Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 43
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Nat Commun ; 12(1): 5068, 2021 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-34417460

RESUMO

p53 regulates several signaling pathways to maintain the metabolic homeostasis of cells and modulates the cellular response to stress. Deficiency or excess of nutrients causes cellular metabolic stress, and we hypothesized that p53 could be linked to glucose maintenance. We show here that upon starvation hepatic p53 is stabilized by O-GlcNAcylation and plays an essential role in the physiological regulation of glucose homeostasis. More specifically, p53 binds to PCK1 promoter and regulates its transcriptional activation, thereby controlling hepatic glucose production. Mice lacking p53 in the liver show a reduced gluconeogenic response during calorie restriction. Glucagon, adrenaline and glucocorticoids augment protein levels of p53, and administration of these hormones to p53 deficient human hepatocytes and to liver-specific p53 deficient mice fails to increase glucose levels. Moreover, insulin decreases p53 levels, and over-expression of p53 impairs insulin sensitivity. Finally, protein levels of p53, as well as genes responsible of O-GlcNAcylation are elevated in the liver of type 2 diabetic patients and positively correlate with glucose and HOMA-IR. Overall these results indicate that the O-GlcNAcylation of p53 plays an unsuspected key role regulating in vivo glucose homeostasis.


Assuntos
Acetilglucosamina/metabolismo , Glucose/metabolismo , Fígado/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Sequência de Bases , Restrição Calórica , Linhagem Celular , Colforsina/farmacologia , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/metabolismo , Epinefrina/metabolismo , Glucagon/metabolismo , Glucocorticoides/metabolismo , Gluconeogênese/efeitos dos fármacos , Glicosilação , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Hidrocortisona/metabolismo , Hiperglicemia/complicações , Hiperglicemia/metabolismo , Resistência à Insulina , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fígado/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Obesidade/complicações , Obesidade/metabolismo , Fosfoenolpiruvato Carboxiquinase (GTP)/metabolismo , Regiões Promotoras Genéticas/genética , Ligação Proteica/efeitos dos fármacos , Estabilidade Proteica/efeitos dos fármacos , Ácido Pirúvico/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transcrição Genética/efeitos dos fármacos , Proteína Supressora de Tumor p53/genética
2.
mBio ; 12(4): e0097221, 2021 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-34225493

RESUMO

Ebola virus (EBOV) VP24 protein is a nucleocapsid-associated protein that inhibits interferon (IFN) gene expression and counteracts the IFN-mediated antiviral response, preventing nuclear import of signal transducer and activator of transcription 1 (STAT1). Proteomic studies to identify additional EBOV VP24 partners have pointed to the nuclear membrane component emerin as a potential element of the VP24 cellular interactome. Here, we have further studied this interaction and its impact on cell biology. We demonstrate that VP24 interacts with emerin but also with other components of the inner nuclear membrane, such as lamin A/C and lamin B. We also show that VP24 diminishes the interaction between emerin and lamin A/C and compromises the integrity of the nuclear membrane. This disruption is associated with nuclear morphological abnormalities, activation of a DNA damage response, the phosphorylation of extracellular signal-regulated kinase (ERK), and the induction of interferon-stimulated gene 15 (ISG15). Interestingly, expression of VP24 also promoted the cytoplasmic translocation and downmodulation of barrier-to-autointegration factor (BAF), a common interactor of lamin A/C and emerin, leading to repression of the BAF-regulated CSF1 gene. Importantly, we found that EBOV infection results in the activation of pathways associated with nuclear envelope damage, consistent with our observations in cells expressing VP24. In summary, here we demonstrate that VP24 acts at the nuclear membrane, causing morphological and functional changes in cells that recapitulate several of the hallmarks of laminopathy diseases. IMPORTANCE The Ebola virus (EBOV) VP24 protein is a nucleocapsid-associated protein with multiple functions. Proteomic studies have identified the cellular nuclear membrane component emerin as a potential VP24 interactor. Here, we demonstrate that VP24 not only interacts with emerin but also with lamin A/C and lamin B, prompting nuclear membrane disruption. This disruption is associated with nuclear morphological abnormalities, activation of a DNA damage response, the phosphorylation of extracellular signal-regulated kinase (ERK), and the induction of interferon-stimulated gene 15 (ISG15). Interestingly, VP24 also promotes the cytoplasmic translocation and downmodulation of barrier-to-autointegration factor (BAF), leading to repression of the BAF-regulated CSF1 gene. Finally, we show that EBOV infection also results in the activation of pathways associated with nuclear envelope damage, consistent with our observations in cells expressing VP24. These results reveal novel activities of EBOV VP24 protein, resulting in a cell phenotype similar to that of most laminopathies, with potential impact on EBOV replication.

3.
Cell Mol Life Sci ; 78(8): 4053-4065, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33834259

RESUMO

Class I PI3K are heterodimers composed of a p85 regulatory subunit and a p110 catalytic subunit involved in multiple cellular functions. Recently, the catalytic subunit p110ß has emerged as a class I PI3K isoform playing a major role in tumorigenesis. Understanding its regulation is crucial for the control of the PI3K pathway in p110ß-driven cancers. Here we sought to evaluate the putative regulation of p110ß by SUMO. Our data show that p110ß can be modified by SUMO1 and SUMO2 in vitro, in transfected cells and under completely endogenous conditions, supporting the physiological relevance of p110ß SUMOylation. We identify lysine residue 952, located at the activation loop of p110ß, as essential for SUMOylation. SUMOylation of p110ß stabilizes the protein increasing its activation of AKT which promotes cell growth and oncogenic transformation. Finally, we show that the regulatory subunit p85ß counteracts the conjugation of SUMO to p110ß. In summary, our data reveal that SUMO is a novel p110ß interacting partner with a positive effect on the activation of the PI3K pathway.


Assuntos
Classe Ia de Fosfatidilinositol 3-Quinase/metabolismo , Sumoilação , Animais , Domínio Catalítico , Classe Ia de Fosfatidilinositol 3-Quinase/química , Ativação Enzimática , Estabilidade Enzimática , Células HEK293 , Humanos , Camundongos , Células NIH 3T3 , Células PC-3 , Transdução de Sinais
4.
Drug Deliv Transl Res ; 11(2): 426-444, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33611769

RESUMO

Fundamental studies performed during the last decades have shown that cell fate is much more plastic than previously considered, and technologies for its manipulation are a keystone for many new tissue regeneration therapies. Transcription factors (TFs) are DNA-binding proteins that control gene expression, and they have critical roles in the control of cell fate and other cellular behavior. TF-based therapies have much medical potential, but their use as drugs depends on the development of suitable delivery technologies that can help them reach their action site inside of the cells. TFs can be used either as proteins or encoded in polynucleotides. When used in protein form, many TFs require to be associated to a cell-penetrating peptide or another transduction domain. As polynucleotides, they can be delivered either by viral carriers or by non-viral systems such as polyplexes and lipoplexes. TF-based therapies have extensively shown their potential to solve many tissue-engineering problems, including bone, cartilage and cardiac regeneration. Yet, their use has expanded beyond regenerative medicine to other prominent disease areas such as cancer therapy and immunomodulation. This review summarizes some of the delivery options for effective TF-based therapies and their current main applications.

6.
Biomaterials ; 247: 120016, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32272302

RESUMO

Gene-activated matrices (GAMs) encoding pivotal transcription factors (TFs) represent a powerful tool to direct stem cell specification for tissue engineering applications. However, current TF-based GAMs activated with pDNA, are challenged by their low transfection efficiency and delayed transgene expression. Here, we report a GAM technology activated with mRNAs encoding TFs SOX9 (cartilage) and MYOD (muscle). We find that these mRNA-GAMs induce a higher and faster TF expression compared to pDNA-GAMs, especially in the case of RNase resistant mRNA sequences. This potent TF expression was translated into a high synthesis of cartilage- and muscle-specific markers, and ultimately, into successful tissue specification in vitro. Additionally, we show that the expression of tissue-specific markers can be further modulated by altering the properties of the mRNA-GAM environment. These results highlight the value of this GAM technology for priming cell lineage specification, a key centerpiece for future tissue engineering devices.


Assuntos
Engenharia Tecidual , Fatores de Transcrição , Diferenciação Celular , RNA Mensageiro/genética , Transfecção
7.
Nat Commun ; 10(1): 4731, 2019 10 21.
Artigo em Inglês | MEDLINE | ID: mdl-31636264

RESUMO

Compounds with specific cytotoxic activity in senescent cells, or senolytics, support the causal involvement of senescence in aging and offer therapeutic interventions. Here we report the identification of Cardiac Glycosides (CGs) as a family of compounds with senolytic activity. CGs, by targeting the Na+/K+ATPase pump, cause a disbalanced electrochemical gradient within the cell causing depolarization and acidification. Senescent cells present a slightly depolarized plasma membrane and higher concentrations of H+, making them more susceptible to the action of CGs. These vulnerabilities can be exploited for therapeutic purposes as evidenced by the in vivo eradication of tumors xenografted in mice after treatment with the combination of a senogenic and a senolytic drug. The senolytic effect of CGs is also effective in the elimination of senescence-induced lung fibrosis. This experimental approach allows the identification of compounds with senolytic activity that could potentially be used to develop effective treatments against age-related diseases.


Assuntos
Apoptose/efeitos dos fármacos , Glicosídeos Cardíacos/farmacologia , Senescência Celular/efeitos dos fármacos , Condrócitos/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Células A549 , Animais , Antibióticos Antineoplásicos/farmacologia , Bleomicina/farmacologia , Neoplasias da Mama , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Digoxina/farmacologia , Feminino , Humanos , Concentração de Íons de Hidrogênio/efeitos dos fármacos , Camundongos , Osteoartrite , Ouabaína/farmacologia , Proscilaridina/farmacologia , Fibrose Pulmonar , Ensaios Antitumorais Modelo de Xenoenxerto
8.
Stem Cell Reports ; 12(5): 1099-1112, 2019 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-31056476

RESUMO

Induction of pluripotency in somatic cells with defined genetic factors has been successfully used to investigate the mechanisms of disease initiation and progression. Cellular reprogramming and oncogenic transformation share common features; both involve undergoing a dramatic change in cell identity, and immortalization is a key step for cancer progression that enhances reprogramming. However, there are very few examples of complete successful reprogramming of tumor cells. Here we address the effect of expressing an active oncogene, RAS, on the process of reprogramming and found that, while combined expression with reprogramming factors enhanced dedifferentiation, expression within the context of neoplastic transformation impaired reprogramming. RAS induces expression changes that promote loss of cell identity and acquisition of stemness in a paracrine manner and these changes result in reprogramming when combined with reprogramming factors. When cells carry cooperating oncogenic defects, RAS drives cells into an incompatible cellular fate of malignancy.


Assuntos
Desdiferenciação Celular/genética , Transformação Celular Neoplásica/genética , Reprogramação Celular/genética , Embrião de Mamíferos/citologia , Fibroblastos/metabolismo , Proteínas ras/genética , Animais , Transformação Celular Neoplásica/metabolismo , Células Cultivadas , Fibroblastos/citologia , Regulação da Expressão Gênica , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos Transgênicos , Proteínas ras/metabolismo
9.
Mol Nutr Food Res ; 63(2): e1801096, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30383332

RESUMO

SCOPE: The tumor suppressor p107, a pocket protein member of the retinoblastoma susceptibility protein family, plays an important role in the cell cycle and cellular adipocyte differentiation. Nonetheless, the mechanism by which it influences whole body Energy homeostasis is unknown. METHODS AND RESULTS: The phenotype of p107 knockout (KO) mixed-background C57BL6/129 mice phenotype is studied by focusing on the involvement of white and brown adipose tissue (WAT and BAT) in energy metabolism. It is shown that p107 KO mice are leaner and have high-fat diet resistence. This phenomenon is explained by an increase of energy expenditure. The higher energy expenditure is caused by the activation of thermogenesis and may be mediated by both BAT and the browning of WAT. Consequently, it leads to the resistance of p107 KO mice to high-fat diet effects, prevention of liver steatosis, and improvement of the lipid profile and glucose homeostasis. CONCLUSION: These data allowed the unmasking of a mechanism by which a KO of p107 prevents diet-induced obesity by increasing energy expenditure via increased thermogenesis in BAT and browning of WAT, indicating the relevance of p107 as a modulator of metabolic activity of both brown and white adipocytes. Therefore, it can be targeted for the development of new therapies to ameliorate the metabolic syndrome.


Assuntos
Tecido Adiposo Marrom/metabolismo , Tecido Adiposo Branco/metabolismo , Metabolismo Energético , Proteína p107 Retinoblastoma-Like/fisiologia , Termogênese , Animais , Dieta Hiperlipídica , Fígado Gorduroso/prevenção & controle , Glucose/metabolismo , Resistência à Insulina , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína p107 Retinoblastoma-Like/deficiência
10.
J Control Release ; 294: 154-164, 2019 01 28.
Artigo em Inglês | MEDLINE | ID: mdl-30529724

RESUMO

So far, the success of anticancer nanomedicines has been moderate due to their lack of adequate targeting properties and/or to their difficulties for penetrating tumors. Here we report a multifunctional drug nanocarrier consisting of hyaluronic acid nanocapsules conjugated with the tumor homing peptide tLyp1, which exhibits both, dual targeting properties (to the tumor and to the lymphatics), and enhanced tumor penetration. Data from a 3D co-culture in vitro model showed the capacity of these nanocapsules to interact with the NRP1 receptors over-expressed in cancer cells. The targeting capacity of the nanocapsules was evidenced in orthotopic lung cancer-bearing mice, using docetaxel as a standard drug. The results showed a dramatic accumulation of docetaxel in the tumor (37-fold the one achieved with Taxotere®). This biodistribution profile correlated with the high efficacy shown in terms of tumor growth regression and drastic reduction of metastasis in the lymphatics. When efficacy was validated in a pancreatic patient-derived tumor, the nanocapsule's activity was comparable to that of a dose ten times higher of Abraxane®. Multi-functionality was found to be the key to the success of this new therapy.


Assuntos
Antineoplásicos/administração & dosagem , Docetaxel/administração & dosagem , Portadores de Fármacos/administração & dosagem , Ácido Hialurônico/administração & dosagem , Neoplasias Pulmonares/tratamento farmacológico , Células A549 , Animais , Antineoplásicos/farmacocinética , Técnicas de Cocultura , Docetaxel/farmacocinética , Portadores de Fármacos/farmacocinética , Feminino , Humanos , Ácido Hialurônico/farmacocinética , Células Jurkat , Neoplasias Pulmonares/metabolismo , Camundongos Nus , Distribuição Tecidual
11.
FASEB J ; 33(1): 643-651, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30024791

RESUMO

The ribosomal protein L11 (RPL11) integrates different types of stress into a p53-mediated response. Here, we analyzed the impact of the ubiquitin-like protein SUMO on the RPL11-mouse double-minute 2 homolog-p53 signaling. We show that small ubiquitin-related modifier (SUMO)1 and SUMO2 covalently modify RPL11. We find that SUMO negatively modulates the conjugation of the ubiquitin-like protein neural precursor cell-expressed developmentally downregulated 8 (NEDD8) to RPL11 and promotes the translocation of the RP outside of the nucleoli. Moreover, the SUMO-conjugating enzyme, Ubc9, is required for RPL11-mediated activation of p53. SUMOylation of RPL11 is triggered by ribosomal stress, as well as by alternate reading frame protein upregulation. Collectively, our data identify SUMO protein conjugation to RPL11 as a new regulator of the p53-mediated cellular response to different types of stress and reveal a previously unknown SUMO-NEDD8 interplay.-El Motiam, A., Vidal, S., de la Cruz-Herrera, C. F., Da Silva-Álvarez, S., Baz-Martínez, M., Seoane, R., Vidal, A., Rodríguez, M. S., Xirodimas, D. P., Carvalho, A. S., Beck, H. C., Matthiesen, R., Collado, M., Rivas, C. Interplay between SUMOylation and NEDDylation regulates RPL11 localization and function.


Assuntos
Proteína NEDD8/metabolismo , Neoplasias/patologia , Processamento de Proteína Pós-Traducional , Proteínas Ribossômicas/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Sumoilação , Ubiquitinas/metabolismo , Células HEK293 , Humanos , Neoplasias/metabolismo , Células Tumorais Cultivadas
12.
Aging Cell ; 17(5): e12834, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30129215

RESUMO

Aging is characterized by a gradual functional decline of tissues with age. Adult stem and progenitor cells are responsible for tissue maintenance, repair, and regeneration, but during aging, this population of cells is decreased or its activity is reduced, compromising tissue integrity and causing pathologies that increase vulnerability, and ultimately lead to death. The causes of stem cell exhaustion during aging are not clear, and whether a reduction in stem cell function is a cause or a consequence of aging remains unresolved. Here, we took advantage of a mouse model of induced adult Sox2+ stem cell depletion to address whether accelerated stem cell depletion can promote premature aging. After a short period of partial repetitive depletion of this adult stem cell population in mice, we observed increased kyphosis and hair graying, and reduced fat mass, all of them signs of premature aging. It is interesting that cellular senescence was identified in kidney after this partial repetitive Sox2+ cell depletion. To confirm these observations, we performed a prolonged protocol of partial repetitive depletion of Sox2+ cells, forcing regeneration from the remaining Sox2+ cells, thereby causing their exhaustion. Senescence specific staining and the analysis of the expression of genetic markers clearly corroborated that adult stem cell exhaustion can lead to cellular senescence induction and premature aging.


Assuntos
Células-Tronco Adultas/metabolismo , Células-Tronco Adultas/patologia , Senilidade Prematura/metabolismo , Senilidade Prematura/patologia , Senescência Celular , Fatores de Transcrição SOXB1/metabolismo , Animais , Camundongos
13.
Angew Chem Int Ed Engl ; 57(19): 5273-5277, 2018 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-29529349

RESUMO

The generation of dendrimers is a powerful tool in the control of the size and biodistribution of polyion complexes (PIC). Using a combinatorial screening of six dendrimers (18-243 terminal groups) and five oppositely charged PEGylated copolymers, a dendrimer-to-PIC hierarchical transfer of structural information was revealed with PIC diameters that increased from 80 to 500 nm on decreasing the dendrimer generation. This rise in size, which was also accompanied by a micelle-to-vesicle transition, is interpreted according to a cone- to rod-shaped progression in the architecture of the unit PIC (uPIC). This precise size tuning enabled dendritic PICs to act as nanorulers for controlled biodistribution. Overall, a domino-like control of the size and biological properties of PIC that is not attainable with linear polymers is feasible through dendrimer generation.

14.
Sci Rep ; 7(1): 14055, 2017 10 25.
Artigo em Inglês | MEDLINE | ID: mdl-29070839

RESUMO

Activated dsRNA-dependent serine/threonine kinase PKR phosphorylates the alpha subunit of eukaryotic initiation factor 2 (eIF2α), resulting in a shut-off of general translation, induction of apoptosis, and inhibition of virus replication. PKR can be activated by binding to dsRNA or cellular proteins such as PACT/RAX, or by its conjugation to ISG15 or SUMO. Here, we demonstrate that PKR also interacts with SUMO in a non-covalent manner. We identify the phosphorylable tyrosine residue 162 in PKR (Y162) as a modulator of the PKR-SUMO non-covalent interaction as well as of the PKR SUMOylation. Finally, we show that the efficient SUMO-mediated eIF2α phosphorylation and inhibition of protein synthesis induced by PKR in response to dsRNA depend on this residue. In summary, our data identify a new mechanism of regulation of PKR activity and reinforce the relevance of both, tyrosine phosphorylation and SUMO interaction in controlling the activity of PKR.


Assuntos
RNA de Cadeia Dupla/metabolismo , Proteína SUMO-1/metabolismo , Tirosina/metabolismo , eIF-2 Quinase/fisiologia , Animais , Apoptose , Ativação Enzimática , Células HEK293 , Humanos , Camundongos , Camundongos Knockout , Fosforilação , Ligação Proteica , Sumoilação , eIF-2 Quinase/metabolismo
16.
Nat Commun ; 8: 15111, 2017 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-28480888

RESUMO

p53 family members control several metabolic and cellular functions. The p53 ortholog p63 modulates cellular adaptations to stress and has a major role in cell maintenance and proliferation. Here we show that p63 regulates hepatic lipid metabolism. Mice with liver-specific p53 deletion develop steatosis and show increased levels of p63. Down-regulation of p63 attenuates liver steatosis in p53 knockout mice and in diet-induced obese mice, whereas the activation of p63 induces lipid accumulation. Hepatic overexpression of N-terminal transactivation domain TAp63 induces liver steatosis through IKKß activation and the induction of ER stress, the inhibition of which rescues the liver functions. Expression of TAp63, IKKß and XBP1s is also increased in livers of obese patients with NAFLD. In cultured human hepatocytes, TAp63 inhibition protects against oleic acid-induced lipid accumulation, whereas TAp63 overexpression promotes lipid storage, an effect reversible by IKKß silencing. Our findings indicate an unexpected role of the p63/IKKß/ER stress pathway in lipid metabolism and liver disease.


Assuntos
Estresse do Retículo Endoplasmático , Fígado Gorduroso/metabolismo , Quinase I-kappa B/metabolismo , Fígado/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Adulto , Animais , Fígado Gorduroso/genética , Fígado Gorduroso/fisiopatologia , Feminino , Hepatócitos/metabolismo , Humanos , Quinase I-kappa B/genética , Metabolismo dos Lipídeos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Transativadores/genética , Transativadores/metabolismo , Fatores de Transcrição/genética , Proteínas Supressoras de Tumor/genética , Proteína 1 de Ligação a X-Box/genética , Proteína 1 de Ligação a X-Box/metabolismo
17.
Eur J Pharm Biopharm ; 114: 69-78, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-28087378

RESUMO

The regeneration of articular cartilage remains an unresolved question despite the current access to a variety of tissue scaffolds activated with growth factors relevant to this application. Further advances might result from combining more than one of these factors; here, we propose a scaffold composition optimized for the dual delivery of BMP-7 and TGF-ß3, two proteins with described chondrogenic activity. First, we tested in a mesenchymal stem cell micromass culture with TGF-ß3 whether the exposure to microspheres loaded with BMP-7 would improve cartilage formation. Histology and qRT-PCR data confirmed that the sustained release of BMP-7 cooperates with TGF-ß3 towards chondrogenic differentiation. Then, we optimized a scaffold prototype for tissue culture and dual encapsulation of BMP-7 and TGF-ß3. The scaffolds were prepared from poly(lactic-co-glycolic acid), and BMP-7/TGF-ß3 were loaded as nanocomplexes with heparin and Tetronic 1107. The scaffolds showed the sustained release of both proteins over four weeks, with minimal burst effect. We finally cultured human mesenchymal stem cells on these scaffolds, in the absence of exogenous chondrogenic factor supplementation. The cells cultured on the scaffolds loaded with BMP-7 and TGF-ß3 showed clear signs of cartilage formation macroscopically and histologically. RT-PCR studies confirmed a clear upregulation of cartilage markers SOX9 and Aggrecan. In summary, scaffolds encapsulating BMP-7 and TGF-ß3 can efficiently deliver a cooperative growth factor combination that drives efficient cartilage formation in human mesenchymal stem cell cultures. These results open attractive perspectives towards in vivo translation of this technology in cartilage regeneration experiments.


Assuntos
Proteína Morfogenética Óssea 7/administração & dosagem , Proteína Morfogenética Óssea 7/farmacologia , Condrócitos/efeitos dos fármacos , Condrogênese/efeitos dos fármacos , Tecidos Suporte/química , Fator de Crescimento Transformador beta3/administração & dosagem , Fator de Crescimento Transformador beta3/farmacologia , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Combinação de Medicamentos , Composição de Medicamentos , Humanos , Ácido Láctico , Células-Tronco Mesenquimais/efeitos dos fármacos , Ácido Poliglicólico , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Regeneração/efeitos dos fármacos , Engenharia Tecidual/métodos
18.
Sci Rep ; 6: 37007, 2016 11 16.
Artigo em Inglês | MEDLINE | ID: mdl-27849057

RESUMO

Cellular senescence is often considered a protection mechanism triggered by conditions that impose cellular stress. Continuous proliferation, DNA damaging agents or activated oncogenes are well-known activators of cell senescence. Apart from a characteristic stable cell cycle arrest, this response also involves a proinflammatory phenotype known as senescence-associated secretory phenotype (SASP). This, together with the widely known interference with senescence pathways by some oncoviruses, had led to the hypothesis that senescence may also be part of the host cell response to fight virus. Here, we evaluate this hypothesis using vesicular stomatitis virus (VSV) as a model. Our results show that VSV replication is significantly impaired in both primary and tumor senescent cells in comparison with non-senescent cells, and independently of the stimulus used to trigger senescence. Importantly, we also demonstrate a protective effect of senescence against VSV in vivo. Finally, our results identify the SASP as the major contributor to the antiviral defense exerted by cell senescence in vitro, and points to a role activating and recruiting the immune system to clear out the infection. Thus, our study indicates that cell senescence has also a role as a natural antiviral defense mechanism.


Assuntos
Senescência Celular , Sistema Imunitário/fisiologia , Estomatite Vesicular/prevenção & controle , Vesiculovirus/patogenicidade , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Células Cultivadas , Fibroblastos/citologia , Fibroblastos/virologia , Humanos , Camundongos , Inoculações Seriadas , Estomatite Vesicular/imunologia , Vesiculovirus/fisiologia , Replicação Viral
19.
J Control Release ; 238: 263-271, 2016 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-27476608

RESUMO

The design of nanomedicines with suitable physicochemical characteristics for the lymphatic targeting of drugs is critical in order to reach the lymph nodes, where metastatic cells often accumulate. Based on the known effect of particle size and surface hydrophilicity on the capacity of nanocarriers to reach the lymph nodes, here we report the formation and characterization of 100nm polyglutamic acid-polyethylene glycol (PGA-PEG) nanocapsules together with the assessment of their potential for the treatment of cancer with lymphatic metastatic spread. To this purpose, we first studied the biodistribution of fluorescently labeled PGA-PEG nanocapsules (100nm), following, either intravenous or subcutaneous administration. The results confirmed the accumulation of nanocapsules in the lymphatic system, especially upon subcutaneous administration. Next, we evaluated the efficacy and toxicity of the docetaxel-loaded nanocapsules in an orthotopic lung cancer model that metastasizes to the lymph nodes. As expected from the rational design, DCX-loaded PGA-PEG nanocapsules exhibited a greatly enhanced antitumoral efficacy and a reduced toxicity when compared with the commercial formulation Taxotere®. Furthermore, the administration of DCX-loaded PGA-PEG nanocapsules resulted in the practical elimination of the metastatic load in the mediastinal lymph nodes, whereas the treatment with the commercial formulation had a minor effect. Overall, these findings underscore the potential of PGA-PEG nanocapsules for the delivery of anticancer drugs to both, the tumor tissue and the metastatic lymph nodes. Therefore, they represent a promising therapy for the treatment of lung metastatic cancer.


Assuntos
Antineoplásicos/administração & dosagem , Neoplasias Pulmonares/tratamento farmacológico , Metástase Linfática/prevenção & controle , Nanocápsulas/química , Ácido Poliglutâmico/química , Taxoides/administração & dosagem , Animais , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Docetaxel , Feminino , Humanos , Pulmão/efeitos dos fármacos , Pulmão/patologia , Neoplasias Pulmonares/patologia , Linfonodos/efeitos dos fármacos , Linfonodos/patologia , Metástase Linfática/patologia , Camundongos SCID , Taxoides/uso terapêutico
20.
J Nanobiotechnology ; 14(1): 51, 2016 Jun 23.
Artigo em Inglês | MEDLINE | ID: mdl-27339609

RESUMO

BACKGROUND: Metastases are the most common reason of cancer death in patients with solid tumors. Lymph nodes, once invaded by tumor cells, act as reservoirs before cancer cells spread to distant organs. To address the limited access of intravenously infused chemotherapeutics to the lymph nodes, we have developed PEGylated polyglutamic acid nanocapsules (PGA-PEG NCs), which have shown ability to reach and to accumulate in the lymphatic nodes and could therefore act as nanotransporters. Once in the lymphatics, the idea is that these nanocapsules would selectively interact with cancer cells, while avoiding non-specific interactions with immune cells and the appearance of subsequent immunotoxicity. RESULTS: The potential of the PGA-PEG NCs, with a mean size of 100 nm and a negative zeta potential, to selectively reach metastatic cancer cells, has been explored in a novel 3D model that mimics an infiltrated lymph node. Our 3D model, a co-culture of cancer cells and lymphocytes, allows performing experiments under dynamic conditions that simulate the lymphatic flow. After perfusion of the nanocarriers, we observe a selective interaction with the tumor cells. Efficacy studies manifest the need to develop specific therapies addressed to treat metastatic cells that can be in a dormant state. CONCLUSIONS: We provide evidence of the ability of PGA-PEG NCs to selectively interact with the tumor cells in presence of lymphocytes, highlighting their potential in cancer therapeutics. We also state the importance of designing precise in vitro models that allow performing mechanistic assays, to efficiently develop and evaluate specific therapies to confront the formation of metastasis.


Assuntos
Linfonodos/efeitos dos fármacos , Modelos Biológicos , Nanocápsulas/química , Polietilenoglicóis/química , Ácido Poliglutâmico/química , Células A549 , Antineoplásicos/química , Antineoplásicos/farmacologia , Carbocianinas/química , Ciclo Celular/efeitos dos fármacos , Técnicas de Cocultura , Docetaxel , Corantes Fluorescentes/química , Humanos , Células Jurkat , Linfonodos/metabolismo , Linfonodos/patologia , Metástase Linfática/patologia , Nanocápsulas/ultraestrutura , Tamanho da Partícula , Reologia , Taxoides/química , Taxoides/farmacologia
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...