U87 Glioblastoma Multiforme Cells Research Articles

Kinin-B1 Receptor Stimulation Promotes Invasion and is Involved in Cell-Cell Interaction of Co-Cultured Glioblastoma and Mesenchymal Stem Cells

Glioblastoma multiforme (GBM) represents the most lethal brain tumour, and these tumours have very limited treatment options. Mesenchymal stem cells (MSC) are considered as candidates for advanced cell therapies, due to their tropism towards GBM, possibly affecting their malignancy, thus also representing a potential therapeutic vector. Therefore, we aimed to compare the effects of bone-marrow-derived versus adipose-tissue-derived MSC (BM-/AT-MSC) on heterogeneous populations of tumour cells. This cells’ interplay was addressed by the in-vitro two-dimensional (monolayer) and three-dimensional (spheroid) co-culture models, using U87 and U373 GBM cell lines, expressing genotypically different mesenchymal transcriptome profiles. U87 cell low mesenchymal profile expressed high levels of kinin receptor 1 (B1R) and their invasion was greatly enhanced by the B1R agonist des-Arg9-bradykinin upon BM-MSC co-culturing in 3D co-cultures. This correlated to significantly higher cell-cell interactions in U87/BM-MSC mixed spheroids. This was not observed with the U373 cells and not in AT-MSC co-cultures. Altogether, these data support the on-going exploration of B1R as target for adjuvant approach in GBM therapy. Secondly, the results emphasize the need for further careful exploration of the selectivity regarding the origin of MSC as potential candidates for cell therapies, particular in cancer, where they may adversely affect heterogeneous tumour cell populations.

Delivery of Exogenous miR-124 to Glioblastoma Multiform Cells by Wharton's Jelly Mesenchymal Stem Cells Decreases Cell Proliferation and Migration, and Confers Chemosensitivity.

MicroRNAs (miRs) are potential therapeutic targets in glioblastoma multiforme (GBM), but the difficulties associated with their delivery to tumor target cells have hampered their widespread use. Mesenchymal stem cells (MSCs) can migrate to the sites of cancers, including GBM and exert anti-tumor effects. In this study, it is shown that Wharton's jelly-MSCs (WJ-MSCs) have the ability to deliver exogenous miRs to GBM cells and the functional impact of this delivery is characterized. It is found that the labeled miR-124, as an example for miR of interest, can be successfully delivered with WJ-MSCs to U87 GBM cells via dependent or exosome-independent processes. It is demonstrated that the delivered exogenous miR-124 significantly decreases the luciferase activity of the target gene CDK6. In addition, the delivered miR-124 enhances the chemosensitivity of GBM cells to temozolomide and decreases the migration of GBM cells. These results suggest that the use of exogenous miRNA delivery with the derived exosomes from WJ-MSCs may provide a novel approach for miRNA replacement therapy in GBM cancers.

Augmented expression of RUNX1 deregulates the global gene expression of U87 glioblastoma multiforme cells and inhibits tumor growth in mice.

Glioblastoma multiforme is the most common and aggressive primary brain tumor in adults. A mesenchymal phenotype was associated with tumor aggressiveness and poor prognosis in glioblastoma multiforme patients. Recently, the transcription factor RUNX1 was suggested as a driver of the glioblastoma multiforme mesenchymal gene expression signature; however, its independent role in this process is yet to be described. Here, we assessed the role of RUNX1 in U87 glioblastoma multiforme cells in correspondence to its mediated transcriptome and genome-wide occupancy pattern. Overexpression of RUNX1 led to diminished tumor growth in nude and severe combined immunodeficiency mouse xenograft tumor model. At the molecular level, RUNX1 occupied thousands of genomic regions and regulated the expression of hundreds of target genes, both directly and indirectly. RUNX1 occupied genomic regions that corresponded to genes that were shown to play a role in brain tumor progression and angiogenesis and upon overexpression led to a substantial down-regulation of their expression level. When overexpressed in U87 glioblastoma multiforme cells, RUNX1 down-regulated key pathways in glioblastoma multiforme progression including epithelial to mesenchymal transition, MTORC1 signaling, hypoxia-induced signaling, and TNFa signaling via NFkB. Moreover, master regulators of the glioblastoma multiforme mesenchymal phenotype including CEBPb, ZNF238, and FOSL2 were directly regulated by RUNX1. The data suggest a central role for RUNX1 as master regulator of gene expression in the U87 glioblastoma multiforme cell line and mark RUNX1 as a potential target for novel future therapies for glioblastoma multiforme.

Identification of new pyrrolo[2,3-d]pyrimidines as Src tyrosine kinase inhibitors in vitro active against Glioblastoma

In the last few years, several pyrrolo-pyrimidine derivatives have been either approved by the US FDA and in other countries for the treatment of different diseases or are currently in phase I/II clinical trials. Herein we present the synthesis and the characterization of a novel series of pyrrolo[2,3-d]pyrimidines, compounds 8a-j, and their activity against Glioblastoma multiforme (GBM). Docking studies and MM-GBSA analysis revealed the ability of such compounds to efficiently interact with the ATP binding site of Src. Enzymatic assays against a mini-panel of kinases (Src, Fyn, EGFR, Kit, Flt3, Abl, AblT315I) have been performed, showing an unexpected selectivity of our pyrrolo[2,3-d]pyrimidines for Src. Finally, the derivatives were tested for their antiproliferative potency on U87 GBM cell line. Compound 8h showed a considerable cytotoxicity effect against U87 cell line with an IC50 value of 7.1 μM.

Selective Toxicity of a Highly Potent Camptothecin Analogue: A Pilot Study with Glioblastoma Multiforme Cells.

We developed a novel camptothecin analogue, CPT417, that yields reduced toxicity compared to other analogues used in chemotherapeutic regimens. In this pilot study, we assessed the activity of CPT417 against glioblastoma multiforme (GBM) cells and glioma stem cells. The human U251 GBM cell line and normal human astrocytes were cultured in parallel for clonogenic survival analysis following exposure to increasing concentrations of CPT417. Cell viability of a glioma stem cell line was assessed 5 days after exposure to a range of CPT417 concentrations. CPT417 completely inhibited clonogenic survival of GBM cells at 10 nM, whereas this concentration only inhibited astrocytes by approximately 50%. Cell viability analysis of glioma stem cell cultures yielded a half-maximal response at 15 nM. CPT417 acts selectively against GBM cells at concentrations that are at least an order of magnitude below reported values for related alkylating agents in clinical use.

Nitidine chloride inhibits the malignant behavior of human glioblastoma cells by targeting the PI3K/AKT/mTOR signaling pathway.

Recent studies have demonstrated that nitidine chloride (NC), a natural bioactive alkaloid, displays potent antitumor activity in various types of cancer. In the present study, NC was examined for efficacy in the treatment of human glioblastoma multiforme (GBM) as well as the molecular basis for its more general inhibitory effects in cancer. U251 and U87 GBM cell lines were exposed to three concentrations of NC (5, 25 and 50µM) invitro, and tumor cell growth was assessed on the basis of proliferation, migration and energy metabolism. Decreases in viability and proliferation reached ~50% for both cell lines with 50µM NC at 24h as assessed by cell viability Cell Counting Kit-8 (CCK-8) and EdU assays. In wound closure and Transwell assays, migration and invasion were inhibited at 50µM after 24h (~20 and 80%, respectively; P<0.05). ATP and L-lactate levels were also decreased after treatment with NC (50µM, 24h; P<0.05 and P<0.01, respectively). Finally, in western blot analysis, phosphorylation of Akt and mTOR was suppressed by NC, but partially restored when cells were treated simultaneously with a novel Akt activator, SC79. Partial restoration was also observed in viability/proliferation (U251 and U87, ~15 vs. 40%; NC vs. NC+SC79; P<0.05) and invasion (U251 and U87, ~30 vs. 60%; NC vs. NC+SC79; P<0.05). Our results demonstrated that NC inhibits development of GBM by targeting the PI3K/Akt/mTOR signaling pathway and provides a potential therapeutic agent for the treatment of GBM.

ATPS-02CONTINUOUS ADMINISTRATION OF THE NOVEL IRON-MIMETIC GALLIUM MALTOLATE INHIBITS GLIOMA GROWTHIN VITROANDIN VIVO

BACKGROUND: Limited treatment options and an aggressive nature make glioblastoma multiforme (GBM) a highly deadly cancer associated with poor prognosis and short survival. Gallium (Ga), an iron-mimetic metal, disrupts tumor iron homeostasis, thus effecting potential antineoplastic activity. Here we present compelling evidence that gallium maltolate (GaM) has a profound antitumor effect on GBM in vitro and in vivo. METHODS: The in vitro cytotoxic effects of GaM were measured by MTT assay in human U87 and D54 GBM cells, and cells isolated from normal rat brain. The in vivo antineoplastic effect of GaM was studied in a xenograft model that involved stereotactic inoculation of U87 cells into the right hemisphere of 8-week old male athymic rats. Following tumor growth confirmation on Day 8 post-inoculation by magnetic resonance imaging (MRI), rats received continuous intravenous (IV) administration of GaM (50 µM) for 10 days. Follow-up MRI occurred on Day 18 post-inoculation. RESULTS: In vitro, GaM profoundly inhibited the proliferation of both U87 and D54 GBM cell lines, but not control cells. The IC50 decreased 46% from 79.8 µM (3-day incubation) to 42.8 µM (5-day incubation) in U87 cells, and 24% from 51.4 µM (3 days) to 38.9 µM (5 days) in D54 cells. Removing GaM on days 3 and 5, and maintaining cells for another 5 days, caused a further decrease in IC50 (U87: 47.4% and 21.8%, respectively; D54: 11.8% and 10.3%, respectively). In vivo, rats receiving IV GaM tolerated the drug well, and no adverse effects were observed. Contrast-agent enhancing tumor volume on T1-weighted MRI increased in both treated (n = 8) and untreated (n = 7) animals, but significantly less so in treated rats. Mean percent increase in cerebral blood volume, determined from steady-state post-MION images, was 45% (controls) and 28% (treated rats) (p < 0.05). CONCLUSION: Gallium maltolate shows great promise for therapeutic potential in GBM.

High metallothionein predicts poor survival in glioblastoma multiforme.

BackgroundGlioblastoma multiforme (GBM) is the most common and aggressive malignant brain tumor. Even with vigorous surgery, radiation and chemotherapy treatment, survival rates of GBM are very poor and predictive markers for prognosis are currently lacking.MethodsWe performed whole genome expression studies of 67 fresh frozen untreated GBM tumors and validated results by 210 GBM samples’ expression data from The Cancer Genome Atlas.Results and discussionHere we show that in GBM patients, high metallothionein (MT) expression is associated with poor survival whereas low MT levels correspond to good prognosis. Furthermore we show that in U87 GBM cell line, p53 is found to be in an inactive mutant-like conformation concurrently with more than 4 times higher MT3 expression level than normal astrocytes and U251GBM cell line. We then show that U87- p53 inactivity can be rescued by zinc (Zn).ConclusionsTaken together, these data suggest that MT expression may be a potential novel prognostic biomarker for GBM, and that U87 cells may be a good model for patients with non active WT p53 resulting from high levels of MTs.

Facile bio-synthesis of gold nanoparticles by using extract of Hibiscus sabdariffa and evaluation of its cytotoxicity against U87 glioblastoma cells under hyperglycemic condition

In the present paper, a facile synthesis of gold nanoparticles is reported with leaf and stem extract of Hibiscus sabdariffa. Structural features of as synthesized nanoparticles are characterized by UV–vis spectroscopy, XRD, FTIR, and XPS. Morphology of the above synthesized gold nanoparticles is investigated by electron microscopy. The stability of the nanoparticles is studied in different concentrations of glucose which suggested their possible application under hyperglycemic condition. As synthesized nanoparticles has shown selective toxicity towards U87 glioblastoma multiforme cell line under normal and hyperglycemic condition, indicating their potential to be used in the development of value-added products in the biochemical industries. The possible mode of activity of the above nanoparticles has been studied by in vitro molecular techniques.

A New Interleukin-13 Amino-Coated Gadolinium Metallofullerene Nanoparticle for Targeted MRI Detection of Glioblastoma Tumor Cells.

The development of new nanoparticles as next-generation diagnostic and therapeutic ("theranostic") drug platforms is an active area of both chemistry and cancer research. Although numerous gadolinium (Gd) containing metallofullerenes as diagnostic magnetic resonance imaging (MRI) contrast agents have been reported, the metallofullerene cage surface, in most cases, consists of negatively charged carboxyl or hydroxyl groups that limit attractive forces with the cellular surface. It has been reported that nanoparticles with a positive charge will bind more efficiently to negatively charged phospholipid bilayer cellular surfaces, and will more readily undergo endocytosis. In this paper, we report the preparation of a new functionalized trimetallic nitride template endohedral metallofullerene (TNT EMF), Gd3N@C80(OH)x(NH2)y, with a cage surface bearing positively charged amino groups (-NH3(+)) and directly compare it with a similar carboxyl and hydroxyl functionalized derivative. This new nanoparticle was characterized by X-ray photoelectron spectroscopy (XPS), dynamic light scattering (DLS), and infrared spectroscopy. It exhibits excellent (1)H MR relaxivity. Previous studies have clearly demonstrated that the cytokine interleukin-13 (IL-13) effectively targets glioblastoma multiforme (GBM) cells, which are known to overexpress IL-13Rα2. We also report that this amino-coated Gd-nanoplatform, when subsequently conjugated with interleukin-13 peptide IL-13-Gd3N@C80(OH)x(NH2)y, exhibits enhanced targeting of U-251 GBM cell lines and can be effectively delivered intravenously in an orthotopic GBM mouse model.

HG-16 * THE ANTI-APOPTOTIC PROTEIN ARC IS OVER-EXPRESSED IN MALIGNANT GLIOMA AND CORRELATES WITH PATIENT OUTCOME

BACKGROUND: Glioblastoma multiforme (GBM) is among the most malignant of cancers with poor long term survival. ARC is an anti-apoptotic protein that we have previously examined published microarray data sets and found that ARC is overexpressed in malignant gliomas with higher mRNA levels correlating with poor survival. We hypothesised that ARC expression on immunohistochemistry may be a novel prognostic marker, that ARC may engender tumour resistance to conventional and targeted therapies. METHODS: We analysed a tissue bank of 94 grade 2-4 gliomas and performed immunohistochemistry (IHC) to assess ARC protein levels in high grade gliomas and correlation with patient outcomes. To assess the biological role of ARC in GBM we used siRNA and shRNA to suppress ARC expression and used an ARC plasmid to induce over-expression in U87 and U118 GBM cells. Following lenti-viral knock down, ARC expression was measured by western blot and PCR on the U87 VIII cells with non-silencing, empty vector and 2 shRNA variants. The biological effects of ARC silencing or overexpression, either alone or in combination with radiotherapy was measured using cell proliferation, clonogenic, caspase 3/7 activity and scratch assays. RESULTS: The IHC analysis of 94 glioma specimens showed that ARC protein levels in malignant gliomas specimens increased significantly with higher tumour grade. Increased protein levels of ARC in all high grade gliomas and separately in GBM, corresponded with reduced patient survival. Functional studies showed no impact on cell proliferation, colony formation, caspase 3/7 activity or sensitivity to radiation therapy or other cytotoxic therapies. CONCLUSION: Our findings indicate that ARC expression by IHC correlates with tumour grade and is a potential prognostic indicator and marker of higher grade malignant tumours. Functional studies showed no impact of ARC expression on tumour progression, apoptosis or treatment sensitivity indicating that increased ARC expression is likely a secondary phenomenon.

Rhodiola crenulata inhibits Wnt/β-catenin signaling in glioblastoma

BackgroundRhodiola crenulata is a perennial plant that grows in the high altitudes of Eastern Europe and Asia. R crenulata has been used for many years in Eastern traditional medicine for a variety of medicinal purposes and it has been shown to elicit antineoplastic effects. The purpose of this study is to determine if R crenulata extract exhibits antitumor properties on glioblastoma multiforme (GBM), the most common and aggressive primary brain tumor. Materials and MethodsHuman U87 GBM cells were treated with 200 μg/mL of R crenulata or vehicle control. Cell proliferation was measured via MTS assay and clonogenic assay. The expressions of glial fibrillary acidic protein, a protein marker of differentiation, E-cadherin, and non–phospho active β-catenin were measured with immunocytochemistry. Neurosphere assay was performed in low attachment plates. Activity of the Wnt/β-catenin transcriptional activation was assessed via a dual-luciferase assay. ResultsMTS and clonogenicity assays revealed a decrease in proliferation with R crenulata therapy with an increased sensitivity to radiation. Immunocytochemistry revealed that R crenulata induced glial fibrillary acidic protein and E-cadherin expression suggestive of a more differentiated state. In agreement with the change in differentiation neurosphere formation was decreased upon treatment with R crenulata. β-Catenin dual reporter assay revealed a decrease in Wnt promoter activity after treatment with R crenulata; this was supported by a decrease in nuclear localization of β-catenin. ConclusionsTreatment with R crenulata extract effectively suppresses proliferation, stimulates differentiation, and eliminates tumorsphere formation of GBM cells in vitro. The observed effects are associated with inhibition of Wnt/β-catenin signaling pathway.

MicroRNA-210 regulates cell proliferation and apoptosis by targeting regulator of differentiation 1 in glioblastoma cells

MicroRNAs (miRNAs) are small noncoding RNAs that negatively regulate protein biosynthesis and participate in the pathogenesis of various tumours. Previous studies have shown that miR-210 is highly expressed in different types of human cancers, including glioblastoma multiforme (GBM). However, the role that miR-210 plays in GBM remains unclear. Here, we detected the expression and examined the function of miRNA-210 in GBM cells. Furthermore, we investigated the possible molecular mechanisms by which miRNA-210 mediates cell proliferation and apoptosis. Fifteen GBM and five normal brain tissues, in addition to the U87MG and U251 GBM cell lines, were analysed in this study. We found that miR-210 was upregulated in GBM tissues and cell lines when compared to normal brain tissue. Cell counting and flow cytometric assay results demonstrated that upregulation of miR-210 induced cell proliferation and decreased cell apoptosis, respectively. In addition, downregulation of miR-210 inhibited cell proliferation and induced apoptosis. We also detected a miR-210 target, regulator of differentiation 1 (ROD1), which is involved in GBM progression. Knockdown of ROD1 reversed the growth arrest and apoptosis that were originally induced by miR-210 inhibition. We propose that miR-210 regulates cell proliferation and apoptosis in GBM cells by targeting ROD1. Our findings may provide a new potential therapeutic target for the treatment of GBM.

Abstract 4914: AKT- and ERK signaling inhibition does not sensitize GBM cells to chemo-/radiation in vitro

Abstract A common feature of Glioblastoma Multiforme (GBM) is an augmented RTK/RAS/PI3K pathway signaling. This pathway has been shown to be involved in DNA repair by both NHEJ and HR in various tumor types and is thought thereby to interfere with its chemo- and radioresistance. Targeting this pathway to sensitize GBM to chemo- and radiotherapy seems to be an attractive therapeutic approach. Proliferation and clonogenic survival of U251, U87 and T98 GBM cells were assessed following exposure to, BKM120 (pan class I PI3K inhibitor), RAD001 (mTORC1 inhibitor), MK-2206 (AKT inhibitor) and/or MEK162 (ERK inhibitor) alone and in combination with either irradiation or Temozolomide (TMZ). Effectivity of each compound on its target pathway was assessed by westernblot. BEZ235 (PI3K/mTOR/DNA-PK inhibitor) was used as a positive control for radiosensitization. Our data show BKM120 and MK-2206 to effectively inhibit AKT phosphorylation at 1 uM and leads to growth inhibition in all tested cell lines. However, these compounds are not toxic on clonogenic cell survival and do not sensitize the cells to TMZ or irradiation. Increasing the dose to 5 uM and 10 uM for BKM120 and MK-2206 respectively an antagonistic effect was found in the combination with TMZ and irradiation. Effectively inhibiting mTOR, the downstream effector of AKT, by 100 nM RAD001 also does not radiosensitize GBM cells. Inhibiting the parallel ERK-pathway by 1 uM MEK162, dephosphorylates ERK1/2 and leads to a slight increase in AKT phosphorylation. Inhibiting both pathways with BKM120 and MEK162 resulted in a synergistic growth inhibition of U251 cells however no radiosensitizing effect was found in a clonogenic assay. In conclusion, selective targeting of key components in the AKT- and/or ERK signaling pathway effectively inhibits proliferation of GBM cells yet does not sensitize them to either TMZ or radiotherapy, indicating that GBM does not depend on the RTK/RAS/PI3K pathway for its DNA-repair. Citation Format: Ravi Narayan, Rogier Dik, Jaap van den Berg, Lukas J.A. Stalpers, Brigitta G. Baumert, Ben J. Slotman, Peter Sminia. AKT- and ERK signaling inhibition does not sensitize GBM cells to chemo-/radiation in vitro. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4914. doi:10.1158/1538-7445.AM2014-4914

CD133 antibody-conjugated immunoliposomes encapsulating gemcitabine for targeting glioblastoma stem cells.

CD133+ cells in glioblastoma multiforme (GBM) display glioma stem cell (GSC) properties and have been considered to be the culprit in tumor recurrence, justifying the exploration of potential therapeutic modalities targeting CD133+ cells. Such strategies require a drug-delivery vehicle, and increasingly, the unique properties of PEGylated liposomes are being exploited for this purpose. More advanced liposomal delivery studies have suggested conjugation of CD133 antibodies as a suitable method for targeting GSCs. For targeting studies, GSCs separated from U87 GBM cells using a magnetic bead separation method were challenged with liposomes encapsulating gemcitabine (GEM) conjugated with a CD133 monoclonal antibody (PEG-lipo-CD133-GEM). An in vitro study showed that conjugation of CD133 antibody significantly enhanced the cytotoxicity of GEM through endocytosis of CD133 surface markers overexpressed on GSCs. The anti-tumor effect of PEG-lipo-CD133-GEM was 15 times higher than that of free GEM, presumably reflecting the specific targeting of the CD133 surface marker by PEG-lipo-CD133-GEM and the enhanced stability and cytotoxicity through the PEGylated liposome formulation in xenograft models. Moreover, monitoring of body weight changes showed that the use of PEGylated liposomes significantly reduced the toxicity of GEM. Taken together, our studies demonstrate that PEG-lipo-CD133-GEM shows promise for the treatment of gliomas in vitro and in xenograft models.

Bortezomib overcomes MGMT-related resistance of glioblastoma cell lines to temozolomide in a schedule-dependent manner

Development of drug resistance after standard chemotherapy for glioblastoma multiforme (GBM) with temozolomide (TMZ) is associated with poor prognosis of GBM patients and is at least partially mediated by a direct DNA repair pathway involving O6-methylguanine methyltransferase (MGMT). This enzyme is under post-translational control by a multisubunit proteolytic cellular machinery, the 26S proteasome. Inhibition of the proteasome by bortezomib (BZ), a boronic acid dipeptide already in clinical use for the treatment of myeloma, has been demonstrated to induce growth arrest and apoptosis in GBM cells. In this study we investigated the effect of sequential treatment with BZ and TMZ on cell proliferation-viability and apoptosis of the human T98G and U87 GBM cell lines. We also tested for an effect of treatment on MGMT expression and important upstream regulators of the latter, including nuclear factor kappa B (NFκB), p44/42 mitogen-activated protein kinase (MAPK), p53, signal transducer and activator of transcription 3 (STAT3) and hypoxia-inducible factor 1α (HIF-1α). The sequence of drug administration for maximal cytotoxicity favored BZ prior to TMZ in T98G cells while the opposite was the case for U87 cells. Maximal efficacy was associated with downregulation of MGMT, reduced IκBα-mediated proteasome-dependent nuclear accumulation of NFκB, attenuation of p44/42 MAPK, AKT and STAT3 activation, and stabilization of p53 and inactive HIF-1α. Collectively, these results suggest that proteasome inhibition by BZ overcomes MGMT-mediated GBM chemoresistance, with scheduling of administration being critical for obtaining the maximal tumoricidal effect of combination with TMZ.

CMV-Independent Lysis of Glioblastoma by Ex Vivo Expanded/Activated Vδ1+ γδ T Cells

Vδ2neg γδ T cells, of which Vδ1+ γδ T cells are by far the largest subset, are important effectors against CMV infection. Malignant gliomas often contain CMV genetic material and proteins, and evidence exists that CMV infection may be associated with initiation and/or progression of glioblastoma multiforme (GBM). We sought to determine if Vδ1+ γδ T cells were cytotoxic to GBM and the extent to which their cytotoxicity was CMV dependent. We examined the cytotoxic effect of ex vivo expanded/activated Vδ1+ γδ T cells from healthy CMV seropositive and CMV seronegative donors on unmanipulated and CMV-infected established GBM cell lines and cell lines developed from short- term culture of primary tumors. Expanded/activated Vδ1+ T cells killed CMV-negative U251, U87, and U373 GBM cell lines and two primary tumor explants regardless of the serologic status of the donor. Experimental CMV infection did not increase Vδ1+ T cell - mediated cytotoxicity and in some cases the cell lines were more resistant to lysis when infected with CMV. Flow cytometry analysis of CMV-infected cell lines revealed down-regulation of the NKG2D ligands ULBP-2, and ULBP-3 as well as MICA/B in CMV-infected cells. These studies show that ex vivo expanded/activated Vδ1+ γδ T cells readily recognize and kill established GBM cell lines and primary tumor-derived GBM cells regardless of whether CMV infection is present, however, CMV may enhance the resistance GBM cell lines to innate recognition possibly contributing to the poor immunogenicity of GBM.

NFAT1 Is Highly Expressed in, and Regulates the Invasion of, Glioblastoma Multiforme Cells

Members of the nuclear factor of activated T cells (NFAT) family have been identified as regulators of oncogenic transformation in several human malignancies. A prominent member of this family, NFAT1, is associated with tumor cell survival, apoptosis, migration and invasion. Here, we investigated the role of NFAT1 in glioma cells. In 111 clinical samples, microarray analysis demonstrated that NFAT1 was over-expressed in glioblastoma multiforme (GBM), compared with low-grade gliomas, a result confirmed by RT-PCR in 24 clinical samples and in the U87 and U251 cell lines. Immunohistochemistry and immunofluorescence stain indicated that over-expressed NFAT1 was mainly located in the nucleus, where it acted as a transcription factor. After treatment with the NFAT antagonist cyclosporin A (CsA) and FK506, levels of NFAT1 in the nuclei of U87 GBM cells were dramatically reduced. The invasive potential of U87 cells was reduced by the same treatment, as well as by inhibition of NFAT1 expression using small hairpin RNA. Proliferation of U87 cells was unaffected by CsA, FK506 and NFAT1 shRNA transfection. Clustering analysis and Pearson correlation analysis of microarray data showed that the expression of NFAT1 correlated with the expression of the invasion-related genes cyclooxygenase-2 (COX-2), matrix metalloproteinase-7 (MMP-7) and MMP-9, a result confirmed by in vitro analysis. These findings demonstrate that NFAT1 contributes to the invasive potential but not the proliferation of GBM cells, and suggest that CsA may find application as an adjuvant in combined treatment strategies for GBM.

MiR-34a functions as a tumor suppressor modulating EGFR in glioblastoma multiforme

Chromosome 1p36.23 is frequently deleted in glioblastoma multiforme (GBM). miR-34a localizes in this region. Our experiments found that miR-34a was often deleted and epidermal growth factor receptor (EGFR) was frequently amplified in genomic DNA of 55 GBMs using single-nucleotide polymorphism DNA microarray. Notably, we found that the mean survival time was significantly shortened for patients whose GBMs had both EGFR amplification and miR-34a deletion. Expression of miR-34a was significantly lower in GBM samples compared with normal brain tissue. Forced expression of miR-34a in GBM cells decreased their ability to migrate and profoundly decreased their levels of cyclin-A1, -B1, -D1, and -D3, as well as cyclin-dependent kinase and increased expression of cyclin kinase inhibitor proteins (p21, p27). Also, human GBM cells (U251) stable overexpressing mir-34a formed smaller tumors when growing as xenografts in immunodeficient mice compared with wild-type U251 GBM cells. Furthermore, the protein expression of EGFR decreased in the cells with forced overexpression of miR-34a. Additional studies showed that mir-34a targeted Yin Yang-1 (YY1) and YY1 is a transcription factor that can stimulate the expression of EGFR. Thus, our data suggest that miR-34a acts as a tumor suppressor by inhibiting growth of GBM cells in vitro and in vivo associated with moderating the expression of cell-cycle proteins and EGFR. Moreover, we discovered for the first time that both deletion of miR-34a and amplification of EGFR were associated with significantly decreased overall survival of GBM patients.

CXCL12 mediates trophic interactions between endothelial and tumor cells in glioblastoma.

Emerging evidence suggests endothelial cells (EC) play a critical role in promoting Glioblastoma multiforme (GBM) cell proliferation and resistance to therapy. The molecular basis for GBM-EC interactions is incompletely understood. We hypothesized that the chemokine CXCL12 and its receptor CXCR4 could mediate direct interactions between GBM cells and tumor-associated endothelial cells and that disruption of this interaction might be the molecular basis for the anti-tumor effects of CXCR4 antagonists. We investigated this possibility in vivo and in an in vitro co-culture model that incorporated extracellular matrix, primary human brain microvascular ECs (HBMECs) and either an established GBM cell line or primary GBM specimens. Depletion of CXCR4 in U87 GBM cells blocked their growth as intracranial xenografts indicating that tumor cell CXCR4 is required for tumor growth in vivo. In vitro, co-culture of either U87 cells or primary GBM cells with HBMECs resulted in their co-localization and enhanced GBM cell growth. Genetic manipulation of CXCL12 expression and pharmacological inhibition of its receptors CXCR4 and CXCR7 revealed that the localizing and trophic effects of endothelial cells on GBM cells were dependent upon CXCL12 and CXCR4. These findings indicate that the CXCL12/CXCR4 pathway directly mediates endothelial cell trophic function in GBMs and that inhibition of CXCL12-CXCR4 signaling may uniquely target this activity. Therapeutic disruption of endothelial cell trophic functions could complement the structural disruption of anti-angiogenic regimens and, in combination, might also improve the efficacy of radiation and chemotherapy in treating GBMs.