Duration of antagonizing effect of RU486 on the agonist induction of tyrosine aminotransferase via glucocorticoid receptor

The duration of the antagonizing activity of RU486 on tyrosine aminotransferase (TAT) induction and the glucocorticoid receptor in rat liver was studied. A single dose of RU486 (10 mg/kg) caused occupation of the cytosol glucocorticoid receptor in rat liver at 1h. During this time no nuclear binding of [³H]dexamethasone ([³H]Dex) receptor complex was recorded, and TAT induction was completely blocked. TAT inducibility recovery parallelled receptor binding in both the cytosol and the nuclei, reaching maximum at 12 h. In contrast, nuclear binding recovered in 24 h, and [³H]Dex receptor binding in cytosol 48 h after RU486 application. It is concluded that the inhibitory effect of a single dose of RU486 on TAT induction is of rather short duration. At concomitant presence of agonist and antagonist in vivo, no direct correlation between agonist receptor occupancy and TAT induction could be observed.     

Non-classical androgenic actions of RU38486 in androgen-responsive Shionogi carcinoma 115 cells in serum-free culture

Antiglucocorticoid and antiprogestin RU38486 (RU486) stimulated the growth of highly androgen- and moderately glucocorticoid-sensitive SC-3 cells (a cloned cell line from Shionogi mouse mammary carcinoma 115) in a dose-dependent manner. A maximal 8-fold stimulation of growth by RU486 has been observed at 10(-7) M in a serum-free medium and its potency has been found to be almost the same as that of dexamethasone (Dex). The growth rate of SC-3 cells treated by triamcinolone acetonide (TA) or Dex combined with RU486 at 10(-9)-10(-7) M was enhanced compared to cells treated by TA or Dex alone, indicating that RU486 had additive rather than antagonistic effects. Our previous study revealed that RU486 could compete with the specific uptake of [3H]testosterone in intact SC-3 cells at relatively low affinity and the present study showed that the stimulatory effect of RU486 on the growth of SC-3 cells was significantly inhibited by pure antiandrogen flutamine and that half-maximal inhibition by flutamine was achieved at 10(-6) M. Moreover, we demonstrated that the conditioned medium from RU486-stimulated SC-3 cells contained growth-promoting activity which caused a 3.5-fold increase in DNA synthesis by SC-3 cells in the absence of RU486 and which was abolished by treatment with heparin-Sepharose. These results indicate that RU486-induced growth of SC-3 cells may be expressed as an androgenic activity through androgen receptor and mediated by a heparin-binding growth factor.     

Evidence from pulse-chase labeling studies that the antiglucocorticoid hormone RU486 stabilizes the nonactivated form of the glucocorticoid receptor in mouse lymphoma cells

A pulse-chase labeling technique was used to determine the properties of glucocorticoid receptors occupied by the antiglucocorticoid hormone RU486 in S49.1 mouse lymphoma cells. Cells were pulse-labeled with [35S]methionine and then at the beginning of the chase, either no hormone (control), dexamethasone, or RU486 was added to cells. At 4 h into the chase, cytosol was prepared and receptors were immunoadsorbed to protein A-Sepharose using the BuGR2 antireceptor antibody. Immunoadsorbed proteins were resolved by gel electrophoresis and analyzed by autoradiography. The 90 kDa heat shock protein (hsp90) coimmunoadsorbed with receptors from control cells when protein A-Sepharose pellets were washed with 250 mM NaCl but not when protein A-Sepharose pellets were washed with 500 mM NaCl, indicating that hsp90-receptor complexes are disrupted by a high concentration of salt in the absence of molybdate. hsp90 coimmunoadsorbed with receptors from RU486-treated cells even when protein A-Sepharose pellets were washed with 500 mM NaCl, indicating that RU486 stabilizes the association of hsp90 with the glucocorticoid receptor. In contrast, hsp90 did not coimmunoadsorb with receptors from dexamethasone-treated cells, consistent with earlier evidence that hsp90 dissociates from the receptor when the receptor binds glucocorticoid hormone. Dexamethasone induced a rapid quantum decrease in the amount of normal receptor recovered from cytosol but did not induce a decrease in the amount of nuclear transfer deficient receptor recovered from cytosol, consistent with tight nuclear binding of normal receptors occupied by dexamethasone. In contrast, RU486 did not induce a quantum decrease in the recovery of normal receptors from cytosol, indicating that receptors occupied by RU486 are not tightly bound in the nuclear fraction. We conclude that the antiglucocorticoid hormone RU486, in contrast to the glucocorticoid hormone dexamethasone, stabilizes the association between the glucocorticoid receptor and hsp90. The decreased affinity of receptors occupied by RU486 for the nuclear fraction may be due to their association with hsp90 and may account for the failure of RU486 to exert agonist activity.     

Interrelationship between RU38486 and the P450 activities in rat liver

Microsomal P450 monooxygenases contribute actively to the biotransformation of the antiglucocorticoid RU38486, an 11 beta-substituted nor-steroid. Pretreatment of adult rats by inducers of specific forms, belonging to different P450 subfamilies, affects the ability of liver microsomes to metabolize RU38486. Phenobarbital and pregnenolone 16 alpha-carbonitrile increase the metabolic activity of liver microsomes whereas methylcholanthrene decreases their capacity to oxidize the steroid. Thus P450 forms IIIA, IIB1,2 and IIC7 are good candidates to be involved in the degradation of this peculiar molecule. Our study has been completed by investigating whether RU38486 would influence the P450 spectrum. Whereas the treatment of rats with either a glucocorticoid (cortisol, dexamethasone) or an antiglucocorticoid (pregnenolone 16 alpha-carbonitrile) has been shown to induce the P450 activity by increasing the hepatic concentration of form IIIA, we observed a slight decrease of the P450 activity by treating the animals with RU38486. Moreover RU38486 was able to antagonize the P450 induction by the other steroids as well as it inhibits the synthesis of various liver enzymes induced by glucocorticoids (for instance tyrosine aminotransferase). These findings may be important for the therapeutic use of RU38486 since its inhibitory effect on P450 activity may be at the origin of drug interactions by modifying the endogenous hormonal status.     

Glucocorticoid receptors take part in the apoptotic process of human lens epithelial cells, but the glucocorticoid receptor antagonist RU486 does not rescue the cells fully

To identify an agent with specific activity against human lens epithelial cells (HLECs), we confirmed the presence of glucocorticoid receptors (GRs) and GR-α genes and evaluated whether GRs have a relationship with the apoptotic process in cultured HLECs. We also determined whether the inhibitor RU486 could rescue the cells from apoptosis when the HLECs were exposed to dexamethasone (Dex), a steroid, in 4 concentrations for 4 periods, or were co-treated with the antagonist RU486. We found that Dex, which has been used as a medical agent for a long time, resulted in increased expression of GRE-luciferase, the GR-α gene and GR-protein and, in contrast, decreased the viability of HLECs. The expression of Bax protein was increased in an earlier stage in contrast to the expression of Bcl-2 protein, which was increased in a later stage. Caspase-3 activity was significantly increased under lower concentrations of Dex in the last stage. The nuclear morphology of HLECs showed an obvious apoptotic phenomenon under greater concentrations of Dex in the last stage. However, RU486, a GR antagonist, could partially inhibit GR and Bax expressions and the expression of caspase-3 was increased so that there was not a decrease in the ratio of apoptotic cells and an increase in the viability of HLECs. Our data showed that GRs had a partial relationship to the apoptotic process of HLECs when exposed to Dex and RU486 did not rescue the cells fully. Because of its toxicity, RU486 did not provide a therapeutic benefit in a glucocorticoid induced cataract (GIC) for the in vitro model, however, its activity and pathway targeting should still be studied further with appropriate drug combinations.     

RU486, a progestin antagonist, binds to progesterone receptors in a human endometrial cancer cell line and reverses the growth inhibition by progestins

The human endometrial cancer cell line, IK-90 cells, contains estrogen-independent progesterone receptors (PR) and is progestin sensitive. Accumulation of glycogen in the cytoplasm of IK-90 cells as well as growth inhibition of the cells in response to progestins are observed. In the present study, the effects of RU486, a progestin antagonist, on IK-90 cells were investigated in a serum-supplemented medium. Scatchard plot analysis of cytoplasmic binding data in the cells revealed a high affinity binding site for RU486 (Kd, 2.6 nM) with maximum binding sites of 169 fmol/mg protein. However, the binding ability to DNA-cellulose of heat activated [3H]RU486-PR complexes was lower when compared with that of the progestin agonist [3H]R5020-PR complexes, suggesting a decrease in progestin activity of RU486 in IK-90 cells. The addition of 1 microM RU486 to culture medium produced periodic acid-Schiff-positive granules in the cytoplasm of the cells. On the other hand, RU486 (1 nM-1 microM) did not significantly inhibit the growth of cells. However, RU486 (0.1-1 microM) totally prevented the growth-inhibitory effect of R5020 (0.1-1 microM) on IK-90 cells. In conclusion, RU486, an antiprogestin, had a dual activity both a progestin antagonist and weak agonist in human endometrial cancer cells, which was not mediated through the estrogen receptor system.     

Luteolytic effect of the antiprogestin and antiglucocorticoid agent RU486 in rats

Ovarian cells of pregnant rats were cultured with synthetic progestins (R5020, R2323), dexamethasone and RU486. Progesterone and 20 alpha-hydroxy-pregn-4-en-3-one (20 alpha-dihydroprogesterone) in the medium were measured by specific radioimmunoassay. Both R5020 and R2323 increased concentrations of these intrinsic progestins. RU486 decreased concentrations of progesterone, however, the addition of R5020 or R2323 counteracted this action. Immature hypophysectomized rats treated with pregnant mare serum gonadotropin (PMS) and human chorionic gonadotropin (hCG) were administered with RU486; the serum levels of progesterone and 20 alpha-dihydroprogesterone tended to decrease. R5020 and R2323 inhibited the effect of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD), whereas RU486 did not. Inhibition of the cholesterol side chain cleavage enzyme (CSCC) by RU486 was more marked than that by R5020 or R2323. These results show that RU486 decreases progesterone synthesis in cultured ovarian cells. A part of the mechanism may involve an inhibition of CSCC.     

Regulation of cytochrome P450scc synthesis and activity in the ovine corpus luteum

The rate-limiting step in luteal biosynthesis of progesterone consists of cleavage of the side chain of cholesterol by mitochondrial cytochrome P450 side-chain cleavage enzyme (P450_scc) to form pregnenolone. Luteal mRNA encoding P450_scc, quantitated on selected days of the 16-day ovine estrous cycle, was similar on days 3 and 6, increased by 2-fold on day 9 (P < 0.05) and remained elevated on day 15. Levels of P450_scc mRNA on day 15 of pregnancy were not different from those found on any day of the cycle (P < 0.05). To determine whether levels of mRNA encoding P450_scc are hormonally regulated, ewes on day 10 of the estrous cycle were injected with hCG or prostaglandin F₂ (PGF₂). P450_scc mRNA was not increased for up to 36 h after injection of hCG, nor decreased within 8 h after injection of PGF₂ (P < 0.05). An assay for P450_scc activity was developed which utilized ovine small and large luteal cells in the presence of 22R-hydroxycholesterol and ovine high density lipoprotein. Enzyme activity was quantitated by measurement of progesterone production. In small luteal cells activation of the protein kinase A (PKA) second-messenger system by treatment with LH resulted in 910% increase in progesterone production without altering activity of P450_scc. Activation of the protein kinase C (PKC) second-messenger system with phorbol 12-myristate 13-acetate caused a 51% reduction in progesterone secretion from large luteal cells but did not alter activity of P450_scc. These findings suggest that in mature luteal tissue steady state levels of mRNA encoding P450_scc, and enzyme activity are independent of acute regulation by activation of PKA or PKC second-messenger systems.     

Effect of the progesterone antagonist RU486 on human myometrial spontaneous contractility and PGI2 release.

We studied the effect of antiprogesterone RU 486 on spontaneous uterine contractility and PGI2 release with human myometrial strips superfused "in vitro". A decrease of PGI2 release into the superfusion medium was observed after 20 min superfusion. The inhibition was dose-dependent and reversible. After 20 min washing with tyrode medium without RU 486, the uterine strips recovered their initial rate of release. R5020, a progesterone agonist, did not affect PGI2 release nor dexamethasone and testosterone. Parallel to the decrease of PGI2 observed during RU 486 superfusion, the uterine spontaneous contraction frequency decreased, while the amplitude and duration of contractions increased. The alteration of uterine contractility was also rapid, dose-dependent and reversible. Modification of uterine strip spontaneous contractility, similar to those induced by RU 486, were also observed with superfusions of R5020 at concentrations as low as 10(-9)M, dexamethasone (10(-8)M), but not with superfusions of testosterone. These observations are not in favour of a progesterone-receptor mediated effect of RU 486 in our model. The mechanism of action may be related to the antiprogesterone specific structure i.e. the bulky substituent at the C-11 position. The RU 486 effect on uterine strip contractility, mimicked by other steroids, could point to a non-specific lipid/membrane interaction. However, the fact that testosterone did not affect motility, may indicate a possible specificity of steroids having a 3 oxo pregnene structure.     

Regioselective metabolism of phenanthrene in Atlantic cod (Gadus morhua): studies on the effects of monooxygenase inducers and role of cytochromes P-450

The polycyclic aromatic hydrocarbon phenanthrene was converted mainly (>90%) to the 1,2-dihydrodiol when metabolized in vivo by the marine teleost cod. This is also found in other bony fishes, but contrary to what is known from cartilaginous fish, crustaceans and mammals, where the K-region 9,10-dihydrodiol is the main metabolite. When liver microsomal preparations from differently pretreated cod were incubated with phenanthrene in vitro, the metabolic profile was dramatically different from the in vivo pattern, as shown by gas chromatography—mass spectrometry. The microsomes from untreated, phenanthrene, phenobarbital and pregnenolone-16-carbonitrile-treated cod converted phenanthrene mainly, but to a varying extent, to the 9,10-dihydrodiol. Treatment with β-naphthoflavone (BNF), however, resulted in a large increase in the oxidation at the 1,2-position, along with a four- to seven-fold increase in specific activity. The major cytochrome P-450 isozyme purified from BNF-treated cod liver (P-450c) showed highest activity with phenanthrene (a turnover of 0.18 nmol/min per nmol P-450), but with about equal selectivity for the 1,2- and 9,10-region of the substrate in a reconstituted system with phospholipid and NADPH-cytochrome P-450 reductase. The low regioselectivity was also observed as a lack of regioselective inhibition of microsomal phenanthrene metabolism with antiserum to cod P-450c. Two of the minor isozymes, cod cytochromes P-450b and d, showed a similar turnover to P-450c, but with a stronger selectivity for the 1,2-position (55–60%). The results indicate that other control systems, in addition to the content of individual P-450-forms in the regulatory systems, in addition to the content of individual P-450-forms in the endoplasmic reticulum, are involved in the in vivo transformation of phenanthrene by cod to the 1,2-dihydrodiol metabolite.     

Plasma concentrations and receptor binding of RU 486 and its metabolites in humans

Using Chromosorb chromatography and HPLC, we measured the plasma concentrations of RU 486, and its monodemethylated (RU 42633), didemethylated (RU 42848) and alcoholic nondemethylated (RU 42698) metabolites up to 72 h following oral ingestion of 100 mg of RU 486 by five female volunteers. The peak plasma level of RU 486 (4.5 mumol/l) occurred within 1 h after ingestion of the compound; at this point significant amounts of the metabolites were also present in the plasma. After the initial redistribution within 6 h the plasma concentrations of RU 486 and three of its metabolites measured remained stable for 24 h. Concentrations of the monodomethylated metabolite exceeded those of the parent steroid during the time period measured, whereas the concentrations of the didemethylated and alcoholic metabolites were lower than those of RU 486, but still notable. At 72 h the concentrations of all the four steroids were still in the micromolar range. The relative binding affinities of these metabolites to human endometrial and myometrial progesterone receptors as well as to human placental glucocorticoid receptors were determined in vitro. The affinity of RU 486 for the human uterine progesterone receptor (Kd = 1.3 X 10(-9) M for RU 486) was higher than that of progesterone but lower than that of ORG-2058, a potent synthetic progestin. The relative binding affinities of the monodemethylated, alcoholic and didemethylated metabolites to the progesterone receptor were 21, 15 and 9%, respectively, compared with the parent compound RU 486; each was lower than that of progesterone (43%). RU 486 had an approx. 4-fold higher relative binding affinity to the glucocorticoid receptor than dexamethasone. Interestingly, the relative binding affinities of the metabolites studied to the human glucocorticoid receptor exceeded those of dexamethasone or cortisol. Compared with the parent compound RU 486, they were 61, 48 and 45% for the monodemethylated, alcoholic and didemethylated metabolites, respectively; each was higher than that of dexamethasone (23%). The affinity of dexamethasone to the human glucocorticoid receptor was 1.6 X 10(-9) M. These data indicate that the pool of certain metabolites of RU 486 may contribute to a significant extent to the antiprogestagenic (23-33%) and even greater extent to the antiglucocorticoid (47-61%) effects of RU 486.