Search

The mitochondria-targeted drugs mitoquinone (Mito-Q) and mitovitamin E (MitoVit-E) are a new class of antioxidants containing the triphenylphosphonium cation moiety that facilitates drug accumulation in mitochondria. In this study, Mito-Q (ubiquinone attached to a triphenylphosphonium cation) and MitoVit-E (vitamin E attached to a triphenylphosphonium cation) were used. The aim of this study was to test the hypothesis that mitochondria-targeted antioxidants inhibit peroxide-induced oxidative stress and apoptosis in bovine aortic endothelial cells (BAEC) through enhanced scavenging of mitochondrial reactive oxygen species, thereby blocking reactive oxygen species-induced transferrin receptor (TfR)-mediated iron uptake into mitochondria. Glucose/glucose oxidase-induced oxidative stress in BAECs was monitored by oxidation of dichlorodihydrofluorescein that was catalyzed by both intracellular H2O2 and transferrin iron transported into cells. Pretreatment of BAECs with Mito-Q (1 μM) and MitoVit-E (1 μM) but not untargeted antioxidants (e.g. vitamin E) significantly abrogated H2O2- and lipid peroxide-induced 2′,7′-dichlorofluorescein fluorescence and protein oxidation. Mitochondria-targeted antioxidants inhibit cytochrome c release, caspase-3 activation, and DNA fragmentation. Mito-Q and MitoVit-E inhibited H2O2- and lipid peroxide-induced inactivation of complex I and aconitase, TfR overexpression, and mitochondrial uptake of 55Fe, while restoring the mitochondrial membrane potential and proteasomal activity. We conclude that Mito-Q or MitoVit-E supplementation of endothelial cells mitigates peroxide-mediated oxidant stress and maintains proteasomal function, resulting in the overall inhibition of TfR-dependent iron uptake and apoptosis. [ABSTRACT FROM AUTHOR]

In this study, we investigated the molecular mechanisms of toxicity of 1-methyl-4-phenylpyridinium (MPP+), an ultimate toxic metabolite of a mitochondrial neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, that causes Parkinson-like symptoms in experimental animals and humans. We used rat cerebellar granule neurons as a model cell system for investigating MPP+ toxicity. Results show that MPP+ treatment resulted in the generation of reactive oxygen species from inhibition of complex I of the mitochondrial respiratory chain, and inactivation of aconitase. This, in turn, stimulated transferrin receptor (TfR)-dependent iron signaling via activation of the iron-regulatory protein/iron-responsive element interaction. MPP+ caused a time-dependent depletion of tetrahydrobiopterin (BH4) that was mediated by H2O2 and trans-ferrin iron. Depletion of BH4 decreased the active, dimeric form of neuronal nitric-oxide synthase (nNOS). MPP+-mediated ’uncoupling’of nNOS decreased NO and increased superoxide formation. Pretreatment of cells with sepiapterin to promote BH4 biosynthesis or cell-permeable iron chelator and TfR antibody to prevent ironcatalyzed BH4 decomposition inhibited MPP+ cytotoxicity. Preincubation of cerebellar granule neurons with nNOS inhibitor exacerbated MPP+-induced iron uptake, BH4 depletion, proteasomal inactivation, and apoptosis. We conclude that MPP+-dependent aconitase inactivation, Tf-iron uptake, and oxidant generation result in the depletion of intracellular BH4, leading to the uncoupling of nNOS activity. This further exacerbates reactive oxygen species-mediated oxidative damage and apoptosis. Implications of these results in unraveling the molecular mechanisms of neurodegenerative diseases (Parkinson's and Alzheimer's disease) are discussed. [ABSTRACT FROM AUTHOR]

Doxorubicin (DOX), a widely used chemotherapeutic agent, exhibits cardiotoxicity as an adverse side effect in cancer patients. DOX-mediated cardiomyopathy is linked to its ability to induce apoptosis in endothelial cells and cardiomyocytes by activation of p53 protein and reactive oxygen species. We evaluated the potential roles of H2O2 and p53 in DOX-induced apoptosis in normal bovine aortic endothelial cells and adult rat cardiomyo. cytes and in tumor cell lines PA-1 (human ovarian teratocarcinoma) and MCF-7 (human breast adenocareinoma). Time course measurements indicated that activation of caspase-3 preceded the stimulation of p53 transcriptional activity in endothelial cells. In contrast, DOX caused early activation of p53 in tumor cells that was followed by caspase-3 activation and DNA fragmenta. tion. These findings suggest that the transcriptional activation of p53 in DOX-induced apoptosis in endothelial cells may not be as crucial as it is in tumor cells. Further evidence was obtained using a p53 inhibitor, pifithrin-α. Pifithrin-α completely suppressed DOX-induced activation of p53 in both normal and tumor cell lines and prevented apoptosis in tumor cell lines but not in endothelial cells and cardiomyocytes. In contrast, detoxification of H2O2, either by redox-active metalloporphyrin or overexpression of glutathione peroxidase, decreased DOX-induced apoptosis in endothelial cells and cardiomyocytes but not in tumor cells. This newly discovered mechanistic difference in DOX-induced apoptotic cell death in normal versus tumor cells will be useful in developing drugs that selectively mitigate the toxic side effects of DOX without affecting its antitumor action. [ABSTRACT FROM AUTHOR]

1-Methyl-4-phenylpyridinium (MPP+) is a neurotoxin that causes Parkinson's disease in experimental animals and humans. Despite the fact that intracellular iron was shown to be crucial for MPP+-induced apoptotic cell death, the molecular mechanisms for the iron requirement remain unclear. We investigated the role of transferrin receptor (TfR) and iron in modulating the expression of α-synuclein (α-syn) in MPP+-induced oxidative stress and apoptosis. Results show that MPP+ inhibits mitochondrial complex-1 and aconitase activities leading to enhanced H2O2 generation, TfR expression and α-syn expression/aggregation. Pretreatment with cellpermeable iron chelators, TfR antibody (that inhibits TfR-mediated iron uptake), or transfection with glutathione peroxidase (GPx1) enzyme inhibits intracellular oxidant generation, α-syn expression/aggregation, and apoptotic signaling as measured by caspase-3 activation. Cells overexpressing α-syn exacerbated MPP+ toxicity, whereas antisense α-syn treatment totally abrogated MPP+-induced apoptosis in neuroblastoma cells without affecting oxidant generation. The increased cytotoxic effects of α-syn in MPP+-treated cells were attributed to inhibition of mitogen-activated protein kinase and proteasomal function. We conclude that MPP+induced iron signaling is responsible for intracellular oxidant generation, α-syn expression, proteasomal dysfunction, and apoptosis. Relevance to Parkinson's disease is discussed. [ABSTRACT FROM AUTHOR]

Endothelial senescence in conjunction with mitochondrial dysfunction orchestrates age-associated cardiovascular disorders. In this study we investigated the causal link between these two processes and studied the molecular mechanisms by which metformin acts to coordinate the delay of endothelial senescence via enhancing mitochondrial biogenesis/function. AMPK activators metformin and AICAR delayed endothelial senescence via SIRT1-mediated upregulation of DOT1L, leading to increased trimethylation of H3K79 (H3K79me3). Treatment of cells with either siAMPK or siSIRT1 repressed DOT1L-mediated enhancement of H3K79me3. Moreover, the increase in SIRT3 expression and mitochondrial biogenesis/function by AMPK activators was H3K79me-dependent as H3K79N mutant or siDOT1L abrogated these effects. This was confirmed by the enrichment of H3K79me3 in the SIRT3 promoter with AMPK activation. Intriguingly, enhanced PGC-1α expression by SIRT3 via AMPK activation was responsible for increased hTERT expression and delayed endothelial senescence. In contrast, SIRT3 knockdown caused increased oxidative stress and premature senescence, possibly by depleting hTERT expression. Furthermore, a chronic low dose administration of metformin significantly attenuated vascular aging and inhibited age-associated atherosclerotic plaque formation in ApoE −/− mice. Overall, the results of this study show a novel regulation of mitochondrial biogenesis/function, and cellular senescence by H3K79me acting through SIRT3, thus providing a molecular basis for metformin-mediated age-delaying effects. [ABSTRACT FROM AUTHOR]