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

Abstract

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 rho 0 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.

(+)α-Tocopheryl succinate inhibits the mitochondrial respiratory chain complex I and is as effective as arsenic trioxide or ATRA against acute promyelocytic leukemia in vivo

“…For example, adaphostin has been characterized as an inhibitor of MRC complex III through an assay which uses decylubiquinol (reduced form of DB) as the electron donor. 27 This may be the reason by which our results disagree with a previous study reporting lack of complex I inhibition by a-TOS. 13 In that case, the assay was performed with a hydrophilic electron carrier, phenazine monosulfate, which is not suitable for evaluating the specific effect of an inhibitor on quinone-binding sites because the activity measured does not depend on quinone concentration; in fact, the assay is suitable to determine succinate dehydrogenase activity in preparations devoid of complex II hydrophobic domains.…”

“…Interestingly, the dihydroquinone derivative adaphostin, which is chemically related to a-TOS and exhibits essentially the same biological properties, was shown to be toxic to leukemic cells but not to normal hematopoietic precursors. 27 In conclusion, we demonstrated an in vivo antileukemic action and a novel mitochondrial target for a-TOS, highlighting MRC complex I as a potential target for anticancer therapy.…”

α-Tocopheryl succinate induces apoptosis by targeting ubiquinone-binding sites in mitochondrial respiratory complex II

“…Our results are not dissimilar to a recent report documenting selective molecular mechanism of apoptosis induction in cancer cells by adaphostin, a compound that binds to the UbQ reduction (Q i ) site of CIII, resulting in ROS accumulation (Le et al, 2007). That CII is of importance in ROS formation is further evidenced by a recent report showing that non-steroid anti-inflammatory drugs caused ROS generation in cells, of which the majority was derived from CII (Soller et al, 2007).…”

“…Although anti-cancer drugs have been described that may target the UbQ sites on the oxidoreductase complexes along the mitochondrial respiratory chain to increase cellular ROS levels and activate apoptosis (Dias and Bailly, 2005;Hall, 2005;Le et al, 2007), a-TOS has an additional feature in that it also disrupts the anti-apoptotic function of Bcl-2 and Bcl-x L by blocking their BH3-binding domain (Shiau et al, 2006). Hence, a-TOS may prove superior for inducing cancer cell death because of its dual action on two important targets, Q P and Q D sites of CII, and the Bcl-2 family proteins, promoting the induction of mitochondrially mediated apoptosis.…”

Oxidative Stress by Targeted Agents Promotes Cytotoxicity in Hematologic Malignancies

“…Once in the cytosol, it forms a complex with dATP, an adaptor molecule called Apaf-1, and caspase-9. This conglomerate, called the apoptosome, functions to activate caspase-9, which then activates caspase-3 (26,58). Other proapoptotic molecules are known to be released from mitochondria to promote caspase activation, but of these, only the exit of cytochrome c from mitochondria has been linked to redox (43).…”