2003 Fiscal Year Final Research Report Summary
Development of anti-cytokine drugs for treatment of endometriosis
| Project/Area Number |
13671715
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Obstetrics and gynecology
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| Research Institution | Tottori University School of Medicine |
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Principal Investigator |
HARADA Tasuku Tottori Univ., Dept.Obstet.Gunecol., Assistant Professor, 医学部, 講師 (40218649)
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| Co-Investigator(Kenkyū-buntansha) |
TANIGUCHI Fuminori Tottori Univ., Dept.Obstet.Gunecol., Research Associates, 医学部附属病院, 助手 (40322218)
YOSHIDA Souichi Tottori Univ., Dept.Obstet.Gunecol., Research Associates, 医学部附属病院, 助手 (70304219)
TERAKAWA Naoki Tottori Univ., Dept.Obstet.Gunecol., Professor, 医学部, 教授 (90163906)
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| Project Period (FY) |
2001 – 2003
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| Keywords | endometriosis / cytokine / interleukin-8 / tumor necrosis factor alpha / anti-TNF alpha antibody / マウスモデル |
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| Research Abstract |
Peritoneal fluid (PF) in women with endometriosis contains an increased number of activated macrophages that secrete a variety of cytokines. We have previously shown that the PF levels of tumor necrosis factor alpha (TNFa) and interleukin-8 (IL-8) were significantly higher in patients with endometriosis. There was a significant correlation between PF levels of TNFa and IL-8. The purpose of the study is to investigate the role of TNFa in proliferation of endometriotic stromal cells. We also examined the activation of nulear factor-kB (NF-kB) during the induction of IL-8 by TNFa.
Chocolate cyst linings of the ovaries in patients with endometriosis (n=39) were a source of endometriotic tissue. The receptors for IL-8 and TNFa were examined by RT-PCR. Proliferation of endometriotic cells was studied using MTT assay. The expression of IL-8 gene and protein was analyzed by Northern blotting and ELISA, respectively. Western blot analysis and electrophoretic mobility shift assay (EMSA) were used
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to detect NF-kB activation by TNFa. Effects of inhibitor for NF-kB, TPCK, on TNFa action were also examined.
Transcripts of IL-8 receptor type A, TNF receptor type I and II were detected in endometriotic stromal cells. TNFa (10 and 100 pg/mL) induced the gene and protein expression of IL-8 in endometriotic stromal cells in a dose-dependent fashion. Addition of IL-8 (25-100 pg/mL) or TNFa (5-100 pg/mL) to the culture medium stimulated the proliferation of stromal cells. The stimulatory effects of TNFa were abolished by adding anti-IL-8 antibody. These results suggest that the action of IL-8 mediates the stimulatory effects of TNFa on stromal cell proliferation. The activation of NF-kB is usually associated with phosphorylation of IkB, followed by its degradation by the proteasome and NF-kB nuclear translocation. In order to detect the activation of NF-kB in stromal cells during inducible expression of IL-8 by TNFa, Western blotting was performed using an antibody for phosphorylated IkB (p-IkB). Addition of TNFa (100 pg/mL) rapidly induced p-IkB in stromal cells. EMSA revealed that adding TNFa promoted translocation of activated NF-kB.
Preincubation with TPCK reduced TNFa inducible expression of IL-8 gene and protein. TNFa action mediated by IL-8 may contribute to progression of endometriosis by promoting the growth of endometriotic cells. We demonstrated here that NF-kB activation was involved in the induction of IL-8 by TNFa in endometriotic tissues. Thus, TNFa and its signal transduction molecule, NF-kB, play important roles in the pathophysiology of endometriosis. Less
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