Human Brain Glioblastoma Research Articles

Determination of optical properties of human brain tumor tissues from 350 to 1000nm to investigate the cause of false negatives in fluorescence-guided resection with 5-aminolevulinic acid.

The optical properties of human brain tumor tissues, including glioblastoma, meningioma, oligodendroglioma, and metastasis, that were classified into "strong," "vague," and "unobservable" fluorescence by a neurosurgeon were measured and compared. The optical properties of the tissues were measured with a double integrating sphere and the inverse Monte Carlo technique from 350 to 1000nm. Using reasons of ex-vivo measurement, the optical properties at around 420nm were potentially affected by the hemoglobin content in tissues. Significant differences were not observed between the optical properties of the glioblastoma regions with "strong" and "unobservable" fluorescence. Sections of human brain tumor tissue with "strong" and "unobservable" fluorescence were stained with hematoxylin and eosin. The cell densities [mean ± standard deviation (S.D.)] in regions with "strong" and "unobservable" fluorescence were 31 ± 9 × 102 per mm2 and 12 ± 4 × 102 per mm2, respectively, which is a statistically significant difference. The higher fluorescence intensity is associated with higher cell density. The difference in cell density modified the scattering coefficient yet it does not lead to significant differences in the reduced scattering coefficient and thus does not affect the propagation of the diffuse fluorescent light. Hence, the false negatives, which mean a brain tumor only shows "unobservable" fluorescence and is hence classified incorrectly as nontumor, in using 5-ALA for detection of human glioblastoma do not result from the differences in optical properties of human brain glioblastoma tissues. Our results suggest that the primary cause of false negatives may be a lack of PpIX or a low accumulation of PpIX.

07 Identifying and prognosticating malignant brain tumors non-invasively using unique metabolomic signatures derived from patient serum and urine samples

BACKGROUND: Metabolomics technology has the potential to revolutionize how we screen, diagnose, and treat cancer, as well as improve upon existing cancer molecular tests that may not sufficiently capture the complexity of most malignancies. In this study, we explore the clinical potential of metabolomics analysis in the diagnosis and risk-stratification of brain tumors. METHODS: To test the hypothesis that brain tumor type and survival could be predicted with metabolomics, we analyzed the pre-operative serum and urine samples of patients with glioblastoma (GBM), oligoastrocytoma (OA2), meningioma (M1) and compared them to healthy controls. (HC). Sera from immune-deficient NOD-SCID mice xenografted with human GBM brain tumor initiating cells were also studied. RESULTS: Metabolomics analysis of patient samples was able to accurately differentiate GBM, OA2, M1 and HC (p = 2.3 x 10-26). Subsequently, a prediction model developed and validated internally was able to diagnose GBM with a sensitivity of 86.7% and specificity of 93.8%, and distinguish whether a GBM patient possess O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation (p = 7.4 x 10-10). Within the MGMT methylated group, the model was able to predict longevity (p = 3.25 x 10-4). The model was also able to predict survival irrespective of MGMT methylation status (p = 2.9 x 10-6). CONCLUSIONS: In this study, we demonstrate that metabolomic analysis of patient biofluids can identify brain tumors, distinguish brain tumor subtypes, and independently predict MGMT status as well as longevity among GBM patients. Metabolomics analysis may facilitate non-invasive diagnosis of aggressive brain tumours.

Polysaccharide sulphated derivative from Aconitumcoreanum induces cell apoptosis in the human brain glioblastoma U87MG cell line via the NF-κB/Bcl-2 cell apoptotic signaling pathway.

In a previous study, our team preliminarily investigated the bioefficacy of an extracted polysaccharide from the medicinal plant Aconitumcoreanum (ACP1). In the present study, we further evaluated the antitumor efficacy of an ACP1 sulphated derivative (ACP1‑s) in the human brain glioblastoma U87MG cell line. Cell viability assay and flow cytometry results demonstrated that 400, 800 and 1,600µg/ml ACP1‑s induced cell growth inhibition and cell apoptosis. We then investigated the underlying molecular mechanism of the ACP1‑s induced cell apoptosis and found that the NF‑κB/Bcl‑2 cell apoptotic signaling pathway was involved. Following treatment with ACP1‑s, the expression of IκB in U87MG cells was significantly upregulated, whereas the level of NF‑κB and the ratio of Bcl‑2/Bax was significantly decreased. The level of cleaved caspase‑3 was increased accordingly. When we introduced exogenous p65 protein into the U87MG cells, the ACP1‑s-induced cell growth inhibition and cell apoptosis were partially neutralized, and the expression of the anti‑apoptotic gene Bcl‑2 was recovered accordingly. These findings suggest the potential value of ACP1‑s as a novel therapeutic agent for the treatment of glioblastoma.

Anti-CD133 monoclonal antibody conjugated immunomagnetic nanosensor for molecular imaging of targeted cancer stem cells

Abstract Magnetic nanosensors are considered as highly attractive platforms for effective treatments in cancer diagnosis and therapy. Herein a smart immunomagnetic nanosensor is novelly developed for molecular imaging of targeted glioblastoma cancer stem cells (CSCs), based on specific interaction between cell-membrane marker antigen CD133 of glioblastoma CSCs and its raised anti-CD133 monoclonal antibody (mAb). Superparamagnetic iron oxide (SPIO) γ-Fe 2 O 3 nanoparticles (NPs) were fabricated as nanosensor cores with approximately 10–15 nm in size, and coated with carboxymethyl chitosan (CMCS) via sodium tripolyphosphate (TPP) crosslinking, and then chemically modified with polyethylenimine (PEI). Anti-CD133 mAb, specific affinity with the cancer stem biomarker CD133 expressed on the membrane surface of glioblastoma CSCs, was subsequently conjugated with the PEI- modified SPIO NPs to form anti-CD133 mAb conjugated immunomagnetic nanosensor. The prepared immunomagnetic nanosensor i.e. anti-CD133 mAb-conjugated nanoscale magnetic sensor (mAb-nano-MSN) was biologically assayed with cellular toxicity, cell cycle and specificity, and finally delivered to the targeted CSCs for fluorescence imaging and magnetic resonance imaging (MRI) within human brain glioblastoma CSCs. The results demenstrated that the developed immunomagnetic nanosensor displayed preferable properties such as excellent biocompatibility, non-toxicity and high specificity. The glioblastoma CSCs treated with anti-CD133 mAb-nano-MSN displayed a strong red fluorescence signal and a negative contrast enhancement compared with the cells treated with non-mAb-functionalized magnetic nanopartitles, probably due to efficient endocytosis mediated by anti-CD133 mAb grafted onto the prepared magnetic nanosensor. Therefore, these results indicated the fabricated anti-CD133 mAb-nano-MSN could be used as a promising nanovehicle for molecular imaging for the targeted CSCs in human brain tumor diagnosis and therapy.

Abstract 388: Transcellular Transport of HDL Bearing Gold Nanoparticles Across the Blood Brain Barrier

The overall objective of this study was to develop a HDL-based multifunctional platform for transport and delivery of highly hydrophobic gold nanoparticles (AuNP) bearing photothermic properties across the blood brain barrier (BBB). We exploited the ability of apolipoprotein E3 (apoE3) to act as a high affinity ligand for the low-density lipoprotein receptor to gain entry into endothelial and glioblastoma cells. The issue of poor aqueous solubility of AuNP of varying diameters (3, 10, or 10 nm) was overcome by integrating them with phospholipids and apoE3, yielding reconstituted rHDL bearing AuNP (rHDL-AuNP). Transmission electron microscopy (TEM) revealed the presence of AuNP embedded in spherical particles. Incubation of human brain microvasculature endothelial cells or glioblastoma cells with rHDL-AuNP bearing unlabeled or FITC-labeled apoE3 revealed robust uptake of particles that were localized in endocytic/lysosomal vesicles. The transport of rHDL-AuNP across an in vitro BBB model developed from primary porcine endothelial cells was examined. The addition of rHDL-AuNP to the luminal side of the cells did not affect the integrity of the BBB as assessed by the localization of key tight junction markers such as occludin, claudins and ZO-1 by immunofluorescence, and, by continual measurement of the transepithelial electrical resistance by impedance spectroscopy under physiological conditions. Lastly, the appearance of fluorescein fluorescence and AuNP in the abluminal side suggested transport of rHDL-AuNP across the neurovascular junction. These findings demonstrate that rHDL bearing apoE3 acts as a detergent in solubilizing and dramatically improving the aqueous solubility of AuNP, facilitates cellular uptake and transcellular transport of rHDL-AuNP across endothelial cells. They are significant since they present rHDL bearing apoE3 as an effective platform for delivering AuNP across the BBB.

Cytotoxic, anti-inflammatory and antioxidant activities of four different extracts of <i>Galega officinalis</i> L (Goat’s rue)

Purpose : To evaluate the cytotoxic, anti-inflammatory and antioxidant activities of four different solvent extracts obtained from the aerial parts of Galega officinalis L. Methods : The hexane, DCM, methanol and water extracts of G. officinalis were successively obtained by soxhlet extraction method. The cytotoxic activity of the extracts was assessed against human lung carcinoma (A-549), human colorectal adenocarcinoma (HT-29), human brain glioblastoma (U-87), and colon adenocarcinoma (DLD-1) by Resazurine test. The antioxidant activity of extracts were determined by Folin-Ciocalteau, oxygen radical absorbing capacity (ORAC), and 2’.7’-dichlorofluorescin-diacetate (DCFH-DA) cell-based assay while their anti-inflammatory activity was determined by nitric oxide (NO) assay. Results : DCM extract showed strong cytotoxic activity against lung adenocarcinoma and brain glioblastoma cell lines, with IC 50 (concentration inhibiting 50 % of cell growth) values of 11 ± 0.4 and 16 ± 3 μg/mL, respectively. The hexane extract showed moderate anticancer activity against the same cell lines (59 ± 13 and 63 ± 16 μg/mL, respectively). DCM extract also showed significant anti-inflammatory activity, inhibiting NO release by 86.7 % at 40 μg/mL in lipopolysaccharide (LPS) - stimulated murine RAW 264.7 macrophages. Of all test extracts, the methanol extract of G. officinalis showed the highest antioxidant activity with 2.33 ± 0.09 μmol Trolox/mg , 7.10 ± 0.9 g tannic acid equivalent (TAE), and IC 50 of 44 ± 4 μg/mL. Conclusion : The findings of this study suggest that DCM extract may possess anticancer effect against lung adenocarcinoma and brain glioblastoma, as well as serve as an anti-inflammatory agent. Keywords : Galega officinalis L, Biological activity, Bioassay, Anticancer, Lung adenocarcinoma, Brain glioblastoma, Goat’s rue

Stereotaxic administrations of allogeneic human Vγ9Vδ2 T cells efficiently control the development of human glioblastoma brain tumors

ABSTRACTGlioblastoma multiforme (GBM) represents the most frequent and deadliest primary brain tumor. Aggressive treatment still fails to eliminate deep brain infiltrative and highly resistant tumor cells. Human Vγ9Vδ2 T cells, the major peripheral blood γδ T cell subset, react against a wide array of tumor cells and represent attractive immune effector T cells for the design of antitumor therapies. This study aims at providing a preclinical rationale for immunotherapies in GBM based on stereotaxic administration of allogeneic human Vγ9Vδ2 T cells. The feasibility and the antitumor efficacy of stereotaxic Vγ9Vδ2 T cell injections have been investigated in orthotopic GBM mice model using selected heterogeneous and invasive primary human GBM cells. Allogeneic human Vγ9Vδ2 T cells survive and patrol for several days within the brain parenchyma following adoptive transfer and can successfully eliminate infiltrative GBM primary cells. These striking observations pave the way for optimized stereotaxic antitumor immunotherapies targeting human allogeneic Vγ9Vδ2 T cells in GBM patients.

Benzyl isothiocyanate alters the gene expression with cell cycle regulation and cell death in human brain glioblastoma GBM 8401 cells.

Glioblastoma multiforme (GBM) is a highly malignant devastating brain tumor in adults. Benzyl isothiocyanate (BITC) is one of the isothiocyanates that have been shown to induce human cancer cell apoptosis and cell cycle arrest. Herein, the effect of BITC on cell viability and apoptotic cell death and the genetic levels of human brain glioblastoma GBM 8401 cells in vitro were investigated. We found that BITC induced cell morphological changes, decreased cell viability and the induction of cell apoptosis in GBM 8401 cells was time-dependent. cDNA microarray was used to examine the effects of BITC on GBM 8401 cells and we found that numerous genes associated with cell death and cell cycle regulation in GBM 8401 cells were altered after BITC treatment. The results show that expression of 317 genes was upregulated, and two genes were associated with DNA damage, the DNA-damage-inducible transcript 3 (DDIT3) was increased 3.66-fold and the growth arrest and DNA-damage-inducible α (GADD45A) was increased 2.34-fold. We also found that expression of 182 genes was downregulated and two genes were associated with receptor for cell responses to stimuli, the EGF containing fibulin-like extracellular matrix protein 1 (EFEMP1) was inhibited 2.01-fold and the TNF receptor-associated protein 1 (TRAP1) was inhibited 2.08-fold. BITC inhibited seven mitochondria ribosomal genes, the mitochondrial ribosomal protein; tumor protein D52 (MRPS28) was inhibited 2.06-fold, the mitochondria ribosomal protein S2 (MRPS2) decreased 2.07-fold, the mitochondria ribosomal protein L23 (MRPL23) decreased 2.08-fold, the mitochondria ribosomal protein S2 (MRPS2) decreased 2.07-fold, the mitochondria ribosomal protein S12 (MRPS12) decreased 2.08-fold, the mitochondria ribosomal protein L12 (MRPL12) decreased 2.25-fold and the mitochondria ribosomal protein S34 (MRPS34) was decreased 2.30-fold in GBM 8401 cells. These changes of gene expression can provide the effects of BITC on the genetic level and are potential biomarkers for glioblastoma therapy.

Phenethyl isothiocyanate alters the gene expression and the levels of protein associated with cell cycle regulation in human glioblastoma GBM 8401 cells

Glioblastoma is the most common and aggressive primary brain malignancy. Phenethyl isothiocyanate (PEITC), a member of the isothiocyanate family, can induce apoptosis in many human cancer cells. Our previous study disclosed that PEITC induces apoptosis through the extrinsic pathway, dysfunction of mitochondria, reactive oxygen species (ROS)-induced endoplasmic reticulum (ER) stress, and intrinsic (mitochondrial) pathway in human brain glioblastoma multiforme (GBM) 8401 cells. To the best of our knowledge, we first investigated the effects of PEITC on the genetic levels of GBM 8401 cells in vitro. PEITC may induce G0/G1 cell-cycle arrest through affecting the proteins such as cdk2, cyclin E, and p21 in GBM 8401 cells. Many genes associated with cell-cycle regulation of GBM 8401 cells were changed after PEITC treatment: 48 genes were upregulated and 118 were downregulated. The cell-division cycle protein 20 (CDC20), Budding uninhibited by benzimidazole 1 homolog beta (BUB1B), and cyclin B1 were downregulated, and clusterin was upregulated in GBM 8401 cells treated with PEITC. These changes of gene expression can provide the effects of PEITC on the genetic levels and potential biomarkers for glioblastoma. © 2015 Wiley Periodicals, Inc. Environ Toxicol 32: 176-187, 2017.

Pyrvinium Targets CD133 in Human Glioblastoma Brain Tumor-Initiating Cells.

Clonal evolution of cancer may be regulated by determinants of stemness, specifically self-renewal, and current therapies have not considered how genetic perturbations or properties of stemness affect such functional processes. Glioblastoma-initiating cells (GICs), identified by expression of the cell surface marker CD133, are shown to be chemoradioresistant. In the current study, we sought to elucidate the functional role of CD133 in self-renewal and identify compounds that can specifically target this CD133(+) treatment-refractory population. Using gain/loss-of-function studies for CD133 we assessed the in vitro self-renewal and in vivo tumor formation capabilities of patient-derived glioblastoma cells. We generated a CD133 signature combined with an in silico screen to find compounds that target GICs. Self-renewal and proliferation assays on CD133-sorted samples were performed to identify the preferential action of hit compounds. In vivo efficacy of the lead compound pyrvinium was assessed in intracranial GIC xenografts and survival studies. Lastly, microarray analysis was performed on pyrvinium-treated GICs to discover core signaling events involved. We discovered pyrvinium, a small-molecule inhibitor of GIC self-renewal in vitro and in vivo, in part through inhibition of Wnt/β-catenin signaling and other essential stem cell regulatory pathways. We provide a therapeutically tractable strategy to target self-renewing, chemoradioresistant, and functionally important CD133(+) stem cells that drive glioblastoma relapse and mortality. Our study provides an integrated approach for the eradication of clonal populations responsible for cancer progression, and may apply to other aggressive and heterogeneous cancers.

PEITC inhibits human brain glioblastoma GBM 8401 cell migration and invasion through the inhibition of uPA, Rho A, and Ras with inhibition of MMP-2, -7 and -9 gene expression.

Glioblastoma is the most aggressive primary brain malignancy, and the efficacy of multimodality treatments remains unsatisfactory. Phenethyl isothiocyanate (PEITC), one member of the isothiocyanate family, was found to inhibit the migration and invasion of many types of human cancer cells. In our previous study, PEITC induced the apoptosis of human brain glioblastoma GBM 8401 cells through the extrinsic and intrinsic signaling pathways. In the present study, we first investigated the effects of PEITC on the migration and invasion of GBM 8401 cells. PEITC decreased the migration of GBM 8401 cells in a dose-dependent manner as determined from scratch wound healing and Transwell migration assays. The percentage of inhibition ranged from 46.89 to 15.75%, and from 27.80 to 7.31% after a 48-h treatment of PEITC as determined from the Transwell migration assay and invasion assay, respectively. The western blot analysis indicated that PEITC decreased the levels of proteins associated with migration and invasion, Ras, uPA, RhoA, GRB2, p-p38, p-JNK, p-ERK, p65, SOS1, MMP-2, MMP-9 and MMP-13, in a dose-dependent manner. Real-time PCR analyses revealed that PEITC reduced the mRNA levels of MMP-2, MMP-7, MMP-9 and RhoA in a dose- and time-dependent manner. PEITC exhibited potent anticancer activities through the inhibition of migration and invasion in the GBM 8401 cells. Our findings elucidate the possible molecular mechanisms and signaling pathways of the anti-metastatic effects of PEITC on human brain glioblastoma cells, and PEITC may be considered as a therapeutic agent.

Benzyl isothiocyanate (BITC) induces apoptosis of GBM 8401 human brain glioblastoma multiforms cells via activation of caspase-8/Bid and the reactive oxygen species-dependent mitochondrial pathway.

Benzyl isothiocyanate (BITC) is one of member of the isothiocyanate family which has been shown to induce cancer cell apoptosis in many human cancer cells. In the present study, we investigated the effects of BITC on the growth of GBM 8401 human brain glioblastoma multiforms cells. Results indicated that BITC-induced cell morphological changes decreased in the percentage of viable GBM8401 cells and these effects are dose-dependent manners. Results from flow cytometric assay indicated that BITC induced sub-G1 phase and induction of apoptosis of GBM 8401 cells. Furthermore, results also showed that BITC promoted the production of reactive oxygen species (ROS) and Ca2+ release, but decreased the mitochondrial membrane potential (ΔΨm ) and promoted caspase-8, -9, and -3 activates. After cells were pretreated with Z-IETD-FMK, Z-LEHD-FMK, and Z-DEVD-FMK (caspase-8, -9, and -3 inhibitors, respectively) led to decrease in the activities of caspase-8, -9, and -3 and increased the percentage of viable GBM 8401 cells that indicated which BITC induced cell apoptosis through caspase-dependent pathways. Western blotting indicated that BITC induced Fas, Fas-L, FADD, caspase-8, caspase -3, and pro-apoptotic protein (Bax, Bid, and Bak), but inhibited the ant-apoptotic proteins (Bcl-2 and Bcl-x) in GBM 8401 cells. Furthermore, BITC increased the release of cytochrome c, AIF, and Endo G from mitochondria that led to cell apoptosis. Results also showed that BITC increased GADD153, GRP 78, XBP-1, and ATF-6β, IRE-1α, IRE-1β, Calpain 1 and 2 in GBM 8401 cells, which is associated with ER stress. Based on these observations, we may suggest that BITC-induced apoptosis might be through Fas receptor, ROS induced ER stress, caspase-3, and mitochondrial signaling pathways. Taken together, these molecular alterations and signaling pathways offer an insight into BITC-caused growth inhibition and induced apoptotic cell death of GBM 8401 cells. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1751-1760, 2016.

Nuclear translocation of fibroblast growth factor-2 (FGF2) is regulated by Karyopherin-β2 and Ran GTPase in human glioblastoma cells.

Human glioblastoma multiforme (GBM) is the most malignant tumor of the central nervous system (CNS). Fibroblast growth factor-2 (FGF2) belongs to the FGF superfamily and functions as a potential oncoprotein in GBM. FGF2 has low molecular weight (18K) and high molecular weight (HMW) isoforms. Nuclear accumulation of HMW-FGF2 strongly promotes glioblastoma cell proliferation, yet mechanism governing such cellular distribution remains unexplored. We investigated the mechanisms regulating FGF2 cellular localization in T98G human brain glioblastoma cells. We found HMW-FGF2, but not 18K-FGF2, is primarily located in the nucleus and interacts with nuclear transport protein Karyopherin-β2/Transportin (Kapβ2). SiRNA-directed Kapβ2 knockdown significantly reduced HMW-FGF2's nuclear translocation. Moreover, inhibiting Ran GTPase activity also resulted in decreased HMW-FGF2 nuclear accumulation. Proliferation of T98G cells is greatly enhanced with transfections HMW-FGF2. Decreased PTEN expression and activated Akt signaling were observed upon HMW-FGF2 overexpression and might mediate pro-survival effect of FGF2. Interestingly, addition of nuclear localization signal (NLS) to 18K-FGF2 forced its nuclear import and dramatically increased cell proliferation and Akt activation. These findings demonstrated for the first time the molecular mechanisms for FGF2's nuclear import, which promotes GBM cell proliferation and survival, providing novel insights to the development of GBM treatments.

PEITC induces apoptosis of Human Brain Glioblastoma GBM8401 Cells through the extrinsic- and intrinsic -signaling pathways

Glioblastoma is the most common and most aggressive primary brain malignancy. The multimodality treatments for this tumor including surgery, radiotherapy, and chemotherapy, are still not completely satisfied. Phenethyl isothiocyanate (PEITC), one member of the isothiocyanate family, has been shown to induce apoptosis in many human cancer cells. In this study, we investigate the pro-apoptotic effects caused by PETIC in human brain glioblastoma multiforme GBM 8401 cells.In our data, PEITC induced the cell morphological changes and decreased the cell viability of GBM8401 cells in a dose- and time-dependent manner. Moreover, the analysis of cell cycle distribution detected by flow cytometry showed that PEITC induced significantly sub-G1 phase (apoptotic population) in GBM 8401 cells. In addition, PEITC promoted the production of reactive oxygen species (ROS) and increase in [Ca2+]I, but decreased the mitochondrial membrane potential (ΔΨm) in treated cells. PEITC also induced caspases activities in GBM 8401 cells. Results from Western blot analysis indicated that PEITC promoted Fas, FasL, FADD, TRAIL, caspase-8, -9, -3, increased the pro-apoptotic protein (Bax, Bid and Bak), and inhibited the anti-apoptotic proteins (Bcl-2 and Bcl-xl) in GBM 8401 cells. Furthermore, PEITC promoted the release of cytochrome c, AIF and Endo G. GADD153, GRP 78, XBP-1 and IRE-1α, Calpain I and II in GBM 8401 cells. PEITC also promoted the expression of associated protein with endoplasmic reticulum (ER) stress. PEITC induces apoptosis through the extrinsic (death receptor) pathway, dysfunction of mitochondria, ROS induced ER stress, intrinsic (mitochondrial) pathway in GBM 8401 cells. The possible molecular mechanisms and signaling pathways of the anti-cancer properties of PEITC for human brain glioblastoma cells were postulated.

Alkaloid extracts of Ficus species and palm oil-derived tocotrienols synergistically inhibit proliferation of human cancer cells

Tocotrienols have been reported to possess anticancer effects other than anti-inflammatory and antioxidant activities. This study explored the potential synergism of antiproliferative effects induced by individual alkaloid extracts of Ficus fistulosa, Ficus hispida and Ficus schwarzii combined with δ- and γ-tocotrienols against human brain glioblastoma (U87MG), lung adenocarcinoma (A549) and colorectal adenocarcinoma (HT-29) cells. Cell viability and morphological results demonstrated that extracts containing a mixture of alkaloids from the leaves and bark of F. schwarzii inhibited the proliferation of HT-29 cells, whereas the alkaloid extracts of F. fistulosa inhibited the proliferation of both U87MG and HT-29 cells and showed synergism in combined treatments with either δ- or γ-tocotrienol resulting in 2.2–34.7 fold of reduction in IC50 values of tocotrienols. The observed apoptotic cell characteristics in conjunction with the synergistic antiproliferative effects of Ficus species-derived alkaloids and tocotrienols assuredly warrant future investigations towards the development of a value-added chemotherapeutic regimen against cancers.

Lentiviral Vector Mediated Delivery of RHBDD1 shRNA down Regulated the Proliferation of Human Glioblastoma Cells

Rhomboid domain containing 1 (RHBDD1) gene, a new member of rhomboid family of proteins is highly responsible for the regulation of apoptosis by cleaving pro-apoptotic Bcl-2 family protein BIK. Therefore, the higher expression levels of RHBDD1 in cancer tissues may have a direct influence on cancer progression by arresting apoptosis. With this background this study was focused to find out the effect of RHBDD1 silencing on the progression of human brain glioblastoma cells, U251 and U87MG. The results indicated that both cell lines show a higher expression level of RHBDD1 and RNA interference (RNA) mediated gene silencing successfully down regulated the RHBDD1 gene expression. As a result of RHBDD1 silencing the proliferation of both cell types was reduced by over 50%, 5 days after silencing. Moreover the colony formation was completely inhibited and there were no cells present following two week RHBDD1 gene silencing. The cell proliferation was inhibited as a result of cell cycle arrest due to RHBDD1 absence. Therefore, these results clearly indicate that, RHBDD1 is essential for the progression of glioblastoma cells and silencing of it is resulting in significant inhibition of cell cycle progression and cell proliferation. Collectively, this study shows that RHBDD1 gene engineering could be used as an effective tool in malignant brain tumor therapy.

Abstract IA6: Misregulation of pre-mRNA splicing in cancer

Abstract Alternative splicing occurs in transcripts produced from ~95% of human genes. Under normal conditions, this process is tightly regulated, but it can go awry in numerous diseases. Cancer cells, for example, take advantage of this mechanism to produce aberrant proteins with added, deleted, or altered functional domains that contribute to tumorigenesis. Misregulation in cancer occurs frequently by overexpression of splicing regulatory factors, but recent studies have for the first time identified mutated components of the splicing machinery in certain cancers. Here I describe our studies relevant to both these pathways. HnRNP proteins are an example of proteins overexpressed in many cancers. Our previous work defined a pathway whereby expression of three of these, hnRNPA1, A2 and PTB, is upregulated by the cMyc oncogenic transcription factor, which leads to a switch in splicing of the pyruvate kinase M (PKM) pre-mRNA such that a form of PKM necessary for tumor cell proliferation is made. Our more recent studies have identified other pathways that contribute to overexpression of the hnRNPs, and begun to elucidate the role that these proteins play in glioblastoma (GBM). In human GBM patients, expression of these proteins inversely correlates with survival, while in rat and mouse models of GBM, the proteins are significantly overexpressed, and reducing their expression inhibits cell proliferation and tumor formation. Comparing RNA-seq data we obtained following knockdown of the proteins in human U87 GBM cells with known changes in alternative splicing in human GBM brain samples, we identified a number of transcripts, which include PKM, that are direct targets of the hnRNP proteins and relevant to GBM. Examples of these proteins, and the pathways they contribute to, will be discussed. Mutations in the genes encoding components of the RNA splicing machinery have been described in myelodysplastic syndromes (MDS), a group of neoplasms characterized by an abnormality in myeloid blood cell production and a propensity for progression into acute myeloid leukemia. The most frequently mutated genes encode splicing factors SF3B1, a U2 snRNP associated protein, U2AF1, SRSF2 and ZRSR2, all of which function in 3' splice site recognition. Mutational frequencies for SF3B1 are particularly high in refractory anemia with ring sideroblasts, and SF3B1 is also frequently mutated in Chronic Lymphocytic Leukemia as well as in uveal melanoma. All SF3B1 mutations described to date are heterozygous missense mutations. We are studying how mutations in SF3B1 affect the protein's function in splicing, and contribute to MDS. Wild-type and mutant SF3B1 behave identically in a number of splicing-related assays, indicating that MDS mutation does not grossly affect protein function. We are also analyzing RNA samples obtained from MDS patients to identify changes in splicing. Remarkably, we observe a sharp increase in intron retention in patient samples, which will lead to production of truncated proteins and frequently mRNA decay by the NMD pathway. We have identified several candidate genes that may contribute to MDS, including one that produces an enzyme in the heme synthesis pathway that when defective may lead to anemia, and another that encodes a protein important for controlling expansion of hematopoietic stem cells, perhaps resulting in clonal expansion of mutant cells. Citation Format: Jian Zhang, Ritam Neupane, Peter Canoll, Yen Lieu, Siddhartha Mukherjee, Raul Rabadan, Azra Raza, James L. Manley. Misregulation of pre-mRNA splicing in cancer. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr IA6.

Human brain glioblastoma cells do not induce but do respond to the bleomycin-induced bystander response from lung adenocarcinoma cells

To determine whether the bleomycin (BLM)-induced bystander response occurs in human brain glioblastoma (BMG-1) cells, the BMG-1 cells were exposed to two different concentrations of BLM. The co-culture methodology was adopted to study the in vitro bystander effects. DNA damage was measured using the micronucleus (MN) and γ-H2AX assays. Cytotoxicity was measured using the trypan blue assay. Cell cycle kinetics was analyzed using flow cytometry. The overall results did not show any significant increase in either genotoxicity or cytotoxicity or a delay in the cell cycle kinetics in BMG-1 bystander cells co-cultured with BLM-exposed cells, suggesting that BLM did not induce a bystander response in the BMG-1 cells. Furthermore, the MN results of the BLM-exposed BMG-1 cells co-cultured with unexposed bystander human lung adenocarcinoma (A549 and NCI-H460) cells and vice versa suggested that the BMG-1 cells do not secrete bystander signals but do respond to those signals. Analyzing the underlying mechanism and pathways involved in preventing the cells from secreting bystander signals will provide new insights that can be applied to inhibit these mechanisms in other cell types, thereby preventing and controlling the bystander response and genomic instability and increasing the therapeutic gain in chemotherapy.

Intracranial elimination of human glioblastoma brain tumors in a mouse xenograft model using the bispecific targeted toxin DTATEGF and convection enhanced delivery

Objective To investigate the anticancer effect of the bispecific immunotoxin DTATEGF in vitro and in vivo when delivered bv convection-enhanced delivery (CED) via an osmotic minipump in a human glioblastoma brain tumor mouse xenograft model.Methods The effects of the immunotoxins were tested for their ability to inhibit the proliferation of Ln229-luc cells in vitro by methyl thiazol tetrazolium (MTT) assay.On a xenograft intracranial model,1 μg of DTATEGF or eontrol Bickel3 was delivered intracranially by CED via an osmotic minipump.The bioluminescent imaging (BLI) was performed and Kaplan-Meier survival curves were generated.The brain tumor samples were stained by hematoxylin and eosin for histopathological assessment.Results In vitro,DTATEGF could kill Ln229-luc cells and showed an 50％ inhibitory dose(IC50) less than 0.001 nmol/L.In vivo,mice with tumors were treated intracranially with drug via CED where the treatment was successful in providing a survival benefit with the median survival of mice treated with DTATEGF being significantly prolonged relative to controls (85 vs.67 days,P ＜0.01).Conclusion DTATEGF kills the Ln229-luc cell line in vitro,and when it is delivered via CED intracranially,it is highly efficacious against human glioblastoma brain tumors. Key words: Glioblastoma; Immunotherapy; Convection-enhanced delivery; Model,animal

Single cell molecular recognition of migrating and invading tumor cells using a targeted fluorescent probe to receptor PTPmu

Detection of an extracellular cleaved fragment of a cell-cell adhesion molecule represents a new paradigm in molecular recognition and imaging of tumors. We previously demonstrated that probes that recognize the cleaved extracellular domain of receptor protein tyrosine phosphatase mu (PTPmu) label human glioblastoma brain tumor sections and the main tumor mass of intracranial xenograft gliomas. In this article, we examine whether one of these probes, SBK2, can label dispersed glioma cells that are no longer connected to the main tumor mass. Live mice with highly dispersive glioma tumors were injected intravenously with the fluorescent PTPmu probe to test the ability of the probe to label the dispersive glioma cells in vivo. Analysis was performed using a unique three-dimensional (3D) cryo-imaging technique to reveal highly migratory and invasive glioma cell dispersal within the brain and the extent of colabeling by the PTPmu probe. The PTPmu probe labeled the main tumor site and dispersed cells up to 3.5 mm away. The cryo-images of tumors labeled with the PTPmu probe provide a novel, high-resolution view of molecular tumor recognition, with excellent 3D detail regarding the pathways of tumor cell migration. Our data demonstrate that the PTPmu probe recognizes distant tumor cells even in parts of the brain where the blood-brain barrier is likely intact. The PTPmu probe has potential translational significance for recognizing tumor cells to facilitate molecular imaging, a more complete tumor resection and to serve as a molecular targeting agent to deliver chemotherapeutics to the main tumor mass and distant dispersive tumor cells.