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.

Combined thioredoxin reductase and glutaminase inhibition exerts synergistic anti-tumor activity in MYC-high high-grade serous ovarian carcinoma.

Approximately 50%-55% of high-grade serous ovarian carcinoma (HGSOC) patients have MYC oncogenic pathway activation. Because MYC is not directly targetable, we have analyzed molecular pathways enriched in MYC-high HGSOC tumors to identify potential therapeutic targets. Here, we report that MYC-high HGSOC tumors show enrichment in genes controlled by NRF2, an antioxidant signaling pathway, along with increased thioredoxin redox activity. Treatment of MYC-high HGSOC tumors cells with US Food and Drug Administration (FDA)-approved thioredoxin reductase 1 (TrxR1) inhibitor auranofin resulted in significant growth suppression and apoptosis in MYC-high HGSOC cells in vitro and also significantly reduced tumor growth in an MYC-high HGSOC patient-derived tumor xenograft. We found that auranofin treatment inhibited glycolysis in MYC-high cells via oxidation-induced GAPDH inhibition. Interestingly, in response to auranofin-induced glycolysis inhibition, MYC-high HGSOC cells switched to glutamine metabolism for survival. Depletion of glutamine with either glutamine starvation or glutaminase (GLS1) inhibitor CB-839 exerted synergistic anti-tumor activity with auranofin in HGSOC cells and OVCAR-8 cell line xenograft. These findings suggest that applying a combined therapy of GLS1 inhibitor and TrxR1 inhibitor could effectively treat MYC-high HGSOC patients.

Auranofin sensitizes breast cancer cells to paclitaxel mediated cell death via regulating FOXO3/Nrf2/Keap1 signaling pathway.

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcriptional factor which acts as a regulator for cellular oxidative stress, and tightly regulated by Kelch-like ECH-associated protein 1 (Keap1). In this study, we found that auranofin and paclitaxel combination treatment increased TUNEL positive apoptotic cells and enhanced the DNA damage marker γ-H2AX in MCF-7 and MDA-MB-231 breast cancer cells. The immunoblotting analysis revealed the combination of auranofin and paclitaxel significantly increased the FOXO3 expression in a concentration dependent manner. Further we observed that auranofin and paclitaxel treatment prevents the translocation of Nrf2 in a concentration dependent manner. The increased FOXO3 expression might be involved in the cytoplasmic degradation of Nrf1-Keap1 complex. Further, the molecular docking results confirm auranofin act as the agonist for Foxo3. Therefore, the present results suggest that auranofin sensitize the breast cancer cells to paclitaxel via regulating FOXO3/Nrf2/Keap1signaling pathway.

Auranofin inhibits virulence pathways in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is widely attributed as the leading cause of hospital-acquired infections. Due to intrinsic antibiotic resistance mechanisms and the ability to form biofilms, P. aeruginosa infections are challenging to treat. P. aeruginosa employs multiple virulence mechanisms to establish infections, many of which are controlled by the global virulence regulator Vfr. An attractive strategy to combat P. aeruginosa infections is thus the use of anti-virulence compounds. Here, we report the discovery that FDA-approved drug auranofin attenuates virulence pathways in P. aeruginosa, including quorum sensing (QS) and Type IV pili (TFP). We show that auranofin acts via multiple targets, one of which being Vfr. Consistent with inhibition of QS and TFP expression, we show that auranofin attenuates biofilm maturation, and when used in combination with colistin, displays strong synergy in eradicating P. aeruginosa biofilms. Auranofin may have immediate applications as an anti-virulence drug against P. aeruginosa infections.

Auranofin sensitizes breast cancer cells to paclitaxel chemotherapy by disturbing the cellular redox system.

The intrinsic redox status of cancer cells limits the efficacy of chemotherapeutic drugs. Auranofin, a Food and Drug Administration-approved gold-containing compound, documented with effective pharmacokinetics and safety profiles in humans, has recently been repurposed for anticancer activity. This study examined the paclitaxel-sensitizing effect of auranofin by targeting redox balance in the MDA-MB-231 and MCF-7 breast cancer cell lines. Auranofin treatment depletes the activities of superoxide dismutase, catalase, and glutathione peroxidase and alters the redox ratio in the breast cancer cell lines. Furthermore, it has been noticed that auranofin augmented paclitaxel-mediated cytotoxicity in a concentration-dependent manner in both MDA-MB-231 and MCF-7 cell lines. Moreover, auranofin increased the levels of intracellular reactive oxygen species (observed using 2, 7-diacetyl dichlorofluorescein diacetate staining) and subsequently altered the mitochondrial membrane potential (rhodamine-123 staining) in a concentration-dependent manner. Further, the expression of apoptotic marker p21 was found to be higher in auranofin plus paclitaxel-treated breast cancer cells compared to paclitaxel-alone treatment. Thus, the present results illustrate the chemosensitizing property of auranofin in MDA-MB-231 and MCF-7 breast cancer cell lines via oxidative metabolism. Therefore, auranofin could be considered a chemosensitizing agent during cancer chemotherapy.

Structure of Leishmania donovani 6-Phosphogluconate Dehydrogenase and Inhibition by Phosphine Gold(I) Complexes: A Potential Approach to Leishmaniasis Treatment.

As unicellular parasites are highly dependent on NADPH as a source for reducing equivalents, the main NADPH-producing enzymes glucose 6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (6PGD) of the pentose phosphate pathway are considered promising antitrypanosomatid drug targets. Here we present the biochemical characterization and crystal structure of Leishmania donovani 6PGD (Ld6PGD) in complex with NADP(H). Most interestingly, a previously unknown conformation of NADPH is visible in this structure. In addition, we identified auranofin and other gold(I)-containing compounds as efficient Ld6PGD inhibitors, although it has so far been assumed that trypanothione reductase is the sole target of auranofin in Kinetoplastida. Interestingly, 6PGD from Plasmodium falciparum is also inhibited at lower micromolar concentrations, whereas human 6PGD is not. Mode-of-inhibition studies indicate that auranofin competes with 6PG for its binding site followed by a rapid irreversible inhibition. By analogy with other enzymes, this suggests that the gold moiety is responsible for the observed inhibition. Taken together, we identified gold(I)-containing compounds as an interesting class of inhibitors against 6PGDs from Leishmania and possibly from other protozoan parasites. Together with the three-dimensional crystal structure, this provides a valid basis for further drug discovery approaches.

Chemical Modification of Auranofin Yields a New Family of Anticancer Drug Candidates: The Gold(I) Phosphite Analogues.

A panel of four novel gold(I) complexes, inspired by the clinically established gold drug auranofin (1-Thio-ß-D-glucopyranosatotriethylphosphine gold-2,3,4,6-tetraacetate), was prepared and characterized. All these compounds feature the replacement of the triethylphosphine ligand of the parent compound auranofin with a trimethylphosphite ligand. The linear coordination around the gold(I) center is completed by Cl-, Br-, I- or by the thioglucose tetraacetate ligand (SAtg). The in-solution behavior of these gold compounds as well as their interactions with some representative model proteins were comparatively analyzed through 31PNMR and ESI-MS measurements. Notably, all panel compounds turned out to be stable in aqueous media, but significant differences with respect to auranofin were disclosed in their interactions with a few leading proteins. In addition, the cytotoxic effects produced by the panel compounds toward A2780, A2780R and SKOV-3 ovarian cancer cells were quantitated and found to be in the low micromolar range, since the IC50 of all compounds was found to be between 1 µM and 10 µM. Notably, these novel gold complexes showed large and similar inhibition capabilities towards the key enzyme thioredoxin reductase, again comparable to those of auranofin. The implications of these results for the discovery of new and effective gold-based anticancer agents are discussed.

Auranofin attenuates Schistosoma mansoni egg-induced liver granuloma and fibrosis in mice.

Schistosomiasis is a serious tropical disease. Despite extensive research into the etiology of liver fibrosis, effective therapeutic options remain limited. This study aims to assess the effectiveness of auranofin in treating hepatic granuloma and fibrogenesis produced by Schistosoma (S.) mansoni eggs. Auranofin is a gold complex that contains thioglucose tetraacetate and triethylphosphine. Eighty BALB/c male mice were divided into four groups (n=20/group): negative control (GI), positive control (GII), and early (GIII) and late (GIV) treatment groups with oral auranofin according to beginning of treatment 4th week and 6th week post-infection. Mice were infected subcutaneously in a dose of 60±10 cercariae/mouse. Worm counts, egg loads, and oogram patterns were determined. Biochemical, histological, and immunostaining of interleukin-1ß (IL-1ß), Sirtuin 3 (SIRT3), and smooth muscle actin (SMA) were assessed. GIII showed a significant decrease in the total S. mansoni worm burden and ova/gram in liver tissue (with reduction percent of 63.07% and 78.26%, respectively). Schistosomal oogram patterns, immature and mature ova, also showed a significant decrease. The reduction in granuloma number and size was 40.63% and 48.66%, respectively, in GIII, whereas in GIV, the reduction percent was 76.63% and 67.08%. In addition, the degree of fibrosis was significantly diminished in both treated groups. GIV showed significant reduction in IL-1ß and SMA expression and increase in SIRT3 expression. These findings reveal how auranofin suppresses the development of liver fibrosis. Therefore, it is crucial to take another look at auranofin as a prospective medication for the treatment of S. mansoni egg-induced hepatic granuloma and consequent fibrosis.

Upgrade of an old drug: Auranofin in innovative cancer therapies to overcome drug resistance and to increase drug effectiveness.

Auranofin is an oral gold(I) compound, initially developed for the treatment of rheumatoid arthritis. Currently, Auranofin is under investigation for oncological application within a drug repurposing plan due to the relevant antineoplastic activity observed both in vitro and in vivo tumor models. In this review, we analysed studies in which Auranofin was used as a single drug or in combination with other molecules to enhance their anticancer activity or to overcome chemoresistance. The analysis of different targets/pathways affected by this drug in different cancer types has allowed us to highlight several interesting targets and effects of Auranofin besides the already well-known inhibition of thioredoxin reductase. Among these targets, inhibitory-κB kinase, deubiquitinates, protein kinase C iota have been frequently suggested. To rationalize the effects of Auranofin by a system biology-like approach, we exploited transcriptomic data obtained from a wide range of cell models, extrapolating the data deposited in the Connectivity Maps website and we attempted to provide a general conclusion and discussed the major points that need further investigation.

Autophagic degradation of IRF3 induced by the small-molecule auranofin inhibits its transcriptional and proapoptotic activities.

The ubiquitously expressed transcription factor interferon (IFN) regulatory factor 3 (IRF3) is critical for the induction of antiviral genes, e.g., type-I IFN. In addition to its transcriptional function, IRF3 also activates a nontranscriptional, proapoptotic signaling pathway. While the proapoptotic function of IRF3 protects against viral infections, it is also involved in harmful immune responses that trigger hepatocyte cell death and promote liver disease. Thus, we hypothesized that a small-molecule inhibitor of the proapoptotic activity of IRF3 could alleviate fatty-acid-induced hepatocyte cell death. We conducted a high-throughput screen, which identified auranofin as a small-molecule inhibitor of the proapoptotic activity of IRF3. In addition to the nontranscriptional apoptotic pathway, auranofin also inhibited the transcriptional activity of IRF3. Using biochemical and genetic tools in human and mouse cells, we uncovered a novel mechanism of action for auranofin, in which it induces cellular autophagy to degrade IRF3 protein, thereby suppressing IRF3 functions. Autophagy-deficient cells were unable to degrade IRF3 upon auranofin treatment, suggesting that the autophagic degradation of IRF3 is a novel approach to regulate IRF3 activities. Using a physiologically relevant in vitro model, we demonstrated that auranofin inhibited fatty-acid-induced apoptotic cell death of hepatocytes. In summary, auranofin is a novel inhibitor of IRF3 functions and may represent a potential therapeutic option in diseases where IRF3 is deleterious.

Targeted inhibition of TXNRD1 prevents cartilage extracellular matrix degeneration by activating Nrf2 pathway in osteoarthritis.

Osteoarthritis, a prevalent orthopedic disease, can affect the elderly and causes impairment. The degradation and aberrant homeostasis of cartilage extracellular matrix figure pivotally in the progression of osteoarthritis. Thioredoxin systems plays a role in a wide range of biological processes, including cell proliferation, apoptosis, and oxidative stress. The present study aimed to investigate the unique function and underlying pathophysiological mechanism of TXNRD1 in chondrocytes. An upregulated expression of TXNRD1 was observed in the articular cartilage of osteoarthritis patients compared with normal articular cartilage. Furthermore, in vitro experiments showed that the expression of TXNRD1 was also abnormally increased in IL-1ß-induced primary mouse chondrocytes. Silencing TXNRD1 using siRNA in chondrocytes could effectively inhibit the expression of ADAMTS5 and MMP13, and enhance the expression of COL2A1 and SOX9. The same was true for auranofin, an inhibitor of TXNRD1. This phenomenon indicated that inhibition of TXNRD1 attenuated il-1ß-induced metabolic imbalance of extracellular matrix (ECM) and the progression of chondrocyte osteoarthritis. Further mechanism analysis revealed that the activation of Nrf2 signaling pathway and the expression of heme oxygenase-1 (HO-1) were increased upon TXNRD1 inhibition. Furthermore, auranofin was found to attenuate DMM-induced osteoarthritis progression in vivo. Therefore, the pharmacological downregulation of TXNRD1 may provide an effective novel therapy for OA.

Revisiting the anticancer properties of phosphane(9-ribosylpurine-6-thiolato)gold(I) complexes and their 9H-purine precursors.

New mono- and di-nuclear thio-purine and thio-purine nucleoside gold(I) complexes were synthesized, characterized, and evaluated in vitro for biological activities in comparison to related known purine complexes. By combining known anti-tumoral thio-purines with R3PAu moieties as present in auranofin, complexes with enhanced effects and selectivities were obtained, which not only act as cytostatics, but also disrupt tumor-specific processes. Their IC50 values in cytotoxicity test with tumor cell lines ranged from three-digit nanomolar to single-digit micromolar, revealing a tentative structure-activity relationship (SAR). Both the residues R2 of the phosphane ligand and R1 at C2 of the pyrimidine ring had a significant impact on the cytotoxicity. In most cases, the introduction of a ribo-furanosyl group at N9 of the purine led to a distinctly more cytotoxic complex. Most complexes were more active against multi-drug-resistant tumor cells or such lacking functional p53 when compared to the respective untreated wild type cell lines. Some nucleoside complexes displayed an interesting dose-dependent dual mode of action regarding cell cycle arrest and DNA repair mechanism. Some phosphane(purine-6-thiolato)gold (I) complexes had a stronger inhibitory effect on the thioredoxin reductase (TrxR) and on the reactive oxygen species (ROS) generation in cancer cells than is typical of other gold complexes. They also led to DNA fragmentation and showed anti-angiogenic effects. Their stability under test conditions was demonstrated by 77Se NMR monitoring of an exemplary selenopurine complex.

Auranofin promotes antibacterial effect of doripenem against carbapenem-resistant Acinetobacter baumannii.

AIMS: This study was performed to identify the potential for repurposing auranofin as an antibiotic adjuvant against carbapenemase-producing Acinetobacter baumannii. METHODS AND RESULTS: The clinically isolated A. baumannii strains used in this study were all resistant to carbapenems and harboured the blaOXA-23 gene. The synergistic effect of auranofin and doripenem against carbapenemase-producing A. baumannii was confirmed through checkerboard and growth kinetic analyses. This study also demonstrated the inhibitory effects of auranofin against A. baumannii biofilms. The anti-biofilm effects of auranofin were visualized by confocal laser scanning microscopy (CLSM). Furthermore, auranofin inhibited motility, one of the virulence factors. Additionally, the changes in the expression of carbapenemase-, biofilm- and efflux pump-related genes induced by auranofin were confirmed via quantitative polymerase chain reaction (qPCR). CONCLUSIONS: Our results demonstrated that auranofin has an antibacterial effect with doripenem and an inhibitory effect on several factors related to carbapenem resistance. SIGNIFICANCE AND IMPACT OF THE STUDY: This study suggests that auranofin is a promising antibiotic adjuvant that can be used to prevent antibiotic resistance in carbapenem-resistant A. baumannii.

Combinatorial Approaches to Enhance DNA Damage following Enzyme-Mediated Depletion of L-Cys for Treatment of Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) represents one of the deadliest forms of cancer with very few available therapeutic options. We previously reported that an engineered human enzyme, cyst(e)inase, which degrades L-cysteine (L-Cys) and cystine, inhibits growth of multiple cancer cells, including PDAC both in vitro and in vivo. Here, we show that cyst(e)inase treatment leads to increased clustered oxidative DNA damage, DNA single-strand breaks, apurinic/apyrimidinic sites, and DNA double-strand breaks (DSBs) in PDAC cells sensitive to intracellular depletion of L-Cys that is associated with reduced survival. BRCA2-deficient PDAC cells exhibited increased DSBs and enhanced sensitivity to cyst(e)inase. The blocking of a second antioxidant pathway (thioredoxin/thioredoxin reductase) using auranofin or inhibiting DNA repair using the poly (ADP-ribose) polymerase (PARP) inhibitor, olaparib, led to significant increases in DSBs following cyst(e)inase treatment in all PDAC cells examined. Cyst(e)inase plus olaparib also synergistically inhibited growth of sensitive and resistant PDAC cells in both xenograft and allograft tumor models. Collectively, these results demonstrate an important role for oxidative DNA damage and ultimately DNA DSBs in the anticancer action of cyst(e)inase. The data further show the potential for combining agents that target alternate antioxidant pathways or by targeting DNA repair pathways or genetic liabilities in DNA repair pathways to enhance the therapeutic action of cyst(e)inase for PDAC.

Menadione Potentiates Auranofin-Induced Glioblastoma Cell Death.

Glioblastoma (GBM) is the most aggressive primary brain tumor. Recently, agents increasing the level of oxidative stress have been proposed as anticancer drugs. However, their efficacy may be lowered by the cytoprotective activity of antioxidant enzymes, often upregulated in neoplastic cells. Here, we assessed the mRNA and protein expression of thioredoxin reductase 1 (TrxR1), a master regulator of cellular redox homeostasis, in GBM and non-tumor brain tissues. Next, we examined the influence of an inhibitor of TrxR1, auranofin (AF), alone or in combination with a prooxidant menadione (MEN), on growth of GBM cell lines, patient-derived GBM cells and normal human astrocytes. We detected considerable amount of TrxR1 in the majority of GBM tissues. Treatment with AF decreased viability of GBM cells and their potential to form colonies and neurospheres. Moreover, it increased the intracellular level of reactive oxygen species (ROS). Pre-treatment with ROS scavenger prevented the AF-induced cell death, pointing to the important role of ROS in the reduction of cell viability. The cytotoxic effect of AF was potentiated by treatment with MEN. In conclusion, our results identify TrxR1 as an attractive drug target and highlights AF as an off-patent drug candidate in GBM therapy.

Anti-Cancer Effects of Auranofin in Human Lung Cancer Cells by Increasing Intracellular ROS Levels and Depleting GSH Levels.

Auranofin, as a thioredoxin reductase (TrxR) inhibitor, has promising anti-cancer activity in several cancer types. However, little is known about the inhibitory effect of auranofin on lung cancer cell growth. We, therefore, investigated the antigrowth effects of auranofin in various lung cancer cells with respect to cell death, reactive oxygen species (ROS), and glutathione (GSH) levels. Treatment with 0~5 µM auranofin decreased cell proliferation and induced cell death in Calu-6, A549, SK-LU-1, NCI-H460, and NCI-H1299 lung cancer cells at 24 h. In addition, 0~5 µM auranofin increased ROS levels, including O2•-, and depleted GSH levels in these cells. N-acetyl cysteine (NAC) prevented growth inhibition and mitochondrial membrane potential (MMP, ∆Ψm) loss in 3 and 5 µM auranofin-treated Calu-6 and A549 cells at 24 h, respectively, and decreased ROS levels and GSH depletion in these cells. In contrast, L-buthionine sulfoximine (BSO) enhanced cell death, MMP (∆Ψm) loss, ROS levels, and GSH depletion in auranofin-treated Calu-6 and A549 cells. Treatment with 3 and 5 µM auranofin induced caspase-3 activation and poly (ADP ribose) polymerase (PARP) cleavage in Calu-6 and A549 cells, respectively. Both were prevented by NAC, but enhanced by BSO. Moreover, TrxR activity was reduced in auranofin-treated Calu-6 and A549 cells. That activity was decreased by BSO, but increased by NAC. In conclusion, these findings demonstrate that auranofin-induced cell death is closely related to oxidative stress resulted from increased ROS levels and GSH depletion in lung cancer cells.

Anti-Staphylococcal Activity of the Auranofin Analogue Bearing Acetylcysteine in Place of the Thiosugar: An Experimental and Theoretical Investigation.

Auranofin (AF, hereafter) is an orally administered chrysotherapeutic agent approved for the treatment of rheumatoid arthritis that is being repurposed for various indications including bacterial infections. Its likely mode of action involves the impairment of the TrxR system through the binding of the pharmacophoric cation [AuPEt3]+. Accordingly, a reliable strategy to expand the medicinal profile of AF is the replacement of the thiosugar moiety with different ligands. Herein, we aimed to prepare the AF analogue bearing the acetylcysteine ligand (AF-AcCys, hereafter) and characterize its anti-staphylococcal activity. Biological studies revealed that AF-AcCys retains an antibacterial effect superimposable with that of AF against Staphylococcus aureus, whereas it is about 20 times less effective against Staphylococcus epidermidis. Bioinorganic studies confirmed that upon incubation with human serum albumin, AF-AcCys, similarly to AF, induced protein metalation through the [AuPEt3]+ fragment. Additionally, AF-AcCys appeared capable of binding the dodecapeptide Ac-SGGDILQSGCUG-NH2, corresponding to the tryptic C-terminal fragment (488-499) of hTrxR. To shed light on the pharmacological differences between AF and AF-AcCys, we carried out a comparative experimental stability study and a theoretical estimation of bond dissociation energies, unveiling the higher strength of the Au-S bond in AF-AcCys. From the results, it emerged that the lower lipophilicity of AF-AcCys with respect to AF could be a key feature for its different antibacterial activity. The differences and similarities between AF and AF-AcCys are discussed, alongside the opportunities and consequences that chemical structure modifications imply.

Human pancreatic cancer cells under nutrient deprivation are vulnerable to redox system inhibition.

Large regions in tumor tissues, particularly pancreatic cancer, are hypoxic and nutrient-deprived because of unregulated cell growth and insufficient vascular supply. Certain cancer cells, such as those inside a tumor, can tolerate these severe conditions and survive for prolonged periods. We hypothesized that small molecular agents, which can preferentially reduce cancer cell survival under nutrient-deprived conditions, could function as anticancer drugs. In this study, we constructed a high-throughput screening system to identify such small molecules and screened chemical libraries and microbial culture extracts. We were able to determine that some small molecular compounds, such as penicillic acid, papyracillic acid, and auranofin, exhibit preferential cytotoxicity to human pancreatic cancer cells under nutrient-deprived compared with nutrient-sufficient conditions. Further analysis revealed that these compounds target to redox systems such as GSH and thioredoxin and induce accumulation of reactive oxygen species in nutrient-deprived cancer cells, potentially contributing to apoptosis under nutrient-deprived conditions. Nutrient-deficient cancer cells are often deficient in GSH; thus, they are susceptible to redox system inhibitors. Targeting redox systems might be an attractive therapeutic strategy under nutrient-deprived conditions of the tumor microenvironment.

Methionine restriction exposes a targetable redox vulnerability of triple-negative breast cancer cells by inducing thioredoxin reductase.

PURPOSE: Tumor cells are dependent on the glutathione and thioredoxin antioxidant pathways to survive oxidative stress. Since the essential amino acid methionine is converted to glutathione, we hypothesized that methionine restriction (MR) would deplete glutathione and render tumors dependent on the thioredoxin pathway and its rate-limiting enzyme thioredoxin reductase (TXNRD). METHODS: Triple (ER/PR/HER2)-negative breast cancer (TNBC) cells were treated with control or MR media and the effects on reactive oxygen species (ROS) and antioxidant signaling were examined. To determine the role of TXNRD in MR-induced cell death, TXNRD1 was inhibited by RNAi or the pan-TXNRD inhibitor auranofin, an antirheumatic agent. Metastatic and PDX TNBC mouse models were utilized to evaluate in vivo antitumor activity. RESULTS: MR rapidly and transiently increased ROS, depleted glutathione, and decreased the ratio of reduced glutathione/oxidized glutathione in TNBC cells. TXNRD1 mRNA and protein levels were induced by MR via a ROS-dependent mechanism mediated by the transcriptional regulators NRF2 and ATF4. MR dramatically sensitized TNBC cells to TXNRD1 silencing and the TXNRD inhibitor auranofin, as determined by crystal violet staining and caspase activity; these effects were suppressed by the antioxidant N-acetylcysteine. H-Ras-transformed MCF-10A cells, but not untransformed MCF-10A cells, were highly sensitive to the combination of auranofin and MR. Furthermore, dietary MR induced TXNRD1 expression in mammary tumors and enhanced the antitumor effects of auranofin in metastatic and PDX TNBC murine models. CONCLUSION: MR exposes a vulnerability of TNBC cells to the TXNRD inhibitor auranofin by increasing expression of its molecular target and creating a dependency on the thioredoxin pathway.

Formation of oxidised (OX) proteins in Entamoeba histolytica exposed to auranofin and consequences on the parasite virulence.

Metronidazole (MNZ), the first line drug for amoebiasis and auranofin (AF), an emerging antiprotozoan drug, are both inhibiting Entamoeba histolytica thioredoxin reductase. The nature of oxidised proteins (OXs) formed in AF- or MNZ-treated E. histolytica trophozoites is unknown. In order to fill this knowledge gap, we performed a large-scale identification and quantification of the OXs formed in AF- or MNZ-treated E. histolytica trophozoites using resin-assisted capture coupled to mass spectrometry (MS). We detected 661 OXs in MNZ-treated trophozoites and 583 OXs in AF-treated trophozoites. More than 50% of these OXs were shared, and their functions include hydrolases, enzyme modulators, transferases, nucleic acid binding proteins, oxidoreductases, cytoskeletal proteins, chaperones, and ligases. Here, we report that the formation of actin filaments (F-actin) is impaired in AF-treated trophozoites. Consequently, their erythrophagocytosis, cytopathic activity, and their motility are impaired. We also observed that less than 15% of OXs present in H2 O2 -treated trophozoites are also present in AF- or MNZ-treated trophozoites. These results strongly suggest that the formation of OXs in AF- or MNZ-treated trophozoites and in H2 O2 -treated trophozoites occurred by two different mechanisms.