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An investigation into the interactions of gold nanoparticles and anti-arthritic drugs with macrophages, and their reactivity towards thioredoxin reductase
Abstract
Gold(I) complexes are an important tool in the arsenal of established approaches for treating rheumatoid arthritis (RA), while some recent studies have suggested that gold nanoparticles (Au NPs) may also be therapeutically efficacious. These observations prompted the current biological studies involving gold(I) anti-RA agents and Au NPs, which are aimed towards improving our knowledge of how they work. The cytotoxicity of auranofin, aurothiomalate, aurothiosulfate and Au NPs towards RAW264.7 macrophages was evaluated using the MTT assay, with the former compound proving to be the most toxic. The extent of cellular uptake of the various gold agents was determined using graphite furnace atomic absorption spectrometry, while their distribution within macrophages was examined using microprobe synchrotron radiation X-ray fluorescence spectroscopy. The latter technique showed accumulation of gold in discrete regions of the cell, and co-localisation with sulfur in the case of cells treated with aurothiomalate or auranofin. Electrospray ionization mass spectrometry was used to characterize thioredoxin reductase (TrxR) in which the penultimate selenocysteine residue was replaced by cysteine. Mass spectra of solutions of TrxR and aurothiomalate, aurothiosulfate or auranofin showed complexes containing bare gold atoms bound to the protein, or protein adducts containing gold atoms retaining some of their initial ligands. These results support TrxR being an important target of gold(I) drugs used to treat RA, while the finding that Au NPs are incorporated into macrophages, but elicit little toxicity, indicates further exploration of their potential for treatment of RA is warranted.
... Another reason for the scarcity of data is the difficulty with the heterologous expression making it necessary to purify selenoproteins for metal binding studies [41]. Note that in some studies the (single) selenocysteine residue was mutated to cysteine to facilitate the study, e.g., 48 SeCys to 48 Cys in SelM [14] and penultimate selenocysteine in TrxR [35]. ...
... However, as gold(III) compounds can easily undergo reduction and lose their original ligands upon protein binding, the results of MALDI MS did not make it possible to accurately assign the number and identity of the bound gold moieties [18]. Regarding ESI MS, the only, to the best of our knowledge, work on TrxR concerned a mutant in which the penultimate selenocysteine residue was replaced by a cysteine [35]. ...
... ESI mass spectra of solutions of a TrxR mutant (in which the penultimate selenocysteine residue was replaced by cysteine) and aurothiomalate, aurothiosulfate or auranofin showed complexes containing bare gold atoms bound to the protein, or protein adducts containing gold atoms retaining some of their initial ligands [35]. ...
The selenol (-SeH) group of selenocysteine (SeCys), a cysteine analogue and a more potent nucleophile, has often been considered as a potential target of toxic trace metals and metalloids (Hg, As) and metallodrugs (Au, Pt), privileged with regard to more abundant aminoacids, such as cysteine or histidine. However, the formal evidence of the occurrence of the Se-metal bond, the involvement of selenol and the exact location of the Se-metal bonds in proteins containing several SeCys residues, especially in vivo, have largely been missing. This review discusses critically the contributions of the state-of-the-art analytical mass spectrometric and atomic spectroscopic techniques to studies of metal-binding sites with the different level of confidence in terms of demonstration of the occurrence of the Se-metal binding and the location of the binding site within the Se-containing molecule. They include methods indicating potential interactions on the basis of evidence of co-elution (HPLC-ICP MS) and co-localisation (laser ablation ICP MS, XRF, nanoSIMS) of selenium and the metal(s), methods confirming the evidence of the formation of Se-containing molecule – metal adducts (electrospray and MALDI MS), methods proving the existence of the actual Se-Me bond (EXAFS and XANES) and, finally, methods able to identify the actual binding site in proteins containing several SeCys residues (top-down MS, MS of tryptic digests of metal-protein adducts). The state of the art of in-vitro studies of metal-ion interactions with model SeCys-containing compounds at different levels of molecular complexity (selenoproteins, mimics selenopeptides, selenocysteine) and oxidation (Se-Se, Se-S bridges) as well as that of in-vivo studies are critically reviewed.
... However, recent investigations have shown a much more complicated effect resulting in down or up-regulation of cytokine production, particularly when gold is associated with cell activators such as TNF-a or lipopolysaccharide [172][173][174]. Various mechanisms of gold (I) agents for anti-RA effects include (1) inhibiting cathepsins K and S; (2) repressing of hydrolytic enzymes such as β-glucuronidase and elastase [169], which play a role in the progression of RA (by aurothiomalate and auranofin) [175]; (3) targeting of thioredoxin reductase (TrxR) enzyme, which modulates cellular processes through the reduction of thioredoxin (Trx) [176,177]; (4) inhibition of leukocyte infiltration [177]; (5) changing macrophage activity [178]; and regulation of the adhesion of neutrophils [179]. Moreover, it also demonstrated that various gold (I) drugs' metabolites modulate the immune system activity through direct binding to the T-cell receptors and then block antigen signaling [180], or may suppress T-cell activation via intervening with IL-2-mediated proliferative responses [181,182]. ...
... However, recent investigations have shown a much more complicated effect resulting in down or up-regulation of cytokine production, particularly when gold is associated with cell activators such as TNF-a or lipopolysaccharide [172][173][174]. Various mechanisms of gold (I) agents for anti-RA effects include (1) inhibiting cathepsins K and S; (2) repressing of hydrolytic enzymes such as β-glucuronidase and elastase [169], which play a role in the progression of RA (by aurothiomalate and auranofin) [175]; (3) targeting of thioredoxin reductase (TrxR) enzyme, which modulates cellular processes through the reduction of thioredoxin (Trx) [176,177]; (4) inhibition of leukocyte infiltration [177]; (5) changing macrophage activity [178]; and regulation of the adhesion of neutrophils [179]. Moreover, it also demonstrated that various gold (I) drugs' metabolites modulate the immune system activity through direct binding to the T-cell receptors and then block antigen signaling [180], or may suppress T-cell activation via intervening with IL-2-mediated proliferative responses [181,182]. ...
... Some studies showed that GNP is a more potent and effective anti-arthritic agent and has significantly less toxicity than gold (I) drugs [163,177,196]. Brown et al. [163] have shown that although colloidal GNPs (Au0) with 27 ± 3 nm size were effective in inhibiting the progress of three different arthritis forms (including mycobacterium-, pristine-, and collagen-induced arthritis), sodium aurothiomalate (I) was only effective against mycobacterium-induced arthritis, common inflammatory arthritis characterized via active leukocytes producing ROS such as hypochlorite (ClO − ) and H 2 O 2 . These results proved that GNPs are a more potent and effective anti-arthritic agent than sodium aurothiomalate (I) to treat rheumatoid arthritis [130] and suggested GNPs as a novel therapeutic tool for treating RA. ...
Gold nanoparticles (GNPs) have been recently applied for various diagnostic and therapeutic purposes. The unique properties of these nanoparticles (NPs), such as relative ease of synthesis in various sizes, shapes and charges, stability, high drug-loading capacity and relative availability for modification accompanied by non-cytotoxicity and biocompatibility, make them an ideal field of research in bio-nanotechnology. Moreover, their potential to alleviate various inflammatory factors, nitrite species, and reactive oxygen production and the capacity to deliver therapeutic agents has attracted attention for further studies in inflammatory and autoimmune disorders. Furthermore, the characteristics of GNPs and surface modification can modulate their toxicity, biodistribution, biocompatibility, and effects. This review discusses in vitro and in vivo effects of GNPs and their functionalized forms in managing various autoimmune disorders (Ads) such as rheumatoid arthritis, type 1 diabetes, and multiple sclerosis.
... AuNPs, due to their variability in shape, size, material hybridization and functionalization, are becoming one of the major agents in cancer therapeutics [1][2][3][4][5][6][7][8][9][10][11][12], diagnostics [13][14][15][16][17], targeted drug delivery [18][19][20][21], cell function regulation [22][23][24][25][26], biolabeling [27][28][29][30][31][32], and immunology [33]. Virtually all these applications require uptake of nanoparticles into the target cells to realise their designed functionality, hence much research has been devoted to controlling the uptake and accurate quantification of nanoparticles [34][35][36][37][38][39][40][41][42][43][44][45][46][47][48][49][50][51][52]. ...
... Alternatively, if α 2 is unknown, then g 1,3 (0,0) or g 2,2 (0,0) can be further calculated from the image, meaning that there are three simultaneous equations to solve for three unknowns (N 1 , α 2 and N 2 ). These are shown in Eq. (6). This can be further extended to higher order equations if there are more unknown species concentrations [22,26]. ...
The application of gold nanoparticles (AuNPs) in cancer therapeutics and diagnostics has recently reached a clinical level. Functional use of the AuNP in theranostics first requires effective uptake into the cells, but accurate quantification of AuNPs cellular uptake in real-time is still a challenge due to the destructive nature of existing characterization methods. The optical imaging-based quantification method is highly desirable. Here, we propose the use of high-order image correlation spectroscopy (HICS) as an optical imaging-based nanoparticle quantification technique. Coupled with dark field microscopy (DFM), a non-destructive and easy quantification method could be achieved. We demonstrate HICS analysis on 80 nm AuNPs coated with cetyltrimethylammonium bromide (CTAB) uptake in HeLa cells to calculate the percentage of aggregate species (dimer) in the total uptake and their relative scattering quantum yield inside the cells, the details of which are not available with other quantification techniques. The total particle uptake kinetics measured were in a reasonable agreement with the literature.
... Recently it has been reported that nanogold can play an important role in the treatment of rheumatoid arthritis. [23][24][25][26] It is suggested that undesired immune system response may contribute to rheumatoid arthritis. Gold may affect this response and decelerate the progression of the disease. ...
... 52,61,62 Injection of organo-gold compounds has been utilized for nearly a century to treat inflammation and only recent reports provide the first analysis of the biochemical pathway in which gold nanoparticles may reduce inflammation. 26 Dykman and Khlebtsov 63 reviewed Immunological properties of gold nanoparticles and gave detailed analysis of data on interaction of gold nanoparticles with immune cells. Noble metal nanoparticles such as gold and silver, interact with both the innate and adaptive immune systems but there are few reports that uncover the mechanism behind AuNPs ability to elicit an immunomodulatory response. ...
Nanotechnology is undoubtedly one of the most significant industrial innovations of the 21st century. Nanomaterials are being used in various industrial applications and products as well as for numerous biomedical purposes. Since the manufacture and use of nanoparticles are increasing exponentially, humans and animals are more likely to be exposed occupationally via consumer products or through environment. In particular, gold nanoparticles (AuNPs) have attracted substantial attention and have been used for various biomedical applications. AuNPs are considered to be non-toxic, similar to that of bulk gold; however, numerous in vitro and in vivo studies have been performed to establish AuNPs toxicity which have revealed conflicting results. The immune system is considered as a sensitive indicator against exposure of such foreign materials. Keeping in view the above scenario, present study was carried out to evaluate immunomodulatory effect of AuNPs in chicken lymphocytes cell culture system. The study revealed maximum non-cytotoxic and minimal cytotoxic dose to be 50µg/ml and 60µg/ml in chicken lymphocytes cell culture system. At minimal cytotoxic dose, immuno-toxic effects were observed through lymphocytes proliferation assay. However, at lower doses of AuNPs, there was increase in lymphocytes proliferation and maximum proliferative dose was determined to be 20µg/ml at which immuno-potentiating effects were observed. Thus, it could be inferred that AuNPs displayed non-linear response in chicken lymphocytes culture system in relation to immunomodulatory effects.
... The bricks are proteinenriched (mainly keratin), whereas the lipid matrix is composed mainly of ceramides, cholesterol, and free fatty acids [13]. GNPs are expected to penetrate SC mainly through intercellular pathways, SR-XRF has been used for mapping and quantification of metals distributed in various biological matrices including culture cells, tumor tissues, and plants [22][23][24]. However, the employment of SR-XRF for metal mapping of skin is rare in the literature. ...
... Contrary to our findings, James et al. used SR-XRF to image gold in single macrophages cells and found that GNPs showed a high degree of "co-localization" with sulfur owing to interactions between gold and sulfur inside macrophages. They concluded that because of the dominating and intense gold M line, it was difficult to determine whether gold derived from GNPs co-localized with sulfur [23]. Many factors could contribute to the disparity in the results obtained such as beamline characteristics, sample preparation, and environmental setup for the experiments. ...
Understanding the distribution of nanoparticles in skin layers is fundamentally important and essential for developing nanoparticle-based dermal drug delivery systems. In the present study, we provide insights into the distribution of gold nanorods (GNRs) functionalized with hydrophobic or hydrophilic ligands in human skin layers using synchrotron X-ray fluorescence (SR-XRF) spectroscopy, confocal microscopy, and transmission electron microscopy. The results confirmed the important role that the surface chemistry of GNRs plays in their penetration into the skin; the GNRs coated with polyethylene glycol were distributed into the skin layers to a greater extent than the GNRs coated with hydrophobic polystyrene thiol. In addition, SR-XRF analysis revealed that the spatial distribution of endogenous elements (phosphorus and sulfur) in skin layers demonstrated a significant "anti-correlation" relationship with that of GNRs. These results suggest possible association (via adsorption) between the GNRs and these two elements localized in skin, which can be valuable for understanding the penetration mechanism of gold nanoparticles into the skin.
... With the increasing popularity and use of nanometer-sized gold particles (nanoGold), the impact of the smaller metal particles on inflammation has received considerable attention (Brown et al. 2007;James et al. 2015;Han et al. 2022). When the gold particles are bigger than 20 µm, they are too big for the macrophages to engulf, and the macrophages thus proceed to secrete cyanide compounds and dissolve the gold Autometallographic (AMG) illustration of the dissolucytotic release of the gold ions from a gold grid. ...
The newest data on metallic gold have placed the noble metal central in the fight for the safe treatment of autoimmune inflammation. There are two different ways to use gold for the treatment of inflammation: gold microparticles > 20 µm and gold nanoparticles. The injection of gold microparticles (µGold) is a purely local therapy. µGold particles stay put where injected, and gold ions released from them are relatively few and taken up by cells within a sphere of only a few millimeters in diameter from their origin particles. The macrophage-induced release of gold ions may continue for years. Injection of gold nanoparticles (nanoGold), on the other hand, is spread throughout the whole body, and the bio-released gold ions, therefore, affect multitudes of cells all over the body, as when using gold-containing drugs such as Myocrisin. Since macrophages and other phagocytotic cells take up and transport nanoGold and remove it after a short period, repeated treatment is necessary. This review describes the details of the cellular mechanisms that lead to the bio-release of gold ions in µGold and nanoGold.
... Attempts have been made to expose macrophages to gold nanoparticles and compare them with gold complexes, which have resulted in a significantly greater uptake of gold without significant cytotoxicity towards macrophages. 33 These results support the potential of these colloidal systems as anti-RA agents. When combining the delivery of gold nanoparticles with MTX to the region of inflammation in collagen-induced arthritic mice, the retention of nanoparticles was enhanced under the external magnetic field, 34 resulting in enhanced therapeutic effects with an MTX dosage of just 0.05% compared to free MTX therapy for the treatment of RA. 34 The use of small interfering RNA (siRNA) has shown therapeutic effects in diverse disease models by silencing the gene responsible for the defects, including RA. 35 In order to enhance the therapeutic efficacy of siRNA, incorporation of siRNA into nanoparticles has shown improved target ability. ...
Rheumatoid arthritis (RA), the most frequent chronic inflammatory autoimmune disease, can lead to pain, bone and articular destruction, and limb deformity and impairment, with great impact on the activities of daily life. Several drug modifiers of the inflammatory process have been used in the treatment of the disease, all with specific patient targets and indications. However, the side effects are a frequent cause of undertreatment and non-adherence. To promote better compliance with the therapy, drug researchers have been trying to develop a new carrier of the immunomodulated molecules to increase their concentration in the target cell (mostly synovial), avoiding side effects for organs that are not targeted, as well as providing an easier manner of administration. The research results from animal models are promising and the clinical applications will show if these results are similarly impressive. This paper aims to explain the major applications of nanomedicine in RA treatment and diagnosis. The use of nanocarriers able to act as a diagnostic imaging agent and targeted drug delivery system, simultaneously, also known as nanotheranostics, can allow an improved efficacy and safety pharmacological profile, earlier detection, and thither monitoring of the disease.1 Commercial successes of RA active targeting of nanomedicine and products under development will be revised.
... 18 As a result of some groundbreaking discoveries, advanced treatments are available for RA patients, for example, methotrexate for RA therapy is signicantly effective but is known to have a wide range of side effects if taken for a long duration. 19 Several cellular receptors and targets have been studied to understand the cellular pathways and molecular targets for targeted therapy, such as using small interfering RNA (siRNA) as a therapeutic substance to downregulate these signalling-specic genes 20,21 . Applied nanotechnology is a promising interdisciplinary research eld that combines the expertise of engineers and scientists, resulting in recent advances demonstrating the tremendous potential of nanoparticles in medical applications, 22 and nanomedicine is rapidly evolving with the ability to target specic and effective drug delivery. ...
Nanotechnology has increasingly emerged as a promising tool for exploring new approaches, from treating complex conditions to early detection of the onset of multiple disease states. Tailored designer nanoparticles can now more comprehensively interact with their cellular targets and various pathogens due to a similar size range and tunable surface properties. The basic goal of drug delivery is to employ pharmaceuticals only where they are needed, with as few adverse effects and off-target consequences as possible. Rheumatoid arthritis (RA) is a chronic inflammatory illness that leads to progressive loss of bone and cartilage, resulting in acute impairment, decreased life expectancy, and increased death rates. Recent advancements in treatment have significantly slowed the progression of the disease and improved the lives of many RA sufferers. Some patients, on the other hand, attain or maintain illness remission without needing to continue immunosuppressive therapy. Furthermore, a large percentage of patients do not respond to current treatments or acquire tolerance to them. As a result, novel medication options for RA therapy are still needed. Nanocarriers, unlike standard medications, are fabricated to transport drugs directly to the location of joint inflammation, evading systemic and negative effects. As a result, researchers are reconsidering medicines that were previously thought to be too hazardous for systemic delivery. This article gives an overview of contemporary nanotechnology-based tactics for treating rheumatoid arthritis, as well as how the nanotherapeutic regimen could be enhanced in the future.
... Some studies showed that compared to gold salts, AuNP is a more potent and effective anti-arthritic agent and has significantly less toxicity [1,8,9]. Specifically, in a small exploratory clinical study published in 1997, ten patients with severe rheumatoid arthritis who failed previous treatments saw drastic improvements in their knee swelling and pain when placed on a colloidal metallic gold treatment for 6 months [10]. ...
The purpose of this clinical study was to determine whether gold nanoparticle (AuNP)
supplementation at a dosage of 0.34 mg elemental gold per day can improve knee joint health,
function, and quality of life for arthritis patients. A total of 51 participants (24 male and 27 female, age
62.1 � 13.1) were followed for 20 weeks through a three-phase longitudinal study. Both subjective
and objective parameters were used to measure changes in joint health and function, as well as quality
of life. The study found patients’ Knee injury and Osteoarthritis Outcome Score (KOOS) improved
with statistical significance. It was reported that 71.42% of the cohort experienced improvements
in their perceived knee pain and 61.22% with improvements in knee stiffness. Majority of objective
measurements such as pain with range of motion and specific exercises requiring proper knee health
and function did not show statistically significant improvement but did show a positive improving
trend in support of AuNP supplement. Study cohort showed statistically significant improvements
in two specific exercises: sit-to-stand and single-leg squat. By the end of the study, 70% of the study
cohort indicated that they would continue to take the supplement even after the study concluded.
Though the study has limitations and is not definitely conclusive, it was the first clinical study to show
that oral micro-dosage of AuNP as low as 0.34 mg daily is safe and effective for both rheumatoid
arthritis and osteoarthritis patients. This study opened way for the use of AuNP in both clinical and
daily settings to improve joint health and function for both average and athletic users.
... Reactive oxygen species, and protein kinase 3 enzymes that interact with the receptors are also influential on the formation of NETs [171]. Although the inflammationreducing effect of gold-based nanoparticles has been used for nearly a century, molecular and biochemical activity mechanisms have only been revealed with recent reports [193,194]. For example, citrate-coated gold nanoparticles, neither cytotoxic nor organotoxic, show anti-inflammatory activity depending on their size by inhibiting the immune responses induced by interleukin one beta (IL-1β) [195]. ...
Nanotechnology has encouraged new and amended materials (metal nanoparticles) for therapeutic applications with specific prominence in healthcare. Metal nanoparticles (NPs) are versatile nanoscale entities, widely used to diagnose and treat cancer. Evidence suggested that metal NPs can modulate the expression of various intracellular and extra-cellular signaling molecules in the tumor microenvironment. Metal nanoparticles possess anti-cancer activities via apoptosis and cell cycle arrest. In addition, metal NPs inhibit tumor angiogenesis, metastasis and inflammation to stop cancer proliferation. Synergistic applications of metal NPs with existing anti-cancer agents showed improvement in their bioactivity and bioavailability. This review explores the synthetic approaches, pharmacokinetics, and the cellular and molecular interactions of metal NPs in cancer.
... thiol, amine, and carboxyl groups that are reactive [104]. GNPs were strategically planted in macrophages to target thioredoxin reductase to evaluate its antiangiogenic impact by binding to the vascular endothelial growth factor (VEGF) [105]. Lee et al. suggested MTX encapsulated RGD-attached gold half-shell NP system for RA treatment [106]. ...
Rheumatoid arthritis (RA) is a progressive autoimmune inflammatory disorder characterized by cellular infiltration in synovium causing joint destruction and bone erosion. The heterogeneous nature of the disease manifests in different clinical forms, hence treatment of RA still remains obscure. Treatments are limited owing to systemic toxicity by dose-escalation and lack of selectivity. To overcome these limitations, Smart drug delivery systems (SDDS) are under investigation to exploit the arthritic microenvironment either by passive targeting or active targeting to the inflamed joints via folate receptor, CD44, angiogenesis, integrins. This review comprehensively deliberates upon understanding the pathophysiology of RA and role of SDDSs, highlighting the emerging trends for RA nanotherapeutics.
... The uptake of TrxR is greater than that of auranofin, which is transported into cells using the sulfhydryl shuttle model. Simultaneously, the surface of Au-NPs can be modified with a Au-sulfur (Au-S) bond via chemical grafting or electrostatic coating, which endows nanomaterials with multiple biological functions (James et al., 2015). In the study conducted by Pandey et al., the Au-NP was modified with a thiolated dendritic polymer to produce nanogold core dendrimer NPs (Au-DEN-NPs). ...
Rheumatoid arthritis (RA) is a chronic, progressive, and systemic inflammatory autoimmune disease, characterized by synovial inflammation, synovial lining hyperplasia and inflammatory cell infiltration, autoantibody production, and cartilage/bone destruction. Macrophages are crucial effector cells in the pathological process of RA, which can interact with T, B, and fibroblast-like synovial cells to produce large amounts of cytokines, chemokines, digestive enzymes, prostaglandins, and reactive oxygen species to accelerate bone destruction. Therefore, the use of nanomaterials to target macrophages has far-reaching therapeutic implications for RA. A number of limitations exist in the current clinical therapy for patients with RA, including severe side effects and poor selectivity, as well as the need for frequent administration of therapeutic agents and high doses of medication. These challenges have encouraged the development of targeting drug delivery systems and their application in the treatment of RA. Recently, obvious therapeutic effects on RA were observed following the use of various types of nanomaterials to manipulate macrophages through intravenous injection (active or passive targeting), oral administration, percutaneous absorption, intraperitoneal injection, and intra-articular injection, which offers several advantages, such as high-precision targeting of the macrophages and synovial tissue of the joint. In this review, the mechanisms involved in the manipulation of macrophages by nanomaterials are analyzed, and the prospect of clinical application is also discussed. The objective of this article was to provide a reference for the ongoing research concerning the treatment of RA based on the targeting of macrophages.
... This phenomenon might occur due to an inhibitory effect of the remaining AuCl 4 − ions. 46,47 Then the cell viability increases to ≥100% again when the samples are the sugars−AuNPs synthesized in 1.0% w/v sugars treated with plasma for 30 min. In other words, the sugars− AuNPs synthesized with this condition exhibit no toxicity to the MRC-5 cells. ...
... Furthermore, recently, a new class of drugs for rheumatoid arthritis has been developed, exploiting the Au NP ability to invade macrophages and stop them from producing inflammation without killing them. The researchers found that by reducing gold into NPs (50 nm), more gold was absorbed into the cells, with much less toxicity in the case of rheumatoid arthritis [131]. Again, Au NPs have been widely used in imaging and diagnosis of many diseases [132], or as intravenous contrast agents for imaging and noninvasive detection of lung cancer and many other topics [133]. ...
The increase of multidrug-resistant bacteria remains a global concern. Among the proposed strategies, the use of nanoparticles (NPs) alone or associated with orthopedic implants represents a promising solution. NPs are well-known for their antimicrobial effects, induced by their size, shape, charge, concentration and reactive oxygen species (ROS) generation. However, this non-specific cytotoxic potential is a powerful weapon effective against almost all microorganisms, but also against eukaryotic cells, raising concerns related to their safe use. Among the analyzed transition metals, silver is the most investigated element due to its antimicrobial properties per se or as NPs; however, its toxicity raises questions about its biosafety. Even though it has milder antimicrobial and cytotoxic activity, TiO2 needs to be exposed to UV light to be activated, thus limiting its use conjugated to orthopedic devices. By contrast, gold has a good balance between antimicrobial activity as an NP and cytocompatibility because of its inability to generate ROS. Nevertheless, although the toxicity and persistence of NPs within filter organs are not well verified, nowadays, several basic research on NP development and potential uses as antimicrobial weapons is reported, overemphasizing NPs potentialities, but without any existing potential of translation in clinics. This analysis cautions readers with respect to regulation in advancing the development and use of NPs. Hopefully, future works in vivo and clinical trials will support and regulate the use of nano-coatings to guarantee safer use of this promising approach against antibiotic-resistant microorganisms.
... The central role of the Trx system in cellular redox homeostasis, antioxidant defense, and DNA synthesis (30) has led to the investigation of TrxR as a target for antimicrobials (31)(32)(33)(34) as well as drugs for the treatment of cancer (35,36) and rheumatoid arthritis (37). In addition, the differing structures and molecular mechanisms of high-and low-MW TrxRs afford the possibility of selectively inhibiting the Trx systems of bacterial and fungal pathogens over the human system. ...
Aspergillus fumigatus infections are associated with high mortality rates and high treatment costs. Limited available antifungals and increasing antifungal resistance highlight an urgent need for new antifungals. Thioredoxin reductase (TrxR) is essential for maintaining redox homeostasis and presents as a promising target for novel antifungals. We show that ebselen (2-phenyl-1,2-benzoselenazol-3(2H)-one) is an inhibitor of A. fumigatus TrxR ( K i = 0.22 μM) and inhibits growth of Aspergillus spp. with in vitro MIC values of 16-64 μg/mL. Mass spectrometry analysis demonstrates that ebselen interacts covalently with a catalytic cysteine of TrxR, Cys148. We also present the X-ray crystal structure of A. fumigatus TrxR, and use in silico modeling of the enzyme-inhibitor complex to outline key molecular interactions. This provides a scaffold for future design of potent and selective antifungal drugs that target TrxR, improving upon the potency of ebselen towards inhbition of A. fumigatus growth.
... IBD is a relapsing and incurable disease that is featured by chronic intestinal inflammation and displays a variety of symptoms, such as persistent diarrhea, abdominal pain, and fatigue. AuNPs have been shown to play an anti-inflammatory role in mouse obesity, rat arthritis, and rat focal cerebral ischemia-reperfusion [8][9][10][11], and AuNPs can inhibit innate immune activation in macrophages in vitro by attenuating nuclear factor kappa beta (NF-κB), interferon-β/signal transducer and activator of transcription 1 (STAT1), and Toll-like receptor 9 signaling pathways [12][13][14]. The oral administration of AuNPs exerts little toxicity [15,16] and, with the gastrointestinal tract as their initial point of contact, AuNPs may have the potential to attenuate IBD. ...
Background
Gold nanoparticles (AuNPs) are attracting interest as potential therapeutic agents to treat inflammatory diseases, but their anti-inflammatory mechanism of action is not clear yet. In addition, the effect of orally administered AuNPs on gut microbiota has been overlooked so far. Here, we evaluated the therapeutic and gut microbiota-modulating effects, as well as the anti-inflammatory paradigm, of AuNPs with three different coatings and five difference sizes in experimental mouse colitis and RAW264.7 macrophages.
Results
Citrate- and polyvinylpyrrolidone (PVP)-stabilized 5-nm AuNPs (Au-5 nm/Citrate and Au-5 nm/PVP) and tannic acid (TA)-stabilized 5-, 10-, 15-, 30- and 60-nm AuNPs were intragastrically administered to C57BL/6 mice daily for 8 days during and after 5-day dextran sodium sulfate exposure. Clinical signs and colon histopathology revealed more marked anti-colitis effects by oral administration of Au-5 nm/Citrate and Au-5 nm/PVP, when compared to TA-stabilized AuNPs. Based on colonic myeloperoxidase activity, colonic and peripheral levels of interleukin-6 and tumor necrosis factor-α, and peripheral counts of leukocyte and lymphocyte, Au-5 nm/Citrate and Au-5 nm/PVP attenuated colonic and systemic inflammation more effectively than TA-stabilized AuNPs. High-throughput sequencing of fecal 16S rRNA indicated that AuNPs could induce gut dysbiosis in mice by decreasing the α-diversity, the Firmicutes/Bacteroidetes ratio, certain short-chain fatty acid-producing bacteria and Lactobacillus. Based on in vitro studies using RAW264.7 cells and electron spin resonance oximetry, AuNPs inhibited lipopolysaccharide (LPS)-triggered inducible nitric oxide (NO) synthase expression and NO production via reduction of Toll-like receptor 4 (TLR4), and attenuated LPS-induced nuclear factor kappa beta activation and proinflammatory cytokine production via both TLR4 reduction and catalytic detoxification of peroxynitrite and hydrogen peroxide.
Conclusions
AuNPs have promising potential as anti-inflammatory agents; however, their therapeutic applications via the oral route may have a negative impact on the gut microbiota.
... Among the transition metals, gold is an essential element due to their complexes having potential biological properties. For instance, auranofin is a gold metal based drug has been used in the treatment of rheumatoid arthritis [11]. Besides, some of the literature reports have been suggested that, gold(III) complexes possess in vitro and in vivo antitumor effects [12][13][14]. of Schiff base ligands. ...
... Among the transition metals, gold is an essential element due to their complexes having potential biological properties. For instance, auranofin is a gold metal based drug has been used in the treatment of rheumatoid arthritis [11]. Besides, some of the literature reports have been suggested that, gold(III) complexes possess in vitro and in vivo antitumor effects [12][13][14]. ...
Novel gold and platinum complexes [AuL2].Cl, 1 and [PtL2].2Cl, 2 with ligand, 2-methoxy-6-((2-(4-(trifluoromethyl)pyrimidin-2-yl)hydrazono)methyl)phenol (HL) have been synthesized and screened for their antimicrobial, antioxidant, DNA binding and anticancer (in vitro) activities. The single crystal of ligand HL was obtained by slow evaporation technique. The molecular structure of HL was confirmed from single crystal X-ray technique. Density functional theory calculations have been performed to gain insights into the electronic structure of these metal complexes. Antimicrobial result shows that, HL and complexes (1 and 2) have good antimicrobial agents against E. coli (bacteria) and C. albicans (fungi) than others bacterial and fungal strains. Antioxidant assay results suggest that, HL and complexes (1 and 2) possess good radical scavenging activity against diverse free radicals (DPPH, SOD, NO and H2O2). The intercalative interactions of HL and complexes (1 and 2) with CT-DNA were confirmed from spectroscopic titrations and viscometric measurements. Furthermore, the interactions of prepared compounds with DNA were confirmed by molecular docking analysis. In order to understand the nature of interactions between these metal complexes and BSA protein results clearly shows that complex 1 binds better than that of complex 2. The antitumor activities of prepared products were tested against single normal and different tumor cell lines by MTT assay. These results reveal that prepared complexes (1 and 2) have significant cytotoxic effect against tumor cell lines.
... GNPs may also be therapeutically efficacious. It was shown that GNPs could be incorporated in the macrophages, target to the thioredoxin reductase and exhibit an antiangiogenic effect by binding vascular endothelial growth factor [92,93]. The utilisation of GNPs in targeted therapy of RA has been reported by Lee et al. [94]. ...
Rheumatoid arthritis (RA) is an immune-mediated inflammatory disease that selectively attacks human joints. The common non-targeted treatment approaches lead to obvious side effect and systemtic complication for RA patients. Therefore, targeted drug delivery for treatment of RA has gained much attetntion in the past few years. In this paper, we reviewed the potential targets (folate receptor, angiogenesis, matrix metalloproteases, selectins, vasoactive intestinal peptide receptor, Fc-γ receptor) that could be utilized to facilitate the specific delivery of drugs to the inflammed synovium, and also presented different drug delivery systems for targeting RA, including the liposomes, various types of nanoparticles, polymeric micelles and the macromolecular prodrugs. The strategies combining nanotechnologies and ligand mediated active targeting for RA would be emphatically illustrated, which was expected to be helpful for identifying technologies and drug delivery methods for targeted treatment of RA.
... The same nanoparticles may be beneficial in one model and/or using one end point, and adverse when using another model or end point (Ilinskaya & Dobrovolskaia, 2014). For example, administration of organo-gold compounds has been utilized for nearly a century to treat inflammation but it was only until recently that the anti-inflammatory activity was attributed to the inhibition of cellular responses induced by interleukin 1 beta (IL-1β) (Table S1), involved both in the innate and the adaptive immunity (James, et al., 2015), which contradicts the GNPs' pro-inflammatory responses described in the previous section. Dependency both on NP size and surface charge was demonstrated, where GNPs with zwitterionic charges were shown to completely block the IL-1β pathway (Fernandez, et al., 2015), while only GNPs of specific sizes and at specific concentrations have been shown to interfere with TLR9 ...
Considering both cancer's serious impact on public health and the side effects of cancer treatments, strategies towards targeted cancer therapy have lately gained considerable interest. Employment of gold nanoparticles (GNPs), in combination with ionizing and non-ionizing radiations, has been shown to improve the effect of radiation treatment significantly. GNPs, as high-Z particles, possess the ability to absorb ionizing radiation and enhance the deposited dose within the targeted tumors. Furthermore, they can convert non-ionizing radiation into heat, due to plasmon resonance, leading to hyperthermic damage to cancer cells. These observations, also supported by experimental evidence both in vitro and in vivo systems, reveal the capacity of GNPs to act as radiosensitizers for different types of radiation. In addition, they can be chemically modified to selectively target tumors, which renders them suitable for future cancer treatment therapies. Herein, a current review of the latest data on the physical properties of GNPs and their effects on GNP circulation time, biodistribution and clearance, as well as their interactions with plasma proteins and the immune system, is presented. Emphasis is also given with an in depth discussion on the underlying physical and biological mechanisms of radiosensitization. Furthermore, simulation data are provided on the use of GNPs in photothermal therapy upon non-ionizing laser irradiation treatment. Finally, the results obtained from the application of GNPs at clinical trials and pre-clinical experiments in vivo are reported.
... Injection of organo-gold compounds has been utilized for nearly a century to treat inflammation, and only recent reports provide the first analysis of the biochemical pathway in which gold nanoparticles may reduce inflammation. 52,53 Citrate-coated gold nanoparticles were reported to not cause detectable cell or organ toxicity in mice, but the report concluded that citratecoated gold nanoparticles showed anti-inflammatory activity and inhibited cellular responses induced by interleukin 1 beta (IL-1b). IL-1b is an inflammatory cytokine that acts as an arbiter between the innate and adaptive immune response; moreover, common inflammatory disorders, for example, rheumatoid arthritis, are mediated by IL-1b production. ...
Targeting the immune system with nanomaterials is an intensely active area of research. Specifically, the capability to induce immunosuppression is a promising complement for drug delivery and regenerative medicine therapies. Many novel strategies for immunosuppression rely on nanoparticles as delivery vehicles for small-molecule immunosuppressive compounds. As a consequence, efforts in understanding the mechanisms in which nanoparticles directly interact with the immune system have been overshadowed. The immunological activity of nanoparticles is dependent on the physiochemical properties of the nanoparticles and its subsequent cellular internalization. As the underlying factors for these reactions are elucidated, more nanoparticles may be engineered and evaluated for inducing immunosuppression and complementing immunosuppressive drugs. This review will briefly summarize the state-of-the-art and developments in understanding how nanoparticles induce immunosuppressive responses, compare the inherent properties of nanomaterials which induce these immunological reactions, and comment on the potential for using nanomaterials to modulate and control the immune system.
... For example, they inhibit production of reactive nitrogen and oxygen species, as well as activation of NF-kB [101]. In addition, Au NPs do not induce secretion of the proinflammatory cytokines TNF-a and IL-1b [101,102]. In one study, tocilizumabloaded hyaluronate-gold nanoparticles (HA-AuNP/TCZ) targeted with a monoclonal antibody against IL-6 has also been tested in CIA mice. ...
Thioredoxin (Trx) is essential in a redox-control system, with many bacteria containing two Trxs: Trx1 and Trx2. Due to a Trx system's critical function, Trxs are targets for novel antibiotics. Here, a 1.20 Å high-resolution structure of Trx2 from Acinetobacter baumannii (abTrx2), an antibiotic resistant pathogenic superbug, is elucidated. By comparing Trx1 and Trx2, it is revealed that the two Trxs possess similar activity, although Trx2 contains an additional N-terminal zinc-finger domain and exhibits more flexible properties in solution. Finally, it is shown that the Trx2 zinc-finger domain might be rotatable and that proper zinc coordination at the zinc-finger domain is critical to abTrx2 activity. This study enhances understanding of the Trx system and will facilitate the design of novel antibiotics.
Gold salts have been used to treat rheumatoid arthritis (RA) since the 1940s, and, with advances in nanotechnology, the use of nanogold provides multiple options for anti-inflammatory therapies. This paper presents the synthesis and characterization of silica-gold nanostructures (SGNs) and their therapeutic effect in collagen-induced arthritis (CIA) in DBA/1 mice. At the end of the treatment, the synovial membranes, kidneys, livers, and spleens were dissected and analyzed by inductively coupled plasma mass spectroscopy (ICP) showing less than 0.0001 and 0.1% of the administered doses of Au and Si, respectively. Remains of the SGNs were visually identified in the synovial membrane by scanning electron microscopy (SEM), and the bone density of the hind paws was observed by computerized tomography (CT) indicating a reduction of porosity in the CIA-experimental group. The DNA microarray analysis carried out with RNA obtained from the hind paws showed 2628 differentially expressed genes (DEGs) by SGNs. The bioinformatic analysis showed that DEGs were significantly associated with several inflammatory signalling pathways including chemokines, cytokine-cytokine receptor interaction, PI3K-Akt, TNF, IL-17, NFκβ, MAPK, and RA. SGNs downregulated relevant inflammatory genes in the arthritic joints, including Tnf, Ifng, Il6, and Cxcl5; immunohistochemistry (IHC) confirmed the reduction of TNFα, IL-6, NFκβ, and VEGF in the joints due to the effect of SGNs. TNFα and IL-6 were also reduced in the serum of DBA/1 mice treated with SGNs.
Osseous tissue repair has advanced due to the introduction of tissue engineering. The key elements required while engineering new tissues involve scaffolds, cells, and bioactive cues. The macrostructural to the nanostructural framework of such complex tissue has engrossed the intervention of nanotechnology for efficient neo‐bone formation. Gold nanoparticles (GNPs) have recently gained interest in bone tissue regeneration due to their multimodal functionality. They are proven to modulate the properties of scaffolds and the osteogenic cells significantly. GNPs also influence different metabolic functions within the body, which directly or indirectly govern the mechanism of bone regeneration. Therefore, this review highlights nanoparticle‐mediated osteogenic development, focusing on different aspects of GNPs ranging from scaffold modulation to cellular stimulation. The toxic aspects of gold nanoparticles studied so far are critically explicated, while further insight into the advancements and prospects of these nanoparticles in bone regeneration is also highlighted. The nanometric scale mimics the microarchitectural arrangement of the osseous tissues. Incorporation of gold nanoparticles to the biomaterials used in bone tissue engineering enhances the mechanical properties of the scaffolds, influences the functions of the cells, and renders antibiotic, immunomodulatory, and angiogenic properties. The comprehensive understanding about the eminence of gold nanoparticles during tissue regeneration is elaborated in this review.
Owing to the encouraging pharmacological action and acceptable toxicity profile, Au(I) complexes have attracted growing interest in the application of disease treatment. In order to investigate their potential target proteins and related bioinformation, herein, we screened four Au(I) complexes and explored the binding proteins utilizing a competitive activity-based protein profiling (ABPP) strategy, including identification experiments and reactivity classification experiments, which offers a simple and robust method to identify the target proteins of Au(I) complexes. We quantified the target proteins of the four Au(I) complexes and found that most of proteins were associated with cancer. In addition, the newly Au(I)-binding proteins and biological gold-protein interaction pathways were exhibited. Furthermore, we estimated the correlation between target proteins of Au(I) complexes and various cancers, which will promote the development of the gold anticancer drugs.
Selenium is known to be an essential trace element with antioxidant activities. Its physiological role is mainly due to its co-translational incorporation into selenoproteins as selenocysteine (SeCys), referred to as the 21st amino acid. selenoprotein P (SELENOP) is the major selenoprotein in plasma with up to 10 SeCys in its sequence. Because of the chemical complexity of the serum matrix, the low abundance of selenoprotein P, and the occurrence of its putative multiple isoforms and post-translational modifications (e.g., glycosylation, Se-S and Se-Se bridges), the characterization of selenoprotein P requires the protein selective pre-concentration and custom-designed optimization of analytical methodology. Containing 10 SeCys residues, SELENOP is a potential preferential target for metallodrugs such as auranofin and cisplatin. The interactions have not been studied yet at the molecular level, despite considerable evidence that free selenols are biding sites for auranofin in another selenoprotein, thioredoxin reductase. The lack of studies of selenoprotein P is likely due to the fact that the protein cannot be heterologously expressed and needs to be purified from serum where it is present at ng/g concentration (as Se). This thesis presents the development of the method of purification of SELENOP from human serum using two-dimensional affinity chromatography. The two chromatographic steps using immobilized metal (cobalt) affinity and heparin affinity chromatography allowed the purification of SELENOP with excellent selectivity. The subsequent characterization of the purified SELENOP by nanoHPLC - electrospray ESI MS/MS allowed accounting for almost all selenocysteine-containing tryptic SELENOP peptides and the elucidation of some glycosylation sites. In addition, the recovery of selenium incorporated in SELENOP was quantitatively monitored, for the first time, at each step of the purification procedure. The recovery of SELENOP was 14% after all the purification steps. Selenium present in SELENOP was found to account for 35% of total selenium in serum.The purification of SELENOP allowed the first study of its interactions with auranofin and cisplatin containing, Au(I) and Pt(II), respectively. The protein metalation was observed after incubation of SELENOP with both metallodrugs. After digestion with trypsin, Au and Pt modifications were observed on the resulting peptides. Either in the case of auranofin and cisplatin, two SELENOP peptides were found to form Au or Pt adducts by Cy and SeCys binding (MS/MS characterization). These four peptides, specific to SELENOP, displayed different sequences.Moreover, to comparatively study the interactions of the Se-Se and S-S bridges with metallodrugs, vasopressin and its di-selenide analogues were used as model peptides. Their reactivity with auranofin and its strict analogues was investigated by LC-electrospray MS/MS. Evidence was obtained of the possible cleavage of the S-S and Se-Se bridges induced by Au(I) in the absence of reducing agents, in contrast to the previous studies requiring a prior reduction of the Se-Se bond to make it react with Au(I) compounds. In addition, we found that at high temperatures (70 °C), the sulfur and selenium atoms were metallated with the preferential binding of gold to selenium, the reaction not taking place under physiological conditions (37 °C).
Thioredoxin (Trx) is a central component of the redox control system that maintains the redox homeostasis critical for organism survival. Owing to its central role in survival, Trx is a prospective target for novel antimicrobial agents. Herein, we report a 1.45 Å high-resolution structure of Trx1 of Acinetobacter baumannii (abTrx1), an antibiotic-resistant pathogenic superbug. Although abTrx1 exhibited the canonical Trx fold, which consists of a four-stranded β-sheet surrounded by four α-helices, structural differences were detected in the loop forming the C-X-X-C redox center and the C-terminal. The unique CAPC sequence of the C-X-X-C motif in the abTrx1 redox center was characterized by mutagenesis. This study contributes to the field of drug designing against superbugs.
A phosphine gold(I) and phosphine‐phosphonium gold(I) complexes bearing a fluorescent coumarin moiety were synthesized and characterized. Both complexes displayed interesting photophysical properties: good molar absorption coefficient, good quantum yield of fluorescence, and ability to be tracked in vitro thanks to two‐photon imaging. Their in vitro and in vivo biological properties were evaluated onto cancer cell lines both human and murine and into CT26 tumor‐bearing BALB/c mice. They displayed moderate to strong antiproliferative properties and the phosphine‐phosphonium gold(I) complex induced significant in vivo anti‐cancer effect.
The gold(I) thiosulfate complex Na3[Au(S2O3)2]·2H2O has been studied in the gas phase by ESI MS, which showed the aggregate ion [Na2Au(S2O3)2]⁻ (m/z 466.78) at moderate capillary exit voltages, while at high capillary exit voltage (240 V) the major ion is the auride anion, Au⁻, (m/z 196.96), together with [AuS]⁻ (m/z 228.93) at low relative intensity. The observation of Au⁻ is surprising, and is related to the importance of relativistic effects in gold chemistry. No auride ions were produced when either [Me4N][AuCl4] or K[Au(SCN)2] were subjected to high capillary exit voltages. The ESI MS study of the corresponding silver thiosulfate complex Na3[Ag(S2O3)2]·2H2O showed a very low relative intensity argentide (Ag⁻) anion, due to the much lesser impact of relativistic effects in silver chemistry.
Purpose
: Methotrexate (MTX) is a first-line drug for rheumatoid arthritis (RA). Targeting of MTX to inflamed joints is essential to the prevention of potential toxicity and improving therapeutic effects. Gold nanoparticles (GNPs) are characterized by controllable particle sizes and good biocompatibilities, therefore, they are promising drug delivery systems. We aimed at developing a GNPs drug delivery system incorporating MTX and FA with strong efficacies against RA.
Methods
: MTX-Cys-FA was synthesized through solid-phase organic synthesis. Then, it was coupled with sulfhydryl groups in GNPs, thereby successfully preparing a GNPs/MTX-Cys-FA nanoconjugate with targeting properties. Physical and chemical techniques were used to characterize it. Moreover, we conducted its stability, release, pharmacokinetics, biodistribution and cell cytotoxicity, cell uptake, cell migration, as well as its therapeutic effect on CIA rats. Histopathology was conducted to investigate anti-RA effects of GNPs/MTX-Cys-FA nanoconjugates.
Results
: The GNPs/MTX-Cys-FA nanoconjugate exhibited a spherical appearance, had a particle size of 103.06 nm, a Zeta potential of -33.68 mV, drug loading capacity of 11.04 %, and an encapsulation efficiency of 73.61%. Cytotoxicity experiments revealed that GNPs had good biocompatibilities while GNPs/MTX-Cys-FA exhibited excellent drug-delivery abilities. Cell uptake and migration experiment showed that nanoconjugates containing folic acid (FA) by LPS activated RAW264.7 cells was significantly increased, and they exerted significant inhibitory effects on human fibroblast-like synoviocytes (HFLS) of RA (p<0.01). In addition, the nanoconjugate prolonged blood circulation time of MTX in collagen-induced arthritis (CIA) rats (p<0.01), enhanced MTX accumulation in inflamed joints (p<0.01), enhanced their therapeutic effects (p<0.01), and reduced toxicity to major organs (p<0.01).
Conclusion
: GNPs/MTX-Cys-FA nanoconjugates provide effective approaches for RA targeted therapeutic strategies.
Thioredoxin reductase (TrxR) is a central component in the thioredoxin system involved in catalyzing the reduction of thioredoxin, which is critical for organism survival. Because this system is essential, it is a promising target for novel antimicrobial agents. Herein, we solved the 1.9 Å high‐resolution structure of TrxR from Acinetobacter baumannii (AbTrxR), which is a gram‐negative, pathogenic bacterium and drug‐resistant superbug. AbTrxR was cofactor‐free and formed a dimer in solution. AbTrxR contained a longer dimerization loop2 and a shorter β7‐β8 connecting loop than other TrxRs. AbTrxR cofactor‐free form exhibited a flavin‐oxidizing (FO) conformation, whose NADPH domain was located close to the dimeric interface. This structural information might be helpful for development of new antibiotic agents targeting superbugs.
Three near-infrared (NIR-I) optical theranostic systems were synthesized, characterized and studied in vitro and in vivo. These original homo-bimetallic gold(I)-based aza-BODIPY complexes proved to be trackable through near-infrared optical imaging in cells and in mice. They display anti-proliferative properties in micromolar range against human and murine cancer cell lines (4T1, MDA-MB-231, CT26, and SW480). Moreover, the injection of the most promising theranostic agent in CT26 tumor-bearing BALB/c mice induced a significant anti-cancer activity.
Real-time monitoring of drug metabolism in vivo is of great significance to drug development and toxicology research. The purpose of this study is to establish a rapid and visual in vivo detection method for the detection of an intermediate metabolite of the gold (I) drug. Gold (I) drugs such as sodium aurothiomalate (AuTM) have anti-inflammatory effects in the treatment of rheumatoid arthritis. Gold(III) ions (Au³⁺) is the intermediate metabolite of gold medicine, and it is also the leading factor of side effects in the treatment of patients. However, the rapid reduction of Au³⁺ to Au⁺ by thiol proteins in organisms limits the in-depth study of metabolism of gold drugs in vivo. Here we describe a luminescence Au³⁺ probe (RA) based on ruthenium (II) complex for detecting Au³⁺ in vitro and in vivo. RA with large Stokes shift, good water solubility and biocompatibility was successfully applied to detect Au³⁺ in living cells and vivo by luminescence imaging, and to trap the fluctuation of Au³⁺ level produced by gold (I) medicine. More importantly, the luminescent probe was used to the detection of the intermediate metabolites of gold (I) drugs for the first time. Overall, this work offers a new detection tool/method for a deeper study of gold (I) drugs metabolite.
Rheumatoid arthritis (RA) is an autoimmune disease that is currently incurable. Clinical practice has shown significant benefits of combined therapies for RA treatment. This study aims to develop and demonstrate an efficient triple therapy for RA in vitro and in vivo. Three anti-inflammatory agents, NF-κB decoy oligodeoxynucleotides (ODNs), gold nanorods (GNRs), and dexamethasone (DEX), were encapsulated into folate (FA) modified liposomes (FA-lip(DEX+GNRs/ODNs)). The FA-lip(DEX+GNRs/ODNs) showed favorable physicochemical properties and efficient intracellular uptake by inflamed macrophages. Combined with laser irradiation, FA-lip(DEX+GNRs/ODNs) greatly reduced the secretion of proinflammatory proteins and oxidative factors in vitro. In adjuvant-induced arthritis (AIA) mice, FA-lip(DEX+GNRs/ODNs) achieved prolonged and enhanced accumulation at inflamed paws. FA-lip(DEX+GNRs/ODNs)+laser treatment reduced clinical arthritis scores and serum cytokine levels and protected cartilage. In summary, the triple therapy demonstrated significantly enhanced anti-inflammatory efficacy and is a promising strategy to treat RA via combined anti-inflammatory mechanisms.
Primary amebic meningoencephalitis, caused by brain infection with a free-living ameba Naegleria fowleri, leads to an extensive inflammation of brain and death within 3-7 days after symptoms begin. Treatment of primary amebic meningoencephalitis relies on amphotericin B in combination with other drugs, but use of amphotericin B is associated with severe adverse effects. Despite a fatality rate of over 97%, economic incentive to invest in development of antiamebic drugs by the pharmaceutical industry is lacking. Development of safe and rapidly acting drugs remains a critical unmet need to avert future deaths. Since FDA-approved anti-inflammatory and anti-arthritic drug auranofin is a known inhibitor of selenoprotein synthesis and thioredoxin reductase and the genome of N. fowleri encodes genes for both selenocysteine biosynthesis and thioredoxin reductases, we tested the effect of auranofin against N. fowleri strains of different genotypes from USA, Europe and Australia. Auranofin was equipotent against all tested strains with an EC50 of 1-2 µM. Our growth inhibition study at different time points demonstrated that auranofin is fast-acting and ~90% growth inhibition was achieved within 16 hours of drug exposure. A short exposure of N. fowleri to auranofin led to the accumulation of intracellular reactive oxygen species. This is consistent with auranofin’s role in inhibiting antioxidant pathways. Further, combination of auranofin and amphotericin B led to 95% of growth inhibition with 2- to 9-fold dose reduction for amphotericin B and 3- to 20-fold dose reduction for auranofin. Auranofin has the potential to be repurposed for the treatment of primary amebic meningoencephalitis.
Taking the articular and periarticular structures as a litmus test for gold-based nanoformulations, the potential of gold nanoparticles in protecting the normal physiological functions of these structures particularly in geriatric patients is one of the research areas of current interest. Aside from its use to make the traditional and fashionable ornaments for human usage, the gold metal is also known for its rich therapeutic activity. This is especially true when the gold is converted from its bulk form into nanosized form before its administering into the human body. Since it is the age of nanocomponents in medical and pharmaceutical research areas, this review is therefore mainly focused on nanoparticulate systems consisting of aurum. Accumulating research reports nevertheless show concrete evidence indicating the potential of gold-based nanoformulations to manage joint syndromes such as osteoarthritis and rheumatoid arthritis. This review embarks from preparation techniques and characterization methods to therapeutical application potentials of gold-based nanoformulations.
Nanoparticles, the building blocks of nanotechnology, have been widely utilized in various biomedical applications, such as detection, diagnosis, imaging, and therapy. However, another emerging, albeit under represented, area is the employment of nanoparticles as tools to understand cellular processes (e.g. oxidative stress-induced signaling cascades). Such investigations have enormous potential to characterize a disease from a different perspective and unravel some new features that otherwise would have remained a mystery. In this topical review, we summarize intrinsic biological properties of unmodified as well surface modified nanoparticles and discuss how such properties could be utilized to interrogate biological processes and provide a perspective for future evolution of this field.
The ability of aurothiomalate and auranofin to alter the production of several cellular mediators of inflammation by RAW264.7 macrophages, was compared with each other and that of gold nanoparticles (Au NPs). Addition of auranofin was found to have a pronounced ability to lower the production of reactive nitrogen and oxygen species (RNS and ROS respectively), as well as interleukin-10 (IL-10) and tumour necrosis factor (TNF), by macrophages that were subsequently treated with lipopolysaccharide (LPS) to stimulate production of the mediators. In contrast, prior treatment of the cells with either aurothiomalate or Au NPs had either little or no significant effect on production of RNS and ROS. Treatment of the macrophages with Au NPs had a small effect on production of TNF by cells that were subsequently stimulated with LPS; however, the effect was much smaller than that elicited by auranofin. Similarly, aurothiomalate had a small but significant effect on production of IL-10. Varying the size of the Au NPs or the identity of the protective sheath surrounding the nanoparticles did not have a significant effect on the production of RNS or ROS by LPS-stimulated macrophages. The results of some of these investigations are discussed in the light of other studies reported in the literature. In addition, results obtained by scanning electron microscopy and energy-dispersive X-ray spectroscopy are presented that provide evidence for the accumulation of gold within macrophages exposed to Au NPs.
The nucleocapsidic protein (NC) of orthoretroviruses has been considered as a promising target for antiretroviral drugs. Compounds, including zinc ejectors and metal derivatives capable to substitute the zinc ion in the nucleocapsidic zinc fingers, have been described. Auranofin, a gold(I) drug used for the treatment of rheumatoid arthritis, restricts the viral reservoir in the monkey AIDS model and induces containment of viral load following anti-retroviral therapy suspension. Here is reported a study of the interactions of auranofin with two synthetic CCHC-type peptides corresponding to the C-terminal zinc finger of the HIV-2 NC protein from two different isolates, using spectroscopic techniques and mass spectrometry. Both apopeptides interact very slowly with auranofin forming an Au-peptide complex. In contrast, as shown by mass spectrometry, the zinc-bound peptides interact on a shorter time-scale by forming an Et3P-Au-peptide complex and an Au-peptide complex. The two peptides show kinetic differences in the formation of zinc complexes, in Zn2+ binding constants as well in Zn2+ displacement by Au+.
An increased understanding in the pathophysiology of chronic inflammatory diseases, such as rheumatoid arthritis, reveals that the diseased tissue and the increased presence of macrophages and other overexpressed molecules within the tissue can be exploited to enhance the delivery of nanomedicine. Nanomedicine can passively accumulate into chronic inflammatory tissues via the enhanced permeability and retention phenomenon, or be surface conjugated with a ligand to actively bind to receptors overexpressed by cells within chronic inflammatory tissues, leading to increased efficacy and reduced systemic side-effects. This review highlights the research conducted over the past decade on using nanomedicine for potential treatment of rheumatoid arthritis and summarizes some of the major findings and promising opportunities on using nanomedicine to treat this prevalent and chronic disease.
Au25 clusters stabilized by tridecapeptides were firstly synthesized, which can well penetrate the cell membrane and exactly locate in the cytoplasm. Hence the Au clusters significantly suppress the TrxR1 activity in cytoplasm, and further induce the up-regulation of activated PARP level which carries the tumor cell apoptosis in Au dose dependent manner
Over the last two decades, native mass spectrometry (MS) has emerged as a valuable tool to study intact proteins and noncovalent protein complexes. Studied experimental systems range from small-molecule (drug)–protein interactions, to nanomachineries such as the proteasome and ribosome, to even virus assembly. In native MS, ions attain high m/z values, requiring special mass analyzers for their detection. Depending on the particular mass analyzer used, instrumental mass resolution does often decrease at higher m/z but can still be above a couple of thousand at m/z 5000. However, the mass resolving power obtained on charge states of protein complexes in this m/z region is experimentally found to remain well below the inherent instrument resolution of the mass analyzers employed. Here, we inquire into reasons for this discrepancy and ask how native MS would benefit from higher instrumental mass resolution. To answer this question, we discuss advantages and shortcomings of mass analyzers used to study intact biomolecules and biomolecular complexes in their native state, and we review which other factors determine mass resolving power in native MS analyses. Recent examples from the literature are given to illustrate the current status and limitations.
Figure
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The seleno-enzymethioredoxin reductase (TrxR) is a putative target for cytotoxic gold complexes. We investigated the mechanism of TrxR inhibition by a group of structurally diverse gold(III) compounds; the antiarthritic gold(I)drugsauranofin and aurothiomalate were also studied for comparison purposes. The tested compounds – either gold(III) or gold(I) – were found to produce potent enzyme inhibition only after pre-reduction of the enzyme with NADPH, indicating that TrxR inhibition is the result of protein structure modifications occurring upon cofactor binding. MALDI-ToF MS experiments on the intact enzyme provided evidence for extensive enzymemetallation, while experiments on trypsinized gold(III)-protein adducts identified a specific protein fragment – namely 236IGEHMEEHGIK246 – bearing an attached gold(I) ion. Independent mechanistic information on the system was derived from BIAM assays capable of monitoring selective metal binding to cysteine and/or selenocysteine residues. While the effects produced by auranofin could be essentially ascribed to gold(I) coordination to the active site selenol, the effects caused by the various gold(III) compounds were better interpreted in terms of oxidative protein damage.
A hard x-ray micro-nanoprobe has commenced operation at the Australian Synchrotron providing versatile x-ray fluorescence microscopy across an incident energy range from 4 to 25 keV. Two x-ray probes are used to collect mu-XRF and mu-XANES for elemental and chemical microanalysis: a Kirkpatrick-Baez mirror microprobe for micron resolution studies and a Fresnel zone plate nanoprobe capable of 60-nm resolution. Some unique aspects of the beamline design and operation are discussed. An advanced energy dispersive x-ray fluorescence detection scheme named Maia has been developed for the beamline, which enables ultrafast x-ray fluorescence microscopy.
Gold salts of monovalent gold (AU I) with a gold-sulfur ligand (aurothiolates) are the only form of gold currently in use for the management of rheumatoid arthritis (RA). Aurothiolates have limited success and are associated with a high incidence of side-effects. Metallic gold (AUo) is non-toxic and used extensively in dentistry. Monoatomic metallic gold is generated in vivo from AU I salts, during oxidation to trivalent gold (AU III). Monoatomic gold tends to form clusters of colloid particles. It is postulated that the active ingredient in aurotherapy is AUo and the side-effects are caused by AU III. To test this postulate, ten RA patients with long-standing erosive bone disease not responding to previous treatment were recruited from a private practice. Clinical and laboratory evaluations were performed prior to oral administration of 30 mg of colloidal AUo daily and thereafter weekly for 4 weeks and monthly for an additional 5 months. There was no clinical or laboratory evidence of toxicity in any of the patients. The effects of the colloidal gold on the tenderness and swelling of joints were rapid and dramatic, with a significant decrease in both parameters after the first week, which persisted during the study period. The mean scores for tenderness and swelling were, respectively, for pre- and post-1 week 58.8 and 18.2 ( p 0.01) and 42.5 and 15.9 ( p 0.01). By 24 weeks of gold administration, the mean scores were ten times lower than the pre-treatment levels being, respectively, 5.4 and 3.3 for tenderness and swelling. As a group, there was a significant improvement of functional status by 24 weeks of gold therapy: three patients were in clinical remission and one patient's status improved from totally disabled to full-time work. Evaluated individually, nine of the ten patients improved markedly after 24 weeks of colloidal gold at 30 mg/day. The cytokines interleukin6 (IL-6) and tumour necrosis factor alpha (TFN- alpha ), the immune complexes IgG and IgM, and rheumatoid factor were significantly suppressed by the colloidal gold. The results of this open trial in ten patients with long-standing erosive RA not responding to previous treatment support the postulate that colloidal gold is indeed the active ingredient in aurothiolate therapy and that the side-effects are mainly due to the AU III generated by oxidation of AU I. Colloidal AUo could become an effective and safer alternative to the aurothiolates in the management of RA patients.
The abundance and activation of macrophages in the inflamed synovial membrane/pannus significantly correlates with the severity of rheumatoid arthritis (RA). Although unlikely to be the 'initiators' of RA (if not as antigen-presenting cells in early disease), macrophages possess widespread pro-inflammatory, destructive, and remodeling capabilities that can critically contribute to acute and chronic disease. Also, activation of the monocytic lineage is not locally restricted, but extends to systemic parts of the mononuclear phagocyte system. Thus, selective counteraction of macrophage activation remains as efficacious approach to diminish local and systemic inflammation, as well as to prevent irreversible joint damage. AP = activator protein; BST = bone marrow stromal antigen; DMARD = disease-modifying antirheumatic drug; EBER(s) = Epstein-Barr virus-encoded small nuclear RNA(s); GM-CSF = granulocyte–macrophage colony-stimulating factor; GRO = growth-related oncogene protein; HLA = human leucocyte antigen; MCP = monocyte chemoattractant protein; MIP = macrophage inflammatory protein; NF-κB = nuclear factor-κB; NO = nitric oxide; RA = rheumatoid arthritis; TGF = transforming growth factor; Th = T-helper (cell); TIMP = tissue inhibitor of metalloproteinase; TNF = tumour necrosis factor.
Silver nanoparticles (AgNPs) and silver (Ag)-based materials are increasingly being incorporated into consumer products, and although humans have been exposed to colloidal Ag in many forms for decades, this rise in the use of Ag materials has spurred interest into their toxicology. Recent reports have shown that exposure to AgNPs or Ag ions leads to oxidative stress, endoplasmic reticulum stress, and reduced cell proliferation. Previous studies have shown that Ag accumulates in tissues as silver sulfides (Ag2S) and silver selenide (Ag2Se).
In this study we investigated whether exposure of cells in culture to AgNPs or Ag ions at subtoxic doses would alter the effective metabolism of selenium, that is, the incorporation of selenium into selenoproteins.
For these studies we used a keratinocyte cell model (HaCat) and a lung cell model (A549). We also tested (in vitro, both cellular and chemical) whether Ag ions could inhibit the activity of a key selenoenzyme, thioredoxin reductase (TrxR).
We found that exposure to AgNPs or far lower levels of Ag ions led to a dose-dependent inhibition of selenium metabolism in both cell models. The synthesis of protein was not altered under these conditions. Exposure to nanomolar levels of Ag ions effectively blocked selenium metabolism, suggesting that Ag ion leaching was likely the mechanism underlying observed changes during AgNP exposure. Exposure likewise inhibited TrxR activity in cultured cells, and Ag ions were potent inhibitors of purified rat TrxR isoform 1 (cytosolic) (TrxR1) enzyme.
Exposure to AgNPs leads to the inhibition of selenoprotein synthesis and inhibition of TrxR1. Further, we propose these two sites of action comprise the likely mechanism underlying increases in oxidative stress, increases endoplasmic reticulum stress, and reduced cell proliferation during exposure to Ag.
Palladium (Pd), platinum (Pt), and gold (Au) are noble metals, two of which have established medical use. Pt has anticancer efficacy, predominantly as cisplatin, whereas the gold compound auranofin is used against arthritis. Both compounds inhibit the selenoprotein thioredoxin reductase (TrxR), but Pd has not been studied in this regard. Using salts of Pd, Pt, and Au as well as cisplatin and auranofin we found that Pd and Au were strikingly more potent inhibitors of recombinant TrxR1 than Pt. The TrxR-related nonselenoprotein glutathione reductase in pure form (but less so in a cellular context), as well as cellular thioredoxin (Trx) activities, were inhibited by the gold salt KAuCl(4) but were little affected by auranofin or the other compounds. In an analysis of three cancer cell lines, the extent of inhibition of TrxR activity and decrease in cell viability depended upon the choice of both noble metal and ligand and also seemed independent of p53 status. During treatment of cells with cisplatin, covalent complexes of TrxR1 with either Trx1 or TRP14 (Trx-related protein of 14kDa) were formed, as verified by Western blot analyses and mass spectrometry. These results reveal that Au and Pd are strong inhibitors of TrxR, but Pt and cisplatin trigger highly specific cellular effects on the Trx system, including covalent cross-linking of TrxR1 with Trx1 and TRP14.
In order to better understand the physical basis of the biological activity of nanoparticles (NPs) in nanomedicine applications and under conditions of environmental exposure, we performed an array of photophysical measurements to quantify the interaction of model gold NPs having a wide range of NP diameters with common blood proteins. In particular, absorbance, fluorescence quenching, circular dichroism, dynamic light scattering, and electron microscopy measurements were performed on surface-functionalized water-soluble gold NPs having a diameter range from 5 to 100 nm in the presence of common human blood proteins: albumin, fibrinogen, gamma-globulin, histone, and insulin. We find that the gold NPs strongly associate with these essential blood proteins where the binding constant, K, as well as the degree of cooperativity of particle--protein binding (Hill constant, n), depends on particle size and the native protein structure. We also find tentative evidence that the model proteins undergo conformational change upon association with the NPs and that the thickness of the adsorbed protein layer (bare NP diameter <50 nm) progressively increases with NP size, effects that have potential general importance for understanding NP aggregation in biological media and the interaction of NP with biological materials broadly.
Nanotechnology has been used to provide advanced biomedical research tools in diagnostic imaging and therapy, which requires targeting of nanoparticles (NPs) to individual cells and subcellular compartments. However, a complete understanding of the intracellular uptake, transport, and subcellular distribution of nanostructured materials remains limited. Hence, gold NPs were explored as a model system to study the intracellular behavior of NPs in real time. Our results show that the cellular uptake of gold NPs is dependent on their size and surface properties. The NPs were transported in vesicles of 300-500 nm diameter within the cytoplasm. The average velocity and diffusion coefficient of the vesicles containing NPs were 10.2 (+/-1.8) microm/hr and 3.33 (+/-0.52) microm 2/hr, respectively. Analysis of the time-dependent intracellular spatial distribution of the NPs demonstrated that they reside in lysosomes (final degrading organelles) within 40 minutes of incubation. These findings can be used to tailor nanoscale devices for effective cell targeting and delivery.
The bacterial pathogen Vibrio parahaemolyticus utilizes a type III secretion system to cause death of host cells within hours of infection. We report that cell death is completely independent of apoptosis and occurs by a mechanism in which injection of multiple type III effectors causes induction of autophagy, cell rounding, and the subsequent release of cellular contents. Autophagy is detected by the appearance of lipidated light chain 3 (LC3) and by increases in punctae and vacuole formation. Electron microscopy reveals the production of early autophagic vesicles during infection. Consistent with phosphoinositide 3 (PI3) kinase playing a role in autophagy, treatment of infected cells with a PI3 kinase inhibitor attenuates autophagy in infected cells. Because many effectors are injected during a V. parahaemolyticus infection, it is not surprising that the presence of a sole PI3 kinase inhibitor does not prevent inevitable host-cell death. Our studies reveal an infection paradigm whereby an extracellular pathogen uses its type III secretion system to cause at least three parallel events that eventually result in the proinflammatory death of an infected host cell.
• host-pathogen
• type III secretion
• apoptosis
• effector
• inflammatory
In a significant fraction of the Escherichia coli cytosolic proteins, the N-terminal methionine residue incorporated during the translation initiation step is excised. The N-terminal methionine excision is catalyzed by methionyl-aminopeptidase (MAP). Previous studies have suggested that the action of this enzyme could depend mainly on the nature of the second amino acid residue in the polypeptide chain. In this study, to achieve a systematic analysis of the specificity of MAP action, each of the 20 amino acids was introduced at the penultimate position of methionyl-tRNA synthetase of E. coli and the extent of in vivo methionine excision was measured. To facilitate variant protein purification and N-terminal sequence determination, an expression shuttle vector based on protein fusion with beta-galactosidase was used. From our results, methionine excision catalyzed by MAP is shown to obey the following rule: the catalytic efficiency of MAP, and therefore the extent of cleavage, decreases in parallel with the increasing of the maximal side-chain length of the amino acid in the penultimate position. This molecular model accounts for the rate of N-terminal methionine excision in E. coli, as deduced from the analysis of 100 protein N-terminal sequences.
Endothelial adhesion molecules play an important role in the tissue recruitment of leukocytes in inflammatory conditions such as rheumatoid arthritis. We have investigated the effect of the antirheumatic drug gold sodium thiomalate on adhesion molecule protein and mRNA expression in cultured human endothelial cells. Gold sodium thiomalate inhibited cytokine (TNF, IL-1, IL-4)-stimulated expression of vascular cell adhesion molecule-1 and E-selectin but not intercellular adhesion molecule-1 on endothelial cells. Gold sodium thiomalate also suppressed TNF-stimulated increases in vascular cell adhesion molecule-1 and E-selectin mRNA levels but had no effect on intercellular adhesion molecule-1 mRNA. Thiomalate (mercaptosuccinate), but not gold thioglucose or D-penicillamine, mimics the effect of gold sodium thiomalate at equimolar concentrations. We propose that the inhibition of vascular cell adhesion molecule-1 and E-selectin expression by gold sodium thiomalate is due to its thiomalate and not its gold component. Gold sodium thiomalate has a direct effect on endothelial adhesion molecule expression, and this may contribute to its antiinflammatory activity.
Human thioredoxin reductase is a pyridine nucleotide-disulfide oxidoreductase closely related to glutathione reductase but
differing from the latter in having a Cys-SeCys (selenocysteine) sequence as an additional redox center. Because selenoproteins
cannot be expressed yet in heterologous systems, we optimized the purification of the protein from placenta with respect to
final yield (1–2 mg from one placenta), specific activity (42 units/mg), and selenium content (0.94 ± 0.03 mol/mol subunit).
The steady state kinetics showed that the enzyme operates by a ping-pong mechanism; the value of k
cat was 3330 ± 882 min−1, and the K
mvalues were 18 μm for NADPH and 25 μm forEscherichia coli thioredoxin. The activation energy of the reaction was found to be 53.2 kJ/mol, which allows comparisons of the steady state
data with previous pre-steady state measurements. In its physiological, NADPH-reduced form, the enzyme is strongly inhibited
by organic gold compounds that are widely used in the treatment of rheumatoid arthritis; for auranofin, the K
i was 4 nm when measured in the presence of 50 μmthioredoxin. At 1000-fold higher concentrations, that is at micromolar levels, the drugs also inhibited human glutathione
reductase and the selenoenzyme glutathione peroxidase.
The mammalian thioredoxin reductases (TrxRs) are a family of selenium-containing pyridine nucleotide-disulphide oxidoreductases with mechanistic and sequence identity, including a conserved -Cys-Val-Asn-Val-Gly-Cys- redox catalytic site, to glutathione reductases. TrxRs catalyse the NADPH-dependent reduction of the redox protein thioredoxin (Trx), as well as of other endogenous and exogenous compounds. The broad substrate specificity of mammalian TrxRs is due to a second redox-active site, a C-terminal -Cys-SeCys- (where SeCys is selenocysteine), that is not found in glutathione reductase or Escherichia coli TrxR. There are currently two confirmed forms of mammalian TrxRs, TrxR1 and TrxR2, and it is possible that other forms will be identified. The availability of Se is a key factor determining TrxR activity both in cell culture and in vivo, and the mechanism(s) for the incorporation of Se into TrxRs, as well as the regulation of TrxR activity, have only recently begun to be investigated. The importance of Trx to many aspects of cell function make it likely that TrxRs also play a role in protection against oxidant injury, cell growth and transformation, and the recycling of ascorbate from its oxidized form. Since TrxRs are able to reduce a number of substrates other than Trx, it is likely that additional biological effects will be discovered for TrxR. Furthermore, inhibiting TrxR with drugs may lead to new treatments for human diseases such as cancer, AIDS and autoimmune diseases.
The enzyme thioredoxin reductase (TrxR) is attracting much interest as a potential target for cancer therapy. The presence of a selenium atom in the catalytic site makes it sensitive to inhibition by electrophilic molecules, including the AuI complex auranofin [2,3,4,6-tetra-O-acetyl-1-thio-β-D-glucopyranosato-S-(triethylphosphane)gold]. The reactions between auranofin and models of thiol and selenol nucleophiles present in TrxR (PhSH and PhSeH) have been investigated in chloroform and methanol by means of 1H, 31P, and 77Se NMR spectroscopy. In chloroform, auranofin undergoes ligand substitution of the tetraacetylthioglucose moiety by a PhS or PhSe group. The reaction is reversible in both cases, but it is characterized by widely different equilibrium constants (ca. 1 for S and at least 103 for Se). In polar solvents, such as methanol, the reaction is more complex, and the phosphane moiety also undergoes ligand exchange. Some features have been clarified through the investigation of Et3PAuCl. The elementary processes involved have been characterized by DFT calculations.
Biological properties of a series of aminophosphine-thiolate gold(I) complexes [Au(SR)(PPh2NHpy)] [Ph2PNHpy=2-(diphenylphosphinoamino)pyridine; HSR=2-mercaptopyridine (2-HSpy) (3), 2-mercaptonicotinic acid (2-H2-mna) (4), 2-thiouracil (2-HTU) (5) or 2-thiocytosine (2-HTC) (6)] and [Au(SR){PPh2NH(Htrz)}] [Ph2PNH(Htrz)=3-(diphenylphosphinoamino)-1,2,4-triazole]; HSR=2-mercaptopyridine (2-HSpy) (7), 2-thiocytosine (2-HTC) (8) or 6-thioguanine (6-HTG) (9) have been studied. Their antitumor properties have been tested in vitro against two tumor human cell lines, HeLa (derived from cervical cancer) and MCF-7 (derived from breast cancer), using a metabolic activity test (3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide, MTT). Some of them showed excellent cytotoxic activity. With the aim to obtain more information about the mechanisms of action of these derivatives, the interactions of complexes 3, 5, 7 and 9 with thioredoxin reductase in HeLa cells were studied. They showed a potent inhibition of thioredoxin reductase activity. In order to complete this study, interactions of the complexes with calf thymus (CT-) DNA and with different bacterial DNAs, namely the plasmid pEMBL9 and the promoter region of the furA (ferric uptake regulator A) gene from Anabaena sp. PCC 7120 were investigated. Although interactions of complexes with CT-DNA have been verified, none of them cause significant changes in its structure.
NMR investigations of isotopically-labelled glycine show that AuIII induces deamination and subsequent decarboxylation of the amino acid with formation of glyoxylic acid, NH4+, formic acid, CO2 and metallic gold.
The effect of selenium compounds on extracellular redox modulating capacity was studied in murine macrophage RAW 264.7 cells and differentiated human THP-1 monocytes. The arylselenium compounds benzeneselenol (PhSeH), dibenzyl diselenide (DBDSe), diphenyl diselenide (DPDSe) and ebselen were capable of inducing extracellular cysteine accumulation via a cystine- and glucose-dependent process. Extracellular cysteine production was dose-dependently inhibited by glutamate, an inhibitor of cystine/glutamate antiporter (Xc- transporter), supporting the involvement of Xc- transporter for cystine uptake in the above process. These arylselenium compounds also induced cellular thioredoxin reductase (TrxR) expression, particularly at the exofacial surface of cells. TrxR1 knockdown using small interfering RNA attenuated TrxR increases and cysteine efflux induced in cells by DPDSe. Sodium selenite (Na2SeO3), selenomethionine (SeMet), seleno-L-cystine (SeCySS), and Se-methylselenocysteine (MeSeCys) did not have these effects on macrophages under the same treatment conditions. The effects of organoselenium compounds on extracellular reodx may contribute to the known, but inadequately understood, biological effects of selenium compounds.
Gold−silver alloy nanoparticles with varying mole fractions are prepared in aqueous solution by the co-reduction of chlorauric acid HAuCl4 and silver nitrate AgNO3 with sodium citrate. As the optical absorption spectra of their solutions show only one plasmon absorption it is concluded that mixing of gold and silver leads to a homogeneous formation of alloy nanoparticles. The maximum of the plasmon band blue-shifts linearly with increasing silver content. This fact cannot be explained by a simple linear combination of the dielectric constants of gold and silver within the Mie theory. On the other hand, the extinction coefficient is found to decrease exponentially rather than linearly with increasing gold mole fraction xAu. Furthermore, the size distribution of the alloy nanoparticles is examined using transmission electron microscopy (TEM). High-resolution TEM (HRTEM) also confirms the formation of homogeneous gold−silver alloy nanocrystals.
Seleno-cysteamine, seleno-cysteine and seleno-ethanoic acid react with aurothiomalate in aqueous solution to give [Au(selenoate)2]– complexes. The13C-n.m.r. chemical shift differences between thiolates and their analogous selenoates complexes with gold(I), as [Au(thiolate)2]– and [Au(selenoate)2]–, are compared and the [Au(Secysteine)2]– complex was found to be the most stable.
In RAW 264.7 cells, a murine macrophage cell line, treatment with lipopolysaccharide (1 to 10 ng/ml) stimulated production of nitric oxide (NO), which was inhibited by l-NG-monomethyl-l-arginine acetate, an inhibitor of NO synthase. Auranofin, an orally active chrysotherapeutic agent, also inhibited the lipopolysaccharide-induced NO production in a concentration-dependent manner (0.3 to 3 μM). Other gold salts such as aurothioglucose and aurothiomalate had no effect. Western blot analysis demonstrated that the lipopolysaccharide (10 ng/ml)-induced expression of inducible NO synthase protein was inhibited by auranofin as well as by the protein synthesis inhibitor cycloheximide. In addition, the lipopolysaccharide-induced increase in the level of mRNA for inducible NO synthase was also lowered by auranofin. Furthermore, auranofin showed no direct effect on the conversion of []arginine to []citrulline by the cell lysate. These findings indicate that auranofin inhibits lipopolysaccharide-induced NO production by suppressing the expression of inducible NO synthase.
Gold compounds are widely used in the treatment of rheumatoid arthritis, but their mechanisms of action remain unclear. We demonstrate here that auranofin (AF) (0.1–3 μM), but neither the hydrophilic gold compounds aurothiomalate (ATM) and aurothioglucose nor methotrexate or D-penicillamine, inhibits the induction of interleukin 1β and tumor necrosis factor (TNF) α mRNA and protein by either zymosan, lipopolysaccharide (LPS), or various bacteria in mouse macrophages. The auranofin-mediated inhibition of the induction of TNF-α mRNA was stronger than that of interleukin (IL) 1β mRNA. AF, but not the other drugs, also inhibited zymosan-induced mobilization of arachidonate. The fact that AF inhibited the induction of mRNA for both these proinflammatory cytokines, irrespective of which stimulus was used, may indicate that it affects some common signal transduction step vital to their induction.
Because disease-modifying antirheumatic drugs might exert part of their effects on adhesion of polymorphonuclear neutrophils (PMN) to endothelial cells, this being the first step for PMN migration to inflammatory lesions, we evaluated such drug effects in vitro. Gold sodium thiomalate (GSTM) impaired the ability of interleukin 1β (IL-1β)-stimulated human umbilical vein endothelial cells (HUVEC) to express E-selectin and to bind PMN but had no effect on the expression of intercellular adhesion molecule 1 (ICAM-1) or on hyperadhesivity of N-formyl-methionyl-leucyl-phenylalanine (fMLP)-stimulated PMN. Auranofin (AF) interacted with HUVEC and PMN adhesiveness but in opposite directions: this drug hampered IL-1β-induced HUVEC hyperadhesiveness and expression of E-selectin and intercellular adhesion molecule 1, but augmented PMN adherence and CD18 expression. The net effect of auranofin was a reduction of cytokine-driven adhesiveness and enhancement of formylpeptide-induced adhesion. Salazopyrin did not affect HUVEC or PMN adhesiveness or E-selectin and intercellular adhesion molecule 1 expression. Thus, the gold-containing drugs modulated HUVEC and PMN adhesiveness by different mechanisms but ones involving surface adhesion molecules.
After a preliminary survey with the electron microscope of various preparations of colloidal gold, a study was made of the process of nucleation and growth in gold colloids. It was shown that nucleating agents may be identified with reducing agents which form a mixed polymer with chlorauric ion before the reduction to the nucleus takes place. It was also shown that the law of growth is exponential. The average size, the deviation from the average size and the character of the particle size distribution curve are determined by the amount of gold, the nucleation process and the law of growth.
Since the dawn of civilization, elemental gold and gold compounds have been revered and utilized by Shamen and medical practitioners alike for many varied pathological problems. In the 20(th) century following the observations of Jacques Forestier, injectable gold compounds were successfully used for the treatment of rheumatoid arthritis. Of the many compounds developed, gold sodium thiomalate has been the most extensively studied by basic scientists and by clinicians. In the1980s, the oral gold compound auranofin showed promise as a therapeutic contender to injectable gold, but the clinical side effect profile and fear of long term effects of immune suppression by auranofin, resulted in gold sodium thiomalate continuing as the preferred gold compound for rheumatoid treatment. However, the increased use and demonstration of effectiveness of low dose Methotrexate (MTX) in rheumatoid treatment over the last 20 years has resulted in a significant decline in the use of injectable gold sodium thiomalate, this despite the claims and evidence that it remains a useful agent in the management of rheumatoid arthritis. Several authors still contend that the injectable gold compounds can still play a valuable role, and indeed may be the correct first choice in the management of rheumatoid arthritis.
Metallic gold (Au degrees ) is a likely biotransformation product of monovalent gold, Au(I) whenever it is dissociated from in vivo ligands, Au degrees being formed either by bioreduction or by spontaneous dismutation (with co-production of trivalent gold). This review discusses the preparation and some biologically relevant properties of colloidal metallic gold (CMG) in its nano-particulate form. Tyndall's purple, a well characterised preparation of CMG, shows potent anti-arthritic activity in rats, approximately 10(3) times that of sodium aurothiomalate (Myocrysin). Even more remarkable is its broader spectrum of action in rats compared to this classic DMARD.
Macrophage-derived nitric oxide (NO) plays an important role in protection against microbial infection in immune responses. Overproduction of NO by inducible nitric synthase (iNOS) is known to be closely correlated with the pathology of a variety of diseases and inflammations. In this study, we investigated the inhibitory effect of polyethylene glycol coated gold nanoparticles (GNP) on NO production and its molecular mechanism in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. It was found that GNP inhibited LPS-induced NO production and iNOS expression in RAW264.7 cells. Furthermore, GNP suppressed LPS-induced activation of NF-kappaB through the inhibition of Akt activity. GNP also inhibited LPS-induced phosphorylation of signal transducer and activator of transcription 1 (STAT1) via down-regulation of interferon-beta (IFN-beta) expression. Our results suggest that GNP inhibits NO production and iNOS expression through blocking the activation of NF-kappaB and STAT1 in LPS-stimulated RAW264.7 cells.
Terpyridine-platinum(II) (TP-Pt(II)) complexes are known to possess DNA-intercalating activity and have been regarded as potential antitumor agents. However, their cytotoxic mechanism remains unclear. To investigate the possible mechanism, a series of TP-Pt(II) compounds were prepared and their biological activities assessed. The DNA binding activities of the aromatic thiolato[TP-Pt(II)] complexes were stronger than the aliphatic 2-hydroxylethanethiolato(2,2':6',2''-terpyridine)platinum(II) [TP(HET)]. TP-Pt(II) complexes inhibited topoisomerase IIalpha or topoisomerase I activity at IC(50) values of about 5 microM and 10-20 microM, respectively, whereas the human thioredoxin reductase 1 (hTrxR1) activity was inhibited with IC(50) values in the range of 58-78 nM. At the cellular level, they possessed cytotoxicity with IC(50) values between 7 and 19 microM against HeLa cells. Additionally, using X-ray crystallography and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry, we elucidated that the TP-Pt(II) complexes inhibited hTrxR1 activity by blocking its C-terminal active-site selenocysteine. Therefore, TP-Pt(II) complexes possess inhibitory activities against multiple biological targets, and they may be further studied as anticancer agents.
Electrospray ionisation mass spectrometry was used to analyse the reactions of metal compounds with mixtures of selected proteins. Three representative medicinally relevant compounds, cisplatin, transplatin and the organometallic ruthenium compound RAPTA-C, were reacted with a pool of three proteins, ubiquitin, cytochrome c and superoxide dismutase, and the reaction products were analysed using high-resolution mass spectrometry. Highly informative electrospray ionisation mass spectra were acquired following careful optimisation of the experimental conditions. The formation of metal-protein adducts was clearly observed for the three proteins. In addition, valuable information was obtained on the nature of the protein-bound metallofragments, on their distribution among the three different proteins and on the binding kinetics. The platinum compounds were less reactive and considerably less selective in protein binding than RAPTA-C, which showed a high affinity towards ubiquitin and cytochrome c, but not superoxide dismutase. In addition, competition studies between cisplatin and RAPTA-C showed that the two metallodrugs have affinities for the same amino acid residues on protein binding.
Aurosomes are lysosomal bodies containing gold. Colloidal gold produces aurosomes containing spherical electron-dense granules. Soluble gold salts produce aurosomes containing lamellar, filamentous and rod-like profiles studded with particles and granules. This morphological pattern is quite distinctive, and is not affected or altered by which particular soluble gold salt is administered, by which route it is administered or in which species or cell type the aurosome occurs. In the skin of patients treated with soluble gold salts the characteristics electron-dense formations indicating the presence of gold are often found in compound melanosomes and other lysosomes in the dermal macrophages; while in the synovial membrane these characteristic electron-dense deposits are seen in the numerous lysosomes that develop in the rheumatoid synovial membrane. Thus it would appear that while aurosomes may vary somewhat in morphology, the electron dense contents indicating the presence of gold have a fairly constant morphology.
Auranofin (AF), a recently introduced oral antirheumatic coordinated gold compound, was investigated for its antitumor potential. Due to certain similarities with the antitumor-coordinated compound, cis-Diamminedichloroplatinum II, we studied the effects of AF on cell proliferation. These studies included assessing DNA, RNA, and protein synthesis as measured by incorporation of 3H-thymidine, 3H-uridine, and 3H-leucine, respectively, into HeLa cells. AF was shown to exert a dose-dependent inhibition on DNA synthesis and to inhibit 3H-thymidine uptake more rapidly and persistently than 3H-uridine or 3H-leucine uptake at a gold concentration of 75--100 micrograms/dl. These three parameters were inhibited with a 24-hour exposure to 100 micrograms/dl. The inhibition of 3H-thymidine uptake in HeLa pretreated for 6 hours with 50 or 100 micrograms/dl of gold was found to be irreversible. No change in tracer uptake was observed in the acid-soluble pool or in the uptake of 3H-2-deoxy-D-glucose in these cells. Furthermore, HeLa cells demonstrated marked reductions in viability and oxygen uptake after exposure to AF. Dose-dependent surface morphological changes, e.g., blebbing, pitting, were noted in these cells after a brief treatment period. These results suggest this coordinated gold compount exerts a significant inhibitory effect on essential biological processes and functions.
Intramuscular injection of sodium aurothiomalate (Myochrysine) produced lysosomes (aurosomes) of a characteristic morphology distinct and different from other lysosomes. The aurosome is characterised by particle and granulestudded electron-dense membranous formations which present as rod-like and curled structures. Intramuscularly injected sodium aurothiomalate in immature rabbits leads to the production of aurosomes in the articular cartilage chondrocytes and synovial intimal cells and subsynovial macrophages. In mature rabbits aurosomes develop only in the synovial intimal cells and subsynovial macrophages, but not in the chondrocytes.
We performed 2 metaanalyses of placebo-controlled and comparative clinical trials to examine the relative efficacy and toxicity of methotrexate (MTX), injectable gold, D-penicillamine (DP), sulfasalazine (SSZ), auranofin (AUR), and antimalarial drugs, the second-line drugs most commonly used to treat rheumatoid arthritis (RA). For the efficacy study, we applied a set of inclusion criteria and focused on trials which provided information on tender joint count, erythrocyte sedimentation rate, or grip strength. We found 66 clinical trials that contained 117 treatment groups of interest, and for each drug, we combined the treatment groups. For each outcome, results showed that AUR tended to be weaker than other second-line drugs. The results of the 3 outcome measures were synthesized into a composite measure of outcomes, and AUR was significantly weaker than MTX (P = 0.006), injectable gold (P < 0.0001), DP (P < 0.0001), and SSZ (P = 0.009) and was slightly, but not significantly, weaker than antimalarial agents (P = 0.11). We also found heterogeneity among antimalarial agents, in that patients treated with chloroquine did better than those treated with hydroxychloroquine. We found little difference in efficacy between MTX, injectable gold, DP, and SSZ. A power analysis showed that a trial should contain at least 170 patients per treatment group to successfully differentiate between more effective and less effective (e.g., AUR) second-line drugs. None of the reported interdrug comparative trials we reviewed were this large.
The roles of anti-arthritic gold(I)-thiolate drugs such as disodium aurothiomalate ('Myocrisin') in the modulation or promotion of oxygen radical-mediated oxidative damage in vivo are reviewed. In particular, the precise molecular mechanisms by which these novel second-line agents exert their therapeutic effects are discussed in terms of (i) the direct and indirect control of enzymes involved in the generation or scavenging of reactive oxygen species (ROS) such as superoxide ion, hydrogen peroxide and hydroxyl radical, (ii) the protection of proteins and relevant enzyme systems against attack by ROS and (iii) their direct involvement in the production (at appropriate 'target' sites) or scavenging of ROS in vivo. In addition, the role of the orally-effective gold(I)-phosphine complex auranofin in the control of oxidative damage in rheumatoid arthritis is also discussed.
Auranofin (AF), an orally active, antiarthritic agent, modulates the functional activities of macrophages in vivo and in vitro. To better understand the molecular mechanism of action of auranofin with macrophages we have investigated its cellular association, intracellular distribution, and efflux with RAW 264.7 cells using auranofin radiolabeled within the triethylphosphine (Et3P) [3H], the gold [195Au] or the tetraacetylthioglucose (TATG)[14C] moieties of the molecule. Evaluation of the effects of auranofin on RAW 264.7 cells demonstrates that (1) cellular association of this compound was concentration, time and temperature dependent; (2) cellular association of AF was inhibited by N-ethylmaleimide but not by 2,4-dinitrophenol and NaF; (3) cellular association and uptake of Au and Et3P into cells was reduced when the drug was preincubated with increasing concentrations of fetal calf serum and albumin; (4) no tetraacetylthioglucose from the auranofin molecule became cell associated whereas the Au and Et3P moieties were internalized and distributed between the nuclear, cytosolic and membrane fractions of cells; and (5) efflux of Au and Et3P from RAW 264.7 cells was time and temperature dependent. Based on these data we propose a model, a sequential ligand exchange process, that describes the molecular interactions of auranofin and possibly other gold compounds with these cells.
A series of gold(I) coordination complexes including analogues of the antiarthritic agent auranofin 1 were evaluated for in vitro cytotoxic potency against both B16 melanoma cells and P388 leukemia cells and in vivo antitumor activity against P388 leukemia in mice. A number of the complexes showed potent cytotoxic activity in vitro and antitumor activity in vivo, with the phosphine-coordinated gold(I) thiosugar complexes demonstrating the greatest in vitro and in vivo activity. The data compiled for 63 complexes of the general structural formula LAuX provide the basis for the following observations: potent in vitro cytotoxic activity is observed for substituted (phosphine) gold complexes, lack of potency in vitro correlates well with lack of antitumor activity, potent cytotoxicity in vitro is not necessarily predictive of activity in vivo, in vivo antitumor activity is generally optimized by ligation of Au(I) with a substituted phosphine and a thiosugar.
Gold sodium thiomalate and auranofin, anti-rheumatic gold-containing compounds, induced some stress proteins in cultured mouse peritoneal macrophages. The enhanced synthesis of two proteins, heme oxygenase (a 34-kDa protein) and a 23-kDa protein, was particularly prominent. The 23-kDa protein induced by the gold compounds was identical to that found in macrophages exposed to oxidative stress and was suggested to have antioxidant activity. Intraperitoneal injection of gold sodium thiomalate and oral administration of auranofin to mice induced enhanced synthesis of these proteins in peritoneal macrophages analyzed ex vivo. These data suggest that increased synthesis of these proteins may have a role in mediating the pharmacologic effect of these agents.
Three new findings are reviewed that help to understand the mechanisms of action of anti-rheumatic gold drugs, such as disodium aurothiomalate (Na2Au(I)TM): i) We found that Na2Au(I)TM selectively inhibits T-cell receptor-mediated antigen recognition by murine CD4+ T-cell hybridomas specific for antigenic peptides containing at least two cysteine residues. Presumably, Au(I) acts as a chelating agent forming linear complexes (Cys-Au(I)-Cys) which prevents correct antigen-processing and/or peptide recognition by the T-cell receptor, ii) We were able to show that Au(I) is oxidized to Au(III) in mononuclear phagocytes, such as macrophages. Because Au(III) rapidly oxidizes protein and itself is re-reduced to Au(I), this may introduce an Au(I)/Au(III) redox system into phagocytes which scavenges reactive oxygen species, such as hypochlorous acid (HOCl) and inactivates lysosomal enzymes, iii) Pretreatment with Au(III) of a model protein antigen, bovine ribonuclease A (RNase A), induced novel antigenic determinants recognized by CD4+ T lymphocytes. Analysis of the fine specificity of these "Au(III)-specific" T-cells revealed that they react to RNase peptides that are not presented to T-cells when the native protein, i.e., not treated with Au(III), is used as antigen. The T-cell recognition of these cryptic peptides did not require the presence of gold. This finding has important implications for understanding the pathogenesis of allergic and autoimmune responses induced by gold drugs. Taken together, our findings indicate that Au(I) and Au(III) each exert specific effects on several distinct functions of macrophages and the activation of T-cells. These effects may explain both the desired anti-inflammatory and the adverse effects of antirheumatic gold drugs.
To examine the expression of the thioredoxin (TRX)-thioredoxin reductase (TR) system in patients with rheumatoid arthritis (RA) and patients with other rheumatic diseases.
Levels of TRX in plasma and synovial fluid (SF) were measured using enzyme-linked immunosorbent assay. Cellular distribution of TRX was determined by flow cytometry and histochemistry. Cellular expression of TR was studied by in situ messenger RNA (mRNA) hybridization. The effect of oxidative stress and tumor necrosis factor alpha (TNF alpha) on TRX expression by cultured rheumatoid fibroblast-like synoviocytes was studied.
Significantly increased TRX levels were found in the SF from 22 patients with RA, when compared with plasma levels in the same patients (P < 0.001) and compared with SF TRX levels in 15 patients with osteoarthritis (P < 0.001), 13 patients with gout (P < 0.05), and 9 patients with reactive arthritis (P < 0.0001). The presence of TRX could be demonstrated within the SF-derived mononuclear cells and synovial tissue (ST) of RA patients. Concordantly, expression of TR mRNA was observed in the ST of these patients. Stimulation of synovial fibroblast-like synoviocytes with either H2O2 or TNF alpha induced an increase in the production of TRX.
The data demonstrate significantly increased concentrations of TRX in the SF and ST of RA patients when compared with the levels in patients with other joint diseases. Evidence is presented that the local environment in the rheumatic joint contributes to increased TRX production. Based on its growth-promoting and cytokine-like properties, it is proposed that increased expression of TRX contributes to the disease activity in RA.
The mammalian thioredoxin reductases (TrxRs) are a family of selenium-containing pyridine nucleotide-disulphide oxidoreductases with mechanistic and sequence identity, including a conserved -Cys-Val-Asn-Val-Gly-Cys- redox catalytic site, to glutathione reductases. TrxRs catalyse the NADPH-dependent reduction of the redox protein thioredoxin (Trx), as well as of other endogenous and exogenous compounds. The broad substrate specificity of mammalian TrxRs is due to a second redox-active site, a C-terminal -Cys-SeCys- (where SeCys is selenocysteine), that is not found in glutathione reductase or Escherichia coli TrxR. There are currently two confirmed forms of mammalian TrxRs, TrxR1 and TrxR2, and it is possible that other forms will be identified. The availability of Se is a key factor determining TrxR activity both in cell culture and in vivo, and the mechanism(s) for the incorporation of Se into TrxRs, as well as the regulation of TrxR activity, have only recently begun to be investigated. The importance of Trx to many aspects of cell function make it likely that TrxRs also play a role in protection against oxidant injury, cell growth and transformation, and the recycling of ascorbate from its oxidized form. Since TrxRs are able to reduce a number of substrates other than Trx, it is likely that additional biological effects will be discovered for TrxR. Furthermore, inhibiting TrxR with drugs may lead to new treatments for human diseases such as cancer, AIDS and autoimmune diseases.
Glutathione reductase (GR) plays a pivotal role in maintaining glutathione (GSH) in its reduced form. We have isolated a cDNA for rat GR and constructed a baculovirus system to produce recombinant GR on a large scale. This protein was purified by simple, two-step chromatographic procedure using DE52 and 2',5'-ADP Sepharose. Tissue distributions of GR were examined by Northern and Western blotting with a rabbit antibody to purified GR. GR was expressed in the order of reactivity; kidney, colon, liver, stomach, etc. Western blot analysis showed that both the cytosolic and the mitochondrial fractions of liver homogenate gave immunoreactive bands of similar size. This indicates that the same gene products exist in these fractions. Since nitric oxide (NO) produced under inflammatory conditions causes nitrosative stress and affects the redox states of surrounding tissues, we investigated the effects of NO donors on the enzymatic activities of purified GR. S-nitrosoglutathione (GSNO), 3-morpholinosydnonimine N-ethylcarbamide (SIN-1), and S-nitroso-N-acetyl-D,L-penicillamine (SNAP) at 1 mM gave 39, 15, and 12% inhibitions, respectively. In RAW 264.7 cells the GR activity was reported to be inhibited by GSNO.