RESUMO
Infantile hemangiomas are the most common benign tumors of infancy, characterized by unregulated angiogenesis and endothelial cells with high mitotic rate. Although spontaneous regression occurs, sometimes treatment is required and alternatives to corticosteroids should be considered to reduce side effects. Imiquimod is an imidazoquinoline, approved for some skin pathologies other than hemangioma. It is proposed that the effectiveness of imiquimod comes from the activation of immune cells at tumor microenvironment. However, the possibility to selectively kill different cell types and to directly impede angiogenesis has been scarcely explored in vitro for endothelial cells. In this work we showed a dramatic cytotoxicity on hemangioma cell, with a significant lower IC50 value in hemangioma compared to normal endothelial cells and melanoma (employed as a non-endothelial tumor cell line). Nuclear morphometric and flow-cytometry assays revealed imiquimod-induced apoptosis on hemangioma and melanoma cells but a small percentage of senescence on normal endothelial cells. At sub-lethal conditions, cell migration, a key step in angiogenesis turned out to be inhibited in a tumor-selective manner along with actin cytoskeleton disorganization on hemangioma cells. Altogether, these findings pointed out the selective cytotoxic effects of imiquimod on transformed endothelial cells, evidencing the potential for imiquimod to be a therapeutic alternative to reduce extensive superficial hemangioma lesions.
Assuntos
Aminoquinolinas/farmacologia , Antineoplásicos/farmacologia , Hemangioma/patologia , Neoplasias Cutâneas/patologia , Aminoquinolinas/uso terapêutico , Animais , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/ultraestrutura , Sobrevivência Celular/efeitos dos fármacos , Senescência Celular/efeitos dos fármacos , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/ultraestrutura , Células Endoteliais/efeitos dos fármacos , Hemangioma/tratamento farmacológico , Humanos , Imiquimode , Melanoma Experimental/tratamento farmacológico , Melanoma Experimental/patologia , Camundongos , Neoplasias Cutâneas/tratamento farmacológico , Fibras de Estresse/efeitos dos fármacos , Fibras de Estresse/ultraestruturaRESUMO
Tumor aggressiveness is usually associated with metastasis. MDA-MB 231, a triple-negative breast cancer (TNBC), is an aggressive type of breast cancer and associated with early metastasis. The Rho/ROCK pathway is a key regulator of cell motility involving cytoskeleton regulation through stabilization of actin filaments and stress fiber formation. In this study we show that Fasudil, a ROCK inhibitor, inhibited the migration of MDA-MB 231 and A549 cells, without altering the viability of these cells at the concentration of 10 µM, modified tumor cell morphology, with disorganization of stress fibers and promotes activation of the canonical-Wnt/beta-catenin pathway. Therefore, Fasudil present a promising approach to the prevention of breast cancer metastasis through a different mechanism of action from the well-known one.
Assuntos
1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , Antineoplásicos/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Quinases Associadas a rho/antagonistas & inibidores , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/farmacologia , Carcinoma/tratamento farmacológico , Carcinoma/metabolismo , Carcinoma/patologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Núcleo Celular/patologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Relação Dose-Resposta a Droga , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Metástase Neoplásica/fisiopatologia , Metástase Neoplásica/prevenção & controle , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/fisiologia , Transdução de Sinais/efeitos dos fármacos , Fibras de Estresse/efeitos dos fármacos , Fibras de Estresse/metabolismo , Fibras de Estresse/patologia , Neoplasias de Mama Triplo Negativas/enzimologia , Neoplasias de Mama Triplo Negativas/patologia , beta Catenina/metabolismo , Quinases Associadas a rho/metabolismoRESUMO
The exposure to particulate matter with a mean aerodynamic diameter ≤10 µm (PM10) from urban zones is considered to be a risk factor in the development of cancer. The aim of this work was to determine if PM10 exposure induces factors related to the acquisition of a neoplastic phenotype, such as cytoskeletal remodeling, changes in the subcellular localization of p21(CIP1/WAF1), an increase in ß-galactosidase activity and changes in cell cycle. To test our hypothesis, PM10 from an industrial zone (IZ) and a commercial zone (CZ) were collected, and human adenocarcinoma lung cell cultures (A549) were exposed to a sublethal PM10 concentration (10 µg/cm(2)) for 24 h and 48 h. The results showed that PM10 exposure induced an increase in F-actin stress fibers and caused the cytoplasmic stabilization of p21(CIP1/WAF1) via phosphorylation at Thr(145) and Ser(146) and the phosphorylation of ERK1/2 on Thr(202). Changes in the cell cycle or apoptosis were not observed, but an increase in ß-galactosidase activity was detected. The PM10 from CZ caused more dramatic effects in lung cells. We conclude that PM10 exposure induced cytoplasmic p21(CIP1/WAF1) retention, ERK1/2 activation, cytoskeleton remodeling and the acquisition of a senescence-like phenotype in lung cells. These alterations could have mechanistic implications regarding the carcinogenic potential of PM10.
Assuntos
Senescência Celular/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Citoesqueleto/efeitos dos fármacos , Pulmão/efeitos dos fármacos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Material Particulado/toxicidade , Actinas/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Citoplasma/enzimologia , Citoesqueleto/enzimologia , Citoesqueleto/patologia , Ativação Enzimática , Humanos , Pulmão/enzimologia , Pulmão/patologia , Tamanho da Partícula , Fenótipo , Fosforilação , Transdução de Sinais/efeitos dos fármacos , Fibras de Estresse/efeitos dos fármacos , Fibras de Estresse/enzimologia , Fibras de Estresse/patologia , Fatores de Tempo , beta-Galactosidase/metabolismoRESUMO
Rotavirus infection induces an increase in [Ca(2+)](cyto), which in turn may affect the distribution of the cytoskeleton proteins in the infected cell. Changes in microfilaments, including the formation of stress fibers, were observed starting at 0.5 h.p.i. using fluorescent phalloidin. Western blot analysis indicated that RhoA is activated between 0.5 and 1 h.p.i. Neither the phosphorylation of RhoA nor the formation of stress fibers were observed in cells infected with virions pre-treated with an anti-VP5* non-neutralizing mAb, suggesting that RhoA activation is stimulated by the interaction of the virus with integrins forming the cell receptor complex. In addition, the structure of the tubulin cytoskeleton was also studied. Alterations of the microtubules were evident starting at 3 h.p.i. and by 7 h.p.i. when microtubules were markedly displaced toward the periphery of the cell cytoplasm. Loading of rotavirus-infected cells with either a Ca(2+) chelator (BAPTA) or transfection with siRNAs to silence NSP4, reversed the changes observed in both the microfilaments and microtubules distribution, but not the appearance of stress fibers. These results indicate that alterations in the distribution of actin microfilaments are initiated early during infection by the activation of RhoA, and that latter changes in the Ca(2+) homeostasis promoted by NSP4 during infection may be responsible for other alterations in the actin and tubulin cytoskeleton.
Assuntos
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Infecções por Rotavirus/enzimologia , Tubulina (Proteína)/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Citoesqueleto de Actina/efeitos dos fármacos , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Cálcio/metabolismo , Células Cultivadas , Quelantes/farmacologia , Chlorocebus aethiops , Ativação Enzimática/efeitos dos fármacos , Inativação Gênica/efeitos dos fármacos , Glicoproteínas/metabolismo , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Modelos Biológicos , Fosforilação/efeitos dos fármacos , RNA Interferente Pequeno/metabolismo , Rotavirus/efeitos dos fármacos , Rotavirus/fisiologia , Fibras de Estresse/efeitos dos fármacos , Fibras de Estresse/metabolismo , Fatores de Tempo , Toxinas Biológicas/metabolismo , Proteínas não Estruturais Virais/metabolismo , Vírion/imunologiaRESUMO
In this study we investigated the anti-inflammatory effects of chronic ethanol (EtOH) treatment on lipopolysaccharide (LPS)-stimulated C6 glioma cells. The cells were chronically treated with 200mM EtOH; coincubation with LPS and EtOH was obtained upon addition of 2µg/ml LPS to the incubation medium in the last 24h of EtOH exposure. We found that EtOH prevented the LPS-induced production of tumor necrosis factor α (TNFα) without decreasing cell viability. Either LPS treated or EtOH plus LPS treated cells presented upregulated glial fibrillary acidic protein (GFAP) and downregulated vimentin levels characterizing a program of reactive astrogliosis. Also, EtOH plus LPS stimulation greatly increased the oxidative stress generation evaluated by DCF-DA measurement, while either EtOH alone or LPS alone was unable to induce oxidative stress. Western blot analysis indicated that either EtOH, LPS or EtOH plus LPS treatments are unable to affect Akt/GSK3ß signaling pathway. However, LPS alone and EtOH plus LPS co-treatment inhibited Erk phosphorylation. A dramatic loss of stress fibers was found in EtOH exposed cells, evaluated by cytochemistry using phalloidin-fluorescein. However, LPS alone was not able to disrupt actin organization. Furthermore, cells co-incubated with LPS and EtOH presented reversion of the disrupted stress fibers provoked by EtOH. Supporting this action, RhoA and vinculin immunocontent were upregulated in response to EtOH plus LPS. Interestingly, EtOH suppresses the inflammatory cascade (TNFα production) in response to LPS. Concomitantly it sustains Erk inhibition, increases oxidative stress generation and induces reactive astrogliosis in the presence of LPS, conditions associated with neurotoxicity. The effects observed were not supported by actin reorganization. Altogether, these findings suggest that Erk signaling inhibition could play a role in both suppressing TNFα production and inducing oxidative stress generation and astrogliosis, therefore modulating a dual action of EtOH plus LPS in glial cells.
Assuntos
Anti-Inflamatórios/farmacologia , Etanol/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Neuroglia/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Animais , Western Blotting , Linhagem Celular Tumoral , Sobrevivência Celular , Eletroforese em Gel de Poliacrilamida , Gliose/induzido quimicamente , Gliose/metabolismo , Gliose/patologia , Imuno-Histoquímica , Inflamação/induzido quimicamente , Inflamação/metabolismo , Lipopolissacarídeos/toxicidade , Neuroglia/metabolismo , Neuroglia/patologia , Ratos , Fibras de Estresse/efeitos dos fármacos , Fibras de Estresse/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Lysophosphatidic acid (LPA) acts as a potent stimulator of tumorigenesis. Cell-cell adhesion disassembly, actin cytoskeletal alterations, and increased migratory potential are initial steps of colorectal cancer progression. However, the role that LPA plays in these events in this cancer type is still unknown. We explored this question by using Caco-2 cells, as colon cancer model, and treatment with LPA or pretreatment with different cell signalling inhibitors. Changes in the location of adherent junction proteins were examined by immunofluorescence and immunoblotting. The actin cytoskeleton organisation and focal adhesion were analysed by confocal microscopy. Rho-GTPase activation was analysed by the pull-down assay, FAK and Src activation by immunoblotting, and cell migration by the wound healing technique. We show that LPA induced adherent junction disassembly, perijunctional actin cytoskeletal reorganisation, and increased cell migration. These events were dependent on Src, Rho and Rock because their chemical inhibitors PP2, toxin A and Y27632, respectively, abrogated the effects of LPA. Moreover, we showed that Src acts upstream of RhoA in this signalling cascade and that LPA induces focal adhesion formation and FAK redistribution and activation in confluent monolayers. Focal adhesion formation was also observed in the front of migrating cells in response to LPA, and Rock inhibitor abolished this effect. In conclusion, our findings show that LPA modulates adherent junction disassembly, actin cytoskeletal disorganisation, and focal adhesion formation, conferring a migratory phenotype in colon tumour cells. We suggest a functional regulatory cascade that integrates RhoA-Rock and Src-FAK signalling to control these events during colorectal cancer progression.
Assuntos
Movimento Celular/efeitos dos fármacos , Neoplasias do Colo/patologia , Lisofosfolipídeos/farmacologia , Proteínas Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteína rhoA de Ligação ao GTP/metabolismo , Junções Aderentes/efeitos dos fármacos , Junções Aderentes/metabolismo , Células CACO-2 , Progressão da Doença , Quinase 1 de Adesão Focal/metabolismo , Adesões Focais/efeitos dos fármacos , Adesões Focais/metabolismo , Humanos , Fenótipo , Proteínas Proto-Oncogênicas pp60(c-src)/metabolismo , Fibras de Estresse/efeitos dos fármacos , Fibras de Estresse/metabolismo , Quinases Associadas a rho/metabolismoRESUMO
Central nervous system dysfunctions are among the most significant effects of exposure to ethanol and the glial cells that play an important role in maintaining neuronal function, are extremely involved with these effects. The actin cytoskeleton plays a crucial role in a wide variety of cellular functions, especially when there is some injury. Therefore the aim of the present study was to analyze the short-term effects of ethanol (50, 100 and 200 mM) on the cytoskeleton of C6 glioma cells. Here we report that acute ethanol exposure profoundly disrupts the actin cytoskeleton in C6 cells decreasing stress fiber formation and downregulating RhoA and vinculin immunocontent. In contrast, microtubule and GFAP networks were not altered. We further demonstrate that anti-oxidants prevent ethanol-induced actin alterations, suggesting that the actions of ethanol on the actin cytoskeleton are related with generation of reactive oxygen species (ROS) in these cells. Our results show that ethanol at concentrations described to be toxic to the central nervous system was able to target the cytoskeleton of C6 cells and this effect could be related with increased ROS generation. Therefore, we propose that the dynamic restructuring of the cytoskeleton of glial cells might contribute to the response to the injury provoked by binge-like ethanol exposure in brain.
Assuntos
Actinas/metabolismo , Fármacos do Sistema Nervoso Central/toxicidade , Citoesqueleto/efeitos dos fármacos , Etanol/toxicidade , Proteínas do Tecido Nervoso/metabolismo , Neuroglia/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Actinas/genética , Animais , Antioxidantes/farmacologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Fármacos do Sistema Nervoso Central/antagonistas & inibidores , Citoesqueleto/metabolismo , Citoesqueleto/ultraestrutura , Regulação para Baixo/efeitos dos fármacos , Etanol/antagonistas & inibidores , Neuroglia/metabolismo , Neuroglia/ultraestrutura , Concentração Osmolar , Estresse Oxidativo/efeitos dos fármacos , RNA Mensageiro/metabolismo , Ratos , Fibras de Estresse/efeitos dos fármacos , Fibras de Estresse/metabolismo , Fatores de Tempo , Vinculina/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
The objective of this work was to evaluate the relative role of the calcium phosphate surface chemistry and surface topography on human osteoblast behavior. Highly dense phosphate ceramics (single-phase hydroxyapatite HA and beta-tricalcium phosphates TCP) presenting two distinct nano roughnesses were produced. Some samples were gold-sputter coated in order to conveniently mask the surface chemical effects (without modification of the original roughness) and to study the isolated effect of surface topography on cellular behavior. Our results indicated that the nanotopography of the studied ceramics had no effect on the cellular adhesion (cell spreading, focal contacts and stress fibers formation). On the contrary, strong topographical effects were verified on cell proliferation and differentiation. Moreover, the phosphate chemistry was responsible for changes in adhesion, proliferation and cell differentiation. On TCP, it was shown that the main influent parameter was surface chemistry, which negatively affected the initial cell adhesion but positively affected the subsequent stage of proliferation and differentiation. On HA, the main influent parameter was surface topography, which increased cell differentiation but lowered proliferation.