Gold(I) somplexes of the type [AuL{κC-2-C6H4P(S)Ph2}] [L = PTA, PPh3, PPh2(C6H4-3-SO3Na) and PPh2(2-py)]: Synthesis, characterisation, crystal structures, and In Vitro and In Vivo anticancer properties.

Four new mononuclear gold (I) compounds of the type [AuL{κC-2-C6H4P(S)Ph2}] {L = PTA (1), PPh3 (2), PPh2(C6H4-3-SO3Na) (3), and PPh2(2-py) (4)} were prepared by scission of the dinuclear compound [Au2{µ-2-C6H4P(S)Ph2}2] by L or via a transmetalation reaction using the organotin reagent 2-Me3SnC6H4P(S)Ph2 and a suitable gold halide precursor. The cytotoxic potential of complexes 1-4 was evaluated against four human cancer cell lines of diverse cellular origin: cervical (HeLa), prostate (PC-3), non-small cell lung adenocarcinoma (A549), and fibrosarcoma (HT-1080). The in vitro cytotoxicity results showed that 1 demonstrated exceptional anticancer activity with IC50 values ranging from 0.08 to 3.5 µM. Complex 3, which contains a sulfonated triphenyl phosphine ligand, displayed the weakest anticancer activity with IC50 values ranging from 3.1 to >50 µM. When compared to the standard chemotherapeutic drug cisplatin, 1 displayed approximately 27-fold greater cytotoxic activity against cervical cancer cells and 3.5- and 7.5-fold greater activities against prostate and fibrosarcoma cancer cells, respectively. Additionally, 1 exhibited 3-fold selectivity for cervical cancer cells compared to non-cancerous HEK-293 cells. Mechanistic investigations revealed that 1 induced apoptosis, which was associated with elevated reactive oxygen species (ROS) and inhibition of the intracellular enzyme thioredoxin reductase. Furthermore, 1 exhibited notable antiangiogenic characteristics in an in vivo model using transgenic zebrafish Tg(fli1a:EGFP). In vivo studies using mouse xenograft models showed that complex 1 displayed superior inhibition of tumour growth (82 %) compared to the clinical drug cisplatin (29 %). Overall, these results highlight the potential of gold (I) compounds as novel antitumour agents.

Thioredoxin System in Insects: Uncovering the Roles of Thioredoxins and Thioredoxin Reductase beyond the Antioxidant Defences.

Increased levels of reactive oxygen species (ROS) produced during aerobic metabolism in animals can negatively affect the intracellular redox status, cause oxidative stress and interfere with physiological processes in the cells. The antioxidant defence regulates ROS levels by interplaying diverse enzymes and non-enzymatic metabolites. The thioredoxin system, consisting of the enzyme thioredoxin reductase (TrxR), the redox-active protein thioredoxin (Trx) and NADPH, represent a crucial component of antioxidant defence. It is involved in the signalling and regulation of multiple developmental processes, such as cell proliferation or apoptotic death. Insects have evolved unique variations of TrxR, which resemble mammalian enzymes in overall structure and catalytic mechanisms, but the selenocysteine-cysteine pair in the active site is replaced by a cysteine-cysteine pair typical of bacteria. Moreover, the role of the thioredoxin system in insects is indispensable due to the absence of glutathione reductase, an essential enzyme of the glutathione system. However, the functions of the Trx system in insects are still poorly characterised. In the present review, we provide a critical overview of the current knowledge on the insect Trx system, focusing mainly on TrxR's role in the antioxidant and immune system of model insect species.

Thioredoxin reductase inhibition and glutathione depletion mediated by glaucocalyxin A promote intracellular disulfide stress in gastric cancer cells.

Thioredoxin reductase 1 (TXNRD1) has been identified as one of the promising chemotherapeutic targets in cancer cells. Therefore, a novel TXNRD1 inhibitor could accelerate chemotherapy in clinical anticancer research. In this study, glaucocalyxin A (GlauA), a natural diterpene extracted from Rabdosia japonica var. glaucocalyx, was identified as a novel inhibitor of TXNRD1. We found that GlauA effectively inhibited recombinant TXNRD1 and reduced its activity in gastric cancer cells without affecting the enzyme's expression level. Mechanistically, the selenocysteine residue (U498) of TXNRD1 was irreversibly modified by GlauA through a Michael addition. Additionally, GlauA formed a covalent adduct with glutathione (GSH) and disrupted cellular redox balance by depleting cellular GSH. The inhibition of TXNRD1 and depletion of GSH by GlauA conferred its cytotoxic effects in spheroid culture and Transwell assays in AGS cells. The disulfide stress induced cytotoxicity of GlauA could be mitigated by adding reducing agents, such as DTT and ß-ME. Furthermore, the FDA-approval drug auranofin, a TXNRD1 inhibitor, triggered oligomerization of the cytoskeletal protein Talin-1 in AGS cells, indicating that inhibiting TXNRD1 triggered disulfide stress. In conclusion, this study uncovered GlauA as an efficient inhibitor of TXNRD1 and demonstrated the potential of TXNRD1 inhibition as an effective anticancer strategy by disrupting redox homeostasis and inducing disulfide stress.

Characterization of thioredoxin-thioredoxin reductase system in Filifactor alocis.

INTRODUCTION: Filifactor alocis is a newly appreciated member of the periodontal community with a strong periodontal disease correlation. Little is known about the survival mechanisms by which F. alocis copes with oxidative stress and establishes the infection within the local inflammatory microenvironment of the periodontal pocket. The aim of this study is to investigate if F. alocis putative peroxiredoxin/AhpC protein FA768 may constitute an alkyl hydroperoxide reductase system utilizing putative thioredoxin reductase protein FA608, and putative thioredoxin/glutaredoxin homolog FA1411/FA455. METHODS: FA768, FA608, FA1411 and FA455 proteins from F. alocis were expressed and purified from Escherichia coli. Insulin and 5,5-dithio-bis-2-nitrobenzoic acid (DTNB) reduction assays were performed to determine if purified FA1411 and FA455 proteins could be a substrate for FA608. The peroxidase activity of FA768 was examined by measuring its ability to reduce hydrogen peroxide (H2O2) with FA608 and FA1411/FA455 provided as the reducing systems. Further, the hydroperoxide substrate specificity of FA768 was analyzed by monitoring the NADPH oxidation in the presence of different peroxides, including H2O2, cumyl hydroperoxide (CHP), and tert-butyl hydroperoxide (t-BHP). RESULTS: In this study, we have demonstrated the existence of a functioning thioredoxin-dependent alkyl hydroperoxide system in F. alocis. This system is comprised of a thioredoxin reductase (FA608), a thioredoxin/glutaredoxin homolog (FA1411/FA455), and a typical 2-cysteine peroxiredoxin/AhpC (FA768). FA608, together with FA1411/FA455, can function as a thioredoxin reductase system to reduce insulin, DTNB, and FA768. FA455 is a glutaredoxin-like protein with thioredoxin functions in F. alocis. Both the FA768/FA608/FA1411 and FA768/FA608/FA455 reductase systems were NADPH-dependent and exhibited specificity for broad hydroperoxide substrates H2O2, CHP, and t-BHP. CONCLUSIONS: This is the first study of a thioredoxin dependent alkyl hydroperoxide system from a periodontal pathogen. This system is proposed to protect F. alocis against oxidative stress due to the likely absence of a catalase or an additional peroxiredoxin homolog.

Synergistic Dual Targeting of Thioredoxin and Glutathione Systems Irrespective of p53 in Glioblastoma Stem Cells.

Glioblastoma (GBM) is an incurable primary brain cancer characterized by increased reactive oxygen species (ROS) production. The redox-sensitive tumor suppressor gene TP53, wild-type (wt) for 70% of patients, regulates redox homeostasis. Glioblastoma stem cells (GSCs) increase thioredoxin (Trx) and glutathione (GSH) antioxidant systems as survival redox-adaptive mechanisms to maintain ROS below the cytotoxic threshold. Auranofin, an FDA-approved anti-rheumatoid drug, inhibits thioredoxin reductase 1 (TrxR1). L-buthionine sulfoximine (L-BSO) and the natural product piperlongumine (PPL) inhibit the GSH system. We evaluated the cytotoxic effects of Auranofin alone and in combination with L-BSO or PPL in GBM cell lines and GSCs with a known TP53 status. The Cancer Genome Atlas/GBM analysis revealed a significant positive correlation between wtp53 and TrxR1 expression in GBM. Auranofin induced ROS-dependent cytotoxicity within a micromolar range in GSCs. Auranofin decreased TrxR1 expression, AKT (Ser-473) phosphorylation, and increased p53, p21, and PARP-1 apoptotic cleavage in wtp53-GSCs, while mutant-p53 was decreased in a mutant-p53 GSC line. Additionally, p53-knockdown in a wtp53-GSC line decreased TrxR1 expression and significantly increased sensitivity to Auranofin, suggesting the role of wtp53 as a negative redox-sensitive mechanism in response to Auranofin in GSCs. The combination of Auranofin and L-BSO synergistically increased ROS, decreased IC50s, and induced long-term cytotoxicity irrespective of p53 in GBM cell lines and GSCs. Intriguingly, Auranofin increased the expression of glutathione S-transferase pi-1 (GSTP-1), a target of PPL. Combining Auranofin with PPL synergistically decreased IC50s to a nanomolar range in GSCs, supporting the potential to repurpose Auranofin and PPL in GBM.

NRF2 and Thioredoxin Reductase 1 as Modulators of Interactions between Zinc and Selenium.

BACKGROUND: Selenium and zinc are essential trace elements known to regulate cellular processes including redox homeostasis. During inflammation, circulating selenium and zinc concentrations are reduced in parallel, but underlying mechanisms are unknown. Accordingly, we modulated the zinc and selenium supply of HepG2 cells to study their relationship. METHODS: HepG2 cells were supplied with selenite in combination with a short- or long-term zinc treatment to investigate intracellular concentrations of selenium and zinc together with biomarkers describing their status. In addition, the activation of the redox-sensitive transcription factor NRF2 was analyzed. RESULTS: Zinc not only increased the nuclear translocation of NRF2 after 2 to 6 h but also enhanced the intracellular selenium content after 72 h, when the cells were exposed to both trace elements. In parallel, the activity and expression of the selenoprotein thioredoxin reductase 1 (TXNRD1) increased, while the gene expression of other selenoproteins remained unaffected or was even downregulated. The zinc effects on the selenium concentration and TXNRD activity were reduced in cells with stable NRF2 knockdown in comparison to control cells. CONCLUSIONS: This indicates a functional role of NRF2 in mediating the zinc/selenium crosstalk and provides an explanation for the observed unidirectional behavior of selenium and zinc.

Comparison of the mechanism of antimicrobial action of the gold(I) compound auranofin in Gram-positive and Gram-negative bacteria.

While highly effective at killing Gram-positive bacteria, auranofin lacks significant activity against Gram-negative species for reasons that largely remain unclear. Here, we aimed to elucidate the molecular mechanisms underlying the low susceptibility of the Gram-negative model organism Escherichia coli to auranofin when compared to the Gram-positive model organism Bacillus subtilis. The proteome response of E. coli exposed to auranofin suggests a combination of inactivation of thiol-containing enzymes and the induction of systemic oxidative stress. Susceptibility tests in E. coli mutants lacking proteins upregulated upon auranofin treatment suggested that none of them are directly involved in E. coli's high tolerance to auranofin. E. coli cells lacking the efflux pump component TolC were more sensitive to auranofin treatment, but not to an extent that would fully explain the observed difference in susceptibility of Gram-positive and Gram-negative organisms. We thus tested whether E. coli's thioredoxin reductase (TrxB) is inherently less sensitive to auranofin than TrxB from B. subtilis, which was not the case. However, E. coli strains lacking the low-molecular-weight thiol glutathione, but not glutathione reductase, showed a high susceptibility to auranofin. Bacterial cells expressing the genetically encoded redox probe roGFP2 allowed us to observe the oxidation of cellular protein thiols in situ. Based on our findings, we hypothesize that auranofin leads to a global disturbance in the cellular thiol redox homeostasis in bacteria, but Gram-negative bacteria are inherently more resistant due to the presence of drug export systems and high cellular concentrations of glutathione.IMPORTANCEAuranofin is an FDA-approved drug for the treatment of rheumatoid arthritis. However, it has also high antibacterial activity, in particular against Gram-positive organisms. In the current antibiotics crisis, this would make it an ideal candidate for drug repurposing. However, its much lower activity against Gram-negative organisms prevents its broad-spectrum application. Here we show that, on the level of the presumed target, there is no difference in susceptibility between Gram-negative and Gram-positive species: thioredoxin reductases from both Escherichia coli and Bacillus subtilis are equally inhibited by auranofin. In both species, auranofin treatment leads to oxidative protein modification on a systemic level, as monitored by proteomics and the genetically encoded redox probe roGFP2. The single largest contributor to E. coli's relative resistance to auranofin seems to be the low-molecular-weight thiol glutathione, which is absent in B. subtilis and other Gram-positive species.

Anticancer Activity of Imidazolyl Gold(I/III) Compounds in Non-Small Cell Lung Cancer Cell Lines.

Lung cancer is a leading cause of cancer-related death worldwide that needs updated therapies to contrast both the serious side effects and the occurrence of drug resistance. A panel of non-small cell lung cancer (NSCLC) cells were herein employed as cancer models. Eight structurally related gold(I) and gold(III) complexes with NHC and halides or triphenylphosphane ligands were investigated as lung cancer cell growth inhibitors. As expected, gold compounds with PPh3 were found to be more cytotoxic than homoleptic [(NHC)2-Au(I)]X or heteroleptic NHC-Au(I)X or NHC-Au(III)X3 complexes. Mixed ligand gold(I) compounds exhibiting the linear NHC-AuPPh3 (compound 7) or the trigonal NHC-Au(Cl)PPh3 (compound 8) arrangements at the central metal were found to be the best lung cancer cytotoxic compounds. Analysis of the TrxR residual activity of the treated cells revealed that these compounds efficiently inhibit the most accredited molecular target for gold compounds, the TrxR, with compound 8 reaching more than 80% activity reduction in lung cells. Some of the current cancer lung therapy protocols consist of specific lung cancer cell cytotoxic agents combined with antifolate drugs; interestingly, the herein gold compounds are both TrxR and antifolate inhibitors. The human DHFR was inhibited with IC50 ranging between 10-21 µM, depending on substrate concentrations, proceeding by a likely allosteric mechanism only for compound 8.

Exploring the Thioredoxin System as a Therapeutic Target in Cancer: Mechanisms and Implications.

Cells constantly face the challenge of managing oxidants. In aerobic organisms, oxygen (O2) is used for energy production, generating reactive oxygen species (ROS) as byproducts of enzymatic reactions. To protect against oxidative damage, cells possess an intricate system of redox scavengers and antioxidant enzymes, collectively forming the antioxidant defense system. This system maintains the redox equilibrium and enables the generation of localized oxidative signals that regulate essential cellular functions. One key component of this defense is the thioredoxin (Trx) system, which includes Trx, thioredoxin reductase (TrxR), and NADPH. The Trx system reverses oxidation of macromolecules and indirectly neutralizes ROS via peroxiredoxin (Prx). This dual function protects cells from damage accumulation and supports physiological cell signaling. However, the Trx system also shields tumors from oxidative damage, aiding their survival. Due to elevated ROS levels from their metabolism, tumors often rely on the Trx system. In addition, the Trx system regulates critical pathways such as proliferation and neoangiogenesis, which tumors exploit to enhance growth and optimize nutrient and oxygen supply. Consequently, the Trx system is a potential target for cancer therapy. The challenge lies in selectively targeting malignant cells without disrupting the redox equilibrium in healthy cells. The aim of this review article is threefold: first, to elucidate the function of the Trx system; second, to discuss the Trx system as a potential target for cancer therapies; and third, to present the possibilities for inhibiting key components of the Trx system, along with an overview of the latest clinical studies on these inhibitors.

Endogenous electrophiles and peroxymonocarbonate can link tyrosine phosphorylation cascades with the cytosolic TXNRD1 selenoprotein and the KEAP1/NRF2 system.

Endogenously formed reactive molecules, such as lipid peroxides, 4-hydroxynonenal, methylglyoxal and other reactive oxygen species, can have major effects on cells. Accumulation of these molecules is counteracted by antioxidant enzymes, including the glutathione (GSH) and thioredoxin (Trx) systems, in turn regulated by the KEAP1/NRF2 system. Receptor tyrosine kinases (RTK) and their counteracting protein tyrosine phosphatases (PTP) are also modulated through redox regulation of PTP activities. The cytosolic selenoprotein thioredoxin reductase (TXNRD1) is particularly prone to attack at its easily accessible catalytic selenocysteine (Sec) residue by reactive electrophilic compounds. Therefore, we here discuss how endogenously formed electrophiles can modulate RTK/PTP signaling in a concentration- and time dependent manner by reactions either directly or indirectly linking TXNRD1 with the KEAP1/NRF2 system. Moreover, recent findings suggest that endogenous formation of peroxymonocarbonate can efficiently inhibit PTP activities and stimulate RTK signaling, seemingly bypassing PTP reduction as otherwise supported by the GSH/Trx systems.

Design, synthesis and biological evaluation of novel curcumin-fluorouracil hybrids as potential anti-cancer agents.

The latest global cancer data statistics report shows that cancer poses a serious threat to human life and health; The number of new cancer and death cases worldwide is severe. Molecular hybridization is considered an effective strategy for developing new anti-cancer drugs. Curcumin (Cur) is a natural active compound containing Michael receptors that target thioredoxin reductase (TrxR). Fluorouracil (5-FU) is the first anti-metabolic drug synthesized based on certain assumptions for tumor treatment, acting on thymidylate synthase (TS). This study synthesized a series of novel hybrid derivatives of Cur and 5-FU, and evaluated their anti-tumor cell proliferation effects. Several compounds with good cytotoxic activity against tumor cells were discovered; and they exhibited high selectivity towards A549 cells, compared to normal THLE cells. Among them, the hybrid derivative F-4 has the best anti-proliferative activity in tumor cells. F-4 can target TrxR, increase reactive oxygen species levels in tumor cells, and lead to tumor cell apoptosis, which may be related to the Michael receptor structure in the chemical structure of F-4; F-4 can also target TS, leading to cell cycle arrest in G0/G1 phase, which may be related to the 5-FU structure in the chemical structure of F-4. Moreover, F-4 can effectively exert anti-tumor activity in mice, significantly reduce tumor volume and weight, and has low toxic side effects. These results indicate that Cur-5-FU hybrid derivative F-4 is a novel lead compound with in vivo anti-tumor activity and minimal side effects, which deserves further investigation.

Evaluating the amoeba thioredoxin reductase selenoprotein as potential drug target for treatment of Acanthamoeba infections.

The genus Acanthamoeba comprises facultative pathogens, causing Acanthamoeba keratitis (AK) and granulomatous amoebic encephalitis (GAE). In both diseases, treatment options are limited, and drug development is challenging. This study aimed to investigate the role of the large thioredoxin reductase selenoprotein of Acanthamoeba (AcTrxR-L) as a potential drug target assessing the effects of the thioredoxin reductase inhibitors auranofin, TRi-1, and TRi-2 on AcTrxR-L activity and on the viability of Acanthamoeba trophozoites. Recombinant expression and purification of AcTrxR-L as a selenoprotein allowed assessments of its enzymatic activity, with reduction of various substrates, including different thioredoxin isoforms. Auranofin demonstrated potent inhibition towards AcTrxR-L, followed by TRi-1, and TRi-2 exhibiting lower effectiveness. The inhibitors showed variable activity against trophozoites in culture, with TRi-1 and TRi-2 resulting in strongly impaired trophozoite viability. Cytotoxicity tests with human corneal epithelial cells revealed lower susceptibility to all compounds compared to Acanthamoeba trophozoites, underscoring their potential as future amoebicidal agents. Altogether, this study highlights the druggability of AcTrxR-L and suggests it to be a promising drug target for the treatment of Acanthamoeba infections. Further research is warranted to elucidate the role of AcTrxR-L in Acanthamoeba pathogenesis and to develop effective therapeutic strategies targeting this redox enzyme.

Unveiling the cytotoxicity of a new gold(I) complex towards hepatocellular carcinoma by inhibiting TrxR activity.

Hepatocellular carcinoma (HCC), the predominant type of liver cancer, is an aggressive malignancy with limited therapeutic options. In this study, we assess a collection of newly designed gold(I) phosphine complexes. Remarkably, the compound GC002 exhibits the greatest toxicity to HCC cells and outperforms established medications, such as sorafenib and auranofin, in terms of antitumor efficacy. GC002 triggers irreversible necroptosis in HCC cells by increasing the intracellular accumulation of reactive oxygen species (ROS). Mechanistically, GC002 significantly suppresses the activity of thioredoxin reductase (TrxR), which plays a crucial role in regulating redox homeostasis and is often overexpressed in HCC by binding directly to the enzyme. Our in vivo xenograft study confirms that GC002 possesses remarkable antitumor activity against HCC without severe side effects. These findings not only highlight the novel mechanism of controlling necroptosis via TrxR and ROS but also identify GC002 as a promising candidate for the further development of antitumor agents targeting HCC.

Enhance the Antimycobacterial Activity of Streptomycin with Ebselen as an Antibiotic Adjuvant Through Disrupting Redox Homeostasis.

Purpose: Tuberculosis (TB) remains a major health threat worldwide, and the spread of drug-resistant (DR) TB impedes the reduction of the global disease burden. Ebselen (EbSe) targets bacterial thioredoxin reductase (bTrxR) and causes an imbalance in the redox status of bacteria. Previous work has shown that the synergistic action of bTrxR and sensitization to common antibiotics by EbSe is a promising strategy for the treatment of DR pathogens. Thus, we aimed to evaluate whether EbSe could enhance anti-TB drugs against Mycobacterium marinum (M. marinum) which is genetically related to Mycobacterium tuberculosis (Mtb) and resistant to many antituberculosis drugs. Methods: Minimum inhibitory concentrations (MIC) of isoniazid (INH), rifampicin (RFP), and streptomycin (SM) against M. marinum were determined by microdilution. The Bliss Independence Model was used to determine the adjuvant effects of EbSe over the anti-TB drugs. Thioredoxin reductase activity was measured using the DTNB assay, and its effects on bacterial redox homeostasis were verified by the elevation of intracellular ROS levels and intracellular GSH levels. The adjuvant efficacy of EbSe as an anti-TB drug was further evaluated in a mouse model of M. marinum infection. Cytotoxicity was observed in the macrophage cells Raw264.7 and mice model. Results: The results reveal that EbSe acts as an antibiotic adjuvant over SM on M. marinum. EbSe + SM disrupted the intracellular redox microenvironment of M. marinum by inhibiting bTrxR activity, which could rescue mice from the high bacterial load, and accelerated recovery from tail injury with low mammalian toxicity. Conclusion: The above studies suggest that EbSe significantly enhanced the anti-Mtb effect of SM, and its synergistic combination showed low mammalian toxicity in vitro and in vivo. Further efforts are required to study the underlying mechanisms of EbSe as an antibiotic adjuvant in combination with anti-TB drug MS.

Unveiling the anticancer potential of Pestalotioprolide E, an unexplored macrolide: Targeting TRXR1-TRX1-ASK1-P38 signaling cascade in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is highly aggressive and metastatic in nature. Existing treatment modalities for TNBC are associated with severe side effects. Thioredoxin reductase (TRXR), the pivotal component of the thioredoxin system, remains overexpressed in various cancer cells including TNBC; promotes cell growth, proliferation, and metastasis, and inhibits apoptosis. Pestalotioprolide E is one of the potent macrolides, a class of secondary metabolites derived from an endophytic fungus Pestalotiopsis microspora with relatively unexplored biological activities. Our study revealed increased expression and activity of TRXR1 in MDA-MB-231 cells compared to the non-cancerous cells. In silico docking analysis and in vitro activity assay demonstrated that Pestalotioprolide E directly interacts with TRXR1 and inhibits its enzymatic activity. This inhibition induces apoptosis via TRX1/ASK1/P38MAPK death signaling cascade and retards metastasis through modulating VEGF, MMP-2, MMP-9, E-cadherin, N-cadherin in MDA-MB-231 cells. Taken together present study establishes TRXR1 as a molecular target for Pestalotioprolide E and its anticancer effect can be attributed to the inhibition of TRXR1 activity in MDA-MB-231.

Pharmacological Inhibition of TXNRD1 by a Small Molecule Flavonoid Butein Overcomes Cisplatin Resistance in Lung Cancer Cells.

Mammalian cytosolic selenoprotein thioredoxin reductase (TXNRD1) is crucial for maintaining the reduced state of cellular thioredoxin 1 (TXN1) and is commonly up-regulated in cancer cells. TXNRD1 has been identified as an effective target in cancer chemotherapy. Discovering novel TXNRD1 inhibitors and elucidating the cellular effects of TXNRD1 inhibition are valuable for developing targeted therapies based on redox regulation strategies. In this study, we demonstrated that butein, a plant-derived small molecule flavonoid, is a novel TXNRD1 inhibitor. We found that butein irreversibly inhibited recombinant TXNRD1 activity in a time-dependent manner. Using TXNRD1 mutant variants and LC-MS, we identified that butein modifies the catalytic cysteine (Cys) residues of TXNRD1. In cellular contexts, butein promoted the accumulation of reactive oxygen species (ROS) and exhibited cytotoxic effects in HeLa cells. Notably, we found that pharmacological inhibition of TXNRD1 by butein overcame the cisplatin resistance of A549 cisplatin-resistant cells, accompanied by increased cellular ROS levels and enhanced expression of p53. Taken together, the results of this study demonstrate that butein is an effective small molecule inhibitor of TXNRD1, highlighting the therapeutic potential of inhibiting TXNRD1 in platinum-resistant cancer cells.

Heteronuclear Complexes with Promising Anticancer Activity against Colon Cancer.

This study investigates the activity of novel gold(I) and copper(I)/zinc(II) heteronuclear complexes against colon cancer. The synthesised heteronuclear Au(I)-Cu(I) and Au(I)-Zn(II) complexes were characterised and evaluated for their anticancer activity using human colon cancer cell lines (Caco-2). The complexes exhibited potent cytotoxicity, with IC50 values in the low micromolar range, and effectively induced apoptosis in cancer cells. In the case of complex [Cu{Au(Spy)(PTA)}2]PF6 (2), its cytotoxicity is ×10 higher than its mononuclear precursor, while showing low cytotoxicity towards differentiated healthy cells. Mechanistic studies revealed that complex 2 inhibits the activity of thioredoxin reductase, a key enzyme involved in redox regulation, leading to an increase in reactive oxygen species (ROS) levels and oxidative stress, in addition to an alteration in DNA's tertiary structure. Furthermore, the complexes demonstrated a strong binding affinity to bovine serum albumin (BSA), suggesting the potential for effective drug delivery and bioavailability. Collectively, these findings highlight the potential of the investigated heteronuclear Au(I)-Cu(I) and Au(I)-Zn(II) complexes as promising anticancer agents, particularly against colon cancer, through their ability to disrupt redox homeostasis and induce oxidative stress-mediated cell death.

Pro-Oxidant Auranofin and Glutathione-Depleting Combination Unveils Synergistic Lethality in Glioblastoma Cells with Aberrant Epidermal Growth Factor Receptor Expression.

Glioblastoma (GBM) is the most prevalent and advanced malignant primary brain tumor in adults. GBM frequently harbors epidermal growth factor receptor (EGFR) wild-type (EGFRwt) gene amplification and/or EGFRvIII activating mutation. EGFR-driven GBM relies on the thioredoxin (Trx) and/or glutathione (GSH) antioxidant systems to withstand the excessive production of reactive oxygen species (ROS). The impact of EGFRwt or EGFRvIII overexpression on the response to a Trx/GSH co-targeting strategy is unknown. In this study, we investigated Trx/GSH co-targeting in the context of EGFR overexpression in GBM. Auranofin is a thioredoxin reductase (TrxR) inhibitor, FDA-approved for rheumatoid arthritis. L-buthionine-sulfoximine (L-BSO) inhibits GSH synthesis by targeting the glutamate-cysteine ligase catalytic (GCLC) enzyme subunit. We analyzed the mechanisms of cytotoxicity of auranofin and the interaction between auranofin and L-BSO in U87MG, U87/EGFRwt, and U87/EGFRvIII GBM isogenic GBM cell lines. ROS-dependent effects were assessed using the antioxidant N-acetylsteine. We show that auranofin decreased TrxR1 activity and increased ROS. Auranofin decreased cell vitality and colony formation and increased protein polyubiquitination through ROS-dependent mechanisms, suggesting the role of ROS in auranofin-induced cytotoxicity in the three cell lines. ROS-dependent PARP-1 cleavage was associated with EGFRvIII downregulation in U87/EGFRvIII cells. Remarkably, the auranofin and L-BSO combination induced the significant depletion of intracellular GSH and synergistic cytotoxicity regardless of EGFR overexpression. Nevertheless, molecular mechanisms associated with cytotoxicity were modulated to a different extent among the three cell lines. U87/EGFRvIII exhibited the most prominent ROS increase, P-AKT(Ser-473), and AKT decrease along with drastic EGFRvIII downregulation. U87/EGFRwt and U87/EGFRvIII displayed lower basal intracellular GSH levels and synergistic ROS-dependent DNA damage compared to U87MG cells. Our study provides evidence for ROS-dependent synergistic cytotoxicity of auranofin and L-BSO combination in GBM in vitro. Unraveling the sensitivity of EGFR-overexpressing cells to auranofin alone, and synergistic auranofin and L-BSO combination, supports the rationale to repurpose this promising pro-oxidant treatment strategy in GBM.

The Many Lives of Auranofin: How an Old Anti-Rheumatic Agent May Become a Promising Antimicrobial Drug.

Auranofin (AF) is a gold-based compound with a well-known pharmacological and toxicological profile, currently used in the treatment of some severe forms of rheumatoid arthritis. Over the last twenty years, AF has also been repurposed as antiviral, antitumor, and antibacterial drug. In this review we focused on the antibacterial properties of AF, specifically researching the minimal inhibitory concentrations (MIC) of AF in both mono- and diderm bacteria reported so far in literature. AF proves to be highly effective against monoderm bacteria, while diderm are far less susceptible, probably due to the outer membrane barrier. We also reported the current mechanistic hypotheses concerning the antimicrobial properties of AF, although a conclusive description of its antibacterial mode of action is not yet available. Even if its mechanism of action has not been fully elucidated yet and further studies are required to optimize its delivery strategy, AF deserves additional investigation because of its unique mode of action and high efficacy against a wide range of pathogens, which could lead to potential applications in fighting antimicrobial resistance and improving therapeutic outcomes in infectious diseases.

Auranofin inhibition of thioredoxin reductase sensitizes lung neuroendocrine tumor cells (NETs) and small cell lung cancer (SCLC) cells to sorafenib as well as inhibiting SCLC xenograft growth.

Thioredoxin Reductase (TrxR) functions to recycle thioredoxin (Trx) during hydroperoxide metabolism mediated by peroxiredoxins and is currently being targeted using the FDA-approved anti-rheumatic drug, auranofin (AF), to selectively sensitize cancer cells to therapy. AF treatment decreased TrxR activity and clonogenic survival in small cell lung cancer (SCLC) cell lines (DMS273 and DMS53) as well as the H727 atypical lung carcinoid cell line. AF treatment also significantly sensitized DMS273 and H727 cell lines in vitro to sorafenib, an FDA-approved multi-kinase inhibitor that depleted intracellular glutathione (GSH). The pharmacokinetic, pharmacodynamic, and safety profile of AF was examined in nude mice with DMS273 xenografts administered AF intraperitoneally at 2 mg/kg or 4 mg/kg (IP) once (QD) or twice daily (BID) for 1-5 d. Plasma levels of AF were 10-20 µM (determined by mass spectrometry of gold), and the optimal inhibition of TrxR activity was obtained at 4 mg/kg once daily, with no effect on glutathione peroxidase 1 activity. This AF treatment extended for 14 d, inhibited TrxR (>75%), and resulted in a significant prolongation of median overall survival from 19 to 23 d (p = .04, N = 30 controls, 28 AF). In this experiment, there were no observed changes in animal bodyweight, complete blood counts (CBCs), bone marrow toxicity, blood urea nitrogen, or creatinine. These results support the hypothesis that AF effectively inhibits TrxR both in vitro and in vivo in SCLC, sensitizes NETs and SCLC to sorafenib, and could be repurposed as an adjuvant therapy with targeted agents that induce disruptions in thiol metabolism.