Inhibition of NF-kappa B activation through targeting I kappa B kinase by celastrol, a quinone methide triterpenoid |
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Authors: | Lee Jeong-Hyung Koo Tae Hyeon Yoon Hyunkyung Jung Haeng Sun Jin Hui Zi Lee Kyeong Hong Young-Soo Lee Jung Joon |
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Affiliation: | Anticancer Research Laboratory, Korea Research Institute of Bioscience and Biotechnology, 52 Aueun-Dong, Yuseong-Gu, Daejeon, Republic of Korea. |
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Abstract: | Celastrol, a quinone methide triterpenoid, was isolated as an inhibitor of NF-kappaB from Celastrus orbiculatus. This compound dose-dependently inhibited a variety of stimuli-induced NF-kappa B-regulated gene expression and the DNA-binding of NF-kappa B in different cell lines without affecting DNA-binding activity of AP-1. Preincubation of celastrol completely blocked the LPS-, TNF-alpha-, or PMA-induced degradation and phosphorylation of I kappa B alpha. Importantly, celastrol inhibited IKK activity and the constitutively active IKK beta activity in a dose-dependent manner without either affecting the NF-kappa B activation induced by RelA over-expression or directly suppressing the DNA-binding of activated NF-kappa B. However, mutation of cysteine 179 in the activation loop of IKK beta abolished sensitivity towards to celastrol, suggesting that celastrol suppressed the NF-kappa B activation by targeting cysteine 179 in the IKK. To verify that celastrol is a NF-kappa B inhibitor, we investigated its effect on some NF-kappa B target genes expressions. Celastrol prevented not only LPS-induced mRNA expression of iNOS and TNF-alpha, but also TNF-alpha-induced Bfl-1/A1 expression, a prosurvival Bcl-2 homologue. Consistent with these results, celastrol significantly suppressed the production of NO and TNF-alpha in LPS-stimulated RAW264.7 cells, and increased the cytotoxicity of TNF-alpha in HT-1080 cells. We also demonstrated that celastrol showed anti-inflammatory and anti-tumor activities in animal models. Taken together, this study extends our understanding on the molecular mechanisms underlying the anti-inflammatory and anti-cancer activities of celastrol and celastrol-containing medicinal plant, which would be a valuable candidate for the intervention of NF-kappa B-dependent pathological conditions. |
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Keywords: | NF-κB, nuclear factor κB AP-1, activator protein-1 LPS, lipopolysaccharide TNF-α, tumor necrosis factor-α PMA, phorbol myristyl acetate MEKK-1, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase-1 IKK, IκB kinase EMSA, electrophoretic mobility shift assay COX-2, cyclooxygenase-2 PGE2, prostaglandin E2 iNOS, inducible nitric oxide synthase NO, nitric oxide MPO, myeloperoxidase DTT, dithiothreitol |
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