Proteomic Analysis Identifies Oxidative Stress Induction by Adaphostin Luke H

Cancer Therapy: Preclinical

Proteomic Analysis Identifies Oxidative Stress Induction by Adaphostin Luke H. Stockwin,1Maja A. Bumke,1Sherry X. Yu,1Simon P. Webb,2 Jack R. Collins,2 Melinda G. Hollingshead,3 and Dianne L. Newton1

Abstract Purpose: Activities distinct from inhibition of Bcr/abl have led to adaphostin (NSC 680410) being described as ‘‘a drug in search of a mechanism.’’ In this study, proteomic analysis of adaphostin- treated myeloid leukemia cell lines was used to further elucidate a mechanism of action. Experimental Design: HL60 and K562 cells treated with adaphostin for 6, 12, or 24 h were analyzed using two-dimensional PAGE. Differentially expressed spots were excised, digested with trypsin, and analyzed by liquid chromatography ^ tandem mass spectrometry.The contribu- tion of the redox-active hydroquinone group in adaphostin was also examined by carrying out proteomic analysis of HL60 cells treated with a simple hydroquinone (1,4-dihydroxybenzene) or H2O2. Results: Analysis of adaphostin-treated cells identified 49 differentially expressed proteins, the majority being implicated in the response to oxidative stress (e.g., CALM, ERP29, GSTP1,PDIA1) or induction of apoptosis (e.g., LAMA, FLNA, TPR, GDIS). Interestingly, modulation of these proteins was almost fully prevented by inclusion of an antioxidant, N-acetylcysteine. Validation of the proteomic data confirmed GSTP1as an adaphostin resistance gene. Subsequent analysis of HL60 cells treated with1,4-dihydroxybenzene or H2O2 showed similar increases in intracellular peroxides and an almost identical proteomic profiles to that of adaphostin treatment. Western blotting of a panel of cell lines identified Cu/Zn superoxide dismutase (SOD) as correlating with adaphostin resistance. The role of SOD as a second adaphostin resistance gene was confirmed by demonstrating that inhibition of SOD using diethyldithiocarbamate increased adaphostin sensitivity, whereas transfection of SOD I attenuated toxicity. Importantly, treatment with 1,4-dihydroxybenzene or H2O2 replicated adaphostin-induced Bcr/abl polypeptide degradation, suggesting that kinase inhibition is a ROS-dependent phenomenon. Conclusion: Adaphostin should be classified as a redox-active ^ substituted dihydroquinone.

Adaphostin (NSC 680410) is a potent anticancer agent with an (1). Adaphostin was originally identified as a more active elusive mechanism of action. As a member of the tyrphostin congener of AG957, a non-ATP–competitive inhibitor of family, this compound belongs to a group of chemically and p210Bcr/abl (2–4). The antiproliferative effects of adaphostin mechanistically diverse inhibitors of protein tyrosine kinases and AG957 are associated with degradation of p210bcr/abl polypeptide and the rapid induction of apoptosis (5, 6). Adaphostin differs from AG957 in its 3- to 4-fold improved ability to promote Bcr/abl degradation in vitro and slightly 1 2 Authors’Affiliations: Developmental Therapeutics Program and Advanced enhanced activity with respect to 3-(4,5-dimethylthiazol-2-yl)- Biomedical Computing Center, Science Applications International Corporation Frederick, and 3DevelopmentalTherapeutics Program, Division of CancerTreatment 2,5-diphenyltetrazolium bromide cytotoxicity assays (4, 5). and Diagnosis, National Cancer Institute at Frederick, Frederick, Maryland This mechanism contrasts with ATP-dependent inhibitors of Received 1/4/07; accepted1/25/07. Bcr/abl, such as STI571 (Gleevec, imatinib mesylate), which Grant support: National Cancer Institute, NIH, under contract no. NO1-CO-12400, prevent autophosphorylation without polypeptide degradation and Developmental Therapeutics Program in the Division of CancerTreatment and Diagnosis of the National Cancer Institute. and induce apoptosis after longer exposure periods (18-48 h; The costs of publication of this article were defrayed in part by the payment of page refs. 6, 7). Further evidence pointing toward mechanistic dif- charges. This article must therefore be hereby marked advertisement in accordance ferences came from the observation that adaphostin and with 18 U.S.C. Section 1734 solely to indicate this fact. STI571 synergize against T-lymphoblastic leukemia cell lines, Note: The content of this publication does not necessarily reflect the views or and that adaphostin can kill STI571-resistant clones (6, 8). The policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. specificity of adaphostin for Bcr/abl was subsequently chal- Government. lenged after it was shown to have activity against leukemias and Requests for reprints: Dianne L. Newton, DevelopmentalTherapeutics Program, glioma cell lines that do not express Bcr/abl (9, 10). This led to Science Applications International Corporation-Frederick, Inc., National Cancer the suggestion that adaphostin was either a ‘‘promiscuous’’ Institute at Frederick, Room 6, Building 320, Frederick, MD 21702. Phone: 301- 846-6809; Fax: 301-846-7021;E-mail: [email protected]. kinase inhibitor or had an entirely unrelated activity. F 2007 American Association for Cancer Research. Insights into an alternative mechanism come from reports doi:10.1158/1078-0432.CCR-07-0025 showing adaphostin-induced cell death is accompanied by

www.aacrjournals.org 3667 Clin Cancer Res 2007;13(12) June 15, 2007 Downloaded from clincancerres.aacrjournals.org on September 30, 2021. © 2007 American Association for Cancer Research. Cancer Therapy: Preclinical increases in reactive oxygen species (ROS; refs. 9, 11). It has h-actin, Sigma. Secondary horseradish peroxidase–conjugated anti- been suggested that because adaphostin contains a substituted bodies were from Jackson Immunoresearch. The Cu/Zn SOD expression 1,4-dihydroquinone, electron transfer has the potential to construct and control base vector pCMV6-XL5 were from Origene generate superoxide radicals and this accounts for the increased Technologies. Unless otherwise indicated in the following methods, all other chemicals and inhibitors were from Sigma. ROS levels (9). Support for this hypothesis comes from Cytotoxicity and cell viability. Assays were conducted as follows: structure-activity relationships showing that the 1,4-dihydro- 104 cells in 100 AL were placed into each well of a 96-well plate quinone motif is essential for activity (4). Another possible 24 h before treatment. Sample or buffer control (10 AL) were added explanation for increased ROS comes from a recent cDNA array to the appropriate wells and the plates were incubated at 37jCina study, which linked activity to increased expression of tran- humidified CO2 incubator for the times indicated in the figure scripts involved in iron metabolism (12). It was subsequently legends. To determine protein synthesis, the serum-containing shown that adaphostin promotes the release of chelatable free medium was replaced with serum- and leucine-free RPMI containing iron (Fe2+) and that this may catalyze the formation of toxic 0.1 mCi of [14C]leucine. Incubation continued for 2 to 3 h at 37jC. 2+ The cells were harvested onto glass fiber filters using a PHD cell hydroxyl radicals through Fenton’s reaction [H2O2 +Fe ! 3+ . harvester, washed with water, dried with methanol, and counted. The Fe +OH ]. In spite of this wealth of experimental data, several 14 observations remain unresolved and as a consequence the results are expressed as % [ C]leucine incorporation into the control-treated cells. To assay for cell viability, 10 AL WST reagent underlying molecular basis for activity has yet to be deter- were added to each well and the plate was incubated for 2 to mined. For instance, adaphostin has been shown to inhibit 4 h followed by reading absorbance of the formazan dye product at secretion of vascular endothelial growth factor, leading to the 450 nm, using a microplate reader (Bio-Tek Instruments). Experi- proposal that, like other tyrphostins (e.g., SU5416, semaxanib), ments were done at least twice with triplicate determinations for each adaphostin has antiangiogenic activity (10). Similarly, alter- point. The IC50 was defined as the concentration of adaphostin ations in signal transduction pathways (RAF-1/mitogen-activat- required to inhibit protein synthesis or cell viability by 50% relative ed protein kinase kinase/extracellular signal-regulated kinase, to control-treated cells. AKT, c-met, and p38 mitogen-activated protein kinase) and Apoptosis and necrosis determination. The percentage of apoptotic adaphostin selectivity for certain malignancies (acute myelog- and necrotic cells in culture was determined using the Vybrant enous leukemia, chronic myelogenous leukemia) requires Apoptosis Assay kit (Molecular Probes) comprising an Annexin V– Alexa488 conjugate and propidium iodide as described by the further attention (13–15). manufacturer. Acquisition and analysis of data was done using a In this study, a proteomics platform based on two- FACScan flow cytometer (Becton Dickinson) controlled by Cellquest dimensional PAGE was used to identify proteins modulated Pro Software. by adaphostin with the intention of gaining further insight into Cell cycle analysis. Treated cells were harvested and washed once a mechanism of action and to identify surrogate markers of with PBS. The samples were resuspended in 5 mL PBS and 5 mL cold activity. Total cell lysate from two adaphostin-treated myeloid 70% ethanol was added drop wise. After 5-min incubation, the cells leukemia cells lines, HL60 (Bcr/abl negative) and K562 (Bcr/abl were centrifuged, resuspended in 10 mL cold 70% ethanol, and stored positive), formed the basis of the study. Results showed a at 4jC for 1 h. The cells were washed twice with 5 mL PBS and A proteomic profile consistent with an oxidative stress response resuspended in 1 mL PBS containing 50 g/mL propidium iodide A j and induction of apoptosis. Transfection of one modulated (Molecular Probes) and 100 g/mL RNase A (Sigma). After 1 h at 37 C, cell cycle analysis was done using the FL3-A channel on a FACScan flow protein, GSTP1, into cells was sufficient to confer resistance to cytometer. adaphostin. The proteomic ‘‘fingerprint’’ of adaphostin treat- Western blotting. Treated cells were washed twice in PBS and lysed ment was then shown to be almost identical to that derived in RIPA-CHAPS buffer [50 mmol/L Tris-HCl (pH 7.4), 1 mmol/L EDTA, through exposure to a simple hydroquinone or H2O2, impli- 1% CHAPS, 1% deoxycholate, 1 complete protease inhibitor]. Lysates cating the redox biology of the dihydroquinone in adaphostin were sonicated, centrifuged to remove insoluble material, and protein as central to the mechanism of action. This prompted further concentration was determined using the BCA Protein assay according analysis of the role of antioxidant enzymes, leading to the to the manufacturer’s instructions. SDS-PAGE was done using 10 Ag identification of Cu/Zn superoxide dismutase (SOD I) as a protein per well on a 10% NUPAGE Tris-glycine gel with subsequent further marker of resistance to adaphostin. Finally, a simple transfer to a polyvinylidene difluoride membrane by electroblotting. Following overnight blocking in 4% milk/TBS, membranes were hydroquinone and H2O2 were also able to affect degradation of Bcr/abl polypeptide in a similar manner to adaphostin incubated with primary antibody for 2 h, washed several times in 4% milk/TBS, and incubated with the secondary, peroxidase-conjugated treatment, suggesting that tyrosine kinase inhibitory activity is antisera for 2 h. Bands were visualized using enhanced chemilumines- an indirect event associated with increased oxidative stress. cence reagents ECL (Amersham, GE Healthcare) according to the manufacturer’s protocol. Protein carbonyl determination. HL60 cells (15 106 in 30 mL) Materials and Methods were treated over a time course with 5 Amol/L adaphostin or 100 Amol/L

H2O2. Where applicable, cells were pretreated with 100 mmol/L Materials. Adaphostin (NSC 680410) was obtained from the Drug N-acetyl-L-cysteine (L-NAC) for 30 min before the addition of Synthesis and Chemistry Branch of the Developmental Therapeutics adaphostin or H2O2. Following treatment, cells were washed with Program, National Cancer Institute (Rockville, MD). All cell lines were PBS, and the 0.5 mL of 0.2 mol/L NaPO4 (pH 6.6), containing 1 mmol/L from the Division of Cancer Treatment and Diagnosis Tumor EDTA, and protease inhibitor (Roche) were added. The cells were Repository (Frederick, MD). Materials were from the following: PBS sonicated twice for 10 s followed by centrifugation to remove any and RPMI, Quality Biologicals; BCA Protein assay, Pierce; polyvinyli- insoluble material. Protein concentration was determined using the dene difluoride membranes and all gels, Invitrogen; complete protease BCA Protein assay according to the manufacturer’s instructions. For inhibitor tablets and WST reagent, Roche. Primary antibodies were carbonyl determination, the method of Levine et al. (16) was modified from the following: SOD I/II, catalase, GSTP1, and myeloperoxidase, as follows: 0.2 mL 2 mg/mL protein was precipitated with 16% Abcam; c-Abl (ab3) used in the detection of p210Bcr/abl, Calbiochem, trichloroacetic acid (final concentration). Pellets were resuspended in

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1 mL 2 mol/L HCl (control) or 1 mL 0.2% 2,4-dinitrophenylhydrazine Amersham Biosciences). Cell lysates were sonicated twice for 10 s and in 2 mol/L HCl, incubated for 30 min in the dark at room temperature, then centrifuged to remove any insoluble material. For first-dimension and vortexed every 5 min. Samples were reprecipitated with 16% electrophoresis, 200 Ag protein sample were adjusted to 450 AL trichloroacetic acid (final concentration), incubated on ice for 5 min, with lysis/rehydration buffer and applied to 24 cm Immobiline and centrifuged for 5 min. Precipitates were washed thrice with ethanol/ DryStrips (pH 3-10 NonLinear, Amersham Biosciences). Rehydration ethyl acetate (1:1, v/v) before being dissolved in 0.1 mL 6 mol/L and isoelectric focusing were done in the Ettan IPGphor apparatus guanidine HCl in 2 mol/L HCl. Insoluble debris was removed by (Amersham Biosciences) at 20jC, maximum of 80 AA per strip, accord- centrifugation for 5 min. The difference spectrum between the 2,4- ing to the following program: 4 h at 0 V, 7 h at 30 V (rehydration); 1 h at dinitrophenylhydrazine–treated sample and the 2 mol/L HCl control 200 V, 1 h at 500 V, 1 h at 1000 V, then 8 to 12 h at 8,000 V (isoelectric sample was determined at 366 nm using a molar extinction coefficient focusing), until reaching 60 to 100 kV-h. After isoelectrofocusing, the for 2,4-dinitrophenylhydrazine of 22,000 (mol/L)-1 cm-1. The results gel strips (pH 3-10 NonLinear, Amersham Biosciences) were cut into were expressed as nanomoles of 2,4-dinitrophenylhydrazine incorpo- three equal pieces, equilibrated twice for 20 min with gentle shaking rated per milligram of protein. Polypeptide carbonylation was in 5 mL each of a solution containing 50 mmol/L Tris-HCl (pH 6.8), measured using the OxyBlot kit (Upstate). Cells were treated with 6 mol/L urea, 30% glycerol, 2% w/v SDS, and a trace of bromophenol varying concentrations of adaphostin or H2O2, harvested into RIPA- blue. Two percent (w/v) dithioerythritol (Sigma) was added to the first CHAPS buffer, sonicated, and the protein concentration was deter- equilibration step, whereas 2.5% w/v iodoacetamide (Sigma) was mined using the BCA Protein assay. Fifteen to 25 Ag protein were added to the second equilibration step. For the second dimension, the labeled according to the manufacturer’s instructions and run on a 10% strips were placed on top of NuPAGE 4% to 12% Bis-Tris ZOOM mini NUPAGE Tris-glycine gel. Western blot analysis was carried out using gels, sealed with 0.5% agarose in NuPAGE MES SDS running buffer and a-DNP primary antibody and a horseradish peroxidase–conjugated run for 40 min at 200 V. For fluorescent staining with SYPRO Ruby, goat a-rabbit secondary antibody. compatible with subsequent tryptic digestion, the gels were washed in

Glutathione S-transferase activity assay. Glutathione S-transferase H2O, fixed in 7% acetic acid, 10% methanol for 30 min, and then activity was measured by following conjugation of the thiol group of stained overnight in SYPRO Ruby protein gel stain (Molecular Probes). glutathione to 1-chloro-2,4-dinitrobenzene at 340 nm in a spectropho- To decrease background fluorescence, the gels were destained in 7% tometric assay. Assays were done according to the manufacturer’s acetic acid, 10% methanol for 30 min before imaging. The gels were instructions (Glutathione S-Transferase Assay kit, Sigma). Cells were imaged with a Typhoon 9200 imager (Amersham Biosciences). collected by centrifugation (suspension cells) or by scraping (adherent Gel cutting and in-gel tryptic digestion of proteins. Protein spots were cells), sonicated for 10 s in cold 100 mmol/L potassium phosphate excised from the SYPRO Ruby stained gels and transferred to a 96-well (pH 6.5) containing 2 mmol/L EDTA and protease inhibitor (Roche), plate. Gel spots were washed twice with 100 mmol/L ammonium and centrifuged to remove any insoluble material. Protein concentra- bicarbonate. The gel spots were dehydrated with acetonitrile and dried tion was determined using the BCA Protein assay according to the in a SpeedVac concentrator SC110A (Savant, Fisher Scientific). The dry manufacturer’s instructions. Each assay contained 10 Ag protein and gel spots were rehydrated with 50 mmol/L ammonium bicarbonate activity was followed over a 6-min period. The linear portion of the buffer containing 12.5 ng/AL sequencing grade porcine trypsin assay (2-6 min) and an extinction coefficient for 1-chloro-2,4- (Promega) for 45 min on ice. After reswelling of the gel spots, the dinitrobenzene at 340 nm of 9.6 (mmol/L)-1 cm-1 was used in the remaining buffer was removed and replaced with 50 mmol/L final calculations. ammonium bicarbonate. Digestion was carried out at 37jC for 16 h. Detection of ROS. Cells were harvested, washed once in PBS, and The supernatant was removed and the tryptic peptides were extracted resuspended at 1 106/mL in serum-containing medium. The cells first with 25 mmol/L ammonium bicarbonate and thereafter with 5% were then incubated for 1 h at 37jC in the presence of varying formic acid for 20 min each. The combined extracts, as well as the adaphostin concentrations. CM-H2DCFDA (Molecular Probes), which supernatant after digestion, were dried in a SpeedVac concentrator and reacts with peroxides to produce green fluorescence, was added to a dissolved in 10 AL 1% formic acid, 5% acetonitrile before mass final concentration of 10 Amol/L. After 1 h, cells were washed twice in spectrometric analysis. PBS and analyzed using the FL1 channel on a FACScan cytometer. Mass spectrometry. The technique used for protein identification SOD I and GSTP1 transfection. Cell transfections were done using involved microcapillary liquid chromatography–tandem mass spec- Amaxa Nucleofector technology (Amaxa). In brief, cells were washed in trometry. Trypsin-digested protein samples were analyzed using a PBS, 2 106 cells were resuspended in 100 AL of transfection solution, Finnigan LTQ ion trap mass spectrometer by Protana, Inc. (Toronto, 2 Ag of plasmid DNA were added, the suspension was mixed and placed Canada) and LPAT (Science Applications International Corporation into an Amaxa cuvette and electroporated using the appropriate Frederick, under contract NO1-CO-12400). The resulting data files were program.4 After transfection, the cells were resuspended in complete analyzed using Mascot (Matrix Science Ltd.). The National Center for medium, plated into a 96-well plate, and incubated for 14 h at 37jC Biotechnology Information database was searched using the following before varying concentrations of adaphostin were added and a protein variables: Variable modifications were carbamidomethylation of synthesis assay was done as described above. cysteine residues and oxidation of methionine residues and maximum Two-dimensional PAGE. Cells (15 106 in 30 mL) were treated for of two missed cleavages. 6, 12, and 24 h with 5 Amol/L (HL60 cells) or 30 Amol/L (K562 cells) adaphostin. In addition, HL60 cells were also treated for 24 h with Results 50 Amol/L hydroquinone, 100 Amol/L H2O2, or adaphostin with 10 mmol/L L-NAC. Following treatment, the cells were washed twice Establishing conditions for proteomic analysis. Adaphostin with PBS. For protein determination, cells from 7 mL cultures were treatment (structure in Fig. 1A) is associated with the inhibition lysed in 250 AL RIPA-CHAPS buffer containing 1 mmol/L sodium of protein synthesis and the rapid induction of apoptosis (5, 6). orthovanadate and analyzed using the BCA Protein assay. For two- dimensional gel electrophoresis, cells from 21 mL cultures were lysed in Several assays were used to monitor the effects of adaphostin 750 AL cell lysis/rehydration buffer (8 mol/L urea, 2% w/v CHAPS, on HL60 (Bcr/abl negative) and K562 (Bcr/abl positive) cells, 0.5% v/v Pharmalyte 3-10, 100 mmol/L dithioerythritol, 0.002% with the aim of elucidating the optimal conditions for bromophenol blue, 1 Amol/L Tris; all except dithioerythritol were from proteome analysis. The first set of assays measured the effect of adaphostin on protein synthesis and cell viability (Table 1) over 72 h. In both assays, the maximal effect of adaphostin on 4 http://www.amaxa.com/celldatabase.html HL60 and K562 cells was achieved within the first 24 h. The

www.aacrjournals.org 3669 Clin Cancer Res 2007;13(12) June 15, 2007 Downloaded from clincancerres.aacrjournals.org on September 30, 2021. © 2007 American Association for Cancer Research. Cancer Therapy: Preclinical concentration of adaphostin required to inhibit protein viability were 2.0, 1.5, and 1.5 Amol/L for HL60 cells and 10, synthesis for days 1, 2, and 3 by 50% was 0.35, 0.3, and 7, and 7 Amol/L for K562 cells for days 1, 2, and 3, re- 0.4 Amol/L for HL60 cells, and 3.5, 2.5, and 3.0 Amol/L for spectively. In both assays, the Bcr/abl–negative cell line, HL60, K562 cells, respectively. The IC50 values determined for cell was f5- to 10-fold more sensitive to adaphostin than the Bcr/abl–positive cell line, K562 (protein synthesis: IC50 0.35 and 3.5 Amol/L; cell viability: IC50 2.0 and 10 Amol/L, HL60 and K562, respectively). In the second set of assays, flow cytometry was used to monitor the induction of apoptosis using Annexin V/propidium iodide (Fig. 1B). A time course of the response of HL60 and K562 cells to 1.0 and 10 Amol/L adaphostin showed detectable increases in Annexin V–positive cells already after 4 h. By 24 h, the majority of cells were Annexin V/propidium iodide positive. In addition, cell cycle analysis of HL60 and K562 cells at 24 h showed an increase in the percentage of cells with subdiploid DNA content without phase-specific inhibition (14-23% and 15-30%, HL60 and K562 cells, respectively) after adaphostin treatment (Fig. 1C). These data support previous observations that Bcr/ abl expression is not an absolute requirement for adaphostin activity (10, 11). Proteomic analysis of adaphostin-treated cells. To character- ize changes in the cellular proteome induced by adaphostin, two-dimensional PAGE was done on whole-cell lysates of control and treated HL60 and K562 cells. A mini two- dimensional gel format, in which the 24 cm isoelectric focusing nonlinear strip (pH 3-10) was divided into three 8-cm pieces and applied to three 4% to 12% Bis-Tris Zoom gels, was chosen for its high-throughput, simplicity in handling, excellent reproducibility, and the ability to perform subsequent Western blot analysis. Initial experiments using 0.1, 1, and 5 Amol/L adaphostin with exposure times of 6, 12, and 24 h showed that optimal responses, in terms of the number of modulated spots observed, were seen using 5 Amol/L adaphostin for HL60 cells. No changes in the proteomic profiles were observed for K562 cells at these concentrations. Increasing the adaphostin concentration to 30 Amol/L with exposure times of 12 and 24 h proved optimal for K562 cells. Several hundred spots could be observed in every composite of three mini gels after staining with SYPRO Ruby. A representative two-dimensional gel composite from an HL60 experiment is shown in Fig. 2. SYPRO Ruby–stained gels were inspected manually for modulated spots, which were only considered legitimate if they were seen in three separate experiments using separate lysate preparations. Only those spots that were either present upon treatment (i.e., up-regulated and absent in control gels) or absent upon treatment (i.e., down-regulated) were chosen for further analysis. Analysis of gel sets showed adaphostin- induced differential expression of 60 spots in HL60 cells and 59 spots in K562. The time course analysis of HL60 cells (data not shown) showed that by 6 h, the majority of changes had already occurred: 69% of down-regulated proteins and 86% of up-regulated proteins were already modulated, pointing to a rapid response to adaphostin treatment. By 12 h, these numbers increased to 92% and 100% down- and up-regulated spots, respectively. One hundred different protein spots Fig. 1. Analysis of adaphostin-treated HL60 and K562 using assays for cell exhibiting expression trends (mainly present versus absent) viability, protein synthesis, apoptosis, and cell cycle. A, the chemical structure of adaphostin (NSC 680410) illustrating the dihydroquinone group (box). B, time and several reference spots were then excised from SYPRO course analysis (0-24 h) of adaphostin-induced apoptosis at 1 Amol/L (HL60) or Ruby–stained gels, digested with trypsin, and subjected to 10 Amol/L (K562) adaphostin assessed by flow cytometry using propidium iodide nanospray microcapillary liquid chromatography–tandem (Yaxis) and AnnexinV-Alexa488 (Xaxis) shows rapid induction of apoptosis. C, cell cycle analysis of HL60 and K562 cells after 24-h adaphostin treatment shows an mass spectrometry for protein identification. This work increase in the percentage of cells with subdiploid DNA content. culminated in the identification of 79 differentially regulated

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Table 1. Cell viability and inhibition of protein synthesis

Time (hrs) IC50 (Mmol/L) Cell Viability (WST) Protein Synthesis (14C-Leu) HL60 K562 HL60 K562

24 2.0 10.0 0.35 3.50 48 1.5 7.0 0.30 2.50 72 1.5 7.0 0.30 3.00

NOTE: HL60 and K562 cells were exposed to increasing concentrations of adaphostin over a 1-, 2-, or 3-d period and assayed for perturbations in protein synthesis using [14C]leucine incorporation or for changes in cell viability using the WST reagent. protein spots. Condensing the list to remove multiple gels from HL60 and K562 cell lysates revealed identical isoforms of the same protein generated a refined list of 49 proteins. A magnified view of several modulated spots with unique proteins that were all modulated by adaphostin protein identification is shown in Fig. 3. Of the protein spots treatment. A comparison of differentially expressed proteins present in both cell lines, 78% of spots (39 of 50) were identified from HL60 and K562, subdivided into molecular modulated in the same manner (23 up-regulated and 16 function and showing the type of spot modulation, are shown down-regulated proteins). in Table 2A. Analysis of spots (some reference and some At the protein level, there were 19 differences between HL60 differentially regulated) occupying the same position on the and K562, eight proteins (proliferating cell nuclear antigen,

Fig. 2. Representative two-dimensional PAGE analysis of adaphostin-treated HL60 cells.Total cell lysate was prepared from HL60 cells treated with 5 Amol/L adaphostin for 12 h. Protein samples were subjected to first-dimension isoelectric focusing separation (pH 3-10 NonLinear, 24 cm).The first-dimension pH strip was then cut into three equal pieces and subjected to the second dimension as described in Materials and Methods using three 4% to 12% BisTris zoom gels. Differentially regulated spots identified from triplicate gels are shown with SwissProt identifiers inTable 2.

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Table 2. Proteins differentially regulated after adaphostin treatment

Molecular Identity ID Swissprot MW pI A B C function HL60 K562 HL60 HL60 HL60 Ada +

Ada Ada Ada H2O2 HQ NAC (%)*

Apoptosis Programmed cell PDCD5 O14737 14.1 <5.78 D ND D D D 38 death protein 5 PDCD5 O14737 14.1 5.78 U U U U U 100 Transitional TERA P55072 89.1 5.14 D ND D D D 2 endoplasmic reticulum ATPase Calcium Binding Calmodulin CALM P62158 16.7 4.09 U U U ND U 47 protein S100 Ca-binding S10A7 P31151 13.2 5.71 D ND D D D 100 protein A7 Cell cycle Proliferating cell PCNA P12004 28.7 4.57 NC U NC ND NC NC nuclear antigen Prothymosin a PTMA P06454 12 3.69 D D D NC D ND PTMA P06454 <12 <3.69 U ND U U U 100 Cell structure Filamin a FLNA P21333 V280 5.73 U U U U ND 100 and motility Gelsolin GELS P06396 85.6 <5.90 D ND D D NC 100 GELS P06396 85.6 5.90 D ND D D NC 66 Lamin B1 LAM1 P25391 66.2 5.11 D D D D D 100 LAM1 P25391 V66.2 5.11 U U U U ND 100 Lamin B2 LAM2 Q03252 67.6 5.29 D ND D D D 100 Lamin A/C LAMA P02545 <74.1 V6.57 U U U U U ND LAMA P02545 <74.1 <6.57 ND U ND ND ND ND Myosin 9 MYH9 P35579 J22.6 5.50 U U U U U 28 Nucleoprotein TPR TPR P12270 <265 5.01 U U U U U 100 Vimentin VIME P08670 53.4 5.06 D D D D D 100 VIME P08670 53.4 <5.06 ND D ND ND ND ND VIME P08670 53.4 V5.06 ND D ND ND ND ND VIME P08670 V53.4 V5.06 ND U ND ND ND ND VIME P08670 V53.4 V5.06 ND U ND ND ND ND VIME P08670 V53.4 V5.06 ND U ND ND ND ND Tubulin h2 TBB2 P07437 49.6 4.78 NC U NC NC NC NC Plectin 1 PLEC1 Q15149 <531 <5.73 ND U ND ND ND ND Vinculin VINC P18206 <123 5.51 ND U ND ND ND ND Chaperones T-complex protein h subunit TCPB P78371 <57.3 >6.02 NC U NC NC NC NC Protein disulfide isomerase PDIA1 P07237 57 4.76 D D D D D ND Endoplasmic reticulum ERP29 P30040 28.9 <6.77 ND U ND ND ND ND protein 29 Glutathione S-transferase P GSTP1 P09211 23.2 5.44 ND U ND ND ND ND Energy metabolism a-Enolase ENOA P06733 47 V6.99 D NC D D D 38 ENOA P06733 <47 <6.99 U U U U/NC U 30 GAPDHG3P2 P04406 35.9 8.58 D ND D D D 13 Pyruvate kinase KPYM P14618 57.8 V7.95 D ND D D D ND

isozymes M1/M2 KPYMc P14618 57.8 <7.95 D ND D D D 100 ATP-AMP transphosphorylase KAD2 P54819 26.3 7.85 U U U U D 100

NOTE: (A) Direct comparison of response in adaphostin-treated HL60 and K562 cells. (B) Comparison of the response between adapostin-,

H2O2-, and hydroquinone-treated HL60 cells. (C) Effect of the antioxidant L-NAC on the adaphostin response in HL60 cells. Predicted values for Mr and isoelectric point are shown; > or < represent deviation from predicted values. Abbreviations: ID, protein identifier; SwissProt, SwissProt accession number; MW, molecular weight; pI, isoelectric point; Ada, adaphostin; HQ, hydroquinone; ND, not detected; NC, no change; U, proteins that are up-regulated; D, proteins that are down-regulated. *Effect of L-NAC on proteomic profile of adaphostin-treated HL60 cells. Percentage values, obtained using the software Progenesis SameSpots (Nonlinear Dynamics), illustrate the extent to which L-NAC reverses adaphostin-associated protein modulation (complete reversal is 100%). cKAD2 and HCD2 identified from the same spot.

TBB2, PLEC1, VINC, T complex protein h subunit, ERP29, not changed in K562 cells). These results show that adaphostin GSTP1, and PRS6A) were up-regulated and one protein treatment induces profound changes in the cellular proteome. (SNP29) was down-regulated in K562 cells only (and either Combined manual and PANTHER database5 directed gene not detected or not changed in HL60 cells), whereas 10 proteins (transitional endoplasmic reticulum ATPase, S10A7, GELS, LAM2, G3P2, KPYM, HNRPC, PCBP1, RLA1, and VDAC2) were down-regulated in HL60 cells (and either not detected or 5 http://www.pantherdb.org

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Table 2. Proteins differentially regulated after adaphostin treatment (Cont’d)

Molecular Identity ID SwissProt MW pI ABC function HL60 K562 HL60 HL60 HL60 Ada +

Ada Ada Ada H2O2 HQ NAC (%)* Gene Acidic nuclear AN32B Q92688 28.7 3.94 U U U U NC ND regulation phosphoprotein 32B dUTP pyrophosphatase DUT P33316 26.5 V9.46 D D/NC D D/NC D 63 Far upstream element-binding FUBP1 Q96AE4 67.4 >7.18 D D D D D 100 protein 1 Far upstream element-binding FUBP2 Q92945 72.6 <8.02 D D/NC D D D 50 protein 2 FUBP2 Q92945 72.6 8.02 D D D D D 10 Heterogeneous HNRPC P07910 33.6 4.95 D ND D D D 0 ribonucleoprotein C Heterogeneous HNRPH1 P31943 49 5.89 D D D D D 61 ribonucleoprotein H1 HNRPH1 P31943 V49 <5.89 ND U ND ND ND ND HNRPH1 P31943 49 >5.89 NC D NC NC NC NC Heterogeneous HNRPK P61978 >50.9 >5.39 D ND D D D 97 ribonucleoprotein K HNRPK P61978 50.9 J5.39 U U U U U 100 HNRPK P61978 >50.9 <5.39 D NC D D D 78 HNRPK P61978 >50.9 <5.39 D NC D D D 15 HNRPK P61978 50.9 J5.39 ND U ND ND ND ND Heterogeneous ribonucleoprotein Q HNRPQ O60506 V69.5 V8.68 D ND D D D 100 HNRPQ O60506 <69.5 <8.68 U U U U U 100 DNA replication licensing MCM4 P33991 96.5 6.28 U U U ND U 100 factor MCM4 Poly (rC) binding protein PCBP1 Q15365 37.4 6.66 D NC D D/NC D 82 PCBP1 Q15365 <37.4 6.66 D ND D D D 100 Heterogeneous ribonucleoprotein A1 ROA1 P09651 34.2 J5.71 U U U U ND 44 Small nuclear ribonucleoprotein F RUXF P62306 9.7 4.70 D U D D D ND RUXFc P62306 <9.7 >4.70 U U U ND ND 100 Lipid metabolism 3-hydroxyacyl-CoA HCD2 Q99714 26.7 7.86 U U U U D 100 dehydrogenase type II Protein synthesis, Nascent polypeptide NACA Q13765 23.4 >4.52 U U U U U 100 metabolism and complex a subunit modification NACA Q13765 >23.4 4.52 D D D D D 40 NACA Q13765 >23.4 4.52 D D D D D 0 Antisecretory factor 1 PSD4 P55036 40.7 4.68 D D D D D 100 60S acidic ribosomal protein P0 RLA0 P05388 34.2 5.71 U U U U U 100 60 S acidic ribosomal protein P1 RLA1 P05386 11.5 4.26 D NC D D D 18 60S acidic ribosomal protein P2 RLA2 P05387 11.6 4.42 D D/NC D D/NC D 0 RLA2 P05387 11.6 4.42 U U U ND U 47 RLA2 P05387 >11.6 <4.42 U U U U U 100 RLA2 P05387 11.6 4.42 U U U U U 100 Eukaryotic translation EIF3S4 O75821 >35.5 5.87 U U U U NC 100 initiation factor 3 SU 4 Proteasome subunit P50 PRS6A P17980 49.1 >5.13 ND U ND ND ND ND Signal transduction Rho GDP-dissociation inhibitor 2 GDIS P52566 22.9 J5.10 U U U U U 18 GDIS P52566 22.9 >5.10 U U U U U 57 GDIS P52566 >22.9 5.10 D D D U NC 11 Transport Chloride ion current ICLN P54105 >26.2 3.97 D D D D D 0 inducer protein Voltage-dependent anion VDAC2 P45880 38 J6.32 D ND D D D 100 channel protein 2 VDAC2 P45880 38 >6.32 NC D NC NC NC NC Synaptosomal-associated SNP29 O95721 28.9 5.56 ND D ND ND ND ND protein 29

ontogeny analysis identified the responses to apoptosis and with GSTP1 to investigate whether an increase in expression oxidative stress as the cellular processes most likely responsible would modulate adaphostin activity (Fig. 4A). As expected from for the majority of spot changes. proteomic results, Western blotting of K562 cells revealed Expression of GSTP1 confers resistance to adaphostin. Up- endogenous expression of GSTP1. After transfection, prevailing regulation of the antioxidant enzyme GSTP1 in K562 cells levels of GSTP1 increased considerably. This elevated expres- prompted further investigation into a potential role in sion translated into a significant increase in adaphostin resistance to adaphostin (Fig. 4). K562 cells were transfected resistance (IC50, 4.5 to 10.2 Amol/L and 17 to >30 Amol/L)

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14 in the context of [ C]leucine (Fig. 4A) and WST viability assays or H2O2 for 24 h, and protein synthesis was measured (data not shown), respectively. Attempts at transfecting the (Fig. 5A). Results show marked similarity between treatments same construct into HL60 cells resulted in unacceptable levels in that K562 cells were more resistant to each of the treatments of cell death (>50%). However, transfection of IGROV1 cells than HL60 cells (IC50 0.8 versus 3, 20 versus 40, and 45 versus with the GSTP1 construct confirmed increased adaphostin 80 Amol/L for adaphostin, 1,4-dihydroxybenzene, and H2O2 14 resistance (data not shown) using both [ C]leucine (IC50 5.0 in HL60, and K562 cells, respectively). To further evaluate the to 15 Amol/L) and WST assays (IC50 15 to >30 Amol/L). role of ROS, the effect of adaphostin on protein synthesis in Western blotting of a panel of cell lines using an anti-GSTP1 the presence of the antioxidant L-NAC was determined. L-NAC antibody was then done to determine whether a correlation attenuated the effect of adaphostin on protein synthesis by exists between GSTP1 expression and adaphostin activity 5-fold for HL60 and 2.2-fold for K562 cells (IC50 0.6 versus (Fig. 4B). Results failed to show any link between GSTP1 3.0 Amol/L for HL60 cells, and 2.5 versus 5.5 Amol/L for expression and activity. However, measurement of total glutathi- K562 cells in the absence and presence of 12 mmol/L L-NAC, one S-transferase activity in the same panel of lines using an assay respectively). In the second set of experiments, HL60 and K562 based around the glutathione S-transferase substrate chloro-2-4- cells were treated with adaphostin (10 Amol/L), hydroquinone dinitrobenene revealed a positive correlation between total (20 Amol/L), or H2O2 (200 Amol/L) for 1 h, followed by the activity and adaphostin resistance (compare Fig. 4C with D). addition of CM-H2DCFDA (Fig. 5B). Reaction of CM- Adaphostin, 1,4-dihydroxybenzene, and H2O2 increase intra- H2DCFDA with peroxides generates the green fluorescent cellular ROSand inhibit protein synthesis. Recent studies molecule 5-chloromethyl-2¶,7¶-dichlorofluorescein. Analysis by implicating ROS as central to the mechanism of adaphostin flow cytometry showed that in both cell lines, fluorescence led to an investigation into the ability of the hydroquinone, increased significantly after incubation with all three reagents. 1,4-dihydroxybenzene, or H2O2 to replicate certain aspects These assays provide the first evidence of potential similarity of biological activity observed with adaphostin (9, 11). We between adaphostin, a simple hydroquinone, and a direct ROS- investigated this hypothesis using two different methods. In the generating agent (H2O2). first set of experiments, HL60 and K562 cells were treated with Adaphostin increases protein carbonylation by a process varying concentrations of adaphostin, 1,4-dihydroxybenzene, prevented by L-NAC. Carbonylation, the addition of an oxygen

Fig. 3. Selected two-dimensional images of up- and down-regulated proteins induced by adaphostin treatment of HL60 and K562 cells. ICLN, ion current inducerprotein; DUT, dUTP pyrophosphatase;TPR, nucleoproteinTPR; PTMA, prothymosin a; LAMA, lamin A/C; RLA1/2, 60S acidic ribosomal protein1/2; CALM, calmodulin; MCM4, DNA replication licensing factor; FUBP2, far upstream element-binding protein 2; GDIS, rho GDP dissociation inhibitor 2; NACA, nascent polypeptide complex a subunit; LAM1, lamin B1.All events shown are conserved between both HL60 and K562 cells. CON, control cells; ADA, adaphostin-treated cells. Molecular weight markers are visible on the left side of the box inTPRand prothymosin a images.

Clin Cancer Res 2007;13(12) June 15, 2007 3674 www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on September 30, 2021. © 2007 American Association for Cancer Research. Adaphostin Induces Oxidative Stress atom to a polypeptide carbon backbone, is the most frequent protein modification arising through increased ROS. To examine the effects of adaphostin treatment on protein carbonyls, lysates derived from HL60 cells treated with adaphostin (5 Amol/L) for varying lengths of time were assayed by a standard 2,4-dinitrophenylhydrazine–coupled spectrophotometric method (Fig. 6A). Results showed time-dependent increases in protein carbonyl groups for adaphostin and the positive control (H2O2), with maximum carbonylation peaking at 8 h. Similarly, reacting adaphostin- or H2O2-exposed lysates with 2,4-dinitrophenylhydrazine and imaging carbonyl groups by Western blotting using an anti-DNP antibody revealed concentration-dependent increases in carbonyls for both treatments (Fig. 6B). In addition, patterns of protein carbonylation were identical in cell lysates prepared from adaphostin and H2O2-treated cells. Last, the ROS dependence of increased protein carbonylation was confirmed by demonstrating that preincubation with the antioxidant L-NAC reduced the amount of protein carbonyls detected (Fig. 6C). The proteomic profile of adaphostin-treated cells is similar to that derived by treatment with hydroquinone or H2O2 and can be reversed by L-NAC. To further elaborate on the role of hydroquinone-derived ROS in adaphostin activity, proteomic profiles of HL60 cells treated with 1,4,dihyroxybenzene or the ROS-generating reagent, H2O2, were compared. In addition, the effects of the antioxidant L-NAC on the proteomic profile of adaphostin-treated cells were investigated. HL60 cells were incubated with either adaphostin (5 Amol/L), 1,4,dihyroxybenzene (50 Amol/L), or H2O2 (100 Amol/L) for 24 h and subjected to two-dimensional gel electrophoresis. Table 2B compares the protein changes induced by adaphostin, 1,4,dihyroxybenzene, or H2O2. For hydroquinone-treated cells 87% (69 of 79) of proteins showed identical changes to adaphostin treatment. Similarly, for H2O2-treated cells, 91% Fig. 4. Expression of GSTP1confers resistance to adaphostin. A, transient (72 of 79) of proteins showed identical changes between the transfection of K562 cells with a construct encoding GSTP1confers resistance to sets. Table 2C shows the effect of the antioxidant L-NAC on adaphostin. Inset,Western blotting of GSTP1expression in base vector and GSTP1construct ^ transfected cells. B, Western blotting of a panel of cell lines adaphostin-induced changes. Here, changes observed in 68% for endogenous expression of GSTP1. C, determination of total glutathione (54 of 79) spots were prevented by 50% or more due to the S-transferase (GST) activity in the same panel of cell lines using a chloro-2-4-dinitrobenene ^ based spectrophotometric assay. D, IC values L 50 addition of -NAC. Only 14% of spots (11 of 79) were obtained from protein synthesis inhibition assays of adaphostin for each cell line. prevented <20% by L-NAC. Thus, similarities between proteomic profiles derived from adaphostin, 1,4,dihyroxybenzene, or H2O2 exposure, and the ability of L-NAC to prevent adaphos- Expression of the SODs, however, had a close correlation with tin-induced changes, supports a role for hydroquinone-derived IC50 values. For SOD II, high expression correlated with ROS in adaphostin activity. resistance to adaphostin in all cases except for the renal cancer Cu/Zn SOD I confers resistance to adaphostin. Proteomic cell pair, where CAKI cells expressed high levels of SOD II but profiling of the response to drug treatment was followed by an were relatively sensitive to adaphostin. The most consistent investigation into a possible link between expression of correlation was found for SOD I, where high expression levels antioxidant enzymes and adaphostin toxicity (Fig. 7). A panel of SOD I correlated with resistance to adaphostin. of nine cell lines (two myelogenous leukemia, HL60 and K562; The association between SOD I expression and adaphostin one T-cell leukemia, MOLT4; two ovarian, IGROV1 and activity was further explored using three different assays. In the SKOV3; two colon, COLO205 and HCC2998; and two renal, first assay, diethyldithiocarbamate, an inhibitor of SOD I, was A498 and CAKI) were chosen based on their high or low used to determine whether pretreatment could modulate susceptibility to adaphostin as defined in the panel of 60 adaphostin activity. In its capacity as a Cu2+ chelator, human tumor cell lines of the Developmental Therapeutics diethyldithiocarbamate is regarded as specific for SOD I (Cu/ Program of the National Cancer Institute. Cell lysates were Zn) rather than manganese-containing SOD II (Fig. 7B). Cells prepared and probed with antibodies specific for catalase, were preincubated in diethyldithiocarbamate for 30 min myeloperoxidase, and SOD I or II (Fig. 7A). The relative followed by the addition of adaphostin at different concen- expression of each protein was then compared with the IC50 trations. After 24 h, treated cells were analyzed for the change in values determined from protein synthesis inhibition assays. protein synthesis (Fig. 7B). Results showed that pretreatment Catalase expression was constant throughout the panel, with the SOD I inhibitor diethyldithiocarbamate increased the whereas myeloperoxidase was only detected in HL60 cells. sensitivity of cells toward adaphostin by 3-fold for HL60 and

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30-fold for K562 cells (IC50 0.3 and 0.1 Amol/L for HL60 cells for transfection on the basis of low intrinsic SOD I expression. and 3 and 0.1 Amol/L for K562 cells in the absence and As shown in the inset to Fig. 7D, cells transiently transfected presence of diethyldithiocarbamate, respectively). The large with the vector encoding SOD I had an increased expression of change in IC50 for K562 cells relative to HL60 may reflect the SOD I, relative to the vector control. To determine what effect higher levels of endogenous SOD I in K562 cells compared with elevated SOD I had on adaphostin activity, protein synthesis HL60 cells. This is further shown by the higher concentration of assays were done on transfected cells. Results showed that there diethyldithiocarbamate required to achieve the maximal effect was a 4.7-fold increase in IC50 for cells transfected with SOD I, in K562 (18 Amol/L) versus HL60 (3 Amol/L) cells. Interest- compared with the vector control (IC50 7.0 versus 1.5 Amol/L, ingly, the IC50 values for both cell lines in the presence of the respectively), suggesting that increasing levels of SOD I confer SOD I inhibitor were similar (0.1 Amol/L each). The second set resistance to adaphostin. Similar results were noted for cell of assays examined the effect of exogenous catalase and SOD I viability (IC50 15 versus 100 Amol/L, control vector and SOD1 on the activity of adaphostin. As shown in Fig. 7C, the addition vector, respectively, data not shown). These results highlight the of either catalase or SOD I attenuated the effect of adaphostin. role of antioxidant enzymes, in particular SOD I, in the cellular In the presence of 2 Amol/L adaphostin, protein synthesis was sensitivity to adaphostin. reduced to 13.9 F 2.8% of control. Addition of exogenous Adaphostin, hydroquinone, and H2O2 affect polypeptide catalase or SOD I decreased protein synthesis inhibition to degradation of the tyrosine kinase p210Bcr/abl. To determine 39.8 F 4.6% and 22.5 F 1.2%, respectively. Similar results were whether induction of oxidative stress alone could explain Bcr/ noted at 1 Amol/L adaphostin (51.7 F 3.2% in the absence of abl polypeptide degradation, K562 cells were treated with any additions and 90 F 2.9% and 82.8 F 4.4% in the presence increasing doses of adaphostin, 1,4,dihyroxybenzene, or H2O2 of added catalase or SOD I, respectively). In the third assay, a for 24 h (Fig. 8A). Results showed that Bcr/abl was preferen- construct encoding SOD I was used to establish a causal tially degraded relative to h-actin in K562 cells after exposure to relationship between SOD I levels and resistance to adaphostin adaphostin (>3 Amol/L), 1,4,dihyroxybenzene (>30 Amol/L), (Fig. 7D). The ovarian carcinoma cell line IGROV1 was selected or H2O2 (>50 Amol/L). In addition, all treatments resulted in

Fig. 5. Adaphostin, hydroquinone (HQ), and H 2O2 have similar effects in terms of protein synthesis inhibition and generation of ROS. A, HL60andK562cellswere 14 treated with increasing concentrations of adaphostin,1,4,dihyroxybenzene, or H2O2, incubated for 24 h, and changes in protein synthesis was determined using a [ C]leucine incorporation assay. Results showed that K562 cells were more resistant to all three reagents. B, HL60 and K562 cells were labeled with the fluorescent peroxide sensor CM-H2DCFDA (1 Ag/mL for 2 h) and treated with adaphostin (5 Amol/L), hydroquinone (50 Amol/L), and H2O2 (200 Amol/L) for 2 h. Flow cytometric analysis of FL1 fluorescence showed rapid increases in peroxides after treatment with all three reagents.

Clin Cancer Res 2007;13(12) June 15, 2007 3676 www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on September 30, 2021. © 2007 American Association for Cancer Research. Adaphostin Induces Oxidative Stress the appearance of c-Abl antibody–reactive high molecular weight aggregates. Second, the effect of antioxidants (L-NAC, TIRON, and ascorbate) on Bcr/Abl degradation induced by adaphostin, 1,4,dihyroxybenzene, and H2O2 was studied (Fig. 8B). Results showed that antioxidant pretreatment was incapable of restoring Bcr/abl expression.

Discussion

In this study, proteomic analysis of myeloid leukemia cell lines was undertaken to provide insights into the biological activity of the putative anticancer agent, adaphostin (NSC 680410). Preliminary assays supported prior observations that adaphostin exposure was associated with inhibition of protein synthesis, oxidative stress, and induction of apoptosis (5, 6, 9, 11). Analysis of HL60 and K562 cells using two-dimensional PAGE revealed altered expression of several proteins involved in several biological pathways, predominantly apoptosis and the response to oxidative stress. With respect to apoptosis, cleavage fragments from known caspase substrates (e.g., nucleoprotein TPR, filamin a, rho GDP-dissociation inhibitor 2, vimentin, and lamins A/C and B1) were identified in both cell lines (17–21). Likewise, down-regulation of the native form of far upstream element–binding protein 2 probably represents one facet of caspase cleavage (22). Further confirmation that apoptotic pathways are active in both lines came from detection of increased levels of programmed cell death protein 5 (PDCD5) and decreased levels of dUTPase. Expression of programmed cell death protein 5 is significantly elevated during initiation of apoptosis whereas dUTPase has been shown to be necessary for cell survival (23, 24). Identification of additional caspase cleavage products (prothymosin a, gelsolin, and lamin B2) only in HL60 cells supports the inference from the cell-based assays and time course proteomics that the response was far more advanced in these cells than in K562 cells (18, 25–27). This is further confirmed by the down-regulation of the transitional endoplasmic reticulum ATPase in HL60 cells. Transitional endoplasmic reticulum ATPase has been shown to be associated with an antiapoptotic function through activation of the nuclear factor-nB signaling pathway (28). Fig. 6. Adaphostin increases protein carbonylation in a process reversible by the Several proteins implicated in the oxidative stress response antioxidant L-NAC. A, a time course of protein carbonylation as determined by a were also modulated in both HL60 and K562 cells. One such spectrophotometric assay using DNP-hydrazone shows that maximal increases are observed after 8 h. B, adaphostin and H2O2 treatment produce identical example was the calcium-binding protein calmodulin. An carbonylation patterns as determined by Western blotting of DNP-hydrazone ^ labeled 2+ extensive and complex interaction between ROS and Ca has cellular proteins using an anti-DNP monoclonal antibody.Three lanes are shown A A been identified (29). Under conditions of oxidative stress, for each treatment: adaphostin 0, 0.1, and 1 mol/L; H2O2 0, 10, and 30 mol/L. C, preincubation (30 min) with L-NAC (100 mmol/L) is sufficient to prevent calmodulin is selectively oxidized at critical methionine adaphostin-induced protein carbonyls to control levels. residues, allowing it to regulate the activity of several protein kinases and phosphatases (30). For example, oxidized calmod- 2+ ulin stabilizes target proteins such as plasma membrane Ca - oxidative stress (H2O2), PDIA1 was found to be a major target ATPase in an inhibited state reducing ATP utilization, thereby of proteasome-dependent oxidative degradation (35). In this regulating cellular metabolism (31). Up-regulation of calmod- project, a similar down-regulation of native PDIA1 was ulin has also been observed in Jurkat cells undergoing FAS- observed in both lines. induced apoptosis, emphasizing the intrinsic link between An important pattern of change restricted to HL60 cells oxidative stress and apoptosis (32). Protein disulfide isomerase concerned down-regulation of the glycolytic enzymes a- (PDIA1) is a chaperone found mainly in the endoplasmic enolase, pyruvate kinase, and glyceraldehyde-3-phosphate reticulum that catalyzes thiol disulfide exchange (33). Increased dehydrogenase. Posttranslational modification of glycolytic oxidative stress is thought to modify PDIA1 at a critical cysteine enzymes is recognized as an intracellular sensor of oxidative residue involved in client protein binding, thereby impairing stress (36). In a recent proteomic study, oxidizing agents, activity (34). In a proteomic project investigating the effects of including H2O2, were shown to carbonylate and induce

www.aacrjournals.org 3677 Clin Cancer Res 2007;13(12) June 15, 2007 Downloaded from clincancerres.aacrjournals.org on September 30, 2021. © 2007 American Association for Cancer Research. Cancer Therapy: Preclinical polypeptide degradation in glycolytic enzymes including a- endoplasmic reticulum chaperones (GRP94, BiP, ERp72; enolase, pyruvate kinase, and glyceraldehyde-3-phosphate ref. 41). The most compelling candidate for an adaphostin dehydrogenase (37). In a study of Escherichia coli, a-enolase response modifier involved the detoxifying enzyme GSTP1, - was one of the primary targets of H2O2 –orO2-mediated which showed elevated expression in K562 cells. This phase II carbonylation (38). Therefore, if ‘‘sensors’’ of oxidative stress metabolizing enzyme plays an important role in protection are being modulated, this suggests that higher prevailing levels from oxidative stress. Transcription of GSTP1 is under the of ROS are present in HL60 cells. control of an antioxidant response element (42). Overexpres- A number of proteomic adaptations exclusive to K562 sion of GSTP1 has been shown to confer resistance to provide a possible molecular basis for increased adaphostin doxorubicin and chlorambucil, whereas clinically, increased resistance relative to HL60 cells. For example, increased levels of GSTP1 are a negative prognostic indicator (43). Here, expression of the T complex protein h subunit was observed the marked decrease in adaphostin activity observed for GSTP1- in only K562 cells. This type II chaperone is indispensable for transfected cells provides strong evidence for a role in drug cell survival because folding of an essential set of cytosolic resistance. Likewise, the correlation observed between overall proteins (a/h actin, a/h tubulin, myosin heavy chain) requires glutathione S-transferase activity and adaphostin activity hints T complex protein h subunit and this function cannot be done at the potential of this family of enzymes in resistance to by other chaperones (39). Likewise, up-regulation of prolifer- adaphostin. ating cell nuclear antigen in K562 cells may represent a Structurally, adaphostin consists of a dihydroquinone and an protective mechanism. In addition to a role in DNA replication, inert adamantyl group. Under alkaline conditions, hydro- PCNA is involved in nucleotide mismatch and base excision quinones are oxidized by molecular oxygen to form superoxide repair (40). Increased levels of PCNA may therefore enhance radicals and the respective quinone (44, 45). Several reports the ability of K562 cells to withstand cellular stresses that result have suggested that hydroquinone redox activity may account in DNA damage. Similarly, the stress-inducible protein ERp29 for some of the biological observations attributed to adaphos- was also up-regulated in K562 cells. Although the biological tin (9). Interestingly, in a study of the effects of benzene function of this chaperone remains unclear, studies using an metabolites on HL60 cells, simple hydroquinones were ERp29-overexpressing rat thyroid cell line suggested that ERp29 confirmed as potent ROS generators (46). Hydroquinone assists in protein folding and secretion in association with other treatment has also been shown to induce apoptosis in HL60

Fig. 7. SODI confers resistance to adaphostin. A, top, analysis of endogenous expression of antioxidant enzymes in a diverse panel of nine cell lines byWestern blotting with antibodies against catalase (CAT), myeloperoxidase (MPO), and SOD I/II. Bottom, IC50 values obtained from protein synthesis inhibition assays of adaphostin for each cell line. B, the SOD I inhibitor, diethyldithiocarbamate (DET), increases the susceptibility of HL60 and K562 cells to adaphostin. C, exogenous catalase or SOD I attenuate adaphostin activity. D, transient transfection of IGROVI cells with a construct encoding SOD I confers resistance to adaphostin. Inset,Western blotting of SOD I expression in control and transfected cells.

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strengthened by the finding that cells transfected with SOD I showed resistance, whereas preincubation with a SOD I inhibitor potentiated toxicity. It is noteworthy that anthracy- clins such as doxorubicin undergo redox conversions to produce superoxide ions and alkylating semiquinone radicals (51). In a study of gastric cancer cell lines, resistance to doxorubicin was also conferred by SOD (52). Degradation of Bcr/abl polypeptide in adaphostin-treated chronic myelogenous leukemia cells is a seminal experiment used to imply tyrosine kinase inhibitor activity (5, 6). Here, we show that a hydroquinone or direct oxidative stress (H2O2) affects degradation of Bcr/abl polypeptide in an identical manner to adaphostin treatment. It has been suggested that because Bcr/abl degradation cannot be reversed with antioxidants, the process is distinct from oxidative stress induction (9). Interestingly, we report an identical result but show that Bcr/abl degradation promulgated by hydroquinone or H2O2 was also unaffected by treatment with antioxidants. These results imply that adaphostin-induced Bcr/abl degradation may yet be related to increased levels of oxidative stress but the effect is independent of antioxidants. Therefore, a wealth of evidence supports the hypothesis that the biological activity of adaphostin is reliant on the redox

Fig. 8. Adaphostin, hydroquinone, and H2O2-mediated Bcr/abl polypeptide properties of the dihydroquinone group. Further evidence can degradation. A, cell lysates were prepared from K562 cells treated for 24 h with be found in recent adaphostin publications. Most importantly, increasing concentrations of adaphostin (0, 3, and10 Amol/L),1,4-dihyroxybenzene structure activity studies showed that the hydroquinone moiety (0, 30, and 100 Amol/L), or H2O2 (0, 50, and 200 Amol/L). Bcr/abl expression was probed using an anti ^ c-abl antibody. Results show similar Bcr/abl polypeptide in adaphostin was essential for activity (4). Also, in a study of degradation after treatment, with the appearance of high molecular weight glioblastoma cell lines, a positive correlation was found aggregates. B, K562 cells were preincubated for 4 h with the antioxidants L-NAC (10 and 50 mmol/L),TIRON (100 and 500Amol/L), and ascorbate (10 and between sensitivities to both adaphostin and ROS (53). This 50 mmol/L) followed by addition of adaphostin (10 Amol/L),1,4,dihyroxybenzene study also noted that catalase expression was not altered with A A (100 mol/L), and H2O2 (200 mol/L) or 24 h. Lysates were prepared and blotted adaphostin treatment. Several other reports have elaborated on for Bcr/abl expression. Results showed that L-NAC,TIRON, and ascorbate were incapable of reversing adaphostin-induced Bcr/abl p210 polypeptide degradation. the role of ROS in adaphostin activity, while speculating on the existence of a molecular target (9, 11, 13, 54). The data presented here, showing hydroquinone- and H2O2-mediated cells and to inhibit protein synthesis and the secretion of Bcr/abl degradation, identical proteomic profiles, and the cytokines (e.g., interleukin-1h) in monocytes (47–49). Here, identification of GSTP1/SODI as resistance markers, undermine we confirm rapid increases in ROS after treatment with the any suggestion that adaphostin operates by direct interaction hydroquinone 1,4-dihydroxybenzene, and show that the with Bcr/abl. However, a primary molecular target for proteomic profile is almost identical to cells treated with adaphostin-derived ROS may yet exist and one plausible adaphostin. A logical extension was therefore to compare candidate is the ubiquitous sarcoplasmic reticulum Ca2+ adaphostin activity with a simple ROS generator, such as ATPase. Evidence to support a role for sarcoplasmic reticulum 2+ hydrogen peroxide (H2O2). HL60 cells were f2-fold more Ca ATPase comes from studies of 2-5-di-(t-butyl)-1,4- sensitive to H2O2 than K562 cells, mirroring the relative hydroquinone, a compound with a very similar chemical susceptibility of these cell lines to adaphostin. Proteomic structure to adaphostin. This simple hydroquinone has been 2+ analysis of H2O2-treated HL60 cells revealed a profile almost shown to mobilize Ca specifically from inositol-sensitive synonymous with that found after adaphostin exposure. These stores by inhibiting sarcoplasmic reticulum Ca2+ ATPase in a results support the concept that direct generation of ROS by the process mediated by superoxide ions (55). dihydroquinone in adaphostin is the predominant effector This hydroquinone-centric mechanism provides possible mechanism. However, the importance of the adamantyl group explanations for several reported observations. For example, in adaphostin should not be overlooked. This inert nonpolar there is direct evidence to show that hydroquinone and the group has been shown to enhance the membrane penetration semiqiunone intermediates of several anticancer agents pro- of several biological molecules, and this may account for the mote the release of iron from ferritin in reactions inhibited by increased activity of adaphostin relative to 1,4-dihydroxyben- SOD (56, 57). An identical mechanism may account for the zene (50). reported increases in free ferrous iron and ferritin mRNA after Given that hydroquinone oxidation is associated with adaphostin treatment (12). Likewise, the increases in protein generation of superoxide ions, we speculated that antioxidant carbonylation observed after adaphostin treatment would place enzymes and in particular, SODs, may act as modifiers of increasing strain on protein catabolism, providing a basis for adaphostin activity. In this regard, a limited survey of previous reports of synergy with proteasome inhibitors (54). antioxidant enzymes in a diverse panel of cell lines was Also, if adaphostin activity were divorced from kinase conducted with the result that expression of Cu/Zn SOD I inhibition, it would provide a basis for the ability to correlated with resistance to adaphostin. This observation was ‘‘overcome’’ resistance to ATP-dependent kinase inhibitors

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(15, 58). From a molecular perspective, adaphostin-associated myelogenous leukemia, chronic myelogenous leukemia, and peturbations in signal transduction pathways (p38 mitogen- chronic lymphocytic leukemia can be explained given that these activated protein kinase, RAF-1/mitogen-activated protein diseases are exquisitely sensitive to other ROS-generating kinase kinase/extracellular signal-regulated kinase, and AKT) reagents, such as arsenic compounds or sesquiterpene lactones cannot be extricated from changes that would also occur with (9, 15, 59, 60). In conclusion, evidence from proteomic and increased oxidative stress (13, 14). Similarly, the selectivity of biochemical studies supports classification of adaphostin as a adaphostin for hemopoietic malignancies such as acute redox-active substituted hydroquinone.

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Luke H. Stockwin, Maja A. Bumke, Sherry X. Yu, et al.

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