Microglial stimulation of glioblastoma invasion involves epidermal growth factor receptor (EGFR) and colony stimulating factor 1 receptor (CSF-1R) signaling

Glioblastoma multiforme is a deadly cancer for which current treatment options are limited. The ability of glioblastoma tumor cells to infiltrate the surrounding brain parenchyma critically limits the effectiveness of current treatments. We investigated how microglia, the resident macrophages of the brain, stimulate glioblastoma cell invasion. We first examined the ability of normal microglia from C57Bl/6J mice to stimulate GL261 glioblastoma cell invasion in vitro. We found that microglia stimulate the invasion of GL261 glioblastoma cells by approximately eightfold in an in vitro invasion assay. Pharmacological inhibition of epidermal growth factor receptor (EGFR) strongly inhibited microglia-stimulated invasion. Furthermore, blockade of colony stimulating factor 1 receptor (CSF-1R) signaling using ribonucleic acid (RNA) interference or pharmacological inhibitors completely inhibited microglial enhancement of glioblastoma invasion. GL261 cells were found to constitutively secrete CSF-1, the levels of which were unaffected by epidermal growth factor (EGF) stimulation, EGFR inhibition or coculture with microglia. CSF-1 only stimulated microglia invasion, whereas EGF only stimulated glioblastoma cell migration, demonstrating a synergistic interaction between these two cell types. Finally, using PLX3397 (a CSF-1R inhibitor that can cross the blood-brain barrier) in live animals, we discovered that blockade of CSF-1R signaling in vivo reduced the number of tumor-associated microglia and glioblastoma invasion. These data indicate that glioblastoma and microglia interactions mediated by EGF and CSF-1 can enhance glioblastoma invasion and demonstrate the possibility of inhibiting glioblastoma invasion by targeting glioblastoma-associated microglia via inhibition of the CSF-1R.     

Mitogen-activated protein kinase, ERK1/2, is essential for the induction of vascular endothelial growth factor by ionizing radiation mediated by activator protein-1 in human glioblastoma cells

Vascular Endothelial Growth Factor (VEGF)/Vascular Permeability Factor plays an important role in angiogenesis and cell proliferation of cancer cells. Glioblastoma cells are most malignant and show resistance to radiation therapy inducing VEGF to cause angiogenesis and brain edema. In the present study, the regulatory mechanism of the expression of VEGF by ionizing radiation was studied in three human glioblastoma cells. Induction of VEGF mRNA by ionizing radiation was dependent on dose and incubation time. Activator protein-1 (AP-1) was activated by 10 Gy of ionizing radiation in 1 h in T98G glioblastoma cells on an electrophoretic mobility shift assay. We constructed chimeric genes containing various regions of the VEGF promoter gene and the coding region for chloramphenicol acetyltransferase (CAT) and transiently transfected them to T98G cells. CAT assay with the VEGF promoter gene containing an AP-1 site demonstrated that the promoter activity of the VEGF gene was enhanced by ionizing radiation. Immunological analysis of the activity of mitogen-activated protein kinase, ERK1/2, showed that this activity is up-regulated by ionizing radiation.

These results suggest that ERK1/2 pathway is involved in the up-regulation of VEGF expression ionizing radiation mediated by AP-1, which may lead to further neovascularization and proliferation of glioblastoma cells resistant to radiation therapy.     

MiR-330-Mediated Regulation of SH3GL2 Expression Enhances Malignant Behaviors of Glioblastoma Stem Cells by Activating ERK and PI3K/AKT Signaling Pathways

MicroRNAs are currently considered as an active and rapidly evolving area for the treatment of tumors. In this study, we elucidated the biological significance of miR-330 in glioblastoma stem cells (GSCs) as well as the possible molecular mechanisms. SH3GL2 is mainly distributed in the central nervous system and considered to be a tumor suppressor in many tumors. In the present study, we identified miR-330 as a potential regulator of SH3GL2 and we found that it was to be inversely correlated with SH3GL2 expression in GSCs which were isolated from U87 cell lines. The expression of miR-330 enhanced cellular proliferation, promoted cell migration and invasion, and dampened cell apoptosis. When the GSCs were co-transfected with the plasmid containing short hairpin RNA directed against human SH3GL2 gene and miR-330 mimic, we found that miR-330 promoted the malignant behavior of GSCs by down-regulating the expression of SH3GL2. Meanwhile, the ERK and PI3K/AKT signaling pathways were significantly activated, leading to the decreased expression of apoptotic protein and increased expression of anti-apoptotic protein. Furthermore, in orthotopic mouse xenografts, the mice given stable over-expressed SH3GL2 cells co-transfected with miR-330 knockdown plasmid had the smallest tumor sizes and longest survival. In conclusion, these results suggested that miR-330 negatively regulated the expression of SH3GL2 in GSCs, which promoted the oncogenic progression of GSCs through activating ERK and PI3K/AKT signaling pathways. The elucidation of these mechanisms will provide potential therapeutic approaches for human glioblastoma.     

MicroRNA-330 Is an Oncogenic Factor in Glioblastoma Cells by Regulating SH3GL2 Gene

MicroRNAs have recently emerged as key regulators of cancers. This study was therefore conducted to investigate the role of miR-330 in biological behaviors of human glioblastoma U87 and U251 cell lines and its molecular mechanism. SH3GL2 gene was identified as the target of miR-330. MiR-330 overexpression was established by transfecting miR-330 precursor into U87 and U251 cells, and its effects on proliferation, migration, invasion, cell cycle and apoptosis were studied. Overexpression of miR-330 can enhance cellular proliferation, promote migration and invasion, activate cell cycle and also inhibit apoptosis in U87 and U251 cells. Collectively, these above-mentioned results suggest that miRNA-330 plays an oncogenic role in human glioblastoma by regulating SH3GL2 gene and might be a new therapeutic target of human glioblastoma.     

Radiation Sensitivity of GL261 Murine Glioma Model and Enhanced Radiation Response by Flavopiridol

Response of a solid tumor to radiation treatment depends, in part, on the intrinsic radiosensitivity of tumor cells, the proliferation rate of tumor cells between radiation treatments and the hypoxic state of the tumor cells. A successful radiosensitizing agent would target S-phase cells and hypoxia. Recently, we demonstrated the anti-tumor effects of flavopiridol in the GL261 murine glioma model might involve 1) recruitment of tumor cells to S-phase (Newcomb et al., Cell Cycle 2004; 3:230-234) and 2) an anti-angiogenic effect on the tumor vasculature by downregulation of hypoxia-inducible factor -1? (HIF-1?) (Newcomb et al., Neuro-Oncology 2005; 7:225-235). Given that flavopiridol has demonstrated radiosensitizing activity in several murine tumor models, we tested whether it would enhance the response of GL261 tumors to radiation. In the present study, we evaluated the intrinsic radiation sensitivity of the GL261 glioma model using the tumor control/cure dose of radiation assay (TCD50). We found that a single dose of 65 Gy (CI 57.1-73.1) was required to cure 50% of the tumors locally. Using the tumor growth delay assay, fractionated radiation (5 fractions of 5 Gy over 10 days) combined with flavopiridol (5 mg/kg) given three times weekly for 3 cycles produced a significant growth delay. Our results indicate that the GL261 murine glioma model mimics the radioresistance encountered in human gliomas, and thus should prove useful in identifying promising new investigational radiosensitizers for use in the treatment of glioma patients.     

Downregulation of Survivin Gene Expression Affects Ionizing Radiation Resistance of Human T98 Glioma Cells

Survivin is a tumor-associated gene, which has been detected in a wide variety of human tumors. Previous research has shown that Survivin can affect hepatoma carcinoma cell radiosensitivity. However, little is known about the role of Survivin in ionizing radiation resistance in glioma cells. In this study, we aimed to identify the effects of Survivin on ionizing radiation resistance in glioma cell line T98. Our results showed that downregulation of Survivin gene expression and ionizing irradiation could both inhibit T98 cell proliferation by assays in vitro including CCK-8 and immunohistochemistry. The inhibitory effect of downregulation of Survivin combined with irradiation was the most significant compared with other groups. Results of Western blotting and flow cytometric analysis also showed that downregulation of Survivin combined with the irradiation group achieved the highest apoptosis rate. Experimental results in vivo by intracranial implanting into nude mice were consistent with those in vitro. These findings indicated that ionizing radiation resistance of human T98 glioma cells can be inhibited effectively after Survivin gene silencing.     

PCDH10 is required for the tumorigenicity of glioblastoma cells

Protocadherin10 (PCDH10)/OL-protocadherin is a cadherin-related transmembrane protein that has multiple roles in the brain, including facilitating specific cell–cell connections, cell migration and axon guidance. It has recently been reported that PCDH10 functions as a tumor suppressor and that its overexpression inhibits proliferation or invasion of multiple tumor cells. However, the function of PCDH10 in glioblastoma cells has not been elucidated. In contrast to previous reports on other tumors, we show here that suppression of the expression of PCDH10 by RNA interference (RNAi) induces the growth arrest and apoptosis of glioblastoma cells in vitro. Furthermore, we demonstrate that knockdown of PCDH10 inhibits the growth of glioblastoma cells xenografted into immunocompromised mice. These results suggest that PCDH10 is required for the proliferation and tumorigenicity of glioblastoma cells. We speculate that PCDH10 may be a promising target for the therapy of glioblastoma.     

A global perspective of radiation-induced signal transduction pathways in cancer therapeutics

Radiation is a well established therapeutic modality for the treatment of solid tumors. By merging molecular biological approaches with radiation biology, a significant number of signaling events elicited by ionizing radiation have been delineated. These signaling pathways include events leading to cell cycle arrest, apoptosis or cell survival. There are two major signaling events that affect radiation response. One is the intrinsic/constitutive pro-survival signaling event that is present in proliferating tumor cells while the other is "induced pro-survival event" in response to radiation, both of these events confer resistance to the killing effects of radiation. In this review, signaling pathways that lead to either apoptosis or survival of cells following ionizing radiation are discussed in detail. In addition, mechanisms of action for gene/drug based inhibitors that modulate the expression and function of various genes and gene products involved in pro-survival signaling pathways are described. Further, novel strategies to abrogate the "induced radiation resistance" leading to enhanced therapeutic efficacy of ionizing radiation have been proposed. These novel strategies include the use of radio-gene therapy, low dose fractionated radiation therapy as a chemopotentiator and therapeutic utility of high radiation dose induced bystander effect. The complete understanding of the molecular pathways leading to apoptosis/survival of cells following ionizing radiation will help in tailoring more effective novel strategies and treatment modalities for complete eradication of cancer.     

LRRC4 inhibits human glioblastoma cells proliferation, invasion, and proMMP-2 activation by reducing SDF-1 alpha/CXCR4-mediated ERK1/2 and Akt signaling pathways

Gliomas take a number of different genetic routes in the progression to glioblastoma multiforme, a highly invasive variant that is mostly unresponsive to current therapies. The alpha-chemokine stromal cell-derived factor (SDF)-1 alpha binds to the seven transmembrane G-protein-coupled CXCR-4 receptor and acts to modulate cell migration and proliferation by activating multiple signal transduction pathways. Leucine-rich repeats containing 4 (LRRC4), a putative glioma suppressive gene, inhibits glioblastoma cells tumorigenesis in vivo and cell proliferation and invasion in vitro. We also previously demonstrated that LRRC4 controlled glioblastoma cells proliferation by ERK/AKT/NF-kappa B signaling pathway. In the present study, we demonstrate that CXC chemokine receptor 4 (CXCR4) is expressed in human glioblastoma U251 cell line, and that SDF-1 alpha increases the proliferation, chemotaxis, and invasion in CXCR4+ glioblastoma U251 cells through the activation of ERK1/2 and Akt. The reintroduction of LRRC4 in U251 cells inhibits the expression of CXCR4 and SDF-1 alpha/CXCR4 axis-mediated downstream intracellular pathways such as ERK1/2 and Akt leading to proliferate, chemotactic and invasive effects. Furthermore, we provide evidence for proMMP-2 activation involvement in the SDF-1 alpha/CXCR4 axis-mediated signaling pathway. LRRC4 significantly inhibits proMMP-2 activation by SDF-1 alpha/CXCR4 axis-mediated ERK1/2 and Akt signaling pathway. Collectively, these results suggest a possible important "cross-talk" between LRRC4 and SDF-1 alpha/CXCR4 axis-mediated intracellular pathways that can link signals of cell proliferation, chemotaxis and invasion in glioblastoma, and may represent a new target for development of new therapeutic strategies in glioma.     

Targeting the NF-κB Pathway as a Combination Therapy for Advanced Thyroid Cancer

NF-κB signaling plays an important role in tumor cell proliferation, cell survival, angiogenesis, invasion, metastasis and drug/radiation resistance. Combination therapy involving NF-κB pathway inhibition is an attractive strategy for the treatment of advanced forms of thyroid cancer. This study was designed to test the efficacy of NF-κB pathway inhibition in combination with cytotoxic chemotherapy, using docetaxel and ionizing radiation in in vitro models of thyroid cancer. We found that while both docetaxel and ionizing radiation activated NF-κB signaling in thyroid cancer cells, there was no synergistic effect on cell proliferation and/or programmed cell death with either genetic (transduction of a dominant negative mutant form of IκBα) or pharmacologic (proteasome inhibitor bortezomib and IKKβ inhibitor GO-Y030) inhibition of the NF-κB pathway in thyroid cancer cell lines BCPAP, 8505C, THJ16T and SW1736. Docetaxel plus bortezomib synergistically decreased in vitro invasion of 8505C cells, but not in the other cell lines. Screening of a panel of clinically relevant targeted therapies for synergy with genetic NF-κB inhibition in a proliferation/cytotoxicity assay identified the histone deacetylase (HDAC) inhibitor suberoylanilide hydroxamic acid (SAHA) as a potential candidate. However, the synergistic effect was confirmed only in the BCPAP cells. These results indicate that NF-κB inhibitors are unlikely to be beneficial as combination therapy with taxane cytotoxic chemotherapy, external radiation therapy or radioiodine therapy. There may be unique circumstances where NF-κB inhibitors may be considered in combination with docetaxel to reduce tumor invasion or in combination with HDAC inhibitors to reduce tumor growth, but this does not appear to be a combination therapy that could be broadly applied to patients with advanced thyroid cancer. Further research may identify which subsets of patients/tumors may respond to this therapeutic approach.     

Intracranial implantation with subsequent 3D in vivo bioluminescent imaging of murine gliomas

The mouse glioma 261 (GL261) is recognized as an in vivo model system that recapitulates many of the features of human glioblastoma multiforme (GBM). The cell line was originally induced by intracranial injection of 3-methyl-cholantrene into a C57BL/6 syngeneic mouse strain (1); therefore, immunologically competent C57BL/6 mice can be used. While we use GL261, the following protocol can be used for the implantation and monitoring of any intracranial mouse tumor model. GL261 cells were engineered to stably express firefly luciferase (GL261-luc). We also created the brighter GL261-luc2 cell line by stable transfection of the luc2 gene expressed from the CMV promoter. C57BL/6-cBrd/cBrd/Cr mice (albino variant of C57BL/6) from the National Cancer Institute, Frederick, MD were used to eliminate the light attenuation caused by black skin and fur. With the use of albino C57BL/6 mice; in vivo imaging using the IVIS Spectrum in vivo imaging system is possible from the day of implantation (Caliper Life Sciences, Hopkinton, MA). The GL261-luc and GL261-luc2 cell lines showed the same in vivo behavior as the parental GL261 cells. Some of the shared histological features present in human GBMs and this mouse model include: tumor necrosis, pseudopalisades, neovascularization, invasion, hypercellularity, and inflammation (1). Prior to implantation animals were anesthetized by an intraperitoneal injection of ketamine (50 mg/kg), xylazine (5 mg/kg) and buprenorphine (0.05 mg/kg), placed in a stereotactic apparatus and an incision was made with a scalpel over the cranial midline. A burrhole was made 0.1 mm posterior to the bregma and 2.3mm to the right of the midline. A needle was inserted to a depth of 3mm and withdrawn 0.4 mm to a depth of 2.6 mm. Two μl of GL261-luc or GL261-luc2 cells (10(7) cells/ml) were infused over the course of 3 minutes. The burrhole was closed with bonewax and the incision was sutured. Following stereotactic implantation the bioluminescent cells are detectable from the day of implantation and the tumor can be analyzed using the 3D image reconstruction feature of the IVIS Spectrum instrument. Animals receive a subcutaneous injection of 150 μg luciferin /kg body weight 20 min prior to imaging. Tumor burden is quantified using mean tumor bioluminescence over time. Tumor-bearing mice were observed daily to assess morbidity and were euthanized when one or more of the following symptoms are present: lethargy, failure to ambulate, hunched posture, failure to groom, anorexia resulting in >10% loss of weight. Tumors were evident in all of the animals on necropsy.     

Rapamycin-mediated mTOR inhibition attenuates survivin and sensitizes glioblastoma cells to radiation therapy

Survivin, an antiapoptotic protein, is elevated in most malignancies and attributes to radiation resistance in tumors including glioblastoma multiforme. The downregulation of survivin could sensitize glioblastoma cells to radiation therapy. In this study, we investigated the effect of rapamycin, an inhibitor of mammalian target of rapamycin (mTOR), in attenuating survivin and enhancing the therapeutic efficacy for glioblastoma cells, and elucidated the underlying mechanisms. Here we tested various concentrations of rapamycin (1-8 nM) in combination with radiation dose 4 Gy. Rapamycin effectively modulated the protein kinase B (Akt)/mTOR pathway by inhibiting the phosphorylation of Akt and mTOR proteins, and this inhibition was further enhanced by radiation. The expression level of survivin was decreased in rapamycin pre-treatment glioblastoma cells followed by radiation; meanwhile, the phosphorylation of H2A histone family member X (H2AX) at serine-139 (γ-H2AX) was increased. p21 protein was also induced on radiation with rapamycin pre-treatment, which enhanced G1 arrest and the accumulation of cells at G0/subG1 phase. Furthermore, the clonogenic cell survival assay revealed a significant dose-dependent decrease in the surviving fraction for all three cell lines pre-treated with rapamycin. Our studies demonstrated that targeting survivin may be an effective approach for radiosensitization of malignant glioblastoma.     

Effective Treatment of Established GL261 Murine Gliomas through Picornavirus Vaccination-Enhanced Tumor Antigen-Specific CD8+ T Cell Responses

Glioblastoma (GBM) is among the most invasive and lethal of cancers, frequently infiltrating surrounding healthy tissue and giving rise to rapid recurrence. It is therefore critical to establish experimental model systems and develop therapeutic approaches that enhance anti-tumor immunity. In the current study, we have employed a newly developed murine glioma model to assess the efficacy of a novel picornavirus vaccination approach for the treatment of established tumors. The GL261-Quad system is a variation of the GL261 syngeneic glioma that has been engineered to expresses model T cell epitopes including OVA_257–264. MRI revealed that both GL261 and GL261-Quad tumors display characteristic features of human gliomas such as heterogeneous gadolinium leakage and larger T2 weighted volumes. Analysis of brain-infiltrating immune cells demonstrated that GL261-Quad gliomas generate detectable CD8+ T cell responses toward the tumor-specific K^b:OVA_257–264 antigen. Enhancing this response via a single intracranial or peripheral vaccination with picornavirus expressing the OVA_257–264 antigen increased anti-tumor CD8+ T cells infiltrating the brain, attenuated progression of established tumors, and extended survival of treated mice. Importantly, the efficacy of the picornavirus vaccination is dependent on functional cytotoxic activity of CD8+ T cells, as the beneficial response was completely abrogated in mice lacking perforin expression. Therefore, we have developed a novel system for evaluating mechanisms of anti-tumor immunity in vivo, incorporating the GL261-Quad model, 3D volumetric MRI, and picornavirus vaccination to enhance tumor-specific cytotoxic CD8+ T cell responses and track their effectiveness at eradicating established gliomas in vivo.     

YKL-40在脑胶质母细胞瘤微环境中的作用

胶质母细胞瘤是大脑及其他中枢神经系统最常见的恶性肿瘤,其复杂的肿瘤微环境是胶质母细胞瘤临床治疗的主要挑战,也是胶质母细胞瘤患者复发率高、生存率低的主要原因。YKL-40,这一分泌性蛋白质与多种类型的癌症预后不良相关,且在高级别胶质瘤尤其是胶质母细胞瘤患者中血清水平与肿瘤组织表达水平显著升高,而在低级别胶质瘤中并未发现这一特征。这提示,YKL-40与胶质瘤分级及胶质母细胞瘤恶性发展过程密切相关。针对YKL-40的抗体治疗也被证明能够与电离辐射协同抑制胶质母细胞瘤血管生成及恶性发展。基于YKL-40的临床价值,本文将从肿瘤微环境的角度,归纳总结YKL-40在恶性肿瘤中的相关研究成果,并讨论其在胶质母细胞瘤发生发展中的相关作用及临床应用前景。     

miR‐200c enhances radiosensitivity of human breast cancer cells

Due to the intrinsic resistance of many tumors to radiotherapy, current methods to improve the survival of cancer patients largely depend on increasing tumor radiosensitivity. It is well‐known that miR‐200c inhibits epithelial–mesenchymal transition (EMT), and enhances cancer cell chemosensitivity. We sought to clarify the effects of miR‐200c on the radiosensitization of human breast cancer cells. In this study, we found that low levels of miR‐200c expression correlated with radiotolerance in breast cancer cells. miR‐200c overexpression could increase radiosensitivity in breast cancer cells by inhibiting cell proliferation, and by increasing apoptosis and DNA double‐strand breaks. Additionally, we found that miR‐200c directly targeted TANK‐binding kinase 1 (TBK1). However, overexpression of TBK1 partially rescued miR‐200c mediated apoptosis induced by ionizing radiation. In summary, miR‐200c can be a potential target for enhancing the effect of radiation treatment on breast cancer cells. J. Cell. Biochem. 114: 606–615, 2013. © 2012 Wiley Periodicals, Inc.     

Low-dose radiation stimulates the proliferation of normal human lung fibroblasts via a transient activation of Raf and Akt

The biological effects of low-dose radiation have been investigated and debated for more than a century, but its cellular effects and regulatory mechanisms remain poorly understood. This study shows the human cellular responses to low-dose radiation in CCD-18 Lu cells, which are derived from normal human lung fibroblasts. We examined a colony-forming assay for cell survival by ionizing radiation. Live cell counting and cell cycle analysis were measured for cell proliferation and cell cycle progression following low-dose irradiation. We examined Raf and Akt phosphorylation to determine the proliferation mechanism resulting from low-dose radiation. We also observed that p53 and p21 were related to cell cycle response. We found that 0.05 Gy of ionizing radiation enhanced cell proliferation and did not change the progression of the cell cycle. In addition, 0.05 Gy of ionizing radiation transiently activated Raf and Akt, but did not change phospho-p53, p53 and p21 in CCD-18 Lu cells. However, 2 Gy of ionizing radiation induced cell cycle arrest, phosphorylation of p53, and expression of p53 and p21. The phosphorylation of Raf and Akt proteins induced by 0.05 Gy of ionizing radiation was abolished by pre-treatment with an EGFR inhibitor, AG1478, or a PI3k inhibitor, LY294002. Cell proliferation stimulated by 0.05 Gy of ionizing radiation was blocked by the suppression of Raf and Akt phosphorylation with these inhibitors. These results suggest that 0.05 Gy of ionizing radiation stimulates cell proliferation through the transient activation of Raf and Akt in CCD-18 Lu cells.     

二甲双胍对胶质母细胞瘤细胞的抑制作用研究

为了探究二甲双胍对不同胶质母细胞瘤U87细胞、GL261细胞及C6细胞增殖的影响,选取小鼠GBM细胞GL261细胞系、大鼠GBM细胞C6细胞系及人源GBM细胞U87MG细胞系,使用二甲双胍处理,通过CCK-8法检测细胞增殖活性;细胞实时荧光检测细胞凋亡水平;平板克隆实验检测GBM细胞克隆形成能力;CCK-L法检测胞内ATP水平;Western blot检测Akt及其磷酸化水平。结果显示,与对照组相比,随着作用浓度增加,二甲双胍显著抑制GBM细胞增殖活性,影响细胞形态;与对照组相比,同一作用浓度下,二甲双胍提高了GBM细胞凋亡水平,抑制了GBM细胞克隆形成能力,降低了GBM胞内ATP的产生;二甲双胍处理24 h后,GBM细胞内p-Akt表达显著下调,Akt无明显变化。结果表明,二甲双胍在体外可抑制多种GBM细胞的增殖、克隆,降低胞内ATP水平,其机制可能与Akt磷酸化水平相关,研究结果为进一步探索二甲双胍对胶质母细胞瘤的作用机制提供了体外研究理论基础。     

COX-2 Inhibition Combined with Radiation Reduces Orthotopic Glioma Outgrowth by Targeting the Tumor Vasculature

Cyclooxygenase 2 (COX-2) inhibitors have been shown to enhance tumor''s response to radiation in several animal models. The strong association of COX-2 and angiogenesis suggests that the tumor vasculature may be involved in this process. The current study investigated whether treatment with the COX-2 inhibitor E-6087 could influence response to local radiation in orthotopically growing murine gliomas and aimed to analyze the involvement of the tumor vasculature. GL261 glioma cells were injected into the cerebrum of C57bl/6 mice. From day 7 after tumor cell injection, mice were treated with COX-2 inhibitor at 50 mg/kg i.p. every third day. Radiation consisted of three fractions of 2 Gy given daily from day 9 to day 11. Mice were killed at day 21. The COX-2 inhibitor significantly enhanced the response to radiation, reducing mean volume to 32% of tumors treated with radiation only. The combination treatment neither increased apoptosis of tumor cells or stromal cells nor affected tumor microvascular density. In vitro, E-6087 and its active metabolite did not affect clonogenic survival of GL261 cells or human umbilical vein endothelial cell after radiation. In vivo, however, there was a nonsignificant increase in Angiopoietin (Ang)-1 and Tie-2 mRNA levels and a decrease of Ang-2 mRNA levels after combination treatment. These changes coincided with a significant increase in α-smooth muscle actin-positive pericyte coverage of tumor vessels. In conclusion, the antitumor effect of radiation on murine intracranial glioma growth is augmented by combining with COX-2 inhibition. Our findings suggest an involvement of the tumor vasculature in the observed effects.