR-IIs reduced SMAD2 phosphorylation and TGF-beta-dependent reporter activity. Furthermore, it enhanced glioma cell lysis by natural killer cells. TbetaR-IIIs alone were inactive in these assays, but enhanced the effects of TbetaR-IIs. Transduction of LN-308 cells with TbetaRs markedly delayed growth of intracerebral xenografts in nude mice in vivo. These data commend TbetaRs for possible experimental therapy of gliomas.

Topics: Animals; Brain Neoplasms; Cell Line, Tumor; Cell Survival; Genetic Therapy; Glioma; Humans; Killer Cells, Natural; Mice; Mice, Nude; Neoplasm Transplantation; Protein Binding; Protein Serine-Threonine Kinases; Proteoglycans; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Transforming Growth Factor beta

Transforming growth factor (TGF)-beta induces various cellular responses principally through Smad-dependent transcriptional regulation. Activated Smad complexes cooperate with transcription factors in regulating a group of target genes. The target genes controlled by the same Smad-cofactor complexes are denoted a synexpression group. We found that an Id-like helix-loop-helix protein, human homologue of Maid (HHM), is a synexpression group-restricted regulator of TGF-beta signalling. HHM suppressed TGF-beta-induced growth inhibition and cell migration but not epithelial-mesenchymal transition. In addition, HHM inhibited TGF-beta-induced expression of plasminogen activator inhibitor-type 1 (PAI-1), PDGF-B, and p21(WAF), but not Snail. We identified a basic-helix-loop-helix protein, Olig1, as one of the Smad-binding transcription factors affected by HHM. Olig1 interacted with Smad2/3 in response to TGF-beta stimulation, and was involved in transcriptional activation of PAI-1 and PDGF-B. HHM, but not Id proteins, inhibited TGF-beta signalling-dependent association of Olig1 with Smad2/3 through physical interaction with Olig1. HHM thus appears to regulate a subset of TGF-beta target genes including the Olig1-Smad synexpression group. HHM is the first example of a cellular response-selective regulator of TGF-beta signalling with clearly determined mechanisms.

Topics: Animals; Basic Helix-Loop-Helix Transcription Factors; Cell Line; Gene Expression Regulation; Glioma; Humans; Mice; Mice, Nude; Neoplasm Transplantation; Nerve Tissue Proteins; Plasminogen Activator Inhibitor 1; Promoter Regions, Genetic; Protein Binding; Protein Structure, Tertiary; Proto-Oncogene Proteins c-sis; RNA Interference; Signal Transduction; Smad Proteins; Transcription Factors; Transcription, Genetic; Transforming Growth Factor beta

The invasion of tumor cells into brain tissue is a pathologic hallmark of malignant gliomas and contributes to treatment failures. Diffuse glioblastomas contain numerous microglial cells, which enhance the progression of gliomas; however, factors responsible for invasion-promoting role of microglia are unknown. Transforming growth factor-beta (TGF-beta) can enhance tumor growth, invasion, angiogenesis and immunosuppression. Antagonizing TGF-beta activity has been shown to inhibit tumor invasion in vitro and tumorigenicity, but a systemic inhibition or lack of TGF-beta signaling results in acute inflammation and disruption of immune system homeostasis. We developed plasmid-transcribed small hairpin RNAs (shRNAs) to downregulate the TGF-beta type II receptor (TbetaIIR) expression, which effectively inhibited cytokine-induced signaling pathways and transcriptional responses in transiently transfected human glioblastoma cells. Silencing of TbetaIIR abolished TGF-beta-induced glioblastoma invasiveness and migratory responses in vitro. Moreover, tumorigenicity of glioblastoma cells stably expressing TbetaIIR shRNAs in nude mice was reduced by 50%. Microglia strongly enhanced glioma invasiveness in the co-culture system, but this invasion-promoting activity was lost in glioma cells stably expressing shTbetaRII, indicating a crucial role of microglia-derived TGF-beta in tumor-host interactions. Our results demonstrate a successful targeting of TGF-beta-dependent invasiveness and tumorigenicity of glioblastoma cells by RNAi-mediated gene silencing.

Topics: Animals; Cell Movement; Coculture Techniques; Collagen; Drug Combinations; Enzyme-Linked Immunosorbent Assay; Flow Cytometry; Gene Silencing; Glioma; Humans; Laminin; Male; Mice; Mice, Nude; Microglia; Neoplasm Invasiveness; Protein Serine-Threonine Kinases; Proteoglycans; Rats; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Transfection; Transforming Growth Factor beta; Tumor Cells, Cultured

Endoglin is a cell-surface adhesion protein as well as a coreceptor for transforming growth factor-beta (TGF-beta). It is located on endothelial and few other cells, but also found on certain tumor cells. Brain metastatic breast tumor cells derived from the MDA-MB-231 cell line heavily express endoglin in contrast to the corresponding parental ones. To clarify whether this determines their invasive phenotype, we compared their biological properties with endoglin-silenced brain-metastatic cells, low-expressing parental cells and these transfected with L- and S-endoglins, isoforms transducing or lacking TGF-beta signals. All L-endoglin-overexpressing cells were characterized by numerous invadopodia where endoglin was preferentially localized. Endoglin-expression resulted in elevated levels of the matrix metalloproteinases (MMP-1 and MMP-19) and downregulation of the plasminogen activator inhibitor-1. In Boyden-chamber and wound-healing assays, endoglin-overexpressing cells showed a considerably higher migration and chemotaxis to TGF-beta. In 3D spheroid confrontation assays between breast tumor cells and TGF-beta-secreting glioma cells, high L-endoglin-expressing cells invaded into the glioma-spheroids whereas low-endoglin-expressing cells dissociated in the culture; invasion was blocked by TGF-beta antibodies. In contrast to parental cells, endoglin-overexpressing cells invaded deeply into mouse brain slices. Thus, endoglin expression on tumor cells enhances their invasive character by formation of invadopodia, extracellular proteolysis, chemotaxis and migration.

Topics: Animals; Antigens, CD; Cell Line, Tumor; Cell Movement; Endoglin; Gene Expression Regulation, Neoplastic; Glioma; Humans; Intracellular Signaling Peptides and Proteins; Matrix Metalloproteinase 1; Matrix Metalloproteinases, Secreted; Mice; Neoplasm Invasiveness; Neoplasm Metastasis; Phenotype; Receptors, Cell Surface; Transforming Growth Factor beta

The transforming growth factor-beta (TGF-beta) plays a pivotal role in the pathobiology of human gliomas: during carcinogenesis, it turns from a tumor suppressor to a tumor promoter. The traditional Smad pathway and the more recently discovered MAPK pathway are the most important pathways for TGF-beta related intracellular signal transduction mediating differential pathobiological effects. In this study, we investigated the effects of TGF-beta2 and the TGF-beta2 antisense phosphorothioate oligodeoxynucleotide (PTO) AS-11 on the functionality of both the Smad and MAPK pathways in high-grade gliomas. We aimed to correlate the imbalance between the pathways with differences in the behaviour of high-grade glioma cells. Gene and protein expression studies were used to detect levels of members of the Smad and MAPK pathways under regulation of TGF-beta2 and AS-11. Proliferation and migration assays were functional readouts for effects caused by these regulating tools. Gene arrays were used to detect yet unknown regulators of these functional effects. The Smad pathway was functional in the tested cell lines. Exogenous TGF-beta2 inhibited proliferation but enhanced migration. Smad 2 mRNA expression and activation were significantly reduced by incubation with AS-11. K-ras was reduced both in gene arrays and quPCR under treatment with AS-11, but there was no influence of K-ras down-regulation on the activity of ERK. Ubiquitination-related genes also were specifically down-regulated with AS-11. Our results indicate the involvement of K-ras in TGF-beta signaling in high-grade gliomas. ERK, which is a member of the MAPK pathway, was not influenced and seems to be activated through RAS independent cascades in glioma. These results suggest that combined antagonization of the TGF-beta and MAPK pathways might be a promising approach for glioma therapy. An imbalance between these two pathways might be responsible for TGF-beta switching to a tumor promoter protein in high-grade gliomas.

Topics: Brain Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; Extracellular Signal-Regulated MAP Kinases; Gene Expression Regulation, Neoplastic; Glioma; Humans; MAP Kinase Signaling System; Models, Biological; Oligonucleotides; Phosphorylation; Promoter Regions, Genetic; Signal Transduction; Smad Proteins; Transforming Growth Factor beta; Transforming Growth Factor beta2

Due to its immunosuppressive properties, the cytokine transforming growth factor (TGF)-beta has become a promising target in the experimental treatment of human malignant gliomas. Here, we report that the antifibrotic drug 5-methyl-1-phenyl-2-(1H)-pyridone (pirfenidone, PFD) elicits growth-inhibitory effects and reduces TGF-beta2 protein levels in human glioma cell lines. This reduction in TGF-beta2 is biologically relevant since PFD treatment reduces the growth inhibition of TGF-beta-sensitive CCL-64 cells mediated by conditioned media of glioma cells. The downregulation of TGF-beta is mediated at multiple levels. PFD leads to a reduction of TGF-beta2 mRNA levels and of the mature TGF-beta2 protein due to decreased expression and direct inhibition of the TGF-beta pro-protein convertase furin. In addition, PFD reduces the protein levels of the matrix metalloproteinase (MMP)-11, a TGF-beta target gene and furin substrate involved in carcinogenesis. These data define PFD or PFD-related agents as promising agents for human cancers associated with enhanced TGF-beta activity.

Topics: Animals; Antineoplastic Agents; Cell Line; Cell Line, Tumor; Gene Expression Regulation, Neoplastic; Glioma; Humans; Mink; Pyridones; Transforming Growth Factor beta

TGFbeta acts as a tumor suppressor in normal epithelial cells and early-stage tumors and becomes an oncogenic factor in advanced tumors. The molecular mechanisms involved in the malignant function of TGFbeta are not fully elucidated. We demonstrate that high TGFbeta-Smad activity is present in aggressive, highly proliferative gliomas and confers poor prognosis in patients with glioma. We discern the mechanisms and molecular determinants of the TGFbeta oncogenic response with a transcriptomic approach and by analyzing primary cultured patient-derived gliomas and human glioma biopsies. The TGFbeta-Smad pathway promotes proliferation through the induction of PDGF-B in gliomas with an unmethylated PDGF-B gene. The epigenetic regulation of the PDGF-B gene dictates whether TGFbeta acts as an oncogenic factor inducing PDGF-B and proliferation in human glioma.

Topics: Adolescent; Adult; Aged; Astrocytoma; Brain Neoplasms; Cell Proliferation; Child; Child, Preschool; DNA Methylation; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Glioblastoma; Glioma; Humans; Infant; Middle Aged; Oligonucleotide Array Sequence Analysis; Phosphorylation; Prognosis; Proto-Oncogene Proteins c-sis; Receptors, Transforming Growth Factor beta; Signal Transduction; Smad2 Protein; Smad7 Protein; Survival Rate; Transforming Growth Factor beta; Tumor Cells, Cultured

TGFbeta functions as a tumor suppressor in some contexts and a tumor promoter in others. In a recent issue of Cancer Cell, Bruna et al. (2007) shed light on an epigenetic mechanism that underlies this schizophrenic behavior in malignant glioma. Their findings highlight a stem cell/cancer link...and a potential blind spot in large-scale cancer genome sequencing projects.

Topics: Animals; Brain Neoplasms; Cell Transformation, Neoplastic; DNA Methylation; Epigenesis, Genetic; Gene Expression Regulation, Neoplastic; Glioma; Humans; Smad Proteins; Stem Cells; Transforming Growth Factor beta

Two immunoresistant (IR) glioma cell variants, 13-06-IR29 and 13-06-IR30, were cloned from 13-06-MG glioma cell populations after receiving continuous immunoselective pressure from multiple alloreactive cytotoxic T lymphocyte (aCTL) preparations. Reapplication of aCTL immunoselective pressure to the IR clones, displaying a partial regain in sensitivity to aCTL after removal of the selective pressure, restored the resistance. The IR variants exhibited cross-resistance to non-human leukocyte antigen (HLA)-restricted effector cells and gamma-irradiation, but not to carmustine. The IR clones were characterized for factors that might contribute to the immunoresistance. The aCTL adhesion to extracellular matrix extracts derived from either the IR clones or the parental cells was similar and not impaired. Furthermore, aCTL binding to parental cells and IR clones was equal. Down-regulation of the cell recognition molecules, class I HLA or intercellular adhesion molecule-1 (ICAM-1), that would inhibit their recognition by aCTL was not observed on the IR clones. The down-regulation of Fas by the IR clones correlated with their resistance to FasL-induced apoptosis. HLA-G or FasL that might provide an immunotolerant environment or provide a means of counterattack to aCTL, respectively, were not associated with the IR phenotype. The aCTL, coincubated with the IR clones and parental cells, displayed up-regulation of multiple secreted cytokines. A significant up-regulation of bioactive transforming growth factor (TGF)-beta was observed in the IR clones compared with the parental cells. These data suggest that increased secretion of bioactive TGF-beta may inhibit aCTL lysis of the IR clones. Disruption of the TGF-beta signaling pathway may circumvent the resistance.

Topics: Antineoplastic Agents, Alkylating; Apoptosis; Brain Neoplasms; Calcium; Carmustine; Cell Adhesion; Cell Line, Tumor; Coculture Techniques; Cytokines; Extracellular Matrix; Fas Ligand Protein; fas Receptor; Gamma Rays; Glioma; HLA-A Antigens; Humans; Intercellular Adhesion Molecule-1; Killer Cells, Lymphokine-Activated; T-Lymphocytes, Cytotoxic; Transforming Growth Factor beta; Up-Regulation

Glioblastoma, one of the most lethal tumors, is paradigmatic for tumor-associated immunosuppression. Lectin-like transcript-1 (LLT1) is a newly identified ligand for the inhibitory natural killer (NK) cell receptor CD161. Here, we report that glioma cells express LLT1 mRNA and protein in vitro and in vivo, whereas expression levels in normal brain are low. LLT1 expression in human gliomas increases with the WHO grade of malignancy. We further show that transforming growth factor-beta (TGF-beta) up-regulates the expression of LLT1 in glioma cells. Small interfering RNA (siRNA)-mediated down-regulation of LLT1 in LNT-229 and LN-428 cells promotes their lysis by NK cells. Thus, LLT1 acts as a mediator of immune escape and contributes to the immunosuppressive properties of glioma cells.

Topics: Antigens, Surface; Cell Line, Tumor; Down-Regulation; Glioma; Humans; Killer Cells, Natural; Lectins, C-Type; NK Cell Lectin-Like Receptor Subfamily B; Receptors, Cell Surface; RNA, Messenger; RNA, Small Interfering; Transforming Growth Factor beta

Studies were conducted to determine whether gene expression profiles following a single dose of radiation would yield equivalent profiles following fractionated radiation in different tumor cell lines. MCF7 (breast), DU145 (prostate), and SF539 (gliosarcoma) cells were exposed to a total radiation dose of 10 Gy administered as a single dose (SD) or by daily multifractions (MF) of 5 x 2 Gy. Following radiation treatment, mRNA was isolated at 1, 4, 10, and 24 h and processed for cDNA microarray analysis. To determine the influence of the tumor microenvironment on gene expression, one cell type (DU145) was evaluated growing as a solid tumor in athymic nude mice for both radiation protocols. Unsupervised hierarchical cluster map analysis showed significant differences in gene expression profiles between SD and MF treatments for cells treated in vitro, with MF yielding a more robust induction compared with SD. Several genes were uniquely up-regulated by MF treatment, including multiple IFN-related genes (STAT1, G1P2, OAS1, OAS3, G1P3, IFITM1) and TGF-beta-associated genes (EGR1, VEGF, THBS1, and TGFB2). DU145 cells grown in vivo exhibited a completely different set of genes induced by both SD and MF compared with the same cells exposed in vitro. The results of the study clearly show distinct differences in the molecular response of cells between SD and MF radiation exposures and show that the tumor microenvironment can significantly influence the pattern of gene expression after radiation exposures.

Topics: Animals; Breast Neoplasms; Cell Cycle; Cell Line, Tumor; Cluster Analysis; Dose Fractionation, Radiation; Female; Gene Expression; Gene Expression Profiling; Genes, p53; Glioma; Humans; Interferons; Male; Mice; Mice, Nude; Neoplasm Transplantation; Oligonucleotide Array Sequence Analysis; Prostatic Neoplasms; Transforming Growth Factor beta; Transplantation, Heterologous; Up-Regulation

Hepatocyte growth factor (HGF) is a pleiotrophic cytokine that stimulates motility and invasion of several cancer cell types and induces angiogenesis, which is known to be expressed in several malignancies including glioma. The effect of transforming growth factor-beta (TGF-beta) isoforrns as well as gangliosides on HGF production was investigated in human glioma cell lines. TGF-beta isoforms and gangliosides were found to differentially stimulate HGF production by these cells. The ganglioside GD3 enhanced this release to the greatest extent and the stimulation was more marked in a glioblastoma cell line than in the two other anaplastic astrocytoma cell lines. These results suggest that both TGF-betas and gangliosides may act as indirect angiogenic factors by stimulating HGF secretion.

Topics: Astrocytoma; Brain Neoplasms; Cell Line, Tumor; Culture Media; Culture Media, Serum-Free; Enzyme-Linked Immunosorbent Assay; Gangliosides; Glioma; Hepatocyte Growth Factor; Humans; Isomerism; Neovascularization, Pathologic; Stimulation, Chemical; Transforming Growth Factor beta

Transforming growth factor-beta (TGF-beta) is a proinvasive and immunosuppressive cytokine that plays a major role in the malignant phenotype of gliomas. One novel strategy of disabling TGF-beta activity in gliomas is to disrupt the signaling cascade at the level of the TGF-beta receptor I (TGF-betaRI) kinase, thus abrogating TGF-beta-mediated invasiveness and immune suppression. SX-007, an orally active, small-molecule TGF-betaRI kinase inhibitor, was evaluated for its therapeutic potential in cell culture and in an in vivo glioma model. The syngeneic, orthotopic glioma model SMA-560 was used to evaluate the efficacy of SX-007. Cells were implanted into the striatum of VM/Dk mice. Dosing began three days after implantation and continued until the end of the study. Efficacy was established by assessing survival benefit. SX-007 dosed at 20 mg/kg p.o. once daily (q.d.) modulated TGF-beta signaling in the tumor and improved the median survival. Strikingly, approximately 25% of the treated animals were disease-free at the end of the study. Increasing the dose to 40 mg/kg q.d. or 20 mg/kg twice daily did not further improve efficacy. The data suggest that SX-007 can exert a therapeutic effect by reducing TGF-beta-mediated invasion and reversing immune suppression. SX-007 modulates the TGF-beta signaling pathway and is associated with improved survival in this glioma model. Survival benefit is due to reduced tumor invasion and reversal of TGF-beta-mediated immune suppression, allowing for rejection of the tumor. Together, these results suggest that treatment with a TGF-betaRI inhibitor may be useful in the treatment of glioblastoma.

Topics: Animals; Antineoplastic Agents; Brain Neoplasms; Cell Line, Tumor; Dose-Response Relationship, Drug; Enzyme Inhibitors; Enzyme-Linked Immunosorbent Assay; Flow Cytometry; Glioma; Humans; Immunoblotting; Immunohistochemistry; Immunologic Surveillance; Mice; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Transforming Growth Factor beta; Xenograft Model Antitumor Assays

Gliomas are the most common primary tumors of the central nervous system, with glioblastomas as the most malignant entity. Rapid proliferation and diffuse brain invasion of these tumors are likely to determine the unfavorable prognosis. Considering its promigratory properties, the transforming growth factor-beta (TGF-beta) signaling pathway has become a major therapeutic target. Analyses of resected glioma tissues revealed an intriguing correlation between tumor grade and the expression of TGF-beta(1-3) as well as their receptors I and II. Here, we analyzed the effects of peroxisome proliferator-activated receptor gamma (PPAR-gamma) agonists on glioma proliferation, migration, and brain invasion. Using an organotypic glioma invasion model, we show that micromolar doses of the PPAR-gamma activator troglitazone blocked glioma progression without neurotoxic damage to the organotypic neuronal environment observed. This intriguing antiglioma property of troglitazone seems to be only partially based on its moderate cytostatic effects. We identified troglitazone as a potent inhibitor of glioma cell migration and brain invasion, which occurred in a PPAR-gamma-independent manner. The antimigratory property of troglitazone was in concordance with the transcriptional repression of TGF-beta(1-3) and their receptors I and II and associated with reduced TGF-beta release. Due to its capacity to counteract TGF-beta release and glioma cell motility and invasiveness already at low micromolar doses, troglitazone represents a promising drug for adjuvant therapy of glioma and other highly migratory tumor entities.

Topics: Animals; Base Sequence; Brain Neoplasms; Cell Line, Tumor; Cell Movement; Chromans; DNA Primers; Down-Regulation; G1 Phase; Glioma; Mice; Neoplasm Invasiveness; PPAR gamma; Thiazolidinediones; Transforming Growth Factor beta; Troglitazone

CD70 (CD27 ligand) promotes the expansion of primed lymphocytes by enhancing cell survival. Surprisingly, we previously observed that CD70 aberrantly expressed on human glioma cells promoted immune cell apoptosis and inhibited alloreactive lysis. Here we report that ectopic expression of CD70 in mouse glioma cells enhances apoptosis of T, B and NK cells in coculture, but nevertheless promotes glioma cell lysis by NK cells in vitro. In nude mice, CD70 expression in SMA-560 gliomas delays the glioma growth upon subcutaneous (s.c.) or intracerebral (i.c.) inoculation, suggesting a role for CD70/CD27-dependent NK cell activity in tumor surveillance. In syngeneic immunocompetent VM/Dk mice, CD70 allows the rejection of s.c. and i.c. implanted SMA-560 tumors. The tumorigenicity of CD70-expressing glioma cells is abrogated when TGF-beta signaling is blocked. Moreover, mice surviving the s.c. CD70 glioma challenge subsequently also reject wild-type glioma cells administered i.c. Similarly, CD70-expressing GL-261 gliomas are rejected in syngeneic C57BL/6 mice, while glioma growth is restored in C57BL/6 CD27(-/-) mice, suggesting that the CD70/CD27 interaction recruits a tumor-specific T-cell repertoire and induces tumor-specific memory. Altogether, these observations indicate that the net effect of aberrant CD70 expression in gliomas is immune stimulatory rather than immune paralytic and encourage its application in tumor immunotherapy.

Topics: Animals; Antigens, CD; Apoptosis; B-Lymphocytes; Brain Neoplasms; CD27 Ligand; Glioma; Humans; Immunotherapy; Killer Cells, Natural; Membrane Proteins; Mice; Mice, Inbred C57BL; Mice, Nude; T-Lymphocytes; Transforming Growth Factor beta; Tumor Necrosis Factors

Conditionally BCL-xL-overexpressing LNT-229 Tet-On glioma cell clones were generated to investigate whether the 'antiapoptosis phenotype' and the 'motility phenotype' mediated by BCL-2 family proteins in glioma cells could be separated. BCL-xL induction led to an immediate and concentration-dependent protection of LNT-229 cells from apoptosis. BCL-xL induction for up to 3 days did not result in altered invasiveness. In contrast, long-term BCL-xL induction for 21 days resulted in increased transforming growth factor-beta2 expression, and in metalloproteinase-2 and -14 dependent, but integrin independent, increased invasiveness. Withdrawal of doxycycline (Dox) abolished the protection from apoptosis whereas the 'invasion phenotype' remained stable. Dox stimulation of BCL-xL-inducible LNT-229 cells conferred infiltrative growth to BCL-xL-positive glioma cells in vivo and reduced the survival of tumor-bearing mice. These data allow to dissect a direct antiapoptotic action of BCL-xL from an indirect effect, presumably mediated by altered gene expression, which modifies tumor cell invasiveness in vitro and in vivo.

Topics: Animals; Apoptosis; bcl-X Protein; Brain Neoplasms; Cell Line; Cell Movement; Cell Survival; Dose-Response Relationship, Drug; Doxycycline; Glioma; Humans; Immunoblotting; Matrix Metalloproteinase 2; Mice; Mice, Nude; Neoplasm Invasiveness; NIH 3T3 Cells; Survival Analysis; Time Factors; Transfection; Transforming Growth Factor beta; Transforming Growth Factor beta2; Xenograft Model Antitumor Assays

Tenascin-C is an extracellular matrix protein known to correlate with prognosis in patients with glioblastoma, probably by stimulation of invasion and neoangiogenesis. Transforming Growth Factor-beta1 (TGF-beta1) plays an important role in the biology of high-grade gliomas, partly by regulating invasion of these tumors into parenchyma. This study was designed to evaluate if TGF-beta1 induces the expression and deposition of Tenascin-C in the extracellular matrix of high-grade gliomas which may be pivotal for the invasion of these tumors into healthy parenchyma.. A series of 20 high-grade gliomas was stained immunohistochemically with Tenascin-C- and TGF-beta1- specific antibodies. Expression levels of both proteins were evaluated and correlated with each other, time to progression and molecular and morphological markers of invasion. A quantitative PCR assay was performed evaluating the induction of Tenascin-C mRNA by treatment with TGF-beta1 in vitro.. Tenascin-C was expressed in 18 of 19 (95%) evaluable tumors, whereas 14 of 20 tumors (70%) expressed TGF-beta1 in a significant percentage of cells. Treatment with TGF-beta1 did induce the expression of Tenascin-C at the mRNA and protein level in vitro. The expression of Tenascin-C and TGF-beta1 did neighter statistically correlate with each other nor with time to progression.. In our series, Tenascin-C and TGF-beta1 were expressed in the vast majority of high-grade gliomas. We could not detect a correlation of one of the proteins with time to progression. Nevertheless, we describe induction of Tenascin-C by TGF-beta1, possibly providing a mechanism for the invasion of high-grade gliomas into healthy parenchyma.

Topics: Adult; Aged; Astrocytoma; Biomarkers, Tumor; Brain Neoplasms; Child; Disease Progression; Extracellular Matrix Proteins; Gene Expression Regulation, Neoplastic; Glioblastoma; Glioma; Gliosarcoma; Humans; Immunohistochemistry; Middle Aged; Neoplasm Invasiveness; RNA, Messenger; Tenascin; Time Factors; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Cells, Cultured

Amyloid beta (Abeta), the major component of the senile plaques of Alzheimer's disease, is implicated in neuronal cell death. We have found that Abeta42, a neurotoxic form of Abeta peptide, induces both neuronal and glial expression of TGFbeta2. We have further demonstrated that the addition into culture media of neutralizing antibody to TGFbeta2 or a large amount of the recombinant soluble amyloid precursor protein alpha, the extracellular domain of amyloid precursor protein (APP) generated by alpha secretase, suppresses death in primary cortical neurons (PCNs) induced by Abeta42 in vitro. Combined with the finding in our recent study indicating that TGFbeta2 is a neuronal cell death-inducing ligand for APP, it is suggested that TGFbeta2 is an autocrinal mediator for Abeta42-induced death in PCNs.

Topics: Amyloid beta-Peptides; Analysis of Variance; Animals; Blotting, Western; Cell Death; Cell Line, Tumor; Cerebral Cortex; Drug Interactions; Embryo, Mammalian; Gene Expression Regulation; Glioma; Humans; Immunohistochemistry; Mice; Neuroblastoma; Neurons; Peptide Fragments; Reverse Transcriptase Polymerase Chain Reaction; Time Factors; Transforming Growth Factor beta; Transforming Growth Factor beta2

Previously we defined a pathway of transforming growth factor beta (TGF-beta) and stromal cell-derived factor-1/CXC chemokine ligand 12 (SDF-1alpha/CXCL12) dependent migration of adult haematopoietic stem and progenitor cells (HPC) towards glioma cells in vitro and their homing to experimental gliomas in vivo. Hypoxia is a critical aspect of the microenvironment of gliomas and irradiation is an essential part of the standard therapy. To evaluate the therapeutic potential of HPC as vectors for a cell-based therapy of gliomas, we investigated the impact of hypoxia and irradiation on the attraction of HPC by glioma cells. Temozolomide (TMZ) treatment and hyperthermia served as controls. Supernatants of irradiated or hypoxic LNT-229 glioma cells promote HPC migration in vitro. Reporter assays reveal that the CXCL12 promoter activity is enhanced in LNT-229 cells at 24 h after irradiation at 8 Gy or after exposure to 1% oxygen for 12 h. The irradiation- and hypoxia-induced release of CXCL12 depends on hypoxia inducible factor-1 alpha (HIF-1alpha), but not on p53. Induction of transcriptional activity of HIF-1alpha by hypoxia or irradiation requires an intact TGF-beta signalling cascade. This delineates a novel stress signalling cascade in glioma cells involving TGF-beta, HIF-1alpha and CXCL12. Stress stimuli can be irradiation, hypoxia or TMZ, but not hyperthermia. Cerebral irradiation of nude mice at 21 days after intracerebral implantation of LNT-229 glioma induces tumour satellite formation and enhances the glioma tropism of HPC to the tumour bulk and even to these satellites in vivo. These data suggest that the use of HPC as cellular vectors in the treatment of glioblastoma may well be combined with irradiation or other anti-angiogenic therapies that induce tumour hypoxia.

Topics: Animals; Antineoplastic Agents, Alkylating; Brain; Brain Neoplasms; Cell Hypoxia; Cell Line, Tumor; Cell Movement; Chemokine CXCL12; Chemokines, CXC; Dacarbazine; Gamma Rays; Glioma; Hematopoietic Stem Cells; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Immunohistochemistry; Mice; Mice, Nude; Neoplasm Proteins; Promoter Regions, Genetic; Signal Transduction; Temozolomide; Transcription, Genetic; Transforming Growth Factor beta

NKG2D ligands (NKG2DL) are expressed by infected and transformed cells. They transmit danger signals to NKG2D-expressing immune cells, leading to lysis of NKG2DL-expressing cells. We here report that the NKG2DL MHC class I-chain-related molecules A and B (MICA/B) and UL16-binding proteins (ULBP) 1-3 are expressed in human brain tumours in vivo, while expression levels are low or undetectable in normal brain. MICA and ULBP2 expression decrease with increasing WHO grade of malignancy, while MICB and ULBP1 are expressed independently of tumour grade. We further delineate two independent mechanisms that can explain these expression patterns: (i) transforming growth factor-beta (TGF-beta) is upregulated during malignant progression and selectively downregulates MICA, ULBP2 and ULBP4 expression, while MICB, ULBP1 and ULBP3 are unaffected. (ii) Cleavage of MICA and ULBP2 is reduced by inhibition of metalloproteinases (MP), whereas no changes in the expression levels of other NKG2DL were detected. Consequently, NKG2DL-dependent NK cell-mediated lysis is enhanced by depletion of TGF-beta or inhibition of MP. Thus, escape from NKG2D-mediated immune surveillance of malignant gliomas in vivo may be promoted by the inhibition of MICA and ULBP2 expression via an autocrine TGF-beta loop and by MP-dependent shedding from the cell surface. Loss of MICA and ULBP2, in contrast to other NKG2DL, may be particularly important in glioma immune escape, and differential regulation of human NKG2DL expression is part of the immunosuppressive properties of human malignant glioma cells.

Topics: Brain Neoplasms; Carrier Proteins; Cell Death; Cell Line, Tumor; Down-Regulation; Glioma; GPI-Linked Proteins; Histocompatibility Antigens Class I; Humans; Immunohistochemistry; Intercellular Signaling Peptides and Proteins; Intracellular Signaling Peptides and Proteins; Killer Cells, Natural; Membrane Proteins; Metalloproteases; NK Cell Lectin-Like Receptor Subfamily K; Receptors, Immunologic; Receptors, Natural Killer Cell; Transforming Growth Factor beta; Up-Regulation

Marfan syndrome (MFS) is a heritable disorder of the connective tissue which has been linked to mutations in the FBN (fibrillin-1) gene. Murine knockouts of the FBN gene show increased interstitial fibrosis and TGF-beta (tumor growth factor-beta) gene activation. Abnormal TGF-beta expression has previously been linked to radiation-induced fibrosis, suggesting a possible link between MFS and increased late effects following radiotherapy. Herein we report two cases in which MFS patients treated with radical radiotherapy without undue acute or late radiotherapy toxicity suggesting that radiotherapy should not be withheld from MFS patients. MFS patients may provide a unique clinico-translational setting to test associations between FBN mutations, TGF-beta activation and the risk of tissue fibrosis.

Topics: Adolescent; Aged; Brain Neoplasms; Female; Fibrillin-1; Fibrillins; Glioma; Humans; Male; Marfan Syndrome; Microfilament Proteins; Prostatic Neoplasms; Radiation Pneumonitis; Transforming Growth Factor beta

Stem and progenitor cells (PCs) of various lineages have become attractive vehicles to improve therapeutic gene delivery to cancers, notably glioblastoma. Here we report that adult human and murine haematopoietic PCs display a tropism for intracerebral gliomas but not for normal brain tissue in mice. Organotypic hippocampal slice culture and spheroid confrontation assays confirm a directed PC migration towards glioma cells ex vivo and in vitro. RNA interference-mediated disruption of transforming growth factor beta (TGF-beta) synthesis by the glioma cells strongly inhibits PC migration. We delineate a CXC chemokine ligand (CXCL) 12-dependent pathway of TGF-beta-induced PC migration that is facilitated by MMP-9-mediated stem cell factor cleavage in vitro. Moreover, neutralizing antibodies to CXCL12 strongly reduce PC homing to experimental gliomas in vivo. Thus, we define here the molecular mechanism underlying the glioma tropism of the probably most easily accessible PC population suitable for cancer therapy, that is, adult haematopoietic PC.

Topics: Adult; Animals; Brain Neoplasms; Chemokine CXCL12; Chemokines, CXC; Chemotaxis; Glioma; Hematopoietic Stem Cell Transplantation; Hematopoietic Stem Cells; Humans; Matrix Metalloproteinase 9; Mice; Mice, Nude; Neoplasm Transplantation; Stem Cell Factor; Transforming Growth Factor beta; Tumor Cells, Cultured

Here, we provide first evidence that long-term continuous infusion of highly purified antisense phosphorothioate oligodeoxynucleotides (S-ODN) into brain parenchyma is well tolerated and thus highly suitable for in vivo application. AP 12009 is an S-ODN for the therapy of malignant glioma. It is directed against human transforming growth factor-beta (TGF-beta2) mRNA. In the clinical setting, AP 12009 is administered intratumorally by continuous infusion directly into the brain tumor. In view of this clinical application, the focus of our data is on local toxicology studies in rabbits and monkeys to evaluate the safety of AP 12009. AP 12009 was administered either by intrathecal bolus injection into the subarachnoidal space of the lumbar region of both cynomolgus monkeys and rabbits or by continuous intraparenchymatous infusion directly into the brain tissue of rabbits. Intrathecal bolus administration of 0.1 ml of 500 microM AP 12009 showed neither clinical signs of toxicity nor macroscopically visible or histomorphologic changes. After a 7-day intraparenchymatous continuous infusion of 500 microM AP 12009 at 1 microl/h in rabbits, there was no evidence of toxicity except for local mild to moderate lymphocytic leptomeningoencephalitis. Additionally, AP 12009 showed good tolerability in safety pharmacology as well as in acute toxicity studies and 4-week subchronic toxicity studies in mice, rats, and monkeys. This favorable safety profile proves the suitability of AP 12009 for local administration in brain tumor patients from the point of view of toxicology.

Topics: Animals; Brain Neoplasms; Drug Evaluation, Preclinical; Glioma; Injections, Intraventricular; Injections, Spinal; Macaca fascicularis; Mice; Oligonucleotides, Antisense; Rabbits; Rats; Rats, Sprague-Dawley; Thionucleotides; Transforming Growth Factor beta; Transforming Growth Factor beta2

High-grade tumors of the brain remain virtually incurable with current therapeutic regimens, new approaches to augment existing therapies need to be explored. The major goal of this pilot study was to evaluate the feasibility of gene therapy using plasmid DNA encoding tumor necrosis factor-alpha and bax together with proton radiation in an immunocompetent animal model with orthotopic brain tumor. C6 glioma cells were stereotactically implanted into the left hemibrain of Wistar rats (day 0). On day 5, the appropriate groups received intratumoral pGL1-TNF-a and pGL1-Bax (10 microg each), parental plasmid pWS4 (20 microg), or PBS. Hemibrain proton irradiation (10 Gy, 90 MeV, single fraction) was delivered 18-20 hr later. Rats were euthanized when signs of illness appeared. In addition, a subset of animals from each group was euthanized on day 9 for immune and other assays. By day 9, 25%, 20%, and 10% of rats treated with PBS, pWS4, or pGL1-TNF-alpha/pGL1-Bax, respectively, had been euthanized due to weight loss or other signs of illness, whereas all rats treated with pGL1-TNF-alpha/pGL1-Bax + radiation or radiation alone were healthy (P<0.05). At this same time, the pGL1-TNF-alpha/pGL1-Bax + radiation group had significantly elevated lymphocyte percentages (P<0.005 or less) and a relatively high level of lymphocytic infiltrate within tumors. Although the rats treated with pGL1-TNF-alpha/pGL1-Bax had the highest levels of activated T helper (CD4+/CD71+) and T cytotoxic (CD8+/CD71+) cells, the values were not significantly different compared to the pWS4-injected control group. Splenocytes in all tumor cell-injected groups had higher mean values for DNA and protein synthesis compared to the non-tumor cell injected control group, whereas oxygen radical production by phagocytes was consistently higher in groups injected with plasmid or treated with radiation. Body, hemibrain, and spleen masses, white blood cell, red blood cell and platelet counts, hemoglobin, hematocrit, and transforming growth factor-beta1 levels in plasma were similar among groups. The results demonstrate that treatment with pGL1-TNF-alpha/pGL1-Bax combined with proton hemibrain irradiation is safe under the conditions used. Overall, these data support further investigation of this unique combination therapy.

Topics: Animals; Antineoplastic Agents; bcl-2-Associated X Protein; Brain Neoplasms; Combined Modality Therapy; Genetic Therapy; Genetic Vectors; Glioma; Lymphocyte Activation; Lymphocytes; Male; Mitogens; Phagocytes; Plasmids; Proto-Oncogene Proteins c-bcl-2; Proton Therapy; Rats; Rats, Wistar; Reactive Oxygen Species; Spleen; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

Transforming growth factor-beta (TGF-beta) is a multifunctional cytokine that promotes malignant glioma invasion, angiogenesis, and immunosuppression. Antisense oligonucleotide suppression of TGF-beta(2) ligand expression has shown promise in preclinical and clinical studies but at least two ligands mediate the effects of TGF-beta in gliomas. Therefore, we examined the effects of SB-431542, a novel, small molecule inhibitor of the type I TGF-beta receptor, on a panel of human malignant glioma cell lines. SB-431542 blocked the phosphorylation and nuclear translocation of the SMADs, intracellular mediators of TGF-beta signaling, with decreased TGF-beta-mediated transcription. Furthermore, SB-431542 inhibited the expression of two critical effectors of TGF-beta-vascular endothelial growth factor and plasminogen activator inhibitor-1. SB-431542 treatment of glioma cultures inhibited proliferation, TGF-beta-mediated morphologic changes, and cellular motility. Together, our results suggest that small molecule inhibitors of TGF-beta receptors may offer a novel therapy for malignant gliomas by reducing cell proliferation, angiogenesis, and motility.

Topics: Active Transport, Cell Nucleus; Activin Receptors, Type I; Benzamides; Cell Line, Tumor; Cell Movement; Cell Proliferation; Dioxoles; DNA-Binding Proteins; Gene Expression Regulation, Neoplastic; Glioma; Humans; Phosphorylation; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; Smad Proteins; Trans-Activators; Transcription, Genetic; Transforming Growth Factor beta

Current therapies for gliomas often fail to address their infiltrative nature. Conventional treatments leave behind small clusters of neoplastic cells, resulting in eventual tumor recurrence. In the present study, we have evaluated the antitumor activity of neural progenitor cells against gliomas when stereotactically injected into nucleus Caudatus of Fisher rats. We show that the rat neural progenitor cell lines HiB5 and ST14A, from embryonic hippocampus and striatum primordium, respectively, are able to prolong animal survival and, in 25% of the cases, completely inhibit the outgrowth of N29 glioma compared with control animals. Delayed tumor outgrowth was also seen when HiB5 cells were inoculated at the site of tumor growth 1 week after tumor inoculation or when a mixture of tumor cells and HiB5 cells were injected s.c. into Fisher rats. HiB5 cells were additionally coinoculated together with two alternative rat gliomas, N32 and N25. N32 was growth inhibited, but rats inoculated with N25 cells did not show a prolonged survival. To evaluate the possibility of the involvement of the immune system in the tumor outgrowth inhibition, we show that HiB5 cells do not evoke an immune response when injected into Fisher rats. Furthermore, the rat neural progenitor cells produce all transforming growth factor beta isotypes, which could explain the observed immunosuppressive nature of these cells. Hence, some neural progenitor cells have the ability to inhibit tumor outgrowth when implanted into rats. These results indicate the usefulness of neural stem cells as therapeutically effective cells for the treatment of intracranial tumors.

Topics: Animals; Brain Neoplasms; Colonic Neoplasms; Corpus Striatum; Glioma; Hippocampus; Humans; Immunosuppressive Agents; Male; Neurons; Rats; Rats, Inbred F344; Rats, Sprague-Dawley; Stem Cells; Survival Rate; Transforming Growth Factor beta; Tumor Cells, Cultured

The cytokine transforming growth factor (TGF)-beta, by virtue of its immunosuppressive and promigratory properties, has become a major target for the experimental treatment of human malignant gliomas. Here we characterize the effects of a novel TGF-beta receptor (TGF-betaR) I kinase inhibitor, SD-208, on the growth and immunogenicity of murine SMA-560 and human LN-308 glioma cells in vitro and the growth of and immune response to intracranial SMA-560 gliomas in syngeneic VM/Dk mice in vivo. SD-208 inhibits the growth inhibition of TGF-beta-sensitive CCL64 cells mediated by recombinant TGF-beta1 or TGF-beta2 or of TGF-beta-containing glioma cell supernatant at an EC(50) of 0.1 mumol/L. SD-208 blocks autocrine and paracrine TGF-beta signaling in glioma cells as detected by the phosphorylation of Smad2 or TGF-beta reporter assays and strongly inhibits constitutive and TGF-beta-evoked migration and invasion, but not viability or proliferation. Peripheral blood lymphocytes or purified T cells, cocultured with TGF-beta-releasing LN-308 glioma cells in the presence of SD-208, exhibit enhanced lytic activity against LN-308 targets. The release of interferon gamma and tumor necrosis factor alpha by these immune effector cells is enhanced by SD-208, whereas the release of interleukin 10 is reduced. SD-208 restores the lytic activity of polyclonal natural killer cells against glioma cells in the presence of recombinant TGF-beta or of TGF-beta-containing glioma cell supernatant. The oral bioavailability of SD-208 was verified by demonstrating the inhibition of TGF-beta-induced Smad phosphorylation in spleen and brain. Systemic SD-208 treatment initiated 3 days after the implantation of SMA-560 cells into the brains of syngeneic VM/Dk mice prolongs their median survival from 18.6 to 25.1 days. Histologic analysis revealed no difference in blood vessel formation, proliferation, or apoptosis. However, animals responding to SD-208 showed an increased tumor infiltration by natural killer cells, CD8 T cells, and macrophages. These data define TGF-beta receptor I kinase inhibitors such as SD-208 as promising novel agents for the treatment of human malignant glioma and other conditions associated with pathological TGF-beta activity.

Topics: Activin Receptors, Type I; Animals; Antineoplastic Agents; Cell Division; Cell Line, Tumor; Cell Movement; Glioma; Humans; Male; Mice; Mice, Inbred BALB C; Neoplasm Invasiveness; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; Signal Transduction; Transforming Growth Factor beta; Transforming Growth Factor beta1; Transforming Growth Factor beta2

Melanoma inhibitory activity (MIA) is related to disease progression in patients with malignant melanoma and to invasion and metastasis of melanoma in vivo and in vitro. An alternative splice product termed MIA(splice) was described recently. In addition to melanoma, both proteins are expressed in a substantial subset of high-grade gliomas. We hypothesize that expression levels of both proteins correlate with early tumor progression and parameters of disseminated disease in patients with high-grade glioma. We examined the correlation of expression levels of MIA and MIA(splice) with time to progression and morphological and clinical markers of disseminated disease (defined as multifocal occurrence, gliomatosis, invasion or metastasis) in a series of 24 newly-diagnosed human high-grade gliomas. Homogenates of surgical specimens, cell cultures and blood samples were analyzed. Significant levels of MIA and MIA(splice) protein were detected in 71% of homogenates of high-grade glioma, but not in the related blood samples. Patients with early tumor progression had lower expression levels of MIA than patients with late progression, and the expression level of MIA was inversely related to time to progression. In addition, MIA expression correlated with a high fiber content of the extracellular matrix, suggesting a role in dissemination as known from malignant melanoma. Expression levels of MIA in homogenates of surgical specimen directly relate to a more benign clinical prognosis in patients with high-grade glioma. While a mechanistic relation has not yet been verified, factors such as a high fiber content of the extracellular matrix may explain this observation.

Topics: Adult; Aged; Alternative Splicing; Biomarkers, Tumor; Blotting, Western; Brain Neoplasms; Cells, Cultured; Child; Disease Progression; Enzyme-Linked Immunosorbent Assay; Extracellular Matrix Proteins; Glioma; Humans; Immunohistochemistry; Middle Aged; Neoplasm Proteins; Prognosis; Proteins; Reverse Transcriptase Polymerase Chain Reaction; Tenascin; Time Factors; Transforming Growth Factor beta; Transforming Growth Factor beta1

Migration and invasion are prerequisites for the neoplastic phenotype of malignant glioma. Ectopic expression of BCL-2 enhances migration and invasion of glioma cells and promotes their synthesis of transforming growth factor-beta2 (TGF-beta2). We here report that BCL-2-expressing cells show enhanced expression and activity of the proprotein convertase, furin, which processes metalloproteinases (MMP) and TGF-beta. Consistent with a biological role for a BCL-2-dependent increase in furin-like protease (FLP) activity, BCL-2-expressing cells exhibit enhanced MMP activity. Both a pseudosubstrate furin inhibitor, decanoyl-Arg-Val-Lys-Arg-chloromethylketone (dec-RVKR-cmk), or alpha 1-anti-trypsin Portland (PDX), a recombinant furin-inhibitory protein, suppress constitutive and BCL-2-mediated MMP activity and invasion. This inhibition is not overcome by TGF-beta or hepatocyte growth factor (HGF). A neutralizing TGF-beta antibody attenuates, but not abrogates, the invasive properties conferred by exogenous expression of BCL-2, whereas the MMP inhibitor o-phenantroline (o-PA) abolishes the pro-invasive action of BCL-2. Exogenous HGF results in enhanced, and expression of dominant-negative ezrin in reduced, FLP activity, and dec-RVKR-cmk blunts the HGF-induced expression of mature TGF-beta2. Consequently, HGF and BCL-2 family proteins use a furin-dependent pathway to promote invasion via TGF-beta and MMP in human malignant glioma cells and the pro-invasive properties of TGF-beta require furin- dependent MMP activity.

Topics: Cell Line, Tumor; Furin; Glioma; Hepatocyte Growth Factor; Humans; Matrix Metalloproteinases; Neoplasm Invasiveness; Peptide Hydrolases; Proto-Oncogene Proteins c-bcl-2; RNA, Messenger; Transforming Growth Factor beta

The study gives a further biochemical description of two different forms of autosomal dominant osteopetrosis (ADO) in relation to murine counterparts, with special attention to osteoblast function and the recent discovery of LRP5 gene mutations in ADO I. Patients and controls were investigated for markers of bone formation and resorption at baseline and following stimulation with thyroid hormone. Moreover, four different well-described murine models of osteopetrosis were investigated. Concerning the human forms, serum TSH levels decreased in all subjects, indicating effects on the target tissue. Osteocalcin and cross-linked collagen (NTx) were without significant differences among the groups. Significant increases in both markers were seen following stimulation. Baseline active TGF-beta1 levels were increased in both types of ADO (60% in ADO I [P = 0.006]; 46% in ADO II [P = 0.001], respectively), whereas fibronectin levels were decreased in both (ADO I 58% and ADO II 63% of normal, respectively [P = 0.012 and P = 0.001]). Following treatment, levels increased temporarily in all groups. In the murine models, active TGF-beta1 was significantly decreased in the tl- and ia-rat, whereas fibronectin levels were decreased in the mi-mouse, however, increased in the ia-rat. In conclusion, both types of ADO showed the same qualitative biochemical differences compared to controls, except that OPG levels were higher in ADO I. The decreased fibronectin levels in both types and in murine models reflect decreased bone resorption; however, this may also indicate hitherto unrecognized alterations in bone formation. Biochemical differences among known syndromes related to mutations in the LRP5 gene indicate different underlying pathogenetic mechanisms.

Topics: Adult; Animals; Bone and Bones; Collagen; Disease Models, Animal; Female; Fibronectins; Genes, Dominant; Glioma; Hemoglobin M; Humans; LDL-Receptor Related Proteins; Low Density Lipoprotein Receptor-Related Protein-5; Male; Mice; Middle Aged; Mutation; Osteocalcin; Osteopetrosis; Rats; Receptors, LDL; Syndrome; Thyroid Hormones; Time Factors; Transforming Growth Factor beta

We have demonstrated that secreted protein acidic and rich in cysteine (SPARC) is highly expressed in human gliomas and it promotes glioma invasion and delays tumor growth in vitro and in vivo. cDNA array analyses were performed to determine whether SPARC, which interacts at the cell surface, has an impact on intracellular signaling and downstream gene expression changes, which might account for some of its effects on invasion and growth. Using a doxycycline (dox)-controlled gene expression system, two cDNA array analyses were performed using a parental U87T2 clone (-SPARC) transfected with the dox-controlled transactivator and a U87T2 parental-derived SPARC-transfected clone, A2b2 (+SPARC). Array analysis performed between the parental and the SPARC-transfected clone (-dox) identified 13 upregulated genes and 14 downregulated genes. With the exception of PAI-1 and MMP2, the identified genes are novel with respect to SPARC's mechanism of action. Array analysis performed using the SPARC-transfected clone ( +/- dox) identified 2 types of gene regulation; one reversible upon SPARC suppression, the other irreversible. Two of the SPARC-induced genes, BIGH3 (irreversible by dox) and PAI-1 (reversible by dox) were further studied in additional SPARC-transfected clones, human astrocytoma tissues, and human glioma cell lines by RT-PCR and Northern blot analyses. The results indicate that: (1) the array results were validated, (2) the dox regulation was validated, and (3) the differential expression identified by the array analyses was present between normal brain and in human astrocytoma tissues and cell lines. Therefore, we conclude that these cDNA array analyses provide candidate genes involved in SPARC-mediated effects on glioma cell cycle progression, signaling, and migration, and that SPARC may induce reversible and irreversible gene expression changes. Further investigation of these candidates may shed insights into SPARC's role in glioma cell proliferation and invasion, and potential use as a therapeutic target.

Topics: Anti-Bacterial Agents; Astrocytes; Astrocytoma; Blotting, Northern; Brain; Cell Transformation, Neoplastic; Doxycycline; Extracellular Matrix Proteins; Fetus; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Glioma; Humans; Neoplasm Invasiveness; Neoplasm Proteins; Oligonucleotide Array Sequence Analysis; Osteonectin; Reverse Transcriptase Polymerase Chain Reaction; RNA-Binding Proteins; Transfection; Transforming Growth Factor beta; Tumor Cells, Cultured

The formation of new microvasculature by capillary sprouting, or angiogenesis, is a prerequisite for solid tumor growth. The genetic alterations required to activate the angiogenic program in tumor angiogenesis are still only vaguely known, but dominantly acting oncoproteins may have a much greater impact than previously realized. Here we have studied the consequences of oncogenic transformation on tumor angiogenesis in a mouse mammary carcinoma model. We provide evidence that the expression of vascular endothelial growth factor (VEGF) and of the VEGF receptor-2 (Flk-1), a signaling system centrally involved in tumor angiogenesis, occurs efficiently in tumors formed by Ras-transformed mammary epithelial cells and that both TGF-beta1 and hypoxia are potent inducers of VEGF expression in these cells. VEGF induction in the tumor periphery is mainly triggered by TGF-beta1, whereas VEGF expression in perinecrotic areas is regulated by both hypoxia and TGF-beta1. As the Ras-transformed tumor cells convert into migrating, fibroblastoid cells that start to produce TGF-beta during tumor progression, the TGF-beta effect on VEGF expression becomes propagated throughout the tumor tissue. Thus, in progressed tumors, areas of TGF-beta1 activation and hypoxia may overlap and hence cooperate to induce VEGF expression and angiogenesis. Nevertheless, the overexpression of VEGF in non-Ras-transformed mouse mammary epithelial cells was not sufficient to promote vascularization in vivo. Based on these findings, we conclude that amongst the multiple mutations that render a normal cell tumorigenic, oncogenic Ras is a major player that in conjunction with the tumor's micro-environment sets the stage for tumor cell invasion and angiogenesis.

Topics: Animals; Cell Movement; Cell Transformation, Neoplastic; Cells, Cultured; Endothelial Growth Factors; Endothelium, Vascular; Epithelial Cells; Fibroblasts; Glioma; Humans; Hypoxia; Immunoblotting; Immunoenzyme Techniques; In Situ Hybridization; Lymphokines; Mammary Neoplasms, Experimental; Mice; Mice, Inbred BALB C; Neoplasm Invasiveness; Neovascularization, Pathologic; Oncogene Protein p21(ras); Rats; Receptor Protein-Tyrosine Kinases; Receptors, Growth Factor; Receptors, Vascular Endothelial Growth Factor; RNA, Messenger; Transforming Growth Factor beta; Umbilical Veins; Up-Regulation; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

The aim of the current study was to determine whether basic fibroblast growth factor (bFGF), regulates the release of transforming growth factor-beta1 (TGF-beta1) from C6 glioma cells. The results of the study show that bFGF (2, 5 and 10 ng/ml) dose dependently induced the release of TGF-beta1 from C6 glioma cells, with the 10 ng/ml dose inducing a 2- to 3-fold increase of TGF-beta1 levels. This effect was evident as early as 6 h following treatment, with maximal levels observed at 18 h. The effect of bFGF was largely on latent TGF-beta1, and was isoform specific, as bFGF had no effect on TGF-beta2 release. The bFGF effect on TGF-beta1 was also glioma specific, as no such stimulatory effect was observed in rat cortical astrocytes.

Topics: Animals; Astrocytes; Cerebral Cortex; Dose-Response Relationship, Drug; Fibroblast Growth Factor 2; Glioma; Protein Isoforms; Rats; Time Factors; Transforming Growth Factor beta; Tumor Cells, Cultured

To investigate the effects of recombinant human bone morphogenic protein-2 (rhBMP-2) and hyaluronic acid (HA) on the invasion and metastasis of rat brain glioma in vivo.. C6 rat glioma cells were transfected with the plasmid vector named pEGFP-N3 which contained an enhanced green fluorescent protein (EGFP) gene. The stable cell clones which expressed EGFP protein were stereotactically injected into the brain parenchyma of SD rats in order to establish a xenotransplanted tumor model. After rhBMP-2 and HA were administered in the xenotransplanted tumor model at different dosages, their effects on glioma invasion and metastasis were observed by pathology, flow cytometry fluoresclnec, and electronmicvoscopy.. EGFP-transfected C6 glioma cells gave off green fluorescence in vivo and in vitro. Area with tumor and area without tumor could be easily distinguished by fluorescence microscopy. Even distant micrometastasis and invasion at single-cell level could be detected. 10 microliter rhBMP-2 of the concentration of 5 microgram/ml inhibited C6 tumor invasion, while 10 microliter hyaluronic acid of the concentration of 100 microgram/ml evidently promoted the tumor invasion.. EGFP-transfected C6 cells can be transplanted into rats' brain so as to establish an excellent experimental animal model in the research on invasion and metastasis of brain glioma in vivo. RhBMP-2 inhibits tumor invasion, while HA evidently promotes its invasion.

Topics: Animals; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Brain Neoplasms; Disease Models, Animal; Female; Glioma; Green Fluorescent Proteins; Humans; Hyaluronic Acid; Luminescent Proteins; Male; Neoplasm Invasiveness; Neoplasm Transplantation; Rats; Rats, Sprague-Dawley; Recombinant Proteins; Transfection; Transforming Growth Factor beta

Processes like cell proliferation, differentiation, and tumor metastasis require a flexible adaptation of cell shape and cell plasticity. A regulator of cell structure and shape is the centrosome and its associated microtubules. Recently, oncogenes like p53, pRB, and the tumor suppressor BRCA1 have been characterized as members of the centrosome. In this communication, we identified rat Nm23-R1/NDPKbeta, a homologue of the human tumor metastasis suppressor Nm23-H1 and a regulator of cell proliferation and differentiation, as a component of the centrosomal complex. We used confocal laser scanning microscopy on different cell types and biochemical analysis of purified centrosomes to demonstrate that Nm23-R1 is located in the centrosome of dividing and nondividing cells. We also showed that the centrosomal enzyme is catalytically active and able to transfer the gamma-phosphate from a nucleoside triphosphate to a nucleoside diphosphate. In addition, Nm23-R1 coimmunoprecipitated with gamma-tubulin, a core centrosomal protein essential for microtubule nucleation. In addition, human Nm23-R1/-H1 was also shown to be present in the centrosome of different human and rat cell types, demonstrating that the presence of Nm23-H1 homologues in the latter organelle is a general event.

Topics: Adenosine Triphosphate; Adrenergic beta-Agonists; Animals; Cell Differentiation; Cell Division; Cells, Cultured; Centrosome; Glioma; Guanosine Triphosphate; Immunohistochemistry; Isoenzymes; Microtubules; Monomeric GTP-Binding Proteins; Neoplasm Metastasis; NM23 Nucleoside Diphosphate Kinases; Nucleoside-Diphosphate Kinase; Precipitin Tests; Rats; Recombinant Proteins; Transcription Factors; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tubulin

Ezrin belongs to the ezrin-radixin-moesin family proteins, which cross-link actin cytoskeleton and plasma membrane. Malignant glioma cells are paradigmatic for their strong migratory and invasive properties. Here, we report that the expression of dominant-negative ezrins inhibits clonogenicity, migration, and invasiveness of human malignant glioma cells. Furthermore, dominant-negative ezrins block hepatocyte growth factor (HGF)-mediated stimulation of clonogenicity and migration, without altering HGF-induced protein kinase B/Akt and focal adhesion kinase phosphorylation. Glioma cells expressing dominant-negative ezrins exhibit a shift of the BCL-2/BAX rheostat toward apoptosis, reduced alpha(V)beta(3) integrin expression and reduced matrix metalloproteinase (MMP) expression and activity. These changes are associated with a dramatic loss of transforming growth factor beta(2) (TGF-beta(2)) release. Exogenous supplementation of TGF-beta(2) overcomes the inhibitory effects of dominant-negative ezrins on migration and clonogenicity. A neutralizing TGF-beta(2) antibody mimics the effects of dominant-negative ezrins on clonogenicity and migration. Exogenous HGF markedly induces TGF-beta(2) protein levels, and a neutralizing TGF-beta(2) antibody abolishes the HGF-mediated increase in glioma cell motility. Finally, TGF-beta(2) does not modulate BCL-2 or BAX expression, but BCL-2 gene transfer increases the levels of latent and active TGF-beta(2). Intracranial xenografts of U87MG glioma cells transfected with the dominant-negative ezrins in athymic mice grow to significantly smaller volumes, and the median survival of these mice is 50 d compared with 28 d in the control group. These data define a novel pathway for HGF-induced glioma cell migration and invasion, which requires ezrin, changes in the BCL-2/BAX rheostat, and the induction of TGF-beta(2) expression in vitro, and underscore the important role of HGF signaling in vivo.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Cell Division; Cell Movement; Cytoskeletal Proteins; Disease Models, Animal; Genes, Dominant; Glioma; Hepatocyte Growth Factor; Humans; Mice; Mice, Nude; Neoplasm Invasiveness; Phosphoproteins; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Rats; Receptors, Vitronectin; Survival Rate; Transfection; Transforming Growth Factor beta; Transforming Growth Factor beta2; Tumor Cells, Cultured; Tumor Stem Cell Assay; Xenograft Model Antitumor Assays

Extensive infiltration of normal brain tissue and suppression of anti-tumor immune surveillance mediated by molecules such as transforming growth factor-beta (TGF-beta) are key biological features that contribute to the malignant phenotype of human gliomas. Tranilast (N-[3,4-dimethoxycinnamoyl]-anthranilic acid) is an anti-allergic compound used clinically to control atopic and fibrotic disorders. These effects are attributed to the suppression of TGF-beta1 synthesis and interference with growth factor-mediated proliferation and migration of fibroblasts and vascular smooth muscle cells. Here, we show that tranilast inhibits DNA synthesis and proliferation of human malignant glioma cells and promotes p21 accumulation in the absence of cytotoxicity. Further, tranilast reduces the release of TGF-beta1 and TGF-beta2 by glioma cells and inhibits migration, chemotactic responses and invasiveness. These effects are not associated with a reduction of alpha(v)beta(3) integrin expression at the cell surface but appear to involve inhibition of matrix metalloproteinase-2 expression and activity. Neither the tranilast-mediated inhibition of proliferation nor the inhibition of migration was counteracted by supplementation with exogenous TGF-beta. Finally, tranilast administered orally inhibited the growth of experimental 9L rat gliomas and reduced expression of TGF-beta2 in vivo. We conclude that tranilast might be a useful therapeutic agent for the treatment of human malignant glioma because of a TGF-beta-independent abrogation of the malignant phenotype of proliferation, migration and invasiveness and because of the antagonism of TGF-beta-associated immunosuppression.

Topics: 3T3 Cells; Adenocarcinoma; Animals; Brain Neoplasms; Cell Division; Chemotaxis; Female; Glioma; Histamine H1 Antagonists; Humans; Kinetics; Matrix Metalloproteinase 2; Mice; Neoplasm Invasiveness; Neuroblastoma; ortho-Aminobenzoates; Ovarian Neoplasms; Platelet Aggregation Inhibitors; Receptors, Vitronectin; Transforming Growth Factor beta; Tumor Cells, Cultured

Malignant glioma cells secrete transforming growth factor-beta (TGF-beta) and can activate latent TGF-beta. However, the mechanism of the latent TGF-beta activation has not yet been determined. This study examined whether thrombospondin-1 (TSP-1) secreted by malignant glioma cell lines participates in the activation of latent TGF-beta secreted by the glioma cells. Western blot analysis revealed that TSP-1 was present in both the cell lysates and the culture supernatants of all three malignant glioma cell lines (T98G, A172, and U251). A bioassay for TGF-beta activity revealed that all malignant glioma cell lines used in this study could activate latent TGF-beta by themselves. Latent TGF-beta 1 activation, evaluated by enzyme-linked immunosorbent assay, was inhibited by more than 50% by the addition of neutralizing anti-TSP-1 monoclonal antibody or anti-TSP-1 polyclonal antibody. These results indicate that TSP-1 has a predominant role in the activation of latent TGF-beta in malignant glioma cells.

Topics: Biomarkers, Tumor; Brain Neoplasms; Glioma; Humans; RNA, Messenger; Thrombospondin 1; Transforming Growth Factor beta; Tumor Cells, Cultured

In this study, the effect of thyroid hormone (triiodothyronine, T(3)) on the secretion of mitogenic growth factors in astrocytes and C6 glioma cells was examined. The proliferating activity of T(3) could be due, at least in part, to the astrocyte secretion of acidic and basic fibroblast growth factor (aFGF and bFGF), tumor necrosis factor-beta, and transforming growth factor-beta. In contrast, the conditioned medium (CM) of T(3)-treated C6 cells was mitogenic to this cell line only after hyaluronidase digestion, suggesting the impairment of growth factor mitogenic activity by hyaluronic acid. Furthermore, the presence of bFGF was significantly greater in the CM of both T(3)-treated astrocytes and T(3)-treated C6 cells than in the corresponding control CM. These data show that T(3) induces cerebellar astrocytes to secrete mitogenic growth factors, predominantly bFGF, that could influence astrocyte and neuronal proliferation via autocrine and paracrine pathways.

Topics: Animals; Astrocytes; Cell Division; Cells, Cultured; Cerebellum; Culture Media, Conditioned; Fibroblast Growth Factor 1; Fibroblast Growth Factor 2; Glioma; Growth Substances; Heparin; Hyaluronic Acid; Hyaluronoglucosaminidase; Lymphotoxin-alpha; Rats; Rats, Wistar; Transforming Growth Factor beta; Triiodothyronine; Tumor Cells, Cultured

Matrix metalloproteinases (MMPs) are a growing family of zinc-dependent endopeptidases that are capable of degrading various components of the extracellular matrix. These enzymes have been implicated in a variety of physiological and pathological conditions including embryogenesis and tumour invasion. The synthesis of many MMPs is thought to be regulated by growth factors, cytokines and hormones. In this study, we investigated the effects of five exogenous growth factors known to be expressed by gliomas [epidermal growth factor (EGF), basic growth factor (bFGF), transforming growth factor beta (TGF-beta1,2) and vascular endothelial growth factor (VEGF)].on MMP-2 and MMP-9 expression in an ependymoma, two grade III astrocytomas, a grade III oligoastrocytoma and a benign meningioma. Zymogram analysis revealed that the effects of the growth factors depended upon the cell lines used in the study. Growth factors generally up-regulated MMP-2 and MMP-9 expression in the gliomas but were least effective in the meningioma; the effect being most prominent with TGF-beta1 and TGF-beta2 in all the cell lines. It is hypothesized that paracrine growth factor interplay may be crucial in the regulation of MMP expression by glioma invasion of the normal brain.

Topics: Brain Neoplasms; Endothelial Growth Factors; Epidermal Growth Factor; Fibroblast Growth Factor 2; Glioma; Growth Substances; Humans; Lymphokines; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Meningeal Neoplasms; Meningioma; Neoplasm Proteins; Transforming Growth Factor alpha; Transforming Growth Factor beta; Tumor Cells, Cultured; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

The migratory behaviour of malignant gliomas relies on the interaction of integrins with extracellular matrix (ECM) components. Transforming growth factor-beta(1) (TGF-beta(1)) potently stimulates glioma cell motility whereas TGF-beta(2) is known for its immunosuppressive properties. Here, we show that both TGF-beta(1) and TGF-beta(2) promote migration of glioma cells. In parallel, TGF-beta(1) and TGF-beta(2) induce alpha(V) and beta(3) intergrin mRNA expression and enhance cell surface expression of alpha(V)beta(3) integrin. TGF-beta-mediated promotion of migration is abrogated by echistatin, a Arg-Gly-Asp (RGD) peptide antagonist of alpha(V)beta(3) integrin, and by a neutralizing anti-alpha(V)beta(3) integrin antibody. Taken together, we report a novel mechanism by which TGF-beta modulates cell ECM interactions and promotes glioma cell motility.

Topics: Cell Movement; Gene Expression Regulation; Glioma; Humans; Integrins; Intercellular Signaling Peptides and Proteins; Peptides; RNA, Messenger; Transcriptional Activation; Transforming Growth Factor beta; Tumor Cells, Cultured; Up-Regulation

We studied the changes of tumor size after gene therapy treatment and its relationship with the changes of vascular volume as measured by dynamic contrast-enhanced magnetic resonance imaging (MRI), to investigate whether the vascular changes is predictive of tumor regression. The study was carried out using a spontaneously regressing rat tumor model (C6 Glioma grown subcutaneously in rats). Three rats were treated with recombinant adenoviruses expressing three genes, mouse interleukin 1-alpha (IL1-alpha), mouse interferon gamma (IFN-gamma), and human transforming growth factor beta (TGF-beta), one from each kind. Two rats were treated with saline as controls. Longitudinal studies were performed to monitor the changes of tumor volume (based on T(2)-weighted images) and the vascular volume (based on dynamic contrast enhanced images). In untreated animals, tumor regression was preceded by several days with a decrease in vascular volume. When the tumor growth was perturbed by expression of mouse IL-1alpha, the increase in vascular volume was correlated with the continuing growth in size, and the decrease in vascular volume was predictive of the onset of tumor regression. As new advances in immunotherapy in cancer treatment emerge, the ability to determine the efficacy of therapy as early as possible will enable optimization of treatment regiments. The vascularity changes measured by dynamic MRI may provide a means to serve for this purpose.

Topics: Adenoviridae; Animals; Gadolinium DTPA; Genetic Therapy; Genetic Vectors; Glioma; Humans; Image Enhancement; Interferon-gamma; Interleukin-1; Liver; Longitudinal Studies; Magnetic Resonance Angiography; Mice; Microcirculation; Neoplasm Regression, Spontaneous; Neoplasm Transplantation; Neovascularization, Pathologic; Nonlinear Dynamics; Rats; Rats, Wistar; Recombinant Fusion Proteins; Recombinant Proteins; Remission Induction; Transforming Growth Factor beta; Tumor Cells, Cultured

Human gliomas express TGF-beta but, so far the expression of downstream mediators has been investigated in only a few cell lines. We have examined tissue specimens of 23 gliomas: 3 astrocytomas grade II (AST), 8 anaplastic astrocytomas grade III (AAST), and 12 glioblastoma multiforme grade IV (GBM). We analyzed the mRNA expression of TGF-beta1, TGF-beta2, TGF-beta3, the TGF-beta receptors type I (TbetaR-I) and type II (TbetaR-II), Smad2, Smad3, and Smad4. mRNA expression of IL-10 and CD95 (FAS/APO-1) were also studied. We detected increased mRNA levels of the 3 TGF-beta isoforms, correlating with the degree of malignancy. TGF-beta3 mRNA was increased, particularly in AST and AAST, while TGF-beta1 and TGF-beta2 mRNAs were strongly expressed in GBM. TGF-beta normally up-regulates the TGF-beta receptors, and TbetaR-I and TbetaR-II showed stronger expression in all gliomas when compared to normal tissues. However, the mRNA expression of Smad2, Smad3, and Smad4 was decreased in GBM. IL-10 mRNA expression was detected in glioma tissues but not in glioma cell lines. No marked increase in the expression of soluble CD95 splicing variants was found in the gliomas compared with normal tissue. However, total CD95 mRNA was elevated among GBM tissues.

Topics: Activin Receptors, Type I; Adolescent; Adult; Astrocytoma; Brain; Brain Neoplasms; DNA-Binding Proteins; DNA, Complementary; Enzyme-Linked Immunosorbent Assay; fas Receptor; Female; Glioblastoma; Glioma; Humans; Interleukin-10; Male; Middle Aged; Protein Isoforms; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type I; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Smad2 Protein; Smad3 Protein; Smad4 Protein; Trans-Activators; Transforming Growth Factor beta; Tumor Cells, Cultured

The authors previously demonstrated the presence of cells in primary human malignant gliomas that intrinsically are resistant to carmustine (BCNU). Numerous studies have identified mechanisms of therapy resistance in these cells; however, the authors' work and that of others suggest that additional mechanisms of resistance exist.. The authors identified a glioma cell line that lacks detectable methylguanine methyltransferase expression and does not alter its expression of glutathione-S-transferase-pi in response to BCNU chemotherapy. This cell line was used in mRNA differential display experiments to identify genes involved in what to the authors' knowledge were previously undescribed mechanisms of resistance.. The overexpression of the gene encoding the transforming growth factor latency binding protein was demonstrated in glioma cells selected for resistance to BCNU, compared with their parental unselected cells.. Transforming growth factor-beta1 has pleiotropic functions in transformed and normal cells. Although activation of TGF-beta1 does not appear to be a causative factor in BCNU resistance in the current study, it may be involved in the growth of these resistant cells.

Topics: Aged; Antineoplastic Agents, Alkylating; Blotting, Northern; Blotting, Southern; Carmustine; Carrier Proteins; Cell Division; Cell Survival; Drug Resistance, Neoplasm; Female; Gene Dosage; Gene Expression Regulation, Neoplastic; Glioma; Humans; Intracellular Signaling Peptides and Proteins; Latent TGF-beta Binding Proteins; Oligonucleotide Array Sequence Analysis; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transforming Growth Factor beta; Tumor Cells, Cultured

The expression of thrombospondin-1 (TSP-1) and its role in gliomas have not been well examined. In the present study TSP-1 expression in a panel of malignant glioma cell lines and the expression of TSP-1 and transforming growth factor (TGF-beta) proteins in low-grade and malignant glioma tissues were investigated. Reverse transcription-polymerase chain reaction analysis showed that nine of nine malignant glioma cell lines expressed TSP-1 mRNA, and seven of nine glioma lines expressed TSP-2 mRNA. Production and secretion of TSP-1 were examined in the T98G glioblastoma cell line by western blot analysis. Total TSP-1 protein content in the supernatant was 10 times higher than that in the cell lysate. Secretion of TSP-1 was examined in these glioma cell lines by western blot analysis. All glioma lines secreted significant levels of TSP-1. Bioassay showed that all tumor lines had the capacity to activate latent TGF-beta. Localization of TSP-1, TGF-beta1, -beta2, and -beta3 was examined immunohistochemically in surgically resected glioma tissues, including 11 glioblastomas, six anaplastic astrocytomas, and eight astrocytomas. Most glioblastomas expressed high levels of both TSP-1 and TGF-beta. Anaplastic astrocytomas expressed moderate levels of TSP-1 and TGF-beta. Most malignant gliomas expressed various levels of TGF-beta1, -beta2, and -beta3. The expression of both proteins, however, was weak in low-grade gliomas. Normal brain tissues around the tumors were negatively or very weakly positively stained for TSP-1 and TGF-beta. These results indicate that most malignant glioma cells express TSP-1 in vitro and in vivo, and the expression of TSP-1 and TGF-beta in vivo correlates with the histologic malignancy of glioma. Overexpression of both TSP-1 and TGF-beta may increase the biologic malignancy of malignant gliomas, through generating the active form of TGF-beta in tumor tissues.

Topics: Astrocytoma; Biomarkers, Tumor; Diagnosis, Differential; Glioblastoma; Glioma; Humans; RNA, Messenger; Thrombospondin 1; Thrombospondins; Transforming Growth Factor beta; Tumor Cells, Cultured

To investigate the effects of recombinant human bone morphogenetic protein (rhBMP-2) on the biological characteristics of human glioma cells (SHG44) in vivo and in vitro.. The growth curve of SHG44 cells was ploted with or without rhBMP-2. The proliferation quantity of SHG44 cells with rhBMP-2 was determined by MTT method. The cell cycle, ultrastructure and DNA fragments of SHG44 cells were detected respectively by flow cytometry (FCM), electron microscope and agarose gel electrophoresis. Tumor growth state of SHG44 gliomas, which grew under the skin of nude mice, was observed with local injection of rhBMP-2.. FCM showed that rhBMP-2 inhibited the growth of SHG44 cell in vitro and its effect depended on dosage. DNA content of SHG44 cells in G1 phase was obviously increased (5 mg/L group, 77.4%; control group, 55.2%, P < 0.05) and the content of its S phase was reduced (5 mg/L group, 15.2%; control group, 33.3%, P < 0.01). The apoptosis spike (12.3%) occurred in the 5 mg/L group. The nucleolus chromatin condensation, bound of aggregation and caryorrhexis were observed under the electron microscope and the apoptotic cells appeared. DNA electrophoresis showed a ladder strap (DNA ladder). SHG44 glioma growth was relatively slower and its volume was smaller with the biquadratic local injection of rhBMP-2 (tumor end volume in average: experimental group 894 mm(3) +/- 145 mm(3), control group 5 163 mm(3) +/- 1 028 mm(3), P < 0.01. rhBMP-2 can suppress the proliferation of SHG44 gliomas and induce its apoptosisN:

Topics: Animals; Apoptosis; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Cell Cycle; DNA Fragmentation; Dose-Response Relationship, Drug; Glioma; Humans; Mice; Mice, Nude; Microscopy, Electron; Neoplasm Transplantation; Recombinant Proteins; Transforming Growth Factor beta; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

The neuropeptide substance P (SP), by stimulating tachykinin NK1 receptors (NK1R), triggers a number of biological responses in human glioma cells which are potentially relevant for tumour growth. First, radioligand binding studies demonstrated the presence of tachykinin NK1R on SNB-19, DBTRG-05 MG and U373 MG, but not on U138 MG and MOG-G-GCM human glioma cell lines. Second, application of SP or neurokinin A (NKA) to NK1R+ glioma cell lines increased the secretion of interleukin 6 (IL-6) and potentiated IL-6 secretion induced by IL-1beta. SP also up-regulated the release of transforming growth factor beta1 (TGF-beta1) by the U373 MG glioma cell line. Third, SP induced new DNA synthesis and enhanced the proliferation rate of NK1R+, but not of NK1R- glioma cell lines. Also, NKA stimulated the proliferation and cytokine secretion in NK1R+ glioma cell lines. All the stimulant effects of SP/NKA on NK1R+ glioma cell lines were completely blocked by a specific tachykinin NK1R antagonist, MEN 11467. These data support the potential use of tachykinin NK1R antagonist for controlling the proliferative rate of human gliomas.

Topics: Cell Division; DNA, Neoplasm; Glioma; Humans; Interleukin-10; Interleukin-6; Neoplasm Proteins; Neurokinin A; Neurokinin-1 Receptor Antagonists; Receptors, Neurokinin-1; Substance P; Transforming Growth Factor beta; Tumor Cells, Cultured

Transforming growth factor-beta1 (TGF-beta1) is widely recognized for its multiple roles in development, cellular maintenance, and protection against injury. In the brain, TGF-beta1 upregulation in microglia/macrophages is a predominant response to lesion and during pathology. However, the precise functions of TGF-beta1 in this context are still enigmatic. The present study investigates changes in astroglial gene expression as a major target of TGF-beta1 signaling in the brain. Differential display reverse transcription-polymerase chain reaction (DDRT-PCR) was used to identify several gene fragments differentially regulated by TGF-beta1 in rat astrocytes and C6 glioma cells. Among the cDNAs regulated by TGF-beta1 in C6 cells two cDNAs showed homology to alpha-tropomyosin and glycerol-3-phosphate dehydrogenase, respectively. Cloning of a full length cDNA corresponding to a differentially regulated gene fragment revealed close homology to latent TGF-beta binding protein (LTBP)-2. Data using antisense LTBP-2 oligonucleotides to decrease LTBP-2 expression suggest that LTBP-2 functions to activate TGF-beta. Therefore, it is likely that upregulation of the rat LTBP-2 homolog mRNA in C6 cells and cortical astrocytes by TGF-1 might lead to self-activation and exaggeration of TGF-beta signaling. These data will extend our current understanding of TGF-beta1 functioning on lesion-related features of glial cells.

Topics: Animals; Animals, Newborn; Astrocytes; Carrier Proteins; Cells, Cultured; Cloning, Molecular; DNA, Complementary; Gene Expression Regulation; Glioma; Latent TGF-beta Binding Proteins; Neuroglia; Rats; Rats, Wistar; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sequence Analysis, DNA; Transforming Growth Factor beta; Tumor Cells, Cultured; Up-Regulation

A panel of 6 human glioma cell lines was examined for TGF-beta1 responsiveness. U-178 MG and U-251 MG AgCl1 were significantly inhibited by TGF-beta1, while U-343 MGa 31L and U-343 MGa 35L were potently stimulated to proliferate. TGF-beta1 induced endogenous PAI-1 protein synthesis, Smad binding element/(CAGA)12-luciferase-reporter activity, as well as mRNA expression of Smad6 and Smad7 in all gliomas. Interestingly, TGF-beta1 differentially stimulated or inhibited the expression of TbetaR-I and TbetaR-II mRNA in the gliomas. Affinity cross-linking studies using 125I-TGF-beta1 revealed that the gliomas expressed TGF-beta-type-I(TbetaR-I) and -type-II(TbetaR-II) receptors, although binding to TbetaR-II in U-343 MGa 31L and U-251 MG AgCl1 was low to undetectable. Smad2 protein was abundantly present in U-178 MG, U-343 MG, and U-343 MGa 35L, while Smad3 was readily detectable in U-178 MG, U-343 MG, U-343 MGa 35L and U-251 MG AgCl1. In all gliomas, TGF-beta1 induced phosphorylation of Smad2. The level to which TGF-beta1 could activate the pathway leading to induction of the (CAGA)12-luciferase reporter seemed to correlate to the expression levels of TGF-beta receptors, Smad3 and Smad4 proteins. However, despite the plethora of data regarding TGF-beta1 signalling in the different glioma cell lines, the mechanism underlying the differential growth effects mediated by TGF-beta1 is still unclear. The results suggest that a complex balance between several components in the TGF-beta signalling pathway controls glioma responsiveness to TGF-beta1, and extend reports indicating that distinct signal transduction pathways are involved in growth inhibition and other cellular responses.

Topics: DNA-Binding Proteins; DNA, Neoplasm; Genes, Reporter; Glioblastoma; Glioma; Humans; Luciferases; Receptors, Transforming Growth Factor beta; Recombinant Fusion Proteins; Smad2 Protein; Smad3 Protein; Smad4 Protein; Trans-Activators; Transcription, Genetic; Transfection; Transforming Growth Factor beta; Tumor Cells, Cultured

Ectopic expression of the proteoglycan, decorin, abrogates the growth of experimental C6 gliomas in the rat. Since gliomas release large amounts of transforming growth factor-beta (TGF-beta) and since decorin is a TGF-beta antagonist, decorin gene transfer-mediated abrogation of glioma growth in vivo may involve enhanced immunogenicity of the tumor cells. Here, we report that human glioma cells stimulate alloreactive immune responses when engineered to express decorin whereas parental glioma cells are non-immunogenic in vitro. The alloreactive immune response is mediated by CD8+ and CD4+ T cells as well as by NK cells. The immunosuppression exerted by parental or mock-transfected glioma cells is mediated by soluble factors and can in part be mimicked by exogenous TGF-beta. However, neutralizing anti-TGF-beta antibodies do not reverse glioma-mediated immunosuppression, suggesting that decorin abrogates glioma-induced immune cell inhibition by interfering with the activity of other, so far unidentified glioma-secreted mediators. We conclude that enhanced immunogenicity may mediate the antineoplastic effects of decorin gene therapy for malignant glioma but that factors other than TGF-beta may be responsible for glioma-induced immunosuppression.

Topics: Adenoviridae; Animals; Cell Line; Cytotoxicity, Immunologic; Decorin; Extracellular Matrix Proteins; Gene Expression Regulation; Gene Transfer Techniques; Genetic Engineering; Genetic Vectors; Glioma; Humans; Immune Tolerance; Immunophenotyping; Leukocytes, Mononuclear; Mice; Mink; Proteoglycans; Transfection; Transforming Growth Factor beta; Tumor Cells, Cultured

Cytokines have roles in tumor biology and induce neurological manifestations. Cytokines produced in response to a brain tumor may generate neurological manifestations via paracrine action. We investigated cytokine modulation in an in vivo brain tumor model with behavioral, morphological, and molecular approaches. Rat C6 glioma cells were implanted into the third cerebral ventricle of Wistar rats, their behavior was monitored, and the development of an intracranial tumor of astrocytic origin was confirmed by histology and positive immunostaining for vimentin, S-100 protein, and glial fibrillary acidic protein. Sensitive and specific RNase protection assays were used to analyze cytokine messenger RNA (mRNA) in brain regions from anorexic brain tumor-bearing animals. Brain tumor formation was associated with significant increased levels of interleukin (IL)-1beta, IL-1 receptor antagonist, IL-1 receptor type I, tumor necrosis factor (TNF)-alpha, and transforming growth factor (TGF)-beta1 mRNAs in the cerebellum, hippocampus, and hypothalamus. IL-1 receptor accessory proteins I and II mRNAs were increased in the cerebellum and hypothalamus. We also examined hypothalamic feeding-associated components: neuropeptide Y and proopiomelanocortin mRNAs were down-regulated, glycoprotein 130 mRNA levels were up-regulated, and leptin receptor (OB-R) mRNA levels were unchanged. These dissimilar profiles of mRNA expression suggest specificity of brain tumor-induced transcriptional changes. The data implicate cytokines as important factors in brain tumor-host interactions in vivo. The data also show that the C6 cell-induced glioma can be used as a behavioral-molecular model to study cytokine and neuropeptide modulation and action during the host biochemical and physiological responses to brain tumor development. Paracrine interactions seem pivotal because cytokine modulation was observed in various brain regions. These results also suggest that cytokine and neuropeptide changes during brain tumor progression are involved in brain tumor-associated neurological and neuropsychiatrical manifestations.

Topics: Animals; Brain Neoplasms; Central Nervous System; Cytokines; Glioma; Immunohistochemistry; Interleukin 1 Receptor Antagonist Protein; Interleukin-1; Male; Neuropeptides; Rats; Rats, Wistar; Receptors, Interleukin-1; RNA, Messenger; Sialoglycoproteins; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Human glioma cell line, Onda 10 produces TGF-beta1. TGF-beta1 has a biological role for the immunosuppression of the host. We have investigated whether suppression of TGF-beta1 on human glioma cell enhanced the susceptibility to lymphokine-activated killer (LAK) cells. In vitro, susceptibility to LAK cells on Onda 10 cell is augmented by retroviral gene transfection with antisense TGF-beta1. Nude mice bearing Onda 10 cells transduced with antisense TGF-beta1 gene has a longer life span compared to mice carrying that of sense TGF-beta1 gene or vector alone. The cytotoxic activity of LAK cells induced from spleen cells of mice carrying antisense TGF-beta1 gene transduced cells is higher against Onda 10 cell than that of LAK cells from mice carrying vector alone transduced cells. Also, antisense TGF-beta1 gene transduced cells are much more sensitive to LAK cells compared to Onda 10. These suggest that the augmented host systemic immunity in mice is one of the mechanisms of the reduced tumorigenicity of antisense TGF-beta1 gene transduced cells and that the increased systemic immunity could be ascribed to the increased immunogenicity of the tumor cells. The gene therapy for malignant glioma with antisense TGF-beta1 gene is expected to be promising.

Topics: Animals; Antisense Elements (Genetics); Cell Division; Cloning, Molecular; Cytotoxicity, Immunologic; Glioma; Humans; Immunosuppression Therapy; Killer Cells, Lymphokine-Activated; Mice; Mice, Nude; Retroviridae; Transduction, Genetic; Transfection; Transforming Growth Factor beta; Tumor Cells, Cultured

Decorin gene therapy for experimental malignant glioma is thought to involve antagonism of immunosuppression induced by glioma-derived transforming growth factor-beta (TGF-beta). TGF-beta is chemotactic for cells of the monocyte macrophage lineage but inhibits their functional activity in many in vitro paradigms. Here, we examined changes in the patterns of microglial infiltration of rat C6 gliomas expressing a decorin transgene. We find that the number of OX42/ED-1-positive microglial cells is reduced rather than enhanced in the presence of decorin. Decorin-expressing gliomas contain lower numbers of MHC class II antigen-expressing microglial cells whereas the relative frequency of MHC I immunoreactivity among microglial cells is increased. Interestingly, the reduction of TGF-beta levels in the tumors by decorin is associated with the de novo expression of inducible nitric oxide synthase (iNOS) in a minority of microglial cells. These data suggest that microglial cells do not participate in the regression of decorin-expressing rat C6 gliomas.

Topics: Animals; Brain Neoplasms; Corpus Striatum; Decorin; Extracellular Matrix Proteins; Female; Glioma; Humans; Immunoenzyme Techniques; Microglia; Neoplasm Invasiveness; Neoplasm Transplantation; Nerve Tissue Proteins; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Proteoglycans; Rats; Rats, Sprague-Dawley; Recombinant Fusion Proteins; Transfection; Transforming Growth Factor beta; Tumor Cells, Cultured

We sought to characterize the pathway by which the multifunctional cytokine transforming growth factor-beta (TGF-beta) inhibits the proliferation of normal astrocytes, and we analyzed the alterations in the TGF-beta pathway in human glioma cell lines. Upon TGF-beta treatment, primary rat astrocytes showed a significant decrease in DNA synthesis upon thymidine incorporation with a cell cycle arrest in the G(1) phase. Western analysis of the astrocytes revealed that the expression of the cyclin-dependent kinase inhibitor (CdkI) p15(INK4B) was significantly up-regulated upon TGF-beta treatment without a change in other CdkI levels. The retinoblastoma protein (Rb) became hypophosphorylated, and Cdk2 activity decreased. Analysis of Smad3 null mouse astrocytes showed a significant loss of both TGF-beta-mediated growth inhibition and p15(INK4B) induction compared with wild-type mouse astrocytes. Infection of rat astrocytes by SMAD3 and SMAD4 adenoviruses failed to induce increased expression of p15(INK4B), implying indirect transcriptional regulation of p15(INK4B) by SMAD3. High-grade human gliomas secrete TGF-beta, yet are resistant to its growth inhibitory effects. Analysis of the effects of TGF-beta on 12 human glioma cell lines showed that TGF-beta mildly inhibited the growth of six lines, had no effect on four lines, and stimulated the growth of two lines. The majority of glioma lines had homozygous deletions of the p15(INK4B) gene, except for two lines that expressed p15(INK4B) protein, which was induced further upon TGF-beta treatment. Three lines mildly induced CdkI p21(WAF1) expression in response to TGF-beta. Most tumor lines retained other TGF-beta-mediated responses, including extracellular matrix protein and angiogenic factor secretion, which may contribute to increased malignant behavior. This suggests that the loss of p15(INK4B) may explain, in part, the selective loss of growth inhibition by TGF-beta in gliomas to form a more aggressive tumor phenotype.

Topics: Animals; Astrocytes; Blotting, Southern; Blotting, Western; Carrier Proteins; CDC2-CDC28 Kinases; Cell Cycle Proteins; Cell Division; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase Inhibitor p15; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinases; Cyclins; DNA-Binding Proteins; Flow Cytometry; G1 Phase; Glioma; Humans; Mice; Protein Kinases; Protein Serine-Threonine Kinases; Rats; Signal Transduction; Smad3 Protein; Trans-Activators; Transforming Growth Factor beta; Tumor Cells, Cultured; Tumor Suppressor Proteins

It has been hypothesized that transforming growth factor beta (TGF-beta) may prevent immune-mediated glioma cell elimination; however, previous work has also indicated that increased TGF-beta may lead to reduced proliferation, induction of apoptosis, and enhancement of Fas-induced apoptosis. We have investigated the role of TGF-beta in the progression of malignant glioma using an immunocompetent murine model. SMA 560 malignant glioma cells were stably transfected with constructs that resulted in over- or underproduction of active TGF-beta1. Using these cell lines, we have shown that (a) TGF-beta1 inhibits induction of antitumor cytotoxicity when the tumor cells are given s.c. but not when they are given intracranially; (b) Fas/APO-1 is expressed on SMA 560 cells in vitro and in vivo, SMA 560 cells are susceptible to TGF-beta1- and Fas-induced apoptosis in vitro, and TGF-beta1 and Fas act synergistically to induce glioma cell death; (c) increased levels of endogenous TGF-beta1 production by SMA 560 cells lead to increased sensitivity to Fas-mediated apoptosis; (d) overproduction of endogenous TGF-beta1 reduces the rate of s.c. SMA 560 tumor growth and also reduces the tumorigenicity of tumors located in the central nervous system, with opposite effects observed with underproduction of TGF-beta1 using antisense cell lines; and (e) the observed changes in growth parameters in vivo were associated with increased rates of apoptosis in TGF-beta1-overproducing cells. Taken together, these results indicate that, despite decreased induction of CTL responses, the dominant net effect of TGF-beta1 on the growth of the SMA 560 murine high-grade glioma in vivo is growth inhibition. This contrasts with results seen with non-central nervous system malignant tumors in immunocompetent animals, in which TGF-beta1 production provides a major growth advantage.

Topics: Animals; Apoptosis; Brain Neoplasms; Cytotoxicity, Immunologic; Disease Models, Animal; DNA Fragmentation; DNA, Neoplasm; Enzyme-Linked Immunosorbent Assay; Fas Ligand Protein; fas Receptor; Genetic Vectors; Glial Fibrillary Acidic Protein; Glioma; Male; Membrane Glycoproteins; Mice; Neoplasms, Experimental; Transfection; Transforming Growth Factor beta; Tumor Cells, Cultured

The aim of this study was to investigate the mechanism by which malignant glioma cells escape from growth inhibition mediated by transforming growth factor-beta (TGF-beta), a ubiquitous cytokine that inhibits cell proliferation by causing growth arrest in the G1 phase of the cell cycle.. The authors measured the response of eight malignant glioma cell lines to the growth-inhibiting activity of TGF-beta in vitro and the expression of TGF-beta Types I and II receptors in malignant glioma cells. The effect of TGF-beta on the expression of a p27Kip1 cyclin-dependent kinase inhibitor was also investigated to assess the downstream signal transmission from TGF-beta receptors. All malignant glioma cell lines were insensitive to growth inhibition by TGF-beta1 and TGF-beta2. Analyses of TGF-beta receptors by means of affinity labeling in which 125I-TGF-beta1 was used showed that six glioma lines had both TGF-beta Types I and II receptors on their cell surfaces, whereas two lines had very small amounts of TGF-beta Type I and/or Type II receptors. Northern blot analysis showed that all tumor lines expressed variable levels of messenger RNAs for both TGF-beta Types I and II receptors. Flow cytometric analyses revealed that treatment of malignant glioma cells with TGF-beta1 significantly downregulated the expression of p27Kip1 protein in all malignant glioma cell lines except one.. The authors suggest that most malignant glioma cells express TGF-beta Types I and II receptors, which can transmit some signals downstream and that the loss of response to TGF-beta growth inhibition may not be caused by an abnormality of the TGF-beta receptors.

Topics: Adenocarcinoma; Affinity Labels; Blotting, Northern; Cell Cycle Proteins; Cell Division; Cell Line; Cyclin-Dependent Kinase Inhibitor p27; Cyclin-Dependent Kinases; Down-Regulation; Drug Resistance, Neoplasm; Enzyme Inhibitors; Epithelial Cells; Fibroblasts; Flow Cytometry; G1 Phase; Gene Expression Regulation, Neoplastic; Genes, Tumor Suppressor; Glioma; Growth Inhibitors; Humans; Iodine Radioisotopes; Lung; Microtubule-Associated Proteins; Radiopharmaceuticals; Receptors, Transforming Growth Factor beta; RNA, Messenger; Signal Transduction; Skin; Transforming Growth Factor beta; Tumor Cells, Cultured; Tumor Suppressor Proteins

p53 immunoreactivity and humoral immune response to p53 were examined in 14 patients with malignant glioma, including 4 patients with leptomeningeal glioma cell dissemination. Twelve patients expressed p53 protein within the tumour tissue. p53 antibodies were detected in the serum in 2 of 14 patients but never in the cerebrospinal fluid (CSF). Soluble p53 protein was detected neither in serum nor in CSF of the glioma patients. CSF levels of the immunosuppressive cytokine, transforming growth factor (TGF)-beta, were elevated in the glioma patients, including those with a humoral response to p53. These preliminary findings raise the possibility of systemic humoral immune responses to antigens, including mutant p53, expressed by glioma cells in the central nervous system.

Topics: Adult; Aged; Autoantibodies; Brain Neoplasms; Female; Glioma; Humans; Male; Middle Aged; Transforming Growth Factor beta; Tumor Suppressor Protein p53

To assess immunohistochemically whether the neural cell adhesion molecule L1, which is a member of the immunoglobulin superfamily and has been shown recently to be a stimulating factor for glioma migration, is expressed in glioma tissues, and to investigate factors that can regulate this expression.. Twenty seven glioma tissue specimens including 13 glioblastomas, seven anaplastic astrocytomas, and seven astrocytomas were examined. Immunohistochemical analyses of L1, p53, and transforming growth cell factor beta (TGF-beta) were performed on each tumour using both polyclonal and monoclonal antibodies.. Nine (33%) specimens (six glioblastomas and three anaplastic astrocytomas) had L1 positive immunostaining. p53 positive staining was detected in 10 (43%) of 23 glioma specimens (seven glioblastomas and three anaplastic astrocytomas). TGF-beta positive immunostaining was observed in 12 (52%) of the 23 glioma specimens (six glioblastomas, four anaplastic astrocytomas, and two astrocytomas). There was a statistical correlation between both p53 and L1 expression and TGF-beta and L1 expression. No such correlation was found between p53 and TGF-beta expression.. These results suggest that mutation of the p53 gene or expression of TGF-beta may upregulate the expression of the L1 gene, thus resulting in high grade migration of glioma cells.

Topics: Antigens, Neoplasm; Astrocytoma; Glioblastoma; Glioma; Humans; Immunoenzyme Techniques; Leukocyte L1 Antigen Complex; Neoplasm Proteins; Neural Cell Adhesion Molecules; Transforming Growth Factor beta; Tumor Suppressor Protein p53

We have previously reported that local secretion of either TNF-alpha or TGF beta1 by intracerebral SMA-560 malignant glioma tumor cells can reduce or eliminate tumor growth in mice. However, the use of TNF-alpha, while improving the overall survival of tumor bearing animals, was associated with early toxic deaths due to cerebral edema. In the present study, we demonstrate that TNF-alpha induces apoptosis of the SMA 560 cell line, as does TGF beta1, and that these two cytokines act in an additive fashion to enhance apoptosis and thus, to inhibit SMA 560 cell growth in vitro. Next, we show that the production of TGF beta1 when added to TNF-alpha production by central nervous system tumors in vivo abrogates any early deaths seen due to TNF-alpha toxicity and leads to a larger percentage of animals surviving CNS tumor challenge. Finally, we demonstrate that the production of TGF beta1 by tumor cells is associated with the abolition of tumor-associated cerebral edema in both TNF-alpha and in non-TNF-alpha producing tumors. These results are important for the development of effective and less toxic therapies for brain tumors, as well as for examining the pathogenesis of tumor-related cerebral edema.

Topics: Animals; Apoptosis; Brain Edema; Brain Neoplasms; Carcinogenicity Tests; Cell Division; Disease Models, Animal; Gene Transfer Techniques; Glioma; Lac Operon; Mice; Mice, Inbred Strains; Retroviridae; Retroviridae Infections; Survival Analysis; Transforming Growth Factor beta; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

Angiopoietin-2 (Ang2) is a ligand for the endothelial cell tyrosine kinase receptor Tie2 and counteracts blood vessel maturation/stability mediated by angiopoietin-1 (Ang1), the other known ligand of Tie2. Using degenerate oligonucleotides and reverse transcriptase-polymerase chain reaction, we have screened bovine microvascular endothelial (BME), aortic, lymphatic, pulmonary artery, and transformed fetal aortic endothelial cells, as well as rat smooth muscle cells for Ang1 and Ang2 expression. Except for high Ang2 mRNA levels found in BME cells, none of the endothelial cell types studied expressed appreciable levels of Ang1 or Ang2 mRNAs, whereas smooth muscle cells expressed both Ang1 and Ang2. BME cell Ang2 mRNA levels were increased by vascular endothelial growth factor (1.9- to 2.9-fold), basic fibroblast growth factor (1.6- to 2-fold), both cytokines in combination (2.9- to 4-fold), and hypoxia (3.1- to 5.6-fold) and were decreased by Ang1 (31% to 70%) or transforming growth factor-ss1 (64% to 81%). Ang2 also decreased (60% to 82%) BME cell Ang2 mRNA. mRNA levels for the Tie1 or Tie2 receptors were only slightly modulated under the conditions described above. These findings suggest that the angiogenic effect of a number of regulators may be achieved in part through the regulation of an autocrine loop of Ang2 activity in microvascular endothelial cells.

Topics: Adrenal Cortex; Amino Acid Sequence; Angiopoietin-1; Angiopoietin-2; Animals; Aorta; Base Sequence; Cattle; Cell Hypoxia; Cells, Cultured; Cytokines; Endothelial Growth Factors; Endothelium, Vascular; Enzyme Inhibitors; Fibroblast Growth Factor 2; Gene Expression Regulation, Enzymologic; Glioma; Humans; Lymphokines; Membrane Glycoproteins; Molecular Sequence Data; Neovascularization, Physiologic; Proteins; Rats; Receptor Protein-Tyrosine Kinases; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transcription, Genetic; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

We previously reported that schwannoma-derived growth factor (SDGF), a member of heparin-binding epidermal growth factor (EGF) family, participates in autocrine pathways and promotes rat glioma cell growth. To investigate the potential role of similar molecules in human gliomas, we examined 7 human glioma cell lines and 11 glioblastoma specimens for expression of the human homologue of SDGF, amphiregulin (AR), as well as heparin-binding EGF-like growth factor (HB-EGF). Northern blot analysis revealed that only one cell line and no tumor specimens expressed AR mRNA. In contrast, HB-EGF mRNA was expressed in all human glioma cell lines and its level of expression was two- to five-fold higher than that of control brain tissues in 8 of 11 glioblastoma cases. Immunohistochemistry demonstrated that membrane-anchored HB-EGF (proHB-EGF) and EGFR were co-expressed in 44% of 34 human malignant gliomas. Introduction of exogenous HB-EGF (10 ng/ml) increased human glioma cell proliferation, and anti-HB-EGF blocking antibodies reduced the growth of glioma cells by 30-40%, confirming the presence of an autocrine loop. When added to the medium, transforming growth factor-alpha, basic fibroblast growth factor, or HB-EGF rapidly induced HB-EGF mRNA expression. These results indicate that HB-EGF and proHB-EGF contribute to the growth of human malignant glioma cells, most likely through autocrine and juxtacrine mechanisms.

Topics: Amphiregulin; Cell Division; EGF Family of Proteins; Epidermal Growth Factor; ErbB Receptors; Glioblastoma; Glioma; Glycoproteins; Growth Substances; Heparin-binding EGF-like Growth Factor; Humans; Intercellular Signaling Peptides and Proteins; Mitosis; Recombinant Proteins; RNA, Messenger; Signal Transduction; Transforming Growth Factor alpha; Transforming Growth Factor beta; Tumor Cells, Cultured

Nitric oxide (NO) is thought to play an important role in neurotransmission, inflammation, and regulation of cell death in the mammalian brain. Here, we examined the synthesis and biological effects of NO in human malignant glioma cells. Exposure to cytokines such as interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha or interleukin (IL)-1beta and lipopolysaccharide (LPS) induced NO synthesis in rat C6 and A172 human glioma cells, but not in LN-229, T98G or LN-18 human malignant glioma cells. Induced release of NO involved enhanced expression of inducible NO synthase (iNOS). Failure to detect NO release in the latter cell lines was not overcome by neutralization of endogenous TGF-beta or by coexposure to cytokines, LPS, and antioxidants. Apoptosis induced by CD95 ligand (CD95L) did not involve NO formation. Neither NOS inhibitors nor NO donators modulated CD95L-induced apoptosis. Dexamethasone (DEX)-mediated protection of glioma cells from CD95L-induced apoptosis was also independent of DEX effects on NO metabolism. DEX inhibited not only cytokine/LPS-evoked NO release but also attenuated the toxicity of NO in three of five cell lines. Forced expression of temperature-sensitive p53 val135 in C6 cells in either mutant or wild-type conformation inhibited cytokine/LPS-induced NO synthesis. Further, accumulation of p53 in both mutant or wild-type conformation protected glioma cells from the toxicity of exogenous NO, consistent with a gain of p53 function associated with p53 accumulation. We conclude that resistance to NO-dependent immune defense mechanisms may contribute to the malignant progression of human cancers with p53 alterations, notably those associated with the accumulation of mutant p53 protein.

Topics: Animals; Antineoplastic Agents, Hormonal; Apoptosis; Cycloheximide; Cytokines; Dexamethasone; fas Receptor; Genes, p53; Glioma; Humans; Interleukin-1; Lipopolysaccharides; Neoplasm Proteins; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Protein Synthesis Inhibitors; Rats; Transforming Growth Factor alpha; Transforming Growth Factor beta; Tumor Cells, Cultured; Tumor Suppressor Protein p53

The effect of treatment with interleukin-1 beta (IL-1 beta), interferon-gamma (IFN-gamma), vincristine, and etoposide was evaluated on the secretion of transforming growth factor-beta (TGF-beta) and IL-10 and the expression of major histocompatibility complex (MHC) class I, intercellular adhesion molecule-1 (ICAM-1), and CD80 molecules by malignant glioma cells. Five malignant glioma cell lines were treated with IL-1 beta, IFN-gamma, and/or anticancer agents (vincristine and etoposide). Combined treatment with IL-1 beta and IFN-gamma caused greater inhibition of TGF-beta secretion compared to treatment with IFN-gamma, and almost the same levels of inhibition as treatment with vincristine and etoposide. The greatest inhibition of TGF-beta secretion was achieved by treatment with all agents. Low levels of IL-10 secretion were determined in two out of five malignant glioma cell lines. This IL-10 secretion was inhibited by treatment with IL-1 beta, IFN-gamma, vincristine, and/or etoposide. Treatment with both cytokines and anticancer agents increased the expression of MHC class I and ICAM-1 in all tumor cell lines. The mean increase of expression of MHC class I was 50% and that of ICAM-1 was 12-fold. No tumor cell lines expressed CD80 molecules on the cell surface, and no treatment caused CD80 expression. These results suggest that TGF-beta and IL-10 secretion by malignant glioma cells can be suppressed by treatment with a combination of IL-1 beta, IFN-gamma, vincristine, and etoposide, and the treatment up-regulates MHC class I and ICAM-1 expression on tumor cells. These results have implications for immunotherapy and chemotherapy in patients with malignant tumors.

Topics: Antigens, Neoplasm; Antineoplastic Agents; B7-1 Antigen; Cytokines; Down-Regulation; Etoposide; Flow Cytometry; Glioma; Histocompatibility Antigens Class I; Humans; Intercellular Adhesion Molecule-1; Interferon-gamma; Interleukin-1; Interleukin-10; Transforming Growth Factor beta; Tumor Cells, Cultured; Vincristine

Using an intracranial rat C6 glioma model, we tested the hypothesis that gene modification of glioma cells to block the expression of the immunosuppressive cytokine TGF-beta (transforming growth factor beta) may enhance anti-tumor immune responses and thereby prolong survival of tumor-bearing animals. The cDNA for simian TGF-beta 2 was ligated in antisense orientation into the episomal plasmid mammalian expression vector pCEP-4. This TGF-beta-antisense vector was transfected into C6 glioma cells by standard electroporation techniques. PCR was used to determine that the rat C6 clones were successfully transfected with the antisense-TGF beta construct. Twenty-nine adult female Wistar rats harboring 7-day-old intracranial C6 tumors were then subcutaneously injected with either saline (n = 9), unmodified C6 glioma cells (n = 10), or TGF-beta-antisense-modified C6 cells (n = 10). Animals were followed for survival, and Fisher's exact method was used to interpret the significance of difference between experimental groups. The survival of tumor-bearing rats injected with TGF-beta-antisense-modified C6 cells was significantly prolonged, relative to the survival of rats receiving injections of saline or unmodified C6 cells alone. Six of the ten (60%) TGF-beta-antisense treated animals survived for 12 weeks, whereas none of the nine (0%) animals treated with saline and none of ten (0%) of those treated with C6 cells alone survived past 5 weeks. These results indicate that the genetic inhibition of immunosuppressive cytokines (such as TGF-beta) may reverse the phenotypic immunosuppression caused by such factors, and thereby prolong the survival of C6 tumor-bearing animals. Future investigations using cytokine gene modifications in other brain tumor models are warranted.

Topics: Animals; Antisense Elements (Genetics); Brain; Brain Neoplasms; Female; Genetic Therapy; Glioma; Necrosis; Phenotype; Polymerase Chain Reaction; Rats; Rats, Wistar; Survival Analysis; Transfection; Transforming Growth Factor beta; Treatment Outcome

Cytokines such as transforming growth factor-beta (TGF-beta) are thought to mediate escape from immune surveillance in human malignant glioma. Here, we report that ectopic expression of the small TGF-beta-binding proteoglycan, decorin, inhibits not only TGF-beta bioactivity but also TGF-beta 1 and TGF-beta 2 mRNA transcription and TGF-beta protein synthesis by human LN-18, LN-229, T98G and rat C6 glioma cells in vitro. Ectopic expression of decorin in C6 rat glioma cells results in strong inhibition of tumor formation in vivo. Decorin-expressing C6 gliomas grow initially but regress to very small residual tumors at 12 weeks after implantation whereas all control animals die or have to be killed within 4 weeks. Decorin-expressing tumors show a four-fold increase of infiltration by activated T cells and a 1.6-fold increase in total B and T cells. Chronic steroid-mediated immunosuppression abrogates the inhibitory effects of decorin gene transfer. We conclude that decorin-induced inhibition of TGF-beta release by glioma cells significantly enhances antiglioma immune responses in vivo. Clinical evaluation of decorin gene therapy for human malignant gliomas may be warranted.

Topics: Animals; Anti-Inflammatory Agents; B-Lymphocytes; Blotting, Northern; Decorin; Dexamethasone; Enzyme-Linked Immunosorbent Assay; Extracellular Matrix Proteins; Female; Gene Expression; Gene Transfer Techniques; Genetic Therapy; Glioma; Humans; Immunoblotting; Lymphocyte Activation; Neoplasm Transplantation; Proteoglycans; Rats; Rats, Sprague-Dawley; T-Lymphocytes; Transforming Growth Factor beta; Tumor Cells, Cultured

Transforming growth factor beta-2 (TGF-B2) is secreted by glioma cells and is known to decrease leukocyte-endothelium interaction. TGF-B2 alone and in conjunction with soluble tumor necrosis factor (TNF) p55 receptor, was found to decrease the expression of TNF induced VCAM-1 on the malignant glioma cell line A-172 and human cerebral microvessel endothelial (CNS-EC) cells. Co-culture of A-172 glioma cells led to a decrease in VCAM-1 expression; this effect on CNS-EC in co-culture could be simulated by glioma supernatant alone. These results suggest that malignant gliomas, by secreting TGF-B2 and releasing soluble TNF receptors, modulate adhesion molecules.

Topics: Antigens, CD; Cell Line; Cerebrovascular Circulation; Coculture Techniques; Endothelium, Vascular; Glioma; Humans; Receptors, Tumor Necrosis Factor; Receptors, Tumor Necrosis Factor, Type I; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha; Vascular Cell Adhesion Molecule-1

Because the prominent neovascularization characteristic of high grade primary brain tumors is composed mostly of vascular smooth muscle cells (VSMC), we studied the expression of the potent smooth muscle mitogen endothelin-1 (ET-1) and one of its secretagogues, transforming growth factor beta 1 (TGF-beta 1) in a series of astrocytic tumors. TGF-beta 1 is also of interest due to its known activity as an angiogenic factor. Using immunohistochemical methods, we examined 30 surgical cases: 10 glioblastoma multiforme, 10 anaplastic astrocytomas, and 10 low-grade astrocytomas. Using a monoclonal antibody to TGF-beta 1 and a polyclonal antibody to ET-1, we detected both growth factors in all cases of glioblastoma examined. In cases of anaplastic astrocytoma, 4 tumors were positive for both factors; 2 contained only ET-1; 2 contained only TGF-beta 1; and 2 exhibited no tumor cell immunoreactivity for either factor. In low-grade astrocytoma, 4 of 10 tumors showed weak ET-1 immunoreactivity; 2 of those contained TGF-beta 1 immunopositive tumor astrocytes: 6 tumors were negative for both factors. In all tumors that expressed both factors, serial sections showed that regions of ET-1 immunopositivity also tended to be positive for TGF-beta 1. Endothelial cells within all tumors were positive for ET-1. ET-1 and TGF-beta 1 are present in human astrocytomas and their expression correlates with tumor vascularity and malignancy. These results suggest roles for both ET-1 and TGF-beta 1 in the growth and progressive angiogenesis of the human glioma.

Topics: Blood Vessels; Brain Neoplasms; Endothelin-1; Glioma; Humans; Immunohistochemistry; Staining and Labeling; Transforming Growth Factor beta

Glioblastoma cells secrete transforming growth factor-beta (TGF-beta), which has a variety of immunosuppressive properties. We investigated the effect of irradiation TGF-beta secretion by malignant glioma cells. Three malignant glioma cell lines (T98G, A172, KG-1-C) were cultured and irradiated using 10 and 50 Gy Linac radiation. After further culture for 36 hours in serum-free culture medium, the supernatants were collected. The TGF-beta activity in the culture supernatants was determined using a specific bioassay. The levels of the active form and total TGF-beta in the supernatants from irradiated malignant glioma cells decreased compared to those from un-irradiated cells. However, since irradiation inhibited the growth of tumor cells, the amount of TGF-beta secretion per cell in irradiated cells tended to increase after irradiation. These results suggest that malignant glioma cells can still secrete TGF-beta and activate latent TGF-beta even after large dose irradiation, despite the inhibition of tumor growth.

Topics: Animals; Culture Media, Serum-Free; Glioblastoma; Glioma; Humans; Immune Tolerance; Transforming Growth Factor beta; Tumor Cells, Cultured

Long-term control of high-grade brain tumors is rarely achieved with current therapeutic regimens. The aim of this study was to determine if low doses of tumor necrosis factor-alpha (TNF-alpha) could augment the effects of radiation in a glioma xenograft model and to evaluate hematological and other parameters that might indicate treatment-related toxicity. Nude mice were injected subcutaneously with C6 rat glioma cells and randomized into groups. Two different time-dose protocols were employed using intravenous human recombinant TNF-alpha and radiation beginning within 24 h after tumor cell implantation. The administration of radiation as a single agent slowed tumor progression, whereas TNF-alpha alone had no effect. However, TNF-alpha, especially when given twice per week before radiation for a total of four doses each, significantly increased the efficacy of the radiation. Low leukocyte counts were associated with combination treatment, whereas transforming growth factor-beta 1 levels were depressed in all treated groups. TNF-alpha did not modulate radiation-induced inhibition of C6 cell proliferation in vitro. The data show that TNF-alpha at relatively nontoxic doses can significantly enhance the antitumor effects of radiation against a rapidly growing glioma. This effect was more than additive, because TNF-alpha alone did not slow tumor progression.

Topics: Animals; Body Weight; Brain Neoplasms; DNA Replication; DNA, Neoplasm; Glioma; Humans; Leukocyte Count; Mice; Mice, Nude; Neoplasm Transplantation; Radiation-Sensitizing Agents; Rats; Recombinant Proteins; Spleen; Transforming Growth Factor beta; Transplantation, Heterologous; Tumor Necrosis Factor-alpha

Transforming growth factors-beta 1 and -beta 2 (TGF-beta 1 and -beta 2) are important growth-regulatory proteins for astroglial neoplasms. We analyzed their role in tumor-cell proliferation in 12 glioma cell lines, employing phosphorothioate antisense oligodeoxynucleotides (S-ODNs, 14 mer), specifically targeted against the coding sequences of TGF-beta 1-mRNA and TGF-beta 2-mRNA. TGF-beta 1-S-ODNs inhibited cell proliferation in 5 of 12 gliomas, whereas TGF-beta 2-S-ODNs reduced the cell proliferation in all glioma cell lines, compared to nonsense-S-ODN-treated and S-ODN-untreated cells as controls. The efficacy and specificity of antisense effects was validated by Northern-blot analysis and determination of protein concentrations in culture supernatants (ELISA). Exogenous hrTGF-beta 1 either stimulated or inhibited the cell lines, whereas pnTGF-beta 2 stimulated the proliferation of most glioma cells. Blocking the extracellular pathway of TGF-beta by neutralizing antibodies only slightly inhibited those cell lines, which were markedly stimulated by TGF-betas. As the effects of TGF-beta 2-S-ODNs were much stronger than those of TGF-beta neutralizing antibodies, we postulate that the endogenously produced TGF-beta 2 control glioma-cell proliferation, in part by an intracellular loop.

Topics: Base Sequence; Cell Division; Glioma; Humans; Molecular Sequence Data; Oligonucleotides, Antisense; Thionucleotides; Transforming Growth Factor beta; Tumor Cells, Cultured

Vascular endothelial growth factor (VEGF) is an angiogenic factor which is known to be expressed in several malignancies including glioma. The effect of transforming growth factor-beta (TGF-beta) isoforms as well as gangliosides on VEGF production was investigated in human glioma cell lines. TGF-beta isoforms and gangliosides were found to differentially stimulate VEGF production by these cells. The ganglioside GD3 enhanced this release to the greatest extent and the stimulation was more marked in a glioblastoma cell line than in the two other anaplastic astrocytoma cell lines. These results suggest that both TGF-betas and gangliosides may act as indirect angiogenic factors by stimulating VEGF secretion.

Topics: Endothelial Growth Factors; Gangliosides; Glioma; Humans; Isomerism; Lymphokines; Stimulation, Chemical; Transforming Growth Factor beta; Tumor Cells, Cultured; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

The distribution of bone morphogenetic protein (BMP) type I receptors and the activin type I receptor (ActR-I) was investigated in 16 cases of human glioma and five cases of non-tumourous gliosis tissue by immunohistochemical technique. Both BMP type IA (BMPR-IA) and the type IB (BMPR-IB) receptors were detected in human glioma cells. A significant increase in BMPR-IB in tumour cells was observed in malignant glioma compared with both low-grade astrocytomas (n=16, P<0.005) and gliosis (n=13, P<0.001). However, enhancement of BMPR-IA staining was moderate and ActR-I staining was only weakly expressed in the malignant glioma tumours. Osteogenic protein (OP)-1/BMP-7, which is known to bind BMPR-IA, BMPR-IB and ActR-I, was expressed in nervous tissue and was also detected in anaplastic areas of malignant glioma. In contrast to the tissue materials, BMPR-IA was expressed to a stronger degree than BMPR-IB in human glioma cell lines; the growth of these cells was suppressed by OP-1. These results suggest the presence of BMP receptors and a functional role for BMPs in malignant glioma.

Topics: Activin Receptors; Animals; Bone Morphogenetic Protein 7; Bone Morphogenetic Protein Receptors; Bone Morphogenetic Protein Receptors, Type I; Bone Morphogenetic Proteins; Glioma; Humans; Immunohistochemistry; Proteins; Rabbits; Receptors, Cell Surface; Receptors, Growth Factor; Transforming Growth Factor beta; Tumor Cells, Cultured

Topics: Animals; Brain Neoplasms; Clinical Trials as Topic; Disease Models, Animal; Genetic Therapy; Glioma; Humans; Rats; Transforming Growth Factor beta

The efficacy of intrathecal treatment of leptomeningeal metastasis (LM) with interleukin-2 (IL-2) was evaluated in an animal model using Wistar rats inoculated intracisternally with 10(7) C6 glioma cells. Prior to the in vivo experiments the antiproliferative effects of human IL-2, and of murine IFN-gamma and TNF-alpha which are cytokines induced by IL-2 were tested in a colony forming assay. Only IFN-gamma caused a dose-dependent inhibition of colony formation. Twelve animals were treated intracisternally with either 10(5) IU IL-2 or control medium on day 0, 2, and 5 after tumor cell inoculation. Both IL-2 treated and sham-treated animals developed LM with a symptom-free survival of 7 to 9 days. There was no significant difference between treated and untreated animals regarding time to onset of symptoms and pattern of tumor growth. Infiltration of the tumor tissue with ED-1+ monocytes and macrophages, and CD8+ lymphocytes, however, was slightly increased in IL-2 treated animals. In a second experiment 4 non tumor-bearing Wistar rats were intracisternally injected with a single dose of 10(5) IU IL-2. These animals also showed slightly enhanced leptomeningeal infiltration with CD8+ lymphocytes compared to controls. We conclude that intrathecal application of high-dose IL-2 although eliciting a slight immune reaction within the leptomeninges does not inhibit leptomeningeal tumor growth or prolong symptom-free survival in our animal model of LM. These results raise doubt about the clinical efficacy of intrathecal IL-2 treatment in patients with LM.

Topics: Animals; Disease Models, Animal; Evaluation Studies as Topic; Glioma; Injections, Spinal; Interleukin-2; Meningeal Neoplasms; Neoplasm Transplantation; Rats; Rats, Wistar; Transforming Growth Factor beta; Transplantation, Isogeneic; Tumor Stem Cell Assay

Malignant glioma cells secrete transforming growth factor-beta (TGF-beta) which has potent immunosuppressive properties. We investigated the effect of interleukin-1 beta (IL-1 beta) on TGF-beta secretion from malignant glioma cells in vitro. T98G glioblastoma cells were treated with various doses of IL-1 beta and the TGF-beta activity in the supernatant was determined using a specific bioassay. Six other human malignant glioma cell lines were also treated with 1000 U/ml of IL-1 beta, and the TGF-beta activity in the supernatants was determined. The effect of IL-1 beta on the growth of tumor cells was also assessed by a bioassay using crystal violet which reflects the actual cell number in the plate wells. IL-1 beta treatment resulted in inhibition of TGF-beta secretion in two malignant glioma cell lines. TGF-beta secretion from T98G cells was suppressed by IL-1 beta in a dose-related manner. However, IL-1 beta treatment resulted in an obvious increase (> 20%) of TGF-beta secretion in two tumor lines, and a slight increase (< 20%) in three tumor lines. IL-1 beta did not affect the growth of four malignant glioma cell lines, and only slightly affected the growth of the other three cell lines. IL-1 beta modulates TGF-beta secretion from malignant glioma cells, but not in a consistent way.

Topics: Cell Division; Glioma; Humans; Interleukin-1; Transforming Growth Factor beta; Tumor Cells, Cultured

The expression of vascular endothelial growth factor (VEGF) has been implicated in brain tumor angiogenesis, and the promoter region for the VEGF gene contains several SP-1 and AP-1 (c-Fos and c-Jun) binding motifs. Among eight human glioma cell lines, cellular mRNA levels of transcription factors SP-1 and AP-1 (c-Fos and c-Jun) were found to be closely correlated with those of VEGF. VEGF expression appears to be highly susceptible to hypoxia or exogenous cytokines and growth factors. Of various cytokines and growth factors, basic fibroblast growth factor (bFGF), tumor necrosis factor alpha (TNF-alpha), and interleukin 1 most potently enhanced VEGF mRNA levels of a glioma cell line, U251. Incubation of the glioma cells with bFGF or TNF-alpha increased both VEGF and SP-1 mRNA at 30 min and c-Fos mRNA at 1-3 h, over 5-fold. Nuclear run-on assays showed an apparent increase of the transcription of the VEGF gene as well as the SP-1 gene by bFGF or TNF-alpha. Gel mobility shift assays demonstrated that only SP-1 binding activity was increased 1 h after exposure to bFGF or TNF-alpha, and also that AP-1, but not SP-1, activity was significantly activated by hypoxia. Mithramycin, an inhibitor of SP-1, at 1-10 nM inhibited activation of the VEGF gene by bFGF or TNF-alpha but not that by hypoxia. Western blot analysis also demonstrated an increase in cellular amounts of VEGF by TNF-alpha and a decrease by co-administration with mithramycin. The promoter activity of the VEGF gene, which contains five SP-1 binding sites and one AP-1 binding site but not hypoxia regulatory elements, was enhanced by bFGF or TNF-alpha but not by hypoxia. The chloramphenicol acetyltransferase assay with VEGF promoter deletion constructs demonstrated that four clusterized SP-1 binding sites in the proximal promoter were essential for the basal transcription and the TNF-alpha-dependent activation. These data indicated that the expression of the VEGF gene enhanced by bFGF or TNF-alpha appeared to be mediated in part through the transcription factor SP-1, suggesting a different mechanism from that for hypoxia-induced activation of the VEGF gene.

Topics: Antineoplastic Agents; DNA-Binding Proteins; Endothelial Growth Factors; Epidermal Growth Factor; Fibroblast Growth Factor 2; Gene Expression Regulation; Glioma; Humans; Lymphokines; Oxygen; Platelet-Derived Growth Factor; Plicamycin; Promoter Regions, Genetic; RNA, Messenger; Sp1 Transcription Factor; Transcription Factor AP-1; Transforming Growth Factor beta; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

The effects of transforming growth factor-beta1 (TGFbeta) on two human neuroblastoma cell lines, LAN-5 and SK-N-AS, and one human glioblastoma cell line, GL15, were evaluated. Of the three cultures, only two, SK-N-AS and GL15, had a complete response to TGFbeta, with induction of the following effects: (i) inhibition of cell proliferation; (ii) up-regulation of the extracellular matrix glycoprotein fibronectin, together with down-regulation of the VLA5 integrin receptor; (iii) up-regulation of histotype-specific cytoskeletal intermediate filaments (neurofilaments for neuroblastoma and GFAP for glioblastoma); and (iv) increase in the glycoprotein CD44, only in SK-N-AS. In the third cell line, neuroblastoma LAN-5, the effects exerted by TGFbeta consisted only of (i) neurofilament increase and (ii) morphological differentiation. The TGFbeta receptor pattern was different in each culture: SK-N-AS expressed low rates of type I and type II receptors and high rates of type III receptor; LAN-5 expressed high rates of type I, low rates of type II, and no type III; GL15 expressed high rates of all three receptors. These data suggest that TGFbeta can induce a histotype-specific cell maturation and that the neuroblastoma expressing low type II and at the same time lacking type III receptor responds only partially to TGFbeta, with induction of neural differentiation but without inhibition of cell growth.

Topics: Cell Differentiation; Cell Division; Culture Media, Conditioned; Fibronectins; Gene Expression Regulation; Glial Fibrillary Acidic Protein; Glioma; Humans; Hyaluronan Receptors; Neuroblastoma; Neurofilament Proteins; Neurons; Phenotype; Receptors, Fibronectin; Receptors, Transforming Growth Factor beta; Transforming Growth Factor beta; Tumor Cells, Cultured

To examine which growth factors correlate with neovascularization in human brain tumors, the mRNA levels of transforming growth factor alpha, transforming growth factor beta, basic fibroblast growth factor, and vascular endothelial growth factor (VEGF) genes were determined by a Northern blot analysis in surgically obtained human gliomas and meningiomas. The vascular development was determined by counting the number of microvessels which were immunostained with von Willebrand factor. We normalized the growth factor mRNA levels versus the glyceraldehyde phosphate dehydrogenase mRNA level. In the 17 gliomas and 16 meningiomas examined, the mRNA of transforming growth factors alpha and beta, basic fibroblast growth factor, and VEGF were expressed at various levels. Among those 4 growth factors, the mRNA levels of VEGF, but not those of transforming growth factors alpha and beta and basic fibroblast growth factor, correlated significantly with vascularity in both gliomas (correlation coefficient r = 0.499; P < 0.05) and meningiomas (correlation coefficient r = 0.779; P < 0.001). These findings thus suggest that VEGF may be a positive factor in tumor angiogenesis in both human gliomas and meningiomas.

Topics: Brain Neoplasms; Endothelial Growth Factors; Fibroblast Growth Factor 2; Glioma; Humans; Lymphokines; Meningioma; Neovascularization, Pathologic; RNA, Messenger; Transforming Growth Factor alpha; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

Neoplastic cells from intrinsic, neuroectodermal tumours may migrate up to several millimeters away from the original tumour mass into normal nervous tissue. The biological mechanisms underlying this local invasive behaviour of gliomas are poorly understood. We have demonstrated recently that growth factors and cell surface gangliosides are positively involved in human glioma cell adhesion, migration and invasion in vitro. In order to study the mechanism of action of gangliosides and growth factors in this process, their role in the production of laminin, the major component of glioma vascular basal lamina, was investigated. Both growth factors and gangliosides stimulated laminin production in vitro suggesting that these factors increase laminin production in order to enable glioma cells to adhere and then migrate and invade in vivo.

Topics: Brain Neoplasms; Epidermal Growth Factor; Fibroblast Growth Factor 2; Gangliosides; Glioma; Growth Substances; Humans; Laminin; Recombinant Proteins; Stimulation, Chemical; Transforming Growth Factor beta; Tumor Cells, Cultured

Growth inhibition assays using radioisotope or dye are used to detect transforming growth factor-beta (TGF-beta). Here, we describe a modified bioassay using crystal violet for the quantitative detection of TGF-beta 1 and TGF-beta 2. The procedure is based on staining Mv1Lu mink lung epithelial cells with crystal violet, followed by measurement of the absorbance at 570 nm in individual wells of a 96-well microtiter plate. The number of Mv1Lu cells correlated with the eluted dye intensity. The sensitivity of the bioassay to recombinant TGF-beta 1 and TGF-beta 2 increased approximately twofold by using only 500 Mv1Lu cells in microtiter wells. The bioassay was used to measure TGF-beta activity in the culture supernatant from glioblastoma cells. Culture supernatants were untreated or acid-activated to quantify the active or total TGF-beta, and neutralized with anti-TGF-beta 1 and/or anti-TGF-beta 2 antibody to measure the activity. Both TGF-beta 1 and TGF-beta 2 were detected in the untreated and acid-activated supernatants, and the amounts were calculated by extrapolating from the known recombinant TGF-beta 1 or TGF-beta 2 dilution curve. Our results show that the modified bioassay using crystal violet can measure the levels of TGF-beta 1 and TGF-beta 2 in culture supernatants from malignant glioma cells.

Topics: Biological Assay; Cell Count; Culture Techniques; Gentian Violet; Glioma; Growth Inhibitors; Humans; Lung; Lung Neoplasms; Transforming Growth Factor beta

Fas/APO-1 is a transmembrane protein of the nerve growth factor/TNF alpha receptor family which signals apoptotic cell death in susceptible target cells. We have investigated the susceptibility of seven human malignant glioma cell lines to Fas/APO-1-dependent apoptosis. Sensitivity to Fas/APO-1 antibody-mediated cell killing correlated with cell surface expression of Fas/APO-1. Expression of Fas/APO-1 as well as Fas/APO-1-dependent cytotoxicity were augmented by preexposure of human malignant glioma cells to IFN gamma and TNF alpha. Further, pretreatment with TGF beta 2, IL1 and IL8 enhanced Fas/APO-1 antibody-induced glioma cell apoptosis whereas other cytokines including TNF beta, IL6, macrophage colony-stimulating factor, IL10 and IL13 had no such effect. None of the human malignant glioma cell lines was susceptible to TNF alpha-induced cytotoxicity. Fas/APO-1 antibody-sensitive glioma cell lines (n = 5), but not Fas/APO-1 antibody-resistant glioma cell lines (n = 2), became sensitive to TNF alpha when co-treated with inhibitors of RNA and protein synthesis. Resistance of human glioma cells to Fas/APO-1 antibody-mediated apoptosis was mainly related to low level expression of Fas/APO-1 and appeared not to be linked to overexpression of the anti-apoptotic protooncogene, bcl-2. Given the resistance of human malignant glioma to surgery, irradiation, chemotherapy and immunotherapy, we propose that Fas/APO-1 may be a promising target for a novel locoregionary approach to human malignant glioma. This strategy gains support from the demonstration of Fas/APO-1 expression in ex vivo human malignant glioma specimens and from the absence of Fas/APO-1 in normal human brain parenchyma.

Topics: Antibodies; Antigens, Surface; Apoptosis; Cell Line; Cytokines; DNA, Neoplasm; fas Receptor; Flow Cytometry; Glioma; Humans; Immunohistochemistry; Interferon-gamma; Interleukins; Lipopolysaccharides; Macrophage Colony-Stimulating Factor; Microscopy, Electron; Receptors, Cell Surface; Recombinant Proteins; Transforming Growth Factor beta; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

We have previously established a human malignant glioma cell line, TM-1. TM-1 cells could proliferate in the serum-free medium. In the present study, immunochemical analysis demonstrated that platelet-derived growth factor (PDGF), transforming growth factor (TGF)-alpha, and TGF-beta are present in the serum-free medium conditioned by growing TM-1 cells. While the cells appeared to possess a single type of binding sites for epidermal growth factor (EGF) with properties comparable to those determined for other tumor cells, the conditioned medium did not contain EGF.PDGF, TGF-alpha, and EGF added exogenously to serum-free media stimulated thymidine incorporation into DNA of TM-1 cells. In addition, antibodies specific for PDGF and TGF-alpha suppressed this activity. These results indicate autocrine and stimulatory roles of PDGF and TGF-alpha for the proliferation of TM-1 cells. As observed for other tumor cells, TGF-beta by itself weakly suppressed thymidine incorporation by TM-1 cells. However, TGF-beta employed in combination with TGF-alpha or EGF appeared to stimulate thymidine incorporation, suggesting that a cooperative action of TGF-beta with different growth factors may be involved in the stimulatory growth regulation at least for TM-1 cells.

Topics: Cell Division; DNA; ErbB Receptors; Glioma; Humans; Platelet-Derived Growth Factor; Transforming Growth Factor alpha; Transforming Growth Factor beta; Tumor Cells, Cultured

Radiation-induced delayed brain injury is a well-documented complication of both standard external beam radiation (teletherapy) and interstitial brachytherapy; however, the cause of this damage has not been determined. Cytokines and growth factors are important regulatory proteins controlling the growth and differentiation of normal and malignant glial cells, which have been implicated in the tissue response to radiation injury. Six snap-frozen brain biopsies showing radiation injury were obtained from four patients harboring malignant gliomas who underwent either postoperative external beam and/or stereotactic interstitial brachytherapy at standard dosages. The specimens showed variable amounts of gliosis, tissue necrosis, calcification, inflammation, and vascular proliferation and hyalinization. Frozen tissue sections were examined for the presence of infiltrating lymphocytes, macrophages, cytokines, and other immunoregulatory molecules by the use of a panel of specific monoclonal and polyclonal antibodies. All specimens showed diffuse T cell infiltration with both CD4+ and CD8+ cells. Infiltrating activated macrophages (CD11c+, HLA-DR+) were prominent in five of six cases. Tumor necrosis factor-alpha and interleukin-6 immunoreactivity was prominent in four of six cases and was predominately localized to macrophages. Transforming growth factor-beta astrocytic and macrophage immunoreactivity was present at moderate levels in all cases. This study suggests that in radiation necrosis, interleukin-1 alpha, tumor necrosis factor-alpha, and interleukin-6 are expressed, predominately by infiltrating macrophages.

Topics: Adult; Aged; Brachytherapy; Brain; Brain Neoplasms; Combined Modality Therapy; Cranial Irradiation; Cytokines; Glioma; Humans; Immunoenzyme Techniques; Interleukin-1; Interleukin-6; Lymphocytes; Macrophages; Male; Middle Aged; Nerve Growth Factors; Radiation Injuries; Radiotherapy Dosage; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

The present study describes identification and partial characterization of a glioma-derived high molecular weight transforming growth factor beta-like molecule (HMW-TGF beta) that requires no activation for biological activity. HMW-TGF beta, constitutively produced by the human glioma cell line, D54MG, is not acid- or heat-labile; is relatively resistant to denaturation, reduction, and high salt treatment. Monoclonal antibody 12A12.D7, produced against partially-purified HMW-TGF beta, was used both to deplete and to neutralize directly a > 158 kDa HMW-TGF beta activity from gel filtration fractions; the antibody also directly neutralized purified mature TGF beta 1. 12A12.D7 recognized a single protein species of 186 kDa from unlabeled glioma cell conditioned media and 35S-labeled lysates. HMW-TGF beta is not due to complex formation between TGF beta and any of the known carrier molecules. Production of HMW-TGF beta by glioma cells could facilitate tumor cell proliferation, and thus contribute to the inexorable and rapid progression that characterizes malignant gliomas.

Topics: Antibodies; Electrophoresis; Glioma; Growth Inhibitors; Humans; Molecular Weight; Transforming Growth Factor beta; Tumor Cells, Cultured

Factors involved in the control of the biological properties of gliomas, the major form of brain tumour in man, are poorly documented. We investigated the role of transforming growth factor beta 1 (TGF-beta 1) in the control of proliferation of human glioma cell lines as well as normal human fetal brain cells. The data presented show that TGF-beta 1 exerts a growth-inhibitory action on both human fetal brain cells and three cell lines derived from human glioma of different grades of malignancy. In addition, this growth-inhibitory effect is dose dependent and serum independent. Since TGF-beta 1 is known to be involved in the control of cell migration during ontogenesis and oncogenesis, we investigated the role of this factor in the motile and invasive behaviour that characterises human gliomas in vivo. TGF-beta 1 was found to elicit a strong stimulation of migration and invasiveness of glioma cells in vitro. In combination with recent data showing an inverse correlation between TGF-beta 1 expression in human gliomas and survival, these findings may suggest that TGF-beta 1 plays an important role in the malignant progression of gliomas in man. A study of the molecular mechanisms involved in the antiproliferative action and the invasion-promoting action of TGF-beta 1 may help to identify new targets in therapy for brain tumours. A combined antiproliferative and anti-invasive therapy could be envisaged.

Topics: Brain Neoplasms; Cell Division; Cell Movement; Dose-Response Relationship, Drug; Female; Glioma; Humans; Infant; Kinetics; Male; Middle Aged; Neoplasm Invasiveness; Transforming Growth Factor beta; Tumor Cells, Cultured

Topics: Animals; Brain Neoplasms; Glioma; Humans; Immunity, Cellular; Major Histocompatibility Complex; Microglia; Phenotype; Rats; Transforming Growth Factor beta

Malignant gliomas are associated with a state of systemic immunosuppression which appears to be partially mediated by transforming growth factor beta (TGF-beta) secreted from glioma cells. In a recently described animal model of malignant glioma, massive activation of local microglial cells and formation of microglia-derived macrophages has been observed in the absence of detectable tumour regression. We have investigated the in situ expression of TGF-beta in rat glioma as a possible cause of ineffective tumour destruction. Two weeks following unilateral injection of glioma cells, large tumours were observed in the affected hemisphere. In situ hybridization for TGF-beta 1 mRNA revealed an intense signal over the entire tumour area. In the peritumoural area, at sites of glial activation, a lower signal was obtained over cellular profiles containing nuclei typical for microglia, as well as other unidentified cellular profiles. No signal was obtained over the contralateral unaffected hemisphere. Northern blot analysis revealed a strong expression of TGF-beta 1 mRNA in tumour tissue, a lesser signal in the peritumoural reactive brain tissue and virtually no signal in normal tissue. Our data indicate that the experimental rat glioma has the potential to secrete TGF-beta in vivo which might render the microglial infiltration ineffective. TGF-beta expressed by activated microglial cells themselves might further inhibit their tumoricidal potential, thus contributing further to unrestrained tumour growth.

Topics: Animals; Blotting, Northern; Brain Neoplasms; Cell Transplantation; Female; Glioma; In Situ Hybridization; Neoplasm Transplantation; Neuroglia; Rats; Rats, Wistar; RNA Probes; RNA, Messenger; RNA, Neoplasm; Transforming Growth Factor beta

We have developed two different models of tumor angiogenesis by human brain tumors: one being tube formation by bovine aortic endothelial (BAE) cells cocultured with tumor cells in vitro, and other being in vivo angiogenesis in mice when tumor cells are transplanted into the dorsal sac. We investigated whether tube formation could be induced in BAE cells in type I collagen gel when these cells were cocultured with seven human glioma cell lines. Four of the seven glioma cell lines, which had high levels of basic fibroblast growth factor (bFGF) mRNA, induced tube formation by BAE cells. The tube formation was blocked by coadministration of anti-bFGF antibody. In in vivo model system of tumor angiogenesis in mice, these four cell lines were highly angiogenic. In contrast, with the other three glioma cell lines, which had poor expression of bFGF, BAE cells showed no apparent tube formation. These three cell lines did not efficiently develop capillary networks in mice. The results demonstrated a correlative relationship in the tubulogenesis of BAE cells, bFGF mRNA levels and angiogenesis in mice. The present study with two model systems of tumor angiogenesis suggests that the angiogenesis of some human glioma cell lines is mediated by bFGF, possibly via paracrine control.

Topics: Animals; Endothelium, Vascular; Fibroblast Growth Factor 2; Gene Expression; Glioma; Growth Substances; Humans; In Vitro Techniques; Mice; Morphogenesis; Neoplasm Transplantation; Neovascularization, Pathologic; RNA, Messenger; Transforming Growth Factor alpha; Transforming Growth Factor beta; Tumor Cells, Cultured

Topics: Astrocytes; Cell Line; Gene Expression Regulation, Neoplastic; Glioma; Humans; Neuroglia; Transforming Growth Factor beta; Tumor Cells, Cultured

We examined transforming growth factor-beta (TGF-beta) activity in cerebrospinal fluid of 39 patients with various brain tumors, and found it in 10 glioma cases that had lesions related to subarachnoid space or ventricle. In one glioma case, TGF-beta detected on admission disappeared after radiation and chemotherapy. We confirmed that five glioma cell lines produced TGF-beta, and that four of them produced active form of TGF-beta directly. The active form of TGF-beta was also identified from cerebrospinal fluid before the acidification treatment in two cases. The calculated contents were 110 ng/ml and 18 ng/ml. These results indicate that active form of TGF-beta is directly produced by tumor cells in patients with glioma, and may contribute to immunodeficiency of the host.

Topics: Brain Neoplasms; Glioma; Humans; Transforming Growth Factor beta; Tumor Cells, Cultured

Brain tumor cells secrete platelet-derived growth factor (PDGF) and transforming growth factor beta (TGF-beta), and through local production of these growth factors, brain tumor cells may stimulate their own proliferation. Previously we have shown that several different clones of canine glioma cells secrete varying amounts of PDGF and TGF-beta which correlate with in vitro cloning efficiency and in vivo tumorigenicity. In this study, intracellular trafficking of PDGF and TGF-beta was assessed by treatment of each clone with agents preventing vesicular degradation and secretion of growth factors. Clone 2 was more sensitive to these agents (chloroquine and monensin) than clone 5, resulting in retention of intracellular 125I-PDGF and 125I-TGF-beta. Furthermore, exogenous TGF-beta inhibited DNA-synthesis dramatically in clone 2 (compared with clone 5), presumably by interfering with intracellular growth factor receptor availability. This is supported by the fact that exogenous TGF-beta increased the number of its receptors on clone 2 cells, whereas surface receptors decreased on clone 5 cells treated with TGF-beta. These results illustrate the potential for autocrine growth factors to interact with their receptors intracellularly during neoplastic cell proliferation.

Topics: Animals; Brain Neoplasms; Cell Division; Dogs; Glioma; Platelet-Derived Growth Factor; Receptors, Cell Surface; Receptors, Transforming Growth Factor beta; Transforming Growth Factor beta; Tumor Cells, Cultured

This in vitro study was aimed at restitution of transforming growth factor (TGF)-beta 2-mediated suppression of T-lymphocyte activation within malignant gliomas. In early-passage tumor cell cultures of two glioblastomas (HTZ-153 and HTZ-209) and one malignant astrocytoma classified as World Health Organization Grade III (HTZ-243), autologous peripheral blood mononuclear cells were activated by interleukin-1 alpha and interleukin-2 in vitro (lymphokine-activated killer cells) and tested for cytotoxic and proliferative activity. In expression studies (Western blot and Northern hybridization) of all three tumors, TGF-beta could be detected at the protein and messenger ribonucleic acid (mRNA) levels. A polyclonal anti-TGF-beta neutralizing antibody did not enhance lymphocyte proliferation upon stimulation with tumor targets (3H-thymidine incorporation) and slightly stimulated lymphocyte cytotoxicity against autologous target cells. Preincubation of target cells for 12 hours with TGF-beta 2-specific phosphorothioate-anti-sense oligodeoxynucleotides (S-ODN's) did, however, enhance lymphocyte proliferation up to 2.5-fold and autologous tumor cytotoxicity up to 60%, compared to controls not treated with S-ODN's. Incubation of tumor cells with TGF-beta 2-specific S-ODN's resulted in decreased TGF-beta-specific immunoreactivity in cultured glioma cells, in reduced TGF-beta 2 protein concentration (Western blot), and in a change in the expression pattern of TGF-beta 2 mRNA's. These observations may have implications for in vivo and in vitro activation of a cellular immune response against autologous malignant glioma cells.

Topics: Antigens, CD; Antigens, Neoplasm; Blotting, Northern; Blotting, Western; Brain Neoplasms; Glioma; Humans; Immunity, Cellular; Immunocompromised Host; Killer Cells, Lymphokine-Activated; Leukocytes, Mononuclear; Oligonucleotides, Antisense; Phenotype; RNA, Neoplasm; T-Lymphocytes, Cytotoxic; Transforming Growth Factor beta; Tumor Cells, Cultured

Adherent lymphokine-activated killer (A-LAK) cells were obtained from peripheral blood lymphocytes of patients with recurrent glioblastoma. In vitro features of A-LAK cultures were assessed in comparison to those of non-adherent lymphokine-activated killer (NA-LAK) cells of the same patients with regard to cytotoxic activity, proliferation and surface markers. Only in a minority of cases did A-LAK cells show a markedly higher cytotoxicity on K562, Daudi and allogeneic glioblastoma cells. Nevertheless, A-LAK cells proliferated significantly better than NA-LAK and contained higher percentages of CD16+, CD56+ and CD25+ cells, indicating that A-LAK cells from these patients represent a subpopulation of lymphocytes enriched for activated natural killer cells. We also investigated whether immunosuppressive factor(s) were present in the tumour bed of recurrent gliomas. To this end, samples of glioblastoma cavity fluid (GCF), which accumulates in the cavity of subtotally removed tumour, were recovered and tested for the presence of immunosuppressive activity. All GCF samples analysed were shown to inhibit in vitro proliferation and antitumour cytotoxicity of 1-week-cultured A-LAK cells in a dose-dependent manner. Such GCF activity was effectively antagonized by a transforming growth factor beta (TGF beta) neutralizing antibody, indicating the involvement of TGF beta in lymphocyte inhibition. These results show that in the tumour cavity remaining after subtotal glioblastoma resection a marked immunosuppressive activity, probably due to local release of TGF beta, is present; such activity may negatively influence the therapeutic effectiveness of local cellular immunotherapy.

Topics: Cell Adhesion; Cytotoxicity, Immunologic; Glioma; Growth Inhibitors; Humans; Immunosuppressive Agents; Killer Cells, Lymphokine-Activated; Lymphocyte Activation; Transforming Growth Factor beta

Production of matrilysin and stromelysin by five human glioma cell lines was investigated by Northern blot and immunoblot analyses. Four cell lines constitutively produced matrilysin. Its production was stimulated by phorbol-12-myristate-13-acetate (PMA) in two cell lines and by transforming growth factor-beta 1 (TGF-beta 1) in two other cell lines. Stromelysin transcript was constitutively expressed in only two cell lines, but enhanced or induced by PMA in four cell lines. These results suggest that these enzymes, especially matrilysin, may be involved in the invasive growth of neoplastic glial cells.

Topics: Blotting, Northern; Enzyme Induction; Glioma; Humans; Matrix Metalloproteinase 3; Matrix Metalloproteinase 7; Metalloendopeptidases; Molecular Weight; RNA, Neoplasm; Tetradecanoylphorbol Acetate; Transcription, Genetic; Transforming Growth Factor beta; Tumor Cells, Cultured

One of the morphologic hallmarks of human gliomas are inflammatory infiltrates with accumulation of macrophages in the tumor site. The signals leading to the macrophage response are only at the beginning of being understood. Novel chemotactic factors that have recently been characterized as secretory products of glioblastoma cells may attract mononuclear cells from the blood. Within the tumor tissue blood-derived monocytes and macrophages of the brain tissue, the microglial cells, may increase in cell numbers due to tumor-derived growth factors. Both astrocytoma cell lines and cultured astrocytes have been shown recently to produce granulocyte-macrophage (GM)-CSF. We show that in vitro not only astrocytoma but also glioblastoma cell lines secrete GM-CSF when stimulated with TNF-alpha or IL-1. However, there is no evidence for GM-CSF production by glioblastoma cells in vivo: fresh tumor samples lack the mRNA for GM-CSF and the protein is not detectable in the tumor cyst fluids or the cerebrospinal fluids of glioblastoma patients. This contrasts IL-1 and IL-6 that are detectable in the tumor cyst fluids and IL-6 also in the cerebrospinal fluids of the patients. Unlike GM-CSF, transforming growth factor-beta 2 mRNA is expressed in ex vivo tested glioblastoma tissues. Absence of GM-CSF in vivo may be explained by the presence of tumor-derived inhibitory factors, such as transforming growth factor-beta 2 and PGE which suppress GM-CSF production by glioblastoma cells in vitro. The accumulation of macrophages at the tumor site may be due to local elaboration of chemoattractants and/or not yet defined growth factors rather than due to GM-CSF production.

Topics: Chemotactic Factors; Cyclic AMP; Dinoprostone; Glioma; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Interleukin-1; RNA, Messenger; Transforming Growth Factor beta; Tumor Cells, Cultured

Topics: Animals; Bradykinin; Cell Line; Enkephalins; Enzyme Activation; Glioma; Insulin-Like Growth Factor I; Interleukin-1; Kinetics; Nerve Growth Factors; Neuroblastoma; Phosphatidylinositol 3-Kinases; Phosphotransferases; Transforming Growth Factor beta; Vasopressins

Protein kinase C (PKC), an enzyme involved in signal transduction, responds to diacyl glycerol and also to phorbol ester, a ligand analogous to diacyl glycerol. We have studied the expression of the major isoforms (alpha, beta I, beta II, and gamma) in eight human glioblastoma cell lines. In all eight lines, PKC-alpha mRNA and protein were expressed. In none of the eight did a probe for PKC-beta I and -beta II mRNA give positive results nor were Western blots for PKC-beta II positive. The half-life for PKC alpha mRNA was approximately 16 h and levels of the mRNA were increased slightly following addition of phorbol myristate acetate (PMA) or transforming growth factor-beta (TGF beta). PKC-gamma was present in most of the glioblastomas. In cell line A172, 82% of the PKC-alpha was present in the cytosol with the remainder evenly divided between plasma membrane and nucleus. Thirty minutes after addition of PMA, 33% of the total original protein was in the plasma membrane and 48% in the nuclear fraction. By 21 h, no PKC-alpha was recovered from any fraction. PKC-gamma was also down-regulated in the presence of PMA, but there was no evidence for translocation to the plasma membrane or nuclear fraction. In a more detailed study, translocation of PKC-alpha in the presence of PMA was complete by 10 min, and a major decrease in the PKC translocated to the plasma-membrane fraction occurred some time between 2 and 4 h after PMA addition, while a major decrease in the translocated nuclear fraction occurred some time after 6 h. cAMP alone had no effect on the PKC alpha protein level or distribution, nor did it alter the translocation and down-regulation due to PMA exposure. In these studies the level of PKC-alpha mRNA in tumors was similar to that in normal glial cells.

Topics: Cyclic AMP; Glioma; Half-Life; Humans; Isoenzymes; Neuroglia; Protein Kinase C; Reference Values; RNA, Messenger; Subcellular Fractions; Tetradecanoylphorbol Acetate; Tissue Distribution; Transforming Growth Factor beta; Tumor Cells, Cultured

Glioblastomas are malignant brain tumors that are attended by an immunosuppressed state. The authors have studied the expression of transforming growth factor-beta 2, which is known to have potent immunosuppressive and angiogenic properties. Transforming growth factor-beta 2 messenger ribonucleic acid and its protein product are both found to be greatly overexpressed in these tumors and are absent from normal brain tissue. The overexpression of this growth factor may contribute to the escape of neoplastic astrocytes from immune surveillance and, furthermore, to the immunosuppressed state that is characteristic of many of these patients.

Topics: Astrocytoma; Blotting, Northern; Brain Neoplasms; Gene Expression; Glioma; Humans; Immune Tolerance; Immunologic Surveillance; Neovascularization, Pathologic; Nucleic Acid Hybridization; RNA, Messenger; Transforming Growth Factor beta

The mitogenic and morphological effects of acidic fibroblast growth factor (aFGF) and transforming growth factor-beta (TGF-beta) were assessed on cultured fetal rat astrocytes and C6 rat glioma cells in the presence and absence of serum. Astrocytes incubated with aFGF exhibited an increase in mitotic activity and characteristic morphological changes involving extensive process formation and rounding of cell bodies. Astrocytes incubated with TGF-beta underwent a slight decrease in mitotic activity and remained morphologically unchanged. Cells exposed to a combination of aFGF and TGF-beta demonstrated an attenuation of both the mitogenic and morphological changes observed in the presence of aFGF alone. The C6 glioma cells cultured in the presence of aFGF underwent a characteristic morphological change from a rounded piling cell mass to a more spindle-shaped bipolar cell layer, accompanied by an increase in mitotic activity. In contrast to the astrocyte cultures, increased growth and similar morphological effects were produced by TGF-beta. The combination of aFGF and TGF-beta did not result in attenuation of the mitogenic and morphological changes (as seen in astrocytic cells). These results suggest that, in normal fetal rat astrocytes, TGF-beta is capable of attenuating the mitogenic and morphological changes induced by aFGF in vitro. In the transformed C6 glioma cell line, aFGF and TGF-beta elicit similar mitogenic and morphological changes, without evidence of an antagonistic interaction as seen in normal astrocytes.

Topics: Animals; Astrocytes; Cells, Cultured; Fibroblast Growth Factor 1; Glioma; Rats; Transforming Growth Factor beta; Tumor Cells, Cultured

The neural cell adhesion molecule (N-CAM) plays a fundamental role in nervous system development and regeneration, yet the regulation of the expression of N-CAM in different brain regions has remained poorly understood. Osteogenic protein 1 (OP-1) is a member of the transforming growth factor beta superfamily that is expressed in the nervous system. Treatment of the neuroblastoma-glioma hybrid cell line NG108-15 for 1-4 days with recombinant human OP-1 (hOP-1) induced alterations in cell shape, formation of epithelioid sheets, and aggregation of cells into multilayered clusters. Immunofluorescence studies and Western blots demonstrated a striking differential induction of the three N-CAM isoforms in hOP-1-treated cells. hOP-1 caused a 6-fold up-regulation of the 140-kDa N-CAM, the isoform showing the highest constitutive expression, and a 29-fold up-regulation of the 180-kDa isoform. The 120-kDa isoform was not detected in control NG108-15 cells but was readily identified in hOP-1-treated cells. Incubation of NG108-15 cells with an antisense N-CAM oligonucleotide reduced the induction of N-CAM by hOP-1 and decreased the formation of multilayered cell aggregates. Anti-N-CAM monoclonal antibodies also diminished the formation of multilayered cell aggregates by hOP-1 and decreased cell-cell adhesion when hOP-1-treated NG108-15 cells were dispersed and replated. Thus, hOP-1 produces morphologic changes in NG108-15 cells, at least in part, by inducing N-CAM. These observations suggest that OP-1 or a homologue may participate in the regulation of N-CAM during nervous system development and regeneration.

Topics: Animals; Antibodies, Monoclonal; Blotting, Western; Bone Morphogenetic Protein 7; Bone Morphogenetic Proteins; Cell Adhesion; Cell Adhesion Molecules, Neuronal; Cell Aggregation; Dose-Response Relationship, Drug; Fluorescent Antibody Technique; Glioma; Kinetics; Molecular Weight; Neuroblastoma; Proteins; Recombinant Proteins; Transforming Growth Factor beta; Tumor Cells, Cultured

Topics: Gene Expression; Glioma; Humans; Platelet-Derived Growth Factor; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-sis; RNA, Messenger; Transforming Growth Factor beta; Tumor Cells, Cultured

Transforming growth factor-beta 1 (TGF-beta 1) has been found to occur as latent high molecular weight complexes, with or without an associated component denoted latent TGF-beta 1-binding protein (LTBP). We show here that a human glioblastoma cell line (U-1240 MG) secretes all isoforms of TGF-beta s found in mammalian cells (TGF-beta 1, -beta 2, and -beta 3). Approximately 26% of the secreted TGF-beta is in an active form. Latent TGF-beta s were partially purified from medium conditioned by the U-1240 MG cell line using anion exchange chromatography. Analysis of the different fractions by immunoblotting using antisera against precursor parts of the different TGF-beta isoforms, and against LTBP, revealed that not only TGF-beta 1 but also other isoforms of TGF-beta may occur in high molecular weight forms containing LTBP. In addition, each one of the TGF-beta isoforms occurred in smaller forms not containing LTBP. Interestingly, each of the TGF-beta isoforms was also seen in complexes of about 210 kDa containing associated component(s) distinct from LTBP. These results indicate that each of the different isoforms of TGF-beta is synthesized and secreted by this glioblastoma cell line in several different high molecular weight latent forms; the biological importance of the various latent TGF-beta complexes is discussed.

Topics: Carrier Proteins; Glioma; Humans; In Vitro Techniques; Intracellular Signaling Peptides and Proteins; Latent TGF-beta Binding Proteins; Macromolecular Substances; Molecular Weight; Transforming Growth Factor beta; Tumor Cells, Cultured

The type beta transforming growth factors (TGF) are potent regulators of the growth and functions of lymphocytes and macrophages. Recently the human glioblastoma cell line 308 was shown to produce TGF-beta 2. The relevance of this finding was evaluated further by comparing human glioblastoma cells with their nontransformed animal counterpart, astrocytes, with regard to the production of the three TGF-beta isoforms observed so far in mammals. In this report astrocytes are demonstrated to secrete also TGF-beta 2 and to express TGF-beta 1, -beta 2, and -beta 3 mRNA in vitro. In contrast, cultured murine brain macrophages release TGF-beta 1 and are positive for TGF-beta 1 mRNA only. Glia cell-derived TGF-beta 1 and -beta 2 are detected in latent form whereas both latent and active TGF-beta are identified in the supernatant of three human glioblastoma cell lines tested. These cell lines, however, show heterogeneity in regard to the isoform of TGF-beta expressed but share with astrocytes the inability to release TGF-beta 3. Provided production and activation of latent TGF-beta occur in vivo, astrocytes and microglia may then be expected to exert regulatory influences on immune mediated diseases of the central nervous system.

Topics: Animals; Astrocytes; Cells, Cultured; Glioma; Mice; Mice, Inbred ICR; Neuroglia; Rats; Ribonucleases; RNA, Messenger; T-Lymphocytes; Transforming Growth Factor beta; Tumor Cells, Cultured

Cytokines are important regulatory proteins controlling growth and differentiation of normal and malignant glial cells. Astrocytes and microglial cells produce and respond to many of the same cytokines employed by cells of the immune system. The authors have analyzed 15 histologically confirmed malignant glial neoplasms for the presence of infiltrating lymphocytes, macrophages, cytokines, and other immunoregulatory molecules using a panel of specific monoclonal and polyclonal antibodies on frozen-tissue sections. All neoplasms showed focal T-cell infiltration with CD8 cells predominating. Infiltration of activated macrophages (positive for CD11c, class II, and interleukin-2 receptor) was marked in all tumors. Within the neoplasm, tumor necrosis factor-alpha (TNF-alpha)- and interleukin (IL)-6-positive macrophages were prominent in five cases, while the tumor cells themselves were only weakly positive. In the other 10 cases, the numerous infiltrating macrophages were only rarely immunoreactive for TNF-alpha or IL-6. Transforming growth factor-beta (TGF-beta) immunoreactivity was most prominent in those tumors with little TNF-alpha-positive macrophage infiltration, although intratumoral variability was present. This study suggests that, in malignant gliomas, the cytokines TNF-alpha and IL-6, although weakly present in neoplastic cells, are most prominent in infiltrating macrophages and in those regions of the tumors that show little immunoreactivity for TGF-beta. The important interactions among neoplastic, reactive glial, and inflammatory cells, which regulate tumor growth, are likely to be in part mediated through these molecules.

Topics: Adult; Aged; Brain Neoplasms; CD4-CD8 Ratio; Cytokines; Female; Glioma; Histocompatibility Antigens Class II; HLA-DR Antigens; Humans; Immunophenotyping; Interleukin-6; Lymphocytes; Male; Middle Aged; Receptors, Interleukin-2; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Tumor cells have been reported to exert inhibitory effects on the activation of T lymphocytes in vitro. We show that the IL-2-stimulated proliferation of a Th cell line is suppressed when the T cells are cocultured with human glioblastoma and melanoma cell lines. The use of two Th cell clones that differ in their responsiveness to growth-inhibition by transforming growth factor-beta (TGF-beta) and the analysis of tumor cell-derived supernatants as well as of TGF-beta 1/TGF-beta 2 gene expression allowed to distinguish two pathways of tumor-induced immunosuppression. Glioblastoma cells exert their immunosuppressive effects by producing biologically active TGF-beta 2, whereas the immunosuppressive state induced by melanoma cells is TGF-beta-independent and requires direct contact between tumor cell and T cell. The TGF-beta-dependent immunosuppression is down-regulated by various protease inhibitors and up-regulated by estradiol via modulation of the production of biologically active TGF-beta 2 by glioblastoma cells leaving total activatable TGF-beta 2 unaffected. No such modulation is functional for the TGF-beta-independent pathway of immunosuppression. We conclude that the production of active TGF-beta by tumor cells is regulated at a posttranslational level by the coordinated action of several proteolytic enzymes.

Topics: Animals; Cell Line; Dexamethasone; Estradiol; Glioma; Immune Tolerance; In Vitro Techniques; Lymphocyte Activation; Melanoma, Experimental; Mice; Ovalbumin; Protease Inhibitors; T-Lymphocytes; Transforming Growth Factor beta; Tumor Cells, Cultured; Tunicamycin

This study was undertaken to evaluate the role of two sets of growth factors, platelet-derived growth factor (PDGF) and transforming growth factor-beta (TGF-beta), in the induction and maintenance of glial tumors and their phenotypic expression. Explants from eight malignant tumors, five benign tumors, and two nontumor glial cells were analyzed for levels of messenger ribonucleic acid (mRNA) expression of PDGFA, PDGFB, TGF-beta 1, and TGF-beta 2. Results were normalized to the mRNA expression of tubulin, a "housekeeping" gene present in glial cells. Of the 15 explants tested, PDGFB was seen in six, all of which were malignant tumors; PDGFA was seen in all 15 with much higher levels expressed in malignant tumors; and TGF-beta 1 and TGF-beta 2 were seen in all 15 without a clear difference between cell types, although expression tended to be higher in malignant tumors. This project supports the theory that the induction and maintenance of glial tumors is likely to be a multifactorial phenomenon.

Topics: Astrocytoma; Cells, Cultured; Gene Expression Regulation, Neoplastic; Glioma; Humans; Neoplasms, Germ Cell and Embryonal; Neuroglia; Nucleic Acid Hybridization; Platelet-Derived Growth Factor; RNA, Messenger; RNA, Neoplasm; Transforming Growth Factor beta; Tumor Cells, Cultured

The multidrug transporting cell membrane molecule P-glycoprotein can be spontaneously expressed in human glioma cells. Transcripts of mdr genes were detected in glial tumor cells by polymerase chain reaction and Northern blotting, expression of P-glycoprotein was analyzed by immunocytochemistry and functional activity by cytofluorometry of fluorescent probe transport. In vitro treatment of glioma cells with vincristine induced coordinate over-expression of both mdr1 and mdr3 genes associated with very high P-glycoprotein-mediated multidrug transport, resistant to the inhibitory activity of chemosensitizers like verapamil. The physiological modulators of multidrug transport are as yet unknown. We therefore initiated a screening program to analyze the effects of cytokines on multidrug transport. We observed, that transforming growth factors (TGF)-beta 1, -beta 2, and -beta 1.2-but not the related bone morphogenetic protein (BMP) 2--inhibited multidrug transport. Interestingly, BMP 2 antagonized the TGF-beta induced inhibition of multidrug transport.

Topics: Adult; Aged; ATP Binding Cassette Transporter, Subfamily B, Member 1; Blotting, Northern; Female; Flow Cytometry; Glioma; Humans; Immunohistochemistry; Male; Membrane Glycoproteins; Middle Aged; Nervous System Neoplasms; Pharmaceutical Preparations; Polymerase Chain Reaction; Transforming Growth Factor beta; Tumor Cells, Cultured

Transforming growth factor-beta (TGF-beta), a regulator of cell growth and differentiation, is secreted by most cultured cells in latent form (L-TGF-beta). Activation of L-TGF-beta can be achieved by various physico-chemical treatments, including acidification, alkalinization, heating and chaotropic agents. Proposed physiological activators include proteinases and glycosidases, which, however, only lead to limited activation (15-20% of the total TGF-beta activity after acidic activation). In the present study L-TGF-beta 1 partially purified from human platelets was not activated by treatment with neuraminidase or the proteinases plasmin, endoproteinase Arg-C, elastase and chymotrypsin. The mechanism of activation of L-TGF-beta was further assessed by using the human glioblastoma cell line 308, which releases biologically active TGF-beta 2. Factor(s) secreted by 308 glioblastoma cells were found to be able to activate partially purified L-TGF-beta 1 from human platelets. Our finding may prove to constitute a physiologically relevant mechanism for the activation of latent forms of TGF-beta in vivo.

Topics: Blood Platelets; Cell Division; Cell Line; Endopeptidases; Fibrinolysin; Glioma; Humans; Kinetics; Neuraminidase; Recombinant Proteins; Transforming Growth Factor beta

Low-grade astrocytomas, anaplastic astrocytomas and glioblastomas in vitro were found to ubiquitously produce the mRNA of transforming growth factor-beta (TGF beta). TGF beta 1 and TGF beta 2 mRNA were expressed to a lesser degree among the hyperdiploid malignant gliomas. By radioreceptor assay of conditioned medium, TGF beta was secreted predominantly in latent form, in both latent and active form, or only in active form within a panel of low-grade and malignant gliomas. The TGF beta receptor (types I, II, and III) was evident among the glioma lines. Many near-diploid gliomas were growth-inhibited by TGF beta 1 and TGF beta 2 in vitro. Most hyperdiploid glioblastomas showed a positive mitogenic response to exogenous TGF beta 1 and TGF beta 2. A synergistic or additive mitogenic interaction with epidermal growth factor and insulin was observed among some. Under serum-free conditions, anti-TGF beta antibody neutralized the expected growth-regulatory effect of endogenous TGF beta, thus establishing the specificity of the response in vitro. TGF beta 1 also enhanced the clonogenicity of certain gliomas which had been growth-stimulated in monolayer. Thus, basic elements in support of an autocrine hypothesis have been demonstrated: TGF beta mRNA was expressed among low-grade and malignant gliomas, TGF beta was secreted in latent and/or active form into conditioned media and appeared to serve as an endogenous regulator of glioma proliferation in vitro. The mitogenic response, either positive or negative, correlated with the degree of anaplasia and karyotypic divergence.

Topics: Blotting, Northern; Cell Division; DNA, Neoplasm; Epidermal Growth Factor; Gene Expression; Glioma; Humans; In Vitro Techniques; Insulin; Radioligand Assay; RNA, Messenger; RNA, Neoplasm; Transforming Growth Factor beta; Tumor Cells, Cultured

To study the regulation of major histocompatibility complex class II antigen by central nervous system cells, the expression of one of these antigens, human leukocyte antigenDR (HLADR) in three human glioblastoma cell lines (HTB14, 16 and 17) and a neuroblastoma cell line (HTB11) was determined. Interferon-gamma (IFN gamma) induced HTB16 and HTB17 cells to express HLADR, and enhanced the antigen expression in HTB14 cells, but it failed to induce HLADR expression in HTB11 cells. Tumor necrosis factor-alpha amplified and accelerated the expression of HLADR induced by IFN gamma in HTB16 cells. Interleukin-1 beta, prostaglandin E2 and transforming growth factor-beta suppressed IFN gamma-induced HLADR expression in HTB16 cells. Several other substances tested did not affect HLADR expression or IFN gamma-induced HLADR. These findings confirm that IFN gamma plays a role in the regulation of HLADR expression in cells derived from the brain and that some other cytokines modify IFN gamma-HLADR interactions.

Topics: Cytokines; Dinoprostone; Fluorescent Antibody Technique; Glioma; HLA-DR Antigens; Humans; Interferon-gamma; Interleukin-1; Transforming Growth Factor beta; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

The transforming growth factors type beta 1, beta 2, and beta 1.2 suppress multidrug transport in human pat-1 glioblastoma cells and even in cells that strongly over-express mdr genes and are resistant to inhibition of multidrug transport by chemosensitizers. Thus, inhibition of multidrug transport by cytokines might be a new approach to increase cellular accumulation of chemotherapeutic agents in multidrug resistant glial tumor cells. Interestingly, a member of the more distantly related decapentaplegic subgroup of transforming growth factors, the bone morphogenetic protein BMP 2, did not inhibit multidrug transport.

Topics: Biological Transport; Drug Antagonism; Flow Cytometry; Fluorescent Dyes; Glioma; Humans; Rhodamine 123; Rhodamines; Transforming Growth Factor beta

We analyzed a series of human glioma cell lines with regard to establishing what variables may contribute to their overall functional immunomodulating capability. We observed that supernatants derived from the gliomas, but not those from non-malignant human astrocyte cultures, suppressed lymphocyte proliferation. The extent of suppression elicited differed between tumors and for the same tumor depending upon its growth phase. For individual gliomas, supernatants from cultures approaching or at confluency elicited maximal lymphocyte suppression. For the series of tumors, levels of production of the immunosuppressive molecules transforming growth factor beta 2 and prostanoids (prostaglandin E2) did not correlate with the levels of functional suppression observed at any of the different growth phases. In some cases, glioma cultures grown in the presence of indomethacin to abolish prostanoid synthesis resulted in supernatants with net stimulatory activity. Our results indicate that malignant transformation of astrocytes is associated with acquisition of immunosuppressive capability which is determined by the combined effect of multiple immunomodulatory soluble factors, inhibitory or enhancing, and is dependent on the growth phase of the tumor.

Topics: Dinoprostone; Glioma; Humans; Immune Tolerance; Immunity; Indomethacin; Lymphocyte Activation; Transforming Growth Factor beta; Tumor Cells, Cultured

Human glioblastoma cells secrete a factor termed glioblastoma derived T cell suppressor factor (G-TsF) or transforming growth factor beta 2 (TGF-beta 2) which inhibits the response of T cells to mitogenic or antigenic stimulation. In the present study we isolated the promoter region of the G-TsF/TGF-beta 2 gene. The promoter region shares no homology to the promoter of the TGF-beta 1 or the 5' region of the TGF-beta 3 gene and harbours several familiar DNA motifs, including the cytokine-1 region, an octamer-like sequence, Sp1- and AP-2-like elements and a putative NF-kappa B site. In contrast to the TGF-beta 1 gene, the G-TsF/TGF-beta 2 gene contains three TATA-like sequences but lacks an AP-1 site. To understand the cell type specificity of expression of G-TsF/TGF-beta 2, the individual contribution of the DNA elements detected in the promoter has to be analysed in further studies.

Topics: Base Sequence; Cytokines; DNA; Female; Genomic Library; Glioma; Humans; Molecular Sequence Data; Oligonucleotide Probes; Placenta; Pregnancy; Promoter Regions, Genetic; Restriction Mapping; Sequence Homology, Nucleic Acid; TATA Box; Transforming Growth Factor beta

Human glioblastoma cells secrete a peptide, termed glioblastoma-derived T cell suppressor factor (G-TsF), which has suppressive effects on interleukin-2-dependent T cell growth. As shown here, complementary DNA for G-TsF reveals that G-TsF shares 71% amino acid homology with transforming growth factor-beta (TGF-beta). In analogy to TGF-beta it is apparently synthesized as the carboxy-terminal end of a precursor polypeptide which undergoes proteolytic cleavage to yield the 112 amino-acid-long mature form of G-TsF. Comparison of the amino-terminal sequence of G-TsF with that of porcine TGF-beta 2 and bovine cartilage-inducing factor B shows complete homology, which indicates that we have cloned the human analogue of these factors. It is tempting to consider a role for G-TsF in tumor growth where it may enhance tumor cell proliferation in an autocrine way and/or reduce immunosurveillance of tumor development.

Topics: Amino Acid Sequence; Base Sequence; Cell Line; Cloning, Molecular; DNA; Genes; Glioma; Humans; Molecular Sequence Data; Neoplasm Proteins; Peptides; Suppressor Factors, Immunologic; T-Lymphocytes; Transforming Growth Factor beta; Transforming Growth Factors