Human Glioblastoma Cell Line U87MG Research Articles

Inhibitory mechanisms of timosaponin AⅢ on proliferation and growth of human glioblastoma cells

To explore the inhibitory effect of timosaponin AⅢ on the proliferation of human glioblastoma cell line U87MG and investigate its related mechanism. As compared with the model group, the tumor weight was significantly reduced in timosaponin AⅢ-treated group. Timosaponin AⅢinhibited the proliferation of U87MG cell line in a dose-dependent manner. It up-regulated the gene and protein expression levels of p21, meanwhile inhibited the protein expression levels of β-Catenin, Cyclin D1 and Bcl-2. It also inhibited the translocation of β-Catenin into nucleus, suppressed the phosphorylation expression of ERK, but increased the phosphorylation expression of p38 and JNK. Combined use of JNK inhibitor SP600125 and p38 inhibitor SB203580 could decrease p21 and increase β-Catenin protein expressions. Timosaponin AⅢ inhibited the proliferation of human glioblastoma cell line U87MG partly by intervening MAPK and Wnt/β-Catenin signal pathways.

Cordycepin inhibits migration of human glioblastoma cells by affecting lysosomal degradation and protein phosphatase activation

Cordycepin, a nucleoside-derivative-isolated form Cordyceps militaris, has been reported to suppress tumor cell proliferation and cause apoptosis. This study investigates the effect of cordycepin on the migration of human glioblastoma cells. Cordycepin suppressed the migration of the human glioblastoma cell lines U87MG and LN229 in transwell and wound healing assays. Cordycepin decreased protein expression of integrin α1, focal adhesion kinase (FAK), p-FAK, paxillin and p-paxillin. The lysosomal inhibitor NH4Cl blocked the ability of cordycepin to inhibit focal adhesion protein expression and glioma cell migration. In addition, the protein phosphatase inhibitors calyculin A and okadaic acid blocked the cordycepin-mediated reduction in p-Akt, p-FAK and migration. Hematoxylin and eosin staining of mouse xenografts demonstrated that cordycepin reduced brain tumor size in vivo. In conclusion, cordycepin inhibited migration of human glioblastoma cells by affecting lysosomal degradation and protein phosphatase activation. This pathway may be a useful target for clinical therapy in the future.

Extract of the sea cucumber, Holothuria scabra, induces apoptosis in human glioblastoma cell lines

Background: Glioblastoma is the most aggressive primary brain tumor resistant to conventional treatment, and has a frequent recurrence. Holothuria scabra is the most widely consumed sea cucumber in Thailand and Asian countries. Sea cucumber extracts have valuable bioactive ingredients that are utilized in Chinese traditional medicine for several therapeutic measures including anti-cancer. In this study, we aimed to test the anticancer activity of H. scabra body wall extract in ethyl acetate fraction against two human glioblastoma cell lines A172 and U87MG.Material and methods: The anticancer effect of the ethyl acetate fraction of body wall extracts from H. scabra (HSBWEA) was tested against two human glioblastoma cell lines A172 and U87MG to determine cell viability, apoptotic cell analysis, mitochondrial membrane potentials, nuclear condensation and expression of apoptotic molecules. The bioactive compound in the extract was characterized by LC-MS/MS.Results: The extract exhibited strong cytotoxic effect to A172 and U87MG cells with IC50 4.23 and 4.46 mg/ml, respectively. The extract also induced both early and late stages of apoptosis, loss of mitochondrial membrane potentials and nuclear condensation and fragmentation. After treatment with the extract for 24 h, the cells expressed increasing amounts of a pro-apoptotic Bax and caspase-3 while an anti-apoptotic Bcl-2 was decreased in a concentration-dependent manner. The major bioactive components in this extract are triterpene glycosides similar to those described previously in H. scabra as well as in other related sea cucumber species.Conclusions: The crude extract from the body wall of H. scabra exhibited anticancer activity against human glioblastoma cells through the mitochondrial-mediated apoptotic pathway, and the compounds in the extract might be a novel candidate for anti-glioblastoma.

Apigenin Inhibits Cancer Stem Cell-Like Phenotypes in Human Glioblastoma Cells via Suppression of c-Met Signaling.

Glioblastoma (GBM) is a highly malignant human brain tumor with limited treatment choices. The extremely aggressive characteristics of GBM result from GBM stem cells (GSCs), a subpopulation in tumor having self-renewal potential and resistance to chemotherapy and radiotherapy. Therefore, eliminating GSCs is an effective strategy to treat this fatal disease. In this study, we investigated the therapeutic effects of dietary flavonoids, including apigenin, quercetin, and naringenin, against cancer stem cell-like phenotypes of human GBM cell lines U87MG and U373MG. Among flavonoids studied, apigenin and quercetin significantly suppressed not only the self-renewal capacity such as cell growth and clonogenicity, but also the invasiveness of GBM stem-like cells. Notably, apigenin blocked the phosphorylation of c-Met and its downstream effectors, transducer and activator of transcription 3, AKT (Protein kinase B), and mitogen-activated protein kinase in the GSCs, thereby reducing the expression levels of GSC markers such as CD133, Nanog, and Sox2. These results suggest that the GSC inhibition effect of apigenin may be caused by downregulation of c-Met signaling pathway.

Haloperidol, but not olanzapine, may affect expression of PER1 and CRY1 genes in human glioblastoma cell line

Background: There is barely any evidence of antipsychotic drugs affecting the molecular clockwork in human, yet it is suggested that clock genes are associated with dopaminergic transmission, i.e. the main target of this therapeutics. We decided to verify if haloperidol and olanzapine affect expression of CLOCK, BMAL1, PER1 and CRY1 in a human central nervous system cell line model. Methods: U-87MG human glioblastoma cell line was used as an experimental model. The cells were incubated with or without haloperidol and olanzapine in the concentration of 5 and 20 μM for 24 h. Real-time quantitative polymerase chain reaction with the ΔCT analysis was used to examine the effect of haloperidol and olanzapine on the mRNA expression of the genes. Results: At 5 μM, haloperidol decreased expression of CRY1 almost 20-fold. There was nearly a 1.5-fold increase in expression of PER1. Considering the 20 μM haloperidol concentration and both olanzapine concentrations, no other statistically significant effect was observed. Conclusions: At certain concentration, haloperidol seems to affect expression of particular clock genes in a human central nervous system cell line model, yet mechanism underlying this phenomenon remains elusive.

Targeting CXCR4 by a selective peptide antagonist modulates tumor microenvironment and microglia reactivity in a human glioblastoma model.

BackgroundThe CXCL12/CXCR4 pathway regulates tumor cell proliferation, metastasis, angiogenesis and the tumor-microenvironment cross-talk in several solid tumors, including glioblastoma (GBM), the most common and fatal brain cancer. In the present study, we evaluated the effects of peptide R, a new specific CXCR4 antagonist that we recently developed by a ligand-based approach, in an in vitro and in vivo model of GBM. The well-characterized CXCR4 antagonist Plerixafor was also included in the study.MethodsThe effects of peptide R on CXCR4 expression, cell survival and migration were assessed on the human glioblastoma cell line U87MG exposed to CXCL12, by immunofluorescence and western blotting, MTT assay, flow cytometry and transwell chamber migration assay. Peptide R was then tested in vivo, by using U87MG intracranial xenografts in CD1 nude mice. Peptide R was administered for 23 days since cell implantation and tumor volume was assessed by magnetic resonance imaging (MRI) at 4.7 T. Glioma associated microglia/macrophage (GAMs) polarization (anti-tumor M1 versus pro-tumor M2 phenotypes) and expressions of vascular endothelial growth factor (VEGF) and CD31 were assessed by immunohistochemistry and immunofluorescence.ResultsWe found that peptide R impairs the metabolic activity and cell proliferation of human U87MG cells and stably reduces CXCR4 expression and cell migration in response to CXCL12 in vitro. In the orthotopic U87MG model, peptide R reduced tumor cellularity, promoted M1 features of GAMs and astrogliosis, and hindered intra-tumor vasculature.ConclusionsOur findings suggest that targeting CXCR4 by peptide R might represent a novel therapeutic approach against GBM, and contribute to the rationale to further explore in more complex pre-clinical settings the therapeutic potential of peptide R, alone or in combination with standard therapies of GBM.Electronic supplementary materialThe online version of this article (doi:10.1186/s13046-016-0326-y) contains supplementary material, which is available to authorized users.

10-Boronic acid substituted camptothecin as prodrug of SN-38

Malignant tumor cells have been found to have high levels of reactive oxygen species such as hydrogen peroxide (H2O2), supporting the hypothesis that a prodrug could be activated by intracellular H2O2 and lead to a potential antitumor therapy. In this study, the 7-ethyl-10-boronic acid camptothecin (B1) was synthesized for the first time as prodrug of SN-38, by linking a cleavable aryl carbon–boron bond to the SN-38. Prodrug B1 selectively activated by H2O2, converted rapidly to the active form SN-38 under favorable oxidative conditions in cancer cells with elevated levels of H2O2. The cell survival assay showed that prodrug B1 was equally or more effective in inhibiting the growth of six different cancer cells, as compared to SN-38. Unexpectedly, prodrug B1 displayed even more potent Topo I inhibitory activity than SN-38, suggesting that it was not only a prodrug of SN-38 but also a typical Topo I inhibitor. Prodrug B1 also demonstrated a significant antitumor activity at 2.0 mg/kg in a xenograft model using human brain star glioblastoma cell lines U87MG.

Antipsychotic Drugs Differentially Affect mRNA Expression of Genes Encoding the Neuregulin 1-Downstream ErbB4-PI3K Pathway

Background/Aims: The PIK3CD gene encodes the delta catalytic subunit of phosphoinositide 3-kinase (PI3K), an element of the neuregulin 1-downstream ErbB4-PI3K signaling pathway, which was recently identified as a molecular target for the treatment of schizophrenia. The aim of the study was to examine the effect of haloperidol (HALO), clozapine (CLO), olanzapine (OLA), quetiapine (QUE) and amisulpride (AMI) on the mRNA and protein expression of genes encoding the elements of ErbB4-PI3K pathway, in a human central nervous system cell line. Methods: The U-87MG human glioblastoma cell line was used as an experimental model. Quantitative polymerase chain reaction was used to examine the expression of mRNA and enzyme-linked immunosorbent assay for protein expression. Results: At concentrations reached in clinical settings in the brain, CLO, as well as OLA and QUE to a lesser extent, but not AMI and probably not HALO, decreased the mRNA expression of PIK3CD. Protein expression of the gene did not confirm the mRNA expression profile. Conclusions: The tested antipsychotic drugs (APDs) in the U-87MG glioblastoma cell line differentially regulates the mRNA expression of PIK3CD; however, the protein expression does not confirm these findings. The results of the study may help deepen the understanding of the mechanism of action of APDs.

New insights into the anticancer activity of carnosol: p53 reactivation in the U87MG human glioblastoma cell line

Glioblastoma multiforme (GBM) is an aggressive brain tumour with high resistance to radio- and chemotherapy. As such, increasing attention has focused on developing new therapeutic strategies to improve treatment responses. Recently, attention has been shifted to natural compounds that are able to halt tumour development. Among them, carnosol (CAR), a phenolic diterpene present in rosemary, has become a promising molecule that is able to prevent certain types of solid cancer. However, no data are available on the effects of CAR in GBM. Here, CAR activity decreased the proliferation of different human glioblastoma cell lines, particularly cells that express wild type p53. The p53 pathway is involved in the control of apoptosis and is often impaired in GBM. Notably, CAR, through the dissociation of p53 from its endogenous inhibitor MDM2, was able to increase the intracellular p53 levels in GBM cells. Accordingly, functional reactivation of p53 was demonstrated by the stimulation of p53 target genes’ transcription, the induction of apoptosis and cell cycle blockade. Most importantly, CAR produced synergistic effects with temozolomide (TMZ) and reduced the restoration of the tumour cells’ proliferation after drug removal. Thus, for the first time, these data highlighted the potential use of the diterpene in the sensitization of GBM cells to chemotherapy through a direct re-activation of p53 pathway. Furthermore, progress has been made in delineating the biochemical mechanisms underlying the pro-apoptotic effects of this molecule.

Evaluation of cytotoxic and tumor targeting capability of (177)Lu-DOTATATE-nanoparticles: a trailblazing strategy in peptide receptor radionuclide therapy.

We propose an innovative strategy of nanoparticle-mediated-peptide receptor radionuclide therapy (PRRT) employing PLGA-nanoparticles together with anti-β-hCG antibodies that can protect kidneys from radiation damage while simultaneously enhancing its tumor targeting and cytotoxic ability for somatostatin receptor (SSR) positive tumors. PEG-coated-(177)Lu-DOTATATE-PLGA-nanoparticles (PEG-LuD-NP) were formulated and characterized. In vitro toxicity of these particles was tested on human glioblastoma cell line U87MG over a radiation dose range of 19-78Gy, using MTT assay and flow cytometry. To further enhance cytotoxicity and test the feasibility of active tumor targeting, apoptosis-inducing anti-β-hCG monoclonal antibodies were employed in vitro, after confirming expression of β-hCG on U87MG. In vivo tumor targeting ability of these particles, in comparison to uncoated particles and un-encapsulated (177)Lu-DOTATATE, was assessed by intravenous administration in tumor-induced wistar rats. Rats were first imaged in a gamma camera followed by euthanasia for organ extraction and counting in gamma counter. The particles were spherical in shape with mean diameter of 300nm. Highest cytotoxicity that could be achieved with PEG-LuD-NP, on radio-resistant U87MG cells, was 35.8% due to complex cellular response triggered by ionizing radiation. Interestingly, synergistic action of antibodies and PEG-LuD-NP doubled the cytotoxicity (80%). PEG-LuD-NP showed the highest tumor uptake (4.3±0.46% ID/g) as compared to (177)Lu-DOTATATE (3.5±0.31%) and uncoated-(177)Lu-DOTATATE-nanoparticles (3.4±0.35%) in tumor-inoculated wistar rats (p<0.001). Renal uptake/retention was decreased 3-4 folds with these particles, resulting in the highest tumor-to-kidney ratio (8.58; p<0.01) while tumor-to-liver and tumor-to-bone ratios were comparable to un-encapsulated-drug. Nanocarrier-mediated-PRRT is an effective way of targeting SSR positive tumors for enhanced cytoxicity and reduced renal radiation dose associated with conventional PRRT. To our knowledge of literature, this is the first study to establish in vitro and in vivo efficacy profile of nanoparticles in PRRT providing a stepping-stone for undergoing and future research endeavors in the direction of abating associated radiation concerns of radionuclide therapy and may offer a paradigm shift in PRRT strategy.

Assessment of cytotoxic damage induced by irradiation combined with hyperthermia and Gemcitabine on cultured glioblastoma spheroid cells

Abstract Background Glioblastoma is a highly lethal tumor. There are several methods for the treatment of glioblastoma including surgery/chemotherapy/radiotherapy/radiosurgery or corticosteroids. However none of them could have improved treatment considerably. Therefore this study assessed the cytotoxicity of the combined treatment with Gemcitabine, radiation and hyperthermia on cultured glioblastoma spheroid cells. Material and methods In this study human glioblastoma cell line U87-MG, was cultured as spheroid using the liquid overlay technique. After reaching spheroid dimensions to 100 µm, cells treated by 100 nM Gemcitabine, 2 Gy gamma rays and then hyperthermia of 43 °C for an hour. Colony assay was used to determine cell induced damage and synergistic effect of Gemcitabine, irradiation and hyperthermia. Results The combined therapy indicated greater percentage of cellular damage, although more damages were seen when Gemcitabine, radiation and hyperthermia induced into U87-MG cell line simultaneously. Combination of those three factors in comparison to the radiation combined with hyperthermia and Gemcitabine separately showed 1.85 and 1.5 times more damages respectively. Conclusion This study revealed the synergistic effect for the combined therapy of Gemcitabine, radiation and hyperthermia in glioblastoma treatment.

Predicting risk of human drug-induced liver injuries from non-clinical studies in R&D

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 (Ki 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 (ΔΨm) substantiates the intracellular pro-apoptotic mechanism activated by ligand binding to TSPO.

Anticancer potential and mechanism of action of mango ginger (Curcuma amada Roxb.) supercritical CO₂ extract in human glioblastoma cells.

Mango ginger (Curcuma amada Roxb.) is among the less-investigated species of Curcuma for anticancer properties. We have investigated the anticancer potential and the mechanism of action of a supercritical CO2 extract of mango ginger (CA) in the U-87MG human glioblastoma cell line. CA demonstrated higher cytotoxicity than temozolomide, etoposide, curcumin, and turmeric force with IC50, IC75, and IC90 values of 4.92 μg/mL, 12.87 μg/mL, and 21.30 μg/mL, respectively. Inhibitory concentration values of CA for normal embryonic mouse hypothalamus cell line (mHypoE-N1) is significantly higher than glioblastoma cell line, indicating the specificity of CA against brain tumor cells. CompuSyn analysis indicates that CA acts synergistically with temozolomide and etoposide for the cytotoxicity with combination index values of <1. CA treatment also induces apoptosis in glioblastoma cells in a dose-dependent manner and downregulates genes associated with apoptosis, cell proliferation, telomerase activity, oncogenesis, and drug resistance in glioblastoma cells.

FTY720 reduces migration and invasion of human glioblastoma cell lines via inhibiting the PI3K/AKT/mTOR/p70S6K signaling pathway.

2-Amino-2-[2-(4-octylphenyl)]-1,3-propanediol hydrochloride (FTY720), a synthetic compound from Isaria sinclairii, has been proven to possess various biological benefits including anti-cancer activity. However, the effects and related mechanisms of FTY720 on the migration and invasion of glioblastoma cells are still unclear. In the present study, we utilized U251MG and U87MG human glioblastoma cell lines to assess the effects of FTY720. We found that FTY720 significantly inhibited migration and invasion of glioblastoma cells. The anti-migration and invasion effects of FTY720 were associated with its down-regulation of matrix metalloproteinase-2 (MMP-2) and MMP-9 while up-regulation of tissue inhibitor of metalloproteinase-1 (TIMP-1) and TIMP-2. Furthermore, FTY720 modulated the expression of roundabouts 1 (ROBO1), Rho-associated kinase-1 (ROCK1), and epithelial-to-mesenchymal transition (EMT)-related factors. In addition, the phosphatidylinositide 3-kinases/protein kinase B/mammalian target of rapamycin/p70S6 kinase (PI3K/AKT/mTOR/p70S6K) signaling pathway participated in FTY720-mediated suppression of migration and invasion. Thus, our findings demonstrated that FTY720 reduced glioblastoma cells migration and invasion via multiple signaling pathways, suggesting that FTY720 is a potential therapeutic agent against glioblastoma.

Synthesis and Biological Activity of Triazole‐Appended N,O‐Nucleosides

Abstract1,2,3‐Triazole‐appended N,O‐nucleosides have been synthesized by an approach combining a 1,3‐dipolar cycloaddition process and an alkyne–azide click chemistry reaction. Biological assays, performed on six tumor cell lines, revealed the antiproliferative activity of the synthesized compounds. The effect was more evident in the U87MG human glioblastoma cell line. Thus, these nucleoside analogues could be promising scaffolds for the construction of new anticancer agents.

Quantification of Protoporphyrin IX Accumulation in Glioblastoma Cells: A New Technique.

Introduction. 5-Aminolevulinic Acid (5-ALA) is a precursor of heme synthesis. A metabolite, protoporphyrin IX (PpIX), selectively accumulates in neoplastic tissue including glioblastoma. Presurgical administration of 5-ALA forms the basis of fluorescence-guided resection (FGR) of glioblastoma (GBM) tumors. However, not all gliomas accumulate sufficient quantities of PpIX to fluoresce, thus limiting the utility of FGR. We therefore developed an assay to determine cellular and pharmacological factors that impact PpIX fluorescence in GBM. This assay takes advantage of a GBM cell line engineered to express yellow fluorescent protein. Methods. The human GBM cell line U87MG was transfected with a YFP expression vector. After treatment with a series of 5-ALA doses, both PpIX and YFP fluorescence were measured. The ratio of PpIX to YFP fluorescence was calculated. Results. YFP fluorescence permitted the quantification of cell numbers and did not interfere with 5-ALA metabolism. The PpIX/YFP fluorescence ratio provided accurate relative PpIX levels, allowing for the assessment of PpIX accumulation in tissue. Conclusion. Constitutive YFP expression strongly correlates with cell number and permits PpIX quantification. Absolute PpIX fluorescence alone does not provide information regarding PpIX accumulation within the cells. Our research indicates that our PpIX/YFP ratio assay may be a promising model for in vitro 5-ALA testing and its interactions with other compounds during FGR surgery.

Heat shock induces expression of OSTC/DC2, a novel subunit of oligosaccharyltransferase, in vitro and in vivo

Mammalian oligosaccharyltransferase complex subunit OSTC/DC2 protein has recently been shown to be a new subunit of the oligosaccharyltransferase; however, its physiological role is still unclear. Here, we report the expression pattern of OSTC/DC2 protein in the context of heat shock stress. Its upregulation was detected both in cells treated with heat shock in vitro and in an animal model of heat shock in vivo. Northern blot analysis indicated that OSTC/DC2 mRNA is ubiquitously expressed in various human tissues, with abundant expression in the placenta and liver. The temporal changes of OSTC/DC2 protein expression following acute heat shock in human malignant glioblastoma cell line U87MG and mice were analyzed by Western blot assay. In general, expression of OSTC/DC2 protein was elevated after heat shock; however, the time courses of the change of OSTC/DC2 protein expression varied in different tissues. In the cerebellum, heat shock induction of OSTC/DC2 protein and activation of AKT, a key regulator of stress response, followed a similar time course. These results suggest that the upregulation of OSTC/DC2, a novel component of the oligosaccharyltransferase complex, is part of the mammalian heat shock response.

Interleukin-1β promotes hypoxia-induced apoptosis of glioblastoma cells by inhibiting hypoxia-inducible factor-1 mediated adrenomedullin production

Glioblastoma is the most common brain tumor in adults. Advanced glioblastomas normally contain hypoxic areas. The primary cellular responses to hypoxia are generally mediated by the transcription factor hypoxia-inducible factor 1 (HIF-1). Interleukin-1β (IL-1β) is a cytokine that is often present in the glioblastoma microenvironment and is known to be a modulator of glioblastoma progression. However, the role of IL-1β in regulating glioblastoma progression is still controversial. In this study, we found that in the human glioblastoma cell lines U87MG and U138MG, IL-1β inhibits the transactivation activity of HIF-1 by promoting the ubiquitin-independent proteasomal degradation of the oxygen-labile α-subunit of HIF-1 and downregulates the expression of the HIF-1 target gene adrenomedullin (AM). Apoptosis and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assays showed that AM protects glioblastoma cells against hypoxia-induced apoptosis in a dose-dependent manner. Thus, in the presence of IL-1β more glioblastoma cells undergo hypoxia-induced cell death. Our findings suggest that when estimating the influence of IL-1β on the prognosis of glioblastoma patients, factors such as the degree of hypoxia, the expression levels of HIF-1 and AM should be taken into consideration. For the AM-producing glioblastoma cells, IL-1β represents a potent apoptosis inducer.

A High-Content Assay Strategy for the Identification and Profiling of ABCG2 Modulators in Live Cells

ABCG2 is a member of the ATP-binding cassette (ABC) family of transporters, the overexpression of which has been implicated in resistance to various chemotherapeutic agents. Though a number of cell-based assays to screen for inhibitors have been reported, they do not provide a content-rich platform to discriminate toxic and autofluorescent compounds. To fill this gap, we developed a live high-content cell-based assay to identify inhibitors of ABCG2-mediated transport and, at the same time, assess their cytotoxic effect and potential optical interference. We used a pair of isogenic U87MG human glioblastoma cell lines, with one stably overexpressing the ABCG2 transporter. JC-1 (J-aggregate-forming lipophilic cation 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazol carbocyanine iodide) was selected as the optimal reporter substrate for ABCG2 activity, and the resulting assay was characterized by a Z' value of 0.50 and a signal-to-noise (S/N) ratio of 14 in a pilot screen of ∼ 7,000 diverse chemicals. The screen led to the identification of 64 unique nontoxic positives, yielding an initial hit rate of 1%, with 58 of them being confirmed activity. In addition, treatment with two selected confirmed positives suppressed the side population of U87MG-ABCG2 cells that was able to efflux the Hoechst dye as measured by flow cytometry, confirming that they constitute potent new ABCG2 transporter inhibitors. Our results demonstrate that our live cell and content-rich platform enables the rapid identification and profiling of ABCG2 modulators, and this new strategy opens the door to the discovery of compounds targeting the expression and/or trafficking of ABC transporters as an alternative to functional inhibitors that failed in the clinic.

Role of Collagen Matrix in Tumor Angiogenesis and Glioblastoma Multiforme Progression

Glioblastoma is a highly vascularized brain tumor, and antiangiogenic therapy improves its progression-free survival. However, current antiangiogenic therapy induces serious adverse effects including neuronal cytotoxicity and tumor invasiveness and resistance to therapy. Although it has been suggested that the physical microenvironment has a key role in tumor angiogenesis and progression, the mechanism by which physical properties of extracellular matrix control tumor angiogenesis and glioblastoma progression is not completely understood. Herein we show that physical compaction (the process in which cells gather and pack together and cause associated changes in cell shape and size) of human glioblastoma cell lines U87MG, U251, and LN229 induces expression of collagen types IV and VI and the collagen crosslinking enzyme lysyl oxidase and up-regulates in vitro expression of the angiogenic factor vascular endothelial growth factor. The lysyl oxidase inhibitor β-aminopropionitrile disrupts collagen structure in the tumor and inhibits tumor angiogenesis and glioblastoma multiforme growth in a mouse orthotopic brain tumor model. Similarly, d-penicillamine, which inhibits lysyl oxidase enzymatic activity by depleting intracerebral copper, also exhibits antiangiogenic effects on brain tumor growth in mice. These findings suggest that tumor microenvironment controlled by collagen structure is important in tumor angiogenesis and brain tumor progression.