Response Of Glioma Cells Research Articles

IGF-1 induced HIF-1α-TLR9 cross talk regulates inflammatory responses in glioma

The insulin-like growth factor (IGF-1) induces hypoxia inducible factor (HIF-1α) regulated genes in glioblastoma multiforme (GBM). As HIF-1α links inflammatory and oncogenic pathways in GBM, we investigated whether IGF-1 affects HIF-1α to regulate inflammatory response in glioma cells under normoxia. IGF-1 induced Ras and Calmodulin-dependent kinase II (CaMKII) regulated HIF-1α transcriptional activity in glioma cells. Increase in HIF-1α was concurrent with decreased Toll-like receptor (TLR9) and CXCR4 expression and elevated suppressor of cytokine signaling (SOCS3) levels. Interestingly, while synthetic CpG containing oligodeoxynucleotide TLR9 agonist (CpG DNA) decreased IGF-1 mediated increase in HIF-1α activity, siRNA mediated knockdown of HIF-1α decreased TLR9 levels. This suggested that IGF-1 induced HIF-1α-TLR9 axis is regulated by both positive and negative feedback loops. Importantly, TLR9 agonist reversed the effect of IGF-1 on CXCR4 and SOCS3 expression. While knockdown of HIF-1α abrogated IGF-1 mediated increase in SOCS3 it elevated IGF-1 induced decrease in CXCR4 levels. Thus HIF-1α positively and negatively regulates SOCS3 and CXCR4 expression respectively, in glioma cells. Though TLR9 agonist had no additive effect on IGF-1 mediated increase in pro-inflammatory cytokines IL-1β, IL-6 and IL-8, treatment with TLR9 agonist alone elevated expression of these pro-inflammatory cytokines. Our studies indicate that a complex HIF-1α-TLR9 cross-talk sustains a self-regulating cycle of inflammatory response through intrinsic negative and positive feedback mechanisms.

Abstract 1951: Inhibition of aurora kinase using VX-680 enhances the radiation response in glioma cells

Abstract The central role of aurora kinases in cell cycle regulation coupled with overexpression of aurora A and B kinases in a wide variety of human malignancies that led to the development of pharmacological inhibitors of aurora kinases. New generations of aurora inhibitors, including VX-680, has been evaluated preclinically and are in phase I and II trials for the treatment of solid tumors and leukemia. In this study, we investigated the role of VX680, Aurora kinase inhibitor in malignant human glioma cells with different p53 statuses as well as cytotoxicity of glioma cells to irradiation. VX-680 inhibits aurora A and B kinases and cell proliferation with nanomolar to low micromolar potency. Endoreduplication, the re-replication of DNA in the absence of mitotic division, ultimately results in polyploidy and inhibition of cell proliferation. VX680 treatment led to an increase in the number of multinucleated cells and the ratios of G2/M phase in p53-mutant (LN229) and p53-deleted cells (LNZ308), but minimal in p53-wild type (A172) glioma cells. FACS analysis revealed that the increase in nuclear size correlated with polyploidization, reaching a >16 DNA content on day 2. During mitosis, the cells rounded normally, condensed their chromosomes on a metaphase-like plate. Our results demonstrate that pretreating cells with VX-680 prior to ionizing radiation led to an enhancement of radiation -induced apoptosis. Accordingly, our results suggested that Aurora kinase inhibition followed by irradiation increases in the appearance of multinucleated cells and increase apoptosis, and this treatment may become a potential therapy against malignant human gliomas. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1951. doi:10.1158/1538-7445.AM2011-1951

Abstract 1642: TIP-1 regulates sensitivity of glioma cells to radio- and chemotherapeutic agents through modulating p53 stability and activation

Abstract Malignant gliomas represent one group of tumors that poorly respond to chemo- and radio-therapies because of the intrinsic or acquired resistance. Identification of novel molecules that are involved in the therapeutics resistance is critical to develop new therapeutic modalities. Our previous studies show that TIP-1 involves in tumor cells response to ionizing radiation (IR). In this study, we found that TIP-1 expression level correlates to the clinical staging and overall patient survival time after diagnosis of malignant gliomas. We hypothesized that TIP-1 is one novel predictive biomarker of glioma cell response to treatment, and targeting TIP-1 represents one novel strategy to combat the disease of high mortality. Studies on cell-based models indicated that TIP-1 deficiency resulted in the enhanced sensitivity of D54 and GL261 glioma cell lines to IR and chemotherapeutic agents, whereas ectopic expression of TIP-1 conferred resistance to the therapeutics in the cells. The data also showed that sensitivity of the glioma cell lines to IR correlates to stability and activation of p53 protein after IR treatment. Yeast two-hybrid screening identified C53/LZAP as one TIP-1 interacting protein within glioma cells. PDZ domain within TIP-1 binds to one classic type I PDZ-binding motif at the carboxyl terminus of C53/LZAP protein. C53/LZAP has been studied as one tumor suppressor and modulator of the cancer cell susceptibility to genotoxic stress including IR and several chemotherapeutic agents. C53/LZAP interacts with ARF and protects p53 from Mdm2-mediated protein degradation. Our data demonstrated that, after irradiation, presence of TIP-1 enhanced C53/LZAP interaction with ARF, thus promoted Mdm2-mediated p53 ubiquitination and resulted in less stable p53 protein. On the other hand, with absence of TIP-1 protein, IR treatment resulted in more stabilized and activated p53 protein that drives IR-induced apoptosis of the glioma cells. Taken together, our data suggests that TIP-1 is one novel modulator of p53 tumor suppressor and contributes to chemo- and radio-resistance of glioma cells. Therefore, targeting TIP-1 represents one novel approach for the improved therapy of malignant glioma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1642. doi:10.1158/1538-7445.AM2011-1642

Activation of p53 by nutlin-3a induces apoptosis and cellular senescence in human glioblastoma multiforme.

Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor in adults. Despite concerted efforts to improve current therapies and develop novel clinical approaches, patient survival remains poor. As such, increasing attention has focused on developing new therapeutic strategies that specifically target the apoptotic pathway in order to improve treatment responses. Recently, nutlins, small-molecule antagonists of MDM2, have been developed to inhibit p53-MDM2 interaction and activate p53 signaling in cancer cells. Glioma cell lines and primary cultured glioblastoma cells were treated with nutlin-3a. Nutlin-3a induced p53-dependent G1- and G2-M cell cycle arrest and apoptosis in glioma cell lines with normal TP53 status. In addition, nutlin-arrested glioma cells show morphological features of senescence and persistent induction of p21 protein. Furthermore, senescence induced by nutlin-3a might be depending on mTOR pathway activity. In wild-type TP53 primary cultured cells, exposure to nutlin-3a resulted in variable degrees of apoptosis as well as cellular features of senescence. Nutlin-3a-induced apoptosis and senescence were firmly dependent on the presence of functional p53, as revealed by the fact that glioblastoma cells with knockdown p53 with specific siRNA, or cells with mutated or functionally impaired p53 pathway, were completely insensitive to the drug. Finally, we also found that nutlin-3a increased response of glioma cells to radiation therapy. The results provide a basis for the rational use of MDM2 antagonists as a novel treatment option for glioblastoma patients.

Validation of an amino-acid-based radionuclide therapy plus external beam radiotherapy in heterotopic glioblastoma models

Malignant gliomas represent a major therapeutic challenge because no efficient treatment is currently available. p-[(131)I]iodo-L-phenylalanine ([(131)I]IPA) is a glioma avid radiopharmaceutical that demonstrated antiproliferative and tumoricidal effects in gliomas. The present study validated the therapeutic efficiency of [(131)I]IPA combined with external beam radiotherapy in experimental gliomas. Glioma cells derived from the primary human A1207, T5135, Tx3868 and M059K glioblastoma cell lines or rat F98 glioma cell line were treated with various doses of [(131)I]IPA, external photon irradiation (RT) or combined [(131)I]IPA/RT treatment. Responsiveness of glioma cells to the different therapy modalities was investigated at 24, 48 and 72 h after treatments by trypan blue, WST-1 assay, propidium iodide and bisbenzimide staining as well as by clonogenic assay. In addition, the therapy-induced DNA damage and repair were evaluated using phosphorylated histone H2AX (γ-H2AX). In vivo, the effectiveness of the combination treatment was validated in human Tx3868 and A1207 glioblastoma xenografts in CD1 nu/nu mice and RNU rats. In vitro, the combination treatment resulted in a greater than additive increase in cytotoxic effect in glioma cell lines. Cell survival rate following a treatment with 1.0 μCi (37 kBq) of [(131)I]IPA amounted to 70%±15% and 60%±10% after 48 and 72 h, respectively, and decreased under 20% after additional RT with 5 Gy. At higher RT doses, cell survival rate decreased below 5%. As a measure of DNA double-strand break, nuclear γ-H2AX foci were determined as a function of time. Within 24 h, the number of γ-H2AX foci per cell was significantly greater after combined modality compared with the individual treatments. In vivo, when combined with RT, the radionuclide therapy with [(131)I]IPA resulted in an extended tumor growth delay, a reduction of the initial tumor volume and an enhanced radiosensitivity in Tx3868 and A1207 glioblastoma xenografts in CD1 nu/nu mice and RNU rats. On day 90 after monotherapy with [(131)I]IPA (20 MBq) or RT (20 Gy), 35%-50% of the treated rats were still alive. In comparison, up to 70%-80% survival rates were registered after combined [(131)I]IPA/RT treatment on day 100 for all animal models. These preclinical data convincingly demonstrated that [(131)I]IPA plus external beam photon radiotherapy is a safe and highly effective treatment for experimental gliomas, which may merit a clinical trial to ascertain its potential as a therapeutic approach in patients. As only a low [(131)I]IPA activity and a low RT dose were applied, further optimization strategies should be pursued experimentally, including application of higher radiation doses and conventional fractionated regimens or use of methods aiming to increase target doses and maximize dose effects.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Inactivation of ataxia telangiectasia mutated gene can increase intracellular reactive oxygen species levels and alter radiation-induced cell death pathways in human glioma cells

Purpose: To investigate the effects of ataxia telangiectasia mutated (ATM)-regulated reactive oxygen species (ROS) and cell death pathways on the response of U87MG glioma cells to ionising radiation (IR) and oxidative stress.Material and methods: ATM expression was blocked in U87MG glioma cells using a small interfering RNA (siRNA) technique. Cell survival, sub-lethal damage (SLD), and potential lethal damage (PLD) repair following IR were assessed by clonogenic assay while changes in intracellular ROS, the apoptosis, and autophagy were followed by flow cytometry and Western blotting.Results: Blocking ATM expression in U87MG cells increased intracellular ROS levels and sensitivity to the cytotoxic effects of IR and oxygen stress; effects that could be partly counteracted by the antioxidant N-acetylcysteine (NAC). Knock down of ATM rendered cells unable to repair sub-lethal or potentially lethal damage and DNA double strand breaks (DSB) after IR exposure; something that NAC could not counteract. ATM did control the pathways a cell used to die following IR and this did seem to be ROS-dependent.Conclusion: ATM is involved in redox control but ROS elevations following ATM knock down seem more involved in the decision as to what cell death pathway is utilised after IR than DSB repair and radiosensitivity.

A Dual PI3K/mTOR Inhibitor, PI-103, Cooperates with Stem Cell–Delivered TRAIL in Experimental Glioma Models

The resistance of glioma cells to a number of antitumor agents and the highly invasive nature of glioma cells that escape the primary tumor mass are key impediments to the eradication of tumors in glioma patients. In this study, we evaluated the therapeutic efficacy of a novel PI3-kinase/mTOR inhibitor, PI-103, in established glioma lines and primary CD133(+) glioma-initiating cells and explored the potential of combining PI-103 with stem cell-delivered secretable tumor necrosis factor apoptosis-inducing ligand (S-TRAIL) both in vitro and in orthotopic mouse models of gliomas. We show that PI-103 inhibits proliferation and invasion, causes G(0)-G(1) arrest in cell cycle, and results in significant attenuation of orthotopic tumor growth in vivo. Establishing cocultures of neural stem cells (NSC) and glioma cells, we show that PI-103 augments the response of glioma cells to stem cell-delivered S-TRAIL. Using bimodal optical imaging, we show that when different regimens of systemic PI-103 delivery are combined with NSC-derived S-TRAIL, a significant reduction in tumor volumes is observed compared with PI-103 treatment alone. To our knowledge, this is the first study that reveals the antitumor effect of PI-103 in intracranial gliomas. Our findings offer a preclinical rationale for application of mechanism-based systemically delivered antiproliferative agents and novel stem cell-based proapoptotic therapies to improve treatment of malignant gliomas.

Radiosensitivity of glioma to Gamma Knife treatment enhanced in vitro and in vivo by RNA interfering Ku70 that is mediated by a recombinant adenovirus

The authors sought to evaluate modification of the radiation response of C6 glioma cells in vitro and in vivo by inhibiting the expression of Ku70. To do so they investigated the effect of gene transfer involving a recombinant replication-defective adenovirus containing Ku70 short hairpin RNA (Ad-Ku70shRNA) combined with Gamma Knife treatment (GKT). First, Ad-Ku70shRNA was transfected into C6 glioma cells and the expression of Ku70 was measured using Western blot analysis. In vitro, phenotypical changes in C6 cells, including proliferation, cell cycle modification, invasion ability, and apoptosis were evaluated using the MTT (3'(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide) assay, Western blot analysis, and cell flow cytometry. In vivo, parental C6 cells transfected with Ad-Ku70shRNA were implanted stereotactically into the right caudate nucleus in Sprague-Dawley rats. After GKS, apoptosis was analyzed using the TUNEL (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling) method. The inhibitory effects on growth and invasion that were induced by expression of proliferating cell nuclear antigen and matrix metalloproteinase-9 were determined using immunohistochemical analyses. The expression of Ku70 was clearly inhibited in C6 cells after transfection with Ad-Ku70shRNA. In vitro following transfection, the C6 cells showed improved responses to GKT, including suppression of proliferation and invasion as well as an increased apoptosis index. In vivo following transfection of Ad-Ku70shRNA, the therapeutic efficacy of GKT in rats with C6 gliomas was greatly enhanced and survival times in these animals were prolonged. Our data support the potential for downregulation of Ku70 expression in enhancing the radiosensitivity of gliomas. The findings of our study indicate that targeted gene therapy-mediated inactivation of Ku70 may represent a promising strategy in improving the radioresponsiveness of gliomas to GKT.

In vitro biological dosimeter modeling of the glioblastoma response to radiation delivered by the Gamma Knife

The goal of this study was to develop an assay that makes possible the assessment of the glioma cell response to single-fraction high-dose Gamma Knife surgery. In this assay, the isolation of radioresistant cell subpopulations facilitates mechanistic studies of radioresistance. A tissue-equivalent paraffin phantom with an aperture capable of holding an Opticell cell culture cassette was developed for treatment with the Leksell Gamma Knife model C. A second apparatus, which the authors also created, uses the manufacturer-supplied polystyrene phantom, thereby allowing this assay to be performed in the Leksell Gamma Knife Perfexion. After treatment, the cells were morphologically assessed to determine their response to radiation treatment. Two specific parameters were used to determine radiosensitivity: 1) the diameter of the clearing zone, defined as the central region of cell death; and 2) the number of surviving colonies within this central high-dose clearing zone. Radioresistance was compared in 2 different cell lines from glioblastomas. The first cell line, ME, was established from a primary tumor before its treatment, and the second cell line, DIV, was established from a tumor that recurred after treatment with chemotherapy and fractionated radiotherapy. The ME cell line had the most robust response to radiosurgery, as characterized by a consistently larger clearing zone (28.33 ± 1.1 mm). In contrast, the clearing zone produced when the DIV cell line was used was 24.0 ± 1 mm, indicating an approximate response difference of 5 Gy. The mean number of surviving colonies within the clearing zone for the ME cell line was 1.33 ± 1 compared with that for the DIV cell line, which was 66.67 ± 2. The authors developed a biological dosimeter to model the response of cells from glioblastomas to single-fraction high-dose radiation. This system also allows the identification and isolation of radioresistant cells.

Induction of Bystander Response in Human Glioma Cells using High-Energy Electrons: A Role for TGF-β1

We examined bystander cell death produced in T98G cells by exposure to irradiated cell conditioned medium (ICCM) produced by high-energy 20 MeV electrons at a dose rate of 10 Gy min(-1) and doses up to 20 Gy. ICCM induced a bystander response in T98G glioma cells, reducing recipient cell survival by more than 25% below controls at 5 and 10 Gy. Higher doses increased survival to near control levels. ICCM was analyzed for the presence of transforming growth factor alpha (TGF-alpha) and transforming growth factor beta1 (TGF-beta1). Monoclonal antibodies for TGF-alpha (mAb TGF-alpha) and TGF-beta1 (mAb TGF-beta1) were added to the ICCM to neutralize any potential effect of the cytokines. The results indicate that TGF-alpha was not present in the ICCM and addition of mAb TGF-alpha to the ICCM had no effect on bystander cell survival. No active TGF-beta1 was present in the ICCM; however, addition of mAb TGF-beta1 completely abolished bystander death of reporter cells at all doses. These results indicate that bystander cell death can be induced in T98G glioma if a large enough radiation stress is applied and that TGF-beta1 plays a downstream role in this response.

Abstract 1250: Inhibition of FAK and engagement of migration leads to similar changes in patterns of transcription factor activation in glioma cells

Abstract Malignant dispersion of glioma cells is a nemesis to clinical management, provoking the need for a better understanding of the underlying molecular drivers of invasion. The changes in signal transduction and consequent alterations in activation/de-activation of Transcription Factors (TFs) in glioma cells were investigated coincident to migration-engagement or impaired FAK kinase function by small molecule inhibition (PF-562,271). Earlier work showed that FAK and Pyk2 affect proliferation and migration in reciprocal ways in glioma cells (Lipinski CA, et al 2008; Loftus JC, et al 2009). The interaction of FAK and Pyk2 with specific downstream signaling effectors and the resultant activation of disparate signaling pathways is likely to script a signaling “signature” that will reveal to what extent glioma cell malignant behaviors are dependent on these kinases. Inhibition of FAK leads to diminution of signaling by pSrc, pJnk, and pNFkB, and variable activation of pAkt, and pp38. Of the 20 TFs assayed (Marligen, Inc.), the activation state of 7 TFs showed significant and consistent patterns of change across 6 primary GBM cell strains following FAK inhibition. Across a panel of 6 human GBM xenograft lines SP1, p53, E2F1-5, and NFκB evidenced strong activation (heightened nuclear localization), while AR (androgen receptor), Myc-Max, and CREB activation levels dropped by more than 2-fold following PF-562,271 treatment. Separate studies of established glioma cell lines under conditions of sparse plating (migration) and of dense crowded plating (migration-constrained), showed that p53, E2F1-5, and NFκB were standouts for activation accompanying migration, also associated by strong reduced activation of AR, Myc-Max, and CREB. (Because glioma cells following FAK knockdown or FAK inhibition show reduced entry into cell cycle, the signaling linkage between FAK and Myc-Max activation and also between FAK signaling and CREB activation suggest mechanistic ties to phenotype. E2F1 is a transcription factor that regulates genes involved in cell proliferation and cell survival and has even been suggested to fit a paradigm of oncogene addiction in glioma cells. Our findings demonstrate phenotypic responses in glioma cells downstream of FAK signaling linked to the activation or inactivation of specific transcription factors. If these patterns of TF activation state, and the signaling pathways upstream of that activation, associate (or account for) patterns of glioma cell invasion or proliferation in situ, they will suggest points of vulnerability in the underlying pathological processes of this disease. Supported by NIH NS042262 (Berens) and NIH CA103956 (Loftus). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1250.

Abstract 3495: Targeting gliomas with a dual kinase inhibitor and stem-cell delivered TRAIL

Abstract Gliomas are difficult to eradicate due to several factors: a) the lack of efficient delivery of anti-tumor agents to the tumors without affecting the normal brain tissue; b) the inherent or acquired resistance of tumor cells to anti-tumor agents; and c) the highly invasive nature of tumor cells that escape the primary tumor mass and subsequently cause recurrence. We previously established that a secreted form of tumor necrosis factor-related apoptosis inducing ligand (S-TRAIL) specifically eradicates glioma cells when delivered by various stem cell types in culture and in orthotopic glioma models. However, recent evidence has shown that a number of established and primary glioma lines have varying degrees of resistance to TRAIL, due to the heterogeneity of genetic alterations and possibly due to the over-activation of phosphatidylinositol 3 kinase (PI3K) pathway. In this study, we report the effect of a novel PI3K inhibitor, PI-103, alone or in combination with neural stem cell (NSC)-delivered S-TRAIL in mouse models of malignant and invasive gliomas. Using established and primary CD133+ glioma initiating cells engineered to express fluorescent and bioluminescent markers, we show that PI-103 alone inhibits proliferation and invasion, and causes G0-G1 arrest in cell cycle of most of the glioma cells tested. Furthermore, PI-103 augments the response of glioma cells to stem cell delivered S-TRAIL in co-cultures of NSCs and glioma cells suggesting that it can synergize with S-TRAIL-induced apoptosis. Using orthotopic malignant glioma models and bioluminescence imaging, PI-103 treatment in combination with NSC-derived S-TRAIL was shown to result in significant reduction in tumor volumes compared to either treatment alone in vivo. Our results reveal that combination of the novel PI3K inhibitor, PI-103, and stem cell delivered TRAIL might represent a novel therapy for malignant gliomas. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3495.

Assay of glioma cell responses to an anticancer drug in a cell-based microfluidic device

Despite progress in research and treatment, the prognosis for patients with gliomas remains relatively dismal. As such, develop a new in vitro model is fast becoming a necessary step in efforts to improve research on malignant gliomas. Microfluidics, a potentially effective tool, has been increasingly applied in neuroscience and oncology studies. However, gliomas, the most common primary brain tumours, have not yet been treated with this application. In the current study, we developed a glioma-related research method using microfluidics. After successfully culturing rat C6 glioma cells for up to 7 days in a microfluidic device, we monitored cellular responses to the anticancer drug, colchicines, after which we analysed cell viability using propidium iodide (PI) staining. We recorded temporal changes in cell morphology at various concentrations of colchicine using an inverted microscope. Results show that the number of injured/dead cancer cells and morphological changes increased relative to the drug’s concentration and treatment frequency. These findings will be helpful in developing microfluidic device applications for future research on brain tumour therapy (for malignant gliomas and other types of tumours), for conducting cytotoxicity research in a biomimetic microenvironment, for developing glioma-related anticancer drugs, and for developing glial cell-based biosensors for glioma detection.

Annexin A2 Is a Molecular Target for TM601, a Peptide with Tumor-targeting and Anti-angiogenic Effects

TM601 is a synthetic form of chlorotoxin, a 36-amino acid peptide derived from the venom of the Israeli scorpion, Leirius quinquestriatus, initially found to specifically bind and inhibit the migration of glioma cells in culture. Subsequent studies demonstrated specific in vitro binding to additional tumor cell lines. Recently, we demonstrated that proliferating human vascular endothelial cells are the only normal cell line tested that exhibits specific binding to TM601. Here, we identify annexin A2 as a novel binding partner for TM601 in multiple human tumor cell lines and human umbilical vein endothelial cell (HUVEC). We demonstrate that the surface binding of TM601 to the pancreatic tumor cell line Panc-1 is dependent on the expression of annexin A2. Identification of annexin A2 as a binding partner for TM601 is also consistent with the anti-angiogenic effects of TM601. Annexin A2 functions in angiogenesis by binding to tissue plasminogen activator and regulating plasminogen activation on vascular endothelial cells. We demonstrate that in HUVECs, TM601 inhibits both vascular endothelial growth factor- and basic fibroblast growth factor-induced tissue plasminogen activator activation, which is required for activation of plasminogen to plasmin. Consistent with inhibition of cell surface protease activity, TM601 also inhibits platelet-derived growth factor-C induced trans-well migration of both HUVEC and U373-MG glioma cells.

Cilengitide modulates attachment and viability of human glioma cells, but not sensitivity to irradiation or temozolomide in vitro

Cilengitide is a cyclic peptide antagonist of integrins alphavbeta3 and alphavbeta5 that is currently being evaluated as a novel therapeutic agent for recurrent and newly diagnosed glioblastoma. Its mode of action is thought to be mainly antiangiogenic but may include direct effects on tumor cells, notably on attachment, migration, invasion, and viability. In this study we found that, at clinically relevant concentrations, cilengitide (1-100 microM) induces detachment in some but not all glioma cell lines, while the effect on cell viability is modest. Detachment induced by cilengitide could not be predicted by the level of expression of the cilengitide target molecules, alphavbeta3 and alphavbeta5, at the cell surface. Glioma cell death induced by cilengitide was associated with the generation of caspase activity, but caspase activity was not required for cell death since ectopic expression of cytokine response modifier (crm)-A or coexposure to the broad-spectrum caspase inhibitor zVAD-fmk was not protective. Moreover, forced expression of the antiapoptotic protein marker Bcl-X(L) or altering the p53 status did not modulate cilengitide-induced cell death. No consistent effects of cilengitide on glioma cell migration or invasiveness were observed in vitro. Preliminary clinical results indicate a preferential benefit from cilengitide added to temozolomide-based radiochemotherapy in patients with O(6)-methylguanine DNA methyltransferase (MGMT) gene promoter methylation. Accordingly, we also examined whether the MGMT status determines glioma cell responses to cilengitide alone or in combination with temozolomide. Neither ectopic expression of MGMT in MGMT-negative cells nor silencing the MGMT gene in MGMT-positive cells altered glioma cell responses to cilengitide alone or to cilengitide in combination with temozolomide. These data suggest that the beneficial clinical effects derived from cilengitide in vivo may arise from altered perfusion, which promotes temozolomide delivery to glioma cells.

Differential Response of Glioma Cells to FOXO1-Directed Therapy

Gliomas are the most common adult primary brain tumors, and the most malignant form, glioblastoma multiforme, is invariably fatal. The phosphatidylinositol 3-kinase (PI3K)-Akt signaling pathway is altered in most glioblastoma multiforme. PTEN, an important negative regulator of the PI3K-Akt pathway, is also commonly mutated in glioma, leading to constitutive activation of Akt. One ultimate consequence is phosphorylation and inactivation of FOXO forkhead transcription factors that regulate genes involved in apoptosis, cell cycle arrest, nutrient availability, DNA repair, stress, and angiogenesis. We tested the ability of a mutant FOXO1 factor that is not subject to Akt phosphorylation to overcome dysregulated PI3K-Akt signaling in two PTEN-null glioma cell lines, U87 and U251. Adenovirus-mediated gene transfer of the mutant FOXO1 successfully restored cell cycle arrest and induced cell death in vitro and prolonged survival in vivo in xenograft models of human glioma (33% survival at 1 year of animals bearing U251 tumors). However, U87 were much more resistant than U251 to mutant FOXO1-induced death, showing evidence of increased nuclear export and Akt-independent phosphorylation of FOXO1 at S249. A cyclin-dependent kinase 2 inhibitor decreased phosphorylation of S249 and rendered U87 cells significantly more susceptible to mutant FOXO1-induced death. Our results indicate that targeting FOXO1, which is at the convergence point of several growth factor receptor tyrosine kinase pathways, can effectively induce glioma cell death and inhibit tumor growth. They also highlight the importance of Akt-independent phosphorylation events in the nuclear export of FOXO1.

Investigation of immunosuppressive mechanisms in a mouse glioma model

The development of an immune competent mouse model for the study of immunosuppressive mechanisms is important for improving the efficacy of brain tumor immunotherapy. In the present study we investigated regulatory T cells (Tregs), TGF-beta1 and other putative immunosuppressive cytokines using GL261 mouse glioma in C57BL mice. We explored whether tumor growth factor-beta1 (TGF-beta1) is expressed and secreted by glioma cells constitutively or in response to a T-cell mediated immunity (simulated by conditioned media from T cells (TCM) activated by anti-CD3 antibody). We also investigated TGF-beta1's role in Treg mediated immunosuppression by quantifying TGF-beta1secretion from T regulatory cells (Tregs) co-incubated with GL261 cells as compared to Tregs alone. Finally, we studied other newly identified cytokines that were secreted preferentially by glioma cells in response to CD3 activated TCM versus cytokines secreted by glioma cells in absence of T-cell activation (naïve TCM). TGF-beta1expression was studied using RT-PCR and secretion was quantified using ELISA. A 308 protein cytokine array was used to identify and quantify cytokine expression. TGF-beta1expression and secretion from glioma cells was found to be up-regulated by conditioned media from CD3-activated T cells, suggesting that this immunosuppressive cytokine is not secreted constitutively but in response to immunity. TGF-beta1 was not found to be differentially secreted by Tregs co-incubated with glioma cells as compared to Tregs alone. This data suggest that TGF-beta1immunosupppression may not be a Treg dependent mechanism in this glioma model. Finally, the cytokine array elucidated several other cytokines which were up-regulated or down-regulated by CD3-activated TCM. These results have several implications for enhancing immunotherapy treatment, including the potential benefit of TGF-beta1inhibition in conjunction with immunotherapy, as well as the illumination of several other potential cytokine targets to be explored as shown by the cytokine array.

The role of autophagy in sensitizing malignant glioma cells to radiation therapy

Malignant gliomas represent the majority of primary brain tumors. The current standard treatments for malignant gliomas include surgical resection, radiation therapy, and chemotherapy. Radiotherapy, a standard adjuvant therapy, confers some survival advantages, but resistance of the glioma cells to the efficacy of radiation limits the success of the treatment. The mechanisms underlying glioma cell radioresistance have remained elusive. Autophagy is a protein degradation system characterized by a prominent formation of double-membrane vesicles in the cytoplasm. Recent studies suggest that autophagy may be important in the regulation of cancer development and progression and in determining the response of tumor cells to anticancer therapy. Also, autophagy is a novel response of glioma cells to ionizing radiation. Autophagic cell death is considered programmed cell death type II, whereas apoptosis is programmed cell death type I. These two types of cell death are predominantly distinctive, but many studies demonstrate a cross-talk between them. Whether autophagy in cancer cells causes death or protects cells is controversial. The regulatory pathways of autophagy share several molecules. PI3K/Akt/mTOR, DNA-PK, tumor suppressor genes, mitochondrial damage, and lysosome may play important roles in radiation-induced autophagy in glioma cells. Recently, a highly tumorigenic glioma tumor subpopulation, termed cancer stem cell or tumor-initiating cell, has been shown to promote therapeutic resistance. This review summarizes the main mediators associated with radiation-induced autophagy in malignant glioma cells and discusses the implications of the cancer stem cell hypothesis for the development of future therapies for brain tumors.

Bee venom suppresses LPS-mediated NO/iNOS induction through inhibition of PKC-α expression

Ethnopharmacological relevance Bee venom (BV) is a traditional Korean medicine that has been widely used with satisfactory results in the treatment of some immune-related diseases, especially rheumatoid arthritis. Aim of the study The purpose of this study is to elucidate the molecular mechanism underlying the anti-inflammatory effects of BV, which is used in the treatment of various inflammatory diseases in traditional Korean medicine. We evaluated the anti-inflammatory effect of BV on NO generation and iNOS expression by LPS in rat C6 glioma cells. Material and methods BV was obtained from the National Institute of Agricultural Science and Technology (NIAST) of Korea. Nitrite measurement, Immuno blot analysis, Reverse transcriptase-PCR and Electrophoretic mobility shift assay (EMSA) were used for assessment. Results BV suppressed the LPS-induced NO generation and iNOS expression, and it also inhibited the expressions of LPS-induced pro-inflammatory molecules including Cox-2 and IL-1β in rat C6 glioma cells. Then, BV inhibited LPS-induced expression of PKC-α and MEK/ERK, not p38 and JNK. Moreover, inhibition of LPS-induced iNOS expression by BV was dependent on transcriptional activities of AP-1/NF-κB through MEK/ERK pathway. Conclusion These results indicate that BV suppresses LPS-induced iNOS activation through regulation of PKC-α. Accordingly, BV exerts a potent suppressive effect on pro-inflammatory responses in rat C6 glioma cells.