RESUMEN
6-Ketocholestanol (kCh) is known as a mitochondrial recoupler, i.e. it abolishes uncoupling of mitochondria by such potent agents as carbonyl cyanide m-chlorophenyl hydrazone (CCCP) and 3,5-di(tert-butyl)-4-hydroxybenzylidenemalononitril (SF6847) [Starkov et al., 1997]. Here, we report data on the kCh-induced inhibition of both NADH-oxidase and NADH-ubiquinone oxidoreductase activities of the respiratory complex I in bovine heart submitochondrial particles (SMP). Based on the absence of such inhibition with hexaammineruthenium (III) (HAR) as the complex I electron acceptor, the kCh effect could be associated with the ubiquinone-binding centre of this respiratory enzyme. In isolated rat liver mitochondria (RLM), kCh inhibited oxygen consumption with the glutamate/malate, substrates of NAD-linked dehydrogenases, while no inhibition of RLM respiration was observed with succinate, in agreement with the absence of the kCh effect on the succinate oxidase activity in SMP. Three kCh analogs (cholesterol, 6α-hydroxycholesterol, and 5α,6α-epoxycholesterol) exhibited no effect on the NADH oxidase activities in both SMP and RLM. Importantly, the kCh analogs were ineffective in the recoupling of RLM treated with CCCP or SF6847. Therefore, interaction of kCh with the complex I may be involved in the kCh-mediated mitochondrial recoupling.
Asunto(s)
Complejo I de Transporte de Electrón , Mitocondrias , Animales , Carbonil Cianuro m-Clorofenil Hidrazona/farmacología , Bovinos , Cetocolesteroles/farmacología , RatasRESUMEN
Mitochondria have emerged as important determinants in cancer progression and malignancy. However, the role of mitochondrial membranes in cancer onset and progression has not been thoroughly investigated. This study compares the structural and functional properties of mitochondrial membranes in prostate and colon cancer cells in comparison to normal mitochondria, and possible therapeutic implications of these membrane changes. Specifically, isolation of cell mitochondria and preparation of inverted sub-mitochondrial particles (SMPs) illuminated significant cancer-induced modulations of membrane lipid compositions, fluidity, and activity of cytochrome c oxidase, one of the key mitochondrial enzymes. The experimental data further show that cancer-associated membrane transformations may account for mitochondria targeting by betulinic acid and resveratrol, known anti-cancer molecules. Overall, this study probes the relationship between cancer and mitochondrial membrane transformations, underlying a potential therapeutic significance for mitochondrial membrane targeting in cancer.
Asunto(s)
Neoplasias del Colon , Lípidos de la Membrana/metabolismo , Mitocondrias , Membranas Mitocondriales , Proteínas Mitocondriales/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias de la Próstata , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Células HCT116 , Humanos , Masculino , Mitocondrias/metabolismo , Mitocondrias/patología , Membranas Mitocondriales/metabolismo , Membranas Mitocondriales/patología , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patologíaRESUMEN
The FDA has approved 31 small-molecule kinase inhibitors (KIs) for human use as of November 2016, with six having black box warnings for hepatotoxicity (BBW-H) in product labeling. The precise mechanisms and risk factors for KI-induced hepatotoxicity are poorly understood. Here, the 31 KIs were tested in isolated rat liver mitochondria, an in vitro system recently proposed to be a useful tool to predict drug-induced hepatotoxicity in humans. The KIs were incubated with mitochondria or submitochondrial particles at concentrations ranging from therapeutic maximal blood concentrations (Cmax) levels to 100-fold Cmax levels. Ten endpoints were measured, including oxygen consumption rate, inner membrane potential, cytochrome c release, swelling, reactive oxygen species, and individual respiratory chain complex (I-V) activities. Of the 31 KIs examined only three including sorafenib, regorafenib and pazopanib, all of which are hepatotoxic, caused significant mitochondrial toxicity at concentrations equal to the Cmax, indicating that mitochondrial toxicity likely contributes to the pathogenesis of hepatotoxicity associated with these KIs. At concentrations equal to 100-fold Cmax, 18 KIs were found to be toxic to mitochondria, and among six KIs with BBW-H, mitochondrial injury was induced by regorafenib, lapatinib, idelalisib, and pazopanib, but not ponatinib, or sunitinib. Mitochondrial liability at 100-fold Cmax had a positive predictive power (PPV) of 72% and negative predictive power (NPV) of 33% in predicting human KI hepatotoxicity as defined by product labeling, with the sensitivity and specificity being 62% and 44%, respectively. Similar predictive power was obtained using the criterion of Cmax ≥1.1 µM or daily dose ≥100 mg. Mitochondrial liability at 1-2.5-fold Cmax showed a 100% PPV and specificity, though the NPV and sensitivity were 32% and 14%, respectively. These data provide novel mechanistic insights into KI hepatotoxicity and indicate that mitochondrial toxicity at therapeutic levels can help identify hepatotoxic KIs.
Asunto(s)
Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Mitocondrias Hepáticas/efectos de los fármacos , Inhibidores de Proteínas Quinasas/toxicidad , Especies Reactivas de Oxígeno/metabolismo , Animales , Citocromos c/metabolismo , Relación Dosis-Respuesta a Droga , Etiquetado de Medicamentos , Femenino , Masculino , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mitocondrias Hepáticas/patología , Oxígeno/metabolismo , Valor Predictivo de las Pruebas , Inhibidores de Proteínas Quinasas/administración & dosificación , Ratas , Ratas Sprague-Dawley , Factores de Riesgo , Sensibilidad y Especificidad , Especificidad de la EspecieRESUMEN
The impact of complex II (succinate:ubiquinone oxidoreductase) on the mitochondrial production of reactive oxygen species (ROS) has been underestimated for a long time. However, recent studies with intact mitochondria revealed that complex II can be a significant source of ROS. Using submitochondrial particles from bovine heart mitochondria as a system that allows the precise setting of substrate concentrations we could show that mammalian complex II produces ROS at subsaturating succinate concentrations in the presence of Q-site inhibitors like atpenin A5 or when a further downstream block of the respiratory chain occurred. Upon inhibition of the ubiquinone reductase activity, complex II produced about 75% hydrogen peroxide and 25% superoxide. ROS generation was attenuated by all dicarboxylates that are known to bind competitively to the substrate binding site of complex II, suggesting that the oxygen radicals are mainly generated by the unoccupied flavin site. Importantly, the ROS production induced by the Q-site inhibitor atpenin A5 was largely unaffected by the redox state of the Q pool and the activity of other respiratory chain complexes. Hence, complex II has to be considered as an independent source of mitochondrial ROS in physiology and pathophysiology.
Asunto(s)
Ciclo del Ácido Cítrico/efectos de los fármacos , Ácidos Dicarboxílicos/metabolismo , Complejo II de Transporte de Electrones/metabolismo , Transporte de Electrón/efectos de los fármacos , Mitocondrias Cardíacas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Ácido Succínico/metabolismo , Animales , Sitios de Unión , Bovinos , Complejo I de Transporte de Electrón/antagonistas & inhibidores , Complejo I de Transporte de Electrón/metabolismo , Complejo II de Transporte de Electrones/efectos de los fármacos , Peróxido de Hidrógeno/metabolismo , Mitocondrias Cardíacas/efectos de los fármacos , Piridonas/farmacología , Superóxidos/metabolismo , Ubiquinona/metabolismoRESUMEN
Apart from complex I (NADH:ubiquinone oxidoreductase) the mitochondrial cytochrome bc1 complex (complex III; ubiquinol:cytochrome c oxidoreductase) has been identified as the main producer of superoxide and derived reactive oxygen species (ROS) within the mitochondrial respiratory chain. Mitochondrial ROS are generally linked to oxidative stress, aging and other pathophysiological settings like in neurodegenerative diseases. However, ROS produced at the ubiquinol oxidation center (center P, Qo site) of complex III seem to have additional physiological functions as signaling molecules during cellular processes like the adaptation to hypoxia. The molecular mechanism of superoxide production that is mechanistically linked to the electron bifurcation during ubiquinol oxidation is still a matter of debate. Some insight comes from extensive kinetic studies with mutated complexes from yeast and bacterial cytochrome bc1 complexes. This review is intended to bridge the gap between those mechanistic studies and investigations on complex III ROS in cellular signal transduction and highlights factors that impact superoxide generation. This article is part of a Special Issue entitled: Respiratory complex III and related bc complexes.