Analyse de l'apoptose spontanée ou induite par le TGF-b1 au niveau d'hépatocytes humains et de rat en culture primaire (effet de la dexamethasone et d'autres xenobiotiques)

Le foie est un des organes clé dans la biotransformation et les effets pharmaco toxicologiques des xénobiotiques. Comme toute cellule, les hépatocytes possèdent la capacité d activer un programme dont l exécution conduit à une mort cellulaire appelée apoptose. Nous avons analysé les mécanismes moléculaires de cette apoptose, spontanée ou induite par le TGF-b1 et la staurosporine, dans les hépatocytes humains et de rat. Leur régulation par la déxamethasone (DEX) et par certains pesticides a également été étudiée. La DEX prévient l apoptose des hépatocytes par une cascade d événements intervenant en amont de la mitochondrie : inhibition de la relocalisation de Bad et de la libération du cytochrome c mitochondrial, induction des protéines Bcl-2 et Bcl-xL, stabilisation de l expression des caspases initiatrices 8 et 9 L induction des protéines Bcl-2 et Bcl-xL suffit à protéger les hépatocytes contre l apoptose induite par le TGF- b1 et la staurosporine. Les voies de signalisation P13K et Erk1/2 apparaissent impliquées dans cette régulation. Enfin, la dieldrine, l aldicarbe et le lindane induisent les mêmes effets anti-apoptotiques que la DEX. Ces résultats pourraient contribuer d une part au plan clinique, pour traiter certaines pathologies hépatiques, - d autre part au plan toxicologique, pour mieux apprécier les risques potentiels des pesticides en santé humaine.The liver is a key organ used for biotransformation and pharmacological studies. Like other cells, hepatocytes possess the inherent machinery necessary to induce programmed cell death lso called apoptosis. We analyzed the molecular mechanisms of spontaneous apoptosis and TGF-b1-, staurosporine-induced cell death in human and rat hepatocytes. Their regulation by dexamethasone (DEX) and pesticides was investigated. DEX prevents apoptosis in these cells by acting upstream of mitochondrial events: inhibiting bcl2-2 and bcl-xL downregulation and Bad translocation from the cytosol to mitochondria, blocking cytochrome c release and caspases activation (initiator caspases-8 and -9 and effector caspases-3). Moreover, up regulation of Bcl-2 and Bcl-xL protects hepatocytes against TGF-b1- and staurosporine induced apoptosis, P13K and Erk1/2 signaling pathways participated in this regulation? Xenobiotics such as dieldrin, aldicarb, and lindane induced similar anti-apoptotic effects as DEX. These results could have clinical applications, in hepatologic pathology treatments, and, toxicologic applications, in evaluation of pesticide risk in human health.NICE-BU Sciences (060882101) / SudocSudocFranceF

La réponse au stress du réticulum endoplasmique dans la physiopathologie des maladies chroniques du foie

La prévalence des maladies chroniques du foie ne cesse d’augmenter, du fait de la pandémie de l’obésité. Ces maladies s’étendent de la bégnine stéatose à la stéatopathie non alcoolique (NASH) qui peut évoluer vers le carcinome hépatocellulaire. Il n’existe pas de traitement pour ces maladies. La transition stéatose-NASH apparaît déterminante dans leur progression. Au cours de l’obésité, l’activation chronique de la réponse au stress du réticulum endoplasmique (RE) jouerait un rôle crucial dans cette transition, conduisant à la mort cellulaire, à l’inflammation et à l’aggravation des désordres métaboliques. Dans cette revue, nous discutons ces aspects et proposons que le ciblage de cette réponse au stress du RE puisse être pertinent dans la prise en charge thérapeutique de la NASH

No association between binge eating disorder and severity of non‐alcoholic fatty liver disease in severely obese patients

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Endoplasmic reticulum stress mediates resistance to BCL-2 inhibitor in uveal melanoma cells

International audienceTo address unmet clinical need for uveal melanomas, we assessed the effects of BH3-mimetic molecules, the ABT family, known to exert pro-apoptotic activities in cancer cells. Our results uncovered that ABT-263 (Navitoclax), a potent and orally bioavailable BCL-2 family inhibitor, induced antiproliferative effects in metastatic human uveal melanoma cells through cell cycle arrest at the G0/G1 phase, loss of mitochondrial membrane potential, and subsequently apoptotic cell death monitored by caspase activation and poly-ADP ribose polymerase cleavage. ABT-263-mediated reduction in tumor growth was also observed in vivo. We observed in some cells that ABT-263 treatment mounted a pro-survival response through activation of the ER stress signaling pathway. Blocking the PERK signaling pathway increased the pro-apoptotic ABT-263 effect. We thus uncovered a resistance mechanism in uveal melanoma cells mediated by activation of endoplasmic reticulum stress pathway. Therefore, our study identifies ABT-263 as a valid therapeutic option for patients suffering from uveal melanoma

Disruption of mitochondrial function during apoptosis is mediated by caspase cleavage of the p75 subunit of complex I of the electron transport chain.

Mitochondrial outer membrane permeabilization and cytochrome c release promote caspase activation and execution of apoptosis through cleavage of specific caspase substrates in the cell. Among the first targets of activated caspases are the permeabilized mitochondria themselves, leading to disruption of electron transport, loss of mitochondrial transmembrane potential (DeltaPsim), decline in ATP levels, production of reactive oxygen species (ROS), and loss of mitochondrial structural integrity. Here, we identify NDUFS1, the 75 kDa subunit of respiratory complex I, as a critical caspase substrate in the mitochondria. Cells expressing a noncleavable mutant of p75 sustain DeltaPsim and ATP levels during apoptosis, and ROS production in response to apoptotic stimuli is dampened. While cytochrome c release and DNA fragmentation are unaffected by the noncleavable p75 mutant, mitochondrial morphology of dying cells is maintained, and loss of plasma membrane integrity is delayed. Therefore, caspase cleavage of NDUFS1 is required for several mitochondrial changes associated with apoptosis

Disruption of mitochondrial function during apoptosis is mediated by caspase cleavage of the p75 subunit of complex I of the electron transport chain.

Mitochondrial outer membrane permeabilization and cytochrome c release promote caspase activation and execution of apoptosis through cleavage of specific caspase substrates in the cell. Among the first targets of activated caspases are the permeabilized mitochondria themselves, leading to disruption of electron transport, loss of mitochondrial transmembrane potential (DeltaPsim), decline in ATP levels, production of reactive oxygen species (ROS), and loss of mitochondrial structural integrity. Here, we identify NDUFS1, the 75 kDa subunit of respiratory complex I, as a critical caspase substrate in the mitochondria. Cells expressing a noncleavable mutant of p75 sustain DeltaPsim and ATP levels during apoptosis, and ROS production in response to apoptotic stimuli is dampened. While cytochrome c release and DNA fragmentation are unaffected by the noncleavable p75 mutant, mitochondrial morphology of dying cells is maintained, and loss of plasma membrane integrity is delayed. Therefore, caspase cleavage of NDUFS1 is required for several mitochondrial changes associated with apoptosis

The mid-infrared spectroscopy: A novel non-invasive diagnostic tool for NASH diagnosis in severe obesity

International audienceThere is an urgent medical need to develop non-invasive tests for non-alcoholic steatohepatitis (NASH). This study evaluates the diagnostic performance of an innovative model based on mid-infrared (MIR) spectroscopy for the diagnosis of NASH.Methods: Severely obese patients who underwent a bariatric procedure at the University Hospital of Nice, France (n = 395) were prospectively recruited. The clinico-biological characteristics were measured prior to surgery. Liver biopsies were collected during the surgical procedure and assessed by a pathologist. A training group (316 patients, NASH: 16.8%) and a validation group (79 patients, NASH: 16.5%) were randomly defined. MIR spectra were acquired by fiber evanescent wave spectroscopy, using chalcogenide glass fiber optic sensors and a spectrometer. This absorption spectroscopic technique delivers a spectrum that identifies the molecular composition of a sample, defining a patient's metabolic fingerprint.Results: The areas under the receiver operating curve (AUROC) for the diagnosis of NASH were 0.82 and 0.77 in the training and validation groups, respectively. The best threshold was 0.15, which was associated with a sensitivity of 0.75 and 0.69, and a specificity of 0.72 and 0.76. Negative predictive values of 0.94 and 0.93 and positive predictive values of 0.35 and 0.36, as well as correctly classified patient rates of 72% and 75% were obtained in the training and validation groups, respectively. A composite model using aspartate aminotransferase level, triglyceride level and waist circumference alongside the MIR spectra led to an increase in AUROC (0.88 and 0.84 for the training and validations groups, respectively).Conclusions: MIR spectroscopy provides good sensitivity and negative predictive values for NASH screening in patients with severe obesity.Lay summary: There is an urgent need for tools to non-invasively diagnose and monitor non-alcoholic steatohepatitis (NASH). This study evaluates the performance of a new tool for fast NASH diagnosis based on mid-infrared (MIR) spectroscopy. Using serum samples from severely obese patients who underwent a bariatric procedure, which enabled a concomitant liver biopsy to be performed, the MIR spectroscopy model performed well in screening patients for NASH compared to a traditional, histological diagnosis