Mitochondria and NADPH oxidases are the major sources of TNF-α/cycloheximide-induced oxidative stress in murine intestinal epithelial MODE-K cells.
TNF-α/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-α/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-α/CHX on mitochondrial membrane potential (Ψ(m)) and cellular oxygen consumption was also studied. TNF-α/CHX time-dependently increased intracellular total ROS and mitochondrial superoxide anion production in MODE-K cells, starting from 2h. 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-α/CHX-induced total ROS and cell death levels. The mitochondrial electron transport chain inhibitors, amytal (IQ site of complex I) and TTFA (Qp site of complex II) showed a pronounced decrease in TNF-α/CHX-induced total ROS, mitochondrial superoxide anion and cell death levels. TNF-α/CHX treatment caused an immediate decrease in mitochondrial respiration, and a loss of Ψ(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-α/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.
Heymans Institute of Pharmacology, Faculty of Medicine and Health Sciences, Ghent University, Belgium. Electronic address: dinesh.babu@ugent.be.
Dinesh
D
Babu
Department of Clinical Chemistry, Microbiology and Immunology, Faculty of Medicine and Health Sciences, Ghent University, Belgium.
Georges
G
Leclercq
Inflammation Research Center, Molecular Signaling and Cell Death Unit, VIB, Ghent, Belgium; Department of Biomedical Molecular Biology, Molecular Signaling and Cell Death Unit, Ghent University, Ghent, Belgium.
Vera
V
Goossens
Inflammation Research Center, Molecular Signaling and Cell Death Unit, VIB, Ghent, Belgium; Department of Biomedical Molecular Biology, Molecular Signaling and Cell Death Unit, Ghent University, Ghent, Belgium.
Tom
T
Vanden Berghe
Inflammation Research Center, Molecular Signaling and Cell Death Unit, VIB, Ghent, Belgium; Department of Biomedical Molecular Biology, Molecular Signaling and Cell Death Unit, Ghent University, Ghent, Belgium.
Evelien
E
Van Hamme
Inflammation Research Center, Molecular Signaling and Cell Death Unit, VIB, Ghent, Belgium; Department of Biomedical Molecular Biology, Molecular Signaling and Cell Death Unit, Ghent University, Ghent, Belgium.
Peter
P
Vandenabeele
Heymans Institute of Pharmacology, Faculty of Medicine and Health Sciences, Ghent University, Belgium.
Romain A
RA
Lefebvre
D000096:Acetone / Q000031:analogs & derivatives / Q000494:pharmacology; D000654:Amobarbital / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D002460:Cell Line; D003513:Cycloheximide / Q000494:pharmacology*; D042967:Electron Transport Complex I / Q000037:antagonists & inhibitors / Q000378:metabolism; D042963:Electron Transport Complex II / Q000037:antagonists & inhibitors / Q000378:metabolism; D004847:Epithelial Cells / Q000378:metabolism; D007422:Intestines / Q000166:cytology; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D051379:Mice; D008928:Mitochondria / Q000378:metabolism*; D019255:NADPH Oxidases / Q000378:metabolism*; D018384:Oxidative Stress / Q000187:drug effects*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology; D013481:Superoxides / Q000378:metabolism; D013876:Thiophenes / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*
D011994:Recombinant Proteins; D013876:Thiophenes; D014409:Tumor Necrosis Factor-alpha; C429269:theonyltrifluoroacetone; D013481:Superoxides; D000096:Acetone; D003513:Cycloheximide; D042963:Electron Transport Complex II; D019255:NADPH Oxidases; D042967:Electron Transport Complex I; D000654:Amobarbital
Apoptosis; Intestinal epithelial cells; Mitochondria; ROS production; TNF; α/CHX