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Title
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Mitochondria and NADPH oxidases are the major sources of TNF-α/cycloheximide-induced oxidative stress in murine intestinal epithelial MODE-K cells
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Author
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Abstract
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| TNF-alpha/cycloheximide (CHX)-induced apoptosis of the mouse intestinal epithelial cell line MODE-K corresponds with the production of reactive oxygen species (ROS). The aim of the study is to investigate the sources of ROS production contributing to apoptotic cell death during TNF-alpha/CHX-induced oxidative stress in MODE-K cells. Total ROS or mitochondrial superoxide anion production was measured simultaneously with cell death in the absence or presence of pharmacological inhibitors of various ROS-producing systems, and of ROS scavengers/antioxidants. The influence of TNF-alpha/CHX on mitochondrial membrane potential (Psi(m)) and cellular oxygen consumption was also studied. TNF-alpha/CHX time-dependently increased intracellular total ROS and mitochondrial superoxide anion production in MODE-K cells, starting from 2 h. Inhibition of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) by a pan-NOX inhibitor (VAS-2870) and a specific inhibitor of Rac1 (NSC23766) significantly reduced TNF-alpha/CHX-induced total ROS and cell death levels. The mitochondrial electron transport chain inhibitors, amytal (I-Q site of complex I) and TTFA (Qp site of complex II) showed a pronounced decrease in TNF-alpha/CHX-induced total ROS, mitochondrial superoxide anion and cell death levels. TNF-alpha/CHX treatment caused an immediate decrease in mitochondrial respiration, and a loss of Psi(m) and increase in mitochondrial dysfunction from 1 h on. The results suggest that mitochondria and NOX are the two major sources of ROS overproduction during TNF-alpha/CHX-induced cell death in MODE-K cells, with superoxide anions being the major ROS species. Particularly, the quinone-binding sites of mitochondrial complex I (site I-Q) and complex II (site Qp) seem to be the major sites of mitochondrial ROS production. (C) 2015 Elsevier Inc. All rights reserved. |
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Language
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English
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Source (journal)
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Cellular signalling. - Oxford
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Publication
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Oxford
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2015
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ISSN
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0898-6568
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Volume/pages
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27
:6
(2015)
, p. 1141-1158
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ISI
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000353096700011
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Full text (Publisher's DOI)
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