The anti-aromatase effect of progesterone and of its natural metabolites 20alpha- and 5alpha-dihydroprogesterone in the MCF-7aro breast cancer cell line.

BACKGROUND: Progesterone is metabolized in the normal breast mainly into 4-ene-pregnenes (e.g. 20alpha-dihydroprogesterone, 20alphaDHP) but, in contrast, in breast cancer tissue the 5alpha-dihydropregnanes (e.g. 5alpha-dihydroprogesterone, 5alphaDHP) are prevalent. In the present study the effect of progesterone and its main metabolites 20alphaDHP and 5alphaDHP on the aromatase activity in a stable aromatase-expressing estrogen receptor-positive human breast cancer cell line, MCF-7aro, was explored. MATERIALS AND METHODS: The MCF-7aro cells were stripped of endogenous steroids and incubated with physiological concentrations of [3H]-testosterone ([3H]-testos: 5 x 10(-9)M) alone or in the presence of progesterone, 20alphaDHP or 5alphaDHP (5 x 10(-6) or 5 x 10(-8)M) for 24 h at 37 degrees C. The cellular radioactivity uptake was determined in the ethanolic supernatant and the DNA content in the remaining pellet. [3H]-Estradiol (E2), [3H]-estrone (E1) and [3H]-testos were characterized by thin layer chromatography and quantified using the corresponding standard. RESULTS: Aromatase activity was present at a high level in the MCF-7aro cells after incubation with [3H]-testos when the concentration of [3H]-E2 was 3.70 pmol/mg DNA; 20alphaDHP at concentrations of 5 x 10(-6)M or 5 x 10(-8)M significantly inhibited this conversion by 50.3% and 36.5%, respectively. No significant effect was found with the metabolite 5alphaDHP or the parent hormone, progesterone. CONCLUSION: The MCF-7aro cell line shows high detectable aromatase activity. The present data indicate that the progesterone metabolite 20alphaDHP, found mainly in normal breast tissue, can act as an anti-aromatase agent.

Estradiol inhibits the estrone sulfatase activity in normal and cancerous human breast tissues.

It is well accepted that estradiol (E2) plays an important role in the genesis and evolution of breast cancer. Quantitative evaluation indicates that in human breast tumor, estrone sulfate (E1S) 'via sulfatase' is a much more likely precursor for E2 than is androstenedione 'via aromatase'. In previous studies, it was demonstrated that in isolated MCF-7 and T-47D breast cancer cell lines, estradiol can block estrone sulfatase activity. In the present study, the effect of E2 was explored using total normal and cancerous breast tissues. This study was carried out with post-menopausal patients with breast cancer. None of the patients had a history of endocrine, metabolic or hepatic diseases or had received treatment in the previous 2 months. Each patient received local anaesthetic (lidocaine 1%) and two regions of the mammary tissue were selected: (A) the tumoral tissue and (B) the distant zone (glandular tissue) which was considered as normal. Samples were placed in liquid nitrogen and stored at -80 degrees C until enzyme activity analysis. Breast cancer histotypes were ductal and post-menopausal stages were T2. Homogenates of tumoral or normal breast tissues (45-75 mg) were incubated in 20 mM Tris-HCl, pH 7.2 with physiological concentrations of [3H]-E1S (5 x 10(-9)M) alone or in the presence of E2 (5 x 10(-5) to 5 x 10(-7) M) during 30 min or 3 h. E1S, E1 and E2 were characterized by thin layer chromatography and quantified using the corresponding standard. The sulfatase activity is significantly more intense with the breast cancer tissue than normal tissue, since the concentration of E1 was 3.20 +/- 0.15 and 0.42 +/- 0.07 pmol/mg protein, respectively after 30 min incubation. The values were 27.8 +/- 1.8 and 3.5 +/- 0.21 pmol/mg protein, respectively after 3 h incubation. Estradiol at the concentration of 5 x 10(-7) M inhibits this conversion by 33% and 31% in cancerous and normal breast tissues, respectively and by 53% and 88% at the concentration of 5 x 10(-5) M after 30 min incubation. The values were 24% and 18% for 5 x 10(-7) M and 49% and 42% for 5 x 10(-5) M, respectively after 3h incubation. It was observed that [3H]-E1S is only converted to [3H]-E1 and not to [3H]-E2 in normal or cancerous breast tissues, which suggests a low or no 17beta-hydroxysteroid dehydrogenase (17beta-HSD) Type 1 reductive activity in these experimental conditions. In conclusion, estradiol is a strong anti-sulfatase agent in cancerous and normal breast tissues. This data can open attractive perspectives in clinical trials using this hormone.

Estradiol as an anti-aromatase agent in human breast cancer cells.

Estradiol (E(2)) is an important risk factor in the development and progression of breast cancer. However, a "direct effect" of E(2) in breast cancerization has not yet been demonstrated. The estrogen receptor complex can mediate the activation of oncogens, proto-oncogens, nuclear proteins and other target genes that can be involved in the transformation of normal to cancerous cells. Breast cancer cells possess all the enzymes (sulfatase, aromatase, 17beta-hydroxysteroid dehydrogenase (17beta-HSD)) necessary for the local bioformation of E(2). In the last years, many studies have shown that treatment of breast cancer patients using anti-aromatase agents has beneficial therapeutic effects. The aromatase activity is very low in most breast cancer cells but was significantly increased in a hormone-dependent breast cancer cell line: the MCF-7aro, using the aromatase cDNA transfection and G-418 (neomycin) selection. In the present study, we explore the effect of E(2) on the aromatase activity of this cell line. The MCF-7aro cell line was a gift from Dr. S. Chen (Beckman Research Institute, Duarte, U.S.A.). For experiments the cells were stripped of endogenous steroids and incubated with physiological concentrations of [(3)H]-testosterone (5 x 10(-9)mol/l) alone or in the presence of E(2) (5 x 10(-5), 5 x 10(-7) and 5 x 10(-9)mol/l) for 24h at 37 degrees C. The cellular radioactivity uptake was determined in the ethanolic supernatant and the DNA content in the remaining pellet. [(3)H]-E(2), [(3)H]-estrone ([(3)H]-E(1)) and [(3)H]-testosterone were characterized by thin layer chromatography and quantified using the corresponding standard. It was observed that [(3)H]-testosterone is converted mainly into [(3)H]-E(2) and not to E(1), which suggests very low or absence of oxidative 17beta-HSD (type 2) activity in these experimental conditions. The aromatase activity, corresponding to the conversion of [(3)H]-testosterone to [(3)H]-E(2) after 24h, is relatively high, since the concentration of E(2) was 2.74+/-0.11pmol/mg DNA in the non-treated cells. E(2) inhibits this conversion by 77, 57 and 21%, respectively, at the concentrations of 5 x 10(-5), 5 x 10(-7) and 5 x 10(-9)mol. In previous studies, it was demonstrated that E(2) exerts a potent anti-sulfatase activity in the MCF-7 and T-47D breast cancer cells. The present data show that E(2) can also block the aromatase activity. The dual inhibition of the aromatase and sulfatase activities, two crucial enzymes for the biosynthesis of E(2) by E(2) itself in breast cancer add interesting and attractive information for the use of estrogen therapeutic treatments.

Enzymes involved in the formation and transformation of steroid hormones in the fetal and placental compartments.

Human fetal and placental compartments have all the enzymatic systems necessary to produce steroid hormones. However, their activities are different and complementary: the fetus is very active in converting acetate into cholesterol, in transforming pregnanes to androstanes, various hydroxylases, sulfotransferases, whilst all these transformations are absent or very limited in the placenta. This compartment can transform cholesterol to C21-steroids, convert 5-ene to 4-ene steroids, and has a high capacity to aromatize C19 precursors and to hydrolyse sulfates. Steroid hormone receptors are present at an early stage of gestation and are functional for important physiological activities. The production rate of some steroids increases drastically with fetal evolution (e.g. estriol increases 500-1000 times in relation to non-pregnant women). We can hypothesize that the control of active steroid hormones could be carried out by fetal and placental factors, which act by stimulating or inhibiting the enzymes involved in their formation and transformation during pregnancy evolution and, consequently, limiting the high levels of the biologically active hormone.

Effect of nomegestrol acetate on estrogen biosynthesis and transformation in MCF-7 and T47-D breast cancer cells.

Although ovaries serve as the primary source of estrogen for pre-menopausal women, after menopause estrogen biosynthesis from circulating precursors occurs in peripheral tissues by the action of several enzymes, 17beta-hydroxysteroid dehydrogenase 1 (17beta-HSD1), aromatase and estrogen sulfatase. In the breast, both normal and tumoral tissues have been shown to be capable of synthesizing estrogens, and this local estrogen production can be implicated in the development of breast tumors. In these tissues, estradiol (E(2)) can be synthesized by three pathways: (1) estrone sulfatase transforms estrogen sulfates into bioactive estrogens, (2) 17beta-HSD1 converts estrone (E(1)) into E(2), (3) aromatase which converts androgens into estrogens is also present and contributes to the in situ synthesis of active estrogens but to a far lesser extent than estrone sulfatase. Quantitative assessment of E(2) formation in human breast tumors indicates that metabolism of estrone sulfate (E(1)S) via the sulfatase pathway produces 100-500 times more E(2) than androgen aromatization. Breast tissue also possesses the estrogen sulfotransferase involved in the conversion of estrogens into their sulfates that are biologically inactive. In the present review, we summarized the action of the 19-nor-progestin nomegestrol acetate (NOMAC) on the sulfatase, 17beta-HSD1 and sulfotransferase activities in the hormone-dependent MCF-7 and T47-D human breast cancer cell lines. Using physiological doses of substrates NOMAC blocks very significantly the conversion of E(1)S to E(2). It inhibits the transformation of E(1) to E(2). NOMAC has a stimulatory effect on sulfotransferase activity in both cell lines, with a strong stimulating effect at low doses but only a weak effect at high concentrations. The effects on the three enzymes are always stronger in the progesterone-receptor rich T47-D cell line as compared with the MCF-7 cell line. Besides, no effect is found for NOMAC on the transformation of androstenedione to E(1) in the aromatase-rich choriocarcinoma cell line JEG-3. In conclusion, the inhibitory effect provoked by NOMAC on the enzymes involved in the biosynthesis of E(2) (sulfatase and 17HSD pathways) in estrogen-dependent breast cancer, as well as the stimulatory effect on the formation of the inactive E(1)S, can open attractive perspectives for future clinical trials.

Differential effects of progestins on breast tissue enzymes.

There is substantial evidence that mammary cancer tissue contains all the enzymes responsible for the local biosynthesis of estradiol (E2) from circulating precursors. Two principal pathways are implicated in the final steps of E2 formation in breast cancer tissue: the 'aromatase pathway' that transforms androgens into estrogens and the 'sulfatase pathway' that converts estrone sulfate (E1S) into estrone (E1) via estrone sulfatase. The final step is the conversion of weak E1 to potent biologically active E2 via reductive 17beta-hydroxysteroid dehydrogenase type 1 activity. It is also well established that steroid sulfotransferases, which convert estrogens into their sulfates, are present in breast cancer tissues. One of the possible means of blocking E2 effects in breast cancer is to use anti-estrogens, which act by binding to the estrogen receptor (ER). Another option is to block E2 using anti-enzymes (anti-sulfatase, anti-aromatase, or anti-17beta-hydroxysteroid dehydrogenase (17beta-HSD). Various progestins (e.g. promegestone, nomegestrol acetate, medrogestone, 17-deacetyl norgestimate, dydrogesterone and its 20-dihydro derivative), as well as tibolone and its metabolites, have been shown to inhibit estrone sulfatase and 17beta-hydroxysteroid dehydrogenase. Some progestins and tibolone can also stimulate sulfotransferase activity. These various progestins may therefore provide a new option for the treatment of breast cancer.

Paradoxical effect of estradiol: it can block its own bioformation in human breast cancer cells.

The great majority of breast cancers are in their early stage hormone-dependent and it is well accepted that estradiol (E(2)) plays an important role in the genesis and evolution of this tumor. Human breast cancer tissues contain all the enzymes: estrone sulfatase, 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD), aromatase, involved in the last steps of E(2) bioformation in this tissue. Quantitative data show that the 'sulfatase pathway', which transforms estrogen sulfates into the bioactive unconjugated E(2), is 100-500 times higher than the 'aromatase pathway' which converts androgens into estrogens. In this paper we explore the effect of E(2) on the sulfatase activity using two hormone-dependent human breast cancer cells: MCF-7 and T-47D. The action of E(2) on the sulfatase activity was evaluated by the conversion of estrone sulfate (E(1)S) into E(2). The cells were incubated in Minimal Essential Medium (MEM) containing 5% steroid-depleted fetal calf serum and incubated with physiological concentrations of [(3)H]E(1)S (5 x 10(-9) M) alone (control) or in the presence of E(2) (5 x 10(-10) to 5 x 10(-5) M) for 24 h at 37 degrees C. It was found that E(2) is a potent inhibitory agent of the estrone sulfatase activity in both cell lines. A low concentration of E(2): 5 x 10(-9) M decreases the sulfatase activity by 67% in MCF-7 cells and 57% in T-47D cells. More than 80% of the decrease in the formation of E(2) was obtained with the dose of 5 x 10(-7) M in both cell lines. It is concluded that this paradoxical effect of E(2) adds a new biological response of this hormone and could be related to estrogen replacement therapy in which it was observed to have either no effect or to decrease breast cancer mortality in postmenopausal women. Preliminary results are indicated in the Proceedings of the 14th International Symposium of the Journal of Steroid Biochemistry & Molecular Biology (Quebec, Canada, 24-27 June 2000) [J. Steroid Biochem. Molec. Biol. 76 (2001) 95-104](1) and presented at the 83rd Annual Meeting of the Endocrine Society (Denver, USA, 20-23 June 2001 (abstract no. P2-615).

The selective estrogen enzyme modulator (SEEM) in breast cancer.

Human breast cancer tissue contains all the enzymes (estrone sulfatase, 17beta-hydroxysteroid dehydrogenase, aromatase) involved in the last steps of estradiol biosynthesis. This tissue also contains sulfotransferase for the formation of the biologically inactive estrogen sulfates. In the last years, it was demonstrated that various progestins (promegestone, nomegestrol acetate, medrogestone), as well as tibolone and its metabolites are potent inhibitors of sulfatase and 17beta-hydroxysteroid dehydrogenase activities. It was also shown that medrogestone, nomegestrol acetate, promegestone or tibolone can stimulate the sulfotransferase activity for the local production of estrogen sulfates. All these data, in addition to numerous agents, which can block the aromatase action, lead to the new concept of selective estrogen enzyme modulators (SEEM), which can largely apply to breast cancer tissue. The exploration of various progestins and other active agents in trials with breast cancer patients, showing an inhibitory effect on sulfatase and 17beta-hydroxysteroid dehydrogenase, or a stimulatory effect on sulfotransferase, will provide a new possibility in the treatment of this disease.

Biological effects of progestins in breast cancer.

The action of progestins is derived from many factors: structure, affinity for the progesterone receptor or for other steroid receptors, the target tissue considered, the biological response, the experimental conditions, the dose and metabolic transformation. The proliferative response to progestins in human breast cancer cells is contradictory: some progestins inhibit, others stimulate, have no effect at all, or have a dual action. For instance, medroxyprogesterone acetate has a stimulatory effect on breast cancer cells after a short period of treatment, but this effect becomes inhibitory when treatment is prolonged. It has been demonstrated that, in hormone-dependent breast cancer cells, various progestins (nomegestrol acetate, medrogestone, promegestone) are potent sulfatase inhibitory agents. The progestins can also involve the inhibition of the mRNA expression of this enzyme. In another series of studies it was also demonstrated that some progestins are very active in inhibiting 17beta-hydroxysteroid dehydrogenase for the conversion of estrone to estradiol. More recently it was observed that the progestins promegestone and medrogestone stimulate sulfotransferase for the formation of estrogen sulfates. Consequently, the action of progestins in blocking estradiol formation via sulfatase, or in stimulating the effect on sulfotransferase activity, can open interesting and new possibilities in clinical applications in breast cancer.

Comparison of estrogen concentrations, estrone sulfatase and aromatase activities in normal, and in cancerous, human breast tissues.

In the present study, the concentrations of estrone (E(1)), estradiol (E(2)) and their sulfates (E(1)S and E(2)S), as well as the sulfatase and aromatase activities, were evaluated in post-menopausal patients with breast cancer. Comparative studies of the evaluation of these parameters were carried out in (a) tumor tissue, (b) areas surrounding the tumor, and (c) areas distant from the tumor (glandular tissue) which were considered as normal tissue. The levels (in pm/g; mean +/- SEM) were: for E(1) in the (a) area: 320+/-95; in (b): 232+/-86; and in (c): 203+/-71; for E(2) in the (a) area: 388+/-106; in (b): 224+/-48; and in (c): 172+/-80; for E(1)S in the (a) area: 454+/-110; in (b): 259+/-90; and in (c): 237+/-65; for E(2)S in the (a) area:318+/-67; in (b): 261+/-72; and in (c): 232+/-75, respectively. The values of E(1)S and E(2) were significantly higher in the tumor tissue than in the area considered as normal. In all the tissues studied, the sulfatase activity was much higher than aromatase (130-200). In addition, the sulfatase levels were significantly higher in the peripheral and in the tumor tissue than in the area considered as normal. The levels of aromatase were significantly higher in tumoral than in normal tissue. The present data extend the "intracrine concept" for breast cancer tumors. The physiopathology and clinical significance as promoter parameters in breast cancer is to be explored.

Control of sulfatase and sulfotransferase activities by medrogestone in the hormone-dependent MCF-7 and T-47D human breast cancer cell lines.

In the present study, we explored the effect of the progestin medrogestone on the sulfatase and sulfotransferase activities in the hormone-dependent MCF-7 and T-47D human breast cancer cell lines. After 24 h incubation at 37 degrees C of physiological concentrations of estrone sulfate ([3H]-E1S: 5x10(-9) mol/l), it was observed that this estrogen was converted in a great proportion to E2 in both cell lines. Medrogestone significantly inhibits this transformation, at all the concentrations tested (5x10(-8) to 5x10(-5) mol/l), in both cell lines. The IC50 values were 1.93 micromol/l and 0.21 micromol/l in MCF-7 and T-47D cells, respectively. In another series of studies, after 24 h incubation at 37 degrees C of physiological concentrations of estrone ([3H]-E1: 5x10(-9) mol/l), the sulfotransferase activity was detectable in both cell lines. Estrogen sulfates (ES) are found exclusively in the culture medium, which suggests that as soon as they are formed they are excreted into the medium. Medrogestone has a biphasic effect on sulfotransferase activity in both cell lines. At low doses: 5x10(-8) and 5x10(-7) mol/l, this compound stimulates the enzyme by +73.5 and 52.7%, respectively, in MCF-7, and by 84.5 and 62.6% in T-47D cells. At high concentrations: 5x10(-6) and 5x10(-5) mol/l, medrogestone has no effect on MCF-7 cells, but inhibits the sulfotransferase activity in T-47D cells by -31.4% at 5x10(-5) mol/l. In conclusion, the inhibitory effect provoked by medrogestone on the enzyme involved in the biosynthesis of E2 (sulfatase pathway) in estrogen-dependent breast cancer, as well as the stimulatory effect on the formation of the inactive ES, support a probable anti-proliferative effect of this progestin in breast tissue. Clinical applications of these findings can open new therapeutic possibilities for this disease.

Estrone sulfatase versus estrone sulfotransferase in human breast cancer: potential clinical applications.

Estrone sulfate (E1S) is concentrated in high levels in human breast cancer tissue. The values are particularly high in postmenopausal women and many times those circulating in the plasma. Also, the tissular concentration of this conjugate are significantly higher in tumoural tissue than in the area of the breast considered as normal. The enzyme which hydrolyzes E1S: sulfatase, as well as the enzyme which biosynthesises this conjugate: sulfotransferase, are present in significant concentrations in breast cancer tissue. Consequently, E1S is a balance between the activities of the two enzymes. As breast cancer tissue has all the enzymes necessary for the synthesis of estradiol (E2), and the formation of E2 from E1S 'via sulfatase' is the main pathway, it was very attractive to explore inhibitory agents of this enzyme. It was observed that different substances including antiestrogens (4-hydroxytamoxifen, ICI 164,384) and various progestins (promegestone, nomegestrol acetate, medrogestone) as well as Org OD14 (tibolone) can block the sulfatase activity. In addition, it was demonstrated that different progestins (medrogestone, nomegestrol acetate, TX-525) and org OD14 can stimulate the sulfotransferase activity for the formation of the biologically inactive E1S. It is concluded that the inhibition of sulfatase and the stimulation of sulfotransferase activity can open interesting possibilities to explore these effects in patients with breast cancer.

Effects of Org OD14 (Livial) and its metabolites on 17 beta-hydroxysteroid dehydrogenase activity in hormone-dependent MCF-7 and T-47D breast cancer cells.

It is well recognized that estradiol (E2) is one of the most important hormones supporting the growth and evolution of breast cancer. Consequently, to block this hormone before it enters the cancer cell, or in the cell itself, has been one of the main targets in recent years. In the present study we explored the effect of Org OD14 (active substance in Livial) and its metabolites (Org 30126, Org 4094, Org OM38) on the 17 beta-hydroxysteroid dehydrogenase (17 beta HSD) activity of MCF-7 and T-47D human breast cancer cells. Using physiological doses of estrone ([3H]-E1: 5 x 10(-9) M) this estrogen is converted in a great proportion to E2 in both cell lines. After 24 hours, Org OD14 significantly inhibits this transformation in a dose-dependent manner, by 32 and 73% at 5 x 10(-7) M and 5 x 10(-5) M respectively, in T-47D cells; the effect is similar in MCF-7 cells. Among the three Org OD14 metabolites tested, Org 4094 and Org 30126 (3 alpha- and 3 beta-hydroxy metabolites) are more potent than their precursor, and Org OM-38 (4-ene isomer) is the weakest of the three steroids. The IC50 values were 0.79, 1.98, 7.12, and 22.84 microM in MCF-7 cells for Org 4094, Org 30126, Org OD14, and Org OM-38, respectively, and 4.83, 1.44, 2.03, and 35.25 microM, respectively, in T-47D cells. As Org OD14 and two of its metabolites, Org 30126 and Org 4094, also strongly decrease the conversion of estrone sulphate to estradiol in the hormone-dependent MCF-7 and T-47D breast cancer cells, it is concluded that the inhibition provoked by these steroids on the enzymes (estrone sulphatase and 17 beta-HSD) involved in the local biosynthesis of the biologically active estrogen estradiol, may reduce the risk of breast cancer in postmenopausal women during long-term hormone replacement treatment with Livial.

Effect of Org OD14 (LIVIAL) and its metabolites on human estrogen sulphotransferase activity in the hormone-dependent MCF-7 and T-47D, and the hormone-independent MDA-MB-231, breast cancer cell lines.

The concentration of estrogen sulphates (ES) is particularly high in tumoural breast tissues in post-menopausal women. It is well known that during the post-menopausal phase the levels of circulating estrogens are very low, suggesting the local production of these hormones in the breast cancer tissue itself. Breast cancer cells possess all the enzymes involved in the last steps of estradiol (E2) formation, as well as in its transformation (e.g.: sulphotransferase). As ES are not biologically active, the formation of this conjugate is an important transformation pathway in the control of E2. Consequently, it was interesting to investigate the factors which regulate the sulphotransferase activity. In the present study, we explored the effect of Org OD14; the active substance in Livial, and of its main metabolites (Org 4094, Org 30126, Org OM-38) on the estrogen sulphotransferase activity in the hormone-dependent: MCF-7, T-47D, and hormone-independent: MDA-MB-231, human breast cancer cell lines. After 24 hours incubation at 37 degrees C of physiological concentrations of estrone ([3H]-E1: 5 x 10(-9) mol/l), it was observed that the sulphotransferase activity is detectable in MCF-7 and T-47D cells, since the concentrations of ES found were 14.90 and 17.30 pmol/mg DNA, respectively, whereas in MDA-MB-231 cells the concentration of ES found was only 2.01 pmol/mg DNA. Sulphates are found exclusively in the culture medium, which suggests that as soon as the sulphate is biosynthesized it is secreted into the medium. Org OD14, Org 30126, and Org 4094 have a biphasic effect on sulphotransferase activity in the hormone-dependent cells only. At low doses (5 x 10(-8) mol/l) these compounds stimulate this enzyme by 63, 101, and 51%, respectively in the MCF-7 cells, and by 41, 102, and 80%, respectively in the T-47D cells. No stimulatory effect was detected with Org OM-38. At high concentrations (5 x 10(-5) mol/l) Org OD14 and its three metabolites inhibit the sulphotransferase activity in MCF-7 and T-47D cells by 50-70%. In conclusion, the stimulatory effect provoked at low doses by Org OD14 and its metabolites (Org 4094, Org 30126) on the estrogen sulphotransferase involved in the biosynthesis of the inactive estrogen sulphates in estrogen-dependent breast cancer cells, can contribute to the protection of breast tissue in postmenopausal women with hormone replacement therapy.

Effect of Medrogestone on 17beta-hydroxysteroid dehydrogenase activity in the hormone-dependent MCF-7 and T-47D human breast cancer cell lines.

Estradiol (E2) is one of the most important hormones supporting the growth and evolution of breast cancer. Consequently, to block this hormone before it enters the cancer cell, or in the cell itself, has been one of the main targets in recent years. In the present study we explored the effect of Medrogestone (Prothil) on 17beta-hydroxysteroid dehydrogenase (17beta-HSD) activities of the hormone-dependent MCF-7 and T-47D human breast cancer cell lines. Using physiological doses of estrone ([3H]-E1: 5 x 10(-9) mol/l) this estrogen is converted in a great proportion to E2 in both cell lines. After 24 h of the cell culture, Medrogestone significantly inhibits this transformation in a dose-dependent manner by 39% and 80% at 5 x 10(-8) M and 5 x 10(-5) M, respectively in T-47D cells; the effect is less intense in MCF-7 cells: 25% and 55% respectively. The IC50 values are 0.45 micromol/l in T-47D and 17.36 micromol/l in MCF-7 cells. It is concluded that the inhibition provoked by Medrogestone on the reductive 17beta-HSD activity involved in the local biosynthesis of the biologically active estrogen estradiol, may constitute a new therapeutic approach for the treatment of breast cancer.

Biological effects of progestins in breast cancer.

Developments in the synthesis of different progestins have opened up new possibilities for the biological effects and therapeutic uses of these compounds. The actions of progestins are a function of their structure, affinity to the progesterone receptor or to other steroid receptors, the target tissue considered, the biological response, the experimental conditions, dose, and metabolic transformation. Data on the action of progestins in breast cancer patients are very limited. A positive response with the progestins medroxyprogesterone acetate and megestrol acetate has been obtained in postmenopausal patients with advanced breast cancer. However, extensive information on the effect of progestins was obtained in in vitro studies using hormone-dependent and hormone-independent human mammary cancer cell lines. It was demonstrated that in hormone-dependent breast cancer cells, various progestins (nomegestrol acetate, medrogestone, promegestone) as well as tibolone, are potent sulfatase-inhibitory agents. Progestins may also be involved in the inhibition of the mRNA of this enzyme. In another series of studies, it was also demonstrated that various progestins are very active in inhibiting the 17 beta-hydroxysteroid dehydrogenase for the conversion of estrone to estradiol. More recently, it has been observed that promegestone or medrogestone stimulates the sulfotransferase for the formation of estrogen sulfates. Clinical trials of these enzymatic effects on the formation and transformation of estradiol in breast cancer patients could be the next step to investigate new therapeutic possibilities for this disease.

Recommendations for estrogen and progestin replacement in the climacteric and postmenopause. European Progestin Club.

The diversity of function that sex steroids have proven to have in the female body, gives them a position of central importance in gynaecology. Scientific research demonstrates not only the well known genital functions of sexual steroids, furthermore, various extragenital organs are influenced and modulated by ovarian hormones. Therefore, the general benefit of HRT for the female organism becomes clearer and the clinical management of menopause is developing to a broad new discipline, the gender specific medicine. In clinical practise, phytosteroids are claimed by the patient and therefore, also of high interest for the scientific research. Also, tissue specificity of the endocrine treatment and the biological relevance of different steroid receptors of HRT are discussed, leading to the development of new HrT preparations. Individualisation, the tailoring of HRT, according to the patients needs, and low dose steroids management, will also become an important aspect in the recommendations for estrogen and progestin replacement therapy.

The SEEM: selective estrogen enzyme modulators in breast cancer.

Human breast cancer tissue contains all the enzymes (estrone sulfatase, 17 beta-hydroxysteroid dehydrogenase, aromatase) involved in the last steps of estradiol biosynthesis. This tissue also contains sulfotransferase for the formation of the biologically inactive estrogen sulfates. In the past years, it has been demonstrated that various progestins (promegestone, nomegestrol acetate, medrogestone) as well as tibolone and its metabolites are potent inhibitors of sulfatase and 17 beta-hydroxysteroid dehydrogenase activities. It was also shown that medrogestone, nomegestrol acetate, promegestone or tibolone can stimulate the sulfotransferase activity for the local production of estrogen sulfates. All these data, in addition to numerous agents which can block the aromatase action, lead to the new concept of Selective Estrogen Enzyme Modulators (SEEM) which can largely apply to breast cancer tissue. The exploration of various progestins and other active agents in trials with breast cancer patients, showing an inhibitory effect on sulfatase and 17 beta-hydroxysteroid dehydrogenase, or a stimulatory effect on sulfotransferase, will provide a new option in the treatment of this disease.

Human estrogen sulfotransferase (hEST1) activities and its mRNA in various breast cancer cell lines. Effect of the progestin, promegestone (R-5020).

Using reverse transcriptase-polymerase chain reaction amplification it was possible to detect the presence of type 1 human estrogen sulfotransferase (hEST1) mRNA in the hormone-dependent: MCF-7 and T-47D, and hormone-independent: MDA-MB-231 and MDA-MB-468, human breast cancer cells. The expression of this mRNA is significantly higher in the MDA-MB-468 cells and a correlation of this mRNA expression with the enzymatic activity was observed. The progestin promegestone (R-5020) at a low concentration (5 x 10(-7) M) can significantly increase the estrogen sulfotransferase activity and its mRNA in the hormone-dependent MCF-7 and T-47D cells. As estrogen sulfates are biologically inactive, the stimulatory effect on sulfotransferase by promegestone may open attractive possibilities in the control of estradiol in human breast cancer.

Progestins and breast cancer.

In the last years there has been an extraordinary development in the synthesis of new progestins. These compounds are classified, in agreement with their structure, in various groups which include progesterone, retroprogesterones, 17alpha-hydroxyprogesterones, 19-norprogesterones, 17alpha-hydroxyprogesterone derivatives, androstane and estrane derivatives. The action of progestins is a function of many factors: its structure, affinity to the progesterone receptor or to other steroid receptors, the target tissue considered, the biological response, the experimental conditions, dose, and metabolic transformation. The information on the action of progestins in breast cancer patients is very limited. Positive response with the progestins: medroxyprogesterone acetate and megestrol acetate was obtained in post-menopausal patients with advanced breast cancer. However, extensive information on the effect of progestins was obtained in in vitro studies using hormone-dependent and hormone-independent human mammary cancer cell lines. It was demonstrated that in the hormone-dependent breast cancer cells, various progestins (nomegestrol acetate, tibolone, medrogestone, promegestone) are potent sulfatase inhibitory agents. The progestins can also involve the inhibition of mRNA of this enzyme. In another series of studies it was also demonstrated that various progestins are very active in inhibiting the 17beta-hydroxysteroid dehydrogenase for the conversion of estrone to estradiol. More recently it was observed that the progestins promegestone or medrogestone stimulate the sulfotransferase for the formation of estrogen sulfates. Consequently, the blockage in the formation of estradiol via sulfatase, or the stimulatory effect on sulfotransferase activity, by progestins can open interesting and new possibilities in clinical applications in breast cancer.