Effects of retinoic acid on estrogen- and thyroid hormone-induced growth in a newly established rat pituitary tumor cell line.

In order to elucidate the complex mechanism(s) of action of steroid hormones, thyroid hormone and retinoic acid in pituitary mammotrophs, a clonal cell line (G3) was isolated from the rat pituitary tumor MtT/F84. G3 cells were found to secrete prolactin constitutively and to contain receptors for estrogen, glucocorticoid, progesterone and thyroid hormone. Stimulation of G3 cells with thyroid hormone resulted in a modest but significant increase in estrogen and progesterone receptor levels, however, retinoic acid treatment had no effect. Simultaneous addition of thyroid hormone and estrogen showed an additive effect on progesterone receptor levels in G3 cells. Thyroid hormone as well as estrogen enhanced the growth of G3 cells. Interestingly, retinoic acid was also found to enhance their growth but its enhancement was less potent than thyroid hormone and estrogen. Low concentrations of estradiol and thyroid hormone showed additive effects, but G3 cells stimulated with high concentrations of thyroid hormone failed to elicit an additive effect with estrogen, suggesting the presence of a common pathway in the growth-stimulatory actions of these hormones. In addition, exposure of G3 cells to retinoic acid completely abolished the effects of estrogen or thyroid hormone in terms of cell growth. These results suggest that there are complex interactions in the signalling pathways for estrogen, thyroid hormone and retinoic acid action in G3 cells.     

The use of the biotinyl estradiol-avidin system for the purification of "nontransformed" estrogen receptor by biohormonal affinity chromatography

Several biotinyl estradiol derivatives have been prepared by coupling estradiol 7 alpha-carboxylic acid to biotin via different linear linkers. All these compounds exhibit a high affinity for the estrogen receptor as determined by competitive binding assays against [3H]estradiol. These compounds also displaced the dye 4-hydroxyazobenzene-2'-carboxylic acid from the biotin-binding sites of avidin free or immobilized on agarose. It was demonstrated that only the derivatives bearing a long spacer chain (greater than 42 A greater than) between estradiol and biotin were able to bind receptor and avidin simultaneously, suggesting some steric hindrance. The biotin-avidin system has been investigated for the purification of the cytosoluble "nontransformed" estrogen receptor stabilized by sodium molybdate. The method relies on: 1) high biohormonal affinity of receptor for biotinyl estradiol derivative; 2) the specific selection by avidin-agarose column of biotinyl estradiol-receptor complexes; and 3) the biohormonal elution step by an excess of radioactive estradiol. Starting from unfractionated cytosol containing molybdate-stabilized nontransformed 8S estrogen receptor with estradiol 7 alpha-(CH2)10-CO-NH-(CH2)2-O-(CH2)2-O-(CH2)2-NH-CO-(CH2)3-NH-biotin, preliminary experiments using avidin-agarose chromatography and then a specific elution step by exchange with free [3H]estradiol, allowed a 500-1,500-fold purification. Further purification of estrogen receptor was obtained by ion exchange chromatography through a DEAE-Sephacel column and led to a congruent to 20% pure protein, assuming one binding site/65,000-Da unit. The hydrodynamic parameters of the purified receptor were essentially identical to those of molybdate-stabilized nontransformed receptor present in crude cytosol. The advantages of this double biotinyl steroid derivative-avidin chromatographic technique over more conventional affinity procedures are discussed and make it applicable to the purification of minute amounts of steroid receptors in a wide variety of tissues.     

17Alpha-alkan (or alkyn) amide derivatives of estradiol as inhibitors of steroid-sulfatase activity

To develop inhibitors of steroid sulfatase without residual estrogenic activity, we have designed a series of estradiol (E2) derivatives bearing an alkan (or alkyn) amide side chain at position 17alpha. A hydrophobic alkyl group was selected from our previous study where 17alpha-octyl-E2 was found to inhibit strongly the steroid-sulfatase activity. Furthermore, it is known that an alkylamide side chain blocks the estrogen-receptor activation. Starting from ethynylestradiol, the chemical synthesis of target compounds was short and efficient with overall yields of 22-42% (3 or 4 steps). Among these compounds, N-octyl,N-methyl-3-(3',17'beta-dihydroxy-1',3',5'(10')-estratrien- 17'alpha-yl)-propanamide (15) was the most potent inhibitor, with an IC50 value of 0.08 microM for the transformation of estrone sulfate (E1S) to estrone (E1) by homogenated JEG-3 cells. N-butyl, N-hexyl, and N,N-dioctyl propanamide derivatives of E2 (IC50 values of 6.4, 2.8, and >20 microM, respectively) were less potent inhibitors than N-octyl analog 15. Furthermore, the unsaturated propynamide analog of 15 gave lower inhibition (four times) than the saturated compound. Compound 15 is also about 100-fold more effective in interacting with the enzyme than substrate E1S itself. The ability of target compounds to bind the estrogen receptor, to stimulate the proliferation of estrogen-sensitive ZR-75-1 cells, or to inhibit the E2-stimulation of ZR-75-1 cells was also evaluated. Although a mixed estrogenic/anti-estrogenic activity was obtained for tested compounds at 1 microM, no estrogenic activity was observed at 0.03 microM for 15. In conclusion, a promising inhibitor of steroid-sulfatase activity was obtained by introducing a hydrophobic octyl group in a 17alpha-propanamide side chain of E2, but further structure-activity relationships (SAR) studies are necessary to minimize the residual estrogenic activity.     

Antiestrogens inhibit the mitogenic effect of growth factors on breast cancer cells in the total absence of estrogens

The antiproliferative effect of antiestrogens in breast cancer is believed to be entirely due to the inhibition of estrogen induced growth. We show here that non-steroidal antiestrogens inhibit the growth of the human breast cancer MCF7 cells in the complete absence of estrogens (phenol-red-free medium) when cell proliferation is stimulated by insulin or epidermal growth factor. This non-antiestrogenic effect of antiestrogens is, however, mediated by accessible estrogen receptor sites, as it is not observed in receptor negative hormone-independent breast cancers, and is rescued by estradiol but not by insulin. We conclude that antiestrogens inhibit cell proliferation by inhibiting growth factor action as well as estrogen action and that in both cases, accessible estrogen receptors are required.     

Melatonin blocks the activation of estrogen receptor for DNA binding.

The present study shows that melatonin prevents, within the first cell cycle, the estradiol-induced growth of synchronized MCF7 breast cancer cells. By using nuclear extracts of these cells, we first examined the binding of estradiol-estrogen receptor complexes to estrogen-responsive elements and found that the addition of estradiol to whole cells activates the binding of the estrogen receptor to DNA whereas melatonin blocks this interaction. By contrast, melatonin neither affects the binding of estradiol to its receptor nor the receptor nuclear localization. Moreover, we also show that addition of estradiol to nuclear extracts stimulates the binding of estrogen receptor to DNA, but this activation is also prevented by melatonin. The inhibitory effect caused by melatonin is saturable at nanomolar concentrations and does not appear to be mediated by RZR nuclear receptors. The effect is also specific, since indol derivatives do not cause significant inhibition. Furthermore, we provide evidence that melatonin does not interact with the estrogen receptor in the absence of estradiol. Together, these results demonstrate that melatonin interferes with the activation of estrogen receptor by estradiol. The effect of melatonin suggests the presence of a receptor that, upon melatonin addition, destabilizes the binding of the estradiol-estrogen receptor complex to the estrogen responsive element.     

2,5-Diphenylfuran-based pure antiestrogens with selectivity for the estrogen receptor alpha

The estrogen receptor alpha (ERalpha) is understood to play an important role in the progression of breast cancer. Therefore, pure antiestrogens with a preference for this receptor form are of interest as new agents for the treatment of this malignancy. Several chemical structures with selective binding affinity for ERalpha have been identified and might be useful for the synthesis of ERalpha-selective pure antiestrogens. In this study we applied the 2,5-diphenylfuran system which is closely related to the triphenylfurans described by others. Various side chains with amino and/or sulfur functions were linked to C3 to convert the furans to estrogen antagonists without residual estrogenic activity. The degree of alpha-selectivity which ranges from 2.5- to 236-fold is strongly influenced by the alkyl group at C4. Antiestrogenic potency was determined in MCF-7/2a breast cancer cells stably transfected with a luciferase gene under the control of an ERE. The 2,5-bis(4-hydroxyphenyl)furan with an ethyl substituent and a 6-[N-methyl-N-(3-pentylthiopropyl)amino]hexyl side chain exerted the strongest antiestrogenic effect in this series with an IC(50) value of 50 nM in cells stimulated with 1 nM estradiol. The RBA values of this derivative were 18% (ERalpha) and 3.4% (ERbeta) of estradiol, respectively. It inhibited the growth of wild-type MCF-7 cells with an IC(50) value of 22 nM. The data show that the 2,5-diphenylfuran system is appropriate for the development of pure antiestrogens with preference for ERalpha.     

Structure-function relationships of the raloxifene-estrogen receptor-alpha complex for regulating transforming growth factor-alpha expression in breast cancer cells.

Amino acid Asp-351 in the ligand binding domain of estrogen receptor alpha (ERalpha) plays an important role in regulating the estrogen-like activity of selective estrogen receptor modulator-ERalpha complexes. 4-Hydroxytamoxifen is a full agonist at a transforming growth factor alpha target gene in situ in MDA-MB-231 human breast cancer cells stably transfected with the wild-type ERalpha. In contrast, raloxifene (Ral), which is also a selective estrogen receptor modulator, is a complete antiestrogen in this system. Because D351G ERalpha allosterically silences activation function-1 activity in the 4-hydroxytamoxifen-ERalpha complex with the complete loss of estrogen-like activity, we examined the converse interaction of amino acid 351 and the piperidine ring of the antiestrogen side chain of raloxifene to enhance estrogen-like action. MDA-MB-231 cells were either transiently or stably transfected with Asp-351 (the wild type), D351E, D351Y, or D351F ERalpha expression vectors. Profound differences in the agonist and antagonist actions of Ralcenter dotERalpha complexes were noted only in stable transfectants. The agonist activity of the Ralcenter dotERalpha complex was enhanced with D351E and D351Y ERalpha, but raloxifene lost its agonist activity with D351F ERalpha. The distance between the piperidine nitrogen of raloxifene and the negative charge of amino acid 351 was critical for estrogen-like actions. The role of the piperidine ring in neutralizing Asp-351 was addressed using compound R1h, a raloxifene derivative replacing the nitrogen on its piperidine ring with a carbon to form cyclohexane. The derivative was a potent agonist with wild type ERalpha. These results support the concept that the side chain of raloxifene shields and neutralizes the Asp-351 to produce an antiestrogenic ERalpha complex. Alteration of either the side chain or its relationship with the negative charge at amino acid 351 controls the estrogen-like action at activating function 2b of the selective estrogen receptor modulator ERalpha complex.     

Estradiol and raloxifene decrease the formation of multinucleate cells in human bone marrow cultures

Estrogen (E2) deficiency is responsible for increased bone turnover in the postmenopausal period, and it can be prevented by estrogen replacement therapy. The way estrogen acts on bone cells is not fully understood. Human bone marrow cell cultures may be a reliable model for studying the action of steroids on osteoclastogenesis in vitro. We examine the effects of estradiol and Raloxifene, a selective estrogen receptor modulator, on human primary bone marrow cells cultured for 15 days. 17beta-estradiol and Raloxifene significantly decreased the number of tartrate-resistant acid phosphatase multinucleate cells from osteoclast precursors on day 15. Estrogen receptor alpha (ER-alpha) mRNA was present in bone marrow mononuclear cells cultured for 5 days, but there was no estrogen receptor beta (ER-beta) mRNA, suggesting that this effect was mediated by ER-alpha. 15-day cultures no longer contained ER-alpha mRNA, suggesting that estrogen acts on early events of osteoclast differentiation. Finally, 10-8 M 17beta-estradiol has no effect on the release of IL-6 and IL-6-sr into the medium of marrow mononuclear cells cultured for 5 or 15 days. Osteoclast apoptosis was not affected by estradiol or Raloxifene after 15 days of culture under our conditions. In conclusion, we have shown that both estradiol and Raloxifene inhibit osteoclast differentiation in human bone marrow mononuclear cultures. The biological effect that can mimic in vivo differentiation could be mediated through ER-alpha.     

Glucocorticoids inhibit estradiol-mediated uterine growth: possible role of the uterine estradiol receptor

Stress-related activation of the hypothalamic-pituitary-adrenal axis (HPA) is associated with suppression of the reproductive axis. This effect has been explained by findings indicating that corticotropin-releasing hormone suppresses hypothalamic gonadotropin-releasing hormone (GnRH) secretion via an opioid peptide-mediated mechanism, and that glucocorticoids suppress both GnRH and gonadotropin secretion and inhibit testosterone and estradiol production by the testis and ovary, respectively. To evaluate whether glucocorticoids suppress the effects of estradiol on its target tissues, we examined the ability of dexamethasone to inhibit estradiol-stimulated uterine and thymic growth in ovariectomized rats. Estradiol alone, given daily for 5 days, caused dose-dependent uterine and thymic growth. Dexamethasone alone, given daily for 5 days, caused a dose-dependent decrease in body weight gain and in thymic growth. When estradiol and dexamethasone were administered simultaneously, however, body weight gain and thymic growth were also inhibited (p less than 0.05). Dexamethasone decreased estradiol-induced uterine cytosolic and nuclear estrogen receptor concentrations (E2 R0, p less than 0.05; E2nR0, respectively), but had no effect on estradiol-induced progesterone receptor concentrations (P4R0, p greater than 0.05). Levels of uterine glucocorticoid receptors were not affected by estrogen and/or dexamethasone treatment. These findings suggest that stress levels of glucocorticoids, administered over a 5-day interval, block the estradiol-stimulated growth of female sex hormone target tissues. This effect may be partially mediated by a glucocorticoid-induced decrease of the estradiol receptor concentration. Thus, another mechanism by which the HPA may influence reproductive function during stress is by a direct effect of glucocorticoids on the target tissues of sex steroids.     

Insulin receptor substrate-1 expression is regulated by estrogen in the MCF-7 human breast cancer cell line

Estrogens can stimulate the proliferation of estrogen-responsive breast cancer cells by increasing their proliferative response to insulin-like growth factors. The mechanism underlying the increased proliferation could involve the induction of components of the insulin-like growth factor signal transduction pathway by estrogen. In this study we have examined the regulation of the expression of insulin receptor substrate-1, a major intracellular substrate of the type I insulin-like growth factor receptor tyrosine kinase. Estradiol increased insulin receptor substrate-1 mRNA and protein levels at concentrations consistent with a mechanism involving the estrogen receptor. Insulin receptor substrate-1 was not induced significantly by the antiestrogens tamoxifen and ICI 182,780, but they inhibited the induction of insulin receptor substrate-1 by estradiol. Analysis of tyrosine-phosphorylated insulin receptor substrate-1 showed that the highest levels were found in cells stimulated by estradiol and insulin-like growth factor-I, whereas low levels were found in the absence of estradiol irrespective of whether type I insulin-like growth factor ligands were present. Insulin receptor substrate-2, -3, and -4 were not induced by estradiol. These results suggest that estrogens and antiestrogens may regulate cell proliferation by controlling insulin receptor substrate-1 expression, thereby amplifying or attenuating signaling through the insulin-like growth factor signal transduction pathway.     

In vivo estrogenic and antiestrogenic activity of phenolphthalein and derivative compounds

The estrogenic activity of phenolphthalein and other related triphenylmethane dyes was evaluated in vivo in the immature rat uterus. Phenolphthalein behaved as a partial agonist of estradiol in stimulating the growth of rat uterus. Other specific estrogenic effects of the dye included an increase of the uterine DNA content, histological changes and induction of estrogen-modulated secretory proteins. The progressive introduction of side chains in the triphenylmethane skeleton concomitantly decreased the estrogenic activity. Triphenylmethanes competed with [3H]estradiol for the binding to the estrogen receptor in vitro, the relative binding affinity being correlated with the estrogenic potency observed in vivo. Phenolphthalein also showed antiestrogenic activity that could be overcome by increasing the dose of estradiol.     

2-Methoxymethylestradiol: a new 2-methoxy estrogen analog that exhibits antiproliferative activity and alters tubulin dynamics

An estradiol metabolite, 2-methoxyestradiol (2-MeOE(2)), has shown antiproliferative effects in both hormone-dependent and hormone-independent breast cancer cells. Previously, a series of 2-hydroxyalkyl estradiol analogs had been synthesized in our laboratories as potential probes for comparison of estrogen receptor (ER)-mediated versus non-ER-mediated effects in breast cancer cells. A methoxy derivative of 2-hydroxymethyl estradiol was prepared for biological evaluation and comparison with 2-MeOE(2). Estrogenic activity of the synthetic analogs was evaluated in two ways, one by examining affinity of the analogs for the estrogen receptor in MCF-7 cells and the other by examining the ability of the analogs to induce estrogen-responsive gene expression. The analog, 2-methoxymethyl estradiol (2-MeOMeE(2)), demonstrated weak affinity for the estrogen receptor (0.9% of estradiol) and weak ability to stimulate estrogen-induced expression of the pS2 gene (0.02% of estradiol). Antitumor activity was evaluated both in vitro and in vivo. The steroidal nucleus seems to be an attractive target for developing novel tubulin polymerization inhibitors. Additionally, such steroidal compounds may have low toxicity compared to the natural products known to interact with tubulin. Interestingly, 2-MeOMeE(2) inhibited tubulin polymerization in vitro at concentrations of 1 and 3 microM and was more effective than 2-MeOE(2). In cells, 2-MeOMeE(2) was effective in suppressing growth and inducing cytotoxicity in MCF-7 and MDA-MB-231 breast cancer cells. The cytotoxic effects of 2-MeOMeE(2) are associated with alterations in tubulin dynamics, with the frequent appearance of misaligned chromosomes, a significant mitotic delay, and the formation of multinucleated cells. In comparison, 2-MeOE(2) was more effective than 2-MeOMeE(2) in producing cytotoxicity and altering tubulin dynamics in intact cells. Assessment of in vivo antitumor activity was performed in athymic mice containing human breast tumor xenografts. Nude mice bearing MDA-MB-435 tumor xenografts were treated i.p. with 50 mg/kg per day of 2-MeOMeE(2) or vehicle control for 45 days. Treatment with 2-MeOMeE(2) resulted in an approximate 50% reduction in mean tumor volume at treatment day 45 when compared to control animals and had no effect on animal weight. Thus, 2-MeOMeE(2) is an estrogen analog with minimal estrogenic properties that demonstrates antiproliferative effects both in vitro and in the human xenograft animal model of human breast cancer.     

Significance of lipoidal estradiol in human mammary tumors

Incubations of p-nitrophenyl fatty acyl esters and estradiol-17 beta fatty acyl 17-esters with porcine esterase, human mammary tumor cytosol and rat uterine cytosol leads to ester hydrolysis of compounds with short chain fatty acids. Esters with long chain fatty acids show no hydrolysis except in the presence of Tween 80. Short chain fatty acid esters have a higher binding potency to the estrogen receptor than long chain fatty acid esters. Extraction of the nuclear receptor peak sedimenting at 4.6S and identification of the steroid showed that about 90% of the radioactivity was associated with estradiol and only 10% with estradiol esters. These studies show that estradiol fatty acyl esters act as a storage form from which estradiol is released by enzymatic hydrolysis.     

Interactions of progesterone and dihydroprogesterone with dihydrotestosterone on estrogen activated sexual receptivity in female mice

Lordosis behavior in response to a mounting stimulation was quantified in ovariectomized female CD-1 and SW mice primed with estradiol benzoate and either progesterone or dihydroprogesterone. Both progestins were effective in stimulating receptivity, in CD-1 females, while only progesterone was effective in SW females. Dihydrotestosterone given concurrently with both the estrogen and progestin injections was found to have no effect on progesterone stimulated receptivity, but it produced a dose related inhibition of receptivity in the dihydroprogesterone treated females.     

Experimental Study on the Estrogen-Like Effect of Boric Acid

There are now considerable evidences that boric acid has reproductive and developmental toxicity, but it is uncertain whether such toxicity is caused by estrogen-like effect. Our objective is to determine the estrogen-like effect of boric acid. Proliferation assay of MCF-7 human breast cancer cells, uterotrophic assay, measure assay of the estradiol (E₂), proliferation assay of mucous membrane cells, and assay of estrogen receptor were conducted in this study. Boric acid could increase the weight of uterus of ovariectomized SD rats and the height of epithelium cells of mucous membrane, enhance the expression of the proliferating cell nucleus antigen, and reduce the density of estrogen receptors. However, boric acid could not affect the level of estradiol in serum and stimulate the proliferation of MCF-7 human breast cancer cells. In this study, boric acid exhibited the estrogen-like effect in vivo.