Biochemical Pharmacology 93 (2015) 266–276
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Biochemical Pharmacology journal homepage: www.elsevier.com/locate/biochempharm
Cellular mechanisms of the cytotoxicity of the anticancer drug elesclomol and its complex with Cu(II) Brian B. Hasinoff a,*, Xing Wu a, Arun A. Yadav a, Daywin Patel a, Hui Zhang b,c, De-Shen Wang b,d, Zhe-Sheng Chen b, Jack C. Yalowich e a
Faculty of Pharmacy, Apotex Centre, University of Manitoba, 750 McDermot Avenue, Winnipeg, MB, Canada R3E 0T5 Department of Pharmaceutical Sciences, College of Pharmacy and Health Sciences, St. John’s University, Queens, New York, NY, USA Sun Yat-Sen University Cancer Center, State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, Guangzhou 510060, China d Department of Medical Oncology, Sun Yat-sen University Cancer Center, Guangzhou, Guangdong, China e Division of Pharmacology, College of Pharmacy, The Ohio State University, 500 West 12th Avenue, Columbus, OH 43210, USA b c
A R T I C L E I N F O
A B S T R A C T
Article history: Received 5 November 2014 Accepted 1 December 2014 Available online 27 December 2014
The potent anticancer drug elesclomol, which forms an extremely strong complex with copper, is currently undergoing clinical trials. However, its mechanism of action is not well understood. Treatment of human erythroleukemic K562 cells with either elesclomol or Cu(II)–elesclomol caused an immediate halt in cell growth which was followed by a loss of cell viability after several hours. Treatment of K562 cells also resulted in induction of apoptosis as measured by annexin V binding. Elesclomol or Cu(II)– elesclomol treatment caused a G1 cell cycle block in synchronized Chinese hamster ovary cells. Elesclomol and Cu(II)–elesclomol induced DNA double strand breaks in K562 cells, suggesting that they may also have exerted their cytotoxicity by damaging DNA. Cu(II)–elesclomol also weakly inhibited DNA topoisomerase I (5.99.1.2) but was not active against DNA topoisomerase IIa (5.99.1.3). Elesclomol or Cu(II)–elesclomol treatment had little effect on the mitochondrial membrane potential of viable K562 cells. NCI COMPARE analysis showed that Cu(II)–elesclomol exerted its cytotoxicity by mechanisms similar to other cytotoxic copper chelating compounds. Experiments with cross-resistant cell lines overexpressing several ATP-binding cassette (ABC) type efflux transporters showed that neither elesclomol nor Cu(II)–elesclomol were cross-resistant to cells overexpressing either ABCB1 (Pgp) or ABCG2 (BCRP), but that cells overexpressing ABCC1 (MRP1) were slightly cross-resistant. In conclusion, these results showed that elesclomol caused a rapid halt in cell growth, induced apoptosis, and may also have inhibited cell growth, in part, through its ability to damage DNA. ß 2014 Elsevier Inc. All rights reserved.
Chemical compounds studied in this article: Elesclomol (PubChem CID: 300471) Cu(II)–elesclomol (PubChem CID: 45273878) Etoposide (PubChem CID: 36462) Keywords: Elesclomol Copper JC-1 Cell cycle Oxidative stress Efflux
1. Introduction Elesclomol is a highly novel anticancer drug that has completed phase 3 clinical trials for patients with advanced melanoma [1] and is currently undergoing Phase 1 and 2 trials for the treatment of a variety of other cancers (http://www.clinicaltrials.gov) [2–4]. Elesclomol and Cu(II)–elesclomol (Fig. 1) are both extremely potent in vitro and typically inhibit cancer cell growth at low nanomolar concentrations [2,5–8]. It has been proposed that elesclomol is cytotoxic through the induction of oxidative stress that is mediated through its Cu2+ complex [2,3,5]. The development of copper
* Corresponding author. E-mail address: [email protected] (B.B. Hasinoff). http://dx.doi.org/10.1016/j.bcp.2014.12.008 0006-2952/ß 2014 Elsevier Inc. All rights reserved.
complexes as anticancer agents has recently been reviewed [9]. A recent report using an HClO-specific fluorescent probe has shown that elesclomol can induce formation of the highly reactive and strongly oxidizing HClO in breast cancer MCF7 cells [10]. However, it is not known whether this is a direct or an indirect effect. Elesclomol strongly binds both Cu2+ [2,3,6,8,11] (Fig. 1) and Cu+ [5]. Elesclomol can scavenge copper from the culture medium and selectively transport it to the mitochondria where it induces oxidative stress [2,3]. It has also been shown that the elesclomol was subsequently effluxed from the cell after it had transported copper into the cell, and was then free to shuttle more copper into the cell [2]. MCF7 cells with a compromised ability to repair oxidative DNA damage have increased sensitivity to elesclomol [7], which suggests that elesclomol may also exert some of its cytotoxicity through DNA-damaging mechanisms. Interestingly, it
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Fig. 1. Structure of elesclomol and its reaction with Cu2+ to form the neutral Cu(II)– elesclomol complex.
has been shown that elesclomol-treated patients with normal serum lactate dehydrogenase levels had improved outcomes compared to patients with high lactate dehydrogenase levels [1]. Yeast gene deletion mutant studies suggested that elesclomol does not work through a specific cellular protein target and is unlike any other currently approved anticancer drugs [3]. In previous studies we showed that elesclomol forms an extremely strong 1:1 neutral complex with Cu2+ (stability constant of 1024.2 M1; conditional stability constant at pH 7.4 of 1017.1 M1) and also forms a 1:1 complex with Cu+ [5,6]. We also showed that ascorbic acid, but not glutathione or NADH, reduces the Cu(II)–elesclomol complex to produce hydrogen peroxide [5]. Electron paramagnetic resonance (EPR) spin trapping experiments showed that the ascorbic acid-reduced Cu(II)–elesclomol complex, in comparison to ascorbic acid-reduced Cu2+, does not directly generate damaging hydroxyl radicals. We also showed that depletion of glutathione levels in K562 cells by treatment with buthionine sulfoximine sensitizes cells to both elesclomol and Cu(II)–elesclomol. Consistent with a role for copper in the cytotoxicity of elesclomol, the highly specific copper chelators tetrathiomolybdate and triethylenetetramine greatly reduce the cytotoxicity of both elesclomol and Cu(II)–elesclomol complex toward K562 cells [5]. These results showed that elesclomol indirectly inhibited cancer cell growth through Cu(II)-mediated oxidative stress. In order to further characterize the cell growth inhibitory effects of elesclomol and Cu(II)–elesclomol we designed experiments to measure their effects on: (1) cell cycle; (2) induction of apoptosis; (3) mitochondrial membrane potential; (4) formation of DNA double strand breaks; and (5) inhibition of DNA topoisomerase I (5.99.1.2) and DNA topoisomerase IIa (5.99.1.3). We also looked for cross-resistance in cells lines overexpressing several ATP-binding cassette (ABC) type efflux transporters in order to determine if either elesclomol or Cu(II)–elesclomol were substrates for these transporters. Finally, GI50 results obtained from submission of Cu(II)–elesclomol for testing in the NCI-60 cell line screen were used to conduct NCI COMPARE analyses in order to determine which compounds in the NCI database had a similar mechanism of action. These results further delineate the mechanisms of the unique cancer cell growth inhibitory effects of elesclomol and Cu(II)–elesclomol. 2. Material and methods 2.1. Materials, cell culture and growth inhibition assays Elesclomol and Cu(II)–elesclomol were synthesized and characterized as we previously described [6]. Unless specified, all other reagents were obtained from Sigma-Aldrich (Oakville, Canada). Human leukemia K562 cells, obtained from the American Type Culture Collection (Manassas, VA), and the acquired etoposideresistant K/VP.5 subline (containing decreased levels of topoisomerase IIa mRNA and protein) [12,13] were maintained as suspension cultures in aMEM (minimal essential medium alpha; Life Technologies, Burlington, Canada) containing 10% fetal calf
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serum and 20 mM HEPES (4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid) (pH 7.2). The spectrophotometric 96-well plate cell (5 104 cell/ml, 0.1 ml/well) growth inhibition 3-(4,5dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) CellTiter 96 AQueous One Solution Cell Proliferation1 assay (Promega, Madison, WI), which measures the ability of the cells to enzymatically reduce MTS after drug treatment, has been described previously [5,6,14]. 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 effect of elesclomol or Cu(II)–elesclomol on the 72 h growth inhibition of other cell lines that were used to test for the role of efflux transporters were assayed with an 3-[4,5-dimethylthiazol-2-yl]2,5-tetrazolium bromide (MTT) assay essentially as we have described [14,15]. The IC50 values for cell growth inhibition were measured by fitting the absorbance-drug concentration data to a four-parameter logistic equation as we described [14]. The errors that were calculated from these four-parameter non-linear least squares fits to the data are the S.E.M.s. 2.2. Cell growth and viability, cell cycle synchronization, cell cycle analysis and annexin V flow cytometry Total cell density and viability using a trypan blue assay were determined on a Bio-Rad (Mississauga, Canada) TC10TM automated cell counter. In these experiments erythroleukemic K562 cells at a density of 50,000 cells/ml in 96-well plates were continuously treated with 200 nM elesclomol or Cu(II)–elesclomol for various times. The cell cycle synchronization experiments were carried out as we previously described [14]. A measure of cells that are necrotic is also obtained since necrotic cell membranes are permeable to propidium iodide yielding high red fluorescence. For the synchronization experiments Chinese hamster ovary (CHO) cells were grown to confluence in a-MEM supplemented with 10% fetal calf serum. Following serum starvation with a-MEM-0% fetal calf serum for 48 h, the cells that were seeded at 2 l04 cells/ml, were repleted with a-MEM-10% fetal calf serum. Directly after repletion they were continuously treated with DMSO vehicle control or 50 nM of either elesclomol or Cu(II)–elesclomol in 35-mm diameter dishes for different periods of time. Cells were fixed in 75% ethanol and stained with a solution containing 20 mg/ml propidium iodide, 100 mg/ml RNase A in 0.1% (v/v) Triton X-100 at room temperature for 30 min. Flow cytometry was carried out on a BD FACSCantoTM II flow cytometry system (BD Biosciences, Mississauga ON, Canada) and analyzed with FlowJo software (Tree Star, Ashland OR) for the proportion of cells in subG0/G1, G0/G1, S, and G2/M phases of the cell cycle. The fraction of apoptotic cells induced by treatment of K562 cells with elesclomol and Cu(II)–elesclomol were quantified by two-color flow cytometry by simultaneously measuring integrated green (annexin V-FITC) fluorescence, and integrated red (propidium iodide) fluorescence as we previously described [14]. The annexin V-FITC binding to phosphatidylserine present on the outer cell membrane was determined using an Apoptosis Detection Kit (BD Biosciences, Mississauga, Canada). Briefly, K562 cells in suspension were untreated or treated with elesclomol or Cu(II)– elesclomol at the concentrations indicated at 37 8C for 5 h. The cells were collected by centrifugation at 1000 g for 3 min and the pooled cells were washed with the manufacturer-supplied binding buffer. Approximately 2.5 105 cells were resuspended in 500 ml of manufacturer-supplied binding buffer, and mixed with 5 ml of annexin V-FITC and 5 ml of propidium iodide at a final concentration of 1 mg/ml. After 15 min of incubation in the dark, the cells were analyzed using flow cytometry.
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2.3. Measurement of mitochondrial membrane potential K562 cells were treated with various concentrations of elesclomol or Cu(II)–elesclomol. The mitochondrial membrane potential sensing dye JC-1 (Life Technologies) [16,17] was then loaded into suspended K562 cells (200,000 cells/well in 96-well black plates) by incubating cells with 8 mM JC-1 in Hank’s buffer (pH 7.4 with 1.3/0.8 mM Ca2+/Mg2+) at 37 8C for 20 min as we previously described [17]. The cells were then gently washed with Hank’s buffer. The average ratio of the red fluorescence (lEx 544 nm, lEm 590 nm) to the green fluorescence (lEx 485 nm, lEm 520 nm), which is a measure of the mitochondrial membrane potential [16,17], was determined for cells treated for 6 h with various concentrations of elesclomol or Cu(II)–elesclomol on a BMG (Cary, NC) Fluostar Galaxy fluorescence plate reader. The ionophore valinomycin (1 mM), which depolarizes the mitochondrial membrane, and doxorubicin (1.6 mM) were used as positive controls as we previously described [17]. 2.4. gH2AX assay for DNA double-strand breaks in K562 cells The gH2AX assay was carried out essentially as described [18]. K562 cells in growth medium (2 ml in a 24-well plate, 1 106 cells/ml) were incubated with drug or with DMSO as a control for 4.5 h at 37 8C. Cell lysates (50 mg protein) were subjected to SDS–polyacrylamide gel electrophoresis on a 14% gel. Separated proteins were transferred to polyvinylidene fluoride (PVDF) membranes and then treated overnight with rabbit antigH2AX primary antibody diluted 1:1000 (Upstate, Charlottesville, VA). This was followed by incubation for 1 h with peroxidaseconjugated goat-anti-rabbit secondary antibody (Cell Signaling Technology, Danvers, MA) diluted 1:2500. After incubation with luminol/enhancer/peroxide solution (Bio-Rad, Mississauga, Canada), chemiluminescence of the gH2AX band was imaged on a Cell Biosciences (Santa Clara, CA) FluorChem1 FC2 imaging system equipped with a charge-coupled-device camera. 2.5. Cellular assays for the detection of covalent DNA–topoisomerase IIa and DNA–topoisomerase I protein complexes The cellular ICE (immunodetection of complexes of enzyme-toDNA), assays for topoisomerase I and topoisomerase IIa covalently bound to DNA were carried out as we previously described [14]. The ICE assay used was a modification of the original cesium
chloride ultracentrifugation gradient assay used to isolate DNA [19]. The modification of this assay instead employed the selective precipitation of genomic DNA using DNAzol1 (Life Technologies). 2.6. Inhibition of topoisomerase I DNA relaxation assay and topoisomerase IIa kDNA decatenation and cleavage assays A gel assay as described [18] was used to determine if elesclomol or Cu(II)–elesclomol inhibited topoisomerase I. The pBR322 DNA was from MBI Fermentas (Burlington, Canada) and the topoisomerase I was from TopoGEN (Port Orange, FL). The topoisomerase I inhibitor camptothecin (20 mM) was used as a positive control. A gel assay as we previously described [20] was used to determine if elesclomol or Cu(II)–elesclomol inhibited the catalytic decatenation activity of topoisomerase IIa. kDNA, which consists of highly catenated networks of circular DNA, is decatenated by topoisomerase IIa in an ATP-dependent reaction to yield individual minicircles of DNA. Topoisomerase II-cleaved DNA covalent complexes produced by anticancer drugs may be trapped by rapidly denaturing the complexed enzyme with sodium dodecyl sulfate (SDS) [20,21]. The drug-induced cleavage of closed circular plasmid pBR322 DNA to form linear DNA at 37 8C was followed by separating the SDS-treated reaction products by ethidium bromide gel electrophoresis, essentially as described, except that all components of the assay mixture were assembled and mixed on ice prior to addition of the drug [14,21]. 3. Results 3.1. Cell growth and viability and cell cycle analysis and two-color flow cytometry A redetermination of the growth inhibitory effects of elesclomol and Cu(II)–elesclomol on K562 cells using an MTS assay yielded IC50 values of 14.3 and 7.5 nM, respectively (Fig. 2), which are close to values we previously determined [6]. It is likely that these IC50 values are similar because elesclomol binds Cu(II) extremely strongly [6] and scavenged copper from the culture medium. This was conclusion is confirmed by the loss of potency we observed when the culture medium was depleted of copper [5]. The effect on cell growth and viability as a function of time on treating K562 cells with 200 nM elesclomol and Cu(II)–elesclomol experiments are shown in Fig. 3A and B. As shown in Fig. 3A treatment with either elesclomol or Cu(II)–elesclomol resulted in a complete cessation in
Fig. 2. Comparison of the growth inhibitory effects of elesclomol and Cu(II)–elesclomol on K562 and K/VP.5 cells with reduced levels of topoisomerase IIa. (A) K562 (*) and K/ VP.5 (~) cells were treated with elesclomol for 72 h prior to the assessment of growth inhibition. Curve fitting yielded IC50-values of 14.3 0.9 nM and 10.6 0.9 nM, respectively. (B) K562 (*) and K/VP.5 (~) cells were treated with Cu(II)–elesclomol for 72 h prior to the assessment of growth inhibition. Curve fitting yielded IC50-values of 7.5 1.0 nM and 8.5 0.9 nM, respectively. The curved lines were calculated from non-linear least squares fits to 4-parameter logistic equations.
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Fig. 3. Effect on K562 cell number and cell viability after treatment with 200 nM elesclomol or Cu(II)–elesclomol. K562 cells were treated with Cu(II)–elesclomol (*), elesclomol (~), or vehicle control (*). (A) Cell density is plotted as a function of time. There was essentially no increase in cell number after treatment with either Cu(II)– elesclomol or elesclomol. (B) Cell viability as measured by trypan blue uptake is plotted as a function of time. Cell viability rapidly decreased at times after 8 h.
cell growth, while control cell numbers increased. Cell viability (Fig. 3B) remained high for about 8 h, after which it rapidly decreased. Elesclomol only slightly lagged the effect of Cu(II)– elesclomol in decreasing cell viability. A continuous 72 h treatment with elesclomol or Cu(II)– elesclomol inhibited the growth of attached CHO cells with IC50 values of 4.1 and 2.8 nM, respectively (results not shown). Experiments were also done in which CHO cells were treated with elesclomol or Cu(II)–elesclomol for 1.5 h, washed and then allowed to grow for a further 72 h. With this reduced treatment time cell growth was inhibited with IC50 values of 14.8 and 9.5 nM for elesclomol and Cu(II)–elesclomol, respectively (results not shown). These results suggest that the processes that ultimately lead to growth inhibition by either elesclomol or Cu(II)–elesclomol are rapidly initiated upon treatment. Cell cycle analysis was carried out on synchronized CHO cells treated with elesclomol and Cu(II)–elesclomol as we previously described [14] (Fig. 4). CHO cells (normal doubling time of 12 h), that were synchronized to G0/G1 through serum starvation, were treated with 50 nM of either elesclomol or Cu(II)–elesclomol in order to determine the effect of these compounds on cell cycle progression. CHO cells were chosen for these experiments as they are easily and effectively synchronized by serum starvation. Subsequent serum repletion resulted in the control (DMSO vehicle) cells advancing to G2/M by 18 h (Fig. 4A). The control cells then went through several complete cell cycles as evidenced by the 12 h periodicity for peaks in the various cell cycle stages. Both elesclomol and Cu(II)–elesclomol caused a delayed exit from G0/ G1 of 18 h without any significant move into S or G2/M. The reduction in the percentage of cells in G0/G1 was largely a result of the progressive increase in the sub-G0/G1 phase, which was indicative of apoptosis or necrosis. Cu(II)–elesclomol was only slightly more potent than elesclomol in inducing sub-G0/G1 cells. The percentage of sub-G0/G1 cell was small in control-treated cells (8 h) there was a rapid decrease in viability. As shown in Fig. 5A and B, treatment of K562 cells with 0.1, 1, 10 or 50 mM Cu(II)–elesclomol for 5 h progressively reduced the proportion of viable cells and increased the proportion of both apoptotic and apoptotic/necrotic cells. In contrast, elesclomol treatment did not, at least over this short time, result in apoptosis compared to controls. 3.2. Effect of elesclomol and Cu(II)–elesclomol on mitochondrial membrane potential It has been shown that the mitochondrial fraction of Cu(II)– elesclomol treated HL-60 cells and isolated mitochondria showed increased levels of copper and oxidative stress [2]. However, no results were reported for elesclomol itself. In order to determine if elesclomol or Cu(II)–elesclomol treatment of cells directly resulted in mitochondrial damage, the effect on the mitochondrial membrane potential of K562 cells was determined using the ratiometric mitochondrial membrane potential sensing dye JC-1 [16,17]. In Fig. 6, K562 cells were treated with elesclomol or Cu(II)– elesclomol for 6 h, when cells were still highly viable (Fig. 3B). Results show that neither eleclomol nor Cu(II)–elesclomol reduced the mitochondrial membrane potential under these experimental conditions. In contrast, the valinomycin and doxorubicin positive controls significantly and strongly reduced the mitochondrial membrane potential. When incubation times were extended to 15 h, Cu(II)–elesclomol (at concentrations of 5 mM or higher), but not elesclomol, caused significant mitochondrial damage (results not shown) paralleling loss of cell viability shown in Fig. 3B at this prolonged treatment time. 3.3. Elesclomol and Cu(II)–elesclomol induced DNA double strand breaks Phosphorylated H2AX (gH2AX), which is a variant of an H2A core histone, rapidly localizes at the site of double-strand DNA breaks upon treatment of cells with DNA damaging drugs or ionizing radiation [24]. The thousands of gH2AX molecules that are localized at the site of DNA double-strand breaks are thought to amplify the DNA damage signal and are a widely accepted marker of doublestrand breaks [24]. Thus, in order to determine if elesclomol or Cu(II)–elesclomol could induce double-strand breaks in intact K562 cells, the level of gH2AX protein was determined by Western blotting with etoposide as the positive control [21]. Experiments, carried out as we previously described, [14] and shown in Fig. 7A, indicate that both elesclomol and Cu(II)–elesclomol, with a 4.5 h
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Fig. 4. Cell cycle effects of treatment of synchronized CHO cells with elesclomol and Cu(II)–elesclomol. CHO cells that had been synchronized in G0/G1 through serum starvation were repleted with serum and were treated with 50 nM Cu(II)–elesclomol (*), elesclomol (~), or the DMSO vehicle control (*) directly after repletion and allowed to grow for the times indicated, after which they were subjected to cell cycle analysis of their propidium iodide-stained DNA. (A) The percentage of the cells in the sub-G0/G1, G0/G1, S and G2/M phases is plotted as a function of time for each of the compounds indicated. The solid lines are a least-squares calculated spline fit to the data. As shown in the plots a high percentage of the serum-starved cells were initially present in G0/G1. After serum repletion the percentage of control cells in each phase varied periodically as the cells progressed through several cell cycles. (B) Representative plots are shown in which the cell counts are displayed on the vertical axis and the DNA content is plotted on the horizontal axis. Unsynchronized cells are labeled as ‘‘unsynch’’.
treatment, increased levels of gH2AX in K562 cells in a concentration dependent manner. Elesclomol was less effective than Cu(II)–elesclomol in increasing gH2AX levels. The potency of Cu(II)–elesclomol in increasing gH2AX levels exceeded that of the topoisomerase II poison etoposide. 3.4. Effect of elesclomol and Cu(II)–elesclomol on the inhibition and poisoning of topoisomerase I and topoisomerase IIa We and others have shown that topoisomerase II is highly sensitive to heavy metal complexes such as cisplatin [25], thimerosal [26] and selenium compounds [27]. Topoisomerase I and topoisomerase II have also been shown to be inhibited by copper complexes [9]. Inhibition of these enzymes have been shown to result in DNA single and double strand breaks [28,29]. Because elesclomol and Cu(II) elesclomol increased levels of gH2AX consistent with DNA double strand breakage (Fig. 7A) the ability of these agents to inhibit topoisomerase I and II was investigated in experiments shown in Fig. 7B–F. The results of Fig. 7B show that Cu(II)–elesclomol, but not elesclomol, inhibited the relaxation activity of topoisomerase I, but only at the highest concentration tested (50 mM). The ability of Cu(II)–elesclomol to
produce a topoisomerase I-covalent complex in K562 cells was carried out using a cellular ICE assay (Fig. 7C). Cu(II)–elesclomol and the positive control camptothecin, a topoisomerase I inhibitor, increased the amount of the topoisomerase I-covalent complex, but only at the highest concentration of Cu(II)–elesclomol tested (50 mM), which is a much higher concentration than the low nanomolar concentrations required for K562 cell growth inhibition (Fig. 2) [5,6]. Treatment of K562 cells with Cu(II)–elesclomol did not produce topoisomerase IIa-covalent complexes in K562 cells carried out using a cellular ICE assay (Fig. 7D). Likewise, neither elesclomol nor Cu(II)–elesclomol inhibited the decatenation activity of topoisomerase IIa (Fig. 7E), nor were they able to induce cleavage complexes using purified topoisomerase IIa (Fig. 7F). We have previously used a clonal K562 cell line selected for resistance to etoposide as a screen to determine the ability of compounds to act as topoisomerase II poisons [14,20]. These K/ VP.5 cells were determined to be 26-fold resistant to etoposide and to contain reduced levels of both topoisomerase IIa (6-fold) and topoisomerase IIß (3-fold) [12,13]. In addition, K/VP.5 cells are cross-resistant to other known topoisomerase II poisons, but are not cross-resistant to camptothecin and other non-topoisomerase IIa targeted drugs [12]. Decreased cellular topoisomerase II
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Fig. 5. Effect of the treatment of K562 cells with elesclomol or Cu(II)–elesclomol on the induction of apoptosis as determined by annexin V-FITC/propidium iodide two-color fluorescence flow cytometry. (A) Representative two-color flow cytometry scatter plots of untreated control K562 cells, and treatments with 10 mM or 50 mM of elesclomol or Cu(II)–elesclomol for 5 h. The lower left quadrant contained viable cells; the upper left quadrant contained cells that were necrotic-only; the lower right quadrant contains cells that were apoptotic-only, but were not necrotic; and the upper right quadrant contained cells that were both apoptotic and necrotic. (B) Changes in relative number of K562 cells that were classified as necrotic, apoptotic/necrotic, apoptotic or viable 5 h after no treatment, or after treatment with 0.1, 1, 10 or 50 mM of elesclomol or Cu(II)– elesclomol as indicated.
translates to fewer DNA strand breaks and reduced cytotoxicity. The cell growth inhibition plots for K562 and K/VP.5 cells for elesclomol and Cu(II)–elesclomol are shown in Fig. 2A and 2B. Elesclomol IC50 values of 14.3 nM and 10.6 nM were obtained for K562 and K/VP.5 cells, respectively, and yielded a relative resistance value of 0.74. Cu(II)–elesclomol IC50 values of 7.5 nM and 8.5 nM were also obtained for K562 and K/VP.5 cells, respectively and yielded a relative resistance value of 1.13. Taken together, the results from Figs. 2 and 7 confirm that elesclomol and Cu(II)–elesclomol did not target topoisomerase IIa. 3.5. Cell growth inhibitory effects of elesclomol and Cu(II)–elesclomol on cell lines overexpressing efflux transporters To assess whether elesclomol and Cu(II)–elesclomol were substrates for several common drug efflux transporters, the IC50 values for elesclomol and Cu(II)–elesclomol were determined in ABCB1- (Pgp), ABCG2- (BCRP/MXR) and ABCC1- (MRP1) overexpressing cell lines and compared to their parental cell lines (Table 1). The growth inhibition curves are shown in Fig. 8. The pairs studied were Madin Darby canine kidney MDCK cells and its
ABCB1-transfected MDCK/MDR derivative [30] (maintained in 0.2 mM colchicine); human epidermoid carcinoma KB-3-1 cells and its colchicine-selected ABCB1-overexpressing KB-C2 derivative [31]; human lung cancer H460 cells and its mitoxantroneselected ABCG2-overexpressing H460/MX20 derivative [32,33]; and human kidney HEK293 cells with an empty vector transfectant and its transfected MRP1-overexpressing HEK293/MRP1 derivative [34]. As determined by a propagation-of-errors analysis using the S.E.M.s [35] the IC50 values were not significantly different between MDCK/MDR and MDCK cells; and between KB-3-1 and KB-C2 cells; and between H460 and H460/MX20 cells, treated with either elesclomol or Cu(II)–elesclomol. However, the HEK293/ MRP1 cells, compared to the HEK293/pcDNA3.1 cells, were significantly, cross-resistant to elesclomol (1.58 0.55 fold). The HEK293/MRP1 cells, were also slightly and significantly crossresistant to Cu(II)–elesclomol (3.2 0.68 fold). These results suggest that elesclomol and Cu(II)–elesclomol may be weak substrates of ABCC1 (MRP1), but not of ABCB1 (Pgp) or ABCG2 (BCRP). A previous report showed that elesclomol was even more potent toward several MDR-overexpressing cell lines that were highly cross-resistant to paclitaxel [8].
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Fig. 6. Effect of elesclomol or Cu(II)–elesclomol treatment on the mitochondrial membrane potential of K562 cells. The ratio of the red fluorescence (lEx 544 nm, lEm 590 nm) to the green fluorescence (lEx 485 nm, lEm 520 nm) of cells that were loaded with the membrane potential sensing dye JC-1 were measured 6 h after treatment. Valinomycin (Val, 1 mM) and doxorubicin (Dox, 1.6 mM) were used as positive controls. Both valinomycin and doxorubicin significantly, and strongly, reduced the mitochondrial membrane potential. The results are an average of 4 wells. The results were typical of two experiments carried out on different days.
3.6. Evaluation of cell growth inhibitory effects of Cu(II)–elesclomol in the NCI-60 human tumor cell line screen Cu(II)–elesclomol was submitted to the National Cancer Institute (http://dtp.nci.nih.gov) to identify which tumor cell types were most sensitive to this compound and to subsequently carry out NCI COMPARE analyses (http://dtp.nci.nih.gov/compare). The average of duplicate results using Cu(II)–elesclomol for NCI-60 cell line GI50 5-dose testing are given in Fig. 9. Leukemia, colon and breast cancer cell lines were, as a group, the most sensitive to Cu(II)–elesclomol, with GI50 values generally in the low nanomolar range. Approximately 25% of the GI50 values were reported as
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Cellular mechanisms of the cytotoxicity of the anticancer drug elesclomol and its complex with Cu(II) Brian B. Hasinoff a,*, Xing Wu a, Arun A. Yadav a, Daywin Patel a, Hui Zhang b,c, De-Shen Wang b,d, Zhe-Sheng Chen b, Jack C. Yalowich e a
Faculty of Pharmacy, Apotex Centre, University of Manitoba, 750 McDermot Avenue, Winnipeg, MB, Canada R3E 0T5 Department of Pharmaceutical Sciences, College of Pharmacy and Health Sciences, St. John’s University, Queens, New York, NY, USA Sun Yat-Sen University Cancer Center, State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, Guangzhou 510060, China d Department of Medical Oncology, Sun Yat-sen University Cancer Center, Guangzhou, Guangdong, China e Division of Pharmacology, College of Pharmacy, The Ohio State University, 500 West 12th Avenue, Columbus, OH 43210, USA b c
A R T I C L E I N F O
A B S T R A C T
Article history: Received 5 November 2014 Accepted 1 December 2014 Available online 27 December 2014
The potent anticancer drug elesclomol, which forms an extremely strong complex with copper, is currently undergoing clinical trials. However, its mechanism of action is not well understood. Treatment of human erythroleukemic K562 cells with either elesclomol or Cu(II)–elesclomol caused an immediate halt in cell growth which was followed by a loss of cell viability after several hours. Treatment of K562 cells also resulted in induction of apoptosis as measured by annexin V binding. Elesclomol or Cu(II)– elesclomol treatment caused a G1 cell cycle block in synchronized Chinese hamster ovary cells. Elesclomol and Cu(II)–elesclomol induced DNA double strand breaks in K562 cells, suggesting that they may also have exerted their cytotoxicity by damaging DNA. Cu(II)–elesclomol also weakly inhibited DNA topoisomerase I (5.99.1.2) but was not active against DNA topoisomerase IIa (5.99.1.3). Elesclomol or Cu(II)–elesclomol treatment had little effect on the mitochondrial membrane potential of viable K562 cells. NCI COMPARE analysis showed that Cu(II)–elesclomol exerted its cytotoxicity by mechanisms similar to other cytotoxic copper chelating compounds. Experiments with cross-resistant cell lines overexpressing several ATP-binding cassette (ABC) type efflux transporters showed that neither elesclomol nor Cu(II)–elesclomol were cross-resistant to cells overexpressing either ABCB1 (Pgp) or ABCG2 (BCRP), but that cells overexpressing ABCC1 (MRP1) were slightly cross-resistant. In conclusion, these results showed that elesclomol caused a rapid halt in cell growth, induced apoptosis, and may also have inhibited cell growth, in part, through its ability to damage DNA. ß 2014 Elsevier Inc. All rights reserved.
Chemical compounds studied in this article: Elesclomol (PubChem CID: 300471) Cu(II)–elesclomol (PubChem CID: 45273878) Etoposide (PubChem CID: 36462) Keywords: Elesclomol Copper JC-1 Cell cycle Oxidative stress Efflux
1. Introduction Elesclomol is a highly novel anticancer drug that has completed phase 3 clinical trials for patients with advanced melanoma [1] and is currently undergoing Phase 1 and 2 trials for the treatment of a variety of other cancers (http://www.clinicaltrials.gov) [2–4]. Elesclomol and Cu(II)–elesclomol (Fig. 1) are both extremely potent in vitro and typically inhibit cancer cell growth at low nanomolar concentrations [2,5–8]. It has been proposed that elesclomol is cytotoxic through the induction of oxidative stress that is mediated through its Cu2+ complex [2,3,5]. The development of copper
* Corresponding author. E-mail address: [email protected] (B.B. Hasinoff). http://dx.doi.org/10.1016/j.bcp.2014.12.008 0006-2952/ß 2014 Elsevier Inc. All rights reserved.
complexes as anticancer agents has recently been reviewed [9]. A recent report using an HClO-specific fluorescent probe has shown that elesclomol can induce formation of the highly reactive and strongly oxidizing HClO in breast cancer MCF7 cells [10]. However, it is not known whether this is a direct or an indirect effect. Elesclomol strongly binds both Cu2+ [2,3,6,8,11] (Fig. 1) and Cu+ [5]. Elesclomol can scavenge copper from the culture medium and selectively transport it to the mitochondria where it induces oxidative stress [2,3]. It has also been shown that the elesclomol was subsequently effluxed from the cell after it had transported copper into the cell, and was then free to shuttle more copper into the cell [2]. MCF7 cells with a compromised ability to repair oxidative DNA damage have increased sensitivity to elesclomol [7], which suggests that elesclomol may also exert some of its cytotoxicity through DNA-damaging mechanisms. Interestingly, it
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Fig. 1. Structure of elesclomol and its reaction with Cu2+ to form the neutral Cu(II)– elesclomol complex.
has been shown that elesclomol-treated patients with normal serum lactate dehydrogenase levels had improved outcomes compared to patients with high lactate dehydrogenase levels [1]. Yeast gene deletion mutant studies suggested that elesclomol does not work through a specific cellular protein target and is unlike any other currently approved anticancer drugs [3]. In previous studies we showed that elesclomol forms an extremely strong 1:1 neutral complex with Cu2+ (stability constant of 1024.2 M1; conditional stability constant at pH 7.4 of 1017.1 M1) and also forms a 1:1 complex with Cu+ [5,6]. We also showed that ascorbic acid, but not glutathione or NADH, reduces the Cu(II)–elesclomol complex to produce hydrogen peroxide [5]. Electron paramagnetic resonance (EPR) spin trapping experiments showed that the ascorbic acid-reduced Cu(II)–elesclomol complex, in comparison to ascorbic acid-reduced Cu2+, does not directly generate damaging hydroxyl radicals. We also showed that depletion of glutathione levels in K562 cells by treatment with buthionine sulfoximine sensitizes cells to both elesclomol and Cu(II)–elesclomol. Consistent with a role for copper in the cytotoxicity of elesclomol, the highly specific copper chelators tetrathiomolybdate and triethylenetetramine greatly reduce the cytotoxicity of both elesclomol and Cu(II)–elesclomol complex toward K562 cells [5]. These results showed that elesclomol indirectly inhibited cancer cell growth through Cu(II)-mediated oxidative stress. In order to further characterize the cell growth inhibitory effects of elesclomol and Cu(II)–elesclomol we designed experiments to measure their effects on: (1) cell cycle; (2) induction of apoptosis; (3) mitochondrial membrane potential; (4) formation of DNA double strand breaks; and (5) inhibition of DNA topoisomerase I (5.99.1.2) and DNA topoisomerase IIa (5.99.1.3). We also looked for cross-resistance in cells lines overexpressing several ATP-binding cassette (ABC) type efflux transporters in order to determine if either elesclomol or Cu(II)–elesclomol were substrates for these transporters. Finally, GI50 results obtained from submission of Cu(II)–elesclomol for testing in the NCI-60 cell line screen were used to conduct NCI COMPARE analyses in order to determine which compounds in the NCI database had a similar mechanism of action. These results further delineate the mechanisms of the unique cancer cell growth inhibitory effects of elesclomol and Cu(II)–elesclomol. 2. Material and methods 2.1. Materials, cell culture and growth inhibition assays Elesclomol and Cu(II)–elesclomol were synthesized and characterized as we previously described [6]. Unless specified, all other reagents were obtained from Sigma-Aldrich (Oakville, Canada). Human leukemia K562 cells, obtained from the American Type Culture Collection (Manassas, VA), and the acquired etoposideresistant K/VP.5 subline (containing decreased levels of topoisomerase IIa mRNA and protein) [12,13] were maintained as suspension cultures in aMEM (minimal essential medium alpha; Life Technologies, Burlington, Canada) containing 10% fetal calf
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serum and 20 mM HEPES (4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid) (pH 7.2). The spectrophotometric 96-well plate cell (5 104 cell/ml, 0.1 ml/well) growth inhibition 3-(4,5dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) CellTiter 96 AQueous One Solution Cell Proliferation1 assay (Promega, Madison, WI), which measures the ability of the cells to enzymatically reduce MTS after drug treatment, has been described previously [5,6,14]. 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 effect of elesclomol or Cu(II)–elesclomol on the 72 h growth inhibition of other cell lines that were used to test for the role of efflux transporters were assayed with an 3-[4,5-dimethylthiazol-2-yl]2,5-tetrazolium bromide (MTT) assay essentially as we have described [14,15]. The IC50 values for cell growth inhibition were measured by fitting the absorbance-drug concentration data to a four-parameter logistic equation as we described [14]. The errors that were calculated from these four-parameter non-linear least squares fits to the data are the S.E.M.s. 2.2. Cell growth and viability, cell cycle synchronization, cell cycle analysis and annexin V flow cytometry Total cell density and viability using a trypan blue assay were determined on a Bio-Rad (Mississauga, Canada) TC10TM automated cell counter. In these experiments erythroleukemic K562 cells at a density of 50,000 cells/ml in 96-well plates were continuously treated with 200 nM elesclomol or Cu(II)–elesclomol for various times. The cell cycle synchronization experiments were carried out as we previously described [14]. A measure of cells that are necrotic is also obtained since necrotic cell membranes are permeable to propidium iodide yielding high red fluorescence. For the synchronization experiments Chinese hamster ovary (CHO) cells were grown to confluence in a-MEM supplemented with 10% fetal calf serum. Following serum starvation with a-MEM-0% fetal calf serum for 48 h, the cells that were seeded at 2 l04 cells/ml, were repleted with a-MEM-10% fetal calf serum. Directly after repletion they were continuously treated with DMSO vehicle control or 50 nM of either elesclomol or Cu(II)–elesclomol in 35-mm diameter dishes for different periods of time. Cells were fixed in 75% ethanol and stained with a solution containing 20 mg/ml propidium iodide, 100 mg/ml RNase A in 0.1% (v/v) Triton X-100 at room temperature for 30 min. Flow cytometry was carried out on a BD FACSCantoTM II flow cytometry system (BD Biosciences, Mississauga ON, Canada) and analyzed with FlowJo software (Tree Star, Ashland OR) for the proportion of cells in subG0/G1, G0/G1, S, and G2/M phases of the cell cycle. The fraction of apoptotic cells induced by treatment of K562 cells with elesclomol and Cu(II)–elesclomol were quantified by two-color flow cytometry by simultaneously measuring integrated green (annexin V-FITC) fluorescence, and integrated red (propidium iodide) fluorescence as we previously described [14]. The annexin V-FITC binding to phosphatidylserine present on the outer cell membrane was determined using an Apoptosis Detection Kit (BD Biosciences, Mississauga, Canada). Briefly, K562 cells in suspension were untreated or treated with elesclomol or Cu(II)– elesclomol at the concentrations indicated at 37 8C for 5 h. The cells were collected by centrifugation at 1000 g for 3 min and the pooled cells were washed with the manufacturer-supplied binding buffer. Approximately 2.5 105 cells were resuspended in 500 ml of manufacturer-supplied binding buffer, and mixed with 5 ml of annexin V-FITC and 5 ml of propidium iodide at a final concentration of 1 mg/ml. After 15 min of incubation in the dark, the cells were analyzed using flow cytometry.
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2.3. Measurement of mitochondrial membrane potential K562 cells were treated with various concentrations of elesclomol or Cu(II)–elesclomol. The mitochondrial membrane potential sensing dye JC-1 (Life Technologies) [16,17] was then loaded into suspended K562 cells (200,000 cells/well in 96-well black plates) by incubating cells with 8 mM JC-1 in Hank’s buffer (pH 7.4 with 1.3/0.8 mM Ca2+/Mg2+) at 37 8C for 20 min as we previously described [17]. The cells were then gently washed with Hank’s buffer. The average ratio of the red fluorescence (lEx 544 nm, lEm 590 nm) to the green fluorescence (lEx 485 nm, lEm 520 nm), which is a measure of the mitochondrial membrane potential [16,17], was determined for cells treated for 6 h with various concentrations of elesclomol or Cu(II)–elesclomol on a BMG (Cary, NC) Fluostar Galaxy fluorescence plate reader. The ionophore valinomycin (1 mM), which depolarizes the mitochondrial membrane, and doxorubicin (1.6 mM) were used as positive controls as we previously described [17]. 2.4. gH2AX assay for DNA double-strand breaks in K562 cells The gH2AX assay was carried out essentially as described [18]. K562 cells in growth medium (2 ml in a 24-well plate, 1 106 cells/ml) were incubated with drug or with DMSO as a control for 4.5 h at 37 8C. Cell lysates (50 mg protein) were subjected to SDS–polyacrylamide gel electrophoresis on a 14% gel. Separated proteins were transferred to polyvinylidene fluoride (PVDF) membranes and then treated overnight with rabbit antigH2AX primary antibody diluted 1:1000 (Upstate, Charlottesville, VA). This was followed by incubation for 1 h with peroxidaseconjugated goat-anti-rabbit secondary antibody (Cell Signaling Technology, Danvers, MA) diluted 1:2500. After incubation with luminol/enhancer/peroxide solution (Bio-Rad, Mississauga, Canada), chemiluminescence of the gH2AX band was imaged on a Cell Biosciences (Santa Clara, CA) FluorChem1 FC2 imaging system equipped with a charge-coupled-device camera. 2.5. Cellular assays for the detection of covalent DNA–topoisomerase IIa and DNA–topoisomerase I protein complexes The cellular ICE (immunodetection of complexes of enzyme-toDNA), assays for topoisomerase I and topoisomerase IIa covalently bound to DNA were carried out as we previously described [14]. The ICE assay used was a modification of the original cesium
chloride ultracentrifugation gradient assay used to isolate DNA [19]. The modification of this assay instead employed the selective precipitation of genomic DNA using DNAzol1 (Life Technologies). 2.6. Inhibition of topoisomerase I DNA relaxation assay and topoisomerase IIa kDNA decatenation and cleavage assays A gel assay as described [18] was used to determine if elesclomol or Cu(II)–elesclomol inhibited topoisomerase I. The pBR322 DNA was from MBI Fermentas (Burlington, Canada) and the topoisomerase I was from TopoGEN (Port Orange, FL). The topoisomerase I inhibitor camptothecin (20 mM) was used as a positive control. A gel assay as we previously described [20] was used to determine if elesclomol or Cu(II)–elesclomol inhibited the catalytic decatenation activity of topoisomerase IIa. kDNA, which consists of highly catenated networks of circular DNA, is decatenated by topoisomerase IIa in an ATP-dependent reaction to yield individual minicircles of DNA. Topoisomerase II-cleaved DNA covalent complexes produced by anticancer drugs may be trapped by rapidly denaturing the complexed enzyme with sodium dodecyl sulfate (SDS) [20,21]. The drug-induced cleavage of closed circular plasmid pBR322 DNA to form linear DNA at 37 8C was followed by separating the SDS-treated reaction products by ethidium bromide gel electrophoresis, essentially as described, except that all components of the assay mixture were assembled and mixed on ice prior to addition of the drug [14,21]. 3. Results 3.1. Cell growth and viability and cell cycle analysis and two-color flow cytometry A redetermination of the growth inhibitory effects of elesclomol and Cu(II)–elesclomol on K562 cells using an MTS assay yielded IC50 values of 14.3 and 7.5 nM, respectively (Fig. 2), which are close to values we previously determined [6]. It is likely that these IC50 values are similar because elesclomol binds Cu(II) extremely strongly [6] and scavenged copper from the culture medium. This was conclusion is confirmed by the loss of potency we observed when the culture medium was depleted of copper [5]. The effect on cell growth and viability as a function of time on treating K562 cells with 200 nM elesclomol and Cu(II)–elesclomol experiments are shown in Fig. 3A and B. As shown in Fig. 3A treatment with either elesclomol or Cu(II)–elesclomol resulted in a complete cessation in
Fig. 2. Comparison of the growth inhibitory effects of elesclomol and Cu(II)–elesclomol on K562 and K/VP.5 cells with reduced levels of topoisomerase IIa. (A) K562 (*) and K/ VP.5 (~) cells were treated with elesclomol for 72 h prior to the assessment of growth inhibition. Curve fitting yielded IC50-values of 14.3 0.9 nM and 10.6 0.9 nM, respectively. (B) K562 (*) and K/VP.5 (~) cells were treated with Cu(II)–elesclomol for 72 h prior to the assessment of growth inhibition. Curve fitting yielded IC50-values of 7.5 1.0 nM and 8.5 0.9 nM, respectively. The curved lines were calculated from non-linear least squares fits to 4-parameter logistic equations.
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Fig. 3. Effect on K562 cell number and cell viability after treatment with 200 nM elesclomol or Cu(II)–elesclomol. K562 cells were treated with Cu(II)–elesclomol (*), elesclomol (~), or vehicle control (*). (A) Cell density is plotted as a function of time. There was essentially no increase in cell number after treatment with either Cu(II)– elesclomol or elesclomol. (B) Cell viability as measured by trypan blue uptake is plotted as a function of time. Cell viability rapidly decreased at times after 8 h.
cell growth, while control cell numbers increased. Cell viability (Fig. 3B) remained high for about 8 h, after which it rapidly decreased. Elesclomol only slightly lagged the effect of Cu(II)– elesclomol in decreasing cell viability. A continuous 72 h treatment with elesclomol or Cu(II)– elesclomol inhibited the growth of attached CHO cells with IC50 values of 4.1 and 2.8 nM, respectively (results not shown). Experiments were also done in which CHO cells were treated with elesclomol or Cu(II)–elesclomol for 1.5 h, washed and then allowed to grow for a further 72 h. With this reduced treatment time cell growth was inhibited with IC50 values of 14.8 and 9.5 nM for elesclomol and Cu(II)–elesclomol, respectively (results not shown). These results suggest that the processes that ultimately lead to growth inhibition by either elesclomol or Cu(II)–elesclomol are rapidly initiated upon treatment. Cell cycle analysis was carried out on synchronized CHO cells treated with elesclomol and Cu(II)–elesclomol as we previously described [14] (Fig. 4). CHO cells (normal doubling time of 12 h), that were synchronized to G0/G1 through serum starvation, were treated with 50 nM of either elesclomol or Cu(II)–elesclomol in order to determine the effect of these compounds on cell cycle progression. CHO cells were chosen for these experiments as they are easily and effectively synchronized by serum starvation. Subsequent serum repletion resulted in the control (DMSO vehicle) cells advancing to G2/M by 18 h (Fig. 4A). The control cells then went through several complete cell cycles as evidenced by the 12 h periodicity for peaks in the various cell cycle stages. Both elesclomol and Cu(II)–elesclomol caused a delayed exit from G0/ G1 of 18 h without any significant move into S or G2/M. The reduction in the percentage of cells in G0/G1 was largely a result of the progressive increase in the sub-G0/G1 phase, which was indicative of apoptosis or necrosis. Cu(II)–elesclomol was only slightly more potent than elesclomol in inducing sub-G0/G1 cells. The percentage of sub-G0/G1 cell was small in control-treated cells (8 h) there was a rapid decrease in viability. As shown in Fig. 5A and B, treatment of K562 cells with 0.1, 1, 10 or 50 mM Cu(II)–elesclomol for 5 h progressively reduced the proportion of viable cells and increased the proportion of both apoptotic and apoptotic/necrotic cells. In contrast, elesclomol treatment did not, at least over this short time, result in apoptosis compared to controls. 3.2. Effect of elesclomol and Cu(II)–elesclomol on mitochondrial membrane potential It has been shown that the mitochondrial fraction of Cu(II)– elesclomol treated HL-60 cells and isolated mitochondria showed increased levels of copper and oxidative stress [2]. However, no results were reported for elesclomol itself. In order to determine if elesclomol or Cu(II)–elesclomol treatment of cells directly resulted in mitochondrial damage, the effect on the mitochondrial membrane potential of K562 cells was determined using the ratiometric mitochondrial membrane potential sensing dye JC-1 [16,17]. In Fig. 6, K562 cells were treated with elesclomol or Cu(II)– elesclomol for 6 h, when cells were still highly viable (Fig. 3B). Results show that neither eleclomol nor Cu(II)–elesclomol reduced the mitochondrial membrane potential under these experimental conditions. In contrast, the valinomycin and doxorubicin positive controls significantly and strongly reduced the mitochondrial membrane potential. When incubation times were extended to 15 h, Cu(II)–elesclomol (at concentrations of 5 mM or higher), but not elesclomol, caused significant mitochondrial damage (results not shown) paralleling loss of cell viability shown in Fig. 3B at this prolonged treatment time. 3.3. Elesclomol and Cu(II)–elesclomol induced DNA double strand breaks Phosphorylated H2AX (gH2AX), which is a variant of an H2A core histone, rapidly localizes at the site of double-strand DNA breaks upon treatment of cells with DNA damaging drugs or ionizing radiation [24]. The thousands of gH2AX molecules that are localized at the site of DNA double-strand breaks are thought to amplify the DNA damage signal and are a widely accepted marker of doublestrand breaks [24]. Thus, in order to determine if elesclomol or Cu(II)–elesclomol could induce double-strand breaks in intact K562 cells, the level of gH2AX protein was determined by Western blotting with etoposide as the positive control [21]. Experiments, carried out as we previously described, [14] and shown in Fig. 7A, indicate that both elesclomol and Cu(II)–elesclomol, with a 4.5 h
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Fig. 4. Cell cycle effects of treatment of synchronized CHO cells with elesclomol and Cu(II)–elesclomol. CHO cells that had been synchronized in G0/G1 through serum starvation were repleted with serum and were treated with 50 nM Cu(II)–elesclomol (*), elesclomol (~), or the DMSO vehicle control (*) directly after repletion and allowed to grow for the times indicated, after which they were subjected to cell cycle analysis of their propidium iodide-stained DNA. (A) The percentage of the cells in the sub-G0/G1, G0/G1, S and G2/M phases is plotted as a function of time for each of the compounds indicated. The solid lines are a least-squares calculated spline fit to the data. As shown in the plots a high percentage of the serum-starved cells were initially present in G0/G1. After serum repletion the percentage of control cells in each phase varied periodically as the cells progressed through several cell cycles. (B) Representative plots are shown in which the cell counts are displayed on the vertical axis and the DNA content is plotted on the horizontal axis. Unsynchronized cells are labeled as ‘‘unsynch’’.
treatment, increased levels of gH2AX in K562 cells in a concentration dependent manner. Elesclomol was less effective than Cu(II)–elesclomol in increasing gH2AX levels. The potency of Cu(II)–elesclomol in increasing gH2AX levels exceeded that of the topoisomerase II poison etoposide. 3.4. Effect of elesclomol and Cu(II)–elesclomol on the inhibition and poisoning of topoisomerase I and topoisomerase IIa We and others have shown that topoisomerase II is highly sensitive to heavy metal complexes such as cisplatin [25], thimerosal [26] and selenium compounds [27]. Topoisomerase I and topoisomerase II have also been shown to be inhibited by copper complexes [9]. Inhibition of these enzymes have been shown to result in DNA single and double strand breaks [28,29]. Because elesclomol and Cu(II) elesclomol increased levels of gH2AX consistent with DNA double strand breakage (Fig. 7A) the ability of these agents to inhibit topoisomerase I and II was investigated in experiments shown in Fig. 7B–F. The results of Fig. 7B show that Cu(II)–elesclomol, but not elesclomol, inhibited the relaxation activity of topoisomerase I, but only at the highest concentration tested (50 mM). The ability of Cu(II)–elesclomol to
produce a topoisomerase I-covalent complex in K562 cells was carried out using a cellular ICE assay (Fig. 7C). Cu(II)–elesclomol and the positive control camptothecin, a topoisomerase I inhibitor, increased the amount of the topoisomerase I-covalent complex, but only at the highest concentration of Cu(II)–elesclomol tested (50 mM), which is a much higher concentration than the low nanomolar concentrations required for K562 cell growth inhibition (Fig. 2) [5,6]. Treatment of K562 cells with Cu(II)–elesclomol did not produce topoisomerase IIa-covalent complexes in K562 cells carried out using a cellular ICE assay (Fig. 7D). Likewise, neither elesclomol nor Cu(II)–elesclomol inhibited the decatenation activity of topoisomerase IIa (Fig. 7E), nor were they able to induce cleavage complexes using purified topoisomerase IIa (Fig. 7F). We have previously used a clonal K562 cell line selected for resistance to etoposide as a screen to determine the ability of compounds to act as topoisomerase II poisons [14,20]. These K/ VP.5 cells were determined to be 26-fold resistant to etoposide and to contain reduced levels of both topoisomerase IIa (6-fold) and topoisomerase IIß (3-fold) [12,13]. In addition, K/VP.5 cells are cross-resistant to other known topoisomerase II poisons, but are not cross-resistant to camptothecin and other non-topoisomerase IIa targeted drugs [12]. Decreased cellular topoisomerase II
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Fig. 5. Effect of the treatment of K562 cells with elesclomol or Cu(II)–elesclomol on the induction of apoptosis as determined by annexin V-FITC/propidium iodide two-color fluorescence flow cytometry. (A) Representative two-color flow cytometry scatter plots of untreated control K562 cells, and treatments with 10 mM or 50 mM of elesclomol or Cu(II)–elesclomol for 5 h. The lower left quadrant contained viable cells; the upper left quadrant contained cells that were necrotic-only; the lower right quadrant contains cells that were apoptotic-only, but were not necrotic; and the upper right quadrant contained cells that were both apoptotic and necrotic. (B) Changes in relative number of K562 cells that were classified as necrotic, apoptotic/necrotic, apoptotic or viable 5 h after no treatment, or after treatment with 0.1, 1, 10 or 50 mM of elesclomol or Cu(II)– elesclomol as indicated.
translates to fewer DNA strand breaks and reduced cytotoxicity. The cell growth inhibition plots for K562 and K/VP.5 cells for elesclomol and Cu(II)–elesclomol are shown in Fig. 2A and 2B. Elesclomol IC50 values of 14.3 nM and 10.6 nM were obtained for K562 and K/VP.5 cells, respectively, and yielded a relative resistance value of 0.74. Cu(II)–elesclomol IC50 values of 7.5 nM and 8.5 nM were also obtained for K562 and K/VP.5 cells, respectively and yielded a relative resistance value of 1.13. Taken together, the results from Figs. 2 and 7 confirm that elesclomol and Cu(II)–elesclomol did not target topoisomerase IIa. 3.5. Cell growth inhibitory effects of elesclomol and Cu(II)–elesclomol on cell lines overexpressing efflux transporters To assess whether elesclomol and Cu(II)–elesclomol were substrates for several common drug efflux transporters, the IC50 values for elesclomol and Cu(II)–elesclomol were determined in ABCB1- (Pgp), ABCG2- (BCRP/MXR) and ABCC1- (MRP1) overexpressing cell lines and compared to their parental cell lines (Table 1). The growth inhibition curves are shown in Fig. 8. The pairs studied were Madin Darby canine kidney MDCK cells and its
ABCB1-transfected MDCK/MDR derivative [30] (maintained in 0.2 mM colchicine); human epidermoid carcinoma KB-3-1 cells and its colchicine-selected ABCB1-overexpressing KB-C2 derivative [31]; human lung cancer H460 cells and its mitoxantroneselected ABCG2-overexpressing H460/MX20 derivative [32,33]; and human kidney HEK293 cells with an empty vector transfectant and its transfected MRP1-overexpressing HEK293/MRP1 derivative [34]. As determined by a propagation-of-errors analysis using the S.E.M.s [35] the IC50 values were not significantly different between MDCK/MDR and MDCK cells; and between KB-3-1 and KB-C2 cells; and between H460 and H460/MX20 cells, treated with either elesclomol or Cu(II)–elesclomol. However, the HEK293/ MRP1 cells, compared to the HEK293/pcDNA3.1 cells, were significantly, cross-resistant to elesclomol (1.58 0.55 fold). The HEK293/MRP1 cells, were also slightly and significantly crossresistant to Cu(II)–elesclomol (3.2 0.68 fold). These results suggest that elesclomol and Cu(II)–elesclomol may be weak substrates of ABCC1 (MRP1), but not of ABCB1 (Pgp) or ABCG2 (BCRP). A previous report showed that elesclomol was even more potent toward several MDR-overexpressing cell lines that were highly cross-resistant to paclitaxel [8].
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Fig. 6. Effect of elesclomol or Cu(II)–elesclomol treatment on the mitochondrial membrane potential of K562 cells. The ratio of the red fluorescence (lEx 544 nm, lEm 590 nm) to the green fluorescence (lEx 485 nm, lEm 520 nm) of cells that were loaded with the membrane potential sensing dye JC-1 were measured 6 h after treatment. Valinomycin (Val, 1 mM) and doxorubicin (Dox, 1.6 mM) were used as positive controls. Both valinomycin and doxorubicin significantly, and strongly, reduced the mitochondrial membrane potential. The results are an average of 4 wells. The results were typical of two experiments carried out on different days.
3.6. Evaluation of cell growth inhibitory effects of Cu(II)–elesclomol in the NCI-60 human tumor cell line screen Cu(II)–elesclomol was submitted to the National Cancer Institute (http://dtp.nci.nih.gov) to identify which tumor cell types were most sensitive to this compound and to subsequently carry out NCI COMPARE analyses (http://dtp.nci.nih.gov/compare). The average of duplicate results using Cu(II)–elesclomol for NCI-60 cell line GI50 5-dose testing are given in Fig. 9. Leukemia, colon and breast cancer cell lines were, as a group, the most sensitive to Cu(II)–elesclomol, with GI50 values generally in the low nanomolar range. Approximately 25% of the GI50 values were reported as
