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Proteomic Analysis Identifies Oxidative Stress Induction by Adaphostin Luke H

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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 degra- dation, 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
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