A Novel Mechanism of Action of Methyl-2-cyano-3,12 Dioxoolean-1,9 Diene-28-oate: Direct Permeabilization of the Inner Mitochondrial Membrane to Inhibit Electron Transport and Induce Apoptosis Ismael Samudio, Marina Konopleva, Helene Pelicano, Peng Huang, Olga Frolova, William Bornmann, Yunming Ying, Randall Evans, Rooha Contractor, and Michael Andreeff Section of Molecular Hematology and Therapy, Departments of Blood and Marrow Transplantation (I.S., M.K., O.F., R.E., R.C., M.A.), Molecular Pathology (H.P., P.H.), and Experimental Diagnostic Imaging (W.B., Y.Y.), The University of Texas M. D. Anderson Cancer Center, Houston, Texas Received August 15, 2005; accepted January 12, 2006 ABSTRACT Methyl-2-cyano-3,12 dioxoolean-1,9 diene-28-oate (CDDO- Me) is a synthetic oleanolic acid derivative that displays antitu- morigenic and anti-inflammatory activities, and we have previ- ously reported that this agent potently activates the intrinsic apoptotic pathway in leukemia cells. In this study, we demon- strate that mitochondrial dysfunction induced by CDDO-Me is mediated by direct permeabilization of the inner mitochondrial membrane, which results in the rapid depletion of mitochondrial glutathione (GSXm), loss of cardiolipin, and inhibition of mito- chondrial respiration. More importantly, we demonstrate that in addition to activating the intrinsic apoptotic pathway, the mito- chondrial effects of CDDO-Me may mediate its anti-inflamma- tory activity by modulating the generation of superoxide anion (O 2 . ). It is noteworthy that CDDO-Me did not increase the generation of O 2 . , and pretreatment of leukemia cells with CDDO-Me prevented the increase of this reactive oxygen spe- cies elicited by inhibition of complex I or III in the absence of de novo protein synthesis. CDDO-Me, but not other inhibitors of respiration, induced a time- and dose-dependent, cyclosporin A-independent permeability transition (PT) of isolated mito- chondria that was sensitive to sulfhydryl antioxidants but not to EDTA. PT induced by CDDO-Me and Ca 2+ was accompanied by loss of GSXm, suggesting that the increased permeability of the inner mitochondrial membrane facilitates the loss of this antioxidant. Finally, transmission electron microscopy revealed that CDDO-Me rapidly induced caspase-independent mito- chondrial swelling and loss of inner membrane structure before the release of cytochrome c. Taken together, our results indi- cate that CDDO-Me is a novel mitochondriotoxic agent that induces apoptosis and inhibits mitochondrial electron transport via perturbations in inner mitochondrial membrane integrity. 2-Cyano-3,12-dioxooleana-1,9-diene-28-oic acid (CDDO) is a synthetic triterpenoid that displays potent anti-inflamma- tory and antitumorigenic activities in vitro and in vivo (Suh et al., 1999; Lapillonne et al., 2003; Konopleva et al., 2004). CDDO and its more potent methyl ester derivative, methyl- 2-cyano-3,12-dioxooleana-1,9-diene-28-oate (CDDO-Me), in- duce apoptosis and differentiation in leukemia cells in cul- ture, and we have demonstrated that apoptosis induced by these agents is initiated by the intrinsic apoptotic pathway possibly through direct perturbation of mitochondrial ho- meostasis (Konopleva et al., 2002, 2004). As an anti-inflam- matory agent, CDDO has been shown to potently prevent This work was supported in part by the National Institutes of Health Core grant CA16672 High Resolution Electron Microscopy Facility, The University of Texas M. D. Anderson Cancer Center. Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.105.018051. ABBREVIATIONS: CDDO, 2-cyano-3,12-dioxooleana-1,9-diene-28-oic acid; CDDO-Me, methyl-2-cyano-3,12-dioxooleana-1,9-diene-28-oate; NFB, nuclear factor B; TNF, tumor necrosis factor ;O 2 . , superoxide anion; ROS, reactive oxygen species; mPT, mitochondrial permeability transition; CsA, cyclosporine A; PT, permeability transition; TMRM, tetramethylrhodamine methyl ester perchlorate; COXIV, cytochrome c oxidase IV; z-, N- benzyloxycarbonyl; fmk, fluoromethyl ketone; HO-1, hemeoxigenase-1; AIF, apoptosis-inducing factor; BB-CDDO, biotinylated derivative of CDDO; PBS, phosphate-buffered saline; DMSO, dimethyl sulfoxide; M, mitochondrial membrane potential; NAO, nonyl acridine orange; GSX, glutathione; GSXm, mitochondrial glutathione; GSXwc, whole-cell glutathione; MTG, MitoTracker Green; TR-S, Texas Red-conjugated streptavidin; CHX, cycloheximide; PTP, permeability transition conductance pore; DTT, dithiothreitol; TEM, transmission electron microscopy; ER, endoplasmic reticulum. 0026-895X/06/6904-1182–1193$20.00 MOLECULAR PHARMACOLOGY Vol. 69, No. 4 Copyright © 2006 The American Society for Pharmacology and Experimental Therapeutics 18051/3098707 Mol Pharmacol 69:1182–1193, 2006 Printed in U.S.A. 1182 at ASPET Journals on March 11, 2017 molpharm.aspetjournals.org Downloaded from