ABSTRACT
Hydrogen peroxide is widely viewed as the main second messenger in redox signaling, and it has been proposed that deactivation of the antioxidant peroxiredoxin (Prdx) enzymes allows free peroxide to accumulate and directly oxidize target proteins (the floodgate model). We assessed the role of cytosolic Prdxs 1 and 2 in peroxide-induced activation of the apoptosis signaling kinase 1 (ASK1)/p38 signaling pathway, in which oxidation of ASK1 is required for phosphorylation of p38. In response to peroxide, Prdx1 catalyzed oxidation of ASK1 to a disulfide-linked multimer, and this occurred via transient formation of a Prdx1-ASK1 mixed disulfide intermediate. Oxidation of ASK1 and phosphorylation of p38 were inhibited by knockdown of Prdx1, but also by overexpression of Prdx2. This suggests that these two cytosolic Prdxs have distinct roles in the cellular peroxide response and compete for available peroxide substrate. These data imply that Prdx1 can function as a peroxide receptor in response to extracellular H(2)O(2), receiving the peroxide signal and transducing it into a disulfide bond that is subsequently transmitted to the substrate, ASK1, resulting in p38 phosphorylation. Interestingly, in response to peroxide, Prdx1 and Prdx3 transiently formed reducible higher molecular weight complexes, suggesting that multiple proteins are targets for Prdx-mediated oxidation via a disulfide-exchange mechanism. This model of active peroxide signal distribution via disulfide exchange is consistent with Prdx function in yeast and explains how peroxides may trigger specific disulfide bond formation in mammalian cells.
Subject(s)
Macrophages/metabolism , Peroxiredoxins/metabolism , Signal Transduction , Cell Line , Disulfides/chemistry , Disulfides/metabolism , Gene Expression Regulation , Gene Knockdown Techniques , Humans , Hydrogen Peroxide/metabolism , Hydrogen Peroxide/pharmacology , MAP Kinase Kinase Kinase 5/genetics , MAP Kinase Kinase Kinase 5/metabolism , Macrophages/cytology , Macrophages/drug effects , Oxidation-Reduction , Peroxiredoxin III/genetics , Peroxiredoxin III/metabolism , Peroxiredoxins/genetics , Phosphorylation , p38 Mitogen-Activated Protein Kinases/genetics , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
The anticancer effects elicited by epigallocatechin gallate (EGCG) are well established in various models of cancer, while raloxifene is as an established selective estrogen receptor modulator (SERM), which is not yet clinically utilized for the treatment of breast cancer. Previous study from this laboratory has demonstrated that the combination of EGCG (25 microM) and raloxifene (4 microM) elicits a strong cytotoxic response in MDA-MB-231 human breast cancer cells, which lack the estrogen receptor (ER) and erbB-2/ Her-2 receptor. This study was therefore designed to probe the mechanism underlying this cytotoxic response, with an emphasis on determining how the combination treatment influenced the total expression and phosphorylation of key signaling proteins. Specifically, following 12 and 18 h of the combination treatment, we observed significant decreases in the phosphorylation of the epidermal growth factor receptor (EGFR), AKT, mammalian target of rapamycin (mTOR) and S-6-kinase (S6K), and significant increases in the phosphorylation of stress activated protein kinases (SAPKs). Furthermore, these changes were associated with a reduction in the nuclear localization of p65, a major subunit of NF-kappaB. These results demonstrate that the combination of EGCG and raloxifene effectively reduced the mitogenic and survival signaling in MDA-MB-231 cells. Thus, this combination warrants further experimentation as a potential treatment for ER-negative breast cancer.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/pharmacology , Breast Neoplasms/metabolism , Signal Transduction/drug effects , Breast Neoplasms/pathology , Catechin/analogs & derivatives , Catechin/pharmacology , Cell Line, Tumor , ErbB Receptors/metabolism , Estrogen Receptor alpha/deficiency , Female , Humans , Mitogen-Activated Protein Kinases/metabolism , Phosphorylation , Proto-Oncogene Proteins c-akt/metabolism , Raloxifene Hydrochloride/pharmacology , Receptor, ErbB-2/deficiency , Ribosomal Protein S6 Kinases/metabolism , Selective Estrogen Receptor Modulators/pharmacology , TOR Serine-Threonine Kinases/metabolism , Time Factors , Transcription Factor RelA/metabolismABSTRACT
Mitochondrial production of reactive oxygen species (ROS) is widely reported as a central effector during TNF-induced necrosis. The effect of a family of mitochondria-targeted antioxidants on TNF-induced necrosis of L929 cells was studied. While the commonly used lipid-soluble antioxidant BHA effectively protected cells from TNF-induced necrosis, the mitochondria-targeted antioxidants MitoQ(3), MitoQ(5), MitoQ(10) and MitoPBN had no effect on TNF-induced necrosis. Since BHA also acts as an uncoupler of mitochondrial membrane potential, two additional uncouplers were tested. FCCP and CCCP both provided dose-dependent inhibition of TNF-induced necrosis. In conclusion, the generation of mitochondrial ROS may not be necessary for TNF-induced necrosis. Instead, these results suggest alternative mitochondrial functions, such as a respiration-dependent process, are critical for necrotic death.
