TY - JOUR
T1 - Activation of NFκB and MnSOD gene expression by free radical scavengers in human microvascular endothelial cells
AU - Murley, Jeffrey S.
AU - Kataoka, Yasushi
AU - Hallahan, Dennis E.
AU - Roberts, Jeanette C.
AU - Grdina, David J.
N1 - Funding Information:
The authors wish to acknowledge the helpful discussions with Dr. David Rubin in the preparation of this manuscript, along with the technical assistance of Ms. Britta H. Wilmore at The Huntsman Cancer Institute, Utah Regional Cancer Center. This work was supported by NIH/NCI RO1 grant CA37435 to D.J. Grdina.
PY - 2001/6/15
Y1 - 2001/6/15
N2 - The effect of nonprotein thiol (NPT) free radical scavengers WR-1065 (SH) and WR-33278 (SS), the active thiol and disulfide metabolites of amifostine, N-acetylcysteine (NAC; both L- and D- isomers), mesna, captopril, and dithiothreitol (DTT) on NFκB activation in human microvascular endothelial cells (HMEC) was investigated and contrasted to TNFα. The use of each of these NPTs at millimolar concentrations independent of oxidative damage-inducing agents resulted in a marked activation of NFκB, with the maximum effect observed between 30 min and 1 h after treatment. Only the SH and SS forms of amifostine, however, were effective in activating NFκB when administered at micromolar levels. Using a supershift assay, SH and SS equally affected the p50-p65 heterodimer, but not homodimers or heterodimers containing p52 or c-Rel subunits of NFκB. Neither catalase nor pyruvate when added to the culture medium to minimize hydrogen peroxide production had an effect on NFκB activation by SH. Thus, while oxidative damage is known to activate NFκB, the intracellular redox environment may also be affected by the addition of free radical scavenging agents such as NPT, and these in turn are capable of activating the redox sensitive transcription factor NFκB. There does not appear to be a significant role, if any, for the production of H2O2 as an intermediate step in the activation of NFκB by either the SH or the SS form of amifostine. Rather, the underlying mechanism of action, especially for the SS form, may be related to the close structural and functional similarities of these agents to polyamines, which have been reported to be capable of activating NFκB. In contrast to TNFα, exposure of cells to either 40 μM or 4 mM of SH for 30 min did not induce intercellular adhesion molecule-1 (ICAM-1) gene expression, but did increase manganese superoxide dismutase (MnSOD) gene expression. MnSOD expression rose by 2-fold and remained elevated from 4 to 22 h following SH exposure.
AB - The effect of nonprotein thiol (NPT) free radical scavengers WR-1065 (SH) and WR-33278 (SS), the active thiol and disulfide metabolites of amifostine, N-acetylcysteine (NAC; both L- and D- isomers), mesna, captopril, and dithiothreitol (DTT) on NFκB activation in human microvascular endothelial cells (HMEC) was investigated and contrasted to TNFα. The use of each of these NPTs at millimolar concentrations independent of oxidative damage-inducing agents resulted in a marked activation of NFκB, with the maximum effect observed between 30 min and 1 h after treatment. Only the SH and SS forms of amifostine, however, were effective in activating NFκB when administered at micromolar levels. Using a supershift assay, SH and SS equally affected the p50-p65 heterodimer, but not homodimers or heterodimers containing p52 or c-Rel subunits of NFκB. Neither catalase nor pyruvate when added to the culture medium to minimize hydrogen peroxide production had an effect on NFκB activation by SH. Thus, while oxidative damage is known to activate NFκB, the intracellular redox environment may also be affected by the addition of free radical scavenging agents such as NPT, and these in turn are capable of activating the redox sensitive transcription factor NFκB. There does not appear to be a significant role, if any, for the production of H2O2 as an intermediate step in the activation of NFκB by either the SH or the SS form of amifostine. Rather, the underlying mechanism of action, especially for the SS form, may be related to the close structural and functional similarities of these agents to polyamines, which have been reported to be capable of activating NFκB. In contrast to TNFα, exposure of cells to either 40 μM or 4 mM of SH for 30 min did not induce intercellular adhesion molecule-1 (ICAM-1) gene expression, but did increase manganese superoxide dismutase (MnSOD) gene expression. MnSOD expression rose by 2-fold and remained elevated from 4 to 22 h following SH exposure.
KW - Free radicals
KW - Gene expression
KW - Intercellular adhesion molecule-1
KW - Manganese superoxide dismutase
KW - Nonprotein thiols
KW - Nuclear transcription factor κB
UR - http://www.scopus.com/inward/record.url?scp=0035874637&partnerID=8YFLogxK
U2 - 10.1016/S0891-5849(01)00554-8
DO - 10.1016/S0891-5849(01)00554-8
M3 - Article
C2 - 11390188
AN - SCOPUS:0035874637
SN - 0891-5849
VL - 30
SP - 1426
EP - 1439
JO - Free Radical Biology and Medicine
JF - Free Radical Biology and Medicine
IS - 12
ER -