Differential Regulation of Bcl-2 and Bax Expression in Cells Infected with Virulent and Nonvirulent Strains of Sindbis Virus

AbstractSindbis virus is an alphavirus that infects cells in either lytic or persistent infection. In this study we examined the effects of Sindbis virus on cell apoptosis and on the expression of Bcl-2 and Bax. Of the two strains studied, SVA and SVNI, only the neurovirulent strain, SVNI, induced apoptosis of astrocytes and PC-12 cells. SVA, which infects cells in a persistent manner, induced up-regulation of bcl-2 mRNA and Bcl-2 protein, whereas SVNI induced an increase in Bax levels. Our results indicate a differential regulation of Bcl2 and Bax expression by SVA and SVNI, which may be associated with the apoptotic potential of the viruses

sPIF promotes myoblast differentiation and utrophin expression while inhibiting fibrosis in Duchenne muscular dystrophy via the H19/miR-675/let-7 and miR-21 pathways

Duchenne muscular dystrophy (DMD) is a progressive, lethal, X-linked disease of skeletal and cardiac muscles caused by mutations in the dystrophin gene. Loss of dystrophin leads to muscle fiber damage and impairment of satellite cell asymmetric division, which are essential for muscle regeneration. These processes ultimately result in muscle wasting and the replacement of the degenerating muscles by fibrogenic cells, a process that leads to the generation of fibrotic tissues. Preimplantation factor (PIF) is an evolutionary conserved 15-amino acid peptide secreted by viable mammalian embryos. Synthetic PIF (sPIF) reproduces the protective/regenerative effects of the endogenous peptide in immune disorders and transplantation models. In this study, we demonstrated that sPIF treatment promoted mouse and human myoblast differentiation and inhibited the expression of collagen 1A1, collagen 1A2, and TGF-β in DMD patient-derived myoblasts. Additionally, sPIF increased the expression of utrophin, a homolog of dystrophin protein. sPIF effects were mediated via the upregulation of lncRNA H19 and miR-675 and downregulation of let-7. sPIF also inhibited the expression of miR-21, a major fibrosis regulator. The administration of sPIF in mdx mice significantly decreased serum creatine kinase and collagen I and collagen IV expression in the diaphragm, whereas it increased utrophin expression in the diaphragm, heart and quadriceps muscles. In conclusion, sPIF promoted the differentiation of DMD myoblasts, increased utrophin expression via the H19/miRNA-675/let-7 pathway, and reduced muscle fibrosis possibly via the upregulation of miR-675 and inhibition of miR-21 expression. These findings strongly support pursuing sPIF as a potential therapeutic agent for DMD. Moreover, the completion of an sPIF phase I safety trial will further promote the use of sPIF for the treatment of muscular dystrophies

Expanding the MECP2 network using comparative genomics reveals potential therapeutic targets for Rett syndrome

Inactivating mutations in the Methyl-CpG Binding Protein 2 (MECP2) gene are the main cause of Rett syndrome (RTT). Despite extensive research into MECP2 function, no treatments for RTT are currently available. Here, we used an evolutionary genomics approach to construct an unbiased MECP2 gene network, using 1028 eukaryotic genomes to prioritize proteins with strong co-evolutionary signatures with MECP2. Focusing on proteins targeted by FDA-approved drugs led to three promising targets, two of which were previously linked to MECP2 function (IRAK, KEAP1) and one that was not (EPOR). The drugs targeting these three proteins (Pacritinib, DMF, and EPO) were able to rescue different phenotypes of MECP2 inactivation in cultured human neural cell types, and appeared to converge on Nuclear Factor Kappa B (NF-κB) signaling in inflammation. This study highlights the potential of comparative genomics to accelerate drug discovery, and yields potential new avenues for the treatment of RTT

Specific Compositions of Cannabis sativa Compounds Have Cytotoxic Activity and Inhibit Motility and Colony Formation of Human Glioblastoma Cells In Vitro

Glioblastoma multiforme (GBM) is the most lethal subtype of glioma. Cannabis sativa is used for the treatment of various medical conditions. Around 150 phytocannabinoids have been identified in C. sativa, among them Δ-9-tetrahydrocannabinol (THC) and cannabidiol (CBD) that trigger GBM cell death. However, the optimal combinations of cannabis molecules for anti-GBM activity are unknown. Chemical composition was determined using high-performance liquid chromatography (HPLC) and gas chromatography mass spectrometry (GC/MS). Cytotoxic activity was determined by XTT and lactate dehydrogenase (LDH) assays and apoptosis and cell cycle by fluorescence-activated cell sorting (FACS). F-actin structures were observed by confocal microscopy, gene expression by quantitative PCR, and cell migration and invasion by scratch and transwell assays, respectively. Fractions of a high-THC cannabis strain extract had significant cytotoxic activity against GBM cell lines and glioma stem cells derived from tumor specimens. A standard mix (SM) of the active fractions F4 and F5 induced apoptosis and expression of endoplasmic reticulum (ER)-stress associated-genes. F4 and F5 inhibited cell migration and invasion, altered cell cytoskeletons, and inhibited colony formation in 2 and 3-dimensional models. Combinations of cannabis compounds exert cytotoxic, anti-proliferative, and anti-migratory effects and should be examined for efficacy on GBM in pre-clinical studies and clinical trials

Propofol Inhibits Glioma Stem Cell Growth and Migration and Their Interaction with Microglia via BDNF-AS and Extracellular Vesicles

Glioblastoma (GBM) is the most common and aggressive primary brain tumor. GBM contains a small subpopulation of glioma stem cells (GSCs) that are implicated in treatment resistance, tumor infiltration, and recurrence, and are thereby considered important therapeutic targets. Recent clinical studies have suggested that the choice of general anesthetic (GA), particularly propofol, during tumor resection, affects subsequent tumor response to treatments and patient prognosis. In this study, we investigated the molecular mechanisms underlying propofol\u27s anti-tumor effects on GSCs and their interaction with microglia cells. Propofol exerted a dose-dependent inhibitory effect on the self-renewal, expression of mesenchymal markers, and migration of GSCs and sensitized them to both temozolomide (TMZ) and radiation. At higher concentrations, propofol induced a large degree of cell death, as demonstrated using microfluid chip technology. Propofol increased the expression of the lncRNA BDNF-AS, which acts as a tumor suppressor in GBM, and silencing of this lncRNA partially abrogated propofol\u27s effects. Propofol also inhibited the pro-tumorigenic GSC-microglia crosstalk via extracellular vesicles (EVs) and delivery of BDNF-AS. In conclusion, propofol exerted anti-tumor effects on GSCs, sensitized these cells to radiation and TMZ, and inhibited their pro-tumorigenic interactions with microglia via transfer of BDNF-AS by EVs

New Biocompatible Nanohydrogels of Predefined Sizes for Complexing Nucleic Acids

The advent of protein expression using m-RNA applied lately for treating the COVID pandemic, and gene editing using CRISPR/Cas9 technology for introducing DNA sequences at a specific site in the genome, are milestones for the urgent need of developing new nucleic acid delivery systems with improved delivery properties especially for in vivo applications. We have designed, synthesized, and characterized novel cross-linked monodispersed nanohydrogels (NHG’s) with well-defined sizes ranging between 50–400 nm. The synthesis exploits the formation of self-assemblies generated upon heating a thermo-responsive mixture of monomers. Self-assemblies are formed and polymerized at high temperatures resulting in NHGs with sizes that are predetermined by the sizes of the intermediate self-assemblies. The obtained NHGs were chemically reduced to lead particles with highly positive zeta potential and low cell toxicity. The NHGs form complexes with DNA, and at optimal charge ratio the size of the complexes is concomitant with the size of the NHG’s. Thus, the DNA is fully embedded inside the NHGs. The new NHGs and their DNA complexes are devoid of cell toxicity which together with their tunned sizes, make them potential tools for gene delivery and foreign protein expression

New Biocompatible Nanohydrogels of Predefined Sizes for Complexing Nucleic Acids

The advent of protein expression using m-RNA applied lately for treating the COVID pandemic, and gene editing using CRISPR/Cas9 technology for introducing DNA sequences at a specific site in the genome, are milestones for the urgent need of developing new nucleic acid delivery systems with improved delivery properties especially for in vivo applications. We have designed, synthesized, and characterized novel cross-linked monodispersed nanohydrogels (NHG’s) with well-defined sizes ranging between 50–400 nm. The synthesis exploits the formation of self-assemblies generated upon heating a thermo-responsive mixture of monomers. Self-assemblies are formed and polymerized at high temperatures resulting in NHGs with sizes that are predetermined by the sizes of the intermediate self-assemblies. The obtained NHGs were chemically reduced to lead particles with highly positive zeta potential and low cell toxicity. The NHGs form complexes with DNA, and at optimal charge ratio the size of the complexes is concomitant with the size of the NHG’s. Thus, the DNA is fully embedded inside the NHGs. The new NHGs and their DNA complexes are devoid of cell toxicity which together with their tunned sizes, make them potential tools for gene delivery and foreign protein expression

Tyrosine Phosphorylation of Protein Kinase Cδ Is Essential for Its Apoptotic Effect in Response to Etoposide

Protein kinase Cδ (PKCδ) is involved in the apoptosis of various cells in response to diverse stimuli. In this study, we characterized the role of PKCδ in the apoptosis of C6 glioma cells in response to etoposide. We found that etoposide induced apoptosis in the C6 cells within 24 to 48 h and arrested the cells in the G(1)/S phase of the cell cycle. Overexpression of PKCδ increased the apoptotic effect induced by etoposide, whereas the PKCδ selective inhibitor rottlerin and the PKCδ dominant-negative mutant K376R reduced this effect compared to control cells. Etoposide-induced tyrosine phosphorylation of PKCδ and its translocation to the nucleus within 3 h was followed by caspase-dependent cleavage of the enzyme. Using PKC chimeras, we found that both the regulatory and catalytic domains of PKCδ were necessary for its apoptotic effect. The role of tyrosine phosphorylation of PKCδ in the effects of etoposide was examined using cells overexpressing a PKCδ mutant in which five tyrosine residues were mutated to phenylalanine (PKCδ5). These cells exhibited decreased apoptosis in response to etoposide compared to cells overexpressing PKCδ. Likewise, activation of caspase 3 and the cleavage of the PKCδ5 mutant were significantly lower in cells overexpressing PKCδ5. Using mutants of PKCδ altered at individual tyrosine residues, we identified tyrosine 64 and tyrosine 187 as important phosphorylation sites in the apoptotic effect induced by etoposide. Our results suggest a role of PKCδ in the apoptosis induced by etoposide and implicate tyrosine phosphorylation of PKCδ as an important regulator of this effect

Mesenchymal stem cells enhance the oncolytic effect of Newcastle disease virus in glioma cells and glioma stem cells via the secretion of TRAIL

BACKGROUND: Newcastle disease virus (NDV) is an avian paramyxovirus, which selectively exerts oncolytic effects in cancer cells. Mesenchymal stem cells (MSCs) have been reported to affect tumor growth and deliver anti-tumor agents to experimental glioblastoma (GBM). Here, we explored the effects of NDV-infected MSCs derived from different sources, on glioma cells and glioma stem cells (GSCs) and the mechanisms involved in their effects. METHODS: The glioma cell lines (A172 and U87) and primary GSCs that were generated from GBM tumors were used in this study. MSCs derived from bone marrow, adipose tissue or umbilical cord were infected with NDV (MTH-68/H). The ability of these cells to deliver the virus to glioma cell lines and GSCs and the effects of NDV-infected MSCs on cell death and on the stemness and self-renewal of GSCs were examined. The mechanisms involved in the cytotoxic effects of the NDV-infected MSCs and their influence on the radiation sensitivity of GSCs were examined as well. RESULTS: NDV induced a dose-dependent cell death in glioma cells and a low level of apoptosis and inhibition of self-renewal in GSCs. MSCs derived from bone marrow, adipose and umbilical cord that were infected with NDV delivered the virus to co-cultured glioma cells and GSCs. Conditioned medium of NDV-infected MSCs induced higher level of apoptosis in the tumor cells compared with the apoptosis induced by their direct infection with similar virus titers. These results suggest that factor(s) secreted by the infected MSCs sensitized the glioma cells to the cytotoxic effects of NDV. We identified TRAIL as a mediator of the cytotoxic effects of the infected MSCs and demonstrated that TRAIL synergized with NDV in the induction of cell death in glioma cells and GSCs. Moreover, conditioned medium of infected MSCs enhanced the sensitivity of GSCs to γ-radiation. CONCLUSIONS: NDV-infected umbilical cord-derived MSCs may provide a novel effective therapeutic approach for targeting GSCs and GBM and for sensitizing these tumors to γ-radiation

Role of Protein Kinase C δ in Reactivation of Kaposi's Sarcoma-Associated Herpesvirus

TPA (12-O-tetradecanoylphorbol-13-acetate), a well-known activator of protein kinase C (PKC), can experimentally induce reactivation of Kaposi's sarcoma-associated herpesvirus (KSHV) in certain latently infected cells. We selectively blocked the activity of PKC isoforms by using GF 109203X or rottlerin and demonstrated that this inhibition largely decreased lytic KSHV reactivation by TPA. Translocation of the PKCδ isoform was evident shortly after TPA stimulation. Overexpression of the dominant-negative PKCδ mutant supported an essential role for the PKCδ isoform in virus reactivation, yet overexpression of PKCδ alone was not sufficient to induce lytic reactivation of KSHV, suggesting that additional signaling molecules participate in this pathway