Binding characteristics of RU 486 with glucocorticoid and progestin receptors

RU 34886 (RU 486) has been proved to fully antagonize the actions of glucocorticoid and progestin at the receptor level. The binding characteristics of RU 486 with the thymic glucocorticoid receptor (GR) and the uterine progestin receptor (PR) were investigated in order to elucidate the mechanism of these antagonistic actions. The ability of RU 486 was studied to promote the "activation" and the "nuclear translocation" of GR and RP. Under heat activation, RU 486 dissociated faster from the activated GR than the non activated, and the binding complex of RU 486 and GR showed lower affinity for DNA-cellulose than the glucocorticoid agonist-GR complex. Nearly undetectable amounts of RU 486 were recovered in the nucleus. Conversely, the RU 486-PR complex showed no difference of dissociation rate between the activated and the non activated condition. Its affinity for DNA-cellulose was the same as that of the activated progestin agonist-PR complex. A large amount of this compound was demonstrated in the nucleus.     

RU486, a progestin and glucocorticoid antagonist, inhibits the growth of breast cancer cells via the progesterone receptor

The progestin and glucocorticoid antagonist RU486 was tested on the growth of several cell lines in culture. RU486 inhibited the growth of two progesterone receptor (RP) positive human breast cancer cell lines (MCF7 and T47D). The antiproliferative effect was dose dependent and its magnitude correlated with the RP content of the tested cells (T47D greater than estradiol-primed MCF7 greater than withdrawn MCF7). Cell growth inhibition was not prevented by the addition of dexamethasone, dihydrotestosterone, or estradiol, but the cells were rescued by low concentrations of the progestin R5020. RU486 had no effect on the growth of two RP negative human breast cancer cell lines and a rat fibroblast cell line. Moreover, RU486 had no progestin agonist activity in T47D cells when evaluated by measuring the 35S-labeling of two progestin-regulated proteins with mol wts of 48,000 and 250,000, but it totally prevented the induction of these two proteins by R5020. In conclusion, RU486 selectively inhibited the growth of human breast cancer cell lines with unoccupied RP sites and its effect was correlated with the RP concentration of these cells. We propose that RU486 is a RP-targeted drug of potential utility in breast cancer treatment.     

Successful treatment of Cushing's syndrome with the glucocorticoid antagonist RU 486

A patient with Cushing's syndrome due to ectopic ACTH secretion was treated successfully with the new glucocorticoid antagonist RU 486 [17 beta-hydroxy-11 beta-(4-dimethylamino phenyl) 17 alpha-(1-propynyl)estra-4,9-dien-3-one]. This compound is a 19-nor steroid with substitutions at positions C11 and C17 which antagonizes cortisol action competitively at the receptor level. Oral RU 486 was given in increasing doses of 5, 10, 15, and 20 mg/kg . day for a 9-week period. Treatment efficacy was monitored by assessment of clinical status and by measuring several glucocorticoid-sensitive variables, including fasting blood sugar, blood sugar 120 min after oral glucose administration, and plasma concentrations of TSH, corticosteroid-binding globulin, LH, testosterone-estradiol-binding globulin, and total and free testosterone. With therapy, the somatic features of Cushing's syndrome (buffalo hump, central obesity, and moon facies) ameliorated, mean arterial blood pressure normalized, suicidal depression resolved, and libido returned. All biochemical glucocorticoid-sensitive parameters normalized. No side-effects of drug toxicity were observed. We conclude that RU 486 may provide a safe, well tolerated, and effective medical treatment for hypercortisolism.     

Effect of chronic treatment with the glucocorticoid antagonist RU 486 in man: toxicity, immunological, and hormonal aspects

Suppression of immune function was traditionally thought to occur only with pharmacological levels of glucocorticoids. However, recent studies in rodents have suggested that glucocorticoids exert tonic antiinflammatory/immunosuppressive effects even at basal nonstress concentrations. To examine whether basal glucocorticoid secretion modulates immune function in man we employed the specific glucocorticoid receptor antagonist RU 486. If a tonic level of inhibition of the immune system by basal glucocorticoid levels was present, then a potentiation or enhancement of immune function might evolve in the absence of glucocorticoid action. To examine this hypothesis, we studied 11 healthy male normal volunteers who received RU 486 (10 mg/kg.day) or placebo vehicle, divided into 2 daily oral doses, for 7-14 days. Blood samples were collected every 2 days for measurement of plasma ACTH and cortisol concentrations along with 24-h urine samples for measurement of 17-hydroxysteroid and free cortisol excretion. Complete and differential blood counts, erythrocyte sedimentation rates, C-reactive protein, antinuclear antibodies, rheumatoid factor, and quantitative immunoglobulins were also determined at 2-day intervals. Leukocytes were obtained by leukopheresis for phenotypic characterization and functional analysis before and 7 days after the initiation of RU 486 or placebo therapy. Blockade of cortisol receptors with RU 486 was associated with marked compensatory elevations of plasma ACTH and cortisol and increases in 24-h urinary excretion of 17-hydroxysteroids and free cortisol. Unexpectedly, 8 of the 11 subjects developed generalized exanthem after 9 days of RU 486 treatment. One subject developed symptoms and signs consistent with the diagnosis of adrenal insufficiency. Total white blood cell counts, absolute lymphocyte, neutrophil and eosinophil counts, erythrocyte sedimentation rate, and quantitative immunoglobulins did not change with RU 486 therapy. Similarly, T-, B-, and natural killer cell subsets did not change during RU 486 treatment. Furthermore, functional evaluation of lymphocyte cytotoxicity and proliferation revealed no changes. We conclude that administration of high doses of RU 486 to normal volunteers does not result in measurable enhancement of immune function. This suggests that in man, glucocorticoids may not exert a tonic inhibitory effect on the immune system as they appear to do in rodents. Alternatively, the compensatory increase in endogenous cortisol may obviate any effect of the glucocorticoid antagonist on the immune system.(ABSTRACT TRUNCATED AT 400 WORDS)     

Differential interaction of RU486 with the progesterone and glucocorticoid receptors

Steroid hormone receptor antagonists are widely used in clinical medicine, but their use is often complicated by the lack of receptor specificity to presently available drugs. We previously demonstrated an important role of a widely conserved helix 3 (H3)-helix 5 (H5) interaction in determining the sensitivity and specificity of steroid hormone receptors to receptor agonists. Intriguingly, the same H3 residues also play a crucial role in receptor antagonism; mutation of these residues alters the response of these receptors to antagonists. Given the close interaction of H3 and H5 residues at this site, we asked whether H5 residues might also play a role in the sensitivity of these receptors to antagonists. We demonstrate here that modification of H5 residues produces marked changes in the sensitivities of the glucocorticoid and progesterone receptor (PR) to RU486 antagonism. Moreover, while we confirm previous reports that alteration of the H3 residue, Gly 722 prevents RU486-mediated inhibition of the PR, we show that the corresponding substitution in the glucocorticoid receptor does not inhibit RU486-mediated receptor antagonism. Taken together, our data support the notion that RU486 binds differently to these two receptors, providing a potential target for the design of more specific antiglucocorticoid and antiprogestin drugs.     

Long-term treatment with RU486 and glucocorticoid receptor resistance Theoretical and therapeutic implications.

Glucocorticoid receptor resistance in humans occurs rarely in hereditary disorders. RU486 blocks progesterone receptors, but at higher doses also blocks glucocorticoid receptors. In this article, the clinical and biochemical effects of long-term treatment with RU486 of patients with meningiomas, breast cancer, and Cushing's syndrome are compared with those present in patients with hereditary glucocorticoid receptor resistance. The potential use of RU486 outside the field of fertility regulation is discussed.     

Parallel determination of glucocorticoid receptors in human mononuclear and polymorphonuclear leukocytes after Percoll separation

Circulating human leukocytes are known to possess glucocorticoid receptors, but the data in the literature on the affinity and capacity widely range. We here report our results on the determination of affinity and capacity of glucocorticoid receptors in both mononuclear and polymorphonuclear leukocytes using a Percoll gradient for separating cells. The mean affinity of mononuclear and polymorphonuclear cells was respectively 4.3 +/- 0.7 and 4.3 +/- 15 nM (mean +/- S.D., n = 6), and the capacity 4135 +/- 1950 and 1810 +/- 832 sites per cell. Previous reports using a Ficoll separation showed a lower affinity under the same conditions of incubation. From these findings we suggest that the separation with Percoll may give values closer to physiological than other separation techniques.     

Blockade of glucocorticoid receptor binding and inhibition of dexamethasone-induced muscle atrophy in the rat by RU38486, a potent glucocorticoid antagonist

Glucocorticoid hormones cause marked muscular atrophy, the mechanism of which is unknown. We employed a potent glucocorticoid antagonist, RU38486 [11 beta-(4-dimethylaminophenyl)17 beta-hydroxy-17 alpha-(prop-1-ynyl)estra-4,9-dien-3-one], to determine whether intracellular glucocorticoid receptors are involved. RU38486 was shown to be an effective blocker of glucocorticoid receptor binding in vivo and in vitro. Furthermore, this compound significantly blocked the loss of body and muscle weight caused by injection of dexamethasone. These data indicate that intracellular glucocorticoid receptors are important in the etiology of steroid myopathy. Studies with glucocorticoid antagonists may lead to the design of specific therapeutic modalities for the treatment of both endogenously and exogenously produced steroid myopathies.     

Effects of the progesterone antagonist RU486 on ovarian activity in the rat

Adult female rats were treated for 2 or 4 weeks with the progesterone antagonist RU486 to study its effect on the regulation of ovarian function. In rats with 5-day ovarian cycles, the vaginal cyclicity disappeared. Uninterrupted vaginal cornification emerged within 4 days after the start of treatment and cornification persisted for the whole period of treatment. It took more than 2 weeks after cessation of 2-4 weeks of treatment before 5-day vaginal cycles reappeared. Ovarian weights increased rapidly resulting from the accumulation of large numbers of corpora lutea. In addition, the ovaries developed occasional follicular cysts which could reach an extremely large size (2 mm or more). Analysis of serial histological sections of ovaries, combined with plasma concentrations of estradiol-17 beta and progesterone, indicated cyclic ovulation and corpus luteum formation together with persistence of functional activity of already existing and newly formed corpora lutea. RU486 seems to have the unique property of dissociating cessation of luteal activity and ovulation in rats. After treatment with RU486, pituitary enlargement and mammary gland alveolar development were observed. It is hypothesized that these effects result from unopposed estrogen action on PRL secretion. The effects of RU486 are reversible: 4 to 5 weeks after the end of treatment ovarian activity seems normal (as evidenced by reduction of ovarian weights and 5-day vaginal cycles) except for the presence of occasional large follicular cysts which may require longer periods for their regression.     

Differences in the interaction of RU 486 and ketoconazole with the second binding site of the glucocorticoid receptor

Ketoconazole and RU 486 are both antagonists of glucocorticoid hormone action at the target cell level. Both inhibit the binding of agonists to the receptor, although RU 486 is many-fold more potent. As reported here, ketoconazole, like all naturally occurring steroidal antiglucocorticoids, enhances the rate at which agonists dissociate from the glucocorticoid receptor. This indicates that this antiglucocorticoid interacts with the receptor at a site other than the agonist-binding site. RU 486, on the other hand, does not accelerate dissociation, suggesting that it does not interact with the putative allosteric regulatory site on the glucocorticoid receptor. Combining these findings with previous reports leads to the hypothesis that glucocorticoid antagonists can be divided into two classes: those that act through a second regulatory site on the glucocorticoid receptor and those that act by direct competition with agonists at the agonist-binding site.     

The antiprogestin RU38 486: receptor-mediated progestin versus antiprogestin actions screened in estrogen-insensitive T47Dco human breast cancer cells

Despite the theoretical promise of synthetic antiprogestational agents as anticancer agents, experimental tools, midcycle contraceptives, and implantation inhibitors, none has been available for either basic or clinical studies. However, a candidate antiprogestin, RU38 486 [17 beta-hydroxy-11 beta-(4-dimethylaminophenyl)17 alpha-(1-propynl)estra-4,9 -dien-3-one], has recently been described that has antiprogestational and antiglucocorticoid activities in early clinical trials. Its mechanisms of action are unclear. Furthermore, development of this drug underscores an old bioassay problem: that biological screening of progestins and antiprogestins is complex because of the physiological requirement that progestational effects must be superimposed upon an estrogenized system. This has made it difficult to distinguish among progestational, antiprogestational, and antiestrogenic properties of unknown agents. Here we describe the use of T47Dco human breast cancer cells to circumvent these problems. T47Dco cells are rich in progesterone receptors (PR), but are resistant to estrogens and antiestrogens. Their PR are estrogen-independent, and this permits progestins to be studied in an estrogen-free system. We have used these cells to assess the receptor-binding properties and the biological actions of RU38 486. Since RU38 486 absorbs UV at approximately 300 nm, this wavelength was used to covalently photolink the drug to PR in situ. Like the synthetic progestin R5020, low concentrations (10 nM) of [3H]RU38 486 bind two PR subunits in nuclei of T47Dco; glucocorticoid receptors are not bound. RU38 486 has a high affinity for PR in vitro (Kd approximately 2 nM at 0-4 C), and in intact cells, low concentrations (6-8 nM) transform more than 95% of PR to a high affinity nuclear binding state. In contrast to progesterone, the compound is not metabolized, so that it chronically (3-6 days) suppresses PR replenishment. These biochemical properties of RU38 486 are typical of synthetic progestins, but distinguish it from pure glucocorticoids. To bioassay RU38 486, we have measured growth and insulin receptors, since in T47Dco, physiological concentrations of progestins inhibit proliferation and increase the number of cell surface insulin-binding sites. Like progestins, RU38 486 is growth inhibitory; unlike progestins, it fails to stimulate insulin receptors and partially blocks their stimulation by R5020. Thus, RU38 486 has dual progestin agonist/antagonist actions depending on the biological response measured.     

Progesterone and RU486: opposing effects on human sperm.

Progesterone induced a rapid influx of calcium in capacitated human sperm, followed by a long-lasting, dose-dependent increase of intracellular free calcium. Thereafter, progesterone increased the fraction of hyperactivated sperm and the acrosome reaction. On the contrary, the progesterone antagonist RU486 (mifepristone) induced an immediate and transient, dose-dependent decrease of intracellular free calcium and a drop in the values of sperm movement parameters related to hyperactivation. Moreover, RU486 counteracted the effects of progesterone on calcium influx, lateral sperm head displacement, and the acrosome reaction. Therefore, RU486 effects were opposite to those of progesterone. The nature of the membrane receptor(s) involved is unknown. Several steroids bearing 11 beta-phenyl substitutions, with different pharmacological profiles, were also investigated. It was concluded that the steroid structure and chemical groups added to the 11 beta-phenyl influence effects on calcium influx.     

The progesterone antagonist RU486 acquires agonist activity upon stimulation of cAMP signaling pathways.

The protein kinase A stimulator cAMP can potentiate the ability of progestins to induce the transactivation function of the human progesterone receptor (hPR). We questioned in the present study whether cAMP could functionally cooperate with the progestin antagonist RU486. In T47D human breast cancer cells, RU486 behaves as a pure antagonist with respect to induction of the progesterone-responsive mouse mammary tumor virus chloramphenicol acetyltransferase (MMTV-CAT) reporter gene. It fails to stimulate MMTV-CAT expression and completely inhibits induction by the synthetic progestin R5020. However, when RU486 is combined with 8-bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP), MMTV-CAT is induced to levels approaching that stimulated by R5020 alone. Also, RU486 in the presence of 8-Br-cAMP is only partially effective in antagonizing R5020 action. The agonist activity exhibited under these conditions appears to be due to RU486 acting through hPR as evidenced by the fact that 8-Br-cAMP alone has no effect on MMTV-CAT, whereas induction by the combination of 8-Br-cAMP and RU486 is dose responsive to RU486 in a saturable manner and can be inhibited by the type I antiprogestin (prevents hPR-DNA binding) ZK98299, which does not exhibit positive functional cooperation with cAMP. Acquisition of agonist activity in the presence of 8-Br-cAMP also extends to the type II antiprogestin (permits hPR-DNA binding) ZK112993. Since RU486 is also a type II antagonist, these results suggest that detection of functional synergism between cAMP and antiprogestins may require binding of the hPR-antagonist complex to DNA. We propose that cross-talk between second messenger and steroid receptor signal transduction pathways may be one mechanism for resistance to steroid antagonists that frequently develops in breast cancer.     

The steroid hormone antagonist RU486. Mechanism at the cellular level and clinical applications.

The cellular and molecular mechanism of RU486, a steroid hormone antagonist, is discussed in detail. Principally, RU486 opposes the action of two types of hormones: progesterone and glucocorticosteroids. The clinical applications are also described, as well as the future outlook.     

A correlative study between glucocorticoid receptor levels in human mononuclear leukocytes and biochemical data in Cushing's disease

We determined glucocorticoid receptors in human mononuclear leukocytes in 9 patients with Cushing's disease, in order to correlate them with laboratory data. Receptors were measured by a whole-cell assay method, after incubation with [3H]-dexamethasone in the presence or absence of excess unlabelled hormone. In Cushing's disease, there were 4425 +/- 364 sites/cell (N = 9), similar to in the controls: 4473 +/- 476 (N = 10); average Kd was 2.42 +/- 0.52 nmol/l (N = 3) similar to in the controls: 2.0 +/- 0.20 nmol/l (N = 3). In Cushing's patients we found significant negative correlations between basal glucocorticoid receptors and: 1) morning blood cortisol (r = -0.67, P less than 0.05), and 2) 17-ketogenic steroids after 2 mg of dexamethasone (r = -0.85, P less than 0.01). No correlations were observed with afternoon blood cortisol, free urinary cortisol, basal and post-8-mg dexamethasone 17-ketogenic steroids, TRH-TSH area, urinary calcium, plasma glucose, or systolic blood pressure. Conclusions: In Cushing's disease, a subtle receptor down-regulation may exist, as suggested by the inverse relationship between glucocorticoid receptors and morning blood cortisol. Secondly, the relationship between basal receptors and 17-ketogenic steroids after 2 mg of dexamethasone suggests that glucocorticoid receptors in human mononuclear leukocytes could reflect the sensitivity of the nervous system-pituitary-adrenal axis to dexamethasone inhibition.     

Modulation of aromatase activity in human endometrial stromal cells by steroids, tamoxifen and RU 486

The regulation of aromatase activity (AA) in human endometrial stromal cells by various steroids was studied in primary cell culture. Various progestins, but not androgens or glucocorticoids, stimulated AA. Medroxyprogesterone acetate (MPA) was the most potent progestin. Estrogen (E) alone did not change the activity but it potentiated the stimulation of AA by progestin. Biphasic regulation of AA by progestin was noted in both time- and dose-dependent manners. Endometrial AA was stimulated by MPA and reached the maximum rate between 2-5 days of incubation with subsequent decline of AA in prolonged culture. When stromal cells were treated with MPA (0.03 to 30 microM) for 3 days, AA was increased over the control at all the concentrations tested. The maximum was found at doses between 0.1-1 microM. The activities reduced steadily from the maximum stimulation to less than 50% when the concentration of MPA increased from 1-30 microM. In addition, initial treatment of stroma cells with MPA (1-3 days) resulted in further increase of activity after progestin withdrawal. The enhancement of the induction of AA by E did not alter the biphasic pattern regulated by progestin alone, i.e. E enhanced both the stimulation and the decay of AA. The time study of the effect of E showed that enhancement of AA required at least 10 h of incubation of E with MPA conditioned cells. The effect of E is dose dependent between 0.04-40 nM and shows the greatest effect in the presence of MPA between 0.01-1 microM. The optimal concentrations of E and progestin that stimulate AA in culture are similar to the plasma concentrations after pregnancy, suggesting that the physiological function of the endometrial aromatase is at the time of decidualization. The effects of antiprogestin, Ru 486, and antiestrogen, tamoxifen (TAM), on AA were studied. Ru 486 or TAM alone did not alter AA. Ru 486 inhibited the MPA stimulated AA in a dose-dependent manner suggesting that the effect of progestin may be mediated through a receptor mechanism. Enhancement, but no inhibitory effect, was observed when cells were treated with TAM + MPA and TAM + MPA + E. The effectiveness of Ru 486 to inhibit the induction of AA in endometrial cells may be of primary importance for contraception.     

Blockade of the spontaneous midcycle gonadotropin surge in monkeys by RU 486: a progesterone antagonist or agonist?

Preovulatory ovarian secretion of progesterone (P4), several hours before the onset of the typical midcycle gonadotropin surge, occurs in humans and monkeys. We investigated the potentially obligatory role of preovulatory P4 secretion in stimulating the midcycle LH surge by administering a potent P4 antagonist, RU 486(17 beta-hydroxy-11 beta-[4-dimethylaminophenyl-1]17 alpha-[prop-1-ynyl]estra-4,9-dien-3-one), to sexually mature, normally ovulatory cynomolgus monkeys on days 10-12 of the menstrual cycle (n = 18). Monkeys were randomized to receive RU 486 alone (5 mg/day, im; group I); RU 486 plus dexamethasone (1 mg/day, im; group II); dexamethasone alone (group III); or vehicle (ethanol; 0.5 ml; group IV). Before drug treatment, the follicular phases were quite similar among groups. The administration of RU 486 blocked (delayed) the expected gonadotropin surge, despite rising estrogen concentrations (greater than 250 pg/ml). The expected LH surge was delayed by RU 486 (n = 5) or RU 486 with dexamethasone (n = 3) until 36 +/- 7 (+/- SEM) and 27 +/- 8 days in groups I and II, respectively. In contrast, groups III (n = 3) and IV (n = 5) had timely midcycle surges after the administration of dexamethasone or vehicle alone (4 +/- 2 and 6 +/- 2 days, respectively). The intermenstrual interval was lengthened by RU 486 administration in both group I and II animals (61 +/- 6 and 54 +/- 6 days) compared to controls (30 +/- 2; P less than 0.0001). In summary, RU 486 effectively blocked imminent midcycle gonadotropin surges, delayed subsequent folliculogenesis, and significantly extended the menstrual cycle length. If RU 486 acted as a pure P4 antagonist, then P4 is necessary for timely midcycle gonadotropin surges to occur. However, recent evidence showing agonistic properties of RU 486 (in the virtual absence of P4) at both endometrial and pituitary levels may favor a P4-like (agonistic) blockade of the estrogen-induced FSH/LH surges by RU 486.     

Insulin-degrading activity in mononuclear and polymorphonuclear circulating leukocytes of nondiabetic and diabetic subjects

Insulin-degrading activity in mononuclear (MN) and polymorphonuclear (PMN) fractions of circulating leukocytes obtained from 7 nondiabetic and 13 insulin-dependent diabetic subjects was studied. Insulin-degrading activity in both MN and PMN fractions was activated by reduced glutathione and was inhibited completely by N-ethylmaleimide. Both fractions had Michaelis-Menten constant (Km) (insulin) values within the range of values reported for purified glutathione-insulin transhydrogenase (GIT). In double immunodiffusion tests with antibody to human liver GIT, the MN fraction showed immunoprecipitin bands continuous with those of purified liver enzyme, but the PMN fraction showed little or no reaction with the antibody. These data indicate that both leukocyte fractions contain thiol-dependent insulin-degrading activity; however, only in the MN fraction was the degrading activity immunologically similar to that of liver GIT. Kinetic studies showed that the insulin-degrading activity of MN and PMN cells from diabetic patients had a 3.6- and 14.5-fold, respectively, higher maximal capacity (Vmax) than the insulin-degrading activity of these cells from nondiabetic subjects, without any change in the half-saturation constant for the substrate (Km for insulin). These results demonstrate that diabetes and/or insulin therapy result in increased leukocyte glutathione-dependent insulin-degrading activity.