interleukin-8 and Glioblastoma

Glioblastoma (GBM) is the most highly malignant brain tumor in the human central nerve system. In this paper, we review new and significant molecular findings on angiogenesis and possible resistance mechanisms. Expression of a number of genes and regulators has been shown to be upregulated in GBM microvessel cells, such as interleukin-8, signal transducer and activator of transcription 3, Tax-interacting protein-1, hypoxia induced factor-1 and anterior gradient protein 2. The regulator factors that may strongly promote angiogenesis by promoting endothelial cell metastasis, changing the microenvironment, enhancing the ability of resistance to anti-angiogenic therapy, and that inhibit angiogenesis are reviewed. Based on the current knowledge, several potential targets and strategies are proposed for better therapeutic outcomes, such as its mRNA interference of DII4-Notch signaling pathway and depletion of b1 integrin expression. We also discuss possible mechanisms underlying the resistance to anti-angiogenesis and future directions and challenges in developing new targeted therapy for GBM.

Topics: Angiogenesis Inhibitors; Antineoplastic Agents; Brain Neoplasms; Gene Expression; Glioblastoma; Humans; Hypoxia-Inducible Factor 1; Integrin beta1; Interleukin-8; Intracellular Signaling Peptides and Proteins; Molecular Targeted Therapy; Mucoproteins; Neovascularization, Pathologic; Oncogene Proteins; Proteins; Receptors, Notch; RNA Interference; RNA, Messenger; Signal Transduction; STAT3 Transcription Factor; Up-Regulation

Malignant gliomas are the most common primary brain tumors. Despite efforts to find effective treatments, these tumors remain incurable. The failure of malignant gliomas to respond to conventional cancer therapies may reflect the unique biology of these tumors, underscoring the need for new approaches in their investigation. Recently, progress has been made in characterization of the molecular pathogenesis of glioblastoma using a developmental neurobiological perspective, by exploring the role of signaling pathways that control the differentiation of neural stem cells along the glial lineage. The transcription factor STAT3, which has an established function in neural stem cell and astrocyte development, has been found to play dual tumor suppressive and oncogenic roles in glial malignancy depending on the mutational profile of the tumor. These findings establish a novel developmental paradigm in the study of glioblastoma pathogenesis and provide the rationale for patient-tailored therapy in the treatment of this devastating disease.

Topics: Animals; Brain Neoplasms; Gene Expression Regulation, Neoplastic; Glioblastoma; Humans; Interleukin-8; Membrane Proteins; Models, Biological; Mutation; PTEN Phosphohydrolase; STAT3 Transcription Factor

Expression of the two types of tumor necrosis factor (TNF) receptor, p55 and p75, in 12 human glioblastoma cell lines was studied. Reverse-transcription polymerase chain reaction detected messenger ribonucleic acid (mRNA) transcripts of p55 TNF receptor in all 12 cell lines tested, but p75 TNF receptor mRNA in only four cell lines. Flow cytometric analysis with anti-p55 and anti-p75 TNF receptor monoclonal antibodies demonstrated both p55 and p75 proteins in these four cell lines, but the level of expression of p75 molecule was very low. Correlation of p55 and p75 TNF receptor expression with TNF-induced growth suppression and production of bioactive molecules (interleukin-6, interleukin-8, manganase-superoxide dismutase, prostaglandin E2) showed that p55 TNF receptor mediates these TNF actions, but none of the responses were influenced by the presence of the p75 TNF receptor, which apparently has no specific role.

Topics: Base Sequence; Cells, Cultured; Culture Techniques; Dinoprostone; Flow Cytometry; Gene Expression; Glioblastoma; Humans; Immunohistochemistry; Interleukin-6; Interleukin-8; Molecular Sequence Data; Polymerase Chain Reaction; Receptors, Tumor Necrosis Factor; RNA, Messenger; Tumor Necrosis Factor-alpha

Topics: Antibody Formation; Antigen Presentation; Antigens, Neoplasm; Brain Neoplasms; Chemotactic Factors; Colony-Stimulating Factors; Cytokines; Glioblastoma; Humans; Immunity, Cellular; Interferons; Interleukin-1; Interleukin-10; Interleukin-6; Interleukin-8; Monocyte Chemoattractant Proteins; T-Lymphocytes

Graphene oxide (GO) is a single layer of carbon atoms with unique properties, which are beneficial due to its surface functionalisation by miRNA. miRNAs are a non-coding small form of RNA that suppress the expression of protein-coding genes by translational repression or degradation of messenger RNA. Antisense miRNA-21 is very promising for future investigation in cancer therapy. This study aimed to detect cytokine expression levels after the administration of GO-antisense miRNA-21 into U87, U118, U251 and T98 glioma cell lines.. U87, U118, U251 and T98 glioma cell line were investigated in term of viability, human cytokine expression level at protein and genes after treatment with GO, GO-antisense miRNA-21 and antisense miRNA-21. The delivery of antisense miRNA-21 into the glioma cell at in vitro investigation were conducted by GO based transfection and electroporation.. The results of the protein microarray and gene expression profile showed that complexes of GO-antisense miRNA-21 modified the metallopeptidase inhibitor 2 (TIMP-2), interleukin-6 (IL-6), interleukin 8 (IL-8), intercellular adhesion molecule 1 (ICAM-1), and monocyte chemoattractant protein-1 (MCP-1) expression level compared to transfection by electroporation of antisense miRNA-21 at investigated glioblastoma cell lines. The TIMP-2 protein and gene expression level was upregulated after antisense miRNA-21 delivery by GO complex into U87, U251 and T98 glioblastoma cell lines comparing to the non-treated control group. The downregulation at protein expression level of ICAM - 1 was observed at U87, U118, U251 and T98 glioma cell lines. Moreover, the IL-8 expression level at mRNA for genes and protein was decreased significantly after delivery the antisense-miRNA-21 by GO compared to electroporation as a transfection method.. This work demonstrated that the graphene oxide complexes with antisense miRNA-21 can effectively modulate the cytokine mRNA and protein expression level at U87, U118, U251 and T98 glioma cell lines.

Topics: Cell Line; Cytokines; Glioblastoma; Glioma; Humans; Interleukin-8; MicroRNAs; Tissue Inhibitor of Metalloproteinase-2

Glioblastoma (GB) is highly invasive and resistant to multimodal treatment partly due to distorted vasculature and exacerbated inflammation. The aggressiveness of brain tumors may be attributed to the dysregulated release of angiogenic and inflammatory factors. The glycoprotein pentraxin-3 (PTX3) is correlated with the severity of some cancers. However, the mechanism responsible for the invasive oncogenic role of PTX3 in GB malignancy remains unclear. In this study, we examined the role of PTX3 in GB growth, angiogenesis, and invasion using in vitro and in vivo GB models, proteomic profiling, molecular and biochemical approaches. Under in vitro conditions, PTX3 over-expression in U87 cells correlated with cell cycle progression, increased migratory potential, and proliferation under hypoxic conditions. Conditioned media containing PTX3 enhanced the angiogenic potential of endothelial cells. While silencing of PTX3 by siRNA decreased the proliferation, migration, and angiogenic potential of U87 cells in vitro. Importantly, PTX3 over-expression increased tumor growth, angiogenesis, and invasion in an orthotopic mouse model. Higher levels of PTX3 in these tumors were associated with the upregulation of inflammatory and angiogenic markers including interleukin-8 (IL-8) and vascular endothelial growth factor (VEGF), but decreased levels of thrombospondin-1, an anti-angiogenic factor. Mechanistically, exogenous production of PTX3 triggered an IKK/NFκB signaling pathway that enhances the expression of the motility genes AHGEF7 and Rac1. Taken together, PTX3 expression is dysregulated in GB. PTX3 may augment invasion through enhanced angiogenesis in the GB microenvironment through the IL8-VEGF axis. Thus, PTX3 may represent a potential therapeutic target to mitigate the aggressive behavior of gliomas.

Topics: Animals; Brain Neoplasms; C-Reactive Protein; Cell Line; Glioblastoma; Interleukin-8; Mice; Neoplasm Invasiveness; Neurons; Serum Amyloid P-Component; Signal Transduction; Vascular Endothelial Growth Factor A

Annexin-1 (ANXA1) is widely reported to be deregulated in various cancers and is involved in tumorigenesis. However, its effects on glioblastoma (GBM) remain unclear. Using immunohistochemistry with tissue microarrays, we showed that ANXA1 was overexpressed in GBM, positively correlated with higher World Health Organization (WHO) grades of glioma, and negatively associated with poor survival. To further explore its role and the underlying molecular mechanism in GBM, we constructed ANXA1shRNA U87 and U251 cell lines for further experiments. ANXA1 downregulation suppressed GBM cell proliferation, migration, and invasion and enhanced their radiosensitivity. Furthermore, we determined that ANXA1 was involved in dendritic cell (DC) maturation in patients with GBM and that DC infiltration was inversely proportional to GBM prognosis. Considering that previous reports have shown that Interleukin-8 (IL-8) is associated with DC migration and maturation and is correlated with NF-κB transcriptional regulation, we examined IL-8 and p65 subunit expressions and p65 phosphorylation levels in GBM cells under an ANXA1 knockdown. These results suggest that ANXA1 significantly promotes IL-8 production and p65 phosphorylation levels. We inferred that ANXA1 is a potential biomarker and a candidate therapeutic target for GBM treatment and may mediate tumour immune escape through NF-kB (p65) activation and IL-8 upregulation.

Topics: Annexin A1; Annexins; Brain Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; Gene Expression Regulation, Neoplastic; Glioblastoma; Humans; Interleukin-8; NF-kappa B; Oncogenes; Tumor Escape; Up-Regulation

Tumor recurrence is the main challenge in glioblastoma (GBM) treatment. Gold standard therapy temozolomide (TMZ) is known to induce upregulation of IL8/CXCL2/CXCR2 signaling that promotes tumor progression and angiogenesis. Our aim was to verify the alterations on this signaling pathway in human GBM recurrence and to investigate the impact of TMZ in particular. Furthermore, a combi-therapy of TMZ and CXCR2 antagonization was established to assess the efficacy and tolerability. First, we analyzed 76 matched primary and recurrent GBM samples with regard to various histological aspects with a focus on the role of TMZ treatment and the assessment of predictors of overall survival (OS). Second, the combi-therapy with TMZ and CXCR2-antagonization was evaluated in a syngeneic mouse tumor model with in-depth immunohistological investigations and subsequent gene expression analyses. We observed a significantly decreased infiltration of tumor-associated microglia/macrophages (TAM) in recurrent tumors, while a high TAM infiltration in primary tumors was associated with a reduced OS. Additionally, more patients expressed IL8 in recurrent tumors and TMZ therapy maintained CXCL2 expression. In mice, enhanced anti-tumoral effects were observed after combi-therapy. In conclusion, high TAM infiltration predicts a survival disadvantage, supporting findings of the tumor-promoting phenotype of TAMs. Furthermore, the combination therapy seemed to be promising to overcome CXCR2-mediated resistance.

Topics: Adult; Aged; Aged, 80 and over; Animals; Antineoplastic Agents, Alkylating; Antineoplastic Combined Chemotherapy Protocols; Brain Neoplasms; Disease Models, Animal; Drug Synergism; Female; Gene Expression Regulation, Neoplastic; Glioblastoma; Humans; Interleukin-8; Male; Mice; Middle Aged; Neoplasm Recurrence, Local; Neovascularization, Pathologic; Phenylurea Compounds; Prognosis; Receptors, Interleukin-8B; Signal Transduction; Survival Analysis; Temozolomide; Tumor Microenvironment; Tumor-Associated Macrophages; Young Adult

This short note presents previous research data supporting a pilot study of metronomic dapsone during the entire course of glioblastoma treatment. The reviewed data indicate that neutrophils are an integral part of human glioblastoma pathophysiology, contributing to or facilitating glioblastoma growth and treatment resistance. Neutrophils collect within glioblastoma by chemotaxis along several chemokine/cytokine gradients, prominently among which is interleukin-8. Old data from dermatology research has shown that the old and inexpensive generic drug dapsone inhibits neutrophils' chemotaxis along interleukin-8 gradients. It is on that basis that dapsone is used to treat neutrophilic dermatoses, for example, dermatitis herpetiformis, bullous pemphigoid, erlotinib-related rash, and others. The hypothesis of this paper is that dapsone will reduce glioblastomas' neutrophil accumulations by the same mechanisms by which it reduces dermal neutrophil accumulations in the neutrophilic dermatoses. Dapsone would thereby reduce neutrophils' contributions to glioblastoma growth. Dapsone is not an ideal drug, however. It generates methemoglobinemia that occasionally is symptomatic. This generation is reduced by concomitant use of the antacid drug cimetidine. Given the uniform lethality of glioblastoma as of 2020, the risks of dapsone 100 mg twice daily and cimetidine 400 mg twice daily is low enough to warrant a judicious pilot study.

Topics: Cimetidine; Dapsone; Glioblastoma; Humans; Interleukin-8; Pemphigoid, Bullous; Pharmaceutical Preparations; Pilot Projects

We aimed to evaluate the angiogenic capacity of CXCL2 and IL8 affecting human endothelial cells to clarify their potential role in glioblastoma (GBM) angiogenesis. Human GBM samples and controls were stained for proangiogenic factors. Survival curves and molecule correlations were obtained from the TCGA (The Cancer Genome Atlas) database. Moreover, proliferative, migratory and angiogenic activity of peripheral (HUVEC) and brain specific (HBMEC) primary human endothelial cells were investigated including blockage of CXCR2 signaling with SB225502. Gene expression analyses of angiogenic molecules from endothelial cells were performed. Overexpression of VEGF and CXCL2 was observed in GBM patients and associated with a survival disadvantage. Molecules of the

Topics: Brain Neoplasms; Chemokine CXCL2; Glioblastoma; Human Umbilical Vein Endothelial Cells; Humans; Interleukin-8; Neoplasm Proteins; Receptors, Interleukin-8B; Signal Transduction; Vascular Endothelial Growth Factor A

Yokukansan is a traditional Japanese herbal medicine that has an antiallodynic effect in patients with chronic pain. However, the mechanisms by which yokukansan inhibits neuropathic pain are unclear.. This study aimed to investigate the molecular effects of yokukansan on neuroinflammation in U373 MG glioblastoma astrocytoma cells, which express a functional high-affinity neurokinin 1 receptor (substance P receptor), and produce interleukin (IL)-6 and IL-8 in response to stimulation by substance P (SP).. We assessed the effect of yokukansan on the expression of ERK1/2, P38 MAPK, nuclear factor (NF)-κB, and cyclooxygenase-2 (COX-2) in U373 cells by western blot assay. Levels of IL-6 and IL-8 in conditioned medium obtained after stimulation of cells with SP for 24 h were measured by enzyme-linked immunosorbent assay. All experiments were conducted in triplicate. Results were analyzed by one-way ANOVA, and significance was accepted at p < 0.05.. Yokukansan suppressed SP-induced production of IL-6 and IL-8 by U373 MG cells, and downregulated SP-induced COX-2 expression. Yokukansan also inhibited phosphorylation of ERK1/2 and p38 MAPK, as well as nuclear translocation of NF-κB, induced by SP stimulation of U373 MG cells.. Yokukansan exhibits anti-inflammatory activity by suppressing SP-induced production of IL-6 and IL-8 and downregulating COX-2 expression in U373 MG cells, possibly via inhibition of the activation of signaling molecules, such as ERK1/2, p38 MAPK, and NF-κB.

Topics: Anti-Inflammatory Agents; Astrocytoma; Brain Neoplasms; Cell Line, Tumor; Drugs, Chinese Herbal; Glioblastoma; Herb-Drug Interactions; Herbal Medicine; Humans; Interleukin-6; Interleukin-8; Japan; Neuritis; Neuroimmunomodulation; Neuroprotective Agents; Signal Transduction; Substance P

Brain tumors are one of the most common causes of cancer-related deaths around the world. Angiogenesis is critical in high-grade malignant gliomas, such as glioblastoma multiforme. The aim of this study is to comparatively analyze the angiogenesis-related genes, namely VEGFA, VEGFB, KDR, CXCL8, CXCR1 and CXCR2 in LGG vs. GBM to identify molecular distinctions using datasets available on The Cancer Genome Atlas (TCGA).. DNA sequencing and mRNA expression data for 514 brain lower grade glioma (LGG) and 592 glioblastoma multiforme (GBM) patients were acquired from The Cancer Genome Atlas (TCGA), and the genetic alterations and expression levels of the selected genes were analyzed.. We identified six distinct KDR mutations in the LGG patients and 18 distinct KDR mutations in the GBM patients, including missense and nonsense mutations, frame shift deletion and altered splice region. Furthermore, VEGFA and CXCL8 were significantly overexpressed within GBM patients.. VEGFA and CXCL8 are important factors for angiogenesis, which are suggested to have significant roles during tumorigenesis. Our results provide further evidence that VEGFA and CXCL8 could induce angiogenesis and promote LGG to progress into GBM. These findings could be useful in developing novel targeted therapeutics approaches in the future.

Topics: Brain Neoplasms; Carcinogenesis; Gene Expression; Glioblastoma; Glioma; Humans; Interleukin-8; Neovascularization, Pathologic; Point Mutation; Receptors, Interleukin-8A; Receptors, Interleukin-8B; Reference Values; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factor B; Vascular Endothelial Growth Factor Receptor-2

Chimeric antigen receptor (CAR) T-cell therapy targeting solid tumors has stagnated as a result of tumor heterogeneity, immunosuppressive microenvironments, and inadequate intratumoral T cell trafficking and persistence. Early (≤3 days) intratumoral presentation of CAR T cells post-treatment is a superior predictor of survival than peripheral persistence. Therefore, we have co-opted IL-8 release from tumors to enhance intratumoral T-cell trafficking through a CAR design for maximal antitumor activity in solid tumors. Here, we demonstrate that IL-8 receptor, CXCR1 or CXCR2, modified CARs markedly enhance migration and persistence of T cells in the tumor, which induce complete tumor regression and long-lasting immunologic memory in pre-clinical models of aggressive tumors such as glioblastoma, ovarian and pancreatic cancer.

Topics: Animals; Antigens, Neoplasm; Brain Neoplasms; Cell Line, Tumor; Cell Proliferation; Cytokines; Disease Models, Animal; Female; Glioblastoma; Humans; Immunotherapy, Adoptive; Interleukin-8; Mice, Inbred NOD; Receptors, Antigen, T-Cell; Receptors, Interleukin-8A; Receptors, Interleukin-8B; T-Lymphocytes; Tumor Microenvironment; Xenograft Model Antitumor Assays

Glioblastoma (GBM), the most aggressive brain tumor, has a poor prognosis due to the ease of migration to surrounding healthy brain tissue. Recent studies have shown that bradykinin receptors are involved in the progression of various cancers. However, the molecular mechanism and pathological role of bradykinin receptors remains unclear. We observed the expressions of two major bradykinin receptors, B1R and B2R, in two different human GBM cell lines, U87 and GBM8901. Cytokine array analysis showed that bradykinin increases the production of interleukin (IL)-8 in GBM via B1R. Higher B1R levels correlate with IL-8 expression in U87 and GBM8901. We observed increased levels of phosphorylated STAT3 and SP-1 in the nucleus as well. Using chromatin immunoprecipitation assay, we found that STAT3 and SP-1 mediate IL-8 expression, which gets abrogated by the inhibition of FAK and STAT3. We further demonstrated that IL-8 expression and cell migration are also regulated by the SP-1. In addition, expression levels of STAT3 and SP-1 positively correlate with clinicopathological grades of gliomas. Interestingly, our results found that inhibition of HDAC increases IL-8 expression. Moreover, stimulation with bradykinin caused increases in acetylated SP-1 and p300 complex formation, which are abrogated by inhibition of FAK and STAT3. Meanwhile, knockdown of SP-1 and p300 decreased the augmentation of bradykinin-induced IL-8 expression. These results indicate that bradykinin-induced IL-8 expression is dependent on B1R which causes phosphorylated STAT3 and acetylated SP-1 to translocate to the nucleus, hence resulting in GBM migration.

Topics: Acetylation; Bradykinin; Brain Neoplasms; Cell Line, Tumor; Cell Movement; Cell Nucleus; E1A-Associated p300 Protein; Gene Expression Regulation, Neoplastic; Glioblastoma; Humans; Interleukin-8; Phosphorylation; Receptor, Bradykinin B1; Receptor, Bradykinin B2; Signal Transduction; Sp1 Transcription Factor; STAT3 Transcription Factor

Exosomes communicate inter-cellularly and miRNAs play critical roles in this scenario. MiR-214-5p was implicated in multiple tumors with diverse functions uncovered. However, whether miR-214-5p is mechanistically involved in glioblastoma, especially via exosomal pathway, is still elusive. Here we sought to comprehensively address the critical role of exosomal miR-214-5p in glioblastoma (GBM) microenvironment.. The relative expression of miR-214-5p was determined by real-time PCR. Cell viability and migration were measured by MTT and transwell chamber assays, respectively. The secretory cytokines were measured with ELISA kits. The regulatory effect of miR-214-5p on CXCR5 expression was interrogated by luciferase reporter assay. Protein level was analyzed by Western blot.. We demonstrated that miR-214-5p was aberrantly overexpressed in GBM and associated with poorer clinical prognosis. High level of miR-214-5p significantly contributed to cell proliferation and migration. GBM-derived exosomal miR-214-5p promoted inflammatory response in primary microglia upon lipopolysaccharide challenge. We further identified CXCR5 as the direct target of miR-214- 5p in this setting.. Overexpression of miR-214-5p in GBM modulated the inflammatory response in microglia via exosomal transfer.

Topics: Cell Line, Tumor; Cell Movement; Cell Survival; Cells, Cultured; Exosomes; Glioblastoma; Humans; Inflammation; Interleukin-6; Interleukin-8; Lipopolysaccharides; Microglia; MicroRNAs; Primary Cell Culture; Receptors, CXCR5; Tumor Necrosis Factor-alpha

Glioblastoma multiforme (GBM) is the most aggressive brain tumor. Current therapeutic strategies are based on the use of Temozolomide (TMZ) and antihuman epidermal growth factor receptor (EGFR) drugs, such as Afatinib. However, clinically relevant drug-resistance events are still present and closely related to a proinflammatory cancer brain microenvironment. The primary aim of this study is the association of Boswellic acid (BA), a molecule derived from Boswellia Serrata, with TMZ and Afatinibin different human GBM cells. We performed cell viability studies evaluating its antioxidant and anti-inflammatory effects analyzing p65/NF-κB and Leukotriene B4 expression and production of interleukins and growth factors (IL-8, IL-6, vascular endothelial growth factor, CXCL-12, and MMP-9). Considering the cardiotoxicity of TMZ and anti-EGFR drugs, we evaluated the putative cardioprotective effects of BA in adult cardiomyocytes. BA significantly increased the anticancer activities of TMZ and Afatinib. These effects are related to its anti-inflammatory and antioxidant effects, based on the inhibition of growth factors and proinflammatory interleukins. Notably, BA exerts also cardioprotective effects in combination to both drugs. This study provides evidences of anti-inflammatory, cardioprotective, and chemo sensitizing effects of BA in glioblastoma cells giving a rationale for new translational studies based on the use of this natural molecule during conventional therapies.

Topics: Afatinib; Anti-Inflammatory Agents; Antineoplastic Agents, Phytogenic; Antioxidants; Boswellia; Cardiotonic Agents; Cell Line, Tumor; Chemokine CXCL12; ErbB Receptors; Glioblastoma; Humans; Interleukin-6; Interleukin-8; Leukotriene B4; Lipid Peroxidation; Myocytes, Cardiac; Reactive Oxygen Species; Temozolomide; Transcription Factor RelA; Triterpenes

Emerging evidence reveals enrichment of glioma-initiating cells (GICs) following therapeutic intervention. One factor known to contribute to this enrichment is cellular plasticity-the ability of glioma cells to attain multiple phenotypes. To elucidate the molecular mechanisms governing therapy-induced cellular plasticity, we performed genome-wide chromatin immunoprecipitation sequencing (ChIP-Seq) and gene expression analysis (gene microarray analysis) during treatment with standard of care temozolomide (TMZ) chemotherapy. Analysis revealed significant enhancement of open-chromatin marks in known astrocytic enhancers for interleukin-8 (IL-8) loci as well as elevated expression during anti-glioma chemotherapy. The Cancer Genome Atlas and Ivy Glioblastoma Atlas Project data demonstrated that IL-8 transcript expression is negatively correlated with GBM patient survival (p = 0.001) and positively correlated with that of genes associated with the GIC phenotypes, such as KLF4, c-Myc, and HIF2α (p < 0.001). Immunohistochemical analysis of patient samples demonstrated elevated IL-8 expression in about 60% of recurrent GBM tumors relative to matched primary tumors and this expression also positively correlates with time to recurrence. Exposure to IL-8 significantly enhanced the self-renewing capacity of PDX GBM (average threefold, p < 0.0005), as well as increasing the expression of GIC markers in the CXCR2 population. Furthermore, IL-8 knockdown significantly delayed PDX GBM tumor growth in vivo (p < 0.0005). Finally, guided by in silico analysis of TCGA data, we examined the effect of therapy-induced IL-8 expression on the epigenomic landscape of GBM cells and observed increased trimethylation of H3K9 and H3K27. Our results show that autocrine IL-8 alters cellular plasticity and mediates alterations in histone status. These findings suggest that IL-8 signaling participates in regulating GBM adaptation to therapeutic stress and therefore represents a promising target for combination with conventional chemotherapy in order to limit GBM recurrence.

Topics: Animals; Brain Neoplasms; Carcinogenesis; Cell Line, Tumor; Cell Plasticity; Drug Resistance, Neoplasm; Gene Knockdown Techniques; Glioblastoma; Histones; Humans; Interleukin-8; Kruppel-Like Factor 4; Mice; Mice, Nude; Neoplasm Recurrence, Local; Receptors, Interleukin-8B; Temozolomide; Xenograft Model Antitumor Assays

Rapid growth and perivascular invasion are hallmarks of glioblastoma (GBM) that have been attributed to the presence of cancer stem-like cells (CSCs) and their association with the perivascular niche. However, the mechanisms by which the perivascular niche regulates GBM invasion and CSCs remain poorly understood due in part to a lack of relevant model systems. To simulate perivascular niche conditions and analyze consequential changes of GBM growth and invasion, patient-derived GBM spheroids were co-cultured with brain endothelial cells (ECs) in microfabricated collagen gels. Integrating these systems with 3D imaging and biochemical assays revealed that ECs increase GBM invasiveness and growth through interleukin-8 (IL-8)-mediated enrichment of CSCs. Blockade of IL-8 inhibited these effects in GBM-EC co-cultures, while IL-8 supplementation increased CSC-mediated growth and invasion in GBM-monocultures. Experiments in mice confirmed that ECs and IL-8 stimulate intracranial tumor growth and invasion in vivo. Collectively, perivascular niche conditions promote GBM growth and invasion by increasing CSC frequency, and IL-8 may be explored clinically to inhibit these interactions.

Topics: Brain Neoplasms; Cell Line, Tumor; Coculture Techniques; Endothelial Cells; Glioblastoma; Humans; Interleukin-8; Neoplasm Invasiveness; Neoplastic Stem Cells

Abnormal activation of NF-κB signalling is a major mechanism of apoptosis resistance in glioblastoma multiforme (GBM). Therefore, better understanding of the regulation of NF-κB signalling has a significant impact for GBM therapy. Here, we uncovered a critical role of the small GTPase RND3 in regulating the p65 subunit of NF-κB and NF-κB signalling in GBM.. Human GBM samples, GBM cells and a human orthotopic GBM-xenografted animal model were used. The mechanisms of RND3 in regulation of NF-κB signalling and GBM cell apoptosis were examined by luciferase assay, quantitative PCR, immunostaining, immunoblotting, immunofluorescence, coimmunoprecipitation, TUNEL staining, JC-1 analysis and flow cytometry.. Overexpression of RND3 led to reduced p65 activity in GBM-cultured cells and a GBM animal model, indicating that the NF-κB pathway is negatively regulated by RND3 in GBM. Mechanistically, we found that RND3 bound p65 and promoted p65 ubiquitination, leading to decreased p65 protein levels. Furthermore, RND3 enhanced cleaved caspase 3 levels and promoted apoptosis in GBM cells, and RND3 expression was positively correlated with cleaved caspase 3 and IL-8 in human GBM samples. The effect of RND3 on promoting apoptosis disappeared when p65 ubiquitination was blocked by protease inhibitor carfilzomib or upon co-expression of ectopic p65.. RND3 binds p65 protein and promotes its ubiquitination, resulting in reduced p65 protein expression and inhibition of NF-κB signalling to induce GBM cell apoptosis.

Topics: Animals; Apoptosis; Brain Neoplasms; Caspase 3; Cell Line, Tumor; Gene Expression Regulation, Neoplastic; Glioblastoma; Humans; Interleukin-8; Mice; Mice, Nude; Oligopeptides; Protein Binding; rho GTP-Binding Proteins; Signal Transduction; Transcription Factor RelA; Transplantation, Heterologous; Ubiquitination

Glioblastoma multiforme is the most common and aggressive primary brain cancer with only ∼3% of patients surviving more than 3 years from diagnosis. Several mechanisms are involved in drug and radiation resistance to anticancer treatments and among them one of the most important factors is the tumor microenvironment status, characterized by cancer cell hypersecretion of interleukins and cytokines. The aim of our research was the synthesis of a nanocarrier of quercetin combined with temozolomide, to enhance the specificity and efficacy of this anticancer drug commonly used in glioblastoma treatment. The nanohydrogel increased the internalization and cytotoxicity of quercetin in human glioblastoma cells and, when co-delivered with temozolomide, contribute to an improved anticancer effect. The nanohydrogel loaded with quercetin had the ability to recognize CD44 receptor, a brain cancer cell marker, through an energy and caveolae dependent mechanism of internalization. Moreover, nanohydrogel of quercetin was able to reduce significantly IL-8, IL-6, and VEGF production in pro-inflammatory conditions with interesting implications on the mechanism of glioblastoma cells drug resistance. In summary, novel CD44 targeted polymeric based nanocarriers appear to be proficient in mediating site-specific delivery of quercetin via CD44 receptor in glioblastoma cells. This targeted therapy lead to an improved therapeutic efficacy of temozolomide by modulating the brain tumor microenvironment.

Topics: Cell Line, Tumor; Cell Proliferation; Drug Carriers; Drug Resistance, Neoplasm; Gene Expression Regulation, Neoplastic; Glioblastoma; Humans; Hyaluronan Receptors; Hyaluronic Acid; Hydrogel, Polyethylene Glycol Dimethacrylate; Interleukin-6; Interleukin-8; Molecular Targeted Therapy; Nanoparticles; Polymers; Quercetin; Temozolomide; Vascular Endothelial Growth Factor A

Glioblastoma multiforme (GBM) is one of the lethal malignant tumors of the central nervous system. Despite advances made in understanding this complex disease, little has been achieved in improving clinical efficacy towards it. Factors such as chemokines play important role in shaping the tumor microenvironment which in turn plays a significant role in deciding course of tumor progression. In this study, we investigated the role of chemokine IL-8 in glioblastoma progression with particular emphasis on immunomodulation, cellular proliferation, invasion and vascular mimicry.. Role of IL-8 in GBM immunology was determined by correlating the expression of IL-8 by immunohistochemistry with other immune cell markers such as CD3 and CD68. Effect of high IL-8 expression on overall survival, the difference in expression level between different GBM subgroups and anatomic structures were analyzed using other databases. Two GBM cell lines -U-87MG and LN-18 were used to study the impact of targeting IL-8-CXCR1/2 signalling using neutralizing antibodies and pharmacological antagonist. Reverse transcriptase-polymerase chain reaction and immunocytochemistry were used to determine the expression of these axes. Impact on cell viability and proliferation was assessed by MTT, proliferation marker-ki-67 and clonogenic survival assays. Multicellular tumor spheroids generated from GBM cell lines were used to study invasion in matrigel.. Weak Positive correlation was observed between IL-8 and CD3 as well as between IL-8 and CD68. High IL-8 expression in GBM patients was found to be associated with dismal survival. No significant difference in IL-8 expression between different molecular subgroups of GBM was observed. In vitro targeting of IL-8-CXCR1/2 signalling displayed a significant reduction in cell viability and proliferation, and spheroid invasion. Furthermore, the presence of CD34-/CXCR1+ vessels in GBM tissues showed the involvement of IL-8/CXCR1 in vascular mimicry structure formation.. These results suggest a direct involvement of IL-8-CXCR1/2 axes in GBM progression by promoting both cell proliferation and invasion and indirectly by promoting neovascularization in the form of vascular mimicry.

Topics: Apoptosis; Cell Line, Tumor; Cell Movement; Cell Proliferation; Disease Progression; Female; Gene Expression Regulation, Neoplastic; Glioblastoma; Humans; Interleukin-8; Male; Neoplasm Invasiveness; Neovascularization, Pathologic; Receptors, Interleukin-8A; Receptors, Interleukin-8B; Signal Transduction

Glioblastoma (GBM) was shown to relapse faster and displayed therapeutic resistance to antiangiogenic therapies (AATs) through an alternative tumor cell-driven mechanism of neovascularization called vascular mimicry (VM). We identified highly upregulated interleukin 8 (IL-8)-CXCR2 axis in tumor cells in high-grade human glioma and AAT-treated orthotopic GBM tumors.. Human GBM tissue sections and tissue array were used to ascertain the clinical relevance of CXCR2-positive tumor cells in the formation of VM. We utilized U251 and U87 human tumor cells to understand VM in an orthotopic GBM model and AAT-mediated enhancement in VM was modeled using vatalanib (anti-VEGFR2) and avastin (anti-VEGF). Later, VM was inhibited by SB225002 (CXCR2 inhibitor) in a preclinical study.. Overexpression of IL8 and CXCR2 in human datasets and histological analysis was identified as a bonafide candidate to validate VM through in vitro and animal model studies. AAT-treated tumors displayed a higher number of CXCR2-positive GBM-stem cells with endothelial-like phenotypes. Stable knockdown of CXCR2 expression in tumor cells led to decreased tumor growth as well as incomplete VM structures in the animal models. Similar data were obtained following SB225002 treatment.. The present study suggests that tumor cell autonomous IL-8-CXCR2 pathway is instrumental in AAT-mediated resistance and VM formation in GBM. Therefore, CXCR2 can be targeted through SB225002 and can be combined with standard therapies to improve the therapeutic outcomes in clinical trials.

Topics: Angiogenesis Inhibitors; Animals; Bevacizumab; Brain Neoplasms; Glioblastoma; Humans; Interleukin-8; Molecular Targeted Therapy; Neovascularization, Pathologic; Phenylurea Compounds; Phthalazines; Pyridines; Rats, Nude; Receptors, Interleukin-8B; Tissue Array Analysis; Tumor Burden; Xenograft Model Antitumor Assays

Gabapentin (GBP) and pregabalin (PGB) exert antinociceptive effects on chronic nociceptive responses with neuropathic or inflammatory conditions. Furthermore, it is considered that GBP and PGB exhibit anti‑inflammatory effects by modulating the substance P (SP)‑mediated neurokinin‑1 receptor (NK1R; a SP receptor) response. Thus, in the present study, the effects of GBP and PGB on SP‑induced activation were investigated in the human glioblastoma astrocytoma U373 MG cell line, which expresses high levels of functional high‑affinity NK1R, and produces interleukin (IL)‑6 and IL‑8 in response to SP. The results indicated that GBP and PGB suppressed the SP‑induced production of IL‑6, and IL‑8 in U373 MG cells. Furthermore, GBP and PGB inhibited the SP‑induced phosphorylation of p38 mitogen‑activated protein kinase (MAPK) and nuclear factor (NF)‑κB, and the nuclear translocation of NF‑κB in U373 MG cells. Together, these observations suggest that GBP and PGB likely prevent SP‑induced IL‑6 and IL‑8 production in U373 MG cells via the inhibition of signaling molecules, including p38 MAPK and NF‑κB, thereby exhibiting antineuroinflammatory effects.

Topics: Amines; Anti-Inflammatory Agents; Astrocytoma; Cell Line, Tumor; Cyclohexanecarboxylic Acids; Gabapentin; gamma-Aminobutyric Acid; Glioblastoma; Humans; Interleukin-6; Interleukin-8; MAP Kinase Signaling System; NF-kappa B; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Pregabalin; Receptors, Neurokinin-1; Substance P

The neuropeptide substance P (SP) is an important mediator of neurogenic inflammation within the central and peripheral nervous systems. SP has been shown to induce the expression of pro-inflammatory cytokines implicated in the pathogenesis of several disorders of the human brain via the neurokinin-1 receptor (NK-1R). Ketamine, an intravenous anesthetic agent, functions as a competitive antagonist of the excitatory neurotransmission N-methyl-D‑aspartate (NMDA) receptor, and also antagonizes the NK-1R by interfering with the binding of SP. In the present study, we investigated the anti-inflammatory effects of ketamine on the SP-induced activation of a human astrocytoma cell line, U373MG, which expresses high levels of NK-1R. The results from our experiments indicated that ketamine suppressed the production of interleukin (IL)-6 and IL-8 by the U373MG cells. Furthermore, ketamine inhibited the SP-induced activation of extracellular signal‑regulated kinase (ERK)1/2, p38 mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB). Taken together, these observations suggest that ketamine may suppress the SP-induced activation (IL-6 and IL-8 production) of U373MG cells by inhibiting the phosphorylation of signaling molecules (namely ERK1/2, p38 MAPK and NF-κB), thereby exerting anti‑inflammatory effects. Thus, ketamine may modulate SP-induced inflammatory responses by NK-1R‑expressing cells through the suppression of signaling molecules (such as ERK1/2, p38 MAPK and NF-κB).

Topics: Astrocytoma; Cell Line, Tumor; Enzyme Activation; Extracellular Signal-Regulated MAP Kinases; Glioblastoma; Humans; Interleukin-6; Interleukin-8; Ketamine; MAP Kinase Signaling System; NF-kappa B; p38 Mitogen-Activated Protein Kinases; Substance P

Glioblastoma multiforme (GBM) is the most common primary intracranial tumor in adults and has poor prognosis. Diffuse infiltration into normal brain parenchyma, rapid growth, and the presence of necrosis are remarkable hallmarks of GBM. However, the effect of necrotic cells on GBM growth and metastasis is poorly understood at present. In this study, we examined the biological significance of necrotic tissues by exploring the molecular mechanisms underlying the signaling network between necrotic tissues and GBM cells. The migration and invasion of the GBM cell line CRT-MG was significantly enhanced by treatment with necrotic cells, as shown by assays for scratch wound healing and spheroid invasion. Incubation with necrotic cells induced IL-8 secretion in CRT-MG cells in a dose-dependent manner. In human GBM tissues, IL-8 positive cells were mainly distributed in the perinecrotic region, as seen in immunohistochemistry and immunofluorescence analysis. Necrotic cells induced NF-κB and AP-1 activation and their binding to the IL-8 promoter, leading to enhanced IL-8 production and secretion in GBM cells. Our data demonstrate that when GBM cells are exposed to and stimulated by necrotic cells, the migration and invasion of GBM cells are enhanced and facilitated via NF-κB/AP-1 mediated IL-8 upregulation.

Topics: Brain Neoplasms; Cell Line; Cell Movement; Gene Expression Regulation; Glioblastoma; Humans; Immunohistochemistry; Interleukin-8; Microscopy, Fluorescence; Models, Biological; Necrosis; Neoplasm Invasiveness; NF-kappa B; Protein Interaction Maps; Transcription Factor AP-1

Natural killer (NK) cells are functionally suppressed in the glioblastoma multiforme (GBM) tumor microenvironment. We have recently shown that survival and differentiation of cancer stem-like cells (CSCs)/poorly differentiated tumors are controlled through two distinct phenotypes of cytotoxic and non-cytotoxic/split anergized NK cells, respectively. In this paper, we studied the function of NK cells against brain CSCs/poorly differentiated GBM and their NK cell-differentiated counterparts. Brain CSCs/poorly differentiated GBM, differentiated by split anergized NK supernatants (supernatants from NK cells treated with IL-2 + anti-CD16mAb) expressed higher levels of CD54, B7H1 and MHC-I and were killed less by the NK cells, whereas their CSCs/poorly differentiated counterparts were highly susceptible to NK cell lysis. Resistance to NK cells and differentiation of brain CSCs/poorly differentiated GBM by split anergized NK cells were mediated by interferon (IFN)-γ and tumor necrosis factor (TNF)-α. Brain CSCs/poorly differentiated GBM expressed low levels of TNFRs and IFN-γRs, and when differentiated and cultured with IL-2-treated NK cells, they induced increased secretion of pro-inflammatory cytokine interleukin (IL)-6 and chemokine IL-8 in the presence of decreased IFN-γ secretion. NK-induced differentiation of brain CSCs/poorly differentiated GBM cells was independent of the function of IL-6 and/or IL-8. The inability of NK cells to lyse GBM tumors and the presence of a sustained release of pro-inflammatory cytokines IL-6 and chemokine IL-8 in the presence of a decreased IFN-γ secretion may lead to the inadequacy of NK cells to differentiate GBM CSCs/poorly differentiated tumors, thus failing to control tumor growth.

Topics: Brain Neoplasms; Cell Communication; Cell Differentiation; Cell Line, Tumor; Cytotoxicity, Immunologic; Glioblastoma; Humans; Interferon-gamma; Interleukin-2; Interleukin-6; Interleukin-8; Killer Cells, Natural; Neoplastic Stem Cells

The expression levels of tissue factor (TF), the clotting initiator protein, have been correlated with angiogenesis and the histological grade of malignancy in glioma patients. The pro-tumor function of TF is linked to a family of G protein-coupled receptors known as protease-activated receptors (PARs), which may be activated by blood coagulation proteases. Activation of PARs elicits a number of responses, including the expression of vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8). In the present study, we analyzed the expression of TF signaling pathway elements (TF, PAR1 and PAR2) and evaluated their correlation with the expression of downstream products (VEGF and IL-8) in human astrocytoma patients. Quantitative PCR (qPCR) showed a significant increase in TF expression in grade IV (glioblastoma) tumors, which was inversely correlated with the expression of the tumor-suppressor PTEN. Immunohistochemistry and qPCR analyses demonstrated a highly significant elevation in the expression of PAR1, but not PAR2, in tumor samples from high-grade astrocytoma patients. The elevated VEGF expression levels detected in the high-grade astrocytoma samples were positively correlated with TF, PAR1 and PAR2 expression. In addition, IL-8 was significantly increased in glioblastoma patients and positively correlated with TF and PAR2 expression. Further in vitro assays employing the human glioma cell lines U87-MG and HOG demonstrated that a synthetic peptide PAR2 agonist stimulated VEGF and IL-8 production. Our findings suggest a role for TF signaling pathway elements in astrocytoma progression, particularly in glioblastoma. Therefore, TF/PAR signaling elements may be suitable targets for the development of new therapies for the treatment of aggressive glioma.

Topics: Brain Neoplasms; Glioblastoma; Humans; Interleukin-8; Neovascularization, Pathologic; PTEN Phosphohydrolase; Receptor, PAR-1; Receptor, PAR-2; Signal Transduction; Thromboplastin; Vascular Endothelial Growth Factors

IL-32 is a multifaceted cytokine with a role in infections, autoimmune diseases, and cancer, and it exerts diverse functions, including aggravation of inflammation and inhibition of virus propagation. We previously identified IL-32 as a critical regulator of endothelial cell (EC) functions, and we now reveal that IL-32 also possesses angiogenic properties. The hyperproliferative ECs of human pulmonary arterial hypertension and glioblastoma multiforme exhibited a markedly increased abundance of IL-32, and, significantly, the cytokine colocalized with integrin αVβ3. Vascular endothelial growth factor (VEGF) receptor blockade, which resulted in EC hyperproliferation, increased IL-32 three-fold. Small interfering RNA-mediated silencing of IL-32 negated the 58% proliferation of ECs that occurred within 24 h in scrambled-transfected controls. Reduction of IL-32 neither affected apoptosis (insignificant changes in Bak-1, Bcl-2, Bcl-xL, lactate dehydrogenase, annexin V, and propidium iodide) nor VEGF or TGF-β levels, but siIL-32-transfected adult and neonatal ECs produced up to 61% less NO, IL-8, and matrix metalloproteinase-9, and up to 3-fold more activin A and endostatin. In coculture-based angiogenesis assays, IL-32γ dose-dependently increased tube formation up to 3-fold; an αVβ3 inhibitor prevented this activity and reduced IL-32γ-induced IL-8 by 85%. In matrigel plugs loaded with IL-32γ, VEGF, or vehicle and injected into live mice, we observed the anticipated VEGF-induced increase in neocapillarization (8-fold versus vehicle), but unexpectedly, IL-32γ was equally angiogenic. A second signal such as IFN-γ was required to render cells responsive to exogenous IL-32γ; importantly, this was confirmed using a completely synthetic preparation of IL-32γ. In summary, we add angiogenic properties that are mediated by integrin αVβ3 but VEGF-independent to the portfolio of IL-32, implicating a role for this versatile cytokine in pulmonary arterial hypertension and neoplastic diseases.

Topics: Activins; Animals; Apoptosis; Cells, Cultured; Endostatins; Familial Primary Pulmonary Hypertension; Glioblastoma; Human Umbilical Vein Endothelial Cells; Humans; Hypertension, Pulmonary; Integrin alphaVbeta3; Interferon-gamma; Interleukin-8; Interleukins; Matrix Metalloproteinase 9; Mice; Neovascularization, Pathologic; Nitrogen Oxides; Receptors, Vascular Endothelial Growth Factor; Transforming Growth Factor beta1; Vascular Endothelial Growth Factor A

Glioblastoma multiforme (GBM) represents a very aggressive brain tumor. Angiogenesis is the formation of a network of new blood vessels, from preexisting ones. It plays an important role in the formation of the tumor, as it supplies it with oxygen and nutrients. Angiogenesis and inflammation play essential roles in glioblastoma development. These processes are regulated by the balance of a few molecules, acting as pro- or antiangiogenic and pro- or anti-inflammatory factors. The purpose of our study was to evaluate the expression of 7 markers involved in angiogenesis and inflammation pathways in patients with glioblastoma. VEGF, PDGF-bb, IGF-1, TGF-β, TNF-α, IL-6 and IL-8 levels were measured using the ELISA method, in the preoperative sera of 14 patients with histopathologically confirmed glioblastoma multiforme and 32 healthy patients. Serum levels of PDGF-bb, IGF-1 and IL-8 were significantly higher in patients with GBM, compared to the control group (p-value < 0.01). A statistically significant correlation has been found between IGF-1 and IL-6 levels (rho= -0.53, p-value < 0.05) and also between TNF-α and IL-6 levels (rho=0.60, p-value < 0.05). Statistically significant associations have been found between the presence of low levels of IL-8 and the development of coagulation necrosis (p-value < 0.05), high levels of VEGF and development of ischemic necrosis (p-value < 0.01) and high levels of IL-8 and the development of endothelial hyperplasia (p-value < 0.05). We have observed no statistically significant associations between the serum levels of the markers and the survival rates.

Topics: Becaplermin; Biomarkers; Brain Neoplasms; Case-Control Studies; Disease-Free Survival; Glioblastoma; Humans; Inflammation; Insulin-Like Growth Factor I; Interleukin-6; Interleukin-8; Neovascularization, Physiologic; Predictive Value of Tests; Proto-Oncogene Proteins c-sis; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha; Vascular Endothelial Growth Factor A

We recently found that neurotensin (NTS) and its primary receptor NTSR1 play a crucial role in glioblastoma cell proliferation and invasion. However, very little is known regarding the functional role of NTS/NTSR1 signaling in glioblastoma stem cells (GSCs). Here, we showed that NTSR1 is highly expressed in GSCs than its non-GSC counterparts. Pharmacological blockade with SR48692 or lentivirus mediated knockdown of NTSR1 efficiently reduced the sphere-forming ability and expression of stem cell markers such as nestin and Sox2 in GSCs isolated from glioblastoma cell line and glioblastoma tissues. Conversely, treated GSCs with NTS led to increase of tumor sphere formation. Mechanistically, we demonstrated that EGFR-dependent enhancement of IL-8 secretion is responsible for the effect of NTS signaling in the regulation of stem-like traits. Finally, we showed that NTSR1 or IL-8 knockdown decreased the phosphorylation of transcriptional factor STAT3 at Tyr705, which is a major transcription factor implicated in the regulation of GSC stem-like traits. Although both CXCR1 and CXCR2 inhibition reduced the tumor sphere formation, we found that CXCR1, but not CXCR2, is primarily responsible for STAT3 phosphorylation. Taken together, our findings suggest that NTS/IL-8/CXCR1/STAT3 signaling is crucial for the maintenance of stem-like traits in GSCs and provides a potential therapeutic target for glioblastoma therapy.

Topics: Brain Neoplasms; Cell Line, Tumor; ErbB Receptors; Gene Expression Regulation, Neoplastic; Glioblastoma; Humans; Interleukin-8; Models, Biological; Neoplastic Stem Cells; Neurotensin; Phosphorylation; Receptors, Interleukin-8A; Receptors, Neurotensin; Signal Transduction; STAT3 Transcription Factor

Glioblastoma multiforme contains a subpopulation of cancer stem-like cells (CSC) believed to underlie tumorigenesis and therapeutic resistance. Recent studies have localized CSCs in this disease adjacent to endothelial cells (EC) in what has been termed a perivascular niche, spurring investigation into the role of EC-CSC interactions in glioblastoma multiforme pathobiology. However, these studies have been limited by a lack of in vitro models of three-dimensional disease that can recapitulate the relevant conditions of the niche. In this study, we engineered a scaffold-based culture system enabling brain endothelial cells to form vascular networks. Using this system, we showed that vascular assembly induces CSC maintenance and growth in vitro and accelerates tumor growth in vivo through paracrine interleukin (IL)-8 signaling. Relative to conventional monolayers, endothelial cells cultured in this three-dimensional system not only secreted enhanced levels of IL-8 but also induced CSCs to upregulate the IL-8 cognate receptors CXCR1 and CXCR2, which collectively enhanced CSC migration, growth, and stemness properties. CXCR2 silencing in CSCs abolished the tumor-promoting effects of endothelial cells in vivo, confirming a critical role for this signaling pathway in GMB pathogenesis. Together, our results reveal synergistic interactions between endothelial cells and CSCs that promote the malignant properties of CSCs in an IL-8-dependent manner. Furthermore, our findings underscore the relevance of tissue-engineered cell culture platforms to fully analyze signaling mechanisms in the tumor microenvironment.

Topics: Animals; Blood Vessels; Cell Culture Techniques; Cells, Cultured; Endothelium, Vascular; Glioblastoma; Humans; Interleukin-8; Male; Mice, Inbred C57BL; Mice, SCID; Neoplastic Stem Cells; Paracrine Communication; Stem Cell Niche; Tissue Scaffolds; Tumor Microenvironment

Sustaining a high growth rate requires tumors to exploit resources in their microenvironment. One example of this is the extensive angiogenesis that is a typical feature of high-grade gliomas. Here, we show that expression of the constitutively active mutant epidermal growth factor receptor, ΔEGFR (EGFRvIII, EGFR*, de2-7EGFR) is associated with significantly higher expression levels of the pro-angiogenic factor interleukin (IL)-8 in human glioma specimens and glioma stem cells. Furthermore, the ectopic expression of ΔEGFR in different glioma cell lines caused up to 60-fold increases in the secretion of IL-8. Xenografts of these cells exhibit increased neovascularization, which is not elicited by cells overexpressing wild-type (wt)EGFR or ΔEGFR with an additional kinase domain mutation. Analysis of the regulation of IL-8 by site-directed mutagenesis of its promoter showed that ΔEGFR regulates its expression through the transcription factors nuclear factor (NF)-κB, activator protein 1 (AP-1) and CCAAT/enhancer binding protein (C/EBP). Glioma cells overexpressing ΔEGFR showed constitutive activation and DNA binding of NF-κB, overexpression of c-Jun and activation of its upstream kinase c-Jun N-terminal kinase (JNK) and overexpression of C/EBPβ. Selective pharmacological or genetic targeting of the NF-κB or AP-1 pathways efficiently blocked promoter activity and secretion of IL-8. Moreover, RNA interference-mediated knock-down of either IL-8 or the NF-κB subunit p65, in ΔEGFR-expressing cells attenuated their ability to form tumors and to induce angiogenesis when injected subcutaneously into nude mice. On the contrary, the overexpression of IL-8 in glioma cells lacking ΔEGFR potently enhanced their tumorigenicity and produced highly vascularized tumors, suggesting the importance of this cytokine and its transcription regulators in promoting glioma angiogenesis and tumor growth.

Topics: Animals; CCAAT-Enhancer-Binding Proteins; Cell Line, Tumor; Cell Proliferation; ErbB Receptors; Female; Gene Expression Regulation, Neoplastic; Glioblastoma; Human Umbilical Vein Endothelial Cells; Humans; Interleukin-8; JNK Mitogen-Activated Protein Kinases; MAP Kinase Signaling System; Mice; Mice, Nude; Neoplasm Transplantation; Neoplastic Stem Cells; Neovascularization, Pathologic; NF-kappa B; Nitriles; Phosphatidylinositol 3-Kinases; Phosphorylation; Protein Kinase Inhibitors; Protein Processing, Post-Translational; ras Proteins; Response Elements; Sulfones; Transcription Factor AP-1; Transcriptional Activation; Tumor Burden

Increasing evidence suggests that an inflammatory microenvironment promotes invasion by glioblastoma (GBM) cells. Together with p38 mitogen-activated protein kinase (MAPK) activation being regarded as promoting inflammation, we hypothesized that elevated inflammatory cytokine secretion and p38 MAPK activity contribute to expansion of GBMs. Here we report that IL-1β, IL-6, and IL-8 levels and p38 MAPK activity are elevated in human glioblastoma specimens and that p38 MAPK inhibitors attenuate the secretion of pro-inflammatory cytokines by microglia and glioblastoma cells. RNAi knockdown and immunoprecipitation experiments suggest that the p38α MAPK isoform drives inflammation in GBM cells. Importantly, p38 MAPK inhibition strongly reduced invasion of U251 glioblastoma cells in an inflammatory microenvironment, providing evidence for a p38 MAPK-regulated link between inflammation and invasiveness in GBM pathophysiology.

Topics: Apoptosis; Blotting, Western; Brain Neoplasms; Case-Control Studies; Cell Movement; Enzyme Inhibitors; Flow Cytometry; Glioblastoma; Humans; Immunoprecipitation; Inflammation Mediators; Interleukin-1beta; Interleukin-6; Interleukin-8; Lipopolysaccharides; Mitogen-Activated Protein Kinase 14; Neoplasm Invasiveness; RNA, Small Interfering; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha; Wound Healing

Glioblastoma constitutes the most aggressive and deadly of brain tumors. As yet, both conventional and molecular-based therapies have met with limited success in treatment of this cancer. Among other explanations, the heterogeneity of glioblastoma and the associated microenvironment contribute to its development, as well as resistance and recurrence in response to treatments. Increased vascularity suggests that tumor angiogenesis plays an important role in glioblastoma progression. However, the molecular crosstalk between endothelial and glioblastoma cells requires further investigation. To examine the effects of glioblastoma-derived signals on endothelial homeostasis, glioblastoma cell secretions were collected and used to treat brain endothelial cells. Here, we present evidence that the glioblastoma secretome provides pro-angiogenic signals sufficient to disrupt VE-cadherin-mediated cell-cell junctions and promote endothelial permeability in brain microvascular endothelial cells. An unbiased angiogenesis-specific antibody array screen identified the chemokine, interleukin-8, which was further demonstrated to function as a key factor involved in glioblastoma-induced permeability, mediated through its receptor CXCR2 on brain endothelia. This underappreciated interface between glioblastoma cells and associated endothelium may inspire the development of novel therapeutic strategies to induce tumor regression by preventing vascular permeability and inhibiting angiogenesis.

Topics: Brain Neoplasms; Capillary Permeability; Cell Line, Tumor; Culture Media, Conditioned; Endothelial Cells; Gene Expression Regulation, Neoplastic; Glioblastoma; Humans; Interleukin-8; Receptors, Interleukin-8B

We have recently reported that in astrocytoma cells the expression of interleukin-8 (IL-8) is upregulated by prostaglandin E2 (PGE2). Unfortunately, the exact mechanism by which this happens has not been clarified yet. Here, we have investigated whether IL-8 activation by PGE2 involves changes in DNA methylation and/or histone modifications in 46 astrocytoma specimens, two astrocytoma cell lines and normal astrocytic cells. The DNA methylation status of the IL-8 promoter was analyzed by bisulphite sequencing and by methylation DNA immunoprecipitation analysis. The involvement of DNA methyltransferases (DNMTs) and histone deacetylases (HDACs), as well as histone acetylation levels, was assayed by chromatin immunoprecipitation. IL-8 expression at promoter, mRNA and protein level was explored by transfection, real-time PCR and enzyme immunoassay experiments in cells untreated or treated with PGE2, 5-aza-2'-deoxycytidine (5-aza-dC) and HDAC inhibitors, alone or in combination. EMSA was performed with crude cell extracts or recombinant protein. We observed that PGE2 induced IL-8 activation through: (1) demethylation of the single CpG site 5 located at position -83 within the binding region for CEBP-β in the IL-8 promoter; (2) C/EBP-β and p300 co-activator recruitment; (3) H3 acetylation enhancement and (4) reduction in DNMT1, DNMT3a, HDAC2 and HDAC3 association to CpG site 5 region. Treatment with 5-aza-dC or HDAC inhibitors of class I HDACs strengthened either basal or PGE2-mediated IL-8 expression. These findings have elucidated an orchestrated mechanism triggered by PGE2 whereby concurrent association of site-specific demethylation and histone H3 hyperacetylation resulted in derepression of IL-8 gene expression in human astrocytoma.

Topics: Acetylation; Adolescent; Adult; Base Sequence; Brain Neoplasms; Cell Line, Tumor; CpG Islands; Dinoprostone; DNA (Cytosine-5-)-Methyltransferase 1; DNA (Cytosine-5-)-Methyltransferases; DNA Methylation; DNA Methyltransferase 3A; DNA, Neoplasm; Female; Gene Expression Regulation, Neoplastic; Glioblastoma; Histone Deacetylase 2; Histone Deacetylase Inhibitors; Histone Deacetylases; Histones; Humans; Interleukin-8; Male; Middle Aged; Molecular Sequence Data; Promoter Regions, Genetic; Protein Processing, Post-Translational; RNA, Messenger; Sequence Analysis, DNA

Although cyclophilin A (CypA) has been reported to be over-expressed in cancer cells and solid tumors, its expression and role in glioblastomas have not been studied. Herein, we show that expression of CypA in human glioblastoma cell lines and tissues is significantly higher than in normal human astrocytes and normal counterparts of brain tissue. To determine the role of over-expressed CypA in glioblastoma, stable RNA interference (RNAi)-mediated knockdown of CypA (CypA KD) was performed in gliobastoma cell line U87vIII (U87MG · ΔEGFR). CypA KD stable single clones decrease proliferation, infiltration, migration, and anchorage-independent growth in vitro and with slower growth in vivo as xenografts in immunodeficient nude mice. We have also observed that knockdown of CypA inhibits expression of interleukin-8 (IL-8), a tumorigenic and proangiogenic cytokine. Conversely, enforced expression of CypA in the CypA KD cell line, Ud-12, markedly enhanced IL-8 transcripts and restored Ud-12 proliferation, suggesting that CypA-mediated IL-8 production provides a growth advantage to glioblastoma cells. CypA knockdown-mediated inhibition of IL-8 is due to reduced activity of NF-κB, which is one of the major transcription factors regulating IL-8 expression. These results not only establish the relevance of CypA to glioblastoma growth in vitro and in vivo, but also suggest that small interfering RNA-based CypA knockdown could be an effective therapeutic approach against glioblastomas.

Topics: Animals; Cell Line, Tumor; Cell Proliferation; Cyclophilin A; Down-Regulation; Electrophoretic Mobility Shift Assay; Enzyme-Linked Immunosorbent Assay; Glioblastoma; Humans; Immunohistochemistry; Interleukin-8; Mice; Mice, Nude; NF-kappa B; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; Tissue Array Analysis

Glioblastoma (GBM) is the most common and deadly form of primary brain tumor with a median survival of eleven months, despite use of extensive chemotherapy, radiotherapy and surgery. We have previously shown that nuclear factor-kappa B (NF-κB) is aberrantly expressed in GBM tumors and primary cell lines derived from tumor tissue. Here we show that IL-8, a chemokine is also aberrantly expressed by GBM cell lines and expression of IL-8 is in large part, attributable to the aberrant activation of NF-κB. We hypothesized that invasiveness of GBM cells is driven at least in part by aberrantly expressed IL-8. In support of the hypothesis we found that treatment of glioma cells with an IL-8 neutralizing antibody markedly decreased their invasiveness compared to cells treated with control IgG or left untreated. Furthermore, downregulation of IL-8 protein production with use of IL-8 targeted siRNA also resulted in decreased invasion in matrigel. We next investigated the presence of IL-8 receptors by FACS analysis and found that GBM cells (U87, U251, D54 and LN229) only express CXCR1 but not CXCR2. Treatment of U87 cells with a blocking CXCR1 antibody reduced their invasion through matrigel. Finally, we found that addition of exogenous IL-8, following downregulation of NF-κB which results in loss of endogenous IL-8 production, incompletely restored tumor cell invasion. Our data indicate that IL-8 is necessary but not solely responsible for glioma cell invasion and mediates its effect in an autocrine manner.

Topics: Antibodies; Cell Line, Tumor; Enzyme-Linked Immunosorbent Assay; Flow Cytometry; Gene Expression Regulation, Neoplastic; Gene Transfer Techniques; Glioblastoma; Humans; Interleukin-8; Neoplasm Invasiveness; NF-kappa B; Receptors, Interleukin-8A; Receptors, Interleukin-8B; RNA, Small Interfering; Time Factors

Angiogenesis is a key pathologic feature of glioblastoma, which is the most common and most lethal primary brain tumor in adults. The degree of angiogenesis has been shown to be inversely related to patient survival. However, the molecular changes leading to angiogenesis in glioblastoma remain poorly understood. In the present study, we found a direct correlation between nuclear factor (NF)-kappaB activation and angiogenesis in glioblastomas. Blockade of NF-kappaB signaling significantly inhibited glioblastoma growth and angiogenesis in nude mice. These effects were consistent with significant inhibition of the expression of multiple angiogenic molecules, including vascular endothelial growth factor, and interleukin-8, in vitro and in vivo. Furthermore, blockade of NF-kappaB signaling also significantly inhibited the angiogenic potential of glioblastoma cells in vitro and angiogenesis of brain tumors in mouse xenograft models. Collectively, these results suggest that NF-kappaB activation plays a critical role in the growth and progression of glioblastoma and is a potential target for therapy for human glioblastoma.

Topics: Animals; Cell Line, Tumor; Female; Gene Expression Regulation, Neoplastic; Glioblastoma; Humans; Interleukin-8; Mice; Mice, Inbred BALB C; Neovascularization, Pathologic; NF-kappa B; RNA, Messenger; Vascular Endothelial Growth Factor A

Exposure of cells to ionising radiation causes the release of several factors, such as cytokines, which are likely to be involved in some biological effects occurring in the irradiated cells and in the neighbouring non-irradiated cells (i.e. bystander effect).. The release of interleukin (IL)-6 and IL-8 in the culture medium of irradiated human glioblastoma cells was investigated using an ELISA technique. Immunocytochemistry was used to investigate the expression of corresponding cell membrane receptors in irradiated cells and in cells cultured with medium collected from irradiated cells.. The exposure to radiation determined an increase of IL-6 concentration which was dose dependent at 20 hours, whereas IL-8 release was lower than control shortly after irradiation but increased with time, in particular at the dose of 0.5.. Our data suggest that these cytokines are differently modulated by radiation and are likely to play a role in the transmission of radiation-induced response, probably orchestrating the inflammatory microenvironment of the tumour.

Topics: Cell Line, Tumor; Culture Media; Enzyme-Linked Immunosorbent Assay; Gamma Rays; Glioblastoma; Humans; Immunohistochemistry; Interleukin-6; Interleukin-8; Signal Transduction

The brain tumor glioblastoma (GBM) remains one of the most aggressive and devastating tumors despite decades of effort to find more effective treatments. A hallmark of GBM is the constitutive activation of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kappaB) signaling pathway, which regulates cell proliferation, inflammation, migration and apoptosis. The prolyl isomerase, Pin1, has been found to bind directly to the NF-kappaB protein, p65, and cause increases in NF-kappaB promoter activity in a breast cancer model. We now present evidence that this interaction occurs in GBM and that it has important consequences on NF-kappaB signaling. We demonstrate that Pin1 levels are enhanced in primary GBM tissues compared with controls, and that this difference in Pin1 expression affects the migratory capacity of GBM-derived cells. Pin1 knockdown decreases the amount of activated, phosphorylated p65 in the nucleus, resulting in inhibition of the transcriptional program of the IL-8 gene. Through the use of microarray, we also observed changes in the expression levels of other NF-kappaB regulated genes due to Pin1 knockdown. Taken together, these data suggest that Pin1 is an important regulator of NF-kappaB in GBM, and support the notion of using Pin1 as a therapeutic target in the future.

Topics: Animals; Brain Neoplasms; Cell Line, Tumor; Cell Movement; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Glioblastoma; Humans; Interleukin-8; Inverted Repeat Sequences; Mice; NF-kappa B; NIMA-Interacting Peptidylprolyl Isomerase; Peptidylprolyl Isomerase; Phosphorylation; Promoter Regions, Genetic; RNA, Messenger; Signal Transduction; Tetracycline; Transcription Factor RelA

Inactivation of the tumor suppressor phosphatase and tensin homolog (mutated in multiple advanced cancers 1) (PTEN) is recognized as a major event in the pathogenesis of the brain tumor glioblastoma. However, the mechanisms by which PTEN loss specifically impacts the malignant behavior of glioblastoma cells, including their proliferation and propensity for invasiveness, remain poorly understood. Genetic studies suggest that the transcription factor signal transducers and activators of transcription 3 (STAT3) harbors a PTEN-regulated tumor suppressive function in mouse astrocytes. Here, we report that STAT3 plays a critical tumor suppressive role in PTEN-deficient human glioblastoma cells. Endogenous STAT3 signaling is specifically inhibited in PTEN-deficient glioblastoma cells. Strikingly, reactivation of STAT3 in PTEN-deficient glioblastoma cells inhibits their proliferation, invasiveness, and ability to spread on myelin. We also identify the chemokine interleukin 8 (IL8) as a novel target gene of STAT3 in human glioblastoma cells. Activated STAT3 occupies the endogenous IL8 promoter and directly represses IL8 transcription. Consistent with these results, IL8 is upregulated in PTEN-deficient human glioblastoma tumors. Importantly, IL8 repression mediates STAT3 inhibition of glioblastoma cell proliferation, invasiveness, and spreading on myelin. Collectively, our findings uncover a novel link between STAT3 and IL8, the deregulation of which plays a key role in the malignant behavior of PTEN-deficient glioblastoma cells. These studies suggest that STAT3 activation or IL8 inhibition may have potential in patient-tailored treatment of PTEN-deficient brain tumors.

Topics: Cell Line; Cell Line, Tumor; Cell Proliferation; Gene Targeting; Glioblastoma; Growth Inhibitors; Humans; Interleukin-8; Neoplasm Invasiveness; PTEN Phosphohydrolase; Signal Transduction; STAT3 Transcription Factor; Tumor Suppressor Proteins

Focal necrosis is a key pathologic feature that distinguishes glioblastoma from lower grade glioma. The presence of necrosis in a glioblastoma could promote its rapid growth and clinical progression. Focal necrosis of glioblastoma seems to be associated with thrombosis that result from hyper-coagulability. In the present study, we found that glioblastoma cells had a high level of constitutive nuclear factor (NF)-kappaB activity, which was directly correlated with necrosis in glioblastomas. We also found a direct correlation between NF-kappaB activity and the expression of tissue factor (TF), a potent procoagulant factor in gliomas. Inhibition of TF by an inhibitory antibody prevented the procoagulant activity of glioblastoma cells, indicating a TF-dependent mechanism. Blockade of NF-kappaB activation significantly inhibited TF expression and the procoagulant activity of glioblastoma cells in vitro. Blockade of NF-kappaB activation also significantly inhibited in vivo expression of TF, which was directly correlated with decreased necrosis formation and tumor growth of glioblastoma cells in nude mice. Collectively, these results suggest that elevated NF-kappaB activity in glioblastomas cells plays a critical role in necrosis formation of glioblastoma and that inhibition of NF-kappaB activity in glioblastoma can suppress necrosis formation and progressive growth.

Topics: Cell Line, Tumor; Glioblastoma; Humans; I-kappa B Proteins; Immunohistochemistry; Interleukin-8; Necrosis; NF-kappa B; NF-KappaB Inhibitor alpha; Thromboplastin; Transfection; Vascular Endothelial Growth Factor A

We recently reported that bcl-xL regulates interleukin 8 (CXCL8) protein expression and promoter activity in glioblastoma cells. In this paper we demonstrate that CXCL8 induction by bcl-xL is mediated through a nuclear factor-kappa B (NF-kB)-dependent mechanism. Mutational studies on the CXCL8 promoter showed that NF-kB binding site was required for bcl-xL-induced promoter activity and an enhanced nuclear expression of NF-kB subunits p65 and p50 was observed after bcl-xL over-expression. Electrophoretic mobility shift assay showed an increased DNA-binding activity of NF-kB in bcl-xL over-expressing cells and the use of specific antibodies confirmed the involvement of p65 and p50 in NF-kB activity on CXCL8 promoter sequence. NF-kB activity regulation by bcl-xL involved IkBalpha and IKK complex signaling pathway. In fact, bcl-xL over-expression induced a decrease of cytoplasmic expression of the IkBalpha protein, paralleled by an increase in the phosphorylation of the same IkBalpha and IKKalpha/beta. Moreover, the down-regulation of the ectopic or endogenous bcl-xL expression through RNA interference confirmed the ability of bcl-xL to modulate NF-kB pathway, and the transient expression of a degradation-resistant form of the cytoplasmic NF-kB inhibitor IkBalpha in bcl-xL transfectants confirmed the involvement of that inhibitor in bcl-xL-induced CXCL8 expression and promoter activity. In conclusion, our results demonstrate the role of NF-kB as the mediator of bcl-xL-induced CXCL8 up-regulation in glioblastoma cells.

Topics: bcl-X Protein; Blotting, Western; Cell Line, Tumor; Electrophoretic Mobility Shift Assay; Enzyme-Linked Immunosorbent Assay; Gene Expression Regulation; Glioblastoma; Humans; Interleukin-8; Neovascularization, Pathologic; NF-kappa B; Promoter Regions, Genetic; Signal Transduction; Transfection; Up-Regulation

Activation of the formylpeptide receptor (FPR), a G-protein-coupled receptor, by its chemotactic peptide ligand N-formylmethionyl-leucyl-phenylalanine (fMLF) promotes the directional migration and survival of human glioblastoma cells. fMLF also stimulates glioblastoma cells to produce biologically active VEGF, an important angiogenic factor involved in tumor progression. In this study, we examined the capacity of FPR to regulate the production of another angiogenic factor, the chemokine IL-8 (CXCL8), in addition to its demonstrated ability to induce VEGF secretion by malignant glioma cells. We showed that the human glioblastoma cell line U87 secreted considerable levels of IL-8 (CXCL8) upon stimulation by the FPR agonist peptide fMLF. Tumor cells transfected with small interference (si)RNA targeting FPR failed to produce IL-8 as well as VEGF in response to fMLF. Glioblastoma cells bearing FPR siRNA exhibited reduced rate of tumorigenicity in nude mice and tumors formed by such tumor cells showed less active angiogenesis and lower level expression of both IL-8 and VEGF. These results suggest that FPR plays an important role in the angiogenesis of human malignant gliomas through increasing the production of angiogenic factors by FPR positive tumor cells.

Topics: Angiogenesis Inducing Agents; Animals; Cell Line, Tumor; Cell Proliferation; Corneal Surgery, Laser; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Gene Expression Regulation, Neoplastic; Glioblastoma; Humans; Interleukin-8; Mice; N-Formylmethionine Leucyl-Phenylalanine; Receptors, Formyl Peptide; Tetrazolium Salts; Thiazoles; Time Factors; Transplantation, Heterologous; Vascular Endothelial Growth Factor A

Chemoattractant receptors participate in essential pathophysiologic processes, including inflammation, wound healing, human immunodeficiency virus infection, and most interestingly in the progression of malignant tumor. This study was to explore the functional expression of formylpeptide receptor (FPR) in human glioblastoma cell line U87.. The expression of FPR in U87 and FPR small interfering RNA (siRNA)-transfected U87 cells (FPR-siRNA-U87 cells) was detected with indirect immunofluorescent staining by confocal laser scanning microscopy. FPR was activated by its ligand formy-Met-Leu-Phe (fMLF). Cell proliferation was assessed by MTT assay. The production of vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8) was measured by ELISA, and IL-8 mRNA was assessed by reverse transcription-polymerase chain reaction (RT-PCR).. FPR was expressed on U87 cells, but not on FPR-siRNA-U87 cells. After activation of FPR by fMLF, the proliferation of U87 cells was enhanced markedly (P<0.05), but that of FPR-siRNA-U87 cells had no obvious change (P>0.05). fMLF (100 nmol/L) elicited a time-dependent increase in the secretion of IL-8 and VEGF. When stimulated with 100 nmol/L fMLF for 36 h, the protein levels of VEGF and IL-8 were significantly higher in stimulated U87 cells than in control cells [(3.13+/-0.23) ng/ml vs. (2.55+/-0.25) ng/ml, P<0.05û (7.54+/-0.53) ng/ml vs. (4.02+/-0.09) ng/ml, P<0.05], but those in FPR-siRNA-U87 cells had no obvious change; the mRNA level of IL-8 was significantly increased in U87 cells, but not changed in FPR-siRNA-U87 cells.. Activating FPR can promote the proliferation of U87 cells and enhance the production of angiogenic factors.

Topics: Angiogenesis Inducing Agents; Cell Line, Tumor; Cell Proliferation; Glioblastoma; Humans; Interleukin-8; N-Formylmethionine Leucyl-Phenylalanine; Receptors, Formyl Peptide; RNA, Messenger; RNA, Small Interfering; Transfection; Vascular Endothelial Growth Factor A

To isolate, culture and identify glioma stem cells from human malignant glioma cell line U87, and investigate the changes of pro-angiogenic factors production by glioma stem cells followed by activation of CXCR4 and observe their tumorigenesis as well as the expression of vascular endothelial growth factor when implanted into nude mice.. The ratio of CD133 positive cells was detected by flow cytometry. Magnetic separation of CD133 positive cells was carried out on the magnetic cell sorting system (MACS). Expression of nestin, glial fibrillary acidic protein (GFAP) and CXCR4 on tumorspheres was detected by indirect immunofluorescence under confocal laser scanning microscopy. The functional activation of CXCR4 was assessed by calcium mobilization experiments. ELISA was used to detect the production of vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8) in conditioned medium. Glioma stem cells were implanted into nude mice to assess their tumorigenesis ability and the expression of VEGF.. The ratio of CD133 positive cells with stem cell property was 0.5% in U87 cells. Activation of CXCR4 on glioma stem cells induced calcium mobilization and increased VEGF and IL-8 protein secretion. CD133 positive cells secreted more VEGF and IL-8 than their negative counterparts in vitro. Tumors derived from CD133 positive cells grew more rapidly and expressed elevated level of VEGF than their negative counterparts.. There are a small fraction of glioma stem cells in human glioblastoma cell line U87. Expressing functional CXCR4 and secreting more pro-angiogenic factors may be involved in tumor angiogenesis mediated by glioma stem cells.

Topics: AC133 Antigen; Animals; Antigens, CD; Brain Neoplasms; Cell Line, Tumor; Female; Gene Expression Regulation, Neoplastic; Glial Fibrillary Acidic Protein; Glioblastoma; Glycoproteins; Humans; Interleukin-8; Intermediate Filament Proteins; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Transplantation; Neoplastic Stem Cells; Neovascularization, Pathologic; Nerve Tissue Proteins; Nestin; Peptides; Receptors, CXCR4; Vascular Endothelial Growth Factor A

In this paper, we investigated whether bcl-xL can be involved in the modulation of the angiogenic phenotype of human tumor cells. Using the ADF human glioblastoma and the M14 melanoma lines, and their derivative bcl-xL-overexpressing clones, we showed that the conditioned medium of bcl-xL transfectants increased in vitro endothelial cell functions, such as proliferation and morphogenesis, and in vivo vessel formation in Matrigel plugs, compared with the conditioned medium of control cells. Moreover, the overexpression of bcl-xL induced an increased expression of the proangiogenic interleukin-8 (CXCL8), both at the protein and mRNA levels, and an enhanced CXCL8 promoter activity. The role of CXCL8 on bcl-xL-induced angiogenesis was validated using CXCL8-neutralizing antibodies, whereas down-regulation of bcl-xL through antisense oligonucleotide or RNA interference strategies confirmed the involvement of bcl-xL on CXCL8 expression. Transient overexpression of bcl-xL led to extend this observation to other tumor cell lines with different origin, such as colon and prostate carcinoma. In conclusion, our results showed that CXCL8 modulation by bcl-xL regulates tumor angiogenesis, and they point to elucidate an additional function of bcl-xL protein.

Topics: bcl-X Protein; Biomarkers, Tumor; Blotting, Northern; Blotting, Western; Cells, Cultured; Collagen; Drug Combinations; Endothelium, Vascular; Enzyme-Linked Immunosorbent Assay; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Glioblastoma; Humans; Interleukin-8; Laminin; Melanoma, Experimental; Neovascularization, Pathologic; Oligonucleotides, Antisense; Promoter Regions, Genetic; Protein Array Analysis; Proteoglycans; RNA, Messenger; Umbilical Veins

Despite recent advances in understanding molecular mechanisms involved in glioblastoma progression, the prognosis of the most malignant brain tumor continues to be dismal. Because the flavonoid kaempferol is known to suppress growth of a number of human malignancies, we investigated the effect of kaempferol on human glioblastoma cells. Kaempferol induced apoptosis in glioma cells by elevating intracellular oxidative stress. Heightened oxidative stress was characterized by an increased generation of reactive oxygen species (ROS) accompanied by a decrease in oxidant-scavenging agents such as superoxide dismutase (SOD-1) and thioredoxin (TRX-1). Knockdown of SOD-1 and TRX-1 expression by small interfering RNA (siRNA) increased ROS generation and sensitivity of glioma cells to kaempferol-induced apoptosis. Signs of apoptosis included decreased expression of Bcl-2 and altered mitochondrial membrane potential with elevated active caspase-3 and cleaved poly(ADP-ribose) polymerase expression. Plasma membrane potential and membrane fluidity were altered in kaempferol-treated cells. Kaempferol suppressed the expression of proinflammatory cytokine interleukin-6 and chemokines interleukin-8, monocyte chemoattractant protein-1, and regulated on activation, normal T-cell expressed and secreted. Kaempferol inhibited glioma cell migration in a ROS-dependent manner. Importantly, kaempferol potentiated the toxic effect of chemotherapeutic agent doxorubicin by amplifying ROS toxicity and decreasing the efflux of doxorubicin. Because the toxic effect of both kaempferol and doxorubicin was amplified when used in combination, this study raises the possibility of combinatorial therapy whose basis constitutes enhancing redox perturbation as a strategy to kill glioma cells.

Topics: Apoptosis; Caspases; Cell Line, Tumor; Chemokine CCL2; Doxorubicin; Glioblastoma; Humans; Interleukin-6; Interleukin-8; Kaempferols; Membrane Potential, Mitochondrial; Mitochondria; Oxidants; Oxidative Stress; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; RNA, Small Interfering; Superoxide Dismutase; Superoxide Dismutase-1; Thioredoxins

C/EBP beta (CCAAT/enhancer binding protein beta) is a transcriptional factor that belongs to the basic region-leucine zipper class DNA-binding proteins and plays a role in cell differentiation and inflammatory reactions. Although high tissue levels of inflammatory cytokines, such as interleukin (IL)-6, IL-8 and transforming growth factor-beta, have been observed in glioma patients, the mechanisms underlying this phenomenon remain to be elucidated. C/EBP beta induces a variety of cytokines and thus may play a role in the pathogenesis of glioma. In this study, we investigated the relationship between C/EBP beta expression, tumor histology, and prognosis in glioma. The expression of C/EBP beta mRNA was examined with quantitative real-time PCR and protein expression was examined with immunohistochemical techniques in 47 glioma tissue samples. Expression of C/EBP beta mRNA and protein was markedly increased in high grade glioma compared with low grade glioma. Patients whose expression of C/EBP beta mRNA and protein in tumor tissue was lower survived longer than those whose expressions were higher. In vitro, C/EBP beta siRNA inhibited glioma cell proliferation and invasion. Moreover, IL-8 production by glioma cells was inhibited by C/EBP beta siRNA transfection. These data suggest that increased expression of C/EBP beta may contribute to the promotion of tumor invasiveness and progression. The data imply that the comparison of C/EBP beta expression could be a prognostic marker for patients with glioma.

Topics: Adolescent; Adult; Aged; Astrocytoma; Brain Neoplasms; CCAAT-Enhancer-Binding Protein-beta; Cell Line, Tumor; Cell Movement; Cell Proliferation; Child; Female; Gene Expression; Glioblastoma; Glioma; Humans; Immunohistochemistry; Interleukin-8; Male; Middle Aged; Prognosis; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; RNA, Messenger; RNA, Small Interfering; Survival Analysis; Transfection

Recent experimental evidence has challenged the paradigm according to which radiation traversal through the nucleus of a cell is a prerequisite for producing genetic changes or biological responses. Thus, unexposed cells in the vicinity of directly irradiated cells or recipient cells of medium from irradiated cultures can also be affected. The aim of the present study was to evaluate, by means of the medium transfer technique, whether interleukin-8 and its receptor (CXCR1) may play a role in the bystander effect after gamma irradiation of T98G cells in vitro. In fact the cell specificity in inducing the bystander effect and in receiving the secreted signals that has been described suggests that not only the ability to release the cytokines but also the receptor profiles are likely to modulate the cell responses and the final outcome. The dose and time dependence of the cytokine release into the medium, quantified using an enzyme linked immunosorbent assay, showed that radiation causes alteration in the release of interleukin-8 from exposed cells in a dose-independent but time-dependent manner. The relative receptor expression was also affected in exposed and bystander cells.

Topics: Bystander Effect; Cell Line; Cell Line, Tumor; Cell Survival; Dose-Response Relationship, Radiation; Gamma Rays; Gene Expression Regulation, Neoplastic; Glioblastoma; Humans; Interleukin-8; Radiation Dosage; Radiation Tolerance; Receptors, Interleukin-8

R(+)WIN 55,212-2 is a synthetic cannabinoid that controls disease progression in models of multiple sclerosis. This is associated with its ability to reduce migration of leukocytes into the central nervous system. Because leukocyte migration is dependent on induction of adhesion molecules and chemokines by pro-inflammatory cytokines, we examined the effects of R(+)WIN 55,212-2 on their expression. Using 1321N1 astrocytoma and A-172 glioblastoma as cell models we show that R(+)WIN 55,212-2, but not its inactive chiral form S(-)WIN 55,212-2, strongly inhibits the interleukin-1 (IL-1) induction of the adhesion molecules intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) and the chemokine IL-8. This inhibition is not mediated via the CB1 or CB2 cannabinoid receptors, because their selective antagonists and pertussis toxin failed to affect the inhibitory effects of R(+)WIN 55,212-2. Furthermore reverse transcription-PCR analysis did not detect the expression of either receptor in 1321N1 cells. R(+)WIN 55,212-2 was shown to inhibit adhesion molecule and chemokine expression at the level of transcription, because it strongly inhibited the IL-1 induction of ICAM-1, VCAM-1, and IL-8 mRNAs and blocked the IL-1 activation of their promoters. The NFkappaB pathway was then assessed as a lead target for R(+)WIN 55,212-2. NFkappaB was measured by expression of a transfected NFkappaB-regulated reporter gene. Using this assay, R(+)WIN 55,212-2 strongly inhibited IL-1 activation of NFkappaB. Furthermore R(+)WIN 55,212-2 inhibited the ability of overexpressed Myd88, Tak-1, and IKK-2 to induce the reporter gene suggesting that R(+)WIN 55,212-2 acts at or downstream of IKK-2 in the IL-1 pathway. However R(+)WIN 55,212-2 failed to inhibit IL-1-induced degradation of IkappaBalpha, excluding IKK-2 as a direct target. In addition electrophoretic mobility shift and chromatin immunoprecipitation assays showed that R(+)WIN 55,212-2 does not regulate the IL-1-induced nuclear translocation of NFkappaB or the ability of the latter to bind to promoters regulating expression of ICAM-1 and IL-8. These data suggest that R(+)WIN 55,212-2 blocks IL-1 signaling by inhibiting the transactivation potential of NFkappaB.

Topics: Active Transport, Cell Nucleus; Astrocytes; Astrocytoma; Benzoxazines; Blotting, Western; Calcium Channel Blockers; Cannabinoids; Cell Adhesion; Cell Line, Tumor; Cell Movement; Chromatin Immunoprecipitation; Cytosol; DNA; Dose-Response Relationship, Drug; Enzyme-Linked Immunosorbent Assay; Genes, Reporter; Glioblastoma; Humans; I-kappa B Proteins; Immunoprecipitation; Intercellular Adhesion Molecule-1; Interleukin-1; Interleukin-8; Leukocytes; Morpholines; Multiple Sclerosis; Naphthalenes; NF-kappa B; NF-KappaB Inhibitor alpha; Phosphorylation; Reverse Transcriptase Polymerase Chain Reaction; RNA; RNA, Messenger; Serine; Signal Transduction; Transcription, Genetic; Transcriptional Activation; Transfection; Vascular Cell Adhesion Molecule-1

Interleukin-8 (IL-8) is a chemokine involved in angiogenesis, a process vital to tumor growth. Previously, we showed that endothelial cells derived from human tumor tissue have different functional and phenotypic properties compared with normal endothelial cells. This study analyzes the role of IL-8 in regulating angiogenesis of tumor-associated brain endothelial cells (TuBEC). Results show that TuBECs have a higher baseline migration rate compared with normal brain endothelial cells (BEC). TuBECs are unaffected when stimulated with IL-8 whereas BECs are activated. This lack of response of TuBECs to IL-8 is due to the constitutive production of IL-8. Endogenously produced IL-8 activates TuBECs in an autocrine manner as shown by IL-8 receptor inhibition. Blocking either CXCR1 or CXCR2 partially reduces TuBEC migration, whereas blocking both receptors further reduces migration. Treatment with antibody against vascular endothelial growth factor (VEGF) shows that production of IL-8 by TuBECs is dependent on VEGF. Transforming growth factor-beta1 (TGF-beta1), shown to down-regulate IL-8 production in BECs, does not inhibit IL-8 production in TuBECs. In summary, these studies show that TuBECs constitutively secrete IL-8 and autocrine activation by IL-8 is the result of VEGF stimulation. Furthermore, TuBECs do not respond to the feedback inhibition normally induced by TGF-beta1. These data emphasize the functional uniqueness of TuBECs. Understanding the functions and regulatory processes of tumor-associated endothelial cells is critical for developing appropriate antiangiogenic therapies.

Topics: Brain; Brain Neoplasms; Cell Movement; Endothelial Cells; Glioblastoma; Humans; Interleukin-8; Receptors, Interleukin-8A; Receptors, Interleukin-8B; Transforming Growth Factor beta; Transforming Growth Factor beta1; Vascular Endothelial Growth Factor A

Gliomas are the most common primary tumours of the central nervous system, with nearly 15,000 diagnosed annually in the United States and a lethality approaching 80% within the first year of glioblastoma diagnosis. The marked induction of angiogenesis in glioblastomas suggests that it is a necessary part of malignant progression; however, the precise molecular mechanisms underlying the regulation of brain tumour growth and angiogenesis remain unresolved. Here we report that a candidate tumour suppressor gene, ING4, is involved in regulating brain tumour growth and angiogenesis. Expression of ING4 is significantly reduced in gliomas as compared with normal human brain tissue, and the extent of reduction correlates with the progression from lower to higher grades of tumours. In mice, xenografts of human glioblastoma U87MG, which has decreased expression of ING4, grow significantly faster and have higher vascular volume fractions than control tumours. We show that ING4 physically interacts with p65 (RelA) subunit of nuclear factor NF-kappaB, and that ING4 regulates brain tumour angiogenesis through transcriptional repression of NF-kappaB-responsive genes. These results indicate that ING4 has an important role in brain tumour pathogenesis.

Topics: Animals; Brain Neoplasms; Cell Cycle Proteins; Cell Division; Cell Line, Tumor; Cyclooxygenase 2; Disease Progression; Down-Regulation; Gene Expression Regulation, Neoplastic; Glioblastoma; Homeodomain Proteins; Humans; Interleukin-6; Interleukin-8; Isoenzymes; Membrane Proteins; Mice; Mice, SCID; Neoplasm Transplantation; Neovascularization, Pathologic; NF-kappa B; Prostaglandin-Endoperoxide Synthases; Protein Binding; Transcription Factor RelA; Tumor Suppressor Proteins

Interleukin (IL)-8 produced from glioblastoma is suggested to contribute to its own proliferation and progression. Since various external stimuli have been shown to increase intracellular Ca(2+) in glioma cells, we investigated Ca(2+) mobilization-dependent IL-8 expression and effect of cyclosporin A (CsA), an inhibitor of calcineurin (Cn), on the expression and invasive potential of human glioblastoma U251MG cells. Combined treatment with Ca(2+)-ionophore and phorbol-myristate-acetate (A23187/PMA) increased IL-8 mRNA and protein levels. This increase was suppressed by CsA and by another Cn inhibitor FK506. Luciferase reporter gene assay and electrophoretic mobility shift assay revealed that activation of p65-containing nuclear factor-kappaB was essential for A23187/PMA-dependent activation of IL-8 promoter. CsA suppressed the promoter activity by attenuating IkappaB-alpha degradation. U251MG cells expressed IL-8 receptors CXCR-1 and -2, and Matrigel invasion assay revealed that CsA attenuated A23187/PMA-dependent stimulation of invasive potential, probably by inhibiting IL-8 production. In addition, IL-8-dependent proliferation was also suppressed by CsA. Taken together, these results demonstrate the novel inhibitory effects of CsA on glioblastoma cell functions, suggesting CsA as a potential therapeutic adjuvant for glioma treatment.

Topics: Calcimycin; Calcium; Cell Division; Cell Line, Tumor; Cyclosporine; Glioblastoma; Humans; I-kappa B Kinase; Interleukin-8; Neoplasm Invasiveness; NF-kappa B; Promoter Regions, Genetic; Protein Serine-Threonine Kinases; Receptors, Interleukin-8A; Receptors, Interleukin-8B; RNA, Messenger; Tetradecanoylphorbol Acetate

This study aims to investigate the role of gastrin-17 (G17) on angiogenesis features in gliomas both in vitro and in vivo.. The influences of G17 and G17 receptor antagonists were characterized in vitro in terms of angiogenesis on human umbilical vein endothelial cell (HUVEC) tubulogenesis processes on Matrigel and in vivo with respect to U373 orthotopic glioma xenografts. The influence of phosphatidylinositol 3'-kinase, protein kinase C, and nuclear factor-kappaB inhibitors was characterized in vitro on G17-mediated HUVEC tubulogenesis. G17-mediated release of interleukin (IL)-8 from HUVECs and G17-induced modifications in nuclear factor-kappaB DNA binding activity were characterized by means of specific enzyme-linked immunosorbent assays. The influence of G17 on E- and P-selectin expression was determined by means of computer-assisted microscopy, whereas the influence of E- and P-selectin on HUVEC migration was approached by means of antisense oligonucleotides. The chemotactic influence of G17 and IL-8 on HUVEC migration was characterized by means of computer-assisted videomicroscopy with Dunn chambers.. Messenger RNAs for cholecystokinin (CCK)A, CCKB, and CCKC receptors were present in HUVECs and microvessels dissected from a human glioblastoma. Whereas G17 significantly increased the levels of angiogenesis in vivo in the U373 experimental glioma model and in vitro in the HUVECs, the CCKB receptor antagonist L365,260 significantly counteracted the G17-mediated proangiogenic effects. G17 chemoattracted HUVECs, whereas IL-8 failed to do so. IL-8 receptor alpha (CXCR1) and IL-8 receptor beta (CXCR2) mRNAs were not detected in these endothelial cells. Gastrin significantly (but only transiently) decreased the level of expression of E-selectin, but not P-selectin, whereas IL-8 increased the expression of E-selectin. Specific antisense oligonucleotides against E- and P-selectin significantly decreased HUVEC tubulogenesis processes in vitro on Matrigel.. The present study shows that gastrin has marked proangiogenic effects in vivo on experimental gliomas and in vitro on HUVECs. This effect depends in part on the level of E-selectin activation, but not on IL-8 expression/release by HUVECs.

Topics: Animals; Benzodiazepinones; Brain Neoplasms; Cell Movement; Collagen; Drug Combinations; E-Selectin; Endothelium, Vascular; Enzyme-Linked Immunosorbent Assay; Female; Gastrins; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Glioblastoma; Humans; In Vitro Techniques; Interleukin-8; Laminin; Mice; Mice, Nude; Neovascularization, Pathologic; NF-kappa B; P-Selectin; Phenylurea Compounds; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Protein Kinase C; Proteoglycans; Rats; Rats, Nude; Receptors, Cholecystokinin; Receptors, Interleukin-8A; Receptors, Interleukin-8B; Transplantation, Heterologous; Tumor Cells, Cultured; Umbilical Veins

AU-rich elements (AREs), located in the 3'-untranslated region of unstable cytokine and chemokine mRNAs, promote rapid decay of otherwise stable mRNAs and may mediate selective mRNA stabilization in response to stimulation with interleukin-1 (IL-1). AREs vary considerably, however, in both size and sequence context. To assess the heterogeneity involved in control of mRNA stability by ARE motifs, human mRNA sequences from IL-1alpha-stimulated HEK293 cells and T98G cells were screened for either instability or stability using both cDNA (950 ARE containing sequences) and Affymetrix oligonucleotide (U95Av2 GeneChip) array analysis. Although ARE-containing mRNAs exhibited a broad range of stability, IL-1alpha promoted stability in a subset of mRNAs that were unstable when transcriptionally induced by tumor necrosis factor alpha. Stabilization of granulocyte/macrophage-colony stimulating factor and IL-8 mRNAs by IL-1alpha was achieved only after 2 h of stimulation, required ongoing protein synthesis, and depended on the activation of p38 MAPK. In contrast, stabilization of Gro3 mRNA in response to IL-1alpha was achieved immediately and was insensitive to inhibitors of protein synthesis and p38 MAPK activation. In concert, these findings demonstrate that ARE sequences are functionally heterogeneous; only a subset of unstable mRNAs is sensitive to stabilization by IL-1alpha. Moreover, IL-1alpha promotes stabilization of unstable mRNAs through distinct mechanistic pathways that distinguish between specific mRNA sequences.

Topics: 3' Untranslated Regions; Genetic Heterogeneity; Glioblastoma; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Interleukin-1; Interleukin-8; Kidney; Mitogen-Activated Protein Kinases; Oligonucleotide Array Sequence Analysis; p38 Mitogen-Activated Protein Kinases; RNA Stability; RNA, Messenger; Transcription, Genetic; Tumor Cells, Cultured

Intracavitary levels of VEGF, bFGF, IL-8 and IL- 12 were evaluated by ELISA in 45 patients, 7 with recurrent anaplastic astrocytoma (rAA), 12 with glioblastoma (GBM) and 26 with recurrent glioblastoma (rGBM). In 25 patients plasma levels of the molecules were also quantitated. Twenty-three healthy controls were also studied for plasma concentrations of the same molecules. Plasma levels of VEGF (mean 33.89 +/- 6.71 pg/ml) and bFGF (mean 11.1 +/- 3.24 pg/ml) were higher in patients than in controls (mean 16.78 +/- 3.7 pg/ml for VEGF, mean 0.21 +/- 0.09 pg/ml for bFGF) (p = 0.04 and p = 0.001, respectively) while plasma IL-12 levels were lower (mean 45.6 +/- 1.5 pg/ml in patients, mean 79.7 +/- 1.3 pg/ml in controls) (p = 0.009). Intracavitary VEGF levels were 5-53.307 fold higher (mean 90,900 +/- 24,789 pg/ml) than in the corresponding plasma. Also IL-8 concentrations were higher in intracavitary fluid (mean 6,349.76 +/- 1,460.93 pg/ml) than in plasma (mean 43.44 +/- 24.82 pg/ml). Maximum VEGF levels were found in tumor fluid of recurrent glioblastoma patients (mean 147,678 +/- 39.903 pg/ml), intermediate levels in glioblastoma patients (mean 20,322 +/- 11,892 pg/ml) and lower levels in rAA patients (mean 9,111 +/- 5,789 pg/ml). The data also suggest that higher intracavitary levels of VEGF and IL-8, and lower IL-12 levels, may be correlated with shorter adjunctive survival times, but more data will need to be collected to establish this correlation clearly.

Topics: Brain Neoplasms; Case-Control Studies; Cerebral Ventricles; Endothelial Growth Factors; Enzyme-Linked Immunosorbent Assay; Fibroblast Growth Factor 2; Glioblastoma; Humans; Intercellular Signaling Peptides and Proteins; Interleukin-12; Interleukin-8; Lymphokines; Neoplasm Recurrence, Local; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

Fas transduces not only apoptotic signals through various pathways but also angiogenic and proinflammatory responses in vivo. Human glioma cells express Fas although sensitivity to Fas-mediated cell death is variable, suggesting that Fas may have functions other than apoptosis in these cells. In this study, we addressed alternative functions of Fas expressed on human gliomas by Fas ligation in three human glioma cell lines, CRT-MG, U373-MG, and U87-MG, and the in vivo expression of Fas and chemokines in human glioblastoma multiforme (GBM). Herein, we demonstrate that: (a) stimulation with agonistic anti-Fas monoclonal antibody CH-11 and human recombinant soluble Fas ligand induces expression of the CC chemokine MCP-1 and the CXC chemokine interleukin-8 by human glioma cell lines at the mRNA and protein levels in a dose- and time-dependent manner; (b) selective pharmacological inhibitors of MEK1 (U0126 and PD98059) and p38 mitogen-activated protein kinase (MAPK) (SB202190) suppress Fas-mediated chemokine expression in a dose-dependent manner; (c) Fas ligation on human glioma cells leads to activation of both extracellular signal-regulated kinases ERK1/ERK2 and p38 MAPK; and (d) GBM samples express higher levels of Fas compared with normal control brain, which correlates with increased interleukin 8 expression. These findings indicate that Fas ligation on human glioma cells leads to the selective induction of chemokine expression, which involves the ERK1/ERK2 and p38 MAPK signaling pathways. Therefore, the Fas-Fas ligand system in human brain tumors may be involved not only in apoptotic processes but also in the provocation of angiogenic and proinflammatory responses.

Topics: Adult; Apoptosis; Chemokines; fas Receptor; Glioblastoma; Glioma; Humans; Interleukin-8; MAP Kinase Signaling System; Middle Aged; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; p38 Mitogen-Activated Protein Kinases; Tumor Cells, Cultured

Oxygen deprivation is an important biological feature of tumor growth. We previously showed that in glioma, anoxia increases expression of IL-8, a chemokine and angiogenic factor. Here, we analysed for the first time the biochemical mechanisms inducing the IL-8 gene upon anoxia in glioma cells, and showed that they differ from those inducing the VEGF gene. Both genes are induced in biologically and genetically heterogenous glioblastoma cell lines (LN-229, LN-Z308, U87MG, T98G), whereas, in gliosarcoma cells (D247MG), only the VEGF gene is induced. The kinetics of IL-8 and VEGF mRNA inductions differ in these cells and reoxygenation experiments showed that the induction is due to the anoxic stress per se. Furthermore, in LN-229 and LN-Z308 cell lines actinomycin D, DRB and nuclear run-on experiments showed that anoxia stimulates increased transcription of both genes. Electromobility shift assays show increased protein binding to the AP-1 site on the IL-8 promoter following anoxia treatment. Finally, in situ hybridization on glioblastoma sections shows that the in vivo expression patterns of IL-8 and VEGF genes overlap, but are not identical. Since intratumoral augmentation of IL-8 and VEGF secretion, following microenvironmental decreases in oxygen pressure, may promote angiogenesis, further definition of these pathways is essential to appropriately target them for antitumoral therapy.

Topics: Animals; Cell Hypoxia; Cobalt; Dactinomycin; Dichlororibofuranosylbenzimidazole; Endothelial Growth Factors; Gene Expression Regulation; Glioblastoma; Humans; Interleukin-8; Lymphokines; Mice; Nucleic Acid Synthesis Inhibitors; Oxidation-Reduction; Oxygen; Response Elements; RNA, Messenger; Transcription Factor AP-1; Transcription, Genetic; Tumor Cells, Cultured; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

Macrophages are key participants in angiogenesis. In this study on human brain tumors, we first investigated whether macrophage infiltration is associated with angiogenesis and malignant histological appearance. Immunostaining of macrophages and small vessels in resected glioma specimens indicated that numbers of infiltrating macrophages and small vessel density were higher in glioblastomas than in astrocytomas or anaplastic astrocytomas. Macrophage infiltration was closely correlated with vascular density in human gliomas. Heme oxygenase-1 (HO-1), which is the rate-limiting enzyme in heme catabolism, was also associated with activated macrophages. Expression of mRNA encoding HO-1 was correlated with macrophage infiltration and vascular density in human glioma samples. Infiltrating macrophages were positively stained with anti-HO-1 antibody by immunohistochemical analysis, and in situ hybridization for HO-1 indicated that HO-1 was expressed in infiltrating macrophages in gliomas. HO-1 gene may be a useful marker for macrophage infiltration as well as neovascularization in human gliomas.

Topics: Adolescent; Adult; Aged; Astrocytoma; Blotting, Northern; Brain Neoplasms; Child; Child, Preschool; Enzyme Induction; Female; Glioblastoma; Glioma; Heme Oxygenase (Decyclizing); Heme Oxygenase-1; Humans; In Situ Hybridization; Interleukin-8; Macrophage Activation; Macrophages; Male; Membrane Proteins; Middle Aged; Neovascularization, Pathologic; Oligodendroglioma; RNA, Messenger

Interleukin-17 (IL-17) has been characterized as a proinflammatory cytokine produced by CD4+ activated memory T cells. In an effort to elucidate the biological effects of IL-17 in glial cells, we investigated the ability of this cytokine in order to activate nuclear factor (NF)-kappaB, which is being discussed as one of the most important transcription factors in the regulation of neuronal and glial cell function. Activation of NF-kappaB involves the degradation of its cytoplasmatic inhibitor IkappaB-alpha, which allows the nuclear translocation of NF-kappaB, and ensures transcriptional activation of genes including IkappaB-alpha itself. Using a competitive RT-PCR, we examined the IL-17-induced IkappaB-alpha mRNA expression in glioblastoma cells, and we examined IL-17 up-regulated IkappaB-alpha mRNA expression in a dose- and time-dependent fashion with a maximum time between 1 and 3 h. This induction could be inhibited by Calphostin C (protein kinase C inhibitor) and genistein (tyrosine kinase inhibitor). After 60 min of IL-17 stimulation, a degradation of the IkappaB-alpha protein was detectable. Furthermore, IL-17 stimulated the secretion of IL-6 and IL-8 in glial cells, and IL-17 and IL-1beta in combination showed a superadditive effect. We suggest IL-17 to play a role as an immune factor, possibly involved in complex pathophysiological interactions of neurodegenerative diseases.

Topics: DNA-Binding Proteins; Glioblastoma; Humans; I-kappa B Proteins; Interleukin-17; Interleukin-6; Interleukin-8; NF-KappaB Inhibitor alpha; RNA, Messenger; Tumor Cells, Cultured

Coculture of T98G glioblastoma cells with the myeloid and monocytic cell lines, HL-60, and THP-1 produced minimal amounts of interleukin-8 (IL-8). Pretreatment of HL-60 or THP-1 cells with phorbol myristate acetate (PMA) enhanced their capacity to induce IL-8 production by T98G cells. In contrast, the murine macrophage cell lines J774 A.1 and RAW 264.7 induced high levels of IL-8 production by T98G cells without PMA activation. To determine the molecules responsible for the induction of IL-8 by T98G cells, we carried out coculture experiments with a membrane fraction prepared from RAW cells and indicated that membrane-associated and free forms of murine IL-1alpha acted on human T98G cells to produce IL-8. RAW cells were unique in that increasing the number of RAW cells relative to the number of T98G cells (RAW/T98G ratio > 4:1) significantly suppressed IL-8 production by T98G cells. Because RAW cells produce large amounts of nitric oxide (NO), we assumed that the suppression of IL-8 production was ascribable to the NO produced by the RAW cells. This was supported by the inverse relationship between increasing concentrations of NO and IL-8 production seen in this coculture system. The involvement of NO in the suppression of IL-8 production was confirmed by the finding that N-monomethyl-L-arginine (NMMA), which inhibits NO production, reversed this suppression, whereas S-nitroso-N-acetyl-D,L-penicillamine (SNAP), a strong NO generator, suppressed IL-8 production. Our results indicate that high levels of NO suppress IL-8 production by T98G cells, and murine IL-1alpha plays a major role in the induction of IL-8 production by T98G cells. It is, therefore, possible that excessive production of NO during the interaction of glioma cells with macrophages may play a regulatory role in chemokine production, thus mitigating inflammatory responses.

Topics: Animals; Cell Line; Coculture Techniques; Glioblastoma; HL-60 Cells; Humans; Interleukin-8; Macrophages; Mice; Monocytes; Nitric Oxide; omega-N-Methylarginine; Penicillamine; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

Leukocyte infiltration and necrosis are two biological phenomena associated with the development of neovascularization during the malignant progression of human astrocytoma. Here, we demonstrate expression of interleukin (IL)-8, a cytokine with chemotactic and angiogenic properties, and of IL-8-binding receptors in astrocytoma. IL-8 expression is first observed in low grade astrocytoma in perivascular tumor areas expressing inflammatory cytokines. In glioblastoma, it further localizes to oxygen-deprived cells surrounding necrosis. Hypoxic/anoxic insults on glioblastoma cells in vitro using anaerobic chamber systems or within spheroids developing central necrosis induced an increase in IL-8 messenger RNA (mRNA) and protein expression. mRNA for IL-8-binding chemokine receptors CXCR1, CXCR2, and the Duffy antigen receptor for chemokines (DARC) were found in all astrocytoma grades by reverse transcription/PCR analysis. In situ hybridization and immunohistochemistry localized DARC expression on normal brain and tumor microvascular cells and CXCR1 and CXCR2 expression to infiltrating leukocytes. These results support a model where IL-8 expression is initiated early in astrocytoma development through induction by inflammatory stimuli and later in tumor progression increases due to reduced microenvironmental oxygen pressure. Augmented IL-8 would directly and/or indirectly promote angiogenesis by binding to DARC and by inducing leukocyte infiltration and activation by binding to CXCR1 and CXCR2.

Topics: Anaerobiosis; Antigens, CD; Blotting, Northern; Cell Hypoxia; Chemotaxis, Leukocyte; Disease Progression; Glioblastoma; Humans; In Situ Hybridization; Interleukin-8; Necrosis; Neovascularization, Physiologic; Receptors, Interleukin; Receptors, Interleukin-8A; RNA, Messenger; Spheroids, Cellular; Up-Regulation

A glucocorticoid, dexamethasone, inhibited the production of a leukocyte chemotactic cytokine, interleukin 8 (IL-8), as well as mRNA expression by a glioblastoma cell line, T98G, stimulated with interleukin 1 (IL-1). Dexamethasone also inhibited IL-8 promoter-driven chloramphenicol acetyltransferase (CAT) activities induced by IL-1, suggesting that dexamethasone inhibited IL-8 production mainly at the transcriptional level. Moreover, CAT assay revealed that the nuclear factor-kappa B (NF-kappa B) binding site was the crucial cis-element required for conferring IL-1 responsiveness in conjunction with the CCAAT enhancer binding protein/nuclear factor-IL-6 (NF-IL6) and/or the AP-1 binding site(s). Mutation of either the AP-1 or NF-IL6 binding site did not abolish IL-8 gene repression by dexamethasone, suggesting that these sites were not targets for dexamethasone. Trimerized kappa B sequence in the IL-8 gene was enough for conferring the induction by IL-1 and inhibition by dexamethasone of CAT activity. Finally, dexamethasone diminished the IL-1-induced formation of NF-kappa B complexes, which were identified immunochemically to consist of p50 and p65, without reducing the amount of translocated factors. Collectively, dexamethasone interfered with the binding of the most essential transcription factor, NF-kappa B, to its cognate cis-element, thereby suppressing the transcription of IL-8 gene.

Topics: Amino Acid Sequence; Base Sequence; Biological Transport; Cell Nucleus; Dexamethasone; Gene Expression Regulation; Glioblastoma; Humans; Interleukin-1; Interleukin-8; Molecular Sequence Data; NF-kappa B; Transcription, Genetic; Tumor Cells, Cultured

The production of interleukin 8 (IL-8), a neutrophil chemotactic factor, and its amino acid sequence were examined in glioblastoma cell lines in vitro. Neutrophil chemotactic activity was demonstrated in 9 conditioned media of 15 human glioblastoma cell lines. Tumor necrosis factor (TNF)-alpha stimulated secretion of the activity in 7 lines and induced secretion in 4 other lines. ELISA quantification disclosed that the conditioned media contained interleukin 8 (IL-8) in an amount equivalent to the chemotactic activity. The IL-8 secretion increased with the stimulation by TNF-alpha. Northern blot analysis and the RT-PCR method confirmed expression of mRNA in the glioblastoma cells and its augmentation by TNF-alpha and/or IL-beta. Reversed-phase HPLC following ion-exchange chromatography revealed that the chemotactic activity was a single peptide, which was determined to be IL-8 by the retention time and ELISA. Furthermore, amino acid analysis disclosed that a major part of the glioblastoma-cell derived IL-8 peptide was 77 amino acid IL-8 (IL-8(77); with the N-terminal sequence AVLPRSAKELRCQCI-).

Topics: Amino Acid Sequence; Blotting, Northern; Brain Neoplasms; Chemotaxis, Leukocyte; Chromatography, Liquid; Cytokines; Enzyme-Linked Immunosorbent Assay; Glioblastoma; Humans; Interleukin-8; Molecular Sequence Data; Neutrophils; Polymerase Chain Reaction; RNA, Messenger; Tumor Cells, Cultured