title: Down-regulation of proliferation and up-regulation of apoptosis by gonadotropin-releasing hormone agonist in cultured uterine leiomyoma cells.
abstract: OBJECTIVE
  To elucidate the direct effects of gonadotropin-releasing hormone agonist (GnRHa) on the growth of human uterine leiomyoma cells, cell proliferation and apoptosis in cultured leiomyoma cells treated with GnRHa were investigated.
  
  
  METHODS
  Isolated leiomyoma cells were subcultured in DMEM supplemented with 10% FBS for 5 days and stepped down to serum-free conditions for an additional 6 days in the presence or absence of graded concentrations of GnRHa (10(-9) mol/l to 10(-12) mol/l). The effects of GnRHa on the number of viable cells, expression of proliferating cell nuclear antigen (PCNA), Fas and Fas ligand, and apoptosis in cultured leiomyoma cells were examined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrasodium bromide) assay, immunocytochemical analysis, Western blot analysis and TUNEL assay respectively. RT-PCR was performed to detect the expression of GnRH receptor mRNA in cultured leiomyoma cells.
  
  
  RESULTS
  Treatment with GnRHa resulted in a decrease in the number of cultured viable leiomyoma cells assessed by MTT assay in a dose-dependent manner compared with that in control cultures (P<0.01). The growth inhibition of cultured leiomyoma cells treated with GnRHa in concentrations higher than 10(-10) mol/l was associated with the suppression of the proliferative potential characterized by a decrease in PCNA-positive rate of the cultured cells (P<0.01) and an increase in the apoptosis-positive rate assessed by TUNEL assay (P<0.05 and P<0.01). GnRHa markedly increased the expression of Fas and induced the expression of Fas ligand in the cultured leiomyoma cells on the basis of Western blot analysis. These direct effects of GnRHa on the number of viable cultured leiomyoma cells, PCNA-positive rate, apoptosis-positive rate and Fas/Fas ligand expression in the cultured leiomyoma cells were only attained after the 4-day treatment. RT-PCR analysis revealed that GnRH receptor mRNA was expressed in cultured leiomyoma cells.
  
  
  CONCLUSIONS
  The present results demonstrate that GnRHa directly inhibits the growth of human uterine leiomyoma cells by suppressing cell proliferation and inducing apoptosis, which might be associated with the increase in Fas expression and the induction of Fas ligand expression in the cells.
authors.affiliation: Department of Obstetrics and Gynecology, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan.
authors.forename: Yin
authors.initials: Y
authors.lastname: Wang
authors.forename: Hiroya
authors.initials: H
authors.lastname: Matsuo
authors.forename: Osamu
authors.initials: O
authors.lastname: Kurachi
authors.forename: Takeshi
authors.initials: T
authors.lastname: Maruo
mesh_terms: D018931:Antineoplastic Agents, Hormonal / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D002064:Buserelin / Q000494:pharmacology*; D002452:Cell Count; D002455:Cell Division / Q000187:drug effects; D002469:Cell Separation; D003124:Colorimetry; D015536:Down-Regulation / Q000187:drug effects*; D005260:Female; D007987:Gonadotropin-Releasing Hormone / Q000819:agonists*; D006801:Humans; D007150:Immunohistochemistry; D020287:In Situ Nick-End Labeling; D007889:Leiomyoma / Q000473:pathology*; D018809:Proliferating Cell Nuclear Antigen / Q000096:biosynthesis / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis; D011966:Receptors, LHRH / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013778:Tetrazolium Salts; D013844:Thiazoles; D014407:Tumor Cells, Cultured; D015854:Up-Regulation / Q000187:drug effects*; D014594:Uterine Neoplasms / Q000473:pathology*; D019014:fas Receptor / Q000096:biosynthesis / Q000235:genetics
chemical_list: D018931:Antineoplastic Agents, Hormonal; D018809:Proliferating Cell Nuclear Antigen; D012333:RNA, Messenger; D011966:Receptors, LHRH; D013778:Tetrazolium Salts; D013844:Thiazoles; D019014:fas Receptor; D007987:Gonadotropin-Releasing Hormone; C022616:thiazolyl blue; D002064:Buserelin