Abstract
Previously we have shown that both Rac1 and c-Jun NH(2)-terminal kinase (JNK1/2) are key proapoptotic molecules in tumor necrosis factor (TNF)-alpha/cycloheximide (CHX)-induced apoptosis in intestinal epithelial cells, whereas the role of reactive oxygen species (ROS) in apoptosis is unclear. The present studies tested the hypothesis that Rac1-mediated ROS production is involved in TNF-alpha-induced apoptosis. In this study, we showed that TNF-alpha/CHX-induced ROS production and hydrogen peroxide (H(2)O(2))-induced oxidative stress increased apoptosis. Inhibition of Rac1 by a specific inhibitor NSC23766 prevented TNF-alpha-induced ROS production. The antioxidant, N-acetylcysteine (NAC), or rotenone (Rot), the mitochondrial electron transport chain inhibitor, attenuated mitochondrial ROS production and apoptosis. Rot also prevented JNK1/2 activation during apoptosis. Inhibition of Rac1 by expression of dominant negative Rac1 decreased TNF-alpha-induced mitochondrial ROS production. Moreover, TNF-alpha-induced cytosolic ROS production was inhibited by Rac1 inhibition, diphenyleneiodonium (DPI, an inhibitor of NADPH oxidase), and NAC. In addition, DPI inhibited TNF-alpha-induced apoptosis as judged by morphological changes, DNA fragmentation, and JNK1/2 activation. Mitochondrial membrane potential change is Rac1 or cytosolic ROS dependent. Lastly, all ROS inhibitors inhibited caspase-3 activity. Thus these results indicate that TNF-alpha-induced apoptosis requires Rac1-dependent ROS production in intestinal epithelial cells.
Publication types
-
Research Support, N.I.H., Extramural
-
Research Support, Non-U.S. Gov't
MeSH terms
-
Acetylcysteine / pharmacology
-
Aminoquinolines / pharmacology
-
Animals
-
Antioxidants / pharmacology
-
Apoptosis / drug effects*
-
Caspase 3 / metabolism
-
Cell Line
-
Cycloheximide / pharmacology*
-
Cytosol / metabolism
-
Dose-Response Relationship, Drug
-
Enzyme Activation
-
Enzyme Inhibitors / pharmacology
-
Epithelial Cells / drug effects*
-
Epithelial Cells / enzymology
-
Epithelial Cells / metabolism
-
Epithelial Cells / pathology
-
Hydrogen Peroxide / pharmacology
-
Intestinal Mucosa / drug effects*
-
Intestinal Mucosa / enzymology
-
Intestinal Mucosa / metabolism
-
Intestinal Mucosa / pathology
-
Membrane Potential, Mitochondrial / drug effects
-
Mitochondria / drug effects
-
Mitochondria / metabolism
-
Mitogen-Activated Protein Kinase 8 / metabolism
-
Mitogen-Activated Protein Kinase 9 / metabolism
-
NADPH Oxidases / antagonists & inhibitors
-
NADPH Oxidases / metabolism
-
Onium Compounds / pharmacology
-
Oxidants / pharmacology
-
Oxidative Stress / drug effects*
-
Pyrimidines / pharmacology
-
Rats
-
Reactive Oxygen Species / metabolism*
-
Rotenone / pharmacology
-
Signal Transduction / drug effects
-
Tumor Necrosis Factor-alpha / metabolism*
-
Uncoupling Agents / pharmacology
-
rac1 GTP-Binding Protein / antagonists & inhibitors
-
rac1 GTP-Binding Protein / genetics
-
rac1 GTP-Binding Protein / metabolism*
Substances
-
Aminoquinolines
-
Antioxidants
-
Enzyme Inhibitors
-
NSC 23766
-
Onium Compounds
-
Oxidants
-
Pyrimidines
-
Reactive Oxygen Species
-
Tumor Necrosis Factor-alpha
-
Uncoupling Agents
-
Rotenone
-
diphenyleneiodonium
-
Cycloheximide
-
Hydrogen Peroxide
-
NADPH Oxidases
-
Mitogen-Activated Protein Kinase 9
-
Mitogen-Activated Protein Kinase 8
-
Caspase 3
-
Rac1 protein, rat
-
rac1 GTP-Binding Protein
-
Acetylcysteine