Elesclomol | Inhibitor library

The cytotoxicity of the anticancer drug elesclomol is due to oxidative stress indirectly mediated through its complex with Cu(II)
Brian B. Hasinoff ⁎, Arun A. Yadav, Daywin Patel, Xing Wu
Faculty of Pharmacy, Apotex Centre, 750 McDermot Avenue, University of Manitoba, Winnipeg, Manitoba R3E 0 T5, Canada

a r t i c l e i n f o

Article history:
Received 6 January 2014
Received in revised form 1 April 2014
Accepted 2 April 2014
Available online 16 April 2014

Keywords:
Elesclomol Copper Ascorbic acid
Dichloroﬂuorescin Oxidative stress
Electron paramagnetic resonance
a b s t r a c t

Elesclomol is an anticancer drug that is currently undergoing clinical trials. Elesclomol forms a strong 1:1 complex with Cu(II) and may exert its anticancer activity through the induction of oxidative stress and/or its ability to transport copper into the cell. A UV–vis spectrophotometric titration showed that Cu(I) also formed a 1:1 complex with elesclomol. Ascorbic acid, but not glutathione or NADH, potently reduced the Cu(II)-elesclomol complex to produce hydrogen peroxide. Even though hydrogen peroxide mediated reoxidation of the copper(I) produced by ascorbic acid reduction has the potential to lead to hydroxyl radical formation, electron paramagnet- ic resonance spin trapping experiments, either with or without added hydrogen peroxide, showed that the ascorbic acid-reduced Cu(II)-elesclomol complex could not directly generate damaging hydroxyl radicals. Both Cu(II)-elesclomol and elesclomol potently oxidized dichloroﬂuorescin in K562 cells. The highly specific copper chelators tetrathiomolybdate and triethylenetetramine were found to greatly reduce the cytotoxicity of both elesclomol and Cu(II)-elesclomol complex towards erythroleukemic K562 cells, consistent with a role for copper in the cytotoxicity of elesclomol. The superoxide dismutating activity of Cu(II)-elesclomol was much lower than that of Cu(II). Depletion of glutathione levels in K562 cells by treatment with buthionine sulfoximine sensitized cells to both elesclomol and Cu(II)-elesclomol. In conclusion, these results showed that elesclomol indirectly inhibited cancer cell growth through Cu(II)-mediated oxidative stress.

⦁ Introduction

Elesclomol is a first-in-class anticancer drug that has just completed Phase 3 clinical trials for patients with advanced melanoma [1]. Elesclomol has also undergone randomized Phase 2 and 3 clinical trials for the treatment of a variety of other cancers (http://www.clinicaltrials. gov) [2–4]. Elesclomol typically inhibits cancer cell growth in vitro at low nanomolar concentrations [2,5–7]. It has been proposed that elesclomol is cytotoxic through the induction of oxidative stress that is mediated through its Cu2+ complex [2,3]. Elesclomol strongly binds Cu2+ [2,3,5,7,8] (Fig. 1) and has been shown to be able to scavenge copper from the culture medium and selectively transport it to the mitochondria where it induces oxidative stress [2,3]. A 3 h treatment of HL-60 cells with 100 nM elesclomol was shown to increase cellular concentrations of copper about 10-fold [2]. It was also shown that the elesclomol was efﬂuxed from the cell after it had transported copper into the cell, and was then free to shuttle more copper into the cell [2]. It has been shown that a Cu(II)-bis(thiosemicarbazone) (bisTSC) complex upon reduction by biological reductants can transfer the Cu+ produced to the copper-binding proteins Atx1 and Ctr1c [9]. Thus this may be a mechanism by which these complexes cause cellular retention

⁎ Corresponding author. Tel.: +1 204 474 8325; fax: +1 204 474 7617.
E-mail address: [email protected] (B.B. Hasinoff).
of copper. The fact that MCF7 cells with a compromised ability to repair oxidative DNA damage displayed increased sensitivity to elesclomol [6] suggests that elesclomol may also exert some of its cytotoxicity through non-mitochondrial targeted mechanisms. It has been shown [1] that elesclomol-treated patients with normal serum lactate dehydrogenase levels had improved outcomes compared to patients with high lactate dehydrogenase levels [1]. It was speculated that high lactate dehydroge- nase levels may be a reﬂection of the state of tumor hypoxia because well-oxygenated non-hypoxic cells would be dependent on oxidative phosphorylation rather than on glycolysis [1–3]. The Cu2+ complex of the structurally similar TSC NSC 689534 that displays antitumor activity has also been shown to induce oxidative stress [10].
In a previous study [5] we showed that elesclomol formed an ex- tremely strong complex with Cu2+ (stability constant of 1024.2 M−1; conditional stability constant at pH 7.4 of 1017.1 M−1). Our X-ray crystal- lographic determination showed that Cu(II)-elesclomol formed a neu-
tral 1:1 slightly distorted square planar complex. We also showed that both Fe2+ and Fe3+ had no detectable affinity for elesclomol. The Ni2+ and Pt2+ complexes of elesclomol that we synthesized and charac- terized were much less cytotoxic than the Cu2+ complex [5]. We also showed that ascorbic acid was oxidized by the Cu(II)-elesclomol complex. These results supported a putative central role for copper- induced oxidative stress in the cytotoxicity and the anticancer activity of elesclomol. Because elesclomol appears to exert its anticancer activity

Fig. 1. Structure of elesclomol and its reaction with Cu2+to form the Cu(II)-elesclomol complex.

through a mechanism unlike any other currently approved anticancer drug [3], it is important that its mechanism of action be characterized. Thus we designed experiments to see if cellular reductants could reduce Cu(II)-elesclomol to produce H2O2, and whether the H2O2 produced could promote hydroxyl radical formation through a Fenton-type reac- tion. We also carried out experiments in which the cell culture medium was depleted of copper with specific copper chelators in order to exam- ine the role of copper on the cytotoxicity of elesclomol. The role of oxi- dative stress on the cytotoxicity of elesclomol and Cu(II)-elesclomol was also probed using cells that had reduced levels of the antioxidant glutathione (GSH). These results further delineate the central role that copper plays as an inducer of oxidative stress in the cytotoxicity and the anticancer activity of elesclomol.

⦁ Experimental

⦁ Materials, spectrophotometry, cell culture and growth inhibition assays

Elesclomol and Cu(II)-elesclomol were synthesized and character- ized as we previously described [5]. Unless specified, all other reagents were obtained from Sigma-Aldrich (Oakville, Canada). The spectropho- tometric experiments were carried out in Tris buffer (10 mM, pH 7.4, 25% DMSO) at 37 °C in 1 cm spectrophotometer cells as we previously described [5] on a Cary 300 double beam spectrophotometer with a thermostated cell holder. It was found necessary to carry out the reactions in 25% (v/v) DMSO due to the low aqueous solubility of the Cu(II)-elesclomol complex. Human leukemia K562 cells, obtained from the American Type Culture Collection, were maintained as suspen- sion cultures in αMEM (minimal essential medium alpha) (Invitrogen, Burlington, Canada) containing 10% fetal calf serum. The spectrophoto- metric 96-well plate cell (5 × 104 cell/ml, 0.1 ml/well) growth inhibi- tion 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4- sulfophenyl)-2H-tetrazolium (MTS) CellTiter 96 AQueous One Solution Cell Proliferation assay (Promega, Madison, WI), which measures the ability of the cells to enzymatically reduce MTS after drug treatment, has been described previously [5,11]. The compounds tested were dissolved in DMSO and the final concentration of DMSO did not exceed an amount (typically 0.5% or less) that had any detectable effect on cell growth. The cells were incubated with the drugs for 72 h and then assayed with MTS. The IC50 values for cell growth inhibition were mea- sured by fitting the absorbance-drug concentration data to a four- parameter logistic equation as we described [5,12]. Where shown, error bars are the S.E.M.s. Where significance is indicated (p b 0.05) an unpaired t-test was used (SigmaPlot, San Rafael, CA).
For an assessment of the effects of lowering cellular GSH levels on
elesclomol and Cu(II)-elesclomol cytotoxicity, K562 cells in exponential growth were pretreated in 35 mm culture dishes with either buthionine sulfoximine (BSO) (100 μM) or vehicle, respectively, for 24 h as we previously described [13]. The cells were then seeded into 96-well plates (5000 cells/well) in triplicate in media that contained either BSO (100 μM) or vehicle, respectively. The cells were then treated with various concentrations of elesclomol or Cu(II)-elesclomol and allowed to grow an additional 72 h.
The kinetics of the trapping of Cu+ produced from the reduction of Cu(II)-elesclomol by ascorbic acid, GSH and NADH were followed spec- trophotometrically at 483 nm in Tris/DMSO buffer by formation of the
Cu(I)-bathocuproinedisulfonic acid (BCS; 2,9-dimethyl-4,7-diphenyl- 1,10-phenanthroline disulfonate disodium salt) complex. The kinetics of the displacement of Cu2+ from Cu(II)-elesclomol by the copper chela- tors triethylenetetramine (TRIEN) and ammonium tetrathiomolybdate (TM) were directly followed spectrophotometrically in Tris/DMSO buffer. The reaction of Cu(II)-elesclomol with TRIEN was followed at 355 nm and the reaction with TM at the 471 nm TM peak maximum. The percentage removal of Cu2+ from Cu(II)-elesclomol that TRIEN effected was estimated as we previously described [5], and that for TM from the total absorbance decrease at 471 nm observed when TM was titrated with CuCl2.

⦁ Spectrophotometric titration of elesclomol with Cu+

The spectrophotometric titration of elesclomol with CuCl was car- ried out similarly to that described for titration of elesclomol with Cu2+ from CuCl2 [5] except that solutions were N2-saturated with puri- fied N2 and they also contained ascorbic acid to maintain reduction of the complex. CuCl (1.4 M) was dissolved in 32% (w/v) NH4Cl in order to solubilize and stabilize it. This stock was diluted to 10 mM into N2- saturated water under N2 positive pressure containing 10 mM ascorbic acid. This stock was assayed with BCS at 483 nm using an extinction co-
efficient of 13,300 M−1 cm−1 [14]. The titration was carried out in a 1 cm serum-capped quartz spectrophotometer cell in N2-saturated
Tris buffer (10 mM, pH 7.4, 37 °C) that contained 25% (v/v) DMSO and 1 mM ascorbic acid under N2 positive pressure by adding microliter amounts of CuCl from a gas-tight syringe that had been ﬂushed with N2.

⦁ Fluorometric assay of DCF oxidation in K562 cells

2′,7′-Dichloroﬂuorescin diacetate (DCFH-DA) is a non-ﬂuorescent compound that when taken up by cells is hydrolyzed by esterases to yield non-permeable 2′,7′-dichloroﬂuorescin (DCFH) [15]. Cellular or exogenous oxidants are then able to oxidize reduced DCFH to the ﬂuo- rescent 2′,7′-dichloroﬂuorescein (DCF). The loading of K562 cells in Hank’s buffered salt saline (HBSS) with DCFH-DA and the oxidation to intracellular DCF assay (50,000 cells/well) was carried out as we previ- ously described [16] on a Fluostar Galaxy (BMG Labtech, Cary, NC) ﬂuo- rescence plate reader (excitation wavelength of 485 nm and emission wavelength of 520 nm, 30 °C) equipped with excitation and emission probes directed to the bottom of the plate. The average rate of ﬂuores- cence increase was computed from data in 6 wells 4 min directly after addition of the drug. Hydrogen peroxide, which rapidly enters cells and oxidizes DCF, was used as a positive control [16].

⦁ Measurement of H2O2 produced by ascorbic acid reduction of Cu(II)- elesclomol in the FOX1 assay

The production of H2O2 by Cu(II)-elesclomol or Cu2+ by ascorbic acid was determined using a slightly modified FOX1 microplate reader assay [17]. In this assay the oxidation of Fe2+ at low pH is determined in the presence of the Fe3+ complexing dye xylenol orange and the chain amplifier sorbitol. Cu(II)-elesclomol or the Cu2+ positive control was incubated with 100 μM ascorbic acid in Tris buffer (10 mM, pH 7.4, 25% DMSO) at 37 °C for 30 min. A 10 μl aliquot of the reaction mix- ture was added to 190 μl of the FOX1 reagent [17] and after 30 min of color development at 30 °C on a microplate reader the absorbance at 560 nm was measured in triplicate. The FOX1 reagent contained sor- bitol (300 mM), xylenol orange (100 μM), ferrous ammonium sulfate (250 μM) and H2SO4 (25 mM). The DMSO concentration (1.3 v/v%) in the FOX1 reagent was kept constant. Ascorbic acid stock solutions were made up fresh daily and stored on ice to reduce autoxidation. The H2O2 concentration produced was determined from an H2O2 stan- dard curve (r2 N 0.99) carried out under identical conditions in which the H2O2 was varied from 0 to 70 μM.

⦁ EPR spin trapping experiments

EPR spin trapping experiments were carried out as described [18] in order to see if the Cu(II)-elesclomol complex could be reductively acti- vated under aerobic conditions by ascorbic acid, either in the presence or absence of added H2O2, to produce hydroxyl radicals. Cu+ produced by the reduction of Cu2+ is a well known hydroxyl radical generating system [19]. The spin trapping systems contained either the Cu(II)- elesclomol complex or CuCl2 as indicated, ascorbic acid (1 mM, pH 7.4), Tris buffer (20 mM, pH 7.4), the secondary spin trap DMSO (5%, v/v) and 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) (100 mM; Enzo Life Sciences, Farmingdale, NY). In the case of the Cu(II)- elesclomol/ascorbic acid/H2O2 reaction system, the solution contained H2O2 (200 μM) in addition to the other components. Fifteen μl of the re- action mixture was added to the gas-permeable Teﬂon tubing, which was then folded at both ends and inserted into a quartz EPR tube open at both ends, and placed in the EPR cavity. Thermostated air (25 °C) was passed over the sample in the EPR cavity. For the EPR experiments carried out on the Bruker EMX (Milton, Canada) spectrometer 5 scans were averaged with the following instrument settings: microwave power 20 mW, modulation frequency 100 kHz, microwave frequency
9.3 GHz, modulation amplitude 2.0 G, 42 s scan time, 1024 data
points/scan, magnetic field centered at 3310 G, and a 100 G scan range.

⦁ Superoxide dismutating activity of Cu(II)-elesclomol

2
The superoxide dismutating activity of Cu(II)-elesclomol, Cu2+ and Cu/Zn superoxide dismutase (SOD) were determined using the nitro blue tetrazolium (NBT) 96-well plate dye reduction assay as described [20]. In this assay O•− that was generated by the xanthine (0.1 mM)/ xanthine oxidase (1.8 mU/ml; from bovine milk) system reacted with
NBT (50 μM) to produce an increase in absorbance at 570 nm (typically
0.005 absorbance units per min). The assay buffer (200 μl/well) contained 50 mM phosphate buffer (pH 7.4) and 100 μM EDTA (no EDTA was used for the CuCl2 experiments). Cu/Zn superoxide dismutase from bovine erythrocytes was run as a positive control. The IC50 values were measured from the inhibition of the triplicate averages of initial rates of absorbance increase fitted to a 4-parameter logistic equation.

⦁ Results

⦁ Reduction of Cu(II)-elesclomol by ascorbic acid, GSH and NADH yields Cu+

We previously showed that Cu(II)-elesclomol oxidized ascorbic acid under aerobic conditions [5]. This result suggested that reactive oxygen species (ROS) production mediated through formation of reduced Cu+ species may have a role in the cytotoxicity of elesclomol. The UV–vis spectral changes observed (Fig. 2A) when 20 μM Cu(II)-elesclomol was added to a solution containing 2 mM ascorbic acid and 100 μM BCS in Tris/DMSO buffer at 37 °C in a 1 cm spectrophotometer cell were consistent with BCS trapping Cu+. BCS is a dimethyl-substituted 1,10-phenanthroline that has a high affinity and specificity for Cu+ plus a high molar absorptivity [14,21] at 483 nm. The initial velocities for the ascorbic acid-, GSH- and NADH-mediated formation of the Cu+(BCS)2 complex are shown in Fig. 2B. These three biological reduc- tants were studied as they are the main non-enzymatic reductants present in the cell. Ascorbic acid-mediated formation of Cu+(BCS)2 displayed saturation behavior. Non-linear least squares curve fitting to this data yielded an apparent maximum velocity of 62 ± 10 mAbs/min and an apparent binding constant of 87 ± 31 μM. In contrast both GSH and NADH reduced Cu(II)-elesclomol much more slowly than did ascorbic acid. It can be estimated from the initial velocities that GSH and NADH reduced Cu(II)-elesclomol 60- and 120-fold slower, respec- tively, than did ascorbic acid.

Fig. 2. The reduction of Cu(II)-elesclomol with ascorbic acid, GSH and NADH followed by trapping Cu+ produced with BCS. A. UV–vis spectral changes observed when 20 μM Cu(II)-elesclomol was added to a solution containing 2 mM ascorbic acid, 100 μM BCS in Tris buffer (10 mM, pH 7.4, 25% DMSO) at 37 °C in a 1 cm spectrophotometer cell. The spectrum indicated by the arrow with the lowest absorbance at 483 nm (solid line) was recorded directly after the addition of Cu(II)-elesclomol and the subsequent spectra were recorded at intervals of 1.5 min. B. Initial velocities for reduction reaction of Cu(II)-
elesclomol with ascorbic acid (○), solid line; GSH (□), long dashed line; and NADH (Δ), short dashed line. The reactions were followed at 483 nm in the presence of 500 μM
BCS. The curved solid line is a saturation-type non-linear least squares fit to the concentration-dependent ascorbic acid reduction reaction. The straight lines for the GSH and NADH results were linear least squares calculated. From the initial velocity slopes it can be estimated that GSH and NADH reduced Cu(II)-elesclomol 60 and 120-fold slower than ascorbic acid did.

⦁ Spectrophotometric titration of elesclomol with Cu+

We previously showed [5] by spectrophotometric titration with CuCl2 that elesclomol formed a strong 1:1 complex with Cu2+. The re- sults above showed that biological reductants reduced the Cu(II)- elesclomol to produce Cu+. In order to determine if elesclomol could also form a complex with Cu+, elesclomol was titrated with CuCl under N2 in the presence of 1 mM ascorbic acid to prevent oxidation. The titration as we previously described for the Cu(II)-elesclomol titra- tion [5] was monitored by spectrophotometry in Tris buffer (10 mM, pH 7.4, 37 °C) that contained 25% (v/v) DMSO and 1 mM ascorbic acid. As shown in Fig. 3A the absorbance at the 475 nm shoulder system- atically increased with the addition of Cu+ up to 100 μM elesclomol. The least squares fits of the two linear segments of the plot of absorbance vs. Cu+ concentration (Fig. 3B) intersected at 102 μM or at a Cu+: elesclomol ratio of 1:1.02. These results were consistent with the forma- tion of a 1:1 complex. The fact that the plot had no significant curvature in the equivalence region was consistent with the formation of a very strong complex. The decrease in the absorbance that occurred after

3.4. EPR spin trapping experiments with ascorbic acid and Cu(II)-elesclomol and Cu2+

Spin trapping EPR experiments were carried out to see if the Cu(II)- elesclomol complex could upon reduction produce HO• in a H2O2- dependent reaction and thus provide a basis for a possible mechanism for the cytotoxicity of Cu(II)-elesclomol. Aqueous Cu+ has been shown to produced HO• in a H2O2-dependent reaction [19]. Likewise Cu(II)-EDTA in the presence of ascorbic acid has been shown to produce HO• [23]. The radicals produced can be trapped with DMPO and detected by EPR. The Cu+ complex may then be aerobically oxi-
dized to form O•−, which in turn dismutates to H O . It is well known
2 2 2
that O•− reacts with DMPO to form DMPO-O•−, which then decays rap-
2
2
2
2 2

Fig. 3. Spectrophotometric titration of elesclomol by Cu+. A. UV–vis spectral changes observed when microliter amounts of CuCl were added to 100 μM elesclomol in N2-saturated Tris buffer (10 mM, pH 7.4, 25% DMSO) containing 1 mM ascorbic acid at 37 °C in a 1 cm spectrophotometer cell. As indicated by the arrow, the lowest trace at the 475 nm shoulder increased in absorbance with the successive total concentrations of Cu+ indicated. B. Spectrophotometric titration of elesclomol by Cu+ at 475 nm. The intersection of the least squares calculated straight lines occurred at 102 μM which was consistent with the formation of a 1:1 complex between Cu+ and elesclomol.

the equivalence point (Fig. 3B) suggested that higher order complexes might be produced at excess Cu+ concentrations. Our previous studies showed that the Cu(II)-elesclomol complex had a conditional stability constant at pH 7.4 of 1017.1 M−1 [5].

⦁ Reduction of Cu(II)-elesclomol and Cu2+ by ascorbic acid produces H2O2

Because the results of Fig. 2 showed that ascorbic acid reduction of Cu(II)-elesclomol produced Cu+ we next investigated whether the Cu+ produced by this reaction yielded H2O2 mediated through an aero- bic redox cycling reoxidation reaction. As measured using the FOX1 assay [17] the results of Fig. 4 show that the Cu(II)-elesclomol/ascorbic acid system produced significant amounts of H2O2, even at the lowest concentration of 0.05 μM tested. At this concentration Cu(II)-elesclomol was even more potent than CuCl2 in generating H2O2. This is in spite of the fact that we previously showed by UV spectrophotometry that Cu2+ oxidizes ascorbic acid 15-fold faster than does Cu(II)-elesclomol [5]. The addition of catalase (0.1 mg/ml) to the incubation mixture reduced the amount of H2O2 produced to levels that were not significantly different than the control. The reduction in H2O2 formed at 20 μM Cu(II)- elesclomol compared to 5 μM Cu(II)-elesclomol could be due to a slight catalase activity of the complex as has been seen for Cu2+ and its 2,2′- bipyridyl complex [22].
idly to DMPO-OH• [24]. Because of the instability of DMPO-O•− and its rapid decay to form DMPO-OH•, a DMPO-O•− signal is generally seen in conjunction with a DMPO-OH• signal which could arise from either decay of DMPO-O•− or by reaction with HO•. Thus, because results in the absence of a secondary spin trap are ambiguous, DMSO was used to measure HO• formation unambiguously. Thus, in order to distinguish hydroxyl radical production from O•− production, spin trapping exper- iments were carried out employing 5% (v/v) DMSO as a secondary rad-
2
2
ical trap. Thus, HO• produced in this system reacts rapidly with DMSO to produce a methyl radical that then reacts with DMPO. Our results showed that ascorbic acid could reduce Cu(II)-elesclomol (Fig. 2) to form Cu(I)-elesclomol (Fig. 3) that was reoxidized to yield H2O2 (Fig. 4). This presumably occurred through dismutation of O•− to yield
H2O2. Thus EPR spin trapping experiments were carried out to see if
the ascorbic acid reduction of Cu(II)-elesclomol itself, or in the presence of added H2O2, could produce HO•. As shown in Fig. 5D and F, neither of these reaction systems produced measurable amounts of HO•. The amount of 6-line HO•-derived signal in Fig. 5F was about equal to that of the ascorbic acid/H2O2 control (Fig. 5E). In contrast, as shown in Fig. 5G the Cu2+/ascorbic acid system used as a positive control strongly promoted the formation of HO•. The 6-line spectrum (Fig. 5G) with a 1:1:1:1:1:1 ratio of peak heights and hyperfine splitting constants of AN = 16.2 G and AH = 23.3 G was characteristic of a carbon-centered radical expected for DMPO-CH3• [25].

⦁ Elesclomol and Cu(II)-elesclomol induce oxidation of DCFH to DCF in K562 cells

In order to investigate whether elesclomol or Cu(II)-elesclomol pro- duced oxidative stress in K562 cells, the oxidation of DCFH-DA loaded into K562 cells was followed in a ﬂuorescence plate reader. As shown in Fig. 6, treatment of K562 cells with both elesclomol and Cu(II)- elesclomol significantly increased the rate of oxidation to DCF at nearly all concentrations tested. The amount of DCF oxidation produced by 5 μM Cu(II)-elesclomol was nearly equal to that produced by 50 μM H2O2. These results show that Cu(II)-elesclomol potently produces ROS in cells.

⦁ Effect of the copper chelators TM and TRIEN on the cell growth inhibitory effects of elesclomol and Cu(II)-elesclomol and the kinetics of the displacement of Cu2+ from Cu(II)-elesclomol by TM and TRIEN

Both TM and TRIEN have very high specificity and affinity for copper ions and both have been clinically used in treating copper toxicity in Wilson’s disease [26]. In order to determine if copper has a role in medi- ating the K562 cell growth inhibitory effects of elesclomol and Cu(II)- elesclomol, experiments were carried out in which either TM or TRIEN were added to the culture medium. Either of these agents can potentially deplete the culture medium of free or loosely bound copper or possibly directly remove Cu2+ from its complex with elesclomol. Preliminary experiments that were carried out showed that treatment with 1 mM TRIEN had no measurable effect on K562 cell growth over 72 h. However, treatment with TM alone

Fig. 4. The reaction of ascorbic acid with Cu(II)-elesclomol produces H2O2. Cu(II)- elesclomol or the Cu2+ positive control was incubated with 100 μM ascorbic acid in Tris buffer (10 mM, pH 7.4, 25% DMSO) at 37 °C for 30 min. The reaction of both Cu(II)- elesclomol and Cu2+ with ascorbic acid produced dose dependent and significant amounts (p b 0.005) of H2O2 compared to the 0 μM control, except where no significance (NS) is indicated.

did marginally increase cell growth (Supplementary Fig. S1). This is also evident from Fig. 7A and B in which the absorbance values at low drug concentrations in the presence of added TM are higher than in its absence. The reason for this stimulation of growth is un- known. The addition of 30 μM TM to the culture medium increased the elesclomol IC50 values 6400-fold from 2.2 to approximately 14,300 nM (Fig. 7A). The effect of the addition of 10 μM TM on Cu(II)-elesclomol growth inhibition was similarly large and
increased the Cu(II)-elesclomol IC50 values 113-fold from 6.2 to 696 nM (Fig. 7B). Similar results were also seen with the addition of 30 μM TM for Cu(II)-elesclomol in which the IC50 was observed to increase 115-fold from 10 nM to 1160 nM (data not shown). Given these large increases in IC50 the stimulation on cell growth that TM induced did not significantly affect our conclusion about the effect of TM on growth inhibition. Treatment with 1 mM TRIEN also decreased the growth inhibitory effects of elesclomol (Fig. 7C) and Cu(II)-elesclomol (Fig. 7D), though the effects were not nearly as dramatic as with TM. As shown in Fig. 7C TRIEN treatment in- creased the elesclomol IC50 values 12-fold from 12 to 129 nM. Like- wise, TRIEN increased Cu(II)-elesclomol IC50 values 13-fold from
6.3 to 83 nM. These results with TM and TRIEN are in accord with previous results in which it was shown that the Cu+ chelator BCS de- creased the cytotoxicity of elesclomol [2]. Thus, the results that showed that the copper chelators TM and TRIEN greatly reduced the growth inhibitory effects of elesclomol and Cu(II)-elesclomol strongly suggests that copper present in the medium has a role in the K562 cell growth inhibitory effects of elesclomol and Cu(II)-elesclomol. In order to determine the mechanism of how TM or TRIEN acted to reduce the cytotoxicity of elesclomol or Cu(II)-elesclomol, spectropho- tometric experiments were carried out to see if TM or TRIEN could di- rectly displace Cu2+ from its complex with elesclomol. We previously showed that the Cu2+ chelator TRIEN could quickly, but only partially, displace Cu2+ from Cu(II)-elesclomol in an equilibrium displacement
reaction [5]. In fact these experiments allowed us to calculate a stability constant of 1024.2 M−1 for Cu(II)-elesclomol and a conditional stability constant of 1017.1 M−1 at pH 7.4 [5]. The results of Fig. 8 show that TRIEN (500 μM) quickly (t1/2 6 min), but only partially, displaced
(57%) Cu2+ from Cu(II)-elesclomol. In contrast, TM (30 μM) only slowly (t1/2 54 min), but completely (~ 100%) displaced Cu2+ from Cu(II)- elesclomol. Thus TM and TRIEN have the ability to displace the Cu2+ that elesclomol scavenges from the culture medium and prevent forma- tion of Cu(II)-elesclomol. Likewise, TM and TRIEN have the ability to di- rectly displace Cu2+ from the Cu(II)-elesclomol added to the medium.

⦁ Cu(II)-elesclomol has moderate superoxide dismutating activity

Both Cu2+ and copper complexes may have the ability to

dismutate O•− into H O
[20]. The results in Fig. 9A show that Cu(II)-

2 2 2

Fig. 5. EPR spectra produced by the Cu(II)-elesclomol complex compared to that of Cu2+ in the DMPO spin trapping reaction systems. A. Control EPR spectrum containing DMPO. B. EPR spectrum of Cu(II)-elesclomol (100 μM). C. EPR spectrum of ascorbic acid (1 mM, pH 7.4). The small 2-line signal at center field was due to the ascorbyl radical normally present in ascorbic acid solution. D. EPR spectrum of Cu(II)-elesclomol/ascorbic acid (100 μM/1 mM) system showing the lack of any detectable hydroxyl radical- derived methyl radical adduct free radical signal. E. EPR spectrum of ascorbic acid/H2O2 (1 mM/200 μM) system. F. EPR spectrum of Cu(II)-elesclomol/ascorbic acid/H2O2 (100 μM/1 mM/200 μM) system showing the lack of any detectable hydroxyl radical- derived methyl radical adduct free radical signal. G. EPR spectrum of Cu2+/ascorbic acid/H2O2 (100 μM/1 mM/200 μM) system showing the six-line 1:1:1:1:1:1 EPR spectrum of the hydroxyl radical-derived methyl radical adduct of DMPO produced. The spectra are an average of 5 scans recorded over 3.5 min at 25 °C. For all spectra the spin trap DMPO (100 mM) was used with 5% (v/v) DMSO as a secondary spin trap. All reaction mixtures contained Tris buffer (20 mM, pH 7.4).

Fig. 6. Both Cu(II)-elesclomol and elesclomol increased the oxidation of DCFH to DCF in K562 cells. K562 cells were loaded with DCFH-DA for 20 min and washed with HBSS. After obtaining a ﬂuorescence baseline measurement for 10 min, cells were treated as indicated and the rate at which DCF ﬂuorescence increased was measured. H2O2 was used as a positive control. Error bars were calculated from averages from six replicate measurements. Significant differences between treated and untreated samples are designated by ***, p b 0.001; **, p b 0.01; *, p b 0.05.

Fig. 7. Effect of the copper chelators TM and TRIEN on the K562 cell growth inhibitory effects of elesclomol and Cu(II)-elesclomol. A. Effect of the addition of TM on the K562 cell growth inhibitory effects of elesclomol; control treatment (○), + 30 μM TM (▲). B. Effect of the addition of TM on the K562 cell growth inhibitory effects of Cu(II)-elesclomol; control treatment (○), + 10 μM TM (▲). C. Effect of the addition of TRIEN on the K562 cell growth inhibitory effects of elesclomol; control treatment (○), + 1 mM TRIEN (▲). D. Effect of the addition of TRIEN on the K562 cell growth inhibitory effects of Cu(II)-elesclomol; control treatment (○), + 1 mM TRIEN (▲). The cells were treated with TM just prior to the addition of either elesclomol or Cu(II)-elesclomol. In the case of TRIEN the cells were pretreated with TRIEN for 1.5 h prior to the addition of either elesclomol or Cu(II)-elesclomol. Cell growth inhibition was evaluated by an MTS assay after 72 h. The curved lines were calculated from non-linear least squares fits to 4-parameter logistic equations to yield IC50 values.

2
2
elesclomol inhibited the O•− reduction of NBT with an IC50 of 7.1 μM compared to 0.24 μM for Cu2+. The rate of reduction of NBT by O•− can be conveniently followed by the decrease in its absorbance at

Fig. 8. Kinetics of the displacement of Cu2+ from Cu(II)-elesclomol by the copper chelators TRIEN and TM. The reaction mixture contained Cu(II)-elesclomol (20 μM) in Tris buffer (10 mM, pH 7.4, 25% DMSO) at 37 °C in a 1 cm spectrophotometer cell. The reaction with 500 μM TRIEN was followed at 355 nm (solid line) where Cu(II)-elesclomol has a shoulder absorbance [5]. The reaction with 30 μM TM (broken dashed line) was followed at 471 nm where TM has a peak absorbance. The percentage of Cu2+ that was displaced from Cu(II)-elesclomol at equilibrium calculated from the absorbance changes at these wavelengths is indicated on the right hand side of the graph.
570 nm in a 96-well plate. If O•− produced by the xanthine/xanthine oxidase system is removed from the reaction system by reaction with
2
SOD or Cu(II)-elesclomol the decrease in the rate of the absorbance change can be used to measure superoxide dismutating activity. The 30-fold lower superoxide dismutating activity of Cu(II)-elesclomol compared to Cu2+ suggests that the Cu2+-promoted dismutating activity from Cu2+ transported into the cells by elesclomol [2] would be the relatively more important mechanism by which H2O2 is produced. From the results of Fig. 9A and B it can be estimated from their respective IC50 values that Cu(II)-elesclomol had a superoxide dismutating activity equal to that of 0.031 mU SOD/ml.

⦁ Reducing intracellular GSH levels increases elesclomol and Cu(II)-elesclomol cytotoxicity in K562 cells

GSH is an intracellular antioxidant that protects cells from oxidative stress. Cells with reduced cellular GSH levels should be more sensitive to the effects of elesclomol and Cu(II)-elesclomol if oxidative stress has a role in their cytotoxicity. As we reported previously [13,16], GSH levels in K562 cells were decreased to one-sixth of their normal value by treat- ment with BSO by a 24 h preincubation with 100 μM BSO, an inhibitor of the rate limiting GSH-synthesizing enzyme γ-glutamylcysteine synthe- tase [27]. The BSO pretreatments were followed by a 72 h-exposure to various concentrations of elesclomol and Cu(II)-elesclomol, after which cell growth was measured with the MTS assay. As shown in

2
Fig. 9. Comparison of the inhibition of O•− reduction of NBT due to the superoxide dismutating activity of Cu(II)-elesclomol, Cu2+ and Cu/Zn bovine erythrocyte superoxide dismutase. The superoxide dismutating activity was measured by following the inhibition of the rate of reduction of NBT at 570 nm in a xanthine/xanthine oxidase superoxide generating system.
A. Inhibition of superoxide dismutating activity by Cu(II)-elesclomol (○) and Cu2+ (●). B. Inhibition of the superoxide dismutating activity of SOD (○) under the same conditions as that for Cu(II)-elesclomol. The left-most data point in each plot are the rates measured in the absence of either added Cu(II)-elesclomol or SOD, respectively. The assay buffer for Cu(II)-elesclomol contained 100 μM EDTA and 1.8 mU/ml of xanthine oxidase, while that for Cu2+ contained no EDTA and 3.6 mU/ml of xanthine oxidase to compensate for the absence of EDTA in the assay buffer. The curved lines are non-linear least squares fits to 4-parameter logistic equations and yield IC50 values.

Fig. 10A, the IC50 value for elesclomol was reduced 5.2-fold from 25 to
4.7 nM for the GSH-depleted BSO-treated cells. Likewise, BSO treatment reduced the IC50 value for Cu(II)-elesclomol 5.5-fold from 18 to 3.2 nM.

⦁ Discussion

Previous studies have shown that elesclomol can preferentially che- late Cu2+ outside the cell and transport it into the cell as its neutral Cu(II)-elesclomol complex causing greatly increased cellular copper levels [2]. It was also shown that Cu(II)-elesclomol is transported to the mitochondria where it causes an accumulation of copper in the mitochondria [2,3]. It is unknown what this accumulated copper is complexed with. It was proposed that after reduction of Cu2+ to Cu+ damaging ROS are produced which leads to mitochondrial apo- ptosis [2,28]. The Cu+ species produced would be readily oxidized by
2
2
2
O2 to produce O•− which would then, in turn, dismutate to H O with the potential to form HO• in a Fenton-type reaction by reaction with
Cu+. It was also proposed that after reduction the complex dissociates and elesclomol is efﬂuxed from the cell where it can continue to shuttle more Cu2+ into the cell [2].
The results of this study showed that Cu(II)-elesclomol was reduced by physiological concentrations of ascorbic acid [29] to produce Cu+
that could be trapped by BCS. We also showed that elesclomol also formed a strong 1:1 complex with Cu+ (Fig. 3). The results of Fig. 2 show that 100 μM BCS is able to displace Cu+ from Cu(I)-elesclomol produced from the ascorbic acid reduction of Cu(II)-elesclomol. The conditional stability constant for the reaction of BCS with Cu+ has re- cently be determined to have a log ß2 of 20.8 at pH 7 [21]. Both GSH and NADH reduced Cu(II)-elesclomol much more slowly (60- and 120-fold, respectively) than did ascorbic acid (Fig. 2B). Thus, in the cell Cu(II)-elesclomol would be preferentially reduced by ascorbic acid at intracellular concentrations of these reductants. Aerobic reoxidation of the Cu(I)-elesclomol produced by the ascorbic acid-mediated reduc- tion of Cu(II)-elesclomol should initially produce O•− which can sponta-
2
neously dismutate to form H2O2. The FOX1 assay was used to show
(Fig. 4) that the ascorbic acid/Cu(II)-elesclomol system extremely effi- ciently generated H2O2 in the low nanomolar range, and in this regard was even more efficient that Cu2+ itself. Thus, ascorbic acid mediated reduction of Cu(II)-elesclomol would be highly capable of producing damaging amounts of H2O2 in the cell.
EDTA complexes of Cu2+ and Fe3+ are well known to be able to re- duced by ascorbic acid to produce H2O2, which can then react with the reduced form of the metal ion to produce HO• [18,23]. EPR spin trapping experiments (Fig. 5) were carried out to determine if ascorbic acid

Fig. 10. Depletion of GSH in K562 cells increases the growth inhibitory effects of elesclomol and Cu(II)-elesclomol. A. Growth inhibitory effects of elesclomol on K562 cells not treated with BSO (○), or treated with 100 μM BSO (▲) (broken dashed line), respectively. B. Growth inhibitory effects of Cu(II)-elesclomol on K562 cells not treated with BSO (○), or treated with 100 μM BSO (▲) (broken dashed line), respectively. The curved lines are non-linear least squares fits to 4-parameter logistic equations and yield IC50 values.

reduction of the Cu(II)-elesclomol complex could also produce HO•. The results of Fig. 5 showed that while ascorbic acid reduction of Cu2+ very efficiently produced HO•, somewhat surprisingly ascorbic acid reduction of Cu(II)-elesclomol produced no measurable formation of HO•. There was also no measurable formation of HO• even when the ascorbic acid/Cu(II)-elesclomol system was supplemented with H2O2 (Fig. 5 F). Thus, it can be concluded that ascorbic acid mediated formation of HO• by Cu(II)-elesclomol is not a significant mechanism by which Cu(II)-elesclomol exerts its cell growth inhibitory activity. Given the strong ability of free Cu2+ to generate HO• (Fig. 5G) it may be that it is the Cu2+ that is shuttled into the cell by elesclomol [2] and released, rather than Cu(II)-elesclomol that may be generating damaging HO•. For iron complexes to be able to generate hydroxyl radicals there is a requirement for a free iron coordination site that can react with H2O2 [30]. The Cu2+ complex [23] of hexadentate EDTA, which has two coordinated water molecules [31], fills this requirement. Because elesclomol is a tetradentate ligand that forms an extremely stable 4- coordinate square planar complex with Cu2+ [5], it likely does not have free coordination sites that can react with H2O2. Consequently, like the iron complexes of diethylenetriaminepentaacetic acid and de- feroxamine [32], the Cu(II)-elesclomol complex may be unable to pro- duce hydroxyl radicals.
Cu(II)-elesclomol and elesclomol were both able to cause oxidation to DCF in K562 cells. The effect was more pronounced with Cu(II)- elesclomol than with elesclomol. This was likely due to the more rapid uptake of Cu(II)-elesclomol into the cell [2]. The ROS generating ability of 5 μM Cu(II)-elesclomol to produce oxidized DCF in K562 cells was sig- nificant, judged by the fact that it produced almost as much oxidized DCF as that produced by a 50 μM H2O2 treatment. The DCF experiments do not, however, distinguish what ROS species are responsible for the oxidation of DCFH to DCF. These results were consistent with the results of Fig. 4 that showed that the ascorbic acid/Cu(II)-elesclomol system was very efficient at producing H2O2.
It has been proposed that elesclomol exerts its in vitro cell growth inhibitory activity by scavenging copper from the culture medium and selectively transporting copper into the cell and inducing oxidative stress [2,3]. In order to test this hypothesis experiments were carried out in which the highly specific copper chelators TM and TRIEN were added to the culture medium. The results of Fig. 7 showed that both TM and TRIEN strongly reduced the cell growth inhibitory effects of elesclomol and Cu(II)-elesclomol. Both TM and TRIEN directly displaced Cu2+ from Cu(II)-elesclomol (Fig. 8) and provide, in part, a mechanism by which TM and TRIEN reduced the cell growth inhibitory effects of elesclomol and Cu(II)-elesclomol. While TRIEN displaced Cu2+ from Cu(II)-elesclomol much faster than TM did, the percentage of Cu2+ it displaced from Cu(II)-elesclomol was less than that caused by TM. These results were consistent with the smaller reduction in growth in- hibitory effects seen with TRIEN as compared to TM (Fig. 7). Together these results support the hypothesis that elesclomol exerted its in vitro cell growth inhibitory activity by scavenging free or loosely bound copper from the medium. The fact that elesclomol and Cu(II)- elesclomol inhibited cell growth about equally well (Fig. 7) was also consistent with a role for copper in its cytotoxicity. Elesclomol, which inhibited cell growth at low nanomolar concentrations (Fig. 7), presum- ably obtained its copper from adventitious copper present in the medi- um and/or from copper binding proteins in the added serum. It should be noted that all the cellular results were obtained in K562 cells. It is possible that other cell types could have a different response to elesclomol or Cu(II)-elesclomol. The plasma copper protein carriers albu- min, transcuprein, and ceruloplasmin are the main source of copper in
blood [33]. Cu(II)-elesclomol displayed only moderate O•− dismutating
GSH is an intracellular antioxidant, which when depleted, leaves the cell susceptible to oxidative stress. The results of Fig. 10 showed that when GSH was depleted 6-fold in K562 cells that the cytotoxicity of both elesclomol and Cu(II)-elesclomol were increased. Thus, these results were also consistent with elesclomol and Cu(II)-elesclomol acting, at least in part, through induction of oxidative stress.
In conclusion, the results of this study showed that Cu(II)-elesclomol was rapidly and efficiently reduced by cellular levels of ascorbic acid, but not by GSH or NADH. Elesclomol was also shown to form a strong complex with Cu+. Upon ascorbic acid reduction Cu(II)-elesclomol effi- ciently generated high levels of H2O2 which would be damaging to the cell. The H2O2 produced would be able to generate damaging HO• by re- action with Cu+ produced by the ascorbic acid reduction of Cu2+ that had been transported into the cell by elesclomol. However, even though ascorbic acid reduction of Cu(II)-elesclomol efficiently produced H2O2, our EPR experiments showed that this system did not result in hydroxyl radical production through a Fenton-type reaction. If Cu(II)-elesclomol was able to generate significant levels of HO•, it would likely be even more cytotoxic and nonspecific than it is. The fact that the highly specif- ic copper chelators TM and TRIEN reduced the cytotoxicity of elesclomol and Cu(II)-elesclomol indicated a central role for copper in their cyto- toxicity. This study did not directly address the proposed mitochondrial basis [2,3] of the cytotoxicity of elesclomol or the possibility that elesclomol interferes with sensitive copper proteins. However, the fact that cytoplasmic levels of ascorbic acid efficiently generated H2O2 sug- gests that there may also be an indirect non-mitochondrial component to the cytotoxicity and the anticancer activity of elesclomol.

Acknowledgments

This work was supported by the Canadian Institutes of Health Re- search, the Canada Research Chairs Program, and a Canada Research Chair in Drug Development for B.B.H.

Appendix A. Supplementary data

Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.jinorgbio.2014.04.004. These data include MOL files and InChiKeys of the most important compounds described in this article.

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