In vivo and in vitro estrogenic and progestagenic actions of Tibolone

Abstract

Estrogen and progestin combination in hormone replacement therapy (HRT) increases the incidence of breast cancer, but decreases the endometrial cancer risk of unopposed estrogen. Therefore, a SERM such as Tibolone, that delivers the beneficial, but not the adverse side effects, of steroid hormones would be clinically advantageous. However, data from the Million Women Study suggests that Tibolone increases the risk of both breast and endometrial cancer. Herein, we assessed the estrogenic and progestagenic actions of Tibolone using transvaginal sonography studies and an in vitro model of breast (ZR-75, MCF7) and endometrial cancer (Ishikawa). The known cancer associated proteins (ER, EGFR, STATS, tissue factor and Bcl-xL) were selected for study. Transvaginal sonography demonstrated that postmenopausal women treated with Tibolone displayed a thinner endometrium than in the late proliferative phase, but had a phenotype characteristic of the secretory phase, thus demonstrating the estrogenic and progestagenic actions of this SERM. In vitro, Tibolone acted as an estrogen in downregulating ER and upregulating Bcl-xL, yet as progesterone, increasing STAT5 and tissue factor in breast cancer cells. The increase in tissue factor by Tibolone correlated with its coagulative potential. Interestingly, EGFR was up-regulated by progesterone in the breast and by estrogen in endometrial cells, while Tibolone increased protein levels in both cell types. In conclusion, this study further demonstrates the estrogenic and progestagenic nature of Tibolone. The pattern of regulation of known oncogenes in cells of breast and endometrial origin dictates caution and vigilance in the prescription of Tibolone and subsequent patient monitoring.

FIGURE 1

Representative ultrasounds obtained with patient consent of (A) Proliferative phase endometrium, (B) Secretory phase endometrium, (C) Postmenopausal endometrium, (D & E) Postmenopausal endometrium with Tibolone. The diameter of the endometrium is shown in the lower left side of each panel.

FIGURE 2

Western blots demonstrating ER expression levels in the MCF-7 cell line, after 24 hours of treatment with Ethanol (C), 17-β–estradiol (E), progesterone (P) and Tibolone (T).

FIGURE 3

Western blots demonstrating a time course of ER expression in the MCF-7 cell line after the administration of Ethanol (C), 17-β–estradiol (E) or Tibolone (T).Protein levels are expressed as relative densitometric units. Percentages represent changes in respect to ethanol treatments within each time points.

FIGURE 4

Western blots demonstrating EGFR expression levels after 24 hours of treatment with Ethanol (C), 17-β–estradiol (E), progesterone (P) and Tibolone (T) in the ZR-75 cell line (Panel A) and the Ishikawa cell line (Panel B).

FIGURE 5

Western blots demonstrating Stat-5a/b (upper panel), erk-2 (internal standard) and Bcl-xL (lower panel) expression levels after the administration of Ethanol (C), 17-β–estradiol (E), progesterone (P) and Tibolone (T) in ZR-75 and Ishikawa cell lines.

FIGURE 6

Western blots demonstrating TF expression levels after 24 hours of treatment with Ethanol (C), 17- β–estradiol (E), progesterone (P) and Tibolone (T) in ZR-75 and Ishikawa cell lines. The lower band in the ZR-75 cell line corresponds to a non-specific band.

FIGURE 7

Western blots demonstrating a time course in the ZR-75 cell line of TF expression after 6, 24, 30 and 48 hours of treatment with Ethanol (C) and progesterone (P) in panel A and with Ethanol (C) and Tibolone (T) in panel B. The lower constitutive band corresponds to a non-specific band.

FIGURE 8

RT-PCR of TF (lower band) and GAPDH (internal loading control, upper band). ZR-75 cells were treated with Ethanol (C), 17- β–estradiol (E), progesterone (P) and Tibolone (T) for 9 hours..

FIGURE 9

Procoagulant assay Effect of ovarian hormones and Tibolone on the induction of TF activity in ZR-75 and Ishikawa cells. ZR-75 and Ishikawa cells were treated with either Ethanol(C), 17–β–estradiol (E), Progesterone (P) or Tibolone (T) for 24 hrs. Procoagulant activity was measured by the formation of Factor Xa in the presence of Factor VIIa.