Elsevier

Biochemical Pharmacology

Volume 72, Issue 10, 15 November 2006, Pages 1311-1321
Biochemical Pharmacology

Inhibition of NF-κB activation through targeting IκB kinase by celastrol, a quinone methide triterpenoid

https://doi.org/10.1016/j.bcp.2006.08.014Get rights and content

Abstract

Celastrol, a quinone methide triterpenoid, was isolated as an inhibitor of NF-κB from Celastrus orbiculatus. This compound dose-dependently inhibited a variety of stimuli-induced NF-κB-regulated gene expression and the DNA-binding of NF-κB in different cell lines without affecting DNA-binding activity of AP-1. Preincubation of celastrol completely blocked the LPS-, TNF-α-, or PMA-induced degradation and phosphorylation of IκBα. Importantly, celastrol inhibited IKK activity and the constitutively active IKKβ activity in a dose-dependent manner without either affecting the NF-κB activation induced by RelA over-expression or directly suppressing the DNA-binding of activated NF-κB. However, mutation of cysteine 179 in the activation loop of IKKβ abolished sensitivity towards to celastrol, suggesting that celastrol suppressed the NF-κB activation by targeting cysteine 179 in the IKK. To verify that celastrol is a NF-κB inhibitor, we investigated its effect on some NF-κB target genes expressions. Celastrol prevented not only LPS-induced mRNA expression of iNOS and TNF-α, but also TNF-α-induced Bfl-1/A1 expression, a prosurvival Bcl-2 homologue. Consistent with these results, celastrol significantly suppressed the production of NO and TNF-α in LPS-stimulated RAW264.7 cells, and increased the cytotoxicity of TNF-α 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-κB-dependent pathological conditions.

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.

References (40)

  • H. Sassa et al.

    The triterpene celastrol as a very potent inhibitor of lipid peroxidation in mitochondria

    Biochem Biophys Res Commun

    (1990)
  • W. He et al.

    Novel cytokine release inhibitors. Part III. Truncated analogs of tripterine

    Bioorg Med Chem Lett

    (1998)
  • V.M. Dirsch et al.

    The triterpenoid quinonemethide pristimerin inhibits induction of inducible nitric oxide synthase in murine macrophages

    Eur J Pharmacol

    (1997)
  • G.F. Pinna et al.

    Celastrol inhibits pro-inflammatory cytokine secretion in Crohn's disease biopsies

    Biochem Biophys Res Commun

    (2004)
  • S. Ghosh et al.

    NF-κB and Rel proteins: evolutionarily conserved mediators of immune responses

    Annu Rev Immunol

    (1998)
  • A.S. Baldwin

    The transcription factor NF-κB and human disease

    J Clin Invest

    (2001)
  • F. Mercurio et al.

    IKK-1 and IKK-2: cytokine-activated IκB kinases essential for NF-κB activation

    Science

    (1997)
  • J.D. Woronicz et al.

    IκB kinase-β: NF-κB activation and complex formation with IκB kinase-α and NIK

    Science

    (1997)
  • Z. Chen et al.

    Signal-induced site-specific phosphorylation targets IκBα to the ubiquitin-proteasome pathway

    Genes Dev

    (1995)
  • D.C. Scherer et al.

    Signal-induced degradation of IκBα requires site-specific ubiquitination

    Proc Natl Acad Sci USA

    (1995)
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