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J. Biol. Chem., Vol. 282, Issue 12, 8860-8872, March 23, 2007

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Inhibition of Mitochondrial Respiration as a Source of Adaphostin-induced Reactive Oxygen Species and Cytotoxicity^*

Son B. Le¹, M. Katie Hailer, Sarah Buhrow, Qi Wang, Karen Flatten, Peter Pediaditakis^¶, Keith C. Bible^||, Lionel D. Lewis^**, Edward A. Sausville, Yuan-Ping Pang^||, Matthew M. Ames^||, John J. Lemasters^¶2, Ekhson L. Holmuhamedov^¶3, and Scott H. Kaufmann^||34

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

Adaphostin is a dihydroquinone derivative that is undergoing extensive preclinical testing as a potential anticancer drug. Previous studies have suggested that the generation of reactive oxygen species (ROS) plays a critical role in the cytotoxicity of this agent. In this study, we investigated the source of these ROS. Consistent with the known chemical properties of dihydroquinones, adaphostin simultaneously underwent oxidation to the corresponding quinone and generated ROS under aqueous conditions. Interestingly, however, this quinone was not detected in intact cells. Instead, high performance liquid chromatography demonstrated that adaphostin was concentrated by up to 300-fold in cells relative to the extracellular medium and that the highest concentration of adaphostin (3000-fold over extracellular concentrations) was detected in mitochondria. Consistent with a mitochondrial site for adaphostin action, adaphostin-induced ROS production was diminished by >75% in MOLT-4 rho⁰ cells, which lack mitochondrial electron transport, relative to parental MOLT-4 cells. In addition, inhibition of oxygen consumption was observed when intact cells were treated with adaphostin. Loading of isolated mitochondria to equivalent adaphostin concentrations caused inhibition of uncoupled oxygen consumption in mitochondria incubated with the complex I substrates pyruvate and malate or the complex II substrate succinate. Further analysis demonstrated that adaphostin had no effect on pyruvate or succinate dehydrogenase activity. Instead, adaphostin inhibited reduced decylubiquinone-induced cytochrome c reduction, identifying complex III as the site of inhibition by this agent. Moreover, adaphostin enhanced the production of ROS by succinate-charged mitochondria. Collectively, these observations demonstrate that mitochondrial respiration rather than direct redox cycling of the hydroquinone moiety is a source of adaphostin-induced ROS and identify complex III as a potential target for antineoplastic agents.

Received for publication, July 12, 2006

^* This work was supported in part by National Institutes of Health Grant R01 CA85972, the Mayo Foundation for Education and Research, and the University of Minnesota Supercomputing Institute. 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 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.

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