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Sample records for glioma cells overexpress

  1. Overexpressed KDM5B is associated with the progression of glioma and promotes glioma cell growth via downregulating p21

    Dai, Bin [Department of Neurosurgery, Beijing Shijitan Hospital, Capital Medical University, Beijing 100038 (China); Hu, Zhiqiang, E-mail: zhiqhutg@126.com [Department of Neurosurgery, Beijing Shijitan Hospital, Capital Medical University, Beijing 100038 (China); Huang, Hui; Zhu, Guangtong; Xiao, Zhiyong [Department of Neurosurgery, Beijing Shijitan Hospital, Capital Medical University, Beijing 100038 (China); Wan, Weiqing; Zhang, Peng; Jia, Wang; Zhang, Liwei [Department of Neurosurgery, Beijing Tian Tan Hospital, Capital Medical University, Beijing 100050 (China)

    2014-11-07

    Highlights: • KDM5B is overexpressed in glioma samples. • KDM5B stimulated proliferation of glioma cells. • Inhibition of p21contributes to KDM5B-induced proliferation. - Abstract: Epigenetic alterations such as aberrant expression of histone-modifying enzymes have been implicated in tumorigenesis. Upregulation of lysine (K)-specific demethylase 5B (KDM5B) has been reported in a variety of malignant tumors. However, the impact of KDM5B in glioma remains unclear. The objective of this study was to investigate the expression and prognostic value of KDM5B in glioma. In clinical glioma samples, we found that KDM5B expression was significantly upregulated in cancer lesions compared with normal brain tissues. Kaplan–Meier analysis showed that patients with glioma and higher KDM5B expression tend to have shorter overall survival time. By silencing or overexpressing KDM5B in glioma cells, we found that KDM5B could promote cell growth both in vitro and in vivo. Moreover, we demonstrated that KDM5B promoted glioma proliferation partly via regulation of the expression of p21. Our study provided evidence that KDM5B functions as a novel tumor oncogene in glioma and may be a potential therapeutic target for glioma management.

  2. Frequent Nek1 overexpression in human gliomas

    Zhu, Jun [School of Biomedical Engineering, Shanghai Jiao Tong University, Shanghai (China); Neurosurgery Department, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai (China); Cai, Yu, E-mail: aihaozuqiu22@163.com [School of Biomedical Engineering, Shanghai Jiao Tong University, Shanghai (China); Neurosurgery Department, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai (China); Liu, Pin [Med-X Research Institute, Shanghai Jiao Tong University, Shanghai (China); Zhao, Weiguo [Neurosurgery Department, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai (China)

    2016-08-05

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

  3. Frequent Nek1 overexpression in human gliomas

    Zhu, Jun; Cai, Yu; Liu, Pin; Zhao, Weiguo

    2016-01-01

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

  4. KDM2B overexpression correlates with poor prognosis and regulates glioma cell growth

    Wang Y

    2018-01-01

    Full Text Available Yiwei Wang,1 Jin Zang,1 Dongyong Zhang,2 Zhenxiang Sun,1 Bo Qiu,2 Xiaojie Wang1 1Department of Human Anatomy, Shenyang Medical College, Huanggu District, Shenyang City, 2Department of Neurosurgery, First Affiliated Hospital of China Medical University, Heping District, Shenyang City, Liaoning Province, ChinaBackground: Gliomas are one of the most lethal cancers in the human central nervous system. Despite clinical treatment advancements, the prognosis of patients with glioma remains poor. KDM2B is a histone lysine demethylase, which has been observed in multiple tumors. But the concrete role of KDM2B in gliomas remains to be further illustrated.Methods: The KDM2B expression in gliomas was detected with immunohistochemistry and Western blot assay. Furthermore, knockdown of KDM2B in U87 and U251 glioma cell lines, the proliferation capacity was evaluated by cell viability assay, colon formation assay and flow cytometry in vitro. Western blot assay was used to analyze the p21, EZH2 and cyclinD1 changes followed by knockdown of KDM2B.Results: KDM2B was upregulated in tissues of glioma patients, and the expression was correlated to cancer progression. Downregulation of KDM2B in U87 and U251 glioma cell lines inhibited cell proliferation and arrested cell cycle in G0/G1 phase. In addition, silencing KDM2B promoted the upregulation of p21 while reduced the expression of EZH2 and cyclinD1.Conclusion: Taken together, our results revealed that KDM2B might influence gliomas growth and act as a novel therapeutic target for glioma patients.Keywords: EZH2, glioma, KDM2B, P21

  5. [Overexpressed miRNA-134b inhibits proliferation and invasion of CD133+ U87 glioma stem cells].

    Liu, Yifeng; Zhang, Baochao; Wen, Changming; Wen, Gongling; Zhou, Guoping; Zhang, Jingwei; He, Haifa; Wang, Ning; Li, Wei

    2017-05-01

    Objective To investigate the role of microRNA-134b (miR-134b) in the tumorigenesis of glioma stem cells (GSCs) and the possible molecular mechanism. Methods Real-time quantitative PCR (qRT-PCR) was used to evalate the expression of miR-134b in CD133 + and CD133 - U87 GSCs. A lentiviral vector overexpressing miR-134b in U87 GSCs was constructed, and the effect of miR-134b overexpression on matrix metalloproteinase-2 (MMP-2), MMP-9 and MMP-12 expressions at both mRNA and protein levels were detected by qRT-PCR and Western blotting, respectively. Transwell TM assay was performed to determine the effect of miR-134b overexpression on GSCs invasion ability. Tumor xenograft models in nude mice were established to evaluate the effect of miR-134b overexpression on tumorgenesis in vivo. Results The qRT-PCR showed that, compared with CD133 - cells, miR-134b was significantly down-regulated in CD133 + cells. Cell line over-expressing miR-134b was successfully established, and miR-134b was up-regulated significantly compared with empty vector control. Overexpression of miR-134b remarkably inhibited the invasion of U87 GSCs and the expression of MMP-12. However, overexpression of miR-134b did not affect MMP-2 and MMP-9 expressions. miR-134b also suppressed U87 GSCs xenograft growth in vivo. Tumor volume in tumor xenograft model group was significantly lower than that in control group, and tumor weight decreased by 42% in the former group. Conclusion Overexpression of miR-134b inhibits the growth and invasion of CD133 + GSCs.

  6. Radiosensitization by overexpression of the nonphosphorylation form of IκB-α in human glioma cells

    Honda, Naoko; Yagi, Kasumi; Ding, Gui-Rong; Miyakoshi, Junji

    2002-01-01

    To assess the role of NF-κB in cellular radiosensitivity, we constructed mutated IκB expression plasmids for SY-IκB (with mutations at residues of 32, 36 and 42) expression in human malignant glioma cells (radiosensitive MO54 and radioresistant T98 cells), giving respective cell types referred to as MO54-SY4 and T98-SY14. Both of the clones expressing SY-IκB became radiosensitive, compared with the parental MO54 and T98 cells. A treatment with herbimycin A or genistein did not change the radiosensitivity of cells expressing SY-IκB, but made both the MO54 and T98 parental cells more sensitive to ionizing radiation. A treatment with TNF-α induced DNA fragmentation and apoptosis in cells expressing SY-IκB, but not in MO54 and T98 cells. The survival after X-ray exposure of the parental MO54 cells was slightly increased by a TNF-α treatment, but that of the parental T98 cells did not change. The change in sensitivity to ultra-violet (UV) radiation and adriamycin in MO54-SY4 cells was very similar to that for X-ray sensitivity, but no change was observed in T98-SY14 cells. Significant sublethal damage repair was observed in T98 cells, whereas MO54 cells showed little repair activity. The expression of p53 was enhanced in the parental MO54 cells, while the p53 levels in the MO54-SY4, and in the parent and clonal T98 cells, did not change. Our data suggest that the serine and tyrosine phosphorylation of IκB-α may play a role in determining the radiosensitivity of malignant glioma cells. (author)

  7. Overexpression of high molecular weight FGF-2 forms inhibits glioma growth by acting on cell-cycle progression and protein translation

    Lemiere, Sylvie; Azar, Rania; Belloc, Francis; Guersel, Demir; Pyronnet, Stephane; Bikfalvi, Andreas; Auguste, Patrick

    2008-01-01

    In order to clarify the role of HMW FGF-2 in glioma development and angiogenesis, we over-expressed different human FGF-2 isoforms in C6 rat glioma cell line using a tetracycline-regulated expression system. Phenotypic modifications were analyzed in vitro and compared to untransfected cells or to cells over-expressing 18 kDa FGF-2 or all FGF-2 isoforms. In particular, we demonstrate that HMW FGF-2 has unique features in inhibiting glioma cell proliferation. HMW FGF-2 expressing cells showed a cell-cycle arrest at the G2M, demonstrating a role of HMW FGF-2 in controlling the entry in mitosis. Moreover, hydroxyurea was ineffective in blocking cells at the G1S boundary when HMW FGF-2 was expressed. We also show that the HMW FGF-2 isoforms inhibit 4E-BP1 phosphorylation at critical sites restoring the translation inhibitory activity of 4E-BP1. In vivo, inhibition of tumor growth was observed when cells expressed HMW FGF-2. This indicates that HMW FGF-2 inhibits tumor growth in glioma cells by acting on cell-cycle progression and protein translation

  8. KDM2B overexpression correlates with poor prognosis and regulates glioma cell growth

    Wang, Yiwei; Zang, Jin; Zhang, Dongyong; Sun, Zhenxiang; Qiu, Bo; Wang, Xiaojie

    2018-01-01

    Yiwei Wang,1 Jin Zang,1 Dongyong Zhang,2 Zhenxiang Sun,1 Bo Qiu,2 Xiaojie Wang1 1Department of Human Anatomy, Shenyang Medical College, Huanggu District, Shenyang City, 2Department of Neurosurgery, First Affiliated Hospital of China Medical University, Heping District, Shenyang City, Liaoning Province, ChinaBackground: Gliomas are one of the most lethal cancers in the human central nervous system. Despite clinical treatment advancements, the prognosis of patients with glioma remains ...

  9. C-MET overexpression and amplification in gliomas.

    Kwak, Yoonjin; Kim, Seong-Ik; Park, Chul-Kee; Paek, Sun Ha; Lee, Soon-Tae; Park, Sung-Hye

    2015-01-01

    We investigated c-Met overexpression and MET gene amplification in gliomas to determine their incidence and prognostic significance. c-Met immunohistochemistry and MET gene fluorescence in situ hybridization were carried out on tissue microarrays from 250 patients with gliomas (137 grade IV GBMs and 113 grade II and III diffuse gliomas). Clinicopathological features of these cases were reviewed. c-Met overexpression and MET gene amplification were detected in 13.1% and 5.1% of the GBMs, respectively. All the MET-amplified cases showed c-Met overexpression, but MET amplification was not always concordant with c-Met overexpression. None of grade II and III gliomas demonstrated c-Met overexpression or MET gene amplification. Mean survival of the GBM patients with MET amplification was not significantly different from patients without MET amplification (P=0.155). However, GBM patients with c-Met overexpression survived longer than patients without c-Met overexpression (P=0.035). Although MET amplification was not related to poor GBM prognosis, it is partially associated with the aggressiveness of gliomas, as MET amplification was found only in grade IV, not in grade II and III gliomas. We suggest that MET inhibitor therapy may be beneficial in about 5% GBMs, which was the incidence of MET gene amplification found in the patients included in this study.

  10. MiR-146b-5p overexpression attenuates stemness and radioresistance of glioma stem cells by targeting HuR/lincRNA-p21/β-catenin pathway

    Yang, Wei; Yu, Hongquan; Shen, Yueming; Liu, Yingying; Yang, Zhanshan; Sun, Ting

    2016-01-01

    A stem-like subpopulation existed in GBM cells, called glioma stem cells (GSCs), might contribute to cancer invasion, angiogenesis, immune evasion, and therapeutic resistance, providing a rationale to eliminate GSCs population and their supporting niche for successful GBM treatment. LincRNA-p21, a novel regulator of cell proliferation, apoptosis and DNA damage response, is found to be downregulated in several types of tumor. However, little is known about the role of lincRNA-p21 in stemness and radioresistance of GSCs and its regulating mechanisms. In this study, we found that lincRNA-p21 negatively regulated the expression and activity of β-catenin in GSCs. Downregulation of lincRNA-p21 in GSCs was resulted from upregulation of Hu antigen R (HuR) expression caused by miR-146b-5p downregulation. MiR-146b-5p overexpression increased apoptosis and radiosensitivity, decreased cell viability, neurosphere formation capacity and stem cell marker expression, and induced differentiation in GSCs. Moreover, knock-down lincRNA-p21 or HuR and β-catenin overexpression could rescue the phenotypic changes resulted from miR-146b-5p overexpression in GSCs. These findings suggest that targeting the miR-146b-5p/HuR/lincRNA-p21/β-catenin signaling pathway may be valuable therapeutic strategies against glioma. PMID:27166258

  11. Glioma cells on the run – the migratory transcriptome of 10 human glioma cell lines

    Holz David

    2008-01-01

    Full Text Available Abstract Background Glioblastoma multiforme (GBM is the most common primary intracranial tumor and despite recent advances in treatment regimens, prognosis for affected patients remains poor. Active cell migration and invasion of GBM cells ultimately lead to ubiquitous tumor recurrence and patient death. To further understand the genetic mechanisms underlying the ability of glioma cells to migrate, we compared the matched transcriptional profiles of migratory and stationary populations of human glioma cells. Using a monolayer radial migration assay, motile and stationary cell populations from seven human long term glioma cell lines and three primary GBM cultures were isolated and prepared for expression analysis. Results Gene expression signatures of stationary and migratory populations across all cell lines were identified using a pattern recognition approach that integrates a priori knowledge with expression data. Principal component analysis (PCA revealed two discriminating patterns between migrating and stationary glioma cells: i global down-regulation and ii global up-regulation profiles that were used in a proband-based rule function implemented in GABRIEL to find subsets of genes having similar expression patterns. Genes with up-regulation pattern in migrating glioma cells were found to be overexpressed in 75% of human GBM biopsy specimens compared to normal brain. A 22 gene signature capable of classifying glioma cultures based on their migration rate was developed. Fidelity of this discovery algorithm was assessed by validation of the invasion candidate gene, connective tissue growth factor (CTGF. siRNA mediated knockdown yielded reduced in vitro migration and ex vivo invasion; immunohistochemistry on glioma invasion tissue microarray confirmed up-regulation of CTGF in invasive glioma cells. Conclusion Gene expression profiling of migratory glioma cells induced to disperse in vitro affords discovery of genomic signatures; selected

  12. Overexpression of NIMA-related kinase 2 is associated with poor prognoses in malignant glioma.

    Liu, Huajie; Liu, Bin; Hou, Xianzeng; Pang, Bo; Guo, Pengbo; Jiang, Wanli; Ding, Qian; Zhang, Rui; Xin, Tao; Guo, Hua; Xu, Shangchen; Pang, Qi

    2017-05-01

    Eleated expression of NIMA-related kinase 2 (NEK2) was frequently observed in a variety of malignant cancers, and it appears to be involved in the initiation, maintenance, progression, metastasis of cancer and is positively associated with poor prognosis. We sought to investigate NEK2 expression and its predictive roles in malignant gliomas, and study the correlation of NEK2 protein expression with proliferation, clinical parameters, overall survival and some other parameters. We investigate NEK2 protein expression in 99 samples of malignant gliomas, including 35 WHO grade II, 22 grade III, and 42 grade IV gliomas, by immunohistochemistry and western blot (n = 50). We then made correlative analysis of protein overexpression using the Kaplan-Meier method, Log rank test, and Cox proportional-hazards model analysis. NEK2 protein was overexpressed in malignant gliomas, but not in normal brain tissues. Overexpression of NEK2 correlated with malignancy, proliferation and adverse overall survival in gliomas. Moreover, chemotherapy, resection extent and WHO grade also correlate with overall survival in gliomas. However, within WHO grade II glioma subgroup, NEK2 overexpression showed no impact on overall survival. The present study firstly reveals that NEK2 protein is widely overexpressed in gliomas. NEK2 overexpression correlates significantly with malignancy (WHO grades), proliferation (Ki-67) and prognosis in malignant gliomas. NEK2 is a potential gene therapy target and prognostic indicator.

  13. Gamma-glutamylcyclotransferase promotes the growth of human glioma cells by activating Notch-Akt signaling

    Shen, Shang-Hang; Yu, Ning; Liu, Xi-Yao; Tan, Guo-Wei; Wang, Zhan-Xiang, E-mail: md_wzx7189@163.com

    2016-03-18

    Glioma as an aggressive type tumor is rapidly growing and has become one of the leading cause of cancer-related death worldwide. γ-Glutamylcyclotransferase (GGCT) has been shown as a diagnostic marker in various cancers. To reveal whether there is a correlation between GGCT and human glioma, GGCT expression in human glioma tissues and cell lines was first determined. We found that GGCT expression was up-regulated in human glioma tissues and cell lines. Further, we demonstrate that GGCT knockdown inhibits glioma cell T98G and U251 proliferation and colony formation, whereas GGCT overexpression leads to oppose effects. GGCT overexpression promotes the expression of Notch receptors and activates Akt signaling in glioma cells, and Notch-Akt signaling is activated in glioma tissues with high expression of GGCT. Finally, we show that inhibition of Notch-Akt signaling with Notch inhibitor MK-0752 blocks the effects of GGCT on glioma proliferation and colony formation. In conclusion, GGCT plays a critical role in glioma cell proliferation and may be a potential cancer therapeutic target. - Highlights: • GGCT expression is up-regulated in human glioma tissues and cell lines. • GGCT promotes glioma cell growth and colony formation. • GGCT promotes the activation of Notch-Akt signaling in glioma cells and tissues. • Notch inhibition blocks the role of GGCT in human glioma cells.

  14. Overexpression of ceramide synthase 1 increases C18-ceramide and leads to lethal autophagy in human glioma

    Wang, Zheng; Wen, Lijun; Zhu, Fei; Wang, Yanping; Xie, Qing; Chen, Zijun; Li, Yunsen

    2017-01-01

    Ceramide synthase 1 (CERS1) is the most highly expressed CERS in the central nervous system, and ceramide with an 18-carbon–containing fatty acid chain (C18-ceramide) in the brain plays important roles in signaling and sphingolipid development. However, the roles of CERS1 and C18-ceramide in glioma are largely unknown. In the present study, measured by electrospray ionization linear ion trap mass spectrometry, C18-ceramide was significantly lower in glioma tumor tissues compared with controls (P overexpression of CERS1, which has been shown to specifically induce the generation of C18-ceramide. Overexpression of CERS1 or adding exogenous C18-ceramide inhibited cell viability and induced cell death by activating endoplasmic reticulum stress, which induced lethal autophagy and inhibited PI3K/AKT signal pathway in U251 and A172 glioma cells. Moreover, overexpression of CERS1 or adding exogenous C18-ceramide increased the sensitivity of U251 and A172 glioma cells to teniposide (VM-26). Thus, the combined therapy of CERS1/C18-ceramide and VM-26 may be a novel therapeutic strategy for the treatment of human glioma. PMID:29262618

  15. Astroglial c-Myc overexpression predisposes mice to primary malignant gliomas

    Jensen, Niels Aagaard; Pedersen, Karen-Marie; Lihme, Frederikke

    2003-01-01

    Malignant astrocytomas are common human primary brain tumors that result from neoplastic transformation of astroglia or their progenitors. Here we show that deregulation of the c-Myc pathway in developing astroglia predisposes mice to malignant astrocytomas within 2-3 weeks of age. The genetically...... engineered murine (GEM) gliomas harbor a molecular signature resembling that of human primary glioblastoma multiforme, including up-regulation of epidermal growth factor receptor and Mdm2. The GEM gliomas seem to originate in an abnormal population of glial fibrillary acidic protein-expressing cells...... the neoplastic process, presumably by inducing the sustained growth of early astroglial cells. This is in contrast to most other transgenic studies in which c-Myc overexpression requires co-operating transgenes for rapid tumor induction....

  16. Overexpression of Transforming Acidic Coiled Coil‑Containing Protein 3 Reflects Malignant Characteristics and Poor Prognosis of Glioma

    Ying Sun

    2017-03-01

    Full Text Available Gliomas are malignant primary brain tumors with poor prognosis. Recently, research was indicative of a tight connection between tumor malignancy and genetic alterations. Here, we propose an oncogenic implication of transforming acidic coiled-coil-containing protein 3 (TACC3 in gliomas. By comprehensively analyzing the Chinese glioma genome atlas (CGGA and publicly available data, we demonstrated that TACC3 were overexpressed along with glioma grade and served as an independent negative prognostic biomarker for glioma patients. Functions’ annotations and gene sets’ enrichment analysis suggested that TACC3 may participate in cell cycle, DNA repair, epithelium-mesenchymal transition and other tumor-related biological processes and molecular pathways. Patients with high TACC3 expression showed CD133+ stem cell properties, glioma plasticity and shorter overall survival time under chemo-/radio-therapy. Additionally, a TACC3 associated the miRNA-mRNA network was constructed based on in silico prediction and expression pattern, which provide a foundation for further detection of TACC3-miRNA-mRNA axis function. Collectively, our observations identify TACC3 as an oncogene of tumor malignancy, as well as a prognostic and motoring biomarker for glioma patients.

  17. MTSS1 is epigenetically regulated in glioma cells and inhibits glioma cell motility

    Daniel Luxen

    2017-02-01

    Full Text Available Epigenetic silencing by DNA methylation in brain tumors has been reported for many genes, however, their function on pathogenesis needs to be evaluated. We investigated the MTSS1 gene, identified as hypermethylated by differential methylation hybridization (DMH. Fifty-nine glioma tissue samples and seven glioma cell lines were examined for hypermethylation of the MTSS1 promotor, MTSS1 expression levels and gene dosage. GBM cell lines were treated with demethylating agents and interrogated for functional consequences of MTSS1 expression after transient transfection. Hypermethylation was significantly associated with IDH1/2 mutation. Comparative SNP analysis indicates higher incidence of loss of heterozygosity of MTSS1 in anaplastic astrocytomas and secondary glioblastomas as well as hypermethylation of the remaining allele. Reversal of promoter hypermethylation results in an increased MTSS1 expression. Cell motility was significantly inhibited by MTSS1 overexpression without influencing cell growth or apoptosis. Immunofluorescence analysis of MTSS1 in human astrocytes indicates co-localization with actin filaments. MTSS1 is down-regulated by DNA methylation in glioblastoma cell lines and is part of the G-CIMP phenotype in primary glioma tissues. Our data on normal astrocytes suggest a function of MTSS1 at focal contact structures with an impact on migratory capacity but no influence on apoptosis or cellular proliferation.

  18. Glutamate/glutamine metabolism coupling between astrocytes and glioma cells: neuroprotection and inhibition of glioma growth.

    Yao, Pei-Sen; Kang, De-Zhi; Lin, Ru-Ying; Ye, Bing; Wang, Wei; Ye, Zu-Cheng

    2014-07-18

    Glioma glutamate release has been shown to promote the growth of glioma cells and induce neuronal injuries from epilepsy to neuronal death. However, potential counteractions from normal astrocytes against glioma glutamate release have not been fully evaluated. In this study, we investigated the glutamate/glutamine cycling between glioma cells and astrocytes and their impact on neuronal function. Co-cultures of glioma cells with astrocytes (CGA) in direct contact were established under different mix ratio of astrocyte/glioma. Culture medium conditioned in these CGAs were sampled for HPLC measurement, for neuronal ratiometric calcium imaging, and for neuronal survival assay. We found: (1) High levels of glutaminase expression in glioma cells, but not in astrocytes, glutaminase enables glioma cells to release large amount of glutamate in the presence of glutamine. (2) Glutamate levels in CGAs were directly determined by the astrocyte/glioma ratios, indicating a balance between glioma glutamate release and astrocyte glutamate uptake. (3) Culture media from CGAs of higher glioma/astrocyte ratios induced stronger neuronal Ca(2+) response and more severe neuronal death. (4) Co-culturing with astrocytes significantly reduced the growth rate of glioma cells. These results indicate that normal astrocytes in the brain play pivotal roles in glioma growth inhibition and in reducing neuronal injuries from glioma glutamate release. However, as tumor growth, the protective role of astrocytes gradually succumb to glioma cells. Copyright © 2014 Elsevier Inc. All rights reserved.

  19. Upregulation of B23 promotes tumor cell proliferation and predicts poor prognosis in glioma

    Chen, Jianguo [Department of Neurosurgery, The Second Affiliated Hospital of Soochow University, 1055 Sanxiang Road, Suzhou, 215004, Jiangsu Province (China); Department of Neurosurgery, Affiliated Hospital of Nantong University, 20 Xisi Road, Nantong, 226001, Jiangsu Province (China); Sun, Jie; Yang, Liu; Yan, Yaohua; Shi, Wei; Shi, Jinlong; Huang, Qingfeng; Chen, Jian [Department of Neurosurgery, Affiliated Hospital of Nantong University, 20 Xisi Road, Nantong, 226001, Jiangsu Province (China); Lan, Qing, E-mail: lanqingsj@163.com [Department of Neurosurgery, The Second Affiliated Hospital of Soochow University, 1055 Sanxiang Road, Suzhou, 215004, Jiangsu Province (China)

    2015-10-09

    B23 (also known as Nucleophosmin, NPM, numatrin or NO38) is a ubiquitously expressed phosphoprotein belonging to the nucleoplasmin family of chaperones. In this study we intended to investigate the clinical significance of B23 expression in human glioma and its biological function in glioma cells. Western blot and immunohistochemistry analysis showed that B23 was overexpressed in glioma tissues and glioma cell lines. In addition, the expression level of B23 was positively correlated with glioma pathological grade and Ki-67 expression. Kaplan–Meier analysis revealed that a higher B23 expression in patients with glioma was associated with a poorer prognosis. In vitro, after the release of glioma cell lines from serum starvation, the expression of B23 was upregulated, as well as PCNA (Proliferating Cell Nuclear Antigen) and cyclin A. In addition, knockdown of B23 by small interfering RNA transfection diminished the expression of PCNA, cyclin D1 and arrested cell growth at G1 phase. Taken together, our results implied that B23 could be a candidate prognostic biomarker as well as a potential therapeutical target of glioma. - Highlights: • B23 expression increased as the malignant degree of glioma increased, which was consistent with Ki-67 expression. • High expression of B23 could be a strong determinant of poor prognosis in glioma. • B23 may be involved in the proliferation of glioma in a cell-cycle-dependent pathway. • Knockdown of B23 expression by siRNA could affect the progression of glioma. • B23 may be a potential prognosis biomarker and a possible therapeutic target for glioma.

  20. Type I collagen gene suppresses tumor growth and invasion of malignant human glioma cells

    Miyata Teruo

    2007-06-01

    Full Text Available Abstract Background Invasion is a hallmark of a malignant tumor, such as a glioma, and the progression is followed by the interaction of tumor cells with an extracellular matrix (ECM. This study examined the role of type I collagen in the invasion of the malignant human glioma cell line T98G by the introduction of the human collagen type I α1 (HCOL1A1 gene. Results The cells overexpressing HCOL1A1 were in a cluster, whereas the control cells were scattered. Overexpression of HCOL1A1 significantly suppressed the motility and invasion of the tumor cells. The glioma cell growth was markedly inhibited in vitro and in vivo by the overexpression of HCOL1A1; in particular, tumorigenicity completely regressed in nude mice. Furthermore, the HCOL1A1 gene induced apoptosis in glioma cells. Conclusion These results indicate that HCOL1A1 have a suppressive biological function in glioma progression and that the introduction of HCOL1A1 provides the basis of a novel therapeutic approach for the treatment of malignant human glioma.

  1. Insulin-like Growth Factor Binding Protein 7 Mediates Glioma Cell Growth and Migration

    Wei Jiang

    2008-12-01

    Full Text Available Insulin-like growth factor binding protein 7 (IGFBP-7 is the only member of the IGFBP superfamily that binds strongly to insulin, suggesting that IGFBP-7 may have different functions from other IGFBPs. Unlike other IGFBPs, the expression and functions of IGFBP-7 in glioma tumors have not been reported. Using cDNA microarray analysis, we found that expression of IGFBP-7 correlated with the grade of glioma tumors and the overall patient survival. This finding was further validated by real-time reverse transcription-polymerase chain reaction and Western blot analysis. We used RNAi to examine the role of IGFBP-7 in glioma cells, inhibiting IGFBP-7 expression by short interfering RNA transfection. Cell proliferation was suppressed after IGFBP-7 expression was inhibited for 5 days, and glioma cell growth was stimulated consistently by the addition of recombinant IGFBP-7 protein. Moreover, glioma cell migration was attenuated by IGFBP-7 depletion but enhanced by IGFBP-7 overexpression and addition. Overexpression of AKT1 in IGFBP-7-overxpressed cells attenuated the IGFBP-7-promoted migration and further enhanced inhibition of IGFBP-7 depletion on the migration. Phosphorylation of AKT and Erk1/2 was also inversely regulated by IGFBP-7 expression. These two factors together suggest that IGFBP-7 can regulate glioma cell migration through the AKT-ERK pathway, thereby playing an important role in glioma growth and migration.

  2. Glioma cell fate decisions mediated by Dll1-Jag1-Fringe in Notch1 signaling pathway.

    Shi, Xiaofei; Wang, Ruiqi

    2017-09-21

    The Notch family of proteins plays a vital role in determining cell fates, such as proliferation, differentiation, and apoptosis. It has been shown that Notch1 and its ligands, Dll1 and Jag1, are overexpressed in many glioma cell lines and primary human gliomas. The roles of Notch1 in some cancers have been firmly established, and recent data implicate that it plays important roles in glioma cell fate decisions. This paper focuses on devising a specific theoretical framework that incorporates Dll1, Jag1, and Fringe in Notch1 signaling pathway to explore their functional roles of these proteins in glioma cells in the tumorigenesis and progression of human gliomas, and to study how glioma cell fate decisions are modulated by both trans-activation and cis-inhibition. This paper presents a computational model for Notch1 signaling pathway in glioma cells. Based on the bifurcation analysis of the model, we show that how the glioma cell fate decisions are modulated by both trans-activation and cis-inhibition mediated by the Fringe protein, providing insight into the design and control principles of the Notch signaling system and the gliomas. This paper presents a computational model for Notch1 signaling pathway in glioma cells based on intertwined dynamics with cis-inhibition and trans-activation involving the proteins Notch1, Dll1, Jag1, and Fringe. The results show that how the glioma cell fate transitions are performed by the Notch1 signaling. Transition from grade III ∼ IV with significantly high Notch1 to grade I ∼ II with high Notch1, and then to normal cells by repressing the Fringe levels or decreasing the strength of enhancement induced by Fringe.

  3. Sirt2 suppresses glioma cell growth through targeting NF-κB–miR-21 axis

    Li, Ya’nan; Dai, Dongwei; Lu, Qiong; Fei, Mingyu; Li, Mengmeng; Wu, Xi

    2013-01-01

    Highlights: •Sirt2 expression is down-regulated in human glioma tissues and cell lines. •Sirt2 regresses glioma cell growth and colony formation via inducing apoptosis. •miR-21 is essential for the functions of Sirt2 in glioma cells. •Sirt2 deacetylates p65 to decrease miR-21 expression. -- Abstract: Sirtuins are NAD + -dependent deacetylases that regulate numerous cellular processes including aging, DNA repair, cell cycle, metabolism, and survival under stress conditions. The roles of sirtuin family members are widely studied in carcinogenesis. However, their roles in glioma remain unclear. Here we report that Sir2 was under expressed in human glioma tissues and cell lines. We found that Sirt2 overexpression decreased cell proliferation and colony formation capacity. In addition, Sirt2 overexpression induced cellular apoptosis via up-regulating cleaved caspase 3 and Bax, and down-regulating anti-apoptotic protein Bcl-2. Sirt2 knockdown obtained opposing results. We showed that Sirt2 overexpression inhibited miR-21 expression, and Sirt2 was not sufficient to reduce cell proliferation and colony formation as well as to induce apoptosis when miR-21 was knocked down in glioma cells. Mechanically, we demonstrated that Sirt2 deacetylated p65 at K310 and blocked p65 binding to the promoter region of miR-21, thus regressing the transcription of miR-21. In summary, Sirt2 is critical in human glioma via NF-κB–miR-21 pathway and Sirt2 activator may serve as candidate drug for glioma therapy

  4. The neural stem cell fate determinant TLX promotes tumorigenesis and genesis of cells resembling glioma stem cells.

    Park, Hyo-Jung; Kim, Jun-Kyum; Jeon, Hye-Min; Oh, Se-Yeong; Kim, Sung-Hak; Nam, Do-Hyun; Kim, Hyunggee

    2010-11-01

    A growing body of evidence indicates that deregulation of stem cell fate determinants is a hallmark of many types of malignancies. The neural stem cell fate determinant TLX plays a pivotal role in neurogenesis in the adult brain by maintaining neural stem cells. Here, we report a tumorigenic role of TLX in brain tumor initiation and progression. Increased TLX expression was observed in a number of glioma cells and glioma stem cells, and correlated with poor survival of patients with gliomas. Ectopic expression of TLX in the U87MG glioma cell line and Ink4a/Arf-deficient mouse astrocytes (Ink4a/Arf(-/-) astrocytes) induced cell proliferation with a concomitant increase in cyclin D expression, and accelerated foci formation in soft agar and tumor formation in in vivo transplantation assays. Furthermore, overexpression of TLX in Ink4a/Arf(-/-) astrocytes inhibited cell migration and invasion and promoted neurosphere formation and Nestin expression, which are hallmark characteristics of glioma stem cells, under stem cell culture conditions. Our results indicate that TLX is involved in glioma stem cell genesis and represents a potential therapeutic target for this type of malignancy.

  5. Aldehyde dehydrogenase 1A1 circumscribes high invasive glioma cells and predicts poor prognosis

    Xu, Sen-Lin; Liu, Sha; Cui, Wei; Shi, Yu; Liu, Qin; Duan, Jiang-Jie; Yu, Shi-Cang; Zhang, Xia; Cui, You-Hong; Kung, Hsiang-Fu; Bian, Xiu-Wu

    2015-01-01

    Glioma is the most aggressive brain tumor with high invasiveness and poor prognosis. More reliable, sensitive and practical biomarkers to reveal glioma high invasiveness remain to be explored for the guidance of therapy. We herein evaluated the diagnostic and prognostic value of aldehyde dehydrogenase 1A1 (ALDH1A1) in the glioma specimens from 237 patients, and found that ADLH1A1 was frequently overexpressed in the high-grade glioma (WHO grade III-IV) as compared to the low-grade glioma (WHO grade I-II) patients. The tumor cells with ALDH1A1 expression were more abundant in the region between tumor and the borderline of adjacent tissue as compared to the central part of the tumor. ALDH1A1 overexpression was associated with poor differentiation and dismal prognosis. Notably, the overall and disease-free survivals of the patients who had ALDH1A1+ tumor cells sparsely located in the adjacent tissue were much worse. Furthermore, ALDH1A1 expression was correlated with the “classical-like” (CL) subtype as we examined GBM specimens from 72 patients. Multivariate Cox regression analysis revealed that ALDH1A1 was an independent marker for glioma patients’ outcome. Mechanistically, both in vitro and in vivo studies revealed that ALDH1A1+ cells isolated from either a glioblastoma cell line U251 or primary glioblastoma cells displayed significant invasiveness, clonogenicity, and proliferation as compared to ALDH1A1- cells, due to increased levels of mRNA and protein for matrix metalloproteinase 2, 7 and 9 (MMP2, MMP7 and MMP9). These results indicate that ALDH1A1+ cells contribute to the progression of glioma including invasion, proliferation and poor prognosis, and suggest that targeting ALDH1A1 may have important implications for the treatment of highly invasive glioma. PMID:26101711

  6. AS1411-Induced Growth Inhibition of Glioma Cells by Up-Regulation of p53 and Down-Regulation of Bcl-2 and Akt1 via Nucleolin.

    Ye Cheng

    Full Text Available AS1411 binds nucleolin (NCL and is the first oligodeoxynucleotide aptamer to reach phase I and II clinical trials for the treatment of several cancers. However, the mechanisms by which AS1411 targets and kills glioma cells and tissues remain unclear. Here we report that AS1411 induces cell apoptosis and cycle arrest, and inhibits cell viability by up-regulation of p53 and down-regulation of Bcl-2 and Akt1 in human glioma cells. NCL was overexpressed in both nucleus and cytoplasm in human glioma U87, U251 and SHG44 cells compared to normal human astrocytes (NHA. AS1411 bound NCL and inhibited the proliferation of glioma cells but not NHA, which was accompanied with up-regulation of p53 and down-regulation of Bcl-2 and Akt1. Moreover, AS1411 treatment resulted in the G2/M cell cycle arrest in glioma cells, which was however abolished by overexpression of NCL. Further, AS1411 induced cell apoptosis, which was prevented by silencing of p53 and overexpression of Bcl-2. In addition, AS1411 inhibited the migration and invasion of glioma cells in an Akt1-dependent manner. Importantly, AS1411 inhibited the growth of glioma xenograft and prolonged the survival time of glioma tumor-bearing mice. These results revealed a promising treatment of glioma by oligodeoxynucleotide aptamer.

  7. Activation of glioma cells generates immune tolerant NKT cells.

    Tang, Bo; Wu, Wei; Wei, Xiaowei; Li, Yang; Ren, Gang; Fan, Wenhai

    2014-12-12

    Therapeutic outcomes of glioma are currently not encouraging. Tumor tolerance plays an important role in the pathogenesis of glioma. It is reported that micro RNAs (miR) are associated with tumor development. This study aims to investigate the role of miR-92a in the development of tolerant natural killer T (NKT) cells. In this study, U87 cells (a human glioma cell line) and primary glioma cells were prepared. The assessment of miR-92a was performed by real time RT-PCR. The expression of interleukin (IL)-10 and IL-6 in NKT cells was evaluated by flow cytometry. Results showed that abundant IL-6(+) IL-10(+) NKT cells were detected in glioma tissue. Cultures of glioma cells and NKT cells induced the expression of IL-6 and IL-10 in NKT cells. Glioma cells expressed miR-92a; the latter played a critical role in the induction of IL-6 and IL-10 expression in NKT cells. The expression of the antitumor molecules, including perforin, Fas ligand, and interferon-γ, was significantly attenuated compared with control NKT cells. The IL-6(+) IL-10(+) NKT cells showed less capability in the induction of apoptosis in glioma cells, but showed the immune suppressor functions on CD8(+) T cell activities. We conclude that glioma-derived miR-92a induces IL-6(+) IL-10(+) NKT cells; this fraction of NKT cells can suppress cytotoxic CD8(+) T cells. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. Regulation of Glioma Cell Migration by Seri ne-Phosphorylated P3111

    Wendy S. McDonough

    2005-09-01

    Full Text Available P311, an 8-kDa polypeptide, was previously shown to be highly expressed in invasive glioma cells. Here, we report the functional characteristics of P311 with regard to influencing glioma cell migration. P311 is constitutively serine-phosphorylated; decreased phosphorylation is observed in migration-activated glioma cells. The primary amino acid sequence of P311 indicates a putative serine phosphorylation site (S59 near the PEST domain. Site-directed mutagenesis of S59A retarded P311 degradation, induced glioma cell motility. In contrast, S59D mutation resulted in the rapid degradation of P311, reduced glioma cell migration. Coimmunoprecipitation coupled with matrixassisted laser desorption/ionization time-of-flight mass spectrometry analysis identified Filamin A as a binding partner of P311, immunofluorescence studies showed that both proteins colocalized at the cell periphery. Moreover, P311-induced cell migration was abrogated by inhibition of β1 integrin function using TACβ1A, a dominant-negative inhibitor of β1 integrin signaling, suggesting that P311 acts downstream of β1 signaling. Finally, overexpression of P311 or P311 S59A mutant protein activates Raci GTPase; small interfering RNA-mediated depletion of Raci suppresses P311-induced motility. Collectively, these results suggest a role for levels of P311 in regulating glioma motility, invasion through the reorganization of actin cytoskeleton at the cell periphery.

  9. Transmigration of neural stem cells across the blood brain barrier induced by glioma cells.

    Mónica Díaz-Coránguez

    Full Text Available Transit of human neural stem cells, ReNcell CX, through the blood brain barrier (BBB was evaluated in an in vitro model of BBB and in nude mice. The BBB model was based on rat brain microvascular endothelial cells (RBMECs cultured on Millicell inserts bathed from the basolateral side with conditioned media (CM from astrocytes or glioma C6 cells. Glioma C6 CM induced a significant transendothelial migration of ReNcells CX in comparison to astrocyte CM. The presence in glioma C6 CM of high amounts of HGF, VEGF, zonulin and PGE2, together with the low abundance of EGF, promoted ReNcells CX transmigration. In contrast cytokines IFN-α, TNF-α, IL-12p70, IL-1β, IL-6, IL-8 and IL-10, as well as metalloproteinases -2 and -9 were present in equal amounts in glioma C6 and astrocyte CMs. ReNcells expressed the tight junction proteins occludin and claudins 1, 3 and 4, and the cell adhesion molecule CRTAM, while RBMECs expressed occludin, claudins 1 and 5 and CRTAM. Competing CRTAM mediated adhesion with soluble CRTAM, inhibited ReNcells CX transmigration, and at the sites of transmigration, the expression of occludin and claudin-5 diminished in RBMECs. In nude mice we found that ReNcells CX injected into systemic circulation passed the BBB and reached intracranial gliomas, which overexpressed HGF, VEGF and zonulin/prehaptoglobin 2.

  10. Transmigration of neural stem cells across the blood brain barrier induced by glioma cells.

    Díaz-Coránguez, Mónica; Segovia, José; López-Ornelas, Adolfo; Puerta-Guardo, Henry; Ludert, Juan; Chávez, Bibiana; Meraz-Cruz, Noemi; González-Mariscal, Lorenza

    2013-01-01

    Transit of human neural stem cells, ReNcell CX, through the blood brain barrier (BBB) was evaluated in an in vitro model of BBB and in nude mice. The BBB model was based on rat brain microvascular endothelial cells (RBMECs) cultured on Millicell inserts bathed from the basolateral side with conditioned media (CM) from astrocytes or glioma C6 cells. Glioma C6 CM induced a significant transendothelial migration of ReNcells CX in comparison to astrocyte CM. The presence in glioma C6 CM of high amounts of HGF, VEGF, zonulin and PGE2, together with the low abundance of EGF, promoted ReNcells CX transmigration. In contrast cytokines IFN-α, TNF-α, IL-12p70, IL-1β, IL-6, IL-8 and IL-10, as well as metalloproteinases -2 and -9 were present in equal amounts in glioma C6 and astrocyte CMs. ReNcells expressed the tight junction proteins occludin and claudins 1, 3 and 4, and the cell adhesion molecule CRTAM, while RBMECs expressed occludin, claudins 1 and 5 and CRTAM. Competing CRTAM mediated adhesion with soluble CRTAM, inhibited ReNcells CX transmigration, and at the sites of transmigration, the expression of occludin and claudin-5 diminished in RBMECs. In nude mice we found that ReNcells CX injected into systemic circulation passed the BBB and reached intracranial gliomas, which overexpressed HGF, VEGF and zonulin/prehaptoglobin 2.

  11. Nuclear FABP7 immunoreactivity is preferentially expressed in infiltrative glioma and is associated with poor prognosis in EGFR-overexpressing glioblastoma

    Liang, Yu; Bollen, Andrew W; Aldape, Ken D; Gupta, Nalin

    2006-01-01

    We previously identified brain type fatty acid-binding protein (FABP7) as a prognostic marker for patients with glioblastoma (GBM). Increased expression of FABP7 is associated with reduced survival. To investigate possible molecular mechanisms underlying this association, we compared the expression and subcellular localization of FABP7 in non-tumor brain tissues with different types of glioma, and examined the expression of FABP7 and epidermal growth factor receptor (EGFR) in GBM tumors. Expression of FABP7 in non-tumor brain and glioma specimens was examined using immunohistochemistry, and its correlation to the clinical behavior of the tumors was analyzed. We also analyzed the association between FABP7 and EGFR expression in different sets of GBM specimens using published DNA microarray datasets and semi-quantitative immunohistochemistry. In vitro migration was examined using SF763 glioma cell line. FABP7 was present in a unique population of glia in normal human brain, and its expression was increased in a subset of reactive astrocytes. FABP7 immunoreactivity in grade I pilocytic astrocytoma was predominantly cytoplasmic, whereas nuclear FABP7 was detected in other types of infiltrative glioma. Nuclear, not cytoplasmic, FABP7 immunoreactivity was associated with EGFR overexpression in GBM (N = 61, p = 0.008). Expression of the FABP7 gene in GBM also correlated with the abundance of EGFR mRNA in our previous microarray analyses (N = 34, p = 0.016) and an independent public microarray dataset (N = 28, p = 0.03). Compared to those negative for both markers, nuclear FABP7-positive/EGFR-positive and nuclear FABP7-positive/EGFR-negative GBM tumors demonstrated shortest survival, whereas those only positive for EGFR had intermediate survival. EGFR activation increased nuclear FABP7 immunoreactivity in a glioma cell line in vitro, and inhibition of FABP7 expression suppressed EGF-induced glioma-cell migration. Our data suggested that in EGFR-positive GBM the presence of

  12. Anti-invasive and antiangiogenic effects of MMI-166 on malignant glioma cells

    Nakabayashi, Hiromichi; Yawata, Toshio; Shimizu, Keiji

    2010-01-01

    The constitutive overexpression of matrix metalloproteinases (MMPs) is frequently observed in malignant tumours. In particular, MMP-2 and MMP-9 have been reported to be closely associated with invasion and angiogenesis in malignant gliomas. Our study aimed to evaluate the antitumour effects of MMI-166 (Nalpha-[4-(2-Phenyl-2H- tetrazole-5-yl) phenyl sulfonyl]-D-tryptophan), a third generation MMP inhibitor, on three human glioma cell lines (T98G, U87MG, and ONS12) in vitro and in vivo. The effects of MMI-166 on the gelatinolytic activity was analysed by gelatine zymography. The anti-invasive effect of MMI-166 was analysed by an in vitro invasion assay. An in vitro angiogenesis assay was also performed. In vitro growth inhibition of glioma cells by MMI-166 was determined by the MTT assay. The effect of MMI-166 on an orthotropic implantation model using athymic mice was also evaluated. Gelatine zymography revealed that MMP-2 and MMP-9 activities were suppressed by MMI-166. The invasion of glioma cells was suppressed by MMI-166. The angiogenesis assay showed that MMI-166 had a suppressive effect on glioma cell-induced angiogenesis. However, MMI-166 did not suppress glioma cell proliferation in the MTT assay. In vivo, MMI-166 suppressed tumour growth in athymic mice implanted orthotropically with T98G cells and showed an inhibitory effect on tumour-induced angiogenesis and tumour growth. This is the first report of the effect of a third generation MMP inhibitor on malignant glioma cells. These results suggest that MMI-166 may have potentially suppressive effects on the invasion and angiogenesis of malignant gliomas

  13. Selective uptake of boronophenylalanine by glioma stem/progenitor cells

    Sun, Ting; Zhou, Youxin; Xie, Xueshun; Chen, Guilin; Li, Bin; Wei, Yongxin; Chen, Jinming; Huang, Qiang; Du, Ziwei

    2012-01-01

    The success of boron neutron capture therapy (BNCT) depends on the amount of boron in cells and the tumor/blood and tumor/(normal tissue) boron concentration ratios. For the first time, measurements of boron uptake in both stem/progenitor and differentiated glioma cells were performed along with measurements of boron biodistribution in suitable animal models. In glioma stem/progenitor cells, the selective accumulation of boronophenylalanine (BPA) was lower, and retention of boron after BPA removal was longer than in differentiated glioma cells in vitro. However, boron biodistribution was not statistically significantly different in mice with xenografts. - Highlights: ► Uptake of BPA was analyzed in stem/progenitor and differentiated glioma cells. ► Selective accumulation of BPA was lower in glioma stem/progenitor cells. ► Retention of boron after BPA removal was longer in glioma stem/progenitor cells. ► Boron biodistribution was not statistically different in mice with xenografts.

  14. Roles of sphingosine-1-phosphate (S1P) receptors in malignant behavior of glioma cells. Differential effects of S1P2 on cell migration and invasiveness

    Young, Nicholas; Van Brocklyn, James R.

    2007-01-01

    Sphingosine-1-phosphate (S1P) is a bioactive lipid that signals through a family of five G-protein-coupled receptors, termed S1P 1-5 . S1P stimulates growth and invasiveness of glioma cells, and high expression levels of the enzyme that forms S1P, sphingosine kinase-1, correlate with short survival of glioma patients. In this study we examined the mechanism of S1P stimulation of glioma cell proliferation and invasion by either overexpressing or knocking down, by RNA interference, S1P receptor expression in glioma cell lines. S1P 1 , S1P 2 and S1P 3 all contribute positively to S1P-stimulated glioma cell proliferation, with S1P 1 being the major contributor. Stimulation of glioma cell proliferation by these receptors correlated with activation of ERK MAP kinase. S1P 5 blocks glioma cell proliferation, and inhibits ERK activation. S1P 1 and S1P 3 enhance glioma cell migration and invasion. S1P 2 inhibits migration through Rho activation, Rho kinase signaling and stress fiber formation, but unexpectedly, enhances glioma cell invasiveness by stimulating cell adhesion. S1P 2 also potently enhances expression of the matricellular protein CCN1/Cyr61, which has been implicated in tumor cell adhesion, and invasion as well as tumor angiogenesis. A neutralizing antibody to CCN1 blocked S1P 2 -stimulated glioma invasion. Thus, while S1P 2 decreases glioma cell motility, it may enhance invasion through induction of proteins that modulate glioma cell interaction with the extracellular matrix

  15. MicroRNAs let-7b/i suppress human glioma cell invasion and migration by targeting IKBKE directly

    Tian, Yuan; Hao, Shaobo; Ye, Minhua; Zhang, Anling; Nan, Yang; Wang, Guangxiu; Jia, Zhifan; Yu, Kai; Guo, Lianmei; Pu, Peiyu; Huang, Qiang; Zhong, Yue

    2015-01-01

    We demonstrated that IKBKE is overexpressed in human gliomas and that the downregulation of IKBKE markedly inhibits the proliferative and invasive abilities of glioma cells, which is consistent with the results reported by several different research groups. Therefore, IKBKE represents a promising therapeutic target for the treatment of glioma. In the present study, we verified that the microRNAs let-7b and let-7i target IKBKE through luciferase assays and found that let-7b/i mimics can knock down IKBKE and upregulate E-cadherin through western blot analysis. Moreover, the expression levels of let-7b/i were significantly lower in glioma cell lines than that in normal brain tissues, as determined by quantitative real-time PCR. Furthermore, let-7b/i inhibit the invasion and migration of glioma cells, as determined through wound healing and Transwell assays. The above-mentioned data suggest that let-7b/i inhibit the invasive ability of glioma cells by directly downregulating IKBKE and indirectly upregulating E-cadherin. - Highlights: • Let-7b and let-7i are downregulated in glioma cell lines. • IKBKE is a target gene of let-7b/i. • Let-7b/i inhibit the invasion and migration of glioma cells. • Let-7b/i upregulate E-cadherin by downregulating IKBKE

  16. MicroRNAs let-7b/i suppress human glioma cell invasion and migration by targeting IKBKE directly

    Tian, Yuan; Hao, Shaobo [Department of Neurosurgery, Tianjin Medical University General Hospital, Tianjin 300052 (China); Laboratory of Neuro-Oncology, Tianjin Neurological Institute, Tianjin 300052 (China); Key Laboratory of Neurotrauma, Variation and Regeneration, Ministry of Education and Tianjin Municipal Government (China); Ye, Minhua [Department of Neurosurgery, Second Affiliated Hospital of Nanchang University, Nanchang, Jiangxi Province 330006 (China); Zhang, Anling [Laboratory of Neuro-Oncology, Tianjin Neurological Institute, Tianjin 300052 (China); Key Laboratory of Neurotrauma, Variation and Regeneration, Ministry of Education and Tianjin Municipal Government (China); Nan, Yang [Department of Neurosurgery, Tianjin Medical University General Hospital, Tianjin 300052 (China); Wang, Guangxiu; Jia, Zhifan [Laboratory of Neuro-Oncology, Tianjin Neurological Institute, Tianjin 300052 (China); Key Laboratory of Neurotrauma, Variation and Regeneration, Ministry of Education and Tianjin Municipal Government (China); Yu, Kai; Guo, Lianmei [Department of Neurosurgery, Tianjin Medical University General Hospital, Tianjin 300052 (China); Pu, Peiyu [Laboratory of Neuro-Oncology, Tianjin Neurological Institute, Tianjin 300052 (China); Key Laboratory of Neurotrauma, Variation and Regeneration, Ministry of Education and Tianjin Municipal Government (China); Huang, Qiang, E-mail: huangqiang209@163.com [Department of Neurosurgery, Tianjin Medical University General Hospital, Tianjin 300052 (China); Zhong, Yue, E-mail: zhongyue2457@sina.com [Department of Neurosurgery, Tianjin Medical University General Hospital, Tianjin 300052 (China)

    2015-03-06

    We demonstrated that IKBKE is overexpressed in human gliomas and that the downregulation of IKBKE markedly inhibits the proliferative and invasive abilities of glioma cells, which is consistent with the results reported by several different research groups. Therefore, IKBKE represents a promising therapeutic target for the treatment of glioma. In the present study, we verified that the microRNAs let-7b and let-7i target IKBKE through luciferase assays and found that let-7b/i mimics can knock down IKBKE and upregulate E-cadherin through western blot analysis. Moreover, the expression levels of let-7b/i were significantly lower in glioma cell lines than that in normal brain tissues, as determined by quantitative real-time PCR. Furthermore, let-7b/i inhibit the invasion and migration of glioma cells, as determined through wound healing and Transwell assays. The above-mentioned data suggest that let-7b/i inhibit the invasive ability of glioma cells by directly downregulating IKBKE and indirectly upregulating E-cadherin. - Highlights: • Let-7b and let-7i are downregulated in glioma cell lines. • IKBKE is a target gene of let-7b/i. • Let-7b/i inhibit the invasion and migration of glioma cells. • Let-7b/i upregulate E-cadherin by downregulating IKBKE.

  17. RAD18 mediates resistance to ionizing radiation in human glioma cells

    Xie, Chen; Wang, Hongwei; Cheng, Hongbin; Li, Jianhua; Wang, Zhi; Yue, Wu

    2014-01-01

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

  18. Resveratrol Represses Pokemon Expression in Human Glioma Cells.

    Yang, Yutao; Cui, Jiajun; Xue, Feng; Overstreet, Anne-Marie; Zhan, Yiping; Shan, Dapeng; Li, Hui; Li, Hui; Wang, Yongjun; Zhang, Mengmeng; Yu, Chunjiang; Xu, Zhi-Qing David

    2016-03-01

    POK erythroid myeloid ontogenic factor (Pokemon), an important proto-oncoprotein, is a transcriptional repressor that regulates the expression of many genes and plays an important role in tumorigenesis. Resveratrol (RSV), a natural polyphenolic compound, has many beneficial biological effects on health. In this study, we investigated the role of Pokemon in RSV-induced biological effects and the effect of RSV on the expression of Pokemon in glioma cells. We found that overexpression of Pokemon decreased RSV-induced cell apoptosis, senescence, and anti-proliferative effects. Moreover, we showed that RSV could efficiently decrease the activity of the Pokemon promoter and the expression of Pokemon. Meanwhile, RSV also inhibited Sp1 DNA binding activity to the Pokemon promoter; whereas, it did not influence the expression and nuclear translocation of Sp1. In addition, we found that RSV could increase the recruitment of HDAC1, but decreased p300 to the Pokemon promoter. Taken together, all these results extended our understanding on the anti-cancer mechanism of RSV in glioma cells.

  19. 17-AAG sensitized malignant glioma cells to death-receptor mediated apoptosis.

    Siegelin, Markus David; Habel, Antje; Gaiser, Timo

    2009-02-01

    17-AAG is a selective HSP90-inhibitor that exhibited therapeutic activity in cancer. In this study three glioblastoma cell lines (U87, LN229 and U251) were treated with 17-AAG, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) or the combination of both. Treatment with subtoxic doses of 17-AAG in combination with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces rapid apoptosis in TRAIL-resistant glioma cells, suggesting that this combined treatment may offer an attractive strategy for treating gliomas. 17-AAG treatment down-regulated survivin through proteasomal degradation. In addition, over-expression of survivin attenuated cytotoxicity induced by the combination of 17-AAG and TRAIL. In summary, survivin is a key regulator of TRAIL-17-AAG mediated cell death in malignant glioma.

  20. TGF-β promotes glioma cell growth via activating Nodal expression through Smad and ERK1/2 pathways

    Sun, Jing; Liu, Su-zhi; Lin, Yan; Cao, Xiao-pan; Liu, Jia-ming

    2014-01-01

    Highlights: •TGF-β promoted Nodal expression in glioma cells. •TGF-β promoted Nodal expression via activating Smad and ERK1/2 pathways. •TGF-β promotes glioma cell growth via activating Nodal expression. -- Abstract: While there were certain studies focusing on the mechanism of TGF-β promoting the growth of glioma cells, the present work revealed another novel mechanism that TGF-β may promote glioma cell growth via enhancing Nodal expression. Our results showed that Nodal expression was significantly upregulated in glioma cells when TGF-β was added, whereas the TGF-β-induced Nodal expression was evidently inhibited by transfection Smad2 or Smad3 siRNAs, and the suppression was especially significant when the Smad3 was downregulated. Another, the attenuation of TGF-β-induced Nodal expression was observed with blockade of the ERK1/2 pathway also. Further detection of the proliferation, apoptosis, and invasion of glioma cells indicated that Nodal overexpression promoted the proliferation and invasion of tumor cells and inhibited their apoptosis, resembling the effect of TGF-β addition. Downregulation of Nodal expression via transfection Nodal-specific siRNA in the presence of TGF-β weakened the promoting effect of the latter on glioma cells growth, and transfecting Nodal siRNA alone in the absence of exogenous TGF-β more profoundly inhibited the growth of glioma cells. These results demonstrated that while both TGF-β and Nodal promoted glioma cells growth, the former might exert such effect by enhancing Nodal expression, which may form a new target for glioma therapy

  1. ER stress inducer tunicamycin suppresses the self-renewal of glioma-initiating cell partly through inhibiting Sox2 translation.

    Xing, Yang; Ge, Yuqing; Liu, Chanjuan; Zhang, Xiaobiao; Jiang, Jianhai; Wei, Yuanyan

    2016-06-14

    Glioma-initiating cells possess tumor-initiating potential and are relatively resistant to conventional chemotherapy and irradiation. Therefore, their elimination is an essential factor for the development of efficient therapy. Here, we report that endoplasmic reticulum (ER) stress inducer tunicamycin inhibits glioma-initiating cell self-renewal as determined by neurosphere formation assay. Moreover, tunicamycin decreases the efficiency of glioma-initiating cell to initiate tumor formation. Although tunicamycin induces glioma-initiating cell apoptosis, apoptosis inhibitor z-VAD-fmk only partly abrogates the reduction in glioma-initiating cell self-renewal induced by tunicamycin. Indeed, tunicamycin reduces the expression of self-renewal regulator Sox2 at translation level. Overexpression of Sox2 obviously abrogates the reduction in glioma-initiating cell self-renewal induced by tunicamycin. Taken together, tunicamycin suppresses the self-renewal and tumorigenic potential of glioma-initiating cell partly through reducing Sox2 translation. This finding provides a cue to potential effective treatment of glioblastoma through controlling stem cells.

  2. Products of cells from gliomas: VIII. Multiple-well immunoperoxidase assay of immunoreactivity of primary hybridoma supernatants with human glioma and brain tissue and cultured glioma cells.

    McKeever, P E; Wahl, R L; Shakui, P; Jackson, G A; Letica, L H; Liebert, M; Taren, J A; Beierwaltes, W H; Hoff, J T

    1990-06-01

    To test the feasibility of primary screening of hybridoma supernatants against human glioma tissue, over 5000 combinations of hybridoma supernatants with glioma tissue, cultured glioma cells, and normal central neural tissue were screened with a new multiple-well (M-well) screening system. This is an immunoperoxidase assay system with visual endpoints for screening 20-30 hybridoma supernatants per single microscope slide. There were extensive differences between specificities to tissue and to cultured glioma cells when both were screened with M-wells and when cultured cells were screened with standard semi-automated fluorescence. Primary M-well screening with glioma tissue detected seven hybridoma supernatants that specifically identified parenchymal cells of glioma tissue and that were not detected with cultured cells. Immunoreactivities of individual supernatants for vascular components (nine supernatants), necrosis (five supernatants), and nuclei (three supernatants) were detected. Other supernatants bound multiple sites on glioma tissue and/or subpopulations of neurons and glia of normal tissue. The results show that primary screening with glioma tissue detects a number of different specificities of hybridoma supernatants to gliomas not detected by conventional screening with cultured cells. These are potentially applicable to diagnosis and therapy.

  3. Monitoring in real time the effect of TLX overexpression on proliferation and migration of C6 cells.

    Li, G L; Fang, S H; Xu, B

    2017-01-01

    Orphan nuclear receptor TLX has been shown to play an essential role in regulating the self-renewal and proliferation of neural stem cells (NSCs). However, TLX overexpression in NSCs induces long-term NSC expansion and further leads to glioma initiation in mouse when combined with p53 mutations. Whether overexpression of TLX plays a role in glioma stem cell (GSC) proliferation and migration still remains largely unknown. In this study, we infected C6 cells, a special glioma cell line which is mainly composed of cancer stem cells(CSCs), with lentiviruses expressing GFP(LV-GFP) or GFP-T2A-TLX(LV-TLX) and then monitored cell proliferation and migration using the real-time analyzer system (RTCA, xCELLigence, Roche). We found that the cell index (CI) observed for the TLX overexpressing C6 cells showed a lower value than that of the LV-GFP transduced cells. And the MTT results correlated highly with the RTCA proliferation assessments. Furthermore, the expression of p21 was decreased while other downstream genes PTEN and p53 were not significantly changed in TLX overexpressing C6 cells . These findings strongly indicate that TLX overexpression has the ability to decrease the proliferating and migratory properties of C6 cells by targeting p21. Further, our results suggest that TLX overexpression may also have a similar inhibitory effect on GSC proliferation and migration.

  4. Glioma cell death induced by irradiation or alkylating agent chemotherapy is independent of the intrinsic ceramide pathway.

    Dorothee Gramatzki

    Full Text Available Resistance to genotoxic therapy is a characteristic feature of glioma cells. Acid sphingomyelinase (ASM hydrolyzes sphingomyelin to ceramide and glucosylceramide synthase (GCS catalyzes ceramide metabolism. Increased ceramide levels have been suggested to enhance chemotherapy-induced death of cancer cells.Microarray and clinical data for ASM and GCS in astrocytomas WHO grade II-IV were acquired from the Rembrandt database. Moreover, the glioblastoma database of the Cancer Genome Atlas network (TCGA was used for survival data of glioblastoma patients. For in vitro studies, increases in ceramide levels were achieved either by ASM overexpression or by the GCS inhibitor DL-threo-1-phenyl-2-palmitoylamino-3-morpholino-1-propanol (PPMP in human glioma cell lines. Combinations of alkylating chemotherapy or irradiation and ASM overexpression, PPMP or exogenous ceramide were applied in parental cells. The anti-glioma effects were investigated by assessing proliferation, metabolic activity, viability and clonogenicity. Finally, viability and clonogenicity were assessed in temozolomide (TMZ-resistant cells upon treatment with PPMP, exogenous ceramide, alkylating chemotherapy, irradiation or their combinations.Interrogations from the Rembrandt and TCGA database showed a better survival of glioblastoma patients with low expression of ASM or GCS. ASM overexpression or PPMP treatment alone led to ceramide accumulation but did not enhance the anti-glioma activity of alkylating chemotherapy or irradiation. PPMP or exogenous ceramide induced acute cytotoxicity in glioblastoma cells. Combined treatments with chemotherapy or irradiation led to additive, but not synergistic effects. Finally, no synergy was found when TMZ-resistant cells were treated with exogenous ceramide or PPMP alone or in combination with TMZ or irradiation.Modulation of intrinsic glioma cell ceramide levels by ASM overexpression or GCS inhibition does not enhance the anti-glioma activity of

  5. Autophagy involved in resveratrol increased radiosensitivity in glioma stem cells

    Long Linmei; Zhang Qingqing; Yang Neng; Ji Wenjun; Song Yunzhen; Zhao Jianghu; Liang Zhongqin

    2012-01-01

    Objective: To investigate the effect of Resveratrol combined with X-ray on radiosensitivity in glioma stem cells. Methods: The proliferation inhibition of glioma stem cells induced by X-rays and Resveratrol was assessed with MTT assay. The activation of proapoptotic effect was characterized by Hoechst 33258 stain. MDC stain and Western blot analysis were used to analyze the autophagy mechanism in X-rays-induced death of glioma stem cells. Results: MTT assay indicated that X-rays and Resveratrol decreased the viability of glioma stem cells (P<0.05); we found the proliferative inhibition of glioma stem cells was declined when we used 3-MA to inhibit autophagy(P<0.05). When the cells were treated by the Resveratrol and x-rays, their spherical shape were changed. Apoptosis was induced in glioma stem cells by combined X-rays and Resveratrol as detected by Hoechst 33258 staining. In addition, autophagy was induced in glioma stem cells in the combined treatment group as detected by MDC staining. Western blotting showed that Bcl-2 expression was decreased. in the combined treatment group (P<0.01), and the LC3-Ⅱ expression was increased in the combined treatment group (P<0.01). Conclusion: Resveratrol can increased the radiation sensitivity of glioma stem cells, the apoptosis and autophagy was induced in the glioma stem cells in the combined treatment X-rays and Resveratrol. Our results suggest that autophagy plays an essential role in the regulation of radiosensitization of glioma stem cells. (authors)

  6. MicroRNA-195 inhibits the proliferation of human glioma cells by directly targeting cyclin D1 and cyclin E1.

    Wang Hui

    Full Text Available Glioma proliferation is a multistep process during which a sequence of genetic and epigenetic alterations randomly occur to affect the genes controlling cell proliferation, cell death and genetic stability. microRNAs are emerging as important epigenetic modulators of multiple target genes, leading to abnormal cellular signaling involving cellular proliferation in cancers.In the present study, we found that expression of miR-195 was markedly downregulated in glioma cell lines and human primary glioma tissues, compared to normal human astrocytes and matched non-tumor associated tissues. Upregulation of miR-195 dramatically reduced the proliferation of glioma cells. Flow cytometry analysis showed that ectopic expression of miR-195 significantly decreased the percentage of S phase cells and increased the percentage of G1/G0 phase cells. Overexpression of miR-195 dramatically reduced the anchorage-independent growth ability of glioma cells. Furthermore, overexpression of miR-195 downregulated the levels of phosphorylated retinoblastoma (pRb and proliferating cell nuclear antigen (PCNA in glioma cells. Conversely, inhibition of miR-195 promoted cell proliferation, increased the percentage of S phase cells, reduced the percentage of G1/G0 phase cells, enhanced anchorage-independent growth ability, upregulated the phosphorylation of pRb and PCNA in glioma cells. Moreover, we show that miR-195 inhibited glioma cell proliferation by downregulating expression of cyclin D1 and cyclin E1, via directly targeting the 3'-untranslated regions (3'-UTR of cyclin D1 and cyclin E1 mRNA. Taken together, our results suggest that miR-195 plays an important role to inhibit the proliferation of glioma cells, and present a novel mechanism for direct miRNA-mediated suppression of cyclin D1 and cyclin E1 in glioma.

  7. Nimotuzumab enhances temozolomide?induced growth suppression of glioma cells expressing mutant EGFR in vivo

    Nitta, Yusuke; Shimizu, Saki; Shishido?Hara, Yukiko; Suzuki, Kaori; Shiokawa, Yoshiaki; Nagane, Motoo

    2016-01-01

    Abstract A mutant form of epidermal growth factor receptor (EGFR), EGFRvIII, is common in glioblastoma (GBM) and confers enhanced tumorigenic activity and drug resistance. Nimotuzumab, an anti?EGFR antibody, has shown preclinical and clinical activity to GBM, but its specific activity against EGFRvIII has not been fully investigated. Human glioma U87MG or LNZ308 cells overexpressing either wild?type (wt) EGFR or EGFRvIII were treated with nimotuzumab, temozolomide, or both. Expression and pho...

  8. Identification of molecular pathways facilitating glioma cell invasion in situ.

    Ido Nevo

    Full Text Available Gliomas are mostly incurable secondary to their diffuse infiltrative nature. Thus, specific therapeutic targeting of invasive glioma cells is an attractive concept. As cells exit the tumor mass and infiltrate brain parenchyma, they closely interact with a changing micro-environmental landscape that sustains tumor cell invasion. In this study, we used a unique microarray profiling approach on a human glioma stem cell (GSC xenograft model to explore gene expression changes in situ in Invading Glioma Cells (IGCs compared to tumor core, as well as changes in host cells residing within the infiltrated microenvironment relative to the unaffected cortex. IGCs were found to have reduced expression of genes within the extracellular matrix compartment, and genes involved in cell adhesion, cell polarity and epithelial to mesenchymal transition (EMT processes. The infiltrated microenvironment showed activation of wound repair and tissue remodeling networks. We confirmed by protein analysis the downregulation of EMT and polarity related genes such as CD44 and PARD3 in IGCs, and EFNB3, a tissue-remodeling agent enriched at the infiltrated microenvironment. OLIG2, a proliferation regulator and glioma progenitor cell marker upregulated in IGCs was found to function in enhancing migration and stemness of GSCs. Overall, our results unveiled a more comprehensive picture of the complex and dynamic cell autonomous and tumor-host interactive pathways of glioma invasion than has been previously demonstrated. This suggests targeting of multiple pathways at the junction of invading tumor and microenvironment as a viable option for glioma therapy.

  9. Molecular and Genetic Determinants of Glioma Cell Invasion

    Kenta Masui

    2017-12-01

    Full Text Available A diffusely invasive nature is a major obstacle in treating a malignant brain tumor, “diffuse glioma”, which prevents neurooncologists from surgically removing the tumor cells even in combination with chemotherapy and radiation. Recently updated classification of diffuse gliomas based on distinct genetic and epigenetic features has culminated in a multilayered diagnostic approach to combine histologic phenotypes and molecular genotypes in an integrated diagnosis. However, it is still a work in progress to decipher how the genetic aberrations contribute to the aggressive nature of gliomas including their highly invasive capacity. Here we depict a set of recent discoveries involving molecular genetic determinants of the infiltrating nature of glioma cells, especially focusing on genetic mutations in receptor tyrosine kinase pathways and metabolic reprogramming downstream of common cancer mutations. The specific biology of glioma cell invasion provides an opportunity to explore the genotype-phenotype correlation in cancer and develop novel glioma-specific therapeutic strategies for this devastating disease.

  10. Effect of inhibition of the ROCK isoform on RT2 malignant glioma cells.

    Inaba, Nobuharu; Ishizawa, Sho; Kimura, Masaki; Fujioka, Kouki; Watanabe, Michiko; Shibasaki, Toshiaki; Manome, Yoshinobu

    2010-09-01

    Malignant glioma is one of the most intractable diseases in the human body. Rho-kinase (ROCK) is overexpressed and has been proposed as the main cause for the refractoriness of the disease. Since efficacious treatment is required, this study investigated the effect of inhibition of ROCK isoforms. The short hairpin RNA transcription vector was transfected into the RT2 rat glioma cell line and the characteristics of the cells were investigated. The effect of nimustine hydrochloride (ACNU) anti-neoplastic agent on cells was also measured. Inhibition of ROCK isoforms did not alter cell growth. Cell cycle analysis revealed that ROCK1 down-regulation reduced the G(0) phase population and ROCK2 down-regulation reduced the G(2)/M phase population. When ROCK1-down-regulated cells were exposed to ACNU, they demonstrated susceptibility to the agent. The roles of ROCK1 and ROCK2 may be different in glioma cells. Furthermore, the combination of ROCK1 down-regulation and an anti-neoplastic agent may be useful for the therapy of malignant glioma.

  11. MicroRNA-145 is downregulated in glial tumors and regulates glioma cell migration by targeting connective tissue growth factor.

    Lee, Hae Kyung; Bier, Ariel; Cazacu, Simona; Finniss, Susan; Xiang, Cunli; Twito, Hodaya; Poisson, Laila M; Mikkelsen, Tom; Slavin, Shimon; Jacoby, Elad; Yalon, Michal; Toren, Amos; Rempel, Sandra A; Brodie, Chaya

    2013-01-01

    Glioblastomas (GBM), the most common and aggressive type of malignant glioma, are characterized by increased invasion into the surrounding brain tissues. Despite intensive therapeutic strategies, the median survival of GBM patients has remained dismal over the last decades. In this study we examined the expression of miR-145 in glial tumors and its function in glioma cells. Using TCGA analysis and real-time PCR we found that the expression of miR-145/143 cluster was downregulated in astrocytic tumors compared to normal brain specimens and in glioma cells and glioma stem cells (GSCs) compared to normal astrocytes and neural stem cells. Moreover, the low expression of both miR-145 and miR-143 in GBM was correlated with poor patient prognosis. Transfection of glioma cells with miR-145 mimic or transduction with a lentivirus vector expressing pre-miR 145 significantly decreased the migration and invasion of glioma cells. We identified connective tissue growth factor (CTGF) as a novel target of miR-145 in glioma cells; transfection of the cells with this miRNA decreased the expression of CTGF as determined by Western blot analysis and the expression of its 3'-UTR fused to luciferase. Overexpression of a CTGF plasmid lacking the 3'-UTR and administration of recombinant CTGF protein abrogated the inhibitory effect of miR-145 on glioma cell migration. Similarly, we found that silencing of CTGF decreased the migration of glioma cells. CTGF silencing also decreased the expression of SPARC, phospho-FAK and FAK and overexpression of SPARC abrogated the inhibitory effect of CTGF silencing on cell migration. These results demonstrate that miR-145 is downregulated in glial tumors and its low expression in GBM predicts poor patient prognosis. In addition miR-145 regulates glioma cell migration by targeting CTGF which downregulates SPARC expression. Therefore, miR-145 is an attractive therapeutic target for anti-invasive treatment of astrocytic tumors.

  12. MicroRNA-145 is downregulated in glial tumors and regulates glioma cell migration by targeting connective tissue growth factor.

    Hae Kyung Lee

    Full Text Available Glioblastomas (GBM, the most common and aggressive type of malignant glioma, are characterized by increased invasion into the surrounding brain tissues. Despite intensive therapeutic strategies, the median survival of GBM patients has remained dismal over the last decades. In this study we examined the expression of miR-145 in glial tumors and its function in glioma cells. Using TCGA analysis and real-time PCR we found that the expression of miR-145/143 cluster was downregulated in astrocytic tumors compared to normal brain specimens and in glioma cells and glioma stem cells (GSCs compared to normal astrocytes and neural stem cells. Moreover, the low expression of both miR-145 and miR-143 in GBM was correlated with poor patient prognosis. Transfection of glioma cells with miR-145 mimic or transduction with a lentivirus vector expressing pre-miR 145 significantly decreased the migration and invasion of glioma cells. We identified connective tissue growth factor (CTGF as a novel target of miR-145 in glioma cells; transfection of the cells with this miRNA decreased the expression of CTGF as determined by Western blot analysis and the expression of its 3'-UTR fused to luciferase. Overexpression of a CTGF plasmid lacking the 3'-UTR and administration of recombinant CTGF protein abrogated the inhibitory effect of miR-145 on glioma cell migration. Similarly, we found that silencing of CTGF decreased the migration of glioma cells. CTGF silencing also decreased the expression of SPARC, phospho-FAK and FAK and overexpression of SPARC abrogated the inhibitory effect of CTGF silencing on cell migration. These results demonstrate that miR-145 is downregulated in glial tumors and its low expression in GBM predicts poor patient prognosis. In addition miR-145 regulates glioma cell migration by targeting CTGF which downregulates SPARC expression. Therefore, miR-145 is an attractive therapeutic target for anti-invasive treatment of astrocytic tumors.

  13. Gene expression of manganese superoxide dismutase in human glioma cells

    Novi S. Hardiany

    2010-02-01

    Full Text Available Aim This study analyze the MnSOD gene expression as endogenous antioxidant in human glioma cells compared with leucocyte cells as control.Methods MnSOD gene expression of 20 glioma patients was analyzed by measuring the relative expression of mRNA and enzyme activity of MnSOD in brain and leucocyte cells. The relative expression of mRNA MnSOD was determined by using quantitative Real Time RT-PCR and the enzyme activity of MnSOD using biochemical kit assay (xantine oxidase inhibition. Statistic analysis for mRNA and enzyme activity of MnSOD was performed using Kruskal Wallis test.Results mRNA of MnSOD in glioma cells of 70% sample was 0.015–0.627 lower, 10% was 1.002-1.059 and 20% was 1.409-6.915 higher than in leucocyte cells. Also the specific activity of MnSOD enzyme in glioma cells of 80% sample showed 0,064-0,506 lower and 20% sample was 1.249-2.718 higher than in leucocyte cells.Conclusion MnSOD gene expression in human glioma cells are significantly lower than its expression in leucocytes cells. (Med J Indones 2010; 19:21-5Keywords : MnSOD, glioma, gene expression

  14. LncRNA TUG1 acts as a tumor suppressor in human glioma by promoting cell apoptosis.

    Li, Jun; Zhang, Meng; An, Gang; Ma, Qingfang

    2016-03-01

    Previous studies have revealed multiple functional roles of long non-coding RNA taurine upregulated gene 1 in different types of malignant tumors, except for human glioma. Here, it was designed to study the potential function of taurine upregulated gene 1 in glioma pathogenesis focusing on its regulation on cell apoptosis. The expression of taurine upregulated gene 1 in glioma tissues was detected by quantitative RT-PCR and compared with that in adjacent normal tissues. Further correlation analysis was conducted to show the relationship between taurine upregulated gene 1 expression and different clinicopathologic parameters. Functional studies were performed to investigate the influence of taurine upregulated gene 1 on apoptosis and cell proliferation by using Annexin V/PI staining and cell counting kit-8 assays, respectively. And, caspase activation and Bcl-2 expression were analyzed to explore taurine upregulated gene 1-induced mechanism. taurine upregulated gene 1 expression was significantly inhibited in glioma and showed significant correlation with WHO Grade, tumor size and overall survival. Further experiments revealed that the dysregulation of taurine upregulated gene 1 affected the apoptosis and cell proliferation of glioma cells. Moreover, taurine upregulated gene 1 could induce the activation of caspase-3 and-9, with inhibited expression of Bcl-2, implying the mechanism in taurine upregulated gene 1-induced apoptosis. taurine upregulated gene 1 promoted cell apoptosis of glioma cells by activating caspase-3 and -9-mediated intrinsic pathways and inhibiting Bcl-2-mediated anti-apoptotic pathways, acting as a tumor suppressor in human glioma. This study provided new insights for the function of taurine upregulated gene 1 in cancer biology, and suggested a potent application of taurine upregulated gene 1 overexpression for glioma therapy. © 2016 by the Society for Experimental Biology and Medicine.

  15. Bex2 regulates cell proliferation and apoptosis in malignant glioma cells via the c-Jun NH2-terminal kinase pathway

    Zhou, Xiuping; Meng, Qingming; Xu, Xuebin; Zhi, Tongle; Shi, Qiong; Wang, Yong; Yu, Rutong

    2012-01-01

    Highlights: ► The expression levels of Bex2 markedly increased in glioma tissues. ► Bex2 over-expression promoted cell proliferation, while its down-regulation inhibited cell growth. ► Bex2 down-regulation promoted cell apoptosis via JNK/c-Jun signaling pathway. -- Abstract: The function of Bex2, a member of the Brain Expressed X-linked gene family, in glioma is controversial and its mechanism is largely unknown. We report here that Bex2 regulates cell proliferation and apoptosis in malignant glioma cells via the c-Jun NH2-terminal kinase (JNK) pathway. The expression level of Bex2 is markedly increased in glioma tissues. We observed that Bex2 over-expression promotes cell proliferation, while down-regulation of Bex2 inhibits cell growth. Furthermore, Bex2 down-regulation promotes cell apoptosis and activates the JNK pathway; these effects were abolished by administration of the JNK specific inhibitor, (SP600125). Thus, Bex2 may be an important player during the development of glioma.

  16. Exploring the regulatory role of isocitrate dehydrogenase mutant protein on glioma stem cell proliferation.

    Lu, H-C; Ma, J; Zhuang, Z; Qiu, F; Cheng, H-L; Shi, J-X

    2016-08-01

    Glioma is the most lethal form of cancer that originates mostly from the brain and less frequently from the spine. Glioma is characterized by abnormal regulation of glial cell differentiation. The severity of the glioma was found to be relaxed in isocitrate dehydrogenase 1 (IDH1) mutant. The present study focused on histological discrimination and regulation of cancer stem cell between IDH1 mutant and in non-IDH1 mutant glioma tissue. Histology, immunohistochemistry and Western blotting techniques are used to analyze the glioma nature and variation in glioma stem cells that differ between IDH1 mutant and in non-IDH1 mutant glioma tissue. The aggressive form of non-IDH1 mutant glioma shows abnormal cellular histological variation with prominent larger nucleus along with abnormal clustering of cells. The longer survival form of IDH1 mutant glioma has a control over glioma stem cell proliferation. Immunohistochemistry with stem cell markers, CD133 and EGFRvIII are used to demonstrate that the IDH1 mutant glioma shows limited dependence on cancer stem cells and it shows marked apoptotic signals in TUNEL assay to regulate abnormal cells. The non-IDH1 mutant glioma failed to regulate misbehaving cells and it promotes cancer stem cell proliferation. Our finding supports that the IDH1 mutant glioma has a regulatory role in glioma stem cells and their survival.

  17. Radiation effects on human glia and glioma cells in vitro

    Nilsson, S.

    1983-01-01

    The radiosensitivity of human glia and glioma cells has been studied in vitro, and a new cloning method has been developed to overcome the difficulties due to the very low cloning efficiency of these cells. The cells were confined to small palladium areas surrounded by agarose, which increased the cell density, but kept the clones separated. Using this method, the glia cells were found to be very sensitive to gamma irradiation (D 0 =1.0-1.5 Gy and n=1) in comparision with the glioma cells (D 0 =1.5-2.5 Gy and n=3.5). The induction and repair of DNA strand breaks were studied with two DNA unwinding techniques. No differences between the two cell-lines were detected when induction and fast repair were studied with the single-labelling method, while the glioma cells showed less unrepaired DNA strand breaks than the glia cells after 1, 2 and 3 hours, when the double-labelling method was used. Detachment, attachment and growth kinetics were studied using the palladium-agarose cloning method. All of the glioma cell-lines studied, detached and attached themselves at rates higher than the normal diploid glia cell-lines. All of the cell-lines contained clones with different properties. Some clones were rapidly growing, others maintained a nearly constant number of cells or even decreased. The effects of chronic hypoxia were tested in a few experiments. Low oxygen tension in the culture medium reduced the rate of growth and the DNA synthesis of the glioma cells. The present study indicates that cultured human glioma cells are less radiosensitive than cultured glia cells. The palladium-agarose technique, enable studying growth kinetics detachment, attachment and radiosensitivity in a quantitative manner for cells with low cloning efficiency. (author)

  18. Metabolic remodeling precedes mitochondrial outer membrane permeabilization in human glioma xenograft cells.

    Ponnala, Shivani; Chetty, Chandramu; Veeravalli, Krishna Kumar; Dinh, Dzung H; Klopfenstein, Jeffrey D; Rao, Jasti S

    2012-02-01

    Glioma cancer cells adapt to changing microenvironment and shift from mitochondrial oxidative phosphorylation to aerobic glycolysis for their metabolic needs irrespective of oxygen availability. In the present study, we show that silencing MMP-9 in combination with uPAR/cathepsin B switch the glycolytic metabolism of glioma cells to oxidative phosphorylation (OXPHOS) and generate reactive oxygen species (ROS) to predispose glioma cells to mitochondrial outer membrane permeabilization. shRNA for MMP-9 and uPAR (pMU) as well as shRNA for MMP-9 and cathepsin B (pMC) activated complexes of mitochondria involved in OXPHOS and inhibited glycolytic hexokinase expression. The decreased interaction of hexokinase 2 with mitochondria in the treated cells indicated the inhibition of glycolysis activation. Overexpression of Akt reversed the pMU- and pMC-mediated OXPHOS to glycolysis switch. The OXPHOS un-coupler oligomycin A altered the expression levels of the Bcl-2 family of proteins; treatment with pMU or pMC reversed this effect and induced mitochondrial outer membrane permeabilization. In addition, our results show changes in mitochondrial pore transition to release cytochrome c due to changes in the VDAC-Bcl-XL and BAX-BAK interaction with pMU and pMC treatments. Taken together, our results suggest that pMU and pMC treatments switch glioma cells from the glycolytic to the OXPHOS pathway through an inhibitory effect on Akt, ROS induction and an increase of cytosolic cytochrome c accumulation. These results demonstrate the potential of pMU and pMC as therapeutic candidates for the treatment of glioma.

  19. Undersulfation of proteoglycans and proteins alter C6 glioma cells proliferation, adhesion and extracellular matrix organization.

    Mendes de Aguiar, Claudia B N; Garcez, Ricardo Castilho; Alvarez-Silva, Marcio; Trentin, Andréa Gonçalves

    2002-11-01

    Proteoglycans are considered to be important molecule in cell-microenvironment interactions. They are overexpressed in neoplastic cells modifying their growth and migration in hosts. In this work we verified that undersulfation of proteoglycans and other sulfated molecules, induced by sodium chlorate treatment, inhibited C6 glioma cells proliferation in a dose-dependent way. This effect was restored by the addition of exogenous heparin. We could not detect significant cell mortality in our culture condition. The treatment also impaired in a dose-dependent manner, C6 cell adhesion to extracellular matrix (ECM) proteins (collagen IV, laminin and fibronectin). In addition, sodium chlorate treatment altered C6 glioma cell morphology, from the fibroblast-like to a more rounded one. This effect was accompanied by increased synthesis of fibronectin and alterations in its extracellular network organization. However, we could not observe modifications on laminin organization and synthesis. The results suggest an important connection between sulfation degree with important tumor functions, such as proliferation and adhesion. We suggest that proteoglycans may modulate the glioma microenvironment network during tumor cell progression and invasion.

  20. FK506 Binding Protein Mediates Glioma Cell Growth and Sensitivity to Rapamycin Treatment by Regulating NF-κB Signaling Pathway

    Wei Jiang

    2008-03-01

    Full Text Available FK506 binding protein 5 (FKBP5 belongs to a family of immunophilins named for their ability to bind immunosuppressive drugs, also known as peptidyl-prolyl cis-trans isomerases, and also with chaperones to help protein folding. Using glioma cDNA microarray analysis, we found that FKBP5 was overexpressed in glioma tumors. This finding was further validated by real-time reverse transcription-polymerase chain reaction and Western blot analysis. The roles of FKBP5 in glioma cells were then examined. We found that cell growth was suppressed after FKBP5 expression was inhibited by short interfering RNA transfection and enhanced by FKBP5 overexpression. Electrophoretic mobility shift assay showed that nuclear factor-kappa B (NF-κB and DNA binding was enhanced by FKBP5 overexpression. The expression level of I-kappa B alpha and phosphorylated NF-κB was regulated by the expression of FKBP5. These data suggest that FKBP5 is involved in NF-κB pathway activation in glioma cells. In addition, FKBP5 overexpression in rapamycin-sensitive U87 cells blocked the cells' response to rapamycin treatment, whereas rapamycin-resistant glioma cells, both PTEN-positive and -negative, were synergistically sensitive to rapamycin after FKBP5 was knocked down, suggesting that the FKBP5 regulates glioma cell response to rapamycin treatment. In conclusion, our study demonstrates that FKBP5 plays an important role in glioma growth and chemoresistance through regulating signal transduction of the NF-κB pathway.

  1. Forkhead box K2 inhibits the proliferation, migration, and invasion of human glioma cells and predicts a favorable prognosis.

    Wang, Bo; Zhang, XueBin; Wang, Wei; Zhu, ZhiZhong; Tang, Fan; Wang, Dong; Liu, Xi; Zhuang, Hao; Yan, XiaoLing

    2018-01-01

    Forkhead box K2 (FOXK2) is a member of the forkhead box family of transcription factors. Recently, researchers discovered that overexpression of FOXK2 inhibits the proliferation and metastasis of breast cancer, non-small cell lung cancer, and colorectal cancer, and is related to the clinical prognosis. However, in hepatocellular carcinoma, FOXK2 results in the opposite phenotypes. Currently, the contribution of FOXK2 to glioma pathogenesis is not clear. We evaluated the expression of FOXK2 in 151 glioma patients using immunohistochemistry assays. The associations among the expression of FOXK2, clinicopathological parameters, and the prognosis of glioma patients were statistically analyzed. We downregulated and upregulated the level of FOXK2 in glioma cells by transfections with small interfering RNA and plasmids. Then, we investigated the effects on tumor cell behavior in vitro by Cell Counting Kit-8 assays, colony-formation assay, transwell assay, and the epithelial-to-mesenchymal transition (EMT) biomarker levels. The clinical data showed that expression of FOXK2 gradually decreased with increasing World Health Organization (WHO) grades and a low level of FOXK2 indicates a poor prognosis. FOXK2 expression is negatively correlated with Ki67 expression and the WHO degree but is not correlated with other clinicopathological parameters, including sex, age, Karnofsky Performance Status, tumor diameter, O -6-methylguanine-DNA methyltransferase, and glutathione S -transferase pi. FOXK2 knockdown enhances glioma cell proliferation, migration, invasion, and EMT process, and, in contrast, FOXK2 overexpression inhibits glioma cell proliferation, migration, invasion, and the EMT process. Expression of FOXK2 gradually decreases with increasing WHO grades. FOXK2 inhibits tumor proliferation, migration, and invasion. FOXK2 is a critical mediator of the EMT process.

  2. Targeting the erythropoietin receptor on glioma cells reduces tumour growth

    Peres, Elodie A.; Valable, Samuel; Guillamo, Jean-Sebastien; Marteau, Lena; Bernaudin, Jean-Francois; Roussel, Simon; Lechapt-Zalcman, Emmanuele; Bernaudin, Myriam; Petit, Edwige

    2011-01-01

    Hypoxia has been shown to be one of the major events involved in EPO expression. Accordingly, EPO might be expressed by cerebral neoplastic cells, especially in glioblastoma, known to be highly hypoxic tumours. The expression of EPOR has been described in glioma cells. However, data from the literature remain descriptive and controversial. On the basis of an endogenous source of EPO in the brain, we have focused on a potential role of EPOR in brain tumour growth. In the present study, with complementary approaches to target EPO/EPOR signalling, we demonstrate the presence of a functional EPO/EPOR system on glioma cells leading to the activation of the ERK pathway. This EPO/EPOR system is involved in glioma cell proliferation in vitro. In vivo, we show that the down-regulation of EPOR expression on glioma cells reduces tumour growth and enhances animal survival. Our results support the hypothesis that EPOR signalling in tumour cells is involved in the control of glioma growth.

  3. ALDH1A3: A Marker of Mesenchymal Phenotype in Gliomas Associated with Cell Invasion.

    Wenlong Zhang

    Full Text Available Aldehyde dehydrogenases (ALDH is a family of enzymes including 19 members. For now, ALDH activity had been wildly used as a marker of cancer stem cells (CSCs. But biological functions of relevant isoforms and their clinical applications are still controversial. Here, we investigate the clinical significance and potential function of ALDH1A3 in gliomas. By whole-genome transcriptome microarray and mRNA sequencing analysis, we compared the expression of ALDH1A3 in high- and low- grade gliomas as well as different molecular subtypes. Microarray analysis was performed to identify the correlated genes of ALDH1A3. We further used Gene Ontology (GO and Kyoto Encyclopedia of Genes and Genomes (KEGG pathways analysis to explore the biological function of ALDH1A3. Finally, by mRNA knockdown we revealed the relationship between ALDH1A3 and the ability of tumor invasion. ALDH1A3 overexpression was significantly associated with high grade as well as the higher mortality of gliomas in survival analysis. ALDH1A3 was characteristically highly expressed in Mesenchymal (Mes subtype gliomas. Moreover, we found that ALDH1A3 was most relevant to extracellular matrix organization and cell adhesion biological process, and the ability of tumor invasion was suppressed after ALDH1A3 knockdown in vitro. In conclusion, ALDH1A3 can serve as a novel marker of Mes phenotype in gliomas with potential clinical prognostic value. The expression of ALDH1A3 is associated with tumor cell invasion.

  4. PAR1 inhibition suppresses the self-renewal and growth of A2B5-defined glioma progenitor cells and their derived gliomas in vivo

    Auvergne, R.; Wu, C.; Connell, A.

    2016-01-01

    Glioblastoma (GBM) remains the most common and lethal intracranial tumor. In a comparison of gene expression by A2B5-defined tumor-initiating progenitor cells (TPCs) to glial progenitor cells derived from normal adult human brain, we found that the F2R gene encoding PAR1 was differentially...... overexpressed by A2B5-sorted TPCs isolated from gliomas at all stages of malignant development. In this study, we asked if PAR1 is causally associated with glioma progression. Lentiviral knockdown of PAR1 inhibited the expansion and self-renewal of human GBM-derived A2B5(+) TPCs in vitro, while pharmacological...

  5. Cyclic hexapeptide-conjugated nanoparticles enhance curcumin delivery to glioma tumor cells and tissue

    Zhang X

    2017-08-01

    Full Text Available Xuemei Zhang,1–3 Xuejuan Li,1,4 Hongchen Hua,1 Aiping Wang,1 Wanhui Liu,1–3 Youxin Li,1–3 Fenghua Fu,1–3 Yanan Shi,5 Kaoxiang Sun1 1School of Pharmacy, Yantai University, Yantai, Shandong Province, People’s Republic of China; 2State Key Laboratory of Long-acting and Targeting Drug Delivery System, Yantai, Shandong Province, People’s Republic of China; 3Luye Pharmaceutical Co., Ltd., Shandong Province, People’s Republic of China; 4National Engineering and Technology Research Center of Chirality Pharmaceutical, Lunan Pharmaceutical Group Co., Ltd., Shandong Province, People’s Republic of China; 5School of Pharmacy, Binzhou Medical University, Shandong Province, People’s Republic of China Abstract: Glioma has one of the highest mortality rates among primary brain tumors. The clinical treatment for glioma is very difficult due to its infiltration and specific growth locations. To achieve improved drug delivery to a brain tumor, we report the preparation and in vitro and in vivo evaluation of curcumin nanoparticles (Cur-NPs. The cyclic hexapeptide c(RGDf(N-meVK-C (cHP has increased affinity for cells that overexpress integrins and was designed to target Cur-NPs to tumors. Functional polyethyleneglycol-modified poly(D,L-lactide-co-glycolide (PEG-PLGA conjugated to cHP was synthesized, and targeted Cur-NPs were prepared using a self-assembly nanoprecipitation process. The physicochemical properties and the in vitro cytotoxicity, accuracy, and penetration capabilities of Cur-NPs targeting cells with high levels of integrin expression were investigated. The in vivo targeting and penetration capabilities of the NPs were also evaluated against glioma in rats using in vivo imaging equipment. The results showed that the in vitro cytotoxicity of the targeted cHP-modified curcumin nanoparticles (cHP/Cur-NPs was higher than that of either free curcumin or non-targeted Cur-NPs due to the superior ability of the cHP/Cur-NPs to target tumor cells

  6. Senescence from glioma stem cell differentiation promotes tumor growth

    Ouchi, Rie; Okabe, Sachiko; Migita, Toshiro; Nakano, Ichiro; Seimiya, Hiroyuki

    2016-01-01

    Glioblastoma (GBM) is a lethal brain tumor composed of heterogeneous cellular populations including glioma stem cells (GSCs) and differentiated non-stem glioma cells (NSGCs). While GSCs are involved in tumor initiation and propagation, NSGCs' role remains elusive. Here, we demonstrate that NSGCs undergo senescence and secrete pro-angiogenic proteins, boosting the GSC-derived tumor formation in vivo. We used a GSC model that maintains stemness in neurospheres, but loses the stemness and differentiates into NSGCs upon serum stimulation. These NSGCs downregulated telomerase, shortened telomeres, and eventually became senescent. The senescent NSGCs released pro-angiogenic proteins, including vascular endothelial growth factors and senescence-associated interleukins, such as IL-6 and IL-8. Conditioned medium from senescent NSGCs promoted proliferation of brain microvascular endothelial cells, and mixed implantation of GSCs and senescent NSGCs into mice enhanced the tumorigenic potential of GSCs. The senescent NSGCs seem to be clinically relevant, because both clinical samples and xenografts of GBM contained tumor cells that expressed the senescence markers. Our data suggest that senescent NSGCs promote malignant progression of GBM in part via paracrine effects of the secreted proteins. - Highlights: • Non-stem glioma cells (NSGCs) lose telomerase and eventually become senescent. • Senescent NSGCs secrete pro-angiogenic proteins, such as VEGFs, IL-6, and IL-8. • Senescent NSGCs enhance the growth of brain microvascular endothelial cells. • Senescent NSGCs enhance the tumorigenic potential of glioma stem cells in vivo.

  7. Senescence from glioma stem cell differentiation promotes tumor growth

    Ouchi, Rie [Division of Molecular Biotherapy, Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, 3-8-31 Ariake, Koto-ku, Tokyo 135-8550 (Japan); Laboratory of Molecular Target Therapy of Cancer, Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo, 3-8-31 Ariake, Koto-ku, Tokyo 135-8550 (Japan); Okabe, Sachiko; Migita, Toshiro [Division of Molecular Biotherapy, Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, 3-8-31 Ariake, Koto-ku, Tokyo 135-8550 (Japan); Nakano, Ichiro [Department of Neurosurgery, Comprehensive Cancer Center, University of Alabama at Birmingham, 1824 6th Avenue South, Birmingham, AL 35233 (United States); Seimiya, Hiroyuki, E-mail: hseimiya@jfcr.or.jp [Division of Molecular Biotherapy, Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, 3-8-31 Ariake, Koto-ku, Tokyo 135-8550 (Japan); Laboratory of Molecular Target Therapy of Cancer, Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo, 3-8-31 Ariake, Koto-ku, Tokyo 135-8550 (Japan)

    2016-02-05

    Glioblastoma (GBM) is a lethal brain tumor composed of heterogeneous cellular populations including glioma stem cells (GSCs) and differentiated non-stem glioma cells (NSGCs). While GSCs are involved in tumor initiation and propagation, NSGCs' role remains elusive. Here, we demonstrate that NSGCs undergo senescence and secrete pro-angiogenic proteins, boosting the GSC-derived tumor formation in vivo. We used a GSC model that maintains stemness in neurospheres, but loses the stemness and differentiates into NSGCs upon serum stimulation. These NSGCs downregulated telomerase, shortened telomeres, and eventually became senescent. The senescent NSGCs released pro-angiogenic proteins, including vascular endothelial growth factors and senescence-associated interleukins, such as IL-6 and IL-8. Conditioned medium from senescent NSGCs promoted proliferation of brain microvascular endothelial cells, and mixed implantation of GSCs and senescent NSGCs into mice enhanced the tumorigenic potential of GSCs. The senescent NSGCs seem to be clinically relevant, because both clinical samples and xenografts of GBM contained tumor cells that expressed the senescence markers. Our data suggest that senescent NSGCs promote malignant progression of GBM in part via paracrine effects of the secreted proteins. - Highlights: • Non-stem glioma cells (NSGCs) lose telomerase and eventually become senescent. • Senescent NSGCs secrete pro-angiogenic proteins, such as VEGFs, IL-6, and IL-8. • Senescent NSGCs enhance the growth of brain microvascular endothelial cells. • Senescent NSGCs enhance the tumorigenic potential of glioma stem cells in vivo.

  8. Treatment Resistance Mechanisms of Malignant Glioma Tumor Stem Cells

    Schmalz, Philip G.R.; Shen, Michael J.; Park, John K.

    2011-01-01

    Malignant gliomas are highly lethal because of their resistance to conventional treatments. Recent evidence suggests that a minor subpopulation of cells with stem cell properties reside within these tumors. These tumor stem cells are more resistant to radiation and chemotherapies than their counterpart differentiated tumor cells and may underlie the persistence and recurrence of tumors following treatment. The various mechanisms by which tumor stem cells avoid or repair the damaging effects of cancer therapies are discussed

  9. A Pilot Feasibility Study of Oral 5-Fluorocytosine and Genetically-Modified Neural Stem Cells Expressing E.Coli Cytosine Deaminase for Treatment of Recurrent High Grade Gliomas

    2017-11-07

    Adult Anaplastic Astrocytoma; Recurrent Grade III Glioma; Recurrent Grade IV Glioma; Adult Anaplastic Oligodendroglioma; Adult Brain Tumor; Adult Giant Cell Glioblastoma; Adult Glioblastoma; Adult Gliosarcoma; Adult Mixed Glioma; Recurrent Adult Brain Tumor; Adult Anaplastic Oligoastrocytoma; Recurrent High Grade Glioma

  10. Pediatric glioma stem cells: biologic strategies for oncolytic HSV virotherapy

    Gregory K Friedman

    2013-02-01

    Full Text Available While glioblastoma multiforme (GBM is the most common adult malignant brain tumor, GBMs in childhood represent less than 10% of pediatric malignant brain tumors and are phenotypically and molecularly distinct from adult GBMs. Similar to adult patients, outcomes for children with high-grade gliomas (HGGs remain poor. Furthermore, the significant morbidity and mortality yielded by pediatric GBM is compounded by neurotoxicity for the developing brain caused by current therapies. Poor outcomes have been attributed to a subpopulation of chemotherapy and radiotherapy resistant cells, termed ‘glioma stem cells’ (GSCs, ‘glioma progenitor cells’, or ‘glioma-initiating cells', which have the ability to initiate and maintain the tumor and to repopulate the recurring tumor after conventional therapy. Future innovative therapies for pediatric HGGs must be able to eradicate these therapy-resistant GSCs. Oncolytic herpes simplex viruses, genetically engineered to be safe for normal cells and to express diverse foreign anti-tumor therapeutic genes, have been demonstrated in preclinical studies to infect and kill GSCs and tumor cells equally while sparing normal brain cells. In this review, we discuss the unique aspects of pediatric GSCs, including markers to identify them, the microenvironment they reside in, signaling pathways that regulate them, mechanisms of cellular resistance, and approaches to target GSCs, with a focus on the promising therapeutic, genetically engineered oncolytic herpes simplex virus (HSV.

  11. Forkhead box P3 regulates ARHGAP15 expression and affects migration of glioma cells through the Rac1 signaling pathway.

    Sun, Zhen; Zhang, Biao; Wang, Chen; Fu, Tao; Li, Lianling; Wu, Qiaoli; Cai, Ying; Wang, Jinhuan

    2017-01-01

    Forkhead box P3 (FOXP3) plays a crucial role in the development and function of regulatory T cells and was recently identified as a tumor suppressor in different cancer types. Forkhead box P3 is expressed in normal brain tissues, but is strongly downregulated or absent in glioblastomas. In order to understand the FOXP3 adjustment mechanisms in glioma cells, we performed a DNA microarray in U87 cells overexpressing FOXP3 and validated the differences using quantitative real-time PCR, Western blot analysis, and immunohistochemistry in vitro and in vivo. We found that FOXP3 can regulate the expression of ARHGAP15. Expression of FOXP3 was also correlated with ARHGAP15 in glioma samples. Overexpression of FOXP3 inhibited glioma cell migration through ARHGAP15 upregulation and Rac1 inactivation. Silencing of FOXP3 promoted migration through ARHGAP15 downregulation and Rac1 activation. ARHGAP15, a GTPase-activating protein for Rac1, inhibits small GTPase signaling in a dual negative manner. We found that there is a correlation between expression of ARHGAP15 and glioma level. The small GTPase Rac1 plays an important role in cell migration. In addition, we found that FOXP3 regulates expression of epithelial-mesenchymal transition markers E-cadherin and N-cadherin, which is important given that epithelial-mesenchymal transition is critically involved in tumor spreading and dissemination. Thus, FOXP3 or ARHGAP15 may serve as a new molecular target for antimetastatic therapies in treating glioma. © 2016 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

  12. Fenofibrate dose not protect glioma cells from irradiation

    Ro, Jae Lim; Kim, Won Dong; Park, Woo Yoon

    2012-01-01

    Fenofibrate(FF) is a ligand for peroxisome proliferator-activated receptor (PPAR) α and used clinically as a hypolipidemic drug. FF has been reported to have a radioprotective effect of newborn cells in the dentate gyrus 1) and inhibit radiation-induced microglial pro-inflammatory response 2). However, if FF also protect tumor cells, it can not be used clinically during radiotherapy. Thus, we're interested in whether FF has an radioprotective effect of brain tumor cells or not Although the radiosensitive G0/G1 phase cells were increased, radiosensitization by FF was not observed in three human glioma cells. This may be due to counterbalance of radiosensitizing and radioprotecting proteins increased by FF. Taken together, FF neither radiosensitize nor radioprotect glioma cells, so it can be used to protect normal neural cells from radiation damage

  13. Autophagy contributes to gefitinib-induced glioma cell growth inhibition

    Chang, Cheng-Yi [Department of Surgery, Fong-Yuan Hospital, Taichung 420, Taiwan (China); Graduate Institute of Pharmaceutical Science and Technology, Central Taiwan University of Science and Technology, Taichung 406, Taiwan (China); Kuan, Yu-Hsiang [Department of Pharmacology, School of Medicine, Chung Shan Medical University, Taichung 402, Taiwan (China); Department of Pharmacy, Chung Shan Medical University Hospital, Taichung 402, Taiwan (China); Ou, Yen-Chuan; Li, Jian-Ri [Division of Urology, Taichung Veterans General Hospital, Taichung 407, Taiwan (China); Wu, Chih-Cheng [Department of Anesthesiology, Taichung Veterans General Hospital, Taichung 407, Taiwan (China); Department of Financial and Computational Mathematics, Providence University, Taichung 433, Taiwan (China); Pan, Pin-Ho [Department of Pediatrics, Tungs’ Taichung MetroHarbor Hospital, Taichung 435, Taiwan (China); Chen, Wen-Ying [Department of Veterinary Medicine, National Chung Hsing University, Taichung 402, Taiwan (China); Huang, Hsuan-Yi [Department of Surgery, Fong-Yuan Hospital, Taichung 420, Taiwan (China); Chen, Chun-Jung, E-mail: cjchen@vghtc.gov.tw [Department of Medical Research, Taichung Veterans General Hospital, Taichung 407, Taiwan (China); Institute of Biomedical Sciences, National Chung Hsing University, Taichung 402, Taiwan (China); Rong Hsing Research Center for Translational Medicine, National Chung Hsing University, Taichung 402, Taiwan (China); Center for General Education, Tunghai University, Taichung 407, Taiwan (China); Department of Nursing, HungKuang University, Taichung 433, Taiwan (China)

    2014-09-10

    Epidermal growth factor receptor tyrosine kinase inhibitors, including gefitinib, have been evaluated in patients with malignant gliomas. However, the molecular mechanisms involved in gefitinib-mediated anticancer effects against glioma are incompletely understood. In the present study, the cytostatic potential of gefitinib was demonstrated by the inhibition of glioma cell growth, long-term clonogenic survival, and xenograft tumor growth. The cytostatic consequences were accompanied by autophagy, as evidenced by monodansylcadaverine staining of acidic vesicle formation, conversion of microtubule-associated protein-1 light chain 3-II (LC3-II), degradation of p62, punctate pattern of GFP-LC3, and conversion of GFP-LC3 to cleaved-GFP. Autophagy inhibitor 3-methyladenosine and chloroquine and genetic silencing of LC3 or Beclin 1 attenuated gefitinib-induced growth inhibition. Gefitinib-induced autophagy was not accompanied by the disruption of the Akt/mammalian target of rapamycin signaling. Instead, the activation of liver kinase-B1/AMP-activated protein kinase (AMPK) signaling correlated well with the induction of autophagy and growth inhibition caused by gefitinib. Silencing of AMPK suppressed gefitinib-induced autophagy and growth inhibition. The crucial role of AMPK activation in inducing glioma autophagy and growth inhibition was further supported by the actions of AMP mimetic AICAR. Gefitinib was shown to be capable of reducing the proliferation of glioma cells, presumably by autophagic mechanisms involving AMPK activation. - Highlights: • Gefitinib causes cytotoxic and cytostatic effect on glioma. • Gefitinib induces autophagy. • Gefitinib causes cytostatic effect through autophagy. • Gefitinib induces autophagy involving AMPK.

  14. Autophagy contributes to gefitinib-induced glioma cell growth inhibition

    Chang, Cheng-Yi; Kuan, Yu-Hsiang; Ou, Yen-Chuan; Li, Jian-Ri; Wu, Chih-Cheng; Pan, Pin-Ho; Chen, Wen-Ying; Huang, Hsuan-Yi; Chen, Chun-Jung

    2014-01-01

    Epidermal growth factor receptor tyrosine kinase inhibitors, including gefitinib, have been evaluated in patients with malignant gliomas. However, the molecular mechanisms involved in gefitinib-mediated anticancer effects against glioma are incompletely understood. In the present study, the cytostatic potential of gefitinib was demonstrated by the inhibition of glioma cell growth, long-term clonogenic survival, and xenograft tumor growth. The cytostatic consequences were accompanied by autophagy, as evidenced by monodansylcadaverine staining of acidic vesicle formation, conversion of microtubule-associated protein-1 light chain 3-II (LC3-II), degradation of p62, punctate pattern of GFP-LC3, and conversion of GFP-LC3 to cleaved-GFP. Autophagy inhibitor 3-methyladenosine and chloroquine and genetic silencing of LC3 or Beclin 1 attenuated gefitinib-induced growth inhibition. Gefitinib-induced autophagy was not accompanied by the disruption of the Akt/mammalian target of rapamycin signaling. Instead, the activation of liver kinase-B1/AMP-activated protein kinase (AMPK) signaling correlated well with the induction of autophagy and growth inhibition caused by gefitinib. Silencing of AMPK suppressed gefitinib-induced autophagy and growth inhibition. The crucial role of AMPK activation in inducing glioma autophagy and growth inhibition was further supported by the actions of AMP mimetic AICAR. Gefitinib was shown to be capable of reducing the proliferation of glioma cells, presumably by autophagic mechanisms involving AMPK activation. - Highlights: • Gefitinib causes cytotoxic and cytostatic effect on glioma. • Gefitinib induces autophagy. • Gefitinib causes cytostatic effect through autophagy. • Gefitinib induces autophagy involving AMPK

  15. Gallic acid suppresses cell viability, proliferation, invasion and angiogenesis in human glioma cells

    Lu, Yong; Jiang, Feng; Jiang, Hao; Wu, Kalina; Zheng, Xuguang; Cai, Yizhong; Katakowski, Mark; Chopp, Michael; To, Shing-Shun Tony

    2010-01-01

    Gallic acid, an organic acid, also known as 3,4,5-trihydroxybenzoic acid, is cytotoxic against certain cancer cells, without harming normal cells. The objective of this study is to evaluate whether gallic acid can inhibit glioma cell viability, proliferation, invasion and reduce glioma cell mediated angiogenesis. Treatment of U87 and U251n glioma cells with gallic acid inhibited cell viability in a dose- and time-dependent manner. BrdU and tube formation assays indicated that gallic acid sign...

  16. Boron neutron capture therapy induces cell cycle arrest and cell apoptosis of glioma stem/progenitor cells in vitro

    Sun, Ting; Zhang, Zizhu; Li, Bin; Chen, Guilin; Xie, Xueshun; Wei, Yongxin; Wu, Jie; Zhou, Youxin; Du, Ziwei

    2013-01-01

    Glioma stem cells in the quiescent state are resistant to clinical radiation therapy. An almost inevitable glioma recurrence is due to the persistence of these cells. The high linear energy transfer associated with boron neutron capture therapy (BNCT) could kill quiescent and proliferative cells. The present study aimed to evaluate the effects of BNCT on glioma stem/progenitor cells in vitro. The damage induced by BNCT was assessed using cell cycle progression, apoptotic cell ratio and apoptosis-associated proteins expression. The surviving fraction and cell viability of glioma stem/progenitor cells were decreased compared with differentiated glioma cells using the same boronophenylalanine pretreatment and the same dose of neutron flux. BNCT induced cell cycle arrest in the G2/M phase and cell apoptosis via the mitochondrial pathway, with changes in the expression of associated proteins. Glioma stem/progenitor cells, which are resistant to current clinical radiotherapy, could be effectively killed by BNCT in vitro via cell cycle arrest and apoptosis using a prolonged neutron irradiation, although radiosensitivity of glioma stem/progenitor cells was decreased compared with differentiated glioma cells when using the same dose of thermal neutron exposure and boronophenylalanine pretreatment. Thus, BNCT could offer an appreciable therapeutic advantage to prevent tumor recurrence, and may become a promising treatment in recurrent glioma

  17. Astrocytes protect glioma cells from chemotherapy and upregulate survival genes via gap junctional communication.

    Lin, Qingtang; Liu, Zhao; Ling, Feng; Xu, Geng

    2016-02-01

    Gliomas are the most common type of primary brain tumor. Using current standard treatment regimens, the prognosis of patients with gliomas remains poor, which is predominantly due to the resistance of glioma cells to chemotherapy. The organ microenvironment has been implicated in the pathogenesis and survival of tumor cells. Thus, the aim of the present study was to test the hypothesis that astrocytes (the housekeeping cells of the brain microenvironment) may protect glioma cells from chemotherapy and to investigate the underlying mechanism. Immunofluorescent and scanning electron microscopy demonstrated that glioma cells were surrounded and infiltrated by activated astrocytes. In vitro co-culture of glioma cells with astrocytes significantly reduced the cytotoxic effects on glioma cells caused by various chemotherapeutic agents, as demonstrated by fluorescein isothiocyanate-propidium iodide flow cytometry. Transwell experiments indicated that this protective effect was dependent on physical contact and the gap junctional communication (GJC) between astrocytes and glioma cells. Microarray expression profiling further revealed that astrocytes upregulated the expression levels of various critical survival genes in the glioma cells via GJC. The results of the present study indicated that the organ microenvironment may affect the biological behavior of tumor cells and suggest a novel mechanism of resistance in glioma cells, which may be of therapeutic relevance clinically.

  18. Efficacy of the HSP90 inhibitor 17-AAG in human glioma cell lines and tumorigenic glioma stem cells.

    Sauvageot, Claire Marie-Elisabeth; Weatherbee, Jessica Leigh; Kesari, Santosh; Winters, Susan Elizabeth; Barnes, Jessica; Dellagatta, Jamie; Ramakrishna, Naren Raj; Stiles, Charles Dean; Kung, Andrew Li-Jen; Kieran, Mark W; Wen, Patrick Yung Chih

    2009-04-01

    Glioblastoma multiforme (GBM) arises from genetic and signaling abnormalities in components of signal transduction pathways involved in proliferation, survival, and the cell cycle axis. Studies to date with single-agent targeted molecular therapy have revealed only modest effects in attenuating the growth of these tumors, suggesting that targeting multiple aberrant pathways may be more beneficial. Heat-shock protein 90 (HSP90) is a molecular chaperone that is involved in the conformational maturation of a defined group of client proteins, many of which are deregulated in GBM. 17-allylamino-17-demethoxygeldanamycin (17-AAG) is a well-characterized HSP90 inhibitor that should be able to target many of the aberrant signal transduction pathways in GBM. We assessed the ability of 17-AAG to inhibit the growth of glioma cell lines and glioma stem cells both in vitro and in vivo and assessed its ability to synergize with radiation and/or temozolomide, the standard therapies for GBM. Our results reveal that 17-AAG is able to inhibit the growth of both human glioma cell lines and glioma stem cells in vitro and is able to target the appropriate proteins within these cells. In addition, 17-AAG can inhibit the growth of intracranial tumors and can synergize with radiation both in tissue culture and in intracranial tumors. This compound was not found to synergize with temozolomide in any of our models of gliomas. Our results suggest that HSP90 inhibitors like 17-AAG may have therapeutic potential in GBM, either as a single agent or in combination with radiation.

  19. Dipeptidyl peptidase IV in two human glioma cell lines

    A Sedo

    2009-12-01

    Full Text Available There is growing evidence that dipeptidyl peptidase IV [DPP-IV, EC 3.4.14.5] takes part in the metabolism of biologically active peptides participating in the regulation of growth and transformation of glial cells. However, the knowledge on the DPP-IV expression in human glial and glioma cells is still very limited. In this study, using histochemical and biochemical techniques, the DPP-IV activity was demonstrated in two commercially available human glioma cell lines of different transformation degree, as represented by U373 astrocytoma (Grade III and U87 glioblastoma multiforme (Grade IV lines. Higher total activity of the enzyme, as well as its preferential localisation in the plasma membrane, was observed in U87 cells. Compared to U373 population, U87 cells were morphologically more pleiomorphic, they were cycling at lower rate and expressing less Glial Fibrillary Acidic Protein. The data revealed positive correlation between the degree of transformation of cells and activity of DPP-IV. Great difference in expression of this enzyme, together with the phenotypic differences of cells, makes these lines a suitable standard model for further 57 studies of function of this enzyme in human glioma cells.

  20. The nitric oxide donor JS-K sensitizes U87 glioma cells to repetitive irradiation.

    Heckler, Max; Osterberg, Nadja; Guenzle, Jessica; Thiede-Stan, Nina Kristin; Reichardt, Wilfried; Weidensteiner, Claudia; Saavedra, Joseph E; Weyerbrock, Astrid

    2017-06-01

    As a potent radiosensitizer nitric oxide (NO) may be a putative adjuvant in the treatment of malignant gliomas which are known for their radio- and chemoresistance. The NO donor prodrug JS-K (O2-(2.4-dinitrophenyl) 1-[(4-ethoxycarbonyl) piperazin-1-yl] diazen-1-ium-1,2-diolate) allows cell-type specific intracellular NO release via enzymatic activation by glutathione-S-transferases overexpressed in glioblastoma multiforme. The cytotoxic and radiosensitizing efficacy of JS-K was assessed in U87 glioma cells in vitro focusing on cell proliferation, induction of DNA damage, and cell death. In vivo efficacy of JS-K and repetitive irradiation were investigated in an orthotopic U87 xenograft model in mice. For the first time, we could show that JS-K acts as a potent cytotoxic and radiosensitizing agent in U87 cells in vitro. This dose- and time-dependent effect is due to an enhanced induction of DNA double-strand breaks leading to mitotic catastrophe as the dominant form of cell death. However, this potent cytotoxic and radiosensitizing effect could not be confirmed in an intracranial U87 xenograft model, possibly due to insufficient delivery into the brain. Although NO donor treatment was well tolerated, neither a retardation of tumor growth nor an extended survival could be observed after JS-K and/or radiotherapy.

  1. Implications of Rho GTPase signaling in glioma cell invasion and tumor progression

    Shannon Patricia Fortin Ensign

    2013-10-01

    Full Text Available Glioblastoma (GB is the most malignant of primary adult brain tumors, characterized by a highly locally-invasive cell population, as well as abundant proliferative cells, neoangiogenesis, and necrosis. Clinical intervention with chemotherapy or radiation may either promote or establish an environment for manifestation of invasive behavior. Understanding the molecular drivers of invasion in the context of glioma progression may be insightful in directing new treatments for patients with GB. Here, we review current knowledge on Rho family GTPases, their aberrant regulation in GB, and their effect on GB cell invasion and tumor progression. Rho GTPases are modulators of cell migration through effects on actin cytoskeleton rearrangement; in non-neoplastic tissue, expression and activation of Rho GTPases are normally under tight regulation. In GB, Rho GTPases are deregulated, often via hyperactivity or overexpression of their activators, Rho GEFs. Downstream effectors of Rho GTPases have been shown to promote invasiveness and, importantly, glioma cell survival. The study of aberrant Rho GTPase signaling in GB is thus an important investigation of cell invasion as well as treatment resistance and disease progression.

  2. Inhibition of autophagy induced by proteasome inhibition increases cell death in human SHG-44 glioma cells.

    Ge, Peng-Fei; Zhang, Ji-Zhou; Wang, Xiao-Fei; Meng, Fan-Kai; Li, Wen-Chen; Luan, Yong-Xin; Ling, Feng; Luo, Yi-Nan

    2009-07-01

    The ubiquitin-proteasome system (UPS) and lysosome-dependent macroautophagy (autophagy) are two major intracellular pathways for protein degradation. Recent studies suggest that proteasome inhibitors may reduce tumor growth and activate autophagy. Due to the dual roles of autophagy in tumor cell survival and death, the effect of autophagy on the destiny of glioma cells remains unclear. In this study, we sought to investigate whether inhibition of the proteasome can induce autophagy and the effects of autophagy on the fate of human SHG-44 glioma cells. The proteasome inhibitor MG-132 was used to induce autophagy in SHG-44 glioma cells, and the effect of autophagy on the survival of SHG-44 glioma cells was investigated using an autophagy inhibitor 3-MA. Cell viability was measured by MTT assay. Apoptosis and cell cycle were detected by flow cytometry. The expression of autophagy related proteins was determined by Western blot. MG-132 inhibited cell proliferation, induced cell death and cell cycle arrest at G(2)/M phase, and activated autophagy in SHG-44 glioma cells. The expression of autophagy-related Beclin-1 and LC3-I was significantly up-regulated and part of LC3-I was converted into LC3-II. However, when SHG-44 glioma cells were co-treated with MG-132 and 3-MA, the cells became less viable, but cell death and cell numbers at G(2)/M phase increased. Moreover, the accumulation of acidic vesicular organelles was decreased, the expression of Beclin-1 and LC3 was significantly down-regulated and the conversion of LC3-II from LC3-I was also inhibited. Inhibition of the proteasome can induce autophagy in human SHG-44 glioma cells, and inhibition of autophagy increases cell death. This discovery may shed new light on the effect of autophagy on modulating the fate of SHG-44 glioma cells.Acta Pharmacologica Sinica (2009) 30: 1046-1052; doi: 10.1038/aps.2009.71.

  3. Paired related homeobox 1 transactivates dopamine D2 receptor to maintain propagation and tumorigenicity of glioma-initiating cells

    Yamu Li; Ying Liu; Shu Li; Xiaobing Jiang; Guangwei Du; Yan Zhou; Wen Wang; Fangyu Wang; Qiushuang Wu; Wei Li; Xiaoling Zhong; Kuan Tian; Tao Zeng; Liang Gao

    2017-01-01

    Glioblastoma multiforme (GBM) is a highly invasive brain tumor with limited therapeutic means and poor prognosis.Recent studies indicate that glioma-initiating cells/glioma stem cells (GICs/GSCs) may be responsible for tumor initiation,infiltration,and recurrence.GlCs could aberrantly employ molecular machinery balancing self-renewal and differentiation of embryonic neural precursors.Here,we find that paired related homeobox 1 (PRRX1),a homeodomain transcription factor that was previously reported to control skeletal development,is expressed in cortical neural progenitors and is required for their self-renewal and proper differentiation.Further,PRRX1 is overrepresented in glioma samples and labels GlCs.Glioma cells and GlCs depleted with PRRX1 could not propagate in vitro or form tumors in the xenograft mouse model.The GIC self-renewal function regulated by PRRX1 is mediated by dopamine D2 receptor (DRD2).PRRX1 directly binds to the DRD2 promoter and transactivates its expression in GlCs.Blockage of the DRD2 signaling hampers GIC self-renewal,whereas its overexpression restores the propagating and tumorigenic potential of PRRX1-depleted GlCs.Finally,PRRX1 potentiates GlCs via DRD2-mediated extracellular signal-related kinase (ERK) and AKT activation.Thus,our study suggests that therapeutic targeting the PRRX1-DRD2-ERK/AKT axis in GlCs is a promising strategy for treating GBMs.

  4. An attempt to understand glioma stem cell biology through centrality analysis of a protein interaction network.

    Mallik, Mrinmay Kumar

    2018-02-07

    Biological networks can be analyzed using "Centrality Analysis" to identify the more influential nodes and interactions in the network. This study was undertaken to create and visualize a biological network comprising of protein-protein interactions (PPIs) amongst proteins which are preferentially over-expressed in glioma cancer stem cell component (GCSC) of glioblastomas as compared to the glioma non-stem cancer cell (GNSC) component and then to analyze this network through centrality analyses (CA) in order to identify the essential proteins in this network and their interactions. In addition, this study proposes a new centrality analysis method pertaining exclusively to transcription factors (TFs) and interactions amongst them. Moreover the relevant molecular functions, biological processes and biochemical pathways amongst these proteins were sought through enrichment analysis. A protein interaction network was created using a list of proteins which have been shown to be preferentially expressed or over-expressed in GCSCs isolated from glioblastomas as compared to the GNSCs. This list comprising of 38 proteins, created using manual literature mining, was submitted to the Reactome FIViz tool, a web based application integrated into Cytoscape, an open source software platform for visualizing and analyzing molecular interaction networks and biological pathways to produce the network. This network was subjected to centrality analyses utilizing ranked lists of six centrality measures using the FIViz application and (for the first time) a dedicated centrality analysis plug-in ; CytoNCA. The interactions exclusively amongst the transcription factors were nalyzed through a newly proposed centrality analysis method called "Gene Expression Associated Degree Centrality Analysis (GEADCA)". Enrichment analysis was performed using the "network function analysis" tool on Reactome. The CA was able to identify a small set of proteins with consistently high centrality ranks that

  5. Investigation of adhesion and mechanical properties of human glioma cells by single cell force spectroscopy and atomic force microscopy.

    Andolfi, Laura; Bourkoula, Eugenia; Migliorini, Elisa; Palma, Anita; Pucer, Anja; Skrap, Miran; Scoles, Giacinto; Beltrami, Antonio Paolo; Cesselli, Daniela; Lazzarino, Marco

    2014-01-01

    Active cell migration and invasion is a peculiar feature of glioma that makes this tumor able to rapidly infiltrate into the surrounding brain tissue. In our recent work, we identified a novel class of glioma-associated-stem cells (defined as GASC for high-grade glioma--HG--and Gasc for low-grade glioma--LG) that, although not tumorigenic, act supporting the biological aggressiveness of glioma-initiating stem cells (defined as GSC for HG and Gsc for LG) favoring also their motility. Migrating cancer cells undergo considerable molecular and cellular changes by remodeling their cytoskeleton and cell interactions with surrounding environment. To get a better understanding about the role of the glioma-associated-stem cells in tumor progression, cell deformability and interactions between glioma-initiating stem cells and glioma-associated-stem cells were investigated. Adhesion of HG/LG-cancer cells on HG/LG-glioma-associated stem cells was studied by time-lapse microscopy, while cell deformability and cell-cell adhesion strengths were quantified by indentation measurements by atomic force microscopy and single cell force spectroscopy. Our results demonstrate that for both HG and LG glioma, cancer-initiating-stem cells are softer than glioma-associated-stem cells, in agreement with their neoplastic features. The adhesion strength of GSC on GASC appears to be significantly lower than that observed for Gsc on Gasc. Whereas, GSC spread and firmly adhere on Gasc with an adhesion strength increased as compared to that obtained on GASC. These findings highlight that the grade of glioma-associated-stem cells plays an important role in modulating cancer cell adhesion, which could affect glioma cell migration, invasion and thus cancer aggressiveness. Moreover this work provides evidence about the importance of investigating cell adhesion and elasticity for new developments in disease diagnostics and therapeutics.

  6. Differential utilization of ketone bodies by neurons and glioma cell lines: a rationale for ketogenic diet as experimental glioma therapy.

    Maurer, Gabriele D; Brucker, Daniel P; Bähr, Oliver; Harter, Patrick N; Hattingen, Elke; Walenta, Stefan; Mueller-Klieser, Wolfgang; Steinbach, Joachim P; Rieger, Johannes

    2011-07-26

    Even in the presence of oxygen, malignant cells often highly depend on glycolysis for energy generation, a phenomenon known as the Warburg effect. One strategy targeting this metabolic phenotype is glucose restriction by administration of a high-fat, low-carbohydrate (ketogenic) diet. Under these conditions, ketone bodies are generated serving as an important energy source at least for non-transformed cells. To investigate whether a ketogenic diet might selectively impair energy metabolism in tumor cells, we characterized in vitro effects of the principle ketone body 3-hydroxybutyrate in rat hippocampal neurons and five glioma cell lines. In vivo, a non-calorie-restricted ketogenic diet was examined in an orthotopic xenograft glioma mouse model. The ketone body metabolizing enzymes 3-hydroxybutyrate dehydrogenase 1 and 2 (BDH1 and 2), 3-oxoacid-CoA transferase 1 (OXCT1) and acetyl-CoA acetyltransferase 1 (ACAT1) were expressed at the mRNA and protein level in all glioma cell lines. However, no activation of the hypoxia-inducible factor-1α (HIF-1α) pathway was observed in glioma cells, consistent with the absence of substantial 3-hydroxybutyrate metabolism and subsequent accumulation of succinate. Further, 3-hydroxybutyrate rescued hippocampal neurons from glucose withdrawal-induced cell death but did not protect glioma cell lines. In hypoxia, mRNA expression of OXCT1, ACAT1, BDH1 and 2 was downregulated. In vivo, the ketogenic diet led to a robust increase of blood 3-hydroxybutyrate, but did not alter blood glucose levels or improve survival. In summary, glioma cells are incapable of compensating for glucose restriction by metabolizing ketone bodies in vitro, suggesting a potential disadvantage of tumor cells compared to normal cells under a carbohydrate-restricted ketogenic diet. Further investigations are necessary to identify co-treatment modalities, e.g. glycolysis inhibitors or antiangiogenic agents that efficiently target non-oxidative pathways.

  7. Differential utilization of ketone bodies by neurons and glioma cell lines: a rationale for ketogenic diet as experimental glioma therapy

    Maurer, Gabriele D; Brucker, Daniel P; Bähr, Oliver; Harter, Patrick N; Hattingen, Elke; Walenta, Stefan; Mueller-Klieser, Wolfgang; Steinbach, Joachim P; Rieger, Johannes

    2011-01-01

    Even in the presence of oxygen, malignant cells often highly depend on glycolysis for energy generation, a phenomenon known as the Warburg effect. One strategy targeting this metabolic phenotype is glucose restriction by administration of a high-fat, low-carbohydrate (ketogenic) diet. Under these conditions, ketone bodies are generated serving as an important energy source at least for non-transformed cells. To investigate whether a ketogenic diet might selectively impair energy metabolism in tumor cells, we characterized in vitro effects of the principle ketone body 3-hydroxybutyrate in rat hippocampal neurons and five glioma cell lines. In vivo, a non-calorie-restricted ketogenic diet was examined in an orthotopic xenograft glioma mouse model. The ketone body metabolizing enzymes 3-hydroxybutyrate dehydrogenase 1 and 2 (BDH1 and 2), 3-oxoacid-CoA transferase 1 (OXCT1) and acetyl-CoA acetyltransferase 1 (ACAT1) were expressed at the mRNA and protein level in all glioma cell lines. However, no activation of the hypoxia-inducible factor-1α (HIF-1α) pathway was observed in glioma cells, consistent with the absence of substantial 3-hydroxybutyrate metabolism and subsequent accumulation of succinate. Further, 3-hydroxybutyrate rescued hippocampal neurons from glucose withdrawal-induced cell death but did not protect glioma cell lines. In hypoxia, mRNA expression of OXCT1, ACAT1, BDH1 and 2 was downregulated. In vivo, the ketogenic diet led to a robust increase of blood 3-hydroxybutyrate, but did not alter blood glucose levels or improve survival. In summary, glioma cells are incapable of compensating for glucose restriction by metabolizing ketone bodies in vitro, suggesting a potential disadvantage of tumor cells compared to normal cells under a carbohydrate-restricted ketogenic diet. Further investigations are necessary to identify co-treatment modalities, e.g. glycolysis inhibitors or antiangiogenic agents that efficiently target non-oxidative pathways

  8. Differential utilization of ketone bodies by neurons and glioma cell lines: a rationale for ketogenic diet as experimental glioma therapy

    Mueller-Klieser Wolfgang

    2011-07-01

    Full Text Available Abstract Background Even in the presence of oxygen, malignant cells often highly depend on glycolysis for energy generation, a phenomenon known as the Warburg effect. One strategy targeting this metabolic phenotype is glucose restriction by administration of a high-fat, low-carbohydrate (ketogenic diet. Under these conditions, ketone bodies are generated serving as an important energy source at least for non-transformed cells. Methods To investigate whether a ketogenic diet might selectively impair energy metabolism in tumor cells, we characterized in vitro effects of the principle ketone body 3-hydroxybutyrate in rat hippocampal neurons and five glioma cell lines. In vivo, a non-calorie-restricted ketogenic diet was examined in an orthotopic xenograft glioma mouse model. Results The ketone body metabolizing enzymes 3-hydroxybutyrate dehydrogenase 1 and 2 (BDH1 and 2, 3-oxoacid-CoA transferase 1 (OXCT1 and acetyl-CoA acetyltransferase 1 (ACAT1 were expressed at the mRNA and protein level in all glioma cell lines. However, no activation of the hypoxia-inducible factor-1α (HIF-1α pathway was observed in glioma cells, consistent with the absence of substantial 3-hydroxybutyrate metabolism and subsequent accumulation of succinate. Further, 3-hydroxybutyrate rescued hippocampal neurons from glucose withdrawal-induced cell death but did not protect glioma cell lines. In hypoxia, mRNA expression of OXCT1, ACAT1, BDH1 and 2 was downregulated. In vivo, the ketogenic diet led to a robust increase of blood 3-hydroxybutyrate, but did not alter blood glucose levels or improve survival. Conclusion In summary, glioma cells are incapable of compensating for glucose restriction by metabolizing ketone bodies in vitro, suggesting a potential disadvantage of tumor cells compared to normal cells under a carbohydrate-restricted ketogenic diet. Further investigations are necessary to identify co-treatment modalities, e.g. glycolysis inhibitors or antiangiogenic

  9. Radiosensitivity and TP 53, EGFR amplification and LOH10 analysis of primary glioma cell cultures

    Gerlach, Bärbel; Harder, Anna H.; Hulsebos, Theo J. M.; Leenstra, Sieger; Slotman, Berend J.; Vandertop, W. Peter; Hartmann, Karl-Axel; Sminia, Peter

    2002-01-01

    Aim: Determination of in-vitro radiosensitivity and genetic alterations of cell cultures derived from human glioma biopsy tissue and established glioma cell lines. Material and Methods: Fresh brain tumor specimens of six patients were processed to early passage cell cultures. In addition the cell

  10. Antitumor Activity of Rat Mesenchymal Stem Cells during Direct or Indirect Co-Culturing with C6 Glioma Cells.

    Gabashvili, A N; Baklaushev, V P; Grinenko, N F; Mel'nikov, P A; Cherepanov, S A; Levinsky, A B; Chehonin, V P

    2016-02-01

    The tumor-suppressive effect of rat mesenchymal stem cells against low-differentiated rat C6 glioma cells during their direct and indirect co-culturing and during culturing of C6 glioma cells in the medium conditioned by mesenchymal stem cells was studied in an in vitro experiment. The most pronounced antitumor activity of mesenchymal stem cells was observed during direct co-culturing with C6 glioma cells. The number of live C6 glioma cells during indirect co-culturing and during culturing in conditioned medium was slightly higher than during direct co-culturing, but significantly differed from the control (C6 glioma cells cultured in medium conditioned by C6 glioma cells). The cytotoxic effect of medium conditioned by mesenchymal stem cells was not related to medium depletion by glioma cells during their growth. The medium conditioned by other "non-stem" cells (rat astrocytes and fibroblasts) produced no tumor-suppressive effect. Rat mesenchymal stem cells, similar to rat C6 glioma cells express connexin 43, the main astroglial gap junction protein. During co-culturing, mesenchymal stem cells and glioma C6 cells formed functionally active gap junctions. Gap junction blockade with connexon inhibitor carbenoxolone attenuated the antitumor effect observed during direct co-culturing of C6 glioma cells and mesenchymal stem cells to the level produced by conditioned medium. Cell-cell signaling mediated by gap junctions can be a mechanism of the tumor-suppressive effect of mesenchymal stem cells against C6 glioma cells. This phenomenon can be used for the development of new methods of cell therapy for high-grade malignant gliomas.

  11. Effects of irradiation on cytokine production in glioma cell lines

    Yamanaka, Ryuya; Tanaka, Ryuichi; Yoshida, Seiichi [Niigata Univ. (Japan). Brain Research Inst.

    1993-11-01

    The effects of irradiation on cytokine production in glioma cell lines, NP1, NP2 and NP3, were studied. Culture supernatants were collected after 6, 24, 48 or 72 hours and the concentrations of interleukin (IL)-6 and IL-8 measured by enzyme-linked immunosorbent assay. Spontaneous and IL-1[beta]-stimulated productions were analyzed. Some cells were given a single dose of Lineac irradiation (10 or 20 Gy). Production of IL-6 (with or without IL-1[beta] stimulation) increased gradually to a maximum after 72 hours, more in the 20 Gy-irradiated cells than 10 Gy cells (p<0.01). Production of IL-8 increased gradually to a maximum after 48 or 72 hours. Spontaneous production of IL-8 increased more in 20 Gy-irradiated cells than 10 Gy cells after 6 and 24 hours (p<0.01), but increased more in 10 Gy cells than 20 Gy cells after 48 and 72 hours (p<0.01). The production of IL-8 stimulated by IL-1[beta] increased more in 10 Gy cells than 20 Gy cells 24 hours later (p<0.01). IL-6 and IL-8 production differed in the response to irradiation. Our data suggest that bidirectional communication between the immune system and glioma cells changes after radiotherapy. (author).

  12. The effects of CD147 on the cell proliferation, apoptosis, invasion, and angiogenesis in glioma.

    Yin, Haoyuan; Shao, Ying; Chen, Xuan

    2017-01-01

    To analyze the effects of extracellular matrix metalloproteinase inducer (CD147) on glioma proliferation, apoptosis, invasion, and angiogenesis. Tissue samples were obtained from 101 glioma cases while normal brain tissues were obtained from 30 brain injury cases. Immunohistochemical assay was performed to detect the expressions of CD147, CD34, and VEGF in tissue samples. QRT-PCR was performed to detect the relative expression of CD147 mRNA in human glioma cell lines. CD147 siRNA was transfected into glioma cell line U251. Cell proliferation, apoptosis, invasion, and angiogenesis were tested by MTT, flow cytometry, Transwell assay, and vasculogenic mimicry assay, respectively. Expressions of relative proteins were analyzed with western blot. CD147 was positively expressed with the percentage of 0, 37.5, 44.8, 67.9, and 85.7 % in normal tissues and glioma tissues with WHO grades I-IV, respectively, and the scores of MVDand VEGF were associated with the expression of CD147. CD147 was significantly upregulated in the human glioma cell lines (P CD147 suppressed cell proliferation, blocked cell cycle, induced apoptosis, inhibited cell invasion and angiogenesis in glioma cells in vitro. The expression of CD147 was significantly associated with WHO tumor grade and angiogenesis; silencing of CD147 contributed to inhibition of glioma proliferation, invasion, and angiogenesis. Our study provided firm evidence that CD 147 is a potential glioma target for anti-angiogenic therapies.

  13. Inhibition of cell growth by EGR-1 in human primary cultures from malignant glioma

    Gagliardi Franco

    2004-01-01

    Full Text Available Abstract Background The aim of this work was to investigate in vitro the putative role of EGR-1 in the growth of glioma cells. EGR-1 expression was examined during the early passages in vitro of 17 primary cell lines grown from 3 grade III and from 14 grade IV malignant astrocytoma explants. The explanted tumors were genetically characterized at the p53, MDM2 and INK4a/ARF loci, and fibronectin expression and growth characteristics were examined. A recombinant adenovirus overexpressing EGR-1 was tested in the primary cell lines. Results Low levels of EGR-1 protein were found in all primary cultures examined, with lower values present in grade IV tumors and in cultures carrying wild-type copies of p53 gene. The levels of EGR-1 protein were significantly correlated to the amount of intracellular fibronectin, but only in tumors carrying wild-type copies of the p53 gene (R = 0,78, p = 0.0082. Duplication time, plating efficiency, colony formation in agarose, and contact inhibition were also altered in the p53 mutated tumor cultures compared to those carrying wild-type p53. Growth arrest was achieved in both types of tumor within 1–2 weeks following infection with a recombinant adenovirus overexpressing EGR-1 but not with the control adenovirus. Conclusions Suppression of EGR-1 is a common event in gliomas and in most cases this is achieved through down-regulation of gene expression. Expression of EGR-1 by recombinant adenovirus infection almost completely abolishes the growth of tumor cells in vitro, regardless of the mutational status of the p53 gene.

  14. Saw Palmetto Extract Inhibits Metastasis and Antiangiogenesis through STAT3 Signal Pathway in Glioma Cell

    Ding, Hong; Shen, Jinglian; Yang, Yang; Che, Yuqin

    2015-01-01

    Signal transducer and activator of transcription factor 3 (STAT3) plays an important role in the proliferation and angiogenesis in human glioma. Previous research indicated that saw palmetto extract markedly inhibited the proliferation of human glioma cells through STAT3 signal pathway. But its effect on tumor metastasis and antiangiogenesis is not clear. This study is to further clear the impact of saw palmetto extract on glioma cell metastasis, antiangiogenesis, and its mechanism. TUNEL ass...

  15. T-lymphokine-activated killer cell-originated protein kinase (TOPK) as a prognostic factor and a potential therapeutic target in glioma

    Duan, Qiuhong; Yuan, Ping; Xue, Peipei; Lu, Hui; Yan, Meng; Guo, Dongsheng; Xu, Sanpeng; Zhang, Xiaohui; Lin, Xuan; Wang, Yong; Dogan, Soner; Zhang, Jianmin; Zhu, Feng; Ke, Changshu; Liu, Lin

    2018-01-01

    TOPK is overexpressed in various types of cancer and associated with poor outcomes in different types of cancer. In this study, we first found that the expression of T-lymphokine-activated killer cell-originated protein kinase (TOPK) was significantly higher in Grade III or Grade IV than that in Grade II in glioma (P = 0.007 and P < 0.001, respectively). Expression of TOPK was positively correlated with Ki67 (P < 0.001). Knockdown of TOPK significantly inhibited cell growth, colony formation and increased sensitivities to temozolomide (TMZ) in U-87 MG or U-251 cells, while TOPK overexpression promoted cell growth and colony formation in Hs 683 or A-172 cells. Glioma patients expressing high levels of TOPK have poor survival compared with those expressing low levels of TOPK in high-grade or low-grade gliomas (hazard ratio = 0.2995; 95% CI, 0.1262 to 0.7108; P = 0.0063 and hazard ratio = 0.1509; 95% CI, 0.05928 to 0.3842; P < 0.0001, respectively). The level of TOPK was low in TMZ-sensitive patients compared with TMZ-resistant patients (P = 0.0056). In TMZ-resistant population, patients expressing high TOPK have two months’ shorter survival time than those expressing low TOPK. Our findings demonstrated that TOPK might represent as a promising prognostic and predictive factor and potential therapeutic target for glioma. PMID:29487691

  16. Downregulation of HIF-1a sensitizes U251 glioma cells to the temozolomide (TMZ) treatment

    Tang, Jun-Hai [Department of Neurosurgery, Xinqiao Hospital, Third Military Medical University, Chongqing 400037 (China); Ma, Zhi-Xiong [National Institute of Biological Sciences, Beijing 102206 (China); Huang, Guo-Hao; Xu, Qing-Fu; Xiang, Yan [Department of Neurosurgery, Xinqiao Hospital, Third Military Medical University, Chongqing 400037 (China); Li, Ningning; Sidlauskas, Kastytis [Division of Neuropathology and Department of Neurodegenerative Disease, Institute of Neurology, University College London, London WC1N 3BG (United Kingdom); Zhang, Eric Erquan [National Institute of Biological Sciences, Beijing 102206 (China); Lv, Sheng-Qing, E-mail: lvsq0518@hotmail.com [Department of Neurosurgery, Xinqiao Hospital, Third Military Medical University, Chongqing 400037 (China)

    2016-05-01

    Purpose: The aim of this study was to investigate the effect of downregulation of HIF-1α gene on human U251 glioma cells and examine the consequent changes of TMZ induced effects and explore the molecular mechanisms. Methods: U251 cell line stably expressing HIF-1α shRNA was acquired via lentiviral vector transfection. The mRNA and protein expression alterations of genes involved in our study were determined respectively by qRT-PCR and Western blot. Cell proliferation was measured by MTT assay and colony formation assay, cell invasion/migration capacity was determined by transwell invasion assay/wound healing assay, and cell apoptosis was detected by flow cytometry. Results: We successfully established a U251 cell line with highly efficient HIF-1α knockdown. HIF-1a downregulation sensitized U251 cells to TMZ treatment and enhanced the proliferation-inhibiting, invasion/migration-suppressing, apoptosis-inducing and differentiation-promoting effects exerted by TMZ. The related molecular mechanisms demonstrated that expression of O{sup 6}-methylguanine DNA methyltransferase gene (MGMT) and genes of Notch1 pathway were significantly upregulated by TMZ treatment. However, this upregulation was abrogated by HIF-1α knockdown. We further confirmed important regulatory roles of HIF-1α in the expression of MGMT and activation of Notch1 pathways. Conclusion: HIF-1α downregulation sensitizes U251 glioma cells to the temozolomide treatment via inhibiting MGMT expression and Notch1 pathway activation. - Highlights: • TMZ caused more significant proliferation inhibition and apoptosis in U251 cells after downregulating HIF-1α. • Under TMZ treatment, HIF-1 downregulated U251 cells exhibited weaker mobility and more differentiated state. • TMZ caused MGMT over-expression and Notch1 pathway activation, which could be abrogated by HIF-1α downregulation.

  17. Eckol suppresses maintenance of stemness and malignancies in glioma stem-like cells

    Hyun, Kyung-Hwan; Yoon, Chang-Hwan; Kim, Rae-Kwon; Lim, Eun-Jung; An, Sungkwan; Park, Myung-Jin; Hyun, Jin-Won; Suh, Yongjoon; Kim, Min-Jung; Lee, Su-Jae

    2011-01-01

    A subpopulation of cancer cells with stem cell properties is responsible for tumor maintenance and progression, and may contribute to resistance to anticancer treatments. Thus, compounds that target cancer stem-like cells could be usefully applied to destroy cancer. In this study, we investigated the effect of Eckol, a phlorotannin compound, on stemness and malignancies in glioma stem-like cells. To determine whether Eckol targets glioma stem-like cells, we examined whether Eckol treatment could change the expression levels of glioma stem-like cell markers and self-renewal-related proteins as well as the sphere forming ability, and the sensitivity to anticancer treatments. Alterations in the malignant properties of sphere-derived cells by Eckol were also investigated by soft-agar colony forming assay, by xenograft assay in nude mice, and by cell invasion assay. Treatment of sphere-forming glioma cells with Eckol effectively decreased the sphere formation as well as the CD133 + cell population. Eckol treatment suppressed expression of the glioma stem-like cell markers and the self-renewal-related proteins without cell death. Moreover, treatment of glioma stem-like cells with Eckol significantly attenuated anchorage-independent growth on soft agar and tumor formation in xenograft mice. Importantly, Eckol treatment effectively reduced the resistance of glioma stem-like cells to ionizing radiation and temozolomide. Treatment of glioma stem-like cells with Eckol markedly blocked both phosphoinositide 3-kinase-Akt and Ras-Raf-1-Erk signaling pathways. These results indicate that the natural phlorotannin Eckol suppresses stemness and malignancies in glioma stem-like cells, and thereby makes glioma stem-like cells more sensitive to anticancer treatments, providing novel therapeutic strategies targeting specifically cancer stem-like cells.

  18. WW domain of BAG3 is required for the induction of autophagy in glioma cells.

    Merabova, Nana; Sariyer, Ilker Kudret; Saribas, A Sami; Knezevic, Tijana; Gordon, Jennifer; Turco, M Caterina; Rosati, Alessandra; Weaver, Michael; Landry, Jacques; Khalili, Kamel

    2015-04-01

    Autophagy is an evolutionarily conserved, selective degradation pathway of cellular components that is important for cell homeostasis under healthy and pathologic conditions. Here we demonstrate that an increase in the level of BAG3 results in stimulation of autophagy in glioblastoma cells. BAG3 is a member of a co-chaperone family of proteins that associates with Hsp70 through a conserved BAG domain positioned near the C-terminus of the protein. Expression of BAG3 is induced by a variety of environmental changes that cause stress to cells. Our results show that BAG3 overexpression induces autophagy in glioma cells. Interestingly, inhibition of the proteasome caused an increase in BAG3 levels and induced autophagy. Further analysis using specific siRNA against BAG3 suggests that autophagic activation due to proteosomal inhibition is mediated by BAG3. Analyses of BAG3 domain mutants suggest that the WW domain of BAG3 is crucial for the induction of autophagy. BAG3 overexpression also increased the interaction between Bcl2 and Beclin-1, instead of disrupting them, suggesting that BAG3 induced autophagy is Beclin-1 independent. These observations reveal a novel role for the WW domain of BAG3 in the regulation of autophagy. © 2014 Wiley Periodicals, Inc.

  19. “...those left behind.” Biology and Oncology of Invasive Glioma Cells

    Michael E Berens

    1999-08-01

    Full Text Available Although significant technical advances in surgical and radiation treatment for brain tumors have emerged in recent years, their impact on clinical outcome for patients has been disappointing. A fundamental source of the management challenge presented by glioma patients is the insidious propensity of the malignant cells to invade into adjacent normal brain. Invasive tumor cells escape surgical removal and geographically dodge lethal radiation exposure. Recent improved understanding of the biochemistry and molecular determinants of glioma cell invasion provide valuable insight to the underlying biological features of the disease, as well as illuminating possible new therapeutic targets. Heightened commitment to migrate and invade is accompanied by a glioma cell's reduced proliferative activity. The microenvironmental manipulations coincident to invasion and migration may also impact the glioma cell's response to cytotoxic treatments. These collateral aspects of the glioma cell invasive phenotype should be further explored and exploited as novel antiglioma therapies.

  20. Capacity of ultraviolet-induced DNA repair in human glioma cells

    Itoh, Hiroji

    1987-04-01

    A DNA repair abnormality is likely related to an increased incidence of neoplasms in several autosomal recessive diseases such as xeroderma pigmentosum, Fanconi's anemia, Bloom's syndrome and ataxia telangiectasia. In human glioma cells, however, there are only a few reports on DNA repair. In this study, an ultraviolet (UV)-induced DNA repair was examined systematically in many human glioma cells. Two human malignant glioma cell lines (MMG-851, U-251-MG) and 7 human glioma cell strains (4, benign; 3, malignant) of short term culture, in which glial fibrillary acidic protein (GFAP) staining were positive, were used. To investigate the capacity of DNA repair, UV sensitivity was determined by colony formation; excision repair by autoradiography and Cytosine Arabinoside (Ara-C) assay; and post-replication repair by the joining rate of newly synthesized DNA. As a result, the colony-forming abilities of malignant glioma cell lines were lower than those of normal human fibroblasts, but no difference was found between two malignant glioma cell lines. The excision repair of the malignant group (2 cell lines and 3 cell strains) was apparently lower than that of the benign group (4 cell strains). In two malignant glioma cell lines, the excision repair of MMG-851 was lower than that of U-251-MG, and the post-replication repair of MMG-851 was higher than that of U-251-MG. These results were considered to correspond well with colony-forming ability. The results indicate that there are some differences in each human malignant glioma cell in its UV-induced DNA repair mechanism, and that the excision repair of the malignant glioma cells is apparently lower than that of the benign glioma cells. These findings may be useful for diagnosis and treatment.

  1. Interference with the HSF1/HSP70/BAG3 Pathway Primes Glioma Cells to Matrix Detachment and BH3 Mimetic-Induced Apoptosis.

    Antonietti, Patrick; Linder, Benedikt; Hehlgans, Stephanie; Mildenberger, Iris C; Burger, Michael C; Fulda, Simone; Steinbach, Joachim P; Gessler, Florian; Rödel, Franz; Mittelbronn, Michel; Kögel, Donat

    2017-01-01

    Malignant gliomas exhibit a high intrinsic resistance against stimuli triggering apoptotic cell death. HSF1 acts as transcription factor upstream of HSP70 and the HSP70 co-chaperone BAG3 that is overexpressed in glioblastoma. To specifically target this resistance mechanism, we applied the selective HSF1 inhibitor KRIBB11 and the HSP70/BAG3 interaction inhibitor YM-1 in combination with the pan-Bcl-2 inhibitor AT-101. Here, we demonstrate that lentiviral BAG3 silencing significantly enhances AT-101-induced cell death and reactivates effector caspase-mediated apoptosis in U251 glioma cells with high BAG3 expression, whereas these sensitizing effects were less pronounced in U343 cells expressing lower BAG3 levels. KRIBB11 decreased protein levels of HSP70, BAG3, and the antiapoptotic Bcl-2 protein Mcl-1, and both KRIBB11 and YM-1 elicited significantly increased mitochondrial dysfunction, effector caspase activity, and apoptotic cell death after combined treatment with AT-101 and ABT-737. Depletion of BAG3 also led to a pronounced loss of cell-matrix adhesion, FAK phosphorylation, and in vivo tumor growth in an orthotopic mouse glioma model. Furthermore, it reduced the plating efficiency of U251 cells in three-dimensional clonogenic assays and limited clonogenic survival after short-term treatment with AT-101. Collectively, our data suggest that the HSF1/HSP70/BAG3 pathway plays a pivotal role for overexpression of prosurvival Bcl-2 proteins and cell death resistance of glioma. They also support the hypothesis that interference with BAG3 function is an effective novel approach to prime glioma cells to anoikis. Mol Cancer Ther; 16(1); 156-68. ©2016 AACR. ©2016 American Association for Cancer Research.

  2. Effect of microenviroment hypoxia on glioma cells radiosensitivity through cancer stem cell pathway

    Tong Liumei; Feng Libo; Lu Xueguan; Chen Liesong; Guo Xinwei; Tian Ye

    2010-01-01

    Objective: To investigate the effect of microenviroment hypoxia on glioma cells radiosensitivity through cancer stem pathway, and to explore the related mechanism. Methods: Glioma cell lines SHG44 and U251 were cultured in normoxia (20% O 2 ) or continuous hypoxia (1% O 2 ) for 12 and 24 h. The fraction of glioma cells with positive expression of CD133 was assayed by flow cytometry. The radiosensitivity of glioma cells was determined by clonogenic cell assay. Western blotting was used to investigate the expressions of HIF-1 α and its downstream gene Notch 1. Results: The fraction of glioma cells with positive expression of CD133 was higher after hypoxic culture for 12 and 24 h than that of the corresponding cells cultured in normoxia. Compared to the cells cultured in normoxia, SF 2 (survival fraction at 2 Gy) were enhanced significantly in SHG44 and U251 cells cultured in hypoxia for 12 and 24 h. The OER (oxygen-enhancement ratio) of SHG44 cells in hypoxia for 12 and 24 h was 1.54 and 1.38, respectively. The OER of U251 cells was 1.44 and 1.23, respectively. The radiosensitivity of these two cell line was decreased in hypoxia. The protein expressions of HIF-1 α and Notch 1 genes were elevated more significantly for cells cultured in hypoxia for 12 and 24 h than for those in normoxia. Conclusions: Microenviroment hypoxia could increase the radioresistance of glioma cells through enrichment of cancer stem cells, and HIF-1 α-Notch 1 signal pathway may play an important role in this process. (authors)

  3. Cloning and characterization of human RTVP-1b, a novel splice variant of RTVP-1 in glioma cells

    Xiang Cunli; Sarid, Ronit; Cazacu, Simona; Finniss, Susan; Lee, Hae-Kyung; Ziv-Av, Amotz; Mikkelsen, Tom; Brodie, Chaya

    2007-01-01

    Here, we report the cloning and characterization of RTVP-1b, a novel splice variant of human RTVP-1, which was isolated from the U87 glioma cell line. Sequence analysis revealed that RTVP-1b contains an additional 71 base exon between exons 2 and 3 that is missing in RTVP-1, leading to a frame-shift and a different putative protein. The deduced protein was 237 amino acids in length, sharing the N-terminal 141 amino acids with RTVP-1. RT-PCR analysis demonstrated that RTVP-1b was expressed in a wide range of tissues and that its expression was different from that of RTVP-1. In contrast, RTVP-1 and RTVP-1b showed similar patterns of expression in astrocytic tumors; highly expressed in glioblastomas as compared to normal brains, low-grade astrocytomas and anaplastic oligodendrogliomas. Overexpression of RTVP-1b increased glioma cell proliferation but did not affect cell migration. Our results suggest that RTVP-1b represents a potential prognostic marker and therapeutic target in gliomas

  4. Resveratrol represses YKL-40 expression in human glioma U87 cells

    Zhang, Wei; Tamiya, Takashi; Murao, Koji; Zhang, Xiang; Matsumoto, Kensuke; Diah, Suwarni; Okada, Masaki; Miyake, Keisuke; Kawai, Nobuyuki; Fei, Zhou

    2010-01-01

    Glioblastoma multiforme (GBM) is the most malignant intracranial tumour that develops in both adults and children. Microarray gene analyses have confirmed that the human YKL-40 gene is one of the most over-expressed genes in these tumours but not in normal brain tissue. Clinical studies have shown that serum YKL-40 levels are positively correlated with tumour burden in addition to being an independent prognostic factor of a short relapse-free interval as well as short overall survival in patients with various cancers. Our previous study revealed that YKL-40 was closely correlated with the pathological grades of human primary astrocytomas and played a crucial role in glioma cell proliferation. Hence, YKL-40 could be an attractive target in the design of anti-cancer therapies. Cell viability and invasion assays were performed to detect the cell proliferation and invasive ability of U87 cells induced by resveratrol (3, 5, 4'-trihydroxystilbene; Res) or YKL-40 small-interfering RNAs (siRNAs). In addition, the luciferase assay, real-time RT-PCR, western blotting, and ELISA were used to measure YKL-40 promoter activity, mRNA, and protein expression, respectively. The expressions of phosphor-ERK1/2 and ERK1/2 were determined by western blotting. Res inhibited U87 cell proliferation and invasion in vitro and repressed YKL-40 in U87 cells by decreasing the activity of its promoter and reducing mRNA transcription and protein expression in vitro. YKL-40 siRNA treatment also impaired the invasiveness of U87 cells. When U87 cells were cultured with 20 μM PD98059 (an ERK1/2 inhibitor) alone, with 20 μM PD98059 and 100 μM Res, or with 100 μM Res alone for 48 h, YKL-40 protein expression decreased most significantly in the Res-treated group. PD98059 partially reversed the decrease of YKL-40 protein expression induced by Res. Furthermore, phosphor-ERK1/2 expression was reduced by Res treatment in a time-dependent manner. We demonstrated for the first time that Res

  5. Morphological characterization of a human glioma cell l ine.

    Machado, Camila Ml; Schenka, André; Vassallo, José; Tamashiro, Wirla Msc; Gonçalves, Estela M; Genari, Selma C; Verinaud, Liana

    2005-05-10

    A human malignant continuous cell line, named NG97, was recently established in our laboratory. This cell line has been serially subcultured over 100 times in standard culture media presenting no sign of cell senescence. The NG97 cell line has a doubling time of about 24 h. Immunocytochemical analysis of glial markers demonstrated that cells are positive for glial fibrillary acidic protein (GFAP) and S-100 protein, and negative for vimentin. Under phase-contrast microscope, cultures of NG97 showed cells with variable morphological features, such as small rounded cells, fusiform cells (fibroblastic-like cells), and dendritic-like cells. However, at confluence just small rounded and fusiform cells can be observed. At scanning electron microscopy (SEM) small rounded cells showed heterogeneous microextentions, including blebs and filopodia. Dendritic-like cells were flat and presented extensive prolongations, making several contacts with small rounded cells, while fusiform cells presented their surfaces dominated by microvilli.We believe that the knowledge about NG97 cell line may be useful for a deeper understanding of biological and immunological characteristics of gliomas.

  6. Nitric oxide donors attenuate clongenic potential in rat C6 glioma cells treated with alkylating chemotherapeutic agents.

    Yang, Jir-Jei; Yin, Jiu-Haw; Yang, Ding-I

    2007-05-11

    1,3-Bis(2-chloroethyl)-1-nitrosourea (BCNU) kills tumor cells via multiple actions including alkylation and carbamoylation. Previously, we have reported that formation of S-nitrosoglutathione (GSNO) in glioma cells overexpressing inducible nitric oxide synthase (iNOS) contributed to nitric oxide (NO)-dependent carbamoylating chemoresistance against BCNU. To further characterize the effects of NO on alkylating cytotoxicity, colony formation assay was applied to evaluate the effects of various NO donors on rat C6 glioma cells challenged with alkylating agents. We demonstrate that NO donors including GSNO, diethylamine NONOate (DEA/NO), and sodium nitroprusside (SNP) substantially reduced the extent of colony formation in glioma cells treated with alkylating agents, namely methyl methanesulfonate (MMS), N-methyl-N-nitrosourea (MNU), and N-ethyl-N-nitrosourea (ENU). Without alkylating agents these NO-releasing agents alone had no effects on clongenic potential of rat C6 glioma cells. Among these three NO donors used, the effectiveness in potentiating alkylating cytotoxicity is in the order of "GSNO>DEA/NO>SNP" when applied at the same dosages. GSNO also exerted similar synergistic actions reducing the extents of colony formation when co-administrated with 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)-hydrazine (compound #1), another alkylating agent that mimics the chloroethylating action of BCNU. Together with our previous findings, we propose that NO donors may be used as adjunct chemotherapy with alkylating agents for such malignant brain tumors as glioblastoma multiforme (GBM). In contrast, production of NO as a result of iNOS induction, such as that occurring after surgical resection of brain tumors, may compromise the efficacy of carbamoylating chemotherapy.

  7. CD147 overexpression promotes tumorigenicity in Chinese hamster ovary cells.

    Yong, Yu-Le; Liao, Cheng-Gong; Wei, Ding; Chen, Zhi-Nan; Bian, Huijie

    2016-04-01

    CD147 overexpresses in many epithelium-originated tumors and plays an important role in tumor migration and invasion. Most studies aim at the role of CD147 in tumor progression using tumor cell models. However, the influence of abnormal overexpression of CD147 on neoplastic transformation of normal cells is unknown. Here, the role of CD147 in malignant phenotype transformation in CHO cells was investigated. Three CHO cell lines that stably overexpressed CD147 (CHO-CD147), EGFP-CD147 (CHO-EGFP-CD147), and EGFP (CHO-EGFP) were generated by transfection of plasmids containing human CD147, EGFP-human CD147, and EGFP genes into CHO cells. Cell migration and invasion were detected by wound healing and transwell matrix penetration assay. Trypan blue exclusion, MTT, cell cycle analysis, and BrdU cell proliferation assay were used to detect cell viability and cell proliferation. Annexin V-FITC analysis was performed to detect apoptosis. We found that CD147 overexpression promoted the migration and invasion of CHO cells. CD147 accelerated the G1 to S phase transition and enhanced the CHO cell proliferation. Overexpression of CD147 inhibited both early- and late-stages of apoptosis of CHO-CD147 cells, which is caused by serum deprivation. CHO-EGFP-CD147 cells showed an increased anchorage-independent growth compared with CHO-EGFP cells as detected by soft-agar colony formation assay. The tumors formed by CHO-CD147 cells in nude mice were larger and coupled with higher expression of proliferating cell nuclear antigen and Ki-67 than that of CHO cells. In conclusion, human CD147 overexpression induces malignant phenotype in CHO cells. © 2015 International Federation for Cell Biology.

  8. hERG1 channels are overexpressed in glioblastoma multiforme and modulate VEGF secretion in glioblastoma cell lines

    Masi, A; Becchetti, A; Restano-Cassulini, R; Polvani, S; Hofmann, G; Buccoliero, A M; Paglierani, M; Pollo, B; Taddei, G L; Gallina, P; Di Lorenzo, N; Franceschetti, S; Wanke, E; Arcangeli, A

    2005-01-01

    Recent studies have led to considerable advancement in our understanding of the molecular mechanisms that underlie the relentless cell growth and invasiveness of human gliomas. Partial understanding of these mechanisms has (1) improved the classification for gliomas, by identifying prognostic subgroups, and (2) pointed to novel potential therapeutic targets. Some classes of ion channels have turned out to be involved in the pathogenesis and malignancy of gliomas. We studied the expression and properties of K+ channels in primary cultures obtained from surgical specimens: human ether a gò-gò related (hERG)1 voltage-dependent K+ channels, which have been found to be overexpressed in various human cancers, and human ether a gò-gò-like 2 channels, that share many of hERG1's biophysical features. The expression pattern of these two channels was compared to that of the classical inward rectifying K+ channels, IRK, that are widely expressed in astrocytic cells and classically considered a marker of astrocytic differentiation. In our study, hERG1 was found to be specifically overexpressed in high-grade astrocytomas, that is, glioblastoma multiforme (GBM). In addition, we present evidence that, in GBM cell lines, hERG1 channel activity actively contributes to malignancy by promoting vascular endothelial growth factor secretion, thus stimulating the neoangiogenesis typical of high-grade gliomas. Our data provide important confirmation for studies proposing the hERG1 channel as a molecular marker of tumour progression and a possible target for novel anticancer therapies. PMID:16175187

  9. Single-wall carbon nanohorns (SWNHs) inhibited proliferation of human glioma cells and promoted its apoptosis

    Li, Yunjun [The Military General Hospital of Beijing PLA, Affiliated Bayi Brain Hospital (China); Zhang, Jinqian, E-mail: jingwanghou@yahoo.com.cn [Capital Medical University, Institute of Infectious Diseases, Beijing Ditan Hospital (China); Zhao, Ming [Peking University, Department of Chemical Biology, School of Pharmaceutical Sciences (China); Shi, Zujin [Peking University, Beijing National Laboratory for Molecular Sciences, State Key Laboratory of Rare Earth Materials Chemistry and Applications, College of Chemistry and Molecular Engineering (China); Chen, Xin; He, Xihui; Han, Nanyin, E-mail: jingwanghou@sina.com [Peking University, Department of Chemical Biology, School of Pharmaceutical Sciences (China); Xu, Ruxiang, E-mail: everbright999@163.com [The Military General Hospital of Beijing PLA, Affiliated Bayi Brain Hospital (China)

    2013-08-15

    Although single-wall carbon nanohorns (SWNHs) have been demonstrated to accumulate to cytotoxic levels within organs of various animal models and cell types, they have been exploited for cancer therapies. The role of SWNHs in human glioma cell lines was unclear. To address this question, the research about direct role of SWNHs on the growth, proliferation, and apoptosis of human glioma cell lines (U87, U251, and U373) had been performed. Our results indicate that particle size of SWNHs in water is between 342 and 712 nm, the films of SEM show that SWNHs on PS surface are individual particles. SWNHs significantly delayed mitotic entry of human glioma cell lines cells, and inhibited its proliferation in a time- and dose-dependent manner. SWNHs induced a significant increase in G1 phase and inhibition of S phase followed the gradually increasing concentrations. SWNHs in human glioma cell lines cells significantly induced apoptosis followed by their gradually increasing concentrations. The TEM images showed that individual spherical SWNHs particles smaller than 100 nm in diameters were localized inside lysosomes of human glioma cell lines. SWNHs inhibited mitotic entry, growth, and proliferation of human glioma cell lines, and promoted its apoptosis. SWNHs may be a novel opportunity or method for the research on treatment of human glioma.

  10. Single-wall carbon nanohorns (SWNHs) inhibited proliferation of human glioma cells and promoted its apoptosis

    Li, Yunjun; Zhang, Jinqian; Zhao, Ming; Shi, Zujin; Chen, Xin; He, Xihui; Han, Nanyin; Xu, Ruxiang

    2013-08-01

    Although single-wall carbon nanohorns (SWNHs) have been demonstrated to accumulate to cytotoxic levels within organs of various animal models and cell types, they have been exploited for cancer therapies. The role of SWNHs in human glioma cell lines was unclear. To address this question, the research about direct role of SWNHs on the growth, proliferation, and apoptosis of human glioma cell lines (U87, U251, and U373) had been performed. Our results indicate that particle size of SWNHs in water is between 342 and 712 nm, the films of SEM show that SWNHs on PS surface are individual particles. SWNHs significantly delayed mitotic entry of human glioma cell lines cells, and inhibited its proliferation in a time- and dose-dependent manner. SWNHs induced a significant increase in G1 phase and inhibition of S phase followed the gradually increasing concentrations. SWNHs in human glioma cell lines cells significantly induced apoptosis followed by their gradually increasing concentrations. The TEM images showed that individual spherical SWNHs particles smaller than 100 nm in diameters were localized inside lysosomes of human glioma cell lines. SWNHs inhibited mitotic entry, growth, and proliferation of human glioma cell lines, and promoted its apoptosis. SWNHs may be a novel opportunity or method for the research on treatment of human glioma.

  11. Estradiol Receptors Regulate Differential Connexin 43 Expression in F98 and C6 Glioma Cell Lines.

    Zahra Moinfar

    Full Text Available Glioma is the most common malignant primary brain tumour with male preponderance and poor prognosis. Glioma cells express variable amounts of connexin 43 (Cx43 and estrogen receptors (ERs. Both, Cx43 and ERs, play important roles in cell proliferation and migration. Therefore, we investigated the effects of 17-ß estradiol (E2 on Cx43 expression in two glioma cell lines with variable native expression of Cx43.F98 and C6 rat glioma cells were cultured for 24 h in the presence of 10 nM or 100 nM E2, and the E2-antagonist, Fulvestrant. An MTT assay was performed to evaluate cell viability. ERα, ERβ and Cx43 protein expressions were analysed by western blotting and Cx43 mRNA expression was analysed by real-time polymerase chain reaction. To quantify cell migration, an exclusive zone migration assay was used. Functional coupling of cells via gap junctions was examined using whole-cell patch-clamp technique.E2 reduced Cx43 expression in C6 cells, but increased Cx43 expression in F98 cultures. These effects were mediated via ERs. Moreover, E2 promoted C6 cell migration, but it did not affect F98 cell migration. The expression level of ERα was found to be high in C6, but low in F98 cells. ERβ was exclusively expressed in C6 cells. In addition, E2 treatment induced a significant decrease of ERβ in C6 cultures, while it decreased ERα expression in F98 glioma cells.These findings show that E2 differentially modulates Cx43 expression in F98 and C6 glioma cells, likely due to the differential expression of ERs in each of these cell lines. Our findings point to the molecular mechanisms that might contribute to the gender-specific differences in the malignancy of glioma and could have implications for therapeutic strategies against glioma.

  12. CD44 Interacts with HIF-2α to Modulate the Hypoxic Phenotype of Perinecrotic and Perivascular Glioma Cells

    Johansson, Elinn; Grassi, Elisa S.; Pantazopoulou, Vasiliki

    2017-01-01

    Hypoxia-inducible factors enhance glioma stemness, and glioma stem cells have an amplified hypoxic response despite residing within a perivascular niche. Still, little is known about differential HIF regulation in stem versus bulk glioma cells. We show that the intracellular domain of stem cell...... marker CD44 (CD44ICD) is released at hypoxia, binds HIF-2α (but not HIF-1α), enhances HIF target gene activation, and is required for hypoxia-induced stemness in glioma. In a glioma mouse model, CD44 was restricted to hypoxic and perivascular tumor regions, and in human glioma, a hypoxia signature...... correlated with CD44. The CD44ICD was sufficient to induce hypoxic signaling at perivascular oxygen tensions, and blocking CD44 cleavage decreased HIF-2α stabilization in CD44-expressing cells. Our data indicate that the stem cell marker CD44 modulates the hypoxic response of glioma cells and that the pseudo-hypoxic...

  13. Intraoperative neuropathology of glioma recurrence: cell detection and classification

    Abas, Fazly S.; Gokozan, Hamza N.; Goksel, Behiye; Otero, Jose J.; Gurcan, Metin N.

    2016-03-01

    Intraoperative neuropathology of glioma recurrence represents significant visual challenges to pathologists as they carry significant clinical implications. For example, rendering a diagnosis of recurrent glioma can help the surgeon decide to perform more aggressive resection if surgically appropriate. In addition, the success of recent clinical trials for intraoperative administration of therapies, such as inoculation with oncolytic viruses, may suggest that refinement of the intraoperative diagnosis during neurosurgery is an emerging need for pathologists. Typically, these diagnoses require rapid/STAT processing lasting only 20-30 minutes after receipt from neurosurgery. In this relatively short time frame, only dyes, such as hematoxylin and eosin (H and E), can be implemented. The visual challenge lies in the fact that these patients have undergone chemotherapy and radiation, both of which induce cytological atypia in astrocytes, and pathologists are unable to implement helpful biomarkers in their diagnoses. Therefore, there is a need to help pathologists differentiate between astrocytes that are cytologically atypical due to treatment versus infiltrating, recurrent, neoplastic astrocytes. This study focuses on classification of neoplastic versus non-neoplastic astrocytes with the long term goal of providing a better neuropathological computer-aided consultation via classification of cells into reactive gliosis versus recurrent glioma. We present a method to detect cells in H and E stained digitized slides of intraoperative cytologic preparations. The method uses a combination of the `value' component of the HSV color space and `b*' component of the CIE L*a*b* color space to create an enhanced image that suppresses the background while revealing cells on an image. A composite image is formed based on the morphological closing of the hue-luminance combined image. Geometrical and textural features extracted from Discrete Wavelet Frames and combined to classify

  14. Neuroglobin Overexpression Inhibits AMPK Signaling and Promotes Cell Anabolism.

    Cai, Bin; Li, Wenjun; Mao, XiaoOu; Winters, Ali; Ryou, Myoung-Gwi; Liu, Ran; Greenberg, David A; Wang, Ning; Jin, Kunlin; Yang, Shao-Hua

    2016-03-01

    Neuroglobin (Ngb) is a recently discovered globin with preferential localization to neurons. Growing evidence indicates that Ngb has distinct physiological functions separate from the oxygen storage and transport roles of other globins, such as hemoglobin and myoglobin. We found increased ATP production and decreased glycolysis in Ngb-overexpressing immortalized murine hippocampal cell line (HT-22), in parallel with inhibition of AMP-activated protein kinase (AMPK) signaling and activation of acetyl-CoA carboxylase (ACC). In addition, lipid and glycogen content was increased in Ngb-overexpressing HT-22 cells. AMPK signaling was also inhibited in the brain and heart from Ngb-overexpressing transgenic mice. Although Ngb overexpression did not change glycogen content in whole brain, glycogen synthase was activated in cortical neurons of Ngb-overexpressing mouse brain and Ngb overexpression primary neurons. Moreover, lipid and glycogen content was increased in hearts derived from Ngb-overexpressing mice. These findings suggest that Ngb functions as a metabolic regulator and enhances cellular anabolism through the inhibition of AMPK signaling.

  15. Effects of irradiation and cisplatin on human glioma spheroids: inhibition of cell proliferation and cell migration

    Fehlauer, Fabian; Muench, Martina; Rades, Dirk; Stalpers, Lukas J. A.; Leenstra, Sieger; van der Valk, Paul; Slotman, Ben; Smid, Ernst J.; Sminia, Peter

    2005-01-01

    Investigation of cell migration and proliferation of human glioma cell line spheroids (CLS) and evaluation of morphology, apoptosis, and immunohistochemical expression of MIB-1, p53, and p21 of organotypic muticellular spheroids (OMS) following cisplatin (CDDP) and irradiation (RT). Spheroids of the

  16. Sensitivity of C6 Glioma Cells Carrying the Human Poliovirus Receptor to Oncolytic Polioviruses.

    Sosnovtseva, A O; Lipatova, A V; Grinenko, N F; Baklaushev, V P; Chumakov, P M; Chekhonin, V P

    2016-10-01

    A humanized line of rat C6 glioma cells expressing human poliovirus receptor was obtained and tested for the sensitivity to oncolytic effects of vaccine strains of type 1, 2, and 3 polioviruses. Presentation of the poliovirus receptor on the surface of C6 glioma cells was shown to be a necessary condition for the interaction of cells with polioviruses, but insufficient for complete poliovirus oncolysis.

  17. Asymmetric Distribution of GFAP in Glioma Multipotent Cells

    Guichet, Pierre-Olivier; Guelfi, Sophie; Ripoll, Chantal; Teigell, Marisa; Sabourin, Jean-Charles; Bauchet, Luc; Rigau, Valérie; Rothhut, Bernard; Hugnot, Jean-Philippe

    2016-01-01

    Asymmetric division (AD) is a fundamental mechanism whereby unequal inheritance of various cellular compounds during mitosis generates unequal fate in the two daughter cells. Unequal repartitions of transcription factors, receptors as well as mRNA have been abundantly described in AD. In contrast, the involvement of intermediate filaments in this process is still largely unknown. AD occurs in stem cells during development but was also recently observed in cancer stem cells. Here, we demonstrate the asymmetric distribution of the main astrocytic intermediate filament, namely the glial fibrillary acid protein (GFAP), in mitotic glioma multipotent cells isolated from glioblastoma (GBM), the most frequent type of brain tumor. Unequal mitotic repartition of GFAP was also observed in mice non-tumoral neural stem cells indicating that this process occurs across species and is not restricted to cancerous cells. Immunofluorescence and videomicroscopy were used to capture these rare and transient events. Considering the role of intermediate filaments in cytoplasm organization and cell signaling, we propose that asymmetric distribution of GFAP could possibly participate in the regulation of normal and cancerous neural stem cell fate. PMID:26953813

  18. Asymmetric Distribution of GFAP in Glioma Multipotent Cells.

    Pierre-Olivier Guichet

    Full Text Available Asymmetric division (AD is a fundamental mechanism whereby unequal inheritance of various cellular compounds during mitosis generates unequal fate in the two daughter cells. Unequal repartitions of transcription factors, receptors as well as mRNA have been abundantly described in AD. In contrast, the involvement of intermediate filaments in this process is still largely unknown. AD occurs in stem cells during development but was also recently observed in cancer stem cells. Here, we demonstrate the asymmetric distribution of the main astrocytic intermediate filament, namely the glial fibrillary acid protein (GFAP, in mitotic glioma multipotent cells isolated from glioblastoma (GBM, the most frequent type of brain tumor. Unequal mitotic repartition of GFAP was also observed in mice non-tumoral neural stem cells indicating that this process occurs across species and is not restricted to cancerous cells. Immunofluorescence and videomicroscopy were used to capture these rare and transient events. Considering the role of intermediate filaments in cytoplasm organization and cell signaling, we propose that asymmetric distribution of GFAP could possibly participate in the regulation of normal and cancerous neural stem cell fate.

  19. Glioma-Associated Oncogene Homolog Inhibitors Have the Potential of Suppressing Cancer Stem Cells of Breast Cancer.

    Jeng, Kuo-Shyang; Jeng, Chi-Juei; Sheen, I-Shyan; Wu, Szu-Hua; Lu, Ssu-Jung; Wang, Chih-Hsuan; Chang, Chiung-Fang

    2018-05-05

    Overexpression of Sonic Hedgehog signaling (Shh) pathway molecules is associated with invasiveness and recurrence in breast carcinoma. Therefore, inhibition of the Shh pathway downstream molecule Glioma-associated Oncogene Homolog (Gli) was investigated for its ability to reduce progression and invasiveness of patient-derived breast cancer cells and cell lines. Human primary breast cancer T2 cells with high expression of Shh signaling pathway molecules were compared with breast cancer line MDA-MB-231 cells. The therapeutic effects of Gli inhibitors were examined in terms of the cell proliferation, apoptosis, cancer stem cells, cell migration and gene expression. Blockade of the Shh signaling pathway could reduce cell proliferation and migration only in MDA-MB-231 cells. Hh pathway inhibitor-1 (HPI-1) increased the percentages of late apoptotic cells in MDA-MB-231 cells and early apoptotic cells in T2 cells. It reduced Bcl2 expression for cell proliferation and increased Bim expression for apoptosis. In addition, Gli inhibitor HPI-1 decreased significantly the percentages of cancer stem cells in T2 cells. HPI-1 worked more effectively than GANT-58 against breast carcinoma cells. In conclusion, HPI-1 could inhibit cell proliferation, reduce cell invasion and decrease cancer stem cell population in breast cancer cells. To target Gli-1 could be a potential strategy to suppress breast cancer stem cells.

  20. Atypical nuclear localization of VIP receptors in glioma cell lines and patients

    Barbarin, Alice; Séité, Paule [Equipe Récepteurs, Régulations et Cellules Tumorales, Université de Poitiers, PBS bât 36, 1 rue Georges Bonnet, TSA 51106, 86073 Poitiers Cedex 9 (France); Godet, Julie [Laboratoire d’anatomie et de cytologie pathologiques, CHU de Poitiers, 2 rue de la Milétrie, 86000 Poitiers (France); Bensalma, Souheyla; Muller, Jean-Marc [Equipe Récepteurs, Régulations et Cellules Tumorales, Université de Poitiers, PBS bât 36, 1 rue Georges Bonnet, TSA 51106, 86073 Poitiers Cedex 9 (France); Chadéneau, Corinne, E-mail: corinne.chadeneau@univ-poitiers.fr [Equipe Récepteurs, Régulations et Cellules Tumorales, Université de Poitiers, PBS bât 36, 1 rue Georges Bonnet, TSA 51106, 86073 Poitiers Cedex 9 (France)

    2014-11-28

    Highlights: • The VIP receptor VPAC1 contains a putative NLS signal. • VPAC1 is predominantly nuclear in GBM cell lines but not VPAC2. • Non-nuclear VPAC1/2 protein expression is correlated with glioma grade. • Nuclear VPAC1 is observed in 50% of stage IV glioma (GBM). - Abstract: An increasing number of G protein-coupled receptors, like receptors for vasoactive intestinal peptide (VIP), are found in cell nucleus. As VIP receptors are involved in the regulation of glioma cell proliferation and migration, we investigated the expression and the nuclear localization of the VIP receptors VPAC1 and VPAC2 in this cancer. First, by applying Western blot and immunofluorescence detection in three human glioblastoma (GBM) cell lines, we observed a strong nuclear staining for the VPAC1 receptor and a weak nuclear VPAC2 receptor staining. Second, immunohistochemical staining of VPAC1 and VPAC2 on tissue microarrays (TMA) showed that the two receptors were expressed in normal brain and glioma tissues. Expression in the non-nuclear compartment of the two receptors significantly increased with the grade of the tumors. Analysis of nuclear staining revealed a significant increase of VPAC1 staining with glioma grade, with up to 50% of GBM displaying strong VPAC1 nuclear staining, whereas nuclear VPAC2 staining remained marginal. The increase in VPAC receptor expression with glioma grades and the enhanced nuclear localization of the VPAC1 receptors in GBM might be of importance for glioma progression.

  1. CXCR7 is induced by hypoxia and mediates glioma cell migration towards SDF-1α

    Esencay, Mine; Sarfraz, Yasmeen; Zagzag, David

    2013-01-01

    Glioblastomas, the most common and malignant brain tumors of the central nervous system, exhibit high invasive capacity, which hinders effective therapy. Therefore, intense efforts aimed at improved therapeutics are ongoing to delineate the molecular mechanisms governing glioma cell migration and invasion. In order to perform the studies, we employed optimal cell culture methods and hypoxic conditions, lentivirus-mediated knockdown of protein expression, Western Blot analysis, migration assays and immunoprecipitation. We determined statistical significance by unpaired t-test. In this report, we show that U87MG, LN229 and LN308 glioma cells express CXCR7 and that exposure to hypoxia upregulates CXCR7 protein expression in these cell lines. CXCR7-expressing U87MG, LN229 and LN308 glioma cells migrated towards stromal-derived factor (SDF)-1α/CXCL12 in hypoxic conditions in the Boyden chamber assays. While shRNA-mediated knockdown of CXCR7 expression did not affect the migration of any of the three cell lines in normoxic conditions, we observed a reduction in the migration of LN229 and LN308, but not U87MG, glioma cells towards SDF-1α in hypoxic conditions. In addition, knockdown of CXCR7 expression in LN229 and LN308 glioma cells decreased levels of SDF-1α-induced phosphorylation of ERK1/2 and Akt. Inhibiting CXCR4 in LN229 and LN308 glioma cells that were knocked down for CXCR7 did not further reduce migration towards SDF-1α in hypoxic conditions and did not affect the levels of phosphorylated ERK1/2 and Akt. Analysis of immunoprecipitated CXCR4 from LN229 and LN308 glioma cells revealed co-precipitated CXCR7. Taken together, our findings indicate that both CXCR4 and CXCR7 mediate glioma cell migration towards SDF-1α in hypoxic conditions and support the development of therapeutic agents targeting these receptors

  2. Mesenchymal Stem Cells Isolated From Human Gliomas Increase Proliferation and Maintain Stemness of Glioma Stem Cells Through the IL-6/gp130/STAT3 Pathway.

    Hossain, Anwar; Gumin, Joy; Gao, Feng; Figueroa, Javier; Shinojima, Naoki; Takezaki, Tatsuya; Priebe, Waldemar; Villarreal, Diana; Kang, Seok-Gu; Joyce, Celine; Sulman, Erik; Wang, Qianghu; Marini, Frank C; Andreeff, Michael; Colman, Howard; Lang, Frederick F

    2015-08-01

    Although mesenchymal stem cells (MSCs) have been implicated as stromal components of several cancers, their ultimate contribution to tumorigenesis and their potential to drive cancer stem cells, particularly in the unique microenvironment of human brain tumors, remain largely undefined. Consequently, using established criteria, we isolated glioma-associated-human MSCs (GA-hMSCs) from fresh human glioma surgical specimens for the first time. We show that these GA-hMSCs are nontumorigenic stromal cells that are phenotypically similar to prototypical bone marrow-MSCs. Low-passage genomic sequencing analyses comparing GA-hMSCs with matched tumor-initiating glioma stem cells (GSCs) suggest that most GA-hMSCs (60%) are normal cells recruited to the tumor (group 1 GA-hMSCs), although, rarely (10%), GA-hMSCs may differentiate directly from GSCs (group 2 GA-hMSCs) or display genetic patterns intermediate between these groups (group 3 GA-hMSCs). Importantly, GA-hMSCs increase proliferation and self-renewal of GSCs in vitro and enhance GSC tumorigenicity and mesenchymal features in vivo, confirming their functional significance within the GSC niche. These effects are mediated by GA-hMSC-secreted interleukin-6, which activates STAT3 in GSCs. Our results establish GA-hMSCs as a potentially new stromal component of gliomas that drives the aggressiveness of GSCs, and point to GA-hMSCs as a novel therapeutic target within gliomas. © 2015 AlphaMed Press.

  3. Forkhead box K2 inhibits the proliferation, migration, and invasion of human glioma cells and predicts a favorable prognosis

    Wang B

    2018-02-01

    Full Text Available Bo Wang,1–3,* XueBin Zhang,2–4,* Wei Wang,1–3 ZhiZhong Zhu,5 Fan Tang,2–4 Dong Wang,6–8 Xi Liu,9 Hao Zhuang,10 XiaoLing Yan2–4 1Department of Neurosurgery, Tianjin Huanhu Hospital, Tianjin, China; 2Tianjin Key Laboratory of Cerebral Vascular and Neurodegenerative Diseases, Tianjin, China; 3Tianjin Neurosurgical Institute, Tianjin, China; 4Department of Pathology, Tianjin Huanhu Hospital, Tianjin, China; 5Department of Rehabilitation, Tianjin Huanhu Hospital, Tianjin, China; 6Department of Neurosurgery, Tianjin Medical University, General Hospital, Tianjin, China; 7Tianjin Key Laboratory of Injuries, Variations and Regeneration of Nervous System, Tianjin, China; 8Tianjin Neurological Institute, Tianjin, China; 9Department of Gastroenterology, Tianjin NanKai Hospital, Tianjin, China; 10Department of Hepatic Biliary Pancreatic Surgery, Cancer Hospital Affiliated to Zhengzhou University, Zhengzhou, China *These authors contributed equally to this work Purpose: Forkhead box K2 (FOXK2 is a member of the forkhead box family of transcription factors. Recently, researchers discovered that overexpression of FOXK2 inhibits the proliferation and metastasis of breast cancer, non-small cell lung cancer, and colorectal cancer, and is related to the clinical prognosis. However, in hepatocellular carcinoma, FOXK2 results in the opposite phenotypes. Currently, the contribution of FOXK2 to glioma pathogenesis is not clear. Patients and methods: We evaluated the expression of FOXK2 in 151 glioma patients using immunohistochemistry assays. The associations among the expression of FOXK2, clinicopathological parameters, and the prognosis of glioma patients were statistically analyzed. We downregulated and upregulated the level of FOXK2 in glioma cells by transfections with small interfering RNA and plasmids. Then, we investigated the effects on tumor cell behavior in vitro by Cell Counting Kit-8 assays, colony-formation assay, transwell assay, and the

  4. Silencing Nrf2 impairs glioma cell proliferation via AMPK-activated mTOR inhibition

    Jia, Yue [Department of Neurosurgery, Jinling Hospital, School of Medicine, Nanjing University, 305 East Zhongshan Road, Nanjing 210002, Jiangsu Province (China); Wang, Handong, E-mail: njhdwang@hotmail.com [Department of Neurosurgery, Jinling Hospital, School of Medicine, Nanjing University, 305 East Zhongshan Road, Nanjing 210002, Jiangsu Province (China); Wang, Qiang [Department of Neurosurgery, Jinling Hospital, School of Medicine, Nanjing University, 305 East Zhongshan Road, Nanjing 210002, Jiangsu Province (China); Ding, Hui [Department of Neurosurgery, Jinling Hospital, School of Medicine, Southern Medical University (Guangzhou), 305 East Zhongshan Road, Nanjing 210002, Jiangsu Province (China); Wu, Heming [Department of Neurosurgery, Nanjing Jingdu Hospital, No. 34, Biao 34, Yanggongjing Road, Nanjing 210002, Jiangsu Province (China); Pan, Hao [Department of Neurosurgery, Jinling Hospital, School of Medicine, Nanjing University, 305 East Zhongshan Road, Nanjing 210002, Jiangsu Province (China)

    2016-01-15

    Gliomas are the leading cause of death among adults with primary brain malignancies. Treatment for malignant gliomas remains limited, and targeted therapies have been incompletely explored. Nuclear factor erythroid 2-related factor 2 (Nrf2), a key transcription regulator for antioxidant and detoxification enzymes, is abundantly expressed in cancer cells. In this study, the role and mechanism of Nrf2 in cancer cell proliferation was investigated in multiple glioma cell lines. We first evaluated the expression patterns of Nrf2 in four glioma cell lines and found all four cell lines expressed Nrf2, but the highest level was observed in U251 cells. We further evaluated the biological functions of Nrf2 in U251 glioma cell proliferation by specific inhibition of Nrf2 using short hairpin RNA (shRNA). We found that Nrf2 depletion inhibited glioma cell proliferation. Nrf2 depletion also decreased colony formation in U251 cells stably expressing Nrf2 shRNA compared to scrambled control shRNA. Moreover, suppression of Nrf2 expression could lead to ATP depletion (with concomitant rise in AMP/ATP ratio) and consequently to AMPK-activated mTOR inhibition. Finally, activation of adenosine monophosphate–activated protein kinase (AMPK) by treated with phenformin, an AMPK agonist, can mimic the inhibitory effect of Nrf2 knockdown in U251 cells. In conclusion, our findings will shed light to the role and mechanism of Nrf2 in regulating glioma proliferation via ATP-depletion-induced AMPK activation and consequent mTOR inhibition, a novel insight into our understanding the role and mechanism of Nrf2 in glioma pathoetiology. To our knowledge, this is also the first report to provide a rationale for the implication of cross-linking between Nrf2 and mTOR signaling.

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

    Chang, Ji Hyun [Department of Radiation Oncology, Graduate School of Medicine, Seoul National University, Seoul (Korea, Republic of); Hwang, Yeo Hyun; Lee, David J.; Kim, Dan Hyo; Park, Ji Min [Medical Science Research Institute, Seoul National University Bundang Hospital, Kyeonggido (Korea, Republic of); Wu, Hong-Gyun [Department of Radiation Oncology, Graduate School of Medicine, Seoul National University, Seoul (Korea, Republic of); Kim, In Ah, E-mail: inah228@snu.ac.kr [Department of Radiation Oncology, Graduate School of Medicine, Seoul National University, Seoul (Korea, Republic of); Medical Science Research Institute, Seoul National University Bundang Hospital, Kyeonggido (Korea, Republic of); Cancer Research Institute, Seoul National University, Seoul (Korea, Republic of)

    2016-02-01

    Purpose: We investigated whether miR-203 could modulate the radiation sensitivity of glioblastoma (GBM) cells and which target gene(s) could be involved. Methods and Materials: 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. Results: 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. Conclusions: 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.

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

    Chang, Ji Hyun; Hwang, Yeo Hyun; Lee, David J.; Kim, Dan Hyo; Park, Ji Min; Wu, Hong-Gyun; Kim, In Ah

    2016-01-01

    Purpose: We investigated whether miR-203 could modulate the radiation sensitivity of glioblastoma (GBM) cells and which target gene(s) could be involved. Methods and Materials: 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. Results: 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. Conclusions: 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.

  7. GCN5 Potentiates Glioma Proliferation and Invasion via STAT3 and AKT Signaling Pathways

    Kun Liu

    2015-09-01

    Full Text Available The general control of nucleotide synthesis 5 (GCN5, which is one kind of lysine acetyltransferases, regulates a number of cellular processes, such as cell proliferation, differentiation, cell cycle and DNA damage repair. However, its biological role in human glioma development remains elusive. In the present study, we firstly reported that GCN5 was frequently overexpressed in human glioma tissues and GCN5 was positively correlated with proliferation of cell nuclear antigen PCNA and matrix metallopeptidase MMP9. Meanwhile, down-regulation of GCN5 by siRNA interfering inhibited glioma cell proliferation and invasion. In addition, GCN5 knockdown reduced expression of p-STAT3, p-AKT, PCNA and MMP9 and increased the expression of p21 in glioma cells. In conclusion, GCN5 exhibited critical roles in glioma development by regulating cell proliferation and invasion, which suggested that GCN5 might be a potential molecular target for glioma treatment.

  8. Saw Palmetto Extract Inhibits Metastasis and Antiangiogenesis through STAT3 Signal Pathway in Glioma Cell

    Hong Ding

    2015-01-01

    Full Text Available Signal transducer and activator of transcription factor 3 (STAT3 plays an important role in the proliferation and angiogenesis in human glioma. Previous research indicated that saw palmetto extract markedly inhibited the proliferation of human glioma cells through STAT3 signal pathway. But its effect on tumor metastasis and antiangiogenesis is not clear. This study is to further clear the impact of saw palmetto extract on glioma cell metastasis, antiangiogenesis, and its mechanism. TUNEL assay indicated that the apoptotic cells in the saw palmetto treated group are higher than that in the control group (p<0.05. The apoptosis related protein is detected and the results revealed that saw palmetto extract inhibits the proliferation of human glioma. Meanwhile pSTAT3 is lower in the experimental group and CD34 is also inhibited in the saw palmetto treated group. This means that saw palmetto extract could inhibit the angiogenesis in glioma. We found that saw palmetto extract was an important phytotherapeutic drug against the human glioma through STAT3 signal pathway. Saw palmetto extract may be useful as an adjunctive therapeutic agent for treatment of individuals with glioma and other types of cancer in which STAT3 signaling is activated.

  9. Saw Palmetto Extract Inhibits Metastasis and Antiangiogenesis through STAT3 Signal Pathway in Glioma Cell.

    Ding, Hong; Shen, Jinglian; Yang, Yang; Che, Yuqin

    2015-01-01

    Signal transducer and activator of transcription factor 3 (STAT3) plays an important role in the proliferation and angiogenesis in human glioma. Previous research indicated that saw palmetto extract markedly inhibited the proliferation of human glioma cells through STAT3 signal pathway. But its effect on tumor metastasis and antiangiogenesis is not clear. This study is to further clear the impact of saw palmetto extract on glioma cell metastasis, antiangiogenesis, and its mechanism. TUNEL assay indicated that the apoptotic cells in the saw palmetto treated group are higher than that in the control group (p saw palmetto extract inhibits the proliferation of human glioma. Meanwhile pSTAT3 is lower in the experimental group and CD34 is also inhibited in the saw palmetto treated group. This means that saw palmetto extract could inhibit the angiogenesis in glioma. We found that saw palmetto extract was an important phytotherapeutic drug against the human glioma through STAT3 signal pathway. Saw palmetto extract may be useful as an adjunctive therapeutic agent for treatment of individuals with glioma and other types of cancer in which STAT3 signaling is activated.

  10. Predominant antitumor effects by fully human anti-TRAIL-receptor2 (DR5) monoclonal antibodies in human glioma cells in vitro and in vivo

    Nagane, Motoo; Shimizu, Saki; Mori, Eiji; Kataoka, Shiro; Shiokawa, Yoshiaki

    2010-01-01

    Tumor necrosis factor–related apoptosis-inducing ligand (TRAIL/Apo2 L) preferentially induces apoptosis in human tumor cells through its cognate death receptors DR4 or DR5, thereby being investigated as a potential agent for cancer therapy. Here, we applied fully human anti-human TRAIL receptor monoclonal antibodies (mAbs) to specifically target one of death receptors for TRAIL in human glioma cells, which could also reduce potential TRAIL-induced toxicity in humans. Twelve human glioma cell lines treated with several fully human anti-human TRAIL receptor mAbs were sensitive to only anti-DR5 mAbs, whereas they were totally insensitive to anti-DR4 mAb. Treatment with anti-DR5 mAbs exerted rapid cytotoxicity and lead to apoptosis induction. The cellular sensitivity was closely associated with cell-surface expression of DR5. Expression of c-FLIPL, Akt, and Cyclin D1 significantly correlated with sensitivity to anti-DR5 mAbs. Primary cultures of glioma cells were also relatively resistant to anti-DR5 mAbs, exhibiting both lower DR5 and higher c-FLIPL expression. Downregulation of c-FLIPL expression resulted in the sensitization of human glioma cells to anti-DR5 mAbs, whereas overexpression of c-FLIPL conferred resistance to anti-DR5 mAb. Treatment of tumor-burden nude mice with the direct agonist anti-DR5 mAb KMTR2 significantly suppressed growth of subcutaneous glioma xenografts leading to complete regression. Similarly, treatment of nude mice bearing intracerebral glioma xenografts with KMTR2 significantly elongated lifespan without tumor recurrence. These results suggest that DR5 is the predominant TRAIL receptor mediating apoptotic signals in human glioma cells, and sensitivity to anti-DR5 mAbs was determined at least in part by the expression level of c-FLIPL and Akt. Specific targeting of death receptor pathway through DR5 using fully human mAbs might provide a novel therapeutic strategy for intractable malignant gliomas. PMID:20511188

  11. Methylation of the ATM promoter in glioma cells alters ionizing radiation sensitivity

    Roy, Kanaklata; Wang, Lilin; Makrigiorgos, G. Mike; Price, Brendan D.

    2006-01-01

    Glioblastomas are among the malignancies most resistant to radiation therapy. In contrast, cells lacking the ATM protein are highly sensitive to ionizing radiation. The relationship between ATM protein expression and radiosensitivity in 3 glioma cell lines was examined. T98G cells exhibited normal levels of ATM protein, whereas U118 and U87 cells had significantly lower levels of ATM and increased (>2-fold) sensitivity to ionizing radiation compared to T98G cells. The ATM promoter was methylated in U87 cells. Demethylation by azacytidine treatment increased ATM protein levels in the U87 cells and decreased their radiosensitivity. In contrast, the ATM promoter in U118 cells was not methylated. Further, expression of exogenous ATM did not significantly alter the radiosensitivity of U118 cells. ATM expression is therefore heterogeneous in the glioma cells examined. In conclusion, methylation of the ATM promoter may account for the variable radiosensitivity and heterogeneous ATM expression in a fraction of glioma cells

  12. Glioma Cells in the Tumor Periphery Have a Stem Cell Phenotype

    Munthe, Sune; Petterson, Stine Asferg; Dahlrot, Rikke Hedegaard

    2016-01-01

    and a panel of markers was used. The panel comprised of six stem cell-related markers (CD133, Musashi-1, Bmi-1, Sox-2, Nestin and Glut-3), a proliferation marker (Ki-67) as well as a chemo-resistance marker (MGMT). Computer-based automated classifiers were designed to measure the mIDH1 positive nucleus area......-fraction of the chosen markers. Moreover, orthotopic glioblastoma xenografts from five different patient-derived spheroid cultures were obtained and the tumor cells identified by human specific immunohistochemical markers. The results showed that tumor cells in the periphery of patient gliomas expressed stem cell...... in the periphery of patient gliomas have a stem cell phenotype, although it is less pronounced than in the tumor core. Novel therapies aiming at preventing recurrence should therefore take tumor stemness into account. Migrating cells in orthotopic glioblastoma xenografts preserve expression and stem cell markers...

  13. Autologous glioma cell vaccine admixed with interleukin-4 gene transfected fibroblasts in the treatment of patients with malignant gliomas

    Torres-Trejo Alejandro

    2007-12-01

    Full Text Available Abstract Background The prognosis for malignant gliomas remains dismal. We addressed the safety, feasibility and preliminary clinical activity of the vaccinations using autologous glioma cells and interleukin (IL-4 gene transfected fibroblasts. Methods In University of Pittsburgh Cancer Institute (UPCI protocol 95-033, adult participants with recurrent glioblastoma multiforme (GBM or anaplastic astrocytoma (AA received gross total resection (GTR of the recurrent tumors, followed by two vaccinations with autologous fibroblasts retrovirally transfected with TFG-IL4-Neo-TK vector admixed with irradiated autologous glioma cells. In UPCI 99-111, adult participants with newly diagnosed GBM or AA, following GTR and radiation therapy, received two intradermal vaccinations with the TFG-IL4-Neo-TK-transfected fibroblasts admixed with type-1 dendritic cells (DC loaded with autologous tumor lysate. The participants were evaluated for occurrence of adverse events, immune response, and clinical response by radiological imaging. Results and Discussion In UPCI 95-033, only 2 of 6 participants received the vaccinations. Four other participants were withdrawn from the trial because of tumor progression prior to production of the cellular vaccine. However, both participants who received two vaccinations demonstrated encouraging immunological and clinical responses. Biopsies from the local vaccine sites from one participant displayed IL-4 dose-dependent infiltration of CD4+ as well as CD8+ T cells. Interferon (IFN-γ Enzyme-Linked Immuno-SPOT (ELISPOT assay in another human leukocyte antigen (HLA-A2+ participant demonstrated systemic T-cell responses against an HLA-A2-restricted glioma-associated antigen (GAA epitope EphA2883–891. Moreover, both participants demonstrated clinical and radiological improvement with no evidence of allergic encephalitis, although both participants eventually succumbed with the tumor recurrence. In 99-111, 5 of 6 enrolled participants

  14. Characterization of NCAM expression and function in BT4C and BT4Cn glioma cells

    Andersson, A M; Moran, N; Gaardsvoll, H

    1991-01-01

    The neural cell adhesion molecule, NCAM, plays an important role in cell-cell adhesion. Therefore, we have studied NCAM expression in the glioma cell lines BT4C and BT4Cn. We demonstrate that the 2 cell lines differ in their metastatic ability; while BT4C cells have a very low capacity for produc...

  15. Molecular Alterations of KIT Oncogene in Gliomas

    Ana L. Gomes

    2007-01-01

    Full Text Available Gliomas are the most common and devastating primary brain tumours. Despite therapeutic advances, the majority of gliomas do not respond either to chemo or radiotherapy. KIT, a class III receptor tyrosine kinase (RTK, is frequently involved in tumourigenic processes. Currently, KIT constitutes an attractive therapeutic target. In the present study we assessed the frequency of KIT overexpression in gliomas and investigated the genetic mechanisms underlying KIT overexpression. KIT (CD117 immunohistochemistry was performed in a series of 179 gliomas of various grades. KIT activating gene mutations (exons 9, 11, 13 and 17 and gene amplification analysis, as defined by chromogenic in situ hybridization (CISH and quantitative real-time PCR (qRT-PCR were performed in CD117 positive cases. Tumour cell immunopositivity was detected in 15.6% (28/179 of cases, namely in 25% (1/4 of pilocytic astrocytomas, 25% (5/20 of diffuse astrocytomas, 20% (1/5 of anaplastic astrocytomas, 19.5% (15/77 of glioblastomas and one third (3/9 of anaplastic oligoastrocytomas. Only 5.7% (2/35 of anaplastic oligodendrogliomas showed CD117 immunoreactivity. No association was found between tumour CD117 overexpression and patient survival. In addition, we also observed CD117 overexpression in endothelial cells, which varied from 0–22.2% of cases, being more frequent in high-grade lesions. No KIT activating mutations were identified. Interestingly, CISH and/or qRT-PCR analysis revealed the presence of KIT gene amplification in 6 glioblastomas and 2 anaplastic oligoastrocytomas, corresponding to 33% (8/24 of CD117 positive cases. In conclusion, our results demonstrate that KIT gene amplification rather than gene mutation is a common genetic mechanism underlying KIT expression in subset of malignant gliomas. Further studies are warranted to determine whether glioma patients exhibiting KIT overexpression and KIT gene amplification may benefit from therapy with anti-KIT RTK

  16. Growth inhibition and chemosensitization of exogenous nitric oxide released from NONOates in glioma cells in vitro.

    Weyerbrock, Astrid; Baumer, Brunhilde; Papazoglou, Anna

    2009-01-01

    Exogenous nitric oxide (NO) from NO donors has cytotoxic, chemosensitizing, and radiosensitizing effects, and increases vascular permeability and blood flow in tumors. Yet little is known about whether these cytotoxic and chemosensitizing effects can be observed in glioma cells at doses that alter tumor physiological characteristics in vivo and whether these effects are tumor selective. The effect of NO released from proline NONOate, diethylamine NONOate, spermine NONOate, and sodium nitrite on cell proliferation, apoptosis, and chemosensitivity to carboplatin of cultured glioma cells was studied in C6, U87 glioma cells, human glioblastoma cells, and human astrocytes and fibroblasts. Although proline NONOate failed to induce cell death, the other NO donors induced growth arrest when present in high concentrations (10(-2) M) in all cell lines. Chemosensitization was observed after concomitant incubation with spermine NONOate and carboplatin in C6 and human glioblastoma cells. There is strong evidence that cell death occurs primarily by necrosis and to a lesser degree by apoptosis. The NO doses, which altered tumor physiology in vivo, were not cytotoxic, indicating that NO alters vascular permeability and cell viability in vivo by different mechanisms. The authors found that NO-generating agents at high concentrations are potent growth inhibitors and might also be useful as chemosensitizers in glioma cells. These data corroborate the theory that the use of NOgenerating agents may play a role in the multimodal treatment of malignant gliomas but that the NO release must be targeted more specifically to tumor cells to improve selectivity and efficacy.

  17. Melatonin inhibits proliferation and invasion via repression of miRNA-155 in glioma cells.

    Gu, Junyi; Lu, Zhongsheng; Ji, Chenghong; Chen, Yuchao; Liu, Yuzhao; Lei, Zhe; Wang, Longqiang; Zhang, Hong-Tao; Li, Xiangdong

    2017-09-01

    Melatonin, an indolamine mostly synthesized in the pineal gland, exerts the anti-cancer effect by various mechanisms in glioma cells. Our previous study showed that miR-155 promoted glioma cell proliferation and invasion. However, the question of whether melatonin may inhibit glioma by regulating miRNAs has not yet been addressed. In this study, we found that melatonin (100μM, 1μM and 1nM) significantly inhibited the expression of miR-155 in human glioma cell lines U87, U373 and U251. Especially, the lowest expression of miR-155 was detected in 1μM melatonin-treated glioma cells. Melatonin (1μM) inhibits cell proliferation of U87 by promoting cell apoptosis. Nevertheless, melatonin had no effect on cell cycle distribution of U87 cells. Moreover, U87 cells treated with 1μM melatonin presented significantly lower migration and invasion ability when compared with control cells. Importantly, melatonin inhibited c-MYB expression, and c-MYB knockdown reduced miR-155 expression and migration and invasion in U87 cells. Taken together, for the first time, our findings show that melatonin inhibits miR-155 expression and thereby represses glioma cell proliferation, migration and invasion, and suggest that melatonin may downregulate the expression of miR-155 via repression of c-MYB. This will provide a theoretical basis for revealing the anti-glioma mechanisms of melatonin. Copyright © 2017. Published by Elsevier Masson SAS.

  18. TRIM8 downregulation in glioma affects cell proliferation and it is associated with patients survival

    Micale, Lucia; Fusco, Carmela; Fontana, Andrea; Barbano, Raffaela; Augello, Bartolomeo; De Nittis, Pasquelena; Copetti, Massimiliano; Pellico, Maria Teresa; Mandriani, Barbara; Cocciadiferro, Dario; Parrella, Paola; Fazio, Vito Michele; Dimitri, Lucia Maria Cecilia; D’Angelo, Vincenzo; Novielli, Chiara; Larizza, Lidia; Daga, Antonio; Merla, Giuseppe

    2015-01-01

    Human gliomas are a heterogeneous group of primary malignant brain tumors whose molecular pathogenesis is not yet solved. In this regard, a major research effort has been directed at identifying novel specific glioma-associated genes. Here, we investigated the effect of TRIM8 gene in glioma. TRIM8 transcriptional level was profiled in our own glioma cases collection by qPCR and confirmed in the independent TCGA glioma cohort. The association between TRIM8 expression and Overall Survival and Progression-free Survival in TCGA cohort was determined by using uni-multivariable Cox regression analysis. The effect of TRIM8 on patient glioma cell proliferation was evaluated by performing MTT and clonogenic assays. The mechanisms causing the reduction of TRIM8 expression were explored by using qPCR and in vitro assays. We showed that TRIM8 expression correlates with unfavorable clinical outcome in glioma patients. We found that a restored TRIM8 expression induced a significant reduction of clonogenic potential in U87MG and patient’s glioblastoma cells. Finally we provide experimental evidences showing that miR-17 directly targets the 3′ UTR of TRIM8 and post-transcriptionally represses the expression of TRIM8. Our study provides evidences that TRIM8 may participate in the carcinogenesis and progression of glioma and that the transcriptional repression of TRIM8 might have potential value for predicting poor prognosis in glioma patients. The online version of this article (doi:10.1186/s12885-015-1449-9) contains supplementary material, which is available to authorized users

  19. Transfection of glioma cells with the neural-cell adhesion molecule NCAM

    Edvardsen, K; Pedersen, P H; Bjerkvig, R

    1994-01-01

    The tumor growth and the invasive capacity of a rat glioma cell line (BT4Cn) were studied after transfection with the human transmembrane 140-kDa isoform of the neural-cell adhesion molecule, NCAM. After s.c. injection, the NCAM-transfected cells showed a slower growth rate than the parent cell...... of the injection site, with a sharply demarcated border between the tumor and brain tissue. In contrast, the parental cell line showed single-cell infiltration and more pronounced destruction of normal brain tissue. Using a 51Cr-release assay, spleen cells from rats transplanted with BT4Cn tumor cells generally...

  20. Demethoxycurcumin Retards Cell Growth and Induces Apoptosis in Human Brain Malignant Glioma GBM 8401 Cells

    Tzuu-Yuan Huang

    2012-01-01

    Full Text Available Demethoxycurcumin (DMC; a curcumin-related demethoxy compound has been recently shown to display antioxidant and antitumor activities. It has also produced a potent chemopreventive action against cancer. In the present study, the antiproliferation (using the MTT assay, DMC was found to have cytotoxic activities against GBM 8401 cell with IC50 values at 22.71 μM and induced apoptosis effects of DMC have been investigated in human brain malignant glioma GBM 8401 cells. We have studied the mitochondrial membrane potential (MMP, DNA fragmentation, caspase activation, and NF-κB transcriptional factor activity. By these approaches, our results indicated that DMC has produced an inhibition of cell proliferation as well as the activation of apoptosis in GBM 8401 cells. Both effects were observed to increase in proportion with the dosage of DMC treatment, and the apoptosis was induced by DMC in human brain malignant glioma GBM 8401 cells via mitochondria- and caspase-dependent pathways.

  1. The role of p97 in iron metabolism in human brain glioma cells

    Xia Chunlin; Chen Guiwen; Qian Zhongming

    2000-01-01

    Objective: To investigate the role of p97 (melanotransferrin) in iron uptake in human brain glioma cells . Methods: Human brain glioma cell lines, GBM and BT325 were incubated in the medium containing 59 Fe-Citrate. The cells were treated with phosphatidylinositol-phospholipase C (PI-PLC) and pronase. The iron uptake of the cells was expressed as relative iron uptake level according to the cpm measured by the gamma scintillation counter. Results: 59 Fe uptake of the cells was significantly declined with the certain concentration of PI-PCL. 59 Fe uptake of the cells treated with pronase tended to coincide with that of the cells treated without pronase in the increasing concentration of PI-PLC. Conclusion: p97 expresses a high level and plays an important role in iron uptake in human brain glioma cells

  2. Arctigenin, a natural lignan compound, induces G0/G1 cell cycle arrest and apoptosis in human glioma cells

    Maimaitili, Aisha; Shu, Zunhua; Cheng, Xiaojiang; Kaheerman, Kadeer; Sikandeer, Alifu; Li, Weimin

    2016-01-01

    The aim of the current study was to investigate the anticancer potential of arctigenin, a natural lignan compound, in malignant gliomas. The U87MG and T98G human glioma cell lines were treated with various concentrations of arctigenin for 48 h and the effects of arctigenin on the aggressive phenotypes of glioma cells were assessed. The results demonstrated that arctigenin dose-dependently inhibited the growth of U87MG and T98G cells, as determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphen...

  3. PTEN-induction in U251 glioma cells decreases the expression of insulin-like growth factor binding protein-2

    Levitt, Randy J.; Georgescu, Maria-Magdalena; Pollak, Michael

    2005-01-01

    PTEN is a tumor suppressor gene whose loss of function is observed in ∼40-50% of human cancers. Although insulin-like growth factor binding protein-2 (IGFBP-2) was classically described as a growth inhibitor, multiple recent reports have shown an association of overexpression and/or high serum levels of IGFBP-2 with poor prognosis of several malignancies, including gliomas. Using an inducible PTEN expression system in the PTEN-null glioma cell line U251, we demonstrate that PTEN-induction is associated with reduced proliferation, increased apoptosis, and a substantial reduction of the high levels of IGFBP-2 expression. The PTEN-induced decrease in IGFBP-2 expression could be mimicked with the PI3-kinase inhibitor LY294002, indicating that the lipid phosphatase activity of PTEN is responsible for the observed effect. However, the rapamycin analog CCI-779 did not affect IGFBP-2 expression, suggesting that the PTEN-induced decrease in IGFBP-2 expression is not attributable to decreased mTOR signalling. Recombinant human IGFBP-2 was unable to rescue U251-PTEN cells from the antiproliferative effects of PTEN, and IGFBP-2 siRNA did not affect the IGF-dependent or -independent growth of this cell line. These results suggest that the clinical data linking IGFBP-2 expression to poor prognosis may arise, at least in part, because high levels of IGFBP-2 expression correlate with loss of function of PTEN, which is well known to lead to aggressive behavior of gliomas. Our results motivate translational research regarding the relationship between IGFBP-2 expression and loss of function of PTEN

  4. Systemic T Cells Immunosuppression of Glioma Stem Cell-Derived Exosomes Is Mediated by Monocytic Myeloid-Derived Suppressor Cells.

    Rossana Domenis

    Full Text Available A major contributing factor to glioma development and progression is its ability to evade the immune system. Nano-meter sized vesicles, exosomes, secreted by glioma-stem cells (GSC can act as mediators of intercellular communication to promote tumor immune escape. Here, we investigated the immunomodulatory properties of GCS-derived exosomes on different peripheral immune cell populations. Healthy donor peripheral blood mononuclear cells (PBMCs stimulated with anti-CD3, anti-CD28 and IL-2, were treated with GSC-derived exosomes. Phenotypic characterization, cell proliferation, Th1/Th2 cytokine secretion and intracellular cytokine production were analysed by distinguishing among effector T cells, regulatory T cells and monocytes. In unfractionated PBMCs, GSC-derived exosomes inhibited T cell activation (CD25 and CD69 expression, proliferation and Th1 cytokine production, and did not affect cell viability or regulatory T-cell suppression ability. Furthermore, exosomes were able to enhance proliferation of purified CD4+ T cells. In PBMCs culture, glioma-derived exosomes directly promoted IL-10 and arginase-1 production and downregulation of HLA-DR by unstimulated CD14+ monocytic cells, that displayed an immunophenotype resembling that of monocytic myeloid-derived suppressor cells (Mo-MDSCs. Importantly, the removal of CD14+ monocytic cell fraction from PBMCs restored T-cell proliferation. The same results were observed with exosomes purified from plasma of glioblastoma patients. Our results indicate that glioma-derived exosomes suppress T-cell immune response by acting on monocyte maturation rather than on direct interaction with T cells. Selective targeting of Mo-MDSC to treat glioma should be considered with regard to how immune cells allow the acquirement of effector functions and therefore counteracting tumor progression.

  5. Cytolytic effects of autologous lymphokine-activated killer cells on organotypic multicellular spheroids of gliomas in vitro

    Kaaijk, P.; Troost, D.; Dast, P. K.; van den Berg, F.; Leenstra, S.; Bosch, D. A.

    1995-01-01

    Knowledge about lymphokine-activated killer (LAK) cell infiltration and LAK cell cytotoxicity is essential to improve the effectiveness of LAK cell therapy against gliomas. In the present study, organotypic multicellular spheroids (OMS) of glioma tissue were used as a culture model to study the

  6. Inhibitory effect of calcium channel blockers on proliferation of human glioma cells in vitro

    Kunert-Radek, J.; Stepien, H.; Lyson, K.; Pawlikowski, M.; Radek, A.

    1989-01-01

    The effects of 2 specific calcium channel blockers, verapamil and nimodipine, on the proliferation of human glioma tumour cells were investigated in vitro. Tumour tissues for primary cell cultures were obtained bioptically from 3 patients with the histopathological diagnosis of glioblastoma. The [ 3 H]-thymidine incorporation into glioma tumour cells DNA was used as a sensitive index of the cell proliferation. It was found that varapamil (10 4 -10 5 M) and nimodipine (10 4 -10 6 M) significantly inhibited the [ 3 H]-thymidine uptake in a dose-related manner. The inhibitory effect of both calcium channel antagonists was reversed by stimultancous addition of calcium chloride (5x10 3 M). These results indicate that verapamil and nimodipine may exert an antiproliferative effect on glioma cells growth acting through a blokade of specific voltage-dependent calcium channels. (author)

  7. Effects of liposome-adriamycin (L-ADM) and thermotherapy on glioma cells: an experimental study

    Zheng-hua SHI; Jian-ning ZHANG

    2013-01-01

    Objective  To observe the effects of liposome-adriamycin (L-ADM) and thermotherapy on proliferation and apoptosis of SWO-38 glioma cells. Methods  The SWO-38 glioma cells were cultivated in vitro. The effects of thermotherapy (43℃), ADM chemotherapy, L-ADM chemotherapy, thermotherapy + ADM chemotherapy, and thermotherapy + L-ADM chemotherapy on the cell proliferation and apoptosis were observed. The working concentration of ADM and L-ADM, and the cell proliferation rate were determined by MTT...

  8. Culture conditions tailored to the cell of origin are critical for maintaining native properties and tumorigenicity of glioma cells.

    Ledur, Pítia F; Liu, Chong; He, Hua; Harris, Alexandra R; Minussi, Darlan C; Zhou, Hai-Yan; Shaffrey, Mark E; Asthagiri, Ashok; Lopes, Maria Beatriz S; Schiff, David; Lu, Yi-Cheng; Mandell, James W; Lenz, Guido; Zong, Hui

    2016-10-01

    Cell culture plays a pivotal role in cancer research. However, culture-induced changes in biological properties of tumor cells profoundly affect research reproducibility and translational potential. Establishing culture conditions tailored to the cancer cell of origin could resolve this problem. For glioma research, it has been previously shown that replacing serum with defined growth factors for neural stem cells (NSCs) greatly improved the retention of gene expression profile and tumorigenicity. However, among all molecular subtypes of glioma, our laboratory and others have previously shown that the oligodendrocyte precursor cell (OPC) rather than the NSC serves as the cell of origin for the proneural subtype, raising questions regarding the suitability of NSC-tailored media for culturing proneural glioma cells. OPC-originated mouse glioma cells were cultured in conditions for normal OPCs or NSCs, respectively, for multiple passages. Gene expression profiles, morphologies, tumorigenicity, and drug responsiveness of cultured cells were examined in comparison with freshly isolated tumor cells. OPC media-cultured glioma cells maintained tumorigenicity, gene expression profiles, and morphologies similar to freshly isolated tumor cells. In contrast, NSC-media cultured glioma cells gradually lost their OPC features and most tumor-initiating ability and acquired heightened sensitivity to temozolomide. To improve experimental reproducibility and translational potential of glioma research, it is important to identify the cell of origin, and subsequently apply this knowledge to establish culture conditions that allow the retention of native properties of tumor cells. © The Author(s) 2016. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  9. Differences in radiosensitivity between three HER2 overexpressing cell lines

    Steffen, Ann-Charlott; Tolmachev, Vladimir; Stenerloew, Bo; Goestring, Lovisa; Palm, Stig; Carlsson, Joergen

    2008-01-01

    HER2 is a potential target for radionuclide therapy, especially when HER2 overexpressing breast cancer cells are resistant to Herceptin registered treatment. Therefore, it is of interest to analyse whether HER2 overexpressing tumour cells have different inherent radiosensitivity. The radiosensitivity of three often used HER2 overexpressing cell lines, SKOV-3, SKBR-3 and BT-474, was analysed. The cells were exposed to conventional photon irradiation, low linear energy transfer (LET), to characterise their inherent radiosensitivity. The analysis was made with clonogenic survival and growth extrapolation assays. The cells were also exposed to alpha particles, high LET, from 211 At decays using the HER2-binding affibody molecule 211 At-(Z HER2:4 ) 2 as targeting agent. Assays for studies of internalisation of the affibody molecule were applied. SKOV-3 cells were most radioresistant, SKBR-3 cells were intermediate and BT-474 cells were most sensitive as measured with the clonogenic and growth extrapolation assays after photon irradiation. The HER2 dependent cellular uptake of 211 At was qualitatively similar for all three cell lines. However, the sensitivity to the alpha particles from 211 At differed; SKOV-3 was most resistant, SKBR-3 intermediate and BT-474 most sensitive. These differences were unexpected because it is assumed that all types of cells should have similar sensitivity to high-LET radiation. The sensitivity to alpha particle exposure correlated with internalisation of the affibody molecule and with size of the cell nucleus. There can be differences in radiosensitivity, which, if they also exist between patient breast cancer cells, are important to consider for both conventional radiotherapy and for HER2-targeted radionuclide therapy. (orig.)

  10. Sox2 is translationally activated by eukaryotic initiation factor 4E in human glioma-initiating cells

    Ge, Yuqing; Zhou, Fengbiao; Chen, Hong; Cui, Chunhong; Liu, Dan [Key Laboratory of Glycoconjuates Research, Ministry of Public Health and Gene Research Center, Shanghai Medical College of Fudan University, Shanghai 200032 (China); Li, Qiuping [Zhongshan Hospital of Fudan University, Shanghai 200032 (China); Yang, Zhiyuan; Wu, Guoqiang [Key Laboratory of Glycoconjuates Research, Ministry of Public Health and Gene Research Center, Shanghai Medical College of Fudan University, Shanghai 200032 (China); Sun, Shuhui [Key Laboratory of Medical Molecular Virology, Ministry of Education and Health, Shanghai Medical College of Fudan University, Shanghai 200032 (China); Gu, Jianxin [Key Laboratory of Glycoconjuates Research, Ministry of Public Health and Gene Research Center, Shanghai Medical College of Fudan University, Shanghai 200032 (China); Institutes of Biomedical Sciences of Fudan University, Shanghai 200032 (China); Wei, Yuanyan, E-mail: yywei@fudan.edu.cn [Key Laboratory of Glycoconjuates Research, Ministry of Public Health and Gene Research Center, Shanghai Medical College of Fudan University, Shanghai 200032 (China); Jiang, Jianhai, E-mail: jianhaijiang@fudan.edu.cn [Key Laboratory of Glycoconjuates Research, Ministry of Public Health and Gene Research Center, Shanghai Medical College of Fudan University, Shanghai 200032 (China)

    2010-07-09

    Sox2, a master transcription factor, contributes to the generation of induced pluripotent stem cells and plays significant roles in sustaining the self-renewal of neural stem cells and glioma-initiating cells. Understanding the functional differences of Sox2 between glioma-initiating cells and normal neural stem cells would contribute to therapeutic approach for treatment of brain tumors. Here, we first demonstrated that Sox2 could contribute to the self-renewal and proliferation of glioma-initiating cells. The following experiments showed that Sox2 was activated at translational level in a subset of human glioma-initiating cells compared with the normal neural stem cells. Further investigation revealed there was a positive correlation between Sox2 and eukaryotic initiation factor 4E (eIF4E) in glioma tissues. Down-regulation of eIF4E decreased Sox2 protein level without altering its mRNA level in glioma-initiating cells, indicating that Sox2 was activated by eIF4E at translational level. Furthermore, eIF4E was presumed to regulate the expression of Sox2 by its 5' untranslated region (5' UTR) sequence. Our results suggest that the eIF4E-Sox2 axis is a novel mechanism of unregulated self-renewal of glioma-initiating cells, providing a potential therapeutic target for glioma.

  11. Sox2 is translationally activated by eukaryotic initiation factor 4E in human glioma-initiating cells

    Ge, Yuqing; Zhou, Fengbiao; Chen, Hong; Cui, Chunhong; Liu, Dan; Li, Qiuping; Yang, Zhiyuan; Wu, Guoqiang; Sun, Shuhui; Gu, Jianxin; Wei, Yuanyan; Jiang, Jianhai

    2010-01-01

    Sox2, a master transcription factor, contributes to the generation of induced pluripotent stem cells and plays significant roles in sustaining the self-renewal of neural stem cells and glioma-initiating cells. Understanding the functional differences of Sox2 between glioma-initiating cells and normal neural stem cells would contribute to therapeutic approach for treatment of brain tumors. Here, we first demonstrated that Sox2 could contribute to the self-renewal and proliferation of glioma-initiating cells. The following experiments showed that Sox2 was activated at translational level in a subset of human glioma-initiating cells compared with the normal neural stem cells. Further investigation revealed there was a positive correlation between Sox2 and eukaryotic initiation factor 4E (eIF4E) in glioma tissues. Down-regulation of eIF4E decreased Sox2 protein level without altering its mRNA level in glioma-initiating cells, indicating that Sox2 was activated by eIF4E at translational level. Furthermore, eIF4E was presumed to regulate the expression of Sox2 by its 5' untranslated region (5' UTR) sequence. Our results suggest that the eIF4E-Sox2 axis is a novel mechanism of unregulated self-renewal of glioma-initiating cells, providing a potential therapeutic target for glioma.

  12. FBXW7/hCDC4 controls glioma cell proliferation in vitro and is a prognostic marker for survival in glioblastoma patients

    Hagedorn Martin

    2007-02-01

    Full Text Available Abstract Background In the quest for novel molecular mediators of glioma progression, we studied the regulation of FBXW7 (hCDC4/hAGO/SEL10, its association with survival of patients with glioblastoma and its potential role as a tumor suppressor gene in glioma cells. The F-box protein Fbxw7 is a component of SCFFbxw7, a Skp1-Cul1-F-box E3 ubiquitin ligase complex that tags specific proteins for proteasome degradation. FBXW7 is mutated in several human cancers and functions as a haploinsufficient tumor suppressor in mice. Any of the identified targets, Cyclin E, c-Myc, c-Jun, Notch1/4 and Aurora-A may have oncogenic properties when accumulated in tumors with FBXW7 loss. Results We tested the expression of FBXW7 in human glioma biopsies by quantitative PCR and compared the transcript levels of grade IV glioma (glioblastoma, G-IV with those of grade II tumors (G-II. In more than 80% G-IV, expression of FBXW7 was significantly reduced. In addition, levels of FBXW7 were correlated with survival indicating a possible implication in tumor aggressiveness. Locus 4q31.3 which carries FBXW7 was investigated by in situ hybridization on biopsy touchprints. This excluded allelic loss as the principal cause for low expression of FBXW7 in G-IV tumors. Two targets of Fbxw7, Aurora-A and Notch4 were preferentially immunodetected in G-IV biopsies. Next, we investigated the effects of FBXW7 misregulation in glioma cells. U87 cells overexpressing nuclear isoforms of Fbxw7 lose the expression of the proliferation markers PCNA and Ki-67, and get counterselected in vitro. This observation fits well with the hypothesis that Fbxw7 functions as a tumor suppressor in astroglial cells. Finally, FBXW7 knockdown in U87 cells leads to defects in mitosis that may promote aneuploidy in progressing glioma. Conclusion Our results show that FBXW7 expression is a prognostic marker for patients with glioblastoma. We suggest that loss of FBXW7 plays an important role in glioma

  13. Berberine enhances inhibition of glioma tumor cell migration and invasiveness mediated by arsenic trioxide

    Lin, Tseng-Hsi; Kuo, Hsing-Chun; Chou, Fen-Pi; Lu, Fung-Jou

    2008-01-01

    Arsenic trioxide (As 2 O 3 ) exhibits promising anticarcinogenic activity in acute promyelocytic leukemic patients and induces apoptosis in various tumor cells in vitro. Here, we investigated the effect of the natural alkaloid berberine on As 2 O 3 -mediated inhibition of cancer cell migration using rat and human glioma cell lines. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was used to determine the viability of rat C6 and human U-87 glioma cells after treatment with As 2 O 3 or berberine, and after co-treatment with As 2 O 3 and berberine. The wound scratch and Boyden chamber assays were applied to determine the effect of As 2 O 3 and berberine on the migration capacity and invasiveness of glioma cancer cells. Zymography and Western blot analyses provided information on the effect of As 2 O 3 and berberine on the intracellular translocation and activation of protein kinase C (PKC), and some PKC-related downstream factors. Most assays were performed three times, independently, and data were analyzed using ANOVA. The cell viability studies demonstrated that berberine enhances As 2 O 3 -mediated inhibition of glioma cell growth after 24 h incubation. Untreated control cells formed a confluent layer, the formation of which was inhibited upon incubation with 5 μM As 2 O 3 . The latter effect was even more pronounced in the presence of 10 μM berberine. The As 2 O 3 -mediated reduction in motility and invasion of glioma cells was enhanced upon co-treatment with berberine. Furthermore, it has been reported that PKC isoforms influence the morphology of the actin cytoskeleton, as well as the activation of metalloproteases MT1-MMP and MMP-2, reported to be involved in cancer cell migration. Treatment of glioma cells with As 2 O 3 and berberine significantly decreased the activation of PKC α and ε and led to actin cytoskeleton rearrangements. The levels of two downstream transcription factors, myc and jun, and MT1-MMP and MMP-2 were also

  14. Effective transvascular delivery of nanoparticles across the blood-brain tumor barrier into malignant glioma cells

    Sharma Kamal

    2008-12-01

    Full Text Available Abstract Background Effective transvascular delivery of nanoparticle-based chemotherapeutics across the blood-brain tumor barrier of malignant gliomas remains a challenge. This is due to our limited understanding of nanoparticle properties in relation to the physiologic size of pores within the blood-brain tumor barrier. Polyamidoamine dendrimers are particularly small multigenerational nanoparticles with uniform sizes within each generation. Dendrimer sizes increase by only 1 to 2 nm with each successive generation. Using functionalized polyamidoamine dendrimer generations 1 through 8, we investigated how nanoparticle size influences particle accumulation within malignant glioma cells. Methods Magnetic resonance and fluorescence imaging probes were conjugated to the dendrimer terminal amines. Functionalized dendrimers were administered intravenously to rodents with orthotopically grown malignant gliomas. Transvascular transport and accumulation of the nanoparticles in brain tumor tissue was measured in vivo with dynamic contrast-enhanced magnetic resonance imaging. Localization of the nanoparticles within glioma cells was confirmed ex vivo with fluorescence imaging. Results We found that the intravenously administered functionalized dendrimers less than approximately 11.7 to 11.9 nm in diameter were able to traverse pores of the blood-brain tumor barrier of RG-2 malignant gliomas, while larger ones could not. Of the permeable functionalized dendrimer generations, those that possessed long blood half-lives could accumulate within glioma cells. Conclusion The therapeutically relevant upper limit of blood-brain tumor barrier pore size is approximately 11.7 to 11.9 nm. Therefore, effective transvascular drug delivery into malignant glioma cells can be accomplished by using nanoparticles that are smaller than 11.7 to 11.9 nm in diameter and possess long blood half-lives.

  15. Arctigenin, a natural lignan compound, induces G0/G1 cell cycle arrest and apoptosis in human glioma cells.

    Maimaitili, Aisha; Shu, Zunhua; Cheng, Xiaojiang; Kaheerman, Kadeer; Sikandeer, Alifu; Li, Weimin

    2017-02-01

    The aim of the current study was to investigate the anticancer potential of arctigenin, a natural lignan compound, in malignant gliomas. The U87MG and T98G human glioma cell lines were treated with various concentrations of arctigenin for 48 h and the effects of arctigenin on the aggressive phenotypes of glioma cells were assessed. The results demonstrated that arctigenin dose-dependently inhibited the growth of U87MG and T98G cells, as determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and bromodeoxyuridine incorporation assays. Arctigenin exposure also induced a 60-75% reduction in colony formation compared with vehicle-treated control cells. However, arctigenin was not observed to affect the invasiveness of glioma cells. Arctigenin significantly increased the proportion of cells in the G 0 /G 1 phase and reduced the number of cells in the S phase, as compared with the control group (Parctigenin increased the expression levels of p21, retinoblastoma and p53 proteins, and significantly decreased the expression levels of cyclin D1 and cyclin-dependent kinase 4 proteins. Additionally, arctigenin was able to induce apoptosis in glioma cells, coupled with increased expression levels of cleaved caspase-3 and the pro-apoptotic BCL2-associated X protein. Furthermore, arctigenin-induced apoptosis was significantly suppressed by the pretreatment of cells with Z-DEVD-FMK, a caspase-3 inhibitor. In conclusion, the results suggest that arctigenin is able to inhibit cell proliferation and may induce apoptosis and cell cycle arrest at the G 0 /G 1 phase in glioma cells. These results warrant further investigation of the anticancer effects of arctigenin in animal models of gliomas.

  16. Effects of irradiation on the expression of the adhesion molecules (NCAM, ICAM-1) by glioma cell lines

    Yamanaka, Ryuya; Tanaka, Ryuichi; Yoshida, Seiichi [Niigata Univ. (Japan). Brain Research Inst.

    1993-11-01

    The expression of the intercellular adhesion molecule-1 (ICAM-1) and neural cell adhesion molecule (NCAM) by glioma cell lines was investigated. The effects of interferon (IFN)-[gamma] or irradiation on the expression was also assessed. Two glioma cell lines showed more than 75% NCAM-positive cells. After treatment with IFN-[gamma] or irradiation, another three cell lines were induced to show more than 50% positive cells. Three glioma cell lines showed more than 50% ICAM-1-positive cells. After treatment with IFN-[gamma], another two cell lines were induced to show more than 50% positive cells. After treatment with irradiation, one more cell line was induced to show more than 50% positive cells. ICAM-1 and NCAM expression by glioma cell lines is susceptible to modulation by IFN-[gamma] or irradiation. (author).

  17. SNEV overexpression extends the life span of human endothelial cells

    Voglauer, Regina; Chang, Martina Wei-Fen; Dampier, Brigitta; Wieser, Matthias; Baumann, Kristin; Sterovsky, Thomas; Schreiber, Martin; Katinger, Hermann; Grillari, Johannes

    2006-01-01

    In a recent screening for genes downregulated in replicatively senescent human umbilical vein endothelial cells (HUVECs), we have isolated the novel protein SNEV. Since then SNEV has proven as a multifaceted protein playing a role in pre-mRNA splicing, DNA repair, and the ubiquitin/proteosome system. Here, we report that SNEV mRNA decreases in various cell types during replicative senescence, and that it is increased in various immortalized cell lines, as well as in breast tumors, where SNEV transcript levels also correlate with the survival of breast cancer patients. Since these mRNA profiles suggested a role of SNEV in the regulation of cell proliferation, the effect of its overexpression was tested. Thereby, a significant extension of the cellular life span was observed, which was not caused by altered telomerase activity or telomere dynamics but rather by enhanced stress resistance. When SNEV overexpressing cells were treated with bleomycin or bleomycin combined with BSO, inducing DNA damage as well as reactive oxygen species, a significantly lower fraction of apoptotic cells was found in comparison to vector control cells. These data suggest that high levels of SNEV might extend the cellular life span by increasing the resistance to stress or by improving the DNA repair capacity of the cells

  18. ROS accumulation and IGF-IR inhibition contribute to fenofibrate/PPARα -mediated inhibition of Glioma cell motility in vitro

    Del Valle Luis

    2010-06-01

    Full Text Available Abstract Background Glioblastomas are characterized by rapid cell growth, aggressive CNS infiltration, and are resistant to all known anticancer regimens. Recent studies indicate that fibrates and statins possess anticancer potential. Fenofibrate is a potent agonist of peroxisome proliferator activated receptor alpha (PPARα that can switch energy metabolism from glycolysis to fatty acid β-oxidation, and has low systemic toxicity. Fenofibrate also attenuates IGF-I-mediated cellular responses, which could be relevant in the process of glioblastoma cell dispersal. Methods The effects of fenofibrate on Glioma cell motility, IGF-I receptor (IGF-IR signaling, PPARα activity, reactive oxygen species (ROS metabolism, mitochondrial potential, and ATP production were analyzed in human glioma cell lines. Results Fenofibrate treatment attenuated IGF-I signaling responses and repressed cell motility of LN-229 and T98G Glioma cell lines. In the absence of fenofibrate, specific inhibition of the IGF-IR had only modest effects on Glioma cell motility. Further experiments revealed that PPARα-dependent accumulation of ROS is a strong contributing factor in Glioma cell lines responses to fenofibrate. The ROS scavenger, N-acetyl-cysteine (NAC, restored cell motility, improved mitochondrial potential, and increased ATP levels in fenofibrate treated Glioma cell lines. Conclusions Our results indicate that although fenofibrate-mediated inhibition of the IGF-IR may not be sufficient in counteracting Glioma cell dispersal, PPARα-dependent metabolic switch and the resulting ROS accumulation strongly contribute to the inhibition of these devastating brain tumor cells.

  19. Doxycycline Impairs Mitochondrial Function and Protects Human Glioma Cells from Hypoxia-Induced Cell Death: Implications of Using Tet-Inducible Systems.

    Luger, Anna-Luisa; Sauer, Benedikt; Lorenz, Nadja I; Engel, Anna L; Braun, Yannick; Voss, Martin; Harter, Patrick N; Steinbach, Joachim P; Ronellenfitsch, Michael W

    2018-05-17

    Inducible gene expression is an important tool in molecular biology research to study protein function. Most frequently, the antibiotic doxycycline is used for regulation of so-called tetracycline (Tet)-inducible systems. In contrast to stable gene overexpression, these systems allow investigation of acute and reversible effects of cellular protein induction. Recent reports have already called for caution when using Tet-inducible systems as the employed antibiotics can disturb mitochondrial function and alter cellular metabolism by interfering with mitochondrial translation. Reprogramming of energy metabolism has lately been recognized as an important emerging hallmark of cancer and is a central focus of cancer research. Therefore, the scope of this study was to systematically analyze dose-dependent metabolic effects of doxycycline on a panel of glioma cell lines with concomitant monitoring of gene expression from Tet-inducible systems. We report that doxycycline doses commonly used with inducible expression systems (0.01⁻1 µg/mL) substantially alter cellular metabolism: Mitochondrial protein synthesis was inhibited accompanied by reduced oxygen and increased glucose consumption. Furthermore, doxycycline protected human glioma cells from hypoxia-induced cell death. An impairment of cell growth was only detectable with higher doxycycline doses (10 µg/mL). Our findings describe settings where doxycycline exerts effects on eukaryotic cellular metabolism, limiting the employment of Tet-inducible systems.

  20. Overexpression of neurofilament H disrupts normal cell structure and function

    Szebenyi, Gyorgyi; Smith, George M.; Li, Ping; Brady, Scott T.

    2002-01-01

    Studying exogenously expressed tagged proteins in live cells has become a standard technique for evaluating protein distribution and function. Typically, expression levels of experimentally introduced proteins are not regulated, and high levels are often preferred to facilitate detection. However, overexpression of many proteins leads to mislocalization and pathologies. Therefore, for normative studies, moderate levels of expression may be more suitable. To understand better the dynamics of intermediate filament formation, transport, and stability in a healthy, living cell, we inserted neurofilament heavy chain (NFH)-green fluorescent protein (GFP) fusion constructs in adenoviral vectors with tetracycline (tet)-regulated promoters. This system allows for turning on or off the synthesis of NFH-GFP at a selected time, for a defined period, in a dose-dependent manner. We used this inducible system for live cell imaging of changes in filament structure and cell shape, motility, and transport associated with increasing NFH-GFP expression. Cells with low to intermediate levels of NFH-GFP were structurally and functionally similar to neighboring, nonexpressing cells. In contrast, overexpression led to pathological alterations in both filament organization and cell function. Copyright 2002 Wiley-Liss, Inc.

  1. Cord blood stem cell-mediated induction of apoptosis in glioma downregulates X-linked inhibitor of apoptosis protein (XIAP.

    Venkata Ramesh Dasari

    2010-07-01

    Full Text Available XIAP (X-linked inhibitor of apoptosis protein is one of the most important members of the apoptosis inhibitor family. XIAP is upregulated in various malignancies, including human glioblastoma. It promotes invasion, metastasis, growth and survival of malignant cells. We hypothesized that downregulation of XIAP by human umbilical cord blood mesenchymal stem cells (hUCBSC in glioma cells would cause them to undergo apoptotic death.We observed the effect of hUCBSC on two malignant glioma cell lines (SNB19 and U251 and two glioma xenograft cell lines (4910 and 5310. In co-cultures of glioma cells with hUCBSC, proliferation of glioma cells was significantly inhibited. This is associated with increased cytotoxicity of glioma cells, which led to glioma cell death. Stem cells induced apoptosis in glioma cells, which was evaluated by TUNEL assay, FACS analyses and immunoblotting. The induction of apoptosis is associated with inhibition of XIAP in co-cultures of hUCBSC. Similar results were obtained by the treatment of glioma cells with shRNA to downregulate XIAP (siXIAP. Downregulation of XIAP resulted in activation of caspase-3 and caspase-9 to trigger apoptosis in glioma cells. Apoptosis is characterized by the loss of mitochondrial membrane potential and upregulation of mitochondrial apoptotic proteins Bax and Bad. Cell death of glioma cells was marked by downregulation of Akt and phospho-Akt molecules. We observed similar results under in vivo conditions in U251- and 5310-injected nude mice brains, which were treated with hUCBSC. Under in vivo conditions, Smac/DIABLO was found to be colocalized in the nucleus, showing that hUCBSC induced apoptosis is mediated by inhibition of XIAP and activation of Smac/DIABLO.Our results indicate that downregulation of XIAP by hUCBSC treatment induces apoptosis, which led to the death of the glioma cells and xenograft cells. This study demonstrates the therapeutic potential of XIAP and hUCBSC to treat malignant

  2. [Vitamin K3-induced activation of molecular oxygen in glioma cells].

    Krylova, N G; Kulagova, T A; Semenkova, G N; Cherenkevich, S N

    2009-01-01

    It has been shown by the method of fluorescent analysis that the rate of hydrogen peroxide generation in human U251 glioma cells under the effect of lipophilic (menadione) or hydrophilic (vikasol) analogues of vitamin K3 was different. Analyzing experimental data we can conclude that menadione underwent one- and two-electron reduction by intracellular reductases in glioma cells. Reduced forms of menadione interact with molecular oxygen leading to reactive oxygen species (ROS) generation. The theoretical model of ROS generation including two competitive processes of one- and two-electron reduction of menadione has been proposed. Rate constants of ROS generation mediated by one-electron reduction process have been estimated.

  3. DNA lability induced by nimustine and ramustine in rat glioma cells.

    Mineura, K; Fushimi, S; Itoh, Y; Kowada, M

    1988-01-01

    The DNA labile sites induced by two nitrosoureas, nimustine (ACNU) and ramustine (MCNU) synthesised in Japan, have been examined in highly reiterated DNA sequences of rat glioma cells. Reiterated fragments of 167 and 203 base pairs (bp), obtained after Hind III and Hae III restriction endonuclease digestion of rat glioma cells DNA, were used as target DNA sequences to determine the labile sites. In vitro reaction with ACNU and MCNU resulted in scission products corresponding to the locations of guanine. Subsequent piperidine hydrolysis produced more frequent breaks of the phosphodiester bonds at guanine positions, thus forming alkali-labile sites. Images PMID:3236017

  4. Fascin overexpression promotes neoplastic progression in oral squamous cell carcinoma

    Alam Hunain

    2012-01-01

    Full Text Available Abstract Background Fascin is a globular actin cross-linking protein, which plays a major role in forming parallel actin bundles in cell protrusions and is found to be associated with tumor cell invasion and metastasis in various type of cancers including oral squamous cell carcinoma (OSCC. Previously, we have demonstrated that fascin regulates actin polymerization and thereby promotes cell motility in K8-depleted OSCC cells. In the present study we have investigated the role of fascin in tumor progression of OSCC. Methods To understand the role of fascin in OSCC development and/or progression, fascin was overexpressed along with vector control in OSCC derived cells AW13516. The phenotype was studied using wound healing, Boyden chamber, cell adhesion, Hanging drop, soft agar and tumorigenicity assays. Further, fascin expression was examined in human OSCC samples (N = 131 using immunohistochemistry and level of its expression was correlated with clinico-pathological parameters of the patients. Results Fascin overexpression in OSCC derived cells led to significant increase in cell migration, cell invasion and MMP-2 activity. In addition these cells demonstrated increased levels of phosphorylated AKT, ERK1/2 and JNK1/2. Our in vitro results were consistent with correlative studies of fascin expression with the clinico-pathological parameters of the OSCC patients. Fascin expression in OSCC showed statistically significant correlation with increased tumor stage (P = 0.041, increased lymph node metastasis (P = 0.001, less differentiation (P = 0.005, increased recurrence (P = 0.038 and shorter survival (P = 0.004 of the patients. Conclusion In conclusion, our results indicate that fascin promotes tumor progression and activates AKT and MAPK pathways in OSCC-derived cells. Further, our correlative studies of fascin expression in OSCC with clinico-pathological parameters of the patients indicate that fascin may prove to be useful in prognostication and

  5. Fascin overexpression promotes neoplastic progression in oral squamous cell carcinoma

    Alam, Hunain; Kannanl, Sadhna; Gude, Rajiv; Kane, Shubhada; Dalal, Sorab N; Vaidya, Milind M; Bhate, Amruta V; Gangadaran, Prakash; Sawant, Sharda S; Salot, Shimul; Sehgal, Lalit; Dange, Prerana P; Chaukar, Devendra A; D'cruz, Anil K

    2012-01-01

    Fascin is a globular actin cross-linking protein, which plays a major role in forming parallel actin bundles in cell protrusions and is found to be associated with tumor cell invasion and metastasis in various type of cancers including oral squamous cell carcinoma (OSCC). Previously, we have demonstrated that fascin regulates actin polymerization and thereby promotes cell motility in K8-depleted OSCC cells. In the present study we have investigated the role of fascin in tumor progression of OSCC. To understand the role of fascin in OSCC development and/or progression, fascin was overexpressed along with vector control in OSCC derived cells AW13516. The phenotype was studied using wound healing, Boyden chamber, cell adhesion, Hanging drop, soft agar and tumorigenicity assays. Further, fascin expression was examined in human OSCC samples (N = 131) using immunohistochemistry and level of its expression was correlated with clinico-pathological parameters of the patients. Fascin overexpression in OSCC derived cells led to significant increase in cell migration, cell invasion and MMP-2 activity. In addition these cells demonstrated increased levels of phosphorylated AKT, ERK1/2 and JNK1/2. Our in vitro results were consistent with correlative studies of fascin expression with the clinico-pathological parameters of the OSCC patients. Fascin expression in OSCC showed statistically significant correlation with increased tumor stage (P = 0.041), increased lymph node metastasis (P = 0.001), less differentiation (P = 0.005), increased recurrence (P = 0.038) and shorter survival (P = 0.004) of the patients. In conclusion, our results indicate that fascin promotes tumor progression and activates AKT and MAPK pathways in OSCC-derived cells. Further, our correlative studies of fascin expression in OSCC with clinico-pathological parameters of the patients indicate that fascin may prove to be useful in prognostication and treatment of OSCC

  6. Nitrosoureas inhibit the stathmin-mediated migration and invasion of malignant glioma cells.

    Liang, Xing-Jie; Choi, Yong; Sackett, Dan L; Park, John K

    2008-07-01

    Malignant gliomas are the most common primary intrinsic brain tumors and are highly lethal. The widespread migration and invasion of neoplastic cells from the initial site of tumor formation into the surrounding brain render these lesions refractory to definitive surgical treatment. Stathmin, a microtubule-destabilizing protein that mediates cell cycle progression, can also regulate directed cell movement. Nitrosoureas, traditionally viewed as DNA alkylating agents, can also covalently modify proteins such as stathmin. We therefore sought to establish a role for stathmin in malignant glioma cell motility, migration, and invasion and determine the effects of nitrosoureas on these cell movement-related processes. Scratch wound-healing recovery, Boyden chamber migration, Matrigel invasion, and organotypic slice invasion assays were performed before and after the down-regulation of cellular stathmin levels and in the absence and presence of sublethal nitrosourea ([1-(2-chloroethyl)-3-cyclohexyl-l-nitrosourea]; CCNU) concentrations. We show that decreases in stathmin expression lead to significant decreases in malignant glioma cell motility, migration, and invasion. CCNU, at a concentration of 10 micromol/L, causes similar significant decreases, even in the absence of any effects on cell viability. The direct inhibition of stathmin by CCNU is likely a contributing factor. These findings suggest that the inhibition of stathmin expression and function may be useful in limiting the spread of malignant gliomas within the brain, and that nitrosoureas may have therapeutic benefits in addition to their antiproliferative effects.

  7. Nitrosoureas Inhibit the Stathmin Mediated Migration and Invasion of Malignant Glioma Cells

    Liang, Xing-Jie; Choi, Yong; Sackett, Dan L.; Park, John K.

    2008-01-01

    Malignant gliomas are the most common primary intrinsic brain tumors and are highly lethal. The widespread migration and invasion of neoplastic cells from the initial site of tumor formation into the surrounding brain render these lesions refractory to definitive surgical treatment. Stathmin, a microtubule destabilizing protein that mediates cell cycle progression, can also regulate directed cell movement. Nitrosoureas, traditionally viewed as DNA alkylating agents, can also covalently modify proteins such as stathmin. We therefore sought to establish a role for stathmin in malignant glioma cell motility, migration, and invasion and determine the effects of nitrosoureas on these cell movement related processes. Scratch-wound healing recovery, Boyden chamber migration, Matrigel invasion, and organotypic slice invasion assays were performed before and after the down regulation of cellular stathmin levels and in the absence and presence of sub-lethal nitrosourea (CCNU; [1-(2-chloroethyl)-3-cyclohexyl-l-nitrosourea]) concentrations. We demonstrate that decreases in stathmin expression lead to significant decreases in malignant glioma cell motility, migration, and invasion. CCNU, at a concentration of 10 μM, causes similar significant decreases, even in the absence of any effects on cell viability. The direct inhibition of stathmin by CCNU is likely a contributing factor. These findings suggest that the inhibition of stathmin expression and function may be useful in limiting the spread of malignant gliomas within the brain and that nitrosoureas may have therapeutic benefits in addition to their anti-proliferative effects. PMID:18593927

  8. RasGRP3 regulates the migration of glioma cells via interaction with Arp3

    Lee, Hae Kyung; Finniss, Susan; Cazacu, Simona; Xiang, Cunli; Poisson, Laila M.; Blumberg, Peter M.; Brodie, Chaya

    2015-01-01

    Glioblastoma (GBM), the most aggressive primary brain tumors, are highly infiltrative. Although GBM express high Ras activity and Ras proteins have been implicated in gliomagenesis, Ras-activating mutations are not frequent in these tumors. RasGRP3, an important signaling protein responsive to diacylglycerol (DAG), increases Ras activation. Here, we examined the expression and functions of RasGRP3 in GBM and glioma cells. RasGRP3 expression was upregulated in GBM specimens and glioma stem cells compared with normal brains and neural stem cells, respectively. RasGRP3 activated Ras and Rap1 in glioma cells and increased cell migration and invasion partially via Ras activation. Using pull-down assay and mass spectroscopy we identified the actin-related protein, Arp3, as a novel interacting protein of RasGRP3. The interaction of RasGRP3 and Arp3 was validated by immunofluorescence staining and co-immunoprecipitation, and PMA, which activates RasGRP3 and induces its translocation to the peri-nuclear region, increased the association of Arp3 and RasGRP3. Arp3 was upregulated in GBM, regulated cell spreading and migration and its silencing partially decreased these effects of RasGRP3 in glioma cells. In summary, RasGRP3 acts as an important integrating signaling protein of the DAG and Ras signaling pathways and actin polymerization and represents an important therapeutic target in GBM. PMID:25682201

  9. 3-Bromopyruvate inhibits cell proliferation and induces apoptosis in CD133+ population in human glioma.

    Xu, Dong-Qiang; Tan, Xiao-Yu; Zhang, Bao-Wei; Wu, Tao; Liu, Ping; Sun, Shao-Jun; Cao, Yin-Guang

    2016-03-01

    The study was aimed to investigate the role of 3-bromopyruvate in inhibition of CD133+ U87 human glioma cell population growth. The results demonstrated that 3-bromopyruvate inhibited the viability of both CD133+ and parental cells derived from U87 human glioma cell line. However, the 3-bromopyruvate-induced inhibition in viability was more prominent in CD133+ cells at 10 μM concentration after 48 h. Treatment of CD133+ cells with 3-bromopyruvate caused reduction in cell population and cell size, membrane bubbling, and degradation of cell membranes. Hoechst 33258 staining showed condensation of chromatin material and fragmentation of DNA in treated CD133+ cells after 48 h. 3-Bromopyruvate inhibited the migration rate of CD133+ cells significantly compared to the parental cells. Flow cytometry revealed that exposure of CD133+ cells to 3-bromopyruvate increased the cell population in S phase from 24.5 to 37.9 % with increase in time from 12 to 48 h. In addition, 3-bromopyruvate significantly enhanced the expression of Bax and cleaved caspase 3 in CD133+ cells compared to the parental cells. Therefore, 3-bromopyruvate is a potent chemotherapeutic agent for the treatment of glioma by targeting stem cells selectively.

  10. Del-1 overexpression potentiates lung cancer cell proliferation and invasion

    Lee, Seung-Hwan; Kim, Dong-Young; Jing, Feifeng; Kim, Hyesoon [Department of Biomedical Sciences, University of Ulsan College of Medicine, Seoul (Korea, Republic of); Yun, Chae-Ok [Department of Bioengineering, College of Engineering, Hanyang University, Seoul (Korea, Republic of); Han, Deok-Jong [Department of Surgery, Asan Medical Center, University of Ulsan College of Medicine, Seoul (Korea, Republic of); Choi, Eun Young, E-mail: choieun@ulsan.ac.kr [Department of Biomedical Sciences, University of Ulsan College of Medicine, Seoul (Korea, Republic of)

    2015-12-04

    Developmental endothelial locus-1 (Del-1) is an endogenous anti-inflammatory molecule that is highly expressed in the lung and the brain and limits leukocyte migration to these tissues. We previously reported that the expression of Del-1 is positively regulated by p53 in lung endothelial cells. Although several reports have implicated the altered expression of Del-1 gene in cancer patients, little is known about its role in tumor cells. We here investigated the effect of Del-1 on the features of human lung carcinoma cells. Del-1 mRNA was found to be significantly decreased in the human lung adenocarcinoma cell lines A549 (containing wild type of p53), H1299 (null for p53) and EKVX (mutant p53), compared to in human normal lung epithelial BEAS-2B cells and MRC-5 fibroblasts. The decrease of Del-1 expression was dependent on the p53 activity in the cell lines, but not on the expression of p53. Neither treatment with recombinant human Del-1 protein nor the introduction of adenovirus expressing Del-1 altered the expression of the apoptosis regulators BAX, PUMA and Bcl-2. Unexpectedly, the adenovirus-mediated overexpression of Del-1 gene into the lung carcinoma cell lines promoted proliferation and invasion of the lung carcinoma cells, as revealed by BrdU incorporation and transwell invasion assays, respectively. In addition, overexpression of the Del-1 gene enhanced features of epithelial–mesenchymal transition (EMT), such as increasing vimentin while decreasing E-cadherin in A549 cells, and increases in the level of Slug, an EMT-associated transcription regulator. Our findings demonstrated for the first time that there are deleterious effects of high levels of Del-1 in lung carcinoma cells, and suggest that Del-1 may be used as a diagnostic or prognostic marker for cancer progression, and as a novel therapeutic target for lung carcinoma. - Highlights: • Developmental Endothelial Locus-1 (Del-1) expression is downregulated in human lung cancer cells.

  11. Del-1 overexpression potentiates lung cancer cell proliferation and invasion

    Lee, Seung-Hwan; Kim, Dong-Young; Jing, Feifeng; Kim, Hyesoon; Yun, Chae-Ok; Han, Deok-Jong; Choi, Eun Young

    2015-01-01

    Developmental endothelial locus-1 (Del-1) is an endogenous anti-inflammatory molecule that is highly expressed in the lung and the brain and limits leukocyte migration to these tissues. We previously reported that the expression of Del-1 is positively regulated by p53 in lung endothelial cells. Although several reports have implicated the altered expression of Del-1 gene in cancer patients, little is known about its role in tumor cells. We here investigated the effect of Del-1 on the features of human lung carcinoma cells. Del-1 mRNA was found to be significantly decreased in the human lung adenocarcinoma cell lines A549 (containing wild type of p53), H1299 (null for p53) and EKVX (mutant p53), compared to in human normal lung epithelial BEAS-2B cells and MRC-5 fibroblasts. The decrease of Del-1 expression was dependent on the p53 activity in the cell lines, but not on the expression of p53. Neither treatment with recombinant human Del-1 protein nor the introduction of adenovirus expressing Del-1 altered the expression of the apoptosis regulators BAX, PUMA and Bcl-2. Unexpectedly, the adenovirus-mediated overexpression of Del-1 gene into the lung carcinoma cell lines promoted proliferation and invasion of the lung carcinoma cells, as revealed by BrdU incorporation and transwell invasion assays, respectively. In addition, overexpression of the Del-1 gene enhanced features of epithelial–mesenchymal transition (EMT), such as increasing vimentin while decreasing E-cadherin in A549 cells, and increases in the level of Slug, an EMT-associated transcription regulator. Our findings demonstrated for the first time that there are deleterious effects of high levels of Del-1 in lung carcinoma cells, and suggest that Del-1 may be used as a diagnostic or prognostic marker for cancer progression, and as a novel therapeutic target for lung carcinoma. - Highlights: • Developmental Endothelial Locus-1 (Del-1) expression is downregulated in human lung cancer cells.

  12. mir-300 promotes self-renewal and inhibits the differentiation of glioma stem-like cells

    Zhang, Daming

    2014-01-28

    MicroRNAs (miRNAs) are small noncoding RNAs that have been critically implicated in several human cancers. miRNAs are thought to participate in various biological processes, including proliferation, cell cycle, apoptosis, and even the regulation of the stemness properties of cancer stem cells. In this study, we explore the potential role of miR-300 in glioma stem-like cells (GSLCs). We isolated GSLCs from glioma biopsy specimens and identified the stemness properties of the cells through neurosphere formation assays, multilineage differentiation ability analysis, and immunofluorescence analysis of glioma stem cell markers. We found that miR-300 is commonly upregulated in glioma tissues, and the expression of miR-300 was higher in GSLCs. The results of functional experiments demonstrated that miR-300 can enhance the self-renewal of GSLCs and reduce differentiation toward both astrocyte and neural fates. In addition, LZTS2 is a direct target of miR-300. In conclusion, our results demonstrate the critical role of miR-300 in GSLCs and its functions in LZTS2 inhibition and describe a new approach for the molecular regulation of tumor stem cells. © 2014 Springer Science+Business Media.

  13. Elemental characterization of individual glia and glioma cells in the nuclear microprobe

    Lindh, U.

    1982-01-01

    To investigate whether variations in levels of microelements are reflected at the cellular level, a study of cultured cells was undertaken. For elemental characterization were chosen human glia and glioma cell lines. The cells were freeze-dried and about 1000 cells of each line were analyzed in the nuclear microprobe with a probe diameter of 10 μm. Scanning of the specimens under the beam made possible heat reduction and the X-ray spectrum induced was continuously recorded and subsequently processed in the computer. Elemental maps of the cells were then generated and the information from each member of the cell populations could be considered as well as the population statistics. Mass determination was accomplished by means of the bremsstrahlung continuum intensity. The main feature resulting from the characterization was that the glioma cells in average held appreciably higher contents of copper and zinc than did the glia cells. (orig.)

  14. Ebselen Induced C6 Glioma Cell Death in Oxygen and Glucose Deprivation

    Shi, Honglian; Liu, Shimin; Miyake, Minoru; Liu, Ke Jian

    2006-01-01

    Studies have shown that ebselen is an anti-inflammatory and antioxidative agent. Its protective effect has been investigated in oxidative stress related diseases such as cerebral ischemia in recent years. However, experimental evidence also shows that ebselen causes cell death in several different cell types. Whether ebselen will have beneficial or detrimental effect on cells under ischemic condition is not known. Herein, we studied the effect of ebselen on C6 glioma cell under oxygen and glu...

  15. CD133 positive U87 glioma stem cell radiosensitivity and DNA double-strand break repair

    Li Ping; Zong Tianzhou; Ji Xiaoqin; Lu Xueguan

    2013-01-01

    Objective: To explore the radiosensitivity and DNA double-strand break repair of CD133 + U87 glioma stem cell. Methods: CD133 + and CD133 - cells were isolated from glioma U87 cell lines by flow cytometry sorter system. After irradiated vertically by 4 Gy X-rays, the radiosensitivity of cells was determined by clonogenic assay. The radiation-induced DNA double-strand break repair of CD133 + and CD133 - cells was determined by the neutral comet assay,and the expression of phosphorylated histone H2AX (γ-H2AX) and Rad51 foci were measured by immunofluorescence. Results: The clone forming rate of CD133 + cells was higher than CD133 - cells (t=3.66, P<0.01) with no radiation. The clone forming rate of CD133 + cells irradiated by 4 Gy X-rays has no significant changes compared to that of the non-irradiation cells (t=0.71, P>0.05), but for CD133 - cells, it decreased compared to non-irradiation cells (t=2.91, P<0.05). The tailmoment between CD133 + cells and CD133 - cells had no difference at 0.5 h after irradiation (t=1.44, P>0.05); the tailmoment of CD133 + cells was lower than CD133 - cells at 6 and 24 h after irradiation,respectively (t=5.31 and 8.09, P<0.01). There was no significant difference in the expression of γ-H2AX foci between CD133 + and CD133 - cells at 0.5 and 6 h after irradiation (t=0.12 and 0.99, P>0.05), γ-H2AX foci of CD133 + cells was significantly decreased compared to CD133 - cells at 24 h after irradiation (t=4.99, P<0.01). For Rad 51 foci, there was no difference between CD133 + and CD133 - cells at 0.5 h after irradiation (t=1.12, P>0.05). The expression of Rad 51 foci of CD133 - cells was decreased compared to that of CD133 + cells at 6 and 24 h after irradiation,respectively (t=22.88 and 12.43, P<0.01). And the expression of Rad51 foci of CD133 + cells had no significant changes at 6-24 h after irradiation. Conclusions: Glioma stem cells is more radioresistive than glioma non-stem cells. The probable mechanism is that the DNA double

  16. A computational model incorporating neural stem cell dynamics reproduces glioma incidence across the lifespan in the human population.

    Roman Bauer

    Full Text Available Glioma is the most common form of primary brain tumor. Demographically, the risk of occurrence increases until old age. Here we present a novel computational model to reproduce the probability of glioma incidence across the lifespan. Previous mathematical models explaining glioma incidence are framed in a rather abstract way, and do not directly relate to empirical findings. To decrease this gap between theory and experimental observations, we incorporate recent data on cellular and molecular factors underlying gliomagenesis. Since evidence implicates the adult neural stem cell as the likely cell-of-origin of glioma, we have incorporated empirically-determined estimates of neural stem cell number, cell division rate, mutation rate and oncogenic potential into our model. We demonstrate that our model yields results which match actual demographic data in the human population. In particular, this model accounts for the observed peak incidence of glioma at approximately 80 years of age, without the need to assert differential susceptibility throughout the population. Overall, our model supports the hypothesis that glioma is caused by randomly-occurring oncogenic mutations within the neural stem cell population. Based on this model, we assess the influence of the (experimentally indicated decrease in the number of neural stem cells and increase of cell division rate during aging. Our model provides multiple testable predictions, and suggests that different temporal sequences of oncogenic mutations can lead to tumorigenesis. Finally, we conclude that four or five oncogenic mutations are sufficient for the formation of glioma.

  17. Nimotuzumab enhances temozolomide-induced growth suppression of glioma cells expressing mutant EGFR in vivo

    Nitta, Yusuke; Shimizu, Saki; Shishido-Hara, Yukiko; Suzuki, Kaori; Shiokawa, Yoshiaki; Nagane, Motoo

    2016-01-01

    A mutant form of epidermal growth factor receptor (EGFR), EGFRvIII, is common in glioblastoma (GBM) and confers enhanced tumorigenic activity and drug resistance. Nimotuzumab, an anti-EGFR antibody, has shown preclinical and clinical activity to GBM, but its specific activity against EGFRvIII has not been fully investigated. Human glioma U87MG or LNZ308 cells overexpressing either wild-type (wt) EGFR or EGFRvIII were treated with nimotuzumab, temozolomide, or both. Expression and phosphorylation status of molecules were determined by Western blot analysis. Methylation status of promoter region of O 6 -methylguanine-DNA methyltransferase (MGMT) was detected by methylation-specific PCR. Antitumor activity was tested using nude mice bearing either subcutaneous or intracerebral xenografts along with analyses of EGFR phosphorylation status, proliferation, apoptosis, and vessel density. Nimotuzumab treatment resulted in reduction of EGFRvIII tyrosine phosphorylation with a decrease in Akt phosphorylation that was greater than that of wtEGFR. Correspondingly, antitumor effects, growth suppression and survival elongation, were more significant in mice bearing either subcutaneous or intracerebral tumor expressing EGFRvIII than in those expressing wtEGFR. These effects were markedly increased when temozolomide was combined with nimotuzumab. The post-treatment recurrent brain tumors exhibited a decrease in expression of the mismatch repair (MMR) proteins, MSH6 and MLH1, but their methylated MGMT status did not changed. Nimotuzumab has in vivo antitumor activity against GBM, especially those expressing EGFRvIII, when combined with temozolomide. This could provide a basis for preselection of patients with GBM by EGFR status who might benefit from the nimotuzumab and temozolomide combination therapy

  18. Nicotine enhances proliferation, migration, and radioresistance of human malignant glioma cells through EGFR activation

    Khalil, A.A.; Jameson, M.J.; Broaddus, W.C.; Lin, P.S.; Chung, T.D.

    2013-01-01

    It has been suggested that continued tobacco use during radiation therapy contributes to maintenance of neoplastic growth despite treatment with radiation. Nicotine is a cigarette component that is an established risk factor for many diseases, neoplastic and otherwise. The hypothesis of this work is that nicotine promotes the proliferation, migration, and radioresistance of human malignant glioma cells. The effect of nicotine on cellular proliferation, migration, signaling, and radiation sensitivity were evaluated for malignant glioma U87 and GBM12 cells by use of the AlamarBlue, scratch healing, and clonogenic survival assays. Signal transduction was assessed by immunoblotting for activated EGFR, extracellular regulated kinase (ERK), and AKT. At concentrations comparable with those found in chronic smokers, nicotine induced malignant glioma cell migration, growth, colony formation, and radioresistance. Nicotine increased phosphorylation of EGFR tyr992 , AKT ser473 , and ERK. These molecular effects were reduced by pharmacological inhibitors of EGFR, PI3K, and MEK. It was therefore concluded that nicotine stimulates the malignant behavior of glioma cells in vitro by activation of the EGFR and downstream AKT and ERK pathways. (author)

  19. Transient increase in neuronal chloride concentration by neuroactive amino acids released from glioma cells

    Cristina eBertollini

    2012-11-01

    Full Text Available Neuronal chloride concentration ([Cl-]i is known to be dynamically modulated and alterations in Cl- homeostasis may occur in the brain at physiological and pathological conditions, being also likely involved in glioma-related seizures. However, the mechanism leading to changes in neuronal [Cl-]i during glioma invasion are still unclear. To characterize the potential effect of glioma released soluble factors on neuronal [Cl-]i, we used genetically encoded CFP/YFP-based ratiometric Cl-Sensor transiently expressed in cultured hippocampal neurons. Exposition of neurons to glioma conditioned medium (GCM caused rapid and transient elevation of [Cl-]i, resulting in the increase of fluorescence ratio, which was strongly reduced by blockers of ionotropic glutamate receptors APV and NBQX. Furthermore, in HEK cells expressing GluR1-AMPA receptors, GCM activated ionic current with efficacy similar to those caused by glutamate, supporting the notion that GCM contains glutamate or glutamatergic agonists, which cause neuronal depolarization, activation of NMDA and AMPA/KA receptors leading to elevation of [Cl-]i. Chromatographic analysis of the GCM showed that it contained several aminoacids, including glutamate, whose release from glioma cells did not occur via the most common glial mechanisms of transport, or in response to hypoosmotic stress. GCM also contained glycine, whose action contrasted the glutamate effect. Indeed, strychnine application significantly increased GCM-induced depolarization and [Cl-]i rise. GCM-evoked [Cl-]i elevation was not inhibited by antagonists of Cl- transporters and significantly reduced in the presence of anion channels blocker NPPB, suggesting that Cl-selective channels are a major route for GCM-induced Cl- influx. Altogether, these data show that glioma released aminoacids may dynamically alter Cl- equilibrium in surrounding neurons, deeply interfering with their inhibitory balance, likely leading to physiological and

  20. Low dose ionizing radiation responses and knockdown of ATM kinase activity in glioma stem cells

    Lim, Y.C.; Roberts, T.; Day, B.; Kozlov, S.; Walker, D.; Lavin, M.; Harding, A.

    2009-01-01

    Genesis of new cells in the mammalian brain has previously been regarded as a negligible event; an assumption that long limited our understanding in the development of neoplasias. The recent discovery of perpetual lineages derived from neural stem cells has resulted in a new approach to studying the cellular behaviour of potential cancer stem cells in the brain. Glioblastoma multiforme (GBM), the most aggressive and lethal brain tumour is derived from a group of cancerous stem cells known as glioma stem cells. GBM cells are impervious to conventional therapies such as surgical resection and ionizing radiation because of their pluripotent and radioresistant properties. Thus in our study, we aim to investigate whether a combination of chemo- and radio- therapies is an effective treatment for glioma stem cells. The study utilizes a specific kinase inhibitor (ATMi) of the ATM (Ataxia-telangiectasia mutated) protein which is an essential protein in DNA-damage responses. In the presence of both low dose radiation and ATMi, glioma stem cells have rapid onset of cell death and reduction in growth. Since DNA damage can be inherited through cell division, accumulated DNA breaks in later generations may also lead to cell death. The limitation of conventional radiation therapy is that administration of fractionated (low) doses to reduce any potential harm to the surrounding healthy cells in the brain outweighs the benefits of high radiation doses to induce actual arrest in the propagation of malignant cells. Our study demonstrates a benefit in using low dose radiation combined with chemotherapy resulting in a reduction in malignancy of glioma stem cells. (author)

  1. Tumor initiating cells in malignant gliomas: biology and implications for therapy.

    Hadjipanayis, Costas G; Van Meir, Erwin G

    2009-04-01

    A rare subpopulation of cells within malignant gliomas, which shares canonical properties with neural stem cells (NSCs), may be integral to glial tumor development and perpetuation. These cells, also known as tumor initiating cells (TICs), have the ability to self-renew, develop into any cell in the overall tumor population (multipotency), and proliferate. A defining property of TICs is their ability to initiate new tumors in immunocompromised mice with high efficiency. Mounting evidence suggests that TICs originate from the transformation of NSCs and their progenitors. New findings show that TICs may be more resistant to chemotherapy and radiation than the bulk of tumor cells, thereby permitting recurrent tumor formation and accounting for the failure of conventional therapies. The development of new therapeutic strategies selectively targeting TICs while sparing NSCs may provide for more effective treatment of malignant gliomas.

  2. Critical role of the FERM domain in Pyk2 stimulated glioma cell migration

    Lipinski, Christopher A.; Tran, Nhan L.; Dooley, Andrea; Pang, Yuan-Ping; Rohl, Carole; Kloss, Jean; Yang, Zhongbo; McDonough, Wendy; Craig, David; Berens, Michael E.; Loftus, Joseph C.

    2006-01-01

    The strong tendency of malignant glioma cells to invade locally into surrounding normal brain precludes effective surgical resection, reduces the efficacy of radiotherapy, and is associated with increased resistance to chemotherapy regimens. We report that the N-terminal FERM domain of Pyk2 regulates its promigratory function. A 3-dimensional model of the Pyk2 FERM domain was generated and mutagenesis studies identified residues essential for Pyk2 promigratory function. Model-based targeted mutations within the FERM domain decreased Pyk2 phosphorylation and reduced the capacity of Pyk2 to stimulate glioma cell migration but did not significantly alter the intracellular distribution of Pyk2. Expression of autonomous Pyk2 FERM domain fragments containing analogous mutations exhibited reduced capacity to inhibit glioma cell migration and Pyk2 phosphorylation relative to expression of an autonomous wild type FERM domain fragment. These results indicate that the FERM domain plays an important role in regulating the functional competency of Pyk2 as a promigratory factor in glioma

  3. Nitrosoureas Inhibit the Stathmin Mediated Migration and Invasion of Malignant Glioma Cells

    Liang, Xing-Jie; Choi, Yong; Sackett, Dan L.; Park, John K.

    2008-01-01

    Malignant gliomas are the most common primary intrinsic brain tumors and are highly lethal. The widespread migration and invasion of neoplastic cells from the initial site of tumor formation into the surrounding brain render these lesions refractory to definitive surgical treatment. Stathmin, a microtubule destabilizing protein that mediates cell cycle progression, can also regulate directed cell movement. Nitrosoureas, traditionally viewed as DNA alkylating agents, can also covalently modify...

  4. 5-Aminolevulinic acid-mediated sonosensitization of rat RG2 glioma cells in vitro

    Krzysztof Bilmin

    2016-10-01

    Full Text Available Sonodynamic therapy (SDT is a promising technique based on the ability of certain substances, called sonosensitizers, to sensitize cancer cells to non-thermal effects of low-energy ultrasound waves, allowing their destruction. Sonosensitization is thought to induce cell death by direct physical effects such as cavitation and acoustical streaming as well as by complementary chemical reactions generating oxygen free radicals. One of the promising sonosensitizers is 5-aminolevulinic acid (ALA which upon selective uptake by cancer cells is metabolized and accumulated as protoporphyrin IX. The objective of the study was to describe ALA-mediated sonodynamic effects in vitro on a rat RG2 glioma cell line. Glioma cells, seeded at the bottom of 96-well plates and incubated with ALA (10 µg/ml for 6 h, were exposed to the sinusoidal US pulses with a resonance frequency of 1 MHz, 1000 µs duration, 0.4 duty-cycle, and average acoustic power varying from 2 W to 6 W. Ultrasound waves were generated by a flat circular piezoelectric transducer with a diameter of 25 mm. Cell viability was determined by MTT assay. Structural cellular changes were visualized with a fluorescence microscope. Signs of cytotoxicity such as a decrease in cell viability, chromatin condensation and apoptosis were found. ALA-mediated SDT evokes cytotoxic effects of low intensity US on rat RG2 glioma cells in vitro . This cell line is indicated for further preclinical assessment of SDT in in vivo conditions.

  5. PERK silence inhibits glioma cell growth under low glucose stress by blockage of p-AKT and subsequent HK2's mitochondria translocation

    Hou, Xu

    2015-03-12

    Glioma relies on glycolysis to obtain energy and sustain its survival under low glucose microenvironment in vivo. The mechanisms on glioma cell glycolysis regulation are still unclear. Signaling mediated by Double-stranded RNA-activated protein kinase (PKR) - like ER kinase (PERK) is one of the important pathways of unfolded protein response (UPR) which is comprehensively activated in cancer cells upon the hypoxic and low glucose stress. Here we show that PERK is significantly activated in human glioma tissues. PERK silencing results in decreased glioma cell viability and ATP/lactate production upon low glucose stress, which is mediated by partially blocked AKT activation and subsequent inhibition of Hexokinase II (HK2)\\'s mitochondria translocation. More importantly, PERK silenced glioma cells show decreased tumor formation capacity. Our results reveal that PERK activation is involved in glioma glycolysis regulation and may be a potential molecular target for glioma treatment.

  6. PERK silence inhibits glioma cell growth under low glucose stress by blockage of p-AKT and subsequent HK2's mitochondria translocation

    Hou, Xu; Liu, Yaohua; Liu, Huailei; Chen, Xin; Liu, Min; Che, Hui; Guo, Fei; Wang, Chunlei; Zhang, Daming; Wu, Jianing; Chen, Xiaofeng; Shen, Chen; Li, Chenguang; Peng, Fei; Bi, Yunke; Yang, Zhuowen; Yang, Guang; Ai, Jing; Gao, Xin; Zhao, Shiguang

    2015-01-01

    Glioma relies on glycolysis to obtain energy and sustain its survival under low glucose microenvironment in vivo. The mechanisms on glioma cell glycolysis regulation are still unclear. Signaling mediated by Double-stranded RNA-activated protein kinase (PKR) - like ER kinase (PERK) is one of the important pathways of unfolded protein response (UPR) which is comprehensively activated in cancer cells upon the hypoxic and low glucose stress. Here we show that PERK is significantly activated in human glioma tissues. PERK silencing results in decreased glioma cell viability and ATP/lactate production upon low glucose stress, which is mediated by partially blocked AKT activation and subsequent inhibition of Hexokinase II (HK2)'s mitochondria translocation. More importantly, PERK silenced glioma cells show decreased tumor formation capacity. Our results reveal that PERK activation is involved in glioma glycolysis regulation and may be a potential molecular target for glioma treatment.

  7. Bmi-1 promotes the aggressiveness of glioma via activating the NF-kappaB/MMP-9 signaling pathway

    Jiang, Lili; Wu, Jueheng; Yang, Yi; Liu, Liping; Song, Libing; Li, Jun; Li, Mengfeng

    2012-01-01

    The prognosis of human glioma is poor, and the highly invasive nature of the disease represents a major impediment to current therapeutic modalities. The oncoprotein B-cell-specific Moloney murine leukemia virus integration site 1 protein (Bmi-1) has been linked to the development and progression of glioma; however, the biological role of Bmi-1 in the invasion of glioma remains unclear. A172 and LN229 glioma cells were engineered to overexpress Bmi-1 via stable transfection or to be silenced for Bmi-1 expression using RNA interfering method. Migration and invasiveness of the engineered cells were assessed using wound healing assay, Transwell migration assay, Transwell matrix penetration assay and 3-D spheroid invasion assay. MMP-9 expression and activity were measured using real-time PCR, ELISA and the gelatin zymography methods. Expression of NF-kappaB target genes was quantified using real-time PCR. NF-kappaB transcriptional activity was assessed using an NF-kappaB luciferase reporter system. Expression of Bmi-1 and MMP-9 in clinical specimens was analyzed using immunohistochemical assay. Ectopic overexpression of Bmi-1 dramatically increased, whereas knockdown of endogenous Bmi-1 reduced, the invasiveness and migration of glioma cells. NF-kappaB transcriptional activity and MMP-9 expression and activity were significantly increased in Bmi-1-overexpressing but reduced in Bmi-1-silenced cells. The reporter luciferase activity driven by MMP-9 promoter in Bmi-1-overexpressing cells was dependent on the presence of a functional NF-kappaB binding site, and blockade of NF-kappaB signaling inhibited the upregulation of MMP-9 in Bmi-1 overexpressing cells. Furthermore, expression of Bmi-1 correlated with NF-kappaB nuclear translocation as well as MMP-9 expression in clinical glioma samples. Bmi-1 may play an important role in the development of aggressive phenotype of glioma via activating the NF-kappaB/MMP-9 pathway and therefore might represent a novel therapeutic

  8. Local delivery of cancer-cell glycolytic inhibitors in high-grade glioma

    Wicks, Robert T.; Azadi, Javad; Mangraviti, Antonella; Zhang, Irma; Hwang, Lee; Joshi, Avadhut; Bow, Hansen; Hutt-Cabezas, Marianne; Martin, Kristin L.; Rudek, Michelle A.; Zhao, Ming; Brem, Henry; Tyler, Betty M.

    2015-01-01

    Background 3-bromopyruvate (3-BrPA) and dichloroacetate (DCA) are inhibitors of cancer-cell specific aerobic glycolysis. Their application in glioma is limited by 3-BrPA's inability to cross the blood-brain-barrier and DCA's dose-limiting toxicity. The safety and efficacy of intracranial delivery of these compounds were assessed. Methods Cytotoxicity of 3-BrPA and DCA were analyzed in U87, 9L, and F98 glioma cell lines. 3-BrPA and DCA were incorporated into biodegradable pCPP:SA wafers, and the maximally tolerated dose was determined in F344 rats. Efficacies of the intracranial 3-BrPA wafer and DCA wafer were assessed in a rodent allograft model of high-grade glioma, both as a monotherapy and in combination with temozolomide (TMZ) and radiation therapy (XRT). Results 3-BrPA and DCA were found to have similar IC50 values across the 3 glioma cell lines. 5% 3-BrPA wafer-treated animals had significantly increased survival compared with controls (P = .0027). The median survival of rats with the 50% DCA wafer increased significantly compared with both the oral DCA group (P = .050) and the controls (P = .02). Rats implanted on day 0 with a 5% 3-BrPA wafer in combination with TMZ had significantly increased survival over either therapy alone. No statistical difference in survival was noted when the wafers were added to the combination therapy of TMZ and XRT, but the 5% 3-BrPA wafer given on day 0 in combination with TMZ and XRT resulted in long-term survivorship of 30%. Conclusion Intracranial delivery of 3-BrPA and DCA polymer was safe and significantly increased survival in an animal model of glioma, a potential novel therapeutic approach. The combination of intracranial 3-BrPA and TMZ provided a synergistic effect. PMID:25053853

  9. DNA fingerprinting of glioma cell lines and considerations on similarity measurements.

    Bady, Pierre; Diserens, Annie-Claire; Castella, Vincent; Kalt, Stefanie; Heinimann, Karl; Hamou, Marie-France; Delorenzi, Mauro; Hegi, Monika E

    2012-06-01

    Glioma cell lines are an important tool for research in basic and translational neuro-oncology. Documentation of their genetic identity has become a requirement for scientific journals and grant applications to exclude cross-contamination and misidentification that lead to misinterpretation of results. Here, we report the standard 16 marker short tandem repeat (STR) DNA fingerprints for a panel of 39 widely used glioma cell lines as reference. Comparison of the fingerprints among themselves and with the large DSMZ database comprising 9 marker STRs for 2278 cell lines uncovered 3 misidentified cell lines and confirmed previously known cross-contaminations. Furthermore, 2 glioma cell lines exhibited identity scores of 0.8, which is proposed as the cutoff for detecting cross-contamination. Additional characteristics, comprising lack of a B-raf mutation in one line and a similarity score of 1 with the original tumor tissue in the other, excluded a cross-contamination. Subsequent simulation procedures suggested that, when using DNA fingerprints comprising only 9 STR markers, the commonly used similarity score of 0.8 is not sufficiently stringent to unambiguously differentiate the origin. DNA fingerprints are confounded by frequent genetic alterations in cancer cell lines, particularly loss of heterozygosity, that reduce the informativeness of STR markers and, thereby, the overall power for distinction. The similarity score depends on the number of markers measured; thus, more markers or additional cell line characteristics, such as information on specific mutations, may be necessary to clarify the origin.

  10. Long Non-coding RNA LINC00339 Stimulates Glioma Vasculogenic Mimicry Formation by Regulating the miR-539-5p/TWIST1/MMPs Axis

    Junqing Guo

    2018-03-01

    Full Text Available Glioma is recognized as a highly angiogenic malignant brain tumor. Vasculogenic mimicry (VM greatly restricts the therapeutic effect of anti-angiogenic tumor therapy for glioma patients. However, the molecular mechanisms of VM formation in glioma remain unclear. Here, we demonstrated that LINC00339 was upregulated in glioma tissue as well as in glioma cell lines. The expression of LINC00339 in glioma tissues was positively correlated with glioma VM formation. Knockdown of LINC00339 inhibited glioma cell proliferation, migration, invasion, and tube formation, meanwhile downregulating the expression of VM-related molecular MMP-2 and MMP-14. Furthermore, knockdown of LINC00339 significantly increased the expression of miR-539-5p. Both bioinformatics and luciferase reporter assay revealed that LINC00339 regulated the above effects via binding to miR-539-5p. Besides, overexpression of miR-539-5p resulted in decreased expression of TWIST1, a transcription factor known to play an oncogenic role in glioma and identified as a direct target of miR-539-5p. TWIST1 upregulated the promoter activities of MMP-2 and MMP-14. The in vivo study showed that nude mice carrying tumors with knockdown of LINC00339 and overexpression of miR-539-5p exhibited the smallest tumor volume through inhibiting VM formation. In conclusion, LINC00339 may be used as a novel therapeutic target for VM formation in glioma.

  11. The Inhibition of microRNA-128 on IGF-1-Activating mTOR Signaling Involves in Temozolomide-Induced Glioma Cell Apoptotic Death.

    Peng-Hsu Chen

    Full Text Available Temozolomide (TMZ, an alkylating agent of the imidazotetrazine series, is a first-line chemotherapeutic drug used in the clinical therapy of glioblastoma multiforme, the most common and high-grade primary glioma in adults. Micro (miRNAs, which are small noncoding RNAs, post-transcriptionally regulate gene expressions and are involved in gliomagenesis. However, no studies have reported relationships between TMZ and miRNA gene regulation. We investigated TMZ-mediated miRNA profiles and its molecular mechanisms underlying the induction of glioma cell death. By performing miRNA microarray and bioinformatics analyses, we observed that expression of 248 miRNAs was altered, including five significantly upregulated and 17 significantly downregulated miRNAs, in TMZ-treated U87MG cells. miR-128 expression levels were lower in different glioma cells and strongly associated with poor survival. TMZ treatment significantly upregulated miR-128 expression. TMZ significantly enhanced miR-128-1 promoter activity and transcriptionally regulated miR-128 levels through c-Jun N-terminal kinase 2/c-Jun pathways. The overexpression and knockdown of miR-128 expression significantly affected TMZ-mediated cell viability and apoptosis-related protein expression. Furthermore, the overexpression of miR-128 alone enhanced apoptotic death of glioma cells through caspase-3/9 activation, poly(ADP ribose polymerase degradation, reactive oxygen species generation, mitochondrial membrane potential loss, and non-protective autophagy formation. Finally, we identified that key members in mammalian target of rapamycin (mTOR signaling including mTOR, rapamycin-insensitive companion of mTOR, insulin-like growth factor 1, and PIK3R1, but not PDK1, were direct target genes of miR-128. TMZ inhibited mTOR signaling through miR-128 regulation. These results indicate that miR-128-inhibited mTOR signaling is involved in TMZ-mediated cytotoxicity. Our findings may provide a better understanding

  12. Monstrous cell in malignant gliomas. In relation to radiation and chemotherapy

    Ogashiwa, M; Takeuchi, K; Akai, K [Kyorin Univ., Mitaka, Tokyo (Japan). School of Medicine

    1981-06-01

    The pathological effects of irradiation and chemotherapy have been studied in 9 autopsy cases of malignant and low grade gliomas. The brains have been examined by means of the complete study technique. Many histological features have been related to surgery, grading of histological classification of gliomas, irradiation and chemotherapy. Following irradiation and chemotherapy, in addition to increased necrosis and vascular response, a variety of characteristic changes were observed in cell and nuclear morphology with prominent formation of monstrous cells in all of 5 malignant gliomas treated with nitrosourea. These monstrous cells had irregular and hyperchromatic multinuclei and showed cytoplasmic degeneration. These cells which had no direct relationship to vessels distributed both in the periphery of tumor or necrosis and in the white matter remote from the main tumor. These changes were more pronounced in autopsy than in biopsy. The features showed here indicate that the monstrous cells may appear due to the result of inhibition of tumor cell division at the late mitotic phase after irradiation and chemotherapy.

  13. LuIII parvovirus selectively and efficiently targets, replicates in, and kills human glioma cells.

    Paglino, Justin C; Ozduman, Koray; van den Pol, Anthony N

    2012-07-01

    Because productive infection by parvoviruses requires cell division and is enhanced by oncogenic transformation, some parvoviruses may have potential utility in killing cancer cells. To identify the parvovirus(es) with the optimal oncolytic effect against human glioblastomas, we screened 12 parvoviruses at a high multiplicity of infection (MOI). MVMi, MVMc, MVM-G17, tumor virus X (TVX), canine parvovirus (CPV), porcine parvovirus (PPV), rat parvovirus 1A (RPV1A), and H-3 were relatively ineffective. The four viruses with the greatest oncolytic activity, LuIII, H-1, MVMp, and MVM-G52, were tested for the ability, at a low MOI, to progressively infect the culture over time, causing cell death at a rate higher than that of cell proliferation. LuIII alone was effective in all five human glioblastomas tested. H-1 progressively infected only two of five; MVMp and MVM-G52 were ineffective in all five. To investigate the underlying mechanism of LuIII's phenotype, we used recombinant parvoviruses with the LuIII capsid replacing the MVMp capsid or with molecular alteration of the P4 promoter. The LuIII capsid enhanced efficient replication and oncolysis in MO59J gliomas cells; other gliomas tested required the entire LuIII genome to exhibit enhanced infection. LuIII selectively infected glioma cells over normal glial cells in vitro. In mouse models, human glioblastoma xenografts were selectively infected by LuIII when administered intratumorally; LuIII reduced tumor growth by 75%. LuIII also had the capacity to selectively infect subcutaneous or intracranial gliomas after intravenous inoculation. Intravenous or intracranial LuIII caused no adverse effects. Intracranial LuIII caused no infection of mature mouse neurons or glia in vivo but showed a modest infection of developing neurons.

  14. SCD1 Confers Temozolomide Resistance to Human Glioma Cells via the Akt/GSK3β/β-Catenin Signaling Axis

    Shuang Dai

    2018-01-01

    Full Text Available Resistance to temozolomide (TMZ, the standard chemotherapy agent for glioblastoma (GBM, poses a major clinical challenge to GBM prognosis. Understanding the mechanisms of TMZ resistance can help to identify novel drug targets and more effective therapies. Recent studies suggest that bioenergetic alterations of cancer cells play important roles in drug resistance. In our study, the altered metabolism of cancer cells was observed using a metabolic PCR array. We found that stearoyl-coenzyme A desaturase 1 (SCD1, a key rate-limiting enzyme for synthesis of monounsaturated fatty acids, was significantly upregulated in TMZ-resistant GBM cells compared to their parental counterparts. Overexpression of SCD1 promoted resistance to TMZ in parental GBM cells, whereas SCD1 downregulation by siRNA could re-sensitize TMZ-resistant cells in vitro. Combinational treatment of TMZ and an SCD1-specific inhibitor showed a combined inhibitory effect on TMZ-resistant glioma cells. We also observed that overexpression of SCD1 promoted Akt/GSK3β/β-catenin signaling, while silencing of SCD1 inhibited the signaling. The combination of an Akt activator with exogenous SCD1 or the combined inhibition of Akt and enforced expression of SCD1 resulted in the most significant changes of Akt signaling. Functionally, significantly lower viability and mobility rates were observed in TMZ-resistant cells when treated with Akt inhibitors and an SCD1 inhibitor simultaneously compared to when treated individually. In conclusion, our study identified SCD1 along with its functional pathway as a novel target in the development of TMZ resistance. SCD1 inhibition used alone or in combination with Akt inhibition could effectively overcome TMZ resistance in gliomas.

  15. The microRNA-302b-inhibited insulin-like growth factor-binding protein 2 signaling pathway induces glioma cell apoptosis by targeting nuclear factor IA.

    Chin-Cheng Lee

    Full Text Available MicroRNAs are small noncoding RNAs that post-transcriptionally control the expression of genes involved in glioblastoma multiforme (GBM development. Although miR-302b functions as a tumor suppressor, its role in GBM is still unclear. Therefore, this study comprehensively explored the roles of miR-302b-mediated gene networks in GBM cell death. We found that miR-302b levels were significantly higher in primary astrocytes than in GBM cell lines. miR-302b overexpression dose dependently reduced U87-MG cell viability and induced apoptosis through caspase-3 activation and poly(ADP ribose polymerase degradation. A transcriptome microarray revealed 150 downregulated genes and 380 upregulated genes in miR-302b-overexpressing cells. Nuclear factor IA (NFIA, higher levels of which were significantly related to poor survival, was identified as a direct target gene of miR-302b and was involved in miR-302b-induced glioma cell death. Higher NFIA levels were observed in GBM cell lines and human tumor sections compared with astrocytes and non-tumor tissues, respectively. NFIA knockdown significantly enhanced apoptosis. We found high levels of insulin-like growth factor-binding protein 2 (IGFBP2, another miR-302b-downregulated gene, in patients with poor survival. We verified that NFIA binds to the IGFBP2 promoter and transcriptionally enhances IGFBP2 expression levels. We identified that NFIA-mediated IGFBP2 signaling pathways are involved in miR-302b-induced glioma cell death. The identification of a regulatory loop whereby miR-302b inhibits NFIA, leading to a decrease in expression of IGFBP-2, may provide novel directions for developing therapies to target glioblastoma tumorigenesis.

  16. The role of drebrin in glioma migration and invasion

    Terakawa, Yuzo [The Arthur and Sonia Labatt Brain Tumour Research Centre, The Hospital for Sick Children, Toronto, Ontario (Canada); Department of Neurosurgery, Osaka City University Graduate School of Medicine, Osaka (Japan); Agnihotri, Sameer; Golbourn, Brian; Nadi, Mustafa; Sabha, Nesrin; Smith, Christian A. [The Arthur and Sonia Labatt Brain Tumour Research Centre, The Hospital for Sick Children, Toronto, Ontario (Canada); Croul, Sidney E. [The Arthur and Sonia Labatt Brain Tumour Research Centre, The Hospital for Sick Children, Toronto, Ontario (Canada); Division of Neuropathology, University Health Network, Department of Laboratory Medicine and Pathobiology (Canada); Rutka, James T., E-mail: james.rutka@sickkids.ca [The Arthur and Sonia Labatt Brain Tumour Research Centre, The Hospital for Sick Children, Toronto, Ontario (Canada); Department of Surgery, University of Toronto, Toronto, Ontario (Canada)

    2013-02-15

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

  17. Interleukin-13 conjugated quantum dots for identification of glioma initiating cells and their extracellular vesicles.

    Madhankumar, A B; Mrowczynski, Oliver D; Patel, Suhag R; Weston, Cody L; Zacharia, Brad E; Glantz, Michael J; Siedlecki, Christopher A; Xu, Li-Chong; Connor, James R

    2017-08-01

    Cadmium selenide (CdSe) based quantum dots modified with polyethylene glycol and chemically linked to interleukin-13 (IL13) were prepared with the aim of identifying the high affinity receptor (IL13Rα2) which is expressed in glioma stem cells and exosomes secreted by these cancer stem cells. IL13 conjugated quantum dots (IL13QD) were thoroughly characterized for their physicochemical properties including particle size and surface morphology. Furthermore, the specific binding of the IL13QD to glioma cells and to glioma stem cells (GSC) was verified using a competitive binding study. The exosomes were isolated from the GSC conditioned medium and the expression of IL13Rα2 in the GSC and exosomes was verified. The binding property of IL13QD to the tumor associated exosomes was initially confirmed by transmission electron microscopy. The force of attraction between the quantum dots and U251 glioma cells and the exosomes was investigated by atomic force microscopy, which indicated a higher force of binding interaction between the IL13QD and IL13Rα2 expressing glioma cells and exosomes secreted by glioma stem cells. Flow cytometry of the IL13QD and exosomes from the culture media and cerebrospinal fluid (CSF) of patients with glioma tumors indicated a distinctly populated complex pattern different from that of non-targeted quantum dots and bovine serum albumin (BSA) conjugated quantum dots confirming specific binding potential of the IL13QD to the tumor associated exosomes. The results of this study demonstrate that IL13QD can serve as an ex vivo marker for glioma stem cells and exosomes that can inform diagnosis and prognosis of patients harboring malignant disease. Functionalized quantum dots are flexible semiconductor nanomaterials which have an immense application in biomedical research. In particular, when they are functionalized with biomolecules like proteins or antibodies, they have the specialized ability to detect the expression of receptors and antigens in

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

    Pei, Jian; Park, In-Ho; Ryu, Hyang-Hwa; Li, Song-Yuan; Li, Chun-Hao; Lim, Sa-Hoe; Wen, Min; Jang, Woo-Youl; Jung, Shin

    2015-01-01

    Glioblastoma 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. The 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. MMP-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. Radiation-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

  19. Resveratrol induces cellular senescence with attenuated mono-ubiquitination of histone H2B in glioma cells

    Gao, Zhen; Xu, Michael S.; Barnett, Tamara L.; Xu, C. Wilson

    2011-01-01

    Research highlights: → Resveratrol induces cellular senescence in glioma cell. → Resveratrol inhibits mono-ubiquitination of histone H2B at K120. → Depletion of RNF20, phenocopies the inhibitory effects of resveratrol. → Mono-ubiquitination of histone H2B at K120 is a novel target of resveratrol. → RNF20 inhibits cellular senescence in proliferating glioma cells. -- Abstract: Resveratrol (3,4',5-trihydroxy-trans-stilbene), a polyphenol naturally occurring in grapes and other plants, has cancer chemo-preventive effects and therapeutic potential. Although resveratrol modulates multiple pathways in tumor cells, how resveratrol or its affected pathways converge on chromatin to mediate its effects is not known. Using glioma cells as a model, we showed here that resveratrol inhibited cell proliferation and induced cellular hypertrophy by transforming spindle-shaped cells to enlarged, irregular and flatten-shaped ones. We further showed that resveratrol-induced hypertrophic cells expressed senescence-associated-β-galactosidase, suggesting that resveratrol-induced cellular senescence in glioma cells. Consistent with these observations, we demonstrated that resveratrol inhibited clonogenic efficiencies in vitro and tumor growth in a xenograft model. Furthermore, we found that acute treatment of resveratrol inhibited mono-ubiquitination of histone H2B at K120 (uH2B) in breast, prostate, pancreatic, lung, brain tumor cells as well as primary human cells. Chronic treatment with low doses of resveratrol also inhibited uH2B in the resveratrol-induced senescent glioma cells. Moreover, we showed that depletion of RNF20, a ubiquitin ligase of histone H2B, inhibited uH2B and induced cellular senescence in glioma cells in vitro, thereby recapitulated the effects of resveratrol. Taken together, our results suggest that uH2B is a novel direct or indirect chromatin target of resveratrol and RNF20 plays an important role in inhibiting cellular senescence programs that are

  20. Resveratrol induces cellular senescence with attenuated mono-ubiquitination of histone H2B in glioma cells

    Gao, Zhen; Xu, Michael S.; Barnett, Tamara L. [Nevada Cancer Institute, Las Vegas, NV 89135 (United States); Xu, C. Wilson, E-mail: wxu@nvcancer.org [Nevada Cancer Institute, Las Vegas, NV 89135 (United States)

    2011-04-08

    Research highlights: {yields} Resveratrol induces cellular senescence in glioma cell. {yields} Resveratrol inhibits mono-ubiquitination of histone H2B at K120. {yields} Depletion of RNF20, phenocopies the inhibitory effects of resveratrol. {yields} Mono-ubiquitination of histone H2B at K120 is a novel target of resveratrol. {yields} RNF20 inhibits cellular senescence in proliferating glioma cells. -- Abstract: Resveratrol (3,4',5-trihydroxy-trans-stilbene), a polyphenol naturally occurring in grapes and other plants, has cancer chemo-preventive effects and therapeutic potential. Although resveratrol modulates multiple pathways in tumor cells, how resveratrol or its affected pathways converge on chromatin to mediate its effects is not known. Using glioma cells as a model, we showed here that resveratrol inhibited cell proliferation and induced cellular hypertrophy by transforming spindle-shaped cells to enlarged, irregular and flatten-shaped ones. We further showed that resveratrol-induced hypertrophic cells expressed senescence-associated-{beta}-galactosidase, suggesting that resveratrol-induced cellular senescence in glioma cells. Consistent with these observations, we demonstrated that resveratrol inhibited clonogenic efficiencies in vitro and tumor growth in a xenograft model. Furthermore, we found that acute treatment of resveratrol inhibited mono-ubiquitination of histone H2B at K120 (uH2B) in breast, prostate, pancreatic, lung, brain tumor cells as well as primary human cells. Chronic treatment with low doses of resveratrol also inhibited uH2B in the resveratrol-induced senescent glioma cells. Moreover, we showed that depletion of RNF20, a ubiquitin ligase of histone H2B, inhibited uH2B and induced cellular senescence in glioma cells in vitro, thereby recapitulated the effects of resveratrol. Taken together, our results suggest that uH2B is a novel direct or indirect chromatin target of resveratrol and RNF20 plays an important role in inhibiting cellular

  1. Long non-coding RNA TUG1 acts as a miR-26a sponge in human glioma cells.

    Li, Jun; An, Gang; Zhang, Meng; Ma, Qingfang

    2016-09-02

    Long non-coding RNA taurine upregulated gene 1 (TUG1) acts as an important regulator in cancer pathogenesis; however, its functional mechanism in glioma development remains unclear. This study aims to explore the potential function of TUG1 in glioma by sponging miR-26a. The expression of TUG1, miR-26a, and phosphatase and tensin homolog (PTEN) in 20 paired glioma tissues was detected by quantitative real-time PCR and subjected to correlation analysis. Bioinformatics analysis was performed by using DIANA Tools. Abnormal TUG1 expression was conducted in two glioma cells to analyze its regulation on miR-26a and PTEN using real-time PCR, western blot, and luciferase reporter assay. TUG1 expression was confirmed to be upregulated in glioma tissues, and showed an inverse correlation with downregulated miR-26a. TUG1 could negatively regulate the expression of miR-26a in glioma cells. The bioinformatics prediction revealed putative miR-26a binding sites within TUG1 transcripts. Further experiments demonstrated the positive regulation of TUG1 on the miR-26a target, PTEN, wherein TUG1 could inhibit the negative regulation of miR-26a on PTEN by binding its 3'UTR. Additionally, the expression of PTEN was also upregulated in glioma tissues, showing a positive or negative correlation with TUG1 or miR-26a, respectively. TUG1 could serve as a miR-26a sponge in human glioma cells, contributing to the upregulation of PTEN. This study revealed a new TUG1/miR-26a/PTEN regulatory mechanism and provided a further understanding of the tumor-suppressive role of TUG1 in glioma development. Copyright © 2016 Elsevier Inc. All rights reserved.

  2. Flavopiridol induces apoptosis in glioma cell lines independent of retinoblastoma and p53 tumor suppressor pathway alterations by a caspase-independent pathway.

    Alonso, Michelle; Tamasdan, Cristina; Miller, Douglas C; Newcomb, Elizabeth W

    2003-02-01

    Flavopiridol is a synthetic flavone, which inhibits growth in vitro and in vivo of several solid malignancies such as renal, prostate, and colon cancers. It is a potent cyclin-dependent kinase inhibitor presently in clinical trials. In this study, we examined the effect of flavopiridol on a panel of glioma cell lines having different genetic profiles: five of six have codeletion of p16(INK4a) and p14(ARF); three of six have p53 mutations; and one of six shows overexpression of mouse double minute-2 (MDM2) protein. Independent of retinoblastoma and p53 tumor suppressor pathway alterations, flavopiridol induced apoptosis in all cell lines but through a caspase-independent mechanism. No cleavage products for caspase 3 or its substrate poly(ADP-ribose) polymerase or caspase 8 were detected. The pan-caspase inhibitor Z-VAD-fmk did not inhibit flavopiridol-induced apoptosis. Mitochondrial damage measured by cytochrome c release and transmission electron microscopy was not observed in drug-treated glioma cells. In contrast, flavopiridol treatment induced translocation of apoptosis-inducing factor from the mitochondria to the nucleus. The proteins cyclin D(1) and MDM2 involved in the regulation of retinoblastoma and p53 activity, respectively, were down-regulated early after flavopiridol treatment. Given that MDM2 protein can confer oncogenic properties under certain circumstances, loss of MDM2 expression in tumor cells could promote increased chemosensitivity. After drug treatment, a low Bcl-2/Bax ratio was observed, a condition that may favor apoptosis. Taken together, the data indicate that flavopiridol has activity against glioma cell lines in vitro and should be considered for clinical development in the treatment of glioblastoma multiforme.

  3. Hydroxyapatite nanoparticles inhibit the growth of human glioma cells in vitro and in vivo

    Chu SH

    2012-07-01

    Full Text Available Sheng-Hua Chu,1 Dong-Fu Feng,1 Yan-Bin Ma,1 Zhi-Qiang Li21Department of Neurosurgery, No 3 People's Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai, China; 2Department of Neurosurgery, Zhongnan Hospital of Wuhan University, Wuhan, ChinaAbstract: Hydroxyapatite nanoparticles (nano-HAPs have been reported to exhibit antitumor effects on various human cancers, but the effects of nano-HAPs on human glioma cells remain unclear. The aim of this study was to explore the inhibitory effect of nano-HAPs on the growth of human glioma U251 and SHG44 cells in vitro and in vivo. Nano-HAPs could inhibit the growth of U251 and SHG44 cells in a dose- and time-dependent manner, according to methyl thiazoletetrazolium assay and flow cytometry. Treated with 120 mg/L and 240 mg/L nano-HAPs for 48 hours, typical apoptotic morphological changes were noted under Hoechst staining and transmission electron microscopy. The tumor growth of cells was inhibited after the injection in vivo, and the related side effects significantly decreased in the nano-HAP-and-drug combination group. Because of the function of nano-HAPs, the expression of c-Met, SATB1, Ki-67, and bcl-2 protein decreased, and the expression of SLC22A18 and caspase-3 protein decreased noticeably. The findings indicate that nano-HAPs have an evident inhibitory action and induce apoptosis of human glioma cells in vitro and in vivo. In a drug combination, they can significantly reduce the adverse reaction related to the chemotherapeutic drug 1,3-bis(2-chloroethyl-1-nitrosourea (BCNU.Keywords: glioma, hydroxyapatite nanoparticles, growth mechanism

  4. Toll-like receptor triggered dendritic cell maturation and IL-12 secretion are necessary to overcome T-cell inhibition by glioma-associated TGF-beta2.

    Grauer, O.M.; Poschl, P.; Lohmeier, A.; Adema, G.J.; Bogdahn, U.

    2007-01-01

    Malignant gliomas are able to secrete large amounts of immunosuppressive cytokines like transforming growth factor beta 2 (TGF-beta2) and regularly escape from immune surveillance. Many strategies have been developed to induce potent anti-glioma responses, among those the use of dendritic cells (DC)

  5. Radiosensitivity and TP 53, EGFR amplification and LOH10 analysis of primary glioma cell cultures

    Gerlach, B.; Harder, A.H.; Slotman, B.J.; Sminia, P.; Hulsebos, T.J.M.; Leenstra, S.; Peter Vandertop, W.; Hartmann, K.A.

    2002-01-01

    Aim: Determination of in-vitro radiosensitivity and genetic alterations of cell cultures derived from human glioma biopsy tissue and established glioma cell lines. Material and Methods: Fresh brain tumor specimens of six patients were processed to early passage cell cultures. In addition the cell lines D 384 and Gli 6 were used. Cell cultures were irradiated with doses from 2 to 10 Gy. Following irradiation, cell survival was determined by clonogenic assay and survival curves were generated. The surviving fractions after 2 Gy (SF2) and 4 Gy (SF4) were used as radiosensitivity parameters. Genetic analysis included determination of the mutational and loss of heterozygosity (LOH) status of TP 53 (exons 5-8), the LOH 10- and epidermal growth factor receptor gene (EGFR) amplification status. Results: The SF2 and SF4 values ranged from 0.54 to 0.88 (mean: 0.70) and from 0.13 to 0.52 (mean: 0.32), respectively. Genetic alterations were found in the Gli 6 cell line and in two primary cell cultures. The genetic profile of Gli 6 showed LOH but no TP 53 mutation, complete LOH 10 and no EGFR amplification. The VU 15 cell culture showed TP 53 mutation but no LOH 10 or EGFR amplification, while VU 24 showed incomplete LOH 10, EGFR amplification and no TP 53 mutation. In the other four cell cultures and D 384 cell line no genetic alterations were diagnosed. Histopathological classification of glioblastoma multiforme and/or genetic alterations resulted in lower radiosensitivity. Conclusion: In this small series of early passage glioma cell cultures low radiosensitivity and alterations in cell regulatory genes were seen. Further testing of biological behavior in larger series of patient-derived material is ongoing. (orig.)

  6. Hyperthermia studies using inductive and ultrasound methods on E. coli bacteria and mouse glioma cells

    Cabral–Prieto, A., E-mail: agustin.cabral@inin.gob.mx; López-Callejas, R., E-mail: regulo.lopez@inin.gob.mx; Rodríguez-Méndez, B. G., E-mail: benjamin.rodriguez@inin.gob.mx; Santos-Cuevas, C. L., E-mail: clara.cuevas@inin.gob.mx [Carretera México-Toluca s/n, La Marquesa, Instituto Nacional de Investigaciones Nucleares (Mexico); Celis-Almazán, J., E-mail: jony-jac-5@hotmail.com; Olea-Mejía, O., E-mail: oleaoscar@yahoo.com.mx [Universidad Autónoma del Estado de México, Centro Conjunto de Investigación en Química Sustentable (Mexico); Gómez-Morales, J. L. [Universidad Autónoma del Estado de México, Campus El Cerrillo, Facultad de Ciencias (Mexico); Peña-Eguiluz, R., E-mail: rosendo.eguiluz@inin.gob.mx; Valencia-Alvarado, R., E-mail: raul.valencia@inin.gob.mx; Mercado-Cabrera, A., E-mail: antonio.mercado@inin.gob.mx; Muñoz-Castro, A. E., E-mail: arturo.munoz@inin.gob.mx [Carretera México-Toluca s/n, La Marquesa, Instituto Nacional de Investigaciones Nucleares (Mexico); García-Santibañez, F., E-mail: fegasa2@yahoo.com.mx [Universidad Autónoma del Estado de México, Campus El Cerrillo, Facultad de Ciencias (Mexico)

    2017-11-15

    The survival of Escherichia coli bacteria and mouse glioma cells were studied under different temperatures using direct heating in water, ultrasound, and magnetic fluid hyperthermia. The survival of these microorganisms depended on whether the heating mode was continuous or discontinuous, surviving more in the former than in the discontinuous heating mode. Whereas Escherichia coli bacteria did not survive at temperatures ≥50{sup ∘}C, the mouse glioma cells did not survive at temperatures ≥48{sup ∘}C. The survival of both these microorganisms was independent of the presence or absence of the magnetic nanoparticles of magnetite, suggesting that these, having mean particle sizes of 9.5, 8.5 and 5, did not show any apparent cytotoxicity effect. Present results also showed that the inductive heating system which used a radiofrequency of 13.56 MHz, providing a maximum magnetic field strength of 160 A/m, the electric rather than magnetic heating predominated.

  7. Hyperthermia studies using inductive and ultrasound methods on E. coli bacteria and mouse glioma cells

    Cabral–Prieto, A.; López-Callejas, R.; Rodríguez-Méndez, B. G.; Santos-Cuevas, C. L.; Celis-Almazán, J.; Olea-Mejía, O.; Gómez-Morales, J. L.; Peña-Eguiluz, R.; Valencia-Alvarado, R.; Mercado-Cabrera, A.; Muñoz-Castro, A. E.; García-Santibañez, F.

    2017-01-01

    The survival of Escherichia coli bacteria and mouse glioma cells were studied under different temperatures using direct heating in water, ultrasound, and magnetic fluid hyperthermia. The survival of these microorganisms depended on whether the heating mode was continuous or discontinuous, surviving more in the former than in the discontinuous heating mode. Whereas Escherichia coli bacteria did not survive at temperatures ≥50"∘C, the mouse glioma cells did not survive at temperatures ≥48"∘C. The survival of both these microorganisms was independent of the presence or absence of the magnetic nanoparticles of magnetite, suggesting that these, having mean particle sizes of 9.5, 8.5 and 5, did not show any apparent cytotoxicity effect. Present results also showed that the inductive heating system which used a radiofrequency of 13.56 MHz, providing a maximum magnetic field strength of 160 A/m, the electric rather than magnetic heating predominated.

  8. Revisit the Candidacy of Brain Cell Types as the Cell(s of Origin for Human High-Grade Glioma

    Fangjie Shao

    2018-02-01

    Full Text Available High-grade glioma, particularly, glioblastoma, is the most aggressive cancer of the central nervous system (CNS in adults. Due to its heterogeneous nature, glioblastoma almost inevitably relapses after surgical resection and radio-/chemotherapy, and is thus highly lethal and associated with a dismal prognosis. Identifying the cell of origin has been considered an important aspect in understanding tumor heterogeneity, thereby holding great promise in designing novel therapeutic strategies for glioblastoma. Taking advantage of genetic lineage-tracing techniques, performed mainly on genetically engineered mouse models (GEMMs, multiple cell types in the CNS have been suggested as potential cells of origin for glioblastoma, among which adult neural stem cells (NSCs and oligodendrocyte precursor cells (OPCs are the major candidates. However, it remains highly debated whether these cell types are equally capable of transforming in patients, given that in the human brain, some cell types divide so slowly, therefore may never have a chance to transform. With the recent advances in studying adult NSCs and OPCs, particularly from the perspective of comparative biology, we now realize that notable differences exist among mammalian species. These differences have critical impacts on shaping our understanding of the cell of origin of glioma in humans. In this perspective, we update the current progress in this field and clarify some misconceptions with inputs from important findings about the biology of adult NSCs and OPCs. We propose to re-evaluate the cellular origin candidacy of these cells, with an emphasis on comparative studies between animal models and humans.

  9. Boron neutron capture therapy induces apoptosis of glioma cells through Bcl-2/Bax

    Wang, Peng; Zhen, Haining; Jiang, Xinbiao; Zhang, Wei; Cheng, Xin; Guo, Geng; Mao, Xinggang; Zhang, Xiang

    2010-01-01

    Boron neutron capture therapy (BNCT) is an alternative treatment modality for patients with glioma. The aim of this study was to determine whether induction of apoptosis contributes to the main therapeutic efficacy of BNCT and to compare the relative biological effect (RBE) of BNCT, γ-ray and reactor neutron irradiation. The neutron beam was obtained from the Xi'an Pulsed Reactor (XAPR) and γ-rays were obtained from [ 60 Co] γ source of the Fourth Military Medical University (FMMU) in China. Human glioma cells (the U87, U251, and SHG44 cell lines) were irradiated by neutron beams at the XAPR or [ 60 Co] γ-rays at the FMMU with different protocols: Group A included control nonirradiated cells; Group B included cells treated with 4 Gy of [ 60 Co] γ-rays; Group C included cells treated with 8 Gy of [ 60 Co] γ-rays; Group D included cells treated with 4 Gy BPA (p-borono-phenylalanine)-BNCT; Group E included cells treated with 8 Gy BPA-BNCT; Group F included cells irradiated in the reactor for the same treatment period as used for Group D; Group G included cells irradiated in the reactor for the same treatment period as used for Group E; Group H included cells irradiated with 4 Gy in the reactor; and Group I included cells irradiated with 8 Gy in the reactor. Cell survival was determined using the 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium (MTT) cytotoxicity assay. The morphology of cells was detected by Hoechst33342 staining and transmission electron microscope (TEM). The apoptosis rate was detected by flow cytometer (FCM). The level of Bcl-2 and Bax protein was measured by western blot analysis. Proliferation of U87, U251, and SHG44 cells was much more strongly inhibited by BPA-BNCT than by irradiation with [ 60 Co] γ-rays (P < 0.01). Nuclear condensation was determined using both a fluorescence technique and electron microscopy in all cell lines treated with BPA-BNCT. Furthermore, the cellular apoptotic rates in Group D and Group E treated with

  10. Boron neutron capture therapy induces apoptosis of glioma cells through Bcl-2/Bax

    Mao Xinggang

    2010-12-01

    Full Text Available Abstract Background Boron neutron capture therapy (BNCT is an alternative treatment modality for patients with glioma. The aim of this study was to determine whether induction of apoptosis contributes to the main therapeutic efficacy of BNCT and to compare the relative biological effect (RBE of BNCT, γ-ray and reactor neutron irradiation. Methods The neutron beam was obtained from the Xi'an Pulsed Reactor (XAPR and γ-rays were obtained from [60Co] γ source of the Fourth Military Medical University (FMMU in China. Human glioma cells (the U87, U251, and SHG44 cell lines were irradiated by neutron beams at the XAPR or [60Co] γ-rays at the FMMU with different protocols: Group A included control nonirradiated cells; Group B included cells treated with 4 Gy of [60Co] γ-rays; Group C included cells treated with 8 Gy of [60Co] γ-rays; Group D included cells treated with 4 Gy BPA (p-borono-phenylalanine-BNCT; Group E included cells treated with 8 Gy BPA-BNCT; Group F included cells irradiated in the reactor for the same treatment period as used for Group D; Group G included cells irradiated in the reactor for the same treatment period as used for Group E; Group H included cells irradiated with 4 Gy in the reactor; and Group I included cells irradiated with 8 Gy in the reactor. Cell survival was determined using the 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium (MTT cytotoxicity assay. The morphology of cells was detected by Hoechst33342 staining and transmission electron microscope (TEM. The apoptosis rate was detected by flow cytometer (FCM. The level of Bcl-2 and Bax protein was measured by western blot analysis. Results Proliferation of U87, U251, and SHG44 cells was much more strongly inhibited by BPA-BNCT than by irradiation with [60Co] γ-rays (P 60Co] γ-rays (P P Conclusions Compared with ��-ray and reactor neutron irradiation, a higher RBE can be achieved upon treatment of glioma cells with BNCT. Glioma cell apoptosis induced by

  11. Metabolic impact of anti-angiogenic agents on U87 glioma cells.

    Tanja Mesti

    Full Text Available BACKGROUND: Glioma cells not only secrete high levels of vascular endothelial growth factor (VEGF but also express VEGF receptors (VEGFR, supporting the existence of an autocrine loop. The direct impact on glioma cells metabolism of drugs targeting the VEGF pathway, such as Bevacizumab (Bev or VEGFR Tyrosine Kinase Inhibitor (TKI, is poorly known. MATERIAL AND METHODS: U87 cells were treated with Bev or SU1498, a selective VEGFR2 TKI. VEGFR expression was checked with FACS flow cytometry and Quantitative Real-Time PCR. VEGF secretion into the medium was assessed with an ELISA kit. Metabolomic studies on cells were performed using High Resolution Magic Angle Spinning Spectroscopy (HR-MAS. RESULTS: U87 cells secreted VEGF and expressed low level of VEGFR2, but no detectable VEGFR1. Exposure to SU1498, but not Bev, significantly impacted cell proliferation and apoptosis. Metabolomic studies with HR MAS showed that Bev had no significant effect on cell metabolism, while SU1498 induced a marked increase in lipids and a decrease in glycerophosphocholine. Accordingly, accumulation of lipid droplets was seen in the cytoplasm of SU1498-treated U87 cells. CONCLUSION: Although both drugs target the VEGF pathway, only SU1498 showed a clear impact on cell proliferation, cell morphology and metabolism. Bevacizumab is thus less likely to modify glioma cells phenotype due to a direct therapeutic pressure on the VEGF autocrine loop. In patients treated with VEGFR TKI, monitoring lipids with magnetic resonance spectroscopic (MRS might be a valuable marker to assess drug cytotoxicity.

  12. In vitro anti-tubulin effects of mebendazole and fenbendazole on canine glioma cells.

    Lai, S R; Castello, S A; Robinson, A C; Koehler, J W

    2017-12-01

    Benzimidazole anthelmintics have reported anti-neoplastic effects both in vitro and in vivo. The purpose of this study was to evaluate the in vitro chemosensitivity of three canine glioma cell lines to mebendazole and fenbendazole. The mean inhibitory concentration (IC 50 ) (±SD) obtained from performing the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay after treating J3T, G06-A, and SDT-3G cells for 72 h with mebendazole were 0.030 ± 0.003, 0.080 ± 0.015 and 0.030 ± 0.006 μM respectively, while those for fenbendazole were 0.550  ± 0.015, 1.530 ± 0.159 and 0.690 ± 0.095 μM; treatment of primary canine fibroblasts for 72 h at IC 50 showed no significant effect. Immunofluorescence studies showed disruption of tubulin after treatment. Mebendazole and fenbendazole are cytotoxic in canine glioma cell lines in vitro and may be good candidates for treatment of canine gliomas. Further in vivo studies are required. © 2017 John Wiley & Sons Ltd.

  13. D-amino acid oxidase gene therapy sensitizes glioma cells to the antiglycolytic effect of 3-bromopyruvate.

    El Sayed, S M; Abou El-Magd, R M; Shishido, Y; Chung, S P; Sakai, T; Watanabe, H; Kagami, S; Fukui, K

    2012-01-01

    Glioma tumors are refractory to conventional treatment. Glioblastoma multiforme is the most aggressive type of primary brain tumors in humans. In this study, we introduce oxidative stress-energy depletion (OSED) therapy as a new suggested treatment for glioblastoma. OSED utilizes D-amino acid oxidase (DAO), which is a promising therapeutic protein that induces oxidative stress and apoptosis through generating hydrogen peroxide (H2O2). OSED combines DAO with 3-bromopyruvate (3BP), a hexokinase II (HK II) inhibitor that interferes with Warburg effect, a metabolic alteration of most tumor cells that is characterized by enhanced aerobic glycolysis. Our data revealed that 3BP induced depletion of energetic capabilities of glioma cells. 3BP induced H2O2 production as a novel mechanism of its action. C6 glioma transfected with DAO and treated with D-serine together with 3BP-sensitized glioma cells to 3BP and decreased markedly proliferation, clonogenic power and viability in a three-dimensional tumor model with lesser effect on normal astrocytes. DAO gene therapy using atelocollagen as an in vivo transfection agent proved effective in a glioma tumor model in Sprague-Dawley (SD) rats, especially after combination with 3BP. OSED treatment was safe and tolerable in SD rats. OSED therapy may be a promising therapeutic modality for glioma.

  14. Immunohistochemical detection and correlation between MHC antigen and cell-mediated immune system in recurrent glioma by APAAP method.

    Miyagi, K; Ingram, M; Techy, G B; Jacques, D B; Freshwater, D B; Sheldon, H

    1990-09-01

    As part of an on-going clinical trial of immunotherapy for recurrent malignant gliomas, using alkaline phosphatase-anti-alkaline phosphatase method with monoclonal antibodies, we investigated the correlation between expression of the major histocompatibility complex (MHC) and the subpopulation of tumor-infiltrating lymphocytes (TILs) in 38 glioma specimens (20 grade IV, 11 grade III, and 7 grade II) from 33 patients. Thirty specimens (78.9%) were positive to class I MHC antigen and 20 (52.6%) were positive to class II MHC antigen. The correlations between class I MHC antigen expression and the number of infiltrating T8 (p less than 0.01), and also between class II MHC antigen expression and the number of infiltrating T4 (p less than 0.05) were significant. We conclude that TILs are the result of immunoreaction (host-defense mechanism). 31.6% of specimens had perivascular infiltration of T cells. The main infiltrating lymphocyte subset in moderate to marked perivascular cuffing was T4. Our results may indicate that lack of MHC antigen on the glioma cell surface has a share in the poor immunogenicity in glioma-bearing patients. In addition, considering the effector/target ratio, the number of infiltrating lymphocytes against glioma cells was too small, so the immunological intervention seems to be essential in glioma therapy. Previous radiation therapy and chemotherapy, including steroid therapy, did not influence lymphocyte and macrophage infiltration.

  15. EG-07CELL CYCLE SIGNATURE AND TUMOR PHYLOGENY ARE ENCODED IN THE EVOLUTIONARY DYNAMICS OF DNA METHYLATION IN GLIOMA

    Mazor, Tali; Pankov, Aleksandr; Johnson, Brett E.; Hong, Chibo; Bell, Robert J.A.; Smirnov, Ivan V.; Reis, Gerald F.; Phillips, Joanna J.; Barnes, Michael; Bollen, Andrew W.; Taylor, Barry S.; Molinaro, Annette M.; Olshen, Adam B.; Song, Jun S.; Berger, Mitchel S.; Chang, Susan M.; Costello, Joseph F.

    2014-01-01

    The clonal evolution of tumor cell populations can be reconstructed from patterns of genetic alterations. In contrast, tumor epigenetic states, including DNA methylation, are reversible and sensitive to the tumor microenvironment, presumably precluding the use of epigenetics to discover tumor phylogeny. Here we examined the spatial and temporal dynamics of DNA methylation in a clinically and genetically characterized cohort of IDH1-mutant low-grade gliomas and their patient-matched recurrences. WHO grade II gliomas are diffuse, infiltrative tumors that frequently recur and may undergo malignant progression to a higher grade with a worse prognosis. The extent to which epigenetic alterations contribute to the evolution of low-grade gliomas, including malignant progression, is unknown. While all gliomas in the cohort exhibited the hypermethylation signature associated with IDH1 mutation, low-grade gliomas that underwent malignant progression to high-grade glioblastoma (GBM) had a unique signature of DNA hypomethylation enriched for active enhancers, as well as sites of age-related hypermethylation in the brain. Genes with promoter hypomethylation and concordant transcriptional upregulation during evolution to GBM were enriched in cell cycle function, evolving in concert with genetic alterations that deregulate the G1/S cell cycle checkpoint. Despite the plasticity of tumor epigenetic states, phyloepigenetic trees robustly recapitulated phylogenetic trees derived from somatic mutations in the same patients. These findings highlight widespread co-dependency of genetic and epigenetic events throughout the clonal evolution of initial and recurrent glioma.

  16. CD4+ and Perivascular Foxp3+ T Cells in Glioma Correlate with Angiogenesis and Tumor Progression

    Luyan Mu

    2017-11-01

    Full Text Available BackgroundAngiogenesis and immune cell infiltration are key features of gliomas and their manipulation of the microenvironment, but their prognostic significance remains indeterminate. We evaluate the interconnection between tumor-infiltrating lymphocyte (TIL and tumor blood-vasculatures in the context of glioma progression.MethodsPaired tumor tissues of 44 patients from three tumor-recurrent groups: diffuse astrocytomas (DA recurred as DA, DA recurred as glioblastomas (GBM, and GBM recurred as GBM were evaluated by genetic analysis, immunohistochemistry for tumor blood vessel density, TIL subsets, and clinical outcomes. These cells were geographically divided into perivascular and intratumoral TILs. Associations were examined between these TILs, CD34+ tumor blood vessels, and clinical outcomes. To determine key changes in TIL subsets, microarray data of 15-paired tumors from patients who failed antiangiogenic therapy- bevacizumab, and 16-paired tumors from chemo-naïve recurrent GBM were also evaluated and compared.ResultsUpon recurrence in primary gliomas, similar kinetic changes were found between tumor blood vessels and each TIL subset in all groups, but only CD4+ including Foxp3+ TILs, positively correlated with the density of tumor blood vessels. CD4 was the predominant T cell population based on the expression of gene-transcripts in primary GBMs, and increased activated CD4+ T cells were revealed in Bevacizumab-resistant recurrent tumors (not in chemo-naïve recurrent tumors. Among these TILs, 2/3 of them were found in the perivascular niche; Foxp3+ T cells in these niches not only correlated with the tumor vessels but were also an independent predictor of shortened recurrence-free survival (RFS (HR = 4.199, 95% CI 1.522–11.584, p = 0.006.ConclusionThe minimal intratumoral T cell infiltration and low detection of CD8 transcripts expression in primary GBMs can potentially limit antitumor response. CD4+ and perivascular Foxp3

  17. EGFRvIII promotes glioma angiogenesis and growth through the NF-κB, interleukin-8 pathway.

    Bonavia, R; Inda, M M; Vandenberg, S; Cheng, S-Y; Nagane, M; Hadwiger, P; Tan, P; Sah, D W Y; Cavenee, W K; Furnari, F B

    2012-09-06

    Sustaining a high growth rate requires tumors to exploit resources in their microenvironment. One example of this is the extensive angiogenesis that is a typical feature of high-grade gliomas. Here, we show that expression of the constitutively active mutant epidermal growth factor receptor, ΔEGFR (EGFRvIII, EGFR*, de2-7EGFR) is associated with significantly higher expression levels of the pro-angiogenic factor interleukin (IL)-8 in human glioma specimens and glioma stem cells. Furthermore, the ectopic expression of ΔEGFR in different glioma cell lines caused up to 60-fold increases in the secretion of IL-8. Xenografts of these cells exhibit increased neovascularization, which is not elicited by cells overexpressing wild-type (wt)EGFR or ΔEGFR with an additional kinase domain mutation. Analysis of the regulation of IL-8 by site-directed mutagenesis of its promoter showed that ΔEGFR regulates its expression through the transcription factors nuclear factor (NF)-κB, activator protein 1 (AP-1) and CCAAT/enhancer binding protein (C/EBP). Glioma cells overexpressing ΔEGFR showed constitutive activation and DNA binding of NF-κB, overexpression of c-Jun and activation of its upstream kinase c-Jun N-terminal kinase (JNK) and overexpression of C/EBPβ. Selective pharmacological or genetic targeting of the NF-κB or AP-1 pathways efficiently blocked promoter activity and secretion of IL-8. Moreover, RNA interference-mediated knock-down of either IL-8 or the NF-κB subunit p65, in ΔEGFR-expressing cells attenuated their ability to form tumors and to induce angiogenesis when injected subcutaneously into nude mice. On the contrary, the overexpression of IL-8 in glioma cells lacking ΔEGFR potently enhanced their tumorigenicity and produced highly vascularized tumors, suggesting the importance of this cytokine and its transcription regulators in promoting glioma angiogenesis and tumor growth.

  18. The effect of γ-tocopherol on proliferation, integrin expression, adhesion, and migration of human glioma cells

    Samandari, Elika; Visarius, Theresa; Zingg, Jean-Marc; Azzi, Angelo

    2006-01-01

    The effect of vitamin E on proliferation, integrin expression, adhesion, and migration in human glioma cells has been studied. γ-tocopherol at 50 μM concentration exerted more inhibitory effect than α-tocopherol at the same concentration on glioma cell proliferation. Integrin α5 and β1 protein levels were increased upon both α- and γ-tocopherol treatments. In parallel, an increase in the α5β1 heterodimer cell surface expression was observed. The tocopherols inhibited glioma cell-binding to fibronectin where γ-tocopherol treatment induced glioma cell migration. Taken together, the data reported here are consistent with the notion that the inhibition of glioma cell proliferation induced by tocopherols may be mediated, at least in part, by an increase in integrin α5 and β1 expression. Cell adhesion is also negatively affected by tocopherols, despite a small increase in the surface appearance of the α5β1 heterodimer. Cell migration is stimulated by γ-tocopherol. It is concluded that α5 and β1 integrin expression and surface appearance are not sufficient to explain all the observations and that other integrins or in general other factors may be associated with these events

  19. The prosurvival role of autophagy in Resveratrol-induced cytotoxicity in human U251 glioma cells

    Li, Jun; Qin, Zhenghong; Liang, Zhongqin

    2009-01-01

    Previous study reported that resveratrol has anti-tumor activity. In this study, we investigated the involvement of autophagy in the resveratrol-induced apoptotic death of human U251 glioma cells. The growth inhibition of U251 cells induced by resveratrol was assessed with methyl thiazolyl tetrazolium (MTT). The activation of autophagy and proapoptotic effect were characterized by monodansylcadaverine labeling and Hoechst stain, respectively. Mitochondrialtransmembrane potential (ΔΨm) was measured as a function of drug treatment using 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide (JC-1). The role of autophagy and apoptosis in the resveratrol-induced death of U251 cells was assessed using autophagic and caspase inhibitors. Immunofluorescence, flow cytometry, and Western blot analysis were used to study the apoptotic and autophagic mechanisms. Methyl thiazolyl tetrazolium (MTT) assays indicated that resveratrol decreased the viability of U251 cells in a dose- and time-dependent manner. Flow cytometry analysis indicated that resveratrol increased cell population at sub-G1 phase, an index of apoptosis. Furthermore, resveratrol-induced cell death was associated with a collapse of the mitochondrial membrane potential. The pan-caspase inhibitor Z-VAD-fmk suppressed resveratrol-induced U251 cell death. Resveratrol stimulated autophagy was evidenced by punctuate monodansylcadaverine(MDC) staining and microtubule-associated protein light chain 3 (LC3) immunoreactivty. Resveratrol also increased protein levels of beclin 1 and membrane form LC3 (LC3-II). Autophagy inhibitors 3-methylademine (3-MA) and bafilomycin A1 sensitized the cytotoxicity of resveratrol. Together, these findings indicate that resveratrol induces autophagy in human U251 glioma cells and autophagy suppressed resveratrol-induced apoptosis. This study thus suggests that autophagy inhibitors can increase the cytotoxicity of resveratrol to glioma cells

  20. Binding of cetuximab to the EGFRvIII deletion mutant and its biological consequences in malignant glioma cells

    Jutten, Barry; Dubois, Ludwig; Li Younan; Aerts, Hugo; Wouters, Bradly G.; Lambin, Philippe; Theys, Jan; Lammering, Guido

    2009-01-01

    Background and purpose: Despite the clinical use of cetuximab, a chimeric antibody against EGFR, little is known regarding its interaction with EGFRvIII, a frequently expressed deletion mutant of EGFR. Therefore, we investigated the interaction and the functional consequences of cetuximab treatment on glioma cells stably expressing EGFRvIII. Materials and methods: The human glioma cell line U373 genetically modified to express EGFRvIII was used to measure the binding of cetuximab and its internalization using flow cytometry and confocal microscopy. Proliferation and cell survival were analyzed by cell growth and clonogenic survival assays. Results: Cetuximab is able to bind to EGFRvIII and causes an internalization of the receptor and decreases its expression levels. Furthermore, in contrast to EGF, cetuximab was able to activate EGFRvIII which was evidenced by multiple phosphorylation sites and its downstream signaling targets. Despite this activation, the growth rate and the radiosensitivity of the EGFRvIII-expressing glioma cells were not modulated. Conclusions: Cetuximab binds to EGFRvIII and leads to the initial activation, internalization and subsequent downregulation of EGFRvIII, but it does not seem to modulate the proliferation or radiosensitivity of EGFRvIII-expressing glioma cells. Thus, approaches to treat EGFRvIII-expressing glioma cells should be evaluated more carefully.

  1. Ibuprofen and Diclofenac Restrict Migration and Proliferation of Human Glioma Cells by Distinct Molecular Mechanisms

    Leidgens, Verena; Seliger, Corinna; Jachnik, Birgit; Welz, Tobias; Leukel, Petra; Vollmann-Zwerenz, Arabel; Bogdahn, Ulrich; Kreutz, Marina; Grauer, Oliver M.; Hau, Peter

    2015-01-01

    Background Non-steroidal anti-inflammatory drugs (NSAIDs) have been associated with anti-tumorigenic effects in different tumor entities. For glioma, research has generally focused on diclofenac; however data on other NSAIDs, such as ibuprofen, is limited. Therefore, we performed a comprehensive investigation of the cellular, molecular, and metabolic effects of ibuprofen and diclofenac on human glioblastoma cells. Methods Glioma cell lines were treated with ibuprofen or diclofenac to investigate functional effects on proliferation and cell motility. Cell cycle, extracellular lactate levels, lactate dehydrogenase-A (LDH-A) expression and activity, as well as inhibition of the Signal Transducer and Activator of Transcription 3 (STAT-3) signaling pathway, were determined. Specific effects of diclofenac and ibuprofen on STAT-3 were investigated by comparing their effects with those of the specific STAT-3 inhibitor STATTIC. Results Ibuprofen treatment led to a stronger inhibition of cell growth and migration than treatment with diclofenac. Proliferation was affected by cell cycle arrest at different checkpoints by both agents. In addition, diclofenac, but not ibuprofen, decreased lactate levels in all concentrations used. Both decreased STAT-3 phosphorylation; however, diclofenac led to decreased c-myc expression and subsequent reduction in LDH-A activity, whereas treatment with ibuprofen in higher doses induced c-myc expression and less LDH-A alteration. Conclusions This study indicates that both ibuprofen and diclofenac strongly inhibit glioma cells, but the subsequent metabolic responses of both agents are distinct. We postulate that ibuprofen may inhibit tumor cells also by COX- and lactate-independent mechanisms after long-term treatment in physiological dosages, whereas diclofenac mainly acts by inhibition of STAT-3 signaling and downstream modulation of glycolysis. PMID:26485029

  2. NUMB does not impair growth and differentiation status of experimental gliomas

    Euskirchen, Philipp; Skaftnesmo, Kai-Ove; Huszthy, Peter C.; Brekkå, Narve; Bjerkvig, Rolf; Jacobs, Andreas H.; Miletic, Hrvoje

    2011-01-01

    The cell fate determinant NUMB orchestrates asymmetric cell division in flies and mammals and has lately been suggested to have a tumor suppressor function in breast and lung cancer. Here, we studied NUMB in the context of malignant gliomas. We used ectopic expression of NUMB in order to inhibit proliferation and induce differentiation in glioma cells by alteration of Notch, Hedgehog and p53 signaling. We found that NUMB is consistently expressed in glioma biopsies with predominance of NUMB2/4 isoforms as determined by isoform-specific real-time PCR and Western blotting. Upon lentiviral overexpression, in vitro proliferation rate and the grade of differentiation as assessed by morphology and expression of neural and glial markers remained unchanged. Orthotopic xenografts of NUMB-transduced human U87 glioma cells could be established in nude rats without impairing engraftment or causing significant changes in morphology based on magnetic resonance imaging (MRI). The previously reported alteration of Hedgehog and p53 signaling by NUMB could not be recapitulated in glioma cells. We thus show that in experimental gliomas, NUMB overexpression most likely does not exert a tumor suppressor function such as seen in epithelial cancers.

  3. Effect of Rad 51 overexpression on chromosomal stability and radiation sensitivity in tumour cells

    Jend, C.; Stuerzbecher, H.W.; Dikomey, E.; Borgmann, K.

    2004-01-01

    The present study was dedicated to examining the effects of Rad51 overexpression on genomic instability, expressed in terms of chromosomal aberrations in G1 and G2 phases following X-ray irradiation. For this purpose an osteosarcoma cell line (Ui-OS) which shows inducing Rad51 overexpression (UiRad5-2) after stable transfection was compared with an isogenetic line (UiLacZ) which overexpresses beta-galactosidase instead of Rad51 [de

  4. TGF-b and a specific TGF-b inhibitor regulate pericentrin B and MYH9 in glioma cell lines

    Óscar Álzate

    2006-01-01

    Full Text Available Malignant gliomas are heterogeneous, highly invasive vascular tumours. The multifunctional cytokine, transforming growth factor-beta (TGF-P, is expressed by grade III/IV gliomas and promotes tumour angiogenesis, invasión and immune escape. It has been shown previously that a small TGF-P receptor type I (TGF-(3-RI molecule inhibitor (SB-431542 blocks TGF-(3-mediated signal transduction, induction of angiogenic factor expression and cellular motility. As glioma cell lines display differential sensitivity to TGF-P, it was expected that they would also be differentially impacted by disruption of TGF-P signalling. Differential in gel expression (DIGE analysis and mass spectrometry was used in this work for determining protein regulation effects of both TGF-P and SB-431542 on human glioma cell lines. It was found that pericentrin B and non muscle myosin were differentially expressed in fragments which likely resulted from protease activation by the tumour growth mechanism. These results suggest that both pericentrin B and non-muscle myosin might be potential glioma biomarkers. Key words: DIGE, proteomics, glioma, TGF-P, mass spectrometry, non muscle myosin, pericentrin B.

  5. Transcriptional differences between normal and glioma-derived glial progenitor cells identify a core set of dysregulated genes.

    Auvergne, Romane M; Sim, Fraser J; Wang, Su; Chandler-Militello, Devin; Burch, Jaclyn; Al Fanek, Yazan; Davis, Danielle; Benraiss, Abdellatif; Walter, Kevin; Achanta, Pragathi; Johnson, Mahlon; Quinones-Hinojosa, Alfredo; Natesan, Sridaran; Ford, Heide L; Goldman, Steven A

    2013-06-27

    Glial progenitor cells (GPCs) are a potential source of malignant gliomas. We used A2B5-based sorting to extract tumorigenic GPCs from human gliomas spanning World Health Organization grades II-IV. Messenger RNA profiling identified a cohort of genes that distinguished A2B5+ glioma tumor progenitor cells (TPCs) from A2B5+ GPCs isolated from normal white matter. A core set of genes and pathways was substantially dysregulated in A2B5+ TPCs, which included the transcription factor SIX1 and its principal cofactors, EYA1 and DACH2. Small hairpin RNAi silencing of SIX1 inhibited the expansion of glioma TPCs in vitro and in vivo, suggesting a critical and unrecognized role of the SIX1-EYA1-DACH2 system in glioma genesis or progression. By comparing the expression patterns of glioma TPCs with those of normal GPCs, we have identified a discrete set of pathways by which glial tumorigenesis may be better understood and more specifically targeted. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

  6. Nrf2 mediates redox adaptation in NOX4-overexpressed non-small cell lung cancer cells

    Wu, Qipeng; Yao, Bei; Li, Ning; Ma, Lei; Deng, Yanchao; Yang, Yang; Zeng, Cheng; Yang, Zhicheng [Department of Clinical Pharmacy, School of Pharmacy, Guangdong Pharmaceutical University, Guangzhou 510006 (China); Liu, Bing, E-mail: liubing520@gdpu.edu.cn [Department of Clinical Pharmacy, School of Pharmacy, Guangdong Pharmaceutical University, Guangzhou 510006 (China); Guangdong Key Laboratory of Pharmaceutical Bioactive Substances, Guangdong Pharmaceutical University, Guangzhou 510006 (China)

    2017-03-15

    The redox adaptation mechanisms in cancer cells are very complex and remain largely unclear. Our previous studies have confirmed that NADPH oxidase 4 (NOX4) is abundantly expressed in non-small cell lung cancer (NSCLC) and confers apoptosis resistance on NSCLC cells. However, the comprehensive mechanisms for NOX4-mediated oxidative resistance of cancer cells remain still undentified. The present study found that NOX4-derived H{sub 2}O{sub 2} enhanced the nuclear factor erythroid 2-related factor 2 (Nrf2) stability via disruption of redox-dependent proteasomal degradation and stimulated its activity through activation of PI3K signaling. Specifically, the results showed that ectopic NOX4 expression did not induce apoptosis of A549 cells; however, inhibition of Nrf2 resulted in obvious apoptotic death of NOX4-overexpressed A549 cells, accompanied by a significant increase in H{sub 2}O{sub 2} level and decrease in GSH content. Besides, inhibition of Nrf2 could suppress cell growth and efficiently reverse the enhancement effect of NOX4 on cell growth. The in vivo data confirmed that inhibition of Nrf2 could interfere apoptosis resistance in NOX4-overexpressed A549 tumors and led to cell growth inhibition. In conclusion, these results reveal that Nrf2 is critically involved in redox adaptation regulation in NOX4-overexpressed NSCLC cells. Therefore, NOX4 and Nrf2 may be promising combination targets against malignant progression of NSCLC. - Highlights: • NOX4-derived H{sub 2}O{sub 2} upregulates Nrf2 expression and activity in NSCLC. • Nrf2 confers apoptosis resistance in NOX4-overexpressed NSCLC cells. • Inhibition of Nrf2 reverses the enhancement effect of NOX4 on cell growth.

  7. Establishment of 9L/F344 rat intracerebral glioma model of brain tumor stem cells

    Zong-yu XIAO

    2015-04-01

    Full Text Available Objective To establish the 9L/F344 rat intracerebral glioma model of brain tumor stem cells.  Methods Rat 9L gliosarcoma stem-like cells were cultured in serum-free suspension. The expression of CD133 and nestin were tested by immunohistochemistry. A total of 48 inbredline male F344 rats were randomly divided into 2 groups, and 9L tumor sphere cells and 9L monolayer cells were respectively implanted into the right caudate nucleus of F344 rats in 2 groups. Survival time was observed and determined using the method of Kaplan-Meier survival analysis. Fourteen days after implantation or when the rats were dying, their brains were perfused and sectioned for HE staining, and CD133 and nestin were detected by immunohistochemistry.  Results Rat 9L tumor spheres were formed with suspension culture in serum-free medium. The gliomas formed in both groups were invasive without obvious capsule. More new vessels, bleeding and necrosis could be detected in 9L tumor spheres group. The tumor cells in both groups were positive for CD133 and nestin. There was no significant difference in the expression of CD133 and nestin between 2 groups (P > 0.05, for all. According to the expression of nestin, the tumors formed by 9L tumor sphere cells were more invasive. The median survival time of the rats bearing 9L tumor sphere cells was 15 d (95%CI: 15.219-15.781, and the median survival time of the rats bearing 9L monolayer cells was 21 d (95%CI: 20.395-21.605. There was significant difference between 2 groups (χ2 = 12.800, P = 0.000.  Conclusions 9L/F344 rat intracerebral glioma model of brain tumor stem cells is successfully established, which provides a glioma model for the future research. DOI: 10.3969/j.issn.1672-6731.2015.04.012

  8. Pankiller effect of prolonged exposure to menadione on glioma cells: potentiation by vitamin C.

    Vita, Marina F; Nagachar, Nivedita; Avramidis, Dimitrios; Delwar, Zahid M; Cruz, Mabel H; Siden, Åke; Paulsson, Kajsa M; Yakisich, Juan Sebastian

    2011-12-01

    Menadione (Vitamin K3) has anti-tumoral effects against a wide range of cancer cells. Its potential toxicity to normal cells and narrow therapeutic range limit its use as single agent but in combination with radiation or other anti-neoplastic agents can be of therapeutic use. In this paper, we first evaluated the early (within 3 h) effect of menadione on ongoing DNA replication. In normal rat cerebral cortex mini-units menadione showed an age dependent anti-proliferative effect. In tissue mini-units prepared from newborn rats, menadione inhibited ongoing DNA replication with an IC (50) of approximately 10 μM but 50 μM had no effect on mini-units from prepared adult rat tissue. The effect of short (72 h) and prolonged exposure (1-2 weeks) to menadione alone in the DBTRG.05MG human glioma cells line and in combination with vitamin C was studied. After short period of exposure data show that menadione alone or in combination with vitamin C provided similar concentration-response curves (and IC(50) values). Prolonged exposure to these drugs was evaluated by their ability to kill 100% of glioma cells and prevent regrowth when cells are re-incubated in drug-free media. In this long-term assay, menadione:vitamin C at a ratio 1:100 showed higher anti-proliferative activity when compared to each drug alone and allowed to reduce each drug concentration between 2.5 to 5-fold. Similar anti-proliferative effect was demonstrated in 8 patient derived glioblastoma cell cultures. Our data should be able to encourage further advanced studies on animal models to evaluate the potential use of this combination therapy for glioma treatment.

  9. Altered expression of long non-coding RNAs during genotoxic stress-induced cell death in human glioma cells.

    Liu, Qian; Sun, Shanquan; Yu, Wei; Jiang, Jin; Zhuo, Fei; Qiu, Guoping; Xu, Shiye; Jiang, Xuli

    2015-04-01

    Long non-coding RNAs (lncRNAs), a recently discovered class of non-coding genes, are transcribed throughout the genome. Emerging evidence suggests that lncRNAs may be involved in modulating various aspects of tumor biology, including regulating gene activity in response to external stimuli or DNA damage. No data are available regarding the expression of lncRNAs during genotoxic stress-induced apoptosis and/or necrosis in human glioma cells. In this study, we detected a change in the expression of specific candidate lncRNAs (neat1, GAS5, TUG1, BC200, Malat1, MEG3, MIR155HG, PAR5, and ST7OT1) during DNA damage-induced apoptosis in human glioma cell lines (U251 and U87) using doxorubicin (DOX) and resveratrol (RES). We also detected the expression pattern of these lncRNAs in human glioma cell lines under necrosis induced using an increased dose of DOX. Our results reveal that the lncRNA expression patterns are distinct between genotoxic stress-induced apoptosis and necrosis in human glioma cells. The sets of lncRNA expressed during genotoxic stress-induced apoptosis were DNA-damaging agent-specific. Generally, MEG3 and ST7OT1 are up-regulated in both cell lines under apoptosis induced using both agents. The induction of GAS5 is only clearly detected during DOX-induced apoptosis, whereas the up-regulation of neat1 and MIR155HG is only found during RES-induced apoptosis in both cell lines. However, TUG1, BC200 and MIR155HG are down regulated when necrosis is induced using a high dose of DOX in both cell lines. In conclusion, our findings suggest that the distinct regulation of lncRNAs may possibly involve in the process of cellular defense against genotoxic agents.

  10. Effect of saw palmetto extract on PI3K cell signaling transduction in human glioma.

    Yang, Yang; Hui, Lv; Yuqin, Che; Jie, Li; Shuai, Hou; Tiezhu, Zhou; Wei, Wang

    2014-08-01

    Saw palmetto extract can induce the apoptosis of prostate cancer cells. The aim of the present study was to investigate the effect of saw palmetto extract on the phosphatidylinositol 3-kinase (PI3K)/Akt signaling transduction pathway in human glioma U87 and U251 cell lines. Suspensions of U87 and U251 cells in a logarithmic growth phase were seeded into six-well plates at a density of 10 4 cells/well. In the experimental group, 1 μl/ml saw palmetto extract was added, while the control group was cultured without a drug for 24 h. The expression levels of PI3K, B-cell lymphoma-extra large (Bcl-xL) and p53 were evaluated through western blot analysis. In the experimental group, the U87 and U251 cells exhibited a lower expression level of PI3K protein as compared with the control group (t=6.849; Psaw palmetto extract induces glioma cell growth arrest and apoptosis via decreasing PI3K/Akt signal transduction.

  11. Stem cells and the origin of gliomas: A historical reappraisal with molecular advancements

    Michael L Levy

    2009-01-01

    Full Text Available Michael L Levy1, Allen L Ho1,2, Samuel Hughes3, Jayant Menon1, Rahul Jandial41Division of Neurosurgery, University of California, San Diego, La Jolla, California, USA; 2Del E Webb Neurosciences, Aging and Stem Cell Research Center, The Burnham Institute for Medical Research, La Jolla, California, USA; 3Department of Neurological Surgery, Oregon Health and Science University, Portland, OR, USA; 4Division of Neurosurgery, Department of Surgery, City of Hope Cancer Center, Duarte, CA, USAAbstract: The biology of both normal and tumor development clearly possesses overlapping and parallel features. Oncogenes and tumor suppressors are relevant not only in tumor biology, but also in physiological developmental regulators of growth and differentiation. Conversely, genes identified as regulators of developmental biology are relevant to tumor biology. This is particularly relevant in the context of brain tumors, where recent evidence is mounting that the origin of brain tumors, specifically gliomas, may represent dysfunctional developmental neurobiology. Neural stem cells are increasingly being investigated as the cell type that originally undergoes malignant transformation – the cell of origin – and the evidence for this is discussed.Keywords: stem cells, gliomas, neural stem cells, brain tumors, cancer stem cells

  12. Survival of irradiated glia and glioma cells studied with a new cloning technique

    Nilsson, S.; Carlsson, J.; Larsson, B.; Ponten, J.

    1980-01-01

    A method allowing cloning of monolayer cultured cells with a low plating efficiency was developed. Cells were grown in several small palladium squares to obtain a high cell density. These squares were surrounded by non-adhesive agarose to prevent large distance migration and thereby mixing of the clones. By using easily-cloned hamster cells for comparison it was found that the survival curves were similar to the curves obtained with conventional cloning. The new method was used to compare the radiosensitivity of cultured human glia and glioma cells which both have a low plating efficiency ( 0 -values (1.5 to 2.5 Gy) and large shoulders (extrapolation numbers around 5) indicating that they were rather resistant and had a high capacity for accumulation of sublethal damage. The survival curves for glia cells had lower D 0 -values (1.3 to 1.5 Gy) and no shoulders at all, indicating that they were more sensitive than the glioma cells. (author)

  13. Irradiation With Carbon Ion Beams Induces Apoptosis, Autophagy, and Cellular Senescence in a Human Glioma-Derived Cell Line

    Jinno-Oue, Atsushi; Shimizu, Nobuaki; Hamada, Nobuyuki; Wada, Seiichi; Tanaka, Atsushi; Shinagawa, Masahiko; Ohtsuki, Takahiro; Mori, Takahisa; Saha, Manujendra N.; Hoque, Ariful S.; Islam, Salequl; Kogure, Kimitaka; Funayama, Tomoo; Kobayashi, Yasuhiko

    2010-01-01

    Purpose: We examined biological responses of human glioma cells to irradiation with carbon ion beams (C-ions). Methods and Materials: A human glioma-derived cell line, NP-2, was irradiated with C-ions. Apoptotic cell nuclei were stained with Hoechst 33342. Induction of autophagy was examined either by staining cells with monodansylcadaverine (MDC) or by Western blotting to detect conversion of microtuble-associated protein light chain 3 (MAP-LC3) (LC3-I) to the membrane-bound form (LC3-II). Cellular senescence markers including induction of senescence-associated β-galactosidase (SA-β-gal) were examined. The mean telomere length of irradiated cells was determined by Southern blot hybridization. Expression of tumor suppressor p53 and cyclin/cyclin-dependent kinase inhibitor p21 WAF1/CIP1 in the irradiated cells was analyzed by Western blotting. Results: When NP-2 cells were irradiated with C-ions at 6 Gy, the major population of the cells died of apoptosis and autophagy. The residual fraction of attached cells ( WAF1/CIP1 was induced in NP-2 cells after irradiation. Furthermore, we found that irradiation with C-ions induced cellular senescence in a human glioma cell line lacking functional p53. Conclusions: Irradiation with C-ions induced apoptosis, autophagy, and cellular senescence in human glioma cells.

  14. Targeting ανβ3 and ανβ5 inhibits photon-induced hypermigration of malignant glioma cells

    Rieken, Stefan; Habermehl, Daniel; Mohr, Angela; Wuerth, Lena; Lindel, Katja; Weber, Klaus; Debus, Jürgen; Combs, Stephanie E

    2011-01-01

    Sublethal photon irradiation was recently suspected to increase tumor cell motility and promote locoregional recurrence of disease. This study was set up to describe mechanisms underlying increased glioma cell migration through photon irradiation and to analyse the modifiability of photon-altered glioma cell motility by integrin inhibition. Eight μm pore size membranes were coated with vitronectin (VN), collagen I and collagen IV. U87 and Ln229 glioma cells were analysed in migration experiments with and without radiotherapy (RT), serum stimulation and addition of monoclonal antibodies directed to human integrins α ν β 3 and α ν β 5 . Quantitative FACS analysis of integrins was performed in U87 and Ln229 glioma cells following RT. Statistical analysis was performed using Student's t-test. Glioma cell migration is serum-dependent and can be increased by photon RT which leads to enhanced expression of Vn receptor integrins. Blocking of either α ν β 3 or α ν β 5 integrins by antibodies inhibits Vn-based migration of both untreated and photon-irradiated glioma cells. Peripheral glioma cells are at risk of attraction into the adjacent healthy brain by serum components leaking through the blood brain barrier (BBB). Radiation therapy is associated with upregulation of Vn receptor integrins and enhanced glioma cell migration at sublethal doses. This effect can be inhibited by specific integrin blockade. Future therapeutical benefit may be derived from pharmacological integrin inhibition in combination with photon irradiation

  15. Sema3C Promotes the Survival and Tumorigenicity of Glioma Stem Cells through Rac1 Activation

    Jianghong Man

    2014-12-01

    Full Text Available Summary: Different cancer cell compartments often communicate through soluble factors to facilitate tumor growth. Glioma stem cells (GSCs are a subset of tumor cells that resist standard therapy to contribute to disease progression. How GSCs employ a distinct secretory program to communicate with and nurture each other over the nonstem tumor cell (NSTC population is not well defined. Here, we show that GSCs preferentially secrete Sema3C and coordinately express PlexinA2/D1 receptors to activate Rac1/nuclear factor (NF-κB signaling in an autocrine/paracrine loop to promote their own survival. Importantly, Sema3C is not expressed in neural progenitor cells (NPCs or NSTCs. Disruption of Sema3C induced apoptosis of GSCs, but not NPCs or NSTCs, and suppressed tumor growth in orthotopic models of glioblastoma. Introduction of activated Rac1 rescued the Sema3C knockdown phenotype in vivo. Our study supports the targeting of Sema3C to break this GSC-specific autocrine/paracrine loop in order to improve glioblastoma treatment, potentially with a high therapeutic index. : Glioma stem cells (GSCs have a high capacity for self-renewal, invasion, and survival. How they communicate with each other to survive and maintain their identity is not clear. Man et al. now show that GSCs have co-opted a neurodevelopmental program to activate Rac1 to promote defining features of GSCs.

  16. Overexpression of Oct4 suppresses the metastatic potential of breast cancer cells via Rnd1 downregulation.

    Shen, Long; Qin, Kunhua; Wang, Dekun; Zhang, Yan; Bai, Nan; Yang, Shengyong; Luo, Yunping; Xiang, Rong; Tan, Xiaoyue

    2014-11-01

    Although Oct4 is known as a critical transcription factor involved in maintaining "stemness", its role in tumor metastasis is still controversial. Herein, we overexpressed and silenced Oct4 expression in two breast cancer cell lines, MDA-MB-231 and 4T1, separately. Our data showed that ectopic overexpression of Oct4 suppressed cell migration and invasion in vitro and the formation of metastatic lung nodules in vivo. Conversely, Oct4 downregulation increased the metastatic potential of breast cancer cells both in vitro and in vivo. Furthermore, we identified Rnd1 as the downstream target of Oct4 by ribonucleic acid sequencing (RNA-seq) analysis, which was significantly downregulated upon Oct4 overexpression. Chromatin immunoprecipitation assays revealed the binding of Oct4 to the promoter region of Rnd1 by ectopic overexpression of Oct4. Dual luciferase assays indicated that Oct4 overexpression suppressed transcriptional activity of the Rnd1 promoter. Moreover, overexpression of Rnd1 partially rescued the inhibitory effects of Oct4 on the migration and invasion of breast cancer cells. Overexpression of Rnd1 counteracted the influence of Oct4 on the formation of cell adhesion and lamellipodia, which implied a potential underlying mechanism involving Rnd1. In addition, we also found that overexpression of Oct4 led to an elevation of E-cadherin expression, even in 4T1 cells that possess a relatively high basal level of E-cadherin. Rnd1 overexpression impaired the promoting effects of Oct4 on E-cadherin expression in MDA-MB-231 cells. These results suggest that Oct4 affects the metastatic potential of breast cancer cells through Rnd1-mediated effects that influence cell motility and E-cadherin expression. Copyright © 2014 Elsevier B.V. All rights reserved.

  17. Attenuation of teratoma formation by p27 overexpression in induced pluripotent stem cells.

    Matsu-ura, Toru; Sasaki, Hiroshi; Okada, Motoi; Mikoshiba, Katsuhiko; Ashraf, Muhammad

    2016-02-15

    Pluripotent stem cells, such as embryonic stem cells or induced pluripotent stem cells, have a great potential for regenerative medicine. Induced pluripotent stem cells, in particular, are suitable for replacement of tissue by autologous transplantation. However, tumorigenicity is a major risk in clinical application of both embryonic stem cells and induced pluripotent stem cells. This study explores the possibility of manipulating the cell cycle for inhibition of tumorigenicity. We genetically modified mouse induced pluripotent stem cells (miPSCs) to overexpress p27 tumor suppressor and examined their proliferation rate, gene expression, cardiac differentiation, tumorigenicity, and therapeutic potential in a mouse model of coronary artery ligation. Overexpression of p27 inhibited cell division of miPSCs, and that inhibition was dependent on the expression level of p27. p27 overexpressing miPSCs had pluripotency characteristics but lost stemness earlier than normal miPSCs during embryoid body and teratoma formation. These cellular characteristics led to none or smaller teratoma when the cells were injected into nude mice. Transplantation of both miPSCs and p27 overexpressing miPSCs into the infarcted mouse heart reduced the infarction size and improved left ventricular function. The overexpression of p27 attenuated tumorigenicity by reducing proliferation and earlier loss of stemness of miPSCs. The overexpression of p27 did not affect pluripotency and differentiation characteristics of miPSC. Therefore, regulation of the proliferation rate of miPSCs offers great therapeutic potential for repair of the injured myocardium.

  18. Enrichment of glioma stem cell-like cells on 3D porous scaffolds composed of different extracellular matrix.

    Wang, Xuanzhi; Dai, Xingliang; Zhang, Xinzhi; Li, Xinda; Xu, Tao; Lan, Qing

    2018-04-15

    Cancer stem cells (CSCs), being tumor-initiating with self-renewal capacity and heterogeneity, are most likely the cause of tumor resistance, reoccurrence and metastasis. To further investigate the role of CSCs in tumor biology, there is a need to develop an effective culture system to grow, maintain and enrich CSCs. Three-dimensional (3D) cell culture model has been widely used in tumor research and drug screening. Recently, researchers have begun to utilize 3D models to culture cancer cells for CSCs enrichment. In this study, glioma cell line was cultured with 3D porous chitosan (CS) scaffolds or chitosan-hyaluronic acid (CS-HA) scaffolds to explore the possibility of glioma stem cells (GSCs)-like cells enrichment, to study the morphology, gene expression, and in vivo tumorigenicity of 3D scaffolds cells, and to compare results to 2D controls. Results showed that glioma cells on both CS and CS-HA scaffolds could form tumor cell spheroids and increased the expression of GSCs biomarkers compared to conventional 2D monolayers. Furthermore, cells in CS-HA scaffolds had higher expression levels of epithelial-to-mesenchymal transition (EMT)-related gene. Specifically, the in vivo tumorigenicity capability of CS-HA scaffold cultured cells was greater than 2D cells or CS scaffold cultured cells. It is indicated that the chemical composition of scaffold plays an important role in the enrichment of CSCs. Our results suggest that CS-HA scaffolds have a better capability to enrich GSCs-like cells and can serve as a simple and effective way to cultivate and enrich CSCs in vitro to support the study of CSCs biology and development of novel anti-cancer therapies. Copyright © 2018 Elsevier Inc. All rights reserved.

  19. Proliferation-dependent changes in amino acid transport and glucose metabolism in glioma cell lines

    Sasajima, Toshio; Miyagawa, Tadashi; Oku, Takamitsu; Gelovani, Juri G.; Finn, Ronald; Blasberg, Ronald

    2004-01-01

    Amino acid imaging is increasingly being used for assessment of brain tumor malignancy, extent of disease, and prognosis. This study explores the relationship between proliferative activity, amino acid transport, and glucose metabolism in three glioma cell lines (U87, Hs683, C6) at different phases of growth in culture. Growth phase was characterized by direct cell counting, proliferation index determined by flow cytometry, and [ 3 H]thymidine (TdR) accumulation, and was compared with the uptake of two non-metabolized amino acids ([ 14 C]aminocyclopentane carboxylic acid (ACPC) and [ 14 C]aminoisobutyric acid (AIB)), and [ 18 F]fluorodeoxyglucose (FDG). Highly significant relationships between cell number (density), proliferation index, and TdR accumulation rate were observed in all cell lines (r>0.99). Influx (K 1 ) of both ACPC and AIB was directly related to cell density, and inversely related to the proliferation index and TdR accumulation in all cell lines. The volume of distribution (V d ) for ACPC and AIB was lowest during rapid growth and highest during the near-plateau growth phase in all cell lines. FDG accumulation in Hs683 and C6 cells was unaffected by proliferation rate, growth phase, and cell density, whereas FDG accumulation was correlated with TdR accumulation, growth phase, and cell density in U87 cells. This study demonstrates that proliferation rate and glucose metabolism are not necessarily co-related in all glioma cell lines. The values of K 1 and V d for ACPC and AIB under different growth conditions suggest that these tumor cell lines can up-regulate amino acid transporters in their cell membranes when their growth conditions become adverse and less than optimal. (orig.)

  20. General applicability of synthetic gene-overexpression for cell-type ratio control via reprogramming.

    Ishimatsu, Kana; Hata, Takashi; Mochizuki, Atsushi; Sekine, Ryoji; Yamamura, Masayuki; Kiga, Daisuke

    2014-09-19

    Control of the cell-type ratio in multistable systems requires wide-range control of the initial states of cells. Here, using a synthetic circuit in E. coli, we describe the use of a simple gene-overexpression system combined with a bistable toggle switch, for the purposes of enabling the wide-range control of cellular states and thus generating arbitrary cell-type ratios. Theoretically, overexpression induction temporarily alters the bistable system to a monostable system, in which the location of the single steady state of cells can be manipulated over a wide range by regulating the overexpression levels. This induced cellular state becomes the initial state of the basal bistable system upon overexpression cessation, which restores the original bistable system. We experimentally demonstrated that the overexpression induced a monomodal cell distribution, and subsequent overexpression withdrawal generated a bimodal distribution. Furthermore, as designed theoretically, regulating the overexpression levels by adjusting the concentrations of small molecules generated arbitrary cell-type ratios.

  1. Delayed cell death associated with mitotic catastrophe in γ-irradiated stem-like glioma cells

    Firat, Elke; Gaedicke, Simone; Tsurumi, Chizuko; Esser, Norbert; Weyerbrock, Astrid; Niedermann, Gabriele

    2011-01-01

    Stem-like tumor cells are regarded as highly resistant to ionizing radiation (IR). Previous studies have focused on apoptosis early after irradiation, and the apoptosis resistance observed has been attributed to reduced DNA damage or enhanced DNA repair compared to non-stem tumor cells. Here, early and late radioresponse of patient-derived stem-like glioma cells (SLGCs) and differentiated cells directly derived from them were examined for cell death mode and the influence of stem cell-specific growth factors. Primary SLGCs were propagated in serum-free medium with the stem-cell mitogens epidermal growth factor (EGF) and fibroblast growth factor-2 (FGF-2). Differentiation was induced by serum-containing medium without EGF and FGF. Radiation sensitivity was evaluated by assessing proliferation, clonogenic survival, apoptosis, and mitotic catastrophe. DNA damage-associated γH2AX as well as p53 and p21 expression were determined by Western blots. SLGCs failed to apoptose in the first 4 days after irradiation even at high single doses up to 10 Gy, but we observed substantial cell death later than 4 days postirradiation in 3 of 6 SLGC lines treated with 5 or 10 Gy. This delayed cell death was observed in 3 of the 4 SLGC lines with nonfunctional p53, was associated with mitotic catastrophe and occurred via apoptosis. The early apoptosis resistance of the SLGCs was associated with lower γH2AX compared to differentiated cells, but we found that the stem-cell culture cytokines EGF plus FGF-2 strongly reduce γH2AX levels. Nonetheless, in two p53-deficient SLGC lines examined γIR-induced apoptosis even correlated with EGF/FGF-induced proliferation and mitotic catastrophe. In a line containing CD133-positive and -negative stem-like cells, the CD133-positive cells proliferated faster and underwent more γIR-induced mitotic catastrophe. Our results suggest the importance of delayed apoptosis, associated mitotic catastrophe, and cellular proliferation for γIR-induced death of

  2. CYTOKINESIS-BLOCK MICRONUCLEUS ASSAY IN HUMAN GLIOMA CELLS EXPOSED TO RADIATION

    Jerzy Slowinski

    2011-05-01

    Full Text Available Biological tests are efficient in reflecting the biological influences of several types of generally harmful exposures. The micronucleus assay is widely used in genotoxicity studies or studies on genomic damage in general. We present methodological aspects of cytokinesis-block micronucleus assay performed in human gliomas irradiated in vitro. Eight human glioblastoma cell lines obtained from DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Germany were gamma-irradiated (60Co over a dose range of 0-10 Gy. Cytokinesis-block micronucleus assay was performed to quantitate cytogenetic damage. The cells were fixed directly on dishes, stained with fluorochrome DAPI and evaluated under fluorescent and phase contrast microscope. The micronucleus frequency was expressed as a micronuclei (MN per binucleated cell (BNC ratio, calculated after scoring at least 100 BNC per dish. The frequency of spontaneous MN ranged from 0.17 to 0.613 (mean: 0.29 ± 0.14. After irradiation increase of MN frequency in the range of 0.312 - 2.241 (mean: 0.98 ± 0.68 was found at 10 Gy. Gliomas are extremely heterogenous in regard to cytogenetic effects of irradiation, as shown in this study by cytokinesis-block micronucleus assay. This test is easily performed on irradiated glioma cell lines and can assist in determining their radiosensitivity. However, in order to obtain reliable and reproducible results, precise criteria for MN scoring must be strictly followed. Simultaneous use of fluorescent and phase contrast equipment improves imaging of morphological details and can further optimize MN scoring.

  3. Hyaluronan synthase 3 (HAS3) overexpression downregulates MV3 melanoma cell proliferation, migration and adhesion

    Takabe, Piia; Bart, Geneviève; Ropponen, Antti; Rilla, Kirsi; Tammi, Markku; Tammi, Raija; Pasonen-Seppänen, Sanna

    2015-01-01

    Malignant skin melanoma is one of the most deadly human cancers. Extracellular matrix (ECM) influences the growth of malignant tumors by modulating tumor cells adhesion and migration. Hyaluronan is an essential component of the ECM, and its amount is altered in many tumors, suggesting an important role for hyaluronan in tumorigenesis. Nonetheless its role in melanomagenesis is not understood. In this study we produced a MV3 melanoma cell line with inducible expression of the hyaluronan synthase 3 (HAS3) and studied its effect on the behavior of the melanoma cells. HAS3 overexpression expanded the cell surface hyaluronan coat and decreased melanoma cell adhesion, migration and proliferation by cell cycle arrest at G1/G0. Melanoma cell migration was restored by removal of cell surface hyaluronan by Streptomyces hyaluronidase and by receptor blocking with hyaluronan oligosaccharides, while the effect on cell proliferation was receptor independent. Overexpression of HAS3 decreased ERK1/2 phosphorylation suggesting that inhibition of MAP-kinase signaling was responsible for these suppressive effects on the malignant phenotype of MV3 melanoma cells. - Highlights: • Inducible HAS3-MV3 melanoma cell line was generated using Lentiviral transduction. • HAS3 overexpression inhibits MV3 cell migration via hyaluronan–receptor interaction. • HAS3 overexpression decreases MV3 melanoma cell proliferation and adhesion. • ERK1/2 phosphorylation is downregulated by 50% in HAS3 overexpressing cells. • The results suggest that hyaluronan has anti-cancer like effects in melanoma

  4. Hyaluronan synthase 3 (HAS3) overexpression downregulates MV3 melanoma cell proliferation, migration and adhesion

    Takabe, Piia, E-mail: piia.takabe@uef.fi [University of Eastern Finland, Institute of Biomedicine, 70211 Kuopio (Finland); Bart, Geneviève [University of Eastern Finland, Institute of Biomedicine, 70211 Kuopio (Finland); Ropponen, Antti [University of Eastern Finland, Institute of Clinical Medicine, 70211 Kuopio (Finland); Rilla, Kirsi; Tammi, Markku; Tammi, Raija; Pasonen-Seppänen, Sanna [University of Eastern Finland, Institute of Biomedicine, 70211 Kuopio (Finland)

    2015-09-10

    Malignant skin melanoma is one of the most deadly human cancers. Extracellular matrix (ECM) influences the growth of malignant tumors by modulating tumor cells adhesion and migration. Hyaluronan is an essential component of the ECM, and its amount is altered in many tumors, suggesting an important role for hyaluronan in tumorigenesis. Nonetheless its role in melanomagenesis is not understood. In this study we produced a MV3 melanoma cell line with inducible expression of the hyaluronan synthase 3 (HAS3) and studied its effect on the behavior of the melanoma cells. HAS3 overexpression expanded the cell surface hyaluronan coat and decreased melanoma cell adhesion, migration and proliferation by cell cycle arrest at G1/G0. Melanoma cell migration was restored by removal of cell surface hyaluronan by Streptomyces hyaluronidase and by receptor blocking with hyaluronan oligosaccharides, while the effect on cell proliferation was receptor independent. Overexpression of HAS3 decreased ERK1/2 phosphorylation suggesting that inhibition of MAP-kinase signaling was responsible for these suppressive effects on the malignant phenotype of MV3 melanoma cells. - Highlights: • Inducible HAS3-MV3 melanoma cell line was generated using Lentiviral transduction. • HAS3 overexpression inhibits MV3 cell migration via hyaluronan–receptor interaction. • HAS3 overexpression decreases MV3 melanoma cell proliferation and adhesion. • ERK1/2 phosphorylation is downregulated by 50% in HAS3 overexpressing cells. • The results suggest that hyaluronan has anti-cancer like effects in melanoma.

  5. The Effects of NDRG2 Overexpression on Cell Proliferation and Invasiveness of SW48 Colorectal Cancer Cell Line.

    Golestan, Ali; Mojtahedi, Zahra; Ghalamfarsa, Ghasem; Hamidinia, Maryam; Takhshid, Mohammad Ali

    2015-09-01

    Colorectal cancer (CRC) is one of the most common causes of cancer-related death in the world. The expression of N-myc downstream-regulated gene 2 (NDRG2) is down-regulated in CRC. The aim of this study was to investigate the effect of NDRG2 overexpression on cell proliferation and invasive potential of SW48 cells. SW48 cells were transfected with a plasmid overexpressing NDRG2. After stable transfection, the effect of NDRG2 overexpression on cell proliferation was evaluated by MTT assay. The effects of NDRG2 overexpression on cell migration, invasion and cell motility and matrix metalloproteinase 9 (MMP9) activities were also investigated using matrigel transwell assay, wound healing assay and gelatin zymography, respectively. MTT assay showed that overexpression of NDRG2 caused attenuation of SW48 cell proliferation. Transwell and wound healing assay revealed that NDRG2 overexpression led to inhibition of migration, invasion, and motility of SW48 cells. The overexpression of NDRG2 also reduced the activity of secreted MMP-9. The results of this study suggest that NDRG2 overexpression inhibits proliferation and invasive potential of SW48 cells, which likely occurs via suppression of MMP-9 activity.

  6. Response-predictive gene expression profiling of glioma progenitor cells in vitro.

    Sylvia Moeckel

    Full Text Available High-grade gliomas are amongst the most deadly human tumors. Treatment results are disappointing. Still, in several trials around 20% of patients respond to therapy. To date, diagnostic strategies to identify patients that will profit from a specific therapy do not exist.In this study, we used serum-free short-term treated in vitro cell cultures to predict treatment response in vitro. This approach allowed us (a to enrich specimens for brain tumor initiating cells and (b to confront cells with a therapeutic agent before expression profiling.As a proof of principle we analyzed gene expression in 18 short-term serum-free cultures of high-grade gliomas enhanced for brain tumor initiating cells (BTIC before and after in vitro treatment with the tyrosine kinase inhibitor Sunitinib. Profiles from treated progenitor cells allowed to predict therapy-induced impairment of proliferation in vitro.For the tyrosine kinase inhibitor Sunitinib used in this dataset, the approach revealed additional predictive information in comparison to the evaluation of classical signaling analysis.

  7. The production of nitric oxide in EL4 lymphoma cells overexpressing growth hormone.

    Arnold, Robyn E; Weigent, Douglas A

    2003-01-01

    Growth hormone (GH) is produced by immunocompetent cells and has been implicated in the regulation of a multiplicity of functions in the immune system involved in growth and activation. However, the actions of endogenous or lymphocyte GH and its contribution to immune reactivity when compared with those of serum or exogenous GH are still unclear. In the present study, we overexpressed lymphocyte GH in EL4 lymphoma cells, which lack the GH receptor (GHR), to determine the role of endogenous GH in nitric oxide (NO) production and response to genotoxic stress. Western blot analysis demonstrated that the levels of GH increased approximately 40% in cells overexpressing GH (GHo) when compared with cells with vector alone. The results also show a substantial increase in NO production in cells overexpressing GH that could be blocked by N(G)-monomethyl-L-arginine (L-NMMA), an L-arginine analogue that competitively inhibits all three isoforms of nitric oxide synthase (NOS). No evidence was obtained to support an increase in peroxynitrite in cells overexpressing GH. Overexpression of GH increased NOS activity, inducible nitric oxide synthase (iNOS) promoter activity, and iNOS protein expression, whereas endothelial nitric oxide synthase and neuronal nitric oxide synthase protein levels were essentially unchanged. In addition, cells overexpressing GH showed increased arginine transport ability and intracellular arginase activity when compared with control cells. GH overexpression appeared to protect cells from the toxic effects of the DNA alkylating agent methyl methanesulfonate. This possibility was suggested by maintenance of the mitochondrial transmembrane potential in cells overexpressing GH when compared with control cells that could be blocked by L-NMMA. Taken together, the data support the notion that lymphocyte GH, independently of the GH receptor, may play a key role in the survival of lymphocytes exposed to stressful stimuli via the production of NO.

  8. Mesenchymal stem cells derived from adipose tissue vs bone marrow: in vitro comparison of their tropism towards gliomas.

    Courtney Pendleton

    Full Text Available INTRODUCTION: Glioblastoma is the most common primary malignant brain tumor, and is refractory to surgical resection, radiation, and chemotherapy. Human mesenchymal stem cells (hMSC may be harvested from bone marrow (BMSC and adipose (AMSC tissue. These cells are a promising avenue of investigation for the delivery of adjuvant therapies. Despite extensive research into putative mechanisms for the tumor tropism of MSCs, there remains no direct comparison of the efficacy and specificity of AMSC and BMSC tropism towards glioma. METHODS: Under an IRB-approved protocol, intraoperative human Adipose MSCs (hAMSCs were established and characterized for cell surface markers of mesenchymal stem cell origin in conjunction with the potential for tri-lineage differentiation (adipogenic, chondrogenic, and osteogenic. Validated experimental hAMSCs were compared to commercially derived hBMSCs (Lonza and hAMSCs (Invitrogen for growth responsiveness and glioma tropism in response to glioma conditioned media obtained from primary glioma neurosphere cultures. RESULTS: Commercial and primary culture AMSCs and commercial BMSCs demonstrated no statistically significant difference in their migration towards glioma conditioned media in vitro. There was statistically significant difference in the proliferation rate of both commercial AMSCs and BMSCs as compared to primary culture AMSCs, suggesting primary cultures have a slower growth rate than commercially available cell lines. CONCLUSIONS: Adipose- and bone marrow-derived mesenchymal stem cells have similar in vitro glioma tropism. Given the well-documented ability to harvest larger numbers of AMSCs under local anesthesia, adipose tissue may provide a more efficient source of MSCs for research and clinical applications, while minimizing patient morbidity during cell harvesting.

  9. Cafestol overcomes ABT-737 resistance in Mcl-1-overexpressed renal carcinoma Caki cells through downregulation of Mcl-1 expression and upregulation of Bim expression.

    Woo, S M; Min, K-J; Seo, B R; Nam, J-O; Choi, K S; Yoo, Y H; Kwon, T K

    2014-11-06

    Although ABT-737, a small-molecule Bcl-2/Bcl-xL inhibitor, has recently emerged as a novel cancer therapeutic agent, ABT-737-induced apoptosis is often blocked in several types of cancer cells with elevated expression of Mcl-1. Cafestol, one of the major compounds in coffee beans, has been reported to have anti-carcinogenic activity and tumor cell growth-inhibitory activity, and we examined whether cafestol could overcome resistance against ABT-737 in Mcl-1-overexpressed human renal carcinoma Caki cells. ABT-737 alone had no effect on apoptosis, but cafestol markedly enhanced ABT-737-mediated apoptosis in Mcl-1-overexpressed Caki cells, human glioma U251MG cells, and human breast carcinoma MDA-MB231 cells. By contrast, co-treatment with ABT-737 and cafestol did not induce apoptosis in normal human skin fibroblast. Furthermore, combined treatment with cafestol and ABT-737 markedly reduced tumor growth compared with either drug alone in xenograft models. We found that cafestol inhibited Mcl-1 protein expression, which is important for ABT-737 resistance, through promotion of protein degradation. Moreover, cafestol increased Bim expression, and siRNA-mediated suppression of Bim expression reduced the apoptosis induced by cafestol plus ABT-737. Taken together, cafestol may be effectively used to enhance ABT-737 sensitivity in cancer therapy via downregulation of Mcl-1 expression and upregulation of Bim expression.

  10. The anti-epidermal growth factor receptor (EGFR) monoclonal antibody, C225, enhances radiation-induced apoptosis in primary glioma cell lines through mediation of MAPK/JNK/p38 signaling pathways

    Chakravarti, A.; Noll, E.; Black, P.M.; Loeffler, J.S.

    2001-01-01

    Purpose: Increasing evidence suggests that signaling mediated by the epidermal growth factor receptor (EGFR) pathway contributes to radiation resistance. The anti-EGFR monoclonal antibody, C225, has been shown to enhance radiation response for several tumor types in preclinical models. Malignant gliomas are known to express, and quite frequently overexpress, EGFR. Our objectives in this study were to 1) Evaluate the efficacy of C225 as a radiation response modifier in EGFR-expressing glioma cell lines and to 2) Investigate the underlying molecular mechanisms mediating C225-induced enhancement of radiation response. Materials and Methods: Twelve EGFR-expressing glioma cells lines, established from patient tumors, were used for this study. Cells were incubated with C225, irradiated, and then evaluated for radiation response. Assays used to evaluate efficacy of C225-mediated radiosensitization included time-course apoptosis assays (Annexin V and TUNEL), viability assays (MTT), and clonogenic survival assays. The changes along MAPK (p44/p42)/JNK/p38-MAPK signal transduction pathways were then investigated using quantitative Western analysis with phospho-specific antibodies to determine the molecular mechanisms by which C225 mediates a given response. Results: C225 clearly enhanced radiation response for 7 of the 12 primary glioma cell lines studied. Enhancement of both immediate and delayed apoptotic responses was evident in these 7 responsive cell lines after C225 administration. The average apoptosis index at 6 hours post-RT+C225 for the 7 responsive lines was 9.5%, compared to 1.2% for the RT-only controls. A pattern of delayed apoptosis was evident in these 7 lines, with secondary apoptotic peaks (∼ 8.0%) occurring at 24 hours post-RT+C225. Time course viability measurements revealed a steady decrease in viable tumor cells in these responsive cell lines from 75% at 6 hours post-RT+C225 to 20% at 7 days. Clonogenic survival was also diminished in these 7 lines

  11. Cytotoxic effect of menadione and sodium orthovanadate in combination on human glioma cells.

    Delwar, Zahid M; Avramidis, Dimitrios; Follin, Elna; Hua, Yan; Siden, Åke; Cruz, Mabel; Paulsson, Kajsa M; Yakisich, Juan Sebastian

    2012-08-01

    Gliomas are the most common primary brain tumor, and their treatment is still a challenge. Here, we evaluated the antiproliferative effect of a novel combination of two potent oxidative stress enhancers: menadione (M) and sodium orthovanadate (SO). We observed both short-term and prolonged growth inhibitory effects of M or SO alone as well as in combination (M:SO) on DBTRG.05MG human glioma cells. A stronger antiproliferative effect was observed in the short-term proliferation assay with the M:SO combination compared to either investigated agent alone. In the long-term proliferation assay, a 10-day exposure to M:SO at concentrations of 10 μM:17.5 μM or 17.5 μM:10 μM was enough to kill 100% of the cells; no cell regrowth was observed after re-incubation in drug-free media. When used in combination, the single concentration of M and SO could be decreased by 2.5- to 5-fold of those used for each experimental drug alone and still obtain a similar antiproliferative effect. The underlying molecular mechanism was investigated by co-incubating M:SO with dithiothreitol (DTT) and genistein. Both substances partially neutralized the effects of the M:SO combination, showing additive effects. This observation suggests a role of oxidative stress and tyrosine kinase stimulation in the M:SO cytotoxic effect. Our results indicate that M:SO combination is an attractive alternative for glioma treatment that encourages further study. The neutralizing effects of genistein and DTT reveal a possibility for their use in the minimization of potential M:SO systemic toxicity.

  12. Antitumor effects of cannabidiol, a nonpsychoactive cannabinoid, on human glioma cell lines.

    Massi, Paola; Vaccani, Angelo; Ceruti, Stefania; Colombo, Arianna; Abbracchio, Maria P; Parolaro, Daniela

    2004-03-01

    Recently, cannabinoids (CBs) have been shown to possess antitumor properties. Because the psychoactivity of cannabinoid compounds limits their medicinal usage, we undertook the present study to evaluate the in vitro antiproliferative ability of cannabidiol (CBD), a nonpsychoactive cannabinoid compound, on U87 and U373 human glioma cell lines. The addition of CBD to the culture medium led to a dramatic drop of mitochondrial oxidative metabolism [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazolium bromide test] and viability in glioma cells, in a concentration-dependent manner that was already evident 24 h after CBD exposure, with an apparent IC(50) of 25 microM. The antiproliferative effect of CBD was partially prevented by the CB2 receptor antagonist N-[(1S)-endo-1,3,3-trimethylbicyclo[2,2,1]heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide (SR144528; SR2) and alpha-tocopherol. By contrast, the CB1 cannabinoid receptor antagonist N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboximide hydrochloride (SR141716; SR1), capsazepine (vanilloid receptor antagonist), the inhibitors of ceramide generation, or pertussis toxin did not counteract CBD effects. We also show, for the first time, that the antiproliferative effect of CBD was correlated to induction of apoptosis, as determined by cytofluorimetric analysis and single-strand DNA staining, which was not reverted by cannabinoid antagonists. Finally, CBD, administered s.c. to nude mice at the dose of 0.5 mg/mouse, significantly inhibited the growth of subcutaneously implanted U87 human glioma cells. In conclusion, the nonpsychoactive CBD was able to produce a significant antitumor activity both in vitro and in vivo, thus suggesting a possible application of CBD as an antineoplastic agent.

  13. The Expression of Connexins and SOX2 Reflects the Plasticity of Glioma Stem-Like Cells

    Joana Balça-Silva

    2017-08-01

    Full Text Available Glioblastoma (GBM is the most malignant primary brain tumor, with an average survival rate of 15 months. GBM is highly refractory to therapy, and such unresponsiveness is due, primarily, but not exclusively, to the glioma stem-like cells (GSCs. This subpopulation express stem-like cell markers and is responsible for the heterogeneity of GBM, generating multiple differentiated cell phenotypes. However, how GBMs maintain the balance between stem and non-stem populations is still poorly understood. We investigated the GBM ability to interconvert between stem and non-stem states through the evaluation of the expression of specific stem cell markers as well as cell communication proteins. We evaluated the molecular and phenotypic characteristics of GSCs derived from differentiated GBM cell lines by comparing their stem-like cell properties and expression of connexins. We showed that non-GSCs as well as GSCs can undergo successive cycles of gain and loss of stem properties, demonstrating a bidirectional cellular plasticity model that is accompanied by changes on connexins expression. Our findings indicate that the interconversion between non-GSCs and GSCs can be modulated by extracellular factors culminating on differential expression of stem-like cell markers and cell-cell communication proteins. Ultimately, we observed that stem markers are mostly expressed on GBMs rather than on low-grade astrocytomas, suggesting that the presence of GSCs is a feature of high-grade gliomas. Together, our data demonstrate the utmost importance of the understanding of stem cell plasticity properties in a way to a step closer to new strategic approaches to potentially eliminate GSCs and, hopefully, prevent tumor recurrence.

  14. miR-21 Is Linked to Glioma Angiogenesis

    Hermansen, Simon Kjær; Nielsen, Boye Schnack; Aaberg-Jessen, Charlotte

    2016-01-01

    MicroRNA-21 (miR-21) is the most consistently over-expressed microRNA (miRNA) in malignant gliomas. We have previously reported that miR-21 is upregulated in glioma vessels and subsets of glioma cells. To better understand the role of miR-21 in glioma angiogenesis and to characterize miR-21......-localized with the hypoxia- and angiogenesis-associated markers HIF-1α (p=0.0020) and VEGF (p=0.0096), whereas the putative miR-21 target, PTEN, was expressed independently of miR-21. Expression of stem cell markers Oct4, Sox2 and CD133 was not associated with miR-21. In six glioblastoma cultures, miR-21 did not correlate...... with the six markers. These findings suggest that miR-21 is linked to glioma angiogenesis, that miR-21 is unlikely to regulate PTEN, and that miR-21-positive tumor cells do not possess stem cell characteristics....

  15. Increased betulinic acid induced cytotoxicity and radiosensitivity in glioma cells under hypoxic conditions

    Bache, Matthias; Taubert, Helge; Vordermark, Dirk; Zschornak, Martin P; Passin, Sarina; Keßler, Jacqueline; Wichmann, Henri; Kappler, Matthias; Paschke, Reinhard; Kaluđerović, Goran N; Kommera, Harish

    2011-01-01

    Betulinic acid (BA) is a novel antineoplastic agent under evaluation for tumor therapy. Because of the selective cytotoxic effects of BA in tumor cells (including gliomas), the combination of this agent with conservative therapies (such as radiotherapy and chemotherapy) may be useful. Previously, the combination of BA with irradiation under hypoxic conditions had never been studied. In this study, the effects of 3 to 30 μM BA on cytotoxicity, migration, the protein expression of PARP, survivin and HIF-1α, as well as radiosensitivity under normoxic and hypoxic conditions were analyzed in the human malignant glioma cell lines U251MG and U343MG. Cytotoxicity and radiosensitivity were analyzed with clonogenic survival assays, migration was analyzed with Boyden chamber assays (or scratch assays) and protein expression was examined with Western blot analyses. Under normoxic conditions, a half maximal inhibitory concentration (IC 50 ) of 23 μM was observed in U251MG cells and 24 μM was observed in U343MG cells. Under hypoxic conditions, 10 μM or 15 μM of BA showed a significantly increased cytotoxicity in U251MG cells (p = 0.004 and p = 0.01, respectively) and U343MG cells (p < 0.05 and p = 0.01, respectively). The combination of BA with radiotherapy resulted in an additive effect in the U343MG cell line under normoxic and hypoxic conditions. Weak radiation enhancement was observed in U251MG cell line after treatment with BA under normoxic conditions. Furthermore, under hypoxic conditions, the incubation with BA resulted in increased radiation enhancement. The enhancement factor, at an irradiation dose of 15 Gy after treatment with 10 or 15 μM BA, was 2.20 (p = 0.02) and 4.50 (p = 0.03), respectively. Incubation with BA led to decreased cell migration, cleavage of PARP and decreased expression levels of survivin in both cell lines. Additionally, BA treatment resulted in a reduction of HIF-1α protein under hypoxic conditions. Our results suggest that BA is capable

  16. Overexpression of metallothioneins, stem cell niches and field cancerization in experimental gliomagenesis Superexpresión de metalotioneínas, nichos de células madre y campos de cancerización en gliomagénesis experimental A superexpressão de metalotioneínas em células-tronco de áreas cancerígenas na gênese de glioma experimental

    Julio César Fernandes da Silva

    2009-12-01

    Full Text Available INTRODUCTION: stem cells may originate and perpetuate the tumor growth, but they are poorly known in gliomagenesis. Metallothioneins (MTs are proteins involved in oncogenesis and immunopositivity, for MT may be used as a stem cell mutation marker. OBJECTIVE: to study the MT expression in the ENU experimental model and to establish an experimental model to track glioma stem cells in early oncogenesis. METHODS: Thirty-six male Wistar rats were divided into two groups; the experimental group was treated within 24 hours after birth (neonate rats with a single dose of subcutaneously injected N-ethyl N-nitrosourea ENU (40 mg/kg body weight. The control animals were injected with the same volume of saline. These experimental animals were subdivided into three groups according to the euthanize time, as follows: the Group 1 (G1 was euthanized at the age of 30 days; the Group 2 (G2, at the age of 180 days and the Group 3 (G3 was euthanized soon after the appearing of signs of the existence of nervous system tumors, at an average age of 321 days. Immunohistochemical detection of MT protein in cold acetone-fixed paraffin embedded spine cord sections was performed by the streptavidin-avidin-biotin-immuno peroxidase complex method. RESULTS: by using the experimental model of gliomagenesis induced by the N-ethyl N-nitrosourea, it was possible to detect putative tumor stem cells in early oncogenesis, to analyze a field cancerization process and to observe a close morphological relationship between MT positive cells and blood vessels. CONCLUSIONS: this reproducible experimental model allows further studies on the origins, development and regulating factors involved in gliomagenesis.INTRODUCCIÓN: células madre pueden originar y perpetuar el crecimiento tumoral, sin embargo son poco conocidas en la gliomagénesis. Las metalotioneínas (MTs son proteínas involucradas en la oncogénesis, y la inmunopositividad de las MTs puede ser utilizada como marcador de

  17. Bmi1 overexpression in the cerebellar granule cell lineage of mice affects cell proliferation and survival without initiating medulloblastoma formation

    Hourinaz Behesti

    2013-01-01

    BMI1 is a potent inducer of neural stem cell self-renewal and neural progenitor cell proliferation during development and in adult tissue homeostasis. It is overexpressed in numerous human cancers – including medulloblastomas, in which its functional role is unclear. We generated transgenic mouse lines with targeted overexpression of Bmi1 in the cerebellar granule cell lineage, a cell type that has been shown to act as a cell of origin for medulloblastomas. Overexpression of Bmi1 in granule cell progenitors (GCPs led to a decrease in cerebellar size due to decreased GCP proliferation and repression of the expression of cyclin genes, whereas Bmi1 overexpression in postmitotic granule cells improved cell survival in response to stress by altering the expression of genes in the mitochondrial cell death pathway and of Myc and Lef-1. Although no medulloblastomas developed in ageing cohorts of transgenic mice, crosses with Trp53−/− mice resulted in a low incidence of medulloblastoma formation. Furthermore, analysis of a large collection of primary human medulloblastomas revealed that tumours with a BMI1high TP53low molecular profile are significantly enriched in Group 4 human medulloblastomas. Our data suggest that different levels and timing of Bmi1 overexpression yield distinct cellular outcomes within the same cellular lineage. Importantly, Bmi1 overexpression at the GCP stage does not induce tumour formation, suggesting that BMI1 overexpression in GCP-derived human medulloblastomas probably occurs during later stages of oncogenesis and might serve to enhance tumour cell survival.

  18. Investigation of effects of a new integrin inhibitor and of its association with radiotherapy on paediatric glioma cell lines; etude des effets d'un nouvel inhibiteur d'integrines et de son association avec la radiotherapie sur des lignees de gliomes pediatriques

    Le Tinier, F.; Meignan, S.; Leblond, P.; Dewitte, A.; Wattez, N.; Lartigau, E.; Lansiaux, A. [Centre Oscar-Lambret, Lille (France)

    2011-10-15

    The authors report the investigation of the effects of Cilengitide (an integrin inhibitor) alone or in association with ionizing radiation on paediatric glioma cell lines. Five grade-II to IV paediatric glioma cell lines and one high grade adult glioma cell line have been studied. It appears that Cilengitide has a direct effect on paediatric glioma cells, notably before irradiation. Short communication

  19. SNAI2/Slug promotes growth and invasion in human gliomas

    Yang, Hong Wei; Menon, Lata G; Black, Peter M; Carroll, Rona S; Johnson, Mark D

    2010-01-01

    Numerous factors that contribute to malignant glioma invasion have been identified, but the upstream genes coordinating this process are poorly known. To identify genes controlling glioma invasion, we used genome-wide mRNA expression profiles of primary human glioblastomas to develop an expression-based rank ordering of 30 transcription factors that have previously been implicated in the regulation of invasion and metastasis in cancer. Using this approach, we identified the oncogenic transcriptional repressor, SNAI2/Slug, among the upper tenth percentile of invasion-related transcription factors overexpressed in glioblastomas. SNAI2 mRNA expression correlated with histologic grade and invasive phenotype in primary human glioma specimens, and was induced by EGF receptor activation in human glioblastoma cells. Overexpression of SNAI2/Slug increased glioblastoma cell proliferation and invasion in vitro and promoted angiogenesis and glioblastoma growth in vivo. Importantly, knockdown of endogenous SNAI2/Slug in glioblastoma cells decreased invasion and increased survival in a mouse intracranial human glioblastoma transplantation model. This genome-scale approach has thus identified SNAI2/Slug as a regulator of growth and invasion in human gliomas

  20. Fascin-1 knock-down of human glioma cells reduces their microvilli/filopodia while improving their susceptibility to lymphocyte-mediated cytotoxicity

    Hoa, Neil T; Ge, Lisheng; Erickson, Kate L; Kruse, Carol A; Cornforth, Andrew N; Kuznetsov, Yurii; McPherson, Alex; Martini, Filippo; Jadus, Martin R

    2015-01-01

    Cancer cells derived from Glioblastoma multiforme possess membranous protrusions allowing these cells to infiltrate surrounding tissue, while resisting lymphocyte cytotoxicity. Microvilli and filopodia are supported by actin filaments cross-linked by fascin. Fascin-1 was genetically silenced within human U251 glioma cells; these knock-down glioma cells lost their microvilli/filopodia. The doubling time of these fascin-1 knock-down cells was doubled that of shRNA control U251 cells. Fascin-1 knock-down cells lost their transmigratory ability responding to interleukin-6 or insulin-like growth factor-1. Fascin-1 silenced U251 cells were more easily killed by cytolytic lymphocytes. Fascin-1 knock-down provides unique opportunities to augment glioma immunotherapy by simultaneously targeting several key glioma functions: like cell transmigration, cell division and resisting immune responses. PMID:25901196

  1. Isolation of plasma membranes from cultured glioma cells and application to evaluation of membrane sphingomyelin turnover

    Cook, H.W.; Palmer, F.B.; Byers, D.M.; Spence, M.W.

    1988-01-01

    A rapid and reliable method for the isolation of plasma membranes and microsomes of high purity and yield from cultured glioma cells is described. The procedure involves disruption by N2 cavitation, preliminary separation by centrifugation in Tricine buffer, and final separation on a gradient formed from 40% Percoll at pH 9.3. Enzyme and chemical markers indicated greater than 60% yield with six- to eightfold enrichment for plasma membranes and greater than 25% yield with three- to fourfold enrichment for a microsomal fraction consisting mainly of endoplasmic reticulum. The final fractions were obtained with high reproducibility in less than 1 h from the time of cell harvesting. Application of this procedure to human fibroblasts in culture is assessed. The isolation procedure was applied to investigations of synthesis and turnover of sphingomyelin and phosphatidylcholine in plasma membranes of glioma cells following incubation for 4-24 h with [methyl- 3 H]choline. These studies indicated that radioactivity from phosphatidylcholine synthesized in microsomes from exogenous choline may serve as a precursor of the head-group of sphingomyelin accumulating in the plasma membrane

  2. Ameliorating replicative senescence of human bone marrow stromal cells by PSMB5 overexpression

    Lu, Li; Song, Hui-Fang; Wei, Jiao-Long; Liu, Xue-Qin; Song, Wen-Hui; Yan, Ba-Yi; Yang, Gui-Jiao; Li, Ang; Yang, Wu-Lin

    2014-01-01

    Highlights: • PSMB5 overexpression restores the differentiation potential of aged hBMSCs. • PSMB5 overexpression enhances the proteasomal activity of late-stage hBMSCs. • PSMB5 overexpression inhibits replicative senescence and improved cell viability. • PSMB5 overexpression promotes cell growth by upregulating the Cyclin D1/CDK4 complex. - Abstract: Multipotent human bone marrow stromal cells (hBMSCs) potentially serve as a source for cell-based therapy in regenerative medicine. However, in vitro expansion was inescapably accompanied with cell senescence, characterized by inhibited proliferation and compromised pluripotency. We have previously demonstrated that this aging process is closely associated with reduced 20S proteasomal activity, with down-regulation of rate-limiting catalytic β-subunits particularly evident. In the present study, we confirmed that proteasomal activity directly contributes to senescence of hBMSCs, which could be reversed by overexpression of the β5-subunit (PSMB5). Knocking down PSMB5 led to decreased proteasomal activity concurrent with reduced cell proliferation in early-stage hBMSCs, which is similar to the senescent phenotype observed in late-stage cells. In contrast, overexpressing PSMB5 in late-stage cells efficiently restored the normal activity of 20S proteasomes and promoted cell growth, possibly via upregulating the Cyclin D1/CDK4 complex. Additionally, PSMB5 could enhance cell resistance to oxidative stress, as evidenced by the increased cell survival upon exposing senescent hBMSCs to hydrogen peroxide. Furthermore, PSMB5 overexpression retained the pluripotency of late-stage hBMSCs by facilitating their neural differentiation both in vitro and in vivo. Collectively, our work reveals a critical role of PSMB5 in 20S proteasome-mediated protection against replicative senescence, pointing to a possible strategy for maintaining the integrity of culture-expanded hBMSCs by manipulating the expression of PSMB5

  3. Ameliorating replicative senescence of human bone marrow stromal cells by PSMB5 overexpression

    Lu, Li, E-mail: luli7300@126.com [Department of Anatomy, Shanxi Medical University, Taiyuan 030001 (China); Song, Hui-Fang; Wei, Jiao-Long; Liu, Xue-Qin [Department of Anatomy, Shanxi Medical University, Taiyuan 030001 (China); Song, Wen-Hui [Department of Orthopaedics, The Second Affiliated Hospital of Shanxi Medical University, Taiyuan 030001 (China); Yan, Ba-Yi; Yang, Gui-Jiao [Department of Anatomy, Shanxi Medical University, Taiyuan 030001 (China); Li, Ang [Department of Medicine, University of Hong Kong Faculty of Medicine, Hong Kong (Hong Kong); Department of Anatomy, University of Hong Kong Faculty of Medicine, Hong Kong (Hong Kong); Yang, Wu-Lin, E-mail: wulinyoung@163.com [School of Biotechnology and Food Engineering, Hefei University of Technology, Hefei 230009 (China); Laboratory of Metabolic Medicine, Singapore Bioimaging Consortium (SBIC), Agency for Science, Technology and Research - A*STAR (Singapore)

    2014-01-24

    Highlights: • PSMB5 overexpression restores the differentiation potential of aged hBMSCs. • PSMB5 overexpression enhances the proteasomal activity of late-stage hBMSCs. • PSMB5 overexpression inhibits replicative senescence and improved cell viability. • PSMB5 overexpression promotes cell growth by upregulating the Cyclin D1/CDK4 complex. - Abstract: Multipotent human bone marrow stromal cells (hBMSCs) potentially serve as a source for cell-based therapy in regenerative medicine. However, in vitro expansion was inescapably accompanied with cell senescence, characterized by inhibited proliferation and compromised pluripotency. We have previously demonstrated that this aging process is closely associated with reduced 20S proteasomal activity, with down-regulation of rate-limiting catalytic β-subunits particularly evident. In the present study, we confirmed that proteasomal activity directly contributes to senescence of hBMSCs, which could be reversed by overexpression of the β5-subunit (PSMB5). Knocking down PSMB5 led to decreased proteasomal activity concurrent with reduced cell proliferation in early-stage hBMSCs, which is similar to the senescent phenotype observed in late-stage cells. In contrast, overexpressing PSMB5 in late-stage cells efficiently restored the normal activity of 20S proteasomes and promoted cell growth, possibly via upregulating the Cyclin D1/CDK4 complex. Additionally, PSMB5 could enhance cell resistance to oxidative stress, as evidenced by the increased cell survival upon exposing senescent hBMSCs to hydrogen peroxide. Furthermore, PSMB5 overexpression retained the pluripotency of late-stage hBMSCs by facilitating their neural differentiation both in vitro and in vivo. Collectively, our work reveals a critical role of PSMB5 in 20S proteasome-mediated protection against replicative senescence, pointing to a possible strategy for maintaining the integrity of culture-expanded hBMSCs by manipulating the expression of PSMB5.

  4. Functional and gene expression analysis of hTERT overexpressed endothelial cells

    Haruna Takano

    2008-09-01

    Full Text Available Haruna Takano1, Satoshi Murasawa1,2, Takayuki Asahara1,2,31Institute of Biomedical Research and Innovation, Kobe, Japan; 2RIKEN Center for Developmental Biology, Kobe 650-0047, Japan; 3Tokai University of School of Medicine, Tokai, JapanAbstract: Telomerase dysfunction contributes to cellular senescence. Recent advances indicate the importance of senescence in maintaining vascular cell function in vitro. Human telomerase reverse transcriptase (hTERT overexpression is thought to lead to resistance to apoptosis and oxidative stress. However, the mechanism in endothelial lineage cells is unclear. We tried to generate an immortal endothelial cell line from human umbilical vein endothelial cells using a no-virus system and examine the functional mechanisms of hTERT overexpressed endothelial cell senescence in vitro. High levels of hTERT genes and endothelial cell-specific markers were expressed during long-term culture. Also, angiogenic responses were observed in hTERT overexpressed endothelial cell. These cells showed a delay in senescence and appeared more resistant to stressed conditions. PI3K/Akt-related gene levels were enhanced in hTERT overexpressed endothelial cells. An up-regulated PI3K/Akt pathway caused by hTERT overexpression might contribute to anti-apoptosis and survival effects in endothelial lineage cells.Keywords: endothelial, telomerase, senescence, oxidative stress, anti-apoptosis, PI3K/Akt pathway

  5. Extracellular diffusion quantified by magnetic resonance imaging during rat C6 glioma cell progression

    G. Song

    Full Text Available Solution reflux and edema hamper the convection-enhanced delivery of the standard treatment for glioma. Therefore, a real-time magnetic resonance imaging (MRI method was developed to monitor the dosing process, but a quantitative analysis of local diffusion and clearance parameters has not been assessed. The objective of this study was to compare diffusion into the extracellular space (ECS at different stages of rat C6 gliomas, and analyze the effects of the extracellular matrix (ECM on the diffusion process. At 10 and 20 days, after successful glioma modeling, gadolinium-diethylenetriamine pentaacetic acid (Gd-DTPA was introduced into the ECS of rat C6 gliomas. Diffusion parameters and half-life of the reagent were then detected using MRI, and quantified according to the mathematical model of diffusion. The main ECM components [chondroitin sulfate proteoglycans (CSPGs, collagen IV, and tenascin C] were detected by immunohistochemical and immunoblot analyses. In 20-day gliomas, Gd-DTPA diffused more slowly and derived higher tortuosity, with lower clearance rate and longer half-life compared to 10-day gliomas. The increased glioma ECM was associated with different diffusion and clearance parameters in 20-day rat gliomas compared to 10-day gliomas. ECS parameters were altered with C6 glioma progression from increased ECM content. Our study might help better understand the glioma microenvironment and provide benefits for interstitial drug delivery to treat brain gliomas.

  6. Mechanisms of MRP over-expression in four human lung-cancer cell lines and analysis of the MRP amplicon

    Eijdems, E. W.; de Haas, M.; Coco-Martin, J. M.; Ottenheim, C. P.; Zaman, G. J.; Dauwerse, H. G.; Breuning, M. H.; Twentyman, P. R.; Borst, P.; Baas, F.

    1995-01-01

    Some multidrug resistant cell lines over-express the gene encoding the multidrug-resistance-associated protein (MRP). In all cell lines reported thus far, over-expression is associated with gene amplification. We have studied the predominant mechanisms of MRP over-expression in 4 human lung-cancer

  7. In vitro neutron irradiation of glioma and endothelial cultured cells

    Menichetti, L. [Department of PET and Radiopharmaceutical Chemistry, C.N.R. Institute of Clinical Physiology, Pisa (Italy)], E-mail: luca.menichetti@ifc.cnr.it; Gaetano, L. [University Scuola Superiore Sant' Anna, Pisa (Italy); Zampolli, A.; Del Turco, S. [Department of PET and Radiopharmaceutical Chemistry, C.N.R. Institute of Clinical Physiology, Pisa (Italy); Ferrari, C. [University of Pavia, Department of Surgery, Laboratory of experimental Surgery, Pavia (Italy); Bortolussi, S.; Stella, S.; Altieri, S. [University of Pavia, Department of Nuclear Physics, Pavia (Italy); National Institute for Nuclear Physics (INFN), Section of Pavia (Italy); Salvadori, P.A. [Department of PET and Radiopharmaceutical Chemistry, C.N.R. Institute of Clinical Physiology, Pisa (Italy); Cionini, L. [Unit of Radiotherapy, AOUP-University Hospital, Pisa (Italy)

    2009-07-15

    To fully develop its potential boron neutron capture therapy (BNCT) requires the combination of a suitable thermal/epithermal neutron flux together with a selective intake of {sup 10}B-boron nuclei in the target tissue. The latter condition is the most critical to be realized as none of the boron carriers used for experimental or clinical purposes proved at the moment an optimal selectivity for cancer cells compared to normal cells. In addition to complex physical factors, the assessment of the intracellular concentration of boron represent a crucial parameter to predict the dose delivered to the cancer cells during the treatment. Nowadays the dosimetry calculation and then the prediction of the treatment effectiveness are made using Monte Carlo simulations, but some of the model assumption are still uncertain: the radiobiological dose efficacy and the probability of tumour cell survival are crucial parameters that needs a more reliable experimental approach. The aim of this work was to evaluate the differential ability of two cell lines to selectively concentrate the boron-10 administered as di-hydroxyboryl-phenylalanine (BPA)-fructose adduct, and the effect of the differential boron intake on the damage produced by the irradiation with thermal neutrons; the two cell lines were selected to be representative one of normal tissues involved in the active/passive transport of boron carriers, and one of the tumour. Recent in vitro studies demonstrated how BPA is taken by proliferating cells, however the mechanism of BPA uptake and the parameters driving the kinetics of influx and the elimination of BPA are still not clarified. In these preliminary studies we analysed the survival of F98 and human umbilical vein endothelial cells (HUVEC) cells line after irradiation, using different thermal fluencies at the same level of density population and boron concentration in the growing medium prior the irradiation. This is first study performed on endothelium model obtained by

  8. Preclinical investigation of ibrutinib, a Bruton's kinase tyrosine (Btk) inhibitor, in suppressing glioma tumorigenesis and stem cell phenotypes

    Wei, Li; Su, Yu-Kai; Lin, Chien-Min; Chao, Tsu-Yi; Huang, Shang-Pen; Huynh, Thanh-Tuan; Jan, Hsun-Jin; Whang-Peng, Jacqueline; Chiou, Jeng-Fong; Wu, Alexander T.H.; Hsiao, Michael

    2016-01-01

    Standard interventions for glioma include surgery, radiation and chemotherapies but the prognosis for malignant cases such as glioblastoma multiforme remain grim. Even with targeted therapeutic agent, bevacitumab, malignant glioma often develops resistance and recurrence. Thus, developing alternative interventions (therapeutic targets, biomarkers) is urgently required. Bruton's tyrosine kinase (Btk) has been long implicated in B cell malignancies but surprisingly it has recently been shown to also play a tumorigenic role in solid tumors such as ovarian and prostate cancer. Bioinformatics data indicates that Btk is significantly higher in clinical glioma samples as compared to normal brain cells and Btk expression level is associated with stage progression. This prompts us to investigate the potential role of Btk as a therapeutic target for glioma. Here, we demonstrate Btk expression is associated with GBM tumorigenesis. Down-regulation of Btk in GBM cell lines showed a significantly reduced abilities in colony formation, migration and GBM sphere-forming potential. Mechanistically, Btk-silenced cells showed a concomitant reduction in the expression of CD133 and Akt/mTOR signaling. In parallel, Ibrutinib (a Btk inhibitor) treatment led to a similar anti-tumorigenic response. Using xenograft mouse model, tumorigenesis was significantly reduced in Btk-silenced or ibrutinib-treated mice as compared to control counterparts. Finally, our glioma tissue microarray analysis indicated a higher Btk staining in the malignant tumors than less malignant and normal brain tissues. Collectively, Btk may represent a novel therapeutic target for glioma and ibrunitib may be used as an adjuvant treatment for malignant GBM. PMID:27564106

  9. Quercetin-induced downregulation of phospholipase D1 inhibits proliferation and invasion in U87 glioma cells

    Park, Mi Hee [Department of Molecular Biology, College of Natural Science, Pusan National University, 30 Jangjeon dong, Geumjeong gu, Busan 609-735 (Korea, Republic of); Min, Do Sik, E-mail: minds@pusan.ac.kr [Department of Molecular Biology, College of Natural Science, Pusan National University, 30 Jangjeon dong, Geumjeong gu, Busan 609-735 (Korea, Republic of)

    2011-09-09

    Highlights: {yields} Quercetin, a bioactive flavonoid, suppresses expression and enzymatic activity of phospholipase D1. {yields} Quercetin abolishes NFkB-induced phospholipase D1 expression via inhibition of NFkB transactivation. {yields} Quercetin-induced suppression of phospholipase D1 inhibits invasion and proliferation of human glioma cells. -- Abstract: Phospholipase D (PLD) has been recognized as a regulator of cell proliferation and tumorigenesis, but little is known about the molecules regulating PLD expression. Thus, the identification of small molecules inhibiting PLD expression would be an important advance in PLD-mediated physiology. Quercetin, a ubiquitous bioactive flavonoid, is known to inhibit proliferation and induce apoptosis in a variety of cancer cells. In the present study, we examined the effect of quercetin on the expression of PLD in U87 glioma cells. Quercetin significantly suppressed the expression of PLD1 at the transcriptional level. Moreover, quercetin abolished the protein expression of PLD1 in a time and dose-dependent manner, as well as inhibited PLD activity. Quercetin suppressed NF{kappa}B-induced PLD1 expression via inhibition of NFkB transactivation. Furthermore, quercetin inhibited activation and invasion of metalloproteinase-2 (MMP-2), a key modulator of glioma cell invasion, induced by phosphatidic acid (PA), a product of PLD activity. Taken together these data demonstrate that quercetin abolishes PLD1 expression and subsequently inhibits invasion and proliferation of glioma cells.

  10. Conditioned Medium from Adipose-Derived Stem Cells (ADSCs) Promotes Epithelial-to-Mesenchymal-Like Transition (EMT-Like) in Glioma Cells In vitro.

    Iser, Isabele C; Ceschini, Stefanie M; Onzi, Giovana R; Bertoni, Ana Paula S; Lenz, Guido; Wink, Márcia R

    2016-12-01

    Mesenchymal stem cells (MSCs) have recently been described to home to brain tumors and to integrate into the tumor-associated stroma. Understanding the communication between cancer cells and MSCs has become fundamental to determine whether MSC-tumor interactions should be exploited as a vehicle for therapeutic agents or considered a target for intervention. Therefore, we investigated whether conditioned medium from adipose-derived stem cells (ADSCs-CM) modulate glioma tumor cells by analyzing several cell biology processes in vitro. C6 rat glioma cells were treated with ADSCs-CM, and cell proliferation, cell cycle, cell viability, cell morphology, adhesion, migration, and expression of epithelial-mesenchymal transition (EMT)-related surface markers were analyzed. ADSCs-CM did not alter cell viability, cell cycle, and growth rate of C6 glioma cells but increased their migratory capacity. Moreover, C6 cells treated with ADSC-CM showed reduced adhesion and underwent changes in cell morphology. Up-regulation of EMT-associated markers (vimentin, MMP2, and NRAS) was also observed following treatment with ADSC-CM. Our findings demonstrate that the paracrine factors released by ADSCs are able to modulate glioma cell biology. Therefore, ADSC-tumor cell interactions in a tumor microenvironment must be considered in the design of clinical application of stem cell therapy. Graphical Abstract Factors released by adipose-derived stem cells (ADSCs) may modulate the biology of C6 glioma cells. When C6 cells are exposed to a conditioned medium from adipose-derived stem cells (ADSCs-CM), some of these cells can undergo an EMT-like process and trans-differentiate into cells with a more mesenchymal phenotype, characterized by enhanced expression of EMT-related surface markers, reduced cell adhesion capacity, increased migratory capacity, as well as changes in cell and nuclei morphology.

  11. CD133+ and Nestin+ Glioma Stem-Like Cells Reside Around CD31+ Arterioles in Niches that Express SDF-1α, CXCR4, Osteopontin and Cathepsin K

    Hira, Vashendriya V. V.; Ploegmakers, Kimberley J.; Grevers, Frederieke; Verbovšek, Urška; Silvestre-Roig, Carlos; Aronica, Eleonora; Tigchelaar, Wikky; Turnšek, Tamara Lah; Molenaar, Remco J.; van Noorden, Cornelis J. F.

    2015-01-01

    Poor survival of high-grade glioma is at least partly caused by glioma stem-like cells (GSLCs) that are resistant to therapy. GSLCs reside in niches in close vicinity of endothelium. The aim of the present study was to characterize proteins that may be functional in the GSLC niche by performing

  12. Radiosensitive effect of hypoxia-inducible factor 1α inhibitor YC-1 on hypoxic glioma SHG44 cell line

    Guo Xinwei; Lu Xueguan; Tong Liumei; Zong Tianzhou; Chen Liesong

    2011-01-01

    Objective: To investigate the radiosensitive effect of hypoxia-inducible factor 1α (HIF-1α) inhibitor YC-1 on hypoxic glioma SHG44 cell line and its related mechanism. Methods: Glioma SHG44 cell line was cultured in normoxic (20% O 2 ), continuous hypoxia (1% O 2 ) for 12 h and 24 h, continuous hypoxia plus YC-1 was performed for 12 h and 24 h, respectively. The expression of HIF-1α was assessed by Western blot. The radiosensitivity was evaluated by the survival curve, and the sublethal damage repair (SLDR) ability was measured by dose-fraction experiment. Results: HIF-1α protein levels of glioma SHG44 cells were significantly increased after hypoxic cultures for 12 h and 24 h than those of the corresponding cells cultured in normoxic, while the radiosensitivity was lower. The OER (oxygen-enhancement ratio) of SHG44 cells in hypoxia for 12 h and 24 h were 1.22 and 1.37, respectively. By the further statistical analysis it was found that SLDR ability of glioma SHG44 was increased at hypoxia, and when irradiation was carried one at the interval of 8, 10, 12 h it was statistically significant (P<0.05). HIF-1α protein levels of glioma SHG44 cells cultured in hypoxia plus YC-1 for 12 h and 24 h were decreased significantly compared to the corresponding cells cultured in hypoxia only, while the radiosensitivity was significantly increased. the EF (enhancement factor) of YC-1 for glioma SHG44 cells at hypoxia for 12 h and 24 h was 1.27. By the further statistical analysis it was also found that SLDR ability was decreased significantly for hypoxic SHG44 cells which was co-cultured with YC-1, and at the interval of 8, 10, 12 h irradiation was statistically significant (P<0.05). Conclusion: YC-1 can increase the radiosensitivity of hypoxic glioma SHG44 cell line, and its mechanism is related to SLDR inhibited by YC-1. (authors)

  13. The Effect of Gabapentin and Tramadol in Cancer Pain Induced by Glioma Cell in Rat Femur.

    Corona-Ramos, Janette Nallely; Déciga-Campos, Myrna; Romero-Piña, Mario; Medina, Luis A; Martínez-Racine, Issac; Jaramillo-Morales, Osmar A; García-López, Patricia; López-Muñoz, Francisco Javier

    2017-08-01

    Preclinical Research The presence of pain as part of the cancer process is variable. Glioblastoma multiform (GBM) can produce bone metastasis, a condition that involves other pathological phenotypes including neuropathic and inflammatory pain. Tramadol and gabapentin are drugs used in the treatment of neuropathic pain. However, there are no studies evaluating their analgesic effects in bone metastasis. We produced a pain model induced by the inoculation of glioma cells (10 5 ) into the rat femur, by perforating the intercodiloid fossa. Painful behavior was evaluated by measuring mechanical allodynia using the Von Frey test while thermal hyperalgesia was assessed in the plantar test. Histopathological features were evaluated and antinociceptive responses were compared using tramadol and gabapentin. The inoculation of cells inside the right femur produced nociceptive behaviors. Tramadol and gabapentin produced an anti-allodynic effect in this condition, but tramadol did not produce an anti-hyperalgesic response. The development of this model will allow us to perform tests to elucidate the pathology of bone metastasis, cancer pain, and in particular the pain produced by glioma. Drug Dev Res 78 : 173-183, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  14. Nrf2 is required to maintain the self-renewal of glioma stem cells

    Zhu, Jianhong; Wang, Handong; Sun, Qing; Ji, Xiangjun; Zhu, Lin; Cong, Zixiang; Zhou, Yuan; Liu, Huandong; Zhou, Mengliang

    2013-01-01

    Glioblastomas are deadly cancers that display a functional cellular hierarchy maintained by self-renewing glioma stem cells (GSCs). Self-renewal is a complex biological process necessary for maintaining the glioma stem cells. Nuclear factor rythroid 2-related factor 2(Nrf2) plays a significant role in protecting cells from endogenous and exogenous stresses. Nrf2 is a key nuclear transcription factor that regulates antioxidant response element (ARE)-containing genes. Previous studies have demonstrated the significant role of Nrf2 in the proliferation of glioblastoma, and in their resistance to radioactive therapies. We examined the effect of knocking down Nrf2 in GSCs. Nrf2 expression was down-regulated by shRNA transinfected with lentivirus. Expression levels of Nestin, Nrf2, BMI-1, Sox2 and Cyclin E were assessed by western blotting, quantitative polymerase chain reaction (qPCR) and immunohistochemistry analysis. The capacity for self-renewal in vitro was assessed by genesis of colonies. The capacity for self-renewal in vivo was analyzed by tumor genesis of xenografts in nude mice. Knockdown of Nrf2 inhibited the proliferation of GSCs, and significantly reduced the expression of BMI-1, Sox2 and CyclinE. Knocking down of Nrf2 changed the cell cycle distribution of GSCs by causing an uncharacteristic increase in the proportion of cells in the G2 phase and a decrease in the proportion of cells in the S phase of the cell cycle. Nrf2 is required to maintain the self-renewal of GSCs, and its down-regulation can attenuate the self-renewal of GSCs significantly

  15. Influence of drugs on gap junctions in glioma cell lines and primary astrocytes in vitro

    Zahra eMoinfar

    2014-05-01

    Full Text Available Gap junctions (GJs are hemichannels on cell membrane. Once they are intercellulary connected to the neighboring cells, they build a functional syncytium which allows rapid transfer of ions and molecules between cells. This characteristic makes GJs a potential modulator in proliferation, migration and development of the cells. So far, several types of GJs are recognized on different brain cells as well as in glioma. Astrocytes, as one of the major cells that maintain neuronal homeostasis, express different types of GJs that let them communicate with neurons, oligodendrocytes and endothelial cells of the blood brain barrier; however, the main GJ in astrocytes is connexin 43. There are different cerebral diseases in which astrocyte GJs might play a role. Several drugs have been reported to modulate gap junctional communication in the brain which can consequently have beneficial or detrimental effects on the course of treatment in certain diseases. However, the exact cellular mechanism behind those pharmaceutical efficacies on GJs is not well-understood. Accordingly, how specific drugs would affect GJs and what some consequent specific brain diseases would be are the interests of the authors of this chapter. We would focus on pharmaceutical effects on GJs on astrocytes in specific diseases where GJs could possibly play a role including: 1 migraine and a novel therapy for migraine with aura, 2 neuroautoimmune diseases and immunomodulatory drugs in the treatment of demyelinating diseases of the central nervous system such as multiple sclerosis, 3 glioma and antineoplastic and anti-inflammatory agents that are used in treating brain tumors and 4 epilepsy and anticonvulsants that are widely used for seizures therapy. All of the above-mentioned therapeutic categories can possibly affect GJs expression of astrocytes and the role is discussed in the upcoming chapter.

  16. Poly(ADP-ribose) polymerase-independent potentiation of nitrosourea cytotoxicity by 3-aminobenzamide in human malignant glioma cells.

    Winter, S; Weller, M

    2000-06-16

    Poly(ADP-ribose) polymerase is a zinc-finger DNA-binding protein that detects specifically DNA strand breaks generated by genotoxic agents and is thought to be involved in DNA repair. Here, we examined the effects of 3-aminobenzamide, a poly(ADP-ribose) polymerase inhibitor, on the chemosensitivity of human malignant glioma cells. 3-Aminobenzamide selectively potentiated the cytotoxicity of the nitrosoureas, nimustine, carmustine and lomustine in 10 of 12 human malignant glioma cell lines. In contrast, 3-aminobenzamide did not modulate the cytotoxic effects of doxorubicine, teniposide, vincristine, camptothecin or cytarabine. The nitrosoureas did not induce poly(ADP-ribose) polymerase activity in the glioma cells. Ectopic expression of truncated poly(ADP-ribose) polymerase containing the poly(ADP-ribose) polymerase DNA-binding domain, which acts as a dominant-negative mutant, in LN-18 or LN-229 cells did not alter the 3-aminobenzamide effect on nitrosourea-mediated cytotoxicity. Thus, 3-aminobenzamide may target another nicotinamide adenine dinucleotide (NAD)-requiring enzyme, but not poly(ADP-ribose) polymerase, when enhancing nitrosourea cytotoxicity in human malignant glioma cells. Carmustine cytotoxicity was associated with a G2/M arrest. Coexposure to carmustine and 3-aminobenzamide overcame this G2/M arrest in T98G cells, which are sensitized to carmustine by 3-aminobenzamide, but not in U251MG cells, which are refractory to 3-aminobenzamide-mediated sensitization to carmustine. Thus, 3-aminobenzamide-mediated sensitization to carmustine cytotoxicity may result from interference with the stable G2/M arrest response to carmustine in human glioma cells.

  17. Effect of SOCS1 overexpression on RPE cell activation by proinflammatory cytokines.

    Bazewicz, Magdalena; Draganova, Dafina; Makhoul, Maya; Chtarto, Abdel; Elmaleh, Valerie; Tenenbaum, Liliane; Caspers, Laure; Bruyns, Catherine; Willermain, François

    2016-09-06

    The purpose of this study was to investigate the in vitro effect of Suppressor Of Cytokine Signaling 1 (SOCS1) overexpression in retinal pigment epithelium (RPE) cells on their activation by pro-inflammatory cytokines IFNγ, TNFα and IL-17. Retinal pigment epithelium cells (ARPE-19) were stably transfected with the control plasmid pIRES2-AcGFP1 or the plasmid pSOCS1-IRES2-AcGFP1. They were stimulated by IFNγ (150ng/ml), TNFα (30ng/ml) or IL-17 (100ng/ml). The levels of SOCS1 mRNA were measured by real-time PCR. Signal Transducer and Activator of Transcription 1 (STAT1) phosphorylation and IκBα expression were analysed by western Blot (WB). IL-8 secretion was analysed by ELISA and expression of MHCII molecules and ICAM-1/CD54 by flow cytometry. Our data show that SOCS1 mRNA overexpression in RPE cells prevents IFNγ-induced SOCS1 mRNA increase and IFNγ-mediated STAT1 phosphorylation. Moreover, SOCS1 overexpression in RPE cells inhibits IFNγ-induced decrease of IL-8 secretion and prevents IFNγ-induced MHC II and ICAM1/CD54 upregulation. However, SOCS1 overexpression does not affect TNFα-induced IκBα degradation nor block TNFα-induced or IL-17-induced IL-8 secretion. On the contrary, IL-17-induced secretion is increased by SOCS1 overexpression. In conclusion, SOCS1 overexpression in RPE cells inhibits some IFNγ-mediated responses that lead to uveitis development. This notion raises the possibility that SOCS1 overexpression could be a novel target for treating non-infectious uveitis. However, some proinflammatory effects of TNFα and IL-17 stimulation on RPE are not blocked by SOCS1 overexpression. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  18. The experimental investigation of glioma-trophic capacity of human umbilical cord-derived mesenchymal stem cells after intraventricular administration

    FAN Cun-gang

    2013-07-01

    Full Text Available Objective To explore the glioma-trophic migration capacity of human umbilical cord-derived mesenchymal stem cells (hUC-MSCs by intraventricular administration. Methods The umbilical cord tissue were obtained during full-term pregnancy cesarean section under sterile conditions. This study was approved by Ethics Committee and got the informed consent of patient. The hUC-MSCs were isolated by trypsin and collagenase digestion, followed by adherent culture methods. The characteristics of isolated hUC-MSCs were demonstrated by cell morphylogy, phenotype analysis and multi-differentiation potentials into adipocytes, osteoblasts and neural cells. Then the hUC-MSCs were labeled with CM-DiI and injected into contralateral ventricle of glioma of the C6 glioma-bearing Sprague-Dawley (SD rats. Two weeks later, the rats were sacrificed and the brains were taken out to examine the migration and distribution of hUC-MSCs in the tumor bed, at the interface of tumor and cerebral parenchyma as well as the tumor satelites infiltrating into the normal brain. Results The hUC-MSCs demonstrated plastic-adherent characterization and homogeneous fibroblastic-like morphylogy in culture, expression of specific surface phenotypes of MSCs (CD13, CD29, CD44, CD90 but not endothelial or hematopoietic markers (CD14, CD31, CD34, CD38, CD45, CD133, and muti-differentiatiation potentials into Oil red O stained adipocytes, Alizarin red S stained osteoblasts, neuron-specific enolase (NSE-positive neurons and glial fibrillary acidic protein (GFAP-positive astrocytes in permissive inducive conditions. Importantly, after labeled hUC-MSCs injection into contralateral ventricle of glioma, the hUC-MSCs migrated from initial injection site to the glioma mass and along the interface of tumor and brain, and some of them "chasing" the glioma satellites infiltrated into the normal parenchyma. Conclusion The hUC-MSCs possess prominent tumor-specific targeting capacity and extensive intratumoral

  19. β-Elemene Selectively Inhibits the Proliferation of Glioma Stem-Like Cells Through the Downregulation of Notch1.

    Feng, Hai-Bin; Wang, Jing; Jiang, Hao-Ran; Mei, Xin; Zhao, Yi-Ying; Chen, Fu-Rong; Qu, Yue; Sai, Ke; Guo, Cheng-Cheng; Yang, Qun-Ying; Zhang, Zong-Ping; Chen, Zhong-Ping

    2017-03-01

    Glioma is the most frequent primary central nervous system tumor. Although the current first-line medicine, temozolomide (TMZ), promotes patient survival, drug resistance develops easily. Thus, it is important to investigate novel therapeutic reagents to solidify the treatment effect. β-Elemene (bELE) is a compound from a Chinese herb whose anticancer effect has been shown in various types of cancer. However, its role in the inhibition of glioma stem-like cells (GSLCs) has not yet been reported. We studied both the in vitro and the in vivo inhibitory effect of bELE and TMZ in GSLCs and parental cells and their combined effects. The molecular mechanisms were also investigated. We also optimized the delivery methods of bELE. We found that bELE selectively inhibits the proliferation and sphere formation of GSLCs, other than parental glioma cells, and TMZ exerts its effects on parental cells instead of GSLCs. The in vivo data confirmed that the combination of bELE and TMZ worked better in the xenografts of GSLCs, mimicking the situation of tumorigenesis of human cancer. Notch1 was downregulated with bELE treatment. Our data also demonstrated that the continuous administration of bELE produces an ideal effect to control tumor progression. Our findings have demonstrated, for the first time, that bELE could compensate for TMZ to kill both GSLCs and nonstem-like cancer cells, probably improving the prognosis of glioma patients tremendously. Notch1 might be a downstream target of bELE. Therefore, our data shed light on improving the outcomes of glioma patients by combining bELE and TMZ. Stem Cells Translational Medicine 2017;6:830-839. © 2016 The Authors Stem Cells Translational Medicine published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.

  20. In vivo detection of c-Met expression in a rat C6 glioma model.

    Towner, R A; Smith, N; Doblas, S; Tesiram, Y; Garteiser, P; Saunders, D; Cranford, R; Silasi-Mansat, R; Herlea, O; Ivanciu, L; Wu, D; Lupu, F

    2008-01-01

    The tyrosine kinase receptor, c-Met, and its substrate, the hepatocyte growth factor (HGF), are implicated in the malignant progression of glioblastomas. In vivo detection of c-Met expression may be helpful in the diagnosis of malignant tumours. The C6 rat glioma model is a widely used intracranial brain tumour model used to study gliomas experimentally. We used a magnetic resonance imaging (MRI) molecular targeting agent to specifically tag the cell surface receptor, c-Met, with an anti-c-Met antibody (Ab) linked to biotinylated Gd (gadolinium)-DTPA (diethylene triamine penta acetic acid)-albumin in rat gliomas to detect overexpression of this antigen in vivo. The anti-c-Met probe (anti-c-Met-Gd-DTPA-albumin) was administered intravenously, and as determined by an increase in MRI signal intensity and a corresponding decrease in regional T(1) relaxation values, this probe was found to detect increased expression of c-Met protein levels in C6 gliomas. In addition, specificity for the binding of the anti-c-Met contrast agent was determined by using fluorescence microscopic imaging of the biotinylated portion of the targeting agent within neoplastic and 'normal'brain tissues following in vivo administration of the anti-c-Met probe. Controls with no Ab or with a normal rat IgG attached to the contrast agent component indicated no non-specific binding to glioma tissue. This is the first successful visualization of in vivo overexpression of c-Met in gliomas.

  1. NY-ESO-1- and survivin-specific T-cell responses in the peripheral blood from patients with glioma

    Liu, Zhenjiang; Poiret, Thomas; Persson, Oscar

    2018-01-01

    The prognosis for patients with glioblastoma is grim. Ex vivo expanded tumor-associated antigen (TAA)-reactive T-cells from patients with glioma may represent a viable source for anticancer-directed cellular therapies. Immunohistochemistry was used to test the survivin (n = 40 samples) and NY-ESO...

  2. Effect of fluoxetine and adenosine receptor NECA agonist on G alpha q/11 protein of C6 glioma cells

    Kovářů, H.; Kovářů, F.; Lisá, Věra

    2012-01-01

    Roč. 33, č. 6 (2012), s. 614-618 ISSN 0172-780X Institutional support: RVO:67985823 Keywords : C6 glioma cells * SSRI antidepressant * G alpha q/11 signalling * G protein coupled receptor Subject RIV: ED - Physiology Impact factor: 0.932, year: 2012

  3. The change of p16 gene expression in glioma cell line C6 after radiation with gamma knife

    Zhao Xingli; Zhao Conghai; Tian Yu

    2002-01-01

    Objective: T observe the change of expression of p16 gene product, P16 protein, after treated by gamma knife on glioma cell line C6. Methods: Glioma C6 cells proliferated in vitro, treated by γ-knife in dose of 5.00 and 6.22 Gy, respectively. P16 protein was detected by immunohistochemical technique and image analysis. Results: The P16 protein in glioma C6 cells was notably increased after treatment with γ knife (P < 0.01). The grey number in C6 group (control group) was 167.1 +- 6.2 and was 155.4 +- 2.0 and 124.9 +- 7.1, respectively, in 5.00 Gy and 6.22 Gy gamma knife treated group. Conclusion: It is suggests that one of the mechanisms of glioma cell C6 apoptosis induced by γ-knife radiation may be associated with activation of p16 gene and increase of P16 protein expression

  4. 'Dipeptidyl peptidase-IV activity and/or structure homologs' (DASH) in growth-modulated glioma cell lines

    Šedo, Aleksi; Bušek, P.; Scholzová, E.; Malík, Radek; Vlašicová, K.; Janáčková, S.; Mareš, Vladislav

    2004-01-01

    Roč. 385, č. 6 (2004), s. 557-559 ISSN 1431-6730 R&D Projects: GA ČR GA301/02/0962 Institutional research plan: CEZ:AV0Z5011922 Keywords : brain tumors * glioma cells * DPP-IV Subject RIV: FD - Oncology ; Hematology Impact factor: 3.598, year: 2004

  5. Cetuximab insufficiently inhibits glioma cell growth due to persistent EGFR downstream signaling

    Hasselbalch, Benedikte; Lassen, Ulrik; Poulsen, Hans S

    2010-01-01

    Overexpression and/or amplification of the epidermal growth factor receptor (EGFR) is present in 35-45% of primary glioblastoma multiforme tumors and has been correlated with a poor prognosis. In this study, we investigated the effect of cetuximab and intracellular signaling pathways downstream...... of EGFR, important for cell survival and proliferation. We show insufficient EGFR downregulation and competition with endogenous EGFR ligands upon cetuximab treatment. Dose-response experiments showed inhibition of EGFR phosphorylation without affecting two of the prominent downstream signaling pathways....... Our results indicate that amplification and/or overexpression of EGFR is an unsatisfactory predictor for response to cetuximab....

  6. β‐Elemene Selectively Inhibits the Proliferation of Glioma Stem‐Like Cells Through the Downregulation of Notch1

    Feng, Hai‐bin; Wang, Jing; Jiang, Hao‐ran; Mei, Xin; Zhao, Yi‐ying; Chen, Fu‐rong; Qu, Yue; Sai, Ke; Guo, Cheng‐cheng; Yang, Qun‐ying; Zhang, Zong‐ping

    2016-01-01

    Abstract Glioma is the most frequent primary central nervous system tumor. Although the current first‐line medicine, temozolomide (TMZ), promotes patient survival, drug resistance develops easily. Thus, it is important to investigate novel therapeutic reagents to solidify the treatment effect. β‐Elemene (bELE) is a compound from a Chinese herb whose anticancer effect has been shown in various types of cancer. However, its role in the inhibition of glioma stem‐like cells (GSLCs) has not yet been reported. We studied both the in vitro and the in vivo inhibitory effect of bELE and TMZ in GSLCs and parental cells and their combined effects. The molecular mechanisms were also investigated. We also optimized the delivery methods of bELE. We found that bELE selectively inhibits the proliferation and sphere formation of GSLCs, other than parental glioma cells, and TMZ exerts its effects on parental cells instead of GSLCs. The in vivo data confirmed that the combination of bELE and TMZ worked better in the xenografts of GSLCs, mimicking the situation of tumorigenesis of human cancer. Notch1 was downregulated with bELE treatment. Our data also demonstrated that the continuous administration of bELE produces an ideal effect to control tumor progression. Our findings have demonstrated, for the first time, that bELE could compensate for TMZ to kill both GSLCs and nonstem‐like cancer cells, probably improving the prognosis of glioma patients tremendously. Notch1 might be a downstream target of bELE. Therefore, our data shed light on improving the outcomes of glioma patients by combining bELE and TMZ. Stem Cells Translational Medicine 2017;6:830–839 PMID:28297578

  7. Quantitative proteomics and transcriptomics reveals metabolic differences in attracting and non-attracting human-in-mouse glioma stem cell xenografts and stromal cells

    Norelle C. Wildburger

    2015-09-01

    Full Text Available Bone marrow-derived human mesenchymal stem cells (BM-hMSCs show promise as cell-based delivery vehicles for anti-glioma therapeutics, due to innate tropism for gliomas. However, in clinically relevant human-in-mouse glioma stem cell xenograft models, BM-hMSCs tropism is variable. We compared the proteomic profile of cancer and stromal cells in GSCXs that attract BM-hMSCs (“attractors” with those to do not (“non-attractors” to identify pathways that may modulate BM-hMSC homing, followed by targeted transcriptomics. The results provide the first link between fatty acid metabolism, glucose metabolism, ROS, and N-glycosylation patterns in attractors. Reciprocal expression of these pathways in the stromal cells suggests microenvironmental cross-talk.

  8. Lactoferrin conjugated iron oxide nanoparticles for targeting brain glioma cells in magnetic particle imaging

    Tomitaka, Asahi; Arami, Hamed; Gandhi, Sonu; Krishnan, Kannan M.

    2015-10-01

    Magnetic Particle Imaging (MPI) is a new real-time imaging modality, which promises high tracer mass sensitivity and spatial resolution directly generated from iron oxide nanoparticles. In this study, monodisperse iron oxide nanoparticles with median core diameters ranging from 14 to 26 nm were synthesized and their surface was conjugated with lactoferrin to convert them into brain glioma targeting agents. The conjugation was confirmed with the increase of the hydrodynamic diameters, change of zeta potential, and Bradford assay. Magnetic particle spectrometry (MPS), performed to evaluate the MPI performance of these nanoparticles, showed no change in signal after lactoferrin conjugation to nanoparticles for all core diameters, suggesting that the MPI signal is dominated by Néel relaxation and thus independent of hydrodynamic size difference or presence of coating molecules before and after conjugations. For this range of core sizes (14-26 nm), both MPS signal intensity and spatial resolution improved with increasing core diameter of nanoparticles. The lactoferrin conjugated iron oxide nanoparticles (Lf-IONPs) showed specific cellular internalization into C6 cells with a 5-fold increase in MPS signal compared to IONPs without lactoferrin, both after 24 h incubation. These results suggest that Lf-IONPs can be used as tracers for targeted brain glioma imaging using MPI.

  9. δ-Opioid receptor-stimulated Akt signaling in neuroblastoma x glioma (NG108-15) hybrid cells involves receptor tyrosine kinase-mediated PI3K activation

    Heiss, Anika; Ammer, Hermann; Eisinger, Daniela A.

    2009-01-01

    δ-Opioid receptor (DOR) agonists possess cytoprotective properties, an effect associated with activation of the 'pro-survival' kinase Akt. Here we delineate the signal transduction pathway by which opioids induce Akt activation in neuroblastoma x glioma (NG108-15) hybrid cells. Exposure of the cells to both [D-Pen 2,5 ]enkephalin and etorphine resulted in a time- and dose-dependent increase in Akt activity, as measured by means of an activation-specific antibody recognizing phosphoserine-473. DOR-mediated Akt signaling is blocked by the opioid antagonist naloxone and involves inhibitory G i/o proteins, because pre-treatment with pertussis toxin, but not over-expression of the G q/11 scavengers EBP50 and GRK2-K220R, prevented this effect. Further studies with Wortmannin and LY294002 revealed that phophoinositol-3-kinase (PI3K) plays a central role in opioid-induced Akt activation. Opioids stimulate Akt activity through transactivation of receptor tyrosine kinases (RTK), because pre-treatment of the cells with inhibitors for neurotrophin receptor tyrosine kinases (AG879) and the insulin-like growth factor receptor IGF-1 (AG1024), but not over-expression of the Gβγ scavenger phosducin, abolished this effect. Activated Akt translocates to the nuclear membrane, where it promotes GSK3 phosphorylation and prevents caspase-3 cleavage, two key events mediating inhibition of cell apoptosis and enhancement of cell survival. Taken together, these results demonstrate that in NG108-15 hybrid cells DOR agonists possess cytoprotective properties mediated by activation of the RTK/PI3K/Akt signaling pathway.

  10. {delta}-Opioid receptor-stimulated Akt signaling in neuroblastoma x glioma (NG108-15) hybrid cells involves receptor tyrosine kinase-mediated PI3K activation

    Heiss, Anika; Ammer, Hermann [Institute of Pharmacology, Toxicology and Pharmacy Ludwig-Maximilians-University of Munich Koeniginstrasse 16 80539 Muenchen Federal Republic of Germany (Germany); Eisinger, Daniela A., E-mail: eisinger@pharmtox.vetmed.uni-muenchen.de [Institute of Pharmacology, Toxicology and Pharmacy Ludwig-Maximilians-University of Munich Koeniginstrasse 16 80539 Muenchen Federal Republic of Germany (Germany)

    2009-07-15

    {delta}-Opioid receptor (DOR) agonists possess cytoprotective properties, an effect associated with activation of the 'pro-survival' kinase Akt. Here we delineate the signal transduction pathway by which opioids induce Akt activation in neuroblastoma x glioma (NG108-15) hybrid cells. Exposure of the cells to both [D-Pen{sup 2,5}]enkephalin and etorphine resulted in a time- and dose-dependent increase in Akt activity, as measured by means of an activation-specific antibody recognizing phosphoserine-473. DOR-mediated Akt signaling is blocked by the opioid antagonist naloxone and involves inhibitory G{sub i/o} proteins, because pre-treatment with pertussis toxin, but not over-expression of the G{sub q/11} scavengers EBP50 and GRK2-K220R, prevented this effect. Further studies with Wortmannin and LY294002 revealed that phophoinositol-3-kinase (PI3K) plays a central role in opioid-induced Akt activation. Opioids stimulate Akt activity through transactivation of receptor tyrosine kinases (RTK), because pre-treatment of the cells with inhibitors for neurotrophin receptor tyrosine kinases (AG879) and the insulin-like growth factor receptor IGF-1 (AG1024), but not over-expression of the G{beta}{gamma} scavenger phosducin, abolished this effect. Activated Akt translocates to the nuclear membrane, where it promotes GSK3 phosphorylation and prevents caspase-3 cleavage, two key events mediating inhibition of cell apoptosis and enhancement of cell survival. Taken together, these results demonstrate that in NG108-15 hybrid cells DOR agonists possess cytoprotective properties mediated by activation of the RTK/PI3K/Akt signaling pathway.

  11. Cyclin D1 overexpression, cell cycle progression and radiosensitivity in MBP cells

    Wu Lijun; Yu Zengliang

    2000-11-01

    Clones that exhibited a minimum of 7-8 fold cyclin D1 level above the parent cell lines or the vector control were obtained after transfected with the entire coding sequence of human 1.1 kb cyclin D1 cDNA. Studies showed that there was no significant difference in Radiosensitivity between over-expressing cyclin D1 and control cultures from either mouse or human origin. Using flow cytometry to access cell cycle distribution in the exponentially growth cultures of MCF10F-D1-21 and MCF10F-V-3, it was found that there was a 50 percent increase in the proportion of G2/M phase cells and 5.3 percent decrease in the proportion of G0/G1 phase cells in MCF10F-D1-21 comparing with MCF10F-V-3, though they were with the same proportion of cells in S phase

  12. The discussion of method for survey the radiosensitivity of human glioma cell line SHG-44

    Li Li; Xu Changshao; Zhou Juying; Xu Xiaoting; Luo Jialin

    2005-01-01

    Objective: To investigate if thiazolyl blue colorimetric assay (MTT) and cell counting kit-8 (CCK-8) can replace clone forming assay for survey the radiosensitivity of SHG-44. Methods; Three assays was applied to examine the growth inhibition of human glioma cell line SHG-44 in eight dose groups of 0, 1, 2, 3, 4, 6, 8 and 10 Gy, and statistical research was applied to analyze the correlation between survival fraction and various doses. Results: Dose was associated with survival fraction in these three assays at some range of irradiation doseage (dose≤3 Gy). If out of the range, the relation is poor. CCK-8 has no rather superiority than MTT. Conclusion: By now clone forming assay is still the 'gold standard'. In some cases, MTT and other assays can give us some reference, but these assays still can not replace clone forming assay. (authors)

  13. Vascular regulation of glioma stem-like cells: a balancing act.

    Brooks, Lucy J; Parrinello, Simona

    2017-12-01

    Glioblastoma (GBM) are aggressive and therapy-resistant brain tumours driven by glioma stem-like cells (GSCs). GSC behaviour is controlled by the microenvironment, or niche, in which the cells reside. It is well-established that the vasculature is a key component of the GSC niche, which drives maintenance in the tumour bulk and invasion at the margin. Emerging evidence now indicates that the specific properties of the vasculature within these two regions impose different functional states on resident GSCs, generating distinct subpopulations. Here, we review these recent findings, focusing on the mechanisms that underlie GSC/vascular communication. We further discuss how plasticity enables GSCs to respond to vascular changes by interconverting bidirectionally between states, and address the therapeutic implications of this dynamic response. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

  14. Cell division cycle 20 overexpression predicts poor prognosis for patients with lung adenocarcinoma.

    Shi, Run; Sun, Qi; Sun, Jing; Wang, Xin; Xia, Wenjie; Dong, Gaochao; Wang, Anpeng; Jiang, Feng; Xu, Lin

    2017-03-01

    The cell division cycle 20, a key component of spindle assembly checkpoint, is an essential activator of the anaphase-promoting complex. Aberrant expression of cell division cycle 20 has been detected in various human cancers. However, its clinical significance has never been deeply investigated in non-small-cell lung cancer. By analyzing The Cancer Genome Atlas database and using some certain online databases, we validated overexpression of cell division cycle 20 in both messenger RNA and protein levels, explored its clinical significance, and evaluated the prognostic role of cell division cycle 20 in non-small-cell lung cancer. Cell division cycle 20 expression was significantly correlated with sex (p = 0.003), histological classification (p overexpression of cell division cycle 20 was significantly associated with bigger primary tumor size (p = 0.0023), higher MKI67 level (r = 0.7618, p Overexpression of cell division cycle 20 is associated with poor prognosis in lung adenocarcinoma patients, and its overexpression can also be used to identify high-risk groups. In conclusion, cell division cycle 20 might serve as a potential biomarker for lung adenocarcinoma patients.

  15. Silibinin induces apoptosis via calpain-dependent AIF nuclear translocation in U87MG human glioma cell death

    Kim Yong K

    2011-04-01

    Full Text Available Abstract Background Silibinin, a natural polyphenolic flavonoid, has been reported to induce cell death in various cancer cell types. However, the molecular mechanism is not clearly defined. Our previous study showed that silibinin induces glioma cell death and its effect was effectively prevented by calpain inhibitor. The present study was therefore undertaken to examine the role of calpain in the silibinin-induced glioma cell death. Methods U87MG cells were grown on well tissue culture plates and cell viability was measured by MTT assay. ROS generation and △ψm were estimated using the fluorescence dyes. PKC activation and Bax expression were measured by Western blot analysis. AIF nuclear translocation was determined by Western blot and immunocytochemistry. Results Silibinin induced activation of calpain, which was blocked by EGTA and the calpain inhibitor Z-Leu-Leu-CHO. Silibinin caused ROS generation and its effect was inhibited by calpain inhibitor, the general PKC inhibitor GF 109203X, the specific PKCδ inhibitor rottlerin, and catalase. Silibinin-induce cell death was blocked by calpain inhibitor and PKC inhibitors. Silibinin-induced PKCδ activation and disruption of △ψm were prevented by the calpain inhibitor. Silibinin induced AIF nuclear translocation and its effect was prevented by calpain inhibitor. Transfection of vector expressing microRNA of AIF prevented the silibinin-induced cell death. Conclusions Silibinin induces apoptotic cell death through a calpain-dependent mechanism involving PKC, ROS, and AIF nuclear translocation in U87MG human glioma cells.

  16. Lessons we learned from high-throughput and top-down systems biology analyses about glioma stem cells.

    Mock, Andreas; Chiblak, Sara; Herold-Mende, Christel

    2014-01-01

    A growing body of evidence suggests that glioma stem cells (GSCs) account for tumor initiation, therapy resistance, and the subsequent regrowth of gliomas. Thus, continuous efforts have been undertaken to further characterize this subpopulation of less differentiated tumor cells. Although we are able to enrich GSCs, we still lack a comprehensive understanding of GSC phenotypes and behavior. The advent of high-throughput technologies raised hope that incorporation of these newly developed platforms would help to tackle such questions. Since then a couple of comparative genome-, transcriptome- and proteome-wide studies on GSCs have been conducted giving new insights in GSC biology. However, lessons had to be learned in designing high-throughput experiments and some of the resulting conclusions fell short of expectations because they were performed on only a few GSC lines or at one molecular level instead of an integrative poly-omics approach. Despite these shortcomings, our knowledge of GSC biology has markedly expanded due to a number of survival-associated biomarkers as well as glioma-relevant signaling pathways and therapeutic targets being identified. In this article we review recent findings obtained by comparative high-throughput analyses of GSCs. We further summarize fundamental concepts of systems biology as well as its applications for glioma stem cell research.

  17. Amplification and overexpression of aurora kinase A (AURKA) in immortalized human ovarian epithelial (HOSE) cells.

    Chung, C M; Man, C; Jin, Y; Jin, C; Guan, X Y; Wang, Q; Wan, T S K; Cheung, A L M; Tsao, S W

    2005-07-01

    Immortalization is an early and essential step of human carcinogenesis. Amplification of chromosome 20q has been shown to be a common event in immortalized cells and cancers. We have previously reported that gain and amplification of chromosome 20q is a non-random and common event in immortalized human ovarian surface epithelial (HOSE) cells. The chromosome 20q harbors genes including TGIF2 (20q11.2-q12), AIB1 (20q12), PTPN1 (20q13.1), ZNF217 (20q13.2), and AURKA (20q13.2-q13.3), which were previously reported to be amplified and overexpressed in ovarian cancers. Some of these genes may be involved in immortalization of HOSE cells and represent crucial premalignant changes in ovarian surface epithelium. Investigation of the involvement of these genes was examined in four pairs of pre-crisis (preimmortalized) and post-crisis (immortalized) HOSE cells. Overexpression of AURKA (Aurora kinase A), also known as BTAK and STK15, by both real time-quantitative polymerase chain reaction (RT-QPCR) and Western blotting was detected in all the four immortalized HOSE cells examined while overexpression of AIB1 and ZNF217 was observed in two of four immortalized HOSE cells examined. Overexpression of TGIF2 and PTPN1 was not significant in our immortalized HOSE cell systems. The degree of overexpression of AURKA was shown to be closely associated with the amplification of chromosome 20q in immortalized HOSE cells. Fluorescence in situ hybridization (FISH) with labeled P1 artificial clone (PAC) confirmed the amplification of the chromosomal region (20q13.2-13.3) where AURKA resides. DNA amplification of AURKA was also confirmed using semi-quantitative PCR. Our study showed that amplification and overexpression of AURKA is a common and significant event during immortalization of HOSE cells and may represent an important premalignant change in ovarian carcinogenesis. Copyright (c) 2005 Wiley-Liss, Inc.

  18. SHMT2 drives glioma cell survival in ischaemia but imposes a dependence on glycine clearance.

    Kim, Dohoon; Fiske, Brian P; Birsoy, Kivanc; Freinkman, Elizaveta; Kami, Kenjiro; Possemato, Richard L; Chudnovsky, Yakov; Pacold, Michael E; Chen, Walter W; Cantor, Jason R; Shelton, Laura M; Gui, Dan Y; Kwon, Manjae; Ramkissoon, Shakti H; Ligon, Keith L; Kang, Seong Woo; Snuderl, Matija; Vander Heiden, Matthew G; Sabatini, David M

    2015-04-16

    Cancer cells adapt their metabolic processes to support rapid proliferation, but less is known about how cancer cells alter metabolism to promote cell survival in a poorly vascularized tumour microenvironment. Here we identify a key role for serine and glycine metabolism in the survival of brain cancer cells within the ischaemic zones of gliomas. In human glioblastoma multiforme, mitochondrial serine hydroxymethyltransferase (SHMT2) and glycine decarboxylase (GLDC) are highly expressed in the pseudopalisading cells that surround necrotic foci. We find that SHMT2 activity limits that of pyruvate kinase (PKM2) and reduces oxygen consumption, eliciting a metabolic state that confers a profound survival advantage to cells in poorly vascularized tumour regions. GLDC inhibition impairs cells with high SHMT2 levels as the excess glycine not metabolized by GLDC can be converted to the toxic molecules aminoacetone and methylglyoxal. Thus, SHMT2 is required for cancer cells to adapt to the tumour environment, but also renders these cells sensitive to glycine cleavage system inhibition.

  19. Regulation of DNA repair mechanism in human glioma xenograft cells both in vitro and in vivo in nude mice.

    Ponnala, Shivani; Veeravalli, Krishna Kumar; Chetty, Chandramu; Dinh, Dzung H; Rao, Jasti S

    2011-01-01

    Glioblastoma Multiforme (GBM) is the most lethal form of brain tumor. Efficient DNA repair and anti-apoptotic mechanisms are making glioma treatment difficult. Proteases such as MMP9, cathepsin B and urokinase plasminogen activator receptor (uPAR) are over expressed in gliomas and contribute to enhanced cancer cell proliferation. Non-homologous end joining (NHEJ) repair mechanism plays a major role in double strand break (DSB) repair in mammalian cells. Here we show that silencing MMP9 in combination with uPAR/cathepsin B effects NHEJ repair machinery. Expression of DNA PKcs and Ku70/80 at both mRNA and protein levels in MMP9-uPAR (pMU) and MMP9-cathepsin B (pMC) shRNA-treated glioma xenograft cells were reduced. FACS analysis showed an increase in apoptotic peak and proliferation assays revealed a significant reduction in the cell population in pMU- and pMC-treated cells compared to untreated cells. We hypothesized that reduced NHEJ repair led to DSBs accumulation in pMU- and pMC-treated cells, thereby initiating cell death. This hypothesis was confirmed by reduced Ku70/Ku80 protein binding to DSB, increased comet tail length and elevated γH2AX expression in treated cells compared to control. Immunoprecipitation analysis showed that EGFR-mediated lowered DNA PK activity in treated cells compared to controls. Treatment with pMU and pMC shRNA reduced the expression of DNA PKcs and ATM, and elevated γH2AX levels in xenograft implanted nude mice. Glioma cells exposed to hypoxia and irradiation showed DSB accumulation and apoptosis after pMU and pMC treatments compared to respective controls. Our results suggest that pMU and pMC shRNA reduce glioma proliferation by DSB accumulation and increase apoptosis under normoxia, hypoxia and in combination with irradiation. Considering the radio- and chemo-resistant cancers favored by hypoxia, our study provides important therapeutic potential of MMP9, uPAR and cathepsin B shRNA in the treatment of glioma from clinical stand

  20. Regulation of DNA repair mechanism in human glioma xenograft cells both in vitro and in vivo in nude mice.

    Shivani Ponnala

    Full Text Available Glioblastoma Multiforme (GBM is the most lethal form of brain tumor. Efficient DNA repair and anti-apoptotic mechanisms are making glioma treatment difficult. Proteases such as MMP9, cathepsin B and urokinase plasminogen activator receptor (uPAR are over expressed in gliomas and contribute to enhanced cancer cell proliferation. Non-homologous end joining (NHEJ repair mechanism plays a major role in double strand break (DSB repair in mammalian cells.Here we show that silencing MMP9 in combination with uPAR/cathepsin B effects NHEJ repair machinery. Expression of DNA PKcs and Ku70/80 at both mRNA and protein levels in MMP9-uPAR (pMU and MMP9-cathepsin B (pMC shRNA-treated glioma xenograft cells were reduced. FACS analysis showed an increase in apoptotic peak and proliferation assays revealed a significant reduction in the cell population in pMU- and pMC-treated cells compared to untreated cells. We hypothesized that reduced NHEJ repair led to DSBs accumulation in pMU- and pMC-treated cells, thereby initiating cell death. This hypothesis was confirmed by reduced Ku70/Ku80 protein binding to DSB, increased comet tail length and elevated γH2AX expression in treated cells compared to control. Immunoprecipitation analysis showed that EGFR-mediated lowered DNA PK activity in treated cells compared to controls. Treatment with pMU and pMC shRNA reduced the expression of DNA PKcs and ATM, and elevated γH2AX levels in xenograft implanted nude mice. Glioma cells exposed to hypoxia and irradiation showed DSB accumulation and apoptosis after pMU and pMC treatments compared to respective controls.Our results suggest that pMU and pMC shRNA reduce glioma proliferation by DSB accumulation and increase apoptosis under normoxia, hypoxia and in combination with irradiation. Considering the radio- and chemo-resistant cancers favored by hypoxia, our study provides important therapeutic potential of MMP9, uPAR and cathepsin B shRNA in the treatment of glioma from

  1. Overexpression of Rac1 in leukemia patients and its role in leukemia cell migration and growth

    Wang, Jiying; Rao, Qing; Wang, Min; Wei, Hui; Xing, Haiyan; Liu, Hang; Wang, Yanzhong; Tang, Kejing; Peng, Leiwen; Tian, Zheng; Wang, Jianxiang

    2009-01-01

    Rac1 belongs to the Rho family that act as critical mediators of signaling pathways controlling cell migration and proliferation and contributes to the interactions of hematopoietic stem cells with their microenvironment. Alteration of Rac1 might result in unbalanced interactions and ultimately lead to leukemogenesis. In this study, we analyze the expression of Rac1 protein in leukemia patients and determine its role in the abnormal behaviours of leukemic cells. Rac1 protein is overexpressed in primary acute myeloid leukemia cells as compared to normal bone marrow mononuclear cells. siRNA-mediated silencing of Rac1 in leukemia cell lines induced inhibition of cell migration, proliferation, and colony formation. Additionally, blocking Rac1 activity by an inhibitor of Rac1-GTPase, NSC23766, suppressed cell migration and growth. We conclude that overexpression of Rac1 contributes to the accelerated migration and high proliferation potential of leukemia cells, which could be implicated in leukemia development and progression.

  2. Overexpression of Rac1 in leukemia patients and its role in leukemia cell migration and growth

    Wang, Jiying [State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, 288 Nanjing Road, Tianjin 300020 (China); Rao, Qing, E-mail: raoqing@gmail.com [State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, 288 Nanjing Road, Tianjin 300020 (China); Wang, Min; Wei, Hui; Xing, Haiyan; Liu, Hang; Wang, Yanzhong; Tang, Kejing; Peng, Leiwen; Tian, Zheng; Wang, Jianxiang [State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, 288 Nanjing Road, Tianjin 300020 (China)

    2009-09-04

    Rac1 belongs to the Rho family that act as critical mediators of signaling pathways controlling cell migration and proliferation and contributes to the interactions of hematopoietic stem cells with their microenvironment. Alteration of Rac1 might result in unbalanced interactions and ultimately lead to leukemogenesis. In this study, we analyze the expression of Rac1 protein in leukemia patients and determine its role in the abnormal behaviours of leukemic cells. Rac1 protein is overexpressed in primary acute myeloid leukemia cells as compared to normal bone marrow mononuclear cells. siRNA-mediated silencing of Rac1 in leukemia cell lines induced inhibition of cell migration, proliferation, and colony formation. Additionally, blocking Rac1 activity by an inhibitor of Rac1-GTPase, NSC23766, suppressed cell migration and growth. We conclude that overexpression of Rac1 contributes to the accelerated migration and high proliferation potential of leukemia cells, which could be implicated in leukemia development and progression.

  3. Cyclin-dependent kinase inhibitor 3 is overexpressed in hepatocellular carcinoma and promotes tumor cell proliferation

    Xing, Chunyang; Xie, Haiyang; Zhou, Lin; Zhou, Wuhua; Zhang, Wu; Ding, Songming; Wei, Bajin; Yu, Xiaobo; Su, Rong; Zheng, Shusen

    2012-01-01

    Highlights: ► CDKN3 is commonly overexpressed in HCC and is associated with poor clinical outcome. ► Overexpression of CDKN3 could stimulate the proliferation of HCC cells by promoting G1/S transition. ► CDKN3 could inhibit the expression of p21 in HCC cells. ► Overexpression of CDKN3 has no effect on apoptosis and invasion of HCC cells. ► We identified 61 genes co-expressed with CDKN3, and BIRC5 was located at the center of the co-expression network. -- Abstract: Cyclin-dependent kinase inhibitor 3 (CDKN3) belongs to the protein phosphatases family and has a dual function in cell cycling. The function of this gene has been studied in several kinds of cancers, but its role in human hepatocellular carcinoma (HCC) remains to be elucidated. In this study, we found that CDKN3 was frequently overexpressed in both HCC cell lines and clinical samples, and this overexpression was correlated with poor tumor differentiation and advanced tumor stage. Functional studies showed that overexpression of CDKN3 could promote cell proliferation by stimulating G1-S transition but has no impact on cell apoptosis and invasion. Microarray-based co-expression analysis identified a total of 61 genes co-expressed with CDKN3, with most of them involved in cell proliferation, and BIRC5 was located at the center of CDKN3 co-expression network. These results suggest that CDKN3 acts as an oncogene in human hepatocellular carcinoma and antagonism of CDKN3 may be of interest for the treatment of HCC.

  4. Effect of flupirtine on the growth and viability of U373 malignant glioma cells

    Panchanathan, Elango; Ramanathan, Gnanasambandan; Lakkakula, Bhaskar Venkata Kameswara Subrahmanya

    2013-01-01

    Flupirtine is a non-opioid analgesic without antipyretic or antiphlogistic properties but with favorable tolerability in humans. This analgesic also exhibits neuroprotective activities. Furthermore, flupirtine antagonizes glutamate- and NMDA-induced intracellular levels of Ca 2+ and counteracts the effects of focal cerebral ischemia. Although flupirtine has been used to relieve pain caused by different diseases and clinical procedures, information on the safety and efficacy of flupirtine is limited. The present study was conducted to investigate the neuroprotective effects of flupirtine on U373 malignant glioma (MG) cell lines. Cell viability and cell cycle analysis was performed by MTT assay and flow cytometry, respectively. Variations in the growth of U373 MG cells in 5 mM N-methyl-D-aspartate (NMDA), 1 mM flupirtine, and combined treatment indicated the antagonistic effects of NMDA and flupirtine on MG cell lines. The variation in the percentage of gated cell population in different cell cycle phases showed significant variations after 48 h of treatment. Flupirtine has neuroprotective effect of on U373 MG cells, which limits its use in the pain management of brain tumors. This property warrants further studies using animal models and large-scale clinical trials

  5. Histone deacetylase inhibitor, 2-propylpentanoic acid, increases the chemosensitivity and radiosensitivity of human glioma cell lines in vitro

    SHAO Cui-jie; WU Ming-wei; CHEN Fu-rong; LI Cong; XIA Yun-fei; CHEN Zhong-ping

    2012-01-01

    Background Treatment for malignant glioma generally consists of cytoreductive surgery followed by radiotherapy and chemotherapy.In this study,we intended to investigate the effects of 2-propylpentanoic acid (VPA),a histone deacetylase inhibitor,on chemosensitivity and radiosensitivity in human glioma cell lines.Methods Human glioma cell lines,T98-G,and SF295,were treated with temozolomide (TMZ) or irradiation (IR),with or without VPA (1.0 mmol/L).Then,cytotoxicity and clonogenic survival assay was performed.Cell cycle stage,apoptosis,and autophagy were also detected using flow cytometry and dansyl monocadaverin (MDC) incorporation assay.One-way analysis of variance (ANOVA) and t-test were used to analyze the differences among variant groups.Results Mild cytotoxicity of VPA was revealed in both cell lines,T98-G and SF295,with the 50% inhibiting concentration (IC50) value of (3.85±0.58) mmol/L and (2.15±0.38) mmol/L,respectively; while the IC50 value of TMZ was (0.20±0.09) mmol/L for T98-G and (0.08±0.02) mmol/L for SF295.Moreover,if combined with VPA (1.0 mmol/L) for 96hours,the sensitivity of glioma cells to TMZ was significant increased (P <0.05).The surviving fractions at 2 Gy (SF2) of T98-G and SF295 cells exposed to IR alone were 0.52 and 0.58.However,when VPA was combined with IR,the SF2 of T98-G and SF295 dropped to 0.39 (P=0.047) and 0.49 (P=-0.049),respectively.Treatment with VPA plus TMZ or IR also resulted in a significant decrease in the proportion of cells in the G2 phase and increased apoptotic rates as well as autophagy in T98-G and SF295 cell lines (P <0.01).Conclusion VPA may enhance the activities of TMZ and IR on glioma cells possibly through cell cycle block and promote autophagy,and thus could be a potential sensitizer of glioma treatment.

  6. Low dose radiation enhance the anti-tumor effect of high dose radiation on human glioma cell U251

    Wang Chang; Wang Guanjun; Tan Yehui; Jiang Hongyu; Li Wei

    2008-01-01

    Objective: To detect the effect on the growth of human glioma cell U251 induced by low dose irradiation and low dose irradiation combined with large dose irradiation. Methods: Human glioma cell line U251 and nude mice carried with human glioma were used. The tumor cells and the mice were treated with low dose, high dose, and low dose combined high dose radiation. Cells growth curve, MTT and flow cytometry were used to detect the proliferation, cell cycle and apoptosis of the cells; and the tumor inhibition rate was used to assess the growth of tumor in vivo. Results: After low dose irradiation, there was no difference between experimental group and control group in cell count, MTT and flow cytometry. Single high dose group and low dose combined high dose group both show significantly the suppressing effect on tumor cells, the apoptosis increased and there was cell cycle blocked in G 2 period, but there was no difference between two groups. In vivo apparent anti-tumor effect in high dose radiation group and the combining group was observed, and that was more significant in the combining group; the prior low dose radiation alleviated the injury of hematological system. There was no difference between single low dose radiation group and control. Conclusions: There is no significant effect on human glioma cell induced by low dose radiation, and low dose radiation could not induce adaptive response. But in vivo experience, low dose radiation could enhance the anti-tumor effect of high dose radiation and alleviated the injury of hematological system. (authors)

  7. Stem-like tumor-initiating cells isolated from IL13Rα2 expressing gliomas are targeted and killed by IL13-zetakine-redirected T Cells.

    Brown, Christine E; Starr, Renate; Aguilar, Brenda; Shami, Andrew F; Martinez, Catalina; D'Apuzzo, Massimo; Barish, Michael E; Forman, Stephen J; Jensen, Michael C

    2012-04-15

    To evaluate IL13Rα2 as an immunotherapeutic target for eliminating glioma stem-like cancer initiating cells (GSC) of high-grade gliomas, with particular focus on the potential of genetically engineered IL13Rα2-specific primary human CD8(+) CTLs (IL13-zetakine(+) CTL) to target this therapeutically resistant glioma subpopulation. A panel of low-passage GSC tumor sphere (TS) and serum-differentiated glioma lines were expanded from patient glioblastoma specimens. These glioblastoma lines were evaluated for expression of IL13Rα2 and for susceptibility to IL13-zetakine(+) CTL-mediated killing in vitro and in vivo. We observed that although glioma IL13Rα2 expression varies between patients, for IL13Rα2(pos) cases this antigen was detected on both GSCs and more differentiated tumor cell populations. IL13-zetakine(+) CTL were capable of efficient recognition and killing of both IL13Rα2(pos) GSCs and IL13Rα2(pos) differentiated cells in vitro, as well as eliminating glioma-initiating activity in an orthotopic mouse tumor model. Furthermore, intracranial administration of IL13-zetakine(+) CTL displayed robust antitumor activity against established IL13Rα2(pos) GSC TS-initiated orthotopic tumors in mice. Within IL13Rα2 expressing high-grade gliomas, this receptor is expressed by GSCs and differentiated tumor populations, rendering both targetable by IL13-zetakine(+) CTLs. Thus, our results support the potential usefullness of IL13Rα2-directed immunotherapeutic approaches for eradicating therapeutically resistant GSC populations. ©2012 AACR.

  8. Glucocorticoids promote a glioma stem cell-like phenotype and resistance to chemotherapy in human glioblastoma primary cells

    Kostopoulou, Ourania N; Mohammad, Abdul-Aleem; Bartek, Jiri

    2018-01-01

    Glioma stem cells (GSCs) are glioblastoma (GBM) cells that are resistant to therapy and can give rise to recurrent tumors. The identification of patient-related factors that support GSCs is thus necessary to design effective therapies for GBM patients. Glucocorticoids (GCs) are used to treat GBM......-associated edema. However, glucocorticoids participate in the physiological response to psychosocial stress, which has been linked to poor cancer prognosis. This raises concern that glucocorticoids affect the tumor and GSCs. Here, we treated primary human GBM cells with dexamethasone and evaluated GC......-driven changes in cell morphology, proliferation, migration, gene expression, secretory activity and growth as neurospheres. Dexamethasone treatment of GBM cells appeared to promote the development of a GSC-like phenotype and conferred resistance to physiological stress and chemotherapy. We also analyzed...

  9. Synergistic Effects of NDRG2 Overexpression and Radiotherapy on Cell Death of Human Prostate LNCaP Cells.

    Alizadeh Zarei, M; Takhshid, M A; Behzad Behbahani, A; Hosseini, S Y; Okhovat, M A; Rafiee Dehbidi, Gh R; Mosleh Shirazi, M A

    2017-09-01

    Radiation therapy is among the most conventional cancer therapeutic modalities with effective local tumor control. However, due to the development of radio-resistance, tumor recurrence and metastasis often occur following radiation therapy. In recent years, combination of radiotherapy and gene therapy has been suggested to overcome this problem. The aim of the current study was to explore the potential synergistic effects of N-Myc Downstream-Regulated Gene 2 (NDRG2) overexpression, a newly identified candidate tumor suppressor gene, with radiotherapy against proliferation of prostate LNCaP cell line. In this study, LNCaP cells were exposed to X-ray radiation in the presence or absence of NDRG2 overexpression using plasmid PSES- pAdenoVator-PSA-NDRG2-IRES-GFP. The effects of NDRG2 overexpression, X-ray radiation or combination of both on the cell proliferation and apoptosis of LNCaP cells were then analyzed using MTT assay and flow cytometery, respectively. Results of MTT assay showed that NDRG2 overexpression and X-ray radiation had a synergistic effect against proliferation of LNCaP cells. Moreover, NDRG2 overexpression increased apoptotic effect of X-ray radiation in LNCaP cells synergistically. Our findings suggested that NDRG2 overexpression in combination with radiotherapy may be an effective therapeutic option against prostate cancer.

  10. The infrared spectrum of human glioma cells is related to their in vitro and in vivo behavior

    Gaigneaux, A.; Decaestecker, C.; Camby, I.; Mijatovic, T.; Kiss, R.; Ruysschaert, J.M.; Goormaghtigh, E.

    2004-01-01

    The present research investigates whether infrared spectra can be related to the biological characteristics of glioma cell lines. We used nine human glioma cell lines for which a series of in vitro and in vivo biological features had already been established [Glia 36 (2001) 375] and were able to show that their characteristic infrared spectra reflect their in vitro migration (i.e., motility and invasiveness) properties and their in vivo aggressiveness. More particularly, the infrared data evidenced correlations at the level of the lipid/protein ratio. These relationships were found to be tissue-dependent when controlled on seven pancreatic carcinoma cell lines. We also showed that oligodendroglial and astrocytic tumor cells, whose identification remains difficult, can easily be identified by their infrared spectra in the lipid acyl chain region as well as in the nucleic acid region. We concluded that infrared spectroscopy could usefully complement information provided by more conventional diagnostic and prognostic (e.g., morphological and molecular) approaches

  11. The role of glioma stem cells in chemotherapy resistance and glioblastoma multiforme recurrence

    Auffinger, Brenda; Spencer, Drew; Pytel, Peter; Ahmed, Atique U.; Lesniak, Maciej S.

    2016-01-01

    Glioma stem cells (GSCs) constitute a slow-dividing, small population within a heterogeneous glioblastoma. They are able to self-renew, recapitulate a whole tumor, and differentiate into other specific GBM subpopulations. Therefore, they have been held responsible for malignant relapse after primary standard therapy and the poor prognosis of recurrent GBM. The failure of current therapies to eliminate specific GSC subpopulations has been considered a major factor contributing to the inevitable recurrence in GBM patients following treatment. Here, we discuss the molecular mechanisms of chemoresistance of GSCs and the reasons why complete eradication of GSCs is so difficult to achieve. We will also describe the targeted therapies currently available towards GSCs and possible mechanisms to overcome such chemoresistance and avoid therapeutic relapse. PMID:26027432

  12. Matrix-Dependent Regulation of AKT in Hepsin-Overexpressing PC3 Prostate Cancer Cells12

    Wittig-Blaich, Stephanie M; Kacprzyk, Lukasz A; Eismann, Thorsten; Bewerunge-Hudler, Melanie; Kruse, Petra; Winkler, Eva; Strauss, Wolfgang S L; Hibst, Raimund; Steiner, Rudolf; Schrader, Mark; Mertens, Daniel; Sültmann, Holger; Wittig, Rainer

    2011-01-01

    The serine-protease hepsin is one of the most prominently overexpressed genes in human prostate carcinoma. Forced expression of the enzyme in mice prostates is associated with matrix degradation, invasive growth, and prostate cancer progression. Conversely, hepsin overexpression in metastatic prostate cancer cell lines was reported to induce cell cycle arrest and reduction of invasive growth in vitro. We used a system for doxycycline (dox)-inducible target gene expression in metastasis-derived PC3 cells to analyze the effects of hepsin in a quantitative manner. Loss of viability and adhesion correlated with hepsin expression levels during anchorage-dependent but not anchorage-independent growth. Full expression of hepsin led to cell death and detachment and was specifically associated with reduced phosphorylation of AKT at Ser473, which was restored by growth on matrix derived from RWPE1 normal prostatic epithelial cells. In the chorioallantoic membrane xenograft model, hepsin overexpression in PC3 cells reduced the viability of tumors but did not suppress invasive growth. The data presented here provide evidence that elevated levels of hepsin interfere with cell adhesion and viability in the background of prostate cancer as well as other tissue types, the details of which depend on the microenvironment provided. Our findings suggest that overexpression of the enzyme in prostate carcinogenesis must be spatially and temporally restricted for the efficient development of tumors and metastases. PMID:21750652

  13. Matrix-Dependent Regulation of AKT in Hepsin-Overexpressing PC3 Prostate Cancer Cells

    Stephanie M Wittig-Blaich

    2011-07-01

    Full Text Available The serine-protease hepsin is one of the most prominently overexpressed genes in human prostate carcinoma. Forced expression of the enzyme in mice prostates is associated with matrix degradation, invasive growth, and prostate cancer progression. Conversely, hepsin overexpression in metastatic prostate cancer cell lines was reported to induce cell cycle arrest and reduction of invasive growth in vitro. We used a system for doxycycline (dox-inducible target gene expression in metastasis-derived PC3 cells to analyze the effects of hepsin in a quantitative manner. Loss of viability and adhesion correlated with hepsin expression levels during anchorage-dependent but not anchorage-independent growth. Full expression of hepsin led to cell death and detachment and was specifically associated with reduced phosphorylation of AKT at Ser473, which was restored by growth on matrix derived from RWPE1 normal prostatic epithelial cells. In the chorioallantoic membrane xenograft model, hepsin overexpression in PC3 cells reduced the viability of tumors but did not suppress invasive growth. The data presented here provide evidence that elevated levels of hepsin interfere with cell adhesion and viability in the background of prostate cancer as well as other tissue types, the details of which depend on the microenvironment provided. Our findings suggest that overexpression of the enzyme in prostate carcinogenesis must be spatially and temporally restricted for the efficient development of tumors and metastases.

  14. The inhibition of superoxide production in EL4 lymphoma cells overexpressing growth hormone.

    Arnold, Robyn E; Weigent, Douglas A

    2003-05-01

    A substantial body of research exists to support the production of growth hormone by cells of the immune system. However, the function and mechanism of action of lymphocyte-derived growth hormone remain largely unelucidated. Since, it has been found that exogenous growth hormone (GH) primes neutrophils for the production of reactive oxygen intermediates (ROI) and in particular superoxide (O2-), we investigated the role of GH on the production of O2- in T cells. Furthermore, we examined whether endogenous and exogenous GH act similarly. Our studies show that overexpression of GH in EL4, a T-cell lymphoma cell line, results in a decrease in the production of O2- compared to control cells, as detected using the fluorescent dye, dihydroethidium. O2- production in control cells was not affected by treatment with inhibitors of xanthine oxidase or a non-specific NADPH-oxidase inhibitor. However, treatment with diallyl sulfide, an inhibitor of cytochrome P450 2E1 mimicked the reduction in O2- production seen in cells overexpressing GH. Although no significant change could be detected in CYP2E1 protein levels, CYP2E1 activity was found to be greater in control EL4 than in cells overexpressing GH. Both the decrease in O2- production and the lower CYP2E1 activity in GH overexpressing cells could be abrogated by treatment with N(G)-monomethyl-L-arginine, an inhibitor of nitric oxide synthase. The overexpression of GH protects cells from apoptosis induced by isoniazid, a CYP2E1 inducer, suggesting a role for nitric oxide as a mediator in the regulation of xenobiotic metabolism and apoptosis-protection by lymphocyte GH.

  15. Use of EF5 to Measure the Oxygen Level in Tumor Cells of Patients Undergoing Surgery or Biopsy for Newly Diagnosed Supratentorial Malignant Glioma

    2013-01-15

    Adult Anaplastic Astrocytoma; Adult Anaplastic Ependymoma; Adult Anaplastic Oligodendroglioma; Adult Diffuse Astrocytoma; Adult Ependymoma; Adult Giant Cell Glioblastoma; Adult Glioblastoma; Adult Gliosarcoma; Adult Mixed Glioma; Adult Myxopapillary Ependymoma; Adult Oligodendroglioma; Adult Pilocytic Astrocytoma; Adult Pineal Gland Astrocytoma; Adult Subependymoma

  16. Serum-dependent effects of tamoxifen and cannabinoids upon C6 glioma cell viability.

    Jacobsson, S O; Rongård, E; Stridh, M; Tiger, G; Fowler, C J

    2000-12-15

    In the present study, the effects of the combination of tamoxifen ((Z)-2[p-(1,2-diphenyl-1-butenyl)phenoxy]-N,N-dimethylamine citrate) and three cannabinoids (Delta(9)-tetrahydrocannabinol [Delta(9)-THC], cannabidiol, and anandamide [AEA]) upon the viability of C6 rat glioma cells was assessed at different incubation times and using different culturing concentrations of foetal bovine serum (FBS). Consistent with previous data for human glioblastoma cells, the tamoxifen sensitivity of the cells was increased as the FBS content of the culture medium was reduced from 10 to 0.4 and 0%. The cells expressed protein kinase C alpha and calmodulin (the concentration of which did not change significantly as the FBS concentration was reduced), but did not express estrogen receptors. Delta(9)-THC and cannabidiol, but not AEA, produced a modest reduction in cell viability after 6 days of incubation in serum-free medium, whereas no effects were seen in 10% FBS-containing medium. There was no observed synergy between the effects of tamoxifen and the cannabinoids upon cell viability.

  17. Bcl-2 overexpression prevents 99mTc-MIBI uptake in breast cancer cell lines

    Aloj, Luigi; Zannetti, Antonella; Caraco, Corradina; Del Vecchio, Silvana; Salvatore, Marco

    2004-01-01

    We have previously shown a correlation between the absence of technetium-99m methoxyisobutylisonitrile ( 99m Tc-MIBI) uptake and overexpression of the anti-apoptotic protein Bcl-2 in human breast carcinoma. To establish a direct cause-effect relationship between Bcl-2 overexpression and reduced 99m Tc-MIBI uptake, MCF-7 and T47D breast cancer cell lines were stably transfected with the human Bcl-2 gene to increase intracellular protein levels and tested for 99m Tc-MIBI uptake. All clones overexpressing Bcl-2 showed a dramatic reduction of 99m Tc-MIBI uptake as compared with mock transfected control cells. Tracer uptake was promptly and partially restored by induction of apoptosis with staurosporine treatment. After 4.5 h of staurosporine treatment, a tenfold increase in 99m Tc-MIBI uptake was observed in treated as compared with untreated Bcl-2 overexpressing cells. Our findings provide a rational basis for the development of an in vivo test to detect Bcl-2 overexpression in human tumours. (orig.)

  18. Overexpression of microRNA-1288 in oesophageal squamous cell carcinoma

    Gopalan, Vinod; Islam, Farhadul; Pillai, Suja; Tang, Johnny Cheuk-On; Tong, Daniel King-Hung; Law, Simon; Chan, Kwok-Wah; Lam, Alfred King-Yin

    2016-01-01

    Purpose: This study aims to examine the expression profiles miR-1288 in oesophageal squamous cell carcinoma (ESCC). The cellular implications and target interactions of ESCC cells following miR-1288 overexpression was also examined. Methods: In total, 120 oesophageal tissues (90 primary ESCCs and 30 non-neoplastic tissues) were recruited for miR-1288 expression analysis using qRT-PCR. An exogenous miR-1288 mimic and its inhibitor were used to explore the in-vitro effects of miR-1288 on ESCC cells by performing cell proliferation, colony formation, cell invasion and migration assays. Localisation and modulatory changes of various miR-1288 regulated proteins such as FOXO1, p53, TAB3, BCL2 and kRAS was examined using immunofluorescence and western blot. Results: Overexpression of miR-1288 was more often noted in ESCC tissues when compared to non-neoplastic oesophageal tissues. High expression was often noted in high grade carcinomas and with metastases. Patients with high levels of miR-1288 expression showed a slightly better survival compared to patients with low miR-1288 levels. Furthermore, overexpression of miR-1288 showed increased cell proliferation and colony formation, improved cell migration and enhanced cell invasion properties in ESCC cells. In addition, miR-1288 overexpression in ESCC cells showed repression of cytoplasmic tumour suppressor FOXO1 protein expression. Inversely, inhibition of miR-1288 expression exhibited remarkable upregulation of FOXO1 protein, while expressions of other tested proteins remain unchanged. Conclusions: Up regulation of miR-1288 expression in ESCC tissues and miR-1288 induced oncogenic features of ESCC cells in-vitro indicates the oncogenic roles of miR-1288 in ESCCs. Overexpression of miR-1288 play a key role in the pathogenesis of ESCCs and its modulation may have potential therapeutic value in patients with ESCC. - Highlights: • miR-1288 was more often noted in neoplastic than non-neoplastic tissue. • miR-1288

  19. Overexpression of microRNA-1288 in oesophageal squamous cell carcinoma

    Gopalan, Vinod; Islam, Farhadul; Pillai, Suja [Cancer Molecular Pathology, School of Medicine and Menzies Health Institute Queensland, Griffith University, Gold Coast (Australia); Tang, Johnny Cheuk-On [State Key Laboratory of Chirosciences, Lo Ka Chung Centre for Natural Anti-cancer Drug Development, Department of Applied Biology and Chemical Technology, the Hong Kong Polytechnic University (Hong Kong); Tong, Daniel King-Hung; Law, Simon [Department of Surgery, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Queen Mary Hospital (Hong Kong); Chan, Kwok-Wah [Department of Pathology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Queen Mary Hospital (Hong Kong); Lam, Alfred King-Yin, E-mail: a.lam@griffith.edu.au [Cancer Molecular Pathology, School of Medicine and Menzies Health Institute Queensland, Griffith University, Gold Coast (Australia)

    2016-11-01

    Purpose: This study aims to examine the expression profiles miR-1288 in oesophageal squamous cell carcinoma (ESCC). The cellular implications and target interactions of ESCC cells following miR-1288 overexpression was also examined. Methods: In total, 120 oesophageal tissues (90 primary ESCCs and 30 non-neoplastic tissues) were recruited for miR-1288 expression analysis using qRT-PCR. An exogenous miR-1288 mimic and its inhibitor were used to explore the in-vitro effects of miR-1288 on ESCC cells by performing cell proliferation, colony formation, cell invasion and migration assays. Localisation and modulatory changes of various miR-1288 regulated proteins such as FOXO1, p53, TAB3, BCL2 and kRAS was examined using immunofluorescence and western blot. Results: Overexpression of miR-1288 was more often noted in ESCC tissues when compared to non-neoplastic oesophageal tissues. High expression was often noted in high grade carcinomas and with metastases. Patients with high levels of miR-1288 expression showed a slightly better survival compared to patients with low miR-1288 levels. Furthermore, overexpression of miR-1288 showed increased cell proliferation and colony formation, improved cell migration and enhanced cell invasion properties in ESCC cells. In addition, miR-1288 overexpression in ESCC cells showed repression of cytoplasmic tumour suppressor FOXO1 protein expression. Inversely, inhibition of miR-1288 expression exhibited remarkable upregulation of FOXO1 protein, while expressions of other tested proteins remain unchanged. Conclusions: Up regulation of miR-1288 expression in ESCC tissues and miR-1288 induced oncogenic features of ESCC cells in-vitro indicates the oncogenic roles of miR-1288 in ESCCs. Overexpression of miR-1288 play a key role in the pathogenesis of ESCCs and its modulation may have potential therapeutic value in patients with ESCC. - Highlights: • miR-1288 was more often noted in neoplastic than non-neoplastic tissue. • miR-1288

  20. Effects of concomitant temozolomide and radiation therapies on WT1-specific T-cells in malignant glioma

    Chiba, Yasuyoshi; Hashimoto, Naoya; Tsuboi, Akihiro

    2010-01-01

    Immunotherapy targeting the Wilms' tumour 1 gene product has been proven safe and effective for treating malignant glioma in a phase II clinical study. Currently, radiation/temozolomide therapy is the standard treatment with only modest benefit. Whether combining radiation/temozolomide therapy with WT1 immunotherapy will have a negating effect on immunotherapy is still controversial because of the significant lymphocytopaenia induced by the former therapy. To address this issue, we investigated the changes in frequency and number of WT1-specific T-cells in patients with malignant gliomas. Twenty-two patients with newly diagnosed malignant glioma who received standard radiation/temozolomide therapy were recruited for the study. Blood samples were collected before treatment and on the sixth week of therapy. The frequencies and numbers of lymphocytes, CD8 + T-cells, WT1-specific T-cells, regulatory T-cells, natural killer cells and natural killer T-cells were measured and analysed using T-tests. Analysis of the frequency of T lymphocytes and its subpopulation showed an increase in regulatory T-cells, but no significant change was noted in the populations of T-cells, WT1-specific T-cells, natural killer (NK) cells and natural killer T (NKT) cells. Reductions in the total numbers of T-cells, WT1-specific T-cells, NK cells and NKT cells were mainly a consequence of the decrease in the total lymphocyte count. Radiation/temozolomide therapy did not significantly affect the frequency of WT1-specific T-cells, suggesting that the combination with WT1 immunotherapy may be possible, although further assessment in the clinical setting is warranted. (author)

  1. RANK (TNFRSF11A Is Epigenetically Inactivated and Induces Apoptosis in Gliomas

    Anna von dem Knesebeck

    2012-06-01

    Full Text Available Alterations of DNA methylation play an important role in gliomas. In a genome-wide screen, we identified a CpG-rich fragment within the 5′ region of the tumor necrosis factor receptor superfamily, member 11A gene (TNFRSF11A that showed de novo methylation in gliomas. TNFRSF11A, also known as receptor activator of NF-κB (RANK, activates several signaling pathways, such as NF-κB, JNK, ERK, p38α, and Akt/PKB. Using pyrosequencing, we detected RANK/TNFRSF11A promoter methylation in 8 (57.1% of 14 diffuse astrocytomas, 17 (77.3% of 22 anaplastic astrocytomas, 101 (84.2% of 120 glioblastomas, 6 (100% of 6 glioma cell lines, and 7 (100% of 7 glioma stem cell-enriched glioblastoma primary cultures but not in four normal white matter tissue samples. Treatment of glioma cell lines with the demethylating agent 5-aza-2′-deoxycytidine significantly reduced the methylation level and resulted in increased RANK/TNFRSF11A mRNA expression. Overexpression of RANK/TNFRSF11A in glioblastoma cell lines leads to a significant reduction in focus formation and elevated apoptotic activity after flow cytometric analysis. Reporter assay studies of transfected glioma cells supported these results by showing the activation of signaling pathways associated with regulation of apoptosis. We conclude that RANK/TNFRSF11A is a novel and frequent target for de novo methylation in gliomas, which affects apoptotic activity and focus formation thereby contributing to the molecular pathogenesis of gliomas.

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

    Albert, Ina; Hefti, Martin; Luginbuehl, Vera

    2014-11-01

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

  3. Characterization of MCF mammary epithelial cells overexpressing the Arylhydrocarbon receptor (AhR)

    Wong, Patrick S; Li, Wen; Vogel, Christoph F; Matsumura, Fumio

    2009-01-01

    Recent reports indicate the existence of breast cancer cells expressing very high levels of the Arylhydrocarbon receptor (AhR), a ubiquitous intracellular receptor best known for mediating toxic action of dioxin and related pollutants. Positive correlation between the degree of AhR overexpression and states of increasing transformation of mammary epithelial cells appears to occur in the absence of any exogenous AhR ligands. These observations have raised many questions such as why and how AhR is overexpressed in breast cancer and its physiological roles in the progression to advanced carcinogenic transformation. To address those questions, we hypothesized that AhR overexpression occurs in cells experiencing deficiencies in normally required estrogen receptor (ER) signaling, and the basic role of AhR in such cases is to guide the affected cells to develop orchestrated cellular changes aimed at substituting the normal functions of ER. At the same time, the AhR serves as the mediator of the cell survival program in the absence of ER signaling. We subjected two lines of Michigan Cancer Foundation (MCF) mammary epithelial cells to 3 different types ER interacting agents for a number of passages and followed the changes in the expression of AhR mRNA. The resulting sublines were analyzed for phenotypical changes and unique molecular characteristics. MCF10AT1 cells continuously exposed to 17-beta-estradiol (E2) developed sub-lines that show AhR overexpression with the characteristic phenotype of increased proliferation, and distinct resistance to apoptosis. When these chemically selected cell lines were treated with a specific AhR antagonist, 3-methoxy-4-nitroflavone (MNF), both of the above abnormal cellular characteristics disappeared, indicating the pivotal role of AhR in expressing those cellular phenotypes. The most prominent molecular characteristics of these AhR overexpressing MCF cells were found to be overexpression of ErbB2 and COX-2. Furthermore, we could

  4. Involvement of triacylglycerol in the metabolism of fatty acids by cultured neuroblastoma and glioma cells

    Cook, H.W.; Clarke, J.T.; Spence, M.W.

    1982-01-01

    The metabolism (chain elongation, desaturation, and incorporation into complex lipids) of thirteen different radiolabeled fatty acids and acetate was examined in N1E-115 neuroblastoma and C-6 glioma cell lines in culture. During 6-hr incubations, all fatty acids were extensively (14-80%) esterified to complex lipids, mainly choline phosphoglycerides and triacylglycerol. With trienoic and tetraenoic substrates, inositol and ethanolamine phosphoglycerides also contained up to 30% of the labeled fatty acids; plasmalogen contained up to half of the label in the ethanolamine phosphoglyceride fraction of neuroblastoma cells. Chain elongation and delta 9, delta 6, and delta 5 desaturation occurred in both cell lines; delta 4 desaturation was not observed. Seemingly anomalous utilization of arachidic acid and some selectivity based on the geometric configuration of double bonds was observed. These studies indicate that these cell lines are capable of modulating cellular membrane composition by a combination of selective exclusion and removal of inappropriate acyl chains and of modification of other acyl chains by desaturation and chain elongation. The time courses and patterns of modification and incorporation of exogenous substrates into phospholipids and triacylglycerol suggest that exogenous unsaturated fatty acid may be incorporated into triacylglycerol and later released for further metabolism and incorporation into phospholipids. This supports a role for triacylglycerol in the synthesis of membrane complex lipids in cell lines derived from neural tissue

  5. Molecular and Microenvironmental Determinants of Glioma Stem-Like Cell Survival and Invasion

    Alison Roos

    2017-06-01

    Full Text Available Glioblastoma multiforme (GBM is the most frequent primary brain tumor in adults with a 5-year survival rate of 5% despite intensive research efforts. The poor prognosis is due, in part, to aggressive invasion into the surrounding brain parenchyma. Invasion is a complex process mediated by cell-intrinsic pathways, extrinsic microenvironmental cues, and biophysical cues from the peritumoral stromal matrix. Recent data have attributed GBM invasion to the glioma stem-like cell (GSC subpopulation. GSCs are slowly dividing, highly invasive, therapy resistant, and are considered to give rise to tumor recurrence. GSCs are localized in a heterogeneous cellular niche, and cross talk between stromal cells and GSCs cultivates a fertile environment that promotes GSC invasion. Pro-migratory soluble factors from endothelial cells, astrocytes, macrophages, microglia, and non-stem-like tumor cells can stimulate peritumoral invasion of GSCs. Therefore, therapeutic efforts designed to target the invasive GSCs may enhance patient survival. In this review, we summarize the current understanding of extrinsic pathways and major stromal and immune players facilitating GSC maintenance and survival.

  6. EMMPRIN overexpression in SVZ neural progenitor cells increases their migration towards ischemic cortex.

    Kanemitsu, Michiko; Tsupykov, Oleg; Potter, Gaël; Boitard, Michael; Salmon, Patrick; Zgraggen, Eloisa; Gascon, Eduardo; Skibo, Galina; Dayer, Alexandre G; Kiss, Jozsef Z

    2017-11-01

    Stimulation of endogenous neurogenesis and recruitment of neural progenitors from the subventricular zone (SVZ) neurogenic site may represent a useful strategy to improve regeneration in the ischemic cortex. Here, we tested whether transgenic overexpression of extracellular matrix metalloproteinase inducer (EMMPRIN), the regulator of matrix metalloproteinases (MMPs) expression, in endogenous neural progenitor cells (NPCs) in the subventricular zone (SVZ) could increase migration towards ischemic injury. For this purpose, we applied a lentivector-mediated gene transfer system. We found that EMMPRIN-transduced progenitors exhibited enhanced MMP-2 activity in vitro and showed improved motility in 3D collagen gel as well as in cortical slices. Using a rat model of neonatal ischemia, we showed that EMMPRIN overexpressing SVZ cells invade the injured cortical tissue more efficiently than controls. Our results suggest that EMMPRIN overexpression could be suitable approach to improve capacities of endogenous or transplanted progenitors to invade the injured cortex. Copyright © 2017 Elsevier Inc. All rights reserved.

  7. Overexpression of protein kinase A - RIalpha reduces lipofection efficiency of cisplatin-resistant human tumor cells.

    Son, K K; Rosenblatt, J

    2001-04-10

    Cisplatin-resistant variant A2780CP/vector cells were 4.0-5.3-fold more transfectable and 7.6-fold more resistant to cisplatin than their parent cisplatin-sensitive human ovarian carcinoma A2780/vector cells. Overexpression of cAMP-dependent protein kinase Type I regulatory alpha subunit (PKA-RIalpha) gene in A2780CP cells significantly reduced (maximum 47.0%) the transfection activity, with a slight reduction (maximum 27.3%) of cisplatin resistance, of A2780CP cells. However, RIalpha-overexpressing A2780CP (A2780CP/RIalpha) cells were still 2.5-to 3.0-fold more transfectable and 5.5-fold more resistant to cisplatin than A2780 cells. This results suggest that gene transfer efficiency is associated with cisplatin resistance, in part, through the PKA-mediated cAMP signal transduction pathway.

  8. BAD overexpression inhibits cell growth and induces apoptosis via mitochondrial-dependent pathway in non-small cell lung cancer.

    Jiang, Li; Luo, Man; Liu, Dan; Chen, Bojiang; Zhang, Wen; Mai, Lin; Zeng, Jing; Huang, Na; Huang, Yi; Mo, Xianming; Li, Weimin

    2013-06-01

    The pro-apoptotic Bcl-2 protein BAD initiated apoptosis in human cells and has been identified as a prognostic marker in non-small cell lung cancer (NSCLC). In this study, we aimed to explore the functions of BAD in NSCLC. Overexpression of BAD was performed by transfecting different NSCLC cell lines with wild-type BAD. Cell proliferation, cell cycle, apoptosis, and invasion were characterized in vitro. Tumorigenicity was analyzed in vivo. Western blot was performed to determine the effects of BAD overexpression on the Bcl-2 family proteins and apoptosis-related proteins. Overexpression of BAD significantly inhibited cell proliferation in H1299, H292, and SPC-A1 but not in SK-MES-1 and H460 cell lines in vitro. BAD overexpression also reduced the tumorigenicity of H1299/SPC-A1 cell in vivo. However, no appreciable effects on cell cycle distribution and invasion were observed in all these cell lines. BAD overexpression also induced apoptosis in all cell types, in which process expression of mitochondrial cytochrom c (cyto-c) and caspase 3 were increased, whereas Bcl-xl, Bcl-2, Bax and caspase 8 expressions did not changed. These findings indicated that a mitochondrial pathway, in which process cyto-c was released from mitochondrial to activate caspase 3, was involved in BAD overexpression-mediated apoptosis. Our data suggested that increased expression of BAD enhance apoptosis and has negative influence on cell proliferation and tumor growth in NSCLC. Bad is a new potential target for tumor interventions.

  9. Overexpression of Hiwi Inhibits the Growth and Migration of Chronic Myeloid Leukemia Cells.

    Wang, Yalin; Jiang, Yan; Ma, Ning; Sang, Bailu; Hu, Xiaolin; Cong, Xiaofeng; Liu, Ziling

    2015-09-01

    Chronic myeloid leukemia (CML) is a hematopoietic malignancy characterized by dysregulated growth and proliferation of hematopoietic stem/progenitor cells in bone marrow and excessive expansion of hematopoietic compartments in peripheral blood. Expression deletion of Hiwi, a human Piwi homolog, has been reported to be implicated in leukemogenesis. We here explored Hiwi's role in CML pathogenesis by determining how and whether its forced overexpression could affect CML cell growth and migration. The present results showed that lentivirus-mediated overexpression of Hiwi significantly suppressed cell proliferation and induced obvious apoptosis in K562 cells, a CML line cell line. Tumors in BALB/c nude mice generated by the K562 cells expressing Hiwi were much smaller than those formed by the control cells. Like in vitro, Hiwi upregulation induced cell apoptosis in the tumor tissues in vivo. Additionally, Hiwi elevation suppressed K562 cell migration and inhibited the activity and expression of matrix metalloproteinase-2 and -9. In summary, our study demonstrates that Hiwi overexpression inhibits CML cell growth and migration, providing insights into its role in CML pathogenesis.

  10. Study of the G2/M cell cycle checkpoint in irradiated mammary epithelial cells overexpressing Cul-4A gene

    Gupta, Anu; Yang, L.-X.; Chen, L.-C.

    2002-01-01

    Purpose: Members of the cullin gene family are known to be involved in cell cycle control. One of the cullin genes, Cul-4A, is amplified and overexpressed in breast cancer cells. This study investigates the effect of Cul-4A overexpression upon G2/M cell cycle checkpoint after DNA damage induced by either ionizing or nonionizing radiation. Methods and Materials: The normal mammary epithelial cell line MCF10A was stably transfected with full-length Cul-4A cDNA. Independent clones of MCF10A cells that overexpress Cul-4A proteins were selected and treated with either 8 Gy of ionizing radiation or 7 J/M 2 of UV radiation. The profile of cell cycle progression and the accumulation of several cell cycle proteins were analyzed. Results: We found that overexpression of Cul-4A in MCF10A cells abrogated the G2/M cell cycle checkpoint in response to DNA damage induced by ionizing irradiation, but not to DNA damage induced by nonionizing radiation. Analysis of cell cycle proteins showed that after ionizing irradiation, p53 accumulated in the mock-transfected MCF10A cells, but not in the Cul-4A transfectants. Conclusion: Our results suggest a role for Cul-4A in tumorigenesis and/or tumor progression, possibly through disruption of cell cycle control

  11. Enhancement of Temozolomide and radiation induced damage in malignant glioma cell lines by 2-deoxy-D-glucose

    Kumari, Kalyani; Shyam, Sai; Chandrasekhar Sagar, B.K.; Jagath Lal, G.; Kalia, Vijay Kumar

    2014-01-01

    Malignant Gliomas are the most common and aggressive CNS tumors. The current standard treatment includes surgery, followed by Temozolomide (TMZ)-Radiotherapy. It leads to increased survival as compared to radiotherapy alone. However hematological toxicities are also increased by the combination treatments. Therefore, it is important to carry out further preclinical studies, to develop more effective treatment for these tumors. 2-deoxy-D-Glucose (2-DG), an inhibitor of glycolytic energy metabolism, has been shown earlier to differentially inhibit growth and survival of tumor cells in vitro. It also increases tumor regression in experimental models; and has been used in a few clinical studies as radiosensitizer. In the present study, effects of combining 2-DG with TMZ on radiation induced damage were studied in established malignant glioma cell lines (U251MG and U87MG); and primary cultures derived from malignant glioma biopsies. Exponentially growing cells were exposed to drugs and radiation. Drugs were removed 4 hours later and cultures were processed further for different assays of damage. Effects on proliferation response, viability and total cellular damage (TCD; micronuclei + apoptosis) were studied after post-treatment growth for 1, 2, 4 or 6 days. Our results showed that combination of 2-DG with TMZ ± Radiation significantly inhibited tumor cell proliferation up to 6 days, at low drug concentrations in primary as well as in established cell lines. The TCD at 24 and 48 hours after Gamma irradiation was also significantly increased by the combination of drugs as compared to individual treatments. Experiments to study proliferation kinetics by flow cytometry and cell survival are in progress. These studies suggest that 2-DG significantly enhances the cytotoxic effect of TMZ + radiation without increasing toxic side effects. Therefore, combining 2-DG with TMZ+ radiation therapy could be a potential strategy to improve the therapeutic outcome for Malignant

  12. Polysialic Acid Neural Cell Adhesion Molecule (PSA-NCAM) is an adverse prognosis factor in glioblastoma, and regulates olig2 expression in glioma cell lines

    Amoureux, Marie-Claude; Coulibaly, Béma; Chinot, Olivier; Loundou, Anderson; Metellus, Philippe; Rougon, Geneviève; Figarella-Branger, Dominique

    2010-01-01

    Glioblastoma multiforme (GBM) is the most aggressive and frequent brain tumor, albeit without cure. Although patient survival is limited to one year on average, significant variability in outcome is observed. The assessment of biomarkers is needed to gain better knowledge of this type of tumor, help prognosis, design and evaluate therapies. The neurodevelopmental polysialic acid neural cell adhesion molecule (PSA-NCAM) protein is overexpressed in various cancers. Here, we studied its expression in GBM and evaluated its prognosis value for overall survival (OS) and disease free survival (DFS). We set up a specific and sensitive enzyme linked immunosorbent assay (ELISA) test for PSA-NCAM quantification, which correlated well with PSA-NCAM semi quantitative analysis by immunohistochemistry, and thus provides an accurate quantitative measurement of PSA-NCAM content for the 56 GBM biopsies analyzed. For statistics, the Spearman correlation coefficient was used to evaluate the consistency between the immunohistochemistry and ELISA data. Patients' survival was estimated by using the Kaplan-Meier method, and curves were compared using the log-rank test. On multivariate analysis, the effect of potential risk factors on the DFS and OS were evaluated using the cox regression proportional hazard models. The threshold for statistical significance was p = 0.05. We showed that PSA-NCAM was expressed by approximately two thirds of the GBM at variable levels. On univariate analysis, PSA-NCAM content was an adverse prognosis factor for both OS (p = 0.04) and DFS (p = 0.0017). On multivariate analysis, PSA-NCAM expression was an independent negative predictor of OS (p = 0.046) and DFS (p = 0.007). Furthermore, in glioma cell lines, PSA-NCAM level expression was correlated to the one of olig2, a transcription factor required for gliomagenesis. PSA-NCAM represents a valuable biomarker for the prognosis of GBM patients

  13. Overexpressed Genes/ESTs and Characterization of Distinct Amplicons on 17823 in Breast Cancer Cells

    Ayse E. Erson

    2001-01-01

    Full Text Available 17823 is a frequent site of gene amplification in breast cancer. Several lines of evidence suggest the presence of multiple amplicons on 17823. To characterize distinct amplicons on 17823 and localize putative oncogenes, we screened genes and expressed sequence tags (ESTs in existing physical and radiation hybrid maps for amplification and overexpression in breast cancer cell lines by semiquantitative duplex PCR, semiquantitative duplex RT-PCR, Southern blot, Northern blot analyses. We identified two distinct amplicons on 17823, one including TBX2 and another proximal region including RPS6KB1 (PS6K and MUL. In addition to these previously reported overexpressed genes, we also identified amplification and overexpression of additional uncharacterized genes and ESTs, some of which suggest potential oncogenic activity. In conclusion, we have further defined two distinct regions of gene amplification and overexpression on 17823 with identification of new potential oncogene candidates. Based on the amplification and overexpression patterns of known and as of yet unrecognized genes on 17823, it is likely that some of these genes mapping to the discrete amplicons function as oncogenes and contribute to tumor progression in breast cancer cells.

  14. PTEN overexpression improves cisplatin-resistance of human ovarian cancer cells through upregulating KRT10 expression

    Wu, Huijuan; Wang, Ke; Liu, Wenxin; Hao, Quan

    2014-01-01

    Highlights: • Overexpression of PTEN enhanced the sensitivity of C13K cells to cisplatin. • KRT10 is a downstream molecule of PTEN involved in the resistance-reversing effect. • Overexpression of KRT10 enhanced the chemosensitivity of C13K cells to cisplatin. - Abstract: Multi-drug resistance (MDR) is a common cause of the failure of chemotherapy in ovarian cancer. PTEN, a tumor suppressor gene, has been demonstrated to be able to reverse cisplatin-resistance in ovarian cancer cell line C13K. However, the downstream molecules of PTEN involved in the resistance-reversing effect have not been completely clarified. Therefore, we screened the downstream molecules of PTEN and studied their interactions in C13K ovarian cancer cells using a 3D culture model. Firstly, we constructed an ovarian cancer cell line stably expressing PTEN, C13K/PTEN. MTT assay showed that overexpression of PTEN enhanced the sensitivity of C13K cells to cisplatin, but not to paclitaxel. Then we examined the differently expressed proteins that interacted with PTEN in C13K/PTEN cells with or without cisplatin treatment by co-immunoprecipitation. KRT10 was identified as a differently expressed protein in cisplatin-treated C13K/PTEN cells. Further study confirmed that cisplatin could induce upregulation of KRT10 mRNA and protein in C13K/PTEN cells and there was a directly interaction between KRT10 and PTEN. Forced expression of KRT10 in C13K cells also enhanced cisplatin-induced proliferation inhibition and apoptosis of C13K cells. In addition, KRT10 siRNA blocked cisplatin-induced proliferation inhibition of C13K/PTEN cells. In conclusion, our data demonstrate that KRT10 is a downstream molecule of PTEN which improves cisplatin-resistance of ovarian cancer and forced KRT10 overexpression may also act as a therapeutic method for overcoming MDR in ovarian cancer

  15. Molecular markers in glioma.

    Ludwig, Kirsten; Kornblum, Harley I

    2017-09-01

    Gliomas are the most malignant and aggressive form of brain tumors, and account for the majority of brain cancer related deaths. Malignant gliomas, including glioblastoma are treated with radiation and temozolomide, with only a minor benefit in survival time. A number of advances have been made in understanding glioma biology, including the discovery of cancer stem cells, termed glioma stem cells (GSC). Some of these advances include the delineation of molecular heterogeneity both between tumors from different patients as well as within tumors from the same patient. Such research highlights the importance of identifying and validating molecular markers in glioma. This review, intended as a practical resource for both clinical and basic investigators, summarizes some of the more well-known molecular markers (MGMT, 1p/19q, IDH, EGFR, p53, PI3K, Rb, and RAF), discusses how they are identified, and what, if any, clinical relevance they may have, in addition to discussing some of the specific biology for these markers. Additionally, we discuss identification methods for studying putative GSC's (CD133, CD15, A2B5, nestin, ALDH1, proteasome activity, ABC transporters, and label-retention). While much research has been done on these markers, there is still a significant amount that we do not yet understand, which may account for some conflicting reports in the literature. Furthermore, it is unlikely that the investigator will be able to utilize one single marker to prospectively identify and isolate GSC from all, or possibly, any gliomas.

  16. In vitro anticancer drug test: A new method emerges from the model of glioma stem cells

    Gabriele Riva

    2014-01-01

    Full Text Available Glioblastoma multiforme (GBM is a grade IV astrocytoma and the most common malignant brain tumor. Current therapies provide a median survival of 12–15 months after diagnosis, due to the high recurrence rate. The failure of current therapies may be due to the presence, within the tumor, of cells characterized by enhanced self-renewal capacity, multilineage differentiation potential and elevated invasive behavior, called glioma stem cells (GSCs. To evaluate the pharmacological efficacy of selected drugs on six GSC lines, we set up a multiple drug responsivity assay based on the combined evaluation of cytomorphological and functional parameters, including the analysis of polymorphic nuclei, mitotic index and cell viability. In order to understand the real pharmacological efficacy of the tested drugs, we assigned a specific drug responsivity score to each GSC line, integrating the data produced by multiple assays. In this work we explored the antineoplastic effects of paclitaxel (PTX, an inhibitor of microtubule depolymerization, utilized as standard treatment in several cancers, and of valproic acid (VPA, an inhibitor of histone deacetylases (HDACs with multiple anticancer properties. We classified the six GSC lines as responsive or resistant to these drugs, on the basis of their responsivity scores. This method can also be useful to identify the best way to combine two or more drugs. In particular, we utilized the pro-differentiating effect of VPA to improve the PTX effectiveness and we observed a significant reduction of cell viability compared to single treatments.

  17. Non-coding RNAs as epigenetic regulator of glioma stem-like cell differentiation

    Keisuke eKatsushima

    2014-02-01

    Full Text Available Glioblastomas show heterogeneous histological features. These distinct phenotypic states are thought to be associated with the presence of glioma stem cells (GSCs, which are highly tumorigenic and self-renewing sub-population of tumor cells that have different functional characteristics. Differentiation of GSCs may be regulated by multi-tiered epigenetic mechanisms that orchestrate the expression of thousands of genes. One such regulatory mechanism involves functional non-coding RNAs (ncRNAs, such as microRNAs (miRNAs; a large number of ncRNAs have been identified and shown to regulate the expression of genes associated with cell differentiation programs. Given the roles of miRNAs in cell differentiation, it is possible they are involved in the regulation of gene expression networks in GSCs that are important for the maintenance of the pluripotent state and for directing differentiation. Here, we review recent findings on ncRNAs associated with GSC differentiation and discuss how these ncRNAs contribute to the establishment of tissue heterogeneity during glioblastoma tumor formation.

  18. Oleic Acid and Hydroxytyrosol Inhibit Cholesterol and Fatty Acid Synthesis in C6 Glioma Cells

    Paola Priore

    2017-01-01

    Full Text Available Recently, the discovery of natural compounds capable of modulating nervous system function has revealed new perspectives for a healthier brain. Here, we investigated the effects of oleic acid (OA and hydroxytyrosol (HTyr, two important extra virgin olive oil compounds, on lipid synthesis in C6 glioma cells. OA and HTyr inhibited both de novo fatty acid and cholesterol syntheses without affecting cell viability. The inhibitory effect of the individual compounds was more pronounced if OA and HTyr were administered in combination. A reduction of polar lipid biosynthesis was also detected, while triglyceride synthesis was marginally affected. To clarify the lipid-lowering mechanism of these compounds, their effects on the activity of key enzymes of fatty acid biosynthesis (acetyl-CoA carboxylase-ACC and fatty acid synthase-FAS and cholesterologenesis (3-hydroxy-3-methylglutaryl-CoA reductase-HMGCR were investigated in situ by using digitonin-permeabilized C6 cells. ACC and HMGCR activities were especially reduced after 4 h of 25 μM OA and HTyr treatment. No change in FAS activity was observed. Inhibition of ACC and HMGCR activities is corroborated by the decrease of their mRNA abundance and protein level. Our results indicate a direct and rapid downregulatory effect of the two olive oil compounds on lipid synthesis in C6 cells.

  19. Bmi-1 promotes the aggressiveness of glioma via activating the NF-kappaB/MMP-9 signaling pathway

    Jiang Lili

    2012-09-01

    Full Text Available Abstract Background The prognosis of human glioma is poor, and the highly invasive nature of the disease represents a major impediment to current therapeutic modalities. The oncoprotein B-cell-specific Moloney murine leukemia virus integration site 1 protein (Bmi-1 has been linked to the development and progression of glioma; however, the biological role of Bmi-1 in the invasion of glioma remains unclear. Methods A172 and LN229 glioma cells were engineered to overexpress Bmi-1 via stable transfection or to be silenced for Bmi-1 expression using RNA interfering method. Migration and invasiveness of the engineered cells were assessed using wound healing assay, Transwell migration assay, Transwell matrix penetration assay and 3-D spheroid invasion assay. MMP-9 expression and activity were measured using real-time PCR, ELISA and the gelatin zymography methods. Expression of NF-kappaB target genes was quantified using real-time PCR. NF-kappaB transcriptional activity was assessed using an NF-kappaB luciferase reporter system. Expression of Bmi-1 and MMP-9 in clinical specimens was analyzed using immunohistochemical assay. Results Ectopic overexpression of Bmi-1 dramatically increased, whereas knockdown of endogenous Bmi-1 reduced, the invasiveness and migration of glioma cells. NF-kappaB transcriptional activity and MMP-9 expression and activity were significantly increased in Bmi-1-overexpressing but reduced in Bmi-1-silenced cells. The reporter luciferase activity driven by MMP-9 promoter in Bmi-1-overexpressing cells was dependent on the presence of a functional NF-kappaB binding site, and blockade of NF-kappaB signaling inhibited the upregulation of MMP-9 in Bmi-1 overexpressing cells. Furthermore, expression of Bmi-1 correlated with NF-kappaB nuclear translocation as well as MMP-9 expression in clinical glioma samples. Conclusions Bmi-1 may play an important role in the development of aggressive phenotype of glioma via activating the NF

  20. Nicotinamide augments the survival and incidence of apoptosis in glioma cells following photodynamic therapy in vitro

    Bisland, Stuart K.; Modi, Nayan; Wilson, Brian C.

    2004-10-01

    The ability to customize photodynamic therapy (PDT) parameters with regards to timing and dosing of administered drug and light can be beneficial in determining target specificity and mode of cell death. Sustained, low level PDT or metronomic PDT (mPDT) may afford enhanced apoptotic cell death. This is of particular importance when considering PDT for the treatment of brain tumors as unlike apoptosis, necrotic cell death often leads to inflammation with increased intracranial pressure. The ability, therefore, to 'fine tune' PDT in favour of apoptosis is paramount. We have studied both acute (one time treatment) PDT (aPDT) and mPDT delivery strategies in combination with nicotinamide (NA) in an attempt to maximize the number of tumor cells dieing by apoptosis. Using several different glioma cell lines (9L, U87-MG and CNS-1) we now confirm that NA provides a dose-dependent (0.1-0.5 mM) increase in apoptotic cells following d-aminolevulinic acid-mediated aPDT or mPDT. Furthermore, using the 9L cell line stably transfected with the luciferase gene, NA was shown to delay the depletion of bioluminscence signal in aPDT and mPDT treated cells, inferring that adenosine triphosphate levels are maintained for longer following NA treatment. NA has previously been reported as promoting neuronal and vascular cell survival in normal brain following a number of neurological insults in which reactive oxygen species are implicated including, stroke, Alzheimer's disease and toxin-induced lesions. It is likely that the effects of NA reflect its capacity as an antioxidant as well as its ability to inhibit poly (adenosine diphosphate-ribose) polymerase-mediated depletion of ATP. Our results indicate that NA may prove therapeutically advantageous when used in combination with PDT treatment of brain tumors.

  1. Overexpression of Drosophila frataxin triggers cell death in an iron-dependent manner.

    Edenharter, Oliver; Clement, Janik; Schneuwly, Stephan; Navarro, Juan A

    2017-12-01

    Friedreich ataxia (FRDA) is the most important autosomal recessive ataxia in the Caucasian population. FRDA patients display severe neurological and cardiac symptoms that reflect a strong cellular and axonal degeneration. FRDA is caused by a loss of function of the mitochondrial protein frataxin which impairs the biosynthesis of iron-sulfur clusters and in turn the catalytic activity of several enzymes in the Krebs cycle and the respiratory chain leading to a diminished energy production. Although FRDA is due to frataxin depletion, overexpression might also be very helpful to better understand cellular functions of frataxin. In this work, we have increased frataxin expression in neurons to elucidate specific roles that frataxin might play in these tissues. Using molecular, biochemical, histological and behavioral methods, we report that frataxin overexpression is sufficient to increase oxidative phosphorylation, modify mitochondrial morphology, alter iron homeostasis and trigger oxidative stress-dependent cell death. Interestingly, genetic manipulation of mitochondrial iron metabolism by silencing mitoferrin successfully improves cell survival under oxidative-attack conditions, although enhancing antioxidant defenses or mitochondrial fusion failed to ameliorate frataxin overexpression phenotypes. This result suggests that cell degeneration is directly related to enhanced incorporation of iron into the mitochondria. Drosophila frataxin overexpression might also provide an alternative approach to identify processes that are important in FRDA such as changes in mitochondrial morphology and oxidative stress induced cell death.

  2. Overexpression of mitochondrial sirtuins alters glycolysis and mitochondrial function in HEK293 cells.

    Michelle Barbi de Moura

    Full Text Available SIRT3, SIRT4, and SIRT5 are mitochondrial deacylases that impact multiple facets of energy metabolism and mitochondrial function. SIRT3 activates several mitochondrial enzymes, SIRT4 represses its targets, and SIRT5 has been shown to both activate and repress mitochondrial enzymes. To gain insight into the relative effects of the mitochondrial sirtuins in governing mitochondrial energy metabolism, SIRT3, SIRT4, and SIRT5 overexpressing HEK293 cells were directly compared. When grown under standard cell culture conditions (25 mM glucose all three sirtuins induced increases in mitochondrial respiration, glycolysis, and glucose oxidation, but with no change in growth rate or in steady-state ATP concentration. Increased proton leak, as evidenced by oxygen consumption in the presence of oligomycin, appeared to explain much of the increase in basal oxygen utilization. Growth in 5 mM glucose normalized the elevations in basal oxygen consumption, proton leak, and glycolysis in all sirtuin over-expressing cells. While the above effects were common to all three mitochondrial sirtuins, some differences between the SIRT3, SIRT4, and SIRT5 expressing cells were noted. Only SIRT3 overexpression affected fatty acid metabolism, and only SIRT4 overexpression altered superoxide levels and mitochondrial membrane potential. We conclude that all three mitochondrial sirtuins can promote increased mitochondrial respiration and cellular metabolism. SIRT3, SIRT4, and SIRT5 appear to respond to excess glucose by inducing a coordinated increase of glycolysis and respiration, with the excess energy dissipated via proton leak.

  3. Cardiac cell therapy: overexpression of connexin43 in skeletal myoblasts and prevention of ventricular arrhythmias

    Fernandes, Sarah; van Rijen, Harold V. M.; Forest, Virginie; Evain, Stéphane; Leblond, Anne-Laure; Mérot, Jean; Charpentier, Flavien; de Bakker, Jacques M. T.; Lemarchand, Patricia

    2009-01-01

    Cell-based therapies have great potential for the treatment of cardiovascular diseases. Recently, using a transgenic mouse model Roell et al. reported that cardiac engraftment of connexin43 (Cx43)-overexpressing myoblasts in vivo prevents post-infarct arrhythmia, a common cause of death in patients

  4. ß-Cell Specific Overexpression of GPR39 Protects against Streptozotocin-Induced Hyperglycemia

    Egerod, Kristoffer Lihme; Jin, Chunyu; Petersen, Pia Steen

    2011-01-01

    and OGTT. Although the overexpression of the constitutively active GPR39 receptor in animals not treated with streptozotocin appeared by itself to impair the glucose tolerance slightly and to decrease the ß-cell mass, it nevertheless totally protected against the gradual hyperglycemia in the steptozotocin...

  5. Hypoxic regulation of the expression of genes encoded estrogen related proteins in U87 glioma cells: eff ect of IRE1 inhibition.

    Minchenko, D O; Riabovol, O O; Ratushna, O O; Minchenko, O H

    2017-01-01

    The aim of the present study was to examine the effect of inhibition of endoplasmic reticulum stress signaling, mediated by IRE1 (inositol requiring enzyme 1), which is a central mediator of the unfolded protein response on the expression of genes encoded estrogen related proteins (NRIP1/RIP140, TRIM16/EBBP, ESRRA/NR3B1, FAM162A/E2IG5, PGRMC2/PMBP, and SLC39A6/LIV-1) and their hypoxic regulation in U87 glioma cells for evaluation of their possible significance in the control of glioma cells proliferation. The expression of NRIP1, EBBP, ESRRA, E2IG5, PGRMC2, and SLC39A6 genes in U87 glioma cells, transfected by empty vector pcDNA3.1 (control) and cells without IRE1 signaling enzyme function (transfected by dnIRE1) upon hypoxia, was studied by a quantitative polymerase chain reaction. Inhibition of both enzymatic activities (kinase and endoribonuclease) of IRE1 signaling enzyme function up-regulates the expression of EBBP, E2IG5, PGRMC2, and SLC39A6 genes is in U87 glioma cells in comparison with the control glioma cells, with more significant changes for E2IG5 and PGRMC2 genes. At the same time, the expression of NRIP1 and ESRRA genes is strongly down-regulated in glioma cells upon inhibition of IRE1. We also showed that hypoxia increases the expression of E2IG5, PGRMC2, and EBBP genes and decreases NRIP1 and ESRRA genes expression in control glioma cells. Furthermore, the inhibition of IRE1 in U87 glioma cells decreases the eff ect of hypoxia on the expression of E2IG5 and PGRMC2 genes, eliminates hypoxic regulation of NRIP1 gene, and enhances the sensitivity of ESRRA gene to hypoxic condition. Furthermore, the expression of SLC39A6 gene is resistant to hypoxia in both the glioma cells with and without IRE1 signaling enzyme function. Results of this investigation demonstrate that inhibition of IRE1 signaling enzyme function affects the expression of NRIP1, EBBP, ESRRA, E2IG5, PGRMC2, and SLC39A6 genes in U87 glioma cells in gene specific manner and these changes

  6. Overexpression of IGF-I receptor in HeLa cells enhances in vivo radioresponse

    Kaneko, Haruna; Yu, Dong; Miura, Masahiko

    2007-01-01

    Insulin-like growth factor I receptor (IGF-IR) is a transmembrane receptor tyrosine kinase whose activation strongly promotes cell growth and survival. We previously reported that IGF-IR activity confers intrinsic radioresistance in mouse embryo fibroblasts in vitro. However, it is still unclear whether tumor cells overexpressing IGF-IR exhibit radioresistance in vivo. For this purpose, we established HeLa cells that overexpress IGF-IR (HeLa-R), subcutaneously transplanted these cells into nude mice, and examined radioresponse in the resulting solid tumors. HeLa-R cells exhibited typical in vitro phenotypes generally observed in IGF-IR-overexpressing cells, as well as significant intrinsic radioresistance in vitro compared with parent cells. As expected, the transplanted HeLa-R tumors grew at a remarkably higher rate than parent tumors. Histological analysis revealed that HeLa-R tumors expressed more VEGF and had a higher density of tumor vessels. Unexpectedly, a marked growth delay was observed in HeLa-R tumors following 10 Gy of X-irradiation. Immunostaining of HeLa-R tumors for the hypoxia marker pimonidazole revealed a significantly lower level of hypoxic cells. Moreover, clamp hypoxia significantly increased radioresistance in HeLa-R tumors. Tumor microenvironments in vivo generated by the IGF-IR expression thus could be a major factor in determining the tumor radioresponse in vivo

  7. Regulation of Id2 expression in EL4 T lymphoma cells overexpressing growth hormone.

    Weigent, Douglas A

    2009-01-01

    In previous studies, we have shown that overexpression of growth hormone (GH) in cells of the immune system upregulates proteins involved in cell growth and protects from apoptosis. Here, we report that overexpression of GH in EL4 T lymphoma cells (GHo) also significantly increased levels of the inhibitor of differentiation-2 (Id2). The increase in Id2 was suggested in both Id2 promoter luciferase assays and by Western analysis for Id2 protein. To identify the regulatory elements that mediate transcriptional activation by GH in the Id2 promoter, promoter deletion analysis was performed. Deletion analysis revealed that transactivation involved a 301-132bp region upstream to the Id2 transcriptional start site. The pattern in the human GHo Jurkat T lymphoma cell line paralleled that found in the mouse GHo EL4 T lymphoma cell line. Significantly less Id2 was detected in the nucleus of GHo EL4 T lymphoma cells compared to vector alone controls. Although serum increased the levels of Id2 in control vector alone cells, no difference was found in the total levels of Id2 in GHo EL4 T lymphoma cells treated with or without serum. The increase in Id2 expression in GHo EL4 T lymphoma cells measured by Id2 promoter luciferase expression and Western blot analysis was blocked by the overexpression of a dominant-negative mutant of STAT5. The results suggest that in EL4 T lymphoma cells overexpressing GH, there is an upregulation of Id2 protein that appears to involve STAT protein activity.

  8. Mesenchymal stem cells overexpressing Ihh promote bone repair.

    Zou, Shasha; Chen, Tingting; Wang, Yanan; Tian, Ruhui; Zhang, Lingling; Song, Pingping; Yang, Shi; Zhu, Yong; Guo, Xizhi; Huang, Yiran; Li, Zheng; Kan, Lixin; Hu, Hongliang

    2014-10-28

    Indian hedgehog (Ihh) signaling pathway is known to play key roles in various aspects of normal endochondral bone development. This study tested the potential roles of high Ihh signaling in the context of injury-induced bone regeneration. A rabbit tibia defect model was established to test the effects of the implant of Ihh/mesenchymal stem cells (MSCs)/scaffold complex. Computed tomography (CT), gross observation, and standard histological and immunohistological techniques were used to evaluate the effectiveness of the treatment. In vitro studies with MSCs and C3H10T1/2 cells were also employed to further understand the cellular and molecular mechanisms. We found that the implanted Ihh/MSCs/scaffold complex promoted bone repair. Consistently, in vitro study found that Ihh induced the upregulation of chondrocytic, osteogenic, and vascular cell markers, both in C3H10T1/2 cells and MSCs. Our study has demonstrated that high Ihh signaling in a complex with MSCs enhanced bone regeneration effectively in a clinically relevant acute injury model. Even though the exact underlying mechanisms are still far from clear, our primary data suggested that enhanced chondrogenesis, osteogenesis, and angiogenesis of MSCs at least partially contribute to the process. This study not only has implications for basic research of MSCs and Ihh signaling pathway but also points to the possibility of direct application of this specific paradigm to clinical bone repair.

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

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

    2013-07-01

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

  10. Induction of reactive oxygen intermediates-dependent programmed cell death in human malignant ex vivo glioma cells and inhibition of the vascular endothelial growth factor production by taurolidine.

    Rodak, Roksana; Kubota, Hisashi; Ishihara, Hideyuki; Eugster, Hans-Pietro; Könü, Dilek; Möhler, Hanns; Yonekawa, Yasuhiro; Frei, Karl

    2005-06-01

    Taurolidine, a derivative of the amino acid taurin, was recently found to display a potent antineoplastic effect both in vitro and in vivo. The authors therefore initiated studies to assess the potential antineoplastic activity of taurolidine in human glioma cell lines and in ex vivo malignant cell cultures. They also studied the mechanisms that induce cell death and the impact of taurolidine on tumor-derived vascular endothelial growth factor (VEGF) production. Cytotoxicity and clonogenic assays were performed using crystal violet staining. In the cytotoxicity assay 100% of glioma cell lines (eight of eight) and 74% of ex vivo glioma cultures (14 of 19) demonstrated sensitivity to taurolidine, with a mean median effective concentration (EC50) of 51 +/- 28 microg/ml and 56 +/- 23 microg/ml, respectively. Colony formation was inhibited by taurolidine, with a mean EC50 of 7 +/- 3 microg/ml for the cell lines and a mean EC50 of 3.5 +/- 1.7 microg/ml for the ex vivo glioma cultures. On observing this high activity of taurolidine in both assays, the authors decided to evaluate its cell death mechanisms. Fragmentation of DNA, externalization of phosphatidylserine, activation of poly(adenosine diphosphate-ribose) polymerase, loss of the mitochondrial membrane potential followed by a release of apoptosis-inducing factor, and typical apoptotic features were found after taurolidine treatment. Cell death was preceded by the generation of reactive O2 intermediates, which was abrogated by N-acetylcysteine but not by benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone. Moreover, taurolidine also induced suppression of VEGF production on the protein and messenger RNA level, as shown by an enzyme-linked immunosorbent assay and by reverse transcription-polymerase chain reaction. Given all these findings, taurolidine may be a promising new agent in the treatment of malignant gliomas; it displays a combination of antineoplastic and antiangiogenic activities, inducing tumor cell

  11. In Silico Neuro-Oncology: Brownian Motion-Based Mathematical Treatment as a Potential Platform for Modeling the Infiltration of Glioma Cells into Normal Brain Tissue

    Antonopoulos, Markos; Stamatakos, Georgios

    2015-01-01

    Intensive glioma tumor infiltration into the surrounding normal brain tissues is one of the most critical causes of glioma treatment failure. To quantitatively understand and mathematically simulate this phenomenon, several diffusion-based mathematical models have appeared in the literature. The majority of them ignore the anisotropic character of diffusion of glioma cells since availability of pertinent truly exploitable tomographic imaging data is limited. Aiming at enriching the anisotropy-enhanced glioma model weaponry so as to increase the potential of exploiting available tomographic imaging data, we propose a Brownian motion-based mathematical analysis that could serve as the basis for a simulation model estimating the infiltration of glioblastoma cells into the surrounding brain tissue. The analysis is based on clinical observations and exploits diffusion tensor imaging (DTI) data. Numerical simulations and suggestions for further elaboration are provided. PMID:26309390

  12. Pregnenolone biosynthesis in C6-2B glioma cell mitochondria: regulation by a mitochondrial diazepam binding inhibitor receptor.

    Papadopoulos, V; Guarneri, P; Kreuger, K E; Guidotti, A; Costa, E

    1992-01-01

    The C6-2B glioma cell line, rich in mitochondrial receptors that bind with high affinity to benzodiazepines, imidazopyridines, and isoquinolinecarboxamides (previously called peripheral-type benzodiazepine receptors), was investigated as a model to study the significance of the polypeptide diazepam binding inhibitor (DBI) and the putative DBI processing products on mitochondrial receptor-regulated steroidogenesis. DBI and its naturally occurring fragments have been found to be present in high...

  13. Newcastle disease virus triggers autophagy in U251 glioma cells to enhance virus replication.

    Meng, Chunchun; Zhou, Zhizhi; Jiang, Ke; Yu, Shengqing; Jia, Lijun; Wu, Yantao; Liu, Yanqing; Meng, Songshu; Ding, Chan

    2012-06-01

    Newcastle disease virus (NDV) can replicate in tumor cells and induce apoptosis in late stages of infection. However, the interaction between NDV and cells in early stages of infection is not well understood. Here, we report that, shortly after infection, NDV triggers the formation of autophagosomes in U251 glioma cells, as demonstrated by an increased number of double-membrane vesicles, GFP-microtubule-associated protein 1 light chain 3 (GFP-LC3) a dot formations, and elevated production of LC3II. Moreover, modulation of NDV-induced autophagy by rapamycin, chloroquine or small interfering RNAs targeting the genes critical for autophagosome formation (Atg5 and Beclin-1) affects virus production, indicating that autophagy may be utilized by NDV to facilitate its own production. Furthermore, the class III phosphatidylinositol 3-kinase (PI3K)/Beclin-1 pathway plays a role in NDV-induced autophagy and virus production. Collectively, our data provide a unique example of a paramyxovirus that uses autophagy to enhance its production.

  14. Repurposing phenformin for the targeting of glioma stem cells and the treatment of glioblastoma

    Jiang, Wei; Finniss, Susan; Cazacu, Simona; Xiang, Cunli; Brodie, Ziv; Mikkelsen, Tom; Poisson, Laila; Shackelford, David B.; Brodie, Chaya

    2016-01-01

    Glioblastoma (GBM) is the most aggressive primary brain tumor with poor prognosis. Here, we studied the effects of phenformin, a mitochondrial complex I inhibitor and more potent chemical analog of the diabetes drug metformin on the inhibition of cell growth and induction of apoptosis of glioma stem cells (GSCs) using both in vitro and in vivo models. Phenformin inhibited the self-renewal of GSCs, decreased the expression of stemness and mesenchymal markers and increased the expression of miR-124, 137 and let-7. Silencing of let-7 abrogated phenformin effects on the self-renewal of GSCs via a pathway associated with inhibition of H19 and HMGA2 expression. Moreover, we demonstrate that phenformin inhibited tumor growth and prolonged the overall survival of mice orthotopically transplanted with GSCs. Combined treatments of phenformin and temozolomide exerted an increased antitumor effect on GSCs in vitro and in vivo. In addition, dichloroacetate, an inhibitor of the glycolysis enzyme pyruvate dehydrogenase kinase, that decreases lactic acidosis induced by biguanides, enhanced phenformin effects on the induction of cell death in GSCs and prolonged the survival of xenograft-bearing mice. Our results demonstrate for the first time that phenformin targets GSCs and can be efficiently combined with current therapies for GBM treatment and GSC eradication. PMID:27486821

  15. Overexpression of Periostin and Lumican in Esophageal Squamous Cell Carcinoma

    Kashyap, Manoj Kumar; Marimuthu, Arivusudar; Peri, Suraj; Kumar, Ghantasala S. Sameer; Jacob, Harrys K.C.; Prasad, Thottethodi Subrahmanya Keshava; Mahmood, Riaz; Kumar, K. V. Veerendra; Kumar, M. Vijaya; Meltzer, Stephen J.; Montgomery, Elizabeth A.; Kumar, Rekha V.; Pandey, Akhilesh

    2010-01-01

    To identify biomarkers for early detection for esophageal squamous cell carcinoma (ESCC), we previously carried out a genome-wide gene expression profiling study using an oligonucleotide microarray platform. This analysis led to identification of several transcripts that were significantly upregulated in ESCC compared to the adjacent normal epithelium. In the current study, we performed immunohistochemical analyses of protein products for two candidates genes identified from the DNA microarray analysis, periostin (POSTN) and lumican (LUM), using tissue microarrays. Increased expression of both periostin and lumican was observed in 100% of 137 different ESCC samples arrayed on tissue microarrays. Increased expression of periostin and lumican was observed in carcinoma as well as in stromal cell in the large majority of cases. These findings suggest that these candidates can be investigated in the sera of ESCC patients using ELISA or multiple reaction monitoring (MRM) type assays to further explore their utility as biomarkers

  16. Limb-bud and Heart Overexpression Inhibits the Proliferation and Migration of PC3M Cells.

    Liu, Qicai; Li, Ermao; Huang, Long; Cheng, Minsheng; Li, Li

    2018-01-01

    Background: The limb-bud and heart gene ( LBH ) was discovered in the early 21st century and is specifically expressed in the mouse embryonic limb and heart development. Increasing evidences have indicated that LBH not only plays an important role in embryo development, it is also closely correlated with the occurance and progression of many tumors. However, its function in prostate cancer (PCa) is still not well understood. Here, we explored the effects of LBH on the proliferation and migration of the PCa cell line PC3M. Methods: LBH expression in tissues and cell lines of PCa was detected by immunohistochemistry and Western blotting. Lentivirus was used to transduct the LBH gene into the PC3M cells. Stable LBH-overexpressing PC3M-LBH cells and PC3M-NC control cells were obtained via puromycin screening. Cell proliferation was examined using the 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Cell cycle distribution and apoptosis rate were investigated using flow cytometry. Cell migration was studied using the Transwell assay. Results: LBH expression level was down-regulated in 3 different PCa cell lines, especially in PC3M cells, compared with the normal prostate epithelial cells(RWPE-1). Cell lines of LBH-upregulated PC3M-LBH and PC3M-NC control were successfully constructed. Significantly increased LBH expression level and decreased cyclin D1 and cyclin E2 expression level was found in PC3M-LBH cells as compared to the PC3M-NC cells. The overexpression of LBH significantly inhibited PC3M cell proliferation in vitro and tumor growth in nude mice. LBH overexpression in PC3M cell, also induced cell cycle G0/G1 phase arrest and decreased the migration of PC3M cells. Conclusions : Our results reveal that LBH expression is down-regulated in the tissue and cell lines of PCa. LBH overexpression inhibits PC3M cell proliferation and tumor growth by inducing cell cycle arrest through down-regulating cyclin D1and cyclin E2 expression. LBH might

  17. Radiosensitization and relative mechanisms of vanillin derivative BVAN08 on human glioma U-251 cells

    Wang Shubin; Zhang Bo; Sun Weijian; Wang Yu; Liu Xiaodan; Xu Qinzhi; Zhou Pingkun

    2010-01-01

    Objective: To provide more convincing evidences and experimental data for exploring vanillin derivative BVAN08, 6-bromine-5-hydroxy-4-methoxy-benzaldehyde, as a new anticancer drug, and to investigate the effect on the growth, radiosensitization of human glioma cell line U-251 and the relative mechanism. Methods: The effect of BVAN08 on cell proliferation of U-251 and radiosensitivity to 60 Co γ-rays (irradiation dose rate 2.3 Gy/min) were analyzed with MTT and colony-forming ability assay. Change in cellular morphology was observed by using light microscope. Change in cell cycle and apoptosis was detected with flow cytometry. The autophagy was observed by using TEM (irradiation dose rate is transmission electron microscope). DNA-PKcs protein level was detected through Western blot analysis. Results: BVAN08 exhibited a dose- and time-dependent inhibition on the proliferation of U-251 cells during the concentration range of 10-100 mol/L (t=1.83-3.07, P 50 at 48 h and 72 h after administration with BVAN08 were 55.3 and 52.7 mol/L, respectively. Obvious G 2 /M arrest was induced in U-251 cells after 4 h administration with BVAN08, and reached peck at 12 h. The G 2 /M population reached 63.3% in U-251 cells after 12 h administration of 60 μmol/L BVAN08 and kept increasing with the time, while both apoptosis and autophagic cell death were induced. The most effective radiosensitization time for BVAN08 treatment was 12 h before irradiation. The enhancement ratio of radiosensitivity was 3.14 for 20 μmol/L of BVAN08 12 h before 2 Gy irradiation. Conclusions: BVAN08 can induce apoptosis as radiosensitizing effect might be associated with the induction of G 2 /M arrest and inhibition of DNA-PKcs expression. BVAN08 seemed to be a promising radiosensitizing anticancer drug. (authors)

  18. Human gliomas contain morphine

    Olsen, Peter; Rasmussen, Mads; Zhu, Wei

    2005-01-01

    BACKGROUND: Morphine has been found in cancer cell lines originating from human and animal cells. Thus, it became important to demonstrate whether or not actual tumours contain this opiate alkaloid. MATERIAL/METHODS: Human glioma tissues were biochemically treated to isolate and separate endogeno...... of the solutions used in the study nor was it present as a residual material in blank HPLC runs. CONCLUSIONS: Morphine is present in human gliomas, suggesting that it may exert an action that effects tumour physiology/pathology.......BACKGROUND: Morphine has been found in cancer cell lines originating from human and animal cells. Thus, it became important to demonstrate whether or not actual tumours contain this opiate alkaloid. MATERIAL/METHODS: Human glioma tissues were biochemically treated to isolate and separate endogenous...

  19. Bcl-2 inhibitors potentiate the cytotoxic effects of radiation in Bcl-2 overexpressing radioresistant tumor cells

    Hara, Takamitsu; Omura-Minamisawa, Motoko; Chao Cheng; Nakagami, Yoshihiro; Ito, Megumi; Inoue, Tomio

    2005-01-01

    Purpose: Bcl-2, an inhibitor of apoptosis frequently shows elevated expression in human tumors, thus resulting in resistance to radiation therapy. Therefore, inhibiting Bcl-2 function may enhance the radiosensitivity of tumor cells. Tetrocarcin A (TC-A) and bcl-2 antisense oligonucleotides exhibit antitumor activity by inhibiting Bcl-2 function and transcription, respectively. We investigated whether these antitumor agents would enhance the cytotoxic effects of radiation in tumor cells overexpressing Bcl-2. Methods and materials: We used HeLa/bcl-2 cells, a stable Bcl-2-expressing cell line derived from wild-type HeLa (HeLa/wt) cells. Cells were incubated with TC-A and bcl-2 antisense oligonucleotides for 24 h after irradiation, and cell viability was then determined. Apoptotic cells were quantified by flow cytometric assay. Results: The HeLa/bcl-2 cells were more resistant to radiation than HeLa/wt cells. At concentrations that are not inherently cytotoxic, both TC-A and bcl-2 antisense oligonucleotides increased the cytotoxic effects of radiation in HeLa/bcl-2 cells, but not in HeLa/wt cells. However, in HeLa/bcl-2 cells, additional treatment with TC-A in combination with radiation did not significantly increase apoptosis. Conclusions: The present results suggest that TC-A and bcl-2 antisense oligonucleotides reduce radioresistance of tumor cells overexpressing Bcl-2. Therefore, a combination of radiotherapy and Bcl-2 inhibitors may prove to be a useful therapeutic approach for treating tumors that overexpress Bcl-2

  20. Nras Overexpression Results in Granulocytosis, T-Cell Expansion and Early Lethality in Mice

    Lassen, Louise Berkhoudt; Gonzalez, Borja Ballarin; Schmitz, Alexander

    2012-01-01

    NRAS is a proto-oncogene involved in numerous myeloid malignancies. Here, we report on a mouse line bearing a single retroviral long terminal repeat inserted into Nras. This genetic modification resulted in an increased level of wild type Nras mRNA giving the possibility of studying the function ...... the increment in immature myeloid cells detected in these mice. The short latency period indicates that Nras overexpression alone is sufficient to cause dose-dependent granulocytosis and T-cell expansion....

  1. Increased catalase activity by all-trans retinoic acid and its effect on radiosensitivity in rat glioma cells

    Jin, Hua; Jeon, Ha Yeun; Park, Woo Yoon; Kim, Won Dong; Ahn, Hee Yul; Yu, Jae Ran

    2005-01-01

    It has been reported that all-trans retinoic acid (ATRA) can inhibit glioma growing in vitro. However, clinical trials with ATRA alone in gliomas revealed modest results. ATRA has been shown to increase radiosensitivity in other tumor types, so combining radiation and ATRA would be one of alternatives to increase therapeutic efficacy in malignant gliomas. Thus, we intended to know the role of catalase, which is induced by ATRA, for radiosensitivity. If radiation-reduced reactive oxygen species (ROS) is removed by catalase, the effect of radiation will be reduced. A rat glioma cell line (36B10) was used for this study. The change of catalase activity and radiosensitivity by ATRA, with or without 3-amino-1, 2, 4-triazole (ATZ), a chemical inhibitor of catalase were measured. Catalase activity was measured by the decomposition of H 2 O 2 spectrophotometrically. Radiosensitivity was measured with clonogenic assay. Also ROS was measured using a 2, 7-dichlorofluores-cein diacetate spectrophotometrically. When 36B10 cells were exposed to 10, 25 and 50 μ M of ATRA for 48 h, the expression of catalase activity were increased with increasing concentration and incubation time of ATRA. Catalase activity was decreased with increasing the concentration of AT (1, 10 mM) dose-dependently. ROS was increased with ATRA and it was augmented with the combination of ATRA and radiation. ATZ decreased ROS production and increased cell survival in combination of ATRA and radiation despite the reduction of catalase. The increase of ROS is one of the reasons for the increased radiosensitivity in combination with ATRA. The catalase that is induced by ATRA doesn't decrease ROS production and radiosensitivity

  2. Overexpression of transcription factor Sp1 leads to gene expression perturbations and cell cycle inhibition.

    Emmanuelle Deniaud

    Full Text Available BACKGROUND: The ubiquitous transcription factor Sp1 regulates the expression of a vast number of genes involved in many cellular functions ranging from differentiation to proliferation and apoptosis. Sp1 expression levels show a dramatic increase during transformation and this could play a critical role for tumour development or maintenance. Although Sp1 deregulation might be beneficial for tumour cells, its overexpression induces apoptosis of untransformed cells. Here we further characterised the functional and transcriptional responses of untransformed cells following Sp1 overexpression. METHODOLOGY AND PRINCIPAL FINDINGS: We made use of wild-type and DNA-binding-deficient Sp1 to demonstrate that the induction of apoptosis by Sp1 is dependent on its capacity to bind DNA. Genome-wide expression profiling identified genes involved in cancer, cell death and cell cycle as being enriched among differentially expressed genes following Sp1 overexpression. In silico search to determine the presence of Sp1 binding sites in the promoter region of modulated genes was conducted. Genes that contained Sp1 binding sites in their promoters were enriched among down-regulated genes. The endogenous sp1 gene is one of the most down-regulated suggesting a negative feedback loop induced by overexpressed Sp1. In contrast, genes containing Sp1 binding sites in their promoters were not enriched among up-regulated genes. These results suggest that the transcriptional response involves both direct Sp1-driven transcription and indirect mechanisms. Finally, we show that Sp1 overexpression led to a modified expression of G1/S transition regulatory genes such as the down-regulation of cyclin D2 and the up-regulation of cyclin G2 and cdkn2c/p18 expression. The biological significance of these modifications was confirmed by showing that the cells accumulated in the G1 phase of the cell cycle before the onset of apoptosis. CONCLUSION: This study shows that the binding to DNA

  3. Discovery of Power-Law Growth in the Self-Renewal of Heterogeneous Glioma Stem Cell Populations.

    Michiya Sugimori

    Full Text Available Accumulating evidence indicates that cancer stem cells (CSCs drive tumorigenesis. This suggests that CSCs should make ideal therapeutic targets. However, because CSC populations in tumors appear heterogeneous, it remains unclear how CSCs might be effectively targeted. To investigate the mechanisms by which CSC populations maintain heterogeneity during self-renewal, we established a glioma sphere (GS forming model, to generate a population in which glioma stem cells (GSCs become enriched. We hypothesized, based on the clonal evolution concept, that with each passage in culture, heterogeneous clonal sublines of GSs are generated that progressively show increased proliferative ability.To test this hypothesis, we determined whether, with each passage, glioma neurosphere culture generated from four different glioma cell lines become progressively proliferative (i.e., enriched in large spheres. Rather than monitoring self-renewal, we measured heterogeneity based on neurosphere clone sizes (#cells/clone. Log-log plots of distributions of clone sizes yielded a good fit (r>0.90 to a straight line (log(% total clones = k*log(#cells/clone indicating that the system follows a power-law (y = xk with a specific degree exponent (k = -1.42. Repeated passaging of the total GS population showed that the same power-law was maintained over six passages (CV = -1.01 to -1.17. Surprisingly, passage of either isolated small or large subclones generated fully heterogeneous populations that retained the original power-law-dependent heterogeneity. The anti-GSC agent Temozolomide, which is well known as a standard therapy for glioblastoma multiforme (GBM, suppressed the self-renewal of clones, but it never disrupted the power-law behavior of a GS population.Although the data above did not support the stated hypothesis, they did strongly suggest a novel mechanism that underlies CSC heterogeneity. They indicate that power-law growth governs the self-renewal of heterogeneous

  4. Targeting Src family kinases inhibits bevacizumab-induced glioma cell invasion.

    Deborah Huveldt

    Full Text Available Anti-VEGF antibody therapy with bevacizumab provides significant clinical benefit in patients with recurrent glioblastoma multiforme (GBM. Unfortunately, progression on bevacizumab therapy is often associated with a diffuse disease recurrence pattern, which limits subsequent therapeutic options. Therefore, there is an urgent need to understand bevacizumab's influence on glioma biology and block it's actions towards cell invasion. To explore the mechanism(s of GBM cell invasion we have examined a panel of serially transplanted human GBM lines grown either in short-term culture, as xenografts in mouse flank, or injected orthotopically in mouse brain. Using an orthotopic xenograft model that exhibits increased invasiveness upon bevacizumab treatment, we also tested the effect of dasatinib, a broad spectrum SFK inhibitor, on bevacizumab-induced invasion.We show that 1 activation of Src family kinases (SFKs is common in GBM, 2 the relative invasiveness of 17 serially transplanted GBM xenografts correlates strongly with p120 catenin phosphorylation at Y228, a Src kinase site, and 3 SFK activation assessed immunohistochemically in orthotopic xenografts, as well as the phosphorylation of downstream substrates occurs specifically at the invasive tumor edge. Further, we show that SFK signaling is markedly elevated at the invasive tumor front upon bevacizumab administration, and that dasatinib treatment effectively blocked the increased invasion induced by bevacizumab.Our data are consistent with the hypothesis that the increased invasiveness associated with anti-VEGF therapy is due to increased SFK signaling, and support testing the combination of dasatinib with bevacizumab in the clinic.

  5. [Role of autophagy in TXNIP overexpression-induced apoptosis of INS-1 islet cells].

    Wang, Jing; Wang, Jin; Wang, Juan-Juan; Zhang, Wei-Fang; Jiao, Xiang-Ying

    2017-08-25

    Thioredoxin (Trx) interacting protein (TXNIP) is a Trx-binding protein that inhibits the antioxidative function of Trx and is highly expressed in the serum and tissue samples from diabetes patients. This study was to explore whether TXNIP overexpression could cause INS-1 cell autophagy under normal glucose and lipid concentrations, and to analyze the role of autophagy in the apoptosis of INS-1 cells. The INS-1 cells cultured under normal conditions were divided into three groups: normal control, empty adenovirus vector (Ad-eGFP) and TXNIP overexpression (Ad-TXNIP-eGFP) groups. Forty-eight hours after transfection, the expression levels of TXNIP mRNA and protein were measured. Western blot was used to examine the protein expression levels of Beclin-1 and P62, as well as LC3-II/LC3-I ratio, which are associated with autophagy. IF/ICC was used to measure the autophagosome. In addition, the cleaved caspase-3/caspase-3 ratio, the apoptosis marker, was also measured, and the apoptotic rates were detected by flow cytometry (FCM). The results showed that the TXNIP mRNA and protein levels were significantly up-regulated in Ad-TXNIP-eGFP group, suggesting that TXNIP overexpression model was successfully established. In Ad-TXNIP-eGFP group, the protein levels of Beclin-1 and LC3-II/LC3-I ratio were increased, while the protein expression of P62 was decreased, compared with those in Ad-eGFP group. Red fluorescent intensity, representing autophagy level, was higher in Ad-TXNIP-eGFP group than that in Ad-eGFP group. These results suggested that TXNIP overexpression can significantly promote INS-1 cell autophagy. Meanwhile, cleaved caspase 3/caspase 3 ratio and the number of apoptotic cells were significantly increased in Ad-TXNIP-eGFP group. The inhibitor of autophagy, 3-MA, reduced TXNIP overexpression-induced apoptosis in INS-1 cells. Taken together, our data demonstrate that autophagy appears to be an important pathway in TXNIP overexpression-induced apoptosis in INS-1 cells.

  6. [Overexpression of liver kinase B1 inhibits the proliferation of lung cancer cells].

    Li, Yang; Zhang, Libin; Wang, Ping

    2017-01-01

    Objective To explore the effect of overexpressed liver kinase B1(LKB1) on the proliferation of lung cancer cell lines. Methods The expression levels of LKB1 and PTEN in A549, NCI-H23, NCI-H157, XWLC-05, NCI-H446 lung cancer cells were detected by immunocytochemistry (ICC) and Western blotting. Plasmid pcDNA3.1 + -LKB1 and empty vector pcDNA3.1 + -null were separately transfected into the above five cell lines, and then the expression of LKB1 mRNA and protein were determined by quantitative real-time PCR and Western blotting, respectively. Finally, CCK-8 assay was used to analyze the proliferation ability of the transfected cells. Results LKB1 and PTEN were positive in NCI-H23 cells; LKB1 was negative while PTEN was positive in A549 and NCI-H446 cells; both LKB1 and PTEN were negative in NCI-H157 and XWLC-05 cells. Quantitative real-time PCR and Western blotting showed that the expression level of LKB1 significantly increased in the above cell lines transfected with plasmid pcDNA3.1 + -LKB1 compared with the ones with empty vector pcDNA3.1 + -null. Besides, CCK-8 assay showed that the overexpression of LKB1 in the lung cancer cells transfected with pcDNA3.1 + -LKB1 had an obvious inhibitory effect on cell proliferation. Conclusion The expression of LKB1 is down-regulated in most of the lung cell lines to different extent and the over-expression of LKB1 can remarkably inhibit the proliferation ability of lung cancer cell lines.

  7. Overexpression of PGC-1α Increases Fatty Acid Oxidative Capacity of Human Skeletal Muscle Cells

    Nataša Nikolić

    2012-01-01

    Full Text Available We investigated the effects of PGC-1α (peroxisome proliferator-activated receptor γ coactivator-1α overexpression on the oxidative capacity of human skeletal muscle cells ex vivo. PGC-1α overexpression increased the oxidation rate of palmitic acid and mRNA expression of genes regulating lipid metabolism, mitochondrial biogenesis, and function in human myotubes. Basal and insulin-stimulated deoxyglucose uptake were decreased, possibly due to upregulation of PDK4 mRNA. Expression of fast fiber-type gene marker (MHCIIa was decreased. Compared to skeletal muscle in vivo, PGC-1α overexpression increased expression of several genes, which were downregulated during the process of cell isolation and culturing. In conclusion, PGC-1α overexpression increased oxidative capacity of cultured myotubes by improving lipid metabolism, increasing expression of genes involved in regulation of mitochondrial function and biogenesis, and decreasing expression of MHCIIa. These results suggest that therapies aimed at increasing PGC-1α expression may have utility in treatment of obesity and obesity-related diseases.

  8. Cyclopamine and jervine induce COX-2 overexpression in human erythroleukemia cells but only cyclopamine has a pro-apoptotic effect

    Ghezali, Lamia; Leger, David Yannick; Limami, Youness; Cook-Moreau, Jeanne; Beneytout, Jean-Louis; Liagre, Bertrand

    2013-01-01

    Erythroleukemia is generally associated with a very poor response and survival to current available therapeutic agents. Cyclooxygenase-2 (COX-2) has been described to play a crucial role in the proliferation and differentiation of leukemia cells, this enzyme seems to play an important role in chemoresistance in different cancer types. Previously, we demonstrated that diosgenin, a plant steroid, induced apoptosis in HEL cells with concomitant COX-2 overexpression. In this study, we investigated the antiproliferative and apoptotic effects of cyclopamine and jervine, two steroidal alkaloids with similar structures, on HEL and TF1a human erythroleukemia cell lines and, for the first time, their effect on COX-2 expression. Cyclopamine, but not jervine, inhibited cell proliferation and induced apoptosis in these cells. Both compounds induced COX-2 overexpression which was responsible for apoptosis resistance. In jervine-treated cells, COX-2 overexpression was NF-κB dependent. Inhibition of NF-κB reduced COX-2 overexpression and induced apoptosis. In addition, cyclopamine induced apoptosis and COX-2 overexpression via PKC activation. Inhibition of the PKC pathway reduced both apoptosis and COX-2 overexpression in both cell lines. Furthermore, we demonstrated that the p38/COX-2 pathway was involved in resistance to cyclopamine-induced apoptosis since p38 inhibition reduced COX-2 overexpression and increased apoptosis in both cell lines. - Highlights: ► Cyclopamine alone but not jervine induces apoptosis in human erythroleukemia cells. ► Cyclopamine and jervine induce COX-2 overexpression. ► COX-2 overexpression is implicated in resistance to cyclopamine-induced apoptosis. ► Apoptotic potential of jervine is restrained by NF-κB pathway activation. ► PKC is involved in cyclopamine-induced apoptosis and COX-2 overexpression

  9. Cyclopamine and jervine induce COX-2 overexpression in human erythroleukemia cells but only cyclopamine has a pro-apoptotic effect

    Ghezali, Lamia; Leger, David Yannick; Limami, Youness [Université de Limoges, FR 3503 GEIST, EA 1069 “Laboratoire de Chimie des Substances Naturelles”, GDR CNRS 3049, Faculté de Pharmacie, Laboratoire de Biochimie et Biologie Moléculaire, 2 rue du Docteur Marcland, 87025 Limoges Cedex (France); Cook-Moreau, Jeanne [Université de Limoges, FR 3503 GEIST, UMR CNRS 7276 “Contrôle de la réponse immune B et lymphoproliférations”, Faculté de Médecine, 2 rue du Docteur Marcland, 87025 Limoges Cedex (France); Beneytout, Jean-Louis [Université de Limoges, FR 3503 GEIST, EA 1069 “Laboratoire de Chimie des Substances Naturelles”, GDR CNRS 3049, Faculté de Pharmacie, Laboratoire de Biochimie et Biologie Moléculaire, 2 rue du Docteur Marcland, 87025 Limoges Cedex (France); Liagre, Bertrand, E-mail: bertrand.liagre@unilim.fr [Université de Limoges, FR 3503 GEIST, EA 1069 “Laboratoire de Chimie des Substances Naturelles”, GDR CNRS 3049, Faculté de Pharmacie, Laboratoire de Biochimie et Biologie Moléculaire, 2 rue du Docteur Marcland, 87025 Limoges Cedex (France)

    2013-04-15

    Erythroleukemia is generally associated with a very poor response and survival to current available therapeutic agents. Cyclooxygenase-2 (COX-2) has been described to play a crucial role in the proliferation and differentiation of leukemia cells, this enzyme seems to play an important role in chemoresistance in different cancer types. Previously, we demonstrated that diosgenin, a plant steroid, induced apoptosis in HEL cells with concomitant COX-2 overexpression. In this study, we investigated the antiproliferative and apoptotic effects of cyclopamine and jervine, two steroidal alkaloids with similar structures, on HEL and TF1a human erythroleukemia cell lines and, for the first time, their effect on COX-2 expression. Cyclopamine, but not jervine, inhibited cell proliferation and induced apoptosis in these cells. Both compounds induced COX-2 overexpression which was responsible for apoptosis resistance. In jervine-treated cells, COX-2 overexpression was NF-κB dependent. Inhibition of NF-κB reduced COX-2 overexpression and induced apoptosis. In addition, cyclopamine induced apoptosis and COX-2 overexpression via PKC activation. Inhibition of the PKC pathway reduced both apoptosis and COX-2 overexpression in both cell lines. Furthermore, we demonstrated that the p38/COX-2 pathway was involved in resistance to cyclopamine-induced apoptosis since p38 inhibition reduced COX-2 overexpression and increased apoptosis in both cell lines. - Highlights: ► Cyclopamine alone but not jervine induces apoptosis in human erythroleukemia cells. ► Cyclopamine and jervine induce COX-2 overexpression. ► COX-2 overexpression is implicated in resistance to cyclopamine-induced apoptosis. ► Apoptotic potential of jervine is restrained by NF-κB pathway activation. ► PKC is involved in cyclopamine-induced apoptosis and COX-2 overexpression.

  10. CENPI is overexpressed in colorectal cancer and regulates cell migration and invasion.

    Ding, Na; Li, Rongxin; Shi, Wenhao; He, Cui

    2018-06-21

    Centromere protein I (CENPI),an important member of centromere protein family, has been suggest to serve as a oncogene in breast cancer, but the clinical significance and biological function of CENPI in colorectal cancer (CRC) is still unclear. In our results, we found CENPI was overexpressed in CRC tissues and cells, and associated with clinical stage, tumor depth, lymph node metastasis, distant metastasis and differentiation in CRC patients. However, there was no significant association between CENPI protein expression and overall survival time in colon cancer patients and rectal cancer patients through analyzing TCGA survival data. Moreover, CENPI mRNA and protein were increased in metastatic lymph nodes compared with primary CRC tissues. Down-regulation of CENPI expression suppresses CRC cell migration, invasion and epithelial mesenchymal transition process. In conclusion, CENPI is overexpressed in CRC and functions as oncogene in modulating CRC cell migration, invasion and EMT process. Copyright © 2018. Published by Elsevier B.V.

  11. EGFR overexpressing cells and tumors are dependent on autophagy for growth and survival

    Jutten, Barry; Keulers, Tom G.; Schaaf, Marco B.E.; Savelkouls, Kim; Theys, Jan; Span, Paul N.; Vooijs, Marc A.; Bussink, Johan; Rouschop, Kasper M.A.

    2013-01-01

    Background and purpose: The epidermal growth factor receptor (EGFR) is overexpressed, amplified or mutated in various human epithelial tumors, and is associated with tumor aggressiveness and therapy resistance. Autophagy activation provides a survival advantage for cells in the tumor microenvironment. In the current study, we assessed the potential of autophagy inhibition (using chloroquine (CQ)) in treatment of EGFR expressing tumors. Material and methods: Quantitative PCR, immunohistochemistry, clonogenic survival, proliferation assays and in vivo tumor growth were used to assess this potential. Results: We show that EGFR overexpressing xenografts are sensitive to CQ treatment and are sensitized to irradiation by autophagy inhibition. In HNSSC xenografts, a correlation between EGFR and expression of the autophagy marker LC3b is observed, suggesting a role for autophagy in EGFR expressing tumors. This observation was substantiated in cell lines, showing high EGFR expressing cells to be more sensitive to CQ addition as reflected by decreased proliferation and survival. Surprisingly high EGFR expressing cells display a lower autophagic flux. Conclusions: The EGFR high expressing cells and tumors investigated in this study are highly dependent on autophagy for growth and survival. Inhibition of autophagy may therefore provide a novel treatment opportunity for EGFR overexpressing tumors

  12. Transplantation of mesenchymal stem cells overexpressing IL10 attenuates cardiac impairments in rats with myocardial infarction.

    Meng, Xin; Li, Jianping; Yu, Ming; Yang, Jian; Zheng, Minjuan; Zhang, Jinzhou; Sun, Chao; Liang, Hongliang; Liu, Liwen

    2018-01-01

    Mesenchymal stem cell (MSC) has been well known to exert therapeutic potential for patients with myocardial infarction (MI). In addition, interleukin-10 (IL10) could attenuate MI through suppressing inflammation. Thus, the combination of MSC implantation with IL10 delivery may extend health benefits to ameliorate cardiac injury after MI. Here we established overexpression of IL10 in bone marrow-derived MSC through adenoviral transduction. Cell viability, apoptosis, and IL10 secretion under ischemic challenge in vitro were examined. In addition, MSC was transplanted into the injured hearts in a rat model of MI. Four weeks after the MI induction, MI, cardiac functions, apoptotic cells, and inflammation cytokines were assessed. In response to in vitro oxygen-glucose deprivation (OGD), IL10 overexpression in MSC (Ad.IL10-MSC) enhanced cell viability, decreased apoptosis, and increased IL10 secretion. Consistently, the implantation of Ad.IL10-MSCs into MI animals resulted in more reductions in myocardial infarct size, cardiac impairment, and cell apoptosis, compared to the individual treatments of either MSC or IL10 administration. Moreover, the attenuation of both systemic and local inflammations was most prominent for Ad.IL10-MSC treatment. IL10 overexpression and MSC may exert a synergistic anti-inflammatory effect to alleviate cardiac injury after MI. © 2017 Wiley Periodicals, Inc.

  13. Cell death is induced by ciglitazone, a peroxisome proliferator-activated receptor γ (PPARγ) agonist, independently of PPARγ in human glioma cells

    Lee, Myoung Woo; Kim, Dae Seong; Kim, Hye Ryung; Kim, Hye Jin; Yang, Jin Mo; Ryu, Somi; Noh, Yoo Hun; Lee, Soo Hyun; Son, Meong Hi; Jung, Hye Lim; Yoo, Keon Hee; Koo, Hong Hoe; Sung, Ki Woong

    2012-01-01

    Highlights: ► Greater than 30 μM ciglitazone induces cell death in glioma cells. ► Cell death by ciglitazone is independent of PPARγ in glioma cells. ► CGZ induces cell death by the loss of MMP via decreased Akt. -- Abstract: Peroxisome proliferator-activated receptor γ (PPARγ) regulates multiple signaling pathways, and its agonists induce apoptosis in various cancer cells. However, their role in cell death is unclear. In this study, the relationship between ciglitazone (CGZ) and PPARγ in CGZ-induced cell death was examined. At concentrations of greater than 30 μM, CGZ, a synthetic PPARγ agonist, activated caspase-3 and induced apoptosis in T98G cells. Treatment of T98G cells with less than 30 μM CGZ effectively induced cell death after pretreatment with 30 μM of the PPARγ antagonist GW9662, although GW9662 alone did not induce cell death. This cell death was also observed when cells were co-treated with CGZ and GW9662, but was not observed when cells were treated with CGZ prior to GW9662. In cells in which PPARγ was down-regulated cells by siRNA, lower concentrations of CGZ (<30 μM) were sufficient to induce cell death, although higher concentrations of CGZ (⩾30 μM) were required to induce cell death in control T98G cells, indicating that CGZ effectively induces cell death in T98G cells independently of PPARγ. Treatment with GW9662 followed by CGZ resulted in a down-regulation of Akt activity and the loss of mitochondrial membrane potential (MMP), which was accompanied by a decrease in Bcl-2 expression and an increase in Bid cleavage. These data suggest that CGZ is capable of inducing apoptotic cell death independently of PPARγ in glioma cells, by down-regulating Akt activity and inducing MMP collapse.

  14. Identification of cytotoxic drugs that selectively target tumor cells with MYC overexpression.

    Anna Frenzel

    Full Text Available Expression of MYC is deregulated in a wide range of human cancers, and is often associated with aggressive disease and poorly differentiated tumor cells. Identification of compounds with selectivity for cells overexpressing MYC would hence be beneficial for the treatment of these tumors. For this purpose we used cell lines with conditional MYCN or c-MYC expression, to screen a library of 80 conventional cytotoxic compounds for their ability to reduce tumor cell viability and/or growth in a MYC dependent way. We found that 25% of the studied compounds induced apoptosis and/or inhibited proliferation in a MYC-specific manner. The activities of the majority of these were enhanced both by c-MYC or MYCN over-expression. Interestingly, these compounds were acting on distinct cellular targets, including microtubules (paclitaxel, podophyllotoxin, vinblastine and topoisomerases (10-hydroxycamptothecin, camptothecin, daunorubicin, doxorubicin, etoposide as well as DNA, RNA and protein synthesis and turnover (anisomycin, aphidicholin, gliotoxin, MG132, methotrexate, mitomycin C. Our data indicate that MYC overexpression sensitizes cells to disruption of specific pathways and that in most cases c-MYC and MYCN overexpression have similar effects on the responses to cytotoxic compounds. Treatment of the cells with topoisomerase I inhibitors led to down-regulation of MYC protein levels, while doxorubicin and the small molecule MYRA-A was found to disrupt MYC-Max interaction. We conclude that the MYC pathway is only targeted by a subset of conventional cytotoxic drugs currently used in the clinic. Elucidating the mechanisms underlying their specificity towards MYC may be of importance for optimizing treatment of tumors with MYC deregulation. Our data also underscores that MYC is an attractive target for novel therapies and that cellular screenings of chemical libraries can be a powerful tool for identifying compounds with a desired biological activity.

  15. MiR-26b Mimic Inhibits Glioma Proliferation In Vitro and In Vivo Suppressing COX-2 Expression.

    Chen, Zheng-Gang; Zheng, Chuan-Yi; Cai, Wang-Qing; Li, Da-Wei; Ye, Fu-Yue; Zhou, Jian; Wu, Ran; Yang, Kun

    2017-08-11

    Glioma is the most common malignant tumor of the nervous system. Studies have shown the microRNA (miR)-26b/cyclooxygenase (COX)-2 axis in the development and progression in many tumor cells. Our study aims to investigate the effect and mechanism of miR-26b/COX-2 axis in glioma. Decreased expression of miR-26b with increased level of COX-2 was found in glioma tissues compared with matched normal tissues. A strong negative correlation was observed between the level of miR-26b and COX-2 in 30 glioma tissues. The miR-26b was then overexpressed by transfecting miR-26b mimic into U-373 cells. The invasive cell number and wounld closing rate were reduced in U-373 cells transfected with miR-26b mimic. Besides, COX2 siRNA enhanced the effect of miR-26b mimic in suppressing the expression of p-ERK1 and p-JNK. Finally, the in vivo experiment revealed that miR-26b mimic transfection strongly reduced the tumor growth, tumor volume and the expression of matrix metalloproteinase (MMP)-2, MMP-9). Taken together, our research indicated a miR-26b/COX-2/ERK/JNK axis in regulating the motility of glioma in vitro and in vivo, providing a new sight for treatment of glioma.

  16. Increased sensitivity to ionizing radiation by targeting the homologous recombination pathway in glioma initiating cells.

    Lim, Yi Chieh; Roberts, Tara L; Day, Bryan W; Stringer, Brett W; Kozlov, Sergei; Fazry, Shazrul; Bruce, Zara C; Ensbey, Kathleen S; Walker, David G; Boyd, Andrew W; Lavin, Martin F

    2014-12-01

    Glioblastoma is deemed the most malignant form of brain tumour, particularly due to its resistance to conventional treatments. A small surviving group of aberrant stem cells termed glioma initiation cells (GICs) that escape surgical debulking are suggested to be the cause of this resistance. Relatively quiescent in nature, GICs are capable of driving tumour recurrence and undergo lineage differentiation. Most importantly, these GICs are resistant to radiotherapy, suggesting that radioresistance contribute to their survival. In a previous study, we demonstrated that GICs had a restricted double strand break (DSB) repair pathway involving predominantly homologous recombination (HR) associated with a lack of functional G1/S checkpoint arrest. This unusual behaviour led to less efficient non-homologous end joining (NHEJ) repair and overall slower DNA DSB repair kinetics. To determine whether specific targeting of the HR pathway with small molecule inhibitors could increase GIC radiosensitivity, we used the Ataxia-telangiectasia mutated inhibitor (ATMi) to ablate HR and the DNA-dependent protein kinase inhibitor (DNA-PKi) to inhibit NHEJ. Pre-treatment with ATMi prior to ionizing radiation (IR) exposure prevented HR-mediated DNA DSB repair as measured by Rad51 foci accumulation. Increased cell death in vitro and improved in vivo animal survival could be observed with combined ATMi and IR treatment. Conversely, DNA-PKi treatment had minimal impact on GICs ability to resolve DNA DSB after IR with only partial reduction in cell survival, confirming the major role of HR. These results provide a mechanistic insight into the predominant form of DNA DSB repair in GICs, which when targeted may be a potential translational approach to increase patient survival. Copyright © 2014. Published by Elsevier B.V.

  17. Human gliomas contain morphine

    Olsen, Peter; Rasmussen, Mads; Zhu, Wei

    2005-01-01

    BACKGROUND: Morphine has been found in cancer cell lines originating from human and animal cells. Thus, it became important to demonstrate whether or not actual tumours contain this opiate alkaloid. MATERIAL/METHODS: Human glioma tissues were biochemically treated to isolate and separate endogenous...

  18. P02.04MICRORNA-MEDIATED DOWN-REGULATION OF NKG2D LIGAND EXPRESSION REDUCES GLIOMA CELL IMMUNOGENICITY

    Codo, P.; Weller, M.; Meister, G.; Szabo, E.; Steinle, A.; Wolter, M.; Reifenberger, G.; Roth, P.

    2014-01-01

    Glioblastoma is a primary brain tumor with a dismal prognosis despite comprehensive therapeutic regimens. It is characterized by diffuse infiltration of the surrounding healthy brain tissue, well-adapted to hypoxic conditions and regarded as paradigmatic for tumor-associated immunosuppression. One of the major activating receptors of natural killer (NK) cells is NKG2D. It binds to at least 8 ligands (NKG2DL) which are induced after malignant transformation and cellular stress. Regulation of NKG2DL expression may be affected by endogenous RNA molecules known as microRNA (miRNA). Here, we aimed at characterizing the role of miRNA in the control of NKG2DL expression in glioma cells. We selected 6 miRNA that were described or predicted to target NKG2DL. Three of the miRNA candidates, miR-20a, miR-93 and miR-106b, were expressed in glioma cell lines and were also detected in glioblastoma tissue specimens. Silencing of these miRNA with locked nucleic acid (LNA) molecules resulted in an up-regulation of NKG2DL cell surface levels which translated into increased sensitivity to immune cell killing. This effect was reversed by neutralizing NKG2D antibodies, confirming that enhanced immune cell lysis upon miRNA silencing was mediated through the NKG2D system. We conclude that the expression of several miRNA may contribute to the immune escape of glioma cells at the level of the NKG2D system. Therapeutic targeting of miRNA that regulate NKG2DL levels may therefore represent a promising approach to allow for more potent immune responses against glioblastoma.

  19. Microglia immunophenotyping in gliomas

    Annovazzi, Laura; Mellai, Marta; Bovio, Enrica; Mazzetti, Samanta; Pollo, Bianca; Schiffer, Davide

    2018-01-01

    Microglia, once assimilated to peripheral macrophages, in gliomas has long been discussed and currently it is hypothesized to play a pro-tumor role in tumor progression. Uncertain between M1 and M2 polarization, it exchanges signals with glioma cells to create an immunosuppressive microenvironment and stimulates cell proliferation and migration. Four antibodies are currently used for microglia/macrophage identification in tissues that exhibit different cell forms and cell localization. The aim of the present work was to describe the distribution of the different cell forms and to deduce their significance on the basis of what is known on their function from the literature. Normal resting microglia, reactive microglia, intermediate and bumpy forms and macrophage-like cells can be distinguished by Iba1, CD68, CD16 and CD163 and further categorized by CD11b, CD45, c-MAF and CD98. The number of microglia/macrophages strongly increased from normal cortex and white matter to infiltrating and solid tumors. The ramified microglia accumulated in infiltration areas of both high- and low-grade gliomas, when hypertrophy and hyperplasia occur. In solid tumors, intermediate and bumpy forms prevailed and there is a large increase of macrophage-like cells in glioblastoma. The total number of microglia cells did not vary among the three grades of malignancy, but macrophage-like cells definitely prevailed in high-grade gliomas and frequently expressed CD45 and c-MAF. CD98+ cells were present. Microglia favors tumor progression, but many aspects suggest that the phagocytosing function is maintained. CD98+ cells can be the product of fusion, but also of phagocytosis. Microglia correlated with poorer survival in glioblastoma, when considering CD163+ cells, whereas it did not change prognosis in isocitrate dehydrogenase-mutant low grade gliomas. PMID:29399160

  20. SIRT1 overexpression is an independent prognosticator for patients with esophageal squamous cell carcinoma.

    Ma, Ming-Chun; Chiu, Tai-Jan; Lu, Hung-I; Huang, Wan-Ting; Lo, Chien-Ming; Tien, Wan-Yu; Lan, Ya-Chun; Chen, Yen-Yang; Chen, Chang-Han; Li, Shau-Hsuan

    2018-04-10

    Sirtuin 1 (SIRT1) regulates DNA repair and metabolism by deacetylating target proteins. SIRT1 may be oncogenic because its overexpression has been detected in many cancers. The aim of the present study was to clarify the prognostic role of SIRT1 in patients with esophageal squamous cell carcinoma (ESCC) and evaluate the effect of SIRT1 inhibitor in vitro. The expression of SIRT1 was evaluated immunohistochemically in 155 surgically resected ESCC and the staining results were evaluated semiquantitatively by the Immunoreactive Scoring System. The clinical features and treatment outcome were analyzed. The effect of SIRT1 inhibitor, SIRT 1 inhibitor IV, (S)-35, was investigated in vitro on ESCC cell lines. The expression of SIRT1 on ESCC did not correlate with age, gender, tumor location, stage, T classification, N classification, surgical margin or histology. Univariate analysis showed that SIRT1 overexpression was associated with inferior overall survival (P = 0.004) and disease-free survival (P = 0.004). In multivariate comparison, SIRT1 overexpression remained independently associated with worse overall survival (P = 0.009, hazard ratio = 1.776) and disease-free survival (P = 0.017, hazard ratio = 1.642). In cell lines, SIRT1 inhibitor inhibited ESCC growth. Our study suggests that SIRT1 overexpression is an independent prognosticator for patients with ESCC and the SIRT1 inhibitor suppressed cell proliferation of ESCC cell lines. Our findings suggest that inhibition of SIRT1 signaling may be a promising novel target for ESCC.

  1. CEP55 overexpression predicts poor prognosis in patients with locally advanced esophageal squamous cell carcinoma.

    Jiang, Wenpeng; Wang, Zhou; Jia, Yang

    2017-01-01

    Development of esophageal squamous cell carcinoma (ESCC) involves alterations in multiple genes with corresponding proteins. Recent studies have demonstrated that centrosomal protein 55 (CEP55) shares certain features with oncogenes, and CEP55 overexpression is associated with the development and progression of malignant tumors. The present study aimed to analyze, for the first time, whether CEP55 expression is related to clinicopothalogic features in the esophageal squamous cell carcinoma (ESCC), as well as patient survival. A total of 110 patients with mid-thoracic ESCC who suffered from Ivor-Lewis were enrolled. The CEP55 expression profile of these patients in tumour tissues and corresponding healthy esophageal mucosa (CHEM) was detected by immunohistochemistry and semi-quantitative reverse transcription-polymerase chain reaction analyses. Correlations between CEP55 expression and clinicopathological factors were analyzed using χ 2 test. The log-rank test was employed to calculate survival rate. A Cox regression multivariate analysis was performed to determine independent prognostic factors. The results demonstrated that CEP55 expression in ESCC was significantly higher than that of CHEM (POverexpression of CEP55 was significantly associated with differentiation degree (P=0.022), T stage (P=0.019), lymph node metastasis (P=0.033), clinicopathological staging (P=0.002) and tumor recurrence (P=0.021) in locally advanced ESCC patients. In addition, CEP55 overexpression was significantly associated with reduced overall survival of patients after surgery (P=0.012). The 5-year survival rate of patients without CEP55 overexpression was significantly higher than that of patients with CEP55 overexpression (P=0.012). Therefore, these findings suggest that CEP55 overexpression correlates with poor prognosis in locally advanced ESCC patients.

  2. Antitumor action of temozolomide, ritonavir and aprepitant against human glioma cells.

    Kast, Richard E; Ramiro, Susana; Lladó, Sandra; Toro, Salvador; Coveñas, Rafael; Muñoz, Miguel

    2016-02-01

    In the effort to find better treatments for glioblastoma we tested several currently marketed non-chemotherapy drugs for their ability to enhance the standard cytotoxic drug currently used to treat glioblastoma- temozolomide. We tested four antiviral drugs- acyclovir, cidofovir, maraviroc, ritonavir, and an anti-emetic, aprepitant. We found no cytotoxicity of cidofovir and discussed possible reasons for discrepancy from previous findings of others. We also found no cytotoxicity from acyclovir or maraviroc also in contradistinction to predictions. Cytotoxicity to glioma cell line GAMG for temozolomide alone was 14%, aprepitant alone 7%, ritonavir alone 14%, while temozolomide + aprepitant was 19%, temozolomide + ritonavir 34%, ritonavir + aprepitant 64 %, and all three, temozolomide + ritonavir + aprepitant 78%. We conclude that a remarkable synergy exists between aprepitant and ritonavir. Given the long clinical experience with these two well-tolerated drugs in treating non-cancer conditions, and the current median survival of glioblastoma of 2 years, a trial is warranted of adding these two simple drugs to current standard treatment with temozolomide.

  3. HIF-2α mediates a marked increase in migration and stemness characteristics in a subset of glioma cells under hypoxia by activating an Oct-4/Sox-2-Mena (INV) axis.

    Bhagat, Mohita; Palanichamy, Jayanth Kumar; Ramalingam, Pradeep; Mudassir, Madeeha; Irshad, Khushboo; Chosdol, Kunzang; Sarkar, Chitra; Seth, Pankaj; Goswami, Sumanta; Sinha, Subrata; Chattopadhyay, Parthaprasad

    2016-05-01

    Hypoxia is a salient feature of most solid tumors and plays a central role in tumor progression owing to its multiple contributions to therapeutic resistance, metastasis, angiogenesis and stemness properties. Reports exist in literature about hypoxia increasing stemness characteristics and invasiveness potential of malignant cells. In order to delineate molecular crosstalk among factors driving glioma progression, we used knockdown and overexpression strategies. We have demonstrated that U87MG and A172 glioma cells inherently have a subset of cells with high migratory potential due to migration-inducing Mena transcripts. These cells also have elevated stemness markers (Sox-2 and Oct-4). There was a significant increase of number in this subset of migratory cells on exposure to hypoxia with corresponding elevation (over 1000 fold) in migration-inducing Mena transcripts. We were able to demonstrate that a HIF-2α-Sox-2/Oct-4-Mena (INV) axis that is strongly activated in hypoxia and markedly increases the migratory potential of the cells. Such cells also formed tumor spheres with greater efficiency. We have correlated our in-vitro results with human glioblastoma samples and found that hypoxia, invasiveness and stemness markers correlated well in native tumor samples. This study identifies a novel signaling mechanism mediated by HIF-2α in regulating invasiveness and stemness characteristics, suggesting that under hypoxic conditions, some tumor cells acquire more migratory potential by increased Pan Mena and Mena INV expression as a consequence of this HIF-2α mediated increase in Oct-4 and Sox-2. These properties would help the cells to form a new nidus after local invasion or metastasis. Copyright © 2016 Elsevier Ltd. All rights reserved.

  4. Photodynamic synchrotron x-ray therapy in Glioma cell using superparamagnetic iron nanoparticle

    Kim, Hong-Tae; Kim, Ki-Hong; Choi, Gi-Hwan; Jheon, Sanghoon; Park, Sung-Hwan; Kim, Bong-Il; Hyodo, Kazuyuki; Ando, Masami; Kim, Jong-Ki

    2009-06-01

    In order to evaluate cytotoxic effects of secondary Auger electron emission(Photon Activation Therapy:PAT) from alginate-coated iron nanoparticles(Alg-SNP), Alg-SNP-uptaken C6 glioma cell lines were irradiated with 6.89/7.2 Kev synchrotron X-ray. 0-125 Gy were irradiated on three experimental groups including No-SNP group incubating without SNP as control group, 6hr-SNP group incubating with SNP for 6hr and ON-SNP group incubating with SNP overnight. Irradiated cells were stained with Acridine Orange(AO) and Edithium Bromide(EB) to count their viability with fluorescent microscopy in comparison with control groups. AO stained in damaged DNA, giving FL color change in X-ray plus SNP group. EB did not or less enter inside the cell nucleus of control group. In contrast, EB entered inside the cell nucleus of Alg-SNP group which means more damage compared with Control groups. The results of MTT assay demonstrated a X-ray dose-dependent reduction generally in cell viability in the experimental groups. 3 or 9 times increase in cell survival loss rate was observed at 6hr-SNP and ON-SNP groups, respectively compared to No-SNP control group in first experiment that was done to test cell survival rate at relatively lower dose, from 0 to 50 Gy. In second experiment X-ray dose was increased to 125 Gy. Survival loss was sharply decreased in a relatively lower dose from 5 to 25 Gy, and then demonstrated an exponentially decreasing behavior with a convergence until 125 Gy for each group. This observation suggests PAT effects on the cell directly by X-ray in the presence of Alg-SNP occurs within lower X-ray dose, and conventional X-ray radiation effect becomes dominant in higher X-ray dose. The cell viability loss of ON-SNP group was three times higher compared with that of 6hr-SNP group. In conclusion, it is possible to design photodynamic X-ray therapy study using a monochromatic x-ray energy and metal nanoparticle as x-ray sensitizer, which may enable new X-ray PDT to

  5. SCCRO Promotes Glioma Formation and Malignant Progression in Mice

    Stephen R. Broderick

    2010-06-01

    Full Text Available Originally identified as an oncogene activated by amplification in squamous cell carcinomas, several lines of evidence now suggest that squamous cell carcinoma-related oncogene (SCCRO; aka DCUN1D1 may play a role in the pathogenesis of a wide range of human cancers including gliomas. SCCRO's oncogenic function is substantiated by its ectopic expression, resulting in transformation of cells in culture and xenograft formation in nude mice. The aim of this study was to assess the in vivo oncogenicity of SCCRO in a murine model. Ubiquitous expression of SCCRO resulted in early embryonic lethality. Because SCCRO overexpression was detected in human gliomas, its in vivo oncogenic activity was assessed in an established murine glioma model. Conditional expression of SCCRO using a replication-competent ASLV long terminal repeat with splice acceptor/nestin-(tumor virus-A tv-a model system was not sufficient to induce tumor formation in a wild-type genetic background, but tumors formed with increasing frequency and decreasing latency in facilitated background containing Ink4a deletion alone or in combination with PTEN loss. Ectopic expression of SCCRO in glial progenitor cells resulted in lower-grade gliomas in Ink4a-/- mice, whereas its expression in Ink4a-/-/PTEN-/- background produced high-grade glioblastoma-like lesions that were indistinguishable from human tumors. Expression of SCCRO with platelet-derived growth factor-beta (PDGF-β resulted in an increased proportion of mice forming glioblastoma-like tumors compared with those induced by PDGF-β alone. This work substantiates SCCRO's function as an oncogene by showing its ability to facilitate malignant transformation and carcinogenic progression in vivo and supports a role for SCCRO in the pathogenesis of gliomas and other human cancers.

  6. Boronated protoporphyrin (BOPP): localization in lysosomes of the human glioma cell line SF-767 with uptake modulated by lipoprotein levels

    Callahan, Daniel E.; Forte, Trudy M.; Javed Afzal, S.M.; Deen, Dennis F.; Kahl, Stephen B.; Bjornstad, Kathleen A.; Bauer, William F.; Blakely, Eleanor A.

    1999-01-01

    Purpose: Boronated protoporphyrin (BOPP) is a candidate for use in both boron neutron capture therapy (BNCT) and photodynamic therapy (PDT) of glioblastoma multiforme (GBM). Our objectives are to identify factors that influence the uptake and retention of BOPP in vitro and to determine BOPP distribution in a human glioma cell line in vitro. This information will aid the development of compounds and treatment strategies that increase the effectiveness of BNCT therapy for GBM. Methods and Materials: The amount, distribution pattern, and site of internalization of BOPP were assessed using fluorescence microscopy. Living human glioma (SF-767) cells were imaged after a 24-h exposure to BOPP (20-135.6 μg/ml, normal serum). Dose-dependent uptake of BOPP was determined using both fluorescence microscopy of individual living cells and inductively-coupled plasma-atomic emission spectroscopy (ICP-AES) analysis of cell pellets. Lysosome- or mitochondria-specific fluorescent probes were used to identify the cellular compartment containing BOPP. Two human fibroblast cell lines, AG-1522 (LDL receptor-positive) and GM019-15C (LDL receptor-deficient), were used to investigate LDL receptor-dependent BOPP uptake. The dependence of BOPP uptake on lipoproteins in the media was determined by exposing each of the three cell types to BOPP in medium containing either normal (NS) or lipoprotein deficient serum (LPDS). Results: BOPP accumulated in the lysosomes of human glioma cells in vitro, and not in the mitochondria, as reported for C6 rat glioma cells in vitro. BOPP uptake was concentration-dependent and was also dependent on the amount of lipoproteins in the medium. Over the range of incubation concentrations studied and at the single exposure duration time point investigated (24 h), all cells retained a similar amount of BOPP. At the lowest incubation concentration (20 μg/ml, NS), the amount of boron retained was near 10 9 atoms per cell (15 μg B/g cells). Lysosomes containing high

  7. Foxp3 overexpression in tumor cells predicts poor survival in oral squamous cell carcinoma

    Song, Jing-Jing; Zhao, Si-Jia; Fang, Juan; Ma, Da; Liu, Xiang-Qi; Chen, Xiao-Bing; Wang, Yun; Cheng, Bin; Wang, Zhi

    2016-01-01

    Forkhead Box P3 (Foxp3) is a regulatory T cells marker, and its expression correlates with prognosis in a number of malignancies. The aim of this study is to determine the relationship of Foxp3 expression with clinicopathological parameters and prognosis in oral squamous cell carcinoma (OSCC). Foxp3 expression was examined using immunohistochemistry (IHC) in paraffin-embedded tissue samples from 273 OSCC patients. Statistical analysis was performed to evaluate the associations between Foxp3 expression, the clinicopathologic characteristics and prognostic factors in OSCC. Foxp3 protein expression was significantly associated with lymph node metastasis (P <0.01). Both univariate and multivariate analyses revealed that Foxp3 was an independent factor for both 5 years overall survival (OS) and relapse-free survival (RFS) (both P <0.01). Patients with Foxp3 overexpression had shorter OS and RFS. Our results determined that elevated Foxp3 protein expression was a predictive factor of outcome in OSCC and could act as a promising therapeutic target

  8. Dek overexpression in murine epithelia increases overt esophageal squamous cell carcinoma incidence

    Cimperman, Katherine A.; Haas, Sarah R.; Guasch, Geraldine; Waclaw, Ronald R.; Komurov, Kakajan; Lane, Adam; Wikenheiser-Brokamp, Kathryn A.

    2018-01-01

    Esophageal cancer occurs as either squamous cell carcinoma (ESCC) or adenocarcinoma. ESCCs comprise almost 90% of cases worldwide, and recur with a less than 15% five-year survival rate despite available treatments. The identification of new ESCC drivers and therapeutic targets is critical for improving outcomes. Here we report that expression of the human DEK oncogene is strongly upregulated in esophageal SCC based on data in the cancer genome atlas (TCGA). DEK is a chromatin-associated protein with important roles in several nuclear processes including gene transcription, epigenetics, and DNA repair. Our previous data have utilized a murine knockout model to demonstrate that Dek expression is required for oral and esophageal SCC growth. Also, DEK overexpression in human keratinocytes, the cell of origin for SCC, was sufficient to cause hyperplasia in 3D organotypic raft cultures that mimic human skin, thus linking high DEK expression in keratinocytes to oncogenic phenotypes. However, the role of DEK over-expression in ESCC development remains unknown in human cells or genetic mouse models. To define the consequences of Dek overexpression in vivo, we generated and validated a tetracycline responsive Dek transgenic mouse model referred to as Bi-L-Dek. Dek overexpression was induced in the basal keratinocytes of stratified squamous epithelium by crossing Bi-L-Dek mice to keratin 5 tetracycline transactivator (K5-tTA) mice. Conditional transgene expression was validated in the resulting Bi-L-Dek_K5-tTA mice and was suppressed with doxycycline treatment in the tetracycline-off system. The mice were subjected to an established HNSCC and esophageal carcinogenesis protocol using the chemical carcinogen 4-nitroquinoline 1-oxide (4NQO). Dek overexpression stimulated gross esophageal tumor development, when compared to doxycycline treated control mice. Furthermore, high Dek expression caused a trend toward esophageal hyperplasia in 4NQO treated mice. Taken together, these

  9. Effects of CD44 and E-cadherin overexpression on the proliferation, adhesion and invasion of ovarian cancer cells.

    Mao, Meiya; Zheng, Xiaojiao; Jin, Bohong; Zhang, Fubin; Zhu, Linyan; Cui, Lining

    2017-12-01

    CD44 is a prognostic indicator of shorter survival time in ovarian cancer. E-cadherin fragmentation promotes the progression of ovarian cancer. However, the effects of CD44 and E-cadherin overexpression on ovarian cancer cells have remained elusive. The present study aimed to investigate the effects of overexpression of CD44 and E-cadherin on cell proliferation, adhesion and invasion of SKOV-3 and OVCAR-3 ovarian cancer cells. Overexpression of CD44 and E-cadherin was achieved by transfecting SKOV-3 and OVCAR-3 cells with viruses carrying the CD44 or E-cadherin gene, respectively. Expression of CD44 and E-cadherin was detected by western blot analysis. The proliferation of SKOV-3 and OVCAR-3 cells was measured by a Cell Counting Kit-8 at 0, 24 and 48 h after viral transfection. The adhesion ability of SKOV-3 and OVCAR-3 cells to the endothelial layer was detected. A Transwell invasion assay was utilized to assess the invasion ability of the cells. Overexpression of CD44 and E-cadherin in SKOV-3 and OVCAR-3 cells was confirmed by western blot. Compared with the blank or negative control groups, the CD44 overexpression groups of SKOV-3 and OVCAR-3 cells exhibited an increased cell proliferation rate at 24 and 48 h, whereas overexpression of E-cadherin did not alter the proliferation of these cells. Furthermore, compared with the blank and negative control groups, the cell adhesion and invasion ability in the CD44 overexpression groups of SKOV-3 and OVCAR-3 cells was markedly higher. There were no significant differences in adhesion ability between the E-cadherin overexpression group and the blank/negative control group. Of note, overexpression of E-cadherin decreased the invasive ability of SKOV-3 and OVCAR-3 cells. In conclusion, Overexpression of CD44 increased the proliferation, adhesion and invasion of ovarian cancer cells, while overexpression of E-cadherin decreased the invasion of ovarian cancer cells.

  10. Lipid-Based Nanovectors for Targeting of CD44-Overexpressing Tumor Cells

    Silvia Arpicco

    2013-01-01

    Full Text Available Hyaluronic acid (HA is a naturally occurring glycosaminoglycan that exists in living systems, and it is a major component of the extracellular matrix. The hyaluronic acid receptor CD44 is found at low levels on the surface of epithelial, haematopoietic, and neuronal cells and is overexpressed in many cancer cells particularly in tumour initiating cells. HA has been therefore used as ligand attached to HA-lipid-based nanovectors for the active targeting of small or large active molecules for the treatment of cancer. This paper describes the different approaches employed for the preparation, characterization, and evaluation of these potent delivery systems.

  11. Overexpressed human metallothionein IIA gene protects Chinese hamster ovary cells from killing by alkylating agents.

    Kaina, B; Lohrer, H; Karin, M; Herrlich, P

    1990-01-01

    Experiments were designed to detect survival advantages that cells gain by overexpressing metallothionein (MT). Chinese hamster ovary K1-2 cells and an x-ray-sensitive derivative were transfected with a bovine papillomavirus (BPV)-linked construct carrying the human metallothionein IIA (hMT-IIA) gene. Transfectants survived 40-fold higher levels of cadmium chloride, harbored at least 30 copies of hMT-IIA, and contained 25- to 166-fold more MT than the parent cells. Even under conditions of re...

  12. Pancreatic beta-cell lipotoxicity induced by overexpression of hormone-sensitive lipase

    Winzell, Maria Sörhede; Svensson, Håkan; Enerbäck, Sven

    2003-01-01

    Lipid perturbations associated with triglyceride overstorage in beta-cells impair insulin secretion, a process termed lipotoxicity. To assess the role of hormone-sensitive lipase, which is expressed and enzymatically active in beta-cells, in the development of lipotoxicity, we generated transgenic...... mice overexpressing hormone-sensitive lipase specifically in beta-cells. Transgenic mice developed glucose intolerance and severely blunted glucose-stimulated insulin secretion when challenged with a high-fat diet. As expected, both lipase activity and forskolin-stimulated lipolysis was increased...

  13. BAK overexpression mediates p53-independent apoptosis inducing effects on human gastric cancer cells

    Liu Jun

    2004-07-01

    Full Text Available Abstract Background BAK (Bcl-2 homologous antagonist/killer is a novel pro-apoptotic gene of the Bcl-2 family. It has been reported that gastric tumors have reduced BAK levels when compared with the normal mucosa. Moreover, mutations of the BAK gene have been identified in human gastrointestinal cancers, suggesting that a perturbation of BAK-mediated apoptosis may contribute to the pathogenesis of gastric cancer. In this study, we explored the therapeutic effects of gene transfer mediated elevations in BAK expression on human gastric cancer cells in vitro. Methods Eukaryotic expression vector for the BAK gene was constructed and transferred into gastric cancer cell lines, MKN-45 (wild-type p53 and MKN-28 (mutant-type p53. RT-PCR and Western Blotting detected cellular BAK gene expression. Cell growth activities were detected by MTT colorimetry and flow cytometry, while apoptosis was assayed by electronic microscopy and TUNEL. Western Blotting and colorimetry investigated cellular caspase-3 activities. Results BAK gene transfer could result in significant BAK overexpression, decreased in vitro growth, cell cycle G0/G1 arrest, and induced apoptosis in gastric cancer cells. In transferred cells, inactive caspase-3 precursor was cleaved into the active subunits p20 and p17, during BAK overexpression-induced apoptosis. In addition, this process occurred equally well in p53 wild-type (MKN-45, or in p53 mutant-type (MKN-28 gastric cancer cells. Conclusions The data presented suggests that overexpression of the BAK gene can lead to apoptosis of gastric cancer cells in vitro, which does not appear to be dependent on p53 status. The action mechanism of BAK mediated apoptosis correlates with activation of caspase-3. This could be served as a potential strategy for further development of gastric cancer therapies.

  14. BAK overexpression mediates p53-independent apoptosis inducing effects on human gastric cancer cells

    Tong, Qiang-Song; Zheng, Li-Duan; Wang, Liang; Liu, Jun; Qian, Wei

    2004-01-01

    BAK (Bcl-2 homologous antagonist/killer) is a novel pro-apoptotic gene of the Bcl-2 family. It has been reported that gastric tumors have reduced BAK levels when compared with the normal mucosa. Moreover, mutations of the BAK gene have been identified in human gastrointestinal cancers, suggesting that a perturbation of BAK-mediated apoptosis may contribute to the pathogenesis of gastric cancer. In this study, we explored the therapeutic effects of gene transfer mediated elevations in BAK expression on human gastric cancer cells in vitro. Eukaryotic expression vector for the BAK gene was constructed and transferred into gastric cancer cell lines, MKN-45 (wild-type p53) and MKN-28 (mutant-type p53). RT-PCR and Western Blotting detected cellular BAK gene expression. Cell growth activities were detected by MTT colorimetry and flow cytometry, while apoptosis was assayed by electronic microscopy and TUNEL. Western Blotting and colorimetry investigated cellular caspase-3 activities. BAK gene transfer could result in significant BAK overexpression, decreased in vitro growth, cell cycle G 0 /G 1 arrest, and induced apoptosis in gastric cancer cells. In transferred cells, inactive caspase-3 precursor was cleaved into the active subunits p20 and p17, during BAK overexpression-induced apoptosis. In addition, this process occurred equally well in p53 wild-type (MKN-45), or in p53 mutant-type (MKN-28) gastric cancer cells. The data presented suggests that overexpression of the BAK gene can lead to apoptosis of gastric cancer cells in vitro, which does not appear to be dependent on p53 status. The action mechanism of BAK mediated apoptosis correlates with activation of caspase-3. This could be served as a potential strategy for further development of gastric cancer therapies

  15. Endothelial Nitric Oxide Synthase Overexpression Restores the Efficiency of Bone Marrow Mononuclear Cell-Based Therapy

    Mees, Barend; Récalde, Alice; Loinard, Céline; Tempel, Dennie; Godinho, Marcia; Vilar, José; van Haperen, Rien; Lévy, Bernard; de Crom, Rini; Silvestre, Jean-Sébastien

    2011-01-01

    Bone marrow-derived mononuclear cells (BMMNCs) enhance postischemic neovascularization, and their therapeutic use is currently under clinical investigation. However, cardiovascular risk factors, including diabetes mellitus and hypercholesterolemia, lead to the abrogation of BMMNCs proangiogenic potential. NO has been shown to be critical for the proangiogenic function of BMMNCs, and increased endothelial NO synthase (eNOS) activity promotes vessel growth in ischemic conditions. We therefore hypothesized that eNOS overexpression could restore both the impaired neovascularization response and decreased proangiogenic function of BMMNCs in clinically relevant models of diabetes and hypercholesterolemia. Transgenic eNOS overexpression in diabetic, atherosclerotic, and wild-type mice induced a 1.5- to 2.3-fold increase in postischemic neovascularization compared with control. eNOS overexpression in diabetic or atherosclerotic BMMNCs restored their reduced proangiogenic potential in ischemic hind limb. This effect was associated with an increase in BMMNC ability to differentiate into cells with endothelial phenotype in vitro and in vivo and an increase in BMMNCs paracrine function, including vascular endothelial growth factor A release and NO-dependent vasodilation. Moreover, although wild-type BMMNCs treatment resulted in significant progression of atherosclerotic plaque in ischemic mice, eNOS transgenic atherosclerotic BMMNCs treatment even had antiatherogenic effects. Cell-based eNOS gene therapy has both proangiogenic and antiatherogenic effects and should be further investigated for the development of efficient therapeutic neovascularization designed to treat ischemic cardiovascular disease. PMID:21224043

  16. N-acetylaspartate (NAA) and N-acetylaspartylglutamate (NAAG) promote growth and inhibit differentiation of glioma stem-like cells.

    Long, Patrick M; Moffett, John R; Namboodiri, Aryan M A; Viapiano, Mariano S; Lawler, Sean E; Jaworski, Diane M

    2013-09-06

    Metabolic reprogramming is a pathological feature of cancer and a driver of tumor cell transformation. N-Acetylaspartate (NAA) is one of the most abundant amino acid derivatives in the brain and serves as a source of metabolic acetate for oligodendrocyte myelination and protein/histone acetylation or a precursor for the synthesis of the neurotransmitter N-acetylaspartylglutamate (NAAG). NAA and NAAG as well as aspartoacylase (ASPA), the enzyme responsible for NAA degradation, are significantly reduced in glioma tumors, suggesting a possible role for decreased acetate metabolism in tumorigenesis. This study sought to examine the effects of NAA and NAAG on primary tumor-derived glioma stem-like cells (GSCs) from oligodendroglioma as well as proneural and mesenchymal glioblastoma, relative to oligodendrocyte progenitor cells (Oli-Neu). Although the NAA dicarboxylate transporter NaDC3 is primarily thought to be expressed by astrocytes, all cell lines expressed NaDC3 and, thus, are capable of NAA up-take. Treatment with NAA or NAAG significantly increased GSC growth and suppressed differentiation of Oli-Neu cells and proneural GSCs. Interestingly, ASPA was expressed in both the cytosol and nuclei of GSCs and exhibited greatest nuclear immunoreactivity in differentiation-resistant GSCs. Both NAA and NAAG elicited the expression of a novel immunoreactive ASPA species in select GSC nuclei, suggesting differential ASPA regulation in response to these metabolites. Therefore, this study highlights a potential role for nuclear ASPA expression in GSC malignancy and suggests that the use of NAA or NAAG is not an appropriate therapeutic approach to increase acetate bioavailability in glioma. Thus, an alternative acetate source is required.

  17. N-Acetylaspartate (NAA) and N-Acetylaspartylglutamate (NAAG) Promote Growth and Inhibit Differentiation of Glioma Stem-like Cells*

    Long, Patrick M.; Moffett, John R.; Namboodiri, Aryan M. A.; Viapiano, Mariano S.; Lawler, Sean E.; Jaworski, Diane M.

    2013-01-01

    Metabolic reprogramming is a pathological feature of cancer and a driver of tumor cell transformation. N-Acetylaspartate (NAA) is one of the most abundant amino acid derivatives in the brain and serves as a source of metabolic acetate for oligodendrocyte myelination and protein/histone acetylation or a precursor for the synthesis of the neurotransmitter N-acetylaspartylglutamate (NAAG). NAA and NAAG as well as aspartoacylase (ASPA), the enzyme responsible for NAA degradation, are significantly reduced in glioma tumors, suggesting a possible role for decreased acetate metabolism in tumorigenesis. This study sought to examine the effects of NAA and NAAG on primary tumor-derived glioma stem-like cells (GSCs) from oligodendroglioma as well as proneural and mesenchymal glioblastoma, relative to oligodendrocyte progenitor cells (Oli-Neu). Although the NAA dicarboxylate transporter NaDC3 is primarily thought to be expressed by astrocytes, all cell lines expressed NaDC3 and, thus, are capable of NAA up-take. Treatment with NAA or NAAG significantly increased GSC growth and suppressed differentiation of Oli-Neu cells and proneural GSCs. Interestingly, ASPA was expressed in both the cytosol and nuclei of GSCs and exhibited greatest nuclear immunoreactivity in differentiation-resistant GSCs. Both NAA and NAAG elicited the expression of a novel immunoreactive ASPA species in select GSC nuclei, suggesting differential ASPA regulation in response to these metabolites. Therefore, this study highlights a potential role for nuclear ASPA expression in GSC malignancy and suggests that the use of NAA or NAAG is not an appropriate therapeutic approach to increase acetate bioavailability in glioma. Thus, an alternative acetate source is required. PMID:23884408

  18. Pancreatic beta-cell overexpression of the glucagon receptor gene results in enhanced beta-cell function and mass

    Gelling, Richard W; Vuguin, Patricia M; Du, Xiu Quan

    2009-01-01

    in vivo, we generated mice overexpressing the Gcgr specifically on pancreatic beta-cells (RIP-Gcgr). In vivo and in vitro insulin secretion in response to glucagon and glucose was increased 1.7- to 3.9-fold in RIP-Gcgr mice compared with controls. Consistent with the observed increase in insulin release...

  19. ER-mitochondria contacts control surface glycan expression and sensitivity to killer lymphocytes in glioma stem-like cells.

    Bassoy, Esen Yonca; Kasahara, Atsuko; Chiusolo, Valentina; Jacquemin, Guillaume; Boydell, Emma; Zamorano, Sebastian; Riccadonna, Cristina; Pellegatta, Serena; Hulo, Nicolas; Dutoit, Valérie; Derouazi, Madiha; Dietrich, Pierre Yves; Walker, Paul R; Martinvalet, Denis

    2017-06-01

    Glioblastoma is a highly heterogeneous aggressive primary brain tumor, with the glioma stem-like cells (GSC) being more sensitive to cytotoxic lymphocyte-mediated killing than glioma differentiated cells (GDC). However, the mechanism behind this higher sensitivity is unclear. Here, we found that the mitochondrial morphology of GSCs modulates the ER-mitochondria contacts that regulate the surface expression of sialylated glycans and their recognition by cytotoxic T lymphocytes and natural killer cells. GSCs displayed diminished ER-mitochondria contacts compared to GDCs. Forced ER-mitochondria contacts in GSCs increased their cell surface expression of sialylated glycans and reduced their susceptibility to cytotoxic lymphocytes. Therefore, mitochondrial morphology and dynamism dictate the ER-mitochondria contacts in order to regulate the surface expression of certain glycans and thus play a role in GSC recognition and elimination by immune effector cells. Targeting the mitochondrial morphology, dynamism, and contacts with the ER could be an innovative strategy to deplete the cancer stem cell compartment to successfully treat glioblastoma. © 2017 The Authors.

  20. Abscisic-acid-induced cellular apoptosis and differentiation in glioma via the retinoid acid signaling pathway.

    Zhou, Nan; Yao, Yu; Ye, Hongxing; Zhu, Wei; Chen, Liang; Mao, Ying

    2016-04-15

    Retinoid acid (RA) plays critical roles in regulating differentiation and apoptosis in a variety of cancer cells. Abscisic acid (ABA) and RA are direct derivatives of carotenoids and share structural similarities. Here we proposed that ABA may also play a role in cellular differentiation and apoptosis by sharing a similar signaling pathway with RA that may be involved in glioma pathogenesis. We reported for the first time that the ABA levels were twofold higher in low-grade gliomas compared with high-grade gliomas. In glioma tissues, there was a positive correlation between the ABA levels and the transcription of cellular retinoic acid-binding protein 2 (CRABP2) and a negative correlation between the ABA levels and transcription of fatty acid-binding protein 5 (FABP5). ABA treatment induced a significant increase in the expression of CRABP2 and a decrease in the expression of peroxisome proliferator-activated receptor (PPAR) in glioblastoma cells. Remarkably, both cellular apoptosis and differentiation were increased in the glioblastoma cells after ABA treatment. ABA-induced cellular apoptosis and differentiation were significantly reduced by selectively silencing RAR-α, while RAR-α overexpression exaggerated the ABA-induced effects. These results suggest that ABA may play a role in the pathogenesis of glioma by promoting cellular apoptosis and differentiation through the RA signaling pathway. © 2015 UICC.

  1. The inhibition of apoptosis in EL4 lymphoma cells overexpressing growth hormone.

    Arnold, Robyn E; Weigent, Douglas A

    2004-01-01

    The antiapoptotic action of exogenous growth hormone (GH) has been reported for several lymphoid cell lines; however, the potential role of endogenous GH in apoptosis has not been thoroughly investigated. This study was designed to investigate the effects of endogenous GH on apoptosis induced by methyl methanesulfonate (MMS) in a T cell lymphoma overexpressing GH (GHo). The results of these experiments have shown that in EL4 lymphoma cells, overexpression of GH sustained viability after exposure to MMS compared to control cells. The extent of DNA fragmentation measured by ladder formation on agarose gels was reduced in GHo cells following treatment with MMS, when compared to control cells. Adding exogenous GH to control cells and treatment of GHo cells with antibodies to GH had no effect on MMS-induced DNA ladder formation. In further studies, DNA microarray analysis suggested a marked decrease in the constitutive expression of bax, BAD, and caspases 3, 8, and 9 in GHo cells compared to controls. In addition, after treatment with MMS, the activities of caspases 2, 3, 6, 8, and 9 were all lower than control in GHo cells. Western blot analysis detected an increase in Bcl-2 while the levels of nuclear factor kappa B (NFkappaB) remained unchanged in GHo cells. Treatment of EL4 cells with antisense deoxyoligonucleotides to GH and specific inhibitors of NFkappaB (SN-50) increased DNA fragmentation. GHo cells show increased levels of phosphorylated Akt and GSK-3, suggesting inactivation of this proapoptotic protein. The results, taken together with our previous data which showed increased nitric oxide formation in GHo cells, suggest a possible mechanism for the antiapoptotic effects of endogenous GH through the production of nitric oxide and support the idea that endogenous GH may play an important role in the survival of lymphocytes exposed to stressful stimuli. Copyright 2004 S. Karger AG, Basel

  2. Overexpression of IL-7R alpha provides a competitive advantage during early T-cell development.

    Laouar, Yasmina; Crispe, I Nicholas; Flavell, Richard A

    2004-03-15

    Critical checkpoints controlling early thymic T-cell development and homeostasis are set by the proper signaling function of the interleukin 7 receptor (IL-7R) and the pre-T-cell antigen receptor. Although alpha beta T-cell development is observed in IL-7- and IL-7R alpha-deficient mice, the number of thymocytes is significantly reduced, implying a role for the IL-7R in controlling the size of the thymic T-cell compartment. Here, we report the overexpression of IL-7R alpha that occurs in the early T-cell compartment from AKR/J mice, animals that are highly susceptible to the spontaneous development of thymoma. Increased IL-7R alpha was revealed by surface staining, and increased IL-7R alpha mRNA was documented by using reverse transcriptase-polymerase chain reaction (RT-PCR). This resulted in increased survival of AKR/J early thymocytes, shown by the decreased frequency of TUNEL(+) (terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate [dUTP]-fluorescein nick end labeling) cells. In an in vivo thymocyte repopulation model, AKR/J thymocytes had a selective advantage over healthy thymocytes. This advantage occurred at early stages of T-cell development. Our findings support the model that overexpression of growth factor receptors can contribute to proliferation and malignancy.

  3. Overexpression of xeroderma pigmentosum group C decreases the chemotherapeutic sensitivity of colorectal carcinoma cells to cisplatin.

    Zhang, Yi; Cao, Jia; Meng, Yanni; Qu, Chunying; Shen, Feng; Xu, Leiming

    2018-05-01

    Xeroderma pigmentosum group C (XPC) is a DNA-damage-recognition gene active at the early stage of DNA repair. XPC also participates in regulation of cell-cycle checkpoint and DNA-damage-induced apoptosis. In the present study, the expression levels of genes involved in nucleotide excision repair (NER) were assessed in human colorectal cancer (CRC) tissue. This analysis revealed that expression of XPC mRNA significantly increased in colorectal carcinoma tissues compared with matched normal controls. Expression of XPC gradually increased along with the degree of progression of CRC. In vitro , an XTT assay demonstrated that small interfering RNA (siRNA) targeting XPC significantly increased the sensitivity of CRC SW480 cells to cisplatin, whereas cells transfected with a XPC-overexpression plasmid became more resistant to cisplatin. Furthermore, flow cytometry revealed that the proportion of apoptotic cells significantly increased in XPC-knockdown cells upon cisplatin treatment. However, the overexpression XPC significantly increased the resistance of cells to cisplatin. In vivo , tumor growth was significantly reduced in tumor-bearing mice when the XPC gene was knocked down. Upregulation of the expression of pro-apoptotic Bcl-associated X and downregulation of the anti-apoptotic B-cell lymphoma 2 proteins was observed in the implanted tumor tissue. In conclusion, XPC serves a key role in chemotherapeutic sensitivity of CRC to cisplatin, meaning that it may be a potential target for chemotherapy of CRC.

  4. FasL and FADD delivery by a glioma-specific and cell cycle-dependent HSV-1 amplicon virus enhanced apoptosis in primary human brain tumors

    Lam Paula Y

    2010-10-01

    Full Text Available Abstract Background Glioblastoma multiforme is the most malignant cancer of the brain and is notoriously difficult to treat due to the highly proliferative and infiltrative nature of the cells. Herein, we explored the combination treatment of pre-established human glioma xenograft using multiple therapeutic genes whereby the gene expression is regulated by both cell-type and cell cycle-dependent transcriptional regulatory mechanism conferred by recombinant HSV-1 amplicon vectors. Results We demonstrated for the first time that Ki67-positive proliferating primary human glioma cells cultured from biopsy samples were effectively induced into cell death by the dual-specific function of the pG8-FasL amplicon vectors. These vectors were relatively stable and exhibited minimal cytotoxicity in vivo. Intracranial implantation of pre-transduced glioma cells resulted in better survival outcome when compared with viral vectors inoculated one week post-implantation of tumor cells, indicating that therapeutic efficacy is dependent on the viral spread and mode of viral vectors administration. We further showed that pG8-FasL amplicon vectors are functional in the presence of commonly used treatment regimens for human brain cancer. In fact, the combined therapies of pG8-FasL and pG8-FADD in the presence of temozolomide significantly improved the survival of mice bearing intracranial high-grade gliomas. Conclusion Taken together, our results showed that the glioma-specific and cell cycle-dependent HSV-1 amplicon vector is potentially useful as an adjuvant therapy to complement the current gene therapy strategy for gliomas.

  5. Long noncoding RNA CASC2 predicts the prognosis of glioma patients and functions as a suppressor for gliomas by suppressing Wnt/β-catenin signaling pathway

    Wang R

    2017-07-01

    Full Text Available Ronglin Wang,1,* Yuqian Li,1,* Gang Zhu,1,* Bo Tian,1 Wen Zeng,1 Yang Yang,2 Zhihong Li1 1Department of Neurosurgery, Tangdu Hospital, The Fourth Military Medical University, 2Department of Neurosurgery, The 451th hospital of PLA, Xi’an, Shaanxi, People’s Republic of China *These authors contributed equally to this work Background: Previous studies have demonstrated that long noncoding RNA cancer susceptibility candidate 2 (lncRNA CASC2 is frequently downregulated in several types of tumors and functions as a tumor-suppressive factor. However, the clinical significance and function of CASC2 in human glioma remain largely unknown. The purpose of this study was to identify the clinical values of CASC2, as well as investigate the potential molecular mechanisms in glioma. Methods: This retrospective study first analyzed the expression levels of CASC2 using quantitative real-time polymerase chain reaction. Then, CASC2 expression levels were associated with various clinicopathologic characteristics and the survival rate of patients with glioma. Finally, the function and underlying molecular mechanisms of CASC2 in human glioma were investigated in U251 cell line. Results: By quantitative real-time polymerase chain reaction analysis, our data showed that CASC2 expression was significantly downregulated in glioma tissues and cell lines (U87 and U251 compared to adjacent normal brain tissues or normal human astrocytes. Moreover, its expression negatively correlated with tumor grade in glioma patients. Furthermore, Kaplan–Meier curves with log-rank analysis revealed a close correlation between downregulated CASC2 and shorter survival time in glioma patients. In addition, Cox regression analysis indicated that CASC2 could be considered as an independent risk factor for poor prognosis. Finally, in vitro experiment demonstrated that CASC2 overexpression remarkably suppressed glioma cell proliferation, migration, and invasion through suppressing Wnt

  6. Overexpression of HIF-1α in mesenchymal stem cells contributes to repairing hypoxic-ischemic brain damage in rats.

    Lin, Deju; Zhou, Liping; Wang, Biao; Liu, Lizhen; Cong, Li; Hu, Chuanqin; Ge, Tingting; Yu, Qin

    2017-01-01

    Preclinical researches on mesenchymal stem cells (MSCs) transplantation, which is used to treat hypoxic-ischemic (HI) brain damage, have received inspiring achievements. However, the insufficient migration of active cells to damaged tissues has limited their potential therapeutic effects. There are some evidences that hypoxia inducible factor-1 alpha (HIF-1α) promotes the viability and migration of the cells. Here, we aim to investigate whether overexpression of HIF-1α in MSCs could improve the viability and migration capacity of cells, and its therapeutic efficiency on HI brain damage. In the study, MSCs with HIF-1α overexpression was achieved by recombinant lentiviral vector and transplanted to the rats subsequent to HI. Our data indicated that overexpression of HIF-1α promoted the viability and migration of MSCs, HIF-1α overexpressed MSCs also had a stronger therapeutic efficiency on HI brain damaged treatment by mitigating the injury on behavioral and histological changes evoked by HI insults, accompanied with more MSCs migrating to cerebral damaged area. This study demonstrated that HIF-1α overexpression could increase the MSCs' therapeutic efficiency in HI and the promotion of the cells' directional migration to cerebral HI area by overexpression may be responsible for it, which showed that transplantation of MSCs with HIF-1α overexpression is an attractive therapeutic option to treat HI-induced brain injury in the future. Copyright © 2016 Académie des sciences. Published by Elsevier SAS. All rights reserved.

  7. Hyperthermia radiosensitization in human glioma cells comparison of recovery of polymerase activity, survival, and potentially lethal damage repair

    Raaphorst, G.P.; Feeley, M.M.

    1994-01-01

    DNA polymerase inactivation is compared to thermal radiosensitization and inhibition of damage recovery in human glioma cells. Two human glioma cell lines (U87MG and U373MG) were exposed to hyperthermia and irradiation. Hyperthermia was given at 43 degrees C and 45 degrees C and DNA polymerase α + δ + ε and β activities were measured. Hyperthermia was given at various times before irradiation and the degree of radiosensitization and polymerase activity was assessed at various times after heating. In addition the ability of cells to undergo repair of potentially lethal radiation damage was assessed for cells irradiated at various times after heating. Polymerase α + δ + ε and polymerase β both recovered after heating but polymerase β was faster and was complete in U373MG but not in the U87MG cell lines after 48 h incubation after heating (45 degrees C, 60 min). Incubation, between hyperthermia and irradiation resulted in a loss of radiosensitization and a loss of inhibition of repair of potentially lethal damage. These changes correlated well with recovery of polymerase β but not with polymerase α + δ + ε. The correlation of polymerase β activity and thermoradiosensitization and its recovery indicate that polymerase β may be one of the mechanisms involved in thermoradiosensitization. 35 refs., 7 figs

  8. Apoptotic block in colon cancer cells may be rectified by lentivirus m