RESUMO
Brain is the major consumer of glucose in the human body, whose pattern of consumption changes through lifetime, decreasing during adolescence up to adulthood. This evidence leads to the hypothesis that, in cerebral developmental stages, glycolysis might be the driving force for the high-energy requirement. Furthermore, several studies claim that neurogenesis process is accompanied by a shift into mitochondrial oxidative metabolism. Herein, we discuss recent work about cell metabolism during neuronal differentiation process, in particular the mitochondrial role in cellular bioenergy dynamics.
Assuntos
Encéfalo/citologia , Encéfalo/metabolismo , Mitocôndrias/metabolismo , Neurogênese/fisiologia , Neurônios/metabolismo , Animais , Sobrevivência Celular/fisiologia , Glicólise/fisiologia , Humanos , Oxirredução , Estresse Oxidativo/fisiologiaRESUMO
The process of cell differentiation goes hand-in-hand with metabolic adaptations, which are needed to provide energy and new metabolites. Carbon monoxide (CO) is an endogenous cytoprotective molecule able to inhibit cell death and improve mitochondrial metabolism. Neuronal differentiation processes were studied using the NT2 cell line, which is derived from human testicular embryonic teratocarcinoma and differentiates into post-mitotic neurons upon retinoic acid treatment. CO-releasing molecule A1 (CORM-A1) was used do deliver CO into cell culture. CO treatment improved NT2 neuronal differentiation and yield, since there were more neurons and the total cell number increased following the differentiation process. CO supplementation enhanced the mitochondrial population in post-mitotic neurons derived from NT2 cells, as indicated by an increase in mitochondrial DNA. CO treatment during neuronal differentiation increased the extent of the classical metabolic change that occurs during neuronal differentiation, from glycolytic to more oxidative metabolism, by decreasing the ratio of lactate production and glucose consumption. The expression of pyruvate and lactate dehydrogenases was higher, indicating an augmented oxidative metabolism. Moreover, these findings were corroborated by an increased percentage of (13) C incorporation from [U-(13) C]glucose into the tricarboxylic acid cycle metabolites malate and citrate, and also glutamate and aspartate in CO-treated cells. Finally, under low levels of oxygen (5%), which enhances glycolytic metabolism, some of the enhancing effects of CO on mitochondria were not observed. In conclusion, our data show that CO improves neuronal and mitochondrial yield by stimulation of tricarboxylic acid cycle activity, and thus oxidative metabolism of NT2 cells during the process of neuronal differentiation. The process of cell differentiation is coupled with metabolic adaptations. Carbon monoxide (CO) is an endogenous cytoprotective gasotransmitter able to prevent cell death and improve mitochondrial metabolism. Herein CO supplementation improved neuronal differentiation yield, by enhancing mitochondrial population and promoting the classical metabolic change that occurs during neuronal differentiation, from glycolytic to oxidative metabolism.
Assuntos
Monóxido de Carbono/farmacologia , Diferenciação Celular/efeitos dos fármacos , DNA Mitocondrial/metabolismo , Mitocôndrias/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Monóxido de Carbono/metabolismo , Linhagem Celular , Ciclo do Ácido Cítrico/efeitos dos fármacos , Humanos , Mitocôndrias/metabolismo , Neurônios/metabolismo , Tretinoína/farmacologiaRESUMO
BACKGROUND: Klebsiella pneumoniae strains are pathogenic to animals and humans, in which they are both a frequent cause of nosocomial infections and a re-emerging cause of severe community-acquired infections. K. pneumoniae isolates of the capsular serotype K2 are among the most virulent. In order to identify novel putative virulence factors that may account for the severity of K2 infections, the genome sequence of the K2 reference strain Kp52.145 was determined and compared to two K1 and K2 strains of low virulence and to the reference strains MGH 78578 and NTUH-K2044. RESULTS: In addition to diverse functions related to host colonization and virulence encoded in genomic regions common to the four strains, four genomic islands specific for Kp52.145 were identified. These regions encoded genes for the synthesis of colibactin toxin, a putative cytotoxin outer membrane protein, secretion systems, nucleases and eukaryotic-like proteins. In addition, an insertion within a type VI secretion system locus included sel1 domain containing proteins and a phospholipase D family protein (PLD1). The pld1 mutant was avirulent in a pneumonia model in mouse. The pld1 mRNA was expressed in vivo and the pld1 gene was associated with K. pneumoniae isolates from severe infections. Analysis of lipid composition of a defective E. coli strain complemented with pld1 suggests an involvement of PLD1 in cardiolipin metabolism. CONCLUSIONS: Determination of the complete genome of the K2 reference strain identified several genomic islands comprising putative elements of pathogenicity. The role of PLD1 in pathogenesis was demonstrated for the first time and suggests that lipid metabolism is a novel virulence mechanism of K. pneumoniae.
Assuntos
Genoma Bacteriano/genética , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/patogenicidade , Família Multigênica , Fosfolipase D/genética , Fatores de Virulência/genética , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sistemas de Secreção Bacterianos/genética , Genes Bacterianos/genética , Ilhas Genômicas/genética , Klebsiella pneumoniae/isolamento & purificação , Metabolismo dos Lipídeos/genética , Camundongos , Anotação de Sequência Molecular , Dados de Sequência Molecular , Mutagênese Insercional/genética , Fosfolipase D/química , Plasmídeos/genética , Alinhamento de Sequência , Virulência/genéticaRESUMO
Modulation of cerebral cell metabolism for improving the outcome of hypoxia-ischemia and reperfusion is a strategy yet to be explored. Because carbon monoxide (CO) is known to prevent cerebral cell death; herein the role of CO in the modulation of astrocytic metabolism, in particular, at the level of mitochondria was investigated. Low concentrations of CO partially inhibited oxidative stress-induced apoptosis in astrocytes, by preventing caspase-3 activation, mitochondrial potential depolarization, and plasmatic membrane permeability. CO exposure enhanced intracellular ATP generation, which was accompanied by an increase on specific oxygen consumption, a decrease on lactate production, and a reduction of glucose use, indicating an improvement of oxidative phosphorylation. Accordingly, CO increased cytochrome c oxidase (COX) enzymatic specific activity and stimulated mitochondrial biogenesis. In astrocytes, COX interacts with Bcl-2, which was verified by immunoprecipitation; this interaction is superior after 24 h of CO treatment. Furthermore, CO enhanced Bcl-2 expression in astrocytes. By silencing Bcl-2 expression with siRNA transfection, CO effects in astrocytes were prevented, namely: (i) inhibition of apoptosis, (ii) increase on ATP generation, (iii) stimulation of COX activity, and (iv) mitochondrial biogenesis. Thus, Bcl-2 expression is crucial for CO modulation of oxidative metabolism and for conferring cytoprotection. In conclusion, CO protects astrocytes against oxidative stress-induced apoptosis by improving metabolism functioning, particularly mitochondrial oxidative phosphorylation.
Assuntos
Apoptose/efeitos dos fármacos , Astrócitos/citologia , Astrócitos/metabolismo , Monóxido de Carbono/farmacologia , Citoproteção/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Astrócitos/efeitos dos fármacos , Hipóxia Celular/efeitos dos fármacos , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Oxirredução/efeitos dos fármacos , Fosforilação Oxidativa/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/genética , Ratos , Traumatismo por Reperfusão/patologiaRESUMO
Immune checkpoint inhibitors are monoclonal antibodies that are used to treat over one in three cancer patients. While they have changed the natural history of disease, prolonging life and preserving quality of life, they are highly active in less than 40% of patients, even in the most responsive malignancies such as melanoma, and cause significant autoimmune side effects. Licenced biomarkers include tumour Programmed Death Ligand 1 expression by immunohistochemistry, microsatellite instability, and tumour mutational burden, none of which are particularly sensitive or specific. Emerging tumour and immune tissue biomarkers such as novel immunohistochemistry scores, tumour, stromal and immune cell gene expression profiling, and liquid biomarkers such as systemic inflammatory markers, kynurenine/tryptophan ratio, circulating immune cells, cytokines and DNA are discussed in this review. We also examine the influence of the faecal microbiome on treatment outcome and its use as a biomarker of response and toxicity.
RESUMO
BACKGROUND AND AIMS: Mutations in DNA mismatch repair (MMR) genes are causative in Lynch syndrome and a significant proportion of sporadic colorectal cancers (CRCs). MMR-deficient (dMMR) CRCs display increased mutation rates, with mutations frequently accumulating at short repetitive DNA sequences throughout the genome (microsatellite instability). The TGFBR2 gene is one of the most frequently mutated genes in dMMR CRCs. Therefore, we generated an animal model to study how the loss of both TGFBR2 signaling impacts dMMR-driven intestinal tumorigenesis in vivo and explore the impact of the gut microbiota. METHODS: We generated VCMsh2/Tgfbr2 mice in which Msh2loxP and Tgfbr2loxP alleles are inactivated by Villin-Cre recombinase in the intestinal epithelium. VCMsh2/Tgfbr2 mice were analyzed for their rate of intestinal cancer development and for the mutational spectra and gene expression profiles of tumors. In addition, we assessed the impact of chemically induced chronic inflammation and gut microbiota composition on colorectal tumorigenesis. RESULTS: VCMsh2/Tgfbr2 mice developed small intestinal adenocarcinomas and CRCs with histopathological features highly similar to CRCs in Lynch syndrome patients. The CRCs in VCMsh2/Tgfbr2 mice were associated with the presence of colitis and displayed genetic and histological features that resembled inflammation-associated CRCs in human patients. The development of CRCs in VCMsh2/Tgfbr2 mice was strongly modulated by the gut microbiota composition, which in turn was impacted by the TGFBR2 status of the tumors. CONCLUSIONS: Our results demonstrate a synergistic interaction between MMR and TGFBR2 inactivation in inflammation-associated colon tumorigenesis and highlight the crucial impact of the gut microbiota on modulating the incidence of inflammation-associated CRCs.
Assuntos
Neoplasias do Colo , Neoplasias Colorretais Hereditárias sem Polipose , Microbiota , Animais , Carcinogênese/genética , Neoplasias do Colo/genética , Neoplasias Colorretais Hereditárias sem Polipose/genética , Neoplasias Colorretais Hereditárias sem Polipose/metabolismo , Neoplasias Colorretais Hereditárias sem Polipose/patologia , Reparo de Erro de Pareamento de DNA , Humanos , Inflamação , Camundongos , Receptor do Fator de Crescimento Transformador beta Tipo II/genética , Receptor do Fator de Crescimento Transformador beta Tipo II/metabolismoRESUMO
The present work demonstrates the ability of CO to prevent apoptosis in a primary culture of astrocytes. For the first time, the antiapoptotic behavior can be clearly attributed to the inhibition of mitochondrial membrane permeabilization (MMP), a key event in the intrinsic apoptotic pathway. In isolated non-synaptic mitochondria, CO partially inhibits (i) loss of potential, (ii) the opening of a nonspecific pore through the inner membrane, (iii) swelling, and (iv) cytochrome c release, which are induced by calcium, diamide, or atractyloside (a ligand of ANT). CO directly modulates ANT function by enhancing ADP/ATP exchange and prevents its pore-forming activity. Additionally, CO induces reactive oxygen species (ROS) generation, and its prevention by beta-carotene decreases CO cytoprotection in intact cells as well as in isolated mitochondria, revealing the key role of ROS. On the other hand, CO induces a slight increase in mitochondrial oxidized glutathione, which is essential for apoptosis modulation by (i) delaying astrocytic apoptosis, (ii) decreasing MMP, and (iii) enhancing ADP/ATP translocation activity of ANT. Moreover, CO and GSSG trigger ANT glutathionylation, a post-translational process regulating protein function in response to redox cellular changes. In conclusion, CO protects astrocytes from apoptosis by preventing MMP, acting on ANT (glutathionylation and inhibition of its pore activity) via a preconditioning-like process mediated by ROS and GSSG.
Assuntos
Apoptose , Monóxido de Carbono/química , Glutationa/metabolismo , Mitocôndrias/metabolismo , Translocases Mitocondriais de ADP e ATP/metabolismo , Membranas Mitocondriais/metabolismo , Animais , Astrócitos/citologia , Encéfalo/metabolismo , Citocromos c/metabolismo , Masculino , Potenciais da Membrana , Oxirredução , Ratos , Ratos Wistar , Espécies Reativas de OxigênioRESUMO
BACKGROUND: Low concentrations of carbon monoxide (CO) protect hepatocytes against apoptosis and confers cytoprotection in several models of liver. Mitochondria are key organelles in cell death control via their membrane permeabilization and the release of pro-apoptotic factors. RESULTS: Herein, we show that CO prevents mitochondrial membrane permeabilization (MMP) in liver isolated mitochondria. Direct and indirect approaches were used to evaluate MMP inhibition by CO: mitochondrial swelling, mitochondrial depolarization and inner membrane permeabilization. Additionally, CO increases mitochondrial reactive oxygen species (ROS) generation, and their scavenging, by ß-carotene addition, decreases CO protection, which reveals the key role of ROS. Interestingly, cytochrome c oxidase transiently responds to low concentrations of CO by decreasing its activity in the first 5 min, later on there is an increase of cytochrome c oxidase activity, which were detected up to 30 min. CONCLUSION: CO directly prevents mitochondrial membrane permeabilization, which might be implicated in the hepatic apoptosis inhibition by this gaseoustransmitter.
Assuntos
Monóxido de Carbono/farmacologia , Citoproteção/efeitos dos fármacos , Mitocôndrias Hepáticas , Membranas Mitocondriais , Animais , Apoptose/efeitos dos fármacos , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Feminino , Hepatócitos/citologia , Hepatócitos/metabolismo , Fígado/citologia , Fígado/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias Hepáticas/efeitos dos fármacos , Mitocôndrias Hepáticas/metabolismo , Membranas Mitocondriais/efeitos dos fármacos , Membranas Mitocondriais/metabolismo , Dilatação Mitocondrial/efeitos dos fármacos , Permeabilidade/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , beta Caroteno/metabolismoRESUMO
Flow cytometry is a popular laser-based technology that allows the phenotypic and functional characterization of individual cells in a high-throughput manner. Here, we describe a detailed procedure for preparing a single-cell suspension from mammary tumors of the mouse mammary tumor virus-polyoma middle T (MMTV-PyMT) and analyzing these cells by multi-color flow cytometry. This protocol can be used to study the following tumor-infiltrating immune cell populations, defined by the expression of cell surface molecules: total leukocytes, tumor-associated macrophages (TAMs), conventional dendritic cells (DCs), CD103-expressing DCs, tumor-associated neutrophils, inflammatory monocytes, natural killer (NK) cells, CD4+ T cells, CD8+ T cells, γδT cells, and regulatory T cells.
RESUMO
Protein glutathionylation is a posttranslational process that regulates protein function in response to redox cellular changes. Furthermore, carbon monoxide-induced cellular pathways involve reactive oxygen species (ROS) signaling and mitochondrial protein glutathionylation. Herein, it is described as a technique to assess mitochondrial glutathionylation due to low concentrations of CO exposure. Mitochondria are isolated from cell culture or tissue, followed by an immunoprecipitation assay, which allows the capture of any glutathionylated mitochondrial protein using a specific antibody coupled to a solid matrix that binds to glutathione antigen. The precipitated protein is further identified and quantified by immunoblotting analysis.
Assuntos
Encéfalo/metabolismo , Monóxido de Carbono/metabolismo , Glutationa/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Processamento de Proteína Pós-Traducional , Animais , Immunoblotting/métodos , Masculino , Proteínas Mitocondriais/química , Estresse Oxidativo/fisiologia , Ratos , Transdução de SinaisRESUMO
C-C chemokine receptor type 2 (CCR2) is expressed on monocytes and facilitates their recruitment to tumors. Though breast cancer cells also express CCR2, its functions in these cells are unclear. We found that Ccr2 deletion in cancer cells led to reduced tumor growth and approximately twofold longer survival in an orthotopic, isograft breast cancer mouse model. Deletion of Ccr2 in cancer cells resulted in multiple alterations associated with better immune control: increased infiltration and activation of cytotoxic T lymphocytes (CTLs) and CD103+ cross-presenting dendritic cells (DCs), as well as up-regulation of MHC class I and down-regulation of checkpoint regulator PD-L1 on the cancer cells. Pharmacological or genetic targeting of CCR2 increased cancer cell sensitivity to CTLs and enabled the cancer cells to induce DC maturation toward the CD103+ subtype. Consistently, Ccr2-/- cancer cells did not induce immune suppression in Batf3-/- mice lacking CD103+ DCs. Our results establish that CCR2 signaling in cancer cells can orchestrate suppression of the immune response.
Assuntos
Imunidade Adaptativa/imunologia , Tolerância Imunológica , Neoplasias Mamárias Experimentais/imunologia , Receptores CCR2/fisiologia , Imunidade Adaptativa/fisiologia , Animais , Apoptose , Antígeno B7-H1/metabolismo , Células Dendríticas/imunologia , Células Dendríticas/fisiologia , Feminino , Antígenos de Histocompatibilidade Classe I/metabolismo , Tolerância Imunológica/imunologia , Tolerância Imunológica/fisiologia , Interferons/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Receptores CCR2/imunologia , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/fisiologiaRESUMO
Over the last decades, the silent-killer carbon monoxide (CO) has been shown to also be an endogenous cytoprotective molecule able to inhibit cell death and modulate mitochondrial metabolism. Neuronal metabolism is mostly oxidative and neurons also use glucose for maintaining their anti-oxidant status by generation of reduced glutathione (GSH) via the pentose-phosphate pathway (PPP). It is established that neuronal differentiation depends on reactive oxygen species (ROS) generation and signalling, however there is a lack of information about modulation of the PPP during adult neurogenesis. Thus, the main goal of this study was to unravel the role of CO on cell metabolism during neuronal differentiation, particularly by targeting PPP flux and GSH levels as anti-oxidant system. A human neuroblastoma SH-S5Y5 cell line was used, which differentiates into post-mitotic neurons by treatment with retinoic acid (RA), supplemented or not with CO-releasing molecule-A1 (CORM-A1). SH-SY5Y cell differentiation supplemented with CORM-A1 prompted an increase in neuronal yield production. It did, however, not alter glycolytic metabolism, but increased the PPP. In fact, CORM-A1 treatment stimulated (i) mRNA expression of 6-phosphogluconate dehydrogenase (PGDH) and transketolase (TKT), which are enzymes for oxidative and non-oxidative phases of the PPP, respectively and (ii) protein expression and activity of glucose 6-phosphate dehydrogenase (G6PD) the rate-limiting enzyme of the PPP. Likewise, whenever G6PD was knocked-down CO-induced improvement on neuronal differentiation was reverted, while pharmacological inhibition of GSH synthesis did not change CO's effect on the improvement of neuronal differentiation. Both results indicate the key role of PPP in CO-modulation of neuronal differentiation. Furthermore, at the end of SH-SY5Y neuronal differentiation process, CORM-A1 supplementation increased the ratio of reduced and oxidized glutathione (GSH/GSSG) without alteration of GSH metabolism. These data corroborate with PPP stimulation. In conclusion, CO improves neuronal differentiation of SH-S5Y5 cells by stimulating the PPP and modulating the GSH system.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Mitocôndrias/metabolismo , Neurônios/metabolismo , Via de Pentose Fosfato/efeitos dos fármacos , Monóxido de Carbono/farmacologia , Glucose/metabolismo , Glucosefosfato Desidrogenase/genética , Glucosefosfato Desidrogenase/metabolismo , Glutationa/metabolismo , Dissulfeto de Glutationa/metabolismo , Glicólise/efeitos dos fármacos , Humanos , Mitocôndrias/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Tretinoína/farmacologiaRESUMO
Rhinoscleroma is a human specific chronic granulomatous infection of the nose and upper airways caused by the Gram-negative bacterium Klebsiella pneumoniae subsp. rhinoscleromatis. Although considered a rare disease, it is endemic in low-income countries where hygienic conditions are poor. A hallmark of this pathology is the appearance of atypical foamy monocytes called Mikulicz cells. However, the pathogenesis of rhinoscleroma remains poorly investigated. Capsule polysaccharide (CPS) is a prominent virulence factor in bacteria. All K. rhinoscleromatis strains are of K3 serotype, suggesting that CPS can be an important driver of rhinoscleroma disease. In this study, we describe the creation of the first mutant of K. rhinoscleromatis, inactivated in its capsule export machinery. Using a murine model recapitulating the formation of Mikulicz cells in lungs, we observed that a K. rhinoscleromatis CPS mutant (KR cps-) is strongly attenuated and that mice infected with a high dose of KR cps- are still able to induce Mikulicz cells formation, unlike a K. pneumoniae capsule mutant, and to partially recapitulate the characteristic strong production of IL-10. Altogether, the results of this study show that CPS is a virulence factor of K. rhinoscleromatis not involved in the specific appearance of Mikulicz cells.
Assuntos
Cápsulas Bacterianas/metabolismo , Klebsiella pneumoniae/patogenicidade , Monócitos/imunologia , Rinoscleroma/fisiopatologia , Fatores de Virulência/metabolismo , Animais , Cápsulas Bacterianas/genética , Modelos Animais de Doenças , Deleção de Genes , Klebsiella pneumoniae/genética , Camundongos , Fatores de Virulência/genéticaRESUMO
Microglia fulfill important immunological functions in the brain by responding to pathological stresses and modulating their activities according to pro- or anti-inflammatory stimuli. Recent evidence indicates that changes in metabolism accompany the switch in microglia activation state, favoring glycolysis over oxidative phosphorylation when cells exhibit a pro-inflammatory phenotype. Carbon monoxide (CO), a byproduct of heme breakdown by heme oxygenase, exerts anti-inflammatory action and affects mitochondrial function in cells and tissues. In the present study, we analyzed the metabolic profile of BV2 and primary mouse microglia exposed to the CO-releasing molecules CORM-401 and CORM-A1 and investigated whether CO affects the metabolic adaptation of cells to the inflammatory stimulus lipopolysaccharide (LPS). Microglia respiration and glycolysis were measured using an Extracellular Flux Analyzer to provide a real-time bioenergetic assessment, and biochemical parameters were evaluated to define the metabolic status of the cells under normal or inflammatory conditions. We show that CO prevents LPS-induced depression of microglia respiration and reduction in ATP levels while altering the early expression of inflammatory markers, suggesting the metabolic changes induced by CO are associated with control of inflammation. CO alone affects microglia respiration depending on the concentration, as low levels increase oxygen consumption while higher amounts inhibit respiration. Increased oxygen consumption was attributed to an uncoupling activity observed in cells, at the molecular level (respiratory complex activities) and during challenge with LPS. Thus, application of CO is a potential countermeasure to reverse the metabolic changes that occur during microglia inflammation and in turn modulate their inflammatory profile.
Assuntos
Monóxido de Carbono/metabolismo , Inflamação/metabolismo , Microglia/metabolismo , Mitocôndrias/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Linhagem Celular , Glicólise , Heme Oxigenase-1/metabolismo , Inflamação/induzido quimicamente , Inflamação/patologia , Lipopolissacarídeos/toxicidade , Camundongos , Microglia/patologia , Mitocôndrias/patologia , Fosforilação Oxidativa , Consumo de Oxigênio , Piroptose/genética , RespiraçãoRESUMO
BACKGROUND: Activated protein C/endothelial protein C receptor (APC/EPCR) axis is physiologically involved in anticoagulant and cytoprotective activities in endothelial cells. Emerging evidence indicates that EPCR also plays a role in breast stemness and human tumorigenesis. Yet, its contribution to breast cancer progression and metastasis has not been elucidated. METHODS: Transcriptomic status of EPCR was examined in a cohort of 286 breast cancer patients. Cell growth kinetics was evaluated in control and EPCR and SPARC/osteonectin, Cwcv, and kazal-like domains proteoglycan (SPOCK1/testican 1) silenced breast cancer cells in 2D, 3D, and in co-culture conditions. Orthotopic tumor growth and lung and osseous metastases were evaluated in several human and murine xenograft breast cancer models. Tumor-stroma interactions were further studied in vivo by immunohistochemistry and flow cytometry. An EPCR-induced gene signature was identified by microarray analysis. RESULTS: Analysis of a cohort of breast cancer patients revealed an association of high EPCR levels with adverse clinical outcome. Interestingly, EPCR knockdown did not affect cell growth kinetics in 2D but significantly reduced cell growth in 3D cultures. Using several human and murine xenograft breast cancer models, we showed that EPCR silencing reduced primary tumor growth and secondary outgrowths at metastatic sites, including the skeleton and the lungs. Interestingly, these effects were independent of APC ligand stimulation in vitro and in vivo. Transcriptomic analysis of EPCR-silenced tumors unveiled an effect mediated by matricellular secreted proteoglycan SPOCK1/testican 1. Interestingly, SPOCK1 silencing suppressed in vitro 3D growth. Moreover, SPOCK1 ablation severely decreased orthotopic tumor growth and reduced bone metastatic osteolytic tumors. High SPOCK1 levels were also associated with poor clinical outcome in a subset breast cancer patients. Our results suggest that EPCR through SPOCK1 confers a cell growth advantage in 3D promoting breast tumorigenesis and metastasis. CONCLUSIONS: EPCR represents a clinically relevant factor associated with poor outcome and a novel vulnerability to develop combination therapies for breast cancer patients.
Assuntos
Neoplasias da Mama/patologia , Carcinoma/secundário , Receptor de Proteína C Endotelial/fisiologia , Proteínas de Neoplasias/fisiologia , Proteoglicanas/fisiologia , Animais , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/secundário , Neoplasias da Mama/metabolismo , Carcinoma/metabolismo , Técnicas de Cultura de Células , Ciclo Celular , Divisão Celular , Linhagem Celular Tumoral , Técnicas de Cocultura , Progressão da Doença , Receptor de Proteína C Endotelial/antagonistas & inibidores , Receptor de Proteína C Endotelial/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Xenoenxertos , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundário , Camundongos , Camundongos Nus , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Transplante de Neoplasias , Organismos Livres de Patógenos Específicos , Transcriptoma , Microambiente TumoralRESUMO
Pancreatic stellate cells (PSCs) differentiate into cancer-associated fibroblasts (CAFs) that produce desmoplastic stroma, thereby modulating disease progression and therapeutic response in pancreatic ductal adenocarcinoma (PDA). However, it is unknown whether CAFs uniformly carry out these tasks or if subtypes of CAFs with distinct phenotypes in PDA exist. We identified a CAF subpopulation with elevated expression of α-smooth muscle actin (αSMA) located immediately adjacent to neoplastic cells in mouse and human PDA tissue. We recapitulated this finding in co-cultures of murine PSCs and PDA organoids, and demonstrated that organoid-activated CAFs produced desmoplastic stroma. The co-cultures showed cooperative interactions and revealed another distinct subpopulation of CAFs, located more distantly from neoplastic cells, which lacked elevated αSMA expression and instead secreted IL6 and additional inflammatory mediators. These findings were corroborated in mouse and human PDA tissue, providing direct evidence for CAF heterogeneity in PDA tumor biology with implications for disease etiology and therapeutic development.
Assuntos
Carcinoma Ductal Pancreático/patologia , Fibroblastos/fisiologia , Miofibroblastos/fisiologia , Neoplasias Pancreáticas/patologia , Actinas/análise , Animais , Carcinoma Ductal Pancreático/imunologia , Carcinoma Ductal Pancreático/metabolismo , Células Cultivadas , Citocinas/biossíntese , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias Pancreáticas/imunologia , Neoplasias Pancreáticas/metabolismo , Fator de Transcrição STAT3/metabolismoRESUMO
Cerebral ischemia and neurodegenerative diseases lead to impairment or death of neurons in the central nervous system. Stem cell based therapies are promising strategies currently under investigation. Carbon monoxide (CO) is an endogenous product of heme degradation by heme oxygenase (HO) activity. Administration of CO at low concentrations produces several beneficial effects in distinct tissues, namely anti-apoptotic and anti-inflammatory. Herein the CO role on modulation of neuronal differentiation was assessed. Three different models with increasing complexity were used: human neuroblastoma SH-S5Y5 cell line, human teratocarcinoma NT2 cell line and organotypic hippocampal slice cultures (OHSC). Cell lines were differentiated into post-mitotic neurons by treatment with retinoic acid (RA) supplemented with CO-releasing molecule A1 (CORM-A1). CORM-A1 positively modulated neuronal differentiation, since it increased final neuronal production and enhanced the expression of specific neuronal genes: Nestin, Tuj1 and MAP2. Furthermore, during neuronal differentiation process, there was an increase in proliferative cell number (ki67 mRNA expressing cells) and a decrease in cell death (lower propidium iodide (PI) uptake, limitation of caspase-3 activation and higher Bcl-2 expressing cells). CO supplementation did not increase the expression of RA receptors. In the case of SH-S5Y5 model, small amounts of reactive oxygen species (ROS) generation emerges as important signaling molecules during CO-promoted neuronal differentiation. CO's improvement of neuronal differentiation yield was validated using OHSC as ex vivo model. CORM-A1 treatment of OHSC promoted higher levels of cells expressing the neuronal marker Tuj1. Still, CORM-A1 increased cell proliferation assessed by ki67 expression and also prevented cell death, which was followed by increased Bcl-2 expression, decreased levels of active caspase-3 and PI uptake. Likewise, ROS signaling emerged as key factors in CO's increasing number of differentiated neurons in OHSC. In conclusion, CO's increasing number of differentiated neurons is a novel biological role disclosed herein. CO improves neuronal yield due to its capacity to reduce cell death, promoting an increase in proliferative population. However, one cannot disregard a direct CO's effect on specific cellular processes of neuronal differentiation. Further studies are needed to evaluate how CO can potentially modulate cell mechanisms involved in neuronal differentiation. In summary, CO appears as a promising therapeutic molecule to stimulate endogenous neurogenesis or to improve in vitro neuronal production for cell therapy strategies.
Assuntos
Boranos/farmacologia , Carbonatos/farmacologia , Morte Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Neurogênese/efeitos dos fármacos , Animais , Caspase 3/metabolismo , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
Neutrophils, the most abundant type of leukocytes in blood, can form neutrophil extracellular traps (NETs). These are pathogen-trapping structures generated by expulsion of the neutrophil's DNA with associated proteolytic enzymes. NETs produced by infection can promote cancer metastasis. We show that metastatic breast cancer cells can induce neutrophils to form metastasis-supporting NETs in the absence of infection. Using intravital imaging, we observed NET-like structures around metastatic 4T1 cancer cells that had reached the lungs of mice. We also found NETs in clinical samples of triple-negative human breast cancer. The formation of NETs stimulated the invasion and migration of breast cancer cells in vitro. Inhibiting NET formation or digesting NETs with deoxyribonuclease I (DNase I) blocked these processes. Treatment with NET-digesting, DNase I-coated nanoparticles markedly reduced lung metastases in mice. Our data suggest that induction of NETs by cancer cells is a previously unidentified metastasis-promoting tumor-host interaction and a potential therapeutic target.
Assuntos
Armadilhas Extracelulares , Metástase Neoplásica , Neutrófilos/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Animais , Linhagem Celular Tumoral , Movimento Celular , Desoxirribonuclease I/química , Humanos , Pulmão/patologia , Neoplasias Pulmonares/secundário , Camundongos , Camundongos Endogâmicos BALB C , Nanopartículas/química , Neutrófilos/citologiaRESUMO
Protein glutathionylation is a posttranslational process that regulates protein function in response to redox cellular changes. Furthermore, carbon monoxide-induced cellular pathways involve reactive oxygen species (ROS) signaling and mitochondrial protein glutathionylation. Herein, it is described a technique to assess mitochondrial glutathionylation due to low concentrations of CO exposure. Mitochondria are isolated from cell culture or tissue, followed by an immunoprecipitation assay, which allows the capture of any glutathionylated mitochondrial protein using a specific antibody coupled to a solid matrix that binds to glutathione antigen. The precipitated protein is further identified and quantified by immunoblotting analysis.
Assuntos
Monóxido de Carbono/metabolismo , Glutationa/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Transdução de Sinais , Animais , Western Blotting , Fracionamento Celular/métodos , Córtex Cerebral/citologia , Córtex Cerebral/metabolismo , Humanos , Imunoprecipitação , Oxirredução , Espécies Reativas de OxigênioRESUMO
Carbon monoxide (CO) is an endogenously produced gasotransmitter, which is associated with cytoprotection and cellular homeostasis in several distinct cell types and tissues. CO mainly targets mitochondria because: (i) mitochondrial heme-proteins are the main potential candidates for CO to bind, (ii) many CO's biological actions are dependent on mitochondrial ROS signaling and (iii) heme is generated in the mitochondrial compartment. Mitochondria are the key cell energy factory, producing ATP through oxidative phosphorylation and regulating cell metabolism. These organelles are also implicated in many cell signaling pathways and the production of reactive oxygen species (ROS). Finally, mitochondria contain several factors activating programmed cell death pathways, which are released from the mitochondrial inter-membrane space upon mitochondrial membrane permeabilization. Therefore, disclosing CO mode of action at mitochondria opens avenues for deeper understanding CO's biological properties. Herein, it is discussed how CO affects the three main aspects of mitochondrial modulation of cell function: metabolism, redox response and cell death.