Inhibition of NF-κB activation through targeting IκB kinase by celastrol, a quinone methide triterpenoid
Introduction
NF-κB regulates the transcription of a large number of genes, particularly those involved in immune, inflammatory, and antiapoptotic responses [1]. Inappropriate regulation of NF-κB is directly involved in a wide range of human disorders, including a variety of cancers, neurodegenerative diseases, ataxiatelangiectasia, arthritis, asthma, inflammatory bowel disease, and numerous other inflammatory conditions [2]. In most cell types, the dimeric transcription factor NF-κB is trapped in the cytoplasm by binding to its inhibitor protein IκBs. IκBs retain NF-κB in the cytoplasm by masking the nuclear localization sequence contained in the Rel homology domain [3], [4]. The latent NF-κB is activated by a variety of cellular stimuli, which trigger site-specific phosphorylation of IκBα by a IκB kinase complex [5], [6]. The phosphorylated IκBα becomes rapidly ubiquitinated and degraded by the proteasome complex [7], [8]. Following IκBα degradation, the NF-κB is translocated to the nucleus, where it activates the transcription of target genes.
NF-κB regulates the transcription of various inflammatory cytokines, such as interleukin-1, -2, -6, -8 and TNF-α, as well as genes encoding COX-2, iNOS, immunoreceptors, cell adhesion molecules, hematopoietic growth factors, and growth factor receptors [9]. In addition to regulating the expression of genes important for immune and inflammatory responses, NF-κB also controls the transcription of genes that confer resistance to death-inducing signals. Target genes include those encoding the caspase inhibitors, c-IAP1s and anti-apoptotic regulators, such as Bfl-1/A1 and GADD45β[10]. Interestingly, recent findings show that activation of NF-κB may function as a tumor promoter in inflammation-associated cancer, suggesting that NF-κB is a key player linking between chronic inflammation and cancer [11], [12].
NF-κB and the signaling pathways that regulate its activity have become a focal point for intense drug discovery and development efforts [13]. Well-known anti-inflammatory substances such as glucocorticoids or nonsteroidal anti-inflammatory drugs (NSAIDs) exert, at least a part of their effects, by inhibiting NF-κB activity [14], [15], [16]. Several anti-inflammatory medicinal plants also contain structurally diverse compounds that inhibit NF-κB activation [17], [18], [19]. In our search for inhibitors of NF-κB activation from natural products, we identified celastrol, a quinone methide triterpenoid as a major component of active principles in the methanol extract of the roots of Celastrus orbiculatus[20], which has been used as a treatment for rheumatoid arthritis and bacterial infection in folk medicine [21].
In this study, we investigated the effect of celastrol on the NF-κB activation and anti-inflammatory and anti-tumor activities. Our results show that celastrol suppressed the NF-κB activation by inhibiting IKK activity, possibly through directly targeting cysteine 179 in the activation loop of IKK. Celastrol prevented not only the expression of iNOS, TNF-α, and Bfl-1/A1, but also the production of NO and TNF-α in LPS-stimulated RAW264.7 cells and increased the cytotoxicity of TNF-α in HT-1080 cells. Furthermore, celastrol showed anti-inflammatory and anti-tumor activities in animal models. Our results suggest that celastrol is a valuable compound for modulation of NF-κB-dependent pathological conditions and support pharmacological basis that the Celastraceae plants have been used as a traditional herbal medicine for the treatments of inflammation and cancer.
Section snippets
Cell culture and reagents
Human Jurkat T leukemia cells and monocyte U937 cells were maintained in RPMI 1640 medium. RAW264.7 cells, HeLa cells, human 293 embryonic kidney cells and HT-1080 cells were maintained in Dulbecco's modified Eagle's medium. MDA-MB-435 cells were the generous gift of Dr. Danny R. Welch (University of Alabama at Birmingham, Birmingham, AL, USA) and maintained in DME-F12 medium. All media were supplemented with penicillin (100 units/ml)–streptomycin (100 μg/ml) (Invitrogen, Carlsbad, CA, USA) and
Celastrol inhibits NF-κB activation by different stimuli
In our effort to identify NF-κB inhibitors from natural resources, celastrol was isolated from C. orbiculatus[20]. This compound dose-dependently suppressed the LPS-, PMA-, and TNF-α-induced expression of NF-κB reporter gene. In this study, we investigated the inhibitory effect of celastrol on the NF-κB activation. Two cell lines, human lymphoma Jurkat, and human monocyte U937, were preincubated with various concentrations of celastrol for 30 min prior to stimulation. Jurkat cells were
Discussion
Plants of the Celestraceae family including Tripterygium wilfordii and Celastrus angulatus are a rich source of quinone methide triterpenoids. Extracts containing quinone methide, celastrol have been used in traditional medicine such as rheumatoid arthritis [21]. It has been reported that celastrol exerts potent anti-inflammatory activities in various experimental models [31] and strong cytotoxicity against various cancer cell-lines [32]. However, how celastrol exerts these pharmacological
Acknowledgments
The authors thank Drs. M. Karin, D.M. Crew, Mira Jung, and S. Hong for the kind gift of expression vectors. This research was partially supported by a grant from Plant Diversity Research Center of 21st Century Frontier Research Program and KRIBB Research Initiative Program funded by Ministry of Science and Technology of Korean government.
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