Nepalolide A inhibits the expression of inducible nitric oxide synthase by modulating the degradation of IkappaB-alpha and IkappaB-beta in C6 glioma cells and rat primary astrocytes.
1 The effects of nepalolide A on the expression of inducible nitric oxide synthase (iNOS) caused by incubation with lipopolysaccharide/interferon-gamma (LPS/IFN-gamma) or tumour necrosis factor-alpha/interleukin-1beta/IFN-gamma (TNF-alpha/IL-1beta/IFN-gamma, mixed cytokines) in C6 glioma cells and primary astrocytes of rat were investigated. The mechanisms by which nepalolide A confers its effect on iNOS expression were also elucidated. 2 Treatment with LPS/IFN-gamma and mixed cytokines for 24 h elicited the induction of iNOS activity as determined by nitrite accumulation in the culture medium and assay of enzyme activity. Nepalolide A at 10 microM abrogated the LPS/IFN-gamma- and mixed cytokines-mediated induction of iNOS by more than 90% in C6 glioma cells, and by 80% for mixed cytokines-induced induction of iNOS in primary astrocytes. The effect of nepalolide A (2-10 microM) was concentration-dependent. 3 The inhibition of iNOS induction by nepalolide A was attributed to decreases in the content of iNOS protein and the level of iNOS mRNA, as measured by immunoblotting and reverse transcriptase-polymerase chain reaction. 4 Electrophoretic mobility shift assay was used to evaluate the effect of nepalolide A on the activation of nuclear factor-kappaB (NF-kappaB). Results showed that nepalolide A diminished the LPS/IFN-gamma-mediated association of NF-kappaB with consensus oligonucleotide in a concentration-dependent manner. The activation of NF-kappaB by mixed cytokines was modulated both in the extent of activation and in its time-course by nepalolide A. 5 The ability of nepalolide A to inhibit NF-kappaB activation was further confirmed by studies on the degradation of the inhibitor of NF-kappaB, IkappaB, as measured by immunoblotting. 6 The present study demonstrates that the attenuation of NF-kappaB activation by nepalolide A was mediated by blockade of the degradation of IkappaB, leading to suppression of the expression of iNOS.
Wang
C N
CN
National Research Institute of Chinese Medicine, NO. 155-1 SEC. 2, LiNung ST., Peitou, Taipei, Taiwan, Republic of China.
Shiao
Y J
YJ
Lin
Y L
YL
Chen
C F
CF
D000818:Animals; D000873:Anthracenes / Q000494:pharmacology*; D001253:Astrocytes / Q000187:drug effects / Q000378:metabolism*; D001711:Biotransformation / Q000187:drug effects; D015153:Blotting, Western; D001932:Brain Neoplasms / Q000378:metabolism*; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000378:metabolism*; D003864:Depression, Chemical; D004586:Electrophoresis; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005910:Glioma / Q000378:metabolism*; D020672:I-kappa B Proteins; D007371:Interferon-gamma / Q000037:antagonists & inhibitors / Q000494:pharmacology; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000096:biosynthesis; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors* / Q000096:biosynthesis*; D052247:Nitric Oxide Synthase Type II; D009573:Nitrites / Q000378:metabolism; D009841:Oligonucleotides / Q000378:metabolism; D011233:Precipitin Tests; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins
D000873:Anthracenes; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; C083487:I kappa B beta protein; D020672:I-kappa B Proteins; D016328:NF-kappa B; C000606206:Nfkbia protein, rat; D009573:Nitrites; D009841:Oligonucleotides; D012333:RNA, Messenger; D011994:Recombinant Proteins; C402342:nepalolide A; D000072000:NF-KappaB Inhibitor alpha; D007371:Interferon-gamma; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496318:Nos2 protein, rat