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1.
Pharmacol Res ; 102: 218-34, 2015 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-26375988

RESUMEN

Selenium supplement has been shown in clinical trials to reduce the risk of different cancers including lung carcinoma. Previous studies reported that the antiproliferative and pro-apoptotic activities of methylseleninic acid (MSA) in cancer cells could be mediated by inhibition of the PI3K pathway. A better understanding of the downstream cellular targets of MSA will provide information on its mechanism of action and will help to optimize its use in combination therapies with PI3K inhibitors. For this study, the effects of MSA on viability, cell cycle, metabolism, apoptosis, protein and mRNA expression, and reactive oxygen species production were analysed in A549 cells. FOXO3a subcellular localization was examined in A549 cells and in stably transfected human osteosarcoma U2foxRELOC cells. Our results demonstrate that MSA induces FOXO3a nuclear translocation in A549 cells and in U2OS cells that stably express GFP-FOXO3a. Interestingly, sodium selenite, another selenium compound, did not induce any significant effects on FOXO3a translocation despite inducing apoptosis. Single strand break of DNA, disruption of tumour cell metabolic adaptations, decrease in ROS production, and cell cycle arrest in G1 accompanied by induction of apoptosis are late events occurring after 24h of MSA treatment in A549 cells. Our findings suggest that FOXO3a is a relevant mediator of the antiproliferative effects of MSA. This new evidence on the mechanistic action of MSA can open new avenues in exploiting its antitumour properties and in the optimal design of novel combination therapies. We present MSA as a promising chemotherapeutic agent with synergistic antiproliferative effects with cisplatin.


Asunto(s)
Antineoplásicos/farmacología , Núcleo Celular/metabolismo , Factores de Transcripción Forkhead/metabolismo , Compuestos de Organoselenio/farmacología , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Células 3T3 , Animales , Apoptosis/efectos de los fármacos , Proteínas Reguladoras de la Apoptosis/metabolismo , Ciclo Celular/efectos de los fármacos , Línea Celular , Línea Celular Tumoral , Núcleo Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Cisplatino/farmacología , Proteína Forkhead Box O3 , Puntos de Control de la Fase G1 del Ciclo Celular/efectos de los fármacos , Humanos , Ratones , Fosfatidilinositol 3-Quinasas/metabolismo , Transporte de Proteínas/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo
2.
Toxicol In Vitro ; 28(5): 1006-15, 2014 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-24809893

RESUMEN

The formation of reactive oxygen species (ROS) could cause cellular damage and eventually lead to apoptosis and necrosis. The ratio between oxidized glutathione and reduced glutathione (GSSG-to-GSH ratio) has been used as an important in vitro and in vivo biomarker of the redox balance in the cell and consequently of cellular oxidative stress. This paper optimizes a LC-MS/MS method for the simultaneous determination of GSH and GSSG. The proposed method is based on the derivatization of reduced GSH using iodoacetic acid (IAA) in order to prevent its rapid oxidation to GSSG during sample preparation. The optimized analytical method was applied to evaluate the effect of different pharmaceutical agents on GSSG-to-GSH ratio in cryopreserved rat and human hepatocytes in culture. Hepatocyte viabilities were also determined at the same time by using the WST-1 assay as a direct measurement of cell mitochondrial respiration. The results obtained demonstrate that cryopreserved rat and human hepatocytes in culture are reliable in vitro models for the evaluation of cellular oxidative stress. In addition, the GSSG-to-GSH ratio measurements could be a biomarker of hepatotoxicity providing similar results to those of cytotoxicity assay.


Asunto(s)
Criopreservación , Disulfuro de Glutatión/metabolismo , Glutatión/metabolismo , Hepatocitos , Aminopirina/toxicidad , Animales , Biomarcadores/metabolismo , Células Cultivadas , Ciclosporina/toxicidad , Flutamida/toxicidad , Humanos , Masculino , Estrés Oxidativo , Ratas Sprague-Dawley , Tolmetina/análogos & derivados , Tolmetina/toxicidad
3.
Metabolomics ; 8(3): 454-464, 2012 Jun 01.
Artículo en Inglés | MEDLINE | ID: mdl-22661920

RESUMEN

Cyclin-dependent kinases CDK4 and CDK6 are essential for the control of the cell cycle through the G(1) phase. Aberrant expression of CDK4 and CDK6 is a hallmark of cancer, which would suggest that CDK4 and CDK6 are attractive targets for cancer therapy. Herein, we report that calcein AM (the calcein acetoxymethyl-ester) is a potent specific inhibitor of CDK4 and CDK6 in HCT116 human colon adenocarcinoma cells, inhibiting retinoblastoma protein (pRb) phosphorylation and inducing cell cycle arrest in the G(1) phase. The metabolic effects of calcein AM on HCT116 cells were also evaluated and the flux between the oxidative and non-oxidative branches of the pentose phosphate pathway was significantly altered. To elucidate whether these metabolic changes were due to the inhibition of CDK4 and CDK6, we also characterized the metabolic profile of a CDK4, CDK6 and CDK2 triple knockout of mouse embryonic fibroblasts. The results show that the metabolic profile associated with the depletion of CDK4, CDK6 and CDK2 coincides with the metabolic changes induced by calcein AM on HCT116 cells, thus confirming that the inhibition of CDK4 and CDK6 disrupts the balance between the oxidative and non-oxidative branches of the pentose phosphate pathway. Taken together, these results indicate that low doses of calcein can halt cell division and kill tumor cells. Thus, selective inhibition of CDK4 and CDK6 may be of greater pharmacological interest, since inhibitors of these kinases affect both cell cycle progression and the robust metabolic profile of tumors.

4.
Biochim Biophys Acta ; 1807(6): 746-54, 2011 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-21185256

RESUMEN

Metabolic adaptations are associated with changes in enzyme activities. These adaptations are characterized by patterns of positive and negative changes in metabolic fluxes and concentrations of intermediate metabolites. Knowledge of the mechanism and parameters governing enzyme kinetics is rarely available. However, the signs-increases or decreases-of many of these changes can be predicted using the signs of metabolic control coefficients. These signs require the only knowledge of the structure of the metabolic network and a limited qualitative knowledge of the regulatory dependences, which is widely available for carbon metabolism. Here, as a case study, we identified control coefficients with fixed signs in order to predict the pattern of changes in key enzyme activities which can explain the observed changes in fluxes and concentrations underlying the metabolic adaptations in oncogenic K-ras transformation in NIH-3T3 cells. The fixed signs of control coefficients indicate that metabolic changes following the oncogenic transformation-increased glycolysis and oxidative branch of the pentose-phosphate pathway, and decreased concentration in sugar-phosphates-could be associated with increases in activity for glucose-6-phosphate dehydrogenase, pyruvate kinase and lactate dehydrogenase, and decrease for transketolase. These predictions were validated experimentally by measuring specific activities. We conclude that predictions based on fixed signs of control coefficients are a very robust tool for the identification of changes in enzyme activities that can explain observed metabolic adaptations in carbon metabolism.


Asunto(s)
Adaptación Biológica , Carbono/metabolismo , Transformación Celular Neoplásica/genética , Genes ras , Redes y Vías Metabólicas , Adaptación Biológica/genética , Adaptación Biológica/fisiología , Animales , Transporte Biológico , Metabolismo de los Hidratos de Carbono/genética , Metabolismo de los Hidratos de Carbono/fisiología , Transformación Celular Neoplásica/metabolismo , Transformación Celular Neoplásica/patología , Regulación Neoplásica de la Expresión Génica/fisiología , Genes ras/fisiología , Redes y Vías Metabólicas/genética , Redes y Vías Metabólicas/fisiología , Ratones , Modelos Biológicos , Células 3T3 NIH , Concentración Osmolar , Transfección
5.
Biochem Soc Trans ; 38(5): 1302-6, 2010 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-20863303

RESUMEN

Metabolite concentrations and fluxes are the system variables that characterize metabolism. The systematic study of metabolite profiles is known as metabolomics; however, knowledge of the complete set of metabolites may not be enough to predict distinct phenotypes. A complete understanding of metabolic processes requires detailed knowledge of enzyme-controlled intracellular fluxes. These can be estimated through quantitative measurements of metabolites at different times or by analysing the stable isotope patterns obtained after incubation with labelled substrates. We have identified distinct intracellular fluxes associated with metabolic adaptations accompanying cancer. The maintenance of an imbalance between fluxes for the oxidative and non-oxidative PPP (pentose phosphate pathway) has been shown to be critical for angiogenesis and cancer cell survival. Mouse NIH 3T3 cells transformed by different mutated K-ras oncogenes have differential routing of glucose to anaerobic glycolysis, the PPP and the Krebs cycle. These results indicate that knowledge of metabolic fingerprints associated with an altered genetic profile could be exploited in the rational design of new therapies. We conclude that the understanding of the multifactorial nature of metabolic adaptations in cancer may open new ways to develop novel multi-hit antitumoral therapies.


Asunto(s)
Redes y Vías Metabólicas/fisiología , Neoplasias/metabolismo , Animales , Antineoplásicos/uso terapéutico , Humanos , Modelos Biológicos , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Biología de Sistemas/métodos
6.
Anal Chem ; 79(13): 5000-5, 2007 Jul 01.
Artículo en Inglés | MEDLINE | ID: mdl-17523595

RESUMEN

The quantitative understanding of the role of sugar phosphates in regulating tumor energetic metabolism at the proteomic and genomic level is a prerequisite for an efficient rational design in combined drug chemotherapy. Therefore, it is necessary to determine accurately the concentration of the main sugar phosphate pools at the lower concentrations present in the often-limited volume of tumor cell samples. Taking as an example the human adenocarcinoma cell line HT29, we here report a fast and reliable quantitative method based on the use of liquid nitrogen, a weak acid extraction, and liquid chromatography-electrospray ionization tandem mass spectrometry to quantify simultaneously the intracellular concentration of sugar phosphate pools. The method was set up using standard addition curves. Thus, it is possible to identify and quantify hexose phosphate, pentose phosphate, and triose phosphate pools up to 0.02-0.10 ng x microL(-1), depending on the analyte. The method developed was here used for the quantitative study of changes in phosphorylated carbohydrates of central carbon metabolism when high or low glucose concentration conditions are induced in vitro in the HT29 human colon adenocarcinoma cell line.


Asunto(s)
Adenocarcinoma/metabolismo , Cromatografía Liquida/métodos , Neoplasias del Colon/metabolismo , Espectrometría de Masa por Ionización de Electrospray/métodos , Fosfatos de Azúcar/análisis , Adenocarcinoma/patología , Línea Celular , Células Cultivadas , Neoplasias del Colon/patología , Glucosa/análisis , Glucosa/metabolismo , Glucosa-6-Fosfato/análisis , Glucosa-6-Fosfato/metabolismo , Células HT29 , Humanos , Vía de Pentosa Fosfato , Estándares de Referencia , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Fosfatos de Azúcar/metabolismo , Factores de Tiempo
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