Characteristics Of Glioma Cells Research Articles

Sevoflurane impedes the progression of glioma through modulating the circular RNA has_circ_0012129/miR-761/TGIF2 axis.

Glioma is a highly aggressive and lethal brain tumor. Anesthetics have been shown to have important effects on the biological characteristics of cancer cells. Nevertheless, the molecular mechanism of anesthetic-mediated glioma cells progression remains unclear. Sevoflurane (sev) was employed to treat glioma cells. The biological characteristics (viability, colony formation, apoptosis, cell cycle, migration, and invasion) of glioma cells were determined via Cell Counting Kit-8 (CCK-8), cell colony formation, flow cytometry, PI cytometry, or transwell assays. The protein levels of Cell Cycle Dependent Kinase (CDK) 2, CDK4, E-cadherin, N-cadherin, Vimentin, and Transforming Growth Factor Beta (TGFB) induced factor homeobox 2 (TGIF2) were assessed through Western blot analysis. Glucose consumption and lactate production were measured using special commercial kits. The expression of circular RNA has_circ_0012129 (circ_0012129) and miR-761 was detected via quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). The relationship between circ_0012129 or TGIF2 and miR-761 was verified with Dual-Luciferase reporter assay. Sevoflurane-mediated molecular mechanisms have been confirmed via xenograft assay. Sevoflurane suppressed viability, colony formation, cell cycle, migration, and invasion and promoted apoptosis of glioma cells in vitro, and impeded tumor growth in vivo. Circ_0012129 and TGIF2 were downregulated and miR-761 was upregulated in sevoflurane-treated glioma cells. Circ_0012129 elevation abolished sevoflurane-mediated biological characteristics of glioma cells. MiR-761 served as target for circ_0012129 and miR-761 targeted TGIF2. Moreover, both miR-761 overexpression and TGIF2 suppression restored circ_0012129 enhancement-mediated biological characteristics of sevoflurane-treated glioma cells. Sevoflurane mediated the progression of glioma via regulating the circ_0012129/miR-761/TGIF2 axis.

MiR-150-5p suppresses the stem cell-like characteristics of glioma cells by targeting the Wnt/β-catenin signaling pathway.

Glioma is the most common brain tumor malignancy with high mortality and poor prognosis. Emerging evidence suggests that cancer stem cells are the key culprit in the development of cancer. MicroRNAs have been reported to be dysregulated in many cancers, while the mechanism underlying miR-150-5p in glioma progression and proportion of stem cells is unclear. The expression levels of miR-150-5p and catenin beta 1 (CTNNB1, which encodes β-catenin) were measured by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot. The expression levels of downstream genes of the Wnt/β-catenin pathway and stem cell markers were detected by qRT-PCR. Tumorigenesis was investigated by cell viability, colony formation, and tumor growth in vitro and in vivo. The interaction between miR-150-5p and β-catenin was explored via bioinformatics analysis and luciferase activity assay. We found that miR-150-5p was downregulated in glioma and its overexpression inhibited cell proliferation, colony formation, and tumor growth. Moreover, miR-150-5p directly suppressed CTNNB1 and negatively regulated the abundances of downstream genes of the Wnt/β-catenin pathway and stem cell markers. Furthermore, miR-150-5p expression was decreased and β-catenin level was enhanced in CD133+ glioma stem cells. Knockdown of miR-150-5p contributed to CD133- cells with stem cell-like phenotype, whereas overexpression of miR-150-5p suppressed CD133+ glioma stem cell-like characteristics. In conclusion, miR-150-5p inhibited the progression of glioma by controlling stem cell-like characteristics via regulating the Wnt/β-catenin pathway, providing a novel target for glioma treatment.

Effect of knockdown of eIF4A1 by shRNA on the biological characteristics of glioma cells

Effect of knockdown of eIF4A1 by shRNA on the biological characteristics of glioma cells

The effect of circ-ZEB1 on the biological characteristics of glioma cells

Objective To investigate the effects of circ-ZEB1 on proliferation, invasion and migration of glioma cells. Methods Real-time quantitative PCR (polymerase chain reaction) was used to detect the expression of circ-ZEB1 in 30 glioma tissues and 8 normal brain tissues(obtained from Department of Neurosurgery, Sun Yat-sen Memorial Hospital, Sun Yat-sen University), glioma cell lines (A172 and U251) and normal glial cells (HA1800). A172 and U251 cells were transfected with si-circ-ZEB1 to knock down circ-ZEB1. According to the type of si-circ-ZEB1, it was divided into si-1 group and si-2 group (transfected with si-circ-ZEB1-1, si-circ-ZEB1-2, respectively), and a control group was established (siRNA-NC as negative groups transfected with siRNA-NC). The effect of circ-ZEB1 on the proliferation of glioma cell lines was detected by CCK-8 assay. Scratch assay and Transwell assay were used to observe the effects of circ-ZEB1 on the migration and invasion in glioma cells respectively. CircNet database was used to examine and evaluate of circRNA-miRNA gene regulatory network of circ-ZEB1. The dual luciferase reporter assay was used to validate the interaction of circ-ZEB1/miR-140-3p in the gene regulatory network of circRNA-miRNA. Results The expression of circ-ZEB1 was higher in glioma tissues than in normal tissues (12.16±1.31 vs. 6.84±0.56, P=0.001). Circ-ZEB1 was over-expressed in high-grade glioma tissues compared with low-grade gliomas (13.77±3.27 vs. 7.50±1.82, P=0.001). The expression levels of circ-ZEB1 in A172 and U251 were 2.91±0.30 and 3.32±0.20, respectively, compared with HA1800 (both P<0.01). The results of CCK-8 showed that the growth of circ-ZEB1 knockdown group (si-1 group and si-2 group) was significantly inhibited compared with the control group. The proliferation abilities of A172 cells were decreased by 40.0% and 57.1%, respectively (both P<0.01) and those of U251 cells were decreased by 35.3% and 48.5%, respectively (both P<0.01). In the scratch test, the migration of circ-ZEB1 knockdown group was significantly inhibited (both P<0.05). Transwell assay showed that the invasive abilities of glioma cells in si-1 group and si-2 group were lower than those of the control group (both P<0.01). The gene regulatory networks of circRNA-miRNAs showed that circ-ZEB1 interacted with a variety of miRNAs, which affected the regulation of multiple miRNAs and their downstream proteins. The dual luciferase reporter assay also confirmed the interaction of circ-ZEB1/miR-140-3p in the gene regulatory network of circRNA-miRNA. Conclusions Circ-ZEB1 is over-expressed in glioma tissues and cell lines. Down-regulation of circ-ZEB1 could decrease cell proliferation and cell migration in gliomas. The gene regulatory network of circRNA-miRNA suggests that circ-ZEB1 functions by interacting with a variety of miRNAs. Key words: Glioma; Circ-ZEB1; Cell proliferation; Cell migration assays

The effect of celecoxib on the biological characteristics of glioma cells

Objective To investigate the effect of celecoxib on glioma cell proliferation, apoptosis, migration and invasion ability. Methods Methyl thiazol tetrazolium (MTT) assay was used to detect glial tumor cell proliferation of U251, T98G, U87 after treatment with 0, 20, 40 or 60 μmol/L celecoxib for 24, 48, 72 or 96 h. Flow cytometry was used to detect cell apoptosis after treatment with 40 μmol/L celecoxib for 48 h. Cell scratch test was used to detect cell migration after treatment with 40 μmol/L celecoxib for 48 h. Transwell was used to detect cell invasion after treatment with 40 μmol/L celecoxib for 48 h. The expression levels of cyclooxygenase-2 (COX-2), B cell lymphoma/leukemia-2 (bcl-2), B cell lymphoma/leukemia-2 associated X protein (bax), matrix metalloproteinase (MMP)-9, MMP-2 in cells was detected by Western blotting. Results Celecoxib can inhibit the proliferation of glioma cells and there is no obvious inhibitory effect on normal astrocytoma. The inhibitory effect of Celecoxib on glioma cell proliferation was depended on the reaction time and drug concentration. The apoptosis rate of glioma cell after 40μmol/L celecoxib was higher than the control group, and the difference was significant [(3.17±0.16)% vs. (16.08±1.57)%, (3.17±0.16)% vs. (16.08±1.57)%, t=14.169, P=0.000]. The migration rate of glioma cell after treatment with 40 μmol/L celecoxib was lower than the control group, the difference was significant . The invasion ability of glioma cell after 40 μmol/L celecoxib was lower than the control group, the difference was significant [(100.00±5.26)% vs. (41.28±5.84)%, t=12.940, P=0.000]. Glioma cells after celecoxib treatment in COX-2, bcl-2, MMP-9, MMP-2 was significantly decreased than control group [(229.5±11.36) vs. (97.32±7.95), t=16.512, P=0.000]. The bax protein of Glioma cells after celecoxib treatmentwas significantly higher than control group (t1=19.696, P1=0.000; t2=17.774, P2=0.000; t3=12.970, P3=0.000; t4=10.569, P4=0.001). Conclusion Celecoxib can inhibit glioma cell proliferation, migration and invasion ability, and promote the apoptosis of glioma cells, the mechanism of which was related to COX-2, bcl-2, bax, MMP-9, MMP-2. Key words: Glioma; Proliferation; Apoptosis; Invasion; Celecoxib

Influence of the MACC1 gene on sensitivity to chemotherapy in human U251 glioblastoma cells.

This study was conducted to determine the influence of MACC1 expression on chemotherapy sensitivity in human U251 glioblastoma cells. Expression of the MACC1 gene in 49 cases of human brain glioma was determined by quantitative real-time PCR. Silencing effects of RNA interference on MACC1 was detected by Western-blotting. Flow cytometry methods and methyl thiazolyl tetrazolium assay (MTT) were used to determine the apoptosis and growth inhibitory rates of the U251 cells with MACC1 silencing. before and after treatment with cisplatin (DDP). MACC1 mRNA in gliomas was up-regulated remarkably, to 158.8% of that in peri-cancerous tissues (P<0.05). The siRNA-MACC1 could inhibit the expression of MACC1 protein significantly (p<0.05), associated with an increase in apoptosis rate from 2.57% to 5.39% in U251 cells and elevation of the growth inhibitory rate from 1.5% to 17.8% (p<0.05 for both). After treatment with DDP at various concentrations (1, 3, 5μg/ml), compared with control U251 cells, the apoptosis rate of MACC1-silenced U251 cells rose from 8.41%, 13.2% and 19.5% to 12.8%, 17.8% and 25.8%; the growth inhibitory rate increased from 16.2%, 19.3% and 24.5% to 23.7%, 28.4% and 36.3%. There is a notable relationship between over-expression of MACC1 and the characteristics of glioma cells. Silencing of MACC1 was found to enhance the apoptosis and growth inhibitory rates of U251 glioma cells, and thereby increase their sensitivity to DDP chemotherapy.

ME-19 * ANALYSIS OF TREG RECRUITMENT AND FOXP3 EXPRESSION IN GLIOMAS REGARDING THE INTRAOPERATIVE FLUORESCENCE OF 5-ALA

Not only are regulatory T cells (Tregs) an essential component of the immune system in maintaining immunologic self-tolerance but they are also involved in the suppression of anti-tumor immune responses. The transcription factor forkhead box P3 (FOXP3) is widely used as a marker for the identification of Tregs being essential for the functionality. Oral 5 aminolevulinic acid (5-ALA) is used to assist surgical resection of high-grade gliomas, being known as an indicator of malignancy as it is able to pass the blood brain barrier in high-grade gliomas. Gaining more knowledge regarding the pathogenesis of gliomas, the tumor microenvironment came to the fore. In this study, patterns of Treg recruitment and FOXP3 expression in the microenvironment of gliomas were characterized, regarding the fluorescence of 5-ALA. Glioma tissue positive and negative for intraoperative fluorescence of 5-ALA was analyzed and compared. Tregs present in the tumor microenvironment were detected by double-immunohistochemistry for FOXP3 and the glioma marker, anti- glial fibrillary acidic protein (GFAP). FOXP3 mRNA expression was examined, using quantitative real-time-PCR. Tregs were found in the tumor microenvironment as well as within tumor cell islets, and FOXP3 mRNA expression in the tumors was significantly higher than in normal brain. Intriguingly, single FOXP3+ cells exhibited morphologic characteristics of glioma cells. 5-ALA positive tumor tissue showed a higher accumulation of Tregs than 5-ALA negative tissue. Gliomas recruit Tregs into their microenvironment, presumably in order to suppress immunosurveillance, thus avoiding destruction by the immune system. Endogenous FOXP3 expression in glioma cells might present a novel mechanism of immune escape. Our study showed differences between the amount of Treg and FOPX3 expression in 5-ALA positive and negative glioma tissue indicating that Tregs are more present in high-grade malignant gliomas. These findings suggest FOXP3 and Tregs as a possible therapeutic target in the therapy of gliomas.

Abstract 4342: Crosstalk between initiating cells with different metabolism in a murine model of malignant glioma

Abstract Background The metabolic preference of malignant glioma for glycolysis as an energy source is a potential therapeutic target. As a result of the cellular heterogeneity of these tumors, however, the relation between glycolytic preference, tumor formation, and tumor cell clonogenicity has remained unknown. To address this issue, we analyzed the metabolic profiles of isogenic glioma-initiating cells (GICs) in a mouse model. Methods GICs were established by overexpression of H-RasV12 in Ink4a/Arf-null neural stem cells. Subpopulations of these cells were obtained by single-cell cloning, and clones differing in extracellular acidification potential were assessed for metabolic characteristics by quantification of intra- and extracellular metabolites. Tumorigenicity was assessed by implantation of 100 cells of each subpopulation into the forebrain of wild-type mice. Tumors were examined for pathological features of glioma and expression of glycolytic enzymes. Results Malignant transformation of neural stem cells resulted in a shift in metabolism characterized by a significant increase in glucose uptake and lactic acid production. Clonal populations of GICs also manifested pronounced differences in their metabolic profiles. Certain GICs consumed more glucose and produced more lactate, while others had higher oxygen consumption. These differences were reflected in the levels of intracellular metabolites and they were paralleled by a differential expression of glycolytic enzymes such as hexokinase 2 and pyruvate kinase M2. GIC clones with different metabolic profiles had the same level of tumorigenic ability and the tumors formed by all types of GICs displayed the histopathological features of glioblastoma. However, the differential expression of the glycolytic enzymes was also evident in the tumors formed by each of the clones. Implantation of a mix of GICs with different metabolic profiles showed that clones with higher glycolytic ability were the major component of the tumor mass and that they provided a scaffold for the less glycolytic clones, supporting their expansion. Conclusions The metabolic characteristics of glioma cells appear early during malignant transformation and persist until the late stages of tumor formation. Even isogenic clones may be heterogeneous in terms of metabolic features, however, and this heterogeneity may play a role in tumor cell proliferation and survival. Our results suggest that a more detailed understanding of the metabolic profile of malignant gliomas is imperative for their effective therapeutic targeting. Citation Format: Oltea Sampetrean, Isako Saga, Shunsuke Shibao, Jun Okubo, Satoru Osuka, Nobuyuki Onishi, Hideyuki Saya. Crosstalk between initiating cells with different metabolism in a murine model of malignant glioma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4342. doi:10.1158/1538-7445.AM2014-4342

INTEGRATED ANALYSIS IDENTIFIES DIFFERENT METABOLIC SIGNATURES WITHIN BOTH TUMOR-INITIATING CELLS AND STEM CELLS IN A MURINE GLIOBLASTOMA MODEL

BACKGROUND: The metabolic preference of malignant gliomas for glycolysis as an energy source is a potential diagnostic and therapeutic target. As a result of the cellular heterogeneity of these tumors, however, the relation between glycolytic preference, tumor formation and tumor maintenance has remained unknown. To address this issue, we analyzed the metabolic profiles of isogenic glioma-initiating cells (GICs) and glioma stem cells (GSCs) in a syngeneic mouse model. METHODS: GICs were established by overexpression of H-RasV12-dsRed in Ink4a/Arf–null neural stem cells. Subpopulations of GICs were obtained by single-cell cloning, and clones differing in extracellular acidification potential were used for quantification of intracellular metabolites and related enzymes. Tumors formed after intracranial implantation of the GIC clones in mice were examined for pathological features of glioma and expression of glycolytic enzymes. GSCs were established by sphere culture of dsRed-positive cells from the primary tumors and assessed for metabolic characteristics. RESULTS: Malignant transformation of neural stem cells resulted in a distinct shift in metabolism, with a significant increase in lactic acid production as the most pronounced characteristic of all malignant cells. Interestingly however, both GICs and GSCs could adopt two different metabolic profiles. Some clones displayed high glucose consumption and lactate production, while others consumed more oxygen, produced more ATP and had a higher respiratory capacity. The glycolytic clones exhibited higher levels of key glycolytic enzymes such as hexokinase 2 and pyruvate kinase M2, and the clones relying on oxidative phosphorylation had lower levels of pyruvate dehydrogenase kinase 1. While all GICs had equal tumorigenic ability, the differential expression of the metabolic enzymes persisted in the tumors formed by these clones in vivo, as well as in the GSCs of the respective tumors. CONCLUSIONS: The metabolic characteristics of glioma cells appear early during malignant transformation and persist until the late stages of tumor formation. However, even isogenic clones may be heterogeneous in terms of metabolic features, with both GICs and GSCs being able to satisfy their bioenergetics needs not only through aerobic glycolysis, but also through oxidative phosphorylation. These results suggest that a more detailed understanding of the metabolic profile of glioma is imperative for effective diagnostic and therapeutic targeting. SECONDARY CATEGORY: n/a.

Assessment of the intrinsic radiosensitivity of glioma cells and monitoring of metabolite ratio changes after irradiation by 14.7-T high-resolution1H MRS

Gliomas are the most common type of primary brain tumor. Radiation therapy (RT) is the primary adjuvant treatment to eliminate residual tumor tissue after surgery. However, the current RT guided by conventional imaging is unsatisfactory. A fundamental question is whether it is possible to further enhance the effectiveness and efficiency of RT based on individual radiosensitivity. In this research, to probe the correlation between radiosensitivity and the metabolite characteristics of glioma cells in vitro, a perchloric acid (PCA) extracting method was used to obtain water-soluble metabolites [such as N-acetylaspartate (NAA), choline (Cho), creatine (Cr) and succinate (Suc)]. Spectral patterns from these processed water-soluble metabolite samples were acquired by in vitro 14.7-T high-resolution ¹H MRS. Survival fraction analysis was performed to test the intrinsic radiosensitivity of glioma cell lines. Good ¹H MRS of PCA extracts from glioma cells was obtained. The radiosensitivity of glioma cells correlated positively with the Cho/Cr and Cho/NAA ratios, but negatively with the Suc/Cr ratio. Irradiation of the C6 cell line at different X-ray dosages led to changes in metabolite ratios and apoptotic rates. A plateau phase of metabolite ratio change and a decrease in apoptotic rate were found in the C6 cell line. We conclude that in vitro high-resolution ¹H MRS possesses the sensitivity required to detect subtle biochemical changes at the cellular level. ¹H MRS may aid in the assessment of the individual radiosensitivity of brain tumors, which is pivotal in the identification of the biological target volume.

Abstract 4305: Invasion of primary glioma- and cell line-derived spheroids implanted into corticostriatal slice cultures

Abstract Gliomas are known for being highly invasive and therefore incurable by surgery explaining a recurrence rate close to 100%. The aim of the present study was to develop and evaluate an in vivo-like in vitro model preserving the invasive features of glioma cells in order to facilitate research using more true models. This study was performed by implantation of glioma spheroids into organotypic corticostriatal slice cultures. The spheroids chosen for implantation were primary glioma spheroids as well as spheroids derived from the U87 glioblastoma cell line. Primary glioma spheroids are cultured small glioma fragments obtained from freshly removed tumor tissue. In contrast to cell line-derived spheroids, primary glioma spheroids preserve the tumor tissue components and structures such as cell-to-cell interactions, blood vessels and macrophages. In the present study, primary glioma spheroids from one anaplastic astrocytoma (WHO grade III) and two glioblastomas (WHO grade IV) were included in addition to U87 spheroids. The spheroids were labelled with the fluorescent dye DiI and implanted into 400 µm thick corticostriatal slice cultures prepared from newborn rat pups. The tumor cell invasion was followed by confocal microscopy. Hereafter the co-cultures were prepared for anti-human vimentin immunohistochemical staining identifying invasive tumor cells. The results showed that the primary glioma spheroid area was constant after implantation, but there was a clear increase in number of invading cells with time. In contrast, the U87 spheroid area increased after implantation, but there was only relatively few invading cells entering the brain tissue. Anti-human vimentin stained paraffin sections of co-cultures confirmed the tumor cell invasion recorded by confocal microscopy. The present study suggests implantation of primary glioma spheroids into corticostriatal slice cultures as an excellent in-vivo like in-vitro model suitable for studies of invasion. Similar to earlier results obtained in vivo, the U87 cell line, when used in vitro as well, appears to have lost the invasive feature of gliomas, preventing the use of this cell line in studies of invasion. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4305. doi:10.1158/1538-7445.AM2011-4305

Abstract 3690: Asiatic acid is an effective alternative agent targeting growth, angiogenesis and invasiveness of malignant gliomas

Abstract Malignant gliomas are one of the most devastating and incurable tumors with approximately 77% of patients dying within first year of diagnosis. This poor prognosis is due to characteristics of glioma cells to proliferate uncontrollably, sustain excessive angiogenesis and invade aggressively. There has been no improvement in survival of glioma patients in past few decades and existing therapies, with tremendous side-effects, have largely failed. This suggests that immediate additional and alternative strategies are warranted to control this deadly malignancy. In this regard, preventing or inhibiting growth and progression of gliomas through non-toxic natural agents is one such strategy. An agent suited ideally against advance glioma is Asiatic Acid (AsA), the active constituent in Centella asiatica; both Centella asiatica extract and pure AsA are extensively used in traditional medicine to treat neurological disorders. Here we analyzed the anti-cancer efficacy of AsA against glioma cells both in vitro and in vivo. AsA treatment (10-100 microM, 6-24 hrs) significantly decreased cell viability in U87, LN18 and U118 glioma cells, together with strong apoptotic cell death. AsA treatment (6 hrs) in glioma cells caused PARP, Bid and caspases (3, 9 and 8) cleavage, activated JNK1/2, and decreased phosphorylated-Akt and survivin. Use of pan-caspase inhibitor (z-VAD.fmk) confirmed that AsA-induced apoptosis is caspase-dependent. Next, we studied whether AsA could target the pro-angiogenic effects of glioma cells. We observed that AsA treatment at non-cytotoxic doses significantly impairs potency of glioma cells (U87 and LN18) to promote capillary formation by HUVEC (human umbilical vein endothelial cells) and HBMEC (human brain microvascular endothelial cells) on matrigel. Further, AsA (5-20 microM) significantly inhibited VEGF-induced growth of HUVEC and HMBEC. We also observed remarkable anti-migratory effects of AsA treatment (5-20 microM) in wound healing assay and anti-invasive potential in Boyden chambers against glioma cells. In a novel assay developed by us, we further established the inhibitory effects of AsA on fibronectin-induced motility in glioma cells. Additional mechanistic studies revealed that AsA strongly increases intracellular Ca++ levels; and antiproliferative and antimigratory effects of AsA were largely compromised in the presence of Ca++ chelating agent BAPTA. We also established for the first time in vivo anti-cancer efficacy of AsA using xenograft model. AsA feeding (30 mg/kg body weight dose in saline) for 5 weeks significantly decreased U87 xenografts volume by 54% (p value less than 0.05) and strongly inhibited tumor vasculature without causing any adverse effects to animals. Together, these results suggest that AsA could be an effective alternative agent against malignant glioma and recommend its clinical use. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3690. doi:10.1158/1538-7445.AM2011-3690