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J. Biol. Chem., Vol. 282, Issue 12, 8860-8872, March 23, 2007
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From the
Departments of Oncology, Physiology and Biomedical Engineering, and ||Molecular Pharmacology, Mayo Clinic College of Medicine, Rochester, Minnesota 55905, the ¶Department of Cell and Developmental Biology, University of North Carolina, Chapel Hill, North Carolina 27599, the **Department of Medicine, Dartmouth Medical School, Lebanon, New Hampshire 03756, and the Greenebaum Cancer Center, Baltimore, Maryland 21201
Received for publication, July 12, 2006
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Previous studies (3–6) have suggested that adaphostin kills target cells through a mechanism that involves up-regulation of reactive oxygen species (ROS).5 Evidence in support of this view includes increased oxidation of the dyes 5(6)-chloromethyldihydro-2',7'-dichlorofluorescein (CM-H2DCF) and dihydroethidium in adaphostin-treated cells, including Bcr/Abl-negative ML-1 and Jurkat cells, as well as the ability of the antioxidants N-acetylcysteine (CysNAc), tiron, and Trolox to inhibit the cytotoxicity of adaphostin (4, 6). On the other hand, it has also been recognized that dyes such as 5(6)-chloromethyl-2',7'-dichlorofluorescein (CM-DCF) are subject to efflux by drug-sensitive transporters (7, 8), raising some doubt regarding the role of ROS in adaphostin cytotoxicity.
In biological systems, quinones are known to undergo enzymatic reduction to semiquinone radicals, which then spontaneously oxidize back to quinones, generating superoxide anion in the process (9–12). These prior observations have led to a general assumption that adaphostin might be rapidly oxidized either extracellularly or intracellularly to a quinone derivative, which could then generate ROS through redox cycling. Direct observations testing this hypothesis have not been reported, however. Moreover, the possibility that leakage of electrons from the mitochondrial electron transport chain, another common source of ROS (13–15), might contribute to the efficacy of adaphostin has not been examined previously.
In this study, we confirmed that adaphostin induces oxidative damage of DNA in intact cells and then followed the fate of adaphostin in solution and in intact cells. The results of this analysis demonstrate that adaphostin is concentrated several thousandfold in mitochondria, where it inhibits complex III of the respiratory chain and leads to mitochondrial ROS generation. These observations not only provide new insight into the mechanism of action of a promising anti-leukemic agent, but also provide additional support for the view that certain inhibitors of mitochondrial respiration might be potential anticancer agents.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-benzyloxycarbonyl-Val-Ala)-Asp(O-methyl ester)-fluoromethyl ketone from BIOMOL (Plymouth Meeting, PA); and formamidopyrimidine-DNA glycosylase from New England Biolabs (Ipswich, MA). All components for manufacturing a tetraphenylphosphonium cation (TPP+)-selective membrane, including polyvinyl chloride, TPP sodium salt, and tetrahydrofuran, were from Fluka (Buchs, Switzerland). 2-[14C]Thymidine (59 mCi/mmol) was from ICN (Irvine, CA). Tissue Culture—K562 human leukemia cells and A549 non-small cell lung cancer cells (American Type Culture Collection, Manassas, VA) were grown in RPMI 1640 medium containing 5% (v/v) heat-inactivated fetal bovine serum, 100 units/ml penicillin G, 100 µg/ml streptomycin, and 2 mM glutamine. HCT116 human colon cancer cells and OVCAR5 human ovarian cancer cells were propagated in RPMI 1640 medium containing 10% (v/v) heat-inactivated fetal bovine serum, 100 units/ml penicillin G, 100 µg/ml streptomycin, and 2 mM glutamine. The MOLT-4 human acute lymphocytic leukemia cell line and its rho0 derivative, which was generated as described previously (16), were grown in the same medium supplemented with 50 µg/ml uridine and 100 µg/ml sodium pyruvate.
To assess cell volume, photos of cells were taken using a Zeiss Axiovert 200M optical morphology microscope. The images were captured with a Zeiss Axiocam high resolution digital camera controlled with Zeiss KS400 software, which was used to measure the diameters of at least 80–100 individual cells from each line. The volumes of the cells were derived from the mean diameters and used in calculations to determine adaphostin whole cell uptake.
Colony-forming assays, which were performed as described previously (17, 18), were utilized to determine the effects of adaphostin on long-term viability of adherent cell lines. In brief, aliquots containing 350–500 cells were plated in replicate 35- or 60-mm tissue culture plates and allowed to adhere overnight. Cells were then treated with the indicated concentrations of BSO or CysNAc for 4 h, at which time adaphostin was added (without removal of BSO or CysNAc) for 24 h. At completion of the incubation, cells were washed, incubated for 7–8 days, stained with Coomassie Blue, and manually examined for colony formation. Diluent-treated plates typically contained 150–250 colonies.
Assessment of Intracellular ROS—CM-H2DCF diacetate (a cell-permeant non-fluorescent compound that diffuses into cells, undergoes de-esterification, and is oxidized to the fluorescent dye CM-DCF) was used to assess changes in relative intracellular ROS levels. After exposure to drugs for the indicated times, cells were collected by centrifugation, incubated with 10 µM CM-H2DCF diacetate in calcium- and magnesium-free Dulbecco's phosphate-buffered saline (DPBS) for 30 min in the dark at 37 °C, washed with DPBS, read on the FL1 channel of a BD Biosciences FACSCalibur, and analyzed using BD Biosciences CellQuest software.
Measurement of Adaphostin-mediated Superoxide and Peroxide Generation in Vitro—Superoxide radical generation was assessed using a cytochrome c reduction assay (19). Briefly, the reaction was initiated by the addition of adaphostin or diluent to a 1-ml mixture containing 50 µM oxidized cytochrome c and 50 mM Tris-HCl (pH 8.0) in the absence or presence of 30 µg/ml bovine erythrocyte superoxide dismutase. The level of superoxide generation was estimated from the difference in absorbance at 550 and 418 nm, which was measured after 1 h of incubation at 21 °C. Generation of H2O2 in solution was assessed using an Amplex Red assay, which monitors peroxidase-catalyzed oxidation of Amplex Red to resorufin (20), as described by the supplier. Known concentrations of H2O2 provided a standard curve.
Alkaline Elution with Formamidopyrimidine-DNA Glycosylase (Adapted from Refs. 21 and 22)—Logarithmically growing cells were labeled for 24 h with 1 µM [14C]thymidine, sedimented, and incubated for 1–2 h at 37 °C in fresh culture medium. After incubation for 30 min at 37 °C with the indicated concentrations of adaphostin, H2O2, or vehicle, cells (
200,000/aliquot) were deposited on Nucleopore phosphocellulose filters (1-µm pore size; VWR Scientific Products, Minneapolis, MN) by gentle suction. Cells were washed with 20 ml of icecold pre-lysis buffer (0.075 M NaCl and 0.024 M Na4EDTA (pH 7.4)) and lysed by allowing 5 ml of buffer A (2% (w/v) SDS, 100 mM glycine, and 25 mM EDTA (pH 10)) to drip through the filters over 30 min. After the filters were washed with 25 ml of buffer B (40 mM HEPES-KOH (pH 8.0), 0.1 M KCl, 0.5 mM EDTA, and 0.2 mg/ml bovine serum albumin), 2 ml of buffer B containing 8 units/ml formamidopyrimidine-DNA glycosylase was added to each filter, which was then incubated at 37 °C for 30 min and washed with an additional 25 ml of buffer B. Prior to elution, 5 ml of buffer A with 0.5 mg/ml proteinase K was allowed to drip through the filter over 30 min. Filters were then washed with 20 mM EDTA (pH 10), and DNA was eluted with 20 mM EDTA (adjusted to pH 12.1 with tetrapropylammonium hydroxide). The distribution of radiolabel in the eluate, the tubing of the elution apparatus, and the filter was analyzed as described by Covey et al. (23). Cells that received 150–900 centigrays of 
-irradiation from a 137Cs source were included in each experiment to provide a standard curve.
Analysis of Adaphostin Degradation in DPBS—Adaphostin was added to DPBS and incubated as indicated below. Aliquots were injected onto a Beckman System Gold HPLC system and separated on a Beckman Ultrasphere ODS 5-µm column (4 mm x 25 cm) by isocratic elution with a mobile phase consisting of 1:1 acetonitrile and 2.5 mM ammonium formate (pH 4). Elution was monitored by absorbance at 305 nm.
Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) Analysis—The LC/MS system consisted of a Shimadzu HPLC system containing two LC-10ADvp pumps and an SIL-10ADvp autoinjector (injection volume of 60 µl) coupled to a Quattro micro triple quadrupole mass spectrometer (Waters Corp., Milford, MA). Separation was performed using a Brownlee NewGuard RP-18 7-µm pre-column (15 x 3.2 mm; Chrom Tech, Apple Valley, MN) and a Genesis Lightning C18 120A 4-µm analytical column (10 cm x 2.1 mm; Jones Chromatography, Lakewood, CO). The mobile phase (flow rate of 0.20 ml/min) consisted of acetonitrile and 0.625 mM formate buffer (pH 4), initially at a 40:60 (v/v) ratio for 40 min and then ramped to 60% acetonitrile and held for 10 min.
Ionizations of adaphostin and the quinone of adaphostin were accomplished using an electrospray ionization probe operating in positive mode. Detection was accomplished by MS/MS using a daughter ion spectrum scan (m/z 45–450), looking for daughters of the specific parent ions at m/z 394 and 392 for adaphostin and the quinone, respectively. The scan time, cone voltage, and collision energy values were, 1.0 s, 30 V, and 15 eV, respectively. The source temperature, desolvation temperature, cone gas flow, and desolvation gas flow were 100 °C, 120 °C, 100 liters/h, and 250 liters/h, respectively. LC-MS/MS data were collected from 5 to 55 min after sample injection. Spectra and chromatograms were processed using Micromass MassLynx Version 3.5 software.
Cell Fractionation and Isolation of Mitochondria from K562 Cells (Adapted from Ref. 24)—All steps were performed at 4 °C. K562 cells (108) were harvested by centrifugation at 100 x g for 5 min, washed twice with ice-cold DPBS, and resuspended in 5 ml of buffer C (20 mM HEPES-KOH (pH 7.5), 10 mM KCl, 3 mM MgSO4, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 0.1 mM -phenylmethylsulfonyl fluoride, and 250 mM sucrose). After 20 min on ice, cells were homogenized in a tight-fitting Dounce homogenizer until lysed (50 strokes). After the homogenate was sedimented at 800 x g, the pellet containing unbroken cells and nuclei was discarded, and the supernatant was sedimented at 10,000 x g for 15 min at 4 °C to obtain the mitochondrial fraction, which was resuspended in 5 ml of ice-cold buffer C and placed on ice for 15–30 min until used. The post-mitochondrial supernatant was sedimented at 100,000 x g for 1 h to separate the microsomal fraction (pellet) from the cytosol (supernatant). Alternatively, rat liver mitochondria were isolated by differential centrifugation as described (25, 26).
Measurement of Adaphostin Content in Cellular Fractions— After 6 volumes of acetonitrile was added to 1 volume of each subcellular fraction, samples were mixed thoroughly and centrifuged at 14,000 x g for 5 min. The supernatant was subjected to HPLC as described above with known concentrations of adaphostin as a standard. The pellet was hydrolyzed in 0.5 M NaOH and assayed for protein.
Measurement of 
m of Isolated Mitochondria Using a TPP+-selective Electrode—The membrane potential and respiration of isolated mitochondria were recorded using a prototype multichannel recorder (ABMT-USA, Inc., Durham, NC). m was measured in incubation medium containing 200 mM mannitol, 75 mM sucrose, 5 mM KH2PO4, 5 mM pyruvate, 5 mM malate, 2 mM MgCl2, 1 mM EGTA, and 10 mM MOPS (pH 7.35) using a home-made TPP+-sensitive mini-electrode as described (27). Briefly, mitochondria (1 mg/ml protein) were added to the incubation medium containing 500 µM TPP chloride. was calculated from the TPP+ electrode reading according to the following equation: = 59 log(
/V) - 59 log(10(E-E0)/59 - 1), where is the mitochondrial membrane potential (in millivolts); is the mitochondrial matrix volume (1.6 µl/mg of mitochondrial protein) (28); V is the volume of the incubation medium (1 ml); and E0 and E are electrode potentials (in millivolts) before and after the addition of mitochondria, respectively (29). Adaphostin up to 25 µM and vehicle (Me2SO) up to 0.5% (v/v) did not interfere with the readings of the TPP+-sensitive electrode. Only electrodes that exhibited a Nernstian response were used.
Oxygen Consumption—Mitochondria were suspended at a concentration of 1 mg/ml protein in mitochondrial incubation buffer (MIB) consisting of 200 mM mannitol, 75 mM sucrose, 5 mM KH2PO4, 2 mM MgCl2, 1 mM EGTA, and 10 mM MOPS-KOH (pH 7.35). K562 cells were suspended in cell culture medium at a concentration of 107 cells/ml. Oxygen consumption was measured using a Clark-type electrode (Model 5300, YSI Inc.). All measurements were performed at room temperature.
Dehydrogenase Assays—Succinate dehydrogenase activity was assayed as described (30). Briefly, mitochondria (1 mg of protein) were suspended in 1 ml of reaction buffer containing 50 mM potassium phosphate (pH 7.4), 50 mM sodium succinate, 25 mM sucrose, 2 mM EDTA, and 0.1% (w/v) p-iodonitrotetrazolium violet and incubated on ice for 30 min. Following the addition of adaphostin, the suspension was incubated at 37 °C for 15 min. After the reaction was terminated with 1 ml of 10% (w/v) trichloroacetic acid, the product was extracted into 4 ml of ethyl acetate and assayed at 490 nm.
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Complex III Activity Measurement—Complex III activity was assessed by monitoring the reduction in cytochrome c measured as changes in absorbance (A550 - A540) in the presence of reduced decylubiquinone (32). To synthesize reduced decylubiquinone, a small crystal of sodium borohydride was added to 200 µl of 10 mM decylubiquinone (oxidized form). 5 µlof0.1 M HCl was added, and the reaction was mixed until the yellow solution became colorless. The reduced decylubiquinone (supernatant) was transferred to a fresh tube, diluted with 5 ml of 1 M HCl, and stored in the dark until used. To assay complex III activity, mitochondria (30 µg/ml protein) were resuspended in reaction buffer containing 250 mM sucrose, 1 mM EDTA, 50 mM Tris-HCl (pH 7.4), 50 µM cytochrome c, and 2 mM KCN at 21 °C. The reaction was initiated by the addition of 50 µM reduced decylubiquinone, and complex III activity was measured by monitoring changes in absorbance (A550 - A540) over 3 min.
ROS Generation by Isolated Mitochondria—Rat liver mitochondria (1 mg/ml protein) in MIB supplemented with 5 mM succinate were snap-frozen in liquid nitrogen and thawed three times at 30 °C. After he addition of adaphostin and/or catalase, samples were incubated for 1 h at 30°C. The concentration of H2O2 generated was assayed using Amplex Red (20) as described above.
Docking of Adaphostin into the Drug-binding Sites of the Cytochrome bc1 Complex in Silico—Four different conformations of adaphostin were docked into two binding sites of the heme bH-containing cytochrome b subunit of the cytochrome bc1 complex using the EUDOC program with rotational and translational increments of 10° of arc and 0.5 Å, respectively (33). The atomic charges of adaphostin were generated according to the RESP procedure (34) with ab initio calculations at the HF/6-31G* level using the Gaussian 03 program (35). The adaphostin conformations were generated by systematically altering the conformation-governing torsions and subsequently optimized by energy minimizations using the force field (parm99.dat) of Cornell et al. (36) and the SANDER module of AMBER5 (37). The energy minimizations were monitored for reaching local minimum conformations with a normal mode analysis using the NMODE module of AMBER8 (37) and then terminated when the analysis showed that adaphostin reached a local minimum potential energy conformation. The first binding site of the holocytochrome b subunit, reported as the quinone reduction (Qi) site (38), was taken from a crystal structure of the subunit containing antimycin A in the Qi site (Protein Data Bank code 1NTK). The docking box (6.0 x 6.0 x 6.0 Å3) at the Qi site was defined to be surrounded by Phe18, Ser35, Leu197, and heme bH. The second binding site of the holosubunit, reported as the quinol oxidation (Qo) site (39), was obtained from a crystal structure of the subunit containing azoxystrobin in the Qo site (Protein Data Bank code 1SQB). The docking box (6.0 x 6.0 x 6.0 Å3) at the Qo site was defined to be surrounded by Phe128, Tyr131, Ile146, Pro270, and Phe274. The EUDOC-generated adaphostin complex with the lowest interaction energy was then refined with 20 different molecular dynamics simulations (1.0 ns for each simulation with a 1.0-fs time step and different initial velocities) according to a published procedure (40) using the PMEMD module of AMBER8. An average of 10,000 trajectories obtained at 1.0-ps intervals during the last 0.5-ns period of the 20 simulations was used as the refined three-dimensional model of the adaphostin complex.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-benzyloxycarbonyl-Val-Ala)-Asp(O-methyl ester)-fluoromethyl ketone (Fig. 1B), placing the ROS generation upstream of mitochondrial permeability transition and caspase activation. Because alternative explanations, including adaphostin-induced inhibition of CM-DCF transporters (7, 8), might account for the increased CM-DCF fluorescence, additional experiments using alternative methods were performed to verify that adaphostin induced an increase in intracellular ROS levels. In particular, after cells were treated with vehicle or adaphostin, DNA was subjected to filter elution in the absence or presence of formamidopyrimidine-DNA glycosylase, an enzyme that recognizes and cleaves at 8-oxoguanine residues (41). As indicated in Fig. 1C, the increase in strand breaks detected in DNA from adaphostin-treated cells after formamidopyrimidine-DNA glycosylase incubation was similar to that detected in DNA from cells treated with 10 µM H2O2, providing independent evidence of adaphostin-induced oxidative stress. Collectively, all of the results in Fig. 1 suggest that cells treated with adaphostin sustain oxidative damage.
Fate of Adaphostin in Aqueous Media—To identify the ROS causing this damage, we initially examined the fate of adaphostin in aqueous buffer. When adaphostin was incubated in DPBS, multiple assays indicated the presence of increased ROS levels. In preliminary experiments, the addition of adaphostin to DPBS containing 2-methyl-6-(4-methoxyphenyl)-3,7-dihydroimidazo[1,2-a]pyrazin-3-one resulted in increased luminescence indicative of increased ROS levels (data not shown). Likewise, the oxidation of dihydroethidium to the fluorescent compound ethidium was increased by adaphostin. Because these assays do not clearly distinguish between superoxide anion and peroxides, two ROS that could potentially be generated during adaphostin oxidation, we employed more selective assays in additional experiments. As shown in Fig. 2A, adaphostin induced a reduction in cytochrome c. The ability of purified superoxide dismutase to blunt this reduction suggests that adaphostin generates superoxide anion in solution. Likewise, adaphostin enhanced the peroxidase-mediated oxidation of Amplex Red to resorufin (Fig. 2B), suggesting that adaphostin also induces generation of H2O2.
To determine the basis for the generation of these ROS, the fate of adaphostin was followed by HPLC. During the course of incubation in DPBS at ambient oxygen tension, the parent compound disappeared, with a half-life of 8 ± 2 h (mean ± S.D., n = 4), and a new peak appeared (Fig. 2, C and E). Based on a 2-Da decrease in the size of the parent ion and the fragmentation observed during LC-MS/MS, the new peak was identified as the quinone derivative of adaphostin (Fig. 2D). Additional experiments indicated that chemically synthesized quinone had an indistinguishable retention time and collision-induced dissociation spectrum during LC-MS/MS (data not shown). Time course experiments indicated that the quinone concentration peaked at 6.2 ± 0.4 h (n = 3) and subsequently declined as this compound was oxidized even further (Fig. 2E). Collectively, these observations indicate that adaphostin in aqueous buffers is oxidized to the corresponding quinone with concomitant formation of superoxide and H2O2.
Fate of Adaphostin in Intact Cells—Armed with the information that adaphostin can be converted to the corresponding quinone, we next examined the fate of adaphostin in intact cells. For this analysis, K562 cells (106/ml) were treated with 2.5 µM adaphostin for periods of up to 4 h, extensively washed, and then extracted for HPLC analysis. In contrast with results obtained when adaphostin was incubated in DPBS (Fig. 2), the quinone could not be detected in the reducing environment of intact cells (Fig. 3A). Instead, only the parent drug was detected (Fig. 3A). Notably, however, this analysis also indicated that adaphostin was concentrated by 300 ± 30-fold (n = 3) in intact K562 cells relative to the extracellular medium (Fig. 3B). As a consequence, 38 ± 8% of the total drug added to the tissue culture medium was recovered with the cell pellet under the conditions of this incubation (Fig. 3C).
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3000-fold concentration of drug in mitochondria. Time course experiments demonstrated that this dramatic uptake of adaphostin into mitochondria occurred in <30 min and persisted for at least 2–3 h (Fig. 3E). Further analysis demonstrated that similar mitochondrial concentrations could be achieved by incubating isolated mitochondria with 200 µM adaphostin under cell-free conditions, providing a means of reproducing the effects of adaphostin under cell-free conditions in the experiments described below.
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To further analyze this possibility, we examined the effect of adaphostin on ADP-induced depolarization of isolated mitochondria using an ion-sensitive electrode (26, 27). These and subsequent experiments utilized mitochondria from rat liver because of the ability to isolate large quantities of highly purified organelles from this tissue. The addition of 300 µM ADP to these mitochondria resulted in transient depolarization during the period of ATP synthesis, followed by repolarization to a steady-state m of -170 mV (Fig. 4B, upper panel). Treatment with 25 µM adaphostin caused prolongation of the ADP-induced mitochondrial depolarization (Fig. 4B, lower panel), suggesting that adaphostin might inhibit some component of the mitochondrial respiratory chain, nucleotide exchange, or mitochondrial F0F1-ATPase.
To examine the action of adaphostin on mitochondria in greater detail, the rate of oxygen consumption by isolated mitochondria was examined in the presence of various respiratory substrates. As indicated in Fig. 4C (inset), when mitochondria were incubated with the complex I substrates pyruvate and malate, adaphostin diminished ADP-induced mitochondrial oxygen consumption in a dose-dependent manner. On average, 200 µM adaphostin, which resulted in the same mitochondrial drug concentration as observed in intact cells treated with 2.5 µM adaphostin, caused a 48 ± 2% (n = 3) decrease in ADP-stimulated respiration (Fig. 4C). When the complex II substrate succinate was substituted for pyruvate and malate, adaphostin likewise caused a 63.2 ± 2.5% (n = 3) decrease in ADP-stimulated respiration (Fig. 4C). Moreover, adaphostin also inhibited CCCP-stimulated oxygen consumption in the presence of the same substrates (Fig. 4C). Because CCCP uncouples electron transport and ATP synthesis, making oxygen consumption independent of ATP generation, the observed ability of adaphostin to inhibit respiration of CCCP-treated mitochondria argued against the possibility that adaphostin inhibits the adenine nucleotide transporter or F0F1-ATPase.
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Collectively, the preceding experiments suggested that adaphostin might inhibit complex III. To test this hypothesis more directly, complex III activity was assessed by monitoring cytochrome c reduction in the presence of reduced decylubiquinone. Like the selective complex III inhibitor antimycin A (46), adaphostin inhibited decylubiquinone-induced cytochrome c reduction (Fig. 6).
Adaphostin Fits into the Qi Site of Complex III—To gain further insight into the potential interaction of adaphostin with complex III, the docking of multiple conformations of adaphostin into two known binding sites of the heme bH-containing cytochrome b subunit of the cytochrome bc1 complex (38, 39) was analyzed in silico. This analysis suggested that adaphostin preferentially binds to the Qi site (-28 kcal/mol for the Qi site versus more than -5 kcal/mol for the Qo site). In the refined three-dimensional model of the adaphostin complex with an intermolecular interaction energy of -47.3 kcal/mol (EvdW = -45.6 kcal/mol and Eele =-1.7 kcal/mol), the adamantyl group of adaphostin has van der Waals interactions with the methyl group of heme bH and with the side chains of Gly38, Leu41, Ile42, Met190, and Met194; the middle and terminal aromatic groups of the drug form 
- interactions with the side chains of Phe18 and Phe220, respectively; the meta-hydroxyl group of the drug has a water-bridged hydrogen bond with Asn32 and Ser35; and the ortho-hydroxyl group has a water-bridged hydrogen bond with Ser205 (Fig. 7A). Interestingly, the binding mode of adaphostin is very similar to that of antimycin A, as shown in Fig. 7 (B and C), even though the adaphostin model was computationally developed without any assumption that adaphostin shares the binding mode of antimycin A.
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An additional series of experiments assessed whether the effects of adaphostin on mitochondrial function (Figs. 4, 5, 6) could contribute to the increased ROS levels observed in intact cells (Fig. 1). For these experiments, H2O2 production (13, 47) was assessed after mitochondria were incubated with diluent or adaphostin in the absence or presence of catalase. Consistent with our previous results (Fig. 2B), adaphostin by itself generated H2O2 in solution (Fig. 8B). In the presence of mitochondria, however, the amount of ROS generated increased at least 7-fold, suggesting that the effects of adaphostin on mitochondrial respiration contribute substantially to ROS generation by this agent.
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ROS-dependent Cytotoxicity in Solid Tumor Cell Lines—Because the ability to inhibit complex III and to increase ROS levels should not be limited to leukemia cell lines, we examined the effect of adaphostin in widely studied solid tumor cell lines. As indicated in Fig. 9A, treatment of HCT116 human colon adenocarcinoma cells with 10 µM adaphostin for 30 min caused an increase in CM-DCF fluorescence to 195 ± 7% of control values (mean ± S.D., n = 4). Additional experiments (not shown) indicated that adaphostin was concentrated 80-fold in these cells relative to the extracellular medium. To assess the impact of the increased ROS levels, cells were exposed for 24 h to adaphostin, washed, and assayed for the ability to subsequently proliferate into colonies. Treatment with adaphostin inhibited HCT116 colony formation in a dose-dependent manner (Fig. 9C), with IC90 = 17 ± 3 µM (mean ± S.D., n = 4). CysNAc at 6 mM, one-fourth of the concentration used previously (4), diminished the anti-proliferative effects at the highest adaphostin concentration tested (Fig. 9C).6 Conversely, glutathione depletion with BSO diminished the IC90 by 4.5 ± 1.9-fold (mean ± S.D., n = 4). In A549 non-small cell lung cancer cells, which took up 8-fold less adaphostin than HCT116 cells, the increase in ROS levels after exposure to 10 µM adaphostin was much lower (Fig. 9B), and the anti-proliferative effects of adaphostin were less potent (Fig. 9D), with IC90 = 33 ± 3 µM (n = 3). Nonetheless, treatment with CysNAc at 24 mM protected the cells at multiple adaphostin concentrations, and BSO treatment again diminished the IC90 by a factor of 7.65 ± 1.2 (n = 3) (Fig. 9D). Similar results were observed in OVCAR5 ovarian cancer cells (data not shown). Collectively, these results indicate that adaphostin is concentrated in solid tumor cell lines, that it generates ROS, and that the ability of solid tumor cells to detoxify these ROS plays an important role in resistance to this agent.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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While this work was in progress, Hose et al. (51) reported that adaphostin treatment results in an increase in cytosolic free iron as assessed by decreased fluorescence of the iron-sensitive dye Phen Green SK and that the iron chelator desferrioxamine diminishes adaphostin-induced cytotoxicity, prompting the authors to conclude that mitochondrial release of iron contributes to the ROS elevation and cytotoxicity of adaphostin. Our subsequent experiments showed, however, that the diminished dye fluorescence might be a result of adaphostin-mediated quenching (26), that increased cytosolic iron could not be detected by mass spectroscopy,7 and that desferrioxamine failed to protect K562 cells from adaphostin-induced cytoxicity.7,8 These observations, which might differ from the results of Hose et al. because of cell type-specific differences in the effects of adaphostin on iron sequestration, prompted us to search further for the source of adaphostin-induced ROS.
Several lines of evidence indicate that this increase in ROS levels is independent of any inhibitory effect of adaphostin on Bcr/Abl. First, increased ROS levels are evident in K562 cells within 15–30 min (Fig. 1) (4), whereas inhibition of Bcr/Abl-mediated phosphorylation is not evident for 4–6 h (2). Second, inhibition of Bcr/Abl and Abl by imatinib mesylate is accompanied by decreased ROS levels (52), whereas adaphostin causes an increase in ROS levels. Finally, adaphostin-induced increases in ROS levels are observed in a variety of Bcr/Abl-negative cells, including the Jurkat, ML-1, MOLT-4, A549, and HCT116 cell lines, as well as in chronic lymphocytic leukemia and acute myelogenous leukemia clinical samples (Figs. 8C and 9) (4–6).
On the basis of the dihydroquinone structure of adaphostin, we anticipated that this compound might undergo oxidation to the corresponding quinone in aqueous buffer at ambient oxygen tension. Consistent with this hypothesis, the quinone derivative of adaphostin was readily detected, along with superoxide radical and H2O2, when adaphostin was incubated in aqueous buffers (Fig. 2, A–D). Under conditions encountered in cells, however, this oxidation was not detectable (Fig. 3A). Instead, adaphostin was concentrated 3000-fold in mitochondria (Fig. 3, B–D), where it inhibited mitochondrial respiration (Fig. 4) and contributed to generation of ROS (Fig. 8B).
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Additional experiments demonstrated that adaphostin attenuated respiration of isolated mitochondria incubated with the complex I substrates pyruvate and malate (Fig. 4, C and D) or the complex II substrate succinate (Fig. 4C). Coupled with the failure of adaphostin to inhibit pyruvate and succinate dehydrogenases (Fig. 5), these results suggested that adaphostin might act downstream of complexes I and II. Consistent with this hypothesis, adaphostin was found to inhibit the ability of reduced decylubiquinone to reduce cytochrome c (Fig. 6). These results indicate that, like antimycin A and certain ubiquinone analogs (53–55), adaphostin is an inhibitor of respiratory complex III. Consistent with this possibility, an in silico docking study performed without any prior assumption about the mode of adaphostin binding to the Qo and Qi sites of cytochrome b demonstrated that the Qi site could accommodate adaphostin in a binding mode similar to that of antimycin A (Fig. 7). Additional analysis demonstrated that, like antimycin A (48, 49), adaphostin enhanced mitochondrial generation of ROS (Fig. 8B), providing an explanation for the increased ROS in adaphostin-treated cells.
Despite the fact that adaphostin and antimycin A both inhibit complex III, there are important differences between these agents. A previous study suggested that antimycin A is a general cellular poison that exhibits little selectivity for neoplastic compared with normal cells (56). In contrast, previous studies have demonstrated that adaphostin is selective for chronic myelogenous leukemia (1, 2) and chronic lymphocytic leukemia (4, 5) compared with normal bone marrow progenitors and normal lymphocytes. Although the basis for this selectivity is currently unknown, previous studies have demonstrated enhanced uptake of a number of aromatic cationic dyes into the mitochondria of neoplastic cells (57–60). Additional experiments examining the mechanism of uptake of adaphostin into mitochondria are required to determine whether the propensity of mitochondria in neoplastic cells to concentrate aromatic cationic compounds contributes to the observed selectivity of adaphostin for leukemic as opposed to normal leukocytes.
Even though this study demonstrated a remarkable concentration of adaphostin within mitochondria, it is important to note that almost 40% of the total cellular drug was also found in nuclei. We cannot at present rule out the possibility that nuclear accumulation of adaphostin also contributes to the cytotoxicity of this agent. Nonetheless, previous results from three laboratories demonstrating the ability of reducing agents such as CysNAc to inhibit the cytotoxicity of adaphostin (4–6) support the view that mitochondrial generation of ROS in the presence of adaphostin (Fig. 8B) plays a predominant role in the cytotoxicity.
These observations distinguish the action of adaphostin from those of a large class of anticancer drugs that are quinones. Previous studies have suggested that the cytotoxicity of these quinones is due to redox cycling between the quinone and semiquinone free radical, which generates ROS in the process (9). In contrast, our results provide little evidence for spontaneous oxidation of adaphostin in intact cells, but instead indicate that mitochondria play a role in the generation of ROS by this antitumor hydroquinone. Whether complex III is a target of other hydroquinones and possibly even quinones remains to be determined.
Finally, because the involvement of complex III in adaphostin-induced increases in ROS levels suggested that the cytotoxic effects of this agent should not be limited to leukemia cells, we examined the effects of this agent in solid tumor cell lines. Drug-induced ROS elevations and sulfhydryl-sensitive killing were observed in HCT116 colon cancer cells, A549 lung cancer cells, and OVCAR5 ovarian cancer cells (Fig. 9) (data not shown). While this manuscript was under review, Long et al. (61) also reported that adaphostin induces ROS-mediated cytotoxicity in glioblastoma cells. Collectively, these observations suggest that adaphostin might be active against a wide variety of cancer cell types.
In summary, adaphostin is concentrated in mitochondria, where its ability to inhibit respiratory complex III appears to contribute to the intracellular generation of ROS. Moreover, this study has demonstrated adaphostin-induced oxidative damage of DNA in intact cells. Based on the unique mechanism of action of this agent and its demonstrated selectivity for leukemia cells in vitro, further study of this agent appears to be warranted.
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1 Supported by a postdoctoral fellowship from the government of the Republic of Vietnam. Present address: Inst. of Biotechnology, Vietnamese Academy of Science & Technology, Hanoi, Vietnam.
2 Present address: College of Pharmacy, Medical University of South Carolina, Charleston, SC 29425.
3 Both authors contributed equally to this work.
4 To whom correspondence should be addressed: Div. of Oncology Research, Guggenheim 1342C, Mayo Clinic, 200 First St. S. W., Rochester, MN 55905. Tel.: 507-284-8950; Fax: 507-284-3906; E-mail: Kaufmann.scott{at}mayo.edu.
5 The abbreviations used are: ROS, reactive oxygen species; CM-H2DCF, 5(6)-chloromethyldihydro-2',7'-dichlorofluorescein; CysNAc, N-acetylcysteine; CM-DCF, 5(6)-chloromethyl-2',7'-dichlorofluorescein; CCCP, carbonyl cyanide 3-chlorophenylhydrazone; BSO, buthionine sulfoximine; TPP+, tetraphenylphosphonium cation; DPBS, calcium- and magnesium-free Dulbecco's phosphate-buffered saline; HPLC, high performance liquid chromatography; LC-MS/MS, liquid chromatography-tandem mass spectrometry; MOPS, 3-(N-morpholino)propanesulfonic acid; MIB, mitochondrial incubation buffer.
6 Higher CysNAc concentrations could not be utilized because they diminished HCT116 cell colony formation by themselves.
7 S. B. Lee and S. H. Kaufmann, unpublished data.
8 J. Chandra, personal communication.
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) was delayed in the presence of 25 µM adaphostin. C, isolated rat liver mitochondria were added to buffer containing either 5 mM succinate and 1 µM rotenone or 5 mM pyruvate (Pyr) and 5 mM malate in the absence or presence of 100 µM adaphostin. The oxygen level was measured using a Clark-type electrode. After 1 min, 300 µM ADP or 0.4 µM CCCP was added. The rate of oxygen consumption by mitochondria was calculated after the addition of respiration stimuli (CCCP or ADP) and is expressed as nanomoles/min/mg of mitochondrial protein. Inset, plot of oxygen level versus time after the addition of CCCP in the presence of diluent or different adaphostin concentrations. Note that treatment of mitochondria with 200 µM adaphostin under cell-free conditions resulted in mitochondrial levels similar to those obtained by treating whole cells with 2.5 µM adaphostin. D, after isolated rat liver mitochondria were subjected to three cycles of freezing in liquid nitrogen, aliquots were added to MIB containing 1 mM NADH, and oxygen levels were measured using a Clark-type electrode. Adaphostin (200 µM) or diluent was added after 1 min. The rate of oxygen consumption by mitochondria after the addition of adaphostin or diluent was calculated. Error bars represent the means ± S.D. of three to five independent determinations.
