RESUMO
Dendrimers are appealing scaffolds for creating carbohydrate mimics with unique multivalent cooperativity. We report here novel bola-amphiphilic glycodendrimers bearing mannose and glucose terminals, and a hydrophobic thioacetal core responsive to reactive oxygen species. The peculiar bola-amphiphilic feature enabled stronger binding to lectin compared to conventional amphiphiles. In addition, these dendrimers are able to target mannose receptors and glucose transporters expressed at the surface of cells, thus allowing effective and specific cellular uptake. This highlights their great promise for targeted delivery.
Assuntos
Dendrímeros , Manose , Manose/química , Dendrímeros/química , Espécies Reativas de Oxigênio , Carboidratos/química , Lectinas/química , GlucoseRESUMO
New blood vessel formation is a key component of the cardiac repair process after myocardial infarction (MI). Hypoxia following MI is a major driver of angiogenesis in the myocardium. Hypoxia-inducible factor 1α (HIF1α) is the key regulator of proangiogenic signaling. The present study found that stearoyl-CoA desaturase (SCD) significantly contributed to the induction of angiogenesis in the hypoxic myocardium independently of HIF1α expression. The pharmacological inhibition of SCD activity in HL-1 cardiomyocytes and SCD knockout in an animal model disturbed the expression and secretion of proangiogenic factors including vascular endothelial growth factor-A, proinflammatory cytokines (interleukin-1ß, interleukin-6, tumor necrosis factor α, monocyte chemoattractant protein-1, and Rantes), metalloproteinase-9, and platelet-derived growth factor in ischemic cardiomyocytes. These disturbances affected the proangiogenic potential of ischemic cardiomyocytes after SCD depletion. Together with the most abundant SCD1 isoform, the heart-specific SCD4 isoform emerged as an important regulator of new blood vessel formation in the murine post-MI myocardium. We also provide evidence that SCD shapes energy metabolism of the ischemic heart by maintaining the shift from fatty acids to glucose as the substrate that is used for adenosine triphosphate production. Furthermore, we propose that the regulation of the proangiogenic properties of hypoxic cardiomyocytes by key modulators of metabolic signaling such as adenosine monophosphate kinase, protein kinase B (AKT), and peroxisome-proliferator-activated receptor-γ coactivator 1α/peroxisome proliferator-activated receptor α depends on SCD to some extent. Thus, our results reveal a novel mechanism that links SCD to cardiac repair processes after MI.
Assuntos
Infarto do Miocárdio , Estearoil-CoA Dessaturase , Monofosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Quimiocina CCL2/metabolismo , Quimiocina CCL5/metabolismo , Metabolismo Energético , Ácidos Graxos/metabolismo , Glucose/metabolismo , Hipóxia/metabolismo , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Camundongos , Infarto do Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , PPAR alfa/metabolismo , Fator de Crescimento Derivado de Plaquetas/metabolismo , Isoformas de Proteínas/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Estearoil-CoA Dessaturase/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
The activation of NFAT (nuclear factor of activated T cells) transcription factors by calcium-dependent phosphatase calcineurin is a key step in controlling T cell activation and plays a vital role during carcinogenesis. NFATs are overexpressed in many cancers, including the most common primary brain tumor, gliomas. In the present study, we demonstrate the expression of NFATs and NFAT-driven transcription in several human glioma cells. We used a VIVIT peptide for interference in calcineurin binding to NFAT via a conserved PxIxIT motif. VIVIT was expressed as a fusion protein with a green fluorescent protein (VIVIT-GFP) or conjugated to cell-penetrating peptides (CPP), Sim-2 or 11R. We analyzed the NFAT expression, phosphorylation, subcellular localization and their transcriptional activity in cells treated with peptides. Overexpression of VIVIT-GFP decreased the NFAT-driven activity and inhibited the transcription of endogenous NFAT-target genes. These effects were not reproduced with synthetic peptides: Sim2-VIVIT did not show any activity, and 11R-VIVIT did not inhibit NFAT signaling in glioma cells. The presence of two calcineurin docking sites in NFATc3 might require dual-specificity blocking peptides. The cell-penetrating peptides Sim-2 or 11R linked to VIVIT did not improve its action making it unsuitable for evaluating NFAT dependent events in glioma cells with high expression of NFATc3.
Assuntos
Neoplasias Encefálicas/patologia , Calcineurina/metabolismo , Glioma/patologia , Fatores de Transcrição NFATC/metabolismo , Oligopeptídeos/farmacologia , Transdução de Sinais , Sequência de Aminoácidos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Neoplasias Encefálicas/genética , Linhagem Celular Tumoral , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Peptídeos Penetradores de Células/farmacologia , Glioma/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Fatores de Transcrição NFATC/química , Oligopeptídeos/química , Peptídeos/farmacologia , Transporte Proteico/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacosRESUMO
BACKGROUND: Malignant tumours release factors, which attract myeloid cells and induce their polarisation to pro-invasive, immunosuppressive phenotypes. Brain-resident microglia and peripheral macrophages accumulate in the tumour microenvironment of glioblastoma (GBM) and induce immunosuppression fostering tumour progression. Macrophage colony stimulating factors (CSFs) control the recruitment of myeloid cells during peripheral cancer progression, but it is disputable, which CSFs drive their accumulation in gliomas. METHODS: The expression of CSF2 (encoding granulocyte-macrophage colony stimulating factor) was determined in TCGA datasets and five human glioma cell lines. Effects of stable CSF2 knockdown in glioma cells or neutralising CSF2 or receptor CSF2Rα antibodies on glioma invasion were tested in vitro and in vivo. RESULTS: CSF2 knockdown or blockade of its signalling reduced microglia-dependent glioma invasion in microglia-glioma co-cultures. CSF2-deficient human glioma cells encapsulated in cell-impermeable hollow fibres and transplanted to mouse brains, failed to attract microglia, but stimulated astrocyte recruitment. CSF2-depleted gliomas were smaller, attracted less microglia and macrophages, and provided survival benefit in tumour-bearing mice. Apoptotic microglia/macrophages were detected in CSF2-depleted tumours. CONCLUSIONS: CSF2 is overexpressed in a subset of mesenchymal GBMs in association with high immune gene expression. Tumour-derived CSF2 attracts, supports survival and induces pro-tumorigenic polarisation of microglia and macrophages.
Assuntos
Neoplasias Encefálicas/patologia , Glioma/patologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Células Mieloides/patologia , Animais , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Linhagem Celular Tumoral , Técnicas de Cocultura , Bases de Dados Genéticas , Progressão da Doença , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Glioma/genética , Glioma/metabolismo , Humanos , Células Jurkat , Masculino , Camundongos , Células Mieloides/metabolismo , Invasividade Neoplásica , Transplante de Neoplasias , Regulação para CimaRESUMO
Cannabinoids are a group of structurally heterogeneous but pharmacologically related compounds, including plant-derived cannabinoids, synthetic substances and endogenous cannabinoids, such as anandamide and 2-arachidonoylglycerol. Cannabinoids elicit a wide range of central and peripheral effects mostly mediated through cannabinoid receptors. There are two types of specific Gi/o-protein-coupled receptors cloned so far, called CB1 and CB2, although an existence of additional cannabinoid-binding receptors has been suggested. CB1 and CB2 differ in their predicted amino acid sequence, tissue distribution, physiological role and signaling mechanisms. Significant alterations of a balance in the cannabinoid system between the levels of endogenous ligands and their receptors occur during malignant transformation in various types of cancer, including gliomas. Cannabinoids exert anti-proliferative action in tumor cells. Induction of cell death by cannabinoid treatment relies on the generation of a pro-apoptotic sphingolipid ceramide and disruption of signaling pathways crucial for regulation of cellular proliferation, differentiation or apoptosis. Increased ceramide levels lead also to ER-stress and autophagy in drug-treated glioblastoma cells. Beyond blocking of tumor cells proliferation cannabinoids inhibit invasiveness, angiogenesis and the stem cell-like properties of glioma cells, showing profound activity in the complex tumor microenvironment. Advances in translational research on cannabinoid signaling led to clinical investigations on the use of cannabinoids in treatments of glioblastomas.
Assuntos
Canabinoides/metabolismo , Glioma/metabolismo , Glioma/patologia , Transdução de Sinais , Animais , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Glioblastoma/metabolismo , Glioblastoma/patologia , Humanos , Receptores de Canabinoides/metabolismo , Microambiente TumoralRESUMO
Integrins are a large family of transmembrane adhesion receptors, which play a key role in interactions of a cell with the surrounding stroma. Integrins are comprised of non-covalently linked α and ß chains, which form heterodimeric receptor complexes. The signals from integrin receptors are combined with those originating from growth factor receptors and participate in orchestrating morphological changes of cells, organization of the cytoskeleton, stimulation of cell proliferation and rescuing cells from programmed cell death induced by extracellular matrix (ECM) detachment. Upon binding to specific ligands or ECM components, integrin dimers activate downstream signaling pathways, including focal adhesion kinase, phosphoinositide-3-kinase (PI3K) and AKT kinases, which regulate migration, invasion, proliferation and survival. Expression of specific integrins is upregulated in both tumor cells and stromal cells in a tumor microenvironment. Therefore, integrins became an attractive therapeutic target for many cancers, including the most common primary brain tumors-gliomas. In this review we provide an overview of the involvement of integrin signaling in glioma pathogenesis, formation of the tumor niche and brain tissue infiltration. We will summarize up-to-date therapeutic strategies for gliomas focused on interference with integrin ligand-receptor signaling.
Assuntos
Glioma/genética , Integrinas/metabolismo , Complexo Glicoproteico GPIb-IX de Plaquetas/metabolismo , Transdução de Sinais , Microambiente Tumoral , Encéfalo/metabolismo , Proliferação de Células , Matriz Extracelular/metabolismo , Glioma/metabolismo , Integrinas/genética , Ligantes , Complexo Glicoproteico GPIb-IX de Plaquetas/genéticaRESUMO
Growing evidence supports a critical role of the tumor-reprogrammed stromal cells in tumor growth and progression. Several extracellular communication networks are hijacked by the tumors to influence the surrounding tumor microenvironment. In malignant gliomas, tumor derived factors attract brain resident microglia and peripheral macrophages. These cells, instead of initiating antitumor responses, are re-educated by tumor cells and participate in matrix remodeling, support invasion and angiogenesis, and induce immunosuppression. Molecular underlining of these mutual and complex interactions in malignant gliomas is the main scope of this review.
Assuntos
Neoplasias Encefálicas/metabolismo , Glioma/metabolismo , Microambiente Tumoral , Neoplasias Encefálicas/imunologia , Neoplasias Encefálicas/patologia , Glioma/imunologia , Glioma/patologia , Humanos , Macrófagos/imunologia , Macrófagos/patologia , Microglia/imunologia , Microglia/patologiaRESUMO
Microglia are myeloid cells residing in the central nervous system that participate in inflammatory responses and could promote injury and repair. Gliomas attract microglia and polarize them into tumor-supporting cells that participate in matrix remodeling, invasion, angiogenesis, and suppression of adaptive immunity. Although signaling pathways and critical regulators underlying classical inflammation are well established, signal transduction and transcriptional circuits underlying the alternative activation of microglia are poorly known. Using primary rat microglial cultures exposed to glioma conditioned medium or lipopolysaccharide (LPS), we demonstrate that microglia adapt different fates and polarize into pro-inflammatory or alternatively activated cells. Glioma-derived factors increased cell motility, phagocytosis, and sustained proliferation of microglial cells that was mediated by enhanced focal adhesion kinase and PI-3K/Akt signaling. The signals from glioma cells induced ERK and p38 MAPK but not JNK signaling and failed to activate pro-inflammatory Stat1 and NFκB signaling in microglial cells. Transcriptome analysis of microglial cultures at 6 h after exposure to glioma-conditioned medium or LPS revealed different patterns of gene expression. Glioma-induced activation was associated with induction of genes coding for ID (inhibitor of DNA binding) 1/3 and c-Myc, markers of the alternative phenotype Arg1, MT1-MMP, CXCL14, and numerous cytokines/chemokines implicated in immune cell trafficking. Many classical inflammation-related genes and signaling pathways failed to be induced. Our study indicates for the first time molecular pathways that direct microglia toward the pro-invasive, immunosuppressive phenotype.
Assuntos
Carcinogênese/genética , Movimento Celular/fisiologia , Glioma/patologia , Microglia/fisiologia , Transdução de Sinais/fisiologia , Animais , Animais Recém-Nascidos , Carcinogênese/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proliferação de Células , Córtex Cerebral/citologia , Meios de Cultivo Condicionados/farmacologia , Citocinas/metabolismo , Inibidores Enzimáticos/farmacologia , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/genética , Lipopolissacarídeos/farmacologia , Análise de Sequência com Séries de Oligonucleotídeos , Fagocitose/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/metabolismo , Ratos , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos , Fator de Crescimento Transformador beta1/metabolismo , Quinase Induzida por NF-kappaBRESUMO
Cannabinoids are a group of structurally heterogeneous but pharmacologically related compounds, including plant-derived cannabinoids, synthetic substances and endogenous cannabinoids, such as anandamide and 2-arachidonoylglycerol. Cannabinoids elicit a wide range of central and peripheral effects mostly mediated through cannabinoid receptors. There are two types of specific G(i/o)-protein-coupled receptors cloned so far, called CB1 and CB2, although an existence of additional cannabinoid-binding receptors has been suggested. CB1 and CB2 differ in their predicted amino acid sequence, tissue distribution, physiological role and signaling mechanisms. Significant alterations of a balance in the cannabinoid system between the levels of endogenous ligands and their receptors occur during malignant transformation in various types of cancer, including gliomas. Cannabinoids exert anti-proliferative action in tumor cells. Induction of cell death by cannabinoid treatment relies on the generation of a pro-apoptotic sphingolipid ceramide and disruption of signaling pathways crucial for regulation of cellular proliferation, differentiation or apoptosis. Increased ceramide levels lead also to ER-stress and autophagy in drug-treated glioblastoma cells.
Assuntos
Neoplasias Encefálicas/metabolismo , Canabinoides/metabolismo , Glioma/metabolismo , Receptores de Canabinoides/metabolismo , Transdução de Sinais , Animais , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Glioma/tratamento farmacológico , Glioma/patologia , HumanosRESUMO
BACKGROUND: Glioblastoma (GBM, WHO grade IV) is an aggressive, primary brain tumor. Despite extensive tumor resection followed by radio- and chemotherapy, life expectancy of GBM patients did not improve over decades. Several studies reported transcription deregulation in GBMs, but regulatory mechanisms driving overexpression of GBM-specific genes remain largely unknown. Transcription in open chromatin regions is directed by transcription factors (TFs) that bind to specific motifs, recruit co-activators/repressors and the transcriptional machinery. Identification of GBM-related TFs-gene regulatory networks may reveal new and targetable mechanisms of gliomagenesis. RESULTS: We predicted TFs-regulated networks in GBMs in silico and intersected them with putative TF binding sites identified in the accessible chromatin in human glioma cells and GBM patient samples. The Cancer Genome Atlas and Glioma Atlas datasets (DNA methylation, H3K27 acetylation, transcriptomic profiles) were explored to elucidate TFs-gene regulatory networks and effects of the epigenetic background. In contrast to the majority of tumors, c-Jun expression was higher in GBMs than in normal brain and c-Jun binding sites were found in multiple genes overexpressed in GBMs, including VIM, FOSL2 or UPP1. Binding of c-Jun to the VIM gene promoter was stronger in GBM-derived cells than in cells derived from benign glioma as evidenced by gel shift and supershift assays. Regulatory regions of the majority of c-Jun targets have distinct DNA methylation patterns in GBMs as compared to benign gliomas, suggesting the contribution of DNA methylation to the c-Jun-dependent gene expression. CONCLUSIONS: GBM-specific TFs-gene networks identified in GBMs differ from regulatory pathways attributed to benign brain tumors and imply a decisive role of c-Jun in controlling genes that drive glioma growth and invasion as well as a modulatory role of DNA methylation.
Assuntos
Neoplasias Encefálicas , Glioblastoma , Glioma , Humanos , Neoplasias Encefálicas/genética , Cromatina/genética , Metilação de DNA , Epigênese Genética , Glioblastoma/genética , Proteínas Proto-Oncogênicas c-jun/metabolismoRESUMO
Glioblastomas (GBMs) are aggressive brain tumors with frequent genetic alterations in TP53 and PTEN tumor suppressor genes rendering resistance to standard chemotherapeutics. Cannabinoid type 1 and 2 (CB1/CB2) receptor expression in GBMs and antitumor activity of cannabinoids in glioma cells and animal models, raised promises for a targeted treatment of these tumors. The susceptibility of human glioma cells to CB2-agonists and their mechanism of action are not fully elucidated. We determined CB1 and CB2 expression in 14 low-grade and 21 high-grade tumor biopsies, GBM-derived primary cultures and established cell lines. The non-selective CB receptor agonist WIN55,212-2 (but not its inactive enantiomer) or the CB2-selective agonist JWH133 induced apoptosis in patient-derived glioma cultures and five established glioma cell lines despite p53 and/or PTEN deficiency. Growth inhibitory efficacy of cannabinoids correlated with CB1/CB2 expression (EC50 WIN55,212-2: 7.36-15.70 µM, JWH133: 12.15-143.20 µM). Treatment with WIN55,212-2 or JWH133 led to activation of the apoptotic mitochondrial pathway and DNA fragmentation. Synthetic cannabinoid action was associated with the induction of autophagy and knockdown of autophagy genes augmented cannabinoid-induced apoptotic cell death. The high susceptibility of human glioblastoma cells to synthetic cannabinoids, despite genetic defects contributing to apoptosis resistance, makes cannabinoids promising anti-glioma therapeutics.
RESUMO
Glioblastomas (GBM) are the common and aggressive primary brain tumors that are incurable by conventional therapies. Immunotherapy with immune checkpoint inhibitors is not effective in GBM patients due to the highly immunosuppressive tumor microenvironment (TME) restraining the infiltration and activation of cytotoxic T cells. Clinical and experimental studies showed the upregulation of expression of the arginase 1 and 2 (ARG1 and ARG2, respectively) in murine and human GBMs. The elevated arginase activity leads to the depletion of L-arginine, an amino-acid required for the proliferation of T lymphocytes and natural killer cells. Inhibition of ARG1/2 in the TME may unblock T cell proliferation and activate effective antitumor responses. To explore the antitumor potential of ARG1/2 inhibition, we analyzed bulk and single-cell RNA sequencing (scRNA-seq) data from human and murine gliomas. We found the upregulation of ARG1/2 expression in GBMs, both in tumor cells and in tumor infiltrating microglia and monocytes/macrophages. We employed selective arginase inhibitors to evaluate if ARG1/2 inhibition in vitro and in vivo exerts the antitumor effects. A novel, selective ARG1/2 inhibitor - OAT-1746 blocked microglia-dependent invasion of U87-MG and LN18 glioma cells in a Matrigel invasion assay better than reference compounds, without affecting the cell viability. OAT-1746 effectively crossed the blood brain barrier in mice and increased arginine levels in the brains of GL261 glioma bearing mice. We evaluated its antitumor efficacy against GL261 intracranial gliomas as a monotherapy and in combination with the PD-1 inhibition. The oral treatment with OAT-1746 did not affect the immune composition of TME, it induced profound transcriptomic changes in CD11b+ cells immunosorted from tumor-bearing brains as demonstrated by RNA sequencing analyses. Treatment with OAT-1746 modified the TME resulting in reduced glioma growth and increased antitumor effects of the anti-PD-1 antibody. Our findings provide the evidence that inhibition of ARG1/2 activity in tumor cells and myeloid cells in the TME unblocks antitumor responses in myeloid cells and NK cells, and improves the efficacy of the PD-1 inhibition.
RESUMO
Using siRNAs to genetically manipulate immune cells is important to both basic immunological studies and therapeutic applications. However, siRNA delivery is challenging because primary immune cells are often sensitive to the delivery materials and generate immune responses. We have recently developed an amphiphilic dendrimer that is able to deliver siRNA to a variety of cells, including primary immune cells. We provide here a protocol for the synthesis of this dendrimer, as well as siRNA delivery to immune cells such as primary T and B cells, natural killer cells, macrophages, and primary microglia. The dendrimer synthesis entails straightforward click coupling followed by an amidation reaction, and the siRNA delivery protocol requires simple mixing of the siRNA and dendrimer in buffer, with subsequent application to the primary immune cells to achieve effective and functional siRNA delivery. This dendrimer-mediated siRNA delivery largely outperforms the standard electroporation technique, opening a new avenue for functional and therapeutic studies of the immune system. The whole protocol encompasses the dendrimer synthesis, which takes 10 days; the primary immune cell preparation, which takes 3-10 d, depending on the tissue source and cell type; the dendrimer-mediated siRNA delivery; and subsequent functional assays, which take an additional 3-6 d.
Assuntos
Linfócitos B/metabolismo , Dendrímeros/química , Interferência de RNA , RNA Interferente Pequeno/administração & dosagem , Linfócitos T/metabolismo , Animais , Linhagem Celular , Células Cultivadas , Química Click , Dendrímeros/síntese química , Humanos , Camundongos Endogâmicos C57BL , RNA Interferente Pequeno/genéticaRESUMO
The Janus kinases (JAK) and signal transducer and activator of transcription (STAT) signaling are strongly activated in many tumors. STAT proteins are activated by phosphorylation at the tyrosine residue, then dimerize, translocate to the nucleus and bind DNA, initiating the transcription of target genes. Activation of JAK-STAT pathway is implicated in the regulation of cell growth, differentiation, survival and cross-talk between cancer and immune cells. The activation of STATs depends on phosphorylation on a single tyrosine residue (e.g., Tyr705 in STAT3 and Tyr694 in STAT5) in the C-terminal domain. Commercially available antibodies discriminate between total and specifically phosphorylated (active) forms of different STATs, which allows to measure directly STATs activation in crude cell extracts. Nuclear translocation and transcriptional activity of STATs can be measured in transfected cells using STAT dependent promoter driving reporter luciferase gene. STAT signaling pathway and STAT-dependent gene expression in cells can be specifically modulated using oligodeoxynucleotide (ODN) STAT decoy which is a double-stranded fragment of DNA containing an overlapping ISRE/GAS binding site.
Assuntos
Ensaio de Desvio de Mobilidade Eletroforética/métodos , Glioma/genética , Inflamação/genética , Fatores de Transcrição STAT/genética , Transdução de Sinais , Western Blotting , Humanos , Luciferases/metabolismo , Fosforilação , Células Tumorais CultivadasRESUMO
Aim: Alterations of microglia, the brain-resident macrophages, are associated with numerous brain pathologies. Genetic manipulation of microglia in diseases using small interfering RNA (siRNA) is hampered by the lack of safe and efficient siRNA delivery methods. We assessed the amphiphilic dendrimer (AD) for functional siRNA delivery and gene knockdown in primary microglia. Materials & methods: We characterized the ability of AD to form nanoparticles with siRNA, and studied their size, surface potential, cell uptake and gene silencing in rodent microglia. Results: AD effectively delivered siRNA to primary microglia and decreased target gene and protein expression, leading to transcriptomic changes without affecting basal microglial functions. Conclusion: The dendrimer AD promises to be an innocuous carrier for siRNA delivery into microglia.
Assuntos
Dendrímeros/química , Portadores de Fármacos/química , Interferência de RNA , RNA Interferente Pequeno/administração & dosagem , Tensoativos/química , Animais , Células Cultivadas , Camundongos Endogâmicos C57BL , Microglia/citologia , Microglia/metabolismo , RNA Interferente Pequeno/genética , Ratos WistarRESUMO
The efficacy of cannabinoids against high-grade glioma in animal models, mediated by two specific receptors, CB1 and CB2, raised promises for targeted treatment of the most frequent and malignant primary brain tumors. Unlike the abundantly expressed CB1, the CB2 receptor shows a restricted distribution in normal brain. Although brain tumors constitute the second most common malignancy in children and the prevalence of histological types of brain tumors vary significantly between the adult and pediatric populations, cannabinoid receptor expression in pediatric tumors remains unknown. In the present study, we compared the expression of the CB2 receptor in paraffin-embedded sections from primary brain tumors of adult and pediatric patients. Most glioblastomas expressed very high levels of CB2 receptors and the expression correlated with tumor grade. Interestingly, some benign pediatric astrocytic tumors, such as subependymal giant cell astrocytoma (SEGA), which may occasionally cause mortality owing to progressive growth, also displayed high CB2 immunoreactivity. The high levels of CB2 expression would predestine those tumors to be vulnerable to cannabinoid treatment. In contrast, all examined cases of embryonal tumors (medulloblastoma and S-PNET), the most frequently diagnosed malignant brain tumors in childhood, showed no or trace CB2 immunoreactivity. Our results suggest that the CB2 receptor expression depends primarily on the histopathological origin of the brain tumor cells and differentiation state, reflecting the tumor grade.
Assuntos
Neoplasias Encefálicas/metabolismo , Regulação Neoplásica da Expressão Gênica/fisiologia , Receptor CB2 de Canabinoide/metabolismo , Adolescente , Adulto , Fatores Etários , Astrocitoma/metabolismo , Neoplasias Encefálicas/classificação , Criança , Glioblastoma/metabolismo , Antígenos de Histocompatibilidade/metabolismo , Humanos , Imuno-Histoquímica , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodosRESUMO
Short peptides have many advantages, such as low molecular weight, selectivity for a specific target, organelles or cells with minimal toxicity. We describe properties of short peptides, which interfere with communication networks in tumor cells and within microenvironment of malignant gliomas, the most common brain tumors. We focus on ligand/receptor axes and intracellular signaling pathways critical for gliomagenesis that could be targeted with interfering peptides. We review structures and efficacy of organelle-specific and cell-penetrating peptides and describe diverse chemical modifications increasing proteolytic stability and protecting synthetic peptides against degradation. We report results of application of short peptides in glioma therapy clinical trials, their rises and falls. The most advanced examples of therapeutics such as short interfering peptides combined with cell-penetrating peptides that show good effectiveness in disease models are presented. It is foreseen that identification of peptides with better clinical properties may improve their success rates in clinical trials.
Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Neoplasias Encefálicas/tratamento farmacológico , Glioma/tratamento farmacológico , Peptídeos/farmacologia , Transdução de Sinais/efeitos dos fármacos , Antineoplásicos/síntese química , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Glioma/metabolismo , Glioma/patologia , Humanos , Peptídeos/síntese química , Peptídeos/químicaRESUMO
Cannabinoids were shown to induce apoptosis of glioma cells in vitro and tumor regression in vivo, but mechanisms of their antiproliferative action remain elusive. In the present studies, C6 cells were exposed to a synthetic cannabinoid, WIN 55,212-2, which produced down-regulation of the Akt and Erk signalling pathways prior to appearance of any sign of apoptosis. We hypothesized that cannabinoid-induced cell death may be mediated by a Bcl-2 family member--Bad, whose function is hampered by these kinases due to control of its phosphorylation state. Using Western blot analysis, we found that levels of phosphorylated Bad, but not total Bad protein, decreased under exposure to WIN 55,212-2. WIN 55,212-2 treatment further resulted in mitochondrial depolarization and activation of caspase cascade. Thus, we suggest that the increase of proapoptotic Bad activity is an important link between the inhibition of survival pathways and an onset of execution phase of cannabinoid-induced glioma cell death.
Assuntos
Apoptose/fisiologia , Canabinoides/farmacologia , Proteínas de Transporte/fisiologia , Sistema de Sinalização das MAP Quinases/fisiologia , Morfolinas/farmacologia , Naftalenos/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Animais , Apoptose/efeitos dos fármacos , Benzoxazinas , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Glioma , Cinética , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt , Ratos , Proteína de Morte Celular Associada a bclRESUMO
Human malignant gliomas are highly resistant to current therapeutic approaches. We previously demonstrated that cyclosporine A (CsA) induces an apoptotic cell death in rat C6 glioma cells. In the present study, we found the induction of growth arrest or cell death of human malignant glioma cells exposed to CsA. In studied glioma cells, an accumulation of p21Cip1/Waf1 protein, a cell cycle inhibitor, was observed following CsA treatment, even in the absence of functional p53 tumour suppressor. CsA induced a senescence-associated growth arrest, in U87-MG glioma cells with functional p53, while in U373 and T98G glioma cells with mutated p53, CsA treatment triggered cell death associated with alterations of cell morphology, cytoplasm vacuolation, and condensation of chromatin. In T98G cells this effect was completely abolished by simultaneous treatment with an inhibitor of protein synthesis, cycloheximide (CHX). Moreover, CsA-induced cell death was accompanied by activation of executory caspases followed by PARP cleavage. CsA treatment did not elevate fasL expression and had no effect on mitochondrial membrane potential. We conclude that CsA triggers either growth arrest or non-apoptotic, programmed cell death in human malignant glioma cells. Moreover, CsA employs mechanisms different to those in the action of radio- and chemotherapeutics, and operating even in cells resistant to conventional treatments. Thus, CsA or related drugs may be an effective novel strategy to treat drug-resistant gliomas or complement apoptosis-based therapies.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias Encefálicas/tratamento farmacológico , Ciclosporina/farmacologia , Glioma/tratamento farmacológico , Apoptose/fisiologia , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/fisiopatologia , Caspases/efeitos dos fármacos , Caspases/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Forma Celular/fisiologia , Senescência Celular/fisiologia , Cromatina/efeitos dos fármacos , Cromatina/metabolismo , Colágeno Tipo XI/efeitos dos fármacos , Colágeno Tipo XI/metabolismo , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Glioma/metabolismo , Glioma/fisiopatologia , Inibidores do Crescimento/farmacologia , Humanos , Imunossupressores/farmacologia , Inibidores da Síntese de Proteínas/farmacologia , Proteína Supressora de Tumor p53/metabolismo , Vacúolos/efeitos dos fármacos , Vacúolos/metabolismoRESUMO
UNLABELLED: Most neurological diseases are associated with chronic inflammation initiated by the activation of microglia, which produce cytotoxic and inflammatory factors. Signal transducers and activators of transcription (STATs) are potent regulators of gene expression but contribution of particular STAT to inflammatory gene expression and STAT-dependent transcriptional networks underlying brain inflammation need to be identified. In the present study, we investigated the genomic distribution of Stat binding sites and the role of Stats in the gene expression in lipopolysaccharide (LPS)-activated primary microglial cultures. Integration of chromatin immunoprecipitation-promoter microarray data and transcriptome data revealed novel Stat-target genes including Jmjd3, Ccl5, Ezr, Ifih1, Irf7, Uba7, and Pim1. While knockdown of individual Stat had little effect on the expression of tested genes, knockdown of both Stat1 and Stat3 inhibited the expression of Jmjd3 and inflammatory genes. Transcriptional regulation of Jmjd3 by Stat1 and Stat3 is a novel mechanism crucial for launching inflammatory responses in microglia. The effects of Jmjd3 on inflammatory gene expression were independent of its H3K27me3 demethylase activity. Forced expression of constitutively activated Stat1 and Stat3 induced the expression of Jmjd3, inflammation-related genes, and the production of pro-inflammatory cytokines as potently as lipopolysacharide. Gene set enrichment and gene function analysis revealed categories linked to the inflammatory response in LPS and Stat1C + Stat3C groups. We defined upstream pathways that activate STATs in response to LPS and demonstrated contribution of Tlr4 and Il-6 and interferon-γ signaling. Our findings define novel direct transcriptional targets of Stat1 and Stat3 and highlight their contribution to inflammatory gene expression. KEY MESSAGE: Combined analysis of genomic Stat occupancy and transcriptome revealed novel Stat target genes in LPS-induced microglia. Jmjd3 transcription factor is a novel transcriptional target of Stat1 and Stat3. Stat1 and Stat3 cooperate with Jmjd3 to induce the expression of pro-inflammatory genes. Constitutively active Stat1 and Stat3 fully mimic the LPS-induced upregulation of inflammatory genes and secretion of cytokines.