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PDF file - 809K, Table S1 List of genes deregulated in IRE1_DN cells. Table S2 Potential IRE1 cleavage sites on PER1 mRNA. Table S3 Characteristics of human glioma biopsies used in this study. Table S4 Primer Pairs used for PCR and qPCR studies. Table S5 siRNA sequences. Figure S1 PER1 mRNA expression in IRE1 signaling deficient cells. Figure S2 Validation of ski2, xrn1/2 siRNA efficacy. Figure S3 Potential IRE1 cleavage sites on PER1 mRNA. Figure S4 Expression of PER1 mRNA in PER1 silenced cells. Figure S5 XBP1s expression in glioma biopsies by immunohistochemistry. Figure S6: stress independent regulation of PER1 by IRE1α. Figure S7: (A) Two-dimensional modeling of potential Per1 mRNA cleavage sites using the M-Fold program. (B) In vitro RNA cleavage assay. Figure S8: Expression of Per1 mRNA in U87 cells upon lentiviral transduction. Figure S9: determination of protein (A-D) and mRNA (E) expression levels of potential PER1 targets in EV and IRE_DN cells transduced or not with shPER1. Figure S10: impact of CXCL3 re-expression on cell proliferation and neurosphere formation in IRE1_DN cells. Figure S11: (A-F) Immunohistochemical analysis of 3 typical glioblastoma paraffin sections using anti sXBP1 antibodies.

Regulation of ROS metabolism plays a major role in cellular adaptation to oxidative stress in cancer cells, but the molecular mechanism that regulates catalase, a key antioxidant enzyme responsible for conversion of hydrogen peroxide to water and oxygen, remains to be elucidated. Therefore, we investigated the transcriptional regulatory mechanism controlling catalase expression in three human mammary cell lines: the normal mammary epithelial 250MK primary cells, the breast adenocarcinoma MCF-7 cells and an experimental model of MCF-7 cells resistant against oxidative stress resulting from chronic exposure to H2O2 (Resox), in which catalase was overexpressed. Here we identify a novel promoter region responsible for the regulation of catalase expression at −1518/−1226 locus and the key molecules that interact with this promoter and affect catalase transcription. We show that the AP-1 family member JunB and retinoic acid receptor alpha (RARα) mediate catalase transcriptional activation and repression, respectively, by controlling chromatin remodeling through a histone deacetylases-dependent mechanism. This regulatory mechanism plays an important role in redox adaptation to chronic exposure to H2O2 in breast cancer cells. Our study suggests that cancer adaptation to oxidative stress may be regulated by transcriptional factors through chromatin remodeling, and reveals a potential new mechanism to target cancer cells.

Alterations in the expression of antioxidant enzymes are associated with changes in cancer cell sensitivity to chemotherapeutic drugs (menadione and β-lapachone). Mechanisms of acquisition of resistance to pro-oxidant drugs were investigated using a model of oxidative stress-resistant MCF-7 breast cancer cells (Resox cells).FISH experiments were performed in tumor biopsy and breast cancer cells to characterize the pattern of the NQO1 gene. SNP-arrays were conducted to detect chromosomal imbalances. Finally, the importance of NQO1 overexpression in the putative acquisition of either drug resistance or an increased sensitivity to quinones by cancer cells was investigated by immunoblotting and cytotoxicity assays.Genomic gain of the chromosomal band 16q22 was detected in Resox cells compared to parental breast cancer MCF-7 cells and normal human mammary epithelial 250MK cells. This genomic gain was associated with amplification of the NQO1 gene in one tumor biopsy as well as in breast cancer cell lines. Using different breast cell models, we found that NQO1 overexpression was a main determinant for a potential chemotherapy resistance or an increased sensitivity to quinone-bearing compounds.Because NQO1 is frequently modified in tumors at genomic and transcriptomic levels, the impact of NQO1 modulation on breast cancer cell sensitivity places NQO1 as a potential link between cancer redox alterations and resistance to chemotherapy. Thus, the NQO1 gene copy number and NQO1 activity should be considered when quinone-bearing molecules are being utilized as potential drugs against breast tumors.

Development of cancer cell resistance against prooxidant drugs limits its potential clinical use. MCF-7 breast cancer cells chronically exposed to ascorbate/menadione became resistant (Resox cells) by increasing mainly catalase activity. Since catalase appears as an anticancer target, the elucidation of mechanisms regulating its expression is an important issue. In MCF-7 and Resox cells, karyotype analysis showed that chromosome 11 is not altered compared to healthy mammary epithelial cells. The genomic gain ofcatalaselocus observed in MCF-7 and Resox cells cannot explain the differential catalase expression. Since ROS cause DNA lesions, the activation of DNA damage signaling pathways may influence catalase expression. However, none of the related proteins (i.e., p53, ChK) was activated in Resox cells compared to MCF-7. The c-abl kinase may lead to catalase protein degradation via posttranslational modifications, but neither ubiquitination nor phosphorylation of catalase was detected after catalase immunoprecipitation. Catalase mRNA levels did not decrease after actinomycin D treatment in both cell lines. DNMT inhibitor (5-aza-2′-deoxycytidine) increased catalase protein level in MCF-7 and its resistance to prooxidant drugs. In line with our previous report, chromatin remodeling appears as the main regulator of catalase expression in breast cancer after chronic exposure to an oxidative stress.

International audience; The unfolded protein response (UPR) is an integrated, adaptive biochemical process that is inextricably linked with cell homeostasis and paramount to maintenance of normal physiological function. Prolonged accumulation of improperly folded proteins in the endoplasmic reticulum (ER) leads to stress. This is the driving stimulus behind the UPR. As such, prolonged ER stress can push the UPR past beneficial functions such as reduced protein production and increased folding and clearance to apoptotic signaling. The UPR is thus contributory to the commencement, maintenance, and exacerbation of a multitude of disease states, making it an attractive global target to tackle conditions sorely in need of novel therapeutic intervention. The accumulation of information of screening tools, readily available therapies, and potential pathways to drug development is the cornerstone of informed clinical research and clinical trial design. Here, we review the UPR's involvement in health and disease and, beyond providing an in-depth description of the molecules found to target the three UPR arms, we compile all the tools available to screen for and develop novel therapeutic agents that modulate the UPR with the scope of future disease intervention.

The unfolded protein response (UPR) mediates the adaptation of the secretory pathway (SP) to fluctuations in cellular protein demand or to environmental variations. Recently, drug screenings have confirmed the therapeutic potential of targeting the UPR in cancer models. However, the UPR may not be the only druggable target of the SP. Moreover, recent studies have revealed other contributions of the SP to cancer development. This article does not intend to describe the well-established implication of UPR signaling pathways in cancer cell life and cell decision, but rather aims at defining the concept of 'tumor cell secretory addiction', from molecular, cellular, and therapeutic perspectives. Furthermore, the implication of UPR modulations in this context will be discussed.

International audience; Background & aims: Postprandial lipemia has been associated with inflammation, oxidative stress and vascular dysfunction. This metabolic disturbance represents a complex process only partly understood. The purpose of this study was to identify variations in plasma proteome after a high-fat challenge in healthy middle-aged men. Methods: Two-dimensional electrophoresis was used to compare plasma from seven subjects, drawn before and 4 h after a high-fat challenge. Results: Among the 231 spots detected and analyzed, 22 were present at different levels in postprandial hyperlipemic plasma compared to preprandial plasma. For 10 of them, corresponding proteins were identified by mass spectrometry. Some of them are related to the hemostatic system (tetranectin and fibrinogen) or the complement system (complement component 3 and 4 and ficollin-3) and have been previously associated to atherothrombosis. Conclusion: These results provide new perspectives and broaden our understanding of the biological processes of postprandial metabolic stress, as well as its links with the development of atherosclerosis. (C) 2012 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.

Cancer cells are particularly vulnerable to treatments impairing redox homeostasis. Reactive oxygen species (ROS) can indeed play an important role in the initiation and progression of cancer, and advanced stage tumors frequently exhibit high basal levels of ROS that stimulate cell proliferation and promote genetic instability. In addition, an inverse correlation between histological grade and antioxidant enzyme activities is frequently observed in human tumors, further supporting the existence of a redox dysregulation in cancer cells. This biochemical property can be exploited by using redox-modulating compounds, which represent an interesting approach to induce cancer cell death. Thus, we have developed a new strategy based on the use of pharmacologic concentrations of ascorbate and redox-active quinones. Ascorbate-driven quinone redox cycling leads to ROS formation and provoke an oxidative stress that preferentially kill cancer cells and spare healthy tissues. Cancer cell death occurs through necrosis and the underlying mechanism implies an energetic impairment (ATP depletion) that is likely due to glycolysis inhibition. Additional mechanisms that participate to cell death include calcium equilibrium impairment and oxidative cleavage of protein chaperone Hsp90. Given the low systemic toxicity of ascorbate and the impairment of crucial survival pathways when associated with redox-active quinones, these combinations could represent an original approach that could be combined to standard cancer therapy.

Numerous studies suggest that generation of oxidative stress could be useful in cancer treatment. In this study, we evaluated, in vitro and in vivo, the antitumor potential of oxidative stress induced by ascorbate/menadione (asc/men). This combination of a reducing agent (ascorbate) and a redox active quinone (menadione) generates redox cycling leading to formation of reactive oxygen species (ROS). Asc/men was tested in several cell types including K562 cells (a stable human-derived leukemia cell line), freshly isolated leukocytes from patients with chronic myeloid leukemia, BaF3 cells (a murine pro-B cell line) transfected with Bcr-Abl and peripheral blood leukocytes derived from healthy donors. Although these latter cells were resistant to asc/men, survival of all the other cell lines was markedly reduced, including the BaF3 cells expressing either wild-type or mutated Bcr-Abl. In a standard in vivo model of subcutaneous tumor transplantation, asc/men provoked a significant delay in the proliferation of K562 and BaF3 cells expressing the T315I mutated form of Bcr-Abl. No effect of asc/men was observed when these latter cells were injected into blood of mice most probably because of the high antioxidant potential of red blood cells, as shown by in vitro experiments. We postulate that cancer cells are more sensitive to asc/men than healthy cells because of their lack of antioxidant enzymes, mainly catalase. The mechanism underlying this cytotoxicity involves the oxidative cleavage of Hsp90 with a subsequent loss of its chaperone function thus leading to degradation of wild-type and mutated Bcr-Abl protein.

Cancer cells show an up-regulation of glycolysis, they readily take up vitamin C, and they appear more susceptible to an oxidative stress than the surrounding normal cells. Here we compare, analyse and discuss these particular hallmarks by performing experiments in murine hepatomas (TLT cells) and freshly isolated mouse hepatocytes. The results show that rates of lactate formation are higher in TLT cells as compared to mouse hepatocytes, but their ATP content represents less than 25% of that in normal cells. The uptake of vitamin C is more important in hepatoma cells as compared to normal hepatocytes. This uptake mainly occurs through GLUT1 transporters. Hepatoma cells have less than 10% of antioxidant enzyme activities as compared to normal hepatocytes. This decrease includes not only the major antioxidant enzymes, namely catalase, superoxide dismutase and glutathione peroxidase, but also the GSH content. Moreover, catalase is almost not expressed in hepatoma cells as shown by western blot analysis. We explored therefore a selective exposure of cancer cells to an oxidative stress induced by pro-oxidant mixtures containing pharmacological doses of vitamin C and a redox active compound such as menadione (vitamin K(3)). Indeed, the combination of vitamin C (which accumulates in hepatoma cells) and a quinone undergoing a redox cycling (vitamin K(3)) leads to an oxidative stress that kills cancer cells in a selective manner. This differential sensitivity between cancer cells and normal cells may have important clinical applications, as it has been observed with other pro-oxidants like Arsenic trioxide, isothiocyanates, Adaphostin.

International audience; The hallmarks of cancer currently define the molecular mechanisms responsible for conferring specific tumor phenotypes. Recently, these characteristics were also connected to the status of the secretory pathway, thereby linking the functionality of this cellular machinery to the acquisition of cancer cell features. The secretory pathway ensures the biogenesis of proteins that are membrane-bound or secreted into the extracellular milieu and can control its own homeostasis through an adaptive signaling pathway named the Unfolded Protein Response (UPR). In the present review, we discuss the specific features of the UPR in various tumor types and the impact of the selective activation of this pathway on cell transformation, tumor development and aggressiveness.

Hepatocellular carcinoma is one of the most frequent tumor types worldwide and oxidative stress represents a major risk factor in pathogenesis of liver diseases leading to HCC. Nuclear factor erythroid 2-related factor (Nrf2) is a transcription factor activated by oxidative stress that governs the expression of many genes which constitute the antioxidant defenses of the cell. In addition, oxidative stress activates AMP-activated protein kinase (AMPK), which has emerged in recent years as a kinase that controls the redox-state of the cell. Since both AMPK and Nrf2 are involved in redox homeostasis, we investigated whether there was a crosstalk between the both signaling systems in hepatocarcinoma cells. Here, we demonstrated that AMPK activator AICAR, in contrary to the A769662 allosteric activator, induces Nrf2 activation and concomitantly modulates the basal redox state of the hepatocarcinoma cells. When the expression of Nrf2 is knocked down, AICAR failed to induce its effect on redox state. These data highlight a major role of Nrf2 signaling pathway in mediating the AICAR effect on basal oxidative state. Furthermore, we demonstrated that AICAR metabolization by the cell is required to induce Nrf2 activation while, the silencing of AMPK does not have any effect on Nrf2 activation. This suggests that AICAR-induced Nrf2 activation is independent of AMPK activity. In conclusion, we identified AICAR as a potent modulator of the redox state of human hepatocarcinoma cells, via the Nrf2 signaling pathway and in an AMPK-independent mechanism. © 2014 Elsevier Inc.

In the past 20 years both the circadian clock and endoplasmic reticulum (ER) stress signaling have emerged as major players in oncogenesis and cancer development. Although several lines of evidence have established functional links between these two molecular pathways, their interconnection and the subsequent functional implications in cancer development remain to be fully characterized. Herein, we provide an extensive review of the literature depicting the molecular connectivity linking ER stress signaling and the circadian clock and elaborate on the potential use of these functional interactions in cancer therapeutics.

Catalase is an antioxidant enzyme that catalyzes mainly the transformation of hydrogen peroxide into water and oxygen. Although catalase is frequently down-regulated in tumors the underlying mechanism remains unclear. Few transcription factors have been reported to directly bind the human catalase promoter. Among them FoxO3a has been proposed as a positive regulator of catalase expression. Therefore, we decided to study the role of the transcription factor FoxO3a and the phosphatidylinositol-3 kinase (PI3K) signaling pathway, which regulates FoxO3a, in the expression of catalase. To this end, we developed an experimental model of mammary breast MCF-7 cancer cells that acquire resistance to oxidative stress, the so-called Resox cells, in which catalase is overexpressed as compared with MCF-7 parental cell line. In Resox cells, Akt expression is decreased but its phosphorylation is enhanced when compared with MCF-7 cells. A similar profile is observed for FoxO3a, with less total protein but more phosphorylated FoxO3a in Resox cells, correlating with its higher Akt activity. The modulation of FoxO3a expression by knockdown and overexpression strategies did not affect catalase expression, neither in MCF-7 nor in Resox cells. Inhibition of PI3K and mTOR by LY295002 and rapamycin, respectively, decreases the phosphorylation of downstream targets (i.e. GSK3β and p70S6K) and leads to an increase of catalase expression only in MCF-7 but not in Resox cells. In conclusion, FoxO3a does not appear to play a critical role in the regulation of catalase expression in both cancer cells. Only MCF-7 cells are sensitive and dependent on PI3K/Akt/mTOR signaling.

The endoplasmic-reticulum (ER) stress response represents a pivotal cellular process, triggered by a variety of stimuli implying incorrect protein folding in the ER. Solid evidence implicates ER stress-induced dysfunction as major contributors in many cancers, among which brain cancers. In this work, an integrative analysis in two transcriptomic datasets scrutinizes the regulatory potency of ER stress mechanisms in glioblastoma. Besides confirming the results of the initial analyses, the integration of both datasets manages to study the effect of cellular stress in conjunction to the dysregulation of the IRE1α branch of the ER stress signaling pathways. Given the respective diversified phenotypic profile of glioblastomas, ranging from increased proliferation to enhanced migration and aggressiveness as a result of the IRE1α molecular switch, this study suggests potential target groups of master regulators of the ER stress pathways, through the application of an established, exhaustive computational framework.

Growing evidence supports a role for the unfolded protein response (UPR) in carcinogenesis; however, the precise molecular mechanisms underlying this phenomenon remain elusive. Herein, we identified the circadian clock PER1 mRNA as a novel substrate of the endoribonuclease activity of the UPR sensor IRE1α. Analysis of the mechanism shows that IRE1α endoribonuclease activity decreased PER1 mRNA in tumor cells without affecting PER1 gene transcription. Inhibition of IRE1α signaling using either siRNA-mediated silencing or a dominant-negative strategy prevented PER1 mRNA decay, reduced tumorigenesis, and increased survival, features that were reversed upon PER1 silencing. Clinically, patients showing reduced survival have lower levels of PER1 mRNA expression and increased splicing of XBP1, a known IRE-α substrate, thereby pointing toward an increased IRE1α activity in these patients. Hence, we describe a novel mechanism connecting the UPR and circadian clock components in tumor cells, thereby highlighting the importance of this interplay in tumor development. Cancer Res; 73(15); 4732–43. ©2013 AACR.

MicroRNAs (miRNA) are generally described as negative regulators of gene expression. However, some evidence suggests that they may also play positive roles. As such, we reported that miR-1291 leads to a GPC3 mRNA expression increase in hepatoma cells through a 3′ untranslated region (UTR)-dependent mechanism. In the absence of any direct interaction between miR-1291 and GPC3 mRNA, we hypothesized that miR-1291 could act by silencing a negative regulator of GPC3 mRNA expression. Based on in silico predictions and experimental validation, we demonstrate herein that miR-1291 represses the expression of the mRNA encoding the endoplasmic reticulum (ER)-resident stress sensor IRE1α by interacting with a specific site located in the 5′ UTR. Moreover, we show, in vitro and in cultured cells, that IRE1α cleaves GPC3 mRNA at a 3′ UTR consensus site independently of ER stress, thereby prompting GPC3 mRNA degradation. Finally, we show that the expression of a miR-1291-resistant form of IRE1α abrogates the positive effects of miR-1291 on GPC3 mRNA expression. Collectively, our data demonstrate that miR-1291 is a biologically relevant regulator of GPC3 expression in hepatoma cells and acts through silencing of the ER stress sensor IRE1α.

Introduction:Glioblastoma multiforme (GBM) is the most lethal form of glioma with an overall survival at 5 years nearly null (< 5%). Increasing evidences point towards the RNase activity of IRE1 as a central player in GBM development, particularly in cancer cell invasion, tumor vascularization and recruitment of inflammatory or immune cells. Indeed, IRE1 RNase promotes XBP1 mRNA splicing, a well-described cytoprotective transcription factor that is required for cancer cell survival and this activity also contributes to the degradation of mRNA and microRNA, an activity called RIDD. Recent studies unraveled the presence of somatic mutations on the IRE1 gene in GBM that could play a driver role but without providing any functional information.Results:Here, we have sequenced IRE1 exons in 23 human GBMs and identified a new somatic mutation on the IRE1 gene. Interestingly, using biochemical approaches and an orthotopic tumor graft mouse model we show that this variant displays an altered IRE1 RNase activity. IRE1-dependent gene expression pattern alterations lead to major functional consequences in terms of tumor phenotype including (i) a magnified induction of the inflammatory and pro-angiogenic pathways based on transcriptome signatures from human tumors, (ii) an increase of the aggressiveness/infiltration potential of U87 derived tumors in mice and (iii) the modulation of immune cells recruitment to the tumor.Conclusions:This study provides the first evidence that the selectivity of IRE1 RNase can be modulated naturally and unravels the first mechanistic example of how a somatic mutation in the IRE gene can provide adaptive advantages to glioblastoma cells by reprogramming the tumor stroma.

The endoplasmic reticulum (ER) is an organelle specialized for the folding and assembly of secretory and transmembrane proteins. ER homeostasis is often perturbed in tumor cells due to dramatic changes in solid tumor microenvironment, thereby leading to the activation of an adaptive mechanism named the Unfolded Protein Response (UPR). The activation of the UPR sensor IRE1α has been described to play an important role in tumor progression. However, the molecular events associated with this phenotype remain poorly characterized. In the present study, we examined the effects of IRE1α signaling on glioma cells adaptation to their microenvironment. We show that the characteristics of U87 cells migration are modified under conditions where IRE1α activity is impaired (DN_IRE1). This is linked to increased stress fiber formation and enhanced RhoA activity. Gene expression profiling also revealed that loss of functional IRE1α signaling mostly resulted in the up-regulation of genes encoding extracellular matrix proteins. Among these genes, SPARC, whose mRNA is a direct target of IRE1α endoribonuclease activity, was in part responsible for the phenotypic changes associated with IRE1α inactivation. Hence, our data demonstrate that IRE1α is a key regulator of SPARC expression in vitro in a glioma model. Our results also further support the critical role of IRE1α contribution to tumor growth and infiltration/invasion and extend the paradigm of secretome control in tumor microenvironment conditioning.

Hsp90 is an essential chaperone that is necessary for the folding, stability and activity of numerous proteins. In this study, we demonstrate that free radicals formed during oxidative stress conditions can cleave Hsp90. This cleavage occurs through a Fenton reaction which requires the presence of redox-active iron. As a result of the cleavage, we observed a disruption of the chaperoning function of Hsp90 and the degradation of its client proteins, for example, Bcr-Abl, RIP, c-Raf, NEMO and hTert. Formation of Hsp90 protein radicals on exposure to oxidative stress was confirmed by immuno-spin trapping. Using a proteomic analysis, we determined that the cleavage occurs in a conserved motif of the N-terminal nucleotide binding site, between Ile-126 and Gly-127 in Hsp90β, and between Ile-131 and Gly-132 in Hsp90α. Given the importance of Hsp90 in diverse biological functions, these findings shed new light on how oxidative stress can affect cellular homeostasis.

Cancer cells generally exhibit high levels of reactive oxygen species (ROS) that stimulate cell proliferation and promote genetic instability. Since this biochemical difference between normal and cancer cells represents a specific vulnerability that can be selectively targeted for cancer therapy, various ROS-generating agents are currently in clinical trials, either as single agents or in combination with standard therapy. However, little is known about the potential consequences of an increased oxidative stress for the efficacy of standard chemotherapeutic agents. In this context, we have assessed the influence of an oxidative stress generated by the combination of ascorbate and the redox-active quinone menadione on the capacity of melphalan, a common alkylating agent, to induce apoptosis in a chronic myelogenous leukemia cell line. Our data show that oxidative stress did not inhibit but rather promoted cancer cell killing by melphalan. Interestingly, we observed that, in the presence of oxidative stress, the type of cell death shifted from a caspase-3 dependent apoptosis to necrosis because of an ATP depletion which prevented caspase activation. Taken together, these data suggest that ROS-generating agents could be useful in combination with standard chemotherapy, even if all the molecular consequences of such an addition remain to be determined.

Because reactive oxygen species (ROS) are naturally produced as a consequence of aerobic metabolism, cells have developed a sophisticated set of antioxidant molecules to prevent the toxic accumulation of these species. However, compared with normal cells, malignant cells often exhibit increased levels of intracellular ROS and altered levels of antioxidant molecules. The resulting endogenous oxidative stress favors tumor growth by promoting genetic instability, cell proliferation and angiogenesis. In this context, we assessed the influence of catalase overexpression on the sensitivity of breast cancer cells towards various anticancer treatments. Our data show that catalase overexpression in MCF-7 cells leads to a 7-fold increase in catalase activity but provokes a 40% decrease in the expression of both glutathione peroxidase and peroxiredoxin II. Interestingly, proliferation and migration capacities of MCF-7 cells were impaired by the overexpression of catalase, as compared to parental cells. Regarding their sensitivity to anticancer treatments, we observed that cells overexpressing catalase were more sensitive to paclitaxel, etoposide and arsenic trioxide. However, no effect was observed on the cytotoxic response to ionizing radiations, 5-fluorouracil, cisplatin or doxorubicin. Finally, we observed that catalase overexpression protects cancer cells against the pro-oxidant combination of ascorbate and menadione, suggesting that changes in the expression of antioxidant enzymes could be a mechanism of resistance of cancer cells towards redox-based chemotherapeutic drugs.

Targeting the altered redox status of cancer cells is emerging as an interesting approach to potentiate chemotherapy. However, to maximize the effectiveness of this strategy and define the correct chemotherapeutic associations, it is important to understand the biological consequences of chronically exposing cancer cells to reactive oxygen species (ROS). Using an H(2)O(2)-generating system, we selected a ROS-resistant MCF-7 breast cancer cell line, namely Resox cells. By exploring different survival pathways that are usually induced during oxidative stress, we identified a constitutive overexpression of the endoplasmic reticulum chaperone, GRP94, in these cells, whereas levels of its cytoplasmic homolog HSP90, or GRP78, were not modified. This overexpression was not mediated by constitutive unfolded protein response (UPR) activation. The increase in GRP94 is tightly linked to an increase in cell proliferation and migration capacities, as shown by GRP94-silencing experiments. Interestingly, we also observed that GRP94 silencing inhibits migration and proliferation of the highly aggressive MDA-MB-231 cells. By immunohistochemistry, we showed that GRP94 expression was higher in recurrent human breast cancers than in their paired primary neoplasias. Similar to the situation in the Resox cells, this increase was not associated with an increase in UPR activation in recurrent tumors. In conclusion, this study suggests that GRP94 overexpression may be a hallmark of aggressiveness and recurrence in breast cancers.

Patients with non-alcoholic fatty liver disease are characterised by a decreased n-3/n-6 polyunsaturated fatty acid (PUFA) ratio in hepatic phospholipids. The metabolic consequences of n-3 PUFA depletion in the liver are poorly understood. We have reproduced a drastic drop in n-3 PUFA among hepatic phospholipids by feeding C57Bl/6J mice for 3 months with an n-3 PUFA depleted diet (DEF) versus a control diet (CT), which only differed in the PUFA content. DEF mice exhibited hepatic insulin resistance (assessed by euglycemic-hyperinsulinemic clamp) and steatosis that was associated with a decrease in fatty acid oxidation and occurred despite a higher capacity for triglyceride secretion. Microarray and qPCR analysis of the liver tissue revealed higher expression of all the enzymes involved in lipogenesis in DEF mice compared to CT mice, as well as increased expression and activation of sterol regulatory element binding protein-1c (SREBP-1c). Our data suggest that the activation of the liver X receptor pathway is involved in the overexpression of SREBP-1c, and this phenomenon cannot be attributed to insulin or to endoplasmic reticulum stress responses. In conclusion, n-3 PUFA depletion in liver phospholipids leads to activation of SREBP-1c and lipogenesis, which contributes to hepatic steatosis. © 2011 Pachikian et al., SCOPUS: ar.j, info:eu-repo/semantics/published

Increase in cytosolic calcium concentration ([Ca(2+)](c)), release of endoplasmic reticulum (ER) calcium ([Ca(2+)](er)) and ER stress have been proposed to be involved in oxidative toxicity. Nevertheless, their relative involvements in the processes leading to cell death are not well defined. In this study, we investigated whether oxidative stress generated during ascorbate-driven menadione redox cycling (Asc/Men) could trigger these three events, and, if so, whether they contributed to Asc/Men cytoxicity in MCF-7 cells. Using microspectrofluorimetry, we demonstrated that Asc/Men-generated oxidative stress was associated with a slow and moderate increase in [Ca(2+)](c), largely preceding permeation of propidium iodide, and thus cell death. Asc/Men treatment was shown to partially deplete ER calcium stores after 90min (decrease by 45% compared to control). This event was associated with ER stress activation, as shown by analysis of eIF2 phosphorylation and expression of the molecular chaperone GRP94. Thapsigargin (TG) was then used to study the effect of complete [Ca(2+)](er) emptying during the oxidative stress generated by Asc/Men. Surprisingly, the combination of TG and Asc/Men increased ER stress to a level considerably higher than that observed for either treatment alone, suggesting that [Ca(2+)](er) release alone is not sufficient to explain ER stress activation during oxidative stress. Finally, TG-mediated [Ca(2+)](er) release largely potentiated ER stress, DNA fragmentation and cell death caused by Asc/Men, supporting a role of ER stress in the process of Asc/Men cytotoxicity. Taken together, our results highlight the involvement of ER stress and [Ca(2+)](er) decrease in the process of oxidative stress-induced cell death in MCF-7 cells.

A single high-fat challenge induces plasmatic pro-inflammatory and pro-oxidative responses in the postprandial state, even in healthy men. This period is also associated with vascular endothelial dysfunction, which is an early event in the development of cardiovascular diseases. However, knowledge about the mechanisms involved in postprandial hyperlipaemia-induced endothelial dysfunction is sparse. An objective of our study was to characterize the behaviour and gene expression of vascular endothelial cells exposed to postprandial hyperlipaemic sera. Human umbilical vein endothelial cells (HUVECs) were cultured in media containing 10% serum from healthy men withdrawn either before or 4 h after a high-fat challenge. Endothelial cell proliferation, adhesion and migration were then assessed. The transcriptomic profiles of endothelial cells exposed to pre and postprandial sera were also compared. Exposure to postprandial hyperlipaemic sera significantly decreased HUVEC proliferation when compared to preprandial serum (P 0.0001), while no changes in migration or endothelial/monocyte interactions were observed. The transcriptomic analysis revealed changes in the expression of 675 genes, of which 431 have a known function. Among them, a set of differentially expressed genes was linked to cell cycle regulation and apoptosis and are regulated in favour of cell cycle arrest or death. This result was confirmed by measuring the induction of apoptosis after postprandial sera exposure (P = 0.011). Taken together, the transcriptomic results and pathway analysis showed that postprandial serum promotes apoptosis in HUVECs, potentially through the activation of the p53 network. We conclude that upon postprandial serum exposure, vascular endothelial cells transcriptionally regulate genes involved in the control of cell cycle and death to favour growth arrest and apoptosis. These findings support the hypothesis that postprandial hyperlipaemia is associated with vascular dysfunction and offer new insights into the mechanisms involved.The online version of this article (doi:10.1007/s12263-010-0166-x) contains supplementary material, which is available to authorized users.

Oxidative stress generated by ascorbate-driven menadione redox cycling kills MCF7 cells by a concerted mechanism including glycolysis inhibition, loss of calcium homeostasis, DNA damage and changes in mitogen activated protein kinases (MAPK) activities. Cell death is mediated by necrosis rather than apoptosis or macroautophagy. Neither 3-methyladenine nor Z-VAD affects cytotoxicity by ascorbate/menadione (Asc/Men). BAPTA-AM, by restoring cellular capacity to reduce MTT, underlines the role of calcium in the necrotic process. Oxidative stress-mediated cell death is shown by the opposite effects of N-acetylcysteine and 3-aminotriazole. Moreover, oxidative stress induces DNA damage (protein poly-ADP-ribosylation and γ-H2AX phosphorylation) and inhibits glycolysis. Asc/Men deactivates extracellular signal-regulated kinase (ERK) while activating p38, suggesting an additional mechanism to kill MCF7 cells. Since ascorbate is taken up by cancer cells and, due to their antioxidant enzyme deficiency, oxidative stress should affect cancer cells to a greater extent than normal cells. This differential sensitivity may have clinical applications.

International audience

Hsp90 is a molecular chaperone involved in the stabilization of many oncoproteins that are required for the acquisition and maintenance of the so-called six major hallmarks of cancer cells. Various strategies have, therefore, been developed to inhibit the chaperone activity of Hsp90 and induce cancer cell death through the destabilization of its client proteins. Among these strategies, we have shown that generation of oxidative stress leads to the cleavage and deactivation of Hsp90. Because cancer cells are often deficient in antioxidant enzymes and exhibit higher basal levels of reactive oxygen species (ROS) than their normal counterparts, inducing a selective oxidative stress may be a promising approach for cancer treatment. Thus, many redox-modulating agents have, therefore, been developed or are undergoing clinical trials and Hsp90 represents a new target for oxidative stress-generating agents. The purpose of this article is to review the current state of knowledge about Hsp90 and the use of oxidative stress-generating agents in cancer treatment. We will illustrate the review with some of our results concerning the effects of oxidative stress on Hsp90 using various oxidative stress-generating systems based on different quinones in combination with a well-known reducing agent (i.e., ascorbate). Our results show that oxidative stress provokes the cleavage of Hsp90 in CML cells, as well as the degradation of its client protein Bcr-Abl and the deactivation of its downstream signaling pathways, namely MAPK and STAT5. Overall, these results highlight the potential interest of using oxidative stress to target Hsp90.