Effect of tibolone and its metabolites on vascular endothelial growth factor isoforms 121 and 165 and thrombospondin-1 mRNA in Ishikawa cells.
OBJECTIVE
To compare the mRNA expression of vascular endothelial growth factor (VEGF) 121 and 165 isoforms and thrombospondin-1 (TSP-1) after incubation with tibolone and tibolone metabolites 3alpha-hydroxytibolone, 3beta-hydroxytibolone, Delta4-tibolone, and 17beta-estradiol (E2) in cultured Ishikawa cells.


METHODS
Ishikawa cells (immortalized from a well-differentiated human adenocarcinoma cell line) were cultured in vitro to confluence. Tibolone, 3alpha-hydroxytibolone, 3beta-hydroxytibolone, Delta4-tibolone and E2 at concentrations of 1.0, 0.1 and 0.01 micromol/L were added to confluent cells and further cultured for an additional 24 h. Control cells were treated with medium in absence of hormone. Total RNA was extracted from control and treated Ishikawa cells. After reverse transcription, VEGF, TSP-1 and the housekeeping gene, beta-actin cDNAs, were amplified in a polymerase chain reaction spiked with 33p-dCTP. Relative abundance of VEGF 121 and 165 isoforms and TSP-1 mRNA was measured by scintillation spectroscopy.


RESULTS
E2, tibolone, 3alpha-hydroxytibolone, and 3beta-hydroxytibolone increased both VEGF 121 and 165 mRNA compared with the control. However, Delta4-tibolone had no effect on either VEGF 121 or 165 mRNA compared with the control. Delta4-Tibolone increased TSP-1 mRNA expression compared with control levels. E2, tibolone, 3alpha-hydroxytibolone, and 3beta-hydroxytibolone did not increase TSP-1 mRNA expression at any concentration.


CONCLUSIONS
Tibolone and the 3alpha- and 3beta-tibolone metabolites with E2 increased VEGF 121 and 165 isoforms. Conversely, Delta4-tibolone, which is reported to have progestational-like activity, did not stimulate VEGF 121 and VEGF 165 but increased TSP-1 mRNA synthesis in cultured Ishikawa cells. We hypothesize, based on these data, that the clinical finding of no endometrial growth in women using tibolone may be partly related to alterations in these angiogenic factors.
Mirkin
Sebastian
S
Clinical Research Center, The Jones Institute for Reproductive Medicine, Eastern Virginia Medical School, Norfolk, VA 23507, USA.
Mahony
Mary C
MC
Archer
David F
DF
D002460:Cell Line; D017931:DNA Primers; D004958:Estradiol / Q000494:pharmacology; D020847:Estrogen Receptor Modulators / Q000494:pharmacology*; D006801:Humans; D009652:Norpregnenes / Q000494:pharmacology*; D016133:Polymerase Chain Reaction; D020033:Protein Isoforms / Q000096:biosynthesis / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis*; D019700:Thrombospondin 1 / Q000235:genetics*; D042442:Vascular Endothelial Growth Factors / Q000032:analysis / Q000235:genetics*
C485705:3-hydroxytibolone; D017931:DNA Primers; D020847:Estrogen Receptor Modulators; D009652:Norpregnenes; D020033:Protein Isoforms; D012333:RNA, Messenger; D019700:Thrombospondin 1; D042442:Vascular Endothelial Growth Factors; C485706:delta(4)-tibolone; D004958:Estradiol; C027385:tibolone