Human U251 Cells Research Articles

All-trans retinoic acid enhances temozolomide-induced autophagy in human glioma cells U251 via targeting Keap1/Nrf2/ARE signaling pathway.

The present study evaluated the retinoic acid (RA) enhancement of temozolomide (TMZ) effects on the human glioma cells U251 and explored its underlying molecular mechanism. The cell growth was detected using the MTT assay and the cell cycle was assessed by flow cytometry. Cell apoptosis was analyzed by Annexin V/propidium iodide staining, and the cell morphology was evaluated using transmission electron microscopy (TEM). Additionally, reverse transcription-PCR and western blot analysis were applied to detect the mRNA and protein levels. The RA treatment in combination with TMZ in the human U251 cells further inhibited cells growth, arresting cell cycle progression at the G0/G1 phase, and significantly induced apoptosis of U251 cells. After the RA+TMZ treatment of U251 cells, autophagy associated proteins Beclin 1 and LC3B were significantly increased, and the TEM analysis were consistent with autophagy protein levels. Moreover, Keap1/Nrf2/ARE expression was downregulated significantly, indicating the involvement in the mechanisms that RA treatment could enhance the TMZ effects on U251 cells. RA treatment in combination with TMZ may provide some experimental evidence for the possible effect of RA+TMZ against the growth and the proliferation of glioma cells. Therefore, the RA+TMZ administration may have potential utility for glioblastoma treatment.

Interstitial photodynamic therapy and glioblastoma: Light fractionation in a preclinical model.

Glioblastoma is a high-grade cerebral tumor with local recurrence and poor outcome. Photodynamic therapy (PDT) is a localized treatment based on the light activation of a photosensitizer (PS) in the presence of oxygen, which results in the formation of cytotoxic species. The delivery of fractionated light may enhance treatment efficacy by reoxygenating tissues. To evaluate the efficiency of two light-fractionation schemes using immunohistological data. Human U87 cells were grafted into the right putamen of 39 nude rats. After PS precursor intake (5-ALA), an optic fiber was introduced into the tumor. The rats were randomly divided into three groups: without light, with light split into 2 fractions and with light split into 5 fractions. Treatment effects were assessed using brain immunohistology. Fractionated treatments induced intratumoral necrosis (P < 0.001) and peritumoral edema (P = 0.009) associated with a macrophagic infiltration (P = 0.006). The ratio of apoptotic cells was higher in the 5-fraction group than in either the sham (P = 0.024) or 2-fraction group (P = 0.01). Peripheral vascularization increased after treatment (P = 0.017), and these likely new vessels were more frequently observed in the 5-fraction group (P = 0.028). Interstitial PDT with fractionated light resulted in specific tumoral lesions. The 5-fraction scheme induced more apoptosis but led to greater peripheral neovascularization. Lasers Surg. Med. 49:506-515, 2017. © 2016 Wiley Periodicals, Inc.

Effect of quercetin on the biological characteristics of human glioma U251 cells

Objectives To Study the effect of quercetin on the biological characteristics of human glioma U251 cells and to investigate its possible mechanisms. Methods The U251 cells were cultured in vitro. The effect of quercetin on the proliferation of U251 cells was detected by CCK8 assay. Flow cytometry was used to detect the effect of quercetin on the U251 cell cycle and apoptosis. Scratch test and Transwell test were used to observe the effect of quercetin on metastasis and invasion of U251 cells. Western blot was used to detect the effects of quercetin on protein expression levels of U251 cell Bax, Bcl-2, MMP-9, and MMP-2. Results Quercetin could inhibit the proliferation ability in vitro of U251 cells. IC50 at 24 h and 48 h were 113.65 μg/ml and 48.61 μg/ml respectively. Flow cytometry showed that quercetin acting on U251 cells might have some changes, such as the upregulation of peak ratio, the downregulation of G0/G1 phase rate, and upregulation of G2/M phase rate. The migration and invasion abilities of the U251 cells pretreated by quercetin for 24 h were decreased significantly compared with the control group, and the 20 μg/ml group was lower than the10 μg/ml group (P<0.05). After the U251 cells being treated with quercetin, the Bax expression level was upregulated, the Bcl-2, MMP-9, and MMP-2 protein expression levels were decreased, and there was significant difference between the 10 μg/ml group and the 20 μg/ml group (P<0.05). Conclusions Quercetin has significant cytotoxic effect in vitro on U251 cells. It can induce apoptosis of U251 cells, causing G2/M phase arrest, decreasing the cell migration and invasion abilities. These effects may be associated with the decrease of Bcl-2/Bax ratio and the downregulation of MMP-9 and MMP-2 protein expression levels. Key words: Glioma; Quercetin; Apoptosis; Cell migration assays; Neoplasm invasiveness

Elevation of Ser9 phosphorylation of GSK3β is required for HERV-W env-mediated BDNF signaling in human U251 cells

Human endogenous retrovirus W family (HERV-W) envelope (env) is known to be associated with neurological and psychiatric disorders, such as multiple sclerosis and schizophrenia. Previous studies showed that overexpression of HERV-W env could induce brain-derived neurotrophic factor (BDNF) gene expression. In human and rat cells, BDNF-mediated signal transduction might be modulated by glycogen synthase kinase 3β (GSK3β). Both BDNF and GSK3β are schizophrenia-related genes. In this paper, we investigated whether GSK3β was involved in the HERV-W env-induced expression of BDNF. We found that HERV-W env increased phosphorylation of GSK3β at Ser9 (p-GSK3β (Ser9)) and the ratio of p-GSK3β (Ser9) to total GSK3β (p<0.05) in U251 cells. Overexpression of HERV-W env led to a 36.2% reduction in GSK3β activity compared to control (p<0.05). The levels of β-catenin, cyclin D1 and TSC2 mRNAs were upregulated (p<0.05). These data suggested that overexpression of HERV-W env might activate the GSK3β signaling pathway in U251 cells. Further, knockdown of GSK3β reduced the expression of total GSK3β, p-GSK3β (Ser9), and the ratio of p-GSK3β (Ser9) to total GSK3β by 28.6%, 50.4%, and 30.2%, respectively (p<0.05). Levels of β-catenin, cyclin D1 and TSC2 mRNAs were also reduced (p<0.05). Interestingly, GSK3β activity increased (p<0.05). Knockdown of GSK3β also decreased mRNA and protein expression of BDNF by 49.9% and 48.5% respectively (p<0.05). These results indicated that phosphorylation of GSK3β at Ser9 might be involved in HERV-W env-induced BDNF expression, and will hopefully improve our understanding of the role of HERV-W env in neurological and psychiatric diseases (schizophrenia, etc).

Valproic Acid Promotes Human Glioma U87 Cells Apoptosis and Inhibits Glycogen Synthase Kinase-3β Through ERK/Akt Signaling

Background: Valproic acid (VPA), an established antiepileptic drug, was assessed for antitumor activity, including its effects on glioblastoma, but its role has not been determined. Methods: In the present study, we investigated VPA-induced apoptosis effects on human U87 cells by cell viability, lactate dehydrogenase (LDH) release, TUNEL/Hoechst staining and flow cytometric in vitro, then we further explored the underlying molecular mechanisms using the selective antagonists PD98059, LY294002 and SB216763. Results: The data showed that VPA dose-dependent induction of glioma U87 cells to undergo apoptosis through the mitochondria-dependent pathway in vitro. VPA activated the ERK/Akt pathways by increasing their protein phosphorylation and in turn inhibited GKS3β activation by the induction of GKS3β phosphorylation. However, the MAPK inhibitor PD98059 and/or PI3K inhibitor LY294002 were able to antagonize the effects of VPA by abolishing ERK/Akt activations and cancelling GSK3β suppression, thus it impaired VPA apoptosis-inducing effects on glioma cells. Furthermore, the GSK3β inhibitor SB216763 caused a strong suppression of GSK3β activity, which showed similar effects of VPA on regulation of protein expression and apoptosis. Conclusion: These findings suggest that GSK3β may be the central hub for VPA-induced apoptosis and VPA can be further evaluated as a novel agent for glioma therapy.

Bis((1,4-dimethyl-9H-carbazol-3-yl)methyl)amine-mediated anticancer effect triggered by sequence-specific cleavage of DNA leading to programmed cell death in the human U87 cell line

Bis((1,4-dimethyl-9H-carbazol-3-yl)methyl)amine (DMCM) has been designed as a novel anti-cancer agent which is believed to have a sequence-specific groove binding interaction with DNA, leading to anticancer effects.

ATPS-64PRECLINICAL STUDIES USING NATIVIS VOYAGER RFE SYSTEM, A NOVEL NON-INVASIVE, LOW ENERGY, NON-THERMAL, NON-IONIZING RADIOFREQUENCY ENERGY (RFE) DEVICE IN GLIOBLASTOMA MOUSE MODELS

BACKGROUND: Cancer treatment using radiofrequency energy (RFE) devices have been used in the past and have recently shown benefit in clinical trials of glioblastoma. Here we report a proof-of-concept application using the Nativis Voyager RFE System, which provides specific, low energy, non-invasive, non-thermal and non-ionizing oscillating electromagnetic signals. These signals inhibit in vitro glioblastoma tumor cell proliferation via an anti-mitotic effect. METHODS: We tested the ability of Voyager RFE System signals to inhibit tumor growth in both immunocompromised and immunocompetent mouse models. The immunocompetent model study, using white noise signal compared to control showed no difference in growth in the two groups. After establishing this, in the immunocompromised model, we implanted human U87 cells subcutaneously into 6-8 week old nude mice. In the immunocompetent model, we implanted mouse GL261 cells subcutaneously into 6-8 week old C57/BL6 mice. Voyager RFE signal was started ∼10-14 days after cells were implanted, when there were small, measurable tumors. There were two treatment groups (N = 10 tumors each group). The control group was housed in a separate room and was not treated with any RFE signal. The treatment group was treated with Voyager RFE signal 24 hours daily. Voyager RFE signal was applied using a rectangular 40mG coil placed under the cage. Tumors were measured ∼weekly. RESULTS: In the immunocompromised U87 model, tumor growth was significantly delayed in Voyager RFE signal group compared to control group (at day 39; p < 0.0001). Moreover, in the immunocompetent GL261 model, tumor growth was significantly delayed in Voyager RFE signal group compared to control group (at day 28; p < 0.0001). CONCLUSIONS: We show that Nativis Voyager RFE System signal delays glioblastoma tumor growth in SQ immunocompromised and immunocompetent mouse models.

ALS mutations in TLS/FUS disrupt target gene expression.

Amyotrophic lateral sclerosis (ALS) is caused by mutations in a number of genes, including the gene encoding the RNA/DNA-binding protein translocated in liposarcoma or fused in sarcoma (TLS/FUS or FUS). Previously, we identified a number of FUS target genes, among them MECP2. To investigate how ALS mutations in FUS might impact target gene expression, we examined the effects of several FUS derivatives harboring ALS mutations, such as R521C (FUS(C)), on MECP2 expression in transfected human U87 cells. Strikingly, FUS(C) and other mutants not only altered MECP2 alternative splicing but also markedly increased mRNA abundance, which we show resulted from sharply elevated stability. Paradoxically, however, MeCP2 protein levels were significantly reduced in cells expressing ALS mutant derivatives. Providing a parsimonious explanation for these results, biochemical fractionation and in vivo localization studies revealed that MECP2 mRNA colocalized with cytoplasmic FUS(C) in insoluble aggregates, which are characteristic of ALS mutant proteins. Together, our results establish that ALS mutations in FUS can strongly impact target gene expression, reflecting a dominant effect of FUS-containing aggregates.

Curcumin regulation of glioma cell proliferation and apoptosis through Wnt/β-catenin signaling pathway: an experimental study

Objective To investigate the effects of curcumin on glioma cell proliferation and apoptosis through Wnt/β-catenin signaling pathway. Methods The research objects were the human glioblastoma U251 cell lines. The experiment was divided into either a blank control group or a curcumin experiment group (the experiment concentrations were 10 μmol/L and 20 μmol/L respectively). The cell toxicity test was used to study cellular toxic effect of curcumin on U251 glioma. Four methyl thiazolyl tetrazolium (MTT) assay was used to observe the cell proliferative capacity; the caspase-3 activity assay and flow cytometry (FCM) test were used to detect cell apoptosis; flow cytometry was used detect the cell cycle; real-time quantitative PCR, Western-blotting, and immunofluorescence assay were used to detect the expression levels of intracellular β-catenin and c-myc. The luciferase assay was used to detect the changes of Wnt/β-catenin signaling pathway activity after the effects of curcumin. Results With the increased effect concentration of curcumin, the cytotoxicity of U251 glioma cells enhanced. In the curcumin experimental group, when the concentrations were 5, 10, 20, and 40 μmol/L, the cells activities were 88.55%±2.35%, 62.78%±3.35%, 35.17%±2.05%, and 7.47%±1.92%, respectively (P<0.05), and the effect of proliferation inhibition enhanced obviously. IC50 of the measured curcumin effect for 24 h was 16.32 μmol/L, therefore, the concentrations of 10 μmol/L and 20 μmol/L in the curcumin experimental group were chosen; the relative activities of caspase-3 in the experimental group (10 μmol/L and 20 μmol/L) were 3.64%±1.49% and 5.41%±0.36% respectively. Compared with the control group 0.91%±0.62%, the activities enhanced significantly (P<0.05). After flow cytometry was used to detect the curcumin effects for 48 h, the U87 apoptotic rates of the experimental group were 14.7%±2.21% and 35.6%±1.37% respectively. Compared with the apoptotic rate of the control group (0.5%±0.67%), there was significant difference (P<0.05). The detection of curcumin with the flow cytometry could block the cell cycle to the G2/M phase. The G2/M phases of the control group and the curcumin experiment group (10 μmol/L and 20 μmol/L) were 7.52%±3.84%, 16.28%±2.73%, and 25.47%±5.61%, respctively P<0.05); After curcumin acting on gliomas, the mRNA level of β-catenin was decreased. The test results of fluorescence quantitative PCR were 93.87%±5.73%, 68.83%±7.67%, and 47.46%±8.52%, respectively (P<0.05); and there were significant differences. Westen blotting showed that the expression levels of β-catenin and c-myc protein were decreased. Immunofluorescence assay found that curcumin suppressed β-catenin into the cell nuclei and reduced the formation of its compound at the same time. Luciferase experiment showed that the activity of Wnt/β-catenin signaling pathway was inhibited obviously after curcumin acting on glioma cells (P<0.01). Conclusion Curcumin may inhibit glioma cell proliferation and promote apoptosis by downreglating Wnt/β-catenin signaling pathway. Key words: Glioma; Curcumin; Wnt/β-catenin signaling pathway; Proliferation; Apoptosis

Effect of 3-methyladenine on cisplatin induced apoptosis in human glioma U251 cells

Objective 3-methyl adenine (3-methyladenine, 3-MA) was used to test the effect on cisplatin induced U251 cell apoptosis. Methods MTT assay was used to test the effect of 3-MA on cisplatin cytotoxicity in U251 cells. Hoechst33342 staining was used to test the effect of 3-MA on cisplatin induced apoptosis. Western bolt was used to detect the expression of ER stress associated proteins, ubiquitinated protein, protein disulfide isomerase (PDI), glucose-regulated protein78 (Grp78), C/EBP homologous protein (CHOP), and activation of caspase-4 and caspase-3. The expression of microtubule-associated protein 1 light chain 3 (LC3) and PDI were observed by immunofluorescence staining and confocal microscopy. Results MTT assay showed that 3-MA could enhance cisplatin induced growth inhibition rate in human glioma U251 cells (P=0.00324 cisplatin troup vs. control group; P=0.02249 3-MA+ cisplatin group vs. cisplatin group). Hoechst33342 staining showed that 3-MA could increase cisplatin induced apoptotic effect in U251 cells. Western blot showed that 3-MA increases the expressions of ubiquitinated proteins, ERS marker proteins PDI, Grp78(P=0.00294, 0.0012, 0.00179 cisplatin group vs. control group; P=0.01896, 0.01515, 0.00734 3-MA+ cisplatin group vs cisplatin group), CHOP, and active Caspase-4 and active Caspase-3 (P=0.00081, 0.00017, 0.00018 cisplatin group vs. control group; P=0.00671, 0.00934, 0.01124 3-MA+ cisplatin group vs. cisplatin group). 3-MA could inhibit the expression of LC3II induced by cisplatin in U251 cell (P=0.00447 cisplatin group vs. control group; P=0.01588 3-MA+ cisplatin group vs. cisplatin group). Indirect immunofluorence staining showed that 3-MA inhibited the aggregation of LC3 and increased the expression of PDI. Conclusion 3-MA combined with cisplatin enhances cisplatin induced apoptosis by increasing ER stress. Key words: Cisplatin; Autophagy; ER stress; Apoptosis; Glioma

Silencing of the TPM1 gene induces radioresistance of glioma U251 cells.

The present study was designed to investigate the relationship between tropomyosin 1 (TPM1) and radioresistance in human U251 cells. Radioresistant U251 (RR-U251) cells were established by repeated small irradiating injury. TPM1 levels in the U251 and RR-U251 cells were inhibited by transfection with TPM1-short hairpin RNA (shRNA) while overexpression was induced by treatment with pcDNA3.1‑TPM1. The radiosensitivity of the U251 and RR-U251 cells and the plasmid-transfected cells was evaluated by cell viability, migration and invasion assays. Cell apoptosis was also examined in vitro. The radiosensitivity of U251 xenografts was observed by tumor growth curve after radiotherapy in an in vivo experiment. Western blotting and immunohistochemistry were used to detect the level of TPM1 in vivo. The expression of TPM1 was significantly decreased in the RR-U251 cells, which may be correlated with the radioresistance of the glioma U251 cells. In the TPM1-silenced RR-U251 and TPM1-silenced U251 cells, cell viability, migration and invasion ability were significantly increased, and the rate of cell apoptosis was decreased. Consistent with these results, in the TPM1-overexpressing U251 and RR-U251 cells, cell viability, migration and invasion abilities were markedly decreased, and increased apoptosis was noted when compared to the control group. Tumor growth of the U251 xenografts was significantly inhibited following treatment with pcDNA3.1‑TPM1 combined with radiotherapy. Taken together, these results indicate that TPM1 may be one mechanism underlying radiation resistance, and TPM1 may be a potential target for overcoming the radiation resistance in glioma.

Effects of B cell lymphoma/leukemia-2 associated X protein gene over-expression on expression of second mitochondria-derived activator of caspase/direct IAP-binding protein with low pI in human glioblastoma U251 cells

Objective To explore the effects of B cell lymphoma/leukemia-2 associated X protein (bax) gene over-expression on the expression of second mitochondria-derived activator of caspase (Smac)/direct IAP-binding protein with low pI (DIABLO) in human glioma cell line.Methods The plasmid of expression vector pEGFP-bax was transfected into human glioblastoma U251 cells with lipofectamine2000,and meanwhile the non-transfected cells and the pEGFP-transfected U251 cells were taken as controls.Cellular bax and Smac/DIABLO expression was determined by Western boltting.Cellular distribution of Smac/DIABLO protein was evaluated by immunofluorescence (IF) assays and observed under inverted fluorescence microscope.Results The bax protein expression level in pEGFP-bax-transfected U251 cells was (0.82 ±0.12),significantly higher than in non-transfected group (0.35 ±0.05) and pEGFP-transfected control group (0.32 ± 0.09,P ＜ 0.05).The expression level of Smac/DIABLO protein in pEGFP-bax-transfected group was (1.09 ± 0.16),significantly higher than in non-transfected group (0.62 ± 0.10) and pEGFP-transfected control group (0.70 ± 0.20) (P ＜ 0.05).There was no significant difference in the expression of bax and Smac/DIABLO between non-transfected and pEGFP-transfected groups (P ＞0.05).IF revealed that Smac/DIABLO was mainly localized in the cytoplasm.Conclusion Transfection of extrinsic bax gene results in its over-expression in U251 cells,which could up-regulate the expression of Smac/DIABLO protein. Key words: Glioma; B cell lymphoma/leukemia-2 associated X protein; Second mitochondria-derived activator of caspase/direct IAP-binding protein with low pI

Synergy of enediyne antibiotic lidamycin and temozolomide in suppressing glioma growth with potentiated apoptosis induction.

The present work evaluated the synergistic efficacy of an enediyne antibiotic lidamycin (LDM) plus temozolomide (TMZ) against glioma in vitro and in vivo. LDM plus TMZ inhibited the proliferations of rat glioma C6 cells and human glioma U87 cells more efficiently than the single usage of LDM or TMZ. In addition, LDM also potentiated the apoptosis inductions by TMZ in rat C6 cells and human U87 cells. Meanwhile, the results of TdT-mediated dUTP Nick End Labeling assay for subcutaneous U87 tumor sections indicated an enhanced apoptosis induction in vivo by LDM plus TMZ, which confirmed the high potency of the combination for glioma therapy. As determined by Western blot, apoptosis signal pathways in C6 cells and U87 cells were markedly affected by the synergistic alteration of P53, bax, procaspase 3, and bcd-2 expression. In both subcutaneous U87 xenograft and C6 intracerebral orthotopic implant model, TMZ-induced glioma growth suppression was dramatically potentiated by LDM. As shown, the combination therapy efficiently reduced the tumor volumes and tumor weights of the human glioma U87 xenograft. Kaplan-Meier assay revealed that LDM plus TMZ dramatically prolonged the life span of C6 intracerebral tumor-bearing rats with decreased tumor size. This study indicates that the combination of LDM with TMZ might be a promising strategy for glioma therapy.

Conditioned media from human adipose tissue-derived mesenchymal stem cells and umbilical cord-derived mesenchymal stem cells efficiently induced the apoptosis and differentiation in human glioma cell lines in vitro.

Human mesenchymal stem cells (MSCs) have an intrinsic property for homing towards tumor sites and can be used as tumor-tropic vectors for tumor therapy. But very limited studies investigated the antitumor properties of MSCs themselves. In this study we investigated the antiglioma properties of two easily accessible MSCs, namely, human adipose tissue-derived mesenchymal stem cells (ASCs) and umbilical cord-derived mesenchymal stem cells (UC-MSCs). We found (1) MSC conditioned media can significantly inhibit the growth of human U251 glioma cell line; (2) MSC conditioned media can significantly induce apoptosis in human U251 cell line; (3) real-time PCR experiments showed significant upregulation of apoptotic genes of both caspase-3 and caspase-9 and significant downregulation of antiapoptotic genes such as survivin and XIAP after MSC conditioned media induction in U 251 cells; (4) furthermore, MSCs conditioned media culture induced rapid and complete differentiation in U251 cells. These results indicate MSCs can efficiently induce both apoptosis and differentiation in U251 human glioma cell line. Whereas UC-MSCs are more efficient for apoptosis induction than ASCs, their capability of differentiation induction is not distinguishable from each other. Our findings suggest MSCs themselves have favorable antitumor characteristics and should be further explored in future glioma therapy.

Phosphoglycerate kinase 1 promotes radioresistance in U251 human glioma cells

Phosphoglycerate kinase 1 (PGK1) has been found to be increased in radioresistant astrocytomas. The present study was designed to investigate the potential role of PGK1 in the radioresistance in U251 human cells. Quantitative PCR and western blot analysis were performed to evaluate PGK1 expression for mRNA levels and protein levels, respectively. The short hairpin RNA (shRNA)-PGK1 and the high expression plasmids were transfected to radioresistant U251 cells (RR-U251 cells) or normal U251 cells using lipofectamine™ 2000. The cell viability was determined by MTT assay. The wound-healing assay (WHA) was used to evaluate cell migration ability. Cell invasion abilities were examined using a Transwell culture chamber system. Our results showed that the expression of PGK1 was significantly increased in RR-U251 cells compared to normal U251 cells. Following irradiation, the cell viability as well as the migration and invasion ability were significantly higher in RR-U251 cells compared with normal U251 cells. Downregulating PGK1 using shRNA induced a significantly downregulated cell viability and decreased migration and invasion ability, and overexpression of PGK1 contributed to upregulated cell viability and increased migration and invasion ability, both in RR-U251 cells and normal U251 cells. These findings suggest that PGK1 could promote radioresistance in U251human cells.

Effect of RhoE on apoptosis of human glioma U251 cells and the mechanism

Objective To investigate the effects of Ras homolog gene family,member E (RhoE)overexpression on the apoptosis of human glioma U251 cells and the mechanism.Methods The Myc-N1 and Myc-RhoE plasmids were transfected into human glioma U251 cells by Lipofectamine technology.The changes of RhoE and Cyclin D1 in transfected glioma cells were detected by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting.The cell proliferaion and apopotosis were analyzed by methyl thiazol tetrazolium (MTF) assay and flow cytometry.Results The RhoE mRNA and protein levels in RhoE-U251 group was significantly up-regulated compared to N1-U251 group and control group (P ＜0.05),while the Cyclin D1 mRNA and protein levels in RhoE-U251 group were significantly suppressed.After overexpression of RhoE was up-regulated,the growth of these cells was inhibited and their apoptosis was obviously enhanced.Conclusion RhoE may be a tumor suppressor gene.Up-regulation of RhoE expression can down-regulate the Cyclin D1 expression,and then promote the apoptosis and inhibit the growth of human glioma U251 cells. Key words: Glioma; Apoptosis ; RhoE ; Cyclin D1

Baculoviral transduction facilitates TALEN-mediated targeted transgene integration and Cre/LoxP cassette exchange in human-induced pluripotent stem cells

Safety and reliability of transgene integration in human genome continue to pose challenges for stem cell-based gene therapy. Here, we report a baculovirus-transcription activator-like effector nuclease system for AAVS1 locus-directed homologous recombination in human induced pluripotent stem cells (iPSCs). This viral system, when optimized in human U87 cells, provided a targeted integration efficiency of 95.21% in incorporating a Neo-eGFP cassette and was able to mediate integration of DNA insert up to 13.5 kb. In iPSCs, targeted integration with persistent transgene expression was achieved without compromising genomic stability. The modified iPSCs continued to express stem cell pluripotency markers and maintained the ability to differentiate into three germ lineages in derived embryoid bodies. Using a baculovirus-Cre/LoxP system in the iPSCs, the Neo-eGFP cassette at the AAVS1 locus could be replaced by a Hygro-mCherry cassette, demonstrating the feasibility of cassette exchange. Moreover, as assessed by measuring γ-H2AX expression levels, genome toxicity associated with chromosomal double-strand breaks was not detectable after transduction with moderate doses of baculoviral vectors expressing transcription activator-like effector nucleases. Given high targeted integration efficiency, flexibility in transgene exchange and low genome toxicity, our baculoviral transduction-based approach offers great potential and attractive option for precise genetic manipulation in human pluripotent stem cells.

Construction of a recombinant lentivirus containing human microRNA-7-3 and its inhibitory effects on glioma proliferation.

In the present study, we constructed a lentivirus, FIV-CMV-GFP-miR-7-3, containing the microRNA-7-3 gene and the green fluorescent protein gene, and used it to transfect human glioma U251 cells. Fluorescence microscopy showed that 80% of U251 cells expressed green fluorescence. Real-time reverse transcription PCR showed that microRNA-7-3 RNA expression in U251 cells was significantly increased. Proliferation was slowed in transfected U251 cells, and most cells were in the G1 phase of the cell cycle. In addition, the expression of the serine/threonine protein kinase 2 was decreased. Results suggested that transfection with a lentivirus carrying microRNA-7-3 can effectively suppress epidermal growth factor receptor pathway activity in U251 cells, arrest cell cycle transition from G1 phase to S phase and inhibit glioma cell growth.

Abstract B14: Characterization of an orthotopic glioblastoma in vivo mouse model with firefly luciferase expressing human U87 cells using in vivo bioluminescence imaging and magnetic resonance imaging (MRI).

Abstract Glioblastoma is the most common and most aggressive type of malignant brain tumors. The prognosis is poor, with a median survival time of several months only. Despite steady progress in treatment of glioblastomas this tumor entity is still not curable. Thus, there is an urgent need for the development of novel effective therapies, and for appropriate in vivo models to characterize them. Most often novel antiglioblastoma drugs are tested in subcutaneous xenograft mouse models using various human or murine glioblastoma cell lines. To generate more relevant, orthotopic brain cancer in vivo models, we stably transduced the human glioblastoma cell line U-87 with fire fly luciferase (U-87-Luci) suitable for in vivo bioluminescence imaging. To initiate orthotopic growth, nude mice were anesthetized and placed in a stereotactic fixation device. Through a burr hole cells were implanted intracranially using a Hamilton syringe. Once to twice a week we used bioluminescence imaging to continually monitor tumor growth in vivo. However, as magnetic resonance imaging (MRI) is the most relevant tool to monitor oncological therapy in glioblastomas clinically, we adapted this translational aspect and analysed the mice in parallel using a dedicated animal MRI System. MRI offers detailed information about growth characteristics, size, blood supply and interaction with the surrounding tissue of the tumor. To achieve this, we performed T2-weighted imaging, and diffusion weighted imaging (DWI) weekly to visualise the tumors as well as necrosis and edemas. To monitor the blood supply of the glioblastomas we performed dynamic contrast enhanced MR imaging (DCE-MRI). The use of both methods allowed independent and complementary characterization of the tumors. Approximately four weeks after implantation, exponential growth of the orthotopic tumors was observed, and another two weeks later necropsy was done. In vivo bioluminescence imaging revealed only a very dominant signal in the region of the brain where the primary tumor was located. To address potential metastases in more detail we analysed a set of different resected organs and tissues for in vitro luciferase activity followed by extensive histological examination. Currently, we test different known anti-glioblastoma agents in this orthotopic model to evaluate an antitumoral effect as well as a potential reduction of the edemas. Next, we plan to test carefully novel compounds in order to discover new generations of drugs suitable to help glioblastoma patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B14.

Ghrelin induces cell migration through GHS-R, CaMKII, AMPK, and NF-κB signaling pathway in glioma cells

Ghrelin is a newly discovered gastric peptide which stimulates food intake, energy balance, and growth hormone release. Recent reports have also shown that circulating ghrelin can efficiently reach the brain. However, the molecular mechanisms and pathophysiologic roles underlying ghrelin-induced glioma migration remain unclear. Glioma is the most common primary adult brain tumor with poor prognosis because of the spreading of tumor cell to the other regions of brain easily. In present study, we found that application of recombinant human ghrelin enhances the glioma cell migration in both rat C6 and human U251 cells. Ghrelin and its receptor GHS-R (growth hormone secretagogue receptor) are expressed in a wide variety of tissues and cell types, including various cancer cells. However, little is known about the expression of ghrelin or GHS-R in brain tumors. Here, we found that ghrelin increased GHS-R receptor up-regulation, and the enhancement of ghrelin-induced glioma cell motility markedly inhibited by a GHS-R antagonist. In addition, ghrelin-mediated migration was attenuated by treatment of CaMKII inhibitor, and AMPK inhibitors and pre-transfection with AMPK siRNA. Moreover, ghrelin stimulation also increased the phosphorylation of CaMKII and AMPK. Treatment with three different types of NF-κB inhibitors or pre-transfection with KM-IKKα, or KM-IKKβ also reduced ghrelin-induced glioma cell migration. Moreover, treatment of ghrelin also induced IKKα/β activation, IκBα phosphorylation, p65 phosphorylation at Ser(536), and increased NF-κB-DNA binding activity and κB-transcriptional activity. These results indicate that ghrelin enhances migration of glioma cells is mainly regulated by the GHS-R, CaMKII, AMPK, and NF-κB pathway.