Human Malignant Glioma Cell Lines Research Articles

Actin Alpha 2, Smooth Muscle (ACTA2) Is Involved in the Migratory Potential of Malignant Gliomas, and Its Increased Expression at Recurrence Is a Significant Adverse Prognostic Factor.

Malignant glioma is a highly invasive tumor, and elucidating the glioma invasion mechanism is essential for developing novel therapies. We aimed to highlight actin alpha 2, smooth muscle (ACTA2) as potential biomarkers of brain invasion and distant recurrence in malignant gliomas. Using the human malignant glioma cell line, U251MG, we generated ACTA2 knockdown (KD) cells treated with small interfering RNA, and the cell motility and proliferation of the ACTA2 KD group were analyzed. Furthermore, tumor samples from 12 glioma patients who underwent reoperation at the time of tumor recurrence were utilized to measure ACTA2 expression in the tumors before and after recurrence. Thereafter, we examined how ACTA2 expression correlates with the time to tumor recurrence and the mode of recurrence. The results showed that the ACTA2 KD group demonstrated a decline in the mean motion distance and proliferative capacity compared to the control group. In the clinical glioma samples, ACTA2 expression was remarkably increased in recurrent samples compared to the primary samples from the same patients, and the higher the change in ACTCA2 expression from the start to relapse, the shorter the progression-free survival. In conclusion, ACTA2 may be involved in distant recurrence in clinical gliomas.

Irreversible Electroporation Mediates Glioma Apoptosis via Upregulation of AP-1 and Bim: Transcriptome Evidence.

The heat-sink effect and thermal damage of conventional thermal ablative technologies can be minimized by irreversible electroporation (IRE), which results in clear ablative boundaries and conservation of blood vessels, facilitating maximal safe surgical resection for glioblastoma. Although much comparative data about the death forms in IRE have been published, the comprehensive genetic regulatory mechanism for apoptosis, among other forms of regulatory cell death (RCD), remains elusive. We investigated the electric field intensity threshold for apoptosis/necrosis (YO-PRO-1/PI co-staining) of the U251 human malignant glioma cell line with stepwise increased uniform field intensity. Time course samples (0-6 h) of apoptosis induction and sham treatment were collected for transcriptome sequencing. Sequencing showed that transcription factor AP-1 and its target gene Bim (Bcl2l11), related to the signaling pathway, played a major role in the apoptosis of glioma after IRE. The sequencing results were confirmed by qPCR and Western blot. We also found that the transcription changes also implicated three other forms of RCD: autophagy, necroptosis, and immunogenic cell death (ICD), in addition to apoptosis. These together imply that IRE possibly mediates apoptosis by the AP-1-Bim pathway, causes mixed RCD simultaneously, and has the potential to aid in the generation of a systemic antitumor immune response.

Prenylaromadendrane-type diterpenoids from the gum resin of Boswellia sacra flueck and their cytotoxic effects

Two new prenylaromadendrane-type diterpenoids, and three known analogues, were isolated from the ethanol extract of the gum resin of B. sacra Flueck. The structures of the new compounds were elucidated using 1 D and 2 D NMR spectroscopic analyses, mass spectrometric data, circular dichroism spectra, and comparison with the other compounds in the literature. One diterpenoid represents the first example of an acetoxyl-substituted prenylaromadendranoid in frankincense. All compounds exhibited notable cytotoxicity against human malignant glioma (U87-MG) cell line, with inhibitory rates exceeding that of the positive control 5-fluorouracil. However, nitric oxide inhibition induced by lipopolysaccarides was not observed in primary mouse peritoneal macrophages.

Carbon Dots‐Cluster‐DOX Nanocomposites Fabricated by a Co‐Self‐Assembly Strategy for Tumor‐Targeted Bioimaging and Therapy

AbstractCarbon‐based nanomaterials could afford versatile potential applications in biomedical optical imaging and as nanoparticle drug carriers, owing to their promising optical and biocompatible capabilities. In this paper, it is first found that amphipathic cetylpyridinium chloride (CPC)‐stabilized oil‐soluble carbon dots (CDs) could self‐assemble into hydrophilic CDs clusters with hydrophobic core under ultrasound, in which CPC acts as carbon source, stabilizer, and phase transfer agent. Next, the size‐control (for size‐dependent passive tumor targeting) and doxorubicin (DOX) uploading of aqueous CDs clusters, and subsequent surface charge modification via overcoating with cRGD‐ and octylamine‐modified polyacrylic acid (cRGD‐PAA‐OA) (reversing their surface charges into negative and introducing active tumor‐targeting ability) are explored systematically. Based on this sequential administration mode, CDs‐cluster‐DOX/cRGD‐PAA‐OA nanocomposites exhibit selective human malignant glioma cell line (U87MG) tumor targeting. In in vitro drug release experiments, the nanocomposites could release DOX timely. Owning to the dual tumor targeting effects and seasonable drug release, CDs‐cluster‐DOX/cRGD‐PAA‐OA show remarkably tumor targetability and enhanced antitumor efficacy (and reduced adverse reaction), comparing to free DOX in animal models. These results indicate that fabricating nanocomposite via co‐self‐assembly strategy is efficient toward drug delivery system for tumor‐targeting theranostic.

Human corticotrophin releasing factor inhibits cell proliferation and promotes apoptosis through upregulation of tumor protein p53 in human glioma.

Corticotropin-releasing factor (CRF) and its receptors have been detected in numerous tumors and have an important role in tumorigenesis and proliferation. However, the role of these peptides has not been established in human glioma and malignant glioma cell lines. The present study evaluated for the first time, the expression of CRF receptor 1 (CRFR1) in 35 human glioma samples, 13 normal brain tissues and human U87 glioma cells using immunohistochemistry, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot analysis. Levels of CRFR1 were identified to be significantly increased in human glioma and U87 cells and higher levels of CRFR1 were observed in glioma tissues of higher grade. The biological functions of human CRF (hCRF) on U87 cells glioma cells were investigated by cell counting, a bromodeoxyuridine assay and flow cytometry. The U87 cells under hCRF treatment exhibited reduced proliferation, increased apoptosis and a cell cycle arrest in S and G2/M phase. The tumor protein p53 (p53) gene may participate in the activation of hCRF via CRFR1 in U87 cells, therefore p53 mRNA and protein were evaluated using RT-qPCR and western blot analysis. Finally, the present results suggest that hCRF inhibits proliferation and induces cell-cycle arrest and apoptosis in U87 cells via the CRFR1-mediated p53 signaling pathway. Therefore, the present study also suggests that hCRF may be used therapeutically, and CRFR1 may be a putative therapeutic target for human glioma.

The role of ANKDR49 in evaluating the prognosis of glioma

ObjectiveTo evaluate the role of ANKRD49 in the prognosis of glioma.MethodsTranscriptome expression data sets and the corresponding clinical information of ANKRD49 gene were downloaded from the website of The Cancer Genome Atlas (TCGA). A total of 558 samples, which contained transcriptional expression data of 548 tumor tissues and 10 normal tissues, were available for this analysis. Human malignant glioma cell lines U251 and U87 were cultured. Total RNA was isolated from indicated cells using TRIzol reagent. Quantitative real‐time PCR was used to analyze ANKRD49 gene expression. Total protein was isolated from indicated cells and West‐blot was used to analyze ANKRD49 protein expression. SPSS 16.0 software was used to analyze experimental data and clinical data.ResultsThe expression of ANKRD49 was predominantly higher in glioma samples compared with matched normal samples(fold change =2.11, P=2.99E‐09). The 5‐year overall survival of patients with highly expressed ANKDR49 was distinctively shorter than that of patients with lowly expressed ANKDR49(P<0.01).ConclusionANKRD49 is overexpressed in human glioma tissues, and high expression of ANKRD49 is significantly associated with poor survival. ANKDR49 might be a indicator for evaluating the prognosis of glioma.Support or Funding InformationThis study was supported by the National Natural Science Foundation Youth Fund (Fund No. 30600637), China Postdoctral Foundation(Fund No.2014M561207), the Shanxi Province Basic Research Program Youth Science and Technology Research Fund (Fund No. 2010021034‐4), the Shanxi Scholarship Council of China (Fund No. 2011‐096 and Key Projects No.4), The science and technology research projects of health department of Shanxi Province(Fund No.2016D011119), The Teaching Reform Program of Shanxi Higher Education (No. 2013‐033), The Education Reform Project of Postgraduate Students in Shanxi Province (No. 2017‐027). The Project of “131” Leading Talents of Shanxi Higher Education, The Education Reform Project of Shanxi Medical University.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The redox couple avarol/avarone in the fight with malignant gliomas: the case study of U-251 MG cells

This study aimed to screen in vitro antitumour activity of the redox couple avarol/avarone against the human malignant glioma cell line U-251 MG for the first time. Compared both with avarol and positive controls used (temozolomide and doxorubicin), avarone was found to be the most active compound with IC50 value below 1 μM (IC50 0.68 ± 0.04 μM, 96 h). Considerable less DNA damage in the cells treated with avarol and avarone vs. doxorubicin (105 & 123% vs. 299%, respectively; untreated U-251 MG cells were used as a control, 100%), coupled with no sign of cytotoxicity against the normal human foetal lung fibroblast MRC-5 cells (IC50 > 100 μM), has actually pointed out the importance of this marine sesquiterpenoid quinone structure as a promising lead compound in development of novel brain chemotherapeutics.

Time-and oxygen-dependent expression and regulation of NDRG1 in human brain cancer cells.

N-myc downstream-regulated gene1 (NDRG1) is a tumor suppressor with the potential to suppress metastasis, invasion and migration of cancer cells. It is regulated under stress conditions such as starvation or hypoxia. NDRG1 regulation is both induced and controlled by HIF-1α-dependent and -independent pathways under hypoxic conditions. However, there are profound differences in the way NDRG1 expression is regulated by HIF-1α and other transcription factors. Therefore, we aimed to define the time-dependent pattern of NDRG1 mRNA and protein expression in human glioblastoma cell lines in extreme hypoxia and after re-oxygenation as well as under normoxic conditions. Furthermore, we ascribe the regulation of NDRG1 to the transcription factors HIF-1α, SP1, CEBPα, YB-1 and Smad7 in a time-dependent manner. The human malignant glioma cell lines U87-MG, U373 and GaMG were cultured for 1, 6 and 24h under hypoxic (0.1%O2) conditions and then they were re-oxygenated. The mRNA expression of NDRG1, HIF-1α SP1, CEBPα, YB-1 and Smad7 was measured using semi-quantitative RT-PCR analysis. Their protein expression was analyzed using western blotting. Our experiments revealed that long-term (24h), but not short-term hypoxia led to the induction of NDRG1 expression in human glioma cell lines. NDRG1 expression was found to correlate with the protein expression of HIF-1α, SP1, CEBPα, YB-1 and Smad7. The present study suggests for the first time that SP1 regulates NDRG1 expression in glioma cells under hypoxia in a time-dependent manner along with HIF-1α, CEBPα, YB-1 and Smad7. These molecules, each separately or in combination, may possess the potential to become target molecules for antitumor therapeutic approaches particularly in human brain tumors.

Hypoxia-induced production of peptidylarginine deiminases and citrullinated proteins in malignant glioma cells

BackgroundRecently, it has been reported that hypoxia highly enhances expression of peptidylarginine deiminase (PAD) 4 and production of citrullinated proteins in some tumor cells. However, little is known about malignant gliomas on this issue. Therefore, we here investigated whether expression of PADs was induced by hypoxia and whether PADs citrullinated intracellular proteins if induced using U-251 MG cells of a human malignant glioma cell line. MethodsExpression of PADs in U-251 MG cells, cultured under hypoxia or normoxia for 24 h, was investigated by quantitative polymerase chain reaction (qPCR). Citrullination of proteins in the cells and the cell lysates incubated for 48 h with or without Ca2+ was detected by western blotting. Citrullinated proteins were identified by mass spectrometry. ResultsThe mRNA levels of PAD1, 2, 3, and 4 were up-regulated by hypoxia in a hypoxia-inducible factor-1-dependent manner in U-251 MG cells. In spite of the increased expression, intracellular proteins were not citrullinated. However, the induced PADs citrullinated U-251 MG cell-derived proteins when the cells were lysed. Multiple proteins citrullinated by hypoxia-induced PADs were identified. In addition, the extracellular domain of vascular endothelial growth factor receptor 2 was citrullinated by human PAD2 in vitro. ConclusionOur data may contribute to understanding of pathophysiology of malignant gliomas from the aspects of protein citrullination.

MiRNA-429 Inhibits Astrocytoma Proliferation and Invasion by Targeting BMI1.

Glioblastoma multiforme (GBM), the most common primary brain cancer in adults, is usually the most lethal type of brain tumor. MicroRNAs (miRNAs) are a class of small, non-coding RNA molecules that deeply involves with the regulation of gene expression and cellular processes, including proliferation, apoptosis, migration and invasion. The objective of the study is to investigate the effect of miRNA-429 on human glioblastoma tissues and cell lines. miRNA-429 expressions in human glioblastoma, normal brain tissue samples, and human malignant glioma cell lines (U87, U251 and LN229) were compared using reverse transcription-quantitative PCR and western blot methods. U251 cell lines were transfected with miRNA-429 mimics, and then the effects of miRNA-429 on cell proliferation and invasion were investigated by CCK8 and Transwell invasion assay, respectively. It was found that miRNA-429 expression was significantly reduced in the examined Glioblastoma samples and human glioma cell lines. Overexpression of miRNA-429 inhibited Glioblastoma cell proliferation and invasion. Additionally, the present study also showed that BMI1 was a functional target of miRNA-429. Overexpression of BMI1 undermined the inhibition effect of miRNA-429 in glioblastoma and U251 cell lines. The current study demonstrated that miRNA-429, as a tumor suppressor gene, was capable of negatively regulating the expression of BMI1 in U251 cells.

Abstract 3565: Dinaciclib, a CDK inhibitor promotes proteasomal degradation of Mcl-1 and enhances ABT-737 mediated cell death in malignant human glioma cell lines

Abstract The prognosis of malignant glioma, the most common brain tumor, is still poor, thus underscoring the need to develop novel treatment strategies. Because glioma cells commonly exhibit genomic alterations involving genes that regulate cell cycle control, there is a strong rationale to examine the potential efficacy of strategies to counteract this process. In this study, we examined the antiproliferative effects of the cyclin-dependent kinase inhibitor dinaciclib in malignant human glioma cell lines, with intact or deleted or mutated p53 or PTEN or p14ARF, or EGFR amplification status. Dinaciclib was found to inhibit cell proliferation and induce cell cycle arrest at the G2/M checkpoint, independent of p53 mutational status. In a standard 72 h MTS assay, at clinically relevant concentrations, dose-dependent antiproliferative effects were observed but cell death was not induced. Moreover, the combination of conventional chemotherapeutic agents and various growth signaling inhibitors with dinaciclib did not yield synergistic cytotoxicity. In contrast, combination of the Bcl-2/Bcl-xL inhibitor ABT-263 or ABT-737 with dinaciclib potentiated the apoptotic response induced by each single drug. The synergistic killing by ABT-737 with dinaciclib led to cell death accompanied by the hallmarks of apoptosis, including an early loss of the mitochondrial transmembrane potential, the release of cytochrome c, smac/DIABLO and apoptosis-inducing factor (AIF), phosphatidylserine exposure on the plasma membrane surface and activation of caspases and PARP. Mechanistic studies revealed that dinaciclib promoted proteasomal degradation of Mcl-1. Citation Format: Esther P. Jane, Daniel Premkumar, Jonathon Cavaleri, Thatchana Rajasekar, Philip Sutera, Ian Pollack. Dinaciclib, a CDK inhibitor promotes proteasomal degradation of Mcl-1 and enhances ABT-737 mediated cell death in malignant human glioma cell lines. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3565.

Dinaciclib, a Cyclin-Dependent Kinase Inhibitor Promotes Proteasomal Degradation of Mcl-1 and Enhances ABT-737-Mediated Cell Death in Malignant Human Glioma Cell Lines.

The prognosis for malignant glioma, the most common brain tumor, is still poor, underscoring the need to develop novel treatment strategies. Because glioma cells commonly exhibit genomic alterations involving genes that regulate cell-cycle control, there is a strong rationale for examining the potential efficacy of strategies to counteract this process. In this study, we examined the antiproliferative effects of the cyclin-dependent kinase inhibitor dinaciclib in malignant human glioma cell lines, with intact, deleted, or mutated p53 or phosphatase and tensin homolog on chromosome 10; intact or deleted or p14ARF or wild-type or amplified epidermal growth factor receptor. Dinaciclib inhibited cell proliferation and induced cell-cycle arrest at the G2/M checkpoint, independent of p53 mutational status. In a standard 72-hour 3-[4,5-dimethylthiazol- 2yl]-5-[3-carboxymethoxyphenyl]-2-[4-sulfophenyl]-2H, tetrazolium (MTS) assay, at clinically relevant concentrations, dose-dependent antiproliferative effects were observed, but cell death was not induced. Moreover, the combination of conventional chemotherapeutic agents and various growth-signaling inhibitors with dinaciclib did not yield synergistic cytotoxicity. In contrast, combination of the Bcl-2/Bcl-xL inhibitors ABT-263 (4-[4-[[2-(4-chlorophenyl)-5,5-dimethylcyclohexen-1-yl]methyl]piperazin-1-yl]-N-[4-[[(2R)-4-morpholin-4-yl-1-phenylsulfanylbutan-2-yl]amino]-3-(trifluoromethylsulfonyl)phenyl]sulfonylbenzamide) or ABT-737 (4-[4-[[2-(4-chlorophenyl)phenyl]methyl]piperazin-1-yl]-N-[4-[[(2R)-4-(dimethylamino)-1-phenylsulfanylbutan-2-yl]amino]-3-nitrophenyl]sulfonylbenzamide) with dinaciclib potentiated the apoptotic response induced by each single drug. The synergistic killing by ABT-737 with dinaciclib led to cell death accompanied by the hallmarks of apoptosis, including an early loss of the mitochondrial transmembrane potential; the release of cytochrome c, smac/DIABLO, and apoptosis-inducing factor; phosphatidylserine exposure on the plasma membrane surface and activation of caspases and poly ADP-ribose polymerase. Mechanistic studies revealed that dinaciclib promoted proteasomal degradation of Mcl-1. These observations may have important clinical implications for the design of experimental treatment protocols for malignant human glioma.

CBIO-12EFFECT OF SONODYNAMIC THERAPY WITH 5-AMINOLEVULINIC ACID ON MALIGNANT GLIOMA

INTRODUCTION: Photodiagnosis (PD) with 5-aminolevulinic acid (5-ALA) is useful to detect malignant glioma tissue during surgery. On the other hand, photodynamic therapy (PDT) with 5-ALA is not yet popular for malignant gliomas, although PDT is widely used for skin or gastrointestinal cancers. One of the problems of PDT for gliomas is the limit of penetrating distance of light. Since glioma grows invasively into deep tissue, its effect does not reach the peripheral of the tumor. Ultrasound instead can conduct its energy into the deep tissue with less attenuation and is known to activate photosensitizing drug like light. We have been investigating its application for glioma treatment and report the different effects on two glioma cell lines. MATERIALS AND METHODS: Two human malignant glioma cell lines (U-87 MG, SNB-19) were cultured in exponential growth phase. The glioma cells were treated with 5-aminolevulimic acid (5-ALA) for 4 hours and then enclosed in plastic tubes. Ultrasonic device UST-770 (ITO Co., Ltd.) was used for all experiments. Ultrasonication (US) was performed at the condition of 2.0W/cm2, 1.0MHz, 5min and the depth of 3 cm. As control, 5-ALA (-) or US(-) groups were set. The number of cells was counted immediately after SDT and after 48 hour-incubation. RESULTS: The SDT with 5-ALA showed suppressive effect (approximately 15% by comparison with a control data) on U-87 MG. Whereas no effect was seen in other groups of U-87 MG, and all groups of SNB-19. CONCLUSION: SDT with 5-ALA has the potential as an option for treatment of malignant glioma. The conditions have to be investigated for its stable effectiveness. The mechanism of action should also be elucidated to understand the differential effects on each cell lines.

MicroRNA-203 Modulates the Radiation Sensitivity of Human Malignant Glioma Cells

We investigated whether miR-203 could modulate the radiation sensitivity of glioblastoma (GBM) cells and which target gene(s) could be involved. Three human malignant glioma (MG) cell lines and normal human astrocytes were transfected with control microRNA, pre-miR-203, or antisense miR-203. Real-time PCR (RT-PCR), clonogenic assays, immunofluorescence, and invasion/migration assays were performed. To predict the target(s), bioinformatics analyses using microRNA target databases were performed. Overexpression of miR-203 increased the radiation sensitivity of all 3 human MG cell lines and prolonged radiation-induced γ-H2AX foci formation. Bioinformatics analyses suggested that miR-203 could be involved in post-transcriptional control of DNA repair, PI3K/AKT, SRC, and JAK/STAT3 and the vascular signaling pathway. Western blot analysis validated the fact that miR-203 downregulated ATM, RAD51, SRC, PLD2, PI3K-AKT, JAK-STAT3, VEGF, HIF-1α, and MMP2. Overexpression of miR-203 inhibited invasion and migration potentials, downregulated SLUG and Vimentin, and upregulated Claudin-1 and ZO1. These data demonstrate that miR-203 potentially controls DNA damage repair via the PI3K/AKT and JAK/STAT3 pathways and may collectively contribute to the modulation of radiation sensitivity in MG cells by inhibiting DNA damage repair, prosurvival signaling, and epithelium-mesenchyme transition. Taken together, these findings demonstrate that miR-203 could be a target for overcoming the radiation resistance of GBM.

Antitumorigenic effect of interferon-β by inhibition of undifferentiated glioblastoma cells.

Glioma stem-like cells (GSCs) are undifferentiated cells that are considered to be an origin of glioblastomas. Furthermore, they may contribute to treatment resistance and recurrence in glioblastomas. GSCs differentiate into differentiated glioma cells (non-glioma stem-like cells: non-GSCs), and interconversion might occur between GSCs and non-GSCs. We investigated whether interferon-beta (IFN-β) could exert any efficacy towards GSCs or such interconversion processes. The neural stem cell marker CD133 and pluripotency marker Nanog in GSCs were analyzed to evaluate their differentiation levels. GSCs were considered to undergo differentiation into non-GSCs upon serum exposure, since the expression of CD133 and Nanog in the GSCs was negatively affected. Furthermore, the cells regained their undifferentiated features upon removal of the serum. However, we verified that IFN-β reduced cell proliferation and tumor sphere formation in GSCs, and induced suppression of the restoration of such undifferentiated features. In addition, we also confirmed that IFN-β suppressed the acquisition process of undifferentiated features in human malignant glioma cell lines. Our data thus suggest that IFN-β could be an effective agent not only through its cell growth inhibitory effect on GSCs but also as a means of targeting the interconversion between GSCs and non-GSCs, indicating the possibility of IFN-β being used to prevent treatment resistance and recurrence in glioblastomas, via the inhibition of undifferentiated features.

IDH1R132H mutation causes a less aggressive phenotype and radiosensitizes human malignant glioma cells independent of the oxygenation status

Background and purposeIn malignant glioma the presence of the IDH1 mutation (IDH1R132H) is associated with better clinical outcome. However, it is unclear whether IDH1 mutation is associated with a less aggressive phenotype or directly linked to increased sensitivity to radiotherapy. Material and methodsWe determined the influence of IDH1R132H mutant protein on proliferation and growth in 3D culture, migration, cell survival and radiosensitivity in vitro under normoxia (21% O2) and hypoxia (<1% O2) in a panel of human malignant glioma cell lines (U-251MG, U-343MG, LN-229) with stable overexpression of wild-type (IDH1wt) and mutated IDH1 (IDH1R132H). ResultsOverexpression of IDH1R132H in glioma cells resulted in slightly decreased cell proliferation, considerably reduced cell migration and caused differences in growth properties in 3D spheroid cultures. Furthermore, IDH1R132H-positive cells consistently demonstrated an increased radiosensitivity in human malignant glioma cells U-251MG (DMF10: 1.52, p<0.01 and 1.42, p<0.01), U-343MG (DMF10: 1.78, p<0.01 and 1.75, p<0.01) and LN-229 (DMF10: 1.41, p<0.05 and 1.68, p<0.01) under normoxia and hypoxia, respectively. ConclusionOur data indicate that IDH1R132H mutation causes both a less aggressive biological behavior and direct radiosensitization of human malignant glioma cells. Targeting IDH1 appears to be an attractive approach in combination with radiotherapy.

Sublethal dose of irradiation enhances invasion of malignant glioma cells through p53-MMP 2 pathway in U87MG mouse brain tumor model.

BackgroundGlioblastoma is a highly lethal neoplasm that frequently recurs locally after radiotherapy, and most of these recurrences originate from near the irradiated target field. In the present study, we identified the effects of radiation on glioma invasion and p53, TIMP-2, and MMP-2 expression through in vitro and in vivo experiments.MethodsThe U87MG (wt p53) and U251 (mt p53) human malignant glioma cell lines were prepared, and the U2OS (wt 53) and Saos2 (del p53) osteosarcoma cell lines were used as p53 positive and negative controls. The four cell lines and p53 knock-downed U87MG cells received radiation (2–6 Gy) and were analyzed for expression of p53 and TIMP-2 by Western blot, and MMP-2 activity was detected by zymography. In addition, the effects of irradiation on directional invasion of malignant glioma were evaluated by implanting nude mice with bioluminescent u87-Fluc in vivo followed by MMP-2, p53, and TIMP-2 immunohisto-chemistry and in situ zymography.ResultsMMP-2 activity and p53 expression increased in proportional to the radiation dose in cell lines with wt p53, but not in the cell lines with del or mt p53. TIMP-2 expression did not increase in U87MG cells. MMP-2 activity decreased in p53 knock-downed U87MG cells but increased in the control group. Furthermore, radiation enhanced MMP-2 activity and increased tumor margin invasiveness in vivo. Tumor cells invaded by radiation overexpressed MMP-2 and p53 and revealed high gelatinolytic activity compared with those of non-radiated tumor cells.ConclusionRadiation-induced upregulation of p53 modulated MMP-2 activity, and the imbalance between MMP-2 and TIMP-2 may have an important role in glioblastoma invasion by degrading the extracellular matrix. Bioluminescent “U87-Fluc”was useful for observing tumor formation without sacrifice after implanting tumor cells in the mouse brain. These findings suggest that the radiotherapy involved field for malignant glioma needs to be reconsidered, and that future trials should investigate concurrent pharmacologic therapies that inhibit invasion associated with radiotherapy.Electronic supplementary materialThe online version of this article (doi:10.1186/s13014-015-0475-8) contains supplementary material, which is available to authorized users.

Effect and mechanism of peroxisome proliferator-activated receptor-γ on the drug resistance of the U-87 MG/CDDP human malignant glioma cell line.

Peroxisome proliferator-activated receptor-γ (PPAR-γ) is important in tumor differentiation, proliferation and apoptosis. However, the effect and mechanism of PPAR-γ on the promotion of cisplatin sensitivity in glioma cells remain to be elucidated. The present study established cisplatin-resistant U-87 MG/CDDP cell lines and U-87 MG/CDDP cell lines overexpressing PPAR-γ. With upregulated expression of PPAR-γ, the sensitivity of cancer cells to cisplatin was increased. Flow cytometry revealed that the intracellular content of rhodamine-123 was increased, expression of P-glycoprotein was downregulated, cell cycle was arrested in G0/G1 phase, apoptosis and oxidative stress was increased, levels of intracellular thymidylate synthase, glutathione and transforming growth factor-β1 were decreased, expression levels of multidrug resistance related gene (MDR), multidrug resistance-associated protein and glutothionine S-transferase-π were downregulated, expression levels of cell proliferation and apoptosis associated genes, including survivin and B-cell lymphoma-2, were downregulated, p53, p21 and caspase-3/8 were significantly upregulated, phosphorylation of extracellular signal-regulated kinase and small mothers against decapentaplegic 2 were downregulated, and the transcriptional activities of Twist and nuclear factor (erythroid-derived 2)-like 2 were significantly reduced. The results suggested that upregulation of PPAR-γ in the U-87 MG/DDP cells increased cisplatin sensitivity, and the underlying mechanisms included the regulation of MDR and apoptosis associated genes, which increased the intracellular accumulation of the drug, inhibited cell proliferation and promoted cell apoptosis.

Overexpression of Nrf2 attenuates Carmustine-induced cytotoxicity in U87MG human glioma cells.

BackgroundMalignant glioma is one of the most devastating tumors in adults with poor patient prognosis. Notably, glioma often exhibits resistance to conventional chemotherapeutic approaches, complicating patient treatments. However, the molecular mediators involved in tumor chemoresistance remain poorly defined, creating a barrier to the successful management of glioma. In the present study, we hypothesized that the antioxidant transcription factor, Nrf2 (nuclear factor erythroid-derived 2 like 2), attenuates glioma cytotoxicity to Carmustine (BCNU), a widely used chemotherapeutic agent known to modulate cellular oxidative balance.MethodsTo test the hypothesis, we employed human malignant glioma cell line, U87MG and overexpression of Nrf2 in glioma cells was achieved using both pharmacological and genetic approaches.ResultsNotably, induction of Nrf2 was associated with increased expression of heme oxygenase-1 (HO-1), a stress inducible enzyme involved in anti-oxidant defense. In addition, over expression of Nrf2 in U87MG cells significantly attenuated the cytotoxicity of Carmustine as evidenced by both cellular viability assay and flow cytometry analysis. Consistent with this, antioxidants such as glutathione and N-acetyl cysteine significantly reduced Carmustine mediated glioma cytotoxicity.ConclusionsTaken together, these data strongly implicate an unexplored role of Nrf2 in glioma resistance to Carmustine and raise the possible use of Nrf2 inhibitors as adjunct to Carmustine for the treatment of malignant glioma.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1134-z) contains supplementary material, which is available to authorized users.

Cucurbitacin-I inhibits Aurora kinase A, Aurora kinase B and survivin, induces defects in cell cycle progression and promotes ABT-737-induced cell death in a caspase-independent manner in malignant human glioma cells

Because STAT signaling is commonly activated in malignant gliomas as a result of constitutive EGFR activation, strategies for inhibiting the EGFR/JAK/STAT cascade are of significant interest. We, therefore, treated a panel of established glioma cell lines, including EGFR overexpressors, and primary cultures derived from patients diagnosed with glioblastoma with the JAK/STAT inhibitor cucurbitacin-I. Treatment with cucurbitacin-I depleted p-STAT3, p-STAT5, p-JAK1 and p-JAK2 levels, inhibited cell proliferation, and induced G2/M accumulation, DNA endoreduplication, and multipolar mitotic spindles. Longer exposure to cucurbitacin-I significantly reduced the number of viable cells and this decrease in viability was associated with cell death, as confirmed by an increase in the subG1 fraction. Our data also demonstrated that cucurbitacin-I strikingly downregulated Aurora kinase A, Aurora kinase B and survivin. We then searched for agents that exhibited a synergistic effect on cell death in combination with cucurbitacin-I. We found that cotreatment with cucurbitacin-I significantly increased Bcl-2/Bcl-xL family member antagonist ABT-737-induced cell death regardless of EGFR/PTEN/p53 status of malignant human glioma cell lines. Although >50% of the cucurbitacin-I plus ABT-737 treated cells were annexin V and propidium iodide positive, PARP cleavage or caspase activation was not observed. Pretreatment of z-VAD-fmk, a pan caspase inhibitor did not inhibit cell death, suggesting a caspase-independent mechanism of cell death. Genetic inhibition of Aurora kinase A or Aurora kinase B or survivin by RNA interference also sensitized glioma cells to ABT-737, suggesting a link between STAT activation and Aurora kinases in malignant gliomas.