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

Projet Euromix. Vers une démarche normalisée, à l’échelle européenne, pour évaluer les risques des mélanges de contaminants chimiques.

Ce numéro est constitué d’articles issus du colloque « Contaminants alimentaires – approches émergentes pour connaître et prévenir le risque » qui s’est tenu à Paris le 19 décembre 2018.Through our environment, we are continuously exposed to chemical mixtures. Answering the questionof this impact on our health and that of the environment remains a challenge. The risk assessment ofchemicals for regulatory purposes is mainly based on the assessment of individual products. Whilemethodologies and guidance for assessing risks related to combined exposure to multiple chemicalshave been developed for different regulatory sectors, there is no harmonised and consistent approachto assessing and managing these risks.The EuroMix program develops a strategy of, testing, bioassays and models to assess the risks ofchemical mixtures with a focus on: - Reduction of uncertainties through the generation of hazard data through in vitro testing,- Prioritisation according to hazard (in-silico tools) and/or exposure considerations,- Study of the extrapolation of in vitro tests as reliable alternatives for animal testing,- Development of PB-TK models for in vitro/in vivo extrapolation in the risk assessment ofmixtures,- Development of hazard and exposure models for risk assessment and application of thesemodels to newly generated data.Par notre environnement, nous sommes continuellement exposés à des mélanges de produitschimiques. Répondre à la question de cet impact sur notre santé et celle de l’environnement reste unchallenge, l'évaluation des risques des produits chimiques à des fins réglementaires reposantprincipalement sur l'évaluation des composés pris individuellement. Si des méthodologies et lesdémarches pour évaluer les risques liés à l'exposition combinée à de multiples produits chimiques ontété élaborées pour différents secteurs réglementaires, il n'existe pas d'approche harmonisée etcohérente pour l'évaluation et la gestion de ces risques.Le programme EuroMix1 développe une stratégie de tests, bio-essais et modèles pour évaluer lesrisques des mélanges chimiques en mettant l'accent sur :- La réduction des incertitudes par la production de données sur les dangers par des essais invitro,- L’établissement des priorités en fonction du danger (outils in-silico) et/ou sur des considérationsrelatives à l'exposition,- L’étude de l’extrapolation des essais in vitro comme solutions de rechange fiables pour lesessais sur les animaux,- Le développement de modèles PB-TK d’extrapolation in vitro/in vivo dans l'évaluation desrisques liés aux mélanges,- L’élaboration de modèles de danger et d'exposition pour l'évaluation des risques et appliquerces modèles sur les données nouvellement générées

Transcriptomic analysis of Spodoptera frugiperda Sf9 cells resistant to Bacillus thuringiensis Cry1Ca toxin reveals that extracellular Ca2+, Mg2+ and production of cAMP are involved in toxicity

Bacillus thuringiensis (Bt) produces pore forming toxins that have been used for pest control in agriculture for many years. However, their molecular and cellular mode of action is still unclear. While a first model – referred to as the pore forming model – is the most widely accepted scenario, a second model proposed that toxins could trigger an Mg2+-dependent intracellular signalling pathway leading to cell death. Although Cry1Ca has been shown to form ionic pores in the plasma membrane leading to cell swelling and death, we investigated the existence of other cellular or molecular events involved in Cry1Ca toxicity. The Sf9 insect cell line, derived from Spodoptera frugiperda, is highly and specifically sensitive to Cry1Ca. Through a selection program we developed various levels of laboratory-evolved Cry1Ca-resistant Sf9 cell lines. Using a specific S. frugiperda microarray we performed a comparative transcriptomic analysis between sensitive and resistant cells and revealed genes differentially expressed in resistant cells and related to cation-dependent signalling pathways. Ion chelators protected sensitive cells from Cry1Ca toxicity suggesting the necessity of both Ca2+ and/or Mg2+ for toxin action. Selected cells were highly resistant to Cry1Ca while toxin binding onto their plasma membrane was not affected. This suggested a resistance mechanism different from the classical ‘loss of toxin binding’. We observed a correlation between Cry1Ca cytotoxicity and the increase of intracellular cAMP levels. Indeed, Sf9 sensitive cells produced high levels of cAMP upon toxin stimulation, while Sf9 resistant cells were unable to increase their intracellular cAMP. Together, these results provide new information about the mechanism of Cry1Ca toxicity and clues to potential resistance factors yet to discover

Resistance to cisplatin-induced cell death conferred by the activity of organic anion transporting polypeptides (OATP) in human melanoma cells

International audienceExpression of organic anion transporting polypeptides (OATP) transporters can be modified with potential incidence in cancers, yet they have not been considered in melanoma. Here, we demonstrate transcriptional and protein expression of OATP members in human melanoma cell lines with sodium-independent organic anion uptake activity. Importantly, uptake of different organic anions over 24 h led to a common resistance signal to apoptotic cell death, induced further by cisplatin in 24 h. The mechanism is not dependent on the transport of cisplatin by the OATP, as it is not an OATP substrate. The resistance signal was modulated by PKC, disclosing it as signal mediator. This study suggests that OATP, which can be constantly activated by endobiotics, may contribute to melanoma chemotherapeutic resistance, thereby justifying the development of OATP targeting strategies

Bacillus thuringiensis toxins divert progenitor cells toward enteroendocrine fate by decreasing cell adhesion with intestinal stem cells in Drosophila

Bacillus thuringiensis subsp. kurstaki (Btk) is a strong pathogen toward lepidopteran larvae thanks to specific Cry toxins causing leaky gut phenotypes. Hence, Btk and its toxins are used worldwide as microbial insecticide and in genetically modified crops, respectively, to fight crop pests. However, Btk belongs to the B. cereus group, some strains of which are well known human opportunistic pathogens. Therefore, ingestion of Btk along with food may threaten organisms not susceptible to Btk infection. Here we show that Cry1A toxins induce enterocyte death and intestinal stem cell (ISC) proliferation in the midgut of Drosophila melanogaster, an organism non-susceptible to Btk. Surprisingly, a high proportion of the ISC daughter cells differentiate into enteroendocrine cells instead of their initial enterocyte destiny. We show that Cry1A toxins weaken the E-Cadherin-dependent adherens junction between the ISC and its immediate daughter progenitor, leading the latter to adopt an enteroendocrine fate. Hence, although not lethal to non-susceptible organisms, Cry toxins can interfere with conserved cell adhesion mechanisms, thereby disrupting intestinal homeostasis and endocrine functions

The PERICLES research program : an original approach to characterize thecombined effects of the main pesticide residue mixtures to which the Frenchpopulation is exposed

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Adverse outcome pathway-driven analysis of liver steatosis in vitro : a case study with cyproconazole

Adverse outcome pathways (AOPs) describe causal relationships between molecular perturbation and adverse cellular effects and are being increasingly adopted for linking in vitro mechanistic toxicology to in vivo data from regulatory toxicity studies. In this work, a case study was performed by developing a bioassay toolbox to assess key events in the recently proposed AOP for chemically induced liver steatosis. The toolbox is comprised of in vitro assays to measure nuclear receptor activation, gene and protein expression, lipid accumulation, mitochondrial respiration, and formation of fatty liver cells. Assay evaluation was performed in human HepaRG hepatocarcinoma cells exposed to the model compound cyproconazole, a fungicide inducing steatosis in rodents. Cyproconazole dose-dependently activated RAR alpha and PXR, two molecular initiating events in the steatosis AOP. Moreover, cyproconazole provoked a disruption of mitochondrial functions and induced triglyceride accumulation and the formation of fatty liver cells as described in the AOP. Gene and protein expression analysis, however, showed expression changes different from those proposed in the AOP, thus suggesting that the current version of the AOP might not fully reflect the complex mechanisms linking nuclear receptor activation and liver steatosis. Our study shows that cyproconazole induces steatosis in human liver cells in vitro and demonstrates the utility of systems-based approaches in the mechanistic assessment of molecular and cellular key events in an AOP. AOP-driven in vitro testing as demonstrated can further improve existing AOPs, provide insight regarding molecular mechanisms of toxicity, and inform predictive risk assessment

An adverse outcome pathway-based approach to assess steatotic mixture effects of hepatotoxic pesticides in vitro

Exposure to complex chemical mixtures requires a tiered strategy for efficient mixture risk assessment. As a part of the EuroMix project we developed an adverse outcome pathway (AOP)-based assay toolbox to investigate the combined effects of the liver steatosis-inducing compounds imazalil, thiacloprid, and clothianidin in human HepaRG hepatocarcinoma cells. Compound-specific relative potency factors were determined using a benchmark dose approach. Equipotent mixtures were tested for nuclear receptor activation, gene and protein expression, and triglyceride accumulation, according to the molecular initiating events and key events proposed in the steatosis AOP. All three compounds affected the activity of nuclear receptors, but not key genes/proteins as proposed. Triglyceride accumulation was observed with three different methods. Mixture effects were in agreement with the assumption of dose additivity for all the combinations and endpoints tested. Compound-specific RPFs remained similar over the different endpoints studied downstream the AOP. Therefore, it might be possible to reduce testing to a smaller battery of key tests. The results demonstrate the suitability of our in vitro assay toolbox, integrated within an AOP framework and combined with the RPF approach, for the analysis of steatotic effects of chemical mixtures. However, mRNA results suggest that the steatosis AOP still needs improvement.</p

The PERICLES research program: An integrated approach to characterize the combined effects of mixtures of pesticide residues to which the French population is exposed.

Due to the broad spectrum of pesticide usages, consumers are exposed to mixtures of residues, which may have combined effects on human health. The PERICLES research program aims to test the potential combined effects of pesticide mixtures, which are likely to occur through dietary exposure. The co-exposure of the French general population to 79 pesticide residues present in the diet was first assessed. A Bayesian nonparametric model was then applied to define the main mixtures to which the French general population is simultaneously and most heavily exposed. Seven mixtures made of two to six pesticides were identified from the exposure assessment. An in vitro approach was used for investigating the toxicological effects of these mixtures and their corresponding individual compounds, using a panel of cellular models, i.e. primary rat and human hepatocytes, liver, intestine, kidney, colon and brain human cell lines. A set of cell functions and corresponding end-points were monitored such as cytotoxicity, real-time cell impedance, genotoxicity, oxidative stress, apoptosis and PXR nuclear receptor transactivation. The mixtures were tested in equimolar concentrations. Among the seven mixtures, two appeared highly cytotoxic, five activated PXR and depending on the assay one or two were genotoxic. In some experiments, the mixture effect was quantitatively different from the effect expected from the addition concept. The PERICLES program shows that, for the most pesticides mixtures to which the French general population is exposed, the toxic effects observed on human cells cannot be easily predicted based on the toxic potential of each compound. Consequently, additional studies should be carried on in order to more accurately define the mixtures of chemicals to which the consumers are exposed, as well as to improve the investigation, prediction and monitoring of their potential human health effects