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Autophagy is an evolutionarily conserved biological process required for the turnover of the cytoplasm of eukaryotic cell. Beyond its catabolic nature, autophagy has a plethora of pro-survival functions, thus combatting hypoxia, nutrient shortage, and unfolded protein accumulation. Here, we introduce the naturally short-lived turquoise killifish Nothobranchius furzeri as an emerging model to study autophagic function in vivo, in response to environmental challenges. We show that starvation in killifish is sufficient to increase autophagic flux in the liver, thus enhancing the lipidation of microtubule-associated protein light chain 3 (LC3) and reducing the abundance of the autophagic substrate sequestosome-1 (SQSTM1). We describe an immunoblot-based comprehensive protocol to monitor fluctuations in autophagy in this model organism.
Assuntos
Fundulidae , Animais , AutofagiaRESUMO
Salicylate, the active derivative of aspirin (acetylsalicylate), recapitulates the mode of action of caloric restriction inasmuch as it stimulates autophagy through the inhibition of the acetyltransferase activity of EP300. Here, we directly compared the metabolic effects of aspirin medication with those elicited by 48 h fasting in mice, revealing convergent alterations in the plasma and the heart metabolome. Aspirin caused a transient reduction of general protein acetylation in blood leukocytes, accompanied by the induction of autophagy. However, these effects on global protein acetylation could not be attributed to the mere inhibition of EP300, as determined by epistatic experiments and exploration of the acetyl-proteome from salicylate-treated EP300-deficient cells. Aspirin reduced high-fat diet-induced obesity, diabetes, and hepatosteatosis. These aspirin effects were observed in autophagy-competent mice but not in two different models of genetic (Atg4b-/- or Bcln1+/-) autophagy-deficiency. Aspirin also improved tumor control by immunogenic chemotherapeutics, and this effect was lost in T cell-deficient mice, as well as upon knockdown of an essential autophagy gene (Atg5) in cancer cells. Hence, the health-improving effects of aspirin depend on autophagy.
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The pharmacological targeting of polyamine metabolism is currently under the spotlight for its potential in the prevention and treatment of several age-associated disorders. Here, we report the finding that triethylenetetramine dihydrochloride (TETA), a copper-chelator agent that can be safely administered to patients for the long-term treatment of Wilson disease, exerts therapeutic benefits in animals challenged with hypercaloric dietary regimens. TETA reduced obesity induced by high-fat diet, excessive sucrose intake, or leptin deficiency, as it reduced glucose intolerance and hepatosteatosis, but induced autophagy. Mechanistically, these effects did not involve the depletion of copper from plasma or internal organs. Rather, the TETA effects relied on the activation of an energy-consuming polyamine catabolism, secondary to the stabilization of spermidine/spermine N1-acetyltransferase-1 (SAT1) by TETA, resulting in enhanced enzymatic activity of SAT. All the positive effects of TETA on high-fat diet-induced metabolic syndrome were lost in SAT1-deficient mice. Altogether, these results suggest novel health-promoting effects of TETA that might be taken advantage of for the prevention or treatment of obesity.
Assuntos
Acetiltransferases/metabolismo , Quelantes/farmacologia , Obesidade/tratamento farmacológico , Trientina/análogos & derivados , Animais , Dieta Hiperlipídica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Obesidade/induzido quimicamenteRESUMO
The protein annexin A1 (ANXA1) belongs to the danger-associated molecular patterns (DAMPs) that alert the innate immune system about tissue perturbations. In the context of immunogenic cell death (ICD), ANXA1 is released from the cytoplasm of dying cells and, once extracellular, acts on formyl peptide receptor 1 (FPR1) expressed on dendritic cells to favor long-term interactions between dying and dendritic cells. As a result, the accumulation of extracellular ANXA1 constitutes one of the hallmarks of ICD. In the past, the detection of ANXA1 was based on semiquantitative immunoblots. More recently, a commercial enzyme-linked immunosorbent assay (ELISA) has been developed to measure ANXA1 in an accurate fashion. Here, we detail the protocol to measure the concentration of ANXA1 in the supernatants of cancer cells treated with chemotherapy.
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Anexina A1/análise , Ensaio de Imunoadsorção Enzimática/métodos , Morte Celular Imunogênica/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Anexina A1/imunologia , Anexina A1/metabolismo , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Citoplasma/efeitos dos fármacos , Citoplasma/imunologia , Citoplasma/metabolismo , Monitoramento de Medicamentos/instrumentação , Monitoramento de Medicamentos/métodos , Ensaio de Imunoadsorção Enzimática/instrumentação , Espaço Extracelular/imunologia , Espaço Extracelular/metabolismo , Humanos , Neoplasias/imunologia , Neoplasias/patologia , Kit de Reagentes para DiagnósticoRESUMO
The metabolite α-ketoglutarate is membrane-impermeable, meaning that it is usually added to cells in the form of esters such as dimethyl -ketoglutarate (DMKG), trifluoromethylbenzyl α-ketoglutarate (TFMKG) and octyl α-ketoglutarate (O-KG). Once these compounds cross the plasma membrane, they are hydrolyzed by esterases to generate α-ketoglutarate, which remains trapped within cells. Here, we systematically compared DMKG, TFMKG and O-KG for their metabolic and functional effects. All three compounds similarly increased the intracellular levels of α-ketoglutarate, yet each of them had multiple effects on other metabolites that were not shared among the three agents, as determined by mass spectrometric metabolomics. While all three compounds reduced autophagy induced by culture in nutrient-free conditions, TFMKG and O-KG (but not DMKG) caused an increase in baseline autophagy in cells cultured in complete medium. O-KG (but neither DMKG nor TFMK) inhibited oxidative phosphorylation and exhibited cellular toxicity. Altogether, these results support the idea that intracellular α-ketoglutarate inhibits starvation-induced autophagy and that it has no direct respiration-inhibitory effect.
Assuntos
Autofagia/efeitos dos fármacos , Ácidos Cetoglutáricos/metabolismo , Autofagia/fisiologia , Linhagem Celular Tumoral , Humanos , Ácidos Cetoglutáricos/farmacologia , Espectrometria de Massas , Fosforilação Oxidativa/efeitos dos fármacosRESUMO
Mouse cancers lacking the expression of annexin A1 (ANXA1) fail to respond to immunogenic chemotherapies. This has been initially explained by the requirement of extracellular ANXA1 (which is released from dying cancer cells) to engage formyl peptide receptor-1 (FPR1) on dendritic cells (DC) for the establishment of corpse/DC synapses. Here, we show that ANXA1-deficent cancer cells exhibit a defect in the exposure of calreticulin (CALR), which is an important "eat-me" signal, facilitating the phagocytic uptake of dead-cell antigens by DC. Of note, intratumoral injection of recombinant CALR protein was able to restore the therapeutic response of ANXA1-deficient cancers to anthracycline-based chemotherapy. Carcinomas developing in patients tend to downregulate ANXA1 expression as compared to their normal tissues of origin. ANXA1-low breast, colorectal, lung and kidney cancers are scarcely infiltrated by DC and cytotoxic T lymphocytes, supporting the idea that ANXA1 deficiency facilitates immune escape. We propose that such ANXA1-low cancers might be particularly suitable to local immunotherapy with CALR protein.
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Starvation is a strong physiological stimulus of macroautophagy/autophagy. In this study, we addressed the question as to whether it would be possible to measure autophagy in blood cells after nutrient deprivation. Fasting of mice for 48 h (which causes â¼20% weight loss) or starvation of human volunteers for up to 4 d (which causes <2% weight loss) provokes major changes in the plasma metabolome, yet induces only relatively minor alterations in the intracellular metabolome of circulating leukocytes. White blood cells from mice and human volunteers responded to fasting with a marked reduction in protein lysine acetylation, affecting both nuclear and cytoplasmic compartments. In circulating leukocytes from mice that underwent 48-h fasting, an increase in LC3B lipidation (as assessed by immunoblotting and immunofluorescence) only became detectable if the protease inhibitor leupeptin was injected 2 h before drawing blood. Consistently, measurement of an enhanced autophagic flux was only possible if white blood cells from starved human volunteers were cultured in the presence or absence of leupeptin. Whereas all murine leukocyte subpopulations significantly increased the number of LC3B+ puncta per cell in response to nutrient deprivation, only neutrophils from starved volunteers showed signs of activated autophagy (as determined by a combination of multi-color immunofluorescence, cytofluorometry and image analysis). Altogether, these results suggest that white blood cells are suitable for monitoring autophagic flux. In addition, we propose that the evaluation of protein acetylation in circulating leukocytes can be adopted as a biochemical marker of organismal energetic status.
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Jejum/sangue , Jejum/metabolismo , Acetilação , Adulto , Animais , Autofagia , Células Cultivadas , Feminino , Humanos , Lisina/metabolismo , Masculino , Metaboloma , Metabolômica , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Neutrófilos/metabolismo , Inanição/sangue , Inanição/metabolismo , Adulto JovemRESUMO
During the past decades, anticancer immunotherapy has evolved from a promising therapeutic option to a robust clinical reality. Many immunotherapeutic regimens are now approved by the US Food and Drug Administration and the European Medicines Agency for use in cancer patients, and many others are being investigated as standalone therapeutic interventions or combined with conventional treatments in clinical studies. Immunotherapies may be subdivided into "passive" and "active" based on their ability to engage the host immune system against cancer. Since the anticancer activity of most passive immunotherapeutics (including tumor-targeting monoclonal antibodies) also relies on the host immune system, this classification does not properly reflect the complexity of the drug-host-tumor interaction. Alternatively, anticancer immunotherapeutics can be classified according to their antigen specificity. While some immunotherapies specifically target one (or a few) defined tumor-associated antigen(s), others operate in a relatively non-specific manner and boost natural or therapy-elicited anticancer immune responses of unknown and often broad specificity. Here, we propose a critical, integrated classification of anticancer immunotherapies and discuss the clinical relevance of these approaches.
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Imunoterapia/métodos , Neoplasias/imunologia , Neoplasias/terapia , Animais , HumanosRESUMO
Immunogenic cell death (ICD) inducers can be defined as agents that exert cytotoxic effects while stimulating an immune response against dead cell-associated antigens. When initiated by anthracyclines, ICD is accompanied by stereotyped molecular changes, including the pre-apoptotic exposure of calreticulin (CRT) on the cell surface, the lysosomal secretion of ATP during the blebbing phase of apoptosis, and the release of high mobility group box 1 (HMGB1) from dead cells. By means of genetically engineered human osteosarcoma U2OS cells, we screened the 879 anticancer compounds of the National Cancer Institute (NCI) Mechanistic Diversity Set for their ability to promote all these hallmarks of ICD in vitro. In line with previous findings from our group, several cardiac glycosides exhibit a robust propensity to elicit the major manifestations of ICD in cultured neoplastic cells. This screen pointed to septacidin, an antibiotic produced by Streptomyces fibriatus, as a novel putative inducer of ICD. In low-throughput validation experiments, septacidin promoted CRT exposure, ATP secretion and HGMB1 release from both U2OS cells and murine fibrosarcoma MCA205 cells. Moreover, septacidin-killed MCA205 cells protected immunocompetent mice against a re-challenge with living cancer cells of the same type. Finally, the antineoplastic effects of septacidin on established murine tumors were entirely dependent on T lymphocytes. Altogether, these results underscore the suitability of the high-throughput screening system described here for the identification of novel ICD inducers.