Cultured Glioma Cells Research Articles

Borneol promotes apoptosis of Human Glioma Cells through regulating HIF-1a expression via mTORC1/eIF4E pathway.

The main reason for the failure of malignant glioma treatment is local tumor recurrence. Tumor cells in hypoxic microenvironment activate HIF-1 α transcription, and thus promoting tumor invasion and metastasis is one of the important reasons. In our previous study, we clearly established that borneol opens the blood-brain tumor barrier and its related mechanism. However, the effects of borneol itself on glioma proliferation have not yet been elucidated. Therefore, in this study, we evaluated the effect of borneol on glioma by constructing in vivo SD rat brain glioma model and in vitro human primary cultured glioma cell model. We found that borneol could suppress the proliferation of primary glioma cells and the tumor volume of SD rat brain glioma. Further, we measured the apoptosis effect induced by borneol in human primary cultured glioma cells. The results showed that the higher the concentration of borneol, the higher the apoptosis rate of human primary cultured glioma cells, but the effect was reversed after transfection of HIF-1 overexpression plasmid; In addition, borneol could downregulate the expression of Bcl-2 and upregulation the expression of Bax and caspase-3, similarly, the effect was also reversed after transfection of HIF-1 overexpression plasmid, suggesting that the apoptosis effect induced by borneol in human primary cultured glioma cells is mediated via HIF-1α. Moreover, the bioinformatics analysis of correlation between HIF-1α and apoptosis-related factors based on CGGA database showed that there was a positive correlation between the expression of eIF4E and HIF-1 α (P < 0.05), and in patients with high expression of eIF4E and HIF-1α had poor survival and prognosis (P<0.001). It was further discovered that in the human primary cultured glioma cells borneol regulated HIF-1a expression via mTORC1/eIF4E pathway. In conclusion, the findings of the present study suggest that HIF-1α may be a key factor in borneol induced apoptosis of glioma cells, and mTORC1 / eIF4E pathway is involved in the HIF-1α regulation by borneol in malignant glioma. Our results not only reveal the target and molecular mechanism and action of borneol leading to promote apoptosis in glioma cells, but also provide experimental basis and theoretical support for the clinical application of borneol.

Expression and function of chondroitin 4‐sulfate and chondroitin 6‐sulfate in human glioma

Key molecules promoting migration and invasion exist in the extracellular matrix, and include chondroitin 4-sulfate (C4S) and chondroitin 6-sulfate (C6S), functionally important carbohydrate chains of chondroitin sulfate proteoglycans that participate in regulating cancer development. Here, we show that C4S and C6S expression is upregulated in human glioma tissues, when compared to normal brain tissue, and that the extent of upregulation positively correlated with glioma malignancy. Treatment of cultured glioma cells with C4S and C6S enhanced cell viability, migration, and invasion, increased MMP-2 and MMP-9 levels, enhanced N-cadherin, but reduced E-cadherin expression. Inhibition of expression of the two CS synthetic enzymes chondroitin 4-O-sulfotransferase-1 (C4ST-1/CHST11) and chondroitin 6-O-sulfotransferase-1 (C6ST-1/CHST3) suppressed cell viability, migration and invasion, reduced MMP-2 and MMP-9 expression, and reduced N-cadherin expression, but increased E-cadherin levels. The C4S- and C6S-enhanced epithelial-to-mesenchymal transition and expression of MMP-2 occurred via activation of the PI3K/AKT signaling pathway, known to be involved in promoting cell migration and invasion. In immune-deficient larval zebrafish, C4S and C6S increased the numbers of viable tumor cells, thereby promoting glioma cell proliferation. The present observations point to a novel role of C4S and C6S in human glioma cell functions, thus possibly representing targets in glioma therapy.

IMT-01 THERAPEUTIC EFFECT AGAINST LOWER GRADE GLIOMA INDUCED BY DENDRITIC CELL BASED IMMUNOTHERAPY

BACKGROUNDThis trial was designed to evaluate the safety and clinical responses to an immunotherapy with fusions of dendritic and glioma cells in patients with lower grade glioma (LGG; WHO grade II-III glioma).METHODAutologous cultured glioma cells obtained from surgical specimens were fused with autologous dendritic cells (DC) using polyethylene glycol. The fusion cells (FC) were inoculated intradermally in the cervical region of subjects. Toxicity, progression-free survival (PFS), overall survival (OS), and MRI findings were evaluated. DNA for whole exome and RNA for whole transcriptome extracted from HLA-A*24:02 positive glioma cells were analyzed by next generation sequencer. Variant peptides showing strong binding affinity to HLA-A*24:02 but not the corresponding wild type peptides were selected as candidate of neo-antigens.RESULTSThe number of subjects of this trial were 24 (initially diagnosed cases: 20, recurrence cases: 4). WHO grade III cases were 20, and grade II cases were 4. Male were 15, and female were 9. Mean of follow up periods were 53.0 months (the longest follow up period: 1322 months). The number of events on PFS and OS were 8 and 6, respectively. Mean of candidate of neo-antigen peptides in HLA-A*24:02 positive patients (n=8) was 34. Among these candidates, twelve types of common neo-antigen peptide were identified. Neo-antigen peptides specifically expressed in the glioma cells from the effective group were not identified.CONCLUSIONSThese results indicate that the efficacy of FC-immunotherapy may not always depend on the number of gene mutations or the expression of the specific neo-antigens. FC-immunotherapy, as a means of producing specific immunity against neo-antigens may safely induce anti-tumor effects in patients with LGG. Analysis of prognostic factor in glioma immunotherapy may be the next area of major interest.

TB-04 TERT PROMOTER MUTATION AS A SUSCEPTIBLE MOLECULAR MARKER OF BCNU LOCAL THERAPY

INTRODUCTIONAlkylating agents, including Temozolomide (TMZ) and CCNU (ACNU) have been widely accepted as a standard treatment in malignant gliomas. Several studies also demonstrated that BCNU wafer placement extended survival in glioblastoma patients. However, little study demonstrated gene-specific efficacy of BCNU local therapy in malignant gliomas. Herein, we investigated BCNU sensitivity for patient-derived primary cultured glioma cells.MATERIALS AND METHODSFrom January 2017 to July 2019, 58 gliomas (grade III, IV) were tested genomic analysis and ATP-based cell viability after BCNU treatment. IDH1/2 mutation and TERT promoter mutation status was determined by Sanger sequencing. MGMT methylation status were evaluated by methylation specific PCR.RESULTSOf 58 cases,10 cases (17.2%) and 32 (55.2%) cases harbored IDH1/2 mutation and TERT mutation (C228T, C250T), respectively. Among them, co-mutation was identified in 5/58 cases (8.6%). MGMT was methylated in 17/58 cases (29.3%). Interestingly, the presence of TERT promoter mutation was positively correlated with BCNU sensitivity, particularly in IDH1/2 wild-type tumors (p<0.05). In contrast, there was no significant relationship between TMZ sensitivity and IDH mutation/MGMT methylation status.CONCLUSIONAlthough sample size is small, our results imply TERT promoter mutations might be a predictive molecular marker for BCNU sensitivity in malignant gliomas. Since TERT mutations are located at two hot spot loci (C228T and C250T), vast majority of TERT promoter mutations can be evaluated during surgery, which may contribute tailored therapeutic strategy in malignant gliomas.

Medium dose intermittent cyclophosphamide induces immunogenic cell death and cancer cell autonomous type I interferon production in glioma models.

Cyclophosphamide treatment on a medium-dose, intermittent chemotherapy (MEDIC) schedule activates both innate and adaptive immunity leading to major regression of implanted gliomas. Here, we show that this MEDIC treatment regimen induces tumor cell autonomous type-I interferon signaling, followed by release of soluble factors that activate interferon-stimulated genes in both tumor cells and tumor-infiltrating immune cells. In cultured GL261 and CT-2A glioma cells, activated cyclophosphamide stimulated production and release of type-I interferons, leading to robust activation of downstream gene targets. Antibody against the type-I interferon receptor IFNAR1 blocked the cyclophosphamide-stimulated induction of these genes in both cultured glioma cells and implanted gliomas. Furthermore, IFNAR1 antibody strongly inhibited the MEDIC cyclophosphamide-stimulated increases in tumor cell infiltration of macrophages, dendritic cells, B-cells, as well as natural killer cells and cytotoxic T-cells and their cytotoxic effectors. Finally, cyclophosphamide-treated dying glioma cells producing type-I interferons were an effective vaccine against drug-naïve glioma cells implanted in vivo. Thus, cyclophosphamide induces local, tumor cell-centric increases in type-I interferon signaling, which activates immunogenic cell death and is essential for the striking antitumor immune responses that MEDIC cyclophosphamide treatment elicits in these glioma models.

EXTH-18. PEPTIDE NANO-STRUCTURES ENHANCE PEDIATRIC BRAIN TUMOR CHEMOTHERAPEUTIC EFFICACY

Abstract Pediatric gliomas, particularly high-grade gliomas, which include diffuse intrinsic pontine gliomas (DIPGs), are among the most formidable and devastating cancers in children. These tumors remain incurable, despite many treatment approaches. We recently identified a small population of glioma cells with stem-like features in pediatric gliomas (glioma stem cells: GSCs), that may be responsible, for therapeutic resistance. Bone morphogenetic protein 4 (BMP4), essential for CNS development, increases GSC therapeutic sensitivity and is a promising adjuvant for glioma treatment. Mechanisms through which BMP4 increases therapeutic sensitivity need to be elucidated, as this can lead to identification of additional treatment targets and delivery systems for BMP4 administration in a clinical setting. Additionally, extension of BMP4 short half-life would enhance its’ clinical application. Here we show that BMP4 increases chemosensitivity by decreasing H3K4me3 at the promoter of multidrug resistant gene 1 (MDR1), resulting in decreased MDR1 expression. BMP4 appears to bring about this effect by decreasing hSETD1A, an H3K4me3 methyltransferase. Our work also demonstrates the first use of a novel sulfated glycopeptide (glyco-PA) nanostructure as a vector for BMP4 delivery. Glyco-PA markedly extended and enhanced BMP4 function, and increased chemotherapeutic anti-tumor activity against pediatric malignant glioma cells in culture. Overall, this work illuminates BMP4 effects on pediatric glioma therapeutic sensitivity through epigenetic mechanisms, and demonstrates the potential of bioactive glyco-PA nanostructures as a delivery mechanism for treating pediatric malignant gliomas and other tumors.

A Novel Highly Selective Cannabinoid CB2 Agonist Reduces in vitro Growth and TGF-beta Release of Human Glial Cell Tumors

Cannabinoid receptors have been detected in human gliomas and cannabinoids have been proposed as novel drug candidates in the treatment of brain tumors. To test the in vitro antitumor activity of COR167, a novel cannabinoid CB2-selective agonist displaying a high binding affinity for human CB2 receptors, on tumor cells isolated from human glioblastoma multiforme and anaplastic astrocytoma. Glioma cell cultures were established from two glioblastoma multiforme and two anaplastic astrocytomas. Proliferation was measured in the presence or absence of COR167 with a bromodeoxyuridine (BrdU) cell proliferation ELISA assay. CB2 receptor expression was detected by western blotting. Apoptosis was assessed with phycoerythrin (PE) annexin V flow cytometry kit. TGF-beta 1 and 2 levels were analyzed in culture supernatants with commercial ELISAs. COR167 was found to significantly reduce the proliferation of both glioblastoma and anaplastic astrocytoma in a dose-dependent manner at lower doses than other known, less specific CB2 agonists. This activity is independent of apoptosis and is associated with a significant reduction of TGF-beta 1 and 2 levels in supernatants of glioma cell cultures. These findings add to the role of cannabinoid CB2 receptor as a possible pharmacological target to counteract glial tumor growth and encourage further work to explore any other pharmacological effect of this novel CB2 agonist useful in the treatment of human gliomas.

Enhanced photothermal-photodynamic therapy for glioma based on near-infrared dye functionalized Fe3O4 superparticles

It is challenging and urgent in neurosurgery to improve therapeutic effect of the most common and lethal malignant glioma in central nervous system. The superadditive therapeutic effect of photothermal therapy and photodynamic therapy holds great promise. Herein, a highly efficient photothermal-photodynamic therapy nanoplatform was developed on the basis of Fe3O4-IR806 superparticles. The introduction of the near-infrared (NIR) dye IR806 and the fabrication of the self-assembled structure of the superparticles warrant 3.5 times increase in the photothermal conversion efficiency compared with the Fe3O4 nanoparticles. The Fe3O4-IR806 superparticles generated reactive oxygen species (ROS) with the NIR light irradiation by virtue of the dye IR806. Both in vitro and in vivo experiments demonstrated that the Fe3O4-IR806 superparticles exhibited negligible toxicity. The Fe3O4-IR806 superparticles efficiently inhibited the cultured glioma cells via photothermal ablation and ROS cytotoxicity in vitro, as well as suppressed the bearing glioma growth in vivo with the NIR light induction. Our results highlight the promise of the Fe3O4-IR806 superparticles for improved photothermal-photodynamic therapy of cancer.

Nasal Administration of Cationic Nanoemulsions as CD73-siRNA Delivery System for Glioblastoma Treatment: a New Therapeutical Approach.

Glioblastoma is the most devastating primary brain tumor. Effective therapies are not available, mainly due to high tumor heterogeneity, chemoresistance, and the difficulties imposed by blood-brain barrier. CD73, an enzyme responsible for adenosine (ADO) production, is overexpressed in cancer cells and emerges as a target for glioblastoma treatment. Indeed, ADO causes a variety of tumor-promoting actions, particularly by inducing tumor immune escape, whereas CD73 inhibition impairs tumor progression. Here, a cationic nanoemulsion to deliver CD73siRNA (NE-siRNA CD73R) via nasal route aiming glioblastoma treatment was developed. NE-siRNA CD73R was uptaken by glioma cells in culture, resulting in a parallel 60-80% decrease in AMPase activity and 30-50% in cell viability. Upon nasal delivery, NE-siRNA CD73R was detected in rat brain and serum. Notably, treatment with CD73siRNA complexes of glioma-bearing Wistar rats reduced tumor growth by 60%. Additionally, NE-siRNA CD73R treatment decreased 95% ADO levels in liquor and tumor CD73 expression, confirming in vivo CD73 silencing. Finally, no toxicity was observed in either primary astrocytes or rats with this cationic nanoemulsion. These results suggest that nasal administration of cationic NE as CD73 siRNA delivery system represents a novel potential treatment for glioblastoma. Graphical Abstract Glioblastoma is the most common and devastating form of primary brain tumor. CD73, a protein involved in cell-cell adhesion and migration processes and also responsible for extracellular adenosine (ADO) production, is overexpressed by glioma cells and emerges as an important target for glioma treatment. Indeed, ADO participates in tumor immune escape, cell proliferation, and angiogenesis, and CD73 inhibition impairs those processes. Here, a cationic nanoemulsion to deliver CD73 siRNA (NE-siRNA CD73R) via nasal route aiming glioblastoma treatment was developed. NE-siRNA CD73R knockdown in vitro and in vivo CD73. Upon nasal delivery of NE-siRNA CD73R, the treatment markedly reduced tumor volume by 60% in a rat preclinical glioblastoma model. The treatment was well tolerated, and did not induce kidney, liver, lung, olfactory, bone marrow, or behavior alterations. These results indicate that the nasal administration of NE as a CD73 siRNA delivery system offered an efficient means of gene knockdown and may represent a potential alternative for glioblastoma treatment.

Abstract 984: Synthesis of mixed, hypoxia-activated phosphoramidate esters for the inhibition of Enolase in ENO1-deleted glioblastoma

Abstract Background: Precision oncology is currently restricted to activated oncogenes as drug targets, with tumor suppressor deletions remaining largely in-actionable. We developed a novel method that targets tumor suppressor deletions by exploiting vulnerabilities generated by the co-deletion of neighboring metabolic housekeeping genes with critical but normally redundant functions. Proof-of-principle studies were conducted for the glycolytic enzyme Enolase. Cancers harboring the homozygous deletion of 1p36-localized ENO1 remain metabolically active and viable through redundant action of its paralogue, ENO2. Inhibition of ENO2 in cancer cells harboring ENO1-homozygous deletion discriminately kills ENO1-deleted cells. In accordance, our synthetic efforts have focused on phosphonate chemistry to generate clinically-useful substrate mimics of 2-phosphoglycerate, the natural substrate for Enolase. We have generated a phosphonohydroxamate ENO2-inhibitor termed HEX and a pro-drug of which, POMHEX, kills ENO1-homozygously deleted glioma cells in culture with strong specificity. However, the pharmacokinetics of POMHEX are poor, due to the presence of carboxyesterase in extracellular fluids, including plasma. Results: We have synthesized mechanistically different pro-drugs of HEX, utilizing nitro-aromatics as protecting groups. Nitroaromatic groups are bioactivated by nitroreductases intracellularly, in a manner that is inversely related to oxygen concentration. As such, removal of nitroaromatic pro-drug moieties and release of active drug is favored under low oxygen (hypoxic) conditions. We synthesized a series of nitroaromatic pro-drugs of HEX (the structures will be disclosed at AACR presentation, pending patent filings) and demonstrate that 1) these exhibit selective toxicity to ENO1-deleted glioma cells in culture and 2) this killing is dramatically enhanced by lowering oxygen concentrations as compared to POMHEX. We further find that redox potential of the nitroaromatic moiety correlates with the degree of hypoxia activation with the lower redox potential being associated with great oxygen dependence. Importantly, unlike POMHEX, nitroaromatic HEX pro-drugs are stable in human plasma ex-vivo. We have thus successfully synthesized a pro-drug inhibitor of Enolase that is hypoxia-activated. Conclusion and future directions: We are gearing up synthesis for pre-clinical evaluation of nitroaromatic-HEX in intracranial tumor xenograft models. While the primary purpose of Nitroaromatic HEX is for the precision oncology treatment of ENO1-homozygously deleted tumors, it has not escaped our notice that such drugs have wider applicability within the context of targeting treatment-resistant tumors cells hiding in hypoxic niches. Indeed, we have observed that virtually all glioma cell lines can be killed by nitroaromatic HEX under hypoxia. Note: This abstract was not presented at the meeting. Citation Format: Florian Muller, Victoria C. Yan, Elliot S. Ballato, Kristine L. Yang, Dimitra K. Georgiou, Kenisha Arthur, Pakriti Shrestha, Sunada Khadka, Jeffrey Ackroyd. Synthesis of mixed, hypoxia-activated phosphoramidate esters for the inhibition of Enolase in ENO1-deleted glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 984.

Abstract 3414: Identifying dysregulated lncRNAs and miRNAs in low grade glioma: Uncovering signaling pathways and novel therapeutic targets

Abstract A molecular subtype of low grade gliomas (LGG) is characterized by mutations in Alpha-Thalassemia Mental Retardation X-linked (ATRX), TP53 and isocitrate dehydrogenase 1 (IDH1R132H, mIDH1). Our lab has generated a genetically engineered mouse model that harbors the characteristic genetic lesions found in this molecular subtype of LGG. We determined that mouse neurospheres (NS) bearing these mutations showed downregulation of signaling pathways involved in oligodendrocyte differentiation. The production of 2-hydroxyglutarate (2HG) by mIDH1 induces histone and DNA hypermethylation, which has been shown to impair neural precursor cells’ differentiation. We aim to investigate how the presence of mIDH1 impacts the epigenetic regulation of long non-coding RNAs (lncRNAs), microRNAs (miRNAs) and mRNAs in human mutant IDH1 glioma cell cultures obtained from patients at the time of surgery. We hypothesize that the dysregulation/remodeling of lncRNAs, miRNAs and mRNAs contributes to the invasiveness, differentiation status and stemness of glioma cells. Only a handful of lncRNAs have been studied in relation to their role in neural cells’ lineage commitment and glioma stem cells’ biology. We identified 127 differentially expressed (DE) lncRNA (FDR&amp;lt;0.01) in a human primary glioma cell culture SJGBM2, which has a ATRX mutation and was stably-transfected to express mIDH1. Of the 14 DE miRNAs identified, hsa-miR-210-3p and hsa-miR-4461, which have been linked to tumor invasiveness. The results from this work will reveal novel mechanistic pathways by which lncRNA/miRNAs and differentially expressed mRNAs impact downstream signaling pathways in LGG and uncover new therapeutic targets for this molecular glioma subtype. SC is supported by the T32 Cancer Biology Training Grant, the work is supported by grants from NIH/NINDS to MGC and PRL. Citation Format: Stephen Carney, Felipe Nuñez, Pedro R. Lowenstein, Maria G. Castro. Identifying dysregulated lncRNAs and miRNAs in low grade glioma: Uncovering signaling pathways and novel therapeutic targets [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3414.

CD146 is highly expressed in glioma stem cells and acts as a cell cycle regulator.

CD146 is highly expressed in various malignant tumors and contributes to their malignancy phenotype, which involves metastatic and tumorigenic activity. However, studies on the expression and function of CD146 in brain tumors are limited. We over-expressed or knocked-down CD146 in both conventionally cultured glioma cells and tumor spheres (TS). The distribution of glioma cells and their stem cells in different cell cycle phases was analyzed by flow cytometry using the stem cell marker CD133 and the glial precursor marker A2B5. CD146 expression was immunohistochemically examined in glioma tissues. The majority of glioma stem cells (GSCs) expressing CD133 were also CD146-positive. CD146 knockdown in GSCs significantly compromised cell growth. Cell cycle analysis revealed that most of the CD146 and CD133 double-positive cells were in the G2/M phase. Ectopic expression of CD146 in parental glioma cells resulted in cell cycle arrest of most differentiated cells in G0/G1 phase. In contrast, ectopic expression of CD146 in GSCs resulted in an increase in the number of CD133-positive cells in the G2/M phase. Furthermore, CD146 knockdown reduced the number of CD133-positive cells in the G2/M phase, which was consistent with effects of cell growth inhibition. Immunohistochemical analysis revealed that CD146 expression was significantly upregulated in World Health Organization (WHO) Grade III and IV glioma and positively correlated with CD133 expression. CD146 is mainly expressed in dividing GSCs and may be a potential target for eradicating glioma stem cells.

Loss of cell-matrix contact increases hypoxia-inducible factor-dependent transcriptional activity in glioma cells

In a variety of malignomas, the acquisition of a mesenchymal phenotype has been linked with anchorage-independent growth and invasiveness. To some extent, glioma cells are able to survive a loss of cell-matrix contact. We here describe that non-adherent culture of glioma cells was accompanied by an increase in hypoxia-inducible factor (HIF)-dependent, but not β-catenin/TCF-induced transcription. Levels of reactive oxygen species decreased in suspension and knockdown of HIF-1α enhanced cell death following detachment. By promoting the adaptation to non-adherent conditions, mechanisms driven by HIF-1α may considerably contribute to the biology and aggressiveness of glioblastoma.

The vitamin K-dependent factor, protein S, regulates brain neural stem cell migration and phagocytic activities towards glioma cells

Malignant gliomas are the most common primary brain tumors. Due to both their invasive nature and resistance to multimodal treatments, these tumors have a very high percentage of recurrence leading in most cases to a rapid fatal outcome. Recent data demonstrated that neural stem/progenitor cells possess an inherent ability to migrate towards glioma cells, track them in the brain and reduce their growth. However, mechanisms involved in these processes have not been explored in-depth.In the present report, we investigated interactions between glioma cells and neural stem/progenitor cells derived from the subventricular zone, the major brain stem cell niche. Our data show that neural stem/progenitor cells are attracted by cultured glioma-derived factors. Using multiple approaches, we demonstrate for the first time that the vitamin K-dependent factor protein S produced by glioma cells is involved in tumor tropism through a mechanism involving the tyrosine kinase receptor Tyro3 that, in turn, is expressed by neural stem/progenitor cells.Neural stem/progenitor cells decrease the growth of both glioma cell cultures and clonogenic population. Cultured neural stem/progenitor cells also engulf, by phagocytosis, apoptotic glioma cell-derived fragments and this mechanism depends on the exposure of phosphatidylserine eat-me signal and is stimulated by protein S.The disclosure of a role of protein S/Tyro3 axis in neural stem/progenitor cell tumor-tropism and the demonstration of a phagocytic activity of neural stem/progenitor cells towards dead glioma cells that is regulated by protein S open up new perspectives for both stem cell biology and brain physiopathology.

Long non-coding RNA LINC01503 predicts worse prognosis in glioma and promotes tumorigenesis and progression through activation of Wnt/β-catenin signaling.

Long-noncoding RNAs (lncRNAs) have been recently shown to be involved in the regulation of numerous biological processes including tumor progression. In this study, we aimed to explore the role of lncRNA LINC01503 (LINC01503) in the development and progression of glioma. Relative levels of LINC01503 were evaluated in tumor tissues from 133 patients with glioma as well as from cultured glioma cell lines. The correlation among LINC01503 levels, pathological types, and survivals of glioma patients were also determined using the Kaplan-Meier method and multivariate analysis. Next, we investigated the effect of LINC01503 on the proliferation, colony formation, apoptosis, migration and invasion in the U251 and LN299 cells. Relative protein expression was analyzed by Western blot assays. We found that LINC01503 expression level was significantly up-regulated in glioma tissue and cells, and that its overexpression was significantly correlated with KPS, tumor size and WHO grade in glioma patients. Kaplan-Meier analysis showed that patients with higher levels of LINC01503 had significantly poorer overall survival and disease-free survival than those with lower expression of this lncRNA in glioma patients. Multivariate analysis further confirmed that LINC01503 is an independent prognostic factor in patients with glioma. Functional assays with in vitro showed that knockdown of LINC01503 in the U251 and LN299 cell lines suppressed cells growth, colony formation, invasion and migration, and promoted apoptosis. Mechanistic investigation showed that LINC01503 can modulate Wnt/β-catenin signaling, as determined by that knockdown of LINC01503 decreased the TOP-FLASH activity and β-catenin, cyclin D1 and c-myc. Our findings suggested that LINC01503 conferred oncogenic function in glioma and may be a new prognostic biomarker and novel therapeutic strategy for this malignancy.

IL-17 induces the proliferation and migration of glioma cells through the activation of PI3K/Akt1/NF-κB-p65

Interleukin 17 (IL-17), as a pro-inflammatory cytokine, is up-regulated in the sera and tumor tissues of glioma patients; however the effects of IL-17 on glioma proliferation and migration remain unclear. In this study, the roles of IL-17 in the proliferation and migration of glioma cells and their potential mechanisms were determined. The results showed that IL-17 could not only enhance the proliferation and migration of cultured glioma cells (in vitro), but also promote the tumor formation of glioma cells in BALB/c nude mice (in vivo). Mechanical exploration revealed that IL-17 stimulation could increase the phosphorylation levels of Akt1 and NF-κB-p65 in glioma cells, and knockdown or inhibition of PI3K, Akt1 and NF-κB-p65 could also reduce the IL-17-induced proliferation and migration of the glioma cells. Moreover, PI3K/Akt1 was the upstream regulator of NF-κB-p65 activation in IL-17-incubated glioma cells. Furthermore, the inhibition of PI3K, Akt1 and NF-κB-p65 markedly suppressed the tumor formation of glioma cells induced by IL-17. Together, these data indicate that IL-17 can promote the proliferation and migration of glioma cells via PI3K/Akt1/NF-κB-p65 activation, and these findings might provide a new insight into glioma pathogenesis.

Hsc70 Interacts with β4GalT5 to Regulate the Growth of Gliomas.

Heat shock cognate protein 70 (Hsc70) is a key mediator for the maintenance of intracellular proteins and regulates cellular activities. And it is elevated in various tumor tissues including glioma, which is closely related to the malignancy and poor prognosis of the tumors. However, the effects of Hsc70 on gliomas and its regulatory mechanism have not yet been elucidated. In the present study, we found that Hsc70 was overexpressed in glioma tissues and cultured glioma cells. Furthermore, Hsc70 expression exhibited positive correlation with the grades of gliomas. Knockdown of Hsc70 could effectively inhibit cell proliferation and increase cell apoptosis. Furthermore, we identified that β4GalT5 was a critical target for Hsc70-mediated anti-glioma effects. Blocking β4GalT5 activity could effectively reverse the anti-tumor effect of Hsc70. Taken together, these data indicate that Hsc70 regulates β4GalT5 levels, and possibly plays a role in cell proliferation and apoptosis of glioma.

Peptide Nanosponges Designed for the Delivery of Perillyl Alcohol to Glioma Cells.

Peptide nanosponges of low polydispersity are spontaneously formed from trigonal supramolecular building blocks in aqueous buffers, which feature cationic and/or anionic oligopeptides (n = 5-20) and a hydrophobic unit. In contrast to classical liposomes/vesicles, nanosponges feature interwoven hydrophilic and hydrophobic nanodomains and are readily taken up by mammalian cells. Perillyl alcohol is known to be a simple, but effective small molecule drug against glioma multiforme. However, its efficacy is limited by a poor bioavailability. In order to make perillyl alcohol bioavailable, two nanosponges consisting of 10 aspartates, to which perillyl alcohol is attached by means of an ester bond, and 20 lysines or arginines (type (D-POH)10K20 and (D-POH)10R20) were synthesized, purified, and characterized by dynamic light scattering (DLS) and atomic force microscopy (AFM). These nanosponges were then tested in cell cultures of murine glioma cells (GL26) and murine neural progenitor cells (NPC) because the latter was previously utilized in cell-based cancer therapy. The two nanosponges exhibited significantly different biophysical properties (size distribution and ζ potentials). Consequently, different efficacies in killing GL26 and NPC were observed in serum-containing culture media. The results from these experiments confirmed that the type (D-POH)10K20 nanosponge is a promising candidate for the (cell-mediated) cytotherapy of glioblastoma.

Pre-treatment or Post-treatment of Human Glioma Cells With BIX01294, the Inhibitor of Histone Methyltransferase G9a, Sensitizes Cells to Temozolomide

Glioblastoma (GBM) is a malignant, primary brain tumor, highly resistant to conventional therapies. Temozolomide (TMZ) is a first line therapeutic agent in GBM patients, however, survival of such patients is poor. High level of DNA repair protein, O6-methylguanine-DNA-methyltransferase (MGMT) and occurrence of glioma stem-like cells contribute to GBM resistance to the drug. Here, we explored a possibility of epigenetic reprograming of glioma cells to increase sensitivity to TMZ and restore apoptosis competence. We combined TMZ treatment with BIX01294, an inhibitor of histone methyltransferase G9a, known to be involved in cancerogenesis. Two treatment combinations were tested: BIX01294 was administered to human LN18 and U251 glioma cell cultures 48 h before TMZ or 48 h after TMZ treatment. Despite their different status of the MGMT gene promoter, there was no correlation with the response to TMZ. The analyses of cell viability, appearance of apoptotic alterations in morphology of cells and nuclei, and markers of apoptosis, such as levels of cleaved caspase 3, caspase 7 and PARP, revealed that both pre-treatment and post-treatment with BIX01294 sensitize glioma cells to TMZ. The additive effect was stronger in LN18 cells. Moreover, BIX01294 enhanced the cytotoxic effect of TMZ on glioma stem-like cells, although it was not associated with modulation of the pluripotency markers (NANOG, SOX2, CD133) expression or methylation of NANOG and SOX2 gene promoters. Accordingly, knockdown of methyltransferase G9a augments TMZ-induced cell death in LN18 cells. We found the significant increases of the LC3-II levels in LN18 cells treated with BIX01294 alone and with drug combination that suggests involvement of autophagy in enhancement of anti-tumor effect of TMZ. Treatment with BIX01294 did not affect methylation of the MGMT gene promoter. Altogether, our results suggest that G9a is a potential therapeutic target in malignant glioma and the treatment with the G9a inhibitor reprograms glioma cells and glioma stem-like cells to increase sensitivity to TMZ and restore apoptosis competence.

Long noncoding RNA TSLNC8 suppresses cell proliferation and metastasis and promotes cell apoptosis in human glioma.

Glioma is among the most common primary brain tumors and one of the most aggressive and lethal forms of human cancer. Long noncoding RNAs (lncRNAs) have demonstrated great importance in the development and progression of cancer. The present study aimed to investigate the role of the novel tumor suppressive lncRNA on Chromosome 8p12 (TSLNC8), in cell proliferation, metastasis and apoptosis in human glioma. It was initially reported that the relative transcript levels of TSLNC8 were significantly decreased in human glioma tissues and cultured glioma cells, as evidenced by RT‑qPCR. Among clinical variables, the expression of TSLNC8 was negatively associated with tumor size, distant metastasis, and tumor, node and metastasis stage. MTT assay demonstrated that overexpression of TSLNC8 in glioma cell lines U25‑MG and SWO38 decreased, whereas knockdown of TSLNC8 in glioma cells SHG‑44 and BT325 increased the cell proliferative rate over 5 consecutive days. Additionally, cell metastasis was inhibited in U251 and SWO38 cells when cells were transfected with TSLNC8‑expressing plasmid as observed via Transwell and wound‑healing assays. Furthermore, cell apoptotic rate was upregulated in TSLNC8 plasmid‑treated U251 and SWO38 cells, and inhibited by siRNA against TSLNC8 in SHG‑44 and BT325 cells by cell apoptotic assay. The relative activities of caspase‑3 and caspase‑9 were increased by TSLNC8 overexpression and decreased by TSLNC depletion; however, the activity of caspase‑8 remained unchanged. The results of the present study demonstrated the inhibitory effects of TSLNC8 in human glioma, which may contribute to advancement in the diagnosis and treatment of patients with glioma in clinic.