Gold(III) Complexes Induce Cancer Cell Death PDF

Chemistry & Biology Article Gold(III)-Dithiocarbamato Complexes Induce Cancer Cell Death Triggered by Thioredoxin Redox System Inhibition and Activation of ERK Pathway Daniela Saggioro,1,7 Mar...

Chemistry & Biology Article Gold(III)-Dithiocarbamato Complexes Induce Cancer Cell Death Triggered by Thioredoxin Redox System Inhibition and Activation of ERK Pathway Daniela Saggioro,1,7 Maria Pia Rigobello,2,7 Lucia Paloschi,3 Alessandra Folda,2 Stephen A. Moggach,4 Simon Parsons,4 Luca Ronconi,4,5 Dolores Fregona,5,8,* and Alberto Bindoli6,8,* 1Molecular Immunology and Diagnostic Oncology, Istituto Oncologico Veneto I.R.C.C.S., Via Gattamelata 64, Padova 35128, Italy 2Department of Biological Chemistry, University of Padova, V.le G. Colombo 3, Padova 35121, Italy 3Department of Oncology and Surgical Sciences, University of Padova, Via Gattamelata 64, Padova 35128, Italy 4School of Chemistry, University of Edinburgh, West Mains Road, Edinburgh EH9 3JJ, United Kingdom 5Department of Chemical Sciences, University of Padova, Via Marzolo 1, Padova 35131, Italy 6Institute of Neurosciences, C.N.R., V.le G. Colombo 3, Padova 35121, Italy 7These authors contributed equally to this work. 8These authors are co-senior authors. *Correspondence: [email protected] (D.F.), [email protected] (A.B.) DOI 10.1016/j.chembiol.2007.08.016 SUMMARY (auranofin), were found to exert significant antitumor activ- ity both in vitro and in vivo [4–10]. Although gold compounds are now recognized So far, while the potential anticancer action of gold(I) de- as promising anticancer agents, so far only rivatives has been widely investigated, the development of gold(I) derivatives have been investigated for gold(III) complexes as alternative antitumor agents has this purpose, whereas the use of gold(III) com- been hampered by their poor stability under physiological plexes has been hampered by their poor stabil- conditions. Although the stability of gold(III) metal center may be enhanced by the coordination of multidentate li- ity under physiological conditions. We have gands, the excess of stabilization may result in a loss of bi- therefore carried out studies on selected gold- ological activity. Nevertheless, recent studies showed (III) anticancer agents, showing enhanced sta- that some selected gold(III) complexes are able to exert bility due to the presence of chelating dithio- outstanding cytotoxic activity toward various tumor cell carbamato ligands. We found that they induce lines with IC50 values falling in the range 1–10 mM [11–16]. cancer cell death through both apoptotic and In order to understand the mechanism of action of anti- nonapoptotic mechanisms. They also inhibit cancer agents, it is of paramount importance to identify thioredoxin reductase activity, generate free the molecular components involved in the cascade of radicals, modify some mitochondrial functions, events that finally trigger cell death. and increase ERK1/2 phosphorylation. Based on In light of the close chemical similarity between platinum our results, we propose and discuss a working and gold compounds, it was believed that both might tar- get DNA. However, in addition to a direct interaction with model suggesting that deregulation of the thiore- DNA in vitro , both gold(I) and gold(III) complexes have doxin reductase/thioredoxin redox system is a been reported to interact with other cellular components. major mechanism involved in the anticancer ac- Recently, we have reported on the antiproliferative and tivity of the investigated gold(III)-dithiocarbamato apoptotic activities of two gold(III)-methylsarcosinedithio- complexes. carbamato derivatives toward a panel of human myeloid leukemia cell lines. These novel gold(III) complexes ap- pear to induce a strong downregulation of the antiapop- INTRODUCTION totic Bcl-2 molecule and an upregulation of the proapop- totic Bax protein, but only weak perturbations of the cell The treatment of a variety of cancers by platinum deriva- cycle. This behavior diverges from that of the classical tives has encouraged the continuous investigation of al- platinum(II) complexes which, instead, induce cell cycle ternative metal-based drugs. Owing to their anti-inflam- alterations resulting in an increase in G2M cell fraction matory and immunosuppressive properties, some gold(I). Furthermore, we have identified the proteasome as compounds used for the treatment of rheumatoid arthritis a possible in vitro and in vivo target of gold(III)-dithiocarba- [1–3] were considered for their possible anticancer activ- mato derivatives such as complexes 1 and 4 (Figure 1A). ity. Indeed, some of the clinically established antiarthritic The inhibition of the proteasomal chymotrypsin-like activ- gold(I) compounds, such as gold(I) thiomalate (myocrisin), ity results in the accumulation of ubiquinated proteins and gold(I) thioglucose (solganol), and 2,3,4,6-tetra-o-acetyl- the proteasome target protein p27 in the highly metastatic 1-thio-b-D-glucopyranosato-(S)-triethylphosphine gold(I) MDA-MB-231 human Caucasian breast adenocarcinoma 1128 Chemistry & Biology 14, 1128–1139, October 2007 ª2007 Elsevier Ltd All rights reserved Chemistry & Biology Gold-Dithiocarbamato Complexes and Cancer Cells Figure 1. Chemical Drawings of the In- vestigated Gold(III)-Dithiocarbamato Complexes and Determination of the Apoptotic Effects on HeLa Cells (A) Investigated complexes: [Au(DMDT)Cl2] (1), [Au(DMDT)Br2] (2), [Au(ESDT)Cl2] (3), [Au- (ESDT)Br2] (4). Structure of the reference drug cisplatin (cis-[PtCl2(NH3)2]) is also reported for clarity. (B) Phase-contrast microscopy representative pictures of cell death after treatment with differ- ent concentrations of complexes 2 (2.5/5/10/ 15 mM) and 4 (5/10/20/30 mM), and cisplatin (60 mM) for 24 h. Original magnification = 100 3. (C) Quantification of apoptosis through PARP cleavage determination. After 24 hr treatment the cells were collected, lysed, and aliquots of total proteins subjected to western blot analy- sis of PARP cleavage. Percentage of PARP cleavage was calculated as the ratio between densitometrical values of cleaved and un- cleaved bands 3 100. Values are given as means ± SE of at least three independent experiments. cell line. In addition, treatment of MDA-MB-231 tumor- as membrane potential and permeability conditions, stim- bearing nude mice resulted in a significant inhibition of tu- ulate ROS generation, and are particularly effective in in- mor growth, associated to the inhibition of proteasome hibiting the activity of selenoenzyme thioredoxin reductase activity and the massive induction of apoptosis in vivo. with similar or even higher efficiency compared to other On the other hand, recent data have identified mito- gold(III) complexes [22, 24]. Intriguingly, we have evidence chondria as suitable targets for gold complexes to exert that treatment of HeLa cells with these gold(III)-dithiocar- their cytotoxicity, as recently reviewed by McKeage bamato complexes induces phosphorylation of ERK, and et al.. In particular, mitochondrial thioredoxin reductase that ERK activation can be completely blocked by the an- appears to be a very specific target for gold(I) complexes, tioxidant N-acetyl-L-cysteine (NAC), whereas treatment such as auranofin, aurothiomalate, and other gold(I)-trie- with Trolox is ineffective. Based on our findings, we pro- thylphosphine derivatives , and gold(III) complexes pose and discuss a working model of the molecular mech- containing diethylendiamino-substituted pyridino and bi- anisms involved in the cell death induced by the investi- pyridino ligands [22, 23]. This finding is highly significant gated gold(III) anticancer agents. as mitochondria are now taken into account as potential targets for chemotherapeutic drugs due to their well- RESULTS known key role in apoptosis. As a natural continuation of our previous studies, we here X-Ray Crystallography report on the effect of some recently synthesized gold(III)- The complexes [Au(DMDT)X2] [X = Cl (1), Br (2)] and dithiocarbamato derivatives, namely [Au(DMDT)X2] and [Au(ESDT)X2] [X = Cl (3), Br (4)] (Figure 1A) have been de- [Au(ESDT)X2] (DMDT = N,N-dimethyldithiocarbamato; signed to reproduce very closely the main features of cis- ESDT = ethylsarcosinedithiocarbamato; X = Cl, Br) platin. Conclusions reached upon application of several (Figure 1A) , on cell cytotoxicity and mitochondrial spectroscopic techniques suggest that coordination of functions. Analyses were carried out at molecular, subcel- both DMDT and ESDT ligands takes place in a near lular, and cellular levels. We found that they trigger cell square-planar geometry through the sulfur-donating death by activating both apoptotic and nonapoptotic atoms, with the NCSS moiety coordinating the metal pathways. They alter some mitochondrial functions such center in a bidentate symmetrical mode. The remaining Chemistry & Biology 14, 1128–1139, October 2007 ª2007 Elsevier Ltd All rights reserved 1129  Chemistry & Biology Gold-Dithiocarbamato Complexes and Cancer Cells coordination positions are occupied by two cis-gold(III)- halogen atoms that may undergo hydrolysis. This structural hypothesis is also supported by density func- tional calculations previously carried out for some analo- gous gold(III)-dithiocarbamato complexes. In addi- tion, we recently determined the X-ray structure of complex 4, which is in agreement with the previously re- ported spectroscopic data (see the Supplemental Data available with this article online). Apoptotic Activity The apoptotic activity of complexes 1–4 was evaluated on human cervical carcinoma HeLa cells incubated for 24 h with increasing drug concentrations. Concentrations ranged from 2.5 to 15 mM and from 5 to 30 mM for DMDT (1 and 2) and ESDT (3 and 4) derivatives, respec- tively, with their IC50 values (concentration of drug result- ing in 50% inhibition of cell growth) being slightly different toward the same human tumor cell lines [15, 16]. Cisplatin was also tested as a reference drug. Analysis of cultures after the treatment showed that both DMDT and ESDT gold(III) derivatives were able to induce cell death in a dose-dependent way (Figure 1B); no substantial differ- ences were observed among chloro- and bromo- derivatives containing the same dithiocarbamato ligand. To determine whether the observed cell death was due to the activation of an apoptotic pathway, we analyzed the poly-(ADP-ribose)-polymerase (PARP) cleavage. PARP is a nuclear enzyme, activated by DNA damage, involved in DNA repair, cell death, and inflammation. Moreover, PARP is a specific substrate for caspases 3 and 7; there- fore, PARP cleavage is generally accepted as a marker for apoptosis. Indeed, apoptosis is a form of cell death characterized by the activation of caspases (cysteinyl as- partate specific protease) that cleave multiple targets in the cell. Thus, after microscope observation, the cells were collected, lysed, and total proteins subjected to western blot analysis. As shown in Figure 1C, the percent- age of PARP cleavage after 24 hr treatment, quantified by densitometric analysis, was nearly the same for all tested gold(III) complexes and, surprisingly, non-dose-depen- dent (except for lower dose treatments). These findings Figure 2. Oxygen Uptake by Rat Liver Mitochondria and Sub- contrast with those obtained from the morphological ex- mitochondrial Particles amination of the cultures, in which the cell death appeared Rat liver mitochondria (A) or submitochondrial particles (B and C) were incubated at 25 C with complexes 1–4 to a final concentration of 5 mM to be dose-dependent (Figure 1B). Thus, it is likely that our for mitochondria and 10 mM for submitochondrial particles in the suit- compounds may trigger cell death by activating not only able medium (see Experimental Procedures). For submitochondrial apoptotic pathways but also other death mechanisms particles either succinate (B) or NADH (C) were used as substrates. Ox- (i.e., necrosis). On the contrary, cells treated with cisplatin ygen consumption was measured as reported in the Experimental Pro- induced a complete PARP cleavage, confirming that the cedures section. Respiration control ratio (RCR) was calculated as res- reference drug leads to cell death only through an apopto- piration rate in state 3/respiration rate in state 4. Values are given as tic pathway. means ± SE of at least three independent experiments. Effects on Mitochondrial Functions tion conditions after treatment with complexes 1–4 were Although mitochondria have long been considered to be studied. As summarized in Figure 2A, the respiratory con- solely a powerhouse for production of cellular energy, re- trol ratio (RCR) measured in whole mitochondria is cently they have been clearly recognized to play a critical scarcely affected, and uncoupled respiration shows a role in the apoptotic process. Therefore, the physio- limited inhibition (data not shown), thus indicating that logical consequences on the mitochondrial respiratory the electron flow along the respiratory chain is not signifi- parameters, membrane potential, and permeability transi- cantly affected. In agreement with these observations, in 1130 Chemistry & Biology 14, 1128–1139, October 2007 ª2007 Elsevier Ltd All rights reserved Chemistry & Biology Gold-Dithiocarbamato Complexes and Cancer Cells Figure 3. Effect on Mitochondrial Mem- brane Potential and Swelling (A) Mitochondrial membrane potential was estimated as reported in the Experimental Pro- cedures. Mitochondria were energized by the addition of 5 mM succinate (arrow). Concentra- tion of gold(III) complexes was: 0 (a), 1 mM (b), 2 mM (c), 3 mM (d), 5 mM (e). Values are reported as relative fluorescence units. (B) Induction of mitochondrial swelling was es- timated as described in the Experimental Pro- cedures with complexes 1–4 to a final concen- tration of 2 mM. submitochondrial particles the respiration is modified by of ESDT derivatives (3 and 4), which take a longer time to approximately 10%–20% after treatment with the various accomplish the swelling. As well as the swelling induced gold(III) complexes [using either succinate (Figure 2B) or by auranofin , also in this case swelling is dependent reduced nicotinamide adenine dinucleotide (NADH, on the presence of a low concentration of calcium ions Figure 2C) as substrates]. Similarly to the observed apo- as observed by the inhibitory effect elicited by calcium ptotic effect toward HeLa cells (see above), negligible dif- chelators (EGTA) or inhibitors of mitochondrial calcium ferences were detected among chloro- and bromo-deriv- uptake such as ruthenium red (data not shown). atives containing the same dithiocarbamato ligand. Interestingly, different results were obtained when the Estimation of ROS Accumulation in Isolated membrane potential was estimated (Figure 3A). In fact, Mitochondria and HeLa Cells whereas complexes 1 and 2 caused a marked drop of The effect of complexes 1–4 on the production of reactive membrane potential after about 20 to 30 min have oxygen species (ROS), in particular hydrogen peroxide, in elapsed, complex 4 was able to depolarize membrane isolated rat liver mitochondria is shown in Figures 4A and after about 32 min of incubation, and complex 3 showed 4B. As apparent, the addition of any of the complexes 1–4 an intermediate behavior. In addition, all the investigated strongly stimulates the formation of hydrogen peroxide, gold(III)-dithiocarbamato derivatives exhibit a marked ef- detected by means of Amplex Red/HRP (10 acetyl-3,7- fect when permeability transition, measured in terms of dihydroxyphenoxazine/horseradish peroxidase) assay. mitochondrial swelling, was taken into account. As re- The formation of hydrogen peroxide is further increased ported in Figure 3B, a large swelling is apparent after incu- when antimycin is added (Figure 4A). By inhibiting the mi- bation with complexes 1–4; nevertheless, it is worth noting tochondrial complex III, antimycin favors the production that swelling is faster in the presence of the two DMDT de- of superoxide anion which, after dismutation, is converted rivatives (1 and 2), whereas a longer lag occurs in the case to hydrogen peroxide. Analogously, when antimycin is Chemistry & Biology 14, 1128–1139, October 2007 ª2007 Elsevier Ltd All rights reserved 1131  Chemistry & Biology Gold-Dithiocarbamato Complexes and Cancer Cells Figure 4. Evolution of Hydrogen Perox- ide in Isolated Rat Liver Mitochondria and in Cultured HeLa Cells Hydrogen peroxide formed by exposure of iso- lated rat liver mitochondria to complexes 1–4. The fluorogenic probe Amplex Red and HRP were used at concentration of 10 mM and 0.1 units/ml, respectively. The extent of hydrogen peroxide evolution was reported as relative fluorescence units. Antimycin (1 mM) was added after (A) or before (B) the various gold(III) complexes (1 mM). (C) ROS formation in HeLa cells was determined using the oxidation-sen- sitive probe DHR-123. Cells were loaded with DHR-123 (5 mM) for 30 min, and then exposed to complexes 2 and 4 (10 mM) for an additional 30 min. For comparison purposes, cisplatin (60 mM) was tested under the same experimen- tal conditions. ROS generation was examined using a Zeiss LSM-510 confocal laser micro- scope equipped with a CO2- and tempera- ture-controlled chamber. added before incubating mitochondria with any of the in- adding complex 4, the oxidation state of the probe was re- vestigated gold(III) complexes (Figure 4B), again they fur- duced to levels comparable to those of nontreated control ther stimulate the formation of hydrogen peroxide. The cells (Figure 5A). However, when we analyzed the survival effectiveness of the four complexes is rather similar, al- of cells cotreated with NAC or Trolox and complex 4, we though the two DMDT derivatives seem to be slightly found that while NAC treatment was able to prevent cell more efficient than the ESDT counterparts. death and PARP cleavage, Trolox was not (Figures 5B As experiments carried out in isolated rat liver mitochon- and 5C). Indeed, the cells exhibited comparable mortality dria showed that complexes 1–4 are able to induce forma- and PARP cleavage regardless of Trolox presence (Fig- tion of hydrogen peroxide, we checked whether these ures 5B–5D). These apparently contradictory data sug- complexes trigger ROS generation also in HeLa cells. gest that caspase activation, that ultimately causes Intracellular ROS were detected using the oxidation- PARP cleavage, is not directly triggered by ROS, and indi- sensitive probe dihydrorhodamine-123 (DHR-123), which cate that gold(III) compounds might promote cell death by is reported to react preferentially with hydrogen peroxide other mechanisms that are counteracted by NAC. and to localize at the mitochondrial level. Results, reported in Figure 4C, showed that the treatment with complexes Molecular Targets at Mitochondrial 2 and 4 increases the oxidized state of the cells, detected and Cytosolic Levels as enhancement of fluorescence intensity of the probe; Thioredoxin reductase (TrxR) appears to be a very specific similar results were obtained with complexes 1 and 3 intracellular target for antitumor agents. In particular, the (data not shown). Conversely, control nontreated cells or mitochondrial isoform may be of potential interest regard- cells treated with cisplatin showed only a weak fluorescent ing the role of these organelles in the apoptotic process, background, most likely due to small DHR-123 oxidation and previous reports indicate that both gold(I) and gold(III) occurring during its manipulation. Thus, these results complexes are highly specific inhibitors of mitochondrial indicate that our gold(III) complexes are able to induce thioredoxin reductase. Thioredoxin reductase is a se- ROS in both isolated mitochondria and cells. lenoenzyme with a selenol group in the active site at the C terminus of the enzyme. Gold compounds preferen- Effect of the Antioxidants Trolox and NAC on ROS tially interact with this site since selenol displays a greater Production and PARP Cleavage in HeLa Cells affinity toward heavy metals. To investigate whether ROS were responsible for the ob- On the basis of these considerations, our gold(III)-di- served cell death, HeLa cells were treated in the presence thiocarbamato derivatives were tested to study their po- of either NAC or the hydrophilic form of vitamin E (Trolox, tential inhibitory effect on this selenoenzyme. The inhibi- 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid). tory activity of complexes 1–4 on both cytosolic (TrxR1) Both are good ROS scavengers. NAC is a reducing thiol and mitochondrial (TrxR2) thioredoxin reductase is re- that helps to maintain the cellular thiol redox balance, ported in Figure 6. The cytosolic isoform (Figure 6A) is whereas Trolox interacts preferentially with free radicals particularly sensitive to the action of gold(III) compounds, such as the peroxyl radicals. Given that all four complexes all of them being inhibitory agents at nanomolar level, ex- exhibited similar apoptotic and oxidative behavior, com- hibiting IC50 values ranging from 5.67 nM (complex 1) to plex 4 was chosen as representative. Using DHR-123 as 17.01 nM (complex 3). Mitochondrial thioredoxin reductase a probe for ROS detection, we found that when NAC (Figure 6B) seems to be less sensitive to the treatment, (1 mM) or Trolox (100 mM) were added to cultures before with IC50 values ranging from 24.74 nM (complex 1) to 1132 Chemistry & Biology 14, 1128–1139, October 2007 ª2007 Elsevier Ltd All rights reserved Chemistry & Biology Gold-Dithiocarbamato Complexes and Cancer Cells Figure 5. Effect of Treatment with the Antioxidants Trolox and NAC on ROS Generation in Cell Death (A) HeLa cells were treated and examined for ROS generation as described in Figure 4C. Graphs report the profile of DHR-123 relative intensity quantified using the Zeiss Profile soft- ware. Profiles are referred to the signals along a line traced across few cells. When present, Trolox (100 mM) or NAC (1 mM) were added for 1 h and 18 h, respectively, before treatment with both DHR-123 and complex 4 (10 mM). (B) Phase-contrast microscopy representative pictures showing HeLa cells pretreated for 1 h with Trolox (100 mM) or 18 h with NAC (1 mM) and then incubated for additional 24 h in medium containing 10 mM complex 4. Origi- nal magnification = 1003. Quantification of ap- optosis through PARP cleavage determination. (C) PARP cleavage was analyzed by western blot as described in the Experimental Proce- dures. (D) Graph showing the percentage of PARP cleavage calculated as the ratio between den- sitometrical values of cleaved and uncleaved bands 3 100. Values are given as means ± SE of at least three independent experiments. 35.87 nM (complex 4). However, in both cases the inhibi- might lead to the activation of extracellular signal-regu- tory effect is massive, and it is likely to be due to the inter- lated kinases (ERKs). action with the selenol residue present in the active site of In order to investigate the influence of our gold(III) com- thioredoxin reductase. In fact, thioredoxin reductase ob- plexes on MAPK signaling, we examined the activation of tained from E. coli, lacking the cysteine/selenocysteine ERK1 (p44) and ERK2 (p42). Kinetic studies showed that motif at the C terminus, is almost totally insensitive to treatment with complex 4 rapidly increases phospho- the gold(III) complexes (Figure 6C). Glutathione reductase ERK levels (Figure 6E). Nevertheless, when cells were is also unaffected by the treatment with complexes 1–4, treated in the presence of Trolox, no significant increase and this is in agreement with the fact that, although gluta- in ERK phosphorylation was observed (Figure 6F). These thione reductase is a protein structurally and functionally results might indicate that hydrogen peroxide accumula- close to thioredoxin reductase and belongs to the same tion influences MAPK signaling. Since the antioxidants family of the pyridine nucleotide oxidoreductase, it lacks Trolox and NAC showed different effects on cell survival selenol at the catalytic site (Figure 6D). In fact, as previ- and PARP cleavage in long-term treatment (Figures 5B– ously reported, it requires much greater concentrations 5D), we also studied the ERK phosphorylation state in of auranofin and other gold compounds to be inhibited. cells treated with complex 4 for 24 hr in the presence or absence of either Trolox or NAC. We found that Trolox Analysis of ERK Phosphorylation was not able to block the increase of phospho-ERK Mammalian thioredoxin reductase is a key enzyme for (Figure 6G); on the contrary, NAC treatment completely maintenance of intracellular reduced environment since blocked the induction of phospho-ERK by complex 4 it acts as a direct antioxidant of thioredoxin (Trx). Impair- (Figure 6H). These data may explain the different behavior ment of TrxR will lead to increased levels of oxidized of the two antioxidants on cell survival. It is worth noting Trx. Trx protects cells from a variety of oxidative stresses that NAC, even at relatively high concentration, does not by regulating the redox state and activity of many cellular alter the inhibitory effect of gold(III)-dithiocarbamato com- proteins that control cell growth. In the reduced form, Trx plexes on thioredoxin reductase. associates with apoptosis signal-regulating kinase-1/ mitogen-activated protein kinase kinase kinase (ASK- DISCUSSION 1/MAPKKK). Oxidation of Trx results in its dissociation and consequent activation of the MAPK pathway. The here investigated gold(III) complexes 1–4 were re- Recent studies suggest that apoptotic stimuli are trans- cently proved to be 1- to 4-fold more cytotoxic in vitro mitted to caspases through the activation of MAPKKK than cisplatin, even toward human tumor cell lines intrinsi- , and it is also believed that, depending on the length cally resistant to cisplatin itself. Moreover, they ap- of MAPKs activation (e.g., transient or prolonged), they peared to induce cell death also on cisplatin-resistant might trigger cell proliferation or death. Furthermore, cell lines, with activity levels comparable to those on the it was shown that ROS, in particular hydrogen peroxide, corresponding cisplatin-sensitive cell lines, ruling out the Chemistry & Biology 14, 1128–1139, October 2007 ª2007 Elsevier Ltd All rights reserved 1133  Chemistry & Biology Gold-Dithiocarbamato Complexes and Cancer Cells Figure 6. Effect on Thioredoxin and Glu- tathione Reductase and Induction of ERK Phosphorylation Cytosolic (A), mitochondrial (B) and E. coli (C) thioredoxin reductases and glutathione reduc- tase (D) were incubated as described in the Experimental Procedures. Gold(III) complexes were added at the indicated concentrations. The final concentrations of the enzymes were 2.1 and 3.8 nM for TrxR1 and TrxR2, respec- tively. E. coli thioredoxin reductase was 40.2 mg/ ml while glutathione reductase was 4.3 nM. The inhibitory effect of each tested compound was evaluated in comparison with the corresponding control (untreated thioredoxin and glutathione reductase). Dose-response curves were calcu- lated over the indicated range of concentrations, enabling IC50 values to be obtained. Values are given as means ± SE of at least three indepen- dent experiments. HeLa cells (1.5 3 105 cells per well) were incubated with complex 4 (10 mM) for 30 min to 3 h in the presence (F) or not (E) of 100 mM Trolox or for 24 h with 100 mM Trolox (G) or 1 mM NAC (H). At the end of treat- ments, aliquots of protein lysate, corresponding to 4.5 3 104 cells, were subjected to SDS-PAGE in 10% gel. Proteins were then transferred to PVDF and immunodecorated using anti-ERK and anti-phospho-ERK antibodies. Blots are representative of at least two independent ex- periments. occurrence of crossresistance phenomena and support- in their mechanism of cytotoxicity and antitumor activity ing the hypothesis of a different mechanism of action. Gold(III) complexes were generally proved to be less. The chemical characterization carried out in aqueous effective than gold(I) derivatives, such as auranofin, in solution allowed us to establish that they are reasonably stimulating mitochondrial swelling, and this might depend stable within a physiologically relevant environment, an on the different permeability characteristics of these com- essential prerequisite for any further pharmacological plexes. Moreover, the production of hydrogen perox- evaluation. Moreover, they show high reactivity to- ide by gold(I) compounds was already observed with aur- ward some biologically relevant isolated macromolecules, anofin both in isolated mitochondria and in cellular and dramatically inhibit both DNA and RNA synthesis, and systems. We demonstrate that also gold(III) dithiocar- induce DNA lesions with a faster kinetics than cisplatin. bamato derivatives are able to spark the production of this Nevertheless, they also induce a strong and fast hemolytic reactive oxygen species. Furthermore, due to the lack of effect (compared to cisplatin), suggesting that intracellular effect of these complexes on respiration it is reasonable DNA might not represent their primary or exclusive biolog- to conclude that the observed increase in hydrogen per- ical target. These encouraging results prompted us to oxide is essentially due to their inhibitory effect on thiore- carry out further investigations aimed at elucidating their doxin reductase rather than to stimulation of the mito- mechanism of action. chondrial electron transport chain. Although gold drugs have been in clinical use for over 70 Besides the strong impairment on thioredoxin reduc- years, their exact mechanism of action is on debate, as tase, we found that gold(III)-dithiocarbamato complexes their specific cellular targets are still unclear. Recent increase the levels of phosphorylated ERK1/2 in HeLa studies showed that mitochondria may play a major role cells. Activation of the ERK1/2 pathway by reactive 1134 Chemistry & Biology 14, 1128–1139, October 2007 ª2007 Elsevier Ltd All rights reserved Chemistry & Biology Gold-Dithiocarbamato Complexes and Cancer Cells Figure 7. Proposed Model of the Molecular Mechanisms Underlying Gold(III)-Dithiocarbamato Derivatives-Induced Cell Death oxygen species has been reported by several groups [37– bility of NAC itself to prevent dissociation of the Trx-ASK 39]. Nevertheless, more recently it has been reported that complex , most likely by maintaining thioredoxin re- ERK1/2 play a crucial role in the signaling downstream duced. In addition, these data strongly indicate that sus- ASK1. Indeed, apoptosis induced by the iron chelator de- tained and long-lasting stimulation of ERK phosphoryla- feroxamine or acrolein is mediated by both p38 tion is directly involved in cell death. Indeed, several and ERK activation in keratinocytes. Interestingly, acrolein groups reported that ERK1/2 play important roles not is a potent inhibitor of thioredoxin reductase. More- only in cell proliferation and survival, but also in the pro- over, it was shown that in cultured cerebellar granule gression of apoptosis [45, 46]. The mechanisms by which neurons in low potassium-induced apoptosis the ERK ERK induces apoptosis are not completely clarified yet, phosphorylation was activated by the ASK1-p38MAPK but it is believed that might occur at many different levels pathway. Activation of MAP kinases as a mechanism involving both the extrinsic and intrinsic apoptotic path- of apoptosis induced by gold(I) compounds was also sug- ways. For instance, inhibition of ERK phosphorylation gested by Park and coworkers. However, they re- decreases Bax expression and, in addition, ERK1/2 ported that in promyelocytic leukemia HL60 cells treated activity is downregulated by the ubiquitin/proteasome with auranofin the major kinase involved was p38- system. These results rationalize recent observations MAPK, whereas activation of ERK was independent of obtained with gold(III)-methylsarcosinedithiocarbamato auranofin concentration. These data, apparently in con- complexes indicating that they both upregulate Bax and flict with our findings, also indicate a direct involvement downregulate Bcl2 , whereas complex 1 exerts an in- of ASK1 in gold compounds-induced cell death. The acti- hibitory action on the proteasome system. Hence, vation of different effector kinases downstream of ASK1 proteasome inhibition can also explain the observed might be explained as the results of the different cell sys- long-lasting persistence of phosphorylated ERK1/2. tem used and the experimental timing. Based on these results, we propose a working model for The fact that incubation with the scavenger Trolox can the mechanism of action of our gold(III)-dithiocarbamato reduce phospho-ERK levels after short-term treatment complexes (Figure 7). As already reported for some orga- (3 h) with the investigated gold(III) complexes but it is un- nogold(III) compounds , complexes 1–4 can inhibit thi- able to block ERK activation, as well as cell toxicity and oredoxin reductase by irreversible covalent binding to its PARP cleavage, when the treatment is prolonged for 24 catalytic site. This hampers the function of both mitochon- h, suggests that our compounds trigger ERK phosphory- drial and cytosolic thioredoxin reductases that act as medi- lation mainly through ASK1 pathway deregulation. This ators of electron flow from NADPH to peroxiredoxins hypothesis is supported by the results obtained by using through Trx, and lead to an increase in the oxidized form NAC as an alternative antioxidant, and the reported capa- of Trx and to the accumulation of hydrogen peroxide. Chemistry & Biology 14, 1128–1139, October 2007 ª2007 Elsevier Ltd All rights reserved 1135  Chemistry & Biology Gold-Dithiocarbamato Complexes and Cancer Cells Both the events can improve the levels of phospho-ERK. carbonylcyanide m-chlorophenylhydrazone (CCCP), horseradish per- Indeed, it has been reported that hydrogen peroxide accu- oxidase (HRP), N-acetyl-L-cysteine (NAC), 5,50 -dithiobis-(2-nitroben- zoic acid) (DTNB) and Rhodamine-123 were purchased from Sigma. mulation can trigger ERK1/2 phosphorylation. On the 6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) other hand, oxidation of Trx will bring on dissociation of the was purchased from Fluka. Dihydrorhodamine-123 (DHR-123) and complex Trx-ASK1 and activation of MAPK system ob- 10-acetyl-3,7-dihydroxyphenoxazine (Amplex Red) were purchased served as subsequent ERK1/2 phosphorylation. Thus, from Invitrogen-Molecular Probes. All reagents and solvents were of we hypothesize that a persistent ERK1/2 activation triggered high purity and were used as purchased without any further purification. at first by accumulation of hydrogen peroxide and then by Gold(III) complexes 1–4 were synthesized as previously described. Before use, gold(III) complexes were dissolved in DMSO just be- activation of ASK-1 may be responsible for cell death. fore the experiments; calculated amounts of drug solutions were then added to the proper medium, to a final organic solvent concentration SIGNIFICANCE lower than 0.5% (v/v). All the tested complexes were proved, by 1H NMR studies, to be stable in DMSO over 48 h. Gold compounds possess a variety of therapeutic po- tentials ranging from attenuation of inflammation, sup- X-Ray Crystallography pression of autoimmunity, and promising antitumor Crystals of complex 4 suitable for X-ray crystal-structure determina- tion were obtained from an acetone/hexane (1:1) solution at 4 C. Dif- activity. Due to their chemical similarity to platinum(II) fraction data were collected using a Bruker Smart Apex CCD diffrac- compounds, it was believed that gold(III) compounds tometer equipped with an Oxford Cryosystems low-temperature might have DNA as their biological target. However, device operating at 150 K. The diffraction pattern was indexed with gold(III) complexes have been reported to interact two orientation matrices related via a 2-fold ration about the di- also with several other cellular components, and recent rection (CELL NOW ). Reflections from both domains were inte- data indicated mitochondria as potential targets. Nev- grated simultaneously and used in refinement (SAINT ). An absorp- tion correction was performed with the multiscan procedure TWINABS ertheless, so far the mechanism of action of gold(III). The structure was solved by Patterson methods (DIRDIF) , and compounds is unclear and there is little understanding refined against F2 using SHELXL-97. H atoms were placed in cal- of how they can elicit their activity. culated positions. The terminal EtO– group is disordered over two ori- By means of cellular, biochemical, and molecular entations to avoid a close contact across an inversion center. The oc- approaches, we analyzed the mechanism of action of cupancies were fixed at 0.5, and similarity restraints were applied to recently synthesized gold(III)-dithiocarbamato deriva- the bond distances and angles in the part-weight fragments. The twin scale factor refined to 0.523(15). X-ray crystallographic data are tives. These compounds are able to trigger cell death, available in the Supplemental Data. The crystal structure of complex to induce ROS generation, to modify some mitochon- 4 has been deposited at the Cambridge Crystallographic Data Centre drial functions, and to inactivate both cytosolic and and allocated the deposition number CCDC641437. These data can mitochondrial thioredoxin reductases. Besides the be obtained free of charge from The Cambridge Crystallographic impairment of TrxR, they induce elevated and long- Data Centre via http://www.ccdc.cam.ac.uk/data_request/cif. lasting levels of phosphorylated ERK1/2; persistent high levels of phosphorylated ERK might be the cause Determination of Apoptotic Activity in HeLa Cells HeLa cells were cultured at 37 C in 5% CO2 atmosphere in Dulbecco’s of cell death. Thus, it is worthwhile to envisage that modified Eagle medium supplemented with 10% fetal bovine serum, 2 gold(III) complexes favor the dissociation of the Trx- mM L-glutamine and antibiotics (200 units/ml penicillin, 150 units/ml ASK complex and the consequent prolonged activa- streptomycin). For apoptosis analysis, cells were seeded into 6-well tion of MAPKinase pathways by inhibiting the TrxR/ plates at a density of 1.5 3 105 cells per well, and exposed to the gold- Trx redox system. This hypothesis is further supported (III) complexes or cisplatin. After 24 h of incubation, cell death was by the finding that both ERK phosphorylation and cell evaluated by visual inspection using an Olympus phase-contrast mi- death were inhibited by treatment with the antioxidant croscope. After microscope observation, cells were collected and subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) NAC, whereas the peroxyl scavenger Trolox was inef- and western blot analysis (see below). fective. Indeed, it has been reported that NAC itself prevents the dissociation of the complex Trx/ASK SDS-PAGE and Western Blot Analysis kinase, most likely by keeping Trx in the reduced state. After treatment, all cells, including floating dead ones, were collected, Drugs that target specifically TrxR are of undoubted and total proteins were extracted in Laemmli buffer (LB-1X: 50 mM therapeutic relevance given that many tumors exhibit Tris-HCl [pH 6.8], 2% SDS, 10% glycerol, 1% b-mercaptoethanol, 0.05% bromophenol blue) supplemented with a cocktail of protease increased levels of TrxR or Trx, which might contribute inhibitors (Roche) and 50 mM sodium fluoride. Aliquots of lysate to resistance of cancer to therapy by scavenging ROS were subjected to SDS-PAGE. Proteins were then transferred to poly- eventually generated by various anticancer agents. vinylidene fluoride (PVDF) membranes (Amersham) and probed with the specific antibodies. The following antibodies were utilized: rabbit EXPERIMENTAL PROCEDURES polyclonal anti-PARP, mouse monoclonal anti-phospho-ERK1/2, and rabbit polyclonal anti-total ERK1/2 (Cell Signaling Technology) at the Materials manufacturer’s indicated concentrations. Secondary HRP-conjugated Human cervical carcinoma HeLa cell line was obtained from the antibodies (sheep anti-mouse or donkey anti-rabbit, Amersham) were American Type Culture Collection. Cisplatin (Bristol-Myers Squibb), used at a concentration of 1:10,000 in PBS-milk (5% powdered Dulbecco’s modified Eagle medium (DMEM, Sigma Aldrich), fetal bo- skimmed milk in PBS with 0.05% Tween). Membranes were developed vine serum (FBS, GIBCO), L-glutamine (GIBCO), penicillin (Pharmacia), using the enhanced chemiluminescence detection system ECL-Plus streptomycin (Bristol-Myers Squibb), phosphate buffered saline solu- (Amersham). Densitometric analysis was carried out using the Multia- tion (PBS, Sigma), low fat milk (Roche), rotenone, oligomycin, antimycin, nalyst Biorad analysis software. 1136 Chemistry & Biology 14, 1128–1139, October 2007 ª2007 Elsevier Ltd All rights reserved Chemistry & Biology Gold-Dithiocarbamato Complexes and Cancer Cells Preparation of Rat Liver Mitochondria Determination of ROS Accumulation in HeLa Cells and Submitochondrial Particles Cells were seeded into Lab-Tek Chambered Glasses, 11 3 22 mm Mitochondria were prepared from rat liver by differential centrifugation 4-well plates at a density of 5 3 104 cells per well. After 24 h of incuba- essentially according to Myers et al. in a medium containing 220 tion, cells were loaded with the oxidation-sensitive probe DHR-123 mM mannitol, 70 mM sucrose, 1 mM EDTA and 5 mM HEPES (pH (5 mM) for 30 min, and then exposed to complexes 1–4 for an additional 7.0). EDTA was omitted in the final washing and in the mitochondrial 30 min, in the presence or absence of the antioxidants Trolox or NAC. suspension. Mitochondrial proteins were estimated with the biuret At the end of treatment, cells were examined for ROS generation using procedure using BSA as standard. a Zeiss LSM-510 confocal laser microscope equipped with a CO2- and To obtain submitochondrial particles, mitochondria were diluted to temperature-controlled chamber. about 20 mg/ml with 25 mM Tris-HCl buffer (pH 8.0) and sonicated twice for 30 s. The suspension was then centrifuged at 10,000 x g Purification of Thioredoxin Reductase and Estimation of for 10 min to remove the unbroken mitochondria, the pellet was dis- Thioredoxin Reductase and Glutathione Reductase Activities carded, and the supernatant again centrifuged at 105,000 x g for 30 Cytosolic thioredoxin reductase (TrxR1) was prepared from rat liver ac- min. The obtained pellet, after washing, was suspended in a small vol- cording to Luthman et al.. Mitochondrial thioredoxin reductase ume of the same medium used for mitochondria. (TrxR2) was prepared from rat liver mitochondria according to Rigo- bello et al.. E. coli thioredoxin reductase was prepared according to Williams et al. , whereas yeast glutathione reductase was used Measurement of Oxygen Uptake as purchased (Sigma). The protein content of isolated enzymes was Rat liver mitochondria (1 mg/ml) or submitochondrial particles (0.5 mg/ estimated according to Lowry et al.. Thioredoxin reductase activity ml) were treated with complexes 1–4 at 25 C in 0.1 M sucrose, 50 mM was measured at 25 C in 0.2 M Na,K-Pi buffer (pH 7.5), 2 mM EDTA, KCl, 1 mM MgCl2, 1 mM NaH2PO4, 20 mM HEPES/Tris-HCl buffer (pH and 0.25 mM NADPH and started by the addition of 2 mM DTNB 7.4), 1 mM EGTA, and 5 mM glutamate plus 5 mM malate as sub- and followed spectrophotometrically at 412 nm. Glutathione reductase strates. State 3 respiration was initiated by adding 0.3 mM ADP. For activity was assayed at 25 C in 0.1 M Tris-HCl (pH 8.1) and 0.2 mM submitochondrial particles either 7.5 mM succinate or 0.75 mM NADPH. Reactions were initiated by 1 mM GSSG and followed spec- NADH were used as substrate. Oxygen uptake was estimated polaro- trophotometrically at 340 nm. In both thioredoxin and glutathione re- graphically at 25 C using a Clark-type oxygen electrode (Yellow ductase assays the various gold(III) complexes were added after 5 Springs) fitted in a water-jacketed chamber with continuous stir- min of incubation and, after 5 additional minutes, the reactions were ring. Mitochondria with respiratory control ratio of 4 or greater were uti- initiated by DTNB and GSSG, respectively. lized. To validate the oxygen uptake experiments, positive controls were run in the presence of 0.5 mM CCCP to obtain the maximal respi- ration and 1 mM antimycin A to completely inhibit oxygen consumption Statistical Analyses (not shown). All the values are given as means ± SE of not less than three measure- ments (unless otherwise stated). Multiple comparisons were made by one-way analysis of variance followed by the Tukey-Kramer multiple Measurement of Membrane Potential comparison test. Mitochondrial membrane potential was estimated by means of the fluorescent dye rhodamine-123 according to Emaus et al.. Mito- Supplemental Data chondrial proteins (0.3 mg/ml) were incubated at 25 C in 100 mM su- The Supplemental Data include details of the intermolecular interac- crose, 50 mM KCl, 20 mM HEPES/Tris-HCl buffer (pH 7.4), 1 mM tions and disorder in the X-ray structure of complex 4 and X-ray crys- NaH2PO4, 1 mM MgCl2, 6 mM rotenone, and 0.3 mM rhodamine-123. tallographic data in CIF format and can be found at http://www. Complexes 1–4 were then added at increasing concentrations. Fluo- chembiol.com/cgi/content/full/14/10/1128/DC1/. rescence was estimated in a microplate reader (Ascent FL, Labsystem) at 485 nm (excitation wavelength) and 527 nm (emission wavelength) in ACKNOWLEDGMENTS a final volume of 250 ml. To validate the estimation, a positive control with the uncoupler CCCP (0.5 mM) showing a rapid and complete col- This work was partially supported by Ministero dell’Università e della lapse of membrane potential was performed (not shown). Ricerca Scientifica e Tecnologica (Pharmacological and diagnostic properties of metal complexes). We thank Fondazione Italiana per la Estimation of Mitochondrial Swelling Ricerca sul Cancro—FIRC (Fellowship for L.P.), and European Union Rat liver mitochondria (0.5 mg/ml) were incubated at 25 C in 0.22 M (Marie Curie Fellowship for L.R.) for financial support. mannitol, 71 mM sucrose, 5 mM HEPES/Tris buffer (pH 7.4), 5 mM suc- cinate, 5 mM rotenone, and 3 mM oligomycin. Swelling was triggered by Received: May 10, 2007 the addition of complexes 1–4. Mitochondrial swelling was estimated Revised: July 26, 2007 as absorbance decrease at 540 nm using a Lambda-2 UV/VIS spectro- Accepted: August 10, 2007 photometer (Perkin-Elmer). 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Chemistry & Biology 14, 1128–1139, October 2007 ª2007 Elsevier Ltd All rights reserved 1139