Critical roles of chemokine receptor CCR5 in regulating glioblastoma proliferation and invasion.

PubMed

Zhao, Lanfu; Wang, Yuan; Xue, Yafei; Lv, Wenhai; Zhang, Yufu; He, Shiming

2015-11-01

Glioblastoma (GBM) is the most prevalent malignant primary brain tumor in adults and exhibits a spectrum of aberrantly aggressive phenotype. Tumor cell proliferation and invasion are critically regulated by chemokines and their receptors. Recent studies have shown that the chemokine CCL5 and its receptor CCR5 play important roles in tumor invasion and metastasis. Nonetheless, the roles of the CCR5 in GBM still remain unclear. The present study provides the evidence that the chemokine receptor CCR5 is highly expressed and associated with poor prognosis in human GBM. Mechanistically, CCL5-CCR5 mediates activation of Akt, and subsequently induces proliferation and invasive responses in U87 and U251 cells. Moreover, down-regulation of CCR5 significantly inhibited the growth of glioma in U87 tumor xenograft mouse model. Finally, high CCR5 expression in GBM is correlated with increased p-Akt expression in patient samples. Together, these findings suggest that the CCR5 is a critical molecular event associated with gliomagenesis. © The Author 2015. Published by ABBS Editorial Office in association with Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences.

Downregulation of ZMYND11 induced by miR-196a-5p promotes the progression and growth of GBM.

PubMed

Yang, Ji-Peng; Yang, Jian-Kai; Li, Chen; Cui, Zhi-Qiang; Liu, Hong-Jiang; Sun, Xiao-Feng; Geng, Shao-Mei; Lu, Sheng-Kui; Song, Jian; Guo, Cheng-Yong; Jiao, Bao-Hua

2017-12-16

ZMYND11 (zinc finger MYND-type containing 11) has been widely regarded to be involved in a variety of cancers as a potential suppressor. However, the biological role and mechanism of ZMYND11 in glioblastoma multiform (GBM) remain unknown. In this study, we found that ZMYND11 expression was remarkably decreased in GBM tissues from 20 cases and cell line (U87) compared to normal brain tissue from 10 cases (P < 0.001). Furthermore, we explored that ZMYND11 upregulation significantly suppressed U87 cells proliferation and invasion, induced cell cycle arrest and apoptosis in vitro. Subsequently, we identified increased ZMYND11 inhibited the tumor growth using tumor cells xenograft experiment on rude mice. Moreover, we explored that ZMYND11 was a new direct and functional target of miR-196a-5p in U87 via luciferase reporter assay. In addition, we confirmed the negative correlation between miR-196a-5p and ZMYND11 in GBM tissue and U87 cells by changing the expression level of miR-196a-5p with lentivirus and plasmid vector. Furthermore, we demonstrated that decreased ZMYND11 could reverse suppressive effect of downregulated miR-196a-5p on U87 by rescue experiment. Taken together, ZMYND11 was demonstrated to be a potential and extremely promising suppressor of GBM, while miRNA-196a-5p was quite an important target of treatment of GBM. Copyright © 2017 Elsevier Inc. All rights reserved.

Oncolytic vesicular stomatitis virus induces apoptosis in U87 glioblastoma cells by a type II death receptor mechanism and induces cell death and tumor clearance in vivo.

PubMed

Cary, Zachary D; Willingham, Mark C; Lyles, Douglas S

2011-06-01

Vesicular stomatitis virus (VSV) is a potential oncolytic virus for treating glioblastoma multiforme (GBM), an aggressive brain tumor. Matrix (M) protein mutants of VSV have shown greater selectivity for killing GBM cells versus normal brain cells than VSV with wild-type M protein. The goal of this research was to determine the contribution of death receptor and mitochondrial pathways to apoptosis induced by an M protein mutant (M51R) VSV in U87 human GBM tumor cells. Compared to controls, U87 cells expressing a dominant negative form of Fas (dnFas) or overexpressing Bcl-X(L) had reduced caspase-3 activation following infection with M51R VSV, indicating that both the death receptor pathway and mitochondrial pathways are important for M51R VSV-induced apoptosis. Death receptor signaling has been classified as type I or type II, depending on whether signaling is independent (type I) or dependent on the mitochondrial pathway (type II). Bcl-X(L) overexpression inhibited caspase activation in response to a Fas-inducing antibody, similar to the inhibition in response to M51R VSV infection, indicating that U87 cells behave as type II cells. Inhibition of apoptosis in vitro delayed, but did not prevent, virus-induced cell death. Murine xenografts of U87 cells that overexpress Bcl-X(L) regressed with a time course similar to that of control cells following treatment with M51R VSV, and tumors were not detectable at 21 days postinoculation. Immunohistochemical analysis demonstrated similar levels of viral antigen expression but reduced activation of caspase-3 following virus treatment of Bcl-X(L)-overexpressing tumors compared to controls. Further, the pathological changes in tumors following treatment with virus were quite different in the presence versus the absence of Bcl-X(L) overexpression. These results demonstrate that M51R VSV efficiently induces oncolysis in GBM tumor cells despite deregulation of apoptotic pathways, underscoring its potential use as a treatment for GBM.

Celecoxib enhances radiosensitivity of hypoxic glioblastoma cells through endoplasmic reticulum stress

PubMed Central

Suzuki, Kenshi; Gerelchuluun, Ariungerel; Hong, Zhengshan; Sun, Lue; Zenkoh, Junko; Moritake, Takashi; Tsuboi, Koji

2013-01-01

Background Refractoriness of glioblastoma multiforme (GBM) largely depends on its radioresistance. We investigated the radiosensitizing effects of celecoxib on GBM cell lines under both normoxic and hypoxic conditions. Methods Two human GBM cell lines, U87MG and U251MG, and a mouse GBM cell line, GL261, were treated with celecoxib or γ-irradiation either alone or in combination under normoxic and hypoxic conditions. Radiosensitizing effects were analyzed by clonogenic survival assays and cell growth assays and by assessing apoptosis and autophagy. Expression of apoptosis-, autophagy-, and endoplasmic reticulum (ER) stress–related genes was analyzed by immunoblotting. Results Celecoxib significantly enhanced the radiosensitivity of GBM cells under both normoxic and hypoxic conditions. In addition, combined treatment with celecoxib and γ-irradiation induced marked autophagy, particularly in hypoxic cells. The mechanism underlying the radiosensitizing effect of celecoxib was determined to be ER stress loading on GBM cells. Conclusion Celecoxib enhances the radiosensitivity of GBM cells by a mechanism that is different from cyclooxygenase-2 inhibition. Our results indicate that celecoxib may be a promising radiosensitizing drug for clinical use in patients with GBM. PMID:23658321

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

PubMed

Kim, Boram; Jung, Narae; Lee, Sanghun; Sohng, Jae Kyung; Jung, Hye Jin

2016-11-01

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. Copyright © 2016 John Wiley & Sons, Ltd.

MicroRNA‑518b functions as a tumor suppressor in glioblastoma by targeting PDGFRB.

PubMed

Xu, Xiaolong; Zhang, Fenglin; Chen, Xianzhen; Ying, Qi

2017-10-01

Glioblastoma (GBM) is the most common and aggressive type of primary human brain tumor in China. Dysregulated microRNA (miRNA/miR) expression has been hypothesized to serve a role in the tumorigenesis and progression of human GBM. To explore the potential mechanisms affecting GBM tumorigenesis, the function of miR‑518b in regulating GBM cell proliferation and angiogenesis was examined in vitro by CCK‑8 and tube formation assay and in vivo by xenograft assay. The present study demonstrated that the expression of miR‑518b was downregulated in GBM tissues and in GBM cell lines (U87 and U251). In addition, the expression levels of miR‑518b were highly associated with tumor size, World Health Organization grade and prognosis. Furthermore, overexpression of miR‑518b suppressed GBM cell proliferation and angiogenesis, and induced GBM cell apoptosis in vitro and in vivo. Overexpression of miR‑518b also inhibited the expression of platelet‑derived growth factor receptor β (PDGFRB), and the present study confirmed that the 3' untranslated region (3'UTR) of PDGFRB was a direct target of miR‑518b. In conclusion, to the best of our knowledge, the present study is the first to present evidence suggesting that miR‑518b may serve as a potential marker and target in GBM treatment.

MicroRNA-518b functions as a tumor suppressor in glioblastoma by targeting PDGFRB

PubMed Central

Xu, Xiaolong; Zhang, Fenglin; Chen, Xianzhen; Ying, Qi

2017-01-01

Glioblastoma (GBM) is the most common and aggressive type of primary human brain tumor in China. Dysregulated microRNA (miRNA/miR) expression has been hypothesized to serve a role in the tumorigenesis and progression of human GBM. To explore the potential mechanisms affecting GBM tumorigenesis, the function of miR-518b in regulating GBM cell proliferation and angiogenesis was examined in vitro by CCK-8 and tube formation assay and in vivo by xenograft assay. The present study demonstrated that the expression of miR-518b was downregulated in GBM tissues and in GBM cell lines (U87 and U251). In addition, the expression levels of miR-518b were highly associated with tumor size, World Health Organization grade and prognosis. Furthermore, overexpression of miR-518b suppressed GBM cell proliferation and angiogenesis, and induced GBM cell apoptosis in vitro and in vivo. Overexpression of miR-518b also inhibited the expression of platelet-derived growth factor receptor β (PDGFRB), and the present study confirmed that the 3′ untranslated region (3′UTR) of PDGFRB was a direct target of miR-518b. In conclusion, to the best of our knowledge, the present study is the first to present evidence suggesting that miR-518b may serve as a potential marker and target in GBM treatment. PMID:28849154

Cytotoxic human peripheral blood-derived γδT cells kill glioblastoma cell lines: implications for cell-based immunotherapy for patients with glioblastoma.

PubMed

Nakazawa, Tsutomu; Nakamura, Mitsutoshi; Park, Young Soo; Motoyama, Yasushi; Hironaka, Yasuo; Nishimura, Fumihiko; Nakagawa, Ichiro; Yamada, Shuichi; Matsuda, Ryosuke; Tamura, Kentaro; Sugimoto, Tadashi; Takeshima, Yasuhiro; Marutani, Akiko; Tsujimura, Takahiro; Ouji, Noriko; Ouji, Yukiteru; Yoshikawa, Masahide; Nakase, Hiroyuki

2014-01-01

Glioblastoma (GBM) is a highly aggressive brain tumor for which novel therapeutic approaches, such as immunotherapy, are urgently needed. Zoledronate (ZOL), an inhibitor of osteoclastic activity, is known to stimulate peripheral blood-derived γδT cells and sensitize tumors to γδT cell-mediated killing. To investigate the feasibility of γδT cell-based immunotherapy for patients with GBM, we focused on the killing of GBM cell lines by γδT cells and the molecular mechanisms involved in these cell-cell interactions. Peripheral blood mononuclear cells were expanded in ZOL and interleukin (IL)-2 for 14 days, and γδT cells were enriched in the expanded cells by the immunomagnetic depletion of αβT cells. Gliomas are resistant to NK cells but susceptible to lymphokine-activated killer cells and some cytotoxic T lymphocytes. When the γδT cell-mediated killing of three GBM cell lines (U87MG, U138MG and A172 cells) and an NK-sensitive leukemia cell line (K562 cells) were tested, 32% U87MG, 15% U138MG, 1% A172, and 50% K562 cells were killed at an effector:target ratio of 5:1. The γδT cell-mediated killing of all three GBM cell lines was significantly enhanced by ZOL and this ZOL-enhanced killing was blocked by an anti-T cell receptor (TcR) antibody. These results indicated that TcR γδ is crucial for the recognition of ZOL-treated GBM cells by γδT cells. Since the low level killing of GBM cells by the γδT cells was enhanced by ZOL, γδT cell-targeting therapy in combination with ZOL treatment could be effective for patients with GBM.

Physiological oxygen concentration alters glioma cell malignancy and responsiveness to photodynamic therapy in vitro.

PubMed

Albert, Ina; Hefti, Martin; Luginbuehl, Vera

2014-11-01

The partial pressure of oxygen (pO2) in brain tumors ranges from 5 to 15%. Nevertheless, the majority of in vitro experiments with glioblastoma multiforme (GBM) cell lines are carried out under an atmospheric pO2 of 19 to 21%. Recently, 5-aminolevulinic acid (5-ALA), a precursor of protoporphyrin IX (PpIX), has been introduced to neurosurgery to allow for photodynamic diagnosis and photodynamic therapy (PDT) in high-grade gliomas. Here, we investigate whether low pO2 affects GBM cell physiology, PpIX accumulation, or PDT efficacy. GBM cell lines (U-87 MG and U-251 MG) were cultured under atmospheric (pO2  =  19%) and physiological (pO2  =  9%) oxygen concentrations. PpIX accumulation and localization were investigated, and cell survival and cell death were observed following in vitro PDT. A physiological pO2 of 9% stimulated GBM cell migration, increased hypoxia-inducible factor (HIF)-1 alpha levels, and elevated resistance to camptothecin in U-87 MG cells compared to cultivation at a pO2 of 19%. This oxygen reduction did not alter 5-ALA-induced intracellular PpIX accumulation. However, physiological pO2 changed the responsiveness of U-87 MG but not of U-251 MG cells to in vitro PDT. Around 20% more irradiation light was required to kill U-87 MG cells at physiological pO2, resulting in reduced lactate dehydrogenase (LDH) release (one- to two-fold) and inhibition of caspase 3 activation. Reduction of oxygen concentration from atmospheric to a more physiological level can influence the malignant behavior and survival of GBM cell lines after in vitro PDT. Therefore, precise oxygen concentration control should be considered when designing and performing experiments with GBM cells.

Gene therapy for human glioblastoma using neurotropic JC virus-like particles as a gene delivery vector.

PubMed

Chao, Chun-Nun; Yang, Yu-Hsuan; Wu, Mu-Sheng; Chou, Ming-Chieh; Fang, Chiung-Yao; Lin, Mien-Chun; Tai, Chien-Kuo; Shen, Cheng-Huang; Chen, Pei-Lain; Chang, Deching; Wang, Meilin

2018-02-02

Glioblastoma multiforme (GBM), the most common malignant brain tumor, has a short period of survival even with recent multimodality treatment. The neurotropic JC polyomavirus (JCPyV) infects glial cells and oligodendrocytes and causes fatal progressive multifocal leukoencephalopathy in patients with AIDS. In this study, a possible gene therapy strategy for GBM using JCPyV virus-like particles (VLPs) as a gene delivery vector was investigated. We found that JCPyV VLPs were able to deliver the GFP reporter gene into tumor cells (U87-MG) for expression. In an orthotopic xenograft model, nude mice implanted with U87 cells expressing the near-infrared fluorescent protein and then treated by intratumoral injection of JCPyV VLPs carrying the thymidine kinase suicide gene, combined with ganciclovir administration, exhibited significantly prolonged survival and less tumor fluorescence during the experiment compared with controls. Furthermore, JCPyV VLPs were able to protect and deliver a suicide gene to distal subcutaneously implanted U87 cells in nude mice via blood circulation and inhibit tumor growth. These findings show that metastatic brain tumors can be targeted by JCPyV VLPs carrying a therapeutic gene, thus demonstrating the potential of JCPyV VLPs to serve as a gene therapy vector for the far highly treatment-refractory GBM.

Erythropoietin Augments Survival of Glioma Cells After Radiation and Temozolomide

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hassouna, Imam; Sperling, Swetlana; Kim, Ella

2008-11-01

Purpose: Despite beneficial effects of irradiation/chemotherapy on survival of glioblastoma (GBM) patients, collateral damage to intact neural tissue leads to 'radiochemobrain' and reduced quality of life in survivors. For prophylactic neuroprotection, erythropoietin (EPO) is a promising candidate, provided that concerns regarding potential tumor promoting effects are alleviated. Methods and Materials: Human GBM-derived cell lines U87, G44, G112, and the gliosarcoma-derived line G28 were treated with EPO, with and without combinations of irradiation or temozolomide (TMZ). Responsiveness of glioma cells to EPO was measured by cell migration from spheroids, cell proliferation, and clonogenic survival. Implantation of U87 cells into brains ofmore » nude mice, followed 5 days later by EPO treatment (5,000 U/kg intraperitoneal every other day for 2 weeks) should reveal effects of EPO on tumor growth in vivo. Reverse transcriptase-polymerase chain reaction was performed for EPOR, HIF-1{alpha}, and epidermal growth factor receptor (EGFR)vIII in cell lines and 22 human GBM specimens. Results: EPO did not modulate basal glioma cell migration and stimulated proliferation in only one of four cell lines. Importantly, EPO did not enhance tumor growth in mouse brains. Preincubation of glioma cells with EPO for 3 h, followed by irradiation and TMZ for another 24 h, resulted in protection against chemoradiation-induced cytotoxicity in three cell lines. Conversely, EPO induced a dose-dependent decrease in survival of G28 gliosarcoma cells. In GBM specimens, expression of HIF-1{alpha} correlated positively with expression of EPOR and EGFRvIII. EPOR and EGFRvIII expression did not correlate. Conclusions: EPO is unlikely to appreciably influence basal glioma growth. However, concomitant use of EPO with irradiation/chemotherapy in GBM patients is not advisable.« less

Targeting TWIST1 through loss of function inhibits tumorigenicity of human glioblastoma.

PubMed

Mikheev, Andrei M; Mikheeva, Svetlana A; Severs, Liza J; Funk, Cory C; Huang, Lei; McFaline-Figueroa, José L; Schwensen, Jeanette; Trapnell, Cole; Price, Nathan D; Wong, Stephen; Rostomily, Robert C

2018-05-13

Twist1 (TW) is a bHLH transcription factor (TF) and master regulator of the epithelial to mesenchymal transition (EMT). In vitro, TW promotes mesenchymal change, invasion and self-renewal in glioblastoma (GBM) cells. However the potential therapeutic relevance of TW has not been established through loss of function studies in human GBM cell xenograft models. The effects of TW loss of function (gene editing and knock down) on inhibition of tumorigenicity of U87MG and GBM4 glioma stem cells were tested in orthotopic xenograft models and conditional knockdown in established flank xenograft tumors. RNAseq and the analysis of tumors investigated putative TW associated mechanisms. Multiple bioinformatics tools revealed significant alteration of ECM, membrane receptors, signaling transduction kinases and cytoskeleton dynamics leading to identification of PI3K/AKT signaling. We experimentally show alteration of AKT activity and periostin (POSTN) expression in vivo and/or in vitro. For the first time we show that effect of TW knockout inhibits AKT activity in U87MG cells in vivo independent of PTEN mutation. The clinical relevance of TW and candidate mechanisms was established by analysis of the TCGA and ENCODE databases. TW expression was associated with decreased patient survival and LASSO regression analysis identified POSTN as one of top targets of TW in human GBM. While we previously demonstrated the role of TW in promoting EMT and invasion of glioma cells, these studies provide direct experimental evidence supporting pro-tumorigenic role of TW independent of invasion in vivo and the therapeutic relevance of targeting TW in human GBM. Further, the role of TW driving POSTN expression and AKT signaling suggests actionable targets, which could be leveraged to mitigate the oncogenic effects of TW in GBM. Molecular Oncology (2018) © 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.

Saponin 6 derived from Anemone taipaiensis induces U87 human malignant glioblastoma cell apoptosis via regulation of Fas and Bcl‑2 family proteins.

PubMed

Ji, Chen-Chen; Tang, Hai-Feng; Hu, Yi-Yang; Zhang, Yun; Zheng, Min-Hua; Qin, Hong-Yan; Li, San-Zhong; Wang, Xiao-Yang; Fei, Zhou; Cheng, Guang

2016-07-01

Glioblastoma multiforme (GBM) is the most common and aggressive type of brain tumor, and is associated with a poor prognosis. Saponin 6, derived from Anemone taipaiensis, exerts potent cytotoxic effects against the human hepatocellular carcinoma HepG2 cell line and the human promyelocytic leukemia HL‑60 cell line; however, the effects of saponin 6 on glioblastoma remain unknown. The present study aimed to evaluate the effects of saponin 6 on human U87 malignant glioblastoma (U87 MG) cells. The current study revealed that saponin 6 induced U87 MG cell death in a dose‑ and time‑dependent manner, with a half maximal inhibitory concentration (IC50) value of 2.83 µM after treatment for 48 h. However, saponin 6 was needed to be used at a lesser potency in HT‑22 cells, with an IC50 value of 6.24 µM. Cell apoptosis was assessed by flow cytometry using Annexin V‑fluorescein isothiocyanate/propidium iodide double staining. DNA fragmentation and alterations in nuclear morphology were examined by terminal deoxynucleotidyl transferase‑mediated dUTP nick end labeling and transmission electron microscopy, respectively. The present study demonstrated that treatment with saponin 6 induced cell apoptosis in U87 MG cells, and resulted in DNA fragmentation and nuclear morphological alterations typical of apoptosis. In addition, flow cytometric analysis revealed that saponin 6 was able to induce cell cycle arrest. The present study also demonstrated that saponin 6‑induced apoptosis of U87 MG cells was attributed to increases in the protein expression levels of Fas, Fas ligand, and cleaved caspase‑3, ‑8 and ‑9, and decreases in the levels of B‑cell lymphoma 2. The current study indicated that saponin 6 may exhibit selective cytotoxicity toward U87 MG cells by activating apoptosis via the extrinsic and intrinsic pathways. Therefore, saponin 6 derived from A. taipaiensis may possess therapeutic potential for the treatment of GBM.

Resveratrol Targets AKT and p53 in Glioblastoma and Glioblastoma Stem-like Cells to Suppress Growth and Infiltration

PubMed Central

Clark, Paul A.; Bhattacharya, Saswati; Elmayan, Ardem; Darjatmoko, Soesiawati R.; Thuro, Bradley A.; Yan, Michael B.; van Ginkel, Paul R.; Polans, Arthur S.; Kuo, John S.

2016-01-01

Object Glioblastoma multiforme (GBM) is an aggressive brain cancer with median survival of less than two years with current treatment. GBM exhibits extensive intra-tumor and inter-patient heterogeneity, suggesting that successful therapies should exert broad anti-cancer activities. Therefore, the natural non-toxic pleiotropic agent, resveratrol, was studied for anti-tumorigenic effects against GBM. Methods Resveratrol’s effects on cell proliferation, sphere-forming ability, and invasion were tested using multiple patient-derived GBM stem-like cell (GSC) lines and established U87 glioma cells, and changes in oncogenic AKT and tumor suppressive p53 were analyzed. Resveratrol was also tested in vivo against U87 glioma flank xenografts using multiple delivery methods, including direct tumor injection. Finally, resveratrol was delivered directly to brain tissue to determine toxicity and achievable drug concentrations in the brain parenchyma. Results Resveratrol significantly inhibited proliferation in U87 glioma and multiple patient-derived GSC lines, demonstrating similar inhibitory concentrations across these phenotypically heterogeneous lines. Resveratrol also inhibited the sphere-forming ability of GSCs, suggesting anti-stem cell effects. Additionally, resveratrol blocked U87 glioma and GSC invasion in an in vitro Matrigel transwell assay at doses similar to those mediating anti-proliferative effects. In U87 glioma cells and GSCs, resveratrol reduced AKT phosphorylation and induced p53 expression and activation that led to transcription of downstream p53 target genes. Resveratrol administration via oral gavage or ad libitum in the water supply significantly suppressed GBM xenograft growth; intra-tumor or peri-tumor resveratrol injection further suppressed growth and approximating tumor regression. Intracranial resveratrol injection resulted in 100-fold higher local drug concentration compared to intravenous delivery, and with no apparent toxicity. Conclusions Resveratrol potently inhibited GBM and GBM stem-like cell growth and infiltration, acting partially via AKT deactivation and p53 induction, and suppressed glioblastoma growth in vivo. The ability of resveratrol to modulate AKT and p53, as well as reportedly many other anti-tumorigenic pathways, is attractive for therapy against a genetically heterogeneous tumor such as GBM. Although resveratrol exhibits low bioavailability when administered orally or intravenously, novel delivery methods such as direct injection (i.e. convection enhanced delivery) could potentially be used to achieve and maintain therapeutic doses in brain. Resveratrol’s non-toxic nature and broad anti-GBM effects make it a compelling candidate to supplement current GBM therapies. PMID:27419830

AshwaMAX and Withaferin A inhibits gliomas in cellular and murine orthotopic models

PubMed Central

Pohling, Christoph; Natarajan, Arutselvan; Witney, Timothy H.; Kaur, Jasdeep; Xu, Lingyun; Gowrishankar, Gayatri; D’Souza, Aloma L; Murty, Surya; Schick, Sophie; Chen, Liyin; Wu, Nicholas; Khaw, Phoo; Mischel, Paul; Abbasi, Taher; Usmani, Shahabuddin; Mallick, Parag

2017-01-01

Glioblastoma multiforme (GBM) is an aggressive, malignant cancer Johnson and O’Neill (J Neurooncol 107: 359–364, 2012). An extract from the winter cherry plant (Withania somnifera), AshwaMAX, is concentrated (4.3 %) for Withaferin A; a steroidal lactone that inhibits cancer cells Vanden Berghe et al. (Cancer Epidemiol Biomark Prev 23: 1985–1996, 2014). We hypothesized that AshwaMAX could treat GBM and that bioluminescence imaging (BLI) could track oral therapy in orthotopic murine models of glioblastoma. Human parietal-cortical glioblastoma cells (GBM2, GBM39) were isolated from primary tumors while U87-MG was obtained commercially. GBM2 was transduced with lentiviral vectors that express Green Fluorescent Protein (GFP)/firefly luciferase fusion proteins. Mutational, expression and proliferative status of GBMs were studied. Intracranial xenografts of glioblastomas were grown in the right frontal regions of female, nude mice (n = 3–5 per experiment). Tumor growth was followed through BLI. Neurosphere cultures (U87-MG, GBM2 and GBM39) were inhibited by AshwaMAX at IC50 of 1.4, 0.19 and 0.22 μM equivalent respectively and by Withaferin A with IC50 of 0.31, 0.28 and 0.25 μM respectively. Oral gavage, every other day, of AshwaMAX (40 mg/kg per day) significantly reduced bioluminescence signal (n = 3 mice, p < 0.02, four parameter non-linear regression analysis) in preclinical models. After 30 days of treatment, bioluminescent signal increased suggesting onset of resistance. BLI signal for control, vehicle-treated mice increased and then plateaued. Bioluminescent imaging revealed diffuse growth of GBM2 xenografts. With AshwaMAX, GBM neurospheres collapsed at nanomolar concentrations. Oral treatment studies on murine models confirmed that AshwaMAX is effective against orthotopic GBM. AshwaMAX is thus a promising candidate for future clinical translation in patients with GBM. PMID:26650066

AshwaMAX and Withaferin A inhibits gliomas in cellular and murine orthotopic models.

PubMed

Chang, Edwin; Pohling, Christoph; Natarajan, Arutselvan; Witney, Timothy H; Kaur, Jasdeep; Xu, Lingyun; Gowrishankar, Gayatri; D'Souza, Aloma L; Murty, Surya; Schick, Sophie; Chen, Liyin; Wu, Nicholas; Khaw, Phoo; Mischel, Paul; Abbasi, Taher; Usmani, Shahabuddin; Mallick, Parag; Gambhir, Sanjiv S

2016-01-01

Glioblastoma multiforme (GBM) is an aggressive, malignant cancer Johnson and O'Neill (J Neurooncol 107: 359-364, 2012). An extract from the winter cherry plant (Withania somnifera ), AshwaMAX, is concentrated (4.3 %) for Withaferin A; a steroidal lactone that inhibits cancer cells Vanden Berghe et al. (Cancer Epidemiol Biomark Prev 23: 1985-1996, 2014). We hypothesized that AshwaMAX could treat GBM and that bioluminescence imaging (BLI) could track oral therapy in orthotopic murine models of glioblastoma. Human parietal-cortical glioblastoma cells (GBM2, GBM39) were isolated from primary tumors while U87-MG was obtained commercially. GBM2 was transduced with lentiviral vectors that express Green Fluorescent Protein (GFP)/firefly luciferase fusion proteins. Mutational, expression and proliferative status of GBMs were studied. Intracranial xenografts of glioblastomas were grown in the right frontal regions of female, nude mice (n = 3-5 per experiment). Tumor growth was followed through BLI. Neurosphere cultures (U87-MG, GBM2 and GBM39) were inhibited by AshwaMAX at IC50 of 1.4, 0.19 and 0.22 µM equivalent respectively and by Withaferin A with IC50 of 0.31, 0.28 and 0.25 µM respectively. Oral gavage, every other day, of AshwaMAX (40 mg/kg per day) significantly reduced bioluminescence signal (n = 3 mice, p < 0.02, four parameter non-linear regression analysis) in preclinical models. After 30 days of treatment, bioluminescent signal increased suggesting onset of resistance. BLI signal for control, vehicle-treated mice increased and then plateaued. Bioluminescent imaging revealed diffuse growth of GBM2 xenografts. With AshwaMAX, GBM neurospheres collapsed at nanomolar concentrations. Oral treatment studies on murine models confirmed that AshwaMAX is effective against orthotopic GBM. AshwaMAX is thus a promising candidate for future clinical translation in patients with GBM.

Targeted Antiepidermal Growth Factor Receptor (Cetuximab) Immunoliposomes Enhance Cellular Uptake In Vitro and Exhibit Increased Accumulation in an Intracranial Model of Glioblastoma Multiforme

PubMed Central

Mortensen, Joachim Høg

2013-01-01

Therapeutic advances do not circumvent the devastating fact that the survival rate in glioblastoma multiforme (GBM) is less than 5%. Nanoparticles consisting of liposome-based therapeutics are provided against a variety of cancer types including GBM, but available liposomal formulations are provided without targeting moieties, which increases the dosing demands to reach therapeutic concentrations with risks of side effects. We prepared PEGylated immunoliposomes (ILs) conjugated with anti-human epidermal growth factor receptor (EGFR) antibodies Cetuximab (α-hEGFR-ILs). The affinity of the α-hEGFR-ILs for the EGF receptor was evaluated in vitro using U87 mg and U251 mg cells and in vivo using an intracranial U87 mg xenograft model. The xenograft model was additionally analyzed with respect to permeability to endogenous albumin, tumor size, and vascularization. The in vitro studies revealed significantly higher binding of α-hEGFR-ILs when compared with liposomes conjugated with isotypic nonimmune immunoglobulin. The uptake and internalization of the α-hEGFR-ILs by U87 mg cells were further confirmed by 3D deconvolution analyses. In vivo, the α-hEGFR-ILs accumulated to a higher extent inside the tumor when compared to nonimmune liposomes. The data show that α-hEGFR-ILs significantly enhance the uptake and accumulation of liposomes in this experimental model of GBM suggestive of improved specific nanoparticle-based delivery. PMID:24175095

A tumor-targeting p53 nanodelivery system limits chemoresistance to temozolomide prolonging survival in a mouse model of glioblastoma multiforme.

PubMed

Kim, Sang-Soo; Rait, Antonina; Kim, Eric; Pirollo, Kathleen F; Chang, Esther H

2015-02-01

Development of temozolomide (TMZ) resistance contributes to the poor prognosis for glioblastoma multiforme (GBM) patients. It was previously demonstrated that delivery of exogenous wild-type tumor suppressor gene p53 via a tumor-targeted nanocomplex (SGT-53) which crosses the blood-brain barrier could sensitize highly TMZ-resistant GBM tumors to TMZ. Here we assessed whether SGT-53 could inhibit development of TMZ resistance. SGT-53 significantly chemosensitized TMZ-sensitive human GBM cell lines (U87 and U251), in vitro and in vivo. Furthermore, in an intracranial GBM tumor model, two cycles of concurrent treatment with systemically administered SGT-53 and TMZ inhibited tumor growth, increased apoptosis and most importantly, significantly prolonged median survival. In contrast TMZ alone had no significant effect on median survival compared to a single cycle of TMZ. These results suggest that combining SGT-53 with TMZ appears to limit development of TMZ resistance, prolonging its anti-tumor effect and could be a more effective therapy for GBM. Using human glioblastoma multiforma cell lines, this research team demonstrated that the delivery of exogenous wild-type tumor suppressor gene p53 via a tumor-targeted nanocomplex limited the development of temozolomide resistance and prolonged its anti-tumor effect, which may enable future human application of this or similar techniques. Copyright © 2015 Elsevier Inc. All rights reserved.

Loss of MYC and E-box3 binding contributes to defective MYC-mediated transcriptional suppression of human MC-let-7a-1~let-7d in glioblastoma

PubMed Central

Wang, Zifeng; Lin, Sheng; Zhang, Ji; Xu, Zhenhua; Xiang, Yu; Yao, Hong; Ge, Lei; Xie, Dan; Kung, Hsiang-fu; Lu, Gang; Poon, Wai Sang; Liu, Quentin; Lin, Marie Chia-mi

2016-01-01

Previously, we reported that MYC oncoprotein down-regulates the transcription of human MC-let-7a-1~let-7d microRNA cluster in hepatocarcinoma (HCC). Surprisingly, in silico analysis indicated that let-7 miRNA expression levels are not reduced in glioblastoma (GBM). Here we investigated the molecular basis of this differential expression. Using human GBM U87 and U251 cells, we first demonstrated that forced over-expression of MYC indeed could not down-regulate the expression of human MC-let-7a-1~let-7d microRNA cluster in GBM. Furthermore, analysis of MC-let-7a-1~let-7d promoter in GBM indicated that MYC failed to inhibit the promoter activity. Pearson's correlation and Linear Regression analysis using the expression data from GSE55092 (HCC) and GSE4290 (GBM) demonstrated a converse relationship of MC-let-7a-1~let-7d and MYC only in HCC but not in GBM. To understand the underlying mechanisms, we examined whether MYC could bind to the non-canonical E-box 3 located in the promoter of MC-let-7a-1~let-7d. Results from both chromatin immune-precipitation (ChIP) and super-shift assays clearly demonstrated the loss of MYC and E-box 3 binding in GBM, suggesting for the first time that a defective MYC and E-box3 binding in GBM is responsible for the differential MYC mediated transcriptional inhibition of MC-let-7a-1~let-7d and potentially other tumor suppressors. MYC and let-7 are key oncoprotein and tumor suppressor, respectively. Understanding the molecular mechanisms of their regulations will provide new insight and have important implications in the therapeutics of GBM as well as other cancers. PMID:27409345

MiR-26a enhances the radiosensitivity of glioblastoma multiforme cells through targeting of ataxia–telangiectasia mutated

DOE Office of Scientific and Technical Information (OSTI.GOV)

Guo, Pin; Lan, Jin; Ge, Jianwei

Glioblastoma multiforme (GBM) is notoriously resistant to radiation, and consequently, new radiosensitizers are urgently needed. MicroRNAs are a class of endogenous gene modulators with emerging roles in DNA repair. We found that overexpression of miR-26a can enhance radiosensitivity and reduce the DNA repair ability of U87 cells. However, knockdown miR-26a in U87 cells could act the converse manner. Mechanistically, this effect is mediated by direct targeting of miR-26a to the 3′UTR of ATM, which leads to reduced ATM levels and consequent inhibition of the homologous recombination repair pathway. These results suggest that miR-26a may act as a new radiosensitizer ofmore » GBM. - Highlights: ●miR-26a directly target ATM in GBM cells. ●miR-26a enhances the radiosensitivity of GBM cells. ●miR-26a could reduce the DNA repair capacity of GBM cells.« less

Low oxygen tension reverses antineoplastic effect of iron chelator deferasirox in human glioblastoma cells.

PubMed

Legendre, Claire; Avril, Sylvie; Guillet, Catherine; Garcion, Emmanuel

2016-02-01

Overcoming resistance to treatment is an essential issue in many cancers including glioblastoma (GBM), the deadliest primary tumor of the central nervous system. As dependence on iron is a key feature of tumor cells, using chelators to reduce iron represents an opportunity to improve conventional GBM therapies. The aim of the present study was, therefore, to investigate the cytostatic and cytotoxic impact of the new iron chelator deferasirox (DFX) on human GBM cells in well-defined clinical situations represented by radiation therapy and mild-hypoxia. Under experimental normoxic condition (21% O2), deferasirox (DFX) used at 10 μM for 3 days reduced proliferation, led cell cycle arrest in S and G2-M phases and induced cytotoxicity and apoptosis in U251 and U87 GBM cells. The abolition of the antineoplastic DFX effects when cells were co-treated with ferric ammonium sulfate supports the hypothesis that its effects result from its ability to chelate iron. As radiotherapy is the main treatment for GBM, the combination of DFX and X-ray beam irradiation was also investigated. Irradiation at a dose of 16 Gy repressed proliferation, cytotoxicity and apoptosis, but only in U251 cells, while no synergy with DFX was observed in either cell line. Importantly, when the same experiment was conducted in mild-hypoxic conditions (3% O2), the antiproliferative and cytotoxic effects of DFX were abolished, and its ability to deplete iron was also impaired. Taken together, these in vitro results could raise the question of the benefit of using iron chelators in their native forms under the hypoxic conditions often encountered in solid tumors such as GBM. Developing new chemistry or a new drug delivery system that would keep DFX active in hypoxic cells may be the next step toward their application.

Identification of radiation responsive genes and transcriptome profiling via complete RNA sequencing in a stable radioresistant U87 glioblastoma model.

PubMed

Doan, Ninh B; Nguyen, Ha S; Alhajala, Hisham S; Jaber, Basem; Al-Gizawiy, Mona M; Ahn, Eun-Young Erin; Mueller, Wade M; Chitambar, Christopher R; Mirza, Shama P; Schmainda, Kathleen M

2018-05-04

The absence of major progress in the treatment of glioblastoma (GBM) is partly attributable to our poor understanding of both GBM tumor biology and the acquirement of treatment resistance in recurrent GBMs. Recurrent GBMs are characterized by their resistance to radiation. In this study, we used an established stable U87 radioresistant GBM model and total RNA sequencing to shed light on global mRNA expression changes following irradiation. We identified many genes, the expressions of which were altered in our radioresistant GBM model, that have never before been reported to be associated with the development of radioresistant GBM and should be concertedly further investigated to understand their roles in radioresistance. These genes were enriched in various biological processes such as inflammatory response, cell migration, positive regulation of epithelial to mesenchymal transition, angiogenesis, apoptosis, positive regulation of T-cell migration, positive regulation of macrophage chemotaxis, T-cell antigen processing and presentation, and microglial cell activation involved in immune response genes. These findings furnish crucial information for elucidating the molecular mechanisms associated with radioresistance in GBM. Therapeutically, with the global alterations of multiple biological pathways observed in irradiated GBM cells, an effective GBM therapy may require a cocktail carrying multiple agents targeting multiple implicated pathways in order to have a chance at making a substantial impact on improving the overall GBM survival.

Methylation Status of the RIZ1 Gene Promoter in Human Glioma Tissues and Cell Lines.

PubMed

Zhang, Chenran; Meng, Wei; Wang, Jiajia; Lu, Yicheng; Hu, Guohan; Hu, Liuhua; Ma, Jie

2017-08-01

Retinoblastoma protein-interacting zinc-finger gene 1 (RIZ1), a strong tumor suppressor, is silenced in many human cancers. Our previous studies showed that RIZ1 expression was negatively correlated with the grade of glioma and was a key predictor of patient survival. Therefore, RIZ1 could be a potential tumor suppressor during glioma pathogenesis, although the mechanism underlying RIZ1 gene inactivation in gliomas is unknown. We investigated the methylation status of the RIZ1 promoter in human glioma tissues and four glioblastoma (GBM) cell lines, and verified the effect of the methyltransferase inhibitor 5-aza-2-deoxycytidine (5-aza-CdR) on RIZ1 transcription and cell proliferation. Methylation-specific PCR (MSP) was performed to determine RIZ1 promoter methylation in human glioma specimens. The correlation between RIZ1 hypermethylation in tumors and clinicopathological features also was analyzed. 5-Aza-CdR treatment was used to reactivate gene expression silenced by hypermethylation in the U87 glioblastoma cell line, and real-time PCR was then used to measure RIZ1 expression. The ability of 5-aza-CdR to inhibit the proliferation of glioma cell lines whose RIZ1 promoters were hypermethylated was measured by bromodeoxyuridine (BrdU) incorporation. Among 51 human glioma specimens, RIZ1 promoter methylation was detected in 23 cases. Clinicopathological evaluation suggested that RIZ1 hypermethylation was negatively associated with tumor grade and patient age (P < 0.05). Hypermethylation of the RIZ1 promoter was detected in the U87 and U251 cell lines. RIZ1 mRNA expression in U87 cells was upregulated after treatment with 5-aza-Cdr, which correlated with inhibition of cell proliferation in a time- and concentration-dependent manner. Promoter hypermethylation may play an important role in the epigenetic silencing of RIZ1 expression in human glioma tissues and GBM cell lines.

Magnolol and honokiol exert a synergistic anti-tumor effect through autophagy and apoptosis in human glioblastomas

PubMed Central

Cheng, Yu-Chen; Hueng, Dueng-Yuan; Huang, Hua-Yin; Chen, Jang-Yi; Chen, Ying

2016-01-01

Glioblastoma (GBM) is a malignant brain tumor associated with a high mortality rate. The aim of this study is to investigate the synergistic effects of honokiol (Hono) and magnolol (Mag), extracted from Magnolia officinalis, on cytotoxicity and inhibition of human GBM tumor progression in cellular and animal models. In comparison with Hono or Mag alone, co-treatment with Hono and Mag (Hono-Mag) decreased cyclin A, D1 and cyclin-dependent kinase 2, 4, 6 significantly, leading to cell cycle arrest in U87MG and LN229 human glioma cells. In addition, phosphorylated phosphoinositide 3-kinase (p-PI3K), p-Akt, and Ki67 were decreased after Hono-Mag treatment, showing proliferation inhibition. Hono-Mag treatment also reduced p-p38 and p-JNK but elevated p-ERK expression. Besides, Hono-Mag treatment induced autophagy and intrinsic and extrinsic apoptosis. Both ERK and autophagy inhibitors enhanced Hono-Mag-induced apoptosis in LN229 cells, indicating a rescuer role of ERK. In human GBM orthotopic xenograft model, the Hono-Mag treatment inhibited the tumor progression and induced apoptosis more efficiently than Temozolomide, Hono, or Mag group. In conclusion, the Hono-Mag exerts a synergistic anti-tumor effect by inhibiting cell proliferation and inducing autophagy and apoptosis in human GBM cells. The Hono-Mag may be applied as an adjuvant therapy to improve the therapeutic efficacy of GBM treatment. PMID:27074557

Distribution of the phosphatidylinositol 3-kinase inhibitors Pictilisib (GDC-0941) and GNE-317 in U87 and GS2 intracranial glioblastoma models-assessment by matrix-assisted laser desorption ionization imaging.

PubMed

Salphati, Laurent; Shahidi-Latham, Sheerin; Quiason, Cristine; Barck, Kai; Nishimura, Merry; Alicke, Bruno; Pang, Jodie; Carano, Richard A; Olivero, Alan G; Phillips, Heidi S

2014-07-01

Glioblastoma multiforme (GBM) is the most common primary brain tumor in adults, and the limited available treatment options have not meaningfully impacted patient survival in the past decades. Such poor outcomes can be at least partly attributed to the inability of most drugs tested to cross the blood-brain barrier and reach all areas of the glioma. The objectives of these studies were to visualize and compare by matrix-assisted laser desorption ionization (MALDI) imaging mass spectrometry the brain and tumor distribution of the phosphatidylinositol 3-kinase (PI3K) inhibitors pictilisib (GDC-0941, 2-(1H-indazol-4-yl)-6-(4-methanesulfonyl-piperazin-1-ylmethyl)-4-morpholin-4-yl-thieno[3,2-d]pyrimidine) and GNE-317 [5-(6-(3-methoxyoxetan-3-yl)-7-methyl-4-morpholinothieno[3,2-d]pyrimidin-2-yl)pyrimidin-2-amine] in U87 and GS2 orthotopic models of GBM, models that exhibit differing blood-brain barrier characteristics. Following administration to tumor-bearing mice, pictilisib was readily detected within tumors of the contrast-enhancing U87 model whereas it was not located in tumors of the nonenhancing GS2 model. In both GBM models, pictilisib was not detected in the healthy brain. In contrast, GNE-317 was uniformly distributed throughout the brain in the U87 and GS2 models. MALDI imaging revealed also that the pictilisib signal varied regionally by up to 6-fold within the U87 tumors whereas GNE-317 intratumor levels were more homogeneous. Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) analyses of the nontumored half of the brain showed pictilisib had brain-to-plasma ratios lower than 0.03 whereas they were greater than 1 for GNE-317, in agreement with their brain penetration properties. These results in orthotopic models representing either the contrast-enhancing or invasive areas of GBM clearly demonstrate the need for whole-brain distribution to potentially achieve long-term efficacy in GBM. Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.

HER2-targeted recombinant protein immuno-caspase-6 effectively induces apoptosis in HER2-overexpressing GBM cells in vitro and in vivo.

PubMed

Zhang, Leiming; Ren, Junlin; Zhang, Hangyu; Cheng, Gang; Xu, Yanming; Yang, Shuangwu; Dong, Chao; Fang, Dandong; Zhang, Jianning; Yang, Angang

2016-11-01

Glioblastoma multiforme (GBM), which is associated with a high rate of morbidity and mortality, is among the most malignant and treatment-refractory neoplasms in human adults. As GBM is highly resistant to conventional therapies, immunotherapies are a promising treatment candidate. HER2 is an attractive target for GBM immunotherapy, as its expression is highly associated with various types of GBM. We previously reported that a novel HER2-targeted recombinant protein e23sFv-Fdt-casp6 has an antitumor effect on HER2-positive gastric cancer cells. In this study, we established a genetically modified Chinese hamster ovary cell line, which produced and secreted e23sFv-Fdt-casp6 proteins. Following specific binding to and internalization into HER2-overexpressing tumor cells, the e23sFv-Fdt-casp6 protein induced tumor cell apoptosis and inhibited the proliferation of HER2-overexpressing A172 and U251MG cells in vitro, but not in U87MG cells with undetectable HER2. The e23sFv-Fdt-casp6 gene was introduced into severe combined immunodeficient mice bearing human glioblastoma xenografts by using intramuscular injections of a liposome-encapsulated vector. The recombinant protein e23sFv-Fdt-casp6 specifically targeted tumor cells and induced apoptosis, thereby leading to potent inhibition of tumor growth and prolonged the survival time of tumor-bearing mice. We concluded that e23sFv‑Fdt‑casp6 represents a promising HER2-targeted treatment option for human gliomas.

HDAC4 and HDAC6 sustain DNA double strand break repair and stem-like phenotype by promoting radioresistance in glioblastoma cells.

PubMed

Marampon, Francesco; Megiorni, Francesca; Camero, Simona; Crescioli, Clara; McDowell, Heather P; Sferra, Roberta; Vetuschi, Antonella; Pompili, Simona; Ventura, Luca; De Felice, Francesca; Tombolini, Vincenzo; Dominici, Carlo; Maggio, Roberto; Festuccia, Claudio; Gravina, Giovanni Luca

2017-07-01

The role of histone deacetylase (HDAC) 4 and 6 in glioblastoma (GBM) radioresistance was investigated. We found that tumor samples from 31 GBM patients, who underwent temozolomide and radiotherapy combined treatment, showed HDAC4 and HDAC6 expression in 93.5% and 96.7% of cases, respectively. Retrospective clinical data analysis demonstrated that high-intensity HDAC4 and/or HDAC6 immunostaining was predictive of poor clinical outcome. In vitro experiments revealed that short hairpin RNA-mediated silencing of HDAC4 or HDAC6 radiosensitized U87MG and U251MG GBM cell lines by promoting DNA double-strand break (DSBs) accumulation and by affecting DSBs repair molecular machinery. We found that HDAC6 knock-down predisposes to radiation therapy-induced U251MG apoptosis- and U87MG autophagy-mediated cell death. HDAC4 silencing promoted radiation therapy-induced senescence, independently by the cellular context. Finally, we showed that p53 WT expression contributed to the radiotherapy lethal effects and that HDAC4 or HDAC6 sustained GBM stem-like radioresistant phenotype. Altogether, these observations suggest that HDAC4 and HDAC6 are guardians of irradiation-induced DNA damages and stemness, thus promoting radioresistance, and may represent potential prognostic markers and therapeutic targets in GBM. Copyright © 2017 Elsevier B.V. All rights reserved.

γ-Glutamyl transferase 7 is a novel regulator of glioblastoma growth.

PubMed

Bui, Timothy T; Nitta, Ryan T; Kahn, Suzana A; Razavi, Seyed-Mostafa; Agarwal, Maya; Aujla, Parvir; Gholamin, Sharareh; Recht, Lawrence; Li, Gordon

2015-04-07

Glioblastoma (GBM) is the most malignant primary brain tumor in adults, with a median survival time of one and a half years. Traditional treatments, including radiation, chemotherapy, and surgery, are not curative, making it imperative to find more effective treatments for this lethal disease. γ-Glutamyl transferase (GGT) is a family of enzymes that was shown to control crucial redox-sensitive functions and to regulate the balance between proliferation and apoptosis. GGT7 is a novel GGT family member that is highly expressed in brain and was previously shown to have decreased expression in gliomas. Since other members of the GGT family were found to be altered in a variety of cancers, we hypothesized that GGT7 could regulate GBM growth and formation. To determine if GGT7 is involved in GBM tumorigenesis, we modulated GGT7 expression in two GBM cell lines (U87-MG and U138) and monitored changes in tumorigenicity in vitro and in vivo. We demonstrated for the first time that GBM patients with low GGT7 expression had a worse prognosis and that 87% (7/8) of primary GBM tissue samples showed a 2-fold decrease in GGT7 expression compared to normal brain samples. Exogenous expression of GGT7 resulted in a 2- to 3-fold reduction in proliferation and anchorage-independent growth under minimal growth conditions (1% serum). Decreasing GGT7 expression using either short interfering RNA or short hairpin RNA consistently increased proliferation 1.5- to 2-fold. In addition, intracranial injections of U87-MG cells with reduced GGT7 expression increased tumor growth in mice approximately 2-fold, and decreased mouse survival. To elucidate the mechanism by which GGT7 regulates GBM growth, we analyzed reactive oxygen species (ROS) levels in GBM cells with modulated GGT7 expression. We found that enhanced GGT7 expression reduced ROS levels by 11-33%. Our study demonstrates that GGT7 is a novel player in GBM growth and that GGT7 can play a critical role in tumorigenesis by regulating anti-oxidative damage. Loss of GGT7 may increase the cellular ROS levels, inducing GBM occurrence and growth. Our findings suggest that GGT7 can be a promising biomarker and a potential therapeutic target for GBM.

Ficus carica latex prevents invasion through induction of let-7d expression in GBM cell lines.

PubMed

Tezcan, Gulcin; Tunca, Berrin; Bekar, Ahmet; Yalcin, Murat; Sahin, Saliha; Budak, Ferah; Cecener, Gulsah; Egeli, Unal; Demir, Cevdet; Guvenc, Gokcen; Yilmaz, Gozde; Erkan, Leman Gizem; Malyer, Hulusi; Taskapilioglu, Mevlut Ozgur; Evrensel, Turkkan; Bilir, Ayhan

2015-03-01

Glioblastoma multiforme (GBM) is one of the deadliest human malignancies. A cure for GBM remains elusive, and the overall survival time is less than 1 year. Thus, the development of more efficient therapeutic approaches for the treatment of these patients is required. Induction of tumor cell death by certain phytochemicals derived from medicinal herbs and dietary plants has become a new frontier for cancer therapy research. Although the cancer suppressive effect of Ficus carica (fig) latex (FCL) has been determined in a few cancer types, the effect of this latex on GBM tumors has not been investigated. Therefore, in the current study, the anti-proliferative activity of FCL and the effect of the FCL-temozolomide (TMZ) combination were tested in the T98G, U-138 MG, and U-87 MG GBM cell lines using the WST-1 assay. The mechanism of cell death was analyzed using Annexin-V/FITC and TUNEL assays, and the effect of FCL on invasion was tested using the chick chorioallantoic membrane assay. To determine the effect of FCL on GBM progression, the expression levels of 40 GBM associated miRNAs were analyzed in T98G cells using RT-qPCR. According to the obtained data, FCL causes cell death in GBM cells with different responses to TMZ, and this effect is synergistically increased in combination with TMZ. In addition, the current study is the first to demonstrate the effect of FCL on modulation of let-7d expression, which may be an important underlying mechanism of the anti-invasive effect of this extract.

Mitochondrial VDAC1-based peptides: Attacking oncogenic properties in glioblastoma

PubMed Central

Shteinfer-Kuzmine, Anna; Arif, Tasleem; Krelin, Yakov; Tripathi, Shambhoo Sharan; Paul, Avijit; Shoshan-Barmatz, Varda

2017-01-01

Glioblastoma multiforme (GBM), a primary brain malignancy characterized by high morbidity, invasiveness, proliferation, relapse and mortality, is resistant to chemo- and radiotherapies and lacks effective treatment. GBM tumors undergo metabolic reprograming and develop anti-apoptotic defenses. We targeted GBM with a peptide derived from the mitochondrial protein voltage-dependent anion channel 1 (VDAC1), a key component of cell energy, metabolism and apoptosis regulation. VDAC1-based cell-penetrating peptides perturbed cell energy and metabolic homeostasis and induced apoptosis in several GBM and GBM-derived stem cell lines. We found that the peptides simultaneously attacked several oncogenic properties of human U-87MG cells introduced into sub-cutaneous xenograft mouse model, inhibiting tumor growth, invasion, and cellular metabolism, stemness and inducing apoptosis. Peptide-treated tumors showed decreased expression of all tested metabolism-related enzymes and transporters, and elevated levels of apoptotic proteins, such as p53, cytochrome c and caspases. Retro-Tf-D-LP4, containing the human transferrin receptor (TfR)-recognition sequence, crossed the blood-brain barrier (BBB) via the TfR that is highly expressed in the BBB to strongly inhibit tumor growth in an intracranial xenograft mouse model. In summary, the VDAC1-based peptides tested here offer a potentially affordable and innovative new conceptual therapeutic paradigm that might overcome GBM stemness and invasiveness and reduce relapse rates. PMID:28412744

Assessment Effects of Resveratrol on Human Telomerase Reverse Transcriptase Messenger Ribonucleic Acid Transcript in Human Glioblastoma.

PubMed

Mirzazadeh, Azin; Kheirollahi, Majid; Farashahi, Ehsan; Sadeghian-Nodoushan, Fatemeh; Sheikhha, Mohammad Hasan; Aflatoonian, Behrouz

2017-01-01

Glioblastoma (GBM) is the most common and aggressive brain tumor, which has a poor prognosis despite the advent of different therapeutic strategies. There are numerous molecular biomarkers to contribute diagnosis, prognosis, and prediction of response to the current therapy in GBM. One of the most important markers that are potentially valuable is immortalization-specific or immortalization-associated marker named "hTERT messenger ribonucleic acid (mRNA)" the key subunit of telomerase enzyme, which is expressed in more than 85% of cancer cells, in spite of the majority of normal somatic cells. In this study, we investigated the effects of resveratrol (RSV) on this mRNA marker level, leading to cancer progression. U-87MG cell line was obtained from Pasteur Institute of Iran and treated with various concentrations of 0-160 μg/mL of RSV and at different time points (24, 48, and 72 h). To evaluate viability of U-87MG cells, standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was performed. Real-time polymerase chain reaction (RT-PCR) was used for comparative and quantitative assessment of human telomerase reverse transcriptase (hTERT) mRNA copy number versus control-untreated group. The results of our investigation suggested that RSV effectively inhibited cell growth and caused cell death in dose-dependent ( P < 0.05) and not in time-dependent manner ( P > 0.05), in vitro . Interestingly, quantitative RT-PCR analysis demonstrated that at half inhibition concentration, RSV dramatically decreased mRNA expression of hTERT, the catalytic subunit of telomerase enzyme, which leads to prevention of cell division and tumor progression. With regard to downregulation of this immortalization-associated marker, RSV may potentially be used as a therapeutic agent against GBM.

GM-CSF production by glioblastoma cells has a functional role in eosinophil survival, activation and growth factor production for enhanced tumor cell proliferation

PubMed Central

Curran, Colleen S.; Evans, Michael D.; Bertics, Paul J.

2011-01-01

Medicinal interventions of limited efficacy are currently available for the treatment of glioblastoma multiforme (GBM), the most common and lethal primary brain tumor in adults. The eosinophil is a pivotal immune cell in the pathobiology of atopic disease that is also found to accumulate in certain tumor tissues. Inverse associations between atopy and GBM risk suggest that the eosinophil may play a functional role in certain tumor immune responses. To assess the potential interactions between eosinophils and GBM, human primary blood eosinophils were cultured with two separate human GBM-derived cell lines (A172, U87-MG) or conditioned media generated in the presence or absence of TNF-α. Results revealed differential eosinophil adhesion and increased survival in response to co-culture with GBM cell lines. Eosinophil responses to GBM cell line-conditioned media included increased survival, activation, CD11b expression and S100A9 release. Addition of GM-CSF neutralizing antibodies to GBM cell cultures or conditioned media reduced eosinophil adhesion, survival and activation, linking tumor cell-derived GM-CSF to the functions of eosinophils in the tumor microenvironment. Dexamethasone, which has been reported to inhibit eosinophil recruitment and shrink GBM lesions on contrast enhanced scans, reduced the production of tumor cell-derived GM-CSF. Furthermore, culture of GBM cells in eosinophil-conditioned media increased tumor cell viability, and generation of eosinophil-conditioned media in the presence of GM-CSF enhanced the effect. These data support the idea of a paracrine loop between GM-CSF producing tumors and eosinophil-derived growth factors in tumor promotion/progression. PMID:21705618

Antitumor Activity and Mechanism of a Reverse Transcriptase Inhibitor, Dapivirine, in Glioblastoma.

PubMed

Liu, Weiwen; Song, Xian-Lu; Zhao, Shan-Chao; He, Minyi; Wang, Hai; Chen, Ziyang; Xiang, Wei; Yi, Guozhong; Qi, Songtao; Liu, Yawei

2018-01-01

Dapivirine is one of reverse transcriptase inhibitors (RTIs). It is the prototype of diarylpyrimidines (DAPY), formerly known as TMC120 or DAPY R147681 (IUPAC name: 4- [[4-(2, 4, 6-trimethylphenyl) amino]-2-pyrimidinyl] amino]-benzonitrile; CAS no.244767-67-7). The purpose of this study is to investigate the antitumor activity of dapivirine, one of the RTIs, on U87 glioblastoma (GBM) cells in vitro and in vivo . U87 GBM cells were cultured and treated with or without dapivirine. Cell viability was evaluated by CCK-8 (Cell Counting Kit 8, CCK-8) assay; apoptosis was analyzed by flow cytometry; cell migration was evaluated by Boyden Chamber assay; Western blotting was performed to detect proteins related to apoptosis, epithelial-to-mesenchymal transition and autophagy. PathScan intracellular signaling array kit was used to detect important and well-characterized signaling molecules. Tumor xenograft model in nude mice was used to evaluate the antitumorigenic effect in vivo . Dapivirine weakened proliferation of glioma cells and induced the apoptosis of U87 glioblastoma cells. Furthermore, dapivirine regulated autophagy and induced Akt, Bad and SAPK/JNK activations. Moreover, the inhibition of glioma cell growth by dapivirine was also observed in nude mice in vivo . In summary, in our study dapivirine exposure induces stress, resulting in JNK and PI3K/Akt pathway activation through diminished inhibition of the apoptosis and autophagy cascade in U87 GBM cells, which inhibits cell growth in vitro and in vivo .

Solid Lipid Curcumin Particles Induce More DNA Fragmentation and Cell Death in Cultured Human Glioblastoma Cells than Does Natural Curcumin

PubMed Central

Al-Gharaibeh, Abeer; Kolli, Nivya

2017-01-01

Despite recent advancements in cancer therapies, glioblastoma multiforme (GBM) remains largely incurable. Curcumin (Cur), a natural polyphenol, has potent anticancer effects against several malignancies, including metastatic brain tumors. However, its limited bioavailability reduces its efficiency for treating GBM. Recently, we have shown that solid lipid Cur particles (SLCPs) have greater bioavailability and brain tissue penetration. The present study compares the efficiency of cell death by Cur and/or SLCPs in cultured GBM cells derived from human (U-87MG) and mouse (GL261) tissues. Several cell viability and cell death assays and marker proteins (MTT assay, annexin-V staining, TUNEL staining, comet assay, DNA gel electrophoresis, and Western blot) were investigated following the treatment of Cur and/or SLCP (25 μM) for 24–72 h. Relative to Cur, the use of SLCP increased cell death and DNA fragmentation, produced longer DNA tails, and induced more fragmented nuclear lobes. In addition, cultured GBM cells had increased levels of caspase-3, Bax, and p53, with decreases in Bcl2, c-Myc, and both total Akt, as well as phosphorylated Akt, when SLCP, rather Cur, was used. Our in vitro work suggests that the use of SLCP may be a promising strategy for reversing or preventing GBM growth, as compared to using Cur. PMID:29359011

Upregulation of miR-181a suppresses the formation of glioblastoma stem cells by targeting the Notch2 oncogene and correlates with good prognosis in patients with glioblastoma multiforme

DOE Office of Scientific and Technical Information (OSTI.GOV)

Huang, Shi-Xiong; Zhao, Zhong-Yan; Weng, Guo-Hu

Glioblastoma stem-like cells (GSCs) are responsible for the initiation and progression of glioblastoma multiforme (GBM), and microRNAs (miRNAs) play an important role in this disease. However, the mechanisms underlying the role of miRNAs in the stemness of GSCs have not been completely elucidated. We previously showed that miR-181a is downregulated in GBM and may predict prognosis in patients with this disease. Here, we demonstrate that the upregulation of miR-181a suppressed GSC formation and inhibited GBM tumorigenesis by targeting the Notch2 oncogene. We found that miR-181a was downregulated in GSCs derived from human glioblastoma U87MG and U373MG cells. The high expressionmore » of miR-181a inhibited the levels of stemness-related markers CD133 and BMI1, attenuated sphere proliferation, promoted cell apoptosis, and reduced the tumorigenicity of GSCs. MiR-181a decreased the expression of Notch2 by targeting the 3’-untranslated region of its mRNA. Notch2 overexpression inhibited the effects of miR-181a downregulation on GSCs, and was negatively correlated with miR-181a expression. Moreover, high Notch2 expression together with low miR-181a expression was correlated with a shorter median overall survival for GBM patients. Together, these data show that miR-181a may play an essential role in GSC formation and GBM progression by targeting Notch2, suggesting that Notch2 and miR-181a have potential prognostic value as tumor biomarkers in GBM patients. - Highlights: • MiR-181a suppressed GSC formation and GBM tumorigenesis by targeting Notch2. • Notch2 and miR-181a expression were correlated with OS for GBM patients. • Notch2 and miR-181a have potential prognostic value in GBM patients.« less

ATM inhibitor KU-55933 increases the TMZ responsiveness of only inherently TMZ sensitive GBM cells.

PubMed

Nadkarni, Aditi; Shrivastav, Meena; Mladek, Ann C; Schwingler, Paul M; Grogan, Patrick T; Chen, Junjie; Sarkaria, Jann N

2012-12-01

Ataxia telangiectasia mutated (ATM) kinase is critical in sensing and repairing DNA double-stranded breaks (DSBs) such as those induced by temozolomide (TMZ). ATM deficiency increases TMZ sensitivity, which suggests that ATM inhibitors may be effective TMZ sensitizing agents. In this study, the TMZ sensitizing effects of 2 ATM specific inhibitors were studied in established and xenograft-derived glioblastoma (GBM) lines that are inherently sensitive to TMZ and derivative TMZ-resistant lines. In parental U251 and U87 glioma lines, the addition of KU-55933 to TMZ significantly increased cell killing compared to TMZ alone [U251 survival: 0.004 ± 0.0015 vs. 0.08 ± 0.01 (p < 0.001), respectively, and U87 survival: 0.02 ± 0.005 vs. 0.04 ± 0.002 (p < 0.001), respectively] and also elevated the fraction of cells arrested in G2/M [U251 G2/M fraction: 61.8 ± 1.1 % vs. 35 ± 0.8 % (p < 0.001), respectively, and U87 G2/M fraction 25 ± 0.2 % vs.18.6 ± 0.4 % (p < 0.001), respectively]. In contrast, KU-55933 did not sensitize the resistant lines to TMZ, and neither TMZ alone or combined with KU-55933 induced a G2/M arrest. While KU-55933 did not enhance TMZ induced Chk1/Chk2 activation, it increased TMZ-induced residual γ-H2AX foci in the parental cells but not in the TMZ resistant cells. Similar sensitization was observed with either KU-55933 or CP-466722 combined with TMZ in GBM12 xenograft line but not in GBM12TMZ, which is resistant to TMZ due to MGMT overexpression. These findings are consistent with a model where ATM inhibition suppresses the repair of TMZ-induced DSBs in inherently TMZ-sensitive tumor lines, which suggests an ATM inhibitor potentially could be deployed with an improvement in the therapeutic window when combined with TMZ.

Antitumor Activity and Mechanism of a Reverse Transcriptase Inhibitor, Dapivirine, in Glioblastoma

PubMed Central

Liu, Weiwen; Song, Xian-lu; Zhao, Shan-chao; He, Minyi; Wang, Hai; Chen, Ziyang; Xiang, Wei; Yi, Guozhong; Qi, Songtao; Liu, Yawei

2018-01-01

Ethnopharmacological relevance: Dapivirine is one of reverse transcriptase inhibitors (RTIs). It is the prototype of diarylpyrimidines (DAPY), formerly known as TMC120 or DAPY R147681 (IUPAC name: 4- [[4-(2, 4, 6-trimethylphenyl) amino]-2-pyrimidinyl] amino]-benzonitrile; CAS no.244767-67-7). Aim: The purpose of this study is to investigate the antitumor activity of dapivirine, one of the RTIs, on U87 glioblastoma (GBM) cells in vitro and in vivo. Materials and Methods: U87 GBM cells were cultured and treated with or without dapivirine. Cell viability was evaluated by CCK-8 (Cell Counting Kit 8, CCK-8) assay; apoptosis was analyzed by flow cytometry; cell migration was evaluated by Boyden Chamber assay; Western blotting was performed to detect proteins related to apoptosis, epithelial-to-mesenchymal transition and autophagy. PathScan intracellular signaling array kit was used to detect important and well-characterized signaling molecules. Tumor xenograft model in nude mice was used to evaluate the antitumorigenic effect in vivo. Results: Dapivirine weakened proliferation of glioma cells and induced the apoptosis of U87 glioblastoma cells. Furthermore, dapivirine regulated autophagy and induced Akt, Bad and SAPK/JNK activations. Moreover, the inhibition of glioma cell growth by dapivirine was also observed in nude mice in vivo. Conclusion: In summary, in our study dapivirine exposure induces stress, resulting in JNK and PI3K/Akt pathway activation through diminished inhibition of the apoptosis and autophagy cascade in U87 GBM cells, which inhibits cell growth in vitro and in vivo. PMID:29290776

Novel model of orthotopic U-87 MG glioblastoma resection in athymic nude mice.

PubMed

Bianco, John; Bastiancich, Chiara; Joudiou, Nicolas; Gallez, Bernard; des Rieux, Anne; Danhier, Fabienne

2017-06-01

In vitro and in vivo models of experimental glioma are useful tools to gain a better understanding of glioblastoma (GBM) and to investigate novel treatment strategies. However, the majority of preclinical models focus on treating solid intracranial tumours, despite surgical resection being the mainstay in the standard care of patients with GBM today. The lack of resection and recurrence models therefore has undermined efforts in finding a treatment for this disease. Here we present a novel orthotopic tumour resection and recurrence model that has potential for the investigation of local delivery strategies in the treatment of GBM. The model presented is simple to achieve through the use of a biopsy punch, is reproducible, does not require specific or expensive equipment, and results in a resection cavity suitable for local drug delivery systems, such as the implantation or injection of hydrogels. We show that tumour resection is well tolerated, does not induce deleterious neurological deficits, and significantly prolongs survival of mice bearing U-87 MG GBM tumours. In addition, the resulting cavity could accommodate adequate amounts of hydrogels for local delivery of chemotherapeutic agents to eliminate residual tumour cells that can induce tumour recurrence. Copyright © 2017 Elsevier B.V. All rights reserved.

Assessment Effects of Resveratrol on Human Telomerase Reverse Transcriptase Messenger Ribonucleic Acid Transcript in Human Glioblastoma

PubMed Central

Mirzazadeh, Azin; Kheirollahi, Majid; Farashahi, Ehsan; Sadeghian-Nodoushan, Fatemeh; Sheikhha, Mohammad Hasan; Aflatoonian, Behrouz

2017-01-01

Background: Glioblastoma (GBM) is the most common and aggressive brain tumor, which has a poor prognosis despite the advent of different therapeutic strategies. There are numerous molecular biomarkers to contribute diagnosis, prognosis, and prediction of response to the current therapy in GBM. One of the most important markers that are potentially valuable is immortalization-specific or immortalization-associated marker named “hTERT messenger ribonucleic acid (mRNA)” the key subunit of telomerase enzyme, which is expressed in more than 85% of cancer cells, in spite of the majority of normal somatic cells. In this study, we investigated the effects of resveratrol (RSV) on this mRNA marker level, leading to cancer progression. Materials and Methods: U-87MG cell line was obtained from Pasteur Institute of Iran and treated with various concentrations of 0–160 μg/mL of RSV and at different time points (24, 48, and 72 h). To evaluate viability of U-87MG cells, standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was performed. Real-time polymerase chain reaction (RT-PCR) was used for comparative and quantitative assessment of human telomerase reverse transcriptase (hTERT) mRNA copy number versus control–untreated group. Results: The results of our investigation suggested that RSV effectively inhibited cell growth and caused cell death in dose-dependent (P < 0.05) and not in time-dependent manner (P > 0.05), in vitro. Interestingly, quantitative RT-PCR analysis demonstrated that at half inhibition concentration, RSV dramatically decreased mRNA expression of hTERT, the catalytic subunit of telomerase enzyme, which leads to prevention of cell division and tumor progression. Conclusion: With regard to downregulation of this immortalization-associated marker, RSV may potentially be used as a therapeutic agent against GBM. PMID:28706881

Anti-miR21 oligonucleotide enhances chemosensitivity of T98G cell line to doxorubicin by inducing apoptosis

PubMed Central

Giunti, Laura; da Ros, Martina; Vinci, Serena; Gelmini, Stefania; Iorio, Anna Lisa; Buccoliero, Anna Maria; Cardellicchio, Stefania; Castiglione, Francesca; Genitori, Lorenzo; de Martino, Maurizio; Giglio, Sabrina; Genuardi, Maurizio; Sardi, Iacopo

2015-01-01

Various signal transduction pathways seem to be involved in chemoresistance mechanism of glioblastomas (GBMs). miR-21 is an important oncogenic miRNA which modulates drug resistance of tumor cells. We analyzed the expression of 5 miRNAs, previously found to be dysregulated in high grade gliomas, in 9 pediatric (pGBM) and in 5 adult (aGBM) GBMs. miR-21 was over-expressed, with a significant difference between pGBMs and aGBMs represented by a 4 times lower degree of expression in the pediatric compared to the adult series (p = 0.001). Doxorubicin (Dox) seems to be an effective anti-glioma agent with high antitumor activity also against glioblastoma stem cells. We therefore evaluated the chemosensitivity to Dox in 3 GBM cell lines (A172, U87MG and T98G). Dox had a cytotoxic effect after 48 h of treatment in A172 and U87MG, while T98G cells were resistant. TUNEL assay verified that Dox induced apoptosis in A172 and U87MG but not in T98G. miR-21 showed a low basal expression in treated cells and was over-expressed in untreated cells. To validate the possible association of miR-21 with drug resistance of T98G cells, we transfected anti-miR-21 inhibitor into the cells. The expression level of miR-21 was significantly lower in T98G transfected cells (than in the parental control cells). Transfected cells showed a high apoptotic rate compared to control after Dox treatment by TUNEL assay, suggesting that combined Dox and miR-21 inhibitor therapy can sensitize GBM resistant cells to anthracyclines by enhancing apoptosis. PMID:25628933

Acetate supplementation as a means of inducing glioblastoma stem-like cell growth arrest.

PubMed

Long, Patrick M; Tighe, Scott W; Driscoll, Heather E; Fortner, Karen A; Viapiano, Mariano S; Jaworski, Diane M

2015-08-01

Glioblastoma (GBM), the most common primary adult malignant brain tumor, is associated with a poor prognosis due, in part, to tumor recurrence mediated by chemotherapy and radiation resistant glioma stem-like cells (GSCs). The metabolic and epigenetic state of GSCs differs from their non-GSC counterparts, with GSCs exhibiting greater glycolytic metabolism and global hypoacetylation. However, little attention has been focused on the potential use of acetate supplementation as a therapeutic approach. N-acetyl-l-aspartate (NAA), the primary storage form of brain acetate, and aspartoacylase (ASPA), the enzyme responsible for NAA catalysis, are significantly reduced in GBM tumors. We recently demonstrated that NAA supplementation is not an appropriate therapeutic approach since it increases GSC proliferation and pursued an alternative acetate source. The FDA approved food additive Triacetin (glyceryl triacetate, GTA) has been safely used for acetate supplementation therapy in Canavan disease, a leukodystrophy due to ASPA mutation. This study characterized the effects of GTA on the proliferation and differentiation of six primary GBM-derived GSCs relative to established U87 and U251 GBM cell lines, normal human cerebral cortical astrocytes, and murine neural stem cells. GTA reduced proliferation of GSCs greater than established GBM lines. Moreover, GTA reduced growth of the more aggressive mesenchymal GSCs greater than proneural GSCs. Although sodium acetate induced a dose-dependent reduction of GSC growth, it also reduced cell viability. GTA-mediated growth inhibition was not associated with differentiation, but increased protein acetylation. These data suggest that GTA-mediated acetate supplementation is a novel therapeutic strategy to inhibit GSC growth. © 2015 Wiley Periodicals, Inc.

Acetate supplementation as a means of inducing glioblastoma stem-like cell growth arrest

PubMed Central

Long, Patrick M.; Tighe, Scott W.; Driscoll, Heather E.; Fortner, Karen A.; Viapiano, Mariano S.; Jaworski, Diane M.

2015-01-01

Glioblastoma (GBM), the most common primary adult malignant brain tumor, is associated with a poor prognosis due, in part, to tumor recurrence mediated by chemotherapy and radiation resistant glioma stem-like cells (GSCs). The metabolic and epigenetic state of GSCs differs from their non-GSC counterparts, with GSCs exhibiting greater glycolytic metabolism and global hypoacetylation. However, little attention has been focused on the potential use of acetate supplementation as a therapeutic approach. N-acetyl-L-aspartate (NAA), the primary storage form of brain acetate, and aspartoacylase (ASPA), the enzyme responsible for NAA catalysis, are significantly reduced in GBM tumors. We recently demonstrated that NAA supplementation is not an appropriate therapeutic approach since it increases GSC proliferation and pursued an alternative acetate source. The FDA approved food additive Triacetin (glyceryl triacetate, GTA) has been safely used for acetate supplementation therapy in Canavan disease, a leukodystrophy due to ASPA mutation. This study characterized the effects of GTA on the proliferation and differentiation of six primary GBM-derived GSCs relative to established U87 and U251 GBM cell lines, normal human cerebral cortical astrocytes, and murine neural stem cells. GTA reduced proliferation of GSCs greater than established GBM lines. Moreover, GTA reduced growth of the more aggressive mesenchymal GSCs greater than proneural GSCs. Although sodium acetate induced a dose-dependent reduction of GSC growth, it also reduced cell viability. GTA-mediated growth inhibition was not associated with differentiation, but increased protein acetylation. These data suggest that GTA-mediated acetate supplementation is a novel therapeutic strategy to inhibit GSC growth. PMID:25573156

Toosendanin Exerts an Anti-Cancer Effect in Glioblastoma by Inducing Estrogen Receptor β- and p53-Mediated Apoptosis

PubMed Central

Cao, Liang; Qu, Dingding; Wang, Huan; Zhang, Sha; Jia, Chenming; Shi, Zixuan; Wang, Zongren; Zhang, Jian; Ma, Jing

2016-01-01

Glioblastoma (GBM) is the most common primary brain tumor with median survival of approximately one year. This dismal poor prognosis is due to resistance to currently available chemotherapeutics; therefore, new cytotoxic agents are urgently needed. In the present study, we reported the cytotoxicity of toosendanin (TSN) in the GBM U87 and C6 cell lines in vitro and in vivo. By using the MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide) assay, flow cytometry analysis, and Western blot, we found that TSN inhibited U87 and C6 cell proliferation and induced apoptosis at a concentration as low as 10 nM. Administration of TSN also reduced tumor burden in a xenograft model of athymic nude mice. Pharmacological and molecular studies suggested that estrogen receptor β (ERβ) and p53 were prominent targets for TSN. GBM cell apoptosis induced by TSN was a stepwise biological event involving the upregulation of ERβ and contextual activation of functional p53. Collectively, our study indicates, for the first time, that TSN is a candidate of novel anti-cancer drugs for GBM. Furthermore, ERβ and p53 could act as predictive biomarkers for the sensitivity of cancer to TSN. PMID:27869737

Synergistic inhibition of glioma cell proliferation by Withaferin A and tumor treating fields.

PubMed

Chang, Edwin; Pohling, Christoph; Beygui, Nooshin; Patel, Chirag B; Rosenberg, Jarrett; Ha, Dong Ho; Gambhir, Sanjiv S

2017-09-01

Glioblastoma (GBM) is the most aggressive and lethal form of brain cancer. Standard therapies are non-specific and often of limited effectiveness; thus, efforts are underway to uncover novel, unorthodox therapies against GBM. In previous studies, we investigated Withaferin A, a steroidal lactone from Ayurvedic medicine that inhibits proliferation in cancers including GBM. Another novel approach, tumor treating fields (TTFields), is thought to disrupt mitotic spindle formation and stymie proliferation of actively dividing cells. We hypothesized that combining TTFields with Withaferin A would synergistically inhibit proliferation in glioblastoma. Human glioblastoma cells (GBM2, GBM39, U87-MG) and human breast adenocarcinoma cells (MDA-MB-231) were isolated from primary tumors. The glioma cell lines were genetically engineered to express firefly luciferase. Proliferative potential was assessed either by bioluminescence imaging or cell counting via hemocytometer. TTFields (4 V/cm) significantly inhibited growth of the four cancer cell lines tested (n = 3 experiments per time point, four measurements per sample, p < 0.02 at least; 2-way ANOVA, control vs. treatment). The combination of Withaferin A (10-100 nM) with TTFields significantly inhibited the growth of the glioma cells to a degree beyond that of Withaferin A or TTFields alone. The interaction of the Withaferin A and TTFields on glioma cells was found to be synergistic in nature (p < 0.01, n = 3 experiments). These findings were validated by both bioluminescence and hemocytometric measurements. The combination of Withaferin A with TTFields represents a novel approach to treat GBM in a manner that is likely better than either treatment alone and that is synergistic.

MiR-9 Promotes Apoptosis Via Suppressing SMC1A Expression in GBM Cell Lines.

PubMed

Zu, Yong; Zhu, Zhichuan; Lin, Min; Xu, Dafeng; Liang, Yongjun; Wang, Yueqian; Qiao, Zhengdong; Cao, Ting; Yang, Dan; Gao, Lili; Jin, Pengpeng; Zhang, Peng; Fu, Jianjun; Zheng, Jing

2017-01-01

Glioblastomas multiforme (GBM) is the most malignant brain cancer, which presented vast genomic variation with complicated pathologic mechanism. MicroRNA is a delicate post-transcriptional tuner of gene expression in the organisms by targeting and regulating protein coding genes. MiR-9 was reported as a significant biomarker for GBM patient prognosis and a key factor in regulation of GBM cancer stem cells. To explore the effect of miR-9 on GBM cell growth, we over expressed miR-9 in U87 and U251 cells. The cell viability decreased and apoptosis increased after miR-9 overexpression in these cells. To identify the target of miR-9, we scanned miR-9 binding site in the 3'UTRs region of expression SMC1A (structural maintenance of chromosomes 1A) genes and designed a fluorescent reporter assay to measure miR-9 binding to this region. Our results revealed that miR-9 binds to the 3'sUTR region of SMC1A and down-regulated SMC1A expression. Our results indicated that miR-9 was a potential therapeutic target for GBM through triggering apoptosis of cancer cells.

Down-regulation of MDR1 by Ad-DKK3 via Akt/NFκB pathways augments the anti-tumor effect of temozolomide in glioblastoma cells and a murine xenograft model.

PubMed

Fujihara, Toshitaka; Mizobuchi, Yoshifumi; Nakajima, Kohei; Kageji, Teruyoshi; Matsuzaki, Kazuhito; Kitazato, Keiko T; Otsuka, Ryotaro; Hara, Keijiro; Mure, Hideo; Okazaki, Toshiyuki; Kuwayama, Kazuyuki; Nagahiro, Shinji; Takagi, Yasushi

2018-05-19

Glioblastoma multiforme (GBM) is the most malignant of brain tumors. Acquired drug resistance is a major obstacle for successful treatment. Earlier studies reported that expression of the multiple drug resistance gene (MDR1) is regulated by YB-1 or NFκB via the JNK/c-Jun or Akt pathway. Over-expression of the Dickkopf (DKK) family member DKK3 by an adenovirus vector carrying DKK3 (Ad-DKK3) exerted anti-tumor effects and led to the activation of the JNK/c-Jun pathway. We investigated whether Ad-DKK3 augments the anti-tumor effect of temozolomide (TMZ) via the regulation of MDR1. GBM cells (U87MG and U251MG), primary TGB105 cells, and mice xenografted with U87MG cells were treated with Ad-DKK3 or TMZ alone or in combination. Ad-DKK3 augmentation of the anti-tumor effects of TMZ was associated with reduced MDR1 expression in both in vivo and in vitro studies. The survival of Ad-DKK3-treated U87MG cells was inhibited and the expression of MDR1 was reduced. This was associated with the inhibition of Akt/NFκB but not of YB-1 via the JNK/c-Jun- or Akt pathway. Our results suggest that Ad-DKK3 regulates the expression of MDR1 via Akt/NFκB pathways and that it augments the anti-tumor effects of TMZ in GBM cells.

Locoregional Confinement and Major Clinical Benefit of 188Re-Loaded CXCR4-Targeted Nanocarriers in an Orthotopic Human to Mouse Model of Glioblastoma.

PubMed

Séhédic, Delphine; Chourpa, Igor; Tétaud, Clément; Griveau, Audrey; Loussouarn, Claire; Avril, Sylvie; Legendre, Claire; Lepareur, Nicolas; Wion, Didier; Hindré, François; Davodeau, François; Garcion, Emmanuel

2017-01-01

Gold standard beam radiation for glioblastoma (GBM) treatment is challenged by resistance phenomena occurring in cellular populations well prepared to survive or to repair damage caused by radiation. Among signals that have been linked with radio-resistance, the SDF1/CXCR4 axis, associated with cancer stem-like cell, may be an opportune target. To avoid the problem of systemic toxicity and blood-brain barrier crossing, the relevance and efficacy of an original system of local brain internal radiation therapy combining a radiopharmaceutical with an immuno-nanoparticle was investigated. The nanocarrier combined lipophilic thiobenzoate complexes of rhenium-188 loaded in the core of a lipid nanocapsule (LNC 188 Re) with a function-blocking antibody, 12G5 directed at the CXCR4, on its surface. The efficiency of 12G5-LNC 188 Re was investigated in an orthotopic and xenogenic GBM model of CXCR4-positive U87MG cells implanted in the striatum of Scid mice. We demonstrated that 12G5-LNC 188 Re single infusion treatment by convection-enhanced delivery resulted in a major clinical improvement in median survival that was accompanied by locoregional effects on tumor development including hypovascularization and stimulation of the recruitment of bone marrow derived CD11b- or CD68-positive cells as confirmed by immunohistochemistry analysis. Interestingly, thorough analysis by spectral imaging in a chimeric U87MG GBM model containing CXCR4-positive/red fluorescent protein (RFP)-positive- and CXCR4-negative/RFP-negative-GBM cells revealed greater confinement of DiD-labeled 12G5-LNCs than control IgG2a-LNCs in RFP compartments. Main conclusion: These findings on locoregional impact and targeting of disseminated cancer cells in tumor margins suggest that intracerebral active targeting of nanocarriers loaded with radiopharmaceuticals may have considerable benefits in clinical applications.

Locoregional Confinement and Major Clinical Benefit of 188Re-Loaded CXCR4-Targeted Nanocarriers in an Orthotopic Human to Mouse Model of Glioblastoma

PubMed Central

Séhédic, Delphine; Chourpa, Igor; Tétaud, Clément; Griveau, Audrey; Loussouarn, Claire; Avril, Sylvie; Legendre, Claire; Lepareur, Nicolas; Wion, Didier; Hindré, François; Davodeau, François; Garcion, Emmanuel

2017-01-01

Purpose: Gold standard beam radiation for glioblastoma (GBM) treatment is challenged by resistance phenomena occurring in cellular populations well prepared to survive or to repair damage caused by radiation. Among signals that have been linked with radio-resistance, the SDF1/CXCR4 axis, associated with cancer stem-like cell, may be an opportune target. To avoid the problem of systemic toxicity and blood-brain barrier crossing, the relevance and efficacy of an original system of local brain internal radiation therapy combining a radiopharmaceutical with an immuno-nanoparticle was investigated. Experiment design: The nanocarrier combined lipophilic thiobenzoate complexes of rhenium-188 loaded in the core of a lipid nanocapsule (LNC188Re) with a function-blocking antibody, 12G5 directed at the CXCR4, on its surface. The efficiency of 12G5-LNC188Re was investigated in an orthotopic and xenogenic GBM model of CXCR4-positive U87MG cells implanted in the striatum of Scid mice. Results: We demonstrated that 12G5-LNC188Re single infusion treatment by convection-enhanced delivery resulted in a major clinical improvement in median survival that was accompanied by locoregional effects on tumor development including hypovascularization and stimulation of the recruitment of bone marrow derived CD11b- or CD68-positive cells as confirmed by immunohistochemistry analysis. Interestingly, thorough analysis by spectral imaging in a chimeric U87MG GBM model containing CXCR4-positive/red fluorescent protein (RFP)-positive- and CXCR4-negative/RFP-negative-GBM cells revealed greater confinement of DiD-labeled 12G5-LNCs than control IgG2a-LNCs in RFP compartments. Main conclusion: These findings on locoregional impact and targeting of disseminated cancer cells in tumor margins suggest that intracerebral active targeting of nanocarriers loaded with radiopharmaceuticals may have considerable benefits in clinical applications. PMID:29158842

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

PubMed

Urbańska, Kaja; Pająk, Beata; Orzechowski, Arkadiusz; Sokołowska, Justyna; Grodzik, Marta; Sawosz, Ewa; Szmidt, Maciej; Sysa, Paweł

2015-01-01

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.

All-trans retinoic acid impairs the vasculogenic mimicry formation ability of U87 stem-like cells through promoting differentiation

PubMed Central

LING, GENG-QIANG; LIU, YI-JING; KE, YI-QUAN; CHEN, LEI; JIANG, XIAO-DAN; JIANG, CHUAN-LU; YE, WEI

2015-01-01

The poor therapeutic effect of traditional antiangiogenic therapy on glioblastoma multiforme (GBM) may be attributed to vasculogenic mimicry (VM), which was previously reported to be promoted by cancer stem-like cells (SLCs). All-trans retinoic acid (ATRA), a potent reagent which drives differentiation, was reported to be able to eradicate cancer SLCs in certain malignancies. The aim of the present study was to investigate the effects of ATRA on the VM formation ability of U87 glioblastoma SLCs. The expression of cancer SLC markers CD133 and nestin was detected using immunocytochemistry in order to identify U87 SLCs. In addition, the differentiation of these SLCs was observed through detecting the expression of glial fibrillary acidic protein (GFAP), β-tubulin III and galactosylceramidase (Galc) using immunofluorescent staining. The results showed that the expression levels of GFAP, β-tubulin III and Galc were upregulated following treatment with ATRA in a dose-dependent manner. Furthermore, ATRA significantly reduced the proliferation, invasiveness, tube formation and vascular endothelial growth factor (VEGF) secretion of U87 SLCs. In conclusion, the VM formation ability of SLCs was found to be negatively correlated with differentiation. These results therefore suggested that ATRA may serve as a promising novel agent for the treatment of GBM due to its role in reducing VM formation. PMID:25760394

Suppression of the Eag1 potassium channel sensitizes glioblastoma cells to injury caused by temozolomide.

PubMed

Sales, Thais Torquato; Resende, Fernando Francisco Borges; Chaves, Natália Lemos; Titze-De-Almeida, Simoneide Souza; Báo, Sônia Nair; Brettas, Marcella Lemos; Titze-De-Almeida, Ricardo

2016-10-01

Glioblastoma multiforme (GBM) is the most aggressive type of human primary brain tumor. The standard treatment protocol includes radiotherapy in combination with temozolomide (TMZ). Despite advances in GBM treatment, the survival time of patients diagnosed with glioma is 14.5 months. Regarding tumor biology, various types of cancer cell overexpress the ether à go-go 1 (Eag1) potassium channel. Therefore, the present study examined the role of Eag1 in the cell damage caused by TMZ on the U87MG glioblastoma cell line. Eag1 was inhibited using a channel blocker (astemizole) or silenced by a short-hairpin RNA expression vector (pKv10.1-3). pKv10.1-3 (0.2 µg) improved the Eag1 silencing caused by 250 µM TMZ, as determined by reverse transcription-quantitative polymerase chain reaction and immunocytochemistry. Additionally, inhibiting Eag1 with the vector or astemizole (5 µM) reduced glioblastoma cell viability and sensitized cells to TMZ. Cell viability decreased by 63% for pKv10.1-3 + TMZ compared with 34% for TMZ alone, and by 77% for astemizole + TMZ compared with 46% for TMZ alone, as determined by MTT assay. In addition, both the vector and astemizole increased the apoptosis rate of glioblastoma cells triggered by TMZ, as determined by an Annexin V apoptosis assay. Collectively, the current data reveal that Eag1 has a role in the damage caused to glioblastoma by TMZ. Furthermore, suppression of this channel can improve the action of TMZ on U87MG glioblastoma cells. Thus, silencing Eag1 is a promising strategy to improve GBM treatment and merits additional studies in animal models of glioma.

EVA1A inhibits GBM cell proliferation by inducing autophagy and apoptosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shen, Xue; Kan, Shifeng; Liu, Zhen

Eva-1 homolog A (EVA1A) is a novel lysosome and endoplasmic reticulum-associated protein involved in autophagy and apoptosis. In this study, we constructed a recombinant adenovirus 5-EVA1A vector (Ad5-EVA1A) to overexpress EVA1A in glioblastoma (GBM) cell lines and evaluated its anti-tumor activities in vitro and in vivo. We found that overexpression of EVA1A in three GBM cell lines (U251, U87 and SHG44) resulted in a suppression of tumor cell growth via activation of autophagy and induction of cell apoptosis in a dose- and time-dependent manner. EVA1A-mediated autophagy was associated with inactivation of the mTOR/RPS6KB1 signaling pathway. Furthermore in vivo, overexpression ofmore » EVA1A successfully inhibited tumor growth in NOD/SCID mice. Our data suggest that EVA1A-induced autophagy and apoptosis play a role in suppressing the development of GBM and their up-regulation may be an effective method for treating this form of cancer. - Highlights: • Overexpression of EVA1A suppresses GBM cell growth. • EVA1A induces autophagy through the mTOR/RPS6KB1 pathway. • EVA1A induces GBM cell apoptosis. • EVA1A inhibits the development of GBM in vivo.« less

DEspR roles in tumor vasculo-angiogenesis, invasiveness, CSC-survival and anoikis resistance: a 'common receptor coordinator' paradigm.

PubMed

Herrera, Victoria L; Decano, Julius L; Tan, Glaiza A; Moran, Ann M; Pasion, Khristine A; Matsubara, Yuichi; Ruiz-Opazo, Nelson

2014-01-01

A priori, a common receptor induced in tumor microvessels, cancer cells and cancer stem-like cells (CSCs) that is involved in tumor angiogenesis, invasiveness, and CSC anoikis resistance and survival, could underlie contemporaneous coordination of these events rather than assume stochasticity. Here we show that functional analysis of the dual endothelin1/VEGFsignal peptide receptor, DEspR, (formerly named Dear, Chr.4q31.2) supports the putative common receptor paradigm in pancreatic ductal adenocarcinoma (PDAC) and glioblastoma (GBM) selected for their invasiveness, CD133+CSCs, and polar angiogenic features. Unlike normal tissue, DEspR is detected in PDAC and GBM microvessels, tumor cells, and CSCs isolated from PDAC-Panc1 and GBM-U87 cells. DEspR-inhibition decreased angiogenesis, invasiveness, CSC-survival and anoikis resistance in vitro, and decreased Panc1-CSC and U87-CSC xenograft tumor growth, vasculo-angiogenesis and invasiveness in nude(nu/nu) rats, suggesting that DEspR activation would coordinate these tumor progression events. As an accessible, cell-surface 'common receptor coordinator', DEspR-inhibition defines a novel targeted-therapy paradigm for pancreatic cancer and glioblastoma.

DEspR Roles in Tumor Vasculo-Angiogenesis, Invasiveness, CSC-Survival and Anoikis Resistance: A ‘Common Receptor Coordinator’ Paradigm

PubMed Central

Herrera, Victoria L.; Decano, Julius L.; Tan, Glaiza A.; Moran, Ann M.; Pasion, Khristine A.; Matsubara, Yuichi; Ruiz-Opazo, Nelson

2014-01-01

A priori, a common receptor induced in tumor microvessels, cancer cells and cancer stem-like cells (CSCs) that is involved in tumor angiogenesis, invasiveness, and CSC anoikis resistance and survival, could underlie contemporaneous coordination of these events rather than assume stochasticity. Here we show that functional analysis of the dual endothelin1/VEGFsignal peptide receptor, DEspR, (formerly named Dear, Chr.4q31.2) supports the putative common receptor paradigm in pancreatic ductal adenocarcinoma (PDAC) and glioblastoma (GBM) selected for their invasiveness, CD133+CSCs, and polar angiogenic features. Unlike normal tissue, DEspR is detected in PDAC and GBM microvessels, tumor cells, and CSCs isolated from PDAC-Panc1 and GBM-U87 cells. DEspR-inhibition decreased angiogenesis, invasiveness, CSC-survival and anoikis resistance in vitro, and decreased Panc1-CSC and U87-CSC xenograft tumor growth, vasculo-angiogenesis and invasiveness in nudenu/nu rats, suggesting that DEspR activation would coordinate these tumor progression events. As an accessible, cell-surface ‘common receptor coordinator’, DEspR-inhibition defines a novel targeted-therapy paradigm for pancreatic cancer and glioblastoma. PMID:24465725

Therapeutic efficacy of aldoxorubicin in an intracranial xenograft mouse model of human glioblastoma.

PubMed

Marrero, Luis; Wyczechowska, Dorota; Musto, Alberto E; Wilk, Anna; Vashistha, Himanshu; Zapata, Adriana; Walker, Chelsey; Velasco-Gonzalez, Cruz; Parsons, Christopher; Wieland, Scott; Levitt, Daniel; Reiss, Krzysztof; Prakash, Om

2014-10-01

Glioblastoma multiforme (GBM) is the most aggressive primary brain tumor with a median survival of 12 to 15 months after diagnosis. Acquired chemoresistance, high systemic toxicity, and low penetration of the blood brain barrier by many anticancer drugs contribute to the failure of anti-GBM therapies. To circumvent some of these obstacles, we tested a novel prodrug approach to evaluate anti-GBM efficacy by utilizing serum albumin-binding doxorubicin (Doxo), aldoxorubicin (Aldoxo), which is less toxic, is released from albumin in an acidic environment and accumulates in tumor tissues. A human GBM cell line that expresses a luciferase reporter (U87-luc) was stereotactically injected into the left striatum of the brain of immunodeficient mice. Following initial tumor growth for 12 days, mice were injected once a week in the tail-vein with Aldoxo [24 mg/kg or 18 mg/kg of doxorubicin equivalents-3/4 maximum tolerated dose (MTD)], Doxo [6 mg/kg (3/4 MTD)], or vehicle. Aldoxo-treated mice demonstrated significantly slower growth of the tumor when compared to vehicle-treated or Doxo-treated mice. Five out of eight Aldoxo-treated mice remained alive more than 60 days with a median survival of 62 days, while the median survival of vehicle- and Doxo-treated mice was only 26 days. Importantly, Aldoxo-treated mice exhibited high levels of Doxo within the tumor tissue, accompanied by low tumor cell proliferation (Ki67) and abundant intratumoral programmed cell death (cleaved caspase-3). Effective accumulation of Aldoxo in brain tumor tissues but not normal brain, its anti-tumor efficacy, and low toxicity, provide a strong rationale for evaluating this novel drug conjugate as a treatment for patients afflicted with GBM.

First-in-human study of PET and optical dual-modality image-guided surgery in glioblastoma using 68Ga-IRDye800CW-BBN.

PubMed

Li, Deling; Zhang, Jingjing; Chi, Chongwei; Xiao, Xiong; Wang, Junmei; Lang, Lixin; Ali, Iqbal; Niu, Gang; Zhang, Liwei; Tian, Jie; Ji, Nan; Zhu, Zhaohui; Chen, Xiaoyuan

2018-01-01

Purpose : Despite the use of fluorescence-guided surgery (FGS), maximum safe resection of glioblastoma multiforme (GBM) remains a major challenge. It has restricted surgeons between preoperative diagnosis and intraoperative treatment. Currently, an integrated approach combining preoperative assessment with intraoperative guidance would be a significant step in this direction. Experimental design : We developed a novel 68 Ga-IRDye800CW-BBN PET/near-infrared fluorescence (NIRF) dual-modality imaging probe targeting gastrin-releasing peptide receptor (GRPR) in GBM. The preclinical in vivo tumor imaging and FGS were first evaluated using an orthotopic U87MG glioma xenograft model. Subsequently, the first-in-human prospective cohort study (NCT 02910804) of GBM patients were conducted with preoperative PET assessment and intraoperative FGS. Results : The orthotopic tumors in mice could be precisely resected using the near-infrared intraoperative system. Translational cohort research in 14 GBM patients demonstrated an excellent correlation between preoperative positive PET uptake and intraoperative NIRF signal. The tumor fluorescence signals were significantly higher than those from adjacent brain tissue in vivo and ex vivo (p < 0.0001). Compared with pathology, the sensitivity and specificity of fluorescence using 42 loci of fluorescence-guided sampling were 93.9% (95% CI 79.8%-99.3%) and 100% (95% CI 66.4%-100%), respectively. The tracer was safe and the extent of resection was satisfactory without newly developed neurologic deficits. Progression-free survival (PFS) at 6 months was 80% and two newly diagnosed patients achieved long PFS. Conclusions: This initial study has demonstrated that the novel dual-modality imaging technique is feasible for integrated pre- and intraoperative targeted imaging via the same molecular receptor and improved intraoperative GBM visualization and maximum safe resection.

Fast Neutron Induced Autophagy Leads To Necrosis In Glioblastoma Multiforme Cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yasui, Linda; Gladden, Samantha; Andorf, Christine

Fast neutrons are highly effective at killing glioblastoma multiforme (GBM), U87 and U251 cells. The mode of cell death was investigated using transmission electron microscopy (TEM) to identify the fraction of irradiated U87 or U251 cells having morphological features of autophagy and/or necrosis. U87 or U251 cells were irradiated with 2 Gy fast neturons or 10 Gy {gamma} rays. A majority of U87 and U251 cells exhibit features of cell death with autophagy after irradiation with either 10 Gy {gamma} rays or 2 Gy fast neutrons. Very few {gamma} irradiated cells had features of necrosis (U87 or U251 cell samplesmore » processed for TEM 1 day after 10 Gy {gamma} irradiation). In contrast, a significant increase was observed in necrotic U87 and U251 cells irradiated with fast neutrons. These results show a greater percentage of cells exhibit morphological evidence of necrosis induced by a lower dose of fast neutron irradiation compared to {gamma} irradiation. Also, the evidence of necrosis in fast neutron irradiated U87 and U251 cells occurs in a background of autophagy. Since autophagy is observed before necrosis, autophagy may play a role in signaling programmed necrosis in fast neutron irradiated U87 and U251 cells.« less

EVA1A inhibits GBM cell proliferation by inducing autophagy and apoptosis.

PubMed

Shen, Xue; Kan, Shifeng; Liu, Zhen; Lu, Guang; Zhang, Xiaoyan; Chen, Yingyu; Bai, Yun

2017-03-01

Eva-1 homolog A (EVA1A) is a novel lysosome and endoplasmic reticulum-associated protein involved in autophagy and apoptosis. In this study, we constructed a recombinant adenovirus 5-EVA1A vector (Ad5-EVA1A) to overexpress EVA1A in glioblastoma (GBM) cell lines and evaluated its anti-tumor activities in vitro and in vivo. We found that overexpression of EVA1A in three GBM cell lines (U251, U87 and SHG44) resulted in a suppression of tumor cell growth via activation of autophagy and induction of cell apoptosis in a dose- and time-dependent manner. EVA1A-mediated autophagy was associated with inactivation of the mTOR/RPS6KB1 signaling pathway. Furthermore in vivo, overexpression of EVA1A successfully inhibited tumor growth in NOD/SCID mice. Our data suggest that EVA1A-induced autophagy and apoptosis play a role in suppressing the development of GBM and their up-regulation may be an effective method for treating this form of cancer. Copyright © 2017 Elsevier Inc. All rights reserved.

Sprouty2 enhances the tumorigenic potential of glioblastoma cells.

PubMed

Park, Jong-Whi; Wollmann, Guido; Urbiola, Carles; Fogli, Barbara; Florio, Tullio; Geley, Stephan; Klimaschewski, Lars

2018-02-23

Sprouty2 (SPRY2), a feedback regulator of receptor tyrosine kinase (RTK) signaling, has been shown to be associated with drug resistance and cell proliferation in glioblastoma (GBM), but the underlying mechanisms are still poorly defined. SPRY2 expression and survival patterns of patients with gliomas were analyzed using publicly available databases. Effects of RNA interference targeting SPRY2 on cellular proliferation in established GBM or patient-derived GBM stemlike cells were examined. Loss- or gain-of-function of SPRY2 to regulate the tumorigenic capacity was assessed in both intracranial and subcutaneous xenografts. SPRY2 was found to be upregulated in GBM, which correlated with reduced survival in GBM patients. SPRY2 knockdown significantly impaired proliferation of GBM cells but not of normal astrocytes. Silencing of SPRY2 increased epidermal growth factor-induced extracellular signal-regulated kinase (ERK) and Akt activation causing premature onset of DNA replication, increased DNA damage, and impaired proliferation, suggesting that SPRY2 suppresses DNA replication stress. Abrogating SPRY2 function strongly inhibited intracranial tumor growth and led to significantly prolonged survival of U87 xenograft-bearing mice. In contrast, SPRY2 overexpression promoted tumor propagation of low-tumorigenic U251 cells. The present study highlights an antitumoral effect of SPRY2 inhibition that is based on excessive activation of ERK signaling and DNA damage response, resulting in reduced cell proliferation and increased cytotoxicity, proposing SPRY2 as a promising pharmacological target in GBM patients.

MARCKS Regulates Growth, Radiation Sensitivity and is a Novel Prognostic Factor for Glioma

PubMed Central

Jarboe, John S.; Anderson, Joshua C.; Duarte, Christine W.; Mehta, Tapan; Nowsheen, Somaira; Hicks, Patricia H.; Whitley, Alexander C.; Rohrbach, Timothy D.; McCubrey, Raymond O.; Chiu, Sherard; Burleson, Tamara M.; Bonner, James A.; Gillespie, G. Yancey; Yang, Eddy S.; Willey, Christopher D.

2013-01-01

Purpose This study assessed whether Myristoylated Alanine Rich C-Kinase Substrate (MARCKS) can regulate glioblastoma (GBM) growth, radiation sensitivity and clinical outcome. Experimental Design MARCKS protein levels were analyzed in five GBM explant cell lines and eight patient-derived xenograft tumors by immunoblot, and these levels were correlated to proliferation rates and intracranial growth rates, respectively. Manipulation of MARCKS protein levels was assessed by lentiviral-mediated shRNA knockdown in the U251 cell line and MARCKS over-expression in the U87 cell line. The effect of manipulation of MARCKS on proliferation, radiation sensitivity and senescence was assessed. MARCKS gene expression was correlated with survival outcomes in the Repository of Molecular Brain Neoplasia Data (REMBRANDT) Database and The Cancer Genome Atlas (TCGA). Results MARCKS protein expression was inversely correlated with GBM proliferation and intracranial xenograft growth rates. Genetic silencing of MARCKS promoted GBM proliferation and radiation resistance, while MARCKS overexpression greatly reduced GBM growth potential and induced senescence. We found MARCKS gene expression to be directly correlated with survival in both the REMBRANDT and TCGA databases. Specifically, patients with high MARCKS expressing tumors of the Proneural molecular subtype had significantly increased survival rates. This effect was most pronounced in tumors with unmethylated O6-methylguanine DNA methyltransferase (MGMT) promoters, a traditionally poor prognostic factor. Conclusions MARCKS levels impact GBM growth and radiation sensitivity. High MARCKS expressing GBM tumors are associated with improved survival, particularly with unmethylated MGMT promoters. These findings suggest the use of MARCKS as a novel target and biomarker for prognosis in the Proneural subtype of GBM. PMID:22619307

Nuclear EGFRvIII resists hypoxic microenvironment induced apoptosis via recruiting ERK1/2 nuclear translocation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xie, Hui; Yang, Jinfeng; Xing, Wenjing

2016-02-05

Glioblastoma (GBM) is the most aggressive type of primary brain tumor. Its interaction with the tumor microenvironment promotes tumor progression. Furthermore, GBM bearing expression of EGFRvIII displays more adaptation to tumor microenvironment related stress. But the mechanisms were poorly understood. Here, we presented evidence that in the human U87MG glioblastoma tumor model, EGFRvIII overexpression led aberrant kinase activation and nuclear translocation of EGFRvIII/ERK1/2 under hypoxia, which induced growth advantage by resisting apoptosis. Additionally, EGFRvIII defective in nuclear entry impaired this capacity in hypoxia adaptation, and partially interrupted ERK1/2 nuclear translocation. Pharmacology or genetic interference ERK1/2 decreased hypoxia resistance triggered bymore » EGFRvIII expression, but not EGFRvIII nuclear translocation. In summary, this study identified a novel role for EGFRvIII in hypoxia tolerance, supporting an important link between hypoxia and subcellular localization alterations of the receptor. - Highlights: • Nuclear translocation of EGFRvIII contributes to GBM cell apoptotic resistance by hypoxia. • Nuclear ERK1/2 facilitates EGFRvIII in hypoxia resistance. • EGFRvIII nuclear translocation is not dependent on ERK1/2.« less

Low Dose of Doxorubicin Potentiates the Effect of Temozolomide in Glioblastoma Cells.

PubMed

Villodre, Emilly Schlee; Kipper, Franciele Cristina; Silva, Andrew Oliveira; Lenz, Guido; Lopez, Patrícia Luciana da Costa

2018-05-01

Glioblastoma (GBM) is an aggressive brain tumor with temozolomide (TMZ)-based chemotherapy as the main therapeutic strategy. Doxorubicin (DOX) is not used in gliomas due to its low bioavailability in the brain; however, new delivery strategies and low doses may be effective in the long term, especially as part of a drug cocktail. Our aim was to evaluate the chronic effects of low doses of DOX and TMZ in GBM. Human U87-ATCC cells and a primary GBM culture were chronically treated with TMZ (5 μM) and DOX (1 and 10 nM) alone or combined. DOX resulted in a reduction in the number of cells over a period of 35 days and delayed the cell regrowth. In addition, DOX induced cell senescence and reduced tumor sphere formation and the proportion of NANOG- and OCT4-positive cells after 7 days. Low doses of TMZ potentiated the effects of DOX on senescence and sphere formation. This combined response using low doses of DOX may pave the way for its use in glioma therapy, with new technologies to overcome its low blood-brain barrier permeability.

Hypermethylation of testis derived transcript gene promoter significantly correlates with worse outcomes in glioblastoma patients.

PubMed

Wang, Li-jia; Bai, Yu; Bao, Zhao-shi; Chen, Yan; Yan, Zhuo-hong; Zhang, Wei; Zhang, Quan-geng

2013-01-01

Glioblastoma is the most common and lethal cancer of the central nervous system. Global genomic hypomethylation and some CpG island hypermethylation are common hallmarks of these malignancies, but the effects of these methylation abnormalities on glioblastomas are still largely unclear. Methylation of the O6-methylguanine-DNA methyltransferase promoter is currently an only confirmed molecular predictor of better outcome in temozolomide treatment. To better understand the relationship between CpG island methylation status and patient outcome, this study launched DNA methylation profiles for thirty-three primary glioblastomas (pGBMs) and nine secondary glioblastomas (sGBMs) with the expectation to identify valuable prognostic and therapeutic targets. We evaluated the methylation status of testis derived transcript (TES) gene promoter by microarray analysis of glioblastomas and the prognostic value for TES methylation in the clinical outcome of pGBM patients. Significance analysis of microarrays was used for genes significantly differently methylated between 33 pGBM and nine sGBM. Survival curves were calculated according to the Kaplan-Meier method, and differences between curves were assessed using the log-rank test. Then, we treated glioblastoma cell lines (U87 and U251) with 5-aza-2-deoxycytidines (5-aza-dC) and detected cell biological behaviors. Microarray data analysis identified TES promoter was hypermethylated in pGBMs compared with sGBMs (P < 0.05). Survival curves from the Kaplan-Meier method analysis revealed that the patients with TES hypermethylation had a short overall survival (P < 0.05). This abnormality is also confirmed in glioblastoma cell lines (U87 and U251). Treating these cells with 5-aza-dC released TES protein expression resulted in significant inhibition of cell growth (P = 0.013). Hypermethylation of TES gene promoter highly correlated with worse outcome in pGBM patients. TES might represent a valuable prognostic marker for glioblastoma.

SOX4 inhibits GBM cell growth and induces G0/G1 cell cycle arrest through Akt-p53 axis.

PubMed

Zhang, Jing; Jiang, Huawei; Shao, Jiaofang; Mao, Ruifang; Liu, Jie; Ma, Yingying; Fang, Xuefeng; Zhao, Na; Zheng, Shu; Lin, Biaoyang

2014-11-01

SOX4 is a transcription factor required for tissue development and differentiation in vertebrates. Overexpression of SOX4 has been reported in many cancers including glioblastoma multiforme (GBM), however, the underlying mechanism of actions has not been studied. In this study, we investigated the role of SOX4 in GBM. Kaplan-Meier analysis was performed to assess the association between SOX4 expression levels and survival times in primary GBM samples. Cre/lox P system was used to generate gain or loss of SOX4 in GBM cells, and microarray analysis uncovered the regulation network of SOX4 in GBM cells. High SOX4 expression was significantly associated with good prognosis of primary GBMs. SOX4 inhibited the growth of GBM cell line LN229, A172G and U87MG, partly via the activation of p53-p21 signaling and down-regulation of phosphorylated AKT1. Gene expression profiling and subsequent gene ontology analysis showed that SOX4 influenced several key pathways including the Wnt/ beta-catenin and TGF-beta signaling pathways. Our study found that SOX4 acts as a tumor suppressor in GBM cells by induce cell cycle arrest and inhibiting cell growth.

Targeting MT1-MMP as an ImmunoPET-Based Strategy for Imaging Gliomas

PubMed Central

Oteo, M.; Romero, E.; Cámara, J. A.; de Martino, A.; Arroyo, A. G.; Morcillo, M. Á.; Squatrito, M.; Martinez-Torrecuadrada, J. L.; Mulero, F.

2016-01-01

Background A critical challenge in the management of Glioblastoma Multiforme (GBM) tumors is the accurate diagnosis and assessment of tumor progression in a noninvasive manner. We have identified Membrane-type 1 matrix metalloproteinase (MT1-MMP) as an attractive biomarker for GBM imaging since this protein is actively involved in tumor growth and progression, correlates with tumor grade and is closely associated with poor prognosis in GBM patients. Here, we report the development of an immunoPET tracer for effective detection of MT1-MMP in GBM models. Methods An anti-human MT1-MMP monoclonal antibody (mAb), LEM2/15, was conjugated to p-isothiocyanatobenzyl-desferrioxamine (DFO-NCS) for 89Zr labeling. Biodistribution and PET imaging studies were performed in xenograft mice bearing human GBM cells (U251) expressing MT1-MMP and non-expressing breast carcinoma cells (MCF-7) as negative control. Two orthotopic brain GBM models, patient-derived neurospheres (TS543) and U251 cells, with different degrees of blood-brain barrier (BBB) disruption were also used for PET imaging experiments. Results 89Zr labeling of DFO-LEM2/15 was achieved with high yield (>90%) and specific activity (78.5 MBq/mg). Biodistribution experiments indicated that 89Zr-DFO-LEM2/15 showed excellent potential as a radiotracer for detection of MT1-MMP positive GBM tumors. PET imaging also indicated a specific and prominent 89Zr-DFO-LEM2/15 uptake in MT1-MMP+ U251 GBM tumors compared to MT1-MMP- MCF-7 breast tumors. Results obtained in orthotopic brain GBM models revealed a high dependence of a disrupted BBB for tracer penetrance into tumors. 89Zr-DFO-LEM2/15 showed much higher accumulation in TS543 tumors with a highly disrupted BBB than in U251 orthotopic model in which the BBB permeability was only partially increased. Histological analysis confirmed the specificity of the immunoconjugate in all GBM models. Conclusion A new anti MT1-MMP-mAb tracer, 89Zr-DFO-LEM2/15, was synthesized efficiently. In vivo validation showed high-specific-contrast imaging of MT1-MMP positive GBM tumors and provided strong evidence for utility of MT1-MMP-targeted immunoPET as an alternate to nonspecific imaging of GBM. PMID:27462980

Metformin inhibits TGF-β1-induced epithelial-to-mesenchymal transition-like process and stem-like properties in GBM via AKT/mTOR/ZEB1 pathway.

PubMed

Song, Yang; Chen, Yong; Li, Yunqian; Lyu, Xiaoyan; Cui, Jiayue; Cheng, Ye; Zhao, Liyan; Zhao, Gang

2018-01-23

Glioblastoma (GBM) is the most frequent and aggressive brain tumor in adults. In spite of advances in diagnosis and therapy, the prognosis is still relatively poor. The invasive property of GBM is the major cause of death in patients. Epithelial-to-mesenchymal transition-like process (EMT-like process) is considered to play an important role in the invasive property. Metformin has been reported as a regulator of EMT-like process. In this study, we confirmed that metformin inhibited TGF-β1-induced EMT-like process and EMT-associated migration and invasion in LN18 and U87 GBM cells. Our results also showed that metformin significantly suppressed self-renewal capacity of glioblastoma stem cells (GSCs), and expression of stem cell markers Bmi1, Sox2 and Musashi1, indicating that metformin can inhibit cancer stem-like properties of GBM cells. We further clarified that metformin specifically inhibited TGF-β1 activated AKT, the downstream molecular mTOR and the leading transcription factor ZEB1. Taken together, our data demonstrate that metformin inhibits TGF-β1-induced EMT-like process and cancer stem-like properties in GBM cells via AKT/mTOR/ZEB1 pathway and provide evidence of metformin for further clinical investigation targeted GBM.

MicroPET/CT Imaging of an Orthotopic Model of Human Glioblastoma Multiforme and Evaluation of Pulsed Low-Dose Irradiation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Park, Sean S.; Chunta, John L.; Robertson, John M.

2011-07-01

Purpose: Glioblastoma multiforme (GBM) is an aggressive tumor that typically causes death due to local progression. To assess a novel low-dose radiotherapy regimen for treating GBM, we developed an orthotopic murine model of human GBM and evaluated in vivo treatment efficacy using micro-positron-emission tomography/computed tomography (microPET/CT) tumor imaging. Methods: Orthotopic GBM xenografts were established in nude mice and treated with standard 2-Gy fractionation or 10 0.2-Gy pulses with 3-min interpulse intervals, for 7 consecutive days, for a total dose of 14 Gy. Tumor growth was quantified weekly using the Flex Triumph (GE Healthcare/Gamma Medica-Ideas, Waukesha, WI) combined PET-single-photon emission CTmore » (SPECT)-CT imaging system and necropsy histopathology. Normal tissue damage was assessed by counting dead neural cells in tissue sections from irradiated fields. Results: Tumor engraftment efficiency for U87MG cells was 86%. Implanting 0.5 x 10{sup 6} cells produced a 50- to 70-mm{sup 3} tumor in 10 to 14 days. A significant correlation was seen between CT-derived tumor volume and histopathology-measured volume (p = 0.018). The low-dose 0.2-Gy pulsed regimen produced a significantly longer tumor growth delay than standard 2-Gy fractionation (p = 0.045). Less normal neuronal cell death was observed after the pulsed delivery method (p = 0.004). Conclusion: This study successfully demonstrated the feasibility of in vivo brain tumor imaging and longitudinal assessment of tumor growth and treatment response with microPET/CT. Pulsed radiation treatment was more efficacious than the standard fractionated treatment and was associated with less normal tissue damage.« less

MicroPET/CT imaging of an orthotopic model of human glioblastoma multiforme and evaluation of pulsed low-dose irradiation.

PubMed

Park, Sean S; Chunta, John L; Robertson, John M; Martinez, Alvaro A; Oliver Wong, Ching-Yee; Amin, Mitual; Wilson, George D; Marples, Brian

2011-07-01

Glioblastoma multiforme (GBM) is an aggressive tumor that typically causes death due to local progression. To assess a novel low-dose radiotherapy regimen for treating GBM, we developed an orthotopic murine model of human GBM and evaluated in vivo treatment efficacy using micro-positron-emission tomography/computed tomography (microPET/CT) tumor imaging. Orthotopic GBM xenografts were established in nude mice and treated with standard 2-Gy fractionation or 10 0.2-Gy pulses with 3-min interpulse intervals, for 7 consecutive days, for a total dose of 14 Gy. Tumor growth was quantified weekly using the Flex Triumph (GE Healthcare/Gamma Medica-Ideas, Waukesha, WI) combined PET-single-photon emission CT (SPECT)-CT imaging system and necropsy histopathology. Normal tissue damage was assessed by counting dead neural cells in tissue sections from irradiated fields. Tumor engraftment efficiency for U87MG cells was 86%. Implanting 0.5 × 10(6) cells produced a 50- to 70-mm(3) tumor in 10 to 14 days. A significant correlation was seen between CT-derived tumor volume and histopathology-measured volume (p = 0.018). The low-dose 0.2-Gy pulsed regimen produced a significantly longer tumor growth delay than standard 2-Gy fractionation (p = 0.045). Less normal neuronal cell death was observed after the pulsed delivery method (p = 0.004). This study successfully demonstrated the feasibility of in vivo brain tumor imaging and longitudinal assessment of tumor growth and treatment response with microPET/CT. Pulsed radiation treatment was more efficacious than the standard fractionated treatment and was associated with less normal tissue damage. Copyright © 2011 Elsevier Inc. All rights reserved.

In vitro evaluation of the cytotoxicity and cellular uptake of CMCht/PAMAM dendrimer nanoparticles by glioblastoma cell models

NASA Astrophysics Data System (ADS)

Pojo, M.; Cerqueira, S. R.; Mota, T.; Xavier-Magalhães, A.; Ribeiro-Samy, S.; Mano, J. F.; Oliveira, J. M.; Reis, R. L.; Sousa, N.; Costa, B. M.; Salgado, A. J.

2013-05-01

Glioblastoma (GBM) is simultaneously the most common and most malignant subtype tumor of the central nervous system. These are particularly dramatic diseases ranking first among all human tumor types for tumor-related average years of life lost and for which curative therapies are not available. Recently, the use of nanoparticles as drug delivery systems (DDS) for tumor treatment has gained particular interest. In an attempt to evaluate the potential of carboxymethylchitosan/poly(amidoamine) (CMCht/PAMAM) dendrimer nanoparticles as a DDS, we aimed to evaluate its cytotoxicity and internalization efficiency in GBM cell models. CMCht/PAMAM-mediated cytotoxicity was evaluated in a GBM cell line (U87MG) and in human immortalized astrocytes (hTERT/E6/E7) by MTS and double-stranded DNA quantification. CMCht/PAMAM internalization was assessed by double fluorescence staining. Both cells lines present similar internalization kinetics when exposed to a high dose (400 μg/mL) of these nanoparticles. However, the internalization rate was higher in tumor GBM cells as compared to immortalized astrocytes when cells were exposed to lower doses (200 μg/mL) of CMCht/PAMAM for short periods (<24 h). After 48 h of exposure, both cell lines present 100 % of internalization efficiency for the tested concentrations. Importantly, short-term exposures (1, 6, 12, 24, and 48 h) did not show cytotoxicity, and long-term exposures (7 days) to CMCht/PAMAM induced only low levels of cytotoxicity in both cell lines ( 20 % of decrease in metabolic activity). The high efficiency and rate of internalization of CMCht/PAMAM we show here suggest that these nanoparticles may be an attractive DDS for brain tumor treatment in the future.

Up-regulation of cholesterol associated genes as novel resistance mechanism in glioblastoma cells in response to archazolid B

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hamm, Rebecca; Zeino, Maen; Frewert, Simon

Treatment of glioblastoma multiforme (GBM), the most common and aggressive lethal brain tumor, represents a great challenge. Despite decades of research, the survival prognosis of GBM patients is unfavorable and more effective therapeutics are sorely required. Archazolid B, a potent vacuolar H{sup +}-ATPase inhibitor influencing cellular pH values, is a promising new compound exerting cytotoxicity in the nanomolar range on wild-type U87MG glioblastoma cells and U87MG.∆EGFR cells transfected with a mutant epidermal growth factor receptor (EGFR) gene. Gene expression profiling using microarray technology showed that archazolid B caused drastic disturbances in cholesterol homeostasis. Cholesterol, a main component of cellular membranes,more » is known to be essential for GBM growth and cells bearing EGFRvIII mutation are highly dependent on exogenous cholesterol. Archazolid B caused excessive accumulation of free cholesterol within intracellular compartments thus depleting cellular cholesterol and leading to up-regulation of SREBP targeted genes, including LDLR and HMGCR, the key enzyme of cholesterol biosynthesis. This cholesterol response is considered to be a novel resistance mechanism induced by archazolid B. We surmise that re-elevation of cholesterol levels in archazolid B treated cells may be mediated by newly synthesized cholesterol, since the drug leads to endosomal/lysosomal malfunction and cholesterol accumulation.« less

Olea europaea leaf extract improves the treatment response of GBM stem cells by modulating miRNA expression.

PubMed

Tezcan, Gulcin; Tunca, Berrin; Bekar, Ahmet; Budak, Ferah; Sahin, Saliha; Cecener, Gulsah; Egeli, Unal; Taskapılıoglu, Mevlut Ozgur; Kocaeli, Hasan; Tolunay, Sahsine; Malyer, Hulusi; Demir, Cevdet; Tumen, Gulendam

2014-01-01

The stem-like cells of Glioblastoma multiforme (GBM) tumors (GSCs) are one of the important determinants of recurrence and drug resistance. The aims of the current study were to evaluate the anticancer effect of Olea europaea leaf extract (OLE) on GBM cell lines, the association between OLE and TMZ responses, and the effect of OLE and the OLE-TMZ combination in GSCs and to clarify the molecular mechanism of this effect on the expression of miRNAs related to cell death. The anti-proliferative activity of OLE and the effect of the OLE-TMZ combination were tested in the T98G, U-138MG and U-87MG GBM cell lines using WST-1 assay. The mechanism of cell death was analyzed with Annexin V/FITC and TUNEL assays. The effects of OLE on the expression levels of miR-181b, miR-153, miR-145 and miR-137 and potential mRNA targets were analyzed in GSCs using RT-qPCR. OLE exhibited anti-proliferative effects via apoptosis and necrosis in the GBM cell lines. In addition, OLE significantly induced the expression of miR-153, miR-145, and miR-137 and decreased the expression of the target genes of these miRNAs in GSCs (p < 0.05). OLE causes cell death in GBM cells with different TMZ responses, and this effect is synergistically increased when the cells are treated with a combination of OLE and TMZ. This is the first study to indicate that OLE may interfere with the pluripotency of GSCs by modulating miRNA expression. Further studies are required, but we suggest that OLE may have a potential for advanced therapeutic cancer drug studies in GBM.

Serine/threonine protein phosphatase 6 modulates the radiation sensitivity of glioblastoma

PubMed Central

Shen, Y; Wang, Y; Sheng, K; Fei, X; Guo, Q; Larner, J; Kong, X; Qiu, Y; Mi, J

2011-01-01

Increasing the sensitivity of glioblastoma cells to radiation is a promising approach to improve survival in patients with glioblastoma multiforme (GBM). This study aims to determine if serine/threonine phosphatase (protein phosphatase 6 (PP6)) is a molecular target for GBM radiosensitization treatment. The GBM orthotopic xenograft mice model was used in this study. Our data demonstrated that the protein level of PP6 catalytic subunit (PP6c) was upregulated in the GBM tissue from about 50% patients compared with the surrounding tissue or control tissue. Both the in vitro survival fraction of GBM cells and the patient survival time were highly correlated or inversely correlated with PP6c expression (R2=0.755 and −0.707, respectively). We also found that siRNA knockdown of PP6c reduced DNA-dependent protein kinase (DNA-PK) activity in three different GBM cell lines, increasing their sensitivity to radiation. In the orthotopic mice model, the overexpression of PP6c in GBM U87 cells attenuated the effect of radiation treatment, and reduced the survival time of mice compared with the control mice, while the PP6c knocking-down improved the effect of radiation treatment, and increased the survival time of mice. These findings demonstrate that PP6 regulates the sensitivity of GBM cells to radiation, and suggest small molecules disrupting or inhibiting PP6 association with DNA-PK is a potential radiosensitizer for GBM. PMID:22158480

Validation of an imageable surgical resection animal model of Glioblastoma (GBM).

PubMed

Sweeney, Kieron J; Jarzabek, Monika A; Dicker, Patrick; O'Brien, Donncha F; Callanan, John J; Byrne, Annette T; Prehn, Jochen H M

2014-08-15

Glioblastoma (GBM) is the most common and malignant primary brain tumour having a median survival of just 12-18 months following standard therapy protocols. Local recurrence, post-resection and adjuvant therapy occurs in most cases. U87MG-luc2-bearing GBM xenografts underwent 4.5mm craniectomy and tumour resection using microsurgical techniques. The cranial defect was repaired using a novel modified cranial window technique consisting of a circular microscope coverslip held in place with glue. Immediate post-operative bioluminescence imaging (BLI) revealed a gross total resection rate of 75%. At censor point 4 weeks post-resection, Kaplan-Meier survival analysis revealed 100% survival in the surgical group compared to 0% in the non-surgical cohort (p=0.01). No neurological defects or infections in the surgical group were observed. GBM recurrence was reliably imaged using facile non-invasive optical bioluminescence (BLI) imaging with recurrence observed at week 4. For the first time, we have used a novel cranial defect repair method to extend and improve intracranial surgical resection methods for application in translational GBM rodent disease models. Combining BLI and the cranial window technique described herein facilitates non-invasive serial imaging follow-up. Within the current context we have developed a robust methodology for establishing a clinically relevant imageable GBM surgical resection model that appropriately mimics GBM recurrence post resection in patients. Copyright © 2014 Elsevier B.V. All rights reserved.

NFκB inhibitors induce cell death in glioblastomas.

PubMed

Zanotto-Filho, Alfeu; Braganhol, Elizandra; Schröder, Rafael; de Souza, Luís Henrique T; Dalmolin, Rodrigo J S; Pasquali, Matheus A Bittencourt; Gelain, Daniel Pens; Battastini, Ana Maria Oliveira; Moreira, José Cláudio Fonseca

2011-02-01

Identification of novel target pathways in glioblastoma (GBM) remains critical due to poor prognosis, inefficient therapies and recurrence associated with these tumors. In this work, we evaluated the role of nuclear-factor-kappa-B (NFκB) in the growth of GBM cells, and the potential of NFκB inhibitors as antiglioma agents. NFκB pathway was found overstimulated in GBM cell lines and in tumor specimens compared to normal astrocytes and healthy brain tissues, respectively. Treatment of a panel of established GBM cell lines (U138MG, U87, U373 and C6) with pharmacological NFκB inhibitors (BAY117082, parthenolide, MG132, curcumin and arsenic trioxide) and NFκB-p65 siRNA markedly decreased the viability of GBMs as compared to inhibitors of other signaling pathways such as MAPKs (ERK, JNK and p38), PKC, EGFR and PI3K/Akt. In addition, NFκB inhibitors presented a low toxicity to normal astrocytes, indicating selectivity to cancerous cells. In GBMs, mitochondrial dysfunction (membrane depolarization, bcl-xL downregulation and cytochrome c release) and arrest in the G2/M phase were observed at the early steps of NFκB inhibitors treatment. These events preceded sub-G1 detection, apoptotic body formation and caspase-3 activation. Also, NFκB was found overstimulated in cisplatin-resistant C6 cells, and treatment of GBMs with NFκB inhibitors overcame cisplatin resistance besides potentiating the effects of the chemotherapeutics, cisplatin and doxorubicin. These findings support NFκB as a potential target to cell death induction in GBMs, and that the NFκB inhibitors may be considered for in vivo testing on animal models and possibly on GBM therapy. Copyright © 2010 Elsevier Inc. All rights reserved.

Intracellular cholesterol level regulates sensitivity of glioblastoma cells against temozolomide-induced cell death by modulation of caspase-8 activation via death receptor 5-accumulation and activation in the plasma membrane lipid raft.

PubMed

Yamamoto, Yutaro; Tomiyama, Arata; Sasaki, Nobuyoshi; Yamaguchi, Hideki; Shirakihara, Takuya; Nakashima, Katsuhiko; Kumagai, Kosuke; Takeuchi, Satoru; Toyooka, Terushige; Otani, Naoki; Wada, Kojiro; Narita, Yoshitaka; Ichimura, Koichi; Sakai, Ryuichi; Namba, Hiroki; Mori, Kentaro

2018-01-01

Development of resistance against temozolomide (TMZ) in glioblastoma (GBM) after continuous treatment with TMZ is one of the critical problems in clinical GBM therapy. Intracellular cholesterol regulates cancer cell biology, but whether intracellular cholesterol is involved in TMZ resistance of GBM cells remains unclear. The involvement of intracellular cholesterol in acquired resistance against TMZ in GBM cells was investigated. Intracellular cholesterol levels were measured in human U251 MG cells with acquired TMZ resistance (U251-R cells) and TMZ-sensitive control U251 MG cells (U251-Con cells), and found that the intracellular cholesterol level was significantly lower in U251-R cells than in U251-Con cells. In addition, treatment by intracellular cholesterol remover, methyl-beta cyclodextrin (MβCD), or intracellular cholesterol inducer, soluble cholesterol (Chol), regulated TMZ-induced U251-Con cell death in line with changes in intracellular cholesterol level. Involvement of death receptor 5 (DR5), a death receptor localized in the plasma membrane, was evaluated. TMZ without or with MβCD and/or Chol caused accumulation of DR5 into the plasma membrane lipid raft and formed a complex with caspase-8, an extrinsic caspase cascade inducer, reflected in the induction of cell death. In addition, treatment with caspase-8 inhibitor or knockdown of DR5 dramatically suppressed U251-Con cell death induced by combination treatment with TMZ, MβCD, and Chol. Combined treatment of Chol with TMZ reversed the TMZ resistance of U251-R cells and another GBM cell model with acquired TMZ resistance, whereas clinical antihypercholesterolemia agents at physiological concentrations suppressed TMZ-induced cell death of U251-Con cells. These findings suggest that intracellular cholesterol level affects TMZ treatment of GBM mediated via a DR5-caspase-8 mechanism. Copyright © 2017 Elsevier Inc. All rights reserved.

Oxymatrine Inhibits Proliferation and Migration While Inducing Apoptosis in Human Glioblastoma Cells

PubMed Central

Wang, Baocheng; Wang, Jiajia; Li, Qifeng; Meng, Wei

2016-01-01

Oxymatrine (OMT), an alkaloid derived from the traditional Chinese medicine herb Sophora flavescens Aiton, has been shown to exhibit anticancer properties on various types of cancer cells. In this study, we investigate the anticancer properties of OMT on human glioblastoma (GBM) cells and evaluate their underlying mechanisms. MTT assays were performed and demonstrated that OMT significantly inhibits the proliferation of GBM cells. Flow cytometry suggested that OMT at a concentration of 10−5 M may induce apoptosis in U251 and A172 cells. Western blot analyses demonstrated a significant increase in the expression of Bax and caspase-3 and a significant decrease in expression of Bcl-2 in both U251 and A172 cells. Additionally, OMT was found by transwell and high-content screening assays to decrease the migratory ability of the evaluated GBM cells. These findings suggest that the antitumor effects of OMT may be the result of inhibition of cell proliferation and migration and the induction of apoptosis by regulating the expression of apoptosis-associated proteins. OMT may represent a novel anticancer therapy for the treatment of GBM. PMID:27957488

Nucleotide sequence of wild-type hepatitis A virus GBM in comparison with two cell culture-adapted variants.

PubMed Central

Graff, J; Normann, A; Feinstone, S M; Flehmig, B

1994-01-01

In order to study cell tropism and attenuation of hepatitis A virus (HAV), the genome of HAV wild-type GBM and two cell culture-adapted variants, GBM/FRhK and GBM/HFS, were cloned and sequenced after amplification by reverse transcriptase-PCR. During virus cultivation, the HAV variant GBM/FRhK had a strict host range for FRhK-4 cells, in contrast to GBM/HFS, which can be grown in HFS and FRhK-4 cells. The HAV variant GBM/HFS was shown to be attenuated when inoculated into chimpanzees (B. Flehmig, R. F. Mauler, G. Noll, E. Weinmann, and J. P. Gregerson, p. 87-90, in A. Zuckerman, ed., Viral Hepatitis and Liver Disease, 1988). On the basis of this biological background, the comparison of the nucleotide sequences of these three HAV GBM variants should elucidate differences which may be of importance for cell tropism and attenuation. The comparison of the genome between the GBM wild type and HAV wild types HM175 (J. I. Cohen, J. R. Ticehurst, R. H. Purcell, A. Buckler-White, and B. M. Baroudy, J. Virol. 61:50-59, 1987) and HAV-LA (R. Najarian, O. Caput, W. Gee, S. J. Potter, A. Renard, J. Merryweather, G. Van Nest, and D. Dina, Proc. Natl. Acad. Sci. USA 82:2627-2631, 1985) showed a 92 to 96.3% identity, whereas the identity was 99.3 to 99.6% between the GBM variants. Nucleotide differences between the wild-type and the cell culture-adapted variants, which were identical in both cell culture-adapted GBM variants, were localized in the 5' noncoding region; in 2B, 3B, and 3D; and in the 3' noncoding region. Our result concerning the 2B/2C region confirms a mutation at position 3889 (C-->T, alanine to valine), which had been shown to be of importance for cell culture adaptation (S. U. Emerson, C. McRill, B. Rosenblum, S. M. Feinstone, and R. H. Purcell, J. Virol. 65:4882-4886, 1991; S. U. Emerson, Y. K. Huang, C. McRill, M. Lewis, and R. H. Purcell, J. Virol. 66:650-654, 1992), whereas other mutations differ from published HAV sequence data and may be cell specific. Further comparison of the two cell culture-adapted GBM variants showed cell-specific mutations resulting in deletions of six amino acids in the VP1 region and three amino acids in the 3A region of the GBM variant GBM/FRhK. PMID:8254770

CRMP2 Phosphorylation Drives Glioblastoma Cell Proliferation.

PubMed

Moutal, Aubin; Villa, Lex Salas; Yeon, Seul Ki; Householder, Kyle T; Park, Ki Duk; Sirianni, Rachael W; Khanna, Rajesh

2018-05-01

Glioblastoma (GBM) is an aggressive primary brain tumor. The rapid growth and the privileged provenance of the tumor within the brain contribute to its aggressivity and poor therapeutic targeting. A poor prognostic factor in glioblastoma is the deletion or mutation of the Nf1 gene. This gene codes for the protein neurofibromin, a tumor suppressor gene that is known to interact with the collapsin response mediator protein 2 (CRMP2). CRMP2 expression and elevated expression of nuclear phosphorylated CRMP2 have recently been implicated in cancer progression. The CRMP2-neurofibromin interaction protects CRMP2 from its phosphorylation by cyclin-dependent kinase 5 (Cdk5), an event linked to cancer progression. In three human glioblastoma cell lines (GL15, A172, and U87), we observed an inverse correlation between neurofibromin expression and CRMP2 phosphorylation levels. Glioblastoma cell proliferation was dependent on CRMP2 expression and phosphorylation by Cdk5 and glycogen synthase kinase 3 beta (GSK3β). The CRMP2 phosphorylation inhibitor (S)-lacosamide reduces, in a concentration-dependent manner, glioblastoma cell proliferation and induced apoptosis in all three GBM cell lines tested. Since (S)-lacosamide is bioavailable in the brain, we tested its utility in an in vivo orthotopic model of GBM using GL261-LucNeo glioma cells. (S)-lacosamide decreased tumor size, as measured via in vivo bioluminescence imaging, by ~54% compared to vehicle control. Our results introduce CRMP2 expression and phosphorylation as a novel player in GBM proliferation and survival, which is enhanced by loss of Nf1.

MiR-224 expression increases radiation sensitivity of glioblastoma cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Upraity, Shailendra; Kazi, Sadaf; Padul, Vijay

Highlights: • MiR-224 expression in established glioblastoma cell lines and sporadic tumor tissues is low. • Exogenous miR-224 expression was found to increase radiation sensitivity of glioblastoma cells. • MiR-224 expression brought about 55–60% reduction in API5 expression levels. • Transfection with API5 siRNA increased radiation sensitivity of glioblastoma cells. • Low miR-224 and high API5 expression correlated with worse survival of GBM patients. - Abstract: Glioblastoma (GBM) is the most common and highly aggressive primary malignant brain tumor. The intrinsic resistance of this brain tumor limits the efficacy of administered treatment like radiation therapy. In the present study, effectmore » of miR-224 expression on growth characteristics of established GBM cell lines was analyzed. MiR-224 expression in the cell lines as well as in primary GBM tumor tissues was found to be low. Exogenous transient expression of miR-224 using either synthetic mimics or stable inducible expression using doxycycline inducible lentiviral vector carrying miR-224 gene, was found to bring about 30–55% reduction in clonogenic potential of U87 MG cells. MiR-224 expression reduced clonogenic potential of U87 MG cells by 85–90% on irradiation at a dose of 6 Gy, a dose that brought about 50% reduction in clonogenic potential in the absence of miR-224 expression. MiR-224 expression in glioblastoma cells resulted in 55–65% reduction in the expression levels of API5 gene, a known target of miR-224. Further, siRNA mediated down-regulation of API5 was also found to have radiation sensitizing effect on glioblastoma cell lines. Analysis of the Cancer Genome Atlas data showed lower miR-224 expression levels in male GBM patients to correlate with poorer survival. Higher expression levels of miR-224 target API5 also showed significant correlation with poorer survival of GBM patients. Up-regulation of miR-224 or down-regulation of its target API5 in combination with radiation therapy, therefore appear as promising options for the treatment of glioblastoma, which is refractory to the existing treatment strategies.« less

Engineering a High-Throughput 3-D In Vitro Glioblastoma Model

PubMed Central

Fan, Yantao; Avci, Naze G.; Nguyen, Duong T.; Dragomir, Andrei; Xu, Feng; Akay, Metin

2015-01-01

Glioblastoma multiforme (GBM) is the most common and malignant primary brain tumor in adults because of its highly invasive behavior. The existing treatment for GBM, which involves a combination of resection, chemotherapy, and radiotherapy, has a very limited success rate with a median survival rate of <1 year. This is mainly because of the failure of early detection and effective treatment. We designed a novel 3-D GBM cell culture model based on microwells that could mimic in vitro environment and help to bypass the lack of suitable animal models for preclinical toxicity tests. Microwells were fabricated from simple and inexpensive polyethylene glycol material for the control of in vitro 3-D culture. We applied the 3-D micropatterning system to GBM (U-87) cells using the photolithography technique to control the cell spheroids’ shape, size, and thickness. Our preliminary results suggested that uniform GBM spheroids can be formed in 3-D, and the size of these GBM spheroids depends on the size of microwells. The viability of the spheroids generated in this manner was quantitatively evaluated using live/dead assay and shown to improve over 21 days. We believe that in vitro 3-D cell culture model could help to reduce the time of the preclinical brain tumor growth studies. The proposed novel platform could be useful and cost-effective for high-throughput screening of cancer drugs and assessment of treatment responses. PMID:27170911

Downregulation of TES by hypermethylation in glioblastoma reduces cell apoptosis and predicts poor clinical outcome.

PubMed

Bai, Yu; Zhang, Quan-Geng; Wang, Xin-Hua

2014-12-11

Gliomas are the most common human brain tumors. Glioblastoma, also known as glioblastoma multiform (GBM), is the most aggressive, malignant, and lethal glioma. The investigation of prognostic and diagnostic molecular biomarkers in glioma patients to provide direction on clinical practice is urgent. Recent studies demonstrated that abnormal DNA methylation states play a key role in the pathogenesis of this kind of tumor. In this study, we want to identify a novel biomarker related to glioma initiation and find the role of the glioma-related gene. We performed a methylation-specific microarray on the promoter region to identify methylation gene(s) that may affect outcome of GBM patients. Normal and GBM tissues were collected from Tiantan Hospital. Genomic DNA was extracted from these tissues and analyzed with a DNA promoter methylation microarray. Testis derived transcript (TES) protein expression was analyzed by immunohistochemistry in paraffin-embedded patient tissues. Western blotting was used to detect TES protein expression in the GBM cell line U251 with or without 5-aza-dC treatment. Cell apoptosis was evaluated by flow cytometry analysis using Annexin V/PI staining. We found that the TES promoter was hypermethylated in GBM compared to normal brain tissues under DNA promoter methylation microarray analysis. The GBM patients with TES hypermethylation had a short overall survival (P <0.05, log-rank test). Among GBM samples, reduced TES protein level was detected in 33 (89.2%) of 37 tumor tissues by immunohistochemical staining. Down regulation of TES was also correlated with worse patient outcome (P <0.05, log-rank test). Treatment on the GBM cell line U251 with 5-aza-dC can greatly increase TES expression, confirming the hypermethylation of TES promoter in GBM. Up-regulation of TES prompts U251 apoptosis significantly. This study demonstrated that both TES promoter hypermethylation and down-regulated protein expression significantly correlated with worse patient outcome. Treatment on the GBM cell line (U251) with 5-aza-dC can highly release TES expression resulting in significant apoptosis in these cells. Our findings suggest that the TES gene is a novel tumor suppressor gene and might represent a valuable prognostic marker for glioblastoma, indicating a potential target for future GBM therapy.

MicroRNA-139-5p acts as a tumor suppressor by targeting ELTD1 and regulating cell cycle in glioblastoma multiforme

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dai, Shouping; Wang, Xianjun; Li, Xiao

MicroRNA-139-5p was identified to be significantly down-regulated in glioblastoma multiform (GBM) by miRNA array. In this report we aimed to clarify its biological function, molecular mechanisms and direct target gene in GBM. Twelve patients with GBM were analyzed for the expression of miR-139-5p by quantitative RT-PCR. miR-139-5p overexpression was established by transfecting miR-139-5p-mimic into U87MG and T98G cells, and its effects on cell proliferation were studied using MTT assay and colony formation assays. We concluded that ectopic expression of miR-139-5p in GBM cell lines significantly suppressed cell proliferation and inducing apoptosis. Bioinformatics coupled with luciferase and western blot assays alsomore » revealed that miR-139-5p suppresses glioma cell proliferation by targeting ELTD1 and regulating cell cycle. - Highlights: • miR-139-5p is downregulated in GBM. • miR-139-5p regulates cell proliferation through inducing apoptosis. • miR-139-5p regulates glioblastoma tumorigenesis by targeting 3′UTR of ELTD1. • miR-139-5p is involved in cell cycle regulation.« less

Temozolomide does not influence the transcription or activity of matrix metalloproteinases 9 and 2 in glioma cell lines.

PubMed

Suzuki, Yuta; Fujioka, Kouki; Ikeda, Keiichi; Murayama, Yuichi; Manome, Yoshinobu

2017-07-01

Glioblastoma multiforme (GBM) is a treatment-resistant malignancy with poor prognosis. Temozolomide (TMZ) is widely used as a first-line drug for GBM. Although this improves patient prognosis, it does not completely eradicate the tumour. Even after total surgical resection, GBM can exhibit uncontrollable invasiveness at the tumour margins owing to activation of matrix metalloproteinases (MMPs) such as MMP-2 and -9; these degrade collagen IV in the basement membrane, which normally prevents cancer invasion. TMZ induces DNA damage and activates transcription factors including c-jun, c-fos, nuclear factor-κβ, and early growth response protein-1, which have putative binding sites on the MMP-9 promoter. TMZ may therefore enhance tumour invasion by stimulating MMP-9 transcription and enzymatic activity. To test this hypothesis, we investigated MMP-2 and -9 mRNA transcription and activity in GBM cell lines treated with TMZ. Human A172 GBM cells were exposed to TMZ (25% and 50% inhibitory concentrations) for 24 or 48h; cell cycle distribution and mRNA levels of MMP-2 and -9 were evaluated using flow cytometry and semi-quantitative reverse transcription PCR, respectively. MMP-2 and -9 enzymatic activities were assessed using gelatin zymography in human A172 and U373 MG GBM cells exposed to TMZ under the same conditions. TMZ altered A172 cell cycle distribution, but not MMP-2 or -9 mRNA levels. TMZ did not affect MMP-2 or -9 enzymatic activities in A172 or U373 MG cells. These findings indicated that TMZ is therefore unlikely to promote GBM invasiveness. Copyright © 2017 Elsevier Ltd. All rights reserved.

Targeted nanoparticle delivery of therapeutic antisense microRNAs presensitizes glioblastoma cells to lower effective doses of temozolomide in vitro and in a mouse model.

PubMed

Malhotra, Meenakshi; Sekar, Thillai Veerapazham; Ananta, Jeyarama S; Devulapally, Rammohan; Afjei, Rayhaneh; Babikir, Husam A; Paulmurugan, Ramasamy; Massoud, Tarik F

2018-04-20

Temozolomide (TMZ) chemotherapy for glioblastoma (GBM) is generally well tolerated at standard doses but it can cause side effects. GBMs overexpress microRNA-21 and microRNA-10b, two known oncomiRs that promote cancer development, progression and resistance to drug treatment. We hypothesized that systemic injection of antisense microRNAs (antagomiR-21 and antagomiR-10b) encapsulated in cRGD-tagged PEG-PLGA nanoparticles would result in high cellular delivery of intact functional antagomiRs, with consequent efficient therapeutic response and increased sensitivity of GBM cells to lower doses of TMZ. We synthesized both targeted and non-targeted nanoparticles, and characterized them for size, surface charge and encapsulation efficiency of antagomiRs. When using targeted nanoparticles in U87MG and Ln229 GBM cells, we showed higher uptake-associated improvement in sensitivity of these cells to lower concentrations of TMZ in medium. Co-inhibition of microRNA-21 and microRNA-10b reduced the number of viable cells and increased cell cycle arrest at G2/M phase upon TMZ treatment. We found a significant increase in expression of key target genes for microRNA-21 and microRNA-10b upon using targeted versus non-targeted nanoparticles. There was also significant reduction in tumor volume when using TMZ after pre-treatment with loaded nanoparticles in human GBM cell xenografts in mice. In vivo targeted nanoparticles plus different doses of TMZ showed a significant therapeutic response even at the lowest dose of TMZ, indicating that preloading cells with antagomiR-21 and antagomiR-10b increases cellular chemosensitivity towards lower TMZ doses. Future clinical applications of this combination therapy may result in improved GBM response by using lower doses of TMZ and reducing nonspecific treatment side effects.

A multi-targeted natural flavonoid myricetin impedes abnormal glioblastoma cell motility and invasiveness via suppressing lamellipodia and focal adhesions formation.

PubMed

Zhao, Hua-Fu; Wang, Gang; Wu, Chang-Peng; Zhou, Xiu-Ming; Wang, Jing; Chen, Zhong-Ping; To, Shing-Shun Tony; Li, Wei-Ping

2018-06-10

Glioblastoma multiforme (GBM) is the most aggressive and malignant primary brain tumor characterized by rapid growth and extensive infiltration to neighboring normal brain parenchyma, which contribute to tumor recurrence and poor prognosis. Myricetin is a natural flavonoid with potent anti-oxidant, anti-inflammatory and anti-cancer activities, which may serve as a potential and harmless agent for GBM treatment. To investigate the anti-glioblastoma effects of myricetin, GBM cells were treated with myricetin alone or in combination with temozolomide. Its effects on GBM cell motility and cytoskeletal structures including lamellipodia, focal adhesions and membrane ruffles were also evaluated. We showed that myricetin alone inhibited glioblastoma U-87 MG cell proliferation, migration and invasion, whereas combination of myricetin and temozolomide did not exhibit any synergistic effect. The inhibitory effect on GBM cell proliferation is independent of PTEN status. Moreover, myricetin showed less cytotoxicity to normal astrocytes than GBM cells. Formation of lamellipodia, focal adhesions, membrane ruffles and vasculogenic mimicry were blocked by myricetin though suppressing ROCK2, paxillin and cortactin phosphorylation. In addition, myricetin could bind to a series of kinases and scaffold proteins including PI3K catalytic isoforms (p110α, p110β and p110δ), PDK1, JNK, c-Jun, ROCK2, paxillin, vinculin and VE-cadherin, leading to inactivation of PI3K/Akt and JNK signaling. In conclusion, myricetin is a multi-targeted drug that has potent anti-migratory and anti-invasive effects on GBM cells via suppressing formation of lamellipodia and focal adhesions, suggesting that it may serve as an alternative option for GBM treatment. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Ibrutinib, a Bruton's tyrosine kinase inhibitor, exhibits antitumoral activity and induces autophagy in glioblastoma.

PubMed

Wang, Jin; Liu, Xiaoyang; Hong, Yongzhi; Wang, Songtao; Chen, Pin; Gu, Aihua; Guo, Xiaoyuan; Zhao, Peng

2017-07-17

Glioblastoma (GBM) is the most common and aggressive primary brain tumor in adults. Ibrutinib, a Bruton's tyrosine kinase (BTK) inhibitor, is a novel anticancer drug used for treating several types of cancers. In this study, we aimed to determine the role of ibrutinib on GBM. Cell proliferation was determined by using cell viability, colony formation, and 5-ethynyl-2'-deoxyuridine (EdU) assays. Cell cycle and cell apoptosis were analyzed by flow cytometry. Cell migratory ability was evaluated by wound healing assays and trans-well migration assays. ATG7 expression was knocked-down by transfection with Atg7-specific small interfering RNA. Overexpression of active Akt protein was achieved by transfecting the cells with a plasmid expressing constitutively active Akt (CA-Akt). Transmission electron microscopy was performed to examine the formation of autophagosomes in cells. Immunofluorescence and western blot analyses were used to analyze protein expression. Tumor xenografts in nude mice and immunohistochemistry were performed to evaluate the effect of ibrutinib on tumor growth in vivo. Ibrutinib inhibited cellular proliferation and migration, and induced apoptosis and autophagy in LN229 and U87 cells. Overexpression of the active Akt protein decreased ibrutinib-induced autophagy, while inhibiting Akt by LY294002 treatment enhanced ibrutinib-induced autophagy. Specific inhibition of autophagy by 3-methyladenine (3MA) or Atg7 targeting with small interfering RNA (si-Atg7) enhanced the anti-GBM effect of ibrutinib in vitro and in vivo. Our results indicate that ibrutinib exerts a profound antitumor effect and induces autophagy through Akt/mTOR signaling pathway in GBM cells. Autophagy inhibition promotes the antitumor activity of ibrutinib in GBM. Our findings provide important insights into the action of an anticancer agent combining with autophagy inhibitor for malignant glioma.

The role of drebrin in glioma migration and invasion

DOE Office of Scientific and Technical Information (OSTI.GOV)

Terakawa, Yuzo; Department of Neurosurgery, Osaka City University Graduate School of Medicine, Osaka; Agnihotri, Sameer

Glioblastoma (GBM) is the most common primary brain tumor in adults. Despite current advances in therapy consisting of surgery followed by chemotherapy and radiation, the overall survival rate still remains poor. Therapeutic failures are partly attributable to the highly infiltrative nature of tumor adjacent to normal brain parenchyma. Recently, evidence is mounting to suggest that actin cytoskeleton dynamics are critical components of the cell invasion process. Drebrin is an actin-binding protein involved in the regulation of actin filament organization, and plays a significant role in cell motility; however, the role of drebrin in glioma cell invasiveness has not yet beenmore » fully elucidated. Therefore, this study was aimed to clarify the role of drebrin in glioma cell morphology and cell motility. Here we show that drebrin is expressed in glioma cell lines and in operative specimens of GBM. We demonstrate that stable overexpression of drebrin in U87 cells leads to alterations in cell morphology, and induces increased invasiveness in vitro while knockdown of drebrin in U87 cells by small interfering RNA (siRNA) decreases invasion and migration. In addition, we show that depletion of drebrin by siRNA alters glioma cell morphology in A172 GBM cell line. Our results suggest that drebrin contributes to the maintenance of cell shape, and may play an important role in glioma cell motility. - Highlights: ► Drebrin is an actin-binding protein aberrantly expressed in several cancers. ► Role of drebrin in glioma cell morphology and motility is previously unknown. ► We demonstrate that drebrin is expressed in 40% of glioblastoma specimens. ► Drebrin plays a significant role in modulating glioma cell migration and invasion.« less

Graphene Functionalized with Arginine Decreases the Development of Glioblastoma Multiforme Tumor in a Gene-Dependent Manner

PubMed Central

Sawosz, Ewa; Jaworski, Sławomir; Kutwin, Marta; Vadalasetty, Krishna Prasad; Grodzik, Marta; Wierzbicki, Mateusz; Kurantowicz, Natalia; Strojny, Barbara; Hotowy, Anna; Lipińska, Ludwika; Jagiełło, Joanna; Chwalibog, André

2015-01-01

Our previous studies revealed that graphene had anticancer properties in experiments in vitro with glioblastoma multiforme (GBM) cells and in tumors cultured in vivo. We hypothesized that the addition of arginine or proline to graphene solutions might counteract graphene agglomeration and increase the activity of graphene. Experiments were performed in vitro with GBM U87 cells and in vivo with GBM tumors cultured on chicken embryo chorioallantoic membranes. The measurements included cell morphology, mortality, viability, tumor morphology, histology, and gene expression. The cells and tumors were treated with reduced graphene oxide (rGO) and rGO functionalized with arginine (rGO + Arg) or proline (rGO + Pro). The results confirmed the anticancer effect of graphene on GBM cells and tumor tissue. After functionalization with amino acids, nanoparticles were distributed more specifically, and the flakes of graphene were less agglomerated. The molecule of rGO + Arg did not increase the expression of TP53 in comparison to rGO, but did not increase the expression of MDM2 or the MDM2/TP53 ratio in the tumor, suggesting that arginine may block MDM2 expression. The expression of NQO1, known to be a strong protector of p53 protein in tumor tissue, was greatly increased. The results indicate that the complex of rGO + Arg has potential in GBM therapy. PMID:26512645

Proscillaridin A is cytotoxic for glioblastoma cell lines and controls tumor xenograft growth in vivo.

PubMed

Denicolaï, Emilie; Baeza-Kallee, Nathalie; Tchoghandjian, Aurélie; Carré, Manon; Colin, Carole; Jiglaire, Carine Jiguet; Mercurio, Sandy; Beclin, Christophe; Figarella-Branger, Dominique

2014-11-15

Glioblastoma is the most frequent primary brain tumor in adults. Because of molecular and cellular heterogeneity, high proliferation rate and significant invasive ability, prognosis of patients is poor. Recent therapeutic advances increased median overall survival but tumor recurrence remains inevitable. In this context, we used a high throughput screening approach to bring out novel compounds with anti-proliferative and anti-migratory properties for glioblastoma treatment. Screening of the Prestwick chemical library® of 1120 molecules identified proscillaridin A, a cardiac glycoside inhibitor of the Na(+)/K(+) ATPase pump, with most significant effects on glioblastoma cell lines. In vitro effects of proscillaridin A were evaluated on GBM6 and GBM9 stem-like cell lines and on U87-MG and U251-MG cell lines. We showed that proscillaridin A displayed cytotoxic properties, triggered cell death, induced G2/M phase blockade in all the glioblastoma cell lines and impaired GBM stem self-renewal capacity even at low concentrations. Heterotopic and orthotopic xenotransplantations were used to confirm in vivo anticancer effects of proscillaridin A that both controls xenograft growth and improves mice survival. Altogether, results suggest that proscillaridin A is a promising candidate as cancer therapies in glioblastoma. This sustains previous reports showing that cardiac glycosides act as anticancer drugs in other cancers.

Small-molecule agonists of mammalian Diaphanous-related (mDia) formins reveal an effective glioblastoma anti-invasion strategy.

PubMed

Arden, Jessica D; Lavik, Kari I; Rubinic, Kaitlin A; Chiaia, Nicolas; Khuder, Sadik A; Howard, Marthe J; Nestor-Kalinoski, Andrea L; Alberts, Arthur S; Eisenmann, Kathryn M

2015-11-01

The extensive invasive capacity of glioblastoma (GBM) makes it resistant to surgery, radiotherapy, and chemotherapy and thus makes it lethal. In vivo, GBM invasion is mediated by Rho GTPases through unidentified downstream effectors. Mammalian Diaphanous (mDia) family formins are Rho-directed effectors that regulate the F-actin cytoskeleton to support tumor cell motility. Historically, anti-invasion strategies focused upon mDia inhibition, whereas activation remained unexplored. The recent development of small molecules directly inhibiting or activating mDia-driven F-actin assembly that supports motility allows for exploration of their role in GBM. We used the formin inhibitor SMIFH2 and mDia agonists IMM-01/-02 and mDia2-DAD peptides, which disrupt autoinhibition, to examine the roles of mDia inactivation versus activation in GBM cell migration and invasion in vitro and in an ex vivo brain slice invasion model. Inhibiting mDia suppressed directional migration and spheroid invasion while preserving intrinsic random migration. mDia agonism abrogated both random intrinsic and directional migration and halted U87 spheroid invasion in ex vivo brain slices. Thus mDia agonism is a superior GBM anti-invasion strategy. We conclude that formin agonism impedes the most dangerous GBM component-tumor spread into surrounding healthy tissue. Formin activation impairs novel aspects of transformed cells and informs the development of anti-GBM invasion strategies. © 2015 Arden et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Sustained Radiosensitization of Hypoxic Glioma Cells after Oxygen Pretreatment in an Animal Model of Glioblastoma and In Vitro Models of Tumor Hypoxia

PubMed Central

Clarke, Ryon H.; Moosa, Shayan; Anzivino, Matthew; Wang, Yi; Floyd, Desiree Hunt; Purow, Benjamin W.; Lee, Kevin S.

2014-01-01

Glioblastoma multiforme (GBM) is the most common and lethal form of brain cancer and these tumors are highly resistant to chemo- and radiotherapy. Radioresistance is thought to result from a paucity of molecular oxygen in hypoxic tumor regions, resulting in reduced DNA damage and enhanced cellular defense mechanisms. Efforts to counteract tumor hypoxia during radiotherapy are limited by an attendant increase in the sensitivity of healthy brain tissue to radiation. However, the presence of heightened levels of molecular oxygen during radiotherapy, while conventionally deemed critical for adjuvant oxygen therapy to sensitize hypoxic tumor tissue, might not actually be necessary. We evaluated the concept that pre-treating tumor tissue by transiently elevating tissue oxygenation prior to radiation exposure could increase the efficacy of radiotherapy, even when radiotherapy is administered after the return of tumor tissue oxygen to hypoxic baseline levels. Using nude mice bearing intracranial U87-luciferase xenografts, and in vitro models of tumor hypoxia, the efficacy of oxygen pretreatment for producing radiosensitization was tested. Oxygen-induced radiosensitization of tumor tissue was observed in GBM xenografts, as seen by suppression of tumor growth and increased survival. Additionally, rodent and human glioma cells, and human glioma stem cells, exhibited prolonged enhanced vulnerability to radiation after oxygen pretreatment in vitro, even when radiation was delivered under hypoxic conditions. Over-expression of HIF-1α reduced this radiosensitization, indicating that this effect is mediated, in part, via a change in HIF-1-dependent mechanisms. Importantly, an identical duration of transient hyperoxic exposure does not sensitize normal human astrocytes to radiation in vitro. Taken together, these results indicate that briefly pre-treating tumors with elevated levels of oxygen prior to radiotherapy may represent a means for selectively targeting radiation-resistant hypoxic cancer cells, and could serve as a safe and effective adjuvant to radiation therapy for patients with GBM. PMID:25350400

Activation of PPARγ mediates icaritin-induced cell cycle arrest and apoptosis in glioblastoma multiforme.

PubMed

Liu, Yongji; Shi, Ling; Liu, Yuan; Li, Peng; Jiang, Guoping; Gao, Xiaoning; Zhang, Yongbin; Jiang, Chuanwu; Zhu, Weiping; Han, Hongxing; Ju, Fang

2018-04-01

Glioblastoma multiforme (GBM) is the most prevalent primary malignancy of the brain. This study was designed to investigate whether icaritin exerts anti-neoplastic activity against GBM in vitro. Cell Counting Kit-8 (CCK-8) assay was utilized to examine the viability of GBM cells. The apoptotic cell population was measured by flow cytometry analysis. Cell cycle distribution was detected by flow cytometry as well. Western blot analysis was performed to examine the level of biomarker proteins in GBM cells. Levels of PPARγ mRNA and protein were detected by qPCR and western blot analysis, respectively. To examine the role of PPARγ in the anti-neoplastic activity of icaritin, PPARγ antagonist GW9662 or PPARγ siRNA was used. The activity of PPARγ was determined by DNA binding and luciferase assays. Our findings revealed that icaritin markedly suppresses cell growth in a dose-dependent and time-dependent fashion. The cell population at the G0/G1 phase of the cell cycle was significantly increased following icaritin treatment. Meanwhile, icaritin promoted apoptotic cell death in T98G and U87MG cells. Further investigation showed upregulation of PPARγ played a key role in the anti-neoplastic activities of icaritin. Moreover, our result demonstrated activation of AMPK signaling by icaritin mediated the modulatory effect of icaritin on PPARγ. Our results suggest the PPARγ may mediate anti-neoplastic activities against GBM. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Impact of mesenchymal stem cells' secretome on glioblastoma pathophysiology.

PubMed

Vieira de Castro, Joana; Gomes, Eduardo D; Granja, Sara; Anjo, Sandra I; Baltazar, Fátima; Manadas, Bruno; Salgado, António J; Costa, Bruno M

2017-10-02

Glioblastoma (GBM) is a highly aggressive primary brain cancer, for which curative therapies are not available. An emerging therapeutic approach suggested to have potential to target malignant gliomas has been based on the use of multipotent mesenchymal stem cells (MSCs), either unmodified or engineered to deliver anticancer therapeutic agents, as these cells present an intrinsic capacity to migrate towards malignant tumors. Nevertheless, it is still controversial whether this innate tropism of MSCs towards the tumor area is associated with cancer promotion or suppression. Considering that one of the major mechanisms by which MSCs interact with and modulate tumor cells is via secreted factors, we studied how the secretome of MSCs modulates critical hallmark features of GBM cells. The effect of conditioned media (CM) from human umbilical cord perivascular cells (HUCPVCs, a MSC population present in the Wharton's jelly of the umbilical cord) on GBM cell viability, migration, proliferation and sensitivity to temozolomide treatment of U251 and SNB-19 GBM cells was evaluated. The in vivo chicken chorioallantoic membrane (CAM) assay was used to evaluate the effect of HUCPVCs CM on tumor growth and angiogenesis. The secretome of HUCPVCs was characterized by proteomic analyses. We found that both tested GBM cell lines exposed to HUCPVCs CM presented significantly higher cellular viability, proliferation and migration. In contrast, resistance of GBM cells to temozolomide chemotherapy was not significantly affected by HUCPVCs CM. In the in vivo CAM assay, CM from HUCPVCs promoted U251 and SNB-19 tumor cells growth. Proteomic analysis to characterize the secretome of HUCPVCs identified several proteins involved in promotion of cell survival, proliferation and migration, revealing novel putative molecular mediators for the effects observed in GBM cells exposed to HUCPVCs CM. These findings provide novel insights to better understand the interplay between GBM cells and MSCs, raising awareness to potential safety issues regarding the use of MSCs as stem-cell based therapies for GBM.

Identification of EGFRvIII-derived CTL epitopes restricted by HLA A0201 for dendritic cell based immunotherapy of gliomas.

PubMed

Wu, An-hua; Xiao, Jing; Anker, Lars; Hall, Walter A; Gregerson, Dale S; Cavenee, Webster K; Chen, Wei; Low, Walter C

2006-01-01

The type III variant of the epidermal growth factor receptor (EGFRvIII) mutation is present in 20-25% of patients with glioblastoma multiforme (GBM). EGFRvIII is not expressed in normal tissue and is therefore a suitable candidate antigen for dendritic cell (DC) based immunotherapy of GBM. To identify the antigenic epitope(s) that may serve as targets for EGFRvIII-specific cytotoxic T lymphocytes (CTLs), the peptide sequence of EGFRvIII was screened with two software programs to predict candidate epitopes restricted by the major histocompatibility complex class I subtype HLA-A0201, which is the predominant subtype in most ethnic groups. Three predicted peptides were constructed and loaded to mature human DCs generated from peripheral blood monocytes. Autologous CD8+ T cells were stimulated in vitro with the EGFRvIII peptide-pulsed DCs. One of the three peptides was found to induce EGFRvIII-specific CTLs as demonstrated by IFN-gamma production and cytotoxicity against HLA-A0201+ EGFRvIII transfected U87 glioma cells. These results suggest that vaccination with EGFRvIII peptide-pulsed DCs or adoptive transfer of in vitro elicited EGFRvIII-specific CTLs by EGFRvIII peptide-pulsed DCs are potential approaches to the treatment of glioma patients.

Selective anti-tumor activity of the novel fluoropyrimidine polymer F10 towards G48a orthotopic GBM tumors.

PubMed

Gmeiner, William H; Lema-Tome, Carla; Gibo, Denise; Jennings-Gee, Jamie; Milligan, Carol; Debinski, Waldemar

2014-02-01

F10 is a novel anti-tumor agent with minimal systemic toxicity in vivo and which displays strong cytotoxicity towards glioblastoma (GBM) cells in vitro. Here we investigate the cytotoxicity of F10 towards GBM cells and evaluate the anti-tumor activity of locally-administered F10 towards an orthotopic xenograft model of GBM. The effects of F10 on thymidylate synthase (TS) inhibition and Topoisomerase 1 (Top1) cleavage complex formation were evaluated using TS activity assays and in vivo complex of enzyme bioassays. Cytotoxicity of F10 towards normal brain was evaluated using cortices from embryonic (day 18) mice. F10 displays minimal penetrance of the blood-brain barrier and was delivered by intra-cerebral (i.c.) administration and prospective anti-tumor response towards luciferase-expressing G48a human GBM tumors in nude mice was evaluated using IVIS imaging. Histological examination of tumor and normal brain tissue was used to assess the selectivity of anti-tumor activity. F10 is cytotoxic towards G48a, SNB-19, and U-251 MG GBM cells through dual targeting of TS and Top1. F10 is not toxic to murine primary neuronal cultures. F10 is well-tolerated upon i.c. administration and induces significant regression of G48a tumors that is dose-dependent. Histological analysis from F10-treated mice revealed tumors were essentially completely eradicated in F10-treated mice while vehicle-treated mice displayed substantial infiltration into normal tissue. F10 displays strong efficacy for GBM treatment with minimal toxicity upon i.c. administration establishing F10 as a promising drug-candidate for treating GBM in human patients.

Differential expression of miR16 in glioblastoma and glioblastoma stem cells: their correlation with proliferation, differentiation, metastasis and prognosis.

PubMed

Tian, R; Wang, J; Yan, H; Wu, J; Xu, Q; Zhan, X; Gui, Z; Ding, M; He, J

2017-10-19

The function of miR16 in multiforme glioblastoma multiforme (GBM) and its stem cells (GSCs) remains elusive. To this end, we investigated the patterns of miR16 expression in these cells and their correlation with malignant behaviors and clinical outcomes. The levels of miR16 and its targeted genes in tumor tissue of GBM and GBM SGH44, U87, U251 cells as well as their stem cell counterparts were measured by qRT-PCR or western blot or immunohistochemistry. Luciferase reporter assay was used to confirm the binding of miR16 to 3'-UTR of its target genes. The effects of miR16 on malignant behaviors were investigated, including tumor cell viability, soft-agar colony formation, GSCs Matrigel colony forming and migration and invasion as well as nude mice xenograft model. Differentially expression patterns of miR16 in glioblastoma cells and GSCs cells were found in this study. Changes of miR16 targeted genes, Bcl2 (B cell lymphoma 2), CDK6 (Cyclin-dependent kinase 6), CCND1 (cyclin D1), CCNE1 (cyclin E1) and SOX5 were confirmed in glioblastoma cell lines and tissue specimens. In vitro and in vivo studies showed that tumor cell proliferation was inhibited by miR16 mimic, but enhanced by miR16 inhibitor. The expression level of miR16 positively correlates with GSCs differentiation, but negatively with the abilities of migration, motility, invasion and colony formation in glioblastoma cells. The inhibitory effects of miR16 on its target genes were also found in nude mice xenograft model. Our findings revealed that the miR16 functions as a tumor suppressor in GSCs and its association with prognosis in GBM.

Genetic modification of mesenchymal stem cells to express a single-chain antibody against EGFRvIII on the cell surface.

PubMed

Balyasnikova, Irina V; Franco-Gou, Rosa; Mathis, J Michael; Lesniak, Maciej S

2010-06-01

Human adult mesenchymal stem cells (hMSCs) are under active investigation as cellular carriers for gene therapy. hMSCs possess natural tropism toward tumours; however, the targeting of hMSCs to specific cell populations within tumours is unexplored. In the case of glioblastoma multiforme (GBM), at least half of the tumours express EGFRvIII on the cell surface, an ideal target for antibody-mediated gene/drug delivery. In this study, we investigated the feasibility of genetically modifying hMSCs to express a single-chain antibody (scFv) to EGFRvIII on their surfaces. Nucleofection was used to transfect hMSCs with cDNA encoding scFv EGFRvIII fused with PDGFR or human B7-1 transmembrane domains. The expression of scFv EGFRvIII on the cell surface was assessed by FACS. A stable population of scFv EGFRvIII-expressing hMSCs was selected, based on antibiotic resistance, and enriched using FACS. We found that nucleofection allows the efficient expression of scFv EGFRvIII on the cell surface of hMSCs. hMSCs transfected with the construct encoding scFv EGFRvIII as a fusion with PDGFRtm showed scFv EGFRvIII expression in up to 86% of cells. Most importantly, human MSCs expressing scFv against EGFRvIII demonstrated enhanced binding to U87-EGFRvIII cells in vitro and significantly increased retention in human U87-EGFRvIII-expressing tumours in vivo. In summary, we provide the first conclusive evidence of genetic modification of hMSCs with a single-chain antibody against an antigen expressed on the surface of tumour cells, thereby opening up a new venue for enhanced delivery of gene therapy applications in the context of malignant brain cancer. Copyright 2009 John Wiley & Sons, Ltd.

Phosphatidylcholine-specific phospholipase C inhibition down- regulates CXCR4 expression and interferes with proliferation, invasion and glycolysis in glioma cells

PubMed Central

Ricci, Alessandro; Pacella, Aurora; Cigliana, Giovanni; Bozzuto, Giuseppina; Podo, Franca; Carpinelli, Giulia

2017-01-01

Background The chemokine receptor CXCR4 plays a crucial role in tumors, including glioblastoma multiforme (GBM), the most aggressive glioma. Phosphatidylcholine-specific phospholipase C (PC-PLC), a catabolic enzyme of PC metabolism, is involved in several aspects of cancer biology and its inhibition down-modulates the expression of growth factor membrane receptors interfering with their signaling pathways. In the present work we investigated the possible interplay between CXCR4 and PC-PLC in GBM cells. Methods Confocal microscopy, immunoprecipitation, western blot analyses, and the evaluation of migration and invasion potential were performed on U87MG cells after PC-PLC inhibition with the xanthate D609. The intracellular metabolome was investigated by magnetic resonance spectroscopy; lactate levels and lactate dehydrogenase (LDH) activity were analyzed by colorimetric assay. Results Our studies demonstrated that CXCR4 and PC-PLC co-localize and are associated on U87MG cell membrane. D609 reduced CXCR4 expression, cell proliferation and invasion, interfering with AKT and EGFR activation and expression. Metabolic analyses showed a decrease in intracellular lactate concentration together with a decrement in LDH activity. Conclusions Our data suggest that inhibition of PC-PLC could represent a new molecular approach in glioma biology not only for its ability in modulating cell metabolism, glioma growth and motility, but also for its inhibitory effect on crucial molecules involved in cancer progression. PMID:28423060

PEGylated Polyamidoamine dendrimer conjugated with tumor homing peptide as a potential targeted delivery system for glioma.

PubMed

Jiang, Yan; Lv, Lingyan; Shi, Huihui; Hua, Yabing; Lv, Wei; Wang, Xiuzhen; Xin, Hongliang; Xu, Qunwei

2016-11-01

Glioblastoma multiforme (GBM) is the most common and aggressive primary central nervous system (CNS) tumor with a short survival time. The failure of chemotherapy is ascribed to the low transport of chemotherapeutics across the Blood Brain Tumor Barrier (BBTB) and poor penetration into tumor tissue. In order to overcome the two barriers, small nanoparticles with active targeted capability are urgently needed for GBM drug delivery. In this study, we proposed PEGylated Polyamidoamine (PAMAM) dendrimer nanoparticles conjugated with glioma homing peptides (Pep-1) as potential glioma targeting delivery system (Pep-PEG-PAMAM), where PEGylated PAMAM dendrimer nanoparticle was utilized as carrier due to its small size and perfect penetration into tumor and Pep-1 was used to overcome BBTB via interleukin 13 receptor α2 (IL-13Rα2) mediated endocytosis. The preliminary availability and safety of Pep-PEG-PAMAM as a nanocarrier for glioma was evaluated. In vitro results indicated that a significantly higher amount of Pep-PEG-PAMAM was endocytosed by U87 MG cells. In vivo fluorescence imaging of U87MG tumor-bearing mice confirmed that the fluorescence intensity at glioma site of targeted group was 2.02 folds higher than that of untargeted group (**p<0.01), and glioma distribution experiment further revealed that Pep-PEG-PAMAM exhibited a significantly enhanced accumulation and improved penetration at tumor site. In conclusion, Pep-1 modified PAMAM was a promising nanocarrier for targeted delivery of brain glioma. Copyright © 2016 Elsevier B.V. All rights reserved.

EGFR gene overexpression retained in an invasive xenograft model by solid orthotopic transplantation of human glioblastoma multiforme into nude mice.

PubMed

Yi, Diao; Hua, Tian Xin; Lin, Huang Yan

2011-03-01

Orthotopic xenograft animal model from human glioblastoma multiforme (GBM) cell lines often do not recapitulate an extremely important aspect of invasive growth and epidermal growth factor receptor (EGFR) gene overexpression of human GBM. We developed an orthotopic xenograft model by solid transplantation of human GBM into the brain of nude mouse. The orthotopic xenografts sharing the same histopathological features with their original human GBMs were highly invasive and retained the overexpression of EGFR gene. The murine orthotopic GBM models constitute a valuable in vivo system for preclinical studies to test novel therapies for human GBM.

Cholecystokinin (CCK) and CCK receptor expression by human gliomas: Evidence for an autocrine/paracrine stimulatory loop.

PubMed

Oikonomou, Eftychia; Buchfelder, Michael; Adams, Eric F

2008-06-01

Cholecystokinin (CCK) is a gut-brain peptide has been described to be able to induce mitosis according to recent studies. Additionally, conflicting data has been published on whether tumours of the central and peripheral nervous system in general, and gliomas in particular, express CCK receptors. In the present in vitro study we employed reverse transcription followed by the polymerase chain reaction (RT-PCR) to investigate whether mRNA for CCK-A and CCK-B receptors as well as CCK peptide itself is present in primary human gliomas and the U-87 MG GBM cell line. The data show that 14/14 (100%) of the primary gliomas exhibited mRNA expression for the CCK peptide gene and the B receptor including the U-87 MG cells, whereas, only 2/14 (14%) showed presence of the CCK-A receptor. The presence of CCK receptors together with CCK peptide expression itself suggests presence of an autocrine loop controlling glioma cell growth. In support of this conclusion, a neutralizing antibody against the CCK peptide exhibited a dose dependent inhibition of cell growth whereas, antagonists to CCK caused a dose depend inhibition of exogenous stimulated glioma cell growth in vitro, via the CCK-B receptor which is PKC activated. Assessment of apoptosis and proteasome activity were undertaken and we report that treatment with CCK antagonists decreased proteasome and increased caspase-3 activity. These data indicate that CCK peptide and CCK-B are abundant in human gliomas and they act to stimulate cell growth in an autocrine manner, primarily via the high affinity CCK-B receptor, which was blocked by antagonists to CCK, perhaps via apoptosis.

Anti-proliferative and anti-migration effects of Polish propolis combined with Hypericum perforatum L. on glioblastoma multiforme cell line U87MG.

PubMed

Borawska, Maria H; Naliwajko, Sylwia K; Moskwa, Justyna; Markiewicz-Żukowska, Renata; Puścion-Jakubik, Anna; Soroczyńska, Jolanta

2016-09-20

Propolis and Hypericum perforatum L. are natural products which contain many active compounds and have numerous beneficial effects, including an antitumor effect. Gliobmastoma multiforme (GBM) is a common primary brain tumor with poor prognosis and limited treatment options. In this study, the effect of propolis (EEP) combined with H. perforatum L. (HPE) on glioblastoma cell line U87MG was investigated for the first time. Anti-proliferative activity of EEP, HPE and their combination (EEP + HPE) was determined by a cytotoxicity test, DNA binding by [(3)H]-thymidine incorporation and cell migration assay. Anti-metastatic properties in U87MG treated with EEP, HPE and EEP + HPE were estimated on cells migration test (scratch assay) and metalloproteinases (MMP2 and MMP9) secretion (gelatin zymography). Combination of HPE and EEP extracts was found to have a time- and dose-dependent inhibitory effect on the viability of U87MG cells. This effect was significantly higher (p < 0.05) when compared to these two extracts applied separately, which was confirmed by the significant reduction of DNA synthesis and significantly higher mitochondrial membrane permeabilization. A significant decreasing in migration cells and in pro-MMP9 and pro-MMP2 secretion in U87MG cells were demonstrated after exposure to combination of EEP (30 μg/ml) with HPE (6.25 μg/ml). In this study, the combination of ethanolic extract from propolis and ethanolic extract of fresh-cut H. perforatum L. was proved the ability to reduce invasiveness of glioma cells through the inhibition of MMP2 and MMP9 secretion and suppression of cell migration. It has a more potent anti-proliferative effect on U87MG glioma cell line compared to using propolis and H. perforatum L. separately. Further studies are required to verify whether the examined extracts can activate apoptotic pathways.

Differential expression of miR16 in glioblastoma and glioblastoma stem cells: their correlation with proliferation, differentiation, metastasis and prognosis

PubMed Central

Tian, R; Wang, J; Yan, H; Wu, J; Xu, Q; Zhan, X; Gui, Z; Ding, M; He, J

2017-01-01

The function of miR16 in multiforme glioblastoma multiforme (GBM) and its stem cells (GSCs) remains elusive. To this end, we investigated the patterns of miR16 expression in these cells and their correlation with malignant behaviors and clinical outcomes. The levels of miR16 and its targeted genes in tumor tissue of GBM and GBM SGH44, U87, U251 cells as well as their stem cell counterparts were measured by qRT–PCR or western blot or immunohistochemistry. Luciferase reporter assay was used to confirm the binding of miR16 to 3′-UTR of its target genes. The effects of miR16 on malignant behaviors were investigated, including tumor cell viability, soft-agar colony formation, GSCs Matrigel colony forming and migration and invasion as well as nude mice xenograft model. Differentially expression patterns of miR16 in glioblastoma cells and GSCs cells were found in this study. Changes of miR16 targeted genes, Bcl2 (B cell lymphoma 2), CDK6 (Cyclin-dependent kinase 6), CCND1 (cyclin D1), CCNE1 (cyclin E1) and SOX5 were confirmed in glioblastoma cell lines and tissue specimens. In vitro and in vivo studies showed that tumor cell proliferation was inhibited by miR16 mimic, but enhanced by miR16 inhibitor. The expression level of miR16 positively correlates with GSCs differentiation, but negatively with the abilities of migration, motility, invasion and colony formation in glioblastoma cells. The inhibitory effects of miR16 on its target genes were also found in nude mice xenograft model. Our findings revealed that the miR16 functions as a tumor suppressor in GSCs and its association with prognosis in GBM. PMID:28628119

L1 stimulation of human glioma cell motility correlates with FAK activation

PubMed Central

Yang, Muhua; Li, Yupei; Chilukuri, Kalyani; Brady, Owen A.; Boulos, Magdy I.; Kappes, John C.

2011-01-01

The neural adhesion/recognition protein L1 (L1CAM; CD171) has been shown or implicated to function in stimulation of cell motility in several cancer types, including high-grade gliomas. Our previous work demonstrated the expression and function of L1 protein in stimulation of cell motility in rat glioma cells. However, the mechanism of this stimulation is still unclear. This study further investigated the function of L1 and L1 proteolysis in human glioblastoma multiforme (GBM) cell migration and invasion, as well as the mechanism of this stimulation. L1 mRNA was found to be present in human T98G GBM cell line but not in U-118 MG grade III human glioma cell line. L1 protein expression, proteolysis, and release were found in T98G cells and human surgical GBM cells by Western blotting. Exosome-like vesicles released by T98G cells were purified and contained full-length L1. In a scratch assay, T98G cells that migrated into the denuded scratch area exhibited upregulation of ADAM10 protease expression coincident with loss of surface L1. GBM surgical specimen cells exhibited a similar loss of cell surface L1 when xenografted into the chick embryo brain. When lentivirally introduced shRNA was used to attenuate L1 expression, such T98G/shL1 cells exhibited significantly decreased cell motility by time lapse microscopy in our quantitative Super Scratch assay. These cells also showed a decrease in FAK activity and exhibited increased focal complexes. L1 binding integrins which activate FAK were found in T98G and U-118 MG cells. Addition of L1 ectodomain-containing media (1) rescued the decreased cell motility of T98G/shL1 cells and (2) increased cell motility of U-118 MG cells but (3) did not further increase T98G cell motility. Injection of L1-attenuated T98G/shL1 cells into embryonic chick brains resulted in the absence of detectable invasion compared to control cells which invaded brain tissue. These studies support a mechanism where glioma cells at the edge of a cell mass upregulate ADAM10 to proteolyze surface L1 and the resultant ectodomain increases human glioma cell migration and invasion by binding to integrin receptors, activating FAK, and increasing turnover of focal complexes. PMID:21373966

L1 stimulation of human glioma cell motility correlates with FAK activation.

PubMed

Yang, Muhua; Li, Yupei; Chilukuri, Kalyani; Brady, Owen A; Boulos, Magdy I; Kappes, John C; Galileo, Deni S

2011-10-01

The neural adhesion/recognition protein L1 (L1CAM; CD171) has been shown or implicated to function in stimulation of cell motility in several cancer types, including high-grade gliomas. Our previous work demonstrated the expression and function of L1 protein in stimulation of cell motility in rat glioma cells. However, the mechanism of this stimulation is still unclear. This study further investigated the function of L1 and L1 proteolysis in human glioblastoma multiforme (GBM) cell migration and invasion, as well as the mechanism of this stimulation. L1 mRNA was found to be present in human T98G GBM cell line but not in U-118 MG grade III human glioma cell line. L1 protein expression, proteolysis, and release were found in T98G cells and human surgical GBM cells by Western blotting. Exosome-like vesicles released by T98G cells were purified and contained full-length L1. In a scratch assay, T98G cells that migrated into the denuded scratch area exhibited upregulation of ADAM10 protease expression coincident with loss of surface L1. GBM surgical specimen cells exhibited a similar loss of cell surface L1 when xenografted into the chick embryo brain. When lentivirally introduced shRNA was used to attenuate L1 expression, such T98G/shL1 cells exhibited significantly decreased cell motility by time lapse microscopy in our quantitative Super Scratch assay. These cells also showed a decrease in FAK activity and exhibited increased focal complexes. L1 binding integrins which activate FAK were found in T98G and U-118 MG cells. Addition of L1 ectodomain-containing media (1) rescued the decreased cell motility of T98G/shL1 cells and (2) increased cell motility of U-118 MG cells but (3) did not further increase T98G cell motility. Injection of L1-attenuated T98G/shL1 cells into embryonic chick brains resulted in the absence of detectable invasion compared to control cells which invaded brain tissue. These studies support a mechanism where glioma cells at the edge of a cell mass upregulate ADAM10 to proteolyze surface L1 and the resultant ectodomain increases human glioma cell migration and invasion by binding to integrin receptors, activating FAK, and increasing turnover of focal complexes.

Protein kinase D2 regulates migration and invasion of U87MG glioblastoma cells in vitro

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bernhart, Eva; Damm, Sabine; Wintersperger, Andrea

Glioblastoma multiforme (GBM) is the most common malignant brain tumor, which, despite combined modality treatment, reoccurs and is invariably fatal for affected patients. Recently, a member of the serine/threonine protein kinase D (PRKD) family, PRKD2, was shown to be a potent mediator of glioblastoma growth. Here we studied the role of PRKD2 in U87MG glioblastoma cell migration and invasion in response to sphingosine-1-phosphate (S1P), an activator of PRKD2 and a GBM mitogen. Time-lapse microscopy demonstrated that random cell migration was significantly diminished in response to PRKD2 silencing. The pharmacological PRKD family inhibitor CRT0066101 decreased chemotactic migration and invasion across uncoatedmore » or matrigel-coated Transwell inserts. Silencing of PRKD2 attenuated migration and invasion of U87MG cells even more effectively. In terms of downstream signaling, CRT0066101 prevented PRKD2 autophosphorylation and inhibited p44/42 MAPK and to a smaller extent p54/46 JNK and p38 MAPK activation. PRKD2 silencing impaired activation of p44/42 MAPK and p54/46 JNK, downregulated nuclear c-Jun protein levels and decreased c-Jun{sup S73} phosphorylation without affecting the NFκB pathway. Finally, qPCR array analyses revealed that silencing of PRKD2 downregulates mRNA levels of integrin alpha-2 and -4 (ITGA2 and -4), plasminogen activator urokinase (PLAU), plasminogen activator urokinase receptor (PLAUR), and matrix metallopeptidase 1 (MMP1). Findings of the present study identify PRKD2 as a potential target to interfere with glioblastoma cell migration and invasion, two major determinants contributing to recurrence of glioblastoma after multimodality treatment. Highlights: • Sphingosine-1-phosphate induces glioma cell migration and invasion. • Part of the effects is mediated by protein kinase D2 (PRKD2) activation. • Inactivation of PRKD2 attenuates glioblastoma cell migration and invasion. • Both, RNAi and pharmacological inhibition of PRKD2 inhibits MAPK signaling. • PRKD2 regulates transcription of gene products implicated in migration and invasion.« less

Preclinical activity of combined HDAC and KDM1A inhibition in glioblastoma

PubMed Central

Singh, Melissa M.; Johnson, Blake; Venkatarayan, Avinashnarayan; Flores, Elsa R.; Zhang, Jianping; Su, Xiaoping; Barton, Michelle; Lang, Frederick; Chandra, Joya

2015-01-01

Background Glioblastoma (GBM) is the most common and aggressive form of brain cancer. Our previous studies demonstrated that combined inhibition of HDAC and KDM1A increases apoptotic cell death in vitro. However, whether this combination also increases death of the glioma stem cell (GSC) population or has an effect in vivo is yet to be determined. Therefore, we evaluated the translational potential of combined HDAC and KDM1A inhibition on patient-derived GSCs and xenograft GBM mouse models. We also investigated the changes in transcriptional programing induced by the combination in an effort to understand the induced molecular mechanisms of GBM cell death. Methods Patient-derived GSCs were treated with the combination of vorinostat, a pan-HDAC inhibitor, and tranylcypromine, a KDM1A inhibitor, and viability was measured. To characterize transcriptional profiles associated with cell death, we used RNA-Seq and validated gene changes by RT-qPCR and protein expression via Western blot. Apoptosis was measured using DNA fragmentation assays. Orthotopic xenograft studies were conducted to evaluate the effects of the combination on tumorigenesis and to validate gene changes in vivo. Results The combination of vorinostat and tranylcypromine reduced GSC viability and displayed efficacy in the U87 xenograft model. Additionally, the combination led to changes in apoptosis-related genes, particularly TP53 and TP73 in vitro and in vivo. Conclusions These data support targeting HDACs and KDM1A in combination as a strategy for GBM and identifies TP53 and TP73 as being altered in response to treatment. PMID:25795306

Inactivation of the ATMIN/ATM pathway protects against glioblastoma formation

PubMed Central

Blake, Sophia M; Stricker, Stefan H; Halavach, Hanna; Poetsch, Anna R; Cresswell, George; Kelly, Gavin; Kanu, Nnennaya; Marino, Silvia; Luscombe, Nicholas M; Pollard, Steven M; Behrens, Axel

2016-01-01

Glioblastoma multiforme (GBM) is the most aggressive human primary brain cancer. Using a Trp53-deficient mouse model of GBM, we show that genetic inactivation of the Atm cofactor Atmin, which is dispensable for embryonic and adult neural development, strongly suppresses GBM formation. Mechanistically, expression of several GBM-associated genes, including Pdgfra, was normalized by Atmin deletion in the Trp53-null background. Pharmacological ATM inhibition also reduced Pdgfra expression, and reduced the proliferation of Trp53-deficient primary glioma cells from murine and human tumors, while normal neural stem cells were unaffected. Analysis of GBM datasets showed that PDGFRA expression is also significantly increased in human TP53-mutant compared with TP53-wild-type tumors. Moreover, combined treatment with ATM and PDGFRA inhibitors efficiently killed TP53-mutant primary human GBM cells, but not untransformed neural stem cells. These results reveal a new requirement for ATMIN-dependent ATM signaling in TP53-deficient GBM, indicating a pro-tumorigenic role for ATM in the context of these tumors. DOI: http://dx.doi.org/10.7554/eLife.08711.001 PMID:26984279

Surprisal analysis of Glioblastoma Multiform (GBM) microRNA dynamics unveils tumor specific phenotype.

PubMed

Zadran, Sohila; Remacle, Francoise; Levine, Raphael

2014-01-01

Gliomablastoma multiform (GBM) is the most fatal form of all brain cancers in humans. Currently there are limited diagnostic tools for GBM detection. Here, we applied surprisal analysis, a theory grounded in thermodynamics, to unveil how biomolecule energetics, specifically a redistribution of free energy amongst microRNAs (miRNAs), results in a system deviating from a non-cancer state to the GBM cancer -specific phenotypic state. Utilizing global miRNA microarray expression data of normal and GBM patients tumors, surprisal analysis characterizes a miRNA system response capable of distinguishing GBM samples from normal tissue biopsy samples. We indicate that the miRNAs contributing to this system behavior is a disease phenotypic state specific to GBM and is therefore a unique GBM-specific thermodynamic signature. MiRNAs implicated in the regulation of stochastic signaling processes crucial in the hallmarks of human cancer, dominate this GBM-cancer phenotypic state. With this theory, we were able to distinguish with high fidelity GBM patients solely by monitoring the dynamics of miRNAs present in patients' biopsy samples. We anticipate that the GBM-specific thermodynamic signature will provide a critical translational tool in better characterizing cancer types and in the development of future therapeutics for GBM.

Irradiation induces glioblastoma cell senescence and senescence-associated secretory phenotype.

PubMed

Jeon, Hee-Young; Kim, Jun-Kyum; Ham, Seok Won; Oh, Se-Yeong; Kim, Jaebong; Park, Jae-Bong; Lee, Jae-Yong; Kim, Sung-Chan; Kim, Hyunggee

2016-05-01

Glioblastoma multiforme (GBM) is one of the most aggressive and fatal primary brain tumors in humans. The standard therapy for the treatment of GBM is surgical resection, followed by radiotherapy and/or chemotherapy. However, the frequency of tumor recurrence in GBM patients is very high, and the survival rate remains poor. Delineating the mechanisms of GBM recurrence is essential for therapeutic advances. Here, we demonstrate that irradiation rendered 17-20 % of GBM cells dead, but resulted in 60-80 % of GBM cells growth-arrested with increases in senescence markers, such as senescence-associated beta-galactosidase-positive cells, H3K9me3-positive cells, and p53-p21(CIP1)-positive cells. Moreover, irradiation induced expression of senescence-associated secretory phenotype (SASP) mRNAs and NFκB transcriptional activity in GBM cells. Strikingly, compared to injection of non-irradiated GBM cells into immune-deficient mice, the co-injection of irradiated and non-irradiated GBM cells resulted in faster growth of tumors with the histological features of human GBM. Taken together, our findings suggest that the increases in senescent cells and SASP in GBM cells after irradiation is likely one of main reasons for tumor recurrence in post-radiotherapy GBM patients.

Fibrin matrices enhance the transplant and efficacy of cytotoxic stem cell therapy for post-surgical cancer

PubMed Central

Bagó, Juli R.; Pegna, Guillaume J.; Okolie, Onyi; Hingtgen, Shawn D.

2016-01-01

Tumor-homing cytotoxic stem cell (SC) therapy is a promising new approach for treating the incurable brain cancer glioblastoma (GBM). However, problems of retaining cytotoxic SCs within the post-surgical GBM resection cavity are likely to significantly limit the clinical utility of this strategy. Here, we describe a new fibrin-based transplant approach capable of increasing cytotoxic SC retention and persistence within the resection cavity, yet remaining permissive to tumoritropic migration. This fibrin-based transplant can effectively treat both solid and post-surgical human GBM in mice. Using our murine model of image-guided model of GBM resection, we discovered that suspending human mesenchymal stem cells (hMSCS) in a fibrin matrix increased initial retention in the surgical resection cavity 2-fold and prolonged persistence in the cavity 3-fold compared to conventional delivery strategies. Time-lapse motion analysis revealed that cytotoxic hMSCs in the fibrin matrix remain tumoritropic, rapidly migrating from the fibrin matrix to co-localize with cultured human GBM cells. We encapsulated hMSCs releasing the cytotoxic agent TRAIL (hMSC-sTR) in fibrin, and found hMSC-sTR/fibrin therapy reduced the viability of multiple 3-D human GBM spheroids and regressed established human GBM xenografts 3-fold in 11 days. Mimicking clinical therapy of surgically resected GBM, intra-cavity seeding of therapeutic hMSC-sTR encapsulated in fibrin reduced post-surgical GBM volumes 6-fold, increased time to recurrence 4-fold, and prolonged median survival from 15 to 36 days compared to control-treated animals. Fibrin-based SC therapy could represent a clinically compatible, viable treatment to suppress recurrence of post-surgical GBM and other lethal cancer types. PMID:26803410

Osthole Suppresses the Migratory Ability of Human Glioblastoma Multiforme Cells via Inhibition of Focal Adhesion Kinase-Mediated Matrix Metalloproteinase-13 Expression

PubMed Central

Tsai, Cheng-Fang; Yeh, Wei-Lan; Chen, Jia-Hong; Lin, Chingju; Huang, Shiang-Suo; Lu, Dah-Yuu

2014-01-01

Glioblastoma multiforme (GBM) is the most common type of primary and malignant tumor occurring in the adult central nervous system. GBM often invades surrounding regions of the brain during its early stages, making successful treatment difficult. Osthole, an active constituent isolated from the dried C. monnieri fruit, has been shown to suppress tumor migration and invasion. However, the effects of osthole in human GBM are largely unknown. Focal adhesion kinase (FAK) is important for the metastasis of cancer cells. Results from this study show that osthole can not only induce cell death but also inhibit phosphorylation of FAK in human GBM cells. Results from this study show that incubating GBM cells with osthole reduces matrix metalloproteinase (MMP)-13 expression and cell motility, as assessed by cell transwell and wound healing assays. This study also provides evidence supporting the potential of osthole in reducing FAK activation, MMP-13 expression, and cell motility in human GBM cells. PMID:24599080

Isolation and characterization of novel RECK tumor suppressor gene splice variants

PubMed Central

Trombetta-Lima, Marina; Winnischofer, Sheila Maria Brochado; Demasi, Marcos Angelo Almeida; Filho, Renato Astorino; Carreira, Ana Claudia Oliveira; Wei, Beiyang; de Assis Ribas, Thais; Konig, Michelle Silberspitz; Bowman-Colin, Christian; Oba-Shinjo, Sueli Mieko; Marie, Suely Kazue Nagahashi; Stetler-Stevenson, William; Sogayar, Mari Cleide

2015-01-01

Glioblastoma multiforme is the most common and lethal of the central nervous system glial-derived tumors. RECK suppresses tumor invasion by negatively regulating at least three members of the matrix metalloproteinase family: MMP-9, MMP-2, and MT1-MMP. A positive correlation has been observed between the abundance of RECK expression in tumor samples and a more favorable prognosis for patients with several types of tumors. In the present study, novel alternatively spliced variants of the RECK gene: RECK-B and RECK-I were isolated by RT-PCR and sequenced. The expression levels and profiles of these alternative RECK transcripts, as well as canonical RECK were determined in tissue samples of malignant astrocytomas of different grades and in a normal tissue RNA panel by qRT-PCR. Our results show that higher canonical RECK expression, accompanied by a higher canonical to alternative transcript expression ratio, positively correlates with higher overall survival rate after chemotherapeutic treatment of GBM patients. U87MG and T98G cells over-expressing the RECK-B alternative variant display higher anchorage-independent clonal growth and do not display modulation of, respectively, MMP-2 and MMP-9 expression. Our findings suggest that RECK transcript variants might have opposite roles in GBM biology and the ratio of their expression levels may be informative for the prognostic outcome of GBM patients. PMID:26431549

Natural killer (NK) cells inhibit systemic metastasis of glioblastoma cells and have therapeutic effects against glioblastomas in the brain.

PubMed

Lee, Se Jeong; Kang, Won Young; Yoon, Yeup; Jin, Ju Youn; Song, Hye Jin; Her, Jung Hyun; Kang, Sang Mi; Hwang, Yu Kyeong; Kang, Kyeong Jin; Joo, Kyeung Min; Nam, Do-Hyun

2015-12-24

Glioblastoma multiforme (GBM) is characterized by extensive local invasion, which is in contrast with extremely rare systemic metastasis of GBM. Molecular mechanisms inhibiting systemic metastasis of GBM would be a novel therapeutic candidate for GBM in the brain. Patient-derived GBM cells were primarily cultured from surgical samples of GBM patients and were inoculated into the brains of immune deficient BALB/c-nude or NOD-SCID IL2Rgamma(null) (NSG) mice. Human NK cells were isolated from peripheral blood mononucleated cells and expanded in vitro. Patient-derived GBM cells in the brains of NSG mice unexpectedly induced spontaneous lung metastasis although no metastasis was detected in BALB/c-nude mice. Based on the difference of the innate immunity between two mouse strains, NK cell activities of orthotopic GBM xenograft models based on BALB/c-nude mice were inhibited. NK cell inactivation induced spontaneous lung metastasis of GBM cells, which indicated that NK cells inhibit the systemic metastasis. In vitro cytotoxic activities of human NK cells against GBM cells indicated that cytotoxic activity of NK cells against GBM cells prevents systemic metastasis of GBM and that NK cells could be effective cell therapeutics against GBM. Accordingly, NK cells transplanted into orthotopic GBM xenograft models intravenously or intratumorally induced apoptosis of GBM cells in the brain and showed significant therapeutic effects. Our results suggest that innate NK immunity is responsible for rare systemic metastasis of GBM and that sufficient supplementation of NK cells could be a promising immunotherapeutic strategy for GBM in the brain.

The Accuracy of GBM GRB Localizations

NASA Astrophysics Data System (ADS)

Briggs, Michael Stephen; Connaughton, V.; Meegan, C.; Hurley, K.

2010-03-01

We report an study of the accuracy of GBM GRB localizations, analyzing three types of localizations: those produced automatically by the GBM Flight Software on board GBM, those produced automatically with ground software in near real time, and localizations produced with human guidance. The two types of automatic locations are distributed in near real-time via GCN Notices; the human-guided locations are distributed on timescale of many minutes or hours using GCN Circulars. This work uses a Bayesian analysis that models the distribution of the GBM total location error by comparing GBM locations to more accurate locations obtained with other instruments. Reference locations are obtained from Swift, Super-AGILE, the LAT, and with the IPN. We model the GBM total location errors as having systematic errors in addition to the statistical errors and use the Bayesian analysis to constrain the systematic errors.

High content screening of patient-derived cell lines highlights the potential of non-standard chemotherapeutic agents for the treatment of glioblastoma.

PubMed

Yu, Kenny Kwok-Hei; Taylor, Jessica T; Pathmanaban, Omar N; Youshani, Amir Saam; Beyit, Deniz; Dutko-Gwozdz, Joanna; Benson, Roderick; Griffiths, Gareth; Peers, Ian; Cueppens, Peter; Telfer, Brian A; Williams, Kaye J; McBain, Catherine; Kamaly-Asl, Ian D; Bigger, Brian W

2018-01-01

Glioblastoma (GBM) is the most common primary brain malignancy in adults, yet survival outcomes remain poor. First line treatment is well established, however disease invariably recurs and improving prognosis is challenging. With the aim of personalizing therapy at recurrence, we have established a high content screening (HCS) platform to analyze the sensitivity profile of seven patient-derived cancer stem cell lines to 83 FDA-approved chemotherapy drugs, with and without irradiation. Seven cancer stem cell lines were derived from patients with GBM and, along with the established cell line U87-MG, each patient-derived line was cultured in tandem in serum-free conditions as adherent monolayers and three-dimensional neurospheres. Chemotherapeutics were screened at multiple concentrations and cells double-stained to observe their effect on both cell death and proliferation. Sensitivity was classified using high-throughput algorithmic image analysis. Cell line specific drug responses were observed across the seven patient-derived cell lines. Few agents were seen to have radio-sensitizing effects, yet some drug classes showed a marked difference in efficacy between monolayers and neurospheres. In vivo validation of six drugs suggested that cell death readout in a three-dimensional culture scenario is a more physiologically relevant screening model and could be used effectively to assess the chemosensitivity of patient-derived GBM lines. The study puts forward a number of non-standard chemotherapeutics that could be useful in the treatment of recurrent GBM, namely mitoxantrone, bortezomib and actinomycin D, whilst demonstrating the potential of HCS to be used for personalized treatment based on the chemosensitivity profile of patient tumor cells.

Role and Mechanism of Structural Variation in Progression of Breast Cancer

DTIC Science & Technology

2012-09-01

models  of  CGR  genesis,  and  strongly  argue...numbers  of  tandem   duplications,  and   GBM  samples  showing  numerous  large-­‐scale  rearrangements.  We  also...higher   incidence  in   GBM  (38.9%)  relative  to  the  other  tumor  types  (8.7%).   This  definitively  shows

Phenotypic characterization of glioblastoma identified through shape descriptors

NASA Astrophysics Data System (ADS)

Chaddad, Ahmad; Desrosiers, Christian; Toews, Matthew

2016-03-01

This paper proposes quantitatively describing the shape of glioblastoma (GBM) tissue phenotypes as a set of shape features derived from segmentations, for the purposes of discriminating between GBM phenotypes and monitoring tumor progression. GBM patients were identified from the Cancer Genome Atlas, and quantitative MR imaging data were obtained from the Cancer Imaging Archive. Three GBM tissue phenotypes are considered including necrosis, active tumor and edema/invasion. Volumetric tissue segmentations are obtained from registered T1˗weighted (T1˗WI) postcontrast and fluid-attenuated inversion recovery (FLAIR) MRI modalities. Shape features are computed from respective tissue phenotype segmentations, and a Kruskal-Wallis test was employed to select features capable of classification with a significance level of p < 0.05. Several classifier models are employed to distinguish phenotypes, where a leave-one-out cross-validation was performed. Eight features were found statistically significant for classifying GBM phenotypes with p <0.05, orientation is uninformative. Quantitative evaluations show the SVM results in the highest classification accuracy of 87.50%, sensitivity of 94.59% and specificity of 92.77%. In summary, the shape descriptors proposed in this work show high performance in predicting GBM tissue phenotypes. They are thus closely linked to morphological characteristics of GBM phenotypes and could potentially be used in a computer assisted labeling system.

Metformin Treatment Inhibits Motility and Invasion of Glioblastoma Cancer Cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Al Hassan, Marwa; Fakhoury, Isabelle; El Masri, Zeinab

Glioblastoma multiforme (GBM) is one of the most common and deadliest cancers of the central nervous system (CNS). GBMs high ability to infiltrate healthy brain tissues makes it difficult to remove surgically and account for its fatal outcomes. To improve the chances of survival, it is critical to screen for GBM-targeted anticancer agents with anti-invasive and antimigratory potential. Metformin, a commonly used drug for the treatment of diabetes, has recently emerged as a promising anticancer molecule. This prompted us, to investigate the anticancer potential of metformin against GBMs, specifically its effects on cell motility and invasion. The results show amore » significant decrease in the survival of SF268 cancer cells in response to treatment with metformin. Furthermore, metformin’s efficiency in inhibiting 2D cell motility and cell invasion in addition to increasing cellular adhesion was also demonstrated in SF268 and U87 cells. Finally, AKT inactivation by downregulation of the phosphorylation level upon metformin treatment was also evidenced. In conclusion, this study provides insights into the anti-invasive antimetastatic potential of metformin as well as its underlying mechanism of action.« less

Metformin Treatment Inhibits Motility and Invasion of Glioblastoma Cancer Cells

DOE PAGES

Al Hassan, Marwa; Fakhoury, Isabelle; El Masri, Zeinab; ...

2018-06-26

Glioblastoma multiforme (GBM) is one of the most common and deadliest cancers of the central nervous system (CNS). GBMs high ability to infiltrate healthy brain tissues makes it difficult to remove surgically and account for its fatal outcomes. To improve the chances of survival, it is critical to screen for GBM-targeted anticancer agents with anti-invasive and antimigratory potential. Metformin, a commonly used drug for the treatment of diabetes, has recently emerged as a promising anticancer molecule. This prompted us, to investigate the anticancer potential of metformin against GBMs, specifically its effects on cell motility and invasion. The results show amore » significant decrease in the survival of SF268 cancer cells in response to treatment with metformin. Furthermore, metformin’s efficiency in inhibiting 2D cell motility and cell invasion in addition to increasing cellular adhesion was also demonstrated in SF268 and U87 cells. Finally, AKT inactivation by downregulation of the phosphorylation level upon metformin treatment was also evidenced. In conclusion, this study provides insights into the anti-invasive antimetastatic potential of metformin as well as its underlying mechanism of action.« less

Dominant Negative Pleiotrophin Induces Tetraploidy and Aneuploidy in U87MG Human Glioblastoma Cells

PubMed Central

Chang, Yunchao; Berenson, James R.; Wang, Zhaoyi; Deuel, Thomas F.

2007-01-01

Summary Pleiotrophin (PTN, Ptn) is an 18 kD secretory cytokine that is expressed in many human cancers, including glioblastoma. In previous experiments, interruption of the constitutive PTN signaling in human U87MG glioblastoma cells that inappropriately express endogenous Ptn reversed their rapid growth in vitro and their malignant phenotype in vivo. To seek a mechanism for the effect of the dominant negative PTN, flow cytometry was used to compare the profiles of U87MG cells and four clones of U87MG cells that express the dominant negative PTN (U87MG/PTN 1–40 cells); here, we report that the dominant negative PTN in U87MG cells induces tetraploidy and aneuploidy and arrests the tetraploid and aneuploid cells in the G1 phase of the cell cycle. The data suggest that PTN signaling may have a critical role in chromosomal segregation and cell cycle progression; the data suggest induction of tetraploidy and aneuploidy in U87MG glioblastoma cells may be an important mechanism that contributes to the loss of the malignant phenotype of U87MG cells. PMID:17067552

Quantitative Analyses of Synergistic Responses between Cannabidiol and DNA-Damaging Agents on the Proliferation and Viability of Glioblastoma and Neural Progenitor Cells in Culture

PubMed Central

Deng, Liting; Ng, Lindsay; Ozawa, Tatsuya

2017-01-01

Evidence suggests that the nonpsychotropic cannabis-derived compound, cannabidiol (CBD), has antineoplastic activity in multiple types of cancers, including glioblastoma multiforme (GBM). DNA-damaging agents remain the main standard of care treatment available for patients diagnosed with GBM. Here we studied the antiproliferative and cell-killing activity of CBD alone and in combination with DNA-damaging agents (temozolomide, carmustine, or cisplatin) in several human GBM cell lines and in mouse primary GBM cells in cultures. This activity was also studied in mouse neural progenitor cells (NPCs) in culture to assess for potential central nervous system toxicity. We found that CBD induced a dose-dependent reduction of both proliferation and viability of all cells with similar potencies, suggesting no preferential activity for cancer cells. Hill plot analysis indicates an allosteric mechanism of action triggered by CBD in all cells. Cotreatment regimens combining CBD and DNA-damaging agents produced synergistic antiproliferating and cell-killing responses over a limited range of concentrations in all human GBM cell lines and mouse GBM cells as well as in mouse NPCs. Remarkably, antagonistic responses occurred at low concentrations in select human GBM cell lines and in mouse GBM cells. Our study suggests limited synergistic activity when combining CBD and DNA-damaging agents in treating GBM cells, along with little to no therapeutic window when considering NPCs. PMID:27821713

Towards precision medicine-based therapies for glioblastoma: interrogating human disease genomics and mouse phenotypes.

PubMed

Chen, Yang; Gao, Zhen; Wang, Bingcheng; Xu, Rong

2016-08-22

Glioblastoma (GBM) is the most common and aggressive brain tumors. It has poor prognosis even with optimal radio- and chemo-therapies. Since GBM is highly heterogeneous, drugs that target on specific molecular profiles of individual tumors may achieve maximized efficacy. Currently, the Cancer Genome Atlas (TCGA) projects have identified hundreds of GBM-associated genes. We develop a drug repositioning approach combining disease genomics and mouse phenotype data towards predicting targeted therapies for GBM. We first identified disease specific mouse phenotypes using the most recently discovered GBM genes. Then we systematically searched all FDA-approved drugs for candidates that share similar mouse phenotype profiles with GBM. We evaluated the ranks for approved and novel GBM drugs, and compared with an existing approach, which also use the mouse phenotype data but not the disease genomics data. We achieved significantly higher ranks for the approved and novel GBM drugs than the earlier approach. For all positive examples of GBM drugs, we achieved a median rank of 9.2 45.6 of the top predictions have been demonstrated effective in inhibiting the growth of human GBM cells. We developed a computational drug repositioning approach based on both genomic and phenotypic data. Our approach prioritized existing GBM drugs and outperformed a recent approach. Overall, our approach shows potential in discovering new targeted therapies for GBM.

Knockdown of NF-E2-related factor 2 inhibits the proliferation and growth of U251MG human glioma cells in a mouse xenograft model.

PubMed

Ji, Xiang-Jun; Chen, Sui-Hua; Zhu, Lin; Pan, Hao; Zhou, Yuan; Li, Wei; You, Wan-Chun; Gao, Chao-Chao; Zhu, Jian-Hong; Jiang, Kuan; Wang, Han-Dong

2013-07-01

NF-E2-related factor 2 (Nrf2) is a pivotal transcription factor of cellular responses to oxidative stress and recent evidence suggests that Nrf2 plays an important role in cancer pathobiology. However, the underlying mechanism has yet to be elucidated, particularly in glioma. In the present study, we investigated the role of Nrf2 in the clinical prognosis, cell proliferation and tumor growth of human glioblastoma multiforme (GBM). We detected overexpression of Nrf2 protein levels in GBM compared to normal brain tissues. Notably, higher protein levels of Nrf2 were significantly associated with poorer overall survival and 1-year survival for GBM patients. Furthermore, we constructed the plasmid Si-Nrf2 and transduced it into U251MG cells to downregulate the expression of Nrf2 and established stable Nrf2 knockdown cells. The downregulation of Nrf2 suppressed cell proliferation in vitro and tumor growth in mouse xenograft models. We performed immunohistochemistry staining to detect the protein levels of Nrf2, Ki-67, caspase-3 and CD31 in the xenograft tumors and found that the expression levels of Nrf2 and Ki-67 were much lower in the Si-Nrf2 group compared to the Si-control group. In addition, the number of caspase-3-positive cells was significantly increased in the Si-Nrf2 group. By analysis of microvessel density (MVD) assessed by CD31, the MVD value in the Si-Nrf2 group decreased significantly compared to the Si-control group. These findings indicate that the knockdown of Nrf2 may suppress tumor growth by inhibiting cell proliferation, increasing cell apoptosis and inhibiting angiogenesis. These results highlight the potential of Nrf2 as a candidate molecular target to control GBM cell proliferation and tumor growth.

The DNA-PK Inhibitor VX-984 Enhances the Radiosensitivity of Glioblastoma Cells Grown In Vitro and as Orthotopic Xenografts.

PubMed

Timme, Cindy R; Rath, Barbara H; O'Neill, John W; Camphausen, Kevin; Tofilon, Philip J

2018-06-01

Radiotherapy is a primary treatment modality for glioblastomas (GBM). Because DNA-PKcs is a critical factor in the repair of radiation-induced double strand breaks (DSB), this study evaluated the potential of VX-984, a new DNA-PKcs inhibitor, to enhance the radiosensitivity of GBM cells. Treatment of the established GBM cell line U251 and the GBM stem-like cell (GSC) line NSC11 with VX-984 under in vitro conditions resulted in a concentration-dependent inhibition of radiation-induced DNA-PKcs phosphorylation. In a similar concentration-dependent manner, VX-984 treatment enhanced the radiosensitivity of each GBM cell line as defined by clonogenic analysis. As determined by γH2AX expression and neutral comet analyses, VX-984 inhibited the repair of radiation-induced DNA double-strand break in U251 and NSC11 GBM cells, suggesting that the VX-984-induced radiosensitization is mediated by an inhibition of DNA repair. Extending these results to an in vivo model, treatment of mice with VX-984 inhibited radiation-induced DNA-PKcs phosphorylation in orthotopic brain tumor xenografts, indicating that this compound crosses the blood-brain tumor barrier at sufficient concentrations. For mice bearing U251 or NSC11 brain tumors, VX-984 treatment alone had no significant effect on overall survival; radiation alone increased survival. The survival of mice receiving the combination protocol was significantly increased as compared with control and as compared with radiation alone. These results indicate that VX-984 enhances the radiosensitivity of brain tumor xenografts and suggest that it may be of benefit in the therapeutic management of GBM. Mol Cancer Ther; 17(6); 1207-16. ©2018 AACR . ©2018 American Association for Cancer Research.

An intrinsic DFF40/CAD endonuclease deficiency impairs oligonucleosomal DNA hydrolysis during caspase-dependent cell death: a common trait in human glioblastoma cells

PubMed Central

Sánchez-Osuna, María; Martínez-Escardó, Laura; Granados-Colomina, Carla; Martínez-Soler, Fina; Pascual-Guiral, Sònia; Iglesias-Guimarais, Victoria; Velasco, Roser; Plans, Gerard; Vidal, Noemi; Tortosa, Avelina; Barcia, Carlos; Bruna, Jordi; Yuste, Victor J.

2016-01-01

Background Glioblastoma (GBM) or grade IV astrocytoma is one of the most devastating human cancers. The loss of DFF40/CAD, the key endonuclease that triggers oligonucleosomal DNA fragmentation during apoptosis, has been linked to genomic instability and cell survival after radiation. Despite the near inevitability of GBM tumor recurrence after treatment, the relationship between DFF40/CAD and GBM remains unexplored. Methods We studied the apoptotic behavior of human GBM-derived cells after apoptotic insult. We analyzed caspase activation and the protein levels and subcellular localization of DFF40/CAD apoptotic endonuclease. DFF40/CAD was also evaluated in histological sections from astrocytic tumors and nontumoral human brain. Results We showed that GBM cells undergo incomplete apoptosis without generating oligonucleosomal DNA degradation despite the correct activation of executioner caspases. The major defect of GBM cells relied on the improper accumulation of DFF40/CAD at the nucleoplasmic subcellular compartment. Supporting this finding, DFF40/CAD overexpression allowed GBM cells to display oligonucleosomal DNA degradation after apoptotic challenge. Moreover, the analysis of histological slices from astrocytic tumors showed that DFF40/CAD immunoreactivity in tumoral GFAP-positive cells was markedly reduced when compared with nontumoral samples. Conclusions Our data highlight the low expression levels of DFF40/CAD and the absence of DNA laddering as common molecular traits in GBM. These findings could be of major importance for understanding the malignant behavior of remaining tumor cells after radiochemotherapy. PMID:26755073

Quantitative Analyses of Synergistic Responses between Cannabidiol and DNA-Damaging Agents on the Proliferation and Viability of Glioblastoma and Neural Progenitor Cells in Culture.

PubMed

Deng, Liting; Ng, Lindsay; Ozawa, Tatsuya; Stella, Nephi

2017-01-01

Evidence suggests that the nonpsychotropic cannabis-derived compound, cannabidiol (CBD), has antineoplastic activity in multiple types of cancers, including glioblastoma multiforme (GBM). DNA-damaging agents remain the main standard of care treatment available for patients diagnosed with GBM. Here we studied the antiproliferative and cell-killing activity of CBD alone and in combination with DNA-damaging agents (temozolomide, carmustine, or cisplatin) in several human GBM cell lines and in mouse primary GBM cells in cultures. This activity was also studied in mouse neural progenitor cells (NPCs) in culture to assess for potential central nervous system toxicity. We found that CBD induced a dose-dependent reduction of both proliferation and viability of all cells with similar potencies, suggesting no preferential activity for cancer cells. Hill plot analysis indicates an allosteric mechanism of action triggered by CBD in all cells. Cotreatment regimens combining CBD and DNA-damaging agents produced synergistic antiproliferating and cell-killing responses over a limited range of concentrations in all human GBM cell lines and mouse GBM cells as well as in mouse NPCs. Remarkably, antagonistic responses occurred at low concentrations in select human GBM cell lines and in mouse GBM cells. Our study suggests limited synergistic activity when combining CBD and DNA-damaging agents in treating GBM cells, along with little to no therapeutic window when considering NPCs. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

Differential role of EGF and BFGF in human GBM-TIC proliferation: relationship to EGFR-tyrosine kinase inhibitor sensibility.

PubMed

Bajetto, A; Porcile, C; Pattarozzi, A; Scotti, L; Aceto, A; Daga, A; Barbieri, F; Florio, T

2013-01-01

Glioblastoma multiforme (GBM) is among the most devastating human tumors being rapidly fatal despite aggressive surgery, radiation and chemotherapies. It is characterized by extensive dissemination of tumor cells within the brain that hinders complete surgical resection. GBM tumor initiating-cells (TICs) are a rare subpopulation of cells responsible for tumor development, growth, invasiveness and recurrence after chemotherapy. TICs from human GBM can be selected in vitro using the same conditions permissive for the growth of normal neural cells, of which share some features including marker expression, self-renewal capacity, long-term proliferation, and ability to differentiate into neuronal and glial cells. EGFR overexpression and its constitutive activation is one of the most important signaling alteration identified in GBM, and its pharmacological targeting represents an attractive therapeutic goal. We previously demonstrated that human GBM TICs have different sensitivity to the EGFR kinase inhibitors erlotinib and gefitinib, depending on the differential modulation of downstream signaling cascades. In this work we investigated the mechanisms of resistance to erlotinib in two human GBM TIC cultures, analyzing EGF and bFGF individual contribution to proliferation, clonogenicity, and migration. We demonstrated the presence of a small cell subpopulation whose proliferation is supported by EGF and a larger one mainly dependent on bFGF. Thus, insensitivity to EGFR kinase inhibitors as far as TIC proliferation results from a predominant FGFR activation that hides the inhibitory effects induced on EGFR signaling. Conversely, EGF and bFGF induced cell migration with similar efficacy. In addition, unlike neural stem/progenitors cells, the removal of chondroitin sulphate proteoglycans from cell surface was unable to discern EGF- and bFGF-dependent subpopulations in GBM TICs.

NF-κB-Induced IL-6 Ensures STAT3 Activation and Tumor Aggressiveness in Glioblastoma

PubMed Central

McFarland, Braden C.; Hong, Suk W.; Rajbhandari, Rajani; Twitty, George B.; Gray, G. Kenneth; Yu, Hao; Benveniste, Etty N.; Nozell, Susan E.

2013-01-01

Glioblastoma (GBM) is the most aggressive, neurologically destructive and deadly tumor of the central nervous system (CNS). In GBM, the transcription factors NF-κB and STAT3 are aberrantly activated and associated with tumor cell proliferation, survival, invasion and chemoresistance. In addition, common activators of NF-κB and STAT3, including TNF-α and IL-6, respectively, are abundantly expressed in GBM tumors. Herein, we sought to elucidate the signaling crosstalk that occurs between the NF-κB and STAT3 pathways in GBM tumors. Using cultured GBM cell lines as well as primary human GBM xenografts, we elucidated the signaling crosstalk between the NF-κB and STAT3 pathways utilizing approaches that either a) reduce NF-κB p65 expression, b) inhibit NF-κB activation, c) interfere with IL-6 signaling, or d) inhibit STAT3 activation. Using the clinically relevant human GBM xenograft model, we assessed the efficacy of inhibiting NF-κB and/or STAT3 alone or in combination in mice bearing intracranial xenograft tumors in vivo. We demonstrate that TNF-α-induced activation of NF-κB is sufficient to induce IL-6 expression, activate STAT3, and elevate STAT3 target gene expression in GBM cell lines and human GBM xenografts in vitro. Moreover, the combined inhibition of NF-κB and STAT3 signaling significantly increases survival of mice bearing intracranial tumors. We propose that in GBM, the activation of NF-κB ensures subsequent STAT3 activation through the expression of IL-6. These data verify that pharmacological interventions to effectively inhibit the activity of both NF-κB and STAT3 transcription factors must be used in order to reduce glioma size and aggressiveness. PMID:24244348

NF-κB-induced IL-6 ensures STAT3 activation and tumor aggressiveness in glioblastoma.

PubMed

McFarland, Braden C; Hong, Suk W; Rajbhandari, Rajani; Twitty, George B; Gray, G Kenneth; Yu, Hao; Benveniste, Etty N; Nozell, Susan E

2013-01-01

Glioblastoma (GBM) is the most aggressive, neurologically destructive and deadly tumor of the central nervous system (CNS). In GBM, the transcription factors NF-κB and STAT3 are aberrantly activated and associated with tumor cell proliferation, survival, invasion and chemoresistance. In addition, common activators of NF-κB and STAT3, including TNF-α and IL-6, respectively, are abundantly expressed in GBM tumors. Herein, we sought to elucidate the signaling crosstalk that occurs between the NF-κB and STAT3 pathways in GBM tumors. Using cultured GBM cell lines as well as primary human GBM xenografts, we elucidated the signaling crosstalk between the NF-κB and STAT3 pathways utilizing approaches that either a) reduce NF-κB p65 expression, b) inhibit NF-κB activation, c) interfere with IL-6 signaling, or d) inhibit STAT3 activation. Using the clinically relevant human GBM xenograft model, we assessed the efficacy of inhibiting NF-κB and/or STAT3 alone or in combination in mice bearing intracranial xenograft tumors in vivo. We demonstrate that TNF-α-induced activation of NF-κB is sufficient to induce IL-6 expression, activate STAT3, and elevate STAT3 target gene expression in GBM cell lines and human GBM xenografts in vitro. Moreover, the combined inhibition of NF-κB and STAT3 signaling significantly increases survival of mice bearing intracranial tumors. We propose that in GBM, the activation of NF-κB ensures subsequent STAT3 activation through the expression of IL-6. These data verify that pharmacological interventions to effectively inhibit the activity of both NF-κB and STAT3 transcription factors must be used in order to reduce glioma size and aggressiveness.

Proteasome inhibitor MG132 induces selective apoptosis in glioblastoma cells through inhibition of PI3K/Akt and NFkappaB pathways, mitochondrial dysfunction, and activation of p38-JNK1/2 signaling.

PubMed

Zanotto-Filho, Alfeu; Braganhol, Elizandra; Battastini, Ana Maria Oliveira; Moreira, José Cláudio Fonseca

2012-12-01

Proteasome inhibitors are emerging as a new class of anticancer agents. In this work, we examined the mechanisms underlying cytotoxicity, selectivity and adjuvant potential of the proteasome inhibitor MG132 in a panel of glioblastoma (GBM) cells (U138MG, C6, U87 and U373) and in normal astrocytes. MG132 markedly inhibited GBM cells growth irrespective of the p53 or PTEN mutational status of the cells whereas astrocytic viability was not affected, suggesting a selective toxicity of MG132 to cancerous glial cells. Mechanistically, MG132 arrested cells in G2/M phase of the cell cycle and increased p21(WAF1) protein immunocontent. Following cell arrest, cells become apoptotic as shown by annexin-V binding, caspase-3 activation, chromatin condensation and formation of sub-G1 apoptotic cells. MG132 promoted mitochondrial depolarization and decreased the mitochondrial antiapoptotic protein bcl-xL; it also induced activation of JNK and p38, and inhibition of NFkappaB and PI3K/Akt survival pathways. Pre-treatment of GBMs with the mitochondrial permeability transition pore inhibitor, bongkrekic acid, or pharmacological inhibitors of JNK1/2 and p38, SP600125 and SB203580, attenuated MG132-induced cell death. Besides its apoptotic effect alone, MG132 also enhanced the antiglioma effect of the chemotherapeutics cisplatin, taxol and doxorubicin in C6 and U138MG cells, indicating an adjuvant/chemosensitizer potential. In summary, MG132 exerted profound and selective toxicity in GBMs, being a potential agent for further testing in animal models of the disease.

The orthotopic xenotransplant of human glioblastoma successfully recapitulates glioblastoma-microenvironment interactions in a non-immunosuppressed mouse model.

PubMed

Garcia, Celina; Dubois, Luiz Gustavo; Xavier, Anna Lenice; Geraldo, Luiz Henrique; da Fonseca, Anna Carolina Carvalho; Correia, Ana Helena; Meirelles, Fernanda; Ventura, Grasiella; Romão, Luciana; Canedo, Nathalie Henriques Silva; de Souza, Jorge Marcondes; de Menezes, João Ricardo Lacerda; Moura-Neto, Vivaldo; Tovar-Moll, Fernanda; Lima, Flavia Regina Souza

2014-12-08

Glioblastoma (GBM) is the most common primary brain tumor and the most aggressive glial tumor. This tumor is highly heterogeneous, angiogenic, and insensitive to radio- and chemotherapy. Here we have investigated the progression of GBM produced by the injection of human GBM cells into the brain parenchyma of immunocompetent mice. Xenotransplanted animals were submitted to magnetic resonance imaging (MRI) and histopathological analyses. Our data show that two weeks after injection, the produced tumor presents histopathological characteristics recommended by World Health Organization for the diagnosis of GBM in humans. The tumor was able to produce reactive gliosis in the adjacent parenchyma, angiogenesis, an intense recruitment of macrophage and microglial cells, and presence of necrosis regions. Besides, MRI showed that tumor mass had enhanced contrast, suggesting a blood-brain barrier disruption. This study demonstrated that the xenografted tumor in mouse brain parenchyma develops in a very similar manner to those found in patients affected by GBM and can be used to better understand the biology of GBM as well as testing potential therapies.

The Human Glioblastoma Cell Culture Resource: Validated Cell Models Representing All Molecular Subtypes.

PubMed

Xie, Yuan; Bergström, Tobias; Jiang, Yiwen; Johansson, Patrik; Marinescu, Voichita Dana; Lindberg, Nanna; Segerman, Anna; Wicher, Grzegorz; Niklasson, Mia; Baskaran, Sathishkumar; Sreedharan, Smitha; Everlien, Isabelle; Kastemar, Marianne; Hermansson, Annika; Elfineh, Lioudmila; Libard, Sylwia; Holland, Eric Charles; Hesselager, Göran; Alafuzoff, Irina; Westermark, Bengt; Nelander, Sven; Forsberg-Nilsson, Karin; Uhrbom, Lene

2015-10-01

Glioblastoma (GBM) is the most frequent and malignant form of primary brain tumor. GBM is essentially incurable and its resistance to therapy is attributed to a subpopulation of cells called glioma stem cells (GSCs). To meet the present shortage of relevant GBM cell (GC) lines we developed a library of annotated and validated cell lines derived from surgical samples of GBM patients, maintained under conditions to preserve GSC characteristics. This collection, which we call the Human Glioblastoma Cell Culture (HGCC) resource, consists of a biobank of 48 GC lines and an associated database containing high-resolution molecular data. We demonstrate that the HGCC lines are tumorigenic, harbor genomic lesions characteristic of GBMs, and represent all four transcriptional subtypes. The HGCC panel provides an open resource for in vitro and in vivo modeling of a large part of GBM diversity useful to both basic and translational GBM research.

Development of Advanced Technologies for Complete Genomic and Proteomic Characterization of Quantized Human Tumor Cells

DTIC Science & Technology

2014-07-01

establishment of Glioblastoma ( GBM ) cell lines from GBM patient’s tumor samples and quantized cell populations of each of the parental GBM cell lines, we... GBM patients are now well established and from the basis of the molecular characterization of the tumor development and signatures presented by these...analysis of these quantized cell sub populations and have begun to assemble the protein signatures of GBM tumors underpinned by the comprehensive

Agrin is a major heparan sulfate proteoglycan in the human glomerular basement membrane.

PubMed

Groffen, A J; Ruegg, M A; Dijkman, H; van de Velden, T J; Buskens, C A; van den Born, J; Assmann, K J; Monnens, L A; Veerkamp, J H; van den Heuvel, L P

1998-01-01

Agrin is a heparan sulfate proteoglycan (HSPG) that is highly concentrated in the synaptic basal lamina at the neuromuscular junction (NMJ). Agrin-like immunoreactivity is also detected outside the NMJ. Here we show that agrin is a major HSPG component of the human glomerular basement membrane (GBM). This is in addition to perlecan, a previously characterized HSPG of basement membranes. Antibodies against agrin and against an unidentified GBM HSPG produced a strong staining of the GBM and the NMJ, different from that observed with anti-perlecan antibodies. In addition, anti-agrin antisera recognized purified GBM HSPG and competed with an anti-GBM HSPG monoclonal antibody in ELISA. Furthermore, both antibodies recognized a molecule that migrated in SDS-PAGE as a smear and had a molecular mass of approximately 200-210 kD after deglycosylation. In immunoelectron microscopy, agrin showed a linear distribution along the GBM and was present throughout the width of the GBM. This was again different from perlecan, which was exclusively present on the endothelial side of the GBM and was distributed in a nonlinear manner. Quantitative ELISA showed that, compared with perlecan, the agrin-like GBM HSPG showed a sixfold higher molarity in crude glomerular extract. These results show that agrin is a major component of the GBM, indicating that it may play a role in renal ultrafiltration and cell matrix interaction. (J Histochem Cytochem 46:19-27, 1998)

TCGA Workshop: Genomics and Biology of Glioblastoma Multiforme (GBM) - TCGA

Cancer.gov

The National Cancer Institute (NCI) and National Human Genome Research Institute (NHGRI) held a workshop entitled, “Genomics and Biology of Glioblastoma Multiforme (GBM),” to review the initial GBM data from the TCGA pilot project.

Blockade of the SNARE Protein Syntaxin 1 Inhibits Glioblastoma Tumor Growth

PubMed Central

Ulloa, Fausto; Gonzàlez-Juncà, Alba; Meffre, Delphine; Barrecheguren, Pablo José; Martínez-Mármol, Ramón; Pazos, Irene; Olivé, Núria; Cotrufo, Tiziana; Seoane, Joan; Soriano, Eduardo

2015-01-01

Glioblastoma (GBM) is the most prevalent adult brain tumor, with virtually no cure, and with a median overall survival of 15 months from diagnosis despite of the treatment. SNARE proteins mediate membrane fusion events in cells and are essential for many cellular processes including exocytosis and neurotransmission, intracellular trafficking and cell migration. Here we show that the blockade of the SNARE protein Syntaxin 1 (Stx1) function impairs GBM cell proliferation. We show that Stx1 loss-of-function in GBM cells, through ShRNA lentiviral transduction, a Stx1 dominant negative and botulinum toxins, dramatically reduces the growth of GBM after grafting U373 cells into the brain of immune compromised mice. Interestingly, Stx1 role on GBM progression may not be restricted just to cell proliferation since the blockade of Stx1 also reduces in vitro GBM cell invasiveness suggesting a role in several processes relevant for tumor progression. Altogether, our findings indicate that the blockade of SNARE proteins may represent a novel therapeutic tool against GBM. PMID:25803850

An intrinsic DFF40/CAD endonuclease deficiency impairs oligonucleosomal DNA hydrolysis during caspase-dependent cell death: a common trait in human glioblastoma cells.

PubMed

Sánchez-Osuna, María; Martínez-Escardó, Laura; Granados-Colomina, Carla; Martínez-Soler, Fina; Pascual-Guiral, Sònia; Iglesias-Guimarais, Victoria; Velasco, Roser; Plans, Gerard; Vidal, Noemi; Tortosa, Avelina; Barcia, Carlos; Bruna, Jordi; Yuste, Victor J

2016-07-01

Glioblastoma (GBM) or grade IV astrocytoma is one of the most devastating human cancers. The loss of DFF40/CAD, the key endonuclease that triggers oligonucleosomal DNA fragmentation during apoptosis, has been linked to genomic instability and cell survival after radiation. Despite the near inevitability of GBM tumor recurrence after treatment, the relationship between DFF40/CAD and GBM remains unexplored. We studied the apoptotic behavior of human GBM-derived cells after apoptotic insult. We analyzed caspase activation and the protein levels and subcellular localization of DFF40/CAD apoptotic endonuclease. DFF40/CAD was also evaluated in histological sections from astrocytic tumors and nontumoral human brain. We showed that GBM cells undergo incomplete apoptosis without generating oligonucleosomal DNA degradation despite the correct activation of executioner caspases. The major defect of GBM cells relied on the improper accumulation of DFF40/CAD at the nucleoplasmic subcellular compartment. Supporting this finding, DFF40/CAD overexpression allowed GBM cells to display oligonucleosomal DNA degradation after apoptotic challenge. Moreover, the analysis of histological slices from astrocytic tumors showed that DFF40/CAD immunoreactivity in tumoral GFAP-positive cells was markedly reduced when compared with nontumoral samples. Our data highlight the low expression levels of DFF40/CAD and the absence of DNA laddering as common molecular traits in GBM. These findings could be of major importance for understanding the malignant behavior of remaining tumor cells after radiochemotherapy. © The Author(s) 2016. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Inhibitor of Nicotinamide Phosphoribosyltransferase Sensitizes Glioblastoma Cells to Temozolomide via Activating ROS/JNK Signaling Pathway.

PubMed

Feng, Jun; Yan, Peng-Fei; Zhao, Hong-Yang; Zhang, Fang-Cheng; Zhao, Wo-Hua; Feng, Min

2016-01-01

Overcoming temozolomide (TMZ) resistance is a great challenge in glioblastoma (GBM) treatment. Nicotinamide phosphoribosyltransferase (NAMPT) is a rate-limiting enzyme in the biosynthesis of nicotinamide adenine dinucleotide and has a crucial role in cancer cell metabolism. In this study, we investigated whether FK866 and CHS828, two specific NAMPT inhibitors, could sensitize GBM cells to TMZ. Low doses of FK866 and CHS828 (5 nM and 10 nM, resp.) alone did not significantly decrease cell viability in U251-MG and T98 GBM cells. However, they significantly increased the antitumor action of TMZ in these cells. In U251-MG cells, administration of NAMPT inhibitors increased the TMZ (100  μ M)-induced apoptosis and LDH release from GBM cells. NAMPT inhibitors remarkably enhanced the activities of caspase-1, caspase-3, and caspase-9. Moreover, NAMPT inhibitors increased reactive oxygen species (ROS) production and superoxide anion level but reduced the SOD activity and total antioxidative capacity in GBM cells. Treatment of NAMPT inhibitors increased phosphorylation of c-Jun and JNK. Administration of JNK inhibitor SP600125 or ROS scavenger tocopherol with TMZ and NAMPT inhibitors substantially attenuated the sensitization of NAMPT inhibitor on TMZ antitumor action. Our data indicate a potential value of NAMPT inhibitors in combined use with TMZ for GBM treatment.

Combination therapy in a xenograft model of glioblastoma: enhancement of the antitumor activity of temozolomide by an MDM2 antagonist.

PubMed

Wang, Haiyan; Cai, Shanbao; Bailey, Barbara J; Reza Saadatzadeh, M; Ding, Jixin; Tonsing-Carter, Eva; Georgiadis, Taxiarchis M; Zachary Gunter, T; Long, Eric C; Minto, Robert E; Gordon, Kevin R; Sen, Stephanie E; Cai, Wenjing; Eitel, Jacob A; Waning, David L; Bringman, Lauren R; Wells, Clark D; Murray, Mary E; Sarkaria, Jann N; Gelbert, Lawrence M; Jones, David R; Cohen-Gadol, Aaron A; Mayo, Lindsey D; Shannon, Harlan E; Pollok, Karen E

2017-02-01

OBJECTIVE Improvement in treatment outcome for patients with glioblastoma multiforme (GBM) requires a multifaceted approach due to dysregulation of numerous signaling pathways. The murine double minute 2 (MDM2) protein may fulfill this requirement because it is involved in the regulation of growth, survival, and invasion. The objective of this study was to investigate the impact of modulating MDM2 function in combination with front-line temozolomide (TMZ) therapy in GBM. METHODS The combination of TMZ with the MDM2 protein-protein interaction inhibitor nutlin3a was evaluated for effects on cell growth, p53 pathway activation, expression of DNA repair proteins, and invasive properties. In vivo efficacy was assessed in xenograft models of human GBM. RESULTS In combination, TMZ/nutlin3a was additive to synergistic in decreasing growth of wild-type p53 GBM cells. Pharmacodynamic studies demonstrated that inhibition of cell growth following exposure to TMZ/nutlin3a correlated with: 1) activation of the p53 pathway, 2) downregulation of DNA repair proteins, 3) persistence of DNA damage, and 4) decreased invasion. Pharmacokinetic studies indicated that nutlin3a was detected in human intracranial tumor xenografts. To assess therapeutic potential, efficacy studies were conducted in a xenograft model of intracranial GBM by using GBM cells derived from a recurrent wild-type p53 GBM that is highly TMZ resistant (GBM10). Three 5-day cycles of TMZ/nutlin3a resulted in a significant increase in the survival of mice with GBM10 intracranial tumors compared with single-agent therapy. CONCLUSIONS Modulation of MDM2/p53-associated signaling pathways is a novel approach for decreasing TMZ resistance in GBM. To the authors' knowledge, this is the first study in a humanized intracranial patient-derived xenograft model to demonstrate the efficacy of combining front-line TMZ therapy and an inhibitor of MDM2 protein-protein interactions.

Potentiation of cytotoxic chemotherapy by growth hormone-releasing hormone agonists.

PubMed

Jaszberenyi, Miklos; Rick, Ferenc G; Popovics, Petra; Block, Norman L; Zarandi, Marta; Cai, Ren-Zhi; Vidaurre, Irving; Szalontay, Luca; Jayakumar, Arumugam R; Schally, Andrew V

2014-01-14

The dismal prognosis of malignant brain tumors drives the development of new treatment modalities. In view of the multiple activities of growth hormone-releasing hormone (GHRH), we hypothesized that pretreatment with a GHRH agonist, JI-34, might increase the susceptibility of U-87 MG glioblastoma multiforme (GBM) cells to subsequent treatment with the cytotoxic drug, doxorubicin (DOX). This concept was corroborated by our findings, in vivo, showing that the combination of the GHRH agonist, JI-34, and DOX inhibited the growth of GBM tumors, transplanted into nude mice, more than DOX alone. In vitro, the pretreatment of GBM cells with JI-34 potentiated inhibitory effects of DOX on cell proliferation, diminished cell size and viability, and promoted apoptotic processes, as shown by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide proliferation assay, ApoLive-Glo multiplex assay, and cell volumetric assay. Proteomic studies further revealed that the pretreatment with GHRH agonist evoked differentiation decreasing the expression of the neuroectodermal stem cell antigen, nestin, and up-regulating the glial maturation marker, GFAP. The GHRH agonist also reduced the release of humoral regulators of glial growth, such as FGF basic and TGFβ. Proteomic and gene-expression (RT-PCR) studies confirmed the strong proapoptotic activity (increase in p53, decrease in v-myc and Bcl-2) and anti-invasive potential (decrease in integrin α3) of the combination of GHRH agonist and DOX. These findings indicate that the GHRH agonists can potentiate the anticancer activity of the traditional chemotherapeutic drug, DOX, by multiple mechanisms including the induction of differentiation of cancer cells.

MERTK Inhibition Induces Polyploidy and Promotes Cell Death and Cellular Senescence in Glioblastoma Multiforme

PubMed Central

Sufit, Alexandra; Lee-Sherick, Alisa B.; DeRyckere, Deborah; Rupji, Manali; Dwivedi, Bhakti; Varella-Garcia, Marileila; Pierce, Angela M.; Kowalski, Jeanne; Wang, Xiaodong; Frye, Stephen V.; Earp, H. Shelton

2016-01-01

Background MER receptor tyrosine kinase (MERTK) is expressed in a variety of malignancies, including glioblastoma multiforme (GBM). Our previous work demonstrated that inhibition of MERTK using RNA interference induced cell death and chemosensitivity in GBM cells, implicating MERTK as a potential therapeutic target. Here we investigate whether a novel MERTK-selective small molecule tyrosine kinase inhibitor, UNC2025, has similar anti-tumor effects in GBM cell lines. Methods Correlations between expression of GAS6, a MERTK ligand, and prognosis were determined using data from the TCGA database. GBM cell lines (A172, SF188, U251) were treated in vitro with increasing doses of UNC2025 (50-400nM). Cell count and viability were determined by trypan blue exclusion. Cell cycle profiles and induction of apoptosis were assessed by flow cytometric analysis after BrdU or Po-Pro-1/propidium iodide staining, respectively. Polyploidy was detected by propidium iodide staining and metaphase spread. Cellular senescence was determined by β-galactosidase staining and senescence-associated secretory cytokine analysis. Results Decreased overall survival significantly correlated with high levels of GAS6 expression in GBM, highlighting the importance of TAM kinase signaling in GBM tumorigenesis and/or therapy resistance and providing strong rationale for targeting these pathways in the clinic. All three GBM cell lines exhibited dose dependent reductions in cell number and colony formation (>90% at 200nM) after treatment with UNC2025. Cell cycle analysis demonstrated accumulation of cells in the G2/M phase and development of polyploidy. After extended exposure, 60–80% of cells underwent apoptosis. The majority of surviving cells (65–95%) were senescent and did not recover after drug removal. Thus, UNC2025 mediates anti-tumor activity in GBM by multiple mechanisms. Conclusions The findings described here provide further evidence of oncogenic roles for MERTK in GBM, demonstrate the importance of kinase activity for MERTK tumorigenicity and validate UNC2025, a novel MERTK inhibitor, as a potential therapeutic agent for treatment of GBM. PMID:27783662

Electrospun nanofibrous scaffolds increase the efficacy of stem cell-mediated therapy of surgically resected glioblastoma

PubMed Central

Bagó, Juli R.; Pegna, Guillaume J.; Okolie, Onyi; Mohiti-Asli, Mahsa; Loboa, Elizabeth G.; Hingtgen, Shawn D.

2017-01-01

Engineered stem cell (SC)-based therapy holds enormous promise for treating the incurable brain cancer glioblastoma (GBM). Retaining the cytotoxic SCs in the surgical cavity after GBM resection is one of the greatest challenges to this approach. Here, we describe a biocompatible electrospun nanofibrous scaffold (bENS) implant capable of delivering and retaining tumor-homing cytotoxic stem cells that suppress recurrence of post-surgical GBM. As a new approach to GBM therapy, we created poly(l-lactic acid) (PLA) bENS bearing drug-releasing human mesenchymal stem cells (hMSCs). We discovered that bENS-based implant increased hMSC retention in the surgical cavity 5-fold and prolonged persistence 3-fold compared to standard direct injection using our mouse model of GBM surgical resection/recurrence. Time-lapse imaging showed cytotoxic hMSC/bENS treatment killed co-cultured human GBM cells, and allowed hMSCs to rapidly migrate off the scaffolds as they homed to GBMs. In vivo, bENS loaded with hMSCs releasing the anti-tumor protein TRAIL (bENSsTR) reduced the volume of established GBM xenografts 3-fold. Mimicking clinical GBM patient therapy, lining the post-operative GBM surgical cavity with bENSsTR implants inhibited the re-growth of residual GBM foci 2.3-fold and prolonged post-surgical median survival from 13.5 to 31 days in mice. These results suggest that nanofibrous-based SC therapies could be an innovative new approach to improve the outcomes of patients suffering from terminal brain cancer. PMID:27016620

PKCδ activated by c-MET enhances infiltration of human glioblastoma cells through NOTCH2 signaling

PubMed Central

Kang, Seok-Gu; Kim, Rae-Kwon; Cui, Yan-Hong; Lee, Hae-June; Kim, Min-Jung; Lee, Jae-Seong; Kim, In-Gyu; Suh, Yongjoon; Lee, Su-Jae

2016-01-01

Poor prognosis of glioblastoma (GBM) is attributable to the propensity of tumor cells to infiltrate into the brain parenchyma. Protein kinase C (PKC) isozymes are highly expressed or aberrantly activated in GBM. However, how this signaling node translates to GBM cell invasiveness remains unknown. Here, we report that among PKC isoforms, PKCδ is strongly associated with infiltration of GBM cells. Notably, PKCδ enhanced Tyr418 phosphorylation of the non-receptor tyrosine kinase SRC, which in turn activated STAT3 and subsequent NOTCH2 signaling, ultimately leading to GBM cell invasiveness. Furthermore, we showed that PKCδ was aberrantly activated in GBM cells by c-MET, a receptor tyrosine kinase hyperactivated in GBM. In agreement, inhibition either component in the c-MET/PKCδ/SRC/STAT3 signaling axis effectively blocked the NOTCH2 signaling and invasiveness of GBM cells. Taken together, our findings shed a light on the signaling mechanisms behind the constitutive activation of PKCδ signaling in GBM. PMID:26700818

MUC4 modulates human glioblastoma cell proliferation and invasion by upregulating EGFR expression.

PubMed

Li, Weihua; Wu, Chunming; Yao, Yiqun; Dong, Bin; Wei, Zhenqing; Lv, Xiupeng; Zhang, Jian; Xu, Yinghui

2014-04-30

Glioblastoma (GBM), the most common primary brain tumor, is the leading cause of deaths related to tumors in the central nervous system. The prognosis of GBM patients is currently poor, and the mechanisms underlying GBM genesis remain unclear. The expression of MUC4, a high-molecular-weight and highly glycosylated protein, has been studied in many cancers. However, information on MUC4 expression in GBM is limited. In this study, we found that MUC4 was overexpressed in GBM cell lines and tissues. The proliferation and invasive potential of GBM cells were significantly increased by the ectopic expression of MUC4. By contrast, RNA interference targeting MUC4 in GBM cells significantly decreased the proliferation and invasive potential of GBM cells. We also found that the expression of epidermal growth factor receptor (EGFR) was modulated by MUC4. EGFR inhibition by siRNA reversed the MUC4-induced proliferation and invasion. These results indicated that MUC4 expression in GBM was important in GBM cell proliferation and invasion, which may be partly associated with EGFR overexpression. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

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

PubMed

Chou, Yu-Cheng; Chang, Meng-Ya; Wang, Mei-Jen; Liu, Hsin-Chung; Chang, Shu-Jen; Harnod, Tomor; Hung, Chih-Huang; Lee, Hsu-Tung; Shen, Chiung-Chyi; Chung, Jing-Gung

2017-01-01

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. © 2015 Wiley Periodicals, Inc.

Uncovering MicroRNA and Transcription Factor Mediated Regulatory Networks in Glioblastoma

PubMed Central

Sun, Jingchun; Gong, Xue; Purow, Benjamin; Zhao, Zhongming

2012-01-01

Glioblastoma multiforme (GBM) is the most common and lethal brain tumor in humans. Recent studies revealed that patterns of microRNA (miRNA) expression in GBM tissue samples are different from those in normal brain tissues, suggesting that a number of miRNAs play critical roles in the pathogenesis of GBM. However, little is yet known about which miRNAs play central roles in the pathology of GBM and their regulatory mechanisms of action. To address this issue, in this study, we systematically explored the main regulation format (feed-forward loops, FFLs) consisting of miRNAs, transcription factors (TFs) and their impacting GBM-related genes, and developed a computational approach to construct a miRNA-TF regulatory network. First, we compiled GBM-related miRNAs, GBM-related genes, and known human TFs. We then identified 1,128 3-node FFLs and 805 4-node FFLs with statistical significance. By merging these FFLs together, we constructed a comprehensive GBM-specific miRNA-TF mediated regulatory network. Then, from the network, we extracted a composite GBM-specific regulatory network. To illustrate the GBM-specific regulatory network is promising for identification of critical miRNA components, we specifically examined a Notch signaling pathway subnetwork. Our follow up topological and functional analyses of the subnetwork revealed that six miRNAs (miR-124, miR-137, miR-219-5p, miR-34a, miR-9, and miR-92b) might play important roles in GBM, including some results that are supported by previous studies. In this study, we have developed a computational framework to construct a miRNA-TF regulatory network and generated the first miRNA-TF regulatory network for GBM, providing a valuable resource for further understanding the complex regulatory mechanisms in GBM. The observation of critical miRNAs in the Notch signaling pathway, with partial verification from previous studies, demonstrates that our network-based approach is promising for the identification of new and important miRNAs in GBM and, potentially, other cancers. PMID:22829753

Human Cytomegalovirus-Infected Glioblastoma Cells Display Stem Cell-Like Phenotypes

PubMed Central

Liu, Che; Clark, Paul A.; Kuo, John S.

2017-01-01

ABSTRACT Glioblastoma multiforme (GBM) is the most common brain tumor in adults. Human cytomegalovirus (HCMV) genomes are present in GBM tumors, yielding hope that antiviral treatments could prove therapeutic and improve the poor prognosis of GBM patients. We discovered that GBM cells infected in vitro with HCMV display properties of cancer stem cells. HCMV-infected GBM cells grow more slowly than mock-infected controls, demonstrate a higher capacity for self-renewal determined by a sphere formation assay, and display resistance to the chemotherapeutic drug temozolomide. Our data suggest that HCMV, while present in only a minority of the cells within a tumor, could contribute to the pathogenesis of GBMs by promoting or prolonging stem cell-like phenotypes, thereby perpetuating tumors in the face of chemotherapy. Importantly, we show that temozolomide sensitivity is restored by the antiviral drug ganciclovir, indicating a potential mechanism underlying the positive effects observed in GBM patients treated with antiviral therapy. IMPORTANCE A role for HCMV in GBMs remains controversial for several reasons. Some studies find HCMV in GBM tumors, while others do not. Few cells within a GBM may harbor HCMV, making it unclear how the virus could be contributing to the tumor phenotype without infecting every cell. Finally, HCMV does not overtly transform cells in vitro. However, tumors induced by other viruses can be treated with antiviral remedies, and initial results indicate that this may be true for anti-HCMV therapies and GBMs. With such a poor prognosis for GBM patients, any potential new intervention deserves exploration. Our work here describes an evidence-based model for how HCMV could contribute to GBM biology while infecting very few cells and without transforming them. It also illuminates why anti-HCMV treatments may be beneficial to GBM patients. Our observations provide blueprints for future in vitro studies examining how HCMV manipulates stem cell-specific pathways and future clinical studies of anti-HCMV measures as GBM therapeutics. PMID:28656174

Activation of Aurora A kinase through the FGF1/FGFR signaling axis sustains the stem cell characteristics of glioblastoma cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hsu, Yi-Chao; Institute of Biomedical Sciences, Mackay Medical College, New Taipei City, Taiwan; Kao, Chien-Yu

Fibroblast growth factor 1 (FGF1) binds and activates FGF receptors, thereby regulating cell proliferation and neurogenesis. Human FGF1 gene 1B promoter (−540 to +31)-driven SV40 T antigen has been shown to result in tumorigenesis in the brains of transgenic mice. FGF1B promoter (−540 to +31)-driven green fluorescent protein (F1BGFP) has also been used in isolating neural stem cells (NSCs) with self-renewal and multipotency from developing and adult mouse brains. In this study, we provide six lines of evidence to demonstrate that FGF1/FGFR signaling is implicated in the expression of Aurora A (AurA) and the activation of its kinase domain (Thr288more » phosphorylation) in the maintenance of glioblastoma (GBM) cells and NSCs. First, treatment of FGF1 increases AurA expression in human GBM cell lines. Second, using fluorescence-activated cell sorting, we observed that F1BGFP reporter facilitates the isolation of F1BGFP(+) GBM cells with higher expression levels of FGFR and AurA. Third, both FGFR inhibitor (SU5402) and AurA inhibitor (VX680) could down-regulate F1BGFP-dependent AurA activity. Fourth, inhibition of AurA activity by two different AurA inhibitors (VX680 and valproic acid) not only reduced neurosphere formation but also induced neuronal differentiation of F1BGFP(+) GBM cells. Fifth, flow cytometric analyses demonstrated that F1BGFP(+) GBM cells possessed different NSC cell surface markers. Finally, inhibition of AurA by VX680 reduced the neurosphere formation of different types of NSCs. Our results show that activation of AurA kinase through FGF1/FGFR signaling axis sustains the stem cell characteristics of GBM cells. Implications: This study identified a novel mechanism for the malignancy of GBM, which could be a potential therapeutic target for GBM. - Highlights: • We report that FGF1 treatment can stimulate AurA kinase expression in human GBM cells. • FGF1/FGFR signaling is involved in the activation of AurA kinase. • FGF1 sustains the self-renewal of human GBM cells and embryonic stem cells via AurA activation.« less

Graphene nanoribbons as a drug delivery agent for lucanthone mediated therapy of glioblastoma multiforme

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chowdhury, Sayan Mullick; Surhland, Cassandra; Sanchez, Zina

We report use of PEG-DSPE coated oxidized graphene nanoribbons (O-GNR-PEG-DSPE) as agent for delivery of anti-tumor drug Lucanthone (Luc) into Glioblastoma Multiformae (GBM) cells targeting base excision repair enzyme APE-1 (Apurinic endonuclease-1). Lucanthone, an endonuclease inhibitor of APE-1, was loaded onto O-GNR-PEG-DSPEs using a simple non-covalent method. We found its uptake by GBM cell line U251 exceeding 67% and 60% in APE-1-overexpressing U251, post 24 hours (h). However, their uptake was ~38% and 29% by MCF-7 and rat glial progenitor cells (CG-4), respectively. TEM analysis of U251 showed large aggregates of O-GNR-PEG-DSPE in vesicles. Luc-O-GNR-PEG-DSPE was significantly toxic to U251more » but showed little / no toxicity when exposed to MCF-7/CG-4 cells. This differential uptake effect can be exploited to use O-GNR-PEG-DSPEs as a vehicle for Luc delivery to GBM, while reducing nonspecific cytotoxicity to the surrounding healthy tissue. In conclusion, cell death in U251 was necrotic, probably due to oxidative degradation of APE-1.« less

Graphene nanoribbons as a drug delivery agent for lucanthone mediated therapy of glioblastoma multiforme

DOE PAGES

Chowdhury, Sayan Mullick; Surhland, Cassandra; Sanchez, Zina; ...

2014-08-13

We report use of PEG-DSPE coated oxidized graphene nanoribbons (O-GNR-PEG-DSPE) as agent for delivery of anti-tumor drug Lucanthone (Luc) into Glioblastoma Multiformae (GBM) cells targeting base excision repair enzyme APE-1 (Apurinic endonuclease-1). Lucanthone, an endonuclease inhibitor of APE-1, was loaded onto O-GNR-PEG-DSPEs using a simple non-covalent method. We found its uptake by GBM cell line U251 exceeding 67% and 60% in APE-1-overexpressing U251, post 24 hours (h). However, their uptake was ~38% and 29% by MCF-7 and rat glial progenitor cells (CG-4), respectively. TEM analysis of U251 showed large aggregates of O-GNR-PEG-DSPE in vesicles. Luc-O-GNR-PEG-DSPE was significantly toxic to U251more » but showed little / no toxicity when exposed to MCF-7/CG-4 cells. This differential uptake effect can be exploited to use O-GNR-PEG-DSPEs as a vehicle for Luc delivery to GBM, while reducing nonspecific cytotoxicity to the surrounding healthy tissue. In conclusion, cell death in U251 was necrotic, probably due to oxidative degradation of APE-1.« less

Selective coexpression of VEGF receptor 2 in EGFRvIII-positive glioblastoma cells prevents cellular senescence and contributes to their aggressive nature.

PubMed

Jones, Karra A; Gilder, Andrew S; Lam, Michael S; Du, Na; Banki, Michael A; Merati, Aran; Pizzo, Donald P; VandenBerg, Scott R; Gonias, Steven L

2016-05-01

In glioblastoma (GBM), the gene for epidermal growth factor receptor (EGFR) is frequently amplified. EGFR mutations also are common, including a truncation mutation that yields a constitutively active variant called EGFR variant (v)III. EGFRvIII-positive GBM progresses rapidly; however, the reason for this is not clear because the activity of EGFRvIII is attenuated compared with EGF-ligated wild-type EGFR. We hypothesized that EGFRvIII-expressing GBM cells selectively express other oncogenic receptors that support tumor progression. Mining of The Cancer Genome Atlas prompted us to test whether GBM cells in culture, which express EGFRvIII, selectively express vascular endothelial growth factor receptor (VEGFR)2. We also studied human GBM propagated as xenografts. We then applied multiple approaches to test the effects of VEGFR2 on GBM cell growth, apoptosis, and cellular senescence. In human GBM, EGFR overexpression and EGFRvIII positivity were associated with increased VEGFR2 expression. In GBM cells in culture, EGFRvIII-initiated cell signaling increased expression of VEGFR2, which prevented cellular senescence and promoted cell cycle progression. The VEGFR-selective tyrosine kinase inhibitor cediranib decreased tumor DNA synthesis, increased staining for senescence-associated β-galactosidase, reduced retinoblastoma phosphorylation, and increased p27(Kip1), all markers of cellular senescence. Similar results were obtained when VEGFR2 was silenced. VEGFR2 expression by GBM cells supports cell cycle progression and prevents cellular senescence. Coexpression of VEGFR2 by GBM cells in which EGFR signaling is activated may contribute to the aggressive nature of these cells. © The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Parecoxib inhibits glioblastoma cell proliferation, migration and invasion by upregulating miRNA-29c.

PubMed

Li, Lin-Yong; Xiao, Jie; Liu, Qiang; Xia, Kun

2017-03-15

Glioblastoma (GBM) is one of the most lethal brain cancers worldwide, and there is an urgent need for development of novel therapeutic approaches. Parecoxib is a well-known cyclooxygenase-2 (COX-2) inhibitor, and had already been developed for postoperative analgesia with high efficacy and low adverse reaction. A recent study has suggested that parecoxib potently enhances immunotherapeutic efficacy of GBM, but its effects on GBM growth, migration and invasion have not previously been studied. In the present study, MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] and BrdU (5-bromo-2-deoxyuridine) incorporation assays were used to evaluate the cell proliferation of GBM cells. Wound-healing and transwell assays were preformed to analyze GBM cell migration and invasion, respectively. The results suggested that parecoxib inhibits cell proliferation, migration and invasion of GBM cells in a dose-dependent manner. RT-qPCR (real-time quantitative PCR) analysis demonstrated that miRNA-29c can be significantly induced by parecoxib. Furthermore, our data suggests that a miRNA-29c inhibitor can significantly attenuate parecoxib's effect on proliferation, migration and invasion of GBM. In conclusion, the present study suggests that parecoxib inhibits GBM cell proliferation, migration and invasion by upregulating miRNA-29c. © 2017. Published by The Company of Biologists Ltd.

Parecoxib inhibits glioblastoma cell proliferation, migration and invasion by upregulating miRNA-29c

PubMed Central

Li, Lin-Yong; Xiao, Jie; Liu, Qiang

2017-01-01

ABSTRACT Glioblastoma (GBM) is one of the most lethal brain cancers worldwide, and there is an urgent need for development of novel therapeutic approaches. Parecoxib is a well-known cyclooxygenase-2 (COX-2) inhibitor, and had already been developed for postoperative analgesia with high efficacy and low adverse reaction. A recent study has suggested that parecoxib potently enhances immunotherapeutic efficacy of GBM, but its effects on GBM growth, migration and invasion have not previously been studied. In the present study, MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] and BrdU (5-bromo-2-deoxyuridine) incorporation assays were used to evaluate the cell proliferation of GBM cells. Wound-healing and transwell assays were preformed to analyze GBM cell migration and invasion, respectively. The results suggested that parecoxib inhibits cell proliferation, migration and invasion of GBM cells in a dose-dependent manner. RT-qPCR (real-time quantitative PCR) analysis demonstrated that miRNA-29c can be significantly induced by parecoxib. Furthermore, our data suggests that a miRNA-29c inhibitor can significantly attenuate parecoxib's effect on proliferation, migration and invasion of GBM. In conclusion, the present study suggests that parecoxib inhibits GBM cell proliferation, migration and invasion by upregulating miRNA-29c. PMID:27895048

Development of Advanced Technologies for Complete Genomic and Proteomic Characterization of Quantized Human Tumor Cells

DTIC Science & Technology

2014-07-01

13. SUPPLEMENTARY NOTES 14. ABSTRACT With the establishment of Glioblastoma ( GBM ) cell lines from GBM patient’s tumor samples and quantized cell...populations of each of the parental GBM cell lines, we have completed most of our major aims of this project. We will continue in our efforts in the...signatures. Whole genome sequencing from two families of GBM patients are now well established and from the basis of the molecular characterization of

Hypoxia-mediated upregulation of MCT1 expression supports the glycolytic phenotype of glioblastomas.

PubMed

Miranda-Gonçalves, Vera; Granja, Sara; Martinho, Olga; Honavar, Mrinalini; Pojo, Marta; Costa, Bruno M; Pires, Manuel M; Pinheiro, Célia; Cordeiro, Michelle; Bebiano, Gil; Costa, Paulo; Reis, Rui M; Baltazar, Fátima

2016-07-19

Glioblastomas (GBM) present a high cellular heterogeneity with conspicuous necrotic regions associated with hypoxia, which is related to tumor aggressiveness. GBM tumors exhibit high glycolytic metabolism with increased lactate production that is extruded to the tumor microenvironment through monocarboxylate transporters (MCTs). While hypoxia-mediated regulation of MCT4 has been characterized, the role of MCT1 is still controversial. Thus, we aimed to understand the role of hypoxia in the regulation of MCT expression and function in GBM, MCT1 in particular. Expression of hypoxia- and glycolytic-related markers, as well as MCT1 and MCT4 isoforms was assessed in in vitro and in vivo orthotopic glioma models, and also in human GBM tissues by immunofluorescence/immunohistochemistry and Western blot. Following MCT1 inhibition, either pharmacologically with CHC (α-cyano-4-hydroxynnamic acid) or genetically with siRNAs, we assessed GBM cell viability, proliferation, metabolism, migration and invasion, under normoxia and hypoxia conditions. Hypoxia induced an increase in MCT1 plasma membrane expression in glioma cells, both in in vitro and in vivo models. Additionally, treatment with CHC and downregulation of MCT1 in glioma cells decreased lactate production, cell proliferation and invasion under hypoxia. Moreover, in the in vivo orthotopic model and in human GBM tissues, there was extensive co-expression of MCT1, but not MCT4, with the GBM hypoxia marker CAIX. Hypoxia-induced MCT1 supports GBM glycolytic phenotype, being responsible for lactate efflux and an important mediator of cell survival and aggressiveness. Therefore, MCT1 constitutes a promising therapeutic target in GBM.

Hypoxia-mediated upregulation of MCT1 expression supports the glycolytic phenotype of glioblastomas

PubMed Central

Miranda-Gonçalves, Vera; Granja, Sara; Martinho, Olga; Honavar, Mrinalini; Pojo, Marta; Costa, Bruno M.; Pires, Manuel M.; Pinheiro, Célia; Cordeiro, Michelle; Bebiano, Gil; Costa, Paulo; Reis, Rui M.; Baltazar, Fátima

2016-01-01

Background Glioblastomas (GBM) present a high cellular heterogeneity with conspicuous necrotic regions associated with hypoxia, which is related to tumor aggressiveness. GBM tumors exhibit high glycolytic metabolism with increased lactate production that is extruded to the tumor microenvironment through monocarboxylate transporters (MCTs). While hypoxia-mediated regulation of MCT4 has been characterized, the role of MCT1 is still controversial. Thus, we aimed to understand the role of hypoxia in the regulation of MCT expression and function in GBM, MCT1 in particular. Methods Expression of hypoxia- and glycolytic-related markers, as well as MCT1 and MCT4 isoforms was assessed in in vitro and in vivo orthotopic glioma models, and also in human GBM tissues by immunofluorescence/immunohistochemistry and Western blot. Following MCT1 inhibition, either pharmacologically with CHC (α-cyano-4-hydroxynnamic acid) or genetically with siRNAs, we assessed GBM cell viability, proliferation, metabolism, migration and invasion, under normoxia and hypoxia conditions. Results Hypoxia induced an increase in MCT1 plasma membrane expression in glioma cells, both in in vitro and in vivo models. Additionally, treatment with CHC and downregulation of MCT1 in glioma cells decreased lactate production, cell proliferation and invasion under hypoxia. Moreover, in the in vivo orthotopic model and in human GBM tissues, there was extensive co-expression of MCT1, but not MCT4, with the GBM hypoxia marker CAIX. Conclusion Hypoxia-induced MCT1 supports GBM glycolytic phenotype, being responsible for lactate efflux and an important mediator of cell survival and aggressiveness. Therefore, MCT1 constitutes a promising therapeutic target in GBM. PMID:27331625

Radiogenomics to characterize regional genetic heterogeneity in glioblastoma

PubMed Central

Hu, Leland S.; Ning, Shuluo; Eschbacher, Jennifer M.; Baxter, Leslie C.; Gaw, Nathan; Ranjbar, Sara; Plasencia, Jonathan; Dueck, Amylou C.; Peng, Sen; Smith, Kris A.; Nakaji, Peter; Karis, John P.; Quarles, C. Chad; Wu, Teresa; Loftus, Joseph C.; Jenkins, Robert B.; Sicotte, Hugues; Kollmeyer, Thomas M.; O'Neill, Brian P.; Elmquist, William; Hoxworth, Joseph M.; Frakes, David; Sarkaria, Jann; Swanson, Kristin R.; Tran, Nhan L.; Li, Jing; Mitchell, J. Ross

2017-01-01

Background Glioblastoma (GBM) exhibits profound intratumoral genetic heterogeneity. Each tumor comprises multiple genetically distinct clonal populations with different therapeutic sensitivities. This has implications for targeted therapy and genetically informed paradigms. Contrast-enhanced (CE)-MRI and conventional sampling techniques have failed to resolve this heterogeneity, particularly for nonenhancing tumor populations. This study explores the feasibility of using multiparametric MRI and texture analysis to characterize regional genetic heterogeneity throughout MRI-enhancing and nonenhancing tumor segments. Methods We collected multiple image-guided biopsies from primary GBM patients throughout regions of enhancement (ENH) and nonenhancing parenchyma (so called brain-around-tumor, [BAT]). For each biopsy, we analyzed DNA copy number variants for core GBM driver genes reported by The Cancer Genome Atlas. We co-registered biopsy locations with MRI and texture maps to correlate regional genetic status with spatially matched imaging measurements. We also built multivariate predictive decision-tree models for each GBM driver gene and validated accuracies using leave-one-out-cross-validation (LOOCV). Results We collected 48 biopsies (13 tumors) and identified significant imaging correlations (univariate analysis) for 6 driver genes: EGFR, PDGFRA, PTEN, CDKN2A, RB1, and TP53. Predictive model accuracies (on LOOCV) varied by driver gene of interest. Highest accuracies were observed for PDGFRA (77.1%), EGFR (75%), CDKN2A (87.5%), and RB1 (87.5%), while lowest accuracy was observed in TP53 (37.5%). Models for 4 driver genes (EGFR, RB1, CDKN2A, and PTEN) showed higher accuracy in BAT samples (n = 16) compared with those from ENH segments (n = 32). Conclusion MRI and texture analysis can help characterize regional genetic heterogeneity, which offers potential diagnostic value under the paradigm of individualized oncology. PMID:27502248

Down-regulation of 14-3-3β exerts anti-cancer effects through inducing ER stress in human glioma U87 cells: Involvement of CHOP–Wnt pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cao, Lei; Lei, Hui; Chang, Ming-Ze

We previously identified 14-3-3β as a tumor-specific isoform of 14-3-3 protein in astrocytoma, but its functional role in glioma cells and underlying mechanisms are poorly understood. In the present study, we investigated the effects of 14-3-3β inhibition in human glioma U87 cells using specific targeted small interfering RNA (siRNA). The results showed that 14-3-3β is highly expressed in U87 cells but not in normal astrocyte SVGp12 cells. Knockdown of 14-3-3β by Si-14-3-3β transfection significantly decreased the cell viability but increased the LDH release in a time-dependent fashion in U87 cells, and these effects were accompanied with G0/G1 cell cycle arrestmore » and apoptosis. In addition, 14-3-3β knockdown induced ER stress in U87 cells, as evidenced by ER calcium release, increased expression of XBP1S mRNA and induction of ER related pro-apoptotic factors. Down-regulation of 14-3-3β significantly decreased the nuclear localization of β-catenin and inhibited Topflash activity, which was shown to be reversely correlated with CHOP. Furthermore, Si-CHOP and sFRP were used to inhibit CHOP and Wnt, respectively. The results showed that the anti-cancer effects of 14-3-3β knockdown in U87 cells were mediated by increased expression of CHOP and followed inhibition of Wnt/β-catenin pathway. In summary, the remarkable efficiency of 14-3-3β knockdown to induce apoptotic cell death in U87 cells may find therapeutic application for the treatment of glioma patients. - Highlights: • Knockdown of 14-3-3β leads to cytotoxicity in human glioma U87 cells. • Knockdown of 14-3-3β induces cell cycle arrest and apoptosis in U87 cells. • Knockdown of 14-3-3β results in ER stress in U87 cells. • Knockdown of 14-3-3β inhibits Wnt/β-catenin pathway via CHOP activation.« less

Secondary Glioblastoma: Molecular and Clinical Factors That Affect Outcome After Malignant Progression of a Lower Grade Tumor.

PubMed

Gessler, Florian; Zappi, Johannes; Konczalla, Juergen; Bernstock, Joshua D; Forster, Marie-Therese; Wagner, Marlies; Mittelbronn, Michel; Seifert, Volker; Senft, Christian

2017-06-01

There is limited information on prognostic factors and outcomes in patients with secondary glioblastoma (sGBM). Herein we report on the outcomes of patients with sGBM and identify clinically relevant prognostic factors. We retrospectively analyzed our institutional database for patients with histologic evidence of World Health Organization (WHO) grade II-III gliomas that went on to develop WHO grade IV sGBM. The assessment of the isocitrate dehydrogenase-1 (IDH1) R132H mutation was performed by immunohistochemical staining. Forty-five patients with sGBM were included within our analysis (median age, 41 years). Mutated IDH1 (R132H) protein was present within the gliomas of 24 patients and was absent in 17. Immunohistochemistry assessment could not be performed for 4 patients. The median time between first diagnosis of glioma and sGBM was 158.9 weeks. Median overall survival (OS) after a diagnosis of sGBM was 63.6 weeks. When assessing patient-specific (i.e., therapy-independent) factors, mutated IDH1 (R132H) protein (P = 0.01; hazard ratio (HR), 0.54; confidence interval (CI) 0.33-0.87), WHO grade II tumor as precursor lesion (P = 0.05; HR, 0.49; CI 0.25-0.97), and a frontal tumor location (P = 0.04; HR, 0.48; CI 0.23-0.99) were found to be associated with better OS by multivariate analysis. Our data further indicate that complete tumor removal is associated with better patient survival in sGBM patients within certain risk groups (time period to development of sGBM, >104 weeks; initial WHO grade II tumor, IDH1 mutation, and time period to development of sGBM, >104 weeks; initial WHO grade II or III tumor, IDH1 wild type, frontal lobe localization). Our retrospective analysis suggested that the presence of an IDH1 (R132H) mutation, frontal tumor location, and WHO grade of the initial tumor are associated with OS after progression to sGBM. In addition, some patients with sGBM may benefit from complete tumor resection depending on these patient-specific parameters. This is a finding that will ultimately need prospective validation. Copyright © 2017 Elsevier Inc. All rights reserved.

Pulsed Versus Conventional Radiation Therapy in Combination With Temozolomide in a Murine Orthotopic Model of Glioblastoma Multiforme

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, David Y.; Chunta, John L.; Park, Sean S.

Purpose: To evaluate the efficacy of pulsed low-dose radiation therapy (PLRT) combined with temozolomide (TMZ) as a novel treatment approach for radioresistant glioblastoma multiforme (GBM) in a murine model. Methods and Materials: Orthotopic U87MG hGBM tumors were established in Nu-Foxn1{sup nu} mice and imaged weekly using a small-animal micropositron emission tomography (PET)/computed tomography (CT) system. Tumor volume was determined from contrast-enhanced microCT images and tumor metabolic activity (SUVmax) from the F18-FDG microPET scan. Tumors were irradiated 7 to 10 days after implantation with a total dose of 14 Gy in 7 consecutive days. The daily treatment was given as amore » single continuous 2-Gy dose (RT) or 10 pulses of 0.2 Gy using an interpulse interval of 3 minutes (PLRT). TMZ (10 mg/kg) was given daily by oral gavage 1 hour before RT. Tumor vascularity and normal brain damage were assessed by immunohistochemistry. Results: Radiation therapy with TMZ resulted in a significant 3- to 4-week tumor growth delay compared with controls, with PLRT+TMZ the most effective. PLRT+TMZ resulted in a larger decline in SUVmax than RT+TMZ. Significant differences in survival were evident. Treatment after PLRT+TMZ was associated with increased vascularization compared with RT+TMZ. Significantly fewer degenerating neurons were seen in normal brain after PLRT+TMZ compared with RT+TMZ. Conclusions: PLRT+TMZ produced superior tumor growth delay and less normal brain damage when compared with RT+TMZ. The differential effect of PLRT on vascularization may confirm new treatment avenues for GBM.« less

The CHAC1-inhibited Notch3 pathway is involved in temozolomide-induced glioma cytotoxicity.

PubMed

Chen, Peng-Hsu; Shen, Wan-Lin; Shih, Chwen-Ming; Ho, Kuo-Hao; Cheng, Chia-Hsiung; Lin, Cheng-Wei; Lee, Chin-Cheng; Liu, Ann-Jeng; Chen, Ku-Chung

2017-04-01

Glioblastoma multiforme (GBM) is the high-grade primary glioma in adults. Temozolomide (TMZ), an alkylating agent of the imidazotetrazine series, is a first-line chemotherapeutic drug for clinical therapy. However, the expense of TMZ therapy and increasing drug resistance to TMZ decreases its therapeutic effects. Therefore, our aim was to investigate the detailed molecular mechanisms of TMZ-mediated cytotoxicity to enhance the efficacy of TMZ in clinical GBM therapy. First, TMZ-mediated gene expression profiles and networks in U87-MG cells were identified by transcriptome microarray and bioinformatic analyses. Cation transport regulator-like protein 1 (CHAC1) was the most highly TMZ-upregulated gene. Overexpression and knockdown of CHAC1 expression significantly influenced TMZ-mediated cell viability, apoptosis, caspase-3 activation, and poly(ADP ribose) polymerase (PARP) degradation. The c-Jun N-terminal kinase (JNK)1/c-JUN pathway was identified to participate in TMZ-upregulated CHAC1 expression via transcriptional control. Furthermore, CHAC1 levels were significantly decreased in GBM cell lines, TCGA array data, and tumor tissues. Overexpression of CHAC1 enhanced glioma apoptotic death via caspase-3/9 activation, PARP degradation, autophagy formation, reactive oxygen species generation, increased intracellular calcium, and loss of the mitochondria membrane potential. Finally, we also identified that TMZ significantly reduced Notch3 levels, which are upregulated in gliomas. TMZ also induced CHAC1 to bind to the Notch3 protein and inhibit Notch3 activation, resulting in attenuation of Notch3-mediated downstream signaling pathways. These results emphasize that CHAC1-inhibited Notch3 signaling can influence TMZ-mediated cytotoxicity. Our findings may provide novel therapeutic strategies for future glioblastoma therapy. Copyright © 2016 Elsevier Ltd. All rights reserved.

Patient-specific orthotopic glioblastoma xenograft models recapitulate the histopathology and biology of human glioblastomas in situ.

PubMed

Joo, Kyeung Min; Kim, Jinkuk; Jin, Juyoun; Kim, Misuk; Seol, Ho Jun; Muradov, Johongir; Yang, Heekyoung; Choi, Yoon-La; Park, Woong-Yang; Kong, Doo-Sik; Lee, Jung-Il; Ko, Young-Hyeh; Woo, Hyun Goo; Lee, Jeongwu; Kim, Sunghoon; Nam, Do-Hyun

2013-01-31

Frequent discrepancies between preclinical and clinical results of anticancer agents demand a reliable translational platform that can precisely recapitulate the biology of human cancers. Another critical unmet need is the ability to predict therapeutic responses for individual patients. Toward this goal, we have established a library of orthotopic glioblastoma (GBM) xenograft models using surgical samples of GBM patients. These patient-specific GBM xenograft tumors recapitulate histopathological properties and maintain genomic characteristics of parental GBMs in situ. Furthermore, in vivo irradiation, chemotherapy, and targeted therapy of these xenograft tumors mimic the treatment response of parental GBMs. We also found that establishment of orthotopic xenograft models portends poor prognosis of GBM patients and identified the gene signatures and pathways signatures associated with the clinical aggressiveness of GBMs. Together, the patient-specific orthotopic GBM xenograft library represent the preclinically and clinically valuable "patient tumor's phenocopy" that represents molecular and functional heterogeneity of GBMs. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

The Combined Influence of Hydrogel Stiffness and Matrix-Bound Hyaluronic Acid Content on Glioblastoma Invasion.

PubMed

Chen, Jee-Wei Emily; Pedron, Sara; Harley, Brendan A C

2017-08-01

Glioblastoma (GBM) is the most common and lethal form of brain cancer. Its high mortality is associated with its aggressive invasion throughout the brain. The heterogeneity of stiffness and hyaluronic acid (HA) content within the brain makes it difficult to study invasion in vivo. A dextran-bead assay is employed to quantify GBM invasion within HA-functionalized gelatin hydrogels. Using a library of stiffness-matched hydrogels with variable levels of matrix-bound HA, it is reported that U251 GBM invasion is enhanced in softer hydrogels but reduced in the presence of matrix-bound HA. Inhibiting HA-CD44 interactions reduces invasion, even in hydrogels lacking matrix-bound HA. Analysis of HA biosynthesis suggests that GBM cells compensate for a lack of matrix-bound HA by producing soluble HA to stimulate invasion. Together, a robust method is showed to quantify GBM invasion over long culture times to reveal the coordinated effect of matrix stiffness, immobilized HA, and compensatory HA production on GBM invasion. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Ultrastructural characterization of primary cilia in pathologically characterized human glioblastoma multiforme (GBM) tumors.

PubMed

Moser, Joanna J; Fritzler, Marvin J; Rattner, Jerome B

2014-01-01

Primary cilia are non-motile sensory cytoplasmic organelles that are involved in cell cycle progression. Ultrastructurally, the primary cilium region is complex, with normal ciliogenesis progressing through five distinct morphological stages in human astrocytes. Defects in early stages of ciliogenesis are key features of astrocytoma/glioblastoma cell lines and provided the impetus for the current study which describes the morphology of primary cilia in molecularly characterized human glioblastoma multiforme (GBM) tumors. Seven surgically resected human GBM tissue samples were molecularly characterized according to IDH1/2 mutation status, EGFR amplification status and MGMT promoter methylation status and were examined for primary cilia expression and structure using indirect immunofluorescence and electron microscopy. We report for the first time that primary cilia are disrupted in the early stages of ciliogenesis in human GBM tumors. We confirm that immature primary cilia and basal bodies/centrioles have aberrant ciliogenesis characteristics including absent paired vesicles, misshaped/swollen vesicular hats, abnormal configuration of distal appendages, and discontinuity of centriole microtubular blades. Additionally, the transition zone plate is able to form in the absence of paired vesicles on the distal end of the basal body and when a cilium progresses beyond the early stages of ciliogenesis, it has electron dense material clumped along the transition zone and a darkening of the microtubules at the proximal end of the cilium. Primary cilia play a role in a variety of human cancers. Previously primary cilia structure was perturbed in cultured cell lines derived from astrocytomas/glioblastomas; however there was always some question as to whether these findings were a cell culture phenomena. In this study we confirm that disruptions in ciliogenesis at early stages do occur in GBM tumors and that these ultrastructural findings bear resemblance to those previously observed in cell cultures. This is the first study to demonstrate that defects in cilia expression and function are a true hallmark of GBM tumors and correlate with their unrestrained growth. A review of the current ultrastructural profiles in the literature provides suggestions as to the best possible candidate protein that underlies defects in the early stages of ciliogenesis within GBM tumors.

Human TERT promoter mutation enables survival advantage from MGMT promoter methylation in IDH1 wild-type primary glioblastoma treated by standard chemoradiotherapy.

PubMed

Nguyen, HuyTram N; Lie, Amy; Li, Tie; Chowdhury, Reshmi; Liu, Fei; Ozer, Byram; Wei, Bowen; Green, Richard M; Ellingson, Benjamin M; Wang, He-Jing; Elashoff, Robert; Liau, Linda M; Yong, William H; Nghiemphu, Phioanh L; Cloughesy, Timothy; Lai, Albert

2017-03-01

Promoter mutation in the human telomerase reverse transcriptase gene (hTERT) occurs in ~75% of primary glioblastoma (GBM). Although the mutation appears to upregulate telomerase expression and contributes to the maintenance of telomere length, its clinical significance remains unclear. We performed hTERT promoter genotyping on 303 isocitrate dehydrogenase 1 wild-type GBM tumors treated with standard chemoradiotherapy. We also stratified 190 GBM patients from the database of The Cancer Genome Atlas (TCGA) by hTERT gene expression. We analyzed overall and progression-free survival by Kaplan-Meier and Cox regression. We detected hTERT promoter mutation in 75% of the patients. When included as the only biomarker, hTERT mutation was not prognostic in our patient cohort by Cox regression analysis. However, when hTERT and O6-DNA methylguanine-methyltransferase (MGMT) were included together, we observed an interaction between these 2 factors. To further investigate this interaction, we performed pairwise comparison of the 4 patient subcohorts grouped by hTERT-MGMT status (MUT-M, WT-M, MUT-U, and WT-U). MGMT methylated patients showed improved survival only in the presence of hTERT promoter mutation: MUT-M versus MUT-U (overall survival of 28.3 vs 15.9 mos, log-rank P < .0001 and progression-free survival of 15.4 vs 7.86 mo, log-rank P < .0001). These results were confirmed by Cox analyses. Analogously, the cohort from TCGA demonstrated survival benefit of MGMT promoter methylation only in patients with high hTERT expression. In addition, hTERT mutation was negatively prognostic in our MGMT unmethylated patients, while the analogous association with high expression was not observed in the cohort from TCGA. The prognostic influence of MGMT promoter methylation depends on hTERT promoter mutation. This interaction warrants further mechanistic investigation. © The Author(s) 2017. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com

GBM secretome induces transient transformation of human neural precursor cells.

PubMed

Venugopal, Chitra; Wang, X Simon; Manoranjan, Branavan; McFarlane, Nicole; Nolte, Sara; Li, Meredith; Murty, Naresh; Siu, K W Michael; Singh, Sheila K

2012-09-01

Glioblastoma (GBM) is the most aggressive primary brain tumor in humans, with a uniformly poor prognosis. The tumor microenvironment is composed of both supportive cellular substrates and exogenous factors. We hypothesize that exogenous factors secreted by brain tumor initiating cells (BTICs) could predispose normal neural precursor cells (NPCs) to transformation. When NPCs are grown in GBM-conditioned media, and designated as "tumor-conditioned NPCs" (tcNPCs), they become highly proliferative and exhibit increased stem cell self-renewal, or the unique ability of stem cells to asymmetrically generate another stem cell and a daughter cell. tcNPCs also show an increased transcript level of stem cell markers such as CD133 and ALDH and growth factor receptors such as VEGFR1, VEGFR2, EGFR and PDGFRα. Media analysis by ELISA of GBM-conditioned media reveals an elevated secretion of growth factors such as EGF, VEGF and PDGF-AA when compared to normal neural stem cell-conditioned media. We also demonstrate that tcNPCs require prolonged or continuous exposure to the GBM secretome in vitro to retain GBM BTIC characteristics. Our in vivo studies reveal that tcNPCs are unable to form tumors, confirming that irreversible transformation events may require sustained or prolonged presence of the GBM secretome. Analysis of GBM-conditioned media by mass spectrometry reveals the presence of secreted proteins Chitinase-3-like 1 (CHI3L1) and H2A histone family member H2AX. Collectively, our data suggest that GBM-secreted factors are capable of transiently altering normal NPCs, although for retention of the transformed phenotype, sustained or prolonged secretome exposure or additional transformation events are likely necessary.

Pim1 kinase is upregulated in glioblastoma multiforme and mediates tumor cell survival

PubMed Central

Herzog, Susann; Fink, Matthias Alexander; Weitmann, Kerstin; Friedel, Claudius; Hadlich, Stefan; Langner, Sönke; Kindermann, Katharina; Holm, Tobias; Böhm, Andreas; Eskilsson, Eskil; Miletic, Hrvoje; Hildner, Markus; Fritsch, Michael; Vogelgesang, Silke; Havemann, Christoph; Ritter, Christoph Alexander; Meyer zu Schwabedissen, Henriette Elisabeth; Rauch, Bernhard; Hoffmann, Wolfgang; Kroemer, Heyo Klaus; Schroeder, Henry; Bien-Möller, Sandra

2015-01-01

Background The current therapy for glioblastoma multiforme (GBM), the most aggressive and common primary brain tumor of adults, involves surgery and a combined radiochemotherapy that controls tumor progression only for a limited time window. Therefore, the identification of new molecular targets is highly necessary. Inhibition of kinases has become a standard of clinical oncology, and thus the oncogenic kinase Pim1 might represent a promising target for improvement of GBM therapy. Methods Expression of Pim1 and associated signaling molecules was analyzed in human GBM samples, and the potential role of this kinase in patients' prognosis was evaluated. Furthermore, we analyzed the in vivo role of Pim1 in GBM cell growth in an orthotopic mouse model and examined the consequences of Pim1 inhibition in vitro to clarify underlying pathways. Results In comparison with normal brain, a strong upregulation of Pim1 was demonstrated in human GBM samples. Notably, patients with short overall survival showed a significantly higher Pim1 expression compared with GBM patients who lived longer than the median. In vitro experiments with GBM cells and analysis of patients' GBM samples suggest that Pim1 regulation is dependent on epidermal growth factor receptor. Furthermore, inhibition of Pim1 resulted in reduced cell viability accompanied by decreased cell numbers and increased apoptotic cells, as seen by elevated subG1 cell contents and caspase-3 and -9 activation, as well as modulation of several cell cycle or apoptosis regulatory proteins. Conclusions Altogether, Pim1 could be a novel therapeutic target, which should be further analyzed to improve the outcome of patients with aggressive GBM. PMID:25155357

HER2-specific T cells target primary glioblastoma stem cells and induce regression of autologous experimental tumors.

PubMed

Ahmed, Nabil; Salsman, Vita S; Kew, Yvonne; Shaffer, Donald; Powell, Suzanne; Zhang, Yi J; Grossman, Robert G; Heslop, Helen E; Gottschalk, Stephen

2010-01-15

Glioblastoma multiforme (GBM) is the most aggressive human primary brain tumor and is currently incurable. Immunotherapies have the potential to target GBM stem cells, which are resistant to conventional therapies. Human epidermal growth factor receptor 2 (HER2) is a validated immunotherapy target, and we determined if HER2-specific T cells can be generated from GBM patients that will target autologous HER2-positive GBMs and their CD133-positive stem cell compartment. HER2-specific T cells from 10 consecutive GBM patients were generated by transduction with a retroviral vector encoding a HER2-specific chimeric antigen receptor. The effector function of HER2-specific T cells against autologous GBM cells, including CD133-positive stem cells, was evaluated in vitro and in an orthotopic murine xenograft model. Stimulation of HER2-specific T cells with HER2-positive autologous GBM cells resulted in T-cell proliferation and secretion of IFN-gamma and interleukin-2 in a HER2-dependent manner. Patients' HER2-specific T cells killed CD133-positive and CD133-negative cells derived from primary HER2-positive GBMs, whereas HER2-negative tumor cells were not killed. Injection of HER2-specific T cells induced sustained regression of autologous GBM xenografts established in the brain of severe combined immunodeficient mice. Gene transfer allows the reliable generation of HER2-specific T cells from GBM patients, which have potent antitumor activity against autologous HER2-positive tumors including their putative stem cells. Hence, the adoptive transfer of HER2-redirected T cells may be a promising immunotherapeutic approach for GBM.

Orthotopic Patient-Derived Glioblastoma Xenografts in Mice.

PubMed

Xu, Zhongye; Kader, Michael; Sen, Rajeev; Placantonakis, Dimitris G

2018-01-01

Patient-derived xenografts (PDX) provide in vivo glioblastoma (GBM) models that recapitulate actual tumors. Orthotopic tumor xenografts within the mouse brain are obtained by injection of GBM stem-like cells derived from fresh surgical specimens. These xenografts reproduce GBM's histologic complexity and hallmark biological behaviors, such as brain invasion, angiogenesis, and resistance to therapy. This method has become essential for analyzing mechanisms of tumorigenesis and testing the therapeutic effect of candidate agents in the preclinical setting. Here, we describe a protocol for establishing orthotopic tumor xenografts in the mouse brain with human GBM cells.

Connexin 43 inhibition sensitizes chemoresistant glioblastoma cells to temozolomide

PubMed Central

Murphy, Susan F; Varghese, Robin T; Lamouille, Samy; Guo, Sujuan; Pridham, Kevin J; Kanabur, Pratik; Osimani, Alyssa M; Sharma, Shaan; Jourdan, Jane; Rodgers, Cara M; Simonds, Gary R; Gourdie, Robert G; Sheng, Zhi

2015-01-01

Resistance of glioblastoma (GBM) to the front-line chemotherapeutic agent temozolomide (TMZ) continues to challenge GBM treatment efforts. The repair of TMZ-induced DNA damage by O-6-methylguanine-DNA methyltransferase (MGMT) confers one mechanism of TMZ resistance. Paradoxically, MGMT-deficient GBM patients survive longer despite still developing resistance to TMZ. Recent studies indicate that the gap junction protein connexin 43 (Cx43) renders GBM cells resistant to TMZ through its carboxyl terminus (CT). In this study, we report insights into how Cx43 promotes TMZ resistance. Cx43 levels were inversely correlated with TMZ sensitivity of GBM cells, including GBM stem cells. Moreover, Cx43 levels inversely correlated with patient survival, including as observed in MGMT-deficient GBM patients. Addition of the C-terminal peptide mimetic αCT1, a selective inhibitor of Cx43 channels, sensitized human MGMT-deficient and TMZ-resistant GBM cells to TMZ treatment. Moreover, combining αCT1 with TMZ blocked AKT/mTOR signaling, induced autophagy and apoptosis in TMZ-resistant GBM cells. Our findings suggest that Cx43 may offer a biomarker to predict the survival of patients with MGMT-independent TMZ resistance, and that combining a Cx43 inhibitor with TMZ could enhance therapeutic responses in GBM and perhaps other TMZ-resistant cancers. PMID:26542214

BIRC3 is a biomarker of mesenchymal habitat of glioblastoma, and a mediator of survival adaptation in hypoxia-driven glioblastoma habitats.

PubMed

Wang, Dapeng; Berglund, Anders E; Kenchappa, Rajappa S; MacAulay, Robert J; Mulé, James J; Etame, Arnold B

2017-08-24

Tumor hypoxia is an established facilitator of survival adaptation and mesenchymal transformation in glioblastoma (GBM). The underlying mechanisms that direct hypoxia-mediated survival in GBM habitats are unclear. We previously identified BIRC3 as a mediator of therapeutic resistance in GBM to standard temozolomide (TMZ) chemotherapy and radiotherapy (RT). Here we report that BIRC3 is a biomarker of the hypoxia-mediated adaptive mesenchymal phenotype of GBM. Specifically, in the TCGA dataset elevated BIRC3 gene expression was identified as a superior and selective biomarker of mesenchymal GBM versus neural, proneural and classical subtypes. Further, BIRC3 protein was highly expressed in the tumor cell niches compared to the perivascular niche across multiple regions in GBM patient tissue microarrays. Tumor hypoxia was found to mechanistically induce BIRC3 expression through HIF1-alpha signaling in GBM cells. Moreover, in human GBM xenografts robust BIRC3 expression was noted within hypoxic regions of the tumor. Importantly, selective inhibition of BIRC3 reversed therapeutic resistance of GBM cells to RT in hypoxic microenvironments through enhanced activation of caspases. Collectively, we have uncovered a novel role for BIRC3 as a targetable biomarker and mediator of hypoxia-driven habitats in GBM.

Feedback Circuit among INK4 Tumor Suppressors Constrains Human Glioblastoma Development

PubMed Central

Wiedemeyer, Ruprecht; Brennan, Cameron; Heffernan, Timothy P.; Xiao, Yonghong; Mahoney, John; Protopopov, Alexei; Zheng, Hongwu; Bignell, Graham; Furnari, Frank; Cavenee, Webster K.; Hahn, William C.; Ichimura, Koichi; Collins, V. Peter; Chu, Gerald C.; Stratton, Michael R.; Ligon, Keith L.; Futreal, P. Andrew; Chin, Lynda

2008-01-01

Summary We have developed a nonheuristic genome topography scan (GTS) algorithm to characterize the patterns of genomic alterations in human glioblastoma (GBM), identifying frequent p18INK4C and p16INK4A codeletion. Functional reconstitution of p18INK4C in GBM cells null for both p16INK4A and p18INK4C resulted in impaired cell-cycle progression and tumorigenic potential. Conversely, RNAi-mediated depletion of p18INK4C in p16INK4A-deficient primary astrocytes or established GBM cells enhanced tumorigenicity in vitro and in vivo. Furthermore, acute suppression of p16INK4A in primary astrocytes induced a concomitant increase in p18INK4C. Together, these findings uncover a feedback regulatory circuit in the astrocytic lineage and demonstrate a bona fide tumor suppressor role for p18INK4C in human GBM wherein it functions cooperatively with other INK4 family members to constrain inappropriate proliferation. PMID:18394558

TRIM28 and β-Actin Identified via Nanobody-Based Reverse Proteomics Approach as Possible Human Glioblastoma Biomarkers

PubMed Central

Jovčevska, Ivana; Zupanec, Neja; Kočevar, Nina; Cesselli, Daniela; Podergajs, Neža; Stokin, Clara Limbaeck; Myers, Michael P.; Muyldermans, Serge; Ghassabeh, Gholamreza Hassanzadeh; Motaln, Helena; Ruaro, Maria Elisabetta; Bourkoula, Evgenia; Turnšek, Tamara Lah; Komel, Radovan

2014-01-01

Malignant gliomas are among the rarest brain tumours, and they have the worst prognosis. Grade IV astrocytoma, known as glioblastoma multiforme (GBM), is a highly lethal disease where the standard therapies of surgery, followed by radiation and chemotherapy, cannot significantly prolong the life expectancy of the patients. Tumour recurrence shows more aggressive form compared to the primary tumour, and results in patient survival from 12 to 15 months only. Although still controversial, the cancer stem cell hypothesis postulates that cancer stem cells are responsible for early relapse of the disease after surgical intervention due to their high resistance to therapy. Alternative strategies for GBM therapy are thus urgently needed. Nanobodies are single-domain antigen-binding fragments of heavy-chain antibodies, and together with classical antibodies, they are part of the camelid immune system. Nanobodies are small and stable, and they share a high degree of sequence identity to the human heavy chain variable domain, and these characteristics offer them advantages over classical antibodies or antibody fragments. We first immunised an alpaca with a human GBM stem-like cell line prepared from primary GBM cultures. Next, a nanobody library was constructed in a phage-display vector. Using nanobody phage-display technology, we selected specific GBM stem-like cell binders through a number of affinity selections, using whole cell protein extracts and membrane protein-enriched extracts from eight different GBM patients, and membrane protein-enriched extracts from two established GBM stem-like cell lines (NCH644 and NCH421K cells). After the enrichment, periplasmic extract ELISA was used to screen for specific clones. These nanobody clones were recloned into the pHEN6 vector, expressed in Escherichia coli WK6, and purified using immobilised metal affinity chromatography and size-exclusion chromatography. Specific nanobody:antigen pairs were obtained and mass spectrometry analysis revealed two proteins, TRIM28 and β-actin, that were up-regulated in the GBM stem-like cells compared to the controls. PMID:25419715

Establishment and Biological Characterization of a Panel of Glioblastoma Multiforme (GBM) and GBM Variant Oncosphere Cell Lines.

PubMed

Binder, Zev A; Wilson, Kelli M; Salmasi, Vafi; Orr, Brent A; Eberhart, Charles G; Siu, I-Mei; Lim, Michael; Weingart, Jon D; Quinones-Hinojosa, Alfredo; Bettegowda, Chetan; Kassam, Amin B; Olivi, Alessandro; Brem, Henry; Riggins, Gregory J; Gallia, Gary L

2016-01-01

Human tumor cell lines form the basis of the majority of present day laboratory cancer research. These models are vital to studying the molecular biology of tumors and preclinical testing of new therapies. When compared to traditional adherent cell lines, suspension cell lines recapitulate the genetic profiles and histologic features of glioblastoma multiforme (GBM) with higher fidelity. Using a modified neural stem cell culture technique, here we report the characterization of GBM cell lines including GBM variants. Tumor tissue samples were obtained intra-operatively and cultured in neural stem cell conditions containing growth factors. Tumor lines were characterized in vitro using differentiation assays followed by immunostaining for lineage-specific markers. In vivo tumor formation was assayed by orthotopic injection in nude mice. Genetic uniqueness was confirmed via short tandem repeat (STR) DNA profiling. Thirteen oncosphere lines derived from GBM and GBM variants, including a GBM with PNET features and a GBM with oligodendroglioma component, were established. All unique lines showed distinct genetic profiles by STR profiling. The lines assayed demonstrated a range of in vitro growth rates. Multipotency was confirmed using in vitro differentiation. Tumor formation demonstrated histologic features consistent with high grade gliomas, including invasion, necrosis, abnormal vascularization, and high mitotic rate. Xenografts derived from the GBM variants maintained histopathological features of the primary tumors. We have generated and characterized GBM suspension lines derived from patients with GBMs and GBM variants. These oncosphere cell lines will expand the resources available for preclinical study.

Anti-Epidermal Growth Factor Receptor Gene Therapy for Glioblastoma

PubMed Central

Hicks, Martin J.; Chiuchiolo, Maria J.; Ballon, Douglas; Dyke, Jonathan P.; Aronowitz, Eric; Funato, Kosuke; Tabar, Viviane; Havlicek, David; Fan, Fan; Sondhi, Dolan; Kaminsky, Stephen M.; Crystal, Ronald G.

2016-01-01

Glioblastoma multiforme (GBM) is the most common and aggressive primary intracranial brain tumor in adults with a mean survival of 14 to 15 months. Aberrant activation of the epidermal growth factor receptor (EGFR) plays a significant role in GBM progression, with amplification or overexpression of EGFR in 60% of GBM tumors. To target EGFR expressed by GBM, we have developed a strategy to deliver the coding sequence for cetuximab, an anti-EGFR antibody, directly to the CNS using an adeno-associated virus serotype rh.10 gene transfer vector. The data demonstrates that single, local delivery of an anti-EGFR antibody by an AAVrh.10 vector coding for cetuximab (AAVrh.10Cetmab) reduces GBM tumor growth and increases survival in xenograft mouse models of a human GBM EGFR-expressing cell line and patient-derived GBM. AAVrh10.CetMab-treated mice displayed a reduction in cachexia, a significant decrease in tumor volume and a prolonged survival following therapy. Adeno-associated-directed delivery of a gene encoding a therapeutic anti-EGFR monoclonal antibody may be an effective strategy to treat GBM. PMID:27711187

¹H MRS characterization of neurochemical profiles in orthotopic mouse models of human brain tumors.

PubMed

Hulsey, Keith M; Mashimo, Tomoyuki; Banerjee, Abhishek; Soesbe, Todd C; Spence, Jeffrey S; Vemireddy, Vamsidhara; Maher, Elizabeth A; Bachoo, Robert M; Choi, Changho

2015-01-01

Glioblastoma (GBM), the most common primary brain tumor, is resistant to currently available treatments. The development of mouse models of human GBM has provided a tool for studying mechanisms involved in tumor initiation and growth as well as a platform for preclinical investigation of new drugs. In this study we used (1) H MR spectroscopy to study the neurochemical profile of a human orthotopic tumor (HOT) mouse model of human GBM. The goal of this study was to evaluate differences in metabolite concentrations in the GBM HOT mice when compared with normal mouse brain in order to determine if MRS could reliably differentiate tumor from normal brain. A TE =19 ms PRESS sequence at 9.4 T was used for measuring metabolite levels in 12 GBM mice and 8 healthy mice. Levels for 12 metabolites and for lipids/macromolecules at 0.9 ppm and at 1.3 ppm were reliably detected in all mouse spectra. The tumors had significantly lower concentrations of total creatine, GABA, glutamate, total N-acetylaspartate, aspartate, lipids/macromolecules at 0.9 ppm, and lipids/macromolecules at 1.3 ppm than did the brains of normal mice. The concentrations of glycine and lactate, however, were significantly higher in tumors than in normal brain. Copyright © 2014 John Wiley & Sons, Ltd.

Absence of giant blood Marseille-like virus DNA detection by polymerase chain reaction in plasma from healthy US blood donors and serum from multiply transfused patients from Cameroon.

PubMed

Phan, Tung Gia; Desnues, Christelle; Switzer, William M; Djoko, Cyrille F; Schneider, Bradley S; Deng, Xutao; Delwart, Eric

2015-06-01

A new Marseilleviridae virus family member, giant blood Marseille-like (GBM) virus, was recently reported in persons from France in the serum of an infant with adenitis, in the blood of 4% of healthy blood donors, and in 9% of multiply transfused thalassemia patients. These results suggested the presence of a nucleocytoplasmic large DNA virus potentially transmissible by blood product transfusion. To investigate this possibility we tested the plasma from 113 US blood donors and 74 multiply transfused Cameroon patients for GBM viral DNA using highly sensitive polymerase chain reaction (PCR) assays. GBM DNA was not detected by nested PCR in any of these 187 human specimens. Further testing is required to confirm the occurrence of human GBM virus infections. © 2015 AABB.

Genomic profiling of a Hepatocyte growth factor-dependent signature for MET-targeted therapy in glioblastoma.

PubMed

Johnson, Jennifer; Ascierto, Maria Libera; Mittal, Sandeep; Newsome, David; Kang, Liang; Briggs, Michael; Tanner, Kirk; Marincola, Francesco M; Berens, Michael E; Vande Woude, George F; Xie, Qian

2015-09-17

Constitutive MET signaling promotes invasiveness in most primary and recurrent GBM. However, deployment of available MET-targeting agents is confounded by lack of effective biomarkers for selecting suitable patients for treatment. Because endogenous HGF overexpression often causes autocrine MET activation, and also indicates sensitivity to MET inhibitors, we investigated whether it drives the expression of distinct genes which could serve as a signature indicating vulnerability to MET-targeted therapy in GBM. Interrogation of genomic data from TCGA GBM (Student's t test, GBM patients with high and low HGF expression, p ≤ 0.00001) referenced against patient-derived xenograft (PDX) models (Student's t test, sensitive vs. insensitive models, p ≤ 0.005) was used to identify the HGF-dependent signature. Genomic analysis of GBM xenograft models using both human and mouse gene expression microarrays (Student's t test, treated vs. vehicle tumors, p ≤ 0.01) were performed to elucidate the tumor and microenvironment cross talk. A PDX model with EGFR(amp) was tested for MET activation as a mechanism of erlotinib resistance. We identified a group of 20 genes highly associated with HGF overexpression in GBM and were up- or down-regulated only in tumors sensitive to MET inhibitor. The MET inhibitors regulate tumor (human) and host (mouse) cells within the tumor via distinct molecular processes, but overall impede tumor growth by inhibiting cell cycle progression. EGFR (amp) tumors undergo erlotinib resistance responded to a combination of MET and EGFR inhibitors. Combining TCGA primary tumor datasets (human) and xenograft tumor model datasets (human tumor grown in mice) using therapeutic efficacy as an endpoint may serve as a useful approach to discover and develop molecular signatures as therapeutic biomarkers for targeted therapy. The HGF dependent signature may serve as a candidate predictive signature for patient enrollment in clinical trials using MET inhibitors. Human and mouse microarrays maybe used to dissect the tumor-host interactions. Targeting MET in EGFR (amp) GBM may delay the acquired resistance developed during treatment with erlotinib.

A quantitative study of shape descriptors from glioblastoma multiforme phenotypes for predicting survival outcome

PubMed Central

Desrosiers, Christian; Hassan, Lama; Tanougast, Camel

2016-01-01

Objective: Predicting the survival outcome of patients with glioblastoma multiforme (GBM) is of key importance to clinicians for selecting the optimal course of treatment. The goal of this study was to evaluate the usefulness of geometric shape features, extracted from MR images, as a potential non-invasive way to characterize GBM tumours and predict the overall survival times of patients with GBM. Methods: The data of 40 patients with GBM were obtained from the Cancer Genome Atlas and Cancer Imaging Archive. The T1 weighted post-contrast and fluid-attenuated inversion-recovery volumes of patients were co-registered and segmented into delineate regions corresponding to three GBM phenotypes: necrosis, active tumour and oedema/invasion. A set of two-dimensional shape features were then extracted slicewise from each phenotype region and combined over slices to describe the three-dimensional shape of these phenotypes. Thereafter, a Kruskal–Wallis test was employed to identify shape features with significantly different distributions across phenotypes. Moreover, a Kaplan–Meier analysis was performed to find features strongly associated with GBM survival. Finally, a multivariate analysis based on the random forest model was used for predicting the survival group of patients with GBM. Results: Our analysis using the Kruskal–Wallis test showed that all but one shape feature had statistically significant differences across phenotypes, with p-value < 0.05, following Holm–Bonferroni correction, justifying the analysis of GBM tumour shapes on a per-phenotype basis. Furthermore, the survival analysis based on the Kaplan–Meier estimator identified three features derived from necrotic regions (i.e. Eccentricity, Extent and Solidity) that were significantly correlated with overall survival (corrected p-value < 0.05; hazard ratios between 1.68 and 1.87). In the multivariate analysis, features from necrotic regions gave the highest accuracy in predicting the survival group of patients, with a mean area under the receiver-operating characteristic curve (AUC) of 63.85%. Combining the features of all three phenotypes increased the mean AUC to 66.99%, suggesting that shape features from different phenotypes can be used in a synergic manner to predict GBM survival. Conclusion: Results show that shape features, in particular those extracted from necrotic regions, can be used effectively to characterize GBM tumours and predict the overall survival of patients with GBM. Advances in knowledge: Simple volumetric features have been largely used to characterize the different phenotypes of a GBM tumour (i.e. active tumour, oedema and necrosis). This study extends previous work by considering a wide range of shape features, extracted in different phenotypes, for the prediction of survival in patients with GBM. PMID:27781499

Proteolysis breaks tolerance toward intact α345(IV) collagen, eliciting novel anti-GBM autoantibodies specific for α345NC1 hexamers

PubMed Central

Olaru, Florina; Wang, Xu-Ping; Luo, Wentian; Ge, Linna; Miner, Jeffrey H; Kleinau, Sandra; Geiger, Xochiquetzal J.; Wasiluk, Andrew; Heidet, Laurence; Kitching, A. Richard; Borza, Dorin-Bogdan

2012-01-01

Goodpasture disease is an autoimmune kidney disease mediated by autoAbs against NC1 monomers of α3(IV) collagen that bind to the glomerular basement membrane (GBM), usually causing rapidly progressive glomerulonephritis. We identified a novel type of human IgG4-restricted anti-GBM autoAbs associated with mild non-progressive glomerulonephritis, which specifically targeted α345NC1 hexamers but not α3NC1 monomers. The mechanisms eliciting these anti-GBM autoAbs were investigated in mouse models recapitulating this phenotype. Wild type and FcγRIIB−/− mice immunized with autologous murine GBM NC1 hexamers produced mouse IgG1-restricted autoAbs specific for α345NC1 hexamers, which bound to the GBM in vivo but did not cause glomerulonephritis. In these mice, intact collagen IV from murine GBM was not immunogenic. However, in Col4a3−/− Alport mice, both intact collagen IV and NC1 hexamers from murine GBM elicited IgG antibodies specific for α3α4α5NC1 hexamers, which were not subclass restricted. As heterologous antigen in COL4A3-humanized mice, murine GBM NC1 hexamers elicited mouse IgG1, IgG2a and IgG2b autoAbs specific for α345NC1 hexamers and induced anti-GBM Ab glomerulonephritis. These findings indicate that tolerance toward autologous intact α3α4α5(IV) collagen is established in hosts expressing this antigen, even though autoreactive B cells specific for α345NC1 hexamers are not purged from their repertoire. Proteolysis selectively breaches this tolerance by generating autoimmunogenic α3α4α5NC1 hexamers. This provides a mechanism eliciting autoAbs specific for α345NC1 hexamers, which are restricted to non-inflammatory IgG subclasses and non-nephritogenic. In Alport syndrome, lack of tolerance toward α3α4α5(IV) collagen promotes production of alloantibodies to α345NC1 hexamers, including pro-inflammatory IgG subclasses which mediate post-transplant anti-GBM nephritis. PMID:23303673

Reduction of MLH1 and PMS2 confers temozolomide resistance and is associated with recurrence of glioblastoma

PubMed Central

Shinsato, Yoshinari; Furukawa, Tatsuhiko; Yunoue, Shunji; Yonezawa, Hajime; Minami, Kentarou; Nishizawa, Yukihiko; Ikeda, Ryuji; Kawahara, Kohichi; Yamamoto, Masatatsu; Hirano, Hirofumi; Tokimura, Hiroshi; Arita, Kazunori

2013-01-01

Although there is a relationship between DNA repair deficiency and temozolomide (TMZ) resistance in glioblastoma (GBM), it remains unclear which molecule is associated with GBM recurrence. We isolated three TMZ-resistant human GBM cell lines and examined the expression of O6-methylguanine-DNA methyltransferase (MGMT) and mismatch repair (MMR) components. We used immunohistochemical analysis to compare MutL homolog 1 (MLH1), postmeiotic segregation increased 2 (PMS2) and MGMT expression in primary and recurrent GBM specimens obtained from GBM patients during TMZ treatment. We found a reduction in MLH1 expression and a subsequent reduction in PMS2 protein levels in TMZ-resistant cells. Furthermore, MLH1 or PMS2 knockdown confered TMZ resistance. In recurrent GBM tumours, the expression of MLH1 and PMS2 was reduced when compared to primary tumours. PMID:24259277

Reduction of MLH1 and PMS2 confers temozolomide resistance and is associated with recurrence of glioblastoma.

PubMed

Shinsato, Yoshinari; Furukawa, Tatsuhiko; Yunoue, Shunji; Yonezawa, Hajime; Minami, Kentarou; Nishizawa, Yukihiko; Ikeda, Ryuji; Kawahara, Kohichi; Yamamoto, Masatatsu; Hirano, Hirofumi; Tokimura, Hiroshi; Arita, Kazunori

2013-12-01

Although there is a relationship between DNA repair deficiency and temozolomide (TMZ) resistance in glioblastoma (GBM), it remains unclear which molecule is associated with GBM recurrence. We isolated three TMZ-resistant human GBM cell lines and examined the expression of O6-methylguanine-DNA methyltransferase (MGMT) and mismatch repair (MMR) components. We used immunohistochemical analysis to compare MutL homolog 1 (MLH1), postmeiotic segregation increased 2 (PMS2) and MGMT expression in primary and recurrent GBM specimens obtained from GBM patients during TMZ treatment. We found a reduction in MLH1 expression and a subsequent reduction in PMS2 protein levels in TMZ-resistant cells. Furthermore, MLH1 or PMS2 knockdown confered TMZ resistance. In recurrent GBM tumours, the expression of MLH1 and PMS2 was reduced when compared to primary tumours.

Targeted therapy of glioblastoma stem-like cells and tumor non-stem cells using cetuximab-conjugated iron-oxide nanoparticles

PubMed Central

Kaluzova, Milota; Bouras, Alexandros; Machaidze, Revaz; Hadjipanayis, Costas G.

2015-01-01

Malignant gliomas remain aggressive and lethal primary brain tumors in adults. The epidermal growth factor receptor (EGFR) is frequently overexpressed in the most common malignant glioma, glioblastoma (GBM), and represents an important therapeutic target. GBM stem-like cells (GSCs) present in tumors are felt to be highly tumorigenic and responsible for tumor recurrence. Multifunctional magnetic iron-oxide nanoparticles (IONPs) can be directly imaged by magnetic resonance imaging (MRI) and designed to therapeutically target cancer cells. The targeting effects of IONPs conjugated to the EGFR inhibitor, cetuximab (cetuximab-IONPs), were determined with EGFR- and EGFRvIII-expressing human GBM neurospheres and GSCs. Transmission electron microscopy revealed cetuximab-IONP GBM cell binding and internalization. Fluorescence microscopy and Prussian blue staining showed increased uptake of cetuximab-IONPs by EGFR- as well as EGFRvIII-expressing GSCs and neurospheres in comparison to cetuximab or free IONPs. Treatment with cetuximab-IONPs resulted in a significant antitumor effect that was greater than with cetuximab alone due to more efficient, CD133-independent cellular targeting and uptake, EGFR signaling alterations, EGFR internalization, and apoptosis induction in EGFR-expressing GSCs and neurospheres. A significant increase in survival was found after cetuximab-IONP convection-enhanced delivery treatment of 3 intracranial rodent GBM models employing human EGFR-expressing GBM xenografts. PMID:25871395

MiR224-3p inhibits hypoxia-induced autophagy by targeting autophagy-related genes in human glioblastoma cells.

PubMed

Guo, Xing; Xue, Hao; Guo, Xiaofan; Gao, Xiao; Xu, Shugang; Yan, Shaofeng; Han, Xiao; Li, Tong; Shen, Jie; Li, Gang

2015-12-08

Human glioblastoma multiforme (GBM) is a malignant solid tumor characterized by severe hypoxia. Autophagy plays a protective role in cancer cells under hypoxia. However, the microRNA (miRNA)-related molecular mechanisms underlying hypoxia-reduced autophagy remain poorly understood in GBM. In this study, we performed a miRNA microarray analysis on GBM cells and found that numerous miRNAs were differentially expressed under hypoxic conditions. Further research showed that miR224-3p, one of the significantly down-regulated miRNAs, was involved in regulating hypoxia-induced autophagy in GBM cells. Overexpression of miR224-3p abolished hypoxia-induced autophagy, whereas knocking down endogenous miR224-3p increased autophagic activity under normoxia. In addition, we demonstrated that miR224-3p inhibited autophagy by directly suppressing the expression of two autophagy-related genes (ATGs), ATG5 and FAK family-interacting protein of 200 kDa (FIP200). Furthermore, in vitro, miR224-3p attenuated cell proliferation and promoted hypoxia-induced apoptosis, and in vivo, overexpression of miR224-3p inhibited tumorigenesis of GBM cells. Collectively, our study identified a novel hypoxia-down-regulated miRNA, miR224-3p, as a key modulator of autophagy by inhibiting ATGs in GBM cells.

Opposing roles of PGD2 in GBM.

PubMed

Ferreira, Matthew Thomas; Gomes, Renata Nascimento; Panagopoulos, Alexandros Theodoros; de Almeida, Fernando Gonçalves; Veiga, José Carlos Esteves; Colquhoun, Alison

2018-01-01

The World Health Organization classifies glioblastoma (GBM) as a grade IV astrocytoma. Despite the advances in chemotherapy, surgery, and radiation treatments that improve a patient's length of survival, the overall trajectory of the disease remains unchanged. GBM cells produce significant levels of various types of bioactive lipids. Prostaglandin D 2 (PGD 2 ) influences both pro- and anti-tumorigenic activities in the cell; however, its role in GBM is unclear. Therefore, this study aimed to identify the impact of PGD 2 on GBM cell activities in vitro. First we looked to identify the presence of the PGD 2 synthesis pathway through RT-PCR, immunohistochemistry, and HPLC-MS/MS in three GBM cell lines. Then, to observe PGD 2 's effects on cell count and apoptosis/mitosis (Hoechst 33342 stain), and migration (Transwell Assay), the cells were treated in vitro with physiological (<1μM) and/or supraphysiological (>1μM) concentrations of PGD 2 over 72h. HPLC-MS/MS was used to identify the lipid composition of patients with either Grade II/III gliomas or GBM. We identified the presence of endogenous PGD 2 with its corresponding enzymes and receptors. Exogenous PGD 2 both increased cell count (<1μM) and decreased cell count (10μM) in a concentration-dependent manner. There were no significant effects on apoptosis. A significant decrease in mitotic activity was seen only in U251MG, and a significant increase was seen in migration with 5μM PGD 2 treatments. A very significant increase of PGD 2 was seen from Grade II/III gliomas to GBM. Our study demonstrates that prostaglandin D 2 possesses a dynamic, concentration-dependent effect in GBM cell activities. The increase of PGD 2 production in GBM patients suggests a pro-tumorigenic role of PGD 2 in glioma growth and invasion. Therefore, prostaglandin signaling in GBM requires further investigation to identify new targets for more effective therapies. Copyright © 2017 Elsevier Inc. All rights reserved.

Qualitative examination of enacted stigma towards gay and bisexual men and related health outcomes in Tajikistan, Central Asia.

PubMed

Ibragimov, Umedjon; Wong, Frank Y

2018-05-01

Gay and bisexual men (GBM) in Tajikistan are an extremely stigmatised group at high risk for sexually transmitted infections and HIV. However, there is a paucity of research on how and in what way stigma affects their lives. We conducted a qualitative study to examine the impact of stigma on GBM's lives in Tajikistan, focusing on stigma enactors, settings, factors affecting vulnerability of GBM and health consequences. Eight individual in-depth interviews and 3 focus-group discussions with 13 participants (N   =   21) from GBM community were conducted in two cities of Tajikistan. Results reveal that police frequently engage in blackmail and perpetrate sexual and physical violence against GBM. Service providers often discriminate against GBM limiting their access to health and legal services. Exposure to stigma results in chronic stress affecting mental health of GBM. Fear of disclosure, low social cohesion, absence of prominent opinion leaders and activists reduce resilience of GBM community to stigma. State-sanctioned violations of human rights of marginalised populations and lack of effective legal protection mechanisms have enabled widespread harassment of GBM. These findings warrant further research on stigma leading to the development of culturally adapted and tailored multilevel structural interventions, including broad legal and policy reforms.

Harnessing the cross-talk between tumor cells and tumor-associated macrophages with a nano-drug for modulation of glioblastoma immune microenvironment.

PubMed

Li, Tong-Fei; Li, Ke; Wang, Chao; Liu, Xin; Wen, Yu; Xu, Yong-Hong; Zhang, Quan; Zhao, Qiu-Ya; Shao, Ming; Li, Yan-Ze; Han, Min; Komatsu, Naoki; Zhao, Li; Chen, Xiao

2017-12-28

Glioblastoma (GBM) is the most frequent and malignant brain tumor with a high mortality rate. The presence of a large population of macrophages (Mφ) in the tumor microenvironment is a prominent feature of GBM and these so-called tumor-associated Mφ (TAM) closely interact with the GBM cells to promote the survival, progression and therapy resistance of the GBM. Various therapeutic strategies have been devised either targeting the GBM cells or the TAM but few have addressed the cross-talks between the two cell populations. The present study was carried out to explore the possibility of exploiting the cross-talks between the GBM cells (GC) and TAM for modulation of the GBM microenvironment through using Nano-DOX, a drug composite based on nanodiamonds bearing doxorubicin. In the in vitro work on human cell models, Nano-DOX-loaded TAM were first shown to be viable and able to infiltrate three-dimensional GC spheroids and release cargo drug therein. GC were then demonstrated to encourage Nano-DOX-loaded TAM to unload Nano-DOX back into GC which consequently emitted damage-associated molecular patterns (DAMPs) that are powerful immunostimulatory agents as well as indicators of cell damage. Nano-DOX was next proven to be a more potent inducer of GC DAMPs emission than doxorubicin. As a result, Nano-DOX-damaged GC exhibited an enhanced ability to attract both TAM and Nano-DOX-loaded TAM. Most remarkably, Nano-DOX-damaged GC reprogrammed the TAM from a pro-GBM phenotype to an anti-GBM phenotype that suppressed GC growth. Finally, the in vivo relevance of the in vitro findings was tested in animal study. Mice bearing orthotopic human GBM xenografts were intravenously injected with Nano-DOX-loaded mouse TAM which were found releasing drug in the GBM xenografts 24h after injection. GC damage was evidenced by the induction of DAMPs emission within the xenografts and a shift of TAM phenotype was detected as well. Taken together, our results demonstrate a novel way with therapeutic potential to harness the cross-talk between GBM cells and TAM for modulation of the tumor immune microenvironment. Copyright © 2017 Elsevier B.V. All rights reserved.

Expression of R132H mutational IDH1 in human U87 glioblastoma cells affects the SREBP1a pathway and induces cellular proliferation.

PubMed

Zhu, Jian; Cui, Gang; Chen, Ming; Xu, Qinian; Wang, Xiuyun; Zhou, Dai; Lv, Shengxiang; Fu, Linshan; Wang, Zhong; Zuo, Jianling

2013-05-01

Sterol regulatory element-binding protein-1a (SREBP1a) is a member of the SREBP family of transcription factors, which mainly controls homeostasis of lipids. SREBP1a can also activate the transcription of isocitrate dehydrogenase 1 (IDH1) by binding to its promoter region. IDH1 mutations, especially R132H mutation of IDH1, are a common feature of a major subset of human gliomas. There are few data available on the relationship between mutational IDH1 expression and SREBP1a pathway. In this study, we investigated cellular effects and SREBP1a pathway alterations caused by R132H mutational IDH1 expression in U87 cells. Two glioma cell lines, stably expressing mutational (U87/R132H) or wild type (U87/wt) IDH1, were established. A cell line, stably transfected with pcDNA3.1(+) (U87/vector), was generated as a control. Click-iT EdU assay, sulforhodamine B assay, and wound healing assay respectively showed that the expression of R132H induced cellular proliferation, cell growth, and cell migration. Western blot revealed that SREBP1 was increased in U87/R132H compared with that in U87/wt. Elevated SREBP1a and several its target genes, but not SREBP1c, were detected by real-time polymerase chain reaction in U87/R132H. All these findings indicated that R132H mutational IDH1 is involved in the regulation of proliferation, growth, and migration of glioma cells. These effects may partially be mediated by SREBP1a pathway.

Dysregulated miR-671-5p / CDR1-AS / CDR1 / VSNL1 axis is involved in glioblastoma multiforme.

PubMed

Barbagallo, Davide; Condorelli, Angelo; Ragusa, Marco; Salito, Loredana; Sammito, Mariangela; Banelli, Barbara; Caltabiano, Rosario; Barbagallo, Giuseppe; Zappalà, Agata; Battaglia, Rosalia; Cirnigliaro, Matilde; Lanzafame, Salvatore; Vasquez, Enrico; Parenti, Rosalba; Cicirata, Federico; Di Pietro, Cinzia; Romani, Massimo; Purrello, Michele

2016-01-26

MiR-671-5p is encoded by a gene localized at 7q36.1, a region amplified in human glioblastoma multiforme (GBM), the most malignant brain cancer. To investigate whether expression of miR-671-5p were altered in GBM, we analyzed biopsies from a cohort of forty-five GBM patients and from five GBM cell lines. Our data show significant overexpression of miR-671-5p in both biopsies and cell lines. By exploiting specific miRNA mimics and inhibitors, we demonstrated that miR-671-5p overexpression significantly increases migration and to a less extent proliferation rates of GBM cells. Through a combined in silico and in vitro approach, we identified CDR1-AS, CDR1, VSNL1 as downstream miR-671-5p targets in GBM. Expression of these genes significantly decreased both in GBM biopsies and cell lines and negatively correlated with that of miR-671-5p. Based on our data, we propose that the axis miR-671-5p / CDR1-AS / CDR1 / VSNL1 is functionally altered in GBM cells and is involved in the modification of their biopathological profile.

Suppression of Peroxiredoxin 4 in Glioblastoma Cells Increases Apoptosis and Reduces Tumor Growth

PubMed Central

Kim, Tae Hyong; Song, Jieun; Alcantara Llaguno, Sheila R.; Murnan, Eric; Liyanarachchi, Sandya; Palanichamy, Kamalakannan; Yi, Ji-Yeun; Viapiano, Mariano Sebastian; Nakano, Ichiro; Yoon, Sung Ok; Wu, Hong; Parada, Luis F.; Kwon, Chang-Hyuk

2012-01-01

Glioblastoma multiforme (GBM), the most common and aggressive primary brain malignancy, is incurable despite the best combination of current cancer therapies. For the development of more effective therapies, discovery of novel candidate tumor drivers is urgently needed. Here, we report that peroxiredoxin 4 (PRDX4) is a putative tumor driver. PRDX4 levels were highly increased in a majority of human GBMs as well as in a mouse model of GBM. Reducing PRDX4 expression significantly decreased GBM cell growth and radiation resistance in vitro with increased levels of ROS, DNA damage, and apoptosis. In a syngenic orthotopic transplantation model, Prdx4 knockdown limited GBM infiltration and significantly prolonged mouse survival. These data suggest that PRDX4 can be a novel target for GBM therapies in the future. PMID:22916164

Immunoadsorption in Anti-GBM Glomerulonephritis: Case Report in a Child and Literature Review.

PubMed

Dorval, Guillaume; Lion, Mathilde; Guérin, Sophie; Krid, Saoussen; Galmiche-Rolland, Louise; Salomon, Rémi; Boyer, Olivia

2017-11-01

Antiglomerular basement membrane glomerulonephritis (anti-GBM GN) is a rare autoimmune disease that is characterized by rapidly progressive glomerulonephritis that may be associated with pulmonary hemorrhage. Anti-GBM GN is caused by autoantibodies (classically type G immunoglobulin) directed against the α3 subunit of type IV collagen. Without any appropriate treatment, the disease is generally fulminant, and patient and kidney survival is poor. The current guidelines recommend the use of plasma exchanges and immunosuppressive drugs. Immunoadsorption (IA) can remove pathogenic IgGs from the circulation and do not require plasma infusions, contrary to plasma exchanges. IA has seldom been used in adult patients with good tolerance and efficiency. We report herein the first pediatric case successfully treated with IA combined with immunosuppressive drugs in a 7-year-old girl who presented acute kidney injury (estimated glomerular filtration rate 38 mL/minute/1.73 m 2 ). A kidney biopsy revealed numerous >80% glomerular crescents and linear IgG deposits along the glomerular basement membrane. Ten IA sessions led to rapid and sustained clearance of autoantibodies and improvement of kidney function until 21 months after onset (glomerular filtration rate 87 mL/minute/1.73 m 2 ). No adverse effect was noted. This report adds to the growing body of evidence suggesting IA as a therapeutic alternative to plasma exchanges in anti-GBM GN. The other 27 published pediatric cases of anti-GBM GN are reviewed. Copyright © 2017 by the American Academy of Pediatrics.

Dexamethasone-mediated oncogenicity in vitro and in an animal model of glioblastoma.

PubMed

Luedi, Markus M; Singh, Sanjay K; Mosley, Jennifer C; Hassan, Islam S A; Hatami, Masumeh; Gumin, Joy; Andereggen, Lukas; Sulman, Erik P; Lang, Frederick F; Stueber, Frank; Fuller, Gregory N; Colen, Rivka R; Zinn, Pascal O

2018-01-12

OBJECTIVE Dexamethasone, a known regulator of mesenchymal programming in glioblastoma (GBM), is routinely used to manage edema in GBM patients. Dexamethasone also activates the expression of genes, such as CEBPB, in GBM stem cells (GSCs). However, the drug's impact on invasion, proliferation, and angiogenesis in GBM remains unclear. To determine whether dexamethasone induces invasion, proliferation, and angiogenesis in GBM, the authors investigated the drug's impact in vitro, in vivo, and in clinical information derived from The Cancer Genome Atlas (TCGA) cohort. METHODS Expression profiles of patients from the TCGA cohort with mesenchymal GBM (n = 155) were compared with patients with proneural GBM by comparative marker selection. To obtain robust data, GSCs with IDH1 wild-type (GSC3) and with IDH1 mutant (GSC6) status were exposed to dexamethasone in vitro and in vivo and analyzed for invasion (Boyden chamber, human-specific nucleolin), proliferation (Ki-67), and angiogenesis (CD31). Ex vivo tumor cells from dexamethasone-treated and control mice were isolated by fluorescence activated cell sorting and profiled using Affymetrix chips for mRNA (HTA 2.0) and microRNAs (miRNA 4.0). A pathway analysis was performed to identify a dexamethasone-regulated gene signature, and its relationship with overall survival (OS) was assessed using Kaplan-Meier analysis in the entire GBM TCGA cohort (n = 520). RESULTS The mesenchymal subgroup, when compared with the proneural subgroup, had significant upregulation of a dexamethasone-regulated gene network, as well as canonical pathways of proliferation, invasion, and angiogenesis. Dexamethasone-treated GSC3 demonstrated a significant increase in invasion, both in vitro and in vivo, whereas GSC6 demonstrated a modest increase. Furthermore, dexamethasone treatment of both GSC3 and GSC6 lines resulted in significantly elevated cell proliferation and angiogenesis in vivo. Patients with mesenchymal GBM had significant upregulation of dexamethasone-regulated pathways when compared with patients with proneural GBM. A prognostic (p = 0.0007) 33-gene signature was derived from the ex vivo expression profile analyses and used to dichotomize the entire TCGA cohort by high (median OS 12.65 months) or low (median OS 14.91 months) dexamethasone signature. CONCLUSIONS The authors present evidence that furthers the understanding of the complex effects of dexamethasone on biological characteristics of GBM. The results suggest that the drug increases invasion, proliferation, and angiogenesis in human GSC-derived orthotopic tumors, potentially worsening GBM patients' prognoses. The authors believe that careful investigation is needed to determine how to minimize these deleterious dexamethasone-associated side effects in GBM.

Glomerular basement membrane injuries in IgA nephropathy evaluated by double immunostaining for α5(IV) and α2(IV) chains of type IV collagen and low-vacuum scanning electron microscopy.

PubMed

Masuda, Yukinari; Yamanaka, Nobuaki; Ishikawa, Arimi; Kataoka, Mitue; Arai, Takashi; Wakamatsu, Kyoko; Kuwahara, Naomi; Nagahama, Kiyotaka; Ichikawa, Kaori; Shimizu, Akira

2015-06-01

The glomerulus contains well-developed capillaries, which are at risk of injury due to high hydrostatic pressure, hyperfiltration, hypertension and inflammation. However, the pathological alterations of the injured glomerular basement membrane (GBM), the main component of the glomerular filtration barrier, are still uncertain in cases of glomerulonephritis. We examined the alterations of the GBM in 50 renal biopsy cases with IgA nephropathy (31.8 ± 17.6 years old) using double immunostaining for the α2(IV) and α5(IV) chains of type IV collagen, and examining the ultrastructural alterations by transmission electron microscopy (TEM) and low-vacuum scanning electron microscopy (LV-SEM). The GBM of IgA nephropathy cases showed various morphological and qualitative alterations. In the TEM findings, thinning, gaps, rupture, thickening with a lamellar and reticular structure and double contours were detected in the GBM. Double immunostaining for α5(IV) and α2(IV) showed thickening of the GBM with reduced α5(IV) and increased α2(IV), or mosaic images of α5(IV) and α2(IV), and holes, fractures, spiny projections and rupture of α5(IV) in the GBM. In addition, LV-SEM showed an etched image and multiple holes in a widening and wavy GBM. These findings might be associated with the development of a brittle GBM in IgA nephropathy. Glomerular basement membrane alterations were frequently noted in IgA nephropathy, and were easily evaluated by double immunostaining for α2(IV) and α5(IV) of type IV collagen and LV-SEM. The application of these analyses to human renal biopsy specimens may enhance our understanding of the alterations of the GBM that occur in human glomerular diseases.

Bcl-w Enhances Mesenchymal Changes and Invasiveness of Glioblastoma Cells by Inducing Nuclear Accumulation of β-Catenin

PubMed Central

Lee, Woo Sang; Woo, Eun Young; Kwon, Junhye; Park, Myung-Jin; Lee, Jae-Seon; Han, Young-Hoon; Bae, In Hwa

2013-01-01

Bcl-w a pro-survival member of the Bcl-2 protein family, is expressed in a variety of cancer types, including gastric and colorectal adenocarcinomas, as well as glioblastoma multiforme (GBM), the most common and lethal brain tumor type. Previously, we demonstrated that Bcl-w is upregulated in gastric cancer cells, particularly those displaying infiltrative morphology. These reports propose that Bcl-w is strongly associated with aggressive characteristic, such as invasive or mesenchymal phenotype of GBM. However, there is no information from studies of the role of Bcl-w in GBM. In the current study, we showed that Bcl-w is upregulated in human glioblastoma multiforme (WHO grade IV) tissues, compared with normal and glioma (WHO grade III) tissues. Bcl-w promotes the mesenchymal traits of glioblastoma cells by inducing vimentin expression via activation of transcription factors, β-catenin, Twist1 and Snail in glioblastoma U251 cells. Moreover, Bcl-w induces invasiveness by promoting MMP-2 and FAK activation via the PI3K-p-Akt-p-GSK3β-β-catenin pathway. We further confirmed that Bcl-w has the capacity to induce invasiveness in several human cancer cell lines. In particular, Bcl-w-stimulated β-catenin is translocated into the nucleus as a transcription factor and promotes the expression of target genes, such as mesenchymal markers or MMPs, thereby increasing mesenchymal traits and invasiveness. Our findings collectively indicate that Bcl-w functions as a positive regulator of invasiveness by inducing mesenchymal changes and that trigger their aggressiveness of glioblastoma cells. PMID:23826359

The Anti-Warburg Effect Elicited by the cAMP-PGC1α Pathway Drives Differentiation of Glioblastoma Cells into Astrocytes.

PubMed

Xing, Fan; Luan, Yizhao; Cai, Jing; Wu, Sihan; Mai, Jialuo; Gu, Jiayu; Zhang, Haipeng; Li, Kai; Lin, Yuan; Xiao, Xiao; Liang, Jiankai; Li, Yuan; Chen, Wenli; Tan, Yaqian; Sheng, Longxiang; Lu, Bingzheng; Lu, Wanjun; Gao, Mingshi; Qiu, Pengxin; Su, Xingwen; Yin, Wei; Hu, Jun; Chen, Zhongping; Sai, Ke; Wang, Jing; Chen, Furong; Chen, Yinsheng; Zhu, Shida; Liu, Dongbing; Cheng, Shiyuan; Xie, Zhi; Zhu, Wenbo; Yan, Guangmei

2017-01-10

Glioblastoma multiforme (GBM) is among the most aggressive of human cancers. Although differentiation therapy has been proposed as a potential approach to treat GBM, the mechanisms of induced differentiation remain poorly defined. Here, we established an induced differentiation model of GBM using cAMP activators that specifically directed GBM differentiation into astroglia. Transcriptomic and proteomic analyses revealed that oxidative phosphorylation and mitochondrial biogenesis are involved in induced differentiation of GBM. Dibutyryl cyclic AMP (dbcAMP) reverses the Warburg effect, as evidenced by increased oxygen consumption and reduced lactate production. Mitochondrial biogenesis induced by activation of the CREB-PGC1α pathway triggers metabolic shift and differentiation. Blocking mitochondrial biogenesis using mdivi1 or by silencing PGC1α abrogates differentiation; conversely, overexpression of PGC1α elicits differentiation. In GBM xenograft models and patient-derived GBM samples, cAMP activators also induce tumor growth inhibition and differentiation. Our data show that mitochondrial biogenesis and metabolic switch to oxidative phosphorylation drive the differentiation of tumor cells. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Frequent Nek1 overexpression in human gliomas

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhu, Jun; Neurosurgery Department, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai; Cai, Yu, E-mail: aihaozuqiu22@163.com

Never in mitosis A (NIMA)-related kinase 1 (Nek1) regulates cell cycle progression to mitosis. Its expression and potential functions in human gliomas have not been studied. Here, our immunohistochemistry (IHC) assay and Western blot assay results showed that Nek1 expression was significantly upregulated in fresh and paraffin-embedded human glioma tissues. Its level in normal brain tissues was low. Nek1 overexpression in human gliomas was correlated with the proliferation marker (Ki-67), tumor grade, Karnofsky performance scale (KPS) and more importantly, patients’ poor survival. Further studies showed that Nek1 expression level was also increased in multiple human glioma cell lines (U251-MG, U87-MG,more » U118, H4 and U373). Significantly, siRNA-mediated knockdown of Nek1 inhibited glioma cell (U87-MG/U251-MG) growth. Nek1 siRNA also sensitized U87-MG/U251-MG cells to temozolomide (TMZ), causing a profound apoptosis induction and growth inhibition. The current study indicates Nek1 might be a novel and valuable oncotarget of glioma, it is important for glioma cell growth and TMZ-resistance. - Highlights: • Nek1 is upregulated in multiple human glioma tissues and cell lines. • Nek1 overexpression correlates with glioma grades and patients’ KPS score. • Nek1 overexpression correlates with patients’ poor overall survival. • siRNA knockdown of Nek1 inhibits glioma cell growth. • siRNA knockdown of Nek1 sensitizes human glioma cells to temozolomide.« less

Dexamethasone alone and in combination with desipramine, phenytoin, valproic acid or levetiracetam interferes with 5-ALA-mediated PpIX production and cellular retention in glioblastoma cells.

PubMed

Lawrence, Johnathan E; Steele, Christopher J; Rovin, Richard A; Belton, Robert J; Winn, Robert J

2016-03-01

Extent of resection of glioblastoma (GBM) correlates with overall survival. Fluorescence-guided resection (FGR) using 5-aminolevulinic acid (5-ALA) can improve the extent of resection. Unfortunately not all patients given 5-ALA accumulate sufficient quantities of protoporphyrin IX (PpIX) for successful FGR. In this study, we investigated the effects of dexamethasone, desipramine, phenytoin, valproic acid, and levetiracetam on the production and accumulation of PpIX in U87MG cells. All of these drugs, except levetiracetam, reduce the total amount of PpIX produced by GBM cells (p < 0.05). When dexamethasone is mixed with another drug (desipramine, phenytoin, valproic acid or levetiracetam) the amount of PpIX produced is further decreased (p < 0.01). However, when cells are analyzed for PpIX cellular retention, dexamethasone accumulated significantly more PpIX than the vehicle control (p < 0.05). Cellular retention of PpIX was not different from controls in cells treated with dexamethasone plus desipramine, valproic acid or levetiracetam, but was significantly less for dexamethasone plus phenytoin (p < 0.01). These data suggest that medications given before and during surgery may interfere with PpIX accumulation in malignant cells. At this time, levetiracetam appears to be the best medication in its class (anticonvulsants) for patients undergoing 5-ALA-mediated FGR.

Identification of repaglinide as a therapeutic drug for glioblastoma multiforme

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xiao, Zui Xuan; Chen, Ruo Qiao; Hu, Dian Xing

Glioblastoma multiforme (GBM) is a highly aggressive brain tumor with a median survival time of only 14 months after treatment. It is urgent to find new therapeutic drugs that increase survival time of GBM patients. To achieve this goal, we screened differentially expressed genes between long-term and short-term survived GBM patients from Gene Expression Omnibus database and found gene expression signature for the long-term survived GBM patients. The signaling networks of all those differentially expressed genes converged to protein binding, extracellular matrix and tissue development as revealed in BiNGO and Cytoscape. Drug repositioning in Connectivity Map by using the genemore » expression signature identified repaglinide, a first-line drug for diabetes mellitus, as the most promising novel drug for GBM. In vitro experiments demonstrated that repaglinide significantly inhibited the proliferation and migration of human GBM cells. In vivo experiments demonstrated that repaglinide prominently prolonged the median survival time of mice bearing orthotopic glioma. Mechanistically, repaglinide significantly reduced Bcl-2, Beclin-1 and PD-L1 expression in glioma tissues, indicating that repaglinide may exert its anti-cancer effects via apoptotic, autophagic and immune checkpoint signaling. Taken together, repaglinide is likely to be an effective drug to prolong life span of GBM patients. - Highlights: • Gene expression signarue in long-term survived GBM patients are identified from Gene Expression Omnibus database. • Repaglinide is identified as a survival-related drug for GBM via drug repositioning in CMap. • Repaglinide effectively kills GBM cells, inhibits GBM cell migration and increases survival of mice bearing orthotopic glioma. • Repaglinide reduces Bcl-2, Beclin-1 and PD-L1 in GBM tissues.« less

Performance evaluation of a novel chemiluminescence assay for detection of anti-GBM antibodies: an international multicenter study.

PubMed

Mahler, Michael; Radice, Antonella; Sinico, Renato A; Damoiseaux, Jan; Seaman, Andrea; Buckmelter, Kristen; Vizjak, Alenka; Buchner, Carol; Binder, Walter L; Fritzler, Marvin J; Cui, Zhao

2012-01-01

Autoantibodies to the non-collagen region (NC1) of the alpha-3 subunit of collagen IV represent a serological hallmark in the diagnosis of Goodpasture's syndrome (GPS). The objective of our study was to carefully analyze the performance characteristics of a novel anti-glomerular basement membrane (GBM) chemiluminescence immunoassay (CIA). Sera from patients with GPS (n = 90) were collected from four clinical centers. Samples from different disease groups (n = 397) and healthy individuals (n = 400) were used as controls. All samples were tested for anti-GBM antibodies by a rapid, random access CIA (QUANTA Flash™ GBM). Most of the samples were also tested using other methods including different commercial anti-GBM IgG assays and research assays for anti-GBM IgA and IgM. The sensitivity and specificity of the novel CIA was 95.6% [95% confidence interval (CI) 89.0-98.8%] and 99.6% (95% CI 98.9-99.9%), respectively. Receiver operating characteristic analysis showed good discrimination between GPS patients and controls. The area under the curve was 0.98 (CI 0.96-1.0). The three anti-GBM antibody-positive samples from the control group were from two healthy individuals and one human immunodeficiency virus (HIV)-infected patient. All three individuals had low levels of anti-GBM antibodies [20, 24 and 25 chemiluminescent unit (CU), cutoff 20 CU]. When the results of the new CIA were compared to other methods, good agreement was observed: 95.8% (kappa = 0.92) versus EliA™ GBM, 97.4% (kappa = 0.95) versus both BINDAZYME™ Anti-GBM and QUANTA Lite® GBM. Anti-GBM IgA was detectable in low concentrations in patients with GPS and was associated with anti-GBM IgG but was less useful in discriminating GPS patients and controls. No discrimination was found for anti-GBM IgM. The novel QUANTA Flash™ GBM CIA demonstrated good sensitivity and specificity and had good agreement with other methods. Our data confirm that ∼5% of patients with GPS do not have detectable levels of anti-GBM antibodies.

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.

PubMed

Shang, Hung-Sheng; Shih, Yung-Luen; Lu, Tai-Jung; Lee, Ching-Hsiao; Hsueh, Shu-Ching; Chou, Yu-Cheng; Lu, Hsu-Feng; Liao, Nien-Chieh; Chung, Jing-Gung

2016-12-01

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 Ca 2+ release, but decreased the mitochondrial membrane potential (ΔΨ m ) 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. © 2015 Wiley Periodicals, Inc.

The interaction of bee products with temozolomide in human diffuse astrocytoma, glioblastoma multiforme and astroglia cell lines.

PubMed

Borawska, Maria H; Markiewicz-Żukowska, Renata; Naliwajko, Sylwia K; Moskwa, Justyna; Bartosiuk, Emilia; Socha, Katarzyna; Surażyński, Arkadiusz; Kochanowicz, Jan; Mariak, Zenon

2014-01-01

In the present study, we investigated the influence of extracts from Salix spp. honey (ESH), beebread (EBB), and royal jelly (ERJ) with and without temozolomide (TMZ) on cell lines derived from a patient with diffuse astrocytoma (DASC), human glioblastoma multiforme (U87MG), and normal human astroglia (SVGp12). DASC was identified by immunocytochemistry. TMZ (20 μM) in combination with ESH (30 μg/mL), EBB (50 μg/mL), and ERJ (30 μg/mL) has stronger cytotoxic activity on U87MG cells after 72 h (20.0, 26.5, and 29.3% of control, respectively) than TMZ alone (about 6% of control). An increase of the cytotoxic effect and inhibition of DNA synthesis in SVGp12 were detected after administering TMZ with the studied extracts. NF-κB p50 subunit was reduced in U87MG cells after treatment with ESH (70.9%) and ESH + TMZ (74.7%). A significant decline of MMP-9 and MMP-2 secretion in cultured U87MG was detected after incubation with EBB (42.9% and 73.0%, respectively) and EBB + TMZ (38.4% and 68.5%, respectively). In conclusion, the use of bee products may increase the cytotoxic effect of TMZ in U87MG but also in SVGp12 cell line. It is important to note that the U87MG cells were sensitive to natural bee products, although there was no influence of natural bee products on the DASC cells.

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

PubMed Central

Shannon, Stephen; Vaca, Connan; Jia, Dongxuan; Entersz, Ildiko; Schaer, Andrew; Carcione, Jonathan; Weaver, Michael; Avidar, Yoav; Pettit, Ryan; Nair, Mohan; Khan, Atif; Foty, Ramsey A.

2015-01-01

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. PMID:26284619

Long non-coding RNA CASP5 promotes the malignant phenotypes of human glioblastoma multiforme.

PubMed

Zhou, Yali; Dai, Wei; Wang, Handong; Pan, Hao; Wang, Qiang

2018-06-12

Long non-coding RNAs (lncRNAs) have been demonstrated to be intensively involved in the development of various carcinomas, including glioblastoma multiforme (GBM). However, only a few of them have been well characterized. LncRNA CASP5 have been found to be up-regulated in GBM tissues compared with normal tissues in a microarray-based lncRNA profiling study. In the present study, we further explored the biological role of lncRNA CASP5 in GBM. We examined the expression level of lncRNA CASP5 in GBM tissues as well as GBM cell lines. CCK-8 assay, flow cytometric analysis, western blotting, orthotopic GBM model as well as transwell assay were performed to investigate the biological role of CASP5. We observed that lncRNA CASP5 was highly expressed in GBM tissues and cell lines. Knockdown of CASP5 greatly inhibited GBM proliferation and resulted in G1 cell cycle arrest along with higher apoptosis ratios in vitro and in vivo, while overexpression led to the opposite phenomenon. Furthermore, the migration and invasion ability of GBM cells were significantly decreased after CASP5 down-regulation, while increased migration and invasion can be observed after CASP5 up-regulation. We demonstrate for the first time the potential oncogenic role of lncRNA CASP5 which may be helpful for identifying novel therapeutic targets in GBM. Copyright © 2018 Elsevier Inc. All rights reserved.

Cellular prion protein controls stem cell-like properties of human glioblastoma tumor-initiating cells.

PubMed

Corsaro, Alessandro; Bajetto, Adriana; Thellung, Stefano; Begani, Giulia; Villa, Valentina; Nizzari, Mario; Pattarozzi, Alessandra; Solari, Agnese; Gatti, Monica; Pagano, Aldo; Würth, Roberto; Daga, Antonio; Barbieri, Federica; Florio, Tullio

2016-06-21

Prion protein (PrPC) is a cell surface glycoprotein whose misfolding is responsible for prion diseases. Although its physiological role is not completely defined, several lines of evidence propose that PrPC is involved in self-renewal, pluripotency gene expression, proliferation and differentiation of neural stem cells. Moreover, PrPC regulates different biological functions in human tumors, including glioblastoma (GBM). We analyzed the role of PrPC in GBM cell pathogenicity focusing on tumor-initiating cells (TICs, or cancer stem cells, CSCs), the subpopulation responsible for development, progression and recurrence of most malignancies. Analyzing four GBM CSC-enriched cultures, we show that PrPC expression is directly correlated with the proliferation rate of the cells. To better define its role in CSC biology, we knocked-down PrPC expression in two of these GBM-derived CSC cultures by specific lentiviral-delivered shRNAs. We provide evidence that CSC proliferation rate, spherogenesis and in vivo tumorigenicity are significantly inhibited in PrPC down-regulated cells. Moreover, PrPC down-regulation caused loss of expression of the stemness and self-renewal markers (NANOG, Sox2) and the activation of differentiation pathways (i.e. increased GFAP expression). Our results suggest that PrPC controls the stemness properties of human GBM CSCs and that its down-regulation induces the acquisition of a more differentiated and less oncogenic phenotype.

Cellular prion protein controls stem cell-like properties of human glioblastoma tumor-initiating cells

PubMed Central

Corsaro, Alessandro; Bajetto, Adriana; Thellung, Stefano; Begani, Giulia; Villa, Valentina; Nizzari, Mario; Pattarozzi, Alessandra; Solari, Agnese; Gatti, Monica; Pagano, Aldo; Würth, Roberto; Daga, Antonio; Barbieri, Federica; Florio, Tullio

2016-01-01

Prion protein (PrPC) is a cell surface glycoprotein whose misfolding is responsible for prion diseases. Although its physiological role is not completely defined, several lines of evidence propose that PrPC is involved in self-renewal, pluripotency gene expression, proliferation and differentiation of neural stem cells. Moreover, PrPC regulates different biological functions in human tumors, including glioblastoma (GBM). We analyzed the role of PrPC in GBM cell pathogenicity focusing on tumor-initiating cells (TICs, or cancer stem cells, CSCs), the subpopulation responsible for development, progression and recurrence of most malignancies. Analyzing four GBM CSC-enriched cultures, we show that PrPC expression is directly correlated with the proliferation rate of the cells. To better define its role in CSC biology, we knocked-down PrPC expression in two of these GBM-derived CSC cultures by specific lentiviral-delivered shRNAs. We provide evidence that CSC proliferation rate, spherogenesis and in vivo tumorigenicity are significantly inhibited in PrPC down-regulated cells. Moreover, PrPC down-regulation caused loss of expression of the stemness and self-renewal markers (NANOG, Sox2) and the activation of differentiation pathways (i.e. increased GFAP expression). Our results suggest that PrPC controls the stemness properties of human GBM CSCs and that its down-regulation induces the acquisition of a more differentiated and less oncogenic phenotype. PMID:27229535

Tryptophan PET Imaging of the Kynurenine Pathway in Patient-Derived Xenograft Models of Glioblastoma.

PubMed

Guastella, Anthony R; Michelhaugh, Sharon K; Klinger, Neil V; Kupsky, William J; Polin, Lisa A; Muzik, Otto; Juhász, Csaba; Mittal, Sandeep

2016-01-01

Increasing evidence demonstrates the immunosuppressive kynurenine pathway's (KP) role in the pathophysiology of human gliomas. To study the KP in vivo, we used the noninvasive molecular imaging tracer α-[(11)C]-methyl-l-tryptophan (AMT). The AMT-positron emission tomography (PET) has shown high uptake in high-grade gliomas and predicted survival in patients with recurrent glioblastoma (GBM). We generated patient-derived xenograft (PDX) models from dissociated cells, or tumor fragments, from 5 patients with GBM. Mice bearing subcutaneous tumors were imaged with AMT-PET, and tumors were analyzed to detect the KP enzymes indoleamine 2,3-dioxygenase (IDO) 1, IDO2, tryptophan 2,3-dioxygenase, kynureninase, and kynurenine 3-monooxygenase. Overall, PET imaging showed robust tumoral AMT uptake in PDX mice with prolonged tracer accumulation over 60 minutes, consistent with AMT trapping seen in humans. Immunostained tumor tissues demonstrated positive detection of multiple KP enzymes. Furthermore, intracranial implantation of GBM cells was performed with imaging at both 9 and 14 days postimplant, with a marked increase in AMT uptake at 14 days and a corresponding high level of tissue immunostaining for KP enzymes. These results indicate that our PDX mouse models recapitulate human GBM, including aberrant tryptophan metabolism, and offer an in vivo system for development of targeted therapeutics for patients with GBM. © The Author(s) 2016.

Triggering of the TRPV2 channel by cannabidiol sensitizes glioblastoma cells to cytotoxic chemotherapeutic agents.

PubMed

Nabissi, Massimo; Morelli, Maria Beatrice; Santoni, Matteo; Santoni, Giorgio

2013-01-01

The aggressive behavior of Glioblastoma multiforme (GBM) is mainly due to high invasiveness and proliferation rate as well as to high resistance to standard chemotherapy. Several chemotherapeutic agents like temozolomide (TMZ), carmustine (BCNU) or doxorubicin (DOXO) have been employed for treatment of GBM, but they display limited efficacy. Therefore, it is important to identify new treatment modalities to improve therapeutic effects and enhance GBM chemosensitivity. Recently, activation of the transient receptor potential vanilloid type 2 (TRPV2) has been found to inhibit human GBM cell proliferation and overcome BCNU resistance of GBM cells. Herein, we evaluated the involvement of cannabidiol (CBD)-induced TRPV2 activation, in the modulation of glioma cell chemosensitivity to TMZ, BCNU and DOXO. We found that CBD increases TRPV2 expression and activity. CBD by triggering TRPV2-dependent Ca(2+) influx increases drug uptake and synergizes with cytotoxic agents to induce apoptosis of glioma cells, whereas no effects were observed in normal human astrocytes. Moreover, as the pore region of transient receptor potential (TRP) channels is critical for ion channel permeation, we demonstrated that deletion of TRPV2 poredomain inhibits CBD-induced Ca(2+) influx, drug uptake and cytotoxic effects. Overall, we demonstrated that co-administration of cytotoxic agents together with the TRPV2 agonist CBD increases drug uptake and parallelly potentiates cytotoxic activity in human glioma cells.

Dihydroartemisinin Exerts Anti-Tumor Activity by Inducing Mitochondrion and Endoplasmic Reticulum Apoptosis and Autophagic Cell Death in Human Glioblastoma Cells

PubMed Central

Qu, Chengbin; Ma, Jun; Liu, Xiaobai; Xue, Yixue; Zheng, Jian; Liu, Libo; Liu, Jing; Li, Zhen; Zhang, Lei; Liu, Yunhui

2017-01-01

Glioblastoma (GBM) is the most advanced and aggressive form of gliomas. Dihydroartemisinin (DHA) has been shown to exhibit anti-tumor activity in various cancer cells. However, the effect and molecular mechanisms underlying its anti-tumor activity in human GBM cells remain to be elucidated. Our results proved that DHA treatment significantly reduced cell viability in a dose- and time-dependent manner by CCK-8 assay. Further investigation identified that the cell viability was rescued by pretreatment either with Z-VAD-FMK, 3-methyladenine (3-MA) or in combination. Moreover, DHA induced apoptosis of GBM cells through mitochondrial membrane depolarization, release of cytochrome c and activation of caspases-9. Enhanced expression of GRP78, CHOP and eIF2α and activation of caspase 12 were additionally confirmed that endoplasmic reticulum (ER) stress pathway of apoptosis was involved in the cytotoxicity of DHA. DHA-treated GBM cells exhibited the morphological and biochemical changes typical of autophagy. Co-treatment with chloroquine (CQ) significantly induced the above effects. Furthermore, ER stress and mitochondrial dysfunction were involved in the DHA-induced autophagy. Further study revealed that accumulation of reactive oxygen species (ROS) was attributed to the DHA induction of apoptosis and autophagy. The illustration of these molecular mechanisms will present a novel insight for the treatment of human GBM. PMID:29033794

Development of Advanced Technologies for Complete Genomic and Proteomic Characterization of Quantized Human Tumor Cells

DTIC Science & Technology

2013-07-01

AVAILABILITY STATEMENT Approved for Public Release; Distribution Unlimited 13. SUPPLEMENTARY NOTES 14. ABSTRACT With the establishment of GBM cell lines...and quantized cell populations form these GBM patients tumor samples we are able to complete some of our aims of our project. We will continue to...collect tumor samples with consent from families of GBM patients in preparation to perform the molecular analysis of these. Our efforts in the development

GLUT-1-independent infection of the glioblastoma/astroglioma U87 cells by the human T cell leukemia virus type 1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jin Qingwen; Agrawal, Lokesh; Walther Cancer Institute, Indianapolis, IN 46208

2006-09-15

The human glucose transporter protein 1 (GLUT-1) functions as a receptor for human T cell leukemia virus (HTLV). GLUT-1 is a twelve-transmembrane cell surface receptor with six extracellular (ECL) and seven intracellular domains. To analyze HTLV-1 cytotropism, we utilized polyclonal antibodies to a synthetic peptide corresponding to the large extracellular domain of GLUT-1. The antibodies caused significant blocking of envelope (Env)-mediated fusion and pseudotyped virus infection of HeLa cells but had no significant effect on infection of U87 cells. This differential effect correlated with the detection of high-level surface expression of GLUT-1 on HeLa cells and very weak staining ofmore » U87 cells. To investigate this in terms of viral cytotropism, we cloned GLUT-1 cDNA from U87 cells and isolated two different versions of cDNA clones: the wild-type sequence (encoding 492 residues) and a mutant cDNA with a 5-base pair deletion (GLUT-1{delta}5) between nucleotides 1329 and 1333. The deletion, also detected in genomic DNA, resulted in a frame-shift and premature termination producing a truncated protein of 463 residues. Transfection of the wild-type GLUT-1 but not GLUT-1{delta}5 cDNA into CHO cells resulted in efficient surface expression of the human GLUT-1. Co-expression of GLUT-1 with GLUT-1{delta}5 produces a trans-inhibition by GLUT-1{delta}5 of GLUT-1-mediated HTLV-1 envelope (Env)-mediated fusion. Co-immunoprecipitation experiments demonstrated physical interaction of the wild-type and mutant proteins. Northern blot and RT-PCR analyses demonstrated lower GLUT-1 RNA expression in U87 cells. We propose two mechanisms to account for the impaired cell surface expression of GLUT-1 on U87 cells: low GLUT-1 RNA expression and the formation of GLUT-1/GLUT-1{delta}5 heterodimers that are retained intracellularly. Significant RNAi-mediated reduction of endogenous GLUT-1 expression impaired HTLV-1 Env-mediated fusion with HeLa cells but not with U87 cells. We propose a GLUT-1-independent mechanism of HTLV-1 infection of U87 cells. The results may have important implications for HTLV-1 neurotropism and pathogenesis.« less

Subcellular boron and fluorine distributions with SIMS ion microscopy in BNCT and cancer research

DOE Office of Scientific and Technical Information (OSTI.GOV)

Subhash Chandra

2008-05-30

The development of a secondary ion mass spectrometry (SIMS) based technique of Ion Microscopy in boron neutron capture therapy (BNCT) was the main goal of this project, so that one can study the subcellular location of boron-10 atoms and their partitioning between the normal and cancerous tissue. This information is fundamental for the screening of boronated drugs appropriate for neutron capture therapy of cancer. Our studies at Cornell concentrated mainly on studies of glioblastoma multiforme (GBM). The early years of the grant were dedicated to the development of cryogenic methods and correlative microscopic approaches so that a reliable subcellular analysismore » of boron-10 atoms can be made with SIMS. In later years SIMS was applied to animal models and human tissues of GBM for studying the efficacy of potential boronated agents in BNCT. Under this grant the SIMS program at Cornell attained a new level of excellence and collaborative SIMS studies were published with leading BNCT researchers in the U.S.« less

Radiogenomics to characterize regional genetic heterogeneity in glioblastoma.

PubMed

Hu, Leland S; Ning, Shuluo; Eschbacher, Jennifer M; Baxter, Leslie C; Gaw, Nathan; Ranjbar, Sara; Plasencia, Jonathan; Dueck, Amylou C; Peng, Sen; Smith, Kris A; Nakaji, Peter; Karis, John P; Quarles, C Chad; Wu, Teresa; Loftus, Joseph C; Jenkins, Robert B; Sicotte, Hugues; Kollmeyer, Thomas M; O'Neill, Brian P; Elmquist, William; Hoxworth, Joseph M; Frakes, David; Sarkaria, Jann; Swanson, Kristin R; Tran, Nhan L; Li, Jing; Mitchell, J Ross

2017-01-01

Glioblastoma (GBM) exhibits profound intratumoral genetic heterogeneity. Each tumor comprises multiple genetically distinct clonal populations with different therapeutic sensitivities. This has implications for targeted therapy and genetically informed paradigms. Contrast-enhanced (CE)-MRI and conventional sampling techniques have failed to resolve this heterogeneity, particularly for nonenhancing tumor populations. This study explores the feasibility of using multiparametric MRI and texture analysis to characterize regional genetic heterogeneity throughout MRI-enhancing and nonenhancing tumor segments. We collected multiple image-guided biopsies from primary GBM patients throughout regions of enhancement (ENH) and nonenhancing parenchyma (so called brain-around-tumor, [BAT]). For each biopsy, we analyzed DNA copy number variants for core GBM driver genes reported by The Cancer Genome Atlas. We co-registered biopsy locations with MRI and texture maps to correlate regional genetic status with spatially matched imaging measurements. We also built multivariate predictive decision-tree models for each GBM driver gene and validated accuracies using leave-one-out-cross-validation (LOOCV). We collected 48 biopsies (13 tumors) and identified significant imaging correlations (univariate analysis) for 6 driver genes: EGFR, PDGFRA, PTEN, CDKN2A, RB1, and TP53. Predictive model accuracies (on LOOCV) varied by driver gene of interest. Highest accuracies were observed for PDGFRA (77.1%), EGFR (75%), CDKN2A (87.5%), and RB1 (87.5%), while lowest accuracy was observed in TP53 (37.5%). Models for 4 driver genes (EGFR, RB1, CDKN2A, and PTEN) showed higher accuracy in BAT samples (n = 16) compared with those from ENH segments (n = 32). MRI and texture analysis can help characterize regional genetic heterogeneity, which offers potential diagnostic value under the paradigm of individualized oncology. © The Author(s) 2016. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

REST Controls Self-Renewal and Tumorigenic Competence of Human Glioblastoma Cells

PubMed Central

Conti, Luciano; Crisafulli, Laura; Brilli, Elisa; Conforti, Paola; Zunino, Franco; Magrassi, Lorenzo; Schiffer, Davide; Cattaneo, Elena

2012-01-01

The Repressor Element 1 Silencing Transcription factor (REST/NRSF) is a master repressor of neuronal programs in non-neuronal lineages shown to function as a central regulator of developmental programs and stem cell physiology. Aberrant REST function has been associated with a number of pathological conditions. In cancer biology, REST has been shown to play a tumor suppressor activity in epithelial cancers but an oncogenic role in brain childhood malignancies such as neuroblastoma and medulloblastoma. Here we examined REST expression in human glioblastoma multiforme (GBM) specimens and its role in GBM cells carrying self-renewal and tumorigenic competence. We found REST to be expressed in GBM specimens, its presence being particularly enriched in tumor cells in the perivascular compartment. Significantly, REST is highly expressed in self-renewing tumorigenic-competent GBM cells and its knock down strongly reduces their self-renewal in vitro and tumor-initiating capacity in vivo and affects levels of miR-124 and its downstream targets. These results indicate that REST contributes to GBM maintenance by affecting its self-renewing and tumorigenic cellular component and that, hence, a better understanding of these circuitries in these cells might lead to new exploitable therapeutic targets. PMID:22701651

Protein Kinase CK2 Content in GL261 Mouse Glioblastoma.

PubMed

Ferrer-Font, Laura; Alcaraz, Estefania; Plana, Maria; Candiota, Ana Paula; Itarte, Emilio; Arús, Carles

2016-07-01

Glioblastoma (GBM) is the most prevalent and aggressive human glial tumour with a median survival of 14-15 months. Temozolomide (TMZ) is the standard chemotherapeutic choice for GBM treatment. Unfortunately, chemoresistence always ensues with concomitant tumour regrowth. Protein kinase CK2 (CK2) contributes to tumour development, proliferation, and suppression of apoptosis in cancer and it is overexpressed in human GBM. Targeting CK2 in GBM treatment may benefit patients. With this translational perspective in mind, we have studied the CK2 expression level by Western blot analysis in a preclinical model of GBM: GL261 cells growing orthotopically in C57BL/6 mice. The expression level of the CK2 catalytic subunit (CK2α) was higher in tumour (about 4-fold) and in contralateral brain parenchyma (more than 2-fold) than in normal brain parenchyma (p < 0.05). In contrast, no significant changes were found in CK2 regulatory subunit (CK2β) expression, suggesting an increased unbalance of CK2α/CK2β in GL261 tumours with respect to normal brain parenchyma, in agreement with a differential role of these two subunits in tumours.

Telomerase inhibition improves tumor response to radiotherapy in a murine orthotopic model of human glioblastoma.

PubMed

Ferrandon, Sylvain; Malleval, Céline; El Hamdani, Badia; Battiston-Montagne, Priscillia; Bolbos, Radu; Langlois, Jean-Baptiste; Manas, Patrick; Gryaznov, Sergei M; Alphonse, Gersende; Honnorat, Jérôme; Rodriguez-Lafrasse, Claire; Poncet, Delphine

2015-07-17

Glioblastoma (GBM) is the most frequent and aggressive type of adult brain tumor. Most GBMs express telomerase; a high level of intra-tumoral telomerase activity (TA) is predictive of poor prognosis. Thus, telomerase inhibitors are promising options to treat GBM. These inhibitors increase the response to radiotherapy (RT), in vitro as well as in vivo. Since typical treatments for GBM include RT, our objective was to evaluate the efficiency of Imetelstat (TA inhibitor) combined with RT. We used a murine orthotopic model of human GBM (N = 8 to11 mice per group) and μMRI imaging to evaluate the efficacy of Imetelstat (delivered by intra-peritoneal injection) alone and combined with RT. Using a clinically established protocol, we demonstrated that Imetelstat significantly: (i) inhibited the TA in the very center of the tumor, (ii) reduced tumor volume as a proportion of TA inhibition, and (iii) increased the response to RT, in terms of tumor volume regression and survival increase. Imetelstat is currently evaluated in refractory brain tumors in young patients (without RT). Our results support its clinical evaluation combined with RT to treat GBM.

MiR-18a regulates the proliferation, migration and invasion of human glioblastoma cell by targeting neogenin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Song, Yichen, E-mail: jeff200064017@163.com; Wang, Ping, E-mail: pingwang8000@163.com; Institute of Pathology and Pathophysiology, China Medical University, Shenyang 110001

MiR-17-92 cluster has recently been reported as an oncogene in some tumors. However, the association of miR-18a, an important member of this cluster, with glioblastoma remains unknown. Therefore, this study aims to investigate the expression of miR-18a in glioblastoma and its role in biological behavior of U87 and U251 human glioblastoma cell lines. Quantitative RT-PCR results showed that miR-18a was highly expressed in glioblastoma tissues and U87 and U251 cell lines compared with that in human brain tissues and primary normal human astrocytes, and the expression levels were increased along with the rising pathological grades of glioblastoma. Neogenin was identifiedmore » as the target gene of miR-18a by dual-luciferase reporter assays. RT-PCR and western blot results showed that its expression levels were decreased along with the rising pathological grades of glioblastoma. Inhibition of miR-18a expression was established by transfecting exogenous miR-18a inhibitor into U87 and U251 cells, and its effects on the biological behavior of glioblastoma cells were studied using CCK-8 assay, transwell assay and flow cytometry. Inhibition of miR-18a expression in U87 and U251 cells significantly up-regulated neogenin, and dramatically suppressed the abilities of cell proliferation, migration and invasion, induced cell cycle arrest and promoted cellular apoptosis. Collectively, these results suggest that miR-18a may regulate biological behavior of human glioblastoma cells by targeting neogenin, and miR-18a can serve as a potential target in the treatment of glioblastoma. - Highlights: • MiR-18a was highly expressed in glioblastoma tissues and U87 and U251 cell lines. • Neogenin was identified as the target gene of miR-18a. • Neogenin expressions were decreased along with the rising pathological grades of glioblastoma. • Inhibition of miR-18a suppressed biological behavior of glioma cells by up-regulating neogenin.« less

Dysregulated miR-671-5p / CDR1-AS / CDR1 / VSNL1 axis is involved in glioblastoma multiforme

PubMed Central

Salito, Loredana; Sammito, Mariangela; Banelli, Barbara; Caltabiano, Rosario; Barbagallo, Giuseppe; Zappalà, Agata; Battaglia, Rosalia; Cirnigliaro, Matilde; Lanzafame, Salvatore; Vasquez, Enrico; Parenti, Rosalba; Cicirata, Federico; Di Pietro, Cinzia; Romani, Massimo; Purrello, Michele

2016-01-01

MiR-671-5p is encoded by a gene localized at 7q36.1, a region amplified in human glioblastoma multiforme (GBM), the most malignant brain cancer. To investigate whether expression of miR-671-5p were altered in GBM, we analyzed biopsies from a cohort of forty-five GBM patients and from five GBM cell lines. Our data show significant overexpression of miR-671-5p in both biopsies and cell lines. By exploiting specific miRNA mimics and inhibitors, we demonstrated that miR-671-5p overexpression significantly increases migration and to a less extent proliferation rates of GBM cells. Through a combined in silico and in vitro approach, we identified CDR1-AS, CDR1, VSNL1 as downstream miR-671-5p targets in GBM. Expression of these genes significantly decreased both in GBM biopsies and cell lines and negatively correlated with that of miR-671-5p. Based on our data, we propose that the axis miR-671-5p / CDR1-AS / CDR1 / VSNL1 is functionally altered in GBM cells and is involved in the modification of their biopathological profile. PMID:26683098

Inhibition of the Autophagy Pathway Synergistically Potentiates the Cytotoxic Activity of Givinostat (ITF2357) on Human Glioblastoma Cancer Stem Cells.

PubMed

Angeletti, Francesca; Fossati, Gianluca; Pattarozzi, Alessandra; Würth, Roberto; Solari, Agnese; Daga, Antonio; Masiello, Irene; Barbieri, Federica; Florio, Tullio; Comincini, Sergio

2016-01-01

Increasing evidence highlighted the role of cancer stem cells (CSCs) in the development of tumor resistance to therapy, particularly in glioblastoma (GBM). Therefore, the development of new therapies, specifically directed against GBM CSCs, constitutes an important research avenue. Considering the extended range of cancer-related pathways modulated by histone acetylation/deacetylation processes, we studied the anti-proliferative and pro-apoptotic efficacy of givinostat (GVS), a pan-histone deacetylase inhibitor, on cell cultures enriched in CSCs, isolated from nine human GBMs. We report that GVS induced a significant reduction of viability and self-renewal ability in all GBM CSC cultures; conversely, GVS exposure did not cause a significant cytotoxic activity toward differentiated GBM cells and normal mesenchymal human stem cells. Analyzing the cellular and molecular mechanisms involved, we demonstrated that GVS affected CSC viability through the activation of programmed cell death pathways. In particular, a marked stimulation of macroautophagy was observed after GVS treatment. To understand the functional link between GVS treatment and autophagy activation, different genetic and pharmacological interfering strategies were used. We show that the up-regulation of the autophagy process, obtained by deprivation of growth factors, induced a reduction of CSC sensitivity to GVS, while the pharmacological inhibition of the autophagy pathway and the silencing of the key autophagy gene ATG7 , increased the cell death rate induced by GVS. Altogether these findings suggest that autophagy represents a pro-survival mechanism activated by GBM CSCs to counteract the efficacy of the anti-proliferative activity of GVS. In conclusion, we demonstrate that GVS is a novel pharmacological tool able to target GBM CSC viability and its efficacy can be enhanced by autophagy inhibitory strategies.

Inhibition of the Autophagy Pathway Synergistically Potentiates the Cytotoxic Activity of Givinostat (ITF2357) on Human Glioblastoma Cancer Stem Cells

PubMed Central

Angeletti, Francesca; Fossati, Gianluca; Pattarozzi, Alessandra; Würth, Roberto; Solari, Agnese; Daga, Antonio; Masiello, Irene; Barbieri, Federica; Florio, Tullio; Comincini, Sergio

2016-01-01

Increasing evidence highlighted the role of cancer stem cells (CSCs) in the development of tumor resistance to therapy, particularly in glioblastoma (GBM). Therefore, the development of new therapies, specifically directed against GBM CSCs, constitutes an important research avenue. Considering the extended range of cancer-related pathways modulated by histone acetylation/deacetylation processes, we studied the anti-proliferative and pro-apoptotic efficacy of givinostat (GVS), a pan-histone deacetylase inhibitor, on cell cultures enriched in CSCs, isolated from nine human GBMs. We report that GVS induced a significant reduction of viability and self-renewal ability in all GBM CSC cultures; conversely, GVS exposure did not cause a significant cytotoxic activity toward differentiated GBM cells and normal mesenchymal human stem cells. Analyzing the cellular and molecular mechanisms involved, we demonstrated that GVS affected CSC viability through the activation of programmed cell death pathways. In particular, a marked stimulation of macroautophagy was observed after GVS treatment. To understand the functional link between GVS treatment and autophagy activation, different genetic and pharmacological interfering strategies were used. We show that the up-regulation of the autophagy process, obtained by deprivation of growth factors, induced a reduction of CSC sensitivity to GVS, while the pharmacological inhibition of the autophagy pathway and the silencing of the key autophagy gene ATG7, increased the cell death rate induced by GVS. Altogether these findings suggest that autophagy represents a pro-survival mechanism activated by GBM CSCs to counteract the efficacy of the anti-proliferative activity of GVS. In conclusion, we demonstrate that GVS is a novel pharmacological tool able to target GBM CSC viability and its efficacy can be enhanced by autophagy inhibitory strategies. PMID:27833530

FoxM1 Promotes Stemness and Radio-Resistance of Glioblastoma by Regulating the Master Stem Cell Regulator Sox2

PubMed Central

Kim, Dong Geon; Cho, Hee Jin; Kim, Yeonghwan; Rheey, Jinguen; Shin, Kayoung; Seo, Yun Jee; Choi, Yeon-Sook; Lee, Jung-Il; Lee, Jeongwu; Joo, Kyeung Min; Nam, Do-Hyun

2015-01-01

Glioblastoma (GBM) is the most aggressive and most lethal brain tumor. As current standard therapy consisting of surgery and chemo-irradiation provides limited benefit for GBM patients, novel therapeutic options are urgently required. Forkhead box M1 (FoxM1) transcription factor is an oncogenic regulator that promotes the proliferation, survival, and treatment resistance of various human cancers. The roles of FoxM1 in GBM remain incompletely understood, due in part to pleotropic nature of the FoxM1 pathway. Here, we show the roles of FoxM1 in GBM stem cell maintenance and radioresistance. ShRNA-mediated FoxM1 inhibition significantly impeded clonogenic growth and survival of patient-derived primary GBM cells with marked downregulation of Sox2, a master regulator of stem cell phenotype. Ectopic expression of Sox2 partially rescued FoxM1 inhibition-mediated effects. Conversely, FoxM1 overexpression upregulated Sox2 expression and promoted clonogenic growth of GBM cells. These data, with a direct binding of FoxM1 in the Sox2 promoter region in GBM cells, suggest that FoxM1 regulates stemness of primary GBM cells via Sox2. We also found significant increases in FoxM1 and Sox2 expression in GBM cells after irradiation both in vitro and in vivo orthotopic tumor models. Notably, genetic or a small-molecule FoxM1 inhibitor-mediated FoxM1 targeting significantly sensitized GBM cells to irradiation, accompanying with Sox2 downregulation. Finally, FoxM1 inhibition combined with irradiation in a patient GBM-derived orthotopic model significantly impeded tumor growth and prolonged the survival of tumor bearing mice. Taken together, these results indicate that the FoxM1-Sox2 signaling axis promotes clonogenic growth and radiation resistance of GBM, and suggest that FoxM1 targeting combined with irradiation is a potentially effective therapeutic approach for GBM. PMID:26444992

FoxM1 Promotes Stemness and Radio-Resistance of Glioblastoma by Regulating the Master Stem Cell Regulator Sox2.

PubMed

Lee, Yeri; Kim, Kang Ho; Kim, Dong Geon; Cho, Hee Jin; Kim, Yeonghwan; Rheey, Jinguen; Shin, Kayoung; Seo, Yun Jee; Choi, Yeon-Sook; Lee, Jung-Il; Lee, Jeongwu; Joo, Kyeung Min; Nam, Do-Hyun

2015-01-01

Glioblastoma (GBM) is the most aggressive and most lethal brain tumor. As current standard therapy consisting of surgery and chemo-irradiation provides limited benefit for GBM patients, novel therapeutic options are urgently required. Forkhead box M1 (FoxM1) transcription factor is an oncogenic regulator that promotes the proliferation, survival, and treatment resistance of various human cancers. The roles of FoxM1 in GBM remain incompletely understood, due in part to pleotropic nature of the FoxM1 pathway. Here, we show the roles of FoxM1 in GBM stem cell maintenance and radioresistance. ShRNA-mediated FoxM1 inhibition significantly impeded clonogenic growth and survival of patient-derived primary GBM cells with marked downregulation of Sox2, a master regulator of stem cell phenotype. Ectopic expression of Sox2 partially rescued FoxM1 inhibition-mediated effects. Conversely, FoxM1 overexpression upregulated Sox2 expression and promoted clonogenic growth of GBM cells. These data, with a direct binding of FoxM1 in the Sox2 promoter region in GBM cells, suggest that FoxM1 regulates stemness of primary GBM cells via Sox2. We also found significant increases in FoxM1 and Sox2 expression in GBM cells after irradiation both in vitro and in vivo orthotopic tumor models. Notably, genetic or a small-molecule FoxM1 inhibitor-mediated FoxM1 targeting significantly sensitized GBM cells to irradiation, accompanying with Sox2 downregulation. Finally, FoxM1 inhibition combined with irradiation in a patient GBM-derived orthotopic model significantly impeded tumor growth and prolonged the survival of tumor bearing mice. Taken together, these results indicate that the FoxM1-Sox2 signaling axis promotes clonogenic growth and radiation resistance of GBM, and suggest that FoxM1 targeting combined with irradiation is a potentially effective therapeutic approach for GBM.

Modeled microgravity suppressed invasion and migration of human glioblastoma U87 cells through downregulating store-operated calcium entry

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shi, Zi-xuan; Rao, Wei; Wang, Huan

Glioblastoma is the most common brain tumor and is characterized with robust invasion and migration potential resulting in poor prognosis. Previous investigations have demonstrated that modeled microgravity (MMG) could decline the cell proliferation and attenuate the metastasis potential in several cell lines. In this study, we studied the effects of MMG on the invasion and migration potentials of glioblastoma in human glioblastoma U87 cells. We found that MMG stimulation significantly attenuated the invasion and migration potentials, decreased thapsigargin (TG) induced store-operated calcium entry (SOCE) and downregulated the expression of Orai1 in U87 cells. Inhibition of SOCE by 2-APB or stromalmore » interaction molecule 1 (STIM1) downregulation both mimicked the effects of MMG on the invasion and migration potentials in U87 cells. Furthermore, upregulation of Orai1 significantly weakened the effects of MMG on the invasion and migration potentials in U87 cells. Therefore, these findings indicated that MMG stimulation inhibited the invasion and migration potentials of U87 cells by downregulating the expression of Orai1 and sequentially decreasing the SOCE, suggesting that MMG might be a new potential therapeutic strategy in glioblastoma treatment in the future. - Highlights: • Modeled microgravity (MMG) suppressed migration and invasion in U87 cells. • MMG downregulated the SOCE and the expression of Orai1. • SOCE inhibition mimicked the effects of MMG on migration and invasion potentials. • Restoration of SOCE diminished the effects of MMG on migration and invasion.« less

Analysis of single nucleotide variants of HFE gene and association to survival in The Cancer Genome Atlas GBM data.

PubMed

Lee, Sang Y; Zhu, Junjia; Salzberg, Anna C; Zhang, Bo; Liu, Dajiang J; Muscat, Joshua E; Langan, Sara T; Connor, James R

2017-01-01

Human hemochromatosis protein (HFE) is involved in iron metabolism. Two major HFE polymorphisms, H63D and C282Y, have been associated with an increased risk of cancers. Previously, we reported decreased gender effects in overall survival based on H63D or C282Y HFE polymorphisms patients with glioblastoma multiforme (GBM). However, the effect of other single nucleotide variation (SNV) in the HFE gene on the cancer development and progression has not been systematically studied. To expand our finding in a larger sample, and to identify other HFE SNV, we analyzed the frequency of somatic SNV in HFE gene and its relationship to survival in GBM patients using The Cancer Genome Atlas (TCGA) GBM (Caucasian only) database. We found 9 SNVs with increased frequency in blood normal of TCGA GBM patients compared to the 1000Genome. Among 9 SNVs, 7 SNVs were located in the intron and 2 SNVs (i.e., H63D, C282Y) in the exon of HFE gene. The statistical analysis demonstrated that blood normal samples of TCGA GBM have more H63D (p = 0.0002, 95% Confidence interval (CI): 0.2119-0.3223) or C282Y (p = 0.0129, 95% CI: 0.0474-0.1159) HFE polymorphisms than 1000Genome. The Kaplan-Meier survival curve for the 264 GBM samples revealed no difference between wild type (WT) HFE and H63D, and WT HFE and C282Y GBM patients. In addition, there was no difference in the survival of male/female GBM patients based on HFE genotype. There was no correlation between HFE expression and survival. In conclusion, the current results suggest that somatic HFE polymorphisms do not impact GBM patients' survival in the TCGA data set of GBM.

[Saponin 6 of Anemone Taipaiensis inhibits proliferation and induces apoptosis of U87 MG cells].

PubMed

Ji, Chenchen; Cheng, Guang; Tang, Haifeng; Zhang, Yun; Hu, Yiyang; Zheng, Minhua; Fei, Zhou

2015-04-01

To investigate the effect of saponin 6 of Anemone Taipaiensis on the proliferation of human U87 MG glioma cells and the possible mechanism. U87 MG cells were treated with different concentrations of saponin 6 (0.0, 1.6, 3.2, 6.4, 12.8 μg/mL) for 24 hours or 48 hours. Cell viability was measured by MTT assay; the apoptosis rate was detected by flow cytometry combined with annexin V-FITC /PI staining; Western blotting was applied to determine the protein level of activated caspase-3. Compared with control groups, saponin 6 significantly inhibited U87 MG cell proliferation in a time- and dose-depended manner. Apoptosis rate of U87 MG cells and the expression of activated caspase-3 were raised with the increasing concentration of saponin 6. Saponin 6 of Anemone Taipaiensis could depress cell proliferation in a dose-depended manner, increase the expression of activated caspase-3 and promote apoptosis in U87 MG cells.

Stereotactic intracranial implantation and in vivo bioluminescent imaging of tumor xenografts in a mouse model system of glioblastoma multiforme.

PubMed

Baumann, Brian C; Dorsey, Jay F; Benci, Joseph L; Joh, Daniel Y; Kao, Gary D

2012-09-25

Glioblastoma multiforme (GBM) is a high-grade primary brain cancer with a median survival of only 14.6 months in humans despite standard tri-modality treatment consisting of surgical resection, post-operative radiation therapy and temozolomide chemotherapy. New therapeutic approaches are clearly needed to improve patient survival and quality of life. The development of more effective treatment strategies would be aided by animal models of GBM that recapitulate human disease yet allow serial imaging to monitor tumor growth and treatment response. In this paper, we describe our technique for the precise stereotactic implantation of bio-imageable GBM cancer cells into the brains of nude mice resulting in tumor xenografts that recapitulate key clinical features of GBM. This method yields tumors that are reproducible and are located in precise anatomic locations while allowing in vivo bioluminescent imaging to serially monitor intracranial xenograft growth and response to treatments. This method is also well-tolerated by the animals with low perioperative morbidity and mortality.

Fate mapping of human glioblastoma reveals an invariant stem cell hierarchy

PubMed Central

Lan, Xiaoyang; Jörg, David J.; Cavalli, Florence M. G.; Richards, Laura M.; Nguyen, Long V.; Vanner, Robert J.; Guilhamon, Paul; Lee, Lilian; Kushida, Michelle; Pellacani, Davide; Park, Nicole I.; Coutinho, Fiona J.; Whetstone, Heather; Selvadurai, Hayden J.; Che, Clare; Luu, Betty; Carles, Annaick; Moksa, Michelle; Rastegar, Naghmeh; Head, Renee; Dolma, Sonam; Prinos, Panagiotis; Cusimano, Michael D.; Das, Sunit; Bernstein, Mark; Arrowsmith, Cheryl H.; Mungall, Andrew J.; Moore, Richard A.; Ma, Yussanne; Gallo, Marco; Lupien, Mathieu; Pugh, Trevor J.; Taylor, Michael D.; Hirst, Martin; Eaves, Connie J.; Simons, Benjamin D.; Dirks, Peter B.

2017-01-01

Summary Human glioblastomas (GBMs) harbour a subpopulation of glioblastoma stem cells (GSCs) that drive tumourigenesis. However, the origin of intra-tumoural functional heterogeneity between GBM cells remains poorly understood. Here we study the clonal evolution of barcoded GBM cells in an unbiased way following serial xenotransplantation to define their individual fate behaviours. Independent of an evolving mutational signature, we show that the growth of GBM clones in vivo is consistent with a remarkably neutral process involving a conserved proliferative hierarchy rooted in GSCs. In this model, slow-cycling stem-like cells give rise to a more rapidly cycling progenitor population with extensive self-maintenance capacity, that in turn generates non-proliferative cells. We also identify rare “outlier” clones that deviate from these dynamics, and further show that chemotherapy facilitates the expansion of pre-existing drug-resistant GSCs. Finally, we show that functionally distinct GSCs can be separately targeted using epigenetic compounds, suggesting new avenues for GBM targeted therapy. PMID:28854171

Personalized regulation of glioblastoma cancer stem cells based on biomedical technologies: From theory to experiment (Review).

PubMed

Bryukhovetskiy, Igor; Ponomarenko, Arina; Lyakhova, Irina; Zaitsev, Sergey; Zayats, Yulia; Korneyko, Maria; Eliseikina, Marina; Mischenko, Polina; Shevchenko, Valerie; Shanker Sharma, Hari; Sharma, Aruna; Khotimchenko, Yuri

2018-08-01

Glioblastoma multiforme (GBM) is one of the most aggressive brain tumors. GBM represents >50% of primary tumors of the nervous system and ~20% of intracranial neoplasms. Standard treatment involves surgery, radiation and chemotherapy. However, the prognosis of GBM is usually poor, with a median survival of 15 months. Resistance of GBM to treatment can be explained by the presence of cancer stem cells (CSCs) among the GBM cell population. At present, there are no effective therapeutic strategies for the elimination of CSCs. The present review examined the nature of human GBM therapeutic resistance and attempted to systematize and put forward novel approaches for a personalized therapy of GBM that not only destroys tumor tissue, but also regulates cellular signaling and the morphogenetic properties of CSCs. The CSCs are considered to be an informationally accessible living system, and the CSC proteome should be used as a target for therapy directed at suppressing clonal selection mechanisms and CSC generation, destroying CSC hierarchy, and disrupting the interaction of CSCs with their microenvironment and extracellular matrix. These objectives can be achieved through the use of biomedical cellular products.

Cytomegalovirus-targeted immunotherapy and glioblastoma: hype or hope?

PubMed

Ferguson, Sherise D; Srinivasan, Visish M; Ghali, Michael Gz; Heimberger, Amy B

2016-01-01

Malignant gliomas, including glioblastoma (GBM), are the most common primary brain tumors. Despite extensive research only modest gains have been made in long-term survival. Standard of care involves maximizing safe surgical resection followed by concurrent chemoradiation with temozolomide. Immunotherapy for GBM is an area of intense research in recent years. New immunotherapies, although promising, have not been integrated into standard practice. Human cytomegalovirus (HCMV) is a DNA virus of the family Herpesviridae. Human seroprevalence is approximately 80%, and in most cases, is associated with asymptomatic infection. HCMV may be an important agent in the initiation, promotion and/or progression of tumorigenesis. Regardless of a possible etiologic role in GBM, interest has centered on exploiting this association for development of immunomodulatory therapies.

Different response of human glioma tumor-initiating cells to epidermal growth factor receptor kinase inhibitors.

PubMed

Griffero, Fabrizio; Daga, Antonio; Marubbi, Daniela; Capra, Maria Cristina; Melotti, Alice; Pattarozzi, Alessandra; Gatti, Monica; Bajetto, Adriana; Porcile, Carola; Barbieri, Federica; Favoni, Roberto E; Lo Casto, Michele; Zona, Gianluigi; Spaziante, Renato; Florio, Tullio; Corte, Giorgio

2009-03-13

Because a subpopulation of cancer stem cells (tumor-initiating cells, TICs) is believed to be responsible for the development, progression, and recurrence of many tumors, we evaluated the in vitro sensitivity of human glioma TICs to epidermal growth factor receptor (EGFR) kinase inhibitors (erlotinib and gefitinib) and possible molecular determinants for their effects. Cells isolated from seven glioblastomas (GBM 1-7) and grown using neural stem cell permissive conditions were characterized for in vivo tumorigenicity, expression of tumor stem cell markers (CD133, nestin), and multilineage differentiation properties, confirming that these cultures are enriched in TICs. TIC cultures were challenged with increasing concentrations of erlotinib and gefitinib, and their survival was evaluated after 1-4 days. In most cases, a time- and concentration-dependent cell death was observed, although GBM 2 was completely insensitive to both drugs, and GBM 7 was responsive only to the highest concentrations tested. Using a radioligand binding assay, we show that all GBM TICs express EGFR. Erlotinib and gefitinib inhibited EGFR and ERK1/2 phosphorylation/activation in all GBMs, irrespective of the antiproliferative response observed. However, under basal conditions GBM 2 showed a high Akt phosphorylation that was completely insensitive to both drugs, whereas GBM 7 was completely insensitive to gefitinib, and Akt inactivation occurred only for the highest erlotinib concentration tested, showing a precise relationship with the antiproliferative effects of the drug. Interestingly, in GBM 2, phosphatase and tensin homolog expression was significantly down-regulated, possibly accounting for the insensitivity to the drugs. In conclusion, glioma TICs are responsive to anti-EGFR drugs, but phosphatase and tensin homolog expression and Akt inhibition seem to be necessary for such effect.

RAD18 mediates resistance to ionizing radiation in human glioma cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xie, Chen; Wang, Hongwei; Cheng, Hongbin

Highlights: • RAD18 is an important mediator of the IR-induced resistance in glioma cell lines. • RAD18 overexpression confers resistance to IR-mediated apoptosis. • The elevated expression of RAD18 is associated with recurrent GBM who underwent IR therapy. - Abstract: Radioresistance remains a major challenge in the treatment of glioblastoma multiforme (GBM). RAD18 a central regulator of translesion DNA synthesis (TLS), has been shown to play an important role in regulating genomic stability and DNA damage response. In the present study, we investigate the relationship between RAD18 and resistance to ionizing radiation (IR) and examined the expression levels of RAD18more » in primary and recurrent GBM specimens. Our results showed that RAD18 is an important mediator of the IR-induced resistance in GBM. The expression level of RAD18 in glioma cells correlates with their resistance to IR. Ectopic expression of RAD18 in RAD18-low A172 glioma cells confers significant resistance to IR treatment. Conversely, depletion of endogenous RAD18 in RAD18-high glioma cells sensitized these cells to IR treatment. Moreover, RAD18 overexpression confers resistance to IR-mediated apoptosis in RAD18-low A172 glioma cells, whereas cells deficient in RAD18 exhibit increased apoptosis induced by IR. Furthermore, knockdown of RAD18 in RAD18-high glioma cells disrupts HR-mediated repair, resulting in increased accumulation of DSB. In addition, clinical data indicated that RAD18 was significantly higher in recurrent GBM samples that were exposed to IR compared with the corresponding primary GBM samples. Collectively, our findings reveal that RAD18 may serve as a key mediator of the IR response and may function as a potential target for circumventing IR resistance in human GBM.« less

Regulation of human glioblastoma cell death by combined treatment of cannabidiol, γ-radiation and small molecule inhibitors of cell signaling pathways

PubMed Central

Ivanov, Vladimir N.; Wu, Jinhua; Hei, Tom K.

2017-01-01

Glioblastoma (GBM) is the most common primary malignant brain tumor in adults. The challenging problem in cancer treatment is to find a way to upregulate radiosensitivity of GBM while protecting neurons and neural stem/progenitor cells in the brain. The goal of the present study was upregulation of the cytotoxic effect of γ-irradiation in GBM by non-psychotropic and non-toxic cannabinoid, cannabidiol (CBD). We emphasized three main aspects of signaling mechanisms induced by CBD treatment (alone or in combination with γ-irradiation) in human GBM that govern cell death: 1) CBD significantly upregulated the active (phosphorylated) JNK1/2 and MAPK p38 levels with the subsequent downregulation of the active phospho-ERK1/2 and phospho-AKT1 levels. MAPK p38 was one of the main drivers of CBD-induced cell death, while death levels after combined treatment of CBD and radiation were dependent on both MAPK p38 and JNK. Both MAPK p38 and JNK regulate the endogenous TRAIL expression. 2) NF-κB p65-P(Ser536) was not the main target of CBD treatment and this transcription factor was found at high levels in CBD-treated GBM cells. Additional suppression of p65-P(Ser536) levels using specific small molecule inhibitors significantly increased CBD-induced apoptosis. 3) CBD treatment substantially upregulated TNF/TNFR1 and TRAIL/TRAIL-R2 signaling by modulation of both ligand and receptor levels followed by apoptosis. Our results demonstrate that radiation-induced death in GBM could be enhanced by CBD-mediated signaling in concert with its marginal effects for neural stem/progenitor cells and astrocytes. It will allow selecting efficient targets for sensitization of GBM and overcoming cancer therapy-induced severe adverse sequelae. PMID:29088769

The PI3K inhibitor GDC-0941 enhances radiosensitization and reduces chemoresistance to temozolomide in GBM cell lines.

PubMed

Shi, Fei; Guo, Hongchuan; Zhang, Rong; Liu, Hongyu; Wu, Liangliang; Wu, Qiyan; Liu, Jialin; Liu, Tianyi; Zhang, Qiuhang

2017-03-27

Glioblastoma multiforme (GBM) is among the most lethal of all human tumors. It is the most frequently occurring malignant primary brain tumor in adults. The current standard of care (SOC) for GBM is initial surgical resection followed by treatment with a combination of temozolomide (TMZ) and ionizing radiation (IR). However, GBM has a dismal prognosis, and survivors have compromised quality of life owing to the adverse effects of radiation. GBM is characterized by overt activity of the phosphoinositide 3-kinase (PI3K) signaling pathway. GDC-0941 is a highly specific PI3K inhibitor with promising anti-tumor activity in human solid tumors. It is being evaluated in Phase II clinical trials for the treatment of breast and non-squamous cell lung cancer. We hypothesized that GDC-0941 may act as an antitumor agent and potentiate the effects of TMZ and IR. In this study, GDC-0941 alone induced cytotoxicity and pro-apoptotic effects. Moreover, combined with the standard GBM therapy (TMZ and IR), it suppressed cell viability, showed enhanced pro-apoptotic effects, augmented autophagy response, and attenuated migratory/invasive capacity in three glioma cell lines. Protein microarray analyses showed that treatment with TMZ+GDC-0941+IR induced higher levels of p53 and glycogen synthase kinase 3-beta (GSK3-β) expression in SHG44GBM cells than those induced by other treatments. This was verified in all cell lines by western blot analysis. Furthermore, the combination of TMZ and GDC-0941 with or without IR reduced the levels of p-AKT and O 6 -methylguanine DNA methyltransferase (MGMT) in T98G cells. The results of this study suggest that the combination of TMZ, IR, and GDC-0941 is a promising choice for future treatments of GBM. Copyright © 2017 IBRO. Published by Elsevier Ltd. All rights reserved.

ATP binding cassette (ABC) transporters: expression and clinical value in glioblastoma.

PubMed

Dréan, Antonin; Rosenberg, Shai; Lejeune, François-Xavier; Goli, Larissa; Nadaradjane, Aravindan Arun; Guehennec, Jérémy; Schmitt, Charlotte; Verreault, Maïté; Bielle, Franck; Mokhtari, Karima; Sanson, Marc; Carpentier, Alexandre; Delattre, Jean-Yves; Idbaih, Ahmed

2018-03-08

ATP-binding cassette transporters (ABC transporters) regulate traffic of multiple compounds, including chemotherapeutic agents, through biological membranes. They are expressed by multiple cell types and have been implicated in the drug resistance of some cancer cells. Despite significant research in ABC transporters in the context of many diseases, little is known about their expression and clinical value in glioblastoma (GBM). We analyzed expression of 49 ABC transporters in both commercial and patient-derived GBM cell lines as well as from 51 human GBM tumor biopsies. Using The Cancer Genome Atlas (TCGA) cohort as a training dataset and our cohort as a validation dataset, we also investigated the prognostic value of these ABC transporters in newly diagnosed GBM patients, treated with the standard of care. In contrast to commercial GBM cell lines, GBM-patient derived cell lines (PDCL), grown as neurospheres in a serum-free medium, express ABC transporters similarly to parental tumors. Serum appeared to slightly increase resistance to temozolomide correlating with a tendency for an increased expression of ABCB1. Some differences were observed mainly due to expression of ABC transporters by microenvironmental cells. Together, our data suggest that the efficacy of chemotherapeutic agents may be misestimated in vitro if they are the targets of efflux pumps whose expression can be modulated by serum. Interestingly, several ABC transporters have prognostic value in the TCGA dataset. In our cohort of 51 GBM patients treated with radiation therapy with concurrent and adjuvant temozolomide, ABCA13 overexpression is associated with a decreased progression free survival in univariate (p < 0.01) and multivariate analyses including MGMT promoter methylation (p = 0.05) suggesting reduced sensitivity to temozolomide in ABCA13 overexpressing GBM. Expression of ABC transporters is: (i) detected in GBM and microenvironmental cells and (ii) better reproduced in GBM-PDCL. ABCA13 expression is an independent prognostic factor in newly diagnosed GBM patients. Further prospective studies are warranted to investigate whether ABCA13 expression can be used to further personalize treatments for GBM.

ErbB2/HER2-Specific NK Cells for Targeted Therapy of Glioblastoma.

PubMed

Zhang, Congcong; Burger, Michael C; Jennewein, Lukas; Genßler, Sabrina; Schönfeld, Kurt; Zeiner, Pia; Hattingen, Elke; Harter, Patrick N; Mittelbronn, Michel; Tonn, Torsten; Steinbach, Joachim P; Wels, Winfried S

2016-05-01

Glioblastoma (GBM) is the most common and malignant intracranial tumor in adults and currently incurable. To specifically target natural killer (NK) cell activity to GBM, we employed NK-92/5.28.z cells that are continuously expanding human NK cells expressing an ErbB2-specific chimeric antigen receptor (CAR). ErbB2 expression in 56 primary tumors, four primary cell cultures, and seven established cell lines was assessed by immunohistochemistry and flow cytometry. Cell killing activity of NK-92/5.28.z cells was analyzed in in vitro cytotoxicity assays. In vivo antitumor activity was evaluated in NOD-SCID IL2Rγ(null) (NSG) mice carrying orthotopic human GBM xenografts (6 to 11 mice per group) and C57BL/6 mice carrying subcutaneous and orthotopic ErbB2-expressing murine GBM tumors (5 to 8 mice per group). Statistical tests were two-sided. We found elevated ErbB2 protein expression in 41% of primary GBM samples and in the majority of GBM cell lines investigated. In in vitro assays, NK-92/5.28.z in contrast to untargeted NK-92 cells lysed all ErbB2-positive established and primary GBM cells analyzed. Potent in vivo antitumor activity of NK-92/5.28.z was observed in orthotopic GBM xenograft models in NSG mice, leading to a marked extension of symptom-free survival upon repeated stereotactic injection of CAR NK cells into the tumor area (median survival of 200.5 days upon treatment with NK-92/5.28.z vs 73 days upon treatment with parental NK-92 cells, P < .001). In immunocompetent mice, local therapy with NK-92/5.28.z cells resulted in cures of transplanted syngeneic GBM in four of five mice carrying subcutaneous tumors and five of eight mice carrying intracranial tumors, induction of endogenous antitumor immunity, and long-term protection against tumor rechallenge at distant sites. Our data demonstrate the potential of ErbB2-specific NK-92/5.28.z cells for adoptive immunotherapy of glioblastoma, justifying evaluation of this approach for the treatment of ErbB2-positive GBM in clinical studies. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

SETI as a part of Big History

NASA Astrophysics Data System (ADS)

Maccone, Claudio

2014-08-01

Big History is an emerging academic discipline which examines history scientifically from the Big Bang to the present. It uses a multidisciplinary approach based on combining numerous disciplines from science and the humanities, and explores human existence in the context of this bigger picture. It is taught at some universities. In a series of recent papers ([11] through [15] and [17] through [18]) and in a book [16], we developed a new mathematical model embracing Darwinian Evolution (RNA to Humans, see, in particular, [17] and Human History (Aztecs to USA, see [16]) and then we extrapolated even that into the future up to ten million years (see 18), the minimum time requested for a civilization to expand to the whole Milky Way (Fermi paradox). In this paper, we further extend that model in the past so as to let it start at the Big Bang (13.8 billion years ago) thus merging Big History, Evolution on Earth and SETI (the modern Search for ExtraTerrestrial Intelligence) into a single body of knowledge of a statistical type. Our idea is that the Geometric Brownian Motion (GBM), so far used as the key stochastic process of financial mathematics (Black-Sholes models and related 1997 Nobel Prize in Economics!) may be successfully applied to the whole of Big History. In particular, in this paper we derive

Nanoparticle engineered TRAIL-overexpressing adipose-derived stem cells target and eradicate glioblastoma via intracranial delivery

PubMed Central

Jiang, Xinyi; Fitch, Sergio; Wang, Christine; Wilson, Christy; Li, Jianfeng; Grant, Gerald A.; Yang, Fan

2016-01-01

Glioblastoma multiforme (GBM) is one of the most intractable of human cancers, principally because of the highly infiltrative nature of these neoplasms. Tracking and eradicating infiltrating GBM cells and tumor microsatellites is of utmost importance for the treatment of this devastating disease, yet effective strategies remain elusive. Here we report polymeric nanoparticle-engineered human adipose-derived stem cells (hADSCs) overexpressing tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) as drug-delivery vehicles for targeting and eradicating GBM cells in vivo. Our results showed that polymeric nanoparticle-mediated transfection led to robust up-regulation of TRAIL in hADSCs, and that TRAIL-expressing hADSCs induced tumor-specific apoptosis. When transplanted in a mouse intracranial xenograft model of patient-derived glioblastoma cells, hADSCs exhibited long-range directional migration and infiltration toward GBM tumor. Importantly, TRAIL-overexpressing hADSCs inhibited GBM growth, extended survival, and reduced the occurrence of microsatellites. Repetitive injection of TRAIL-overexpressing hADSCs significantly prolonged animal survival compared with single injection of these cells. Taken together, our data suggest that nanoparticle-engineered TRAIL-expressing hADSCs exhibit the therapeutically relevant behavior of “seek-and-destroy” tumortropic migration and could be a promising therapeutic approach to improve the treatment outcomes of patients with malignant brain tumors. PMID:27849590

MiR-615 inhibits cell proliferation, migration and invasion by targeting EGFR in human glioblastoma.

PubMed

Ji, Yanwei; Sun, Qingshan; Zhang, Jianbin; Hu, Haoran

2018-05-15

MiR-615 and epidermal growth factor receptor (EGFR) are associated with a number of disease processes and pathogenesis. However, little is known about the mechanisms of miR-615 and EGFR in human glioblastoma multiforme (GBM). Here, we found that down-regulation of miR-615 expression occurred in GBM tissues and cells, and was inversely correlated with overall survival, relapse-free survival, WHO grade as well as EGFR expression. We further determined that miR-615 functions as a tumor suppressor by inhibiting GBM cell proliferation, cell cycle, migration and invasion, and promoting cell apoptosis. In-vivo assay validated the inhibition effect of miR-615 on tumor growth and EGFR expression. Luciferase reporter assays demonstrated that miR-615 targeted the 3'-untranslated region (3'-UTR) of EGFR. Besides, over-expression of EGFR reversed the inhibition effects of miR-615, while silencing of EGFR aggravated these inhibition effects. In conclusions, we identified that miR-615 plays a tumor suppressor role in GBM cell proliferation, migration and invasion by targeting EGFR expression, and miR-615 may act as a novel biomarker for early diagnosis or therapeutic targets of GBM. Copyright © 2018 Elsevier Inc. All rights reserved.

Combinatorial Drug Testing in 3D Microtumors Derived from GBM Patient-Derived Xenografts Reveals Cytotoxic Synergy in Pharmacokinomics-informed Pathway Interactions.

PubMed

Gilbert, Ashley N; Anderson, Joshua C; Duarte, Christine W; Shevin, Rachael S; Langford, Catherine P; Singh, Raj; Gillespie, G Yancey; Willey, Christopher D

2018-05-30

Glioblastoma multiforme (GBM), the most common form of primary malignant brain cancer in adults, is a devastating disease for which effective treatment has remained elusive for over 75 years. One reason for the minimal progress during this time is the lack of accurate preclinical models to represent the patient's tumor's in vivo environment, causing a disconnect in drug therapy effectiveness between the laboratory and clinic. While patient-derived xenografts (PDX's or xenolines) are excellent human tumor representations, they are not amenable to high throughput testing. Therefore, we developed a miniaturized xenoline system (microtumors) for drug testing. Nineteen GBM xenolines were profiled for global kinase (kinomic) activity revealing actionable kinase targets associated with intracranial tumor growth rate. Kinase inhibitors for these targets (WP1066, selumetinib, crizotinib, and cediranib) were selected for single and combination therapy using a fully human-derived three-dimensional (3D) microtumor model of GBM xenoline cells embedded in HuBiogel for subsequent molecular and phenotype assays. GBM microtumors closely resembled orthotopically-implanted tumors based on immunohistochemical analysis and displayed kinomic and morphological diversity. Drug response testing could be reproducibly performed in a 96-well format identifying several synergistic combinations. Our findings indicate that 3D microtumors can provide a suitable high-throughput model for combination drug testing.

Decitabine Nano-conjugate Sensitizing Human Glioblastoma Cells to Temozolomide

PubMed Central

Cui, Yi; Naz, Asia; Thompson, David H.; Irudayaraj, Joseph

2015-01-01

In this study we developed and characterized a delivery system for the epigenetic demethylating drug, decitabine, to sensitize temozolomide-resistant human glioblastoma multiforme (GBM) cells to alkylating chemotherapy. A poly(lactic-co-glycolic acid) (PLGA) and polyethylene glycol (PEG) based nano-conjugate was fabricated to encapsulate decitabine and achieved a better therapeutic response in GBM cells. After synthesis, the highly efficient uptake process and intracellular dynamics of this nano-conjugate was monitored by single-molecule fluorescence tools. Our experiments demonstrated that, under an acidic pH due to active glycolysis in cancer cells, the PLGA-PEG nano-vector could release the conjugated decitabine at a faster rate, after which the hydrolyzed lactic acid and glycolic acid would further acidify the intracellular microenvironment, thus providing a “positive feedback” to increase the effective drug concentration and realize growth inhibition. In temozolomide-resistant GBM cells, decitabine can potentiate the cytotoxic DNA alkylation by counteracting cytosine methylation and reactivating tumor suppressor genes, such as p53 and p21. Owing to excellent internalization and endo-lysosomal escape enabled by the PLGA-PEG backbone, the encapsulated decitabine exhibited a better anti-GBM potential than free drug molecules. Hence, the synthesized nano-conjugate and temozolomide could act in synergy to deliver a more potent and long-term anti-proliferation effect against malignant GBM cells. PMID:25751281

A preclinical orthotopic model for glioblastoma recapitulates key features of human tumors and demonstrates sensitivity to a combination of MEK and PI3K pathway inhibitors.

PubMed

El Meskini, Rajaa; Iacovelli, Anthony J; Kulaga, Alan; Gumprecht, Michelle; Martin, Philip L; Baran, Maureen; Householder, Deborah B; Van Dyke, Terry; Weaver Ohler, Zoë

2015-01-01

Current therapies for glioblastoma multiforme (GBM), the highest grade malignant brain tumor, are mostly ineffective, and better preclinical model systems are needed to increase the successful translation of drug discovery efforts into the clinic. Previous work describes a genetically engineered mouse (GEM) model that contains perturbations in the most frequently dysregulated networks in GBM (driven by RB, KRAS and/or PI3K signaling and PTEN) that induce development of Grade IV astrocytoma with properties of the human disease. Here, we developed and characterized an orthotopic mouse model derived from the GEM that retains the features of the GEM model in an immunocompetent background; however, this model is also tractable and efficient for preclinical evaluation of candidate therapeutic regimens. Orthotopic brain tumors are highly proliferative, invasive and vascular, and express histology markers characteristic of human GBM. Primary tumor cells were examined for sensitivity to chemotherapeutics and targeted drugs. PI3K and MAPK pathway inhibitors, when used as single agents, inhibited cell proliferation but did not result in significant apoptosis. However, in combination, these inhibitors resulted in a substantial increase in cell death. Moreover, these findings translated into the in vivo orthotopic model: PI3K or MAPK inhibitor treatment regimens resulted in incomplete pathway suppression and feedback loops, whereas dual treatment delayed tumor growth through increased apoptosis and decreased tumor cell proliferation. Analysis of downstream pathway components revealed a cooperative effect on target downregulation. These concordant results, together with the morphologic similarities to the human GBM disease characteristics of the model, validate it as a new platform for the evaluation of GBM treatment. © 2015. Published by The Company of Biologists Ltd.

A preclinical orthotopic model for glioblastoma recapitulates key features of human tumors and demonstrates sensitivity to a combination of MEK and PI3K pathway inhibitors

PubMed Central

El Meskini, Rajaa; Iacovelli, Anthony J.; Kulaga, Alan; Gumprecht, Michelle; Martin, Philip L.; Baran, Maureen; Householder, Deborah B.; Van Dyke, Terry; Weaver Ohler, Zoë

2015-01-01

Current therapies for glioblastoma multiforme (GBM), the highest grade malignant brain tumor, are mostly ineffective, and better preclinical model systems are needed to increase the successful translation of drug discovery efforts into the clinic. Previous work describes a genetically engineered mouse (GEM) model that contains perturbations in the most frequently dysregulated networks in GBM (driven by RB, KRAS and/or PI3K signaling and PTEN) that induce development of Grade IV astrocytoma with properties of the human disease. Here, we developed and characterized an orthotopic mouse model derived from the GEM that retains the features of the GEM model in an immunocompetent background; however, this model is also tractable and efficient for preclinical evaluation of candidate therapeutic regimens. Orthotopic brain tumors are highly proliferative, invasive and vascular, and express histology markers characteristic of human GBM. Primary tumor cells were examined for sensitivity to chemotherapeutics and targeted drugs. PI3K and MAPK pathway inhibitors, when used as single agents, inhibited cell proliferation but did not result in significant apoptosis. However, in combination, these inhibitors resulted in a substantial increase in cell death. Moreover, these findings translated into the in vivo orthotopic model: PI3K or MAPK inhibitor treatment regimens resulted in incomplete pathway suppression and feedback loops, whereas dual treatment delayed tumor growth through increased apoptosis and decreased tumor cell proliferation. Analysis of downstream pathway components revealed a cooperative effect on target downregulation. These concordant results, together with the morphologic similarities to the human GBM disease characteristics of the model, validate it as a new platform for the evaluation of GBM treatment. PMID:25431423

Human herpesvirus multiplex ddPCR detection in brain tissue from low- and high-grade astrocytoma cases and controls.

PubMed

Lin, Cheng-Te Major; Leibovitch, Emily C; Almira-Suarez, M Isabel; Jacobson, Steven

2016-01-01

Glioblastoma (GBM) is a fatal CNS malignancy, representing 50 % of all gliomas with approximately 12-18 months survival time after initial diagnosis. Recently, the human herpesvirus cytomegalovirus (CMV) has been suggested to have an oncogenic role, yet this association remains controversial. In addition, human herpesvirus 6 (HHV-6) and Epstein-Barr virus (EBV) have also been associated with low-grade gliomas, but few studies have examined HHV-6 and EBV in glioblastomas. Droplet digital PCR (ddPCR) is a highly precise diagnostic tool that enables the absolute quantification of target DNA. This study examines the association between multiple human herpesviruses and astrocytomas. This study analyzed 112 brain tissue specimens, including 45 glioblastoma, 12 astrocytoma grade III, 2 astrocytoma grade II, 4 astrocytoma grade I, and 49 controls. All brain tissue samples were de-identified and pathologically confirmed. Each tissue block was sectioned for DNA extraction and CMV, EBV, HHV-6A and HHV-6B, and a cellular housekeeping gene were amplified by ddPCR. Neither CMV nor HHV-6A were detected in any of the astrocytoma samples. However, HHV-6B (p = 0.147) and EBV (p = 0.049) had a higher positivity frequency in the GBM compared to the controls. The undetectable CMV DNA in the astrocytoma cohort does not support the observation of an increased prevalence of CMV DNA in GBM, as reported in other studies. EBV has a significantly higher positivity in the GBM cohort compared to the controls, while HHV-6B has a higher but not statistically significant positivity in the case cohort. Whether these viruses play an oncogenic role in GBM remains to be further investigated.

CXCL12 modulation of CXCR4 and CXCR7 activity in human glioblastoma stem-like cells and regulation of the tumor microenvironment.

PubMed

Würth, Roberto; Bajetto, Adriana; Harrison, Jeffrey K; Barbieri, Federica; Florio, Tullio

2014-01-01

Chemokines are crucial autocrine and paracrine players in tumor development. In particular, CXCL12, through its receptors CXCR4 and CXCR7, affects tumor progression by controlling cancer cell survival, proliferation and migration, and, indirectly, via angiogenesis or recruiting immune cells. Glioblastoma (GBM) is the most prevalent primary malignant brain tumor in adults and despite current multimodal therapies it remains almost incurable. The aggressive and recurrent phenotype of GBM is ascribed to high growth rate, invasiveness to normal brain, marked angiogenesis, ability to escape the immune system and resistance to standard of care therapies. Tumor molecular and cellular heterogeneity severely hinders GBM therapeutic improvement. In particular, a subpopulation of chemo- and radio-therapy resistant tumorigenic cancer stem-like cells (CSCs) is believed to be the main responsible for tumor cell dissemination to the brain. GBM cells display heterogeneous expression levels of CXCR4 and CXCR7 that are overexpressed in CSCs, representing a molecular correlate for the invasive potential of GBM. The microenvironment contribution in GBM development is increasingly emphasized. An interplay exists between CSCs, differentiated GBM cells, and the microenvironment, mainly through secreted chemokines (e.g., CXCL12) causing recruitment of fibroblasts, endothelial, mesenchymal and inflammatory cells to the tumor, via specific receptors such as CXCR4. This review covers recent developments on the role of CXCL12/CXCR4-CXCR7 networks in GBM progression and the potential translational impact of their targeting. The biological and molecular understanding of the heterogeneous GBM cell behavior, phenotype and signaling is still limited. Progress in the identification of chemokine-dependent mechanisms that affect GBM cell survival, trafficking and chemo-attractive functions, opens new perspectives for development of more specific therapeutic approaches that include chemokine-based drugs.

DW-MRI as a Predictive Biomarker of Radiosensitization of GBM through Targeted Inhibition of Checkpoint Kinases.

PubMed

Williams, Terence M; Galbán, Stefanie; Li, Fei; Heist, Kevin A; Galbán, Craig J; Lawrence, Theodore S; Holland, Eric C; Thomae, Tami L; Chenevert, Thomas L; Rehemtulla, Alnawaz; Ross, Brian D

2013-04-01

The inherent treatment resistance of glioblastoma (GBM) can involve multiple mechanisms including checkpoint kinase (Chk1/2)-mediated increased DNA repair capability, which can attenuate the effects of genotoxic chemotherapies and radiation. The goal of this study was to evaluate diffusion-weighted magnetic resonance imaging (DW-MRI) as a biomarker for Chk1/2 inhibitors in combination with radiation for enhancement of treatment efficacy in GBM. We evaluated a specific small molecule inhibitor of Chk1/2, AZD7762, in combination with radiation using in vitro human cell lines and in vivo using a genetically engineered GBM mouse model. DW-MRI and T1-contrast MRI were used to follow treatment effects on intracranial tumor cellularity and growth rates, respectively. AZD7762 inhibited clonal proliferation in a panel of GBM cell lines and increased radiosensitivity in p53-mutated GBM cell lines to a greater extent compared to p53 wild-type cells. In vivo efficacy of AZD7762 demonstrated a dose-dependent inhibitory effect on GBM tumor growth rate and a reduction in tumor cellularity based on DW-MRI scans along with enhancement of radiation efficacy. DW-MRI was found to be a useful imaging biomarker for the detection of radiosensitization through inhibition of checkpoint kinases. Chk1/2 inhibition resulted in antiproliferative activity, prevention of DNA damage-induced repair, and radiosensitization in preclinical GBM tumor models, both in vitro and in vivo. The effects were found to be maximal in p53-mutated GBM cells. These results provide the rationale for integration of DW-MRI in clinical translation of Chk1/2 inhibition with radiation for the treatment of GBM.

Co-amplification of phosphoinositide 3-kinase enhancer A and cyclin-dependent kinase 4 triggers glioblastoma progression | Office of Cancer Genomics

Cancer.gov

Glioblastoma (GBM) is the most common primary brain tumor and has a dismal prognosis. Amplification of chromosome 12q13-q15 (Cyclin-dependent kinase 4 (CDK4) amplicon) is frequently observed in numerous human cancers including GBM. Phosphoinositide 3-kinase enhancer (PIKE) is a group of GTP-binding proteins that belong to the subgroup of centaurin GTPase family, encoded by CENTG1 located in CDK4 amplicon. However, the pathological significance of CDK4 amplicon in GBM formation remains incompletely understood.

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

PubMed

Tang, Nou-Ying; Chueh, Fu-Shin; Yu, Chien-Chih; Liao, Ching-Lung; Lin, Jen-Jyh; Hsia, Te-Chun; Wu, King-Chuen; Liu, Hsin-Chung; Lu, Kung-Wen; Chung, Jing-Gung

2016-04-01

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.

Identification of repaglinide as a therapeutic drug for glioblastoma multiforme.

PubMed

Xiao, Zui Xuan; Chen, Ruo Qiao; Hu, Dian Xing; Xie, Xiao Qiang; Yu, Shang Bin; Chen, Xiao Qian

2017-06-17

Glioblastoma multiforme (GBM) is a highly aggressive brain tumor with a median survival time of only 14 months after treatment. It is urgent to find new therapeutic drugs that increase survival time of GBM patients. To achieve this goal, we screened differentially expressed genes between long-term and short-term survived GBM patients from Gene Expression Omnibus database and found gene expression signature for the long-term survived GBM patients. The signaling networks of all those differentially expressed genes converged to protein binding, extracellular matrix and tissue development as revealed in BiNGO and Cytoscape. Drug repositioning in Connectivity Map by using the gene expression signature identified repaglinide, a first-line drug for diabetes mellitus, as the most promising novel drug for GBM. In vitro experiments demonstrated that repaglinide significantly inhibited the proliferation and migration of human GBM cells. In vivo experiments demonstrated that repaglinide prominently prolonged the median survival time of mice bearing orthotopic glioma. Mechanistically, repaglinide significantly reduced Bcl-2, Beclin-1 and PD-L1 expression in glioma tissues, indicating that repaglinide may exert its anti-cancer effects via apoptotic, autophagic and immune checkpoint signaling. Taken together, repaglinide is likely to be an effective drug to prolong life span of GBM patients. Copyright © 2017. Published by Elsevier Inc.

Anti-GD2-ch14.18/CHO coated nanoparticles mediate glioblastoma (GBM)-specific delivery of the aromatase inhibitor, Letrozole, reducing proliferation, migration and chemoresistance in patient-derived GBM tumor cells.

PubMed

Tivnan, Amanda; Heilinger, Tatjana; Ramsey, Joanne M; O'Connor, Gemma; Pokorny, Jenny L; Sarkaria, Jann N; Stringer, Brett W; Day, Bryan W; Boyd, Andrew W; Kim, Ella L; Lode, Holger N; Cryan, Sally-Ann; Prehn, Jochen H M

2017-03-07

Aromatase is a critical enzyme in the irreversible conversion of androgens to oestrogens, with inhibition used clinically in hormone-dependent malignancies. We tested the hypothesis that targeted aromatase inhibition in an aggressive brain cancer called glioblastoma (GBM) may represent a new treatment strategy. In this study, aromatase inhibition was achieved using third generation inhibitor, Letrozole, encapsulated within the core of biodegradable poly lactic-co-glycolic acid (PLGA) nanoparticles (NPs). PLGA-NPs were conjugated to human/mouse chimeric anti-GD2 antibody ch14.18/CHO, enabling specific targeting of GD2-positive GBM cells. Treatment of primary and recurrent patient-derived GBM cells with free-Letrozole (0.1 μM) led to significant decrease in cell proliferation and migration; in addition to reduced spheroid formation. Anti-GD2-ch14.18/CHO-NPs displayed specific targeting of GBM cells in colorectal-glioblastoma co-culture, with subsequent reduction in GBM cell numbers when treated with anti-GD2-ch14.18-PLGA-Let-NPs in combination with temozolomide. As miR-191 is an estrogen responsive microRNA, its expression, fluctuation and role in Letrozole treated GBM cells was evaluated, where treatment with premiR-191 was capable of rescuing the reduced proliferative phenotype induced by aromatase inhibitor. The repurposing and targeted delivery of Letrozole for the treatment of GBM, with the potential role of miR-191 identified, provides novel avenues for target assessment in this aggressive brain cancer.

Anti-GD2-ch14.18/CHO coated nanoparticles mediate glioblastoma (GBM)-specific delivery of the aromatase inhibitor, Letrozole, reducing proliferation, migration and chemoresistance in patient-derived GBM tumor cells

PubMed Central

Tivnan, Amanda; Heilinger, Tatjana; Ramsey, Joanne M; O’Connor, Gemma; Pokorny, Jenny L; Sarkaria, Jann N; Stringer, Brett W; Day, Bryan W; Boyd, Andrew W; Kim, Ella L; Lode, Holger N; Cryan, Sally-Ann; Prehn, Jochen H.M

2017-01-01

Aromatase is a critical enzyme in the irreversible conversion of androgens to oestrogens, with inhibition used clinically in hormone-dependent malignancies. We tested the hypothesis that targeted aromatase inhibition in an aggressive brain cancer called glioblastoma (GBM) may represent a new treatment strategy. In this study, aromatase inhibition was achieved using third generation inhibitor, Letrozole, encapsulated within the core of biodegradable poly lactic-co-glycolic acid (PLGA) nanoparticles (NPs). PLGA-NPs were conjugated to human/mouse chimeric anti-GD2 antibody ch14.18/CHO, enabling specific targeting of GD2-positive GBM cells. Treatment of primary and recurrent patient-derived GBM cells with free-Letrozole (0.1 μM) led to significant decrease in cell proliferation and migration; in addition to reduced spheroid formation. Anti-GD2-ch14.18/CHO-NPs displayed specific targeting of GBM cells in colorectal-glioblastoma co-culture, with subsequent reduction in GBM cell numbers when treated with anti-GD2-ch14.18-PLGA-Let-NPs in combination with temozolomide. As miR-191 is an estrogen responsive microRNA, its expression, fluctuation and role in Letrozole treated GBM cells was evaluated, where treatment with premiR-191 was capable of rescuing the reduced proliferative phenotype induced by aromatase inhibitor. The repurposing and targeted delivery of Letrozole for the treatment of GBM, with the potential role of miR-191 identified, provides novel avenues for target assessment in this aggressive brain cancer. PMID:28178667

Inhibition of LSD1 sensitizes glioblastoma cells to histone deacetylase inhibitors

PubMed Central

Singh, Melissa M.; Manton, Christa A.; Bhat, Krishna P.; Tsai, Wen-Wei; Aldape, Kenneth; Barton, Michelle C.; Chandra, Joya

2011-01-01

Glioblastoma multiforme (GBM) is a particularly aggressive brain tumor and remains a clinically devastating disease. Despite innovative therapies for the treatment of GBM, there has been no significant increase in patient survival over the past decade. Enzymes that control epigenetic alterations are of considerable interest as targets for cancer therapy because of their critical roles in cellular processes that lead to oncogenesis. Several inhibitors of histone deacetylases (HDACs) have been developed and tested in GBM with moderate success. We found that treatment of GBM cells with HDAC inhibitors caused the accumulation of histone methylation, a modification removed by the lysine specific demethylase 1 (LSD1). This led us to examine the effects of simultaneously inhibiting HDACs and LSD1 as a potential combination therapy. We evaluated induction of apoptosis in GBM cell lines after combined inhibition of LSD1 and HDACs. LSD1 was inhibited by targeted short hairpin RNA or pharmacological means and inhibition of HDACs was achieved by treatment with either vorinostat or PCI-24781. Caspase-dependent apoptosis was significantly increased (>2-fold) in LSD1-knockdown GBM cells treated with HDAC inhibitors. Moreover, pharmacologically inhibiting LSD1 with the monoamine oxidase inhibitor tranylcypromine, in combination with HDAC inhibitors, led to synergistic apoptotic cell death in GBM cells; this did not occur in normal human astrocytes. Taken together, these results indicate that LSD1 and HDACs cooperate to regulate key pathways of cell death in GBM cell lines but not in normal counterparts, and they validate the combined use of LSD1 and HDAC inhibitors as a therapeutic approach for GBM. PMID:21653597

Identification of Novel Tumor-Associated Cell Surface Sialoglycoproteins in Human Glioblastoma Tumors Using Quantitative Proteomics

PubMed Central

Autelitano, François; Loyaux, Denis; Roudières, Sébastien; Déon, Catherine; Guette, Frédérique; Fabre, Philippe; Ping, Qinggong; Wang, Su; Auvergne, Romane; Badarinarayana, Vasudeo; Smith, Michael; Guillemot, Jean-Claude; Goldman, Steven A.; Natesan, Sridaran; Ferrara, Pascual; August, Paul

2014-01-01

Glioblastoma multiform (GBM) remains clinical indication with significant “unmet medical need”. Innovative new therapy to eliminate residual tumor cells and prevent tumor recurrences is critically needed for this deadly disease. A major challenge of GBM research has been the identification of novel molecular therapeutic targets and accurate diagnostic/prognostic biomarkers. Many of the current clinical therapeutic targets of immunotoxins and ligand-directed toxins for high-grade glioma (HGG) cells are surface sialylated glycoproteins. Therefore, methods that systematically and quantitatively analyze cell surface sialoglycoproteins in human clinical tumor samples would be useful for the identification of potential diagnostic markers and therapeutic targets for malignant gliomas. In this study, we used the bioorthogonal chemical reporter strategy (BOCR) in combination with label-free quantitative mass spectrometry (LFQ-MS) to characterize and accurately quantify the individual cell surface sialoproteome in human GBM tissues, in fetal, adult human astrocytes, and in human neural progenitor cells (NPCs). We identified and quantified a total of 843 proteins, including 801 glycoproteins. Among the 843 proteins, 606 (72%) are known cell surface or secreted glycoproteins, including 156 CD-antigens, all major classes of cell surface receptor proteins, transporters, and adhesion proteins. Our findings identified several known as well as new cell surface antigens whose expression is predominantly restricted to human GBM tumors as confirmed by microarray transcription profiling, quantitative RT-PCR and immunohistochemical staining. This report presents the comprehensive identification of new biomarkers and therapeutic targets for the treatment of malignant gliomas using quantitative sialoglycoproteomics with clinically relevant, patient derived primary glioma cells. PMID:25360666

Identification of novel tumor-associated cell surface sialoglycoproteins in human glioblastoma tumors using quantitative proteomics.

PubMed

Autelitano, François; Loyaux, Denis; Roudières, Sébastien; Déon, Catherine; Guette, Frédérique; Fabre, Philippe; Ping, Qinggong; Wang, Su; Auvergne, Romane; Badarinarayana, Vasudeo; Smith, Michael; Guillemot, Jean-Claude; Goldman, Steven A; Natesan, Sridaran; Ferrara, Pascual; August, Paul

2014-01-01

Glioblastoma multiform (GBM) remains clinical indication with significant "unmet medical need". Innovative new therapy to eliminate residual tumor cells and prevent tumor recurrences is critically needed for this deadly disease. A major challenge of GBM research has been the identification of novel molecular therapeutic targets and accurate diagnostic/prognostic biomarkers. Many of the current clinical therapeutic targets of immunotoxins and ligand-directed toxins for high-grade glioma (HGG) cells are surface sialylated glycoproteins. Therefore, methods that systematically and quantitatively analyze cell surface sialoglycoproteins in human clinical tumor samples would be useful for the identification of potential diagnostic markers and therapeutic targets for malignant gliomas. In this study, we used the bioorthogonal chemical reporter strategy (BOCR) in combination with label-free quantitative mass spectrometry (LFQ-MS) to characterize and accurately quantify the individual cell surface sialoproteome in human GBM tissues, in fetal, adult human astrocytes, and in human neural progenitor cells (NPCs). We identified and quantified a total of 843 proteins, including 801 glycoproteins. Among the 843 proteins, 606 (72%) are known cell surface or secreted glycoproteins, including 156 CD-antigens, all major classes of cell surface receptor proteins, transporters, and adhesion proteins. Our findings identified several known as well as new cell surface antigens whose expression is predominantly restricted to human GBM tumors as confirmed by microarray transcription profiling, quantitative RT-PCR and immunohistochemical staining. This report presents the comprehensive identification of new biomarkers and therapeutic targets for the treatment of malignant gliomas using quantitative sialoglycoproteomics with clinically relevant, patient derived primary glioma cells.

Analysis of single nucleotide variants of HFE gene and association to survival in The Cancer Genome Atlas GBM data

PubMed Central

Zhang, Bo; Liu, Dajiang J.; Muscat, Joshua E.; Langan, Sara T.; Connor, James R.

2017-01-01

Human hemochromatosis protein (HFE) is involved in iron metabolism. Two major HFE polymorphisms, H63D and C282Y, have been associated with an increased risk of cancers. Previously, we reported decreased gender effects in overall survival based on H63D or C282Y HFE polymorphisms patients with glioblastoma multiforme (GBM). However, the effect of other single nucleotide variation (SNV) in the HFE gene on the cancer development and progression has not been systematically studied. To expand our finding in a larger sample, and to identify other HFE SNV, we analyzed the frequency of somatic SNV in HFE gene and its relationship to survival in GBM patients using The Cancer Genome Atlas (TCGA) GBM (Caucasian only) database. We found 9 SNVs with increased frequency in blood normal of TCGA GBM patients compared to the 1000Genome. Among 9 SNVs, 7 SNVs were located in the intron and 2 SNVs (i.e., H63D, C282Y) in the exon of HFE gene. The statistical analysis demonstrated that blood normal samples of TCGA GBM have more H63D (p = 0.0002, 95% Confidence interval (CI): 0.2119–0.3223) or C282Y (p = 0.0129, 95% CI: 0.0474–0.1159) HFE polymorphisms than 1000Genome. The Kaplan-Meier survival curve for the 264 GBM samples revealed no difference between wild type (WT) HFE and H63D, and WT HFE and C282Y GBM patients. In addition, there was no difference in the survival of male/female GBM patients based on HFE genotype. There was no correlation between HFE expression and survival. In conclusion, the current results suggest that somatic HFE polymorphisms do not impact GBM patients’ survival in the TCGA data set of GBM. PMID:28358914

A Chimeric Antibody against ACKR3/CXCR7 in Combination with TMZ Activates Immune Responses and Extends Survival in Mouse GBM Models.

PubMed

Salazar, Nicole; Carlson, Jeffrey C; Huang, Kexin; Zheng, Yayue; Oderup, Cecilia; Gross, Julia; Jang, Andrew D; Burke, Thomas M; Lewén, Susanna; Scholz, Alexander; Huang, Serina; Nease, Leona; Kosek, Jon; Mittelbronn, Michel; Butcher, Eugene C; Tu, Hua; Zabel, Brian A

2018-05-02

Glioblastoma (GBM) is the least treatable type of brain tumor, afflicting over 15,000 people per year in the United States. Patients have a median survival of 16 months, and over 95% die within 5 years. The chemokine receptor ACKR3 is selectively expressed on both GBM cells and tumor-associated blood vessels. High tumor expression of ACKR3 correlates with poor prognosis and potential treatment resistance, making it an attractive therapeutic target. We engineered a single chain FV-human FC-immunoglobulin G1 (IgG 1 ) antibody, X7Ab, to target ACKR3 in human and mouse GBM cells. We used hydrodynamic gene transfer to overexpress the antibody, with efficacy in vivo. X7Ab kills GBM tumor cells and ACKR3-expressing vascular endothelial cells by engaging the cytotoxic activity of natural killer (NK) cells and complement and the phagocytic activity of macrophages. Combining X7Ab with TMZ allows the TMZ dosage to be lowered, without compromising therapeutic efficacy. Mice treated with X7Ab and in combination with TMZ showed significant tumor reduction by MRI and longer survival overall. Brain-tumor-infiltrating leukocyte analysis revealed that X7Ab enhances the activation of M1 macrophages to support anti-tumor immune response in vivo. Targeting ACKR3 with immunotherapeutic monoclonal antibodies (mAbs) in combination with standard of care therapies may prove effective in treating GBM. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Fisetin suppresses ADAM9 expression and inhibits invasion of glioma cancer cells through increased phosphorylation of ERK1/2.

PubMed

Chen, Chien-Min; Hsieh, Yi-Hsien; Hwang, Jin-Ming; Jan, Hsun-Jin; Hsieh, Shu-Ching; Lin, Shin-Huey; Lai, Chung-Yu

2015-05-01

Fisetin (3,3',4',7-tetrahydroxyflavone) is a naturally occurring flavonoid which is widely distributed in plants. It has been reported to possess some anticancer and anti-invasive capabilities. We set out to explore the effects of fisetin on antimetastatic and its mechanism of action in GBM8401 cells. The results indicated that fisetin exhibited effective inhibition of cell migration and inhibited the invasion of GBM8401 cells under non-cytotoxic concentrations. To identify the potential targets of fisetin, human proteinase antibody array analysis was performed, and the results indicated that the fisetin treatment inhibited the expression of ADAM9 protein and mRNA, which are known to contribute to the progression of glioma cancer. Our results showed that fisetin phosphorylated ERK1/2 in a sustained way that contributed to the inhibited ADAM9 protein and mRNA expression determined by Western blot and RT-PCR. Moreover, inhibition of ERK1/2 by U0126 or transfection with the siERK plasmid significantly abolished the fisetin-inhibited migration and invasion through activation of the ERK1/2 pathway. In summary, our results suggest that fisetin might be a potential therapeutic agent against human glioma cells based on its capacity to activate ERK1/2 and to inhibit ADAM9 expression.

Valganciclovir and bevacizumab for recurrent glioblastoma: A single-institution experience

PubMed Central

PENG, CHENGWEI; WANG, JIALING; TANKSLEY, JARRED P.; MOBLEY, BRET C.; AYERS, GREGORY D.; MOOTS, PAUL L.; CLARK, STEPHEN W.

2016-01-01

Prolonged treatment with adjuvant valganciclovir has been shown in one retrospective study to exert a significant effect on overall survival (OS) in newly diagnosed patients with glioblastoma multiforme (GBM). However, studies evaluating the effectiveness of valganciclovir in the treatment of recurrent GBM have not been performed. We evaluated the effect of valganciclovir in the recurrent setting in combination with bevacizumab therapy. A retrospective analysis was performed on patients treated for recurrent GBM with off-label valganciclovir and bevacizumab at Vanderbilt University. We identified 13 patients who received valganciclovir plus bevacizumab at some point during their treatment, 8 of whom were started on valganciclovir and bevacizumab concurrently upon first recurrence, whereas 5 had valganciclovir added to their bevacizumab regimen prior to a second recurrence. of these patients, 12 were pathologically confirmed to have GBM, and 1 patient was diagnosed with gliosarcoma. We also identified an institutional cohort of 50 patients who had not been exposed to valganciclovir, but were treated with bevacizumab for first recurrence. The progression-free survival (PFS) at 6 months (PF6) and median OS (mOS) in the valganciclovir plus bevacizumab group was 62% and 13.1 months, respectively, for all 13 patients, and 50% and 11.3 months, respectively, for the 8 concurrently treated patients. In the institutional bevacizumab cohort, the PF6 and mOS were 34% and 8.7 months, respectively. In this retrospective analysis, valganciclovir in combination with bevacizumab exhibited a trend toward improved survival in patients with recurrent GBM. However, given the small sample size and the retrospective nature of this study, a larger prospective study is required to confirm these results. PMID:26893852

Observations of Accreting Pulsars with the FERMI-GBM

NASA Technical Reports Server (NTRS)

Wilson-Hodge, Colleen

2012-01-01

The Gamma-ray Burst Monitor (GBM) on-board Fermi comprises 12 NaI detectors spanning the 8-1000 keV band and 2 BGO detectors spanning the 100 keV to 40 MeV band. These detectors view the entire unocculted sky, providing long (approximately 40 ks/day) observations of accreting pulsars daily, which allow long-term monitoring of spin-frequencies and pulsed uxes via epoch-folded searches plus daily blind searches for new pulsars. Phase averaged uxes can be measured using the Earth occultation technique. In this talk I will present highlights of GBM accretion-powered pulsar monitoring such as the discovery of a torque reversal in 4U1626-67, a high-energy QPO in A0535+26, and evidence for a stable accretion disk in OAO 1657-415.

Disulfiram, a drug widely used to control alcoholism, suppresses self-renewal of glioblastoma and overrides resistance to temozolomide

PubMed Central

Triscott, Joanna; Lee, Cathy; Hu, Kaiji; Fotovati, Abbas; Berns, Rachel; Pambid, Mary; Luk, Margaret; Kast, Richard E.; Kong, Esther; Toyota, Eric; Yip, Stephen; Toyota, Brian; Dunn, Sandra E.

2012-01-01

Glioblastomas (GBM) are associated with high rates of relapse. These brain tumors are often resistant to chemotherapies like temozolomide (TMZ) and there are very few treatment options available to patients. We recently reported that polo-like kinase-1 (PLK1) is associated with the proliferative subtype of GBM; which has the worst prognosis. In this study, we addressed the potential of repurposing disulfiram (DSF), a drug widely used to control alcoholism for the past six decades. DSF has good safety profiles and penetrates the blood-brain barrier. Here we report that DSF inhibited the growth of TMZ resistant GBM cells, (IC90=100 nM), but did not affect normal human astrocytes. At similar DSF concentrations, self-renewal was blocked by ~100% using neurosphere growth assays. Likewise the drug completely inhibited the self-renewal of the BT74 and GBM4 primary cell lines. Additionally, DSF suppressed growth and self-renewal of primary cells from two GBM tumors. These cells were resistant to TMZ, had unmethylated MGMT, and expressed high levels of PLK1. Consistent with its role in suppressing GBM growth, DSF inhibited the expression of PLK1 in GBM cells. Likewise, PLK1 inhibition with siRNA, or small molecules (BI-2536 or BI-6727) blocked growth of TMZ resistant cells. Our studies suggest that DSF has the potential to be repurposed for treatment of refractory GBM. PMID:23047041

PM-07LOSS OF ATRX DECREASES SURVIVAL AND IMPROVES RESPONSE TO DNA DAMAGING AGENTS IN A NOVEL MOUSE MODEL OF GLIOBLASTOMA

PubMed Central

Koschmann, Carl; Calinescu, Alexandra; Thomas, Daniel; Kamran, Neha; Nunez-Aguilera, Felipe; Dzaman, Marta; Lemons, Rosie; Li, Youping; Roh, Haeji; Lowenstein, Pedro; Castro, Maria

2014-01-01

Pediatric glioblastoma (GBM) remains one of the most difficult childhood tumors to treat. ATRX is a histone chaperone protein that is mutated primarily in younger patients with GBM. No previous animal model has demonstrated the effect of ATRX loss on GBM formation. We cloned an ATRX knockdown sequence into a Sleeping Beauty (SB) transposase-responsive plasmid (shATRX) for insertion into host genomic DNA. Glioblastomas were induced in mice by injecting plasmids encoding SB transposase/ luciferase, shp53 and NRAS, with or without shATRX, into the ventricle of neonatal mice. Tumors in both groups (with or without shATRX) showed histological hallmarks of human glioblastoma. The loss of ATRX was specifically localized only within tumors generated with the shATRX plasmid and not in the adjacent cortex. Notably, loss of ATRX reduced median survival of mice by 43% (p = 0.012). ATRX-deficient tumors were significantly more likely to develop microsatellite instability (p = 0.014), a hallmark of impaired DNA-damage repair. Analysis of three human GBM sequencing datasets confirmed increased number of somatic nucleotide mutations in ATRX-deficient tumors. Treatment of primary cell cultures generated from mouse GBMs showed that ATRX-deficient tumor cells are significantly more sensitive to DNA damaging agents. In addition, mice with ATRX-deficient GBM treated with whole brain irradiation had trend towards improved survival (p= 0.06), with some long-term survivors. Treated ATRX-deficient tumor cells showed greater evidence of double-stranded DNA breakage, by gH2A.X. In summary, this mouse model prospectively validates ATRX as a tumor suppressor in human GBM for the first time in an animal model. In addition, loss of ATRX leads to increased mutation frequency and response to DNA-damaging therapy. We have generated the hypothesis that ATRX loss leads to a genetically unstable tumor; which is more aggressive when untreated, but more responsive to DNA-damaging therapy, ultimately resulting in equivalent or improved overall survival.

Human alpha-lactalbumin made lethal to tumor cells (HAMLET) kills human glioblastoma cells in brain xenografts by an apoptosis-like mechanism and prolongs survival.

PubMed

Fischer, Walter; Gustafsson, Lotta; Mossberg, Ann-Kristin; Gronli, Janne; Mork, Sverre; Bjerkvig, Rolf; Svanborg, Catharina

2004-03-15

Malignant brain tumors present a major therapeutic challenge because no selective or efficient treatment is available. Here, we demonstrate that intratumoral administration of human alpha-lactalbumin made lethal to tumor cells (HAMLET) prolongs survival in a human glioblastoma (GBM) xenograft model, by selective induction of tumor cell apoptosis. HAMLET is a protein-lipid complex that is formed from alpha-lactalbumin when the protein changes its tertiary conformation and binds oleic acid as a cofactor. HAMLET induces apoptosis in a wide range of tumor cells in vitro, but the therapeutic effect in vivo has not been examined. In this study, invasively growing human GBM tumors were established in nude rats (Han:rnu/rnu Rowett, n = 20) by transplantation of human GBM biopsy spheroids. After 7 days, HAMLET was administered by intracerebral convection-enhanced delivery for 24 h into the tumor area; and alpha-lactalbumin, the native, folded variant of the same protein, was used as a control. HAMLET reduced the intracranial tumor volume and delayed the onset of pressure symptoms in the tumor-bearing rats. After 8 weeks, all alpha-lactalbumin-treated rats had developed pressure symptoms, but the HAMLET-treated rats remained asymptomatic. Magnetic resonance imaging scans revealed large differences in tumor volume (456 versus 63 mm(3)). HAMLET caused apoptosis in vivo in the tumor but not in adjacent intact brain tissue or in nontransformed human astrocytes, and no toxic side effects were observed. The results identify HAMLET as a new candidate in cancer therapy and suggest that HAMLET should be additionally explored as a novel approach to controlling GBM progression.

Encapsulated Stem Cells Loaded With Hyaluronidase-expressing Oncolytic Virus for Brain Tumor Therapy

PubMed Central

Martinez-Quintanilla, Jordi; He, Derek; Wakimoto, Hiroaki; Alemany, Ramon; Shah, Khalid

2015-01-01

Despite the proven safety of oncolytic viruses (OV) in clinical trials for glioblastoma (GBM), their efficacy has been hindered by suboptimal spreading within the tumor. We show that hyaluronan or hyaluronic acid (HA), an important component of extracellular matrix (ECM), is highly expressed in a majority of tumor xenografts established from patient-derived GBM lines that present both invasive and nodular phenotypes. Intratumoral injection of a conditionally replicating adenovirus expressing soluble hyaluronidase (ICOVIR17) into nodular GBM, mediated HA degradation and enhanced viral spread, resulting in a significant antitumor effect and mice survival. In an effort to translate OV-based therapeutics into clinical settings, we encapsulated human adipose-derived mesenchymal stem cells (MSC) loaded with ICOVIR17 in biocompatible synthetic extracellular matrix (sECM) and tested their efficacy in a clinically relevant mouse model of GBM resection. Compared with direct injection of ICOVIR17, sECM-MSC loaded with ICOVIR17 resulted in a significant decrease in tumor regrowth and increased mice survival. This is the first report of its kind revealing the expression of HA in GBM and the role of OV-mediated HA targeting in clinically relevant mouse model of GBM resection and thus has clinical implications. PMID:25352242

Antitumor activity of (2E,5Z)-5-(2-hydroxybenzylidene)-2-((4-phenoxyphenyl)imino) thiazolidin-4-one, a novel microtubule-depolymerizing agent, in U87MG human glioblastoma cells and corresponding mouse xenograft model.

PubMed

Zhang, Qiu; Liu, Xiaojun; Li, Xiue; Li, Changlong; Zhou, Hongyu; Yan, Bing

2013-01-01

Glioblastoma is the most lethal brain cancer. In spite of intensive therapy, the prognosis of patients with glioblastoma is very poor. To discover novel therapeutic agents, we screened a combinatorial compound library containing 372 thiazolidinone compounds using U87MG human glioblastoma cells. (2E,5Z)-5-(2-hydroxybenzylidene)-2-((4-phenoxyphenyl)imino) thiazolidin-4-one (HBPT) was identified as the most potent anti-glioblastoma compound. HBPT inhibits U87MG human glioblastoma cell proliferation with an IC50 of 20 μM, which is almost 5-fold more potent than temozolomide (a widely used drug for treating malignant glioma in the clinic). Mechanistic investigation demonstrated that HBPT is a novel microtubule-depolymerizing agent, which arrests cancer cells at the G2/M phase of the cell cycle and induces cell apoptosis. In the mouse U87MG xenograft model, HBPT elicits a robust tumor inhibitory effect. More importantly, no obvious toxicity was observed for HBPT therapy in animal experiments. These findings indicate that HBPT has the potential to be developed as a novel agent for the treatment of glioblastoma. [Supplementary Tables: available only at http://dx.doi.org/10.1254/jphs.13064FP].

Quercetin sensitizes human glioblastoma cells to temozolomide in vitro via inhibition of Hsp27.

PubMed

Sang, Dong-Ping; Li, Ru-Jun; Lan, Qing

2014-06-01

Quercetin is an effective Hsp27 inhibitor and has been reported to facilitate tumor cell apoptosis. The aim of this study was to investigate whether quercetin could sensitize human glioblastoma cells to temozolomide (TMZ) in vitro. Both U251 and U87 human glioblastoma cells were treated with quercetin and/or TMZ for 48 h. Cell viability was detected using the MTT assay. Cell apoptosis was analyzed with caspase-3 activity kits and flow cytometry. Hsp27 expression and phosphorylation were examined using Western blot analysis. RNA interference using Hsp27 siRNA oligos was performed to knock down the gene expression of Hsp27. TMZ (200 or 400 μmol/L) alone effectively inhibited the viability of U251 and U87 cells. When combined with quercetin (30 μmol/L), TMZ (100 μmol/L) significantly inhibited the cell viability, and the inhibition of TMZ (200 and 400 μmol/L) was enhanced. TMZ or quercetin anole did not affect caspase-3 activity and cell apoptosis, while TMZ combined with quercetin significantly increased caspase-3 activity and induced cell apoptosis. TMZ anole significantly increased Hsp27 phosphorylation in U251 and U87 cells, while quercetin or Hsp27 siRNA oligos combined with TMZ attenuated TMZ-induced Hsp27 phosphorylation and significantly inhibited Hsp27 expression. Combined treatment with TMZ and quercetin efficiently suppressed human glioblastoma cell survival in vitro.

Laminin-521 Protein Therapy for Glomerular Basement Membrane and Podocyte Abnormalities in a Model of Pierson Syndrome.

PubMed

Lin, Meei-Hua; Miller, Joseph B; Kikkawa, Yamato; Suleiman, Hani Y; Tryggvason, Karl; Hodges, Bradley L; Miner, Jeffrey H

2018-05-01

Background Laminin α 5 β 2 γ 1 (LM-521) is a major component of the GBM. Mutations in LAMB2 that prevent LM-521 synthesis and/or secretion cause Pierson syndrome, a rare congenital nephrotic syndrome with diffuse mesangial sclerosis and ocular and neurologic defects. Because the GBM is uniquely accessible to plasma, which permeates endothelial cell fenestrae, we hypothesized that intravenous delivery of LM-521 could replace the missing LM-521 in the GBM of Lamb2 mutant mice and restore glomerular permselectivity. Methods We injected human LM-521 (hLM-521), a macromolecule of approximately 800 kD, into the retro-orbital sinus of Lamb2 -/- pups daily. Deposition of hLM-521 into the GBM was investigated by fluorescence microscopy. We assayed the effects of hLM-521 on glomerular permselectivity by urinalysis and the effects on podocytes by desmin immunostaining and ultrastructural analysis of podocyte architecture. Results Injected hLM-521 rapidly and stably accumulated in the GBM of all glomeruli. Super-resolution imaging showed that hLM-521 accumulated in the correct orientation in the GBM, primarily on the endothelial aspect. Treatment with hLM-521 greatly reduced the expression of the podocyte injury marker desmin and attenuated the foot process effacement observed in untreated pups. Moreover, treatment with hLM-521 delayed the onset of proteinuria but did not prevent nephrotic syndrome, perhaps due to its absence from the podocyte aspect of the GBM. Conclusions These studies show that GBM composition and function can be altered in vivo via vascular delivery of even very large proteins, which may advance therapeutic options for patients with abnormal GBM composition, whether genetic or acquired. Copyright © 2018 by the American Society of Nephrology.

Inhibition of glioblastoma tumorspheres by combined treatment with 2-deoxyglucose and metformin.

PubMed

Kim, Eui Hyun; Lee, Ji-Hyun; Oh, Yoonjee; Koh, Ilkyoo; Shim, Jin-Kyoung; Park, Junseong; Choi, Junjeong; Yun, Mijin; Jeon, Jeong Yong; Huh, Yong Min; Chang, Jong Hee; Kim, Sun Ho; Kim, Kyung-Sup; Cheong, Jae-Ho; Kim, Pilnam; Kang, Seok-Gu

2017-02-01

Deprivation of tumor bioenergetics by inhibition of multiple energy pathways has been suggested as an effective therapeutic approach for various human tumors. However, this idea has not been evaluated in glioblastoma (GBM). We hypothesized that dual inhibition of glycolysis and oxidative phosphorylation could effectively suppress GBM tumorspheres (TS). Effects of 2-deoxyglucose (2DG) and metformin, alone and in combination, on GBM-TS were evaluated. Viability, cellular energy metabolism status, stemness, invasive properties, and GBM-TS transcriptomes were examined. In vivo efficacy was tested in a mouse orthotopic xenograft model. GBM-TS viability was decreased by the combination of 2DG and metformin. ATP assay and PET showed that cellular energy metabolism was also decreased by this combination. Sphere formation, expression of stemness-related proteins, and invasive capacity of GBM-TS were also significantly suppressed by combined treatment with 2DG and metformin. A transcriptome analysis showed that the expression levels of stemness- and epithelial mesenchymal transition-related genes were also significantly downregulated by combination of 2DG and metformin. Combination treatment also prolonged survival of tumor-bearing mice and decreased invasiveness of GBM-TS. The combination of 2DG and metformin effectively decreased the stemness and invasive properties of GBM-TS and showed a potential survival benefit in a mouse orthotopic xenograft model. Our findings suggest that targeting TS-forming cells by this dual inhibition of cellular bioenergetics warrants expedited clinical evaluation for the treatment of GBM. © The Author(s) 2016. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com

[RITA combined with temozolomide inhibits the proliferation of human glioblastoma U87 cells].

PubMed

He, Xiao-Yan; Feng, Xiao-Li; Song, Xin-Pei; Zeng, Huan-Chao; Cao, Zhong-Xu; Xiao, Wei-Wei; Zhang, Bao; Wu, Qing-Hua

2016-10-20

To observe the effect of RITA, a small molecule that targets p53, combined with temozolomide (TMZ) on proliferation, colony formation and apoptosis of human glioblastoma U87 cells and explore the underlying mechanism. Cultured U87 cells were treated with RITA (1, 5, 10, 20 µmol/L), TMZ, or RITA+TMZ (half dose) for 24, 48 or 72 h. MTS assay were used to detect the cell proliferation, and the cell proliferation rate and inhibitory rate were calculated. The effect of combined treatments was evaluated by the q value. The expressions of p53, p21 and other apoptosis-associated genes were detected by qRT-PCR and Western blotting; cell apoptosis was assayed using flow cytometry with Annexin V/PI double staining; colony formation of the cells was detected with crystal violet staining. MTS assay showed that RITA at the 4 doses more potently inhibited U87 cell viability than TMZ at 72 h (P=0.000) with inhibitory rates of 25.94%-41.38% and 3.84%-8.20%, respectively. RITA combined with TMZ caused a more significant inhibition of U87 cells (29.21%-52.11%) than RITA (P<0.01) and TMZ (P=0.000) alone. At the doses above 5 µmol/L, the combined treatments with RITA+TMZ for 48 h resulted in q values exceeding 1.2 and showed an obvious synergistic effect of the drugs. Both RITA and TMZ, especially the latter, significantly increased the expressions of p53, p21, puma, and other apoptosis-associated genes to accelerate apoptosis and inhibit the growth and colony formation of U87 cells, and the effect was more obvious with a combined treatment. RITA inhibits the growth of human glioblastoma cells and enhance their sensitivity to TMZ by up-regulating p53 expression, and when combined, RITA and TMZ show a synergistic effect to cause a stronger cell inhibition.

Prospective Isolation and Comparison of Human Germinal Matrix and Glioblastoma EGFR+ Populations with Stem Cell Properties.

PubMed

Tome-Garcia, Jessica; Tejero, Rut; Nudelman, German; Yong, Raymund L; Sebra, Robert; Wang, Huaien; Fowkes, Mary; Magid, Margret; Walsh, Martin; Silva-Vargas, Violeta; Zaslavsky, Elena; Friedel, Roland H; Doetsch, Fiona; Tsankova, Nadejda M

2017-05-09

Characterization of non-neoplastic and malignant human stem cell populations in their native state can provide new insights into gliomagenesis. Here we developed a purification strategy to directly isolate EGFR +/- populations from human germinal matrix (GM) and adult subventricular zone autopsy tissues, and from de novo glioblastoma (GBM) resections, enriching for cells capable of binding EGF ligand ( LB EGFR + ), and uniquely compared their functional and molecular properties. LB EGFR + populations in both GM and GBM encompassed all sphere-forming cells and displayed proliferative stem cell properties in vitro. In xenografts, LB EGFR + GBM cells showed robust tumor initiation and progression to high-grade, infiltrative gliomas. Whole-transcriptome sequencing analysis confirmed enrichment of proliferative pathways in both developing and neoplastic freshly isolated EGFR + populations, and identified both unique and shared sets of genes. The ability to prospectively isolate stem cell populations using native ligand-binding capacity opens new doors onto understanding both normal human development and tumor cell biology. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

TCH1036, a indeno[1,2-c]quinoline derivative, potentially inhibited the growth of human brain malignant glioma (GBM) 8401 cells via suppression of the expression of Suv39h1 and PARP.

PubMed

Liao, Hsien-Feng; Lee, Chien-Chin; Hsiao, Pei-Chi; Chen, Yi-Fong; Tseng, Chih-Hua; Tzeng, Cherng-Chyi; Chen, Yeh-Long; Chen, Jui-Chang; Chang, Ya-Sian; Chang, Jan-Gowth

2016-08-01

A newly synthesized Indeno[1,2-c]quinoline derivative, which has previously been found to potentially trap DNA-topoisomerase cleavage complexes more effectively than camptothecin, could effectively inhibit the proliferation of a variety of cancers, such as breast cancer treated with TCH1030. In this study, we further explore the activity of the TCH1036, TCH1259 and TCH1030 compounds in suppressing the growth of human brain malignant glioma (GBM) 8401 cells, in addition to elucidating the related mechanisms. According to tests of cytotoxicity, the GBM cells were more sensitive to the inhibitory effects of the TCH1036 compound than to those of the other two compounds. Moreover, the accumulation of GBM cells in the sub-G1 and G2/M phases was clearly induced by the TCH1036 compound in a dose-dependent manner. A screening of the majority of histone-modifier enzymes indicated that the expression of Suv39h1 in the GBM cells was attenuated by treatment with each of the TCH compounds, an observation which was further confirmed by Western blotting. The increase in active-form caspase 3 in the GBM cells treated with TCH compounds caused a high degree of poly (ADP-ribose) polymerase (PARP) cleavage and also enhanced the high ratio of hypodiploid GBM cells in the sub-G1 phase. In molecular docking simulations, it was observed that the stable forms of the TCH compounds could successfully insert into the catalytic pocket of PARP, with the highest affinity being between PARP and the TCH1036 compound. These findings suggested that the TCH1036 compound would be a promising compound in the treatment of brain malignant glioma. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Virotherapy of the Malignant U87 Human Glioblastoma in the Orthotopic Xenotransplantation Mouse SCID Model.

PubMed

Shchelkunov, S N; Razumov, I A; Kolosova, I V; Romashchenko, A V; Zavjalov, E L

2018-01-01

The possibility of glioblastoma virotherapy at intravenous injection of the LIVP-GFP recombinant virus was studied in experimental model of orthotopic xenotransplantation of human glioblastoma cell line U87 to SCID laboratory mice. The LIVP-GFP recombinant virus deficient for thymidine kinase exhibited a significantly greater oncolytic capacity than the original LIVP virus, and an intravenous injection of LIVP-GFP at the early stages of tumorigenesis in mouse brain in most cases resulted in the lysis of the tumor.

Dose-dense temozolomide for newly diagnosed glioblastoma: a randomized phase III clinical trial.

PubMed

Gilbert, Mark R; Wang, Meihua; Aldape, Kenneth D; Stupp, Roger; Hegi, Monika E; Jaeckle, Kurt A; Armstrong, Terri S; Wefel, Jeffrey S; Won, Minhee; Blumenthal, Deborah T; Mahajan, Anita; Schultz, Christopher J; Erridge, Sara; Baumert, Brigitta; Hopkins, Kristen I; Tzuk-Shina, Tzahala; Brown, Paul D; Chakravarti, Arnab; Curran, Walter J; Mehta, Minesh P

2013-11-10

Radiotherapy with concomitant and adjuvant temozolomide is the standard of care for newly diagnosed glioblastoma (GBM). O(6)-methylguanine-DNA methyltransferase (MGMT) methylation status may be an important determinant of treatment response. Dose-dense (DD) temozolomide results in prolonged depletion of MGMT in blood mononuclear cells and possibly in tumor. This trial tested whether DD temozolomide improves overall survival (OS) or progression-free survival (PFS) in patients with newly diagnosed GBM. This phase III trial enrolled patients older than age 18 years with a Karnofsky performance score of ≥ 60 with adequate tissue. Stratification included clinical factors and tumor MGMT methylation status. Patients were randomly assigned to standard temozolomide (arm 1) or DD temozolomide (arm 2) for 6 to 12 cycles. The primary end point was OS. Secondary analyses evaluated the impact of MGMT status. A total of 833 patients were randomly assigned to either arm 1 or arm 2 (1,173 registered). No statistically significant difference was observed between arms for median OS (16.6 v 14.9 months, respectively; hazard ratio [HR], 1.03; P = .63) or median PFS (5.5 v 6.7 months; HR, 0.87; P = .06). Efficacy did not differ by methylation status. MGMT methylation was associated with improved OS (21.2 v 14 months; HR, 1.74; P < .001), PFS (8.7 v 5.7 months; HR, 1.63; P < .001), and response (P = .012). There was increased grade ≥ 3 toxicity in arm 2 (34% v 53%; P < .001), mostly lymphopenia and fatigue. This study did not demonstrate improved efficacy for DD temozolomide for newly diagnosed GBM, regardless of methylation status. However, it did confirm the prognostic significance of MGMT methylation. Feasibility of large-scale accrual, prospective tumor collection, and molecular stratification was demonstrated.

Role for Macrophage Metalloelastase in Glomerular Basement Membrane Damage Associated with Alport Syndrome

PubMed Central

Rao, Velidi H.; Meehan, Daniel T.; Delimont, Duane; Nakajima, Motowo; Wada, Takashi; Ann Gratton, Michael; Cosgrove, Dominic

2006-01-01

Alport syndrome is a glomerular basement membrane (GBM) disease caused by mutations in type IV collagen genes. A unique irregular thickening and thinning of the GBM characterizes the progressive glomerular pathology. The metabolic imbalances responsible for these GBM irregularities are not known. Here we show that macrophage metalloelastase (MMP-12) expression is >40-fold induced in glomeruli from Alport mice and is markedly induced in glomeruli of both humans and dogs with Alport syndrome. Treatment of Alport mice with MMI270 (CGS27023A), a broad spectrum MMP inhibitor that blocks MMP-12 activity, results in largely restored GBM ultrastructure and function. Treatment with BAY-129566, a broad spectrum MMP inhibitor that does not inhibit MMP-12, had no effect. We show that inhibition of CC chemokine receptor 2 (CCR2) receptor signaling with propagermanium blocks induction of MMP-12 mRNA and prevents GBM damage. CCR2 receptor is expressed in glomerular podocytes of Alport mice, suggesting MCP-1 activation of CCR2 on podocytes may underlie induction of MMP-12. These data indicate that the irregular GBM that characterizes Alport syndrome may be mediated, in part, by focal degradation of the GBM due to MMP dysregulation, in particular, MMP-12. Thus, MMP-12/CCR2 inhibitors may provide a novel and effective therapeutic strategy for Alport glomerular disease. PMID:16816359

Efficient Mitochondrial Glutamine Targeting Prevails Over Glioblastoma Metabolic Plasticity.

PubMed

Oizel, Kristell; Chauvin, Cynthia; Oliver, Lisa; Gratas, Catherine; Geraldo, Fanny; Jarry, Ulrich; Scotet, Emmanuel; Rabe, Marion; Alves-Guerra, Marie-Clotilde; Teusan, Raluca; Gautier, Fabien; Loussouarn, Delphine; Compan, Vincent; Martinou, Jean-Claude; Vallette, François M; Pecqueur, Claire

2017-10-15

Purpose: Glioblastoma (GBM) is the most common and malignant form of primary human brain tumor in adults, with an average survival at diagnosis of 18 months. Metabolism is a new attractive therapeutic target in cancer; however, little is known about metabolic heterogeneity and plasticity within GBM tumors. We therefore aimed to investigate metabolic phenotyping of primary cultures in the context of molecular tumor heterogeneity to provide a proof of concept for personalized metabolic targeting of GBM. Experimental Design: We have analyzed extensively several primary GBM cultures using transcriptomics, metabolic phenotyping assays, and mitochondrial respirometry. Results: We found that metabolic phenotyping clearly identifies 2 clusters, GLN High and GLN Low , mainly based on metabolic plasticity and glutamine (GLN) utilization. Inhibition of glutamine metabolism slows the in vitro and in vivo growth of GLN High GBM cultures despite metabolic adaptation to nutrient availability, in particular by increasing pyruvate shuttling into mitochondria. Furthermore, phenotypic and molecular analyses show that highly proliferative GLN High cultures are CD133 neg and display a mesenchymal signature in contrast to CD133 pos GLN Low GBM cells. Conclusions: Our results show that metabolic phenotyping identified an essential metabolic pathway in a GBM cell subtype, and provide a proof of concept for theranostic metabolic targeting. Clin Cancer Res; 23(20); 6292-304. ©2017 AACR . ©2017 American Association for Cancer Research.

Cyclophilin B Supports Myc and Mutant p53 Dependent Survival of Glioblastoma Multiforme Cells

PubMed Central

Choi, Jae Won; Schroeder, Mark A.; Sarkaria, Jann N.; Bram, Richard J.

2014-01-01

Glioblastoma multiforme (GBM) is an aggressive, treatment-refractory type of brain tumor for which effective therapeutic targets remain important to identify. Here we report that cyclophilin B (CypB), a prolyl isomerase residing in the endoplasmic reticulum (ER), provides an essential survival signal in GBM cells. Analysis of gene expression databases revealed that CypB is upregulated in many cases of malignant glioma. We found that suppression of CypB reduced cell proliferation and survival in human GBM cells in vitro and in vivo. We also found that treatment with small molecule inhibitors of cyclophilins, including the approved drug cyclosporine, greatly reduced the viability of GBM cells. Mechanistically, depletion or pharmacologic inhibition of CypB caused hyperactivation of the oncogenic RAS-MAPK pathway, induction of cellular senescence signals, and death resulting from loss of MYC, mutant p53, Chk1 and JAK/STAT3 signaling. Elevated reactive oxygen species, ER expansion and abnormal unfolded protein responses in CypB-depleted GBM cells indicated that CypB alleviates oxidative and ER stresses and coordinates stress adaptation responses. Enhanced cell survival and sustained expression of multiple oncogenic proteins downstream of CypB may thus contribute to the poor outcome of GBM tumors. Our findings link chaperone-mediated protein folding in the ER to mechanisms underlying oncogenic transformation, and they make CypB an attractive and immediately targetable molecule for GBM therapy. PMID:24272483

Sulforaphane suppresses the growth of glioblastoma cells, glioblastoma stem cell-like spheroids, and tumor xenografts through multiple cell signaling pathways.

PubMed

Bijangi-Vishehsaraei, Khadijeh; Reza Saadatzadeh, M; Wang, Haiyan; Nguyen, Angie; Kamocka, Malgorzata M; Cai, Wenjing; Cohen-Gadol, Aaron A; Halum, Stacey L; Sarkaria, Jann N; Pollok, Karen E; Safa, Ahmad R

2017-12-01

OBJECTIVE Defects in the apoptotic machinery and augmented survival signals contribute to drug resistance in glioblastoma (GBM). Moreover, another complexity related to GBM treatment is the concept that GBM development and recurrence may arise from the expression of GBM stem cells (GSCs). Therefore, the use of a multifaceted approach or multitargeted agents that affect specific tumor cell characteristics will likely be necessary to successfully eradicate GBM. The objective of this study was to investigate the usefulness of sulforaphane (SFN)-a constituent of cruciferous vegetables with a multitargeted effect-as a therapeutic agent for GBM. METHODS The inhibitory effects of SFN on established cell lines, early primary cultures, CD133-positive GSCs, GSC-derived spheroids, and GBM xenografts were evaluated using various methods, including GSC isolation and the sphere-forming assay, analysis of reactive oxygen species (ROS) and apoptosis, cell growth inhibition assay, comet assays for assessing SFN-triggered DNA damage, confocal microscopy, Western blot analysis, and the determination of in vivo efficacy as assessed in human GBM xenograft models. RESULTS SFN triggered the significant inhibition of cell survival and induced apoptotic cell death, which was associated with caspase 3 and caspase 7 activation. Moreover, SFN triggered the formation of mitochondrial ROS, and SFN-triggered cell death was ROS dependent. Comet assays revealed that SFN increased single- and double-strand DNA breaks in GBM. Compared with the vehicle control cells, a significantly higher amount of γ-H2AX foci correlated with an increase in DNA double-strand breaks in the SFN-treated samples. Furthermore, SFN robustly inhibited the growth of GBM cell-induced cell death in established cell cultures and early-passage primary cultures and, most importantly, was effective in eliminating GSCs, which play a major role in drug resistance and disease recurrence. In vivo studies revealed that SFN administration at 100 mg/kg for 5-day cycles repeated for 3 weeks significantly decreased the growth of ectopic xenografts that were established from the early passage of primary cultures of GBM10. CONCLUSIONS These results suggest that SFN is a potent anti-GBM agent that targets several apoptosis and cell survival pathways and further preclinical and clinical studies may prove that SFN alone or in combination with other therapies may be potentially useful for GBM therapy.

EMMPRIN expression positively correlates with WHO grades of astrocytomas and meningiomas.

PubMed

Tsai, Wen-Chiuan; Chen, Ying; Huang, Li-Chun; Lee, Herng-Sheng; Ma, Hsin-I; Huang, Shih-Ming; Sytwu, Huey-Kang; Hueng, Dueng-Yuan

2013-09-01

High-grade primary brain tumors possessed poor outcome due to invasiveness. Extracellular matrix metalloproteinase inducer (EMMPRIN) stimulates peri-tumoral fibroblasts to secrete matrix metalloproteinase and promote invasiveness. This study hypothesized that high-grade brain tumors overexpress EMMPRIN. Analyzing the public delinked database from the Gene Expression Omnibus profile, the results showed that the EMMPRIN mRNA level was higher in WHO grade IV (n = 81) than in grade III (n = 19, p < 0.0005) astrocytomas and non-tumor brain tissue controls (n = 23, p < 0.00001). The results of tissue microarray-based immunohistochemical (IHC) staining revealed that EMMPRIN levels positively correlated with WHO grades for astrocytomas (p = 0.008) and meningiomas (p = 0.048). EMMPRIN mRNA levels in conventional glioma cell lines (n = 36) was not less than those in glioma primary culture cells (n = 27) and glioblastoma stem-like cells (n = 12). The GBM8401, U87MG, and LN229 human glioma cell lines also overexpressed EMMPRIN. Hematoxylin and eosin, IHC, and immunofluorescence staining of xenografts confirmed that high-grade brain tumors overexpressed EMMPRIN. Lastly, Kaplan-Meier analysis revealed poorer survival in WHO grade IV (n = 56) than in grade III astrocytomas (n = 21, by log-rank test; p = 0.0001, 95 % CI: 1.842-3.053). However, in high-grade astrocytomas, there was no difference in survival between high and low EMMPRIN mRNA levels. Thus, this study identified that high-grade brain tumors overexpress EMMPRIN, which positively correlates with WHO grades in human astrocytomas and meningiomas, and suggests that EMMPRIN may be a therapeutic target of brain tumor.

Bacoside A Induces Tumor Cell Death in Human Glioblastoma Cell Lines through Catastrophic Macropinocytosis

PubMed Central

John, Sebastian; Sivakumar, K. C.; Mishra, Rashmi

2017-01-01

Glioblastoma multiforme (GBM) is a highly aggressive type of brain tumor with an extremely poor prognosis. Recent evidences have shown that the “biomechanical imbalances” induced in GBM patient-derived glioblastoma cells (GC) and in vivo via the administration of synthetic small molecules, may effectively inhibit disease progression and prolong survival of GBM animal models. This novel concept associated with de novo anti-GBM drug development has however suffered obstacles in adequate clinical utility due to the appearance of unrelated toxicity in the prolonged therapeutic windows. Here, we took a “drug repurposing approach” to trigger similar physico-chemical disturbances in the GBM tumor cells, wherein, the candidate therapeutic agent has been previously well established for its neuro-protective roles, safety, efficacy, prolonged tolerance and excellent brain bioavailability in human subjects and mouse models. In this study, we show that the extracts of an Indian traditional medicinal plant Bacopa monnieri (BM) and its bioactive component Bacoside A can generate dosage associated tumor specific disturbances in the hydrostatic pressure balance of the cell via a mechanism involving excessive phosphorylation of calcium/calmodulin-dependent protein kinase IIA (CaMKIIA/CaMK2A) enzyme that is further involved in the release of calcium from the smooth endoplasmic reticular networks. High intracellular calcium stimulated massive macropinocytotic extracellular fluid intake causing cell hypertrophy in the initial stages, excessive macropinosome enlargement and fluid accumulation associated organellar congestion, cell swelling, cell rounding and membrane rupture of glioblastoma cells; with all these events culminating into a non-apoptotic, physical non-homeostasis associated glioblastoma tumor cell death. These results identify glioblastoma tumor cells to be a specific target of the tested herbal medicine and therefore can be exploited as a safe anti-GBM therapeutic. PMID:28663722

Bacoside A Induces Tumor Cell Death in Human Glioblastoma Cell Lines through Catastrophic Macropinocytosis.

PubMed

John, Sebastian; Sivakumar, K C; Mishra, Rashmi

2017-01-01

Glioblastoma multiforme (GBM) is a highly aggressive type of brain tumor with an extremely poor prognosis. Recent evidences have shown that the "biomechanical imbalances" induced in GBM patient-derived glioblastoma cells (GC) and in vivo via the administration of synthetic small molecules, may effectively inhibit disease progression and prolong survival of GBM animal models. This novel concept associated with de novo anti-GBM drug development has however suffered obstacles in adequate clinical utility due to the appearance of unrelated toxicity in the prolonged therapeutic windows. Here, we took a "drug repurposing approach" to trigger similar physico-chemical disturbances in the GBM tumor cells, wherein, the candidate therapeutic agent has been previously well established for its neuro-protective roles, safety, efficacy, prolonged tolerance and excellent brain bioavailability in human subjects and mouse models. In this study, we show that the extracts of an Indian traditional medicinal plant Bacopa monnieri (BM) and its bioactive component Bacoside A can generate dosage associated tumor specific disturbances in the hydrostatic pressure balance of the cell via a mechanism involving excessive phosphorylation of calcium/calmodulin-dependent protein kinase IIA (CaMKIIA/CaMK2A) enzyme that is further involved in the release of calcium from the smooth endoplasmic reticular networks. High intracellular calcium stimulated massive macropinocytotic extracellular fluid intake causing cell hypertrophy in the initial stages, excessive macropinosome enlargement and fluid accumulation associated organellar congestion, cell swelling, cell rounding and membrane rupture of glioblastoma cells; with all these events culminating into a non-apoptotic, physical non-homeostasis associated glioblastoma tumor cell death. These results identify glioblastoma tumor cells to be a specific target of the tested herbal medicine and therefore can be exploited as a safe anti-GBM therapeutic.

Targeted Proteomics to Assess the Response to Anti-Angiogenic Treatment in Human Glioblastoma (GBM).

PubMed

Demeure, Kevin; Fack, Fred; Duriez, Elodie; Tiemann, Katja; Bernard, Amandine; Golebiewska, Anna; Bougnaud, Sébastien; Bjerkvig, Rolf; Domon, Bruno; Niclou, Simone P

2016-02-01

Glioblastoma (GBM) is a highly aggressive primary brain tumor with dismal outcome for affected patients. Because of the significant neo-angiogenesis exhibited by GBMs, anti-angiogenic therapies have been intensively evaluated during the past years. Recent clinical studies were however disappointing, although a subpopulation of patients may benefit from such treatment. We have previously shown that anti-angiogenic targeting in GBM increases hypoxia and leads to a metabolic adaptation toward glycolysis, suggesting that combination treatments also targeting the glycolytic phenotype may be effective in GBM patients. The aim of this study was to identify marker proteins that are altered by treatment and may serve as a short term readout of anti-angiogenic therapy. Ultimately such proteins could be tested as markers of efficacy able to identify patient subpopulations responsive to the treatment. We applied a proteomics approach based on selected reaction monitoring (SRM) to precisely quantify targeted protein candidates, selected from pathways related to metabolism, apoptosis and angiogenesis. The workflow was developed in the context of patient-derived intracranial GBM xenografts developed in rodents and ensured the specific identification of human tumor versus rodent stroma-derived proteins. Quality control experiments were applied to assess sample heterogeneity and reproducibility of SRM assays at different levels. The data demonstrate that tumor specific proteins can be precisely quantified within complex biological samples, reliably identifying small concentration differences induced by the treatment. In line with previous work, we identified decreased levels of TCA cycle enzymes, including isocitrate dehydrogenase, whereas malectin, calnexin, and lactate dehydrogenase A were augmented after treatment. We propose the most responsive proteins of our subset as potential novel biomarkers to assess treatment response after anti-angiogenic therapy that warrant future analysis in clinical GBM samples. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Human fibulin-3 protein variant expresses anti-cancer effects in the malignant glioma extracellular compartment in intracranial xenograft models

PubMed Central

Li, Yanyan; Hu, Yuan; Liu, Chuanjin; Wang, Qingyue; Han, Xiaoxiao; Han, Yong; Xie, Xue-Shun; Chen, Xiong-Hui; Li, Xiang; Siegel, Eric R.; Afrasiabi, Kambiz; Linskey, Mark E.; Zhou, You-Xin; Zhou, Yi-Hong

2017-01-01

Background Decades of cytotoxic and more recently immunotherapy treatments for malignant glioma have had limited success due to dynamic intra-tumoral heterogeneity. The dynamic interplay of cancer cell subpopulations has been found to be under the control of proteins in the cancer microenvironment. EGF-containing fibulin-like extracellular matrix protein (EFEMP1) (also fibulin-3) has the multiple functions of suppressing cancer growth and angiogenesis, while promoting cancer cell invasion. EFEMP1-derived tumor suppressor protein (ETSP) retains EFEMP1’s anti-growth and anti-angiogenic functions while actually inhibiting cancer cell invasion. Methods In this study, we examined the therapeutic effect on glioblastoma multiforme (GBM) of an in vitro synthesized protein, ZR30, which is based on the sequence of ETSP, excluding the signaling peptide. Results ZR30 showed the same effects as ETSP in blocking EGFR/NOTCH/AKT signaling pathways, when applied to cultures of multiple GBM cell lines and primary cultures. ZR30’s inhibition of MMP2 activation was shown not only for GBM cells, but also for other types of cancer cells having overexpression of MMP2. A significant improvement in survival of mice with orthotopic human GBM xenografts was observed after a single, intra-tumoral injection of ZR30. Using a model mimicking the intra-tumoral heterogeneity of GBM with cell subpopulations carrying different invasive and proliferative phenotypes, we demonstrated an equal and simultaneous tumor suppressive effect of ZR30 on both tumor cell subpopulations, with suppression of FOXM1 and activation of SEMA3B expressions in the xenografts. Conclusion Overall, the data support a complementary pleiotrophic therapeutic effect of ZR30 acting in the extracellular compartment of GBM. PMID:29290950

Targeted Proteomics to Assess the Response to Anti-Angiogenic Treatment in Human Glioblastoma (GBM)*

PubMed Central

Demeure, Kevin; Fack, Fred; Duriez, Elodie; Tiemann, Katja; Bernard, Amandine; Golebiewska, Anna; Bougnaud, Sébastien; Bjerkvig, Rolf; Domon, Bruno; Niclou, Simone P.

2016-01-01

Glioblastoma (GBM) is a highly aggressive primary brain tumor with dismal outcome for affected patients. Because of the significant neo-angiogenesis exhibited by GBMs, anti-angiogenic therapies have been intensively evaluated during the past years. Recent clinical studies were however disappointing, although a subpopulation of patients may benefit from such treatment. We have previously shown that anti-angiogenic targeting in GBM increases hypoxia and leads to a metabolic adaptation toward glycolysis, suggesting that combination treatments also targeting the glycolytic phenotype may be effective in GBM patients. The aim of this study was to identify marker proteins that are altered by treatment and may serve as a short term readout of anti-angiogenic therapy. Ultimately such proteins could be tested as markers of efficacy able to identify patient subpopulations responsive to the treatment. We applied a proteomics approach based on selected reaction monitoring (SRM) to precisely quantify targeted protein candidates, selected from pathways related to metabolism, apoptosis and angiogenesis. The workflow was developed in the context of patient-derived intracranial GBM xenografts developed in rodents and ensured the specific identification of human tumor versus rodent stroma-derived proteins. Quality control experiments were applied to assess sample heterogeneity and reproducibility of SRM assays at different levels. The data demonstrate that tumor specific proteins can be precisely quantified within complex biological samples, reliably identifying small concentration differences induced by the treatment. In line with previous work, we identified decreased levels of TCA cycle enzymes, including isocitrate dehydrogenase, whereas malectin, calnexin, and lactate dehydrogenase A were augmented after treatment. We propose the most responsive proteins of our subset as potential novel biomarkers to assess treatment response after anti-angiogenic therapy that warrant future analysis in clinical GBM samples. PMID:26243272

Dual targeting of glioblastoma with chimeric antigen receptor-engineered natural killer cells overcomes heterogeneity of target antigen expression and enhances antitumor activity and survival.

PubMed

Genßler, Sabrina; Burger, Michael C; Zhang, Congcong; Oelsner, Sarah; Mildenberger, Iris; Wagner, Marlies; Steinbach, Joachim P; Wels, Winfried S

2016-04-01

Epidermal growth factor receptor (EGFR) and its mutant form EGFRvIII are overexpressed in a large proportion of glioblastomas (GBM). Immunotherapy with an EGFRvIII-specific vaccine has shown efficacy against GBM in clinical studies. However, immune escape by antigen-loss variants and lack of control of EGFR wild-type positive clones limit the usefulness of this approach. Chimeric antigen receptor (CAR)-engineered natural killer (NK) cells may represent an alternative immunotherapeutic strategy. For targeting to GBM, we generated variants of the clinically applicable human NK cell line NK-92 that express CARs carrying a composite CD28-CD3ζ domain for signaling, and scFv antibody fragments for cell binding either recognizing EGFR, EGFRvIII, or an epitope common to both antigens. In vitro analysis revealed high and specific cytotoxicity of EGFR-targeted NK-92 against established and primary human GBM cells, which was dependent on EGFR expression and CAR signaling. EGFRvIII-targeted NK-92 only lysed EGFRvIII-positive GBM cells, while dual-specific NK cells expressing a cetuximab-based CAR were active against both types of tumor cells. In immunodeficient mice carrying intracranial GBM xenografts either expressing EGFR, EGFRvIII or both receptors, local treatment with dual-specific NK cells was superior to treatment with the corresponding monospecific CAR NK cells. This resulted in a marked extension of survival without inducing rapid immune escape as observed upon therapy with monospecific effectors. Our results demonstrate that dual targeting of CAR NK cells reduces the risk of immune escape and suggest that EGFR/EGFRvIII-targeted dual-specific CAR NK cells may have potential for adoptive immunotherapy of glioblastoma.

Cytotoxic effect of disulfiram/copper on human glioblastoma cell lines and ALDH-positive cancer-stem-like cells

PubMed Central

Liu, P; Brown, S; Goktug, T; Channathodiyil, P; Kannappan, V; Hugnot, J-P; Guichet, P-O; Bian, X; Armesilla, A L; Darling, J L; Wang, W

2012-01-01

Background: Glioblastoma multiforme (GBM) cells are resistant to anticancer drugs. Cancer stem cells (CSCs) are a key mediator of chemoresistance. We have reported that disulfiram (DS), an aldehyde dehydrogenase (ALDH) inhibitor, targets breast CSC-like cells. In this study, the effect of DS and combination of DS and gemcitabine (dFdC) on GBM cells and GBM stem-like cells was investigated. Methods: 1-(4,5-Dimethylthiazol-2-yl)-3,5-diphenylformazan (MTT), combination index (CI)-isobologram, western blot, luciferase reporter gene assay, electrophoretic mobility-shift assay and ALDH analysis were used in this study. Results: Disulfiram is cytotoxic in GBM cell lines in a copper (Cu)-dependent manner. Disulfiram/copper enhances the cytotoxicity of dFdC. Combination index-isobologram analysis indicates a synergistic effect between DS/Cu and dFdC. Disulfiram/copper induces reactive oxygen species (ROS), activates JNK and p38 pathways and inhibits nuclear factor-kappa B activity in GBM cell lines. Disulfiram/copper may trigger intrinsic apoptotic pathway via modulation of the Bcl2 family. Disulfiram/copper abolishes stem-like cell population in GBM cell lines. Conclusion: Our findings indicate that the cytotoxicity of DS/Cu and the enhancing effect of DS/Cu on the cytotoxicity of dFdC in GBM stem-like cells may be caused by induction of ROS and inhibition of both ALDH and the NFkB pathway. Both DS and dFdC can traverse the blood–brain barrier. Further study may lead them into GBM chemotherapy. PMID:23033007

A novel NFIA-NFκB feed-forward loop contributes to glioblastoma cell survival

PubMed Central

Lee, JunSung; Hoxha, Edlira

2017-01-01

Abstract Background. The nuclear factor I-A (NFIA) transcription factor promotes glioma growth and inhibits apoptosis in glioblastoma (GBM) cells. Here we report that the NFIA pro-survival effect in GBM is mediated in part via a novel NFIA–nuclear factor-kappaB (NFκB) p65 feed-forward loop. Methods. We examined effects of gain- and loss-of-function manipulations of NFIA and NFκB p65 on each other’s transcription, cell growth, apoptosis and sensitivity to chemotherapy in patient-derived GBM cells and established GBM cell lines. Results. NFIA enhanced apoptosis evasion by activating NFκB p65 and its downstream anti-apoptotic factors tumor necrosis factor receptor-associated factor 1 (TRAF1) and cellular inhibitor of apoptosis proteins (cIAPs). Induction of NFκB by NFIA was required to protect cells from apoptosis, and inhibition of NFκB effectively reversed the NFIA anti-apoptotic effect. Conversely, NFIA knockdown decreased expression of NFκB and anti-apoptotic genes TRAF1 and cIAPs, and increased baseline apoptosis. NFIA positively regulated NFκB transcription and NFκB protein level. Interestingly, NFκB also activated the NFIA promoter and increased NFIA level, and knockdown of NFIA was sufficient to attenuate the NFκB pro-survival effect, suggesting a reciprocal regulation between NFIA and NFκB in governing GBM cell survival. Supporting this, NFIA and NFκB expression levels were highly correlated in human GBM and patient-derived GBM cells. Conclusions. These data define a previously unknown NFIA-NFκB feed-forward regulation that may contribute to GBM cell survival. PMID:27994064

A single dose of peripherally infused EGFRvIII-directed CAR T cells mediates antigen loss and induces adaptive resistance in patients with recurrent glioblastoma.

PubMed

O'Rourke, Donald M; Nasrallah, MacLean P; Desai, Arati; Melenhorst, Jan J; Mansfield, Keith; Morrissette, Jennifer J D; Martinez-Lage, Maria; Brem, Steven; Maloney, Eileen; Shen, Angela; Isaacs, Randi; Mohan, Suyash; Plesa, Gabriela; Lacey, Simon F; Navenot, Jean-Marc; Zheng, Zhaohui; Levine, Bruce L; Okada, Hideho; June, Carl H; Brogdon, Jennifer L; Maus, Marcela V

2017-07-19

We conducted a first-in-human study of intravenous delivery of a single dose of autologous T cells redirected to the epidermal growth factor receptor variant III (EGFRvIII) mutation by a chimeric antigen receptor (CAR). We report our findings on the first 10 recurrent glioblastoma (GBM) patients treated. We found that manufacturing and infusion of CAR-modified T cell (CART)-EGFRvIII cells are feasible and safe, without evidence of off-tumor toxicity or cytokine release syndrome. One patient has had residual stable disease for over 18 months of follow-up. All patients demonstrated detectable transient expansion of CART-EGFRvIII cells in peripheral blood. Seven patients had post-CART-EGFRvIII surgical intervention, which allowed for tissue-specific analysis of CART-EGFRvIII trafficking to the tumor, phenotyping of tumor-infiltrating T cells and the tumor microenvironment in situ, and analysis of post-therapy EGFRvIII target antigen expression. Imaging findings after CART immunotherapy were complex to interpret, further reinforcing the need for pathologic sampling in infused patients. We found trafficking of CART-EGFRvIII cells to regions of active GBM, with antigen decrease in five of these seven patients. In situ evaluation of the tumor environment demonstrated increased and robust expression of inhibitory molecules and infiltration by regulatory T cells after CART-EGFRvIII infusion, compared to pre-CART-EGFRvIII infusion tumor specimens. Our initial experience with CAR T cells in recurrent GBM suggests that although intravenous infusion results in on-target activity in the brain, overcoming the adaptive changes in the local tumor microenvironment and addressing the antigen heterogeneity may improve the efficacy of EGFRvIII-directed strategies in GBM. Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Transgenic nude mouse with green fluorescent protein expression-based human glioblastoma multiforme animal model with EGFR expression and invasiveness.

PubMed

Tan, Guo-Wei; Lan, Fo-Lin; Gao, Jian-Guo; Jiang, Cai-Mou; Zhang, Yi; Huang, Xiao-Hong; Ma, Yue-Hong; Shao, He-Dui; He, Xue-Yang; Chen, Jin-Long; Long, Jian-Wu; Xiao, Hui-Sheng; Guo, Zhi-Tong; Diao, Yi

2012-08-01

Previously, we developed an orthotopic xenograft model of human glioblastoma multiforme (GBM) with high EGFR expression and invasiveness in Balb/c nu/nu nude mice. Now we also developed the same orthotopic xenograft model in transgenic nude mice with green fluorescent protein (GFP) expression. The present orthotopic xenografts labeled by phycoerythrin fluorescing red showed high EGFR expression profile, and invasive behavior under a bright green-red dual-color fluorescence background. A striking advantage in the present human GBM model is that the change of tumor growth can be observed visually instead of sacrificing animals in our further antitumor therapy studies.

Osthole suppresses the proliferation and accelerates the apoptosis of human glioma cells via the upregulation of microRNA-16 and downregulation of MMP-9.

PubMed

Lin, Kai; Gao, Zhiyu; Shang, Bin; Sui, Shaohua; Fu, Qiang

2015-09-01

Osthole (7-methoxy-8-isoamyl alkenyl coumarin) has been reported to exhibit marked anticancer effects on several types of cancer. The expression levels of matrix metalloproteinase-9 (MMP-9) are closely associated with the pathogenesis of glioma. Furthermore, it is reported that the upregulation of microRNA‑16 (miR‑16) by the MMP‑9 signaling pathway can restrain the proliferation of cancer cells. To examine whether osthole increases the anticancer effect on human glioma cells in the present study, the common glioma cell line, U87, was treated with osthole at concentrations of 0, 50, 100 and 200 µΜ. The effects of osthole on cell viability were determined using a 3‑(4,5‑dimethylthiazol‑2‑thiazolyl)‑2,5‑diphenyl‑tetrazolium bromide assay. The rate of cellular apoptosis was analyzed by measuring the activity of caspase‑3 and using flow cytometry. The expression of MMP‑9 was determined using gelatin zymography assays and the expression of miR‑16 was determined using reverse transcription‑quantitative polymerase chain reaction. The results demonstrated that osthole significantly suppressed the proliferation and accelerated the apoptosis of the U87 cells. Furthermore, increased expression levels of miR‑16 and reduced protein expression levels of MMP‑9 were found in the U87 cells. In addition, miR‑16 was found to regulate the expression of MMP‑9 in the U87 cells through transfection of miR‑16 precursor and anti‑miR‑16 into the U87 cells. In conclusion, these observations indicated that osthole suppressed the proliferation and accelerated the apoptosis of human glioma cells through upregulation of the expression of miR‑16 and downregulation of the expression of MMP-9.

A Heterogeneous Network Based Method for Identifying GBM-Related Genes by Integrating Multi-Dimensional Data.

PubMed

Chen Peng; Ao Li

2017-01-01

The emergence of multi-dimensional data offers opportunities for more comprehensive analysis of the molecular characteristics of human diseases and therefore improving diagnosis, treatment, and prevention. In this study, we proposed a heterogeneous network based method by integrating multi-dimensional data (HNMD) to identify GBM-related genes. The novelty of the method lies in that the multi-dimensional data of GBM from TCGA dataset that provide comprehensive information of genes, are combined with protein-protein interactions to construct a weighted heterogeneous network, which reflects both the general and disease-specific relationships between genes. In addition, a propagation algorithm with resistance is introduced to precisely score and rank GBM-related genes. The results of comprehensive performance evaluation show that the proposed method significantly outperforms the network based methods with single-dimensional data and other existing approaches. Subsequent analysis of the top ranked genes suggests they may be functionally implicated in GBM, which further corroborates the superiority of the proposed method. The source code and the results of HNMD can be downloaded from the following URL: http://bioinformatics.ustc.edu.cn/hnmd/ .

A Nanoparticle Carrying the p53 Gene Targets Tumors Including Cancer Stem Cells, Sensitizes Glioblastoma to Chemotherapy and Improves Survival

PubMed Central

2015-01-01

Temozolomide (TMZ)-resistance in glioblastoma multiforme (GBM) has been linked to upregulation of O6-methylguanine-DNA methyltransferase (MGMT). Wild-type (wt) p53 was previously shown to down-modulate MGMT. However, p53 therapy for GBM is limited by lack of efficient delivery across the blood brain barrier (BBB). We have developed a systemic nanodelivery platform (scL) for tumor-specific targeting (primary and metastatic), which is currently in multiple clinical trials. This self-assembling nanocomplex is formed by simple mixing of the components in a defined order and a specific ratio. Here, we demonstrate that scL crosses the BBB and efficiently targets GBM, as well as cancer stem cells (CSCs), which have been implicated in recurrence and treatment resistance in many human cancers. Moreover, systemic delivery of scL-p53 down-modulates MGMT and induces apoptosis in intracranial GBM xenografts. The combination of scL-p53 and TMZ increased the antitumor efficacy of TMZ with enhanced survival benefit in a mouse model of highly TMZ-resistant GBM. scL-p53 also sensitized both CSCs and bulk tumor cells to TMZ, increasing apoptosis. These results suggest that combining scL-p53 with standard TMZ treatment could be a more effective therapy for GBM. PMID:24811110

c-Met–mediated endothelial plasticity drives aberrant vascularization and chemoresistance in glioblastoma

PubMed Central

Huang, Menggui; Liu, Tianrun; Ma, Peihong; Mitteer, R. Alan; Zhang, Zhenting; Kim, Hyun Jun; Yeo, Eujin; Zhang, Duo; Cai, Peiqiang; Li, Chunsheng; Zhang, Lin; Zhao, Botao; Roccograndi, Laura; O’Rourke, Donald M.; Dahmane, Nadia; Gong, Yanqing; Koumenis, Constantinos

2016-01-01

Aberrant vascularization is a hallmark of cancer progression and treatment resistance. Here, we have shown that endothelial cell (EC) plasticity drives aberrant vascularization and chemoresistance in glioblastoma multiforme (GBM). By utilizing human patient specimens, as well as allograft and genetic murine GBM models, we revealed that a robust endothelial plasticity in GBM allows acquisition of fibroblast transformation (also known as endothelial mesenchymal transition [Endo-MT]), which is characterized by EC expression of fibroblast markers, and determined that a prominent population of GBM-associated fibroblast-like cells have EC origin. Tumor ECs acquired the mesenchymal gene signature without the loss of EC functions, leading to enhanced cell proliferation and migration, as well as vessel permeability. Furthermore, we identified a c-Met/ETS-1/matrix metalloproteinase–14 (MMP-14) axis that controls VE-cadherin degradation, Endo-MT, and vascular abnormality. Pharmacological c-Met inhibition induced vessel normalization in patient tumor–derived ECs. Finally, EC-specific KO of Met inhibited vascular transformation, normalized blood vessels, and reduced intratumoral hypoxia, culminating in suppressed tumor growth and prolonged survival in GBM-bearing mice after temozolomide treatment. Together, these findings illustrate a mechanism that controls aberrant tumor vascularization and suggest that targeting Endo-MT may offer selective and efficient strategies for antivascular and vessel normalization therapies in GBM, and possibly other malignant tumors. PMID:27043280

The small molecule SI113 synergizes with mitotic spindle poisons in arresting the growth of human glioblastoma multiforme

PubMed Central

Abbruzzese, Claudia; Catalogna, Giada; Gallo, Enzo; di Martino, Simona; Mileo, Anna M.; Carosi, Mariantonia; Dattilo, Vincenzo; Schenone, Silvia; Musumeci, Francesca; Lavia, Patrizia; Perrotti, Nicola; Amato, Rosario; Paggi, Marco G.

2017-01-01

Glioblastoma multiforme (GBM) is the deadliest brain tumor. State-of-art GBM therapy often fails to ensure control of a disease characterized by high frequency of recurrences and progression. In search for novel therapeutic approaches, we assayed the effect of compounds from a cancer drug library on the ADF GBM cell line, establishing their elevated sensitivity to mitotic spindle poisons. Our previous work showed that the effectiveness of the spindle poison paclitaxel in inhibiting cancer cell growth was dependent on the expression of RANBP1, a regulatory target of the serine/threonine kinase SGK1. Recently, we developed the small molecule SI113 to inhibit SGK1 activity. Therefore, we explored the outcome of the association between SI113 and selected spindle poisons, finding that these drugs generated a synergistic cytotoxic effect in GBM cells, drastically reducing their viability and clonogenic capabilities in vitro, as well as inhibiting tumor growth in vivo. We also defined the molecular bases of such a synergistic effect. Because SI113 displays low systemic toxicity, yet strong activity in potentiating the effect of radiotherapy in GBM cells, we believe that this drug could be a strong candidate for clinical trials, with the aim to add it to the current GBM therapeutic approaches. PMID:29340013

The small molecule SI113 synergizes with mitotic spindle poisons in arresting the growth of human glioblastoma multiforme.

PubMed

Abbruzzese, Claudia; Catalogna, Giada; Gallo, Enzo; di Martino, Simona; Mileo, Anna M; Carosi, Mariantonia; Dattilo, Vincenzo; Schenone, Silvia; Musumeci, Francesca; Lavia, Patrizia; Perrotti, Nicola; Amato, Rosario; Paggi, Marco G

2017-12-19

Glioblastoma multiforme (GBM) is the deadliest brain tumor. State-of-art GBM therapy often fails to ensure control of a disease characterized by high frequency of recurrences and progression. In search for novel therapeutic approaches, we assayed the effect of compounds from a cancer drug library on the ADF GBM cell line, establishing their elevated sensitivity to mitotic spindle poisons. Our previous work showed that the effectiveness of the spindle poison paclitaxel in inhibiting cancer cell growth was dependent on the expression of RANBP1, a regulatory target of the serine/threonine kinase SGK1. Recently, we developed the small molecule SI113 to inhibit SGK1 activity. Therefore, we explored the outcome of the association between SI113 and selected spindle poisons, finding that these drugs generated a synergistic cytotoxic effect in GBM cells, drastically reducing their viability and clonogenic capabilities in vitro , as well as inhibiting tumor growth in vivo . We also defined the molecular bases of such a synergistic effect. Because SI113 displays low systemic toxicity, yet strong activity in potentiating the effect of radiotherapy in GBM cells, we believe that this drug could be a strong candidate for clinical trials, with the aim to add it to the current GBM therapeutic approaches.

RSK2 activity mediates glioblastoma invasiveness and is a potential target for new therapeutics.

PubMed

Sulzmaier, Florian J; Young-Robbins, Shirley; Jiang, Pengfei; Geerts, Dirk; Prechtl, Amanda M; Matter, Michelle L; Kesari, Santosh; Ramos, Joe W

2016-11-29

In glioblastoma (GBM), infiltration of primary tumor cells into the normal tissue and dispersal throughout the brain is a central challenge to successful treatment that remains unmet. Indeed, patients respond poorly to the current therapies of tumor resection followed by chemotherapy with radiotherapy and have only a 16-month median survival. It is therefore imperative to develop novel therapies. RSK2 is a kinase that regulates proliferation and adhesion and can promote metastasis. We demonstrate that active RSK2 regulates GBM cell adhesion and is essential for cell motility and invasion of patient-derived GBM neurospheres. RSK2 control of adhesion and migration is mediated in part by its effects on integrin-Filamin A complexes. Importantly, inhibition of RSK2 by either RSK inhibitors or shRNA silencing impairs invasion and combining RSK2 inhibitors with temozolomide improves efficacy in vitro. In agreement with the in vitro data, using public datasets, we find that RSK2 is significantly upregulated in vivo in human GBM patient tumors, and that high RSK2 expression significantly correlates with advanced tumor stage and poor patient survival. Together, our data provide strong evidence that RSK inhibitors could enhance the effectiveness of existing GBM treatment, and support RSK2 targeting as a promising approach for novel GBM therapy.

Vascular targeting of LIGHT normalizes blood vessels in primary brain cancer and induces intratumoural high endothelial venules.

PubMed

He, Bo; Jabouille, Arnaud; Steri, Veronica; Johansson-Percival, Anna; Michael, Iacovos P; Kotamraju, Venkata Ramana; Junckerstorff, Reimar; Nowak, Anna K; Hamzah, Juliana; Lee, Gabriel; Bergers, Gabriele; Ganss, Ruth

2018-06-01

High-grade brain cancer such as glioblastoma (GBM) remains an incurable disease. A common feature of GBM is the angiogenic vasculature, which can be targeted with selected peptides for payload delivery. We assessed the ability of micelle-tagged, vascular homing peptides RGR, CGKRK and NGR to specifically bind to blood vessels in syngeneic orthotopic GBM models. By using the peptide CGKRK to deliver the tumour necrosis factor (TNF) superfamily member LIGHT (also known as TNF superfamily member 14; TNFSF14) to angiogenic tumour vessels, we have generated a reagent that normalizes the brain cancer vasculature by inducing pericyte contractility and re-establishing endothelial barrier integrity. LIGHT-mediated vascular remodelling also activates endothelia and induces intratumoural high endothelial venules (HEVs), which are specialized blood vessels for lymphocyte infiltration. Combining CGKRK-LIGHT with anti-vascular endothelial growth factor and checkpoint blockade amplified HEV frequency and T-cell accumulation in GBM, which is often sparsely infiltrated by immune effector cells, and reduced tumour burden. Furthermore, CGKRK and RGR peptides strongly bound to blood vessels in freshly resected human GBM, demonstrating shared peptide-binding activities in mouse and human primary brain tumour vessels. Thus, peptide-mediated LIGHT targeting is a highly translatable approach in primary brain cancer to reduce vascular leakiness and enhance immunotherapy. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Accelerating glioblastoma drug discovery: Convergence of patient-derived models, genome editing and phenotypic screening.

PubMed

O'Duibhir, Eoghan; Carragher, Neil O; Pollard, Steven M

2017-04-01

Patients diagnosed with glioblastoma (GBM) continue to face a bleak prognosis. It is critical that new effective therapeutic strategies are developed. GBM stem cells have molecular hallmarks of neural stem and progenitor cells and it is possible to propagate both non-transformed normal neural stem cells and GBM stem cells, in defined, feeder-free, adherent culture. These primary stem cell lines provide an experimental model that is ideally suited to cell-based drug discovery or genetic screens in order to identify tumour-specific vulnerabilities. For many solid tumours, including GBM, the genetic disruptions that drive tumour initiation and growth have now been catalogued. CRISPR/Cas-based genome editing technologies have recently emerged, transforming our ability to functionally annotate the human genome. Genome editing opens prospects for engineering precise genetic changes in normal and GBM-derived neural stem cells, which will provide more defined and reliable genetic models, with critical matched pairs of isogenic cell lines. Generation of more complex alleles such as knock in tags or fluorescent reporters is also now possible. These new cellular models can be deployed in cell-based phenotypic drug discovery (PDD). Here we discuss the convergence of these advanced technologies (iPS cells, neural stem cell culture, genome editing and high content phenotypic screening) and how they herald a new era in human cellular genetics that should have a major impact in accelerating glioblastoma drug discovery. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Core Canonical Pathways Involved in Developing Human Glioblastoma Multiforme (GBM).

PubMed

Ghosh, Somiranjan; Dutta, Sisir; Thorne, Gabriel; Boston, Ava; Barfield, Alexis; Banerjee, Narendra; Walker, Rayshawn; Banerjee, Hirendra Nath

2017-02-01

Glioblastoma multiforme (GBM) is the most common and aggressive type of the primary brain tumors with pathologic hallmarks of necrosis and vascular proliferation. The diagnosis of GBM is currently mostly based on histological examination of brain tumor tissues, after radiological characterization and surgical biopsy. The ability to characterize tumors comprehensively at the molecular level raises the possibility that diagnosis can be made based on molecular profiling with or without histological examination, rather than solely on histological phenotype. The development of novel genomic and proteomic techniques will foster in the identification of such diagnostic and prognostic molecular markers. We analyzed the global differential gene expression of a GBM cell line HTB15 in comparison to normal human Astrocytes, and established a few canonical pathways that are important in determining the molecular mechanisms of cancer using global gene expression microarray, coupled with the Ingenuity Pathway Analysis ( IPA ®). Overall, we revealed a discrete gene expression profile in the experimental model that resembled progression of GBM cancer. The canonical pathway analysis showed the involvement of genes that differentially expressed in such a disease condition that included Inositol pathway, Polo like kinases, nNOS signaling , and Tetrapyrrole biosynthesis . Our findings established that the gene expression pattern of this dreaded brain cancer will probably help the cancer research community by finding out newer therapeutic strategies to combat this dreaded cancer type that leads to the identification of high-risk population in this category, with almost hundred percent mortality rate.

New molecules and old drugs as emerging approaches to selectively target human glioblastoma cancer stem cells.

PubMed

Würth, Roberto; Barbieri, Federica; Florio, Tullio

2014-01-01

Despite relevant progress obtained by multimodal treatment, glioblastoma (GBM), the most aggressive primary brain tumor, is still incurable. The most encouraging advancement of GBM drug research derives from the identification of cancer stem cells (CSCs), since these cells appear to represent the determinants of resistance to current standard therapies. The goal of most ongoing studies is to identify drugs able to affect CSCs biology, either inducing selective toxicity or differentiating this tumor cell population into nontumorigenic cells. Moreover, the therapeutic approach for GBM could be improved interfering with chemo- or radioresistance mechanisms, microenvironment signals, and the neoangiogenic process. During the last years, molecular targeted compounds such as sorafenib and old drugs, like metformin, displayed interesting efficacy in preclinical studies towards several tumors, including GBM, preferentially affecting CSC viability. In this review, the latest experimental results, controversies, and prospective application concerning these promising anticancer drugs will be discussed.

Prospective of curcumin, a pleiotropic signalling molecule from Curcuma longa in the treatment of Glioblastoma.

PubMed

Luthra, Pratibha Mehta; Lal, Neetika

2016-02-15

GBM (Glioblastoma) is the most malignant human brain tumor with median survival of one year. The treatment involves surgery, radiotherapy and adjuvant chemotherapy mostly with the alkylation agents such as temozolomide (TMZ). Dietary polyphenol curcumin, isolated from the rhizome of the Curcuma longa (turmeric), has emerged as remarkable anti-cancer agent in the treatment of various peripheral cancers such as blood, lymphomas, multiple myeloma, melanoma as well as skin, lung, prostate, breast, ovarian, bladder, liver, gastrointestinal tract, pancreatic and colorectal epithelial cancers with a pleiotropic mode of action and also showed promise in alleviation of GBM. In this review, the mechanism of anticancer effect of curcumin in GBM has been discussed extensively. The clinical safety and pharmacokinetics of curcumin has been scrutinized to combat the challenges for the treatment of GBM. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

Decreased miR-106a inhibits glioma cell glucose uptake and proliferation by targeting SLC2A3 in GBM.

PubMed

Dai, Dong-Wei; Lu, Qiong; Wang, Lai-Xing; Zhao, Wen-Yuan; Cao, Yi-Qun; Li, Ya-Nan; Han, Guo-Sheng; Liu, Jian-Min; Yue, Zhi-Jian

2013-10-14

MiR-106a is frequently down-regulated in various types of human cancer. However the underlying mechanism of miR-106a involved in glioma remains elusive. The association of miR-106a with glioma grade and patient survival was analyzed. The biological function and target of miR-106a were determined by bioinformatic analysis and cell experiments (Western blot, luciferase reporter, cell cycle, ntracellular ATP production and glucose uptake assay). Finally, rescue expression of its target SLC2A3 was used to test the role of SLC2A3 in miR-106a-mediated cell glycolysis and proliferation. Here we showed that miR-106a was a tumor suppressor miRNA was involved in GBM cell glucose uptake and proliferation. Decreased miR-106a in GBM tissues and conferred a poor survival of GBM patients. SLC2A3 was identified as a core target of miR-106a in GBM cells. Inhibition of SLC2A3 by miR-106a attenuated cell proliferation and inhibited glucose uptake. In addition, for each biological process we identified ontology-associated transcripts that significantly correlated with SLC2A3 expression. Finally, the expression of SLC2A3 largely abrogated miR-106a-mediated cell proliferation and glucose uptake in GBM cells. Taken together, miR-106a and SLC2A3 could be potential therapeutic approaches for GBM.

Targeting Hypoxia-Inducible Factor 1α in a New Orthotopic Model of Glioblastoma Recapitulating the Hypoxic Tumor Microenvironment.

PubMed

Nigim, Fares; Cavanaugh, Jill; Patel, Anoop P; Curry, William T; Esaki, Shin-ichi; Kasper, Ekkehard M; Chi, Andrew S; Louis, David N; Martuza, Robert L; Rabkin, Samuel D; Wakimoto, Hiroaki

2015-07-01

Tissue hypoxia and necrosis represent pathophysiologic and histologic hallmarks of glioblastoma (GBM). Although hypoxia inducible factor 1α (HIF-1α) plays crucial roles in the malignant phenotypes of GBM, developing HIF-1α-targeted agents has been hampered by the lack of a suitable preclinical model that recapitulates the complex biology of clinical GBM. We present a new GBM model, MGG123, which was established from a recurrent human GBM. Orthotopic xenografting of stem-like MGG123 cells reproducibly generated lethal tumors that were characterized by foci of palisading necrosis, hypervascularity, and robust stem cell marker expression. Perinecrotic neoplastic cells distinctively express HIF-1α and are proliferative in both xenografts and the patient tissue. The xenografts contain scattered hypoxic foci that were consistently greater than 50 μm distant from blood vessels, indicating intratumoral heterogeneity of oxygenation. Hypoxia enhanced HIF-1α expression in cultured MGG123 cells, which was abrogated by the HIF-1α inhibitors digoxin or ouabain. In vivo, treatment of orthotopic MGG123 xenografts with digoxin decreased HIF-1α expression, vascular endothelial growth factor mRNA levels, and CD34-positive vasculature within the tumors, and extended survival of mice bearing the aggressive MGG123 GBM. This preclinical tumor model faithfully recapitulates the GBM-relevant hypoxic microenvironment and stemness and is a suitable platform for studying disease biology and developing hypoxia-targeted agents.

Preparation and characterization of teniposide PLGA nanoparticles and their uptake in human glioblastoma U87MG cells.

PubMed

Mo, Liqian; Hou, Lianbing; Guo, Dan; Xiao, Xiaoyan; Mao, Ping; Yang, Xixiao

2012-10-15

Many studies have demonstrated the uptake mechanisms of various nanoparticle delivery systems with different physicochemical properties in different cells. In this study, we report for the first time the preparation and characterization of teniposide (VM-26) poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles (NPs) and their cellular uptake pathways in human glioblastoma U87MG cells. The nanoparticles prepared with oil-in-water (O/W) single-emulsion solvent evaporation method had a small particle size and spherical shape and provided effective protection against degradation of teniposide in PBS solution. Differential scanning calorimeter (DSC) thermograms concluded that VM-26 was dispersed as amorphous or disordered crystalline phase in the PLGA matrix. A cytotoxicity study revealed that, in a 24h period, blank PLGA NPs had no cytotoxicity, whereas teniposide-loaded PLGA NPs (VM-26-NPs) had U87MG cytotoxicity levels similar to free teniposide. Confocal laser scanning microscopy (CLSM) and transmission electron microscopy (TEM) images showed the distribution and degradation processes of nanoparticles in cells. An endocytosis inhibition test indicated that clathrin-mediated endocytosis and macropinocytosis were the primary modes of engulfment involved in the internalization of VM-26-NPs. Our findings suggest that PLGA nanoparticles containing a sustained release formula of teniposide may multiplex the therapeutic effect and ultimately degrade in lysosomal within human glioblastoma U87MG cells. Copyright © 2012 Elsevier B.V. All rights reserved.

Cytotoxic Effects of Temozolomide and Radiation are Additive- and Schedule-Dependent

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chalmers, Anthony J., E-mail: a.j.chalmers@sussex.ac.u; Genome Damage and Stability Centre, University of Sussex, Falmer; Ruff, Elliot M.

2009-12-01

Purpose: Despite aggressive therapy comprising radical radiation and temozolomide (TMZ) chemotherapy, the prognosis for patients with glioblastoma multiforme (GBM) remains poor, particularly if tumors express O{sup 6}-methylguanine-DNA-methyltransferase (MGMT). The interactions between radiation and TMZ remain unclear and have important implications for scheduling and for developing strategies to improve outcomes. Methods and Materials: Factors determining the effects of combination therapy on clonogenic survival, cell-cycle checkpoint signaling and DNA repair were investigated in four human glioma cell lines (T98G, U373-MG, UVW, U87-MG). Results: Combining TMZ and radiation yielded additive cytotoxicity, but only when TMZ was delivered 72 h before radiation. Radiosensitization wasmore » not observed. TMZ induced G2/M cell-cycle arrest at 48-72 h, coincident with phosphorylation of Chk1 and Chk2. Additive G2/M arrest and Chk1/Chk2 phosphorylation was only observed when TMZ preceded radiation by 72 h. The ataxia-telangiectasia mutated (ATM) inhibitor KU-55933 increased radiation sensitivity and delayed repair of radiation-induced DNA breaks, but did not influence TMZ effects. The multiple kinase inhibitor caffeine enhanced the cytotoxicity of chemoradiation and exacerbated DNA damage. Conclusions: TMZ is not a radiosensitizing agent but yields additive cytotoxicity in combination with radiation. Our data indicate that TMZ treatment should commence at least 3 days before radiation to achieve maximum benefit. Activation of G2/M checkpoint signaling by TMZ and radiation has a cytoprotective effect that can be overcome by dual inhibition of ATM and ATR. More specific inhibition of checkpoint signaling will be required to increase treatment efficacy without exacerbating toxicity.« less

A cytoplasmic long noncoding RNA LINC00470 as a new AKT activator to mediate glioblastoma cell autophagy.

PubMed

Liu, Changhong; Zhang, Yan; She, Xiaoling; Fan, Li; Li, Peiyao; Feng, Jianbo; Fu, Haijuan; Liu, Qing; Liu, Qiang; Zhao, Chunhua; Sun, Yingnan; Wu, Minghua

2018-06-04

Despite the overwhelming number of investigations on AKT, little is known about lncRNA on AKT regulation, especially in GBM cells. RNA-binding protein immunoprecipitation assay (RIP) and RNA pulldown were used to confirm the binding of LINC00470 and fused in sarcoma (FUS). Confocal imaging, co-immunoprecipitation (Co-IP) and GST pulldown assays were used to detect the interaction between FUS and AKT. EdU assay, CCK-8 assay, and intracranial xenograft assays were performed to demonstrate the effect of LINC00470 on the malignant phenotype of GBM cells. RT-qPCR and Western blotting were performed to test the effect of LINC00470 on AKT and pAKT. In this study, we demonstrated that LINC00470 was a positive regulator for AKT activation in GBM. LINC00470 bound to FUS and AKT to form a ternary complex, anchoring FUS in the cytoplasm to increase AKT activity. Higher pAKT activated by LINC00470 inhibited ubiquitination of HK1, which affected glycolysis, and inhibited cell autophagy. Furthermore, higher LINC00470 expression was associated with GBM tumorigenesis and poor patient prognosis. Our findings revealed a noncanonical AKT activation signaling pathway, i.e., LINC00470 directly interacts with FUS, serving as an AKT activator to promote GBM progression. LINC00470 has an important referential significance to evaluate the prognosis of patients.

Genome-wide transcriptional profiling of human glioblastoma cells in response to ITE treatment.

PubMed

Kang, Bo; Zhou, Yanwen; Zheng, Min; Wang, Ying-Jie

2015-09-01

A ligand-activated transcription factor aryl hydrocarbon receptor (AhR) is recently revealed to play a key role in embryogenesis and tumorigenesis (Feng et al. [1], Safe et al. [2]) and 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE) (Song et al. [3]) is an endogenous AhR ligand that possesses anti-tumor activity. In order to gain insights into how ITE acts via the AhR in embryogenesis and tumorigenesis, we analyzed the genome-wide transcriptional profiles of the following three groups of cells: the human glioblastoma U87 parental cells, U87 tumor sphere cells treated with vehicle (DMSO) and U87 tumor sphere cells treated with ITE. Here, we provide the details of the sample gathering strategy and show the quality controls and the analyses associated with our gene array data deposited into the Gene Expression Omnibus (GEO) under the accession code of GSE67986.

Pulsed low-dose irradiation of orthotopic glioblastoma multiforme (GBM) in a pre-clinical model: effects on vascularization and tumor control.

PubMed

Dilworth, Joshua T; Krueger, Sarah A; Dabjan, Mohamad; Grills, Inga S; Torma, John; Wilson, George D; Marples, Brian

2013-07-01

To compare dose-escalated pulsed low-dose radiation therapy (PLRT) and standard radiation therapy (SRT). Intracranial U87MG GBM tumors were established in nude mice. Animals received whole brain irradiation with daily 2-Gy fractions given continuously (SRT) or in ten 0.2-Gy pulses separated by 3-min intervals (PLRT). Tumor response was evaluated using weekly CT and [(18)F]-FDG-PET scans. Brain tissue was subjected to immunohistochemistry and cytokine bead array to assess tumor and normal tissue effects. Median survival for untreated animals was 18 (SE±0.5) days. A significant difference in median survival was seen between SRT (29±1.8days) and PLRT (34.2±1.9days). Compared to SRT, PLRT resulted in a 31% (p<0.01), 38% (p<0.01), and 53% (p=0.01) reduction in normalized tumor volume and a 48% (p<0.01), 51% (p<0.01), and 70% (p<0.01) reduction in tumor growth rate following the administration of 10Gy, 20Gy, and 30Gy, respectively. Compared to untreated tumors, PLRT resulted in similar tumor vascular density, while SRT produced a 40% reduction in tumor vascular density (p=0.05). Compared to SRT, PLRT was associated with a 28% reduction in degenerating neurons in the surrounding brain parenchyma (p=0.05). Compared to SRT, PLRT resulted in greater inhibition of tumor growth and improved survival, which may be attributable to preservation of vascular density. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Study of interaction of GNR with glioblastoma cells

NASA Astrophysics Data System (ADS)

Hole, Arti; Cardoso-Avila, P. E.; Sridharan, Sangita; Sahu, Aditi; Nair, Jyothi; Dongre, Harsh; Goda, Jayant S.; Sawant, Sharada; Dutt, Shilpee; Pichardo-Molina, J. L.; Murali Krishna, C.

2018-01-01

Radiation resistance is one of the major causes of recurrence and failure of radiotherapy. Different methods have been used to increase the efficacy of radiation therapy and at the same time restrict the radiation resistivity. From last few years nanoparticles have played a key role in the enhancement of radiosensitization. The densely packed nanoparticles can selectively scatter or absorb the high radiations, which allow better targeting of cellular components within the tumor hence resulting in increased radiation damage to the cancer cells. Glioblastoma multiforme (GBM) is one of the highly radioresistant brain cancer. Current treatment methods are surgical resection followed by concurrent chemo and radiation therapy. In this study we have used in-house engineered gold nano rodes (GNR) and analyzed their effect on U-87MG cell lines. MTT assay was employed to determine the cytotoxic concentration of the nanoparticles. Raman spectroscopy was used to analyze the effect of gold nanoparticles on glioma cells, which was followed by transmission electron microscopic examinations to visualize their cellular penetration. Our data shows that GNR were able to penetrate the cells and induce cytotoxicity at the concentration of 198 μM as determined by MTT assay at 24 post GNP treatment. Additionally, we show that Raman spectroscopy, could classify spectra between untreated and cells treated with nanoparticles. Taken together, this study shows GNR penetration and cytotoxicity in glioma cells thereby providing a rationale to use them in cancer therapeutics. Future studies will be carried out to study the biological activity of the formulation as a radiosensitizer in GBM.

A novel 3D human glioblastoma cell culture system for modeling drug and radiation responses

PubMed Central

Stevenson, Katrina; Gilmour, Lesley; Hamilton, Graham; Chalmers, Anthony J

2017-01-01

Abstract Background. Glioblastoma (GBM) is the most common primary brain tumor, with dismal prognosis. The failure of drug–radiation combinations with promising preclinical data to translate into effective clinical treatments may relate to the use of simplified 2-dimensional in vitro GBM cultures. Methods. We developed a customized 3D GBM culture system based on a polystyrene scaffold (Alvetex) that recapitulates key histological features of GBM and compared it with conventional 2D cultures with respect to their response to radiation and to molecular targeted agents for which clinical data are available. Results. In 3 patient-derived GBM lines, no difference in radiation sensitivity was observed between 2D and 3D cultures, as measured by clonogenic survival. Three different molecular targeted agents, for which robust clinical data are available were evaluated in 2D and 3D conditions: (i) temozolomide, which improves overall survival and is standard of care for GBM, exhibited statistically significant effects on clonogenic survival in both patient-derived cell lines when evaluated in the 3D model compared with only one cell line in 2D cells; (ii) bevacizumab, which has been shown to increase progression-free survival when added to standard chemoradiation in phase III clinical trials, exhibited marked radiosensitizing activity in our 3D model but had no effect on 2D cells; and (iii) erlotinib, which had no efficacy in clinical trials, displayed no activity in our 3D GBM model, but radiosensitized 2D cells. Conclusions. Our 3D model reliably predicted clinical efficacy, strongly supporting its clinical relevance and potential value in preclinical evaluation of drug–radiation combinations for GBM. PMID:27576873

Stage-Specific Action of Matrix Metalloproteinases Influences Progressive Hereditary Kidney Disease

PubMed Central

Zeisberg, Michael; Khurana, Mona; Rao, Velidi H; Cosgrove, Dominic; Rougier, Jean-Philippe; Werner, Michelle C; Shield, Charles F; Werb, Zena; Kalluri, Raghu

2006-01-01

Background Glomerular basement membrane (GBM), a key component of the blood-filtration apparatus in the in the kidney, is formed through assembly of type IV collagen with laminins, nidogen, and sulfated proteoglycans. Mutations or deletions involving α3(IV), α4(IV), or α5(IV) chains of type IV collagen in the GBM have been identified as the cause for Alport syndrome in humans, a progressive hereditary kidney disease associated with deafness. The pathological mechanisms by which such mutations lead to eventual kidney failure are not completely understood. Methods and Findings We showed that increased susceptibility of defective human Alport GBM to proteolytic degradation is mediated by three different matrix metalloproteinases (MMPs)—MMP-2, MMP-3, and MMP-9—which influence the progression of renal dysfunction in α3(IV) −/− mice, a model for human Alport syndrome. Genetic ablation of either MMP-2 or MMP-9, or both MMP-2 and MMP-9, led to compensatory up-regulation of other MMPs in the kidney glomerulus. Pharmacological ablation of enzymatic activity associated with multiple GBM-degrading MMPs, before the onset of proteinuria or GBM structural defects in the α3(IV) −/− mice, led to significant attenuation in disease progression associated with delayed proteinuria and marked extension in survival. In contrast, inhibition of MMPs after induction of proteinuria led to acceleration of disease associated with extensive interstitial fibrosis and early death of α3(IV) −/− mice. Conclusions These results suggest that preserving GBM/extracellular matrix integrity before the onset of proteinuria leads to significant disease protection, but if this window of opportunity is lost, MMP-inhibition at the later stages of Alport disease leads to accelerated glomerular and interstitial fibrosis. Our findings identify a crucial dual role for MMPs in the progression of Alport disease in α3(IV) −/− mice, with an early pathogenic function and a later protective action. Hence, we propose possible use of MMP-inhibitors as disease-preventive drugs for patients with Alport syndrome with identified genetic defects, before the onset of proteinuria. PMID:16509766

Measuring Mental Workload: A Performance Battery.

DTIC Science & Technology

1987-09-01

1982). Subjective mental workload. Human Factors, 24, 25-40. Neisser , U ., Novick, R., & Lazar, R. (1963). Searching for ten targets 0...818 MES~A [G MENTAL WORKLOAD: A PERFORMANCE BATTERY( U ) 1/ • i NUN ENGINEERING LAS ABERDEEN PROVING GROUND MO L A MHITAKER ET AL .EP 87 HEL-TH-21-87...UNCLASSIFIED FG 5/9IIEEEEEEIE EhhIEllE~lllEE I.E.E.E I1.2 P e- - ,uIII j .. - - I ,,, 65 -S S p *q 0 0 0 0 0 0 0 0 0 u -; 5 . . ~qqr AD-A187 118

Umbilical Cord Blood-Derived Mesenchymal Stem Cells Inhibit, But Adipose Tissue-Derived Mesenchymal Stem Cells Promote, Glioblastoma Multiforme Proliferation

PubMed Central

Akimoto, Keiko; Kimura, Kenichi; Nagano, Masumi; Takano, Shingo; To'a Salazar, Georgina; Yamashita, Toshiharu

2013-01-01

Mesenchymal stem cells (MSCs) possess self-renewal and multipotential differentiation abilities, and they are thought to be one of the most reliable stem cell sources for a variety of cell therapies. Recently, cell therapy using MSCs has been studied as a novel therapeutic approach for cancers that show refractory progress and poor prognosis. MSCs from different tissues have different properties. However, the effect of different MSC properties on their application in anticancer therapies has not been thoroughly investigated. In this study, to characterize the anticancer therapeutic application of MSCs from different sources, we established two different kinds of human MSCs: umbilical cord blood-derived MSCs (UCB-MSCs) and adipose-tissue-derived MSCs (AT-MSCs). We used these MSCs in a coculture assay with primary glioblastoma multiforme (GBM) cells to analyze how MSCs from different sources can inhibit GBM growth. We found that UCB-MSCs inhibited GBM growth and caused apoptosis, but AT-MSCs promoted GBM growth. Terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick-end labeling assay clearly demonstrated that UCB-MSCs promoted apoptosis of GBM via tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). TRAIL was expressed more highly by UCB-MSCs than by AT-MSCs. Higher mRNA expression levels of angiogenic factors (vascular endothelial growth factor, angiopoietin 1, platelet-derived growth factor, and insulin-like growth factor) and stromal-derived factor-1 (SDF-1/CXCL12) were observed in AT-MSCs, and highly vascularized tumors were developed when AT-MSCs and GBM were cotransplanted. Importantly, CXCL12 inhibited TRAIL activation of the apoptotic pathway in GBM, suggesting that AT-MSCs may support GBM development in vivo by at least two distinct mechanisms—promoting angiogenesis and inhibiting apoptosis. The opposite effects of AT-MSCs and UCB-MSCs on GBM clearly demonstrate that differences must be considered when choosing a stem cell source for safety in clinical application. PMID:23231075

Nutraceutical phycocyanin nanoformulation for efficient drug delivery of paclitaxel in human glioblastoma U87MG cell line

NASA Astrophysics Data System (ADS)

Agrawal, Madhunika; Yadav, Sanjeev Kumar; Agrawal, Satyam Kumar; Karmakar, Surajit

2017-08-01

To enhance the therapeutic efficacy of chemotherapy on glioblastoma U87MG cell line, paclitaxel-loaded phycocyanin nanoparticles (PTX-PcNPs) were prepared by modified desolvation process. PTX-PcNPs were characterised in terms of size, zeta potential, drug loading efficiency and drug release. Confocal laser scanning microscopy showed PTX-PcNPs could be internalised by U87MG cells. The anti-cancer activity was investigated in vitro by 3-(4,5-dimethylthizol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay with and without photodynamic therapy. It was observed that formulation could significantly inhibit growth of U87MG cells as compared to PTX alone and also induced apoptosis, which was evidenced by presence of apoptotic bodies and nuclear fragmentation in treated cells. The present study suggests that PTX-PcNPs can act as a promising drug delivery system for cancer treatment. [Figure not available: see fulltext.

Human cytomegalovirus inhibits apoptosis by regulating the activating transcription factor 5 signaling pathway in human malignant glioma cells

PubMed Central

WANG, TONGMEI; QIAN, DONGMENG; HU, MING; LI, LING; ZHANG, LI; CHEN, HAO; YANG, RUI; WANG, BIN

2014-01-01

The activating transcription factor 5 (ATF5), also termed ATFx, is a member of the ATF/cAMP response element-binding protein (CREB) family of basic zipper proteins. ATF5 is an anti-apoptotic protein that is highly expressed in malignant glioma and is essential for glioma cell survival. Accumulating evidence indicates that human malignant gliomas are universally infected with human cytomegalovirus (HCMV). Recent studies have shown that HCMV may be resistant to the induction of apoptosis by disrupting cellular pathways in glioblastoma. To investigate the potential anti-apoptotic function of HCMV in glioma, malignant U87 glioma cells were infected with HCMV. The present study showed that HCMV infection suppressed apoptosis in glioblastoma U87 cells by regulating the expression of ATF5. Furthermore, in glioblastoma U87 cells, HCMV infection induced cellular proliferation in parallel with an increase in the expression level of ATF5 and B-cell lymphoma/leukemia-2 to Bcl-2-associated X protein ratio. Loss of ATF5 function was achieved using a dominant-negative form of ATF5 in U87 cells, whereby cells appeared to grow marginally following HCMV infection when compared with the control. However, the anti-apoptotic ability was appeared to decline in the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay. These results indicate that ATF5 signaling pathways may be important in the anti-apoptotic activity of HCMV-infected glioblastoma cells; therefore, the anti-apoptotic molecular mechanisms of HCMV in human glioblastoma cells were investigated in the current study. Prevention of HCMV infection may present a potential and promising approach for the treatment of malignant gliomas. PMID:25120656

The Fermi-GBM Three-year X-Ray Burst Catalog

NASA Astrophysics Data System (ADS)

Jenke, P. A.; Linares, M.; Connaughton, V.; Beklen, E.; Camero-Arranz, A.; Finger, M. H.; Wilson-Hodge, C. A.

2016-08-01

The Fermi Gamma-ray Burst Monitor (GBM) is an all-sky gamma-ray monitor well known in the gamma-ray burst (GRB) community. Although GBM excels in detecting the hard, bright extragalactic GRBs, its sensitivity above 8 keV and its all-sky view make it an excellent instrument for the detection of rare, short-lived Galactic transients. In 2010 March, we initiated a systematic search for transients using GBM data. We conclude this phase of the search by presenting a three-year catalog of 1084 X-ray bursts. Using spectral analysis, location, and spatial distributions we classified the 1084 events into 752 thermonuclear X-ray bursts, 267 transient events from accretion flares and X-ray pulses, and 65 untriggered gamma-ray bursts. All thermonuclear bursts have peak blackbody temperatures broadly consistent with photospheric radius expansion (PRE) bursts. We find an average rate of 1.4 PRE bursts per day, integrated over all Galactic bursters within about 10 kpc. These include 33 and 10 bursts from the ultra-compact X-ray binaries 4U 0614+09 and 2S 0918-549, respectively. We discuss these recurrence times and estimate the total mass ejected by PRE bursts in our Galaxy.

D-Amino acid oxidase-induced oxidative stress, 3-bromopyruvate and citrate inhibit angiogenesis, exhibiting potent anticancer effects.

PubMed

El Sayed, S M; El-Magd, R M Abou; Shishido, Y; Yorita, K; Chung, S P; Tran, D H; Sakai, T; Watanabe, H; Kagami, S; Fukui, K

2012-10-01

Angiogenesis is critical for cancer growth and metastasis. Steps of angiogenesis are energy consuming, while vascular endothelial cells are highly glycolytic. Glioblastoma multiforme (GBM) is a highly vascular tumor and this enhances its aggressiveness. D-amino acid oxidase (DAO) is a promising therapeutic protein that induces oxidative stress upon acting on its substrates. Oxidative stress-energy depletion (OSED) therapy was recently reported (El Sayed et al., Cancer Gene Ther, 19, 1-18, 2012). OSED combines DAO-induced oxidative stress with energy depletion caused by glycolytic inhibitors such as 3-bromopyruvate (3BP), a hexokinase II inhibitor that depleted ATP in cancer cells and induced production of hydrogen peroxide. 3BP disturbs the Warburg effect and antagonizes effects of lactate and pyruvate (El Sayed et al., J Bioenerg Biomembr, 44, 61-79, 2012). Citrate is a natural organic acid capable of inhibiting glycolysis by targeting phosphofructokinase. Here, we report that DAO, 3BP and citrate significantly inhibited angiogenesis, decreased the number of vascular branching points and shortened the length of vascular tubules. OSED delayed the growth of C6/DAO glioma cells. 3BP combined with citrate delayed the growth of C6 glioma cells and decreased significantly the number and size of C6 glioma colonies in soft agar. Human GBM cells (U373MG) were resistant to chemotherapy e.g. cisplatin and cytosine arabinoside, while 3BP was effective in decreasing the viability and disturbing the morphology of U373MG cells.

Modulation of miR-21 signaling by MPS1 in human glioblastoma

PubMed Central

Maachani, Uday B.; Tandle, Anita; Shankavaram, Uma; Kramp, Tamalee; Camphausen, Kevin A.

2016-01-01

Monopolar spindle 1 (MPS1) is an essential spindle assembly checkpoint (SAC) kinase involved in determining spindle integrity. Beyond its mitotic functions, it has been implicated in several other signaling pathways. Our earlier studies have elaborated on role of MPS1 in glioblastoma (GBM) radiosensitization. In this study using reverse phase protein arrays (RPPAs), we assessed MPS1 mediated cell signaling pathways and demonstrated that inhibiting MPS1 could upregulate the expression of the tumor suppressor PDCD4 and MSH2 genes, by down regulating micro RNA-21 (miR-21). In GBMs miR-21 expression is significantly elevated and is associated with chemo and radioresistance. Both MPS1 and miR-21 depletion suppressed GBM cell proliferation, whereas, ectopic expression of miR-21 rescued GBM cell growth from MPS1 inhibition. Further, we demonstrate that MPS1 mediates phosphorylation of SMAD3 but not SMAD2 in GBM cells; A possible mechanism behind miR-21 modulation by MPS1. Collectively, our results shed light onto an important role of MPS1 in TGF-β/SMAD signaling via miR-21 regulation. We also, show the prognostic effect of miR-21, PDCD4 and MSH2 levels to patient survival across different GBM molecular subtypes. This scenario in which miR-21 is modulated by MPS1 inhibition may be exploited as a potential target for effective GBM therapy. PMID:25991676

Modulation of miR-21 signaling by MPS1 in human glioblastoma.

PubMed

Maachani, Uday B; Tandle, Anita; Shankavaram, Uma; Kramp, Tamalee; Camphausen, Kevin

2016-08-16

Monopolar spindle 1 (MPS1) is an essential spindle assembly checkpoint (SAC) kinase involved in determining spindle integrity. Beyond its mitotic functions, it has been implicated in several other signaling pathways. Our earlier studies have elaborated on role of MPS1 in glioblastoma (GBM) radiosensitization. In this study using reverse phase protein arrays (RPPAs), we assessed MPS1 mediated cell signaling pathways and demonstrated that inhibiting MPS1 could upregulate the expression of the tumor suppressor PDCD4 and MSH2 genes, by down regulating micro RNA-21 (miR-21). In GBMs miR-21 expression is significantly elevated and is associated with chemo and radioresistance. Both MPS1 and miR-21 depletion suppressed GBM cell proliferation, whereas, ectopic expression of miR-21 rescued GBM cell growth from MPS1 inhibition. Further, we demonstrate that MPS1 mediates phosphorylation of SMAD3 but not SMAD2 in GBM cells; A possible mechanism behind miR-21 modulation by MPS1. Collectively, our results shed light onto an important role of MPS1 in TGF-β/SMAD signaling via miR-21 regulation. We also, show the prognostic effect of miR-21, PDCD4 and MSH2 levels to patient survival across different GBM molecular subtypes. This scenario in which miR-21 is modulated by MPS1 inhibition may be exploited as a potential target for effective GBM therapy.

Chronic activation of wild-type epidermal growth factor receptor and loss of Cdkn2a cause mouse glioblastoma formation.

PubMed

Acquaviva, Jaime; Jun, Hyun Jung; Lessard, Julie; Ruiz, Rolando; Zhu, Haihao; Donovan, Melissa; Woolfenden, Steve; Boskovitz, Abraham; Raval, Ami; Bronson, Roderick T; Pfannl, Rolf; Whittaker, Charles A; Housman, David E; Charest, Al

2011-12-01

Glioblastoma multiforme (GBM) is characterized by overexpression of epidermal growth factor receptor (EGFR) and loss of the tumor suppressors Ink4a/Arf. Efforts at modeling GBM using wild-type EGFR in mice have proven unsuccessful. Here, we present a unique mouse model of wild-type EGFR-driven gliomagenesis. We used a combination of somatic conditional overexpression and ligand-mediated chronic activation of EGFR in cooperation with Ink4a/Arf loss in the central nervous system of adult mice to generate tumors with the histopathologic and molecular characteristics of human GBMs. Sustained, ligand-mediated activation of EGFR was necessary for gliomagenesis, functionally substantiating the clinical observation that EGFR-positive GBMs from patients express EGFR ligands. To gain a better understanding of the clinically disappointing EGFR-targeted therapies for GBM, we investigated the molecular responses to EGFR tyrosine kinase inhibitor (TKI) treatment in this model. Gefitinib treatment of primary GBM cells resulted in a robust apoptotic response, partially conveyed by mitogen-activated protein kinase (MAPK) signaling attenuation and accompanied by BIM(EL) expression. In human GBMs, loss-of-function mutations in the tumor suppressor PTEN are a common occurrence. Elimination of PTEN expression in GBM cells posttumor formation did not confer resistance to TKI treatment, showing that PTEN status in our model is not predictive. Together, these findings offer important mechanistic insights into the genetic determinants of EGFR gliomagenesis and sensitivity to TKIs and provide a robust discovery platform to better understand the molecular events that are associated with predictive markers of TKI therapy.

Identification of a novel set of genes reflecting different in vivo invasive patterns of human GBM cells.

PubMed

Monticone, Massimiliano; Daga, Antonio; Candiani, Simona; Romeo, Francesco; Mirisola, Valentina; Viaggi, Silvia; Melloni, Ilaria; Pedemonte, Simona; Zona, Gianluigi; Giaretti, Walter; Pfeffer, Ulrich; Castagnola, Patrizio

2012-08-17

Most patients affected by Glioblastoma multiforme (GBM, grade IV glioma) experience a recurrence of the disease because of the spreading of tumor cells beyond surgical boundaries. Unveiling mechanisms causing this process is a logic goal to impair the killing capacity of GBM cells by molecular targeting.We noticed that our long-term GBM cultures, established from different patients, may display two categories/types of growth behavior in an orthotopic xenograft model: expansion of the tumor mass and formation of tumor branches/nodules (nodular like, NL-type) or highly diffuse single tumor cell infiltration (HD-type). We determined by DNA microarrays the gene expression profiles of three NL-type and three HD-type long-term GBM cultures. Subsequently, individual genes with different expression levels between the two groups were identified using Significance Analysis of Microarrays (SAM). Real time RT-PCR, immunofluorescence and immunoblot analyses, were performed for a selected subgroup of regulated gene products to confirm the results obtained by the expression analysis. Here, we report the identification of a set of 34 differentially expressed genes in the two types of GBM cultures. Twenty-three of these genes encode for proteins localized to the plasma membrane and 9 of these for proteins are involved in the process of cell adhesion. This study suggests the participation in the diffuse infiltrative/invasive process of GBM cells within the CNS of a novel set of genes coding for membrane-associated proteins, which should be thus susceptible to an inhibition strategy by specific targeting.Massimiliano Monticone and Antonio Daga contributed equally to this work.

New insights into the genetics of glioblastoma multiforme by familial exome sequencing

PubMed Central

Backes, Christina; Harz, Christian; Fischer, Ulrike; Schmitt, Jana; Ludwig, Nicole; Petersen, Britt-Sabina; Mueller, Sabine C.; Kim, Yoo-Jin; Wolf, Nadine M.; Katus, Hugo A.; Meder, Benjamin; Furtwängler, Rhoikos; Franke, Andre; Bohle, Rainer; Henn, Wolfram; Graf, Norbert; Keller, Andreas; Meese, Eckart

2015-01-01

Glioblastoma multiforme (GBM) is the most aggressive and malignant subtype of human brain tumors. While a family clustering of GBM has long been acknowledged, relevant hereditary factors still remained elusive. Exome sequencing of families offers the option to discover respective genetic factors. We sequenced blood samples of one of the rare affected families: while both parents were healthy, both children were diagnosed with GBM. We report 85 homozygous non-synonymous single nucleotide variations (SNVs) in both siblings that were heterozygous in the parents. Beyond known key players for GBM such as ERBB2, PMS2, or CHI3L1, we identified over 50 genes that have not been associated to GBM so far. We also discovered three accumulative effects potentially adding to the tumorigenesis in the siblings: a clustering of multiple variants in single genes (e.g. PTPRB, CROCC), the aggregation of affected genes on specific molecular pathways (e.g. Focal adhesion or ECM receptor interaction) and genomic proximity (e.g. chr22.q12.2, chr1.p36.33). We found a striking accumulation of SNVs in specific genes for the daughter, who developed not only a GBM at the age of 12 years but was subsequently diagnosed with a pilocytic astrocytoma, a common acute lymphatic leukemia and a diffuse pontine glioma. The reported variants underline the relevance of genetic predisposition and cancer development in this family and demonstrate that GBM has a complex and heterogeneous genetic background. Sequencing of other affected families will help to further narrow down the driving genetic causes for this disease. PMID:25537509

A PTEN-COL17A1 fusion gene and its novel regulatory role in Collagen XVII expression and GBM malignance.

PubMed

Yan, Xiaoyan; Zhang, Chuanbao; Liang, Tingyu; Yang, Fan; Wang, Haoyuan; Wu, Fan; Wang, Wen; Wang, Zheng; Cheng, Wen; Xu, Jiangnan; Jiang, Tao; Chen, Jing; Ding, Yaozhong

2017-10-17

Collagen XVII expression has recently been demonstrated to be correlated with the tumor malignance. While Collagen XVII is known to be widely distributed in neurons of the human brain, its precise role in pathogenesis of glioblastoma multiforme (GBM) is unknown. In this study, we identified and characterized a new PTEN-COL17A1 fusion gene in GMB using transcriptome sequencing. Although fusion gene did not result in measurable fusion protein production, its presence is accompanied with high levels of COL17A1 expression, revealed a novel regulatory mechanism of Collagen XVII expression by PTEN-COL17A1 gene fusion. Knocked down Collagen XVII expression in glioma cell lines resulted in decreased tumor invasiveness, along with significant reduction of MMP9 expression, while increased Collagen XVII expression promotes invasive activities of glioma cells and associated with GBM recurrences. Together, our results uncovered a new PTEN-COL17A1 fusion gene and its novel regulatory role in Collagen XVII expression and GBM malignance, and demonstrated that COL17A1 could serve as a useful prognostic biomarker and therapeutic targets for GBM.

Up-regulation of MSH6 is associated with temozolomide resistance in human glioblastoma.

PubMed

Sun, Quanye; Pei, Chunying; Li, Qiuyuan; Dong, Tianxiu; Dong, Yucui; Xing, Wenjing; Zhou, Peng; Gong, Yujiao; Zhen, Ziqi; Gao, Yifan; Xiao, Yun; Su, Jun; Ren, Huan

2018-02-19

The impact of DNA mismatch repair (MMR) on resistance to temozolomide (TMZ) therapy in patients with glioblastoma (GBM) is recently reported but the mechanisms are not understood. We aim to analyze the correlation between MMR function and the acquired TMZ resistance in GBM using both relevant clinical samples and TMZ resistant cells. First we found increased expression of MSH6, one of key components of MMR, in recurrent GBM patients' samples who underwent TMZ chemotherapy, comparing with those matched samples collected at the time of diagnosis. Using the cellular models of acquired resistance to TMZ, we further confirmed the up-regulation of MSH6 in TMZ resistant cells. Moreover, a TCGA dataset contains a large cohort of GBM clinical samples with or without TMZ treatment reinforced the increased expression of MSH6 and other MMR genes after long-term TMZ chemotherapy, which may resulted in MMR dysfunction and acquired TMZ resistance. Our results suggest that increased expression of MSH6, or other MMR, may be a new mechanism contributing to the acquired resistance during TMZ therapy; and may serve as an indicator to the resistance in GBM. Copyright © 2018 Elsevier Inc. All rights reserved.

Effective treatment of glioblastoma requires crossing the blood–brain barrier and targeting tumors including cancer stem cells: The promise of nanomedicine

PubMed Central

Kim, Sang-Soo; Harford, Joe B.; Pirollo, Kathleen F.; Chang, Esther H.

2015-01-01

Glioblastoma multiforme (GBM) is the most aggressive and lethal type of brain tumor. Both therapeutic resistance and restricted permeation of drugs across the blood–brain barrier (BBB) play a major role in the poor prognosis of GBM patients. Accumulated evidence suggests that in many human cancers, including GBM, therapeutic resistance can be attributed to a small fraction of cancer cells known as cancer stem cells (CSCs). CSCs have been shown to have stem cell-like properties that enable them to evade traditional cytotoxic therapies, and so new CSC-directed anti-cancer therapies are needed. Nanoparticles have been designed to selectively deliver payloads to relevant target cells in the body, and there is considerable interest in the use of nanoparticles for CSC-directed anti-cancer therapies. Recent advances in the field of nanomedicine offer new possibilities for overcoming CSC-mediated therapeutic resistance and thus significantly improving management of GBM. In this review, we will examine the current nanomedicine approaches for targeting CSCs and their therapeutic implications. The inhibitory effect of various nanoparticle-based drug delivery system towards CSCs in GBM tumors is the primary focus of this review. PMID:26116770

MicroRNA-106a-5p facilitates human glioblastoma cell proliferation and invasion by targeting adenomatosis polyposis coli protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Dazhi; Wang, Zengliang; Chen, Zigui

The invasive behavior of glioblastoma multiforme (GBM) cells is an important reason for its poor prognosis. Tumor cells acquire an ability to digest the extracellular matrix and infiltrate the adjacent normal tissue during invasion. Restraining GBM invasion by changing effector molecules can significantly improve the patient's prognosis. MiRNAs are involved in multiple biological functions via suppressing target genes. In this study, we found that miR-106a-5p expression was high in GBM tissues and cells. The data showed an inverse correlation in GBM tissues between the levels of miR-106a-5p and adenomatosis polyposis coli (APC) mRNAs.Additionally, ectopic expression of miR-106a-5pfacilitated the invasion ofmore » GBM cells whereas inhibition of miR-106a-5p expression weakened the invasive ability. Numerous transcription factors are downstream effectors of the Wnt/β-catenin pathway. Target prediction databases and luciferase data showed that APC is a new direct target of miR-106a-5p. Importantly, westernblot assays demonstrated that miR-106a-5p can reduce APC protein level and enhance target proteins of Wnt/β-catenin pathway. Thus, we hypothesize that miR-106a-5p directly targets APC, resulting in the activation of Wnt/β-catenin pathway. Our results suggest that miR-106a-5p is involved in the invasive behavior of GBM cells and by targeting APC and activating Wnt/β-catenin pathway, it provides a theoretical basis for developing potential clinical strategies. - Highlights: • miR-106a-5p is upregulated in human glioblastoma. • Upregulation of miR-106a-5p promotes glioma cell proliferation and invasion. • miR-106a-5p inactivates the Wnt/β-catenin pathway by directly targeting APC.« less

Hacking macrophage-associated immunosuppression for regulating glioblastoma angiogenesis.

PubMed

Cui, Xin; Morales, Renee-Tyler Tan; Qian, Weiyi; Wang, Haoyu; Gagner, Jean-Pierre; Dolgalev, Igor; Placantonakis, Dimitris; Zagzag, David; Cimmino, Luisa; Snuderl, Matija; Lam, Raymond H W; Chen, Weiqiang

2018-04-01

Glioblastoma (GBM) is the most lethal primary adult brain tumor and its pathology is hallmarked by distorted neovascularization, diffuse tumor-associated macrophage infiltration, and potent immunosuppression. Reconstituting organotypic tumor angiogenesis models with biomimetic cell heterogeneity and interactions, pro-/anti-inflammatory milieu and extracellular matrix (ECM) mechanics is critical for preclinical anti-angiogenic therapeutic screening. However, current in vitro systems do not accurately mirror in vivo human brain tumor microenvironment. Here, we engineered a three-dimensional (3D), microfluidic angiogenesis model with controllable and biomimetic immunosuppressive conditions, immune-vascular and cell-matrix interactions. We demonstrate in vitro, GL261 and CT-2A GBM-like tumors steer macrophage polarization towards a M2-like phenotype for fostering an immunosuppressive and proangiogenic niche, which is consistent with human brain tumors. We distinguished that GBM and M2-like immunosuppressive macrophages promote angiogenesis, while M1-like pro-inflammatory macrophages suppress angiogenesis, which we coin "inflammation-driven angiogenesis." We observed soluble immunosuppressive cytokines, predominantly TGF-β1, and surface integrin (α v β 3 ) endothelial-macrophage interactions are required in inflammation-driven angiogenesis. We demonstrated tuning cell-adhesion receptors using an integrin (α v β 3 )-specific collagen hydrogel regulated inflammation-driven angiogenesis through Src-PI3K-YAP signaling, highlighting the importance of altered cell-ECM interactions in inflammation. To validate the preclinical applications of our 3D organoid model and mechanistic findings of inflammation-driven angiogenesis, we screened a novel dual integrin (α v β 3 ) and cytokine receptor (TGFβ-R1) blockade that suppresses GBM tumor neovascularization by simultaneously targeting macrophage-associated immunosuppression, endothelial-macrophage interactions, and altered ECM. Hence, we provide an interactive and controllable GBM tumor microenvironment and highlight the importance of macrophage-associated immunosuppression in GBM angiogenesis, paving a new direction of screening novel anti-angiogenic therapies. Copyright © 2018 Elsevier Ltd. All rights reserved.

Analysis of tumor metabolism reveals mitochondrial glucose oxidation in genetically diverse, human glioblastomas in the mouse brain in vivo

PubMed Central

Marin-Valencia, Isaac; Yang, Chendong; Mashimo, Tomoyuki; Cho, Steve; Baek, Hyeonman; Yang, Xiao-Li; Rajagopalan, Kartik N.; Maddie, Melissa; Vemireddy, Vamsidhara; Zhao, Zhenze; Cai, Ling; Good, Levi; Tu, Benjamin P.; Hatanpaa, Kimmo J.; Mickey, Bruce E.; Matés, José M.; Pascual, Juan M.; Maher, Elizabeth A.; Malloy, Craig R.; DeBerardinis, Ralph J.; Bachoo, Robert M.

2012-01-01

SUMMARY Dysregulated metabolism is a hallmark of cancer cell lines, but little is known about the fate of glucose and other nutrients in tumors growing in their native microenvironment. To study tumor metabolism in vivo, we used an orthotopic mouse model of primary human glioblastoma (GBM). We infused 13C-labeled nutrients into mice bearing three independent GBM lines, each with a distinct set of mutations. All three lines displayed glycolysis, as expected for aggressive tumors. They also displayed unexpected metabolic complexity, oxidizing glucose via pyruvate dehydrogenase and the citric acid cycle, and using glucose to supply anaplerosis and other biosynthetic activities. Comparing the tumors to surrounding brain revealed obvious metabolic differences, notably the accumulation of a large glutamine pool within the tumors. Many of these same activities were conserved in cells cultured ex vivo from the tumors. Thus GBM cells utilize mitochondrial glucose oxidation during aggressive tumor growth in vivo. PMID:22682223

Implantation of GL261 neurospheres into C57/BL6 mice: a more reliable syngeneic graft model for research on glioma-initiating cells.

PubMed

Yi, Liang; Zhou, Chun; Wang, Bing; Chen, Tunan; Xu, Minhui; Xu, Lunshan; Feng, Hua

2013-08-01

Recent studies have demonstrated that inflammatory cells and inflammatory mediators are indispensable components of the tumor-initiating cell (TIC) niche and regulate the malignant behavior of TICs. However, conventional animal models for glioma-initiating cell (GIC) studies are based on the implantation of GICs from human glioblastoma (GBM) into immunodeficient mice without the regulation of immune system. Whether animal models can mimic the cellular microenvironment of malignancy and evaluate the biological features of GICs accurately is unclear. Here, we detected the biological features of neurosphere-like tumor cells derived from the murine GBM cell line GL261 (GL261-NS) and from primary human GBM (PGBM-NS) in vitro, injected GL261-NS into syngeneic C57/BL6 mouse brain and injected PGBM-NS into NOD/SCID mouse brain, respectively. The tumorigenic characteristics of the two different orthotopic transplantation models were analyzed and the histological discrepancy between grafts and human primary GBM was compared. We found that GICs enriched in GL261-NS, GL261-NS and PGBM-NS exhibited increased GIC potential and enhanced chemoresistance in vitro. GL261-NS was significantly more aggressive compared to GL261 adhesive cells (GL261-AC) in vivo and the enhanced aggression was more significant in syngeneic mice compared to immunodeficient mice. The discrepancy of tumorigenicity between GL261-NS and GL261-AC in C57/BL6 mice was also larger compared to that between PGBM-NS and PGBM-AC in immunodeficient mice. Syngrafts derived from GL261-NS in C57/BL6 mice corresponded to the human GBM histologically better, compared with xenografts derived from PGBM-NS in NOD/SCID mice, which lack inflammatory cells and inflammatory mediators. We conclude that the inflammatory niche is involved in the tumorigenicity of GICs and implantation of GL261-NS into C57/BL6 mice is a more reliable syngeneic graft model for in vivo study on GICs relative to the immunodeficiency model.

Dexamethasone-mediated inhibition of Glioblastoma neurosphere dispersal in an ex vivo organotypic neural assay

PubMed Central

Meleis, Ahmed M.; Mahtabfar, Aria; Danish, Shabbar

2017-01-01

Glioblastoma is highly aggressive. Early dispersal of the primary tumor renders localized therapy ineffective. Recurrence always occurs and leads to patient death. Prior studies have shown that dispersal of Glioblastoma can be significantly reduced by Dexamethasone (Dex), a drug currently used to control brain tumor related edema. However, due to high doses and significant side effects, treatment is tapered and discontinued as soon as edema has resolved. Prior analyses of the dispersal inhibitory effects of Dex were performed on tissue culture plastic, or polystyrene filters seeded with normal human astrocytes, conditions which inherently differ from the parenchymal architecture of neuronal tissue. The aim of this study was to utilize an ex-vivo model to examine Dex-mediated inhibition of tumor cell migration from low-passage, human Glioblastoma neurospheres on multiple substrates including mouse retina, and slices of mouse, pig, and human brain. We also determined the lowest possible Dex dose that can inhibit dispersal. Analysis by Two-Factor ANOVA shows that for GBM-2 and GBM-3, Dex treatment significantly reduces dispersal on all tissue types. However, the magnitude of the effect appears to be tissue-type specific. Moreover, there does not appear to be a difference in Dex-mediated inhibition of dispersal between mouse retina, mouse brain and human brain. To estimate the lowest possible dose at which Dex can inhibit dispersal, LogEC50 values were compared by Extra Sum-of-Squares F-test. We show that it is possible to achieve 50% reduction in dispersal with Dex doses ranging from 3.8 x10-8M to 8.0x10-9M for GBM-2, and 4.3x10-8M to 1.8x10-9M for GBM-3, on mouse retina and brain slices, respectively. These doses are 3-30-fold lower than those used to control edema. This study extends our previous in vitro data and identifies the mouse retina as a potential substrate for in vivo studies of GBM dispersal. PMID:29040322

Nanoscale protein architecture of the kidney glomerular basement membrane

PubMed Central

Suleiman, Hani; Zhang, Lei; Roth, Robyn; Heuser, John E; Miner, Jeffrey H; Shaw, Andrey S; Dani, Adish

2013-01-01

In multicellular organisms, proteins of the extracellular matrix (ECM) play structural and functional roles in essentially all organs, so understanding ECM protein organization in health and disease remains an important goal. Here, we used sub-diffraction resolution stochastic optical reconstruction microscopy (STORM) to resolve the in situ molecular organization of proteins within the kidney glomerular basement membrane (GBM), an essential mediator of glomerular ultrafiltration. Using multichannel STORM and STORM-electron microscopy correlation, we constructed a molecular reference frame that revealed a laminar organization of ECM proteins within the GBM. Separate analyses of domains near the N- and C-termini of agrin, laminin, and collagen IV in mouse and human GBM revealed a highly oriented macromolecular organization. Our analysis also revealed disruptions in this GBM architecture in a mouse model of Alport syndrome. These results provide the first nanoscopic glimpse into the organization of a complex ECM. DOI: http://dx.doi.org/10.7554/eLife.01149.001 PMID:24137544

Acrylamide-induced apoptosis in rat primary astrocytes and human astrocytoma cell lines.

PubMed

Lee, Jiann-Gwu; Wang, Yan-Shiu; Chou, Chin-Cheng

2014-06-01

This study aimed to evaluate the acrylamide (ACR)-induced apoptotic effects on rat primary astrocytes and three human astrocytoma-derived cell lines (U-1240 MG, U-87 MG, and U-251 MG). As determined through the MTT assay, treatment with 1 and 2 mM ACR for 24-72 h resulted in decreased cell viability in all cells. Decreases in cell viability could be blocked in all cells with the exception of U-251 MG cells by Z-DEVD FMK. ACR-induced dose-dependent apoptotic effects were also demonstrated by increases in the sub-G1 phase cell population in all cells. The decreased expressions of pro-caspase 3, 8, and 9 and the interruption of the mitochondrial membrane potential were observed in all cells. Exposure to 2 mM ACR for 48 h resulted in increased Bax/Bcl-2 ratios in primary astrocytes and U-87 MG cells, whereas the overexpression of Bcl-2 was observed in U-1240 MG and U-251 MG cells. The ACR-induced increases in the levels of p53 and pp53 in primary astrocytes could be attenuated by caffeine. These results suggest the existence of a common apoptotic pathway among all cell types and that U-87 MG cells may be a suitable substitute in vitro model for primary astrocytes in future studies on ACR-induced neurotoxicity. Copyright © 2014 Elsevier Ltd. All rights reserved.

MicroRNA-223 Enhances Radiation Sensitivity of U87MG Cells In Vitro and In Vivo by Targeting Ataxia Telangiectasia Mutated

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liang, Liping; Zhu, Ji; Zaorsky, Nicholas G.

2014-03-15

Purpose: Ataxia telangiectasia mutated (ATM) protein is important in the DNA damage response because it repairs radiation-induced damage in cancers. We examined the effect of microRNA-223 (miR-223), a regulator of ATM expression, on radiation sensitivity of cancer cells. Methods and Materials: Human embryonic kidney 293 T (293T) cells were infected with pLL3.7-miR-223 plasmid to generate the pLL3.7-miR-223 and -empty virus (EV) lentivirus (miR-223 and EV). A dual luciferase assay in which the reporter contained wild-type 3′ untranslated region (UTR) of ATM was performed. U87MG cells were infected with miR-223 or EV to establish the overexpressed stable cell lines (U87-223 or U87-EV, respectively).more » Cells were irradiated in vitro, and dose enhancement ratios at 2 Gy (DER{sub 2}) were calculated. Hind legs of BALB/c athymic mice were injected with U87-223 or U87-EV cells; after 2 weeks, half of the tumors were irradiated. Tumor volumes were tracked for a total of 5 weeks. Results: The dual luciferase reporter assay showed a significant reduction in luciferase activity of 293T cells cotransfected with miR-223 and the ATM 3′UTR compared to that in EV control. Overexpression of miR-223 in U87MG cells showed that ATM expression was significantly downregulated in the U87-223 cells compared to that in U87-EV (ATM/β-actin mRNA 1.0 vs 1.5, P<.05). U87-223 cells were hypersensitive to radiation compared to U87-EV cells in vitro (DER{sub 2} = 1.32, P<.01). Mice injected with miR-223-expressing tumors had almost the same tumors after 3 weeks (1.5 cm{sup 3} vs 1.7 cm{sup 3}). However, irradiation significantly decreased tumor size in miR-223-expressing tumors compared to those in controls (0.033 cm{sup 3} vs 0.829 cm{sup 3}). Conclusions: miR-223 overexpression downregulates ATM expression and sensitizes U87 cells to radiation in vitro and in vivo. MicroRNA-223 may be a novel cancer-targeting therapy, although its cancer- and patient-specific roles are currently undefined.« less

Establishment, maintenance and in vitro and in vivo applications of primary human glioblastoma multiforme (GBM) xenograft models for translational biology studies and drug discovery.

PubMed

Carlson, Brett L; Pokorny, Jenny L; Schroeder, Mark A; Sarkaria, Jann N

2011-03-01

Development of clinically relevant tumor model systems for glioblastoma multiforme (GBM) is important for advancement of basic and translational biology. One model that has gained wide acceptance in the neuro-oncology community is the primary xenograft model. This model entails the engraftment of patient tumor specimens into the flank of nude mice and subsequent serial passage of these tumors in the flank of mice. These tumors are then used to establish short-term explant cultures or intracranial xenografts. This unit describes detailed procedures for establishment, maintenance, and utilization of a primary GBM xenograft panel for the purpose of using them as tumor models for basic or translational studies.

In vivo bioluminescence imaging validation of a human biopsy-derived orthotopic mouse model of glioblastoma multiforme.

PubMed

Jarzabek, Monika A; Huszthy, Peter C; Skaftnesmo, Kai O; McCormack, Emmet; Dicker, Patrick; Prehn, Jochen H M; Bjerkvig, Rolf; Byrne, Annette T

2013-05-01

Glioblastoma multiforme (GBM), the most aggressive brain malignancy, is characterized by extensive cellular proliferation, angiogenesis, and single-cell infiltration into the brain. We have previously shown that a xenograft model based on serial xenotransplantation of human biopsy spheroids in immunodeficient rodents maintains the genotype and phenotype of the original patient tumor. The present work further extends this model for optical assessment of tumor engraftment and growth using bioluminescence imaging (BLI). A method for successful lentiviral transduction of the firefly luciferase gene into multicellular spheroids was developed and implemented to generate optically active patient tumor cells. Luciferase-expressing spheroids were injected into the brains of immunodeficient mice. BLI photon counts and tumor volumes from magnetic resonance imaging (MRI) were correlated. Luciferase-expressing tumors recapitulated the histopathologic hallmarks of human GBMs and showed proliferation rates and microvessel density counts similar to those of wild-type xenografts. Moreover, we detected widespread invasion of luciferase-positive tumor cells in the mouse brains. Herein we describe a novel optically active model of GBM that closely mimics human pathology with respect to invasion, angiogenesis, and proliferation indices. The model may thus be routinely used for the assessment of novel anti-GBM therapeutic approaches implementing well-established and cost-effective optical imaging strategies.

Genome-wide transcriptional profiling of human glioblastoma cells in response to ITE treatment

PubMed Central

Kang, Bo; Zhou, Yanwen; Zheng, Min; Wang, Ying-Jie

2015-01-01

A ligand-activated transcription factor aryl hydrocarbon receptor (AhR) is recently revealed to play a key role in embryogenesis and tumorigenesis (Feng et al. [1], Safe et al. [2]) and 2-(1′H-indole-3′-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE) (Song et al. [3]) is an endogenous AhR ligand that possesses anti-tumor activity. In order to gain insights into how ITE acts via the AhR in embryogenesis and tumorigenesis, we analyzed the genome-wide transcriptional profiles of the following three groups of cells: the human glioblastoma U87 parental cells, U87 tumor sphere cells treated with vehicle (DMSO) and U87 tumor sphere cells treated with ITE. Here, we provide the details of the sample gathering strategy and show the quality controls and the analyses associated with our gene array data deposited into the Gene Expression Omnibus (GEO) under the accession code of GSE67986. PMID:26484269

Cerebrospinal fluid dissemination of high-grade gliomas following boron neutron capture therapy occurs more frequently in the small cell subtype of IDH1R132H mutation-negative glioblastoma

PubMed Central

Barth, Rolf F.; Miyatake, Shin-Ichi; Kawabata, Shinji; Suzuki, Minoru; Ono, Koji

2017-01-01

We have used boron neutron capture therapy (BNCT) to treat patients in Japan with newly diagnosed or recurrent high-grade gliomas and have observed a significant increase in median survival time following BNCT. Although cerebrospinal fluid dissemination (CSFD) is not usually seen with the current standard therapy of patients with glioblastoma (GBM), here we report that subarachnoid or intraventricular CSFD was the most frequent cause of death for a cohort of our patients with high-grade gliomas who had been treated with BNCT. The study population consisted of 87 patients with supratentorial high-grade gliomas; 41 had newly diagnosed tumors and 46 had recurrent tumors. Thirty of 87 patients who were treated between January 2002 and July 2013 developed CSFD. Tumor histology before BNCT and immunohistochemical staining for two molecular markers, Ki-67 and IDH1R132H, were evaluated for 20 of the 30 patients for whom pathology slides were available. Fluorescence in situ hybridization (FISH) was performed on 3 IDH1R132H-positive and 1 control IDH1R132H-negative tumors in order to determine chromosome 1p and 19q status. Histopathologic evaluation revealed that 10 of the 20 patients’ tumors were IDH1R132H-negative small cell GBMs. The remaining patients had tumors consisting of other IDH1R132H-negative GBM variants, an IDH1R132H-positive GBM and two anaplastic oligodendrogliomas. Ki-67 immunopositivity ranged from 2 to 75%. In summary, IDH1R132H-negative GBMs, especially small cell GBMs, accounted for a disproportionately large number of patients who had CSF dissemination. This suggests that these tumor types had an increased propensity to disseminate via the CSF following BNCT and that these patients are at high risk for this clinically serious event. PMID:28534152

Cerebrospinal fluid dissemination of high-grade gliomas following boron neutron capture therapy occurs more frequently in the small cell subtype of IDH1R132H mutation-negative glioblastoma.

PubMed

Kondo, Natsuko; Barth, Rolf F; Miyatake, Shin-Ichi; Kawabata, Shinji; Suzuki, Minoru; Ono, Koji; Lehman, Norman L

2017-05-01

We have used boron neutron capture therapy (BNCT) to treat patients in Japan with newly diagnosed or recurrent high-grade gliomas and have observed a significant increase in median survival time following BNCT. Although cerebrospinal fluid dissemination (CSFD) is not usually seen with the current standard therapy of patients with glioblastoma (GBM), here we report that subarachnoid or intraventricular CSFD was the most frequent cause of death for a cohort of our patients with high-grade gliomas who had been treated with BNCT. The study population consisted of 87 patients with supratentorial high-grade gliomas; 41 had newly diagnosed tumors and 46 had recurrent tumors. Thirty of 87 patients who were treated between January 2002 and July 2013 developed CSFD. Tumor histology before BNCT and immunohistochemical staining for two molecular markers, Ki-67 and IDH1 R132H , were evaluated for 20 of the 30 patients for whom pathology slides were available. Fluorescence in situ hybridization (FISH) was performed on 3 IDH1 R132H -positive and 1 control IDH1 R132H -negative tumors in order to determine chromosome 1p and 19q status. Histopathologic evaluation revealed that 10 of the 20 patients' tumors were IDH1 R132H -negative small cell GBMs. The remaining patients had tumors consisting of other IDH1 R132H -negative GBM variants, an IDH1 R132H -positive GBM and two anaplastic oligodendrogliomas. Ki-67 immunopositivity ranged from 2 to 75%. In summary, IDH1 R132H -negative GBMs, especially small cell GBMs, accounted for a disproportionately large number of patients who had CSF dissemination. This suggests that these tumor types had an increased propensity to disseminate via the CSF following BNCT and that these patients are at high risk for this clinically serious event.

Convection-enhanced delivery of an anti-miR is well-tolerated, preserves anti-miR stability and causes efficient target de-repression: a proof of concept.

PubMed

Halle, Bo; Marcusson, Eric G; Aaberg-Jessen, Charlotte; Jensen, Stine S; Meyer, Morten; Schulz, Mette K; Andersen, Claus; Kristensen, Bjarne W

2016-01-01

Over-expressed microRNAs (miRs) are promising new targets in glioblastoma (GBM) therapy. Inhibition of over-expressed miRs has been shown to diminish GBM proliferation, invasion and angiogenesis, indicating a significant therapeutic potential. However, the methods utilized for miR inhibition have had low translational potential. In clinical trials convection-enhanced delivery (CED) has been applied for local delivery of compounds in the brain. The aim of this study was to determine if safe and efficient miR inhibition was possible by CED of an anti-miR. We used a highly invasive GBM orthotopic xenograft model and targeted a well-validated miR, let-7a, with a 2'-O-methoxyethyl anti-miR with a combined phosphodiester/phosphorothioate backbone to establish an initial proof of concept. In vitro, anti-let-7a was delivered unassisted to the patient-derived T87 glioblastoma spheroid culture. In vivo, anti-let-7a or saline were administered by CED into orthotopic T87-derived tumors. After 1 month of infusion, tumors were removed and tumor mRNA levels of the target-gene High-mobility group AT-hook 2 (HMGA2) were determined. In vitro, 5 days inhibition was superior to 1 day at de-repressing the let-7a target HMGA2 and the inhibition was stable for 24 h. In vivo, anti-miR integrity was preserved in the pumps and no animals showed signs of severe adverse effects attributable to the anti-miR treatment. HMGA2 tumor level was significantly de-repressed in the anti-miR treated animals. The results showed-as an initial proof of concept-that miRs can be efficiently inhibited using CED delivery of anti-miR. The next step is to apply CED for anti-miR delivery focusing on key oncogenic miRs.

Dose-Dense Temozolomide for Newly Diagnosed Glioblastoma: A Randomized Phase III Clinical Trial

PubMed Central

Gilbert, Mark R.; Wang, Meihua; Aldape, Kenneth D.; Stupp, Roger; Hegi, Monika E.; Jaeckle, Kurt A.; Armstrong, Terri S.; Wefel, Jeffrey S.; Won, Minhee; Blumenthal, Deborah T.; Mahajan, Anita; Schultz, Christopher J.; Erridge, Sara; Baumert, Brigitta; Hopkins, Kristen I.; Tzuk-Shina, Tzahala; Brown, Paul D.; Chakravarti, Arnab; Curran, Walter J.; Mehta, Minesh P.

2013-01-01

Purpose Radiotherapy with concomitant and adjuvant temozolomide is the standard of care for newly diagnosed glioblastoma (GBM). O6-methylguanine-DNA methyltransferase (MGMT) methylation status may be an important determinant of treatment response. Dose-dense (DD) temozolomide results in prolonged depletion of MGMT in blood mononuclear cells and possibly in tumor. This trial tested whether DD temozolomide improves overall survival (OS) or progression-free survival (PFS) in patients with newly diagnosed GBM. Patients and Methods This phase III trial enrolled patients older than age 18 years with a Karnofsky performance score of ≥ 60 with adequate tissue. Stratification included clinical factors and tumor MGMT methylation status. Patients were randomly assigned to standard temozolomide (arm 1) or DD temozolomide (arm 2) for 6 to 12 cycles. The primary end point was OS. Secondary analyses evaluated the impact of MGMT status. Results A total of 833 patients were randomly assigned to either arm 1 or arm 2 (1,173 registered). No statistically significant difference was observed between arms for median OS (16.6 v 14.9 months, respectively; hazard ratio [HR], 1.03; P = .63) or median PFS (5.5 v 6.7 months; HR, 0.87; P = .06). Efficacy did not differ by methylation status. MGMT methylation was associated with improved OS (21.2 v 14 months; HR, 1.74; P < .001), PFS (8.7 v 5.7 months; HR, 1.63; P < .001), and response (P = .012). There was increased grade ≥ 3 toxicity in arm 2 (34% v 53%; P < .001), mostly lymphopenia and fatigue. Conclusion This study did not demonstrate improved efficacy for DD temozolomide for newly diagnosed GBM, regardless of methylation status. However, it did confirm the prognostic significance of MGMT methylation. Feasibility of large-scale accrual, prospective tumor collection, and molecular stratification was demonstrated. PMID:24101040

Combinatorial Effects of VEGFR Kinase Inhibitor Axitinib and Oncolytic Virotherapy in Mouse and Human Glioblastoma Stem-Like Cell Models.

PubMed

Saha, Dipongkor; Wakimoto, Hiroaki; Peters, Cole W; Antoszczyk, Slawomir J; Rabkin, Samuel D; Martuza, Robert L

2018-03-29

Purpose: Glioblastoma (GBM), a fatal brain cancer, contains a subpopulation of GBM stem-like cells (GSCs) that contribute to resistance to current therapy. Angiogenesis also plays a key role in GBM progression. Therefore, we developed a strategy to target the complex GBM microenvironment, including GSCs and tumor vasculature. Experimental Design: We evaluated the cytotoxic effects of VEFGR tyrosine kinase inhibitor (TKI) axitinib in vitro and then tested antitumor efficacy of axitinib in combination with oncolytic herpes simplex virus (oHSV) expressing antiangiogenic cytokine murine IL12 (G47Δ-mIL12) in two orthotopic GSC-derived GBM models: patient-derived recurrent MGG123 GSCs, forming vascular xenografts in immunodeficient mice; and mouse 005 GSCs, forming syngeneic tumors in immunocompetent mice. Results: GSCs form endothelial-like tubes and were sensitive to axitinib. G47Δ-mIL12 significantly improved survival, as did axitinib, while dual combinations further extended survival significantly compared with single therapies alone in both models. In MGG123 tumors, axitinib was effective only at high doses (50 mg/kg), alone and in combination with G47Δ-mIL12, and this was associated with greatly decreased vascularity, increased macrophage infiltration, extensive tumor necrosis, and PDGFR/ERK pathway inhibition. In the mouse 005 model, antiglioma activity, after single and combination therapy, was only observed in immunocompetent mice and not the T-cell-deficient athymic mice. Interestingly, immune checkpoint inhibition did not improve efficacy. Conclusions: Systemic TKI (axitinib) beneficially combines with G47Δ-mIL12 to enhance antitumor efficacy in both immunodeficient and immunocompetent orthotopic GBM models. Our results support further investigation of TKIs in combination with oHSV for GBM treatment. Clin Cancer Res; 1-14. ©2018 AACR. ©2018 American Association for Cancer Research.

Combination of systemic chemotherapy with local stem cell delivered S-TRAIL in resected brain tumors.

PubMed

Redjal, Navid; Zhu, Yanni; Shah, Khalid

2015-01-01

Despite advances in standard therapies, the survival of glioblastoma multiforme (GBM) patients has not improved. Limitations to successful translation of new therapies include poor delivery of systemic therapies and use of simplified preclinical models which fail to reflect the clinical complexity of GBMs. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis specifically in tumor cells and we have tested its efficacy by on-site delivery via engineered stem cells (SC) in mouse models of GBM that mimic the clinical scenario of tumor aggressiveness and resection. However, about half of tumor lines are resistant to TRAIL and overcoming TRAIL-resistance in GBM by combining therapeutic agents that are currently in clinical trials with SC-TRAIL and understanding the molecular dynamics of these combination therapies are critical to the broad use of TRAIL as a therapeutic agent in clinics. In this study, we screened clinically relevant chemotherapeutic agents for their ability to sensitize resistant GBM cell lines to TRAIL induced apoptosis. We show that low dose cisplatin increases surface receptor expression of death receptor 4/5 post G2 cycle arrest and sensitizes GBM cells to TRAIL induced apoptosis. In vivo, using an intracranial resection model of resistant primary human-derived GBM and real-time optical imaging, we show that a low dose of cisplatin in combination with synthetic extracellular matrix encapsulated SC-TRAIL significantly decreases tumor regrowth and increases survival in mice bearing GBM. This study has the potential to help expedite effective translation of local stem cell-based delivery of TRAIL into the clinical setting to target a broad spectrum of GBMs. © 2014 AlphaMed Press.

Ccl5 establishes an autocrine high-grade glioma growth regulatory circuit critical for mesenchymal glioblastoma survival

PubMed Central

Pan, Yuan; Smithson, Laura J.; Ma, Yu; Hambardzumyan, Dolores; Gutmann, David H.

2017-01-01

Glioblastoma (GBM) is the most common malignant brain tumor in adults, with a median survival of 15 months. These poor clinical outcomes have prompted the development of drugs that block neoplastic cancer cell growth; however, non-neoplastic cell-derived signals (chemokines and cytokines) in the tumor microenvironment may also represent viable treatment targets. One such chemokine, Ccl5, produced by low-grade tumor-associated microglia, is responsible for maintaining neurofibromatosis type 1 (NF1) mouse optic glioma growth in vivo. Since malignant gliomas may achieve partial independence from growth regulatory factors produced by non-neoplastic cells in the tumor microenvironment by producing the same cytokines secreted by the stromal cells in their low-grade counterparts, we tested the hypothesis that CCL5/CCL5-receptor signaling in glioblastoma creates an autocrine circuit important for high-grade glioma growth. Herein, we demonstrate that increased CCL5 expression was restricted to both human and mouse mesenchymal GBM (M-GBM), a molecular subtype characterized by NF1 loss. We further show that the NF1 protein, neurofibromin, negatively regulates Ccl5 expression through suppression of AKT/mTOR signaling. Consistent with its role as a glioblastoma growth regulator, Ccl5 knockdown in M-GBM cells reduces M-GBM cell survival in vitro, and increases mouse glioblastoma survival in vivo. Finally, we demonstrate that Ccl5 operates through an unconventional CCL5 receptor, CD44, to inhibit M-GBM apoptosis. Collectively, these findings reveal an NF1-dependent CCL5-mediated pathway that regulates M-GBM cell survival, and support the concept that paracrine factors important for low-grade glioma growth can be usurped by high-grade tumors to create autocrine regulatory circuits that maintain malignant glioma survival. PMID:28380429

Selective sensitiveness of mesenchymal stem cells to shock waves leads to anticancer effect in human cancer cell co-cultures.

PubMed

Foglietta, Federica; Duchi, Serena; Canaparo, Roberto; Varchi, Greta; Lucarelli, Enrico; Dozza, Barbara; Serpe, Loredana

2017-03-15

Mesenchymal stem cells (MSC) possess the distinctive feature of homing in on and engrafting into the tumor stroma making their therapeutic applications in cancer treatment very promising. Research into new effectors and external stimuli, which can selectively trigger the release of cytotoxic species from MSC toward the cancer cells, significantly raises their potential. Shock waves (SW) have recently gained recognition for their ability to induce specific biological effects, such as the local generation of cytotoxic reactive oxygen species (ROS) in a non-invasive and tunable manner. We thus investigate whether MSC are able to generate ROS and, in turn, affect cancer cell growth when in co-culture with human glioblastoma (U87) or osteosarcoma (U2OS) cells and exposed to SW. MSC were found to be the cell line that was most sensitive to SW treatment as shown by SW-induced ROS production and cytotoxicity. Notably, U87 and U2OS cancer cell growth was unaffected by SW exposure. However, significant decreases in cancer cell growth, 1.8 fold for U87 and 2.3 fold for U2OS, were observed 24h after the SW treatment of MSC co-cultures with cancer cells. The ROS production induced in MSC by SW exposure was then responsible for lipid peroxidation and cell death in U87 and U2OS cells co-cultured with MSC. This experiment highlights the unique ability of MSC to generate ROS upon SW treatment and induce the cell death of co-cultured cancer cells. SW might therefore be proposed as an innovative tool for MSC-mediated cancer treatment. Copyright © 2017 Elsevier Inc. All rights reserved.

Autocrine VEGF–VEGFR2–Neuropilin-1 signaling promotes glioma stem-like cell viability and tumor growth

PubMed Central

Hamerlik, Petra; Lathia, Justin D.; Rasmussen, Rikke; Wu, Qiulian; Bartkova, Jirina; Lee, MyungHee; Moudry, Pavel; Bartek, Jiri; Fischer, Walter; Lukas, Jiri

2012-01-01

Although vascular endothelial growth factor (VEGF) receptor 2 (VEGFR2) is traditionally regarded as an endothelial cell protein, evidence suggests that VEGFRs may be expressed by cancer cells. Glioblastoma multiforme (GBM) is a lethal cancer characterized by florid vascularization and aberrantly elevated VEGF. Antiangiogenic therapy with the humanized VEGF antibody bevacizumab reduces GBM tumor growth; however, the clinical benefits are transient and invariably followed by tumor recurrence. In this study, we show that VEGFR2 is preferentially expressed on the cell surface of the CD133+ human glioma stem-like cells (GSCs), whose viability, self-renewal, and tumorigenicity rely, at least in part, on signaling through the VEGF-VEGFR2–Neuropilin-1 (NRP1) axis. We find that the limited impact of bevacizumab-mediated VEGF blockage may reflect ongoing autocrine signaling through VEGF–VEGFR2–NRP1, which is associated with VEGFR2–NRP1 recycling and a pool of active VEGFR2 within a cytosolic compartment of a subset of human GBM cells. Whereas bevacizumab failed to inhibit prosurvival effects of VEGFR2-mediated signaling, GSC viability under unperturbed or radiation-evoked stress conditions was attenuated by direct inhibition of VEGFR2 tyrosine kinase activity and/or shRNA-mediated knockdown of VEGFR2 or NRP1. We propose that direct inhibition of VEGFR2 kinase may block the highly dynamic VEGF–VEGFR2–NRP1 pathway and inspire a GBM treatment strategy to complement the currently prevalent ligand neutralization approach. PMID:22393126

Estimation of the effectiveness ratio (α/β) for resistant cancer cells in U87MG human glioblastoma.

PubMed

Marmolejo-León, Perla; Azorín-Vega, Erika Patricia; Jiménez-Mancilla, Nallely; Mendoza-Nava, Héctor Javier; Mitsoura, Eleni; Pineda, Benjamín; Torres-García, Eugenio

2018-01-11

Glioblastoma contains self-renewing, tumorigenic cancer stem-like cells that contribute to tumor initiation and therapeutic resistance. The aim of this research was to estimate and compare the effectiveness ratio (α/β) of stem-like cells and differentiated glioma cells derived from the U87MG glioblastoma cell line. Cell survival experiments were obtained in a dose range of 0-20 Gy (13.52 ± 0.09 Gy/h) as a hyperfractionationated accelerated radiotherapy scheme. Biochemical characterization of the post-irradiated cells was performed by flow cytometry analysis and the percentage of stem-like cells that resisted irradiation was determined by the CD133 expression. Results showed that U87MG stem-like cells are highly proliferative and more radioresistant than the U87MG adherent group (with a lesser stem-like character), this in association with the calculated α/β ratio of 17 and 14.1, respectively. Copyright © 2018 Elsevier Ltd. All rights reserved.

Desmethylanhydroicaritin isolated from Sophora flavescens, shows antitumor activities in U87MG cells via inhibiting the proliferation, migration and invasion.

PubMed

Kang, Chang-Won; Kim, Nan-Hee; Jung, Huyn Ah; Choi, Hyung-Wook; Kang, Min-Jae; Choi, Jae-Sue; Kim, Gun-Do

2016-04-01

This study is the first report of the antitumor activities of desmethylanhydroicaritin (DMAI) isolated from Sophora flavescens on U87MG cells. Human glioblastoma is one of the most aggressive malignant type of brain tumors and highly diffuses to around normal brain tissues. DMAI showed anti-proliferation effects on U87MG cells at the concentration of 30μM, however did not affect to HEK-293 cells. DMAI induced anti-proliferation effects via ERK/MAPK, PI3K/Akt/mTOR signal pathway and G2/M phase cell cycle arrest. DMAI led to morphological change and inhibition of filapodia formation through regulation of Rac 1 and Cdc 42. In addition, migration and invasion of U87MG cells were inhibited by DMAI via down-regulation of matrix metalloproteinase (MMP) -2 and MMP -9 expressions and activities. Our results suggest that DMAI has a potential as a therapeutic agent against glioblastoma cells. Copyright © 2016 Elsevier B.V. All rights reserved.

Alternative Polyadenylation in Glioblastoma Multiforme and Changes in Predicted RNA Binding Protein Profiles

PubMed Central

Shao, Jiaofang; Zhang, Jing; Zhang, Zengming; Jiang, Huawei; Lou, Xiaoyan; Foltz, Gregory; Lan, Qing; Huang, Qiang

2013-01-01

Abstract Alternative polyadenylation (APA) is widely present in the human genome and plays a key role in carcinogenesis. We conducted a comprehensive analysis of the APA products in glioblastoma multiforme (GBM, one of the most lethal brain tumors) and normal brain tissues and further developed a computational pipeline, RNAelements (http://sysbio.zju.edu.cn/RNAelements/), using covariance model from known RNA binding protein (RBP) targets acquired by RNA Immunoprecipitation (RIP) analysis. We identified 4530 APA isoforms for 2733 genes in GBM, and found that 182 APA isoforms from 148 genes showed significant differential expression between normal and GBM brain tissues. We then focused on three genes with long and short APA isoforms that show inconsistent expression changes between normal and GBM brain tissues. These were myocyte enhancer factor 2D, heat shock factor binding protein 1, and polyhomeotic homolog 1 (Drosophila). Using the RNAelements program, we found that RBP binding sites were enriched in the alternative regions between the first and the last polyadenylation sites, which would result in the short APA forms escaping regulation from those RNA binding proteins. To the best of our knowledge, this report is the first comprehensive APA isoform dataset for GBM and normal brain tissues. Additionally, we demonstrated a putative novel APA-mediated mechanism for controlling RNA stability and translation for APA isoforms. These observations collectively lay a foundation for novel diagnostics and molecular mechanisms that can inform future therapeutic interventions for GBM. PMID:23421905

Preclinical efficacy of immune-checkpoint monotherapy does not recapitulate corresponding biomarkers-based clinical predictions in glioblastoma

PubMed Central

Vandenberk, Lien; Van Woensel, Matthias; Schaaf, Marco; De Vleeschouwer, Steven; Agostinis, Patrizia

2017-01-01

ABSTRACT Glioblastoma (GBM) is resistant to most multimodal therapies. Clinical success of immune-checkpoint inhibitors (ICIs) has spurred interest in applying ICIs targeting CTLA4, PD1 or IDO1 against GBM. This amplifies the need to ascertain GBM's intrinsic susceptibility (or resistance) toward these ICIs, through clinical biomarkers that may also “guide and prioritize” preclinical testing. Here, we interrogated the TCGA and/or REMBRANDT human patient-cohorts to predict GBM's predisposition toward ICIs. We exploited various broad clinical biomarkers, including mutational or predicted-neoantigen burden, pre-existing or basal levels of tumor-infiltrating T lymphocytes (TILs), differential expression of immune-checkpoints within the tumor and their correlation with particular TILs/Treg-associated functional signature and prognostic impact of differential immune-checkpoint expression. Based on these analyses, we found that predictive biomarkers of ICI responsiveness exhibited inconsistent patterns in GBM patients, i.e., they either predicted ICI resistance (as compared with typical ICI-responsive cancer-types like melanoma, lung cancer or bladder cancer) or susceptibility to therapeutic targeting of CTLA4 or IDO1. On the other hand, our comprehensive literature meta-analysis and preclinical testing of ICIs using an orthotopic GL261-glioma mice model, indicated significant antitumor properties of anti-PD1 antibody, whereas blockade of IDO1 or CTLA4 either failed or provided very marginal advantage. These trends raise the need to better assess the applicability of ICIs and associated biomarkers for GBM. PMID:28507806

Combined expressional analysis, bioinformatics and targeted proteomics identify new potential therapeutic targets in glioblastoma stem cells.

PubMed

Stangeland, Biljana; Mughal, Awais A; Grieg, Zanina; Sandberg, Cecilie Jonsgar; Joel, Mrinal; Nygård, Ståle; Meling, Torstein; Murrell, Wayne; Vik Mo, Einar O; Langmoen, Iver A

2015-09-22

Glioblastoma (GBM) is both the most common and the most lethal primary brain tumor. It is thought that GBM stem cells (GSCs) are critically important in resistance to therapy. Therefore, there is a strong rationale to target these cells in order to develop new molecular therapies.To identify molecular targets in GSCs, we compared gene expression in GSCs to that in neural stem cells (NSCs) from the adult human brain, using microarrays. Bioinformatic filtering identified 20 genes (PBK/TOPK, CENPA, KIF15, DEPDC1, CDC6, DLG7/DLGAP5/HURP, KIF18A, EZH2, HMMR/RHAMM/CD168, NOL4, MPP6, MDM1, RAPGEF4, RHBDD1, FNDC3B, FILIP1L, MCC, ATXN7L4/ATXN7L1, P2RY5/LPAR6 and FAM118A) that were consistently expressed in GSC cultures and consistently not expressed in NSC cultures. The expression of these genes was confirmed in clinical samples (TCGA and REMBRANDT). The first nine genes were highly co-expressed in all GBM subtypes and were part of the same protein-protein interaction network. Furthermore, their combined up-regulation correlated negatively with patient survival in the mesenchymal GBM subtype. Using targeted proteomics and the COGNOSCENTE database we linked these genes to GBM signalling pathways.Nine genes: PBK, CENPA, KIF15, DEPDC1, CDC6, DLG7, KIF18A, EZH2 and HMMR should be further explored as targets for treatment of GBM.

Spatiotemporal prediction of continuous daily PM2.5 concentrations across China using a spatially explicit machine learning algorithm

NASA Astrophysics Data System (ADS)

Zhan, Yu; Luo, Yuzhou; Deng, Xunfei; Chen, Huajin; Grieneisen, Michael L.; Shen, Xueyou; Zhu, Lizhong; Zhang, Minghua

2017-04-01

A high degree of uncertainty associated with the emission inventory for China tends to degrade the performance of chemical transport models in predicting PM2.5 concentrations especially on a daily basis. In this study a novel machine learning algorithm, Geographically-Weighted Gradient Boosting Machine (GW-GBM), was developed by improving GBM through building spatial smoothing kernels to weigh the loss function. This modification addressed the spatial nonstationarity of the relationships between PM2.5 concentrations and predictor variables such as aerosol optical depth (AOD) and meteorological conditions. GW-GBM also overcame the estimation bias of PM2.5 concentrations due to missing AOD retrievals, and thus potentially improved subsequent exposure analyses. GW-GBM showed good performance in predicting daily PM2.5 concentrations (R2 = 0.76, RMSE = 23.0 μg/m3) even with partially missing AOD data, which was better than the original GBM model (R2 = 0.71, RMSE = 25.3 μg/m3). On the basis of the continuous spatiotemporal prediction of PM2.5 concentrations, it was predicted that 95% of the population lived in areas where the estimated annual mean PM2.5 concentration was higher than 35 μg/m3, and 45% of the population was exposed to PM2.5 >75 μg/m3 for over 100 days in 2014. GW-GBM accurately predicted continuous daily PM2.5 concentrations in China for assessing acute human health effects.

Targeting delivery of etoposide to inhibit the growth of human glioblastoma multiforme using lactoferrin- and folic acid-grafted poly(lactide-co-glycolide) nanoparticles.

PubMed

Kuo, Yung-Chih; Chen, Yu-Chun

2015-02-01

Lactoferrin (Lf) and folic acid (FA) were crosslinked on poly(lactide-co-glycolide) (PLGA) nanoparticles (NPs) for transporting etoposide across the blood-brain barrier (BBB) and treating human brain malignant glioblastoma. Lf- and FA-grafted PLGA NPs (Lf/FA/PLGA NPs) were employed to permeate the monolayer of human brain-microvascular endothelial cells (HBMECs) regulated by human astrocytes and to inhibit the multiplication of U87MG cells. Lf/FA/PLGA NPs showed a satisfactory entrapment efficiency of etoposide and characteristics of sustained drug release. When compared with PLGA NPs, the permeability coefficient for etoposide across the BBB using Lf/FA/PLGA NPs increased about twofold. The antiproliferative efficacy against the growth of U87MG cells was in the following order: Lf/FA/PLGA NPs>FA/PLGA NPs>PLGA NPs>free etoposide solution. In addition, the targeting ability of Lf/FA/PLGA NPs was evidenced by immunostaining of Lf receptor on HBMECs and folate receptor on U87MG cells during endocytosis. Lf/FA/PLGA NPs with loaded etoposide can be a promising anticancer pharmacotherapy to enhance the delivery of etoposide to malignant brain tumors for preclinical trials. Copyright © 2014 Elsevier B.V. All rights reserved.

Phostine PST3.1a Targets MGAT5 and Inhibits Glioblastoma-Initiating Cell Invasiveness and Proliferation.

PubMed

Hassani, Zahra; Saleh, Ali; Turpault, Soumaya; Khiati, Salim; Morelle, Willy; Vignon, Jacques; Hugnot, Jean-Philippe; Uro-Coste, Emmanuelle; Legrand, Philippe; Delaforge, Marcel; Loiseau, Séverine; Clarion, Ludovic; Lecouvey, Marc; Volle, Jean-Noël; Virieux, David; Pirat, Jean-Luc; Duffau, Hugues; Bakalara, Norbert

2017-10-01

Glioblastoma multiforme (GBM) is the most common primary malignant brain tumor and accounts for a significant proportion of all primary brain tumors. Median survival after treatment is around 15 months. Remodeling of N-glycans by the N-acetylglucosamine glycosyltransferase (MGAT5) regulates tumoral development. Here, perturbation of MGAT5 enzymatic activity by the small-molecule inhibitor 3-hydroxy-4,5-bis-benzyloxy-6-benzyloxymethyl-2-phenyl2-oxo-2λ5-[1,2]oxaphosphinane (PST3.1a) restrains GBM growth. In cell-based assays, it is demonstrated that PST3.1a alters the β1,6-GlcNAc N-glycans of GBM-initiating cells (GIC) by inhibiting MGAT5 enzymatic activity, resulting in the inhibition of TGFβR and FAK signaling associated with doublecortin (DCX) upregulation and increase oligodendrocyte lineage transcription factor 2 (OLIG2) expression. PST3.1a thus affects microtubule and microfilament integrity of GBM stem cells, leading to the inhibition of GIC proliferation, migration, invasiveness, and clonogenic capacities. Orthotopic graft models of GIC revealed that PST3.1a treatment leads to a drastic reduction of invasive and proliferative capacity and to an increase in overall survival relative to standard temozolomide therapy. Finally, bioinformatics analyses exposed that PST3.1a cytotoxic activity is positively correlated with the expression of genes of the epithelial-mesenchymal transition (EMT), while the expression of mitochondrial genes correlated negatively with cell sensitivity to the compound. These data demonstrate the relevance of targeting MGAT5, with a novel anti-invasive chemotherapy, to limit glioblastoma stem cell invasion. Mol Cancer Res; 15(10); 1376-87. ©2017 AACR . ©2017 American Association for Cancer Research.

Development of Advanced Technologies for Complete Genomic and Proteomic Characterization of Quantized Human Tumor Cells

DTIC Science & Technology

2013-07-01

extending the period of performance soon. The Ivy Center for Advanced Brain Tumor Treatment at the Swedish Neuroscience Institute (SNI) has...markers: (A) GFAP/astrocytes, (B), TUJ-1/neurons and (C) O4/oligodendrocytes. Cells were grown in NSA media without growth factors (EGF and FGF-2...Treatment at the Swedish Neuroscience Institute (SNI) has collected potentially eligible tumor tissue from over forty GBM patients. • Primary GBM cell

Genome-wide identification of epithelial-mesenchymal transition-associated microRNAs reveals novel targets for glioblastoma therapy

PubMed Central

Zhang, Yong; Zeng, Ailiang; Liu, Shuheng; Li, Rui; Wang, Xiefeng; Yan, Wei; Li, Hailin; You, Yongping

2018-01-01

MicroRNAs (miRNA) regulate a number of cellular processes. Recent studies have indicated that these molecules function in the epithelial-mesenchymal transition (EMT). However, the crucial systematic role of EMT and miRNAs together in glioblastoma (GBM) remains poorly understood. The present study demonstrated that EMT was closely associated with malignant progression and clinical outcome using three independent glioma databases (GSE16011, Rembrandt and The Cancer Genome Atlas). Furthermore, integrated analysis of miRNAs and mRNA profiling in 491 GBM samples revealed an EMT biological process associated with an miRNA profile (19 positively and 18 negatively correlated miRNAs). Among these miRNAs, miR-95 and miR-223 indicated a high level of functional validation, reflecting their positive correlation with EMT. Additionally, the upregulation of miR-95, which was negatively correlated with EMT, inhibited cellular invasion in glioma U251 and LN229 cells and decreased the expression of the mesenchymal marker N-catenin, whereas an miRNA positively correlated with EMT, miR-223, exhibited the opposite effect. Therefore, the results of the present study could further enhance the current understanding of the functions of miRNAs in GBM, indicating that the EMT-specific miRNA signature may represent a novel target for GBM therapy. PMID:29740486

Disruption of prion protein-HOP engagement impairs glioblastoma growth and cognitive decline and improves overall survival.

PubMed

Lopes, M H; Santos, T G; Rodrigues, B R; Queiroz-Hazarbassanov, N; Cunha, I W; Wasilewska-Sampaio, A P; Costa-Silva, B; Marchi, F A; Bleggi-Torres, L F; Sanematsu, P I; Suzuki, S H; Oba-Shinjo, S M; Marie, S K N; Toulmin, E; Hill, A F; Martins, V R

2015-06-01

Glioblastomas (GBMs) are resistant to current therapy protocols and identification of molecules that target these tumors is crucial. Interaction of secreted heat-shock protein 70 (Hsp70)-Hsp90-organizing protein (HOP) with cellular prion protein (PrP(C)) triggers a large number of trophic effects in the nervous system. We found that both PrP(C) and HOP are highly expressed in human GBM samples relative to non-tumoral tissue or astrocytoma grades I-III. High levels of PrP(C) and HOP were associated with greater GBM proliferation and lower patient survival. HOP-PrP(C) binding increased GBM proliferation in vitro via phosphatidylinositide 3-kinase and extracellular-signal-regulated kinase pathways, and a HOP peptide mimicking the PrP(C) binding site (HOP230-245) abrogates this effect. PrP(C) knockdown impaired tumor growth and increased survival of mice with tumors. In mice, intratumor delivery of HOP230-245 peptide impaired proliferation and promoted apoptosis of GBM cells. In addition, treatment with HOP230-245 peptide inhibited tumor growth, maintained cognitive performance and improved survival. Thus, together, the present results indicate that interfering with PrP(C)-HOP engagement is a promising approach for GBM therapy.

Multifuntional Nanotherapeutics for the Combinatorial Drug and Gene Therapy in the Treatment of Glioblastoma Multiforme

NASA Astrophysics Data System (ADS)

Hourigan, Breanne

Glioblastoma multiforme (GBM), a grade IV glioma, is the most common primary brain tumor, affecting about 3 out of 100,000 persons per year in the United States. GBM accounts for about 80% of primary malignant brain tumors, and is also the most aggressive of malignant brain tumors. With exhaustive treatment, survival only averages between 12 and 15 months, with a 2-year survival rate less than 25%. New therapeutic strategies are necessary to improve the outcomes of this disease. Chemotherapy with temozolomide (TMZ), a DNA alkylating agent, is used as a first-line of treatment for GBM. However, GBM tumors develop resistance to TMZ over time due to increased expression of O6-methylguanine-DNA methyltransferase (MGMT), a gene responsible for DNA repair. We previously developed cationic, amphiphilic copolymer poly(lactide-co-glycolide)-g-polyethylenimine (PgP) and demonstrated its utility for nucleic acid delivery. Here, we examine the ability of PgP polyplexes to overcome TMZ resistance and improve therapeutic efficacy through combination drug and gene therapy for GBM treatment. In this study, we evaluated the ability of PgP to deliver siRNA targeting to MGMT (siMGMT), a gene responsible for drug resistance in GBM. Our results demonstrated that PgP effectively forms stable complex with siRNA and protects siRNAs from heparin competition assay, serum- and ribonuclease-mediated degradation, confirming the potential of the polyplex for in vivo delivery. Results from MTT assays showed that PgP/siRNA polyplexes exhibited minimal cytotoxicity compared to untreated cells when incubated with T98G human GBM cells. We also demonstrated that PgP/siMGMT polyplexes mediate knockdown of MGMT protein as well as a significant ˜56% and ˜68% knockdown of MGMT mRNA in T98G GBM cells compared to cells treated with PgP complexed with non-targeting siRNA (siNT) at a 60:1 and 80:1 nitrogen:phosphate (N:P) ratio, respectively. Further, co-incubation of PgP/siMGMT polyplexes with TMZ enhanced therapeutic efficacy in T98G GBM cells compared to treatment with the polyplex or TMZ alone. After generation of athymic mouse GBM model, PgP/siMGMT polyplexes were locally injected into the tumor. Relative to untreated injury only, PgP/siMGMT polyplexes significantly reduced MGMT mRNA and protein expression at 3 days post-injection. These studies demonstrate that PgP is an efficient non-viral delivery carrier for therapeutic siMGMT to the tumor cells and may be a promising platform for the combinatorial siRNA/drug therapy for GBM treatment. In the future, we will study the therapeutic efficacy of combination of PgP/siMGMT and TMZ in athymic mouse GBM model.

Effect of doublecortin on self-renewal and differentiation in brain tumor stem cells

PubMed Central

Santra, Manoranjan; Santra, Sutapa; Buller, Ben; Santra, Kastuv; Nallani, Ankita; Chopp, Michael

2011-01-01

Analysis of Affymetrix Probe data from glioma patient samples in conjuction with patient Kaplan-Meier Survival Plot indicate that expression of a glioma suppressor gene doublecortin (DCX) favors glioma patient survival. From neurosphere formation in culture, Time-Lapse Microscopy video recording and tumor xenograft, we show that DCX synthesis significantly reduces self-renewal of brain tumor stem cells (BTSCs) in human primary glioma (YU-PG, HF66) cells from surgically-removed human glioma specimens and U87 cells in vitro and in vivo. Time-Lapse Microscopic video recording revealed that double transfection of YU-PG, HF66 and U87 cells with DCX and neurabin II caused incomplete cell cycle with failure of cytokinesis, i.e. endomitosis by dividing into three daughter cells from one mother BTSC. Activation of c-jun NH2-terminal kinase 1 (JNK1) after simvastatin (10nM) treatment of DCX+neurabin II+ BTSCs from YU-PG, HF66 and U87 cells induced terminal differentiation into neuron-like cells. TUNEL staining data demonstrated that JNK1 activation also induced apoptosis only in double transfected BTSCs with DCX and neurabin II, but not in single transfected BTSCs from YU-PG, HF66 and U87 cells. Western blot analysis showed that procaspase-3 was induced after DCX transfection and activated after simvastatin treatment in YU-PG, HF66 and U87 BTSCs. Sequential immunoprecipitation and Western blot data revealed that DCX synthesis blocked protein phosphatase-1 (PP1)/caspase-3 protein-protein interaction and increased PP1-DCX interaction. These data demonstrate that DCX synthesis induces apoptosis in BTSCs via a novel JNK1/neurabin II/DCX/PP1/caspase-3 pathway. PMID:21477071

Effect of doublecortin on self-renewal and differentiation in brain tumor stem cells.

PubMed

Santra, Manoranjan; Santra, Sutapa; Buller, Ben; Santra, Kastuv; Nallani, Ankita; Chopp, Michael

2011-07-01

Analysis of microarray probe data from glioma patient samples, in conjunction with patient Kaplan-Meier survival plots, indicates that expression of a glioma suppressor gene doublecortin (DCX) favors glioma patient survival. From neurosphere formation in culture, time-lapse microscopic video recording, and tumor xenograft, we show that DCX synthesis significantly reduces self-renewal of brain tumor stem cells (BTSC) in human primary glioma (YU-PG, HF66) cells from surgically removed human glioma specimens and U87 cells in vitro and in vivo. Time-lapse microscopic video recording revealed that double transfection of YU-PG, HF66, and U87 cells with DCX and neurabin II caused incomplete cell cycle with failure of cytokinesis, that is, endomitosis by dividing into three daughter cells from one mother BTSC. Activation of c-jun NH2-terminal kinase 1 (JNK1) after simvastatin (10 nM) treatment of DCX(+) neurabin II(+) BTSC from YU-PG, HF66, and U87 cells induced terminal differentiation into neuron-like cells. dUTP nick end labeling data indicated that JNK1 activation also induced apoptosis only in double transfected BTSC with DCX and neurabin II, but not in single transfected BTSC from YU-PG, HF66, and U87 cells. Western blot analysis showed that procaspase-3 was induced after DCX transfection and activated after simvastatin treatment in YU-PG, HF66, and U87 BTSC. Sequential immunoprecipitation and Western blot data revealed that DCX synthesis blocked protein phosphatase-1 (PP1)/caspase-3 protein-protein interaction and increased PP1-DCX interaction. These data show that DCX synthesis induces apoptosis in BTSC through a novel JNK1/neurabin II/DCX/PP1/caspase-3 pathway. © 2011 Japanese Cancer Association.

Near-infrared optical imaging in glioblastoma xenograft with ligand targeting α3 integrin

PubMed Central

Xiao, Wenwu; Yao, Nianhuan; Peng, Li; Liu, Ruiwu; Lam, Kit S

2010-01-01

Purpose Patients with glioblastoma usually have a very poor prognosis. Even with a combination of radiotherapy plus temozolomide, the median survival of these patients is only 14.6 months. New treatment approaches to this cancer are needed. Our purpose is to develop new cell-surface binding ligands for glioblastoma cells, and use them as targeted imaging and therapeutic agents for this deadly disease. Methods One-bead one-compound combinatorial cyclic peptide libraries were screened with live human glioblastoma U-87MG cells. The binding affinity and targeting specificity of peptides identified were tested with in vitro experiments on cells and in vivo, and ex vivo experiments on U-87MG xegnograft mouse model. Results A cyclic peptide, LXY1, was identified and shown to be binding to the α3 integrin of U-87MG cells with moderately high affinity (Kd = 0.5+/−0.1 μM) and high specificity. Biotinylated LXY1, when complexed with streptavidin-Cy5.5 (SA-Cy5.5) conjugate, targeted both subcutaneous and orthotopic U-87MG xenograft implants in nude mice. The in vivo targeting specificity was further verified by strong inhibition of tumor uptake of LXY1-biotin-SA-Cy5.5 complex when intravenously injecting the animals with anti-α3 integrin antibody or excess unlabeled LXY1 prior to administrating the imaging probe. The smaller univalent LXY1-Cy5.5 conjugate (2279 Da) was found to have a faster accumulation in the U-87MG tumor and shorter retention time compared with the larger tetravalent LXY1-biotin-SA-Cy5.5 complex (~ 64 KDa). Conclusions Collectively, the data reveals that LXY1 has the potential to be developed into an effective imaging and therapeutic targeting agent for human glioblastoma. PMID:18712382

Immature myeloid cells in the tumor microenvironment: Implications for immunotherapy.

PubMed

Kamran, Neha; Chandran, Mayuri; Lowenstein, Pedro R; Castro, Maria G

2018-04-01

Various preclinical studies have demonstrated that the success of immunotherapeutic strategies in inhibiting tumor progression in animal models of Glioblastoma multiforme (GBM). It is also evident that tumor-induced immune suppression drastically impacts the efficacy of immune based therapies. Among the mechanisms employed by GBM to induce immunosuppression is the accumulation of regulatory T cells (Tregs) and Myeloid derived suppressor cells (MDSCs). Advancing our understanding about the pathways regulating the expansion, accumulation and activity of MDSCs will allow for the development of therapies aimed at abolishing the inhibitory effect of these cells on immunotherapeutic approaches. In this review, we have focused on the origin, expansion and immunosuppressive mechanisms of MDSCs in animal models and human cancer, in particular GBM. Copyright © 2016 Elsevier Inc. All rights reserved.

Targeting miR-381-NEFL axis sensitizes glioblastoma cells to temozolomide by regulating stemness factors and multidrug resistance factors.

PubMed

Wang, Zeyou; Yang, Jing; Xu, Gang; Wang, Wei; Liu, Changhong; Yang, Honghui; Yu, Zhibin; Lei, Qianqian; Xiao, Lan; Xiong, Jing; Zeng, Liang; Xiang, Juanjuan; Ma, Jian; Li, Guiyuan; Wu, Minghua

2015-02-20

MicroRNA-381 (miR-381) is a highly expressed onco-miRNA that is involved in malignant progression and has been suggested to be a good target for glioblastoma multiforme (GBM) therapy. In this study, we employed two-dimensional fluorescence differential gel electrophoresis (2-D DIGE) and MALDI-TOF/TOF-MS/MS to identify 27 differentially expressed proteins, including the significantly upregulated neurofilament light polypeptide (NEFL), in glioblastoma cells in which miR-381 expression was inhibited. We identified NEFL as a novel target molecule of miR-381 and a tumor suppressor gene. In human astrocytoma clinical specimens, NEFL was downregulated with increased levels of miR-381 expression. Either suppressing miR-381 or enforcing NEFL expression dramatically sensitized glioblastoma cells to temozolomide (TMZ), a promising chemotherapeutic agent for treating GBMs. The mechanism by which these cells were sensitized to TMZ was investigated by inhibiting various multidrug resistance factors (ABCG2, ABCC3, and ABCC5) and stemness factors (ALDH1, CD44, CKIT, KLF4, Nanog, Nestin, and SOX2). Our results further demonstrated that miR-381 overexpression reversed the viability of U251 cells exhibiting NEFL-mediated TMZ sensitivity. In addition, NEFL-siRNA also reversed the proliferation rate of U251 cells exhibiting locked nucleic acid (LNA)-anti-miR-381-mediated TMZ sensitivity. Overall, the miR-381-NEFL axis is important for TMZ resistance in GBM and may potentially serve as a novel therapeutic target for glioma.

Demographic variation in incidence of adult glioma by subtype, United States, 1992-2007.

PubMed

Dubrow, Robert; Darefsky, Amy S

2011-07-29

We hypothesized that race/ethnic group, sex, age, and/or calendar period variation in adult glioma incidence differs between the two broad subtypes of glioblastoma (GBM) and non-GBM. Primary GBM, which constitute 90-95% of GBM, differ from non-GBM with respect to a number of molecular characteristics, providing a molecular rationale for these two broad glioma subtypes. We utilized data from the Surveillance, Epidemiology, and End Results Program for 1992-2007, ages 30-69 years. We compared 15,088 GBM cases with 9,252 non-GBM cases. We used Poisson regression to calculate adjusted rate ratios and 95% confidence intervals. The GBM incidence rate increased proportionally with the 4th power of age, whereas the non-GBM rate increased proportionally with the square root of age. For each subtype, compared to non-Hispanic Whites, the incidence rate among Blacks, Asians/Pacific Islanders, and American Indians/Alaskan Natives was substantially lower (one-fourth to one-half for GBM; about two-fifths for non-GBM). Secondary to this primary effect, race/ethnic group variation in incidence was significantly less for non-GBM than for GBM. For each subtype, the incidence rate was higher for males than for females, with the male/female rate ratio being significantly higher for GBM (1.6) than for non-GBM (1.4). We observed significant calendar period trends of increasing incidence for GBM and decreasing incidence for non-GBM. For the two subtypes combined, we observed a 3% decrease in incidence between 1992-1995 and 2004-2007. The substantial difference in age effect between GBM and non-GBM suggests a fundamental difference in the genesis of primary GBM (the driver of GBM incidence) versus non-GBM. However, the commonalities between GBM and non-GBM with respect to race/ethnic group and sex variation, more notable than the somewhat subtle, albeit statistically significant, differences, suggest that within the context of a fundamental difference, some aspects of the complex process of gliomagenesis are shared by these subtypes as well. The increasing calendar period trend of GBM incidence coupled with the decreasing trend of non-GBM incidence may at least partly be due to a secular trend in diagnostic fashion, as opposed to real changes in incidence of these subtypes.

Over-expression of tetraspanin 8 in malignant glioma regulates tumor cell progression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pan, Si-Jian; Wu, Yue-Bing; Cai, Shang

Tumor cell invasion and proliferation remain the overwhelming causes of death for malignant glioma patients. To establish effective therapeutic methods, new targets implied in these processes have to be identified. Tetraspanin 8 (Tspn8) forms complexes with a large variety of trans-membrane and/or cytosolic proteins to regulate several important cellular functions. In the current study, we found that Tspn8 was over-expressed in multiple clinical malignant glioma tissues, and its expression level correlated with the grade of tumors. Tspn8 expression in malignant glioma cells (U251MG and U87MG lines) is important for cell proliferation and migration. siRNA-mediated knockdown of Tspn8 markedly reduced in vitromore » proliferation and migration of U251MG and U87MG cells. Meanwhile, Tspn8 silencing also increased the sensitivity of temozolomide (TMZ), and significantly increased U251MG or U87MG cell death and apoptosis by TMZ were achieved with Tspn8 knockdown. We observed that Tspn8 formed a complex with activated focal adhesion kinase (FAK) in both human malignant glioma tissues and in above glioma cells. This complexation appeared required for FAK activation, since Tspn8 knockdown inhibited FAK activation in U251MG and U87MG cells. These results provide evidence that Tspn8 contributes to the pathogenesis of glioblastoma probably by promoting proliferation, migration and TMZ-resistance of glioma cells. Therefore, targeting Tspn8 may provide a potential therapeutic intervention for malignant glioma. - Highlights: • Tspn8 is over-expressed in multiple clinical malignant glioma tissues. • Tspn8 expression is correlated with the grade of malignant gliomas. • Tspn8 knockdown suppresses U251MG/U87MG proliferation and in vitro migration. • Tspn8 knockdown significantly increases TMZ sensitivity in U251MG/U87MG cells. • Tspn8 forms a complex with FAK, required for FAK activation.« less

A PML/Slit Axis Controls Physiological Cell Migration and Cancer Invasion in the CNS.

PubMed

Amodeo, Valeria; A, Deli; Betts, Joanne; Bartesaghi, Stefano; Zhang, Ying; Richard-Londt, Angela; Ellis, Matthew; Roshani, Rozita; Vouri, Mikaella; Galavotti, Sara; Oberndorfer, Sarah; Leite, Ana Paula; Mackay, Alan; Lampada, Aikaterini; Stratford, Eva Wessel; Li, Ningning; Dinsdale, David; Grimwade, David; Jones, Chris; Nicotera, Pierluigi; Michod, David; Brandner, Sebastian; Salomoni, Paolo

2017-07-11

Cell migration through the brain parenchyma underpins neurogenesis and glioblastoma (GBM) development. Since GBM cells and neuroblasts use the same migratory routes, mechanisms underlying migration during neurogenesis and brain cancer pathogenesis may be similar. Here, we identify a common pathway controlling cell migration in normal and neoplastic cells in the CNS. The nuclear scaffold protein promyelocytic leukemia (PML), a regulator of forebrain development, promotes neural progenitor/stem cell (NPC) and neuroblast migration in the adult mouse brain. The PML pro-migratory role is active also in transformed mouse NPCs and in human primary GBM cells. In both normal and neoplastic settings, PML controls cell migration via Polycomb repressive complex 2 (PRC2)-mediated repression of Slits, key regulators of axon guidance. Finally, a PML/SLIT1 axis regulates sensitivity to the PML-targeting drug arsenic trioxide in primary GBM cells. Taken together, these findings uncover a drug-targetable molecular axis controlling cell migration in both normal and neoplastic cells. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

EG-10LONG NON-CODING RNAs IN GLIOBLASTOMA

PubMed Central

Pastori, Chiara; Kapranov, Philipp; Penas, Clara; Laurent, Georges St.; Ayad, Nagi; Wahlestedt, Claes

2014-01-01

Glioblastoma (GBM) is the most common, aggressive and incurable primary brain tumor in adults. Genome studies have confirmed that GBM is extremely heterogeneous with many genetically different subgroups. Consequently, there is much current interest in epigenetic drugs that may be active across genetically distinct tumors. In support of this, some epigenetic drugs has recently shown efficacy against several cancers including glioblastoma. Much recent interest has also been devoted to long non-coding RNAs (lncRNAs), which can modulate gene expression regulating chromatin architecture, in part through the interaction with epigenetic protein machineries. To date, however, only a few lncRNAs have been studied in human cancer. We therefore embarked on a comprehensive genomic and functional analysis of lncRNAs in GBM. Using the Helicos Single Molecule Sequencing platform glioblastoma samples were sequenced resulting in the identification of hundreds of dysregulated lncRNAs. Among these the lncRNA HOTAIR was found massively increased in GBM. This observation parallels findings in other cancers where HOTAIR's increased expression has been linked to poor prognosis due to metastatic events. Interestingly, here we show that in glioblastoma HOTAIR does not promote metastasis, but instead sustains the ability of these cells to proliferate. In fact, we demonstrate that HOTAIR knockdown in GBM strongly impairs cell proliferation and induces apoptosis in vitro and in vivo. Further, we implicate HOTAIR in the mechanism of action of certain epigenetic drugs. In summary, long noncoding RNAs (newly discovered epigenomic factors) play a vital role in GBM and deserve attention as entirely novel drug targets as well as biomarkers.

Investigating the Link between Molecular Subtypes of Glioblastoma, Epithelial-Mesenchymal Transition, and CD133 Cell Surface Protein

PubMed Central

Zarkoob, Hadi; Taube, Joseph H.; Singh, Sheila K.; Mani, Sendurai A.; Kohandel, Mohammad

2013-01-01

In this manuscript, we use genetic data to provide a three-faceted analysis on the links between molecular subclasses of glioblastoma, epithelial-to-mesenchymal transition (EMT) and CD133 cell surface protein. The contribution of this paper is three-fold: First, we use a newly identified signature for epithelial-to-mesenchymal transition in human mammary epithelial cells, and demonstrate that genes in this signature have significant overlap with genes differentially expressed in all known GBM subtypes. However, the overlap between genes up regulated in the mesenchymal subtype of GBM and in the EMT signature was more significant than other GBM subtypes. Second, we provide evidence that there is a negative correlation between the genetic signature of EMT and that of CD133 cell surface protein, a putative marker for neural stem cells. Third, we study the correlation between GBM molecular subtypes and the genetic signature of CD133 cell surface protein. We demonstrate that the mesenchymal and neural subtypes of GBM have the strongest correlations with the CD133 genetic signature. While the mesenchymal subtype of GBM displays similarity with the signatures of both EMT and CD133, it also exhibits some differences with each of these signatures that are partly due to the fact that the signatures of EMT and CD133 are inversely related to each other. Taken together these data shed light on the role of the mesenchymal transition and neural stem cells, and their mutual interaction, in molecular subtypes of glioblastoma multiforme. PMID:23734191

First clinical results of a personalized immunotherapeutic vaccine against recurrent, incompletely resected, treatment-resistant glioblastoma multiforme (GBM) tumors, based on combined allo- and auto-immune tumor reactivity.

PubMed

Schijns, Virgil E J C; Pretto, Chrystel; Devillers, Laurent; Pierre, Denis; Hofman, Florence M; Chen, Thomas C; Mespouille, Pascal; Hantos, Peter; Glorieux, Philippe; Bota, Daniela A; Stathopoulos, Apostolos

2015-05-28

Glioblastoma multiforme (GBM) patients have a poor prognosis. After tumor recurrence statistics suggest an imminent death within 1-4.5 months. Supportive preclinical data, from a rat model, provided the rational for a prototype clinical vaccine preparation, named Gliovac (or ERC 1671) composed of autologous antigens, derived from the patient's surgically removed tumor tissue, which is administered together with allogeneic antigens from glioma tissue resected from other GBM patients. We now report the first results of the Gliovac treatment for treatment-resistant GBM patients. Nine (9) recurrent GBM patients, after standard of care treatment, including surgery radio- and chemotherapy temozolomide, and for US patients, also bevacizumab (Avastin™), were treated under a compassionate use/hospital exemption protocol. Gliovac was given intradermally, together with human GM-CSF (Leukine(®)), and preceded by a regimen of regulatory T cell-depleting, low-dose cyclophosphamide. Gliovac administration in patients that have failed standard of care therapies showed minimal toxicity and enhanced overall survival (OS). Six-month (26 weeks) survival for the nine Gliovac patients was 100% versus 33% in control group. At week 40, the published overall survival was 10% if recurrent, reoperated patients were not treated. In the Gliovac treated group, the survival at 40 weeks was 77%. Our data suggest that Gliovac has low toxicity and a promising efficacy. A phase II trial has recently been initiated in recurrent, bevacizumab naïve GBM patients (NCT01903330). Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

Colony stimulating factor 1 receptor inhibition delays recurrence of glioblastoma after radiation by altering myeloid cell recruitment and polarization

PubMed Central

Stafford, Jason H.; Hirai, Takahisa; Deng, Lei; Chernikova, Sophia B.; Urata, Kimiko; West, Brian L.; Brown, J. Martin

2016-01-01

Background Glioblastoma (GBM) may initially respond to treatment with ionizing radiation (IR), but the prognosis remains extremely poor because the tumors invariably recur. Using animal models, we previously showed that inhibiting stromal cell–derived factor 1 signaling can prevent or delay GBM recurrence by blocking IR-induced recruitment of myeloid cells, specifically monocytes that give rise to tumor-associated macrophages. The present study was aimed at determining if inhibiting colony stimulating factor 1 (CSF-1) signaling could be used as an alternative strategy to target pro-tumorigenic myeloid cells recruited to irradiated GBM. Methods To inhibit CSF-1 signaling in myeloid cells, we used PLX3397, a small molecule that potently inhibits the tyrosine kinase activity of the CSF-1 receptor (CSF-1R). Combined IR and PLX3397 therapy was compared with IR alone using 2 different human GBM intracranial xenograft models. Results GBM xenografts treated with IR upregulated CSF-1R ligand expression and increased the number of CD11b+ myeloid-derived cells in the tumors. Treatment with PLX3397 both depleted CD11b+ cells and potentiated the response of the intracranial tumors to IR. Median survival was significantly longer for mice receiving combined therapy versus IR alone. Analysis of myeloid cell differentiation markers indicated that CSF-1R inhibition prevented IR-recruited monocyte cells from differentiating into immunosuppressive, pro-angiogenic tumor-associated macrophages. Conclusion CSF-1R inhibition may be a promising strategy to improve GBM response to radiotherapy. PMID:26538619

Effect of saw palmetto extract on PI3K cell signaling transduction in human glioma.

PubMed

Yang, Yang; Hui, Lv; Yuqin, Che; Jie, Li; Shuai, Hou; Tiezhu, Zhou; Wei, Wang

2014-08-01

Saw palmetto extract can induce the apoptosis of prostate cancer cells. The aim of the present study was to investigate the effect of saw palmetto extract on the phosphatidylinositol 3-kinase (PI3K)/Akt signaling transduction pathway in human glioma U87 and U251 cell lines. Suspensions of U87 and U251 cells in a logarithmic growth phase were seeded into six-well plates at a density of 10 4 cells/well. In the experimental group, 1 μl/ml saw palmetto extract was added, while the control group was cultured without a drug for 24 h. The expression levels of PI3K, B-cell lymphoma-extra large (Bcl-xL) and p53 were evaluated through western blot analysis. In the experimental group, the U87 and U251 cells exhibited a lower expression level of PI3K protein as compared with the control group (t=6.849; P<0.001). In addition, the two cell lines had a higher expression level of p53 protein in the experimental group as compared with the control group (t=40.810; P<0.001). Protein expression levels of Bcl-xL decreased significantly in the experimental group as compared with the control group (t=19.640; P=0.000). Therefore, saw palmetto extract induces glioma cell growth arrest and apoptosis via decreasing PI3K/Akt signal transduction.

SI113, a SGK1 inhibitor, potentiates the effects of radiotherapy, modulates the response to oxidative stress and induces cytotoxic autophagy in human glioblastoma multiforme cells

PubMed Central

Talarico, Cristina; Dattilo, Vincenzo; D'Antona, Lucia; Barone, Agnese; Amodio, Nicola; Belviso, Stefania; Musumeci, Francesca; Abbruzzese, Claudia; Bianco, Cataldo; Trapasso, Francesco; Schenone, Silvia; Alcaro, Stefano; Ortuso, Francesco; Florio, Tullio; Paggi, Marco G.; Perrotti, Nicola; Amato, Rosario

2016-01-01

Glioblastoma multiforme (GBM) is the most aggressive CNS tumor and is characterized by a very high frequency of clinical relapse after therapy and thus by a dismal prognosis, which strongly compromises patients survival. We have recently identified the small molecule SI113, as a potent and selective inhibitor of SGK1, a serine/threonine protein kinase, that modulates several oncogenic signaling cascades. The SI113-dependent SGK1 inhibition induces cell death, blocks proliferation and perturbs cell cycle progression by modulating SGK1-related substrates. SI113 is also able to strongly and consistently block, in vitro and in vivo, growth and survival of human hepatocellular-carcinomas, either used as a single agent or in combination with ionizing radiations. In the present paper we aim to study the effect of SI113 on human GBM cell lines with variable p53 expression. Cell viability, cell death, caspase activation and cell cycle progression were then analyzed by FACS and WB-based assays, after exposure to SI113, with or without oxidative stress and ionizing radiations. Moreover, autophagy and related reticulum stress response were evaluated. We show here, that i) SGK1 is over-expressed in highly malignant gliomas and that the treatment with SI113 leads to ii) significant increase in caspase-mediated apoptotic cell death in GBM cell lines but not in normal fibroblasts; iii)enhancement of the effects of ionizing radiations; iv) modulation of the response to oxidative reticulum stress; v) induction of cytotoxic autophagy. Evidence reported here underlines the therapeutic potential of SI113 in GBM, suggesting a new therapeutic strategy either alone or in combination with radiotherapy. PMID:26908461

SI113, a SGK1 inhibitor, potentiates the effects of radiotherapy, modulates the response to oxidative stress and induces cytotoxic autophagy in human glioblastoma multiforme cells.

PubMed

Talarico, Cristina; Dattilo, Vincenzo; D'Antona, Lucia; Barone, Agnese; Amodio, Nicola; Belviso, Stefania; Musumeci, Francesca; Abbruzzese, Claudia; Bianco, Cataldo; Trapasso, Francesco; Schenone, Silvia; Alcaro, Stefano; Ortuso, Francesco; Florio, Tullio; Paggi, Marco G; Perrotti, Nicola; Amato, Rosario

2016-03-29

Glioblastoma multiforme (GBM) is the most aggressive CNS tumor and is characterized by a very high frequency of clinical relapse after therapy and thus by a dismal prognosis, which strongly compromises patients survival. We have recently identified the small molecule SI113, as a potent and selective inhibitor of SGK1, a serine/threonine protein kinase, that modulates several oncogenic signaling cascades. The SI113-dependent SGK1 inhibition induces cell death, blocks proliferation and perturbs cell cycle progression by modulating SGK1-related substrates. SI113 is also able to strongly and consistently block, in vitro and in vivo, growth and survival of human hepatocellular-carcinomas, either used as a single agent or in combination with ionizing radiations. In the present paper we aim to study the effect of SI113 on human GBM cell lines with variable p53 expression. Cell viability, cell death, caspase activation and cell cycle progression were then analyzed by FACS and WB-based assays, after exposure to SI113, with or without oxidative stress and ionizing radiations. Moreover, autophagy and related reticulum stress response were evaluated. We show here, that i) SGK1 is over-expressed in highly malignant gliomas and that the treatment with SI113 leads to ii) significant increase in caspase-mediated apoptotic cell death in GBM cell lines but not in normal fibroblasts; iii)enhancement of the effects of ionizing radiations; iv) modulation of the response to oxidative reticulum stress; v) induction of cytotoxic autophagy. Evidence reported here underlines the therapeutic potential of SI113 in GBM, suggesting a new therapeutic strategy either alone or in combination with radiotherapy.

Bioactivity and Safety of IL13Rα2-Redirected Chimeric Antigen Receptor CD8+ T Cells in Patients with Recurrent Glioblastoma.

PubMed

Brown, Christine E; Badie, Behnam; Barish, Michael E; Weng, Lihong; Ostberg, Julie R; Chang, Wen-Chung; Naranjo, Araceli; Starr, Renate; Wagner, Jamie; Wright, Christine; Zhai, Yubo; Bading, James R; Ressler, Julie A; Portnow, Jana; D'Apuzzo, Massimo; Forman, Stephen J; Jensen, Michael C

2015-09-15

A first-in-human pilot safety and feasibility trial evaluating chimeric antigen receptor (CAR)-engineered, autologous primary human CD8(+) cytotoxic T lymphocytes (CTL) targeting IL13Rα2 for the treatment of recurrent glioblastoma (GBM). Three patients with recurrent GBM were treated with IL13(E13Y)-zetakine CD8(+) CTL targeting IL13Rα2. Patients received up to 12 local infusions at a maximum dose of 10(8) CAR-engineered T cells via a catheter/reservoir system. We demonstrate the feasibility of manufacturing sufficient numbers of autologous CTL clones expressing an IL13(E13Y)-zetakine CAR for redirected HLA-independent IL13Rα2-specific effector function for a cohort of patients diagnosed with GBM. Intracranial delivery of the IL13-zetakine(+) CTL clones into the resection cavity of 3 patients with recurrent disease was well-tolerated, with manageable temporary brain inflammation. Following infusion of IL13-zetakine(+) CTLs, evidence for transient anti-glioma responses was observed in 2 of the patients. Analysis of tumor tissue from 1 patient before and after T-cell therapy suggested reduced overall IL13Rα2 expression within the tumor following treatment. MRI analysis of another patient indicated an increase in tumor necrotic volume at the site of IL13-zetakine(+) T-cell administration. These findings provide promising first-in-human clinical experience for intracranial administration of IL13Rα2-specific CAR T cells for the treatment of GBM, establishing a foundation on which future refinements of adoptive CAR T-cell therapies can be applied. ©2015 American Association for Cancer Research.

Melatonin inhibits proliferation and invasion via repression of miRNA-155 in glioma cells.

PubMed

Gu, Junyi; Lu, Zhongsheng; Ji, Chenghong; Chen, Yuchao; Liu, Yuzhao; Lei, Zhe; Wang, Longqiang; Zhang, Hong-Tao; Li, Xiangdong

2017-09-01

Melatonin, an indolamine mostly synthesized in the pineal gland, exerts the anti-cancer effect by various mechanisms in glioma cells. Our previous study showed that miR-155 promoted glioma cell proliferation and invasion. However, the question of whether melatonin may inhibit glioma by regulating miRNAs has not yet been addressed. In this study, we found that melatonin (100μM, 1μM and 1nM) significantly inhibited the expression of miR-155 in human glioma cell lines U87, U373 and U251. Especially, the lowest expression of miR-155 was detected in 1μM melatonin-treated glioma cells. Melatonin (1μM) inhibits cell proliferation of U87 by promoting cell apoptosis. Nevertheless, melatonin had no effect on cell cycle distribution of U87 cells. Moreover, U87 cells treated with 1μM melatonin presented significantly lower migration and invasion ability when compared with control cells. Importantly, melatonin inhibited c-MYB expression, and c-MYB knockdown reduced miR-155 expression and migration and invasion in U87 cells. Taken together, for the first time, our findings show that melatonin inhibits miR-155 expression and thereby represses glioma cell proliferation, migration and invasion, and suggest that melatonin may downregulate the expression of miR-155 via repression of c-MYB. This will provide a theoretical basis for revealing the anti-glioma mechanisms of melatonin. Copyright © 2017. Published by Elsevier Masson SAS.

Recruiting a U.S. national sample of HIV-negative gay and bisexual men to complete at-home self-administered HIV/STI testing and surveys: Challenges and Opportunities

PubMed Central

Grov, Christian; Cain, Demetria; Whitfield, Thomas H. F.; Rendina, H. Jonathon; Pawson, Mark; Ventuneac, Ana; Parsons, Jeffrey T.

2015-01-01

We describe enrollment for the One Thousand Strong panel, present characteristics of the panel relative to other large U.S. national studies of gay and bisexual men (GBM), and examine demographic and behavioral characteristics that were associated with passing enrollment milestones. A U.S. national sample of HIV-negative men were enrolled via an established online panel of over 22,000 GBM. Participants (n = 1071) passed three milestones to join our panel. Milestone 1 was screening eligible and providing informed consent. Milestone 2 involved completing an hour-long at-home computer-assisted self-interview (CASI) survey. Milestone 3 involved completing at-home self-administered rapid HIV testing and collecting/returning urine and rectal samples for gonorrhea and chlamydia testing. Compared to those who completed milestones: those not passing milestone 1 were more likely to be non-White and older; those not passing milestone 2 were less likely to have insurance or a primary care physician; and those not passing milestone 3 were less educated, more likely to be bisexual as opposed to gay, more likely to live in the Midwest, had fewer male partners in the past year, and less likely to have tested for HIV in the past year. Effect sizes for significant findings were small. We successfully enrolled a national sample of HIV-negative GBM who completed at-home CASI assessments and at-home self-administered HIV and urine and rectal STI testing. This indicates high feasibility and acceptability of incorporating self-administered biological assays into otherwise fully online studies. Differences in completion of study milestones indicate a need for further investigation into the reasons for lower engagement by certain groups. PMID:26858776

Cytopathic Effects of X-ray Irradiation and MnO Nanoparticles on Human Glioblastoma (U87)

NASA Astrophysics Data System (ADS)

Kuper, K. E.; Zavjalov, E. L.; Razumov, I. A.; Romaschenko, A. V.; Stupak, A. S.; Troicky, S. Yu; Goldenberg, B. G.; Legkodymov, A. G.; Lemzyakov, A. A.; Moshkin, M. P.

Glioblastoma is a leader among the most malignant brain tumors with the average lifespan of patients around 9-12 months. For prevention and treatment of neuropathology, a variety of therapeutic and surgical approaches are being developed and improved, including radiation and chemical therapy methods. In our work, we investigated cytopathic effect of X-ray irradiation with application of metal oxides nanoparticles such as manganese oxide (MnO) on U87 human glioblastoma cells. We used the X-ray irradiation dose of 0.5, 4, 40 and 100 Gy in combination with nanoparticles at the concentration of 0.5 ng/ml. The irradiation of glioma cell was carried out at the synchrotron radiation source VEPP-4. After cells treatments with nanoparticles for about 24 h and radiation the results were assessed by MTT assay test with 106/ml cells densities. We demonstrate that preincubation of the glioblastoma cell lines U87 with MnO nanoparticles allows reducing dose of irradiation. This combination of nanoparticles and X-ray irradiation provides new possibilities for the treatment of brain tumors.

The delayed luminescence spectroscopy as tool to investigate the cytotoxic effect on human cancer cells of drug-loaded nanostructured lipid carrier

NASA Astrophysics Data System (ADS)

Grasso, R.; Gulino, M.; Scordino, A.; Musumeci, F.; Campisi, A.; Bonfanti, R.; Carbone, C.; Puglisi, G.

2016-05-01

The first results concerning the possibility to use Delayed Luminescence spectroscopy to evaluate the in vitro induction of cytotoxic effects on human glioblastoma cells of nanostructured lipid carrier and drug-loaded nanostructured lipid carrier are showed in this contribution. We tested the effects of nanostructured lipid carrier, ferulic acid and ferulic acidloaded nanostructured lipid carrier on U-87MG cell line. The study seems to confirm the ability of Delayed Luminescence to be sensible indicator of alterations induced on functionality of the mitochondrial respiratory chain complex I in U-87MG cancer cells when treated with nanostructured lipid carriers.

Reversing glioma malignancy: a new look at the role of antidepressant drugs as adjuvant therapy for glioblastoma multiforme.

PubMed

Bielecka-Wajdman, Anna M; Lesiak, Marta; Ludyga, Tomasz; Sieroń, Aleksander; Obuchowicz, Ewa

2017-06-01

The role of glioma stem cells (GSCs) in cancer progression is currently debated; however, it is hypothesised that this subpopulation is partially responsible for therapeutic resistance observed in glioblastoma multiforme (GBM). Recent studies have shown that the current treatments not only fail to eliminate the GSC population but even promote GSCs through reprogramming of glioma non-stem cells to stem cells. Since the standard GBM treatment often requires supplementation with adjuvant drugs such as antidepressants, their role in the regulation of the heterogeneous nature of GSCs needs evaluation. We examined the effects of imipramine, amitriptyline, fluoxetine, mirtazapine, agomelatine, escitalopram, and temozolomide on the phenotypic signature (CD44, Ki67, Nestin, Sox1, and Sox2 expression) of GSCs isolated from a human T98G cell line. These drugs were examined in several models of hypoxia (1% oxygen, 2.5% oxygen, and a hypoxia-reoxygenation model) as compared to the standard laboratory conditions (20% oxygen). We report that antidepressant drugs, particularly imipramine and amitriptyline, modulate plasticity, silence the GSC profile, and partially reverse the malignant phenotype of GBM. Moreover, we observed that, in contrast to temozolomide, these tricyclic antidepressants stimulated viability and mitochondrial activity in normal human astrocytes. The ability of phenotype switching from GSC to non-GSC as stimulated by antidepressants (primarily imipramine and amitriptyline) sheds new light on the heterogeneous nature of GSC, as well as the role of antidepressants in adjuvant GBM therapy.

Glioblastoma progression is assisted by induction of immunosuppressive function of pericytes through interaction with tumor cells

PubMed Central

Valdor, Rut; García-Bernal, David; Bueno, Carlos; Ródenas, Mónica; Moraleda, José M.; Macian, Fernando; Martínez, Salvador

2017-01-01

The establishment of immune tolerance during Glioblastoma Multiforme (GBM) progression, is characterized by high levels expression of anti-inflammatory cytokines, which suppress the function of tumor assocciated myeloid cells, and the activation and expansion of tumor antigen specific T cells. However, the mechanisms underlying the failed anti-tumor immune response around the blood vessels during GBM, are poorly understood. The consequences of possible interactions between cancer cells and the perivascular compartment might affect the tumor growth. In this work we show for the first time that GBM cells induce immunomodulatory changes in pericytes in a cell interaction-dependent manner, acquiring an immunosuppresive function that possibly assists the evasion of the anti-tumor immune response and consequently participates in tumor growth promotion. Expression of high levels of anti-inflammatory cytokines was detected in vitro and in vivo in brain pericytes that interacted with GBM cells (GBC-PC). Furthermore, reduction of surface expression of co-stimulatory molecules and major histocompatibility complex molecules in GBC-PC correlated with a failure of antigen presentation to T cells and the acquisition of the ability to supress T cell responses. In vivo, orthotopic xenotransplant of human glioblastoma in an immunocompetent mouse model showed significant GBM cell proliferation and tumor growth after the establishment of interspecific immunotolerance that followed GMB interaction with pericytes. PMID:28978142

Establishment and partial characterization of a human tumor cell line, GBM-HSF, from a glioblastoma multiforme.

PubMed

Qu, Jiagui; Rizak, Joshua D; Fan, Yaodong; Guo, Xiaoxuan; Li, Jiejing; Huma, Tanzeel; Ma, Yuanye

2014-07-01

This paper outlines the establishment of a new and stable cell line, designated GBM-HSF, from a malignant glioblastoma multiforme (GBM) removed from a 65-year-old Chinese woman. This cell line has been grown for 1 year without disruption and has been passaged over 50 times. The cells were adherently cultured in RPMI-1640 media with 10% fetal bovine serum supplementation. Cells displayed spindle and polygonal morphology, and displayed multi-layered growth without evidence of contact inhibition. The cell line had a high growth rate with a doubling time of 51 h. The cells were able to grow without adhering to the culture plates, and 4.5% of the total cells formed colonies in soft agar. The cell line has also been found to form tumors in nude mice and to be of a highly invasive nature. The cells were also partially characterized with RT-PCR. The RT-PCR revealed that Nestin, β-tubulin III, Map2, Klf4, Oct4, Sox2, Nanog, and CD26 were positively transcribed, whereas GFAP, Rex1, and CD133 were negatively transcribed in this cell line. These results suggest that the GBM-HSF cell line will provide a good model to study the properties of cancer stem cells and metastasis. It will also facilitate more detailed molecular and cellular studies of GBM cell division and pathology.

Eva1 Maintains the Stem-like Character of Glioblastoma-Initiating Cells by Activating the Noncanonical NF-κB Signaling Pathway.

PubMed

Ohtsu, Naoki; Nakatani, Yuka; Yamashita, Daisuke; Ohue, Shiro; Ohnishi, Takanori; Kondo, Toru

2016-01-01

Glioblastoma (GBM)-initiating cells (GIC) are a tumorigenic subpopulation that are resistant to radio- and chemotherapies and are the source of disease recurrence. Therefore, the identification and characterization of GIC-specific factors is critical toward the generation of effective GBM therapeutics. In this study, we investigated the role of epithelial V-like antigen 1 (Eva1, also known as myelin protein zero-like 2) in stemness and GBM tumorigenesis. Eva1 was prominently expressed in GICs in vitro and in stem cell marker (Sox2, CD15, CD49f)-expressing cells derived from human GBM tissues. Eva1 knockdown in GICs reduced their self-renewal and tumor-forming capabilities, whereas Eva1 overexpression enhanced these properties. Eva1 deficiency was also associated with decreased expression of stemness-related genes, indicating a requirement for Eva1 in maintaining GIC pluripotency. We further demonstrate that Eva1 induced GIC proliferation through the activation of the RelB-dependent noncanonical NF-κB pathway by recruiting TRAF2 to the cytoplasmic tail. Taken together, our findings highlight Eva1 as a novel regulator of GIC function and also provide new mechanistic insight into the role of noncanonical NF-κB activation in GIC, thus offering multiple potential therapeutic targets for preclinical investigation in GBM. ©2015 American Association for Cancer Research.

Culture on 3D Chitosan-Hyaluronic Acid Scaffolds Enhances Stem Cell Marker Expression and Drug Resistance in Human Glioblastoma Cancer Stem Cells.

PubMed

Wang, Kui; Kievit, Forrest M; Erickson, Ariane E; Silber, John R; Ellenbogen, Richard G; Zhang, Miqin

2016-12-01

The lack of in vitro models that support the growth of glioblastoma (GBM) stem cells (GSCs) that underlie clinical aggressiveness hinders developing new, effective therapies for GBM. While orthotopic patient-derived xenograft models of GBM best reflect in vivo tumor behavior, establishing xenografts is a time consuming, costly, and frequently unsuccessful endeavor. To address these limitations, a 3D porous scaffold composed of chitosan and hyaluronic acid (CHA) is synthesized. Growth and expression of the cancer stem cell (CSC) phenotype of the GSC GBM6 taken directly from fresh xenogratfs grown on scaffolds or as adherent monolayers is compared. While 2D adherent cultures grow as monolayers of flat epitheliod cells, GBM6 cells proliferate within pores of CHA scaffolds as clusters of self-adherent ovoid cells. Growth on scaffolds is accompanied by greater expression of genes that mediate epithelial-mesenchymal transition and maintain a primitive, undifferentiated phenotype, hallmarks of CSCs. Scaffold-grown cells also display higher expression of genes that promote resistance to hypoxia-induced oxidative stress. In accord, scaffold-grown cells show markedly greater resistance to clinically utilized alkylating agents compared to adherent cells. These findings suggest that our CHA scaffolds better mimic in vivo biological and clinical behavior and provide insights for developing novel individualized treatments. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Vaccine-preventable anal human papillomavirus in Australian gay and bisexual men.

PubMed

Poynten, I Mary; Tabrizi, Sepehr N; Jin, Fengyi; Templeton, David J; Machalek, Dorothy A; Cornall, Alyssa; Phillips, Samuel; Fairley, Christopher K; Garland, Suzanne M; Law, Carmella; Carr, Andrew; Hillman, Richard J; Grulich, Andrew E

2017-06-01

HPV causes ~90% of anal cancer and HPV16 is the type most commonly associated with anal cancer. Gay and bisexual men (GBM) are at greatly increased risk. We investigated patterns of vaccine-preventable anal HPV in older GBM. The Study of the Prevention of Anal Cancer (SPANC) is an ongoing, prospective cohort study of HIV-positive and HIV-negative Australian GBM. Participants completed questionnaires and underwent an anal swab for HPV genotyping using Roche Linear Array. We analysed baseline data from SPANC by HPV type, mean number of types, stratified by age and HIV status. Anal HPV results from 606 (98.2%) of 617 participants (median age 49 years, 35.7% HIV-positive) showed 525 (86.7%) had ≥1 HPV type and 178 (29.4%) had HPV16. Over one third of participants (214, 35.3%) had no nonavalent vaccine-preventable types detected. Two (0.3%) participants had all quadrivalent types and none had all nonavalent vaccine types. HIV-positive participants (p<0.001) and younger participants (p=0.059) were more likely to have more vaccine-preventable HPV types detected. Anal HPV was highly prevalent in this largely community-based GBM cohort. Vaccine-preventable HPV16 was detected in approximately one third of participants. These findings suggest that the potential efficacy of HPV vaccination of older GBM should be explored. Crown Copyright © 2016. Published by Elsevier B.V. All rights reserved.

EG-11DYSREGULATION OF MGMT IN GLIOBLASTOMA: FRIEND OR FOE?

PubMed Central

Rapkins, Robert W.; Hitchins, Megan P.; McDonald, Kerrie L.

2014-01-01

Glioblastoma (GBM) is the most common and lethal form of brain cancer (median survival <15 months). The DNA alkylating agent, temozolomide, is used as the standard chemotherapeutic agent, resulting in mispairing of guanine with thymidine that leads to cellular arrest. However, in GBM patients the O6-methylguanine-DNA methyltransferase (MGMT) protein protects DNA from damage induced by temozolomide. Nevertheless, loss of MGMT expression is a frequent event in human malignancies and typically the result of MGMT promoter methylation. MGMT methylation has been strongly associated with the T-allele of the rs16906252 SNP (C/T) in colorectal carcinoma, pleural mesothelioma, and lung cancers. We therefore examined the T-allele and MGMT methylation in temozolmide-treated GBM patients. In 255 temozolomide-treated GBM patients, we found that the T-allele was significantly more frequent in patients with a methylated MGMT promoter. The unadjusted hazard ratio for death in carriers of the T-allele compared to wild-type, irrespective of methylation status, was 0.39 (95%CI:0.21-0.73; p = 0.003), indicating a 61% relative reduction in the risk for death of T-allele carriers. Surprisingly, GBM patients harboring the T-allele in the absence of MGMT methylation showed a survival benefit comparable to those with MGMT methylation (median survival: 15.5 months) and significantly better than the median survival of wild-type, unmethylated patients (median survival: 10.3 months). This suggests that the T-allele may reduce MGMT activity by mechanisms independent of methylation. Genotyping of 451 healthy controls indicated the frequency of carriage of the T-allele was 13% (MAF 0.065). In contrast, carriage of the T-allele in 160 GBM patients was 17%. Significantly, elevated risks were associated with carriage of the T-allele and development of GBM (odds ratio of 2.62 [95%CI:1.7-4.2]). We report that the T-allele (rs16906252) has predictive (response to temozolomide) and prognostic value (MGMT methylation and longer survival), but conversely may be a predisposing factor for the development of GBM.

In vitro antineoplastic effects of brivaracetam and lacosamide on human glioma cells.

PubMed

Rizzo, Ambra; Donzelli, Sara; Girgenti, Vita; Sacconi, Andrea; Vasco, Chiara; Salmaggi, Andrea; Blandino, Giovanni; Maschio, Marta; Ciusani, Emilio

2017-06-06

Epilepsy is a frequent symptom in patients with glioma. Although treatment with antiepileptic drugs is generally effective in controlling seizures, drug-resistant patients are not uncommon. Multidrug resistance proteins (MRPs) and P-gp are over-represented in brain tissue of patients with drug-resistant epilepsy, suggesting their involvement in the clearance of antiepileptic medications. In addition to their anticonvulsant action, some drugs have been documented for cytotoxic effects. Aim of this study was to evaluate possible in vitro cytotoxic effects of two new-generation antiepileptic drugs on a human glioma cell line U87MG. Cytotoxicity of brivaracetam and lacosamide was tested on U87MG, SW1783 and T98G by MTS assay. Expression of chemoresistance molecules was evaluated using flow cytometry in U87MG and human umbilical vein endothelial cells (HUVECs). To investigate the putative anti-proliferative effect, apoptosis assay, microRNA expression profile and study of cell cycle were performed. Brivaracetam and lacosamide showed a dose-dependent cytotoxic and anti-migratory effects. Cytotoxicity was not related to apoptosis. The exposure of glioma cells to brivaracetam and lacosamide resulted in the modulation of several microRNAs; particularly, the effect of miR-195-5p modulation seemed to affect cell cycle, while miR-107 seemed to be implicated in the inhibition of cells migration. Moreover, brivaracetam and lacosamide treatment did not modulate the expression of chemoresistance-related molecules MRPs1-3-5, GSTπ, P-gp on U87MG and HUVECs. Based on antineoplastic effect of brivaracetam and lacosamide on glioma cells, we assume that patients with glioma could benefit by the treatment with these two molecules, in addition to standard therapeutic options.

Cox-2-derived PGE2 induces Id1-dependent radiation resistance and self-renewal in experimental glioblastoma.

PubMed

Cook, Peter J; Thomas, Rozario; Kingsley, Philip J; Shimizu, Fumiko; Montrose, David C; Marnett, Lawrence J; Tabar, Viviane S; Dannenberg, Andrew J; Benezra, Robert

2016-10-01

In glioblastoma (GBM), Id1 serves as a functional marker for self-renewing cancer stem-like cells. We investigated the mechanism by which cyclooxygenase-2 (Cox-2)-derived prostaglandin E2 (PGE2) induces Id1 and increases GBM self-renewal and radiation resistance. Mouse and human GBM cells were stimulated with dimethyl-PGE2 (dmPGE2), a stabilized form of PGE2, to test for Id1 induction. To elucidate the signal transduction pathway governing the increase in Id1, a combination of short interfering RNA knockdown and small molecule inhibitors and activators of PGE2 signaling were used. Western blotting, quantitative real-time (qRT)-PCR, and chromatin immunoprecipitation assays were employed. Sphere formation and radiation resistance were measured in cultured primary cells. Immunohistochemical analyses were carried out to evaluate the Cox-2-Id1 axis in experimental GBM. In GBM cells, dmPGE2 stimulates the EP4 receptor leading to activation of ERK1/2 MAPK. This leads, in turn, to upregulation of the early growth response1 (Egr1) transcription factor and enhanced Id1 expression. Activation of this pathway increases self-renewal capacity and resistance to radiation-induced DNA damage, which are dependent on Id1. In GBM, Cox-2-derived PGE2 induces Id1 via EP4-dependent activation of MAPK signaling and the Egr1 transcription factor. PGE2-mediated induction of Id1 is required for optimal tumor cell self-renewal and radiation resistance. Collectively, these findings identify Id1 as a key mediator of PGE2-dependent modulation of radiation response and lend insight into the mechanisms underlying radiation resistance in GBM patients. © The Author(s) 2016. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Cucurbitacin B purified from Ecballium elaterium (L.) A. Rich from Tunisia inhibits α5β1 integrin-mediated adhesion, migration, proliferation of human glioblastoma cell line and angiogenesis.

PubMed

Touihri-Barakati, Imen; Kallech-Ziri, Olfa; Ayadi, Wiem; Kovacic, Hervé; Hanchi, Belgacem; Hosni, Karim; Luis, José

2017-02-15

Integrins are essential protagonists in the complex multistep process of cancer progression and are thus attractive targets for the development of anticancer agents. Cucurbitacin B, a triterpenoid purified from the leaves of Tunisian Ecballium elaterium exhibited an anticancer effect and displayed anti-integrin activity on human glioblastoma U87 cells, without being cytotoxic at concentrations up to 500nM. Here we show that cucurbitacin B affected the adhesion and migration of U87 cells to fibronectin in a dose-dependent manner with IC50 values of 86.2nM and 84.6nM, respectively. Time-lapse videomicroscopy showed that cucurbitacin B significantly reduced U87 cells motility and affected directional persistence. Cucurbitacin B also inhibited proliferation with IC50 value of 70.1nM using Crystal Violet assay. Moreover, cucurbitacin B efficiently inhibited in vitro human microvascular endothelial cells (HMEC) angiogenesis with concentration up to 10nM. Interestingly, we demonstrate for the first time that this effect was specifically mediated by α5β1 integrins. These findings reveal a novel mechanism of action for cucurbitacin B, which displays a potential interest as a specific anti-integrin drug. Copyright © 2017 Elsevier B.V. All rights reserved.

Suppression of STIM1 inhibits human glioblastoma cell proliferation and induces G0/G1 phase arrest

PubMed Central

2013-01-01

Background Depletion of calcium (Ca2+) from the endoplasmic reticulum (ER) activates the ubiquitous store-operated Ca2+ entry (SOCE) pathway which sustains long-term Ca2+ signals and is critical for cellular functions. Stromal interacting molecule 1 (STIM1) serves a dual role as an ER Ca2+ sensor and activator of SOCE. Aberrant expression of STIM1 could be observed in several human cancer cells. However, the role of STIM1 in regulating tumorigenesis of human glioblastoma still remains unclear. Methods Expression of STIM1 protein in a panel of human glioblastoma cell lines (U251, U87 and U373) in different transformation level were evaluated by Western blot method. STIM1 loss of function was performed on U251 cells, derived from grade IV astrocytomas-glioblastoma multiforme with a lentvirus-mediated short harpin RNA (shRNA) method. The biological impacts after knock down of STIM1 on glioblastoma cells were investigated in vitro and in vivo. Results We discovered that STIM1 protein was expressed in U251, U87 and U373 cells, and especially higher in U251 cells. RNA interference efficiently downregulated the expression of STIM1 in U251 cells at both mRNA and protein levels. Specific downregulation of STIM1 inhibited U251 cell proliferation by inducing cell cycle arrest in G0/G1 phase through regulation of cell cycle-related genes, such as p21Waf1/Cip1, cyclin D1 and cyclin-dependent kinase 4 (CDK4), and the antiproliferative effect of STIM1 silencing was also observed in U251 glioma xenograft tumor model. Conclusion Our findings confirm STIM1 as a rational therapeutic target in human glioblastoma, and also indicate that lentivirus-mediated STIM1 silencing is a promising therapeutic strategy for human glioblastoma. PMID:23578185

Suppression of STIM1 inhibits human glioblastoma cell proliferation and induces G0/G1 phase arrest.

PubMed

Li, Guilin; Zhang, Zhenxing; Wang, Renzhi; Ma, Wenbin; Yang, Ying; Wei, Junji; Wei, Yanping

2013-04-11

Depletion of calcium (Ca2+) from the endoplasmic reticulum (ER) activates the ubiquitous store-operated Ca2+ entry (SOCE) pathway which sustains long-term Ca2+ signals and is critical for cellular functions. Stromal interacting molecule 1 (STIM1) serves a dual role as an ER Ca2+ sensor and activator of SOCE. Aberrant expression of STIM1 could be observed in several human cancer cells. However, the role of STIM1 in regulating tumorigenesis of human glioblastoma still remains unclear. Expression of STIM1 protein in a panel of human glioblastoma cell lines (U251, U87 and U373) in different transformation level were evaluated by Western blot method. STIM1 loss of function was performed on U251 cells, derived from grade IV astrocytomas-glioblastoma multiforme with a lentvirus-mediated short harpin RNA (shRNA) method. The biological impacts after knock down of STIM1 on glioblastoma cells were investigated in vitro and in vivo. We discovered that STIM1 protein was expressed in U251, U87 and U373 cells, and especially higher in U251 cells. RNA interference efficiently downregulated the expression of STIM1 in U251 cells at both mRNA and protein levels. Specific downregulation of STIM1 inhibited U251 cell proliferation by inducing cell cycle arrest in G0/G1 phase through regulation of cell cycle-related genes, such as p21Waf1/Cip1, cyclin D1 and cyclin-dependent kinase 4 (CDK4), and the antiproliferative effect of STIM1 silencing was also observed in U251 glioma xenograft tumor model. Our findings confirm STIM1 as a rational therapeutic target in human glioblastoma, and also indicate that lentivirus-mediated STIM1 silencing is a promising therapeutic strategy for human glioblastoma.

Podoplanin increases migration and angiogenesis in malignant glioma

PubMed Central

Grau, Stefan J; Trillsch, Fabian; Tonn, Joerg-Christian; Goldbrunner, Roland H; Noessner, Elfriede; Nelson, Peter J; von Luettichau, Irene

2015-01-01

Expression of podoplanin in glial brain tumors is grade dependent. While serving as a marker for tumor progression and modulating invasion in various neoplasms, little is known about podoplanin function in gliomas. Therefore we stably transfected two human glioma cell lines (U373MG and U87MG) with expression plasmids encoding podoplanin. The efficacy of transfection was confirmed by FACS analysis, PCR and immunocytochemistry. Cells were then sorted for highly podoplanin expressing cells (U373Phigh/U87Phigh). Transfection did not influence the production of pro-angiogenic factors including VEGF, VEGF-C and D. Also, expression of VEGF receptors (VEGFR) remained unchanged except for U87Phigh, where a VEGFR3 expression was induced. U373Phigh showed significantly reduced proliferation as compared to mock transfected group. By contrast, podoplanin significantly increased migration and invasion into collagen matrix. Furthermore, conditioned media from Phigh glioma cells strongly induced tube formation on matrigel. In conclusion, podoplanin increased migration of tumor cells and enhanced tube formation activity in endothelial cells independent from VEGF. Thus, podoplanin expression may be an important step in tumor progression. PMID:26339454

The synthetic ligand of peroxisome proliferator-activated receptor-gamma ciglitazone affects human glioblastoma cell lines.

PubMed

Strakova, Nicol; Ehrmann, Jiri; Dzubak, Petr; Bouchal, Jan; Kolar, Zdenek

2004-06-01

Glioblastoma multiforme is the most common malignant brain tumor in adults, and it is among the most lethal of all cancers. Recent studies have shown that ligand activation of peroxisome proliferator-activated receptor (PPAR)-gamma can induce differentiation and inhibit proliferation of several cancer cells. In this study, we have investigated whether one PPARgamma ligand in particular, ciglitazone, inhibits cell viability and, additionally, whether it affects the cell cycle and apoptosis of human glioblastoma cell lines T98G, U-87 MG, A172, and U-118 MG. All glioblastoma cell lines were found to express PPARgamma protein, and following treatment with ciglitazone, localization was unchanged. Ciglitazone inhibited viability in a dose-dependent manner in all four tested glioblastoma cells after 24 h of treatment. Analysis of the cell cycle showed arrest in the G(1) phase and partial block in G(2)/M phase of the cell cycle. Cyclin D1 and cyclin B expression was decreased. Phosphorylation of Rb protein dropped as well. We found that ciglitazone was followed by increased expression of p27(Kip1) and p21(Waf1/Cip1). It also led to apoptosis induction: bax expression in T98G was elevated. Expression of the antiapoptotic protein bcl-2 was reduced in U-118 MG and U-87 MG and showed a slight decrease in A172 cells. Flow cytometry confirmed the induction of apoptosis. Moreover, PPARgamma ligand decreased telomerase activity in U-87 MG and U-118 MG cell lines. Our results demonstrate that ciglitazone inhibits the viability of human glioblastoma cell lines via induction of apoptosis; as a result, this ligand may offer potential new therapy for the treatment of central nervous system neoplasms.

A recombinant lentiviral PDGF-driven mouse model of proneural glioblastoma.

PubMed

Rahme, Gilbert J; Luikart, Bryan W; Cheng, Chao; Israel, Mark A

2018-02-19

Mouse models of glioblastoma (GBM), the most aggressive primary brain tumor, are critical for understanding GBM pathology and can contribute to the preclinical evaluation of therapeutic agents. Platelet-derived growth factor (PDGF) signaling has been implicated in the development and pathogenesis of GBM, specifically the proneural subtype. Although multiple mouse models of PDGF-driven glioma have been described, they require transgenic mice engineered to activate PDGF signaling and/or impair tumor suppressor genes and typically represent lower-grade glioma. We designed recombinant lentiviruses expressing both PDGFB and a short hairpin RNA targeting Cdkn2a to induce gliomagenesis following stereotactic injection into the dentate gyrus of adult immunocompetent mice. We engineered these viruses to coexpress CreERT2 with PDGFB, allowing for deletion of floxed genes specifically in transduced cells, and designed another version of this recombinant lentivirus in which enhanced green fluorescent protein was coexpressed with PDGFB and CreERT2 to visualize transduced cells. The dentate gyrus of injected mice showed hypercellularity one week post-injection and subsequently developed bona fide tumors with the pathologic hallmarks of GBM leading to a median survival of 77 days post-injection. Transcriptomic analysis of these tumors revealed a proneural gene expression signature. Informed by the genetic alterations observed in human GBM, we engineered a novel mouse model of proneural GBM. While reflecting many of the advantages of transgenic mice, this model allows for the facile in vivo testing of gene function in tumor cells and makes possible the rapid production of large numbers of immunocompetent tumor-bearing mice for preclinical testing of therapeutics. © The Author(s) 2017. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com

The Error-Prone DNA Polymerase κ Promotes Temozolomide Resistance in Glioblastoma through Rad17-Dependent Activation of ATR-Chk1 Signaling.

PubMed

Peng, Chenghao; Chen, Zhengxin; Wang, Shuai; Wang, Hong-Wei; Qiu, Wenjin; Zhao, Lin; Xu, Ran; Luo, Hui; Chen, Yuanyuan; Chen, Dan; You, Yongping; Liu, Ning; Wang, Huibo

2016-04-15

The acquisition of drug resistance is a persistent clinical problem limiting the successful treatment of human cancers, including glioblastoma (GBM). However, the molecular mechanisms by which initially chemoresponsive tumors develop therapeutic resistance remain poorly understood. In this study, we report that Pol κ, an error-prone polymerase that participates in translesion DNA synthesis, was significantly upregulated in GBM cell lines and tumor tissues following temozolomide treatment. Overexpression of Pol κ in temozolomide-sensitive GBM cells conferred resistance to temozolomide, whereas its inhibition markedly sensitized resistant cells to temozolomide in vitro and in orthotopic xenograft mouse models. Mechanistically, depletion of Pol κ disrupted homologous recombination (HR)-mediated repair and restart of stalled replication forks, impaired the activation of ATR-Chk1 signaling, and delayed cell-cycle re-entry and progression. Further investigation of the relationship between Pol κ and temozolomide revealed that Pol κ inactivation facilitated temozolomide-induced Rad17 ubiquitination and proteasomal degradation, subsequently silencing ATR-Chk1 signaling and leading to defective HR repair and the reversal of temozolomide resistance. Moreover, overexpression of Rad17 in Pol κ-depleted GBM cells restored HR efficiency, promoted the clearance of temozolomide-induced DNA breaks, and desensitized cells to the cytotoxic effects of temozolomide observed in the absence of Pol κ. Finally, we found that Pol κ overexpression correlated with poor prognosis in GBM patients undergoing temozolomide therapy. Collectively, our findings identify a potential mechanism by which GBM cells develop resistance to temozolomide and suggest that targeting the DNA damage tolerance pathway may be beneficial for overcoming resistance. Cancer Res; 76(8); 2340-53. ©2016 AACR. ©2016 American Association for Cancer Research.

Downregulation of RND3/RhoE in glioblastoma patients promotes tumorigenesis through augmentation of notch transcriptional complex activity.

PubMed

Liu, Baohui; Lin, Xi; Yang, Xiangsheng; Dong, Huimin; Yue, Xiaojing; Andrade, Kelsey C; Guo, Zhentao; Yang, Jian; Wu, Liquan; Zhu, Xiaonan; Zhang, Shenqi; Tian, Daofeng; Wang, Junmin; Cai, Qiang; Chen, Qizuan; Mao, Shanping; Chen, Qianxue; Chang, Jiang

2015-09-01

Activation of Notch signaling contributes to glioblastoma multiform (GBM) tumorigenesis. However, the molecular mechanism that promotes the Notch signaling augmentation during GBM genesis remains largely unknown. Identification of new factors that regulate Notch signaling is critical for tumor treatment. The expression levels of RND3 and its clinical implication were analyzed in GBM patients. Identification of RND3 as a novel factor in GBM genesis was demonstrated in vitro by cell experiments and in vivo by a GBM xenograft model. We found that RND3 expression was significantly decreased in human glioblastoma. The levels of RND3 expression were inversely correlated with Notch activity, tumor size, and tumor cell proliferation, and positively correlated with patient survival time. We demonstrated that RND3 functioned as an endogenous repressor of the Notch transcriptional complex. RND3 physically interacted with NICD, CSL, and MAML1, the Notch transcriptional complex factors, promoted NICD ubiquitination, and facilitated the degradation of these cofactor proteins. We further revealed that RND3 facilitated the binding of NICD to FBW7, a ubiquitin ligase, and consequently enhanced NICD protein degradation. Therefore, Notch transcriptional activity was inhibited. Forced expression of RND3 repressed Notch signaling, which led to the inhibition of glioblastoma cell proliferation in vitro and tumor growth in the xenograft mice in vivo. Downregulation of RND3, however, enhanced Notch signaling activity, and subsequently promoted glioma cell proliferation. Inhibition of Notch activity abolished RND3 deficiency-mediated GBM cell proliferation. We conclude that downregulation of RND3 is responsible for the enhancement of Notch activity that promotes glioblastoma genesis. © 2015 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

Downregulation of RND3/RhoE in glioblastoma patients promotes tumorigenesis through augmentation of notch transcriptional complex activity

PubMed Central

Liu, Baohui; Lin, Xi; Yang, Xiangsheng; Dong, Huimin; Yue, Xiaojing; Andrade, Kelsey C; Guo, Zhentao; Yang, Jian; Wu, Liquan; Zhu, Xiaonan; Zhang, Shenqi; Tian, Daofeng; Wang, Junmin; Cai, Qiang; Chen, Qizuan; Mao, Shanping; Chen, Qianxue; Chang, Jiang

2015-01-01

Activation of Notch signaling contributes to glioblastoma multiform (GBM) tumorigenesis. However, the molecular mechanism that promotes the Notch signaling augmentation during GBM genesis remains largely unknown. Identification of new factors that regulate Notch signaling is critical for tumor treatment. The expression levels of RND3 and its clinical implication were analyzed in GBM patients. Identification of RND3 as a novel factor in GBM genesis was demonstrated in vitro by cell experiments and in vivo by a GBM xenograft model. We found that RND3 expression was significantly decreased in human glioblastoma. The levels of RND3 expression were inversely correlated with Notch activity, tumor size, and tumor cell proliferation, and positively correlated with patient survival time. We demonstrated that RND3 functioned as an endogenous repressor of the Notch transcriptional complex. RND3 physically interacted with NICD, CSL, and MAML1, the Notch transcriptional complex factors, promoted NICD ubiquitination, and facilitated the degradation of these cofactor proteins. We further revealed that RND3 facilitated the binding of NICD to FBW7, a ubiquitin ligase, and consequently enhanced NICD protein degradation. Therefore, Notch transcriptional activity was inhibited. Forced expression of RND3 repressed Notch signaling, which led to the inhibition of glioblastoma cell proliferation in vitro and tumor growth in the xenograft mice in vivo. Downregulation of RND3, however, enhanced Notch signaling activity, and subsequently promoted glioma cell proliferation. Inhibition of Notch activity abolished RND3 deficiency-mediated GBM cell proliferation. We conclude that downregulation of RND3 is responsible for the enhancement of Notch activity that promotes glioblastoma genesis. PMID:26108681

Targeting MPS1 Enhances Radiosensitization of Human Glioblastoma by Modulating DNA Repair Proteins.

PubMed

Maachani, Uday Bhanu; Kramp, Tamalee; Hanson, Ryan; Zhao, Shuping; Celiku, Orieta; Shankavaram, Uma; Colombo, Riccardo; Caplen, Natasha J; Camphausen, Kevin; Tandle, Anita

2015-05-01

To ensure faithful chromosome segregation, cells use the spindle assembly checkpoint (SAC), which can be activated in aneuploid cancer cells. Targeting the components of SAC machinery required for the growth of aneuploid cells may offer a cancer cell-specific therapeutic approach. In this study, the effects of inhibiting Monopolar spindle 1, MPS1 (TTK), an essential SAC kinase, on the radiosensitization of glioblastoma (GBM) cells were analyzed. Clonogenic survival was used to determine the effects of the MPS1 inhibitor NMS-P715 on radiosensitivity in multiple model systems, including GBM cell lines, a normal astrocyte, and a normal fibroblast cell line. DNA double-strand breaks (DSB) were evaluated using γH2AX foci, and cell death was measured by mitotic catastrophe evaluation. Transcriptome analysis was performed via unbiased microarray expression profiling. Tumor xenografts grown from GBM cells were used in tumor growth delay studies. Inhibition of MPS1 activity resulted in reduced GBM cell proliferation. Furthermore, NMS-P715 enhanced the radiosensitivity of GBM cells by decreased repair of DSBs and induction of postradiation mitotic catastrophe. NMS-P715 in combination with fractionated doses of radiation significantly enhanced the tumor growth delay. Molecular profiling of MPS1-silenced GBM cells showed an altered expression of transcripts associated with DNA damage, repair, and replication, including the DNA-dependent protein kinase (PRKDC/DNAPK). Next, inhibition of MPS1 blocked two important DNA repair pathways. In conclusion, these results not only highlight a role for MPS1 kinase in DNA repair and as prognostic marker but also indicate it as a viable option in glioblastoma therapy. Inhibition of MPS1 kinase in combination with radiation represents a promising new approach for glioblastoma and for other cancer therapies. ©2015 American Association for Cancer Research.

Targeting MPS1 Enhances Radiosensitization of Human Glioblastoma by Modulating DNA Repair Proteins

PubMed Central

Maachani, Uday B.; Kramp, Tamalee; Hanson, Ryan; Zhao, Shuping; Celiku, Orieta; Shankavaram, Uma; Colombo, Riccardo; Caplen, Natasha J.; Camphausen, Kevin; Tandle, Anita

2015-01-01

To ensure faithful chromosome segregation, cells use the spindle assembly checkpoint (SAC), which can be activated in aneuploid cancer cells. Targeting the components of SAC machinery required for the growth of aneuploid cells may offer a cancer cell specific therapeutic approach. In this study, the effects of inhibiting Monopolar spindle 1, MPS1 (TTK), an essential SAC kinase, on the radiosensitization of glioblastoma (GBM) cells was analyzed. Clonogenic survival was used to determine the effects of the MPS1 inhibitor, NMS-P715 on radiosensitivity in multiple model systems including: GBM cell lines, a normal astrocyte and a normal fibroblast cell line. DNA double strand breaks (DSBs) were evaluated using γH2AX foci and cell death was measured by mitotic catastrophe evaluation. Transcriptome analysis was performed via unbiased microarray expression profiling. Tumor xenografts grown from GBM cells were used in tumor growth delay studies. Inhibition of MPS1 activity resulted in reduced GBM cell proliferation. Further, NMS-P715 enhanced the radiosensitivity of GBM cells by decreased repair of DSBs and induction of post-radiation mitotic catastrophe. MNS-P715 in combination with fractionated doses of radiation significantly enhanced the tumor growth delay. Molecular profiling of MPS1 silenced GBM cells showed an altered expression of transcripts associated with DNA damage, repair and replication including the DNA-dependent protein kinase (PRKDC/DNAPK). Next, inhibition of MPS1 blocked two important DNA repair pathways. In conclusion, these results not only highlight a role for MPS1 kinase in DNA repair and as prognostic marker but also indicate it as a viable option in glioblastoma therapy. PMID:25722303

Efficacy of PARP inhibitor rucaparib in orthotopic glioblastoma xenografts is limited by ineffective drug penetration into the central nervous system

PubMed Central

Parrish, Karen E.; Cen, Ling; Murray, James; Calligaris, David; Kizilbash, Sani; Mittapalli, Rajendar K.; Carlson, Brett L.; Schroeder, Mark A.; Sludden, Julieann; Boddy, Alan V.; Agar, Nathalie Y.R.; Curtin, Nicola J.; Elmquist, William F.; Sarkaria, Jann N.

2015-01-01

Poly (ADP-ribose) polymerase (PARP) inhibition can enhance the efficacy of temozolomide (TMZ) and prolong survival in orthotopic glioblastoma (GBM) xenografts. The aim of this study was to evaluate the combination of the PARP inhibitor rucaparib with TMZ and to correlate pharmacokinetic and pharmacodynamic studies with efficacy in patient-derived GBM xenograft models. The combination of rucaparib with TMZ was highly effective in vitro in short-term explant cultures derived from GBM12, and similarly, the combination of rucaparib and TMZ (dosed for 5 days every 28 days × 3 cycles) significantly prolonged the time to tumor regrowth by 40% in heterotopic xenografts. In contrast, the addition of rucaparib had no impact on the efficacy of TMZ in GBM12 or GBM39 orthotopic models. Using Madin-Darby canine kidney (MDCK) II cells stably expressing murine BCRP1 or human MDR1, cell accumulation studies demonstrated that rucaparib is transported by both transporters. Consistent with the influence of these efflux pumps on central nervous system drug distribution, Mdr1a/b−/−Bcrp1−/− knockout mice had a significantly higher brain to plasma ratio for rucaparib (1.61 ± 0.25) than wild-type mice (0.11 ± 0.08). A pharmacokinetic and pharmacodynamic evaluation after a single dose confirmed limited accumulation of rucaparib in the brain associated with substantial residual PARP enzymatic activity. Similarly, matrix-assisted laser desorption/ionization mass spectrometric imaging demonstrated significantly enhanced accumulation of drug in flank tumor compared to normal brain or orthotopic tumors. Collectively, these results suggest that limited drug delivery into brain tumors may significantly limit the efficacy of rucaparib combined with TMZ in GBM. PMID:26438157

Efficacy of PARP Inhibitor Rucaparib in Orthotopic Glioblastoma Xenografts Is Limited by Ineffective Drug Penetration into the Central Nervous System.

PubMed

Parrish, Karen E; Cen, Ling; Murray, James; Calligaris, David; Kizilbash, Sani; Mittapalli, Rajendar K; Carlson, Brett L; Schroeder, Mark A; Sludden, Julieann; Boddy, Alan V; Agar, Nathalie Y R; Curtin, Nicola J; Elmquist, William F; Sarkaria, Jann N

2015-12-01

PARP inhibition can enhance the efficacy of temozolomide and prolong survival in orthotopic glioblastoma (GBM) xenografts. The aim of this study was to evaluate the combination of the PARP inhibitor rucaparib with temozolomide and to correlate pharmacokinetic and pharmacodynamic studies with efficacy in patient-derived GBM xenograft models. The combination of rucaparib with temozolomide was highly effective in vitro in short-term explant cultures derived from GBM12, and, similarly, the combination of rucaparib and temozolomide (dosed for 5 days every 28 days for 3 cycles) significantly prolonged the time to tumor regrowth by 40% in heterotopic xenografts. In contrast, the addition of rucaparib had no impact on the efficacy of temozolomide in GBM12 or GBM39 orthotopic models. Using Madin-Darby canine kidney (MDCK) II cells stably expressing murine BCRP1 or human MDR1, cell accumulation studies demonstrated that rucaparib is transported by both transporters. Consistent with the influence of these efflux pumps on central nervous system drug distribution, Mdr1a/b(-/-)Bcrp1(-/-) knockout mice had a significantly higher brain to plasma ratio for rucaparib (1.61 ± 0.25) than wild-type mice (0.11 ± 0.08). A pharmacokinetic and pharmacodynamic evaluation after a single dose confirmed limited accumulation of rucaparib in the brain is associated with substantial residual PARP enzymatic activity. Similarly, matrix-assisted laser desorption/ionization mass spectrometric imaging demonstrated significantly enhanced accumulation of drug in flank tumor compared with normal brain or orthotopic tumors. Collectively, these results suggest that limited drug delivery into brain tumors may significantly limit the efficacy of rucaparib combined with temozolomide in GBM. ©2015 American Association for Cancer Research.

EGFRvIII-specific chimeric antigen receptor T cells migrate to and kill tumor deposits infiltrating the brain parenchyma in an invasive xenograft model of glioblastoma.

PubMed

Miao, Hongsheng; Choi, Bryan D; Suryadevara, Carter M; Sanchez-Perez, Luis; Yang, Shicheng; De Leon, Gabriel; Sayour, Elias J; McLendon, Roger; Herndon, James E; Healy, Patrick; Archer, Gary E; Bigner, Darell D; Johnson, Laura A; Sampson, John H

2014-01-01

Glioblastoma (GBM) is the most common primary malignant brain tumor in adults and is uniformly lethal. T-cell-based immunotherapy offers a promising platform for treatment given its potential to specifically target tumor tissue while sparing the normal brain. However, the diffuse and infiltrative nature of these tumors in the brain parenchyma may pose an exceptional hurdle to successful immunotherapy in patients. Areas of invasive tumor are thought to reside behind an intact blood brain barrier, isolating them from effective immunosurveillance and thereby predisposing the development of "immunologically silent" tumor peninsulas. Therefore, it remains unclear if adoptively transferred T cells can migrate to and mediate regression in areas of invasive GBM. One barrier has been the lack of a preclinical mouse model that accurately recapitulates the growth patterns of human GBM in vivo. Here, we demonstrate that D-270 MG xenografts exhibit the classical features of GBM and produce the diffuse and invasive tumors seen in patients. Using this model, we designed experiments to assess whether T cells expressing third-generation chimeric antigen receptors (CARs) targeting the tumor-specific mutation of the epidermal growth factor receptor, EGFRvIII, would localize to and treat invasive intracerebral GBM. EGFRvIII-targeted CAR (EGFRvIII+ CAR) T cells demonstrated in vitro EGFRvIII antigen-specific recognition and reactivity to the D-270 MG cell line, which naturally expresses EGFRvIII. Moreover, when administered systemically, EGFRvIII+ CAR T cells localized to areas of invasive tumor, suppressed tumor growth, and enhanced survival of mice with established intracranial D-270 MG tumors. Together, these data demonstrate that systemically administered T cells are capable of migrating to the invasive edges of GBM to mediate antitumor efficacy and tumor regression.

Gene therapy-mediated delivery of targeted cytotoxins for glioma therapeutics

PubMed Central

Candolfi, Marianela; Xiong, Weidong; Yagiz, Kader; Liu, Chunyan; Muhammad, A. K. M. G.; Puntel, Mariana; Foulad, David; Zadmehr, Ali; Ahlzadeh, Gabrielle E.; Kroeger, Kurt M.; Tesarfreund, Matthew; Lee, Sharon; Debinski, Waldemar; Sareen, Dhruv; Svendsen, Clive N.; Rodriguez, Ron; Lowenstein, Pedro R.; Castro, Maria G.

2010-01-01

Restricting the cytotoxicity of anticancer agents by targeting receptors exclusively expressed on tumor cells is critical when treating infiltrative brain tumors such as glioblastoma multiforme (GBM). GBMs express an IL-13 receptor (IL13Rα2) that differs from the physiological IL4R/IL13R receptor. We developed a regulatable adenoviral vector (Ad.mhIL-4.TRE.mhIL-13-PE) encoding a mutated human IL-13 fused to Pseudomonas exotoxin (mhIL-13-PE) that specifically binds to IL13Rα2 to provide sustained expression, effective anti-GBM cytotoxicity, and minimal neurotoxicity. The therapeutic Ad also encodes mutated human IL-4 that binds to the physiological IL4R/IL13R without interacting with IL13Rα2, thus inhibiting potential binding of mhIL-13-PE to normal brain cells. Using intracranial GBM xenografts and syngeneic mouse models, we tested the Ad.mhIL-4.TRE.mhIL-13-PE and two protein formulations, hIL-13-PE used in clinical trials (Cintredekin Besudotox) and a second-generation mhIL-13-PE. Cintredekin Besudotox doubled median survival without eliciting long-term survival and caused severe neurotoxicity; mhIL-13-PE led to ∼40% long-term survival, eliciting severe neurological toxicity at the high dose tested. In contrast, Ad-mediated delivery of mhIL-13-PE led to tumor regression and long-term survival in over 70% of the animals, without causing apparent neurotoxicity. Although Cintredekin Besudotox was originally developed to target GBM, when tested in a phase III trial it failed to achieve clinical endpoints and revealed neurotoxicity. Limitations of Cintredekin Besudotox include its short half-life, which demanded frequent or continued administration, and binding to IL4R/IL13R, present in normal brain cells. These shortcomings were overcome by our therapeutic Ad, thus representing a significant advance in the development of targeted therapeutics for GBM. PMID:21030678

Inhibition of glioblastoma cell invasion by hsa-miR-145-5p and hsa-miR-31-5p co-overexpression in human mesenchymal stem cells.

PubMed

Kurogi, Ryota; Nakamizo, Akira; Suzuki, Satoshi O; Mizoguchi, Masahiro; Yoshimoto, Koji; Amano, Toshiyuki; Amemiya, Takeo; Takagishi, So; Iihara, Koji

2018-03-09

OBJECTIVE Human bone marrow-derived mesenchymal stem cells (hMSCs) show tropism for brain tumors and may be a useful vehicle for drug or gene delivery to malignant gliomas. Recently, some microRNAs (miRNAs) have been shown to suppress the invasiveness of malignant gliomas. METHODS To test their potential to become vehicles for the delivery of miRNA to malignant gliomas, hMSCs were engineered so that hMSC secretion of miRNAs that inhibit glioma cell invasion was enabled without altering the hMSC tropism for glioma cells. RESULTS In coculture, hMSCs cotransfected with hsa-miR-145-5p and -31-5p miRNAs showed markedly reduced invasion by U87 glioma cells in a contact-dependent manner both in vitro and ex vivo, with invasion of hMSCs cotransfected with these 2 miRNAs by the U87 cells reduced to 60.7% compared with control cells. According to a Matrigel invasion assay, the tropism of the hMSCs for U87 cells was not affected. In glioma cell lines U251 and LN229, hMSCs exhibited tropism in vivo, and invasion of hMSCs cotransfected with hsa-miR-145-5p and -31-5p was also significantly less than that of control cells. When U87 cells were coimplanted into the striatum of organotypic rat brain slices with hMSCs cotransfected with hsa-miR-145 and -31-5p, the relative invasive area decreased by 37.1%; interestingly, these U87 cells showed a change to a rounded morphology that was apparent at the invasion front. Whole-genome microarray analysis of the expression levels of 58,341 genes revealed that the co-overexpression of hsa-miR-145-5p and -31-5p downregulated FSCN1 expression in U87 cells. CONCLUSIONS This study demonstrates that miRNA overexpression in hMSCs can alter the function of glioma cells via contact-dependent transfer. Co-overexpression of multiple miRNAs may be a useful and novel therapeutic strategy. The study results suggest that hMSCs can be applied as a delivery vehicle for miRNAs.

Effect of saw palmetto extract on PI3K cell signaling transduction in human glioma

PubMed Central

YANG, YANG; HUI, LV; YUQIN, CHE; JIE, LI; SHUAI, HOU; TIEZHU, ZHOU; WEI, WANG

2014-01-01

Saw palmetto extract can induce the apoptosis of prostate cancer cells. The aim of the present study was to investigate the effect of saw palmetto extract on the phosphatidylinositol 3-kinase (PI3K)/Akt signaling transduction pathway in human glioma U87 and U251 cell lines. Suspensions of U87 and U251 cells in a logarithmic growth phase were seeded into six-well plates at a density of 104 cells/well. In the experimental group, 1 μl/ml saw palmetto extract was added, while the control group was cultured without a drug for 24 h. The expression levels of PI3K, B-cell lymphoma-extra large (Bcl-xL) and p53 were evaluated through western blot analysis. In the experimental group, the U87 and U251 cells exhibited a lower expression level of PI3K protein as compared with the control group (t=6.849; P<0.001). In addition, the two cell lines had a higher expression level of p53 protein in the experimental group as compared with the control group (t=40.810; P<0.001). Protein expression levels of Bcl-xL decreased significantly in the experimental group as compared with the control group (t=19.640; P=0.000). Therefore, saw palmetto extract induces glioma cell growth arrest and apoptosis via decreasing PI3K/Akt signal transduction. PMID:25009620

Nanotechnology Applications for Glioblastoma

PubMed Central

Nduom, Edjah; Bouras, Alexandros; Kaluzova, Milota; Hadjipanayis, Costas G.

2012-01-01

Synopsis Glioblastoma remains one of the most difficult cancers to treat and represents the most common primary malignancy of the brain. While conventional treatments have found modest success in reducing the initial tumor burden, infiltrating cancer cells beyond the main mass are responsible for tumor recurrence and ultimate patient demise. Targeting the residual infiltrating cancer cells requires the development of new treatment strategies. The emerging field of cancer nanotechnology holds much promise in the use of multifunctional nanoparticles for the imaging and targeted therapy of GBM.. Nanoparticles have emerged as potential “theranostic” agents that can permit the diagnosis and therapeutic treatment of GBM tumors. A recent human clinical trial with magnetic nanoparticles has provided feasibility and efficacy data for potential treatment of GBM patients with thermotherapy. Here we examine the current state of nanotechnology in the treatment of glioblastoma and interesting directions of further study. PMID:22748656

AMP kinase promotes glioblastoma bioenergetics and tumour growth.

PubMed

Chhipa, Rishi Raj; Fan, Qiang; Anderson, Jane; Muraleedharan, Ranjithmenon; Huang, Yan; Ciraolo, Georgianne; Chen, Xiaoting; Waclaw, Ronald; Chow, Lionel M; Khuchua, Zaza; Kofron, Matthew; Weirauch, Matthew T; Kendler, Ady; McPherson, Christopher; Ratner, Nancy; Nakano, Ichiro; Dasgupta, Nupur; Komurov, Kakajan; Dasgupta, Biplab

2018-06-18

Stress is integral to tumour evolution, and cancer cell survival depends on stress management. We found that cancer-associated stress chronically activates the bioenergetic sensor AMP kinase (AMPK) and, to survive, tumour cells hijack an AMPK-regulated stress response pathway conserved in normal cells. Analysis of The Cancer Genome Atlas data revealed that AMPK isoforms are highly expressed in the lethal human cancer glioblastoma (GBM). We show that AMPK inhibition reduces viability of patient-derived GBM stem cells (GSCs) and tumours. In stressed (exercised) skeletal muscle, AMPK is activated to cooperate with CREB1 (cAMP response element binding protein-1) and promote glucose metabolism. We demonstrate that oncogenic stress chronically activates AMPK in GSCs that coopt the AMPK-CREB1 pathway to coordinate tumour bioenergetics through the transcription factors HIF1α and GABPA. Finally, we show that adult mice tolerate systemic deletion of AMPK, supporting the use of AMPK pharmacological inhibitors in the treatment of GBM.

Chemical Screening Identifies EUrd as a Novel Inhibitor Against Temozolomide-Resistant Glioblastoma-Initiating Cells.

PubMed

Tsukamoto, Yoshihiro; Ohtsu, Naoki; Echizenya, Smile; Otsuguro, Satoko; Ogura, Ryosuke; Natsumeda, Manabu; Isogawa, Mizuho; Aoki, Hiroshi; Ichikawa, Satoshi; Sakaitani, Masahiro; Matsuda, Akira; Maenaka, Katsumi; Fujii, Yukihiko; Kondo, Toru

2016-08-01

Glioblastoma (GBM), one of the most malignant human cancers, frequently recurs despite multimodal treatment with surgery and chemo/radiotherapies. GBM-initiating cells (GICs) are the likely cell-of-origin in recurrences, as they proliferate indefinitely, form tumors in vivo, and are resistant to chemo/radiotherapies. It is therefore crucial to find chemicals that specifically kill GICs. We established temozolomide (the standard medicine for GBM)-resistant GICs (GICRs) and used the cells for chemical screening. Here, we identified 1-(3-C-ethynyl-β-d-ribopentofuranosyl) uracil (EUrd) as a selective drug for targeting GICRs. EUrd induced the death in GICRs more effectively than their parental GICs, while it was less toxic to normal neural stem cells. We demonstrate that the cytotoxic effect of EUrd on GICRs partly depended on the increased expression of uridine-cytidine kinase-like 1 (UCKL1) and the decreased one of 5'-nucleotidase cytosolic III (NT5C3), which regulate uridine-monophosphate synthesis positively and negatively respectively. Together, these findings suggest that EUrd can be used as a new therapeutic drug for GBM with the expression of surrogate markers UCKL1 and NT5C3. Stem Cells 2016;34:2016-2025. © 2016 AlphaMed Press.

Glioblastoma stem cells exploit the αvβ8 integrin-TGFβ1 signaling axis to drive tumor initiation and progression

PubMed Central

Guerrero, PA; Tchaicha, JH; Chen, Z; Morales, JE; McCarty, N; Wang, Q; Sulman, EP; Fuller, G; Lang, FF; Rao, G; McCarty, JH

2018-01-01

Glioblastoma (GBM) is a primary brain cancer that contains populations of stem-like cancer cells (GSCs) that home to specialized perivascular niches. GSC interactions with their niche influence self-renewal, differentiation and drug resistance, although the pathways underlying these events remain largely unknown. Here, we report that the integrin αvβ8 and its latent transforming growth factor β1 (TGFβ1) protein ligand have central roles in promoting niche co-option and GBM initiation. αvβ8 integrin is highly expressed in GSCs and is essential for self-renewal and lineage commitment in vitro. Fractionation of β8high cells from freshly resected human GBM samples also reveals a requirement for this integrin in tumorigenesis in vivo. Whole-transcriptome sequencing reveals that αvβ8 integrin regulates tumor development, in part, by driving TGFβ1-induced DNA replication and mitotic checkpoint progression. Collectively, these data identify the αvβ8 integrin-TGFβ1 signaling axis as crucial for exploitation of the perivascular niche and identify potential therapeutic targets for inhibiting tumor growth and progression in patients with GBM. PMID:28783169

Juglone reduces growth and migration of U251 glioblastoma cells and disrupts angiogenesis.

PubMed

Wang, Jian; Liu, Ke; Wang, Xiao-Feng; Sun, Dian-Jun

2017-10-01

Accumulating data show that prolylisomerase (Pin1) is overexpressed in human glioblastoma multiforme (GBM) specimens. Therefore, Pin1 inhibitors should be investigated as a new chemotherapeutic drug that may enhance the clinical management of human gliomas. Recently, juglone, a Pin1 inhibitor, was shown to exhibit potent anticancer activity in various tumor cells, but its role in human glioma cells remains unknown. In the present study, we determined if juglone exerts antitumor effects in the U251 human glioma cell line and investigated its potential underlying molecular mechanisms. Cell survival, apoptosis, migration, angiogenesis and molecular targets were identified with multiple detection techniques including the MTT cell proliferation assay, dual acridine orange/ethidium bromide staining, electron microscopy, transwell migration assay, chick chorioallantoic membrane assay, quantitative real-time polymerase chain reaction and immunoblotting. The results showed that 5-20 µM juglone markedly suppressed cell proliferation, induced apoptosis, and enhanced caspase-3 activity in U251 cells in a dose- and time-dependent manner. Moreover, juglone inhibited cell migration and the formation of new blood vessels. At the molecular level, juglone markedly suppressed Pin1 levels in a time-dependent manner. TGF-β1/Smad signaling, a critical upstream regulator of miR-21, was also suppressed by juglone. Moreover, the transient overexpression of Pin1 reversed its antitumor effects in U251 cells and inhibited juglone-mediated changes to the TGF-β1/miR-21 signaling pathway. These findings suggest that juglone inhibits cell growth by causing apoptosis, thereby inhibiting the migration of U251 glioma cells and disrupting angiogenesis; and that Pin1 is a critical target for juglone's antitumor activity. The present study provides evidence that juglone has in vitro efficacy against glioma. Therefore, additional studies are warranted to examine the clinical potential of juglone in human gliomas.

Imaging Heat Shock Protein 90 (Hsp90) Activity in Hormone-Refractory Prostate Cancer

DTIC Science & Technology

2009-03-01

proteins. The quantitative PET imaging of EGFR expression with 64Cu- DOTA -cetuximab is successful for monitoring the early therapeutic response upon 17...activated DOTA ester to afford DOTA -TD) for 64Cu labeling. Mice bearing human glioma U87MG tumors were then subjected to microPET scans at various...time points post-injection (p.i.) of 64Cu- DOTA -TD. The coronal slices that contain the tumor are shown in Fig. 1. The uptake of 64Cu- DOTA -TD into U87MG

Apigenin inhibits glioma cell growth through promoting microRNA-16 and suppression of BCL-2 and nuclear factor-κB/MMP‑9.

PubMed

Chen, Xin-Jun; Wu, Mian-Yun; Li, Deng-Hui; You, Jin

2016-09-01

The present study aimed to investigate the effect of apigenin on glioma cells and to explore its potential mechanism. U87 human glioma cells treated with apigenin were used in the current study. Cell Counting Kit‑8 solution and Annexin V-fluorescein isothiocyanate/propidium iodide Apoptosis Detection kit were used to analyze the effect of apigenin on U87 cell viability and apoptotic cell death. Reverse transcription‑quantitative polymerase chain reaction analysis was also used to determine microRNA‑16 (miR‑16) and MMP‑9 gene expression levels. Nuclear factor‑κB (NF‑κB) and B‑cell CLL/lymphoma 2 (BCL2) protein expression levels were determined using western blot analysis. An anti‑miR‑16 plasmid was constructed and transfected into U87 cells. The current study demonstrated that apigenin significantly decreased cell viability and induced apoptotic cell death of U87 cells in a dose‑dependent manner. Additionally, it was demonstrated that apigenin significantly increased miR‑16 levels, suppressed BCL2 protein expression and suppressed the NF‑κB/MMP9 signaling pathway in U87 cells. Furthermore, downregulation of miR‑16 using the anti‑miR‑16 plasmid reversed the effect of apigenin on cell viability, BCL2 protein expression and the NF‑κB/MMP‑9 pathway in U87 cells. The results of the present study suggested that apigenin inhibits glioma cell growth through promoting miR‑16 and suppression of BCL2 and NF-κB/MMP-9. In conclusion, the present study demonstrated the potential anticancer effects of apigenin on glioma cells.

Development and Characterization of a Novel Single-Cycle Recombinant-Virus Assay To Determine Human Immunodeficiency Virus Type 1 Coreceptor Tropism▿

PubMed Central

Whitcomb, Jeannette M.; Huang, Wei; Fransen, Signe; Limoli, Kay; Toma, Jonathan; Wrin, Terri; Chappey, Colombe; Kiss, Linda D. B.; Paxinos, Ellen E.; Petropoulos, Christos J.

2007-01-01

Most human immunodeficiency virus type 1 (HIV-1) strains require either the CXCR4 or CCR5 chemokine receptor to efficiently enter cells. Blocking viral binding to these coreceptors is an attractive therapeutic target. Currently, several coreceptor antagonists are being evaluated in clinical trials that require characterization of coreceptor tropism for enrollment. In this report, we describe the development of an automated and accurate procedure for determining HIV-1 coreceptor tropism (Trofile) and its validation for routine laboratory testing. HIV-1 pseudoviruses are generated using full-length env genes derived from patient virus populations. Coreceptor tropism is determined by measuring the abilities of these pseudovirus populations to efficiently infect CD4+/U87 cells expressing either the CXCR4 or CCR5 coreceptor. Viruses exclusively and efficiently infecting CXCR4+/CD4+/U87 cells are designated X4-tropic. Conversely, viruses exclusively and efficiently infecting CCR5+/CD4+/U87 cells are designated R5-tropic. Viruses capable of infecting both CXCR4+/CD4+/U87 and CCR5+/CD4+/U87 cells are designated dual/mixed-tropic. Assay accuracy and reproducibility were established by evaluating the tropisms of well-characterized viruses and the variability among replicate results from samples tested repeatedly. The viral subtype, hepatitis B virus or hepatitis C virus coinfection, and the plasma viral load did not affect assay performance. Minority subpopulations with alternate tropisms were reliably detected when present at 5 to 10%. The plasma viral load above which samples can be amplified efficiently in the Trofile assay is 1,000 copies per ml of plasma. Trofile has been automated for high-throughput use; it can be used to identify patients most likely to benefit from treatment regimens that include a coreceptor inhibitor and to monitor patients on treatment for the emergence of resistant virus populations that switch coreceptor tropism. PMID:17116663

Oleanolic acid induces p53-dependent apoptosis via the ERK/JNK/AKT pathway in cancer cell lines in prostatic cancer xenografts in mice.

PubMed

Kim, Gyeong-Ji; Jo, Hyeon-Ju; Lee, Kwon-Jai; Choi, Jeong Woo; An, Jeung Hee

2018-05-29

We evaluated oleanolic acid (OA)-induced anti-cancer activity, apoptotic mechanism, cell cycle status, and MAPK kinase signaling in DU145 (prostate cancer), MCF-7 (breast cancer), U87 (human glioblastoma), normal murine liver cell (BNL CL.2) and human foreskin fibroblast cell lines (Hs 68). The IC50 values for OA-induced cytotoxicity were 112.57 in DU145, 132.29 in MCF-7, and 163.60 in U87 cells, respectively. OA did not exhibit toxicity in BNL CL. 2 and Hs 68 cell lines in our experiments. OA, at 100 µg/mL, increased the number of apoptotic cells to 27.0% in DU145, 27.0% in MCF-7, and 15.7% in U87, when compared to control cells. This enhanced apoptosis was due to increases in p53, cytochrome c, Bax, PARP-1 and caspase-3 expression in DU145, MCF-7 and U87 cell lines. OA-treated DU145 cells were arrested in G2 because of the activation of p-AKT, p-JNK, p21 and p27, and the decrease in p-ERK, cyclin B1 and CDK2 expression; OA-treated MCF-7 cells were arrested in G1 owing to the activation of p-JNK, p-ERK, p21, and p27, and the decrease in p-AKT, cyclin D1, CDK4, cyclin E, and CDK2; and OA-treated U87 cells also exhibited G1 phase arrest caused by the increase in p-ERK, p-JNK, p-AKT, p21, and p27, and the decrease in cyclin D1, CDK4, cyclin E and CDK2. Thus, OA arrested the cell cycle at different phases and induced apoptosis in cancer cells. These results suggested that OA possibly altered the expression of the cell cycle regulatory proteins differently in varying types of cancer.

Cytotoxicity and apoptotic activities of alpha-, gamma- and delta-tocotrienol isomers on human cancer cells.

PubMed

Lim, Su-Wen; Loh, Hwei-San; Ting, Kang-Nee; Bradshaw, Tracey D; Zeenathul, Nazariah A

2014-12-06

Tocotrienols, especially the gamma isomer was discovered to possess cytotoxic effects associated with the induction of apoptosis in numerous cancers. Individual tocotrienol isomers are believed to induce dissimilar apoptotic mechanisms in different cancer types. This study was aimed to compare the cytotoxic potency of alpha-, gamma- and delta-tocotrienols, and to explore their resultant apoptotic mechanisms in human lung adenocarcinoma A549 and glioblastoma U87MG cells which are scarcely researched. The cytotoxic effects of alpha-, gamma- and delta-tocotrienols in both A549 and U87MG cancer cells were first determined at the cell viability and morphological aspects. DNA damage types were then identified by comet assay and flow cytometric study was carried out to support the incidence of apoptosis. The involvements of caspase-8, Bid, Bax and mitochondrial membrane permeability (MMP) in the execution of apoptosis were further expounded. All tocotrienols inhibited the growth of A549 and U87MG cancer cells in a concentration- and time-dependent manner. These treated cancer cells demonstrated some hallmarks of apoptotic morphologies, apoptosis was further confirmed by cell accumulation at the pre-G1 stage. All tocotrienols induced only double strand DNA breaks (DSBs) and no single strand DNA breaks (SSBs) in both treated cancer cells. Activation of caspase-8 leading to increased levels of Bid and Bax as well as cytochrome c release attributed by the disruption of mitochondrial membrane permeability in both A549 and U87MG cells were evident. This study has shown that delta-tocotrienol, in all experimental approaches, possessed a higher efficacy (shorter induction period) and effectiveness (higher induction rate) in the execution of apoptosis in both A549 and U87MG cancer cells as compared to alpha- and gamma-tocotrienols. Tocotrienols in particular the delta isomer can be an alternative chemotherapeutic agent for treating lung and brain cancers.

The Pluripotent Stem-Cell Marker Alkaline Phosphatase is Highly Expressed in Refractory Glioblastoma with DNA Hypomethylation.

PubMed

Iwadate, Yasuo; Suganami, Akiko; Tamura, Yutaka; Matsutani, Tomoo; Hirono, Seiichiro; Shinozaki, Natsuki; Hiwasa, Takaki; Takiguchi, Masaki; Saeki, Naokatsu

2017-02-01

Hypomethylation of genomic DNA induces stem-cell properties in cancer cells and contributes to the treatment resistance of various malignancies. To examine the correlation between the methylation status of stem-cell-related genes and the treatment outcomes in patients with glioblastoma (GBM). The genome-wide DNA methylation status was determined using HumanMethylation450 BeadChips, and the methylation status was compared between a group of patients with good prognosis (survival > 4 yr) and a group with poor prognosis (survival < 1 yr). Immunohistochemistry for proteins translated from hypomethylated genes, including alkaline phosphatase (ALPL), CD133, and CD44, was performed in 70 GBMs and 60 oligodendroglial tumors. The genomic DNA in refractory GBM was more hypomethylated than in GBM from patients with relatively long survival (P = .0111). Stem-cell-related genes including ALPL, CD133, and CD44 were also significantly hypomethylated. A validation study using immunohistochemistry showed that DNA hypomethylation was strongly correlated with high protein expression of ALPL, CD133, and CD44. GBM patients with short survival showed high expression of these stem-cell markers. Multivariate analysis confirmed that co-expression of ALPL + CD133 or ALPL + CD44 was a strong predictor of short survival. Anaplastic oligodendroglial tumors without isocitrate dehydrogenase 1 mutation were significantly correlated with high ALPL expression and poor survival. Accumulation of stem-cell properties due to aberrant DNA hypomethylation is associated with the refractory nature of GBM. Copyright © 2017 by the Congress of Neurological Surgeons

Osthole inhibits insulin-like growth factor-1-induced epithelial to mesenchymal transition via the inhibition of PI3K/Akt signaling pathway in human brain cancer cells.

PubMed

Lin, Ying-Chao; Lin, Jia-Ching; Hung, Chao-Ming; Chen, Yeh; Liu, Liang-Chih; Chang, Tin-Chang; Kao, Jung-Yie; Ho, Chi-Tang; Way, Tzong-Der

2014-06-04

Glioblastoma multiforme (GBM) is one of the most lethal types of tumors and highly metastatic and invasive. The epithelial-to-mesenchymal transition (EMT) is the crucial step for cancer cells to initiate the metastasis and could be induced by many growth factors. In this study, we found that GBM8401 cells were converted to fibroblastic phenotype and the space between the cells became expanded in response to insulin-like growth factor-1 (IGF-1) treatment. Epithelial markers were downregulated and mesenchymal markers were upregulated simultaneously after IGF-1 treatment. Our results illustrate that IGF-1 was able to induce EMT in GBM8401 cells. Osthole would reverse IGF-1-induced morphological changes, upregulated the expression of epithelial markers, and downregulated the expression of mesenchymal markers. Moreover, wound-healing assay also showed that osthole could inhibit IGF-1-induced migration of GBM8401 cells. By using dual-luciferase reporter assay and real-time PCR, we demonstrated that osthole inhibited IGF-1-induced EMT at the transcriptional level. Our study found that osthole decreased the phosphorylation of Akt and GSK3β and recovered the GSK3β bioactivity in inhibiting EMT transcription factor Snail and Twist expression. These results showed that osthole inhibited IGF-1-induced EMT by blocking PI3K/Akt pathway. We hope that osthole can be used in anticancer therapy and be a new therapeutic medicine for GBM in the future.

Treating brain tumor–initiating cells using a combination of myxoma virus and rapamycin

PubMed Central

Zemp, Franz J.; Lun, Xueqing; McKenzie, Brienne A.; Zhou, Hongyuan; Maxwell, Lori; Sun, Beichen; Kelly, John J.P.; Stechishin, Owen; Luchman, Artee; Weiss, Samuel; Cairncross, J. Gregory; Hamilton, Mark G.; Rabinovich, Brian A.; Rahman, Masmudur M.; Mohamed, Mohamed R.; Smallwood, Sherin; Senger, Donna L.; Bell, John; McFadden, Grant; Forsyth, Peter A.

2013-01-01

Background Intratumoral heterogeneity in glioblastoma multiforme (GBM) poses a significant barrier to therapy in certain subpopulation such as the tumor-initiating cell population, being shown to be refractory to conventional therapies. Oncolytic virotherapy has the potential to target multiple compartments within the tumor and thus circumvent some of the barriers facing conventional therapies. In this study, we investigate the oncolytic potential of myxoma virus (MYXV) alone and in combination with rapamycin in vitro and in vivo using human brain tumor–initiating cells (BTICs). Methods We cultured fresh GBM specimens as neurospheres and assayed their growth characteristics in vivo. We then tested the susceptibility of BTICs to MYXV infection with or without rapamycin in vitro and assessed viral biodistribution/survival in vivo in orthotopic xenografts. Results The cultured neurospheres were found to retain stem cell markers in vivo, and they closely resembled human infiltrative GBM. In this study we determined that (i) all patient-derived BTICs tested, including those resistant to temozolomide, were susceptible to MYXV replication and killing in vitro; (ii) MYXV replicated within BTICs in vivo, and intratumoral administration of MYXV significantly prolonged survival of BTIC-bearing mice; (iii) combination therapy with MYXV and rapamycin improved antitumor activity, even in mice bearing “advanced” BTIC tumors; (iv) MYXV treatment decreased expression of stem cell markers in vitro and in vivo. Conclusions Our study suggests that MYXV in combination with rapamycin infects and kills both the BTICs and the differentiated compartments of GBM and may be an effective treatment even in TMZ-resistant patients. PMID:23585629

Inhibition of CXCL12/CXCR4 autocrine/paracrine loop reduces viability of human glioblastoma stem-like cells affecting self-renewal activity.

PubMed

Gatti, Monica; Pattarozzi, Alessandra; Bajetto, Adriana; Würth, Roberto; Daga, Antonio; Fiaschi, Pietro; Zona, Gianluigi; Florio, Tullio; Barbieri, Federica

2013-12-15

Cancer stem cells (CSCs) or tumor initiating cells (TICs) drive glioblastoma (GBM) development, invasiveness and drug resistance. Distinct molecular pathways might regulate CSC biology as compared to cells in the bulk tumor mass, representing potential therapeutic targets. Chemokine CXCL12 and its receptor CXCR4 control proliferation, invasion and angiogenesis in GBM cell lines and primary cultures, but little is known about their activity in GBM CSCs. We demonstrate that CSCs, isolated from five human GBMs, express CXCR4 and release CXCL12 in vitro, although different levels of expression and secretion were observed in individual cultures, as expected for the heterogeneity of GBMs. CXCL12 treatment induced Akt-mediated significant pro-survival and self-renewal activities, while proliferation was induced at low extent. The role of CXCR4 signaling in CSC survival and self-renewal was further demonstrated using the CXCR4 antagonist AMD3100 that reduced self-renewal and survival with greater efficacy in the cultures that released higher CXCL12 amounts. The specificity of CXCL12 in sustaining CSC survival was demonstrated by the lack of AMD3100-dependent inhibition of viability in differentiated cells derived from the same GBMs. These findings, although performed on a limited number of tumor samples, suggest that the CXCL12/CXCR4 interaction mediates survival and self-renewal in GBM CSCs with high selectivity, thus emerging as a candidate system responsible for maintenance of cancer progenitors, and providing survival benefits to the tumor. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

A human functional protein interaction network and its application to cancer data analysis

PubMed Central

2010-01-01

Background One challenge facing biologists is to tease out useful information from massive data sets for further analysis. A pathway-based analysis may shed light by projecting candidate genes onto protein functional relationship networks. We are building such a pathway-based analysis system. Results We have constructed a protein functional interaction network by extending curated pathways with non-curated sources of information, including protein-protein interactions, gene coexpression, protein domain interaction, Gene Ontology (GO) annotations and text-mined protein interactions, which cover close to 50% of the human proteome. By applying this network to two glioblastoma multiforme (GBM) data sets and projecting cancer candidate genes onto the network, we found that the majority of GBM candidate genes form a cluster and are closer than expected by chance, and the majority of GBM samples have sequence-altered genes in two network modules, one mainly comprising genes whose products are localized in the cytoplasm and plasma membrane, and another comprising gene products in the nucleus. Both modules are highly enriched in known oncogenes, tumor suppressors and genes involved in signal transduction. Similar network patterns were also found in breast, colorectal and pancreatic cancers. Conclusions We have built a highly reliable functional interaction network upon expert-curated pathways and applied this network to the analysis of two genome-wide GBM and several other cancer data sets. The network patterns revealed from our results suggest common mechanisms in the cancer biology. Our system should provide a foundation for a network or pathway-based analysis platform for cancer and other diseases. PMID:20482850

Are gadolinium contrast agents suitable for gadolinium neutron capture therapy?

PubMed

De Stasio, Gelsomina; Rajesh, Deepika; Casalbore, Patrizia; Daniels, Matthew J; Erhardt, Robert J; Frazer, Bradley H; Wiese, Lisa M; Richter, Katherine L; Sonderegger, Brandon R; Gilbert, Benjamin; Schaub, Sebastien; Cannara, Rachel J; Crawford, John F; Gilles, Mary K; Tyliszczak, Tolek; Fowler, John F; Larocca, Luigi M; Howard, Steven P; Mercanti, Delio; Mehta, Minesh P; Pallini, Roberto

2005-06-01

Gadolinium neutron capture therapy (GdNCT) is a potential treatment for malignant tumors based on two steps: (1) injection of a tumor-specific (157)Gd compound; (2) tumor irradiation with thermal neutrons. The GdNC reaction can induce cell death provided that Gd is proximate to DNA. Here, we studied the nuclear uptake of Gd by glioblastoma (GBM) tumor cells after treatment with two Gd compounds commonly used for magnetic resonance imaging, to evaluate their potential as GdNCT agents. Using synchrotron X-ray spectromicroscopy, we analyzed the Gd distribution at the subcellular level in: (1) human cultured GBM cells exposed to Gd-DTPA or Gd-DOTA for 0-72 hours; (2) intracerebrally implanted C6 glioma tumors in rats injected with one or two doses of Gd-DOTA, and (3) tumor samples from GBM patients injected with Gd-DTPA. In cell cultures, Gd-DTPA and Gd-DOTA were found in 84% and 56% of the cell nuclei, respectively. In rat tumors, Gd penetrated the nuclei of 47% and 85% of the tumor cells, after single and double injection of Gd-DOTA, respectively. In contrast, in human GBM tumors 6.1% of the cell nuclei contained Gd-DTPA. Efficacy of Gd-DTPA and Gd-DOTA as GdNCT agents is predicted to be low, due to the insufficient number of tumor cell nuclei incorporating Gd. Although multiple administration schedules in vivo might induce Gd penetration into more tumor cell nuclei, a search for new Gd compounds with higher nuclear affinity is warranted before planning GdNCT in animal models or clinical trials.

Upregulation of ASCL1 and inhibition of Notch signaling pathway characterize progressive astrocytoma.

PubMed

Somasundaram, Kumaravel; Reddy, Sreekanth P; Vinnakota, Katyayni; Britto, Ramona; Subbarayan, Madhavan; Nambiar, Sandeep; Hebbar, Aparna; Samuel, Cini; Shetty, Mitesh; Sreepathi, Hari Kishore; Santosh, Vani; Hegde, Alangar Sathyaranjandas; Hegde, Sridevi; Kondaiah, Paturu; Rao, M R S

2005-10-27

Astrocytoma is the most common type of brain cancer constituting more than half of all brain tumors. With an aim to identify markers describing astrocytoma progression, we have carried out microarray analysis of astrocytoma samples of different grades using cDNA microarray containing 1152 cancer-specific genes. Data analysis identified several differentially regulated genes between normal brain tissue and astrocytoma as well as between grades II/III astrocytoma and glioblastoma multiforme (GBM; grade IV). We found several genes known to be involved in malignancy including Achaete-scute complex-like 1 (Drosophila) (ASCL1; Hash 1). As ASCL has been implicated in neuroendocrine, medullary thyroid and small-cell lung cancers, we chose to examine the role of ASCL1 in the astrocytoma development. Our data revealed that ASCL1 is overexpressed in progressive astrocytoma as evidenced by increased levels of ASCL1 transcripts in 85.71% (6/7) of grade II diffuse astrocytoma (DA), 90% (9/10) of grade III anaplastic astrocytoma (AA) and 87.5% (7/8) of secondary GBMs, while the majority of primary de novo GBMs expressed similar to or less than normal brain levels (66.67%; 8/12). ASCL1 upregulation in progressive astrocytoma is accompanied by inhibition of Notch signaling as seen by uninduced levels of HES1, a transcriptional target of Notch1, increased levels of HES6, a dominant-negative inhibitor of HES1-mediated repression of ASCL1, and increased levels of Notch ligand Delta1, which is capable of inhibiting Notch signaling by forming intracellular Notch ligand autonomous complexes. Our results imply that inhibition of Notch signaling may be an important early event in the development of grade II DA and subsequent progression to grade III AA and secondary GBM. Furthermore, ASCL1 appears to be a putative marker to distinguish primary GBM from secondary GBM.

Combined radiotherapy and chemotherapy for high-grade brain tumours

NASA Astrophysics Data System (ADS)

Barazzuol, Lara

Glioblastoma (GBM) is the most common primary brain tumour in adults and among the most aggressive of all tumours. For several decades, the standard care of GBM was surgical resection followed by radiotherapy alone. In 2005, a landmark phase III clinical trial coordinated by the European Organization for Research and Treatment of Cancer (EORTC) and the National Cancer Institute of Canada (NCIC) demonstrated the benefit of radiotherapy with concomitant and adjuvant temozolomide (TMZ) chemotherapy. With TMZ, the median life expectancy in optimally managed patients is still only 12-14 months, with only 25% surviving 24 months. There is an urgent need for new therapies in particular in those patients whose tumour has an unmethylated methylguanine methyltransferase gene (MGMT) promoter, which is a predictive factor of benefit from TMZ. In this dissertation, the nature of the interaction between TMZ and radiation is investigated using both a mathematical model, based on in vivo population statistics of survival, and in vitro experimentation on a panel of human GBM cell lines. The results show that TMZ has an additive effect in vitro and that the population-based model may be insufficient in predicting TMZ response. The combination of TMZ with particle therapy is also investigated. Very little preclinical data exists on the effects of charged particles on GBM cell lines as well as on the concomitant application of chemotherapy. In this study, human GBM cells are exposed to 3 MeV protons and 6 MeV alpha particles in concomitance with TMZ. The results suggest that the radiation quality does not affect the nature of the interaction between TMZ and radiation, showing reproducible additive cytotoxicity. Since TMZ and radiation cause DNA damage in cancer cells, there has been increased attention to the use of poly(ADP-ribose) polymerase (PARP) inhibitors. PARP is a family of enzymes that play a key role in the repair of DNA breaks. In this study, a novel PARP inhibitor, ABT-888, is used in combination with both TMZ and radiation. The results show that ABT-888 significantly enhances TMZ and radiation cell killing, regardless of the MGMT status. In summary, the findings of this research demonstrate that the use of particle therapy and PARP inhibitors are particularly promising and might improve the treatment outcome in patients with GBM.

A nutrient mixture inhibits glioblastoma xenograft U-87 MG growth in male nude mice.

PubMed

Roomi, M W; Kalinovsky, T; Rath, M; Niedzwiecki, A

2016-03-01

Brain tumors are highly aggressive tumors characterized by secretions of high levels of matrix metalloproteinase-2 and -9, leading to tumor growth, invasion and metastasis by digesting the basement membrane and extracellular matrix components. We previously demonstrated the effectiveness of a nutrient mixture (NM) containing ascorbic acid, lysine, proline, and green tea extract in vitro: on activity of urokinase plasminogen activator, matrix metalloproteinases and TIMPs in various human glioblastoma (LN-18, T-98G and A-172) cell lines and on glioblastoma A-172 cell proliferation and Matrigel invasion. Our main objective in this study was to investigate the effect of the NM in vivo on human glioblastoma U-87 MG cell line. Athymic male nude mice inoculated with 3·10(6) U-87 MG cells subcutaneously and were fed a regular diet or a regular diet supplemented with 0.5% NM. Four weeks later, the mice were sacrificed, the tumors were weighed and measured. The samples were studied histologically. NM inhibited tumor weight and tumor burden by 53% (p = 0.015) and 48% (p = 0.010), respectively. These results suggest the therapeutic potential of NM as an adjuvant in the treatment of glioblastoma.

Imp2 regulates GBM progression by activating IGF2/PI3K/Akt pathway.

PubMed

Mu, Qingchun; Wang, Lijun; Yu, Fengbo; Gao, Haijun; Lei, Ting; Li, Peiwen; Liu, Pengfei; Zheng, Xu; Hu, Xitong; Chen, Yong; Jiang, Zhenfeng; Sayari, Arash J; Shen, Jia; Huang, Haiyan

2015-01-01

Glioblastomas multiforme (GBM) are the most frequently occurring malignant brain cancers. Treatment for GBM consists of surgical resection and subsequent adjuvant radiation therapy and chemotherapy. Despite this, GBM patient survival is limited to 12-15 months, and researchers are continually trying to develop improved therapy options. Insulin-like growth factor 2 mRNA-binding protein 2 (Imp2) is known to be upregulated in many cancers and is known to regulate the signaling activity of insulin-like growth factor 2 (IGF2). However, relatively little is known about its role in malignant development of GBM. In this study, we first found Imp2 is upregulated in GBM tissues by using clinical samples and public database search. Studies with loss and gain of Imp2 expression in in vitro GBM cell culture system demonstrated the role of Imp2 in promoting GBM cell proliferation, migration, invasion and epithelial-to-mesenchymal transition (EMT). Additionally, our results show that Imp2 regulates the activity of IGF2, which further activates PI3K/Akt signaling, thereby to promote GBM malignancy. Inhibition of Imp2 was also found to sensitize GBM to temozolomide treatment. These observations add to the current knowledge of GBM biology, and may prove useful in development of more effective GBM therapy.

Imp2 regulates GBM progression by activating IGF2/PI3K/Akt pathway

PubMed Central

Mu, Qingchun; Wang, Lijun; Yu, Fengbo; Gao, Haijun; Lei, Ting; Li, Peiwen; Liu, Pengfei; Zheng, Xu; Hu, Xitong; Chen, Yong; Jiang, Zhenfeng; Sayari, Arash J; Shen, Jia; Huang, Haiyan

2015-01-01

Glioblastomas multiforme (GBM) are the most frequently occurring malignant brain cancers. Treatment for GBM consists of surgical resection and subsequent adjuvant radiation therapy and chemotherapy. Despite this, GBM patient survival is limited to 12–15 months, and researchers are continually trying to develop improved therapy options. Insulin-like growth factor 2 mRNA-binding protein 2 (Imp2) is known to be upregulated in many cancers and is known to regulate the signaling activity of insulin-like growth factor 2 (IGF2). However, relatively little is known about its role in malignant development of GBM. In this study, we first found Imp2 is upregulated in GBM tissues by using clinical samples and public database search. Studies with loss and gain of Imp2 expression in in vitro GBM cell culture system demonstrated the role of Imp2 in promoting GBM cell proliferation, migration, invasion and epithelial-to-mesenchymal transition (EMT). Additionally, our results show that Imp2 regulates the activity of IGF2, which further activates PI3K/Akt signaling, thereby to promote GBM malignancy. Inhibition of Imp2 was also found to sensitize GBM to temozolomide treatment. These observations add to the current knowledge of GBM biology, and may prove useful in development of more effective GBM therapy. PMID:25719943

Comparative analyses identify molecular signature of MRI-classified SVZ-associated glioblastoma

PubMed Central

Lin, Chin-Hsing Annie; Rhodes, Christopher T.; Lin, ChenWei; Phillips, Joanna J.; Berger, Mitchel S.

2017-01-01

ABSTRACT Glioblastoma (GBM) is a highly aggressive brain cancer with limited therapeutic options. While efforts to identify genes responsible for GBM have revealed mutations and aberrant gene expression associated with distinct types of GBM, patients with GBM are often diagnosed and classified based on MRI features. Therefore, we seek to identify molecular representatives in parallel with MRI classification for group I and group II primary GBM associated with the subventricular zone (SVZ). As group I and II GBM contain stem-like signature, we compared gene expression profiles between these 2 groups of primary GBM and endogenous neural stem progenitor cells to reveal dysregulation of cell cycle, chromatin status, cellular morphogenesis, and signaling pathways in these 2 types of MRI-classified GBM. In the absence of IDH mutation, several genes associated with metabolism are differentially expressed in these subtypes of primary GBM, implicating metabolic reprogramming occurs in tumor microenvironment. Furthermore, histone lysine methyltransferase EZH2 was upregulated while histone lysine demethylases KDM2 and KDM4 were downregulated in both group I and II primary GBM. Lastly, we identified 9 common genes across large data sets of gene expression profiles among MRI-classified group I/II GBM, a large cohort of GBM subtypes from TCGA, and glioma stem cells by unsupervised clustering comparison. These commonly upregulated genes have known functions in cell cycle, centromere assembly, chromosome segregation, and mitotic progression. Our findings highlight altered expression of genes important in chromosome integrity across all GBM, suggesting a common mechanism of disrupted fidelity of chromosome structure in GBM. PMID:28278055

p16-Cdk4-Rb axis controls sensitivity to a cyclin-dependent kinase inhibitor PD0332991 in glioblastoma xenograft cells

PubMed Central

Cen, Ling; Carlson, Brett L.; Schroeder, Mark A.; Ostrem, Jamie L.; Kitange, Gaspar J.; Mladek, Ann C.; Fink, Stephanie R.; Decker, Paul A.; Wu, Wenting; Kim, Jung-Sik; Waldman, Todd; Jenkins, Robert B.; Sarkaria, Jann N.

2012-01-01

Deregulation of the p16INK4a-Cdk4/6-Rb pathway is commonly detected in patients with glioblastoma multiforme (GBM) and is a rational therapeutic target. Here, we characterized the p16INK4a-Cdk4/6-Rb pathway in the Mayo panel of GBM xenografts, established from primary tissue samples from patients with GBM, and evaluated their response to PD0332991, a specific inhibitor of Cdk4/6. All GBM xenograft lines evaluated in this study had disruptions in the p16INK4a-Cdk4/6-Rb pathway. In vitro evaluation using short-term explant cultures from selected GBM xenograft lines showed that PD0332991 effectively arrested cell cycle in G1-phase and inhibited cell proliferation dose-dependently in lines deleted for CDKN2A/B-p16INK4a and either single-copy deletion of CDK4 (GBM22), high-level CDK6 amplification (GBM34), or deletion of CDKN2C/p18INK4c (GBM43). In contrast, 2 GBM lines with p16INK4a expression and either CDK4 amplification (GBM5) or RB mutation (GBM28) were completely resistant to PD0332991. Additional xenograft lines were screened, and GBM63 was identified to have p16INK4a expression and CDK4 amplification. Similar to the results with GBM5, GBM63 was resistant to PD0332991 treatment. In an orthotopic survival model, treatment of GBM6 xenografts (CDKN2A/B-deleted and CDK4 wild-type) with PD0332991 significantly suppressed tumor cell proliferation and prolonged survival. Collectively, these data support the concept that GBM tumors lacking p16INK4a expression and with nonamplified CDK4 and wild-type RB status may be more susceptible to Cdk4/6 inhibition using PD0332991. PMID:22711607

ATRX Loss Promotes Tumor Growth and Impairs Non-Homologous End Joining DNA Repair in Glioma

PubMed Central

Koschmann, Carl; Calinescu, Anda-Alexandra; Nunez, Felipe J.; Mackay, Alan; Fazal-Salom, Janet; Thomas, Daniel; Mendez, Flor; Kamran, Neha; Dzaman, Marta; Mulpuri, Lakshman; Krasinkiewicz, Johnathon; Doherty, Robert; Lemons, Rosemary; Brosnan-Cashman, Jackie A.; Li, Youping; Roh, Soyeon; Zhao, Lili; Appelman, Henry; Ferguson, David; Gorbunova, Vera; Meeker, Alan; Jones, Chris; Lowenstein, Pedro R.; Castro, Maria G.

2017-01-01

Recent work in human glioblastoma (GBM) has documented recurrent mutations in the histone chaperone protein ATRX. We developed an animal model of ATRX-deficient GBM and show that loss of ATRX reduces median survival and increases genetic instability. Further, analysis of genome-wide data for human gliomas showed that ATRX mutation is associated with increased mutation rate at the single nucleotide variant (SNV) level. In mouse tumors, ATRX deficiency impairs non-homologous end joining (NHEJ) and increases sensitivity to DNA-damaging agents that induce double-stranded DNA breaks. We propose that ATRX loss results in a genetically unstable tumor, which is more aggressive when left untreated, but is more responsive to double-stranded DNA-damaging agents, resulting in improved overall survival. PMID:26936505

Contact-independent cell death of human microglial cells due to pathogenic Naegleria fowleri trophozoites.

PubMed

Kim, Jong-Hyun; Kim, Daesik; Shin, Ho-Joon

2008-12-01

Free-living Naegleria fowleri leads to a fatal infection known as primary amebic meningoencephalitis in humans. Previously, the target cell death could be induced by phagocytic activity of N. fowleri as a contact-dependent mechanism. However, in this study we investigated the target cell death under a non-contact system using a tissue-culture insert. The human microglial cells, U87MG cells, co-cultured with N. fowleri trophozoites for 30 min in a non-contact system showed morphological changes such as the cell membrane destruction and a reduction in the number. By fluorescence-activated cell sorter (FACS) analysis, U87MG cells co-cultured with N. fowleri trophozoites in a non-contact system showed a significant increase of apoptotic cells (16%) in comparison with that of the control or N. fowleri lysate. When U87MG cells were co-cultured with N. fowleri trophozoites in a non-contact system for 30 min, 2 hr, and 4 hr, the cytotoxicity of amebae against target cells was 40.5, 44.2, and 45.6%, respectively. By contrast, the cytotoxicity of non-pathogenic N. gruberi trophozoites was 10.2, 12.4, and 13.2%, respectively. These results suggest that the molecules released from N. fowleri in a contact-independent manner as well as phagocytosis in a contact-dependent manner may induce the host cell death.

Contact-Independent Cell Death of Human Microglial Cells due to Pathogenic Naegleria fowleri Trophozoites

PubMed Central

Kim, Jong-Hyun

2008-01-01

Free-living Naegleria fowleri leads to a fatal infection known as primary amebic meningoencephalitis in humans. Previously, the target cell death could be induced by phagocytic activity of N. fowleri as a contact-dependent mechanism. However, in this study we investigated the target cell death under a non-contact system using a tissue-culture insert. The human microglial cells, U87MG cells, co-cultured with N. fowleri trophozoites for 30 min in a non-contact system showed morphological changes such as the cell membrane destruction and a reduction in the number. By fluorescence-activated cell sorter (FACS) analysis, U87MG cells co-cultured with N. fowleri trophozoites in a non-contact system showed a significant increasse of apoptotic cells (16%) in comparison with that of the control or N. fowleri lysate. When U87MG cells were co-cultured with N. fowleri trophozoites in a non-contact system for 30 min, 2 hr, and 4 hr, the cytotoxicity of amebae against target cells was 40.5, 44.2, and 45.6%, respectively. By contrast, the cytotoxicity of non-pathogenic N. gruberi trophozoites was 10.2, 12.4, and 13.2%, respectively. These results suggest that the molecules released from N. fowleri in a contact-independent manner as well as phagocytosis in a contact-dependent manner may induce the host cell death. PMID:19127326

Biomimetic strategies for the glioblastoma microenvironment

NASA Astrophysics Data System (ADS)

Cha, Junghwa; Kim, Pilnam

2017-12-01

Glioblastoma multiforme (GBM) is a devastating type of tumor with high mortality, caused by extensive infiltration into adjacent tissue and rapid recurrence. Most therapies for GBM have focused on the cytotoxicity, and have not targeted GBM spread. However, there have been numerous attempts to improve therapy by addressing GBM invasion, through understanding and mimicking its behavior using three-dimensional (3D) experimental models. Compared with two-dimensional models and in vivo animal models, 3D GBM models can capture the invasive motility of glioma cells within a 3D environment comprising many cellular and non-cellular components. Based on tissue engineering techniques, GBM invasion has been investigated within a biologically relevant environment, from biophysical and biochemical perspectives, to clarify the pro-invasive factors of GBM. This review discusses the recent progress in techniques for modeling the microenvironments of GBM tissue and suggests future directions with respect to recreating the GBM microenvironment and preclinical applications.

¹⁸F-Fluoromisonidazole positron emission tomography may differentiate glioblastoma multiforme from less malignant gliomas.

PubMed

Hirata, Kenji; Terasaka, Shunsuke; Shiga, Tohru; Hattori, Naoya; Magota, Keiichi; Kobayashi, Hiroyuki; Yamaguchi, Shigeru; Houkin, Kiyohiro; Tanaka, Shinya; Kuge, Yuji; Tamaki, Nagara

2012-05-01

Glioblastoma multiforme (GBM) is the most aggressive primary brain tumor and its prognosis is significantly poorer than those of less malignant gliomas. Pathologically, necrosis is one of the most important characteristics that differentiate GBM from lower grade gliomas; therefore, we hypothesized that (18)F fluoromisonidazole (FMISO), a radiotracer for hypoxia imaging, accumulates in GBM but not in lower grade gliomas. We aimed to evaluate the diagnostic value of FMISO positron emission tomography (PET) for the differential diagnosis of GBM from lower grade gliomas. This prospective study included 23 patients with pathologically confirmed gliomas. All of the patients underwent FMISO PET and (18)F-fluorodeoxyglucose (FDG) PET within a week. FMISO images were acquired 4 h after intravenous administration of 400 MBq of FMISO. Tracer uptake in the tumor was visually assessed. Lesion to normal tissue ratios and FMISO uptake volume were calculated. Of the 23 glioma patients, 14 were diagnosed as having GBM (grade IV glioma in the 2007 WHO classification), and the others were diagnosed as having non-GBM (5 grade III and 4 grade II). In visual assessment, all GBM patients showed FMISO uptake in the tumor greater than that in the surrounding brain tissues, whereas all the non-GBM patients showed FMISO uptake in the tumor equal to that in the surrounding brain tissues (p ≤ 0.001). One GBM patient was excluded from FDG PET study because of hyperglycemia. All GBM patients and three of the nine (33%) non-GBM patients showed FDG uptake greater than or equal to that in the gray matter. The sensitivity and specificity for diagnosing GBM were 100 and 100% for FMISO, and 100 and 66% for FDG, respectively. The lesion to cerebellum ratio of FMISO uptake was higher in GBM patients (2.74 ± 0.60, range 1.71-3.81) than in non-GBM patients (1.22 ± 0.06, range 1.09-1.29, p ≤ 0.001) with no overlap between the groups. The lesion to gray matter ratio of FDG was also higher in GBM patients (1.46 ± 0.75, range 0.91-3.79) than in non-GBM patients (1.07 ± 0.62, range 0.66-2.95, p ≤ 0.05); however, overlap of the ranges did not allow clear differentiation between GBM and non-GBM. The uptake volume of FMISO was larger in GBM (27.18 ± 10.46%, range 14.02-46.67%) than in non-GBM (6.07 ± 2.50%, range 2.12-9.22%, p ≤ 0.001). These preliminary data suggest that FMISO PET may distinguish GBM from lower grade gliomas.

High expression of B7-H6 in human glioma tissues promotes tumor progression.

PubMed

Jiang, Tianwei; Wu, Wei; Zhang, Huasheng; Zhang, Xiangsheng; Zhang, Dingding; Wang, Qiang; Huang, Lei; Wang, Ye; Hang, Chunhua

2017-06-06

B7-H6, a new member of B7-family ligand, also known as NCR3LG1, plays an important role in NK cells mediated immune responses. Many studies have shown that it is highly expressed in various human cancers, and its expression levels are significantly associated with cancer patients' clinicopathological parameters and postoperative prognoses. But, still the exact role of B7-H6 expression in human glioma remains elusive. In the present study, we have characterized the B7-H6 expression in the human glioma tissues as well as glioma cell lines, U87 and U251. We observed that B7-H6 was highly expressed in the human glioma tissues, and its expression was significantly associated with cancer progression. By using the RNA interference technology, we successfully ablated B7-H6 expression in human glioma cell lines to further study its contribution towards various biological features of this malignancy. Our study identified that the B7-H6 knockdown in U87 and U251 glioma cells significantly suppressed cell proliferation, migration, invasion, and enhanced apoptosis along with induction of cell cycle arrest. It thus suggested that B7-H6 play an important role in the regulation of the biological behavior of these glioma cells. However, the detailed mechanism of B7-H6 mediated regulation of glioma cancer cell transformation and its prognostic value merits further investigation.

Intracranial AAV-IFN-β gene therapy eliminates invasive xenograft glioblastoma and improves survival in orthotopic syngeneic murine model.

PubMed

GuhaSarkar, Dwijit; Neiswender, James; Su, Qin; Gao, Guangping; Sena-Esteves, Miguel

2017-02-01

The highly invasive property of glioblastoma (GBM) cells and genetic heterogeneity are largely responsible for tumor recurrence after the current standard-of-care treatment and thus a direct cause of death. Previously, we have shown that intracranial interferon-beta (IFN-β) gene therapy by locally administered adeno-associated viral vectors (AAV) successfully treats noninvasive orthotopic glioblastoma models. Here, we extend these findings by testing this approach in invasive human GBM xenograft and syngeneic mouse models. First, we show that a single intracranial injection of AAV encoding human IFN-β eliminates invasive human GBM8 tumors and promotes long-term survival. Next, we screened five AAV-IFN-β vectors with different promoters to drive safe expression of mouse IFN-β in the brain in the context of syngeneic GL261 tumors. Two AAV-IFN-β vectors were excluded due to safety concerns, but therapeutic studies with the other three vectors showed extensive tumor cell death, activation of microglia surrounding the tumors, and a 56% increase in median survival of the animals treated with AAV/P2-Int-mIFN-β vector. We also assessed the therapeutic effect of combining AAV-IFN-β therapy with temozolomide (TMZ). As TMZ affects DNA replication, an event that is crucial for second-strand DNA synthesis of single-stranded AAV vectors before active transcription, we tested two TMZ treatment regimens. Treatment with TMZ prior to AAV-IFN-β abrogated any benefit from the latter, while the reverse order of treatment doubled the median survival compared to controls. These studies demonstrate the therapeutic potential of intracranial AAV-IFN-β therapy in a highly migratory GBM model as well as in a syngeneic mouse model and that combination with TMZ is likely to enhance its antitumor potency. © 2016 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.

NK Cells with KIR2DS2 Immunogenotype Have a Functional Activation Advantage To Efficiently Kill Glioblastoma and Prolong Animal Survival

PubMed Central

Gras Navarro, Andrea; Kmiecik, Justyna; Leiss, Lina; Zelkowski, Mateusz; Engelsen, Agnete; Bruserud, Øystein; Zimmer, Jacques; Enger, Per Øyvind

2014-01-01

Glioblastomas (GBMs) are lethal brain cancers that are resistant to current therapies. We investigated the cytotoxicity of human allogeneic NK cells against patient-derived GBM in vitro and in vivo, as well as mechanisms mediating their efficacy. We demonstrate that KIR2DS2 immunogenotype NK cells were more potent killers, notwithstanding the absence of inhibitory killer Ig–like receptor (KIR)-HLA ligand mismatch. FACS-sorted and enriched KIR2DS2+ NK cell subpopulations retained significantly high levels of CD69 and CD16 when in contact with GBM cells at a 1:1 ratio and highly expressed CD107a and secreted more soluble CD137 and granzyme A. In contrast, KIR2DS2− immunogenotype donor NK cells were less cytotoxic against GBM and K562, and, similar to FACS-sorted or gated KIR2DS2− NK cells, significantly diminished CD16, CD107a, granzyme A, and CD69 when in contact with GBM cells. Furthermore, NK cell–mediated GBM killing in vitro depended upon the expression of ligands for the activating receptor NKG2D and was partially abrogated by Ab blockade. Treatment of GBM xenografts in NOD/SCID mice with NK cells from a KIR2DS2+ donor lacking inhibitory KIR-HLA ligand mismatch significantly prolonged the median survival to 163 d compared with vehicle controls (log-rank test, p = 0.0001), in contrast to 117.5 d (log-rank test, p = 0.0005) for NK cells with several inhibitory KIR-HLA ligand mismatches but lacking KIR2DS2 genotype. Significantly more CD56+CD16+ NK cells from a KIR2DS2+ donor survived in nontumor-bearing brains 3 wk after infusion compared with KIR2DS2− NK cells, independent of their proliferative capacity. In conclusion, KIR2DS2 identifies potent alloreactive NK cells against GBM that are mediated by commensurate, but dominant, activating signals. PMID:25381437

A Large-Scale RNAi Screen Identifies SGK1 as a Key Survival Kinase for GBM Stem Cells.

PubMed

Kulkarni, Shreya; Goel-Bhattacharya, Surbhi; Sengupta, Sejuti; Cochran, Brent H

2018-01-01

Glioblastoma multiforme (GBM) is the most common type of primary malignant brain cancer and has a very poor prognosis. A subpopulation of cells known as GBM stem-like cells (GBM-SC) have the capacity to initiate and sustain tumor growth and possess molecular characteristics similar to the parental tumor. GBM-SCs are known to be enriched in hypoxic niches and may contribute to therapeutic resistance. Therefore, to identify genetic determinants important for the proliferation and survival of GBM stem cells, an unbiased pooled shRNA screen of 10,000 genes was conducted under normoxic as well as hypoxic conditions. A number of essential genes were identified that are required for GBM-SC growth, under either or both oxygen conditions, in two different GBM-SC lines. Interestingly, only about a third of the essential genes were common to both cell lines. The oxygen environment significantly impacts the cellular genetic dependencies as 30% of the genes required under hypoxia were not required under normoxic conditions. In addition to identifying essential genes already implicated in GBM such as CDK4, KIF11 , and RAN , the screen also identified new genes that have not been previously implicated in GBM stem cell biology. The importance of the serum and glucocorticoid-regulated kinase 1 (SGK1) for cellular survival was validated in multiple patient-derived GBM stem cell lines using shRNA, CRISPR, and pharmacologic inhibitors. However, SGK1 depletion and inhibition has little effect on traditional serum grown glioma lines and on differentiated GBM-SCs indicating its specific importance in GBM stem cell survival. Implications: This study identifies genes required for the growth and survival of GBM stem cells under both normoxic and hypoxic conditions and finds SGK1 as a novel potential drug target for GBM. Mol Cancer Res; 16(1); 103-14. ©2017 AACR . ©2017 American Association for Cancer Research.

The MicroRNA and MessengerRNA Profile of the RNA-Induced Silencing Complex in Human Primary Astrocyte and Astrocytoma Cells

PubMed Central

Moser, Joanna J.; Fritzler, Marvin J.

2010-01-01

Background GW/P bodies are cytoplasmic ribonucleoprotein-rich foci involved in microRNA (miRNA)-mediated messenger RNA (mRNA) silencing and degradation. The mRNA regulatory functions within GW/P bodies are mediated by GW182 and its binding partner hAgo2 that bind miRNA in the RNA-induced silencing complex (RISC). To date there are no published reports of the profile of miRNA and mRNA targeted to the RISC or a comparison of the RISC-specific miRNA/mRNA profile differences in malignant and non-malignant cells. Methodology/Principal Findings RISC mRNA and miRNA components were profiled by microarray analysis of malignant human U-87 astrocytoma cells and its non-malignant counterpart, primary human astrocytes. Total cell RNA as well as RNA from immunoprecipitated RISC was analyzed. The novel findings were fourfold: (1) miRNAs were highly enriched in astrocyte RISC compared to U-87 astrocytoma RISC, (2) astrocytoma and primary astrocyte cells each contained unique RISC miRNA profiles as compared to their respective cellular miRNA profiles, (3) miR-195, 10b, 29b, 19b, 34a and 455-3p levels were increased and the miR-181b level was decreased in U-87 astrocytoma RISC as compared to astrocyte RISC, and (4) the RISC contained decreased levels of mRNAs in primary astrocyte and U-87 astrocytoma cells. Conclusions/Significance The observation that miR-34a and miR-195 levels were increased in the RISC of U-87 astrocytoma cells suggests an oncogenic role for these miRNAs. Differential regulation of mRNAs by specific miRNAs is evidenced by the observation that three miR34a-targeted mRNAs and two miR-195-targeted mRNAs were downregulated while one miR-195-targeted mRNA was upregulated. Biological pathway analysis of RISC mRNA components suggests that the RISC plays a pivotal role in malignancy and other conditions. This study points to the importance of the RISC and ultimately GW/P body composition and function in miRNA and mRNA deregulation in astrocytoma cells and possibly in other malignancies. PMID:20976148

The microRNA and messengerRNA profile of the RNA-induced silencing complex in human primary astrocyte and astrocytoma cells.

PubMed

Moser, Joanna J; Fritzler, Marvin J

2010-10-18

GW/P bodies are cytoplasmic ribonucleoprotein-rich foci involved in microRNA (miRNA)-mediated messenger RNA (mRNA) silencing and degradation. The mRNA regulatory functions within GW/P bodies are mediated by GW182 and its binding partner hAgo2 that bind miRNA in the RNA-induced silencing complex (RISC). To date there are no published reports of the profile of miRNA and mRNA targeted to the RISC or a comparison of the RISC-specific miRNA/mRNA profile differences in malignant and non-malignant cells. RISC mRNA and miRNA components were profiled by microarray analysis of malignant human U-87 astrocytoma cells and its non-malignant counterpart, primary human astrocytes. Total cell RNA as well as RNA from immunoprecipitated RISC was analyzed. The novel findings were fourfold: (1) miRNAs were highly enriched in astrocyte RISC compared to U-87 astrocytoma RISC, (2) astrocytoma and primary astrocyte cells each contained unique RISC miRNA profiles as compared to their respective cellular miRNA profiles, (3) miR-195, 10b, 29b, 19b, 34a and 455-3p levels were increased and the miR-181b level was decreased in U-87 astrocytoma RISC as compared to astrocyte RISC, and (4) the RISC contained decreased levels of mRNAs in primary astrocyte and U-87 astrocytoma cells. The observation that miR-34a and miR-195 levels were increased in the RISC of U-87 astrocytoma cells suggests an oncogenic role for these miRNAs. Differential regulation of mRNAs by specific miRNAs is evidenced by the observation that three miR34a-targeted mRNAs and two miR-195-targeted mRNAs were downregulated while one miR-195-targeted mRNA was upregulated. Biological pathway analysis of RISC mRNA components suggests that the RISC plays a pivotal role in malignancy and other conditions. This study points to the importance of the RISC and ultimately GW/P body composition and function in miRNA and mRNA deregulation in astrocytoma cells and possibly in other malignancies.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jenke, P. A.; Linares, M.; Connaughton, V.

The Fermi Gamma-ray Burst Monitor (GBM) is an all-sky gamma-ray monitor well known in the gamma-ray burst (GRB) community. Although GBM excels in detecting the hard, bright extragalactic GRBs, its sensitivity above 8 keV and its all-sky view make it an excellent instrument for the detection of rare, short-lived Galactic transients. In 2010 March, we initiated a systematic search for transients using GBM data. We conclude this phase of the search by presenting a three-year catalog of 1084 X-ray bursts. Using spectral analysis, location, and spatial distributions we classified the 1084 events into 752 thermonuclear X-ray bursts, 267 transient eventsmore » from accretion flares and X-ray pulses, and 65 untriggered gamma-ray bursts. All thermonuclear bursts have peak blackbody temperatures broadly consistent with photospheric radius expansion (PRE) bursts. We find an average rate of 1.4 PRE bursts per day, integrated over all Galactic bursters within about 10 kpc. These include 33 and 10 bursts from the ultra-compact X-ray binaries 4U 0614+09 and 2S 0918-549, respectively. We discuss these recurrence times and estimate the total mass ejected by PRE bursts in our Galaxy.« less

Discovery of potent and selective cytotoxic activity of new quinazoline-ureas against TMZ-resistant glioblastoma multiforme (GBM).

PubMed

Elkamhawy, Ahmed; Viswanath, Ambily Nath Indu; Pae, Ae Nim; Kim, Hyeon Young; Heo, Jin-Chul; Park, Woo-Kyu; Lee, Chong-Ock; Yang, Heekyoung; Kim, Kang Ho; Nam, Do-Hyun; Seol, Ho Jun; Cho, Heeyeong; Roh, Eun Joo

2015-10-20

Herein, we report new quinazoline-urea based compounds with potent cytotoxic activities against TMZ-resistant glioblastoma multiforme (GBM) cells. Low micromolar IC₅₀ values were exhibited over a panel of three primary GBM patient-derived cell cultures belonging to proneural (GBM-1), mesenchymal (GBM-2), and classical (GBM-3) subtypes. Eight compounds showed excellent selectivity indices for GBM cells comparing to a normal astrocyte cell line. In JC-1 assay, analogues 11, 12, 20, 22, and 24 exerted promising rates of mPTP opening induction towards proneural GBM subtype. Compounds 11, 20, and 24 bound to the translocator protein 18 kDa (TSPO) in submicromolar range using [(3)H] PK-11195 binding affinity assay. A homology model was built and docked models of 11, 12, 20, 22 and 24 were generated for describing their plausible binding modes in TSPO. In 3D clonogenic assay, compound 20 manifested potent tumoricidal effects on TMZ-resistant GBM cells even at submicromolar concentrations. In addition, CYP450 and hERG assays presented a safe toxicity profile of 20. Taken as a whole, this report presents compound 20 as a potent, selective and safe GBM cytotoxic agent which constitutes a promising direction against TMZ-resistant GBM. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

Strategies of Mesenchymal Invasion of Patient-derived Brain Tumors: Microenvironmental Adaptation.

PubMed

Cha, Junghwa; Kang, Seok-Gu; Kim, Pilnam

2016-04-25

The high mortality in glioblastoma multiforme (GBM) patients is primarily caused by extensive infiltration into adjacent tissue and subsequent rapid recurrence. There are no clear therapeutic strategies that target the infiltrative subpopulation of GBM mass. Using mesenchymal mode of invasion, the GBM is known to widely infiltrate by interacting with various unique components within brain microenvironment such as hyaluronic acid (HA)-rich matrix and white matter tracts. However, it is unclear how these GBM microenvironments influence the strategies of mesenchymal invasion. We hypothesize that GBM has different strategies to facilitate such invasion through adaptation to their local microenvironment. Using our in vitro biomimetic microenvironment platform for three-dimensional GBM tumorspheres (TSs), we found that the strategies of GBM invasion were predominantly regulated by the HA-rich ECM microenvironment, showing marked phenotypic changes in the presence of HA, which were mainly mediated by HA synthase (HAS). Interestingly, after inhibition of the HAS gene, GBM switched their invasion strategies to a focal adhesion (FA)-mediated invasion. These results demonstrate that the microenvironmental adaptation allowed a flexible invasion strategy for GBM. Using our model, we suggest a new inhibitory pathway for targeting infiltrative GBM and propose an importance of multi-target therapy for GBM, which underwent microenvironmental adaptation.

Identification of a Druggable Pathway Controlling Glioblastoma Invasiveness.

PubMed

Pencheva, Nora; de Gooijer, Mark C; Vis, Daniel J; Wessels, Lodewyk F A; Würdinger, Tom; van Tellingen, Olaf; Bernards, René

2017-07-05

Diffuse and uncontrollable brain invasion is a hallmark of glioblastoma (GBM), but its mechanism is understood poorly. We developed a 3D ex vivo organotypic model to study GBM invasion. We demonstrate that invading GBM cells upregulate a network of extracellular matrix (ECM) components, including multiple collagens, whose expression correlates strongly with grade and clinical outcome. We identify interferon regulatory factor 3 (IRF3) as a transcriptional repressor of ECM factors and show that IRF3 acts as a suppressor of GBM invasion. Therapeutic activation of IRF3 by inhibiting casein kinase 2 (CK2)-a negative regulator of IRF3-downregulated the expression of ECM factors and suppressed GBM invasion in ex vivo and in vivo models across a panel of patient-derived GBM cell lines representative of the main molecular GBM subtypes. Our data provide mechanistic insight into the invasive capacity of GBM tumors and identify a potential therapy to inhibit GBM invasion. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Hsf-1 affects podocyte markers NPHS1, NPHS2 and WT1 in a transgenic mouse model of TTRVal30Met-related amyloidosis.

PubMed

Petrakis, Ioannis; Mavroeidi, Vasiliki; Stylianou, Kostas; Andronikidi, Eva; Lioudaki, Eirini; Perakis, Kostas; Stratigis, Spyridon; Vardaki, Eleftheria; Zafeiri, Maria; Giannakakis, Kostantinos; Plaitakis, Andreas; Amoiridis, George; Saraiva, Maria Joao; Daphnis, Eugene

2013-09-01

Familial amyloid polyneuropathy is characterized by transthyretin (TTR) deposition in various tissues, including the kidneys. While deposition induces organ dysfunction, renal involvement in TTR-related amyloidosis could manifest from proteinuria to end-stage kidney failure. As proteinuria is considered result of glomerular filtration barrier injury we investigated whether TTR deposition affects either glomerular basement membrane (GBM) or podocytes. Immunohistochemistry, immunoblot and gene expression studies for nephrin, podocin and WT1 were run on renal tissue from human-TTRV30M transgenic mice hemizygous or homozygous for heat shock factor one (Hsf-1). Transmission electron microscopy was used for evaluation of podocyte foot process width (PFW) and GBM thickness in Hsf-1 hemizygous mice with or without TTRV30M or amyloid deposition. Glomeruli of hsf-1 hemizygous transgenic mice showed lower nephrin and podocin protein levels but an increased podocyte number when compared to Hsf-1 homozygous transgenic mice. Nephrin, podocin and WT1 gene expression levels were unaffected by the Hsf-1 carrier status. TTRV30M deposition was associated with increased PFW and GBM thickness. Under the effect of Hsf-1 hemizygosity, TTRV30M deposition has deleterious effects on GBM thickness, PFW and slit diaphragm composition, without affecting nephrin and podocin gene expression.

The Role of Wnt Signal in Glioblastoma Development and Progression: A Possible New Pharmacological Target for the Therapy of This Tumor

PubMed Central

Zuccarini, Mariachiara; Giuliani, Patricia; Ziberi, Sihana; Carluccio, Marzia; Di Iorio, Patrizia; Caciagli, Francesco

2018-01-01

Wnt is a complex signaling pathway involved in the regulation of crucial biological functions such as development, proliferation, differentiation and migration of cells, mainly stem cells, which are virtually present in all embryonic and adult tissues. Conversely, dysregulation of Wnt signal is implicated in development/progression/invasiveness of different kinds of tumors, wherein a certain number of multipotent cells, namely “cancer stem cells”, are characterized by high self-renewal and aggressiveness. Hence, the pharmacological modulation of Wnt pathway could be of particular interest, especially in tumors for which the current standard therapy results to be unsuccessful. This might be the case of glioblastoma multiforme (GBM), one of the most lethal, aggressive and recurrent brain cancers, probably due to the presence of highly malignant GBM stem cells (GSCs) as well as to a dysregulation of Wnt system. By examining the most recent literature, here we point out several factors in the Wnt pathway that are altered in human GBM and derived GSCs, as well as new molecular strategies or experimental drugs able to modulate/inhibit aberrant Wnt signal. Altogether, these aspects serve to emphasize the existence of alternative pharmacological targets that may be useful to develop novel therapies for GBM. PMID:29462960

Targeting Protein Kinase CK2: Evaluating CX-4945 Potential for GL261 Glioblastoma Therapy in Immunocompetent Mice

PubMed Central

Ferrer-Font, Laura; Villamañan, Lucia; Arias-Ramos, Nuria; Vilardell, Jordi; Plana, Maria; Ruzzene, Maria; Pinna, Lorenzo A.; Itarte, Emilio; Arús, Carles; Candiota, Ana Paula

2017-01-01

Glioblastoma (GBM) causes poor survival in patients even with aggressive treatment. Temozolomide (TMZ) is the standard chemotherapeutic choice for GBM treatment but resistance always ensues. Protein kinase CK2 (CK2) contributes to tumour development and proliferation in cancer, and it is overexpressed in human GBM. Accordingly, targeting CK2 in GBM may benefit patients. Our goal has been to evaluate whether CK2 inhibitors (iCK2s) could increase survival in an immunocompetent preclinical GBM model. Cultured GL261 cells were treated with different iCK2s including CX-4945, and target effects evaluated in vitro. CX-4945 was found to decrease CK2 activity and Akt(S129) phosphorylation in GL261 cells. Longitudinal in vivo studies with CX-4945 alone or in combination with TMZ were performed in tumour-bearing mice. Increase in survival (p < 0.05) was found with combined CX-4945 and TMZ metronomic treatment (54.7 ± 11.9 days, n = 6) when compared to individual metronomic treatments (CX-4945: 24.5 ± 2.0 and TMZ: 38.7 ± 2.7, n = 6) and controls (22.5 ± 1.2, n = 6). Despite this, CX-4945 did not improve mice outcome when administered on every/alternate days, either alone or in combination with 3-cycle TMZ. The highest survival rate was obtained with the metronomic combined TMZ+CX-4945 every 6 days, pointing to the participation of the immune system or other ancillary mechanism in therapy response. PMID:28208677

Laminin α2-Mediated Focal Adhesion Kinase Activation Triggers Alport Glomerular Pathogenesis

PubMed Central

Delimont, Duane; Dufek, Brianna M.; Meehan, Daniel T.; Zallocchi, Marisa; Gratton, Michael Anne; Phillips, Grady; Cosgrove, Dominic

2014-01-01

It has been known for some time that laminins containing α1 and α2 chains, which are normally restricted to the mesangial matrix, accumulate in the glomerular basement membranes (GBM) of Alport mice, dogs, and humans. We show that laminins containing the α2 chain, but not those containing the α1 chain activates focal adhesion kinase (FAK) on glomerular podocytes in vitro and in vivo. CD151-null mice, which have weakened podocyte adhesion to the GBM rendering these mice more susceptible to biomechanical strain in the glomerulus, also show progressive accumulation of α2 laminins in the GBM, and podocyte FAK activation. Analysis of glomerular mRNA from both models demonstrates significant induction of MMP-9, MMP-10, MMP-12, MMPs linked to GBM destruction in Alport disease models, as well as the pro-inflammatory cytokine IL-6. SiRNA knockdown of FAK in cultured podocytes significantly reduced expression of MMP-9, MMP-10 and IL-6, but not MMP-12. Treatment of Alport mice with TAE226, a small molecule inhibitor of FAK activation, ameliorated fibrosis and glomerulosclerosis, significantly reduced proteinuria and blood urea nitrogen levels, and partially restored GBM ultrastructure. Glomerular expression of MMP-9, MMP-10 and MMP-12 mRNAs was significantly reduced in TAE226 treated animals. Collectively, this work identifies laminin α2-mediated FAK activation in podocytes as an important early event in Alport glomerular pathogenesis and suggests that FAK inhibitors, if safe formulations can be developed, might be employed as a novel therapeutic approach for treating Alport renal disease in its early stages. PMID:24915008

Atypical nuclear localization of VIP receptors in glioma cell lines and patients

DOE Office of Scientific and Technical Information (OSTI.GOV)

Barbarin, Alice; Séité, Paule; Godet, Julie

Highlights: • The VIP receptor VPAC1 contains a putative NLS signal. • VPAC1 is predominantly nuclear in GBM cell lines but not VPAC2. • Non-nuclear VPAC1/2 protein expression is correlated with glioma grade. • Nuclear VPAC1 is observed in 50% of stage IV glioma (GBM). - Abstract: An increasing number of G protein-coupled receptors, like receptors for vasoactive intestinal peptide (VIP), are found in cell nucleus. As VIP receptors are involved in the regulation of glioma cell proliferation and migration, we investigated the expression and the nuclear localization of the VIP receptors VPAC1 and VPAC2 in this cancer. First, bymore » applying Western blot and immunofluorescence detection in three human glioblastoma (GBM) cell lines, we observed a strong nuclear staining for the VPAC1 receptor and a weak nuclear VPAC2 receptor staining. Second, immunohistochemical staining of VPAC1 and VPAC2 on tissue microarrays (TMA) showed that the two receptors were expressed in normal brain and glioma tissues. Expression in the non-nuclear compartment of the two receptors significantly increased with the grade of the tumors. Analysis of nuclear staining revealed a significant increase of VPAC1 staining with glioma grade, with up to 50% of GBM displaying strong VPAC1 nuclear staining, whereas nuclear VPAC2 staining remained marginal. The increase in VPAC receptor expression with glioma grades and the enhanced nuclear localization of the VPAC1 receptors in GBM might be of importance for glioma progression.« less

NG2 expression in glioblastoma identifies an actively proliferating population with an aggressive molecular signature

PubMed Central

Al-Mayhani, M. Talal F.; Grenfell, Richard; Narita, Masashi; Piccirillo, Sara; Kenney-Herbert, Emma; Fawcett, James W.; Collins, V. Peter; Ichimura, Koichi; Watts, Colin

2011-01-01

Glioblastoma multiforme (GBM) is the most common type of primary brain tumor and a highly malignant and heterogeneous cancer. Current conventional therapies fail to eradicate or curb GBM cell growth. Hence, exploring the cellular and molecular basis of GBM cell growth is vital to develop novel therapeutic approaches. Neuroglia (NG)-2 is a transmembrane proteoglycan expressed by NG2+ progenitors and is strongly linked to cell proliferation in the normal brain. By using NG2 as a biomarker we identify a GBM cell population (GBM NG2+ cells) with robust proliferative, clonogenic, and tumorigenic capacity. We show that a significant proportion (mean 83%) of cells proliferating in the tumor mass express NG2 and that over 50% of GBM NG2+ cells are proliferating. Compared with the GBM NG2− cells from the same tumor, the GBM of NG2+ cells overexpress genes associated with aggressive tumorigenicity, including overexpression of Mitosis and Cell Cycling Module genes (e.g., MELK, CDC, MCM, E2F), which have been previously shown to correlate with poor survival in GBM. We also show that the coexpression pattern of NG2 with other glial progenitor markers in GBM does not recapitulate that described in the normal brain. The expression of NG2 by such an aggressive and actively cycling GBM population combined with its location on the cell surface identifies this cell population as a potential therapeutic target in a subset of patients with GBM. PMID:21798846

Redox-Responsive Magnetic Nanoparticle for Targeted Convection-Enhanced Delivery of O6-Benzylguanine to Brain Tumors

PubMed Central

2015-01-01

Resistance to temozolomide (TMZ) based chemotherapy in glioblastoma multiforme (GBM) has been attributed to the upregulation of the DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT). Inhibition of MGMT using O6-benzylguanine (BG) has shown promise in these patients, but its clinical use is hindered by poor pharmacokinetics that leads to unacceptable toxicity. To improve BG biodistribution and efficacy, we developed superparamagnetic iron oxide nanoparticles (NP) for targeted convection-enhanced delivery (CED) of BG to GBM. The nanoparticles (NPCP-BG-CTX) consist of a magnetic core coated with a redox-responsive, cross-linked, biocompatible chitosan-PEG copolymer surface coating (NPCP). NPCP was modified through covalent attachment of BG and tumor targeting peptide chlorotoxin (CTX). Controlled, localized BG release was achieved under reductive intracellular conditions and NPCP-BG-CTX demonstrated proper trafficking of BG in human GBM cells in vitro. NPCP-BG-CTX treated cells showed a significant reduction in MGMT activity and the potentiation of TMZ toxicity. In vivo, CED of NPCP-BG-CTX produced an excellent volume of distribution (Vd) within the brain of mice bearing orthotopic human primary GBM xenografts. Significantly, concurrent treatment with NPCP-BG-CTX and TMZ showed a 3-fold increase in median overall survival in comparison to NPCP-CTX/TMZ treated and untreated animals. Furthermore, NPCP-BG-CTX mitigated the myelosuppression observed with free BG in wild-type mice when administered concurrently with TMZ. The combination of favorable physicochemical properties, tumor cell specific BG delivery, controlled BG release, and improved in vivo efficacy demonstrates the great potential of these NPs as a treatment option that could lead to improved clinical outcomes. PMID:25247850

Redox-responsive magnetic nanoparticle for targeted convection-enhanced delivery of O6-benzylguanine to brain tumors.

PubMed

Stephen, Zachary R; Kievit, Forrest M; Veiseh, Omid; Chiarelli, Peter A; Fang, Chen; Wang, Kui; Hatzinger, Shelby J; Ellenbogen, Richard G; Silber, John R; Zhang, Miqin

2014-10-28

Resistance to temozolomide (TMZ) based chemotherapy in glioblastoma multiforme (GBM) has been attributed to the upregulation of the DNA repair protein O(6)-methylguanine-DNA methyltransferase (MGMT). Inhibition of MGMT using O(6)-benzylguanine (BG) has shown promise in these patients, but its clinical use is hindered by poor pharmacokinetics that leads to unacceptable toxicity. To improve BG biodistribution and efficacy, we developed superparamagnetic iron oxide nanoparticles (NP) for targeted convection-enhanced delivery (CED) of BG to GBM. The nanoparticles (NPCP-BG-CTX) consist of a magnetic core coated with a redox-responsive, cross-linked, biocompatible chitosan-PEG copolymer surface coating (NPCP). NPCP was modified through covalent attachment of BG and tumor targeting peptide chlorotoxin (CTX). Controlled, localized BG release was achieved under reductive intracellular conditions and NPCP-BG-CTX demonstrated proper trafficking of BG in human GBM cells in vitro. NPCP-BG-CTX treated cells showed a significant reduction in MGMT activity and the potentiation of TMZ toxicity. In vivo, CED of NPCP-BG-CTX produced an excellent volume of distribution (Vd) within the brain of mice bearing orthotopic human primary GBM xenografts. Significantly, concurrent treatment with NPCP-BG-CTX and TMZ showed a 3-fold increase in median overall survival in comparison to NPCP-CTX/TMZ treated and untreated animals. Furthermore, NPCP-BG-CTX mitigated the myelosuppression observed with free BG in wild-type mice when administered concurrently with TMZ. The combination of favorable physicochemical properties, tumor cell specific BG delivery, controlled BG release, and improved in vivo efficacy demonstrates the great potential of these NPs as a treatment option that could lead to improved clinical outcomes.

High expression of N-myc (and STAT) interactor predicts poor prognosis and promotes tumor growth in human glioblastoma

PubMed Central

Yun, Dapeng; Zhao, Yingjie; Wang, Jingkun; Xu, Tao; Li, Xiaoying; Wang, Yuqi; Yuan, Li; Sun, Ruochuan; Song, Xiao; Huai, Cong; Hu, Lingna; Yang, Song; Min, Taishan; Chen, Juxiang; Chen, Hongyan; Lu, Daru

2015-01-01

Glioma is the most malignant brain tumor and glioblastoma (GBM) is the most aggressive type. The involvement of N-myc (and STAT) interactor (NMI) in tumorigenesis was sporadically reported but far from elucidation. This study aims to investigate roles of NMI in human glioma. Three independent cohorts, the Chinese tissue microarray (TMA) cohort (N = 209), the Repository for Molecular Brain Neoplasia Data (Rembrandt) cohort (N = 371) and The Cancer Genome Atlas (TCGA) cohort (N = 528 or 396) were employed. Transcriptional or protein levels of NMI expression were significantly increased according to tumor grade in all three cohorts. High expression of NMI predicted significantly unfavorable clinical outcome for GBM patients, which was further determined as an independent prognostic factor. Additionally, expression and prognostic value of NMI were associated with molecular features of GBM including PTEN deletion and EGFR amplification in TCGA cohort. Furthermore, overexpression or depletion of NMI revealed its regulation on G1/S progression and cell proliferation (both in vitro and in vivo), and this effect was partially dependent on STAT1, which interacted with and was regulated by NMI. These data demonstrate that NMI may serve as a novel prognostic biomarker and a potential therapeutic target for glioblastoma. PMID:25669971

High expression of N-myc (and STAT) interactor predicts poor prognosis and promotes tumor growth in human glioblastoma.

PubMed

Meng, Delong; Chen, Yuanyuan; Yun, Dapeng; Zhao, Yingjie; Wang, Jingkun; Xu, Tao; Li, Xiaoying; Wang, Yuqi; Yuan, Li; Sun, Ruochuan; Song, Xiao; Huai, Cong; Hu, Lingna; Yang, Song; Min, Taishan; Chen, Juxiang; Chen, Hongyan; Lu, Daru

2015-03-10

Glioma is the most malignant brain tumor and glioblastoma (GBM) is the most aggressive type. The involvement of N-myc (and STAT) interactor (NMI) in tumorigenesis was sporadically reported but far from elucidation. This study aims to investigate roles of NMI in human glioma. Three independent cohorts, the Chinese tissue microarray (TMA) cohort (N = 209), the Repository for Molecular Brain Neoplasia Data (Rembrandt) cohort (N = 371) and The Cancer Genome Atlas (TCGA) cohort (N = 528 or 396) were employed. Transcriptional or protein levels of NMI expression were significantly increased according to tumor grade in all three cohorts. High expression of NMI predicted significantly unfavorable clinical outcome for GBM patients, which was further determined as an independent prognostic factor. Additionally, expression and prognostic value of NMI were associated with molecular features of GBM including PTEN deletion and EGFR amplification in TCGA cohort. Furthermore, overexpression or depletion of NMI revealed its regulation on G1/S progression and cell proliferation (both in vitro and in vivo), and this effect was partially dependent on STAT1, which interacted with and was regulated by NMI. These data demonstrate that NMI may serve as a novel prognostic biomarker and a potential therapeutic target for glioblastoma.

Quantitative Analysis of Signaling Networks across Differentially Embedded Tumors Highlights Interpatient Heterogeneity in Human Glioblastoma

PubMed Central

2015-01-01

Glioblastoma multiforme (GBM) is the most aggressive malignant primary brain tumor, with a dismal mean survival even with the current standard of care. Although in vitro cell systems can provide mechanistic insight into the regulatory networks governing GBM cell proliferation and migration, clinical samples provide a more physiologically relevant view of oncogenic signaling networks. However, clinical samples are not widely available and may be embedded for histopathologic analysis. With the goal of accurately identifying activated signaling networks in GBM tumor samples, we investigated the impact of embedding in optimal cutting temperature (OCT) compound followed by flash freezing in LN2 vs immediate flash freezing (iFF) in LN2 on protein expression and phosphorylation-mediated signaling networks. Quantitative proteomic and phosphoproteomic analysis of 8 pairs of tumor specimens revealed minimal impact of the different sample processing strategies and highlighted the large interpatient heterogeneity present in these tumors. Correlation analyses of the differentially processed tumor sections identified activated signaling networks present in selected tumors and revealed the differential expression of transcription, translation, and degradation associated proteins. This study demonstrates the capability of quantitative mass spectrometry for identification of in vivo oncogenic signaling networks from human tumor specimens that were either OCT-embedded or immediately flash-frozen. PMID:24927040

Vitamin D receptor expression is associated with improved overall survival in human glioblastoma multiforme.

PubMed

Salomón, Débora G; Fermento, María E; Gandini, Norberto A; Ferronato, María J; Arévalo, Julián; Blasco, Jorge; Andrés, Nancy C; Zenklusen, Jean C; Curino, Alejandro C; Facchinetti, María M

2014-05-01

Vitamin D and its analogs have been shown to display anti-proliferative effects in a wide variety of cancer types including glioblastoma multiforme (GBM). These anticancer effects are mediated by its active metabolite, 1α, 25-dihydroxyvitamin D3 (calcitriol) acting mainly through vitamin D receptor (VDR) signaling. In addition to its involvement in calcitriol action, VDR has also been demonstrated to be useful as a prognostic factor for some types of cancer. However, to our knowledge, there are no studies evaluating the expression of VDR protein and its association with outcome in gliomas. Therefore, we investigated VDR expression by using immunohistochemical analysis in human glioma tissue microarrays, and analyzed the association between VDR expression and clinico-pathological parameters. We further investigated the effects of genetic and pharmacologic modulation of VDR on survival and migration of glioma cell lines. Our data demonstrate that VDR is increased in tumor tissues when compared with VDR in non-malignant brains, and that VDR expression is associated with an improved outcome in patients with GBM. We also show that both genetic and pharmacologic modulation of VDR modulates GBM cellular migration and survival and that VDR is necessary for calcitriol-mediated effects on migration. Altogether these results provide some limited evidence supporting a role for VDR in glioma progression.

CXCR4 expression varies significantly among different subtypes of glioblastoma multiforme (GBM) and its low expression or hypermethylation might predict favorable overall survival.

PubMed

Ma, Xinlong; Shang, Feng; Zhu, Weidong; Lin, Qingtang

2017-09-01

CXCR4 is an oncogene in glioblastoma multiforme (GBM) but the mechanism of its dysregulation and its prognostic value in GBM have not been fully understood. Bioinformatic analysis was performed by using R2 and the UCSC Xena browser based on data from GSE16011 in GEO datasets and in GBM cohort in TCGA database (TCGA-GBM). Kaplan Meier curves of overall survival (OS) were generated to assess the association between CXCR4 expression/methylation and OS in patients with GBM. GBM patients with high CXCR4 expression had significantly worse 5 and 10 yrs OS (p < 0.05). Across different GBM subtypes, there was an inverse relationship between overall DNA methylation and CXCR4 expression. CXCR4 expression was significantly lower in CpG island methylation phenotype (CIMP) group than in non CIMP group. Log rank test results showed that patients with high CXCR4 methylation (first tertile) had significantly better 5 yrs OS (p = 0.038). CXCR4 expression is regulated by DNA methylation in GBM and its low expression or hypermethylation might indicate favorable OS in GBM patients.

Selective Estrogen Receptor β Agonist LY500307 as a Novel Therapeutic Agent for Glioblastoma

PubMed Central

Sareddy, Gangadhara R.; Li, Xiaonan; Liu, Jinyou; Viswanadhapalli, Suryavathi; Garcia, Lauren; Gruslova, Aleksandra; Cavazos, David; Garcia, Mike; Strom, Anders M.; Gustafsson, Jan-Ake; Tekmal, Rajeshwar Rao; Brenner, Andrew; Vadlamudi, Ratna K.

2016-01-01

Glioblastomas (GBM), deadly brain tumors, have greater incidence in males than females. Epidemiological evidence supports a tumor suppressive role of estrogen; however, estrogen as a potential therapy for GBM is limited due to safety concerns. Since GBM express ERβ, a second receptor for estrogen, targeting ERβ with a selective agonist may be a potential novel GBM therapy. In the present study, we examined the therapeutic effect of the selective synthetic ERβ agonist LY500307 using in vitro and in vivo GBM models. Treatment with LY500307 significantly reduced the proliferation of GBM cells with no activity on normal astrocytes in vitro. ERβ agonists promoted apoptosis of GBM cells, and mechanistic studies using RNA sequencing revealed that LY500307 modulated several pathways related to apoptosis, cell cycle, and DNA damage response. Further, LY500307 sensitized GBM cells to several FDA-approved chemotherapeutic drugs including cisplatin, lomustine and temozolomide. LY500307 treatment significantly reduced the in vivo tumor growth and promoted apoptosis of GBM tumors in an orthotopic model and improved the overall survival of tumor-bearing mice in the GL26 syngeneic glioma model. Our results demonstrate that LY500307 has potential as a therapeutic agent for GBM. PMID:27126081

Efficacy of protracted temozolomide dosing is limited in MGMT unmethylated GBM xenograft models.

PubMed

Cen, Ling; Carlson, Brett L; Pokorny, Jenny L; Mladek, Ann C; Grogan, Patrick T; Schroeder, Mark A; Decker, Paul A; Anderson, S Keith; Giannini, Caterina; Wu, Wenting; Ballman, Karla V; Kitange, Gaspar J; Sarkaria, Jann N

2013-06-01

Temozolomide (TMZ) is important chemotherapy for glioblastoma multiforme (GBM), but the optimal dosing schedule is unclear. The efficacies of different clinically relevant dosing regimens were compared in a panel of 7 primary GBM xenografts in an intracranial therapy evaluation model. Protracted TMZ therapy (TMZ daily M-F, 3 wk every 4) provided superior survival to a placebo-treated group in 1 of 4 O(6)-DNA methylguanine-methyltransferase (MGMT) promoter hypermethylated lines (GBM12) and none of the 3 MGMT unmethylated lines, while standard therapy (TMZ daily M-F, 1 wk every 4) provided superior survival to the placebo-treated group in 2 of 3 MGMT unmethylated lines (GBM14 and GBM43) and none of the methylated lines. In comparing GBM12, GBM14, and GBM43 intracranial specimens, both GBM14 and GBM43 mice treated with protracted TMZ had a significant elevation in MGMT levels compared with placebo. Similarly, high MGMT was found in a second model of acquired TMZ resistance in GBM14 flank xenografts, and resistance was reversed in vitro by treatment with the MGMT inhibitor O(6)-benzylguanine, demonstrating a mechanistic link between MGMT overexpression and TMZ resistance in this line. Additionally, in an analysis of gene expression data, comparison of parental and TMZ-resistant GBM14 demonstrated enrichment of functional ontologies for cell cycle control within the S, G2, and M phases of the cell cycle and DNA damage checkpoints. Across the 7 tumor models studied, there was no consistent difference between protracted and standard TMZ regimens. The efficacy of protracted TMZ regimens may be limited in a subset of MGMT unmethylated tumors by induction of MGMT expression.

Alantolactone, a natural sesquiterpene lactone, has potent antitumor activity against glioblastoma by targeting IKKβ kinase activity and interrupting NF-κB/COX-2-mediated signaling cascades.

PubMed

Wang, Xun; Yu, Zhenlong; Wang, Chao; Cheng, Wei; Tian, Xiangge; Huo, Xiaokui; Wang, Yan; Sun, Chengpeng; Feng, Lei; Xing, Jinshan; Lan, Yulong; Sun, Dongdong; Hou, Qingjuan; Zhang, Baojing; Ma, Xiaochi; Zhang, Bo

2017-07-12

Glioblastoma multiforme (GBM) is one of the most refractory and palindromic central nervous system (CNS) neoplasms, and current treatments have poor effects in GBM patients. Hence, the identification of novel therapeutic targets and the development of effective treatment strategies are essential. Alantolactone (ATL) has a wide range of pharmacological activities, and its anti-tumor effect is receiving increasing attention. However, the molecular mechanism underlying the anti-GBM activity of ATL remains poorly understood. The biological functions of ATL in GBM cells were investigated using migration/invasion, colony formation and cell cycle/apoptosis assays. The localization of nuclear factor kappa B (NF-κB) p50/p65 and its binding to the cyclooxygenase 2 (COX-2) promoter were determined using confocal immunofluorescence, a streptavidin-agarose pulldown assay and a chromatin immunoprecipitation (ChIP) assay. IKKβ kinase activity was determined using a cell IKKβ kinase activity spectrophotometry quantitative detection kit and a molecular docking study. LC-MS/MS analysis was performed to determine the ability of ATL to traverse the blood-brain barrier (BBB). The in vivo anti-tumor efficacy of ATL was also analyzed in xenografted nude mice. Western blot analysis was performed to detect the protein expression levels. ATL significantly suppressed the growth of GBM in vivo and in vitro. ATL significantly reduced the expression of COX-2 by inhibiting the kinase activity of IKKβ by targeting the ATP-binding site and then attenuating the binding of NF-κB to the COX-2 promoter region. Furthermore, ATL induced apoptosis by activating the cytochrome c (cyt c)/caspase cascade signaling pathway. Moreover, ATL could penetrate the BBB. ATL exerts its anti-tumor effects in human GBM cells at least in part via NF-κB/COX-2-mediated signaling cascades by inhibiting IKKβ kinase activity. ATL, which is a natural small molecule inhibitor, is a promising candidate for clinical applications in the treatment of CNS tumors.

Inhibition of Notch signaling alters the phenotype of orthotopic tumors formed from glioblastoma multiforme neurosphere cells but does not hamper intracranial tumor growth regardless of endogene Notch pathway signature.

PubMed

Kristoffersen, Karina; Nedergaard, Mette Kjølhede; Villingshøj, Mette; Borup, Rehannah; Broholm, Helle; Kjær, Andreas; Poulsen, Hans Skovgaard; Stockhausen, Marie-Thérése

2014-07-01

Brain cancer stem-like cells (bCSC) are cancer cells with neural stem cell (NSC)-like properties found in the devastating brain tumor glioblastoma multiforme (GBM). bCSC are proposed a central role in tumor initiation, progression, treatment resistance and relapse and as such present a promising target in GBM research. The Notch signaling pathway is often deregulated in GBM and we have previously characterized GBM-derived bCSC cultures based on their expression of the Notch-1 receptor and found that it could be used as predictive marker for the effect of Notch inhibition. The aim of the present project was therefore to further elucidate the significance of Notch pathway activity for the tumorigenic properties of GBM-derived bCSC. Human-derived GBM xenograft cells previously established as NSC-like neurosphere cultures were used. Notch inhibition was accomplished by exposing the cells to the gamma-secretase inhibitor DAPT prior to gene expression analysis and intracranial injection into immunocompromised mice. By analyzing the expression of several Notch pathway components, we found that the cultures indeed displayed different Notch pathway signatures. However, when DAPT-treated neurosphere cells were injected into the brain of immunocompromised mice, no increase in survival was obtained regardless of Notch pathway signature and Notch inhibition. We did however observe a decrease in the expression of the stem cell marker Nestin, an increase in the proliferative marker Ki-67 and an increased number of abnormal vessels in tumors formed from DAPT-treated, high Notch-1 expressing cultures, when compared with the control. Based on the presented results we propose that Notch inhibition partly induces differentiation of bCSC, and selects for a cell type that more strongly induces angiogenesis if the treatment is not sustained. However, this more differentiated cell type might prove to be more sensitive to conventional therapies.

The Role of Factor Inhibiting HIF (FIH-1) in Inhibiting HIF-1 Transcriptional Activity in Glioblastoma Multiforme

PubMed Central

Liu, Fengming; Wu, Gang; Schroeder, Mark A.; Lau, Julie S.; Mukhopadhyay, Debabrata; Jiang, Shi-Wen; O'Neill, Brian Patrick; Datta, Kaustubh; Li, Jinping

2014-01-01

Glioblastoma multiforme (GBM) accounts for about 38% of primary brain tumors in the United States. GBM is characterized by extensive angiogenesis induced by vascular growth factors and cytokines. The transcription of these growth factors and cytokines is regulated by the Hypoxia-Inducible-Factor-1(HIF-1), which is a key regulator mediating the cellular response to hypoxia. It is known that Factor Inhibiting HIF-1, or FIH-1, is also involved in the cellular response to hypoxia and has the capability to physically interact with HIF-1 and block its transcriptional activity under normoxic conditions. Delineation of the regulatory role of FIH-1 will help us to better understand the molecular mechanism responsible for tumor growth and progression and may lead to the design of new therapies targeting cellular pathways in response to hypoxia. Previous studies have shown that the chromosomal region of 10q24 containing the FIH-1 gene is often deleted in GBM, suggesting a role for the FIH-1 in GBM tumorigenesis and progression. In the current study, we found that FIH-1 is able to inhibit HIF-mediated transcription of GLUT1 and VEGF-A, even under hypoxic conditions in human glioblastoma cells. FIH-1 has been found to be more potent in inhibiting HIF function than PTEN. This observation points to the possibility that deletion of 10q23-24 and loss or decreased expression of FIH-1 gene may lead to a constitutive activation of HIF-1 activity, an alteration of HIF-1 targets such as GLUT-1 and VEGF-A, and may contribute to the survival of cancer cells in hypoxia and the development of hypervascularization observed in GBM. Therefore FIH-1 can be potential therapeutic target for the treatment of GBM patients with poor prognosis. PMID:24465898

Enhanced In Vivo Tumor Detection by Active Tumor Cell Targeting Using Multiple Tumor Receptor-Binding Peptides Presented on Genetically Engineered Human Ferritin Nanoparticles.

PubMed

Kwon, Koo Chul; Ko, Ho Kyung; Lee, Jiyun; Lee, Eun Jung; Kim, Kwangmeyung; Lee, Jeewon

2016-08-01

Human ferritin heavy-chain nanoparticle (hFTH) is genetically engineered to present tumor receptor-binding peptides (affibody and/or RGD-derived cyclic peptides, named 4CRGD here) on its surface. The affibody and 4CRGD specifically and strongly binds to human epidermal growth factor receptor I (EGFR) and human integrin αvβ3, respectively, which are overexpressed on various tumor cells. Through in vitro culture of EGFR-overexpressing adenocarcinoma (MDA-MB-468) and integrin-overexpressing glioblastoma cells (U87MG), it is clarified that specific interactions between receptors on tumor cells and receptor-binding peptides on engineered hFTH is critical in active tumor cell targeting. After labeling with the near-infrared fluorescence dye (Cy5.5) and intravenouse injection into MDA-MB-468 or U87MG tumor-bearing mice, the recombinant hFTHs presenting either peptide or both of affibody and 4CRGD are successfully delivered to and retained in the tumor for a prolonged period of time. In particular, the recombinant hFTH presenting both affibody and 4CRGD notably enhances in vivo detection of U87MG tumors that express heterogeneous receptors, integrin and EGFR, compared to the other recombinant hFTHs presenting either affibody or 4CRGD only. Like affibody and 4CRGD used in this study, other multiple tumor receptor-binding peptides can be also genetically introduced to the hFTH surface for actively targeting of in vivo tumors with heterogenous receptors. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The Interplanetary Network Supplement to the Fermi GBM Catalog - An AO-2 and AO-3 Guest Investigator Project

NASA Technical Reports Server (NTRS)

Hurley, K.; Briggs, M.; Connaughton, V.; Meegan, C.; von Kienlin, A.; Rau, A.; Zhang, X.; Golenetskii, S.; Aptekar, R.; Mazets, E.;

Glioblastoma Recurrence Patterns After Radiation Therapy With Regard to the Subventricular Zone

DOE Office of Scientific and Technical Information (OSTI.GOV)

Adeberg, Sebastian, E-mail: Sebastian.adeberg@med.uni-heidelberg; König, Laila; Bostel, Tilman

Purpose: We evaluated the influence of tumor location and tumor spread in primary glioblastoma (GBM), with respect to the subventricular zone (SVZ), on recurrence behavior, progression-free survival (PFS), and overall survival (OS). Methods and Materials: 607 patients (376 male and 231 female) with a median age of 61.3 years (range, 3.0-87.9 years) and primary GBM treated with radiation therapy (RT) from 2004 to 2012 at a single institution were included in this retrospective study. Preoperative images and follow-up examination results were assessed to evaluate tumor location. Tumors were classified according to the tumor location in relation to the SVZ. Results: The medianmore » PFS of the study population was 5.2 months (range, 1-91 months), and the median OS was 13.8 months (range, 1-102 months). Kaplan-Meier analysis showed that tumor location in close proximity to the SVZ was associated with a significant decline in PFS and OS (4.8 and 12.3 months, respectively; each P<.001). Furthermore, in cases where tumors were involved with the SVZ, distant cerebral progression (43.8%; P=.005) and multifocal progression (39.8%; P=.008) were more common. Interestingly, opening of the ventricle during the previous surgery showed no impact on PFS and OS. Conclusion: GBM in close proximity to the SVZ was associated with decreased survival and had a higher risk of multifocal or distant progression. Ventricle opening during surgery had no effect on survival rates.« less

Induction of brain tumor stem cell apoptosis by FTY720: a potential therapeutic agent for glioblastoma.

PubMed

Estrada-Bernal, Adriana; Palanichamy, Kamalakannan; Ray Chaudhury, Abhik; Van Brocklyn, James R

2012-04-01

FTY720 is a sphingosine analogue that down regulates expression of sphingosine-1-phosphate receptors and causes apoptosis of multiple tumor cell types, including glioma cells. This study examined the effect of FTY720 on brain tumor stem cells (BTSCs) derived from human glioblastoma (GBM) tissue. FTY720 treatment of BTSCs led to rapid inactivation of ERK MAP kinase, leading to upregulation of the BH3-only protein Bim and apoptosis. In combination with temozolomide (TMZ), the current standard chemotherapeutic agent for GBM, FTY720 synergistically induced BTSC apoptosis. FTY720 also slowed growth of intracranial xenograft tumors in nude mice and augmented the therapeutic effect of TMZ, leading to enhanced survival. Furthermore, the combination of FTY720 and TMZ decreased the invasiveness of BTSCs in mouse brains. FTY720 is known to cross the blood-brain barrier and recently received Food and Drug Administration approval for treatment of relapsing multiple sclerosis. Thus, FTY720 is an excellent potential therapeutic agent for treatment of GBM.

Experimental anti-GBM nephritis as an analytical tool for studying spontaneous lupus nephritis.

PubMed

Du, Yong; Fu, Yuyang; Mohan, Chandra

2008-01-01

Systemic lupus erythematosus (SLE) is an autoimmune disease that results in immune-mediated damage to multiple organs. Among these, kidney involvement is the most common and fatal. Spontaneous lupus nephritis (SLN) in mouse models has provided valuable insights into the underlying mechanisms of human lupus nephritis. However, SLN in mouse models takes 6-12 months to manifest; hence there is clearly the need for a mouse model that can be used to unveil the pathogenic processes that lead to immune nephritis over a shorter time frame. In this article more than 25 different molecules are reviewed that have been studied both in the anti-glomerular basement membrane (anti-GBM) model and in SLN and it was found that these molecules influence both diseases in a parallel fashion, suggesting that the two disease settings share common molecular mechanisms. Based on these observations, the authors believe the experimental anti-GBM disease model might be one of the best tools currently available for uncovering the downstream molecular mechanisms leading to SLN.

Glioblastoma with oligodendroglioma component (GBM-O): molecular genetic and clinical characteristics.

PubMed

Appin, Christina L; Gao, Jingjing; Chisolm, Candace; Torian, Mike; Alexis, Dianne; Vincentelli, Cristina; Schniederjan, Matthew J; Hadjipanayis, Costas; Olson, Jeffrey J; Hunter, Stephen; Hao, Chunhai; Brat, Daniel J

2013-07-01

Glioblastoma (GBM) is an aggressive primary brain tumor with an average survival of approximately 1 year. A recently recognized subtype, glioblastoma with oligodendroglioma component (GBM-O), was designated by the World Health Organization (WHO) in 2007. We investigated GBM-Os for their clinical and molecular characteristics as compared to other forms of GBM. Tissue samples were used to determine EGFR, PTEN, and 1p and 19q status by fluorescence in situ hybridization (FISH); p53 and mutant IDH1 protein expression by immunohistochemistry (IHC); and MGMT promoter status by methylation-specific polymerase chain reaction (PCR). GBM-Os accounted for 11.9% of all GBMs. GBM-Os arose in younger patients compared to other forms of GBMs (50.7 years vs. 58.7 years, respectively), were more frequently secondary neoplasms, had a higher frequency of IDH1 mutations and had a lower frequency of PTEN deletions. Survival was longer in patients with GBM-Os compared to those with other GBMs, with median survivals of 16.2 and 8.1 months, respectively. Most of the survival advantage for GBM-O appeared to be associated with a younger age at presentation. Among patients with GBM-O, younger age at presentation and 1p deletion were most significant in conferring prolonged survival. Thus, GBM-O represents a subset of GBMs with distinctive morphologic, clinical and molecular characteristics. © 2013 The Authors; Brain Pathology © 2013 International Society of Neuropathology.

DNM3, p65 and p53 from exosomes represent potential clinical diagnosis markers for glioblastoma multiforme

PubMed Central

Yang, Jian-kai; Song, Jian; Huo, Hao-ran; Zhao, Yin-long; Zhang, Guang-yu; Zhao, Zong-mao; Sun, Guo-zhu; Jiao, Bao-hua

2017-01-01

Background: Glioblastoma multiforme (GBM) is the most aggressive and deadly primary brain cancer that arises from astrocytes and classified as grade IV. Recently, exosomes have been reported as an essential mediator in diverse cancer carcinogenesis and metastasis. However, their role in GBM is still unclear. In this study, we aimed to investigate whether blood exosomes can be potential clinical diagnostic markers for GBM. Methods: We used a xenograft orthotopic mouse model to detect the differentially expressed genes in the brain and blood exosomes of original/recurrent GBM. Results: We found that recurrent GBM had stronger growth capacity and lethality than original GBM in the mouse model. A gene microarray of original tumors and blood exosomes from GBM orthotopic xenografts results showed that DNM3, p65 and CD117 expressions increased, whereas PTEN and p53 expressions decreased in both original tumors and blood exosomes. In the recurrent GBM tumor model, DNM3 and p65 showed increased expressions, whereas ST14 and p53 showed decreased expressions in tumor and blood exosomes of the recurrent GBM mouse model. Conclusion: In summary, we found that DNM3, p65 and p53 had a similar trend in brain and blood exosomes both for original and recurrent GBM, and could serve as potential clinical diagnostic markers for GBM. PMID:29449895

Polish Natural Bee Honeys Are Anti-Proliferative and Anti-Metastatic Agents in Human Glioblastoma multiforme U87MG Cell Line

PubMed Central

Moskwa, Justyna; Borawska, Maria H.; Markiewicz-Zukowska, Renata; Puscion-Jakubik, Anna; Naliwajko, Sylwia K.; Socha, Katarzyna; Soroczynska, Jolanta

2014-01-01

Honey has been used as food and a traditional medicament since ancient times. However, recently many scientists have been concentrating on the anti-oxidant, anti-proliferative, anti-inflammatory and other properties of honey. In this study, we investigated for the first time an anticancer effect of different honeys from Poland on tumor cell line - glioblastoma multiforme U87MG. Anti-proliferative activity of honeys and its interferences with temozolomide were determined by a cytotoxicity test and DNA binding by [H3]-thymidine incorporation. A gelatin zymography was used to conduct an evaluation of metalloproteinases (MMP-2 and MMP-9) expression in U87MG treatment with honey samples. The honeys were previously tested qualitatively (diastase activity, total phenolic content, lead and cadmium content). The data demonstrated that the examined honeys have a potent anti-proliferative effect on U87MG cell line in a time- and dose-dependent manner, being effective at concentrations as low as 0.5% (multifloral light honey - viability 53% after 72 h of incubation). We observed that after 48 h, combining honey with temozolomide showed a significantly higher inhibitory effect than the samples of honey alone. We observed a strong inhibition of MMP-2 and MMP-9 for the tested honeys (from 20 to 56% and from 5 to 58% compared to control, respectively). Our results suggest that Polish honeys have an anti-proliferative and anti-metastatic effect on U87MG cell line. Therefore, natural bee honey can be considered as a promising adjuvant treatment for brain tumors. PMID:24594866

64Cu-Labeled Lissamine Rhodamine B: A Promising PET Radiotracer Targeting Tumor Mitochondria

PubMed Central

Zhou, Yang; Kim, Young-Seung; Yan, Xin; Jacobson, Orit; Chen, Xiaoyuan; Liu, Shuang

2011-01-01

The enhanced mitochondrial potential in carcinoma cells is an important characteristic of cancer. It is of great current interest to develop a radiotracer that is sensitive to the mitochondrial potential changes at the early stage of tumor growth. In this report, we present the synthesis and evaluation of 64Cu-labeled Lissamine Rhodamine B (LRB), 64Cu(DOTA-LRB) (DOTA-LRB = 2-(6-(diethylamino)-3-(diethyliminio)-3H-xanthen-9-yl)-5-(N-(2-(2-(4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclo-dodecan-1-yl)acetamido)ethyl)-sulfamoyl)benzenesulfonate), as a new radiotracer for imaging tumors in athymic nude mice bearing U87MG human glioma xenografts by positron emission tomography (PET). We also explored its localization mechanism using Cu(DOTA-LRB) as the fluorescent probe in both U87MG human glioma cell line and the cultured primary U87MG glioma cells. It was found that 64Cu(DOTA-LRB) had the highest tumor uptake (6.54 ± 1.50, 6.91 ± 1.26, 5.68 ± 1.13, 7.58 ± 1.96, and 5.14 ± 1.50 %ID/g at 0.5, 1, 2, 4 and 24 h post-injection, respectively) among many 64Cu-labeled organic cations evaluated in the same animal model. The cellular staining study indicated that Cu(DOTA-LRB) was able to localize in mitochondria of U87MG glioma cells due to the enhanced negative mitochondrial potential. This statement is completely supported by the results from decoupling experiment with carbonylcyanide-m-chlorophenylhydrazone (CCCP). MicroPET data showed that the U87MG glioma tumors were clearly visualized as early as 30 min post-injection with 64Cu(DOTA-LRB). 64Cu(DOTA-LRB) remained stable during renal excretion, but underwent extensive degradation during hepatobiliary excretion. On the basis of the results from this study, it was concluded that 64Cu(DOTA-LRB) represents a new class of promising PET radiotracers for noninvasive imaging of the MDR-negative tumors. PMID:21545131

Liposomal TriCurin, A Synergistic Combination of Curcumin, Epicatechin Gallate and Resveratrol, Repolarizes Tumor-Associated Microglia/Macrophages, and Eliminates Glioblastoma (GBM) and GBM Stem Cells.

PubMed

Mukherjee, Sumit; Baidoo, Juliet N E; Sampat, Samay; Mancuso, Andrew; David, Lovena; Cohen, Leah S; Zhou, Shuiqin; Banerjee, Probal

2018-01-18

Glioblastoma (GBM) is a deadly brain tumor with a current mean survival of 12-15 months. Despite being a potent anti-cancer agent, the turmeric ingredient curcumin (C) has limited anti-tumor efficacy in vivo due to its low bioavailability. We have reported earlier a strategy involving the use two other polyphenols, epicatechin gallate (E) from green tea and resveratrol (R) from red grapes at a unique, synergistic molar ratio with C (C:E:R: 4:1:12.5, termed TriCurin) to achieve superior potency against HPV+ tumors than C alone at C:E:R (μM): 32:8:100 (termed 32 μM+ TriCurin). We have now prepared liposomal TriCurin (TrLp) and demonstrated that TrLp boosts activated p53 in cultured GL261 mouse GBM cells to trigger apoptosis of GBM and GBM stem cells in vitro. TrLp administration into mice yielded a stable plasma concentration of 210 nM C for 60 min, which, though sub-lethal for cultured GL261 cells, was able to cause repolarization of M2-like tumor (GBM)-associated microglia/macrophages to the tumoricidal M1-like phenotype and intra-GBM recruitment of activated natural killer cells. The intratumor presence of such tumoricidal immune cells was associated with concomitant suppression of tumor-load, and apoptosis of GBM and GBM stem cells. Thus, TrLp is a potential onco-immunotherapeutic agent against GBM tumors.

Diagnosis and classification of Goodpasture's disease (anti-GBM).

PubMed

Hellmark, Thomas; Segelmark, Mårten

2014-01-01

Goodpasture's disease or anti-glomerular basement membrane disease (anti-GBM-disease) is included among immune complex small vessel vasculitides. The definition of anti-GBM disease is a vasculitis affecting glomerular capillaries, pulmonary capillaries, or both, with GBM deposition of anti-GBM autoantibodies. The disease is a prototype of autoimmune disease, where the patients develop autoantibodies that bind to the basement membranes and activate the classical pathway of the complement system, which start a neutrophil dependent inflammation. The diagnosis of anti-GBM disease relies on the detection of anti-GBM antibodies in conjunction with glomerulonephritis and/or alveolitis. Overt clinical symptoms are most prominent in the glomeruli where the inflammation usually results in a severe rapidly progressive glomerulonephritis. Despite modern treatment less than one third of the patients survive with a preserved kidney function after 6 months follow-up. Frequencies vary from 0.5 to 1 cases per million inhabitants per year and there is a strong genetic linkage to HLA-DRB1(∗)1501 and DRB1(∗)1502. Essentially, anti-GBM disease is now a preferred term for what was earlier called Goodpasture's syndrome or Goodpasture's disease; anti-GBM disease is now classified as small vessel vasculitis caused by in situ immune complex formation; the diagnosis relies on the detection of anti-GBM in tissues or circulation in conjunction with alveolar or glomerular disease; therapy is effective only when detected at an early stage, making a high degree of awareness necessary to find these rare cases; 20-35% have anti-GBM and MPO-ANCA simultaneously, which necessitates testing for anti-GBM whenever acute test for ANCA is ordered in patients with renal disease. Copyright © 2014 Elsevier Ltd. All rights reserved.

MAP30 promotes apoptosis of U251 and U87 cells by suppressing the LGR5 and Wnt/β-catenin signaling pathway, and enhancing Smac expression

PubMed Central

Jiang, Yilin; Miao, Junjie; Wang, Dongliang; Zhou, Jingru; Liu, Bo; Jiao, Feng; Liang, Jiangfeng; Wang, Yangshuo; Fan, Cungang; Zhang, Qingjun

2018-01-01

Significant antitumor activity of Momordica anti-human immunodeficiency virus protein of 30 kDa (MAP30) purified from Momordica charantia has been the subject of previous research. However, the effective mechanism of MAP30 on malignant glioma cells has not yet been clarified. The aim of the present study was to investigate the effects and mechanism of MAP30 on U87 and U251 cell lines. A Cell Counting Kit-8 assay, wound healing assay and Transwell assay were used to detect the effects on U87 and U251 cells treated with different concentrations of MAP30 (0.5, 1, 2, 4, 8 and 16 µM) over different periods of time. Proliferation, migration and invasion of each cell line were markedly inhibited by MAP30 in a dose- and time-dependent manner. Flow cytometry and fluorescence staining demonstrated that apoptosis increased and the cell cycle was arrested in S-phase in the two investigated cell lines following MAP30 treatment. Western blot analysis demonstrated that leucine-rich-repeat-containing G-protein-coupled receptor 5 (LGR5) expression and key proteins in the Wnt/β-catenin signaling pathway were apparently decreased, whereas second mitochondria-derived activator of caspase (Smac) protein expression significantly increased with MAP30 treatment in the same manner. These results suggest that MAP30 markedly induces apoptosis in U87 and U251 cell lines by suppressing LGR5 and the Wnt/β-catenin signaling pathway, and enhancing Smac expression in a dose- and time-dependent manner. PMID:29556310

microRNA-598 inhibits cell proliferation and invasion of glioblastoma by directly targeting metastasis associated in colon cancer-1.

PubMed

Wang, Ning; Zhang, Yang; Liang, Huaxin

2018-02-14

The dysregulation of microRNAs (miRNAs) expression is closely related with tumorigenesis and tumour development in glioblastoma (GBM). In this study, we found that miRNA-598 (miR-598) expression was significantly downregulated in GBM tissues and cell lines. Restoring miR-598 expression inhibited cell proliferation and invasion in GBM. Moreover, we validated that metastasis associated in colon cancer-1 (MACC1) is a novel target of miR-598 in GBM. Recovered MACC1 expression reversed the inhibitory effects of miR-598 overexpression on GBM cells. In addition, miR-598 overexpression suppressed the Met/AKT pathway activation in GBM. Our results provided compelling evidence that miR-598 serves tumour suppressive roles in GBM and that its anti-oncogenic effects are mediated chiefly through the direct suppression of MACC1 expression and regulation of the Met/AKT signalling pathway. Therefore, miR-598 is a potential target in the treatment of GBM.

Discovery of cell surface vimentin targeting mAb for direct disruption of GBM tumor initiating cells.

PubMed

Noh, Hyangsoon; Yan, Jun; Hong, Sungguan; Kong, Ling-Yuan; Gabrusiewicz, Konrad; Xia, Xueqing; Heimberger, Amy B; Li, Shulin

2016-11-01

Intracellular vimentin overexpression has been associated with epithelial-mesenchymal transition, metastasis, invasion, and proliferation, but cell surface vimentin (CSV) is less understood. Furthermore, it remains unknown whether CSV can serve as a therapeutic target in CSV-expressing tumor cells. We found that CSV was present on glioblastoma multiforme (GBM) cancer stem cells and that CSV expression was associated with spheroid formation in those cells. A newly developed monoclonal antibody against CSV, 86C, specifically and significantly induced apoptosis and inhibited spheroid formation in GBM cells in vitro. The addition of 86C to GBM cells in vitro also led to rapid internalization of vimentin and decreased GBM cell viability. These findings were associated with an increase in caspase-3 activity, indicating activation of apoptosis. Finally, treatment with 86C inhibited GBM progression in vivo. In conclusion, CSV-expressing GBM cells have properties of tumor initiating cells, and targeting CSV with the monoclonal antibody 86C is a promising approach in the treatment of GBM.

Genome-wide methylation profiling identifies an essential role of reactive oxygen species in pediatric glioblastoma multiforme and validates a methylome specific for H3 histone family 3A with absence of G-CIMP/isocitrate dehydrogenase 1 mutation

PubMed Central

Jha, Prerana; Pia Patric, Irene Rosita; Shukla, Sudhanshu; Pathak, Pankaj; Pal, Jagriti; Sharma, Vikas; Thinagararanjan, Sivaarumugam; Santosh, Vani; Suri, Vaishali; Sharma, Mehar Chand; Arivazhagan, Arimappamagan; Suri, Ashish; Gupta, Deepak; Somasundaram, Kumaravel; Sarkar, Chitra

2014-01-01

Background Pediatric glioblastoma multiforme (GBM) is rare, and there is a single study, a seminal discovery showing association of histone H3.3 and isocitrate dehydrogenase (IDH)1 mutation with a DNA methylation signature. The present study aims to validate these findings in an independent cohort of pediatric GBM, compare it with adult GBM, and evaluate the involvement of important functionally altered pathways. Methods Genome-wide methylation profiling of 21 pediatric GBM cases was done and compared with adult GBM data (GSE22867). We performed gene mutation analysis of IDH1 and H3 histone family 3A (H3F3A), status evaluation of glioma cytosine–phosphate–guanine island methylator phenotype (G-CIMP), and Gene Ontology analysis. Experimental evaluation of reactive oxygen species (ROS) association was also done. Results Distinct differences were noted between methylomes of pediatric and adult GBM. Pediatric GBM was characterized by 94 hypermethylated and 1206 hypomethylated cytosine–phosphate–guanine (CpG) islands, with 3 distinct clusters, having a trend to prognostic correlation. Interestingly, none of the pediatric GBM cases showed G-CIMP/IDH1 mutation. Gene Ontology analysis identified ROS association in pediatric GBM, which was experimentally validated. H3F3A mutants (36.4%; all K27M) harbored distinct methylomes and showed enrichment of processes related to neuronal development, differentiation, and cell-fate commitment. Conclusions Our study confirms that pediatric GBM has a distinct methylome compared with that of adults. Presence of distinct clusters and an H3F3A mutation–specific methylome indicate existence of epigenetic subgroups within pediatric GBM. Absence of IDH1/G-CIMP status further indicates that findings in adult GBM cannot be simply extrapolated to pediatric GBM and that there is a strong need for identification of separate prognostic markers. A possible role of ROS in pediatric GBM pathogenesis is demonstrated for the first time and needs further evaluation. PMID:24997139

3-Bromopyruvate treatment induces alterations of metabolic and stress-related pathways in glioblastoma cells.

PubMed

Chiasserini, Davide; Davidescu, Magdalena; Orvietani, Pier Luigi; Susta, Federica; Macchioni, Lara; Petricciuolo, Maya; Castigli, Emilia; Roberti, Rita; Binaglia, Luciano; Corazzi, Lanfranco

2017-01-30

Glioblastoma (GBM) is the most common and aggressive brain tumour of adults. The metabolic phenotype of GBM cells is highly dependent on glycolysis; therefore, therapeutic strategies aimed at interfering with glycolytic pathways are under consideration. 3-Bromopyruvate (3BP) is a potent antiglycolytic agent, with a variety of targets and possible effects on global cell metabolism. Here we analyzed the changes in protein expression on a GBM cell line (GL15 cells) caused by 3BP treatment using a global proteomic approach. Validation of differential protein expression was performed with immunoblotting and enzyme activity assays in GL15 and U251 cell lines. The results show that treatment of GL15 cells with 3BP leads to extensive changes in the expression of glycolytic enzymes and stress related proteins. Importantly, other metabolisms were also affected, including pentose phosphate pathway, aminoacid synthesis, and glucose derivatives production. 3BP elicited the activation of stress response proteins, as shown by the phosphorylation of HSPB1 at serine 82, caused by the concomitant activation of the p38 pathway. Our results show that inhibition of glycolysis in GL15 cells by 3BP influences different but interconnected pathways. Proteome analysis may help in the molecular characterization of the glioblastoma response induced by pharmacological treatment with antiglycolytic agents. Alteration of the glycolytic pathway characterizes glioblastoma (GBM), one of the most common brain tumours. Metabolic reprogramming with agents able to inhibit carbohydrate metabolism might be a viable strategy to complement the treatment of these tumours. The antiglycolytic agent 3-bromopyruvate (3BP) is able to strongly inhibit glycolysis but it may affect also other cellular pathways and its precise cellular targets are currently unknown. To understand the protein expression changes induced by 3BP, we performed a global proteomic analysis of a GBM cell line (GL15) treated with 3BP. We found that 3BP affected not only the glycolytic pathway, but also pathways sharing metabolic intermediates with glycolysis, such as the pentose phosphate pathway and aminoacid metabolism. Furthermore, changes in the expression of proteins linked to resistance to cell death and stress response were found. Our work is the first analysis on a global scale of the proteome changes induced by 3BP in a GBM model and may contribute to clarifying the anticancer potential of this drug. Copyright © 2016 Elsevier B.V. All rights reserved.

Neurosphere and adherent culture conditions are equivalent for malignant glioma stem cell lines.

PubMed

Rahman, Maryam; Reyner, Karina; Deleyrolle, Loic; Millette, Sebastien; Azari, Hassan; Day, Bryan W; Stringer, Brett W; Boyd, Andrew W; Johns, Terrance G; Blot, Vincent; Duggal, Rohit; Reynolds, Brent A

2015-03-01

Certain limitations of the neurosphere assay (NSA) have resulted in a search for alternative culture techniques for brain tumor-initiating cells (TICs). Recently, reports have described growing glioblastoma (GBM) TICs as a monolayer using laminin. We performed a side-by-side analysis of the NSA and laminin (adherent) culture conditions to compare the growth and expansion of GBM TICs. GBM cells were grown using the NSA and adherent culture conditions. Comparisons were made using growth in culture, apoptosis assays, protein expression, limiting dilution clonal frequency assay, genetic affymetrix analysis, and tumorigenicity in vivo. In vitro expansion curves for the NSA and adherent culture conditions were virtually identical (P=0.24) and the clonogenic frequencies (5.2% for NSA vs. 5.0% for laminin, P=0.9) were similar as well. Likewise, markers of differentiation (glial fibrillary acidic protein and beta tubulin III) and proliferation (Ki67 and MCM2) revealed no statistical difference between the sphere and attachment methods. Several different methods were used to determine the numbers of dead or dying cells (trypan blue, DiIC, caspase-3, and annexin V) with none of the assays noting a meaningful variance between the two methods. In addition, genetic expression analysis with microarrays revealed no significant differences between the two groups. Finally, glioma cells derived from both methods of expansion formed large invasive tumors exhibiting GBM features when implanted in immune-compromised animals. A detailed functional, protein and genetic characterization of human GBM cells cultured in serum-free defined conditions demonstrated no statistically meaningful differences when grown using sphere (NSA) or adherent conditions. Hence, both methods are functionally equivalent and remain suitable options for expanding primary high-grade gliomas in tissue culture.

Neurosphere and adherent culture conditions are equivalent for malignant glioma stem cell lines

PubMed Central

Reyner, Karina; Deleyrolle, Loic; Millette, Sebastien; Azari, Hassan; Day, Bryan W.; Stringer, Brett W.; Boyd, Andrew W.; Johns, Terrance G.; Blot, Vincent; Duggal, Rohit; Reynolds, Brent A.

2015-01-01

Certain limitations of the neurosphere assay (NSA) have resulted in a search for alternative culture techniques for brain tumor-initiating cells (TICs). Recently, reports have described growing glioblastoma (GBM) TICs as a monolayer using laminin. We performed a side-by-side analysis of the NSA and laminin (adherent) culture conditions to compare the growth and expansion of GBM TICs. GBM cells were grown using the NSA and adherent culture conditions. Comparisons were made using growth in culture, apoptosis assays, protein expression, limiting dilution clonal frequency assay, genetic affymetrix analysis, and tumorigenicity in vivo. In vitro expansion curves for the NSA and adherent culture conditions were virtually identical (P=0.24) and the clonogenic frequencies (5.2% for NSA vs. 5.0% for laminin, P=0.9) were similar as well. Likewise, markers of differentiation (glial fibrillary acidic protein and beta tubulin III) and proliferation (Ki67 and MCM2) revealed no statistical difference between the sphere and attachment methods. Several different methods were used to determine the numbers of dead or dying cells (trypan blue, DiIC, caspase-3, and annexin V) with none of the assays noting a meaningful variance between the two methods. In addition, genetic expression analysis with microarrays revealed no significant differences between the two groups. Finally, glioma cells derived from both methods of expansion formed large invasive tumors exhibiting GBM features when implanted in immune-compromised animals. A detailed functional, protein and genetic characterization of human GBM cells cultured in serum-free defined conditions demonstrated no statistically meaningful differences when grown using sphere (NSA) or adherent conditions. Hence, both methods are functionally equivalent and remain suitable options for expanding primary high-grade gliomas in tissue culture. PMID:25806119

Directly visualized glioblastoma-derived extracellular vesicles transfer RNA to microglia/macrophages in the brain

PubMed Central

van der Vos, Kristan E.; Abels, Erik R.; Zhang, Xuan; Lai, Charles; Carrizosa, Esteban; Oakley, Derek; Prabhakar, Shilpa; Mardini, Osama; Crommentuijn, Matheus H. W.; Skog, Johan; Krichevsky, Anna M.; Stemmer-Rachamimov, Anat; Mempel, Thorsten R.; El Khoury, Joseph; Hickman, Suzanne E.; Breakefield, Xandra O.

2016-01-01

Background To understand the ability of gliomas to manipulate their microenvironment, we visualized the transfer of vesicles and the effects of tumor-released extracellular RNA on the phenotype of microglia in culture and in vivo. Methods Extracellular vesicles (EVs) released from primary human glioblastoma (GBM) cells were isolated and microRNAs (miRNAs) were analyzed. Primary mouse microglia were exposed to GBM-EVs, and their uptake and effect on proliferation and levels of specific miRNAs, mRNAs, and proteins were analyzed. For in vivo analysis, mouse glioma cells were implanted in the brains of mice, and EV release and uptake by microglia and monocytes/macrophages were monitored by intravital 2-photon microscopy, immunohistochemistry, and fluorescence activated cell sorting analysis, as well as RNA and protein levels. Results Microglia avidly took up GBM-EVs, leading to increased proliferation and shifting of their cytokine profile toward immune suppression. High levels of miR-451/miR-21 in GBM-EVs were transferred to microglia with a decrease in the miR-451/miR-21 target c-Myc mRNA. In in vivo analysis, we directly visualized release of EVs from glioma cells and their uptake by microglia and monocytes/macrophages in brain. Dissociated microglia and monocytes/macrophages from tumor-bearing brains revealed increased levels of miR-21 and reduced levels of c-Myc mRNA. Conclusions Intravital microscopy confirms the release of EVs from gliomas and their uptake into microglia and monocytes/macrophages within the brain. Our studies also support functional effects of GBM-released EVs following uptake into microglia, associated in part with increased miRNA levels, decreased target mRNAs, and encoded proteins, presumably as a means for the tumor to manipulate its environs. PMID:26433199

Glomerular Epithelial Cells-Targeted Heme Oxygenase-1 Over Expression in the Rat: Attenuation of Proteinuria in Secondary But Not Primary Injury.

PubMed

Atsaves, Vassilios; Makri, Panagiota; Detsika, Maria G; Tsirogianni, Alexandra; Lianos, Elias A

2016-01-01

Induction of heme oxygenase 1 (HO-1) in glomerular epithelial cells (GEC) in response to injury is poor and this may be a disadvantage. We, therefore, explored whether HO-1 overexpression in GEC can reduce proteinuria induced by puromycin aminonucleoside (PAN) or in anti-glomerular basement membrane (GBM) antibody (Ab)-mediated glomerulonephritis (GN). HO-1 overexpression in GEC (GECHO-1) of Sprague-Dawley rats was achieved by targeting a FLAG-human (h) HO-1 using transposon-mediated transgenesis. Direct GEC injury was induced by a single injection of PAN. GN was induced by administration of an anti-rat GBM Ab and macrophage infiltration in glomeruli was assessed by immunohistochemistry and western blot analysis, which was also used to assess glomerular nephrin expression. In GECHO-1 rats, FLAG-hHO-1 transprotein was co-immunolocalized with nephrin. Baseline glomerular HO-1 protein levels were higher in GECHO-1 compared to wild type (WT) rats. Administration of either PAN or anti-GBM Ab to WT rats increased glomerular HO-1 levels. Nephrin expression markedly decreased in glomeruli of WT or GECHO-1 rats treated with PAN. In anti-GBM Ab-treated WT rats, nephrin expression also decreased. In contrast, it was preserved in anti-GBM Ab-treated GECHO-1 rats. In these, macrophage infiltration in glomeruli and the ratio of urine albumin to urine creatinine (Ualb/Ucreat) were markedly reduced. There was no difference in Ualb/Ucreat between WT and GECHO-1 rats treated with PAN. Depending on the type of injury, HO-1 overexpression in GEC may or may not reduce proteinuria. Reduced macrophage infiltration and preservation of nephrin expression are putative mechanisms underlying the protective effect of HO-1 overexpression following immune injury. © 2016 S. Karger AG, Basel.

A Proposal to Localize Fermi GBM GRBs Through Coordinated Scanning of the GBM Error Circle via Optical Telescopes

NASA Technical Reports Server (NTRS)

Ukwatta, T. N.; Linnemann, J. T.; Tollefson, K.; Abeysekara, A. U.; Bhat, P. N.; Sonbas, E.; Gehrels, N.

2011-01-01

We investigate the feasibility of implementing a system that will coordinate ground-based optical telescopes to cover the Fermi GBM Error Circle (EC). The aim of the system is to localize GBM detected GRBs and facilitate multi-wavelength follow-up from space and ground. This system will optimize the observing locations in the GBM EC based on individual telescope location, Field of View (FoV) and sensitivity. The proposed system will coordinate GBM EC scanning by professional as well as amateur astronomers around the world. The results of a Monte Carlo simulation to investigate the feasibility of the project are presented.

A regulatory circuit of miR-125b/miR-20b and Wnt signalling controls glioblastoma phenotypes through FZD6-modulated pathways

PubMed Central

Huang, Tianzhi; Alvarez, Angel A.; Pangeni, Rajendra P.; M. Horbinski, Craig; Lu, Songjian; Kim, Sung-Hak; James, C. David; J. Raizer, Jeffery; A. Kessler, John; Brenann, Cameron W.; Sulman, Erik P.; Finocchiaro, Gaetano; Tan, Ming; Nishikawa, Ryo; Lu, Xinghua; Nakano, Ichiro; Hu, Bo; Cheng, Shi-Yuan

2016-01-01

Molecularly defined subclassification is associated with phenotypic malignancy of glioblastoma (GBM). However, current understanding of the molecular basis of subclass conversion that is often involved in GBM recurrence remain rudimentary at best. Here we report that canonical Wnt signalling that is active in proneural (PN) but inactive in mesenchymal (MES) GBM, along with miR-125b and miR-20b that are expressed at high levels in PN compared with MES GBM, comprise a regulatory circuit involving TCF4-miR-125b/miR-20b-FZD6. FZD6 acts as a negative regulator of this circuit by activating CaMKII–TAK1–NLK signalling, which, in turn, attenuates Wnt pathway activity while promoting STAT3 and NF-κB signalling that are important regulators of the MES-associated phenotype. These findings are confirmed by targeting differentially enriched pathways in PN versus MES GBM that results in inhibition of distinct GBM subtypes. Correlative expressions of the components of this circuit are prognostic relevant for clinical GBM. Our findings provide insights for understanding GBM pathogenesis and for improving treatment of GBM. PMID:27698350

MicroRNA Regulation of Glycolytic Metabolism in Glioblastoma

PubMed Central

McIntyre, Alan; Smith, Stuart

2017-01-01

Glioblastoma (GBM) is the most aggressive and common malignant brain tumour in adults. A well-known hallmark of GMB and many other tumours is aerobic glycolysis. MicroRNAs (miRNAs) are a class of short nonprotein coding sequences that exert posttranscriptional controls on gene expression and represent critical regulators of aerobic glycolysis in GBM. In GBM, miRNAs regulate the expression of glycolytic genes directly and via the regulation of metabolism-associated tumour suppressors and oncogenic signalling pathways. This review aims to establish links between miRNAs expression levels, the expression of GBM glycolytic regulatory genes, and the malignant progression and prognosis of GBM. In this review, the involvement of 25 miRNAs in the regulation of glycolytic metabolism of GBM is discussed. Seven of these miRNAs have been shown to regulate glycolytic metabolism in other tumour types. Further eight miRNAs, which are differentially expressed in GBM, have also been reported to regulate glycolytic metabolism in other cancer types. Thus, these miRNAs could serve as potential glycolytic regulators in GBM but will require functional validation. As such, the characterisation of these molecular and metabolic signatures in GBM can facilitate a better understanding of the molecular pathogenesis of this disease. PMID:28804724

The Role of Hypoxia in Glioblastoma Invasion

PubMed Central

Monteiro, Ana Rita; Pilkington, Geoffrey J.

2017-01-01

Glioblastoma multiforme (GBM), a grade IV astrocytoma, is the most common and deadly type of primary malignant brain tumor, with a patient’s median survival rate ranging from 15 to 17 months. The current treatment for GBM involves tumor resection surgery based on MRI image analysis, followed by radiotherapy and treatment with temozolomide. However, the gradual development of tumor resistance to temozolomide is frequent in GBM patients leading to subsequent tumor regrowth/relapse. For this reason, the development of more effective therapeutic approaches for GBM is of critical importance. Low tumor oxygenation, also known as hypoxia, constitutes a major concern for GBM patients, since it promotes cancer cell spreading (invasion) into the healthy brain tissue in order to evade this adverse microenvironment. Tumor invasion not only constitutes a major obstacle to surgery, radiotherapy, and chemotherapy, but it is also the main cause of death in GBM patients. Understanding how hypoxia triggers the GBM cells to become invasive is paramount to developing novel and more effective therapies against this devastating disease. In this review, we will present a comprehensive examination of the available literature focused on investigating how GBM hypoxia triggers an invasive cancer cell phenotype and the role of these invasive proteins in GBM progression. PMID:29165393