Gold(I) complexes determine apoptosis with limited oxidative stress in Jurkat T cells

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European Journal of Pharmacology 582 (2008) 26 – 34 www.elsevier.com/locate/ejphar

Gold(I) complexes determine apoptosis with limited oxidative stress in Jurkat T cells Maria Pia Rigobello a , Alessandra Folda a , Barbara Dani a , Roberta Menabò b , Guido Scutari a , Alberto Bindoli b,⁎ b

a Department of Biological Chemistry, University of Padova, Viale G. Colombo 3, Padova, Italy Institute of Neurosciences (CNR), Unit of Padova c/o Department of Biological Chemistry, University of Padova, Padova, Italy

Received 10 August 2007; received in revised form 9 December 2007; accepted 20 December 2007 Available online 4 January 2008

Abstract In Jurkat T cells, S-triethylphosphinegold(I)-2,3,4,6-tetra-O-acetyl-1-thio-β-D-glucopyranoside (auranofin) and triethylphosphine gold(I) chloride (TepAu) induced apoptosis, as estimated by DNA fragmentation and visualised by fluorescence microscopy. Apoptosis was characterised by mitochondrial cytochrome c release which was not prevented by cyclosporin A. Apoptosis appeared to be triggered by inhibition exerted by gold(I) compounds on the cytosolic and mitochondrial isoforms of thioredoxin reductase, which determined a definite increase in hydrogen peroxide, whereas glutathione and its redox state were not modified. Total thiols showed a slight decrease, particularly in the presence of auranofin. However, no significant lipid peroxidation or nitric oxide formation were observed after incubation with gold(I) complexes, indicating that the cells had not been subjected to extensive oxidative stress. Interestingly, the gold(I) compound aurothiomalate was poorly effective, both in inhibiting thioredoxin reductase and in inducing apoptosis. These results demonstrate that the increased production of hydrogen peroxide determines an oxidative shift responsible for the occurrence of apoptosis and not involving lipid peroxidation. © 2007 Elsevier B.V. All rights reserved. Keywords: Apoptosis; Gold(I) complexes; Jurkat T cells; Mitochondria; Reactive oxygen species; Thiols; Thioredoxin reductase

1. Introduction In clinical practice, gold(I) drugs have been extensively used to treat rheumatoid arthritis (Kean et al., 1997), but they have also been studied for their potential antitumour properties, particularly in cultured cell systems, where they show marked cytotoxicity (Simon et al., 1981; Mirabelli et al., 1986; Mc Keage et al., 2002; Tiekink, 2002). S-triethylphosphinegold(I)-2,3,4,6-tetra-Oacetyl-1-thio-β-D-glucopyranoside (auranofin), a gold(I) lipophilic complex, has been shown to induce cell death, with varying characteristics depending on cell type. According to Liu et al. (2000), although auranofin stimulates necrosis at relatively high

⁎ Corresponding author. Dipartimento di Chimica Biologica, Viale G. Colombo 3, 35121 Padova, Italy. Tel.: +39 049 827 6138; fax: +39 049 807 3310. E-mail address: [email protected] (A. Bindoli). 0014-2999/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2007.12.026

concentrations (5 μM), at low concentrations (below 1 μM) it prevents spontaneous neutrophil apoptosis, probably by activation of a survival signal transduction pathway. Other authors suggest that auranofin is able to induce apoptosis in HL-60 cells (Park and Kim, 2005), ovarian cancer cells (Marzano et al., 2007) and cardiac tissue (Venardos et al., 2004). A central role in the apoptotic process is played by mitochondria, which act by releasing several factors leading to cell death (Green and Reed, 1998). This function is added to those already known for mitochondria such as energy production, ion homeostasis control and hydrogen peroxide production. The level of the latter is modulated by two systems, present both in the cytosol and in mitochondria which depend on glutathione, glutathione reductase and peroxidase (Schirmer et al., 1989) and thioredoxin, thioredoxin reductase and peroxidase (Arnér and Holmgren, 2000). Both glutathione peroxidase and thioredoxin reductase are endowed with a selenol group at their active site. Thioredoxin reductase is especially reactive with metal complexes,

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particularly of platinum (Sasada et al., 1999; Arnér et al., 2001; Witte et al., 2005) and gold (Hill et al., 1997; Gromer et al., 1998; Rigobello et al., 2002), and is considered to be a highly specific target for antitumour agents (Urig and Becker, 2006; Barnard and Berners-Price, 2007). We have previously observed that auranofin and other gold compounds cause marked inhibition of mitochondrial thioredoxin reductase in both the purified enzyme and isolated mitochondria (Rigobello et al., 2002) which, after treatment with auranofin, undergo permeability transition, decrease in membrane potential, release of cytochrome c, and stimulation of hydrogen peroxide production (Rigobello et al., 2002; Rigobello et al., 2004; Rigobello et al., 2005). In the present paper, the apoptotic effects of auranofin, triethylphosphine gold (TepAu) and aurothiomalate were examined and compared in the human T-cell lymphoma Jurkat cell line. Auranofin and TepAu are linear monomeric compounds, whereas aurothiomalate is a small polymeric gold compound with hydrophilic properties (Roberts and Shaw, 1998). Consequently, the various gold compounds show different solubility in aqueous solutions and different reactivity with biological targets. Apoptosis appears to depend on the inhibition of thioredoxin reductase and the consequent increase in hydrogen peroxide which, however, does not cause extensive oxidative stress in the cell.

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the cytosolic fraction, supernatant from the mitochondrial step was centrifuged at 100,000 ×g for 1 h. 2.4. Thioredoxin reductase activity Aliquots of cytosolic (TrxR1) or mitochondrial (TrxR2) thioredoxin reductase fractions (0.1 mg/ml) were tested at 25 °C in 0.2 M Na, K-phosphate buffer (pH 7.4) containing 2 mM EDTA and 0.4 mM NADPH. Reactions were started by the addition of 2 mM 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB) and its reduction was followed spectrophotometrically at 412 nm (Luthman and Holmgren, 1982). 2.5. Assay of total thiol groups After incubation with gold(I) compounds, cells were washed twice in PBS and centrifuged. Guanidine (7 M) in 0.2 M Tris– HCl (pH 8.1) containing 10 mM EDTA was added to the pellets. Samples underwent two freeze/thaw cycles, and were then vigorously vortexed and treated with 3 mM DTNB to determine the content of total thiol groups. The increase in absorbance was estimated spectrophotometrically at 412 nm. 2.6. Estimation of total and oxidised glutathione

2. Materials and methods 2.1. Drugs and chemicals TepAu, aurothiomalate, Hoechst dye 33258, propidium iodide and RPMI-1640 medium were obtained from Sigma (St. Louis, MO, USA). Auranofin and benzyloxycarbonyl-Val-Ala-Asp (oMe)-fluoromethylketone (z-VAD) were obtained from Alexis Biochemicals (Lausen, Switzerland). 5-(and -6) chloromethyl-2', 7'-dichlofluorescein diacetate acetyl ester (CM-DCFDA) and dihydrorhodamine 123 (DHR 123) were purchased from Molecular Probes-Invitrogen (Eugene, OR, USA). 5,6-diaminofluorescein diacetate (DAF-2 DA) was obtained from Fluka (Switzerland). Auranofin and TepAu were dissolved in dimethyl sulfoxide and aurothiomalate in the appropriate buffer. The purity of gold complexes was greater than 97–98%. Fetal calf serum was purchased from Biochrom-Seromed (Berlin, Germany). Antiprotease cocktail “Complete” and Cell Death Detection ELISAplus kit were from Roche (Penzberg, Germany). 2.2. Cell culture Human leukaemia Jurkat T cells were cultured in RPMI 1640 medium, supplemented with 10% heat-inactivated fetal calf serum and 2 mM glutamine, in an atmosphere of 5% CO2 in air at 37 °C.

After incubation with gold(I) compounds, cells were washed twice in PBS, treated with 6% metaphosphoric acid, and centrifuged. Supernatant was neutralised with Na3PO4 and assayed for total glutathione following the procedure of Tietze (1969), modified according to Anderson (1985) for determination of oxidised glutathione. 2.7. Estimation of cell reactive oxygen species and nitric oxide The generation of reactive oxygen species was assessed by the fluorogenic freely permeable probe CM-DCFDA (Royall and Ischiropoulos, 1993). After entering the cell, CM-DCFDA is entrapped by deacetylation catalysed by intracellular esterases and oxidised to a fluorescent compound by reactive oxygen species. Alternatively, their formation was followed fluorometrically with DHR123, which is a specific probe for mitochondrial production of reactive oxygen species (Royall and Ischiropoulos, 1993). Nitric oxide (NO) formation was estimated with the cell membranepermeable NO-sensitive dye DAF-2 DA (Kojima et al., 1998). Jurkat T cells were preincubated in the presence of fluorescent probes CM-DCFDA, DHR 123 or DAF-2 DA, and then treated with gold(I) compounds. The increase in fluorescence was followed spectrofluorometrically (Cary Eclipse, Varian; λEx =485 nm and λEm = 538 nm for CM-DCFDA; λEx =500 nm and λEm = 536 nm for DHR 123; λEx = 491 nm and λEm =513 nm for 5,6DAF-2 DA).

2.3. Cell subfractionation 2.8. Measurement of lipid peroxidation After incubation of Jurkat T cells with gold(I) compounds, cells were washed twice in PBS containing 10 mM glucose, and mitochondria were then isolated with the Focus™-Mitochondria kit (GENO-Technology Inc., St. Louis, MO, USA). To separate

Malondialdehyde (MDA) formation in cells was estimated according to Yagi (1984), with some modifications. After incubation in the presence of gold(I) compounds in PBS/10 mM glucose, cells

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were rapidly treated with 5 mM EDTA and 0.024% butylated hydroxytoluene (BHT) and then frozen. They were then thawed, sonicated for 30 s, treated with 3 ml of 0.1 N sulphuric acid and 0.5 ml of 10% phosphotungstic acid, vigorously mixed and, after about 5 min at room temperature, centrifuged at 2,500 ×g for 10 min. Supernatant was discarded and the pellet treated again in the same way. The final pellet was dissolved in 2.76 ml of distilled water, 1 ml of 0.67% thiobarbituric acid (TBA; dissolved in 50% acetic acid), 0.2 ml of 5% Nonidet P-40 and 0.04 ml of 1% BHT, and heated for 60 min at 95 °C. After cooling, the reaction mixture was extracted with 3 ml of n-butanol previously saturated with distilled water, and centrifuged at 7,000 ×g for 15 min. The TBAMDA adduct was then estimated spectrofluorometrically (Cary Eclipse, Varian) in the clear supernatant. The emission spectrum was recorded between 540 and 600 nm, with excitation at 532 nm. The various samples showed a fluorescence peak at about 550 nm, which was quantified by comparison with MDA standard (tetraethoxypropane).

preincubated with z-VAD. They were then centrifuged at 540 ×g, the supernatant removed, and the pellet treated according to the manufacturer's instructions contained in the Cell Death Detection ELISAplus kit. The extent of nuclear fragmentation was measured on a plate reader, following absorbance at 405 minus 492 nm. 2.12. Visualisation of apoptotic and necrotic cells Jurkat T cells were incubated in a 24-well plate (5 × 105 cells/ well) in RPMI without calf serum. They were then treated with 1 µM auranofin, TepAu or aurothiomalate for 18 h, and stained with 10 μM Hoechst dye 33258 and 1 μM propidium iodide for 5 min. After washing in PBS, the cells were visualised under a fluorescence microscope with λEx/λEm at 340/440 and 568/585 for Hoechst 33258 and propidium iodide, respectively. Cell fluorescence images were obtained with an Olympus IMT-2

2.9. Estimation of cytochrome c release After treatment with gold(I) compounds, cells were collected, washed twice in PBS, and centrifuged at 540 ×g for 10 min. They were then treated with a hypotonic lysis buffer formed by 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, and 20 mM Hepes/Tris (pH 7.4); an antiprotease cocktail was also added. The suspension was centrifuged at 12,500 ×g for 10 min at 4 °C and, the resulting supernatant treated with 0.5 mM EGTA and 2.5 mM PMSF and centrifuged for 1 h at 100,000 ×g to obtain the cytosolic fraction. Aliquots were then subjected to SDS-PAGE (gradient 8 to 18%) followed by electroblotting to nitrocellulose membranes. After the saturating step, membranes were treated with the primary monoclonal antibody (clone 7H8.2C12, Biosource International) followed, after rinsing, by incubation with a horseradish peroxidase-conjugated secondary antibody. Bound antibodies were detected by using enhanced chemiluminescence assay. Protein content was estimated according to Lowry et al. (1951). 2.10. Measurement of caspase activity Caspases 3 and 8 activities were measured by Caspase-Glo® 3/7 and Caspase-Glo® 8 assays (Promega, Madison, WI, USA), based on luminescence enhancement due to the cleavage of the luminogenic substrates containing the DEVD sequence (for caspase 3) and LETD sequence (for caspase 8) which, in turn, generate a luminescent signal produced by luciferase. Jurkat T cells were incubated in the conditions indicated in Fig. 4. At the end of the incubation time, the cells were directly treated with the Caspase-Glo® 3/7 or Caspase-Glo® 8 reagents, according to the manufacturer's instructions, and incubated at room temperature for approximately 45 min. Luminescence was recorded on a Fluoroskan Ascent FL (Labsystem, Finland). 2.11. Nuclear DNA fragmentation Jurkat T cells were treated at 37 °C in RPMI without phenol red for 18 h with gold(I) compounds and, when indicated,

Fig. 1. Inhibitory effect of gold(I) compounds on cytosolic (A) and mitochondrial (B) thioredoxin reductases in Jurkat T cells. Jurkat T cells (2 × 107) were incubated for 2 h in PBS/10 mM glucose medium in presence of 2 μM auranofin, TepAu or aurothiomalate at 37 °C. Excess of gold compounds was then washed out with PBS and cells were permeabilised and subjected to subfractionation in order to separate mitochondria from cytosol and estimate enzyme activity of the two isoforms of thioredoxin reductase (see also Materials and methods). *P b 0.05, ***P b 0.001 relative to control.

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microscope. The bright-field images of randomly selected fields were acquired and analysed with Metamorph software (Universal Imaging Corporation, West Chester, PA, USA). 2.13. Statistical analysis All values are the means ±S.D. of not less than four measurements. Data were analysed by one-way ANOVA, followed by the Tukey–Kramer multiple comparison test to assess differences between experimental groups (InStat, GraphPad Software Inc., San Diego, CA, USA). A value of P b 0.05 was considered statistically significant. 3. Results 3.1. Effect of gold(I) compounds on cytosolic (A) and mitochondrial (B) thioredoxin reductase As shown in Fig. 1, in Jurkat T cells treated with 2 μM auranofin or TepAu for 2 h, both cytosolic (TrxR1) and mitochondrial (TrxR2) isoforms of thioredoxin reductase are markedly inhibited,

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whereas aurothiomalate, in the same conditions, is far less efficient. These results indicate that gold(I) compounds such as auranofin and TepAu are able to cross both plasma and mitochondrial membranes and, consequently, inhibit both isoforms of thioredoxin reductase in their own environment, suggesting that the inhibitory effect is not restricted to isolated enzymes. 3.2. Role of gold(I) compounds in production of reactive oxygen species, nitric oxide and malondialdehyde Oxidant production with CM-DCFDA or DHR123 as probes was assessed by fluorometric assay in cells treated with gold(I) compounds. As shown in Fig. 2 (panels A and B), an increase in oxidising species, as observed by CM-DCFDA and DHR123 fluorescence enhancement, takes place after treatment with 2 μM gold(I) compounds. Auranofin appears to be the most efficient and the fluorescence elicited by DHR123 is suggestive of mitochondrial production of hydrogen peroxide. Aurothiomalate, although poorly effective in inhibiting thioredoxin reductase activity (Fig. 1), has previously been shown to act as a potent inhibitor of glutathione peroxidase (Chaudière and Tappell, 1984; Rigobello et al.,

Fig. 2. Effect of gold(I) compounds on hydrogen peroxide(A, B), nitric oxide (C) and malondialdehyde (D) formation in Jurkat T cells. For H2O2 estimation, Jurkat T cells (1 × 107) were preincubated in PBS/10 mM glucose medium for 20 min at 37 °C in presence of 1.5 µM CM-DCFDA or 1.5 µM DHR (freshly prepared), and then treated with 2 μM gold(I) compounds. For nitric oxide (NO) estimation, Jurkat T cells (1 × 107) were preincubated in PBS/10 mM glucose medium for 20 min at 37 °C in presence of 1.5 µM DAF-2 DA, and then treated with 2 μM gold(I) compounds. Spermine-NO (50 µM) was used as positive control. For malondialdehyde (MDA) estimation, Jurkat T cells (1 × 107) in 2 ml PBS/10 mM glucose medium were incubated in presence of gold(I) compounds at increasing concentrations (2, 5 and 10 μM) for 4 h at 37 °C. Positive control was obtained by incubating cells with 10 μM FeSO4 and 0.1 mM ascorbate. At end of incubation, 50 μl of 1% BHT and 50 μl of 0.2 M EDTA were added, and samples were immediately frozen at − 80 °C. After thawing, MDA content of samples was measured as indicated in Materials and methods. *P b 0.05 relative to control.

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2004) and, consequently the removal of hydrogen peroxide through the glutathione system is hampered and its intracellular concentration increases (Fig. 2A and B). In order to define better the nature of the oxidants produced, the formation of nitric oxide (NO) was also examined. However, as Fig. 2, panel C shows, no formation of NO was observed with any of the three gold(I) complexes (2 μM), whereas spermine-NO, a nitric oxide-releasing agent used as a positive control, markedly enhanced the fluorescence of the probe. The cell increase in hydrogen peroxide acts as a signalling event but, according to concentration and site of production, it may also lead to oxidative stress which can irreversibly damage the various cell components. However, as shown in Fig. 3, panel D, no significant lipid peroxidation was measured after 4 h of incubation with auranofin, TepAu or aurothiomalate at 2 and 5 μM concentrations. A slight increase in lipid peroxidation was observed when relatively higher concentrations (up to10 μM) of

gold(I) compounds were used. However, the extent of this lipid peroxidation was far lower than that obtained after incubation of the cells with the iron/ascorbate system used as positive control. 3.3. Effect of gold(I) compounds on glutathione and total thiol groups As glutathione and thiol groups are potential targets of gold(I) compounds (Chaudière and Tappell, 1984; Christodoulou et al., 1995; Roberts and Shaw, 1998; Sadler, 1982; Snyder et al., 1986), their concentration was estimated after cell treatment with 1 μM auranofin, TepAu and aurothiomalate for 2 h. Unlike the enzyme activity of thioredoxin reductase, total glutathione content was not modified, nor were changes in the GSH/GSSG ratio apparent (Fig. 3A). Total thiol groups slightly decreased in the presence of auranofin and TepAu, indicating that only a limited number of thiol groups are involved (Fig. 3B). 3.4. Cytochrome c release and nucleosome formation As a consequence of the oxidative unbalance induced by auranofin, release of cytochrome c from mitochondria to the cytosol was observed (Fig. 4A). Auranofin and TepAu (30 μM/ 3.3 × 107 cells) markedly stimulated the release of cytochrome c, but aurothiomalate was almost ineffective. Interestingly, cyclosporin A was not only unable to prevent the release of this protein but even induced a slight stimulation. The similar effect of cyclosporin has been observed in isolated mitochondria treated with auranofin (Rigobello et al., 2002). This indicates that the release of proapoptotic factors, including cytochrome c, is accomplished by increase in permeability of the outer mitochondrial membrane, probably mediated by components of the Bcl-2 family. The lack of inhibitory effect observed in the presence of cyclosporin A may indicate that permeability transition pore opening is not necessarily involved. The apoptotic effect induced by gold(I) compounds was further analysed in order to evaluate how caspases are involved. As shown in Fig. 4B, auranofin (1 μM/3 × 104 cells) markedly stimulated the formation of nucleosomes, a process inhibited by zVAD, which acts as a caspase inhibitor. In the same experimental conditions, TepAu was slightly less effective than auranofin, and aurothiomalate was completely unable to induce DNA fragmentation. As may be seen by comparing Fig. 4A and B, the extent of nucleosome formation is proportional to the amount of cytochrome c released. 3.5. Effect of gold(I) compounds on caspase-3 and caspase-8

Fig. 3. Estimation of glutathione (A) and total thiols (B) and in Jurkat T cells after treatment with gold(I) compounds. Jurkat T cells (1 × 106) were incubated in PBS/10 mM glucose medium for 2 h at 37 °C in presence of 1 µM auranofin, TepAu or aurothiomalate. Glutathione and total thiols were estimated as indicated in Materials and methods.

Fig. 4C shows that both auranofin and TepAu (0.5 μM/3 × 104 cells) markedly stimulate caspase-3 activity, whereas aurothiomalate, at the same concentration, is poorly effective. Staurosporine was used as positive control, and z-VAD, a pancaspase inhibitor, was effective in inhibiting the activating effect of staurosporine and gold compounds. Unlike caspase-3, caspase-8 was not stimulated by auranofin and TepAu (0.5 μM/104 cells), and a moderate effect was only elicited by aurothiomalate in the same experimental conditions (Fig. 4D).

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Fig. 4. Effect of gold(I) compounds on cytochrome c release (A), nucleosome formation(B) and caspase activation(C, D) in Jurkat T cells. Cytochrome c release (A) was estimated by Western blotting in cytosolic fraction obtained from 3.3 × 107 cells after 5 h of incubation in presence of 30 μM gold(I) compounds, as described in Materials and methods. When present, cyclosporin A (CsA) was 10 μM. For DNA fragmentation experiments (B), Jurkat T cells (3 × 104) were incubated in RPMI without phenol red for 18 h at 37 °C in presence of 1 μM gold(I) compounds. When indicated, z-VAD was 100 μM. Caspase-3 activity (C) was estimated in a 96-well plate (3 × 104 cells/well) incubated for 16 h; for caspase-8 activity (D), cells were 104/well and incubation lasted for 8 h. Gold(I) complexes were 0.5 µM. Positive control for caspase-3 was obtained by incubating cells with 0.2 µM staurosporine; for caspase-8, cells were treated with 80 ng/ml Fas ligand. When indicated, cells were preincubated with 100 μM z-VAD for 1 h. ***P b 0.001 relative to control.

3.6. Visualisation of apoptotic and necrotic cell death after treatment with gold(I) compounds The apoptotic and necrotic morphology of cells treated with 1 μM gold(I) compounds for 18 h was examined by fluorescence microscopy. As shown in Fig. 5A, the large number of cells stained with Hoechst 33258 (blue) indicates that auranofin is the most efficient in inducing apoptosis, although some of the cells also appear necrotic (stained with propidium iodide, red). However, in auranofin-incubated cells, z-VAD, a classical inhibitor of caspases, is also able to inhibit necrosis, indicating that it is probably secondary to an initial apoptotic process. 4. Discussion As both cytosolic and mitochondrial isoforms of isolated and purified thioredoxin reductase were markedly inhibited by gold(I) compounds (Gromer et al., 1998; Rigobello et al., 2004), it seemed of interest to know if and to what extent the same enzymes are inhibited in their cellular environment. We therefore measured the activity of thioredoxin reductase in cells pretreated with gold(I)

compounds. Auranofin and TepAu caused rapid and extensive inhibition of thioredoxin reductase in both cytosolic and mitochondrial compartments, and the extent of this effect was similar to that observed with the purified enzyme (Rigobello et al., 2004). However, aurothiomalate showed a lower efficiency, probably due to differential reactivity towards thioredoxin reductase(s), and previous results have shown that aurothiomalate is less efficient than auranofin and TepAu in inhibiting isolated thioredoxin reductase (Rigobello et al., 2004; Omata et al., 2006). The cell uptake of gold(I) complexes and particularly of auranofin has previously been examined in ovarian cancer cells and found to be concentration- and time-dependent. In addition, its accumulation inside cells correlates well with cytotoxicity data (Marzano et al., 2007). Differential and contrasting effects between auranofin and aurothiomalate have been observed in other systems. For instance, auranofin has no effect on the conversion of xanthine dehydrogenase to xanthine oxidase, whereas aurothiomalate can catalyse the transformation (Sakuma et al., 2004). Instead, jejunal perfusion with auranofin causes severe injury, but aurothiomalate (Myochrysine) has no effect (Ammon et al., 1987). In addition,

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Fig. 5. Induction of apoptosis by gold(I) compounds in Jurkat T cells. Panel A. Apoptotic and necrotic morphology appearance of cells stained with Hoechst dye 33258 and propidium iodide after treatment with 1 μM gold(I) compounds for 18 h. Cells (5 × 105/well) were examined, as described in Materials and methods, with fluorescence microscopy. Control (a); auranofin (b); TepAu (c); aurothiomalate (d). Hoechst 33258 (blue) is freely permeable to membrane and stains condensed, i.e. apoptotic nuclei; propidium iodide (red) penetrates cells with damaged membranes, indicating extent of necrosis. Panel B. Percentage of apoptosis and necrosis by fluorescence microscopy analysis is reported. When indicated, cells were preincubated with 100 μM z-VAD for 1 h. Necrosis was estimated by quantification of the nuclei positive to propidium iodide; apoptosis was determined by observing appropriate changes in nuclei stained with Hoechst 33258.

auranofin can activate MAP kinases (Park and Kim, 2005) but aurothiomalate is ineffective (Seitz et al., 2003). Unlike the effect on TrxR, total glutathione and the GSH/ GSSG ratio are not altered by gold compounds, in accordance with the fact that glutathione reductase is not inhibited by these

compounds, at least in the range of concentrations used (Rigobello et al., 2002). Total thiol groups are only slightly decreased by gold compounds, and auranofin, by reducing the total thiol concentration by about 10%, proves to be the most efficient. This small decrease in total thiols may be a consequence of the inhibition of

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thioredoxin reductase which, unable to maintain thioredoxin reduced, leads to an increase in the oxidation state of protein thiols. Thioredoxin reductase plays a central role in determining the fate of several cell processes (Arnér and Holmgren, 2000) and its inhibition creates dramatic consequences in cells, as the prevention of hydrogen peroxide removal and the consequent increase in its concentration cause an unbalance in cell redox conditions. It is well-known that mitochondria can generate hydrogen peroxide (Sauer et al., 2001) and they also act as both producers and metabolisers of hydrogen peroxide (Zoccarato et al., 2004; Andreyev et al., 2005; Rigobello et al., 2005), therefore modulating its level. The inhibition of mitochondrial thioredoxin reductase leads to increased contents of hydrogen peroxide, both inside and outside the mitochondrion, and this has several consequences for cell signalling systems. It has been shown in HL-60 cells that auranofin stimulates the p38 pathway of the MAPK system (Park and Kim, 2005) thus activating an apoptotic pathway. The main process observed after treatment of cells with auranofin and TepAu is marked release of cytochrome c, not inhibited by cyclosporin A. This indicates that mitochondrial inner membrane permeability transition is not strictly required for the release of cytochrome c and the permeability increase of the outer membrane is sufficient if accompanied by detachment of cytochrome c from the outer face of the inner membrane. In fact, in our experimental conditions, cyclosporin A not only failed to prevent apoptosis induced by auranofin, but in itself increased the release of cytochrome c (Fig. 4A). Cyclosporin A is considered the be a specific inhibitor of permeability transition pore opening, since it interacts with cyclophilin and thus prevents the release of proapoptotic factors. However, it has a biphasic effect as, according to its concentration and exposure time, it can act either as an antiapoptotic or proapoptotic factor (Roy et al., 2006). In addition, like auranofin, silver ions (Almofti et al., 2003) and pristimerin (Wu et al., 2005) induce release of cytochrome c and apoptosis not inhibited by cyclosporin A. Furthermore, in isolated mitochondria, cyclosporin A has been shown to be unable to prevent the release of cytochrome c stimulated by gold(I) compounds (Rigobello et al., 2004). As a consequence, cells undergo apoptosis, as observed with both the nucleosome test and fluorescence microscope analysis. When apoptosis is stimulated by auranofin and TepAu, caspase-3 but not caspase-8 is activated, indicating that a major role in inducing apoptosis is played by the (intrinsic) mitochondrial pathway. Interestingly, the production of hydrogen peroxide does not elicit significant lipid peroxidation, indicating that generalised oxidative stress is not responsible for the observed events. In addition, no stimulation of nitric oxide production by gold(I) complexes has been observed, nor alterations in the content of total glutathione or its redox state. In accordance with these data, Pallis et al. (2003) observed that targeting tumour cells with diamide (a dithiol oxidant) or with specific inhibitors of thioredoxin can give rise to antiproliferative and proapoptotic effects without the occurrence of a “redox catastrophe” due to excessive reactive oxygen species production. The formation of hydrogen peroxide which occurs after thioredoxin reductase inhibition and which appears to be mostly of mitochondrial origin is critical for the induction of apoptosis. In addition to the increased level of hydrogen peroxide, the oxidation

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of components of the thioredoxin system downstream from thioredoxin reductase also play a major role in the apoptotic process, as both thioredoxin and peroxiredoxin are shifted to a more oxidised condition. Through peroxiredoxin, thioredoxin is largely oxidised by hydrogen peroxide and cannot be reduced back by thioredoxin reductase, which is inhibited by gold(I) complexes. In conclusion, our results reveal a mechanism which shows that inhibition of cytosolic and mitochondrial thioredoxin reductases cooperates as sparking events leading to apoptosis. However, exact identification of all the members connecting this initial event to programmed cell death requires further study. Acknowledgments This work was partially supported by grants from the National Research Council of Italy and Progetto di AteneoUniversity of Padova (CPDA 065113-2006), Italy. References Almofti, M.R., Ichikawa, T., Yamashita, K., Terada, H., Shinoara, Y., 2003. Silver ion induces cyclosporin A-insensitive permeability transition in rat liver mitochondria and release of apoptogenic cytochrome c. J. Biochem. 134, 43–49. Ammon, H.V., Fowle, S.A., Cunningham, J.A., Komorowsky, R.A., Loeffler, R.F., 1987. Effects of auranofin and myochrysine on intestinal transport and morphology in the rat. Gut 28, 829–834. Anderson, M.E., 1985. Determination of glutathione and glutathione disulfide in biological samples. Methods Enzymol. 113, 548–555. Andreyev, A.Yu., Kushnareva, Yu.E., Starkov, A.A., 2005. Mitochondrial metabolism of reactive oxygen species. Biochemistry (Moscow) 70, 200–214. Arnér, E.S.J., Holmgren, A., 2000. Physiological functions of thioredoxin and thioredoxin reductase. Eur. J. Biochem. 267, 6102–6109. Arnér, E.S.J., Nakamura, H., Sasada, T., Yodoi, J., Holmgren, A., Spyrou, G., 2001. Analysis of the inhibition of mammalian thioredoxin, thioredoxin reductase, and glutaredoxin by cis-diamminedichloroplatinum (II) and its major metabolite, the glutathione–platinum complex. Free Radic. Biol. Med. 31, 1170–1178. Barnard, P.J., Berners-Price, S.J., 2007. Targeting the mitochondrial cell death pathway with gold compounds. Coord. Chem. Rev. 251, 1889–1902. Chaudière, J., Tappell, A.L., 1984. Interaction of gold(I) with the active site of selenium glutathione–glutathione peroxidase. J. Inorg. Biochem. 20, 313–325. Christodoulou, J., Sadler, P.J., Tucker, A., 1995. 1H NMR of albumin in human blood plasma: drug binding and redox reactions at Cys34. FEBS Lett. 376, 1–5. Green, D.R., Reed, J.C., 1998. Mitochondria and apoptosis. Science 281, 1309–1312. Gromer, S., Arscott, L.D., Williams Jr, C.H., Schirmer, R.H., Becker, K., 1998. Human placenta thioredoxin reductase. Isolation of the selenoenzyme, steady state kinetics, and inhibition by therapeutic gold compounds. J. Biol. Chem. 273, 20096–20101. Hill, K.E., McCollum, G.W., Boeglin, M.E., Burk, R.F., 1997. Thioredoxin reductase activity is decreased by selenium deficiency. Biochem. Biophys. Res. Commun. 234, 293–295. Kean, W.F., Hart, L., Buchanan, W.W., 1997. Auranofin. Br. J. Rheumatol. 36, 560–562. Kojima, H., Nakatsubo, N., Kikuchi, K., Kawahara, S., Kirino, Y., Nagoshi, H., et al., 1998. Detection and imaging of nitric oxide with novel fluorescent indicators: diaminofluoresceins. Anal. Chem. 70, 2446–2453. Liu, J., Akahoshi, T., Namai, R., Matsui, T., Kondo, H., 2000. Effect of auranofin, an antirheumatic drug, on neutrophil apoptosis. Inflamm. Res. 49, 445–451. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265–275. Luthman, M., Holmgren, A., 1982. Rat liver thioredoxin and thioredoxin reductase: Purification and characterization. Biochemistry 21, 6628–6633.

34

M.P. Rigobello et al. / European Journal of Pharmacology 582 (2008) 26–34

Marzano, C., Gandin, V., Folda, A., Scutari, G., Bindoli, A., Rigobello, M.P., 2007. Inhibition of thioredoxin reductase by auranofin induces apoptosis in cisplatin-resistant human ovarian cancer cells. Free Radic. Biol. Med. 42, 872–881. Mc Keage, M.J., Maharaj, L., Berners-Price, S.J., 2002. Mechanisms of cytotoxicity and antitumor activity of gold(I) phosphine complexes: the possible role of mitochondria. Coord. Chem. Rev. 232, 127–135. Mirabelli, C.K., Johnson, R.K., Hill, D.T., Faucette, L.F., Girard, G.R., Kuo, G.Y., Sung, C.M., Crooke, S.T., 1986. Correlation of the in vitro cytotoxic and in vivo antitumor activities of gold(I) coordination complexes. J. Med. Chem. 29, 218–223. Omata, Y., Folan, M., Shaw, M., Messer, R.L., Lockwood, P.E., Hobbs, D., Bouillaguet, S., Sano, H., Lewis, J.B., Wataha, J.C., 2006. Sublethal concentrations of diverse gold compounds inhibit mammalian cytosolic thioredoxin reductase (TrxR1). Toxicol. Vitro 20, 882–890. Pallis, M., Bradshaw, T.D., Westwell, A.D., Grundy, M., Stevens, M.F.G., Russell, N., 2003. Induction of apoptosis without redox catastrophe by thioredoxin-inhibitory compounds. Biochem. Pharm. 66, 1695–1705. Park, S.J., Kim, I.-S., 2005. The role of p38 MAPK activation in auranofininduced apoptosis of human promyelocytic leukaemia HL-60 cells. Br. J. Pharmacol. 146, 506–513. Rigobello, M.P., Scutari, G., Boscolo, R., Bindoli, A., 2002. Induction of mitochondrial permeability transition by auranofin, a gold(I)-phosphine derivative. Br. J. Pharmacol. 136, 1162–1168. Rigobello, M.P., Scutari, G., Folda, A., Bindoli, A., 2004. Mitochondrial thioredoxin reductase inhibition by gold(I) compounds and concurrent stimulation of permeability transition and release of cytochrome c. Biochem. Pharmacol. 67, 689–696. Rigobello, M.P., Folda, A., Scutari, G., Bindoli, A., 2005. The modulation of thiol redox state affects the production and metabolism of hydrogen peroxide by heart mitochondria. Arch. Biochem. Biophys. 441, 112–122. Roberts, J.R., Shaw III., F.C., 1998. Inhibition of erythrocyte selenium-glutathione peroxidase by auranofin analogues and metabolites. Biochem. Pharmacol. 55, 1291–1299. Roy, M.K., Takenaka, M., Kobori, M., Nakahara, K., Isobe, S., Tsushida, T., 2006. Apoptosis, necrosis and cell proliferation-inhibition by cyclosporin A in U937 cells (a human monocytic cell line). Pharmacol. Res. 53, 293–302. Royall, J.A., Ischiropoulos, H., 1993. Evaluation of 2',7'-dichlorofluorescein and dihydrorhodamine 123 as florescent probes for intracellular H2O2 in cultured endothelial cells. Arch. Biochem. Biophys. 302, 348–355. Sadler, P.J., 1982. The comparative evaluation of the physical and chemical properties of gold compounds. J. Rheumatol. Suppl. 8, 71–78. Sakuma, S., Gotoh, K., Sadatoku, N., Fujita, T., Fujimoto, Y., 2004. Effects of antirheumatic gold compounds on the conversion of xanthine dehydrogenase to oxidase in rabbit liver cytosol in vitro. Life Sci. 75, 1211–1218.

Sasada, T., Nakamura, H., Ueda, S., Sato, N., Kitaoka, Y., Gon, Y., et al., 1999. Possible involvement of thioredoxin reductase as well as thioredoxin in cellular sensitivity to cis-diamminedichloroplatinum (II). Free Radic. Biol. Med. 27, 504–514. Sauer, H., Wartenberg, M., Hescheler, J., 2001. Reactive oxygen species as intracellular messengers during cell growth and differentiation. Cell Physiol. Biochem. 11, 173–186. Schirmer, R.H., Krauth-Siegel, R.L., Schulz, G.E., 1989. Glutathione reductase. In: Dolphin, D., Avramovich, O., Poulson, R. (Eds.), Glutathione. Chemical, Biochemical, and Medical Aspects. Part A. John Wiley and Sons, New York, pp. 553–596. Seitz, M., Valbracht, J., Quach, J., Lotz, M., 2003. Gold sodium thiomalate and chloroquine inhibit cytokine production in monocytic THP-1 cells through distinct transcriptional and posttranslational mechanisms. J. Clin. Immunol. 23, 477–484. Simon, T.M., Kunishima, D.H., Vibert, G.J., Lorber, A., 1981. Screening trial with the coordinated gold compound auranofin using mouse lymphocyte leukemia p388. Cancer Res. 41, 94–97. Snyder, R.M., Mirabelli, C.K., Crooke, S.T., 1986. Cellular association, intracellular distribution, and efflux of auranofin via sequential ligand exchange reactions. Biochem. Pharmacol. 35, 923–932. Tiekink, E.R., 2002. Gold derivatives for the treatment of cancer. Crit. Rev. Oncol. Hematol. 42, 225–248. Tietze, F., 1969. Enzymatic method for quantitative determination of nanogram amounts of total and oxidized glutathione. Anal. Biochem. 27, 502–522. Urig, S., Becker, K., 2006. On the potential of thioredoxin reductase inhibitors for cancer therapy. Semin. Cancer Biol. 16, 452–465. Venardos, K., Harrison, G., Headrick, J., Perkins, A., 2004. Auranofin increases apoptosis and ischemia-reperfusion injury in the rat isolated heart. Clin. Exp. Pharmacol. Physiol. 31, 289–294. Witte, A.B., Anestål, K., Jerremalm, E., Ehrsson, H., Arnér, E.S.J., 2005. Inhibition of thioredoxin reductase but not of glutathione reductase by the major classes of alkylating and platinum-containing anticancer compounds. Free Radic. Biol. Med. 39, 696–703. Wu, C.C., Chan, M.L., Chen, W.Y., Tsai, C.Y., Chang, F.R., Wu, Y.C., 2005. Pristimerin induces caspase-dependent apoptosis in MDA-MB-231 cells via direct effects on mitochondria. Mol. Cancer Ther. 4, 1277–1285. Yagi, K., 1984. Lipid peroxidation. Assay for blood plasma or serum. Methods Enzymol. 105, 328–331. Zoccarato, F., Cavallini, L., Alexandre, A., 2004. Respiration-dependent removal of exogenous H2O2 in brain mitochondria. Inhibition by Ca2+. J. Biol. Chem. 279, 4166–4174.