Portrait of Ependymoma Recurrence in Children: Biomarkers of Tumor Progression Identified by Dual-Color Microarray-Based Gene Expression Analysis

BACKGROUND: Children with ependymoma may experience a relapse in up to 50% of cases depending on the extent of resection. Key biological events associated with recurrence are unknown. METHODOLOGY/PRINCIPAL FINDINGS: To discover the biology behind the recurrence of ependymomas, we performed CGHarray and a dual-color gene expression microarray analysis of 17 tumors at diagnosis co-hybridized with the corresponding 27 first or subsequent relapses from the same patient. As treatment and location had only limited influence on specific gene expression changes at relapse, we established a common signature for relapse. Eighty-seven genes showed an absolute fold change ≥2 in at least 50% of relapses and were defined as the gene expression signature of ependymoma recurrence. The most frequently upregulated genes are involved in the kinetochore (ASPM, KIF11) or in neural development (CD133, Wnt and Notch pathways). Metallothionein (MT) genes were downregulated in up to 80% of the recurrences. Quantitative PCR for ASPM, KIF11 and MT3 plus immunohistochemistry for ASPM and MT3 confirmed the microarray results. Immunohistochemistry on an independent series of 24 tumor pairs at diagnosis and at relapse confirmed the decrease of MT3 expression at recurrence in 17/24 tumor pairs (p = 0.002). Conversely, ASPM expression was more frequently positive at relapse (87.5% vs 37.5%, p = 0.03). Loss or deletion of the MT genes cluster was never observed at relapse. Promoter sequencing after bisulfite treatment of DNA from primary tumors and recurrences as well as treatment of short-term ependymoma cells cultures with a demethylating agent showed that methylation was not involved in MT3 downregulation. However, in vitro treatment with a histone deacetylase inhibitor or zinc restored MT3 expression. CONCLUSIONS/SIGNIFICANCE: The most frequent molecular events associated with ependymoma recurrence were over-expression of kinetochore proteins and down-regulation of metallothioneins. Metallothionein-3 expression is epigenetically controlled and can be restored in vitro by histone deacetylase inhibitors

Mécanismes cellulaires et moléculaires de la transition epithelio-mesenchymateuse au niveau hépatique : effets des contaminants environnementaux

La transition épithélio-mésenchymateuse (TEM) est un processus qui interviendrait lors des étapes précoces (fibrose) et tardives (métastases) de cancérogenèse. Elle consiste en la perte des caractères cellulaires épithéliaux tels que les jonctions adhérentes, pour le gain de propriétés mésenchymateuses, et confère ainsi aux cellules la capacité de migration et d'invasion. Nos travaux ont eu pour objectifs d'identifier et de caractériser, sur des cellules d'origine hépatique, des biomarqueurs de TEM, afin d'évaluer de manière prédictive et fiable les effets pro-tumoraux de contaminants chimiques environnementaux. Nous avons d'abord décrypté les évènements cellulaires et moléculaires intervenant en réponse à des inducteurs de TEM, tels que le TPA sur la lignée HepG2 et le TGFβ sur des hépatocytes humains en culture primaire. Puis, combinés à l'utilisation d'une technologie innovante de mesure de l'impédance cellulaire en temps réel et à d'autres biomarqueurs (apoptose, cycle cellulaire…), ces travaux nous ont permis d'estimer l'impact de pesticides dans l'initiation de la TEM et leur potentiel cancérogène sur le foie.The epithelial to mesenchymal transition (EMT) is a process that occurs during the early (fibrosis) and late (metastases) stages of carcinogenesis. It is defined as the loss of epithelial characteristics such as cell adherent junctions and the gain of mesenchymal properties, thereby conferring to cells the ability to migrate and invade. Our objectives were to identify and characterize, in hepatic cells, EMT biomarkers that could reliably predict pro-tumoral effects of environmental chemical contaminants. We first characterized the cellular and molecular events that occur in response to EMT inducers, such as TPA on cell line HepG2 and TGFβ on human hepatocytes in primary culture. Then, using an innovative technology capable of measuring real time cellular impedance alongside other biomarkers of such as apoptosis and the cell cycle, we estimated the impact of pesticides on EMT and assessed their carcinogenic potential on liver

Contribution au développement d'une méthode quantitative de détachement de cellules adhérentes par ultrasons : application à la caractérisation de l'adhésion cellulaire sur biomatériaux

L'adhésion cellulaire est un processus essentiel dans de nombreux phénomènes physiologiques et notamment en ce qui concerne l'intégration biologique des biomatériaux. Les développements constants des applications biotechnologiques réclament une bonne connaissance du phénomène d'adhésion. L'objectif de ce travail est de proposer une contribution au développement d'une méthode ultrasonore de caractérisation de l'adhésion cellulaire sur substrat. L'originalité de la méthode réside dans l'utilisation d'ondes ultrasonores longitudinales basses fréquences pour générer l'application d'une force sur des cellules en culture. Cette méthode globale peut être adaptée pour différents types de cellules ou de substrats. Des insonificateurs adaptés à l'utilisation de boîtes de culture cellulaire ont été développés et étalonnés. Enfin, une approche théorique de l'effet du passage des ultrasons sur l'équilibre ligand-récepteur a été proposée. Outre le développement de la méthode, nous avons mené des essais sur deux types de substrats que sont le verre et l'hydroxyapatite. Une technique originale de mise en forme de l'hydroxyapatite a été utilisée pour conduire des tests sur ce type de matériau Le verre a été testé pour différents conditionnements. Les cellules utilisées sont des ostéoblastes de souris de la lignée MC3T3-E1. Nous obtenons une force nécessaire au détachement cellulaire de 1616+-157 nN sur du verre conditionné et de 2702+-80 nN pour l'hydroxyapatite dans les mêmes conditions. Ces résultats montrent la sensibilité de la méthode aux conditions de culture et au substrat utilisé. Enfin, nous proposons un protocole permettant d'utiliser la méthode mise au point dans ce travail.Many physiological phenomena depend on cell adhesion and especially biological integration of biomaterials. Constant developments of biotechnological applications claim a good knowledge of adhesion phenomenon. The aim of this work is to propose a contribution to the development of an ultrasonic characterisation method of cellular adhesion on substrates. Originality of the method dwells in the use of low frequencies ultrasonic waves to produce force application on cultured cells. This global method can be adapted for different cell types or substrates. Insonificators adapted to the use of cell culture boxes were developed and calibrated. Lastly, a theoretical approach of ultrasounds passage effect on ligand-receiver balance was proposed. In addition to the development of the method, we carried out tests on two types of substrates that are glass and hydroxyapatite. An original shaping technique of hydroxyapatite was used to lead tests on this type of material. Different conditionings were tested on glass substrate. We used MC3T3-E1 osteoblast cell line. We obtain a necessary force to provoke cell detachment of 1616+-157 nN for glass substrate and 2702+-80 nN for hydroxyapatite in the same conditions. Finally, we propose a protocol allowing to use the method developed in this work.VALENCIENNES-BU Sciences Lettres (596062101) / SudocSudocFranceF

Crosstalk between Beta-Catenin and Snail in the Induction of Epithelial to Mesenchymal Transition in Hepatocarcinoma: Role of the ERK1/2 Pathway

Epithelial to mesenchymal transition (EMT) is an integral process in the progression of many epithelial tumors. It involves a coordinated series of events, leading to the loss of epithelial features and the acquisition of a mesenchymal phenotype, resulting in invasion and metastasis. The EMT of hepatocellular carcinoma (HCC) cells is thought to be a key event in intrahepatic dissemination and distal metastasis. In this study, we used 12-O-tet-radecanoylphorbol-13-acetate (TPA) to dissect the signaling pathways involved in the EMT of HepG2 hepatocarcinoma cells. The spectacular change in phenotype induced by TPA, leading to a pronounced spindle-shaped fibroblast-like cell morphology, required ERK1/2 activation. This ERK1/2-dependent EMT process was characterized by a loss of E-cadherin function, modification of the cytoskeleton, the acquisition of mesenchymal markers and profound changes to extracellular matrix composition and mobility. Snail was essential for E-cadherin repression, but was not sufficient for full commitment of the TPA-triggered EMT. We found that TPA triggered the formation of a complex between Snail and β-catenin that activated the Wnt pathway. This study thus provides the first evidence for the existence of a complex network governed by the ERK1/2 signaling pathway, converging on the coregulation of Snail and the Wnt/β-catenin pathway and responsible for the onset and the progression of EMT in hepatocellular carcinoma cells

A simple GC–MS method for the determination of diphenylamine, tolylfluanid propargite and phosalone in liver fractions

International audienceIn this study, an accurate and robust gas chromatography/mass spectrometry method was developed for quantitative analysis of diphenylamine, tolylfluanid, propargite and phosalone in liver fractions. Different injector parameters were optimized by an experimental design technique (central composite design). An optimal combination of injector temperature (°C), splitless time (min) and overpressure (kPa) values enabled to maximize the chromatographic responses. Sample preparation was based on protein precipitation using trichloroacetic acid followed by liquid-liquid extraction (LLE) of the pesticides with hexane. All compounds and endrin as internal standard were quantified without interference in selected ion monitoring mode. The calibration curves for diphenylamine, tolylfluanid, propargite and phosalone compounds were linear over the concentration range of 0.1 to 25 μM with determination coefficients (R2) higher than 0.999. A lower limit of quantification of 0.1 μM was obtained for all analytes, i.e. 422.5, 868.0, 876.2 and 919.5 μg/kg of liver fraction (hepatocytes) for diphenylamine, tolylfluanid, propargite and phosalone, respectively. All compounds showed extraction recoveries higher than 93%, with a maximum RSD of 3.4%. Intra- and inter-day accuracies varied from 88.4 to 102.9% and, imprecision varied from 1.1 to 6.7%. Stability tests demonstrated that all pesticides were stable in liver extracts during instrumental analysis (20 °C in the autosampler tray for 72 h) following three successive freeze-thaw cycles and, at −20 °C for up to 12 months. This simple and efficient analytical procedure is thus suitable for metabolism studies or for assessing mammals liver contamination

TRAIL receptor-induced features of epithelial-to-mesenchymal transition increase tumour phenotypic heterogeneity: potential cell survival mechanisms

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Effects of endosulfan on hepatoma cell adhesion: Epithelial-mesenchymal transition and anoikis resistance

Endosulfan is an organochlorine pesticide commonly used in agriculture yet classified by the Stockholm Convention in 2011 as a persistent organic pollutant (POP). Its potential toxicity makes its continued use a major public health concern. Despite studies in laboratory animals, the molecular mechanisms underlying the carcinogenic effects of endosulfan in human liver remain poorly understood. In this study, we investigated the phenotypical effects of endosulfan on HepG2 liver cells. First, we found that endosulfan disrupted the anoikis process. Indeed, cells exposed to endosulfan were initially sensitized to anoikis and thereafter recovered their resistance to this process. This phenomenon occurred in parallel to the induction of the epithelial to mesenchymal (EMT) process, as demonstrated by: (1) reorganization of the actin cytoskeleton together with activation of the FAK signaling pathway; (2) repression of E-cadherin expression; (3) induction of Snail and Slug; (4) activation of the WNT/beta-catenin pathway; and (5) induction and reorganization of mesenchymal markers (S100a4, vimentin, fibronectin, MMP-7). Secondly, despite the acquisition of mesenchymal characteristics, HepG2 cells exposed to endosulfan failed to migrate. This incapacity to acquire a motile phenotype could be attributed to a disruption of the interaction between the ECM and the cells. Taken together, these results indicate that endosulfan profoundly alters the phenotype of liver cells by inducing cell detachment and partial EMT as well as disrupting the anoikis process. All these events account, at least in part, for the carcinogenic potential of endosulfan in liver

Is bisphenol S a safe substitute for bisphenol A in terms of metabolic function? An in vitro study

International audienceAs bisphenol A (BPA) has been shown to induce adverse effects on human health, especially through the activation of endocrine pathways, it is about to be withdrawn from the European market and replaced by analogues such as bisphenol S (BPS). However, toxicological data on BPS is scarce, and so it is necessary to evaluate the possible effects of this compound on human health. We compared the effect of BPA and BPS on obesity and hepatic steatosis processes using low doses in the same range as those found in the environment. Two in vitro models were used, the adipose cell line 3T3-L1 and HepG2 cells, representative of hepatic functions. We analyzed different parameters such as lipid and glucose uptakes, lipolysis, leptin production and the modulation of genes involved in lipid metabolism and energy balance. BPA and BPS induced an increase in the lipid content in the 3T3-L1 cell line and more moderately in the hepatic cells. We also observed a decrease in lipolysis after bisphenol treatment of adipocytes, but only BPS was involved in the increase in glucose uptake and leptin production. These latter effects could be linked to the modulation of SREBP-1c, PPAR gamma, aP2 and ERR alpha and gamma genes after exposure to BPA, whereas BPS seems to target the PGC1 alpha and the ERR gamma genes. The findings suggest that both BPA and BPS could be involved in obesity and steatosis processes, but through two different metabolic pathways

Evidence of in vitro metabolic interaction effects of a chlorfenvinphos, ethion and linuron mixture on human hepatic detoxification rates

General population exposure to pesticides mainly occurs via food and water consumption. However, their risk assessment for regulatory purposes does not currently consider the actual co-exposure to multiple substances. To address this concern, relevant experimental studies are needed to fill the lack of data concerning effects of mixture on human health. For the first time, the present work evaluated on human microsomes and liver cells the combined metabolic effects of, chlorfenvinphos, ethion and linuron, three pesticides usually found in vegetables of the European Union. Concentrations of these substances were measured during combined incubation experiments, thanks to a new analytical methodology previously developed. The collected data allowed for calculation and comparison of the intrinsic hepatic clearance of each pesticide from different combinations. Finally, the results showed clear inhibitory effects, depending on the association of the chemicals at stake. The major metabolic inhibitor observed was chlorfenvinphos. During co-incubation, it was able to decrease the intrinsic clearance of both linuron and ethion. These latter also showed a potential for metabolic inhibition mainly cytochrome P450-mediated in all cases. Here we demonstrated that human detoxification from a pesticide may be severely hampered in case of co-occurrence of other pesticides, as it is the case for drugs interactions, thus increasing the risk of adverse health effects. These results could contribute to improve the current challenging risk assessment of human and animal dietary to environmental chemical mixtures