RESUMO
Reactive oxygen species are well-known mediators of various biological responses. Recently, new homologues of the catalytic subunit of NADPH oxidase have been discovered in non-phagocytic cells. These new homologues (Nox1-Nox5) produce low levels of superoxides compared to the phagocytic homologue Nox2/gp91phox. Using Nox1 siRNA, we show that Nox1-dependent superoxide production affects the migration of HT29-D4 colonic adenocarcinoma cells on collagen-I. Nox1 inhibition or down-regulation led to a decrease of superoxide production and alpha 2 beta 1 integrin membrane availability. An addition of arachidonic acid stimulated Nox1-dependent superoxide production and HT29-D4 cell migration. Pharmacological evidences using phospholipase A2, lipoxygenases and protein kinase C inhibitors show that upstream regulation of Nox1 relies on arachidonic acid metabolism. Inhibition of 12-lipoxygenase decreased basal and arachidonic acid induced Nox1-dependent superoxide production and cell migration. Migration and ROS production inhibited by a 12-lipoxygenase inhibitor were restored by the addition of 12(S)-HETE, a downstream product of 12-lipoxygenase. Protein kinase C delta inhibition by rottlerin (and also GO6983) prevented Nox1-dependent superoxide production and inhibited cell migration, while other protein kinase C inhibitors were ineffective. We conclude that Nox1 activation by arachidonic acid metabolism occurs through 12-lipoxygenase and protein kinase C delta, and controls cell migration by affecting integrin alpha 2 subunit turn-over.
Assuntos
Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Movimento Celular , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , NADPH Oxidases/metabolismo , Superóxidos/metabolismo , Araquidonato 12-Lipoxigenase/metabolismo , Ácidos Araquidônicos/farmacologia , Membrana Celular/metabolismo , Movimento Celular/efeitos dos fármacos , Células HT29 , Humanos , Integrina alfa2beta1/metabolismo , NADPH Oxidase 1 , Proteína Quinase C-delta/metabolismo , Superóxidos/antagonistas & inibidoresRESUMO
The motile behavior of endothelial cells is a crucial event for neoangiogenesis. We previously showed that noncytotoxic concentrations of vinflunine inhibit capillary-like tube formation on Matrigel and endothelial cell migration with a concomitant increase in interphase microtubule dynamic instability. In this article, we further investigated the effects of vinflunine on migration and cytoskeleton interaction dynamics in HMEC-1 endothelial cells. We confirmed that vinflunine, at low and noncytotoxic concentrations (0.01-1 nmol/L), inhibited endothelial cell random motility by 54%. This effect was associated with a decrease in the percentage of stable microtubules and in the mean duration of pauses for dynamic ones. Moreover, we found that vinflunine altered adhesion site targeting by microtubules and suppressed the microtubule (+) end pause that occurs at adhesion sites during cell migration (from 151 +/- 20 seconds in control cells to 38 +/- 7 seconds in vinflunine-treated cells, P < 0.001). This effect was associated with the inhibition of adhesion site dynamics and the formation of long-lived stress fibers. Importantly, we found that vinflunine altered EB1 localization at microtubule (+) ends. These results highlight a new mechanism of action of vinflunine, which act by disrupting the mutual control between microtubule and adhesion site dynamics and strengthen the role of +TIPs proteins such as EB1 as key regulators of endothelial cell motility.
Assuntos
Inibidores da Angiogênese/farmacologia , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Vimblastina/análogos & derivados , Adesão Celular/efeitos dos fármacos , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Movimento Celular/efeitos dos fármacos , Polaridade Celular/efeitos dos fármacos , Humanos , Transporte Proteico/efeitos dos fármacos , Fibras de Estresse/efeitos dos fármacos , Vimblastina/farmacologiaRESUMO
The catalytic subunit of the NADPH oxidase complex, Nox1 (homologue of gp91phox/Nox2), expressed mainly in intestinal epithelial and vascular smooth muscle cells, functions in innate immune defense and cell proliferation. The molecular mechanisms underlying these functions, however, are not completely understood. We measured Nox1-dependent O2- production during cell spreading on Collagen IV (Coll IV) in colon carcinoma cell lines. Knocking down Nox1 by shRNA, we showed that Nox1-dependent O2- production is activated during cell spreading after 4 hr of adhesion on Collagen IV. Nox1 activation during cell spreading relies on Rac1 activation and arachidonic metabolism. Our results showed that manoalide (a secreted phospholipase A2 inhibitor) and cinnamyl-3,4-dihydroxy-alpha-cyanocinnamate (a 12-lipoxygenase inhibitor) inhibit O2- production, cell spreading and cell proliferation in these colonic epithelial cells. 12-Lipoxygenase inhibition of ROS production and cell spreading can be reversed by adding 12-HETE, a 12-lipoxygenase product, supporting the specific effect observed with cinnamyl-3,4-dihydroxy-alpha-cyanocinnamate. In contrast, Nox1 shRNA and DPI (NADPH oxidase inhibitor) weakly affect cell spreading while inhibiting O2- production and cell proliferation. These results suggest that the 12-lipoxygenase pathway is upstream of Nox1 activation and controls cell spreading and proliferation, while Nox1 specifically affects cell proliferation.
Assuntos
Araquidonato 12-Lipoxigenase/metabolismo , Carcinoma/metabolismo , Carcinoma/patologia , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , NADPH Oxidases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/farmacologia , Western Blotting , Ácidos Cafeicos/farmacologia , Carcinoma/enzimologia , Linhagem Celular Tumoral , Proliferação de Células , Neoplasias do Colo/enzimologia , Ativação Enzimática , Humanos , Inibidores de Lipoxigenase , NADPH Oxidase 1 , Inibidores de Fosfodiesterase/farmacologia , Inibidores de Fosfolipase A2 , RNA/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Terpenos/farmacologiaRESUMO
A series of syn-restricted polymethoxylated 4-heteroarylcoumarins--the isostuctural analogs of combretastatin A-4--was synthesized by Suzuki-Miyaura cross-coupling reaction and evaluated for antiproliferative activity. The 4-(1-methyl-1H-indol-5-yl)chromen-2-ones exhibit a potent cytotoxicity against HBL100 epithelial cell line with an IC(50) value amounting to 0.098 and 0.078 microM, respectively. The two compounds, having an indolyl moiety, potent inhibit in vitro microtubule assembly with a substoichiometric mode of action. A structure-activity relationship was discussed and the indolyl moiety was proved to be a good surrogate for the 3-hydroxy-4-methoxyphenyl ring of CA-4.
Assuntos
Antineoplásicos/farmacologia , Cumarínicos/farmacologia , Estilbenos/farmacologia , Moduladores de Tubulina/farmacologia , Antineoplásicos/síntese química , Sítios de Ligação , Neoplasias da Mama , Linhagem Celular Tumoral , Cumarínicos/síntese química , Reagentes de Ligações Cruzadas/química , Humanos , Concentração Inibidora 50 , Estilbenos/síntese química , Relação Estrutura-Atividade , Moduladores de Tubulina/síntese química , Ensaio Tumoral de Célula-TroncoRESUMO
Angiogenesis is a key event in tumor progression and metastasis. This complex process, which constitutes a potent target for cancer therapy, is inhibited by very low concentrations of microtubule-targeting drugs (MTD). However, the intimate mechanisms of the antiangiogenic activity of MTDs remain unclear. Recently, we have shown that low antiangiogenic and noncytotoxic concentrations of paclitaxel induced an unexpected increase in microtubule dynamics in endothelial cells. In this study, we showed that vinflunine, the newest Vinca alkaloid, increased microtubule dynamic instability in human endothelial cells after 4-hour incubation at low concentrations (29% and 54% at 0.1 and 2 nmol/L). The growth and shortening rates were increased, and the percentage of time spent in pause and the mean duration of pauses were decreased, as previously observed with paclitaxel. As opposed to paclitaxel, the transition frequencies were not significantly disturbed by vinflunine. Moreover, low concentrations of vinflunine did not affect mitotic index and anaphase/metaphase ratio. Interestingly, these low vinflunine concentrations that increased microtubule dynamics exhibited an antiangiogenic effect through the inhibition of both morphogenesis and random motility. Capillary tube formation on Matrigel was decreased up to 44%. The cell speed and the random motility coefficient were decreased (13% and 19% and 13% and 33% at 0.1 and 2 nmol/L, respectively) and the persistent time was statistically increased. Altogether, our results confirm that the increase in microtubule dynamics is involved in MTD antiangiogenic activity and highlight the crucial role of interphase microtubule dynamics in angiogenesis.
Assuntos
Inibidores da Angiogênese/farmacologia , Movimento Celular/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Microtúbulos/efeitos dos fármacos , Vimblastina/análogos & derivados , Capilares/efeitos dos fármacos , Capilares/crescimento & desenvolvimento , Processos de Crescimento Celular/efeitos dos fármacos , Colágeno , Relação Dose-Resposta a Droga , Combinação de Medicamentos , Células Endoteliais/citologia , Humanos , Interfase/efeitos dos fármacos , Interfase/fisiologia , Laminina , Microtúbulos/fisiologia , Mitose/efeitos dos fármacos , Proteoglicanas , Vimblastina/farmacologiaRESUMO
Vinflunine, a new microtubule-targeting drug, has a marked antitumor activity in vitro and in vivo. Here, we studied the mechanisms mediating resistance to vinflunine. We investigated the response to vinflunine of ovarian cancer cells initially selected as paclitaxel-resistant cells (A2780-TC1 cells). By comparison with A2780-wild-type (wt) cells, we showed that A2780-TC1 cells were highly resistant to vinflunine, with resistance factors reaching 800 and 1,830 for IC(50) and IC(70), respectively. We showed that P-glycoprotein minimally participated in this cell resistance. The examination of tubulin composition revealed increased levels of acetylated alpha-tubulin, betaII-tubulin, and betaIII-tubulin in A2780-TC1 cells before vinflunine treatment. As a consequence, vinflunine unequally affected microtubule network organization and function in A2780-wt and A2780-TC1 cells. Whereas the drug depolymerized microtubules and induced a mitotic block in A2780-wt cells, it did not depolymerize microtubules and induced a G(2) block in A2780-TC1 cells. Elsewhere, the mitochondrial protein Bcl-2 was down-regulated in A2780-TC1 cells. This down-regulation was related to resistance, as A2780-TC1 cells stably transfected with a Bcl-2 construct recovered a partial sensitivity to vinflunine. Lastly, we confirmed the role played by Bcl-2 by showing that the mitochondrial membrane potential was only disrupted by vinflunine in cells expressing Bcl-2. Altogether, our results indicate that modifications acquired during treatment (i.e., paclitaxel) have significant consequences on cell response to the following drug (i.e., vinflunine). Especially, this study shows that a specific pool of tubulin subtypes and a down-regulation of Bcl-2 are associated with resistance of ovarian cancer cells to vinflunine.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Regulação para Baixo , Neoplasias Ovarianas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Moduladores de Tubulina/farmacologia , Tubulina (Proteína)/metabolismo , Vimblastina/análogos & derivados , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Relação Dose-Resposta a Droga , Resistencia a Medicamentos Antineoplásicos , Feminino , Humanos , Microtúbulos/fisiologia , Microtúbulos/ultraestrutura , Neoplasias Ovarianas/tratamento farmacológico , Proteínas Proto-Oncogênicas c-bcl-2/genética , Tubulina (Proteína)/classificação , Tubulina (Proteína)/genética , Células Tumorais Cultivadas , Vimblastina/farmacologiaRESUMO
The cytochrome P450 3A5 (CYP3A5) has been shown to be highly involved in the metabolism of many therapeutic agents. To date, several polymorphisms affecting the CYP3A5 gene have been identified but few studies have shown a complete description of the variability of the CYP3A5 in the French population. Therefore, the extent of CYP3A5 genetic polymorphism was investigated in a French population of 114 patients. The screening of the coding region with their intron-exon boundaries and the proximal flanking regions was performed using a PCR-SSCP strategy. Eighteen polymorphisms were identified, including four new mutations. They correspond to -19 T>C upstream of the exon 1, 7360 T>C in intron 4, 12991 T>C in intron 5 and 29788 delG in exon 12. We also identified 13 alleles including six new alleles. As expected, the most frequent allelic variant is CYP3A5*3, with a frequency of 87% of all alleles. These data confirmed that CYP3A5 gene is highly polymorphic. Furthermore, it will be now interesting to evaluate the impact of this polymorphism on the pharmacokinetic parameters of different drugs.
Assuntos
Sistema Enzimático do Citocromo P-450/genética , Genética Populacional/métodos , Polimorfismo Conformacional de Fita Simples , Citocromo P-450 CYP3A , Sistema Enzimático do Citocromo P-450/classificação , França , Frequência do Gene , HumanosRESUMO
Microtubule-targeting agents (MTAs) are largely administered in adults and children cancers. Better deciphering their mechanism of action is of prime importance to develop more convenient therapy strategies. Here, we addressed the question of how reactive oxygen species (ROS) generation by mitochondria can be necessary for MTA efficacy. We showed for the first time that EB1 associates with microtubules in a phosphorylation-dependent manner, under control of ROS. By using phospho-defective mutants, we further characterized the Serine 155 residue as critical for EB1 accumulation at microtubule plus-ends, and both cancer cell migration and proliferation. Phosphorylation of EB1 on the Threonine 166 residue triggered opposite effects, and was identified as a requisite molecular switch in MTA activities. We then showed that GSK3ß activation was responsible for MTA-triggered EB1 phosphorylation, resulting from ROS-mediated inhibition of upstream Akt. We thus disclosed here a novel pathway by which generation of mitochondrial ROS modulates microtubule dynamics through phosphorylation of EB1, improving our fundamental knowledge about this oncogenic protein, and pointing out the need to re-examine the current dogma of microtubule targeting by MTAs. The present work also provides a strong mechanistic rational to the promising therapeutic strategies that currently combine MTAs with anti-Akt targeted therapies.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/metabolismo , Neoplasias Pulmonares/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Antineoplásicos/farmacologia , Western Blotting , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Técnica Indireta de Fluorescência para Anticorpo , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Humanos , Imunoprecipitação , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/fisiologia , Transfecção , Moduladores de Tubulina/farmacologiaRESUMO
Bcl-2 is commonly overexpressed in tumors, where it is often associated with unfavorable outcome. However, it has also been linked to a favorable sensitivity to microtubule-targeting agents (MTAs). We show that Bcl-2-overexpressing lung and breast cancer cells were more sensitive to both paclitaxel and vinorelbine. Bcl-2 over-expression also significantly potentiated in vivo efficacy of paclitaxel, in terms of tumor volume decrease and survival benefits, in models of nude mice bearing lung cancer xenografts. To further investigate this favorable effect of Bcl-2, a genomic approach was taken. It revealed that Bcl-2 overexpression induced up-regulation of the proapoptotic protein Bim in lung cancer cells and that, conversely, Bcl-2 silencing decreased Bim expression level. A gene regulation study implicated the transcription factor Forkhead box-containing protein, class O3a in Bim up-regulation. Lastly, we show that Bim was responsible for MTA-triggered lung cancer cell death through a dynamin-related protein 1-mediated mitochondrial fragmentation. The Bcl-2-governed Bim induction evidence offers for the first time an explanation for the favorable higher sensitivity to treatment shown by Bcl-2-overexpressing cells. We suggest that Bim could be a powerful predictive factor for tumor response to MTA chemotherapy. Our data also give new insight into some failures in the efficacy of therapies targeted against Bcl-2.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Proteínas de Membrana/metabolismo , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Animais , Proteínas Reguladoras de Apoptose/genética , Proteína 11 Semelhante a Bcl-2 , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Morte Celular/efeitos dos fármacos , Morte Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Células MCF-7 , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Microtúbulos/genética , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Paclitaxel/farmacologia , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Distribuição Aleatória , Regulação para Cima/efeitos dos fármacos , Vimblastina/análogos & derivados , Vimblastina/farmacologia , Vinorelbina , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PURPOSE: The aim of this study was to investigate the impact of plasma and intracellular exposure and CYP3A4, CYP3A5, and ABCB1 polymorphisms on vincristine neurotoxicity. We subsequently assessed the impact of ABCB1 polymorphisms on intracellular vincristine accumulation. METHODS: Children treated for solid tumors were enrolled in the study (n = 26) and received 1.5 mg/m² of vincristine per course. Individual pharmacokinetic parameters and CYP3A4, CYP3A5, and ABCB1 genotypes were available from a previous analysis. A global toxicity score (pain, peripheral neurotoxicity, and gastrointestinal toxicity) was collected at each course. Vincristine in plasma and PBMCs were quantified by LC-MS/MS. RESULTS: Vincristine plasma and intracellular concentrations ranged from 0.40 to 89.6 ng/ml and from 0.00225 to 1.85 ng/10(6) cells over a 24-h interval, respectively. The global toxicity score ranged from 0 to 6 and was not correlated with individual pharmacokinetics parameters. Neurotoxicity events (global score ≥ 3) were observed in 8 patients but the incidence was not influenced by the different studied polymorphisms. The global toxicity score was correlated with age, body surface area, and dose in mg. A trend to higher intracellular/plasma ratio of vincristine was found for patients with heterozygous diplotype (CGC-TTT) of ABCB1. CONCLUSIONS: None of the different genetic covariates nor plasma and intracellular exposure was predictive of the observed neurotoxicity in our pediatric population. Nevertheless, the heterozygote diplotype of ABCB1 appears to influence the intracellular accumulation of vincristine. Owing to the small sample size, further evaluations are needed in a larger patient cohort.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Antineoplásicos Fitogênicos/efeitos adversos , Citocromo P-450 CYP3A/genética , Neoplasias/tratamento farmacológico , Síndromes Neurotóxicas/etiologia , Polimorfismo Genético , Vincristina/efeitos adversos , Subfamília B de Transportador de Cassetes de Ligação de ATP , Adolescente , Criança , Pré-Escolar , Feminino , Genótipo , Humanos , Masculino , Vincristina/sangueRESUMO
A series of A-ring variously methoxylated 4-(3-hydroxy-4-methoxyphenyl)coumarins related to combretastatin A-4 was prepared by cross-coupling reactions. Cytotoxicity studies indicated a potent activity against HBL100 cell line. Substitution patterns on A-ring had only a slight effect on antiproliferative activity. For most cytotoxic compounds, the activity as potential modulators of P-gp and BCRP efflux pumps was evaluated. The results show that compounds 2 and 7 were able to restore mitoxantrone accumulation (BCRP) at concentrations similar to that of cyclosporine A. Compound 7 was the most efficient to reverse P-gp activity. All compounds were found to potently inhibit in vitro microtubule formation via a substoichiometric mode of action for the most part. Compounds 1 and 2 were found to have an apparent affinity binding constant similar to that of combretastatin A-4, i.e., 1 × 10 (6) M(-1). The molecular modeling of coumarin derivatives was performed on the basis of the molecular structure of 7, as determined by single-crystal X-ray crystallography. The calculations suggested that the presence of a methoxy group out of the plane of the chromenone moiety is an important steric hindrance factor embedding the accessibility of those molecules inside the binding pocket on tubulin.
Assuntos
Antineoplásicos/síntese química , Cumarínicos/síntese química , Estilbenos/síntese química , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/biossíntese , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/biossíntese , Antineoplásicos/química , Antineoplásicos/farmacologia , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Linhagem Celular Tumoral , Cumarínicos/química , Cumarínicos/farmacologia , Cristalografia por Raios X , Daunorrubicina/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Fase G2/efeitos dos fármacos , Humanos , Mitoxantrona/farmacologia , Modelos Moleculares , Proteínas de Neoplasias/biossíntese , Estilbenos/química , Estilbenos/farmacologia , Relação Estrutura-Atividade , Tubulina (Proteína)/química , Moduladores de Tubulina/síntese química , Moduladores de Tubulina/química , Moduladores de Tubulina/farmacologiaRESUMO
The Bcl-2 family contains a panel of proteins which are conserved regulators of apoptosis in mammalian cells, like the anti-apoptotic protein Bcl-2. According to its significant role in altering susceptibility to apoptosis, the deciphering of the mechanism of Bcl-2 expression modulation may be crucial for identifying therapeutics strategies for cancer. Treatment with microtubule-targeting agents, including taxanes and Vinca alkaloids, generally leads to a decrease in Bcl-2 intracellular amounts. Whereas the interest for these chemotherapeutics is accompanied by advances in the fundamental understanding of their anticancer properties, the molecular mechanism underlying changes in Bcl2 expression remains poorly understood. We report here that p53 contributes to vinorelbine-induced Bcl-2 down-regulation. Indeed, the decrease in Bcl-2 protein levels observed during vinorelbine-induced apoptosis was correlated to the decrease in mRNA levels, as a result of the inhibition of Bcl-2 transcription and promoter activity. In this context, we evaluated p53 contribution in the Bcl-2 transcriptional down-regulation. We identified, by chromatin immunoprecipitation, a novel p53 binding site in the Bcl-2 promoter, within a region upstream P(1) promoter. We showed that vinorelbine treatment increased this interaction in A549 cells. This work strengthens the links between p53 and Bcl-2 at a transcriptional level, upon microtubule-targeting agent treatment. Our study also provides answers that will be useful to assess microtubule-targeting agents' mechanism of action and that may help to better understand and increase their effectiveness.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Regulação para Baixo/efeitos dos fármacos , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-bcl-2/genética , Transcrição Gênica/efeitos dos fármacos , Proteína Supressora de Tumor p53/metabolismo , Vimblastina/análogos & derivados , Sequência de Bases , Sítios de Ligação , Western Blotting , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Primers do DNA , Humanos , Vimblastina/farmacologia , VinorelbinaRESUMO
2-(methoxymethoxymethyl)aryllead triacetates, obtained in situ from the corresponding arylboronic acids, reacted with 4-hydroxycoumarins, leading to 3-(2-methoxymethoxymethyl)aryl-4-hydroxycoumarin derivatives in good to high yields. These compounds underwent a cascade sequence of reactions, deprotection-halogenation-annulation, to afford polyoxygenated tetracyclic 6H,11H-[2]benzopyrano-[4,3-c] [1]benzopyran-11-ones in good yields. Some compounds showed a moderate cytotoxicity against human epithelial mammary HBL100 cells.
Assuntos
Benzopiranos/química , Química Orgânica/métodos , 4-Hidroxicumarinas/química , Antineoplásicos/química , Antineoplásicos/farmacologia , Benzopiranos/farmacologia , Boranos/química , Células Cultivadas , Ensaios de Seleção de Medicamentos Antitumorais , Células Epiteliais , Humanos , Oxigênio/química , Testes de ToxicidadeRESUMO
AIMS: The protease inhibitor indinavir is characterized by an important interindividual pharmacokinetic variability, which results from the actions of the metabolizing enzymes cytochrome P450 (CYP) 3A and the multidrug efflux pump P-glycoprotein (P-gp), encoded by MDR1. Using a population pharmacokinetic approach, we investigated the effect of several MDR1 and CYP3A5 polymorphisms on the pharmacokinetic parameters of indinavir in HIV-infected patients. METHODS: Twenty-eight patients receiving indinavir alone or together with ritonavir were included. Indinavir pharmacokinetics were studied over a 12 h interval. Genetic polymorphisms were assessed by real-time PCR assays and direct sequencing for MDR1 and by PCR-SSCP analysis for CYP3A5. RESULTS: The pharmacokinetics of indinavir were best described by a one-compartment model with first-order absorption. In the final model, the MDR1 C3435T genotype and ritonavir were identified as statistically significant covariates (P = 0.001) for the absorption rate constant (95% confidence interval on the difference between CC and CT genotype 0.37, 5.53) and for clearance (95% confidence interval on the difference 5.8, 26.2), respectively. Patients with the CYP3A5*3/*3 genotype receiving indinavir alone had a 31% decrease in the indinavir clearance rate compared with patients carrying the CYP3A5*1/*3 genotype. CONCLUSIONS: The MDR1 C3435T genotype affects the absorption constant of indinavir suggesting that P-gp may be implicated in its pharmacokinetic variability. Through its inhibition of CYP3A and P-gp, ritonavir could attenuate the pharmacokinetic variability linked to genetic differences, reducing significantly the interindividual variability of indinavir. However, genotyping MDR1 and/or CYP3A5 to optimize protease inhibitor boosted regimens does not seem clinically relevant.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Citocromo P-450 CYP3A/genética , Sistema Enzimático do Citocromo P-450/genética , Infecções por HIV/metabolismo , Inibidores da Protease de HIV/farmacocinética , Indinavir/farmacocinética , Subfamília B de Transportador de Cassetes de Ligação de ATP , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Adulto , Idoso , Citocromo P-450 CYP3A/metabolismo , Feminino , Infecções por HIV/tratamento farmacológico , Inibidores da Protease de HIV/uso terapêutico , Humanos , Indinavir/uso terapêutico , Masculino , Taxa de Depuração Metabólica , Pessoa de Meia-Idade , Modelos Biológicos , Polimorfismo Genético , Estudos Prospectivos , Ritonavir/uso terapêuticoRESUMO
The c-myc oncogene encodes for a transcriptional factor involved in many cellular processes such as proliferation, differentiation and apoptosis. According to these different functions, the role of c-Myc protein in cellular sensitivity to anti-cancer drugs is controversial. We defined the role of c-Myc in cancer cell sensitivity to vinblastine (VLB) using human colon cancer cells: LoVo wild-type or transfected with a plasmid containing the human c-myc gene in antisense orientation (LoVo-mycANS). Analysis of VLB cytotoxicity demonstrated a 3-fold increase in VLB sensitivity in LoVo-mycANS cells. Comparison between cells revealed different apoptosis kinetics: accumulation of cells in sub-G1 phase and poly(ADP-ribose) polymerase cleavage occurred earlier in LoVo-mycANS. Then, we demonstrated a mitochondrial membrane potential disruption followed by cytochrome c release that indicates the involvement of mitochondria in this apoptotic signaling pathway. This earlier apoptosis was accompanied by a Bcl-2 decrease and a p53 increase. In conclusion, the decrease in c-Myc expression enhanced the VLB sensitivity, triggering earlier apoptosis through induction of the intrinsic pathway. Thus, c-myc induction is a resistance factor and our findings suggest that tumors carrying low levels of c-Myc protein could be more responsive to vinca alkaloids treatment. Moreover, the downregulation of c-myc oncogene by an antisense strategy might represent a useful goal for improving the efficacy of this anti-neoplastic drug family.
Assuntos
Antineoplásicos Fitogênicos/uso terapêutico , Neoplasias do Colo/tratamento farmacológico , Proteínas Proto-Oncogênicas c-myc/metabolismo , Vimblastina/uso terapêutico , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/metabolismo , Apoptose/efeitos dos fármacos , Neoplasias do Colo/metabolismo , Citocromos c/metabolismo , DNA Antissenso/farmacologia , Regulação para Baixo , Tolerância a Medicamentos , Fase G1/efeitos dos fármacos , Humanos , Potenciais da Membrana/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Poli(ADP-Ribose) Polimerases/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/metabolismo , Proteína X Associada a bcl-2/metabolismoRESUMO
The synthesis of different 4-arylcoumarin analogues of combretastatin A-4 led to the identification of two new compounds (1 and 2) with potent cytotoxic activity on a CEM leukemia cell line and a third one completely inactive (compound 3). It was suggested that the cytotoxicity of compounds 1 and 2 may be related to their interaction with microtubules and tubulin, since these compounds inhibit microtubule formation from purified tubulin in vitro [Bailly et al. (2003) J. Med. Chem. 46 (25), 5437-5444]. In the present study, tubulin was identified as the main target of these molecules. We studied structure-activity relationships of these compounds using biological experiments specific for tubulin binding. The modification of cell cycle progression induced by compounds 1 and 2 was characterized by an apoptotic induction on human breast cells (HBL100). In addition, these two molecules disturbed cell survival by depolymerizing the microtubule network, leading to a mitotic block. We then determined the thermodynamic parameters of their interaction with purified tubulin by fluorescence spectroscopy and isothermal microcalorimetry. These results, together with a superimposition of the molecule on colchicine in the X-ray-determined three-dimensional structure model of tubulin-colchicine complex, allowed us to identify the pharmacophore of the combretastatin A-4 analogues responsible for their biological activity.
Assuntos
Antineoplásicos Fitogênicos/metabolismo , Cumarínicos/metabolismo , Microtúbulos/metabolismo , Estilbenos/metabolismo , Tubulina (Proteína)/metabolismo , Antineoplásicos Fitogênicos/química , Apoptose , Calorimetria , Ciclo Celular , Linhagem Celular Tumoral , Cumarínicos/química , Humanos , Microtúbulos/química , Modelos Moleculares , Ligação Proteica , Espectrometria de Fluorescência , Estilbenos/química , Relação Estrutura-Atividade , Termodinâmica , Tubulina (Proteína)/químicaRESUMO
The synthesis of an analog of caulerpenyne having a trimethoxyaryl moiety was achieved in 11% overall yield over 11 steps. Its biological activity has been evaluated as inhibitor of in vitro tubulin polymerization or angiogenesis.
Assuntos
Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Colchicina/farmacologia , Neovascularização Fisiológica/efeitos dos fármacos , Sesquiterpenos/farmacologia , Tubulina (Proteína)/efeitos dos fármacos , Linhagem Celular Tumoral , Colchicina/análogos & derivados , Colchicina/síntese química , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Neovascularização Fisiológica/fisiologia , Polímeros/química , Sesquiterpenos/síntese química , Relação Estrutura-Atividade , Tubulina (Proteína)/metabolismoRESUMO
Vinflunine, the newest fluorinated Vinca alkaloid, currently in phase III clinical trials, targets the microtubule network to induce mitotic block and apoptosis by mechanisms that remain unclear. In the current study, we investigated the apoptotic pathways induced by a wide range of vinflunine concentrations in SK-N-SH neuroblastoma cells. The concentrations of vinflunine that inhibited 50 and 70% of cell growth (IC(50) and IC(70)) induced high extents of apoptosis but failed to depolymerize microtubule network and to block cells in G(2)/M. It is interesting that the IC(50) and IC(70) concentrations suppressed microtubule dynamics, slowed down mitotic progression from metaphase to anaphase, and induced a postmitotic G(1) arrest. This G(1) arrest was associated with an increase in p53 and p21 expression and with their nuclear translocation. A high concentration of vinflunine (500 nM) induced both microtubule depolymerization and a canonical G(2)/M block. Mitochondria were involved in apoptotic pathways because all studied concentrations induced cytochrome c release. Bcl-2 family members were differently modulated by the different drug concentrations. Bax was up-regulated and translocated to mitochondria at the IC(50) and IC(70) concentrations, whereas Bcl-2 was phosphorylated only at the highest vinflunine concentration examined (500 nM). Our findings can be extended to other Vinca alkaloids, because similar results were obtained with vinblastine. All together, our results show that low concentrations of vinflunine fail to promote a G(2)/M arrest but are sufficient to induce suppression of microtubule dynamics and subsequent apoptosis. Moreover, mitochondria constitute the point of convergence of apoptotic signals induced by both low and high concentrations of vinflunine.
Assuntos
Apoptose , Fase G1/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Vimblastina/análogos & derivados , Vimblastina/farmacologia , Fase G2/efeitos dos fármacos , Humanos , Microtúbulos , Mitocôndrias/fisiologia , Mitose/efeitos dos fármacos , Neuroblastoma/patologia , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/metabolismo , Regulação para Cima , Vimblastina/administração & dosagem , Proteínas rho de Ligação ao GTP/metabolismoRESUMO
CYP1A1 isoform is mainly regulated by the transcription factor AhR and to a lesser extent by the nuclear receptor RAR. The effect of a coexposure with 3MC, a AhR ligand, and RA, a RAR ligand, which are, respectively, strong and weak CYP1A1 inducers, is poorly known. We showed in Caco-2 cells that addition of RA significantly decreased 3MC-induced CYP1A1 expression by -55% for mRNA level and -30% for promoter and enzymatic activities. We further showed that RA decreased AhR protein level. Moreover, a physical interaction between AhR and the RAR-corepressor SMRT has been described in vitro. Using the corepressor inhibitor TSA, transfected-cells with SMRT cDNA, and coimmunoprecipitation experiments, we demonstrated that RA addition repressed AhR function through a marked AhR/SMRT physical interaction. This interaction explains the decrease of 3MC-induced CYP1A1 expression. This new mechanism involving the repression of AhR-induced CYP1A1 expression by retinoids allows better knowledge of the CYP1A1 regulation.
Assuntos
Citocromo P-450 CYP1A1/metabolismo , Proteínas de Ligação a DNA/metabolismo , Receptores de Hidrocarboneto Arílico/metabolismo , Proteínas Repressoras/metabolismo , Tretinoína/metabolismo , Células CACO-2 , Humanos , Metilcolantreno/metabolismo , Correpressor 2 de Receptor NuclearRESUMO
Human immunodeficiency virus (HIV) infection and the progression to AIDS are characterized by the depletion of CD4(+) T-cells. HIV-1 infection leads to apoptosis of uninfected bystander cells and the direct killing of HIV-infected cells. This is mediated, in part, by the HIV-1 Tat protein, which is secreted by virally infected cells and taken up by uninfected cells. We chemically synthesized two 86-residue subtype D Tat proteins, Ug05RP and Ug11LTS, from two Ugandan patients who were clinically categorized as either rapid progressor or long-term survivor, with non-conservative mutations located essentially in the glutamine-rich region. Structural heterogeneities were revealed by CD, which translate into differing trans-activational and apoptotic effects. CD data analysis and molecular modeling indicated that the short alpha-helix observed in subtype D Tat proteins from rapid progressor patients such as Tat Mal and Tat Ug05RP is not present in Ug11LTS. We show that Tat Ug05RP is more efficient than Tat Ug11LTS in its trans-activational role and in inducing apoptosis in binding tubulin via the mitochondrial pathway. The glutamine-rich region of Tat appears to be involved in the Tat-mediated apoptosis of T-cells.