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Nan Fang Yi Ke Da Xue Xue Bao = Journal... Apr 2022To construct a HEK293 cell line stably overexpressing TrxR1 as a cell model for functional study of TrxR1 and screening of TrxR1-targeting drugs.
OBJECTIVE
To construct a HEK293 cell line stably overexpressing TrxR1 as a cell model for functional study of TrxR1 and screening of TrxR1-targeting drugs.
METHODS
gene was amplified by PCR and ligated with the lentivirus expression vector pLVX-Puro, which was transformed into and identified by Sanger dideoxy sequencing. HEK293 cells were infected with the recombinant lentivirus vector (pLVX-Puro-TXNRD1) and screened with Puromycin for cell clones with stable TrxR1 overexpression (HEK293-TrxR1-OE cells). HEK293-TrxR1-OE cells, along with HEK293 cells infected with pLVX-Puro vector (HEK293-NC) and normal HEK293 cells, were tested for mRNA and protein expression levels of TrxR1 using RT-qPCR and Western blotting. TrxR1 enzyme activity in the cells was evaluated with insulin endpoint assay and TRFS-green probe imaging. The sensitivity of the cells to auranofin, a specific TrxR1 inhibitor, was determined with CCK8 assay.
RESULTS
gene was successfully inserted into the lentiviral vector pLVX-Puro as confirmed by DNA sequencing. The enzyme activity and mRNA and protein expression levels of TrxR1 were significantly higher in HEK293-TrxR1-OE cells than in HEK293 and HEK293-NC cells ( < 0.005). The inhibitory effects of auranofin on proliferation and cellular TrxR1 enzyme activity were significantly attenuated in HEK293-TrxR1-OE cells as compared with HEK293 and HEK293-NC cells ( < 0.005).
CONCLUSION
We successfully obtained a HEK293 cell line with stable TrxR1 overexpression, which shows resistance to auranofin and can be used for screening TrxR1 targeting drugs.
Topics: Auranofin; Cell Line, Tumor; Genetic Vectors; HEK293 Cells; Humans; Lentivirus; RNA, Messenger; Transfection
PubMed: 35527491
DOI: 10.12122/j.issn.1673-4254.2022.04.11 -
Cells Jun 2023(1) Background: Tuberous sclerosis complex (TSC) mutations directly affect mTORC activity and, as a result, protein synthesis. In several cancer types, TSC mutation is...
(1) Background: Tuberous sclerosis complex (TSC) mutations directly affect mTORC activity and, as a result, protein synthesis. In several cancer types, TSC mutation is part of the driver mutation panel. TSC mutations have been associated with mitochondrial dysfunction, tolerance to reactive oxygen species due to increased thioredoxin reductase (TrxR) enzyme activity, tolerance to endoplasmic reticulum (ER) stress, and apoptosis. The FDA-approved drug rapamycin is frequently used in clinical applications to inhibit protein synthesis in cancers. Recently, TrxR inhibitor auranofin has also been involved in clinical trials to investigate the anticancer efficacy of the combination treatment with rapamycin. We aimed to investigate the molecular background of the efficacy of such drug combinations in treating neoplasia modulated by TSC mutations. (2) Methods: TSC2 mutant and TSC2 wild-type (WT) cell lines were exposed to rapamycin and auranofin in either mono- or combination treatment. Mitochondrial membrane potential, TrxR enzyme activity, stress protein array, mRNA and protein levels were investigated via cell proliferation assay, electron microscopy, etc. (3) Results: Auranofin and rapamycin normalized mitochondrial membrane potential and reduced proliferation capacity of TSC2 mutant cells. Database analysis identified peroxiredoxin 5 (Prdx5) as the joint target of auranofin and rapamycin. The auranofin and the combination of the two drugs reduced Prdx5 levels. The combination treatment increased the expression of heat shock protein 70, a cellular ER stress marker. (4) Conclusions: After extensive analyses, Prdx5 was identified as a shared target of the two drugs. The decreased Prdx5 protein level and the inhibition of both TrxR and mTOR by rapamycin and auranofin in the combination treatment made ER stress-induced cell death possible in TSC2 mutant cells.
Topics: Humans; Tumor Suppressor Proteins; Tuberous Sclerosis Complex 2 Protein; Tuberous Sclerosis; Auranofin; Sirolimus; Antioxidants; Thioredoxin-Disulfide Reductase; Mutation
PubMed: 37443747
DOI: 10.3390/cells12131713 -
Frontiers in Cellular and Infection... 2022Given the increasing prevalence of antibiotic resistance, there is an urgent need to repurpose approved drugs with known pharmacology and toxicology as an alternative...
Given the increasing prevalence of antibiotic resistance, there is an urgent need to repurpose approved drugs with known pharmacology and toxicology as an alternative therapeutic strategy. We have reported that the sustained monotherapy of auranofin (AUR) inevitably resulted in reduced susceptibility or even the emergence of resistance to AUR in However, whether drug combination could increase antibacterial activity while preventing AUR resistance is still unknown. Here, we focused on the important role of AUR combined with phenethyl isothiocyanate (PEITC) in skin infection and determined the synergistic antimicrobial effect on by using checkerboard assays and time-kill kinetics analysis. This synergistic antimicrobial activity correlated with increased reactive oxygen species (ROS) generation, disruption of bacterial cell structure, and inhibition of biofilm formation. We also showed that AUR synergized with PEITC effectively restored the susceptibility to AUR regulating thioredoxin reductase (TrxR) and rescued mice from subcutaneous abscesses through eliminating pathogens, including methicillin-resistant (MRSA). Collectively, our study indicated that the AUR and PEITC combination had a synergistic antimicrobial impact on and . These results suggest that AUR and PEITC treatment may be a promising option for infection.
Topics: Animals; Anti-Bacterial Agents; Auranofin; Isothiocyanates; Methicillin-Resistant Staphylococcus aureus; Mice; Microbial Sensitivity Tests; Staphylococcal Infections; Staphylococcus aureus
PubMed: 35774400
DOI: 10.3389/fcimb.2022.927289 -
Frontiers in Cellular and Infection... 2023Microbe entry through catheter ports can lead to biofilm accumulation and complications from catheter-related bloodstream infection and ultimately require antimicrobial...
Microbe entry through catheter ports can lead to biofilm accumulation and complications from catheter-related bloodstream infection and ultimately require antimicrobial treatment and catheter replacement. Although strides have been made with microbial prevention by applying standardized antiseptic techniques during catheter implantation, both bacterial and fungal microbes can present health risks to already sick individuals. To reduce microbial adhesion, murine and human catheters were coated with polyurethane and auranofin using a dip coating method and compared to non-coated materials. Upon passage of fluid through the coated material , flow dynamics were not impacted. The unique antimicrobial properties of the coating material auranofin has shown inhibitory activity against bacteria such as and fungi such as . Auranofin coating on catheters at 10mg/mL reduced . accumulation from 2.0 x 10 to 7.8 x 10 CFU for mouse catheters and from 1.6 x 10 to 2.8 x 10 for human catheters, showing an impact to mature biofilms. Assessment of a dual microbe biofilm on auranofin-coated catheters resulted in a 2-log reduction in . and a 3-log reduction in . compared to uncoated catheters. assessment in a murine subcutaneous model demonstrated that catheters coated with 10 mg/mL auranofin reduced independent . and . accumulation by 4-log and 1-log, respectively, compared to non-coated catheters. In conclusion, the auranofin-coated catheters demonstrate proficiency at inhibiting multiple pathogens by decreasing . and . biofilm accumulation.
Topics: Humans; Animals; Mice; Auranofin; Staphylococcus aureus; Bacteria; Biofilms; Candida albicans; Catheters
PubMed: 37313344
DOI: 10.3389/fcimb.2023.1135942 -
Biomolecules Jan 2022Breast cancers (BCs) may present dramatic diagnoses, both for ineffective therapies and for the limited outcomes in terms of lifespan. For these types of tumors, the... (Review)
Review
Breast cancers (BCs) may present dramatic diagnoses, both for ineffective therapies and for the limited outcomes in terms of lifespan. For these types of tumors, the search for new drugs is a primary necessity. It is widely recognized that gold compounds are highly active and extremely potent as anticancer agents against many cancer cell lines. The presence of the metal plays an essential role in the activation of the cytotoxicity of these coordination compounds, whose activity, if restricted to the ligands alone, would be non-existent. On the other hand, gold exhibits a complex biochemistry, substantially variable depending on the chemical environments around the central metal. In this review, the scientific findings of the last 6-7 years on two classes of gold(I) compounds, containing phosphane or carbene ligands, are reviewed. In addition to this class of Au(I) compounds, the recent developments in the application of Auranofin in regards to BCs are reported. Auranofin is a triethylphosphine-thiosugar compound that, being a drug approved by the FDA-therefore extensively studied-is an interesting lead gold compound and a good comparison to understand the activities of structurally related Au(I) compounds.
Topics: Antineoplastic Agents; Auranofin; Breast Neoplasms; Female; Gold; Humans; Structure-Activity Relationship
PubMed: 35053228
DOI: 10.3390/biom12010080 -
Communications Biology Jun 2021Demand for a cure of liver fibrosis is rising with its increasing morbidity and mortality. Therefore, it is an urgent issue to investigate its therapeutic candidates....
Demand for a cure of liver fibrosis is rising with its increasing morbidity and mortality. Therefore, it is an urgent issue to investigate its therapeutic candidates. Liver fibrosis progresses following 'multi-hit' processes involving hepatic stellate cells, macrophages, and hepatocytes. The NOD-like receptor protein 3 (NLRP3) inflammasome is emerging as a therapeutic target in liver fibrosis. Previous studies showed that the anti-rheumatic agent auranofin inhibits the NLRP3 inflammasome; thus, this study evaluates the antifibrotic effect of auranofin in vivo and explores the underlying molecular mechanism. The antifibrotic effect of auranofin is assessed in thioacetamide- and carbon tetrachloride-induced liver fibrosis models. Moreover, hepatic stellate cell (HSC), bone marrow-derived macrophage (BMDM), kupffer cell, and hepatocyte are used to examine the underlying mechanism of auranofin. Auranofin potently inhibits activation of the NLRP3 inflammasome in BMDM and kupffer cell. It also reduces the migration of HSC. The underlying molecular mechanism was inhibition of cystine-glutamate antiporter, system Xc. Auranofin inhibits system Xc activity and instantly induced oxidative burst, which mediated inhibition of the NLRP3 inflammasome in macrophages and HSCs. Therefore, to the best of our knowledge, we propose the use of auranofin as an anti-liver fibrotic agent.
Topics: Amino Acid Transport System y+; Animals; Apoptosis; Auranofin; Carbon Tetrachloride; Cells, Cultured; Humans; Inflammasomes; Interleukin-1beta; Kupffer Cells; Liver; Liver Cirrhosis; Macrophages; Male; Mice, Inbred BALB C; Mice, Inbred C57BL; NLR Family, Pyrin Domain-Containing 3 Protein; Pyroptosis; Thioacetamide; Mice
PubMed: 34193972
DOI: 10.1038/s42003-021-02345-1 -
Microbiology Spectrum Feb 2024Auranofin, an FDA-approved drug for rheumatoid arthritis, has emerged as a promising antiparasitic medication in recent years. The gold(I) ion in auranofin is postulated...
Auranofin, an FDA-approved drug for rheumatoid arthritis, has emerged as a promising antiparasitic medication in recent years. The gold(I) ion in auranofin is postulated to be responsible for its antiparasitic activity. Notably aurothiomalate and aurothioglucose also contain gold(I), and, like auranofin, they were previously used to treat rheumatoid arthritis. Whether they have antiparasitic activity remains to be elucidated. Herein, we demonstrated that auranofin and similar derivatives, but not aurothiomalate and aurothioglucose, inhibited the growth of . We found that auranofin affected the biological cycle (lytic cycle) by inhibiting invasion and triggering its egress from the host cell. However, auranofin could not prevent parasite replication once resided within the host. Auranofin treatment induced apoptosis in parasites as demonstrated by its reduced size and elevated phosphatidylserine externalization (PS). Notably, the gold from auranofin enters the cytoplasm of as demonstrated by scanning transmission electron microscopy-energy dispersive X-ray spectroscopy (STEM-EDS) and Inductively Coupled Plasma-Mass Spectrometry (ICP-MS).IMPORTANCEToxoplasmosis, caused by , is a devastating disease affecting the brain and the eyes, frequently affecting immunocompromised individuals. Approximately 60 million people in the United States are already infected with , representing a population at-risk of developing toxoplasmosis. Recent advances in treating cancer, autoimmune diseases, and organ transplants have contributed to this at-risk population's exponential growth. Paradoxically, treatments for toxoplasmosis have remained the same for more than 60 years, relying on medications well-known for their bone marrow toxicity and allergic reactions. Discovering new therapies is a priority, and repurposing FDA-approved drugs is an alternative approach to speed up drug discovery. Herein, we report the effect of auranofin, an FDA-approved drug, on the biological cycle of and how both the phosphine ligand and the gold molecule determine the anti-parasitic activity of auranofin and other gold compounds. Our studies would contribute to the pipeline of candidate anti- agents.
Topics: Humans; Auranofin; Gold; Toxoplasma; Ligands; Aurothioglucose; Arthritis, Rheumatoid; Gold Sodium Thiomalate; Toxoplasmosis; Antiparasitic Agents; Phosphines
PubMed: 38206030
DOI: 10.1128/spectrum.02968-23 -
Redox Biology Jun 2022The effects of Auranofin (AF) on protein expression and protein oxidation in A2780 cancer cells were investigated through a strategy based on simultaneous expression...
The effects of Auranofin (AF) on protein expression and protein oxidation in A2780 cancer cells were investigated through a strategy based on simultaneous expression proteomics and redox proteomics determinations. Bioinformatics analysis of the proteomics data supports the view that the most critical cellular changes elicited by AF treatment consist of thioredoxin reductase inhibition, alteration of the cell redox state, impairment of the mitochondrial functions, metabolic changes associated with conversion to a glycolytic phenotype, induction of ER stress. The occurrence of the above cellular changes was extensively validated by performing direct biochemical assays. Our data are consistent with the concept that AF produces its effects through a multitarget mechanism that mainly affects the redox metabolism and the mitochondrial functions and results into severe ER stress. Results are discussed in the context of the current mechanistic knowledge existing on AF.
Topics: Auranofin; Cell Line, Tumor; Female; Humans; Ovarian Neoplasms; Oxidation-Reduction; Proteome; Thioredoxin-Disulfide Reductase
PubMed: 35358852
DOI: 10.1016/j.redox.2022.102294 -
Cell Death & Disease Aug 2021Pediatric gliomas comprise a broad range of brain tumors derived from glial cells. While high-grade gliomas are often resistant to therapy and associated with a poor...
Pediatric gliomas comprise a broad range of brain tumors derived from glial cells. While high-grade gliomas are often resistant to therapy and associated with a poor outcome, children with low-grade gliomas face a better prognosis. However, the treatment of low-grade gliomas is often associated with severe long-term adverse effects. This shows that there is a strong need for improved treatment approaches. Here, we highlight the potential for repurposing disulfiram to treat pediatric gliomas. Disulfiram is a drug used to support the treatment of chronic alcoholism and was found to be effective against diverse cancer types in preclinical studies. Our results show that disulfiram efficiently kills pediatric glioma cell lines as well as patient-derived glioma stem cells. We propose a novel mechanism of action to explain disulfiram's anti-oncogenic activities by providing evidence that disulfiram induces the degradation of the oncoprotein MLL. Our results further reveal that disulfiram treatment and MLL downregulation induce similar responses at the level of histone modifications and gene expression, further strengthening that MLL is a key target of the drug and explaining its anti-oncogenic properties.
Topics: Alcoholism; Auranofin; Cell Line, Tumor; Cell Proliferation; Child; Disulfiram; Down-Regulation; Drug Repositioning; Drug Synergism; Gene Expression Regulation, Neoplastic; Glioma; Histone-Lysine N-Methyltransferase; Histones; Humans; Lysine; Methylation; Myeloid-Lymphoid Leukemia Protein; Neoplasm Grading; Protein Processing, Post-Translational; Proteolysis; Transcription, Genetic
PubMed: 34381018
DOI: 10.1038/s41419-021-04078-9 -
Molecules (Basel, Switzerland) Jul 2023Gold compounds form a new class of promising anticancer agents with innovative modes of action. It is generally believed that anticancer gold compounds, at variance with... (Review)
Review
Gold compounds form a new class of promising anticancer agents with innovative modes of action. It is generally believed that anticancer gold compounds, at variance with clinically established platinum drugs, preferentially target proteins rather than nucleic acids. The reactions of several gold compounds with a few model proteins have been systematically explored in recent years through ESI MS measurements to reveal adduct formation and identify the main features of those reactions. Here, we focus our attention on a group of five gold compounds of remarkable medicinal interest, i.e., Auranofin, Au(NHC)Cl, [Au(NHC)]PF, Aubipyc, and Auoxo6, and on their reactions with four different biomolecular targets, i.e., the proteins HEWL, hCA I, HSA and the C-terminal dodecapeptide of the enzyme thioredoxin reductase. Complete ESI MS data are available for those reactions due to previous experimental work conducted in our laboratory. From the comparative analysis of the ESI MS reaction profiles, some characteristic trends in the metallodrug-protein reactivity may be identified as detailed below. The main features are described and analyzed in this review. Overall, all these observations are broadly consistent with the concept that cytotoxic gold drugs preferentially target cancer cell proteins, with a remarkable selectivity for the cysteine and selenocysteine proteome. These interactions typically result in severe damage to cancer cell metabolism and profound alterations in the redox state, leading to eventual cancer cell death.
Topics: Gold Compounds; Gold; Auranofin; Antineoplastic Agents; Thioredoxin-Disulfide Reductase
PubMed: 37446857
DOI: 10.3390/molecules28135196