Effects of tibolone and its metabolites on Angiopoietin-1, Tie-2 and tumor necrosis factor-alpha mRNA in Ishikawa cells. Implication for tibolone's effects on the endometrium.
OBJECTIVE
To determine the effect of 17beta-estradiol, medroxyprogesterone acetate, tibolone and tibolone metabolites on Angiopoietin-1, Tie-2, and tumor necrosis factor-alpha in Ishikawa cells, in vitro. We hypothesized that differential effects on angiogenic factors or inflammatory cytokines by individual hormones may be related to the endometrial bleeding in postmenopausal women using hormone therapy.


METHODS
Ishikawa cells were cultured to 80% confluence, in vitro. After 24h incubation in serum-free media, 1.0, 0.1 and 0.01 microM of 17beta-estradiol, medroxyprogesterone acetate, tibolone, 3alpha-hydroxytibolone, 3beta-hydroxytibolone, and Delta4-tibolone were added to the Ishikawa cells. The cells plus steroids were then incubated for a further 24h. Total RNA was extracted from control and treated Ishikawa cells. After reverse transcription, Angiopoietin-1, Tie-2, tumor necrosis factor-alpha, and beta-actin cDNAs were amplified in a polymerase chain reaction spiked with 33p-dCTP. Relative abundance of Angiopoietin-1, Tie-2, and tumor necrosis factor-alpha mRNA was measured by scintillation spectroscopy.


RESULTS
17Beta-estradiol and medroxyprogesterone acetate increased Angiopoietin-1 mRNA significantly higher than control, tibolone and tibolone hydroxy metabolites. Delta4-Tibolone at all concentrations tested did not increase Angiopoietin-1. None of the steroids tested at any concentration altered Tie-2 mRNA expression compared to control. 17Beta-Estradiol at 1.0 and 0.1 microM increased tumor necrosis factor-alpha mRNA significantly higher than control. Medroxyprogesterone acetate only at 1.0 microM increased tumor necrosis factor-alpha mRNA above control levels. Tibolone, 3alpha-hydroxytibolone, 3beta-hydroxytibolone, and Delta4-tibolone at every concentration had no effect on tumor necrosis factor-alpha mRNA abundance.


CONCLUSIONS
Delta4-Tibolone did not stimulate Angiopoietin-1, while the other steroids had differential effects greater than control. None of the steroids changed the expression of Tie-2 mRNA. Tumor necrosis factor-alpha was increased by 17beta-estradiol and by the highest concentration of medroxyprogesterone acetate. We interpret these results as supportive of our hypothesis that differential effects on angiogenic factors or inflammatory cytokines by individual steroids may be related to the clinical occurrence of endometrial bleeding in postmenopausal women using hormone therapy.
Mirkin
Sebastian
S
CONRAD Clinical Research Center, Jones Institute for Reproductive Medicine, Eastern Virginia Medical School, USA.
Archer
David F
DF
D000230:Adenocarcinoma / Q000378:metabolism / Q000473:pathology; D042683:Angiopoietin-1 / Q000235:genetics* / Q000378:metabolism; D045744:Cell Line, Tumor; D003271:Contraceptive Agents, Female / Q000494:pharmacology; D016889:Endometrial Neoplasms / Q000378:metabolism / Q000473:pathology; D004717:Endometrium / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D004958:Estradiol / Q000494:pharmacology; D020847:Estrogen Receptor Modulators / Q000378:metabolism / Q000494:pharmacology*; D005260:Female; D006801:Humans; D017258:Medroxyprogesterone Acetate / Q000494:pharmacology; D009652:Norpregnenes / Q000378:metabolism / Q000494:pharmacology*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D042787:Receptor, TIE-2 / Q000235:genetics* / Q000378:metabolism; D011960:Receptors, Estrogen / Q000235:genetics / Q000378:metabolism; D011980:Receptors, Progesterone / Q000235:genetics / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics* / Q000378:metabolism
D042683:Angiopoietin-1; D003271:Contraceptive Agents, Female; D020847:Estrogen Receptor Modulators; D009652:Norpregnenes; D012333:RNA, Messenger; D011960:Receptors, Estrogen; D011980:Receptors, Progesterone; D014409:Tumor Necrosis Factor-alpha; D004958:Estradiol; D017258:Medroxyprogesterone Acetate; D042787:Receptor, TIE-2; C027385:tibolone