Antioxidant effect of the active metabolites of tibolone.

Certain steroidal compounds have an antioxidant effect in humans. Our aim was to test whether the synthetic steroid tibolone and its metabolites are also able to display such a property. For this, granulocytes from healthy men and women were incubated for two hours with different concentrations (10(-7), 10(-8), 10(-9 )M) of either estradiol, tibolone, 3α-hydroxytibolone, 3ß-hydroxytibolone, Δ(4)-tibolone, 3α-sulfated-tibolone, 3α-17ß-disulfated-tibolone, 3ß-sulfated-tibolone or 3ß-17ß-disulfated-tibolone. Superoxide anion generation of neutrophils was measured by photometry. Results of different steroids were given as percentages of their controls. A more simple superoxide generating system, the xanthine-xanthine oxidase reaction was also tested. We found that granulocyte superoxide production did not differ from the control using 10(-9 )M of steroids. Using 10(-8 )M concentration: estradiol (80.9 ± 2.5%); 3ß-sulfated-tibolone (83.3 ± 4.7%); 3ß-17ß-disulfated-tibolone (81.0 ± 4.2%) caused a significant decrease in superoxide production, compared to the control. In addition at 10(-7 )M, 3ß-hydroxytibolone and 3α-sulfated-tibolone also showed antioxidant effects. In the xanthine-xanthine oxidase system estradiol (67.4 ± 1.0%), 3α-sulfated-tibolone (85.8 ± 5.3%), 3α-17ß-disulfated-tibolone (71.9 ± 2.5%), 3ß-sulfated-tibolone (73.9 ± 5.0%), and 3ß-17ß-disulfated-tibolone (65.8 ± 3.4%) caused a significant decrease in superoxide production. Conclusively, although tibolone itself did not show significant antioxidant capacity, most of its active metabolites have antioxidant effects.

Metabolism of the synthetic progestogen norethynodrel by human ketosteroid reductases of the aldo-keto reductase superfamily.

Human ketosteroid reductases of the aldo-keto reductase (AKR) superfamily, i.e. AKR1C1-4, are implicated in the biotransformation of synthetic steroid hormones. Norethynodrel (NOR, 17α-ethynyl-17ß-hydroxy-estra-5(10)-en-3-one), the progestin component of the first marketed oral contraceptive, is known to undergo rapid and extensive metabolism to 3α- and 3ß-hydroxymetabolites. The ability of the four human AKR1C enzymes to catalyze the metabolism of NOR has now been characterized. AKR1C1 and AKR1C2 almost exclusively converted NOR to 3ß-hydroxy NOR, while AKR1C3 gave 3ß-hydroxy NOR as the main product and AKR1C4 predominantly formed 3α-hydroxy NOR. Individual AKR1C enzymes also displayed distinct kinetic properties in the reaction of NOR. In contrast, norethindrone (NET), the Δ(4)-isomer of NOR and the most commonly used synthetic progestogen, was not a substrate for the AKR1C enzymes. NOR is also structurally identical to the hormone replacement therapeutic tibolone (TIB), except TIB has a methyl group at the 7α-position. Product profiles and kinetic parameters for the reduction of NOR catalyzed by each individual AKR1C isoform were identical to those for the reduction of TIB catalyzed by the respective isoform. These data suggest that the presence of the 7α-methyl group has a minimal effect on the stereochemical outcome of the reaction and kinetic behavior of each enzyme. Results indicate a role of AKR1C in the hepatic and peripheral metabolism of NOR to 3α- and 3ß-hydroxy NOR and provide insights into the differential pharmacological properties of NOR, NET and TIB.

Estetrol prevents and suppresses mammary tumors induced by DMBA in a rat model.

BACKGROUND: Estetrol (E4) is a pregnancy-specific estrogenic steroid hormone produced by the human fetal liver in both male and female fetuses. During pregnancy, E4 plasma values increase exponentially until parturition and decrease thereafter. The synthesis of E4 in the liver of a newborn ceases during the first weeks after birth. MATERIALS AND METHODS: Here we report the effect of E4 on the initiation and growth of mammary tumors chemically induced by 7,12 dimethylbenz(a)anthracene (DMBA) in female Sprague-Dawley rats in two different protocols. Two prevention studies to test the effect on initiation and growth of induced tumors and one intervention study to test the effect on tumor growth were performed. In the prevention studies, the effect of oral doses of E4 over a dose range of 0.5-3.0 mg/kg was investigated. In the intervention study, oral doses of 1, 3 and 10 mg/kg E4 were used. The anti-estrogen tamoxifen (TAM) and ethinylestradiol (EE) were used as reference compounds. In all studies, a group with ovariectomized animals (OVX) was included. RESULTS: In the prevention studies, 2.5 mg and 3 mg/kg E4 showed a significant effect on the number and growth of induced tumors by DMBA, and the effects were comparable to those of TAM, whereas EE had no effect. In the intervention study, the effect of a high dose of E4 (10 mg/kg) on tumor number was similar to that of OVX and better than TAM and high-dose EE. The 3 mg/kg E4 had an effect comparable to high-dose EE. The treatment effects were largely due to complete regression of existing tumors. CONCLUSIONS: The natural fetal estrogen E4 prevents tumor initiation by DMBA and inhibits the growth of existing DMBA-induced tumors.

Hormone replacement therapy dependent changes in breast cancer-related gene expression in breast tissue of healthy postmenopausal women.

Risk assessment of future breast cancer risk through exposure to sex steroids currently relies on clinical scorings such as mammographic density. Knowledge about the gene expression patterns in existing breast cancer tumors may be used to identify risk factors in the breast tissue of women still free of cancer. The differential effects of estradiol, estradiol together with gestagens, or tibolone on breast cancer-related gene expression in normal breast tissue samples taken from postmenopausal women may be used to identify gene expression profiles associated with a higher breast cancer risk. Breast tissue samples were taken from 33 healthy postmenopausal women both before and after a six month treatment with either 2mg micronized estradiol [E2], 2mg micronized estradiol and 1mg norethisterone acetate [E2+NETA], 2.5mg tibolone [T] or [no HRT]. Except for [E2], which was only given to women after hysterectomy, the allocation to each of the three groups was randomized. The expression of 102 mRNAs and 46 microRNAs putatively involved in breast cancer was prospectively determined in the biopsies of 6 women receiving [no HRT], 5 women receiving [E2], 5 women receiving [E2+NETA], and 6 receiving [T]. Using epithelial and endothelial markers genes, non-representative biopsies from 11 women were eliminated. Treatment of postmenopausal women with [E2+NETA] resulted in the highest number of differentially (p<0.05) regulated genes (16.2%) compared to baseline, followed by [E2] (10.1%) and [T] (4.7%). Among genes that were significantly down-regulated by [E2+NETA] ranked estrogen-receptor-1 (ESR1, p=0.019) and androgen receptor (AR, p=0.019), whereas CYP1B1, a gene encoding an estrogen-metabolizing enzyme, was significantly up-regulated (p=0.016). Mammary cells triggered by [E2+NETA] and [E2] adjust for steroidogenic up-regulation through down-regulation of the estrogen-receptor pathway. In this prospective study, prolonged administration of [E2+NETA] and to a lesser extent of [E2] but not [T] were associated in otherwise healthy breast tissue with a change in the expression of genes putatively involved in breast cancer. Our data suggest that normal mammary cells triggered by [E2+NETA] adjust for steroidogenic up-regulation through down-regulation of the estrogen-receptor pathway. This feasibility study provides the basis for whole genome analyses to identify novel markers involved in increased breast cancer risk.

Progesterone inhibition of Wnt/beta-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 (E(2)) and E(2) + 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 beta-catenin and CD44. RESULTS: In vivo, targets and components of the Wnt signaling pathway (among them DKK1 and FOXO1) are regulated by E(2) 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.

Progestins induce catalase activities in breast cancer cells through PRB isoform: correlation with cell growth inhibition.

Reactive oxygen species (ROS) have been suggested to participate in tumor emergence due to their mitogenic and apoptotic signaling, and as contributors to DNA structural damage. Here we report that progesterone and various synthetic steroids with progestin potencies (norethisterone acetate, MPA, and Tibolone) counteract cell growth induced by hydrogen peroxide (H(2)O(2)), through a potent induction of catalase activities, in breast cancer cells and normal human epithelial breast cells. At physiological concentrations, progesterone and the pure progestin, Org2058, displayed the most potent H(2)O(2) detoxification ability suggesting its effect was characteristic of its progestin potency. We also report on the enhancement of catalase activities by progesterone receptor isoform B (PRB), as determined from experiments using antiprogestins and MDA-MB-231, cells engineered for the selective expression of progesterone receptor isoform A or B. The potent action of progesterone on catalase activities indicates its contribution to a beneficial role in breast cell homeostasis.

Human cytosolic hydroxysteroid dehydrogenases of the aldo-ketoreductase superfamily catalyze reduction of conjugated steroids: implications for phase I and phase II steroid hormone metabolism.

Aldo-ketoreductase 1C (AKR1C) enzymes catalyze the NADPH-dependent reduction of ketosteroids to hydroxysteroids. They are Phase I metabolizing enzymes for natural and synthetic steroid hormones. They convert 5alpha-dihydrotestosterone (Dht, potent androgen) to 3alpha/beta-androstanediols (inactive androgens) and the prodrug tibolone (Tib) to estrogenic 3alpha/beta-hydroxytibolones. Herein we demonstrate for the first time that human AKR1C enzymes (AKR1C1-4) are able to reduce conjugated steroids such as Dht-17beta-glucuronide (DhtG), Dht-17beta-sulfate (DhtS), and Tib-17beta-sulfate (TibS). Product identities were characterized by liquid chromatography-mass spectrometry, and kinetic parameters of the reactions were determined. The product profile of the reduction of each steroid conjugate by the individual AKR1C isoform was similar to that of the corresponding free steroid except for the reduction of DhtG catalyzed by AKR1C2, where a complete inversion in stereochemical preference to 3beta-reduction (with DhtG) from 3alpha-reduction (with Dht and DhtS) was observed. The catalytic efficiency of 3-keto reduction was modestly affected by the presence of a 17-sulfate group but severely impaired by the presence of a 17-glucuronide group for AKR1C1-3 isoforms. AKR1C4, however, showed superior catalytic efficiencies versus the other isoforms, and those were unaffected by steroid conjugation. Our findings provide evidence for alternative pathways of steroid metabolism where the phase I reaction (reduction) occurs after the phase II reaction (conjugation). Specifically, it is indicated that Dht is metabolized to its metabolite 3alpha-androstanediol-17-glucuronide via the previously unrecognized "conjugation pathway" involving the sequential reactions of UGT2B17 and AKR1C4 in liver but via the conventional "reduction pathway" involving the sequential reactions of AKR1C2 and UGT2B15/17 in prostate.

The estrogenic component of tibolone reduces adiposity in female aromatase knockout mice.

OBJECTIVE: To explore the effects of tibolone on adiposity in the absence of aromatase and determine which of the hormonal properties of tibolone are exerting these effects. METHODS: In this study, vehicle; tibolone; estrogenic (ethinyl estradiol [EE]), progestogenic (ORG2058), or androgenic (dihydrotestosterone) compounds; or a combination of ORG2058 + EE was administered to 6-month-old ovariectomized aromatase knockout (ArKO) mice for a period of 6 weeks. RESULTS: In response to tibolone or EE-alone treatments, omental adipose tissue and infrarenal adipose tissue weights were significantly reduced (P = 0.004 and P = 0.01; P = 0.009 and P = 0.014, respectively) compared with those in ovariectomized and vehicle-treated ArKO mice. In contrast, adipose tissue weight tended to increase after ORG2058-alone treatment. Furthermore, EE in the presence of ORG2058 (ORG2058 + EE group) results in little effect on adiposity when compared with that in ovariectomized and vehicle-treated ArKO mice, showing that ORG2058 can negate the effect of EE. Dihydrotestosterone treatment did not have an impact on adipose tissue mass. Adipocyte volume and numbers followed the same treatment trends. CONCLUSIONS: In summary, our study in the ArKO mouse has confirmed the efficacy of tibolone as a hormone therapy to reduce adipose tissue accumulation after menopause and also shows that aromatization of tibolone is not required to elicit these estrogenic effects.

Effects of tibolone metabolites on human endometrial cell lines in co-culture.

In human endometrium, cell proliferation is regulated by ovarian steroids through heterotypic interactions between stromal and epithelial cells populating this tissue. The authors test the proliferative effects of tibolone and its metabolites using endometrial co-cultures that mimic the normal proliferative response to hormones. They found that both the Delta(4)-tibolone metabolite and the pure progestin ORG2058 counteract estradiol-driven epithelial cell proliferation. Surprisingly, the estrogen receptor binding 3-hydroxyl-metabolites of tibolone also counteracted estradiol-driven proliferation. Inhibition of proliferation by 3beta-OH-tibolone was abrogated by low doses of the progesterone receptor antagonist mifepristone. This suggests that 3beta-OH-tibolone is converted to a progestagenic metabolite. The authors found that the stromal cells used in the co-cultures express high levels of the ketosteroid dehydrogenase AKR1C2, which is able to oxidize 3beta-OH-tibolone back to tibolone. Thus, the unexpected progestagenic effect of 3beta-OH-tibolone in these co-cultures may be due to metabolic activity present in the stromal cells of the co-cultures.

Difference in signalling between various hormone therapies in endometrium, myometrium and upper part of the vagina.

BACKGROUND: Combined hormone treatments in post-menopausal women have different clinical responses on uterus and vagina; therefore, we investigated differences in steroid signalling between various hormone therapies in these tissues. METHODS: A total of 30 post-menopausal women scheduled for hysterectomy were distributed into four subgroups: control-group (n = 9), Tibolone-group (n = 8); estradiol (E(2))-group (n = 7); E(2) + medroxyprogesterone acetate (MPA)-group (n = 6). Medication was administered orally every day for 21 days prior to removal of uterus and upper part of the vagina. Tissue RNA was isolated, and gene expression profiles were generated using GeneChip technology and analysed by cluster analysis and significance analysis of microarrays. Apoptosis and cell proliferation assays, as well as immunohistochemistry for hormone receptors were performed. RESULTS: 21-days of treatment with E(2), E(2) + MPA or tibolone imposes clear differential gene expression profiles on endometrium and myometrium. Treatment with E(2) only results in the most pronounced effect on gene expression (up to 1493 genes differentially expressed), proliferation and apoptosis. Tibolone, potentially metabolized to both estrogenic and progestagenic metabolites, shows some resemblance to E(2) signalling in the endometrium and, in contrast, shows significant resemblance to E(2) + MPA signalling in the myometrium. In the vagina the situation is entirely different; all three hormonal treatments result in regulation of a small number (4-73) of genes, in comparison to signalling in endometrium and myometrium. CONCLUSION: Endometrium and myometrium differentially respond to the hormone therapies and use completely different sets of genes to regulate similar biological processes, while in this experiment the upper part of the vagina is hardly hormone responsive.