Glioblastoma Multiforme Growth Research Articles

Delivery of doxorubicin to glioblastoma multiforme in vitro using solid lipid nanoparticles with surface aprotinin and melanotransferrin antibody for enhanced chemotherapy

Solid lipid nanoparticles (SLNs) conjugated with aprotinin (Apr) and melanotransferrin antibody (Anti-MTf) were used to carry doxorubicin (Dox) across the blood–brain barrier (BBB) for glioblastoma multiforme (GBM) chemotherapy. Dox-entrapped SLNs with grafted Apr and Anti-MTf (Apr-Anti-MTf-Dox-SLNs) were applied to a cellular monolayer comprising human brain-microvascular endothelial cells (HBMECs) with a regulation of human astrocyte (HAs) and to a proliferated colony of U87MG cells. Based on the average particle diameter, zeta potential, entrapping efficiency of Dox, and grafting efficiency of Apr and Anti-MTf, we found that 1,2-dipalmitoyl-sn-glycero-3-phosphocholine of 40% (w/w) in lipids was appropriate for fabricating Apr-Anti-MTf-Dox-SLNs. In addition, Apr-Anti-MTf-Dox-SLNs could prevent Dox from fast dissolution and did not induce a serious cytotoxicity to HBMECs and HAs when compared with free Dox. Moreover, the treatment with Apr-Anti-MTf-Dox-SLNs enhanced the ability of Dox to infuse the BBB and to inhibit the growth of GBM. The current Apr-Anti-MTf-Dox-SLNs can be a promising pharmacotherapeutic preparation to penetrate the BBB for malignant brain tumor management.

Computational Trials: Unraveling Motility Phenotypes, Progression Patterns, and Treatment Options for Glioblastoma Multiforme.

Glioblastoma multiforme is a malignant brain tumor with poor prognosis and high morbidity due to its invasiveness. Hypoxia-driven motility and concentration-driven motility are two mechanisms of glioblastoma multiforme invasion in the brain. The use of anti-angiogenic drugs has uncovered new progression patterns of glioblastoma multiforme associated with significant differences in overall survival. Here, we apply a mathematical model of glioblastoma multiforme growth and invasion in humans and design computational trials using agents that target angiogenesis, tumor replication rates, or motility. The findings link highly-dispersive, moderately-dispersive, and hypoxia-driven tumors to the patterns observed in glioblastoma multiforme treated by anti-angiogenesis, consisting of progression by Expanding FLAIR, Expanding FLAIR + Necrosis, and Expanding Necrosis, respectively. Furthermore, replication rate-reducing strategies (e.g. Tumor Treating Fields) appear to be effective in highly-dispersive and moderately-dispersive tumors but not in hypoxia-driven tumors. The latter may respond to motility-reducing agents. In a population computational trial, with all three phenotypes, a correlation was observed between the efficacy of the rate-reducing agent and the prolongation of overall survival times. This research highlights the potential applications of computational trials and supports new hypotheses on glioblastoma multiforme phenotypes and treatment options.

Abstract B13: Growth kinetics in glioblastome multiforme: Response to radiation and the quantification of treatment response

Abstract High-grade gliomas, commonly known as glioblastoma multiforme (GBM), exhibit linear radial expansion. In untreated patients, these growth kinetics are believed to be constant. Not much is known about the changes in growth kinetics that different treatment regimes induce. In this study we sought to compare pre-treatment growth kinetics to post-treatment growth kinetics; investigate correlations between pre-, during, and post-treatment growth kinetics survival, and time-to-progression; and elucidate measures of response to treatment that define post-treatment growth characteristics. Patient information and MRIs were obtained from 62 newly diagnosed patients with glioblastoma from Northwestern University, University of Washington, and University of California, Los Angeles who had at least 2 pre-treatment MRIs and 1 post-treatment MRI. The tumor growth kinetics were calculated by selecting 2 imaging events, separated by at least 5 days, and calculating the rate of growth according to: Velocity=(T1Gdradius2-T1Gdradius1)/(t2-t1) Velocities were compared with survival and time-to-progression using log-rank survival analysis in Prism 6.0 (Graphpad Software). There was no significant correlation between pre-treatment velocity and overall survival or time-to-progression (n=50). The velocity during radiation treatment was correlated with overall survival. A growth rate of less than 9.125 mm/year during the course of radiation portended greater overall survival (p-value < 0.001). To take into account the previous rate of growth, the pre-treatment velocity was compared to the growth rate during radiation. A relative change of greater than 110% (i.e. the tumor's growth was faster during the course of radiation) correlated with the time to recurrence (p-value= 0.016). The post-treatment velocity was correlated with overall survival. If the tumor grew at a rate greater than 2.87 mm/year after finishing radiation, the patient's survival was significantly less than those patients whose tumors grew at less than 2.87 mm/year. Interestingly, the growth kinetics prior to treatment do not correlate with response metrics. Instead, the response to radiation stratifies patients into survival categories that allow providers to more accurately predict clinical progression. Rather than the current ‘wait-and-see’ scheme of clinical decision-making, metrics of GBM growth kinetics that are calculated off of standard-of-care MRIs could allow for implementation of salvage therapies and secondary options in patients with low treatment response. Citation Format: Corbin Rayfield, Russ Rockne, Andrea Hawkins-Daruud, Joshua Jacobs, Kristin R. Swanson. Growth kinetics in glioblastome multiforme: Response to radiation and the quantification of treatment response. [abstract]. In: Proceedings of the AACR Special Conference: Advances in Brain Cancer Research; May 27-30, 2015; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2015;75(23 Suppl):Abstract nr B13.

Inhibition of SHP2-mediated dephosphorylation of Ras suppresses oncogenesis.

Ras is phosphorylated on a conserved tyrosine at position 32 within the switch I region via Src kinase. This phosphorylation inhibits the binding of effector Raf while promoting the engagement of GTPase-activating protein (GAP) and GTP hydrolysis. Here we identify SHP2 as the ubiquitously expressed tyrosine phosphatase that preferentially binds to and dephosphorylates Ras to increase its association with Raf and activate downstream proliferative Ras/ERK/MAPK signalling. In comparison to normal astrocytes, SHP2 activity is elevated in astrocytes isolated from glioblastoma multiforme (GBM)-prone H-Ras(12V) knock-in mice as well as in glioma cell lines and patient-derived GBM specimens exhibiting hyperactive Ras. Pharmacologic inhibition of SHP2 activity attenuates cell proliferation, soft-agar colony formation and orthotopic GBM growth in NOD/SCID mice and decelerates the progression of low-grade astrocytoma to GBM in a spontaneous transgenic glioma mouse model. These results identify SHP2 as a direct activator of Ras and a potential therapeutic target for cancers driven by a previously ‘undruggable' oncogenic or hyperactive Ras.

ST3GAL1-Associated Transcriptomic Program in Glioblastoma Tumor Growth, Invasion, and Prognosis.

Background:Cell surface sialylation is associated with tumor cell invasiveness in many cancers. Glioblastoma is the most malignant primary brain tumor and is highly infiltrative. ST3GAL1 sialyltransferase gene is amplified in a subclass of glioblastomas, and its role in tumor cell self-renewal remains unexplored.Methods:Self-renewal of patient glioma cells was evaluated using clonogenic, viability, and invasiveness assays. ST3GAL1 was identified from differentially expressed genes in Peanut Agglutinin–stained cells and validated in REMBRANDT (n = 390) and Gravendeel (n = 276) clinical databases. Gene set enrichment analysis revealed upstream processes. TGFβ signaling on ST3GAL1 transcription was assessed using chromatin immunoprecipitation. Transcriptome analysis of ST3GAL1 knockdown cells was done to identify downstream pathways. A constitutively active FoxM1 mutant lacking critical anaphase-promoting complex/cyclosome ([APC/C]-Cdh1) binding sites was used to evaluate ST3Gal1-mediated regulation of FoxM1 protein. Finally, the prognostic role of ST3Gal1 was determined using an orthotopic xenograft model (3 mice groups comprising nontargeting and 2 clones of ST3GAL1 knockdown in NNI-11 [8 per group] and NNI-21 [6 per group]), and the correlation with patient clinical information. All statistical tests on patients’ data were two-sided; other P values below are one-sided.Results:High ST3GAL1 expression defines an invasive subfraction with self-renewal capacity; its loss of function prolongs survival in a mouse model established from mesenchymal NNI-11 (P < .001; groups of 8 in 3 arms: nontargeting, C1, and C2 clones of ST3GAL1 knockdown). ST3GAL1 transcriptomic program stratifies patient survival (hazard ratio [HR] = 2.47, 95% confidence interval [CI] = 1.72 to 3.55, REMBRANDT P = 1.92x10-8; HR = 2.89, 95% CI = 1.94 to 4.30, Gravendeel P = 1.05x10-11), independent of age and histology, and associates with higher tumor grade and T2 volume (P = 1.46x10-4). TGFβ signaling, elevated in mesenchymal patients, correlates with high ST3GAL1 (REMBRANDT gliomacor = 0.31, P = 2.29x10-10; Gravendeel gliomacor = 0.50, P = 3.63x10-20). The transcriptomic program upon ST3GAL1 knockdown enriches for mitotic cell cycle processes. FoxM1 was identified as a statistically significantly modulated gene (P = 2.25x10-5) and mediates ST3Gal1 signaling via the (APC/C)-Cdh1 complex.Conclusions:The ST3GAL1-associated transcriptomic program portends poor prognosis in glioma patients and enriches for higher tumor grades of the mesenchymal molecular classification. We show that ST3Gal1-regulated self-renewal traits are crucial to the sustenance of glioblastoma multiforme growth.

CBIO-43ROLE OF BASIGIN-3 IN MMP EXPRESSION IN GBM

Glioblastoma multiforme (GBM) is one of the most aggressive forms of brain tumor. With the current standard of care, the five year survival rate for GBM patients is less than 3%. An understanding of the molecular mechanisms promoting growth and survival of GBMs may result in novel treatments for this disease. A recent report indicated that the expression of the protein Basigin is a significant prognostic indicator for tumor grade in GBM patients, with tumors expressing highest levels of basigin protein correlating with the most aggressive tumors (Yang 2013). The Basigin gene family contains four isoforms, with Basigin-2 the predominant isoform expressed in both normal and diseased tissue. In 2011, Liao et al, suggested that Basigin-3 interacts with Basigin-2 in Human Hepatocellular Carcinoma (HHC), leading to decreased tumor growth and invasion. Because the Basigin-2 protein (CD-147/EMMPRIN) is a known inducer of matrix metalloproteinase (MMP) the inhibition of Basigin-2 through its interaction with Basigin-3 should decrease both tumor growth and MMP expression. To test this hypothesis, a recombinant Basigin-3/mKate2 fluorescent protein fusion was overexpressed in a GBM cell line (LN229), and RT-qPCR was used to measure MMP gene expression. Confocal microscopy and western blot analysis confirmed the over-expression of Basigin-3 in the transfected populations. However, the overexpression of Basigin-3 did not reduce the expression of MMP-1, MMP-2, and MMP-3. Our data suggest that the role of Basigin-3 in GBM may be different from what was shown in HHCs. Currently, we are performing FRET analysis between Basigin-2 and Basigin-3 to confirm their interaction in our model.

CBIO-46SPINDLE CELLS IN EARLY C6-GBM GROWTH ARE STEM CELLS - FUNCTIONAL STUDY WITH MITOCHONDRIAL VOLUME FOR THERAPEUTIC PURPOSE

OBJECTIVE: Recurrence of glioblastoma multiforme (GBM) after total resection is postulated to originate from precursor cells existing in surrounding brain tissue. The authors have found spindle cells in the early stage of rat C6 GBM growth, resembling spongioblasts, the origin of GBM (Bailey and Cushing, 1926). They are stem-like cells with positive CD15 staining, and should be the primary target for treatment in experimental animals (Poster at SPNO meeting in May). Currently, spindle cells are functionally evaluated by mitochondrial volume studies for future development of therapeutic tactics. METHODS: 1. C6 cells were cultured in MEM solution, and morphological cell changes were studied with and without temozolomide (TMZ) treatment. 2. Mitochondrial volume of C6 cell in these conditions was measured by mitochondrial mass specific fluorescence probe. 3. C6 cells were implanted into rat's brain and observed the growth in the normal brain tissue. RESULTS: 1. Spindle cells grew in early period, and formed syncytiums. As syncytiums multiplied, mitotic cells began to appear, and typical GBM features developed. During TMZ treatment, multiform cells were mostly destroyed, while spindle cells survived. 2. By TMZ treatment, the number of survived C6 cells decreased, but the volume of mitochondria per unit of C6 cells increased, as the concentration of TMZ increased. 3. The same cell growth pattern was identified in implanted tumor, as seen in cultured cells growth. DISCUSSION AND CONCLUSION: 1. Histologically CD15-positive spindle cells have much higher resistance for survival against TMZ than fully developed multiform cells. 2. Increased mitochondrial volume in survived C6 cells is likely related to metabolic activation of spindle cells under TMZ treatment. 3. The control of mitochondrial hyperactivity of C6 cells should be one of the important means for GBM treatment in experimental GBM, and probably in humans.

MiR-429 induces apoptosis of glioblastoma cell through Bcl-2.

An essential role of microRNAs (miRNAs) has been acknowledged in the tumorigenesis of glioblastoma multiforme (GBM). Very recently, miR-429 was reported to have a potential of suppressing cancer growth. However, whether miR-429 may similarly regulate growth of GBM remains unknown. Here, we analyzed the levels of miR-429 and anti-apoptotic protein Bcl-2 in GBM specimens. We combined bioinformatics analyses and luciferase reporter assay to determine the relationship between miR-429 and Bcl-2 in GBM cells. Cell survival upon temozolomide treatment was analyzed in a CCK assay. Cell apoptosis was measured by fluorescein isothiocyanate (FITC) Annexin V apoptosis detection assay. We found that miR-429 levels were significantly decreased and Bcl-2 levels were significantly increased in GBM specimens, compared to the paired adjacent non-tumor brain tissue. Moreover, the levels of miR-429 and Bcl-2 inversely correlated. Low-miR-429 subjects had an overall inferior survival, compared to high-miR-429 subjects. MiR-429 targeted the 3'-UTR of Bcl-2 mRNA to inhibit its translation. Overexpression of miR-429 inhibited Bcl-2-mediated cell survival against temozolomide-induced apoptosis, while depletion of miR-429 augmented it. Together, our data suggest that miR-429 suppression in GBM promotes Bcl-2-mediated cancer cell survival against chemotherapy-induced cell death. Re-expression of miR-429 levels in GBM cells may improve the outcome of chemotherapy.

Dexamethasone-Mediated Activation of Fibronectin Matrix Assembly Reduces Dispersal of Primary Human Glioblastoma Cells.

Despite resection and adjuvant therapy, the 5-year survival for patients with Glioblastoma multiforme (GBM) is less than 10%. This poor outcome is largely attributed to rapid tumor growth and early dispersal of cells, factors that contribute to a high recurrence rate and poor prognosis. An understanding of the cellular and molecular machinery that drive growth and dispersal is essential if we are to impact long-term survival. Our previous studies utilizing a series of immortalized GBM cell lines established a functional causation between activation of fibronectin matrix assembly (FNMA), increased tumor cohesion, and decreased dispersal. Activation of FNMA was accomplished by treatment with Dexamethasone (Dex), a drug routinely used to treat brain tumor related edema. Here, we utilize a broad range of qualitative and quantitative assays and the use of a human GBM tissue microarray and freshly-isolated primary human GBM cells grown both as conventional 2D cultures and as 3D spheroids to explore the role of Dex and FNMA in modulating various parameters that can significantly influence tumor cell dispersal. We show that the expression and processing of fibronectin in a human GBM tissue-microarray is variable, with 90% of tumors displaying some abnormality or lack in capacity to secrete fibronectin or assemble it into a matrix. We also show that low-passage primary GBM cells vary in their capacity for FNMA and that Dex treatment reactivates this process. Activation of FNMA effectively “glues” cells together and prevents cells from detaching from the primary mass. Dex treatment also significantly increases the strength of cell-ECM adhesion and decreases motility. The combination of increased cohesion and decreased motility discourages in vitro and ex vivo dispersal. By increasing cell-cell cohesion, Dex also decreases growth rate of 3D spheroids. These effects could all be reversed by an inhibitor of FNMA and by the glucocorticoid receptor antagonist, RU-486. Our results describe a new role for Dex as a suppressor of GBM dispersal and growth.

Targeting Glioblastoma with the Use of Phytocompounds and Nanoparticles.

Glioblastoma multiforme (GBM) are extremely lethal and still poorly treated primary brain tumors, characterized by the presence of highly tumorigenic cancer stem cell (CSC) subpopulations, considered responsible for tumor relapse. In order to successfully eradicate GBM growth and recurrence, new anti-cancer strategies selectively targeting CSCs should be designed. CSCs might be eradicated by targeting some of their cell surface markers and transporters, inducing their differentiation, impacting their hyper-glycolytic metabolism, inhibiting CSC-related signaling pathways and/or by targeting their microenvironmental niche. In this regard, phytocompounds such as curcumin, isothiocyanates, resveratrol and epigallocatechin-3-gallate have been shown to prevent or reverse cancer-related epigenetic dysfunctions, reducing tumorigenesis, preventing metastasis and/or increasing chemotherapy and radiotherapy efficacy. However, the actual bioavailability and metabolic processing of phytocompounds is generally unknown, and the presence of the blood brain barrier often represents a limitation to glioma treatments. Nowadays, nanoparticles (NPs) can be loaded with therapeutic compounds such as phytochemicals, improving their bioavailability and their targeted delivery within the GBM tumor bulk. Moreover, NPs can be designed to increase their tropism and specificity toward CSCs by conjugating their surface with antibodies specific for CSC antigens, with ligands or with glucose analogues. Here we discuss the use of phytochemicals as anti-glioma agents and the applicability of phytochemical-loaded NPs as drug delivery systems to target GBM. Additionally, we provide some examples on how NPs can be specifically formulated to improve CSC targeting.

Abstract 5222: Endothelial plasticity generates aberrant angiogenesis and therapy resistance in glioblastoma

Abstract Overgrown, abnormal vasculature characterizes the microenvironment that fuels cancer progression and induces therapeutic resistance. Here we reveal that endothelial plasticity drives excessive and abnormal tumor angiogenesis. Glioblastoma multiforme (GBM) is among the most lethal of human malignancies, distinguished by prominent vascularity and extreme vascular abnormality. We identify endothelial fibro-transformation (Endo-FT) in GBM, contributing significantly to aberrant vascularization, tumor progression, and therapeutic resistance. Utilizing human GBM specimens and allograft and genetic murine GBM models driven by RCAS/Ntv-a-mediated gene transfer of PDGF in Ink4a-Arf-/-PTEN-/- mice, we reveal robust Endo-FT in the tumor vasculature, characterized by endothelial cell (EC) expression of the fibroblast and mesenchymal cell markers including FSP-1, α-smooth muscle actin, N-cadherin. Moreover, genetic lineage tracing shows a prominent population of GBM-associated fibroblast-like cells with EC origin. Furthermore, glioma-conditioned medium induces EC in vitro acquisition of the fibroblast signature with decreased expression of EC-specific proteins including CD31, VE-cadherin, Tie2, VEGFR2, leading to enhanced cell proliferation, motility, and invasiveness, and vessel permeability. Interestingly, the fibroblast-like ECs retain the key EC-specific functions including Ac-LDL absorption and tube formation, suggesting Endo-FT without complete EC transition to fibroblasts. Proteomic analysis identifies c-Met is a regulator of Endo-FT, requisite for the vascular abnormality. Pharmacological inhibition and siRNA-mediated knockdown of c-Met or neutralization with a HGF antibody remarkably abrogate tumor-conditioned medium-induced Endo-FT and EC malfunctions, suggesting a critical role of HGF/c-Met in Endo-FT. Furthermore, c-Met activation induces ETS-1-dependent matrix metalloproteinase (MMP)-14 expression, leading to VE-cadherin cleavage and Endo-FT. Finally, we test the in vivo role of c-Met-mediated Endo-FT in GBM progression by using mice with Tie2-Cre-driven, EC-specific Met knockout. Met deletion in ECs significantly improves mouse survival and inhibits GBM growth after temozolomide (TMZ) treatment. Moreover, Met deletion in ECs abolishes Endo-FT and vascular abnormality, as shown by remarkable decreases in CD31+FSP-1+ cell population and intratumor hypoxia, and increases in vasculature-mediated perfusion and vascular pericyte coverage, suggesting that c-Met-mediated Endo-FT is required for vascular abnormality and GBM progression and therapeutic resistance. In sum, these findings illustrate a novel mechanism controlling aberrant vascularization and GBM progression, and suggest that targeting Endo-FT may offer selective and efficient therapeutic strategies for the treatment-resistant GBM, and possibly other malignant tumors. Citation Format: Menggui Huang, Yi Fan. Endothelial plasticity generates aberrant angiogenesis and therapy resistance in glioblastoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5222. doi:10.1158/1538-7445.AM2015-5222

Ginsenoside Rh2 inhibits invasiveness of glioblastoma through modulation of VEGF-A.

The malignancy of glioblastoma multiforme (GBM) is largely due to its local invasion and the presence of the tumor in the relatively restrained region in the brain. Hence, effective prevention of the cancer cell invasion is substantially critical for controlling the growth and deterioration of GBM. We have recently reported the role of ginsenoside Rh2 (GRh2) in suppressing the growth of GBM through EGFR/PI3k/Akt/mTor signaling pathways. Here, we further showed that GRh2 efficiently inhibited the cancer vascularization in vivo. In vitro, GRh2 dose-dependently inhibited the protein, but not messenger RNA (mRNA) of vascular endothelial growth factor A (VEGF-A) in GBM cells. We then examined the underlying mechanisms and found that GRh2 increased the levels of miR-497, which bound to 3'UTR of VEGF-A mRNA to inhibit its translation. Together, our data demonstrate a previously unappreciated role for GRh2 in inhibition of GBM-associated cancer vascularization, which may contribute to the effects of GRh2 on suppression of GBM cancer growth and invasion.

Histone deacetylase 6 promotes growth of glioblastoma through inhibition of SMAD2 signaling.

Histone deacetylases (HDACs) play a role in the tumorigenesis of glioblastoma multiforme (GBM), whereas the underlying mechanism has not been elucidated. Here, we reported significantly higher HDAC6 levels in GBM from the patients. GBM cell growth was significantly inhibited by ACY-1215, a specific HDAC6 inhibitor. Further analyses show that HDAC6 may promote growth of GBM cells through inhibition of SMAD2 phosphorylation to downregulate p21. Thus, our data demonstrate a previously unrecognized regulation pathway in that HDAC6 increases GBM growth through attenuating transforming growth factor β (TGFβ) receptor signaling.

Growth of glioblastoma is inhibited by miR-133-mediated EGFR suppression.

Glioblastoma multiforme (GBM) is a severe and highly lethal brain cancer, which malignancy largely stems from its growing in a relatively restrained area in the brain. Hence, the understanding of the molecular regulation of the growth of GBM is critical for improving its treatment. Dysregulation of microRNAs (miRNAs) has recently been shown to contribute to the development of GBM, whereas the role of miR-133 in GBM is unknown. Here, by qualitative reverse transcription polymerase chain reaction (RT-qPCR), we detected lower miR-133 levels in GBM tissues, compared to the paired normal brain tissue. We overexpressed or inhibited miR-133 in GBM cells. Cell growth and apoptosis were analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and flow cytometry, respectively. We found that overexpression of miR-133 decreased GBM cell growth and increased cell apoptosis, while depletion of miR-133 increased cell growth and decreased cell apoptosis. Bioinformatic analysis was performed, showing that miR-133 may target the 3'-untranslated region (3'-UTR) of the epidermal growth factor receptor (EGFR) that transduces cell growth signals. Further, the protein translation inhibition of EGFR by miR-133 was confirmed by a dual luciferase reporter assay. Together, these data suggest that reduced miR-133 levels in GBM tissues promotes cell growth and decreases cell apoptosis, possibly through targeting mRNA of EGFR to suppress its translation.

The Bromodomain protein BRD4 controls HOTAIR, a long noncoding RNA essential for glioblastoma proliferation

Bromodomain and extraterminal (BET) domain proteins have emerged as promising therapeutic targets in glioblastoma and many other cancers. Small molecule inhibitors of BET bromodomain proteins reduce expression of several oncogenes required for Glioblastoma Multiforme (GBM) progression. However, the mechanism through which BET protein inhibition reduces GBM growth is not completely understood. Long noncoding RNAs (lncRNAs) are important epigenetic regulators with critical roles in cancer initiation and malignant progression, but mechanistic insight into their expression and regulation by BET bromodomain inhibitors remains elusive. In this study, we used Helicos single molecule sequencing to comprehensively profile lncRNAs differentially expressed in GBM, and we identified a subset of GBM-specific lncRNAs whose expression is regulated by BET proteins. Treatment of GBM cells with the BET bromdomain inhibitor I-BET151 reduced levels of the tumor-promoting lncRNA HOX transcript antisense RNA (HOTAIR) and restored the expression of several other GBM down-regulated lncRNAs. Conversely, overexpression of HOTAIR in conjunction with I-BET151 treatment abrogates the antiproliferative activity of the BET bromodomain inhibitor. Moreover, chromatin immunoprecipitation analysis demonstrated binding of Bromodomain Containing 4 (BRD4) to the HOTAIR promoter, suggesting that BET proteins can directly regulate lncRNA expression. Our data unravel a previously unappreciated mechanism through which BET proteins control tumor growth of glioblastoma cells and suggest that modulation of lncRNA networks may, in part, mediate the antiproliferative effects of many epigenetic inhibitors currently in clinical trials for cancer and other diseases.

Inhibition Against Growth of Glioblastoma Multiforme In Vitro Using Etoposide-Loaded Solid Lipid Nanoparticles with ρ-Aminophenyl-α-D-Manno-Pyranoside and Folic Acid

Solid lipid nanoparticles (SLNs) grafted with p-aminophenyl-α-D-manno-pyranoside (APMP) and folic acid (FA) (APMP-FA-SLNs) were applied to encapsulate 4'-demethylepipodophyllotoxin 9-(4,6-O-ethylidene-β-D-glucopyranoside) (etoposide) (ETP) for promoting the antiproliferation of malignant glioblastoma multiforme. ETP-loaded APMP-FA-SLNs (APMP-FA-ETP-SLNs) were used to penetrate the blood-brain barrier (BBB) and retard the propagation of U87MG cells. An incorporation of APMP and FA increased the particle size, the cytotoxicity to U87MG cells, and the permeability coefficient for propidium iodide and ETP across the BBB. In addition, an increase in the APMP and FA concentration reduced the zeta potential, the grafting efficiency of APMP and FA, the dissolution rate of ETP, and the transendothelial electrical resistance. Immunochemical staining images evidenced that APMP-FA-ETP-SLNs could infiltrate the BBB via glucose transporter 1 and recognize U87MG cells via folate receptor. APMP-FA-ETP-SLNs can be an effective pharmacotherapeutic formulation in targeting delivery to the brain and in inhibitory efficacy against tumorous cells for cancer therapy.

286. Systemic Delivery of AAV9-IFNβ Vectors for Glioblastoma Gene Therapy

Introduction: Glioblastoma multiforme (GBM) is the most prevalent primary brain tumor, with a median survival (MS) of only 14.6 months from diagnosis despite providing multimodal treatment including maximal surgical resection, radiation and chemotherapy. Due to the highly invasive properties of GBM cells, complete surgical resection is practically impossible, leading to tumor recurrence and death. Thus, new approaches are needed to prevent tumor recurrence.IFNb is a pleiotropic cytokine with potent anti-tumor effects. Previous studies have shown that local delivery of AAV-IFNb in a non-invasive GBM mouse model had a remarkable therapeutic effect. However local treatment may be insufficient to treat GBM where long distance migration is a common feature. Therefore the therapeutic approach should be matched to the tumor properties, which may be accomplished with a disperse therapeutic network generated by global brain gene delivery. Here we tested the therapeutic efficacy of an AAV9-IFNb vector infused via the tail vein in an orthotopic xenograft GBM model in athymic nude mice implanted in the striatum with 50,000 highly invasive GBM8 human glioblastoma cells stably expressing firefly luciferase (GBM8-fluc) for in vivo bioluminescence monitoring of tumor growth. AAV9-IFNb vector was infused via the tail vein at different times after tumor implantation.We performed a dose response study by infusing AAV9-IFNb vectors at different doses (1E11vg to 1E12vg/mouse) on d14 posttumor implantation. The improvement in survival with AAV9-IFNb was dose dependent and ranged from an increase in median survival of 111 days to complete tumor regression compared to 46 days in PBS-treated controls. Treatment with 3e11vg AAV9-IFNb resulted in the survival of 87.5% of animals for at least 8 months with no remaining evidence of GBM cells in the brain upon histological examination. This dose was considered the minimal effective dose (MED) and used in subsequent experiments. Next, we examined the impact of time between tumor implantation and treatment by infusion AAV9-IFNb at MED on d14, d21, d25 or d28. We found that d21 is the longest interval showing 100% long-term survival. Interestingly, the survival benefit was drastically reduced when treated on d25 or later, corroborated by Fluc imaging results and histological analysis showing untreated GBM growth accelerates considerably after d21.Finally, we tested the importance of global gene delivery in a model of multifocal GBM model with tumors implanted in both hemispheres. On d14, AAV9-IFNb was infused systemically at MED or injected intratumorally on one side of the brain. We found that systemic delivery resulted in reduction of tumor size in both hemispheres while intra-tumoral injection only impacted tumor growth on the injected side. All animals treated systemically with AAV9-IFNb survived long-term, while the median survival for animals receiving a unilateral injection of AAV9-IFNb was 57 days compared to 46 days for PBS-treated controls.Conclusion: Global delivery of AAV9-IFNb can be beneficial particularly to treat invasive or multifocal GBM and can provide long-term survival in the xenograft GBM model when treated before the onset of highly accelerated tumor growth.

Ginsenoside Rh2 inhibits metastasis of glioblastoma multiforme through Akt-regulated MMP13.

Glioblastoma multiforme (GBM) is the most malignant type of primary brain tumor. Although the growth of the tumor cells in a relatively closed space may partially account for its malignancy, highly invasive nature of glioblastoma cells has been suggested to be the main reason for the failure of current therapeutic approaches. Ginsenoside Rh2 (GRh2) has recently been shown to significantly suppress the growth and survival of GBM through inhibiting epidermal growth factor receptor signaling, whereas its effects on the invasion and metastasis have not been examined. Here, we showed that GRh2 dose-dependently decreased GBM cell invasiveness in both scratch wound healing assay and Transwell cell migration assay. Moreover, the inhibitory effects of GRh2 on cell migration seemed to be conducted through decreased expression of matrix metalloproteinase (MMP)-13. Furthermore, using specific inhibitors, we found that GRh2 inhibited MMP13 through PI3k/Akt signaling pathway. Finally, high MMP13 levels were detected in GBM specimen from the patients. Together, these data suggest that GRh2 may suppress GBM migration through inhibiting Akt-mediated MMP13 activation. Thus, our data highlight a previous unappreciated role for GRh2 in suppressing GBM cell metastasis.

The effect of silver nanoparticles (AgNPs) on proliferation and apoptosis of in ovo cultured glioblastoma multiforme (GBM) cells.

Recently, it has been shown that silver nanoparticles (AgNPs) provide a unique approach to the treatment of tumors, especially those of neuroepithelial origin. Thus, the aim of this study was to evaluate the impact of AgNPs on proliferation and activation of the intrinsic apoptotic pathway of glioblastoma multiforme (GBM) cells cultured in an in ovo model. Human GBM cells, line U-87, were placed on chicken embryo chorioallantoic membrane. After 8 days, the tumors were divided into three groups: control (non-treated), treated with colloidal AgNPs (40 μg/ml), and placebo (tumors supplemented with vehicle only). At the end of the experiment, all tumors were isolated. Assessment of cell proliferation and cell apoptosis was estimated by histological, immunohistochemical, and Western blot analyses. The results show that AgNPs can influence GBM growth. AgNPs inhibit proliferation of GBM cells and seem to have proapoptotic properties. Although there were statistically significant differences between control and AgNP groups in the AI and the levels of active caspase 9 and active caspase 3, the level of these proteins in GBM cells treated with AgNPs seems to be on the border between the spontaneous apoptosis and the induced. Our results indicate that the antiproliferative properties of silver nanoparticles overwhelm proapoptotic ones. Further research focused on the cytotoxic effect of AgNPs on tumor and normal cells should be conducted.Electronic supplementary materialThe online version of this article (doi:10.1186/s11671-015-0823-5) contains supplementary material, which is available to authorized users.

Nuclear SMAD2 Restrains Proliferation of Glioblastoma

Aims: Although TGFβ receptor signaling has been shown to play a role in regulation of the growth and metastasis of glioblastoma multiforme (GBM), the downstream pathway through either SMAD2 or SMAD3 has not been elucidated. In this study, we investigate whether nuclear SMAD2 can restrain the proliferation of glioblastoma. Methods: A total of 23 resected specimens from GBM patients were collected for SMAD2 detection. Human GBM cell line A172, U87mg, D341m and Hs683 were maintained in Dulbecco's modified Eagle's medium and transfected with SMAD2 and SMAD3 shRNA plasmids. Gene expression was detected by RT-qPCR and Western and cell growth were detected by MTT assay. Results: Our results showed that the phosphorylated SMAD2 (pSMAD2, the nuclear and functional form of SMAD2) levels in GBM were significantly lower than the paired normal brain tissue in patients. Depletion of SMAD2, but not SMAD3, significantly abolished the inhibitory effects of TGFβ1 on the growth of GBM cells, possibly through pSMAD2-mediated increases in cell-cycle inhibitor, p27. Conclusion: Our data suggest that TGFβ/SMAD2 signaling cascades restrains growth of GBM.