Targeting the redox vulnerability of acute myeloid leukaemia cells with a combination of auranofin and vitamin C.

Acute myeloid leukaemia (AML) is a heterogeneous disease characterized by complex molecular and cytogenetic abnormalities. Pro-oxidant cellular redox status is a common hallmark of AML cells, providing a rationale for redox-based anticancer strategy. We previously discovered that auranofin (AUF), initially used for the treatment of rheumatoid arthritis and repositioned for its anticancer activity, can synergize with a pharmacological concentration of vitamin C (VC) against breast cancer cell line models. In this study, we observed that this drug combination synergistically and efficiently killed cells of leukaemic cell lines established from different myeloid subtypes. In addition to an induced elevation of reactive oxygen species and ATP depletion, a rapid dephosphorylation of 4E-BP1 and p70S6K, together with a strong inhibition of protein synthesis were early events in response to AUF/VC treatment, suggesting their implication in AUF/VC-induced cytotoxicity. Importantly, a study on 22 primary AML specimens from various AML subtypes showed that AUF/VC combinations at pharmacologically achievable concentrations were effective to eradicate primary leukaemic CD34+ cells from the majority of these samples, while being less toxic to normal cord blood CD34+ cells. Our findings indicate that targeting the redox vulnerability of AML with AUF/VC combinations could present a potential anti-AML therapeutic approach.

Iron-sulfur protein odyssey: exploring their cluster functional versatility and challenging identification.

Iron-sulfur (Fe-S) clusters are an essential and ubiquitous class of protein-bound prosthetic centers that are involved in a broad range of biological processes (e.g. respiration, photosynthesis, DNA replication and repair and gene regulation) performing a wide range of functions including electron transfer, enzyme catalysis, and sensing. In a general manner, Fe-S clusters can gain or lose electrons through redox reactions, and are highly sensitive to oxidation, notably by small molecules such as oxygen and nitric oxide. The [2Fe-2S] and [4Fe-4S] clusters, the most common Fe-S cofactors, are typically coordinated by four amino acid side chains from the protein, usually cysteine thiolates, but other residues (e.g. histidine, aspartic acid) can also be found. While diversity in cluster coordination ensures the functional variety of the Fe-S clusters, the lack of conserved motifs makes new Fe-S protein identification challenging especially when the Fe-S cluster is also shared between two proteins as observed in several dimeric transcriptional regulators and in the mitoribosome. Thanks to the recent development of in cellulo, in vitro, and in silico approaches, new Fe-S proteins are still regularly identified, highlighting the functional diversity of this class of proteins. In this review, we will present three main functions of the Fe-S clusters and explain the difficulties encountered to identify Fe-S proteins and methods that have been employed to overcome these issues.

Regulations of mitoNEET by the key redox homeostasis molecule glutathione.

Human mitoNEET (mNT) and CISD2 are two NEET proteins characterized by an atypical [2Fe-2S] cluster coordination involving three cysteines and one histidine. They act as redox switches with an active state linked to the oxidation of their cluster. In the present study, we show that reduced glutathione but also free thiol-containing molecules such as ß-mercaptoethanol can induce a loss of the mNT cluster under aerobic conditions, while CISD2 cluster appears more resistant. This disassembly occurs through a radical-based mechanism as previously observed with the bacterial SoxR. Interestingly, adding cysteine prevents glutathione-induced cluster loss. At low pH, glutathione can bind mNT in the vicinity of the cluster. These results suggest a potential new regulation mechanism of mNT activity by glutathione, an essential actor of the intracellular redox state.

[The glutathionylation of the human mitochondrial protein MIA40 regulates ROS homeostasis]. / La glutathionylation de la protéine mitochondriale humaine MIA40 régule l'homéostasie des ROS.

Title: La glutathionylation de la protéine mitochondriale humaine MIA40 régule l'homéostasie des ROS. Abstract: Dans le cadre du module d'enseignement « Physiopathologie de la signalisation ¼ proposé par l'université Paris-Saclay, les étudiants du Master « Biologie Santé ¼ se sont confrontés à l'écriture scientifique. Ils ont sélectionné des articles scientifiques dans le domaine de la signalisation cellulaire présentant des résultats originaux, via des approches expérimentales variées, sur des thèmes allant de l'exploration des sites de contacts membranaires aux mécanismes moléculaires de la ferroptose, en passant par la signalisation hépatique et tumorale. Après un travail préparatoire réalisé avec l'équipe pédagogique, les étudiants, organisés en binômes/trinômes, ont ensuite rédigé, guidés par des chercheurs, une Nouvelle soulignant les résultats majeurs et l'originalité de l'article étudié. Ils ont beaucoup apprécié cette initiation à l'écriture d'articles scientifiques et, comme vous pourrez le lire, se sont investis dans ce travail avec enthousiasme !

Biochemical and cellular characterization of the CISD3 protein: Molecular bases of cluster release and destabilizing effects of nitric oxide.

The NEET proteins, an important family of iron-sulfur (Fe-S) proteins, have generated a strong interest due to their involvement in diverse diseases such as cancer, diabetes, and neurodegenerative disorders. Among the human NEET proteins, CISD3 has been the least studied, and its functional role is still largely unknown. We have investigated the biochemical features of CISD3 at the atomic and in cellulo levels upon challenge with different stress conditions i.e., iron deficiency, exposure to hydrogen peroxide, and nitric oxide. The redox and cellular stability properties of the protein agree on a predominance of reduced form of CISD3 in the cells. Upon the addition of iron chelators, CISD3 loses its Fe-S clusters and becomes unstructured, and its cellular level drastically decreases. Chemical shift perturbation measurements suggest that, upon cluster oxidation, the protein undergoes a conformational change at the C-terminal CDGSH domain, which determines the instability of the oxidized state. This redox-associated conformational change may be the source of cooperative electron transfer via the two [Fe2S2] clusters in CISD3, which displays a single sharp voltammetric signal at -31 mV versus SHE. Oxidized CISD3 is particularly sensitive to the presence of hydrogen peroxide in vitro, whereas only the reduced form is able to bind nitric oxide. Paramagnetic NMR provides clear evidence that, upon NO binding, the cluster is disassembled but iron ions are still bound to the protein. Accordingly, in cellulo CISD3 is unaffected by oxidative stress induced by hydrogen peroxide but it becomes highly unstable in response to nitric oxide treatment.

Viruses traverse the human proteome through peptide interfaces that can be biomimetically leveraged for drug discovery.

We present a drug design strategy based on structural knowledge of protein-protein interfaces selected through virus-host coevolution and translated into highly potential small molecules. This approach is grounded on Vinland, the most comprehensive atlas of virus-human protein-protein interactions with annotation of interacting domains. From this inspiration, we identified small viral protein domains responsible for interaction with human proteins. These peptides form a library of new chemical entities used to screen for replication modulators of several pathogens. As a proof of concept, a peptide from a KSHV protein, identified as an inhibitor of influenza virus replication, was translated into a small molecule series with low nanomolar antiviral activity. By targeting the NEET proteins, these molecules turn out to be of therapeutic interest in a nonalcoholic steatohepatitis mouse model with kidney lesions. This study provides a biomimetic framework to design original chemistries targeting cellular proteins, with indications going far beyond infectious diseases.

The Apelinergic System in Pregnancy.

The apelinergic system is a highly conserved pleiotropic system. It comprises the apelin receptor apelin peptide jejunum (APJ) and its two peptide ligands, Elabela/Toddler (ELA) and apelin, which have different spatiotemporal localizations. This system has been implicated in the regulation of the adipoinsular axis, in cardiovascular and central nervous systems, in carcinogenesis, and in pregnancy in humans. During pregnancy, the apelinergic system is essential for embryo cardiogenesis and vasculogenesis and for placental development and function. It may also play a role in the initiation of labor. The apelinergic system seems to be involved in the development of placenta-related pregnancy complications, such as preeclampsia (PE) and intrauterine growth restriction, but an improvement in PE-like symptoms and birth weight has been described in murine models after the exogenous administration of apelin or ELA. Although the expression of ELA, apelin, and APJ is altered in human PE placenta, data related to their circulating levels are inconsistent. This article reviews current knowledge about the roles of the apelinergic system in pregnancy and its pathophysiological roles in placenta-related complications in pregnancy. We also discuss the challenges in translating the actors of the apelinergic system into a marker or target for therapeutic interventions in obstetrics.

Redox-Based Strategies against Infections by Eukaryotic Pathogens.

Redox homeostasis is an equilibrium between reducing and oxidizing reactions within cells. It is an essential, dynamic process, which allows proper cellular reactions and regulates biological responses. Unbalanced redox homeostasis is the hallmark of many diseases, including cancer or inflammatory responses, and can eventually lead to cell death. Specifically, disrupting redox balance, essentially by increasing pro-oxidative molecules and favouring hyperoxidation, is a smart strategy to eliminate cells and has been used for cancer treatment, for example. Selectivity between cancer and normal cells thus appears crucial to avoid toxicity as much as possible. Redox-based approaches are also employed in the case of infectious diseases to tackle the pathogens specifically, with limited impacts on host cells. In this review, we focus on recent advances in redox-based strategies to fight eukaryotic pathogens, especially fungi and eukaryotic parasites. We report molecules recently described for causing or being associated with compromising redox homeostasis in pathogens and discuss therapeutic possibilities.

NANO-SBT-PEDF delivery system: A promising approach against ovarian cancer?

Pigment epithelium-derived factor (PEDF) is a secreted glycoprotein involved in various biological processes. Its expression declines during ovarian carcinogenesis where it could decrease macrophages polarization, inhibit angiogenesis and induce apoptosis. Altogether, PEDF represents an ideal anti-cancer agent against ovarian cancer. We previously proposed the non-viral Sleeping Beauty transposon (SBT) system to stably integrate the PEDF transgene into ovarian cancer cells. Here, we report the development of liposomes and lipid nanoparticles for SBT-PEDF gene therapy. We determined that the SBT-PEDF nanolipid delivery system was the best system to increase the expression of PEDF in ovarian cancer spheroids. We also developed an ex vivo model of ovarian tumors which allowed us to show that nanolipoplexe in combination to paclitaxel exhibits synergistic and effective anti-tumor efficacy on ovarian tumors. These findings demonstrate that lipid nanoparticle for SBT-PEDF gene therapy may be a promising therapeutic approach for ovarian cancer.

Prevention of acute and delayed chemotherapy-induced nausea and vomiting in pediatric cancer patients: A clinical practice guideline.

This clinical practice guideline provides recommendations for preventing acute and delayed phase chemotherapy-induced nausea and vomiting (CINV) in pediatric patients. The recommendations are based on two systematic reviews of randomized controlled trials evaluating interventions to prevent (1) acute phase CINV and (2) delayed phase CINV. Recommendations for acute phase and delayed phase CINV prophylaxis are made for patients receiving chemotherapy of varying emetogenicity, as well as for patients not able to receive dexamethasone or a neurokinin-1 receptor antagonist. Evidence gaps, including antiemetic safety and optimal dosing, were identified.

Novel Insights into Redox-Based Mechanisms for Auranofin-Induced Rapid Cancer Cell Death.

Auranofin (Ridaura®, AUF) is a gold complex originally approved as an antirheumatic agent that has emerged as a potential candidate for multiple repurposed therapies. The best-studied anticancer mechanism of AUF is the inhibition of thioredoxin reductase (TrxR). However, a number of reports indicate a more complex and multifaceted mode of action for AUF that could be cancer cell type- and dose-dependent. In this study, we observed that AUF displayed variable cytotoxicity in five triple-negative breast cancer cell lines. Using representative MDA-MB-231 cells treated with moderate and cytotoxic doses of AUF, we evidenced that an AUF-mediated TrxR inhibition alone may not be sufficient to induce cell death. Cytotoxic doses of AUF elicited rapid and drastic intracellular oxidative stress affecting the mitochondria, cytoplasm and nucleus. A "redoxome" proteomics investigation revealed that a short treatment with a cytotoxic dose AUF altered the redox state of a number of cysteines-containing proteins, pointing out that the cell proliferation/cell division/cell cycle and cell-cell adhesion/cytoskeleton structure were the mostly affected pathways. Experimentally, AUF treatment triggered a dose-dependent S-phase arrest and a rapid disintegration of the actin cytoskeleton structure. Our study shows a new spectrum of AUF-induced early effects and should provide novel insights into the complex redox-based mechanisms of this promising anticancer molecule.

Interventions for the prevention of acute phase chemotherapy-induced nausea and vomiting in adult and pediatric patients: a systematic review and meta-analysis.

PURPOSE: To identify effective and safe interventions to prevent acute phase chemotherapy-induced nausea and vomiting (CINV) in adult and pediatric patients. METHODS: We conducted a systematic review of randomized trials evaluating interventions to prevent acute CINV. Outcomes assessed were complete chemotherapy-induced vomiting (CIV) control, complete chemotherapy-induced nausea (CIN) control, complete CINV control, and discontinuation of antiemetics due to adverse effects. RESULTS: The search identified 65,172 citations; 744 were evaluated at full-text, and 295 (25 pediatric) met eligibility criteria. In patients receiving highly emetogenic chemotherapy (HEC), complete CIV (risk ratio (RR) 1.23, 95% confidence interval (CI) 1.05-1.44) and CIN (RR 1.34, 95% CI 1.10-1.62) control improved when olanzapine was added. The addition of a neurokinin-1 receptor antagonist (NK1RA) to a corticosteroid plus a serotonin-3 receptor antagonist (5HT3RA) also improved complete CIV (RR 1.11, 95% CI 1.08-1.14) and CIN (RR 1.05, 95% CI 1.01-1.08) control. Compared to granisetron/ondansetron, palonosetron provided improved complete CIV control when the 5HT3RA was given alone or when combined with dexamethasone. In patients receiving moderately emetogenic chemotherapy (MEC), dexamethasone plus a 5HT3RA improved complete CIV control compared to a 5HT3RA alone (RR 1.29, 95% CI 1.21-1.39). Only a single meta-analysis evaluating the safety outcome was possible. CONCLUSIONS: For patients receiving HEC, various antiemetic regimens improved CIV and CIN control. For patients receiving MEC, administration of a 5HT3RA plus dexamethasone improved CIV control. Analysis of antiemetic safety was constrained by lack of data.

Linking unfolded protein response to ovarian cancer cell fusion.

BACKGROUND: Polyploid giant cancer cells (PGCCs) have been observed in epithelial ovarian tumors. They can resist antimitotic drugs, thus participating in tumor maintenance and recurrence. Although their origin remains unclear, PGCC formation seems to be enhanced by conditions that trigger the unfolded protein response (UPR) such as hypoxia or chemotherapeutic drugs like paclitaxel. Hypoxia has been shown to promote the formation of ovarian PGCCs by cell fusion. We thus hypothesized that the UPR could be involved in EOC cell fusion, possibly explaining the occurrence of PGCCs and the aggressiveness of EOC. METHODS: The UPR was induced in two ovarian cancer cell lines (SKOV3 and COV318). The UPR activation was assessed by Western blot and polyploidy indexes were calculated. Then, to confirm the implication of cell fusion in PGCC formation, two populations of SKOV3 cells were transfected with plasmids encoding for two distinct nuclear fluorescent proteins (GFP and mCherry) associated with different antibiotic resistance genes, and the two cell populations were mixed in co-culture. The co-culture was submitted to a double-antibiotic selection. The resulting cell population was characterized for its morphology, cyclicity, and proliferative and tumorigenic capacities, in addition to transcriptomic characterization. RESULTS: We demonstrated that cell fusion could be involved in the generation of ovarian PGCCs and this process was promoted by paclitaxel and the UPR activation. Double-antibiotic treatment of PGCCs led to the selection of a pure population of cells containing both GFP- and mCherry-positive nuclei. Interestingly, after 3 weeks of selection, we observed that these cells were no longer polynucleated but displayed a single nucleus positive for both fluorescent proteins, suggesting that genetic material mixing had occurred. These cells had reinitiated their normal cell cycles, acquired an increased invasive capacity, and could form ovarian tumors in ovo. CONCLUSIONS: The UPR activation increased the in vitro formation of PGCCs by cell fusion, with the newly generated cells further acquiring new properties. The UPR modulation in ovarian cancer patients could represent an interesting therapeutic strategy to avoid the formation of PGCCs and therefore limit cancer relapse and drug resistance.

Determination of the Absolute Molar Mass of [Fe-S]-Containing Proteins Using Size Exclusion Chromatography-Multi-Angle Light Scattering (SEC-MALS).

Size Exclusion Chromatography coupled with Multi-Angle Light Scattering (SEC-MALS) is a technique that determines the absolute molar mass (molecular weight) of macromolecules in solution, such as proteins or polymers, by detecting their light scattering intensity. Because SEC-MALS does not rely on the assumption of the globular state of the analyte and the calibration of standards, the molar mass can be obtained for proteins of any shape, as well as for intrinsically disordered proteins and aggregates. Yet, corrections need to be made for samples that absorb light at the wavelength of the MALS laser, such as iron-sulfur [Fe-S] cluster-containing proteins. We analyze several examples of [2Fe-2S] and [4Fe-4S] cluster-containing proteins, for which various corrections were applied to determine the absolute molar mass of both the apo- and holo-forms. Importantly, the determination of the absolute molar mass of the [2Fe-2S]-containing holo-NEET proteins allowed us to ascertain a change in the oligomerization state upon cluster binding and, thus, to highlight one essential function of the cluster.

Dual effect of nifedipine on pregnant human myometrium contractility: Implication of TRPC1.

Nifedipine, an L-type voltage-gated Ca2+ channel (L-VGCC) blocker, is one of the most used tocolytics to treat preterm labor. In clinical practice, nifedipine efficiently decreases uterine contractions, but its efficacy is limited over time, and repeated or maintained nifedipine-based tocolysis appears to be ineffective in preventing preterm birth. We aimed to understand why nifedipine has short-lasting efficiency for the inhibition of uterine contractions. We used ex vivo term pregnant human myometrial strips treated with cumulative doses of nifedipine. We observed that nifedipine inhibited spontaneous myometrial contractions in tissues with high and regular spontaneous contractions. By contrast, nifedipine appeared to increase contractions in tissues with low and/or irregular spontaneous contractions. To investigate the molecular mechanisms activated by nifedipine in myometrial cells, we used the pregnant human myometrial cell line PHM1-41 that does not express L-VGCC. The in vitro measurement of intracellular Ca2+ showed that high doses of nifedipine induced an important intracellular Ca2+ entry in myometrial cells. The inhibition or downregulation of the genes encoding for store-operated Ca2+ entry channels from the Orai and transient receptor potential-canonical (TRPC) families in PHM1-41 cells highlighted the implication of TRPC1 in nifedipine-induced Ca2+ entry. In addition, the use of 2-APB in combination with nifedipine on human myometrial strips tends to confirm that the pro-contractile effect induced by nifedipine on myometrial tissues may involve the activation of TRPC channels.

Mechanisms of Immunomodulation and Cytoprotection Conferred to Pancreatic Islet by Human Amniotic Epithelial Cells.

Inhibiting pro-inflammatory cytokine activity can reverse inflammation mediated dysfunction of islet grafts. Human amniotic epithelial cells (hAECs) possess regenerative, immunomodulatory and anti-inflammatory properties. We hypothesized that hAECs could protect islets from cellular damage induced by pro-inflammatory cytokines. To verify our hypothesis, hAEC monocultures, rat islets (RI), or RI-hAEC co-cultures where exposed to a pro-inflammatory cytokine cocktail (Interferon γ: IFN-γ, Tumor necrosis factor α: TNF-α and Interleukin-1ß: IL-1ß). The secretion of anti-inflammatory cytokines and gene expression changes in hAECs and viability and function of RI were evaluated. The expression of non-classical Major Histocompatibility Complex (MHC) class I molecules by hAECs cultured with various IFN-γ concentrations were assessed. Exposure to the pro-inflammatory cocktail significantly increased the secretion of the anti-inflammatory cytokines IL6, IL10 and G-CSF by hAECs, which was confirmed by upregulation of IL6, and IL10 gene expression. HLA-G, HLA-E and PDL-1 gene expression was also increased. This correlated with an upregulation of STAT1, STAT3 and NF-κB1gene expression levels. RI co-cultured with hAECs maintained normal function after cytokine exposure compared to RI cultured alone, and showed significantly lower apoptosis rate. Our results show that exposure to pro-inflammatory cytokines stimulates secretion of anti-inflammatory and immunomodulatory factors by hAECs through the JAK1/2 - STAT1/3 and the NF-κB1 pathways, which in turn protects islets against inflammation-induced damages. Integrating hAECs in islet transplants appears as a valuable strategy to achieve to inhibit inflammation mediated islet damage, prolong islet survival, improve their engraftment and achieve local immune protection allowing reducing systemic immunosuppressive regimens. This study focuses on the cytoprotective effect of isolated hAECs on islets exposed to pro-inflammatory cytokines in vitro. Exposure to pro-inflammatory cytokines stimulated secretion of anti-inflammatory and immunomodulatory factors by hAECs putatively through the JAK1/2 - STAT1/3 and the NF-κB1 pathways. This had protective effect on islets against inflammation-induced damages. Taken together our results indicate that incorporating hAECs in islet transplants could be a valuable strategy to inhibit inflammation mediated islet damage, prolong islet survival, improve their engraftment and achieve local immune protection allowing to reduce systemic immunosuppressive regimens.

Linking cell-surface GRP78 to cancer: From basic research to clinical value of GRP78 antibodies.

Glucose-related protein 78 (GRP78) is a chaperone protein localized primarily in the endoplasmic reticulum (ER) lumen, where it helps in proper protein folding by targeting misfolded proteins and facilitating protein assembly. In stressed cells, GRP78 is translocated to the cell surface (csGRP78) where it binds to various ligands and triggers different intracellular pathways. Thus, csGRP78 expression is associated with cancer, involved in the maintenance and progression of the disease. Extracellular exposition of csGRP78 leads to the production of autoantibodies as observed in patients with prostate or ovarian cancer, in which the ability to target csGRP78 affects the tumor development. Present on the surface of cancer cells and not normal cells in vivo, csGRP78 represents an interesting target for therapeutic antibody strategies. Here we give an overview of the csGRP78 function in the cell and its role in oncogenesis, thereby providing insight into the clinical value of GRP78 monoclonal antibodies for cancer prognosis and treatment.

Role of the Uteroplacental Renin-Angiotensin System in Placental Development and Function, and Its Implication in the Preeclampsia Pathogenesis.

Placental development and function implicate important morphological and physiological adaptations to thereby ensure efficient maternal-fetal exchanges, as well as pregnancy-specific hormone secretion and immune modulation. Incorrect placental development can lead to severe pregnancy disorders, such as preeclampsia (PE), which endangers both the mother and the infant. The implication of the systemic renin-angiotensin system (RAS) in the pregnancy-related physiological changes is now well established. However, despite the fact that the local uteroplacental RAS has been described for several decades, its role in placental development and function seems to have been underestimated. In this review, we provide an overview of the multiple roles of the uteroplacental RAS in several cellular processes of placental development, its implication in the regulation of placental function during pregnancy, and the consequences of its dysregulation in PE pathogenesis.

The Role and Clinical Interest of Extracellular Vesicles in Pregnancy and Ovarian Cancer.

Ovarian cancer and pregnancy are two states in which the host immune system is exposed to novel antigens. Indeed, both the tumor and placenta must invade tissues, remodel vasculature to establish a robust blood supply, and evade detection by the immune system. Interestingly, tumor and placenta tissue use similar mechanisms to induce these necessary changes. One mediator is emerging as a key player in invasion, vascular remodeling, and immune evasion: extracellular vesicles (EVs). Many studies have identified EVs as a key mediator of cell-to-cell communication. Specifically, the cargo carried by EVs, which includes proteins, nucleic acids, and lipids, can interact with cells to induce changes in the target cell ranging from gene expression to migration and metabolism. EVs can promote cell division and tissue invasion, immunosuppression, and angiogenesis which are essential for both cancer and pregnancy. In this review, we examine the role of EVs in ovarian cancer metastasis, chemoresistance, and immune modulation. We then focus on the role of EVs in pregnancy with special attention on the vascular remodeling and regulation of the maternal immune system. Lastly, we discuss the clinical utility of EVs as markers and therapeutics for ovarian cancer and pre-eclampsia.