Regulation of DNA Repair Mechanism in Human Glioma Xenograft Cells both In Vitro and In Vivo in Nude Mice

Glioblastoma Multiforme (GBM) is the most lethal form of brain tumor. Efficient DNA repair and anti-apoptotic mechanisms are making glioma treatment difficult. Proteases such as MMP9, cathepsin B and urokinase plasminogen activator receptor (uPAR) are over expressed in gliomas and contribute to enhanced cancer cell proliferation. Non-homologous end joining (NHEJ) repair mechanism plays a major role in double strand break (DSB) repair in mammalian cells.Here we show that silencing MMP9 in combination with uPAR/cathepsin B effects NHEJ repair machinery. Expression of DNA PKcs and Ku70/80 at both mRNA and protein levels in MMP9-uPAR (pMU) and MMP9-cathepsin B (pMC) shRNA-treated glioma xenograft cells were reduced. FACS analysis showed an increase in apoptotic peak and proliferation assays revealed a significant reduction in the cell population in pMU- and pMC-treated cells compared to untreated cells. We hypothesized that reduced NHEJ repair led to DSBs accumulation in pMU- and pMC-treated cells, thereby initiating cell death. This hypothesis was confirmed by reduced Ku70/Ku80 protein binding to DSB, increased comet tail length and elevated γH2AX expression in treated cells compared to control. Immunoprecipitation analysis showed that EGFR-mediated lowered DNA PK activity in treated cells compared to controls. Treatment with pMU and pMC shRNA reduced the expression of DNA PKcs and ATM, and elevated γH2AX levels in xenograft implanted nude mice. Glioma cells exposed to hypoxia and irradiation showed DSB accumulation and apoptosis after pMU and pMC treatments compared to respective controls.Our results suggest that pMU and pMC shRNA reduce glioma proliferation by DSB accumulation and increase apoptosis under normoxia, hypoxia and in combination with irradiation. Considering the radio- and chemo-resistant cancers favored by hypoxia, our study provides important therapeutic potential of MMP9, uPAR and cathepsin B shRNA in the treatment of glioma from clinical stand point

The contribution of 7q33 copy number variations for intellectual disability

Copy number variations (CNVs) at the 7q33 cytoband are very rarely described in the literature, and almost all of the cases comprise large deletions affecting more than just the q33 segment. We report seven patients (two families with two siblings and their affected mother and one unrelated patient) with neurodevelopmental delay associated with CNVs in 7q33 alone. All the patients presented mild to moderate intellectual disability (ID), dysmorphic features, and a behavioral phenotype characterized by aggressiveness and disinhibition. One family presents a small duplication in cis affecting CALD1 and AGBL3 genes, while the other four patients carry two larger deletions encompassing EXOC4, CALD1, AGBL3, and CNOT4. This work helps to refine the phenotype and narrow the minimal critical region involved in 7q33 CNVs. Comparison with similar cases and functional studies should help us clarify the relevance of the deleted genes for ID and behavioral alterations.FEDER funds, through the Competitiveness Factors Operational Programme (COMPETE), and by National funds, through the Foundation for Science and Technology (FCT), under the scope of the projects PIC/IC/83026/2007, PIC/IC/83013/2007, and POCI-01-0145-FEDER-007038. This work has also been funded by the project NORTE-01-0145-FEDER-000013, supported by the Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (FEDER)info:eu-repo/semantics/publishedVersio

ZEB2 Mediates Multiple Pathways Regulating Cell Proliferation, Migration, Invasion, and Apoptosis in Glioma

BACKGROUND: The aim of the present study was to analyze the expression of Zinc finger E-box Binding homeobox 2 (ZEB2) in glioma and to explore the molecular mechanisms of ZEB2 that regulate cell proliferation, migration, invasion, and apoptosis. METHODOLOGY/PRINCIPAL FINDINGS: Expression of ZEB2 in 90 clinicopathologically characterized glioma patients was analyzed by immunohistochemistry. Furthermore, siRNA targeting ZEB2 was transfected into U251 and U87 glioma cell lines in vitro and proliferation, migration, invasion, and apoptosis were examined separately by MTT assay, Transwell chamber assay, flow cytometry, and western blot. RESULTS: The expression level of ZEB2 protein was significantly increased in glioma tissues compared to normal brain tissues (P<0.001). In addition, high levels of ZEB2 protein were positively correlated with pathology grade classification (P = 0.024) of glioma patients. Knockdown of ZEB2 by siRNA suppressed cell proliferation, migration and invasion, as well as induced cell apoptosis in glioma cells. Furthermore, ZEB2 downregulation was accompanied by decreased expression of CDK4/6, Cyclin D1, Cyclin E, E2F1, and c-myc, while p15 and p21 were upregulated. Lowered expression of ZEB2 enhanced E-cadherin levels but also inhibited β-Catenin, Vimentin, N-cadherin, and Snail expression. Several apoptosis-related regulators such as Caspase-3, Caspase-6, Caspase-9, and Cleaved-PARP were activated while PARP was inhibited after ZEB2 siRNA treatment. CONCLUSION: Overexpression of ZEB2 is an unfavorable factor that may facilitate glioma progression. Knockdown ZEB2 expression by siRNA suppressed cell proliferation, migration, invasion and promoted cell apoptosis in glioma cells

MMP-9, uPAR and Cathepsin B Silencing Downregulate Integrins in Human Glioma Xenograft Cells In Vitro and In Vivo in Nude Mice

Involvement of MMP-9, uPAR and cathepsin B in adhesion, migration, invasion, proliferation, metastasis and tumor growth has been well established. In the present study, MMP-9, uPAR and cathepsin B genes were downregulated in glioma xenograft cells using shRNA plasmid constructs and we evaluated the involvement of integrins and changes in their adhesion, migration and invasive potential.MMP-9, uPAR and cathepsin B single shRNA plasmid constructs were used to downregulate these molecules in xenograft cells. We also used MMP-9/uPAR and MMP-9/cathepsin B bicistronic constructs to evaluate the cumulative effects. MMP-9, uPAR and cathepsin B downregulation significantly inhibits xenograft cell adhesion to several extracellular matrix proteins. Treatment with MMP-9, uPAR and cathepsin B shRNA of xenografts led to the downregulation of several alpha and beta integrins. In all the assays, we noticed more prominent effects with the bicistronic plasmid constructs when compared to the single plasmid shRNA constructs. FACS analysis demonstrated the expression of alphaVbeta3, alpha6beta1 and alpha9beta1 integrins in xenograft cells. Treatment with bicistronic constructs reduced alphaVbeta3, alpha6beta1 and alpha9beta1 integrin expressions in xenograft injected nude mice. Migration and invasion were also inhibited by MMP-9, uPAR and cathepsin B shRNA treatments as assessed by spheroid migration, wound healing, and Matrigel invasion assays. As expected, bicistronic constructs further inhibited the adhesion, migration and invasive potential of the xenograft cells as compared to individual treatments.Downregulation of MMP-9, uPAR and cathespin B alone and in combination inhibits adhesion, migration and invasive potential of glioma xenografts by downregulating integrins and associated signaling molecules. Considering the existence of integrin inhibitor-resistant cancer cells, our study provides a novel and effective approach to inhibiting integrins by downregulating MMP-9, uPAR and cathepsin B in the treatment of glioma

Recurrent stroke as a presenting feature of acquired partial lipodystrophy

Acquired partial lipodystrophy (PL) (Barraquer-Simons syndrome) is a rare condition with onset in childhood, and it is characterized by progressive loss of subcutaneous fat in a cephalocaudal fashion. This report describes a case of acquired PL in a 16-year-old girl, who had progressive loss of facial fat since 3 years. Systemic lupus erythematosus (SLE), anticardiolipin antibody, primary hypothyroidism, diabetes, and dyslipidemia may antedate the development of complications such as cerebrovascular stroke and cardiovascular disease. The girl had developed recurrent left hemiparesis, and withdrawn from school due to poor performance

Catalytic functionalities of nano Ru catalysts supported on TiO2-ZrO2 mixed oxide for vapor phase hydrogenolysis of glycerol to propanediols

Vapor phase hydrogenolysis of glycerol was studied over Ru catalysts supported on TiO2-ZrO2 binary oxide. Ru catalysts with various ruthenium loadings from 1.0 to 6.0 wt% were prepared by deposition-precipitation method on the TiO2-ZrO2 mixed oxide support. These catalysts were characterized by X-ray diffraction, H-2 temperature-programmed reduction, NH3 temperature-programmed desorption, transmission electron microscopy, BET surface area, XPS and CO chemisorption measurements. The catalysts exhibited superior performance for the vapor phase hydrogenolysis of glycerol at moderate temperature and atmospheric pressure. The mixed oxide support plays a significant role in improving the catalytic activity for the production of propanediols. The glycerol conversion and the selectivity of various products depend on the catalyst preparation method and also on the Ru content. The influence of acidity of the catalyst and its correlation to the catalytic performance (selectivity and conversion) has been studied. The weak and strong acidic sites of the catalysts measured by NH3-TPD play a key role in selective formation of 1,2-propanediol and 1,3-propanediol. XRD, TEM, XPS and CO chemisorption studies revealed that ruthenium was well dispersed on TiO2-ZrO2 which further contributed to the superior catalytic activity for glycerol hydrogenolysis

Activity and Selectivity of Platinum–Copper Bimetallic Catalysts Supported on Mordenite for Glycerol Hydrogenolysis to 1,3-Propanediol

Biomass derived glycerol is considered an ideal feedstock with a prospective to be converted into a number of valuable compounds. Catalytic glycerol hydrogenolysis to produce 1,3-propanediol is one of the pioneering biosustainable pathways. Bimetallic Pt–Cu catalysts supported on H-mordenite were synthesized with various copper loadings and applied in the selective glycerol hyrogenolysis to 1,3-propanediol in a continuous fixed bed reactor performed in vapor phase under atmospheric pressure. Several techniques such as XRD, ICP-AES, NH₃-TPD, Pyr FTIR, BET, TPR, HR-TEM, XPS, and solid state NMR were employed to characterize the physical and chemical properties of Pt–Cu/Mor catalysts. A detailed reaction parametric study has been carried out. The results designated that well dispersed Pt–Cu catalysts with small particle size, supported on a Brønsted acidic H-mordenite with a multiple pore system and strong bimetallic phase-support interaction, promote the selectivity to 1,3-propanediol. Over the Pt–Cu/Mor catalyst of optimum composition (2% Pt and 5% Cu by weight) and under the optimum reaction conditions (210 °C, H₂ flow rate of 80 mL min^–1, and gly concentration of 10 wt %), the glycerol conversion and 1,3-PD selectivity reached 90% and 58.5%, respectively. Structural characterizations and reusability of the Pt-5Cu/Mor catalyst were also performed. With evident advantages of selective C–O hydrogenolysis with low C–C cleavages, the bimetallic Pt–Cu/Mor catalysts hold great potential as high-performance catalysts for glycerol conversion to 1,3-propanediol