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1.
Nat Commun ; 11(1): 4640, 2020 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-32934232

RESUMO

Small molecule inhibitors are prime reagents for studies in microtubule cytoskeleton research, being applicable across a range of biological models and not requiring genetic engineering. However, traditional chemical inhibitors cannot be experimentally applied with spatiotemporal precision suiting the length and time scales inherent to microtubule-dependent cellular processes. We have synthesised photoswitchable paclitaxel-based microtubule stabilisers, whose binding is induced by photoisomerisation to their metastable state. Photoisomerising these reagents in living cells allows optical control over microtubule network integrity and dynamics, cell division and survival, with biological response on the timescale of seconds and spatial precision to the level of individual cells within a population. In primary neurons, they enable regulation of microtubule dynamics resolved to subcellular regions within individual neurites. These azobenzene-based microtubule stabilisers thus enable non-invasive, spatiotemporally precise modulation of the microtubule cytoskeleton in living cells, and promise new possibilities for studying intracellular transport, cell motility, and neuronal physiology.


Assuntos
Microtúbulos/química , Paclitaxel/química , Linhagem Celular Tumoral , Citoesqueleto/química , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Humanos , Isomerismo , Microtúbulos/metabolismo , Neurônios/química , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Paclitaxel/farmacologia
2.
Phys Rev Lett ; 125(5): 058101, 2020 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-32794890

RESUMO

Diffusion of tracer particles in the cytoplasm of mammalian cells is often anomalous with a marked heterogeneity even within individual particle trajectories. Despite considerable efforts, the mechanisms behind these observations have remained largely elusive. To tackle this problem, we performed extensive single-particle tracking experiments on quantum dots in the cytoplasm of living mammalian cells at varying conditions. Analyses of the trajectories reveal a strong, microtubule-dependent subdiffusion with antipersistent increments and a substantial heterogeneity. Furthermore, particles stochastically switch between different mobility states, most likely due to transient associations with the cytoskeleton-shaken endoplasmic reticulum network. Comparison to simulations highlight that all experimental observations can be fully described by an intermittent fractional Brownian motion, alternating between two states of different mobility.


Assuntos
Citoplasma/metabolismo , Modelos Biológicos , Citoesqueleto de Actina/metabolismo , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Simulação por Computador , Citocalasina D/farmacologia , Citoplasma/efeitos dos fármacos , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Difusão , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Células HeLa , Humanos , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Nocodazol/farmacologia , Pontos Quânticos , Processos Estocásticos , Tiazolidinas/farmacologia
3.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 36(3): 193-197, 2020 Mar.
Artigo em Chinês | MEDLINE | ID: mdl-32389165

RESUMO

Objective To investigate the role of Ras homolog gene (Rho) A/Rho-associated coiled-coil containing protein kinase (ROCK) signaling pathway in tumor necrosis factor α (TNF-α) promoting hyper-permeability of vascular endothelial cells infected by Listeria monocytogenes (Lm) . Methods The cultured human umbilical vein endothelial cells (HUVECs) were divided into a control group (uninfected cells), TNF-α treatment group (100 ng/mL TNF-α, for 2 hours), Lm infection group (infected with MOI=10 Lm for 2 hours, then added gentamicin for 0.5 hour), Lm infection and TNF-α treatment group (infected with Lm and then treated with 100 ng/mL TNF-α for 2 hours), and Y-27632 inhibitor group combined with Lm infection and TNF-α treatment (treated with 50 µmol/L ROCK inhibitor Y-27632 for 30 minutes, and then Lm infection and TNF-α treatment as above). The protein levels of RhoA, zonula occluden-1 (ZO-1), occludin and ROCK in HUVECs were detected by Western blot analysis; the permeability of HUVECs was analyzed by the horseradish peroxidase (HRP) leakage; and the distribution of F-actin in HUVECs was detected by fluorescein isothiocyanate (FITC)-labeled phalloidine staining. Results TNF-α reduced the expression of tight junction protein ZO-1 and occludin in Lm-infected HUVECs, promoted its hyper-permeability and cytoskeletal rearrangement, and up-regulated the expression of RhoA and ROCK. ROCK inhibitor Y-27632 obviously inhibited the cytoskeleton rearrangement and hyper-permeability of HUVECs induced by TNF-α. Conclusion TNF-α can enhance hyper-permeability of HUVECs infected by Lm, which may be regulated by RhoA/Rock signaling pathway.


Assuntos
Células Endoteliais da Veia Umbilical Humana/microbiologia , Listeria monocytogenes , Transdução de Sinais , Fator de Necrose Tumoral alfa/farmacologia , Quinases Associadas a rho/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Células Cultivadas , Citoesqueleto/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Humanos , Permeabilidade
4.
Int J Nanomedicine ; 15: 2971-2986, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32431496

RESUMO

Background: Due to their extraordinary physical and chemical properties, MoS2 nanosheets (MSNs) are becoming more widely used in nanomedicine. However, their influence on immune systems remains unclear. Materials and Methods: Two few-layered MSNs at sizes of 100-250 nm (S-MSNs) and 400-500 nm (L-MSNs) were used in this study. Bone marrow-derived dendritic cells (DCs) were exposed to both MSNs at different doses (0, 8, 16, 32, 64, 128 µg/mL) for 48 h and subjected to analyses of surface marker expression, cytokine secretion, lymphoid homing and in vivo T cell priming. Results: Different-sized MSNs of all doses did not affect the viability of DCs. The expression of CD40, CD80, CD86 and CCR7 was significantly higher on both S-MSN- and L-MSN-treated DCs at a dose of 128 µg/mL. As the dose of MSN increased, the secretion of IL-12p70 remained unchanged, the secretion of IL-1ß decreased, and the production of TNF-α increased. A significant increase in IL-6 was observed in the 128 µg/mL L-MSN-treated DCs. In particular, MSN treatment dramatically improved the ex vivo movement and in vivo homing ability of both the local resident and blood circulating DCs. Furthermore, the cytoskeleton rearrangement regulated by ROS elevation was responsible for the enhanced homing ability of the MSNs. More robust CD4+ and CD8+ T cell proliferation and activation (characterized by high expression of CD107a, CD69 and ICOS) was observed in mice vaccinated with MSN-treated DCs. Importantly, exposure to MSNs did not interrupt LPS-induced DC activation, homing and T cell priming. Conclusion: Few-layered MSNs ranging from 100 to 500 nm in size could play an immunostimulatory role in enhancing DC maturation, migration and T cell elicitation, making them a good candidate for vaccine adjuvants. Investigation of this study will not only expand the applications of MSNs and other new transition metal dichalcogenides (TMDCs) but also shed light on the in vivo immune-risk evaluation of MSN-based nanomaterials.


Assuntos
Diferenciação Celular , Movimento Celular , Células Dendríticas/citologia , Células Dendríticas/imunologia , Dissulfetos/farmacologia , Molibdênio/farmacologia , Nanopartículas/química , Linfócitos T/citologia , Linfócitos T/imunologia , Animais , Diferenciação Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Células Dendríticas/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Masculino , Camundongos Endogâmicos C57BL , Nanopartículas/ultraestrutura , Espécies Reativas de Oxigênio/metabolismo , Linfócitos T/efeitos dos fármacos
5.
Chem Biol Interact ; 325: 109109, 2020 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-32376239

RESUMO

A series of 10 natural and semisynthetic flavonoids (1 to 10) were obtained from Gardenia oudiepe (Rubiaceae), an endemic plant from New Caledonia. Most of them were polymethoxylated flavones (PMFs) of rare occurrence. After a cell viability screening test, PMFs 2 and 3 showed significant cytotoxic activity against A2058 human melanoma cells (IC50 = 3.92 and 8.18 µM, respectively) and were selected for in-depth pharmacological assays. Both compounds inhibited cell migration and induced apoptosis and cell cycle arrest after 72h of treatment. Immunofluorescence assays indicated that these outcomes were possibly related to the induction of cytoskeleton disruption associated to actin and tubulin depolymerization. These data were confirmed by molecular docking studies, which showed a good interaction between PMFs 2 and 3 and tubulin, particularly at the colchicine binding site. As A2058 are considered as chemoresistant to conventional chemotherapy, compounds 2 and 3 (½IC50) were associated to clinically-used antimelanoma drugs (vemurafenib and dacarbazine) and combined therapies efficacy was assessed by the MTT assay. PMFs 2 restored the sensitivity of A2058 cells to dacarbazine treatment (IC50 = 49.38 µM vs. >100 µM). Taken together, these data suggest that PMFs from G. oudiepe could be potential leaders for the design of new antimelanoma drugs.


Assuntos
Apoptose/efeitos dos fármacos , Citoesqueleto/efeitos dos fármacos , Flavonas/farmacologia , Gardenia/química , Melanoma/patologia , Mutação , Proteínas Proto-Oncogênicas B-raf/genética , Antineoplásicos/farmacologia , Caspase 3/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Citoesqueleto/metabolismo , Sinergismo Farmacológico , Ativação Enzimática/efeitos dos fármacos , Flavonas/química , Flavonas/metabolismo , Humanos , Simulação de Acoplamento Molecular , Conformação Proteica , Relação Estrutura-Atividade , Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismo
6.
Nat Methods ; 17(6): 587-593, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32341544

RESUMO

The mechanical phenotype of a cell is an inherent biophysical marker of its state and function, with many applications in basic and applied biological research. Microfluidics-based methods have enabled single-cell mechanophenotyping at throughputs comparable to those of flow cytometry. Here, we present a standardized cross-laboratory study comparing three microfluidics-based approaches for measuring cell mechanical phenotype: constriction-based deformability cytometry (cDC), shear flow deformability cytometry (sDC) and extensional flow deformability cytometry (xDC). All three methods detect cell deformability changes induced by exposure to altered osmolarity. However, a dose-dependent deformability increase upon latrunculin B-induced actin disassembly was detected only with cDC and sDC, which suggests that when exposing cells to the higher strain rate imposed by xDC, cellular components other than the actin cytoskeleton dominate the response. The direct comparison presented here furthers our understanding of the applicability of the different deformability cytometry methods and provides context for the interpretation of deformability measurements performed using different platforms.


Assuntos
Citometria de Fluxo/métodos , Microfluídica/métodos , Actinas/efeitos dos fármacos , Compostos Bicíclicos Heterocíclicos com Pontes/administração & dosagem , Forma Celular/efeitos dos fármacos , Tamanho Celular/efeitos dos fármacos , Citoesqueleto/efeitos dos fármacos , Relação Dose-Resposta a Droga , Células HL-60 , Humanos , Processamento de Imagem Assistida por Computador , Tiazolidinas/administração & dosagem
7.
Chem Biol Interact ; 323: 109063, 2020 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-32224134

RESUMO

Exposure to TiO2 NPs induces several cellular alterations after NPs uptake including disruption of cytoskeleton that is crucial for lung physiology but is not considered as a footprint of cell damage. We aimed to investigate cytoskeleton disturbances and the impact on cell migration induced by an acute TiO2 NPs exposure (24 h) and the recovery capability after 6 days of NPs-free treatment, which allowed investigating if cytoskeleton damage was reversible. Exposure to TiO2 NPs (10 µg/cm2) for 24 h induced a decrease 20.2% and 25.1% in tubulin and actin polymerization. Exposure to TiO2 NPs (10 µg/cm2) for 24 h followed by 6 days of NPs-free had a decrease of 26.6% and 21.3% in tubulin and actin polymerization, respectively. The sustained exposure for 7 days to 1 µg/cm2 and 10 µg/cm2 induced a decrease of 22.4% and 30.7% of tubulin polymerization respectively, and 28.7% and 46.2% in actin polymerization. In addition, 24 h followed 6 days of NPs-free exposure of TiO2 NPs (1 µg/cm2 and 10 µg/cm2) decreased cell migration 40.7% and 59.2%, respectively. Cells exposed (10 µg/cm2) for 7 days had a decrease of 65.5% in cell migration. Ki67, protein surfactant B (SFTPB) and matrix metalloprotease 2 (MMP2) were analyzed as genes related to lung epithelial function. The results showed a 20% of Ki67 upregulation in cells exposed for 24 h to 10 µg/cm2 TiO2 NPs while a downregulation of 20% and 25.8% in cells exposed to 1 µg/cm2 and 10 µg/cm2 for 24 h followed by 6 days of NPs-free exposure. Exposure to 1 µg/cm2 and 10 µg/cm2 for 24 h and 7 days upregulates SFTPB expression in 53% and 59% respectively, MMP2 expression remain unchanged. In conclusion, exposure of TiO2 NPs affected cytoskeleton of lung epithelial cells irreversibly but this damage was not cumulative.


Assuntos
Citoesqueleto/patologia , Células Epiteliais/patologia , Pulmão/patologia , Nanopartículas/toxicidade , Titânio/toxicidade , Células A549 , Actinas/metabolismo , Movimento Celular/efeitos dos fármacos , Tamanho Celular , Sobrevivência Celular/efeitos dos fármacos , Citoesqueleto/efeitos dos fármacos , Endocitose , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/ultraestrutura , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Antígeno Ki-67/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Nanopartículas/ultraestrutura , Polimerização , Precursores de Proteínas/metabolismo , Proteínas Associadas a Surfactantes Pulmonares/metabolismo , Tubulina (Proteína)/metabolismo
8.
Nat Commun ; 11(1): 1435, 2020 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-32188843

RESUMO

Regeneration of corneal stroma has always been a challenge due to its sophisticated structure and keratocyte-fibroblast transformation. In this study, we fabricate grid poly (ε-caprolactone)-poly (ethylene glycol) microfibrous scaffold and infuse the scaffold with gelatin methacrylate (GelMA) hydrogel to obtain a 3 D fiber hydrogel construct; the fiber spacing is adjusted to fabricate optimal construct that simulates the stromal structure with properties most similar to the native cornea. The topological structure (3 D fiber hydrogel, 3 D GelMA hydrogel, and 2 D culture dish) and chemical factors (serum, ascorbic acid, insulin, and ß-FGF) are examined to study their effects on the differentiation of limbal stromal stem cells to keratocytes or fibroblasts and the phenotype maintenance, in vitro and in vivo tissue regeneration. The results demonstrate that fiber hydrogel and serum-free media synergize to provide an optimal environment for the maintenance of keratocyte phenotype and the regeneration of damaged corneal stroma.


Assuntos
Substância Própria/fisiologia , Gelatina/farmacologia , Hidrogéis/farmacologia , Metacrilatos/farmacologia , Poliésteres/farmacologia , Polietilenoglicóis/farmacologia , Regeneração , Animais , Substância Própria/efeitos dos fármacos , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Limbo da Córnea/citologia , Masculino , Ratos Sprague-Dawley , Regeneração/efeitos dos fármacos , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Estresse Mecânico , Células Estromais/citologia , Células Estromais/efeitos dos fármacos , Suínos , Tecidos Suporte/química , Vimentina/metabolismo
9.
Nat Biotechnol ; 38(4): 460-470, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32094658

RESUMO

Generation of pancreatic ß cells from human pluripotent stem cells (hPSCs) holds promise as a cell replacement therapy for diabetes. In this study, we establish a link between the state of the actin cytoskeleton and the expression of pancreatic transcription factors that drive pancreatic lineage specification. Bulk and single-cell RNA sequencing demonstrated that different degrees of actin polymerization biased cells toward various endodermal lineages and that conditions favoring a polymerized cytoskeleton strongly inhibited neurogenin 3-induced endocrine differentiation. Using latrunculin A to depolymerize the cytoskeleton during endocrine induction, we developed a two-dimensional differentiation protocol for generating human pluripotent stem-cell-derived ß (SC-ß) cells with improved in vitro and in vivo function. SC-ß cells differentiated from four hPSC lines exhibited first- and second-phase dynamic glucose-stimulated insulin secretion. Transplantation of islet-sized aggregates of these cells rapidly reversed severe preexisting diabetes in mice at a rate close to that of human islets and maintained normoglycemia for at least 9 months.


Assuntos
Engenharia Celular/métodos , Citoesqueleto/metabolismo , Células Secretoras de Insulina/citologia , Células-Tronco Pluripotentes/citologia , Actinas/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Diferenciação Celular/efeitos dos fármacos , Linhagem da Célula/efeitos dos fármacos , Terapia Baseada em Transplante de Células e Tecidos , Células Cultivadas , Citoesqueleto/efeitos dos fármacos , Diabetes Mellitus/terapia , Endoderma/citologia , Endoderma/metabolismo , Proteínas de Homeodomínio/metabolismo , Humanos , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/transplante , Camundongos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Células-Tronco Pluripotentes/metabolismo , Tiazolidinas/farmacologia , Transativadores/metabolismo , Moduladores de Tubulina/farmacologia
10.
Environ Res ; 183: 109236, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32062183

RESUMO

Cylindrospermopsin (CYN) is a widely distributed cyanobacterial toxin in water bodies and is considered to pose growing threats to human and environmental health. Although its potential toxicity has been reported, its effects on the vascular system are poorly understood. In this study, we examined the toxic effects of CYN on vascular development and the possible mechanism of vascular toxicity induced by CYN using zebrafish embryos and human umbilical vein endothelial cells (HUVECs). CYN exposure induced abnormal vascular development and led to an increase in the growth of common cardinal vein (CCV), in which CCV remodeling was delayed as reflected by the larger CCV area and wider ventral diameter. CYN decreased HUVECs viability, inhibited HUVECs migration, promoted HUVECs apoptosis, destroyed cytoskeleton, and increased intracellular ROS levels. Additionally, CYN could promote the expression of Bax, Bcl-2, and MLC-1 and inhibit the expression of ITGB1, Rho, ROCK, and VIM-1. Taken together, CYN may induce cytoskeleton damage and promote vascular endothelial cell apoptosis by the Rho/ROCK signaling pathway, leading to abnormal vascular development. The current results provide potential insight into the mechanism of CYN toxicity in angiocardiopathy and are beneficial for understanding the environmental risks of CYN for aquatic organisms and human health.


Assuntos
Apoptose , Toxinas Bacterianas , Uracila/análogos & derivados , Animais , Apoptose/efeitos dos fármacos , Toxinas Bacterianas/toxicidade , Citoesqueleto/efeitos dos fármacos , Humanos , Transdução de Sinais , Cordão Umbilical/citologia , Uracila/toxicidade
11.
Chemosphere ; 248: 126066, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32050317

RESUMO

Silver nanoparticles (AgNPs) are an emerging contaminant, currently considered to be a significant potential risk to the coastal environment. To further test potential risk, and to determine effect concentrations and sensitive response parameters, toxic effects of environmentally relevant AgNP concentrations on the seagrass Cymodocea nodosa were evaluated. Alterations of the cytoskeleton, endoplasmic reticulum, ultrastructure, photosystem II function, oxidative stress markers, cell viability, and leaf, rhizome and root elongation in C. nodosa exposed to AgNP concentrations (0.0002-0.2 mg L-1) under laboratory conditions for 8 days were examined. An increase in H2O2 level, indicating oxidative stress, occurred after the 4th day even at 0.0002 mg L-1. Increased antioxidant enzyme activity, potentially contributing to H2O2 level decline at the end of the experiment, and reduced protein content were also observed. Actin filaments started to diminish on the 6th day at 0.02 mg L-1; microtubule, endoplasmic reticulum, chloroplast and mitochondrion disturbance appeared after 8 days at 0.02 mg L-1, while toxic effects were generally more acute at 0.2 mg L-1. A dose-dependent leaf elongation inhibition was also observed; as for juvenile leaves, toxicity index increased from 2.8 to 40.7% with concentration. Hydrogen peroxide (H2O2) overproduction and actin filament disruption appeared to be the most sensitive response parameters, and thus could be utilized as early warning indicators of risk to seagrass meadows. A risk quotient of 1.33 was calculated, confirming previous findings, that AgNPs may pose a significant risk to the coastal environment.


Assuntos
Alismatales/fisiologia , Nanopartículas Metálicas/toxicidade , Prata/toxicidade , Poluentes Químicos da Água/toxicidade , Alismatales/efeitos dos fármacos , Alismatales/ultraestrutura , Antioxidantes/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Citoesqueleto/efeitos dos fármacos , Peróxido de Hidrogênio/metabolismo , Microtúbulos/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Complexo de Proteína do Fotossistema II/metabolismo , Folhas de Planta/metabolismo
12.
EBioMedicine ; 51: 102583, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31901866

RESUMO

BACKGROUND: Heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1 is an important RNA-binding protein that affects the RNA processing, splicing, transport and stability of many genes. hnRNPA2/B1 is expressed during proliferation and metastasis of various cancer types and promotes such processes. However, the precise role and mechanism of hnRNPA2/B1 in breast cancer remain unclear. METHODS: The association of hnRNPA2/B1 with breast cancer metastasis was assessed using tissue chips, mouse models and publicly available data. The role and mechanism of hnRNPA2/B1 in breast cancer metastasis were studied in cell lines and mouse models. FINDINGS: In contrast to other cancer research findings, hnRNPA2/B1 expression was negatively correlated with breast cancer metastasis. hnRNPA2/B1 inhibited MDA-MB-231 triple-negative breast cancer (TNBC) cell metastasis in vitro and in vivo. hnRNPA2/B1 knockout activated ERK-MAPK/Twist and GR-beta/TCF4 pathways but inhibited STAT3 and WNT/TCF4 signalling pathways. Profilin 2 (PFN2) promoted breast cancer cell migration and invasion, whereas hnRNPA2/B1 bound directly to the UAGGG locus in the 3'-untranslated region of PFN2 mRNA and reduced the stability of PFN2 mRNA. INTERPRETATION: Our data supported the role of hnRNPA2/B1 in tumour metastasis risk and survival prediction in patients with breast cancer. The inhibitory role of hnRNPA2/B1 in metastasis was a balance of downstream multiple genes and signalling pathways. PFN2 downregulation by hnRNPA2/B1 might partly explain the inhibitory mechanism of hnRNPA2/B1 in breast cancer metastasis. Therefore, hnRNPA2/B1 might be used as a new prognostic biomarker and valuable molecular target for breast cancer treatments.


Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Genes Neoplásicos , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/metabolismo , Transdução de Sinais , Regiões 3' não Traduzidas/genética , Actinas/metabolismo , Animais , Neoplasias da Mama/diagnóstico , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Regulação para Baixo/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Técnicas de Inativação de Genes , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/genética , Humanos , Camundongos Endogâmicos BALB C , Camundongos Nus , Invasividade Neoplásica , Metástase Neoplásica , Profilinas/genética , Profilinas/metabolismo , Prognóstico , Estabilidade de RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Análise de Sobrevida
13.
Int J Mol Sci ; 21(2)2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31952224

RESUMO

Neuroindole melatonin, a hormone synthesized during the night mainly-but not exclusively-by the pineal gland of all vertebrates, functions as an adapting signal to the light-dark cycle. Its antioxidant, neuroprotective, anti-inflammatory, and antitumor properties are all well-known and widely reported. Melanoma is one of the most common carcinomas among developed countries and a type of tumor particularly difficult to fight back in medium/advanced stages. In contrast to other types of cancer, influence of melatonin on melanoma has been scarcely investigated. Thus, we have chosen the murine melanoma model B16-F10 cell line to study antiproliferative and antitumoral actions of melatonin. For this purpose, we combined both, cell culture and in vivo models. Melatonin reduced either, growth rate or migration of B16-F10 cells. Furthermore, melanin synthesis was altered by melatonin, promoting its synthesis. Melatonin also induced a G2/M cell cycle arrest and altered the cytoskeletal organization. To corroborate these results, we tested the effect of melatonin in the in vivo model of B16-F10 cell injection in the tail vein, which causes numerous lung metastases. Two different strategies of melatonin administration were used, namely, in drinking water, or daily intraperitoneal injection. However, contrary to what occurred in cell culture, no differences were observed between control and melatonin treated groups. Results obtained led us to conclude that melatonin exerts an antiproliferative and anti-migrating effect on this melanoma model by interfering with the cytoskeleton organization, but this pharmacological effect cannot be translated in vivo as the indole did not prevent metastasis in the murine model, suggesting that further insights into the effects of the indole in melanoma cells should be approached to understand this apparent paradox.


Assuntos
Proliferação de Células/efeitos dos fármacos , Citoesqueleto/efeitos dos fármacos , Melanoma Experimental/metabolismo , Melatonina/farmacologia , Actinas/genética , Actinas/metabolismo , Animais , Antioxidantes/administração & dosagem , Antioxidantes/farmacologia , Catalase/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Citoesqueleto/genética , Citoesqueleto/metabolismo , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundário , Melaninas/metabolismo , Melanoma Experimental/genética , Melanoma Experimental/patologia , Melatonina/administração & dosagem , Camundongos Endogâmicos C57BL , Superóxido Dismutase/metabolismo , Tiorredoxinas/metabolismo , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo
14.
Ecotoxicol Environ Saf ; 189: 109925, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31855841

RESUMO

Information on silver nanoparticle (AgNP) phytotoxicity on seagrasses is provided for the first time. Toxic effects of environmentally relevant AgNP concentrations on Halophila stipulacea were assessed to identify sensitive biomarkers, to determine threshold effect concentrations and to evaluate potential risks. Potential alterations in the cytoskeleton, endoplasmic reticulum, cell ultrastructure and viability, oxidative stress parameters and elongation in H. stipulacea leaves exposed to AgNP concentrations ranging from 0.0002 to 0.2 mg L-1 for 8 days were examined. The first signs of actin filament (AF) response in differentiating cells, exhibiting disorientation and slight bundling, were observed on the 4th day at 0.0002 mg L-1, while at the end of the experiment and at the higher concentrations, AFs were extremely bundled. Endoplasmic reticulum was affected in meristematic and differentiating cells; massive aggregations and loss of the "grainy" structure were observed, initially on the 6th day at 0.002 mg L-1. Effects on microtubules were detected on the last day at 0.2 mg L-1. An increase in H2O2 levels on the 4th and/or 6th day even at 0.0002 mg L-1 was followed by a decrease on, or up to the last day. On the 6th day at the lowest concentration, elevated malondialdehyde content, and superoxide dismutase and peroxidase activity were detected, indicating oxidative damage and antioxidant defense mechanism activation. Dead epidermal cells mainly occurred at 0.02 and 0.2 mg L-1, while no dead vein cells were detected. A significant inhibition in leaf elongation was observed only at 0.2 mg L-1. Therefore, AF disturbance in differentiating leaf cells, being a susceptible response parameter, could be regarded as an early warning indicator of risk posed by AgNPs to H. stipulacea meadows, while most of the remaining parameters examined also constitute useful biomarkers. The lowest observed effect concentration (0.0002 mg L-1), being within the range of environmentally relevant AgNPs concentrations, suggests the possibility of negative impacts of AgNPs on seagrass health. A risk quotient of 1.33 was calculated, indicating that AgNPs may pose a significant potential risk to the coastal environment. The data presented highlight the importance of future research to further investigate the seagrass-AgNP interactions, stress the need for a refinement of the environmental risk assessment of AgNPs and could be utilized for the design of biomonitoring programs for rational management of the coastal environment.


Assuntos
Hydrocharitaceae/fisiologia , Nanopartículas Metálicas/toxicidade , Prata/toxicidade , Antioxidantes/farmacologia , Citoesqueleto/efeitos dos fármacos , Hydrocharitaceae/efeitos dos fármacos , Peróxido de Hidrogênio , Malondialdeído/farmacologia , Microtúbulos/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Folhas de Planta/efeitos dos fármacos , Superóxido Dismutase/análise
15.
Mater Sci Eng C Mater Biol Appl ; 107: 110212, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31761208

RESUMO

A critical challenge to the development of tissue engineering small-diameter vascular grafts is to achieve rapid endothelialization and long-term anticoagulation. It is necessary to graft both adhesion and antithrombus factors onto the surface of polycaprolactone without burst release to promote endothelial cell affinity and antithrombogenicity. A bionic structure with a nanocoating that allows a biologically responsive, long-term release was employed in this work to enable the grafting of various bioactive molecules such as gelatin, polylysine, and heparin. This approach involved orienting the biomimetic vascular structures; the self-assembly grafting of gelatin, polylysine, and heparin nanoparticles; and genipin crosslinking to form a multiphase crosslinked nanocoating. In this biologically inspired design, vascular endothelialization and long-term anticoagulation were successfully induced through a matrix metallopeptidase 2 regulative mechanism by delivering both adhesion and antithrombus factors with a responsive, long-term release without burst release. The method provided a simple and effective approach for delivering dual factors for tissue engineering small-diameter vascular grafts.


Assuntos
Materiais Biomiméticos/química , Nanotecnologia , Materiais Biomiméticos/farmacologia , Coagulação Sanguínea/efeitos dos fármacos , Plaquetas/citologia , Prótese Vascular , Adesão Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Citoesqueleto/efeitos dos fármacos , Gelatina/química , Heparina/química , Heparina/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Metaloproteinase 2 da Matriz/química , Metaloproteinase 2 da Matriz/metabolismo , Nanofibras/química , Poliésteres/química , Polilisina/química , Propriedades de Superfície
16.
Biochim Biophys Acta Mol Basis Dis ; 1866(1): 165581, 2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-31672549

RESUMO

Exposure to environmental toxins, including hydrocarbon solvents, increases the risk of developing Parkinson's disease. An emergent hypothesis considers microtubule dysfunction as one of the crucial events in triggering neuronal degeneration in Parkinson's disease. Here, we used 2,5-hexanedione (2,5-HD), the toxic metabolite of n-hexane, to analyse the early effects of toxin-induced neurodegeneration on the cytoskeleton in multiple model systems. In PC12 cells differentiated with nerve growth factor for 5 days, we found that 2,5-HD treatment affected all the cytoskeletal components. Moreover, we observed alterations in microtubule distribution and stability, in addition to the imbalance of post-translational modifications of α-tubulin. Similar defects were also found in vivo in 2,5-HD-intoxicated mice. Interestingly, we also found that 2,5-HD exposure induced significant changes in microtubule stability in human skin fibroblasts obtained from Parkinson's disease patients harbouring mutations in PRKN gene, whereas it was ineffective in healthy donor fibroblasts, suggesting that the genetic background may really make the difference in microtubule susceptibility to this environmental Parkinson's disease-related toxin. In conclusion, by showing the imbalance between dynamic and stable microtubules in hydrocarbon-induced parkinsonism, our data support the crucial role of microtubule defects in triggering neurodegeneration.


Assuntos
Hexanonas/farmacologia , Microtúbulos/efeitos dos fármacos , Doença de Parkinson/etiologia , Doença de Parkinson/metabolismo , Animais , Linhagem Celular , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Camundongos , Microtúbulos/metabolismo , Fatores de Crescimento Neural/metabolismo , Células PC12 , Transtornos Parkinsonianos/induzido quimicamente , Transtornos Parkinsonianos/metabolismo , Ratos , Tubulina (Proteína)/metabolismo , Ubiquitina-Proteína Ligases/metabolismo
17.
Int J Nanomedicine ; 14: 9325-9336, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31819434

RESUMO

Background: During the past few years, immune cell therapy for malignant cancer has benefited a considerable amount of patients worldwide. As one of several promising candidates for immunotherapy, Vγ9Vδ2 γδ T cells have many unique biological advantages, such as non-MHC restriction and have been noted as the earliest source of IFN-γ. However, potentiating anti-tumor functions of γδ T cells has become of particular interest to researchers studying γδ T cell applications. Purpose: In this study, we proposed a nanotechnology-based methodology for strengthening γδ T cell functions. Methods: As a type of reliable, biocompatible material, chitosan nanoparticles (CSNPs) were used to enhance anti-tumor immunity of γδ T cells. Results: First, we found that the size of prepared CSNPs distributed 50 to 100 nm, and that CSNPs had optimal immunocompatibility. Then, we observed that CSNPs could induce α-tubulin cytoskeleton polarization and rearrangement, correlating with a higher killing ability of γδ T cells. Furthermore, we revealed that CSNPs could enhance Vγ9Vδ2 T cell anti-tumor functions by upregulating killing of related receptors, including NKG2D, CD56, FasL, and perforin secretion. Conclusion: Our work provided evidence of application for CSNPs based bio-carrier in immunotherapy. More importantly, we proposed a new strategy for enhancing γδ T cell anti-tumor activity using nanobiomaterial, which could benefit future clinical applications of γδ T cells.


Assuntos
Quitosana/farmacologia , Citoesqueleto/metabolismo , Nanopartículas/química , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Linfócitos T Citotóxicos/imunologia , Regulação para Cima , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Polaridade Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Citoesqueleto/efeitos dos fármacos , Citotoxicidade Imunológica/efeitos dos fármacos , Humanos , Ativação Linfocitária , Nanopartículas/ultraestrutura , Perforina/metabolismo , Tubulina (Proteína)/metabolismo
18.
Int J Mol Sci ; 20(23)2019 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-31817026

RESUMO

In children, ketamine sedation is often used during radiological procedures. Combined exposure of ketamine and radiation at doses that alone did not affect learning and memory induced permanent cognitive impairment in mice. The aim of this study was to elucidate the mechanism behind this adverse outcome. Neonatal male NMRI mice were administered ketamine (7.5 mg kg-1) and irradiated (whole-body, 100 mGy or 200 mGy, 137Cs) one hour after ketamine exposure on postnatal day 10. The control mice were injected with saline and sham-irradiated. The hippocampi were analyzed using label-free proteomics, immunoblotting, and Golgi staining of CA1 neurons six months after treatment. Mice co-exposed to ketamine and low-dose radiation showed alterations in hippocampal proteins related to neuronal shaping and synaptic plasticity. The expression of brain-derived neurotrophic factor, activity-regulated cytoskeleton-associated protein, and postsynaptic density protein 95 were significantly altered only after the combined treatment (100 mGy or 200 mGy combined with ketamine, respectively). Increased numbers of basal dendrites and branching were observed only after the co-exposure, thereby constituting a possible reason for the displayed alterations in behavior. These data suggest that the risk of radiation-induced neurotoxic effects in the pediatric population may be underestimated if based only on the radiation dose.


Assuntos
Região CA1 Hipocampal/patologia , Ketamina/toxicidade , Neurônios/patologia , Neurônios/efeitos da radiação , Radiação Ionizante , Animais , Animais Recém-Nascidos , Forma Celular/efeitos dos fármacos , Forma Celular/efeitos da radiação , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Citoesqueleto/efeitos da radiação , Masculino , Camundongos , Plasticidade Neuronal/efeitos dos fármacos , Plasticidade Neuronal/efeitos da radiação , Neurônios/efeitos dos fármacos , Proteoma/metabolismo
19.
Biomater Sci ; 7(12): 5451-5466, 2019 Nov 19.
Artigo em Inglês | MEDLINE | ID: mdl-31642822

RESUMO

Although surgical management of peripheral nerve injuries (PNIs) has improved over time, autografts are still the current "gold standard" treatment for PNIs, which presents numerous limitations. In an attempt to improve natural biomaterial-based nerve guidance conduits (NGCs), chitosan (CHT), a derivative of the naturally occurring biopolymer chitin, has been explored for peripheral nerve regeneration (PNR). In addition to CHT, keratin has gained enormous attention as a biomaterial and tissue engineering scaffolding. In this study, biomimetic CHT/keratin membranes were produced using a solvent casting technique. These membranes were broadly characterized in terms of their surface topography and physicochemical properties, with techniques such as Fourier Transform Infrared Spectroscopy (FTIR), Differential Scanning Calorimetry (DSC), contact angle, weight loss and water uptake measurements, Scanning Electron Microscopy (SEM) and Atomic Force Microscopy (AFM). Biological in vitro assays were also performed, where a preliminary cytotoxicity screening with the L929 fibroblast cell line revealed that the membranes and respective materials are suitable for cell culture. In addition, Schwann cells, fibroblasts and endothelial cells were directly seeded in the membranes. Quantitative and qualitative assays revealed that the addition of keratin enhanced cell viablity and adhesion. Based on the encouraging in vitro results, the in vivo angiogenic/antiangiogenic potential of CHT and CHT/keratin membranes was assessed, using an optimized chick embryo chorioallantoic membrane assay, where higher angiogenic responses were seen in keratin-enriched materials. Overall, the obtained results indicate the higher potential of CHT/keratin membranes for guided tissue regeneration applications in the field of PNR.


Assuntos
Quitosana/química , Queratinas/química , Membranas Artificiais , Nervos Periféricos/efeitos dos fármacos , Nervos Periféricos/fisiologia , Animais , Adesão Celular/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Embrião de Galinha , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Humanos , Camundongos , Neovascularização Fisiológica/efeitos dos fármacos , Regeneração Nervosa/efeitos dos fármacos
20.
Mar Drugs ; 17(10)2019 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-31623201

RESUMO

The purpose of the present paper is to investigate the mechanism of action of a pyroglutamate-modified peptide (pE-K092D) on in vitro growth inhibition of MDA-Pca-2b prostate cancer cells. This peptide was derived from a peptide previously isolated from the testis of the lesser spotted dogfish and identified as QLTPEALADEEEMNALAAR (K092D). The effect of the peptide on cell proliferation and cell death mechanisms was studied by flow cytometry. Cellular morphology and cytoskeleton integrity of peptide-treated cells were observed by immunofluorescence microscopy. Results showed the onset of peptide induced early cytoskeleton perturbation, inhibition of autophagy, inhibition of cell proliferation and, at the end, non-apoptotic cell death mechanisms (membrane destabilization and necrosis). All those mechanisms seem to contribute to MDA-Pca-2b growth inhibition by a main cytostatic fate.


Assuntos
Antineoplásicos/farmacologia , Cação (Peixe)/metabolismo , Peptídeos/farmacologia , Neoplasias da Próstata/tratamento farmacológico , Animais , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Citoesqueleto/efeitos dos fármacos , Humanos , Masculino , Pessoa de Meia-Idade , Ácido Pirrolidonocarboxílico/farmacologia
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