BT325 Cells Research Articles

FKBP51 acts as a biomarker of early metastasis and is related to carmustine sensitivity in human glioma cells.

Given that FK506 binding protein 51 (FKBP51) is upregulated in multiple cancers, we designed the present study to characterize its role as well as underlying regulatory mechanisms in glioma in the presence and absence of the chemotherapeutic carmustine (BCNU). Through lentiviral overexpression and shRNA knockdown of FKBP51, we examined the effects on BT325 glioma cell proliferation, migration and invasion using quantitative reverse transcription PCR (qRT-PCR), CCK-8 assay, flow cytometry, and transwell assay. The upregulation of FKBP51 resulted in significantly decreased BT325 cell proliferation and cell viability, cell cycle arrest, reduced BCNU chemosensitivity and AKT pathway inactivation. However, FKBP51-overexpressed BT325 cells showed enhanced migration and invasion, which was supported by corresponding increase in phosphorylated IKKα (p-IKKα), MMP-2, and MMP-9 levels, as well as increased NF-κB p65 nuclear translocation. By contrast, FKBP51-suppressed BT325 cells showed excessive proliferation and BCNU resistance due to increased p-AKT activation and attenuated migration and invasion. We demonstrated that the effects of FKBP51 on BT325 glioma cell proliferation, migration, invasion and BCNU chemosensitization are modulated via the AKT and NF-κB pathways. Furthermore, our findings suggest the potential of FKBP51 as a prognostic glioma biomarker and an indicator of patient response to chemotherapy.

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

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

Chemotherapeutic effect of tamoxifen on temozolomide-resistant gliomas.

Tamoxifen, a selective estrogen receptor modulator, is widely used in the chemotherapy of estrogen receptor-positive breast cancer. Recent studies have indicated that tamoxifen might have a potential chemotherapeutic effect on glioma. In the present study, we determined the chemotherapeutic action of tamoxifen on human glioma cell lines. Methylation of 06-methylguanine-DNA methyltransferase was identified in A172, U251, and BT325 glioma cell lines, but not in the U87 cell line. Consistently, A172, U251, and BT325 cell lines are resistant to temozolomide. Tamoxifen induced significant cytotoxic action in A172, U251, BT325, and U87 cell lines. Further, Hoechst 33342 staining and apoptosis flow cytometric analysis demonstrated that tamoxifen induced apoptosis in the BT325 cell line. Mitochondrial complex analysis indicated that tamoxifen, but not other estrogen receptor modulators, dose-dependently inhibits complex I activity. In summary, our study suggests that tamoxifen might have a chemotherapeutic effect on temozolomide-resistant glioma through its direct action on mitochondrial complex I inhibition and could provide further evidence to support future clinical trials of tamoxifen for the treatment of glioblastoma.

Effect of Flt-1 gene on the expression of VEGF and proliferation in BT325 cells

Objective To study the Effects of Flt-1 gene on the expression of VEGF and proliferation in BT325 cells.Methods The total RNA was extracted from human umbilical vein endothelial cells as the template and Flt-1 gene was amplified by reverse transcription polymerase chain reaction(RT-PCR).By using gene recombination technique,the Flt-1 cDNA was inserted into eukaryotic expression vector pEGFP-C1.The recombinant plasmid was identified by restriction enzyme digestion and sequence analysis.It was infected into BT325 cells.The quantity of VEGF protein in supernatant was detected by ELISA.The proliferation abilities were measured by MTT and FCM.Results The Flt-1 gene could be obtained by RT-PCR.The restriction enzyme digestion and PCR suggested that the recombinant plasmids were constructed successfully.72 hours after transfection,the VEGF protein levels were decreased (transfection group (56.3 ±41.2) pg/ml,untransfection group (95.5 ±35.3) pg/ml(P ＜0.05) and the proliferation rate of cells was inhibited transfection group (42.6 ± 3.7) ％ and untransfection group (92.8 ± 6.6) ％ (P ＜ 0.05).Conclusions Flt-1 gene could inhibit the expression of VEGF and BT325 cells proliferation and induce their apoptosis in vitro. Key words: Flt-1 gene; Gene recombination; BT325 cell line

Abnormal expression of an ADAR2 alternative splicing variant in gliomas downregulates adenosine-to-inosine RNA editing

BackgroundRNA editing is catalyzed by adenosine deaminases acting on RNA (ADARs). ADAR2 is the main enzyme responsible for recoding editing in humans. Adenosine-to-inosine (A-to-I) editing at the Q/R site is reported to be decreased in gliomas; however, the expression of ADAR2 mRNA was not greatly affected.MethodsWe determined ADAR2 mRNA expression in human glioblastoma cell lines and in normal human glial cells by real-time RT-PCR. We also determined ADAR2 mRNA expression in 44 glioma tissues and normal white matter. After identifying an alternative splicing variant (ASV) of ADAR2 in gliomas, we performed sequencing. We then classified glioblastomas based on the presence (+) or absence (–) of the ASV to determine the correlations between ASV + and malignant features of glioblastomas, such as invasion, peritumoral brain edema, and survival time.ResultsThere were no significant differences in ADAR2 mRNA expression among human glioblastoma cell lines or in gliomas compared with normal white matter (all p > 0.05). The ASV, which contained a 47-nucleotide insertion in the ADAR2 mRNA transcript, was detected in the U251 and BT325 cell lines, and in some glioma tissues. The expression rate of ASV differed among gliomas of different grades. ASV + glioblastomas were more malignant than ASV – glioblastomas.ConclusionsADAR2 is a family of enzymes in which ASVs result in differences in enzymatic activity. The ADAR2 ASV may be correlated with the invasiveness of gliomas. Identification of the mechanistic characterization of ADAR2 ASV may have future potential for individualized molecular targeted-therapy for glioma.

Reversal of multidrug resistance by magnetic chitosan-Fe3O4 nanoparticle-encapsulated MDR1 siRNA in glioblastoma cell line

Objective: To investigate the reversal effects of MDR1 gene on multidrug resistance in the glioblastoma cell line BT325 by magnetic chitosan-Fe3O4 nanoparticle-encapsulated MDR1 siRNA.Methods: The shRNA expression vector was constructed and the recombinant plasmids were cloned. Magnetic chitosan-Fe3O4 nanoparticles were prepared and the encapsulation rate was determined. After transfection, the BT325 cells were cultured to assay the transfection efficiency. The changing of MDR1 mRNA level and P-gp protein was evaluated. And the sensitivity to different chemotherapeutic drugs was assessed in BT325-siRNA transfected cell and untransfected cell by IC50 values.Results: The MDR1 RNAi plasmid was successfully designed and preparation. The encapsulation efficiency of the magnetic chitosan-Fe3O4 nanoparticle was 98–99%. The transfection efficiency of the siRNA-nanoparticles in BT325 cells was 70–80%. And the MDR1 mRNA levels were downregulated by reverse transcription (RT)-PCR assay. Furthermore, the results of P-gp protein expression decreased on immunocytochemical assay, Western blot and flow cytometry compared with control group. The IC50 values of DOX and VCR were decreased between the transfected cell and normal BT325 cell.Conclusion: After targeted transfection of the glioblastoma cell line with magnetic chitosan-Fe3O4 nanoparticle-encapsulated MDR1 siRNA, the expression of MDR1 at both the mRNA and protein level decreased, which increased sensitivity to chemotherapy in vitro. It might provide a basis for investigation of the mechanism involved in multidrug resistance in glioma.

Activation of CD40 by soluble recombinant human CD40 ligand inhibits human glioma cells proliferation via nuclear factor-κB signaling pathway

As CD40 transduces activation signals involved in inflammatory and immune disorders, we explored the expression and response to CD40 engagement in human glioma cell lines in this study. The CD40 expression in BT-325 and U251 cells was flow cytometrically detected. The cells were incubated with srhCD40L for 72 h to assess its effects on cell growth in vitro. TNF-α expression was quantified by real-time PCR, and protein expression was analyzed by ELISA. The I-κb mRNA was detected by RT-PCR. I-κB expression decreased after stimulation with 1 μg/mL srhCD40L, but it was upregulated after the cells were pretreated with CD40 antibody. srhCD40L significantly inhibited the proliferation of the CD40+ human glioma cells. The stimulation of CD40+ glioma cells with soluble CD40L (CD154) up-regulated the expression of TNF-α at both mRNA and protein levels. We are led to conclude that CD40L/CD40 could inhibit human glioma cells through I-κb signaling pathway. Interferon-γ can augment CD40 expression and the inhibitory effect of CD40 ligand on cell growth in vitro. These results suggest that srhCD40L may benefit the therapy strategy of glioma.

Reversion of multidrug resistance in human glioma by RNA interference

Objective: To explore whether the vector construction of short hairpin in vivo could make human glioma cell line BT325 produce RNA interference (RNAi) duplexes and reverse the expression of the MDR1 gene.Methods: Three 62nt oligonucleotide fragments (shRNA) were constructed according to GeneBank MDR1 sequence and were cloned to the retrovirus-delivered vectors. After these vectors were transfected directly to the BT325 cell by Lipofectamine 2000 with enhanced green fluorescent protein (EGFP) co-transfection, the MDR1 gene silence effects were detected by the changing levels of mRNA and P-glycoprotein (P-gp) including real-time PCR (RT-PCR), Northern blot and Western blot analysis. For assessing multidrug resistance against doxorubicin (DOX) and vincristine (VCR), cell proliferation assays were performed by cell counting kit-8 and IC50 was calculated.Results: The RNAi plasmid vectors were constructed successfully. The gene silence became the strongest after 48 hour transfection from Northern blot; Western blot analysis demonstrated that P-gp expression reduced to 12.9, 30.3 and 4.8%. The chemosensitivity assays showed that the transfected cell could increase the sensitivity of DOX and VCR. Based on the value of IC50, BT325 cells increased sensitivity to drugs obviously. The sequence specific RNAi could inhibit MDR1 mRNA and P-gp expression of the glioma cell line. And it may reverse multidrug resistance phenotype, which may provide promising therapeutic modalities in the treatment of human glioma.

Construction of 6HRE-GFAP-Baxα system specific for glioma gene therapy

The aim of this paper is to construct a specific and high-performance gene therapy system for glioma. We constructed a combined promoter 6HRE-GFAP (Hypoxia Responsive Element, Glial Fibrillary Acidic Protein) by gene recombination techniques according to the hypoxia microenvironment in glioma and tested its efficacy and specificity in cultured cells, and then constructed GFAP-Baxα gene expressing system and determined its promoting of apoptosis in glioma cells. Our primary results showed that in U251 and BT325 cell lines, the activity of GFAP promoter was 16.40 and 4.73-fold of the promoter of hTERT (Human Telomerase Reverse Transcriptase), respectively. The activities of 6HRE-GFAP-promoter increased by 3.08 and 1.30-fold under 2% O2 condition compared with those under 18% O2 condition, while under 0.2% O2 condition increased by 8.90 and 2.69-fold, respectively. The glioma cells showed typical apoptotic signs 90 hours after the transient transfection of GFAP-Baxα. In these primary experiments, it showed that 6HRE-GFAP-Baxα system could promote glioma cell apoptosis. It was specific and effective for glioma gene therapy.

Expression of RNA Editing Deaminase on Human Glioma Cell Lines

To study the expression of RNA editing deaminases ADAR2 and ADAR3 in different malignant glioma cell lines and the effect of phenylacetate on the expression of these genes, the primarily glial cells of human brain tissue were isolated and cultured. The human glioma SHG-44, U-251, BT-325 cell lines were maintained in culture. The expressions of ADAR2 and ADAR3 mRNA were detected by the semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). The changes in ADAR2 mRNA expression before and after phenylacetate treatment were detected by RT-PCR and image analysis. The level of ADAR gene expression is expressed as the ratio expression rate (RER) of ADAR gene to β-actin according to computer image analysis. ADAR2 displays moderate expression in glial cells, low expression in low-grade malignant glioma SHG-44 cells, and high level expression in high-grade malignant glioma U-251 and BT-325 cells. The expression of ADAR2 can be decreased by phenylacetate treatment in glioma U-251 cells. ADAR3 is not expressed in normal brain glial cells, or glioma SHG-44, U-251 and BT-325 cells before and after phenylacetate treatment. The enhanced expression of ADAR2 may be involved in the tumor progression of malignant glioma. Phenylacetate can decrease the expression of ADAR2 in glioma cells, suggesting that it may act on the RNA editing process in glioma.

Suppression of nitric oxide on cytotoxicity of Lomustine in glioma cell line BT- 325

To investigate the effect of nitric oxide (NO) on cytotoxicity of Lomustine (CCNU) in vitro. CCNU was used to treat human glioma cell line BT-325 with different concentration of cytokines or NO donors, NO levels was measured by Griess assay and cell survival was evaluated by MTT assay. (1) Pretreatment with IL-1 beta and LPS markedly suppressed CCNU cytotoxicity in BT-325 cells with a significant increase in NO production (P<0.05). This function could be inhibited by L-NAME. (2) DETA NONOate suppressed cytotoxicity of CCNU to BT -325 cells in a dose-dependent manner (P<0.05). (3) CCNU co-cultured with SNAP for 24 h showed higher cytotoxic to BT-325 cells(P<0.05). NO partly suppresses cytotoxicity of Lomustine, which might be associated with chemoresistance of BT-325 cells against CCNU in vitro. NO can also slow the degradation of CCNU in water solution.

Effects of ferroxidase activity and species on ceruloplasmin mediated iron uptake by BT325 cells

In a previous study, we found that human ceruloplasmin (hCP) promotes iron uptake rather than release in BT325 cells, a human glioma cell line. In this study, we investigated the effect of ferroxidase activity of hCP and different species of ceruloplasmins on CP-mediated iron uptake by the cells. The cells were incubated for 30 min at 37 °C with 1 μM 59Fe 2+ with or without 150 μg/ml of the untreated and the ferroxidase-defective hCPs (apohCP and heat-inactivated hCP) or different species of ceruloplasmins (human CP, rabbit CP and bovine CP). The untreated hCP induced a significant increase in iron uptake by BT325 cells, while ferroxidase-defective hCPs with (heat-inactivated hCP) or without cooper ions (apohCP) had no such role. The untreated hCP increases significantly internalized iron but not membrane-bound iron, implying that hCP stimulated iron entry into the cell rather than increased extracellular binding of iron to the cell surface. All species of ceruloplasmins could promote iron uptake by the cells and the difference in degree of stimulatory effect among them was insignificant. These results suggested that ferroxidase activity of hCP is essential for the hCP-mediated iron uptake process and also that CP-stimulated iron uptake is not associated with copper ions in the protein, and that the effect of ceruloplasmin on iron uptake by BT325 cells is not species specific.

Ceruloplasmin promotes iron uptake rather than release in BT325 cells.

This study was initiated to investigate the mechanism of ceruloplasmin (CP)-mediated iron release from brain cells using BT325 cells (a glioblastoma cell line); however, negative results were obtained. The BT325 cells were preloaded with 1 microM 59Fe2+ in sucrose (pH 5.8) for 60 min, and then with CP (0-300 microg/ml) for 30 min at 37 degrees C. The addition of CP, either at low (25 microg/ml) or high (300 microg/ml) concentrations, did not lead to a significant change in iron release from iron-loaded BT325 cells. No significant difference in total iron of cells was found between all CP treated groups and the control (P>0.05). Although apotransferrin (apoTf) was shown to have a role in iron release from the cells, the effect of apoTf was not significantly affected by the addition of different concentrations of CP. When the cells were incubated with 1 microM 59Fe2+ in the presence of varying amounts of CP for 30 min at 37 degrees C, it was found that CP increased iron uptake. The total iron uptake by BT325 cells in CP treatment groups (25, 75, 150, 300 microg/ml) was significantly higher than that in the control (no CP addition) (all P<0.01). Furthermore, in contrast to our expectation, CP was shown to promote significantly iron uptake in not only iron-sufficient but also iron-deficient cells. These results showed that CP had a role in iron uptake rather than release in BT325 cells.

Cloning and identification of a novel human glioma differentiation related geneGDR1

In order to identify the genes associated with glioblastoma differentiation, some ESTs, expressed differentially in the control cell and the differentiated human glioblastoma cell line BT-325 induced by the all-trans retinoid acid, have been isolated by the method of DDRT-PCR. Of the 46 ESTs sequenced, 19 are from new genes. A full-length 1 535-bp cDNA, termed geneGDR1, has been isolated from the human cDNA library using the probe designed according to one of the novel ESTs, HGBB098. The open reading frame ofGDR1 gene encodes a putative protein containing 334 amino acid residues. Blast against the current GenBank DNA and protein sequence database did not reveal significant homology with any known proteins. RT-PCR shows thatGDR1 mRNA level increased in the differentiated BT-325 cells after being treated with RA. The different expression patterns ofGDR1 mRNA in human tissues have been detected through the multiple tissue Northern blot hybridization.

Pleiotropic Signaling Pathways in Rapid, Nongenomic Action of Glucocorticoid

The traditional genomic theory of steroid action does not fully explain the rapid effects of hormonal steroids, and it is thought that the nongenomic actions mediated by a putative membrane receptor may provide a plausible explanation. Although there is a rich body of evidence to substantiate the rapid, nongenomic effects of steroid hormones, the signal transduction pathways involved have proved to be complex and pleiotropic. Based on previous studies on the rapid, nongenomic actions of glucocorticoid (GC) and the G-protein–protein kinase pathways involved, including our own studies on PC12, SK-N-SH, BT-325 cells, and synaptosomes, in this review we will discuss the issue of multiple signal transduction pathways involved in the rapid, nongenomic effects of GC.