Helenius J. Kloosterboer

Tibolone: a steroid with a tissue-specific mode of action ☆

In postmenopausal women tibolone has proved to prevent bone-loss and relieve climacteric symptoms as effectively as estrogens, but it does not stimulate the endometrium and the breast. This clinical profile strongly suggests that tibolone is a compound with tissue-specific action. Tibolone is quickly metabolized into its main active metabolites, 3alpha and 3beta-OH, which are also present in an inactive, sulphated, form. In addition a Delta4-metabolite is found in circulation. The 3-OH-metabolites bind only to the estrogen receptor while the Delta4-isomer shows affinity only to the progesterone and androgen receptors. Tibolone prevents bone loss in a similar way to estrogens. Studies on bone mass using anti-estrogen, antiprogestin and anti-androgen in combination with tibolone, confirmed the sole involvement of the estradiol receptor. Increases in skin temperature as well as vaginal atrophy can be prevented by tibolone in a similar way to estrogens. Breast safety studies showed that tibolone clearly inhibited the growth of tumors in a DMBA model. In breast cell lines, tibolone profoundly inhibited sulphatase activity and an increase in apoptosis and decrease in cell proliferation was found. The stimulation of the endometrium is prevented by the local formation of the Delta4-isomer from tibolone or the 3beta-OH-metabolite. We conclude that tibolone acts as a tissue-specific compound by mediating its effects via steroid receptors and enzymatic pathways. This dual effect of tibolone explains its positive clinical effects on bone, vagina and brain, and avoids stimulation of the endometrium and breast tissue.

Hormonal regulation of apoptosis in breast cells and tissues.

Few studies have referred to the implication of apoptotic processes following hormonal treatment. No data are available on the effects of progesterone in breast cells. In order to gain insights on the effects of the gonadal steroids and antiestrogens in breast cells, we have carried out studies on apoptosis in different breast materials. We have developed a model of normal breast cells in cultures that remain hormone-dependent. On these cells and in some hormone-dependent breast cancer cell lines (T-47-D, ZR75-1, MCF-7) we have observed an antiapoptotic effect of estradiol (E(2)) and a potent proapoptotic effect of some antiestrogens. Progestins were also proapoptotic in normal as well as in hormone-dependent breast cancer cells. In order to understand the mechanisms of these hormones on apoptosis, we studied the bcl-2 family proteins. We demonstrated that E(2) increased the antiapoptotic proteins, bcl-2 and bclx(L), whereas, the progestins drastically decreased bcl-2 expression and weakly bclx(L) levels. We investigated the mechanisms by which E(2) increased bcl-2 expression. Our results using quantitative RT-PCR showed that E(2) increased bcl-2 mRNA levels at 48 h of treatment via a transcriptional mechanism. None of the hormone treatments altered the proapoptotic protein levels, bax and bak. We also studied the in vivo expression of bcl-2 and other members of its family in biopsies of normal breast tissues according to the menstrual cycle. Bcl-2 displayed a strong cyclical variation and seemed to be the most hormone-dependent member of the family.

Effects of seven low-dose combined oral contraceptives on sex hormone binding globulin, corticosteroid binding globulin, total and free testosterone

The effect of seven low-dose oral contraceptive preparations on sex hormone binding globulin (SHBG), cortisol binding globulin (CBG), total and absolute free testosterone were investigated in groups of 10 healthy volunteers. All preparations contained about the same amount of ethinylestradiol but they differed in type and/or dose of progestagen. The progestagens studied were: levonorgestrel (LNG; in mono- and triphasic preparations), norethisterone (NET; in monophasic preparation), desogestrel (DSG; in mono- and biphasic preparations) and gestodene (GSD; in triphasic preparation), all 19-nortestosterone derivatives, and the anti-androgen cyproterone acetate (CPA) in a monophasic preparation. Differences observed in SHBG level, which reflect the estrogen-androgen balance, can be attributed to the intrinsic androgenic (or anti-androgenic) properties of the progestagens, and were in agreement with the results of published receptor binding studies, performed in vitro. Based on our results the following ranking (high to low) can be made with respect to the androgenicity of the preparations: monophasic LNG greater than or equal to monophasic NET = triphasic LNG greater than or equal to triphasic GSD = biphasic DSG = monophasic DSG greater than monophasic CPA. An anti-estrogenic effect of the 19-nortestosterone derived progestagens can be excluded by the effect on CBG, a marker for estrogenic activity. All preparations containing a 19-nortestosterone derived progestagen, independent of their type and dose, induce a similar rise in CBG, whereas the preparation with cyproterone acetate induced an even higher CBG level. Irrespective of the effect on total testosterone, which varies between the preparations, the absolute free testosterone level decreased to a comparable degree for all preparations.(ABSTRACT TRUNCATED AT 250 WORDS)

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 tibolones 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.

Development of Time-Resolved Immunofluorometric Assays for Rat Follicle-Stimulating Hormone and Luteinizing Hormone and Application on Sera of Cycling Rats

Abstract The aim of this study was to develop time-resolved immunofluorometric assays (TR-IFMA) for measuring rat (r)FSH and rLH. The advantages of these IFMAs are higher sensitivity due to lower background values, higher specificity as only intact molecules of FSH and LH can be measured, and a very long shelf life of the nonradioactive biotin antigens compared with radiolabeled iodine antigens. For rFSH, IFMAs are lacking, while for rLH, if present, the resources for antibodies are scarce or the mouse monoclonal antibodies (mMAbs) against LHα are inactive with FSH. Thus specific antibodies need to be obtained. With the final TR-IFMAs, rFSH and rLH levels were assessed during the estrous cycle and compared with those obtained with the more classical RIAs and fluoroimmunoassays (FIAs). Two IFMAs for rFSH were developed with mMAbs against the recombinant human (rec h)FSHβ subunit (FSH56A) attached to the wall and two different rabbit polyclonal antibodies (PAbs) against the α subunit of rec hFSH (R93–2705) or recombinant rat (rec r)LH (R95–2715) conjugated with biotin as signal antibody. With both IFMAs, rFSH holo-molecules can be measured. Rat FSH standards could be assessed between 0.02 and 10 ng/ml with a detection limit of 0.05 and 0.24 ng/ml in buffer and serum, respectively. These detection limits in four IFMAs were 8- to 16-fold lower than those in RIAs and FIAs. This detection level allowed the measurement of FSH levels in serum of hypophysectomized (HYPEX) rats at 0.18 ng/ml. In serum of cycling rats, the FSH levels of the IFMA were 2-fold lower than those of the FIA, while in ovariectomized (OVX) rats the IFMA levels were comparable. A peak level of FSH was found during proestrus of Day 2 and gestation with both RIA and FIA, but with IFMAs at gestation only. An IFMA for rLH was set up with mMAb (hCG77A) reacting with rLHβ as capture and rabbit PAb to rec rLHα (R95–2712) as signal antibody. Rat LH standard could be assessed between 0.001 and 10 ng/ml with a detection limit of 0.012 and 0.1 ng/ml in buffer and serum, respectively, which was 8-fold lower than that in RIA/FIA. In serum of HYPEX rats, LH was undetectable (< 0.04 ng/ml), whereas a high background level of 2.5 ng/ml was measured in the FIA. In serum of cycling rats, only a very low LH level of 0.14 ng/ml was measured, which strongly deviated from the level of 3.46 ng/ml with an FIA. The load of LH in serum of OVX rats was 2.91 ng/ml, which was 12-fold lower than that for the FIA. The peak level of LH was detected on proestrus Day 2 with RIA, FIA, and IFMA. In conclusion, two IFMAs for rFSH and one for rLH have been developed with high sensitivity and specificity for intact gonadotropins. The LH pattern during the estrous cycle was comparable between IFMA, RIA, and FIA, although the overall level in the IFMA was much lower, as were HYPEX levels. The FSH pattern differed only on proestrus Day 2 in the IFMA from that of RIA/FIA, showing a peak level with RIA/FIA and a basal level with the IFMA. This implies that in RIA/FIA measurements, proteins other than intact FSH and LH interfere with the analysis at proestrus Day 2 for FSH and in HYPEX, cycling, and OVX rats for LH.

Human endometrial 3β-hydroxysteroid dehydrogenase/isomerase can locally reduce intrinsic estrogenic/progestagenic activity ratios of a steroidal drug (Org OD 14)

In vitro conversion in human endometrial tissue of Org OD 14 [17 alpha-hydroxy-7 alpha-methyl-19-norpregn-5(10)-en-20-yn-3-one, a 3-keto-delta 5-10-19-nortestosterone derivative structurally related to norethynodrel] to its 4-ene isomer was demonstrated and measured spectrophotometrically and by chromatographic separation of the labeled metabolite from the tritiated precursor. The endometrial isomerase catalyzing this conversion is the 3 beta-hydroxy-steroid dehydrogenase/isomerase (3 beta HSD/isomerase), detected by Western blotting as a 42 kDa band, as confirmed by the inhibition of Org OD 14 isomerization with an antibody against this enzyme. The endometrial isomerase activity was found to be higher in secretory than in proliferative tissue and to be influenced by progestins, as suggested by the small but significant increase in activity resulting from exposure of proliferative endometrium to medroxyprogesterone acetate under organotypic culture conditions. In addition to the expected physiologic importance of endometrial 3 beta HSD/isomerase in the local metabolism of circulating steroids of adrenal origin, its presence in the endometrium is likely to have pharmacologic relevance, as illustrated by the local conversion of Org OD 14 to the 4-ene isomer, a metabolite with higher progestagenic and lower estrogenic potencies than those of its precursor. The local, tissue-specific, modification of the precursor would yield intracellular concentration ratios of Org OD 14 to 4-ene isomer in the endometrium significantly lower than those in blood. As a result, the estrogenic effects of Org OD 14 or of its 3-hydroxy metabolites on endometrial cell proliferation are minimized by the local formation of the progestagenic 4-ene isomer. This is a favorable feature of Org OD 14 since it selectively prevents undesirable proliferative stimulation of the endometrium in postmenopausal users while preserving its beneficial effects on other tissues, including bone.

Selectivity in progesterone and androgen receptor binding of progestagens used in oral contraceptives.

The relative binding affinities (RBAs) of four progestational compounds (norethisterone, levonorgestrel, 3-keto-desogestrel and gestodene) for the human progesterone and androgen receptors were measured in MCF-7 cytosol and intact MCF-7 cells. For the binding to the progesterone receptor, both Org 2058 and Org 3236 (or 3-keto-desogestrel) were used as labelled ligands. The following ranking (low to high) for the RBA of the nuclear (intact cells) progesterone receptor irrespective of the ligand used is found: norethisterone much less than levonorgestrel less than 3-keto-destogestrel less than gestodene. The difference between the various progestagens is significant with the exception of that between 3-keto-desogestrel and gestodene, when Org 2058 is used as ligand. For the cytosolic progesterone receptor, the same order is found with the exception that similar RBAs are found for gestodene and 3-keto-desogestrel. The four progestagens clearly differ with respect to binding to the androgen receptor using dihydrotestosterone as labelled ligand in intact cells; the ranking (low to high) is: norethisterone less than 3 keto-desogestrel less than levonorgestrel and gestodene. The difference between 3-keto-desogestrel and levonorgestrel or gestodene is significant. The selectivity indices (ratio of the mean RBA for the progesterone receptor to that of androgen receptor) in intact cells are significantly higher for 3-keto-desogestrel and gestodene than for levonorgestrel and norethisterone. From these results we conclude that the introduction of the 18-methyl in norethisterone (levonorgestel) increases both the binding to the progesterone and androgen receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of progestagens and Org OD14 in in vitro and in vivo tumor models.

Sex steroids, in particular estradiol (E2) and progesterone (P4), play, together with other hormones and growth factors, a role in the development of normal breast tissue. The effect of four progestagens (norethisterone, 3-ketodesogestrel, gestodene and P4) and Org OD14, a steroid with weak estrogenic, progestagenic and androgenic properties were studied on growth of breast tumor cells in vitro using two subclones of MCF-7 (H and A) and T47D (S and A) cells. In addition, we investigated the effects of 3-ketodesogestrel, gestodene and Org OD14 on the growth of 7,12-dimethylbenz(a)anthracene(DMBA)-induced mammary tumors in rats. In the in vitro assays with MCF-7 cells norethisterone, 3-ketodesogestrel and gestodene stimulated growth only at high doses (> or = 10(-7) M), whereas P4 had no effect. Gestodene was more potent than 3-ketodesogestrel and norethisterone. Org OD14, stimulated cell growth at a dose of 10(-8) M, while E2 is active at 10(-10) M. In T47D-A cells similar effects were found, but the subclone S did not respond to the progestagens and Org OD14. The two T47D subclones also reacted differently to progestagens during growth stimulation with E2. In T47D-S the progestagens and Org OD14 inhibited, while in T47D-A these compounds did not modulate the effect of E2. In the DMBA model we found that gestodene and 3-ketodesogestrel were able to inhibit tumor growth to the same extent. Surprisingly, Org OD14 was even more effective in the DMBA model using the therapeutic approach. Using the prophylaxic approach tumor development was delayed and tumor growth was strongly suppressed. The inhibitory effects of Org OD14 on tumor growth in the DMBA model may be attributed to its mixed hormonal profile. From these studies we conclude that different cell lines and even subclones thereof respond quite differently to steroids. Both in vitro and in vivo studies are required to judge whether synthetic steroids might be involved in an increased risk for the development of breast tumors.

Human chorionic gonadotropin in commercial human menopausal gonadotropin preparations.

Several studies indicated the presence of an hCG-immunoreactive substance in commercial hMG preparations. Because hCG represents LH activity with a relatively very long half-life, differences between hMG preparations with respect to hCG content could imply clinical differences. To investigate whether there is any difference in this respect between the two most widely used hMG preparations, we measured the hCG content of ampules of Humegon and Pergonal retrieved from the market, together representing 51 separate production batches, with one or more different specific immunological assays. There are no significant differences between Humegon and Pergonal with respect to the mean hCG level per ampule as measured by RIA, ELISA, and Delfia. The batch-to-batch consistency for Humegon is higher.

Progesterone inhibition of Wnt/β-catenin signaling in normal endometrium and endometrial cancer

Purpose. Wnt signaling regulates the fine balance between stemness and differentiation. Here, the role of Wnt signaling to maintain the balance between estrogen-induced proliferation and progesterone-induced differentiation during the menstrual cycle, as well as during the induction of hyperplasia and carcinogenesis of the endometrium, was investigated. Experimental Design: Endometrial gene expression profiles from estradiol (E2) and E2 + medroxyprogesterone acetate–treated postmenopausal patients were combined with profiles obtained during the menstrual cycle (PubMed; GEO DataSets). Ishikawa cells were transfected with progesterone receptors and Wnt inhibitors dickkopf homologue 1 (DKK1) and forkhead box O1 (FOXO1), measuring Wnt activation. Expression of DKK1 and FOXO1 was inhibited by use of sequence-specific short hairpins. Furthermore, patient samples (hormone-treated endometria, hyperplasia, and endometrial cancer) were stained for Wnt activation using nuclear β-catenin and CD44. Results: In vivo, targets and components of the Wnt signaling pathway (among them DKK1 and FOXO1) are regulated by E2 and progesterone. In Wnt-activated Ishikawa cells, progesterone inhibits Wnt signaling by induction of DKK1 and FOXO1. Furthermore, using siRNA-mediated knockdown of both DKK1 and FOXO1, progesterone inhibition of Wnt signaling was partly circumvented. Subsequently, immunohistochemical analysis of the Wnt target gene CD44 showed that progesterone acted as an inhibitor of Wnt signaling in hyperplasia and in well-differentiated endometrial cancer. Conclusion: Progesterone induction of DKK1 and FOXO1 results in inhibition of Wnt signaling in the human endometrium. This Wnt inhibitory effect of progesterone is likely to play a rate-limiting role in the maintenance of endometrial homeostasis and, on its loss, in tumor onset and progression toward malignancy. (Clin Cancer Res 2009;15(18):5784–93)