[CANCER RESEARCH 53. 1072-1078. March I. 1993] Mitochondrial Cytochrome c Oxidase as a Target Site for Daunomycin in K-562 Cells and Heart Tissue1 Lefkothea C. Papadopoulou and Asterios S. Tsiftsoglou2 iMhoratory of Pharmacology, Department of Pharmaceutical Sciences. Aristotle University' of Thesstiloniki. Thesstiloniki 540 (ÃŒ6, Greece ABSTRACT Daunomycin and other structurally related anthracyclines can cause myelosuppression and cardiomyopathy. We explored the possible mecha- nism(s) by which daunomycin (DAU) interacts with target sites in neo- plastic hemopoietic cells and heart tissue. We observed that | 'lli(. i|l) VI interacts selectively with mitochondria! hemoproteins isolated from K-562 cells and rat and bovine heart and forms relatively stable protein com plexes. Isolation, purification, and Chromatographie analysis of the mito chondria! components complexed with [JH(G)]DAU revealed that one of the major components involved is cytochrome c oxidase (COX). Both DAU and ADR caused a dose-dependent inhibition of COX activity in vitro, an event prevented by exogenous hemin. The interaction of DAL with COX appears to occur via more than one site, one of which at least appears to be the prosthetic group of heme. Therefore, mitochondria! COX, a pivotal mitochondria! enzyme for cell respiration, may serve as a potential target site for DAU and other related anthracvclines. INTRODUCTION DAU3 and other structurally related anthracyclines like ADR are potent antitumor agents with wide clinical applications in the therapy of a large variety of neoplasms (1). Unfortunately, their long-term clinical use is limited due to a cumulative dose-dependent cardiovas cular toxicity (2) and severe bone marrow suppression (3). Although several different mechanisms have been proposed so far to explain anthracycline-induced cytotoxicity, it is as yet unknown which of these mechanisms is most responsible for cardiovascular toxicity, myelosuppression and antitumor activity of anthracyclines. The original hypothesis that anthracyclines kill cells primarily by interacting directly with the genome, promoting DNA damage and retarding RNA synthesis (4) has been challenged over the years. Evidence now exists to indicate that anthracyclines can also kill neo- plastic cells by interacting with cellular components other than DNA. In particular, ADR (a) interacts with the plasma membrane phospho- lipid bilayer (5); (b) promotes lipid peroxidation (6, 7); (c) stimulates formation of free radical species with destructive capacity (8-11); (d) stabilizes the DNA-topoisomerase cleavage complex (12); (e) forms a quite stable complex with iron (Fe3+) (13, 14); and (/) deteriorates mitochondrial structural and functional integrity (15-19). Earlier studies from our laboratory have shown that hemin (iron- protoporphyrin IX) (a) selectively counteracts the induced cytotoxic ity of ADR in normal and transformed hemopoietic cells (20) and (b) interacts directly with DAU (21). In addition, DAU was found to form relatively stable complexes with mitochondrial proteins enriched in hemoproteins (22). These findings prompted us to further explore the effects of DAU on mitochondria. In this study, we used [3H(G)]DAU and intact mitochondria pre pared from human K-562 cells and bovine and rat heart and demon strated that («)hemin counteracts DAU-induced cytotoxicity in K-562 Received 7/31/92; accepted 12/22/92. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported in part by a grant from the Greek National Drug Organization to A. S. T. 2 To whom requests for reprints should be addressed. 'The abbreviations used are: DAU. daunomycin; ADR. Adriamycin; COX. cyto chrome c oxidase; PMSF, phenylmethylsulfonyl fluoride; PBS, phosphate-buffered saline; NP-40, Nonidet P-40; SDS-PAGE. sodium dodecyl sulfate-polyacrylamide gel electro- phoresis. cells like ADR (20); (/;) DAU interacts selectively with mitochondrial COX; these interactions appear to be specific in nature and may occur in part via the prosthetic group of heme located in two of the several subunits of this enzyme; (c) DAU and ADR inhibited COX activity in a dose-dependent fashion that was prevented by exogenously added hemin. In light of these observations, we propose that mitochondrial COX may serve as a target site for DAU and presumably other anthracyclines on highly proliferating neoplastic cells and heart tissue. MATERIALS AND METHODS Chemicals and Biologicals. Hemin was purchased from Eastman Kodak (Rochester. NY) and dissolved in slightly alkaline solution. ADR-HCI. DAU-HC1, cytochrome c (bovine heart type V), cytochrome c oxidase (ferro- cytochrome c: oxygen oxidoreductase. EC 19.3.1 from bovine heart), cyto chrome c-agarose (type VIA from horse heart. 6.6 mg/ml gel). DEAE-cellu- lose. NP-40. and PMSF were purchased from Sigma Chemical Co. (St. Louis. MO). Pyrromycin, kindly donated by Bristol Laboratories (Syracuse, NY), was dissolved in 0.1 N HC1 solution. Protoporphyrin IX was purchased from Por phyrin Products, Inc. (Logan. Utah) and dissolved like hemin. |'H(G)]DAU (specific activity. 5.0 Ci/mmol) was purchased from New England Nuclear Corp. (Boston. MA). Bio-Gel P-I50 and gel filtration standards were pur chased from Bio-Rad Laboratories (Richmond, CA). Sephadex G-25 and Sephadex LH-20 were purchased from Pharmacia Fine Chemicals AB (Upp sala, Sweden). Protein molecular weight standards were purchased from Gibco. Life Technologies, Inc. (Paisley, Scotland). Collagenase II (150 units/mg protein) was purchased from Worthington Biochemical Corp. (Free hold. NJ), proteinase K (27 m Anson units/mg protein) from E. Merck (Darm stadt, Germany), and Triton X-100 from Serva (NY). Phosphate-buffered saline (PBS) containing NaCI (8 g/liter), KC1 (0.2 g/liter), Na2HPO4-2H2O (1.15 g/liter), and KH2PO4 (0.2 g/liter) was prepared in our laboratory. Cell Cultures. Human K-562 erythroleukemia cells, originally developed by Lozzio and Lozzio (23), were seeded in culture at a concentration of 2-3 X IO5 cells/ml in RPMI 1640 supplemented with 10% fetal calf serum, strepto mycin (100 ug/ml). and penicillin (100 units/ml) (Gibco, Life Technologies, Inc.). Cells were kept in exponential growth at 37°Cin 5% CO2 humidified atmosphere by replenishing the cultures with fresh medium every 48-72 h. Cell growth was determined at various time intervals by measuring the number of cells with a hemocytometer under a light microscope. Animals. Adult albino Wistar rats, used throughout this study, were bred in our animal house. Bovine heart was kindly donated by the local slaughter house. Preparation of Mitochondria from K-562 Cells and Heart Tissue. Intact mitochondria were prepared from drug-treated and untreated K-562 cells as well as from heart tissue. In the case of K-562 cells, mitochondria were isolated by homogenizing cells in buffer solution A ( 10 HIMTris-HCI, I ITIMCaCU, 0.5 imi PMSF. 17c w/v sucrose. pH 7.0) for 30 min at 4°C.The homogenate was centrifuged at 600 x g for 10 min (4°C)to remove nuclei, and the postnuclear supernatant was spun at 9000 x g for 30 min (4°C)in order to collect intact mitochondria as previously described (24). Isolated mitochondria were resus- pended into buffer solution (0.25 M sucrose/1 mw PMSF). Mitochondria from bovine and rat heart tissue were prepared according to the method of Vercesi et al. (25). Briefly, l g of bovine or rat heart tissue was placed in a Petri dish carrying ice-cold 0.25 M sucrose solution and dissected in small pieces. The mixture was then treated with 10 ml buffer solution B (0.25 M sucrose. 0.5 ITIMEGTA, 3 mm4-(2-hydroxyethyl)-l-pipera/ineethane- sulfonic acid-HCl, pH 7.25) and 100 ug collagenase for 15 min at 4°C.The dissected and collagenase-treated parts of the heart tissue were then washed twice with 0.25 Msucrose solution to remove collagenase. Finally, the mixture was resuspended in 0.25 Msucrose solution containing PMSF ( 1 m.M),homog enized in a Dounce Potter homogenizer (10 strokes), and centrifuged at 600 x 1072 Research. on October 24, 2015. © 1993 American Association for Cancer cancerres.aacrjournals.org Downloaded from