Harald Seeger

Comparison of the proliferative effects of estradiol and conjugated equine estrogens on human breast cancer cells and impact of continuous combined progestogen addition

Objectives: So far, most epidemiological studies investigating breast cancer risk and hormone replacement therapy have been conducted with conjugated equine estrogens (CEE). Recent trials indicate that the addition of progestogens may increase breast cancer risk. In the present study, we compared the effects of the human estrogen 17β-estradiol (E2) with those of the main equine components of CEE, i.e. equilin (Eq) and 17α-dihydroequilin (Dheq) on the proliferation of human breast cancer cells. The proliferative effect of progestogen addition was also investigated. Materials and methods: The well-established human breast cancer cell line MCF-7 was used as an in vitro model. The proliferative effect of E2, Eq and Dheq was tested in the concentration range 0.01-10 nmol/l. The progestogens progesterone, medroxyprogesterone acetate (MPA) and norethisterone (NET) were continuously combined with 0.1 nmol/l estrogen at concentrations of 0.01 nmol/l, 1 nmol/l, 0.1 μmol/l and 10 μmol/l. Proliferation was measured after 7 days by the adenosine triphosphate (ATP) chemosensitivity test. Results: All three estrogens increased the proliferation of MCF-7 cells by between 40 and 180%. The most proliferatively potent estrogen was E2, followed by Eq and Dheq, which showed a slightly lower proliferative activity than E2. The addition of progesterone inhibited E2-induced proliferation by about 30%, but only at the high non-physiological concentration of 10 μmol/l. All three progestogens inhibited Eq-induced proliferation, although their effect tended to be low, with values between 5 and 40%. No progestogen reduced Dheq-induced proliferation by more than 20%. In contrast, MPA slightly increased the proliferation rate by about 5% at the high physiological concentration of 0.1 μmol/l when combined with Dheq. The same held true when MPA and NET were added at the high pharmacological concentration of 10 μmol/l, causing increases of about 10%. Conclusions: Our results indicate that equine estrogens have a proliferative action similar to that of 17β-estradiol. Continuous addition of progestogens does not result in any major reduction of proliferative potency. Some progestogens may even enhance the estrogen-induced proliferation of pre-existing breast cancer cells, particularly when combined with certain equine estrogens. However, in none of the tested circumstances do progestogens increase the proliferative effect of estradiol, and progesterone has no deleterious effect even at pharmacological levels, in contrast to progestogens.

Progestogens and membrane-initiated effects on the proliferation of human breast cancer cells

ABSTRACT Objectives Evidence is accumulating that progestogens may play a crucial role in the development of breast cancer under contraception and hormone therapy in reproductive and menopausal women. Progesterone receptor membrane component 1 (PGRMC1) expressed in breast cancer may be important in tumorigenesis and thus may increase breast cancer risk. The aim of this project was to investigate the influence of progesterone and nine synthetic progestins on MCF-7 breast cancer cells overexpressing PGRMC1. Methods MCF-7 cells were stably transfected with PGRMC1 expression plasmid (WT-12). To test the effects of progestogerone (P) and the synthetic progestins chlormadinone acetate (CMA), desogestrel (DSG), drospirenone (DRSP), dydrogesterone (DYD), levonorgestrel (LNG), medroxyprogesterone acetate (MPA), nomegestrol (NOM) and norethisterone (NET) on cell proliferation, MCF-7 and WT-12 cells were stimulated with different concentrations (0.01–1 µmol/l). Results In MCF-7 cells, DRSP, DSG, DYD, LNG and NET increased the proliferation at 1 µmol/l, the effect being highest for NET with about 20%. In WT-12 cells, the same progestins, but additionally MPA, showed a significant increase, which was much higher (30–245%) than in MCF-7 cells. Here again, NET showed the highest proliferative effect. No effect was found for CMA, NOM and P. Conclusion Some synthetic progestins trigger a proliferative response of PGRMC1-overexpressed MCF-7 cancer cells. The effect of progestogens on breast cancer tumorigenesis may clearly depend on the specific pharmacology of the various synthetic progestins.

Possible role of PGRMC1 in breast cancer development

Abstract Hormone therapy may increase the risk of breast cancer. Thus, especially the addition of synthetic progestins may play a decisive role according to the results of clinical studies. Overexpression of a special receptor, i.e. the progesterone receptor membrane component-1 (PGRMC1), may offer a potential new pathway to explain the observed increase in breast cancer risk in the combined arm of the Womens Health Initiative. PGRMC1 is expressed in breast cancer tissue and may be important in tumorigenesis. The expression of PGRMC1 in breast cancer tissue is significantly different from that in normal mammary glands. Certain synthetic progestins can increase the proliferation of PGRMC1-overexpressing breast cancer cells and may thus be involved in tumorigenesis, while progesterone and certain synthetic progestins such as nomegestrol or chlormadinone acetate react neutrally. Our investigations point towards an important role of estrogen receptor-α in the signaling cascade, resulting in the proliferative effect induced by progestins. Thus, activation of PGRMC1 may explain the increased breast cancer risk observed during treatment with certain progestins. Very recently, PGRMC1 was investigated in serum samples of lung cancer patients and matched healthy patients; significantly higher concentrations were shown in the cancer patients. Therefore, PGRMC1 might be a predictor for other cancers as well but, according to clinical trials, its importance for a possible screening tool, particularly for breast cancer risk during hormone therapy, seems of interest.

Lipid-independent effects of an estrogen–statin combination: inhibition of expression of adhesion molecules and plasminogen activator inhibitor-1 in human endothelial cell cultures

Objective Estrogens have been shown to elicit beneficial effects on the cardiovascular system by modulating the lipid profile as well as by direct vascular actions. Since estrogenic cardioprotection has still been not confirmed by randomized interventional trials, the combination with statins, which have been proved to act in a cardioprotective fashion, is of special interest. Statins are lipid-lowering drugs, but direct vascular effects also seem to be of importance in this case. Apart from some clinical studies investigating the effect of such combination therapy on the lipid profile, no reports of a possible influence on biochemical markers of vascular function are yet available. The aim of the present study was to investigate, for the first time, lipid-independent effects of an estrogen–statin combination on markers of vascular endothelial function. Methods Experiments were conducted using endothelial cell cultures from human umbilical veins. Markers of endothelial function chosen were adhesion molecules, which are involved in the early stages of atherosclerosis, and plasminogen activator inhibitor-1 (PAI-1), which is an independent risk factor for the development of cardiovascular disease. The concentrations of soluble forms of the adhesion molecules E-selectin and intercellular adhesion molecule (ICAM) as well as concentrations of PAI-1 were measured after the addition of 17β-estradiol, fluvastatin and equimolar combinations of the two in concentrations of 0.01, 0.1 and 1 μmol/l. Results Both drugs significantly reduced concentrations of E-selectin and ICAM, and the effect of the combination was not superior to that of the monosubstances. In addition, both substances were able to inhibit the synthesis of PAI-1. In this case the effect of the combination was significantly greater than that of the monosubstances. Conclusion In summary, an estrogen–statin therapy is able to elicit lipid-independent positive modulations on the expression of cell adhesion molecules and the synthesis of PAI-1. The estrogen–statin combination showed partially additive effects and no negative, antagonistic actions. Since the combination of statins with hormone replacement therapy may attain clinical significance in preventing cardiovascular disease, it seems worthwhile to elucidate further the direct vascular effects of this combination therapy, especially in postmenopausal women with pre-existing coronary disease.

Effects of drospirenone on cardiovascular markers in human aortic endothelial cells

Objectives The effect of the new progestogen drospirenone on biochemical markers in terms of cardiovascular effects was investigated in the presence and absence of aldosterone and compared to progesterone and the progestogens medroxyprogesterone acetate (MPA) and promegestone (R5020), and the antimineralocorticoid spironolactone. Methods Human female aortic endothelial cells were used for the experiments. The progestogens were tested alone at 0.1 and 10 μmol/l and in combination with aldosterone at a concentration of 10 μmol/l. The adhesion molecule E-selectin, the chemokine monocyte attracting protein-1 (MCP-1) and plasminogen activator inhibitor-1 (PAI-1) were chosen as markers. Results In combination with aldosterone, spironolactone, drospirenone, progesterone and R5020 were able to inhibit the aldosterone-induced increase in MCP-1 concentration, the effect being greatest for spironolactone. In contrast, MPA did not show any significant effect. For E-selectin, similar results were found; however, R5020 and MPA were not able to act antagonistically. Spironolactone, drospirenone and progesterone were able to significantly reduce the aldosterone-induced stimulation of PAI-1. For MPA and R5020, no significant effect was found. Conclusions The new progestogen drospirenone seems to have favorable effects on the cardiovascular system due to its antimineralocorticoid property. Clinical studies must prove the results of this in vitro experiment.

Progestogens and Breast Cancer Risk – In Vitro Investigations with Human Benign and Malignant Epithelial Breast Cells

Two recent studies, the Women’s Health Initiative (WHI) and the Million Women Study (MWS), have above all raised concerns over the relationship between progestogens and increased risk of breast cancer in the climacteric and postmenopause (Million Women Study collaborators, 2003; Writing Group, 2002). The Women’s Health Initiative study was terminated early after five years, due to an increased incidence of breast cancer in the group treated with combined estrogen and progestogen therapy (EPT). The MWS concluded that breast cancer risk was increased two-fold in current users of combined HRT compared to a factor of 1.3 for estrogen-only therapy. A crucial role of progestogens in increasing breast cancer risk was supported by the WHI estrogen mono-arm showing no increase but rather a reduction of breast cancer risk, which was significant for patients with more than 80% adherence to study medication (The Women’s Health Initiative Steering Committee, 2004). However, in the French E3N-EPIC trial of over 80 000 postmenopausal women it was reported that hormone therapy containing the progestin medroxyprogesterone acetate or norethisterone was associated with a significant increase in risk of breast cancer, whereas hormone therapy including progesterone and certain other progestins did not induce an increased risk (Fournier et al., 2008). By stimulating the production of survival factors, estradiol (E2) and other steroid hormones may influence cell proliferation. These survival factors include growth factors and cytokines. Epithelial and stromal cell-derived growth factors are understood to be significant in the regulation of breast epithelial cells directly via autocrine, paracrine, juxtacrine or intracrine pathways. Further responses stimulated by growth factors may activate signalling pathways which support the growth of cancer cells (Dickson & Lippman, 1995). Progestogens are conventionally thought to act via the activation of the intracellularlylocated progesterone receptors (PR), PR-A and PR-B. Several in vitro studies indicate that progestogens may exert an antiproliferative effect by activation of these receptors in human breast cancer cells (Cappellatti et al. 1995; Kramer et al., 2006; Schoonen et al., 1995). These data are in contrast to the above mentioned clinical data. Other data suggested a proliferative effect of synthetic progestogens (Catherino et al., 1995; Franke & Vermes, 2003). Thus the mechanisms by which progestogens act on human breast cells remain unclear.