Antiestrogen action of 2-hydroxyestrone on MCF-7 human breast cancer cells

The estrogen responsive human breast cancer MCF-7 cell culture was examined for its response to 2-hydroxyestrone a principal metabolite of estradiol. Addition of 2-hydroxyestrone to the cell cultures in concentration of 10(-9) - 10(-6) M had no effect on cell growth and proliferation because of rapid O-methylation of the catechol estrogen by catechol O-methyltransferase which is highly active in these cells. In the presence of quinalizarin, a potent catechol O-methyltransferase inhibitor which reduces the O-methylation of the steroid, 10(-7) M and 10(-8) M 2-hydroxyestrone markedly suppresses the growth and proliferation of the cells. The tumor cell growth-inhibitory action of the catechol estrogen was neutralized by the presence of 10(-9) M estradiol. The catechol estrogen inhibition of cell growth is not observed in the estrogen receptor-negative human breast cancer cell lines MDA-MB-231 and MDA-MB-330 providing evidence that the inhibition is specific and is estrogen receptor-mediated. In contrast, the 16 alpha-hydroxylated metabolites of estradiol, estriol and 16 alpha-hydroxyestrone, are effective stimulators of MCF-7 cell proliferation with the latter exhibiting potency in excess of that expected from its estrogen receptor affinity. The present results represent the first observation of a specific receptor-mediated antiestrogenic action of 2-hydroxyestrone and suggest that the physiological regulation of the agonist activity of the primary estrogen may involve in situ generation of catechol estrogen.     

Antiestrogen action in mammary cancer and in fetal cells

The present data confirm the very complicity of the response of antiestrogen when this compound is studied in different experimental conditions. The new and potent antiestrogen ICI 164,384, which is considered as a full antagonist in most models studied, concerning the progesterone receptor in the isolated cells of the uterus and vagina of guinea-pig acts as a real agonist. However, this compound antagonizes cell proliferation, progesterone receptor, and decreases the concentration of estradiol in different hormone-dependent mammary cancer cell lines. Another interesting aspect is the response of the antiestrogen 4-hydroxytamoxifen which in isolated cells of very close tissues such as the uterus and vagina is an antagonist for the former and agonist for the latter concerning the progesterone receptor. In conclusion, the present data added new information in the complicity of the mechanism of action of antiestrogens, but using new models interesting possibilities are opened to understand their responses and their mechanism.     

Antiestrogen action: Differential nuclear retention and extractability of the estrogen receptor

Since there is a much longer uterine nuclear retention of the U-11,100A (antiestrogen) receptor complex (UARC) than of the estradiol receptor complex (ERC) at 4–12 hrs after injection, experiments were designed to determine if there is a difference between the relative nuclear affinities for the two RCs as determined by extraction with various ionic strength mediums. Although the UARC was retained longer in the nuclear fraction $\text{in}$$\text{vivo}$, the UARC was completely extractable with 0.3M KC1 or 50mM spermine, whereas the ERC demonstrates a saltresistant form. This suggests that the ERC is more tightly bound to nuclear components through this salt-resistant form of the receptor. In addition, various intercalating agents were used to distinguish the different nuclear chromatin DNA sites where the UARC and ERC may be binding. With actinomycin D (50 uM) more ERC than UARC was retained in the nuclear fraction. However, with ethidium bromide (100uM) less ERC than UARC was retained. Also, the ERC selectively released by ethidium bromide is precisely that fraction not released by salt. These results indicate that the UARC and ERC bind to different chromatin loci.     

Aromatase activity and concentrations of cortisol, progesterone and testosterone in breast and abdominal adipose tissue

Aromatase activity and concentrations of cortisol, progesterone and testosterone were measured in samples of breast and abdominal adipose tissue obtained from both pre- and postmenopausal subjects. Enzyme activity was determined by the incorporation of tritium from [1 beta-3H]androstenedione into water and found to be in close agreement to that measured when tritium labelled oestrone (E1) and oestradiol (E2) were isolated. No significant difference in enzyme activity was noted between breast and abdominal adipose tissue. Increased aromatase activity was not observed in adipose tissue taken from a subject with endometrial cancer. Cortisol concentrations were found to be significantly higher (P less than 0.05) in abdominal as compared to breast tissue. Without attaining statistical significance progesterone concentrations were higher in abdominal as compared to breast adipose tissue. Aromatase activity was not related to either cortisol or testosterone tissue concentration, but an inverse relationship between progesterone concentration and aromatase activity was observed (r = 0.542, P less than 0.02). On the basis of results obtained a hypothesis for the increased conversion of androgen to oestrogen as seen after the menopause has been proposed.     

Metabolism of progesterone in mouse vaginal tissue

The $\text{in vitro}$ and $\text{in vivo}$ metabolism of 1,2- ³H-progesterone was studied in estrogen-stimulated and control vaginae of ovariectomized mice. Employing two-dimensional thin-layer chromatography, gas-liquid chromatography and metabolite “trapping” techniques, the major and minor pathways for progesterone metabolism were determined $\text{in vitro}$ and shown to involve saturation of the Δ⁴-double bond to yield 5α-pregnane compounds and reduction of the C20 and C3 ketone groups to form 20α- and 3α- and 3β-hydroxy derivatives, respectively. The quantities of 20β-hydroxy metabolites and 5β-epimers that were detected were considered not to be significant. The major metabolites formed by untreated tissues following $\text{in vitro}$ incubation in the presence of both high (10^?6M) and low (10^?8M) progesterone concentrations were 3α-hydroxy-5α-pregnan-20-one and 5α-pregnane-3,20-dione. Although these two derivatives were also found in sizable quantities in estrogen-treated tissues, a marked increase (5-fold) in the rate of C20 ketone reduction at high progesterone concentrations (10^?6M) to yield 20α-hydroxy-4-pregnen-3-one was demonstrated. Following intravaginal administration of ³H-progesterone $\text{in vivo}$, only progesterone and 3α-hydroxy-5α-pregnan-20-one were retained in appreciable quantities through 2 hr, suggesting rapid loss of 20α-hydroxy-4-pregnen-3-one and the 5α-pregnanediols from this tissue under $\text{in vivo}$ conditions.     

Natural progesterone and antihypertensive action.

In a placebo controlled, double blind crossover study natural progesterone was given by mouth, in increasing doses, to six men and four postmenopausal women with mild to moderate hypertension who were not receiving any other antihypertensive drugs. When compared with values recorded before treatment and during administration of placebo progesterone caused a significant reduction in blood pressure, suggesting that progesterone has an antihypertensive action rather than a hypertensive one as has been previously thought. This possible protective effect of progesterone should be investigated further.     

Involvement of phosphoinositide-3-kinase and phospholipase C transduction systems in the non-genomic action of progesterone in vascular tissue

We investigate the participation of tyrosine kinase, phosphatidylinositol-3-kinase, phospholipase C systems in the intracellular transduction pathways involved in the non-genomic stimulation of vasodilators compounds synthesis induced by progesterone (Pg). Using aortic strips isolated from female fertile Wistar rats, we showed that physiological concentrations of progesterone markedly increase prostacyclin synthesis in a very short time interval (45 s to 10 min) as well as nitric oxide release (5-30 min). The stimulatory action of progesterone on nitric oxide synthase (NOS) activity was maintained even in the presence of an antagonist of progesterone receptor, compound RU486. In contrast, in the presence of tyrosine kinase inhibitor (1 microM genistein) or phosphatidylinositol-3-kinase inhibitor (1 microM LY294002), the enhancement of nitric oxide elicited by 10-100 nM progesterone was completely suppressed. The steroid stimulates phopholipase C activity, inducing significant increase in diacylglycerol generation (5-15-min treatment). The presence of an inhibitor of protein kinase C (PKC) impaired the anti-aggregatory action of the hormone. Due to the fact that phospholipase C activation implies calcium mobilization, we investigate the role of changes in calcium fluxes on progesterone nitric oxide generation. We found that calcium influx from extracellular medium and calcium mobilization from internal pools was required. The present results suggest that, tyrosine kinase and phosphatidylinositol-3-kinase cascades are involved in progesterone nitric oxide synthase stimulation and that diacilglicerol/protein kinase C system may be relevant for physiological regulation of platelet aggregation process.     

Antiestrogen use in breast cancer patients reduces the risk of subsequent lung cancer: A population-based study

BackgroundThere is accumulating epidemiological and preclinical evidence that estrogen might be a driver of lung cancer. Breast cancer survivors can offer a unique patient cohort to examine the effect of antiestrogen therapy on lung cancer carcinogenesis because many of these women would have received long-term selective estrogen receptor modulators (SERMs) and/or aromatase inhibitors (AIs) as adjuvant treatment. Our hypothesis is that estrogens play a role in lung cancer development, and that antiestrogen therapy would affect the incidence of subsequent lung cancer among breast cancer survivors.MethodsUsing the Taiwan National Health Insurance (NHI) database, the study included 40,900 survivors of non-metastatic breast cancer after primary surgery, and most antiestrogen users complied well with the medication regimen. We evaluate the effect of antiestrogen therapy on the incidence of subsequent lung cancers.ResultsThis population-based study revealed that antiestrogen use in breast cancer patients was associated with a reduced risk of subsequent lung cancer in older patients (≥50 years) (HR 0.73, 95%CI 0.54–0.99) when compared with breast cancer survivors who did not use antiestrogens.ConclusionThe study supports the hypothesis that antiestrogen therapy modifies lung cancer carcinogenesis in older women. Further well-designed clinical trials to explore the potential of antiestrogens in lung cancer prevention and treatment would be worthwhile.     

Antiestrogen therapies affect tissue homeostasis of the gerbil (Meriones unguiculatus) female prostate and ovaries

The present work aims to evaluate the response of the adult gerbil female prostate (paraurethral glands) and ovaries to short-term exposure to antiestrogenic agents, consisting of daily oral doses of letrozole (1 mg kg(-1) day(-1)) or intradermal doses of tamoxifen (1 mg/kg) every other day for 21 days. The serum levels of testosterone and estradiol were monitored, and the prostates and ovaries collected for structural, ultrastructural, and immunocytochemical analyses. The letrozole treatment resulted in increases of serum testosterone levels and secretory activity as well as in glandular hyperplasia and dysplastic growth, simulating the effects caused by the exogenous androgens. The effects caused by tamoxifen indicate that this endocrine agent acted as an estrogenic agonist on the prostate, causing glandular hypertrophy, secretory activity decrease, and the development of prostatic lesions. Therefore, it is possible to conclude that the letrozole and tamoxifen therapies result in a series of complex effects that endanger the physiology of hormone-dependent organs, including the female prostate and ovaries. The hormonal imbalance caused by administration of these drugs resulted in considerable changes in prostatic morphology, in a manner very similar to what occurs during the development of prostatic lesions in aged postmenopausal women. Thus, these therapies must be chosen carefully since long-term treatments can result in female prostate dysplasic lesions.     

Induction of progesterone receptor in an estrogen, progesterone receptor-negative breast cancer cell line

In MCF-7 cell culture, some sera endow estradiol-17 beta with strong growth promoting properties ("active" sera) while other fail to display this property ("inactive" sera). Passage from "inactive" to "active" sera are shown here to induce the appearance of a progestin binding capacity in the receptor negative line Evsa-T. Competition with various unlabeled steroids established the specificity of this binding reaction. The induction of progesterone receptor required neither estrogens, nor ER and failed to confer major growth sensitivity to hormonal steroids: only medroxyprogesterone acetate was slightly inhibitory at high concentration. These observations disclose the influence of seric factors independent of estrogens and of ER-related mechanisms on PgR induction.     

Hypothalamic site of progesterone action on gonadotropin release

Tonic gonadotropin secretion was monitored at 20 min intervals for a total of 9 hours in 3 female volunteers during the mid-luteal phase of an ovulatory cycle. This control period was followed by repeated LH-RH stimulation (12 micrograms LH-RH as i.v. bolus once every hour for another 5 hours). During the control period spontaneous albeit low-frequent pulsatile secretion was observed for LH (a pulse occurring once every 3-8 hours) but not for FSH. While intermittent exogenous LH-RH stimulation was being performed at circhoral LH-RH pulse frequency pulsatile gonadotropin release was established at synchronous episodicity and systemic gonadotropin levels consecutively increased. These data provide indirect evidence that the pituitary gland is not rendered refractory to LH-RH by luteal progesterone secretion but readily responds to LH-RH stimuli even when these simulate a follicular phase LH-RH pulse frequency. Thus, it is concluded that spontaneous pulsatile LH release at low frequency during the luteal phase of the cycle reflects low frequent LH-RH discharges from the hypothalamus. Underlying mechanisms are discussed.     

Specific progesterone receptors in human breast cancer.

We have identified a specific progesterone receptor in 11 of 33 human breast cancer cytosols. Since progesterone itself binds to glucocorticoid receptor, to corticosteroid binding globulin (CBG), and to nonspecific components as well as to its own receptor, we have used a synthetic progestin, R5020 (17,21-dimethyl-19-nor-4,9-pregnadiene-3,20-dione), whose binding specificity is restricted to progesterone receptor. Bound R5020 sediments at 8 S in sucrose gradients; binding is competed by excess unlabeled R5020 or progesterone. The receptor is distinct from glucocorticoid receptor and CBG as determined by competition studies using dexamethasone and hydrocortisone. The dissociation constant for R5020 obtained by Scatchard analysis of dextran-coated charcoal assays is approximately 2 times 10- minus 9 M.