Human Glioblastoma Cell Line U87MG Research Articles

Ginsenoside Rg3 induces apoptosis in the U87MG human glioblastoma cell line through the MEK signaling pathway and reactive oxygen species

Ginsenoside is known to have potential cancer-preventive activities. The major active components in red ginseng consist of a variety of ginsenosides including Rg3, Rg5 and Rk1, each of which has different pharmacological activities. Among these, Rg3 has been reported to exert anticancer activities through inhibition of angiogenesis and cell proliferation. However, the effects of Rg3 and its molecular mechanism on glioblastoma multiforme (GBM) remain unclear. Therefore, it is essential to develop a greater understanding of this novel compound. In the present study, we investigated the effects of Rg3 on a human glioblastoma cell line and its molecular signaling mechanism. The mechanisms of apoptosis by ginsenoside Rg3 were related with the MEK signaling pathway and reactive oxygen species. Our data suggest that ginsenoside Rg3 is a novel agent for the chemotherapy of GBM.

Abstract 4400: The levels of Prion protein and its ligand HOP modulate glioblastoma proliferation and predict a lower survival outcome.

Abstract A main concern in glioblastoma (GBM) therapy is the resistance of these tumors to current treatment protocols. The identification of molecules that can be target to treat these tumors is of major interest. The prion protein (PrPC) is a glycosylphosphatidylinositol-anchored protein very abundant in neurons and astrocytes and its interaction with a diverse number of ligands modulates cell survival and differentiation. One of these ligands is the secreted co-chaperone Hsp70-Hsp90 organizing protein (HOP), which reaches the extracellular space in exosome-like microvesicles. HOP is secreted by a number of cancer cells and seems to be a relevant biomarker in ovarian tumors. However, the role of PrPC-HOP complex in tumor biology is largely unknown. Herein, 185 cases o gliomas (33 grade I, 46 grade II, 15 grade II and 91 GBM) arranged in tissue microarrays were evaluated for the expression of PrPC and its ligand HOP. Both proteins were highly expressed in GBM when compared to tumors of grade I, II or III and also when compared to non-tumor samples (p<0.05 for all comparisons). High expression levels of both proteins in GBMs were direct correlated with a higher tumor proliferation, measured by the number of cells positive for Ki-67. Patients with high levels of both proteins presented a lower survival than those with high levels of HOP but lower levels of PrPC (p=0.017). The treatment of a human GBM cell line U87MG, which presents high expression of PrPC, with soluble HOP increases proliferation by three times however any effect was observed when cells were treated with a soluble HOP lacking the PrPC binding site (HOPΔ230-245). Both HOP or HOPΔ230-245 have no effect in U87MG cells whose PrPC expression was knocked down by 98%. In accordance, the intra-tumor infusion of HOP, but not HOPΔ230-245, increased tumor growth in nude mice bearing U87MG orthotopic xenografts. Remarkably, U87MG cells in which PrPC expression was knocked down were unable to growth tumors in nude mice. Together these results demonstrated that PrPC and HOP are significantly expressed in GBMs and higher levels of these proteins are associated to the patient group presenting a lower survival. The manipulation of PrPC and HOP interaction in cell cultures or in vivo affects tumor growth indicating that the interference with their engagement may represent a therapeutic target in GBMs. Supported by The State of São Paulo Foundation. Citation Format: Marilene H. Lopes, Nicole Gilda Queiroz, Tiago Goss Santos, Bruna R. Rodrigues, Isabela W. Cunha, Bruno Costa Silva, Vilma R. Martins. The levels of Prion protein and its ligand HOP modulate glioblastoma proliferation and predict a lower survival outcome. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4400. doi:10.1158/1538-7445.AM2013-4400

Isolation and in vitro cytotoxic activity of 11-methylixoside isolated from bark of Randia dumetorum Lamk

Summary Randia dumetorum (family Rubiaceae) is highly reputed ayurvedic medicinal tree commonly known as the Mainphal. A large deciduous thorny shrub grows up to 5 m of height. It occurs almost throughout India up to 1200 m of altitude. It is found in Himalaya from Jammu East ward ascending to 400 m and from Kashmir to East ward up to 1200 m. 11-methylixoside (compound 1), an iridoid glucoside, was isolated from the bark of this plant. The structure was characterized by using spectroscopic methods including 1D-1HNMR,13C-NMR and 2D-NMR (HSQC,HMBC, DQF-COSY) experiments and confirmed by comparison of their NMR data with those from the literature. This compound has been reported for the first time in Randia dumetorum bark. The 11-methylixoside was subjected to cytotoxic activity against MDA-MB-231 (breast cancer cell line) and SK-MEL-2 (human skin melanoma cell line), BE(2)C (neuroblastoma cell line derived from human bone marrow) and U87MG (human neuronale glioblastoma (astrozytom) cell line showed appreciable cytotoxic effect with IC50 value 63.10 µg/ml concentration for SK-MEL-2 (human skin melanoma cell line).

Combination therapy of cilengitide with belotecan against experimental glioblastoma

The prognosis of patients diagnosed with glioblastoma remains dismal in spite of the current concomitant chemoradiotherapy with temozolomide. In particular, the resistance to temozolomide appears to be the greatest obstacle to the treatment of glioblastoma. In the present study, we evaluated in vitro and in vivo the antitumor effects of combination therapy of cilengitide with belotecan, a camptothecin derivate, to treat experimental glioblastoma. The therapeutic effects of the drugs on the U87MG and U251MG human glioblastoma cell lines were assessed using in vitro cell viability and apoptosis assays. The combination treatment group with cilengitide and belotecan enhanced the cytotoxic effects to the glioblastoma cell lines and increased the apoptosis of the tumor cells compared to monotherapy with either drug alone in vitro. Nude mice with established U87MG glioblastoma were assigned to the following four groups: control, cilengitide, belotecan and combination treatment. The volume of tumors and length of survival were also measured. Animals in the combination therapy group demonstrated a significant reduction of tumor volume and an increase in survival (p < 0.05). Immunohistochemistry revealed a decrease in angiogenesis by cilengitide and an increase in apoptosis by cilengitide and belotecan in vivo. The combination therapy of cilengitide with belotecan presented more cytotoxic effects compared to the monotherapy of either drug in vitro and in vivo. This combination protocol may serve as an alternative treatment option for glioblastoma.

A transfection method for short interfering RNA with the lipid-like self-assembling nanotube, A6K

The aim of the present study was to develop a novel transfection method for short interfering RNA (siRNA). A nanotube with surfactant activity, A6K, consisting of six alanine residues and a hydrophilic head, lysine, was compared to the conventional cationic transfectant reagents siFECTOR and Lipofectamine 2000. Cytotoxicity for the human glioblastoma cell lines U87MG, A172, and T98G was examined with the MTS assay. Transfection efficiency was analyzed with FITC-labeled siRNA targeting matrix metalloproteinase (MMP)-2 mRNA by fluorescent activity on microscopy. The ultrastructure of A6K was evaluated by electron microscopy. The level of cytotoxicity associated with A6K in the U87MG cells was significantly lower than with siFECTOR and Lipofectamine 2000. Transfection efficiency for siRNA was increased in a dose- and time-dependent fashion. The relative expression of MMP-2 mRNA to β-actin was reduced in a dose-dependent manner by real-time RT-PCR analysis. The ultrastructure of the A6K was transformed to micelle formation when mixed with the siRNA. The lipid-like self-assembling peptide, A6K, has genes in the micelle associated with the hydrophilic tail. This transfection method is a novel and stable technique with lower cytotoxicity than the current standard methods.

Acute and Fractionated Irradiation Differentially Modulate Glioma Stem Cell Division Kinetics

Glioblastoma multiforme (GBM) is one of the most aggressive human malignancies with a poor patient prognosis. Ionizing radiation either alone or adjuvant after surgery is part of standard treatment for GBM but remains primarily noncurative. The mechanisms underlying tumor radioresistance are manifold and, in part, accredited to a special subpopulation of tumorigenic cells. The so-called glioma stem cells (GSC) are bestowed with the exclusive ability to self-renew and repopulate the tumor and have been reported to be less sensitive to radiation-induced damage through preferential activation of DNA damage checkpoint responses and increased capacity for DNA damage repair. During each fraction of radiation, non-stem cancer cells (CC) die and GSCs become enriched and potentially increase in number, which may lead to accelerated repopulation. We propose a cellular Potts model that simulates the kinetics of GSCs and CCs in glioblastoma growth and radiation response. We parameterize and validate this model with experimental data of the U87-MG human glioblastoma cell line. Simulations are conducted to estimate GSC symmetric and asymmetric division rates and explore potential mechanisms for increased GSC fractions after irradiation. Simulations reveal that in addition to their higher radioresistance, a shift from asymmetric to symmetric division or a fast cycle of GSCs following fractionated radiation treatment is required to yield results that match experimental observations. We hypothesize a constitutive activation of stem cell division kinetics signaling pathways during fractionated treatment, which contributes to the frequently observed accelerated repopulation after therapeutic irradiation.

The Anti-Tumor Effect of A3 Adenosine Receptors Is Potentiated by Pulsed Electromagnetic Fields in Cultured Neural Cancer Cells

A3 adenosine receptors (ARs) play a pivotal role in the development of cancer and their activation is involved in the inhibition of tumor growth. The effects of pulsed electromagnetic fields (PEMFs) on cancer have been controversially discussed and the detailed mechanisms are not yet fully understood. In the past we have demonstrated that PEMFs increased A2A and A3AR density and functionality in human neutrophils, human and bovine synoviocytes, and bovine chondrocytes. In the same cells, PEMF exposure increased the anti-inflammatory effect mediated by A2A and/or A3ARs. The primary aim of the present study was to evaluate if PEMF exposure potentiated the anti-tumor effect of A3ARs in PC12 rat adrenal pheochromocytoma and U87MG human glioblastoma cell lines in comparison with rat cortical neurons. Saturation binding assays and mRNA analysis revealed that PEMF exposure up-regulated A2A and A3ARs that are well coupled to adenylate cyclase activity and cAMP production. The activation of A2A and A3ARs resulted in the decrease of nuclear factor-kappa B (NF-kB) levels in tumor cells, whilst only A3ARs are involved in the increase of p53 expression. A3AR stimulation mediated an inhibition of tumor cell proliferation evaluated by thymidine incorporation. An increase of cytotoxicity by lactate dehydrogenase (LDH) release and apoptosis by caspase-3 activation in PC12 and U87MG cells, but not in cortical neurons, was observed following A3AR activation. The effect of the A3AR agonist in tumor cells was enhanced in the presence of PEMFs and blocked by using a well-known selective antagonist. Together these results demonstrated that PEMF exposure significantly increases the anti-tumor effect modulated by A3ARs.

Identification of allele-specific alternative mRNA processing via transcriptome sequencing

Establishing the functional roles of genetic variants remains a significant challenge in the post-genomic era. Here, we present a method, allele-specific alternative mRNA processing (ASARP), to identify genetically influenced mRNA processing events using transcriptome sequencing (RNA-Seq) data. The method examines RNA-Seq data at both single-nucleotide and whole-gene/isoform levels to identify allele-specific expression (ASE) and existence of allele-specific regulation of mRNA processing. We applied the methods to data obtained from the human glioblastoma cell line U87MG and primary breast cancer tissues and found that 26–45% of all genes with sufficient read coverage demonstrated ASE, with significant overlap between the two cell types. Our methods predicted potential mechanisms underlying ASE due to regulations affecting either whole-gene-level expression or alternative mRNA processing, including alternative splicing, alternative polyadenylation and alternative transcriptional initiation. Allele-specific alternative splicing and alternative polyadenylation may explain ASE in hundreds of genes in each cell type. Reporter studies following these predictions identified the causal single nucleotide variants (SNVs) for several allele-specific alternative splicing events. Finally, many genes identified in our study were also reported as disease/phenotype-associated genes in genome-wide association studies. Future applications of our approach may provide ample insights for a better understanding of the genetic basis of gene regulation underlying phenotypic diversity and disease mechanisms.

Nontoxic concentrations of PEGylated graphene nanoribbons for selective cancer cell imaging and photothermal therapy

Reduced graphene oxide nanoribbons functionalized by amphiphilic polyethylene glycol (rGONR–PEG) were applied to attach arginine-glycine-aspartic acid (RGD)-based peptide and cyanine dye 3 (cy3) for targeting ανβ3 integrin receptors on human glioblastoma cell line U87MG and its selective fluorescence imaging, respectively. The rGONR–PEG suspension with a concentration of 100 μg mL−1 showed ∼14 and 2.4-fold higher near infrared (NIR) absorption at 808 nm than GONR (with dimensions of ∼80 nm × 1 μm) and rGO–PEG sheets (with lateral dimensions of ∼2 μm), respectively. The rGONR–PEG–cy3–RGD exhibited highly efficient NIR photothermal therapy performance (concentrations ≥1.0 μg mL−1 resulted in ≥97% cell destruction in vitro under 7.5 W cm−2 NIR irradiation for 8 min). However, the rGONR–PEG exhibited concentration-dependent cyto- and geno-toxicity, so that it initiated at 1.0 μg mL−1 and presented strong effects at concentrations ≥100 μg mL−1 (resulting in >72% cell destruction and >29% DNA fragmentation after 24 h in the dark). Therefore, the concentration of 1.0 μg mL−1 (with <11% cell destruction and 7% DNA fragmentation) is the most effective concentration which can present low cyto- and especially geno-toxic effects. This work can provide insights for simultaneously efficient and biocompatible applications of nano-sized graphene in future photothermal nanotherapy.

Tertiary amides with a five-membered heteroaromatic ring as new probes for the translocator protein

In this study novel ligands of the translocator protein (TSPO), characterized by a five-membered aromatic heterocycle (i.e. oxazole, isoxazole, oxadiazole), a phenyl ring, and an amide side chain of carboxy or acetic type, were designed using a previously reported pharmacophore/topological model. Most of compounds showed significant TSPO binding affinity (K i values in the nanomolar/submicromolar range), the highest being displayed by oxazolacetamides 6. A number of compounds were tested for their ability to inhibit the proliferation/viability of human glioblastoma cell line U87MG. The dose-time dependent cell response to treatment with 6d demonstrated the specificity of the observed effect. The ability of 6d to induce mitochondrial membrane dissipation (Δ Ψm) substantiates the intracellular pro-apoptotic mechanism activated by ligand binding to TSPO.

The treatment of Glioblastoma Xenografts by surfactant conjugated dendritic nanoconjugates

Polysorbate 80 (P80) anchored poly(propyleneimine) (PPI) dendritic nanoconjugate was developed and evaluated for targeting anti-cancer drug, docetaxel (DTX) to the brain tumor. In vitro cytotoxicity studies of free DTX, DTX–PPI and DTX–P80-PPI dendrimers were carried out using U87MG human glioblastoma cell line. The in vivo anti-cancer activity in brain tumor bearing rats revealed that DTX loaded P80 conjugated dendrimers reduced the tumor volume extremely significantly ( p < 0.0001; more than 50%). The median survival time for brain tumor bearing rats treated with DTX–P80-PPI dendrimers (42 days) was extended very significantly as compared to DTX–PPI (23 days; p < 0.001), receptor blocked group (15 days; p < 0.001) and free DTX (18 days; p < 0.001). Gamma scintigraphy and biodistribution studies further confirmed the targeting efficiency and higher biodistribution of ligand conjugated dendrimer into the brain. The results concluded that the developed nanoconjugate has potential to deliver significantly higher amount of drug to brain tumor for improved therapeutic outcome.

A Torilis japonica extract exerts anti-proliferative activities on the U87MG human glioblastoma cell line

Torilis japonica is a wild biennial herb and has been used as a traditional medicine for the treatment of inflammation, skin disease and impotence. Here, we studied the effects of a T. japonica extract on the proliferation of the U87MG human glioblastoma cell line. The extract inhibited cell proliferation in a dose- and time-dependent manner, as determined using the MTT assay. We next investigated the molecular mechanisms underlying its anti-proliferation properties by examining cell cycle progression and cell death. T.japonica extract induced S-phase cell cycle arrest and inhibited the expression of cell cycle-regulatory proteins, including cyclinA, cyclin-dependent protein kinase 2 and E2F1. The extract also induced apoptotic cell death as evaluated by nuclear morphology and flow cytometry using Annexin-V/PI dual staining. Furthermore, Western blot analysis showed that apoptotic cell death was mediated by both mitochondria-independent and caspase-dependent pathways. Together, our findings indicate that the T. japonica extract contains bioactive compounds with anti-cancer effects. These materials may be useful in the chemotherapy of human glioblastoma.

A correlation of endocrine and anticancer effects of some antagonists of GHRH

GHRH receptor antagonists inhibit growth and metastasis of a large number of experimental tumors expressing the pituitary GHRH receptor (pGHRH-R) and its major splice variant SV1. In this study, using Western blot, we demonstrated that DBTRG-05 and U-87MG human glioblastoma cell lines express pGHRH-R at levels 6–15 times higher than SV1. To reveal a correlation between the anticancer activity and the endocrine potency on inhibition of GH release, we compared the antitumor effect of GHRH antagonists JV-1-63 and MZJ-7-138 on growth of DBTRG-05 human glioblastomas grafted into athymic nude mice with their inhibitory potency on GH release. JV-1-63 strongly suppressed the stimulated GH secretion induced by clonidine in rats and inhibited the exogenous GHRH-induced GH surge by 88–99% in vivo and in vitro. MZJ-7-138 decreased the stimulated GH secretion by 58% in vitro and showed only a tendency to inhibit GH secretion in vivo. The strong inhibitor of GH release JV-1-63 reduced tumor growth of DBTRG-05 glioblastomas in nude mice by 46%, while the weak GH release suppressor MZJ-7-138 did not have an effect. Exposure of DBTRG-05 cells to the GHRH antagonists in vitro caused an upregulation of mRNA expression for pGHRH-R and a downregulation of SV1 expression, with JV-1-63 having significantly greater effects than MZJ-7-138. Our results demonstrate that a positive correlation exists between the endocrine potency and the antiproliferative efficacy of GHRH antagonists in tumors strongly expressing pGHRH-R.

The G-protein-coupled formylpeptide receptor FPR confers a more invasive phenotype on human glioblastoma cells

Background:The G-protein-coupled formylpeptide receptor (FPR) that mediates chemotaxis of phagocytic leucocytes induced by bacterial and host-derived chemotactic peptides is selectively expressed by highly malignant human gliomas and contributes to tumour growth and angiogenesis. As invasion of surrounding normal tissues is one of the important features of tumour malignancy, we investigated the function of FPR in the invasive behaviour of human glioblastoma cells.Methods:Cells (FPR+ and FPR−) were isolated by single-cell cloning from a human glioblastoma cell line U-87MG. The FPR expression was assayed by flow cytometry and reverse transcription PCR. The function of FPR was investigated by chemotaxis and calcium flux induced by FPR agonist fMLF. Tumour cell motility was assayed by a wound-healing model in vitro. The growth and invasive phenotype were observed with subcutaneous implantation of tumour cells in nude mice. Over-expression of FPR in FPR− cells was performed by transfection of a plasmid vector-containing human FPR gene.Results:One of the glioma clones F9 that expressed high level of FPR showed a more ‘motile’ phenotype in vitro as compared with a clone G3 without FPR expression. Although F9 and G3 clones both formed subcutaneous tumours in nude mice, only F9 tumours invaded surrounding mouse connective tissues. Over-expression of FPR in G3 clone (G3F) increased the cell motility in vitro and the capacity of the cells to form more rapidly growing and invasive tumours in nude mice. We further found that, in addition to supernatant of necrotic tumour cells, foetal calf serum and human serum used in culture media contained FPR agonist activity and increased the motility of FPR-expressing glioblastoma cells.Conclusion:The expression of FPR is responsible for increased motility of human glioblastoma cells and their formation of highly invasive tumours.

Potential involvement of F0F1-ATP(synth)ase and reactive oxygen species in apoptosis induction by the antineoplastic agent erucylphosphohomocholine in glioblastoma cell lines

Erucylphosphohomocholine (ErPC3, Erufosine™) was reported previously to induce apoptosis in otherwise highly apoptosis-resistant malignant glioma cell lines while sparing their non-tumorigenic counterparts. We also previously found that the mitochondrial 18 kDa Translocator Protein (TSPO) is required for apoptosis induction by ErPC3. These previous studies also suggested involvement of reactive oxygen species (ROS). In the present study we further investigated the potential involvement of ROS generation, the participation of the mitochondrial respiration chain, and the role of the mitochondrial FOF1-ATP(synth)ase in the pro-apoptotic effects of ErPC3 on U87MG and U118MG human glioblastoma cell lines. For this purpose, cells were treated with the ROS chelator butylated hydroxyanisole (BHA), the mitochondrial respiration chain inhibitors rotenone, antimycin A, myxothiazol, and the uncoupler CCCP. Also oligomycin and piceatannol were studied as inhibitors of the FO and F1 subunits of the mitochondrial FOF1-ATP(synth)ase, respectively. BHA was able to attenuate apoptosis induction by ErPC3, including mitochondrial ROS generation as determined with cardiolipin oxidation, as well as collapse of the mitochondrial membrane potential (Δψm). Similarly, we found that oligomycin attenuated apoptosis and collapse of the Δψm, normally induced by ErPC3, including the accompanying reductions in cellular ATP levels. Other inhibitors of the mitochondrial respiration chain, as well as piceatannol, did not show such effects. Consequently, our findings strongly point to a role for the FO subunit of the mitochondrial FOF1-ATP(synth)ase in ErPC3-induced apoptosis and dissipation of Δψm as well as ROS generation by ErPC3 and TSPO.

Glutamate Promotes Cell Growth by EGFR Signaling on U-87MG Human Glioblastoma Cell Line

Accumulating evidences suggest that glutamate plays a key role in the proliferation and invasion of malignant glioblastoma (GBM) tumors. It has been shown that GBM cells release and exploit glutamate for proliferation and invasion through AMPA glutamate receptors. Additionally, amplification of the epidermal growth factor receptor (EGFR) gene occurs in 40-50% of GBM. Since, PI3K/Akt is considered one of the main intracellular pathways involved in EGFR activation, AKT functions could trigger EGFR signaling. Thus, we investigated whether EGFR-phospho-Akt pathway is involved on the glutamate inducing U-87MG human GBM cell line proliferation. For these purpose, we treated the U-87MG cell line with 5 to 200 mM of glutamate and assessed the number of viable cells by trypan blue dye exclusion test. An increase in cell number (50%) was found at 5 mM glutamate, while the addition of DNQX (500 microM), an antagonist of AMPA receptor, inhibited the effect of glutamate on the U87-MG cells proliferation. Also, at 5 mM glutamate we observed an increase on the EGFR and phospho-Akt contents evaluated by immunohistochemistry. Moreover, U-87MG cells treated with glutamate exhibited an increase about 2 times in the EGFR mRNA expression. While, in the presence of the anti-EGFR gefitinib (50 muM) or the PI3K inhibitor wortmannin (5 muM), the U-87MG proliferation was restored to control levels. Together, our data suggest that glutamate signaling mediated by AMPA receptor induces U-87MG human GBM cell line proliferation via EGFR-phospho-Akt pathway.

Egr-1 is not upregulated in response to hypoxic and oxygenation conditions in human glioblastoma in vitro

The early growth response factor 1 (Egr-1) gene (also known as krox24, NGFI-A, TIS8 or zif268) belongs to a family of immediate early response genes. This family of proteins contains a conserved zinc finger DNA-binding domain and can bind to a GC-rich sequence in the promoter region of target genes. Egr-1 expression is rapidly and transiently activated in many different cell types during development. In adult tissues, a variety of signals, including serum, growth factors, cytokines and hormones, stimulate Egr-1 expression. In several studies, it was demonstrated that the transcription factor Egr-1 is regulated by hypoxia, and it is hypothesized that Egr-1 is responsible for the hypoxia-induced regulation of the N-Myc downregulated gene 1 (NDRG1) in human tumor cells. In the present study, Egr-1 regulation was examined in the human glioblastoma cell lines U373, U251, GaMG and U87-MG under extreme hypoxic aeration conditions (0.1% O2) for 1, 6 and 24 h, 24-h extreme hypoxia with reoxygenation for 24 and 48 h, respectively, as well as oxygenated conditions (21% O2 and 5% CO2) in vitro. Protein and mRNA levels were detected in the lysates by Western blotting and RT-PCR, respectively. Egr-1 expression under hypoxic conditions was compared with the well-known and characterized hypoxia-induced gene regulator hypoxia-inducible factor-1α (HIF-1α) in parallel experimental sets. Cells incubated for 24 h with 100 µM desferroxamine served as a positive control for hypoxia, and β-tubulin and β-actin were used as loading controls. The experimental data indicate that Egr-1 was not upregulated under extreme hypoxic conditions (0.1% O2) or by reoxygenation after hypoxia in different glioblastoma cells in vitro. In conclusion, the regulation of Egr-1 in reaction to hypoxic development, at both the protein and mRNA levels, is not a general phenomenon. In contrast to previously published data, no Egr-1 regulatory events were observed in glioblastoma under hypoxic conditions in vitro. We suggest that Egr-1 regulation in human tumors in reaction to hypoxia could be a cell-specific post-translational event. Therefore, at least in glioblastoma, HIF-1α can be considered a major regulator of NDRG1 under hypoxic conditions. Further extensive analysis of tumor cells from different origins under similar physiological conditions is necessary to increase our knowledge of the conditions and functional role of Egr-1 in the regulation of hypoxia-induced gene expression.

Ixolaris, a tissue factor inhibitor, blocks primary tumor growth and angiogenesis in a glioblastoma model.

The expression levels of the clotting initiator protein Tissue Factor (TF) correlate with vessel density and the histological malignancy grade of glioma patients. Increased procoagulant tonus in high grade tumors (glioblastomas) also indicates a potential role for TF in progression of this disease, and suggests that anticoagulants could be used as adjuvants for its treatment. We hypothesized that blocking of TF activity with the tick anticoagulant Ixolaris might interfere with glioblastoma progression. TF was identified in U87-MG cells by flow-cytometric and functional assays (extrinsic tenase). In addition, flow-cytometric analysis demonstrated the exposure of phosphatidylserine in the surface of U87-MG cells, which supported the assembly of intrinsic tenase (FIXa/FVIIIa/FX) and prothrombinase (FVa/FXa/prothrombin) complexes, accounting for the production of FXa and thrombin, respectively. Ixolaris effectively blocked the in vitro TF-dependent procoagulant activity of the U87-MG human glioblastoma cell line and attenuated multimolecular coagulation complexes assembly. Notably, Ixolaris inhibited the in vivo tumorigenic potential of U87-MG cells in nude mice, without observable bleeding. This inhibitory effect of Ixolaris on tumor growth was associated with downregulation of VEGF and reduced tumor vascularization. Our results suggest that Ixolaris might be a promising agent for anti-tumor therapy in humans.

Chondroitin sulphate‐modified neuropilin 1 is expressed in human tumour cells and modulates 3D invasion in the U87MG human glioblastoma cell line through a p130Cas‐mediated pathway

Neuropilin 1 (NRP1), a non-tyrosine kinase receptor for vascular endothelial growth factor and class 3 Semaphorins, is highly expressed in many human tumour cell lines, but its function is poorly understood. Here, we describe the expression of a new chondroitin sulphate-modified NRP1 (NRP1-CS) in human tumour cell lines. Expression of a non-modifiable NRP1 mutant (S612A) in U87MG human glioma cells results in enhanced invasion in three dimensions (3D), whereas wild-type NRP1 has no effect. Furthermore, the S612A NRP1 cells show a significant increase in p130Cas tyrosine phosphorylation compared with control and wild-type NRP1 cells. Silencing of p130Cas in S612A NRP1 cells resulted in a loss of increased invasive phenotype. Interestingly, p130Cas silencing does not inhibit basal 3D invasion, but leads to a mesenchymal to amoeboid transition. Biopsies from both low- and high-grade human gliomas show strong expression of NRP1, and little expression of NRP1-CS. Our data establish distinct roles for NRP1 and NRP1-CS in modulating a new NRP1-p130Cas signalling pathway contributing to glioblastoma cell invasion in 3D.

Δ9-Tetrahydrocannabinol inhibits cell cycle progression by downregulation of E2F1 in human glioblastoma multiforme cells

Background. The active components of Cannabis sativa L., Cannabinoids, traditionally used in the field of cancer for alleviation of pain, nausea, wasting and improvement of well-being have received renewed interest in recent years due to their diverse pharmacologic activities such as cell growth inhibition, anti-inflammatory activity and induction of tumor regression. Here we used several experimental approaches, which identified delta-9-tetrahydrocannabinol (Δ9-THC) as an essential mediator of cannabinoid antitumoral action. Methods and results. Administration of Δ9-THC to glioblastoma multiforme (GBM) cell lines results in a significant decrease in cell viability. Cell cycle analysis showed G0/1 arrest and did not reveal occurrence of apoptosis in the absence of any sub-G1 populations. Western blot analyses revealed a THC altered cellular content of proteins that regulate cell progression through the cell cycle. The cell content of E2F1 and Cyclin A, two proteins that promote cell cycle progression, were suppressed in both U251-MG and U87-MG human glioblastoma cell lines, whereas the level of p16INK4A, a cell cycle inhibitor was upregulated. Transcription of thymidylate synthase (TS) mRNA, which is promoted by E2F1, also declined as evident by QRT-PCR. The decrease in E2F1 levels resulted from proteasome mediated degradation and was prevented by proteasome inhibitors. Conclusions. Δ9-THC is shown to significantly affect viability of GBM cells via a mechanism that appears to elicit G1 arrest due to downregulation of E2F1 and Cyclin A. Hence, it is suggested that Δ9-THC and other cannabinoids be implemented in future clinical evaluation as a therapeutic modality for brain tumors.