Differential activation of nuclear transcription factor kappaB, gene expression, and proteins by amifostine's free thiol in human microvascular endothelial and glioma cells.
The effects of WR1065 (SH), the free thiol form of amifostine, on nuclear transcription factor kappaB (NFkappaB) activation, manganese superoxide dismutase (MnSOD) gene expression, and secretion of human vascular endothelial cell growth factor (hVEGF), basic fibroblast growth factor (bFGF), tumor necrosis factor-alpha (TNF-alpha), vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), E-selectin, P-selectin, and interleukins IL-1alpha, IL-6, and IL-8 were investigated and compared in human microvascular endothelial (HMEC) and human glioma cells. WR1065 was evaluated at 2 concentrations, 4 mmol/L, ie, its most effective cytoprotective dose, and 40 micromol/L, a noncytoprotective but highly effective dose capable of preventing radiation and chemotherapeutic drug-induced mutations in exposed cells. A 30-minute exposure of HMEC and glioma cell lines U87 and U251 to WR1065 at either of the concentrations resulted in a marked activation of NFkappaB as determined by a gel shift assay, with the maximum effect observed between 30 minutes and 1 hour after treatment. Using a supershift assay, WR1065 exposure was observed to affect only the p50-p65 heterodimer, and not the homodimers or heterodimers containing p52 or c-Rel subunits of NFkappaB. WR1065 was also found to enhance MnSOD gene expression in both HMEC and glioma cells. Gene expression was enhanced 1.8-fold over control levels in HMEC over a period ranging from 12 to 24 hours after the time of maximum activation of NFkappaB. In contrast, MnSOD gene expression in U87 cells rose 3.5 times above control levels over this same period. WR1065 had no effect on the levels of adhesion molecules, cytokines, and growth factors secreted by cells exposed for up to 24 hours as measured by enzyme-linked immunosorbent assay.
Grdina
David J
DJ
Department of Radiation and Cellular Oncology, University of Chicago, Chicago, IL 60637, USA.
Murley
Jeffrey S
JS
Kataoka
Yasushi
Y
Calvin
Douglas P
DP
D004999:Amifostine / Q000494:pharmacology*; D015815:Cell Adhesion Molecules / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000378:metabolism* / Q000528:radiation effects; D015870:Gene Expression / Q000187:drug effects*; D005910:Glioma / Q000378:metabolism* / Q000532:radiotherapy; D006133:Growth Substances / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008624:Mercaptoethylamines / Q000494:pharmacology*; D015846:Monokines / Q000235:genetics / Q000378:metabolism; D016328:NF-kappa B / Q000235:genetics* / Q000378:metabolism; D011829:Radiation Dosage; D011837:Radiation-Protective Agents / Q000494:pharmacology*; D013482:Superoxide Dismutase / Q000235:genetics / Q000378:metabolism; D015533:Transcriptional Activation / Q000187:drug effects*; D014407:Tumor Cells, Cultured / Q000528:radiation effects
D015815:Cell Adhesion Molecules; D006133:Growth Substances; D008624:Mercaptoethylamines; D015846:Monokines; D016328:NF-kappa B; D011837:Radiation-Protective Agents; C020174:N-(2-mercaptoethyl)-1,3-diaminopropane; D013482:Superoxide Dismutase; D004999:Amifostine