Abstract
Interleukin (IL)-6 is a key mediator in the regulation and coordination of the immune response and participates in pathogenesis of cancer cachexia, autoimmune disease, and postmenopausal osteoporosis. In the course of a screening program aimed at IL-6 inhibitor from natural products, we isolated 20S,21-epoxy-resibufogenin-3-formate (ERBF) from bufadienolide and examined the effect of ERBF on activities of various cytokines. ERBF dose dependently suppressed IL-6 activity and caused a parallel rightward shift of dose-response curves to IL-6 at concentrations of 0.03 to 10 ng/ml. Analysis of data yields a pA2 of 5.12 and a slope of 0.99. Selectivity of ERBF on activity of cytokines was examined using cytokine-dependent cell lines. ERBF did not affect IL-2-dependent growth of CTLL-2 cells, IL-3-dependent growth of Baf3 cells, or tumor necrosis factor (TNF)α-induced growth suppression in TNFα-sensitive L929 cells. ERBF also did not affect IL-4-stimulated expression of FcεR II receptor (CD23) in U-937 cells, the IL-8-induced chemotaxis of human neutrophils, or nerve growth factor-stimulated neuronal differentiation in PC-12 cells. In contrast, ERBF dose dependently suppressed IL-6-induced neuronal differentiation in PC-12 cells. Furthermore, ERBF suppressed only IL-6-induced osteoclast formation without affecting osteoclast formation induced by IL-11, leukemia inhibitory factor, and 1α,25-dihydroxyvitamin D3. In receptor binding assay, unbound (free) IL-6 was increased in a dose-dependent manner by pretreatment with ERBF on IL-6 receptor (IL-6R), suggesting that ERBF suppresses binding of IL-6 to IL-6R. These results clearly indicate that ERBF is a novel specific small molecule to show IL-6 receptor antagonist activity.
Footnotes
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This work was supported in part by a grant-in-aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan.
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DOI: 10.1124/jpet.102.036137
- Abbreviations:
- IL
- interleukin
- IL-6R
- interleukin-6 receptor
- gp
- glycoprotein
- ERBF
- 20S,21-epoxy-resibufogenin-3-formate
- rmIL
- recombinant murine interleukin
- rhIL
- recombinant human interleukin
- NGF
- nerve growth factor
- 1α,25(OH)2D3
- 1α,25-dihydroxyvitamin D3
- LIF
- leukemia inhibitory factor
- FITC
- fluorescein isothiocyanate
- FCS
- fetal calf serum
- PMNL
- polymorphonuclear leukocyte
- TRAP
- tartrate-resistant acid phosphatase
- TNF
- tumor necrosis factor
- MDL-A
- madindoline A
- sIL-6R
- soluble IL-6 receptor
- OSM
- Oncostatin M
- STAT
- signal transducer and activator of transcription
- Received March 14, 2002.
- Accepted May 9, 2002.
- The American Society for Pharmacology and Experimental Therapeutics
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