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1.
Artigo em Inglês | MEDLINE | ID: mdl-38628622

RESUMO

Nerve injuries present a substantial challenge within the medical domain due to their prevalent occurrence and significant impact. In nerve injuries, a range of physiopathological and metabolic responses come into play to stabilize and repair the resulting damage. A critical concern arises from the disruption of connections at neuromuscular junctions, leading to profound degeneration and substantial loss of muscle function, thereby hampering motor tasks. While end-to-end neurorrhaphy serves as the established technique for treating peripheral nerve injuries, achieving comprehensive morphofunctional recovery remains a formidable challenge. In pursuit of enhancing the repair process, alternative and supportive methods are being explored. A promising candidate is the utilization of heterologous fibrin biopolymer, a sealant devoid of human blood components. Notably, this biopolymer has showcased its prowess in establishing a stable and protective microenvironment at the site of use in multiple scenarios of regenerative medicine. Hence, this scoping review is directed towards assessing the effects of associating heterologous fibrin biopolymer with neurorrhaphy to treat nerve injuries, drawing upon findings from prior studies disseminated through PubMed/MEDLINE, Scopus, and Web of Science databases. Further discourse delves into the intricacies of the biology of neuromuscular junctions, nerve injury pathophysiology, and the broader utilization of fibrin sealants in conjunction with sutures for nerve reconstruction procedures. The association of the heterologous fibrin biopolymer with neurorrhaphy emerges as a potential avenue for surmounting the limitations associated with traditional sealants while also mitigating degeneration in nerves, muscles, and NMJs post-injury, thereby fostering a more conducive environment for subsequent regeneration. Indeed, queries arise regarding the long-term regenerative potential of this approach and its applicability in reconstructive surgeries for human nerve injuries.

2.
Cell Rep Med ; 4(12): 101339, 2023 12 19.
Artigo em Inglês | MEDLINE | ID: mdl-38118405

RESUMO

Rhabdomyosarcoma (RMS) is the main form of pediatric soft-tissue sarcoma. Its cure rate has not notably improved in the last 20 years following relapse, and the lack of reliable preclinical models has hampered the design of new therapies. This is particularly true for highly heterogeneous fusion-negative RMS (FNRMS). Although methods have been proposed to establish FNRMS organoids, their efficiency remains limited to date, both in terms of derivation rate and ability to accurately mimic the original tumor. Here, we present the development of a next-generation 3D organoid model derived from relapsed adult and pediatric FNRMS. This model preserves the molecular features of the patients' tumors and is expandable for several months in 3D, reinforcing its interest to drug combination screening with longitudinal efficacy monitoring. As a proof-of-concept, we demonstrate its preclinical relevance by reevaluating the therapeutic opportunities of targeting apoptosis in FNRMS from a streamlined approach based on transcriptomic data exploitation.


Assuntos
Antineoplásicos , Rabdomiossarcoma , Adulto , Humanos , Criança , Recidiva Local de Neoplasia/tratamento farmacológico , Rabdomiossarcoma/tratamento farmacológico , Rabdomiossarcoma/patologia , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Organoides/patologia , Morte Celular
3.
Medicina (Kaunas) ; 59(11)2023 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-38003989

RESUMO

Background and Objectives: Nandrolone decanoate (ND) is the most widely used among the anabolic androgenic steroids (AAS), synthetic substances derived from testosterone, to improve muscular and health gains associated with exercises. The AAS leads to physical performance enhancement and presents anti-aging properties, but its abuse is associated with several adverse effects. Supraphysiological doses of AAS with or without physical exercise can cause morphological and functional alterations in neuromuscular interactions. This study aims to investigate the effects of ND supraphysiological doses in neuromuscular interactions, focusing on the soleus muscle and its neuromuscular junctions (NMJs) in rats, associated or not with physical exercise. Materials and Methods: Forty male Sprague Dawley rats were divided into four groups: sedentary and exercised groups, with or without ND at the dose of 10 mg/kg/week. The animals were treated for eight weeks, with intramuscular injections, and the soleus muscle was collected for morphological analyses. Results: The supraphysiological doses of ND in the sedentary group caused muscle degeneration, evidenced by splitting fibers, clusters of small fibers, irregular myofibrils, altered sarcomeres, an increase in collagen deposition and in the number of type I muscle fibers (slow-twitch) and central nuclei, as well as a decrease in fibers with peripheral nuclei. On the other hand, in the ND exercise group, there was an increase in the NMJs diameter with scattering of its acetylcholine receptors, although no major morphological changes were found in the skeletal muscle. Thus, the alterations caused by ND in sedentary rats were partially reversed by physical exercise. Conclusions: The supraphysiological ND exposure in the sedentary rats promoted an increase in muscle oxidative pattern and adverse morphological alterations in skeletal muscle, resulting from damage or post-injury regeneration. In the ND-exercised rats, no major morphological changes were found. Thus, the physical exercise partially reversed the alterations caused by ND in sedentary rats.


Assuntos
Anabolizantes , Nandrolona , Ratos , Masculino , Animais , Decanoato de Nandrolona/farmacologia , Nandrolona/efeitos adversos , Anabolizantes/efeitos adversos , Ratos Wistar , Ratos Sprague-Dawley , Músculo Esquelético/fisiologia , Junção Neuromuscular
4.
EBioMedicine ; 95: 104752, 2023 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-37572644

RESUMO

BACKGROUND: Pharmacological synergisms are an attractive anticancer strategy. However, with more than 5000 approved-drugs and compounds in clinical development, identifying synergistic treatments represents a major challenge. METHODS: High-throughput screening was combined with target deconvolution and functional genomics to reveal targetable vulnerabilities in glioblastoma. The role of the top gene hit was investigated by RNA interference, transcriptomics and immunohistochemistry in glioblastoma patient samples. Drug combination screen using a custom-made library of 88 compounds in association with six inhibitors of the identified glioblastoma vulnerabilities was performed to unveil pharmacological synergisms. Glioblastoma 3D spheroid, organotypic ex vivo and syngeneic orthotopic mouse models were used to validate synergistic treatments. FINDINGS: Nine targetable vulnerabilities were identified in glioblastoma and the top gene hit RRM1 was validated as an independent prognostic factor. The associations of CHK1/MEK and AURKA/BET inhibitors were identified as the most potent amongst 528 tested pairwise drug combinations and their efficacy was validated in 3D spheroid models. The high synergism of AURKA/BET dual inhibition was confirmed in ex vivo and in vivo glioblastoma models, without detectable toxicity. INTERPRETATION: Our work provides strong pre-clinical evidence of the efficacy of AURKA/BET inhibitor combination in glioblastoma and opens new therapeutic avenues for this unmet medical need. Besides, we established the proof-of-concept of a stepwise approach aiming at exploiting drug poly-pharmacology to unveil druggable cancer vulnerabilities and to fast-track the identification of synergistic combinations against refractory cancers. FUNDING: This study was funded by institutional grants and charities.


Assuntos
Antineoplásicos , Glioblastoma , Animais , Camundongos , Glioblastoma/tratamento farmacológico , Glioblastoma/genética , Aurora Quinase A , Sinergismo Farmacológico , Linhagem Celular Tumoral , Antineoplásicos/farmacologia , Combinação de Medicamentos
5.
Muscle Nerve ; 67(6): 522-536, 2023 06.
Artigo em Inglês | MEDLINE | ID: mdl-36905197

RESUMO

INTRODUCTION/AIMS: Peripheral nerve injuries result in impaired neuromuscular interactions, leading to morphological and functional alterations. Adjuvant suture repair methods have been used to improve nerve regeneration and modulate the immune response. Heterologous fibrin biopolymer (HFB), a scaffold with adhesive properties, plays a critical role in tissue repair. The aim of this study is to evaluate neuroregeneration and immune response focusing on neuromuscular recovery, using suture-associated HFB for sciatic nerve repair. METHODS: Forty adult male Wistar rats were distributed into four groups (n = 10): C (control), only sciatic nerve location; D (denervated), neurotmesis and 6-mm gap removal and fixation stumps in subcutaneous tissue; S (suture), neurotmesis followed by suture; and SB (suture + HFB), neurotmesis followed by suture and HFB. Analysis of M2 macrophages (CD206+ ), as well as the morphology and morphometry of nerves, soleus muscle, and neuromuscular junctions (NMJs), were performed at 7 and 30 days after surgery. RESULTS: The SB group had the highest M2 macrophage area in both periods. After 7 days, SB was the only group similar to the C group regarding the number of axons; furthermore, after 30 days, the SB group was closer to the C group concerning blood vessel and central myonuclear numbers, NMJ angle, and connective tissue volume. After 7 days, increases in nerve area, as well as the number and area of blood vessels, were also observed in SB. DISCUSSION: HFB potentiates the immune response, increases axonal regeneration, induces angiogenesis, prevents severe muscle degeneration, and assists in NMJ recovery. In conclusion, suture-associated HFB has major implications for improved peripheral nerve repair.


Assuntos
Adesivo Tecidual de Fibrina , Fibrina , Ratos , Animais , Masculino , Adesivo Tecidual de Fibrina/farmacologia , Ratos Wistar , Nervo Isquiático/lesões , Biopolímeros , Regeneração Nervosa , Suturas
6.
Muscle Nerve ; 67(6): 537-547, 2023 06.
Artigo em Inglês | MEDLINE | ID: mdl-36975763

RESUMO

INTRODUCTION/AIMS: The mechanisms that underlie the pathogenesis of statin-associated muscle symptoms (SAMS) remain unclear. Pregnancy is associated with increased cholesterol levels. Statins may be useful during pregnancy, but their safety is uncertain. Hence, we investigated the postpartum effects of exposure to rosuvastatin and simvastatin during pregnancy in Wistar rats, targeting the neuromuscular structures. METHODS: Twenty-one pregnant Wistar rats were divided into three groups: control (C) treated with vehicle (dimethylsulfoxide + dH20), simvastatin (S) 62.5 mg/kg/day, and rosuvastatin (R) 10 mg/kg/day. Gavage was performed daily from the gestational days 8 to 20. At weaning, the postpartum mother tissues were collected and subjected to morphological and morphometric analysis of the soleus muscle, associated neuromuscular junctions (NMJs), and the sciatic nerve; protein quantification; quantification of the cholesterol and creatine kinase in the serum; and intramuscular collagen analysis. RESULTS: An increase in morphometric parameters (area, maximum and minimum diameters, Feret diameter, and minimum Feret) was observed in NMJs from the S and R groups in comparison with the C group, and there was also a loss of common NMJ circularity. The number of myofibers with central nuclei was higher in S (17 ± 3.9, P = .0083) and R (18.86 ± 14.42, P = .0498) than in C (6.8 ± 2.6). DISCUSSION: Gestational exposure to statins induced postpartum NMJ morphology alterations in soleus muscle, which may be caused by the remodeling of clusters of nicotinic acetylcholine receptors. This may be associated with the development and progression of SAMS observed in clinical practice.


Assuntos
Inibidores de Hidroximetilglutaril-CoA Redutases , Ratos , Gravidez , Humanos , Feminino , Animais , Ratos Wistar , Inibidores de Hidroximetilglutaril-CoA Redutases/toxicidade , Rosuvastatina Cálcica , Junção Neuromuscular/metabolismo , Músculo Esquelético/metabolismo , Sinvastatina/efeitos adversos , Período Pós-Parto
7.
Injury ; 54(2): 345-361, 2023 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-36446670

RESUMO

Peripheral nerve injuries (PNI) lead to alterations in the Agrin-LRP4-MuSK pathway. This results in disaggregation of AChRs and change from epsilon (mature, innervated) to gamma (immature, denervated) subunit. Tubulization technique has been shown to be effective for PNI repair and it also allows the use of adjuvants, such as fibrin biopolymer (FB). This study evaluated the effect of the association of tubulization with FB after PNI on AChRs and associated proteins. Fifty-two adults male Wistar rats were used, distributed in 4 experimental groups: Sham Control (S), Denervated Control (D); Tubulization (TB) and Tubulization + Fibrin Biopolymer (TB+FB). Catwalk was performed every 15 days. Ninety days after surgery the right soleus muscles and ischiatic nerves were submitted to the following analyses: (a) morphological and morphometric analysis of AChRs by confocal microscopy; (b) morphological and morphometric analysis of the ischiatic nerve; (c) protein quantification of AChRs: alpha, gama, and epsilon, of Schwann cells, agrin, LRP4, MuSK, rapsyn, MMP3, MyoD, myogenin, MURF1 and atrogin-1. The main results were about the NMJs that in the TB+FB group presented morphological and morphometric approximation (compactness index; area of the AChRs and motor plate) to the S group. In addition, there were also an increase of S100 and AChRε protein expression and a decrease of MyoD. These positive association resulted in AChRs stabilization that potentiate the neuromuscular regeneration, which strengthens the use of TB for severe injuries repair and the beneficial effect of FB, along with tubulization technique.


Assuntos
Traumatismos dos Nervos Periféricos , Ratos , Animais , Masculino , Agrina/farmacologia , Agrina/metabolismo , Fibrina/metabolismo , Distribuição Normal , Ratos Wistar , Junção Neuromuscular/metabolismo , Receptores Colinérgicos/metabolismo
8.
J Proteomics ; 269: 104742, 2022 10 30.
Artigo em Inglês | MEDLINE | ID: mdl-36174952

RESUMO

Snakes of the genus Bothrops are responsible the most snakebites in the Brazil, causing a diverse and complex pathophysiological condition. Bothrops erythromelas is the main specie of medical relevance found in the Caatinga from the Brazilian Northeast region. The pathophysiological effects involving B. erythromelas snakebite as well as the organism reaction in response to this envenomation are not so explored. Thus, edema was induced in mice paws using 2.5 µg or 5.0 µg of B. erythromelas venom, and the percentage of edema was measured. Plasma was collected 30  minutes after the envenomation-induced in mice and analyzed by mass spectrometry. It was identified a total of 112 common plasma proteins differentially abundant among experimental groups, which are involved with the complement system and coagulation cascades, oxidative stress, neutrophil degranulation, platelets degranulation and inflammatory response. Apolipoprotein A1 (Apoa), serum amyloid protein A-4 (Saa4), adiponectin (Adipoq) showed up-regulated in mice plasma after injection of venom, while fibulin (Fbln1), factor XII (F12) and vitamin K-dependent protein Z (Proz) showed down-regulated. The results indicate a protein pattern of thrombo-inflammation to the B. erythromelas snakebite, evidencing potential biomarkers for monitoring this snakebite, new therapeutic targets and its correlations with the degree of envenomation once showed modulations in the abundance among the different groups according to the amount of venom injected into the mice.


Assuntos
Bothrops , Venenos de Crotalídeos , Mordeduras de Serpentes , Adiponectina , Animais , Apolipoproteína A-I , Bothrops/metabolismo , Venenos de Crotalídeos/metabolismo , Edema , Fator XII , Camundongos , Plasma/química , Proteoma/análise , Proteína Amiloide A Sérica , Venenos de Serpentes , Vitamina K
9.
Cancers (Basel) ; 14(9)2022 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-35565457

RESUMO

Ewing sarcoma (EwS) is an aggressive primary bone cancer in children and young adults characterized by oncogenic fusions between genes encoding FET-RNA-binding proteins and ETS transcription factors, the most frequent fusion being EWSR1-FLI1. We show that EGR2, an Ewing-susceptibility gene and an essential direct target of EWSR1-FLI1, directly regulates the transcription of genes encoding key enzymes of the mevalonate (MVA) pathway. Consequently, Ewing sarcoma is one of the tumors that expresses the highest levels of mevalonate pathway genes. Moreover, genome-wide screens indicate that MVA pathway genes constitute major dependencies of Ewing cells. Accordingly, the statin inhibitors of HMG-CoA-reductase, a rate-limiting enzyme of the MVA pathway, demonstrate cytotoxicity in EwS. Statins induce increased ROS and lipid peroxidation levels, as well as decreased membrane localization of prenylated proteins, such as small GTP proteins. These metabolic effects lead to an alteration in the dynamics of S-phase progression and to apoptosis. Statin-induced effects can be rescued by downstream products of the MVA pathway. Finally, we further show that statins impair tumor growth in different Ewing PDX models. Altogether, the data show that statins, which are off-patent, well-tolerated, and inexpensive compounds, should be strongly considered in the therapeutic arsenal against this deadly childhood disease.

10.
Cell Death Differ ; 28(5): 1733-1752, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33335289

RESUMO

Oleate, the most abundant endogenous and dietary cis-unsaturated fatty acid, has the atypical property to cause the redistribution of microtubule-associated proteins 1A/1B light chain 3B (referred to as LC3) to the trans-Golgi network (TGN), as shown here. A genome-wide screen identified multiple, mostly Golgi transport-related genes specifically involved in the oleate-induced relocation of LC3 to the Golgi apparatus. Follow-up analyses revealed that oleate also caused the retention of secreted proteins in the TGN, as determined in two assays in which the secretion of proteins was synchronized, (i) an assay involving a thermosensitive vesicular stomatitis virus G (VSVG) protein that is retained in the endoplasmic reticulum (ER) until the temperature is lowered, and (ii) an isothermic assay involving the reversible retention of the protein of interest in the ER lumen and that was used both in vitro and in vivo. A pharmacological screen searching for agents that induce LC3 aggregation at the Golgi apparatus led to the identification of "oleate mimetics" that share the capacity to block conventional protein secretion. In conclusion, oleate represents a class of molecules that act on the Golgi apparatus to cause the recruitment of LC3 and to stall protein secretion.


Assuntos
Lactosilceramidas/metabolismo , Ácido Oleico/metabolismo , Transporte Proteico/genética , Rede trans-Golgi/metabolismo , Animais , Autofagia , Humanos , Camundongos
11.
Open Heart ; 7(1)2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32595140

RESUMO

OBJECTIVES: To assess the potential occupational radiation reduction and technical feasibility in patients rotated 180° (upside-down) when requiring neck access for transcervical or trans-subclavian catheterisation. METHODS: Upside-down positioning is defined as rotating patients in supine position by 180°, so that the feet come to rest where the head would otherwise be. We retrospectively evaluated all these procedures performed between March 2016 and May 2019. Furthermore, two different phantoms (paediatric and adult) were used prospectively to quantify the occupational dose between conventional or upside-down positioning. In this context, ambient dose equivalents were measured using real-time dosimeters. Three different projection angles were applied. RESULTS: 44 patients with median age and body weight of 1.0 year (range 0-56) and 9.5 kg (range 1.3-74.3) underwent 63 procedures positioned upside-down. This position proved advantageous for practical reasons, since the length of the examination table could be optimally used. Additionally, it resulted in a significantly lower overall ambient dose equivalent for the primary operator (PO) of 94.8% (mean: 2569±807 vs 135±23 nSv; p<0.01) in the adult, and of 65.5% (mean: 351±104 vs 121±56 nSv; p<0.01) in the paediatric phantom, respectively. CONCLUSION: Upside-down positioning facilitates handling in a straightforward manner when access from the neck is required. Moreover, it significantly reduces local radiation exposure for the PO in the paediatric and, most impressively, in the adult phantom.


Assuntos
Cateterismo Venoso Central , Pescoço/irrigação sanguínea , Exposição Ocupacional/prevenção & controle , Saúde Ocupacional , Posicionamento do Paciente , Exposição à Radiação/prevenção & controle , Radiografia Intervencionista , Artéria Subclávia/diagnóstico por imagem , Decúbito Dorsal , Adolescente , Adulto , Cateterismo Venoso Central/efeitos adversos , Criança , Pré-Escolar , Estudos de Viabilidade , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Exposição Ocupacional/efeitos adversos , Exposição à Radiação/efeitos adversos , Radiografia Intervencionista/efeitos adversos , Estudos Retrospectivos , Medição de Risco , Fatores de Risco , Fatores de Tempo , Adulto Jovem
12.
Comput Biol Med ; 107: 227-234, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30852249

RESUMO

The microscopic assessment of the colocalization of fluorescent signals has been widely used in cell biology. Although imaging techniques have drastically improved over the past decades, the quantification of colocalization by measures such as the Pearson correlation coefficient or Manders overlap coefficient, has not changed. Here, we report the development of an R-based application that allows to (i) automatically segment cells and subcellular compartments, (ii) measure morphology and texture features, and (iii) calculate the degree of colocalization within each cell. Colocalization can thus be studied on a cell-by-cell basis, permitting to perform statistical analyses of cellular populations and subpopulations. ColocalizR has been designed to parallelize tasks, making it applicable to the analysis of large data sets. Its graphical user interface makes it suitable for researchers without specific knowledge in image analysis. Moreover, results can be exported into a wide range of formats rendering post-analysis adaptable to statistical requirements. This application and its source code are freely available at https://github.com/kroemerlab/ColocalizR.


Assuntos
Processamento de Imagem Assistida por Computador/métodos , Espaço Intracelular , Microscopia de Fluorescência/métodos , Software , Linhagem Celular Tumoral , Humanos , Espaço Intracelular/diagnóstico por imagem , Espaço Intracelular/fisiologia , Biologia de Sistemas , Interface Usuário-Computador
13.
EBioMedicine ; 30: 261-272, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29606629

RESUMO

Depending on the length of their carbon backbone and their saturation status, natural fatty acids have rather distinct biological effects. Thus, longevity of model organisms is increased by extra supply of the most abundant natural cis-unsaturated fatty acid, oleic acid, but not by that of the most abundant saturated fatty acid, palmitic acid. Here, we systematically compared the capacity of different saturated, cis-unsaturated and alien (industrial or ruminant) trans-unsaturated fatty acids to provoke cellular stress in vitro, on cultured human cells expressing a battery of distinct biosensors that detect signs of autophagy, Golgi stress and the unfolded protein response. In contrast to cis-unsaturated fatty acids, trans-unsaturated fatty acids failed to stimulate signs of autophagy including the formation of GFP-LC3B-positive puncta, production of phosphatidylinositol-3-phosphate, and activation of the transcription factor TFEB. When combined effects were assessed, several trans-unsaturated fatty acids including elaidic acid (the trans-isomer of oleate), linoelaidic acid, trans-vaccenic acid and palmitelaidic acid, were highly efficient in suppressing autophagy and endoplasmic reticulum stress induced by palmitic, but not by oleic acid. Elaidic acid also inhibited autophagy induction by palmitic acid in vivo, in mouse livers and hearts. We conclude that the well-established, though mechanistically enigmatic toxicity of trans-unsaturated fatty acids may reside in their capacity to abolish cytoprotective stress responses induced by saturated fatty acids.


Assuntos
Autofagia/efeitos dos fármacos , Ácidos Graxos/farmacologia , Ácidos Graxos trans/farmacologia , Animais , Linhagem Celular Tumoral , Drosophila melanogaster/efeitos dos fármacos , Drosophila melanogaster/fisiologia , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Feminino , Complexo de Golgi/efeitos dos fármacos , Complexo de Golgi/metabolismo , Humanos , Cinética , Longevidade/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Ácido Oleico/farmacologia , Ácidos Oleicos , Saccharomyces cerevisiae/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos
15.
Cell Death Differ ; 23(12): 2031-2041, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27588704

RESUMO

LTX-401 is an oncolytic amino acid derivative with potential immunogenic properties. Here, we demonstrate that LTX-401 selectively destroys the structure of the Golgi apparatus, as determined by means of ultrastructural analyses and fluorescence microscopic observation of cells expressing Golgi-targeted GFP reporters. Subcellular fractionation followed by mass spectrometric detection revealed that LTX-401 selectively enriched in the Golgi rather than in mitochondria or in the cytosol. The Golgi-dissociating agent Brefeldin A (BFA) reduced cell killing by LTX-401 as it partially inhibited LTX-401-induced mitochondrial release of cytochrome c and the activation of BAX. The cytotoxic effect of LTX-401 was attenuated by the double knockout of BAX and BAK, as well as the mitophagy-enforced depletion of mitochondria, yet was refractory to caspase inhibition. LTX-401 induced all major hallmarks of immunogenic cell death detectable with biosensor cell lines including calreticulin exposure, ATP release, HMGB1 exodus and a type-1 interferon response. Moreover, LTX-401-treated tumors manifested a strong lymphoid infiltration. Altogether these results support the contention that LTX-401 can stimulate immunogenic cell death through a pathway in which Golgi-localized LTX-401 operates upstream of mitochondrial membrane permeabilization.


Assuntos
Antineoplásicos/farmacologia , Complexo de Golgi/metabolismo , beta-Alanina/análogos & derivados , Animais , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Brefeldina A/farmacologia , Linhagem Celular Tumoral , Complexo de Golgi/efeitos dos fármacos , Complexo de Golgi/ultraestrutura , Humanos , Camundongos Endogâmicos C57BL , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Membranas Mitocondriais/efeitos dos fármacos , Membranas Mitocondriais/metabolismo , Oligopeptídeos/química , Oligopeptídeos/farmacologia , Permeabilidade , Frações Subcelulares/metabolismo , beta-Alanina/química , beta-Alanina/farmacologia
16.
Beilstein J Org Chem ; 11: 1960-72, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26664616

RESUMO

Three new ruthenium alkylidene complexes (PCy3)Cl2(H2ITap)Ru=CHSPh (9), (DMAP)2Cl2(H2ITap)Ru=CHPh (11) and (DMAP)2Cl2(H2ITap)Ru=CHSPh (12) have been synthesized bearing the pH-responsive H2ITap ligand (H2ITap = 1,3-bis(2',6'-dimethyl-4'-dimethylaminophenyl)-4,5-dihydroimidazol-2-ylidene). Catalysts 11 and 12 are additionally ligated by two pH-responsive DMAP ligands. The crystal structure was solved for complex 12 by X-ray diffraction. In organic, neutral solution, the catalysts are capable of performing standard ring-opening metathesis polymerization (ROMP) and ring closing metathesis (RCM) reactions with standard substrates. The ROMP with complex 11 is accelerated in the presence of two equiv of H3PO4, but is reduced as soon as the acid amount increased. The metathesis of phenylthiomethylidene catalysts 9 and 12 is sluggish at room temperature, but their ROMP can be dramatically accelerated at 60 °C. Complexes 11 and 12 are soluble in aqueous acid. They display the ability to perform RCM of diallylmalonic acid (DAMA), however, their conversions are very low amounting only to few turnovers before decomposition. However, both catalysts exhibit outstanding performance in the ROMP of dicyclopentadiene (DCPD) and mixtures of DCPD with cyclooctene (COE) in acidic aqueous microemulsion. With loadings as low as 180 ppm, the catalysts afforded mostly quantitative conversions of these monomers while maintaining the size and shape of the droplets throughout the polymerization process. Furthermore, the coagulate content for all experiments stayed <2%. This represents an unprecedented efficiency in emulsion ROMP based on hydrophilic ruthenium alkylidene complexes.

17.
Cell Cycle ; 14(21): 3506-12, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26566869

RESUMO

The oncolytic peptide LTX-315 has been designed for killing human cancer cells and turned out to stimulate anti-cancer immune responses when locally injected into tumors established in immunocompetent mice. Here, we investigated the question whether LTX-315 induces apoptosis or necrosis. Transmission electron microscopy or morphometric analysis of chromatin-stained tumor cells revealed that LTX-315 failed to induce apoptotic nuclear condensation and rather induced a necrotic phenotype. Accordingly, LTX-315 failed to stimulate the activation of caspase-3, and inhibition of caspases by means of Z-VAD-fmk was unable to reduce cell killing by LTX-315. In addition, 2 prominent inhibitors of regulated necrosis (necroptosis), namely, necrostatin-1 and cycosporin A, failed to reduce LTX-315-induced cell death. In conclusion, it appears that LTX-315 triggers unregulated necrosis, which may contribute to its pro-inflammatory and pro-immune effects.


Assuntos
Antineoplásicos/farmacologia , Neoplasias Ósseas/tratamento farmacológico , Oligopeptídeos/farmacologia , Osteossarcoma/tratamento farmacológico , Clorometilcetonas de Aminoácidos/farmacologia , Apoptose , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/ultraestrutura , Inibidores de Caspase/farmacologia , Linhagem Celular Tumoral , Ciclosporina/farmacologia , Relação Dose-Resposta a Droga , Humanos , Imidazóis/farmacologia , Indóis/farmacologia , Microscopia Eletrônica de Transmissão , Necrose , Osteossarcoma/metabolismo , Osteossarcoma/ultraestrutura , Fatores de Tempo
18.
Oncotarget ; 6(29): 26599-614, 2015 Sep 29.
Artigo em Inglês | MEDLINE | ID: mdl-26378049

RESUMO

LTX-315 has been developed as an amphipathic cationic peptide that kills cancer cells. Here, we investigated the putative involvement of mitochondria in the cytotoxic action of LTX-315. Subcellular fractionation of LTX-315-treated cells, followed by mass spectrometric quantification, revealed that the agent was enriched in mitochondria. LTX-315 caused an immediate arrest of mitochondrial respiration without any major uncoupling effect. Accordingly, LTX-315 disrupted the mitochondrial network, dissipated the mitochondrial inner transmembrane potential, and caused the release of mitochondrial intermembrane proteins into the cytosol. LTX-315 was relatively inefficient in stimulating mitophagy. Cells lacking the two pro-apoptotic multidomain proteins from the BCL-2 family, BAX and BAK, were less susceptible to LTX-315-mediated killing. Moreover, cells engineered to lose their mitochondria (by transfection with Parkin combined with treatment with a protonophore causing mitophagy) were relatively resistant against LTX-315, underscoring the importance of this organelle for LTX-315-mediated cytotoxicity. Altogether, these results support the notion that LTX-315 kills cancer cells by virtue of its capacity to permeabilize mitochondrial membranes.


Assuntos
Potencial da Membrana Mitocondrial , Neoplasias/metabolismo , Oligopeptídeos/química , Peptídeos/química , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína X Associada a bcl-2/metabolismo , Animais , Antineoplásicos/química , Apoptose , Linhagem Celular Tumoral , Citosol/metabolismo , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Humanos , Membranas Intracelulares/efeitos dos fármacos , Espectrometria de Massas , Camundongos , Camundongos Knockout , Microscopia de Fluorescência , Mitocôndrias/metabolismo , Mitofagia , Permeabilidade , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ubiquitina-Proteína Ligases/metabolismo
19.
Mol Cell ; 58(6): 1001-14, 2015 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-26004228

RESUMO

Apoptosis-inducing factor (AIF) is a mitochondrial flavoprotein that, beyond its apoptotic function, is required for the normal expression of major respiratory chain complexes. Here we identified an AIF-interacting protein, CHCHD4, which is the central component of a redox-sensitive mitochondrial intermembrane space import machinery. Depletion or hypomorphic mutation of AIF caused a downregulation of CHCHD4 protein by diminishing its mitochondrial import. CHCHD4 depletion sufficed to induce a respiratory defect that mimicked that observed in AIF-deficient cells. CHCHD4 levels could be restored in AIF-deficient cells by enforcing its AIF-independent mitochondrial localization. This modified CHCHD4 protein reestablished respiratory function in AIF-deficient cells and enabled AIF-deficient embryoid bodies to undergo cavitation, a process of programmed cell death required for embryonic morphogenesis. These findings explain how AIF contributes to the biogenesis of respiratory chain complexes, and they establish an unexpected link between the vital function of AIF and the propensity of cells to undergo apoptosis.


Assuntos
Fator de Indução de Apoptose/metabolismo , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Sequência de Aminoácidos , Animais , Fator de Indução de Apoptose/genética , Linhagem Celular Tumoral , Transporte de Elétrons/genética , Complexo de Proteínas da Cadeia de Transporte de Elétrons/genética , Embrião de Mamíferos/embriologia , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário/genética , Humanos , Immunoblotting , Camundongos Knockout , Mitocôndrias/genética , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Dados de Sequência Molecular , Ligação Proteica , Transporte Proteico/genética , Interferência de RNA , Fatores de Tempo
20.
Oncotarget ; 6(11): 9467-75, 2015 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-25816366

RESUMO

Cellular viability is usually determined by measuring the capacity of cells to exclude vital dyes such as 4',6-diamidino-2-phenylindole (DAPI), or by assessing nuclear morphology with chromatinophilic plasma membrane-permeant dyes, such as Hoechst 33342. However, a fraction of cells that exclude DAPI or exhibit normal nuclear morphology have already lost mitochondrial functions and/or manifest massive activation of apoptotic caspases, and hence are irremediably committed to death. Here, we developed a protocol for the simultaneous detection of plasma membrane integrity (based on DAPI) or nuclear morphology (based on Hoechst 33342), mitochondrial functions (based on the mitochondrial transmembrane potential probe DiOC6(3)) and caspase activation (based on YO-PRO®-3, which can enter cells exclusively upon the caspase-mediated activation of pannexin 1 channels). This method, which allows for the precise quantification of dead, dying and healthy cells, can be implemented on epifluorescence microscopy or flow cytometry platforms and is compatible with a robotized, high-throughput workflow.


Assuntos
Sobrevivência Celular , Citometria de Fluxo/métodos , Ensaios de Triagem em Larga Escala/métodos , Microscopia de Fluorescência/métodos , Coloração e Rotulagem/métodos , Clorometilcetonas de Aminoácidos/farmacologia , Apoptose/efeitos dos fármacos , Benzimidazóis/análise , Carbocianinas/análise , Carbocianinas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Caspases/metabolismo , Linhagem Celular Tumoral , Permeabilidade da Membrana Celular , Sobrevivência Celular/efeitos dos fármacos , Conexinas/metabolismo , Sinergismo Farmacológico , Corantes Fluorescentes/análise , Corantes Fluorescentes/metabolismo , Humanos , Indóis/análise , Indóis/metabolismo , Neoplasias Pulmonares/patologia , Potencial da Membrana Mitocondrial , Proteínas do Tecido Nervoso/metabolismo , Compostos Organoplatínicos/farmacologia , Oxaliplatina , Robótica , Estaurosporina/farmacologia , Fluxo de Trabalho
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