Receptor profiling and endocrine interactions of tibolone

The receptor profiles and in vivo activity of tibolone, and its primary metabolites, Delta(4)-isomer, and 3alpha- and 3beta-hydroxytibolone, were studied and compared to those of structurally related compounds. The Delta(4)-isomer was the strongest binder and activator of the progesterone receptor (PR); tibolone was 10 times weaker in binding and half as potent in transactivation of PR; 3alpha- and 3beta-hydroxytibolone did not bind or activate PR. In rabbits oral tibolone produced a minor progestagenic effect in the endometrium, whereas co-administration of tibolone and the anti-estrogen ICI 164,384 unmasked tibolone's progestagenic effect. 3-Hydroxytibolones were the strongest binders and activators of the estrogen receptors (ERs), with greater affinity for ERalpha than for ERbeta. Tibolone showed weaker binding and activation of both ERs and the Delta(4)-isomer has a binding and activation activity of less than 0.1% of E2 for ERalpha or ERbeta. Tamoxifen and 4-hydroxytamoxifen showed partial ERalpha agonistic effects with a maximal response of 12% and raloxifene of 3-5%. Oral administration of 1mg tibolone to ovariectomized rats induced an estrogenic effect on vaginal epithelium. The Delta(4)-isomer was a stronger binder and activator of the androgen receptor (AR) than tibolone; both 3-hydroxytibolones did not bind or activate AR. Introducing a 7alpha-methyl group decreased progestagenic and increased androgenic activity. We conclude that the progestagenic and androgenic activities of tibolone are mediated by the Delta(4)-isomer, and the estrogenic activity, by the 3-hydroxytibolones. The estrogenic activity of the 3-hydroxytibolones masked the progestagenic activity of tibolone in rabbit endometrium. Full estrogenic response was observed in rat vaginal tissue after oral administration of tibolone.     

Biological characterization of non-steroidal progestins from botanicals used for women's health

Progesterone plays a central role in women's reproductive health. Synthetic progestins, such as medroxyprogesterone acetate (MPA) are often used in hormone replacement therapy (HRT), oral contraceptives, and for the treatment of endometriosis and infertility. Although MPA is clinically effective, it also promiscuously binds to androgen and glucocorticoid receptors (AR/GR) leading to many undesirable side effects including cardiovascular diseases and breast cancers. Therefore, identifying alternative progestins is clinically significant. The purpose of this study was to biologically characterize non-steroidal progestins from botanicals by investigating theirinteraction and activation of progesterone receptor (PR). Eight botanicals commonly used to alleviate menopausal symptoms were investigated to determine if they contain progestins using a progesterone responsive element (PRE) luciferase reporter assay and a PR polarization competitive binding assay. Red clover extract stimulated PRE-luciferase and bound to PR. A library of purified compounds previously isolated from red clover was screened using the luciferase reporter assay. Kaempferol identified in red clover and a structurally similar flavonoid, apigenin, bound to PR and induced progestegenic activity and P4 regulated genes in breast epithelial cells and human endometrial stromal cells (HESC). Kaempferol and apigenin demonstrated higher progestegenic potency in the HESC compared to breast epithelial cells. Furthermore, phytoprogestins were able to activate P4 signaling in breast epithelial cells without downregulating PR expression. These data suggest that botanical extracts used for women's health may contain compounds capable of activating progesterone receptor signaling.     

In vitro characterization of trimegestone: a new potent and selective progestin

Trimegestone (TMG) is a novel 19-norpregnane progestin under development for hormone replacement therapy and oral contraception. The objective of the current study was to characterize the potency and steroid receptor selectivity of TMG in several in vitro assays and to compare its activity to that of medroxyprogesterone acetate (MPA). TMG and MPA had a similar competitive binding affinity for human and rabbit progesterone receptor (PR). However, TMG had a significantly higher affinity for rat PR (IC(50) = 3.3 nM) than MPA (IC(50) = 53.3 nM). In T47D cells, both compounds increased alkaline phosphatase activity and cell proliferation with comparable potencies (EC(50s) of 0.1 nM and of 0.02 nM, respectively). To further characterize the progestational activity and steroid receptor selectivity, we established an immortalized human endometrial stromal cell line (HESC-T). This cell line lacks endogenous estrogen receptor (ER) and PR but does have functional glucocorticoid receptors (GR). When ER is transiently expressed in the cells, 17beta-estradiol (E(2)) induces PR, allowing the study of PR-regulated genes. In HESC-T cells expressing exogenous ER, and therefore PR, both TMG and MPA increased HRE-tk-luciferase activity tenfold with an EC(50) of 0.2 nM. In HESC-T cells without exogenous ER, and therefore no PR, TMG did not induce HRE-tk-luciferase activity, whereas MPA induced the reporter activity with an EC(50) of about 10 nM. This MPA-induced reporter activity is believed to be mediated through GR. The steroid receptor selectivity of TMG was further evaluated using the HRE-tk-luciferase assay in the human lung carcinoma cell line A549, which contains GR but no PR. In these cells TMG had no effect on luciferase activity, whereas MPA increased the reporter activity in a dose-dependent manner with an EC(50) of approximately 30 nM. Furthermore, HRE-tk-luciferase assay in mouse fibroblast cell line L929, which expresses androgen receptor (AR) but no PR, showed that TMG had weak antiandrogenic activity whereas MPA had androgenic activity. In summary, data from several in vitro assays demonstrate that TMG is a potent progestin with a better receptor selectivity profile than MPA.     

Receptor binding and transactivation activities of red clover isoflavones and their metabolites

Red clover extracts contain a variety of isoflavones, which have affinity toward estrogen receptor alpha (ERalpha), estrogen receptor beta (ERbeta), androgen receptor (AR), and progesterone receptor (PR). Upon ingestion, they undergo various metabolic transformations. For a complete evaluation of red clover extracts and possible health benefits, the resulting metabolites should also be investigated. Biochanin A, formononetin, genistein, daidzein, dihydrobiochanin A, dihydroformononetin, dihydrogenistein, dihydrodaidzein, 3'-hydroxygenistein, 6-hydroxydaidzein, 6-hydroxydesmethylangolensin, equol, O-desmethylangolensin, angolensin, and p-ethylphenol were tested for their transactivation potential toward ERalpha, AR, and PR in yeast. Competitive binding assays with radiolabeled 17beta-estradiol, 17alpha-methyltrienolone or progesterone assessed binding to the respective ERalpha and ERbeta, AR, and PR. The compounds showed only weak binding affinity to AR and PR, with IC(50) values being greater (i.e., lesser affinity) than 10(-5)M for the respective receptor. So far, beneficial health effects have been attributed to the production of equol. We propose that other metabolites can also contribute to these effects. However, more detailed information for the formation of these metabolites in humans and for bioavailability data are required to confirm our assumptions.     

Design,synthesis, and structure–activity relationships of dihydrofuran-2-one and dihydropyrrol-2-one derivatives as novel benzoxazin-3-one-based mineralocorticoid receptor antagonists

Dihydrofuran-2-one and dihydropyrrol-2-one derivatives were identified as novel, potent and selective mineralocorticoid receptor (MR) antagonists by the structure-based drug design approach utilizing the crystal structure of MR/compound complex. Introduction of lipophilic substituents directed toward the unfilled spaces of the MR and identification of a new scaffold, dihydropyrrol-2-one ring, led to potent in vitro activity. Among the synthesized compounds, dihydropyrrol-2-one 11i showed an excellent in vitro activity (MR binding IC₅₀ = 43 nM) and high selectivity over closely related steroid receptors such as the androgen receptor (AR), progesterone receptor (PR) and glucocorticoid receptor (GR) (>200-fold for AR and PR, 100-fold for GR).     

Regulation of progesterone receptors and decidualization in uterine stroma of the estrogen receptor-alpha knockout mouse

Regulation of progesterone receptor (PR) in uterine stroma (endometrial stroma plus myometrium) by estrogen was investigated in estrogen receptor-alpha (ERalpha) knockout (alphaERKO) mice. 17 beta-Estradiol (E(2)) increased PR levels in uterine stroma of ovariectomized alphaERKO mice, and ICI 182 780 (ICI) inhibited this E(2)-induced PR expression. Estrogen receptor-beta(ER beta) was detected in both uterine epithelium and stroma of wild-type and alphaERKO mice by immunohistochemistry. In organ cultures of alphaERKO uterus, both E(2) and diethylstilbestrol induced stromal PR, and ICI inhibited this induction. These findings suggest that estrogen induces stromal PR via ERbeta in alphaERKO uterus. However, this process is not mediated exclusively by ERbeta+, because in ERbeta knockout mice, which express ERalpha, PR was up-regulated by E(2) in uterine stroma. In both wild-type and alphaERKO mice, progesterone and mechanical traumatization were essential and sufficient to induce decidual cells, even though E(2) and ERalpha were also required for increase in uterine weight. Progesterone receptor was strongly expressed in decidual cells in alphaERKO mice, and ICI did not inhibit decidualization or PR expression. This study suggests that up-regulation of PR in endometrial stroma is mediated through at least three mechanisms: 1) classical estrogen signaling through ERalpha, 2) estrogen signaling through ERbeta, and 3) as a result of mechanical stimulation plus progesterone, which induces stromal cells to differentiate into decidual cells. Each of these pathways can function independently of the others.     

Synthesis and biological effect of halogen substituted phenyl acetic acid derivatives of progesterone as potent progesterone receptor antagonists

In this paper, we report the relative binding affinities to the progesterone receptor (PR) of several progesterone derivatives containing an acetoxyphenyl substituent at C-17 and their structure-bioactivity relationship. The inhibitory effect to ovulation as well as their function as interrupters of endometrial maturation is also described. The biological activity of the novel steroids was determined in vivo and in vitro experiments using female cycling mice, which were synchronized for estrus with luteinizing hormone-releasing hormone (LHRH) and injected with the steroidal compounds. The cytosol used for the determination of the PR, was obtained from the uteri of adult estrogen-primed rabbits and the androgen (AR), mineralocorticoid (MR) and glucocorticoid (GR) receptors were determined in the cytosolic fractions from the prostate of castrated rats and from the kidneys and livers of adenalectomized male rats. We evaluated six related steroidal compounds 8a-8f differing in the nature of the 17alpha ester side chain for the inhibition of [(3)H] R5020 binding to the PR. The IC(50) values for the displacement of [(3)H] R5020 binding to the PR and its relative binding affinities (RBAs) were determined. Progesterone and R5020 had similar IC(50) values; steroids 8a, 8f and 8c bind to the progesterone receptor with RBAs of 100%, whereas 8e, 8b and 8d have RBA values <100%. These data indicate that there is a relationship between the structure of these steroids and their binding activity to the progesterone receptors. Having demonstrated in this study that steroids 8a-8f bind to the PR, we also evaluated the receptor's selectivity, since some progesterone derivatives bind to AR, MR, GR receptors. We demonstrated that the tested steroids did not bind to the AR, MR, GR, since none of the steroids inhibited the labeled mibolerone, aldosterone or dexamethasone binding to the AR, MR or GR, respectively. These results show that the novel compounds have certain selectivity for the PR. After LHRH treatment, the mice of the control group showed the presence of ova in the oviduct, whereas the animals treated with steroids 8a, 8f, 8e and 8c with RBAs of 92-100%, did not exhibit any ovum in the oviducts. As a result of this study, it is evident that the novel steroids 8a, 8f, 8e and 8c inhibited the ovulation in these animals at dose of 0.22mg/kg. After the treatment with LHRH, the uterus of the control group showed the typical progestational activity with an enlarged endometrial thickness with secretory activity. However, the endometrium of the mice treated with steroids 8a, 8f, 8e and 8c (with RBAs of 92-100%) neither did show any enlargement of the endometrium, nor a secretory activity could be detected. The diameter of the uterus was also significantly reduced compared to those of the control group, thus indicating that compounds 8a, 8f, 8e and 8c had antagonistic activity in this tissue. The overall data showed that steroids 8a, 8f, 8e and 8c have a high and selective binding activity to the PR. Furthermore there is a relationship between the structure of these steroids and their binding activity, since the presence of fluorine atom in meta position in the acetoxyphenyl substituent at C-17, improved the binding activity as compared to that for the ortho and para positions. These data also demonstrated that 8a-8f have an anti-progestational activity in vivo, and therefore they have better characteristics than the compounds previously reported.     

Acteoside and martynoside exhibit estrogenic/antiestrogenic properties

Acteoside and martynoside are plant phenylpropanoid glycosides exhibiting anticancer, cytotoxic and antimetastatic activities. We investigated their potential to activate estrogen receptor isoforms ERalpha and ERbeta in HeLa cells transfected with an estrogen response element (ERE)-driven luciferase (Luc) reporter gene and an ERalpha or ERbeta expression vector. Their estrogenic/antiestrogenic effects were also assessed in breast cancer cells (MCF7), endometrial cancer cells (Ishikawa) and osteoblasts (KS483), by measuring IGFBP3 levels, cell viability and number of mineralized nodules, respectively, seeking for a natural selective estrogen receptor modulator (SERM). Acteoside and martynoside antagonized both ERalpha and ERbeta (p<0.001), whereas they reversed the effect of E(2) mainly via ERalpha (p<0.001). Martynoside was a potent antiestrogen in MCF-7 cells, increasing, like ICI182780, IGFBP3 levels via the ER-pathway. In osteoblasts, martynoside induced nodule mineralization, which was abolished by ICI182780, implicating an ER-mediated mechanism. Furthermore, its antiproliferative effect on endometrial cells suggests that martynoside may be an important natural SERM. Acteoside was an antiestrogen in breast cancer cells and osteoblasts, without any effect on endometrial cells. Our study suggests that the nature is rich in selective ERalpha and ERbeta ligands, the discovery of which may lead to the development of novel neutraceutical agents.     

Activities of a non-classical estrogen,Z-bis-dehydrodoisynolic acid,with ERalpha and ERbeta

(+/-)-Z-bis-Dehydrodoisynolic acid [(+/-)-Z-BDDA] is highly estrogenic in vivo, yet binds to estrogen receptor (ER) poorly. This paradox has raised the possibility of alternative ERs and/or molecular mechanisms. To address the possibility of high activities of Z-BDDA with ERbeta, we determined the activities of (+)-Z-BDDA and (-)-Z-BDDA, in cell culture and in vitro, comparing ERbeta to ERalpha. Transfectional analysis in Hela cells showed (-)-Z-BDDA is an agonist for gene activation with both ERalpha (EC(50) congruent with 0.3nM) and ERbeta (EC(50) congruent with 5nM), while little to no activity was observed with (+)-Z-BDDA. Similarly, in gene repression assays, (-)-Z-BDDA was active (EC(50) congruent with 0.2nM), but again minimal activity was exhibited by (+)-Z-BDDA. Binding to ERalpha and ERbeta in vitro used both competition and a direct binding assay. For ERalpha, the relative affinity of (-)-Z-BDDA was approximately 6% by competition and 1.7% by direct binding versus 17beta-estradiol (E2; 100%), while (+)-Z-BDDA also demonstrated binding, but with relative affinities of only 0.08% by competition and 0.3% by the direct assay. For ERbeta, the affinity of (-)-Z-BDDA was approximately 7% by competition and 1.5% by the direct assay relative to E2 (100%), while (+)-Z-BDDA had lower affinity, approximately 0.2% that of E2 by both assays.The paradox of potent in vivo activity but lower activity in receptor binding and in cell culture reporter gene assays, previously seen with ERalpha is now also associated with ERbeta. The failure of ERbeta to explain the activity-binding paradox indicates the need for additional in vivo metabolic and pharmacokinetic studies and continued consideration of alternative mechanisms.     

Messenger RNA levels of estrogen receptors alpha and beta and progesterone receptors in the cyclic and inseminated/early pregnant sow uterus

The aim of the present study was to investigate differences in the expression of mRNAs for ERalpha, ERbeta and PR in the sow uterus at different stages of the estrous cycle as well as in inseminated sows at estrus and during early pregnancy by use of solution hybridization and in relation to plasma levels of estradiol and progesterone. Uterine samples were collected at different stages of the estrous cycle and after insemination/early pregnancy. In the endometrium, the expression of ERalpha mRNA and PR mRNA was similar for cyclic and early pregnant groups. Both were highest at early diestrus/70 h after ovulation and ERalpha mRNA was lowest at late diestrus/d 19 while PR mRNA was lowest at diestrus and late diestrus/d 11 and d 19. The expression of endometrial ERbeta was constantly low during the estrous cycle but higher expression was found in inseminated/early pregnant sows at estrus and 70 h after ovulation. In the myometrium, high expression of ERalpha mRNA and PR mRNA was observed at proestrus and estrus in cyclic sows and at estrus in newly inseminated sows. Higher expression of myometrial ERbeta mRNA was found in inseminated/early pregnant sows compared with cyclic sows, although significant only at estrus. In conclusion, the expression of mRNAs for ERalpha, ERbeta and PR in the sow uterus differed between endometrium and myometrium as well as with stages of the estrous cycle and early pregnancy. In addition to plasma steroid levels, the differences between cyclic and inseminated/early pregnant sows suggest that other factors, e.g. insemination and/or the presence of embryos, influence the expression of these steroid receptor mRNAs in the sow uterus.     

Differential expression of estrogen receptor-alpha and -beta and androgen receptor in the ovaries of marmosets and humans

Estrogens and androgens are essential for the maturation of the ovarian follicle and normal fertility in the female. We have used antibodies specific for both forms of estrogen receptor (alpha [ERalpha] and beta [ERbeta]) and androgen receptor (AR) to investigate the pattern of receptor expression in ovaries obtained from women and from a New World primate, the Common marmoset (Callthrix jacchus). On Western blots, three antibodies directed against different peptides within human ERbeta all recognized recombinant human (h) ERbeta but did not bind to recombinant hERalpha. The ERbeta protein was extracted from human ovary and prostate and marmoset ovary. In marmoset and human ovaries, ERbeta protein was detected in the nuclei of granulosa cells in all sizes of follicle (both before and after formation of the antrum), and it was also detected in thecal cells, corpora lutea, surface epithelium, and stroma. In contrast, ERalpha protein was not detected in the nuclei of granulosa cells in preantral follicles, was low/absent from stromal and thecal cells, but was expressed in granulosa cells of antral follicles and in the surface epithelium. The pattern of expression of AR protein more closely resembled that of ERbeta than ERalpha. In conclusion, three independent antibodies have demonstrated convincingly that ERbeta is expressed in a wide range of cells in the primate ovary. Granulosa cells in preantral follicles could contain ERbeta:beta dimers. In antral follicles, however, ERalpha is also expressed, and the formation of homo- or heterodimers containing ERalpha may influence the pattern of gene activation within these cells.