Glioma Cell Colony Formation Research Articles

CD13 expression affects glioma patient survival and influences key functions of human glioblastoma cell lines in vitro

CD13 (APN) is an Alanyl-Aminopeptidase with diverse functions. The role of CD13 for gliomas is still unknown. In this study, data of glioma patients obtained by TCGA and CGGA databases were used to evaluate the survival rate and prognostic value of CD13 expression level. Protein expression of CD13 was confirmed by immunofluorescence staining of fresh patient tissues. Eight human glioblastoma cell lines were studied by RT-PCR, Western Blot, immunofluorescence staining and flow cytometry to define CD13 expression. Cell lines with different CD13 expression status were treated with a CD13 inhibitor, bestatin, and examined by MTT, scratch and colony formation assaysas well as by apoptosis assay and Western Blots. Bioinformatics analysis indicated that patients with high expression of CD13 had poor survival and prognosis. Additionally, CD13 protein expression was positively associated with clinical malignant characteristics. Investigated glioblastoma cell lines showed distinct expression levels and subcellular localization of CD13 with intracellular enrichment. Bestatin treatment reduced proliferation, migration and colony formation of glioma cells in a CD13-dependent manner while apoptosis was increased. In summary, CD13 has an impact on glioma patient survival and is important for the main function of specific glioma cells.

Systematic pan-cancer analysis identifies SLC35C1 as an immunological and prognostic biomarker

GDP-amylose transporter protein 1 (SLC35C1) plays an important role in many types of cancer. Therefore, it is clinically important to further investigate the expression profile of SLC35C1 in human tumors to provide new molecular clues for the pathogenesis of glioma. In this study, we performed a comprehensive pan-cancer analysis of SLC35C1 using a series of bioinformatics approaches and validated its differential tissue expression and biological function. The results showed that SLC35C1 was aberrantly expressed in different types of tumors and significantly correlated with overall survival (OS) and progression-free interval (PFI). More importantly, the expression level of SLC35C1 was closely correlated with Tumor Microenvironment (TME), immune infiltration and immune-related genes. In addition, we found that SLC35C1 expression was also closely related to Tumor Mutation Burden (TMB), Microsatellite Instability (MSI) and antitumor drug sensitivity in various cancer types. Functional bioinformatics analysis indicated that SLC35C1 may be involved in multiple signaling pathways and biological processes in glioma. Based on SLC35C1 expression, a risk factor model was found to predict OS of glioma. In addition, in vitro experiments showed that SLC35C1 knockdown significantly inhibited the proliferation, migration and invasive ability of glioma cells, while SLC35C1 overexpression promoted proliferation, migration, invasion and colony formation of glioma cells. Finally, quantitative real-time PCR confirmed that SLC35C1 was highly expressed in gliomas.

OS10.7.A Activation of the CCR8-ACP5 axis by human microglia/macrophage derived CCL18 promotes glioma growth

Abstract Background Glioblastoma multiforme is a highly malignant primary brain tumor with an average survival of 14 months and very limited therapeutic options. Glioma associated microglia and macrophages (GAMs) foster tumor growth by releasing several cytokines, which have only partly been identified. Here, we studied the chemokine (C-C motif) ligand 18 (CCL18), a chemokine which is only expressed in human, but not rodent GAMs, in a novel ex-vivo brain slice model including transplantation of human induced pluripotent stem cells (iPSC) derived human microglia (iMGL) and human glioma cells in to murine brain slices, which had been depleted of intrinsic murine microglia before. Material and Methods After establishing the humanized ex-vivo brain slice model, we performed immunohistochemical analysis (IHC) of growth and invasiveness, qrtPCR on glioma cells isolated by magnetic-activated cell sorting (MACS), functional assays measuring invasiveness, proliferation, migration and colony formation of glioma cells in vitro and in slice experiments. Corresponding studies on tumor growth and invasiveness were performed after treatment with a CCL18 neutralizing antibody, a CCR8 neutralizing antibodies and knockdown of CCR8, ACP5 (Acid Phosphatase 5) and PITPNM3 with small interfering RNA (siRNA) and short hairpin RNA (shRNA). QrtPCR, IHC and Westernblot analysis were performed on primary glioma specimens. We also conducted bioinformatic analyses, based on the TCGA GBM, GLIOVIS and GEPIA databases. Results We observed that CCL18 was highly expressed in GAMs, whereas CCR8 was only expressed in glioma cells. We identified the chemokine (C-C motif) receptor 8 (CCR8) as a functional receptor for CCL18 and ACP5 as an important down-stream signaling component in glioma cells. Activation of the CCL18/CCR8/ACP5 signaling pathway in human glioblastoma was associated with enhanced tumor growth and invasiveness. Conclusion GAMs derived CCL18 promoted glioma growth by activation of the CCR8/ACP5 axis in human glioma cells and therefore is a potential therapeutic target.

An in vitro and in vivo study of the role of long non-coding RNA-HOST2 in the proliferation, migration, and invasion of human glioma cells.

Gliomas are the most commonly occurring primary malignant brain tumors in the central nervous system of adults. They are rarely curable and the prognosis for high grade gliomas is generally poor. Recently, long non-coding RNA (lncRNA) human ovarian cancer-specific transcript 2 (HOST2) has been reported to be expressed at high levels in human ovarian cancer, involving tumorigenesis. However, little is still known about whether and how HOST2 regulates glioma development and progression. Therefore, this study aims to investigate the role of HOST2 in human glioma cells. Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) was used to determine the expression of lncRNA HOST2, let-7b, and PBX3 in human glioma cells. Cultured human glioma cells were treated with siRNA (si)-lncRNA HOST2, let-7b mimic, si-lncRNA HOST2 + let-7b inhibitor, and si-PBX3. Parameters including cell viability, colony formation, cell migration, and cell invasion were detected by cell counting kit-8 assay, colony formation assay, scratch test, and Transwell assay respectively to determine the effects of down-regulated HOST2 on glioma cells. Tumor formation in nude mice was evaluated by subcutaneous tumor formation experiment. Results showed that HOST2 and PBX3 were highly expressed in glioma tissue whereas let-7b was expressed at much lower levels. In response to treatment with si-lncRNA HOST2, si-PBX3, and let-7b mimic, glioma cell lines exhibited decreased cell viability, suppressed cell migration, invasion, and reduced colony formation of glioma cells. This was accompanied by an attenuated tumor formation with smaller volume and weight in nude mice, suggesting that down-regulated HOST2 could inhibit the tumorigenicity of glioma cells. Lastly, we found that lncRNA HOST2 was highly expressed in glioma tissues and its down-regulation could inhibit the growth and invasion of glioma cells. © 2018 IUBMB Life, 71(1):93-104, 2019.

GLUT5 increases fructose utilization and promotes tumor progression in glioma

Fructose is now such an important component of human diets, and several studies have found that some cancer cells could utilize fructose to overcome low glucose micro-environment, but the study on the role of fructose in glioma is rare. To explore the role of fructose in glioma, we detected the proliferation and colony formation ability of glioma cells in fructose medium, and found that the abilities of proliferation and colony formation of glioma cells in fructose medium were similar with abilities in glucose medium, however, fructose just partly restored proliferation ability of normal glial cells. To explore the mechanism, we compared the expression level of GLUT5 (Glucose transporter type 5) in these cell lines, and the results showed that glioma cell lines had higher GLUT5 expression than normal glial cell lines. And knockdown of GLUT5 could significantly inhabit cell proliferation of glioma cells in fructose medium. Furthermore, we found that GLUT5 was also higher expressed in glioma tissues, and GLUT5 expression correlated significantly with glioma malignancy and poor survival of glioma patients (p < 0.01). In addition, we also demonstrated that knockdown of GLUT5 could significantly inhabit tumor proliferation in vivo, and intake fructose could increase tumor volume prominently. Taken together, our data show that fructose can be used by glioma cells, and restrict the fructose intake or targeting GLUT5 could be efficacious strategies in glioma.

MiR-96/HBP1/Wnt/β-catenin regulatory circuitry promotes glioma growth.

We found that miR-96 is overexpressed in glioma, and its level inversely correlates with the survival of patients. The reduction in miR-96 abundance suppresses the proliferation and colony formation of glioma cells. The tumorigenicity of U-87 MG cells is reduced by miR-96 silencing. miR-96 contributes to the activation of Wnt/β-catenin pathway in glioma cells. HMG-box transcription factor 1 (HBP-1), a Wnt/β-catenin pathway inhibitor, is suppressed by miR-96. The reactivation of Wnt/β-catenin signaling causes an increase in the proliferation of glioma cells, and a decrease in miR-96 expression. On the other hand, HBP1 silencing promotes miR-96 expression. Collectively, miR-96 contributes to the progression of glioma by enhancing the activation of the Wnt/β-catenin pathway, and the miR-96/HBP1/Wnt/β-catenin regulatory circuitry promotes the proliferation of glioma cells.

MiR-122/Wnt/β-catenin regulatory circuitry sustains glioma progression

Malignant glioma is the most common type of human intracranial cancer and has poor prognosis due to its high recurrence and invasiveness. However, the molecular mechanisms underlying its malignant phenotypes have still not been completely explored yet. miR-122 has been well documented to act as a tumor suppressor for hepatocellular carcinoma and breast cancer, but the implication of miR-122 in the progression of glioma is not clear yet. In this study, we found that miR-122 was underexpressed in glioma specimens and glioma cell lines, compared with normal brain tissues and cell lines. The expression of miR-122 levels is inversely correlated with the survival of patients after surgery. Overexpression of miR-122 by an adenoviral vector suppressed the proliferation and colony formation of glioma cells. The in vivo tumorigenicity of U-87 MG cells was also greatly compromised by restoring miR-122. miR-122 suppressed the activation of Wnt/β-catenin pathway in glioma cells. Interestingly, Wnt/β-catenin signaling conversely reduced the expression of miR-122 in glioma cells, maybe in a hepatocyte nuclear factor (HNF)-dependent mechanism. Taken together, we revealed that there is a miR-122/Wnt/β-catenin regulatory circuitry existing in glioma cells that contributes to glioma progression.

Abstract 674: The lysine demethylase KDM1 is a novel therapeutic target for the treatment of gliomas.

Abstract Glioma development is a multistep process, involving alterations in genetic and epigenetic mechanisms. Understanding the mechanisms and enzymes that promote epigenetic changes in gliomas are urgently needed to identify novel therapeutic targets. In the present study we explored the significance of histone demethylase KDM1 in glioma progression. In order to know the status of expression of KDM1, we utilized glioma tissue microarrays which consist of different grades of astrocytomas, oligodendrogliomas, ependymomas and normal brain tissues. Immunohistochemical analysis showed that KDM1 expression is overexpressed in the gliomas compared to normal brain, and KDM1 expression levels were positively correlated with histological malignancy. KDM1 expression was also found to be elevated in various established glioma cell lines. To study the functional significance of KDM1 in gliomas, KDM1 expression was silenced using siRNA or its pharmacological inhibition using pargyline or NCL-1. Silencing of KDM1 or inhibition of its enzyme activity significantly reduced the proliferation and colony formation of glioma cells. Mechanistic studies showed that inhibition of KDM1 promoted the acetylation of p53 and up regulation of its target genes p21 and PUMA. Patient-derived primary GBM cells expressed high levels of KDM1 and pharmacological inhibition of KDM1 decreased their proliferation. Further, KDM1 inhibition reduced the expression of stemness markers CD133 and nestin in GBM cells. Mouse xenograft assays revealed that inhibition of KDM1 using pargyline significantly reduced U87 glioma xenograft tumor growth. Inhibition of KDM1 increased levels of H3K4-me2 and H3K9-Ac histone modifications, reduced H3K9-me2 modification and promoted expression of p53 target genes (p21 and PUMA), leading to apoptosis of glioma xenograft tumors. Our results suggest that KDM1 is overexpressed in gliomas and could be a potential therapeutic target for the treatment of gliomas. Citation Format: Gangadhara R. Sareddy, Sandeep Saran, Binoj C. Nair, Samaya R. Krishnan, Vijay K. Gonugunta, Andrew J. Brenner, Ratna K. Vadlamudi. The lysine demethylase KDM1 is a novel therapeutic target for the treatment of gliomas. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 674. doi:10.1158/1538-7445.AM2013-674

MicroRNA-200b targets CREB1 and suppresses cell growth in human malignant glioma

MicroRNAs can coordinately repress multiple target genes and interfere with the biological functions of the cell, such as proliferation and apoptosis. In the present study, we report that miR-200b was downregulated in malignant glioma cell lines and specimens. Overexpression of miR-200b suppressed the proliferation and colony formation of glioma cells. An oncogene encoding cAMP responsive element-binding protein 1 (CREB1), which has been shown to be an important transcription factor involved in the proliferation, survival, and metastasis of tumor cells, was here confirmed as a direct target gene of miR-200b. CREB1 was also found to be present at a high level in human glioma tissues. This was inversely correlated with miR-200b expression. Ectopic expression of CREB1 attenuated the growth suppressive phenotypes of glioma cells caused by miR-200b. These results indicate that miR-200b targets the CREB1 gene and suppresses glioma cell growth, suggesting that miR-200b shows tumor-suppressive activity in human malignant glioma.