Développement de biomarqueur Sentinelle en réponse à la pollution aquatique à partir de l'expression de protéines de phénotype "Multidrug Resistance" dans les érythrocytes de la truite Salmo trutta fario

La pollution croissante des milieux aquatiques nécessite la mise au point de nouvelles technologies permettant d optimiser la surveillance de la qualité de l eau. Dans ce contexte, nous avons développé un biomarqueur de susceptibilité du degré de la pollution globale des milieux aquatiques intitulé Sentinelle . Le principe du biomarqueur Sentinelle est basé sur le niveau de coexpression de deux protéines Multidrug Resistance (MDR), la protéine ABCG2-like et la P-gp, dans les érythrocytes de la truite Salmo trutta fario. Le biomarqueur sentinelle a été validé en conditions in vitro grâce au développement des cultures primaires d érythrocytes de truite. Après l exposition des globules rouges de truites à des concentrations croissantes d un polluant modèle, le Benzo-a-pyrène, l expression de la protéine ABCG2-like et de la P-gp augmente d une manière dose dépendante. Le biomarqueur Sentinelle a ensuite été validé en milieu naturel sur des truites fario en provenance de différents cours d eau d Auvergne. En milieu naturel, les deux protéines MDR sont exprimées différemment dans les érythrocytes de truites fario selon le degré de contamination du cours d eau. En effet, dans une rivière où la pollution est faible voire nulle, seule la protéine ABCG2-like est exprimée, alors que dans une rivière présentant une contamination plus importante, la P-gp et l ABCG2-like sont toutes les deux coexprimées par une réponse de type relais. Les expériences menées en conditions in vitro et en milieu naturel, laissent supposer que la protéine ABCG2-like assure une fonction de garde alors que la P-gp assurerait une fonction de protection défensive. En conséquence, selon le niveau d expression de la protéine de garde et de la protéine de défense, le degré de contamination de la rivière pourrait être évalué. L intérêt de l utilisation du biomarqueur Sentinelle a aussi été validé sur des Salmonidés en provenance de pisciculture. Ce nouvel outil biologique apporte des informations plus intégratives et plus précoces sur la qualité des milieux aquatiques, informations essentielles pour une meilleure gestion des ressources en eau.Increasing aquatic pollution requires the development of new technologies for to optimize the monitoring of water quality. In this context, we have developed a biomarker of susceptibility designating the degree of global pollution in aquatic medium, entitled "Sentinel". The Sentinel biomarker is based on the co-expression level of two major "Multidrug resistance" (MDR) proteins, such as ABCG2-like protein and P-gp, in erythrocytes of brown trout s in response to aquatic pollution. After developing a primary erythrocyte culture, the Sentinel biomarker was validated in a controlled medium. Trout erythrocytes exposure to increasing concentrations of Benzo-a-pyrene, a model pollutant, induced an increase expression of ABCG2-like protein and P-gp by a dose-dependent response. The Sentinel biomarker was then developed in a natural environment, using the erythrocytes of brown trout collected from the various rivers located in the Auvergne region of France. In the natural environment, both MDR proteins are differentially expressed in the erythrocytes of brown trout depending on the degree of contamination of rivers. Indeed, wild brown trout erythrocytes in an uncontaminated river, expressed only the ABCG2-like protein, whereas, in a river presenting a higher contamination, P-gp and ABCG2-like were both coexpressed with a relay response. Experiments in vitro conditions and natural environment, suggest that ABCG2-like protein acts as a vanguard protective protein, in complement to P-gp which acts as a defensive protective protein. Consequently, according to the expression level of the vanguard and defense proteins, the degree of contamination of the river could be evaluated. The use advantage of the Sentinel biomarker has also been validated on Salmonidae erythrocytes form farmed fish. This new tool provides biological information more early and integrative on the quality of aquatic environments. These informations are essential for better management of water resources.CLERMONT FD-Bib.électronique (631139902) / SudocSudocFranceF

Etude de la toxicite de la nigericine, antibiotique polyether carboxylique, en relation avec ses proprietes ionophores, sur le protozoaire cilie Tetrahymena pyriformis

Available from INIST (FR), Document Supply Service, under shelf-number : T 83002 / INIST-CNRS - Institut de l'Information Scientifique et TechniqueSIGLEFRFranc

Le complexe P-gp/mdr de la drosophile (un bio-marqueur potentiel de la pollution atmosphérique due aux hydrocarbures aromatiques polycycliques)

Les hydrocarbures aromatiques polycycliques (HAP) sont des polluants, émis lors de combustion incomplètes, qui peuvent engendrer des effets cytotoxiques, génotoxiques ou cancérigène. Dans le milieu atmosphérique la teneur de ces composés a été récemment réglementée au niveau européen. La surveillance de leurs concentrations et de leur impacts sur la santé, doit être effectuée par des mesures physico-chimiques et par le suivi de bio-indicateurs. Cette bio-surveillance est encore peu développée et nécessite la recherche de bio-marqueurs répondant spécifiquement à ces polluants. L'utilisation du complexe P-gp/mdr bien connu chez les mammifères pour son implication dans des phénomènes de " multidrug resistance ", a été évaluée pour le développement d'un bio-marqueur chez le modèle drosophile. Chez cet insecte nous avons trouvé que la P-gp était exprimée dans chaque stade de développement et qu'elle participait très activement à la protection des cellules en rejetant dans le milieu extracellulaire les HAP. De plus, l'exposition des drosophiles, en laboratoire ou sur le terrain, à différentes concentrations en HAP a très clairement été à l'origine d'une augmentation dose dépendante du taux d'expression de la P-gp. Nous avons montré que les transcrits des 3 gènes de type mdr répertoriés chez la mouche, étaient présents chez l'adulte d'une manière ubiquitaire et étaient exprimés au cours des différents stades de développement de cet insecte. L'analyse plus approfondie du gène mdr49 nous a permis de mettre en évidence une augmentation rapide et dose dépendante du taux de ses transcrits en réponse à une exposition des drosophiles à différents HAP. Les résultats obtenus ont permis de démontrer le rôle du complexe Pgp/mdr dans le système de protection de la drosophile vis-à-vis de HAP et sont en faveur de son utilisation potentielle en tant que bio-marqueur.Polycyclic aromatic hydrocarbons (PAHs) are cytotoxic, genotoxic and carcinogenic contaminants resulting from incomplete combustion. In the European community today, the release of PAHs in the atmosphere is tightly controlled. The monitoring of their concentrations and their impacts on health must be carried out by physicochemical measurements and the surveillance of biological indicators. Bio-monitoring is still poorly developed and requires the search for bio-markers responding specifically to these pollutants. We wished to develop a biomarker using Drosophila model. Therefore we evaluated the use of the P-gp/mdr complex known to be involved multidrug resistance in mammals. In Drosophila we found that P-gp is expressed at every developmental stage and is actively implicated in cell protection through the efflux of PAHs, either in the laboratory or in a natural environment, P-gp expression clearly increased in a dose dependant way. We also demonstrated that 3 identified mdr genes transcripts in Drosophila homologous of mammalian mdr, are ubiquitously expressed in the adult fly and are expressed during various stages of fly development. A thorough analysis of the mdr49 gene showed a rapid and dose-dependent increase of its expression after drosophila exposure to various PAHs. The results demonstrate the role of the Pgp/mdr complex in drosophila's defense system against PAHs and are in favour of its use as a bio-marker.CLERMONT FD-BCIU-Santé (631132104) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF

P-gp expression in brown trout erythrocytes: evidence of a detoxification mechanism in fish erythrocytes.

International audienceBlood is a site of physiological transport for a great variety of molecules, including xenobiotics. Blood cells in aquatic vertebrates, such as fish, are directly exposed to aquatic pollution. P-gp are ubiquitous "membrane detoxification proteins" implicated in the cellular efflux of various xenobiotics, such as polycyclic aromatic hydrocarbons (PAHs), which may be pollutants. The existence of this P-gp detoxification system inducible by benzo [a] pyrene (BaP), a highly cytotoxic PAH, was investigated in the nucleated erythrocytes of brown trout. Western blot analysis showed the expression of a 140-kDa P-gp in trout erythrocytes. Primary cultures of erythrocytes exposed to increasing concentrations of BaP showed no evidence of cell toxicity. Yet, in the same BaP-treated erythrocytes, P-gp expression increased significantly in a dose-dependent manner. Brown trout P-gp erythrocytes act as membrane defence mechanism against the pollutant, a property that can be exploited for future biomarker development to monitor water quality

Development and validation of a high-performance liquid chromatography method for the quantitation of intracellular PARP inhibitor Olaparib in cancer cells

International audienceOlaparib is a potent PARP inhibitor in clinical use for cancer therapy. A bioanalytical assay was developed and validated for quantitation of intracellular level of olaparib in cells exposed to the drug. The assay involves an optimized and straightforward sample pretreatment with acetonitrile for olaparib solubilization, cell lysis and protein precipitation, and a high performance liquid chromatography (HPLC) method with ultraviolet detection. Several parameters in both the sample preparation and the detection steps were investigated. Optimal chromatographic conditions were achieved with a 5 μL injection on a Nova-Pak® C18 column (150 × 3.9 mm, 4 μm) using a mobile phase consisting of acetonitrile and ultra-pure water in gradient mode, at a constant 1.2 mL/min flow rate, at 35 °C. Detection was carried out at 254 nm and a diode array detector was used to insure purity of the olaparib peak. The method was validated according to Food and Drug Administration guidelines. Linearity, accuracy and precisions were satisfactory over the concentration range of 200–2000 ng/mL. Limits of detection and quantification for olaparib were 50 ng/mL and 200 ng/mL, respectively. Good stability was showed in three relevant analytical conditions. Finally, the validated analytical method was successfully used to estimate the intracellular level of olaparib in SUM1315 breast cancer cells. A significant difference was observed in intracellular drug level after 1 and 3 h incubations. This method permitting measurement of drug level in tumor cells would allow dosage optimization and improvement of treatment response predictions

Co-targeting EGFR and mTOR with gefitinib and everolimus in triple-negative breast cancer cells

International audienceTriple-negative breast cancers (TNBC) are unlikely to respond to hormonal therapies and anti-HER2-targeted therapies. TNBCs overexpress EGFR and exhibit constitutive activation of the PI3K/AKT/mTOR signalling pathway. We hypothesized that simultaneously blocking EGFR and mTOR could be a potential therapeutic strategy for the treatment of TNBC. We examined the antitumour activity of the mTOR inhibitor everolimus combined with the EGFR tyrosine kinase inhibitor gefitinib in TNBC cell with or without activating mutations in the PI3K/AKT/mTOR signalling pathway. We demonstrated that everolimus and gefitinib induced synergistic growth inhibition in the PI3K and PTEN-mutant CAL-51 cell line but not in the PTEN-null HCC-1937 cell line. The antiproliferative effect was associated with synergistic inhibition of mTOR and P70S6K phosphorylation, as well as a significant reduction in 4E-BP1 activation in the CAL-51 cell line. We also showed that combination therapy significantly inhibited cell cycle progression and increased apoptosis in this cell line. Gene and protein expression analysis revealed significant downregulation of cell cycle regulators after exposure to combined treatment. Collectively, these results suggested that dual inhibition of mTOR and EGFR may be an effective treatment for TNBC with activating mutations of PI3K

Mini-P-gp and P-gp Co-Expression in Brown Trout Erythrocytes: A Prospective Blood Biomarker of Aquatic Pollution

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PARP1 inhibitor derived fluorescent probes: synthesis, characterization and in vitro biological evaluation in breast cancer cell lines

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Anti-EGFR monoclonal antibodies and EGFR tyrosine kinase inhibitors as combination therapy for triple-negative breast cancer

International audienceTriple-negative breast cancer (TNBC) is characterized by overexpression of epidermal growth factor receptor (EGFR) and activation of its downstream signaling pathways. Dual targeting of EGFR using one monoclonal antibody (mAb; cetuximab or panitumumab) and one tyrosine kinase inhibitor (EGFR-TKI; gefitinib or erlotinib) is a potential therapeutic approach. We investigated the effect of these therapies in EGFR-expressing TNBC cell lines that do or do not harbor the main activating mutations of EGFR pathways. Cell lines were sensitive to EGFR-TKIs, whereas mAbs were active only in MDA-MB-468 (EGFR amplification) and SUM-1315 (KRAS and PTEN wild-type) cells. MDA-MB-231 (KRAS mutated) and HCC-1937 (PTEN deletion) cells were resistant to mAbs. The combined treatment resulted in a synergistic effect on cell proliferation and superior inhibition of the RAS/MAPK signaling pathway in mAb-sensitive cells. The anti-proliferative effect was associated with G1 cell cycle arrest followed by apoptosis. Sensitivity to therapies was characterized by induction of positive regulators and inactivation of negative regulators of cell cycle. These results suggest that dual EGFR inhibition might result in an enhanced antitumor effect in a subgroup of TNBC. The status of EGFR, KRAS and PTEN could be used as a molecular marker for predicting the response to this therapeutic strategy

Méthode de détection, localisation et quantification de protéines MDR.

Méthode de détection, localisation et quantification de protéines MDR Méthode in vitro de détection, de localisation et/ou de quantification de protéines de multirésistance aux médicaments (MDR) associées à des cellules, dans un échantillon contenant des cellules procaryotes ou eucaryotes exprimant ou susceptibles d’exprimer des protéines MDR. La méthode comprend : l’ajout à l’échantillon en milieu liquide ou comprenant une phase liquide d’un traceur MDR apte à se fixer aux protéines MDR, l’incubation de l’échantillon avec ce traceur MDR, ce grâce à quoi le traceur MDR se fixe aux protéines MDR lorsqu’elles sont présentes, l’élimination du traceur non fixé à des protéines MDR, la détection, la localisation et/ou la quantification du traceur fixé à des protéines MDR