Synergistic combination of cannabidiol and celecoxib or 2,5-dimethylcelecoxib exerts oxidative stress-mediated cytotoxicity and mitigates glioblastoma invasiveness

Glioblastoma multiforme is the most aggressive primary brain cancer in adults. Therefore, it is important to investigate the mechanisms associated with cell viability and invasion ability of the cells in glioblastoma multiforme. The opposite strand of the glial cell line-derived neurotrophic factor (GDNF) gene is used to transcribe the cis-antisense GDNF opposite strand (GDNFOS) gene, which belongs to the long noncoding RNAs. The current study assessed the effects of GDNFOS1 overexpression and interference on GDNF expression, cell viability and invasion ability in U87 and U251 MG glioblastoma cells. Overexpression and interference were performed using constructed lentiviral vectors, including long non-coding RNA GDNFOS1 overexpression vector, pL-short hairpin RNA (shRNA)-GDNFOS1-9, pL-shRNA-GDNFOS1-49, pL-shRNA-GDNFOS1-248, pL-shRNA-GDNFOS1-9+49, pL-shRNA-GDNFOS1-9+248 and pL-shRNA-GDNFOS1-49+248. Reverse transcription-quantitative PCR was used to determine the efficiency of interference and overexpression of GDNFOS1 in U87 and U251 MG cells. GDNF protein expression in U87 and U251 MG cells was detected using western blot analysis. In addition, cell viability was detected using a cell counting kit-8 assay at 24, 48 and 72 h after GDNFOS1 overexpression or interference. A transwell invasion assay was used to detect invasion ability. Different shRNA sequences were tested and the results revealed that a combination (pL-shRNA-GDNFOS1-49+248) was most effective in the knock-down GDNFOS1. Compared with the control group, GDNF expression in U87 MG cells was significantly increased in the GDNFOS1 overexpression group and decreased in the shRNA-GDNFOS1-248 group. U87 MG cell viability was significantly increased in the GDNFOS1 overexpression group at 24, 48 and 72 h compared with the negative control group. The viability of U87 MG cells was decreased in the GDNFOS1 interference group at 72 h when compared with the control group. The relative invasive ability was significantly increased in the GDNFOS1 overexpression group when compared with the negative control group. The invasive ability was significantly decreased in the GDNFOS1 interference group when compared with the negative control group. Similar results were exhibited by the U251 MG cells. Overall, GDNF expression, cell viability and invasion ability of glioblastoma cells significantly increased with GDNFOS1 overexpression and decreased with GDNFOS1 interference.

TGF-α Differentially Regulates GFAP, Vimentin, and Nestin Gene Expression in U-373 MG Glioblastoma Cells: Correlation with Cell Shape and Motility

The antiproliferative activity of three benzopyrans isolated from the chloroform extract of the aerial parts of Hypericum polyanthemum was analysed in order to determine their effect on the growth and cell cycle in the U-373 MG glioblastoma cell line. Compound 1 was less cytotoxic than compounds 2 and 3. A synergistic effect was noticed when the three benzopyrans were used simultaneously. The cytotoxicity noted could be related to an arrest in G2/M phase, leading to apoptosis in the U-373 MG glioblastoma cell line.

Substance P (SP) acting through substance P receptor (SPR) increases the proliferation of glioblastoma cells. At the molecular level, stimulation of SPR in human U373 MG glioblastoma cells results in phosphorylation of mitogen-activated protein kinases ERK1/2. Examination of the underlying mechanism reveals that SPR mediates ERK1/2 phosphorylation in a calcium-dependent manner. Surprisingly, blockade of epidermal growth factor receptor (EGFR), which is transactivated by SPR, has a minimal effect on SPR-mediated ERK1/2 phosphorylation. However, SPR-mediated ERK1/2 phosphorylation is significantly reduced by the Src kinase inhibitor PP2. Interestingly, ERK1/2 in U373 MG cells is also activated by several other mitogenic G protein-coupled receptors (GPCRs) including alpha(1B)-adrenergic, M(3)-muscarinic, and H(1)-histaminergic in an Src-dependent manner. We conclude that c-Src is a mediator of SP-stimulated ERK1/2 phosphorylation in human U373 MG glioblastoma cells.

Effects of Growth Factors and Basement Membrane Proteins on the Phenotype of U-373 MG Glioblastoma Cells as Determined by the Expression of Intermediate Filament Proteins

NSC 680410, the novel adamantyl ester of AG957, an inhibitor of the p210bcr/abl tyrosine kinase (CML, Ph(+)) and possibly other kinases, was tested for antitumor activity in ten human leukemia and human glioblastoma cell lines. CEM/0, seven ara-C- and/or ASNase-resistant clones, Jurkat/0, the myelomonocytic line U937 and U87 MG glioblastoma cell lines were used for these studies. The drug-resistant leukemic clones lack p53, express bcl-2 and VEGF-R1, and thus are refractory to apoptosis. Since tyrosine kinases drive many proliferative pathways and these activities are increased in many leukemic cells, we hypothesized that NSC 680410 may induce cytotoxicity in drug-resistant leukemia clones, independently of p210bcr/abl expression. NSC 680410 exhibited significant antileukemic activity in CEM/0, Jurkat E6-1, and in the drug-resistant leukemic cell lines. The IC(50) values in nine leukemia lines ranged from 17 to 216 n M. Western blot analyses after NSC 680410 treatment demonstrated caspase-3 cleavage and ELISAs showed a fivefold upregulation of its activity in cellular extracts. In addition, U87 MG human glioblastoma cells, which express VEGF-R1, were treated with the Flt-1/Fc chimera, a specific inhibitor of VEGF, and showed 30-43% cell kill in the MTT assay. Furthermore, the combination of NSC 680410 plus Flt-1/Fc chimera demonstrated an eightfold synergism against U87 MG cells in vitro. To verify this observation in vivo, athymic mice were inoculated orthotopically into the caudate putamen with 10(6) U87 MG cells. On day 3, five mice per group were treated i.p. with either 8.3 mg/kg NSC 680410 daily for three doses per week for 4 weeks alone or in combination with one dose of Flt-1/Fc chimera 100 mg/kg subcutaneously. Treatment with NSC 680410 alone produced no weight changes and increased the median survival to 133%, whereas treatment with NSC680410 plus Flt-1/Fc chimera increased survival to 142% over control. Control animals had large intra- and extracranial tumors while the NSC 680410-treated mice had small, only intracranial tumors with necrotic centers. The combination treatment resulted in small residual tumors around the needle track, indicating significant inhibition of tumor growth. These studies demonstrate that the tyrosine kinase inhibitor NSC 680410 has significant antileukemic activity in p53-null, drug-resistant human leukemia cell lines, as well as significant antitumor activity in combination with Flt-1/Fc chimera against U87 MG glioblastoma brain tumors implanted in situ in athymic mice.

A C2-symmetric bicyclic peptide bearing two RGD motifs was developed as a dimeric ligand, and it displayed enhanced inhibition of ECM protein binding to purified integrin receptors as compared to monomeric RGD analogues. Moreover, the dimeric bicyclic ligand induced cell detachment and inhibited FAK phosphorylation in U-373 MG glioblastoma cells.

The time-sequential change in immune-related gene expression of the glioblastoma cell line after irradiation was evaluated to speculate the effect of combined immunotherapy with radiotherapy. The U373 MG glioblastoma cell line was irradiated with 6 Gy single dose. Next-generation sequencing (NGS) transcriptome data was generated before irradiation (control), and at 6, 24, and 48 h post-irradiation. Immune-related pathways were analyzed at each time period. The same analyses were also performed for A549 lung cancer and U87 MG glioblastoma cell lines. Western blotting confirmed the programmed death-ligand 1 (PD-L1) expression levels over time. In the U373 MG cell line, neutrophil-mediated immunity, type I interferon signaling, antigen cross-presentation to T cell, and interferon-γ signals began to increase significantly at 24 h and were upregulated until 48 h after irradiation. The results were similar to those of the A549 and U87 MG cell lines. Without T cell infiltration, PD-L1 did not increase even with upregulated interferon-γ signaling in cancer cells. In conclusions, in the glioblastoma cell line, immune-related signals were significantly upregulated at 24 and 48 h after irradiation. Therefore, the time interval between daily radiotherapy might not be enough to expect full immune responses by combined immune checkpoint inhibitors and newly infiltrating immune cells after irradiation.

Lipidomics can complement genomics and proteomics by providing new insight into dynamic changes in biomembranes; however, few reports in the literature have explored, on an organism-wide scale, the functional link between nonenzymatic proteins and cellular lipids. Here, we report changes induced by adenovirus-delivered wild-type p53 gene and chemotherapy of U87 MG glioblastoma cells, a treatment known to trigger apoptosis and cell cycle arrest. We compare polar lipid changes in treated cells and control cells by use of a novel, sensitive method that employs lipid extraction, one-step liquid chromatography separation, high-resolution mass analysis, and Kendrick mass defect analysis. Nano-LC FT-ICR MS and quadrupole linear ion trap MS/MS analysis of polar lipids yields hundreds of unique assignments of glyco- and phospholipids at sub-ppm mass accuracy and high resolving power (m/Deltam50% = 200 000 at m/z 400) at 1 s/scan. MS/MS data confirm molecular structures in many instances. Sulfatides are most highly modulated by wild-type p53 treatment. The treatment also leads to an increase in phospholipids such as phosphatidyl inositols, phosphatidyl serines, phosphatidyl glycerols, and phosphatidyl ethanolamines. An increase in hydroxylated phospholipids is especially noteworthy. Also, a decrease in the longer chain gangliosides, GD1 and GM1b, is observed in wild-type p53 (treated) cells.

Glioblastoma (GBM) is a frequent as well as violent type of brain tumor. In this research work, different carmustine formulations were prepared and screened for their cytotoxic activity against U-87 MG glioblastoma and normal human fibroblast L-929 cell lines. The flexible liposomes embedded in situ nasal gel exhibited maximum percentage of growth inhibition against U-87 MG glioblastoma cell line, and in situ nasal gel exhibited lowest percentage of growth inhibition against U-87 MG glioblastoma cell lines. Flexible liposomes embedded in situ nasal gel is observed to be safe and biocompatible against normal human fibroblast L-929 cell line. In cellular uptake study, U-87 MG cell line treated with flexible liposomes embedded in situ thermoreversible intranasal gel emitted stronger and higher intensity fluorescence. It seems that the presence of flexible liposomes embedded in situ thermoreversible nasal gel inside the tumor cells, and would be the best carmustine delivery approach for the management of GBM.

lncRNA BBOX1-AS1 regulates the miR-382-5p/CBX3 Signalling pathway to affect the proliferation and apoptosis of glioblastoma cells.

BackgroundInterleukin-1beta (IL-1β) is a pro-inflammatory cytokine that can be produced in the central nervous system during inflammatory conditions. We have previously shown that IL-1β expression is altered in the rat brain during a morphine tolerant state, indicating that this cytokine may serve as a convergent point between the immune challenge and opiate mediated biological pathways. We hypothesized that IL-1β up-regulates opioid receptors in human astrocytes in both untreated and morphine-desensitized states.MethodsTo test this hypothesis, we compared the basal expression of the mu (MOR), delta (DOR), and kappa (KOR) opioid receptors in the human U87 MG astrocytic cell line to SH-SY5Y neuronal and HL-60 immune cells using absolute quantitative real time RT-PCR (AQ-rt-RT-PCR). To demonstrate that IL-1β induced up-regulation of the MOR, DOR and KOR, U87 MG cells (2 x 105 cells/well) were treated with IL-1β (20 ng/mL or 40 ng/mL), followed by co-treatment with interleukin-1 receptor antagonist protein (IL-1RAP) (400 ng/mL or 400 ng/mL). The above experiment was repeated in the cells desensitized with morphine, where U87 MG cells were pre-treated with 100 nM morphine. The functionality of the MOR in U87 MG cells was then demonstrated using morphine inhibition of forksolin-induced intracellular cAMP, as determined by radioimmunoassay.ResultsU87 MG cells treated with IL-1β for 12 h showed a significant up-regulation of MOR and KOR. DOR expression was also elevated, although not significantly. Treatment with IL-1β also showed a significant up-regulation of the MOR in U87 MG cells desensitized with morphine. Co-treatment with IL-1β and interleukin-1 receptor antagonist protein (IL-1RAP) resulted in a significant decrease in IL-1β-mediated MOR up-regulation.ConclusionOur results indicate that the pro-inflammatory cytokine, IL-1β, affects opiate-dependent pathways by up-regulating the expression of the MOR in both untreated and morphine-desensitized U87 MG.

The PI3K signaling pathway is frequently activated in cancer and has been implicated in many aspects of tumor growth and survival. Inhibition of this pathway represents a potential therapeutic path for the treatment of cancer. This study evaluated the in vitro characteristics of AMG 511, a potent and selective pan class I PI3K inhibitor exhibiting IC50 values of 8, 11, 2, and 6 nM against the PI3K β, α, β, and ≤ isoforms respectively. AMG 511 was shown to be inactive against members of the closely related phosphoinositide 3 kinase related kinases (PIKK) family of kinases and did not inhibit mTOR, hVPS34, PI4Kα or PI4Kα in-vitro (IC50 values > 1 μM). In addition, AMG 511 was inactive against a majority of protein kinases (372) in the human kinome as measured by in-vitro binding assays. AMG 511 inhibited PI3K pathway signaling in U87 MG glioblastoma cells as determined by dose-dependent reduction in AKT S473 phosphorylation (IC50 = 4 nM). AKT inhibition resulted in a concomitant reduction in PRAS40 phosphorylation (IC50 = 23 nM), a downstream effector of AKT. Reduced phosphorylation of mTORC1 substrates p70S6K (IC50 = 30 nM) and S6 (IC50 = 70 nM) but not 4EBP1 (T37/46), was also detected in U87 MG cells, suggesting that upstream blockade of PI3K pathway signaling with AMG 511 treatment leads to a selective reduction in downstream mTORC1 activity. Given the well documented role of mTORC1 in cap-dependent translation we profiled AMG 511 in a methionine-analog incorporation assay in U87 MG cells. However, no significant inhibition of bulk translation was observed following treatment with AMG 511 in U87 MG cells. Treatment of U87 MG cells with AMG 511 revealed a pronounced G1 arrest with a concurrent reduction in BrdU+ cells, detectable within 8 hours of treatment. This anti-proliferative effect was fully reversible by 18 hours following washout. In line with these anti-proliferative effects, reduced cyclin D1 levels and elevated p27 levels were detected within 4 hours of treatment. Minimal cell killing effects were detected with AMG 511 treatment in U87 MG cells as measured by induction of cleaved caspase-3 and DNA content < 2N. AMG 511 was profiled across a large panel of tumor cell lines encompassing several tumor types including breast and lung. A majority of the cell lines tested exhibited sensitivity to AMG 511 with a subset exhibiting evidence for cell death upon treatment with AMG 511. Breast cancer cell lines harboring activating mutations in PI3Kα, loss of PTEN, or amplification of Her2 tended to show greater sensitivity to AMG 511 treatment. In conclusion, AMG 511 is a potent and selective pan class I PI3K inhibitor, capable of inhibiting PI3K signaling and inducing robust anti-proliferative effects via a G1 arrest in many tumor cell lines, with evidence of cell killing in a subset of lines. 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 2805. doi:1538-7445.AM2012-2805

Tumor angiogenesis involves multiple signaling pathways that provide potential therapeutic targets to inhibit tumor growth and metastasis. Regarding the significant role of vascular endothelial growth factor (VEGF) in angiogenesis and tumor progression, VEGF sequence-specific small interfering RNA (siRNA) for anti-angiogenic tumor therapy are under development. In the present study, dual-modified liposomes (At-Lp) was designed by attaching two receptorspecific peptides, Angiopep and tLyP-1, which specifically targeting low-density lipoprotein receptor (LRP) for brain tumor targeting and neuropilin-1 receptor (NRP-1) for tumor penetration, respectively. Gene transfection and silencing, and antitumor effect of the At-Lp loaded with VEGF siRNA were evaluated in vitro and in orthotopic xenograft models of U87 MG tumor. The At-Lp significantly enhanced cellular uptake (2-fold) and down-regulated expression of VEGF in U87 MG glioblastoma cells compared with non-modified and single-modified liposomes. The internalization of the At-Lp into tumor cells was taken via the enhanced permeability and retention effect and receptor-mediated endocytosis, followed by an effective endosomal escape of loaded siRNA into the cytoplasm. The At-Lp showed great superiority in inhibition of tumor growth, anti-angiogenesis, expression of VEGF and apoptosis effect after in vivo application against nude mice bearing U87 MG glioblastoma without activation of system-associated toxicity and the innate immune response. These results demonstrated that the combination of two receptor-specific peptides-mediated liposomes presented a promising platform for effective targeting delivery of siRNA for cancer anti-angiogenic therapy.

Evaluation of [18F]-ATRi as PET tracer for in vivo imaging of ATR in mouse models of brain cancer