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1.
PLoS One ; 8(3): e57461, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23505430

RESUMO

Amitozyn (Am) is a semi-synthetic drug produced by the alkylation of major celandine (Chelidonium majus L.) alkaloids with the organophosphorous compound N,N'N'-triethylenethiophosphoramide (ThioTEPA). We show here that the treatment of living cells with Am reversibly perturbs the microtubule cytoskeleton, provoking a dose-dependent cell arrest in the M phase. Am changed the dynamics of tubulin polymerization in vitro, promoted the appearance of aberrant mitotic phenotypes in HeLa cells and induced apoptosis by the activation of caspase-9, caspase-3 and PARP, without inducing DNA breaks. Am treatment of HeLa cells induced changes in the phosphorylation of the growth suppressor pRb that coincided with maximum mitotic index. The dose-dependent and reversible anti-proliferative effect of Am was observed in several transformed cell lines. Importantly, the drug was also efficient against multidrug-resistant, paclitaxel-resistant or p53-deficient cells. Our results thus open the way to further pre-clinical evaluation of Am.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Apoptose/efeitos dos fármacos , Benzofenantridinas/farmacologia , Segregação de Cromossomos/efeitos dos fármacos , Pontos de Checagem da Fase M do Ciclo Celular/efeitos dos fármacos , Compostos Organotiofosforados/farmacologia , Benzofenantridinas/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Células HeLa , Humanos , Compostos Organotiofosforados/química , Fenótipo , Multimerização Proteica/efeitos dos fármacos , Proteína do Retinoblastoma/metabolismo , Transdução de Sinais/efeitos dos fármacos , Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismo
2.
J Biochem ; 143(6): 821-32, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18343874

RESUMO

Specifically expressed at intercellular adherens junctions of endothelial cells, VE-cadherin is a receptor that exhibits particular self-association properties. Indeed, in vitro studies demonstrated that the extracellular part of VE-cadherin elaborates Ca(++)-dependent hexameric structures. We hypothesized that this assembly could be at the basis of a new cadherin-mediated cell-cell adhesion mechanism. To verify this assumption, we first demonstrated that VE-cadherin can elaborate hexamers at the cell surface of confluent endothelial cells. Second, mutations were introduced within the extracellular part of VE-cadherin to destabilize the hexamer. Following an in vitro screening, three mutants were selected, among which, one is able to elaborate only dimers. The selected mutations were expressed as C-terminal green fluorescent protein fusions in CHO cells. Despite their capacity to elaborate nascent cell-cell contacts, the mutants seem to be rapidly degraded and/or internalized. Altogether, our results suggest that the formation of VE-cadherin hexamers protects this receptor and might allow the elaboration of mature endothelial cell-cell junctions.


Assuntos
Junções Aderentes/metabolismo , Antígenos CD/química , Antígenos CD/metabolismo , Caderinas/química , Caderinas/metabolismo , Adesão Celular/fisiologia , Junções Intercelulares/metabolismo , Animais , Antígenos CD/genética , Células CHO , Caderinas/genética , Cálcio/metabolismo , Comunicação Celular , Cricetinae , Cricetulus , Reagentes de Ligações Cruzadas/farmacologia , Citoplasma/metabolismo , Dimerização , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Imunofluorescência , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Imunoprecipitação , Microscopia de Fluorescência , Mutação/genética , Veias Umbilicais/citologia , Veias Umbilicais/metabolismo
3.
BMC Biotechnol ; 7: 20, 2007 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-17437635

RESUMO

BACKGROUND: Angiogenesis assays are important tools for the identification of regulatory molecules and the potential development of therapeutic strategies to modulate neovascularization. Although numerous in vitro angiogenesis models have been developed in the past, they exhibit limitations since they do not recapitulate the entire angiogenic process or correspond to multi-step procedures that are not easy to use. Convenient, reliable, easily quantifiable and physiologically relevant assays are still needed for pharmacological screenings of angiogenesis. RESULTS: Here, we have optimized an angiogenesis model based on ES cell differentiation for screening experiments. We have established conditions leading to angiogenic sprouting of embryoid bodies during ES cell differentiation in type I three-dimensional collagen gels. Immunostaining experiments carried out during these cultures showed the formation of numerous buds comprising CD31 positive cells, after 11 days of culture of ES cells. Moreover, this one-step model has been validated in response to activators and inhibitors of angiogenesis. Sprouting was specifically stimulated in the presence of VEGF and FGF2. Alternatively, endothelial sprouting induced by angiogenic activators was inhibited by angiogenesis inhibitors such as angiostatin, TGFbeta and PF4. Sprouting angiogenesis can be easily quantified by image analysis after immunostaining of endothelial cells with CD31 pan-endothelial marker. CONCLUSION: Taken together, these data clearly validate that this one-step ES differentiation model constitutes a simple and versatile angiogenesis system that should facilitate, in future investigations, the screening of both activators and inhibitors of angiogenesis.


Assuntos
Indutores da Angiogênese/administração & dosagem , Inibidores da Angiogênese/administração & dosagem , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Neovascularização Fisiológica/efeitos dos fármacos , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Animais , Bioensaio/métodos , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Células Endoteliais/fisiologia , Camundongos , Neovascularização Fisiológica/fisiologia , Células-Tronco/fisiologia
4.
J Biol Chem ; 278(16): 14002-12, 2003 Apr 18.
Artigo em Inglês | MEDLINE | ID: mdl-12584200

RESUMO

Transmigration of neutrophils across the endothelium occurs at the cell-cell junctions where the vascular endothelium cadherin (VE cadherin) is expressed. This adhesive receptor was previously demonstrated to be involved in the maintenance of endothelium integrity. We propose that neutrophil transmigration across the vascular endothelium goes in parallel with cleavage of VE cadherin by elastase and cathepsin G present on the surface of neutrophils. This hypothesis is supported by the following lines of evidence. 1) Proteolytic fragments of VE cadherin are released into the culture medium upon adhesion of neutrophils to endothelial cell monolayers; 2) conditioned culture medium, obtained after neutrophil adhesion to endothelial monolayers, cleaves the recombinantly expressed VE cadherin extracellular domain; 3) these cleavages are inhibited by inhibitors of elastase; 4) VE cadherin fragments produced by conditioned culture medium or by exogenously added elastase are identical as shown by N-terminal sequencing and mass spectrometry analysis; 5) both elastase- and cathepsin G-specific VE cadherin cleavage patterns are produced upon incubation with tumor necrosis factor alpha-stimulated and fixed neutrophils; 6) transendothelial permeability increases in vitro upon addition of either elastase or cathepsin G; and 7) neutrophil transmigration is reduced in vitro in the presence of elastase and cathepsin G inhibitors. Our results suggest that cleavage of VE cadherin by neutrophil surface-bound proteases induces formation of gaps through which neutrophils transmigrate.


Assuntos
Caderinas/química , Caderinas/metabolismo , Endotélio Vascular/metabolismo , Neutrófilos/metabolismo , Animais , Antígenos CD , Western Blotting , Células CHO , Caderinas/fisiologia , Catepsina G , Catepsinas/metabolismo , Adesão Celular , Movimento Celular , Células Cultivadas , Cricetinae , Meios de Cultura/farmacologia , Meios de Cultivo Condicionados/farmacologia , Endotélio/metabolismo , Endotélio Vascular/citologia , Humanos , Leucócitos/metabolismo , Espectrometria de Massas , Microscopia de Fluorescência , Neutrófilos/enzimologia , Elastase Pancreática/metabolismo , Estrutura Terciária de Proteína , Proteínas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Serina Endopeptidases , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Fatores de Tempo , Veias Umbilicais/citologia
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