A Novel Mechanism of Action of Methyl-2-cyano-3,12 dioxoolean-1,9 diene-28-oate (CDDO-Me): Direct Permeabilization of the Inner Mitochondrial Membrane To Inhibit Electron Transport and Induce Apoptosis.

CDDO-Me is a synthetic oleanolic acid derivative that displays antitumorigenic and anti-inflammatory activities, and we have previously reported that this agent potently activates the intrinsic apoptotic pathway in leukemia cells. Here we demonstrate that mitochondrial dysfunction induced by CDDO-Me in leukemia cells is mediated by direct permeabilization of the inner mitochondrial membrane that results in the rapid depletion of mitochondrial glutathione (GSXm), loss of cardiolipin, and inhibition of mitochondrial respiration. More importantly, we demonstrate that in addition to activating the intrinsic apoptotic pathway, the mitochondrial effects of CDDO-Me may mediate its anti-inflammatory activity by modulating the generation of superoxide anion (O₂⁻). Interestingly, CDDO-Me did not increase the generation of O₂⁻, and in fact pretreatment of leukemia cells with CDDO-Me prevented the increase of this reactive oxygen species 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 (mPT) of isolated mitochondria that was sensitive to sulfhydryl antioxidants but not to EDTA. PT induced by CDDO-Me and Ca²⁺ 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 mitochondrial swelling and loss of inner membrane structure prior to the release of cytochrome c. Taken together, our results indicate that CDDO-Me is a novel antileukemic agent that induces apoptosis and inhibits mitochondrial electron transport via perturbations in inner mitochondrial membrane integrity.