Gold Compound Auranofin Research Articles

Mercury binding to proteins disclosed by ESI MS experiments: The case of three organomercurials

Solution interactions of three organomercury compounds, i.e., methylmercury chloride, thimerosal and phenylmercury acetate, with a group of biochemically relevant proteins, namely cytochrome c (Cyt c), ribonuclease A (RNase A), carbonic anhydrase I (hCA I), superoxide dismutase (SOD), and serum albumin (HSA), were investigated using an established ESI MS approach. Temporal analysis of sample aliquots provided insight into the binding kinetics, while comparative analysis of the obtained mass spectra disclosed adduct formation of each mercurial with the tested proteins and the relative abundance of the species. The three organomercurials bind, exclusively and tightly, to free cysteine residues as no binding was observed in the case of proteins lacking such groups. hCA I, SOD and HSA formed distinct mercury adducts, preserving the Hg bound alkyl/aryl ligands; yet, the three organomercurials displayed significant differences in reactivity in relation to their chemical structure. The investigation was then extended to analyze the reactions with the C-terminal dodecapeptide of the enzyme human thioredoxin reductase, which contains a characteristic selenol-thiol moiety: tight Hg binding was observed. Notably, this peptide was able to remove effectively and completely the alkyl/aryl ligands of the three tested organomercurials; this behavior may be relevant to the detoxification mechanism of organomercurials in mammals. Finally, a competition experiment was carried out to establish whether protein bound mercury centers may be displaced by other competing metals. Interestingly, and quite unexpectedly, we observed that a protein bound mercury fragment may be partially displaced from its coordination site in hCA I by the medicinal gold compound auranofin.

Auranofin Modulates Thioredoxin Reductase/Nrf2 Signaling in Peripheral Immune Cells and the CNS in a Mouse Model of Relapsing-Remitting EAE.

Multiple sclerosis (MS) is one of the most prevalent chronic inflammatory autoimmune diseases. It causes the demyelination of neurons and the subsequent degeneration of the central nervous system (CNS). The infiltration of leukocytes of both myeloid and lymphoid origins from the systemic circulation into the CNS triggers autoimmune reactions through the release of multiple mediators. These mediators include oxidants, pro-inflammatory cytokines, and chemokines which ultimately cause the characteristic plaques observed in MS. Thioredoxin reductase (TrxR) and nuclear factor erythroid 2-related factor 2 (Nrf2) signaling plays a crucial role in the regulation of inflammation by modulating the transcription of antioxidants and the suppression of inflammatory cytokines. The gold compound auranofin (AFN) is known to activate Nrf2 through the inhibition of TrxR; however, the effects of this compound have not been explored in a mouse model of relapsing-remitting MS (RRMS). Therefore, this study explored the influence of AFN on clinical features, TrxR/Nrf2 signaling [heme oxygenase 1 (HO-1), superoxide dismutase 1 (SOD-1)] and oxidative/inflammatory mediators [IL-6, IL-17A, inducible nitric oxide synthase (iNOS), myeloperoxidase (MPO), nitrotyrosine] in peripheral immune cells and the CNS of mice with the RR type of EAE. Our results showed an increase in TrxR activity and a decrease in Nrf2 signaling in SJL/J mice with RR-EAE. The treatment with AFN caused the amelioration of the clinical features of RR-EAE through the elevation of Nrf2 signaling and the subsequent upregulation of the levels of antioxidants as well as the downregulation of oxidative/pro-inflammatory mediators in peripheral immune cells and the CNS. These data suggest that AFN may be beneficial in the treatment of RRMS.

Abstract 1014: Repurposing the anti-rheumatic gold compound auranofin for high-grade serous ovarian cancer therapy

Abstract Auranofin (AF) is a gold compound approved in 1985 as a main treatment against rheumatoid arthritis. However, as new anti-rheumatoid agents displaced the use of AF, there have been studies aiming to repurpose the drug to treat other diseases, including cancer. In this study, we investigated the potentiality of AF to impair the growth of high-grade serous ovarian cancer (HGSOC) cells regardless of platinum (CDDP) sensitivity. Preliminary studies utilizing a colorimetric proliferation assay revealed that HGSOC cells that are clinically sensitive (PEO1) or resistant (PEO4) to CDDP-based chemotherapy are both equally sensitive to the growth inhibition induced by AF. The resistant factor estimated for AF-defined as the ratio between IC50 values calculated for the resistant cells and for the sensitive ones—was close to one, suggesting that CDDP-resistant PEO4 cells are equally sensitive to AF than CDDP-sensitive PEO1 cells. As expected, the resistant factor for CDDP was 12-fold higher in PEO4 compared to PEO1. The toxicity of AF against HGSOC cells was further studied exposing PEO1 to various concentrations of AF and assessing cell number, cell viability, and cell cycle traverse after 72 h. Results showed that AF, in a dose-dependent manner, blocked cell proliferation and caused accumulation of cells with hypo-diploid DNA content, suggesting that AF-induced cytotoxicity was associated not only with inhibition of cell proliferation but also with cell death likely driven via apoptosis. We further studied the long-term or residual toxicity of AF by exposing PEO1 cells that were alive, after 72 h treatment with AF, to a clonogenic survival assay. The results clearly demonstrated that even if not dying within 72 h, the cells were affected in a manner not compatible with long-term cell cycle progression as demonstrated by the formation of ‘abortive' colonies (i.e. colonies with few cells that show abnormal phenotypes). Finally, as AF is a pro-oxidant agent, we evaluated whether AF-induced cell death was mediated by oxidative stress by incubating PEO1 cells with AF in the presence or absence of the anti-oxidant N-acetyl-cysteine (NAC). PEO1 cells treated with AF and NAC showed no decrease in cell viability and cell number in comparison to PEO1 cells treated with AF alone. This data reveals that AF most likely induces cell death in a reactive oxygen species (ROS)-dependent manner. In conclusion, our results suggest that AF can equally impair the growth of CDDP-sensitive or -resistant HGSOC cells, and that its toxicity is likely mediated by oxidative stress. Citation Format: Farah H. Abdalbari, Alicia A. Goyeneche, Elvis Martinez-Jaramillo, Siham Sabri, Carlos M. Telleria. Repurposing the anti-rheumatic gold compound auranofin for high-grade serous ovarian cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1014.

Comparative reactivity of medicinal gold(i) compounds with the cyclic peptide vasopressin and its diselenide analogue

The reactions of the medicinal gold(i) compound auranofin and its close analogues with vasopressin and the diselenide analogue were comparatively investigated by LC-electrospray MS/MS.

Antibiofilm efficacy of the gold compound auranofin on dual species biofilms of Staphylococcus aureus and Candida sp.

Staphylococcus aureus (a bacterial pathogen) and Candida sp. (opportunistic fungi) are two clinically relevant biofilm-forming microbes responsible for a majority of community- and nosocomial-acquired infections. Dual species biofilm formation between S. aureus and Candida sp. extremely enhances the antimicrobial resistance of the micro-organisms and is difficult to treat with antibiotic therapy. Hence, it is crucial to explore new antimicrobial agents. Auranofin (AF) is a mixed ligand gold compound and has recently been repurposed as an antibacterial and antifungal agent. However, the effects of AF against dual species biofilm have remained largely untested. In the present study, by constructing biofilms on microplates and urinary catheter surfaces, AF showed strong planktonic cells and biofilm inhibitory effects against mono- and dual culture models of S. aureus and Candida albicans but only exhibited moderate antibiofilm effects on Candida parapsilosis. Auranofin could be synergistic with subminimal inhibitory concentrations of amphotericin B against S. aureus+C. albicans/C. parapsilosis dual biofilms. Auranofin also showed effective antimicrobial effects on vancomycin-resistant strains. However, the antimicrobial effects of AF were decreased in the presence of heat-inactivated foetal bovine serum. In summary, AF could effectively inhibit S. aureus and C. albicans mono- and dual biofilm formation in vitro. Coexistence between Staphylococcus aureus and Candida sp. in dual biofilms leads to increased resistance to some conventionally used antimicrobials, indicating a need for alternative treatments. This study demonstrates the potential for the Au-containing compound AF in the treatment of dual biofilm infections and encourages further investigation of this treatment for clinical use.

The electrochemical profiles of Auranofin and Aubipyc, two representative medicinal gold compounds: A comparative study

A micro-electrochemical reaction cell was coupled to an electrospray mass spectrometer in order to track redox transformations for two representative medicinal gold compounds - i.e. [(2,3,4,6-tetra-O-acetyl-1-thio-β-D-glucopyranosato-S)(triethylphosphine)gold(I)] and [Au(bipydmb-H)(OH)][PF6] (where bipydmb-H is deprotonated 6-(1,1-dimethylbenzyl)-2,2′-bipyridine), known as Auranofin and Aubipyc respectively - in parallel to square wave voltammetry (SWV) measurements. Irreversible oxidation of thio-glucose tetraacetate was the dominant reaction for the gold(I) compound Auranofin; oxidation was accompanied by hydrolysis leading to progressive deacetylation. Two main active forms were identified for this prodrug: the triethylphosphinegold(I) cation and a gold(I) thioglucose species, with a variable number of acetyl groups. For the gold(III) complex Aubipyc irreversible reduction of the gold(III) center was highlighted, accompanied by a ligand exchange process. The free gold(I) ion is proposed to be the final species that subsequently binds transport proteins in the bloodstream. Molecule specific mass spectrometry determinations provide complementary data to square wave voltammetry helping to understand the nature of the electrochemical conversions of complex or unstable compounds. Finally, it was possible to establish that oxidizing conditions during drug preparation and administration should be avoided in the case of Auranofin; conversely, reduction conditions typical for the blood or the cytosol environment are suitable to obtain the active gold(I) species from the gold(III) complex Aubipyc.

Antiproliferative effects of two gold(I)-N-heterocyclic carbene complexes in A2780 human ovarian cancer cells: a comparative proteomic study

Au(NHC) and Au(NHC)2, i.e. a monocarbene gold(I) complex and the corresponding bis(carbene) complex, are two structurally related compounds, endowed with cytotoxic properties against several cancer cell lines. Herein, we explore the molecular and cellular mechanisms at the basis of their cytotoxicity in A2780 human ovarian cancer cells. Through a comparative proteomic analysis, we demonstrated that the number of modulated proteins is far larger in Au(NHC)2-treated than in Au(NHC)-treated A2780 cells. Both gold compounds mainly affected proteins belonging to the following functional classes: protein synthesis, metabolism, cytoskeleton and stress response and chaperones. Particularly, Au(NHC)2 gave rise to an evident upregulation of several glycolytic enzymes. Moreover, only Au(NHC)2 triggered a net impairment of respiration and a metabolic shift towards glycolysis, suggesting that mitochondria are relevant cellular targets. We also found that both carbenes, similarly to the gold(I) compound auranofin, caused a strong inhibition of the seleno-enzyme thioredoxin reductase (TrxR). In conclusion, we highlighted that coordination of two carbene ligands to the same gold(I) center greatly enhances the antiproliferative effects of the resulting compound in comparison to the monocarbene derivative. Moreover, TrxR inhibition and metabolic impairment seem to play a major role in the Au(NHC)2 cytotoxicity. Overall, these antiproliferative effects were also confirmed on other two human ovarian cancer cell lines (i.e. SKOV3 and IGROV1).

Differential expression of disulfide reductase enzymes in a free-living platyhelminth (Dugesia dorotocephala).

A search of the disulfide reductase activities expressed in the adult stage of the free-living platyhelminth Dugesia dorotocephala was carried out. Using GSSG or DTNB as substrates, it was possible to obtain a purified fraction containing both GSSG and DTNB reductase activities. Through the purification procedure, both disulfide reductase activities were obtained in the same chromatographic peak. By mass spectrometry analysis of peptide fragments obtained after tryptic digestion of the purified fraction, the presence of glutathione reductase (GR), thioredoxin-glutathione reductase (TGR), and a putative thioredoxin reductase (TrxR) was detected. Using the gold compound auranofin to selectively inhibit the GSSG reductase activity of TGR, it was found that barely 5% of the total GR activity in the D. dorotocephala extract can be assigned to GR. Such strategy did allow us to determine the kinetic parameters for both GR and TGR. Although It was not possible to discriminate DTNB reductase activity due to TrxR from that of TGR, a chromatofocusing experiment with a D. dorotocephala extract resulted in the obtention of a minor protein fraction enriched in TrxR, strongly suggesting its presence as a functional protein. Thus, unlike its parasitic counterparts, in the free-living platyhelminth lineage the three disulfide reductases are present as functional proteins, albeit TGR is still the major disulfide reductase involved in the reduction of both Trx and GSSG. This fact suggests the development of TGR in parasitic flatworms was not linked to a parasitic mode of life.

A new thioredoxin reductase with additional glutathione reductase activity in Haemonchus contortus

We report, herein, the purification to homogeneity and the biochemical and kinetic characterization of HcTrxR3, a new isoform of thioredoxin reductase (TrxR) from Haemonchus contortus. HcTrxR3 was found to have a relative molecular weight of 134,000, while the corresponding value per subunit obtained under denaturing conditions, was of 67,000. By peptide mass spectrophotometric analysis, HcTrxR3 was determined to have 99% identity with the H. contortus HcTrxR1 although, and most importantly, they are different in their amino acid sequence in two amino acid positions: 48 (isoleucine instead of leucine) and 460 (leucine instead of proline). The enzyme catalyzes NADPH-dependent reduction of DTNB and, unexpectedly, it follows the pattern of glutathione reductases (GR) performing the reduction of oxidized glutathione (GSSG) to reduced glutathione using NADPH as the reducing cofactor. Hence, it is important to highlight this enzyme's new and unexpected condition that makes so special and one our main finding. Enzyme Kcat values for DTNB, GSSG and NADPH were 12, 3 and 8 s−1, respectively. HcTrxR3 developed, into specific TrxR substrates: ebselen and sodium selenite, with activity at 0.5 and 0.068 (U/mg), respectively; and 0.044 (U/mg) for S-nitrosoglutathione through its GR activity. The enzyme was inhibited by gold compound auranofin (AU), a selective inhibitor of thiol-dependent flavoreductases. Although HcTrxR3 has both TrxR and GR activity as thioredoxin glutathione reductase (TGR) does, it is a TrxR because it has no glutaredoxin domain and it does not develop any hysteretic behavior as does TGR. The importance of this new enzyme is potential to further clarify the detoxification and haemostasis redox mechanism in H. contortus. Likewise, this enzyme could also be a protein model to recognize more differences between TrxR and GR.

Auranofin-induced oxidative stress causes redistribution of the glutathione pool in Taenia crassiceps cysticerci

Previously, we have studied the effect of the gold-compound auranofin (AF) on both thioredoxin-glutathione reductasa (TGR) activity and viability of Taenia crassiceps cysticerci. It was demonstrated that micromolar concentrations of AF were high enough to fully inhibit TGR and kill the parasites. In this work, the dynamics of changes in the glutathione pool of T. crassiceps cysticerci following the addition of AF, was analyzed. A dose-dependent decrease in the internal glutathione concentration, concomitant with an increase in ROS production was observed. These changes were simultaneous with the formation of glutathione-protein complexes and the export of glutathione disulfide (GSSG) to the culture medium. Incubation of cysticerci in the presence of both AF and N-acetyl cysteine (NAC) prevents all the above changes, maintaining cysticerci viability. By contrast, the presence of both AF and buthionine sulfoximine (BSO) resulted in a potentiation of the effects of the gold compound, jeopardizing cysticerci viability. These results suggest the lethal effect of AF on T. crassiceps cysticerci, observed at micromolar concentrations, can be explained as a consequence of major changes in the glutathione status, which results in a significant increase in the oxidative stress of the parasites.

Novel use of old drug: Anti-rheumatic agent auranofin overcomes imatinib-resistance of chronic myeloid leukemia cells

Patients with chronic myeloid leukemia (CML) are commonly treated with a specific inhibitor of BCR-ABL tyrosine kinase, imatinib mesylate (IM). Unfortunately, CML patients develop IM-resistance, which has emerged as a significant clinical problem. Somatic mutations, especially T315I mutation, in BCR-ABL kinase domain represent the most common mechanism underlying drug resistance to tyrosine kinase inhibitors (TKI), including imatinib. Thus, it is urgent to develop novel therapeutic strategies to overcome TKI-resistance. The anti-rheumatic gold (I) compound Auranofin (AF), was recently approved by US Food and Drug Administration for Phase II clinical trials to treat leukemia. In a recent study, we discovered that AF can selectively inhibit 19S proteasome-associated deubiquitinases (UCHL5 and USP14), which mediates its anticancer effects. More recently studies we have shown that AF inhibits the growth of both Bcr-Abl wild-type cells and IM-resistant Bcr-Abl-T315I mutation cells in vitro and in vivo. AF-induced Bcr-Abl down regulation is associated with diminished mRNA expression and caspase-dependent Bcr-Abl cleavage. More importantly, we unraveled that AF cytotoxicity is mediated by proteasome inhibition rather than previously suspected reactive oxygen species (ROS) generation. These findings support that AF overcomes IM-resistance through Bcr/Abl-dependent and -independent mechanisms, identifying a potentially new strategy for cancer treatment.

Purification and kinetic analysis of cytosolic and mitochondrial thioredoxin glutathione reductase extracted from Taenia solium cysticerci

Thioredoxin glutathione reductases (TGRs) (EC 1.8.1.9) were purified to homogeneity from the cytosolic (cTsTGR) and mitochondrial (mTsTGR) fractions of Taenia solium, the agent responsible for neurocysticercosis, one of the major central nervous system parasitic diseases in humans. TsTGRs had a relative molecular weight of 132,000, while the corresponding value per subunit obtained under denaturing conditions, was of 62,000. Specific activities for thioredoxin reductase and glutathione reductase substrates for both TGRs explored were in the range or lower than values obtained for other platyhelminths and mammalian TGRs. cTsTGR and mTsTGR also showed hydroperoxide reductase activity using hydroperoxide as substrate. Km(DTNB) and Kcat(DTNB) values for cTsTGR and mTsTGR (88 µM and 1.9 s−1; 45 µM and 12.6 s−1, respectively) and Km(GSSG) and Kcat(GSSG) values for cTsTGR and mTsTGR (6.3 µM and 0.96 s−1; 4 µM and 1.62 s−1, respectively) were similar to or lower than those reported for mammalian TGRs. Mass spectrometry analysis showed that 12 peptides from cTsTGR and seven from mTsTGR were a match for gi|29825896 thioredoxin glutathione reductase [Echinococcus granulosus], confirming that both enzymes are TGRs. Both T. solium TGRs were inhibited by the gold compound auranofin, a selective inhibitor of thiol-dependent flavoreductases (I50 = 3.25, 2.29 nM for DTNB and GSSG substrates, respectively for cTsTGR; I50 = 5.6, 25.4 nM for mTsTGR toward the same substrates in the described order). Glutathione reductase activity of cTsTGR and mTsTGR exhibited hysteretic behavior with moderate to high concentrations of GSSG; this result was not observed either with thioredoxin, DTNB or NADPH. However, the observed hysteretic kinetics was suppressed with increasing amounts of both parasitic TGRs. These data suggest the existence of an effective substitute which may account for the lack of the detoxification enzymes glutathione reductase and thioredoxin reductase in T. solium, as has been described for very few other platyhelminths.

Antiproliferative effect of gold(I) compound auranofin through inhibition of STAT3 and telomerase activity in MDA-MB 231 human breast cancer cells

Signal transducer and activator of transcription 3 (STAT3) and telomerase are considered attractive targets for anticancer therapy. The in vitro anticancer activity of the gold(I) compound auranofin was investigated using MDA-MB 231 human breast cancer cells, in which STAT3 is constitutively active. In cell culture, auranofin inhibited growth in a dose-dependent manner, and N-acetyl-L-cysteine (NAC), a scavenger of reactive oxygen species (ROS), markedly blocked the effect of auranofin. Incorporation of 5-bromo-2’-deoxyuridine into DNA and anchorage-independent cell growth on soft agar were decreased by auranofin treatment. STAT3 phosphorylation and telomerase activity were also attenuated in cells exposed to auranofin, but NAC pretreatment restored STAT3 phosphorylation and telomerase activity in these cells. These findings indicate that auranofin exerts in vitro antitumor effects in MDA-MB 231 cells and its activity involves inhibition of STAT3 and telomerase. Thus, auranofin shows potential as a novel anticancer drug that targets STAT3 and telomerase. [BMB Reports 2013; 46(1): 59-64]

Stimulation of human TRPA1 channels by clinical concentrations of the antirheumatic drug auranofin

Gold compounds, which were widely used to treat rheumatoid arthritis, have been recently used as experimental agents for tumor treatment. Transient receptor potential (TRP) ankyrin repeat 1 (TRPA1) is a Ca(2+)-permeable ion channel that senses acute and inflammatory pain signals. Electrophilic compounds such as mustard oil and cinnamaldehyde activate TRPA1 by interacting with TRPA1 cysteine residues. Here we investigate the effects of the gold compound auranofin (AUR) on TRPA1 channels. Intracellular Ca(2+) and whole cell patch-clamp recordings were performed on human embryonic kidney cells transiently expressed with TRPA1, TRP melastatin 8 (TRPM8), and vanilloid type TRP (TRPV1-4) channels. AUR stimulated TRPA1 in a concentration-dependent manner with a half-maximum potency of around 1.0 μM. The AUR-induced response was effectively blocked by HC030031, a TRPA1 antagonist. On the other hand, AUR failed to activate TRPM8 and TRPV1-4 channels, which are highly expressed in sensory neurons as nociceptors. The stimulatory effect on TRPA1 channels depended on the C414, C421, C621, and C633 cysteine residues and not on the inhibition of thioredoxin reductase by AUR. Moreover, AUR effectively activated TRPA1 channels expressed in human differentiated neuroblastoma cell lines. The study shows that AUR is a potent stimulator of TRPA1 channels.

The Rheumatoid Arthritis Drug Auranofin Has Significant in Vitro Activity in MCL and DLCL and Is Synergistic with a Glutathione Depleting Agent

Abstract 1658There is great interest in repurposing drugs currently available in the pharmacopeia to other indications like cancer as such an approach would significantly shorten the time needed and cost encumbered to provide new and effective cancer therapeutics. One such drug is the gold compound Auranofin (AF), which is FDA approved as a treatment for rheumatoid arthritis (RA). We demonstrate that AF induces MCL cell death with an LD50 of 1000 nM, 500 nM and 90 nM for Granta and Jeko cell lines, and cells derived from a patient biopsy, respectively. The increased sensitivity of primary MCL patient specimens to AF is confirmed on 4 additional patient samples tested. AF similarly induced DLBCL cell death with an LD50 of 500 nM, 500 nM and 1000 nM for OCI Ly-10, SUDHL-6 and −4 cell lines, respectively. Exposure of Granta cells to an AF concentration that induced cell death resulted in the generation of reactive oxygen species (ROS). Pre-treatment of these cells with N-acetyl-cysteine (NAC) or glutathione (GSH) abrogated both ROS generation and the induction of cell death supporting the notion that AF induces NHL cell death through a redox dependent pathway. Although AF does increase mitochondrial membrane permeability (although not through the classical permeability transition pore), the major mechanism through which AF induces NHL cell death is activation of the extrinsic apoptotic pathway. In this regard, AF induces the activation of caspases 7 and 8 and results in PARP cleavage, all of which are blocked by NAC. Despite the fact that AF is a known inhibitor of thioredoxin reductase (TR), its cytotoxic effect is independent of TR inhibition, as we observe no difference in the response to AF in U266 multiple myeloma cells transfected with an expression vector which results in the over-expression of TR. Given the redox dependence of AF-induced cytotoxicity we hypothesized that inhibition of the glutathione system with buthionine sulphoximine (BSO) would further augment AF induced cell death. To test this hypothesis, Granta cells were exposed to both AF and BSO. Significant synergistic interactions of these drugs were seen when tested in a Laska analysis of synergy. For example, whereas the LD50 for AF alone in Granta cells was 1000 nM, the LD50 for AF in combination with 5 μM BSO was 200nm; for Ly-10 cells, the LD50 for AF±BSO was 400 nm vs. 190 nM, respectively, and for SUDHL-6, 411 nM vs. 185 nM, respectively. Similar results were seen in MCL cells flow cytometrically sorted from patient biopsy specimens. As observed in studies using AF alone, the cytotoxic effects of the combination were blocked with both NAC and GSH. Similar to results with AF alone, the synergistic effects of AF and BSO on NHL cytotoxicity were independent of their effects on TR. Exposure of Granta cells to AF resulted in NF-κB inhibition. NF-κB was further inhibited with concomitant exposure to BSO over-expression of relA in Granta cells, however, had no effect on AF and BSO induced cell death, suggesting the synergistic effects of AF and BSO on NHL cell death may be NF-κB-independent. Finally, we demonstrate that AF and BSO modify free thiols on the plasma membrane. As we have recently shown that the redox agent parthenolide induces NHL death in part by modifying surface protein thiols, AF and BSO may induce NHL cell death through a similar mechanism. In summary, AF induces both MCL and DLCL cell death through a redox-dependent pathway that involves the extrinsic apoptotic pathway. Profound synergy is seen with concomitant depletion of glutathione by BSO. The data presented above, along with the fact that AF is a drug having a favorable safety profile in patients, and is an FDA-approved drug for the treatment of RA makes it an attractive candidate for further study as a single agent and in rational combination with other redox active drugs. Disclosures:No relevant conflicts of interest to declare.

The Gold Compound Auranofin Induces Oxidative Stress and Apoptosis in Primary CLL Cells Independent of Classic Prognostic Markers and the Protective Effect of the Tissue Microenvironment

AbstractAbstract 865Chronic lymphocytic leukemia (CLL), remains incurable, except by allogeneic stem cell transplantation. While multiple chemotherapeutic options are available, relapse and eventually development of chemotherapy resistance are common. Drug resistance of CLL cells is mediated by genetic alterations including deletion of chromosomes 17p (p53 locus) and 11q (ATM locus), and by pro-survival signals emanating from the tissue microenvironment. Thus new therapies are needed that are active against tumor cells in protective tissue sites and those with adverse cytogenetic features. To identify possible new therapies for CLL, we conducted a high throughput screening assay of primary CLL cells exposed to 7 step-wise dilutions of each member of a 2,816 compound library (including FDA-approved drugs and known bio-actives from commercial suppliers). As a control for “unspecific” cytotoxicity, we used PBMCs from normal donors. Of 102 compounds efficacious against CLL cells of all 6 patients tested, 5 were not or minimally toxic in normal lymphocytes. One of these 5 compounds is Auranofin (AF), an FDA-approved oral disease-modifying anti-rheumatic drug.Next, we investigated the efficacy of AF against an expanded cohort of CLL samples (n=50) and investigated its mechanism of action. Samples were characterized for IGHV mutational status, ZAP70 expression, CD38 expression, and FISH cytogenetics. PBMCs were tested against serial dilutions of AF (final concentrations: 0.125–4μM) for 24 hours in flat bottom 96-well plates. MTS assay was performed to quantify cell viability. A dose-dependent response was observed in all samples. The inhibitory concentration at 50% (IC50) was calculated using GraphPad Prism Software. For a majority of samples, the IC50 was <1μM, a concentration easily achieved in vivo. AF cytotoxicity was independent of the IGHV mutational status, ZAP70 and CD38 expression, and cytogenetic subgroups (including 17p and 11q deletion). To assess whether AF is selectively cytotoxic for CLL cells, we determined drug-induced apoptosis of PBMCs of CLL patients for CLL cells (identified as CD19+/CD3-; >95% of those are CLL cells) and T-cells (CD19-/CD3+) separately using Annexin V staining. AF reduced viability of T-cells on average by 10% and of CLL cells by 50% (n=8; P<.05 for comparison between T and CLL cells). To determine the effect of the microenvironment on AF toxicity, we cultured primary CLL cells in the presence or absence of Nurse-Like Cells (NLC), a model of the protective effect of the microenvironment. NCLs enhance CLL cell viability and confer resistance to chemotherapeutic agents such as fludarabine. However, co-culture on NLCs did not protect CLL cells from the cytotoxic effect of AF. To investigate the effect of AF on tumor biology, total RNA (2.5 μg) from CLL PBMCs treated for 4 and 10 hours with AF in vitro was profiled on Human Genome U133 Plus 2.0 arrays (Affymetrix) and compared to untreated controls. There were 81 genes whose expression changed >2-fold at P<.01, most prominently several genes encoding heat-shock proteins and antioxidant enzymes such as heme oxygenase and thiroredoxin reductase. In Ingenuity Pathway analysis, the most significantly upregulated pathway in response to AF was the NRF2-mediated Oxidative Stress Response (P<.001). Combined, this suggests that AF induced oxidative stress resulting in the upregulation of a detoxifying response. To study the effect of AF on redox balance we used dihydroethidium (DHE) and concomitantly measured cell viability using 3,3′-dihexyloxacarbocyanine iodide (DiOC6) by flow cytometry. AF induced a time- and dose-dependent increase in ROS production in CLL cells that correlated with the onset of apoptosis. In conclusion, AF is selectively cytotoxic for CLL cells and its activity is independent of classic mechanisms of chemotherapy resistance. Based on these observations AF is now being studied in a clinical proof of concept phase IIa clinical trial for patients with relapsed/refractory CLL (NCT01419691). Disclosures:No relevant conflicts of interest to declare.

Strategies for the Biological Evaluation of Gold Anticancer Agents

Since the introduction of the monomeric orally bioavailable anti-arthritic gold compound auranofin in 1985, and the success of the platinum-based anti-cancer drugs, there has been a great deal of interest in the use of gold compounds for cancer therapy. However this early promise has not materialized into an approved drug in spite of extensive and innovative efforts in gold chemistry. Therefore, in the light of this lack of success, the strategies for the biological evaluation of potential gold-based anti-cancer drugs are discussed. It is proposed that the biological testing strategy should be multi-faceted incorporating an understanding of the molecular properties of the compounds under investigation related to their behaviour in a biological environment, an evaluation of their comparative in vitro potency against tumor cells, ascertaining the biochemical mechanism of action and target identification to aid in medicinal chemistry design, evaluation of in vivo activity in relevant tumor models, and an understanding of their toxicological and pharmacokinetic properties. This strategy will be exemplified with work on Au(III) cyclometallated complexes in which an integrated approach to the search for new metal-based anticancer drugs was adopted, incorporating in vitro screening, in vivo human tumor xenograft models, and mechanistic studies. The importance of mechanistic studies which have led to the identification of new molecular targets for gold drugs, and in vivo evaluation are emphasized.

Substrate and inhibitor specificities differ between human cytosolic and mitochondrial thioredoxin reductases: Implications for development of specific inhibitors

The cytosolic and mitochondrial thioredoxin reductases (TrxR1 and TrxR2) and thioredoxins (Trx1 and Trx2) are key components of the mammalian thioredoxin system, which is important for antioxidant defense and redox regulation of cell function. TrxR1 and TrxR2 are selenoproteins generally considered to have comparable properties, but to be functionally separated by their different compartments. To compare their properties we expressed recombinant human TrxR1 and TrxR2 and determined their substrate specificities and inhibition by metal compounds. TrxR2 preferred its endogenous substrate Trx2 over Trx1, whereas TrxR1 efficiently reduced both Trx1 and Trx2. TrxR2 displayed strikingly lower activity with dithionitrobenzoic acid (DTNB), lipoamide, and the quinone substrate juglone compared to TrxR1, and TrxR2 could not reduce lipoic acid. However, Sec-deficient two-amino-acid-truncated TrxR2 was almost as efficient as full-length TrxR2 in the reduction of DTNB. We found that the gold(I) compound auranofin efficiently inhibited both full-length TrxR1 and TrxR2 and truncated TrxR2. In contrast, some newly synthesized gold(I) compounds and cisplatin inhibited only full-length TrxR1 or TrxR2 and not truncated TrxR2. Surprisingly, one gold(I) compound, [Au(d2pype) 2]Cl, was a better inhibitor of TrxR1, whereas another, [(iPr 2Im) 2Au]Cl, mainly inhibited TrxR2. These compounds also inhibited TrxR activity in the cytoplasm and mitochondria of cells, but their cytotoxicity was not always dependent on the proapoptotic proteins Bax and Bak. In conclusion, this study reveals significant differences between human TrxR1 and TrxR2 in substrate specificity and metal compound inhibition in vitro and in cells, which may be exploited for development of specific TrxR1- or TrxR2-targeting drugs.

Noble metal targeting of thioredoxin reductase — covalent complexes with thioredoxin and thioredoxin-related protein of 14 kDa triggered by cisplatin

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 14 kDa) 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.

The gold compound auranofin induces apoptosis of human multiple myeloma cells through both down-regulation of STAT3 and inhibition of NF-κB activity

Constitutive activation of NF-κB and STAT3 plays an important role in the cellular proliferation and survival of multiple myeloma cells. We first found that auranofin (AF), a coordinated gold compound, induced a significant level of cell cycle arrest at G1 phase and subsequent apoptosis of myeloma cells. Further, AF inhibited constitutive and IL-6-induced activation of JAK2 and phosphorylation of STAT3 followed by the decreased expression of Mcl-1. AF down-regulated the activation of NF-κB, and the combination of AF and a specific NF-κB inhibitor resulted in a marked decrease of Mcl-1 expression. These results suggest that AF inhibits both IL-6 induced-JAK/STAT pathway and NF-κB activation in myeloma cells.