Retracted: Long noncoding RNA MEG3 inhibits proliferation and migration but induces autophagy by regulation of Sirt7 and PI3K/AKT/mTOR pathway in glioma cells

Glioma is a common primary brain tumor with high mortality rate and poor prognosis. Long noncoding RNA maternally expressed gene 3 (MEG3) is a tumor suppressor in diverse cancer types. However, the role of MEG3 in glioma remains unclear. We aimed to explore the effects of MEG3 on U251 cells as well as the underlying mechanisms. U251 cells were stably transfected with different recombined plasmids to overexpress or silence MEG3. Effects of aberrantly expressed MEG3 on cell viability, migration, apoptosis, expressions of apoptosis-associated and autophagy-associated proteins, and phosphorylated levels of key kinases in the PI3K/AKT/mTOR pathway were all evaluated. Then, messenger RNA (mRNA) and protein expression of Sirt7 in cells abnormally expressing MEG3 were estimated. In addition, effects of abnormally expressed MEG3 and Sirt7 on U251 cells were determined to reveal the underlying mechanism of MEG3-associated modulation. Cell viability and migration were significantly reduced by MEG3 overexpression whereas cell apoptosis as well as Bax and cleaved caspase-3/-9 proteins were obviously induced. Beclin-1 and LC3-II/LC3-I were upregulated and p62 was downregulated in MEG3 overexpressed cells. In addition, the autophagy pharmacological inhibitor (3-methyladenine, 3-MA) affected the effect of MEG3 overexpression on cell proliferation. Furthermore, the phosphorylated levels of key kinases in the PI3K/AKT/mTOR pathway were all reduced by MEG3 overexpression. Sirt7 was positively regulated by MEG3 expression, and effects of MEG3 overexpression on U251 cells were ameliorated by Sirt7 silence. MEG3 suppressed cell proliferation and migration but promoted autophagy in U251 cells through positively regulating Sirt7, involving in the inhibition of the PI3K/AKT/mTOR pathway.     

N-cadherin expression level as a critical indicator of invasion in non-epithelial tumors

Cancer cell dissemination away from the primary tumor and their ability to form metastases remain the major causes of death from cancer. Understanding the molecular mechanisms triggering this event could lead to the design of new cancer treatments. The establishment and the maintenance of tissue architecture depend on the coordination of cell behavior within this tissue. Cell-cell interactions must form adhesive structures between neighboring cells while remaining highly dynamic to allow and control tissue renewal or remodeling. Among intercellular junctions, cadherin-based adherens junctions mediate strong physical interactions and transmit information from the cell microenvironment to the cytoplasm. Disruption of these cell-cell contacts perturbs the polarity of epithelial tissues leading to their disorganization and ultimately to aggressive carcinomas. In non-epithelial tissues, the role of cadherins in the development of cancer is still debated. We recently found that downregulation of N-cadherin in malignant glioma—the most frequent primary brain tumor—results in cell polarization defects leading to abnormal motile behavior with increased cell speed and decreased persistence in directionality. Re-expression of N-cadherin in glioma cells restores cell polarity and limits glioma cell migration, providing a potential therapeutic tool for diffuse glioma.     

Characterization by [3H]Dihydroergocryptine Binding of Alpha-Adrenergic Receptors in Neuroblastoma × Glioma Hybrid Cells

[3H]Dihydroergocryptine ([3H]DHE) was shown to bind to sites in membranes from neuroblastoma X glioma hybrid cells (NG 108-15) that had the characteristics expected of alpha-adrenergic receptors. The binding was saturable with 0.3 pmol [3H]DHE bound per mg of protein and of high affinity, with an apparent dissociation constant (KD) of 1.8 nM. The specificity of the binding site for various ligands was more similar to that of alpha 2 receptors than to that of alpha 1. No specific binding of [3H]WB-4101 was found in the membranes derived from NG 108 cells. This finding also indicated that the [3H]DHE binding site in the cell is the alpha 2 receptor. GTP lowered the affinity of agonists for the [3H]DHE binding site, although the nucleotide hardly affected the affinity of antagonists including [3H]DHE.     

Regulation by a β-adrenergic receptor of a Ca2+-independent adenosine 3′,5′-(cyclic)monophosphate phosphodiesterase in C6 glioma cells

The hormonal control of cyclic nucleotide phosphodiesterase (EC 3.1.4.17) activity has been studied by using as a model the isoproterenol stimulation of cyclic AMP phosphodiesterase activity in C6 glioma cells. A 2-fold increase in cyclic AMP phosphodiesterase specific activity was observed in homogenates of isoproterenol-treated cells relative to control. This increase reached a maximum 3 h after addition of isoproterenol, was selective for cyclic AMP hydrolysis, was reproduced by incubation with 8-Br cyclic AMP but not with 8-Br cyclic GMP and was limited to the soluble enzyme activity. The presence of 0.1 mM EGTA did not alter the magnitude of the increase in phosphodiesterase activity. Moreover, the calmodulin content in the cell extracts was not changed after isoproterernol. DEASE-Sephacel chromatography of the 100 000×g supernatant resolved two peaks of phosphodiesterase activity. The first peak hydrolyzed both cyclic nucleotides and was activated by Ca²⁺ and purified calmodulin. The second peak was specific for cyclic AMP but it was Ca²⁺- and calmodulin-insensitive. Isoproterenol selectively increased the specific activity of the second peak. Kinetic analysis of the cyclic AMP hydrolysis by the induced enzyme reveled a non-linear Hofstee plot with apparent K_m values of 2–5 μM. Cyclic GMP was not hydrolyzed by this enzyme in the absence or presence of calmodulin and failed to affect the kinetics of the hydrolysis of cyclic AMP. Gel filtration chromatography of the induced DEASE-Sephacel peak resolved a single peak of enzyme activity with an apparent molecular weight of 54 000.     

Two-hit wonders: The expanding universe of multitargeting epigenetic agents

Multitargeting involves the application of molecules that are deliberately intended to bind to two or more unrelated cellular targets with high affinity. In epigenetic chemical biology and drug discovery, the rational design of multitargeting agents has evolved to a sophisticated level, and there are now five examples that have reached clinical trials. This review covers recent developments in the field and future prospects.     

Use of synchrotron medical microbeam irradiation to investigate radiation-induced bystander and abscopal effects in vivo

The question of whether bystander and abscopal effects are the same is unclear. Our experimental system enables us to address this question by allowing irradiated organisms to partner with unexposed individuals. Organs from both animals and appropriate sham and scatter dose controls are tested for expression of several endpoints such as calcium flux, role of 5HT, reporter assay cell death and proteomic profile. The results show that membrane related functions of calcium and 5HT are critical for true bystander effect expression. Our original inter-animal experiments used fish species whole body irradiated with low doses of X-rays, which prevented us from addressing the abscopal effect question. Data which are much more relevant in radiotherapy are now available for rats which received high dose local irradiation to the implanted right brain glioma. The data were generated using quasi-parallel microbeams at the biomedical beamline at the European Synchrotron Radiation Facility in Grenoble France. This means we can directly compare abscopal and “true” bystander effects in a rodent tumour model. Analysis of right brain hemisphere, left brain and urinary bladder in the directly irradiated animals and their unirradiated partners strongly suggests that bystander effects (in partner animals) are not the same as abscopal effects (in the irradiated animal). Furthermore, the presence of a tumour in the right brain alters the magnitude of both abscopal and bystander effects in the tissues from the directly irradiated animal and in the unirradiated partners which did not contain tumours, meaning the type of signal was different.     

A novel approach to the discovery of survival biomarkers in glioblastoma using a joint analysis of DNA methylation and gene expression

Glioblastoma multiforme (GBM) is the most aggressive of all brain tumors, with a median survival of less than 1.5 years. Recently, epigenetic alterations were found to play key roles in both glioma genesis and clinical outcome, demonstrating the need to integrate genetic and epigenetic data in predictive models. To enhance current models through discovery of novel predictive biomarkers, we employed a genome-wide, agnostic strategy to specifically capture both methylation-directed changes in gene expression and alternative associations of DNA methylation with disease survival in glioma. Human GBM-associated DNA methylation, gene expression, IDH1 mutation status, and survival data were obtained from The Cancer Genome Atlas. DNA methylation loci and expression probes were paired by gene, and their subsequent association with survival was determined by applying an accelerated failure time model to previously published alternative and expression-based association equations. Significant associations were seen in 27 unique methylation/expression pairs with expression-based, alternative, and combinatorial associations observed (10, 13, and 4 pairs, respectively). The majority of the predictive DNA methylation loci were located within CpG islands, and all but three of the locus pairs were negatively correlated with survival. This finding suggests that for most loci, methylation/expression pairs are inversely related, consistent with methylation-associated gene regulatory action. Our results indicate that changes in DNA methylation are associated with altered survival outcome through both coordinated changes in gene expression and alternative mechanisms. Furthermore, our approach offers an alternative method of biomarker discovery using a priori gene pairing and precise targeting to identify novel sites for locus-specific therapeutic intervention.     

全反式维甲酸协同DAPT抑制胶质瘤干细胞的自我更新

前期研究脑表明,脑胶质瘤干细胞（glioma stem cells,GSCs）是胶质瘤发生和发展的重要因素,探索靶向干预GSCs生长有可能成为脑胶质瘤治疗的有效途径之一。该研究旨在阐明两种药物ATRA和Y-分泌酶抑制剂DAPT协同抑制GSCs自我更新的生物学效应。通过用台盼蓝排染法、克隆球形成试验和免疫印迹分析了两种药物的单独使用或联用对GSC样细胞PGCl和PGC2生长、成球能力和自我更新以及干细胞标志物表达的影响。结果发现,单独使用ATRA对PGCl生长有一定的抑制作用,而对PGC2生长几乎没有影响;DAPT对PGCs的生长抑制作用明显强于ATRA。高浓度ATRA（80μmol/L）能诱导PGCs的分化,降低PGCs成球大小,且成球效率降至5％～8％,而正常对照组为32％～35％;同样,DAPT（40μmol／L）也能降低PGCs成球大小,且成球效率降至2％～3％;低浓度ATRA（20μmol／L）和DAPT（5gmol／L）对PGCs自我更新能力和干性没有明显影响,而联合使用后其明显降低PGCs的成球大小,且成球效率降至3％～5％,促进细胞凋亡,并且明显抑制了干细胞标志物Nestin、CDl33、Sox2、Oct4的表达,提高了分化标志物GFAP的表达。该研究证明了低浓度的ATRA和DAPT能协同抑制脑胶质瘤干细胞PGCs的自我更新。研究结果将为脑胶质瘤的临床研究提供实验依据。     

Light-controlled inhibition of malignant glioma by opsin gene transfer

Glioblastomas are aggressive cancers with low survival rates and poor prognosis because of their highly proliferative and invasive capacity. In the current study, we describe a new optogenetic strategy that selectively inhibits glioma cells through light-controlled membrane depolarization and cell death. Transfer of the engineered opsin ChETA (engineered Channelrhodopsin-2 variant) gene into primary human glioma cells or cell lines, but not normal astrocytes, unexpectedly decreased cell proliferation and increased mitochondria-dependent apoptosis, upon light stimulation. These optogenetic effects were mediated by membrane depolarization-induced reductions in cyclin expression and mitochondrial transmembrane potential. Importantly, the ChETA gene transfer and light illumination in mice significantly inhibited subcutaneous and intracranial glioma growth and increased the survival of the animals bearing the glioma. These results uncover an unexpected effect of opsin ion channels on glioma cells and offer the opportunity for the first time to treat glioma using a light-controllable optogenetic approach.