Potentialisation de la chimiothérapie en milieu oxygéné (implication des radicaux libres dans l'effet des anthracyclines)

L hypoxie tumorale peut induire une résistance aux traitements par l adriamycine (ADR), et l effet anti-cancéreux de l anthracycline peut augmenter sous oxygénation hyperbare. Cependant, les mécanismes d action impliqués dans ce gain d efficacité thérapeutique restent à élucider.Nous avons évalué l implication de la production d espèces réactives de l oxygène (ROS) dans l amélioration de l effet de l ADR sur des cellules lymphoblastiques humaines CCRF-CEM sous hypoxie (2% O2) et sous normoxie (21% O2). Nous avons utilisé une nouvelle méthode de détection de ROS basée sur la mesure de la durée de vie de fluorescence de l'acide 1-pyrène butyrique. L analyse d images numériques des populations cellulaires après triple-marquage a fourni des informations morphométriques (tailles cellulaire et nucléaire) et fonctionnelles (activité mitochondriale, teneur en ADN) permettant (i) de quantifier l induction de l apoptose, et (ii) d établir la distribution du cycle cellulaire par analyse multiparamétrique des données.Nous avons observé que le blocage du cycle cellulaire par l'ADR ne dépend pas des conditions d'oxygénation, alors que l induction de l apoptose et la production de ROS dues au traitement sont plus importantes sous condition oxygénée (21% O2). Si on admet que la condition normoxique est une hyperoxygénation comparée à l état d hypoxie tumorale in vivo, alors le gain d efficacité thérapeutique de l ADR fournit par une oxygénation hyperbare pourrait résulter d'une plus forte production de ROS par l'anthracycline, qui entraînerait une induction des processus apoptotiques plus importante.Tumour hypoxia is causally related with resistance to adriamycin (ADR) treatment. However, how hyperbaric oxygen therapy leads to therapeutic gain of the drug is unclear.We investigated the relation of reactive oxygen species (ROS) generation with anti-tumoural effect of ADR on human lymphoblastic CCRF-CEM cells under hypoxic (2% O2) and normoxic (21% O2) conditions. A new method was used to measure intracellular ROS variations through the fluorescence lifetime of 1-pyrenebutyric acid. Numerical image analysis of cell populations labelled with different vital stains allowed to collect morphometric (cellular and nuclear sizes) and physiological (mitochondrial activity, DNA content) informations used to (i) quantify apoptosis induction, and (ii) determine the cell cycle distribution through multiparametric analysis of collected data.We observed that oxygen level has no effect on the cell cycle arrest induced by ADR, whereas apoptosis induction and ROS production resulting from treatment are higher under oxygenated conditions (i.e. normoxia). Considering normoxia as a hyperoxygenated condition compared to in vivo hypoxic tumour level, we suggested that improvement of anti-cancerous effect of ADR due to hyperbaric oxygen therapy results from higher intracellular ROS generation by the drug, leading to a greater induction of apoptosis.PERPIGNAN-BU Sciences (661362101) / SudocSudocFranceF

Ascorbic acid alters cell fate commitment of human neural progenitors in a WNT/β-catenin/ROS signaling dependent manner

Abstract Background Improving the neuronal yield from in vitro cultivated neural progenitor cells (NPCs) is an essential challenge in transplantation therapy in neurological disorders. In this regard, Ascorbic acid (AA) is widely used to expand neurogenesis from NPCs in cultures although the mechanisms of its action remain unclear. Neurogenesis from NPCs is regulated by the redox-sensitive WNT/β-catenin signaling pathway. We therefore aimed to investigate how AA interacts with this pathway and potentiates neurogenesis. Methods Effects of 200 μM AA were compared with the pro-neurogenic reagent and WNT/β-catenin signaling agonist lithium chloride (LiCl), and molecules with antioxidant activities i.e. N-acetyl-L-cysteine (NAC) and ruthenium red (RuR), in differentiating neural progenitor ReNcell VM cells. Cells were supplemented with reagents for two periods of treatment: a full period encompassing the whole differentiation process versus an early short period that is restricted to the cell fate commitment stage. Intracellular redox balance and reactive oxygen species (ROS) metabolism were examined by flow cytometry using redox and ROS sensors. Confocal microscopy was performed to assess cell viability, neuronal yield, and levels of two proteins: Nucleoredoxin (NXN) and the WNT/β-catenin signaling component Dishevelled 2 (DVL2). TUBB3 and MYC gene responses were evaluated by quantitative real-time PCR. DVL2-NXN complex dissociation was measured by fluorescence resonance energy transfer (FRET). Results In contrast to NAC which predictably exhibited an antioxidant effect, AA treatment enhanced ROS metabolism with no cytotoxic induction. Both drugs altered ROS levels only at the early stage of the differentiation as no changes were held beyond the neuronal fate commitment stage. FRET studies showed that AA treatment accelerated the redox-dependent release of the initial pool of DVL2 from its sequestration by NXN, while RuR treatment hampered the dissociation of the two proteins. Accordingly, AA increased WNT/β-catenin signaling output i.e. MYC mRNA level, whereas RuR attenuated it. Moreover, AA improved neurogenesis as much as LiCl as both TUBB3-positive cell yield and TUBB3 mRNA level increased, while NAC or RuR attenuated neurogenesis. Markedly, the neurogenesis outputs between the short and the full treatment with either NAC or AA were found unchanged, supporting our model that neuronal yield is altered by events taking place at the early phase of differentiation. Conclusions Our findings demonstrate that AA treatment elevates ROS metabolism in a non-lethal manner prior to the NPCs commitment to their neuronal fate. Such effect stimulates the redox-sensitive DVL2 activation and WNT/β-catenin signaling response that would enhance the ensuing neuronal cell differentiation

<i>β</i>-catenin activation in response to transient and continuous WNT stimuli.

<div><p>(A-B) Comparison of simulation results (<i>β</i>-catenin concentration fold change) between the newly derived WNT/<i>β</i>-catenin signaling model (red line) and the Lee model (blue, dashed line) [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004106#pcbi.1004106.ref027" target="_blank">27</a>] in response to a transient WNT stimulus. Without adaptation both models expose a similar excitation level, but the temporal scale differs significantly (A). Adopting the temporal scale of our WNT/<i>β</i>-catenin signaling model yields similar simulation results for both models (B).</p> <p>(C) <i>β</i>-catenin accumulation after 2 hours of WNT stimulation with varying concentrations, compared between our simulation results (red line) and experimental in-vitro measurements by <i>Hannoush</i> (blue line) [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004106#pcbi.1004106.ref049" target="_blank">49</a>]. Parametrization of the <i>β</i>-catenin model is exactly the same as listed in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004106#pcbi.1004106.t001" target="_blank">Table 1</a>, despite the WNT production rate (<i>k</i>1), which has been parameterized in accordance to the varying WNT stimuli applied by <i>Hannoush</i>, cf. <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004106#pcbi.1004106.t002" target="_blank">Table 2</a>. The simulation results match almost perfectly with the experimental data for all WNT concentrations applied. Note that the in-silico <i>β</i>-catenin concentration values are scaled by a linear scaling factor to allow a comparison with the experimentally derived values, that measure the <i>β</i>-catenin accumulation based on fluorescence intensities, instead of concentration or fold changes. Simulation results for our model corresponds to mean simulation trajectory (red) with 95% confidence interval (gray error bars).</p></div

Oxidative stress does not play a primary role in the toxicity induced with clinical doses of doxorubicin in myocardial H9c2 cells

International audienceThe implication of oxidative stress as primary mechanism inducing doxorubicin (DOX) cardiotoxicity is still questionable as many in vitro studies implied supra-clinical drug doses or unreliable methodologies for reactive oxygen species (ROS) detection. The aim of this study was to clarify whether oxidative stress is involved in compliance with the conditions of clinical use of DOX, and using reliable tools for ROS detection. We examined the cytotoxic mechanisms of 2 μM DOX 1 day after the beginning of the treatment in differentiated H9c2 rat embryonic cardiac cells. Cells were exposed for 2 or 24 h with DOX to mimic a single chronic dosage or to favor accumulation, respectively. We found that apoptosis was prevalent in cells exposed for a short period with DOX: cells showed typical hallmarks as loss of anchorage ability, mitochondrial hyperpolarization followed by the collapse of mitochondrial activity, and nuclear condensation. Increasing the exposure period favored a shift to necrosis as the cells preferentially exhibited early DNA impairment and nuclear swelling. In either case, measuring the fluorescence lifetime of 1-pyrenebutyric acid or the intensities of dihydroethidium or amplex red showed a consistent pattern in ROS production which was a slight increased level far from representative of an oxidative stress. Moreover, pre-treatment with dexrazoxane provided a cytoprotective effect although it failed to detoxify ROS. Our data support that oxidative stress is unlikely to be the primary mechanism of DOX cardiac toxicity in vitro

Spatio-temporal Model of Endogenous ROS and Raft-Dependent WNT/Beta-Catenin Signaling Driving Cell Fate Commitment in Human Neural Progenitor Cells

<div><p>Canonical WNT/β-catenin signaling is a central pathway in embryonic development, but it is also connected to a number of cancers and developmental disorders. Here we apply a combined in-vitro and in-silico approach to investigate the spatio-temporal regulation of WNT/β-catenin signaling during the early neural differentiation process of human neural progenitors cells (hNPCs), which form a new prospect for replacement therapies in the context of neurodegenerative diseases. Experimental measurements indicate a second signal mechanism, in addition to canonical WNT signaling, being involved in the regulation of nuclear β-catenin levels during the cell fate commitment phase of neural differentiation. We find that the biphasic activation of β-catenin signaling observed experimentally can only be explained through a model that combines Reactive Oxygen Species (ROS) and raft dependent WNT/β-catenin signaling. Accordingly after initiation of differentiation endogenous ROS activates DVL in a redox-dependent manner leading to a transient activation of down-stream β-catenin signaling, followed by continuous auto/paracrine WNT signaling, which crucially depends on lipid rafts. Our simulation studies further illustrate the elaborate spatio-temporal regulation of DVL, which, depending on its concentration and localization, may either act as direct inducer of the transient ROS/β-catenin signal or as amplifier during continuous auto-/parcrine WNT/β-catenin signaling. In addition we provide the first stochastic computational model of WNT/β-catenin signaling that combines membrane-related and intracellular processes, including lipid rafts/receptor dynamics as well as WNT- and ROS-dependent β-catenin activation. The model’s predictive ability is demonstrated under a wide range of varying conditions for in-vitro and in-silico reference data sets. Our in-silico approach is realized in a multi-level rule-based language, that facilitates the extension and modification of the model. Thus, our results provide both new insights and means to further our understanding of canonical WNT/β-catenin signaling and the role of ROS as intracellular signaling mediator.</p></div

Additional file 1: Figure S1. of Ascorbic acid alters cell fate commitment of human neural progenitors in a WNT/β-catenin/ROS signaling dependent manner

Changes in DVL2 and NXN protein amounts do not correlate with variations in FRETeff. Confocal images of DVL2 (a) and NXN proteins (b) were acquired in proliferating and differentiating cells treated or not with 200 μM AA, 0.5 μM RuR or 1 mM H2O2. Mean fluorescence intensities were then quantified at 0 h, 0.5 h, 1 h, 2 h, 3 h and 4 h of differentiation for each protein (see respective bar graphs). These data demonstrate that decreases in FRETeff values (see Fig. 7) do not result from any reduction in protein amounts which rather increase when DVL2-NXN complexes begin to dissociate. Scale: 10 μm. n = ~ 50 cells per time point and condition. Values are means ± SD of three independent experiments. *P ≤ 0.05 compared with untreated differentiating cells at t = 0 h; # P ≤ 0.05 each treatment condition at each time point; ns, non-significant. (JPEG 5532 kb

Experimental data vs. Simulation results.

<p>Nuclear <i>β</i>-catenin concentration fold changes in comparison between experimental data and the validated WNT/<i>β</i>-catenin model. The simulation result (red) of the WNT/<i>β</i>-catenin model (cf. <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004106#pcbi.1004106.g002" target="_blank">Fig. 2</a>, parametrized according to <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004106#pcbi.1004106.t001" target="_blank">Table 1</a>) matches all experimental values (blue) in untreated control cells (A). Though, in its current state it is not capable of reproducing the immediate early <i>β</i>-catenin activation in raft-deficient cells (B). Simulation results correspond to the mean simulation trajectory (red) with 95% confidence interval (gray error bars).</p