The formation and transformation of hormones in maternal, placental and fetal compartments: biological implications.

The fetal endocrine system constitutes the earliest system developing in fetal life and operates during all the steps of gestation. Its regulation is in part dependent on the secretion of placental and/or maternal precursors emanating across the feto-maternal interface. Human fetal and placental compartments possess 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, while 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 hydrolyze 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 greatly increases with fetal evolution (e.g. estriol increases 500-1000 times in relation to non-pregnant women). Other hormones, such as glucocorticoids, in particular the stress hormone cortisol, adipokines (e.g. leptin, adiponectin), insulin-like growth factors, are also a key factor for regulating reproduction, metabolism, appetite and may be significant in programming the fetus and its growth. We can hypothesize that the fetal and placental factors controlling hormonal levels in the fetal compartment can be of capital importance in the normal development of extra-uterine life.

European Progestin Club Guidelines for prevention and treatment of threatened or recurrent (habitual) miscarriage with progestogens.

This guideline has been developed based on studied and clinical investigations. Therefore, it appears to be appropriate to use all the available evidence, which are very encouraging, in a summarized form to propose guidelines by a group of European experts in order to give the gynecologists, obstetricians and reproductive medicine specialists have direction with regard to the prevention or treatment of miscarriage for the benefit of the endangered pregnancies. There are a number of statements, opinions and guidelines already published for this topic, which are not entirely in agreement.

Hormonal enzymatic systems in normal and cancerous human breast: control, prognostic factors, and clinical applications.

The bioformation and transformation of estrogens and other hormones in the breast tissue as a result of the activity of the various enzymes involved attract particular attention for the role they play in the development and pathogenesis of hormone-dependent breast cancer. The enzymatic process concerns the aromatase, which transforms androgens into estrogens; the sulfatase, which hydrolyzes the biologically inactive sulfates to the active hormone; the 17ß-hydroxysteroid dehydrogenases, which are involved in the interconversion estradiol/estrone or testosterone/androstenedione; hydroxylases, which transform estrogens into mitotic and antimitotic derivatives; and sulfotransferases and glucuronidases, which, respectively convert into the biologically inactive sulfates and glucuronides. These enzymatic activities are more intense in the carcinoma than in the normal tissue. Concerning aromatase, the application of antiaromatase agents has been largely developed in the treatment of breast cancer patients, with very positive results. Various studies have shown that the activity levels of these enzymes and their mRNA can be involved as interesting prognostic factors for breast cancer. In conclusion, the application of new antienzymatic molecules can open attractive perspectives in the treatment of hormone-dependent breast cancer.

Effect of tibolone and its principal metabolites (3α- and 3ß-hydroxy, 3α-sulfate, and 4-ene derivatives) on estrone sulfatase activity in normal and cancerous human breast tissue.

BACKGROUND: Tibolone (Org-OD14) is the active substance of Livial®, a synthetic steroid with the structure 7α,17α-17-hydroxy-7-methyl-19-norpregn-5(10)-en-20-yn-3-one, possessing weak tissue-specific estrogenic, progestogenic, and androgenic properties, used to treat menopausal complaints. After oral administration, tibolone is extensively metabolized into the 3α-(Org-4904) and 3ß-(Org-30126) hydroxy derivatives with estrogenic properties, its 4-ene (Org-OM38) isomer with progestogenic/androgenic activities, and the 3α-sulfate (Org-34322) derivative, a major biologically inactive circulating form. We compared the dose response of tibolone and its metabolites on estrone sulfatase activity [conversion of estrone sulfate (E1S) to estrone (E1)] in normal and cancerous human breast tissues. MATERIALS AND METHODS: Tissue minces were incubated with physiological concentrations of [3H]-E1S (5×10-9M) alone or in the presence of tibolone and its metabolites (concentration range: 5×10-7to 5×10-5M) for 4 h. Tritiated E1, estradiol (E2), and E1S were separated and evaluated quantitatively by thin-layer chromatography. RESULTS: The sulfatase activity was significantly higher in cancerous breast but strongly inhibited by tibolone and the different metabolites, whereas 3α- and 3ß-hydroxy derivatives were the most potent inhibitors. CONCLUSION: This very significant inhibitory effect of tibolone and its principal metabolites on the enzyme involved in E2biosynthesis in the human breast provides interesting perspectives to study the biological responses of these compounds in trials with breast cancer patients.

Recent advances on the action of estrogens and progestogens in normal and pathological human endometrium.

Hormonal control in the development of the normal endometrium is of the utmost importance. It is well established that the two main hormones involved in this process are estradiol and progesterone, which are also implicated in the pathological conditions concerning endometriosis and endometrial carcinoma. There are two types of endometrial carcinoma: type I which represents 80%-90% is hormone-dependent, whereas the remainder is type II and is hormone-independent. The endometrial tissue contains all the enzymatic systems in the formation and transformation of the various hormones, including aromatases, sulfatases, sulfotransferases, hydroxysteroid dehydrogenases, hydroxylases, and glucuronidases. It is interesting to note that increased sulfatase activity is correlated with severity of endometriosis. An increased sulfatase/sulfotransferase ratio represents a poor prognosis in patients with endometrial carcinoma. Treatment with hormone replacement therapy (estrogens+progestogens), as well as with tibolone, is most effective in protecting this tissue by climacteric alterations, owing to the significant decrease of ovarian hormones. In conclusion, enzymatic control can open appealing perspectives to protect this organ from possible pathological alterations.

Nomegestrol acetate is an anti-aromatase agent in human MCF-7aro breast cancer cells.

BACKGROUND: The progestogen nomegestrol acetate (NOMAC), a 17α-hydroxy-nor-progesterone derivative (LUTENYL®) is largely used as an oral contraceptive and to treat menopausal complaints. In previous studies, we demonstrated that NOMAC is an anti-sulfatase agent in MCF-7 and T-47D breast cancer cells. In this study, we explore the effect of NOMAC on aromatase activity in a stable aromatase-expressing human breast cancer cell line: MCF-7aro. MATERIALS AND METHODS: Cells were incubated with physiological concentrations of androgen substrates [3H]-testosterone or [3H]-androstenedione (5×10-9 mol/L) alone, or in the presence of NOMAC (5×10-5 mol/L-5×10-8 mol/L) for 24 h at 37°C. [3H]-Estradiol (E2), [3H]-estrone (E1), [3H]-testosterone and [3H]-androstenedione were characterized by thin layer chromatography and quantified using the corresponding standard. RESULTS: Aromatase activity levels are high in MCF-7aro cells because the [3H]-E2 concentration after incubation of [3H]-testosterone was 5.8±0.31 pmol/mg DNA in non-treated cells. At concentrations of 5×10-5 mol/L, 5×10-6 mol/L and 5×10-7 mol/L NOMAC significantly inhibits this conversion by 49.7%, 29.9% and 18.1%, respectively. After [3H]-androstenedione incubation, similar inhibition levels were observed with NOMAC for [3H]-E1 formation; whereas, inhibition of [3H]-E2 production, which implicates 17ß-hydroxysteroid dehydrogenase activity in this pathway, is greater because NOMAC also inhibits this enzyme. CONCLUSION: The MCF-7aro cell line shows high aromatase activity and NOMAC can act as an anti-aromatase agent by inhibiting this activity. This is an important new effect of this progestogen. Because NOMAC can also inhibit sulfatase activity in breast cancer cells, we suggest that this dual effect of NOMAC has attractive possibilities for clinical trials.

Biological responses of progestogen metabolites in normal and cancerous human breast.

At present, more than 200 progestogen molecules are available, but their biological response is a function of various factors: affinity to progesterone or other receptors, their structure, the target tissues considered, biological response, experimental conditions, dose, method of administration and metabolic transformations. Metabolic transformation is of huge importance because in various biological processes the metabolic product(s) not only control the activity of the maternal hormone but also have an important activity of its own. In this regard, it was observed that the 20-dihydro derivative of the progestogen dydrogesterone (Duphaston®) is significantly more active than the parent compound in inhibiting sulfatase and 17ß-hydroxysteroid dehydrogenase in human breast cancer cells. Estrone sulfatase activity is also inhibited by norelgestromin, a norgestimate metabolite. Interesting information was obtained with a similar progestogen, tibolone, which is rapidly metabolized into the active 3α/3ß-hydroxy and 4-ene metabolites. All these metabolites can inhibit sulfatase and 17ß-hydroxysteroid dehydrogenase and stimulate sulfotransferase in human breast cancer cells. Another attractive aspect is the metabolic transformation of progesterone itself in human breast tissues. In the normal breast progesterone is mainly converted to 4-ene derivatives, whereas in the tumor tissue it is converted mostly to 5α-pregnane derivatives. 20α-Dihydroprogesterone is found mainly in normal breast tissue and possesses antiproliferative properties as well as the ability to act as an anti-aromatase agent. Consequently, this progesterone metabolite could be involved in the control of estradiol production in the normal breast and therefore implicated in one of the multifactorial mechanisms of the breast carcinogenesis process. In conclusion, a better understanding of both natural and synthetic hormone metabolic transformations and their control could potentially provide attractive new therapies for the treatment of hormone-dependent pathologies.

Inhibition of aromatase activity in MCF-7aro human breast cancer cells by the natural androgens testosterone and androstenedione.

BACKGROUND: The human breast contains all the enzymes responsible for local bioformation of estradiol (E2). Two principal pathways are implicated in the last steps of E2 formation: the 'aromatase' which transforms androgens into estrogens, and the 'sulfatase' which converts estrogen sulfates into active unconjugated estrogens; activities found in both normal and cancerous breast. Aromatase inhibition by anti-aromatase agents is largely used with very positive results in the treatment of breast cancer patients. In this study, the effects of the natural androgens androstenedione and testosterone were explored on aromatase activity in a stable aromatase-expressing estrogen receptor-positive human breast cancer cell line MCF-7aro. MATERIALS AND METHODS: The cells were incubated with physiological concentrations of [3H]-testosterone (5 nmol/L) alone or in the presence of either testosterone or androstenedione (0.5 and 50 µmol/L) 24 h at 37°C. Cellular radioactivity uptake was determined. [3H]-E2 was characterized by thin-layer chromatography. RESULTS: The MCF-7aro cells have a very high aromatase activity because conversion of [3H]-testosterone to [3H]-E2 was 3.02±0.17 pmol/mg DNA in non-treated cells. Testosterone, at concentrations of 0.5 and 50 µmol/L, provoked inhibition of E2 formation of 36% and 79%, respectively. The effect of androstenedione at 0.5 and 50 µmol/L was 56% and 76%, respectively. CONCLUSION: In breast cancer cells, the natural androgens testosterone and androstenedione, have the capacity to control bioformation of estradiol by blocking aromatase activity. The data can provide important information on the control mechanism of estrogen intratumoral levels and open new possibilities in breast cancer treatment.

Breast cancer and steroid metabolizing enzymes: the role of progestogens.

It is well documented that breast tissue, both normal and cancerous, contains all the enzymatic systems necessary for the bioformation and metabolic transformation of estrogens, androgens and progesterone. These include sulfatases, aromatase, hydroxysteroid-dehydrogenases, sulfotransferases, hydroxylases and glucuronidases. The control of these enzymes plays an important role in the development and pathogenesis of hormone-dependent breast cancer. As discussed in this review, various progestogens including dydrogesterone and its 20alpha-dihydro-derivative, medrogestone, promegestone, nomegestrol acetate and norelgestromin can reduce intratissular levels of estradiol in breast cancer by blocking sulfatase and 17beta-hydroxysteroid-dehydrogenase type 1 activities. A possible correlation has been postulated between breast cell proliferation and estrogen sulfotransferase activity. Progesterone is largely transformed in the breast; normal breast produces mainly 4-ene derivatives, whereas 5alpha-derivatives are most common in breast cancer tissue. It has been suggested that this specific conversion of progesterone may be involved in breast carcinogenesis. In conclusion, treatment with anti-aromatases combined with anti-sulfatase or 17beta-hydroxysteroid-dehydrogenase type 1 could provide new therapeutic possibilities in the treatment of patients with hormone-dependent breast cancer.

Estrogen sulfotransferases in breast and endometrial cancers.

Estrogen sulfotransferase is significantly more active in the normal breast cell (e.g., Human 7) than in the cancer cell (e.g., MCF-7). The data suggest that in breast cancer sulfoconjugated activity is carried out by another enzyme, the SULT1A, which acts at high concentration of the substrates. In breast cancer cells sulfotransferase (SULT) activity can be stimulated by various progestins: medrogestone, promegestone, and nomegestrol acetate, as well as by tibolone and its metabolites. SULT activities can also be controlled by other substances including phytoestrogens, celecoxib, flavonoids (e.g., quercetin, resveratrol), and isoflavones. SULT expression was localized in breast cancer cells, which can be stimulated by promegestone and correlated with the increase of the enzyme activity. The estrogen sulfotransferase (SULT1E1), which acts at nanomolar concentration of estradiol, can inactivate most of this hormone present in the normal breast; however, in the breast cancer cells, the sulfotransferase denoted as SULT1A1 is mainly present, and this acts at micromolar concentrations of E(2). A correlation was postulated among breast cancer cell proliferation, the effect of various progestins, and sulfotransferase stimulation. In conclusion, it is suggested that factors involved in the stimulation of the estrogen sulfotransferases could provide new possibilities for the treatment of patients with hormone-dependent breast and endometrial cancers.

Progestins in the menopause in healthy women and breast cancer patients.

At present, more than 200 progestin compounds are synthetized, but their biological effects are different: this is function of their structure, receptor affinity, metabolic transformations, the target tissues considered, dose. The action of progestins in breast cancer is controversial; some studies indicate an increase in breast cancer incidence, others show no differences, and yet others indicate a decrease. Many studies agree that treatment with progestins plus estrogens at a low dose and during a limited period (less than 5 years) can have beneficial effects in peri- and post-menopausal women. It was demonstrated that various progestins (e.g. nomegestrol acetate, medrogestone, promegestone), as well as tibolone and its metabolites, can block the enzymes involved in estradiol bioformation (sulfatase, 17beta-hydroxysteroid dehydrogenase) in breast cancer. Progesterone is converted into various metabolic products: in normal breast tissue the transformation is mainly to 4-ene derivatives, whereas in the tumor tissue 5alpha-pregane derivatives are predominant. Aromatase activity is the last step in the formation of estrogens by the conversion of androgens. In recent studies it was shown that 20alpha-dihydroprogesterone, a metabolite found mainly in normal breast tissue and having anti-proliferative properties, can act as an anti-aromatase agent. The data suggest the possible utilization of this compound in breast cancer prevention. In conclusion, in order to clarify and better understand the response of progestins in breast cancer (incidence and mortality), as well as in hormone replacement therapy or in endocrine dysfunction, new clinical trials are necessary using other progestins in function of the dose and period of treatment.

Classification and pharmacology of progestins.

Besides the natural progestin, progesterone, there are different classes of progestins, such as retroprogesterone (i.e. dydrogesterone), progesterone derivatives (i.e. medrogestone) 17alpha-hydroxyprogesterone derivatives (i.e. chlormadinone acetate, cyproterone acetate, medroxyprogesterone acetate, megestrol acetate), 19-norprogesterone derivatives (i.e. nomegestrol, promegestone, trimegestone, nesterone), 19-nortestosterone derivatives norethisterone (NET), lynestrenol, levonorgestrel, desogestrel, gestodene, norgestimate, dienogest) and spironolactone derivatives (i.e. drospirenone). Some of the synthetic progestins are prodrugs, which need to be metabolized to become active compounds. Besides the progestogenic effect, which is in common for all progestins, there is a wide range of biological effects, which are different for the various progestins and have to be taken into account, when medical treatment is considered.

Progestins and breast cancer.

Progestins exert their progestational activity by binding to the progesterone receptor (form A, the most active and form B, the less active) and may also interact with other steroid receptors (androgen, glucocorticoid, mineralocorticoid, estrogen). They can have important effects in other tissues besides the endometrium, including the breast, liver, bone and brain. The biological responses of progestins cover a very large domain: lipids, carbohydrates, proteins, water and electrolyte regulation, hemostasis, fibrinolysis, and cardiovascular and immunological systems. At present, more than 200 progestin compounds have been synthesized, but the biological response could be different from one to another depending on their structure, metabolism, receptor affinity, experimental conditions, target tissue or cell line, as well as the biological response considered. There is substantial evidence that mammary cancer tissue contains all the enzymes responsible for the local biosynthesis of estradiol (E(2)) from circulating precursors. Two principal pathways are implicated in the final steps of E(2) formation in breast cancer tissue: the 'aromatase pathway', which transforms androgens into estrogens, and the 'sulfatase pathway', which converts estrone sulfate (E(1)S) into estrone (E(1)) via estrone sulfatase. The final step is the conversion of weak E(1) to the potent biologically active E(2) 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. It has been demonstrated that various progestins (e.g. nomegestrol acetate, medrogestone, promegestone) as well as tibolone and their metabolites can block the enzymes involved in E(2) bioformation (sulfatase, 17beta-hydroxysteroid dehydrogenase) in breast cancer cells. These substances can also stimulate the sulfotransferase activity which converts estrogens into the biologically inactive sulfates. The action of progestins in breast cancer is very controversial; some studies indicate an increase in breast cancer incidence, others show no difference and still others a significant decrease. Progestin action can also be a function of combination with other molecules (e.g. estrogens). In order to clarify and better understand the response of progestins in breast cancer (incidence, mortality), as well as in hormone replacement therapy or endocrine dysfunction, new clinical trials are needed studying other progestins as a function of the dose and period of treatment.

Correlation of estrogen sulfotransferase activity and proliferation in normal and carcinomatous human breast. A hypothesis.

BACKGROUND: Sulfotransferases are present in normal and cancerous human breast tissues. The purpose of this article is to present a hypothetical correlation of sulfotransferase activity with proliferation in breast cancer. MATERIALS AND METHODS: Sulfotransferases were evaluated in breast cancer cells by determining the transformation of non-conjugated estrogens to the sulfates. Proliferation was evaluated by the action on cell growth or the size of a transplanted tumor. The effect was obtained using the progestins: nomegestrol acetate, promegestone, and medrogestone, as well as tibolone and its metabolites at concentrations of 5 x 10(-5) to 5 x 10(-9) M. RESULTS: A possible correlation of sulfotransferase activity stimulation and cell growth inhibition provoked by the various progestins used, or by tibolone and its metabolites was shown. CONCLUSION: It is suggested that the antiproliferative effect of these compounds could be related to the decrease of bioactive estradiol by the formation of its biologically inactive sulfate as a consequence of the stimulatory effect by the various progestins or tibolone on sulfotransferase activity.

Control of sulfatase activity by nomegestrol acetate in normal and cancerous human breast tissues.

Nomegestrol acetate (NOMAC), a 17alpha-hydroxy-nor-progesterone derivative (17alpha-acetoxy-6-methyl-19-nor-4,6-pregnadiene-3,20-dione, the active substance in Lutenyl), is a potent and useful clinical synthetic progestin for the treatment of menopausal complaints and is under current development for oral contraception. Previous studies in this laboratory demonstrated that NOMAC can block sulfatase and 17beta-hydroxysteroid dehydrogenase, the enzymes involved in the biosynthesis and transformation of estradiol (E2) in hormone-dependent MCF-7 and T-47D breast cancer cells. In the present study, the effect of NOMAC on sulfatase activity using total breast cancer tissue, compared to the effect in normal breast tissue, was explored. Slices of tumoral or normal breast tissues (45-65 mg) were incubated in buffer (20 mM Tris-HCl, pH 7.2) with physiological concentrations of [3H]-estrone sulfate (5x10(-9) M), alone or in the presence of nomegestrol acetate (5x10(-5) - 5x10(-7) - 5x10(-9) M), for 4 h at 37 degrees C. Estrone sulfate (E1S), estrone (E1) and E2 were characterized by thin layer chromatography and quantified using the corresponding standard. It was observed that [3H]- E1S was only converted to [3H]- E1 and not to [3H]- E2, in normal or cancerous breast tissues, which suggests a low or no 17beta-HSD activity under these experimental conditions. The sulfatase activity was more intense with breast cancer tissue than normal tissue, since the concentrations of E1 were 42.5 +/- 3.4 and 27.2 +/- 2.5 pg/mg tissue, respectively. NOMAC, at the concentration of 5x10(-5) M, inhibited this conversion by 49.2% and 40.8% in cancerous and normal breast tissues, respectively. The sulfatase inhibition at low concentration (5x10(-7) M) was 32.5% and 22.8%, respectively. It is concluded that sulfatase activity is almost twice as potent in cancerous breast tissues than in normal tissues. Nomegestrol acetate is a strong anti-sulfatase agent, in particular with cancerous breast tissues. The inhibition of estrone sulfatase activity by NOMAC in total normal or cancerous breast tissues can open attractive perspectives for future clinical trials.

Recent insight on the control of enzymes involved in estrogen formation and transformation in human breast cancer.

The great majority of breast cancers are in their early stage hormone-dependent and it is well accepted that estradiol (E2) plays an important role in the genesis and evolution of this tumor. Human breast cancer tissues contain all the enzymes: estrone sulfatase, 17beta-hydroxysteroid dehydrogenase, aromatase involved in the last steps of E2 bioformation. Sulfotransferases which convert estrogens into the biologically inactive estrogen sulfates are also present in this tissue. Quantitative data show that the 'sulfatase pathway', which transforms estrogen sulfates into the bioactive unconjugated E2, is 100-500 times higher than the 'aromatase pathway', which converts androgens into estrogens. The treatment of breast cancer patients with anti-aromatases is largely developed with very positive results. However, the formation of E2 via the 'sulfatase pathway' is very important in the breast cancer tissue. In recent years it was found that antiestrogens (e.g. tamoxifen, 4-hydroxytamoxifen), various progestins (e.g. promegestone, nomegestrol acetate, medrogestone, dydrogesterone, norelgestromin), tibolone and its metabolites, as well as other steroidal (e.g. sulfamates) and non-steroidal compounds, are potent sulfatase inhibitors. In another series of studies, it was found that E2 itself has a strong anti-sulfatase action. This paradoxical effect of E2 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. Interesting information is that high expression of steroid sulfatase mRNA predicts a poor prognosis in patients with +ER. These progestins, as well as tibolone, can also block the conversion of estrone to estradiol by the inhibition of the 17beta-hydroxysteroid dehydrogenase type I (17beta-HSD-1). High expressison of 17beta-HSD-1 can be an indicator of adverse prognosis in ER-positive patients. It was shown that nomegestrol acetate, medrogestone, promegestone or tibolone, could stimulate the sulfotransferase activity for the local production of estrogen sulfates. This is an important point in the physiopathology of this disease, as it is well known that estrogen sulfates are biologically inactive. A possible correlation between this stimulatory effect on sulfotransferase activity and breast cancer cell proliferation is presented. In agreement with all this information, we have proposed the concept of selective estrogen enzyme modulators (SEEM). In conclusion, the blockage in the formation of estradiol via sulfatase, or the stimulatory effect on sulfotransferase activity in combination with anti-aromatases can open interesting and new possibilities in clinical applications in breast cancer.