Notch signaling pathway suppresses mRNA expression of hexokinase 2 under nutrient-poor conditions in U87-MG glioma cells.

Control of nutrient homeostasis plays a central role in cell proliferation/survival during embryonic development and tumor growth. Activation of the Notch signaling pathway, a major contributor to cell-cell interactions, is a potential mechanism for cell adaptation to nutrient-poor conditions. Our previous study also demonstrated that during embryogenesis when nutrients such as glutamine and growth factors are potentially maintained at lower levels, Notch signaling suppresses mRNA expression of hexokinase 2 (hk2), which is a glycolysis-associated gene, in the central nervous system. However, whether and how the genetic regulation of HK2 via Notch signaling contributes to cellular adaptability to nutrient-poor environments remains unknown. In this study, we performed gene expression analysis using a U87-MG human glioma cell line and revealed that under conditions where both glutamine and serum were absent, Notch signaling was activated and HK2 expression was downregulated by Notch signaling. We also found that Notch-mediated HK2 suppression was triggered in a Notch ligand-selective manner. Furthermore, HK2 was shown to inhibit cell proliferation of U87-MG gliomas, which might depend on Notch signaling activity. Together, our findings suggest the involvement of Notch-mediated HK2 suppression in an adaptive mechanism of U87-MG glioma cells to nutrient-poor conditions.

TAK-242 inhibits glioblastoma invasion, migration, and proneural-mesenchymal transition by inhibiting TLR4 signaling.

Resatorvid (TAK-242), a small-molecule inhibitor of Toll-like receptor 4 (TLR4), has the ability to cross the blood-brain barrier (BBB). In this study, we explored the role of TAK-242 on glioblastoma (GBM) invasion, migration, and proneural-mesenchymal transition (PMT). RNA sequencing (RNA-Seq) data and full clinical information of glioma patients were downloaded from the Chinese Glioma Genome Atlas (CGGA) and the Cancer Genome Atlas (TCGA) cohorts and then analyzed using R language; patients were grouped based on proneural (PN) and mesenchymal (MES) subtypes. Bioinformatics analysis was used to detect the difference in survival and TLR4-pathway expression between these groups. Cell viability assay, wound-healing test, and transwell assay, as well as an intracranial xenotransplantation mice model, were used to assess the functional role of TAK-242 in GBM in vitro and in vivo. RNA-Seq, Western blot, and immunofluorescence were employed to investigate the possible mechanism. TLR4 expression in GBM was significantly higher than in normal brain tissue and upregulated the expression of MES marker genes. Moreover, TAK-242 inhibited GBM progression in vitro and in vivo via linking with PMT, which could be a novel treatment strategy for inhibiting GBM recurrence.

ERN1 dependent impact of glucose and glutamine deprivations on PBX3, PBXIP1, PAX6, MEIS1, and MEIS2 genes expression in U87 glioma cells.

Objective. Homeobox genes play a fundamental role in the embryogenesis, but some of them have been linked to oncogenesis. The present study is aimed to investigate the impact of glucose and glutamine deprivations on the expression of homeobox genes such as PAX6 (paired box 6), PBX3 (PBX homeobox 3), PBXIP1 (PBX homeobox interacting protein 1), MEIS1 (MEIS homeobox 1), and MEIS2 in ERN1 knockdown U87 glioma cells with the intent to reveal the role of ERN1 (endoplasmic reticulum to nucleus signaling 1) signaling pathway on the endoplasmic reticulum stress dependent regulation of homeobox genes. Methods. The control (transfected by empty vector) and ERN1 knockdown (transfected by dominant-negative ERN1) U87 glioma cells were exposed to glucose and glutamine deprivations for 24 h. The cells RNA was extracted and reverse transcribed. The expression level of PAX6, PBX3, PBXIP1, MEIS1, and MEIS2 genes was evaluated by a real-time quantitative polymerase chain reaction analysis and normalized to ACTB. Results. It was found that glucose deprivation down-regulated the expression level of PAX6, MEIS1, and MEIS2 genes in control glioma cells, but did not significantly alter PBX3 and PBXIP1 genes expression. At the same time, ERN1 knockdown significantly modified the sensitivity of all studied genes to glucose deprivation. Other changes in gene expression were detected in control glioma cells under the glutamine deprivation. The expression of PBX3 and MEIS2 genes was down- while PAX6 and PBXIP1 genes up-regulated. Furthermore, ERN1 knockdown significantly modified the effect of glutamine deprivation on the majority of studied genes expression in U87 glioma cells. Conclusion. The results of the present study demonstrate that the exposure of U87 glioma cells under glucose and glutamine deprivations affected the expression of the majority of the studied homeobox genes and that the sensitivity of PAX6, PBX3, PBXIP1, MEIS1, and MEIS2 genes expression under these experimental conditions is mediated by ERN1, the major pathway of the endoplasmic reticulum stress signaling.

Co-delivery of paclitaxel and etoposide prodrug by human serum albumin and PLGA nanoparticles: synergistic cytotoxicity in brain tumour cells.

The aims of this study were to develop co-delivery systems of paclitaxel (PTX) and etoposide prodrug (4'-O-benzyloxycarbonyl-etoposide, ETP-cbz) based on non-cross-linked human serum albumin (HSA) and poly(lactide-co-glycolide) nanoparticles and to evaluate the synergistic potential of these drugs in vitro. The nanoformulations were prepared by the high-pressure homogenisation technique and characterised using DLS, TEM, SEM, AFM, HPLC, CZE, in-vitro release, and cytotoxicity in human and murine glioma cells. All nanoparticles had 90-150 nm in size and negative ζ-potentials. The Neuro2A cells were the most sensitive to both HSA- and PLGA-based co-delivery systems (IC50 0.024 µM and 0.053 µM, respectively). The drugs' synergistic effect (combination index < 0.9) was observed in the GL261 cells for both types of co-delivery formulations and in the Neuro2A cells for the HSA-based system. These nanodelivery systems may be useful to improve combination chemotherapy for brain tumour treatment. To our knowledge, this is the first report describing the non-cross-linked HSA-based co-delivery nanosuspension which was prepared using nab™ technology.

Radiosensitizing Effect of Dextran-Coated Iron Oxide Nanoparticles on Malignant Glioma Cells.

The potential of standard methods of radiation therapy is limited by the dose that can be safely delivered to the tumor, which could be too low for radical treatment. The dose efficiency can be increased by using radiosensitizers. In this study, we evaluated the sensitizing potential of biocompatible iron oxide nanoparticles coated with a dextran shell in A172 and Gl-Tr glioblastoma cells in vitro. The cells preincubated with nanoparticles for 24 h were exposed to ionizing radiation (X-ray, gamma, or proton) at doses of 0.5-6 Gy, and their viability was assessed by the Resazurin assay and by staining of the surviving cells with crystal violet. A statistically significant effect of radiosensitization by nanoparticles was observed in both cell lines when cells were exposed to 35 keV X-rays. A weak radiosensitizing effect was found only in the Gl-Tr line for the 1.2 MeV gamma irradiation and there was no radiosensitizing effect in both lines for the 200 MeV proton irradiation at the Bragg peak. A slight (ca. 10%) increase in the formation of additional reactive oxygen species after X-ray irradiation was found when nanoparticles were present. These results suggest that the nanoparticles absorbed by glioma cells can produce a significant radiosensitizing effect, probably due to the action of secondary electrons generated by the magnetite core, whereas the dextran shell of the nanoparticles used in these experiments appears to be rather stable under radiation exposure.

Poly-guanidine shows high cytotoxicity in glioma cell cultures and glioma stem cells.

Glioblastoma multiforme (GBM) is a malignant CNS tumor with a poor prognosis. GBM shows aberrant glycosylation with hypersialylation. This property is a potential target for therapy. This study investigates the growth inhibitory efficacy of poly-guanidine (GuaDex), with an affinity for sialic acid (Sia). Glioma cell cultures and patient-derived glioma cell lines (PDGCLs) expressing Prominin-1 (CD133) were used. Human fibroblasts and astrocyte-derived cells were used as controls. Temozolomide (standard GBM drug, TMZ) and DMSO were used as a comparison. GuaDex at 1-10 µM concentrations, were incubated for 3.5-72 h and with PDGCLs cells for 6-24 h. The cytotoxicity was estimated with a fluorometric cytotoxicity assay (FMCA). Fluorescence-labelled GuaDex was used to study the cell interactions. Sia expression was confirmed with a fluorescence labelled Sia binding lectin. Expression of glial fibrillary acidic protein was determined. GuaDex induction of growth inhibition was fast, showing after less than 5 min incubation while the control cells were not affected even after 50 min incubation. The growth inhibitory effect on PDGCLs spheroids was persistent still showing after 4 weeks post-treatment. The growth inhibition of GuaDex was induced at low µM concentrations while TMZ induced only a slight inhibition at mM concentrations. GuaDex efficacy appears significant and warrants further studies.

Knockdown of PHLDA2 promotes apoptosis and autophagy of glioma cells through the AKT/mTOR pathway.

Pleckstrin homology like domain family A member 2 (PHLDA2) is an imprinted gene expressed in placenta and has been shown to be associated with tumor progression. However, the effect of PHLDA2 on glioma cell growth has not been reported yet. Data based on TCGA database showed that PHLDA2 was up-regulated in glioma tissues. Moreover, PHLDA2 was also elevated in glioma cells. Functional assays showed that siRNA-mediated knockdown of PHLDA2 reduced cell viability of glioma cells and suppressed the cell proliferation. Cell apoptosis of glioma cells was promoted by silencing of PHLDA2 with increased Bax and decreased Bcl-2. Silencing of PHLDA2 reduced protein expression of p62, enhanced LC3 and Beclin1 to promote autophagy. Phosphorylated AKT and mTOR were down-regulated in glioma cells by interference of PHLDA2. In conclusion, downregulation of PHLDA2 inhibited glioma cell proliferation, and promoted cell apoptosis and autophagy through inactivation of AKT/mTOR signaling.

The impact of glutamine deprivation on the expression of MEIS3, SPAG4, LHX1, LHX2, and LHX6 genes in ERN1 knockdown U87 glioma cells.

Objective. The aim of the current study was to investigate the expression of genes encoded homeobox proteins such as MEIS3 (Meis homeobox 3), SPAG4 (sperm associated antigen 4), LHX1 (LIM homeobox 1), LHX2, and LHX6 in U87 glioma cells in response to glutamine deprivation in control glioma cells and cells with knockdown of ERN1 (endoplasmic reticulum to nucleus signaling 1), the major pathway of the endoplasmic reticulum stress signaling, for evaluation of a possible dependence on the expression of these important regulatory genes from glutamine supply and ERN1 signaling. Methods. The expression level of MEIS3, SPAG4, LHX, LHX2, and LHX6 genes was studied by real-time quantitative polymerase chain reaction in control U87 glioma cells (transfected by vector) and cells with ERN1 knockdown after exposure to glutamine deprivation. Results. It was shown that the expression level of MEIS3 and LHX1 genes was up-regulated in control glioma cells treated by glutamine deprivation. At the same time, the expression level of three other genes (LHX2, LHX6, and SPAG4) was down-regulated. Furthermore, ERN1 knockdown significantly modified the effect of glutamine deprivation on LHX1 gene expression in glioma cells, but did not change significantly the sensitivity of all other genes expression to this experimental condition. Conclusion. The results of this investigation demonstrate that the exposure of U87 glioma cells under glutamine deprivation significantly affected the expression of all genes studied encoding the homeobox proteins and that this effect of glutamine deprivation was independent of the endoplasmic reticulum stress signaling mediated by ERN1, except LHX1 gene.

ERN1 dependent impact of glutamine and glucose deprivations on the pyruvate dehydrogenase genes expression in glioma cells.

Objective. The aim of the present study was to investigate the expression of pyruvate dehydrogenase genes such as PDHA1, PDHB, DLAT, DLD, and PDHX in U87 glioma cells in response to glutamine and glucose deprivations in control glioma cells and endoplasmic reticulum to nucleus signaling 1 (ERN1) knockdown cells, the major endoplasmic reticulum (ER) stress signaling pathway, to find out whether there exists a possible dependence of these important regulatory genes expression on both glutamine and glucose supply as well as ERN1 signaling. Methods. The expression level of PDHA1, PDHB, DLAT, DLD, and PDHX genes was studied by real-time quantitative polymerase chain reaction in control U87 glioma cells (transfected by empty vector) and cells with inhibition of ERN1(transfected by dnERN1) after cells exposure to glucose and glutamine deprivations. Results. The data showed that the expression level of PDHA1, PDHB, DLAT, and DLD genes was down-regulated (more profound in PDHB gene) in control glioma cells treated with glutamine deprivation. At the same time, ERN1 knockdown modified the impact of glutamine deprivation on the expression level of all these genes in glioma cells: suppressed the sensitivity of PDHB and DLD genes expression and removed the impact of glutamine deprivation on the expression of PDHA1 and DLAT genes. Glucose deprivation did not significantly change the expression level of all studied genes in control glioma cells, but ERN1 knockdown is suppressed the impact of glucose deprivation on PDHX and DLD genes expression and significantly enhanced the expression of PDHA1 and PDHB genes. No significant changes were observed in the sensitivity of PDHX gene expression to glutamine deprivation neither in control nor ERN1 knock-down glioma cells. The knock-down of ERN1 removed the sensitivity of DLAT gene expression to glucose deprivation. Conclusion. The results of this investigation demonstrate that the exposure of control U87 glioma cells under glutamine deprivation significantly affected the expression of PDHA1, PDHB, DLAT, and DLD genes in a gene specific manner and that impact of glutamine deprivation was modified by inhibition of the ER stress signaling mediated by ERN1. At the same time, glucose deprivation affected the expression of PDHA1, PDHB, PDHX, and DLD genes in ERN1 knockdown glioma cells only. Thus, the expression of pyruvate dehydrogenase genes under glutamine and glucose deprivation conditions appears to be controlled by the ER stress signaling through ERN1.

Recombinantly expressed MeICT, a new toxin from Mesobuthus eupeus scorpion, inhibits glioma cell proliferation and downregulates Annexin A2 and FOXM1 genes.

Gliomas are highly invasive and lethal malignancy that do not respond to current therapeutic approaches. Novel therapeutic agents are required to target molecular mechanisms involved in glioma progression. MeICT is a new short-chain toxin isolated from Mesobuthus eupeus scorpion venom. This toxin contained 34 amino acid residues and belongs to chloride channels toxins. In this study, the coding sequence of MeICT was cloned into the pET32Rh vector and a high yield of soluble recombinant MeICT was expressed and purified. Recombinant MeICT-His significantly inhibited the proliferation and migration of glioma cells at low concentration. In vivo studies showed that MeICT was not toxic when administrated to mice at high doses. We also determined the effect of MeICT on the mRNA expression of MMP-2, Annexin A2 and FOXM-2 that are key molecules in the progression and invasion of glioma. Expression of Annexin A2 and FOXM1 mRNA was significantly down-regulated following treatment with MeICT. However, no significant decrease in the expression of MMP-2 gene was identified. In this study a short toxin with four disulfide bonds was successfully produced and its anti-cancer effects was detected. Our findings suggest that recombinant MeICT can be considered as a new potent agent for glioma targeting.

A zinc ferrite nanodrug carrier for delivery of docetaxel: synthesis, characterization, and in vitro tests on C6 glioma cells.

AIM: Docetaxel (DTX) loaded bio-compatible PLGA-PEG encapsulated zinc ferrite nanoparticles (ZFNP) formulation was developed and evaluated against C6 glioma cells. METHODS: The ZFNP were characterised using XRD, FE-SEM, TEM, etc. A series of drug formulations were fabricated by conjugating hydrothermally synthesised ZFNP with DTX in a PLGA-PEG matrix and optimised for drug loading. FTIR and DLS analysis of the formulation along with in vitro drug release, cytotoxicity, cellular uptake, and haemolytic effect were evaluated. RESULTS: Spherical, monodisperse, crystalline ZFNP with an average size of ∼28 nm were formed. The optimised formulation showed a hydrodynamic diameter of ∼147 nm, a surface charge of -34.8 mV, a drug loading of 6.9% (w/w) with prolonged drug release properties, and higher toxicity in C6 glioma cells compared to free DTX along with good internalisation and negligible haemolysis. CONCLUSION: The results indicate ZFNP could be effectively used as nanodrug carrier for delivery of docetaxel to glioma cells.

The Protein L-Isoaspartyl (D-Aspartyl) Methyltransferase Regulates Glial-to-Mesenchymal Transition and Migration Induced by TGF-ß1 in Human U-87 MG Glioma Cells.

The enzyme PIMT methylates abnormal aspartyl residues in proteins. U-87 MG cells are commonly used to study the most frequent brain tumor, glioblastoma. Previously, we reported that PIMT isoform I possessed oncogenic features when overexpressed in U-87 MG and U-251 MG glioma cells. Higher levels of wild-type PIMT stimulated migration and invasion in both glioma cell lines. Conversely, PIMT silencing reduced these migratory abilities of both cell lines. These results indicate that PIMT could play a critical role in glioblastoma growth. Here, we investigated for the first time, molecular mechanisms involving PIMT in the regulation of epithelial to mesenchymal transition (EMT) upon TGF-ß1 treatments. Gene array analyses indicated that EMT genes but not PIMT gene were regulated in U-87 MG cells treated with TGF-ß1. Importantly, PIMT silencing by siRNA inhibited in vitro migration in U-87 MG cells induced by TGF-ß1. In contrast, overexpressed wild-type PIMT and TGF-ß1 had additive effects on cell migration. When PIMT was inhibited by siRNA, this prevented Slug induction by TGF-ß1, while Snail stimulation by TGF-ß1 was increased. Indeed, overexpression of wild-type PIMT led to the opposite effects on Slug and Snail expression dependent on TGF-ß1. These data highlighted the importance of PIMT in the EMT response dependent on TGF-ß1 in U-87 MG glioma cells by an antagonist regulation in the expression of transcription factors Slug and Snail, which are critical players in EMT.

Microbeam Irradiation as a Simultaneously Integrated Boost in a Conventional Whole-Brain Radiotherapy Protocol.

Microbeam radiotherapy (MRT), an experimental high-dose rate concept with spatial fractionation at the micrometre range, has shown a high therapeutic potential as well as good preservation of normal tissue function in pre-clinical studies. We investigated the suitability of MRT as a simultaneously integrated boost (SIB) in conventional whole-brain irradiation (WBRT). A 174 Gy MRT SIB was administered with an array of quasi-parallel, 50 µm wide microbeams spaced at a centre-to-centre distance of 400 µm either on the first or last day of a 5 × 4 Gy radiotherapy schedule in healthy adult C57 BL/6J mice and in F98 glioma cell cultures. The animals were observed for signs of intracranial pressure and focal neurologic signs. Colony counts were conducted in F98 glioma cell cultures. No signs of acute adverse effects were observed in any of the irradiated animals within 3 days after the last irradiation fraction. The tumoricidal effect on F98 cell in vitro was higher when the MRT boost was delivered on the first day of the irradiation course, as opposed to the last day. Therefore, the MRT SIB should be integrated into a clinical radiotherapy schedule as early as possible.

B-Myb participated in ionizing radiation-induced apoptosis and cell cycle arrest in human glioma cells.

As a common treatment of human glioma, ionizing radiation (IR) was reported to result in cell cycle arrest. However, the mechanisms underlying IR-induced abnormal cell cycle remain largely unclear. Here we found that IR caused an elevated expression of B-Myb and cell cycle-related proteins, as well as G2/M phase arrest in U251 cells instead of U87 cells. However, the knockdown of B-Myb by small interfering RNAs ameliorated the increasing of cell cycle-related proteins and G2/M phase arrest induced by IR. Further analysis demonstrated that decreased-B-Myb enhanced the sensitivity of U251 cells to IR. Moreover, the establishment of H1299 cell line proved that B-Myb expression was associated with the status of p53. Immunoprecipitation (IP) and chromatin immunoprecipitation (CHIP) assay results indicated that mutant p53 and SP1 regulated the expression of B-Myb via different mechanisms. This study not only elucidated the role of B-Myb in IR-induced cell cycle alternation, but also provided insight into mechanism of B-Myb expression.

Arbutin exerts anticancer activity against rat C6 glioma cells by inducing apoptosis and inhibiting the inflammatory markers and P13/Akt/mTOR cascade.

Gliomas are a type of brain cancer that occurs in the supporting glial cells of the brain. It is highly malignant and accounts for 80% of brain tumors with high mortality and morbidity. Phytomedicines are potent alternatives for allopathic drugs which cause side effects. They have been used from ancient times by traditional Chinese, Ayurveda, and Siddha medicine. Arubtin is a glycoside phytochemical extracted from plants and belongs to the family of Ericaceae. Arbutin possesses various pharmacological properties such as anti-inflammatory, antioxidant, antitumor, and so on. Hence in the present study, we analyzed the anticancer potency of arbutin against rat C6 glioma cells. Rat C6 glioma cells were procured from American Type Culture Collection and the cells were cultured in Roswell Park Memorial Institute-1640 medium. To assess the cytotoxicity effect of the arbutin against C6 glioma cells, an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide test was performed with different doses from 10 to 60 µM. Arbutin effectively induced apoptosis in the cells and the IC50 dose was obtained at 30 µM. For further studies, we selected the 30 µM IC50 dose and a higher dose of 40 µM. Reactive oxygen species (ROS) generated were analyzed with DCFDA/H2DCFDA stain and the destruction of mitochondrial membrane permeability which is the initiator of apoptosis was analyzed with a cationic stain Rhodamine 123. Dual staining with acridine orange and ethidium bromide was performed to assess the viable and dead cells. Cell adhesion properties of glioma cells were analyzed with Matrigel assay. The apoptotic, inflammatory, and phosphoinositide 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) signaling molecules were analyzed with quantitative polymerase chain reaction (qPCR) analysis to confirm the anticancer effect of arbutin. Arbutin generated excessive ROS and disrupted the mitochondrial membrane, which induced apoptosis in cells, it also inhibited the cell adhesion property of C6 glioma cells. qPCR analysis clearly indicates arbutin increases the apoptotic genes and decreased the inflammatory and PI3K/mTOR signaling molecules. Overall, our results authentically confirm that arbutin can be a potent alternative for treating glioma.

ERN1 knockdown modifies the impact of glucose and glutamine deprivations on the expression of EDN1 and its receptors in glioma cells.

Objective. The aim of the present investigation was to study the impact of glucose and gluta-mine deprivations on the expression of genes encoding EDN1 (endothelin-1), its cognate receptors (EDNRA and EDNRB), and ECE1 (endothelin converting enzyme 1) in U87 glioma cells in response to knockdown of ERN1 (endoplasmic reticulum to nucleus signaling 1), a major signaling pathway of endoplasmic reticulum stress, for evaluation of their possible implication in the control of glioma growth through ERN1 and nutrient limitations. Methods. The expression level of EDN1, its receptors and converting enzyme 1 in control U87 glioma cells and cells with knockdown of ERN1 treated by glucose or glutamine deprivation by quantitative polymerase chain reaction was studied. Results. We showed that the expression level of EDN1 and ECE1 genes was significantly up-regulated in control U87 glioma cells exposure under glucose deprivation condition in comparison with the glioma cells, growing in regular glucose containing medium. We also observed up-regulation of ECE1 gene expression in U87 glioma cells exposure under glutamine deprivation as well as down-regulation of the expression of EDN1 and EDNRA mRNA, being more significant for EDN1. Furthermore, the knockdown of ERN1 signaling enzyme function significantly modified the response of most studied gene expressions to glucose and glutamine deprivation conditions. Thus, the ERN1 knockdown led to a strong suppression of EDN1 gene expression under glucose deprivation, but did not change the effect of glutamine deprivation on its expression. At the same time, the knockdown of ERN1 signaling introduced the sensitivity of EDNRB gene to both glucose and glutamine deprivations as well as completely removed the impact of glucose deprivation on the expression of ECE1 gene. Conclusions. The results of this study demonstrated that the expression of endothelin-1, its receptors, and ECE1 genes is preferentially sensitive to glucose and glutamine deprivations in gene specific manner and that knockdown of ERN1 significantly modified the expression of EDN1, EDNRB, and ECE1 genes in U87 glioma cells. It is possible that the observed changes in the expression of studied genes under nutrient deprivation may contribute to the suppressive effect of ERN1 knockdown on glioma cell proliferation and invasiveness.

Biscoumarin-pyrimidine conjugates as potent anticancer agents and binding mechanism of hit candidate with human serum albumin.

In our continuing efforts to develop therapeutically active coumarin-based compounds, a series of new C4-C4' biscoumarin-pyrimidine conjugates (1a-l) was synthesized via SN 2 reaction of substituted 4-bromomethyl coumarin with thymine. All compounds were characterized using spectroscopic techniques, that is, attenuated total reflection infrared (ATR-IR), CHN elemental analysis, and 1 H and 13 C NMR (nuclear magnetic resonance). In addition, the structure of compound 1d (1,3-bis[(7-chloro-2-oxo-2H-chromen-4-yl)methyl]-5-methylpyrimidine-2,4(1H,3H)-dione) was established through X-ray crystallography. Compounds 1a-l were screened for in vitro anticancer activity against C6 rat glioma cells. Among the screened compounds, 1,3-bis[(6-chloro-2-oxo-2H-chromen-4-yl)methyl]-5-methylpyrimidine-2,4(1H,3H)-dione (1c) was identified as the best antiproliferative candidate, exhibiting an IC50 value of 4.85 µM. All the compounds (1a-l) were found to be nontoxic toward healthy human embryonic kidney cells (HEK293), indicating their selective nature. In addition, the most active compound (1c) displayed strong binding interactions with the drug carrier protein, human serum albumin, and exhibited good solution stability at biological pH conditions. Fluorescence, UV-visible spectrophotometry and molecular modeling methodologies were employed for studying the interaction mechanism of compound 1c with protein.

Glioblastoma Stem Cell-Derived Exosomes Enhance Stemness and Tumorigenicity of Glioma Cells by Transferring Notch1 Protein.

Exosomes contain plenty of bioactive information, playing an important role in intercellular communication by transfer their bioactive molecular contents to recipient cells. Glioblastoma stem cells (GSCs) and non-GSC glioma cells coexist in GBM microenvironment; GSC-released exosomes contain intracellular signaling molecules, which may affect the biological phenotypes of recipient cells. However, whether GSC exosomes could affect the biological phenotype of non-GSC glioma cells has not yet been defined. To explore whether GSC exosomes could reprogramme non-GSC glioma cells into GSCs and its possible mechanism involved, non-GSC glioma cells were treated with GSCs released exosomes; the potential mechanisms of action were studied with RNA interference, Notch inhibitors and Western blot analysis. The proliferation, neurosphere formation, invasive capacities, and tumorigenicity of non-GSC glioma cells were increased significantly after GSC exosome treatment; Notch1 signaling pathway was activated in GSCs; Notch1 protein was highly enriched in GSC exosomes; Notch1 signaling pathway and stemness-related protein expressions were increased in GSC exosome treated non-GSC glioma cells and these cell generated tumor tissues; Notch1 protein expression in GSCs and their exosomes, and the neurosphere formation of GSCs were decreased by Notch1 RNA interference; Notch1 signaling pathway protein and stemness protein expressions were decreased in GSC exosome treated non-GSC glioma cells by Notch1 RNA interference and Notch inhibitors. The findings in this study indicated that GSC exosomes act as information carriers, mediated non-GSC glioma cell dedifferentiation into GSCs by delivering Notch1 protein through Notch1 signaling activation, and enhanced stemness and tumorigenicity of non-GSC glioma cells.

Polycomb-like 2 regulates PRC2 components to affect proliferation in glioma cells.

INTRODUCTION: The Polycomb group (PcG) is an important family of transcriptional regulators that controls growth and tumorigenesis. The PcG mainly consists of two complexes, PRC1 and Polycomb Repressive Complex 2 (PRC2). Polycomb-like 2 (PCL2) is known to interact with the PRC2 protein. The role of PCL2 in the development and progression of glioma is unclear. METHODS: We use The Cancer Genome Atlas (TCGA) database to detect the expression of PCL2 in various tumors. 117 cases of clinical glioma (WHOI-IV) were collected, and PCL2 expression and localization were detected by immunohistochemical staining. Glioma cells U87/U251 were infected with overexpressed and interfered PCL2. CCK8 assay, colony formation assay, EdU method, cell cycle and apoptosis were used to detect cell proliferation and apoptosis. Western blot was used to detect the expression of PRC2-related core proteins. After DZNeP intervention, PRC2 protein expression was again measured to discuss the mechanism of PCL2 action. RESULTS: TCGA database results and immunohistochemical staining results suggest that PCL2 is highly expressed in gliomas. We found that the PCL2 gene promoted tumor cell proliferation, enhanced the colony formation ability, and increased S phase in the cell cycle. The overexpression of PCL2 upregulated the expression levels of EZH2 and EED (two core members of PRC2), decreased the expression of SUZ12, increased the level of H3K27 trimethylation (H3K27me3), H3K4 dimethylation (H3K4me2), and decreased H3K9 dimethylation (H3K9me2). The result after interfering with PCL2 was the opposite. CONCLUSIONS: As an important accessory protein of PRC2, PCL2 can not only change the expression of PRC2 components, but also affect the expression level of Histone methylation. Therefore, PCL2 may be an important hub for regulating the synergy among PRC2 members. This study revealed PCL2 as a new target for tumor research and open up a new avenue for future research in glioma.

Expression of IDE and PITRM1 genes in ERN1 knockdown U87 glioma cells: effect of hypoxia and glucose deprivation.

OBJECTIVE: The aim of the present investigation was to study the expression of genes encoding polyfunctional proteins insulinase (insulin degrading enzyme, IDE) and pitrilysin metallopeptidase 1 (PITRM1) in U87 glioma cells in response to inhibition of endoplasmic reticulum stress signaling mediated by ERN1/IRE1 (endoplasmic reticulum to nucleus signaling 1) for evaluation of their possible significance in the control of metabolism through ERN1 signaling as well as hypoxia, glucose and glutamine deprivations. METHODS: The expression level of IDE and PITRM1 genes was studied in control and ERN1 knockdown U87 glioma cells under glucose and glutamine deprivations as well as hypoxia by quantitative polymerase chain reaction. RESULTS: It was found that the expression level of IDE and PITRM1 genes was down-regulated in ERN1 knockdown (without ERN1 protein kinase and endoribonuclease activity) glioma cells in comparison with the control glioma cells, being more significant for PITRM1 gene. We also found up-regulation of microRNA MIR7-2 and MIRLET7A2, which have specific binding sites in 3'-untranslated region of IDE and PITRM1 mRNAs, correspondingly, and can participate in posttranscriptional regulation of these mRNA expressions. Only inhibition of ERN1 endoribonuclease did not change significantly the expression of IDE and PITRM1 genes in glioma cells. The expression of IDE and PITRM1 genes is preferentially regulated by ERN1 protein kinase. We also showed that hypoxia down-regulated the expression of IDE and PITRM1 genes and that knockdown of ERN1 signaling enzyme function modified the response of these gene expressions to hypoxia. Glucose deprivation increased the expression level of IDE and PITRM1 genes, but ERN1 knockdown enhanced only the effect of glucose deprivation on PITRM1 gene expression. Glutamine deprivation did not affect the expression of IDE gene in both types of glioma cells, but up-regulated PITRM1 gene and this up-regulation was stronger in ERN1 knockdown cells. CONCLUSIONS: Results of this investigation demonstrate that ERN1 knockdown significantly decreases the expression of IDE and PITRM1 genes by ERN1 protein kinase mediated mechanism. The expression of both studied genes was sensitive to hypoxia as well as glucose deprivation and dependent on ERN1 signaling in gene-specific manner. It is possible that the level of these genes expression under hypoxia and glucose deprivation is a result of complex interaction of variable endoplasmic reticulum stress related and unrelated regulatory factors and contributed to the control of the cell metabolism.