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Purpose: Two main estradiol metabolites have different biological behavior with tumorigenic features of 16-OHE1 and antiproliferative characteristics of 2-OHE1. We investigated the ratio of these estradiol metabolites in pre- and postmenopausal patients with breast cancer (BC) within two case-control studies., Methods: From 41 premenopausal patients with (cases) and without (controls N = 211) BC and 207 postmenopausal patients with and without BC (N = 206), urine samples were collected. Urine samples were collected prior to surgery and stored at -20 °C until measurement by ELISA. The multiple linear regression test with two interactions was performed to evaluate the influence of different factors on the metabolic ratio., Results: In premenopausal patients, log ratio of 2-OHE1/16-OHE1 was 0.25 (CI 0.20;0.29) and 0.21 (CI 0.11;0.31) for controls and cases without significant difference. In postmenopausal patients, log ratio was 0.22 (CI 0.17;0.26) and 0.11 (CI 0.07;0.15) in controls and cases, respectively, and was statistically significantly lower (p = 0.0002). Log ratio was significantly influenced by BMI, but only in postmenopausal patients, an increased BMI resulted in a significantly (p < 0.042) decreased ratio., Conclusions: Our case control studies suggest that in postmenopausal women a different metabolism of estrogens may play a role in the tumorigenesis of breast cancer. This genetically determined metabolism could be influenced by the exogenic factor BMI. In premenopausal women different hormone levels at different time points of the menstrual cycle may be an explanation that why we could not find an influence of estrogen metabolism.

Background: Epidemiological data suggest an enhanced breast cancer risk during estrogen/progestogen therapy as compared to estrogen monotherapy in postmenopausal women. The underlying mechanism, however, still remains unknown. Estrogens are known to be mitogenic agents for benign and cancerous breast epithelial cells whereas the role of progestogens is unclear. Tumor-associated macrophages play a crucial role in tumor growth and metastasis due to the synthesis of various cytokines such as tumor necrosis factor-alpha (TNF-alpha), which can stimulate the synthesis of proliferative and angiogenic factors in tumor cells. In an in vitro model we investigated the influence of estradiol and estradiol/progestogens combinations on the changes of TNF-alpha- induced markers., Methods: MCF-7 cells, a human estrogen- and progesterone-receptor-positive human breast cancer cell line, were used for the experiments. Estradiol (E(2)), at a concentration of 0.1 nM, and the progestogens progesterone (P), norethisterone (NET) and medroxyprogesterone acetate (MPA), each at concentrations of 0.01 to 1 microM, were tested alone and in combination. The cells were incubated for 4 days and the markers monocyte chemoattractant protein-1 (MCP-1), vascular endothelial growth factor (VEGF) and matrix metalloproteinase-9 (MMP-9) were measured in the supernatant by enzyme-linked immunosorbent assay., Results: E(2) in combination with TNF-alpha elicited significant increases in MCP-1 and VEGF concentrations compared with TNF-alpha alone. For the progestogens alone an increase of MCP-1 was observed for NET, whereas MPA induced a decrease. An increase of VEGF was observed for all progestogens, the effect being greatest for MPA. No changes were found for MMP-9. In combinations with E(2), the E(2)-induced increase of MCP-1 was reduced by NET and MPA and the increase of VEGF was diminished by P and NET, but not by MPA. The E(2)-induced decrease of MMP-9 was not antagonized by P and NET, but completely abolished by MPA., Conclusion: Our results indicate that E(2) may have a stimulating effect on pre-existing tumor growth and metastasis. This effect seems to be influenced by progestogens in a different manner. Thus the choice of progestogen addition to estrogen therapy may be important, especially since different effects can occur in the case of pre-existing tumor cells.

Objectives: Certain estradiol metabolites may play a pivotal role in breast carcinogenesis. Of special interest are the metabolites 2-hydroxyestradiol (2-OHE2), which can react anti-carcinogenically, and 4-hydroxyestradiol (4-OHE1) and 16a-hydroxyestrone (16-OHE1), which may have procarcinogenic potential. In the present study, we have compared for the first time the effect of these metabolites and their parent substance 17beta-estradiol (E2) on proliferation, apoptosis, apoptosis markers and markers of metastatic property of human breast cancer cells., Methods: MCF-7 cells (human estrogen-receptor positive metastatic breast cancer cell line) were incubated with the estrogens at concentrations of 0.1-100 nM. Cell proliferation rate was measured by the ATP-assay. Apoptosis was measured by cell death assay and the apoptosis markers cytochrome C, Bcl-2, Fasl and p53 were determined in cell lysates by ELISAs. The markers of metastatic property of the cell line, VEGF and MCP-1 were measured in the cell supernatant by ELISAs., Results: The estrogens E2, 4-OHE2 and 16-OHE1 display a proliferative effect on MCF-7 cells which is accompanied by a down-regulation of apoptosis. Various markers of apoptosis such as Bcl-2, cytochrome C and p53 appear to be involved. No significant effect was found for the metabolite 2-OHE2. VEGF and MCP-1 were up-regulated by E2 and 16-OHE1, whereas 2-OHE2 and 4-OHE2 did not show any effect., Conclusions: The most potent estrogen regarding proliferation, apoptosis and metastasis of breast cancer cells seems to be estradiol. However, the estradiol metabolites 4-OHE2 and 16-OHE1 elicit similar properties on cell proliferation, apoptosis and metastasis as compared to estradiol but only at higher concentrations. In contrast 2-OHE2 did not show any significant effect on these parameters. Thus, intracellular estradiol metabolism may determine an individual's risk for breast carcinogenesis.

In the field of estrogen therapy breast cancer risk is one of the most controversially discussed topic. Actually, the as yet largest placebo-controlled study, the Women's Health Initiative, rather showed a risk reduction, in contrast to observational studies. In the present study we have investigated the effect of estradiol on TNF-alpha-induced changes of various markers in human breast cancer cells and compare it with the effect of the antiestrogens tamoxifen and 2-methoxyestradiol. MCF-7 cells were used for the experiments, the incubation time was 96 h. TNF-alpha elicited a 3-4-fold increase of monocyte-attracting protein-1 (MCP-1) and interleukin-8 (IL-8) as compared to the control value, matrix metalloproteinase-9 (MMP-9) and vascular endothelial growth factor (VEGF) were enhanced by 30 to 40%. E2 alone had no effect on MCP-1, slightly reduced the synthesis of MMP-9 and increased VEGF concentrations by about 20%. In combination with TNF, E2 induced a further stimulation of MCP-1, IL-8 and VEGF, whereby the MMP-9 synthesis was not changed. Tamoxifen and the endogenous estradiol metabolite 2-methoxyestradiol seem to be able to partly inhibit the action of TNF-alpha and estradiol. Our results suggest that estrogens may slightly increase tumor growth and spreading beyond the effect of chemokines such as TNF-alpha. However, the magnitude of this E2 effect seems to be marginal as compared to the effect of TNF-alpha alone. The risk of recurrence of breast cancer in patients taking hormone therapy after breast cancer may be slightly enhanced by estrogens, but seems mainly to be driven by the potency of still existing tumor cells to secrete chemokines which can stimulate tumor growth and spreading.

Purpose of Investigation: The etiology of ovarian cancer appears to be associated with a long-term influence of estrogens. However, evidence is accumulating that certain estradiol metabolites may play a decisive role in the carcinogenesis of estrogen-dependent diseases. In the present study we examined the effect of estradiol metabolites on the proliferation and apoptosis of human ovarian cancer cells in comparison to the effect of their parent substance., Methods: The ovarian cancer cell line OVCAR-3 was used for the experiments. 17Beta-estradiol (E2), 2-hydroxyestradiol (2-OHE2), 4-hydroxyestradiol (4-OHE2) and 16alpha-hydroxyestrone (16-OHE1) were incubated for seven days in the concentration range of 0.01 nM to 10 nM. Proliferation and apoptosis were measured by commercially available assay kits., Results: E2 enhanced proliferation rate and concomitantly reduced apoptotic rate of the ovarian cancer cells at physiological concentrations. 2-OHE2 had no significant effect, whereas 4-OHE2 elicited similar effects on proliferation and apoptotic rate as E2. The greatest proliferative and antiapoptotic effect was observed for 16-OHE1, the values being significantly different to the effects of E2., Conclusion: The pattern of endogenous estradiol metabolism may play a role in defining ovarian cancer risk. This may be of importance in certain predisposed women who are treated with hormone therapy in postmenopause.

Objectives: Long-term hormone replacement therapy is associated with an increased breast cancer risk. Evidence is accumulating that estradiol metabolites are involved in carcinogenesis. These metabolites may have proliferating and anti-proliferative properties. We have investigated the effect of 14 metabolites on the proliferation of human breast cancer cells and on the proliferation of human vascular endothelial cells., Methods: As cell model, human umbilical vein endothelial cells (HUVEC) and the human breast cancer cell line MCF-7 were used. The relationship between dosage and effect was tested over the pharmacological concentration range of 10(-8) to 10(-5) M., Results: In HUVECs, all of 10 A-ring metabolites tested stimulated the growth of the endothelial cells at the lower concentrations. At the highest concentration, some A-ring metabolites caused significant inhibitions. The D-ring metabolites showed no marked effects compared to the A-ring metabolites. In MCF-7 cells also, nearly all A-ring metabolites demonstrated a biphasic reaction behaviour on cell proliferation. For the D-ring metabolites, this biphasic pattern was only found for 16 alpha-hydroxyestrone, but the inhibitory effect of this metabolite was weak., Conclusion: These results indicate that certain endogenous estradiol metabolites are able to stimulate breast cancer cell proliferation, and others may be suitable for breast cancer treatment when used in high dosages, since they inhibit cancer cell growth as well as neoangiogensis. This may be of special importance for therapy, since some of these metabolites are virtually devoid of any oestrogenic activity.

The anti-estrogenic substance tamoxifen is effective in the adjuvant therapy applied in human breast cancer. Since it partly exhibits estrogenic activity and has serious side-effects, however, pure anti-estrogenic compounds are being sought. In our experimental study, we compared the anti-proliferative effect of estradiol and 13 endogenous estradiol metabolites on human breast cancer cells with the effect of tamoxifen. We used MCF-7 and MDA-MB 231, the well-established estrogen receptor-positive and -negative cell lines. 4-hydroxytamoxifen, the active metabolite of tamoxifen, estradiol and 13 estradiol metabolites were tested in concentrations ranging from 3.1 to 100 microM. Incubation time was 4 days and cell proliferation was measured by means of the ATP chemosensitivity test. 4-hydroxytamoxifen showed an IC50 value of 27 microM and 18 microM in MCF-7 and MDA-MB 231 cells, respectively. Estradiol and its metabolites were anti-proliferative in both cell lines. A few A-ring metabolites were more effective in inhibiting cell proliferation than D-ring metabolites and the parent substance 17beta-estradiol. 4-OHE1, 2-MeOE1 and 2-MeOE2 were as effective in both cell lines as tamoxifen. For the first time it has been demonstrated that endogenous estradiol metabolites are equally anti-proliferative as tamoxifen in the context of human breast cancer cells. Since some of these metabolites exhibit no estrogenic activity, they are likely to be valuable in clinical studies of chemoprevention and adjuvant therapy of breast cancer.

Objective: Recent clinical studies indicate that an increase in D-ring estradiol metabolites over A-ring metabolites may be a risk factor for breast cancer. The present work was aimed to investigate the effect of oral contraceptives (OC) on the endogenous estradiol metabolism in premenopausal women., Methods: Two studies were conducted, firstly comparing 2 different progestins, i.e. norethisterone and dienogest, each in combination with a constant ethinyl estradiol dosage (study A) and secondly comparing a single progestin, i.e. levonorgestrel in 2 ethinyl estradiol/progestin dosage combinations (study B). The main A- and D-ring metabolites, i.e. 2-OHE1 and 16-OHE1, were measured by enzyme immunoassay in 8-h night-urine collected before and after 3 cycles of OC administration., Results: In study A, i.e. ethinyl estradiol plus dienogest or norethisterone acetate, the ratios of 16-OHE1 to 2-OHE1 before administration were 0.62 and 0.68, and after 3 months 0.31 and 0.54, respectively. The ratio after ethinyl estradiol and dienogest was significantly lower after treatment. In study B, i.e. ethinyl estradiol plus levonorgestrel (0.03 mg/0.15 mg and 0.02 mg/0.1 mg), the ratios before treatment were 0.71 and 0.75 for the higher and the lower dosages, respectively, which changed not significantly to 0.73 and 0.71 after 3 cycles., Conclusion: OCs containing norethisterone acetate, dienogest or levonorgestrel did not have a negative effect on estradiol metabolism, i.e. they did not elicit a higher D-ring metabolism, which is considered to increase breast cancer risk.

Purpose of Investigation: The newly available nasal estradiol application serum concentrations measure up to 10-fold higher compared to common hormone replacement therapy (HRT). Since this has only a short-time peak it has been suggested that breast cancer risk may be reduced, because of lower activation of the estradiol-receptor-transcription cascade., Methods: Estradiol was tested at 10(-9) and 10(-8) M corresponding to the serum levels achieved with nasal application. In addition also comparable exposure times were investigated: Incubation time was 10, 20, 40, 60 and 180 min every day for four days, compared with continuous non-stop incubation for four days. The well-recognized MCF-7 breast cancer cell line was used. Cell proliferation was measured by the ATP-assay., Results: Estradiol in concentrations achieved with 'pulsed' HT was able to elicit proliferation of MCF-7 cells already after an incubation time of ten minutes which correlates with the minimum of exposure time using nasal estradiol application. Compared to non-stop continuous estradiol added in the same high concentrations over four days no significant differences were observed., Conclusions: Short-time application of estradiol is able to stimulate breast cell proliferation to the same degree as continuously administered estradiol in high concentrations. Thus it remains questionable, if a 'pulsed' estradiol therapy may reduce breast cancer risk during HRT. Clinical studies are urgently needed to reveal the breast cancer risk during nasal estradiol application especially since this risk might be increased depending on the concentrations used.

Purpose of Investigation: The endogenous estradiol metabolite, 2-methoxyestradiol (2ME), has been shown to be a potent inhibitor of cell growth and a strong anti-angiogenic substance. We investigated for the first time whether in vitro combinations of 2ME with various chemotherapeutic compounds may result in an additive inhibitory effect on the proliferation of human ovary cancer cells., Method: As a model two different human ovary cancer cell lines were used. All cell lines were incubated with equimolar concentrations of 2ME (0.8-25 microM) and the chemotherapeutics epirubicine, doxorubicine, paclitaxel, docetaxel, carboplatin, vinorelbine, 5-fluorouracil and mafosfamide. Proliferation was measured after four days using the ATP-chemosensitivity test., Results: For both ovary cancer cell lines a significant additive effect of 2ME with epirubicine and carboplatin was observed at the lower concentration range of these chemotherapeutic substances., Conclusion: 2ME is able to enhance the antiproliferative activity of certain chemotherapeutics at pharmacological relevant concentrations. This estradiol metabolite is currently in a phase II trial in patients with refractary metastatic breast cancer and the tolerability has been shown to be very good. The combination of 2ME with chemotherapeutics may therefore offer a new clinically relevant treatment regimen for hormone-dependent cancer.

Purpose of Investigation: Estradiol is the main proliferating substance for human epithelial breast cells. There is evidence that proliferation is also stimulated by growth factors mainly synthesized by the surrounding stroma. In the present in vitro study we have compared the proliferating activity of three growth factors alone and in combination with estradiol in human breast cancer cells., Method: As a cell model the well-recognized human breast cancer cell line MCF-7 was used. The growth factors tested were basic fibroblast growth factor (bFGF), epithelial growth factor (EGF) and insulin-growth factor-1 (IGF-I) at the concentrations 10(-10), 10(-9) and 10(-8) M alone and in equimolar combination with 10(-10) M estradiol. Cell proliferation was measured after seven days incubation by the ATP-assay., Results: The growth factors alone significantly stimulated proliferation of MCF-7 cells in the tested concentration range. The increases were about 40% for EGF, between 40 and 60% for FGF and between 30 and 90% for IGF-I. The strongest proliferating factor was estradiol alone with values between 70 and 130%. For all three growth factors an additive effect was seen in combination with estradiol, which was greatest for IGF-I., Conclusion: These data indicate that estradiol is the strongest proliferating factor for human breast cancer cells. Therefore the predictive value of cell models using estradiol as a proliferative agent seems to be highly reliable. However, stromal influence by growth factors should not be disregarded, since proliferation of pre-existing malignant cells may be accelerated by the concomitant presence of estrogens and growth factors.

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 17beta-estradiol (E(2)) with those of the main equine components of CEE, i.e. equilin (Eq) and 17alpha-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 E(2), 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 mumol/l and 10 mumol/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 E(2), followed by Eq and Dheq, which showed a slightly lower proliferative activity than E(2). The addition of progesterone inhibited E(2)-induced proliferation by about 30%, but only at the high non-physiological concentration of 10 mumol/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 mumol/l when combined with Dheq. The same held true when MPA and NET were added at the high pharmacological concentration of 10 mumol/l, causing increases of about 10%., Conclusions: Our results indicate that equine estrogens have a proliferative action similar to that of 17beta-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.

Objective: Combination of a statin plus estrogen may reveal benefits on the cardiovascular system in postmenopausal women by additively ameliorating both the lipid profile and vascular function. Long-term therapy with estrogens, however, is associated with an increase of breast cancer risk. In contrast, evidence is accumulating that statins may inhibit carcinogenesis because of their central action on important cellular functions. It is of special clinical interest whether a statin/estrogen combination may reduce the most undesired side effect of estrogen therapy, that is, an increase in breast cancer risk. Therefore, in the present in vitro study, for the first time we have compared the effect of five statins on the proliferation of human breast cancer cells alone and in the presence of stimulatory estradiol (E(2))., Design: As cell models, the receptor-positive cell line MCF-7 and the receptor-negative cell line MDA-MB 231 were used. The statins atorvastatin, fluvastatin, lovastatin, pravastatin, and simvastatin were tested in the concentration range of 1.6 microm to 50 microm alone and in the range of 0.01 nm to 10 microm in combination with E(2). Cell proliferation was measured after 4 days by the adenosinetriphosphate-chemosensitivity test., Results: All statins except pravastatin were able to significantly inhibit dose dependently the cell proliferation of both cell lines. The inhibitory values were between 10% and 90%, whereby the potency was greater in the case of receptor-negative cancer cells. A significant difference in the efficacy of the statins was observed for MCF-7 cells, in which atorvastatin was less effective than the other statins. In contrast, in the presence of E(2), the statins showed similar antiproliferative actions in MCF-7 cells when tested in the concentration range of 0.01 nm to 10 microm. A reduction of cell proliferation of less than 10% was observed at the lower concentrations and between 15% and 25% at the highest concentration of 10 microm., Conclusions: The present data indicate that statins can inhibit the proliferation of receptor-positive and -negative human breast cancer cells but failed to completely abrogate the E(2)-induced proliferation of receptor-positive breast cancer cells. Clinical trials, however, are necessary to prove this anticarcinogenic action of statins.

Endocrine therapy is widely accepted for the treatment of hormone receptor-positive breast cancer. However, in many cases eventually resistance will develop and tumor regrows. Combination therapy may be one way to resolve this problem. In the present study we investigated the effect of a combination of the widely used antiestrogen tamoxifen with the endogenous estradiol metabolite 2-methoxyestradiol (2-ME) on the proliferation of human estrogen receptor-positive and receptor-negative breast cancer cells. The receptor-positive cell line MCF-7 and the receptor-negative cell line BM were treated with 4-hydroxytamoxifen (4-OHTam) and 2-methoxyestradiol in the concentration range of 0.8-25 microM alone and equimolar combinations for 4 days. The proliferation of the cells was determined using the ATP-chemosensitivity test.4-Hydroxytamoxifen inhibited proliferation of MCF-7 and BM cells with IC(50) values of 31 and 10 microM, the corresponding figures for 2-methoxyestradiol were 52 and 8 microM. The combination showed IC(50) values of 6 microM and 4 microM. These results indicate that a combination of tamoxifen with 2-methoxyestradiol showed an additive inhibitory effect concerning the proliferation of estrogen receptor-positive and receptor-negative breast cancer cell lines. Thus a combination of these substances may allow ameliorating of adverse events of tamoxifen by reducing its concentrations and probably also drug resistance and should be tested in clinical trials.

Progestogens are added to oestrogen in hormone replacement therapy regimens to reduce the risk of endometrial cancer. We have performed in vitro studies analysing gene expression of isolated normal endometrial epithelia cells (NEE) treated with estradiol and the progestogen norethisterone acetate (NETA). We report here for the first time upregulation of the Wnt-7a gene by NETA in estrogen treated NEE. Wnt genes are a large family of developmental genes associated with cellular responses such as oncogenesis. We therefore suggest that upregulation of Wnt-7a may be associated with the antineoplastic effects of progestogens on the endometrium.

Objectives: Changes in estradiol metabolism may play a role in the pathophysiology of different diseases, of special interest being an increase in D-ring over A-ring metabolites for the risk of breast cancer. In the present work we investigated the effect of exogenous estradiol therapy on endogenous estradiol metabolism in postmenopausal women., Methods: Three different studies were carried out in 126 women: in study A the women were treated for 4 weeks either with oral or with transdermal 17beta-estradiol, in study B for 4 weeks with oral or transdermal 17beta-estradiol sequentially combined with oral or transdermal norethisterone acetate, and in study C for 12 weeks either with oral 17beta-estradiol or with an oral continuous combination of 17beta-estradiol with the new progestin dienogest. As main representatives of the A- and D-ring metabolism, 2-hydroxyestrone (2-OHE1) and 16 alpha-hydroxyestrone (16-OHE1), respectively, were measured in 8 h night urine using enzyme immunoassay technique., Results: Oral estradiol treatment resulted in a significant higher excretion of estradiol metabolites compared to transdermal treatment. Neither oral nor transdermal estradiol induced a significant change in the ratio of 16-OHE1 to 2-OHE1. The addition of oral or transdermal norethisterone acetate to estradiol did not alter on average the endogenous estradiol metabolism, although in individual patients a significant increase in 16-OHE1 metabolism was observed only with oral norethisterone. The continuous oral addition of dienogest did not lead to any significant change in estradiol metabolism., Conclusions: These results indicate that oral estradiol replacement therapy enhances the quantity of circulating estradiol metabolites. This may have a more negative impact on estrogenic target cells as compared to transdermal application. Progestin addition to estradiol replacement therapy seems to have no major impact on endogenous estradiol metabolism. Further studies, however, are necessary to evaluate the progestin effect in possible pre-disposed patients.

Tibolone is a synthetic progestin with estrogenic and progestogenic properties, widely used for alleviation of menopausal syndromes and for osteoporosis prophylaxis in postmenopausal women. Since only little data are available on tibolone and breast cancer risk the present study investigates the effect of tibolone on the growth of the human breast cancer cell line, MCF-7. Tibolone is clinically comparable to an estradiol/norethisterone combination, therefore we included this hormone combination in our experiments. Tibolone was examined alone and in the presence of 0.1 nM estradiol in the concentration range from 0.001 microM to 1 microM. Norethisterone was studied using the same concentration range in combination with 0.1 nM estradiol. Tibolone led to significant cell growth in the concentration range of 0.01 to 1 microM and was able to significantly stimulate estradiol-induced proliferation at the concentrations 0.01 and 0.1 microM. In contrast, the estradiol/norethisterone combination elicited significant inhibition of cell growth at the concentrations 0.001 and 0.01 microM. These data suggest that tibolone does have tumor cell-growth promoting effects in vitro whereas the estradiol/norethisterone combination partially inhibits cell growth. Therefore no differences in risk profile are to be expected between conventional hormone substitution using estradiol and norethisterone acetate and tibolone. Drawing a clinical consequence from our experiments would result in not recommending the use of tibolone in postmenopausal women at high risk for breast cancer development until long-term controlled clinical studies have been performed on the effect of tibolone administration and breast cancer risk.

Objective: Little is known about the direct comparison between 17alpha-ethinylestradiol (EE2) and 17beta-estradiol (E2) on pre-existing breast cancer cells and on their angiogenetic effects. In this study we investigated the effect of both estrogens on the proliferation of MCF-7 cells, a human breast cancer cell model, and on human umbilical vascular endothelial cells (HUVECs)., Methods: The steroids were tested in the concentration range of 10(-10) to 10(-5) M. The proliferation of MCF-7 and HUVEC cells was measured after five and six days by the crystal violet staining technique., Results: In the concentration range from 10(-10) to 10(-5) M both estrogens showed a proliferative effect in the MCF-7 cells, E2 having a gradually declining effect on proliferation with increasing concentration while EE2 showed a constant proliferative effect over a large concentration range. At the highest concentration tested E2 had no effect on proliferation while EE2 even inhibited growth. In the HUVEC cells both estrogens showed a slightly significant stimulatory effect at the lowest concentration, and no significant effect at the remaining concentrations. EE2 again inhibited cell growth at the highest concentration., Conclusions: At the serum concentrations seen in hormone replacement therapy, EE2 appears to have less proliferative effect on breast cancer cells compared with E2, while both estrogens appear to have similar effects on endothelial cells.

Objectives: Tibolone, a synthetic norethynodrel derivative, is effective at alleviating climacteric complaints and for osteoporosis prophylaxis. Concerning the cardiovascular system little data are available on possible direct effects on the vasculature compared with hormone replacement therapy. Since tibolone is pharmacologically best comparable to an estradiol/norethisterone combination (E2/NET), the aim of the present study was to compare these treatment regimens with respect to their effects on human vascular cells, this being the first study to investigate this question. -, Material and Methods: Human female coronary arteries were used, the most appropriate vessels for our issue in question. The experiments were conducted with endothelial cells as well as smooth muscle cells. Tibolone and E2/NET were tested at the concentrations 0.1, 1 und 10 microM. Incubation time was 24 h for endothelial cell cultures and 7 days for smooth muscle cell cultures. The effects on markers of pathophysiologically different problems were studied in a comparable manner: Markers of endothelial function such as prostacyclin, endothelin and plasminogen-activator-inhibitor-1 (PAI-1), markers of plaque initiation such as the adhesion molecules E-Selectin, ICAM-1 and the monocyte attraction protein-1 (MCP-1) and markers of plaque stability such as the precursor of the matrix-metalloproteinase-1 (pro-MMP-1). -, Results: Tibolone reduced the synthesis of endothelin as well as the concentrations of E-Selectin, PAI-1 and pro-MMP-1. The magnitude of the effects on these markers varied and was in the range of 11-42 %. similar inhibitions were seen for E2/NET; yet a significantly stronger inhibitory effect was found for pro-MMP-1. Moreover, E2/NET also reduced concentrations of ICAM-1 and MCP-1. Concerning smooth muscle cells only E2/NET was able to elicit an anti-proliferative effect. -, Conclusions: Tibolone can positively influence the synthesis of markers in cell cultures of human female coronary artery which modulate vascular tone and which play a decisive role in the various stages of atherosclerosis. In this respect estradiol combined with norethisterone is as potent as tibolone, in addition this combination is partially more effective and able to influence a wider range of markers which are important in the early stages of atherogenesis or for the stability of atherosclerotic plaques. Experimental studies such as the present one are useful to explore mechanisms, but clearly cannot replace clinical studies.

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 17 beta-estradiol, fluvastatin and equimolar combinations of the two in concentrations of 0.01, 0.1 and 1 mumol/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.

Objectives: Aim was to compare the effects of estradiol-only therapy with combined estradiol/progestin treatment on the excretion of vasoactive mediators surrogating on possible effects in the vascular system. The progestin used was dienogest, a new C19-progestin with antiandrogenic properties., Methods: Prospective, randomized trial, 25 healthy postmenopausal women treated for 3 months with estradiol valerate (2 mg/day) and 27 women with estradiol valerate (2 mg/day) continuously combined with dienogest (2 mg/day). Assessment of the following markers or their stable metabolites in nocturnal urine: cGMP, serotonin, prostacyclin and thromboxane, and urodilatin., Results: Estradiol alone increased the excretion of cGMP and serotonin significantly suggesting vasodilating effects. The prostacyclin/thromboxane ratio known to be crucial for the relation of vasorelaxation to vasoconstriction significantly increased. No significant changes were found for urodilatin, which is known to elicit different effects in the cardiovascular and renal system, respectively. Combined estradiol/dienogest therapy also led to significant increases in cGMP and serotonin excretion suggesting that progestin addition for three months does not affect these markers. However, in contrast to estrogen-only treatment, there was no significant increase for the prostacyclin/thromboxane ratio, which can be explained by antagonistic action of the progestin. The excretion of urodilatin was increased significantly, which might be due to counterbalancing progestin effects in the renal vascular system., Conclusions: The changes in vasoactive markers suggest an estrogen effect that is vasorelaxant. Since there were no significant differences between the two groups, possible vascular effects of the progestin dienogest, for the first time evaluated, might not be of clinical relevance, at least not in women without cardiovascular diseases.

Objective: The combination of an estrogen with a statin for therapy of postmenopausal women is of interest because both substance classes exert beneficial effects on the lipid profile. However, both substance classes also elicit positive direct effects on the vasculature. Therefore in the present in vitro study an estrogen/statin combination was investigated for its effect on biochemical markers of endothelial function., Material and Methods: In endothelial cell cultures from human coronary arteries, the effect of estradiol/fluvastatin, alone and in equimolar combinations, were tested at the concentrations 0.01, 0.1, and 1 microM. The vasodilator prostacyclin, the vasoconstrictor endothelin, endothelial nitric oxide synthase (responsible for synthesis of the vasodilator nitric oxide), the procoagulatory factor plasminogen activator inhibitor-1, and the monocyte chemoattractant protein were chosen as markers., Results: The estradiol/fluvastatin combination was able to increase prostacyclin production (25-100%) in an additive manner. The reduction of endothelin synthesis in the range of 21-46% was higher with the combination than with the monosubstances; the reduction, however, was not statistically significant. The expression of endothelial nitric oxide synthase was not significantly increased by the combination compared with the monosubstances, however, a tendency to an additive increase was observed. For the synthesis of plasminogen activator inhibitor-1, no significant changes were seen for either the monosubstances or the combination. The synthesis of monocyte chemoattractant protein-1 was decreased by the combination between 21% and 40%; the decrease, however, was not statistically significant compared with the effect of the monosubstances. The effective estradiol concentrations are higher than can be achieved by replacement therapy; in contrast, fluvastatin was effective at concentrations that can be reached during clinical treatment., Conclusions: These results indicate that an estrogen/statin combination exerts beneficial effects on the vasculature that seem to be superior to the effects of the monosubstances. The changes found for the biochemical markers can improve endothelial function. The presented results should encourage the performance of clinical studies in cardiovascular risk patients with estrogen/statin combinations.

Objective: Little is known on the type of progestin and regimen type in relation to breast cancer risk. We have compared the effect of medroxyprogesterone acetate (MPA) and norethisterone (NET) on the estradiol stimulated proliferation in MCF-7 cells with respect to different regimens used in combined hormone replacement therapy (HRT)., Design: To approximate the in vivo conditions in HRT, MCF-7 cultures were pretreated with estradiol followed by estradiol/progestin treatment to represent the sequential combined model and compared with non pretreated cultures followed by estradiol/progestin treatment for the continuous combined model., Results: When using progestins in the continuous combined form with estradiol (10(-10) M) both progestins showed a significant reduction in the estradiol stimulated proliferation of the MCF-7 cells. In the sequential combined model the addition of MPA led to a stronger significant reduction of MCF-7 proliferation but in a narrower concentration range (from 10(-8) to 10(-6) M) compared to the continuous treatment. NET did not show any significant effect on proliferation in the SC model., Conclusion: Different regimen types and different progestins do lead to significantly different effects on the proliferation of a breast cancer cell line. These findings might be useful in the elucidation of potential mechanisms involved in the clinical situation.

Objective: The addition of progestogen to estrogen replacement therapy is thought to antagonize, at least in part, the beneficial effects of estrogens on the vasculature. The aim of this study was to investigate the effect of two progestogens mostly used in clinical practice on the proliferation of vascular smooth muscle cells, which has been demonstrated to be a crucial step in the development of atherosclerosis., Material and Methods: The effect of medroxyprogesterone acetate (MPA) and norethisterone (NET), which represent the two different classes of C21- and C19-progestogens, respectively, was investigated on proliferation of smooth muscle cells from human coronary artery in vitro. The steroids were tested in the concentration range 10(-8) to 10(-5) M, which is in the upper range of that reached during hormonal replacement therapy, and compared with control values., Results: Estradiol significantly inhibited serum-stimulated cell growth at the concentrations 10(-6) and 10(-5) M by 18% and 34%, respectively. MPA significantly enhanced serum-stimulated growth at the concentrations 10(-6) and 10(-5) M by 29% and 47%, respectively. In equimolar combinations of MPA and estradiol, proliferation of the cells was significantly stimulated at the concentrations 10(-6) and 10(-5) M by 26% and 44%, respectively. In contrast, NET had no significant effect on serum-stimulated cell growth and had no impact on the estradiol-inhibited proliferation., Conclusions: These data suggest that MPA may antagonize beneficial antiatherosclerotic estradiol effects on the vasculature, whereas NET may be neutral in this respect. However, these effects occurred at supraphysiological concentrations. Because these concentrations might be reached in the target tissues, the clinical relevance for treatment of patients with cardiovascular risk cannot be excluded.

Objective: The aim of the study was to examine the effects of two different, clinically relevant, hormone replacement therapy (HRT) regimen types, continuous combined and sequential combined, on breast cancer cells by means of an in vitro model. The study was carried out using the C21-progestin medroxyprogesterone acetate (MPA) in combination with estradiol, and with the proliferation of MCF-7, a human breast cancer cell-line, as end-point., Methods: Proliferation of MCF-7 cells was measured by means of a crystal violet staining technique. Growth was triggered using a constant estradiol concentration of 10(-10) mol/l, while varying the MPA concentration from 10(-11) to 10(-6) mol/l., Results: The continuous combined model of treatment led to the inhibition of estradiol-induced growth of MCF-7 with MPA concentrations of 10(-10) mol/l and upwards, compared with estradiol-alone-induced growth. The sequential combined model showed a greater inhibition at the higher MPA concentrations of 10(-8)-10(-6) mol/l, with reduced sensitivity to inhibition at the lower MPA concentrations tested, of 10(-11)-10(-9) mol/l. The different treatment types resulted in significantly different sensitivities of the MCF-7 cells to inhibition of estradiol-induced proliferation at the higher MPA concentrations of 10(-8)-10(-6) mol/l., Conclusions: The results demonstrate the importance of considering in vivo factors in an in vitro model with regard to improving the cell culture techniques used, to obtain a clearer picture of the possible mechanisms involved in the potential breast cancer risk with different HRT regimens.

Objective: The effect of estradiol on the renal excretion of vasoactive substances was studied in postmenopausal women. The following markers surrogating an estrogenic effect on the cardiovascular system were measured: prostacyclin and thromboxane, cGMP, which reflects systemic nitric monoxide production, serotonin and relaxin., Methods: The effect of estradiol was compared using two clinical forms of administration, transdermal and oral, in two groups of 20 postmenopausal women each. The treatment was carried out for two and four weeks, respectively. Nocturnal urine was collected over 8 hours before and after estradiol treatment. The quantity of markers excreted during the experiment was determined., Results: Excretion of prostacyclin and thromboxane, calculated as prostacyclin/thromboxane ratio, was increased using both forms of administration. Both forms of treatment brought about only slight non-significant changes in renal cGMP excretion compared with values before treatment. The production of serotonin and relaxin was only increased using transdermal treatment., Conclusion: The resulting data show that estradiol replacement in postmenopausal women is able to increase renal excretion of various vasoactive substances implying a vasodilative effect of estrogen. This was seen, both in transdermal and oral administration, transdermal application having a more pronounced effect on the markers than oral administration.

The in vitro effect of a statin and an estrogen on oxidation of human LDL was investigated for the first time, comparing the monosubstances fluvastatin and 17beta-estradiol as well as the effect of the combination. Fluvastatin significantly delayed the onset of LDL oxidation (controls = 90 min) by 23 min at 1 microM, by 110 min and by 221 min at 5 and 10 microM, respectively. 17Beta-estradiol significantly impeded the onset by 70 min at 1 microM and by more than 300 min at 5 and 10 microM. The combination of 1 microM 17beta-estradiol with 1, 5 and 10 microM fluvastatin showed an additive antioxidative effect; the onset was delayed by 100 min, by 167 min and by more than 300 min, respectively. It can be concluded that treatment of postmenopausal women with fluvastatin and 17beta-estradiol may have not only beneficial effects on lipid disorders but may also elicit a direct potent anti-atherosclerotic action on the vasculature.