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1.
Pharmacol Res ; 128: 167-178, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-28970178

RESUMO

Neuronal injury plays a major role in diabetic retinopathy (DR). Our hypothesis was that the balance between neuronal death and survival may depend on a similar equilibrium between apoptosis and autophagy and that a neuroprotectant may act by influencing this equilibrium. Ex vivo mouse retinal explants were treated with high glucose (HG) for 10days and the somatostatin analog octreotide (OCT) was used as a neuroprotectant. Chloroquine (CQ) was used as an autophagy inhibitor. Apoptotic and autophagic markers were evaluated using western blot and immunohistochemistry. HG-treated explants displayed a significant increase of apoptosis paralleled by a significant decrease of the autophagic flux, which was likely to be due to increased activity of the autophagy regulator mTOR (mammalian target of rapamycin). Treatment with OCT rescued HG-treated retinal explants from apoptosis and determined an increase of autophagic activity with concomitant mTOR inhibition. Blocking the autophagic flux with CQ completely abolished the anti-apoptotic effect of OCT. Immunohistochemical observations showed that OCT-induced autophagy is localized to populations of bipolar and amacrine cells and to ganglion cells. These observations revealed the antithetic role of apoptosis and autophagy, highlighting their equilibrium from which neuronal survival is likely to depend. These data suggest the crucial role covered by autophagy, which could be considered as a molecular target for DR neuroprotective treatment strategies.


Assuntos
Fármacos Neuroprotetores/farmacologia , Octreotida/farmacologia , Retina/efeitos dos fármacos , Animais , Autofagia/efeitos dos fármacos , Retinopatia Diabética , Feminino , Glucose/farmacologia , Masculino , Camundongos Endogâmicos C57BL , Neuroproteção
2.
Bioorg Med Chem ; 26(9): 2561-2572, 2018 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-29678535

RESUMO

Monoacylglycerol lipase (MAGL) has an essential role in the catabolic pathway of the endocannabinoid 2-arachidonoylglycerol, which makes it a potential target for highly specific inhibitors for the treatment of a number of diseases. We designed and synthesized a series of carbamate analogues of URB602. We evaluated their inhibitory activity toward human MAGL in vitro both in cell culture and lysates. The target compounds exhibited moderate to excellent inhibitory activity against MAGL. The most promising compound 2b showed good inhibitory activity with IC50 value of 4.5 ±â€¯0.70 µM reducing MAGL activity to 82% of controls at 10 µM compared to 66% for the parent compound URB602. Interestingly, compounds 2b and 2c induce cell death through the inhibition of MAGL. Molecular modelling approaches and docking studies, used to investigate inhibitory profiles, indicated that trifluoromethyl substitutions of the aryl group and the benzene ring present at the oxygen side of the carbamate molecule had a significant impact on the activity.


Assuntos
Antineoplásicos/farmacologia , Carbamatos/farmacologia , Inibidores Enzimáticos/farmacologia , Monoacilglicerol Lipases/antagonistas & inibidores , Amidoidrolases/antagonistas & inibidores , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Sítios de Ligação , Compostos de Bifenilo/síntese química , Compostos de Bifenilo/química , Compostos de Bifenilo/farmacologia , Carbamatos/síntese química , Carbamatos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ensaios Enzimáticos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Células HEK293 , Humanos , Camundongos , Simulação de Acoplamento Molecular , Estrutura Molecular , Monoacilglicerol Lipases/química , Relação Estrutura-Atividade
3.
Cells ; 10(11)2021 11 05.
Artigo em Inglês | MEDLINE | ID: mdl-34831250

RESUMO

Skeletal muscle regeneration is a complex process involving crosstalk between immune cells and myogenic precursor cells, i.e., satellite cells. In this scenario, macrophage recruitment in damaged muscles is a mandatory step for tissue repair since pro-inflammatory M1 macrophages promote the activation of satellite cells, stimulating their proliferation and then, after switching into anti-inflammatory M2 macrophages, they prompt satellite cells' differentiation into myotubes and resolve inflammation. Here, we show that acid sphingomyelinase (ASMase), a key enzyme in sphingolipid metabolism, is activated after skeletal muscle injury induced in vivo by the injection of cardiotoxin. ASMase ablation shortens the early phases of skeletal muscle regeneration without affecting satellite cell behavior. Of interest, ASMase regulates the balance between M1 and M2 macrophages in the injured muscles so that the absence of the enzyme reduces inflammation. The analysis of macrophage populations indicates that these events depend on the altered polarization of M1 macrophages towards an M2 phenotype. Our results unravel a novel role of ASMase in regulating immune response during muscle regeneration/repair and suggest ASMase as a supplemental therapeutic target in conditions of redundant inflammation that impairs muscle recovery.


Assuntos
Macrófagos/metabolismo , Macrófagos/patologia , Músculo Esquelético/fisiologia , Regeneração/fisiologia , Esfingomielina Fosfodiesterase/metabolismo , Animais , Diferenciação Celular , Polaridade Celular , Proliferação de Células , Ativação Enzimática , Inflamação/patologia , Camundongos Knockout , Músculo Esquelético/enzimologia , Músculo Esquelético/patologia , Fenótipo , Células Satélites de Músculo Esquelético/metabolismo , Transdução de Sinais , Esfingomielina Fosfodiesterase/deficiência
4.
Autophagy ; 15(1): 58-77, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30081710

RESUMO

Macroautophagy/autophagy is emerging as an important process in adult muscle stem cells functions: it regulates metabolic reprogramming during activation from a quiescent state, maintains stemness and prevents senescence. We now show that autophagy is specifically required for neonatal myogenesis and muscle development. Specific deletion of Atg7 in PAX7+ (paired box 7) precursors led in mice to a dwarf phenotype, with an effect restricted to the neonatal phase of muscle development. Atg7 knockdown suppressed neonatal satellite cell (nSC) proliferation and differentiation, downregulating the GH-IGF1 functions. When we disrupted autophagy, NFE2L2/NRF2 (nuclear factor, erythroid 2 like 2) accumulated in muscle and nSCs and negatively modulated DDIT3/CHOP (DNA-damage inducible transcript 3) expression. Lower levels of DDIT3 were responsible for reduced GHR expression leading to impaired local production of IGF1. Our results conclusively identify a novel autophagy-dependent pathway that regulates nSC behavior and indicate that autophagy is required for skeletal muscle development in the neonatal phase. Abbreviations: AKT/protein kinase B: Thymoma viral proto-oncogene; ASCs: adult stem cells; ATF4: activating transcription factor 4; ATG7: autophagy related 7; BAT: brown adipose tissue; BMP: bone morphogenetic protein; CEBPB: CCAAT/enhancer binding protein (C/EBP), beta; CSA: cross sectional area; CTNNB1: catenin (cadherin associated protein), beta 1; DDIT3: DNA-damage inducible transcript 3; DM: differentiation medium; E: embryonic stage; EIF2AK3/PERK; EIF4EBP1: eukaryotic translation initiation factor 2 alpha kinase 3; eukaryotic translation initiation factor 4E binding protein 1; ER: endoplasmic reticulum; FGF21: fibroblast growth factor 21; GH: growth hormone; GHR: growth hormone receptor; HSCs: hematopoietic stem cells; IGF1: insulin-like growth factor 1; ITGAM: integrin alpha M; KEAP1: kelch-like ECH-associated protein 1; LY6A/Sca-1; MAP1LC3: lymphocyte antigen 6 complex, locus A; microtubule-associated protein 1 light chain 3; MAPK1/ERK2: mitogen-activated protein kinase 1; MAPK3/ERK1: mitogen-activated protein kinase 3; miRNAs: microRNAs; MSCs: mesenchymal stem cells; MTOR: mechanistic target of rapamycin kinase; mtUPR: mitochondrial unfolded protein response; MYF5: myogenic factor 5; MYH: myosin, heavy polypeptide; MYOD1: myogenic differentiation 1; MYOG: myogenin; NFE2L2: nuclear factor, erythroid derived 2, like 2; nSC: neonatal satellite cells; NSCs: neuronal stem cells; P: postnatal day; PAX7: paired box 7; PECAM1: platelet/endothelial cell adhesion molecule 1; PPARG: peroxisome proliferator activated receptor gamma; PTPRC: protein tyrosine phosphatase, receptor type, C; ROS: reactive oxygen species; RPS6: ribosomal protein S6; SCs: adult satellite cells; SQSTM1: sequestosome 1; STAT5: signal transducer and activator of transcription 5; TGFB1: transforming growth factor beta 1; WAT: white adipose tissue; WT: wild type.


Assuntos
Autofagia/genética , Fator de Crescimento Insulin-Like I/metabolismo , Desenvolvimento Muscular/genética , Músculo Esquelético/crescimento & desenvolvimento , Fator 1 Relacionado a NF-E2/genética , Fator de Transcrição CHOP/genética , Animais , Autofagia/fisiologia , Proteína 7 Relacionada à Autofagia/genética , Proteína 7 Relacionada à Autofagia/metabolismo , Proteínas de Transporte/metabolismo , Diferenciação Celular/genética , Células Cultivadas , Camundongos , Camundongos Knockout , Desenvolvimento Muscular/fisiologia , Músculo Esquelético/embriologia , Músculo Esquelético/metabolismo , Fator 1 Relacionado a NF-E2/metabolismo , Fator de Transcrição PAX7/genética , Fator de Transcrição PAX7/metabolismo , Células Satélites de Músculo Esquelético/citologia , Células Satélites de Músculo Esquelético/metabolismo , Transdução de Sinais/genética , Fator de Transcrição CHOP/metabolismo
5.
Cell Death Dis ; 10(1): 10, 2018 12 19.
Artigo em Inglês | MEDLINE | ID: mdl-30584259

RESUMO

Autophagy occurs at a basal level in all eukaryotic cells and may support cell survival or activate death pathways. Due to its pathophysiologic significance, the autophagic machinery is a promising target for the development of multiple approaches for anti-neoplastic agents. We have recently described the cytotoxic and pro-apoptotic mechanisms, targeting the tumour suppressor p53, of climacostol, a natural product of the ciliated protozoan Climacostomum virens. We report here on how climacostol regulates autophagy and the involvement of p53-dependent mechanisms. Using both in vitro and in vivo techniques, we show that climacostol potently and selectively impairs autophagy in multiple tumour cells that are committed to die by apoptosis. In particular, in B16-F10 mouse melanomas climacostol exerts a marked and sustained accumulation of autophagosomes as the result of dysfunctional autophagic degradation. We also provide mechanistic insights showing that climacostol affects autophagosome turnover via p53-AMPK axis, although the mTOR pathway unrelated to p53 levels plays a role. In particular, climacostol activated p53 inducing the upregulation of p53 protein levels in the nuclei through effects on p53 stability at translational level, as for instance the phosphorylation at Ser15 site. Noteworthy, AMPKα activation was the major responsible of climacostol-induced autophagy disruption in the absence of a key role regulating cell death, thus indicating that climacostol effects on autophagy and apoptosis are two separate events, which may act independently on life/death decisions of the cell. Since the activation of p53 system is at the molecular crossroad regulating both the anti-autophagic action of climacostol and its role in the apoptosis induction, it might be important to explore the dual targeting of autophagy and apoptosis with agents acting on p53 for the selective killing of tumours. These findings also suggest the efficacy of ciliate bioactive molecules to identify novel lead compounds in drug discovery and development.


Assuntos
Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Resorcinóis/farmacologia , Proteína Supressora de Tumor p53/metabolismo , Animais , Linhagem Celular Tumoral , Feminino , Camundongos , Neoplasias/metabolismo , Neoplasias/patologia
6.
Front Immunol ; 9: 1186, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29896202

RESUMO

Tumor microenvironment is fundamental for cancer progression and chemoresistance. Among stromal cells tumor-associated macrophages (TAMs) represent the largest population of infiltrating inflammatory cells in malignant tumors, promoting their growth, invasion, and immune evasion. M2-polarized TAMs are endowed with the nitric oxide (NO)-generating enzyme inducible nitric oxide synthase (iNOS). NO has divergent effects on tumors, since it can either stimulate tumor cells growth or promote their death depending on the source of it; likewise the role of iNOS in cancer differs depending on the cell type. The role of NO generated by TAMs has not been investigated. Using different tumor models in vitro and in vivo we found that NO generated by iNOS of M2-polarized TAMs is able to protect tumor cells from apoptosis induced by the chemotherapeutic agent cisplatin (CDDP). Here, we demonstrate that the protective effect of NO depends on the inhibition of acid sphingomyelinase (A-SMase), which is activated by CDDP in a pathway involving the death receptor CD95. Mechanistic insights indicate that NO actions occur via generation of cyclic GMP and activation of protein kinase G (PKG), inducing phosphorylation of syntaxin 4 (synt4), a SNARE protein responsible for A-SMase trafficking and activation. Noteworthy, phosphorylation of synt4 at serine 78 by PKG is responsible for the proteasome-dependent degradation of synt4, which limits the CDDP-induced exposure of A-SMase to the plasma membrane of tumor cells. This inhibits the cytotoxic mechanism of CDDP reducing A-SMase-triggered apoptosis. This is the first demonstration that endogenous NO system is a key mechanism through which TAMs protect tumor cells from chemotherapeutic drug-induced apoptosis. The identification of the pathway responsible for A-SMase activity downregulation in tumors leading to chemoresistance warrants further investigations as a means to identify new anti-cancer molecules capable of specifically inhibiting synt4 degradation.


Assuntos
Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos/imunologia , Glioma/imunologia , Macrófagos/imunologia , Proteínas de Neoplasias/imunologia , Óxido Nítrico/imunologia , Proteínas Qa-SNARE/imunologia , Esfingomielina Fosfodiesterase/imunologia , Animais , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Glioma/tratamento farmacológico , Glioma/genética , Glioma/patologia , Humanos , Macrófagos/patologia , Camundongos , Camundongos Knockout , Proteínas de Neoplasias/genética , Óxido Nítrico/genética , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/imunologia , Proteínas Qa-SNARE/genética , Esfingomielina Fosfodiesterase/genética
7.
Cell Rep ; 17(11): 3010-3023, 2016 12 13.
Artigo em Inglês | MEDLINE | ID: mdl-27974213

RESUMO

Mitochondrial dysfunction occurs in many muscle degenerative disorders. Here, we demonstrate that mitochondrial biogenesis was impaired in limb-girdle muscular dystrophy (LGMD) 2D patients and mice and was associated with impaired OxPhos capacity. Two distinct approaches that modulated histones or peroxisome proliferator-activated receptor-gamma coactivator 1 α (PGC-1α) acetylation exerted equivalent functional effects by targeting different mitochondrial pathways (mitochondrial biogenesis or fatty acid oxidation[FAO]). The histone deacetylase inhibitor Trichostatin A (TSA) changed chromatin assembly at the PGC-1α promoter, restored mitochondrial biogenesis, and enhanced muscle oxidative capacity. Conversely, nitric oxide (NO) triggered post translation modifications of PGC-1α and induced FAO, recovering the bioenergetics impairment of muscles but shunting the defective mitochondrial biogenesis. In conclusion, a transcriptional blockade of mitochondrial biogenesis occurred in LGMD-2D and could be recovered by TSA changing chromatin conformation, or it could be overcome by NO activating a mitochondrial salvage pathway.


Assuntos
Ácidos Graxos/metabolismo , Mitocôndrias/genética , Distrofia Muscular do Cíngulo dos Membros/metabolismo , Processamento de Proteína Pós-Traducional/genética , Acetilação , Animais , Cromatina/genética , Cromatina/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Metabolismo dos Lipídeos/genética , Camundongos , Mitocôndrias/patologia , Distrofia Muscular do Cíngulo dos Membros/genética , Distrofia Muscular do Cíngulo dos Membros/patologia , Óxido Nítrico/metabolismo , Biogênese de Organelas , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo
8.
Oncotarget ; 7(18): 24995-5009, 2016 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-27107419

RESUMO

The sphingolipid metabolising enzyme Acid Sphingomyelinase (A-SMase) has been recently shown to inhibit melanoma progression and correlate inversely to tumour grade. In this study we have investigated the role of A-SMase in the chemo-resistance to anticancer treatmentusing mice with melanoma allografts and melanoma cells differing in terms of expression/activity of A-SMase. Since autophagy is emerging as a key mechanism in tumour growth and chemo-resistance, we have also investigated whether an action of A-SMase in autophagy can explain its role. Melanoma sensitivity to chemotherapeutic agent cisplatin in terms of cell viability/apoptosis, tumour growth, and animal survival depended directly on the A-SMase levels in tumoural cells. A-SMase action was due to inhibition of autophagy through activation of Akt/mammalian target of rapamycin (mTOR) pathway. Treatment of melanoma-bearing mice with the autophagy inhibitor chloroquine restored sensitivity to cisplatin of tumours expressing low levels of A-SMase while no additive effects were observed in tumours characterised by sustained A-SMase levels. The fact that A-SMase in melanomas affects mTOR-regulated autophagy and plays a central role in cisplatin efficacy encourages pre-clinical testing on the modulation of A-SMase levels/activity as possible novel anti-neoplastic strategy.


Assuntos
Antineoplásicos/farmacologia , Autofagia/efeitos dos fármacos , Cisplatino/farmacologia , Melanoma/enzimologia , Esfingomielina Fosfodiesterase/metabolismo , Animais , Resistencia a Medicamentos Antineoplásicos/fisiologia , Humanos , Melanoma/patologia , Camundongos , Serina-Treonina Quinases TOR/metabolismo
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