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Drugs containing the quinone group were tested on hyperproliferative leukemia T cells (HLTC: Jhp and Jws) and parental Jurkat cells. Doxorubicin, menadione and adaphostin produced different effects on these cell lines. Rapid doxorubicin-induced cell death in Jurkat cells was mediated by caspase activation. Doxorubicin-induced cell death of HLTCs was delayed due to the absence of caspase-3 and -8 expression. Delayed HLTC cell death was mediated and triggered by the generation of reactive oxygen species (ROS). Other drugs containing quinone groups, such as menadione and adaphostin, were also tested on HLTC and both were toxic by a caspase-independent mechanism. The toxicity of these drugs correlated with the generation of the superoxide anion, which increased and was more effective in HLTCs than in parental Jurkat cells. Accordingly, SOD1 activity was much lower in HLTCs than in Jurkat cells. This lower SOD1 activity in HLTCs was associated not only with the absence of the wild-type (16 kDa) SOD1 monomer but also with the presence of a shortened (14 kDa) SOD1 monomer isoform. Moreover, the cytotoxicity of drugs containing the quinone group was prevented by incubation with manganese(III) tetrakis (4-benzoic acid) porphyrin (MnTBAP), a cell-permeable superoxide dismutase mimetic and a potent inhibitor of oxidation. These findings could explain the sensitivity of HLTCs to drugs containing the quinone group using a mechanism dependent on oxidative stress. These observations can also be useful to target hyperproliferative leukemias that are resistant to the classical caspase-dependent apoptotic pathway., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.) more...

Melatonin and steroid hormones are cytochrome P450 (CYP or P450; EC 1.14.14.1) substrates that have antioxidant properties and mitochondrial protective activities. The mitochondrial intermembrane space (IMS) Cu,Zn-superoxide dismutase (SOD1) is activated after oxidative modification of its critical thiol moieties by superoxide anion (O₂(•-)). This study was aimed at investigating the potential association between the hormonal protective antioxidant actions in mitochondria and the regulation of IMS SOD1 activity. Melatonin, testosterone, dihydrotestosterone, estradiol, and vitamin D induced a sustained activation over time of SOD1 in intact mitochondria, showing a bell-shaped enzyme activation dose response with a threshold at 50nM and a maximum effect at 1μM concentration. Enzyme activation was not affected by furafylline, but it was inhibited by omeprazole, ketoconazole, and tiron, thereby supporting the occurrence of a mitochondrial P450 activity and O₂(•-) requirements. Mitochondrial P450-dependent activation of IMS SOD1 prevented O₂(•-)-induced loss of aconitase activity in intact mitochondria respiring in State 3. Optimal protection of aconitase activity was observed at 0.1μM P450 substrate concentration, evidencing a likely oxidative effect on the mitochondrial matrix by higher substrate concentrations. Likewise, enzyme activation mediated by mitochondrial P450 activity delayed CaCl₂-induced loss of transmembrane potential and decreased cytochrome c release. Omeprazole and ketoconazole abrogated both protecting mitochondrial functions promoted by melatonin and steroid hormones., (Copyright © 2011 Elsevier Inc. All rights reserved.) more...

IMS (intermembrane space) SOD1 (Cu/Zn-superoxide dismutase) is inactive in isolated intact rat liver mitochondria and is activated following oxidative modification of its critical thiol groups. The present study aimed to identify biochemical pathways implicated in the regulation of IMS SOD1 activity and to assess the impact of its functional state on key mitochondrial events. Exogenous H2O2 (5 microM) activated SOD1 in intact mitochondria. However, neither H2O2 alone nor H2O2 in the presence of mitochondrial peroxiredoxin III activated SOD1, which was purified from mitochondria and subsequently reduced by dithiothreitol to an inactive state. The reduced enzyme was activated following incubation with the superoxide generating system, xanthine and xanthine oxidase. In intact mitochondria, the extent and duration of SOD1 activation was inversely correlated with mitochondrial superoxide production. The presence of TxrR-1 (thioredoxin reductase-1) was demonstrated in the mitochondrial IMS by Western blotting. Inhibitors of TxrR-1, CDNB (1-chloro-2,4-dinitrobenzene) or auranofin, prolonged the duration of H2O2-induced SOD1 activity in intact mitochondria. TxrR-1 inactivated SOD1 purified from mitochondria in an active oxidized state. Activation of IMS SOD1 by exogenous H2O2 delayed CaCl2-induced loss of transmembrane potential, decreased cytochrome c release and markedly prevented superoxide-induced loss of aconitase activity in intact mitochondria respiring at state-3. These findings suggest that H2O2, superoxide and TxrR-1 regulate IMS SOD1 activity reversibly, and that the active enzyme is implicated in protecting vital mitochondrial functions. more...

The localization of Cu,Zn-superoxide dismutase in the mitochondrial intermembrane space suggests a functional relationship with superoxide anion (O2*-) released into this compartment. The present study was aimed at examining the functionality of Cu,Zn-superoxide dismutase and elucidating the molecular basis for its activation in the intermembrane space. Intact rat liver mitochondria neither scavenged nor dismutated externally generated O2*-, unless the mitochondrial outer membrane was disrupted selectively by digitonin. The activation of the intermembrane space Cu,Zn-superoxide dismutase following the disruption of mitochondrial outer membrane was largely inhibited by bacitracin, an inhibitor of protein disulphide-isomerase. Thiol alkylating agents, such as N-methylmaleimide or iodoacetamide, decreased intermembrane space Cu,Zn-superoxide dismutase activation during, but not after, disruption of the outer membrane. This inhibitory effect was overcome by exposing mitochondria to low micromolar concentrations of H2O2 before disruption of the outer membrane in the presence of the alkylating agents. Moreover, H2O2 treatment alone enabled intact mitochondria to scavenge externally generated O2*-. These findings suggest that intermembrane space Cu,Zn-superoxide dismutase is inactive in intact mitochondria and that an oxidative modification of its critical thiol groups is necessary for its activation. more...