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

Abstract

Methyl-2-cyano-3,12 dioxoolean-1,9 diene-28-oate (CDDOMe) 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. In this study, we demonstrate 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 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 (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 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 (PT) of isolated mitochondria that was sensitive to sulfhydryl antioxidants but not to EDTA. PT induced by CDDO-Me and Ca 2ϩ 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 before the release of cytochrome c. Taken together, our results indicate 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-inflammatory 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), induce apoptosis and differentiation in leukemia cells in culture, and we have demonstrated that apoptosis induced by these agents is initiated by the intrinsic apoptotic pathway possibly through direct perturbation of mitochondrial homeostasis (Konopleva et al., 2002(Konopleva et al., , 2004. As an anti-inflammatory agent, CDDO has been shown to potently prevent

Platelets Promote Mitochondrial Uncoupling and Resistance to Apoptosis in Leukemia Cells: A Novel Paradigm for the Bone Marrow Microenvironment

“…Moreover, as evidenced in Fig. 3a and c, in MOLT4 cells 2.5 % PL also promoted significant (p<0.0005) resistance to apoptosis and mitochondrial damage induced by CDDO-Me -a synthetic triterpenoid that directly permeabilizes the inner mitochondrial membrane to induce swelling and rupture of the outer membrane with subsequent release of apoptogenic factors [26] -supporting the hypothesis that platelet derived factors also antagonize mitochondrial permeability transition-induced apoptosis. Comparable antiapoptotic and mitoprotective effects were observed in OCI-AML3 cells exposed to increasing doses of CDDO-Me in combination with 2.5 % PL ( Fig.…”

“…**= p<0.0005 from untreated RPMI without platelet components; #= p<0.0005 from CDDO-Me treated RPMI without platelet components. *= p<0.05 from RPMI only control mitochondrial membrane, inducing matrix swelling and rupture of the outer mitochondrial membrane [26]. Mechanistically, we demonstrate that platelets antagonize Bax/Bak oligomerization, although it is clear that this may not be the only mechanism since the expression of these proteins is not required for CDDO-Me citotoxicity [26].…”

“…*= p<0.05 from RPMI only control mitochondrial membrane, inducing matrix swelling and rupture of the outer mitochondrial membrane [26]. Mechanistically, we demonstrate that platelets antagonize Bax/Bak oligomerization, although it is clear that this may not be the only mechanism since the expression of these proteins is not required for CDDO-Me citotoxicity [26]. Moreover, our results evidence a greater antiapoptotic effect of platelet contents against CDDO-Me than ABT-737, perhaps indicating a direct protective effect over the inner mitochondrial membrane that may in turn antagonize Bax/Bak oligomerization on the outer mitochondrial membrane.…”

Inhibition of mitochondrial metabolism by methyl-2-cyano-3,12-dioxooleana-1,9-diene-28-oate induces apoptotic or autophagic cell death in chronic myeloid leukemia cells

“…CDDO and CDDO-Me reportedly disrupted intracellular redox balance in U937 cells and multiple myeloma cells, thereby activating the intrinsic apoptotic pathway (11,15), and CDDO-Me exhibited some selectivity in apoptosis induction between tumor and normal cells (19). Interestingly, recent evidence from our group indicates that CDDO induced the release of cytochrome c from isolated mitochondria via a cyclosporine A -independent permeability transition, suggesting that this organelle may be a direct target of this agent (14,20).…”

CDDO induces granulocytic differentiation of myeloid leukemic blasts through translational up-regulation of p42 CCAAT enhancer–binding protein alpha

“…CDDO induces apoptosis through both caspase-independent and -dependent mechanisms, the latter involving caspase-8 activation, Bid cleavage, cytochrome c release, and caspase-3 activation. [3][4][5][6] Furthermore, JNK, p38, and ERK pathways are involved in CDDO-induced apoptosis of tumor cell lines [7][8][9] mediated by disrupted intracellular redox balance and involving decreased glutathione and increased reactive oxygen species [9][10][11][12] CDDO-induced growth arrest of breast cancer cell lines correlates with transactivated PPARgamma and leads to up-regulation of p21 cip1waf1 , GADD153, CCAAT enhancer-binding proteins (CEBPs), and proteasome-regulatory factors, and to down-regulation of cyclin D1, PCNA, and IRS1. 13 CDDO and CDDO-Im activate the TGF␤ pathway through activation of Smad2/3, 14,15 which is required for the repression of inflammatory molecules by CDDO.…”