Human Glioma Cell Line U251 Research Articles

MicroRNA-34a inhibits human brain glioma cell growth by down-regulation of notch1

The effects of microRNA-34a (miR-34a)-regulated Notch1 gene on the proliferation and apoptosis of the human glioma cell line U87 were investigated in this study. The U87 cells were divided into miR-34a mimics, negative control, mock transfection and blank control groups in terms of different treatments. In miR-34a mimics group, human U87 glioma cells were transfected with miR-34a mimics by using lipofectamine 2000. The cells transfected with nonsense microRNA were set up as negative control group. Those treated with lipofectamine 2000 only were designated to the mock tranfection group. In the blank control group, the cells were cultured routinely and no treatment was given. The expression of miR-34a and Notch1 was detected by using real-time RT-PCR. Western blotting was employed to monitor the change in Notch1 protein. Cell proliferation and apoptosis were measured by CCK-8 and flow cytometry. The results showed that the proliferative ability of U87 cells was significantly reduced and the apoptotic cells increased in miR-34a mimics group relative to control groups. The expression of miR-34a was significantly up-regulated in mimics group as compared with control groups (P<0.05). Furthermore, Notch1 protein levels were significantly decreased in miR-34a mimics group when compared with control groups (P<0.05), but the mRNA expression of Notch1 showed no significant difference among these groups. It was concluded that miR-34a may suppress the proliferation and induce apoptosis of U87 cells by decreasing the expression of target gene Notch1, suggesting that miR-34a may become a promising gene therapeutic target for brain glioma.

Effect of interferon-α/β on temozolomide activity against MGMT-positive glioma stem-like cells in vitro and in vivo.

e12503 Background: O6-methylguanine DNA methyltranferase (MGMT) is one of the main mechanisms of chemoresistance for alkylating agents in malignant gliomas. Recent studies have showed that glioma stem-like cells (GSCs) could be the main reason for tumor recurrence and chemoresistance. In this study, we aimed to explore the effects of interferon-α/β against MGMT-positive glioma stem-like cells, and to investigate whether interferon-α/β can enhance the efficiency of temozolomide (TMZ) and the possible mechanism Methods: The growth inhibition effect of TMZ, with interferon-α or interferon-β, against the MGMT-positive GSCs from human glioma cell lines U251 and SKMG-4 (U251G and SKMG-4G) was evaluated by using Cell Counting Kit-8 (CCK-8) assay in vitro, and in xenograft models. MGMT and NF-κB expression in the tumor samples were determined by RT-PCR and Western blot analysis. Results: Our results revealed that the anti-tumor activity of TMZ was significantly enhanced by combined using interferon-α/β in vitro. In xenograft models, the tumor growth inhibit rate (IR) of TMZ to SKMG-4G and U251G was 35.2%±2.28% and 16.7%±1.96%, respectively. When TMZ combined with interferon-α or interferon-β, the IR to SKMG-4G was 58.4%±4.34% and 63.4%±1.08%; and to U251G was 41.1%±8.66% and 44.5%±1.90%, respectively(P&lt;0.05). The expression of NF-κB and MGMT in MGMT-positive GSCs decreased significantly in both mRNA and protein levels after using interferon-α/β. Conclusions: Our results indicate that IFN-α/β can enhance the sensitivity of TMZ, possibly through down-regulate NF-κB expression resulting in lower MGMT transcription expression.

Survivin small interfering RNA suppresses glioblastoma growth by inducing cellular apoptosis.

A survivin small interfering RNA sequence specific for a human and mouse homogenous sequence was constructed. Survivin small interfering RNA could significantly inhibit glioma cell proliferation and induce apoptosis when it was transfected into either a human glioma cell line U251 or rat glioma C6 cells in vitro. In addition, treatment of rat orthotopic glioma models with survivin small interfering demonstrated the inhibition of glioma growth in vivo. Our experimental findings suggest that the use of RNA interference techniques to target the survivin sequence may be useful in the treatment of glioma.

Abstract 1471: Role of ferritin heavy chain in therapeutic sensitivity of human glioblastoma cells

Abstract Elevated iron requirements and heightened iron metabolism is key to the survival and proliferation of cancer cells. As one of the major iron regulatory proteins, ferritin is elevated in cancer cells and we have previously shown that decreasing ferritin expression in human glioblastoma cells will increase the sensitivity to BCNU and radiation both in vitro and in vivo. In this study, we investigate the underlying mechanism of this finding. Through a nanotechnology-based transfection platform, the human glioma cell line U251 was transfected with H-ferritin siRNA and an effective knockdown efficiency of protein was achieved within 48 hours. H-ferritin down-regulation in glioma cells was associated with a transient decrease in transferrin receptor, suggesting a release of intracellular iron with loss of ferritin. This iron release in turn generated intracellular oxidative stress, which was demonstrated by protein oxidation. The stability of hypoxia-inducible factors (HIFs), which is an indicator of radioresistance, was decreased in glioma cells with H-ferritin down-regulation. This observation is consistent with the increased sensitivity to treatment. Exposure to radiation resulted in increased protein oxidation which could be exacerbated by H-ferritin down-regulation. Thus there is a synergistic effect of radiation and H-ferritin down-regulation; possibly also including the destabilization of HIFs. Additionally, we have previously demonstrated a DNA protection role for H-ferritin. Down-regulation of H-ferritin is associated with increased DNA vulnerability when cells were exposed to ionizing radiation. Moreover, the activation of DNA repair mechanisms was impaired when H-ferritin was absent. Finally, we expanded the vulnerability studies to include the first-line chemotherapeutic agent temozolomide and found that H-ferritin down-regulation increased the sensitivity of glioma cells to this DNA alkylating agent. This study suggests that the iron management protein, H-ferritin, has a cytoprotective role in human glioma cells, which renders them more resistant to conventional therapies. Therefore down-regulation of H-ferritin via nanoliposomes delivering siRNA is a potential adjuvant therapy in glioma treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1471. doi:1538-7445.AM2012-1471

Overexpression of the long non‐coding RNA MEG3 impairs in vitro glioma cell proliferation

Gliomas are the most common type of primary brain tumor in the central nervous system of adults. Maternally Expressed Gene 3 (MEG3) is an imprinted gene located at 14q32 that encodes a non-coding RNA (ncRNA) associated with tumorigenesis. However, little is known about whether and how MEG3 regulates glioma development. In the present study we assayed the expression of MEG3 in glioma tissue samples by real-time polymerase chain reaction assay, and defined the biological functions and target genes by CCK-8 assay, flow cytometry, and RNA immunoprecipitation. We first demonstrated that MEG3 expression was markedly decreased in glioma tissues compared with adjacent normal tissues. Moreover, ectopic expression of MEG3 inhibited cell proliferation and promoted cell apoptosis in U251 and U87 MG human glioma cell lines. We further verified that MEG3 was associated with p53 and that this association was required for p53 activation. These data suggest an important role of MEG3 in the molecular etiology of glioma and implicate the potential application of MEG3 in glioma therapy.

Changes in fractal dimension and lacunarity as early markers of UV-induced apoptosis

The aim of our study was to employ fractal analysis for evaluation of ultrastructural changes during early stages of apoptosis. Apoptosis was induced in U251 human glioma cell line by exposure to UVB light. The cells were visualized by optical phase-contrast microscopy and photographed before the UV treatment, immediately after the treatment, as well as at 30min intervals during 5h observation period. For each of the 32 cells analyzed, cellular and nuclear fractal dimension, as well as nuclear lacunarity, were determined at each time point. Our data demonstrate that cellular ultrastructural complexity determined by fractal dimension and lacunarity significantly decreases after the UV irradiation, with the nuclear lacunarity being a particularly sensitive parameter in detecting early apoptosis. Importantly, fractal analysis was able to detect cellular apoptotic changes earlier than conventional flow cytometric analysis of phosphatidylserine exposure, DNA fragmentation and cell membrane permeabilization. These results indicate that fractal analysis might be a powerful and affordable method for non-invasive early identification of apoptosis in cell cultures.

Ursolic Acid Inhibits Proliferation and Induces Apoptosis in Human Glioblastoma Cell Lines U251 by Suppressing TGF‐β1/miR‐21/PDCD4 Pathway

New chemotherapeutic strategy should be investigated to enhance clinical management in human gliomas. Recently, ursolic acid (UA), as a naturally occurring pentacyclic triterpene, has exhibited a potent anticancer activity in various tumour cells but remains uncertain in human glioma cells. Here, we examined whether UA could suppress the proliferation of human glioma cell line U251, and if so, its possible molecular targets. Cell survival, apoptosis and molecular targets were identified by multiple detecting techniques, including trypan blue dye exclusion assay, electron microscopy, AO/EB staining, Real-time PCR and immunoblotting in U251 cells. The results showed that 5-20μM of UA suppressed proliferation and induced apoptosis of U251 cells in dose- and time-dependent manners. UA increased the activation of caspase-3 and markedly suppressed levels of microRNA-21 (miR-21) in a time-dependent manner. The expression of programmed cell death 4 (PDCD4), which is a miR-21 targeting apoptotic gene, has also been enhanced by UA. And over-expression of miR-21 in U251 cells abolished the enhancement of PDCD4 protein by UA. More importantly, TGF-β1/smads signalling, as critical upstream regulators of miR-21, has also been suppressed by UA. These findings suggest that UA inhibits cell growth via causing apoptosis in U251 cells by a UA-triggered TGF-β1/miR-21/PDCD4 pathway. This study provides an evidence for testing UA efficacy in vivo and warranting future investigations examining the clinical potential of UA in human gliomas.

Role of Rac1 in the SDF-1-induced migration and invasion of human glioma cell line U251

To explore the role of Rac1 in the SDF-1-induced migration and invasion of glioma cells with a specific Rac1 inhibitor. Human glioma cell lines U251 treated with SDF-1 or/and specific Rac1 inhibitor were used. The migration and invasion capacities of cells in 2D cell migration/3D invasion assay were assessed. Western blot was employed to detect the levels of Rac1 and GAPDH in cell lysates and the Rac1 activity measured by Rac1 activation assays. Immunofluorescence was used to identify the expression and intracellular location of Rac1 in U251 cells. SDF-1 significantly increased the migration and invasion capacities of U251 cells (P < 0.05). The stimulation of SDF-1 boosted the activity of Rac1 versus the unstimulated cells (P < 0.05). And Rac1 was recruited to protruding edge in SDF-1-stimulated cells. Inhibition of Rac1 with specific Rac1 inhibitor decreased the migration and invasion capacities of SDF-1-induced U251 cells (P < 0.05). In comparison with the SDF-1 treated group, the activity of Rac1 significantly decreased (P < 0.05) and the recruitment of Rac1 to protruding edge significantly decreased in the NSC23766 pre-treated group. This study provides novel evidence that Rac1 modulates the SDF-1-induced migration and invasion of glioma cells. It suggests that the inhibition of Rac1 activation may be a new therapeutic target for glioma.

Effects of LRIG1 expression on radiosensitivity of human glioma cells and the possible mechanism

Objective To investigate the effect of the expression of leucine-rich repeats and immunoglobulin-like domains 1 （LRIG1） on the radiosensitivity of human glioma cell line U251 cells and the possible mechanism. Methods The PEGFP-N1, PEGFP-LRIG1 plasmids were transfected into U251 cells with application of liposome-mediated gene transfer technology. And the cells stably transfected were selected by G418. The radiosensitivity of N1-U251 and LRIG1-U251 cells was analyzed by clonogenic assay. Western blotting demonstrated the expression of LRIG1 and RAD51. Repairment of double-strand （ds） DNA breaks was measured by comet assay. Results The LRIG1 protein level in LRIG1-U251 group （0. 352±0. 011 ） was significantly up-regulated compared to NI-U251 group （0. 071±0. 003 ） （ P 〈 0. 01 ）. Overexpression of LRIG1 up-regulated the radiosensitivity of U251 cells with the sensitive enhancement ratio （SER） being 1. 448. The RAD51 protein levels were significantly suppressed before （P 〈0.01 ） and after irradiation （P 〈 0.01） by up-regulating the LRIG1 expression. Comet assay demonstrated the overexpression of LRIG1 increased the Olive Tail Moment of U251 cells from 21.14±3.42 to 32. 81±5.61 after 8-h radiation （P 〈 0.05）. Conclusion Up-regulation of the LRIG1 expression can down-regulate the RAD51 expression, and then inhibit the repairment of dsDNA breaks, increase the radiosensitivity of U251 cells. Key words: Glioma; Leucine-rich repeats and immunoglobulin-like domains 1; RAD51; Radiosensitivity

The Zfx gene is expressed in human gliomas and is important in the proliferation and apoptosis of the human malignant glioma cell line U251

BackgroundZfx is a zinc finger protein of the Zfy family, whose members are highly conserved in vertebrates. Zfx is a shared transcriptional regulator of both embryonic stem cells (ESC) and hematopoietic stem cells (HSC), which suggests a common genetic basis of self-renewal in embryonic and adult stem cells. The level of Zfx expression correlates with aggressiveness and severity in many cancer types, including prostate cancer, breast cancer, and leukemia. However, the importance of Zfx in human glioma is largely unknown. In the present study, we examined the role of Zfx in human glioma.MethodsWe detected expression levels of Zfx mRNA in U251 cells, U87 cells, U373 cells, and A172 cells by semi-quantitative RT-PCR. To analyze the expression of Zfx mRNA in glioma tissues, we performed real-time quantitative PCR on 35 pathologically confirmed glioma samples (Grade I-4cases, Grade II-13cases, Grade III-11cases, and Grade IV-7cases) and on 5 noncancerous brain tissue samples. We used lentivirus-mediated small interfering RNAs (siRNAs) to knock down Zfx expression in the human malignant glioma cell line U251. Changes in Zfx target gene expression were determined by real-time RT-PCR. Cell proliferation was examined by a High Content Screening assay. DNA synthesis in proliferating cells was determined by BrdU incorporation. Cell cycle distribution and apoptosis were detected by flowcytometric analysis.ResultsWe discovered that Zfx mRNA was expressed in U251 cells, U87 cells, U373 cells, and A172 cells. The expression level of Zfx is significantly higher in gliomas compared to noncancerous brain tissue. Using a lentivirus-based RNAi approach, Zfx expression was significantly inhibited in human glioblastoma U251 cells. The effects of Zfx knockdown on cell proliferation, cell cycle distribution, and apoptosis were assessed. Inhibition of Zfx expression in U251 cells by RNAi significantly impaired cell proliferation, increased apoptosis, and arrested cells in S phase.ConclusionsThe results of our study demonstrate that the Zfx gene is highly expressed in glioma tissue and in glioma cell lines. Furthermore, Zfx may play a critical role in cell proliferation, cell cycle distribution, and apoptosis of human malignant glioma cells.

Fasciculols H and I, Two Lanostane Derivatives from Chinese Mushroom Naematoloma fasciculare

Two new highly oxygenated fasciculol-type lanostane derivatives, fasciculols H and I (1 and 2, resp.), together with five known compounds, 3-7, were isolated from the fruiting bodies of the Chinese toxic mushroom Naematoloma fasciculare. Their structures were elucidated on the basis of spectroscopic analyses including 2D-NMR (HMBC, HSQC, NOESY, (1)H,(1)H-COSY). Compounds 5-7 were identified for the first time in this mushroom. Compounds 1, 3, 4, and 5 were evaluated for inhibitory activity against human glioma cell line U87 and nuclear factor-κB.

Effect of over-expressed LRIG3 on cell cycle and survival of glioma cells

This study examined the effects of over-expression of leucine-rich repeats and immunoglobulin-like domains 3 (LRIG3) on the cell cycle and survival of human glioma cell line U87 and U251 and explored the possible mechanisms. The LRIG3 gene was transduced into U87 and U251 cells respectively by using lentivirus and the transduced cells were selected by puromycin. The changes in LRIG3 mRNA and protein levels were measured by RT-PCR and Western blotting. The apoptosis rate was detected by Annexin V-FITC/PI double labeling and the cell cycle was flow cytometrically analyzed. Compared with control cells, LRIG3 mRNA expression in U251 and U87 cells transduced with pLVX-DsRed-LRIG3-Monomer-N1 were increased by 77.6% and 129.7%, and LRIG3 protein expression was raised by 141.3% and 322.7%, respectively. Cell cycle analysis showed that LRIG3 over-expression increased the percentage of cells at G(0)/G(1) phase (P<0.01). Over-expressed LRIG3 could significantly promote the apoptosis of U87 and U251 cells (P<0.05). These findings suggest that the over-expression of LRIG3 could arrest the cell cycle in G(0)/G(1) phase, and promote apoptosis of U87 and U251 cells.

Role of chemotactic factors in glioma stem cells inducing the migration of neural stem cells in vitro

Objective To observe the migration of neural stem cells (NSCs) to glioma stem cells (GSC), and to investigate the chemotactic mechanism. Methods GSCs were cultured and expanded in stem cell medium from human glioma cell line U251 and surgically resected specimens of human gliomas. All the GSCs were identified by using flow cytometry and Western blotting. Migrations of NSCs to GSCs,differentiated cells and concentration gradient fluid of growth factors were detected by Transwell assay. Concentrations of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) secreted by GSCs and differentiated cells were determined by using enzyme linked immunosorbent assay (ELISA). The in vitro migration of NSCs (marked by Dio) to GSCs (marked by Dil) was observed by using fluorescent microscopy. Results Neurospheres of GSCs, with high expression of stemness markers (CD133: 11.02％-33.55％ ), were cultured in stem cell medium and could be differentiated in serumcontaining medium. After differentiation, the expression of stemness markers was decreased, while the marker of glial fibrillary acidic protein (GFAP) was increased ( P ＜ 0.05). Meanwhile, the concentrations of VEGF and bFGF secreted by GSCs were higher than those by differentiated cells. Both GSCs and differentiated cells could induce the migration of NSCs, while GSCs showed significantly stronger chemotactic effect (P ＜ 0. 05). The migratory ability of NSCs might be positively correlated with the concentration of growth factors in chemotactic fluid ( P ＜ 0. 05 ). NSCs could migrate to GSCs and surround the neurosphere of GSCs, which displays cytostatic effect in vitro. Conclusion Gliona stem cells could induce the migration of NSCs and show enhanced chemotaxis compared with the differentiated cells. This chemotactic mechanism might be related to high concentrations of growth factors secreted by GSCs. Migration of NSCs to GSCs displays cytostatic effect in vitro. Key words: Neural stem cells; Glioma stem cells; Migration; Chemotactic factors

Chemoresistance to Temozolomide in Human Glioma Cell Line U251 is Associated with Increased Activity of O 6-methylguanine-DNA Methyltransferase and Can be Overcome by Metronomic Temozolomide Regimen

Temozolomide (TMZ) is a novel cytotoxic alkylating agent for chemotherapy of malignant gliomas. However, intrinsic or acquired resistance to TMZ often defines poor efficacy of chemotherapy in malignant gliomas. A growing number of studies indicate that expression of O(6)-methylguanine-DNA methyltransferase (MGMT) is one of the principal mechanisms responsible for this chemoresistance. In the present study, we evaluated the relationship between expression of MGMT and resistance to TMZ. We generated a TMZ-resistant cell line, U251/TR, by stepwise (8 months) exposure of parental U251 cells to TMZ. The resistance to TMZ was quantified using SRB assay. MGMT expression was evaluated at mRNA (RT-PCR) and protein (Western blot) levels. U251/TR cells showed increased (~ sevenfold) resistance to TMZ. The MGMT expression (both mRNA and protein) was significantly (P < 0.01) increased in U251/TR cells compared with parental U251 cells. Further, MGMT expression fluctuated during exposure of U251/TR cells to TMZ. The resistance of U251/TR cells to TMZ could be overcome by application of elevated doses of TMZ when MGMT expression was at the lowest level. In conclusion, our results demonstrate that the primary mechanism responsible for resistance of U251/TR cells to TMZ is associated with increased expression of MGMT. Resistance of malignant gliomas to TMZ can be overcome by synchronizing metronomic TMZ regimen with MGMT expression.

RNA interference‐mediated silencing of focal adhesion kinase inhibits growth of human malignant glioma xenograft in nude mice

FAK (focal adhesion kinase), which plays a pivotal role in mediating cell proliferation, survival and migration, is frequently overexpressed in human malignant glioma. The expression of FAK increases with the advance of tumour grade and stage. Based on these observations, we hypothesized that attenuation of FAK expression may have inhibitory effects on the growth of malignant glioma. In the present study, human glioma cell line U251 was transfected with plasmids containing U6 promoter-driven shRNAs (small-hairpin RNAs) against human FAK using cationic liposome. The effects of FAK knockdown in U251 cells in vitro were analysed by using flow cytometry and PI (propidium iodide)-staining assays. Based on the encouraging in vitro results with FAK silencing, plasmids encoding FAK-targeted shRNA were encapsulated by DOTAP (dioleoyltrimethylammonium propane):Chol (cholesterol) cationic liposome and injected via tail vein to evaluate its therapeutic efficiency on suppressing tumour growth in a human glioma xenograft model. PCNA (proliferating-cell nuclear antigen), CD34 immunostaining and TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling) assay were used to assess the changes in tumour angiogenesis, apoptosis and proliferation respectively. The results indicated that DOTAP:Chol cationic liposome could deliver therapeutic plasmids systemically to tumour xenografts, resulting in suppression of tumour growth. Treatment with plasmid encoding FAK-targeted shRNA reduced mean tumour volume by approx. 70% compared with control groups (P<0.05), accompanied with angiogenesis inhibition (P<0.05), tumour cell proliferation suppression (P<0.05) and apoptosis induction (P<0.05). Taken together, our results demonstrated that shRNA-mediated silencing of FAK might be a potential therapeutic approach against human malignant glioma.

Influence of expression of miR-221/222 on expression of genome in glioblastoma U251 cells

Objective To study the cell signaling regulation of miR - 221/222 in U251 cell line by microarray analysis and bioinformation method. Method As - miR -221/222 was transfected into U251 human glioma cell line, and the total RNA was extracted for texting gene expression profile. Differentially expressed genes were analyzed by bio - information method and the core transcriptional factors were identified by Western blot and immunofluorescence. Results After down - regulation of miR -221/222 in glioma cell lines, 158 differentially expressed genes were confirmed by microarray analysis. It was found that INF - α signaling pathway was the most significant one regulated by the differentially experssed genes. Moreover, STAT1 and STAT2 were core proteins in INF - α signaling pathway. It was revealed by Western blot and immunofluorescence that the expression of STAT1 and STAT2 were up - regulated in U251 cells after knocked -down miR - 221/222. Conclusions Knockdown of miR -221/222 would up - regulate STAT1 and STAT2 expression partially through IFN - α pathway and result in the change of mRNA expression profiles in U251 cell line. Key words: miR-221; miR-222; Glioma; INF - α signaling pathway

MMP-2 siRNA Inhibits Radiation-Enhanced Invasiveness in Glioma Cells

BackgroundOur previous work and that of others strongly suggests a relationship between the infiltrative phenotype of gliomas and the expression of MMP-2. Radiation therapy, which represents one of the mainstays of glioma treatment, is known to increase cell invasion by inducing MMP-2. Thus, inhibition of MMP-2 provides a potential means for improving the efficacy of radiotherapy for malignant glioma.Methodology/Principal FindingsWe have tested the ability of a plasmid vector-mediated MMP-2 siRNA (p-MMP-2) to modulate ionizing radiation-induced invasive phenotype in the human glioma cell lines U251 and U87. Cells that were transfected with p-MMP-2 with and without radiation showed a marked reduction of MMP-2 compared to controls and pSV-transfected cells. A significant reduction of proliferation, migration, invasion and angiogenesis of cells transfected with p-MMP-2 and in combination with radiation was observed compared to controls. Western blot analysis revealed that radiation-enhanced levels of VEGF, VEGFR-2, pVEGFR-2, p-FAK, and p-p38 were inhibited with p-MMP-2-transfected cells. TUNEL staining showed that radiation did not induce apoptosis in U87 and U251 cells while a significant increase in TUNEL-positive cells was observed when irradiated cells were simultaneously transfected with p-MMP-2 as compared to controls. Intracranial tumor growth was predominantly inhibited in the animals treated with p-MMP-2 alone or in combination with radiation compared to controls.Conclusion/SignificanceMMP-2 inhibition, mediated by p-MMP-2 and in combination with radiation, significantly reduced tumor cell migration, invasion, angiogenesis and tumor growth by modulating several important downstream signaling molecules and directing cells towards apoptosis. Taken together, our results demonstrate the efficacy of p-MMP-2 in inhibiting radiation-enhanced tumor invasion and progression and suggest that it may act as a potent adjuvant for radiotherapy in glioma patients.

Expression of NANOG in human gliomas and its relationship with undifferentiated glioma cells

Stemness genes, including NANOG, which have been reported to play a significant role in embryonic stem cells (ESCs), are purported to be expressed in specific human tumor types. In the present study, we explored the expression of NANOG in gliomas to demonstrate its key roles in maintaining the undifferentiated state of glioma cells. Brain tumor stem cells (BTSCs) were isolated from the human glioma cell line U87 and cultured in simplified serum-free medium. Significantly higher NANOG mRNA and protein expression levels were demonstrated in U87 parental attached cells and suspended BTSCs as well as in 69 glioma specimens of different pathological grades. The relative levels of NANOG mRNA and protein expression were higher in the BTSCs as compared to those in the U87 parental attached cells and were significantly positively correlated with pathological grade. The coexpression and relationship of NANOG, CD133 and GFAP in situ in the cellular levels was determined through double-label immunohistochemical staining in the gliomas. A positive correlation of NANOG and CD133 expression with pathological grade of the samples was noted, while NANOG and GFAP expression correlated negatively with the pathological grade (P<0.01). Overexpression of NANOG in gliomas and its close relationship with the undifferentiated state of glioma cells invivo and in vitro indicated that NANOG may contribute to the existence of BTSCs and may be related to tumorigenesis of the cerebrum by maintaining the undifferentiated state of glioma cells, which provides a foundation to further explore its role in the biological behavior of gliomas.

Transfer of sodium iodide symporter gene into glioma cells by recombinant adenovirus and radioiodine therapy for the glioma cells in vitro and in nude mice

To explore the possibility of tranfecting hNIS and hTPO genes into gliomas cells by recombinant adenovirus for radioactive iodide treatment. To tranfect hNIS gene into human glioma cell line U251 by recombinant adenovirus. The biological functions of the cells stably expressing hNIS and hTPO genes were assessed by (125)I uptake assay, (125)I influx-course and (125)I-efflux-course. A glioma model was established with inoculation of the U251 cells in nude mice, and the inhibiting effect of (131)I on the tumor growth was tested in the mouse models. The hNIS and hTPO genes were successfully transfected into human gliomas cell line U251 cells by recombinant adenovirus. The radioactive iodide could be intaken by the tumor cells mediated by hNIS gene. The uptake of (125)I was higher in cell lines hNIS-U251 and hNIS-hTPO-U251 cells than in cell line U251 cells. The tumor volume of the mice after (131)I treatment was significantly decreased in comparison with that before treatment. Radioactive (131)I treatment after HNIS-based gene transfer can be enhanced and effectively inhibit the tumor growth in nude mice.

Effects of adriamycin and bone morhogenetic proteins 4 in human brain glioma cell line U251 and probable signal pathway

Objective To investigate the effects of bone morhogenetic proteins 4 （BMP4） and the probable signal pathway in human brain glioma cell line U251.Method Adriamycin or plasmid of BMP4 was given to U251 cells and BMP4 siRNA was given to U251 cells which had been given adriamycin for 48 h.MTT was used to detect the activity of U251 cell growth in control group and treatment group,the formula（ 1 -treatment group OD/control group OD） × 100% was used to calculate U251 cells growth inhibition rate. Reverse transcription - PCR （ RT - PCR ）, fluorescence quantitative PCR （ FQ - PCR ） and Western blot were used to observe whether the translation was sucessful as well as the expression of BMP4 and mothers against decapentaplegic homolog 4 （ SMAD4 ）.Results Compared to control group, U251 cells were inhibited when adriamycin or plasmid of BMP4 was given to U251 cells.The expression of BMP4 was up -regulated and SMAD4was down-regulated.Compared to the adriamycin group,the cells inhibition was increased when BMP4 siRNA was given to adriamycin -treated U251 cells for 48 h.The expression of BMP4 was decreased and the SMAD4 was increased.Conclusions Adriamycin could inhibit U251 cells.BMP4 is associated with the inhibition of U251cells through BMP4 - Smad signal pathway.BMP4 may be a new target for glioma therapy. Key words: Glioma; Bone morphogenetic protein 4; SMAD4; Target therapy; U251