Biological Activity of a Novel Nonpeptide Antagonist to the Interleukin-6 Receptor 20S,21-Epoxy-resibufogenin-3-formate

  1. Masahiko Hayashi1,
  2. Mun-Chual Rho1,2,
  3. Akiko Fukami1,
  4. Akiko Enomoto1,
  5. Shinobu Nonaka1,
  6. Yoshino Sekiguchi1,
  7. Tadashi Yanagisawa3,
  8. Ayano Yamashita4,
  9. Toshihiko Nogawa4,
  10. Yoshiaki Kamano4 and
  11. Kanki Komiyama1
  1. 1The Kitasato Institute, Tokyo, Japan (M.H., M.-C.R., A.F., A.E., S.N., Y.S., K.K.); 2Cardiovascular Research Laboratory, Korea Research Institute of Bioscience and Biotechnology, Taejon, Korea (M.-C.R.);3Department of Applied Biochemistry, Utsunomiya University, Utsunomiya, Japan (T.Y.); and 4Faculty of Science, Kanagawa University, Hiratsuka, Japan (A.Y., T.N., Y.K.)
  1. Masahiko Hayashi, The Kitasato Institute, 5-9-1 Shirokane, Minato-Ku, Tokyo 108-8642, Japan. E-mail: mhayashi{at}lisci.kitasato-u.ac.jp

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

  • 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.

  • 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.
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  1. doi: 10.1124/jpet.102.036137 JPET October 1, 2002 vol. 303 no. 1 104-109
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