Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 6 de 6
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
J Pharm Pharm Sci ; 13(2): 175-90, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-20816004

RESUMO

PURPOSE: We investigated the involvement of MAPK signaling in the cell death mechanisms of classical microtubule interfering agents (MIA) and aryl-3-(2-chloroethyl)ureas (CEU) acting as antimitotics, along with CEU that don't affect directly microtubules (non-MIA CEU). METHODS: To ascertain the activated signaling pathway profile of MIA and non-MIA CEU, Western blot, immunoprecipitation and transfection experiments were performed. RESULTS: Non-MIA CEU do not activate p38, as opposed to MIA, and the extent of ERK and JNK activation is lower than in response to MIA. The effect of MIA and non-MIA CEU on focal adhesion associated protein was also studied; MIA were shown to induce focal adhesion dismantlement associated with a sustained increase in paxillin phosphorylation and FAK cleavage, as opposed to non-MIA CEU. In addition, bcl-2 phosphorylation and AKT cleavage, induced by all MIA tested, was not observed in response to non-MIA CEU further emphasizing the differential cell death mechanisms induced by MIA and non-MIA CEU. Pharmacologic and genetic approaches emphasize that the ASK1-p38 pathway activation contributes to the cytotoxic mechanism of MIA, in contrast to non-MIA CEU. ASK1-p38 is important for increased paxillin phosphorylation and FAK cleavage, suggesting that ASK-1-p38 is an upstream event of FA structure dismantlement induced by MIA. Moreover, the endogen inhibitor of ASK-1, thioredoxin, is released from ASK-1 in response to MIA as opposed to non-MIA CEU. CONCLUSION: Our study supports that ASK1-p38 activation is an important signaling event, induced by MIA, which impairs focal adhesion structure and induces anchorage-dependent apoptosis or anoikis.


Assuntos
Antineoplásicos/farmacologia , MAP Quinase Quinase Quinase 5/metabolismo , Moduladores de Tubulina/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Anoikis/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Western Blotting , Linhagem Celular , Linhagem Celular Tumoral , Adesões Focais/metabolismo , Humanos , Camundongos , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Fosforilação/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Transfecção , Ureia/análogos & derivados , Ureia/farmacologia
2.
Diagn Mol Pathol ; 19(1): 20-7, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-20186008

RESUMO

Up to 30% of patients with stage II (pN0) colon cancer develop recurrences, suggesting that the presence of lymph node (LN) metastases escaped detection at histopathologic staging. A simple way to overcome this limitation and to improve staging accuracy is to use reverse transcription-polymerase chain reaction (RT-PCR) to examine a larger fraction or an entire specimen. The Guanylyl cyclase C (GCC) gene is uniquely expressed in apical cells of the gastrointestinal tract. Its expression in colon cancer cells and metastases is conserved. Therefore, detection of GCC mRNA in LNs has been shown to be indicative of the presence of colon cancer metastases. As the current processing of LNs involves formalin fixation and paraffin embedding, we developed a method for extracting RNA from formalin-fixed paraffin-embedded LN specimens and detecting GCC mRNA by quantitative RT-PCR. The assay has a dynamic range of 5 logs, an average amplification efficiency of 98.4% (95% confidence interval, 96.6-100.3), a reaction linearity of 0.998 (95% confidence interval, 0.997-0.999), and also intraplate and interplate CVs of <1% and <5%, respectively. The test specificity was 98% with LNs collected from patients affected by conditions other than colon cancer (n=380). Sensitivity was 97% for patients with stage III colon cancer (n=34), whereas 35% of patients with stages I and II disease (n=51) had at least 1 GCC mRNA-positive LN. The high specificity of GCC mRNA suggests that routine utilization of the quantitative RT-PCR test has the potential to improve the detection of colon cancer metastases in LNs.


Assuntos
Neoplasias do Colo/diagnóstico , Neoplasias do Colo/secundário , Guanilato Ciclase/genética , Linfonodos/patologia , Patologia Molecular/métodos , Receptores de Peptídeos/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Fixadores/farmacologia , Formaldeído/farmacologia , Humanos , Inclusão em Parafina , Receptores de Enterotoxina , Receptores Acoplados a Guanilato Ciclase , Sensibilidade e Especificidade , Fixação de Tecidos
3.
Cancer Res ; 67(5): 2306-16, 2007 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-17332362

RESUMO

Chloroethylureas (CEU) are soft alkylating agents that covalently bind to beta-tubulin (betaTAC) and affect microtubule polymerization dynamics. Herein, we report the identification of a CEU subset and its corresponding oxazolines, which induce cell growth inhibition, apoptosis, and microtubule disruption without alkylating beta-tubulin (N-betaTAC). Both betaTAC and N-betaTAC trigger the collapse of mitochondrial potential (DeltaPsi(m)) and modulate reactive oxygen species levels, following activation of intrinsic caspase-8 and caspase-9. Experiments using human fibrosarcoma HT1080 respiratory-deficient cells (rho(0)) and uncoupler of the mitochondrial respiratory chain (MRC) showed that betaTAC and N-betaTAC impaired the MRC. rho(0) cells displayed an increased sensitivity toward N-betaTAC as compared with rho(+) cells but, in contrast, were resistant to betaTAC or classic chemotherapeutics, such as paclitaxel. Oxazoline-195 (OXA-195), an N-betaTAC derivative, triggered massive swelling of isolated mitochondria. This effect was insensitive to cyclosporin A and to Bcl-2 addition. In contrast, adenine nucleotide translocator (ANT) antagonists, bongkrekic acid or atractyloside, diminished swelling induced by OXA-195. The antiproliferative activities of the N-betaTACs CEU-025 and OXA-152 were markedly decreased in the presence of atractyloside. Conversely, pretreatment with cyclosporin A enhanced growth inhibition induced by betaTAC and N-betaTAC. One of the proteins alkylated by N-betaTAC was identified as the voltage-dependent anion channel isoform-1, an ANT partner. Our results suggest that betaTAC and N-betaTAC, despite their common ability to affect the microtubule network, trigger different cytotoxic mechanisms in cancer cells. The role of mitochondria in these mechanisms and the potential of N-betaTAC as a new therapeutic approach for targeting hypoxia-resistant cells are discussed.


Assuntos
Antineoplásicos Alquilantes/uso terapêutico , Hipóxia Celular , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Compostos de Fenilureia/uso terapêutico , Animais , Antineoplásicos Alquilantes/química , Células CHO , Cricetinae , Cricetulus , Ensaios de Seleção de Medicamentos Antitumorais , Transporte de Elétrons/efeitos dos fármacos , Células HT29 , Humanos , Microtúbulos/efeitos dos fármacos , Modelos Biológicos , Tubulina (Proteína)/metabolismo , Células Tumorais Cultivadas
4.
J Pharmacol Exp Ther ; 320(2): 853-64, 2007 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-17099073

RESUMO

Microtubule disruption provokes cytoskeleton and cell adhesion changes whose importance for apoptosis induction remains unclear. The present study focuses on the functional and the molecular adhesion kinetics that are induced by microtubule disruption-mediated apoptosis. We showed that antimicrotubules induce a biphasic sequence of adhesion response that precedes the onset of apoptosis and focal adhesion kinase hydrolysis. Antimicrotubules first induced an increase of the cellular adhesion paralleled by the raise of focal adhesion sites and actin contractility, which was followed by a sharp decrease of cell adhesion and disorganization of focal adhesion and actin stress fibers. The latter sequence of events ends by cell rounding, detachment from the extracellular matrix, and cell death. Microtubule-disrupting agents induced a sustained paxillin phosphorylation, before the activation of apoptosis, that requires the prior activation of extracellular signal-regulated kinase and p38 but not c-Jun NH(2)-terminal kinase. Interestingly, integrin-linked kinase overexpression rescued the antimicrotubule-mediated loss of cell viability. Altogether, these results propound that antimicrotubule agents induce anoikis through the loss of focal adhesion structure integrity.


Assuntos
Anoikis/efeitos dos fármacos , Adesões Focais/efeitos dos fármacos , Microtúbulos/efeitos dos fármacos , Moduladores de Tubulina/farmacologia , Animais , Adesão Celular , Linhagem Celular Tumoral , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Humanos , Camundongos , Paclitaxel/farmacologia , Paxilina/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/fisiologia , Vimblastina/farmacologia , Vincristina/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
5.
Cancer Res ; 64(13): 4654-63, 2004 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-15231678

RESUMO

The development of new anticancer agents with lower toxicity, higher therapeutic index, and weaker tendency to induce resistant phenotypes in tumor cells is a continuous challenge for the scientific community. Toward that end, we showed previously that a new class of soft alkylating agents designed as phenyl-3-(2-chloroethyl)ureas (CEUs) inhibits tumor cell growth in vitro and that their efficiency is not altered by clinically relevant mechanisms of resistance such as overexpression of multidrug resistance proteins, increase in intracellular concentration of glutathione and/or glutathione S-transferase activity, alteration of topoisomerase II, and increased DNA repair. Mechanistic studies have showed recently that the cytotoxic activity of several CEUs was mainly related to the disruption of microtubules. Here, we present results supporting our assumption that 4-tert-butyl-[3-(2-chloroethyl)ureido]phenyl (tBCEU) (and its bioisosteric derivative 4-iodo-[3-(2-chloroethyl)ureido]phenyl (ICEU) are potent antimicrotubule agents both in vitro and in vivo. They covalently bind to beta-tubulin, leading to a microtubule depolymerization phenotype, consequently disrupting the actin cytoskeleton and altering the nuclear morphology. Accordingly, tBCEU and ICEU also inhibited the migration and proliferation of endothelial and tumor cells in vitro in a dose-dependent manner. It is noteworthy that ICEU efficiently blocked angiogenesis and tumor growth in three distinct animal models: (a) the Matrigel plug angiogenesis assay; (b) the CT-26 tumor growth assay in mice; and (c) the chick chorioallantoic membrane tumor assay. In addition, we present evidence that CEU cytotoxicity is unaffected by additional resistance mechanisms impeding tumor response to DNA alkylating agents such as cisplatin, namely the cell adhesion mediated-drug resistance mechanism, which failed to influence the cytocidal activity of CEUs. On the basis of the apparent innocuousness of CEUs, on their ability to circumvent many classical and recently described tumor cell resistance mechanisms, and on their specific biodistribution to organs of the gastrointestinal tract, our results suggest that CEUs represent a promising new class of anticancer agents.


Assuntos
Inibidores da Angiogênese/farmacologia , Antineoplásicos Alquilantes/farmacologia , Microtúbulos/efeitos dos fármacos , Ureia/análogos & derivados , Actinas/metabolismo , Animais , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Células Cultivadas , Embrião de Galinha , Colchicina/farmacologia , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/patologia , Cricetinae , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Paclitaxel/farmacologia , Tubulina (Proteína)/metabolismo , Ureia/farmacologia
6.
Biochem J ; 372(Pt 2): 631-41, 2003 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-12646044

RESUMO

Cell transformation by growth-promoting oncoproteins renders cells extremely sensitive to apoptosis through an unknown mechanism affecting the mitochondrial pathway of apoptosis. We have shown previously that sensitization to apoptosis also correlated with the activation of the stress-activated protein kinase p38. In the present study, we investigated the role of p38 in c-Myc-dependent apoptosis induced by the anticancer agent cisplatin. Cisplatin treatment of Rat1 cells with deregulated expression of c-Myc resulted in nuclear fragmentation that was accompanied in all cells by the activation of Bax and the translocation of cytochrome c from the mitochondria to the cytoplasm. None of these features of apoptosis was induced in control Rat-1 cells. p38 was also activated by cisplatin only in cells with deregulated expression of c-Myc, but, in contrast with all features of apoptosis, this activation was not affected by Bcl-2. Remarkably, overexpression of an interfering mutant of the p38alpha isoform, but not p38beta, blocked cisplatin-induced Bax activation or cytochrome c release and nuclear fragmentation. Analysis of the kinase cascade upstream of p38 revealed a c-Myc-dependent activation by cisplatin of mitogen-activated protein kinase kinase (MKK) 3/6 and apoptosis signal-regulating kinase 1 (Ask1). Inhibition of Ask1 blocked p38 activation by cisplatin and all features of apoptosis. Several of these data were confirmed using other DNA-damaging agents. The findings indicated that c-Myc potentiation of the mitochondrial pathway of apoptosis results, at least in part, from a sensitization of Ask1 activation, allowing DNA-damaging agents to induce in cascade Ask1, p38alpha and Bax.


Assuntos
Apoptose , MAP Quinase Quinase Quinases/genética , Mitocôndrias/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Animais , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Western Blotting , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Caspases/metabolismo , Células Cultivadas , Cisplatino/farmacologia , Grupo dos Citocromos c/metabolismo , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Técnicas Imunoenzimáticas , MAP Quinase Quinase 3 , MAP Quinase Quinase 6 , MAP Quinase Quinase Quinase 5 , MAP Quinase Quinase Quinases/metabolismo , Mitocôndrias/efeitos dos fármacos , Proteína Quinase 11 Ativada por Mitógeno , Proteína Quinase 14 Ativada por Mitógeno , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Ratos , Transdução de Sinais/efeitos dos fármacos , Frações Subcelulares , Transfecção , Proteína X Associada a bcl-2
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...