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.

Redox modifications of cysteine-containing proteins, cell cycle arrest and translation inhibition: Involvement in vitamin C-induced breast cancer cell death.

Vitamin C (VitC) possesses pro-oxidant properties at high pharmacologic concentrations which favor repurposing VitC as an anti-cancer therapeutic agent. However, redox-based anticancer properties of VitC are yet partially understood. We examined the difference between the reduced and oxidized forms of VitC, ascorbic acid (AA) and dehydroascorbic acid (DHA), in terms of cytotoxicity and redox mechanisms toward breast cancer cells. Our data showed that AA displayed higher cytotoxicity towards triple-negative breast cancer (TNBC) cell lines in vitro than DHA. AA exhibited a similar cytotoxicity on non-TNBC cells, while only a minor detrimental effect on noncancerous cells. Using MDA-MB-231, a representative TNBC cell line, we observed that AA- and DHA-induced cytotoxicity were linked to cellular redox-state alterations. Hydrogen peroxide (H2O2) accumulation in the extracellular medium and in different intracellular compartments, and to a lesser degree, intracellular glutathione oxidation, played a key role in AA-induced cytotoxicity. In contrast, DHA affected glutathione oxidation and had less cytotoxicity. A "redoxome" approach revealed that AA treatment altered the redox state of key antioxidants and a number of cysteine-containing proteins including many nucleic acid binding proteins and proteins involved in RNA and DNA metabolisms and in energetic processes. We showed that cell cycle arrest and translation inhibition were associated with AA-induced cytotoxicity. Finally, bioinformatics analysis and biological experiments identified that peroxiredoxin 1 (PRDX1) expression levels correlated with AA differential cytotoxicity in breast cancer cells, suggesting a potential predictive value of PRDX1. This study provides insight into the redox-based mechanisms of VitC anticancer activity, indicating that pharmacologic doses of VitC and VitC-based rational drug combinations could be novel therapeutic opportunities for triple-negative breast cancer.

GDF15 secreted by senescent endothelial cells improves vascular progenitor cell functions.

Endothelial dysfunction (ED) is part of the first steps in the development of cardiovascular diseases (CVD). Growth Differentiation Factor 15 (GDF15) is a cytokine belonging to the Transforming Growth Factor ß superfamily and its expression is increased both during ED and in CVD. Because high blood levels of GDF15 have been reported during ED, we hypothesized that GDF15 could be produced by endothelial cells in response to a vascular stress, possibly to attenuate endothelial function loss. Since senescence is mainly involved in both vascular stress and endothelial function loss, we used Endothelial Colony Forming Cells generated from adult blood (AB-ECFCs) as a model of endothelial cells to investigate GDF15 expression during cellular senescence. Then, we analyzed the potential role of GDF15 in AB-ECFC functions and senescence. When AB-ECFCs become senescent, they secrete increased levels of GDF15. We investigated GDF15 paracrine effects on non-senescent AB-ECFCs and showed that GDF15 enhanced proliferation, migration, NO production and activated several signaling pathways including AKT, ERK1/2 and SMAD2 without triggering any oxidative stress. Taken together, our results suggest that GDF15 production by senescent AB-ECFCs could act in a paracrine manner on non-senescent AB-ECFCs, and that this interaction could be beneficial to its model cells. Therefore, GDF15 could play a beneficial role in a dysfunctional vascular system as previously reported in patients with CVD, by limiting ED related to vascular stress occurring in these diseases.

Auranofin/Vitamin C: A Novel Drug Combination Targeting Triple-Negative Breast Cancer.

BACKGROUND: Cancer cells from different origins exhibit various basal redox statuses and thus respond differently to intrinsic or extrinsic oxidative stress. These intricate characteristics condition the success of redox-based anticancer therapies that capitalize on the ability of reactive oxygen species to achieve selective and efficient cancer cell killing. METHODS: Redox biology methods, stable isotope labeling by amino acids in cell culture (SILAC)-based proteomics, and bioinformatics pattern comparisons were used to decipher the underlying mechanisms for differential response of lung and breast cancer cell models to redox-modulating molecule auranofin (AUF) and to combinations of AUF and vitamin C (VC). The in vivo effect of AUF, VC, and two AUF/VC combinations on mice bearing MDA-MB-231 xenografts (n = 5 mice per group) was also evaluated. All statistical tests were two-sided. RESULTS: AUF targeted simultaneously the thioredoxin and glutathione antioxidant systems. AUF/VC combinations exerted a synergistic and hydrogen peroxide (H2O2)-mediated cytotoxicity toward MDA-MB-231 cells and other breast cancer cell lines. The anticancer potential of AUF/VC combinations was validated in vivo on MDA-MB-231 xenografts in mice without notable side effects. On day 14 of treatments, mean (SD) tumor volumes for the vehicle-treated control group and the two AUF/VC combination-treated groups (A/V1 and A/V2) were 197.67 (24.28) mm3, 15.66 (10.90) mm3, and 10.23 (7.30)mm3, respectively; adjusted P values of the differences between mean tumor volumes of vehicle vs A/V1 groups and vehicle vs A/V2 groups were both less than .001. SILAC proteomics, bioinformatics analysis, and functional experiments linked prostaglandin reductase 1 (PTGR1) expression levels with breast cancer cell sensitivity to AUF/VC combinations. CONCLUSION: The combination of AUF and VC, two commonly available drugs, could be efficient against triple-negative breast cancer and potentially other cancers with similar redox properties and PTGR1 expression levels. The redox-based anticancer activity of this combination and the discriminatory potential of PTGR1 expression are worth further assessment in preclinical and clinical studies.

Auranofin/Vitamin C: A Novel Drug Combination Targeting Triple-Negative Breast Cancer.

BACKGROUND: Cancer cells from different origins exhibit various basal redox statuses and thus respond differently to intrinsic or extrinsic oxidative stress. These intricate characteristics condition the success of redox-based anticancer therapies that capitalize on the ability of reactive oxygen species to achieve selective and efficient cancer cell killing. METHODS: Redox biology methods, stable isotope labeling by amino acids in cell culture (SILAC)-based proteomics, and bioinformatics pattern comparisons were used to decipher the underlying mechanisms for differential response of lung and breast cancer cell models to redox-modulating molecule auranofin (AUF) and to combinations of AUF and vitamin C (VC). The in vivo effect of AUF, VC, and two AUF/VC combinations on mice bearing MDA-MB-231 xenografts (n = 5 mice per group) was also evaluated. All statistical tests were two-sided. RESULTS: AUF targeted simultaneously the thioredoxin and glutathione antioxidant systems. AUF/VC combinations exerted a synergistic and hydrogen peroxide (H2O2)-mediated cytotoxicity toward MDA-MB-231 cells and other breast cancer cell lines. The anticancer potential of AUF/VC combinations was validated in vivo on MDA-MB-231 xenografts in mice without notable side effects. On day 14 of treatments, mean (SD) tumor volumes for the vehicle-treated control group and the two AUF/VC combination-treated groups (A/V1 and A/V2) were 197.67 (24.28) mm3, 15.66 (10.90) mm3, and 10.23 (7.30)mm3, respectively; adjusted P values of the differences between mean tumor volumes of vehicle vs A/V1 groups and vehicle vs A/V2 groups were both less than .001. SILAC proteomics, bioinformatics analysis, and functional experiments linked prostaglandin reductase 1 (PTGR1) expression levels with breast cancer cell sensitivity to AUF/VC combinations. CONCLUSION: The combination of AUF and VC, two commonly available drugs, could be efficient against triple-negative breast cancer and potentially other cancers with similar redox properties and PTGR1 expression levels. The redox-based anticancer activity of this combination and the discriminatory potential of PTGR1 expression are worth further assessment in preclinical and clinical studies.

Multifaceted Roles of Glutathione and Glutathione-Based Systems in Carcinogenesis and Anticancer Drug Resistance.

SIGNIFICANCE: Glutathione is the most abundant antioxidant molecule in living organisms and has multiple functions. Intracellular glutathione homeostasis, through its synthesis, consumption, and degradation, is an intricately balanced process. Glutathione levels are often high in tumor cells before treatment, and there is a strong correlation between elevated levels of intracellular glutathione/sustained glutathione-mediated redox activity and resistance to pro-oxidant anticancer therapy. Recent Advances: Ample evidence demonstrates that glutathione and glutathione-based systems are particularly relevant in cancer initiation, progression, and the development of anticancer drug resistance. CRITICAL ISSUES: This review highlights the multifaceted roles of glutathione and glutathione-based systems in carcinogenesis, anticancer drug resistance, and clinical applications. FUTURE DIRECTIONS: The evidence summarized here underscores the important role played by glutathione and the glutathione-based systems in carcinogenesis and anticancer drug resistance. Future studies should address mechanistic questions regarding the distinct roles of glutathione in different stages of cancer development and cancer cell death. It will be important to study how metabolic alterations in cancer cells can influence glutathione homeostasis. Sensitive approaches to monitor glutathione dynamics in subcellular compartments will be an indispensible step. Therapeutic perspectives should focus on mechanism-based rational drug combinations that are directed against multiple redox targets using effective, specific, and clinically safe inhibitors. This new strategy is expected to produce a synergistic effect, prevent drug resistance, and diminish doses of single drugs. Antioxid. Redox Signal. 27, 1217-1234.

Fe-S Clusters Emerging as Targets of Therapeutic Drugs.

Fe-S centers exhibit strong electronic plasticity, which is of importance for insuring fine redox tuning of protein biological properties. In accordance, Fe-S clusters are also highly sensitive to oxidation and can be very easily altered in vivo by different drugs, either directly or indirectly due to catabolic by-products, such as nitric oxide species (NOS) or reactive oxygen species (ROS). In case of metal ions, Fe-S cluster alteration might be the result of metal liganding to the coordinating sulfur atoms, as suggested for copper. Several drugs presented through this review are either capable of direct interaction with Fe-S clusters or of secondary Fe-S clusters alteration following ROS or NOS production. Reactions leading to Fe-S cluster disruption are also reported. Due to the recent interest and progress in Fe-S biology, it is very likely that an increasing number of drugs already used in clinics will emerge as molecules interfering with Fe-S centers in the near future. Targeting Fe-S centers could also become a promising strategy for drug development.

RNA-binding proteins are a major target of silica nanoparticles in cell extracts.

Upon contact with biological fluids, nanoparticles (NPs) are readily coated by cellular compounds, particularly proteins, which are determining factors for the localization and toxicity of NPs in the organism. Here, we improved a methodological approach to identify proteins that adsorb on silica NPs with high affinity. Using large-scale proteomics and mixtures of soluble proteins prepared either from yeast cells or from alveolar human cells, we observed that proteins with large unstructured region(s) are more prone to bind on silica NPs. These disordered regions provide flexibility to proteins, a property that promotes their adsorption. The statistical analyses also pointed to a marked overrepresentation of RNA-binding proteins (RBPs) and of translation initiation factors among the adsorbed proteins. We propose that silica surfaces, which are mainly composed of Si-O- and Si-OH groups, mimic ribose-phosphate molecules (rich in -O- and -OH) and trap the proteins able to interact with ribose-phosphate containing molecules. Finally, using an in vitro assay, we showed that the sequestration of translation initiation factors by silica NPs results in an inhibition of the in vitro translational activity. This result demonstrates that characterizing the protein corona of various NPs would be a relevant approach to predict their potential toxicological effects.

PRX1 knockdown potentiates vitamin K3 toxicity in cancer cells: a potential new therapeutic perspective for an old drug.

BACKGROUND: Many promising anticancer molecules are abandoned during the course from bench to bedside due to lack of clear-cut efficiency and/or severe side effects. Vitamin K3 (vitK3) is a synthetic naphthoquinone exhibiting significant in vitro and in vivo anticancer activity against multiple human cancers, and has therapeutic potential when combined with other anticancer molecules. The major mechanism for the anticancer activity of vitK3 is the generation of cytotoxic reactive oxygen species (ROS). We thus reasoned that a rational redox modulation of cancer cells could enhance vitK3 anticancer efficiency. METHODS: Cancer cell lines with peroxiredoxin 1 (PRX1) gene transiently or stably knocked-down and corresponding controls were exposed to vitK3 as well as a set of anticancer molecules, including vinblastine, taxol, doxorubicin, daunorubicin, actinomycin D and 5-fluorouracil. Cytotoxic effects and cell death events were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)-based assay, cell clonogenic assay, measurement of mitochondrial membrane potential and annexin V/propidium iodide double staining. Global ROS accumulation and compartment-specific H2O2 generation were determined respectively by a redox-sensitive chemical probe and H2O2-sensitive sensor HyPer. Oxidation of endogenous antioxidant proteins including TRX1, TRX2 and PRX3 was monitored by redox western blot. RESULTS: We observed that the PRX1 knockdown in HeLa and A549 cells conferred enhanced sensitivity to vitK3, reducing substantially the necessary doses to kill cancer cells. The same conditions (combination of vitK3 and PRX1 knockdown) caused little cytotoxicity in non-cancerous cells, suggesting a cancer-cell-selective property. Increased ROS accumulation had a crucial role in vitK3-induced cell death in PRX1 knockdown cells. The use of H2O2-specific sensors HyPer revealed that vitK3 lead to immediate accumulation of H2O2 in the cytosol, nucleus, and mitochondrial matrix. PRX1 silencing significantly up-regulated mRNA and protein levels of NRH:quinone oxidoreductase 2, which was partially responsible for vitK3-induced ROS accumulation and consequent cell death. CONCLUSION: Our data suggest that PRX1 inactivation could represent an interesting strategy to enhance cancer cell sensitivity to vitK3, providing a potential new therapeutic perspective for this old molecule. Conceptually, a combination of drugs that modulate intracellular redox states and drugs that operate through the generation of ROS could be a new therapeutic strategy for cancer treatment.

A renal angiomyolipoma with extension to the renal vein and asymptomatic fat pulmonary embolus.

Renal angiomyolipoma (AML) is a rare benign tumor that can extend into the renal vein, inferior vena cava and the right atrium. AML is a mesenchymal tumor composed of smooth muscle, fat and vascular elements. In rare instances, the tumor may release a fatty tissue to the pulmonary vasculature, which can lead to cardiopulmonary collapse and death. Only four cases of fat pulmonary embolism secondary to AML have been reported in the literature but our case was the first to present as asymptomatic. Our patient had left renal AML extending to the renal vein that was associated with fat pulmonary embolus. The patient underwent uncomplicated radical nephrectomy and was discharged home on no anticoagulation. Follow-up chest computed tomography showed no extension of the pulmonary embolism. Whether embolectomy or anticoagulation is necessary in asymptomatic pulmonary embolism secondary to renal AML is unclear. Although controversial, some surgeons prefer to place an inferior vena cava filter prior to radical nephrectomy to prevent dislodgement of new intraoperative emboli, which can lead to catastrophic outcome.

Peroxiredoxin 1 knockdown sensitizes cancer cells to reactive oxygen species-generating drugs - an alternative approach for chemotherapy.

Peroxiredoxins have multiple cellular functions as major antioxidants, signaling regulators and tumor suppressors. Peroxiredoxin 1 (PRX1) is the most abundant among the six isoforms of human peroxiredoxins, catalyzing the reduction of peroxides utilizing thioredoxin 1as an electron donor. PRX1 is frequently over-expressed in various cancer cells, which is thought to be associated with carcinogenesis, metastasis and resistance to radiotherapy or chemotherapy. We investigated how modulations of intracellular redox system, especially PRX1, affect cancer cell sensitivity to reactive oxygen species (ROS)-generating drugs. We observed that stable and transient Prx1 knockdown (Prx1-) significantly enhances HeLa cell sensitivity to ß-lapachone (ß-lap), a potential anticancer agent, and to other ROS-generating molecules. ROS accumulation played a crucial role in drug-enhanced Prx1- cell death. For ß-lap, Prx1- cells sensitization is achieved through combined action of accumulation of ROS and enhancement of mitogen-activated protein kinase pathway activation. The effect of other ROS-inducing drugs on Prx1- cell survival will also be presented and discussed. Taken together, our data provide evidence that PRX1 could be an interesting anticancer target and modulation of intracellular redox states through PRX1 inhibition could be an alternative approach to enhance cancer cell sensitivity to ROS-generating drugs.

Glutathione is essential to preserve nuclear function and cell survival under oxidative stress.

Organisms growing in aerobic environments must cope with Reactive Oxygen Species (ROS). Although ROS damage all the cellular macromolecules, they play a central role in a range of biological processes requiring a tight control of redox homeostasis. It is achieved by antioxidant systems involving a large collection of enzymes that scavenge or degrade the ROS produced endogenously during cell growth. In addition to this enzymatic protection against ROS, cells also contain small antioxidant molecules, such as glutathione (GSH). With an intracellular concentration between 1 and 10mM, GSH is the most abundant non-protein thiol in the cell and is considered as the major redox buffer of the cell. To better characterize its essential function during oxidative stress conditions, we studied the physiological response of H2O2-treated yeast cells containing different amounts of GSH. We showed that the transcriptional response of GSH-depleted cells is severely impaired, despite an efficient nuclear accumulation of the transcription factor Yap1. Moreover, oxidative stress generates high genome instability in GSH-depleted cells, but does not activate the checkpoint kinase Rad53. Surprisingly, scarce amounts of intracellular GSH are sufficient to preserve cell viability under H2O2 treatment. In these cells, oxidative stress still causes the accumulation of oxidized proteins and the inactivation of the translational activity, but nuclear DNA and nuclear functions are protected against oxidative injury, as exemplified by low mutation frequency, moderate histone carbonylation, activation of the checkpoint kinase Rad53 and of the H2O2 transcriptional response. We conclude that the essential role of GSH is to preserve nuclear function, allowing cell survival and growth resumption after oxidative stress release. We propose that cytosolic proteins are part of a protective machinery that shields the nucleus by scavenging reactive oxygen species before they can cross the nuclear membrane.

Glutathione is essential to preserve nuclear function and cell survival under oxidative stress.

Glutathione (GSH) is considered the most important redox buffer of the cell. To better characterize its essential function during oxidative stress conditions, we studied the physiological response of H2O2-treated yeast cells containing various amounts of GSH. We showed that the transcriptional response of GSH-depleted cells is severely impaired, despite an efficient nuclear accumulation of the transcription factor Yap1. Moreover, oxidative stress generates high genome instability in GSH-depleted cells, but does not activate the checkpoint kinase Rad53. Surprisingly, scarce amounts of intracellular GSH are sufficient to preserve cell viability under H2O2 treatment. In these cells, oxidative stress still causes the accumulation of oxidized proteins and the inactivation of the translational activity, but nuclear components and activities are protected against oxidative injury. We conclude that the essential role of GSH is to preserve nuclear function, allowing cell survival and growth resumption after oxidative stress release. We propose that cytosolic proteins are part of a protective machinery that shields the nucleus by scavenging reactive oxygen species before they can cross the nuclear membrane.

Genetic variations creating microRNA target sites in the FXN 3'-UTR affect frataxin expression in Friedreich ataxia.

Friedreich's ataxia (FRDA) is a severe neurodegenerative disease caused by GAA repeat expansion within the first intron of the frataxin gene. It has been suggested that the repeat is responsible for the disease severity due to impaired transcription thereby reducing expression of the protein. However, genotype-phenotype correlation is imperfect, and the influence of other gene regions of the frataxin gene is unknown. We hypothesized that FRDA patients may harbor specific regulatory variants in the 3'-UTR. We sequenced the 3'-UTR region of the frataxin gene in a cohort of 57 FRDA individuals and 58 controls. Seven single nucleotide polymorphisms (SNPs) out of 19 were polymorphic in our case-control sample. These SNPs defined several haplotypes with one reaching 89% of homozygosity in patients versus 24% in controls. In another cohort of 47 FRDA Reunionese patients, 94% patients were found to be homozygous for this haplotype. We found that this FRDA 3'-UTR conferred a 1.2-fold decrease in the expression of a reporter gene versus the alternative haplotype configuration. We established that differential targeting by miRNA could account for this functional variability. We specifically demonstrated the involvement of miR-124 (i.e hsa-mir-124-3p) in the down-regulation of FRDA-3'-UTR. Our results suggest for the first time that post-transcriptional regulation of frataxin occurs through the 3'-UTR and involves miRNA targeting. We propose that the involvement of miRNAs in a FRDA-specific regulation of frataxin may provide a rationale to increase residual levels of frataxin through miRNA-inhibitory molecules.

Thrombus in Transit through Patent Foramen Ovale.

A thrombus in transit through a patent foramen ovale (PFO) with impending paradoxical embolism is an extremely rare event. Due to its transient nature, it is unable to identify the thrombus, and most of the cases have been reported at autopsy. We are reporting a case of thrombus straddling the foramen ovale which was diagnosed by echocardiography and treated surgically. Through this personal case, an exhaustive review of the literature was performed. There were 88 cases reported. We concluded that there is no medical consensus about the best option for treatment. Nevertheless, surgery, which is associated with fewer complications of recurrent embolic events than those of thrombolysis and anticoagulation, appeared to be the best approach in patients who are not at a high surgical risk. Anticoagulant treatment appears to be an acceptable therapeutic alternative to surgery, particularly in patients with comorbidities who are at high surgical risk and for patients with small PFO. Thrombolysis is linked to the highest mortality, which could be explained by the severity of the patient's initial presentation. In conclusion, and after the cumulative effects of these case reports, we propose a diagram consisting of the use of the three therapeutic options in the different clinical scenarios.